A P P L I C AT I O N N O T E

Liquid Chromatography/
Mass Spectrometry

Authors:
Sharanya Reddy
PerkinElmer, Inc.
Shelton, CT

Workflow for
Quantification of Introduction

Benzodiazepines in Urine There is a great need by

forensic toxicologists to develop

Using UHPLC- TOF robust analytical methods to

accurately and quickly measure
benzodiazepines in biological
fluids including urine in cases such as sexual assault and fatalities.
Immunoassays have traditionally been used for testing of benzodiazepines but this
approach can be challenging giving false positive results – needing confirmation
by techniques such as GC/MS. Immunoassays are not always sensitive enough to
detect low levels of the drug in matrices such as urine and blood.
 GC/MS offers its own challenges in the analysis of Experimental
benzodiazepines as most of the compound classes are polar A workflow for the quantification of benzodiazepines is shown
and often thermally labile, thus requiring derivatization prior to in Figure 1.
analysis. Unlike GC/MS, LC/MS does not require time consuming
derivatization of samples and is ideally suited for the rapid analysis Calibration Curve(s)
of these compounds. Among the LC techniques, LC/MS/MS is Urine (0.5 mL) was diluted with 0.5 mL of methanol containing
often used to quantitate drugs of abuse (non-clinical applications) varying concentrations of a mixture of benzodiazepines. 5 µL of
in biological fluids due to its sensitivity and selectivity. the sample was injected on column. Each calibration level was
injected five times.
We present an alternative technique to quantitate
benzodiazepines in urine utilizing a rapid dilute and shoot LC LC conditions:
method in combination with time-of-flight mass spectrometry Pump: PerkinElmer Flexar™ FX-15 UHPLC pump
(TOF MS). The detection limits of benzodiazepines analyzed by Flow: 0.4 mL/min
the TOF were 100-200 times lower than those required by Mobile phase A: Water (0.1% formic acid)
non-specific immunoassays. In addition to the wide quantitative Mobile phase B: Acetonitrile (0.1% formic acid)
dynamic range of the AxION® 2 TOF MS, which rivals Gradient conditions: 20% B to 90% B over four minutes
capabilities of the triple quadrupole instruments, the TOF also Injection volume: 5 μL in partial fill mode
provides full spectral information, allowing for screening of Column: PerkinElmer Brownlee™ SPP C-18, 2.1x50 mm,
non-target compounds. 2.7 μm (part number N9308402), 25 °C

In this application note we present a rapid workflow for the MS conditions:

quantification of benzodiazepines in urine. Mass spectrometer: PerkinElmer AxION 2 TOF MS
Ionization source: PerkinElmer Ultraspray™ 2 (Dual ESI source)
Ionization mode: Positive
Internal calibration was performed using m/z 195.0876 and
622.02896 as lock mass ions.

Targeted Compounds to Screen

ANALYTE
N DIAZEPAM
N OXAZEPAM
N
N
TEMAZEPAM
ALPRALOZAM
NO Negative
N FLUNITRAZEPAM

Dilute and Compound Quantification & Confirmation Interrogate data

shoot detected YES Accurate mass LC/MS ID unknowns
sample Y/N?
Y CHLONAZEPAM
Y LORAZEPAM
Y MIDAZOLAM
Y NORDIAZEPAM
Y α-HYDROXY
ALPRALOZAM
Y α-HYDROXY
TRIAZOLAN

Figure 1. Workflow for identification and quantification of benzodiazepines in urine.

2
 Results
To rapidly identify the presence or absence of compounds in large Quantification
batches of samples, AxION Solo™ software was used. AxION Solo The overall assay sensitivity was determined to be in the
provides quick visualization of the presence or absence of analytes 1- 2 ng/mL range for all of the drugs spiked into urine, (Table 1).
in the samples (Figure 2). The presence of an individual compound The limit of quantification (LOQs) measured by the TOF instrument
can be coded with a specific color for ease of identification. The were 100-200 times more sensitive than what is required by the
software identifies the presence of a compound based on non-specific EMIT immunoassays.
accurate mass and isotope profile ratio as shown in Figure 3. In
When analyzing such low levels of compound, carryover must be
addition to searching against spectral information, the software
assessed to ensure that the assay is suitable for use. In spite of the
can also search for target analytes based on user defined retention
low LOQs provided by the TOF MS, 0% carryover was observed
time windows which further improves the specificity of detection.
after an injection of the upper limit of quantification (ULOQ)
The list of target analytes can be quickly and easily added to as
mixture of the benzodiazepines.
previously unknown analytes are detected in samples. The analysis
of benzodiazepines was completed in < 3 min. (Figure 4) with all The linearity of a representative drug, diazepam, is shown in
peaks eluting before 2.7 minutes. Figure 5. The assay showed linearity over four orders with an r2
value of 0.998. The majority of the benzodiazepines analyzed
showed linearity between 3-4 orders of dynamic range with r2
values of 0.99 (Table 2). Multiple injections (n=5) of each
calibration level showed excellent reproducibility (RSDs< 15%) for
each of the drugs. The presence of a given drug in a urine sample
can be confirmed by accurate mass and isotope profile provided
by TOF MS. As shown in Table 3, the accurate masses of each of
the benzodiazepines are < 3 ppm.

Table 1. Shows the LOQs of the benzodiazepines.

Analyte LOQ (ng /mL)

Figure 2. AxION Solo Software: The top left hand corner shows the presence (pink)
Diazepam 1
and the absence (grey) of chlonazepam in different samples (vials). The remaining Oxazepam 2
benzodiazepines detected in the selected vial are displayed in the table (lower left).
Temazepam 2
Alpralozam 2
Flunitrazepam 2
Chlonazepam 2
Lorazepam 2
Midazolam 2
Nordiazepam 2
α-Hydroxy Alpralozam 2
α-Hydroxy Triazolan 2

Figure 3. The
Fig 3. The accurate accurate
mass mass of chlonazepam
of chlonazepam for A,are
for A, A+1, A+1,
< are < 2 ppm. The isotope
2ppm.
ratios for A+1, A+2 are within 5% of expected ratios.
The isotope ratios for A+1, A+2 are within 5% of expected ratio
nordiazepam

diazepam
midalozam

alpralozam

flunitrazepam
α-hydroxyalpralozam

temazepam
α-hydroxytriazolan

oxazepam

chlonazepam
lorazepam

Figure 5. Shows linearity for diazepam spiked in urine over 1-10,000 ng/mL
concentration
Fig. 5. Shows range
linearity for (r2 = 0.998).
diazepam spiked in urine over 1- 10,000 ng/mL concentration
range(r2 = 0.998).

Fig. 4.4.Analysis
Figure Analysisofofbenzodiazepams
benzodiazepinesbybyUHPLC-TOF
UHPLC-TOFMS spiked in urine
MS spiked < 3 mins.
in urine < 3 mins.

3
 Table 2. Shows the linear dynamic range and regression for each of the Table 3. Shows the theoretical
Analyte Theoriticalmass,
mass ofobserved mass and
Observed massmass
of error of benzodiazepines.
ppm Structure
Analyte Theoritical mass of
benzodiazepams Observed mass of
benzodiazepams ppm
error Structure
benzodiazepines spiked in urine as matrix. Analyte Analyte Theoritical mass of
Theoritical massof Observed mass
ofbenzodiazepams
Observed of of ppm
mass ppm Structure
Structure
Analyte Theoretical Mass
benzodiazepams
Theoritical
benzodiazepams of
mass Observed Mass
Observed massof
of ppm
error
ppm Structure
benzodiazepams benzodiazepams
benzodiazepams errorerror
Analyte Concentration range (ng/mL) r2 Analyte benzodiazepams
Benzodiazepines benzodiazepams
Benzodiazepines error
Error Structure
Diazepam 1-10,000 0.998 α-OH triazolan 359.0461 359.0470 -2.5 ppm
Oxazepam 2-10,000 0.995 α-OH α-OH
α-OH Triazolan
triazolan
triazolan
α-OH triazolan 359.0461
α-OHtriazolan 359.0461
359.0461 359.0470
359.0461 359.0470
359.0470
359.0470
359.0470 -2.5
-2.5
-2.5 ppm
-2.5ppm
-2.5 ppm
ppm
ppm

Temazepam 2-10,000 0.996 α-OH alpralozam 325.0851 325.0856 -1.5 ppm

α-OH
α-OH Alpralozam
alpralozam
α-OH
alpralozam325.0851
α-OHalpralozam
α-OH
alpralozam 325.0851
325.0856
325.0856
325.0851 325.0856
325.0856
325.0856
-1.5 ppm
-1.5
-1.5
-1.5
-1.5
ppm
ppm
ppm
ppm
Alpralozam 2-10,000 0.995 325.0851
325.0851

Flunitrazepam 2-10,000 0.997 nordiazepam

Nordiazepam
nordiazepam
nordiazepam 271.0633
271.0633
271.0633
271.0633
271.0640
271.0640
271.0640
271.0640
-2.5
-2.5 ppm
ppm
-2.5
-2.5 ppm
ppm
nordiazepam
nordiazepam 271.0633
271.0633 271.0640
271.0640 -2.5
-2.5 ppm
ppm
Chlonazepam 2-5,000 0.997
Lorazepam 2-5,000 0.998 Midalozam
midalozam
midalozam
midalozam 326.0855
326.0855
326.0855
326.0855 326.0848
326.0848
326.0848
326.0848 2.1
2.1ppm
2.1
2.1 ppm
ppm
ppm
midalozam
midalozam 326.0855 326.0848
326.0848 2.1
2.1 ppm
ppm
326.0855
Midazolam 2-2,500 0.999
Lorazepam
lorazepam
lorazepam
lorazepam 321.0192
321.0192
321.0192
321.0192 321.0185
321.0185
321.0185
321.0185 2.2
2.2ppm
2.2 ppm
2.2 ppm
ppm
Nordiazepam 2-10,000 0.997 lorazepam 321.0192 321.0185 2.2 ppm
lorazepamchlonazepam 316.0483
321.0192
316.0481
321.0185 0.6 ppm
2.2 ppm
chlonazepam
chlonazepam 316.0483 316.0481
316.0481 0.6
0.6ppm
ppm
α-Hydroxy Alpralozam 2-5,000 0.997 chlonazepam316.0483316.0483
Chlonazepam 316.0483 316.0481
316.0481 0.6
0.6 ppm
ppm
chlonazepam 316.0483 316.0481 0.6 ppm
chlonazepam 316.0483 316.0481 0.6 ppm
α-Hydroxy Triazolan 2-10,000 0.991 flunitrazepam 314.0935 314.0941 -1.9 ppm
flunitrazepam
flunitrazepam 314.0935 314.0941
314.0941 -1.9
-1.9ppm
ppm
Flunitrazepam 314.0935314.0935
flunitrazepam
flunitrazepam 314.0935
314.0935
314.0941
314.0941
314.0941 -1.9
-1.9ppm
ppm
-1.9 ppm
flunitrazepam 314.0935 314.0941 -1.9 ppm
Conclusions alpralozam
alpralozam 309.0902 309.0898
309.0898
1.3 ppm
1.3
alpralozam 309.0902 309.0898 1.3ppm
ppm
Alpralozamalpralozam 309.0902309.0902
alpralozam 309.0902 309.0898
309.0902 309.0898
309.0898 1.3
1.3 ppm
1.3 ppm
ppm
The method required little to no sample preparation or method temazepam
alpralozam 309.0902 309.0898 1.3 ppm
temazepam
temazepam
development, saving hours of time and the use of costly reagents temazepam 301.0738
temazepam
temazepam 301.0738
301.0738
301.0735
301.0735
301.0735
301.0735
0.9
0.9ppm
0.9 ppm
ppm
0.9 ppm
Temazepam 301.0738 301.0738
301.0738 301.0735
301.0738
301.0735
301.0735 0.9
0.9 ppm
0.9 ppm
ppm
and consumables. The AxION 2 TOF was easily able to identify
1-2 ng/mL concentration of benzodiazepines spiked in urine. The oxazepam
oxazepam
oxazepam
oxazepam
oxazepam
287.0582287.0582287.0587
287.0582
287.0582 287.0587
287.0587
287.0587
287.0587 -1.7
-1.7ppm
-1.7ppm
-1.7ppm
ppm
-1.7 ppm
Oxazepamoxazepam 287.0582 287.0582
287.0582 287.0587
287.0587 -1.7
-1.7ppm
ppm
detection limits of these drugs were 100-200 times lower than
diazepamdiazepam 285.0783 2.1 ppm
285.0789 285.0783 2.1 ppm
diazepam
diazepam 285.0789285.0783
285.0789 285.0783 2.1
2.1 ppm
ppm
that required by immunoassays. The AxION 2 TOF with the ADC diazepam 285.0789
285.0789
Diazepamdiazepam 285.0789 285.0789
285.0783
285.0783
285.0783
2.1 ppm
2.1 ppm
2.1 ppm

detector technology provides wide dynamic range capabilities Table

Table 3.
3. Shows
Shows the
the theoritical
theoritical mass,
mass, observed
observed mass
mass and
and mass
mass error
error of
of benzodiazepams
benzodiazepams
TableTable
Table
3. 3.3. Shows
Shows the the
Shows thetheoritical
theoritical
theoritical mass,
mass, observed
mass, observed
observed mass
mass
mass and
andandmass error
masserror
mass ofofbenzodiazepams
errorof benzodiazepams
benzodiazepams
similar to that of a triple quadrupole mass spectrometer and also Table 3. Shows the theoritical mass, observed mass and mass error of benzodiazepams

offers the testing of untargeted compounds. For rapid large scale

testing of batches of samples PerkinElmer AxION Solo software
provides a quick and easy platform to detect the presence or
absence of benzodiazepines.

For Research Use Only. Not for Use in Diagnostic Procedures.

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