Quantification of flunitrazepam's oxidative metabolites, 3-hydroxyflunitrazepam and desmethylflunitrazepam, in hepatic microsomal incubations by high-performance liquid chromatography

https://doi.org/10.1016/S0378-4347(98)00383-1Get rights and content

Abstract

A high-performance liquid chromatographic assay for the quantification of the oxidative metabolites of flunitrazepam, 3-hydroxyflunitrazepam and desmethylflunitrazepam, in human liver microsomal incubations was developed. Both metabolites were quantifiable in a single assay following a solvent extraction and reversed-phase high-performance liquid chromatography with UV detection. Standard curve concentrations for both metabolites ranged from 0.2 to 10 μM. Assay performance was determined using quality control samples and the intra- and inter-day accuracy and precision as determined by the coefficient of variations which were less than 15% (0.5–6 μM) for both metabolites. This method provides good precision and accuracy for use in kinetic studies of the oxidative metabolism of flunitrazepam in human liver microsomes.

Introduction

Flunitrazepam (Rohypnol) is a 7-nitrobenzodiazepine that has been used as a hypnotic and for the induction of anaesthesia [1]for over 20 years. It is also a widely abused benzodiazepine [2]. It undergoes oxidative metabolism to 3-hydroxyflunitrazepam and desmethylflunitrazepam (Fig. 1), mediated by the cytochrome P450 (CYP450) mixed-function oxidase system. However it is not known which CYP450 isoforms catalyse these reactions. It also undergoes reductive metabolism to 7-aminoflunitrazepam.

Several methods for the analysis of flunitrazepam and its metabolites have been developed for pharmacokinetic investigations, therapeutic drug monitoring and forensic testing. Cirimele and co-workers developed gas chromatographic methods for the detection of flunitrazepam and its 7-amino metabolite in human hair 3, 4. Although these mass spectrometry assays are sensitive (quantification limit of ∼17 pg/mg flunitrazepam and 3 pg/mg 7-aminoflunitrazepam) and selective, they do not detect the oxidative metabolites of flunitrazepam. ElSohly et al. also developed a gas chromatography–mass spectrometry (GC–MS) method for quantification in urine [5]. The disadvantage with this method is that the quantification of individual metabolites is not possible, as the acid hydrolysis of the urine converts related metabolites to a common benzophenone which is then quantified by GC–MS. Four individual benzophenones were identified by these authors from: (a) flunitrazepam, 3-hydroxyflunitrazepam and norflunitrazepam; (b) 3-hydroxynorflunitrazepam; (c) 7-aminoflunitrazepam and 7-amino-3-hydroxyflunitrazepam; (d) 7-aminonorflunitrazepam and 7-amino-3-hydroxynorflunitrazepam, respectively. Thus the method can only be used to confirm the presence of flunitrazepam, its metabolites and their 3-hydroxy derivatives collectively.

Immunoassays have also been developed to detect the presence of flunitrazepam and its metabolites in urine [2]. The OnLine and OnTrak immunoassay are used as kits and can detect concentrations as low as 26 ng/ml, however the metabolites are confirmed collectively, preventing the quantification of each metabolite alone.

Robertson and Drummer [6]used reversed-phase high-performance liquid chromatography (HPLC) to quantify flunitrazepam and its 7-amino metabolite in blood, with a limit of quantification of 10 ng/ml. Berthault et al. used a HPLC method which was the first to quantify flunitrazepam and all its known metabolites [7]. The disadvantages of this method were the low extraction efficiency for some metabolites (30%) and the high intra-assay variability (17–27%).

No method has yet been reported for quantification of flunitrazepam and its metabolites in hepatic microsomal incubations. The considerable time and expense associated with GC, and poor reproducibility with previous HPLC methods have led to the development of a method that involves solvent extraction followed by reversed-phase HPLC with UV detection for quantification of the oxidative metabolites of flunitrazepam. The method is suitable for investigation of the CYP450 isoforms involved in flunitrazepam metabolism in human liver microsomes.

Section snippets

Chemicals

Flunitrazepam, 3-hydroxyflunitrazepam, desmethylflunitrazepam, and dextromethorphan were supplied by Roche Products (Dee Why, Australia). Lorazepam was a kind donation from Wyeth Labs. (Sydney, Australia). Furafylline was a kind donation from Professor Wolfgang Pfleiderer (Chemogen, University of Konstanz, Germany). Other materials were obtained from the following sources: S(+)-mephenytoin from Ultrafine Chemicals (Manchester, UK); omeprazole from Astra Pharmaceuticals (Sydney, Australia); dl

Results and discussion

Chromatograms resulting from the extraction of a substrate-free microsomal mixture (blank), an incubated low QC sample (0.5 μM 3-hydroxyflunitrazepam, desmethylflunitrazepam and lorazepam), and a microsomal incubation of 500 μM flunitrazepam are shown in Fig. 2. The microsomal incubation resulted in two peaks with identical retention times to 3-hydroxyflunitrazepam and desmethylflunitrazepam in the QC sample. Under the HPLC conditions described, the retention times for 3-hydroxyflunitrazepam,

Acknowledgements

J.K.C. is a recipient of an Australian Postgraduate Award. The authors acknowledge the kind donation of flunitrazepam and its metabolites by Roche Products, Australia. This work was presented in part at the meeting of the Australasian Society of Clinical and Experimental Pharmacologists and Toxicologists in December 1997.

References (8)

  • V Cirimele et al.

    Forensic Sci. Int.

    (1997)
  • V Cirimele et al.

    J. Chromatogr. B

    (1997)
  • M.D Robertson et al.

    J. Chromatogr. B

    (1995)
  • F Berthault et al.

    J. Chromatogr. B

    (1996)
There are more references available in the full text version of this article.

Cited by (12)

  • A rapid UPLC-MS/MS method for simultaneous determination of flunitrazepam, 7-aminoflunitrazepam, methadone and EDDP in human, rat and rabbit plasma

    2012, Talanta
    Citation Excerpt :

    Given that the toxicity mechanisms of the MET–FNZ association as well as the pharmacological and metabolic interactions between these drugs are only poorly understood, further studies are needed. FNZ is extensively metabolized in the body, by reduction to 7-AFNZ as the main metabolite, but also by hydroxylation and demethylation (oxidative metabolic pathways involving CYP450) [43,46–51]. After ingestion of FNZ, the parent compound and 7-AFNZ (active metabolite) are the main components found in plasma.

  • Characterization of Escherichia coli nitroreductase NfsB in the metabolism of nitrobenzodiazepines

    2009, Biochemical Pharmacology
    Citation Excerpt :

    In humans, metabolism of NBDZ occurs mainly in liver, which involves oxidative and reductive pathways [9]. For example, FZ undergoes oxidative metabolism to N-desmethyl-flunitrazepam (DMFZ) and 3-hydroxy-flunitrazepam (3-OHFZ), or is reduced to 7-amino-flunitrazepam (7AFZ) within liver microsomes (Fig. 1B) [10]. The oxidation of FZ to DMFZ and 3-OHFZ is catalyzed by the hepatic CYP2C9 and CYP3A4, respectively [11], where DMFZ is the main active metabolite correlating to the respiratory defect [8] and anesthetic effect [9].

  • Drug facilitated sexual assault and that the problems in Turkey

    2012, Journal of Clinical and Analytical Medicine
View all citing articles on Scopus
View full text