Chiang Mai Med J 2007;46(1):23-30.

Address requests for reprints: Pattaravadee Pongraveevongsa, M.D., Department of Forensic Medicine, Facultyof Medicine, Chiang Mai University, Chiang Mai 50200, Thailand. E-mail:ppongrav@mail.med.cmu.ac.th

Received 23 October 2006, and in revised form 13 January 2007

Original article

DETERMINATION OF BENZODIAZEPINE IN SERUM BYTHE HIGH-PERFORMANCE LIQUID CHROMATOGRAPHIC

METHOD WITH SOLID PHASE EXTRACTION

Pattaravadee Pongraveevongsa, M.Sc, Kanda Vichairat, M.D.,Pongruk Sribanditmongkol, M.D., Warachate Khobjai, M.Sc.

Department of Forensic Medicine, Faculty of Medicine, Chiang Mai University

Abstract

Benzodiazepines are among the most prescribed drugs for the treatment of a wide spectrumof clinical disorders. They have been used as anticonvulsants, anxiolytics, hypnotics ormuscle relaxants with different durations of action. In this paper, the determination of sixfrequently used benzodiazepines and metabolites: flunitrazepam, oxazepam, alprazolam,chlordiazepoxide, diazepam and desmethyldiazepam in serum was developed by high-per-formance liquid chromatography (HPLC) with solid phase extraction (SPE). Quantificationwas performed with gradient elution of a C8 reversed- phase column, with an alkaline water-acetonitrile-methanol eluent, free of buffer salts. Carbamazepine was detected as an internalstandard by ultraviolet absorbance at 239 nm. The determination of flunitrazepam, oxazepam,alprazolam, chlordiazepoxide, desmethyldiazepam and diazepam was possible in a concen-tration range of 250-4,000 ng/mL, and the mean recovery by this analysis was 91, 91, 93, 84,110 and 92%, respectively. This method allows rapid detection, a purity check, identifica-tion, and quantitation of the eluting peaks. Sensitivity and specificity data are acceptable.The validated procedure has been applied routinely in forensic toxicological analysis. ChiangMai Med J 2007;46(1):23-30.

Keywords: benzodiazepine, solid phase extraction, high-performance liquid chromatography

Benzodiazepine (BZP) drugs are widelyprescribed for their anxiolytic, hypnotic, anti-convulsive and muscle relaxing properties; theyare not only important in treating a variety ofmedical disorders, but also abused by somepeople.(1-3) Thus, extraction and identificationof benzodiazepines in serum is very important

for forensic and clinical toxicology. However,an analysis of benzodiazepines in body fluidsis quite complex because of the diversity ofthose available on the market and the fact thateach drug has a particular therapeutic and toxicrange.

24 Pongraveevongsa P, et al.

Several analytical techniques for the isola-tion and quantitation of benzodiazepines inbiosamples have already been published. Boththin-layer chromatography (TLC) and theimmunoassay approach remain useful for a firstrapid screening. However, both techniqueshave a lack off high specificity. In the case ofimmuno-analysis, they could not discriminatebetween a parent drug and metabolites. Onthe other hand, gas chromatography withflame-ionization (GC/FID) or mass-spectro-metric detection (GC/MS) is much moresensitive and specific, but sample derivatizationand complex equipment are needed.(4-5) Thesereasons have resulted in an increasing popu-larity of high-performance liquid chromatog-raphy (HPLC) for screening and quantitationof benzodiazepines in biosamples. However,several extraction methods for benzodiazepineswith liquid-liquid extraction were used forserum and whole blood sample preparation.These methods were not satisfactory becausethey were too tedious and time consuming.

In this study, we described a rapid andsimple solid-phase extraction method anddeveloped an HPLC procedure based ongradient elution of a reversed-phase C8column with a salt-free eluent. The effluentwas monitored by photodiode array detection,with multi-wavelength allowing for identifica-tion and quantitation of six different benzodi-azepines and their metabolites in postmortemserum by UV detection.

Materials and methodsApparatusA high-pressure gradient system was used,

consisting of a high-performance liquid chro-matography 9012Q Pump Solvent DeliverySystem, Polychromâ 9065 Diod Array Detec-tor and ProStar 310 UV/Vis Detector, a

Rheodyne Injector equipped with a 20 mL loop,and star chromatography workstation (VarianâChromatography system, USA). The solidphase extraction system consisted of theBaker-10 SPETM system, JT Baker, Phillips-burg, NJ., Aspirator A-3S, EYELA Tokyo,Japan., and Bond Elutâ HF bond Elut- C18,500 mg 3 mL, Varian, USA. The HPLCcolumn was Luna C8 (5 mm 100 A 125x4.6mm.) from Phenomenex, USA, and thesecurity guard holder and guard cartridge C8(Octyl,MOS: 4 mmLx3.0 mm ID) was alsofrom Phenomenex.

Solvents and reagentsCarbamazepine, flunitrazepam, oxazepam,

alprazolam, chlordiazepoxide HCl, desmethy-ldiazepam, diazepam purity 99% and isopro-pylamine (99%) were purchased from SigmaUSA. Methanol and acetonitrile HPLC gradecame from Fisher Chemical UK., potassiumdihydrogen phosphate was from Fluka Swit-zerland., and potassium hydroxide pellets andammonia solution 25% were from Merck,Germany.

Stock solutionAll benzodiazepine: flunitrazepam (FNZ),

oxazepam (OZP), alprazolam (AZL), chlordiaz-epoxide (CDZ), desmethyldiazepam (DDZP)and diazepam (DZP) and carbamazepine stocksolutions were prepared in methanol (1 mg/mL) and stored at -20OC. They were found tobe stable for several months.(3,6) The workingsolution was prepared at the appropriate dilu-tion of the stock solution, with a methanol:ultrapure water ratio of 1 : 1 (v/v).

Chromatographic conditionsThe gradient elution, consisting of water

containing 0.125% isopropylamine (v/v) (A)

Serum benzodiazepine determination 25

acetonitrile (B) and methanol (C), was appliedwith the following profile: 0 to 23 min, from 45(A): 5 (B) : 50 (C) to 20 (A) : 12 (B) : 68 (C)and 23 to 25 min was 20 (A) : 12 (B) : 68 (C).A flow rate of 0.7 mL/min signals was moni-tored at 230, 235 and 239 nm, consecutively.The total run time was 25 min. After eachrun, the system was allowed to equilibrate onthe initial solvent conditions for 5 min beforeinjection of the next sample.

SubjectsThis study was approved by the Ethical

Conduct of Research Involving Humans Com-mittee of the Faculty of Medicine, Chiang MaiUniversity. A serum blank was prepared frompooled serum of twenty healthy volunteers,who had not consumed alcohol or other drugsfor 1 month.

Whole blood samples were collected fromtwenty-five autopsy cases, in which serumscreening for benzodiazepine was positive, atthe Department of Forensic Medicine, Facultyof Medicine, Chiang Mai University.

The serum samples were stored at -20OCuntil analyzed. Benzodiazepine and their me-tabolites were stable for at least 12 months.(6)

Samples preparationAfter 100 mL of 10 mg/mL carbamazepine

in methanol : ultrapure water (1:1, v/v) wasadded to 1 mL of serum as an internal standard,the samples were diluted with 1 mL of 0.2 Mphosphate buffer pH 6.0, and the solution wasbriefly mixed. The mixture was applied to abond Elut- C18 cartridge that had previouslybeen activated with 3 mL of MeOH and 3 mLof 0.2 M phosphate buffer pH 6.0. The car-tridge was then washed with 3 mL of 50 mL/Lof methanol in 0.2 M phosphate buffer pH 6.0,and air vacuumed for 10 seconds. The desired

fraction was eluted with 1.2 mL of methanol :10%NH3 solution (5 : 1, v/v). The elutate wasevaporated to dryness under N2 gas (99.99%).The residue was dissolved in 200 mL of metha-nol : ultrapure water (1 : 1, v/v). The sampleswere injected into the HPLC apparatus.

Method evaluationCalibration and linearityCalibration samples were prepared in blank

serum by adding the appropriate amount ofrespective benzodiazepines (250-4,000 ng/mL).Carbamazepine (100 mL of a 10 mg/mL) wasused as an internal standard for the differentsamples. The calibration samples were pro-cessed in the same way as the unknownsamples. Calibration curves were obtained byplotting the peak area ratio of the analyses tothat of the internal standard against the con-centration of the respective benzodiazepines.

Extraction recoveryExtraction recovery was evaluated by com-

paring the peak area of each benzodiazepinein spiked serum with that obtained after injec-tion of a known amount of a standard in mobilephase.

PrecisionWithin run and between run coefficients of

variation were determined by replicate analy-sis of an aliquot of a sample, in either the sameday or on separate days.

Assay detection limitThe limit of detection (LOD) was estimated

from extracted serum samples spiked withdecreasing concentrations of the benzodi-azepines, where the response was equivalentto three times the baseline noise.

26 Pongraveevongsa P, et al.

The limit of quantification (LOQ) was ob-tained by the same procedure used for LOD, butestimated as ten times the signal to noise ratio.

Results and discussionThe retention time of internal standard and

benzodiazepines: flunitrazepam (FNZ),oxazepam (OZP), alprazolam (AZL), chlordiaz-epoxide (CDZ), desmethyldiazepam (DDZP)and diazepam (DZP) were 7.6, 8.7, 9.1, 10.6,

11.9, 12.7 and 14.3 min, respectively. The chro-matographic run time selected was 20 min.Representative chromatograms are shown inFig. 1. In all drug-free serum samples, onlyinternal standard peaks were found, andendogenous peaks were not observed at theretention time of benzodiazepines (Fig.1A).

The linearity of the extracted FNZ, OZP,AZL, CDZ, DDZP and DZP was determinedby spiking blank serum with known amounts

Figure 1. Representative chromatograms of benzodiazepines: flunitrazepam (FZP), oxazepam (OZP),alprazolam (AZL), chlordiazepoxide (CDZ), desmethyldiazepam (DDZP) and diazepam (DZP) detected byultraviolet absorption at 239 nm. (A) Drug-free serum with carbamazepine (I.S.). (B) Drug-free serum withI.S. and 6 standard benzodiazepines (1,000 ng/mL) added. Chromatograms (C) and (D) are serum samplesfrom autopsy cases (C) chlordiazepoxide (CDZ: 17,069 ng/mL) and (D) desmethyldiazepam (DDZP: 2,493ng/mL) and diazepam (DZP: 1,622 ng/mL).

Serum benzodiazepine determination 27

of each analyst, in order to obtain concentra-tions of 250, 500, 1,000, 2,000 and 4,000 ng/mL. Fig. 2 shows the standard concentrationcurves which presented analytical procedurethat proved to be linear (squared correlationcoefficient g2 > 0.995).

The recovery of FNZ, OZP, AZL, CDZ,DDZP and DZP was determined by addingthree different concentrations (250, 1,000 and3,000 ng/mL) to drug-free serum, and therecovery values were 91% (range 89-94%),91% (range 89-93%), 93% (range 89-97%),84% (range 81-87%), 110% (range 93-144%)and 92% (range 84-97%), respectively.

The quantitative evaluation was carried outin serum using six benzodiazepines: FNZ, OZP,AZL, CDZ, DDZP and DZP. The instrumen-tal detection limit of these benzodiazepines was1.25 to 5.0 ng/injection, and the lower limit ofquantitation was 150, 83, 286, 340, 213 and 507

ng/mL, respectively. The examined range was250 to 4,000 ng/mL for all six benzodiazepines.

The coefficient of variaton and relativeaccuracy values of the six benzodiazepinesobtained in the within run and between runassays at a concentration of 500 ng/mL arepresented in Table 1. The results show thatthe within run data were accurate for all ben-zodiazepines within the acceptance interval ofâ 20% of the nominal concentration at thequantitation limit. The precision valuesexpressed as coefficient of variation werewithin the excepted limits of 20% at thequantitation limit. The precision data of DZPwere over the excepted quatitation limit(22.1%), because the determined concentra-tion was lower than the LOQ and the non-homogenous of the spiking serum. Thebetween run data on the accuracy values ofDDZP and DZP were not acceptable (%RA:

Figure 2. Standard concentration curves for FNZ, OZP, AZL, CDZ, DDZP and DZP in serum.FNZ: y = 1581.6x – 28.219, OZP: y = 794.17x – 2.7842, AZL: y = 1031.8x + 0.2575CDZ: y = 868.19x – 5.0003, DDZP: y = 662.08x – 70.542, DZP: y = 721.6x + 27.273.

28 Pongraveevongsa P, et al.

78.4 and 77.0%). This may be due to decreasedbenzodiazepines measuring at a low concen-tration.(6)

Table 2 shows the results of analysis forbenzodiazepines in 25 autopsy cases. Benzo-diazepines were tested positive. More than onebenzodiazepine was detected in two cases,where there was chlordiazepoxide with diaze-pam, desmethyldiazepam and oxazepam. Inthis study, the oxazepam level was higher thanthat in other benzodiazepines in 18 cases(72%). Two cases were found to have alpra-zolam and chlordiazepoxide with diazepam overa toxic range.

ConclusionsThis high-performance liquid chromato-

graphic procedure was developed for the si-multaneous determination and quantification ofsix benzodiazepines: flunitrazepam, oxazepam,alprazolam, chlordiazepoxide, desmethyldiaze-pam and diazepam. It appears that the tech-nique is rapid, simple and suitable for routineanalysis. The mobile phase contains no buffer.Isopropylamine for the modification of pH canavoid some problems, which often happenedin an HPLC assay when the buffer is used.For example, crystal formed in connecting tub-ing and detector cells, as well as damage tothe pump seals.(5) This solid phase extraction

Serum benzodiazepine determination 29

procedure provided cleaner extracts and betterrecovery than traditional liquid-liquid extrac-tion.(4,5,8,9) The solid phase extraction (SPE)method(10) is easy to use and optimized for iso-lation of a broad range of drugs from serum.The blank extract from serum gave no peaksto interfere with any benzodiazepines andcarbamazepine on the chromatogram. The limitof quantitation was lower than the therapeuticrange of the benzodiazepines (except forflunitrazepam and alprazolam). Therefore, themethod can be used in routine forensic appli-cations. It can also be used to quantitate knownbenzodiazepines.

Although there have been more than 35types of benzodiazepines, the protocol devel-oped in this study shows detection of only 6most commonly used benzodiazepines.(1,4) Inour pilot study, this protocol could determineother types of benzodiazepines, such asclonazepam, lorazepam, temazepam, triazolamand midazolam at the retention times of 5.5,8.8, 10.0, 11.2 and 14.2 min, respectively. How-ever, we could not quantify them in this studybecause of the lack of pure standards.

AcknowledgmentsThis work was supported by the Faculty of

Medicine Research Fund, Chiang Mai Univeristy,Chiang Mai, Thailand. The authors would like tothank Dr. Suwiwek Lipigorngoson for kindly sup-plying ultrapure water. We also thank the head ofdepartment, staff, residents and graduated studentsof the Department of Forensic Medicine, ChiangMai University, for their kind support.

References1. Recommended Methods for the Detection and

Assay of Barbiturates and Benzodiazepines in Bio-logical Specimens. National Laboratories. UnitedNations International Drug Control Programme.Vienna Austria 1997. p. 45-92.

2. Karch SB. Drug Abuse Handbook. San Francisco,California. 1998;36:181-4.

3. Mahjoub AEl, Staub C. Simultaneous determina-tion of benzodiazepines in whole blood or serumby HPLC/DAD with a semi-micro column. J PharmBiomed Anal 2000;23:447-58.

4. Lambert WE, Meyer E, Xue-Ping Y, De LeenheerAP. Screening, Identification, and Quantitation ofBenzodiazepines in Postmortem Samples by HPLCwith Photodiode Array Detection. J Anal Tox 1995;19:35-40.

5. He W, Parissis N. Simultaneous determination offlunitrazepam and its metabolites in plasma andurine by HPLC/DAD after solid phase extraction. JPharm Biomed Anal 1997;16:707-15.

6. Mahjoub AEl, Staub C. Stability of benzodiazepinesin whole blood samples stored at varying tempera-tures. J Pharm Biomed Anal 2000;23:1057-63.

7. Winek CL, Wahba WW. Drugs and Chemical blood-level data 2001. Forensic Science International2001;122:107-23.

8. He H, Sun C, Wang XR, et al. Solid-phase extrac-tion of methadone enantiomers and benzodiazepinesin biological fluids by two polymeric cartridges forliquid chromatographic analysis. J Chrom B 2005;814:385-91.

9. Drummer OH. Methods for the measurement ofbenzodiazepines in biological samples. J Chrom B1998;713:201-25.

10. Lai C, Lee T, Au K, Chan A. Uniform solid-phaseextraction procedure for toxicological drugs screen-ing in serum and urine by HPLC with photodiode-array detection. Clin Chem 1997;34:312-25.

30 Pongraveevongsa P, et al.

การตรวจวเคราะหหายาเบนโซไดอะซปนในซรม ผานการสกดดวย solidphase extraction โดยวธ high-performance liquid chromatography

ภทรวด พงษระววงศา, วท.ม., กานดา วชยรตน, พ.บ., พงษรกษ ศรบณฑตมงคล, พ.บ.,วรเชษฐ ขอบใจ, วท.ม.

ภาควชานตเวชศาสตร คณะแพทยศาสตร มหาวทยาลยเชยงใหม

บทคดยอ ปจจบนพบวายากลมเบนโซไดอะซปนถกนำมาใชในทางทผดอยางแพรหลาย ยาในกลมนมคณสมบตคอลดอาการวตกกงวลและซมเศรา รกษาการเกรงตวของกลามเนอ ควบคมกลามเนอสนจากการชก การถอนเหลา และใชรกษาอาการนอนไมหลบทมสาเหตจากความวตกกงวล งานวจยนทำการพฒนาการตรวจวเคราะหยาเบนโซไดอะซปนทพบใชบอย 6 ชนด และสารเมตาบอไลทในซรม ไดแก flunitrazepam, oxazepam, alprazolam, chlordiazepoxide, diazepam และ desmethyldia-zepam โดยใชวธสกดตวอยางโดยใช solid phase extraction (SPE) และวธ high-performance liquidchromatography (HPLC) การตรวจวเคราะหหายาในเชงปรมาณทความยาวคลนแสง 239 นาโนเมตรโดยอาศยคอลมนบรรจสารชนด C8 และตวทำละลายทประกอบดวยสารละลายดางทปราศจากเกลอบฟเฟอรรวมกบ acetonitrile และ methanol ในระบบปรบเปลยนอตราสวนตวทำละลาย ใชสารคารบามาซปนเปนสารมาตรฐานภายในตรวจวเคราะห ยาทง 6 ชนด คอ flunitrazepam, oxazepam,alprazolam, chlordiazepoxide, desmethyl diazepam และ diazepam จะถกตรวจแยกยาออกจากกนตามลำดบ โดยวเคราะหในเชงเสนตรงไดทความเขมขน 250-4,000 นาโนกรม/มลลลตร มคาเฉลยของการตรวจพบยา (recovery) เทากบรอยละ 91, 91, 93, 84, 110 และ 92 ตามลำดบ วธนสามารถตรวจแยกยาไดชดเจน มความไวและความจำเพาะด สามารถนำมาปรบใชในงานประจำของหองปฏบตการพษวทยาได เชยงใหมเวชสาร 2550;46:(1):23-30.คำสำคญ: กลมยาเบนโซไดอะซปน ซรมเบนโซไดอะซปน