Chem. Anal. (Warsaw), 49, 71 (2004) Identification and Determination of Flunitrazepam and Its Metabolites in Blood by Gas Chromatography by Zofia Ch³obowska1, Czes³awa wiegoda1, Pawe³ Kocielniak1,2* and Wojciech Piekoszewski1 1 Institute of Forensic Research, ul. Westerplatte 9, 31033 Cracow 2 Faculty of Chemistry, Jagiellonian University, ul. R. Ingardena 3, 30060 Cracow Key words: flunitrazepam, metabolites, gas chromatography Analytical method for the isolation of flunitrazepam and its metabolites (desmethyl- flunitrazepam and 7-amino-flunitrazepam) from blood involving liquid-liquid extraction with diethyl ether, diisopropyl ether and toluene-isoamyl alcohol mixture (95:5) has been developed. In the latter case the extraction yield approached 80%. The analysis of the com- pounds was performed using gas chromatograph with electron capture detector, and a HP1 column. The developed method allowed one to determine flunitrazepam and its metabolites at the level as low as few nanograms. Opracowano metodê analityczn¹ pozwalaj¹c¹ na ekstrakcyjne wyizolowanie flunitrazepamu i jego metabolitów (desmetyloflunitrazepamu i 7-aminoflunitrazepamu) z krwi przy u¿yciu eteru dietylowego, eteru diizpropylowego lub mieszaniny toluenu z alkoholem izoamylowym (95:5). W ostatnim przypadku osi¹gniêto wydajnoæ na poziomie 80%. Do analizy wymie- nionych substancji zastosowano chromatografiê gazow¹ z detektorem wychwytu elektronów oraz kolumnê HP1. Opracowana metoda pozwala wykrywaæ zawartoæ flunitrazepamu i jego metabolitów we krwi na poziomie kilku nanogramów. * Corresponding author. E-mail: [email protected] 72 Z. Ch³obowska, C. wiegoda, P. Kocielniak and W. Piekoszewski Flunitrazepam (Rohypnol), the fluoro derivative of benzodiazepine, is a sedative and hypnotic drug. It is easily absorbed from alimentary tract and undergoes biotrans- formation via N-dimethylation, hydroxylation and reduction with moderate rapidity [1]. It is used for the treatment of insomnia, as a preanesthetic agent in surgical opera- tions, and in intensive therapy. Flunitrazepam acts synergistically with muscle rela- xants and intensifies depressive effect of drugs inhibiting the central nervous system activity [2]. When taken with alcohol may cause anterograde amnesia and even death. Therefore its influence on humans is investigated in forensic toxicology [35]. Since its introduction to the pharmaceutical market in 1975 flunitrazepam was overused in many countries in America, Australia, Asia and Europe mostly by heroin addicts, since it boosts the effect of heroin or even replaces it when the latter is not available. In order to cause intoxication, flunitrazepam may be taken independently, or in com- bination with alcohol or other drugs orally, intravenously or by inhalation in the form of powdered tablets. Although flunitrazepam is a legal medicament in Poland, it is also smuggled from abroad. Police statistics indicate that the drug is used to intoxicate women before the rape. For that purpose the drug is given secretly, usually mixed with alcohol. Gradu- ally, depending on the doses administered, a loss of consciousness appears, and in consequence of anterograde amnesia victims do not remember what has happened to them. These circumstances necessitated the determination of flunitrazepam in biologi- cal material to meet the needs of forensic investigations and clinical toxicology. Flunitrazepam and its metabolites may be detected and determined in biological fluids (blood, urine) and hair. The most commonly used analytical methods for the detection are high-pressure liquid and gas chromatographies. Gas chromatography with electron capture detector is sensitive towards flunitrazepam and desmethyl-fluni- trazepam, but not to 7-amino-flunitrazepam. GCMS technique requires derivatisation of 7-amino-flunitrazepam before its determination. HPLC with UV detection is not enough sensitive to perform determination of these drugs at therapeutical concentra- tions [510]. Among sample preparation methods the leading group involves liquid- liquid and solid phase extraction techniques. The aim of this study was to develop a suitable analytical procedure for the detec- tion of traces of flunitrazepam and its metabolites in blood. The work includes optimisation of the separation conditions of flunitrazepam, desmethyl-flunitrazepam, and 7-amino-flunitrazepam, elaboration of their extraction procedure from blood, and evaluation of the influence of biological matrix putrefaction on the results of the analyses. Identification and determination of flunitrazepam in blood 73 EXPERIMENTAL Chromatographic separation of flunitrazepam and its metabolites At first it was indispensable to work out the optimum conditions for chromatographic separation of flunitrazepam and its metabolites desmethyl-flunitrazepam and 7-amino-flunitrazepam. In order to do so one prepared 50 ng mL-1 methanolic solutions of the given drug and its metabolites, individually and in the mixture. The solutions were introduced into a megabore chromatographic column HP1 30 m × 0.53 mm (1.5 µm). The analyses were performed using a FISONS GC 8000 gas chromatograph equipped with elec- tron capture detector (EC). Helium was passed through the system as a carrier gas at a flow rate of 15 mL min-1. The temperature of EC detector was kept at 300°C and of the injector at 250°C. The separation of the investigated drugs was carried out at the temperature of 240°C. Development of a blood extraction procedure To develop the extraction procedure of the examined compounds from blood, standard blood samples containing 50 ng mL-1 of flunitrazepam, desmethyl-flunitrazepam, and 7-amino-flunitrazepam have been prepared. The applicability of diethyl ether, diisopropyl ether and a mixture of toluene with isoamyl alcohol (95:5) for the extraction has been examined. 5 µL of 50 ng mL-1 flurazepam solution (internal standard), 200 µL of phosphate buffer, and 1 mL of the extracting agent were added to 0.2 mL of blood sample containing flunitrazepam and its metabolites. The buffer provided the pH at the level of 7.4 in the case of diethyl ether and diisopropyl ether, and at 7.8 when the mixture of tolueneisoamyl alcohol was used. The contents were shaken for 60 s, next centrifuged, and finally the organic phase was separated and extracted again with 400 µL of phosphate buffer solution for 30 s. After that the aqueous phase was removed, while the organic one was evaporated under the stream of nitrogen at a temperature of 37°C. The residue was dissolved in 500 µL of methanol before introducing it to the sample injector. Evaluation of the influence of biological matrix on the determination of flunitrazepam A composition of a matrix of fresh blood sample taken from a living person and a corpse, and a decayed blood sample taken from a corpse were examined employing the extraction procedure described above. An autopsic blood sample with added flunitrazepam was also analysed. In each case five different blood samples have been analysed. Validation of the determination procedure of flunitrazepam In order to estimate the extraction recovery of flunitrazepam from blood and to examine the repeatabil- ity of the proposed analytical procedure, blood samples free from examined drugs and containing the analyte at three different concentrations (20, 50, and 100 ng mL-1) were prepared and extracted according to the procedures described above. The samples of a given concentration of the analyte were subjected to the extraction with diethyl ether, diisopropyl ether and a mixture of toluene with isoamyl alcohol. After extrac- tion flurazepam was introduced to the solutions as an internal standard. Moreover, three methanolic standard solutions containing the analyte and the internal standard at the same concentrations as the extracted samples have been prepared. The signals obtained for the extracted and standard solutions were compared in order to calculate the extraction recovery. The measurement was performed five-fold for each sample extracted and the results were averaged to get the final value. 74 Z. Ch³obowska, C. wiegoda, P. Kocielniak and W. Piekoszewski Detection limits of the examined drugs were determined as usual from the peak heights of the internal standard, and the noise signals referring to five different points on a peak baseline of each drug [11]. Next step was to estimate the concentration range of flunitrazepam within which the measured analyti- cal signals changed linearly. Thus, blood samples free from the examined drugs and containing flurazepam as the internal standard and the analyte at the concentration ranging from 10 to 200 ng mL-1 were subjected to the extraction (using diethyl ether, diisopropyl ether and a mixture of toluene with isoamyl alcohol as extracting agents). After the extraction of each sample was completed, the analyte and the internal standard were determined in few consecutive analyses, and the average value was taken for further calculations. RESULTS AND DISCUSSION Under the optimum conditions, the retention times and the relative retention times for flunitrazepam and its metabolites were determined and compared with the respec- tive values for flurazepam (internal standard). Alcoholic solution containing the stud- ied compounds at the concentration of 50 ng mL-1 was directly injected to the chro- matographic column. Exemplary chromatogram of the sample is shown in Figure 1. 7-amino-flunitrazepam 5.100 flunitrazepam desmetyl-flunitrazepam flurazepam 2 4 6 8 10 12 14 16 min Detector signal, AU 0 2 4 6 8 10 12 14 16