液相色谱-串联质谱法测定大鼠血浆中脱水穿心莲内酯琥珀酸半酯的含量(英文)

Development of a sensitive liquid chromatography/tandem mass spectrometry method for the determination of dehydroandrographolide succinate in rat plasma

目的建立了测定大鼠血浆中脱水穿心莲内酯琥珀酸半酯（DAS）的液相色谱-串联质谱法。方法血浆样品经液-液萃取后,以甲醇-水（70∶30,V/V）为流动相,通过Restek PinnacleⅡC18柱分离,格列吡嗪为内标,选择负离子扫描方式,以多反应监测（MRM）方式进行检测。用于定量分析的离子反应分别为m/z 531→m/z 431（DAS）和m/z 444→m/z319（格列吡嗪,内标）。结果DAS血浆浓度测定方法的线性范围为5～2500 ng/ml,定量下限为5 ng/ml。日内、日间精密度（RSD）均小于9.51%,准确度（RE）在-0.18%~1.93%。样品提取回收率为79.73%~85.99%,每个样品的测试时间为3 min。应用此法测试了大鼠口服或静注穿琥宁（DAS的单钾盐）后DAS的血药浓度,计算出其绝对生物利用度为3.69%。结论本方法灵敏度高、专属性强,适合于DAS的临床前药动学研究。

Objective To develop and validate a rapid and sensitive LC-MS/MS method for the quantification of dehydroandrographolide succinate（DAS） in rat plasma.Methods Plasma samples were prepared by liquid-liquid extraction with a mixture of diethyl ether-dichloromethane（3∶2,V/V）.Isocratic chromatographic separation was performed on a reversed-phase Pinnacle Ⅱ C 18 column（150 mm × 2.1 mm,5 μm）.The mobile phase was methanol-water（70∶30,V/V）.Detection of DAS and glipizide,used as internal standard（IS）,was achieved by ESI MS/MS in the negative ion mode using m/z 531→m/z 431 and m/z 444→m/z 319 transitions,respectively.Results The method was linear from 5 to 2500 ng/ml when 100 μl plasma was analyzed,and the lower limit of quantification was 5 ng/ml.The intra-and inter-day precision values were below 9.51%,and accuracy ranged from-0.18% to 1.93% in all quality control samples.The extraction recovery was from 79.73% to 85.99% for DAS.Total run time for each sample analysis was 3.0 min.The validated method was successfully applied to a pharmacokinetic study in rats after oral and intravenous administration of potassium DAS.DAS could be absorbed as its intact form in rats after oral administration and its absolute bioavailability was 3.69%.Conclusion The method,with the advantages of high sensitivity and specificity,is suitable for the pharmacokinetic studies of DAS.