Identification and quantitative determination of benproperine metabolites in human plasma and urine by liquid chromatography–tandem mass spectrometry

Two novel metabolites of benproperine (BPP), 1-[1-methyl-2-[2-(phenylmethyl)phenoxy]ethyl]-3-piperidinol (3-OH-BPP) and 1-[1-methyl-2-[2-(phenylmethyl)phenoxy]ethyl]-4-piperidinol (4-OH-BPP), were confirmed by comparison of retention times and mass spectra with those of synthetic standards using liquid chromatography–tandem mass spectrometry. Selective and sensitive procedures were developed for the simultaneous determination of BPP, 3-OH-BPP and 4-OH-BPP in human plasma and urine. The analytes were extracted from plasma sample and enzymatically hydrolyzed urine samples by liquid–liquid extraction, separated through a Diamonsil C18 column (150 mm × 4.6 mm i.d.) and determined by tandem mass spectrometry with an electrospray ionization interface in selected reaction monitoring mode. Dextromethorphan was used as internal standard. The mobile phase consisted of acetonitrile–water–formic acid (34:66:1, v/v/v), and flow-rate was 0.5 ml min−1. This method has a lower limit of quantification (LLOQ) of 60, 4.0 and 4.0 nmol l−1for BPP, 3-OH-BPP and 4-OH-BPP in plasma, 4.9, 4.7 and 2.4 nmol l−1 in urine, respectively. The intra- and inter-run precision were measured to be below 9.2%, and the accuracy was within ±4.3% for the analytes. The method was successfully used to determine BPP, 3-OH-BPP and 4-OH-BPP in plasma and urine for pharmacokinetic investigation. The results indicated residue of 3-OH-BPP in the body at least 192 h after an oral dose of BPP.