Anal Bioanal Chem (2014) 406:803–818
DOI 10.1007/s00216-013-7513-x
RESEARCH PAPER
Detection and quantification of benzodiazepines and Z-drugs
in human whole blood, plasma, and serum samples as part
of a comprehensive multi-analyte LC-MS/MS approach
Deborah Montenarh & Markus Hopf & Hans H. Maurer &
Peter Schmidt & Andreas H. Ewald
Received: 14 August 2013 / Revised: 17 October 2013 / Accepted: 14 November 2013 / Published online: 6 December 2013
# Springer-Verlag Berlin Heidelberg 2013
Abstract For the first time, a liquid chromatography-tandem
mass spectrometry (LC-MS/MS) multi-analyte approach
based on a simple liquid–liquid extraction was developed
and validated for fast target screening and quantification of
benzodiazepines and Z-drugs in case of driving ability and
crime responsibility in the three most important biosamples
whole blood, plasma, and serum. Whole blood, plasma, and
serum (500 μL each) were extracted twice at pH 7.4 and at pH
10 with ether/ethyl acetate (1:1). Separation, detection, and
quantification were performed using LC-MS/MS with
electrospray ionization in positive mode. The method was
validated with respect to selectivity, ion suppression/
enhancement of co-eluting analytes, matrix effects, recovery,
process efficiency, accuracy and precision, stabilities, and
limits of detection and quantification. For accuracy and pre-
cision, full calibration was performed with ranges from sub-
therapeutic to toxic concentrations. The presented LC-MS/MS
approach as part of a universal multi-analyte concept for over
100 drugs was applicable for selective detection as well as
accurate and precise quantification in whole blood, plasma,
and serum. The approach was selective, sensitive, accurate,
and precise for 16 of the 19 tested drugs in whole blood, 18 in
plasma, and 17 in serum. Only semiquantitative results could
D. Montenarh : M. Hopf : P. Schmidt : A. H. Ewald (*)
Institute of Legal Medicine, Saarland University, Kirrberger Str.
Building 42, 66421 Homburg, Saarland, Germany
e-mail: andreas.ewald@uks.eu
H. H. Maurer
Department of Experimental and Clinical Toxicology, Institute of
Experimental and Clinical Pharmacology and Toxicology, Saarland
University, Kirrberger Str. Building 46, 66421 Homburg, Saarland,
Germany
be obtained for zopiclone because of its instability in all tested
biosamples.
Keywords HPLC-MS/MS . Benzodiazepines . Plasma .
Serum . Whole blood . Z-drugs
Introduction
In clinical and forensic toxicology, screening and quantifica-
tion of different drug classes is a very important task. For
assessing toxic effects, blood is the sample of choice because
blood levels correlate best with clinical effects. However,
depending on sampling, storing, and/or analysis type, whole
blood, plasma, or serum is used. Whole blood includes all
cellular particles such as erythrocytes and leukocytes, and it is
not stable and coagulates within a few minutes into a half solid
mass named coagulum [1]. Coagulum includes modified plas-
ma, blood cells, and fibrin. After centrifugation, the liquid
phase named serum contains less fibrinogen than plasma but
more phosphate and sodium ions. Plasma is formed by cen-
trifugation of blood treated with anticoagulants [1]. Thus, new
approaches should allow accurate quantification in all these
samples. For saving time and resources, multi-analyte proce-
dures for screening and quantification of various drugs using
liquid chromatography-tandem mass spectrometry (LC-MS/
MS) in body fluids have been published, but dealt only with
one body sample each [2–9]. Therefore, a new LC-MS/MS
multi-analyte procedure should be developed and validated
for screening and quantification of various drugs such as
benzodiazepines, antidepressants, neuroleptics, opioids, new
synthetic drugs, or phosphodiesterase type 5 (PDE-5) inhibi-
tors in the three different blood samples after simple liquid–
liquid extraction (LLE). The first part should cover
804
benzodiazepines and Z-drugs. These drugs may lead to seda-
tive, hypnotic, anxiolytic, anticonvulsant, and muscle-
relaxing effects. These effects may result in slow and uncer-
tain reflexes and can therefore influence the driving ability
[10]. In combination with alcohol and other CNS-active sub-
stances, they can lead to severe poisonings or even death [11,
12]. In addition, it may result in poor judgment, which could
lead to diminished responsibility in cases of crime. So far, LC-
MS multi-analyte procedures for benzodiazepines have been
described using LC-MS [13, 14] or LC-MS/MS [3, 14, 15],
but only for one type of blood sample. Therefore, in the
following, an LC-MS/MS multi-analyte approach for benzo-
diazepines and Z-drugs in whole blood, plasma, and serum
will be presented after standard LLE approved for various
applications [13, 16, 17].
Experimental
Chemicals
The reference substances (1 mg/mL) of the studied analytes
were obtained from Promochem (Wesel, Germany).
Ammonium formate (analytical grade) was from Fluka
(Neu-Ulm, Germany), formic acid (mass spec grade) from
AppliChem (Darmstadt, Germany), acetonitrile from Sigma-
Aldrich (Seelze, Germany), and methanol and water (all LC/
MS grade) from VWR (Darmstadt, Germany).
Biosamples
Human blank plasma, serum, and whole blood samples were
used for the development and validation of the procedure and
were obtained from a local blood bank. The whole blood,
plasma, and serum samples were stored at −20 °C until use.
Apparatus
The LC-MS/MS system consisted of a Shimadzu Prominence
LC 20 AC system which consisted of a degasser, a binary
pump, and an autosampler coupled to an AB SCIEX 3200
Q-TRAP® linear ion trap quadrupole mass spectrometer
(AB SCIEX, Darmstadt, Germany) operated in the positive
electrospray ionization (ESI+) mode. Separation was achieved
with gradient elution on a Waters SunFire C18 column
(2.1×150 mm, 3.5 μm). The mobile phase consisted of
10 mM aqueous ammonium formate plus 0.1 % formic acid
pH 3.4 (eluent A) and acetonitrile plus 0.1 % formic acid
(eluent B). The flow rate was set to 500 μL/min and the
gradient was programmed as follows: 0–3 min 100 % A,
3–4 min 90 % A, 4–12 min 65 % A, 12–22.5 min 5 % A, and
22.5–30 min 100 % A. The column oven was set at 30 °C and
the autosampler was operated at room temperature.
D. Montenarh et al.
For detection, the following ESI inlet conditions were
applied: gas 1, nitrogen (100 psi); gas 2, nitrogen (100 psi);
ion spray voltage, + 5,500 V; ion source temperature, 600 °C;
and curtain gas, nitrogen (60 psi). The mass spectrometer was
operated in the multiple reaction monitoring (MRM) mode
with the collision gas set at medium. The dwell times were
optimized using scheduled MRM algorithm incorporated in
the AB SCIEX Analyst® software 1.6. All other settings were
analyte specific and were determined using Analyst® software
in the quantitative optimization mode (Table 1). An MRM
method with 396 transitions in positive ionization mode for
130 analytes was established as a survey scan. The MRM
transitions were only analyzed at a time window of 60 s, and
the total cycle time of the MRM mode was 2.1 s including the
pause time between the MRM transitions of 2 ms.
For data evaluation, the Analyst® software 1.6 was used to
obtain peak areas. The chromatograms of the 16 benzodiaze-
pines and 3 Z-drugs are shown in Fig. 1.
Whole blood, plasma, and serum sample extraction
Samples were extracted using a modified LLE as described
elsewhere [18]. Briefly, to 50 μL of the stable isotope-labeled
internal standard (SIL-IS; 0.02 mg/mL trimipramine-d3),
300 μL Sørensen phosphate buffer (10 mM), 300 μL sodium
bicarbonate, 500 μL whole blood, plasma, or serum, and 1,
000 μL diethyl ether-ethyl acetate (50/50) were added; the
mixture was vortexed for 30 s and centrifuged (8 min, 1,
780×g). The organic layer was transferred to a vial, 150 μL
sodium hydroxide was added to the aqueous layer, pH was
adjusted to 10, and 1,000 μL diethyl ether-ethyl acetate
(50/50) was added; the mixture was vortexed and centrifuged
(8 min, 1,780×g). The organic layer was combined and evap-
orated to dryness under N2 at 60 °C. The residue was dis-
solved in 100 μL methanol and used for LC-MS/MS screen-
ing and quantification.
Preparation of stock solutions, calibration standards,
and control samples
Separate methanolic stock solutions of each analyte for cali-
bration and quality control samples (QC samples), respective-
ly, were diluted with methanol to obtain working solutions
(0.01 and 0.1 mg/mL). The spiking solutions for calibration
standards and QC samples were prepared by adding the cal-
culated amount of the corresponding stock or working solu-
tion to volumetric flasks and filled up to 5 mL to get the
corresponding whole blood, plasma, and serum concentration
(Tables 2 and 3). All solutions were stored at +4 °C.
Calibration standards and QC samples were prepared freshly
every day using 500 μL of blank whole blood, plasma, and
serum and 50 μL of the corresponding spiking solution. QC
Detection and quantification of benzodiazepines and Z-drugs 805

Table 1 Drugs, Q1 and Q3 mass , declustering potential, entrance potential, collision cell entrance potential, collision energy, and collision cell exit
potential

Drug Q1 Q3 Declustering Entrance Collision cell entrance Collision Collision cell exit
mass mass potential (V) potential (V) potential (V) energy (V) potential (V)

Alprazolam 309.132 281.200 31 4.5 22 37 4

Alprazolam 309.132 205.000 31 4.5 22 57 4
Alprazolam 309.132 274.200 31 4.5 22 35 10
Bromazepam 316.015 182.200 36 7.0 18 41 4
Bromazepam 316.015 209.200 36 7.0 18 33 4
Bromazepam 316.015 181.200 36 7.0 18 57 4
Chlordiazepoxide 300.191 283.300 51 3.0 14 21 4
Chlordiazepoxide 300.191 282.000 51 3.0 14 37 4
Chlordiazepoxide 300.191 227.200 51 3.0 14 37 8
Clonazepam 316.002 270.100 41 6.0 12 35 6
Clonazepam 316.002 214.000 41 6.0 12 55 4
Clonazepam 316.002 241.000 41 6.0 12 45 4
Diazepam 285.162 193.100 46 6.0 8 39 4
Diazepam 285.162 154.100 46 6.0 8 39 4
Diazepam 285.162 222.200 46 6.0 8 37 4
Flunitrazepam 314.149 268.300 41 11.0 10 35 4
Flunitrazepam 314.149 239.100 41 11.0 10 47 4
Flunitrazepam 314.149 183.100 41 11.0 10 63 4
N-Desalkylflurazepam 289.064 140.200 66 10.5 14 39 2
N-Desalkylflurazepam 289.064 226.200 66 10.5 14 37 6
N-Desalkylflurazepam 289.064 104.100 66 10.5 14 69 4
Lorazepam 321.077 275.000 31 5.0 14 29 6
Lorazepam 321.077 303.000 31 5.0 14 21 4
Lorazepam 321.077 229.000 31 5.0 14 39 4
Lormetazepam 335.063 289.100 26 5.0 18 29 4
Lormetazepam 335.063 317.200 26 5.0 18 19 12
Lormetazepam 335.063 177.100 26 5.0 18 53 4
Midazolam 326.156 291.200 56 11.0 18 35 4
Midazolam 326.156 249.100 56 11.0 18 55 8
Midazolam 326.156 222.200 56 11.0 18 65 6
Nitrazepam 282.021 236.100 46 10.5 20 37 6
Nitrazepam 282.021 180.000 46 10.5 20 47 4
Nitrazepam 282.021 207.200 46 10.5 20 45 4
Nordazepam 271.182 140.100 36 3.0 30 35 6
Nordazepam 271.182 165.300 36 3.0 30 33 4
Nordazepam 271.182 208.200 36 3.0 30 35 4
Oxazepam 287.200 241.100 46 5.5 28 25 4
Oxazepam 287.200 269.300 46 5.5 28 19 6
Oxazepam 287.200 104.200 46 5.5 28 43 4
Temazepam 301.094 255.100 31 5.0 18 29 4
Temazepam 301.094 283.100 31 5.0 18 19 10
Temazepam 301.094 177.100 31 5.0 18 51 4
Tetrazepam 289.098 197.100 41 8.0 34 49 4
Tetrazepam 289.098 225.200 41 8.0 34 33 8
Tetrazepam 289.098 115.000 41 8.0 34 97 2
Zaleplon 306.038 236.100 51 5.5 24 39 4
Zaleplon 306.038 264.200 51 5.5 24 29 4
806 D. Montenarh et al.

Table 1 (continued)

Drug Q1 Q3 Declustering Entrance Collision cell entrance Collision Collision cell exit
mass mass potential (V) potential (V) potential (V) energy (V) potential (V)

Zaleplon 306.038 209.100 51 5.5 24 45 4

Zolpidem 308.102 235.100 51 9.0 26 41 4
Zolpidem 308.102 236.100 51 9.0 26 37 4
Zolpidem 308.102 263.200 51 9.0 26 35 6
Zopiclone 388.944 244.900 16 5.5 26 27 4
Zopiclone 388.944 111.900 16 5.5 26 83 4
Zopiclone 388.944 217.000 16 5.5 26 43 4
7-Aminoflunitrazepam 284.125 135.200 51 10.0 12 39 4
7-Aminoflunitrazepam 284.125 227.200 51 10.0 12 31 4
7-Aminoflunitrazepam 284.125 226.200 51 10.0 12 39 4

samples were prepared at three different concentrations PDE-5 inhibitors to check for interfering signals in the ben-
(low, med, and high). zodiazepines, Z-drugs, and SIL-IS transitions.

Assay validation Recovery, matrix effects, and process efficiencies

Selectivity As proposed by Matuszewski et al. [19], extraction efficien-

Ten blank whole blood, plasma, and serum samples from
different sources were analyzed for peaks interfering with
the detection of the analytes and the SIL-IS. Two zero samples
(blank sample plus internal standards) were analyzed to check
for the absence of signals in the respective analyte peaks
caused by the internal standard. Two blank samples spiked
with the spiking solutions of calibration standard 6 were
analyzed to check for the absence of interfering signals of
the analytes in the internal standard peaks. In addition, blank
whole blood, plasma, and serum samples were spiked with
spiking solutions of QC high of the drug classes of antide-
pressants, neuroleptics, opioids, new synthetic drugs, and
cies and matrix effects were calculated using three different
sets of samples. For the analysis of recovery (RE), matrix
effect (ME), and process efficiencies (PE), three sets of
samples were prepared at QC low and high concentra-
tions. Set 1 represents the neat standard containing the
analytes, set 2 represents blank sample spiked after extrac-
tion, and set 3 consists of blank sample spiked before extrac-
tion. RE results were obtained by comparison of the absolute
peak areas of sample set 3 with those of the corresponding
peaks in sample set 2. MEs were estimated by comparing the
peak areas of sample set 2 with those in set 1. For PE, peak
areas of set 3 were compared with the corresponding peaks in
set 1.

Fig. 1 Chromatograms of 16
benzodiazepines and 3 Z-drugs in
low concentration. From front to
back: alprazolam (black),
bromazepam (brown),
chlordiazepoxide (green),
clonazepam (dark blue),
diazepam (orange),
desalkylflurazepam (red),
flunitrazepam (indigo),
lorazepam (gray), lormetazepam
(pink), midazolam (steel blue),
nitrazepam (yellow green),
nordazepam (teal), oxazepam
(yellow), temazepam (purple),
tetrazepam (steel blue), zaleplon
(chocolate), zolpidem (olive),
zopiclone (lime green), and 7-
aminoflunitrazepam (dark slate
gray)
Detection and quantification of benzodiazepines and Z-drugs 807

Table 2 Lower and upper therapeutic plasma concentrations [24, 25], LOD, LLOQ, and plasma concentrations for calibration levels 1–6 and QC
samples

Drugs Lower Upper Plasma concentration (mg/L)

therapeutic therapeutic
plasma conc. plasma conc. LOD LLOQ Cal. 1 Cal. 2 Cal. 3 Cal. 4 Cal. 5 Cal. 6 QC QC QC
(mg/L) (mg/L) low med high

Alprazolam 0.005 0.08 0.0025 0.005 0.005 0.01 0.025 0.075 0.15 0.3 0.006 0.05 0.25
Bromazepam 0.08 0.2 0.01 0.04 0.04 0.06 0.08 0.1 0.2 0.4 0.05 0.09 0.3
Chlordiazepoxide 0.4 4.0 0.005 0.2 0.2 0.4 0.8 1.6 3.0 4.0 0.3 1.0 3.5
Clonazepam 0.02 0.08 0.01 0.02 0.02 0.04 0.06 0.08 0.09 0.15 0.03 0.07 0.1
Diazepam 0.1 2.0 0.05 0.1 0.1 0.2 0.4 0.8 1.6 3.2 0.125 0.7 2.5
Flunitrazepam 0.005 0.015 0.003 0.01 0.01 0.02 0.03 0.04 0.05 0.06 0.015 0.035 0.055
N-Desalkylflurazepam 0.04 0.15 0.003 0.02 0.02 0.03 0.04 0.08 0.1 0.3 0.035 0.060.25
Lorazepam 0.02 0.25 0.005 0.03 0.03 0.04 0.06 0.12 0.25 0.5 0.035 0.080.4
Lormetazepam 0.002 0.025 0.005 0.0075 0.0075 0.01 0.02 0.03 0.04 0.06 0.0085 0.025
0.05
Midazolam 0.04 0.25 0.001 0.02 0.02 0.04 0.08 0.15 0.25 0.5 0.03 0.1 0.4
Nitrazepam 0.03 0.12 0.01 0.02 0.02 0.03 0.04 0.06 0.12 0.4 0.025 0.050.3
Nordazepam 0.2 1.8 0.04 0.1 0.1 0.2 0.4 0.8 1.6 3.2 0.125 0.7 2.5
Oxazepam 0.2 2.0 0.1 0.2 0.2 0.4 0.8 1.0 2.0 3.2 0.3 0.9 2.5
Temazepam 0.02 0.9 0.008 0.01 0.01 0.02 0.1 0.5 1.0 2.0 0.015 0.3 1.5
Tetrazepam 0.05 1.0 0.002 0.025 0.025 0.05 0.1 0.5 1.0 2.0 0.03 0.3 1.5
Zaleplon 0.001 0.1 0.01 0.02 0.02 0.03 0.04 0.05 0.1 0.2 0.025 0.045
0.15
Zolpidem 0.08 0.3 0.001 0.04 0.04 0.08 0.1 0.2 0.3 0.6 0.05 0.2 0.5
Zopiclone 0.01 0.05 0.3 0.45 0.9 0.8
7-Aminoflunitrazepam 0.01 0.03 0.002 0.0075 0.0075 0.01 0.02 0.03 0.04 0.06 0.0085 0.025 0.05

Ion suppression and enhancement of co-eluting analytes
The effect of ion suppression/enhancement was tested at a
medium concentration by comparing peak areas of the ana-
lyte, when the co-eluting analyte is present, to the peak area in
the absence of the co-eluting analyte.
Linearity of calibration
For 6 days, aliquots of blank whole blood, plasma, and serum
(500 μL) were spiked with 50 μL of the calibration standard
spiking solutions (calibrator 1–calibrator 6), extracted, and
analyzed as described above to perform linearity experiments.
The regression lines were calculated using non-weighted, a
weighted [1/concentration], and a weighted [1/(concentration)2]
least-squares regression model. Second-order models with the
same weighting factors were also calculated. The best combi-
nation was used in the following experiments.
The back-calculated concentrations of all calibration sam-
ples were compared with their respective nominal values.
Calibrators whose back-calculated concentrations deviated
more than 20 % from their respective nominal values were
excluded from the calculations of the daily calibration curves.
If more than two calibrators were outside these limits, the
whole calibration curve and the corresponding results were
excluded from the calculations.
Calibration
Quantification of all analytes was carried out by comparison
of peak area ratios (analyte vs. SIL-ISs) to calibration curves
in which the peak areas of spiked calibrators had been plotted
against their theoretical concentrations (see above).
Accuracy and precision
The QC samples (low, med, high) were analyzed according to
the extraction procedures described above in duplicate on each
of 8 days. The concentrations of the analytes in the QC
samples were calculated via the daily calibration curves and
using the different possible combinations of weighting, gained
during calibration model experiments.
Accuracy was calculated for each analyte in terms of bias
as the percent deviation of the mean calculated concentration
at each concentration level from the corresponding theoretical
concentration. Repeatability (within-day precision) and time-
different intermediate precision were calculated (as relative
standard deviation) according to Peters et al. [20].
808 D. Montenarh et al.

Table 3 Lower and upper therapeutic plasma concentrations [24, 25], LOD, LLOQ, and serum and whole blood concentrations for calibration levels 1–
6 and QC samples

Drugs Lower therapeutic Upper therapeutic Serum/whole blood concentration (mg/L)

plasma conc. plasma conc.
(mg/L) (mg/L) LOD LLOQ Cal. 1 Cal. 2 Cal. 3 Cal. 4 Cal. 5 Cal. 6 QC QC QC
low med high

Alprazolam 0.005 0.08 0.0025 0.005 0.005 0.01 0.025 0.075 0.15 0.3 0.006 0.05 0.25
Bromazepam 0.08 0.2 0.01 0.04 0.04 0.06 0.08 0.1 0.2 0.4 0.05 0.09 0.3
Chlordiazepoxide 0.4 4.0 0.005 0.2 0.2 0.4 0.8 1.6 3.0 4.0 0.3 1.0 3.5
Clonazepam 0.02 0.08 0.02 0.04 0.04 0.05 0.06 0.08 0.09 0.15 0.03 0.07 0.1
Diazepam 0.1 2.0 0.05 0.1 0.1 0.2 0.4 0.8 1.6 3.2 0.125 0.7 2.5
Flunitrazepam 0.005 0.015 0.003 0.01 0.01 0.02 0.03 0.04 0.05 0.06 0.015 0.035 0.055
N-Desalkylflurazepam 0.04 0.15 0.003 0.02 0.02 0.03 0.04 0.08 0.1 0.3 0.035 0.060.25
Lorazepam 0.02 0.25 0.005 0.03 0.03 0.04 0.06 0.12 0.25 0.5 0.035 0.080.4
Lormetazepam 0.002 0.025 0.006 0.0075 0.0075 0.01 0.02 0.03 0.04 0.06 0.0085 0.025
0.05
Midazolam 0.04 0.25 0.001 0.02 0.02 0.04 0.08 0.15 0.25 0.5 0.03 0.1 0.4
Nitrazepam 0.03 0.12 0.01 0.02 0.02 0.03 0.04 0.06 0.12 0.4 0.025 0.050.3
Nordazepam 0.2 1.8 0.04 0.1 0.1 0.2 0.4 0.8 1.6 3.2 0.125 0.7 2.5
Oxazepam 0.2 2.0 0.1 0.2 0.2 0.4 0.8 1.0 2.0 3.2 0.3 0.9 2.5
Temazepam 0.02 0.9 0.008 0.01 0.01 0.02 0.1 0.5 1.0 2.0 0.015 0.3 1.5
Tetrazepam 0.05 1.0 0.002 0.025 0.025 0.05 0.1 0.5 1.0 2.0 0.03 0.3 1.5
Zaleplon 0.001 0.1 0.01 0.02 0.02 0.03 0.04 0.05 0.1 0.2 0.025 0.045
0.15
Zolpidem 0.08 0.3 0.001 0.04 0.04 0.08 0.1 0.2 0.3 0.6 0.05 0.2 0.5
Zopiclone 0.01 0.05 0.3 0.45 0.9 0.8
7-Aminoflunitrazepam 0.01 0.03 0.002 0.0075 0.0075 0.01 0.02 0.03 0.04 0.06 0.0085 0.025 0.05

The regression lines were calculated using non-weighted, a 21 h, thawed, and kept at room temperature for 3 h. The
weighted [1/concentration], and a weighted [1/(concentration)2] concentrations of the QC samples were calculated based on
least-squares regression model. Second-order models with the the daily calibration curves. The internal standard was added
same weighting factors were also calculated. just before extraction. To assure stability, the ratio of means
(stability/control) has to be within 90–110 %, and the 90 %
Processed sample stability confidence interval has to be within 80–120 % from the
control sample.
For the estimation of the stability of processed samples under
the conditions of LC-MS/MS analysis, low and high QC
Lower limit of quantification and limit of detection
samples (n =10 each) were extracted as described above.
The resulting extracts at each concentration level were pooled.
The lowest point of the calibration curve was defined as the
Aliquots of these pooled extracts at each concentration level
lowest limit of quantification (LLOQ) of the method (concen-
were transferred to autosampler vials and injected under the
tration listed in Tables 2 and 3). The limit of detection (LOD)
conditions of a regular analytical run at time intervals of 5 h
was determined as this concentration where the signal-to-
over a total run time of 25 h. Stability of the extracted drugs
noise ratio of 3:1 for quantifier and qualifier was given and
was tested by regression analysis plotting absolute peak areas
the peak could be clearly detected.
of each drug at each concentration vs. injection time.

Freeze/thaw stability and bench top stability Statistical data evaluation

For the evaluation of freeze/thaw stability, QC samples (low Variances of the recovery, matrix effects, and process efficien-
and high) were analyzed before (control samples, n =6) and cy data between plasma and serum, plasma, and whole blood
after three freeze/thaw cycles (stability samples, n =6). For and between serum and whole blood were tested with an
each freeze/thaw cycle, the samples were frozen at -20 °C for unpaired two-tailed Student’s t test followed by an f test to
Detection and quantification of benzodiazepines and Z-drugs
compare the variances between the groups using GraphPad
Prism 5.00 (GraphPad Software, San Diego, CA, USA).
Proof of applicability
Whole blood, plasma, and serum samples from authentic
cases and proficiency tests were assayed with the described
method. The calculated concentrations of the external QC
samples were compared with the certified concentrations.
Results and discussion
The aim of this study was to develop and validate an LC-MS/
MS method for accurate and precise quantification of benzo-
diazepines in whole blood, plasma, and serum as part of a
multi-analyte method for 130 analytes of different drug clas-
ses such as benzodiazepines, neuroleptics, antidepressants,
PDE-5 inhibitors, and opioids. The described method was
validated according to recommendations on method valida-
tion published by the Society of Toxicological and Forensic
Chemistry (GTFCh) [20] and other international guidelines
[21, 22].
Extraction and separation
Simple LLE was used as universal approach allowing work-
up for various drugs, methods, and in all types of blood
samples. In contrast to the original approach for plasma ex-
traction [Kratzsch, MPW 2011], the pH (7.4 and 10) had to be
adjusted for whole blood and (stored) serum. For plasma and
freshly serum samples, the original approach from Kratzsch
et al. with double-distillated water could be used because the
pH of these samples was always around 7. For a lot of tested
whole blood samples or long stored serum samples, the pH
ranged from 5–9; so for these samples, our modified LLE had
to be used. Whole blood includes all cellular parts, which
could be the reason of different pH values. For simplicity,
we used our modified LLE for all samples. This LLE proce-
dure was preferred in contrast to that described by Remane
et al. [2, 3] because all validated GC-MS procedures in the
authors’ laboratory were also based on this.
Reversed-phase (C18) separation with gradient elution
(27 min) was performed for the 130 analytes. Chromatograms
of the MRM transitions for the studied analytes (Table 1) of QC
low after LLE are shown in Fig. 1.
Selectivity
Ten blank whole blood, plasma, and serum samples were
analyzed for interfering signals in MRM transitions of
analytes and SIL-IS. All biosamples showed no interfering
809
signals in the analyte and the SIL-IS transitions. Two zero
samples were analyzed to check for interfering peaks in the
analyte MRM transitions caused by the internal standard. The
two blank samples spiked with the spiking solutions of cali-
bration standard 6 showed no interferences in the internal
standard transitions caused by the analytes. No interfering
signals were observed in the blank samples spiked with spik-
ing solutions of QC high of the drug classes of antidepres-
sants, neuroleptics, opioids, new synthetic drugs, and PDE-5
inhibitors.
Recovery, matrix effects, and process efficiencies
Having shown that there were no selectivity problems, RE,
ME, and PE were determined. Standard deviation for RE, ME,
and PE should be within the limits of 20 % for low concen-
trations and 15 % for high concentration. ME values below
100 % indicate ion suppression, while values above 100 %
indicate ion enhancement. The full RE, ME, and PE data for
QC low and high for whole blood, plasma, and serum are
listed in Table 4. For all biosamples, the median RE for QC
low was 98 % (72–235 %) for QC high 88 % (74–97 %), with
two analytes above or below the acceptance criteria for
QC low coefficient variation (CV); the median ME for
QC low was 93 % (78–133 %) and for QC high 98 %
(55–142 %) with two (QC high and low) analytes above
or below the acceptance criteria for the CV. The analytes
above or below the acceptance criteria regarding RE were
lormetazepam (plasma, serum) and tetrazepam (plasma)
(both QC low). For ME in whole blood, zolpidem and
7-aminoflunitrazepam (QC low and high) were the analytes
above or below the acceptance criteria as also shown in Fig. 2
for high concentration.
Ion suppression and enhancement of co-eluting analytes
No significant enhancements or suppression of co-eluting
analytes from the same drug class were observed for the
analyte pairs: alprazolam/nitrazepam and flunitrazepam/
nordazepam. If these combinations of co-eluting analytes are
both present in the same sample, quantification can be done
using calibration solutions including both analytes.
Calibration model
The calibration curves should range from half of the lower to
double of the upper therapeutic concentration for all analytes.
These limits were chosen to detect the lower therapeutic
concentrations and to cover poisoning as well. For alprazo-
lam, clonazepam, flunitrazepam, lorazepam, lormetazepam,
nitrazepam, oxazepam, and zaleplon, 7-aminoflunitrazepam
calibrator 1 could not be set at the half of the lower therapeutic
 810

Table 4 Drugs, recovery, matrix effects, and process efficiency for low and high concentrations in plasma, serum and whole blood. Values above or below the acceptance criteria are marked in bold

Drug Low High

Plasma Serum Whole blood Plasma Serum Whole blood

RE % ME % PE % RE % ME % PE % RE % ME % PE % RE % ME % PE % RE % ME % PE % RE % ME % PE %
(CV %) (CV %) (CV %) (CV %) (CV %) (CV %) (CV %) (CV %) (CV %) (CV %) (CV %) (CV %) (CV %) (CV %) (CV %) (CV %) (CV %) (CV %)

Alprazolam 76 (12) 92 (11) 74 (12) 83 (8) 88 (13) 65 (9) 83 (10) 103 (11) 85 (4) 88 (6) 91 (9) 80 (3) 86 (7) 96 (3) 83 (4) 87 (11) 109 (10) 94 (11)
Bromazepam 89 (7) 94 (14) 82 (7) 76 (8) 102 (11) 78 (12) 83 (8) 104 (0.7) 87 (8) 87 (7) 96 (5) 84 (2) 83 (3) 98 (6) 82 (5) 88 (15) 98 (812) 95 (8)
Chlordiazepoxide 84 (6) 88 (7) 81 (12) 83 (5) 95 (13) 100 (0.1) 91 (4) 102 (4) 92 (3) 92 (3) 97 (2) 90 (2) 89 (5) 104 (1) 92 (5) 89 (11) 101 (13) 91 (13)
Clonazepam 83 (12) 95 (11) 80 (9) 103 (16) 108 (6) 106 (21) 87 (7) 96 (7) 83 (9) 86 (4) 100 (3) 87 (6) 97 (6) 96 (8) 96 (11) 81 (9) 87 (15) 67 (23)
Diazepam 124 (19) 78 (13) 95 (10) 87 (7) 89 (15) 78 (15) 85 (6) 88 (7) 76 (8) 87 (5) 75 (12) 66 (7) 91 (3) 94 (2) 87 (2) 89 (7) 93 (13) 84 (16)
Flunitrazepam 76 (10) 88 (11) 68 (5) 86 (16) 115 (14) 82 (15) 90 (10) 96 (5) 86 (6) 90 (6) 93 (8) 86 (8) 88 (5) 105 (7) 89 (10) 80 (12) 125 (14) 101 (12)
N-Desalkylflurazepam 78 (9) 95 (17) 75 (9) 85 (5) 109 (8) 93 (11) 88 (5) 98 (6) 88 (8) 96 (5) 85 (10) 80 (6) 90 (2) 96 (3) 87 (4) 93 (9) 98 (4) 100 (13)
Lorazepam 86 (10) 90 (11) 76 (4) 98 (10) 93 (8) 91 (4) 89 (9) 97 (8) 86 (11) 90 (3) 96 (3) 87 (4) 91 (4) 96 (3) 87 (4) 81 (6) 99 (15) 86 (15)
Lormetazepam 74 (9) 95 (18) 71 (13) 72 (18) 94 (16) 68 (19) 86 (4) 99 (6) 86 (4) 93 (6) 81 (3) 76 (7) 84 (4) 92 (2) 78 (3) 88 (15) 111 (7) 109 (9)
Midazolam 82 (7) 95 (12) 77 (6) 81 (8) 100 (6) 81 (9) 89 (6) 100 (4) 90 (4) 93 (3) 95 (5) 89 (4) 95 (2) 97 (1) 92 (3) 74 (13) 701 (14) 103 (12)
Nitrazepam 97 (6) 88 (12) 81 (9) 81 (9) 99 (6) 81 (4) 90 (4) 105 (5) 94 (10) 91 (3) 95 (4) 88 (3) 95 (4) 89 (3) 85 (3) 96 (15) 109 (11) 106 (9)
Nordazepam 79 (6) 89 (16) 67 (6) 86 (7) 94 (5) 82 (8) 87 (3) 93 (5) 81 (3) 92 (2) 92 (4) 84 (4) 94 (1) 97 (4) 92 (4) 90 (11) 100 (11) 92 (10)
Oxazepam 96 (5) 89 (9) 81 (9) 100 (42) 85 (11) 89 (44) 97 (10) 97 (10) 85 (9) 89 (4) 97 (3) 86 (3) 90 (11) 101 (3) 89 (9) 82 (9) 113 (11) 91 (14)
Temazepam 78 10) 85 (11) 65 (8) 83 (16) 94 (17) 82 (7) 87 (1) 98 (5) 85 (5) 91 (3) 89 (3) 81 (1) 93 (7) 97 (4) 91 (6) 92 (15) 100 (10) 101 (5)
Tetrazepam 235 (16) 91 (19) 196 (31) 85 (14) 81 (20) 57 (16) 78 (7) 81 (20) 273 (32) 96 (4) 85 (5) 82 (9) 87 (7) 95 (7) 82 (6) 90 (11) 107 (11) 95 (8)
Zaleplon 90 (12) 90 (10) 81 (10) 87 (8) 118 (18) 88 (16) 95 (13) 100 (6) 96 (14) 87 (5) 94 (6) 81 (2) 88 (5) 97 (7) 85 (5) 78 (13) 102 (12) 102 (10)
Zolpidem 115 (9) 91 (12) 104 (10) 81 (8) 104 (13) 84 (12) 89 (5) 133 (9) 118 (6) 90 (2) 99 (3) 889 (3) 88 (9) 108 (5) 97 (7) 81 (11) 142 (20) 113 (26)
Zopiclone – – – – – – – – – – – – – – – – – –
7-Aminoflunitrazepam 80 (12) 93 (9) 75 (9) 80 (5) 91 (16) 79 (16) 100 (14) 74 (9) 74 (4) 92 (11) 76 (12) 75 (12) 83 (8) 88 (13) 83 (32) 88 (21) 55 (17) 86 (80)

RE recovery, ME matrix effects, PE process efficiency

D. Montenarh et al.
Detection and quantification of benzodiazepines and Z-drugs
Fig. 2 Acceptance criteria
of zolpidem and 7-
aminoflunitrazepam
concentration because they are too low due to the low drug
doses. The therapeutic concentrations and chosen calibrator
concentrations are listed in Tables 2 and 3. As there were
several possibilities using different calibration models, the
final decision was made after the evaluation of the accuracy
and precision data.
All analytes from calibrators 1 to 6 were extracted using
LLE from whole blood, plasma, and serum as described above
and checked for outliers. Two outliers in different calibration
levels are allowed. Outliers were detected for chlordiazepox-
ide in calibration level 2, for diazepam in calibration levels 1
and 2, for dealkyl-flunitrazepam in calibration level 5, for
flunitrazepam in calibration level 3, and for nitrazepam in
calibration level 2. The calibration model for all analytes in
all biosamples with the exception of nordazepam (in plasma
and serum) and zolpidem (in whole blood, serum, and plasma)
was linear using different weighted [1/concentration, 1/
concentration2] or non-weighted models. For nordazepam
and zolpidem, a quadratic-weighted [1/concentration] regres-
sion model was used. The calibration model for diazepam,
tetrazepam, and oxazepam in all three biosamples was linear,
but different weighting models had to be used [1/concentra-
tion, 1/concentration2] for the different biosamples with the
exception of oxazepam in plasma, where a non-weighted
model was used.
Accuracy and precision
QC samples (low, med, and high) were analyzed in duplicate
on each of 8 days as it has been proposed by Peters et al. [23].
The concentrations in the QC samples were calculated based
on the daily calibration curves. All calibration samples ful-
filled the acceptance limits (not more than 15 % deviation
from the respective nominal concentrations; 20 % near the
LOQ). Accuracy, repeatability, and time different intermediate
precision were calculated as described above. The results
811
obtained using full calibrations are shown in Tables 5, 6, and
7. The results above the mentioned acceptance criteria of 20 %
for QC low and 15 % for high are marked in bold. With the
exception of tetrazepam in plasma, tetrazepam and 7-
aminoflunitrazepam in serum, and alprazolam, tetrazepam,
and 7-aminoflunitrazepam in whole blood, all analytes ful-
filled the validation parameter range. As examples, the accu-
racy data for midazolam in low and high concentrations for all
three biosamples are shown in Fig. 3.
Statistical data evaluation
The results of the statistical evaluation of the variances of the
recovery, matrix effects, and process efficiency data between
plasma and serum, plasma and whole blood, and between
serum and whole blood are summarized in Table 8.
The ME for serum vs. plasma differs not significant with
the exception of diazepam in high concentration. This might
be due to the similar composition, but as figured out in
Table 8, ME for serum vs. whole blood showed more signif-
icant variances than for plasma vs. whole blood. A reason for
that could be found in the slight differences between plasma
and serum, e.g., fibrinogen is present in plasma and whole
blood, but not in serum. There are some differences for RE
between whole blood, serum, and plasma samples. The RE for
tetrazepam in QC low was 78 and 85 % for serum and whole
blood samples, but these differences are not significant as
shown in Table 8, which means we found less tetrazepam in
an extracted sample than in a methanolic solution. In plasma
samples, the RE for tetrazepam was 235 %, so the signal of
tetrazepam in plasma samples was enhanced, and the result is
significantly different to those for serum and whole blood. By
calculating a tetrazepam concentration in a plasma sample via
a serum or whole blood calibration curve, this would give a
false result. For lormetazepam, suppression was shown in all
three samples, but for serum and plasma samples, we
 812

Table 5 Drugs, used fitting, used weighting, nominal concentration, mean measured concentration, accuracy, repeatability, and intermediate precision for QC low, med, and high calculated using full
calibration for whole blood (results above the validation acceptance criteria marked in bold)

Drugs Fitting Weighting QC low QC med QC high

Nominal Mean Accuracy Repeatability Intermediate Nominal Mean Accuracy Repeatability Intermediate Nominal Mean Accuracy Repeatability Inter-
conc. cal. conc. bias [%] CV [%] precision conc. cal. conc. bias [%] CV [%] precision conc. cal. conc. bias [%] CV [%] mediate
[μg/L] [μg/L] CV [%] [μg/L] [μg/L] CV [%] [μg/L] [μg/L] precision
CV [%]

Alprazolam L 1/x 2 6 4.63 −22.84 6.22 18.98 60 56.66 −5.56 4.26 10.78 250 199.44 −20.23 9.28 11.53
Bromazepam L 1/x 2 50 55.79 11.59 8.81 13.05 90 83.38 −7.35 6.17 8.85 300 284.0 −5.33 6.04 10.70
Chlordiazepoxide L 1/x 300 310.81 3.60 6.03 10.47 1,000 1,016.69 1.67 4.05 6.27 3,500 3,205.63 −8.41 6.47 8.63
Clonazepam L eq 45 49.23 9.39 9.24 19.57 70 69.05 −4.21 7.87 7.46 90 87.56 −2.72 7.78 12.04
Diazepam L 1/x 2 125 106.14 −15.09 7.69 18.22 700 716.31 2.33 5.59 6.18 2,500 2,205.63 −11.78 6.07 11.30
Flunitrazepam L 1/x 15 14.45 −3.67 10.69 13.93 25 27.63 10.53 3.83 13.39 40 39.68 −0.81 6.68
N-Desalkylflurazepam L 1/x 2 35 32.24 −7.88 7.55 10.24 60 58.17 −3.06 8.38 14.34 250 250.38 0.95 10.99 13.43
Lorazepam L 1/x 2 35 35.04 0.11 5.60 13.77 70 75.89 8.41 5.36 7.82 400 400.13 0.03 8.04 11.69
Lormetazepam L 1/x 8.5 8.09 −4.83 11.08 14.37 25 24.01 −3.95 9.23 12.49 50 48.11 −3.78 9.96 9.66
Midazolam L 1/x 30 32.84 9.46 5.71 8.58 100 99.61 −0.39 4.01 6.34 400 370.63 −7.34 7.28 12
Nitrazepam L 1/x 2 20 19.78 −1.09 8.71 16.10 50 48.71 −2.59 5.07 5.55 400 384.56 −3.86 6.54 9.55
Nordazepam L 1/x 2 125 100.01 −19.99 11.77 11.19 700 735.0 5.0 5.59 8.47 2,500 2,178.13 −12.88 6.71 9.15
Oxazepam L 1/x 300 288.75 −3.75 6.42 14.61 900 800.44 −11.06 4.58 4.89 2,500 2,341.88 −6.33 5.79 10.14
Temazepam L 1/x 2 15 15.83 5.54 8.42 17.25 300 317.69 5.90 5.62 5.72 1,500 1,275.63 −14.96 6.61 8.23
Tetrazepam L 1/x 2 30 28.40 −5.33 8.99 17.31 300 303.31 1.10 7.92 7.31 1,500 2,243.75 49.58 8.99 12.41
Zaleplon L 1/x 2 25 25.26 1.03 8.56 10.83 45 42.88 −4.72 6.68 8.04 150 148.69 −0.88 4.81 10.76
Zolpidem Q 1/x 50 45.82 −8.36 7.55 15.13 200 194.75 −2.63 5.31 6.52 500 478.81 −4.24 8.03 12.43
Zopiclone L 1/x – – – – – – – – – – 800 850.31 6.25 10.31 7.51
7-Aminoflunitrazepam L 1/x 2 8.5 9.92 16.71 9.17 23.49 25 26.19 4.78 9.95 14.17 50 53.13 6.26 6.56 10.67

L linear, Q quadratic
D. Montenarh et al.
Table 6 Drugs, used fitting, used weighting, nominal concentration, mean measured concentration, accuracy, repeatability, and intermediate precision for QC low, med, and high calculated using full
calibration for plasma (results above the validation acceptance criteria marked in bold)

Drugs Fitting Weighting QC low QC med QC high

Nominal Mean Accuracy Repeatability Intermediate Nominal Mean Accuracy Repeatability Inter-mediate Nominal Mean Accuracy Repeatability Intermediate
conc. cal. conc. bias [%] CV [%] precision conc. cal. conc. bias [%] CV [%] precision conc. cal. conc. bias [%] CV [%] precision
[μg/L] [μg/L] CV [%] [μg/L] [μg/L] CV [%] [μg/L] [μg/L] CV [%]

Alprazolam L 1/x 2 6 6.37 6.09 10.05 12.61 50 55.10 10.20 10.56 12.90 250 212.75 −14.90 3.66 7.85
Bromazepam L 1/x 2 50 49.50 −1.00 14.81 13.11 90 89.91 −0.10 10.95 10.14 300 301.06 0.35 6.46 8.95
Chlordiazepoxide L 1/x 300 317.13 5.71 13.34 12.91 1,000 1,123.75 12.38 7.44 7.90 3,500 3,195.63 3.72 4.66
Detection and quantification of benzodiazepines and Z-drugs

−8.70
Clonazepam L eq 45 24.84 −0.65 19.36 17.94 70 47.61 −4.79 11.41 13.37 90 96.13 6.81 7.75 12.74
Diazepam L 1/x 2 125 128.56 2.85 9.86 11.08 700 732.88 4.70 11.45 8.81 2,500 2,226.25 −10.95 5.92 12.88
Flunitrazepam L 1/x 15 16.26 8.42 10.09 13.48 25 35.87 2.48 8.51 7.91 40 55.53 0.97 6.02 8.11
N-Desalkylflurazepam L 1/x 35 33.48 −4.36 6.54 11.10 60 56.54 −5.77 6.04 6.33 250 253.19 0.88 5.56 6.67
Lorazepam L 1/x 2 35 16.14 7.63 9.66 12.12 70 82.69 3.37 9.49 9.13 400 419.94 4.98 3.71 6.12
Lormetazepam L 1/x 8.5 7.53 0.44 12.03 14.38 25 26.81 7.25 12.26 12.91 50 55.38 10.76 5.77 11.97
Midazolam L 1/x 30 30.23 0.77 10.84 14.58 100 108.99 8.99 8.64 9.22 400 396.81 −0.80 3.42 6.63
Nitrazepam L 1/x 2 20 22.66 −9.35 7.91 6.72 50 46.36 7.29 6.17 6.82 400 420.44 5.11 4.32 8.91
Nordazepam Q 1/x 125 122.75 −1.80 8.67 8.52 700 719.69 2.81 6.35 8.94 2,500 2,273.75 −9.05 6.45 11.29
Oxazepam L eq 300 282.44 −5.85 12.52 18.02 900 1,022.88 13.65 8.62 8.32 2,500 2492.50 −0.30 6.81 11.06
Temazepam L 1/x 2 15 14.80 −1.33 11.30 17.12 300 302.44 0.81 11.55 13.66 1,500 1,360.00 −9.33 9.30 10.68
Tetrazepam L 1/x 2 30 31.32 4.40 12.98 18.43 300 330.56 10.19 5.64 10.10 1,500 1,852.50 23.50 5.09 9.64
Zaleplon L 1/x 2 25 15.19 1.25 11.93 10.90 45 39.15 −2.13 8.90 7.85 150 146.69 −2.21 6.67 7.46
Zolpidem Q 1/x 50 52.69 5.38 5.82 9.90 200 196.25 −1.88 5.84 10.22 500 499.25 −0.15 6.44 11.87
Zopiclone L 1/x – – – – – – – – – – 800 849.21 −0.08 4.42 3.32
7-Aminoflunitrazepam L 1/x 2 6.5 6.25 −3.80 13.88 14.15 15 15.84 5.58 7.50 11.78 50 49.80 0-40 4.08 6.64

L linear, Q quadratic
813
 814

Table 7 Drugs, used fitting, used weighting, nominal concentration, mean measured concentration, accuracy, repeatability, and intermediate precision for QC low, med, and high calculated using full
calibration for serum (results above the validation acceptance criteria marked in bold)

Drugs Fitting Weighting QC low QC med QC high

Nominal Mean Accuracy Repeatability Intermediate Nominal Mean Accuracy Repeatability Intermediate Nominal Mean Accuracy Repeatability Intermediate
conc. cal. conc. bias [%] CV [%] precision conc. cal. conc. bias [%] CV [%] precision conc. cal. conc. bias [%] CV [%] precision
[μg/L] [μg/L] CV [%] [μg/L] [μg/L] CV [%] [μg/L] [μg/L] CV [%]

Alprazolam L 1/x 2 6 4.99 −16.88 13.47 18.32 60 57.15 −4.75 8.43 7.84 250 233.44 −6.63 8.9 10.93
Bromazepam L 1/x 2 50 59.11 18.23 6.40 6.87 90 82.66 −8.16 4.84 6.94 300 330.25 10.08 5.79 10.69
Chlordiazepoxide L 1/x 300 341.25 13.75 6.40 9.43 1,000 1,083.94 8.39 5.22 9.61 3,500 3,548.13 1.38 6.00 10.51
Clonazepam L eq 45 49.88 10.85 5.15 7.98 70 61.82 −11.69 4.86 6.11 90 99.99 11.10 7.82 12.04
Diazepam L 1/x 125 123.33 −1.33 16.47 18.85 700 710.63 1.52 4.67 8.39 2,500 2,528.75 1.15 7.86 13.69
Flunitrazepam L 1/x 15 16.09 7.29 8.95 9.07 25 26.06 4.25 5.51 4.37 40 41.88 4.70 5.53 5.94
N-Desalkylflurazepam L 1/x 35 39.27 12.20 4.94 8.03 60 51.49 −14.19 3.29 5.79 250 244.06 −2.38 13.63 13.83
Lorazepam L 1/x 2 35 40.53 15.80 8.73 11.33 70 71.59 2.28 4.73 9.41 400 435.06 8.77 6.76 9.24
Lormetazepam L 1/x 8.5 9.52 12.04 8.61 19.36 25 24.08 −3.68 7.84 10.42 50 51.65 3.30 5.10 9.61
Midazolam L 1/x 30 30.48 1.58 9.84 15.69 100 104.24 4.24 7.34 8.77 400 411.38 2.84 6.51 9.72
Nitrazepam L 1/x 2 20 22.27 −10.93 10.67 15.02 50 44.76 −10.49 4.77 5.48 400 434.56 8.64 5.41 10.24
Nordazepam Q 1/x 125 135.00 8.00 10.18 13.68 700 691.00 −1.29 9.35 8.25 2,500 2,331.88 −6.73 6.89 13.13
Oxazepam L 1/x 2 300 344.75 14.92 9.24 14.51 900 790.81 −12.13 5.15 7.30 2,500 2,656.88 6.28 7.41 8.67
Temazepam L 1/x 2 15 14.76 −1.62 13.12 18.51 300 325.44 8.48 7.88 10.99 1,500 1,476.88 −1.54 8.39 11.77
Tetrazepam L 1/x 30 31.53 5.08 12.88 12.66 300 289.75 −3.42 9.18 10.12 1,500 2,253.13 50.21 7.07 11.88
Zaleplon L 1/x 2 25 25.89 3.58 7.94 11.97 45 39.81 −11.54 3.23 5.70 150 173.0 15.0 5.16 10.17
Zolpidem Q 1/x 50 51.26 12.51 18.69 19.57 200 185.44 −7.28 6.40 6.61 500 491.50 −1.70 5.98 8.16
Zopiclone L 1/x – – – – – – – – – – 800 854.54 6.25 10.31 7.51
7-Aminoflunitrazepam L 1/x 2 8.5 14.37 69.04 13.27 13.18 25 27.83 11.30 8.49 12.83 50 55.83 11.66 4.65 6.90

L linear, Q quadratic
D. Montenarh et al.
Detection and quantification of benzodiazepines and Z-drugs
Fig. 3 Accuracy data for midazolam in the three biosamples
observed more suppression than in whole blood samples, so
whole blood samples containing lormetazepam cannot be
calculated via a serum or plasma calibration and vice versa.
As shown in Table 8, the variances between plasma vs. whole
blood as well as serum vs. whole blood are significant. This is
only the fact for low concentration, so this effect seems to
depend on the concentration of lormetazepam in a sample. PE
of plasma vs. whole blood showed significant differences in
low concentration only for chlordiazepoxid and lorazepam,
but in high concentration for additional 11 substances, so that
a comparison of PE depends on the concentration.
In conclusion, whole blood concentrations of these
analytes could not be calculated via a serum or plasma cali-
bration curve.
Stability
All analytes were stable in extracts for a period of 25 h at
autosampler conditions of room temperature. During three
freeze/thaw cycles, all analytes were stable. Long-term stabil-
ity tests showed 4-month stability at −20 °C.
Limits of quantification and limits of detection
The LLOQs and LODs of all analytes are listed in Tables 2
and 3. The LLOQ was set at calibrator 1. For the determina-
tion of the LOD, the signal-to-noise ratio of quantifier and
815
qualifier MRM transitions has to be 3:1. The LLOQs were for
almost all analytes half of the lower therapeutic plasma con-
centration. The LLOQ of oxazepam was at the lower thera-
peutic concentration [24, 25], whereas the LLOQs of clonaz-
epam, flunitrazepam, lorazepam, and zaleplon were slightly
above the lower therapeutic concentrations.
Applicability
The new approach was tested for applicability by analyzing
more than 20 authentic serum samples from driving ability
cases. For example, Fig. 4 shows chromatograms of the
MRM transitions for the SIL-ISs and for nordazepam
(446 μg/mL), diazepam (29 μg/L), and alprazolam
(66 μg/L) identified in an authentic serum sample after LLE
(a) and for the SIL-ISs and for flunitrazepam (19 μg/mL),
7-aminoflunitrazepam (29 μg/mL), oxazepam (139 μg/mL),
midazolam (115 μg/mL), and lorazepam (139 μg/mL) in
the external QC plasma sample BZ 1/13 B after LLE
(b).
The quantitative results (mean of two measurements) of the
proficiency test (BZ 1/13) of the GTFCh along with the
respective target values and acceptance intervals are reported
in Table 9. All measured concentrations lay within the accep-
tance intervals, which further proved the applicability and
reliability of the described method. As zopiclone could not
be detected and did not fulfill the validation criteria for the
816 D. Montenarh et al.

Table 8 Drugs, F test to compare variances for recovery, matrix effects, and process efficiency for low and high concentrations for plasma vs. serum,
plasma vs. whole blood, serum vs. whole blood P value

Drug Low High

Plasma vs. Plasma vs. whole Serum vs. whole Plasma vs. Plasma vs. whole Serum vs. whole
serum blood blood serum blood blood

RE ME PE RE ME PE RE ME PE RE ME PE RE ME PE RE ME PE
P value summary P value summary P value summary P value summary P value summary P value summary

Alprazolam ns ns –* ns ns ns ns ns –*** ns ns ns ns ns –* ns –* –*
Bromazepam ns ns ns ns –*** ns ns –*** ns ns ns ns ns ns –* ns ns ns
Chlordiazepoxide ns ns –* ns ns –* ns –* –*** ns ns ns –* –** –** ns –** ns
Clonazepam ns ns –** ns ns ns ns ns –** ns ns ns –* –* –* ns ns ns
Diazepam ns ns ns –* ns ns ns ns ns ns –* ns ns ns –* ns –** –**
Flunitrazepam ns ns –** ns ns ns ns ns –** ns ns ns ns ns ns ns ns ns
N-Desalkylflurazepam –* ns ns –* ns ns ns ns ns ns ns ns ns ns ns –* ns ns
Lorazepam ns ns ns ns ns –* ns ns ns ns ns ns ns –** –* ns –* –**
Lormetazepam ns ns ns –** ns ns –* ns –* ns ns ns ns ns ns ns –** –*
Midazolam ns ns ns ns ns ns ns ns ns ns ns ns –* ns –* ns –*** –*
Nitrazepam ns ns ns ns ns ns ns ns ns ns ns ns –** ns –* ns –* –*
Nordazepam ns ns ns –** ns ns ns ns ns ns ns ns –** ns ns ns ns ns
Oxazepam –** ns –** ns ns ns –*** ns –** –* ns ns ns ns –* –** –* ns
Temazepam –* ns ns –*** ns ns –*** –* ns ns ns –* –** ns –* ns ns ns
Tetrazepam –* ns ns –* ns ns ns –* ns ns ns ns –* ns ns ns ns ns
Zaleplon ns ns ns ns ns ns ns –* ns ns ns ns ns ns –* ns ns ns
Zolpidem ns ns ns ns ns ns ns ns ns –* ns ns –* ns –*** ns –** –**
Zopiclone – – – – – – – – – – – – – – – – – –
7-Aminoflunitrazepam –* ns ns ns ns ns –* ns ns ns ns ns –* ns –* –* –* ns

RE recovery, ME matrix effects, PE process efficiency, ns not significant

*P =0.05–0.01, **P =0.01–0.001, ***P <0.001

Table 9 Drugs included in the proficiency tests, samples, nominal, target, calculated concentrations (in micrograms per liter, mean of two measurements),
bias (percent) from nominal and target concentrations, and accepted concentrations (in micrograms per liter)

Drug Sample Nominal Target conc. Calculated Bias from nominal Bias from target Accepted Proficiency
conc. [μg/L] [μg/L] conc. [μg/L] conc. [%] conc. [%] conc. [μg/L] test passed?

Proficiency test BZ 1/13

Alprazolam A 30.0 29.5 29.4 −2 −0.3 13.3–45.7 Yes
Bromazepam A 110.0 104.0 116.2 5.6 11.7 56.0–152 Yes
Clonazepam A 95.0 91.2 106.4 12 16.6 49.2–133.2 Yes
Diazepam A 310.0 318.0 236.5 −23.7 −25.6 196.0–440.0 Yes
Flunitrazepam B 15.0 15.0 19.0 26.6 26.6 5.8–24.2 Yes
Lorazepam B 125.0 119.0 139.6 11.6 17.3 65.0–173 Yes
Midazolam B 110.0 107.0 115.6 5.0 8.0 59.0–155.0 Yes
Nordazepam A 285.0 285.0 246.7 −13.4 −13.4 173.0–397.0 Yes
Oxazepam B 240.0 219.0 139.7 −41.7 −36.2 129.0–309.0 Yes
Temazepam A 85.0 84.3 87.5 2.9 3.7 45.1–123.5 Yes
Zolpidem A 120.0 125.0 107.6 −10.3 −13.9 69.0–181.0 Yes
Zopiclone B 50.0 50.3 – – – 24.9–75.7 No
7-Aminoflunitrazepam B 25.0 23.1 29.5 18.0 27.7 9.9–36.3 Yes
Detection and quantification of benzodiazepines and Z-drugs 817

Fig. 4 Chromatograms of the

MRM transitions for the
SIL-ISs and for nordazepam,
diazepam, and alprazolam
identified in an authentic serum
sample after LLE (a) and for the
SILISs and for flunitrazepam, 7-
aminoflunitrazepam, oxazepam,
midazolam, and lorazepam in the
external QC plasma sample BZ 1/
13 B after LLE (b)

intermediate precision, the proficiency test was failed. The
nominal concentration was lower than our LOD for zopiclone.
In summary, selectivity problems could not be observed
for any of the three biosamples. For clonazepam, the LOD
in whole blood could not be set as low as for plasma and
serum. For zolpidem and 7-aminoflunitrazepam, matrix
effects were observed only for whole blood, but not for
plasma and serum. Whole blood includes more matrix
compounds such as cellular particles and can therefore
lead to higher matrix effects. In whole blood, accurate
and precise quantification for 16 of the tested drugs, in
serum for 18, and in plasma for 17 of the 19 tested drugs
was possible. Because of significant variances for ME,
RE, and PE between the three biosamples, a calibration
curve from the same biosample is needed for quantifica-
tion. So, whole blood samples including benzodiazepines
and Z-drugs should not be calculated over a serum or
plasma calibration curve and vice versa.
818
Conclusions
This method fulfilled the requirements for a fully validated
assay. This method allowed for the first time the quantification
of benzodiazepines in whole blood, plasma, and serum in
cases of driving ability and crime responsibility for example.
This LC-MS/MS method allowed accurate and precise quan-
tification of 16 benzodiazepines in whole blood; for serum,
the approach is selective, sensitive, accurate, and precise for
18 of the tested drugs and for plasma for 17 of the 19 tested
drugs. Since in all biosamples zopiclone was instable and
could not be measured over a six-point calibration, only
semiquantitative results could be obtained at least for high
concentrations. As shown in the statistical data, because of
significant variances for ME, RE, and PE, each biosample
should be calculated over a calibration curve made in the same
biosample.
Being part of a multi-analyte method for 130 analytes of
different drug classes, validation data of other drug classes
will be published in the near future.
Acknowledgments The authors would like to thank Dr. Andrea E.
Steuer for the helpful discussion.
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