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 200μL aliquot of incubation solution were precipitated by adding 40μL 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 10-1000nM, with a lower limit of quantification of 10nM. 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 24h at room temperature and 48h 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. IC(50) 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.