A gas chromatography-mass spectrometry method was developed for the analysis of flunitrazepam (FN) and its major metabolite, 7-amino-flunitrazepam (7-amino-FN), in both plasma and whole blood. The method was based on acid hydrolysis of the samples after dilution with HPLC water followed by extraction and derivatization (heptafluorobutyrate) of the resulting benzophenones. Analysis of plasma and whole blood samples from subjects administered 2-mg doses of FN showed that FN was only detected in whole blood (LOD 5 ng/mL) and not in plasma. However, 7-Amino-FN was detected in both plasma and whole blood, although the levels were much higher in plasma. 7-Amino-FN was detected for the entire period of specimen collection (12 h), but FN was only detected in whole blood for 4 h after ingestion with peak levels after 1 

Brenneisen, R.

ElSohly, M.A.

Feng, S.

Salamone, S.J.

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Journal of Analytical Toxicology, Vol. 23, October 1999 GC-MS Determination of Flunitrazepam and its Major Metabolite in Whole Blood and Plasma M.A. EISohly 1, S. Feng 1, S.I. Salamone 2, and R. Brenneisen 3 I EISohly Laboratories, Incorporated, 5 Industrial Park Drive, Oxford, Mississippi 38655; 2Roche Diagnostics Systems, .Somerville, New Jersey 08876; and 3University of Bern, Bern, Switzerland I Abstract [ A gas chromatography-mass spectrometry method was developed for the analy~s of t]unJtrazepam (FN) and its major metabolite, 7-amino-flunitrazepam (7-amino-FN), in both plasma and whole blood. The method was based on acid hydrolysis of the samples after dilution with HPLC water followed by extraction and derivatization (heptafluorobutyrate) of the resulting benzophenones. Analysis of plasma and whole blood samples from subjects administered 2-mg doses of FN showed that FN was only detected in whole blood (rOD 5 ng/mL) and not in plasma. However, 7-amino-FN was detected in both plasma and whole blood, although the levels were much higher in plasma. 7-Amino-FN was detected for the entire period of specimen collection (12 h), but FN was only detected in whole blood for 4 h after ingestion with peak levels after I h. the time course of detection of the parent drug versus its metabo- lite in both plasma nd blood samples collected at the same time. Experimental Materials 7-Amino-FN-ds was purchased from Lipomed, Inc. (Cambridge, MA). FN, nor-FN, and 7-amino-FN were obtained from Radian Corp. (Austin, TX). 7-Amino-nor-FN was provided by Roche Diagnostics Systems (Somerville, N J). All solvents were high-per- formance liquid chromatography (HPLC) grade, and the chemi- cals were American Chemical Society reagent grade. Introduction Flunitrazepam (FN), or Rohypnol ~, is a sleeping aid that belongs to a group of newer-generation benzodiazepines that are admin- istered in small dosage. Although it is not legally available in the United States, it has appeared onU.S. streets and been alleged to have been involved in some date-rape cases (1,2). Studies (3,4) on excretion patterns of FN showed that 7-amino- flunitrazeparn (7-arnino-FN) is the most predominant metabolite in urine with the parent drug detected in only a few cases. The detection window of 7-amino-FN in urine can be extended to at least 72 h after ingestion (4). In the meantime, there are some questions about he concentration f FN and its metabolites in blood. It was reported (5,6) that he concentration f 7-amino-FN can greatly exceed that of unchanged parent drug in stored blood samples as a consequence of an in vitro reduction of the 7-nitro group of FN. In a previous communication (4), we reported on a sensitive gas chromatography-mass spectrometry (GC-MS) pro- cedure for the analysis of FN metabolites in urine samples to detect prior ingestion of the drug. Because serum or, more often, blood specimens are commonly collected for forensic toxicological analysis, this communication was initiated to present a simple extraction method and a sensi- tive GC--MS procedure for the detection of both 7-amino-FN and FN in both serum (or plasma) and whole blood and to ascertain Extraction, hydrolysis, and derivatization To 1 mL of plasma or blood sample in a 15-mL centrifuge tube, 25 I~L of 7-amino-FN-d3 at1 I~g/mL (25 ng/mL of plasma or blood concentration) was added. Three milliliters of water (HPLC grade) was used to dilute the sample, which was then vortex mixed briefly, followed by the addition of 1 mL of concentrated HCI. The tube was tightly capped and placed in an oven at 100~ for I h. After the tube was taken out of the oven and cooled to room temperature, 1.5 mL of 10N KOH was added, and the sample was vortex mixed. Six milliliters of chloroform was used to extract the sample by shaking ently for 2 min. After the sample separated into two layers (centrifugation maybe needed to help phase separation), thetop aqueous layer was aspirated to waste. The chloroform layer was washed with water twice (1mL each) and then poured into a 13 x 100-mm test ube. The solvent was evaporated under N2 at 50~ in a water bath. The residue was dis- solved in 0.5 mL of chloroform containing 20 IJg/mL of 4-pyrro- lidinopyridine, and 100 IJL of heptafluorobutyric anhydride was added. The sample was vortex mixed and allowed to stand at room temperature (65-75~ for 1 h before the addition of 0.2 mL of 2N NaOH and 1 mL of 1.5M carbonate buffer (pH 11). After vortex mixing for 30 s, the aqueous layer was aspirated to waste. One milliliter of deionized water was added, and the sample was vortex mixed for 30 s. The organic layer was then transferred to a GC vial, and the solvent was evaporated under N~ at 50~ The residue was dissolved in 50 I~L of ethyl acetate for GC-MS analysis. Two microliters of sample was injected. 486 Reproduction (photocopying) of editorial content of this journal is prohibited without publisher's permission. Journal of Analytical Toxicology, Vol. 23, October 1999 6oo00 l'" A ~- 60000 ' 0  ,- 40000 ['[l '" 0 _ I, . .  , I , ,  . l .. ';' 1 O0 200 300 400 m/z 0 -~ I _o B co 1600000 "~ 1200000 '" " 400000 [ i . , I .  0 100 200 300 400 m/z 600 r" i' 7ooooo1 I . , .  ~oooooI I ,,, f, ~= 3oooooj I l l  ,., I ,,, I I  < lOOOooL[.ll/... I . .  L, . . .  , I ~1 I , ,  U 100 200 " 300 " 400  61)0 m/z C 6)a  ~"' t, l 9 8~ 16~176176176176 "  D ~ 1400000 -[ [ ,s, ,,, '~176176176176176 / I i == 6oooo0 .,: 9 * 2ooooot J I 'i' .,. ,.,3. I , I ", . . . , ,  I 9 . ; I  . . . .  = . . , , -  . . . , .  , , j  . 100 200 300 400 600 800 m/z Figure 1. Full-scan mass spectra of the heptafluorobutyrate derivatives of FN (A), nor-FN (B), 7-amino-FN (C), and 7-amino-nor-FN (D). Table I. Concentrations (ng/mL) of FN and Metabolites in Blood Collected from Two Subjects Administered a Single 2-rag Oral Dose Time 7-Amino- Subject postdose (h) FN 7-Amino-FN Nor-FN Nor-FN A 0 0.00 0.00 0.00 0.00 A 0.5 5,66 5,13 0.00 0.00 A 1 8.06 5,89 0.00 0.00 A 2 6.21 6.06 0.00 0.00 A 4 < LOD 6.35 < LOD 0.00 A 8 < LOD 5.78 < LOD 0.00 A 12 < LOD 5.62 0.00 0,00 B 0 0.00 0.00 0.00 0,00 B 0.5 < LOD 1.70 0.00 0.00 B 1 7.27 2.13 0.00 0.00 B 2 5.84 3.88 0.00 0.00 B 4 5.94 5.23 0.00 0.00 B 8 0.00 5.17 0.00 0.00 B 12 0.00 4.20 0.00 0.00 GC-MS conditions GC-MS conditions were similar to those used in the analysis of urine samples (4) but required a relatively onger un time for plasma or blood analysis. A Hewlett Packard (Palo Alto, CA) 5890 GC interfaced with a Hewlett Packard 5970 mass selective detector was used. The electron multi- plier voltage was set at 200 V above the tune value. The GC was equipped with a 25-m x 0.2- ram (0.33-pro film thickness) DB-5 MS column operated in the splittess mode with purge valve closed for 0.2 min. Helium carrier flow was 36.2 cm/s under 25 psi column head pressure. Oven temperature was set at 180~ held for 0.5 rain, then increased to 260~ at 20~ where it was held for 1 min, then finally increased to 280~ at 30~ and held for 8 min. The injector and detector temperatures were 250 and 280~ respectively. A Hewlett Packard G1034C version C.02.00 ChemStation data system was used to record the data. SIM ions for the various analytes were as follows (underlined ions are quantitation ions): 516 and 639 for 7-amino-FN- d3 (Rt = 6.30 rain); 513, 636, and 439 for 7- amino-FN (Rt = 6.32 rain); 274 and 257 for flunitrazepam (Rt = 8.53 rain); 453, 622, and 499 for 7-amino-nor-FN (Rt = 5.92 rain); and 456 and 333 for nor-FN (Rt = 6.20 min). Clinical studies Four subjects were recruited forthese studies with the proper consent forms and protocols approved by the institutional IRB. Two clinical studies in which two subjects were administered a 2-rag oral dose f flunitrazepam were conducted. In the first study, blood speci- mens were collected from subjects A and B at 0, 0.5, 1, 2, 4, 8, and 12 h postingestion. In the second study, subjects C and Dwere each admin- istered a 2-mg dose of flunitrazepam, and blood samples were collected at 0, 0.25, 0.5, 1, 2, 4, 8, and 12 h after dosing. However, in the second study, half of the blood samples were centrifuged and the plasma separated for comparison of the drug and metabolites level in plasma versus whole blood. Results and Discussion In developing analytical methods for drugs and metabolites inplasma or whole blood, extraction procedures often involve protein precipitation with methanol r acetonitrile. In our efforts to develop a procedure for flunitrazepam and its metabolites, an initial protein-precipitation step was carried out using 3 mL of methanol per 487 Journal of Analytical Toxicology, Vol. 23, October 1999 milliliter of plasma or whole blood. This was followed by centrifu- gation, evaporation of the methanol, and acid hydrolysis of the extract. This extraction procedure was only partially successful because of the strong protein binding of the analytes. This resulted in inconsistent extraction efficiency, and in some cases, particularly with whole blood, neither the drugs nor the internal standards were recovered. Other extraction methods such as liquid-liquid extraction using butyl chloride (6,7) and solid-phase extrac- tion using Bond-Eiut Certify ~ columns (8) or CleanScreen | extraction cartridges (9) produced unsatisfactory results for whole blood samples. After several trials with different extraction con- ditions, a procedure was developed for both plasma and whole blood in which 1 mL (plasma or whole blood) was diluted with 3 mL of water (HPLC grade) first and hydrolyzed with hydro- chloric acid directly without separation fthe pro- teins. Most of the precipitate formed during the hydrolysis redissolved into the aqueous phase after basification. The analytes were then parti- tioned into CHCI3. Washing twice with water resulted in a clean CHC13 extract. This process resulted in the formation of specific benzophe- nones corresponding tothe individual drug and metabolites a previously described y E1Sohly et al. (4). These benzophenones are then deriva- tized (heptafluorobutyrate) prior to analysis. Full scan mass pectra of the derivatized benzophe- nones corresponding to FN, nor-FN, 7-amino-FN, and 7-amino-nor-FN are shown in Figure 1. The average recovery (n = 5) for 7-amino-FN and FN in whole blood and plasma was 93% and 53%, respectively, at 25 ng/mL This was determined by comparing the absolute peak area from extracted plasma specimens spiked with the analytes to the peak areas ofthe analytes spiked in HPLC-grade water at the same levels. The limits of detection (LOD) for 7-amino-FN, 7-amino-nor-FN, FN, and nor-FN in plasma were 1.0, 1.0, 5, and 5 ng/mL, respectively. The LOD was established as the lowest concentration of each analyte that provided a signal-to-noise ratio greater th n 5 for the quan- titation ion with the presence ofthe required con- firming ions. The limit of quantitation (LOQ) for o all analytes was found to be the same as the LOD. o All curves were linear up to at least 100 ng/mL, m "a The precision of the method was determined at " 5 ng/mL of all analytes, and the coefficient ofvari- .n ation ranged from 5.1% for 7-amino-FN to 11.7% < for FN. Figure 2 shows a typical chromatogram from a whole blood specimen analyzed by this method, with approximately 6 ng/mL 7-amino-FN and 8 ng/mL FN. Concentration of flunitrazepam and metabolites in whole blood Clinical whole blood samples from two subjects (A and B) administered a 2-mg dose of flunitrazeparn were ana- lyzed by this method. Table I shows that nor-FN and 7-arnino-nor- FN were either too l w to detect or not present. The method was able to detect the presence of 7-amino-FN at concentrations ranging from 1.70 to 6.35 ng/mL over a 12-h period with t e peak concentrations appearing t 4 h.The parent drug, flunitrazepam, was also detected between 0.5 h to 4 h with the peak concentra- tion appearing at1 h in both subjects. I n  1Ol.lll l l l l , P l  to l l l .  l l ) l  V l l l - l l l l . l  I l l :  ' " ' "  , 8000 l ie  9 $ 600o A 4o0o < 2ooo o 6.00 6.10 6.20 6.30 5.40 6.50 6.60 6.70 Time (mln) l~  I I l oW l l l l . IO  te Stl. le]l l  ISIS-fIN.If 250001 " ~"'N ( " ' l  ~' "m}'  "~~ .~ 10000 < '~176176 0 ~ " 6.00 6.10 6.20 6.30 6,40 6.50 6.60 6.70 Time (min) 11000 9ooo 7ooo 5o00 3ooo 10o0 o OIA i l l : l l  | l l | : l t  I! l l ) l - l l l l  V|II'III:l 8.40 8.45 8.50 8.55 8.60 6.65 8.70 Time (min) Figure 2. GC--MS SIM chromatograms of a whole blood sample from Subject A at I h. A, 5.89 nglmL of AFN; B, 25 nglmL of AFN-d3; and C, 8.06 ng/nL of FN. Journal of Analytical Toxicology, Vol. 23, October 1999 Table II. Comparison of FN and 7-Amino-FN Concentrations in Blood and Plasma Whole blood Plasma Time FN 7-Amino-FN FN 7-Amino-FN Subject (h) (ng/mL) (ng/mL) (ng/mL) (ng/mL) C 0 0 0 0 0 C 0.25 4.60* 0.95 0 2.16 C 0.5 5.70 1.49 0 4.11 C 1 6.77 2.83 0 10.4 C 2 4.64* 2.38 0 12.2 C 4 < LOD 2.94 0 11.8 C 8 < LOD 2.93 0 5.21 C 12 0 3.16 0 6.81 D 0 0 0 0 0 D 0.25 0 < LOD 0 < LOD D 0.5 < LOD < LOD 0 8.11 D 1 3.47* 1.00 0 < LOD D 2 < LOD 1.67 0 7.58 D 4 0 2.31 0 10.3 D 8 0 2.80 0 4.21 D 12 0 2.80 0 6.74 *Lower than established LOD but quantitated because these samples howed acceptable ion ratios. Comparison of FN and 7-amino-FN concentrations in whole blood versus plasma in the same samples Blood samples were collected fromtwo subjects (C and D) at dif- ferent times after oral ingestion of a 2-rag dose of flunitrazepam. Each sample was then divided into two portions. One portion was kept as is and used for whole blood analysis. The second portion was centrifuged (1000rpm) to separate he plasma which was analyzed for FN and 7-amino-FN. This experi- ment was carried out to determine the relative levels of FN and 7-amino-FN in plasma versus whole blood. We have previously observed (unpublished data) that FN was absent (below LOD of 10 ng/mL) in plasma samples. Table II shows the concentration f FN and 7-amino-FN in whole blood versus plasma from the same samples. The data show that FN was present only in whole blood and ot in plasma. However, 7-amino-FN was present in both plasma nd whole blood, although the levels were much higher in plasma, sug- gesting the predominance of the metabolite in plasma versus cells, possibly because of matrix effect. The absence of FN from plasma samples in this study is not in total agreement with previous reports (10,11).This could be due to the differences in the sensitivities of the methods u ed (GC-electrochemical detection and liquid chromatography-mass spectrometry in the previous work with sensitivity of 0.2-1.0 ng/mL versus GC-MS at 5 ng/mL in this report). However, the blood levels found i  this study were in agreement with those reported by Bogusz et al. (11) for the clinical samples. Conclusions The GC-MS procedure described in this communication has a high sensitivity to detect flunitrazepam and its major metabolite, 7-amino-FN, in plasma and in whole blood. The parent drug, however, was only detectable in whole blood for approximately 4 h after ingestion of 2-rag doses of flunitrazepam, whereas 7-amino-FN was detectable for the entire period of collection (12 h). Therefore, for forensic purposes, detection of FN in whole blood would indicate recent ingestion of the drug, and 7-amino-FN could be monitored for a longer period of time. References 1. J.D. Ropero-Miller and D.B. Fraser. GHB and Roofies: drugs of the 1990s. Forensic Urine Drug Testing, AACC Educational Newsletter for Labora- tories, June 1997. 2. C. McKissick. Rohypnol: the new date rape party drug of 90's. U.S. Department of Justice Drug Enforcement Administration, public information pamphlet (1997). 3. S.J. Salamone, S. Honasoge, C. Brenner, A.J. McNally, J. Passarelli, K. Goc-Szkutnicka, R. Brenneisen, M.A. EISohly, and S. Feng. Flunitrazepam excretion patterns using the Abuscreen OnTrak and OnLine immunoassays: comparison with GC-MS. J. AnaL ToxicoL 21: 341-345 (1997). 4. M.A. EISohly, S. Feng, S.J. Salamone, and R. Wu. A sensitive GC-MS procedure for the analysis of flunitrazepam and its metabolites in urine. J. AnaL ToxicoL 21 : 335-340 (1997). 5. P.X. Iten. No flunitrazepam in postmortem blood in fatal fluni- trazepam intoxications? Proceedings of the 30th International TIAFT meeting, T. Nagata, Ed. The International Association f Forensic Toxicologists, Fukuoka, Japan, 1992, pp 343-346. 6. I.M. Mclntyre, M.L. Syrjanen, K. Crump, S. Horomidis, A.W. Peace, and O.H. Drummer. Simultaneous HPLC gradient analysis of 15 benzodiazepines and selected metabolites in postmortem blood. J. Anal Toxicol. 17:202-207 (1993). 7. I.M. Mclntyre and T.R. Norman. Analysis and identification of ben- zodiazepines in biological specimens. In Analysis of Drugs of Abuse: Instrumental Methods and Techniques, T.W. Ryan, Ed. Marcel Dekker Inc, New York, NY, 1998. 8. I. Deinl, G. Mahr, and L. yon Meyer. Determination f flunitrazepam and its main metabolites in serum and urine by HPLC after mixed mode solid-phase extraction. J. AnaL Toxicol. 22:197-202 (1997). 9. Clean Screen Extraction Column: Application Manual. Worldwide Monitoring Corporation, PA, pp 30-31. 10. H.G. Boxenbaum, H.N. Posmanter, T. Macasieb, K.A. Geitner, R.E. Weinfeld, J.D. Moore, A. Darragh, D.A. O'Kelly, L. Weissman, and S.A. Kaplan. Pharmacokinetics of flunitrazepam following single-and multiple-dose oral administration to healthy human subjects. J. Pharmacokinet. Biopharm. 6:283-293 (1978). 11. M.J. Bogusz, R.D. Marier, K.D. Kruger, and W. Fruchmicht. Determination of flunitrazepam and its metabolites in blood by high- performance liquid chromatography-atmospheric pr ssure chemical ionization mass spectrometry. J. Chromatogr. B 713:361-369 (1998). Manuscript received April 1, 1999; revision received June 1 O, 1999. 489 

GC-MS Determination of Flunitrazepam and its Major Metabolite in Whole Blood and Plasma

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