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目的:建立灵敏、可靠的衍生化LC-MS/MS新方法测定大鼠血浆中帕拉米韦的浓度。方法:血浆样品前处理包括蛋白沉淀和用盐酸(10mol/L)-甲醇(10:90,体积比)为衍生化试剂的衍生化反应,测定采用LC-MS/MS。通过电喷雾电离源以选择反应监测(SRM)方式进行正离子检测,用于定量分析的离子反应分别为m/z343→284(帕拉米韦衍生物)和m/z299→152(内标Ro64-0802衍生物)。色谱分离采用ZorbaxRX-C8柱(2.1mm×150mm,5μm),以乙腈-水-甲酸(30:70:0.1,体积比,0.2ml/min)为流动相。结果:测定帕拉米韦的线性范围为10～10000ng/ml,相关系数r2为0.9940,定量下限为10ng/ml,批内和批间RSD%分别在5.0%和7.1%以内,准确度控制在89.9%～106.1%。结论:本方法通过衍生化反应,使帕拉米韦保留时间增加,基质抑制降低并使检测灵敏度提高。该方法成功应用于帕拉米韦非临床和临床研究中。
OBJECTIVE: To establish a sensitive and reliable derivatization LC-MS / MS method for the determination of the concentration of peramivir in rat plasma. METHODS: Pretreatment of plasma samples consisted of protein precipitation and derivatization with derivatizing reagent of hydrochloric acid (10mol / L) -methanol (10:90, volume ratio). LC-MS / MS was used to determine the plasma samples. The positive ion detection was performed by electrospray ionization source with selective reaction monitoring (SRM). The ion reactions for quantitative analysis were m / z343 → 284 (peramivir derivative) and m / z299 → 152 (internal standard Ro64 -0802 derivatives). The chromatographic separation was performed on a Zorbax RX-C8 column (2.1 mm × 150 mm, 5 μm) using acetonitrile-water-formic acid (30: 70: 0.1, by volume, 0.2 ml / min) as the mobile phase. Results: The linear range was 10 ~ 10000 ng / ml, the correlation coefficient r2 was 0.9940, the lower limit of quantification was 10 ng / ml, the RSD% within and between batches were within 5.0% and 7.1% respectively, and the accuracy was controlled at 89.9% ~ 106.1%. Conclusion: This method through the derivatization reaction, so that the retention of peramivir increased matrix inhibition decreased and the detection sensitivity increased. This method has been successfully used in non-clinical and clinical studies of peramivir.