Department of Clinical Pharmacology & DMPK, AstraZeneca Pharmaceuticals, Wilmington, DE 19803, USA.
J Pharm Biomed Anal. 2011 Jul 15;55(5):964-71. doi: 10.1016/j.jpba.2011.03.034. Epub 2011 Mar 29.
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.
尼古丁被认为是 UGT2B10（人尿苷二磷酸葡萄糖醛酸基转移酶（UGT）的同工酶）的特异性底物。在本研究中，建立并验证了一种灵敏、专一地定量人肝微粒体孵育液中烟碱 N-葡萄糖醛酸苷的液相色谱/串联质谱（LC-MS-MS）方法。在 200μL 孵育液等分试样中加入 40μL 35%高氯酸沉淀蛋白质。总体提取效率大于 98%。烟碱 N-葡萄糖醛酸苷和内标物分别采用正离子电喷雾，选择反应监测记录，离子转移 m/z 339-163 和 m/z 342-166。在 10-1000nM 的浓度范围内获得线性校准曲线，定量下限为 10nM。该方法在所有质控水平的日内和日间精密度（%CV）和准确度（%偏倚）均在 15%以内。根据本研究中的稳定性实验，处理后的样品中烟碱葡萄糖醛酸在室温下 24h 和 4°C 下 48h 稳定。该方法用于评估五种目标化合物（阿米替林、海柯苷元、丙咪嗪、拉莫三嗪和三氟丙嗪）对 UGT2B10 酶活性的抑制作用。计算了阿米替林、丙咪嗪、拉莫三嗪和三氟丙嗪抑制烟碱 N-葡萄糖醛酸化的 IC50 值。发现三氟丙嗪是人 UGT2B10 的非底物抑制剂。
