Liquid chromatography–tandem mass spectrometry method for measurement of nicotine N-glucuronide: A marker for human UGT2B10 inhibition

Nicotine is considered to be a specific substrate for UGT2B10, an isoform of human uridine diphosphate glucuronosyltransferase (UGT). In the present study, a sensitive and selective liquid chromatography/tandem mass spectrometry (LC–MS–MS) method for quantification of nicotine N-glucuronide in pooled human liver microsomal incubates was developed and validated. Proteins in a 200 μL aliquot of incubation solution were precipitated by adding 40 μL 35% perchloric acid. The overall extraction efficiency was greater than 98%. Nicotine N-glucuronide and internal standard were recorded using selected reaction monitoring in positive ion electrospray with ion transitions of m/z 339–163 and m/z 342–166, respectively. The linear calibration curve was obtained over the concentration range of 10–1000 nM, with a lower limit of quantification of 10 nM. The intra-day and inter-day precision (% CV) and accuracy (% bias) of the method were within 15% at all quality control levels. Nicotine glucuronide in processed samples was stable for 24 h at room temperature and 48 h at 4 °C based on the stability experiments performed in this study. This established method was employed to evaluate the inhibitory effects of five target compounds including amitriptyline, hecogenin, imipramine, lamotrigine, and trifluoperazine on enzymatic activity of UGT2B10. IC50 values for inhibition of nicotine N-glucuronidation by amitriptyline, imipramine, lamotrigine, and trifluoperazine were calculated. Trifluoperazine was found to be a non-substrate inhibitor for human UGT2B10.