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

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.