A sensitive, simple and rapid HPLC-MS/MS method for simultaneous quantification of buprenorpine and its N-dealkylated metabolite norbuprenorphine in human plasma.

A sensitive, simple and rapid high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was developed and fully validated for the simultaneous quantification of buprenorphine (BUP) and its N-dealkylated metabolite norbuprenorphine (NBUP) in 200 μL human plasma. Human plasma samples were prepared using liquid-liquid extraction, and then separated on a Shiseido MG C18 (5 μm, 2.0 mm×50 mm) via 4.1 min gradient elution. Following electrospray ionization, the analytes were quantified on a triple-quadrupole mass spectrometer in multiple-reaction-monitoring (MRM) positive ion mode. Linearity was achieved from 25.0 to 10000 pg/mL for buprenorphine, from 20.0 to 8000 pg/mL for norbuprenorphine with >0.99. The method was demonstrated with acceptable accuracy, precision and specificity for the detection of buprenorphine and norbuprenorphine. Recovery was 81.8-88.8% for buprenorphine and 77.0-84.6% for norbuprenorphine, and the matrix effect was 95.6-97.4% for buprenorphine and 94.0-96.9% for norbuprenorphine; all were not concentration dependent. With validated matrix and autosampler stability data, this method was successfully applied in a bioequivalence study to support abbreviated new drug application.