Buprenorphine and norbuprenorphine quantification in human plasma by simple protein precipitation and ultra-high performance liquid chromatography tandem mass spectrometry

Abstract

A highly sensitive ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC–MS/MS) method was developed for the quantification of buprenorphine and its major metabolite norbuprenorphine in human plasma. In order to speed up the process and decrease costs, sample preparation was performed by simple protein precipitation with acetonitrile. To the best of our knowledge, this is the first application of this extraction technique for the quantification of buprenorphine in plasma. Matrix effects were strongly reduced and selectivity increased by using an efficient chromatographic separation on a sub-2μm column (Acquity UPLC BEH C18 1.7μm, 2.1×50mm) in 5min with a gradient of ammonium formate 20mM pH 3.05 and acetonitrile as mobile phase at a flow rate of 0.4ml/min. Detection was made using a tandem quadrupole mass spectrometer operating in positive electrospray ionization mode, using multiple reaction monitoring. The procedure was fully validated according to the latest Food and Drug Administration guidelines and the Société Française des Sciences et Techniques Pharmaceutiques. Very good results were obtained by using a stable isotope-labeled internal standard for each analyte, to compensate for the variability due to the extraction and ionization steps. The method was very sensitive with lower limits of quantification of 0.1ng/ml for buprenorphine and 0.25ng/ml for norbuprenorphine. The upper limit of quantification was 250ng/ml for both drugs. Trueness (98.4–113.7%), repeatability (1.9–7.7%), intermediate precision (2.6–7.9%) and internal standard-normalized matrix effects (94–101%) were in accordance with international recommendations. The procedure was successfully used to quantify plasma samples from patients included in a clinical pharmacogenetic study and can be transferred for routine therapeutic drug monitoring in clinical laboratories without further development.