Development and validation of a gas chromatography–mass spectrometry method for the simultaneous determination of buprenorphine, flunitrazepam and their metabolites in rat plasma: application to the pharmacokinetic study

Abstract

Buprenorphine (BUP), a synthetic opioid analgesic, is frequently abused alone, and in association with benzodiazepines. Fatalities involving buprenorphine alone seem very unusual while its association with benzodiazepines, such as flunitrazepam (FNZ), has been reported to result in severe respiratory depression and death. The quantitative relationship between these drugs remain, however, uncertain. Our objective was to develop an analytical method that could be used as a means to study and explore, in animals, the toxicity and pharmacological interaction mechanisms between buprenorphine, flunitrazepam and their active metabolites. A procedure based on gas chromatography–mass spectrometry (GC–MS) is described for the simultaneous analysis of buprenorphine, norbuprenorphine (NBUP), flunitrazepam, N-desmethylflunitrazepam (N-DMFNZ) and 7-aminoflunitrazepam (7-AFNZ) in rat plasma. The method was set up and adapted for the analysis of small plasma samples taken from rats. Plasma samples were extracted by liquid–liquid extraction using Toxi-tubes A. Extracted compounds were derivatized with N,O-bis-(trimethylsilyl)trifluoroacetamide (BSTFA), using trimethylchlorosilane (TMCS) as a catalyst. They were then separated by GC on a crosslinked 5% phenyl-methylpolysiloxane analytical column and determined by a quadrupole mass spectrometer detector operated under selected ion monitoring mode. Excellent linearity was found between 0.125 and 25 ng/μl plasma for BUP, 0.125 and 12.5 ng/μl for NBUP and N-DMFNZ, 0.125 and 5 ng/μl for FNZ, and between 0.025 and 50 ng/μl for 7-AFNZ. The limit of quantification was 0.025 ng/μl plasma for 7-AFNZ and 0.125 ng/μl for the four other compounds. A good reproducibility (intra-assay CV=0.32–11.69%; inter-assay CV=0.63–9.55%) and accuracy (intra-assay error=2.58–12.73%; inter-assay error=0.83–11.07%) were attained. Recoveries were 71, 67 and 81%, for BUP, FNZ and N-DMFNZ, respectively, and 51% for NBUP and 7-AFNZ, with CV ranging from 5.4 to 13.9%, and were concentration-independent. The GC–MS method was successfully applied to the pharmacokinetic study of BUP, NBUP, FNZ, DMFNZ and 7-AFNZ in rats, after administration of BUP and FNZ.

Introduction

Buprenorphine (BUP), a semi-synthetic opioid derivative, is a powerful analgesic, 25–40 times more potent than morphine. Buprenorphine at low dosages (typically 0.3–0.6 mg intravenous or intramuscular) is widely prescribed for the treatment of moderate to severe pain, and also for premedication in anaesthesiology [1]. Additionally, buprenorphine has been recognized as an effective medication for the substitutive maintenance in opioid dependent-patients [2], [3], [4]. A high dosage formulation of buprenorphine (0.4, 2 and 8 mg tablets for sublingual use) is available in this specific medication in France since 1996. High-dose buprenorphine has been reported to substantially decrease heroin self-administration [2], [5]. The limited respiratory effects of high-dose buprenorphine is of utmost importance regarding the safety of this drug for use in substitution treatment. However, numerous buprenorphine-related deaths have been reported by forensic toxicologists and other sources of information (e.g. intensive care units) since 1996. These fatalities may result from misuse (intravenous injection of crushed tablets) or overdose with substitution treatment [6], [7], or a concomitant intake of psychotropics, mostly benzodiazepines [8], [9]. Benzodiazepines are extensively prescribed to patients with insomnia in many countries, and are considered as relatively safe drugs since deaths involving benzodiazepines solely, in the absence of other pathologies, are very uncommon [10], [11]. Some benzodiazepines, such as flunitrazepam (FNZ), nordiazepam and diazepam, have become popular among heroin addicts. The association of these drugs with substitution products (buprenorphine or methadone) has been found in many fatal intoxications [9], [10], [12]. Experimental studies further suggest that the combination of opioids and benzodiazepines is a major risk factor for lethal outcomes. Severe respiratory depression has been observed in rats when flunitrazepam is administered concurrently and/or acutely with opioids, as assessed by arterial blood gas measurements [13]. Furthermore, studying the acute toxicity of various combinations of opioids and flunitrazepam in rats, some of us observed that lethality was significantly increased in buprenorphine-treated rats relative to rats treated with methadone or morphine: there was both a large decrease in the median lethal dose of buprenorphine and time to death [14].

The toxicity mechanisms of the buprenorphine–flunitrazepam association as well as the pharmacological and metabolic interactions between these drugs are poorly understood, calling for further investigations. The availability of diagnostic means of study and exploration in the animal is, however, a prerequisite for such work. Various analytical methods, including gas chromatography–mass spectrometry (GC–MS) assay methods, have been described for the analysis of buprenorphine [15], [16], [17], [18], [19], [20], [21], [22], [23], [24], [25], [26], [27], [28], of flunitrazepam [29], [30], [31], [32] and their major metabolites. However and until now, there had been no reported method for the simultaneous determination of these drugs.

Here, we describe a GC–MS analytical method which allows the simultaneous determination of buprenorphine and its major metabolite, norbuprenorphine (NBUP), as well as flunitrazepam and its active metabolites, N-desmethylflunitrazepam (N-DMFNZ) and 7-aminoflunitrazepam (7-AFNZ), in rat plasma. The assay method validation is also presented. We used previous studies from our laboratory [33], [34] as a starting point for the extraction, derivatization and quadrupole MS analysis of the compounds under study. The method was set up and adapted for the analysis of the small plasma samples taken from rats. Its development then allowed us to explore the pharmacokinetics of these five drugs after administration of buprenorphine and flunitrazepam.