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目的建立超高效液相色谱-串联质谱法(UPLC-MS/MS)测定大鼠血浆中淫羊藿苷(ICA)浓度的方法,并用于研究大鼠灌胃ICA的药代动力学。方法采用乙酸乙酯提取血浆中ICA,以香豆雌酚(CMT)为内标,在BEH C18(50 mm×2.1 mm,1.7μm)色谱柱上以含0.1%(V/V)甲酸的乙腈-2 mmol/L甲酸铵的水溶液为流动相梯度洗脱,流速0.3 m L/min;以负离子电喷雾多反应监测方式定量分析ICA(m/z675.6→351.1)和CMT(m/z 267.0→211.1)。大鼠多次灌胃50 mg/kg ICA,采集不同时刻血样并测定药物浓度。结果 ICA的线性范围为0.5~50 ng/m L,方法定量下限为0.5 ng/m L。ICA日内和日间精密度(RSD)在11.3%以内,准确度在94.3%~98.7%,提取回收率为81.3%~85.2%,基质效应为94.3%~103.2%。大鼠灌胃ICA的t1/2为(1.68±0.29)h,C_(max)为(29.6±5.3)ng/m L,t_(max)为(1.00±0.00)h,AUC0-t为(88.4±13.9)(h·ng)/m L。结论 UPLC-MS/MS法专属性好、准确度高,适用于ICA临床前药代动力学研究。
Objective To establish a method for the determination of icariin (ICA) in rat plasma by ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) and to study the pharmacokinetics of ICA in rats. Methods The ICA in plasma was extracted with ethyl acetate. The coumarin (CMT) was used as internal standard. The column was on a BEH C18 (50 mm × 2.1 mm, 1.7 μm) column with 0.1% (V / V) formic acid in acetonitrile The aqueous solution of 2 mmol / L ammonium formate was eluted with a mobile phase gradient of 0.3 m L / min. The ICA (m / z 675.6 → 351.1) and CMT (m / z 267.0 → 211.1). The rats were orally gavaged 50 mg / kg ICA, blood samples were collected at different times and the drug concentration was determined. Results The linear range of ICA was 0.5-50 ng / m L and the limit of quantification was 0.5 ng / m L. Intra-day and inter-day ICA precision was less than 11.3% with accuracy of 94.3% -98.7%, extraction recovery was 81.3% -85.2% and matrix effect was 94.3% -103.2%. The IC50 of tibia was (1.68 ± 0.29) h, the max was (29.6 ± 5.3) ng / m L, the t max was (1.00 ± 0.00) h and the AUC0-t was ± 13.9) (ng · m) / m L Conclusion The UPLC-MS / MS method has good specificity and high accuracy, and is suitable for preclinical pharmacokinetic studies in ICA.