Rapid, hydrolysis-free, dilute-and-shoot method for the determination of buprenorphine, norbuprenorphine and their glucuronides in urine samples using UHPLC-MS/MS

Buprenorphine and its metabolites are routinely monitored to assess patient compliance with drug detoxification programs or as pain killers. A rapid method for the simultaneous analysis of buprenorphine, norbuprenorphine, and glucuronides in urine using ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) was developed. Urine samples were diluted in water containing formic acid 0.1% and directly injected into the UHPLC-MS/MS system without any sample pretreatment. Quality control (QC) samples, prepared using 20 different urine matrices, fortified at 3 concentration levels, were quantified using four deuterated internal standards. The accuracy values obtained spanned from 90 to 114% with repeatability lower than 10% also in the inter-day between batch experiments. Matrix effects (ME), evaluated before correction with internal standards using Matuszewski procedure, mainly affected the analysis of buprenorphine glucuronide. The use of deuterated internal standards (IS) for each analyte was necessary to compensate for ME and was essential in the determination of glucuronides. The method was applied to 30 real urine samples from patients under a detoxification therapy. Duplicate analyses were performed with the presented method and compared with another method which involves a standard hydrolysis procedure. Real sample results were compared showing a good performance agreement, with differences between the two methods lower than ±20% in the quantification results.