目的 建立了灵敏快速的同时测定大鼠血浆中丹酚酸B及其主要代谢产物丹参素的LC-MS/MS方法。方法 以氯霉素为内标,用醋酸乙酯萃取,色谱柱为Symmetry C₁₈柱,流动相为甲醇-乙腈-0.5%甲酸(55︰5︰40),体积流量为0.4 mL/min。选用电喷雾电离(ESI)三重四极杆串联质谱仪,在负离子模式下以选择反应监测(SRM)方式进行检测,用于定量分析的离子反应分别为m/z 717→519(丹酚酸B)、m/z 197→135(丹参素)和m/z 321→152(氯霉素)。结果 在该测定条件下丹酚酸B、丹参素和内标物的保留时间分别为3.12、2.60、3.98 min。丹酚酸B的线性范围为10～5 000 ng/mL,r＞0.995;丹参素的线性范围为5～5 000 ng/mL,r＞0.995,丹酚酸B和丹参素定量限分别为10、5 ng/mL。批内、批间精密度(RSD)均小于12.6%。大鼠ig给予丹酚酸B后,迅速吸收并逐渐转化成丹参素,丹酚酸B和丹参素的C_max分别为(1.21±0.31)、(0.27±0.05)μg/mL,t_max分别为(0.50±0.00)、(0.56±0.18)h,t1/2分别为(1.20±0.11)、(1.57±0.16)h,AUC0～t分别为(1.31±0.30)、(0.39±0.05)μg.mL^-1.h。结论 本方法可用于大鼠ig丹酚酸B后的血浆中丹酚酸B及其代谢产物丹参素药动学研究。

Objective A rapid and sensitive method using liquid chromatography–tandem mass spectroscopy (LC-MS/MS) was developed and validated for the simultaneous quantitative determination of salvianolic acid B (Sal B) and its main active metabolite danshensu(DSS) in rat plasma. Methods The analytes were extracted by liquid–liquid extraction with ethyl acetate after IS (IS, chloramphenicol) spiked. The separation was performed on a Symmetry C18 column with methanol-acetonitrile-0.5% formic acid (55:5:40) as mobile phase at a flowrate of 0.4 mL/min. The triple quadrupole LC-MS system was operated under selected reaction monitoring (SRM) mode using the electrospray ionization technique in negative mode. Quantification was performed using SRM of the transitions m/z 717→519 for Sal B, 197→135 for DSS, and 321→152 for the IS, respectively. Results The nominal retention times for Sal B, DSS , and IS were 3.12, 2.60, and 3.98 min, respectively. The standard calibration curve for spiked rat plasma containing Sal B was linear over the range 10-5 000 ng/mL with a correlation coefficient (r >0.995). And DSS was linear over the range 5-5000 ng/mL with a correlation coefficient (r>0.995). The lower limits of quantification (LLOQ) of Sal B and DSS of the method were 10 and 5 ng/mL, respectively. The intra- and inter-day accuracy and precision of the assay were less than 12.6%. After Sal B was ig administered to rats, absorption of Sal B was rapidly metabolized to DSS. Pharmacokinetic parameters of Sal B and DSS after ig administration Sal B to Wistar rats were: Cmax (1.21±0.31) and (0.27±0.05) μg/mL, tmax (0.50±0.00) and (0.56±0.18) h, t1/2 (1.20±0.11) and (1.57±0.16) h, AUC0-t (1.31±0.30) and (0.39±0.05) μg·mL?1·h. Conclusion This method has been applied successfully to a pharmacokinetic study of Sal B and its metabolite DSS involving the ig administration of Sal B to rats.

国家自然科学基金资助项目(306030075,20675056);国家“973”计划基金项目(2004CB518902)