Wang Shuanghu, Wu Haiya, Huang Xueli, Geng Peiwu, Wen Congcong, Ma Jianshe, Zhou Yunfang, Wang Xianqin
The Laboratory of Clinical Pharmacy, The People's Hospital of Lishui, Wenzhou Medical University, Lishui 323000, China.
Department of Anesthesiology, Critical Care and Pain Medicine, the Second Affiliated Hospital, Wenzhou Medical University, Wenzhou 325000, China.
J Chromatogr B Analyt Technol Biomed Life Sci. 2015 May 15;990:118-24. doi: 10.1016/j.jchromb.2015.03.025. Epub 2015 Apr 2.
In this work, a sensitive and selective UPLC-MS/MS method for determination of N-methylcytisine in rat plasma is developed. After addition of hordenine as an internal standard (IS), protein precipitation by acetonitrile-methanol (9:1, v/v) was used to prepare samples. Chromatographic separation was achieved on a UPLC BEH HILIC (2.1 mm×100mm, 1.7μm) with acetonitrile (containing 10mM ammonium formate) and water (containing 0.1% formic acid and 10mM ammonium formate) as the mobile phase with gradient elution. An electrospray ionization source was applied and operated in positive ion mode; multiple reaction monitoring (MRM) mode was used for quantification using target fragment ions m/z 205.1→58.0 for N-methylcytisine, and m/z 166.1→121.0 for IS. Calibration plots were linear throughout the range 2-2000ng/mL for N-methylcytisine in rat plasma. Mean recoveries of N-methylcytisine in rat plasma ranged from 86.1% to 94.8%. RSD of intra-day and inter-day precision were both<13%. The accuracy of the method was between 94.5% and 109.4%. The method was successfully applied to pharmacokinetic study of N-methylcytisine after either oral or intravenous administration. For the first time, the absolute bioavailability of N-methylcytisine was reported as high as 55.5%.
在本研究中，建立了一种灵敏且具选择性的超高效液相色谱-串联质谱法（UPLC-MS/MS）用于测定大鼠血浆中的N-甲基野靛碱。加入去甲乌药碱作为内标（IS）后，采用乙腈-甲醇（9:1，v/v）进行蛋白沉淀来制备样品。在超高效液相色谱BEH HILIC柱（2.1 mm×100mm，1.7μm）上进行色谱分离，以乙腈（含10mM甲酸铵）和水（含0.1%甲酸和10mM甲酸铵）作为流动相进行梯度洗脱。采用电喷雾电离源，以正离子模式运行；采用多反应监测（MRM）模式进行定量分析，N-甲基野靛碱的目标碎片离子为m/z 205.1→58.0，内标的目标碎片离子为m/z 166.1→121.0。大鼠血浆中N-甲基野靛碱在2 - 2000ng/mL范围内校准曲线呈线性。大鼠血浆中N-甲基野靛碱的平均回收率在86.1%至94.8%之间。日内和日间精密度的相对标准偏差（RSD）均<13%。该方法的准确度在94.5%至109.4%之间。该方法成功应用于N-甲基野靛碱口服或静脉给药后的药代动力学研究。首次报道N-甲基野靛碱的绝对生物利用度高达55.5%。
