Chem. Anal. (Warsaw), 49, 71 (2004)

Identification and Determination of Flunitrazepam and Its Metabolites in Blood by Gas Chromatography

by Zofia Ch³obowska1, Czes³awa Œwiegoda1, Pawe³ Koœcielniak1,2* and Wojciech Piekoszewski1

1 Institute of Forensic Research, ul. Westerplatte 9, 31–033 Cracow 2 Faculty of Chemistry, Jagiellonian University, ul. R. Ingardena 3, 30–060 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 HP–1 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 wydajnoœæ na poziomie 80%. Do analizy wymie- nionych substancji zastosowano chromatografiê gazow¹ z detektorem wychwytu elektronów oraz kolumnê HP–1. Opracowana metoda pozwala wykrywaæ zawartoœæ flunitrazepamu i jego metabolitów we krwi na poziomie kilku nanogramów.

* Corresponding author. E-mail: [email protected] 72 Z. Ch³obowska, C. Œwiegoda, P. Koœcielniak 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 [3–5]. 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. GC–MS 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 [5–10]. 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 HP–1 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 toluene–isoamyl 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. Œwiegoda, P. Koœcielniak 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 Retention time, min

Figure 1. Chromatogram of a standard mixture containing 7-amino-flunitrazepam, flunitrazepam, desme- thyl-flunitrazepam and flurazepam (internal standard) Identification and determination of flunitrazepam in blood 75

For flunitrazepam present in the sample at three different concentrations one com- pared its recoveries obtained using diethyl ether, diisopropyl ether and a mixture of toluene with isoamyl alcohol as extracting agents. The results occurred to be very consistent (Tab. 1), which was additionally confirmed by statistical analysis (analysis of variance in simple classification at the significance level α = 0.05). The results differed insignificantly between each other.

Table 1. The comparison of the extraction recoveries of the examined compounds

Extraction recovery, % Concentration Diethyl ether Diisopropyl ether Toluene–isoamyl alcohol ng mL-1 Mean RSD, % Mean RSD, % Mean RSD, %

100 62.7 ± 5.3 3.2 77.9 ± 7.0 4.3 80.5 ±7.8 4.8

50 70.8 ± 8.5 6.6 69.6 ± 8.4 6.8 72.9 ± 6.6 4.6

20 61.8 ± 14.5 6.1 69.0 ± 14.4 6.9 68.4 ± 12.9 6.6

In order to study the influence a biological matrix of the blood sample may have on flunitrazepam determination, the blood samples of different putrefaction extent have been used. It has been found that none of the retention times of the constituents of the matrix overlapped with the retention times of flunitrazepam, desmethyl fluni- trazepam, 7-amino-flunitrazepam, and of the internal standard. However, some differen- ces in the matrix composition due to the extraction method used and the progress of putrefaction process have been observed. The application of isopropyl alcohol revea- led even greater number of constituents. This complicates the interpretation of the ob- tained results, so the use of isopropyl alcohol as extractant is not recommended. Ex- emplary chromatogram of the blood sample containing flunitrazepam and its ma- trix is shown in Figure 2. The estimated detection limits of the examined drugs were close to 10 ng mL-1. This allows one to apply the developed method to the detection of eden therapeutic concentration levels of flunitrazepam, desmethyl-flunitrazepam, and 7-amino-flunitra- zepam in blood. 76 Z. Ch³obowska, C. Œwiegoda, P. Koœcielniak and W. Piekoszewski flunitrazepam flunitrazepam

Detector signal, AU

0 2 4 6 8 10 12 14 16 RiiRetention time, min i

Figure 2. Chromatogram of a fresh autopsic blood sample with added flunitrazepam

For flunitrazepam concentration ranging from 10 to 200 ng mL-1 linear signal vs concentration dependencies have been obtained for diethyl ether, diisopropyl ether, and toluene–isoamyl alcohol mixture serving respectively as extractants:

Y = 0.0063x + 0.0043 (1) Y = 0.0069x + 0.0239 (2) Y = 0.0058x + 0.0471 (3)

Since relative standard deviations (RDSs) of the determination of 20, 50, and 100 ng mL-1 of flunitrazepam did not exceed 10% (Tab. 1), one could assume the results to be almost free from random error.

CONCLUSIONS

The application of gas chromatograph equipped with electron capture detector and extraction procedure with diethyl ether, diisopropyl ether and toluene–isoamyl mixture as extractants allows one to determine flunitrazepam, desmethyl-flunitrazepam and 7-amino-flunitrazepam in fresh and decayed blood at the concentration level of 10 ng mL-1. The measured responses change linearly within the concentration range of flunitrazepam and its metabolites: 10–200 ng mL-1. One may benefit from the developed method in forensic toxicology investogations, when gas (liquid) chromato- graph coupled with mass spectrometer is not available. Identification and determination of flunitrazepam in blood 77

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Received April 2003 Accepted September 2003