Li Hui-Liang, Zhang Wei-Dong, Liu Run-Hui, Zhang Chuan, Han Ting, Wang Xiang-Wei, Wang Xiao-Lin, Zhu Jian-Bao, Chen Chun-Lin
Department of Natural Medicinal Chemistry, Second Military Medical University, 325 Guohe Rd., Shanghai 200433, PR China.
J Chromatogr B Analyt Technol Biomed Life Sci. 2006 Feb 2;831(1-2):140-6. doi: 10.1016/j.jchromb.2005.11.049. Epub 2005 Dec 13.
A sensitive rapid method for the simultaneous determination of four major active alkaloids (dehydrocavidine, coptisine, dehydroapocavidine, and tetradehydroscoulerine, in abbreviation thereafter called YHL-I, YHL-II, YHL-III, and YHL-IV, respectively) from a Chinese traditional medicine Corydalis saxicola Bunting. (Yanhuanglian) in rat plasma and urine was established and validated. The assay for these substances in plasma and urine was based on HPLC coupled with tandem mass spectrometry (MS/MS) detection using multiple reaction monitoring mode (MRM) with berberine and clenbuterol as internal standards. The plasma and urine sample were deproteinated by adding methanol prior to liquid chromatography where separation was performed on a Luna column (5 microm, 100 x 2.00 mm) and an Agilent Zorbax SB-C18 guard column (5 microm, 20 x 4 mm). The method was validated with the concentration range 1-1000 ng/mL in plasma and 10-1000 ng/mL in urine for the four test compounds, and the calibration curves were linear with correlation coefficients >0.999. The lowest limits of quantitation for all four substances were 1 ng/mL in 0.1 mL rat plasma and 10 ng/mL in 0.1 mL urine. The intra-assay accuracy and precision in plasma ranged from 88.1 to 115.7% and 1.4 to 10.8%, respectively, while inter-assay accuracy and precision for YHL-I, YHL-II, YHL-III, and YHL-IV ranged from 96.2 to 113.2% and 0.4 to 16.9%, respectively. The intra-assay accuracy and precision for YHL-I, YHL-II, YHL-III, and YHL-IV in rat urine ranged from 96.1 to 112.9% and 1.2 to 8.3%, respectively, while inter-assay accuracy and precision ranged from 95.0 to 106.8% and 2.2 to 10.3%, respectively. The method was further applied to assess pharmacokinetics and urine excretion of the four alkaloids after oral and intravenous administration to rats. Practical utility of this new LC-MS-MS method was confirmed in pilot pharmacokinetic studies in rats following both intravenous and oral administration.
建立并验证了一种灵敏、快速的方法，用于同时测定大鼠血浆和尿液中来自中药岩黄连（Corydalis saxicola Bunting.）的四种主要活性生物碱（分别简称为YHL-I、YHL-II、YHL-III和YHL-IV的脱氢卡维丁、黄连碱、去氢阿朴卡维丁和四脱氢紫堇碱）。血浆和尿液中这些物质的测定基于高效液相色谱-串联质谱（MS/MS）检测，采用多反应监测模式（MRM），以小檗碱和克伦特罗作为内标。在进行液相色谱分析前，通过加入甲醇使血浆和尿液样品脱蛋白，分离在Luna柱（5μm，100×2.00mm）和安捷伦Zorbax SB-C18保护柱（5μm，20×4mm）上进行。该方法在血浆中四种测试化合物的浓度范围为1-1000 ng/mL，尿液中为10-1000 ng/mL时得到验证，校准曲线呈线性，相关系数>0.999。所有四种物质在0.1 mL大鼠血浆中的最低定量限为1 ng/mL，在0.1 mL尿液中为10 ng/mL。血浆中的批内准确度和精密度分别为88.1%至115.7%和1.4%至10.8%，而YHL-I、YHL-II、YHL-III和YHL-IV的批间准确度和精密度分别为96.2%至113.2%和0.4%至16.9%。大鼠尿液中YHL-I、YHL-II、YHL-III和YHL-IV的批内准确度和精密度分别为96.1%至112.9%和1.2%至8.3%，批间准确度和精密度分别为95.0%至106.8%和2.2%至10.3%。该方法进一步应用于评估大鼠口服和静脉给药后这四种生物碱的药代动力学和尿液排泄情况。在大鼠静脉和口服给药后的初步药代动力学研究中，证实了这种新的液相色谱-质谱联用方法的实际应用价值。
