Determination of buprenorphine and norbuprenorphine in whole blood by liquid chromatography mass spectrometry

A sensitive and highly specific method for the determination of buprenorphine and norbuprenorphine in postmortem and hemolyzed whole blood using combined liquid chromatography and mass spectrometry (LC-MS) with electrospray ionization was developed. After enzymatic hydrolysis and deproteinization with acetonitrile, Extrelut-3 cartridges were used for a preliminary basic sample cleanup. Elution by toluene-ether (50:50, v/v) was followed by an acid wash with 0.05M H3PO4 and a basic re-extraction into ether. A Nucleosil C18 (150 x 1-mm i.d.) reversed-phase column was used for the chromatographic separation together with a mixture of 2mM ammonium formate buffer (pH 3) and acetonitrile (55:45, v/v) as the mobile phase. Recoveries were between 56 and 60%, and detection limits were 0.05 ng/mL for both analyses. The coefficient of variation for repeatability was lower than 4%. Limits of quantitation were 0.1 ng/mL for buprenorphine and norbuprenorphine. Reproducibility was good, and linearity was excellent in the range of 0.1 to 100 ng/mL (r > 0.9999, n = 7).