Ngo Lien, Tran Phuong, Ham Seong-Ho, Cho Jung-Hee, Cho Hea-Young, Lee Yong-Bok
College of Pharmacy, Chonnam National University, 77, Yongbong-ro, Buk-gu, Gwangju, 61186, Republic of Korea.
Division of Traditional Korean Medicine Resource, National Development Institute of Korean Medicine, 288, Woodland-gil, Anyang-myun, Jangheung, Jeonnam, 59338, Republic of Korea.
J Chromatogr B Analyt Technol Biomed Life Sci. 2017 Feb 15;1044-1045:30-38. doi: 10.1016/j.jchromb.2016.12.037. Epub 2016 Dec 28.
An accurate, precise, selective, and sensitive liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) method was developed for the simultaneous determination of imperatorin (IMP) and its metabolite, xanthotoxol (XAN), in rat plasma and urine samples. The analytes, along with psoralen as an internal standard, were determined by multiple reaction monitoring (MRM) operated in the positive electrospray ionization (ESI) mode. Chromatographic separation was performed on an Acquity UPLC BEH C18 column (50mm×2.1mm, 1.7μm) with a mobile phase consisting of 0.1% formic acid solution and 0.1% formic acid in methanol at a flow rate of 0.3mL/min. The run time was 6min per sample and the injection volume was 5μL. The method had a lower limit of quantification (LLOQ) of 0.25ng/mL for IMP in plasma and urine, and 1ng/mL for XAN in urine. The linear calibration curves were fitted over the range of 0.25-1000ng/mL for IMP in plasma, 0.25-1000ng/mL for IMP in urine, and 1-1000ng/mL for XAN in urine, with correlation coefficients greater than 0.995. The inter- and intra-day accuracies (relative error, RE%) were between -8.5% and 3.5%, and the precisions (relative standard deviation, RSD%) were less than 10.0% for all quality control samples (QCs). The analytes were extracted from rat plasma and urine samples using a liquid-liquid extraction method with the extraction recovery in the range of 60.3-79.1%. A good stability of the analytes was observed in all the analysis procedures. The method was successfully validated and applied to determine the pharmacokinetics of IMP in rat plasma and, for the first time, the metabolite kinetics of IMP to XAN in rat urine after IMP administration.
建立了一种准确、精密、选择性好且灵敏的液相色谱-串联质谱（LC-MS/MS）方法，用于同时测定大鼠血浆和尿液样本中蛇床子素（IMP）及其代谢产物花椒毒素（XAN）。将分析物与补骨脂素作为内标物，通过在正电喷雾电离（ESI）模式下运行的多反应监测（MRM）进行测定。在Acquity UPLC BEH C18柱（50mm×2.1mm，1.7μm）上进行色谱分离，流动相由0.1%甲酸溶液和含0.1%甲酸的甲醇组成，流速为0.3mL/min。每个样品的运行时间为6分钟，进样体积为5μL。该方法对血浆和尿液中IMP的定量下限（LLOQ）为0.25ng/mL，对尿液中XAN的定量下限为1ng/mL。血浆中IMP的线性校准曲线在0.25 - 1000ng/mL范围内拟合，尿液中IMP的线性校准曲线在0.25 - 1000ng/mL范围内拟合，尿液中XAN的线性校准曲线在1 - 1000ng/mL范围内拟合，相关系数大于0.995。所有质量控制样品（QCs）的日间和日内准确度（相对误差，RE%）在-8.5%至3.5%之间，精密度（相对标准偏差，RSD%）小于10.0%。采用液-液萃取法从大鼠血浆和尿液样本中提取分析物，萃取回收率在60.3 - 79.1%范围内。在所有分析过程中观察到分析物具有良好的稳定性。该方法成功得到验证，并应用于测定大鼠血浆中IMP的药代动力学，以及首次测定IMP给药后大鼠尿液中IMP向XAN的代谢物动力学。
