液相色谱-串联质谱法测定大鼠血浆中千层纸素的浓度

Determination of oroxylin A in rat plasma by LC-MS/MS

目的建立快速且灵敏的液相色谱-串联质谱法(LC-MS/MS)测定大鼠血浆中千层纸素的浓度。方法色谱分离采用C_(18)色谱柱,甲醇-10 mmol·L^(-1)醋酸铵(77∶23,V/V)为流动相,流速1.05 m L·min^(-1)。大鼠血浆加入β-葡萄糖醛酸酶/硫酸酯酶,37℃孵化过夜,再加入内标染料木素及0.1%磷酸溶液,用乙酸乙酯提取,上清液40℃水浴中氮气流吹干,以流动相200μL溶解残渣,进行分析。检测器采用四极杆串联质谱仪,离子源采用电喷雾电离离子源(ESI),检测方式采用选择反应检测扫描(SRM),检测离子:千层纸素[M+H]^+284.9→269.9,内标[M+H]^+270.9→270.9。结果千层纸素在5~1 000μg·L^(-1)范围内,线性关系良好,回收率大于85%,日内和日间的相对标准偏差(RSD)小于15%。结论建立的LC-MS/MS法符合生物样品分析的要求,可用于研究大鼠灌胃千层纸素后血浆中千层纸素的测定。

Objective To develop a rapid and sensitive liquid chromatography-tandem mass spectrometry（ LC-MS / MS） method for determination of oroxylin A in rat plasma. Methods Chromatographic separation was achieved on a C18 column and the mobile phase consisted of methanol and 10 mmol·L^-1 ammonium acetate buffer（ 77∶ 23,V / V） pumped at a flow rate of 1. 05 m L·min^-1. The rat plasma samples by enzymatic hydrolysis using β-glucuronidase / sulfatase under 37 ℃ for the night were partitioned with ethyl acetate and centrifuged for sample clean-up after genistin used as internal standard（ I. S.） and 0. 1% phosphoric acid were added. The supernatant was then evaporated to dryness,and the residue was dissolved in 200 μL of mobile phase and analyzed. The quadrupole tandem mass spectrometer with electrospray ionization ion source（ ESI） was performed in the selected reaction monitoring（ SRM） mode and the transitions selected for quantitation were [M ＋ H]-＋284. 9→269. 9 for oroxylin A and [M ＋ H]-＋270. 9→270. 9 for genistin（ I. S.）,respectively. Results A linear calibration curve for assay of oroxylin A in rat plasma was obtained in the range of 5 - 1 000 μg·L^-1 with the recoveries of at least 85% and intra-day and inter-day RSD of 15%. Conclusion The method can be used for thedetermination of oroxylin A in rat plasma after intragastric administration.