LC-(TOF) MS Analysis of Benzodiazepines in Urine from Alleged Victims of Drug-Facilitated Sexual Assault

Journal of Analytical Toxicology, Vol. 31, October 2007 LC-(TOF) MS Analysisof Benzodiazepinesin Urine from Alleged Victims of Drug-Facilitated Sexual Assault Mahmoud A. EISohly 1,2,3,*, Waseem Gul 1,2, Timothy P. Murphy 1, Bharathi Avula 2, and Ikhlas A. Khan 2,4 I Abstract I The presentstudy employs a recently reported liquid chromatography(time of flight) massspectrometry procedure for the simultaneousanalysis of 22 benzodiazepines in human urine specimens. The analysisfocused on the most commonly prescribed benzodiazepines and/or their metabolites. Using this method, the limit of quantitation for the benzodiazepines tested ranged from 2 to 10 ng/mL, while the limit of detection range was 0.5 to 3.0 ng/mL. Urine specimenscollected from alleged victims of drug-facilltated sexual assault (156 specimens) were tested. Only 19 out of the 22 benzodiazepinesanalyzed were detected in these specimens.These samespecimens were previously screened for benzodiazepines by various immunoassaytechniques using a 50 ng/mL cut-off level and confirmed by a gas chromatography--massspectrometry method after acid hydrolysisto their benzophenone skeletons,thus making the identification of the specific benzodiazepine(s) involved impossible for most specimens. This study aims to offer an alternative methodology that would allow such identification for similar specimens.Additionally, the distribution of the individual benzodiazepines of interest among the 156 specimensas well as their prevalence in specimensoriginating in different U.S. statesis presented. Introduction last few years to the area of drug-facilitated sexual assault. One of the benzodiazepines that is most often implicated in drug-facilitated sexual assault cases is flunitrazepam (3,4), a drug that has been marketed for over 20 years for the treatment of severe sleep disorders. In the late 1990s, F. Hoffrnan-LaRoche (Roche), the manufacturer of flunitrazepam (brand name Rohypnol), embarked on a program to determine the use of drugs, including flunitrazeparn, in alleged cases of sexual assault, by sponsoring the development of a sensitive method [gas chromatography-mass spectrometry (GC-MS)] to detect flunitrazepam rnetabolite in urine at a level below 1 ng/mL (5) and by initiating and offering a testing program of independent urine analyses to victims of alleged sexual assault in the United States. The program included testing for a wide range of drugs of abuse including alcohol. A total of 3303 urine specimens were submitted and tested under this program, and the results of the testing have been previously reported (6). Although the testing showed only a few urine specimens to be positive for flunitrazepam (9 specimens), over 300 specimens were positive for the benzodiazepines class with a high percentage of specimens testing positive for more than one drug, indicating exposure to polypharmacy. The GC-MS method used in that study involved acid hydrolysis of the benzodiazepines to their benzophenone skeleton in order to increase the sensitivity (5), thus making impossible the determination of the benzodiazepine(s) involved in many cases. Benzodiazepines comprise a class of clinically important drugs with therapeutic activities covering a wide range of indications thus rendering them useful as tranquilizers, anticonvulsants, muscle relaxants, and sleep aids (1). The pharmacological properties of benzodiazepines, as valuable as they are in clinical practice, are cause for serious abuse problems all over the world (2). Such abuse has extended over the * Author to whom correspondenceshould be addressed. In a previous publication (7), the authors of the present study presented a newly developed procedure for the simultaneous analysis of 22 different benzodiazepines using liquid chromatography coupled with (time-of-flight) mass spectrometry, LC-(TOF) MS. The sensitivity of the new LC-(TOF) MS procedure was such that the limit of detection ranged from 0.5 to 3.0 ng/mL for all benzodiazepinestested. For those benzodiazepines where the SOFT DFSA Committee has recommended a maximum detection limit, the detection limit of this procedure is 5-10 times lower, except for 7-aminofluni- Reproduction Iphotocopying) of editorial content of this journal is pn~hibited without publisher's permission. 505 IEISohlyLaboratories, Incorporated, 5 Industrial Park Drive, Oxford, Mississippi38655; 2National Center for Natural Products Research(NCNPR),School of Pharmacy, University of Mississippi, University, Mississippi38677; 3Department of Pharmaceutics,School of Pharmacy, University of Mississippi, University, Mississippi38677; and 4Department of Pharmacognosy,School of Pharrnacy, University of Mississippi, University, Mississippi38677 Journal of Analytical Toxicology, Vol. 31, October 2007 trazepam, where this procedure's LOD is only 2.5 times lower. The method was used to re-analyze 13 specimens that screened positive by the immunoassay used in the study mentioned but were not confirmed by the existing GC-MS method. All 13 specimens were confirmed for one or more benzodiazepines using the LC-(TOF) MS method. The present study describes the use of the LC-(TOF) MS procedure for the analysis of 156 specimens collected nationwide from alleged victims of drug-facilitated sexual assault in an attempt to ascertain the involvement of benzodiazepines in these cases as well as the regional distribution of individual benzodiazepines within the 35 U.S. states included in this study. Materials Standard solutions 7-Aminonorflunitrazepam,7-acetamido-norflunitrazepam, bromazepam, 3-OH-flunitrazepam, 2-OH-flurazepam, diazepam, 7-aminoclonazepam,desmethylflunitrazepam, lorazepam, norfludiazepam, flunitrazepam, 7-aminoflunitrazepam, a-OH-alprazolam-ds,flunitrazepam-dT,temazepam, iormetazepam, triazolam, nitrazepam, a-OH-triazolam, nordiazepam, midazolam,and alprazolamwere purchased from Cerilliant (RoundRock,"IX).Oxazepam,a-hydroxyalprazolam, oxazepam-ds, and nordiazepam-d5 were purchased from Sigma, and 7-aminoflunitrazepam-d3 was obtained from Total # of State Specimens 1' AK AL AR CA CO CT DE FL HI IL IN LA MA MD MI MN MO NC NE NH NJ NM NV NY OH OK OR PA SC TN TX UT VA WA WI WV Total % 2 4 2 19 3 1 1 7 5 5 8 1 4 5 7 3 1 4 2 1 2 3 4 I 2 I 7 I 2 2 8 I 2 I 7 I 2 2 8 1 1 5 3 1 1 5 3 1 1 5 2 1 1 2 2 2 I 1 1 2 2 2 I 2 1 1 1 2 I 1 1 5 3 1 2 1 2 2 2 I 1 1 1 1 1 1 4 2 2 2 2 2 4 3 2 6 3 7 38 4 2 4 1 1 156 1 1 1 2 5 I 6 7 8 2 2 I I 2 I 2 2 I 3 2 2 2 2 3 2 5 5 1 1 2 2 1 1 1 1 2 1 1 2 2 2 1 1 1 4 2 6 11 2 1 2 1 1 1 2 1 1 5 2 6 15 2 1 2 1 1 1 2 1 1 5 1 6 12 1 1 2 1 1 1 2 1 1 5 2 6 17 1 1 2 1 67 43.1 1 75 48.1 1 1 63 78 40.4 50.0 9 10 11 12 13 14 15 16 17 2 18 19 1 1 I 2 1 2 1 1 1 2 1 1 3 1.92 1 1 1 2 I 7 1 1 1 1 2 I 7 1 1 1 9 5 2 1 35 22.4 34 21.8 24 15.4 1 1 1 1 18 2 3 11.5 1.28 1.92 1.92 3 < LOQ 2 1 1 0 4 1 1 1 1 0.64 0 2.56 0.64 0.64 0.64 0.64 * (...truncated)