Bull Vet Inst Pulawy 53, 731-739, 2009

MULTI-RESIDUE SCREENING METHOD

FOR THE DETERMINATION OF NON-STEROIDAL
ANTI-INFLAMMATORY DRUG RESIDUES
IN COWS MILK WITH HPLC-UV AND ITS APPLICATION
TO MELOXICAM RESIDUE DEPLETION STUDY

PIOTR JEDZINIAK, TERESA SZPRENGIER-JUSZKIEWICZ,
AND MAGORZATA OLEJNIK

Department of Pharmacology and Toxicology,
National Veterinary Research Institute, 24-100 Pulawy, Poland
[email protected]

Received for publication July 20, 2009


Abstract

A simple and short HPLC-UV screening method for the determination of residues for seven NSAIDs (carprofen,
diclofenac, flunixin, meloxicam, phenylbutazone, tolfenamic acid, vedaprofen) and their three metabolites (4-methylaminoantipyrine,
5-hydroxyflunixin, oxyphenbutazone) in cows milk has been developed. The sample preparation was based on liquid-liquid
extraction with acetonitrile in the presence of sodium chloride. The separation of analytes was performed on a C18 column with a
gradient of acetonitrile and the ammonium acetate buffer pH 5.0. UV-detector wavelength was programmed in order to improve
sensitivity. The method was validated according to the CD/2002/657 criteria. For most analytes, relatively high recoveries were
observed (76%-98%). Within-laboratory reproducibility levels were in the range of 3.6%-17.8% (CV, %). For phenylbutazone,
oxyphenbutazone, and 4-methylaminoantipyrine recoveries were considerably lower (44%-68%) and reproducibility was up to
41.9%, which was probably caused by the instability of analytes. The robustness of the method for different fat content was
successfully investigated. The method was verified by its use in the determination of meloxicam residues in milk samples obtained
from meloxicam-treated cows. The obtained results confirm the usefulness of the developed method for the analysis of NSAIDs
residues in milk.

Key words: milk, NSAIDs, residues, meloxicam, HPLC.


Non-steroidal anti-inflammatory drugs
(NSAIDs) are a group of compounds which show anti-
inflammatory, analgesic, and antipyretic properties.
Most of them are not chemically related but have a
common mechanism of action based on the inhibition of
the metabolism of arachidonic acid. NSAIDs are used in
veterinary medicine in the treatment of colic and
musculoskeletal disorders in horses, infectious diseases
in cattle, and as an aid in the treatment of mastitis
metritis agalactia syndrome in sows and mastitis in dairy
cows (5). A few NSAIDs are authorised for use in
milking cows. In the European Union, for some
NSAIDs, maximum residue limits (MRLs) in milk have
been established (10): flunixin (marker residue 5-
hydroxyflunixin) 40 g/kg, meloxicam 15 g/kg,
tolfenamic acid 50 g/kg, and metamizole (marker
residue 4-methylaminoantipyrine) 50 g/kg. For
ketoprofen and carprofen MRL value in bovine milk has
not been established because of the negligible risk
related to its authorised use. Most of NSAIDs used in
human medicine (diclofenac, ibuprofen, naproxen, etc.)
or approved for pets (vedaprofen, phenylbutazone) are
prohibited in the treatment of dairy cows.
According to European Union law (9), residues
of NSAIDs in milk have to be monitored because of the
potential risk to consumers health.
The methods for the determination of NSAIDs
in different matrices are widely described in the
literature. Sample preparation is based on liquid-liquid
extraction (21) with SPE clean-up (23). The
determination of these drugs in plasma (6), tissues (4),
and in environmental samples (22) is performed using
different chromatographic techniques: HPLC-UV (12,
14), HPLC-DAD (16, 18), LC-MS/MS (3, 7, 15), and
GC-MS (31). There are also several published methods
for the determination of NSAIDs in milk and most of
them are single-residue procedures. Single-step liquid
extraction was used in the determination of
phenylbutazone (13, 24), ketoprofen (25), ketoprofen
and flunixin (11) by LC-MS/MS, and metamizole and its
metabolites (28) by LC-MS.

732
Multi-residue methods for the determination of
NSAIDs in milk have also been published on. Stoyke et
al. (30) developed a multi-residue procedure for the
determination of 11 NSAIDs in milk. In this method,
samples were extracted with acetonitrile in the presence
of sodium chloride solution and cleaned-up with C18
columns. Final extracts were analysed by HPLC-DAD.
The same procedure was applied by Gallo et al. (17) for
the determination and confirmation of 16 NSAIDs in
milk by HPLC-DAD and LC/ESI-MS/MS. Despite the
wide range of analytes, none of the multi-residue
methods allows the determination of the residues of two
important drugs with MRL values in milk: meloxicam
and 4-methylaminoantipyrine simultaneously.
The objective of this study, therefore, was to
develop a simple screening method allowing
simultaneous determination of a wide spectrum of
NSAIDs (especially all substances with established
MRL values) in milk and to validate it according to the
requirements of residue control methods.


Material and Methods

Reagents. The analytical standards for NSAIDs
and some of their metabolites were supplied by the
following manufacturers: carprofen (CPF), diclofenac
(DC), meloxicam sodium (MEL), phenylbutazone
(PBZ), tolfenamic acid (TOL), oxyphenbutazone
hydrate (OPB) Sigma (USA), flunixin meglumine
(FLU) ISP (USA), vedaprofen (VED) - Akzo Nobel
(Netherlands), 4-methylaminoantipyrine (4MAA)
Hoechst (Germany), and 5-hydroxyflunixin (5OHFLU) -
Xenobiotic Laboratories (USA). CPF, TOL, VED,
4MAA, and 5OHFLU were donated by the Community
Reference Laboratory for Drug Residues in Berlin.
Acetonitrile (ACN), HPLC grade, was purchased from
Merck (Germany). Methanol (MeOH), HPLC grade, and
acetic acid (HPLC grade, <99%) was purchased from
J.T. Baker (Germany). Sodium chloride (NaCl) and
ammonium acetate (CH
3
COONH
4
) were purchased from
POCh (Poland). Ultrapure water (resistance >18 m)
was obtained from Mili-Q system (Millipore, France).
Standard solutions. All standard solutions
were prepared in methanol. Stock-standard solutions
(1,000 g/mL) were prepared by weighing 10.0 (0.1)
mg of standard substances and dissolving in 10 ml of
solvent. Stock-standard solutions were stable for 12
months when stored at 2-10C except of PBZ and OPB
solutions, which were stable for 1 month. Intermediate-
standard solutions (100 g/mL) were prepared by
diluting suitable aliquots of stock standard solutions.
Working-standard solution containing: 1.0 g/mL of
4MAA, DC, CPF, PBZ, TOL, and VED; 2.0 g/mL of
OPB, 0.8 g/mL of FLU, and 5OHFLU; 0.3 g/mL of
MEL used for milk sample fortification, was prepared
by diluting suitable aliquots of working-standard
solutions and was stable for 1 month.
Milk samples. For the method development
and validation, raw milk (free of NSAIDs residues) was
used. Mixed portions of compliant milk samples were
prepared from samples obtained and analysed in the
residue-control programme. Milk was checked for
possible interferences by HPLC, transferred to screw-
capped polypropylene tubes, and stored in the freezer at
a temperature below 18C until analysis.
For the verification of the possible influence of
the milks fat content on NSAIDs recovery, samples
(n=2) with different fat percentages: 2.17; 3.01; 4.04;
and 5.44 (analysed on milk analyser MilkoScan 4000)
were used. For the verification of the developed method,
milk samples with incurred meloxicam were used. Six
cows received a single intravenous dose of 0.5 mg/kg
b.w. of meloxicam (Metacam, Boehringer Ingelheim).
The milk samples were collected in the morning and
evening milking during six consecutive days. Every
yield of milk was mixed and samples of 50 ml were
transferred into polypropylene screw-capped tubes and
stored at a temperature below -18C until analysis.
Sample preparation. The sample of 5 (0.01)
g was weighed in a polypropylene tube. Five millilitres
of ACN and 1 g of NaCl was added and samples were
vigorously vortex-mixed for 1 min. Next, the tube was
centrifuged (4,500 g, 15 min, -5C) and the supernatant
was transferred to a glass tube and evaporated under a
gentle stream of nitrogen at 40C. The dry residue was
reconstituted in 0.5 ml of ACN:MeOH:0.05 M
CH
3
COONH
4
pH 5.0 (1+1+1, v+v+v) solution,
transferred to the autosampler vial (2 ml), centrifuged
(4,500 g, 5 min) and injected (50 l) into the HPLC
column.
Chromatographic conditions. The
instrumental analysis of NSAIDs was performed using
the Varian Prostar HPLC system, equipped with
quaternary pump, autosampler, column oven, and UV-
Vis detector, controlled by Galaxie Workstation
software. For the analysis of UV spectra of NSAIDs, the
Agilent 1100 system, equipped with quaternary pump,
degasser, autosampler, column oven, and diode-array
detector (DAD) and controlled by Chemstation software
was used. Chromatographic separation of the
compounds was performed on Inertsil ODS-3 column
(1504.6 mm, 5 m, GL Science, Japan) connected with
precolumn (43 mm, SecurityGuard, Phenomenex,
USA). The column oven temperature was controlled at
30C. The HPLC column was prepared with the mobile
phase consisting of 10% of acetonitrile (component A)
and 90% of 0.05 M CH
3
COONH
4
, pH 5.0 adjusted with
acetic acid (component B) at flow rate 1.2 ml/min. The
following 30 min gradient elution programme was
applied: 10% of A was held for 3 min, increased to 60%
of A at 15 min and held for 2 min, then increased to
80% of A at 20 min, and reduced to 10% of A at 25 min.
For the next 5 min 10% of A was pumped. The UV-Vis
detector was programmed to monitor the effluent of the
column for absorbance at 265 nm from 0.0 to 11.0 min,
365 nm from 11.0 to 12.4 min, and 290 nm from 12.4 to
30.0 min.
Validation method. A validation experiment
was performed to fulfil the requirements described in the
Commission Decision 2002/657/EC (8). Linearity was
verified by the preparation of calibration curves.

733
Table 1
Spiking levels used in validation of the method
Analyte* MRL
I spiking level
(g/kg)
II spiking level
(g/kg)
III spiking level
(g/kg)
4MAA 50 g/kg 25.0 50.0 75.0
5OHFLU 40 g/kg 20.0 40.0 60.0
CPF - 25.0 50.0 75.0
DC - 25.0 50.0 75.0
FLU - 20.0 40.0 60.0
MEL 15 g/kg 7.5 15.0 22.5
OPB - 50.0 100.0 150.0
PBZ - 25.0 50.0 75.0
TOL 50 g/kg 25.0 50.0 75.0
VED - 25.0 50.0 75.0
4MAA - 4-methylaminoantipyrine, 5OHFLU - 5-hydroxyflunixin, CPF carprofen, DC- diclofenac, FLU - flunixin meglumine,
MEL - meloxicam sodium, OPB - oxyphenbutazone hydrate, PBZ phenylbutazone, TOL - tolfenamic acid, VED vedaprofen.


Table 2
The absorption maxima of NSAIDs and wavelength programme for the UV-Vis detector
Compound
Maximum absorbance,
nm*
Wavelength programme
Retention time, min
(2.5%)
4MAA 245 265 (0.0-11.0 min ) 9.20 0.23
MEL 365 365 (11.0-12.4 min ) 12.00 0.3
5OHFLU 290 12.74 0.32
FLU 285 14.00 0.35
OPB 242 14.40 0.36
DC 280 15.30 0.38
CPF 300 15.60 0.39
PBZ 260 16.55 0.41
TOL 290 17.00 0.43
VED 285
290 (12.4-30.0 min)
21.00 0.53
* wavelengths obtained by analysis of standard of NSAIDs with HPLC-DAD. Abbreviations are explained in the footnote to
Fig. 1.

The mixed-standard solutions on five suitable
concentration levels were analysed and recorded peak
areas of each analyte were plotted versus concentration.
The equations and regression coefficients of the curves
were calculated. Linearity of calibration curves was
demonstrated with the F-test lack-of-fit and the working
range was established.
In order to investigate the specificity of the
method (potential interferences from the matrix and
other veterinary drugs), 20 blank milk samples from
different regions of Poland and standard mixtures of
sulfonamides, nitroimidazoles, and tetracyclines were
analysed.
The precision and accuracy of the method were
evaluated by the analysis of blank milk samples fortified
with NSAIDs on three different levels, specific for each
analyte. For MRL substances (5OHFLU, MEL, 4MAA,
TOL) 0.5MRL, 1MRL and 1.5MRL levels were
applied. For other compounds, spiking levels were
chosen by the authors (Table 1).
For the repeatability study, three series were
analysed (six samples for each spiking level). Standard
deviation (SD) and coefficient of variation (CV, %)
were calculated for each level. Additionally, two series
(on three levels) were analysed in reproducibility
conditions (two different occasions, another technician)
and overall SD and CV were calculated. The overall
mean value of concentration obtained in the
reproducibility study was used to calculate recovery
expressed as percentage. Decision limit (CC) and
detection capability (CC) were calculated based on the
procedure described in ISO 11843 (20). The limit of
detection (LOD) and limit of quantification (LOQ) were
calculated on the basis of the signal to noise ratio
(S/N=3 for LOD and S/N=10 for LOQ) on the
chromatograms of 20 blank milk samples.


Results

Chromatograms of a blank milk sample and a
sample fortified at II spiking level are presented in Fig.
1. The gradient elution of the mobile phase, consisting
of acetonitrile and ammonium acetate buffer (pH 5.0),
allows good separation of NSAIDs from matrix
components in 30 min run time (Fig. 1B). Despite the
simple and short sample preparation, chromatograms of
blank milk samples did not reveal any peaks from the
matrix in the retention times of analytes (Fig. 1A).


734
An analysis of UV spectra showed differences
in the absorption maxima of the respective NSAIDs;
therefore a programme of wavelengths in the UV
detector was introduced (Table 2).
The results of the method validation are
presented in Table 3. The calibration curves were
characterised by high values of regression coefficients
(r
2
>0.998). The results show relatively high recoveries
in the range of 44%-98% for most analytes. The
precision of the method was acceptable; CV values for
repeatability were in the range of 1.2%-20.1% and for
within-laboratory reproducibility 3.5%-41.9%. The
limits of detection (1.5-10 g/kg) show the high
sensitivity of the method.







Fig. 1. Chromatograms (A) - blank milk sample, (B) - milk sample spiked with NSAIDs on II spiking level.

4MAA - 4-methylaminoantipyrine, 5OHFLU - 5-hydroxyflunixin, CPF - carprofen, DC - diclofenac, FLU - flunixin
meglumine, MEL - meloxicam sodium, OPB - oxyphenbutazone hydrate, PBZ - phenylbutazone, TOL - tolfenamic
acid, VED - vedaprofen.




735



























Fig. 2. Recoveries of NSAIDs determined in milk with different fat levels.
Abbreviations are explained in the footnote to Fig. 1.






Fig. 3. Elimination profile of meloxicam in the milk of treated cows (n=6, average SD).

A full range of concentrations, B meloxicam concentrations around the MRL, LOD, and LOQ values.

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LOQ = 2.5 g/kg
LOD = 1.0 g/kg

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736


737
The results of the experiment on the influence
of milk fat content on NSAIDs recovery are presented in
Fig. 2. The analysis of spiked milk samples containing
different fat levels (2.17%-5.44%) shows similar
recoveries of the analytes, and their variability was
comparable with those obtained in the validation study.
The developed and validated method was
verified by the determination of meloxicam residues in
milk from the cows treated intravenously with Metacam.
The elimination profile of meloxicam in milk is
presented in Fig. 3. The highest concentration of
meloxicam determined in milk collected 12 h after the
treatment was 361 78 g/kg (average SD) (Fig. 3A).
The concentration dropped down in further milkings and
decreased below the MRL value (15 g/kg) between
days 2 and 3 after the treatment. Residues of meloxicam
were undetectable 4 d after the treatment (LOD 1.0
g/kg) (Fig. 3B).


Discussion

The developed chromatographic conditions
allow good separation and quantitative analysis of 7
NSAIDs and their three metabolites. According to the
authors knowledge, it is one of the first methods
allowing simultaneous HPLC determination of 4MAA
and the rest of NSAIDs.
The choice of the mobile phase pH was a
significant stage in the method optimisation. Some
authors (2) used acids solutions or buffers at low pH
value (2.0-3.0) for mobile phase, which was useful in
the analysis of most NSAIDs, but not suitable for the
separation of metamizole metabolites. The buffer at pH
5.0 significantly improved the peak shape of 4MAA and
additionally shortened the retention times (RTs) of
NSAIDs (1). The obtained RTs were stable for all
analytes and they fitted into the 2.5% interval as
required by Commission Decision 2002/657/EC (8).
The use of the wavelength programme for the
UV-Vis detector greatly improved the sensitivity of the
method for MEL and 4MAA (Table 3). Additionally, the
use of the mobile phase at pH of 5.0 resulted in a lower
LOD value for PBZ because it has a higher absorption
maximum at higher pH values (240 nm at pH 3.0 against
264 nm at pH 7.0) (24).
Relatively simple and fast sample preparation
was developed. The authors have taken advantage of the
experience of Neto et al. (26), who used acetonitrile for
the deproteinisation of plasma and Daeselaire et al. (11)
for deproteinisation of milk samples. Acetonitrile is a
very useful solvent due to its good solubility of drugs
and ability to precipitate proteins in the sample.
Nevertheless, because of its miscibility with water,
sodium chloride was added to allow separation of
phases. Stoyke et al. (30) extracted NSAIDs from milk
with acetonitrile and NaCl solution. Peck et al. (27)
observed that the addition of NaCl (crystal) to serum or
plasma before the extraction provides cleaner extracts in
comparison to acetonitrile alone. A low temperature (-
5C) during centrifugation additionally improved the
separation of phases. For the evaporation of extracts, a
relatively low temperature (40C) was applied because
the tendency to degradation of some the analytes (PBZ
and OPB) had been previously reported (21). The
sample preparation described above allows the
extraction of up to 30 samples concurrently. Some
described methods are based on solid-phase extraction,
e.g. Stoyke et al. (30) and Rupp et al. (29) with C18
cartridges. Our trials with different SPE cartridges (C18,
Oasis HLB, anion and cation exchange, mixed mode)
indicated problems with developing one clean-up
procedure for the acidic NSAIDs, MEL, and 4-MAA
(non published data). Single-step liquid extraction
ensured good sample clean-up without SPE technique
and allowed the determination of a wider spectrum of
analytes in comparison to the other published methods.
The results of validation show the good
precision and accuracy of the described method.
However, for the three analytes recoveries were
considerably lower: 4MAA - 44-53%, OPB - 62-68%,
and PBZ - 66-67%. In our opinion, it can be explained
by the instability of these compounds. Some authors
reported the fact of PBZ and OPB degradation (6, 19);
however, we have not found any published data on
4MAA stability. This was also indirectly confirmed by
the visibly lower precision of determination of the
mentioned analytes (CV up to 41.9%). Calculated CC
and CC values are used in the residue control for the
correct interpretation of results, according to
Commission Decision 2002/657/EC. The LOD and LOQ
values characterised the sensitivity of the method, and
are useful in pharmacokinetic and residue elimination
studies. The obtained values prove the high sensitivity of
the method and its usefulness for the mentioned
purposes. CC and CC values were comparable to
those obtained by Stoyke et al. (30).
Fat content can be an important factor
influencing the methods performance, because of
lipophilicity of some drugs. Martin et al. (24) performed
a study of phenylbutazone recovery dependence on fat
content in milk sample (0.5%, 3.0%, 3.5%-4.4%). The
robustness of our method for this factor was investigated
for all determined analytes. Stable recoveries of all
NSAIDs have confirmed the independence of the
method for fat content.
The comparison of the results of meloxicam
elimination from milk is difficult because we have not
found any data, except for a summary report published
by the European Medicine Agency (EMEA) (32). The
EMEA report described longer elimination of
meloxicam from milk: concentrations above MRL were
noticed after 5 d post dosing and residues were detected
to 9 d post dosing (LOD 2.5 g/kg). Meloxicam
concentrations in milk collected 6 h post dosing were
347 g/kg and 325 g/kg for high and low milk yield,
respectively. The values obtained in our study had good
correlation with values reported by the EMEA.
In conclusion, the presented method is one of
the first published screening multi-residue procedures
for the determination of NSAIDs residues in milk.
Simple sample preparation and liquid chromatography
with UV detection allows determining drugs with


738
sufficient sensitivity. The method has been successfully
validated and verified in the determination of meloxicam
in the tested samples of cows milk.


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