Quantitative Analysis of Buprenorphine and Norbuprenorphine in Urine using Liquid Chromatography Tandem Mass Spectrometry

Buprenorphine is an opioid analgesic drug that is used as an alternative to methadone to treat heroin addiction. Established methods for the analysis of buprenorphine and its metabolites in urine such as gas chromatography-mass spectrometry (GC-MS) involve complicated sample extraction procedures. The aim of the present study was to develop a sensitive yet straightforward method for the simultaneous analysis of buprenorphine and norbuprenorphine in urine using liquid chromatography-MS-MS. The method comprised an enzymatic hydrolysis using Patella vulgata b-glucuronidase, followed by centrifugation and direct analysis of the supernatant. The limits of detection and quantitation were < 1 microg/L for buprenorphine and < 1 and 4 microg/L, respectively, for norbuprenorphine. Assay coefficients of variation (CVs) were < 15%, with the exception of concentrations close to the limit of quantitation, where CVs were below 20%. In direct comparison with an established GC-MS protocol, the method showed minimal negative bias (8.7% for buprenorphine and 1.8% for norbuprenorphine) and was less susceptible to sample carryover. The extent of conjugation in unhydrolyzed urine was investigated and found to be highly variable, with proportions of unconjugated buprenorphine and norbuprenorphine of 6.4% [range 0% to 67%; standard deviation (SD) 9.7%] and 34% (range 0% to 100%; SD 23.8%), respectively.