a validated method for simultaneous screening and quantification

An ultra-performance liquid chromatography–tandem massspectrometry (UPLC–MS–MS) method for detection of 23benzodiazepines and related compounds in whole blood wasdeveloped and validated. The method is used for screening andquantitation of benzodiazepines in whole blood received fromautopsy cases and living persons. The detected compounds werealprazolam, bromazepam, brotizolam, chlordiazepoxide,demoxepam, clobazam, clonazepam, 7-aminoclonazepam,diazepam, nordiazepam, estazolam, flunitrazepam,7-aminoflunitrazepam, lorazepam, lormetazepam, midazolam,nitrazepam, 7-aminonitrazepam, oxazepam, temazepam,triazolam, zaleplon, and zopiclone. Whole blood from drug-freevolunteers was used for all experiments. Blood samples (0.200 g)were extracted with ethyl acetate at pH 9. Target drugs werequantified using aWaters ACQUITY UPLC system coupled to aWaters Quattro Premier XE triple quadrupole in positiveelectrospray ionization, multiple reaction monitoring mode. Theuse of deuterated internal standards for most compounds verifiedthat the accuracy of the method was not influenced by matrixeffects. Extraction recoveries were 73–108% for all analytes.Lower limits of quantification ranged from 0.002 to 0.005 mg/kg.Long-term imprecision (CV%) ranged from 6.0 to 18.7%.We present a fully validated UPLC–MS–MS method for 23benzodiazepines in whole blood with a run-time of only 5 min andusing only 0.200 g of whole blood.

Introduction

Benzodiazepines belong to the group of tranquilizers thatare used to treat conditions with anxiety and unrest. Althoughbenzodiazepines may be used as hypnotics, the so-called ben-zodiazepine agonists (zolpidem, zopiclone, and zaleplone) areprimarily used for this purpose (z-hypnotics). Most benzodi-azepines and z-hypnotics are safe when taken in larger doses,but some (e.g., clonazepam, triazolam, flunitrazepam, andzopiclone) can cause toxicity or even death (1–3). Because oftheir lethargic effects, benzodiazepines can be a problem whenused by drivers. Other CNS depressants, such as alcohol ormorphine (heroin), can potentiate or add to the effect of ben-zodiazepines, causing considerable impairment of driving (4),and benzodiazepines can contribute to a fatal outcome takenin combination with CNS depressants.

Recently, fixed limits for blood concentrations of drugs ofabuse for drivers have been introduced into the Danish legis-lation. Seventeen different benzodiazepines as well as zopi-clone and zolpidem were considered. Thus to aid enforcementof these regulations, a fast and sensitive analytical methodcovering the most frequently used benzodiazepines is required.In this context, we considered development of an ultra-per-formance liquid chromatography–tandem mass spectrometry(UPLC–MS–MS) method for this class of compounds. We areonly familiar with a single earlier paper describing separationand detection of benzodiazepines with the UPLC–MS–MS tech-nique (5). In this method, solid-phase extraction (SPE) of 43benzodiazepines was performed from 1 mL plasma. A gradientsystem with formic acid in water and acetonitrile, respectively,

AValidated Method for Simultaneous Screening andQuantification of Twenty-Three Benzodiazepines andMetabolites Plus Zopiclone and Zaleplone inWhole Blood by Liquid–Liquid Extraction andUltra-Performance Liquid Chromatography–Tandem Mass Spectrometry

KirstenWiese Simonsen1,*, Sigurd Hermansson2, Anni Steentoft1, and Kristian Linnet11Section of Forensic Chemistry, Department of Forensic Medicine, Faculty of Health Sciences, University of Copenhagen,Frederik V’s vej 11, 3. DK-2100, Denmark and 2Waters Sweden AB, Djupdalsvagen 12-14, SE-191-24 Sollentuna, Sweden

Reproduction (photocopying) of editorial content of this journal is prohibited without publisher’s permission.332

Journal of Analytical Toxicology, Vol. 34, July/August 2010

Abstract

* Author to whom correspondence should be addressed: Section of Forensic Chemistry,Department of Forensic Medicine, Faculty of Health Sciences, University of Copenhagen,Frederik V’s vej 11, 3. DK-2100, Denmark. E-mail: [email protected].

Downloaded from https://academic.oup.com/jat/article-abstract/34/6/332/861472by gueston 07 April 2018

was used for separation of the benzodiazepines with a run-timeof 17 min. The lower limit of quantification (LLOQ) rangedfrom 0.5 to 10 ng/mL. Herein, we describe our UPLC–MS–MSmethod for measurement in blood of 23 benzodiazepines (in-cluding zopiclone and zaleplone) and their metabolites basedon liquid–liquid extraction with ethyl acetate and a short chro-matographic gradient finished within 5 min.

Materials and Methods

Chemicals and reagentsThe following compounds were purchased from Lipomed

(Bad Säckingen, Germany): bromazepam, flunitrazepam, lo-razepam, nordiazepam, 7-aminonitrazepam, 7-aminoclon-azepam, 7-aminoflunitrazepam, zopiclone, 7-aminofluni-trazepam-d3, and flunitrazepam-d3. Alprazolam and triazolamwere received from Pfizer (Ballerup, Denmark). Temazepamand clonazepam were obtained from Roche (Hvidovre, Den-mark). Oxazepam and diazepam came from Durascan MedicalProducts (Odense, Denmark). Midazolam and demoxepamwere obtained from Alpharma (Copenhagen, Denmark). Ni-trazepam and chlordiazepoxide were purchased from NycomedDanmark (Roskilde, Denmark). The following benzodiazepineswere received from different pharmaceutical companies: bro-tizolam (Boehringer Ingelheim, Copenhagen, Denmark),clobazam (Hoechst, Hørsholm, Denmark), estazolam (Lund-beck, Valby, Denmark), lormetazepam (Schering, Albertslund,Denmark), and zaleplone (Wyeth, Glostrup, Denmark). FromCerilliant (Round Rock, TX), we obtained the following sub-stances: diazepam-d5, demethyldiazepam-d5, nitrazepam-d5,oxazepam-d5, alprazolam-d5, clonazepam-d4, 7-aminoclon-azepam-d4, estazolam- d5, and triazolam-d4. 7-Aminoni-trazepam-d5 and zopiclone-d8 were both obtained from TorontoResearch Chemicals (Toronto, ON, Canada). All the referencestandards were of ≥ 98% purity.

Ethyl acetate and LC–MS-grade methanol and acetonitrilewere obtained from Fisher Scientific (Leicestershire, U.K.).Boric acid was purchased from VWR (Albertslund, Denmark).Aqueous ammonia (25%) and sodium carbonate used for ad-justment of the borate buffer solution were obtained fromMerck (Darmstadt, Germany). Purified water was obtainedwith a Milli-Q system (Millipore, Copenhagen, Denmark). Themobile phase used for the LC system was prepared weekly.

We performed the analyses on whole blood stabilized withsodium fluoride and potassium oxalate or CDP adenine. Mostof the experiments were carried out using pooled human bloodobtained from blood donors. Investigations of matrix effectsand extraction efficiency were based on authentic samplesnegative for all kinds of licit and illicit drugs received by the de-partment, either from autopsy cases or from living persons.The whole blood was stored at –20°C until use.

Preparation of standard solutionsAll standard compounds and deuterated analogs were dis-

solved in methanol or acetonitrile as recommended by themanufacturer to concentrations of either 100 or 1000 mg/L

and stored in ampoules at −20°C before mixing. A singleworking solution in purified water containing all of the com-pounds at concentrations of 10 mg/L (except for zopiclone,which was stored in acetonitrile and diluted separately just be-fore extraction) were prepared every three weeks and stored at4°C. On the day of analysis, diluted standard solutions con-taining all 23 compounds for spiking of calibrators and qualitycontrols (QCs) were prepared by further dilution of the10 mg/L stock solution in purified water. Calibrators weremade by spiking 0.200 g of whole blood with 25 µL of standardsolutions, yielding a final calibration range of 0.0025, 0.025,0.25, and 0.5 mg/kg. QCs containing all compounds were pre-pared in pooled whole blood and stored at −20°C.

An internal standard (IS) solution including all of the deuter-ated standards was prepared in purified water monthly andstored at 4°C. The IS solution was adjusted to a concentrationof 0.125 mg/L and 30 µL of IS was added to 0.200 g of wholeblood. The IS concentration was chosen based on the secondcalibrator concentration. The IS solution was used for all val-idation experiments, calibrators, QCs, and samples.

LC chromatographic conditionsThe chromatography was performed using an ACQUITY

UPLC system (Waters, Milford, MA). The column used was anAcquity UPLC BEH C18 (100 mm × 2.1 mm,1.7 µm), which was maintained at a column temperature of65°C and a constant flow rate of 0.4 mL/min. The mobilephase was composed of solvents A [0.1% aqueous ammonia(25%) in purified water] and B [0.1% aqueous ammonia (25%)in methanol]. The gradient program is shown in Table I. Theinjection volume was 10 µL.

MSMS was performed using a Quattro Premier XE triple qua-

drupole (Waters). Positive electrospray ionization mode (ESI+)was used for all MS analyses. The ionization parameters werea capillary voltage of 3.6 kV and source and desolvation tem-peratures of 120 and 450°C, respectively. Cone and desolvationgas (N2) flows were set at 50 and 1100 L /h, respectively. Argonwas used as the collision gas at a pressure of 9.25 × 10−3 mBar,corresponding to a flow of 0.20 mL/min. Determination of themost suitable multiple reaction monitoring (MRM) transi-tions, cone voltages, and collision energies for all analytes anddeuterated analogues were obtained by tuning on the analytesin standard solutions in the concentration of 1 mg /L dis-solved in Milli-Q water and methanol (60:40, v/v). The com-

333


Table I. UPLC Gradient Program*

Time (min) %A %B

0.0 60 400.20 60 404.00 25 754.50 5 955.00 60 40

* Total run-time 5.5 min.


pounds were injected into the MS using thesyringe pump coupled to the UPLC systemwith a Tee fitting. The UPLC system deliv-ered a constant flow of 0.4 mL /min.

MassLynx 4.1 (Waters) software with auto-mated data processing (QuanLynx) was usedrunning in the MRM mode. The analyteswere identified by two characteristic MRMtransitions: their ion ratio and retentiontime. Tolerance was set to ± 20% for the ionratio and ± 1% for the retention time. Quan-tification was performed by integration of thearea under the curve from the specific MRMchromatograms of the analytes and their IS.The response (the ratio of the integrated areaof the analyte and the corresponding IS) wascompared to the calibration curve. The ISchosen for each analyte, retention times, andMRM transitions are shown in Table II.

Sample preparationWhole blood samples (0.200 g) were spiked

with 30 µL of IS solution, and then 125 µL0.63 M borate buffer (pH 9) was added beforeextraction with 1500 µL ethyl acetate. Aftercentrifugation, 500 µL organic phase wastaken, evaporated at room temperature undera stream of nitrogen, and redissolved in 250µL 40% methanol in Milli-Q water.

Method validationCalibration curve. To determine the lin-

earity for each compound in whole blood, weprepared the calibration curves with sevenconcentration points including a blank. Theconcentration points ranged from 0.0015 to1.0 mg/kg. The samples were prepared in0.200 g of whole blood spiked with 25 µL ofstock solution diluted in Milli-Q water to ap-propriate concentrations, and subsequently,30 µL of IS solution was added.

Precision and accuracy. To evaluate preci-sion and accuracy, we analyzed four repli-cates at four concentration levels on two dif-ferent days. The four concentration levelsanalyzed were 0.002, 0.005, 0.05, and 0.5mg/kg. A calibrator series was freshly pre-pared for every run based on 0.200 g of wholeblood spiked with all analytes, yielding theconcentration points 0, 0.0025, 0.025, 0.25,and 0.50 mg/kg. Prior to analysis, four dif-ferent stock samples (5 g each) representingthe four concentration levels were preparedby spiking pooled whole blank blood with allof the analytes. On day one of analysis, foursamples (0.200 g of blood) were taken fromeach of the four stock samples. All 16 samples(four replicates for each concentration level)

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Table II. Retention Time, MRMTransitions, and Operating Parameters for theAnalyzed Drugs*

Retention MRM Cone CollisionTime Transitions Voltage Energy

Compound (min) (m/z) (V) (eV) Internal Standard

Function 17-Aminoclonazepam 1.11 286.1 > 121.0 40 30 7-Aminoclonazepam-d4

286.1 > 222.2 257-Aminoflunitrazepam 1.31 284.1 > 135.1 40 25 7-Aminoflunitrazepam-d3

284.1 > 227.3 257-Aminonitrazepam 1.10 252.15 > 121.0 40 25 7-Aminonitrazepam-d5

252.15 > 94.0 40

Function 2Demoxepam 2.20 287.0 > 180.0 35 20 Diazepam-d5

287.0 > 104.9 20Clonazepam 2.27 316.1 > 270.1 45 25 Clonazepam-d4

316.1 > 214.1 40Zaleplone 1.99 306.1 > 236.1 40 25 Diazepam-d5

306.1 > 264.1 20

Function 3Bromazepam 2.30 318.0 > 182.1 45 30 Diazepam-d5

318.0 > 209.2 25Nitrazepam 2.30 282.1 > 236.2 40 25 Nitrazepam-d5

282.1 > 180.1 35Zopiclone 2.37 389.0 > 245.0 20 20 Zopiclone-d8

389.0 > 217.1 35

Function 4Flunitrazepam 2.57 314.1 > 268.1 45 25 Flunitrazepam-d3

314.1 > 239.2 35

Function 5Clobazam 2.85 301.0 > 224.1 35 30 Diazepam-d5

301.0 > 104.9 36Estazolam 2.80 295.1 > 267.1 40 25 Estazolam-d5

295.1 > 205.1 40Oxazepam 2.89 287.0 > 241.2 30 20 Oxazepam-d5

287.0 > 104.0 35

Function 6Chlordiazepoxide 3.36 300.1 > 283.1 25 15 Diazepam-d5

300.1 > 227.1 25Nordiazepam 3.44 271.1 > 139.9 60 25 Nordiazepam-d5

271.1 > 164.9 28Lorazepam 2.89 321.0 > 275.1 30 20 Diazepam-d5

321.0 > 229.1 33

Function 7Alprazolam 3.05 309.05 > 281.1 45 25 Alprazolam-d5

309.05 > 205.1 40Triazolam 3.01 343.0 > 308.1 50 25 Diazepam-d5

343.0 > 315.0 25

Function 8Brotizolam 3.19 394.9 > 279.1 40 30 Diazepam-d5

394.9 > 314.0 25Lormetazepam 3.26 335.0 > 289.0 30 20 Diazepam-d5

335.0 > 177.1 40Temazepam 3.16 301.0 > 255.1 30 20 Diazepam-d5

301.0 > 177.1 40

Function 9Diazepam 3.70 285.1 > 154.0 45 25 Diazepam-d5

285.1 > 193.1 30Midazolam 3.70 326.1 > 291.1 45 25 Diazepam-d5

326.1 > 249.2 35

* MRM transitions are listed for each analyte with quantifier transition at top and qualifier transitions below.


335


Table III. Validation Parameters for Benzodiazepines in Whole Blood

Fixed MeasuredConcentration Calibration Correlation Theoretical Concentration Extraction

Limit Range Coefficient Polynomal LOD LLOQ Concentration (n = 8) Bias Precision Recovery MEAnalyte (mg/kg) (mg/kg) (n = 7) Regression (mg/kg) (mg/kg) (mg/kg) (mg/kg) (%) (%CV) (%) (%)

Alprazolam 0.005 0–1.0 0.9976 0.375 0.0002 0.002 0.002 0.0021 5 13.5 85 –1.50.005 0.0051 2 10

` 0.050 0.051 1 5.60.50 0.49 –2.7 6

Bromazepam 0.050 0–1.0 0.9942 0.638 0.0004 0.002 0.002 0.0022 9 11 84 –15.20.005 0.0044 –11 16.60.050 0.046 –9 6.30.50 0.45 –10 2.8

Brotizolam 0.002 0–1.0 0.9974 0.173 0.0005 0.002 0.002 0.0016 –19 20.8 81 3.60.005 0.0051 2 14.20.050 0.052 3 70.50 0.49 –2.4 8

Chlordiazepoxide 0.20 0–1.0 0.9949 0.590 0.0011 0.002 0.002 0.0025 24 19 79 –0.20.005 0.0056 11.5 120.050 0.058 15 70.5 0.54 8 6

Demoxepam 0–1.0 0.9924 0.827 0.0012 0.002 0.002 0.0016 –18 11.8 93 –3.10.005 0.0047 –7 9.30.050 0.048 –5 11.30.50 0.46 –8 5.0

Clobazam 0.10 0–1.0 0.9968 0.058 0.0008 0.005 0.005 0.0055 10 15 89 –0.60.050 0.054 7 40.50 0.51 3 4

Clonazepam 0.005 0–1.0 0.9949 0.020 0.0007 0.002 0.002 0.0019 –6 23 85 –0.90.005 0.0049 –2 80.050 0.055 9.7 70.50 0.45 –9.7 7

7-Aminoclonazepam 0–1.0 0.9959 0.577 0.0003 0.002 0.002 0.0016 –21 13 105 15.40.005 0.0048 –5 40.050 0.046 –8.2 40.50 0.50 0 4.1

Diazepam 0.10 0–1.0 0.9962 0.610 0.0003 0.002 0.002 0.0020 0 9.6 82.5 –0.20.005 0.0050 0 6.50.050 0.052 3 3.70.50 0.48 –4 2.2

Estazolam 0.050 0–1.0 0.9963 0.802 0.0007 0.002 0.002 0.0021 5.6 19 87 3.30.005 0.0054 7.2 60.050 0.053 5.5 6.70.50 0.47 –5.2 11

Flunitrazepam 0.005 0–1.0 0.9976 0.078 0.0007 0.002 0.002 0.002 1 15 73 –6.10.005 0.0053 6 150.050 0.055 9 130.50 0.49 –2 8

7-Aminoflunitrazepam 0–1.0 0.9960 0.537 0.0004 0.002 0.002 0.0019 –5.6 11 108 15.80.005 0.0046 –7.2 20.050 0.043 –15 4.10.50 0.48 –4 4.2

Lorazepam 0.020 0–1.0 0.9833 0.027 0.0015 0.005 0.005 0.0050 0 20 85 –3.40.050 0.049 –2 90.50 0.49 –2.4 11

Lormetazepam 0.005 0–1.0 0.9951 0.112 0.0008 0.002 0.002 0.0024 20 8 83 –1.30.005 0.0052 3 7.60.050 0.049 –1.2 50.50 0.46 –7 4

Midazolam 0.050 0–1.0 0.9951 0.069 0.0004 0.002 0.002 0.002 0 4.6 82 –1.00.005 0.0053 6.2 40.050 0.057 14.2 4.20.50 0.46 –8.5 2

Table III continued on next page


and the calibrators were spiked with 30 µL of internal standard,as described earlier, and subjected to liquid–liquid extraction(LLE). The procedure was repeated on day two of analysis. An-other spiked sample at a concentration level of 0.001 mg/kgwas prepared and used for determination of LLOD. Four repli-cates were analyzed. This was repeated the next day, and LODwas calculated from the eight results as 3 × SD.

High and low control samples were included in each run.The long-term precision was calculated from these measure-ments. Uncertainty (coefficient variation, CV) was calculatedfor all drugs as a combination of uncertainty related to drugpurity, preparation of calibrator, and long-term precision:

CVresult = [CVpurity2 + CVcal. preparation

2 + CVlong-term precision2]½ Eq.1

Matrix effects, extraction recoveries, and ion suppression.We evaluated the matrix effects (ME) of whole blood on thepeak-area responses. The experiments were performed as de-scribed by Matuszewski et al. (6,7). Two sets of six whole bloodsamples, obtained from six different authentic samples (autopsycases and living cases), received by our department, andscreened negative for a broad variety of drugs including ben-

zodiazepines, were extracted according to the LLE procedure.Set 1 was spiked with all analytes after extraction (B), and set2 was spiked before extraction (C) to a corresponding concen-tration of 0.03 mg/kg in whole blood. All blood samples had afinal concentration of 0.008 mg/L after extraction and re-so-lution in mobile phase. Three replicates of 0.008 mg/L refer-ence solutions in mobile phase (A) were analyzed directly withthe UPLC–MS–MS system. We calculated the ME for each an-alyte by comparison of the absolute peak areas. The ME resultsobtained in this study were calculated as follows:

ME = (1− (B/A)) × 100% Eq. 2

where A equals the peak area of standards in mobile phaseand B is the peak area obtained for blank whole blood samplesspiked with analytes after extraction. An ME value > 0 indicatesionization suppression and a value < 0 indicates ionizationenhancement.

Extraction recoveries (RE%) were calculated as the mean ab-solute peak areas of all six samples spiked before LLE (C) andcompared with absolute peak areas from samples spiked afterLLE (B):

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Table III. Validation Parameters for Benzodiazepines in Whole Blood (Continued)

Fixed MeasuredConcentration Calibration Correlation Theoretical Concentration Extraction

Limit Range Coefficient Polynomal LOD LLOQ Concentration (n = 8) Bias Precision Recovery MEAnalyte (mg/kg) (mg/kg) (n = 7) Regression (mg/kg) (mg/kg) (mg/kg) (mg/kg) (%) (%CV) (%) (%)

Nitrazepam 0.020 0–1.0 0.9953 0.780 0.0002 0.002 0.002 0.0024 20 8 85 0.70.005 0.0056 11.2 90.050 0.0536 7.2 120.50 0.49 –2.4 10

7-Aminonitrazepam 0–1.0 0.9914 0.168 0.0004 0.002 0.002 0.0020 0.6 5 106 11.30.005 0.0051 2.3 9.20.050 0.048 –3.3 9.50.50 0.49 –2.7 10

Nordiazepam 0.10 0–1.0 0.9910 0.231 0.0007 0.002 0.002 0.0021 6 14.3 88 6.80.005 0.0050 0 8.30.050 0.051 1 6.80.50 0.47 –6 4.1

Oxazepam 0.10 0–1.0 0.9964 0.107 0.001 0.002 0.002 0.0016 –18 19.6 82 4.50.005 0.0048 –4.2 15.50.050 0.052 4.5 8.00.50 0.52 4.1 10.2

Temazepam 0.020 0–1.0 0.9920 0.601 0.0003 0.002 0.002 0.0020 –1.9 7.7 94 –1.40.005 0.0047 –5.7 40.050 0.044 –12.6 5.30.50 0.44 –12.8 4.9

Triazolam 0.002 0–1.0 0.9956 0.129 0.0006 0.002 0.002 0.0020 0 19.7 85 1.50.005 0.0050 –1 5.30.050 0.053 7 2.00.50 0.50 –0.5 5.3

Zaleplone 0–1.0 0.9953 0.037 0.0005 0.002 0.002 0.0025 23.8 7.3 89 –0.90.005 0.0048 –3.7 80.050 0.043 –14.6 13.30.50 0.50 0.3 15

Zopiclone 0.010 0–1.0 0.9982 0.845 0.0005 0.002 0.002 0.0023 14 19.8 91 –200.005 0.0048 –5 7.60.050 0.048 –5 11.90.50 0.48 –5 5.7


RE (%) = (C/B) × 100% Eq. 3

We tested the impact of ion suppression and enhancementfrom ionization of components for all analytes and IS (7). Theanalytes and IS were injected continuously into the MS in mix-tures of a maximum of six compounds, which were selected sothat all of the compounds in the mix had similar responses, and

so that none of the compounds had a ∆[M+H]+ less than m/z 3.Furthermore, all of the compounds had baseline resolutionchromatography. To produce a constant elevated response inboth MRM channels for each analyte, the compounds wereinjected post-column (0.1 mg/L at a constant flow rate of2 µL/min) using the syringe pump and “Tee-fitting” connectedto the UPLC system (delivering a constant flow of 0.4 mL/min).

The slightly elevated baseline responseswere monitored following injection (10µL) from the autosampler with extractedblank blood samples from the pooleddrug-free volunteers. Furthermore, all an-alytes in mobile phase (0.13 mg/L) wereinjected individually from the autosam-pler simultaneously with the flow of ana-lytes from the syringe pump. We per-formed individual injection of the analytesto evaluate possible enhancement or de-pression from co-eluting analytes. Wethen compared the acquired post-injec-tion baseline responses to the baseline re-sponse after injection of a blank mobilephase. We also looked for interference inall traces of the analytes after the indi-vidual injection of the analytes.

Results

Calibration curveWe investigated the analyte/IS peak-

area response ratio in whole blood (TableIII). The calibration curve was fitted to alinear regression curve (1/x). The lin-earity was tested using a polynomial re-gression approach (8). Non-linearity wasindicated by a coefficient for thequadratic term that deviated significantlyfrom zero (p < 0.01). The calibrationrange obtained for all analytes in bloodwas 0.0015–1.0 mg/kg except for loraze-pam and zaleplone, which had a range of0.0025–1.0 mg/kg (Table III). Eventhough the calibration curves were testedlinear up to 1.0 mg/kg, the correlationcoefficient for all compounds was below0.999 (Table III). In the interval 0.0025–0.50 mg/kg, the calibration curves havecorrelation coefficients > 0.999, so we de-cided to evaluate this as the measuringinterval. All of the samples with concen-trations higher than the upper limit ofquantification (ULOQ) were diluted withpurified water (1+9). Figure 1 shows achromatogram of a whole blood samplespiked with 0.0025 mg/kg of the com-pounds. Two cases with detection of sev-

Figure 1. Quantitative transitions for a spiked whole blood sample at 0.0025 mg/kg.

337



eral benzodiazepines are presented in Figure 2 and 3. Figure 2was an autopsy case where the cause of death was a fall from awindow. The following benzodiazepines were detected: alpra-zolam (0.054 mg/kg), bromazepam (2.1 mg/kg), clonazepam(0.49 mg/kg), 7-aminoclonazepam (0.22 mg/kg), diazepam (0.10mg/kg), nordiazepam (0.22 mg/kg), oxazepam (0.27 mg/kg),temazepam (0.010 mg/kg), nitrazepam (0.038 mg/kg), and 7-aminonitrazepam (0.10 mg/kg). Besides the benzodiazepines,methadone, ketamine, mirtazapine, and phenobarbital weredetected. Nordiazepam, oxazepam, and temazepam are pre-sumably metabolites originating from diazepam as nor-diazepam, and temazepam are not marketed as medical drugsin Denmark. Figure 3 is an example of a fixed concentrationlimit case. The benzodiazepines bromazepam (0.58 mg/kg),clonazepam (0.068 mg/kg), 7-aminoclonazepam (0.094 mg/kg),flunitrazepam (0.002 mg/kg), and 7-aminoflunitrazepam (0.007mg/kg), were detected. Bromazepam and clonazepam exceeded

the fixed concentration limit. The corresponding deuteratedinternal standards are also shown in Figure 3.

Limits of quantification, precision, and truenessThe LLOQ was determined as the lowest concentration

yielding precision (CV) of ≤ 20% and bias of ±20% with fulfill-ment of retention time and ion ratio tolerances (9,10). LLOQwas determined to be 0.002 mg/kg for all analytes, exceptclobazam and lorazepam (0.005 mg/kg) (Table III). The LLOQwas low enough to be in compliance with the traffic limitsand to meet the lower therapeutic limit of the compounds.

The CV and accuracy were determined at four concentrationlevels, including the LLOQ and ULOQ and two intermediateconcentrations. The CV and bias were generally accepted at amaximum of 15% (LLOQ 20%) (9). All analytes fulfilled theprecision criteria at all concentration levels, except lorazepam.Lorazepam had a CV estimate exceeding the limit (35%) at the

0.002 mg/kg level (Table III). Clonazepamhad a CV slightly above the criteria atLLOQ (23%). The accuracy values weresatisfactory and not significantly differentfrom the limits for all tested concentra-tions, except clobazam, which had a biasof 30% at the LLOQ, and chlor-diazepoxide and zaleplone, which had abias slightly above the criteria at LLOQ(24%). The LLOQ of lorazepam andclobazam was then set to 0.005 mg/kg.

ME, ion suppression, extractionrecoveries, and carryover

The ME provided as percentages for allanalytes are listed in Table III. All ana-lytes had ME within ±20%, and so weconcluded that ME was of minor signifi-cance. Minor ME will be compensated bythe deuterated IS because ME for thedeuterated IS are of the same levels ascorresponding analytes.

The extraction recoveries (RE) were de-termined in six different sources of wholeblood. Extraction recoveries were allabove 70% (Table III). The recoveries forthe IS were of the same order of magni-tude as the corresponding compounds.

Ion suppression was also tested by in-fusion experiments for all analytes usingpooled whole blood from blood donors.The experiments showed that there wasno major ion suppression or enhance-ment in whole blood. No interference wasobserved for co-eluting compounds. Tria-zolam gave rise to a signal in its corre-sponding deuterated IS (triazolam-d4)trace. We also found a depression per-formed by triazolam-d4 in the MRM traceof triazolam. These interferences betweentriazolam and its deuterated IS gave rise

Figure 2. A case with alprazolam (0.054 mg/kg), bromazepam (2.1 mg/kg), clonazepam (0.49 mg/kg), 7-aminoclonazepam (0.22 mg/kg), diazepam (0.10 mg/kg), nordiazepam (0.22 mg/kg), oxazepam (0.27mg/kg), temazepam (0.010 mg/kg), nitrazepam (0.038 mg/kg), and 7-aminonitrazepam (0.10 mg/kg).

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to problems in the precision and trueness study, which couldnot fulfill the demands. Triazolam-d4 was therefore excluded asan IS, and diazepam-d5 was used instead.

QC samples and uncertaintyThe system ran very stable as indicated by the long-term im-

pression of the controls (Table IV). At the low control level, theCVs ranged from 6.3 to 18.7%. At the high levels, the CVsranged from 4.2 to 14.5% (N = 30–45). Uncertainty calculatedas an average of the two QCs varied from 5.7 to 15.5% (TableIV).

Discussion

Several methods have been described for extraction andquantification or screening of one or more benzodiazepines inurine, plasma, or whole blood. The techniques used have in-cluded gas chromatography (GC) with dual column (11), GC–MS (12–15), high-performance liquid chromatography (HPLC)(16), LC–MS–MS (17–20), and UPLC–MS–MS (5). Few papershave covered a broad variety of benzodiazepines and benzodi-azepine-like hypnotics (z-compounds) in whole blood. A multi-method analyzing 22 benzodiazepines inblood and urine was described by Pernayand colleagues (14). The importance ofusing derivatization was shown, and theuse of trimethylsilylate lowered the de-tection threshold considerably. Recently,a GC–MS method covering the determi-nation of 14 benzodiazepines, includingzaleplone and zolpidem, in whole bloodwas published; after extraction of thebenzodiazepines with butyl acetate, dif-ferent derivatization reagents were tested(15). El Mahjoub and Staub (16) devel-oped a column-switching HPLC–DADmethod and analyzed five different ben-zodiazepines in serum/plasma by directinjection.

The introduction of LC–MS–MS toforensic toxicology has resulted in easiersample preparation and better sensitivity.A screening and quantification methodof 23 benzodiazepines, flumazenil, andz-hypnotics in plasma by use of LC–MSand APCI has been described (17). Analiquot (0.5 mL) was used for LLE withdiethyl ether/ethyl acetate (1:1). TheLLOQ ranged from 0.5 to 200 ng/mL, andthe run-time was 10 min. Laloup et al.(18) also used LLE for extraction of 26benzodiazepines and metabolites inblood, urine, and hair. Blood (250 µL)was extracted with 1-chlorobutane. Byemploying a gradient with a total run-time of 35 min, these investigators wereable to detect the benzodiazepines using

an LC equipped with a quatro Premier tandem MS system(Waters). The LLOQ ranged from 1 to 2 ng/mL in blood. Sminket al. (19) used SPE for extraction of 33 benzodiazepines,metabolites, zolpidem, and zopiclone in whole blood. An iontrap LC–MS technique, which was completed in 45 min, wasused for identification and quantification. LLOQ ranged from0.4 to 41.9 ng/mL with recoveries of 40–114%. A recent paperdescribed an SPE extraction method for 22 benzodiazepinesfrom 100 µL serum, which were then detected by an LC–MS–MS method with a run-time of 25 min (20). Thus, the LC–MS–MS technique has shown its applicability for benzodiazepineanalysis in blood. Except for one method with a short run-timeof 10 min (17), all these methods had rather long analysistimes (25–45 min) (18–20). Low LLOQs were reported, for ex-ample, 1–2 ng/mL (18), 0.5–200 ng/mL (17), and 0.4–41.9ng/mL (19).

The next generation of LC–MS, UPLC–MS, came on themarket a few years ago. The small particle size columns usedgive narrow peaks, making the system useful for sensitive de-tection and separation of many compounds in a short time.Rapid detection methods are important in the detection ofdrugs in blood specimens collected from potentially impairedindividuals in DUI cases, which requires a quick response for

Figure 3. A fixed concentration limit case. Bromazepam (0.58 mg/kg), clonazepam (0.068 mg/kg),7-aminoclonazepam (0.094 mg/kg), flunitrazepam (0.002 mg/kg), and 7-aminoflunitrazepam (0.007mg/kg). Bromazepam and clonazepam exceeded the fixed concentration limit.


the investigating police, thus creating a demand for multi-methods with a short run-time. As mentioned earlier, oneUPLC–MS–MS method for benzodiazepines has been published(5).

The method described in this paper meets the requirementsfor the fixed concentration limits according to the Danish law(Table III), and the LLOQs were low enough to meet the lowertherapeutic limit of the compounds. Our LLE is very simpleand yielded extraction recoveries higher than 70%. This featurecombined with low LLOQs and a short run-time of 5 minmakes our method suitable for routine traffic analysis. LLOQwas 0.002 mg/kg for all compounds except lorazepam andclobazam, which had an LLOQ of 0.005 mg/kg. We chose to val-idate the most common benzodiazepines and metabolites seenin our laboratory. The method may also detect more seldomlyused benzodiazepines like flurazepam, halazepam, medazepam,phenazepam, prazepam, quazepam, tetrazepam, and zo-lazepam. Additionally, flumazenil and zolpidem are also de-tected by the method. Prescription and use of zolpidem isquite frequent in Denmark, but as zolpidem is analyzed in an-other accreditated method in our laboratory, we only screen forzolpidem with the present method.

References

1. A. Steentoft and K. Linnet. Blood concentrations of clonazepamand 7-aminoclonazepam in forensic cases in Denmark for the

period 2002–2007. Forensic Sci. Int. 184: 74–79 (2009).2. A. Steentoft and K. Worm. Cases of fatal triazolam poisoning.

J. Forensic. Sci. Soc. 33: 45–48 (1993).3. H. Druid and P. Holmgren. A compilation of fatal and control

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4. M.W. van Laar and E.R. Volkerts. Driving and benzodiazepineuse. Evidence that they do not mix. CNS Drugs 10: 383–396(1998).

5. T. Ishida, K. Kudo, M. Hayshida, and N. Ikeda. Rapid and quan-titative screening method for 43 benzodiazepines and theirmetabolites, zolpidem and zopiclone in human plasma by liquidchromatography/mass spectrometry with a small particle column.J. Chromatogr. B 877: 2652–2657 (2009).

6. B.K. Matuszewski, M.L. Constanzer, and C.M. Chavez-Eng. Strate-gies for the assessment of matrix effect in quantitative bioanalyt-ical methods based on HPLC–MS–MS. Anal. Chem. 75: 3019–3030 (2003).

7. T.M. Annesley. Ion suppression in mass spectrometry. Clin. Chem.49: 1041–1044 (2003).

8. Clinical and Laboratory Standards Institute/NCCLS. Evaluationof the Linearity of Quantitative Measurement Procedures: A Sta-tistical Approach. Approved Guideline. CLSI/NCCLS DocumentEP6-A. Clinical and Laboratory Standards Institute, Wayne, PA,2003.

9. F.T. Peters, O.H. Drummer, and F. Musshoff. Validation of newmethods. Forensic Sci. Int. 165: 216–224 (2007).

10. V.P. Shah, K.K. Midha, J.W.A. Findlay, H.L Hill, J.D. Hulse,I.J. McGilveray, G McKay, K.J. Miller, R.N. Patnaik, M.L. Powell,A. Tonelli, C.T. Viswanathan, and A. Yacobi. Bioanalytical methodvalidation—a revisit with a decade of progress. Pharm. Res. 17:1551–1557 (2000).

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12. D. Borrey, E. Meyer, W. Lambert, C. Van Peteghem, and A.P. DeLeenheer. Simultaneous determination of fifteen low-dosed ben-zodiazepines in human urine by solid-phase extraction and gaschromatography–mass spectrometry. J. Chromatogr. B 765:187–197 (2001).

13. U. Staerk and W.R. Külpmann. High-temperature solid-phasemicroextraction procedure for the detection of drugs by gaschro-matography–mass spectrometry. J. Chromatogr. B 745: 399–411(2000).

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15. T. Gunnar, K. Ariniemi, and P. Lillsunde. Determination of 14 ben-zodiazepines and hydroxy metabolites, zaleplon, and zolpidemas tert-butyldimethylsilyl derivatives compared with othercommon silylating reagents in whole blood by gas chromatog-raphy–mass spectrometry. J. Chromatogr. B 818: 175–189 (2005).

16. A. El Mahjoub and C. Staub. High-performance liquid chro-matographic method for the determination of benzodiazepines inplasma or serum using the column-switching technique. J. Chro-matogr. B 742: 381–390 (2000).

17. C. Kratzsch, O. Tenberken, F.T. Peters, A.A. Weber, T. Kraemer, andH. Maurer. Screening, library-assisted identification and vali-dated quantification of 23 benzodiazepines, flumazenil, zale-plone, zolpidem, and zopiclone in plasma by liquid chromatog-raphy–mass spectrometry with atmospheric pressure chemicalionization. J. Mass Spectrom. 39: 856–872 (2004).

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Table IV. Long-Term Precision (Quality Controls) andUncertainty in the Measuring Interval Determined inBlankWhole Blood

Tested Levels Long-Term UncertaintyCompound (mg/kg) Precision (%)

Alprazolam 0.005, 0.050 10.2, 14.5 12.32Bromazepam 0.05, 0.50 13.9, 10.8 12.32Brotizolam 0.005, 0.050 15.7, 7.9 11.82Chloridazepoxi 0.05, 0.50 15.2, 10.7 12.92Demoxepam 0.05, 0.50 15.3, 13.6 15.37Clobazam 0.05, 0.50 10.8, 6.0 8.79Clonazepam 0.005, 0.050 11.4, 6.8 9.177-AMC 0.005, 0.050 9.0, 8.8 9.14Diazepam 0.05, 0.50 6.3, 4.2 5.81Estazolam 0.05, 0.50 12.5, 9.9 11.23Flunitrazepam 0.005, 0.050 12.2, 10.5 11.427-AMF 0.005, 0.050 10.9, 9.4 10.40Lorazepam 0.005, 0.050 18.7, 12.3 15.52Lormetazepam 0.005, 0.050 9.5, 8.7 9.14Midazolam 0.05, 0.50 7.0, 4.3 5.69Nitrazepam 0.05, 0.50 7.6, 5.2 6.427-AMN 0.005, 0.050 11.2, 9.5 11.31Nordiazepam 0.05, 0.50 10.2, 10.59 10.44Oxazepam 0.05, 0.50 11.8, 9.9 11.05Temazepam 0.05, 0.50 11.5, 8.3 10.13Triazolam 0.005, 0.050 15.9, 10.7 13.35Zaleplon 0.005, 0.050 8.7, 9.0 8.87Zopiclon 0.005, 0.050 16.1, 8.8 12.62

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19. B.E. Smink, J.E. Brandsma, A. Dijkhuizen, K.J. Lusthof, J.J. deGier, A.C.G. Egberts, and D.R.A. Uges. Quantitative analysis of 33benzodiazepines, metabolites and benzodiazepine-like sub-stances in whole blood by liquid chromatography–tandem massspectrometry. J. Chromatogr. B 811: 13–20 (2004).

20. M. Nakamura, T. Ohmori, Y. Itoh, M. Terashita, and K. Hirano.Simultaneous determination of benzodiazepines and theirmetabolites in human serum by liquid chromatography–tandem

mass spectrometry using a high-resolution octadecyl silica columncompatible with aqueous compounds. Biomed. Chromatogr. 23:357–364 (2009).

Manuscript received January 26, 2010;revision received March 2, 2010.


a validated method for simultaneous screening and quantification

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