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 Veterinary Drugs

With increased regulations and growing consumer awareness, the need to

quickly identify antibiotics, steroids, and other growth hormones present in
meat and poultry has never been greater. Agilent has significant experience
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 Transfer and Optimization of Existing
Methods for Analysis of Antibiotics in
Meat to Agilent Poroshell 120 EC-C18
Columns using MS/MS Detection

Application Note
Food

Authors Abstract
William J. Long, Anne E. Mack and In this work, a generic gradient method with UV detection is used to evaluate mobile
James R. Evans phase choices for fast method optimization of antibiotics analysis in meat, with the
Agilent Technologies, Inc. ultimate goal of producing a mass spectrometer compatible method. This evaluation
2850 Centerville Road included four buffers and two organic choices. The mobile phase combination that
Wilmington, DE 19809 yielded the best separation is transferred and optimized to an Agilent Poroshell 120
USA EC-C18 2.1 mm × 100 mm, 2.7 µm column. Gradient time was decreased from 45 min
to 12 min. Time can be further reduced using a 3 mm × 50 mm column, at the cost of
some resolution. The method is demonstrated on an Agilent 6410 triple quadrupole
LC/MS System coupled with an Agilent 1200 Series Rapid Resolution LC.
Table 1. Method Parameters for Various Column Dimensions
4.6 × 50 mm Poroshell 120 EC-C18 3.0 × 50 mM Poroshell 120 EC-C18 2.1 × 100 mM Poroshell 120 EC-C18
Mobile Phase A: Buffer, varies A: 10 mm ammonium formate pH 3.8 A: 10 mm ammonium formate pH 3.8
B: Organic, varies B: Acetonitrile B: Acetonitrile
Gradient 10-40% B 10-40% B 10-40% B
Gradient Time 12 min 12 min 12 min
Flow Rate 2 mL/min 0.85 mL/min 0.42 mL/min
Injection Volume 0.5 µL 5 µL 2.5 µL or 10 µL
Sample 0.1 mg/mL antibiotics 1 µg/mL antibiotics 1 µg/mL or 10 ng/mL antibiotics
TCC Temperature 30 °C 30 °C 30 °C
Detector DAD: Sig = 270, 4 nm; MS/MS: See Table 2 MS/MS: See Table 2
Ref = 360, 100 nm

Introduction Table 2. MRM Transitions for Antibiotic Compounds.

Administration of antibiotics is a common practice in chicken, Compound Precursor Product Fragmentor Collision
name ion ion voltage energy
pork, beef and fish farming. Many domestic cattle receive vari-
ous antibiotics in their feed for the prevention and control of dis- Sulfamerazine 265 172 100 25
Sulfamerazine 265 108 100 25
ease caused by fungi and bacteria. Many countries regulate
Thiamphenicol 338 308 140 10
acceptable residue levels of these compounds in agricultural Thiamphenicol 338 118 140 50
and animal products. In this work, an older method is transferred
Sulfamethazine 279 124 100 25
from a 5 µm, 250 mm column to a new superficially porous col- Sulfamethazine 279 108 100 30
umn to increase the speed of the analysis and change the Furazolidone 226 137 140 25
method of detection from UV to MS/MS. An increase in Furazolidone 226 122 140 25
throughput of 5 to 10 times is demonstrated, while minimally Sulfamonomethoxine 281 126 100 25
impacting sample preparation. Since the analysis time is short- Sulfamonomethoxine 281 108 100 25
ened dramatically, time is available for optimization of mobile Oxolinic acid 262 160 100 40
phase selectivity (pH, buffer types and organic modifier). Oxolinic acid 262 130 100 45
Pyrimethamine 249 198 140 45
Transition methods can be developed by modifying an existing Pyrimethamine 249 128 140 60
method or starting fresh. In this case, the objective was to Sulfadimethoxine 311 156 140 25
develop a new MS-compatible separation from an existing UV Sulfadimethoxine 311 108 140 55
separation. Consequently, a change in the mobile phase was Sulfaquinoxaline 301 129 100 50
required because 0.7 % phosphoric acid is not a desirable sol- Sulfaquinoxaline 301 108 100 40
vent for MS detection. A generic screening method using 0.1 % Difurazone 361 222 100 15
formic acid was investigated, but additional MS-compatible sol- Difurazone 361 154 100 45
vent systems were also evaluated. In this work a method is
developed by first screening different mobile phase combina-
tions using a short Agilent Poroshell 120 column using UV
detection, then transferring that method to an Agilent 6410 triple
Experimental
quadrupole LC/MS System. A major advantage of the Agilent Method development is based upon the use of a generic gradi-
Poroshell 120 EC-C18 is that it uses the same 2 µm frit as the ent. Using a short 4.6 mm × 50 mm Poroshell 120 EC-C18,
original 5 um column, negating the need for sample preparation 2.7 µm column, several different mobile phases can be quickly
method development. evaluated. The generic gradient is run at 2.0 mL/min, starts at
Agilent Poroshell 120 EC-C18 4.6 mm × 50 mm, 2.7 µm columns 10% and proceeds to 40% organic over 12 min. This gradient is
have similar performance to 1.8-µm totally porous Agilent later transferred to 2.1 mm × 100 mm and 3 mm × 50 mm
ZORBAX Eclipse Plus C18 columns, but since they use 2-µm col- columns by changing the gradient according to Equation 1. The
umn frits similar to those found on 5-µm columns, they require three gradients used are listed in Table 1 with MRM transitions
no additional sample preparation. This allows for a more seam- shown in Table 2. MS-compatible mobile phases consisting of
less method transfer. While some previous work demonstrates volatile buffer components such as formic acid, ammonium for-
the use of Agilent Poroshell 120 columns on older Agilent 1100 mate buffer and ammonium acetate buffer are used.
systems, they are ideally used on more modern systems such as
the Agilent 1200 or 1260 series UHPLC’s.

2
An Agilent 1200 Rapid Resolution LC (RRLC) system was Three Agilent Poroshell 120 EC-C18 columns were used in
used for this work: this work:

• G1312B Binary Pump SL. • 4.6 mm × 50 mm, 2.7 µm p/n 699975-902

• G1367C Automatic Liquid Sampler (ALS) SL. • 3.0 mm × 50 mm, 2.7 µm p/n 699975-302
• G1316C Agilent 1290 Infinity Thermostatted Column • 2.1 mm × 100 mm, 2.7 µm p/n 695775-902
Compartment (TCC) SL.
The compounds of interest are shown in Figure 1, with their
• G1315C Agilent Diode Array Detector (DAD) SL using a respective structures. Compounds were dissolved in water at
G1315-60024 micro flow cell (3-mm path, 2-µL 1 mg/mL. Equal aliquots were combined to produce a mixed
volume). sample. Compounds were purchased from Sigma Aldrich
(Bellefonte, PA). Additionally, methanol, acetonitrile, ammoni-
• G6410 Agilent Triple Quadrupole LC/MS System with
um formate, ammonium acetate, formic acid, and glacial
Electrospray (ESI).
acetic acid were purchased from Sigma Aldrich. Water used
• ChemStation version B.04.01 was used to control the was 18 M-W Milli-Q water (Bedford, MA).
HPLC and process the data. Agilent MassHunter Version
2.0 was also used to control the Agilent 6410 Triple Buffers used in this work were prepared by dissolving an
Quadrupole LC/MS System, the Agilent 1200 Rapid appropriate amount of the ammonium salt to produce a
Resolution LC (RRLC), and to analyze the data. 10 mM solution, adding 950 mL water and titrating the solu-
tion with either formic acid (for the ammonium formate
buffers) or glacial acetic acid (for the ammonium acetate
buffers). The buffer solutions were then brought to a 1 L
volume.

CH3 CH3

N NH2
N
O
HN N HN N CH3
Cl Cl
O S O OH HO S OH N O O
O S
S S NH
HN O N NH O
H3C O
OH N
NH2
NH2 OH NH2 H3CO N OCH3
Sulfamerazine Thiamphenicol Sulfadimidine Sulfadimethoxine Sulfamonomethoxine
(SMR) (TCP) (SDD) (SDMX) (SMMX)
O OH
Cl CH3 H 2N
O O HO Na
N NO 2 +
O
O N N N
N O N S
N
O Furazolidone O O O
H2N N NH2 O N
CH3 (FZD)
Pyrimethamine Oxolinic acid
Sulfaquinoxaline
(PYM) (OXA)
(SQX)
O
+ O NH NH 2
N N
HO
NH

Difurazone
(DFZ)

Figure 1. Compounds of interest.

3
Results and Discussion pump or the degasser. These columns can be used for LC/MS
but typically smaller diameter columns such as 3.0 or 2.1 mm
The original method published in 2002 by Kumagai and columns are used.
Onigbinde provides an effective method for the analysis of
As discussed in reference 5, once a separation has been opti-
antibiotics in meat using UV detection. As seen in Figure 2,
mized according to selectivity and retention index, it is possi-
the method separates the analytes in approximately 45 min.
ble to further improve the chromatography by varying column
However the nonvolatile phosphoric acid in the mobile phase
length, particle size and flow rate. However the k* value must
is not compatible with MS detection.
be maintained, while varying these column conditions so as
In many cases, simple scaling of a method will allow for a fast not to lose selectivity.
method transfer. In this case, however, a change in the mobile
Equation 1: k* = (tgF)/(d/2)2L(D%B)
phase was required for LC/MS compatibility. The use of short
Poroshell 120 EC-18 4.6 mm × 50 mm, 2.7 µm columns for Where:
assessing mobile phase changes has several advantages. One tg is the gradient time
advantage is that they allow quick separations without sacri- F is the flow rate
ficing resolving power. In addition, since they are used at 2 L is the column length
mL/min with a generic gradient, the solvent is rapidly purged d is the column internal diameter
through the system. This ensures that the solvent screening D%B is the change in organic content across the gradient
experiment can be quickly performed by changing solvent bot- segment
tles, with no concerns about residual solvents in the HPLC

Original Method Kumagai and Onigbinde 5988-7135 June 2002

Only 338 and 360 wavelengths are shown for brevity.

mAU

20 at 224 nm

10

0
0 5 10 15 20 25 30 35 min

mAU

20 at 360 nm DFZ
10 FZD

0
0 5 10 15 20 25 30 35 min
Instrument: Agilent 1100 Series HPLC
Column: 250 mm × 4 mm id, RP-18 Purospher, 5 µm, p/n 79925PU-584
Mobile phase: A = 0.7% Phosphoric acid, B = CH3CN
Gradient: 0.0 min 5% B; 10.0 min 5% B; 40.0 min 65% B; 45.0 min
65% B; Post Time 7.0 min 5% B
Flow rate: 1.0 mL/min
Temperature: 40 °C
Injection volume: 20 µL
Diode array detector: A-338/10 nm, reference wavelength off
B-264/8 nm, reference wavelength off
C-360/8 nm, reference wavelength off

Figure 2. Original method produces excellent results on a 250 mm column with UV detection.

4
As illustrated in Figure 3, generic gradients using methanol or Many selectivity improvements and changes can be produced
acetonitrile are used to separate the compounds of interest. by choice of pH or organic modifier. As noted earlier, the peak
The gradients using methanol generate 50% higher pressure shape of many basic compounds are improved when using
(300 bar instead of 200 bar). While this is not critical when methanol, however Poroshell 120 EC-C18 yields excellent
using a 50 mm column, this does become more important as peak shape for all compounds in this study. By adjusting the
the length of the column is increased to 100 or 150 mm. pH even slightly, both the elution order and peak spacing can
be changed. This is most evident in Figure 3, where methanol
With methanol, the last compound elutes later due to the and pH act to dramatically change the elution order. For the
lower solvent strength. Formic acid, while a convenient compounds in this study the best mobile phase combination
mobile phase additive, produces less optimal results than is found at pH 3.8, ammonium formate with acetonitrile.
10 mM ammonium formate buffer (pH =3), particularly for
pyrimethamine. In addition to peak shape improvements,
elution order changes also occur most notably with
pyrimethamine.

Fast evaluation of two low pH MS friendly mobile phases and two

organic modifiers using Agilent Poroshell 120 EC-C18

300 Bar
1 4 5
3
7 6,8,9 0.1 % HCOOH pH 2.7 CH3OH
2 10
1 SMR
2 TPC
1
4 3 SDD
3 5 4 FZD
7 8,9 10 mM NH 4HCO 2 pH 3.0 CH 3OH
6 5 SMMX
2 10 6 PYM
7 OXA
8 SDMX
205 Bar 9 SQZ
1 10 DFZ
3 5
2 4 8 9 0.1 % HCOOH pH 2.7 CH3CN
7
6 10

1
3
5 10 mM NH 4HCO 2 pH 3.0 CH 3CN
2 4 6 8 9
7 10

0 2 4 6 8 10 12 min

10% to 40% B/12 min at 2 mL/min

Agilent Poroshell 120 EC-C18 4.6 mm × 50 mm, 2.7 µm

Figure 3. Comparison of chromatographic conditions: buffer, 0.1 % formic acid, CH3OH, CH3CN.

5
 Acetonitrile with ammonium formate buffer yields excellent peak
shape and selectivity with pH 3.8 being optimal for these analytes

Vary mobile phase additive, CH3CN solvent 205 Bar

1 3 5
2 4 7 8 9 0.1 % HCOOH pH 2.7
6
10
1 SMR
2 TPC
1
3 5
3 SDD
2 4 7 8 9
10 mM NH 4HCO 2 pH 3.0 4 FZD
6
10 5 SMMX
6 PYM
7 OXA
1 3 5 8 SDMX
2 4 7,6 8 9 10 mM NH 4HCO 2 pH 3.4 9 SQZ
10 10 DFZ

1 3 5
2 10 mM NH 4HCO 2 pH 3.8
4 7 8 9
6
10

1
3 5
2
4 89 10 mM CH 3COONH 4 pH 4.8
7 6
10

0 1 2 3 4 5 6 7 8 min

10% to 40% B/12 min at 2 mL/min

Agilent Poroshell 120 EC-C18 4.6 mm × 50 mm, 2.7 µm

Figure 4. Comparison of buffers with CH3CN.

6
 Methanol with ammonium acetate buffer yields excellent peak
shape with pH 4.8 being optimal for these analytes

Vary mobile phase additive, CH3OH solvent 300 Bar

1
4 5
3
7 6,8,9 0.1 % HCOOH pH 2.7
2
10
1 SMR
1 2 TPC
4 3 SDD
3 5 4 FZD
7 8,9 10 mM NH 4HCO 2 pH 3.0
6 5 SMMX
2 10 6 PYM
7 OXA
1 8 SDMX
4 8,9 9 SQZ
5
3 7 10 DFZ
6 10 mM NH 4HCO 2 pH 3.8
2
10

1
4 5
3 6
7,8
9 10 mM CH 3COONH 4 pH 4.8
2
10

0 2 4 6 8 10 12 min

10% to 40% B/12 min at 2 mL/min

Agilent Poroshell 120 EC-C18 4.6 mm × 50 mm, 2.7 µm

Figure 5. Comparison of buffers with CH3OH.

7
Figure 6 illustrates a total ion chromatogram based on the with the same gradient with only the flow rate changed. If the
scouting work shown in Figures 3, 4 and 5. Conditions were gradient had been exactly scaled, the analysis time would
scaled according to Equation 1 for the 3.0 mm × 50 mm col- have been twice as long, but as illustrated, the resolution is
umn. This easy change demonstrates that the 3 mm column adequate. Figure 7 shows an MRM chromatogram of the
can be easily used for both conventional UV and more sensi- antibiotic mixture. The compounds are sufficiently separated
tive MS. In addition, a 2.1 mm × 100 mm column is also used even with a large sample volume injected on-column.
Conditions are listed in Tables 1 and 2.

Overlay of 3.0 × 50 and 2.1 × 100 mm columns using the same gradient parameters
×104
4 3
8 Agilent Poroshell 120 EC-C18 3.0 × 50 mm 1. SMR
10-40 %B/12 min @ 0.85 mL/min 2. TPC
Sample: 5 µL of 1 µg/mL antibiotics 3. SDD
3 5
MS Source: Gas Temp = 350 °C 4. FZD
1 6 5. SMMX
2 Gas Flow = 12 L/min
6. PYM
Nebulizer = 40 psi 7. OXA
7 9
1 Capillary = 4000 V 8. SDMX
2 4
10
MRM transitions found in Table 2 9. SQZ
10. DFZ

Agilent Poroshell 120 EC-C18 2.1 × 100 mm

×104 8
5 10-40 %B/12 min @ 0.42 mL/min
3 Sample: 2.5 µL of 1 µg/mL antibiotics
4 MS Source: Gas Temp = 350 °C
5 Gas Flow = 12 L/min
3
6 Nebulizer = 40 psi
1
2 9 Capillary = 4000 V
7
MRM transitions found in Table 2
1 2 4
10

1 2 3 4 5 6 7 8 9 10 11
Counts vs. Acquisition Time (min)

Figure 6. Total ion chromatograms of antibiotic mixture on 3 × 50 mm, and 2.1 × 100 mm Agilent Poroshell 120 EC-C18 columns.

8
 Optimized MRM of 10 antibiotics in less than 11 minutes on Agilent Poroshell 120 EC-C18

×102 3 5
9
1
1 SMR
8 2 TPC
3 SDD
4 FZD
7 5 SMMX
89 6 PYM
7 OXA
6 7 8 SDMX
9 SQZ
10 DFZ
5

4
6

4
2 2

1
10

1 2 3 4 5 6 7 8 9 10
Counts vs. acquisition time (min)

Agilent Poroshell 120 EC-C18 2.1 mm × 100 mm, 2.7 µm

10% CH 3CN at t 0, ramp to 40% CH 3CN in 12 min (buffer 10 mM NH 4HCO 2 pH 3.8 adjusted with concentrated formic acid), 0.42 mL/min
Sample: 10 uL of 10 ng/mL antibiotics
**using dynamic MRM mode on MS/MS**

Figure 7. Dynamic MRM of antibiotic mixture on Agilent Poroshell 120.

9
Conclusions References
This work shows that in method migration, modern colums 1. W. Long, and A. Mack, “Fast Analysis of Sulfa Drugs
and fast liquid chromatographs make it easier to start fresh. using the Agilent 1100 Series LC with Agilent Poroshell
Using a generic gradient on short columns, 10 mobile phase 120 EC-C18 columns,” Agilent Technologies publication
combinations are quickly evaluated. Following basic scaling 5990-5572EN, 2010.
equations, a method can easily be transferred to a column of
2. A. Gratzfeld-Hüsgen, and E. Naegele, “Maximizing effi-
another dimension. By optimizing the mobile phase using a
ciency using Agilent Poroshell 120 columns,” Agilent
UV detector, the method is partially developed on an instru-
Technologies publication 5990-5602EN,2010.
ment that may be commonly used in a lab rather than the
more expensive and possibly less available instrument that 3. W. Long, and A. Mack, “Fast, Low Pressure Analysis of
the method will be transferred to. Food and Beverage Additives Using a Superficially Porous
Agilent Poroshell 120 EC-C18 Column,” Agilent
Poroshell 120 columns are good to use for LC/MS of complex Technologies publication 5990-6082EN, 2010.
samples at low pressure. Regardless of the analytical power
of the triple quadrupole mass spectrometer, a better separa- 4. A. Mack, and W. Long, “Fast Analysis of Environmental
tion simplifies data analysis, which may shorten cycle time. Phenols with Agilent Poroshell 120 EC-C18 columns,”
Baseline separated compounds also allow the mass spec- Agilent Technologies publication 5990-6156EN, 2010.
trometer to maximize dwell time for a given peak to yield 5. Snyder, Kirkland, Glach “Practical HPLC Method
more accurate and reproducible results. This ensures the best Development,” Chapter 8, 2nd ed. John Wiley & Sons,
possible quantitation. Additionally, less chance of ion sup- 1997
pression is possible caused by coeluting compounds.
6. The Agilent 1200 Series Rapid Resolution LC Method
Several additional factors are also demonstrated. Optimal Translator and Cost Savings Calculator
conditions for this mixture are found using the fast scouting http://www.chem.agilent.com/en-us/products/instru-
method in acetonitrile ammonium formate buffer pH 3.8 ments/lc/pages/gp60931.aspx
(8 min). The analysis also works in methanol with pH 4.8,
ammonium acetate (13 min). This could easily be shortened For More Information
by changing the gradient to elute the last peak more quickly.
For example, ramp organic more quickly at the end with a sec- For more information on our products and services, visit our
ond step; however this would increase pressure further. The Web site at www.agilent.com/chem.
use of a “true buffer” such as 10 mM ammonium formate pro-
vides better peak shape for bases than a buffering solution
such as 0.1 % formic acid at similar pH. The method as shown
is chromatographically optimized and work is in progress to
optimize detection conditions.

www.agilent.com/chem
Agilent shall not be liable for errors contained herein or
for incidental or consequential damages in connection
with the furnishing, performance, or use of this material.

Information, descriptions, and specifications in this

publication are subject to change without notice.

© Agilent Technologies, Inc., 2010

Printed in the USA
August 31, 2010
5990-6238EN
 Determination of Sulfonamide
Residues in Chicken Muscle by
Agilent SampliQ QuEChERS AOAC
Kit and HPLC-FLD

Application Note
Food

Authors Abstract
Bellah O. Pule, Lesego C. Mmualefe, An HPLC-Florescence detection (FLD) method has been developed and validated for
Nelson Torto the determination of nine sulfonamides in chicken muscle, after precolumn derivatiza-
Department of Chemistry tion with fluorescamine. The analyzed drugs include sulfadiazine, sulfathiazole,
Rhodes University sulfamerazine, sulfamethazine, sulfamethizole, sulfamethoxypyridazine, sulfachloropy-
P. O. Box 94, Grahamstown 6140 ridazine, sulfamethoxazole and sulfadimethoxine with sulfapyridine as an internal
South Africa standard. The derivatives were separated on an Agilent ZORBAX Eclipse Plus C18
column (4.6 mm × 75 mm, 3.5 µm) using a gradient elution with a binary system of
methanol–0.05 M acetate buffer (pH 4.5) and fluorescence detection at excitation and
emission wavelengths of 406 and 496 nm, respectively. The method employs a mul-
tiresidue sample preparation procedure based on QuEChERS (quick, easy, cheap,
effective, rugged and safe) which was adopted from the Association of Analytical
Communities (AOAC) Official method 2007.01 for extraction and cleanup. The recover-
ies ranged from 76.8% to 95.2% with relative standard deviation from 1.5% to 4.7% at
the 50, 100 and 150 ng/g fortification levels. The limits of detection and quantification
ranged from 0.02 to 0.39 and 0.25 to 1.30 ng/g respectively.
Introduction The AOAC QuEChERS method has been widely applied in the
analysis of pesticides in food since it was introduced by
Sulfonamides are a broad spectrum of antimicrobial drugs USDA scientists [8]. In general, there are two major steps:
used mainly in veterinary practice for prophylactic, therapeu- extraction and dispersive SPE cleanup. The method uses a
tic or growth promoting purposes [1]. They are the treatment single-step buffered acetonitrile (1% HOAc) extraction while
of choice for disease control of coccidiosis in poultry manage- simultaneously salting out from the aqueous sample using
ment [2-3]. Their use in human therapy has since become lim- anhydrous magnesium sulphate (MgSO4) to induce liquid-liq-
ited due to the advent of antibiotics [4]. There is a health risk uid partitioning. For cleanup, a dispersive solid phase extrac-
associated with consumption of animal products contaminat- tion (dSPE) step is employed using a combination of primary
ed with sulfonamide residues. The residues are usually the secondary amine (PSA) to remove fatty acids and other com-
result of inappropriate administration or withdrawal from ponents, and anhydrous MgSO4 to reduce the remaining
these drugs. The presence of sulfonamide residues can trig- water in the extract. Other sorbents may be added in this
ger adverse side effects such as allergic reactions in hyper- step, such as graphitized carbon black (GCB), to remove pig-
sensitive individuals and are potential carcinogens in the long ments and sterol, or C18 to remove more lipids and waxes.
term. Furthermore, prolonged exposure to sulfonamide
residues may give rise to an increase in drug-resistant bacte- This application note presents a method for the determination
ria [5]. In order to protect consumers from risks related to the of sulfonamide drugs in chicken muscle with HPLC-FLD after
drug residues, maximum residue limits (MRL) have been a precolumn derivatization with fluorescamine, which is a flu-
established by law in many countries. In Europe (EU orogenic reagent specific for primary aliphatic and aromatic
Regulation 1999), Canada and USA (FDA Regulation 1991) the amines [9–10] such as the sulfonamides in the study
MRL for the total sulfonamide concentration in edible tissue (Figure 1). The method includes sample preparation with
is 100 µg/kg while it is 20 µg/kg in Japan [6-7]. SampliQ AOAC Buffered Extraction kit (p/n 5982-5755) and
SampliQ AOAC Fatty Dispersive SPE 15 ml kit (p/n 5982-
5158).

O O O N O O N
O
H2N S N S S
NH S NH N CH3
NH
N H2N H2N

Sulfadizine Sulfathiazole Sulfamerazine

O O N O O N O
CH3 NH N
S S S N
NH S NH N
O
O
H2N H2 N H2N

Sulfamethizole Sulfamethazine Sulfamethoxypridazine

H2N
O
OH
N O N
S O O O O O O
NH
NH S S S
NH NH N O
O N
N
Cl N H2N H2 N H2N

Sulfachloropyridazine Sulfamethoxzole Sulfadimethoxin Sulfapyridine (IS)

Figure 1. Chemical structures for the sulfonamide drugs used in the study.

2
Experimental Instrument conditions
HPLC conditions
Reagents and Chemicals Table 1. HPLC Conditions Used for Separation and Analysis
All reagents were analytical or HPLC grade. Methanol (MeOH) Column: Agilent ZORBAX Eclipse Plus C18
was purchased from Merck KGaA (Darmstadt, Germany) 4.6 × 75 mm, 3.5 µm
Flow rate: 1 mL/min
while acetonitrile (ACN), acetone and glacial acetic acid Column temperature: 25 °C
(HOAc) were obtained from Sigma-Aldrich (St. Louis, MO, Injection volume: 5 µL
USA). Sodium acetate (NaOAc) was from Saarchem Mobile phase: A = 0.05 M Sodium Acetate pH 4.5
B = MeOH
Analytical (Krugersdorp, South Africa). Fluorescamine (98%) Gradient:
and sulfonamide drugs including the internal standard were T (min) %B
purchased from Sigma-Aldrich (St. Louis, MO, USA). The 0 35
35 41
water used was from a MilliQ system (Milford, Mass, USA). 50 55
Detection: Ex = 405 nm Em = 495 nm
Solutions and Standards
Sample preparation
A stock solution of 0.05 M sodium acetate was prepared by
dissolving 4.1 g NaOAc in 1.0 L of ultrapure water and filtered The chicken muscle was purchased from a local food store,
through a Whatman membrane filter (47 mm diameter and minced and deep frozen until analysis.
2 µm pore size). The pH was adjusted using HOAc.
Fluorescamine reagent (0.02%) was prepared by dissolving
Extraction
20 mg Fluram in 10 mL of acetone. The solution was stored at Figure 2 outlines the methodology used in the QuEChERS
4 °C. A 1% HOAc in ACN solution was prepared by diluting experiments. A 2-g portion of chicken muscle homogenate
10 mL HOAc to 1.0 L with ACN. was placed into a 50-mL centrifuge tube from the SampliQ
QuEChERS AOAC Extraction kit. The tube was centrifuged for
Standard and internal standard primary stock solutions 20 s. Samples were then spiked with appropriate spiking solu-
(1 mg/mL) were prepared in ACN and stored at –20 °C. From tions to yield 50, 100, and 150 ng/g sample concentrations for
the primary stock solution, 10 µg/mL standard mixtures also recoveries and reproducibility studies. A 100-µL IS spiking
in ACN were prepared for the calibration curves. All working solution was added to all the samples except the blank. After
solutions were prepared daily by serial dilution in 0.05 M shaking vigorously for 1 min, 8 mL Milli-Q water was added
NaOAc (pH 3.5). All the solution vials were wrapped with followed by further shaking for 30 s. Next, 10 mL 1% HOAc in
aluminium foil because some of the sulfonamide drugs are ACN was added followed by the Agilent SampliQ QuEChERS
light-sensitive. AOAC Extraction salt packet (p/n 5982-5755). The packet
contained 6 g of anhydrous MgSO4 and 1.5 g of anhydrous
Equipment and Material NaOAc. The sample tubes were hand shaken vigorously for 1
The analysis was performed on an Agilent 1200 Series HPLC min then further centrifuged at 4000 rpm for 5 min.
(Agilent Technologies Inc., Santa Clara, CA, USA) equipped
with a binary pump and a fluorescence detector (FLD) set at Dispersive SPE cleanup
λex = 405 nm and λem = 495 nm. Separation of the compounds A 6-ml aliquot of the upper ACN layer was transferred into a
was achieved on an Agilent ZORBAX Eclipse Plus C18 column SampliQ QuEChERS AOAC Dispersive SPE 15 mL tube. This
(4.6 mm × 75 mm, 3.5 µm, p/n 959933-902). The data was SPE tube contained 400 mg of PSA, 400 mg of C18EC, and
processed by HPLC 2D Chemstation software. 1200 mg of anhydrous MgSO4. The tubes were then cen-
trifuged at 4000 rpm for 5 min. Next, 4 mL of the extract was
Extraction and cleanup were carried out with an Agilent
transferred to a test tube and dried with N2 gas at 35 ºC.
SampliQ Buffered QuEChERS AOAC Extraction kit, p/n 5982-
Samples (200 µL) were reconstituted into 600 µL of 0.05 M
5755 and an Agilent SampliQ QuEChERS AOAC Dispersive
NaOAc (pH 3.5).
SPE kit, p/n 5982-5158, (Agilent Technologies).

A Jenway 3510 pH meter (Jenway, London, UK) monitored the Derivatization

pH of the solutions, and a Kenwood grinder (Kenwood, Aliquots of 400 µL working standard mixtures of sulfon-
Grahamstown, South Africa) homogenized the chicken amides, dissolved in 0.05 M acetate buffer (pH 3.4), were
sample. filtered through a 0.45 µm PVDF syringe filter then transferred

3
 Weigh 2 g homogenized chicken muscle into a 50 mL centrifuge tube to reaction vials. A 200 µL 0.02% w/v amount of fluo-
rescamine solution in acetone was added. The mixtures were
shaken for 1 min and the reaction left to proceed for 60 min at
Spike samples with 1000 µL 20 µg/mL IS, 1000 µL of 10 µg/mL ambient temperature. Aliquots of 10 µL of the derivatized
spiking solution solutions were directly injected into the liquid chromatograph.
Shake vigorously 1 min
Add 8 mL water Results and Discussion
Shake vigorously 30 s Derivatization of sulfonamide drugs
Add 10 mL 1% HOAc in ACN
Fluorescamine is a fluorogenic reagent specific for primary
Shake vigorously 1 min aliphatic and aromatic amines that produce fluorophors of a
high fluorescence yield [9]. This reagent and its hydrolysis
Add SampliQ QuEChERS AOAC salt packet
products do not fluoresce, which eliminates the extensive
Shake 1 min, centrifuge at 4000 rpm cleanup step. Fluorescamine was therefore used in this appli-
5 min
cation note to derivatize sulfonamides in the precolumn mode.
Transfer 6 mL aliquot to SampliQ QuEChERS dispersive SPE 15 mL tube The results indicated that the reaction time is the most impor-
Shake 1 min, centrifuge at 4000 rpm tant factor. The reaction was complete within 60 – 100 min
5 min and for reproducibility 60 min was the chosen time. The
Transfer 4 mL extract to a tube; blow down at 35 °C with N2 derivatised sulfonamides were detected with a single pair of
wavelengths, λex = 405 nm and λem = 495 nm.

Reconstitute 200 µL into 600 µl 0.05M NaOAc pH 3.5; add 200 µL Chromatographic results
0.02% fluorescamine
The chromatogram of the standard mixture of these sulfon-
Shake 1 min, incubate 60 min amide derivatives is shown in Figure 3. Figure 4 is the chro-
Samples are ready for HPLC-FLD analysis matogram for the blank chicken muscle extract, and Figure 5
is that of the spiked chicken muscle. All chromatograms of
Figure 2. Flow chart for the QuEChERS AOAC sample preparation
standards, blanks, and spiked extracts were run using the
procedure. conditions outlined in Table I.

Standard mixture of the ten sulfonamides (100 ng/g)

0.56

0.51
10
0.46
0.41

0.36 9
3
LU

2
0.31 4 6
0.26 1 5
7
0.21 8
0.16

0.11
0 5 10 15 20 25 30 35 40 45 50

Time (min)

Figure 3. Chromatogram of the standard mixture of the sulfonamides (100 ng/g): 1. Sulfadiazine; 2. Sulfathiazole; 3. Sulfapyridine (IS); 4. Sulfamerazine;
5. Sulfamethazine; 6. Sulfamethizole; 7. Sulfamethoxypyridazine; 8. Sulfachloropyridazine; 9. Sulfamethoxazole; 10. Sulfadimethoxine.

4
 Chicken muscle blank extract
0.155

0.15

0.145
Intensity (LU)

0.14

0.135

0.13

0.125
0 5 10 15 20 25 30
Time (min)

Figure 4. Chromatogram of the blank chicken muscle extract.

50 ng/g spiked chicken muscle

0.2
2 10
0.19

0.18
3

0.17 6
0.16 5 9
4
LU

7
0.15 1
8
0.14

0.13

0.12

0.11
0 5 10 15 20 25 30 35 40 45 50
Time (min)

Figure 5. Chromatogram of the spiked chicken muscle extract at 50 ng/g level: 1. Sulfadiazine ; 2. Sulfathiazole; 3. Sulfapyridine (IS); 4. Sulfamerazine;
5. Sulfamethazine; 6. Sulfamethizole; 7. Sulfamethoxypyridazine; 8. Sulfachloropyridazine; 9. Sulfamethoxazole; 10. Sulfadimethoxine.

5
Linearity, Limit of Detection (LOD) and Limit of from the concentration of sulfonamides required to give
Quantification (LOQ) signal-to-noise ratios of 3 and 10 respectively. Table 2 shows
the regression equation, correlation coefficients, and very
Linearity acceptable limits of detection and quantification.
The linear calibration curves were obtained by plotting the rel-
ative responses of analytes (peak area of analyte/peak area Recovery and Reproducibility
of IS) verses the relative concentration of analytes (concen-
The recovery and reproducibility (RSD) were evaluated on
tration of analyte/concentration of IS). They were generated
spiked samples at MRL (100 µg/kg), half MRL (50 µg/kg) and
by spiking the sample blanks at levels of 10, 50, 100, 150, 200,
one and a half times the MRL (150 µg/kg). The analysis was
300 and 400 ng/g.
performed in replicates of six (n = 6) at each level. Table 3
shows the recoveries and RSD values for the nine
Limits of Detection and Quantification
sulfonamides.
The limits of detection and quantification were estimated

Table 2. Linearity, LOD and LOQ for the Nine Sulfonamides

Sulfonamide Regression equation R2 LOD ng/g LOQ ng/g

Sulfadiazine Y = 0.4154x + 0.0112 0.9995 0.26 0.87
Sulfathiozole Y = 1.0231x – 0.0757 0.9991 0.02 0.27
Sulfamerazine Y = 0.6735x + 0.0184 0.9993 0.14 0.46
Sulfamethazine Y = 0.6735x + 0.0042 0.9996 0.08 0.26
Sulfamethizole Y = 0.9751x + 0.0115 0.9995 0.30 1.00
Sulfamethoxypyridine Y = 0.4713x – 0.0069 0.9994 0.24 0.80
Sulfachloropyridazine Y = 0. 2769x + 0.0190 0.9992 0.33 1.10
Sulfamethoxazole Y = 0.6996x + 0.0421 0.9991 0.39 1.30
Sulfadimethoxine Y = 0.5008x + 0.0329 0.9991 0.08 0.25

Table 3. Recovery and Repeatability for Sulfonamides in Spiked Chicken Muscle (n = 6)

Sulfonamide Level of spiking (ng/g)

50 100 150
%Recovery %RSD %Recovery %RSD %Recovery %RSD
Sulfadiazine 77.8 2.6 78.1 2.1 78 1.9
Sulfathiazole 83.0 3.9 88.2 2.4 85.2 2.2
Sulfamerazine 85.7 2.9 85.5 3.1 88.4 1.6
Sulfamethazine 80.3 3.1 81.3 2.2 80.3 1.5
Sulfamethizole 95.2 4.1 89.5 2.6 87.2 2.5
Sulfamethoxypyridazine 91.4 2.3 90.7 2.1 90.5 1.7
Sulfachloropyridazine 78.1 3.0 89.4 2.6 80.7 2.2
Sulfamethoxazole 76.8 3.5 87.2 2.5 89.1 1.5
Sulfadimethoxine 90.3 4.7 92.8 2.2 89.2 2.0

6
Conclusions
A simple and fast mulitiresidue method based on SampliQ
QuEChERS AOAC Method 2007.01 and HPLC-FLD with pre-
column derivatization has been developed for the simultane-
ous determination of nine sulfonamide residues in spiked
chicken muscle. The recoveries were good with excellent RSD
and the LOQs were well below the MRL in animal food prod-
ucts. This method can therefore be recommended for residue
control purposes.

References
1. A. Posyniak, J. Zmudzki, K. Mitrowska, J. Chromatogr. A
1087 (2005) 259–264.
2. I. Pecorelli, R. Bibi, L. Fioroni, R. Galarini, J. Chromatogr.
A 1032 (2004) 23–29.
3. A. R. Long, L.C Hsieh, M. S. Malborough, C. R. Short,
S. A. Barker, J. Agric. Food Chem. 38 (1990) 423–426.
4. D. Kim, J. Choi, J. S. Kim, D. W. Lee, Bull. Korean Chem.
Soc 23 (2003).
5. V. Gamba, C. Terzano, L. Fioroni, S. Moretti, G Dusi,
R. Galarini, Anal. Chim. Acta 637 (2009) 18–23.
6. S. Wang, H.Y. Zhang, L. Wang, Z.J Duan, Food Additive
and Contaminants, April 2006; 23 (4): 362–384.
7. X. Wang, K. Li, D. Shi, N. Xiong, X. Jin, J. Yi, D. Bi J. Agric.
Food Chem. 55 (2007) 2077.
8. L. Zhao, J. Stevens, “Analysis of Pesticides Residues in
Spinach Using Agilent SampliQ QuEChERS AOAC Kits by
LC/MS/MS,” Agilent Technologies publication,
5990-4248EN.
9. G.Stoev, A. Michailova, J. Chromatogr. A 871 (2000)
37–42.
10. N. Takeda, Y. Akiyama, J. Chromatogr. B 558 (1991)
175–180.

For More Information

For more information on our products and services, visit our
Web site at www.agilent.com/chem.

7
www.agilent.com/chem
Agilent shall not be liable for errors contained herein or
for incidental or consequential damages in connection
with the furnishing, performance, or use of this material.

Information, descriptions, and specifications in this

publication are subject to change without notice.

© Agilent Technologies, Inc., 2010

Printed in the USA
July 6, 2010
5990-5395EN
 Extraction of Sulfa Drugs in
Honey with Polymeric SPE Cation
Exchange, Bond Elut Plexa PCX

Application Note

Authors Introduction
William Hudson and Rich Motyka Antibiotics and other drugs in agricultural foods is a recurrent problem that can
Agilent Technologies, Inc. cause serious harm or death to the allergic or sensitive consumer. In recent years,
various sources of honey have been shown to be contaminated with residues of
antibiotics and sulfonamides. These contaminants may occur after direct treatment
of bacterial diseases of honey bees, such as American or European foulbrood
and nosemosis. To address this issue, several countries have regulated use of
sulfonamides and require routine testing of honey for their presence.
Honey is a complex matrix consisting of mostly carbohydrates and water. In contrast
to less viscous samples, for example milk, in order to utilize SPE with a viscous
matrix like honey, it is necessary to take additional steps including sonication and
acidification prior to SPE. Herein, we describe a method to extract and analyze sulfa
drugs in honey using cation exchange SPE and LC/MS/MS analysis.
Materials and Methods Results and Discussion
Table 1. SPE Reagents and Solutions LC Conditions The procedure describes a method for
Mobile Phase: A: 5 mM Formic acid extracting four sulfonamide antibiotics
4% Phosphoric Acid Add 40 µL of B: Methanol
concentrated H3PO4 to
from honey (Figure 1). The limit of
Gradient: t = 0-0.5 min 90% A : 10% B
1 mL of DI water t = 5.0-6.0 min 50% A : 50% B detection (LOD) of the combined solid
Methanol Reagent grade or t = 6.01-7.0 min 90% A : 10% B phase extraction and LC/MS/MS
better Column: Pursuit C18 3 µm, 50 x 2.0 mm analysis was 25 ng/g. Recoveries were
(part number A3051050X020) calculated from a 1st order regression
2% Formic Acid Add 20 µL of
concentrated formic MS Conditions with RSD values based on a sampling
acid to 1 mL of DI Transition ions and collision energy were: of n = 6. Excellent absolute recoveries
water Compound Q1 Q3 CE were achieved demonstrating good
Methanol:acetonitrile Add 1 mL of methanol Sulfathiazole 256.0 156.0 18.0V retention and elution, as well as
(1:1, v/v) to 1 mL of acetonitrile Sulfamethazine 279.0 186.0 21.0V
minimal ion suppression. Response for
Sulfaquinoxaline 301.0 156.0 19.5V
5% NH4OH Add 50 µL of
Sulfadimethoxine 311.0 92.1 37.0V all the compounds evaluated was linear
Methanol:acetonitrile concentrated up to three orders of magnitude from
Capillary = 70 V, Dry gas temp = 350 °C, 30 psi,
(1:1, v/v) ammonium
hydroxide to 1 mL of
CID = Argon 1.0 ng/mL to 5.0 mg/mL with
Polarity: Negative correlation coefficients all above 0.999.
methanol:acetonitrile
(1:1, v/v) To demonstrate reproducibility, samples
Bond Elut Plexa 10 mg 96 well plate kCounts Sample ID: 500 ng
were analyzed at two concentrations
(n = 6). As shown in Table 3, the
256.0>155.9 [-18.0V]

(part number A4968010) 30

Sulfathiazole
extractions produced reproducibly high
20

10

recoveries.
0
kCounts Sample ID: 500 ng
100 279.0>186.0 [-21.0V]

Table 2. SPE Method 75

50 Sulfamethazine
25

Sample Pre-treatment 1.0 g Honey. Add 0

kCounts Sample ID: 500 ng
50 301.1>155.9 [-19.0V]
1 mL 4% H3PO4 and
Sulfaquinoxaline
40

sonicate for 20 mins.

30
20

Dilute with 3 mL of
10
0
kCounts Sample ID: 500 ng

2% H3PO4.
100 311.0>91.9 [-37.0V]

75
Sulfadimethoxine
Condition 1. 500 µL CH3OH
50

25

2. 500 µL DI H2O 0
1 2 3 4 5 6
minutes

Wash 1 500 µL 2% formic acid

!"#$%&'()'*+%,-./,#%.-0',1'.'23'4#5-6'&7/%.8/
Wash 2 500 µL
(acids, neutral) methanol:acetonitrile
(1:1, v/v) Table 3. Analyte relative recoveries
Elution 500 µL 5% NH3 Analyte log P pKa % Rec % RSD % Rec % RSD
(bases) methanol:acetonitrile (50 ng/mL) (500 ng/mL)
Sulfathiazole 0.05 7.2 102 6 93 7
All samples are evaporated to dryness and Sulfamethazine 0.89 7.4 103 7 87 4
reconstituted in 100 µL of 80:20 0.1% Aq formic Sulfaquinoxaline 1.68 5.9 107 7 100 7
acid: CH3OH.
Sulfadimethoxine 1.63 5.9 100 4 103 5

!
Conclusions
With excellent flow characteristics
Bond Elut Plexa PCX is an ideal choice
when working with difficult to process
samples such as honey. Similar to
other widely used cation exchangers,
standard adjustments of pH and
organic content with Bond Elut Plexa
PCX yields fast and effective analyte
recoveries approaching 100% with
the four sulfa drugs present in honey.
These data suggest Bond Elut Plexa
PCX lends itself to the extraction of
sulfa and similar drugs from foods and
other complex matrices.

www.agilent.com/chem
This information is subject to change without notice.
© Agilent Technologies, Inc. 2010
Published in UK, August 27, 2010
SI-01120
 Analysis of tetracyclines
by HPLC

Rainer Schuster

Food

Abstract
Tetracyclines are used worldwide as oral or parenteral medication in the form of additives in
animal feed. In food-producing animals, these drugs exhibit a high degree of activity toward a
wide range of bacteria.1, 2

Sample preparation
After homogenization or mincing and addition of mineral acids to dissociate tetracyclines from
proteins, the samples were extracted using liquid/liquid extraction followed by degreasing and/or
deproteinization, purification, and concentration.3

Chromatographic conditions
The HPLC method presented here for the analysis of meat is based on reversed-phase
chromatography and UV-visible diode-array detection.
UV spectra were evaluated as an additional identification Conditions
tool.
Column: 100 ˘ 4 mm Hypersil BDS, 3 µm
Mobile phase:
A = water, pH = 2.1 with sulfuric acid
Absorbance
[mAU]
B = ACN
Gradient: start with 15 % B at 10min 60% B
1 Oxytetracycline Flow rate: 0.5 ml/min
6 2 Tetracycline Column compartment: 25 ºC
2 7
3 epi-Tetracycline Detector:
5 4 Demeclocycline
6 UV-DAD detection wavelength 355 nm/20 nm,
4 1 4 5 epi- Demeclocycline
6 Chlortetracycline reference wavelength 600/100 nm
3 7 Doxycycline

2
Sample preparation
3 5
1 10 ng each 1. 1 g sample was mixed with citric acid
(100 mg).
0
2. add 1 ml nitric acid (30 %)
-1 1 ng each or 0.1 m oxalic acid
3. add 4 ml methanol 5 min in the ultrasonic
2 4 6 8 10 12 14 Time [min]
bath
4. add water up to 10 ml total volume
Figure 1 5. centrifuge
Analysis of tetracyclines by HPLC 6. inject

Agilent Technologies
Innovating the HP Way
 Equipment
Area
Area = 2.59596374*Amt -0.0821516 Agilent 1100 Series
25
• vacuum degasser
1
Rel. Res%(3): 2.461 • quaternary pump
20 • autosampler
• thermostatted column
15
compartment
10
• diode array detector,
2
Agilent ChemStation
5 3 + software
Correlation: 0.99996
0
HPLC method performance
0 5 Amount [ng/ul]
Limit of detection
for UV-DAD 100 ppb
Figure 2 Repeatability
Linearity for oxytetracycline 1-10 ng of RT over 10 runs <0.2 %
of areas over 10 runs <2 %
Absorbance
[mAU] References
0.8

0.7 1.
0.6 Library Tetracycline H. Malisch et al.,
“Determination of residues
0.5
of chemotherapeutic and
0.4 antiparasitic drugs in food
0.3 stuffs of anomaly origin with
0.2
HPLC and UV-Vis diode-
1 ng Tetracycline array detection”
0.1
J. Liq. Chromatogr., 1988,
0 11 (13), 2801–2827.14.
200 250 300 350 400 Wavelength [nm]
2.
M.H. Thomas, J. Assoc. Off.
Figure 3 Anal.; 1989, 72 (4) 564.
Analysis of tetracyclines at 100 ppb by HPLC
3.
Rainer Schuster is application
chemist at Agilent Farrington et. al.,
Technologies, Waldbronn, “Food Additives and
Germany. Contaminants”, 1991, Vol. 8,
No. 1, 55-64.
For more information on our
products and services, visit
© Copyright 1997 Agilent Technologies
our worldwide website at Released 09/97
http://www.agilent.com/chem Publication Number 5966-1619E

Agilent Technologies
Innovating the HP Way
 Analysis of Residual Synthetic
Antibacterials in Meat by HPLC
Application

Food
Hiroki Kumagai, Adebayo Onigbinde

Many domestic cattle receive various antibacterials in their feed for the preven- Highlights
tion and control of disease caused by fungi and bacteria. Residues of antibacterials
are found in food made from the meat of these animals. Since many antibiotics • Separation of 10 antibacterials in
are toxic, many countries regulate acceptable residue levels of compounds meat at low pH
allowable in agricultural and animal products. Many alkyl-C18 columns tail with • Excellent and rapid resolution of
basic compounds and have a shorter life time at low pH. Purospher® column antibacterials at low sample con-
separated basic antibacterials with good resolution, peak shape,and efficiency. centration
Absorbance
2 • Elution of antibacterials from the
[mAU]
25 1 SMr 6 SMMX column with good peak shape and
2 PYM 7 DFZ
20 at 224 nm
3 TCP 8 SDMX
narrow peak width
15 3 8 9 10
4 67 4 SDD 9 SQX
10
5
1
5 5 FZD 10 OXA
• Separation of low level amounts of
0 a wide range of pharmaceutical
0 5 10 15 20 25 30 35 Time (min) compounds with differing struc-
Absorbance tures in a single analysis by
[mAU]
25
Purospher® column
20 DFZ
at 360 nm
15
10 FZD Analyzed Compounds
5
0 • Sulfamerazine (SMR)
0 5 10 15 20 25 30 35 Time (min) • Sulfadimidine (SDD)
Figure 1. Chromatogram of standard solution, 2 µg/mL each analyte.
• Sulfamonomethoxine (SMMX)
Absorbance • Sulfadimethoxine (SDMX
[mAU]
• Sulfaquinoxaline (SQX)
4 at 224 nm
3 • Pyrimethamine (PYM)
2 SDD
1 • Thiamphenicol (TPC)
0
-1 • Furazolidone (FZD)
0
Absorbance
5 10 15 20 25 30 35 Time (min) • Difurazone (DFZ)
[mAU] • Oxolinic acid (OXA)
4
3
2
at 360 nm Sample: Extracts from bovine muscle
1
0 Sample preparation: According to the
-1
official procedure of the Japanese
0 5 10 15 20 25 30 35 Time (min)
food sanitation law.
Figure 1. Chromatogram of extract of bovine muscle.

Instrument: Agilent 1100 Series HPLC; Column: 250 mm × 4 mm id, RP-18 Purospher®, 5 µm, Part no. 79925PU-584;
Mobile phase: A = 0.7 % Phosphoric acid, B = CH3CN; Gradient: 0.0 min 5% B; 10.0 min 65% B; 40.0 min 65% B; 45.0 min
65% B; Post Time 7.0 min 5% B; Flow rate: 1.0 mL/min; Temperature: 40 °C; Injection volume: 20 µL; Diode array detector:
A–338/10 nm, reference wavelength off; B–264/8 nm, reference wavelength off; C–360/8 nm, reference wavelength off
www.agilent.com/chem

Hiroki Kumagai is the PHS Support

manager based at Yokogawa Analytical
Systems Inc., Tokyo, Japan.
Adebayo Onigbinde is an application
chemist based at Agilent Technologies,
Wilmington, Delaware, USA.

For More Information

For more information on our products
and services, visit our Web site at
www.agilent.com/chem.

Agilent shall not be liable for errors contained herein or

for incidental or consequential damages in connection
with the furnishing, performance, or use of this
material.

Information, descriptions, and specifications in this

publication are subject to change without notice.

© Agilent Technologies, Inc. 2002

Printed in the USA

June 18, 2002
5988-7135EN
 HPLC Analysis of Antibacterial Drugs with
Penicillin-Like Structure
Application

Drug Development
Udo Huber, Adebayo O. Onigbinde

Penicillins can be isolated from the culture medium of certain fungi-producing Highlights
natural penicillin, such as Penicillium notatumor and P. chrysogenum. Other
penicillins can be synthesized semisynthetically or by precursor-indicated • There is excellent resolution of
biosynthesis. Total synthesis would not be economical. penicillin analogs without ion
pairing agent.
Penicillin inhibits the polymerization of murin, which is responsible for the stabil-
ity of the bacteria's cell wall. Because many antibacterials are toxic, various • There is rapid resolution of the
countries regulate the level of antibacterial residues in agricultural, veterinary, penicillin analogs on the SB-C18
dairy, and meat-based food products. column at low pH and buffer
concentration.
Figure 1 shows the HPLC separation of four common antibacterial drugs with • Penicillins are eluted from the
pencillin-like structure (amoxicillin, ampicillin, penicillin G, and penicillin V) on column with good and narrow
an SB-C18 reversed phase column. peak shape.
• Extreme stability of sterically pro-
1 Amoxicillin tected SB-C18 bonded phases
1000 2 Ampicillin allows for excellent separation at
3 Penicillin G low pH.
800 2 3 4 Penicillin V
Absorbance (mAU)

• The SB-C18 column provides

600 excellent peak shape and selectiv-
400 1
4 ity for antibacterial drugs.

200 • The HPLC method shows an easy

but reliable and precise analysis of
0 the antibacterial drugs.
0 2 4 6 8 10 12
Time (min)
• The values for limit of detection
(LOD), precision of retention time
(RT) and area show the good per-
Figure 1. Separation of four penicillin analogs. formance of the HPLC analysis.

Experimental Conditions
Equipment: Agilent 1100 Series HPLC; UV Detector: Variable wavelength detector, 204 nm, standard cell; Column: Zorbax
SB-C18, 3. 5 µm, 4.6 × 75 mm (part number 866953-902); Mobile phase: A = 0.025 M KH2PO4 in water (pH = 3), B = acetoni-
trile; Injection volume: 5 µL; Temp: 40 °C; Flow rate: 1.0 mL/min; Gradient: at 5% B to 60% in 10 min; Column wash: 60% B
to 5% B in 2 min; Stop time: 12 min; Post time: 5 min
 www.agilent.com/chem
Table 1. HPLC Method Performance of Antibacterial Drugs with
Penicillin-Like Structure

LOD for Precision of RT Precision of area

S/N = 2 (RSD of 10 runs) (RSD of 10 runs)
Compound (mg/L)* (100 mg/L)* (100 mg/L)*
Ampicillin 1.0 0.32 0.54
Amoxicillin 1.0 0.32 0.55
Penicillin G 1.0 0.32 0.49
Penicillin V 1.0 0.25 0.48

*Injection volume: 5 µL

Udo Huber is an application chemist

based at Agilent Technologies,
Waldbronn, Germany.
Adebayo Onigbinde is an applications
chemist based at Agilent Technologies,
Wilmington, Delaware, USA.

For More Information

For more information on our products
and services, visit our Web site at
www.agilent.com/chem.

Agilent shall not be liable for errors contained herein or

for incidental or consequential damages in connection
with the furnishing, performance, or use of this
material.

Information, descriptions, and specifications in this

publication are subject to change without notice.

© Agilent Technologies, Inc. 2002

Printed in the USA

September 10, 2002
5988-7629EN
 HPLC Separation of Antibacterial Drugs
with Tetracycline Structure
Application

Drug Development
Udo Huber, Adebayo O. Onigbinde

Tetracyclines occur naturally in some streptomyces species. Besides being used Highlights
in human and veterinary medicine, they are fed as nutritional antibiotics in pig
and poultry farming. Because of their long half-life and resistance, there is a high • The SB-C18 column provides
restriction on their usage in some European countries, such as Germany. excellent peak shape and selectiv-
Figure 1 shows the HPLC separation of three common tetracycline analogs on a ity for basic antibacterial drugs.
Zorbax SB-C18 reversed phase column. • The SB-C18 column shows excel-
This application demonstrates separation without ion pairing and the use of an lent stability at low pH.
alternative mobile phase to TFA in separating antibacterial drugs. • The SB-C18 column shows excel-
lent and rapid resolution of antibi-
otics at low pH and buffer
2 concentration.
120 1
• The HPLC method shows an easy
3 but reliable and precise analysis of
100
the antibacterial drugs.
Absorbance (mAU)

80
• The values for limit of detection
1 Minocycline (LOD), precision of retention time
60
2 Tetracycline (RT), and area show the good per-
3 Doxycycline formance of the HPLC analysis.
40

20

0 2 4 6 8 10
Time (min)

Figure 1. Separation of three antibacterial drugs with tetracycline structure.

Experimental Conditions
Equipment: Agilent 1100 Series HPLC; UV Detector: Variable wavelength detector, 350 nm, standard cell; Column: Zorbax
SB-C18, 3. 5 µm, 4.6 × 75 mm (part number 866953-902); Mobile phase: A = 0.025 M KH2PO4 in water (pH = 3), B = acetoni-
trile; Injection volume: 5 µL; Temp: 25 °C; Flow rate: 1.0 mL/min; Gradient: at 5% B to 60% B in 10 min; Column wash: 60%
B to 5% B in 2 min
 www.agilent.com/chem
Table 1. HPLC Method Performance of Antibacterial Drugs with
Tetracycline Structure

LOD for Precision of RT Precision of area

S/N = 2 (RSD of 10 runs) (RSD of 10 runs)
Compound (mg/L)* (100 mg/L)* (100 mg/L)*
Minocycline 0.1 0.06 0.14
Tetracycline 0.1 0.05 0.13
Doxycycline 0.1 0.04 0.21

*Injection volume: 5 µL

Udo Huber is an application chemist

based at Agilent Technologies,
Waldbronn, Germany.
Adebayo Onigbinde is an applications
chemist based at Agilent Technologies,
Wilmington, Delaware, USA.

For More Information

For more information on our products
and services, visit our Web site at
www.agilent.com/chem.

Agilent shall not be liable for errors contained herein or

for incidental or consequential damages in connection
with the furnishing, performance, or use of this
material.

Information, descriptions, and specifications in this

publication are subject to change without notice.

© Agilent Technologies, Inc. 2002

Printed in the USA

September 10, 2002
5988-7630EN
 Determination of the Metabolites of
Nitrofuran Antibacterial Drugs in Chicken
Tissue by Liquid Chromatograph-Electrospray
Ionization-Mass Spectrometry (LC-ESI-MS)
Application

Food, Environmental

Author additives to prevent bacterial enteritis by

Escherichia coli and Salmonella in cattle, fish,
swine, and poultry. The occurrence of furazolidone
Masahiko Takino residue in edible tissue is a major human health
Yokogawa Analytical Systems Inc concern. Effective June 1995, these drugs were
banned from use in food animal production in the
2-11-13, Nakacho, Musashino, Tokyo, 180-8453
European Union (EU) because of concerns about
Japan their carcinogenicity and mutagenicity (Commission
Regulation 1442/95).
Abstract Nitrofuran antibacterial drugs are characterized by
their rapid metabolism, with in vivo half-lives of
A liquid chromatography-electrospray ionization-mass less than a few hours. Therefore, the detection of
spectrometry (LC-ESI-MS) method was developed for the parent drugs in animal tissue is not practical.
simultaneous determination of the metabolites of four Studies using radioactive-labeled furazolidone
nitrofuran antibacterial drugs in chicken tissues: furazoli- have shown that protein-bound metabolites are
done, furaltadone, nitrofurazone, and nitrofurantoin. formed in tissues [1-3]. The tissue-bound metabo-
Sample clean-up and analyte enrichment were performed lites are detectable for several weeks after admin-
by liquid-liquid extraction with ethyl acetate followed by istration. Hence, the analysis of nitrofuran drugs is
solvent washing, hydrolysis of the protein-bound drug based on the detection of the tissue-bound
metabolites, and derivatization with 2-nitrobenzaldehyde metabolites of the parent drugs.
(2-NBA). ESI parameters were optimized, and the chro-
matographic separation of all metabolites was examined. These tissue-bound metabolites are very small mol-
Each metabolite produced a simple mass spectrum con- ecules which are not UV absorbing, and they elute
taining a strong signal corresponding to [M+H]+. too quickly out of a column. To induce UV absorp-
Metabolite calibration curves, in the 0.25 to 1 ng/mL tion in the molecule and to be reasonably retained
range, exhibited correlation coefficients greater than on a column, they are derivatized. It is possible to
0.999. The limit of detection (LOD) for each analyte ranged release these metabolites from the proteins under
from 0.02 to 0.06 ng/mL. moderately acidic conditions and derivatize the
metabolites with 2-nitrobenzaldehyde (2-NBA) to
produce 2-NBA-derivatives for liquid chromatogra-
Introduction phy (LC), UV detection, and mass spectrometry
The four drugs shown in Figure 1, furazolidone, (MS) confirmation. The goal of this study is to
furaltadone, nitrofurazone, and nitrofurantoin, develop a routine analytical method to simultane-
belong to the group of nitrofuran antibacterial ously detect the target nitrofuran metabolites.
drugs. These drugs have been widely used as feed Because no maximum residue limit (MRL) has
 Drugs Metabolites

O O

N H2N
N2O O N O N O

Furazolidone
3-amino-2-oxazolidinone (AOZ)

O O

N H2N
N2O O N O N O

N O N O

Furaltadone 5-morpholinomethyl-3-amino-oxazolidinone (AMOZ)

O O

N H2N
N2O O N NH2 N NH2
H H

Nitrofurazone Semicarbazide (SEM)

O O

N H2N
N2O O N NH N NH

O O

Nitrofurantoin 1-aminohydantoin (AHD)

Figure 1. Structure of the nitrofuran antibacterial drugs and their metabolites .

been set by any regulatory agency, the goal of the Department in Thailand (Palm Thani, Thailand)
analytical method was to estimate the lowest using the procedure described by Leitner [4].
possible detection limit. Stock solutions of these three 2-NBA derivatives
were prepared in methanol at 1000 ng/mL and
stored in the dark at 4 °C. The stock solution was
Experimental diluted to the desired concentration just prior to
its use for the optimization of ESI parameters.
Chemicals and Solvents
Acetonitrile, ethyl acetate, formic acid, and
Three metabolites: 3-amino-2-oxazolidinone (AOZ), dimethyl sulfoxide (DMSO) were supplied by Wako
semicarbazide (SEM), and 1-aminohydantoin Chemical (Osaka, Japan). Hydrochloric acid and
(AHD) were purchased from Sigma Aldrich Japan 2-NBA were purchased from Tokyo Kasei Kogyo
(Tokyo, Japan). The purity of these compounds (Tokyo, Japan). Water was purified with a Milli-Q
was greater than 99%. The 2-NBA derivatives of system (Millipore, Tokyo, Japan).
these metabolites were prepared by the Livestock

2
Sample Preparation Instrument and Experimental Conditions

Sample preparation procedures included solvent An Agilent 1100 series LC, with a solvent
wash and acid extraction by homogenization and degassing unit, a binary high-pressure gradient
derivatization with 2-NBA. Chicken muscle and pump, an automatic sample injector, and a column
liver were prepared by the Livestock Department thermostat, was used for separation. An 1100
in Thailand. series diode array detector (DAD) was connected
in line with an 1100 MSD for detection and confir-
Calibration curves for the four nitrofuran metabo- mation. The column and MS conditions are
lites (from the Livestock Department) were con- described in Table 1.
structed in the range 0.25 to 1.0 ng/mL. The
derivatization and sample preparation procedures Table 1. Instrument Parameters
used by the Livestock Department are the
following: LC: Agilent 1100 series
1. The four metabolite solutions in water, 12.5, Column: Inertsil ODS3, 150 mm × 2.1 mm, 5 µm
25.0, 37.5, and 50 µL at 100 ng/mL, were trans- (GL Science, Tokyo, Japan)
ferred to separate 40 mL glass vials with screw Solvent A: Acetonitrile
caps.
Solvent B: Aqueous 0.5% formic acid
2. A solution of 10 mL HCl (125 mM in water) and
200 µL 2-NBA (50 mM in DMSO) were added to Gradient: 20/80 A/B to 70/30 A/B in 20 min
each vial. Column temp 20 °C
3. The reaction mixtures were kept in a water Sample volume 30 µL
bath at 37 °C for 16 hours. Flow rate: 200 µL/min
4. The solutions were cooled to room temperature.
5. The pH was adjusted to about 7.4 by adding MS: Agilent 1100 MSD, SL
0.1 M aqueous KHPO4 or 0.8 M aqueous NaOH.
Ionization: ESI (Positive)
6. A 5-mL measure of ethyl acetate was added to Scan range: 100–500 m/z for optimization
each reaction mixture, and shaken for 2 min.
SIM ion: Base peak for quantitation
7. Each ethyl acetate phase was transferred to a
separate glass vial and evaporated under a Drying gas: Nitrogen, 10 L/min at 350 °C
stream of nitrogen. Nebulizer gas: Nitrogen, 50 psi
8. Finally, each residue was reconstituted in 5 mL Fragmentor: 120 or 140 V
of 1:1 methanol:water (V/V). Vcap 2000 V
The calibration curve was based on the metabolite
concentration in clean solvent and derivatization
using 2-NBA. Previous studies done by the Live- Quantitative analysis was carried out using selec-
stock Department showed that recovery of all tive ion monitoring (SIM) of the base peak ions
metabolites from chicken extracts was above 80%. according to the program shown in Table 2. To con-
Therefore, the amounts of metabolite in chicken firm the presence of the target analytes in chicken
extract can be calculated by comparing the extract, the sodium adduct ions (qualifier ions) of
responses of 2-NBA derivatives from the samples all target analytes were also monitored.
against the calibration curve.

Table 2. SIM Program

Time Target Qualifier Dwell time Fragmentor
Group window min Analyte(s) ion ion msec voltage, V
1 0–6 2-NBA-AMOZ 335 357 500 140
2 6–12.5 2-NBA-SEM and 209 and 231 and 250 and 120 and
2-NBA-AHD 249 271 250 140
3 12.5–14 2-NBA-AOZ 236 258 500 140

3
System Optimization transmission significantly. Fragmentor voltage also
affected the fragmentation of sample ions. In gen-
Positive ion mass spectra were acquired over the eral, higher fragmentor voltage helps the transmis-
scan range m/z 100–500 using a step size of 0.1 amu sion of ions through the relatively high-pressure
and a scan rate of 2 seconds per scan for the opti- region between the exit of the capillary and the
mization of fragmentor voltage. Ion lens voltages in entrance of the skimmer. High fragmentor voltage
the MS were automatically optimized using a Cali- can cause fragmentation to occur which provides
brant Delivery System and the AutoTune program. structural information of the ion. For compounds
that do not fragment easily, higher fragmentor volt-
Using the analytical column and three 2-NBA
age often results in better ion transmission. Opti-
derivatives (AOZ, SEM, and AHD) at 100 ng/mL,
mal fragmentor voltage is compound dependent.
instrument performance was optimized by adjust-
Evaluation of the fragmentor voltages for the three
ing the four major ESI parameters: the capillary
2-NBA-metabolites was done under the same chro-
voltage, fragmentor voltage, the nebulizer gas pres-
matographic conditions as the analysis. Mass spec-
sure, and the drying gas flow rate. However, signif-
tra of three 2-NBA-metabolites are shown in
icant variation in the intensity of analytes was not
Figure 2. Each mass spectrum exhibited [M+H]+ as
observed when the drying gas flow rate and nebu-
the base peak. Adducts ions [M+NH4]+ and [M+Na]+
lizer gas pressure were varied from 4 L/min to
were observed at lower fragmentor voltage (120 V)
13 L/min and 20 psi to 60 psi, respectively.
and some fragment ions (m/z=178 and 192) were
Capillary and fragmentor voltages applied to the observed at higher fragmentor voltage (180 V).
inlet and exit end of the capillary affected the ion Interestingly, the [M+NH4]+ ion was not observed at

Fragmentor = 120 V Fragmentor = 180 V

800000 209(M+H)+
600000
2-NBA-SEM 2-NBA-SEM
209(M+H)+

600000 231(M+Na)+
231(M+Na)+

400000
400000
192

200000
200000
166

100000 0
100 150 200 250 300 350 100 150 200 250 300 350

500000 400000
+
249(M+H)+

249(M+H)

400000
271(M+Na)+

2-NBA-AHD 300000 2-NBA-AHD

271(M+Na)+
266(M+NH4)+

300000
200000
200000
178

100000 100000

0 0
100 150 200 250 300 350 100 150 200 250 300 350
236(M+H)+
236(M+H)+
253(M+NH4)+

2-NBA-AOZ 60000 2-NBA-AOZ

258(M+Na)+
258(M+Na)+

40000
40000

20000
20000
192

0 0
100 150 200 250 300 350 100 150 200 250 300 350

Figure 2. Mass spectra of 2-NBA-SEM, 2-NBA-AHD, and 2-NBA-AOZ from two ESI fragmentor voltages.

4
 180 V fragmentor voltage due to its stability. As Linearity, Detection Limits, and Precision
seen in Figure 3, in order to ensure the best sensi-
tivity, the fragmentor voltage for 2-NBA-SEM was In order to achieve optimal sensitivity, all quantita-
set to 120 V and that of 2-NBA-AHD and 2-NBA-AOZ tion experiments were carried out under SIM con-
was set to 140 V for the analysis. Although ditions, and the [M+H]+ ions were monitored for all
2-NBA-AMOZ was not examined, fragmentor volt- 2-NBA-metabolites. To evaluate the linearity of the
age of this compound was set to 140 V because of calibration curves, various metabolite solutions
its structural similarity to 2-NBA-AOZ. For the cap- ranging from 0.25 ng/mL to 1 ng/mL were deriva-
illary voltage varied between 1500 and 4500 V, the tized and then analyzed. As shown in Table 3, the
optimal voltage was found to be 2000 V for all linearity was very good for all 2-NBA-metabolites
three metabolites. with correlation coefficients (r2) greater than 0.999.

1000000

800000

700000

600000
Peak intensity

500000

400000

2-NBA-SEM
300000
2-NBA-AOZ

2-NBA-AHD
200000

100000

0
100 120 140 160 180 200
Fragmentor voltage/V

Figure 3. Effect of fragmentor voltage on peak intensity. Mobile phase, 20% acetonitrile/80% water 0.1% formic acid;
Analyte concentration, 100 ng/mL.

5
The LOD for all 2-NBA-metabolites was estimated instrument precision (repeatability) was deter-
by extrapolating to a signal-to-noise ratio (S/N) of mined by injecting aqueous standard solutions
3 using the signal from the standard solution at containing all of the 2-NBA-metabolites at
0.25 ng/mL. These SIM chromatograms are shown 0.5 ng/mL five times during a working day. The
in Figure 4. The LODs of the metabolites were in interday instrument precision (reproducibility)
the range of 0.02 ng/mL to 0.06 ng/mL. These was evaluated by analyzing the same sample three
LODs were lower than those of the LC/MS/MS times over 3 working days. The precision for all
method developed by Leitner [4]. The intraday analytes ranged from 3.1% to 8.2%, as seen in Table 3.

6000

6000

5500

5500
2-NBA-AMOZ 2-NBA-AHD
5000

5000

4500

4500
m/z = 357 m/z = 271
4000
4 6 8 10 12 14 4 6 8 10 12 14

60000
8000

7000
2-NBA-SEM 56000 2-NBA-AOZ

6000

52000 m/z = 258

5000

4000
m/z = 231 48000

4 6 8 10 12 14 4 6 8 10 12 14
Retention time(min) Retention time(min)

Figure 4. SIM chromatograms of aqueous 2-NBA nitrofuran metabolites solution at 0.25 ng/mL.

Table 3. Linearity, LOD, and Instrument Precision of Metabolites in Aqueous Solutions

Instrument precision (%RSD)

Metabolites r2 LOD* (ng/mL) Repeatability** Reproducibility***
AMOZ 0.9999 0.04 5.0 7.3
SEM 0.9998 0.02 4.7 8.1
AHD 0.9989 0.06 4.9 7.9
AOZ 0.9997 0.06 3.1 8.2
*Detection limit is LOD defined as S/N = 3 for standard solution at 0.25 ng/mL
**Repeatability was calculated based on five replicates at 0.5 ng/mL within 1 day
***Reproducibility was calculated based on once per day for 3 days at 0.5 ng/mL

6
Evaluation of Chromatographic Separation

Several reverse-phase columns were evaluated for

HPLC performance. In terms of minimizing the
inherent matrix suppression effects on the ESI
process, Inertsil ODS3 column provided the best
separation between analytes and the majority of
the matrix components with the given mobile
phase. Further, the linear solvent gradient gave the
best compromise between short analysis time and
sufficient matrix and analytes separation. Figure 5
shows individual SIM chromatograms for the four
metabolite derivatives in spiked chicken muscle at
0.2 ng/g. No interference peaks were observed for
2-NBA-AMOZ and 2-NBA-AOZ, but it was difficult
to separate 2-NBA-SEM and 2-NBA-AHD from the
interfering matrix peaks. However, these peaks
could still be identified by comparison with the
blank sample, and the analyte amounts could then
be calculated.

25000
2-NBA-AMOZ
15000

5000
m/z = 335

0 2 4 6 8 10 12 14 16

30000
2-NBA-SEM
20000

10000

0
m/z = 209
0 2 4 6 8 10 12 14 16
40000
2-NBA-AHD
30000
20000
10000 m/z = 249
0
0 2 4 6 8 10 12 14 16
50000
2-NBA-AOZ
40000
30000
20000 m/z = 236
10000
0
0 2 4 6 8 10 12 14 16
Retention time(min)

Figure 5. SIM chromatograms of a spiked chicken muscle tissue sample containing 0.2 ng/g of each of the four
2-NBA nitrofuran metabolites.

7
Application of the Method to Chicken Liver Samples

It has been reported that AOZ concentrations in

liver tissue are several times higher than in muscle
tissue [1,2]. This indicates that detection of nitro-
furan metabolites in liver would be possible over
an even longer period of time. Since the nature of
the liver matrix is considered to be different from
muscle and more difficult to separate target com-
pounds from the interfering matrix, the developed
LC/MS muscle method was also tested for the
applicability to liver matrix. Figure 6 shows indi-
vidual SIM chromatograms of the four metabolite
derivatives in spiked chicken liver tissue. AMOZ,
SEM, and AOZ derivatives were identified unam-
biguously and quantified down to 0.2 ppb. How-
ever, the AHD derivative overlapped with the
matrix component and was difficult to quantify.

35000 2-NBA-AMOZ (0.84 ppb)

25000

15000
m/z = 335
5000

0 2 4 6 8 10 12 14 16
30000
2-NBA-SEM (1.21 ppb)
20000

10000 m/z = 209

0
0 2 4 6 8 10 12 14 16
25000
20000 2-NBA-AHD (0.33 ppb)
15000
10000
5000 m/z = 249
0
0 2 4 6 8 10 12 14 16
70000
50000
30000 2-NBA-AOZ (0.72 ppb)
m/z = 236
10000

0 2 4 6 8 10 12 14 16
Retention time (min)

Figure 6. SIM chromatograms of a spiked chicken liver tissue sample containing 0.2 ng/g of each of the four
2-NBA nitrofuran metabolites.

8
Conclusion
The development of a routine and sensitive LC/MS
method allows for the simultaneous detection of
four nitrofuran metabolite derivatives. The detec-
tion limit of each analyte ranges from 0.05 to
0.2 ng/g in chicken muscle and liver tissues.

References
1. L.A.P. Hoogenboom, M. van Kammen,
M.C.J. Berghmans, J.H. Koeman and
H.A. Kuiper, Food Chem. Toxicol. 1991; 28: 321.
2. L.A.P. Hoogenboom, M.C.J. Berghmans,
T.H.G. Polman, R. Paker and I.C. Shaw, Food
Addit. Contam. 1992; 9: 623.
3. D.W. Gottschall and R. Wang, J. Agric. Food.
Chem. 1995; 43: 2520.
4. A. Leitner, P. Zollner, W. Lindner,
J. Chromatogr. A 2001; 939: 49.

Acknowledgement
The author would like to acknowledge the Live-
stock Department staff in Thailand for providing
the four 2-NBA-metabolite standards and chicken
muscle and liver extracts.

For More Information

For more information on our products and services,
visit our Web site at www.agilent.com/chem.

9
www.agilent.com/chem

Agilent shall not be liable for errors contained herein or for incidental or consequential
damages in connection with the furnishing, performance, or use of this material.

Information, descriptions, and specifications in this publication are subject to change

without notice.

© Agilent Technologies, Inc. 2003

Printed in the USA

March 19, 2003
5988-8903EN
 Determination of Chloramphenicol in Fish
Meat by Liquid Chromatograph-Atmospheric
Pressure Photo Ionization-Mass Spectrometry
(LC-APPI-MS)
Application

Foods, Environmental

Author therapy can result in a well-understood and irre-

versible type bone marrow depression called apla-
Masahiko Takino and Shigeki Daishima sia or hypolasia. This, in turn, can lead to aplastic
Yokogawa Analytical Systems Inc anemia and although uncommon, it is often fatal.
2-11-13, Nakacho, Musashino Because of these health concerns, a joint Food and
Tokyo, 180-8453 Agriculture Organization/World Health Organiza-
Japan tion (FAO/WHO) Expert Committee on Food Addi-
tives has proclaimed that CAP residues in the
human food supply are unacceptable [1]. The use
Abstract of CAP in food products has been banned in EU
and U.S.A. However, CAP’s broad-spectrum activ-
A liquid chromatography-atmospheric pressure ity, ready availability, and low cost attract its use
photoionization-mass spectrometry method was devel- by some third world countries. Admittedly, when-
oped for the determination of chloramphenicol antibiotics ever CAP is accessible, indiscriminate and illegal
in fish meats. For the optimization of APPI, several ion use potentially exists. In fact, the presence of CAP
source parameters were examined. Using the optimized has been detected in shrimp imported from China
parameters, simple mass spectra and a strong signal cor- and Vietnam that was intended for human
–
responding to [M-H] was observed. The samples were consumption.
extracted with ethylacetate and evaporated to dryness
followed by a clean-up step using liquid-liquid distribu- Liquid chromatography/mass spectrometry
tion by acetonitrile and n-hexane. Mean recoveries of (LC/MS) methods are very useful in analyzing CAP
chloramphenicol from young yellowtail meat and flatfish in food because of the high selectivity and sensitiv-
meat spiked at 0.1–2 ng/g were 89.3%–102.5% and ity of MS detection [2-7]. Atmospheric pressure
87.4%–94.8%, respectively. The limit of detection ionization (API) interfaces, represented by atmos-
(signal-to-noise = 3) of the young yellowtail meat and the pheric pressure chemical ionization (APCI) and
flatfish meat were 0.27 and 0.10 ng/g. electrospray ionization (ESI), are commonly used
in LC/MS.
Introduction Atmospheric pressure photoionization (APPI) is a
new ionization technique for LC/MS [8, 9]. The
Chloramphenicol (CAP) is a broad-spectrum antibi- APPI source is based on a high-fluence gas dis-
otic, that exhibits activity against a variety of aero- charge lamp that generates vacuum-ultraviolet
bic and anaerobic microorganisms. Its action works (VUV) photons of 10 and 10.6 eV energy. The
through interference with or inhibition of protein energy of this discharge lamp is normally greater
synthesis. However, weeks or months of CAP
than a first ionization potential (IP) of an analyte step was repeated with another 1 mL of n-hexane.
because many organic compounds have IPs in the Finally, the acetonitrile phase was evaporated to
range of 7–10 eV. On the other hand, the IPs of the dryness under a stream of dry nitrogen using a
most common LC solvents, which are used as a heating block at 50 °C, redissolved in 5 mL of a
mobile phase, have higher values (water, IP = 12.6 10% acetonitrile in 10 mM ammonium acetate
eV; methanol, IP = 10.8 eV; acetonitrile, IP = 12.2 water solution, and filtered through a 0.22 µm
eV). This provides ionization of many analytes with nylon centrifuge filter. The samples were spiked
lower IPs without interference from the mobile with 0.1–100 ng/mL of CAP after the homogena-
phase. To our knowledge, APPI has not yet been tion step to generate a calibration by LC/APPI-MS
applied to residual analysis in food. selected ion monitoring (SIM).

This application note describes how parameters LC/MS

affect the ionization efficiency of APPI for the
An Agilent 1100 series LC, consisting of a vacuum
analysis of CAP. In addition, the suitability of
solvent degassing unit, a binary high-pressure gra-
LC/MS and liquid-liquid extraction using the APPI
dient pump, a standard automatic sample injector,
technique is evaluated for the determination of and a column thermostat, was used for the separa-
CAP in fish meat. tion. An 1100 series diode array detector (DAD)
was connected in line with an 1100 MSD for detec-
Experimental tion and confirmation. See Table 1. The separation
was performed on a 150 × 3 mm id column packed
with 5 µm Zorbax Eclipse XDB C18 (Agilent
Chemicals and Solvents
Technologies, Palo Alto, USA). A 15-min linear sol-
CAP was purchased from Sigma-Aldrich Japan vent gradient was used for elution with the mobile
(Tokyo, Japan). The purity of this compound was phase. Quantitative analysis was carried out using
greater than 99%. Stock solutions at 1 mg/mL were SIM of m/z 321 with a dwell time of 500 msec.
prepared in methanol, stored in the dark at 4 °C,
The following six parameters were optimized using
and diluted to the desired concentrations prior to
use. Ammonium acetate, pesticide-grade ethyl
Table 1. Instrument Parameters
acetate, anhydrous sodium sulfate, acetonitrile,
HPLC-grade methanol and n-hexane were obtained LC: 1100 series LC
from Wako Chemical (Osaka, Japan). Water was Column: Zorbax Eclipse XDB C18 (150 mm × 3 mm,
purified with a Milli-Q system (Millipore, Tokyo, 5 µm)
Japan). A nylon-type 0.22 µm centrifuge filter was Solvent A: Water with 10 mM ammonium acetate
obtained from Toyo Soda (Tokyo, Japan).
Solvent B: Methanol

Sample Preparation Dopant: Acetone at 0.05 mL/min

Gradient: 90/10 A/B 15 min to 70/30 A/B
The samples analyzed (young yellowtail and flat- Column temp: 40 °C
fish) were obtained from a local market. To a cen-
Sample volume: 20 µL
trifuge tube, 5 g fish meat and 5 g anhydrous
sodium sulfate were weighed and 10 mL ethyl Flow rate: 0.5 mL/min
acetate was added. The mixture was homogenized
for 20 s with an Ultra-Turrax TP 18/10 (Janke & MS: 1100 MSD, SL
Kunkel KG, Staufen, Germany). After centrifuga-
Ionization: APPI (Negative)
tion for 5 min at 6000 rpm, the supernatant was
removed and transferred to a round flask. The Scan range: m/z 100–400 for optimization
_
extraction step was repeated twice, each with SIM ion: m/z 321; (M-H)
10 mL ethyl acetate. The combined ethyl acetate Drying gas: Nitrogen, 7 L/min at 350 °C
extract was then evaporated in a rotary evaporator Nebulizer gas: Nitrogen, 50 psi
at 40 °C under vacuum. One mL acetonitrile and 1
Fragmentor: 120 V
mL n-hexane was added to the residue, transferred
into a graduated glass stopper reagent bottle, and Capillary: 3500 V
shaken. The n-hexane phase was discarded. The Vaporizer temp: 350 °C

2
 the analytical column with CAP at 100 ng/mL: the was found that modification of drying gas flow
voltages for in-source-fragmentation (the fragmen- rate and nebulizer gas pressure did not drastically
tor voltage), the capillary voltage (Vcap), the drying improve the sensitivity of CAP. In addition, the
gas flow rate, the nebulizer pressure, the mobile fragmentor voltage was included in optimization
phase composition, and the mobile phase flow because of its compound dependence and its
rate. The ion lens voltages in the MS were automat- significant effect on the mass spectral response.
ically optimized using a Calibrant Delivery System
and the AutoTune program. Negative ion mass Effect of Capillary Voltage
spectra were acquired over the scan range
m/z 100–400 using a step size of 0.1 amu and a The capillary voltage is applied to the inlet of the
scan rate of 2 s per scan for the optimization of capillary and influences the transmission effi-
fragmentor voltage. ciency of the ions through the capillary sampling
orifice. To establish the optimum capillary voltage,
this parameter was varied from 1000 to 4000 V. As
Results and Discussion shown in Figure 1, 1500 V was found optimum. A
tremendous effect of this parameter on the inten-
Optimization of the APPI Parameters sity of CAP was observed in the case where ace-
tone was not used as the dopant. On the other
To optimize the APPI conditions, parameters that hand, when acetone was introduced into the APPI
influence the ionization efficiency were investi- source as the dopant, the maximum intensity of
gated. The drying gas flow, the nebulizer gas pres- the ion was found at 3500 V. The intensity found at
sure, the vaporizer temperature, the capillary 3500 V with the dopant was higher than the maxi-
voltage, and the mobile phase composition were mum intensity without the dopant. Based on the
evaluated under the chromatographic conditions above results, the capillary voltage was set at
mentioned in the Experimental section by SIM 3500 V with acetone.
mode using the m/z 321 ion as the target ion. It

3000000

2500000

2000000
Peak intensity

1500000
Without acetone as the dopant

With acetone as the dopant

1000000

500000

0
1000 1500 2000 2500 3000 3500 4000
Capillary voltage [V]

Figure 1. The effect of the capillary voltage on the peak intensity of CAP concentration : 1 ng/mL. For the other conditions, see
Experimental section.

3
 Effect of Vaporizer Temperature Optimization of Fragmentor Voltage

In APPI, the vaporizer temperature plays a key role The fragmentor voltage is applied to the exit of the
for the complete evaporation of CAP because ion- capillary and affects the transmission and frag-
ization occurs in the vapor state like APCI. Thus, mentation of sample ions between the exit of the
in the case of using linear gradient elution, this capillary and the skimmer at relatively high pres-
temperature must be kept sufficiently high so that sure (3 torr). In general, the higher the fragmentor
the change of mobile phase composition does not voltage (which helps the transfer of ions), the more
influence the ion intensity of CAP. Under high fragmentation will occur. To establish the optimum
temperature, however, the risk of thermal degrada- fragmentor voltage for the analysis of CAP, the
tion occurs. In this study, the vaporizer tempera- intensity of this compound versus the fragmentor
ture was modified between 250 and 450 °C to voltage was studied in the range from 80 to 200 V.
optimize the intensity and the S/N ratio. The high- As shown in Figure 2, the optimum fragmentor
est temperature for a maximum intensity and S/N voltage was found at 120 V, whereas at higher
ratio of CAP was observed at 350 °C. The intensity values a significant intensity reduction was
of CAP decreased as the vaporizer temperature observed. Further, the best S/N ratio was also
was increased over 400 °C. In addition, intense observed at 120 V. The mass spectra of CAP at opti-
fragmentation was observed in the mass spectrum mal and higher fragmentor voltages are shown in
–
at 400 °C. Therefore, the decrease in intensity Figure 3. The deprotonated molecule (M-H) was
above 400 °C seems to be a result of the thermal the predominant ion at 120 V, and this included
degradation. Based on the above results, the vapor- isotopic ions (m/z 321, Cl35 Cl35; m/z 323, Cl35 Cl37;
izer temperature was set at 350 °C. m/z 325, Cl37 Cl37) because CAP includes two

2500000

2000000

1500000
Peak intensity

1000000

500000

0
80 100 120 140 160 180 200
Fragmentor voltage [V]

Figure. 2. The effect of the fragmentor voltage on the peak intensity of CAP concentration : 1 ng/mL. For the other conditions,
see Experimental section.

4
chlorines. A higher fragmentor voltage (180 V) Optimization of the Chromatographic Conditions
generated structurally relevant fragment ions. The
m/z 152 fragment ion gives the greatest intensity The separation of CAP from sample matrix peaks
and might be produced by the cleavage of the was optimized using acetonitrile, methanol, water,
carbon-carbon bond on the alkyl branch as shown and ammonium acetate. The combination of
in Figure 3. Other fragment ions are observed at methanol and ammonium acetate was found opti-
m/z 121 and 257. The m/z 121 may be the nitro- mum for the separation of CAP. When methanol
phenyl fragment. The m/z 257 fragment might be was replaced with acetonitrile, a significant signal
explained by a charge migration hydrogen shift intensity and S/N decrease was observed. This
with a concerted loss of HCl and CO. These result indicates that methanol may be a source of
observed fragment ions in the APPI source corre- electrons for the hydrogen abstraction from CAP.
sponded with the fragment ions in an ESI source Therefore, methanol and 10 mM ammonium
and an APCI source. Based on the above results, acetate was used as the mobile phase in this study.
the fragmentor voltage was set to 120 V. The flow rate was set at 0.5 mL/min considering
the size of the used column.

321

(A). Low fragmentor voltage (120 V)

600000

400000

200000

100000
100 150 200 250 300 350 400

(B). High fragmentor voltage (180 V) 321

250000
152
H
200000
121 NH CO CHCl2

O2N CH CHCH2OH
150000
OH
100000 152

257
50000
121
0
100 150 200 250 300 350 400

Figure 3. The mass spectra of CAP at two different fragmentor voltages.

5
Linearity, Detection Limit and Precision of LC/APPI-MS
System

The analytical performance characteristics of the

optimized LC/APPI-MS were first determined on
standard solutions of CAP in pure solvent. See
Figure 4. In order to achieve optimum sensitivity,
all experiments were carried out under SIM mode
–
using the mass corresponding to the [M-H] ions
for CAP. To test the linearity of the calibration
curves, various concentrations of CAP ranging
from 0.1 to 100 ng/mL were analyzed. The calibra-
tion curves of APPI showed good linearity with
correlation coefficients (r2) = 0.9998. The repeata-
bility of APPI for a standard solution was calcu-
lated on the basis of five replicates at 0.5 ng/mL in
the same day. The limit of detection (LOD) was
calculated by using a S/N ratio of 3 at 0.1 ng/mL.
The SIM chromatogram of CAP with APPI is shown
in Figure 4 (the S/N ratio of this chromatogram
was 4.2); LOD and RSD were 0.07 ng/mL and 2.1%.

900

700

500

300

0 5 10 15 20

Retention time [min]

Figure 4. SIM chromatogram of CAP in pure solvent at 0.1 ng/mL with APPI.

6
APPI Method Evaluation

To evaluate recoveries, the proposed method was

applied to the analysis of spiked CAP-free samples
of young yellowtail and flatfish meat. Eighteen
samples of two different fish were each spiked
with CAP and each sample was spiked at three
levels. The spiking levels ranged from 0.1 to 2 ng/g.
Typical chromatograms from the fish meat
extracts spiked at 1 ng/g and 0.1 ng/g are shown in
Figure 5.

20000 (A)

15000

10000

5000
0 5 10 15 20
1
16000 (B)

12000

8000

0 5 10 15 20

12000
(C)

8000

4000

0 5 10 15 20

10000 (D) 1

6000

2000

0 5 10 15 20
Retention time [min]

Figure 5. SIM chromatograms of A) Young yellowtail meat, B) Spiked young yellowtail meat at 1 ng/g CAP,
C) a flatfish meat, and D) a spiked flatfish meat at 0.1 ng/g CAP.

7
Data from 18 spiked samples led to recoveries and
RSD are summarized in Table 2.

Table 2 Recovery of CAP for Spiked Fish Meat

Spiking levels Recovery [±RSD (%)]*

(ng/g) Young yellowtail Flatfish
0.1 89.3 ±5.1 87.4 ±6.1
0.5 102.5 ±4.9 94.8 ±6.7
2.0 96.1 ±4.3 91.8 ±4.9
*Three spiked samples at the same amount were analyzed.

Mean recoveries ranged from 87.4% to 102.5% with

RSD of 4.3% to 6.7%. The LODs of CAP in fish meats
were determined by the signal corresponding to
three times the background noise on SIM chro-
matogram of spiked sample at 1 ng/g and 0.1 ng/g
and shown in Table 3. The intraday precision
(repeatability) was estimated by injecting the same
spiked fish meat extract at 0.1 ng/g five times during
a working day. The interday precision (reproducibil-
ity) was evaluated by analyzing the same sample
over 5 working days. The repeatability and repro-
ducibility for CAP in fish meats were 4.8%, 9.4% and
2.1%, 7.3%, respectively. These results indicate that
this LC/APPI-MS method is suitable for the analysis
of residues of CAP in fish meats.

Table 3. LODs, Repeatability, and Reproducibility of CAP in Standard Solution Using APPI
LODs* Repeatability** Reproducibility***
Fish meats (ng/g) (RSD, %) (RSD, %)
Young yellowtail 0.27 4.8 9.4
Flatfish 0.10 2.1 7.3

*Detection limit is LOD defined as S/N = 3 at 0.1 ng/mL.

**Repeatability was calculated on the basis of five replicates at 0.5 ng/mL within 1 day.
***Reproducibility was calculated by analyzing one fish meat spiked at 0.1 ng mL–1 per day during 5 days.

8
Conclusion
APPI is an ideal ionization technique because of
high sensitivity and high selectivity for the deter-
mination of CAP in fish meats. An important
advantage of using APPI for CAP content of fish
meats is that sample matrix did not significantly
affect ion intensity of CAP. The data presented
here demonstrate that this method is convenient
for routine analysis of CAP residues in fish meats
at trace levels, as excellent recoveries and preci-
sion for different samples were obtained.

References
1. A. H. Allen J. AOAC Int. 1985, 68, 990.
2. T. L. Li; Y.J. Chung-Wang; Y. C. Shih J. Food
Science 2001, 67, 21.
3. B. Roudaut J. Liq. Chrom. & Rel. Technol.
1994, 19, 1097.
4. C. N. Kenyon; A. Melera; F. Mrmi J. Anal.
Toxicol. 1981, 5, 216.
5. V. Hormazabal; M. Yndestad J. Liq. Chrom. &
Rel. Technol. 2001, 24, 2477.
6. K. Richard; V. Kruft; H. Sommer LaborParaxis
2000, 24, 91.
7. D. G. Kennedy; R. J. McCracken; A. Cannavan;
S. A. Hewitt J. Chromatogr. A. 1998, 812, 77.
8. D. B. Robb; T. R. Covey; A. P. Bruins Anal.
Chem. 2000, 72, 3653.
9. J. A. Syage; M. D. Evans; K. A. Hanold Amer.
Lab. 2000, 32, 24.

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9
www.agilent.com/chem

Agilent shall not be liable for errors contained herein or for incidental or consequential
damages in connection with the furnishing, performance, or use of this material.

Information, descriptions, and specifications in this publication are subject to change

without notice.

© Agilent Technologies, Inc. 2003

Printed in the USA

April 9, 2003
5988-8999EN
 Detection, Confirmation, and Quantification
of Chloramphenicol in Honey and Shrimp at
Regulatory Levels Using Quadrupole and
Ion Trap LC/MS
Application

Foods, Environmental

Authors and shrimp. Because it has displayed significant

toxicological effects on humans, it has been
Gerd Vanhoenacker, Frank David, Pat Sandra
banned from foods in the European community
Research Institute for Chromatography and the United States at levels greater than
Kennedypark 20, B-8500 0.1 ppb. Analytical methods used to determine this
Kortrijk, Belgium limit must achieve both the required sensitivity
Non-Author Contact and maintain sufficient selectively. LC/MS has
Jerry Zweigenbaum been demonstrated by the US Food and Drug
Administration for these analysis [1-3]. In addi-
Agilent Technologies, Inc.
tion, the Commission of European Communities
2850 Centerville Road,
has issued guidelines stipulating that for mass
Wilmington DE 19808-1610
spectral detection, a molecular ion (or quasimolec-
USA ular ion) and at least two fragment ions are needed
e-mail: [email protected] for positive confirmation [4]. For quantitative
analysis the Agilent 1100 LC/MSD provides excel-
lent results and can give some confirmation infor-
Abstract mation. The Agilent 1100 LC/MSD Trap gives
excellent full spectrum confirmation at the
Methodology capable of meeting regulatory requirements
regulated concentration.
has been developed for the determination of chloram-
phenicol in honey and shrimp. Samples of the two food-
stuffs are extracted with Isolute HN-M cartridges and Experimental
analyzed with both the Agilent 1100 LC/MSD Trap (SL)
and the Agilent 1100 LC/MSD (SL) quadrupole with nega- Reagents and Materials
tive mode electrospray ionization. Using deuterated inter-
nal standard and one simple sample extraction procedure, ISOLUTE HM-N cartridges from IST (Hengoed,
both instruments provide a limit of detection at or below UK, Part-nr. 800-1300-FM)
0.1 ppb in both shrimp and honey. Detection limits are Ethyl acetate from Vel (Merck Eurolab, Leuven,
lower using the ion trap for shrimp because of less matrix Belgium)
interference. The Agilent 1100 LC/MSD gives quantitative Methanol HPLC-grade from Merck (LiChrosolv,
results and the Agilent 1100 LC/MSD Trap gives full Darmstadt, Germany)
spectrum confirmation.
Deuterated (d5) CAP internal standard from
Cambridge Isotope Laboratories (CIL, Andover,
Introduction MA, USA)
Syringe filters (0.2 µm, PTFE) from Alltech
Chloramphenicol is a broad range antibiotic that
Associates Inc. (Lokeren, Belgium)
has found its way into foodstuffs such as honey
Sample Preparation 1 ng/µL IS is added. This portion is centrifuged for
10 minutes (2000 rpm). A 20-mL portion of the
For honey, 5 g of sample is diluted to 20 mL with supernatant is loaded on the cartridge and allowed
water and 5 µL of 1 ng/µL internal standard (IS) is to stand for 5 minutes. Elution is performed with
added. The solution is loaded on the cartridge and 50 mL ethyl acetate. The eluate is collected and the
allowed to stand for 5 minutes. Elution is per- solvent evaporated under a nitrogen stream at
formed with 50 mL ethyl acetate. The eluate is col- 40 °C. The residue is redissolved in 1 mL
lected and the solvent is evaporated under a water/methanol (9/1, v/v) and put in an ultrasonic
nitrogen stream at 40 °C. The residue is redis- bath for 1 minute. The solution is filtered before
solved in 1 mL water/methanol (9/1, v/v) and put injection.
in an ultrasonic bath for 1 minute. The solution is
filtered, using a syringe filter, before injection. No LC/MS Conditions
additional clean-up of the sample solution is
performed. The LC/MS systems were the Agilent 1100 LC/MSD
quadrupole mass spectrometer and the Agilent
For shrimp, a portion of at least 10 g of frozen 1100 LC/MSD Trap. Both were equipped with
shrimp is defrosted and mixed in a blender. To 10 g Agilent 1100 binary pumps and 1100 well plate
of the mixed shrimp, 30 mL of water and 10 µL of autosamplers. See Table 1.

Table 1. LC/MS Conditions

HPLC
Column Eclipse XDB C18, 4.6 mm × 150 mm, 5 µm (p/n 993967.902)
Flow-rate 0.9 mL/min
Mobile phase 10 mM ammonium acetate in water (solvent A)
Methanol/acetonitrile 1/9 (solvent B)
both from Merck (LiChrosolv, Darmstadt, Germany)
Gradient 0–1 min 30% B
1–8 min 30%–70% B
8–8.5 min 70%–100% B
8.5–12 min 100% B
Post time 4 min at 30% B
Injection 100 µL with needle wash (methanol)
Injection solvent Water/methanol (9/1 v/v) for both standards and samples
Column temperature 30 °C
MSD source settings
Source ESI
Ion polarity Negative
Drying gas temperature 340 °C
Drying gas flow-rate 11 L/min
Nebulizer pressure 50 psig
Vcap 3500 V
Quadrupole MSD
MSD acquisition on Between 3 and 7.5 min
Fragmentor 160 V
SIM settings m/z 257, 321, 323 (CAP)
m/z 262, 326, 328 (CAP-d5)

2
Table 1. LC/MS Conditions (continued)

Trap MSD
MSD acquisition on Between 3 and 7.5 min
Target mass (SPS) 323 m/z
Trap parameters
Max. accumulation time 300 ms
ICC target 30,000
Scan range 160–340
Averaging 2
Fragmentation parameters (MS/MS)
Smart Frag On, 30%–200% (default)
Isolation mass m/z 325.0
Isolation width 10.0 m/z
Fragmentation amplitude 1.0 V
Fragmentation cutoff m/z 88

Results and Discussion Table 2 Structure and Fragment Ions and Identity of CAP and
CAP-d5 (* Indicates Deuterated Positions for the
CAP-d5 IS)
Spectral Quality and Sensitivity of Standards
Chloramphenicol structure
For analysis with the quadrupole LC/MSD,
OH Cl
selected ion monitoring (SIM) was used to obtain H
the required sensitivity. Table 2 shows the struc- * N
ture, fragment ions and identity of CAP and * * Cl
CAP-d5. Figure 1 shows the analysis of a standard O O
*
mixture containing 2.5 pg/µL CAP and 5 pg/µL N+ OH
*
CAP-d5. By applying a fragmentor voltage of
O-
160 V, fragment ions at m/z 257 and 262 are
detected for confirmation purposes. Lowering the m/z Identity
fragmentor voltage to optimize for the m/z 321 and CAP 257 [M-H-HCOCl]–
m/z 326 and monitoring those ion alone would 249 [M-H-2HCl]–
obtain greater sensitivity. However, the confirma-
194 [M-H-NH2CoCl2H]–
tion of the fragment ions would be lost. For screen-
176 [M-H-NH2CoCl2H-H2O]–
ing analysis without confirmation this would be
acceptable and provide a much lower limit of CAP-d5 262 [M-H-HCOCl]–
detection (LOD). 254 [M-H2HCl]–
199 [M-H-NH2COCl2H]–
180 [M-H-NH2COCl2H-HDO]–

3
 7000 CAP
(2.5 pg/µL) 321 m/z

4000 323 m/z

257 m/z
1000

14000 CAP-d5 326 m/z

(5 pg/µL)

8000 328 m/z

2000 262 m/z

4 5
Time (min)

Figure 1. Analysis of a standard solution containing 2.5 ppb of CAP and 5 ppb of CAP-d5 (IS) on the quadrupole MSD. The
extracted ion chromatogram for the corresponding ions are shown.

Using the LC/MSD Trap in MS/MS mode both the

needed sensitivity (through reduction in chemical
noise) and selectivity (for confirmation) is
obtained. The compound shows a clear and repro-
ducible fragmentation pattern. An example of the
analysis of the standard mixture together with the
corresponding MS/MS spectra is shown in Figure 2.
Optimizing the fragmentation energy [turning off
Smart Frag] and fragmentation cutoff in the ion
trap will increase sensitivity even further than
shown here. Using an isolation width of 10 m/z
allows inclusion of the chlorine isotopes in the
resulting full scan mass spectra of the analyte and
the Cl35 isotope of the internal standard. Contact
Agilent for more details on these and other ion
trap settings.

4
 CAP: EIC 176, 194, 249, 257 m/z
(2.5 pg/µL)

1000

CAP-d5: EIC 180, 199, 254, 262

2000
(5 pg/µL)

1000

0
4 5
Time
(min)

2.5 pg/µL CAP 257

194
5 pg/µL d5-CAP6
262
249
176

160 240

0.2 pg/µL CAP

257
(LOD) 199 254
180
160 240 320 m/z

176 194

249

160 240 320 m/z

Figure 2. Analysis of a standard solution containing 2.5 pg/µL CAP and 5 pg/µL CAP-d5 (IS) on the LC/MSD Trap together with
the corresponding MS/MS spectra and the MS/MS spectrum resulting from an analysis of a standard solution
containing 0.2 pg/µL CAP.

Method Performance A calibration line was constructed by injecting

standard solutions of CAP with a concentration of
Standard solutions of CAP containing 5 pg/µL of 0 to 25 pg/µL with 5 pg/µL of the IS added to each
CAP-d5 were injected six consecutive times to test solution. One injection was performed per concen-
repeatability of injection on the mass selective tration. The quadrupole showed a linear response
detector (MSD) quadrupole instrument. This was for CAP in this concentration range. Calibration
done at two concentration levels. Each time, the curves and correlation coefficients are shown in
response of CAP relative to CAP-d5 was recorded. Figure 3. The LOD with this method was deter-
For a solution containing 0.5 pg/µL CAP the rela- mined to be ca. 0.2 pg/µL in a standard solution
tive standard deviations (RSDs) on the relative for both mass spectrometers. With the 100-µL
response were 5.05%. This 0.5-pg/µL level would injection used, this corresponds with 20 pg
correspond to a sample containing approximately on-column.
0.1 ppb CAP with the five-fold concentration step.
When a solution containing 5 pg/µL CAP was ana-
lyzed, RSDs on the relative response were 1.28%
for the quadrupole.

5
8.E+05 CAP (without IS) 5 CAP/CAP d5 (with IS)
R2 = 0.9994
R2 = 1

4
6.E+05

3
4.E+05
2

2.E+05
1

0.E+00 0
0 5 10 15 20 25 30 0 5 10 15 20 25 30
Concentration CAP (ppb) Concentration CAP (ppb)

Figure 3. Calibration graphs for standard solutions of CAP on the quadrupole with and without CAP-d5 (IS).

Extraction Recovery and Repeatability of Extraction

The extraction procedure was evaluated on

repeatability and linearity with the quadrupole
instrument. Blank honey was spiked with 1 ppb
CAP and 1 ppb CAP-d5. The extraction procedure
was carried out six times and the recovery was cal-
culated. The recovery for CAP varied from 85.31%
to 94.94% and the mean recovery was 90.60%. The
RSD on the recovery was 4.34% for CAP and 3.39%
when the IS was taken into account. An analysis of
blank honey spiked only with the IS is shown in
Figure 4 run on both instruments. With the
quadrupole, LC/MSD matrix interferences are pre-
sent but chromatographically separated from the
CAP signal. The ion trap results show that no
matrix interference is present in the isolation
window from m/z 318 to 328. The data suggest that
other endogenous compounds in honey produce
fragments at the same m/z as CAP. This supports
an even lower detection limit for this matrix if a
screening analysis were conducted with a lower
fragmentor voltage monitoring only the
m/z 321.

6
60000

TIC

30000

15000
EIC 257, 321, 323 m/z (CAP)

QUADRUPOLE
7500

12000

EIC 262, 326, 328 m/z (CAP-d5)

6000

0
4 5 6 7
Time (min)

15000
EIC 176, 194, 249, 257 m/z (CAP)

TRAP
15000
EIC 180, 199, 254, 262 m/z (CAP-d5)

0
4 5 6 7
Time (min)

Figure 4. Analysis of a blank honey sample containing 1 ppb CAP-d5.

A calibration curve was constructed with blank Spectra on the trap were similar for standard solu-
honey samples spiked with 0, 0.1, 0.2, 0.5, 1.0, and tions and real samples. An example of an MS/MS
2.0 ppb CAP. The samples also contained 1 ppb of spectrum of an extract of a honey sample spiked
the IS. The correlation coefficients were 0.9997 with 0.5 ppb CAP and 1 ppb CAP-d5 is shown in
and 0.9998 without and with correction with the Figure 5. Since the analyte and the IS coelute, a
IS, respectively. The slope for the calibration curve mixed spectrum is obtained. This could be avoided
constructed with these extracts for CAP with cor- by using a smaller isolation width and the multiple
rection with the IS was 0.1822. This is in good reaction monitoring (MRM) function of the ion
agreement with the slope obtained with the trap. Note that the chlorine isotope for Cl35Cl37 is
standard solutions, which is 0.1758 (see Figure 3). not observed for the deuterated internal
standard because its precursor ion is at the edge
of the isolation width and thus not trapped.

7
 262

257
194
249
176
199 254
180
160 240 320 m/z

Figure 5. Ion trap MS/MS spectrum from analysis of a honey sample spiked with 0.5 ppb CAP and 1 ppb CAP-d5.

Analysis of Honey

The extraction procedure and LC/MS methods

were applied to the analysis of honey samples that
were known to contain CAP. Sample results
obtained with the quadrupole and trap MSD were
compared (Figure 6).

TIC
80000

40000

0
14000
EIC 257, 321, 323 m/z (CAP)

QUADRUPOLE
8000

2000

15000
EIC 262, 326, 328 m/z (CAP-d5)
7500

0
4 5 6 7
Time (min)

EIC 176, 194, 249, 257 m/z (CAP)

15000
TRAP

0
15000 EIC 180, 199, 254, 262 m/z (CAP-d5)

0
4 5 6 7
Time (min)

Figure 6. Analysis of a honey sample containing 0.5 ppb CAP and 1 ppb CAP-d5.

8
The LOD for the honey samples varies between Analysis of Shrimp
detectors. For the quadrupole, it is found to be
0.5 pg/µL in the analytical solution. This corre- The same sample preparation method was applied
sponds with 50 pg on-column. Taking into account to the analysis of shrimp. The total volume of
the sample preparation with a five-fold concentra- shrimp and water added was about 40 mL. Taking
tion, samples containing 0.1 ppb CAP can be 20 mL of the 10 g shrimp aliquot for the Isolute
detected. It is obvious that the sample matrix inter- sample preparation and reconstituting the dried
feres with the sensitivity (Figures 4 and 6). Due to extract in 1 mL produced a five-fold concentration
the increased selectivity using MS/MS in the trap, as with the honey. This sample preparation shows
the LOD with this MS is similar for honey samples less matrix interference with the analysis com-
as for the standard solutions and is ca. 0.2 pg/µL in pared to honey samples. An example of an analysis
the analytical solution. This is equivalent to of shrimp is shown in Figure 7. Due to the reduced
0.04 ppb CAP in the sample because of the five-fold matrix effect, the LOD with the quadrupole is low-
concentration step. ered to nearly the same level as for the trap
(0.05 ppb in the sample with the five-fold concen-
tration). A concentration of 0.35 ppb was recov-
ered in the shrimp sample by both the quadrupole
and the trap MSD. Extraction recovery was
approximately 85%.

30000
TIC
15000

7000

QUADRUPOLE
EIC 257, 321, 323 m/z (CAP)
4000

16000

EIC 262, 326, 328 m/z (CAP-d5)

8000

4 5 6 7
Time (min)

10000
EIC 176, 194, 249, 257 m/z (CAP)
TRAP

0
10000
EIC 180, 199, 254, 262 m/z (CAP-d5)

0
4 5 6 7
Time (min)

Figure 7. Analysis of a shrimp containing 0.35 ppb CAP and 1 ppb CAP-d5.

9
www.agilent.com/chem
Conclusion
Honey and shrimp samples were successfully ana-
lyzed for CAP with both the quadrupole and trap
MSD. A simple liquid-liquid extraction procedure
using ISOLUTE HM-N cartridges was found to per-
form excellently in view of recovery and repeatabil-
ity. The LC method used a standard 4.6-mm id
column and produced the required sensitivity on
both instruments. The LC/MSD quadrupole instru-
ment produced excellent linearity and demon-
strated its quantitative ability. The LC/MSD Trap
showed the needed sensitivity with excellent full
scan capability below the regulated limit in both
sample matrices. The use of a broad isolation
window for full scan spectra using the ion trap pro-
duced more transition ions than required for
confirmation.

References
1 S. Turnipseed, et al. (2002) Confirmation of
Multiple Phenicol Residues in Honey by Electro-
spray LC/MS, Laboratory Information Bulletin
(4281) U.S. Food and Drug Administration.
2 A. Pfenning, et al. (2002) Confirmation of Multi-
ple Phenicol Residues in Shrimp by Electro-
spray LC/MS, Laboratory Information Bulletin
(4284) Food and Drug Administration.
3 B. K. Neuhaus, et al. (2002) LC/MS/MS Analysis
of Chloramphenicol in Shrimp, Laboratory
Information Bulletin (4290) Food & Drug
Administration.
4 D. Byrne, (2002) Performance Criteria, other
Requirements and Procedures for Analytical
Methods. Official Journal of European
Communities L221, 14–17.

For More Information

For more information on our products and services
visit our web site at www.agilent.com/chem

Agilent shall not be liable for errors contained herein or for incidental or consequential
damages in connection with the furnishing, performance, or use of this material.

Information, descriptions, and specifications in this publication are subject to change

without notice.

© Agilent Technologies, Inc. 2003

Printed in the USA

August 6, 2003
5988-9920EN
 A Validated Atmospheric Pressure
Chemical Ionization Method for Analyzing
Sulfonamides in Pork Muscle
Application

Food

Author Introduction
Ralph Hindle Meat, edible organs, animal feed, and animal waste
Access Analytical Labs may contain antibiotics, growth hormones, and
#3, 2616 - 16 Street N.E. other chemicals that can enter the food supply.
Calgary, AB. T2E 7J8 These compounds are added to maintain animal
Canada health, to increase animal growth rate, and to
reduce stress. Human exposure can result from
Agilent contact:
eating contaminated meat, or contacting runoff
Chin-Kai Meng
and leaching from manure and compost. Health
Agilent Technologies, Inc. specialists warn that there may be reduced options
2850 Centerville Road for effectively treating disease with antibiotics,
Wilmington, DE 19808-1610 such as penicillin and sulfa drugs, since antibiotic-
USA resistant strains of bacteria may develop from the
low-level exposure.
Abstract Sulfonamides are broad-spectrum antimicrobials
used in both humans and animals. The maximum
This application note presents a simple method for the residue limit (MRL) in Canada for sulfonamides in
analysis of sulfonamide antibiotics in pork muscle. Sam- meat is 100 ppb (ng/g), and 10 ppb in milk, while
ples were extracted with acidified methanol, centrifuged, the MRL in the European Union is 100 ppb for
and a portion of the extract was diluted with water. This both of these matrices. The Canadian Food Inspec-
dilution was analyzed directly by HPLC mass spectrome- tion Agency method for sulfonamides in meat
try using chemical ionization, with all compounds eluting tissue calls for extraction in ethyl acetate, parti-
in less than 5 minutes. Using an internal standard, recov- tioning with glycine buffer, followed by a
eries for seven sulfonamides ranged from 84%–118% at a pH-adjusted back extraction into methylene chlo-
spiking level of 50 ppb (ng/g). The statistically derived ride [1]. Extracts are evaporated, reconstituted,
detection limit was 10–25 ppb. A comparison was made to then separated by thin layer chromatography
the cleaned extracts using solid phase extraction, as well (TLC), derivatized, and quantitated by densitome-
as a comparison of mass selective detector settings for try. Alberta Agriculture has improved the quanti-
both screening (maximum sensitivity) and confirmation tative and qualitative aspects by using liquid
(greater fragmentation). The enhanced sensitivity of the chromatography/mass spectrometry (LC/MS) with
Agilent quadrupole mass selective detector allows this atmospheric pressure chemical ionization (APCI)
dilution cleanup technique to be used in labs where high for the final analysis [2]. There are a number of
throughput is required.
extraction steps in the Alberta method, and a Sample Preparation
faster method would greatly benefit laboratories
monitoring the food supply for residues. 1. For pork muscle, 3 g samples were weighed
directly into 50-mL polypropylene centrifuge
The goal of this method was to reliably quantitate tubes.
the sulfa drugs at one-half of the regulatory limit 2. The samples were homogenized for 3 minutes
or lower, with minimal sample preparation, and a with 10 mL acidified methanol using the Ultra-
maximum injection cycle time of 10 minutes. Maxi- Turrax homogenizer.
mum sensitivity is generally obtained by forming
as many parent ions [M+H]+ as possible and mini- 3. The samples were then centrifuged for 10 min-
mizing fragmentation. Due to the operational com- utes, and the supernatant decanted into a clean
plexity of triple quadrupole instruments, it is also test tube.
desirable to confirm positive findings on a single 4. The samples were then re-extracted with a fur-
quadrupole. This could be achieved by using colli- ther 10 mL acidified methanol, and centrifuged
sion induced dissociation (CID) to enhance fragment again.
ions characteristic of the compounds.
5. The supernatants were combined, and 1 mL IS
(2 mg) was added to the combined extract.
Experimental
6. The extract was diluted with de-ionized water
Chemicals and Materials 1 in 4 (250 µL extract + 750 µL water), filtered
through a 0.2 µm PVDF filter into an autosampler
All sulfonamide standards were purchased from vial, and analyzed directly by LC/MS.
Sigma Aldrich Canada, with a minimum purity of
99%. Stock solutions were prepared at 2 mg/mL in By adding an accurately known amount of IS to the
acetone, with the exceptions of sulfadiazine and combined extracts, there is no need to measure the
the sodium salt of sulfaquinoxaline. Three mL of final volume of the extract. The IS calculations per-
0.2N NaOH was added in order to completely dis- formed by the ChemStation measure the relative
solve these compounds. Standard solutions at dif- amounts of the analytes and IS. This corrects for
ferent concentrations were prepared for spiking any concentration or dilution effects in the samples.
and quantitation by diluting with de-ionized water. Sample extracts were also taken through SPE
Internal standard (IS): sulfachloropyridazine cleanup cartridges in order to compare with the
(SCPD) at 2 mg/mL in de-ionized water. dilution-only extracts. The 60-mg Oasis HLB car-
tridges are prewashed by eluting 1.5 mL acidified
HPLC-grade methanol and acetonitrile were pur- methanol, followed by 1.5 mL de-ionized water. The
chased from Caledon Labs (Georgetown, Ontario). 1 mL extract was diluted to 10 mL with de-ionized
water, eluted through the cartridges, and the
Formic acid (min. 98%), was purchased from EM eluant was discarded. The sulfa drugs were then
Science. eluted with 1.5 mL acidified methanol. This eluant
was evaporated to near dryness under nitrogen.
Acidified methanol was prepared by adding about Samples were reconstituted in 1 mL of 25% methanol
100 µL of 98% formic acid to 100-mL methanol. in water, filtered, and analyzed by LC/MS.
Ultra-Turrax T8 homogenizer with 8-mm diameter A further comparison was done by evaporating
dispersing element, 50-mL polypropylene cen- 1 mL methanol extract to near dryness, and recon-
trifuge tubes, and 13-mm polyvinylidene fluoride stituting it in 1 mL of 25% methanol in water with-
(PVDF) syringe filters (0.2 µm), were purchased out the SPE cleanup. This gave the sample extract
from VWR Scientific. the proper solvent composition for HPLC analysis,
Oasis HLB (3 cc, 60 mg) solid phase extraction but without the dilution step to negatively affect
(SPE) cartridges were purchased from Waters. the detection limits (DL) of the compounds.

2
LC/MS Conditions ion [M+H]+ in LC electrospray ionization (ESI) or
APCI in target ion mode. However, once a positive
The LC/MS system was made up of Agilent is detected, a confirmation must be made as to
Technologies 1100 Series solvent degasser, binary whether the suspect peak is actually the target
pump, autosampler, column oven, diode array analyte, or simply a co-eluting compound that pro-
detector, and quadrupole mass selective detector duces the same ion. There are a number of ways to
(MSD) (Table 1). perform the confirmation: re-extract the sample
with a different solvent system; further clean up
the sample to a higher final concentration, to allow
Compound Identification and Confirmation detection of additional confirmation ions or analy-
sis in scan mode; derivatize and analyze by gas
In general, the goal of a monitoring method for
chromatography/mass spectrometry (GC/MS); or
target analytes is to separate the compounds from
re-analyze the extract on a triple quadrupole
potential interferences and maximize sensitivity
LC/MS/MS. All of these techniques are useful, but
on the instrument. Using mass spectrometry (MS),
the drawback is the additional time and expense
maximum sensitivity is achieved by the production
involved, especially with LC/MS/MS.
of a single ion, for example, the protonated parent

Table 1. LC/MSD Conditions

HPLC
Column Zorbax Eclipse XDB-C8, 150 mm × 4.6 mm, 5 µm (p/n 993967-906)
Solvent A 0.1% Formic acid in water
Solvent B 0.1% Formic acid in acetonitrile
Gradient t0 = 20% B
t1 = 20% B
t3 = 90% B
t6.5 = 90% B
Post time = 1.5 min
Flow rate 1.0 mL/min
Injection volume 50 µL
Column temp 30 °C
MSD
Source APCI (positive ion mode)
Ion dwell time 8 Ions at 63 ms each
Fragmentor 70 V
Drying gas 6.0 L/min
Nebulizer pressure 60 psi
Drying gas temperature 350 °C
Vaporizer temperature 400 °C
Capillary voltage 3000 V
Corona current 4 µA

3
The Agilent 1100 MSD has the capability of acquir-
ing up to four separate MS signals during the same
run, where each signal can be made up of a number
of selected ions (SIM) or a full scan spectrum. For
example, Signal 1, with a low fragmentor voltage to
maximize parent ion response, can include each of
the [M+H]+ ions in the target list, while Signal 2, at
higher fragmentor voltages can acquire the confir-
matory fragment ions. For analytes expected at
higher concentrations, Signal 1 could acquire in
SIM mode for quantitation, while Signal 2 could be
set for scan mode for identification. Figure 1
demonstrates the former example, with the Frag-
mentor set to 70 V for Signal 1 (MSD1), and 200 V
for Signal 2 (MSD2).

MSD1 250, EIC=249.7:250.7 (SULFAMSD\SSSCI01.D) APCI, Pos, SIM, Frag: 70, "Quantitation"

8000 Simultaneous 2-signal aquisition; Fragmentor at 70 V or 200 V

3.365 - SPY
4000

0
2.5 3.0 3.5 4.0 4.5 5.0 5.5 min

MSD1 285, EIC=284.7:285.7 (SULFAMSD\SSSCI01.D) APCI, Pos, SIM, Frag: 70, "Quantitation"
8000

4000 4.344 - SCPD (IS)

0
2.5 3.0 3.5 4.0 4.5 5.0 5.5 min

MSD2 108, EIC=107.7:108.7 (SULFAMSD\SSSCI01.D) APCI, Pos, SIM, Frag: 200, "Confirmation"
3000

2000

1000
2.5 3.0 3.5 4.0 4.5 5.0 5.5 min

MSD2 156, EIC=155.7:156.7 (SULFAMSD\SSSCI01.D) APCI, Pos, SIM, Frag: 200, "Confirmation"

700
500
300
2.5 3.0 3.5 4.0 4.5 5.0 5.5 min

Figure 1. Dual MSD acquisition signals (Masses 108 and 156 are class-specific fragments for sulfonamides).

4
Table 2 shows the mass spectra for the sulfon-
amides using various fragmentor voltages. Masses
108 and 156 are class-specific fragments for sulfon-
amides (H2N+=[C6H4]=O and H2N+=[C6H4]=SO2,
respectively), and, as such, are very useful diag-
nostic ions, when acquired along with the
protonated molecular ion.

Table 2. APCI Spectra of Sulfonamides, Using Various Fragmentor Voltages

Compound Fragmentor 70 V Fragmentor 160 V Fragmentor 200 V
*MSD1 SPC, time=2.859:3.513 of SFCISCAN\SULFA006.D *MSD1 SPC, time=2.891:3.337 of SFCISCAN\SULFA008.D *MSD1 SPC, time=2.907:3.258 of SFCISCAN\SULFA009.D
APCI, Pos, Scan, Frag: 70 APCI, Pos, Scan, Frag: 160 APCI, Pos, Scan, Frag: 200

100

156.1

108.1
256.1 Max: 151737 100 Max: 69275 100 Max: 32290

256.1
80 80 80
Sulfathiazole (STZ)
C9H9N3S2O2 60 60 60

101.1
MW = 255

101.1 108.2

156.1
RT = 3.05 min 40 40 40

107.5
257.1

120.1
257.1
20

157.1
20 20

256.1
107.5
0 0 0
100 200 300 m/z 100 200 300 m/z 100 150 200 250 m/z

*MSD1 SPC, time=2.971:3.369 of SFCISCAN\SULFA010.D *MSD1 SPC, time=2.986:3.369 of SFCISCAN\SULFA012.D *MSD1 SPC, time=2.987:3.242 of SFCISCAN\SULFA013.D
APCI, Pos, Scan, Frag: 70 APCI, Pos, Scan, Frag: 160 APCI, Pos, Scan, Frag: 200
100 Max: 66929 100 Max: 23650 100 Max: 10375

251.1
251.1

108.1
80 80 80
Sulfadiazine (SDZ)
156.1

C10H10N4SO2 60 60 60
MW = 250

156.1
RT = 3.09 min 40 40 40
107.5 108.2

107.5

185.1
252.1

252.1

120.1
158.1

20

109.1

158.0
20 20

251.1
185.1

0 0 0
100 200 300 m/z 100 200 300 m/z 100 150 200 m/z

*MSD1 SPC, time=3.212:3.467 of SFCISCAN\SULFA002.D *MSD1 SPC, time=3.210:3.608 of SFCISCAN\SULFA004.D *MSD1 SPC, time=3.210:3.529 of SFCISCAN\SULFA005.D
APCI, Pos, Scan, Frag: 70 APCI, Pos, Scan, Frag: 160 APCI, Pos, Scan, Frag: 200

100 Max: 35037 100 Max: 16213 100 Max: 2943

108.1
250.1

184.1
250.1

80 80 80
Sulfapyridine (SPY) 156.1
C11H11N3SO2 60 60 60

250.1
MW = 249
40 40
107.4

40
183.4
156.1

RT = 3.33 min
120.1

157.1
251.1
101.2

251.1
184.1
108.1

20 20 20

0 0 0
100 150 200 250 m/z 100 200 300 m/z 100 150 200 m/z
*MSD1 SPC, time=3.626:3.865 of SFCISCAN\SULFA002.D *MSD1 SPC, time=3.608:3.991 of SFCISCAN\SULFA004.D *MSD1 SPC, time=3.672:3.927 of SFCISCAN\SULFA005.D
APCI, Pos, Scan, Frag: 70 APCI, Pos, Scan, Frag: 160 APCI, Pos, Scan, Frag: 200

100 Max: 20181 100 Max: 9216 100 Max: 4083

265.1

265.1

110.2

Sulfamerazine (SMR) 80 80 80
C11H12N4SO2
108.2

60 60 60
MW = 264
RT = 3.78 min 40 40 40
156.1
101.2

265.1
117.1

110.2

250.1
266.2

109.4

172.1
199.1
266.1

172.1

120.1

20 20 20
155.3

184.1

0 0 0
100 200 300 m/z 100 200 300 m/z 100 200 300 m/z

5
Table 2. APCI Spectra of Sulfonamides, Using Various Fragmentor Voltages (Continued)
Compound Fragmentor 70 V Fragmentor 160 V Fragmentor 200 V

*MSD1 SPC, time=3.993:4.168 of SFCISCAN\SULFA002.D *MSD1 SPC, time=3.991:4.262 of SFCISCAN\SULFA004.D *MSD1 SPC, time=4.007:4.246 of SFCISCAN\SULFA005.D
APCI, Pos, Scan, Frag: 70 APCI, Pos, Scan, Frag: 160 APCI, Pos, Scan, Frag: 200
100 Max: 57189 100 Max: 35356 100 Max: 11707

279.1

279.1

124.2
80 80 80
Sulfamethazine (SMZ)

279.1
C12H14N4SO2 60 60 60
MW = 278
40 40 40

186.1
108.1
RT = 4.06 min

156.1
280.1
124.2
280.1

123.5

213.2

280.1
20 20 20

186.1
0 0 0
100 200 300 m/z 100 200 300 m/z 100 200 300 m/z
*MSD1 SPC, time=4.184:4.423 of SFCISCAN\SULFA002.D *MSD1 SPC, time=4.262:4.453 of SFCISCAN\SULFA004.D *MSD1 SPC, time=4.262:4.438 of SFCISCAN\SULFA005.D
APCI, Pos, Scan, Frag: 70 APCI, Pos, Scan, Frag: 160 APCI, Pos, Scan, Frag: 200
100 Max: 15461 100 Max: 6222 Max: 3906
100

285.0

156.0

285.0

108.1
Sulfachloropyridazine 80 80
80
(SCPD); istd 60 60
C10H9N4SO2Cl 60

123.3 124.2

156.0
287.1

284.3 287.0
111.2
135.3 138.1
101.2

MW = 284 40 40 40

130.1
115.2

107.4
RT = 4.31 min

120.1
265.1
163.1
179.1
20 20

184.2
20
0 0 0
100 200 300 m/z 100 200 300 m/z 100 150 200 250 m/z

*MSD1 SPC, time=4.470:4.725 of SFCISCAN\SULFA014.D *MSD1 SPC, time=4.389:4.756 of SFCISCAN\SULFA016.D *MSD1 SPC, time=4.469:4.772 of SFCISCAN\SULFA017.D
APCI, Pos, Scan, Frag: 70 APCI, Pos, Scan, Frag: 160 APCI, Pos, Scan, Frag: 200
301.1

301.1

108.2
100 Max: 148192 100 Max: 63906 100 Max: 16120

156.1
80 80 80
Sulfaquinoxaline (SQ)
60 60 60

301.1
C14H12N4SO2

129.1
MW = 300
156.1

40 40 40

107.5

145.3
RT = 4.51 min
302.1

302.1

235.1
120.1
108.2

302.1
146.1

288.4
20 20 20
129.1

0 0 0
100 200 300 m/z 100 200 300 m/z 100 200 300 m/z

*MSD1 SPC, time=4.485:4.740 of SFCISCAN\SULFA018.D *MSD1 SPC, time=4.454:4.900 of SFCISCAN\SULFA020.D *MSD1 SPC, time=4.485:4.900 of SFCISCAN\SULFA021.D
APCI, Pos, Scan, Frag: 70 APCI, Pos, Scan, Frag: 160 APCI, Pos, Scan, Frag: 200
311.1

311.1

156.1

311.1
100 Max: 467058 100 Max: 261189 100 Max: 80490

Sulfadimethoxine 80 80 80
(SDMX) 60 60 60
C14H12N4SO2
MW = 310
155.3

40 40 40
108.1
156.1

312.1

RT = 4.54 min
312.1

312.1
245.2
156.1

157.1

20 20 20
218.1

0 0 0
100 200 300 m/z 100 200 300 m/z 100 200 300 m/z

6
Chromatography analysis; the chromatogram is easier to interpret;
and the amount of SDZ is not underestimated due
While complete separation of target compounds is to co-elution of SPY in the standard mix.
not always necessary when using mass spectral
detection, it is, however, essential when common A recently published application shows four sul-
ions are present. For example, the protonated mol- fonamides were analyzed with an injection cycle
ecular ion of SPY is 250 mass units. Due to the time of 1.1 minutes, using a 2-position 10-port
naturally-occurring C13 isotope, ions 251 coexist valve, two analytical columns in parallel, and a
with the parent ions 250. Separating SPY from SDZ second binary pump [3]. Since most labs do not
(m/z = 251) was, therefore, important when trying have such high sample volume requirements, the
to optimize the chromatographic conditions, and method described in this application note was
was achieved as shown in Figure 2. While this developed using more conventional techniques,
results in a slightly longer chromatographic run without the additional hardware costs. Conditions
than would otherwise be necessary, there is more were set up to provide good chromatographic sepa-
consistent integration of the peaks during data ration in a relatively short time of 6 minutes (total
cycle time was 10 minutes).

MSD1 256, EIC=255.7:256.7 (SULFAMSD\SSS_CI5.D) APCI, Pos, SIM, Frag: 70

3.051 - STZ

1 2 3 4 5 min
MSD1 251, EIC=250.7:251.7 (SULFAMSD\SSS_CI5.D) APCI, Pos, SIM, Frag: 70
3.084 - SDZ

1 2 3 4 5 min
MSD1 250, EIC=249.7:250.7 (SULFAMSD\SSS_CI5.D) APCI, Pos, SIM, Frag: 70
3.332 - SPY

1 2 3 4 5 min
MSD1 265, EIC=264.7:265.7 (SULFAMSD\SSS_CI5.D) APCI, Pos, SIM, Frag: 70
3.783 - SMR

1 2 3 4 5 min
MSD1 279, EIC=278.7:279.7 (SULFAMSD\SSS_CI5.D) APCI, Pos, SIM, Frag: 70
4.056 - SMZ

1 2 3 4 5 min
MSD1 285, EIC=284.7:285.7 (SULFAMSD\SSS_CI5.D) APCI, Pos, SIM, Frag: 70
4.314 - SCPD (IS)

1 2 3 4 5 min
MSD1 301, EIC=300.7:301.7 (SULFAMSD\SSS_CI5.D) APCI, Pos, SIM, Frag: 70
4.514 - SQ

1 2 3 4 5 min
MSD1 311, EIC=310.7:311.7 (SULFAMSD\SSS_CI5.D) APCI, Pos, SIM, Frag: 70
4.537 - SDMX

1 2 3 4 5 min

Figure 2. Sulfonamide standard mix, 500 pg each (SIM).

7
Sample Cleanup In order to develop a high-throughput method,
keep the number of required steps to a minimum.
The total ion chromatograms (TIC) in Figure 3 The Agilent liquid chromatography/mass selective
show that there is considerable matrix background detector (LC/MSD) has enough sensitivity to allow
from the samples. A simple solvent exchange was simple dilution of the extracts with water to act as
performed, where 1 mL of extract was evaporated a cleanup technique. This eliminates the need for
under nitrogen, and reconstituted in 25% methanol costly SPE cartridges and analyst time to further
in water. One of the problems with solvent exchange prepare the samples. Minimal sample handling can
only is the amount of matrix material that is also improve recoveries, since losses are possible
injected onto the HPLC column. Peak shape can be at each step.
negatively affected by overloading, and eventually
the performance of the column will deteriorate. All The third chromatogram in Figure 3 shows how
of this matrix material is also introduced into the the use of SPE cleanup techniques can remove the
MSD. Frequent cleaning and maintenance may be majority of co-extracted materials, allowing for a
required for the MSD, further reducing more concentrated final extract and ultimately
productivity. lower DLs. This also results in a simpler chro-
matogram for integration and interpretation.

MSD1 TIC, MS File (SF030817\SULFA018.D) APCI, Pos, SIM, Frag: 70

200000 Spiked pork extract - solvent exchange to 25% MeOH in water
150000
100000
50000
0
1 2 3 4 5 6 min

MSD1 TIC, MS File (SF030816\SULFA011.D) APCI, Pos, SIM, Frag: 70

200000
150000
100000 Spiked pork extract - Diluted 1 in 4 with water
50000
0
1 2 3 4 5 6 min

MSD1 TIC, MS File (SF030817\SULFA010.D) APCI, Pos, SIM, Frag: 70

200000
150000 Spiked pork extract - Oasis HLB cleanup
100000
50000
0
1 2 3 4 5 6 min

Figure 3. TIC comparisons of various cleanup techniques.

8
However, where the goal of a method is to screen
large numbers of samples to find potential viola-
tions of MRLs, a simple dilution technique may be
preferred. Dilution could offer enough cleanup for
good chromatographic separation, while remaining
concentrated enough to meet DL requirements.
The second chromatogram in Figure 3 shows a
much improved baseline. Figures 4 through 6 show
the same analyses with all the target ions in SIM
mode.

MSD1 256, EIC=255.7:256.7 (SF030817\SULFA018.D) APCI, Pos, SIM, Frag: 70

3 .0 5 4 - S T Z

3.0 3.5 4.0 4.5 5.0 5.5 min

MSD1 251, EIC=250.7:251.7 (SF030817\SULFA018.D) APCI, Pos, SIM, Frag: 70

3.094 - SDZ

3.0 3.5 4.0 4.5 5.0 5.5 min

MSD1 250, EIC=249.7:250.7 (SF030817\SULFA018.D) APCI, Pos, SIM, Frag: 70

3.340 - SPY

3.0 3.5 4.0 4.5 5.0 5.5 min

MSD1 265, EIC=264.7:265.7 (SF030817\SULFA018.D) APCI, Pos, SIM, Frag: 70
3.794 - SMR

3.0 3.5 4.0 4.5 5.0 5.5 min

MSD1 279, EIC=278.7:279.7 (SF030817\SULFA018.D) APCI, Pos, SIM, Frag: 70
3.180
4.083 - SMZ 4.760
3.0 3.5 4.0 4.5 5.0 5.5 min

MSD1 285, EIC=284.7:285.7 (SF030817\SULFA018.D) APCI, Pos, SIM, Frag: 70

4.345 - SCPD (IS)

3.0 3.5 4.0 4.5 5.0 5.5 min

MSD1 301, EIC=300.7:301.7 (SF030817\SULFA018.D) APCI, Pos, SIM, Frag: 70

4.549 - SQ

3.0 3.5 4.0 4.5 5.0 5.5 min

MSD1 311, EIC=310.7:311.7 (SF030817\SULFA018.D) APCI, Pos, SIM, Frag: 70

4.572 - SDMX

3.0 3.5 4.0 4.5 5.0 5.5 min

Figure 4. Solvent exchange only (SIM)

9
MSD1 256, EIC=255.7:256.7 (SF030816\SULFA011.D) APCI, Pos, SIM, Frag: 70

3.044 - STZ

3.0 3.5 4.0 4.5 5.0 5.5 min

MSD1 251, EIC=250.7:251.7 (SF030816\SULFA011.D) APCI, Pos, SIM, Frag: 70
3.078 - SDZ

3.0 3.5 4.0 4.5 5.0 5.5 min

MSD1 250, EIC=249.7:250.7 (SF030816\SULFA011.D) APCI, Pos, SIM, Frag: 70

3.324 - SPY

3.0 3.5 4.0 4.5 5.0 5.5 min

MSD1 265, EIC=264.7:265.7 (SF030816\SULFA011.D) APCI, Pos, SIM, Frag: 70

3.782 - SMR

3.0 3.5 4.0 4.5 5.0 5.5 min

MSD1 279, EIC=278.7:279.7 (SF030816\SULFA011.D) APCI, Pos, SIM, Frag: 70

3.139
4.056 - SMZ
3.0 3.5 4.0 4.5 5.0 5.5 min
MSD1 285, EIC=284.7:285.7 (SF030816\SULFA011.D) APCI, Pos, SIM, Frag: 70

4.316 - SCPD (IS)

3.0 3.5 4.0 4.5 5.0 5.5 min

MSD1 301, EIC=300.7:301.7 (SF030816\SULFA011.D) APCI, Pos, SIM, Frag: 70

4.517 - SQ

3.0 3.5 4.0 4.5 5.0 5.5 min

MSD1 311, EIC=310.7:311.7 (SF030816\SULFA011.D) APCI, Pos, SIM, Frag: 70
4.541 - SDMX

3.0 3.5 4.0 4.5 5.0 5.5 min

Figure 5. Diluted 1 in 4 with water.

10
MSD1 256, EIC=255.7:256.7 (SF030817\SULFA010.D) APCI, Pos, SIM, Frag: 70

3.042 - STZ

3.0 3.5 4.0 4.5 5.0 5.5 min

MSD1 251, EIC=250.7:251.7 (SF030817\SULFA010.D) APCI, Pos, SIM, Frag: 70

3.078 - SDZ

3.0 3.5 4.0 4.5 5.0 5.5 min

MSD1 250, EIC=249.7:250.7 (SF030817\SULFA010.D) APCI, Pos, SIM, Frag: 70

3.322 - SPY

3.0 3.5 4.0 4.5 5.0 5.5 min

MSD1 265, EIC=264.7:265.7 (SF030817\SULFA010.D) APCI, Pos, SIM, Frag: 70

3.777 - SMR

3.0 3.5 4.0 4.5 5.0 5.5 min

MSD1 279, EIC=278.7:279.7 (SF030817\SULFA010.D) APCI, Pos, SIM, Frag: 70

3.166 4.077 - SMZ

3.0 3.5 4.0 4.5 5.0 5.5 min

MSD1 285, EIC=284.7:285.7 (SF030817\SULFA010.D) APCI, Pos, SIM, Frag: 70

4.343 - SCPD (IS)

3.0 3.5 4.0 4.5 5.0 5.5 min

MSD1 301, EIC=300.7:301.7 (SF030817\SULFA010.D) APCI, Pos, SIM, Frag: 70

4.546 - SQ

3.0 3.5 4.0 4.5 5.0 5.5 min

MSD1 311, EIC=310.7:311.7 (SF030817\SULFA010.D) APCI, Pos, SIM, Frag: 70

4.569 - SDMX

3.0 3.5 4.0 4.5 5.0 5.5 min

Figure 6. After HLB cleanup (SIM).

Results and Discussion In both cases, a five-point IS calibration with SCPD

was used, with 20 to 200 pg of each target com-
The recoveries obtained for seven samples spiked pound injected, plus 2,000 pg SCPD. The five stan-
at a level of 50 ppb (150 ng of each sulfonamide in dards were injected both before and after the set of
3 g sample) appear in the following tables. The seven spikes, and the curves were created by using
spiking solutions were added before homogeniza- the average responses of the two sets of standards.
tion and allowed to stand for at least 30 minutes Peak height was used to measure response, as
before extraction. SMR (sulfamerazine) was added there was less variability compared to peak area,
separately at 300 ng per sample before homoge- due to the noticeable tailing of these compounds.
nization, and could be used as a surrogate. Results The linearity results (R2) are tabulated in
in Table 3 were obtained by simply diluting the Tables 3 and 4.
extracts 4-fold with water (recovery 84%–118%),
while results in Table 4 are from extracts taken
through SPE cleanup (recovery 79%–104%).

11
Table 3. Recoveries of Sulfonamides by Diluting Extracts 1 in 4 with Water
Amount recovered (ng)
Description STZ SDZ SPY SMR SMZ SCPD(IS) SQ SDMX
Pork spike 1 167 172 164 317 151 2,000 148 130
Pork spike 2 168 197 68 343 164 2,000 169 137
Pork spike 3 160 183 158 315 157 2,000 133 121
Pork spike 4 158 189 167 336 156 2,000 138 129
Pork spike 5 151 169 154 295 169 2,000 133 129
Pork spike 6 147 161 144 322 143 2,000 120 112
Pork spike 7 144 72 141 272 151 2,000 124 125
Amount spiked (ng) 150 150 150 300 150 2,000 150 150
Mean 156 178 157 314 156 2,000 138 126
SD (Precision) 9 13 11 24 9 – 17 8
MDL (SD × t-stat) ng 29 40 34 77 28 – 53 26
LOQ (SD × 10) ng 94 126 108 245 88 – 167 82
RSD (SD × 100/Mean) 6 7 7 8 6 – 12 7
Accuracy (%) 104 118 104 105 104 100 92 84
Linearity (R2) 0.9997 0.9996 0.9997 0.9972 0.9996 1.0000 0.9984 0.9992
t-stat (N=7) 3.14 3.14 3.14 3.14 3.14 3.14 3.14 3.14

Table 4. Recoveries of Sulfonamides Using Oasis HLB Cleanup Cartridges

Amount recovered (ng)
Description STZ SDZ SPY SMR SMZ SCPD(IS) SQ SDMX
Pork spike 1 161 157 132 273 149 2,000 139 126
Pork spike 2 154 156 132 293 157 2,000 153 131
Pork spike 3 149 158 124 267 155 2,000 132 113
Pork spike 4 145 152 122 279 144 2,000 119 111
Pork spike 5 151 162 127 294 149 2,000 127 121
Pork spike 6 136 147 127 274 136 2,000 116 108
Pork spike 7 148 161 128 275 155 2,000 124 116
Amount spiked (ng) 150 150 150 300 150 2,000 150 150
Mean 149 156 127 279 149 2,000 130 118
SD (Precision) 8 5 4 10 7 – 13 8
MDL (SD × t-stat) ng 24 17 11 33 23 – 40 26
LOQ (SD × 10) ng 76 53 36 104 73 – 128 82
RSD (SD × 100/Mean) 5 3 3 4 5 – 10 7
Accuracy (%) 99 104 85 93 100 100 87 79
Linearity (R )
2
0.9994 0.9994 0.9997 0.9979 0.9998 1.0000 0.9989 0.9989
t-stat (N=7) 3.14 3.14 3.14 3.14 3.14 3.14 3.14 3.14

12
Table 5 summarizes the comparison of recoveries when SPE cleanup is used. Instrumental condi-
when diluted with water versus using Oasis HLB tions allow injection cycle-time of 10 minutes using
cartridge cleanup. Generally there is a greater dif- typical columns and conditions for most labs.
ference in recoveries for the early eluting com-
pounds, as one might expect. Since the samples are
loaded onto the cartridge with a mostly aqueous References
phase (10% methanol in water), the water-soluble 1. TLC-Densitometric Procedure for Sulfonamide
matrix components would tend to pass through the Residues in Animal Tissue, SUL-SP08, Canadian
cartridge to waste. Because these early eluting Food Inspection Agency, Saskatoon,
compounds were removed prior to injection on the Saskatchewan, Canada; 2001/04.
HPLC column, the chromatograms are cleaner with
more reproducible chromatography, as shown by 2. Sulfonamides in Tissue by LC/MS, Alberta
the smaller standard deviations in recoveries. The Agriculture, Edmonton, Alberta, Canada,
results from the HLB cleanup exhibited smaller Standard Operating Procedure TX-0278-01.
standard deviations and lower minimum detection 3. Mark Stahl, “High-throughput analysis with the
levels (MDLs). Agilent 1100 Series high-throughput LC/MS
system”, Agilent Technologies, publication
5988-9638EN. www.agilent.com/chem
Conclusion
A fast and sensitive single quadrupole LC/APCI/MS
method was developed and validated for detection
of sulfonamide residues in pork. The DL ranged
For More Information
from 10 to 25 ng/g of tissue when analyzed by For more information on our products and services,
simple dilution of the extracts, and 4 to 13 ng/g visit our Web site at www.agilent.com/chem.

Table 5. Comparison of Recoveries Obtained by Dilution vs Oasis HLB Cleanup

Description STZ SDZ SPY SMR SMZ SCPD(IS) SQ SDMX
Accuracy % (1 in 4 dilution) 104 118 104 105 104 100 92 84
SD (Precision) 9.4 12.6 10.8 24.5 8.8 – 16.7 8.2
MDL (ng) 29 40 34 77 28 – 53 26
Accuracy % (HLB cleanup) 99 104 85 93 100 100 87 79
SD (Precision) 7.6 5.3 3.6 10.4 7.3 – 12.8 8.2
MDL (ng) 24 17 11 33 23 – 40 26

13
www.agilent.com/chem

Agilent shall not be liable for errors contained herein or for incidental or consequential
damages in connection with the furnishing, performance, or use of this material.

Information, descriptions, and specifications in this publication are subject to change

without notice.

© Agilent Technologies, Inc. 2003

Printed in the USA

October 20, 2003
5989-0182EN
 The Analysis of Fluoroquinolones in Beef
Kidney Using HPLC Electrospray Mass
Spectrometry
Application

Food

Authors Introduction
Ralph Hindle Fluoroquinolones are synthetic antibacterial com-
Access Analytical Labs pounds derived from nalidixic acid, and are useful
#3, 2616 - 16 Street N.E. to treat animal infections that are resistant to
Calgary, Alberta T2E 7J8 other antibacterial agents. They have a broad spec-
Canada trum of activity, acting against both gram-positive
and gram-negative bacteria. The maximum residue
Agilent contact:
limit (MRL) for enrofloxacin (as the sum of
Chin-Kai Meng
enrofloxacin and ciprofloxacin) was entered into
Agilent Technologies, Inc. Annex 1 of Council Regulation (EEC) No. 2377/90
2850 Centerville Road for kidney at 200 µg/kg in bovine and ovine
Wilmington, DE 19808-1610 species, and 300 µg/kg for porcine, poultry, and
USA rabbits. For all other food producing species, the
MRL is 200 µg/kg in kidney [1].
Abstract There are a number of methods describing the
analysis of fluoroquinolones in various tissues,
A fast and simple screening method was validated for the with HPLC coupled with fluorescence and mass
analysis of three fluoroquinolone antibiotics in beef spectrometric detection being very popular. Most
kidney. Samples were extracted with acidified methanol, methods involve extraction into acidic or basic
centrifuged, diluted with water, and filtered. The diluted organic solvents, followed by some type of cleanup,
extract was analyzed directly by HPLC mass spectrometry most notably solid phase extraction (SPE). The
using electrospray ionization in positive ion mode. Using Canadian Food Inspection Agency extracts animal
an internal standard, mean recoveries were 73%–96% at tissue with acidic ethanol, followed by strong
spiking levels of 33 µg/kg (ppb), with statistically derived cation exchange SPE cleanup, and HPLC fluores-
detection limits of 8–19 µg/kg. This is below the European cence analysis [2]. Chen and Schneider [3]
Union maximum residue limit of 200 µg/kg for enrofloxacin described a screening method for enrofloxacin in
and ciprofloxacin in bovine kidney. The method is evaluated chicken, where extracts were detected by fluores-
relative to the requirements of the European Commission cence without cleanup, following extraction and
Decision 2002/657/EC for use as a confirmatory method. centrifugation.
European Community Commission Decision Acidified methanol was prepared by adding 100 µL
2002/657/EC allows the use of HPLC coupled with of 98% formic acid to 100 mL of methanol.
fluorescence detection [4] for substances in Group
B of Annex I to Directive 96/23/EC. Quinolones Acidified deionized water was prepared by adding
and other veterinary drugs fall into Group B, 100 µL of 98% formic acid to 100 mL deionized
where three identification points are required for water.
confirmation by Selected Ion Monitoring (SIM)
Ultra-Turrax T25 homogenizer, 50-mL polypropy-
using mass spectrometry (MS). With low resolution
lene centrifuge tubes, and 13-mm polyvinylidene
HPLC/MS, one point can be earned for each ion
fluoride (PVDF) syringe filters (0.2 µm), were
detected, provided that the ion ratios meet relative
purchased from VWR Scientific.
intensity criteria. Additional requirements of
Directive 2002/657/EC, based on spiking levels of All fluoroquinolones, including the IS, were pro-
33 µg/kg carried out in this study, are as follows: vided as a gift from the Canadian Food Inspection
• The internal standard (IS) shall be added to the Agency, Calgary, Alberta, Canada, as stock solu-
test portion at the beginning of the extraction tions of 100 ng/µL (ppm) in 1% acetic acid in
procedure. methanol. Solutions were stored at 4 °C. Standard
solutions at different concentrations were pre-
• In order to allow the use of data corrected for pared for spiking by dilution with acidified
mean recovery, the range of recoveries allowed methanol solution. The analytes ciprofloxacin,
are –20% to +10%. enrofloxacin, and sarafloxacin were chosen as tar-
• The reproducibility of coefficient variation (CV) gets since these compounds are included in the
(%) is expected to be about one-half to two- Canadian Food Inspection Agency’s proficiency
thirds of the 100 µg/kg CV, which is 23%, at a check samples. The spiking standard for these
concentration of half the permitted limit. compounds (1 ng/µL) was prepared by diluting
100 µL of each the stock solutions to a 10-mL volu-
• For liquid chromatography/mass spectrometry metric flask, and made to volume with acidified
(LC/MS) procedures, the minimum acceptable deionized water. A separate IS solution at 1 ng/µL
retention time (RT) for the analyte under exam- was prepared the same way, except that it only
ination is twice the RT corresponding to the contained norfloxacin and danofloxacin.
void volume of the column.
• The ratio of the chromatographic RT of the ana- Sample Preparation
lyte to that of the IS, that is, the relative RT of 1. For beef kidney, 3 g samples were weighed
the analyte, shall correspond to that of the cali- directly into 50-mL polypropylene centrifuge
bration solution at a tolerance of 2.5% for LC. tubes.
• The molecular ion shall preferably be one of the 2. For spiked samples, 100 µL of the 1-ng/µL
selected diagnostic ions. (100 ng) spiking solution was added, resulting
• The maximum permitted tolerances for relative in fortification levels of 33 µg/kg. Samples were
ion intensities shall meet the criteria in the allowed to stand for 1 hour before subsequent
Annex, (in this case, either ±25% or 30%), as extraction.
reproduced in Table 6. 3. For the sample blank, 100 µL of acidified
methanol solution was added.
Experimental 4. For all spiked samples, 100 µL of the 1-ng/µL
(100 ng) IS solution was added just prior to
Chemicals and Materials extraction. Norfloxacin was included in this
solution at the same level, to be used as an
HPLC-grade methanol and acetonitrile were pur- alternate IS, if required due to potential
chased from Caledon Labs (Georgetown, Ontario). interferences for danofloxacin.
Formic acid, min. 98%, was purchased from EM 5. The samples were homogenized for 2 min
Science. with 15 mL of acidified methanol using the
Ultra-Turrax homogenizer.
Acidified methanol solution: 30% methanol in
pH 3 deionized water (100 µL of formic acid per 6. The samples were then centrifuged for 10 min,
100 mL of water). and the supernatant decanted into a clean test
tube.

2
7. The extract was diluted with acidified deion- LC/MS Conditions
ized water 1 in 4 (250 µL of extract + 750 µL of
water), filtered through a 0.2-µm PVDF filter The HPLC system was made up of an Agilent
into an autosampler vial, and analyzed directly Technologies 1100 series solvent degasser, binary
by LC/MS. pump, autosampler, column oven, diode array detec-
tor (DAD), and quadrupole mass selective detector
By adding an accurately known amount of IS to the (MSD) (Table 2).
initial sample before extraction, there is no need to
measure the final volume of the extracts, nor the
Table 2. LC/MSD Conditions
aliquot to be diluted. The IS calculations, per-
formed by the ChemStation, measure the relative HPLC
amounts of the analytes and IS. This corrects for Column Zorbax Eclipse XDB-C8,
any concentration or dilution effects in the 150 mm × 4.6 mm, 5 µm
samples. (P/N 993967-906)
Solvent A 0.1% Formic acid in water
Standard Preparation
Solvent B 0.1% Formic acid in acetonitrile
A 5-point calibration curve was used for the deter- Gradient t0 = 20% B
mination of each of the three target compounds, t1 = 20% B
and a 1-point curve was used for norfloxacin, the t8 = 90% B
alternate IS. Table 1 gives the volumes of the IS t15 = 90% B
and target solutions added (1 ng/µL each) to each Post time = 2.0 min
of five test tubes. The standards were prepared by Flow rate 0.4 mL/min
adding 250 µL of the blank extract and 750 µL of Injection volume 50 µL
acidified deionized water to the tubes containing
the analytes, after which the solutions were fil- Column temp 30 °C
tered through 0.2-µm PVDF filters. MSD
Source Electrospray Ionization (ESI) (positive
The final solution of each standard contained 5 ng ion mode)
of IS per mL of diluted extract, or 5 pg/µL. With
50 µL injected, this results in 250 pg injected. The Ion dwell time 14 ions at 40 ms each
amount of target analyte in each of the five solu- Fragmentation Varies by ion, see Table 3
tions varies to produce the calibration curves, as Drying gas flow 12 L/min
shown in Table 1.
Nebulizer pressure 30 psi
The correlation coefficient (R2) for the target ana- Drying gas temperature 350 °C
lytes ranged from 0.9987 to 0.9992, as shown in Capillary voltage 4000 V
Table 4.

Preparation of the standards in this fashion will

Table 3. Fragmentor Voltages for Acquired Ions in SIM (single
compensate for any ion suppression or enhance- acquisition group)
ment that may occur, due to the presence of
co-eluting material at the MS source, which may not Compound Ion Fragmentor (V)
otherwise occur if pure solvents alone are used. Norfloxacin (IS) 320 120
302 200
276 200
Table 1. Preparation of Analytical Standards (50-µL Injections
Ciprofloxacin 332 120
into LC/MSD)
314 200
Target Target 288 200
IS Volume volume IS Amount amount Danofloxacin (IS) 358 120
added added injected injected 340 220
Standard (µL) (µL) (pg) (pg) Enrofloxacin 360 120
1 5 1 250 50 342 220
2 5 2 250 100 316 220
3 5 5 250 250 Sarafloxacin 386 120
4 5 10 250 500 368 220
5 5 20 250 1,000 342 220

3
All ions were included in a single acquisition Chromatography
group, which started at injection (time = 0). An
alternative approach would be to set the group All compounds eluted between 5 and 9 minutes,
start time to a value around half a minute before however the total run time was set to 15 minutes
the elution of the first compound, as this will keep with 90% organic solvent to allow co-extractives to
the eluant stream diverted to waste as long as pos- elute from the column. Otherwise, their eventual
sible. This will reduce the amount of co-extracted elution could interfere with subsequent injections.
material being introduced into the source, reduc- This is more of a potential problem when methods
ing contamination. with abbreviated cleanups, such as dilution-only,
are used. The following figures compare the blank
Another alternative is to add an additional time- beef kidney sample to a sample fortified at 33 µg/kg.
programmed acquisition group to the method, and In each case, the selected ions are the protonated
only include the ions for compounds eluting within forms of the parent ion, as well as the protonated
the group times. This will take on more signifi- ions resulting from the loss of H2O (M-18) and CO2
cance as the overall number of compounds in a (M-44).
method increases, and with three ions per com-
pound required for identity confirmation. The qualifier ion for danofloxacin, the compound
used as the IS for this study, is mass 340. The matrix
Fragmentor voltages were chosen that maximized causes an interference at mass 340. The interfer-
the response for each selected ion. For each ence is shown as a small peak in the beef kidney
fluoroquinolone, a value of 120 V produced only blank as shown in Figure 1. Since a diagnostic
the protonated parent ion, while higher voltages qualifier ion is not required for the IS calculations,
were required to induce fragmentation to confir- it had no impact on the results. It does, however,
matory ions. The ions monitored corresponded to indicate that there is elution of co-extractive mate-
the neutral losses of water and carbon dioxide in rial in the samples, and that without further
each case. cleanup, ion suppression may result from its pres-
ence. All standards were prepared in blank beef
Note that although mass 342 is acquired for both kidney extract in order to compensate for these
enrofloxacin and sarafloxacin, it is only added to potential effects.
the MSD acquisition table once.

Norfloxacin (IS) 12000 5.530 - NOR

8000
320 = [M + H]+ 4000 320
302 = [M - H2O + H]+ 0
12000
276 = [M - CO2 + H]+ 8000
5.531- NORq1
4000 302
0
12000
8000
4000 5.532 - NORq2 276
0
5.5 6 6.5 7 7.5 8 8.5 9 min

Beef kidney blank 12000

8000 5.484 - NOR
4000
320
0
12000
8000
302
4000 5.485 - NORq1
0
12000
8000
5.482 - NORq2 276
4000
0
5.5 6 6.5 7 7.5 8 8.5 9 min

Figure 1. Comparative extracted ion chromatograms for fluoroquinolones spiked into beef kidney.

4
 Ciprofloxacin spike 6000 5.854 - CIPRO
4000
332 = [M + H]+ 2000 332
314 = [M – H2O + H]+ 0
6000
288 = [M – CO2 + H]+ 4000 5.850 - CIPROq1
2000 314
0
6000
4000
2000 5.846 - CIPROq2 288
0
5.5 6 6.5 7 7.5 8 8.5 9 min

Beef kidney blank 6000

4000
2000 332
0
6000
4000
2000 314
0
6000
4000
2000 288
0
5.5 6 6.5 7 7.5 8 8.5 9 min

Danofloxacin (IS) 8000

6000 6.009 - DANO
358 = [M + H]+ 4000
340 = [M - H2O + H]+ 2000 358
0
8000
6000 6.012 - DANOq1
4000
2000 340
0
5.5 6 6.5 7 7.5 8 8.5 9 min

8000
Beef kidney blank 6000
4000
2000 358
0
8000
6000
4000 340
2000 6.04 - DANOq1
0
5.5 6 6.5 7 7.5 8 8.5 9 min

Figure 1. Comparative extracted ion chromatograms for fluoroquinolones spiked into beef kidney (Continued).

5
 6000 6.910 - ENRO
4000
2000 360
Enrofloxacin spike 0
6000
360 = [M + H]+ 4000 6.914 - ENROq1 342
342 = [M – H2O + H]+ 2000
0
316 = [M – CO2 + H]+ 6000
4000
2000 6.917 - ENROq2 316
0
5.5 6 6.5 7 7.5 8 8.5 9 min

6000
4000 360
2000
Beef kidney blank 0
6000
4000 342
2000
0
6000
4000
2000 316
0
5.5 6 6.5 7 7.5 8 8.5 9 min

8000
8.604 - SARA
4000 386
Sarafloxacin spike 0
8000
386 = [M + H]+ 8.601- SARAq1
2000 368
368 = [M – H2O + H]+ 0
342 = [M – CO2 + H]+ 8000
8.594 - SARAq2
4000 342
0
5.5 6 6.5 7 7.5 8 8.5 9 min

8000
4000
386
Beef kidney blank 0
8000
4000
368
0
8000
4000
342
0

Figure 1. Comparative extracted ion chromatograms for fluoroquinolones spiked into beef kidney (Continued).

6
Sarafloxacin elutes from the column in the same mean recovery. With a CV of only 8% for this
region as a number of other co-extractives, making compound, it looks as though the method may still
identification and quantitation more difficult. produce acceptable results for screening purposes,
However, as shown in Table 7, the qualifier ions but some additional work may be required to pro-
still meet the identification criteria for relative duce higher recoveries. Since the work presented
responses of the qualifiers, and so further cleanup here involves spiked samples only, recovery-
of the samples may not be necessary. The effect of correction calculations do not apply.
these co-extractives will also be reduced at higher
incurred residue levels, closer to those permitted Norfloxacin was added along with danofloxacin as
by the European Union MRL. an additional IS. However, examination of the
blank beef kidney used in this study shows nor-
Recoveries floxacin to be present as an incurred residue, at a
concentration approximately one half of the spik-
In order to allow results to be corrected for recov- ing level. Assuming a linear response through the
eries, where the determined incurred levels are origin, this would mean that norfloxacin was
divided by the percent recovered from certified detected at approximately 15–20 µg/kg, which is
reference materials or spiked samples, Table 2 of about 10% of the permitted level for enrofloxacin
the Annex requires that the recoveries for analytes in bovine kidney. Recoveries for norfloxacin are
at levels greater than 10 µg/kg be within the range included in Table 4, even though they were calcu-
of 80% to 110%. Table 4 shows that recoveries for lated with a single point calibration, and not cor-
ciprofloxacin and enrofloxacin meet this require- rected for incurred residues. However, there is
ment, with 96.3% and 86.0%, respectively. However, some compensation for this since the standards
sarafloxacin fails the requirement, with only 72.6% used for calibration were prepared by addition of
the targets to the blank extracts.

Table 4. Recoveries of Fluoroquinolones from Beef Kidney

Amount recovered (ng)

Description Norfloxacin Ciprofloxacin Enrofloxacin Sarafloxacin
Kidney spike 1 111.8 96.3 84.9 68.5
Kidney spike 2 93.1 94.0 85.6 64.1
Kidney spike 3 88.0 89.6 83.8 77.6
Kidney spike 4 98.9 95.4 86.2 75.2
Kidney spike 5 82.2 93.8 85.4 82.1
Kidney spike 6 143.0 109.3 87.9 72.9
Kidney spike 7 102.6 101.3 83.3 73.0
Kidney spike 8 110.6 90.8 91.3 67.7
Amount spiked (ng) 100.0 100.0 100.0 100.0
Mean 103.8 96.3 86.0 72.6
SD (Precision) ng 18.9 6.3 2.5 5.8
MDL (SD × t-stat) ng 56.7 19.0 7.6 17.4
LOQ (SD × 10) ng 189.1 63.4 25.4 58.1
CV (SD/Mean) % 18.2 6.6 3.0 8.0
Accuracy (%) 103.8 96.3 86.0 72.6
Linearity (R2) 0.9895 0.9987 0.9992 0.9987
t-stat (N = 8) 3.00 3.00 3.00 3.00

7
Compound Identification

For chromatographic separation, Section 2.3.3.1 of

the Annex to 2002/657/EC requires that the mini-
mum acceptable RT for the analyte under investi-
gation be at least twice the RT corresponding to
the void volume of the column (k'=1). The first
compound to elute under these conditions is
norfloxacin, with a k' of 2.6, therefore this condi-
tion is easily met. The second condition is that the
ratio of the RT of the analyte to that of the IS, that
is the relative RT, shall correspond to that of the
calibration solution at a tolerance of ±2.5% for LC.
Table 5 shows the RT times of each analyte in the
spiked samples, compared to those of the stan-
dards, and that they are well within the allowable
tolerance.

Table 5. Relative RTs of Analytes in Samples, Compared to Standards

Average RRT in CV (%) RRT in RRT in samples, relative

Compound standards (N = 15) standards (N = 15) to standards (N = 8)
Norfloxacin 0.922 0.12% 99.8%–100.1%
Ciprofloxacin 0.975 0.05% 99.9%–100.1%
Enrofloxacin 1.150 0.16% 99.8%–100.2%
Sarafloxacin 1.439 0.47% 99.5%–100.3%

Compound Confirmation

Section 2.3.3.2 of the Annex to 2002/657/EC gives

the maximum permitted tolerances for relative ion
intensities, which is reproduced in Table 6.

Table 6. Maximum Permitted Tolerances for Relative Ion Intensities Using a

Range of Mass Spectrometric Techniques
GC/MS(CI), GC/MSn,
Relative intensity GC/MS(EI) LC/MS, LC/MSn
(% of base peak) (relative) (relative)
>50% ±10% ±20%
>20% to 50% ±15% ±25%
>10% to 20% ±20% ±30%
≤10% ±50% ±50%

Note MSn equals MS/MS if n = 2

8
Table 7 shows the relative intensities for each of sample dilutions and standards. As previously
the qualifier ions for the three target compounds, mentioned, the standards are prepared by accu-
as well as norfloxacin and danofloxacin (one ion). rately measuring the relative amounts of target
As expected, norfloxacin meets the criteria in each and IS compounds into a tube or vial, followed by
of the eight spiked samples, even though it had addition of blank kidney extract and water. The
incurred residues. The presence of additional nor- exact proportions of extract and water do not have
floxacin should not negatively affect this qualita- to be known, since the IS calculations uses amount
tive aspect of performance, and it does not. and response ratios, rather than absolute amount
Danofloxacin, however, showed an interference for and response, in determining concentrations in
the single qualifier ion monitored, and so the rela- unknowns. An accurate measurement of extract
tive amount of this signal would be expected to and water volumes can, however, reduce
vary to a larger degree, depending upon the exact interference variability.
amount of blank extract used in preparing the

Table 7. Relative Intensities of Qualifier Ions for Fluoroquinolones in Beef Kidney, Compared to Permitted Tolerances

Relative intensities (%) of qualifier ions

Norfloxacin Ciprofloxacin Danofloxacin Enrofloxacin Sarafloxacin
Sample Q1 = 302 Q2 = 276 Q1 = 314 Q2 = 288 Q1 = 340 Q1 = 342 Q2 = 316 Q1 = 368 Q2 = 342
Spike 1 49 15 47 17 64 44 28 50 15
Spike 2 45 15 48 17 58 46 24 41 17
Spike 3 48 16 46 20 63 44 28 45 14
Spike 4 42 15 46 17 60 43 30 43 13
Spike 5 49 17 45 21 65 44 26 45 11
Spike 6 50 19 39 17 72 45 29 43 12
Spike 7 49 17 41 18 65 46 29 46 13
Spike 8 47 17 42 19 62 39 24 46 12
Average for Stds 49 20 44 20 86 43 26 47 15
Std Dev for Stds 2 1 3 2 22 2 2 6 1
Tolerance(Table 7) 25 30 25 30 20 25 25 25 30
Lower 37 14 33 14 69 32 19 35 11
Allowable
(calculated)
Upper 62 26 55 25 103 53 32 59 20
Allowable
(calculated)

9
 www.agilent.com/chem
Conclusion
A fast and sensitive single quadrupole LC/ESI/MS
method was validated for the detection of three
fluoroquinolone antibiotics (ciprofloxacin,
enrofloxacin, and sarafloxacin) in beef kidney. The
detection limits ranged from 8 to 19 µg/kg (ppb),
with direct analysis of sample extracts after dilu-
tion with water. All qualitative requirements were
met with respect to the Annex to EU Directive
2002/657/EC for spiked samples, and recoveries of
two of the three compounds met the quantitative
requirements. Recovery of sarafloxacin was
slightly lower than the level required to allow
correction for recoveries in reported results.

References
1. The European Agency for the Evaluation of
Medicinal Products, Veterinary Medicines and
Inspections, EMEA/MRL/820/02-FINAL,
January 2002.
2. Determination of Fluoroquinolones in Bovine,
Porcine and Avian Tissues by Liquid Chro-
matography with Fluorescence Detection,
FQL-SP04, Canadian Food Inspection Agency,
Saskatoon, Saskatchewan, Canada; 2001/03.
3. Chen, G., Schneider, M. J., (2003) A Rapid
Spectrofluorometric Screening Method for
Enrofloxacin in Chicken Muscle. J. Agric. Food
Chem., 51(11), 3249-3253.
4. Annex of Commission Decision 2002/657/EC,
Commission Decision of 12 August 2002, imple-
menting Council Directive 96/23/EC concerning
the performance of analytical methods and the
interpretation of results, Official Journal of the
European Communities, 17.8.2002, L 221/8-36,
Table 5, Footnote 4.

For More Information

For more information on our products and services,
visit our Web site at www.agilent.com/chem.

Agilent shall not be liable for errors contained herein or for incidental or consequential
damages in connection with the furnishing, performance, or use of this material.

Information, descriptions, and specifications in this publication are subject to change

without notice.

© Agilent Technologies, Inc. 2004

Printed in the USA

February 10, 2004
5989-0596EN
 Determining Malachite Green and
Leucomalachite Green in Food by
LC/MS/MS
Application

Food Safety

Authors have already banned MG in fishery. Due to its low

Feng Liang, Peibin Hu, and Ping Li
cost and antifungal effectiveness, MG is still being
used illegally as indicated in the European Rapid
Agilent Technologies
Alert System for Food and Feed.2
Beijing, China
HPLC with UV detection has been used to analyze
MG and LMG. Figure 1 shows the structure of the
Abstract two compounds. Loss of conjugation by reduction
changes the chromaphore of LGM significantly. To
This application note demonstrates a complete method to
obtain the sum of both, the method employs post-
rapidly and precisely determine residue levels of mala-
column oxidation with PbO2 to convert LMG to
chite green and leucomalachite green in fish with the new
MG, thus providing a sum of both comounds.3 Most
Agilent 6410 LC/MS triple quadrupole system. Using pos-
recently, LC/MS has been used to both meet the
itive mode electrospray ionization (ESI+) and multiple
EU confirmation criteria and provide quantitative
reaction monitoring (MRM), qualification and quantifica-
results for both compounds without the need for
tion were accomplished without the traditional tedious
post-column oxidation. In this application, a
PbO2 oxidation process. The LC/MS/MS method’s LOQ is
simple and sensitive method for simultaneously
0.01 µg/Kg, which easily meets the import requirement of
determining MG and LMG is presented.4, 5 The
2 µg/Kg set by Japan and the EU.
LC/MS/MS method’s LOQ is 0.01 µg/Kg, which
easily meets the import requirement set by Japan
Introduction or the EU.6

Malachite green (MG) is a metallic-looking crystal. Experimental

It dissolves in water easily as a blue-green solution.
It is a toxic chemical primarily used as a dye and Reagents
has been found very effective in treating parasites,
fungal infections, and bacterial infections in fish MG Sigma-Aldrich,
and fish eggs.1 On uptake, MG is rapidly reduced CAS 569-64-2, USA
into leucomalachite green (LMG) and deposited in LMG Dr. Ehreastorfer's lab,
the fatty tissue of the fish with little MG remaining. D-86199, 99% pure,
Augsburg, Germany
MG can cause significant health risk for humans Acetonitrile CAS 75-05-8; Burdick &
who eat contaminated fish. For example, it can Jackson; Morristown,
cause liver tumor formation and is suspected of New Jersey, USA
carcinogenesis.1 The United States, Japan, China, Acetic acid Merck, Germany
the European Union, and many other countries Ammonium acetate CAS 631-61-8, Acros
Organics, Morris Plains,
New Jersey, USA
 H
C N C N

_
N Cl N
+

Malachite green Leucomalachite green

Figure 1. Molecular structure of malachite green and leucomalachite green.

Calibration Solutions Instrumentation

A stock standard solution of MG and LMG in ace- LC 1100 LC
tonitrile was prepared at 100 µg/mL and stored at Column C18, 2.1 x 150 mm, 5 µm
%18 oC, avoiding light. The stock solution was Column temp. 40 oC
diluted in 50:50 acetonitrile:water to make the cali- Mobile phase A % 10 mmol/L ammonium acetate
bration solutions+10, 50, 100, 500, 1000, 5000, and (adjust to pH 4.5 with acetic acid)
10,000 fg/µL. B % acetonitrile
Column flow 0.3 mL/min
Sample Preparation Gradient Time %B
0 30
To 5 g tilapia tissue was added 1 mL (0.25 mg/mL) 1 50
hydroxylamine, 2 mL 1 M toluene sulfonic acid, 2 95
2 mL of 0.1 M ammonium acetate buffer (pH 4.5), 8 95
and 40 mL acetonitrile. The mixture was then 8.01 30
homogenized for 2 min. The supernatant was 13 30
decanted, and to the precipitate was added 20 mL Injection vol. 10 µL
acetonitrile. This was filtered and added to the
supernatant. To the combined acetonitrile MS Agilent 6410 LC/MS Triple
extracts, 35 mL water and 30 mL methylene chlo- Quadrupole
ride were added. The solution was shaken and the Ionization ESI(+)
methylene chloride layer collected. A second Capillary 4000 V
extract of 20 mL methylene chloride was made, Nebulizer P. 35 psi
and this layer added to the first extract. The meth- Drying gas 11 L/min
ylene chloride was taken to dryness with a gentle Gas temp. 350 oC
stream of nitrogen and the extract reconstituted in Skimmer 15 V
100 µL of acetonitrile OctDc1 (Skim2) 45 V
Oct RF 500 V
Q1 resolution Unit
Q3 resolution Unit
Collision gas Nitrogen

The MRM parameters are listed in Table 1.

2
Table 1. MRM Method Parameters
Dwell Fragmentor Collision
Time Compound Precursor Product (ms) (V) Energy (V)

0 MG 329.3 313.3 40 100 40

329.3 208.2 40 100 40
7 LMG 331.3 316.3 40 100 30
331.3 239.2 40 100 30

Results and Discussion (a) 20 V, (b) 30 V, and (c) 40 V. Due to their struc-
tural differences, the voltage required for optimum
To obtain the most sensitive results, optimization fragmentation of each compound is different. For
of certain fragmentor voltages is important. MG, the optimum fragmentation was observed at
Figure 2 shows the EICs of both target compounds 40 V. The ion m/z 313 was due to the neutral loss
at fragmentor values of 70 V, 90 V, and 100 V. The of methane. The ion at m/z 208 was due to the neu-
results show that the three different fragmentor tral loss of N,N-dimethylaniline. For LMG, the opti-
values have little effect on the intensity of [M+H]+ mum fragmentation was observed at 30 V. The ion
ions. Thus, 100 V was chosen for this study. at m/z 316 was due to the loss of a methyl radical.
The ion at m/z 239 resulted from a subsequent loss
In addition, an optimal collision energy for the of a benzene radical or, more likely, the rearrange-
MS/MS must be set. Figure 3 shows the MS/MS ment and neutral loss of toluene.
spectra from three different collisional voltages,

x107 + EIC(329.4, 331.4 m/z) Scan optimizing FRG70_3.d

5
3 70 V
1

+ EIC(329.4, 331.4 m/z) Scan optimizing FRG90_4.d

x107
5
90 V
3
1

x107 + EIC(329.4, 331.4 m/z) Scan optimizing FRG100_5.d

5
3 100 V
1
0 1 2 3 4 5 6 7 8 9 10 11 12
Abundance vs. acquisition time (min)

Figure 2. EICs of malachite green and leucomalachite green at fragmentor values of 70 V, 90 V, and 100 V.

3
 329.3
x105 + Product Ion (5.499-5.633 min, 17 scans) (329.3, 331.4 ≥ **) optimizing MS2_FRG100_CE20_2.d
1.2
1.0
0.8
0.6
0.4
0.2
284.3 313.4
0.0 193.1 208.3 236.9 268.4

331.8
x105 + Product Ion (8.349-8.466 min, 15 scans) (331.4, 329.3 ≥ **) optimizing MS2_FRG100_CE20_2.d
2.0 239.8

1.6 316.7
1.2

0.8

0.4 209.8 272.8

120.8 134.5 165.6 195.8 223.9 286.6 301.8
0.0
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340

Abundance vs. mass-to-charge (m/z)

Figure 3a. MS/MS spectra of MG and LMG at collisional voltage of 20 V.

x104
329.3
7 + Product Ion (5.457-5.591 min, 17 scans) (331.4, 329.3 ≥ **) optimizing MS2_FRG100_CE30_3.d
6
5
4
313.4
3
2 208.2
1
134.3 165.1 192.8 217.4 237.2 251.4 270.3 285.3298.9
0

+ Product Ion (8.349-8.457 min, 14 scans) (331.4, 329.3 ≥ **) optimizing MS2_FRG100_CE30_3.d
239.8
x105
3.5
3.0
2.5
2.0
1.5
1.0 315.8
0.5 209.8 223.9 272.7 286.8
134.5 165.8 180.6 194.7 301.9 331.8
0.0
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340
Abundance vs. mass-to-charge (m/z)

Figure 3b. MS/MS spectra of MG and LMG at collisional voltage of 30 V.

4
 x104 + Product Ion (5.474-5.591 min, 15 scans) (331.4, 329.3 ≥ **) optimizing MS2_FRG100_CE40_4.d 313.3

4.0

3.0

2.0 208.2

1.0 329.4
165.3 241.4 284.2
91.5 117.9 134.1 192.1 221.0 270.3 299.4
0.0

+ Product Ion (8.340-8.499 min, 20 scans) (329.3, 331.4 ≥ **) optimizing MS2_FRG100_CE40_4.d
x105 239.8
2.5

2.0

1.5

1.0

0.5 194.7 315.8

165.8 180.7 208.7 223.8
91.6 120.6 134.5 257.9 272.7 286.7
0.0
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340
Abundance vs. mass-to-charge (m/z)

Figure 3c MS/MS spectra of MG and LMG at collisional voltage of 40 V.

Figure 4 shows the calibration curves for both MG Figure 5 shows MS/MS spectra of a blank sample
(4a) and LMG (4b). Calibration solution concentra- extract (5a) and sample extract spiked with
tions were from 10 to 10,000 fg/µL. The linear cali- 10 ppt of each compound (5b). A sample of
bration range is 100 to 100,000 fg on column for tilapia spiked at 100 ppt MG and LMG before
both compounds. The R2 for both compounds was extraction was made to demonstrate method
> 0.999 (origin ignored and no weighting). To performance. The MRM results after extraction
demonstrate the sensitivity of the instrument, and cleanup are shown in Figure 6. The recover-

x105
Malachite green - 7 levels, 7 levels used, 14 points, 14 points used, 0 QCs
2.6
y = 23363.3374 * × - 1766.9951
2.4 R 2 = 0.99946103
2.2
2.0
1.8
1.6
Response

1.4
1.2
1.0
R 2 = 0.9995
0.8
0.6
0.4
0.2
0.0
0 1 2 3 4 5 6 7 8 9 10 11 12
Concentration (ng/mL)

Figure 4a. Calibration curve of malachite green, linear range: 10 ppt to 10 ppb.

5
 x10 6
Leucomalachite green - 7 Levels, 7 Levels Used, 14 Points, 14 Points Used, 0 QCs
1.0 y = 93199.4712 * × - 7543.3588
R 2 = 0.99942595
0.9

0.8

0.7

0.6
Response

0.5

0.4 R 2 = 0.9994
0.3

0.2

0.1

0.0
0 1 2 3 4 5 6 7 8 9 10 11 12
Concentration (ng/mL)

Figure 4b. Calibration curve of leucomalachite green, linear range: 10 ppt to 10 ppb.

8.433

x101 12
+ MRM MRM (331.3 ≥ 239.2) malachite green_200606121.d
2.8

2.4

2.0

1.6

1.2

0.8

0.4

0.0
0 1 2 3 4 5 6 7 8 9 10 11 12
Abundance vs. acquisition time (min)

Figure 5a. MG and LMG MRM of a blank sample.

x102
+ MRM MRM (329.3 ≥ 313.3) malac hite green_200606121.d 12
1.4

1.2

1.0

0.8

0.6 5.440

0.4

0.2

0.0
0 1 2 3 4 5 6 7 8 9 10 11 12
Abundance vs. acquisition time (min)

ppt spiked sample.

Figure 5b. MG and LMG MRM of a 10-ppt spiked sample.

6
 8.315
x102 12
+ MRM MRM (331.3 ≥ 239.2) Spike_100 ppt_1.d
3.2
2.8
2.4
2.0
1.6
1.2
0.8
0.4
0.0
0 1 2 3 4 5 6 7 8 9 10 11 12
Abundance vs. acquisition time (min)

Figure 6. MRM result of talapia extract spiked with 100-ppt MG and LMG.

ies for MG were 48% and 23% for LMG. A mixture 4. M. D. Hernando, M. Mezcua, J. M. Suarez-
of MG and LMG at 100 fg/µL in 50:50 acetonitrile: Barcena, and A. R. Fernandez-Alba, Liquid
ammonium acetate was used for the repeatability chromatography with time-of-flight mass spec-
study for instrument performance. The RSD from trometry for simultaneous determination of
eight injections for MG was 3.52% (S/N > 20). The chemotherapeutant residues in salmon. Analyt-
RSD from eight injections for LMG was 2.25% ica Chimica Acta 2006, 562, (2), 176%184.
(S/N > 40).
5. K.-C. Lee, J.-L. Wu, and Z. Cai, Determination of
malachite green and leucomalachite green in
Conclusions edible goldfish muscle by liquid chromatogra-
phy-ion trap mass spectrometry. Journal of
This application note demonstrates a complete Chromatography B 2006, In Press, Corrected
method to rapidly and precisely determine residue Proof.
levels of malachite green and leuco-malachite
6. 2004/25/EC: Commission Decision of 22 Decem-
green in fish. Using positive mode electrospray
ber 2003 amending Decision 2002/657/EC as
ionization (ESI+) and multiple reaction monitoring
regards the setting of minimum required perfor-
(MRM) technique, the LC/MS/MS method shows
mance limits (MRPLs) for certain residues in
detection limit of 10 ppt, which easily meets the
food of animal origin (Text with EEA relevance)
import requirement set by Japan or EU.
(notified under document number C [2003]
4961). 2003.
References
1. S. Srivastava, R. Sinha, and D. Roy, Toxicologi- Acknowledgement
cal effects of malachite green. Aquatic Toxicol-
ogy 2004, 66, (3), 319%329. The authors would like to thank Dr. Yanqin Liu for
providing the standard solutions and sample
2. The Rapid Alert System for Food and Feed extracts.
(RASFF) Annual Report 2005. 2005, 29.
3. C. A. Hajee and N. Haagsma, Simultaneous
determination of malachite green and its
For More Information
metabolite leucomalachite green in eel plasma For more information on our products and services,
using post-column oxidation. Journal of Chro- visit our Web site at www.agilent.com/chem.
matography B Biomed Appl. 1995, 669, (2),
219%227.

7
www.agilent.com/chem

Agilent shall not be liable for errors contained herein or for incidental or consequential
damages in connection with the furnishing, performance, or use of this material.

Information, descriptions, and specifications in this publication are subject to change

without notice.

© Agilent Technologies, Inc. 2006

Printed in the USA

October 25, 2006
5989-5807EN
 Analysis of Nitrofuran Metabolites
in Tilapia Using Agilent 6410 Triple
Quadrupole
Application

Food Safety

Authors analytical detection of the metabolites and not the

parent compounds are required in samples of
Feng Liang, Peibin Hu, and Ping Li animal origin.
Agilent Technologies, Inc.
Beijing, China The criteria for detection and confirmation of
veterinary drugs in animal and animal products
established by the European Union (EU) [2] has
Abstract been accepted in much of the world. This criteria
mandates a separation technique combined with a
The metabolites of nitrofuran antibiotics banned in meat spectrometric technique. For banned substances
and meat products are analyzed by LC/MS/MS with the such as the nitrofurans, no maximum residue limit
new Agilent 6410 triple quadrupole. The method is shown (MRL) could be set. Therefore a minimum required
to be highly sensitive, to 0.01 ppb (10 ppt), for each of the performance level (MRPL) was set at 1 µg/kg for
four analytes. Calibration from 0.1 ppb to 10 ppb is pre- each metabolite [3]. Only LC/MS could meet these
sented with all criteria for confirmation as set by the criteria, and very good methods have been
European Union decisions for analytical method perfor- reported [4–6]. However, the most widely accepted
mance. Extracts of tilapia are used to show the perfor- methodology employs triple quadrupole tandem
mance of the LC/MS/MS method for aquaculture mass spectrometers. This is the first report show-
samples. ing analysis of these metabolites using the new
Agilent triple quadrupole LC/MS system.
Introduction
Nitrofurans are inexpensive antibiotics used for
Experimental
Gram positive and Gram negative bacteria. They
have been used to treat gastrointestinal and derma- Chemicals
tological infections in farm animals and fish. In Derivatized standards of nitrofuran metabolites
addition, they have been used to treat bacteria in and all chemicals for sample preparation were
bees. Because both parent compounds and their received from a food manufacturing company.
metabolites are suspect carcinogens, they have Acetonitrile was HPLC grade from Merck (Darm-
been banned around the world. The Rapid Alert stadt, Germany). Formic acid was reagent grade
System for Food and Feed Annual report for 2005 from Merck (Darmstadt, Germany).
[1] shows that these compounds continue to be
detected in food samples and remains a major con-
Sample Preparation
cern for food safety. The four compounds–furazoli-
done, furaltadone, nitrofurantoin, and nitrofura- The accepted procedure for sample preparation
zone–have been found to metabolize rapidly, and was followed. To 2 g of tilapia was added
the metabolites bind to muscle tissue. Thus the 15 mL 0.125 M HCl and the mixture homogenized.
To this solution, a 50-µL solution of 2-nitroben- Quantitation
zaldehyde (50 mM in DMSO) was added and
shaken. The solution was then incubated at 37 °C Quantitative analysis was done with the first tran-
for 16 hours. This was followed by neutralization sition listed in the MRM parameter table. The
to pH ~7 with NaOH and K2HPO4. The neutral second transition was used as a qualifier ion for
derivatized sample was then extracted with ethyl confirmation as per the confirmation criteria.
acetate, concentrated to dryness, and reconsti- Quantitative results were performed with the new
tuted in 100 L of initial LC mobile phase. Stan- MassHunter quantitative analysis software.
dards of the four metabolites were spiked into
0.125 M HCl, derivatized, and extracted for calibra-
tion using the same procedure as was used for the
Results and Discussion
samples of tilapia. The instrument sensitivity is an important perfor-
mance parameter for this analysis when consider-
ing the derivatization and extraction needed to
LC/MS/MS Method
meet the required detection limit of 1 ppb for each
LC Conditions metabolite, aminohydantoin (AH), 3-amino-5-mor-
Instrument: Agilent 1100 LC pholinomethyl-2-oxazolidinone (AMOZ), 3-amino-
Column: C18, 2.1 mm × 150 mm, 3 µm 2-oxazolidinone (AOZ), and semicarbazide (SC). To
Column temp.: 40 °C demonstrate this performance, a standard of each
Mobile phase: A = 0.1% formic acid in water 2-nitrobenzaldehyde (2-NBA) derivatized metabo-
B = acetonitrile lite is shown at 0.01 ppb (10 ppt) in Figure 1. The
Gradient: 22% B at 0 min structure of each derivatized metabolite is given
99% B at 6 min and each is shown with a signal-to-noise ratio of
99% B at 9 min
greater than 3:1. Another indicator of performance
Flow rate: 0.3 mL/min
Injection volume: 50 nL
is linearity. Calibration curves from this concentra-
tion (10 ppt) to 10 ppb are displayed in Figure 2
MS Conditions showing the linearity for each compound.
Instrument: Agilent 6410 LC/MS Triple Quadrupole Treatment of fish with nitrofurans is a continual
Ionization mode: Positive ESI
problem for food safety and import into EU
Drying gas flow: 10 L/min
member countries. To demonstrate the capability
Nebulizer: 35 psig
Drying gas temp.: 350 °C of the Agilent triple quadrupole LC/MS, tilapia
Vcap: 5000 V samples were spiked with the four metabolites,
hydrolyzed, derivatized, and extracted. An analy-
sis of a tilapia extract at 500 ppt is shown in
Figure 3 and demonstrates the signal obtained at
half the MRPL. In addition to meeting the sensitiv-

MRM Mode Parameters

Dwell Fragmentor Collision MS2

Compound Transition time (ms) voltage (V) energy (V) resolution
AMOZ 335.1 & 291.4 60 100 5 Unit
335.1 & 262.4 60 100 5 Unit
SC 209.1 & 192.3 60 100 5 Unit
209.1 & 166.3 60 100 5 Unit
AH 249.1 & 134.2 60 100 5 Unit
249.1 & 104.2 60 100 5 Unit
AOZ 236.0 & 134.1 60 100 5 Unit
236.0 & 104.1 60 100 5 Unit

2
ity requirement, the analysis must also meet the were used as the calibrants, so the final concentra-
confirmation criteria, including both chromato- tion is obtained from the curve. The table is pro-
graphic retention time match with the standards duced as the batch using the MassHunter software
and measuring a qualifying ion with a relative results with outliers highlighted in blue (low) and
intensity ratio within a specified tolerance of the red (high). The table shows that in the blank a
quantitation ion. This tolerance is set by the ratio peak is found within the tolerance set for the
obtained when analyzing standards and increasing retention time of AMOZ but the qualifier ratio is
as that ratio decreases. This tolerance ranges from low. For AOZ and SC, retention times for suspect
20% for ions with relative ratio intensities above peaks are below the specified retention in the
0.5 and to 50% for ratios below 0.1. same blank. For AH, the 0.5 ppb spike, the qualifier
ion ratio is outside the 35% tolerance limit set for
Table 1 shows tilapia samples spiked with the this ion (again low).
metabolites, derivatized and extracted. The spikes

+ MRM (335.1 ≥ 291.4) 10ppt_1.d Smooth (1) O

x10 2 12 23 34 _
0.9 O N+ O
A O O
0.7
N N N
0.5
_
O
x10 1 + MRM (209.1 ≥ 192.3) 10ppt_1.d Smooth (1) 12 23 34 +
O N N O
7
B NH
6 NH2
5

x10 1 _
6.0 + MRM (249.1 ≥ 134.2) 10ppt_1.d Smooth (1) O
12 23 34
O N+ N O
5.6
C N
5.2
O
4.8
_
O
x10 2 O N+ N O
+ MRM (236.0 ≥ 134.3) 10ppt_1.d Smooth (1) 12 23 34
1.2 N
1.0 NH
0.8
D
0.6 O

1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2
Abundance vs. acquisition time (min)

Figure 1. The MRM quant ion chromatogram for each derivatized metabole at 10 ppt of A) 2-NBA AMOZ,
B) 2-NBA SC, C) 2-NBA AH, and D) 2-NBA-AOZ

3
 y = 49247.4270 * × - 1850.8024 x105 y = 11978.9741 * × - 1213.1580
x105 R 2 = 0.99989213 1.2 R 2 = 0.99852795
4.5 1.1
4.0 1.0
0.9
3.5 AMOZ 0.8 SEM
3.0
Response

Response
0.7
2.5 0.6
2.0 0.5
R 2 > 0.999 0.4
R 2 > 0.998
1.5
1.0 0.3
0.2
0.5 0.1
0.0 0.0
_0.1
0 1 2 3 4 5 6 7 8 9 10 0 1 2 3 4 5 6 7 8 9 10
Concentration (ng/mL) Concentration (ng/mL)

x104 y = 5697.5004 * × - 565.5495 y = 45077.4032 * × + 4420.7065

x105 R 2 = 0.99523855
5.5 R 2 = 0.99879989
5.0 4.0
4.5 3.5
4.0
3.5
AHD 3.0 AOZ
Response

Response
3.0 2.5
2.5 2.0
2.0 R 2 > 0.998 1.5 R 2 > 0.995
1.5
1.0
1.0
0.5 0.5
0.0 0.0
_0.5
0 1 2 3 4 5 6 7 8 9 10 0 1 2 3 4 5 6 7 8 9 10
Concentration (ng/mL) Concentration (ng/mL)

Figure 2. Calibration curves of nitrofuran metabolites linear range from 10 ppt to 10 ppb.

+ TIC MRM (** ≥ **) 0619_500ppt_spike_MRM_50uL_4seg_4.d Smooth (1)

x10 3
12 23 34

AOZ
AMOZ

1.0

SC

AH

1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2
Abundance vs. acquisition time (min)

Figure 3. Spiked tilapia sample extract at 500 ppt each metabolite.

4
Table 1. Analysis of Talapia Spikes Self-Calibrated. Note Qualifier Ratios and Retention Times Reported.

AH Qualifier AMOZ Qualifier AOZ Qualifier SC Qualifier

Name RT Final Conc. Ratio RT Final Conc. Ratio RT Final Conc. Ratio RT Final Conc. Ratio
Nitrofuran Blank 0.00 2.72 0.02 8.70 8.71 0.00 7.94 0.00
Nitrofuran 0.5 ppb 8.66 0.54 6.07 2.62 0.51 17.44 9.19 0.37 7.99 8.44 0.50 65.32
Nitrofuran 1 ppb 8.66 1.07 15.83 2.64 0.92 20.42 9.19 1.06 10.19 8.44 1.05 70.85
Nitrofuran 3 ppb 8.67 3.42 13.78 2.65 3.41 18.67 9.20 3.18 9.51 8.44 2.89 67.47
Nitrofuran 5 ppb 8.66 5.71 14.24 2.66 4.66 19.28 9.19 4.89 8.61 8.44 5.05 69.06

Conclusions 4. B. Wüst, C. Sauber, and H. J. A. van Rhijn,

“Quantitation of Nitrofuran Metabolites in
This work shows the high performance of the new Shrimp and Poultry by LC/MS/MS Using the
Agilent 6410 LC/MS triple quadrupole system for Agilent LC/MSD Trap XCT,” Agilent Technolo-
the sensitive analysis of the nitrofuran metabolites gies publication, 5989-0738EN: March 25, 2004.
in fish samples. The system readily meets the per-
5. M. Takino, “Determination of the Metabolites of
formance requirements and provides advanced
Antibacterial Drugs in Chicken Tissue by Liquid
quantitation software for calculating and reporting
Chromatography Electrospray Ion ization Mass
all confirmation parameters specified by the
Spectrometry (LC-ESI-MS),” Agilent Technolo-
European Commission decision.
gies publication, 5988-8903EN: March 19, 2003.
6. F. Mandel, B. Wüst, and C. Sauber, “High Reso-
References lution Quantitative Analysis of Nitrofuran
1. The Rapid Alert System for Food and Feed Derivatives in Poultry and Shrimp Using a New
(RASFF) Annual Report 2005, p 29, oa-ESI-TOF,” Agilent Technologies publication,
http://ec.europa.eu/food/food/rapidalert/ 5989-1302EN: July 9, 2004.
index_en.htm.
2. E. Commission, 2002/657/EC: Commission For More Information
Decision of 12 August 2002 implementing Coun-
cil Directive 96/23/EC concerning the perfor- For more information on our products and services,
mance of analytical methods and the visit our Web site at www.agilent.com/chem.
interpretation of results (Text with EEA rele-
vance) (notified under document number C
[2002] 3044) 2002.
3. E. Commission, 2003/181/EC: Commission
Decision of 13 March 2003 amending Decision
2002/657/EC as regards the setting of minimum
required performance limits (MRPLs) for cer-
tain residues in food of animal origin (Text with
EEA relevance) (notified under document
number C[2003] 764) 2003.

5
www.agilent.com/chem

Agilent shall not be liable for errors contained herein or for incidental or consequential
damages in connection with the furnishing, performance, or use of this material.

Information, descriptions, and specifications in this publication are subject to change

without notice.

© Agilent Technologies, Inc. 2006

Printed in the USA

October 31, 2006
5989-5808EN
 Detection, Confirmation, and Quantification
of Chloramphenicol in Honey, Shrimp and
Chicken Using the Agilent 6410 LC/MS
Triple Quadrupole

Application

Food Safety

Authors meet the EU identification points needed for confirmation.

This study is a valuable indicator of the ability of the QQQ
Yanyan Fang for routine quantitative trace analysis of chloramphenicol
Agilent Technologies (Shanghai), Co. in honey, shrimp, and chicken.
412 Yin Lun Road 200131
China
Introduction
Jerry Zweigenbaum
Agilent Technologies, Inc. Chloramphenicol (CAP) is a broad-spectrum
2850 Centerville Road antibiotic. It was concluded that human exposure
Wilimington DE 19808 to CAP can cause aplastic anemia [1]. Chloram-
USA phenicol and other bacterial inhibitors have
arguably been the biggest issue facing interna-
Zhuwei Wang tional seafood trade over the past year. Because
Agilent Technologies (China) chloramphenicol has displayed significant toxico-
Beijing logical effects on humans, it has been banned from
foods in the European community, Japan, and the
United States at levels of 0.3 ppb.
Abstract
LC/MS has been demonstrated for this analysis by
This method was developed using the Agilent G6410AA the U.S. Food and Drug Administration[2-4] and
Triple Quadrupole Mass Spectrometer (QQQ) for chloram- others[5]. In this application, the Agilent G6410AA
phenicol in honey, shrimp, and chicken. The sensitivity QQQ is used. This method employs negative ion
obtained exceeds the minimum required performance mode with electrospray ionization. An internal
level (MRPL) established by the European Union regula- standard (IS), CAP-d5, is added at the beginning of
tion for food monitoring programs. Using a deuterated the extraction. The use of this IS self-corrects for
internal standard and one simple sample solid phase any extraction variability from sample to sample
extraction (SPE) procedure can provide a limit of detec- and response variability caused by the matrix.
tion at 10 ppt in sample matrix. The analytical perfor- With the use of this IS, 50 parts per trillion (ppt)
mance of the method was evaluated for three different CAP levels can be reliably quantified. A solid phase
matrixes and the results show little or no matrix effects. extraction (SPE) procedure is used along with a
Linearity of response over 2 orders of magnitude was mobile phase of only methanol and water without
demonstrated (r > 0.99). In addition, good reproducibility salt buffers, which should help minimize MS main-
of the two required product ion ratios was obtained to tenance.
Experimental Sample Preparation

All SPE cartridges are conditioned with 2 mL of

Reagents and Materials
water before use.
Agilent AccuBond SPE ENV PS DVB Cartridges 1. Honey, 1 g of sample is diluted to 5 mL with
(P/N 188-3060) water and 25 µL 10 ppb IS is added. The solu-
Ethyl acetate from Burdick and Jackson tion is loaded onto the SPE cartridge and
(Morristown, NJ) allowed to stand for 5 min. Elution is performed
Methanol HPLC-Grade from Burdick and Jackson with 10 mL ethyl acetate. The eluate is collected
Water (18 MW) from Milli-Q Synthesis System and the solvent is evaporated under a nitrogen
Chloramphenicol (CAP) from Aldrich Chemical Co. stream at 40 °C. The residue is redissolved in
(Milwaulkee, WI) 1 mL methanol and put in an ultrasonic bath for
Deuterated (d5) CAP internal standard from Cam- 1 min. The solution is filtered, using a syringe
bridge Isotope Laboratories (CIL, Andover, MA, U.S.) filter, before injection. No additional clean-up of
Syringe filter (0.2 µm, PTFE) from Agilent the sample solution is performed.
(P/N 5185-5843)
2. Shrimp, 1 g of shrimp is defrosted and mixed
Overview of Method in a blender. To the 1 g of the mixed shrimp,
3 mL of water and 25 µL 10 ppb IS is added.
The portion is centrifuged for 5 min (8,000 rpm).
Internal Standard Preparation
The supernatant is loaded on the cartridge and
1. A 100-µg/mL (100 ppm) stock standard CAP-d5 allowed to stand for 5 min. Elution is performed
solution in methanol (MeOH) is purchased from with 5 mL ethyl acetate. The eluate is collected
Cambridge Isotope Laboratories, Inc. and the solvent evaporated under a nitrogen
(Lot SCCE-005) stream at 40 °C. The residue is redissolved in
1 mL methanol and put in an ultrasonic bath for
2. A 1:100 dilution in MeOH of the stock standard
1 min; the solution is filtered before injection.
gives an intermediate standard concentration of
1 µg/mL (1 ppm) or 1000 ng/mL CAP-d5 3. Chicken, 1 g of chicken is defrosted and mixed
in a blender. To the 1 g of the mixed chicken,
3. A 1:100 dilution in MeOH gives a diluent solu-
3 mL of water and 25 µL 10 ppb IS is added.
tion (This diluent solution is used to prepare
The portion is centrifuged for 5 min (8,000 rpm).
the samples) concentration of 10 ppb.
The supernatant is loaded on the cartridge and
4. Every 1-g sample is fortified with 25 µL of allowed to stand for 5 min. Elution is performed
CAP-d5 diluent solution for a 0.25 ppb IS with 5 mL ethyl acetate. The eluate is collected
(internal standard) concentration and the solvent evaporated under a nitrogen
stream at 40 °C. The residue is redissolved in
Standard Solution Preparation 1 mL methanol and put in an ultrasonic bath for
1 min.; the solution is filtered before injection.
1. A 100-µg/mL stock standard CAP solution in
methanol (MeOH) is prepared by weighing
LC/MS conditions
5.0 mg CAP std into 50 mL methanol.
2. A 1:100 dilution with methanol of the stock The LC system was the Agilent 1200-SL binary
standard gives an intermediate standard pump with the ALS-SL autosampler. The MS was
concentration of 1 µg/mL (1 ppm) or an Agilent 6410 LC/MS triple quadrupole mass
1000 ng/mL CAP spectrometer. See Table 1 for conditions.

3. Add 25 µL CAP-d5 diluent solution into each

vial.
4. Prepare standard solutions in these vials: 1 ppb,
0.2 ppb, 0.1 ppb, 0.02 ppb, and 0.01 ppb, with
IS at 0.25 ppb level.

2
Table1. LC/MS Conditions Table 2. Structure and Fragment Ions of CAP and CAP-d5
HPLC (* indicates deuterated positions for the CAP-d5 IS)
Column ZORBAX SB-C18, 2.1 × 50 mm, 1.8 µm Chloramphenicol structure
(p/n 827700-902) OH CI
Flow rate 0.4 mL/min H
Mobile phase A: water * N
B: methanol * * CI
Gradient 0-5 min, 30~70% B
O
5-6 min, 70~100% B
O2N
* OH
8 min, 100% B
*
Post time 4 min
Temperature 45 °C m/z
CAP 257 Qualifier ion
Injection 5 µL
152 Quantitiation ion
MS Source Settings CAP-d5 262
Source ESI 157
Ion polarity Negative
Drying gas temperature 350 °C
Optimization of MS Condition
Drying gas flow rate 10 L/min Figure 1 shows the results of varying the Vcap. For
Nebulizer 45 psi this analyte there was little effect from varying
Vcap 3500 V this parameter. Only a slight increase in signal is
Fragmentor 100 V observed at 3,500 V, and this voltage was used.
Collision energy 10 V for m/z 257(qualifier ion) The fragmentor was varied from 90 V to 160 V.
15 V for m/z 152 (quantitation ion) Above 120 V, fragment ions are observed and the
precursor ion signal drops significantly. At 160 V
on the fragmentor almost no m/z 321 is
observed. This results show that 100 V on the
Results and Discussion fragmentor provided the highest precursor ion
signal. Finally, using a product ion scan of the
Spectral Quality and Sensitivity of Standard precursor, m/z 321, the collision energy (CE) was
varied from 2 V, 5 V, 8 V, 10 V, 15 V, 18 V to 40 V.
Table 2 lists the structure of the CAP and the frag-
ment ions used for quantitation and confirmation Response
as described by the identification point system.[6] 4.00E+06
To obtain the most sensitivity, only two or three
parameters need to be optimized on this instru- 3.00E+06
ment. They are the fragmentor, to provide highest
transmission of the precursor ion, the collision 2.00E+06
energy, to maximize signal for the quantitation and
qualifier ion, and possibly the Vcap (electrospray 1.00E+06
voltage), to maximize the number of ions generated.
0.00E+00
4500 4000 3500 3000 2500 Vcap

Figure 1. Plot of Vcap voltage vs. response of precursor ion at

m/z 321.

3
Comparison of extracted ion chromatograms of
the quantitation and qualifier ions showed that
response maximized at 10 V for m/z 257 and at
15 V for m/z 152. The product ion spectra for these
two collision energy experiments are shown in
Figure 2 and Figure 3. As shown in Table 3, the
same CE were used for the deuterated internal
standard.

×104

1.0 321.0
152.1
0.9

0.8

0.7 257.1
0.6

0.5
194.0
0.4

0.3
176.0
0.2 249.0
0.1 219.1
127.0 164.3 206.9 237.0
0
100 120 140 160 180 200 220 240 260 280 300 320 340
Abundance vs. Mass-to-Charge (m/z)

Figure 2. Product ion spectrum of m/z 321 at 10 V collision energy.

x104
1.1 152.1

0.9

0.8

0.7

0.6

0.5
176.0
0.4

0.3
194.1
0.2 257.1
0.1 320.8
121.0 136.2 158.9 207.1 237.2 248.7
105.8
0
100 120 140 160 180 200 220 240 260 280 300 320 340
Abundance vs. Mass-to-Charge (m/z)

Figure 3. Product ion spectrum of m/z 321 at 15 V collision energy.

4
Table 3. MRM Mode Parameters
Compound Transition Dwell time Fragmentor Collision) MS2
(ms) Voltage (V) Energy (V) resolution
CAP 321–257 200 100 10 Unit
321–152 200 100 15 Unit
CAP-d5 326–262 200 100 10 Unit
326–157 200 100 15 Unit

Repeatability

Using honey matrix spiked at 0.1 ppb level as an

example, the repeatability was tested by running
the extract 15 times. Table 4 shows the area of the
qualifier and quantitation ions in both the analyte
and the IS. On average the areas of each ion vary
about 8% and the ratios 5%, well within the 20%
required for ratios 50% and above. Masshunter
quantitation software tabulates these results and
gives a graphic representation as shown in
Figure 4.

Table 4. Integrated Areas of the Quantitation Ion and Qualifier Ion and Their Associated Internal Standard Ion

Chloramphenicol d5-chloramphenicol
Quantitative Qualifier Ratio Quantitative Qualifier Ratio
ion (321–152) ion (321–257) ion (326–157) ion (326–262)
1 350 165 47.1 262 121 50.4
2 346 157 45.2 258 114 55.3
3 346 5 44.6 259 118 49.4
4 313 164 52.3 267 127 47.6
5 301 154 49.5 261 121 46.4
6 313 168 53.6 253 124 49.0
7 320 160 50.1 228 111 48.6
8 326 145 44.5 225 113 50.4
9 317 141 44.5 241 117 48.6
10 290 135 46.6 226 107 47.1
11 300 138 46.2 253 90 45.7
12 281 136 48.4 240 90 47.6
13 303 143 47.3 220 101 45.9
14 290 140 48.3 214 107 49.8
15 261 131 50.3 217 101 46.6
RSD 8.11% 8.30% 5.91% 7.67% 9.99% 4.83%

5
 - MRM (321.0 -> 152.0) 321 -> 152.0 , 257.0

x102 1.849 x102

Ratio = 46.8
5 5
Abundance

Abundance
4 4

3 3

A C
2 2

1 1

0 0

1 1.2 1.4 1.6 1.8 2 2.2 2.4 2.6 2.8 1 1.2 1.4 1.6 1.8 2 2.2 2.4 2.6 2.8
Acquisition Time (min) Acquisition Time (min)

- MRM (326.0 -> 157.0) 326 -> 157.0 , 262.0

x101 1.818 x101
7 Ratio = 50.3
7
Abundance

Abundance
6 6
5 5

4 4

3 B 3 D
2 2

1 1

0 0

1 1.2 1.4 1.6 1.8 2 2.2 2.4 2.6 2.8 1 1.2 1.4 1.6 1.8 2 2.2 2.4 2.6 2.8
Acquisition Time (min) Acquisition Time (min)

Figure 4. Panels A and B show the CAP and IS peak for the quantitation transition. Panels C and D are the graphic representation
of quantitation ion and qualifier ion ratio as shown by MassHunter software.

Linearity

The linearity of the method was determined for

CAP in solvent and each of the matrices. This was
done from 10 ppt to 1 ppb, well below the mini-
mum required performance level (MRPL) and
above that concentration. Figures 5 through 8 show
the graphic representation of those results. Each
was well above an r2 value of 0.99.
Relative Responses
Relative Responses
6 y = 1.3871 * x + 0.3325
8 y = 1.9565 * x + 0.0669
R^2 = 0.99967712 R^2 = 0.99854947
7 5
6
4
5
4 3
3
2
2
1 1
0
0
-0.2 0.2 0.6 1 1.4 1.8 2.2 2.6 3 3.4 3.8 4.2 -0.2 0.2 0.6 1 1.4 1.8 2.2 2.6 3 3.4 3.8 4.2
Relative Concentration Relative Concentration

Figure 5. Linearity of CAP in solvent from 10 ppt to 1 ppb. Figure 6. Linearity of CAP in honey from 10 ppt to 1 ppb.
6
Relative Responses Relative Responses
8
y = 1.7891 * x + 0.2085 7
y = 1.7027 * x + 0.0150
7 R^2 = 0.99890473 6 R^2 = 0.99985348
6
5
5
4
4
3
3
2
2
1
1
0
0

-0.2 0.2 0.6 1 1.4 1.8 2.2 2.6 3 3.4 3.8 4.2 -0.2 0.2 0.6 1 1.4 1.8 2.2 2.6 3 3.4 3.8 4.2
Relative Concentration Relative Concentration

Figure 7. Linearity of CAP in shrimp from 10 ppt to 1 ppb. Figure 8. Linearity of CAP in chicken from 10 ppt to 1 ppb.

Sensitivity Recovery

The sensitivity of CAP standard in solvent is Recovery was determined by spiking CAP into
observed at 10 ppt with an injection volume of three samples of matrix and extracting using the
5 µL. The MRM chromatogram is shown in specified SPE. Table 5 shows both the repeatability
Figure 9. Although this demonstrates the sensitiv- of extraction and analysis and the mean recovery.
ity of the instrument, it is also important to deter- Using the internal standard spiked before extrac-
mine the sensitivity in real sample matrix. This is tion, recovery is automatically compensated. Thus
shown in Figure 10 with a spike concentration of accuracy of the quantification is very good using
CAP at 10 ppt with a 5-µL injection. Not only is the this methodology. The recovery results show the
analyte detectable, but the ratio of the qualifier ion overall effectiveness of the method.
is within the specified tolerance so confirmation
can be obtained.

x10 2 1.724
- TIC MRM (**¡**) CAP 10 ppt
2.0
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 2.2 2.4 2.6 2.8 3 3.2 3.4 3.6 3.8
Abundance vs. Acquisition Time (min)

Figure 9. MRM chromatogram of 10 ppt CAP in solvent with injection volume of 5 µL.

Table 5. Recovery of CAP at 0.1 ppb Where Three Sample quantitation and confirmation well below the
Aliquots of Each Matrix Were Spiked and Determined required 0.3 ppb MRPL. Optimization of the
Honey Shrimp Chicken method was simple, as few parameters in the mass
(n=3) (n=3) (n=3) spectrometer need adjustment. In addition, the
RSD (%) 6.29 3.93 3.29 requirements for a validated method have been
Recovery (%) 89.5 85.4 86.4 shown. These include sensitivity, repeatability, lin-
earity, and recovery. The Agilent 6410 LC/MS
Conclusions triple quadrupole instrument has been shown to be
a highly effective instrument for the analysis of
The method described herein for the analysis of chloramphenicol.
CAP in three important matrices has been shown
to be highly effective and meet the criteria for

7
 www.agilent.com/chem
×10 1 - MRM (321 ¡ 152.0) CAP in Honey.d ×10 1 321 ¡ 152.0, 257.0
1.0 Ratio=57.8
1.751 1.0
0.9
0.9
0.8
0.8
0.7
0.7
0.6 0.6

Abundance
Abundance

0.5 0.5
0.4 0.4
0.3 0.3

0.2 0.2

0.1 0.1

0.0 0.0
_ 0.1 _ 0.1
1 1.2 1.4 1.6 1.8 2 2.2 2.4 2.6 1 1.2 1.4 1.6 1.8 2 2.2 2.4 2.6
Acquisition Time (min) Acquisition Time (min)

Figure 10. MRM chromatogram of quantitation ion and ratio of qualifier ion for 10 ppt CAP in honey.

References For More Information

1. Chemical Safety Information from Intergovern- For more information on our products and services,
mental Organisations (IPCS-INCHEM), Web visit our Web site at www.agilent.com/chem.
page http://www.inchem.org/documents/
jecfa/jecmono/v33je03.htm
2. S. Turnipseed, et al. (2002) Confirmation of
Multiple Phenicol Residues in Honey by Electro-
spray LC/MS, Laboratory Information Bulletin
(4281) U.S. Food and Drug Administration.
3. A. Pfenning, et al. (2002) Confirmation of Multi-
ple Phenicol Residues in Shrimp by Electro-
spray LC/MS, Laboratory Information Bulletin
(4284) Food and Drug Administration.
4. B. K. Neuhaus, et al. (2002) LC/MS/MS Analysis
of Chloramphenicol in Shrimp, Laboratory
Information Bulletin (4290) Food & Drug
Administration
The information contained in this publication is intended for research use only and is
5. P. Mottier, V. Parisod, E. Gremaud, P. A. Guy, not to be followed as a diagnostic procedure.
and R. H. Stadler. Determination of the antibi- Agilent shall not be liable for errors contained herein or for incidental or consequential
otic chloramphenicol in meat and seafood prod- damages in connection with the furnishing, performance, or use of this material.
ucts by liquid chromatography - electrospray
Information, descriptions, and specifications in this publication are subject to change
ionization tandem mass spectrometry. Journal without notice.
of Chromatography A 2003, 994, (1-2), 75-84.
© Agilent Technologies, Inc. 2007
6. Commission, E., 2002/657/EC: Commission
Decision of 12 August 2002 implementing Coun- Printed in the USA
August 21, 2007
cil Directive 96/23/EC concerning the perfor- 5989-5975EN
mance of analytical methods and the
interpretation of results (Text with EEA rele-
vance) (notified under document number
C(2002) (3044) 2002.
 Detection of Recombinant Bovine
Somatotropin in Milk by LC-ESI-MS/MS

Application

Food Safety

Authors exist regarding its use, but the lack of confirma-

tory methods [1] for its detection makes it difficult
Marie-Hélène Le Breton, Sandrine Rochereau-Roulet, Gaud Pinel, to apply these regulations. It turns out to be an
Fabrice Monteau, and Bruno Le Bizec international issue in terms of animal doping and
Laboratoire d'Etude des Résidus et Contaminants dans les in food safety as well. Indeed, residues of rbST can
Aliments (LABERCA) be present in the milk of dairy animals treated
Ecole Nationale Vétérinaire de Nantes (ENVN) with this hormone.
BP 50707, 44307, Nantes
In order to detect residues of rbST in milk, the
France
choice has been made to focus the analysis on the
Marie-Hélène Le Breton tryptic N-terminal peptide of the protein, specifi-
2 Nestlé Research Center cally the difference between the endogenous and
Nestec Ltd., P.O. Box 44 recombinant forms. The N-terminal amino acid ala-
CH-1000 Lausanne 26 nine that is present in the endogenous form is
Switzerland replaced by a methionine in the recombinant one
[2].

Abstract This application describes a method for the detec-

tion of rbST by ESI(+) LC-MS/MS. The method was
Recombinant bovine somatotropin (rbST), also called successfully applied to extracts from milk samples
growth hormone, is a protein hormone used in dairy farming spiked with rbST.
to enhance milk production. A method has been developed
for the detection of rbST in milk by ESI(+)-LC-MS/MS. This
method allowed a detection limit of 20 pg of tryptic
Experimental
N-terminal peptide rbST in standard solution injected on-
Standards of Proteins and Peptides
column and was successfully applied to extracts obtained
from milk samples spiked with 50 ng/mL-1 (2.3 pmol/mL-1) Protein standards of rbST and recombinant equine
rbST. somatotropin, reST (EquiGen-5), were obtained
from the Harbor-UCLA Medical Center, National
Introduction Hormone and Pituitary Program (Torrance, CA,
USA) and Bresagen Limited (Thebarton, Aus-
Recombinant bovine somatotropin (rbST), also tralia), respectively.
called growth hormone, is used in lactating cows to
increase milk production. Different regulations
The peptides used as standards, with the following Table 1. SRM Method Parameters
amino acid sequence MFPAMSLSGLFANAVLR Transitions Collision
(N-terminal tryptic rbST), MFPAMPLSSLFANAVLR Compound RT Charge monitored energy (V)
(N-terminal tryptic reST), and AFPAMSLSGLFAN- Nterm rbST 8.33 z=2 913.2 & 1047.7 30
AVLR (N-terminal tryptic bST) were synthesized 913.2 & 774.1 20
from Millegen (Labege, France). z=3 609.3 & 774 10
609.3 & 643.5 20
Instrumentation Nterm reST 8.39 z=2 933.2 & 1287.9 30
933.2 & 794.1 20
The detail of the instrumentation used for the
detection of the N-terminal peptides is described Nterm bST 8.20 z=2 883.2 & 1047.8 20
883.2 & 774.1 20
in the following tables.
LC Results and Discussion
Instrument Agilent 1200
Column Column Interchrom ModuloCart QS Application of Triple Quadrupole MS-MS and
Uptisphere 3HDO 150 mm × 2 mm
Electrospray Ionization Mode Methodology
Mobile phase A: Acetonitrile + 0.1% formic acid
B: H2O + 0.1% formic acid In this method, the choice has been made to use
Flow rate 0.3 mL/min electrospray ionization in positive mode. Indeed,
this ionization mode presented as a “soft” ioniza-
Injection volume 20 µL
tion technique is optimal for peptides. The ioniza-
Gradient Time (min) %A tion of the N-terminal peptide rbST leads to two
0 10
main forms (z = 2 and z = 3).
5 55
10 60 This use of a triple quadrupole based methodology
15 100
enabled very good sensitivity and selectivity and
17 10
20 10
also a possible quantification of the monitored sig-
nals.
MS
Instrument Agilent 6410 LC/MS Triple Quadrupole
Ionization mode ESI (+) Separation of the Different Compounds
Capillary 5000 V
The detection method was developed for the detec-
Nebulizer 55 psi
Gas flow 13 L/min tion of the tryptic N-terminal peptide of rbST and
Gas temperature 300 °C also for the tryptic N-terminal peptide of endoge-
nous pituitary bovine somatrotropin (bST) and
reST as well. Due to the high homology in the
amino acid sequence with rbST, reST was used as
Selected Reaction Monitoring (SRM) Method Parameters
the internal standard.
In order to obtain a better specificity, the detection
The three compounds were separated chromato-
was performed in SRM mode. The transitions mon-
graphically and analyzed utilizing the transitions
itored are displayed in Table 1.
described in Table 1. The chromatogram corre-
sponding to the injection of 0.2 ng of N-terminal
peptide bST, rbST, and reST is shown in Figure 1.

2
 x10 2
2
(a) * 8.39
815

1 933.2 & 794.1

0
x10 2
* 8.33
(b) 550
1

913.2 & 774.1

0
x10 2 3
* 8.20
(c) 1362

2
883.2 & 774.1
1

0
5.4 5.8 6.2 6.6 7.0 7.4 7.8 8.2 8.6 9.0 9.4 9.8 10.2 10.6
Counts vs. acquisition time (min)

Figure 1. SRM ion chromatograms of standard solutions of tryptic N-terminal peptide of (a) reST, (b) rbST
and (c) bST. The injection aliquot used corresponded to 0.2 ng on-column.

Even with an optimized gradient, due to their high

homology in terms of sequence, the three com-
pounds eluted with very similar retention times.

Linearity and Sensitivity of the Method

The method described allowed detection of

the three peptides with very good sensitivity. A
limit of detection of 20 pg injected on-column
(~900 femtomole) was reached. Quantification was
possible as shown by the good linearity of the cali-
bration curves (Figure 2).

3
 20000

18000 933.2 & 794.1

R 2 = 0.9993
913.2 & 774.1
16000
883.2 & 774.1
14000

12000
R 2 = 0.9972
Area

10000

8000

6000
R 2 = 0.996
4000

2000

0
0 500 1000 1500 2000
Qty injected on-column (pg)

Figure 2. Calibration curve of tryptic N-terminal peptides rbST (913.2 & 774.1), reST (933.2 & 794.1)
and bST (883.2 & 774.1).

Results of Spiked Samples Figure 3 shows the chromatogram of a milk sample

spiked with 50 ng.mL–1 of rbST, in accordance with
The detection method was applied to extracts guidelines for the identification of rbST according
obtained from milk samples spiked with rbST. The to 2002/657 criteria [4]. The chromatogram shows
purification procedure used is described in [3]. excellent peak shape, and above all, nearly null
x10 3
(a) 6 + MRM (913.2 & 774.1)

4
Blank milk 3

0
(b) + MRM (933.2 & 794.1) *
x10 3 1515
3
Milk spiked
_
with
50 ng.mL 1 reST (ISTD) 2

0
(c) x10 3 + MRM (913.2 & 774.1)
5
Milk spiked
_
with
4
50 ng.mL 1 rbST *
3 12

0
5.2 5.6 6.0 6.4 6.8 7.2 7.6 8.0 8.4 8.8 9.2 9.6 10.0 10.4 10.8

Counts vs. acquisition time

Figure 3. SRM ion chromatograms obtained from milk samples. The different signals correspond to
(a) blank milk, (b) the same milk spiked with 50 ng.mL-1 reST (internal standard), and
(c) 50 ng.mL-1 rbST.

4
background noise, demonstrating the selectivity of
the method. The intensity of the signal, although
lower than the internal standard, is, however, sig-
nificant, and shows a clear and distinct signal. The
method clearly allows for unambiguous identifica-
tion of rbST in milk.

Conclusions
The detection of rbST in milk was performed with
detection by ESI(+) LC-MS/MS. The method
showed very good sensitivity, specificity, and
robustness. It was successfully applied to milk
samples spiked with rbST at 50 ng.mL-1, in accor-
dance with criteria outlined by the 2002/657 Coun-
cil Directive.

References
1. L. Bailly-Chouriberry, E. Chu-Van, G. Pinel,
P. Garcia, M.-A. Popot, G. André-Fontaine,
Y. Bonnaire, and B. Le Bizec, Analyst, 133
(2008) 270
2. G. Pinel, F. André, and B. Le Bizec (2004) Jour-
nal of Agricultural and Food Chemistry, 52,
407-414
3. M. H. Le Breton, S. Rochereau-Roulet, G. Pinel,
L. Bailly-Chouriberry, G. Rychen, S. Jurjanz,
T. Goldmann, and B. Le Bizec (2008) Analytical
and Bioanalytical Chemistry, submitted
4. Commission Decision of 12 August 2002 imple-
menting Council Directive 96/23/EC concerning
the performance of analytical methods and the
interpretation of results, Off. J. Eur. Commun.
2002/657/EC, 2002

For More Information

For more information on this application, you may
also contact Agilent Technologies, Inc., Attention:
Lea Bonnington, European Field Support Centre,
Hewlett-Packard Strasse 8, 76337 Waldbronn, Ger-
many.

For more information on our products and services,

visit our Web site at www.agilent.com/chem.

5
www.agilent.com/chem

Agilent shall not be liable for errors contained herein or for incidental or consequential
damages in connection with the furnishing, performance, or use of this material.

Information, descriptions, and specifications in this publication are subject to change

without notice.

© Agilent Technologies, Inc. 2008

Printed in the USA

June 24, 2008
5989-8481EN
 Development of an LC-MS/MS Method for
the Determination of 20-Hydroxyecdysone
and Its Metabolites in Calf Urine
Application to the Control of Its Potential
Misuse in Cattle
Application Note
Food Safety

Authors Abstract
Blandine Destrez, Gaud Pinel, Fabrice
Ecdysteroids, which are steroid hormones present in invertebrates and in plants,
Monteau, and Bruno Le Bizec
could be potentially used as anabolic agents in food-producing animals. The control
Laboratoire d'Etude des Résidus of ecdysteroid misuse in cattle relies on the development of an efficient method for
et Contaminants dans les Aliments their detection in biological matrices at trace levels (µg.L-1). In this context, an ana-
(LABERCA), Ecole Nationale Vétérinaire lytical procedure dedicated to the identification of 20-hydroxy-ecdysone and its
de Nantes (ENVN), metabolites in urine samples, based on purification on two solid-phase extraction
BP 50707, 44307 NANTES, Cedex 3, cartridges (SPE C18 and SPE SiOH) and LC-(ESI+)-MS/MS measurements has been
France developed. The performance of tandem quadrupole MS/MS, in terms of sensitivity
and specificity, allowed measurements at trace levels in both spiked and incurred
samples. Good linearity was observed for all analytes from 0.12 ng to 12 ng on
column.
Introduction Instrumentation
LC:
Ecdysteroids are steroid hormones present both in inverte-
brate species (mainly Arthropods) and plants (belonging to Column: GEMINI C18, Phenomenex
Asteraceae, Caryophyllaceae, or Polypodiaceae). In arthro- (3 µm, 110 Å, 50 × 2 mm)/Agilent
pods, ecdysteroids act as moulting hormones, whereas these equivalent: ZORBAX Extend-C18 3.5 µm,
2.1 mm × 50 mm (p/n 735700-902)
molecules are thought to protect plants against nonadapted
Column temperature: 40 °C
phytophagous insects. The archetypal ecdysteroid in both Mobile phases: A: MeOH
kingdoms is 20-hydroxyecdysone (20E), and several studies B: 0.5% acetic acid in water
have underlined its possible growth-promoting effects in vari- Flow rate: 0.3 mL/min
ous animal species (rats, mice, and Japanese quail), including Gradient: Time (min) %B
humans and cattle [1–3]. Clinical studies demonstrated that 0 90
20E is more anabolic than methandrostanolone (dianabol), 8 0
with no androgenic or other undesirable side effects usually 10 0
observed with classical steroids [4]. However, despite its 12 90
growth-promoting properties, only a few methods have been 16 90
reported for its detection in biological matrices, and no infor- Injection volume: 10 µL
MS: G 6410A QQQ, Agilent Technologies
mation is available concerning its metabolism in cattle [5]. In
Ionization: ESI (+)
this application, the development of a method able to detect Fragmentor: 120 V
and identify 20E and its main metabolites at trace levels (ppb) Mass range: 100–500 amu
in calf urine is described [6]. This method was applied to the Scan time: 300 ms
analysis of calf urine samples after 20E oral administration Capillary: 4000 V
and used to assess the kinetic of elimination of these sub- Nebulizer: 35 psi
stances. Drying gas: 11 L/min
Gas temperature: 325 °C
The monitored transitions for each target compound are
Experimental
reported in Table 1. The first transition corresponds to the
Compound Standards most sensitive signal.
Standard reference 22S,23S-homobrassinolide (belonging to
brassinosteroids, vegetable steroid hormones) was from
Sigma-Aldrich (St. Quentin Fallavier, France); 20-hydrox- Results and Discussion
yecdysone, 14-deoxy, 20-hydroxyecdysone, and 20,26-dihy- Standard solutions of target compounds were analyzed
droxyecdysone were a kind gift from Pr. Lafont. according to the LC-MS/MS parameters described in the
Experimental section, which allowed us to obtain the ion
chromatograms of 20E, M1, M2, and IS, each at 5 ng on col-
Sample Preparation umn (Figure 1). All the compounds are eluted within less than
Twenty-five nanograms of 22S,23S-homobrassinolide were 10 min with very good chromatographic resolution and peak
added as internal standard (IS) to 5 mL of calf urine, cen- shape.
trifuged at 3,500 g for 15 min, then purified on SPE C18. The
C18-SPE cartridges were conditioned with 10 mL methanol,
×10 4 M1 (465.4&303.3)
then 10 mL water, following which the urine samples were
applied. The columns were then washed with 6 mL of a
water/methanol (80/20) mixture, and the ecdysteroids were
subsequently eluted with 10 mL methanol. The eluant was
then evaporated to dryness under a gentle stream of nitrogen.
The residue was reconstituted in 50 µL ethanol and 150 µL 1
cyclohexane before loading onto a SPE SiOH, previously acti-
vated with 25 mL cyclohexane. The phase was washed with
6 mL ethyl acetate/cyclohexane (80/20) and the compounds M2 (497.4&461.4)
of interest were then eluted with 10 mL of a mixture of chlo-
roform/methanol/acetone (6/2/1). The solvent was evapo-
rated to dryness under nitrogen and the final extract was 20E (481.3&445.4) IS (495.5&109.1)
redissolved in 50 µL of methanol/water (30/70) containing
0.5% acetic acid. From this extract 10 µL was injected onto 0
the HPLC column. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
Counts vs. acquisition time (min)
Figure 1. Overlaid extracted ion chromatograms (EICs) for the most sen-
sitive transitions monitored for 20E and its metabolites in posi-
tive ion mode.

2
Table 1. Monitored SRM Transitions for 20E and Its Main Urinary Metabolites and Parameters of Acquisition for Their Analysis by LC-MS/MS (QQQ)
Collision Collision Collision
energy energy energy RT
Analytes Transition 1 (eV) Transition 2 (eV) Transition 3 (eV) (min ± 0.2)
22S,23S-homobrassinolide (IS) 495.5&109.1 20 495.5&127.1 10 495.5&459.1 5 9.8
20-hydroxyecdysone 481.3&445.4 10 481.3&371.4 10 481.3&165.1 20 7.5
14-deoxy,20-hydroxyecdysone (M1) 465.4&303.3 20 465.4&285.3 25 – – 7.9
20,26-dihydroxyecdysone (M2) 497.3&461.4 5 497.3&351.1 15 497.3&371.2 20 6.8

To assess the specificity of the method, a blank urine and a The method has been successfully applied to incurred calf
urine sample fortified with 20E (1 µg.L-1) were analyzed. urine samples after 20E oral administration over four days.
Figure 2 shows the blank traces without any interference at 20-hydroxyecdysone was detected in urine as rapidly as
the expected retention time for 20E, demonstrating the good 30 minutes after its administration and up until 24 hours after
selectivity of the monitored signals. The target analyte 20E the last administration. 20E metabolism was investigated and
was identified in the spiked urine sample with three SRM two main metabolites, 14-deoxy,20-hydroxyecdysone (M1) and
transitions. The monitored signals are detected with good 20,26-dihydroxyecdysone (M2), could be identified [8]. Both
sensitivity and show high signal-to-noise (s/n) ratios. These M1 and M2 were monitored by LC-MS/MS (Table 1). Figure 3
results were in accordance with Decision 2002/657/EC crite- presents the ion chromatograms for M1 in the urine samples
ria, which require more than four identification points [7] in collected before and two days after the last 20E administration.
order to validate an identified compound.
As can be observed, M1 was not detected in the urine collect-
The linearity and the repeatability of the method were ed before 20E administration, whereas it was throughout the
assessed with the analysis of a pool of urine samples fortified four-day administration period. Furthermore, it could still be
at different concentration levels: the calibration curve was detected and identified (in accordance with the four identifica-
established with five concentration points (0.2, 0.5, 1, 5, and tion points required) two days after the last administration of
20 ng.mL-1). The calibration curve correlation coefficients (R2) 20E. This result is of prime interest in the context of potential
were better than 0.99, thus demonstrating the good linearity misuse of ecdysteroids since it offers the longest period for
of the method for 20E. detection, following administration, and therefore enables a
more efficient control mechanism.

3
a) b)
×10 2 ×9.75 ×10 2 ×9.75
5 5
4 4
495.5&109.3 495.5&109.3
3 3
2 2
1 1
0
×10 2 ×9.76 ×10 2
3
6 495.5&127.1
2 495.5&127.1 IS
4 ×9.76 _
1 2
1 µg.L 1
0
×10 2
×10 2
4 9
3 495.5&459.1 7
×9.76 495.5&459.1
2 5
3 ×9.76
1
1
×10 2 ×10 2 ×7.50
9
2
481.3&165.1 7 481.3&165.1
5
1
3
1
0
×10 3
×10 3
1 481.3&371.4 6 481.3&371.4
4 20E
2 ×7.51
0 0
×10 2 ×7.50
3 ×10 3
481.3&445.4 481.3&445.4
2 1

0 0
5 6 7 8 9 10 11 12 13 5 6 7 8 9 10 11 12 13
Counts vs. acquisition time (min) Counts vs. acquisition time (min)

Figure 2. SRM ion chromatograms for a) the blank urine sample and b) the spiked urine sample (1 µg.L-1). LC-(ESI+)-MS/MS measurements.

4
a) b)
×10 2 ×9.75 ×9.76
×10 3
5
1 495.5&109.3
3 495.5&109.3
1
0
×10 2 ×9.76 ×10 2 ×9.76
3 6
2 495.5&127.1 4
1 IS 2
495.5&127.1 IS
0
×10 2
495.5&459.1 ×10 2 ×9.76
4
3 ×9.76 4
2 2 495.5&459.1
1
0

×10 2
×10 3 ×7.97
465.4&285.3
1 1
465.4&285.3

0 0
×10 2
M1 M1
×10 3 ×7.97
5 465.4&303.3
3 465.4&303.3
1
1

5 6 7 8 9 10 11 12 13 5 6 7 8 9 10 11 12 13
Counts vs. acquisition time (min) Counts vs. acquisition time (min)

Figure 3. SRM ion chromatograms of IS and M1 in urine sample collected a) before 20E administration and b) two days after the last 20E administration. LC-
(ESI+)-MS/MS measurements.

Conclusions 2. V. N. Syrov and A. G. Kurmukov, “On the Anabolic Activity

This work demonstrates the performance of LC-MS/MS, of the Phytoecdysone-Ecdysterone Isolated from
which provides efficient identification of 20E and its main Rhaponticum carthamoides,” Farmakologiia I
metabolites in calf urine. The monitoring of these compounds Toksikologiia, 39(6), 690—693, (1976)
facilitates the control of the potential misuse of 20E in meat- 3. K. Slama, K. Koudela, J. Tenora, and A. Mathova, “Insect
producing animals. Tandem quadrupole MS/MS is an analyti- Hormones in Vertebrates: Anabolic Effects of 20-hydrox-
cal technique very well suited to this purpose, since it yecdysone in Japanese Quail,” Experientia, 52 (7): 702—6,
increases confidence in the unambiguous identification of the (1996)
target compounds, in accordance to the criteria fixed by
Decision 2002/657/EC. The successful analysis of the calf 4. N. S. Chermnykh, N. L. Shimanovskii, G. V. Shutko and V.
urine samples proved the robustness of the developed proto- N. Syrov, “The Action of Methandrostenolone and
col. Application of this methodology also enabled the determi- Ecdysterone on the Physical Endurance of Animals and on
nation of the first elimination kinetics and the main metabo- Protein Metabolism in the Skeletal Muscles,”
lites of 20E in calf urine. Farmakologiya IToksikologiya, 51, 57—60, (1988)
5. B. Le Bizec, J. P. Antignac, F. Monteau, and F. Andre,
“Ecdysteroids: One Potential New Anabolic Family in
References Breeding Animals,” Analytica Chimica Acta, 473, 89—97,
1. W. J. Burdette and R. C. Richards, “Alteration of the (2002)
Growth of Mammalian Cells In Vitro by Ecdysone Extract,”
Nature, 189, 666-668, (1961)

5
6. B. Destrez, G. Pinel, E. Bichon, F. Monteau, For More Information
R. Lafont, and B. Le Bizec, “Detection of For more information on our products and services, visit our
20-Hydroxyecdysone in Calf Urine by Comparative LC- Web site at www.agilent.com/chem or contact Lea
HRMS and LC-MS/MS Measurements, Application to the Bonnington at Agilent Technologies, Inc., Waldbronn,
Control of Their Potential Misuse,” Rapid Com. Mass Germany.
Spectrom., submitted 2008
7. Commission Decision of 12 August 2002 Implementing
Council Directive 96/23/EC Concerning the Performance
of Analytical Methods and the Interpretation of Results,
Off. J. Eur. Commun. 2002/657/EC, 2002
8. B. Destrez, G. Pinel, F. Monteau, R. Lafont, and B. Le
Bizec, “Detection and Identification of
20-Hydroxyecdysone Metabolites in Calf Urine by LC-
HRMSn Measurements and Establishment of Their
Kinetics of Elimination After 20E Administration,” Anal.
Chim. Acta, submitted June 2008

www.agilent.com/chem
Agilent shall not be liable for errors contained herein or
for incidental or consequential damages in connection
with the furnishing, performance, or use of this material.

Information, descriptions, and specifications in this

publication are subject to change without notice.

© Agilent Technologies, Inc., 2008

Printed in the USA
September 9, 2008
5989-8879EN
 Determination of Tetracyclines in
Chicken by Solid-Phase Extraction
and High-Performance Liquid
Chromatography
Application Note

Food Safety

Authors Abstract
Chen-Hao Zhai and Yun Zou A method for the simultaneous determination of the seven antibiotic residues of
Agilent Technologies Co. Ltd. minocycline, oxytetracycline, tetracycline, demeclocycline, chlortetracycline, methacy-
412 Ying Lun Road cline, and doxycycline in chicken has been developed. In this method, solid-phase
Waigaoqiao Free Trade Zone extraction (SPE) and HPLC/UV are used consistent with Chinese regulatory methods.
Shanghai, 200131 Samples are prepared in EDTA-Mcllvaine buffer solution (pH 4.0), the clean up is done
China with an Agilent SampliQ OPT cartridge, and the HPLC separation is performed with an
Agilent ZORBAX column (5 µm, 250 mm × 4.6 mm id). The flow rate is 1.5 mL/min,
the detector wavelength is 350 nm, and the injection volume is 100 µL. The limits of
detection are between 2.5 and 5 µg/kg. Linear calibration curves are obtained over
the range of 25 to 500 µg/kg. Overall recoveries range from 59.0 to 99.0%, with RSD
values between 1.0 and 6.5%.
Introduction set maximum residue limits (MRLs) for tetracyclines in mus-
cle (100 µg/kg) and promulgated a government standard
"Tetracyclines" is the common name for a group of antimicro- (GB/T 21317-2007) that established a method for the determi-
bials with a hydronaphthacene structure (Table 1). Tetracy- nation of tetracyclines in animal tissues. This application note
clines are used against a wide range of gram-negative and describes the implementation and optimization of the method
gram-positive microorganisms. The Chinese government has described in GB/T 21317-2007 and the results of validation.

Table 1. Tetracyclines Used in This Study

No. Name pKa log P Structure

1 Minocycline CH3 CH3 CH3 CH3

CAS # 10118-90-8 3.3/7.2/9.3 +0.5 N N
OH

O
OH
OH O OH O NH2
2 Oxytetracycline CH3 CH3
CAS # 6153-64-6 3.3/7.3/9.1 –0.9 OH CH3 OH N
OH

O
OH
OH O OH O NH2
3 Tetracycline CH3 CH3
CAS # 60-54-8 3.3/7.7/9.7 –1.3 OH CH N
3
OH

O
OH
OH O OH O NH2
4 Demeclocycline CH3 CH3
CAS # 127-33-3 3.3/7.2/9.3 +0.2 CI OH N
OH

O
OH
OH O OH O NH2
5 Chlortetracycline CH3 CH3
CAS # 57-62-5 3.3/7.4/9.3 –0.62 Cl OH CH N
3
OH

O
OH
OH O OH O NH2
6 Methacycline CH3 CH3
CAS # 914-00-1 3.5/7.6/9.2 –0.3 CH2 OH N
OH

O
OH
OH O OH O NH2

Continued

2
Table 1. Tetracyclines Used in This Study (continued)

No. Name pKa log P Structure

7 Doxycycline CH3 CH3

CAS# 564-25-0 3.1/7.7/9.3 –0.02 CH3 OH N
OH

O
OH
OH O OH O NH2

Experimental extraction in an ice bath. The sample was then centrifuged at

a rotate speed of 3,000 r/min for 5 minutes (below 15 °C).
Materials and Chemicals
The supernatant was removed and saved in a clean tube. The
All reagents and solvents were HPLC or analytical grade.
extraction was repeated twice with 20 mL and 10 mL succes-
Tetracycline standards were purchased from Sigma-Aldrich or
sively. The combined supernatant fluid was brought to 50 mL
from China’s National Institute for the Control of Pharma-
with buffer, mixed well, centrifuged at a rotate speed of
ceutical and Biological Products (NICPBP).
4,000 r/min for 10 min (below 15 °C), and filtered with fast
Stock solution (0.1 mg/mL) was prepared in methanol and filter paper.
kept in the freezer (–20 °C). Working solutions were prepared
using the stock solution diluted with a mixture of methanol/
10 mmol/L trifluoroacetic acid solution (1/19). The working SPE Purification
solutions were prepared daily. The procedure used for the SPE extraction is shown in
Figure 1. Agilent SampliQ OPT cartridges were preconditioned
The SPE cartridges were Agilent SampliQ OPT 3 mL, 60 mg with 5 mL of methanol, then 5 mL of a 10 mmol/L TFA solu-
(p/n 5982-3036). The analysis was performed on an Agilent tion. A 10-mL extract (equivalent to a 1-g sample) was passed
1200 HPLC with DAD. The analytical column was an Agilent through the SampliQ OPT cartridge at a speed of 1 mL/min.
ZORBAX SB-C8 5 µm, 250 mm × 4.6 mm id (p/n 880975-906). After the sample effused completely, the cartridge was
McIlvaine buffer, mix 1000 mL 0.1 mol/L citric acid with washed with 3 mL of water (pH adjusted to 4.5 with TFA). The
625 mL 0.2 M disodium hydrogen phosphate. Adjust pH to 4.0 entire effluent was discarded. The cartridge was dried under
± 0.05 with NaOH or HCl as needed. negative pressure below 2.0 kPa for 3 minutes. Finally, the
Na2EDTA-McIlvaine buffer (0.1 mol/L), mix 60.5g Na2EDTA. cartridge was eluted with 10 mL of 10 mmol/L oxalic acid in
2H2O into 1625 mL McIlvaine buffer. methanol. The eluent was collected and dried under nitrogen
below 40 °C. The resulting residue was dissolved and made to
a constant volume of 0.5 mL using the methanol/10 mmol/L
HPLC Conditions TFA solution (1/19). Then the residue was filtered through a
Column: Agilent ZORBAX SB-C8 250 mm × 4.6 mm, 5 µm
Flow rate: 1.5 mL/min
0.45-µm filter membrane (p/n 5185-5836) and analyzed.
Column temperature: 30 °C
Injection volume: 100 µL Condition:
Detector wavelength: 350 nm 5 mL methanol
Mobile phase: Methanol-acetonitrile-10 mmol/L TFA solution,
gradient elution Rinse:
Time (minutes) % methanol % acetonitrile % 10 mmol TFA 5 mL 10 mM TFA (pH = 2.16)
0 1 4 95
7.5 6 24 70 Load:
13.5 7 28 65 10 mL sample at 1 mL/min
15 1 4 95
Wash:
Sample Preparation 3 mL TFA solution (pH = 4.5)
A 200-g sample of chicken was homogenized with a tissue
disintegrator, placed in a clean, sealed container, and stored Dry cartridge under vacuum 3 minutes
Elute:
in a freezer below –18 °C. 8 mL 10 mmol oxalic acid in
methanol
A 5-g homogeneous sample (accurate to 0.01 g) was placed
into a 50-mL polypropylene centrifuge tube with 20 mL Dry under N2, reconstitute
0.1 mol/L Na2EDTA-Mcllvaine buffer solution and vortex residue
mixed for 1 minute followed by a 10-minute ultrasonic Figure 1. Tetracycline SPE procedure.

3
Results and Discussion Table 2. Linearity and LODs of Tetracyclines
Regression Correlation
Linearity, Limits of Detection Compound equation coefficient LOD (µg/kg)
Stock solutions were diluted to different concentrations and Minocycline Y = 86.313 × –0.1491 0.9996 2.5
Oxytetracycline Y = 95.965 × +0.0261 0.9999 2.5
analyzed by HPLC. Linear regressions were calculated for the Tetracycline Y = 103.97 × –0.4698 0.9999 2.5
tetracyclines using the areas and the solution concentrations. Demeclocycline Y = 68.659 × –0.1172 0.9998 5
The limit of detection (LOD) was the injection concentration Chlortetracycline Y = 51.752 × –0.0284 0.9999 5
Methacycline Y = 98.243 × +1.2567 0.9985 2.5
whose signal-to-noise ratio was between 2 and 3. The linear Doxycycline Y = 76.408 × +1.0756 0.9987 5
range was between 25 and 500 µg/kg. The linearity and LOD
are shown in Table 2.

Figure 2. Chromatogram of a chicken blank.

Figure 3. Chromatogram of a chicken sample spiked at 50 µg/kg. (1-Minocycline, 2-Oxytetracycline, 3-Tetracycline, 4-Demeclocycline, 5-Chlortetracycline, 6-Methacycline, and
7-Doxycycline)

4
Recovery and Reproducibility References
The precision of the method was determined as recoveries
GB/T 21317-2007, Determination of tetracyclines residues in
of spiked tetracycline standards in chicken at 50 µg/kg,
food of animal origin-LC-MS/MS method and HPLC method.
100 µg/kg, and 200 µg/kg levels. The analysis was performed
in replicates of six at each level. The chromatograms of the
blank and spiked standard (50 µg/kg) are shown in Figure 2 For More Information
and Figure 3. The recovery and reproducibility data are shown For more information on our products and services, visit our
in Table 3. Web site at www.agilent.com/chem.

Table 3. Recoveries and RSDs of Tetracyclines in Chicken by SPE

Compound Spiked level Recovery RSD

(µg/kg) (%) (%)
Minocycline 50 87.6 4.13
100 80.8 5.68
200 81.3 4.19
Oxytetracycline 50 68.8 6.49
100 63.0 4.87
200 59.4 4.35
Tetracycline 50 81.0 4.46
100 70.0 3.47
200 72.3 4.38
Demeclocycline 50 92.0 2.06
100 94.8 3.78
200 92.9 1.92
Chlortetracycline 50 93.3 3.16
100 92.4 4.01
200 87.7 2.54
Methacycline 50 93.3 2.89
100 91.9 2.51
200 86.6 3.39
Doxycycline 50 95.6 4.38
100 96.4 1.00
200 92.0 3.02

Conclusions
Agilent SampliQ provides a simplified and effective single-
cartridge method for the purification and enrichment of multi-
ple tetracycline compounds in chicken. The recovery and
reproducibility results based on solution standards are
acceptable for tetracycline residue determination in chicken
under the Chinese regulation. The impurities from chicken
were minimal and did not interfere with any of the tetracy-
clines analyzed. The LODs of the seven tetracyclines were
significantly lower than the MRL (of 100 ug/kg).

Part number Description

5982-3013 OPT Polymer - Box, 100 × 1 mL tubes, 30 mg
5982-3036 OPT Polymer - Box, 50 × 3 mL tubes, 60 mg
5982-3067 OPT Polymer - Box, 30 × 6 mL tubes, 150 mg
5982-3096 OPT Polymer - 96 Well Plate, 10 mg

5
www.agilent.com/chem
Agilent shall not be liable for errors contained herein or
for incidental or consequential damages in connection
with the furnishing, performance, or use of this material.

Information, descriptions, and specifications in this

publication are subject to change without notice.

© Agilent Technologies, Inc., 2008

Published in the USA
October 17, 2008
5989-9735EN
 Determination of Penicillins in
Meat by High Performance Liquid
Chromatography (HPLC/UV) and
HPLC/MS/MS

Application Note
Food

Author Abstract

Carol Ball Penicillins are antibiotics widely used to treat diseases in animals. They are occa-
Agilent Technologies, Inc. sionally found in animal products destined for human consumption. In this paper, a
200 Regency Forest Drive, Suite 330 solid phase extraction method with a high performance liquid chromatograph tandem
Cary, NC 27511 mass spectrometer (HPLC/MS/MS) is shown for the simultaneous determination of
USA six antibiotic residues: azlocillin, penicillin G, oxacillin, cloxacillin, nafcillin, and
dicloxacillin in animal tissues (porcine muscle). In the method, the reversed phase col-
umn Agilent ZORBAX Eclipse Plus C18 (3.5 µm, 100 mm × 2. 1 mm) and an Agilent
mixed mode polymer solid phase extraction cartridge (Agilent SampliQ OPT) were
combined to give a total solution to the analysis of residual penicillins. The perfor-
mance of the solid phase extraction procedure on trace residues is quantitatively eval-
uated by HPLC/MS/MS.
Introduction by HPLC/MS/MS. This application will show the develop-
ment of a sample extraction and cleanup method and the
ß-lactam antibiotics (penicillins and cephalosporins) repre- quantification by LC/MS/MS
sent some of the most important antibacterial agents used in
animals. However, serious reactions are known to occur in The structures and chemical constants for the compounds
some individuals exposed to penicillins and, as a result, these used in this study are shown in Table 1. ß-lactam antibiotics
compounds are carefully monitored in foods. Maximum are readily decomposed in acid or base [1] and experimentally
residue limits (MRLs) for pencillins in a variety of foods are show a 20 percent loss in 70 hours at 4 °C (data not shown).
established worldwide and are generally in the range of Thus, it is necessary to perform the extractions, cleanup, and
1ng/g. These regulations require detection and quantification analysis within 36 hours.

Table 1. The Compounds in This Study

No. Name log P. pKa Structure

1 Azlocillin 0.2 2.8 O
HN H H CH3
S
NH CONHCHCONH
CH3
N
C 6 H5 COOH
O

2 Penicillin G 1.5 2.74

H CH3
NH S
O CH3
N
COOH
O

3 Oxacillin 2.4 2.72 O CH3

N H H CH3
S
CONH
CH3
N
COOH
O
4 Cloxacillin 2.6 2.78
H H CH3
S
CONH
CH3
Cl N
N COOH
O CH3 O
5 Nafcillin 3.3 2.65
H H CH3
S
CONH
CH3
N
COOH
O
OC 2 H5
6 Dicloxacillin 3.7 2.8
Cl H H CH3
S
CONH
CH3
Cl N
N COOH
O CH3 O

2
Experimental Purification
Reagents and Chemicals The procedure for SPE extraction is shown in Figure 1. Load
Water, acetonitrile, and methanol are all HPLC grade 10 mL of the extract onto the conditioned and equilibrated
(Honeywell Burdick & Jackson). The standards and other cartridge. The cartridge is washed with 0.1 percent formic
chemicals were purchased from Sigma-Aldrich (Saint Louis, acid in water and then pH 8.5 potassium phosphate buffer.
MO). Finally, the sample is eluted with 3 mL acetonitrile. The sam-
ple is filtered with a 13 mm, 45 µm PTFE syringe filter (Agilent
Phosphate buffer (pH = 8.5), 0.05 mol/L: dissolve 8.7 g of p/n 5185-5836). The eluent is dried under nitrogen at room
potassium phosphate dibasic in HPLC grade water to make temperature. The residue is resuspended in mobile phase to
1 L. 1.0 mL. The sample is vortexed for 2 minutes and then trans-
ferred to a 2 mL autosampler vial (Agilent p/n 5182-0864).
Standard stock solutions (1 mg/mL) were made fresh daily
in methanol. Spiking solutions were made by appropriate
dilution of the stock solutions in phosphate buffer. Condition 3 mL methanol

Equipment
Agilent 1100 HPLC with diode array detector (Agilent Equilibrate 3 mL 0.1% formic in water
Technologies, Inc., Santa Clara, CA, USA)

Agilent 6410 triple quadrupole LC/MS system with electro- Load 10 mL of 5 g sample extract (20 mL final vol)
spray ionization source (Agilent Technologies, Inc., Santa
Clara, CA, USA) Dry 30 seconds

Polytron homogenizer (Brinkman Instruments, Inc., PT10-35, Wash 2 mL 0.1% formic acid in water
USA)

Refrigerated centrifuge (Sorvall Instruments, RC-5B, rotor Wash 2 mL phosphate buffer pH 8.5 in water
SA-600)
Dry 3 minutes
Rotary evaporator (BÜCHI, Switzerland/USA)
Sample Preparation Elute with 3 mL ACN
Extraction

Weigh 5 g of raw ground pork (accurate to 0.01 g) into a Dry, reconstitute in mobile phase, vortex
50 mL capped polypropylene centrifuge tube, add 15 mL of
acetonitrile/water (15:2). Homogenize completely using the Figure 1. Agilent SampliQ OPT solid phase extraction of penicillins from pork.
Brinkman Polytron (1 minute). Centrifuge at 4,000 rpm and
4 °C for 5 minutes. Save the supernatant. Add 10 mL acetoni- Instrument Setting
trile/water (15:2) to pellet, mix with a spatula to resuspend The HPLC conditions are shown in Tables 2 and 3.
the ground meat. Homogenize for 1 minute. Centrifuge at
4,000 rpm and 4 °C for five minutes. Combine supernatants. Table 2. HPLC Conditions
Repeat one additional time. HPLC

Take the combined supernatants and place into a round bot- Column Agilent ZORBAX Eclipse Plus, 2.1 mm × 100 mm,
3.5 µm (p/n 959793-902)
tom flask and evaporate the acetonitrile at 37 °C. There
Flow rate 0.6 mL/min
should be approximately 6 mL of water remaining in the flask.
Mobile phase A: water/10 mM ammonium acetate
Bring the total volume to 20 mL using pH 8.5 phosphate B: acetonitrile
buffer. Filter with a regenerated cellulose, 25 mm, 45 µm Run time 12 minutes
syringe filter (Agilent p/n 5185-5831). Load 10 mL of extract Post run 3 minutes
onto the Agilent SampliQ OPT 6 mL/150 mg SPE cartridge Temperature 30 °C
(Agilent p/n 5982-3067).
Injection 10 µL

3
Table 3. HPLC Gradient strates that the sample is extremely clean after the sample
Time %B extraction and SPE cleanup.
0 2
1.2 2 Matrix blank material is prepared by taking the meat through
2.0 10 the entire extraction and sample cleanup procedure. External
6.0 30
8.0 40 standard calibration curves in spiked matrix blanks are made
8.5 80 at concentrations of 0.2, 1.0, 10, and 20 ng/g. Table 4 shows
11.9 80 the calculated recoveries of spiked meat taken through the
12.0 2
entire sample preparation and SPE procedures. All data were
calculated automatically with the Agilent MassHunter
Results and Discussion Quantitative Data Analysis software. Figure 4 shows these
results graphically. All of the compounds show acceptable
Figure 2 shows the chromatograms of the meat spike sample recovery and low relative standard deviation (%RSD).
at the limit of quantification (LOQ), 1.0 ng/g (2a), the matrix
spiked blank at the LOQ (2b) and the meat extract blank (2c).
The cleaned-up pork extract does not show any interferences
Conclusions
with the target analytes. HPLC/UV is a significantly less spe- The results of this study show that the Agilent SampliQ OPT
cific detector than HPLC/MS/MS so the impurities remaining cartridge provides an effective method for cleaning up com-
after cleanup are more visible using the general UV detector. plex food samples such as porcine muscle. This is demon-
As shown in Figure 3, the HPLC/UV chromatogram demon- strated using penicillins as target compounds. HPLC/UV is

5
2a: 1 ng/g meat
1
1. Azlocillin 4. Cloxacillin
3
4 2. Penicillin G 5. Nafcillin
2 6
3. Oxacillin 6. Dicloxacillin

2b: 1 ng/g standard

2 1. Azlocillin 4. Cloxacillin
1 3
4 6 2. Penicillin G 5. Nafcillin
3. Oxacillin 6. Dicloxacillin

2c: Blank

Figure 2. Meat spiked at 1 ng/g taken through extraction and SPE clean-up (2a), meat taken through extraction and clean-up then spiked at 1 ng/g (2b), and unspiked meat
taken through extraction and cleanup (2c).

4
 Table 4. Calibration Results for Spiked Meat Blanks

1.0 ng/g 20 ng/g

% recovery %RSD % recovery %RSD
Name Linear regression R2 n=6 n=6 n=6 n=6
Azlocillin y = 6089x –1283 0.9590 77.8 24.2 75.5 6.7
Penicillin G y = 2690x –513 0.9924 43.6 6.3 48.8 6.8
Oxacillin y = 2319x +1487 0.9794 96.5 6.0 102.8 8.9
Cloxacillin y = 2229x +128 0.9848 86.3 7.3 95.3 8.1
Nafcillin y = 16654x –1264 0.9891 98.5 6.6 101.9 3.1
Dicloxacillin y = 1899x –113 0.9870 105.7 13.5 103.9 3.6

Figure 3. HPLC/UV chromatogram of an unspiked meat sample taken through extraction and SPE cleanup. Wavelength 230 nm.

Penicillin Recoveries
110.0

90.0
% Recovery

70.0

50.0

30.0

10.0

Azlocillin Penicillin g Oxacillin Cloxacillin Nafcillin Dicloxacillin

1 ng/g 20 ng/g

Figure 4. Recovery data for meat extracts at 1.0 and 20 ng/g.

used as a detector to demonstrate the extent of cleanup, Reference

which is found to be excellent. The LC/MS/MS is used to
demonstrate the recovery of the penicillins at trace level. Even 1. Xinbo Lu, Huabin Xing, Baogen Su, and Qilong Ren, Effect
with extensive extraction and SPE cleanup, recoveries are of Buffer Solution and Temperature on the Stability of
acceptable and reproducibilities are excellent. Penicillin G, J. Chem Eng. 53 (2), 543–547, 2008

For More Information

For more information on our products and services, visit our
Web site at www.agilent.com/chem.

5
www.agilent.com/chem
Agilent shall not be liable for errors contained herein or
for incidental or consequential damages in connection
with the furnishing, performance, or use of this material.

Information, descriptions, and specifications in this

publication are subject to change without notice.

© Agilent Technologies, Inc., 2008

Published in the USA
December 29, 2008
5990-3364EN
 Determination of Chloramphenicol,
Florfenicol, and Thiamphenicol in Honey
Using Agilent SampliQ OPT Solid-Phase
Extraction Cartridges and Liquid
Chromatography-Tandem Mass Spectrometry
Application Note
Food Safety

Authors Abstract
Limian Zhao A method for the simultaneous determination of three antibiotic residues of chloram-
Agilent Technologies, Inc. phenicol (CAP), florfenicol (FF), and thiamphenicol (TAP) in honey has been developed
2850 Centerville Road and validated. The analytes are purified by liquid/ liquid extraction and solid-phase
Wilmington, DE 19808 extraction (SPE) and are quantified by liquid chromatography coupled to electrospray
USA ionization tandem mass spectrometry (LC-ESI-MS / MS) operating in negative ion mul-
tiple reaction monitoring (MRM) mode. Chloramphenicol-D5 is used as the internal
Carol Haney Ball standard. The method is validated by achieving reproducible, satisfactory, quantitative
Agilent Technologies, Inc. results. The method provides a sub-ng/ g to ng/ g level of limit of quantitation (LOQ)
200 Regency Forest Drive for all three antibiotics in honey. The overall recoveries range from 74.9 to 107% with
Cary, NC 27511 RSD values between 0.5 and 9.7%. The dynamic calibration ranges for chlorampheni-
USA col and florfenicol are obtained over 0.1 to 20.0 ng/ g and 1.0 to 20.0 ng/ g for
thiamphenicol. The method is demonstrated to be fast, simple, and efficient for
monitoring chloramphenicol, florfenicol, and thiamphenicol residues in honey.
Introduction application note describes a method for the simultaneous
determination of three phenicols in honey, and the results of
Chloramphenicol (CAP) is a broad-spectrum bacteriostatic validation.
antibiotic, obtained originally from the bacterium
Streptomyces venezuelae. Due to potential side effects in
humans, the drug is not recommended for the treatment of
Experimental
minor diseases, but is reserved for the treatment of serious Reagents and Chemicals
infections. In veterinary medicine, CAP has been shown to be
All reagents and solvents were HPLC or analytical grade.
a highly effective, well-tolerated antibiotic; the potential side
Acetonitrile and methanol were from Honeywell, Burdick &
effects observed in humans have not been reported in ani-
Jackson (Muskegon, MI); ethyl acetate was from J.T.Baker
mals. However, because of its toxicity in humans, the use of
(Phillipsburg, NJ). Dimethyl sulfoxide was from Sigma-Aldrich
CAP in animal-derived foods, including honey from honey-
(St. Louis, MO). The standards and other chemicals were pur-
bees, has been strictly regulated. The European Union (EU)
chased from Sigma-Aldrich.
has defined a maximum residue limit (MRL) for CAP in food of
animal origin at a level of 0.3 µg / kg [1], while China has an Water (pH 8.5) was prepared by pH adjustment of Milli-Q
MRL level of 0.5 µg / kg [2]. Thiam-phenicol (TAP) and florfeni- water with 0.05% NH4OH in water solution monitored by a pH
col (FF) are the analogue compounds of CAP. They can be meter. A solution of 20:80 methanol / ethyl acetate was pre-
used as a replacement veterinary antibiotic for CAP in many pared by combining 40 mL of methanol and 160 mL of ethyl
countries. The MRLs have been set for TAP (50 ng / g) and FF acetate and mixing well. A solution of 20:80 acetonitrile / H2O
(100 ng / g) in food to date [3]. Table 1 shows the chemical was prepared by adding 40 mL of acetonitrile into 160 mL of
structure and properties of these three compounds. This Milli-Q water.

Table 1. Chemical Structure and Properties of Target Analytes

Compounds Log P pKa Structure

Chloramphenicol 1.02 9.61 OH OH

Cl
O + HN
N Cl
_
O O

Florfenicol –0.12 9.03

MeO 2 S F
O
Cl
N
H
OH Cl

Thiamphenicol –0.27 9.76 OH

OH
O
HN O
S
H3C
O
Cl Cl

2
Standard stock solutions (1.0 mg / mL) were made in dimethyl Accurately weigh 5 g honey (± 0.05 g) in 50 mL centrifuge tube
sulfoxide (DMSO) individually, and stored in the refrigerator at
4 ºC. A combined working solution (2,500 ng / mL) was made
weekly in 20:80 ACN / H2O, and also stored at 4 ºC. The spik- Spike 0.5 mL of IS solution (50 ng / mL of chloramphenicol-D5
ing solutions were then made daily by appropriate dilution of in H2O), vortex 1 min for mixing
the combined working solution in Milli-Q water or 20:80
ACN / H2O. Add 5 mL of H2O, vortex vigorously for 3 min

Internal standard (IS) stock solution (0.1 mg / mL) was made

in DMSO and stored in the refrigerator at 4 ºC. An IS spiking Add 5 mL of ethyl acetate, then shake for 5 min
solution (50 ng / mL) was made weekly by appropriate dilution
of stock solution into Milli-Q water, and stored at 4 ºC.
Centrifuge at 3200 rpm for 5 min, transfer the Repeat 2×
Equipment and Materials upper organic layer to another tube successively
Agilent 1200 Series HPLC (Agilent Technologies Inc., Santa
Clara, CA, USA) Combine all of transferred organic layer (~ 14 mL),
blow down with N2 flow at 50 ºC
Agilent 6410 Triple Quadrupole LC / MS / MS system with elec-
trospray ionization source (Agilent Technologies Inc., Santa
Clara, CA, USA) Reconstitute into 5 mL of H2O, vortex for 3 min, and
sonicate for 2 min
Agilent SampliQ OPT solid-phase extraction cartridges, 50 ×
3 mL tubes, 60 mg (p / n 5982-3036) (Agilent Technologies Inc.,
Wilmington, DE, USA) Ready for SPE

CentraCL3R centrifuge (Thermo IEC, Needham Heights, MA, Figure 1. Sample preparation – liquid liquid extraction of phenicols in honey.
USA)

N2 dryer (Glas-Col, Terre Haute, IN, USA)

Solid-Phase Extraction
Sample Preparation The procedure for SPE extraction is shown in Figure 2. Agilent
Liquid-Liquid Extraction SampliQ OPT cartridges were preconditioned with 3 mL of
MeOH, and then equilibrated with 5 mL of water. The 5 mL
5g of honey (± 0.05 g) was weighed into a 50 mL capped
sample extract was then loaded onto a cartridge and passed
polypropylene tube. 0.5 mL of IS spiking solution (50 ng/ mL)
through the cartridge slowly by gravity (0.5 mL/ min). The
was added to the tube and vortexed until mixed. This was fol-
tubes were rinsed with 5 mL of Milli-Q water twice. Repeat
lowed by the addition of 5 mL of Milli-Q water and vortexing
the above wash procedure once. The entire effluent was dis-
for 3 minutes to mix the sample thoroughly. 5 mL of ethyl
carded. Apply full vacuum to the cartridge for 3 minutes to
acetate was then added, capped tightly, and the tubes shaken
completely dry the resin. Finally, the compounds were eluted
for 5 minutes. The tubes were then centrifuged at 3,200 rpm
with 5 mL of 20:80 MeOH / ethyl acetate (2.5 mL × 2) at a rate
for 5 minutes, before the upper organic layer was carefully
of 1 mL/ min. The eluent was collected into clean tubes and
transferred to another tube. Ethyl acetate addition, shaking,
dried under N2 flow at 50 ºC. The residue was reconstituted in
centrifuging, and organic layer transfer was repeated two
0.5 mL of 20:80 AcN / H2O. The sample was vortexed and soni-
more times with all supernatants combined. Samples were
cated to completely dissolve the residue in the tubes. The
evaporated to dryness with a controlled N2 flow drier at 50 ºC
sample was transferred to a centrifuge tube and centrifuged
before being reconstituted into 5 mL of Milli-Q water, vor-
at 3,200 rpm for 2 minutes. The samples were then transferred
texed, and sonicated to completely dissolve the residue. The
to 2 mL autosampler vials for analysis.
sample was then ready for SPE purification. Figure 1 shows
the extraction procedure flowchart.

3
 Table 2. Masses Monitored in the Multiple Reaction Monitoring (MRM)
Condition 3 mL methanol Experiment
Analyte MRM (m / z & m / z) Dwell time (ms)
Equilibrate 5 mL Milli-Q H2O (2.5 mL × 2) Thiamphenicol 354.0 & 184.9 (quantifier) 50
354.0 & 290.0 (qualifier) 25
Florfenicol 355.8 & 185.0 (quantifier) 50
Load 5 mL extract (from previous sample preparation, 2.5 mL × 2), 355.8 & 336.0 (qualifier) 25
have sample pass through cartridge slowly with gravity
Chloramphenicol 320.9 & 152.0 (quantifier) 50
320.9 & 176.0 (qualifier) 25
Rinse the sample tubes and wash cartridge with 5 mL × 2 water Chloramphenicol-D5 (IS) 325.9 & 156.8 25

Apply full vacuum for 3 min, dry the needle tip, put the collection Results and Discussion
tubes below
Linearity, Limit of Detection
The extracted ion chromatograms of fortified honey at a con-
Elute with 5 mL 2:8 ethyl acetate / MeOH (2.5 mL × 2) centration of 0.2 ng / g are shown in Figure 3. The extracted
honey blank was clean and free from any analytes, indicating
that the cleaned-up honey extract does not contribute any
Blow down at 50 °C, reconstitute into 0.5 mL of 20:80 AcN / H2O, interference with the target analysis.
vortex 3 min for mixing, then sonicate 2 min
The concentration ranges studied here are significantly below
Centrifuge at 3200 rpm for 2 min, transfer to a 2 mL autosampler
the limit of quantitation (LOQ) defined by the MRL for TAP
vial for injection (50 ng / g) [3]. In this study the limit of quantitation (LOQ)
found for TAP is 1.0 ng / g, and the linear calibration range
Figure 2. Sample clean-up – Agilent SampliQ solid-phase extraction. used for TAP is 1.0 to 20.0 ng / g. The linear calibration range
for CAP (LOQ 0.1 ng / g, MRL 0.3 ng / g) and FF (LOQ 0.1 ng / g,
Instrument Conditions MRL 100 ng / g) was 0.1 to 20.0 ng / g.
HPLC Conditions Calibration curves spiked in matrix blanks were made at levels
Column: Agilent ZORBAX Eclipse Plus 150 mm × of 0.1, 0.2, 1.0, 5.0, 10.0, 15.0, and 20.0 ng / g for CAP and FF.
2.1 mm, 5 µm (PN: 959701-906)
While for TAP they were spiked at a level of 1.0, 5.0, 10.0,
Flow rate: 0.3 mL/ min 15.0, and 20.0 ng / g. The chloramphenicol-D5 was used as
Column temperature: 30 ºC internal standard at 5 ng / g level. The calibration curves were
Injection volume: 20 µL generated by plotting the relative responses of analytes (peak
Mobile phase: pH 8.5 H2O (A), Acetonitrile (B) area of analyte / peak area of IS) to the relative concentration
Gradient: Time % Acetonitrile Flow rate (mL/ min) of analytes (concentration of analyte / concentration of IS).
0 20 0.3 The limit of detection (LOD) was determined with a signal-to-
0.5 20 0.3 noise ratio between 4 and 5. Table 3 shows the linearity equa-
6.0 80 0.3 tion, correlation coefficient (R2) and LOD. The calibration
6.01 100 0.5 curve for chloramphenicol is shown in Figure 4.
6.50 100 0.5
6.51 20 0.3
Table 3. Linearity and LODs of Phenicols
7.00 STOP
Analytes Regression equation R2 LOD (ng / g)
MS Conditions
Chloramphenicol Y = 0.5643X – 0.0001 0.9957 0.02
The three compounds were monitored in the negative ionization mode. The
multiple reaction monitoring channels are shown in Table 2. Florfenicol Y = 0.8790X + 0.0006 0.9932 0.02
Thiamphenicol Y = 0.1510X – 0.0018 0.9953 0.20

4
 MRM (355.8 & 185.0)

Florfenicol

MRM (354.0 & 184.9)

Thiamphenicol

MRM (325.9 & 156.8)

Chloramphenicol-D5

MRM (320.9 & 152.0)

Chloramphenicol

Figure 3. Chromatograms of 0.2 ng / g fortified honey extract.

2.6
Chloramphenicol - 7 Levels, 7 Levels Used, 14 Points, 14 Points Used, 53 QCs
2.4
y = 0.5643 * x - 1.2945E-004
2.2 R^2 = 0.99589657

1.8

1.6
Relative responses

1.4

1.2

0.8

0.6

0.4

0.2

0
_0.2
_0.2 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 2.2 2.4 2.6 2.8 3 3.2 3.4 3.6 3.8 4 4.2
Relative concentration
Figure 4. Calibration curve of chloramphenicol (0.1 to 20.0 ng / g). Dots (•) indicate sample results of calibration curve points, and trianges (∆) indicate
sample results of quality controls.

5
Recovery and Reproducibility Conclusions
The recovery and reproducibility were evaluated by spiking
phenicol standards in honey at levels of 0.1 ng / g (1.0 ng / g Agilent SampliQ OPT SPE cartridges provide a simple and
for TAP), 5.0 ng / g and 20.0 ng / g as quality control samples effective method for the purification and enrichment of chlo-
(QCs), and quantifying those QCs against the matrix spiked ramphenicol, florfenicol, and thiamphenicol in honey. The
calibration curve. The analysis was performed in replicates recovery and reproducibility results based on matrix spiked
of six at each level, except four replicates for TAP low level. standards are acceptable for chloramphenicol residue deter-
The recovery and reproducibility (shown as %RSD) data are mination in honey under EU or Chinese regulations. The impu-
shown in Table 4. CAP and FF show excellent recovery and rities and matrix effect from honey are minimal and do not
reproducibility at all QC levels. The recovery of TAP is ade- interfere with the quantitation of any target compound. The
quate at all concentrations and the reproducibility is excel- LOQs of the three phenicols are significantly lower than the
lent. MRLs.

Table 4. Recoveries and Reproducibility of Phenicols in Fortified Honey References

Spiking Level Recovery RSD (%)
Analytes (ng / g honey) (%) n=6 1. GB / T 18932.19-2003, “Determination of Chloramphenicol
Chloramphenicol 0.10 96.94 3.51 Residues in Honey -LC / MS / MS Method.”
5.00 98.88 0.87
20.00 107.32 0.46 2. “Commission Decision” 2003 / 181 / ED of 13 March 2003,
Florfenicol 0.10 100.67 9.77
Off. J. Eur. Commun. L71 / 17 (2003).
5.00 100.28 2.84 3. “Handbook of Food Analysis: Residue and Other Food
20.00 107.49 2.55
Component Analysis,” CRC Press, 2004, pgs 937, 940.
Thiamphenicol 1.00 76.00 4.39*
5.00 74.89 2.34
20.00 89.81 3.83 For More Information
* The experiment was done in replicates of four.
For more information on our products and services, visit our
Web site at www.agilent.com/ chem.

www.agilent.com/ chem
Agilent shall not be liable for errors contained herein or
for incidental or consequential damages in connection
with the furnishing, performance, or use of this material.

Information, descriptions, and specifications in this

publication are subject to change without notice.

© Agilent Technologies, Inc., 2009

Printed in the USA
February 16, 2009
5990-3615EN
 Determination of Sulfonamides in Milk
Using Solid-Phase Extraction and
Liquid Chromatography-Tandem Mass
Spectrometry

Application Note
Pharmaceuticals

Authors Abstract
Carol A. Gonzalez and Karyn M. Usher The extraction of trace levels of nine nitrogen-containing sulfa drugs (sulfamethoxa-
Department of Chemistry zole, sulfadiazine, sulfathiazole, sulfamerazine, sulfamethizole, sulfamethazine, sul-
West Chester University famethoxypyridazine, sulfachloropyridazine, and sulfadimethoxine) in milk samples by
West Chester, PA 19383 solid-phase extraction was studied using Agilent SampliQ polymeric strong cation
USA exchange (SCX) cartridges. An Agilent 6410 triple quadrupole LC/MS-MS System was
used for the separation and determination of the sulfa drugs. For reversed-phase chro-
Anne E. Brooks and Ronald E. Majors matography, an Agilent ZORBAX Eclipse Plus column (C18, 3.0 mm × 50 mm, 1.8 µm)
Agilent Technologies, Inc. with a 0.1% formic acid/acetonitrile gradient was used. Overall recoveries from the
2850 Centerville Road milk samples ranged from 73 to 99%, with %RSD values less than 10%. Limits of
Wilmington, DE 19808 detection ranged from 0.2 to 2.0 ng/mL in milk (S/N = 3) depending on the sulfa drug,
USA below the U.S. Food and Drug Administration acceptable levels in milk.
Introduction microorganisms have become resistant to these compounds.
Of growing concern are drug-resistant bacteria that may be
Since the discovery of the effective antimicrobial properties of passed from animals to humans. One major cause of the
sulfonamides in the early 1900s, they have been used to treat resistance to these compounds is that feed animals are being
a variety of diseases. In the dairy farming industry, sulfa drugs fed antimicrobial drugs at low levels to treat diseases. In the
are administered to dairy cattle to prevent infection. This 1990s, the United States Food and Drug Administration (FDA)
leads to the possibility of the drugs being excreted in the milk began conducting tests on several milk supplies [3]. If dairy
and passed on to the consumer. This ingestion can cause a cattle were given sulfa drugs, low levels of these compounds
drug resistance, making the drugs ineffective in later uses to could be found in the milk, leading to allergic reactions in
treat illness [1,2]. some consumers, as well as an increase in drug-resistant
organisms. This application demonstrates a complete solu-
Sulfonamides, commonly known as sulfa drugs, have proven tion to the analysis in milk of nine important sulfa drugs: sul-
to be effective antimicrobial agents since their discovery in famethoxazole, sulfadiazine, sulfathiazole, sulfamerazine, sul-
1929 by Gerhard Domagk. Today, β-lactam antibiotics are famethizole, sulfamethazine, sulfamethoxypyridazine, sul-
much more commonly used to prevent infection than sulfon- fachloropyridazine, and sulfadimethoxine (see Figure 1 for
amides, but sulfonamides are still routinely used in different structures and chemical properties).
parts of the world due to their low cost. Over the years, many

S
N
O HN N
N
-
HN S O O S O
H2N N
HN
S N
O
NH2 NH2 O

1. Sulfadiazine 2. Sulfathiazole 3. Sulfamerazine

pKa 6.50 pKa 7.24 pKa 6.98
logP 0.2 logP 0.35 logP 0.44

H2N N H2N N H2N N N

N
HN HN HN O
S N S S S
O O O
O O O

4. Sulfamethazine 5. Sulfamethizole 6. Sulfamethoxypyridazine

pKa 7.45 pKa 5.51 pKa 7.19
logP 0.43 logP 0.53 logP 1.01

O
O
N
O HN S O O
O H2N
S N
Cl NH HN N
N N NH2 S
O O
NH2 O

7. Sulfachloropyridazine 8. Sulfamethoxazole 9. Sulfadimethoxine

pKa 5.90 pKa 5.81 pKa 6.21
logP 1.36 logP 1.58 logP 1.56

Figure 1. Structures and chemical constants for sulfa drugs used in this study.

2
Experimental Sample Preparation

Materials and Chemicals 20 µL of a 45% solution of formic acid in water (prepared by

mixing 10 mL of 90% formic acid with 10 mL of water) solu-
Water (EMD Chemicals, Gibbstown, NJ), acetonitrile, and tion was added to each 1 mL of whole milk to precipitate pro-
methanol (Burdick and Jackson, Muskegan, MI) were HPLC teins and lipids. The milk samples were then centrifuged at
grade. Sulfa drugs were analytical grade and purchased from 8000 rpm for 10 minutes (Eppendorf 5810R 15 amp, Westbury,
Sigma-Aldrich (Saint Louis, MO). The stock solution NY ). Alternatively, the samples may be centrifuged at
(~1.19 mg/mL) was prepared in 25 mL of methanol and kept 3500 rpm for 20 minutes. An aliquot of the supernatant (pre-
refrigerated for up to 14 days. Working solutions were made pared whole milk extract) was removed and used to load onto
daily by dilution of the stock solution in water. SampliQ SCX cartridges.

The SPE cartridges were Agilent SampliQ SCX, 3 mL/60 mg SPE Purification
p/n 5982-3236), a polymeric cation exchanger with a 30-µm
average particle size. The analysis was performed on an After the SPE cartridge was conditioned and equilibrated as
Agilent 1200 Series HPLC coupled to a 6410 triple quadrupole described in Figure 2, 5 mL of prepared whole milk extract
mass spectrometer with electrospray ion source. The analyti- was loaded onto the column. Care was taken that the flow
cal column was an Agilent ZORBAX Eclipse Plus C18, 3.0 mm rate during the load step did not exceed 1.5 mL/min. During
× 50 mm, 1.8 µm (p/n 959941-302). Formic acid was pur- both drying steps, the cartridge was dried under vacuum at
chased from J.T. Baker (Phillipsburg, NJ) (Baker PCS reagent, 15 in Hg for the time indicated. The eluate was dried under
90%) for use in mobile phase preparation and for precipitation nitrogen and then reconstituted in 1 mL of solvent (9:1
of proteins and lipids in the milk. water:methanol). The samples were then sonicated (Branson
1200, Danbury, CT) for 5 minutes and analyzed using the
Agilent 6410 Triple Quad LC/MS-MS system.

Condition: 2 mL of 0.1% formic acid in MeOH

Using 5 mL of whole milk, precipitate
protein/lipids with 45% formic
acid; centrifuge and further process
Equilibrate: 2 mL of 2% formic acid in water supernatant (prepared whole milk extract)

Load: 5 mL prepared whole milk

extract onto SPE cartridge

Dry under vacuum for 1 minute

Wash 1: 2 mL of 5% MeOH in water

Wash 2: 1 mL of 0.5 M HCl

Wash 3: 2 mL of 20% MeOH in water

Dry under vacuum for 3 minutes

Elute: 2.5 mL of 5% ammonia in MeOH

Figure 2. SPE procedure.

3
Due to the low concentrations of sulfa drugs being analyzed, Table 3. Conditions for Electrospray Ionization Source
and the very low LOD that can be achieved with Agilent 6410 Gas temperature 350 °C
Triple Quad LC/MS-MS system, extra care must be taken to Gas flow 12 L/min
keep the SPE manifold system clean to prevent contamination
Nebulizer 40 psi
of samples. The manifold system must be thoroughly cleaned
Capillary 4000 V
between uses or, if the option is available, needle inserts may
be newly installed.

Separation and Analysis Results and Discussion

The chromatographic and MS/MS experimental setup is Linearity and Limits of Detection
shown in Tables 1, 2, and 3.
Solutions used to create external calibration curves were pre-
Table 1. HPLC Setup pared by using a stock solution to spike matrix blanks. Matrix
Column Agilent ZORBAX Eclipse Plus C18, 3.0 × 50 mm, blanks were created by taking the milk through the entire pro-
1.8 µm (p/n 959941-302)
cedure, including the precipitation, centrifugation, and SPE
Flow rate 0.42 mL/min procedures. The results for the calibration curves are summa-
Column temperature 35 °C rized in Table 4. The regression results were used to calculate
Injection volume 1.7 µL w/ needle wash; wash for 30 s in flush port the recoveries. The limits of detection were chosen as the
with MeOH/H2O (5:1)
concentration of each drug that gave a signal-to-noise (S/N)
Mobile phase A: H2O/acetonitrile (9:1) w/ 0.1% formic acid
B: Acetonitrile w/ 0.1% formic acid ratio greater than 3:1. The limits of detection are given in
Run time 8 min
Table 4.
Post time 3 min
Table 4. Calibration Curve Regression Analysis for Sulfa Drugs
Gradient Time 0 3.5 8
%B 0 0 65 LOD in milk
Compound Regression equation R2 (ng/mL)

Table 2. MS/MS Conditions Sulfadiazine y = 282.62x + 225.59 1.0000 0.4

TR (min) Compound Precursor ion Product ion Sulfathiazole y = 440.38x + 246.43 0.9996 1.0

1.69 Sulfadiazine 251.2 156.0 Sulfamerazine y = 358.34x + 485.54 0.9998 1.0

108.0 Sulfamethazine y = 539.09x + 576.81 0.9989 1.0
1.93 Sulfathiazole 256.1 156.0 Sulfamethizole y = 499.57x + 333.03 0.9994 2.0
108.0 Sulfamethoxypyridazine y = 494.61x + 139.66 0.9970 1.0
2.41 Sulfamerazine 265.2 156.0 Sulfachloropyridazine y = 343.78x + 92.808 0.9999 2.0
108.0
Sulfamethoxazole y = 260.05x – 351.97 0.9901 2.0
3.44 Sulfamethazine 279.2 186.1
124.1 Sulfadimethoxine y = 956.97x + 1420.9 0.9973 0.2

3.89 Sulfamethizole 271.1 156.0

108.0
Recovery and Reproducibility
4.10 Sulfamethoxypyridazine 281.2 156.0
108.0 The recoveries and precision for the method were determined
5.99 Sulfachloropyridazine 285.1 156.0 at two levels, milk spiked to a concentration of 5 ng/mL and
108.1 10 ng/mL. Since the procedure for the SPE used 5 mL of pre-
6.42 Sulfamethoxazole 254.2 156.1 pared whole milk extract to load the SPE cartridge and the
108.1 extract was reconstituted in 1 mL of mobile phase, the sam-
7.17 Sulfadimethoxine 311.2 156.0 ple analyzed is five times as concentrated as the milk sam-
108.0
ples. The analyzed solutions are therefore 25 ng/mL for the
samples that were spiked with 5 ng/mL in milk, and
50 ng/mL for the samples that were spiked with 10 ng/mL.
The analysis was performed with five replicates at each level.
The recovery and reproducibility data are shown in Table 5.
The chromatograms for the blank and spiked milk extracts
(5 ng/mL) are shown in Figure 3.

4
Table 5. Recovery and Precision Data for Nine Sulfa Drugs Used in This enrichment of multiple sulfonamides in complex samples
Study such as whole milk. The impurities remaining after the SPE
Level spiked cleanup step were minimal and did not interfere with the
in milk RSD quantitation of the sulfonamides. The levels at which the
Compound (ng/mL) Recovery (%)
quantitation was performed are below the levels of sulfon-
Sulfadiazine 5 74.2 8.3 amides that are considered by the FDA as safe in milk for con-
10 99.7 5.7
sumption (10 ng/mL). The LOD for the method was also well
Sulfathiazole 5 76.8 4.4 below these levels (3 ng/mL in milk).
10 83.2 4.7
Sulfamerazine 5 73.2 6.3
10 84.8 0.6 References
Sulfamethazine 5 78.3 7.5
10 89.0 3.1 1. G. G. Khachatourians, Canadian Medical Association
Sulfamethizole 5 78.4 7.0 Journal, 159 (9): 1129, 1998
10 94.5 5.3
2. M. K. Glynn, C. Bopp, W. Dewitt, P. Dabney, M. Mokhtar,
Sulfamethoxypyridazine 5 76.3 6.2 and F. J. Angulo, New England Journal of Medicine,
10 86.9 2.2
Volume 338 (19): 1333–1339, 1998
Sulfachloropyridazine 5 78.3 9.4
10 84.3 6.0 3. U.S. Food and Drug Administration Center for Food Safety
Sulfamethoxazole 5 74.0 4.3 Applied Nutrition Food Compliance Program Chapter
10 87.7 6.4 03 – Foodborne Biological Hazards (10-01-97)
Sulfadimethoxine 5 75.4 3.1 http://www.cfsan.fda.gov/~comm/cp03039.html
10 82.5 5.4 accessed 2/13/09

Conclusions For More Information

For more information on our products and services, visit our
The results of this study show that Agilent SampliQ SCX car-
Web site at www.agilent.com/chem.
tridges can be used as an effective method of purification and

Figure 3. Total ion chromatograms of (3a) milk taken through extraction and cleanup, then spiked with sulfa drugs; (3b) milk spiked at 5 ng/mL, then taken
through extraction and SPE cleanup; and (3c) milk blank.

5
www.agilent.com/chem
Agilent shall not be liable for errors contained herein or
for incidental or consequential damages in connection
with the furnishing, performance, or use of this material.

Information, descriptions, and specifications in this

publication are subject to change without notice.

© Agilent Technologies, Inc., 2009

Printed in the USA
March 11, 2009
5990-3713EN
 Determination, Confirmation, and
Quantification of Trace ß-Lactam
Antibiotics in Milk by LC/MS/MS

Pei-Bin Hu, Xin Ma, and Tao Bo

Application Brief
Food Safety

The ß-lactam antibiotics are widely used in veterinary medicine for the treatment Highlights
and prevention of disease. This use can result in the presence of residues in milk
and edible tissues which can lead to problems in the fermentation processes and to • Ease of use for method optimization
health problems for individuals who are hypersensitive to ß-lactams. • Good linearity R2 ! 0.98 in real milk
To protect the consumer, MRL values were laid down in EU regulation 2377/90. For samples
milk these values range from 4 µg/kg for penicillin G, ampicillin, and amoxicillin to • Good separation to get almost all the
30 µg/kg for dicloxacillin, cloxacillin, and oxacillin. compounds separated well within
9 minutes
In practice, screening is performed by microbiological and immunological methods.
In this way it is possible to give, for a positive sample, the specification of the group
to which the residue belongs. Confirmation, however, has to be performed by an
independent physicochemical technique. Therefore, a highly sensitive LC/MS/MS
method was developed for the detection of penicillins, etc., in milk samples.

Experimental
Sample Preparation
An aliquot of 2 mL of milk (half skimmed consumption milk or raw milk) was mixed
with 4 mL of acetonitrile for protein precipitation. The sample was vortexed and
then centrifuged for 10 minutes at 3500 rpm. After filtration, 20 µL of the super-
natant was injected into the LC/MS/MS system.
LC Conditions
Instrument Agilent 1200SL
Column Agilent ZORBAX SB-C18, 2.1 × 150 mm, 3.5 µm
(p/n 830990-902)
Mobile phase A: Water/0.3% acetic acid
B: Acetonitrile/0.3% acetic acid
Flow rate 0.3 mL/min
Gradient 0–2 min/A 90%; 2.01–8 min/A 35%; 8.01–9/A 5%
Column compartment temperature 30 °C
Stop time 9 min
Post time 6 min
Injection volume 10 µL
MS Conditions
Instrument Agilent 6410A triple quadrupole LC/MS system
Source ESI +
Drying gas temperature 350 °C
Drying gas flow 10 L/min
Nebulizer pressure 45 psi

MRM Setting
Dwell Frag CE
TS Compound Precursor Product (ms) (V) (V)
0 Amoxicillin 366 114 100 110 15
208 100 110 5
Ampicillin 350 160 100 100 5
192 100 110 10
6 Dicloxacillin 470 311 70 110 10
160 70 110 10
Nafcillin 415 256 70 110 15
160 70 110 10
Oxacillin 402 243 70 110 10
160 70 110 10
Penicillin V 351 192 70 100 5
160 70 100 10
Penicillin G 335 176 70 100 10
160 70 100 5

2
Results
x10 2
1 1 1 2 2
+ MRM (335.00000 -> 176.00000) 100ppt-r002.d
0.95
4
0.9
0.85 6
0.8 1
0.75
0.7
3
0.65
0.6
0.55
0.5
0.45 5
0.4
0.35
7
0.3
0.25
0.2 2
0.15
0.1
0.05
0
0.2 0.6 1 1.4 1.8 2.2 2.6 3 3.4 3.8 4.2 4.6 5 5.4 5.8 6.2 6.6 7 7.4 7.8 8.2 8.6 9
Counts (%) vs. acquisition time (min)

1. Amoxicillin 5. Oxacillin
2. Ampicillin 6. Penicillin V
3. Dicloxacillin 7. Penicillin G
4. Nafcillin

S/N
Compounds (Conc = 0.1 pppb) R2
Amoxicillin 366-114 224 0.992
Ampicillin 350-160 61.6 0.984
Dicloxacillin 470-160 48.5 0.981
Nafcillin 415-199 52.6 0.998
Oxacillin 402-160 70.9 0.993
Penicillin V 351-160 225.9 0.998
Penicillin G 335-160 33.2 0.981

3
Pei-Bin Hu is an application engineer based at Agilent Technologies, Chengdu,
China. Xi Ma is working in the Training Centre and Tao Bo is an application engineer
based at Agilent Technologies, Beijing, China.

For More Information

For more information on our products and services, visit our Web site at
www.agilent.com/chem.

www.agilent.com/chem
Agilent shall not be liable for errors contained herein or
for incidental or consequential damages in connection
with the furnishing, performance, or use of this material.

Information, descriptions, and specifications in this

publication are subject to change without notice.

© Agilent Technologies, Inc., 2009

Printed in the USA
March 25, 2009
5990-3722EN
 Using LC/MS/MS 6410 for Analysis of
Chloramphenicol, Thiamphenicol, and
Florfenicol in Fish Samples
Application Brief

Xiaorong Ran, Tao Bo

Chloramphenicol is a banned compound in the EU. It is a zero tolerance compound, Highlights

and some methods have already been developed for this analysis. The LC/MS/MS
is often used for its greater sensitivity and higher selectivity. • Using a simple RRLC method can
separate the compounds well within
In many cases, not only chloramphenicol but also thiamphenicol and florfenicol are 6 minutes
also found. This method uses a simple method for detecting all three compounds
within less than 6 minutes. Furthermore, the results gave the good results, showing • Quite high sensitivity in negative
the performance at the negative mode. mode
• The ISTD method can remove
Experimental the matrix effect and minimumize
the sample preparation interference
LC Conditions
Column Agilent ZORBAX Eclipse Plus, 2.1 mm × 50 mm, 1.8 µm
Mobile phase A: Water
B: Methanol
Flow rate 0.4 mL/min
Gradient 0–2 min/B 10% to 90%; 2–3 min/B 90%; 3.01/B 10%
Stop time 6 min
Column compartment temperature 45 °C
Injection 5 µL
MSD Condition
Instrument Agilent 6410A triple quadrupole LC/MS system
Source ESI –
Sample Preparation
All SPE cartridges are conditioned with 2 mL of water before use.

1. Honey, 1 g sample is diluted to 5 mL with water and 25 µL 10 ppb IS is added.

The solution is loaded onto the SPE cartridge and allowed to stand for 5 min.
Elution is performed with 10 mL ethyl acetate. The eluate is collected and the
solvent is evaporated under a nitrogen stream at 40 °C. The residue is redis-
solved in 1 mL methanol and put in an ultrasonic bath for 1 min. The solution is
filtered, using a syringe filter, before injection. No additional cleanup of the sam-
ple solution is performed.
2. Shrimp, 1 g of shrimp is defrosted and mixed in a blender. To the 1 g of the
mixed shrimp, 3 mL of water and 25 µL 10 ppb IS are added. The portion is cen-
trifuged for 5 min (8,000 rpm). The supernatant is loaded on the cartridge and
allowed to stand for 5 min. Elution is performed with 5 mL ethyl acetate. The
eluate is collected and the solvent evaporated under a nitrogen stream at 40 °C.
The residue is redissolved in 1 mL methanol and put in an ultrasonic bath for
1 min; the solution is filtered before injection.
3. Chicken, 1 g of chicken is defrosted and mixed in a blender. To the 1 g of the
mixed chicken, 3 mL of water and 25 µL 10 ppb IS are added. The portion is cen-
trifuged for 5 min (8,000 rpm). The supernatant is loaded on the cartridge and
allowed to stand for 5 min. Elution is performed with 5 mL ethyl acetate. The
eluate is collected and the solvent evaporated under a nitrogen stream at 40 °C.
The residue is redissolved in 1 mL methanol and put in an ultrasonic bath for
1 min; the solution is filtered before injection.

MRM Setting
Precursor Product Frag CE Dwell
Name ion ion (V) (V) (ms)

TAP 354.1 185.1* 120 20 60

354.1 289.9 120 10 60

FF 356 185.1* 120 20 60

356 335.8 120 5 60

CAP 321.2 152.1* 120 10 60

321.2 257.1 120 5 60

D5-CAP (ISTD) 326.2 157.2 130 15 60

Results
Name Linearity (0.5–20 ppb)

TAP 0.994
FF 0.992
CAP 0.994

2
Sensitivity Xiaorong Ran, Tao Bo are application
1. CAP: 0.5 ppb S/N = 81 chemist based at Agilent Technologies,
Beijing, China
- MRM (321.2000 & 152.0996) MIX500ppt-r001.d

×10 2
Noise (PeakToPeak) = 1.00; SNR (2.0 min) = 81.0
For More Information
8 1 2.0 1
For more information on our products
7 and services, visit our Web site at
www.agilent.com/chem.
6

1
2.9
0
0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6
Counts vs. acquisition time (min)

2. FF: 0.5 ppb S/N = 56

- MRM (356.0000 & 185.0996) MIX500ppt-r001.d

Noise (PeakToPeak) = 1.00; SNR (1.9 min) = 56.0
×10 1
5.5 1 1.9 1
5
4.5
4
3.5

3
2.5
2
1.5

1
0.5 2.9 3.8 4.1
0

0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6

Counts vs. acquisition time (min)

3
3. TAP 1 ppb, S/N = 12

- MRM (354.1000 & 185.0996) MIX1ppt-r005.d

Noise (PeakToPeak) = 1.00; SNR (1.543 min) = 12.4
×10 1
1 1.543 1
1.2

0.8

0.6

0.4

0.2

0
0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6
Counts vs. acquisition time (min)

www.agilent.com/chem
Agilent shall not be liable for errors contained herein or
for incidental or consequential damages in connection
with the furnishing, performance, or use of this material.

Information, descriptions, and specifications in this

publication are subject to change without notice.

© Agilent Technologies, Inc., 2009

Printed in the USA
June 15, 2009
5990-3734EN
 Detection, Monitoring, and Quantitation
of Trace Sulfonamides in Pork Muscle
Using the Agilent 6410A LC/MS/MS
Application Brief
Jian-qiu Mi

Sulfonamides are the one of the oldest groups of veterinary medicines in use today. Highlights
All sulfonamide drugs are currently included in Annex 1 of the Council Regulation
2377/90. The existing EU maximum residue level (MRL) for all drugs of the sulfon- • The pH of the mobile phase played
amide group is 100 µg/kg in all food-producing species. an important role in the LC separa-
tion because the retention behaviors
A variety of methods have been used to measure sulfonamide residue in biological of the drugs were dependent on the
materials, including thin-layer chromatography (TLC), high-performance liquid chro- ionization of the sulfonamides.
matography (HPLC), high-performance capillary electrophoresis (HPCE), gas chro-
matography (GC), and enzyme-linked immunosorbent assay (ELISA). Now, the • Different kinds of sulfonamide drugs
LC/MS/MS method is used more widely. can be analyzed within one run.
• Using the RRLC can get all 14 com-
In this study, a multiresidue analysis was performed to simultaneously determine
pounds to elute within 10 minutes.
sulfonamides in pork by the Agilent 6410A LC/MS/MS. This multiresidue analysis
for sulfonamides can detect different kinds of sulfonamides within one run. • High sensitivity easily meets the EU
Compared with the classic methods, this method can achieve greater sensitivity and requirements.
be used for screening, confirmation, and quantification.

Experimental
Sample Preparation
1. Weigh – 3-g samples of pork muscle were weighed directly into 50-mL
polypropylene centrifuge tubes.
2. Homogenize – The samples were homogenized for 3 minutes with 10 mL
acidified methanol.
3. Centrifuge – The samples were then centrifuged for 10 minutes.
3. Extract – 10 mL acidified methanol was extracted, filtered, and injected
Instrument Settings
LC Conditions
LC Agilent 1200 Series LC
Column Agilent ZORBAX SB-C18 (2.1 × 50 mm, 1.8 µm)
Mobile phase A: 0.1% TFA, B: Acetonitrile
0 min: 5% B
6 min: 23% B
9 min: 23% B
9.01 min: 90% B
Stop time 10 min
Column temperature 30 °C
Injection volume 1 µL
Flow rate 0.3 mL/min

MSD Conditions
Ionization ESI (positive)
Scan range m/z 100 to 450
Drying gas 7 L/min at 350 °C
Nebulizer gas 30 psi

MRM setting
Compound MRM Frag CE (V)
Sulfachloropyridazine (SCP) 285–156 100 15
285–108 20
Sulfadiazine (SD) 251–156 120 10
251–185 10
Sulfamethazine (SDM) 311–156 140 15
311–218 15
Sulfamethoxypyridazine (SMP) 281–156 120 10
281–215 15
Sulfamerazine (SM1) 265–156 120 15
265–172 15
Sulfamethazin (SM2) 279–156 140 15
279–204 15
Sulfalmethoxazole (SMZ) 254–156 120 15
254–147 20
Sulfamonomethoxine (SMM) 281–156 120 10
281–126 20
Sulfathiazole (ST) 256–156 120 15
256–107 15
Sulfaquinoxaline (SQX) 301–156 140 15
301–208 15
Sulfadoxine (SDM) 311–156 140 15
311–108 20
Sulfaphenazole (SPP) 315–156 140 20
315–160 20
Sulfaclozine 285–156 100 15
285–131 20
Sulfafurazole (SIZ) 268–156 120 5
268–113 10

2
Results Jian-qiu Mi is an application chemist
based at Agilent Technologies, Beijing,
Good separation and response China.

+ MRM (279.00000 & 186.00000) 080425_008.d

50 ppb level in the pork
For More Information
×10 3
1
8
7.5
For more information on our products
7 and services, visit our Web site at
6.5 www.agilent.com/chem.
6
5.5
5
4.5
4
3.5
3
2.5
2
1.5
1
0.5
0
0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5 10 10.5
Counts vs. acquisition time (min)

3
www.agilent.com/chem
Agilent shall not be liable for errors contained herein or
for incidental or consequential damages in connection
with the furnishing, performance, or use of this material.

Information, descriptions, and specifications in this

publication are subject to change without notice.

© Agilent Technologies, Inc., 2009

Printed in the USA
June 15, 2009
5990-3761EN
 Determination of Multi-Residue
Tetracyclines and their Metabolites
in Milk by High Performance Liquid
Chromatography - Tandem Mass
Spectrometry
Application Note
Food

Authors Abstract
Yanyan Fang, Hao Zhai, and Yun Zou Tetracyclines are probably the most frequently used antibiotics in animal husbandry.
Agilent Technologies (Shanghai), Co. In this paper, a high performance liquid chromatography tandem mass spectrometric
412 Yin Lun Road 200131 (HPLC / MS / MS) method is developed for the simultaneous determination of 10
China antibiotic residues: minocycline, 4-epioxytetracycline, 4-epitetracycline, tetracycline,
4-epichlortetracycline, demeclocycline, chlortetracycline, methacycline, doxycycline,
Jerry Zweigenbaum oxytetracycline in milk and animal tissues. In the method, Agilent’s novel solid phase
Agilent Technologies, Inc. extraction cartridge and a reversed phase Agilent ZORBAX RX C8 column (5 µm, 150
2850 Centerville Road mm × 2.1 mm) are used for purification and separation. The limit of detection (LOD) is
Wilmington, DE 19809 between 0.5 and 10.0 µg/ kg and the limit of quantitation (LOQ) is less than 50 µg/ kg.
USA The linearity is obtained from 5 to 1000 µg/ kg. Overall recoveries are between 76.4%
and 101% with a relative standard deviation (RSD, n = 6) less than 8.4%. The method
is rapid, sensitive, convenient and robust, and can be used to simultaneously confirm
multi-residues of tetracyclines and their metabolites in milk.
Introduction ance of 300 ppb for the sum of residues of tetracyclines, a tol-
erance of 300 ppb for each of the three tetracyclines is also
Antibiotics are used worldwide to control bacterial infection accepted.
and promote healthy farm animals for milk production.
Tetracyclines are broad-spectrum antibiotics, so they are In the EU, the maximum residue limit (MRL) for antibiotics is
widely used. However, it is undesirable to have them in the established according to (EEC) 2377 / 90, and for tetracyclines
milk supply. in milk is at 100 µg / kg (100 ppb). In China, the Government
Standard (GB / T 21317-2007) also establishes the method for
FDA's regulations for tetracyclines including oxytetracycline determination of these compounds in milk and animal tissues.
and chlortetracycline are set to provide an acceptable daily This regulation took effective April 1, 2008.
intake (ADI) and a tolerance for residues in milk. The ADI for
total residues of these compounds is 25 micrograms per kilo- The purpose of this study is to develop a method for the
gram of body weight per day. Sixty percent (60 %) of the ADI Agilent 6410 LC / MS / MS to determine the presence of tetra-
is reserved for milk and 40 % for edible tissues. Based on the cyclines and their metabolite residues in milk. The method is
ADI, a tolerance of 300 ppb is set for the sum of residues of rapid and easy to use. The tetracyclines and their metabolites
the tetracyclines including chlortetracycline, oxytetracycline, are given in Table 1.
and tetracycline in milk. With the establishment of a toler-

Table 1. The Compounds in this Study

No. Name CAS No. Structure

1 Minocycline 10118-90-8 H3C CH3 H3C CH3
N N
H H
OH

NH2
OH
OH O OH O O

2 Oxytetracycline 6153-64-6 CH3

OH NH3C
H3C H H
OH

C NH2
OH
OH O OH O O

3 Tetracycline 60-54-8
H3C CH3
H3C OH N
H
H OH

O
OH
OH O OH O NH2

4 Demeclocycline 127-33-3 H3 C CH3

Cl OH N
H H
OH

NH2
OH
OH O OH O O

(Continued)

2
Table 1. The Compounds in this Study

No. Name CAS No. Structure

5 chlortetracycline 57-62-5
H3C CH3
Cl HO CH3 N
H
H OH

NH2
OH
OH O OH O O

6 methacycline 914-00-1 H3 C CH3

CH2 OH N
H H
OH

NH2
OH
OH O OH O O

7 doxycycline 564-25-0 H3C CH3

H2O CH3 OH N
H H
OH

NH2
OH
OH O OH O O

8 H3 C CH3
8 4-epitetracycline 64-75-5 N
HO CH3 H
OH

NH2
OH
OH O OH O O

9 4-epi oxytetracycline 35259-39-3 H3 C CH3

N OH H3C OH
HO

O
OH
NH2 O OH O

10 4-epichlortetracycline 14297-93-9 OH O OH OH
O
OH
H2 N

O
N H3C OH C l
H3 C CH3

3
Experimental Sample Preparation
Extraction:
Reagents and Chemicals 1. Weigh a 5 g-milk sample (accurate to 0.01 g) into a 50-mL
Water and methanol are HPLC grade, and they, along with colorimetric tube, and dissolve with 0.1 mol / L Na2EDTA-
formic acid were all purchased from Fluka. The standards Mcllvaine buffer solution and bring volume to 50 mL.
were purchased from Sigma-Aldrich.
2. Vortex for 1 min and ultrasonicate the extract in an ice
Instrument Settings water bath for 10 min.
Table 2. LC / MS / MS Conditions
3. Transfer the sample to a 50-mL polypropylene centrifuge
HPLC tube and cool to 0 °C ~ 4 °C.
Column ZORBAX RX-C8, 2.1 mm × 150 mm, 5 µm
(p / n 883700-906) 4. Centrifuge the sample at a speed of 5000 rpm for 10 min
Flow rate 0.3 mL/ min (below 15 °C).
Mobile phase A: Water / 0.1 % Formic Acid
B: Methanol 5. Filter with fast filter paper.
Gradient 0–10 min, B from 5% to 30%
10–12 min, B from 30% to 40%
Purification:
12.5–18 min, B 65%
18.5–25 min, B 95% 1. Accurately draw 10 mL of the extract (equivalent to 1 g
25.5 min, B 5.0% sample) and put it through the SampliQ OPT cartridge
Total run 28 min (p / n 5982-3036) at a speed of 1 drop / s.
Post time 5 min
Temp 30 °C 2. After it elutes completely, clean the cartridge with 3 mL
injection 5 µL water adjusted to pH 4.5 with trifluoroacetic acid and then
MS Source settings discard the entire effluent.
Source ESI
Ion polarity Positive 3. Under a negative pressure below 2.0 kPa, drain the
Drying Gas temp. 350 °C cartridge for 5 min.
Drying gas flow rate 10 L/ min
Nebulizer 45 psi 4. Elute with 10 mL of 10 mmol oxalic acid in methanol.
Vcap 4000V
5. Collect the eluent and dry with nitrogen below 40 °C.
MRM Setting

Precursor Product Rt. 6. Dissolve the residue with 1.0 mL of the initial mobile
Name Frag. ion ion CE (min) phase.
Minocycline 120 458 352 35
441 20 8.58 7. Filter with a 0.45-µm filter membrane and inject.
4-Epitetracycline 120 445 410 20
427 10 8.60
4-Epioxytetracycline 120 461 426 20
Results and Discussion
444 15 9.47
Tetracycline 120 445 410 20
Optimization and Separation
427 15 9.90
Fragmentor and Collision Energy (CE) optimization
Oxytetracycline 120 461 426 20
443 10 9.95
It is well known that the LC / MS / MS QQQ is the best tool to
Demethylclocycline 120 465 430 25
448 15 11.25
identify, confirm and quantify target analytes in food matrices.
4-Epichlortetracycline 120 479 444 22 In order to get the best response, only two parameters need
462 15 11.59 to be optimized for each compound on this instrument, the
Chlortetracycline 120 479 444 22 fragmentor and the collision energy. The correct fragmentor
462 15 12.95 voltage allows the highest transmission of the precursor ion
Methacycline 120 443 381 25 into the mass analyzer. The correct collision energy provides
426 15 13.98 the highest intensity of quantitation of the qualifier product
Doxycycline 120 445 154 30
ion.
428 15 14.08

4
One method of optimization is to inject the sample multiple sor ion, and optimize the fragmentor voltage by stepping
times at the different fragmentor voltages set within seg- through the increments that the user selected for one injec-
ments of a single run. This is shown in Figure 1 for minocy- tion. The program then selects the voltage producing the
cline. For this compound, there is a small increase in detec- highest intensity for the precursor ion. For tetracycline, this is
tion as the voltage is increased. Collision energy is optimized shown in Figure 3.
in the same way and the results for tetracycline is shown in
Figure 2. The program then performs a product ion scan on a second
injection of the sample, and chooses the four most prevalent
Recently, Agilent introduced the "Optimizer" program that product ions. It reinjects the sample again and performs
automatically determines the optimum fragmentor voltage MRMs of each ion collected with collision energies in incre-
and collision energy and stores the results in the Optimizer ments covering the range of voltage selected by the user. The
Database. Using this program and flow injection with or with- collision energy that generates the maximum signal for each
out a column, the user enters the compounds to be optimized product ion is then automatically determined and can be
and their molecular formulas. The nominal mass of the com- stored in the database. The data from this collision energy
pound is automatically calculated from the formula. The user optimization for tetracycline is shown in Figure 4 along with
then specifies the adducts expected for positive and negative the ion breakdown curve shown in Figure 5. Compounds with
modes, the low mass cutoff, any ions to be excluded, and the product ions can be imported directly into the users’ acquisi-
method to be used (mobile phase conditions etc.). Once start- tion method.
ed the program will inject the sample, determine the precur-

×10 8 + TIC Scan erjiaan_Frag_01.d

2 1 1 2 2 3 3 4 4 5 5 6 6
1.9
1.8
1.7
1.6
1.5
1.4
1.3
1.2
1.1
1
0.9
0.8
0.7
0.6
0.5
0.4

0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 2.2 2.4 2.6 2.8 3 3.2 3.4 3.6 3.8 4 4.2 4.4 4.6 4.8 5 5.2
Counts vs. acquisition time (min)

Figure 1. Optimization of fragmentor voltage for minocycline from 60-160 by steps of 20 V.

5
 ×10 5 + TIC MRM (** & **) sihuansuCE_01.d
1 1 2 2 3 3 4 4 5 5 6 6 7 7 8 8
7
6.6
6.2
5.8
5.4
5
4.6
4.2
3.8
3.4
3
2.6
2.2
1.8
1.4
1
0.6
0.2

0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 2.2 2.4 2.6 2.8 3 3.2 3.4 3.6 3.8 4 4.2 4.4 4.6 4.8 5 5.2 5.4
Counts vs. acquisition time (min)

Figure 2. Manual collision energy optimization of tetracycline.

+ESI EIC:3 (444.7-445.4) SIM Frag = 90.0V tetracycline_OPTIMIZE_MS2SIM.d

+ESI EIC:1 (444.9-445.4) SIM Frag = 50.0V tetracycline_OPTIMIZE_MS2SIM.d
+ESI EIC:2 (444.7-445.5) SIM Frag = 70.0V tetracycline_OPTIMIZE_MS2SIM.d
+ESI EIC:4 (444.7-445.5) SIM Frag = 110.0V tetracycline_OPTIMIZE_MS2SIM.d
+ESI EIC:5 (444.6-445.7) SIM Frag = 130.0V tetracycline_OPTIMIZE_MS2SIM.d
+ESI EIC:6 (444.1-445.8) SIM Frag =150.0V tetracycline_OPTIMIZE_MS2SIM.d
×10 5

1.8

1.6

1.4

1.2

0.8

0.6

0.4

0.2

0.2 0.22 0.24 0.26 0.28 0.3 0.32 0.34 0.36 0.38 0.4 0.42 0.44 0.46 0.48 0.5 0.52 0.54 0.56 0.58 0.6 0.62
Counts vs. acquisition time (min)

Figure 3. Single injection automatic determination of fragmentor voltage for tetracycline using the Optimizer program.

6
 +ESI MRM Frag=82.0V [email protected] (445.2 & 154.1) tetracycline_OPTIMIZE_MRM.d
×10 4
1.5

1.4

1.3

1.2

1.1

0.9

0.8

0.7

0.6

0.5

0.4

0.3

0.2

0.1

0.2 0.22 0.24 0.26 0.28 0.3 0.32 0.34 0.36 0.38 0.4 0.42 0.44 0.46 0.48 0.5 0.52 0.54 0.56 0.58 0.6 0.62
Counts vs. acquisition time (min)

Figure 4. Single injection automated collision energy determination using Optimizer program for tetracycline.

Figure 5. Ion breakdown profile for tetracycline as determined by the

Optimizer program.

7
Separation
Sample preparation and separation of tetracycline, chlortetra-
cyline and oxytetracycline is important. The challenge in sep-
arating these kinds of compounds is that they easily degrade
under conditions of weak acid, strong acid, strong base, and
heat converting the diasteriomer to its diaxial epimer.

The typical process is shown below with tetracycline:

Figure 6. The degradation of tetracycline to 4-epitetracycline.

8
Tetracyclines and their degradants are diasteriomers with the
same formula and the same fragment ions are formed in
MS / MS. Therefore, they have the same precursor ions, quali-
tative ions, and quantitation ions. In order to identify and con-
firm them in the Rapid Resolution liquid chromatograph
(RRLC), separation is important for this analysis. Using the
Agilent ZORBAX Rx-C8, 2.1 mm × 150 mm, 5-µm particle size
column and a simple gradient, the three epimer pairs of these
compounds are well separated. This is shown with the reten-
tion times given in Table 2. Figure 7 shows the graphic repre-
sentation of the separation of tetracycline and its epimer.

×10 5
+ TIC MRM (** & **) chaxiangsihuansu_01.d
2.4 1 1
2.2
2
1.8
1.6
1.4 a. 4-epitetracycline
1.2
1
0.8
0.6
0.4
0.2

+ TIC MRM (** & **) sihuansu_01.d

×10 5
1 1
5
4.5
4
3.5
3
b. Tetracycline
2.5
2
1.5
1
0.5

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27
Counts vs. acquisition time (min)

Figure 7. The separation of tetracycline and its degradation product 4-epitetracycline.

9
Linearity, LOD and LOQ detection (LOD) for each are still in the low pictograms on-
column. The limit of quantitation is typically set at a signal to
Linearity, LOD and LOQ were evaluated in both solvent and a
noise (S / N) of 10:1 but we report twice that in the solvent.
milk matrix. The results are given in Table 3 and show that lin-
The graphic representation of the calibration curve for
earity is similar for both solvent and milk matrix and generally
minocycline is shown in Figure 8.
provide greater than 0.99 coefficient of variance. The tetracy-
clines do not ionize well with electrospray but the limits of

Table 3. Quantitative Performance of Tetracyclines in Solvent and Milk Matrix

Standards in solvent* Standards in Milk matrix*
Name R2 LOQ LOD R2 LOD
(S / N=20) (S / N=3) (S / N=3)
pg on column pg on column pg on column
Minocycline 0.999 41.5 6.2 0.990 16.3
4-epitetracycline 0.991 10.8 1.6 0.994 8.7
4-epioxytetracycline 0.996 14.7 2.2 0.996 12.8
Tetracycline 0.998 9.4 1.4 0.994 10.2
Oxytetracycline 0.996 10.7 1.6 0.991 8.6
Demethylclocycline 0.999 22.8 3.4 0.993 8.1
4-epichlortetracycline 0.986 38.2 5.7 0.987 11.9
Chlortetracycline 0.986 8.1 1.2 0.994 7.6
Methacycline 0.999 20.8 3.1 0.994 12.3
Doxycycline 0.999 32.2 4.8 0.995 11.2
Note: *The calibration curve range is from 1 ppb-1 ppm with injection volume of 5 uL

×10 5

1.5 jiaxitumeisu - 7 Levels, 7 Levels Used, 21 Points, 21 Points Used, 0 QCs

1.4 y = 143.4386 * × - 442.6564
R 2 = 0.99960865
1.3
1.2
1.1
1
0.9
0.8
Responses

0.7

0.6
0.5
0.4
0.3
0.2
0.1
0
-0.1

-50 0 50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050
Concentration (µg/L)

Figure 8. Tetracycline calibration curve from 1 ppb to 1000 ppb.

10
Recovery and Repeatability centration. Ion ratios for confirmation are a very important
The recovery and repeatability of the method was evaluated performance criterion and these results show excellent
and the results shown in Table 4. All recoveries were greater repeatability. A graphic representation of the ion ratios for
than 80 % which is generally accepted as sufficient. In addi- methacycline is shown in Figure 9. The ratios combined with
tion the precision, as shown in the table, is 5 % or better for matching retention time provide the necessary information for
the lower concentration and less than 2 % for the higher con- confirmation.

Table 4. Recovery and Repeatability in Milk Matrix

Recovery in milk RSD % RSD % Recovery in milk RSD % RSD %

(Conc. 50 ppb (Signal response (Ion ratio (Conc. 100 ppb (Signal response (Ion ratio
Name n=6) n=6) n=6) n=6) n=6) n=6)
Minocycline 96.5 4.9 2.1 101.4 1.6 1.0
4-epitetracycline 89.2 3.8 1.5 96.3 1.6 0.9
4-epioxytetracycline 84.4 5.4 1.3 88.2 0.9 0.6
Tetracycline 86.1 2.5 1.2 90.7 1.1 1.2
Oxytetracycline 77.6 3.8 1.6 82.5 1.2 0.9
Demethylclocycline 79.2 2.0 3.1 84.7 0.9 0.6
4-epichlortetracycline 76.4 5.5 5.4 84.3 1.1 0.5
Chlortetracycline 94.3 4.5 1.5 100.9 1.8 1.1
Methacycline 86.3 1.0 1.9 91.2 1.2 0.8
Doxycycline 78.7 3.6 6.7 82.4 1.0 0.8

+ MRM (443.0 & 426.0) std11-r002.d 443.0 -> 426.0, 443.0 & 381.0 + MRM:10 (7.846-9.272 min, 22 scans) (445.0 & ...
×10 4 ×10 2 Ratio = 12.7
14.021 ×10 2
1 1.25 410.0
0.95 1.2 5.75
0.9 1.15 5.5
1.1 5.25
0.85 1.05
0.8 5
1
0.95 4.75
0.75
0.9 4.5
0.7
0.85 4.25
0.65 0.8 4
Relatieve abundance (%)

0.6 0.75 3.75

0.55 0.7 3.5
0.5 0.65
3.25
Counts

0.6
Counts

0.45 0.55 3
0.4 0.5 2.75
0.35 0.45 2.5
0.3 0.4 2.25
0.35 2
0.25 0.3 1.75
0.2 0.25
1.5
0.15 0.2
0.15 1.25
0.1
0.1 1
0.05 0.05 0.75
0 0 0.5 154.0
_0.05 _0.05
_0.1 0.25 445.0
_0.1 0
12 13 14 15 16 12 13 14 15 16 150 200 250 300 350 400 450
Acquisition time (min) Acquisition time (min) Mass-to-charge (m/z)

Figure 9. Shows the ion ratios for qualifier ion and the quantitation ion of methacycline.

11
Study of Ion Suppression Because of the strong suppression observed, using the exter-
In general, tandem MS can remove chemical noise to get a nal standard method (ESTD) for calibration, matrix matched
"clean" spectrum even in dirty and complex food matrices. standards should be prepared in antibiotic-free milk, or milk
However, the matrix may contain components that suppress known to not contain the analytes. In this way, the calibration
the ionization of the analyte. Figure 10 shows the comparison curve is generated with the same matrix effects as the sam-
of the response of methacycline and tetracycline in solvent ples.
and milk. The difference in the slope of each curve demon-
strates the suppression effect of the milk matrix.

1600000
160000
1
Methacycline 1 140000 Tetracycline
1200000
120000

100000

800000 80000

60000

400000 40000 2

20000
2

0 0
0 500 1000 1500 0 200 400 600

Figure 10. Ion suppression of two of the tetracyclines in milk; 1) response in solvent, 2) response in milk.

12
Conclusions
The results of this work show that the Agilent 6410 triple
quadrupole LC / MS System is a robust, sensitive, and repeat-
able instrument for the study of tetracyclines residues in a
milk matrix. In China, the government standard requirement
(GB / T 21317-2007) sets the detection limit at 50 ppb with a
100 µL injection. This method easily meets these require-
ments. Additionally, these types of antibiotics readily degrade
under the conditions of weak acid, base etc. The preparation
method used here avoids this reaction, allowing the LC
method to separate these isomers for reliable confirmation
and quantitation. Finally, ion suppression is considered for the
LC / MS / MS method when comparing different compounds in
the same matrix to their response in solvent. Using the ESTD
method, the preparation of a matrix-matched calibration curve
is necessary to obtain accurate results, even though the
recoveries measured for the sample preparation are better
than 80%.

For More Information

For more information on our products and services, visit our
Web site at www.agilent.com/ chem.

13
www.agilent.com/ chem
Agilent shall not be liable for errors contained herein or
for incidental or consequential damages in connection
with the furnishing, performance, or use of this material.

Information, descriptions, and specifications in this

publication are subject to change without notice.

© Agilent Technologies, Inc., 2009

Printed in the USA
April 9, 2009
5990-3816EN
 Determination of Hormones in Fish
(Carassius Carassius) by SampliQ-
OPT Solid Phase Extraction with High
Performance Liquid Chromatography

Application Note
Food Safety

Authors Abstract
Chen-Hao Zhai and Yun Zou Solid-phase extraction (SPE) coupled with high performance liquid chromatography
Agilent Technologies Co. Ltd. (HPLC) was optimized for the extraction and determination of sixteen hormones (estri-
412 Ying Lun Road ol, prednisolone, hydrocortisone, prednisone, methylprednisolone, betamethasone,
Waigaoqiao Free Trade Zone dexamethasone, triamcinolone acetate, gestrinone, prednisolone acetate, hydrocorti-
Shanghai 200131 sone acetate, prednisone acetate, estradiol, cortisone acetate, methyltestosterone,
China estrone) in crucian carp (Carassius carassius) meat. Results indicate that SPE using
an Agilent SampliQ OPT (60 mg, 3 mL) and HPLC using an Agilent ZORBAX Eclipse
Rou-Nan Jin Plus C18 column (4.6 mm × 250 mm, 5 µm) is suitable for extraction of these com-
Second Military Medical University pounds. Recoveries ranged from 76.2 to 106.1 % with relative standard deviations
259 Changhai Road (RSDs) between 1.7 and 8.9 %.
Shanghai 200093
China
Introduction androgen, and progesterone are carcinogenic and can lead to
breast cancer, ovarian cancer and cell carcinoma. Many coun-
Food safety has increasingly become an important concern of tries' regulations clearly define residual limits for these com-
people worldwide. Many chemicals added to food create pounds in food.
potential hazards to human health. Hormones are a common
food additive. Long-term consumption of glucocorticoid can An Agilent SampliQ OPT SPE cartridge was used to extract
lead to hyperglycemia, osteoporosis, birth defects, and 16 kinds of hormones (Table 1) from crucian meat and an
immune function decline. Other hormones such as estrogen, HPLC method was established to detect these 16 compounds.

Table 1. Hormones Used in this Study

No. Name CAS No. Log P Structure

1 Estriol 50-27-1 2.45 OH

H OH

H H
HO

2 Prednisolone 50-24-8 1.66 O

HO OH
HO
H

H H

O
3 Hydrocortisone 50-23-7 1.79 O OH

HO OH
H

H H

4 Prednisone 53-03-2 2.07 O

HO OH
O
H

H H

O
5 Methylprednisolone 83-43-2 2.06 O OH

HO OH
H

H H

(Continued)

2
Table 1. Hormones Used in this Study

No. Name CAS No. Log P Structure

6 Betamethasone 378-44-9 1.93 O

HO
OH OH
H

F H
O
7 Dexamethasone 50-02-2 1.93 O OH

HO OH
H

F H
O
8 Triamcinolone acetate 67-78-7 1.9 O

O O

HO OH
H O
O
F H
O

9 Gestrinone 16320-04-0 NA
HO

10 Prednisolone acetate 52-21-1 NA O

O
HO OH
H

H H

O
11 Methylprednisolone 83-43-2 NA O

O
O
HO OH
H

H H
O

(Continued)

3
Table 1. Hormones Used in this Study

No. Name CAS No. Log P Structure

12 Prednisone acetate 125-10-0 NA

O O
O
O OH
H

H H
O

13 Estradiol 50-28-2 3.57

OH

H H

HO

14 Cortisone acetate 50-04-4 2.35 O

O
CH3
O OH O
CH3
CH3 H

H H

O
15 Methylestosterone 58-18-4 NA
HO

H H
O

16 Estrone 53-16-7 4.03

O

H H

HO

4
Experimental SPE Purification
The procedure used for the SPE extraction is shown in
Reagents and Chemicals Figure 1. Agilent SampliQ OPT cartridges are preconditioned
All reagents and solvents were HPLC or analytical grade. with 3 mL of methanol then 5 mL of water. The 5-mL extract
Hormone standards were purchased from NICPBP (National (equivalent to 0.6 g sample) is passed through the SampliQ
Institute for the Control of Pharmaceutical and Biological OPT cartridge at a speed of 1 mL/min. After it effuses com-
Products). Crucian was purchased from a local market. pletely, the cartridge is washed with 5 mL of 30% methanol in
water and the entire effluent is discarded. The cartridge is
Stock solutions (1 mg/mL) were prepared in methanol and dried under negative pressure (below 2.0 kPa) for 3 minutes.
kept in the freezer (–20 °C). Working solutions were prepared The sample is then eluted with 6 mL of methanol, and the elu-
using the stock solution diluted with methanol. The working ent is collected and dried under nitrogen below 40 °C. The
solutions should be prepared every week and need to be residue is dissolved and brought to a constant volume of 1.0
stored below 4 °C. mL using methanol, filtered through a 0.45 µm PTFE filter
membrane, and analyzed by HPLC.
The SPE cartridges were Agilent SampliQ OPT (3 mL, 60 mg,
p/n 5982-3036). The analysis was performed on an Agilent
Condition: 3 mL methanol
1200 Series HPLC with a diode array detector (DAD). The ana-
lytical column was an Agilent ZORBAX Eclipse Plus C18
(5 µm 250 mm × 4.6 mm id, p/n 959990-902). An Agilent Equilibrate 5 mL water
0.45-µm PTFE Premium Syringe Filter (p/n 5185-5836) was
used to filter the sample solution before HPLC.
Load 5 mL extract (equivalent to 0.6 g meat)
HPLC conditions
Column: ZORBAX Eclipse Plus C18 250 mm × 4.6 mm, 5 µm
Flow rate: 1.0 mL/min Wash 5 mL 30 % methanol in water
Injection volume: 5 µL Dry 3 minutes
under vacuum
Column temperature: 18 °C
Detection wavelength: 230 nm Elute with 6 mL methanol

Mobile phase: Water-Acetonitrile Gradient

Time (minutes) % Water % Acetonitrile
Dry, reconstitute in methanol
0 70 30
10 65 35
23 50 50 Filter
30 20 80

Figure 1. Hormones in crucian meat SPE procedure.

Separation
1. Weigh 200 grams of crucian meat, homogenize, and store Results and Discussion
in a clean, sealed container at –18 °C.
2. Place 1 g of homogeneous sample (accurate to 0.01 g) into Linearity, Limits of Detection
a 10-mL polypropylene centrifuge tube with 5 mL of
Stock solutions were diluted to different concentrations and
methanol.
analyzed by HPLC. Linear regressions were calculated for the
3. Vortex for 1 minute.
hormones based on the areas and the solution concentra-
4. Extract ultrasonically for 10 minutes in an ice bath.
tions. Limit of detection (LOD) signifies the injection concen-
5. Centrifuge the sample at a speed of 4000 r/min for 5 min-
tration at which the signal to noise ratio was between 2 and
utes and remove the 3 mL of supernatant.
3. Linear range was between 1–100 mg/kg. The linearity and
6. Save in a clean tube and evaporate with N2 below 40 °C.
LOD are shown in Table 2.
7. Reconstitute the residue in 5 mL of 5 % methanol in water.

5
Table 2. Linearity and LODs of Hormones.

Correlation LOD
No. Compound Regression equation coefficient (mg/kg)
1 Estriol Y = 8.096 × –0.824 0.9998 0.5
2 Prednisolone Y = 17.418 × –2.088 0.9999 0.2
3 Hydrocortisone Y = 15.746 × –1.518 0.9999 0.3
4 Prednisone Y = 20.192 × –2.152 0.9998 0.2
5 Methylprednisolone Y = 16.986 × –1.894 0.9999 0.4
6 Betamethasone Y = 20.439 × –1.106 0.9997 0.2
7 Dexamethasone Y = 20.176 × –2.176 0.9999 0.2
8 Triamcinolone acetate Y = 16.374 × –1.558 0.9997 0.4
9 Gestrinone Y = 6.370 × –0.668 0.9998 1.0
10 Prednisolone acetate Y = 15.589 × –1.627 0.9999 0.4
11 Hydrocortisone acetate Y = 15.051 × –1.584 0.9999 0.4
12 Prednisone acetate Y = 24.106 × –2.401 0.9997 0.2
13 Estradiol Y = 8.709 × –0.635 0.9999 0.8
14 Cortisone acetate Y = 19.826 × –2.336 0.9996 0.4
15 Methyltestosterone Y = 19.980 × –2.209 0.9996 0.3
16 Estrone Y = 10.701 × –0.847 0.9999 0.4

Recovery and Repeatability

The precision of the method was determined in terms of the
recovery of spiked hormone standards in crucian meat at
2, 5, and 10 mg/kg levels. The analysis was repeated six
times at each level. The chromatograms of the blank, the
standards, and the spiked standard (2 mg/kg) sample are
shown in Figures 2 through 4. The recovery and reproducibili-
ty data are shown in Table 3.

Figure 2. Chromatogram of crucian meat blank.

6
1 Estriol 5 Methylprednisolone 9 Gestrinone 13 Estradiol
2 Prednisolone 6 Betamethasone 10 Prednisolone acetate 14 Cortisone acetate
3 Hydrocortisone 7 Dexamethasone 11 Hydrocortisone acetate 15 Methyltestosterone
4 Prednisone 8 Triamicinolone acetate 12 Prednisone acetate 16 Estrone

Figure 3. Chromatogram of hormone standards at 2 mg/kg.

1 Estriol 5 Methylprednisolone 9 Gestrinone 13 Estradiol

2 Prednisolone 6 Betamethasone 10 Prednisolone acetate 14 Cortisone acetate
3 Hydrocortisone 7 Dexamethasone 11 Hydrocortisone acetate 15 Methyltestosterone
4 Prednisone 8 Triamicinolone acetate 12 Prednisone acetate 16 Estrone

Figure 4. Chromatogram of crucian meat sample spiked hormone standards at 2 mg/kg.

7
Table 3. Recoveries and RSDs of Hormones in Crucian Meat by SPE

Compund Spiked level (mg/kg) Recovery (%) RSD (n = 6, %)

Estriol 2 100.4 2.2
5 106.1 1.9
10 102.4 4.4
Prednisolone 2 89.4 3.8
5 90.9 7.6
10 100.7 2.9
Hydrocortisone 2 85.3 6.7
5 91.4 7.6
10 101.4 3.4
Prednisone 2 82.5 7.2
5 92.1 5.2
10 100.7 2.9
Methylprednisolone 2 83.2 8.3
5 93.6 3.2
10 97.4 1.7
Betamethasone 2 88.3 8.9
5 99.6 4.9
10 100.8 3.8
Dexamethasone 2 79.1 4.3
5 98.4 5.3
10 98.4 3.9
Triamcinolone acetate 2 86.7 8.4
5 97.6 5.9
10 97.9 4.1
Gestrinone 2 78.0 6.6
5 78.8 8.1
10 85.3 8.0
Prednisolone acetate 2 86.9 7.3
5 101.2 4.3
10 101.9 5.7
Hydrocortisone acetate 2 87.3 6.8
5 102.7 5.1
10 101.5 7.9
Prednisone acetate 2 76.7 7.7
5 94.1 3.5
10 97.7 4.3
Estradiol 2 78.7 4.2
5 94.7 3.5
10 97.4 4.8
Cortisone acetate 2 82.8 6.9
5 87.8 6.5
10 94.4 4.1
Methyltestosterone 2 82.9 3.4
5 91.9 4.9
10 93.6 4.6
Estrone 2 76.2 6.4
5 90.0 8.7
10 93.9 5.9

8
Conclusions
Agilent's SampliQ OPT, a polymeric sorbent with combined
hydrophilic and lipophilic characteristics that allows retention
of both polar and non-polar compounds, provides a simplified
and effective single cartridge method for the purification and
enrichment of multiple hormone compounds in crucian carp.
Recovery and reproducibility (routinely below 10%) based on
solution standards are acceptable for hormone residue deter-
mination in crucian meat. Impurities from crucian were mini-
mal and did not interfere with any of the hormones analyzed.

Product Information
Part number Description
5982-3013 OPT Polymer - Box, 100x 1 mL tubes, 30 mg
5982-3036 OPT Polymer - Box, 50x 3 mL tubes, 60 mg
5982-3067 OPT Polymer - Box, 30x 6 mL tubes, 150 mg
5982-3096 OPT Polymer - 96 Well Plate, 10 mg
95990-902 Agilent ZORBAX Eclipse Plus C18 250 mm × 4.6 mm, 5 µm
5185-5836 Agilent PTFE 0.45 µm Premium Syringe Filter

For More Information

For more information on our products and services, visit our
Web site at www.agilent.com/chem.

9
www.agilent.com/chem
Agilent shall not be liable for errors contained herein or
for incidental or consequential damages in connection
with the furnishing, performance, or use of this material.

Information, descriptions, and specifications in this

publication are subject to change without notice.

© Agilent Technologies, Inc., 2009

Printed in the USA
April 1, 2009
5990-3845EN
 Determination of β2-Agonists in Pork
Using Agilent SampliQ SCX Solid-Phase
Extraction Cartridges and Liquid
Chromatography-Tandem Mass
Spectrometry
Application Note
Food Safety

Authors Abstract
Chenhao Zhai A method for simultaneous determination of four β2-agonist residues of terbutaline,
Agilent Technologies Co., Ltd. salbutamol, clenbuterol and formoterol in pork has been developed and validated. The
412 Ying Lun Road analytes are purified by liquid-liquid extraction (LLE) and solid-phase extraction (SPE)
Waigaoqiao Free Trade Zone and quantified by liquid chromatography coupled to electrospray ionization tandem
Shanghai 200131 mass spectrometry (LC-ESI-MS/MS) operating in positive ion multiple reaction moni-
China toring (MRM) mode. The method provides a sub-ng/g level of limit of detection (LOD)
for all four β2-agonists in pork. The dynamic calibration ranges for these compounds
Jianzhong Li and Yue Song are obtained from 0.25 to 5 ng/g. The overall recoveries range from 78 to 101% with
Agilent Technologies Co., Ltd. RSD values between 1.8 and 7.2%.
11/F Cross Tower
318 Fu Zhou Road
Shanghai 200001
China
Introduction Equipment and Materials
Agilent 1200 HPLC system
The β2-agonists have been used worldwide as illegal growth
promoters in pork production. Recent incidences of poisoning Agilent 6460 Triple Quadrupole LC-MS/MS system
have occurred due to high levels of the β-agonist (clen-
buterol) in pork. This application note used Agilent's new SPE Agilent SamliQ SCX Polymer cartridges, 50 × 3 mL tubes,
products to extract and enrich four β-agonists from pork and 60 mg (p/n: 5982-3236)
analysis by LC-MS/MS. Table 1 shows the name and struc-
Agilent ZORBAX Eclipse Plus C18, 50 × 2.1 mm, 1.8 µm
ture of the four β-agonist compounds.
(p/n: 959741-906)
Table 1. β2-Agonist Compounds Used in this Study Agilent Vaccum Manifold processing station (p/n: 5982-9120)
Compound Log P Structure
OH
Sample Preparation
Terbutaline 0.55 H
HO N Liquid-Liquid Extraction

A 2 g amount of pork (±0.01 g) was weighed into a 15 mL

OH capped polypropylene tube. To the pork, 8 mL of 0.2 M sodium
Salbutamol 0.44 OH acetate (pH 5.2) solution were added and mixed in a vortex.
H
HO
N Next, 100 µL β-glucuronidase (1000 U/mL) were added and
the tube vortexed thoroughly for 2 minutes. The sample was
HO
OH
hydrolyzed at 37 °C for 16 hours.
Clenbuterol 2.94 OH
H
The hydrolysate was shaken for 15 minutes and centrifuged
Cl N
at 4000 rpm for 10 minutes. A 4 mL amount of supernatant
H2N
was transferred to another centrifuge tube. A 5 mL amount of
Cl 0.1 M perchloric acid solution was added and the pH was
adjusted to 1 ± 0.3. The tubes were then centrifuged at
Formoterol 1.91 OH
H 4000 rpm for 10 minutes. The supernatant was transferred to
N O
another tube, and the pH was adjusted to 11 with 10 M sodi-
NH
OH um hydroxide.
O
Ten milliliters each of a saturated sodium chloride solution
isopropanol-ethyl acetate (60:40) were added to the tubes.
The tubes were shaken for 5 minutes. The tubes were cen-
Experimental trifuged at 4000 rpm for 5 minutes before the organic layer
was carefully transferred to another tube. Isopropanol-ethyl
Reagents and Chemicals acetate addition, shaking, centrifuging and organic layer
All reagents were MS, HPLC or analytical grade. transfer were repeated twice, and all supernants were com-
bined.
Acetonitrile and water were from Scharlau. Ethyl acetate and
isopropanol were from Fisher. The standards were purchased Samples were evaporated to dryness with nitrogen at 40 °C.
from National Institute for the Control of Pharmaceutical and The residue was dissolved in 5 mL of 0.2 M sodium acetate
Biological Products (NICPBP). Pork was purchased from a (pH 5.2). The sample was then ready for SPE purification.
local market.
Solid-Phase Extraction
Standard solutions (1.0 mg/mL) were made in methanol indi- The SPE procedure is shown in Figure 1. Agilent SampliQ SCX
vidually, and refrigerated at 4 °C. A combined working solu- cartridges were preconditioned with 3 mL of methanol and
tion (10 µg/mL) was made in methanol-water (10:90) and also then equilibrated with 3 mL water. Five milliliters of the sam-
stored at 4 °C. The spiked solutions were then made weekly ple solution were then loaded onto a cartridge and passed
by appropriately diluting the combined working solution in through the cartridge by gravity (about 1 mL/min). The tubes
water. were rinsed with 2 mL of water and 2 mL 2% formic acid in
water. The entire effluent was discarded. Full vacuum was

2
applied to the cartridge for 3 minutes to completely dry the
resin. Finally, the compounds were eluted with 5 mL of 5%
ammonia solution in methanol at a rate of 1 mL/min. The elu-
ent was dried with nitrogen flow at 40 °C. The residue was
reconstituted in 1 mL of 0.1% formic acid in water/acetoni-
trile (90:10). The sample was vortexed and ultrasonicated to
completely dissolve the residue. The sample was transferred
to a 1.5 mL tube and centrifuged at 3000 rpm for 5 minutes.
The sample was transferred to a 2 mL chromatography vial for
analysis.
Condition: 3 mL methanol

Equilibrate: 3 mL water

Load: 5 mL pork extract Figure 2. MS source parameters for these four compounds.

Wash A: 2 mL water Table 2. Masses Monitored in the MRM

Compound MRM for quantification MRM for confirmation
Terbutaline 226.1 & 152.1 226.1 & 125
Wash B: 2 mL 2% formic acid in water
Salbutamol 240.1 & 148.1 240.1 & 222.1
Clenbuterol 227 & 203 227 & 259.1
Dry cartridge by vacuum for 3 min Formoterol 345.1 & 149.1 345.1 & 327.1

Elute: 5 mL 5% ammonia solution in methanol Results and Discussion

Linearity and Limit of Detection
Evaporate to dryness at 40 °C under nitrogen flow
Solutions used to create external calibration curves were pre-
pared by using a combined working solution to spike matrix
Reconstitute to 1 mL of 0.1% formic acid in water/acetonitrile (90:10) blanks (0.25, 0.5, 1.0, 2.0 and 5.0 ng/g). Matrix blanks were
created by taking pork through the hydrolysis, LLE and SPE
Figure 1. Pork clean up and enrichment – SPE procedure. procedures. The results for the calibration curves are shown
in Table 3. The limits of detection (LOD) were chosen as the
Instrument Conditions concentration of each compound that gave a signal to noise
HPLC Conditions (S/N) ratio greater than 3:1. The LODs are also shown in
Column: Agilent ZORBAX Eclipse Plus C18 2.1 mm × 50 mm Table 3.
1.8 µm (p/n: 959741-906)
Flow rate: 0.4 mL/min
Table 3. Linearity and LODs of β2-Agonists
Column temperature: 40 °C
Injection volume: 5 µL Compound Regression equation R2 LOD in pork (ng/g)
Mobile phase: Water (0.1% FA+2 mM NH4Ac, A), Terbutaline Y = 3470x + 1325.4 0.9972 0.05
Acetonitrile (0.1% FA, B) Salbutamol Y = 13099x + 2900.3 0.9921 0.05
Gradient: Time (min) %A %B Clenbuterol Y = 27028x + 1143.7 1 0.02
0 90 10
0.5 90 10 Formoterol Y = 23251x + 487.44 0.9983 0.02
1.8 20 80
2 90 10
3.5 90 10
MS Conditions
These four compounds were monitored in the positive mode. The source con-
ditions are shown in Figure 2 and the MRM channels are shown in Table 2.

3
Recovery and Reproducibility Table 4. Recoveries and Reproducibility of β2-agonists in Pork After SPE
Employing Agilent's SampliQ SCX; (p/n: 5982-3236), Recovery
The recovery and reproducibility of the method were deter- 90% and RSD 4.4% on Average
mined at three levels: pork spiked to a concentration of 0.5, Compound Spiked level Recovery RSD
1.0, and 2.0 ng/g. The analysis was performed with six repli- (ng/g pork) (%) (n=6)
cates at each level. The recovery and reproducibility data is Terbutaline 0.5 88.7 5.4
shown in Table 4. The chromatograms of spiked pork extracts 1 98.0 7.2
(1.0 ng/g) are shown in Figure 3. 2 100.8 5.9
Salbutamol 0.5 100.6 1.8
1 92.9 2.1
2 97.4 3.9
Clenbuterol 0.5 82.3 5.0
1 91.5 6.3
2 90.6 4.3
Formoterol 0.5 85.1 1.9
1 83.0 4.0
2 77.9 2.5

Chromatograms for a 1.0 ng/mL spiked pork sample after SPE Clean-up on Agilent's SampliQ SCX (p/n: 5982-3236)

Figure 3. Chromatograms of 1.0 ng/g spiked pork sample extract.

4
Conclusions
The result of this study show that Agilent SampliQ SCX can
be used as an effective method for purification and enrich-
ment of multiple β2-agonists in a complex matrix such as
pork. The recovery and reproducibility results based on matrix
spiked standards are acceptable for β2-agonists residue
determination in pork under Chinese regulations. The impuri-
ties and matrix effects are minimal and do not interfere with
the quantification of any target compound. The LOQ are
significantly lower than the MRLs [1,2].

References
1. GB/T 21313-2007 "Analysis of β2-agonists in Foods of
Animal Origin by High Performance Liquid
Chromatography Tandem Mass Spectrometry"
2. SN/T 1924-2007 "Determination of Clenbuterol,
Ractopamine, Salbutamol and Terbutalin Residues in
Foodstuffs of Animal Origin for Import and Export -HPLC-
MS/MS Method."

For More Information

For more information on our products and services, visit our
Web site at www.agilent.com/chem.

5
www.agilent.com/chem
Agilent shall not be liable for errors contained herein or
for incidental or consequential damages in connection
with the furnishing, performance, or use of this material.

Information, descriptions, and specifications in this

publication are subject to change without notice.

© Agilent Technologies, Inc., 2009

Printed in the USA
October 2, 2009
5990-4180EN
 Profiling medications for decorative or
aquarium fish using the Agilent 1290
Infinity LC system and Agilent ZORBAX
Poroshell 120 2.7 µm columns

Application Note

Consumer Products

Author Abstract
Michael Woodman The Agilent 1290 Infinity LC system has significant capabilities for a wide range of
HPLC and UHPLC applications. It exhibits a broader power range (for example, the
Agilent Technologies, Inc.
combination of pressure and flow capabilities), and the flexibility to operate a wide
2850 Centerville Road range of column dimensions and particle sizes than any other commercially avail-
Wilmington, Delaware 19808 able system. Advanced optical design in the diode array detector allows a wide
dynamic range and high sensitivity, both of which are critical in the monitoring of
USA
small impurities in fine chemicals.

The combined benefits are demonstrated by a separation of primary components

and related impurities including sulfa drugs, nitrofurans and malachite green found
in samples of fish medications. A broad range of products for treating ailments in
decorative or pet fish are available. Many of these medications are banned or
restricted for use in edible fish. If present in edible fish, the levels would be very low
or undetectable by HPLC with UV based detection. These examples show a few
medications and detail the rapid method development used to establish a rapid MS-
compatible separation environment. Many fish medications advertise the use of
“pharmaceutical quality” ingredients, and may imply pharmaceutical quality manu-
facturing and quality control procedures. When profiling these products one should
expect to see very low levels of related impurities, consistent with the goals of
pharmaceutical quality manufacturing

The high pressure capability of the system allows the use of methanol, and acetoni-
trile, to explore the selectivity of the two solvents. At 1 mL/min, using a simple
3 minute gradient and a 3.0 mm x 50 mm Poroshell 120 column, the analysis time is
only less than 5 minutes including the late eluting phthalate artifact. The separation
of the main components of a medicated powder with acetonitrile and methanol is
shown in Figure 1, and the extraction of a medicated feed is shown in Figure 2.
The speed, resolution and flexibility of Configuration Conclusion
the system are further demonstrated by
a separation of a sulfa standard mix • Agilent 1290 Infinity Binary Pump with Taking advantage of flexible solvent and
using solvent, gradient and temperature Integrated Vacuum Degasser column selection features, and high
optimization with a 100 mm length (G4220A) pressure capability, of the system allows
Poroshell 120 column ( see Figure 3). one to use highly efficient columns to
• Agilent 1290 Infinity Autosampler rapidly develop separations with remark-
After further optimization of the sulfa (G4226A) able resolution while conserving solvent
mix using methanol with the elevated over the use of 4.6 mm id columns.
• Agilent 1290 Infinity Thermostatted
temperature, all of the samples were Column Compartment (G1316C)
run with the final method configuration,
as shown in Figure 4. • Agilent 1200 Diode Array Detector
(G1315C)

Poroshall 120, 2.7 µm, 3 mm × 50 mm 30 °C First 100 mm uses same Poroshall 120, 2.7 µm, 3 mm × 50 mm
ACN 5% to 90% organic v. 0.05% formic acid gradient as 50 mm 30 °C 5% to 90% organic v. 0.05% formic
1 mL/min, 3 min. gradient, 1 µL injection column, now gradient acid 1 mL/min, 3 min. gradient, 1 µL
is too steep injection

Decreased gradient Poroshall 120, 2.7 µm, 3 mm × 100 mm

slope by ~40% 30 °C 5% to 60% ACN at 3, to 90% ACN at 4
Nitrofurazone 1 mL/ min.
Malachite green

MeOH Primary gradient impurity

Changed column temp
from 30 to 45 °C
* Poroshall 120, 2.7 µm, 3 mm × 100 mm 45 °C
5% to 60% ACN at 3, to 90% ACN at 4
1 mL/ min.

*Confirmed by spectral comparison

Figure 1 Figure 3
“Super Ick” medicated powder. Sulfa standard mixture (Agilent p/n 59987-20033).

Poroshall 120, 2.7 µm, 3 mm × 50 mm 30 °C Poroshall 120, 2.7 µm, 3 mm × 100 mm 45 °C

ACN Super Ick packet 5% to 60% MeOH at 3, to 90% at 4
5% to 90% organic v. 0.05% formic acid
1 mL/min, 3 min. gradient, 1 µL injection Nitrofurazone 1 mL/min, 3 min. gradient, 1 µL injection
Malachite green
Sulfathiazole
Nitrofurazone

Jungle food
Sulfathiazole Nitrofurazone

Sulfathiazole Nitrofurazone
MeOH

Sulfa mix

Figure 2 Figure 4
Separation of “Jungle” medicated fish food after methanol/water/formic Final method configuration.
acid extraction.

www.agilent.com/chem/1290
© Agilent Technologies, Inc., 2010
July 1, 2010
Publication Number 5990-5993EN
 Determination of Sulfonamide
Antibiotics in Bovine Liver Using
Agilent SampliQ QuEChERS EN Kits
by LC/MS/MS

Application Note
Food

Author Abstract
Limian Zhao, and Joan Stevens
This paper presents an analytical method for the determination of nine sulfonamide
Agilent Technologies, Inc.
antibiotic residues in bovine liver: sulfadizine, sulfathiazole, sulfamerazine, sulfamethi-
2850 Centerville Road
zole, sulfamethazine, sulfamethoxypyridazine, sulfachloropyridazine, sulfamethoxa-
Wilmington, DE 19808
zole, and sulfadimethoxin. The procedure involves a rapid and efficient pretreatment
with SampliQ QuEChERS kits. The homogenized liver sample was initially extracted in
a buffered aqueous/1% acetic acid acetonitrile system with an extraction and parti-
tioning step after the addition of salts. Finally, the sample was cleaned up using dis-
persive solid-phase extraction (dispersive-SPE). The final extracts were analyzed by
the sensitive and selective determination of all compounds in a single run using LC-
ESI-MS-MS operating in positive ion multiple reaction monitoring (MRM) mode.
Sulfapyridine was selected as the internal standard. The accuracy of the method,
expressed as recovery, was between 53 and 93%. The precision, expressed as RSD,
was between 2.1 and 16.8%. The established 5 ng/g limits of quantification (LOQ)
were much lower than the respective Maximum Residue Limit (MRL) for sulfonamide
in animal food products (20-100 ng/g).
Introduction ences in food matrices and the chemical properties of the tar-
get analytes, modifications to the method were also investi-
Sulfonamides (SAs) are a very important class of antibacterial gated to achieve efficient extraction and cleanup.
compounds widely used in veterinary practice for therapeutic,
prophylactic, and growth-promoting purposes. Residues of
SAs may remain in animal tissues if adequate withdrawal
Experimental
time is not observed or if the SAs have been improperly
Reagents and Chemicals
administered. The maximum residue limit (MRL) in the
European Union countries and United States for SAs in animal All reagents and solvents were HPLC or analytical grade.
muscle tissue is 100 ng/g, while in Japan it is 20 ng/g. [1] Methanol (MeOH) was from Honeywell (Muskegon, MI,
USA). Acetonitrile (ACN), dimethyl sulfoxide (DMSO), and
The QuEChERS EN method is an important variation, which glacial acetic acid (HAc) were from Sigma-Aldrich (St Louis,
has been officially accepted by the European Committee for MO, USA). Ammonium acetate (NH4OAc) was from Fisher
Standardization and is widely applied to pesticide analysis in Chemicals (Fair Lawn, NJ, USA). Formic acid (FA) was from
foods of plant origin [2]. The original EN buffered method was Fluka (Sleinheim, Germany). The sulfonamides standards and
designed mostly for multiresidue pesticide analysis in plant the internal standard were from Sigma (St Louis, MO, USA).
food products. In summary, the method uses acetonitrile
extraction followed by the salting out of water from the sam- Solutions and Standards
ple using anhydrous magnesium sulfate (MgSO4), NaCl, and A 1 M ammonium acetate stock solution was made by dis-
buffering citrate salts to induce liquid-liquid partitioning. For solving 19.27 g NH4OAc powder in 250 mL Milli-Q water. The
cleanup, a dispersive solid-phase extraction (dispersive-SPE) solution was stored at 4 °C. A 5 mM ammonium acetate solu-
is conducted using a combination of primary secondary amine tion in water, pH 3, was made by adding 5 mL of 1 M ammoni-
(PSA) to remove fatty acids from other components and anhy- um acetate stock solution into 1 L of Milli-Q water. The pH
drous MgSO4 to reduce the remaining water in the extract. was then adjusted to 3 by adding glacial acetic acid and mon-
After mixing and centrifuging, the upper layer is ready for itoring with a pH meter. A 1:1 MeOH/ACN solution was made
analysis. Fatty dispersive-SPE, which contains 25 mg C18EC by combining 500 mL of MeOH and ACN, and mixing well. A
per mL of ACN extract, is employed to remove more lipids 1% acetic acid in ACN solution was prepared by adding 5 mL
from the matrix when using fruits and vegetables with fats of glacial acetic acid to 500 mL of ACN, and mixing well. A 1:1
and waxes. ACN/H2O solution with 0.1% FA was prepared by combining
50 mL of ACN and Milli-Q water, then adding 100 µL of formic
Food matrices from animal origin contain a substantial
acid. A 1:9 MeOH/H2O solution with 0.1% FA was prepared
amount of proteins and lipids. Therefore, they are very differ-
by combining 10 mL of MeOH and 90 mL of Milli-Q water,
ent from food matrices of plant origin such as fruits and veg-
then adding 100 µL of formic acid.
etables. In this study, a SampliQ QuEChERS EN buffered
extraction kit (p/n 5982-5650) and EN fatty dispersive-SPE Standard and internal standard (IS) stock solutions
15 mL kit (p/n 5982-5165) were tested for the analysis of sul- (1.0 mg/mL for all) were all made in DMSO and stored at
fonamide antibiotics in bovine liver. Because of the differ- 4 °C. Six of the sulfonamides are light-sensitive, so the stock

Sulfadizine Sulfathiazole Sulfamerazine Sulfamethazine Sulfamethizole

Sulfamethoxypyridazine Sulfachloropyridazine Sulfamethoxazole Sulfadimethoxin Sulfapyridine (IS)

Figure 1. Chemical structures of the quinolone antibiotics investigated in this study.

2
solutions were kept in amber vials and wrapped in aluminum Instrument conditions
foil. Three combined QC spiking solutions of 0.2, 4, and 16 HPLC conditions
µg/mL were made fresh daily in 1:1 ACN/H2O containing Column Agilent ZORBAX Solvent Saver HT Eclipse Plus C18
0.1% FA. A 20 µg/mL standard spiking solution in 1:1 50 × 3.0 mm, 1.8 µm (p/n: 959941-302)
ACN/H2O containing 0.1% FA was made for the preparation Flow rate 0.3 mL/min
of calibration curves in the matrix blank extract. Due to light
Column Temperature 30 °C
sensitivity of certain sulfonamides, all of the combined solu-
Injection volume 10 µL
tions were kept in amber vials and wrapped in aluminum foil.
A 20 µg/mL IS spiking solution of Sulfapyridine was made in Mobile Phase A: 5 mM ammonium acetate, pH 3.0 in H2O
1:1 ACN/H2O containing 0.1% FA. B: 1:1 MeOH/ACN
Needle wash 1:1:1:1 ACN/ MeOH/ IPA/ H2O with 0.2% FA.
Equipment and Material Gradient Time %B Flow rate (mL/min)
• Agilent 1200 Series HPLC with Diode Array Detector 0 15 0.3
(Agilent Technologies Inc., CA, USA) 0.2 15 0.3
• Agilent 6410 Triple Quadrupole LC/MS system with 6.0 60 0.3
Electrospray Ionization (Agilent Technologies Inc., CA, 6.01 100 0.3
USA) 7.0 STOP

• Agilent SampliQ QuEChERS EN Extraction kit p/n 5982- Post run 3.5 min
5650 (Agilent Technologies Inc., DE, USA) Total cycle time ~11 min.
MS conditions
• Agilent SampliQ QuEChERS EN fatty dispersive-SPE kit
for 15 mL p/n 5982-5156 (Agilent Technologies Inc., DE, Polarity positive
USA) Gas Temperature 325 °C
Gas Flow 8 L/min
• CentraCL3R Centrifuge (Thermo IEC, MA, USA)
Nebulizer 50 Psi
• Eppendorf microcentrifuge (Brinkmann Instruments, Capillary 4000 V
Westbury, NY, USA)
Other conditions relating to the analytes are listed in Table 1.
• 2010 Geno Grinder (Spex SamplePrep LLC, Metuchen, NJ,
USA)
• Multitube Vortexer (Henry Troemner LLC, Thorofare, NJ,
USA)

3
Table 1. Instrument Acquisition Data for the Analysis of 9 Sulfonamide added to each tube. Sample tubes were capped tightly and
Antibiotics by LC/MS/MS.
shaken vigorously for 1 min by the 2010 Geno Grinder at 4 °C.
MRM channels Fragmentor CE RT
Analyte (m/z) (V) (V) (min) A 6 mL aliquot of the upper ACN layer was transferred into an
Sulfadizine 1) 251.1 → 108.0 100 25 2.1 Agilent SampliQ EN QuEChERS fatty dispersive-SPE 15 mL
2) 251.1 → 156.0 13 tube (p/n 5982-5156). This 15 mL dispersive-SPE tube con-
Sulfathiazole 1) 256.0 → 156.0 94 13 2.3 tained 150 mg of PSA, 150 mg of C18EC, and 900 mg of anhy-
2) 256.0 → 92.1 29 drous MgSO4. The tubes were tightly capped and vortexed for
Sulfamerazine 1) 265.1 → 92.1 125 29 2.9 2 min. The 15 mL tubes were centrifuged at 4000 rpm for
2) 265.1 → 108.1 25 5 min. A 4 mL amount of extract was then transferred into
Sulfamethizole 1) 271.0 → 156.0 112 9 3.7 another tube and dried by N2 flow at 40 °C. Samples were
2) 271.0 → 92.1 29 reconstituted into 800 µL of 1:9 MeOH/H2O solution with
Sulfamethazine 1) 279.1 → 124.0 116 21 3.8 0.1% FA. After vortexing and sonicating for 10 min, the sam-
2) 279.1 → 92.1 33 ple was filtered by a 0.22 µm Cellulose Acetate Spin Filter
Sulfamethoxypyridazine 1) 281.1 → 156.0 128 13 3.9 (p/n 5185-5990). The clear filtered sample was transferred
2) 281.1 → 92.1 29 into an autosampler vial. The samples were capped and vor-
Sulfachloropyridazine 1) 285.0 → 156.0 106 9 4.5 texed thoroughly, in preparation for LC/MS/MS analysis.
2) 285.0 → 92.1 29
Sulfamethoxazole 1) 254.1 → 92.1 113 25 4.8 See Figure 2 for the flow chart of the extraction procedure for
2) 254.1 → 108.0 21 a bovine liver sample.
Sulfadimethoxin 1) 311.1 → 156.0 141 17 6.0
2) 311.1 → 92.1 37
Sulfapyridine (IS) 250.1 → 92.1 113 29 2.7 Weigh 2 g homogenized liver sample (± 0.05 g) in 50 mL centrifuge tube.

1) Quantifier transition channel 2) Qualifier transition channel

Spike 50 µL of IS spike solution, 50 µL of QC spike solution if necessary. Vortex 30 s.

Add 8 mL of water. Vortex.

Sample preparation
Sample preparation includes sample homogenization, extrac-
tion and partitioning, and dispersive-SPE cleanup. Since the Add 10 mL of 1% AA in ACN, and shake vigorously for 30 s.

main focus of existing QuEChERS methodology has been the

extraction of pesticides from plant and vegetable matrices, Add SampliQ EN QuEChERS extraction kit and shake vigorously for 1 min.
certain modifications were adapted in order to optimize
results for the determination of sulfonamides in bovine liver.
Centrifuge @ 4000 rpm for 5 min.
These modifications will be discussed in detail later.

Bovine liver was purchased from a local grocery store. It was Transfer 6 mL of upper ACN layer to SampliQ EN QuEChERS fatty
dispersive-SPE 15 mL tube.
then washed and chopped into small pieces. The chopped
liver sample was homogenized thoroughly with a food grinder,
Vortex 2 min. Centrifuge @ 4000 rpm for 5 min.
then stored at -20 °C. Two gram (±0.05 g) amounts of homo-
geneous sample were placed into 50 mL centrifuge tubes.
Sample tubes were centrifuged 30 s to move any sample Transfer 4 mL extract to another tube. Blow down @ 40 °C with N2 .

sticking to the inside wall of tube to the bottom. Samples

were then fortified with appropriate QC spiking solutions Reconstitute into 800 µL 1:9 MeOH/H2O with 0.1% FA. Vortex and sonicate.
(50 µL) when necessary. Then 50 µL of IS spiking solution
(20 µg/mL of sulfapyridine) were added. After vortexing sam-
Filter samples with 0.22 µm cellulose acetate spin filter.
ple for 30 s, 8 mL of Milli-Q water were added. Tubes were
then vortexed another 10 s for mixing. Ten milliliters of 1% AA
in ACN were added to each tube. Tubes were capped and Samples are ready for LC/MS/MS analysis.
shaken by a 2010 Geno Grinder for 30 s. An Agilent SampliQ
QuEChERS EN extraction salt packet (p/n 5982-5650) was Figure 2. Flow chart of QuEChERS procedure for the determination of
sulfonamides in bovine liver.

4
Results and Discussion better recoveries for all the analytes relative to methods 1 or
2. The matrix effect values show that the sample processed
Method Optimization in the Liver Matrix by method 3 is as clean as the sample processed by method
2, but not as clean as the sample processed by method 1. The
As mentioned previously, modifications of the QuEChERS EN
addition of acid in the ACN partitioning step impedes the per-
method were investigated relative to extraction efficiency. An
formance of PSA in the dispersive step preventing the loss of
EN buffered extraction system provides a solution with a pH
analytes. It also decreases the interaction of PSA with other
of 5.0 – 5.5, which illicits neutral sulfonamide analytes (pKa ~
matrix interferences, producing a greater matrix effect. This is
6-7). PSA sorbent used in dispersive-SPE can strongly inter-
also shown by the color of the ACN extract. Although PSA
act with acid compounds and remove various co-extractive
was used in the dispersive step, the presence of acidified
interferences, such as polar organic acids, sugars, and fatty
ACN extract elicited a light brown-red color, rather than the
acids. However, it may also interact with target compounds
light yellow extract in method 1.
and cause the loss of analytes. Therefore, the QuEChERS
fatty EN dispersive-SPE kit with PSA was compared to the Table 2. Method Optimization Results for the Analysis of Sulfonamides in
Bovine Liver
dispersive-SPE kit without PSA. A previous study [4] showed
that the addition of acid to acetonitrile can inhibit the absorp- Method 1 Method 2 Method 3
tion of PSA, weakening the attraction of PSA to the com- Matrix Matrix Matrix
pounds of interest. Therefore, a 1% AA in ACN was evaluated Analytes Recovery effect Recovery effect Recovery effect
in addition to standard ACN in the first partitioning step. Sulfadiazine 91.9 -33.0 85.2 -65.2 85.6 -57.9
Sulfathiazole 39.9 -35.9 42.0 -57.3 87.7 -65.7
To evaluate the original EN and modified method, a 50 ng/g
Sulfamerazine 77.0 -19.3 43.9 -23.9 89.0 -51.7
of fortified liver sample was extracted with the following pro-
cedures: Sulfamethizole 15.3 -33.6 46.5 -46.9 63.2 -49.8
Sulfamethazine 85.7 -23.1 51.4 -31.6 87.3 -42.0
1. EN buffered extraction kit with ACN and dispersive-SPE Sulfamethoxy- 76.6 -32.6 49.0 -31.7 86.1 -49.1
kit with PSA (25 mg PSA and C18EC per mL) pyridazine
2. EN buffered extraction kit with ACN and dispersive-SPE Sulfachloro- 29.6 -38.5 51.1 -42.3 84.8 -50.6
pyridazine
kit without PSA (25 mg C18EC per mL)
Sulfamethoxa- 60.0 -40.9 53.4 -46.9 87.5 -54.5
3. EN buffered extraction kit with 1% AA in ACN and zole
dispersive-SPE kit with PSA (25 mg PSA and C18EC per Sulfadimethoxin 67.4 -35.3 56.9 -43.0 89.6 -51.9
mL).
The corresponding matrix blanks were extracted at the same Method 1 EN buffered extraction kit + ACN + Fatty dispersive-
SPE kit (25 mg PSA + 25 mg C18EC + 150 mg MgSO4
time, then post-spiked with the same amount of sulfonamide
per mL)
standards. Neat solution post-spiked at the same concentra-
Method 2 EN buffered extraction kit + ACN + Dispersive-SPE kit
tion was also compared to the matrix post-spiked samples. without PSA (25 mg C18EC + 150 mg MgSO4 per mL)
The results are shown in Table 2. The results of method 1 and Method 3 EN buffered extraction kit + 1% AA ACN + Fatty
dispersive-SPE kit (25 mg PSA + 25 mg C18EC +
method 2 show that PSA does contribute to the matrix
150 mg MgSO4 per mL)
cleanup during the dispersive-SPE step. The matrix effect val-
ues for all of the analytes in method 1 were lower than those Responseextracted sample
% Recovery = × 100
in method 2 indicating that the sample processed by method Responsepost-extracted spiked sample
1 was cleaner than the sample processed by method 2. This
was also demonstrated by the color of the ACN extract. After Responsepost-extracted spiked sample
% Matrix Effects = - 1 × 100
the extraction step, the ACN extract was a brownish-red Responsenon-extracted neat sample
color. Using PSA in the dispersive step produced an ACN
extract that was light yellow in color. Removing PSA from the
dispersive step maintained the previously observed brownish-
red ACN extract. Unfortunately, the presence of PSA in the
dispersive step also caused the loss of certain analytes and
produced very low recovery for sulfachloropyridazine (30%)
and sulfamethizole (15%). Method 3 produced substantially

5
Method 3 was selected for the final study. In this study, liver Recovery and Reproducibility
sample was extracted by the EN buffered extraction kit (p/n The recovery and reproducibility were evaluated by fortifying
5982-5650) with 1% AA in ACN. After centrifuging, the ACN sulfonamides standards in homogenized liver sample at levels
extract was further cleaned by EN fatty dispersive-SPE 15mL of 5, 100, and 400 ng/g. These QC samples were quantified
tube (p/n 5982-5156). Figure 3 shows the MRM chro- against the matrix spiked calibration curve. The analysis was
matograms of the liver control blank and 100 ng/g fortified performed in replicates of six at each level. The recovery and
liver extract. The liver control blank chromatogram indicated reproducibility (shown as RSD) data are shown in Table 4. The
that the target analytes were free from any interference. results show that all of the sulfonamides were somewhat low
but still at acceptable recoveries (average of 77.8%) and good
Linearity and limit of quantification (LOQ)
precision (average of 7.2% RSD). Samples were concentrated
The linear calibration range for all the sulfonamide antibiotics during the procedure to optimize sensitivity, which also
was 5 - 400 ng/g. Matrix blanks were prepared for evaluation. caused additional matrix effects that possibly contributed to a
Calibration curves, made from spiked matrix blanks, were higher RSD of target compounds at low levels.
made at levels of 5, 10, 50, 100, 200, 300 and 400 ng/g for
each analyte. The sulfapyridine was used as an internal stan- Table 4. Recovery and Repeatability of Sulfonamides in Fortified Liver
dard at 200 ng/g. The calibration curves were generated by Homogenate
plotting the relative responses of analytes (peak area of ana- 5 ng/g fortified 100 ng/g fortified 400 ng/g fortified
lyte / peak area of IS) versus the relative concentration of QC QC QC
analytes (concentration of analyte/concentration of IS). The Analytes Recovery RSD Recovery RSD Recovery RSD
5 ng/g limits of quantification (5 ppb) of the sulfonamides is (n=6) (n=6) (n=6)
far below the MRLs for residues of these antibiotics in animal Sulfadiazine 73.9 15.6 90.0 13.7 81.9 5.3
food products (20 - 100 ng/g). Table 3 shows the regression Sulfathiazole 62.9 16.8 75.3 8.4 67.9 5.8
equation and correlation coefficient (R2). Linear regression fit Sulfamerazine 77.6 11.5 92.8 6.6 82.0 4.2
was used with 1/x2 weight. Results indicated excellent linear- Sulfamethizole 62.8 4.7 60.7 6.5 53.0 2.1
ity for all of analytes calibration curves over a broad quantifi- Sulfamethazine 87.4 6.9 90.0 10.7 83.4 3.4
cation range.
Sulfamethoxy- 81.8 9.4 84.8 8.1 76.4 2.9
Table 3. Linearity of Sulfonamide Antibiotics in Bovine Liver pyridazine
Sulfachloro- 84.2 10.0 78.6 6.3 73.8 3.6
Analytes Regression Equation R2
pyridazine
Sulfadiazine Y = 1.6585X + 0.0002 0.9963
Sulfamethoxazole 85.9 7.6 82.3 5.9 78.1 3.3
Sulfathiazole Y = 1.3899X + 0.0002 0.9942
Sulfadimethoxin 77.8 8.4 80.9 4.9 75.6 3.3
Sulfamerazine Y = 3.5019X – 0.0001 0.9962
Sulfamethizole Y = 2.3064X + 0.0001 0.9963
Sulfamethazine Y = 4.3780X – 0.0004 0.9977
Sulfamethoxypyridazine Y = 4.4044X + 0.0003 0.9941
Sulfachloropyridazine Y = 1.5869X – 0.0005 0.9971
Sulfamethoxazole Y = 1.9047X – 0.0001 0.9936
Sulfadimethoxin Y = 4.5106X + 0.0020 0.9922

6
 ×103 1 12 23 34
A
4
1.5

0.5

1 IS 12 23 34 4
×103
B
2

1.5 9
5 6

1 3
4
8
0.5 1 2 7

1 2 3 4 5 6
Counts vs. Acquisition Time (min)
Figure 3. LC/MS/MS Chromatograms of A) liver blank extract, and B) 100 ng/g fortified liver extract. Peaks identification: 1. sulfadizine, 2. sulfathiazole,
3. sulfamerazine, 4. sulfamethizole, 5. sulfamethazine, 6. sulfamethoxypyridazine, 7. sulfachloropyridazine, 8. sulfamethoxazole, 9. sulfadimethoxin,
IS (internal standard), sulfapyridine.

Conclusions References:
The Agilent SampliQ EN Buffered Extraction kit and SampliQ [1] Sun H., Ai L., Wang F., Quantitative Analysis of
EN fatty dispersive-SPE kit provide a simple, fast, and effec- Sulfonamide residues in Natural Animal Casings by HPLC,
tive method for the purification of sulfonamide antibiotics in Chromatographia 2007, 66, Sept, (no. 5/6), 333-337.
bovine liver. When compared to other sample preparation
[2] European Council Regulation 2377/90/EC of 26 June
methods, such as LLE and SPE, QuEChERS methodology is
1990 laying down a community procedure for the estab-
easy, fast, low cost and does not require automation. In addi-
lishment of maximum residue limits of veterinary medici-
tion, it is labor saving and a “greener” technology. The recov-
nal products in foodstuffs of animal origin, OJ L, 1990, p.
ery and reproducibility, based on matrix-spiked standards,
224.
were acceptable for multiresidue sulfonamide determination
in bovine liver. The impurities and matrix effects from liver [3] European Committee for Standardization/Technical
were minimal and did not interfere with the quantification of Committee CEN/TC 275 (2007), Foods of plant origin:
any target compound. The LOQs of the quinolones were much Determination of pesticide residues using GC-MS and/or
lower than their regulated MRLs in animal food products (20- LC-MS/MS following acetonitrile extraction/partitioning
100 ng/g). This modified QuEChERS procedure is a very and cleanup by dispersive SPE-QuEChERS method.
promising methodology for the quantitative analysis of sulfon- European Committee for Standardization, Brussels.
amides in food products of animal origin. This application [4] Zhao L., Stevens J., Determination of quinolone antibi-
demonstrates great potential of SampliQ QuEChERS extrac- otics in bovine liver using Agilent SampliQ QuEChERS kits
tion and dispersive-SPE kits, and extend far beyond plant by LC/MS/MS, Agilent application note, 5990-5085EN.
matrices to bio-matrices, such as animal food products and
bio-fluid.

7
www.agilent.com/chem
Agilent shall not be liable for errors contained herein or
for incidental or consequential damages in connection
with the furnishing, performance, or use of this material.

Information, descriptions, and specifications in this

publication are subject to change without notice.

© Agilent Technologies, Inc., 2010

Printed in the USA
January 25, 2010
5990-5086EN
 Determination of Quinolone
Antibiotics in Bovine Liver Using
Agilent SampliQ QuEChERS Kits by
LC/MS/MS

Application Note
Food

Author Abstract
Limian Zhao, and Joan Stevens This paper presents an analytical method which allows the determination of 11
Agilent Technologies, Inc. quinolone antibiotic residue in bovine liver: pipemidic acid, ofloxacin, ciprofloxacin,
2850 Centerville Road danofloxacin, lomefloxacin, enrofloxacin, sarafloxacin, cinoxacin, oxolinic acid, nalidix-
Wilmington, DE 19808 ic acid, and flumequine.
USA
The procedure involves a rapid and efficient pretreatment by SampliQ QuEChERS kits.
The homogenized liver sample was initially extracted in a buffered aqueous, 5%
formic acid acetonitrile system. An extraction and partitioning step was performed
after the addition of salts. Cleanup was done using dispersive solid phase extraction
(dispersive-SPE). The final extracts allowed determination of all compounds in a sin-
gle run using LC-ESI-MS-MS operating in positive ion multiple reaction monitoring
(MRM) mode. Norfloxacin was selected as the internal standard. The accuracy of the
method, expressed as recovery, was between 62 and 113%. The precision, expressed
as RSD, was between 2.2 and 13.4%. The established limit of quantification (LOQ) was
5 ng/g and is significantly lower than the respective Maximum Residue Limit (MRL)
for quinolones in food producing animals.
Introduction a combination of both. Although they have been widely used,
these traditional methods have inherent limitations.
Quinolones are a family of synthetic broad-spectrum antibi- Traditional methods are labor intensive, time consuming,
otics. They prevent bacterial DNA from unwinding and dupli- require a large amount of solvent and waste disposal. In
cating. There is evidence that quinolones in food animals lead 2003, the QuEChERS (Quick, Easy, Cheap, Effective, Rugged,
to the emergence of quinolone-resistant bacteria in animals. and Safe) method for pesticide residue analysis in fruit and
The resistant organisms are transmitted to humans via direct vegetable matrices was introduced. [5] There are two validat-
contact with the animal or through the consumption of conta- ed QuEChERS methodologies: the AOAC and EN versions.
minated food and water. Quinolone-resistant campylobacter is Both are widely accepted and effective for the multiresidue
an example of animal-to-human transmission and has been analysis of pesticides in fruit, vegetables and other plant food
observed in many European countries since the early 1990s matrices. The QuEChERS method contains significant advan-
[1]. Therefore, public health agencies in many countries such tages over traditional methods, including high recoveries for a
as the EU commission [2], the USA FDA administration [3], wide range of pesticides, high sample throughput, minimal
and the Chinese Ministry of Agriculture [4] have established labor, time savings, limited solvent usage, and low waste. In
maximum residue limits (MRLs) of veterinary drugs in food- addition, the method is manually accommodating which has
producing animals. Given the different drugs in different food made QuEChERS a very popular methodology for the analysis
origins and in different countries, the MRLs of quinolones in of pesticide residues in fruits and vegetables in recent years.
food products of animal origin are usually at the level of
100 µg/kg or higher. Although the current QuEChERS methodology has been
designed for removing matrix interferences in food products
As animal food origins, such as muscle, liver, and eggs, are of plant origin, such as polar organic acids, sugars, and lipids,
complicated matrices, it is critical to use an efficient sample it also has potential for other food matrices such as meat.
pretreatment method for analyte extraction and concentra- Based upon the chemical properties of the compounds of
tion, and matrix cleanup. The established sample pretreat- interest and food matrices, some modifications of the original
ment methods used for determination of quinolones include method might be necessary to obtain accurate and precise
traditional solvent extraction, solid phase extraction (SPE), or results. The purpose of this work is to extend the QuEChERS
methodology to veterinary drug residues in food-producing
Fluoroquinolones (FQ) used in food animal animals. Agilent SampliQ QuEChERS EN buffered extraction
kits (p/n 5982-5650) and dispersive-SPE 2 mL kits for drug
residues in meat (p/n 5982-4921) were used for the analysis
HO O
HO
H H3C OH of 11 quinolone antibiotics in bovine liver: pipemidic acid,
H3C H

H
ofloxacin, ciprofloxacin, danofloxacin, lomefloxacin,
H
O enrofloxacin, sarafloxacin, cinoxacin, oxolinic acid, nalidixic
acid and flumequine (Figure 2). The method was validated in
Zoonotic infection in FQ-resistant E coli from Horizontal transfer of Residual antibiotics
terms of recovery and reproducibility.
human by FQ-resistant animal colonizes resistant genes from exert selective
Campylobacter and human gastrointestinal zoonotic to human pressure for resistant
Salmonella tract flora mutant in human flora

Figure 1. Animal to human transmission of resistant bacteria [1].

Pipemidic acid Ofloxacin

Ofl i Ciprofloxacin Danofloxacin Lomefloxacin Enrofloxacin

S fl i
Sarafloxacin Cinoxacin Oxolinic acid N lidi i acid
Nalidixic id Flumequine
Fl i Norfloxacin (IS)

Figure 2. Chemical structures of the quinolone antibiotics investigated in this study.

2
Experimental Equipment and Material
• Agilent 1200 Series HPLC with Diode Array Detector
Reagents and Chemicals (Agilent Technologies Inc., CA, USA).
All reagents and solvents were HPLC or analytical grade. • Agilent 6410 Series triple quadrupole LC/MS system with
Methanol (MeOH) was from Honeywell (Muskegon, MI, USA). Electrospray Ionization (Agilent Technologies Inc., CA,
Acetonitrile (ACN), dimethyl sulfoxide (DMSO) and glacial USA).
acetic acid (HAc) were from Sigma-Aldrich (St Louis, MO,
USA). Ammonium acetate (NH4OAc) was from Fisher • Agilent SampliQ QuEChERS EN Extraction kits, p/n 5982-
Chemicals (Fair Lawn, NJ, USA). Formic acid (FA) was from 5650, and SampliQ QuEChERS dispersive-SPE kits for
Fluka (Sleinheim, Germany). The quinolone standards and Drug Residues in Meat, 2 mL, p/n 5982-4921 (Agilent
internal standard were purchased from Sigma-Aldrich (St Technologies Inc., DE, USA).
Louis, MO, USA). Potassium phosphate, monobasic (KH2PO4), • CentraCL3R Centrifuge (Thermo IEC, MA, USA)
was from J.T. Baker (Phillipsburg, NJ, USA).
• Eppendorf microcentrifuge (Brinkmann Instruments,
Solutions and Standards Westbury, NY, USA)
1M ammonium acetate stock solution was made by dissolv- • 2010 Geno Grinder (Spex SamplePrep LLC, Metuchen, NJ,
ing 19.27 g NH4OAc powder in 250 mL Milli-Q water. The USA)
solution was stored at 4 ºC. A 5 mM ammonium acetate in
• Multi-tube Vortexer (Henry Troemner LLC, Thorofare, NJ,
water solution with pH 3 was made by adding 5 mL of 1M
USA)
ammonium acetate stock solution into 1 L of Milli-Q water,
then adjusting the pH to 3 with glacial acetic acid. A 1:1 Instrument conditions
MeOH/ACN solution was made by combining 500 mL of HPLC conditions
MeOH and ACN, then mixing well. A 5% formic acid solution
Column Agilent ZORBAX Solvent Saver Eclipse Plus Phenyl-
in ACN was made fresh daily by adding 10 mL of formic acid Hexyl 150 × 3.0 mm, 3.5 µm (p/n 959963-312)
to 190 mL of ACN, then mixing well. A 30 mM KH2PO4 buffer, Flow rate 0.3 mL/min
pH 7.0, was made by dissolving 4.08 g KH2PO4 powder into 1 L
Column Temperature 30 °C
Milli-Q water and adjusting the pH to 7.0 with 1 M KOH solu-
tion. A 1:1 ACN/H2O with 0.1% FA was prepared by combin- Injection volume 10 µL
ing 50 mL of ACN and Milli-Q water, then adding 100 µL of Mobile Phase A: 5 mM ammonium acetate, pH 3.0 in H2O
B: 1:1 MeOH/ACN
formic acid. A 1:9 MeOH/H2O solution with 0.1% FA was pre-
pared by combining 10 mL of MeOH and 90 mL of Milli-Q Needle wash 1:1:1:1 ACN/ MeOH/ IPA/ H2O with 0.2% FA.
water, then adding 100 µL of formic acid. Gradient Time %B Flow rate (mL/min)
0 15 0.3
Standard and internal standard (IS) stock solutions (1.0 0.2 15 0.3
mg/mL for all, except 0.25 mg/mL for ciprofloxacin) were 8.0 75 0.3
9.0 100 0.3
made in DMSO and stored at 4 ºC. Due to the solubility of 11.5 STOP
quinolones, it is essential to sonicate stock solutions to
Post run 4 min
ensure they completely dissolve. Three combined QC spiking
Total cycle time ~16 min.
solutions of 0.2, 8 and 16 µg/mL were made fresh daily in 1:1
ACN/H2O containing 0.1% FA. A 10 µg/mL standard spiking MS conditions
solution in 1:1 ACN/H2O containing 0.1% FA was made for Polarity positive
the preparation of calibration curves in the matrix blank Gas Temp. 325 °C
extract. A 20 µg/mL IS spiking solution of norfloxacin was Gas Flow 8 L/min
made in 1:1 ACN/H2O containing 0.1% FA. Nebulizer 50 Psi
Capillary 4000 V
Solvent cut 5 min
Other conditions relating to the analytes are listed in Table 1.

3
Table 1. Instrument Acquisition Data for the Analysis of 11 Quinolone packet (p/n 5982-5650) was added to each tube. Sample
Antibiotics by LC/MS/MS
tubes were capped tightly and shaken vigorously for 1 min by
MRM channels Fragmentor CE RT a 2010 Geno Grinder. Tubes were centrifuged at 4,000 rpm for
Analyte (m/z) (V) (V) (min) 5 min at 4 °C.
Pipemidic acid 1) 304.1 → 286.1 128 17 5.9
2) 304.1 → 215.1 37 A 1 mL aliquot of the upper ACN layer was transferred into an
Ofloxacin 1) 362.2 → 318.1 150 17 6.7 Agilent SampliQ QuEChERS dispersive-SPE 2 mL tube for
2) 362.2 → 344.1 21 Drug Residues in Meat (p/n 5982-4921). This 2 mL dispersive-
Ciprofloxacin 1) 332.1 → 314.1 131 21 6.8 SPE tube contained 25 mg of C18 and 150 mg of anhydrous
2) 332.1 → 231.0 41 MgSO4. The tubes were tightly capped and vortexed for 1 min.
Danofloxacin 1) 358.2 → 340.2 159 25 6.9 The 2 mL tubes were centrifuged with a microcentrifuge at
2) 358.2 → 82.1 49 13,000 rpm for 3 min. An 800 µL volume of extract was trans-
Lomefloxacin 1) 352.2 → 265.2 144 21 7.0 ferred into another tube and dried by N2 flow at 40 °C.
2) 352.2 → 334.1 21 Samples were reconstituted into 800 µL of 1:9 MeOH/H2O
Enrofloxacin 1) 360.2 → 342.2 159 21 7.3 with 0.1% FA. After vortexing and sonicating for 10 min, the
2) 360.2 → 316.2 17 sample was filtered by a 0.22 µm Cellulose Acetate Spin Filter
Sarafloxacin 1) 386.1 → 368.1 144 21 7.9 (p/n 5185-5990). The clear filtered sample was transferred
2) 386.1 → 348.2 33 into an autosampler vial. The samples were capped and vor-
Cinoxacin 1) 263.1 → 217.1 103 21 8.8 texed thoroughly in preparation for LC/MS/MS analysis.
2) 263.1 → 189.0 29 Figure 2 shows the flow chart of the entire extraction proce-
Oxolinic acid 1) 262.1 → 216.0 106 29 9.2 dure for bovine liver sample.
2) 262.1 → 160.0 41
Nalidixic acid 1) 233.1 → 104.1 94 45 10.3
2) 233.1 → 159.1 33
Weigh 2 g homogenized liver sample (± 0.05 g) in 50 mL centrifuge tube.
Flumequine 1) 262.1 → 202.0 106 33 10.8
2) 262.1 → 126.0 50
Norfloxacin (IS) 320.1 → 302.1 134 17 6.6 Spike 50 µL of IS spike solution, 50 µL of QC spike solution if necessary. Vortex 30 s.

1) Quantifier transition channel 2) Qualifier transition channel

Add 8 mL of 30 mM KH2PO4, pH 7.0. Vortex.

Sample preparation
Add 10 mL of 5% FA in ACN, and shake vigorously for 30 s.
The sample preparation procedure includes sample homoge-
nization, extraction/partitioning, and dispersive-SPE cleanup.
As mentioned previously the QuEChERS methods were Add SampliQ EN QuEChERS extraction kit and shake vigorously for 1 min.

designed for pesticides analysis in fruit and vegetable matri-

ces; therefore modifications were necessary to optimize the Centrifuge @ 4000 rpm for 5 min at 4 °C.
results for the determination of quinolones in bovine liver.
Transfer 1 mL of ACN layer to SampliQ QuEChERS dispersive-SPE 2 mL tube,
Bovine liver was purchased from a local grocery store. It was drug residues in meat.
washed and chopped into small pieces. The chopped liver
was homogenized thoroughly with a food grinder and stored Vortex 1 min, centrifuge @ 13,000 rpm for 3 min with microcentrifuge.
at -20 °C. Two-gram (±0.05g) samples of homogenized liver
were placed into 50 mL centrifuge tubes. The tubes were cen-
Transfer 800 µL extract to another tube, blow down @ 40 °C with N2.
trifuged for 30 s to move the sample from the inside tube wall
to the bottom of the tube. Samples were then fortified with
appropriate QC spiking solutions (50 µL) when necessary, Reconstitute into 800 µL 1:9 MeOH/H2O with 0.1% FA. Vortex and sonicate 10 min.
then 50 µL of IS spiking solution (20 µg/mL of norfloxacin).
After vortexing the sample for 30 s, 8 mL of 30 mM KH2PO4
Filter samples with 0.22 µm cellulose acetate spin filter.
buffer, pH 7.0, were added. Tubes were then vortexed for 10 s
to mix. A 10 mL volume of 5% FA in ACN was added to each
tube. Tubes were capped and shaken by a 2010 Geno Grinder Samples are ready for LC/MS/MS analysis.

for 30 s. An Agilent SampliQ QuEChERS EN extraction salt Figure 3. Flow chart of QuEChERS procedure for the determination of
quinolones in bovine liver.

4
Results and Discussion ACN extracts after the extraction step using different extrac-
tion kits. The ACN extracts using the EN extraction kit (p/n
Feasibility Test 5982-5650) showed much higher responses than those using
the AOAC extraction kit (p/n 5982-5755) and the original
Quinolones are a group of relatively new antibacterials syn-
extraction kit (p/n 5982-5550). The buffer system in the
thesized from 3-quinolone carboxylic acid. As shown in
extraction/partitioning step provided by the addition of salts
Figure 2, they all contain the carboxylic group, and are weakly
plays a key role in the extraction efficiency. The pH when the
acidic (pKa 4-6). Since this is the first time for quinolones
acidic analytes exist in their neutral forms facilitates the
determination by the QuEChERS method, the feasibility test
extraction. Both the EN and AOAC extraction kits provide a
was done by extracting 50 ng/mL of neat quinolone solution
buffer system of approximately pH 5.0 [6, 7], which is the
(prepared in water) with different SampliQ QuEChERS kits,
point where most quinolones are neutral. Therefore, these
including the SampliQ AOAC extraction kit, SampliQ EN
extraction kits generate better extraction efficiency than the
extraction kit, and SampliQ Original extraction kit. In addition,
original nonbuffered extraction kit. However, it is unknown
bovine liver is a very different matrix than fruit and vegeta-
why the neat extract from the EN extraction buffer system
bles. Therefore, the cleanup was followed by the correspond-
produced higher responses than that from the AOAC extrac-
ing fatty dispersive-SPE kit (AOAC and EN fatty dispersive-
tion buffer system, especially for the early eluted analytes.
SPE kit) because these fatty dispersive-SPE kits contain C18
From these results, the SampliQ EN buffered QuEChERS
which is critical for removing lipids from liver matrix.
extraction kit was selected for future work.
However, the test results were initially very disappointing. All
of the analytes had extremely low or nonexistent recoveries.
The ACN extracts were tested at two points in the procedure
to investigate where the analytes were being lost. The first
test was made after the extraction step. The second test was
made after both the extraction and the dispersive-SPE steps.
Figure 3 shows the chromatogram comparison for the neat

×103
1 12 23 34 4

Neat extracts by EN
3 extraction kit only

Neat extracts by AOAC

2.5 extraction kit only

Neat extracts by Original

2
extraction kit only

1.5

0.5

1 2 3 4 5 6 7 8 9 10
Counts vs. Acquisition Time (min)

Figure 4. Feasibility test results 1: chromatogram comparison of the neat extracts (no dispersive-SPE) obtained by SampliQ QuEChERS EN extraction kit ,
AOAC extraction kit, and original extraction kit.

5
The addition of acid to acetonitrile during the extraction/par- get analytes, the quinolones, leading to the loss of analytes.
titioning step was also investigated. Acetonitrile only, used in When acetonitrile was used in the extraction step, PSA from
the original EN method, and acidified acetonitrile with 5% the dispersive-SPE kit caused almost total loss of all of ana-
formic acid were evaluated for their efficiency. As demon- lytes (Figure 5, columns D and E). When acidified acetonitrile
strated in Figure 5 by comparing the results from columns A was used in the extraction step, the existence of PSA in the
and D, better analyte recoveries were achieved (10-30% high- dispersive-SPE kit still caused a 10-40% loss of analytes
er) with the acidified acetonitrile. The addition of formic acid (Figure 5, columns A and C). Because of these results, a brand
into solvent extraction inhibits the acid dissociation for new SampliQ dispersive-SPE kit for Drug Residues in Meat
quinolones. Therefore, their protonated neutral form can be (p/n 5982-4921) was used for this study. This new SampliQ
extracted easily into the solvent phase [8]. Furthermore, the dispersive-SPE kit contains 25 mg C18 and 150 mg MgSO4 per
addition of acid into acetonitrile greatly decreased the nega- mL of ACN extract. The new dispersive-SPE kit’s effect on the
tive impact caused by PSA in the dispersive-SPE step analytes recovery is negligible (Figure 5, columns A and B).
(Figure 5, columns C and E). The formic acid in ACN extract
interacts with PSA in the dispersive-SPE step, greatly According to the above feasibility test results, a QuEChERS
decreasing the binding of PSA with the target quinolones. method was developed and applied for the subsequent study
From these results, 5% (vol/vol) formic acid in acetonitrile in the liver matrix. This method uses the SampliQ EN buffered
was chosen as an extraction solvent for further study. extraction kit and 5% FA in ACN for the extraction/ partition-
ing step as well as the new SampliQ dispersive-SPE kit for
Although the EN extraction kit generated better recovery, the drug residues in meat for the following cleanup.
cleanup using the fatty dispersive-SPE kit in step two signifi-
cantly lowered extraction efficiency (Figure 5). The selected
fatty dispersive-SPE kit contains PSA (primary secondary
amine), C18, and MgSO4; however the loss of quinolones was
mostly due to the PSA. In the QuEChERS method, PSA is used
in all dispersive-SPE kits, because it acts as a weak anion
exchanger. It strongly interacts with acidic interferences from
fruits and vegetables such as polar organic acids, sugars, and
fatty acids. However, it can also strongly interact with the tar-

30000
A) 5% FA ACN, No dispersive-SPE
B) 5% FA ACN, C18 dispersive-SPE
C) 5% FA ACN, C18 + PSA dispersive-SPE
25000 D) ACN, No dispersive-SPE
E) ACN, C18 + PSA dispersive-SPE

20000

15000

10000

5000

0
id
ac
ic
id
em
p
Pi

Figure 5. Feasibility test 2. Analytes peak area comparison for the neat extract processed by different procedures. Comparisons include pure ACN and acidi-
fied ACN, with and without PSA dispersive-SPE.

6
Method Optimization in the Liver Matrix After the extraction/partitioning step, the sample was cen-
The QuEChERS method established from the results of the trifuged at 4,000 rpm and 4 °C for 5 min. The low temperature
feasibility test was applied to the determination of quinolones helped to remove lipids from the ACN extracts. After cen-
in bovine liver. trifuging, a thin layer of lipids might show up on the surface of
the ACN layer. Additional lipids will be removed by C18 in the
The homogenized liver sample was very thick and could not dispersive-SPE step. A 1 mL amount of ACN extract was
be used directly for the extraction. Therefore, it was neces- transferred into a 2 mL dispersive-SPE tube containing 25 mg
sary to dilute the liver sample with an aqueous buffer (30 mM C18 and 150 mg MgSO4 for cleanup. An 800 µL amount of
KH2PO4 in water, pH 7.0) before the extraction. Different sam- upper solvent was transferred into another tube by vortexing
ple/buffer ratios including 1:4, 3:7, 1:1, were investigated by and centrifuging. This was the final extract after the
adding 8 mL, 7 mL and 5 mL of buffer to 2 g, 3 g, and 5 g of QuEChERS extraction and cleanup. It appeared light brown to
homogeneous liver sample. After dilution, 10 mL of 5% FA in red in color and was transparent. In order to get sufficient
ACN was added. Visually, the more sample used, the more sensitivity and integrity of peak shape, the sample was dried
foam was generated during the extraction/partitioning step under N2 flow and reconstituted into 800 µL 1:9 MeOH/H2O
resulting in a darker red ACN extract. Although more sample with 0.1% FA. The reconstituted sample was cloudy and filtra-
should lead to a lower detection limit, it simultaneously intro- tion was necessary, which was done by a 0.22 µm cellulose
duced more matrix interferences and higher matrix effect. acetate spin filter. The sample became colorless and clear
Since the addition of 5% FA ACN to the liver sample is also a after filtering, and was ready for LC/MS/MS injection.
protein precipitation procedure, a sample/ACN ratio of 1:4 to
1:5 usually provides the best precipitation effect and suffi- Figure 6 shows the MRM chromatograms of liver control
cient cleanup for proteins. Therefore, a sample/buffer ratio of blank and 5 ng/g fortified liver extract (LOQ). The liver control
1:4 (2 g of liver sample and 8 mL of buffer) was employed. blank chromatogram indicated that it was free from any inter-
ference to the target analytes. The 5 ng/g fortified liver
extract chromatogram demonstrated that the 5 ng/g limits of
quantitation (LOQ) for all of analytes were well established
with a signal-to-noise ratio (S/N) greater than 5.

1 12 23 34
70 A4

60

50

40
70
1 12 23 34 10 11 B 4
4
6

60 3
7
2
5 9
1 8
50

40
1 2 3 4 5 6 7 8 9 10 11
Counts vs. Acquisition Time (min)

Figure 6. LC/MS/MS chromatograms of A) liver blank extract, and B) 5 ng/g fortified liver extract (LOQ). Peaks identification: 1. Pipemidic acid, 2. Ofloxacin,
3. Ciprofloxacin, 4. Danofloxacin, 5. Lomefloxacin, 6. Enrofloxacin, 7. Sarafloxacin, 8. Cinoxacin, 9. Oxolinoc acid, 10. Nalidixic acid, 11. Flumequine.

7
Linearity and limit of quantification (LOQ) Table 3. Recovery and Repeatability of Pesticides in Fortified Liver with
2 mL Dispersive-SPE Tube (p/n 5982-4921)
The linear calibration range for all of the quinolone antibiotics
5 ng/g 200 ng/g 400 ng/g
was 5 – 400 ng/g and matrix blanks were prepared for evalu-
fortified QC fortified QC fortified QC
ation. Calibration curves spiked in matrix blanks were made at RSD RSD RSD
levels of 5, 10, 50, 100, 200, 300, and 400 ng/g for each ana- Analytes Recovery (n=6) Recovery (n=6) Recovery (n=6)
lyte. The norfloxacin was used as an internal standard at Pipemidic acid 71.6 8.1 62.0 6.8 66.4 2.2
200 ng/g. The calibration curves were generated by plotting Ofloxacin 72.9 9.7 101.0 7.7 102.4 5.7
the relative responses of analytes (peak area of analyte /
Ciprofloxacin 108.2 8.3 101.4 4.2 98.9 2.3
peak area of IS) to the relative concentration of analytes (con-
Danofloxacin 88.2 7.9 109.3 7.8 114.0 6.1
centration of analyte/concentration of IS). The 5 ng/g limit of
quantification LOQ (5 ppb) established for all of the quino- Lomefloxacin 82.6 13.4 96.8 8.5 97.8 5.3
lones is far below the MRLs for residues of these antibiotics Enrofloxacin 88.6 7.5 109.5 8.3 113.1 5.8
in animal food products. Table 2 shows the regression equa- Sarafloxacin 99.6 9.0 97.7 8.4 97.0 4.6
tion and correlation coefficient (R2). Linear regression fit was Cinoxacin 92.3 9.3 95.1 7.9 93.5 2.6
used with 1/x2 weight. Results indicated excellent linearity Oxolinic acid 95.1 9.8 92.7 4.3 87.6 2.9
for all of the analytes calibration curves over a broad
Nalidixic acid 92.7 6.0 90.2 5.3 87.7 3.5
quantification range.
Flumequine 91.6 6.6 93.3 5.3 89.9 2.9
Table 2. Linearity of Quinolone Antibiotics in Bovine Liver.

Analytes Regression equation R2

Pipemidic acid Y = 0.2081X – 0.00002 0.9966
Ofloxacin Y = 0.2221X + 0.00001 0.9964
Ciprofloxacin Y = 0.2971X – 0.00005 0.9975
Danofloxacin Y = 0.6861X – 0.0039 0.9957
Lomefloxacin Y = 0.1702X – 0.00003 0.9958
Enrofloxacin Y = 0.6530X – 0.0020 0.9962
Sarafloxacin Y = 0.2132X – 0.0004 0.9937
Cinoxacin Y = 0.0933X – 0.0004 0.9959
Oxolinic acid Y = 0.1043X + 0.0003 0.9939
Nalidixic acid Y = 0.3223X + 0.0005 0.9974
Flumequine Y = 0.3232X + 0.0003 0.9966

Recovery and Reproducibility

The recovery and reproducibility were evaluated by fortifying
quinolone standards in homogenized liver sample at levels of
5, 200 and 400 ng/g. These QC samples were quantified
against the matrix spiked calibration curve. The analysis was
performed in replicates of six at each level. The recovery and
reproducibility (shown as RSD) data are shown in Table 3. It
can be seen from the results that all of quinolones except
pipemidic acid gave excellent recoveries (average of 95.9%)
and precision (average of 6.6% RSD). Pipemidic acid gave
lower recovery (average of 66.7%) but great precision (aver-
age of 5.7% RSD). Additionally, it still meets the 5 ng/g LOQ
requirement. Therefore, the results are acceptable.

8
Conclusions 6. Lehotay S.J., et al; Use of Buffering and Other Means to
Improve Results of Problematic Pesticides in a Fast and
The Agilent SampliQ Buffered Extraction EN kit and the Easy Method for Residue Analysis of Fruits and
SampliQ dispersive-SPE kit for Drug Residues in Meat provide Vegetables, J. AOAC Int., 2005, 88, 615-629.
a simple, fast and effective method for the purification of 7. Payá P., Anastassiades M.; Analysis of pesticide residues
quinolone antibiotics in bovine liver. Compared to the other using the Quick Easy Cheap Effective Rugged and Safe
sample pretreatment methods, such as LLE and SPE, the (QuEChERS) pesticide multiresidue method in combina-
QuEChERS method is easier to handle, faster, labor-saving, tion with gas and liquid chromatography and tandem
and cheaper. The recovery and reproducibility, based on mass spectrometric detection. Anal Bioanal Chem., 2007,
matrix spiked standards, were acceptable for multiresidue 389, 1697-1714.
quinolone determination in bovine liver. The impurities and
matrix effects from liver were minimal and did not interfere 8. Koesukwiwat U., et al; Rapid determination of phenoxy
with the quantification of any target compound. The LOQs of acid residues in rice by modified QuEChERS extraction
the quinolones were much lower than their regulated MRLs in and liquid chromatography-tandem mass spectrometry.
animal food products. On the whole, the QuEChERS proce- Analytical Chim. Acta, 2008, 626, 10-20.
dures presented here appear to be a promising reference
method for the quantitative analysis of quinolones in food
products of animal origin. This method also has the potential
to extend the applications of SampliQ QuEChERS extraction
and dispersive-SPE kits to the quantitative analysis in other
bio-matrices, such as animal food products and bio-fluids,
rather than just plant matrices.

References
1. Fluoroquinolone Antibiotics, A.R. Ronald and D.E. Low pg
58, Birkhauser Verlag, Basil Switzerland, ISBN 3-7643-
6591
2. Commission Regulation (EC) No 508/1999 of 4 March
1999 amending Annexes I to IV to Council Regulation
(EEC) No 2377/90 laying down a Community procedure
for the establishment of maximum residue limits of vet-
erinary medicinal products in foodstuffs of animal origin.
Official Journal L 060, 09/03/1999, 16.
3. Code of Federal Regulation, Title 21 (Food and Drugs),
Vol. 6, Part 556, Revised April 1, 2006.
4. Ministry of Agriculture of the People’s Republic of China,
Announcement 2002/235 concerning the maximum
residue limit of veterinary drug of animal foodstuff.
http://www.agri.gov.cn/blgg/t20030226_59300.htm.
5. Anastassiades M., Lehotay S.J.; Fast and Easy
Multiresidue Method Employment Acetonitrile
Extraction/Partitioning and “dispersive Solid-Phase
Extraction” for the Determination of Pesticide Residues
in Produce, J. AOAC Int., 2003, 86, 412- 431.

9
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