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目的建立准确、灵敏的高效液相色谱-串联质谱法(HPLC-MS/MS)测定人血清中伏立诺他(SHA)及其M2代谢物浓度,以用于SHA药物代谢动力学研究。方法采用API3000LC-MS/MS液质联用系统,Gemini C18(3μm,50 mm×3 mm)色谱柱,流动相为甲醇︰乙腈︰水︰1 mol/L氨水︰甲酸(25︰15︰60︰0.1︰0.05),流速0.23 m L/min,血清样品经酸化后用3.5 m L含3%异丙醇重蒸乙醚萃取浓集后进样,采用电喷雾离子源,正离子多反应离子监测模式检测,d5-SHA作内标。结果 SHA、M2和内标的保留时间分别为4.1、3.1和4.0 min。SHA和M2线性范围分别为1~1 000 ng/m L和2~2 000 ng/m L,最低定量限分别为1.0和2.0 ng/m L,方法回收率分别为93.0%~99.3%和88.11%~104.12%,基质效应均<9%,SHA批内和批间精密度分别<5.5%和<9.0%,M2批内和批间精密度均<11%。血清样品在冰箱冷冻保存63 d,反复冻融3次,在室温放置6 h,处理后的样品在进样室(8℃)放置16 h、萃取物在冰箱(4℃)中放置36 h均稳定。结论 HPLC-MS/MS法具有快速、简便、灵敏、准确等特点,适用于SHA和M2代谢物的血药浓度的测定。
Objective To establish an accurate and sensitive HPLC-MS / MS method for the determination of valencean (SHA) and its metabolite M2 in human serum for SHA pharmacokinetic study. Methods The mobile phase consisted of methanol: acetonitrile: water: 1 mol / L ammonia: formic acid (25:15:60:50) by using API3000LC-MS / MS system with Gemini C18 (3μm, 50 mm × 3 mm) 0.1: 0.05) at a flow rate of 0.23 m L / min. The serum samples were acidified and extracted with 3.5 mL of distilled water containing 3% isopropyl alcohol and distilled with ether. Electrospray ionization (ESI) Detection, d5-SHA as an internal standard. Results The retention time of SHA, M2 and internal standard were 4.1, 3.1 and 4.0 min respectively. The linear ranges of SHA and M2 were 1 ~ 1 000 ng / m L and 2 ~ 2 000 ng / m L, respectively, with the minimum limits of quantitation of 1.0 and 2.0 ng / m L, respectively. The recovery rates were 93.0% -99.3% and 88.11 % ~ 104.12%, matrix effects were less than 9%, SHA intra-assay and inter-assay precision were <5.5% and <9.0%, respectively. Serum samples were stored in the freezer for 63 days, frozen and thawed repeatedly for 3 times, and then placed at room temperature for 6 hours. The treated samples were placed in the sample chamber (8 ° C) for 16 h, and the extracts were placed in the refrigerator (4 ° C) for 36 h stable. Conclusion The HPLC-MS / MS method is rapid, simple, sensitive and accurate and is suitable for the determination of plasma concentrations of SHA and M2 metabolites.