Simultaneous determination of flunitrazepam and its metabolites in plasma and urine by HPLC/DAD after solid phase extraction

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Abstract

A high performance liquid chromatography (HPLC) assay was developed for the determination of flunitrazepam (FNZ) and its metabolites in urine and plasma. The analytes and the internal standard (triazolam, TRZ) were extracted by Sep-Pak C18 SPE-cartridge and separated utilizing a 5 μm ChromSpher C8 glass column with a gradient mobile phase containing methanol and 0.125% (v/v) of isopropylamine in water. Diode array detection (DAD) was carried out at a monitoring wavelength of 240 nm and a reference wavelength of 550 nm. Standard curves were linear from their quantitation limits until 200 ng ml−1 urine or 250 ng ml−1 plasma for 7-amino-desmethyl-flunitrazepam (ADF), 7-amino-flunitrazepam (AF), 7-acetamino-flunitrazepam (ACF) and until 400 ng ml−1 urine or 500 ng ml−1 plasma for FNZ, 1-desmethyl-flunitrazepam (DF), and 3-hydroxyl-flunitrazepam (HF). The intraday and interday coefficients of variation ranged from 2.04 to 9.07% and from 2.64 to 14.10%, respectively in urine and from 5.13 to 8.60% and from 7.27 to 10.46%, respectively in plasma. The developed method is used in forensic toxicology and is also applicable to pharmacokinetic studies in man.

Introduction

Flunitrazepam (FNZ), RohypnolR, 5-(2-fluorophenyl)-1,3-dihydro-1- methyl-7-nitro-2H-1,4-benzodiazepin-2-one, is a benzodiazepine derivative and pharmacologically a full agonist whose hypnotic effect predominates over the sedative, anxiolytic, muscle relaxing and anticonvulsant effects, characteristics of benzodiazepines. It is used in many hospitals and its abuse and intoxication was reported by several authors 1, 2, 3, 4.The equivalent oral dose of FNZ relating to 10 mg of diazepam, is 2 mg [5]. Following oral doses of 2 mg daily for 28 days given to seven subjects, a mean peak plasma concentration of 20 ng ml−1 (63.8 nmol l−1) was reported [6]. According to the literature 7, 8toxic symptoms have been observed at serum concentrations of FNZ above 50 ng ml−1 (159.5 nmol l−1).

The still maintaining popularity of the benzodiazepines is mainly caused by their wide therapeutic index, minimal serious adverse reactions and the absence of undesirable autonomic nervous side effects especially when compared with formerly used anti-anxiety agents, e.g. meprobamate or phenobarbital [9]. The American Association of Poison Control Centers, in its 1994 annual report, cites benzodiazepines in third position (after analgesics and antidepressants) among the categories of substances with highest number of reported deaths [10].

Although benzodiazepines are well tolerated, prolonged regular use can lead to physical dependence which results from a kind of habituation, the functional counterparts of which become manifest during abstinence. With the long-acting benzodiazepines, this problem is less obvious, because of the delayed appearance of withdrawal symptoms. The severity of this syndrome is inversely related to the elimination half-life. There are indications that benzodiazepines with intermediate elimination half-lives, e.g. FNZ, lorazepam, bromazepam, are most frequently abused [11].

The structures of FNZ, its metabolites and triazolam (TRZ) are shown in Fig. 1. Abbreviations are explained in the paragraph of drug standards.

Numerous methods are currently available for the determination of FNZ in biological samples. Thin-layer chromatography (TLC) 12, 13, 14(W. He, A. Heyndrickx, A two-dimensional thin-layer chromatography for determination of flunitrazepam and its metabolites, unpublished result), polarography [15], radioimmunoassay (RIA) [16], differential pulse voltametry (DPV) [17], and flow injection analysis (FIA) [18]methods have been described. By far the most of the published methods useful in clinical and forensic laboratories are utilizing gas chromatography (GC) 19, 20, 21, 22and high performance liquid chromatography (HPLC) 23, 24, 25, 26.

This paper describes a developed HPLC method with a photo diode array detector (DAD) which facilitates either quantification or qualification of sample. The gradient elution provided a good separation of FNZ and its six metabolites. The solid phase extraction (SPE) was used for sample pretreatment, which may enable to extract large amount of sample in order to increase the sensitivity of determination.

Section snippets

Liquid chromatography system and chromatographic conditions

A Hewlett Packard model 1090 M liquid chromatograph, equipped with a HP 1040 A DAD, was used. The system was controlled by a HP 79994 A HPLC work station which consists of HP 9000-300 computer, HP 9133 disc driver, HP 2225AB Thinkjet printer and HP 7440A Color Pro Graphics plotter. A Rheodyne injector with a 50 μl loop was fixed on the instrument. The column was a 100×3 mm i.d. ChromSep glass column packed with ChromSpher C8, 5 μm (Cat. No. 28262) protected by a 10×2 mm i.d. high efficiency

Chromatography

Under the described chromatographic conditions, FNZ and its metabolites are well separated. Note that at 7 min, a step gradient elution starts and so baseline increases. Fig. 2 shows the chromatographic separation of these compounds after injecting directly a mixture containing 100–200 ng per 50 μl of each of them. The retention times are shown on Table 1.

Linearity and assay detection limits

The linearity for FNZ, DF and HF was checked in the concentration range 25–500 ng ml−1 plasma, 20–400 ng ml−1 urine and for ADF, AF and ACF

Discussion

FNZ has only two pharmacologically active metabolites, AF and DF which appear in blood or plasma [28]. The urinary excretion of unchanged FNZ is less than 1%. To be able to correlate the effects or symptoms with the plasma concentrations we must take into account not only the main compound but also its active metabolites. It has to be kept in mind that only the free-drug is pharmacologically active.

In urine, the metabolites of FNZ are present mainly as glucuronide conjugates which have high

Acknowledgements

The author gratefully acknowledges the Hoffmann-LaRoche and Upjohn companies for the standards donation and thanks Mr R. Vereecke and Mr J. Vanderverren for their help in this study. This study is supported by the Algemeen Bestuur van Ontwikkelingssamenwerking (Grant No. 118603).

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