液相色谱-质谱联用法测定人血浆中西洛他唑的浓度

Determination of cilostazol concentration in human plasma by HPLC-MS/MS

目的建立液相色谱-质谱联用法(HPLC-MS/MS)测定人血浆中西洛他唑浓度。方法用乙腈蛋白沉淀法处理血浆,以西洛他唑-d4为内标,色谱柱:ACE Excel 5 C18-AR(2.1 mm×50 mm,5μm),柱温:40℃,梯度洗脱,流动相:乙腈(B)-5 mmol·L^-1乙酸铵水溶液(含0.1%甲酸;A),流速:0.45 mL·min^-1,进样量10μL,分析时间3.5 min。用电喷雾电离子化源,正离子方式、多反应监测(MRM)扫描进行监测,用于定量分析的离子反应分别为m/z 370.2→m/z 288.2(西洛他唑)和m/z 374.2→m/z 288.2(内标西洛他唑-d4)。考察该方法的专属性、标准曲线和定量下限、精密度和回收率、基质效应及稳定性。结果血浆样品中西洛他唑回归方程为Y=5.12×10^-3X+4.86×10^-3(r=0.9954),西洛他唑在5.00~2000 ng·mL^-1内线性关系良好,定量下限为5.00 ng·mL-1,批内、批间精密度均在3.2%~5.8%,相对偏差(RE)在-1.9%~6.6%;低、中、高3个浓度西洛他唑质控样本内标化归一的基质效应分别为(98.1±2.2)%,(98.2±1.3)%和(99.9±1.0)%;提取回收率分别为(90.9±6.3)%,(90.9±9.0)%和(89.3±7.4)%。结论本方法快速、准确、灵敏且高效,适用于人血浆中西洛他唑的检测和药代动力学研究。

Objective To determine the cilostazol concentration in human plasma by established HPLC-MS/MS method.Methods Plasma samples were pretreated by acetonitrile precipitation,cilostazol-d4 was used as internal standard,the sample was separated on ACE Excel 5 C18-AR(2.1 mm×50 mm,5μm)column using gradient elution with a mobile phase of acetonitrile(B)-5 mmol·L^-1 ammonium acetate,isocratic elution,flow rate was 0.45 mL·min^-1 and the column temperature was 40℃.Detection of the analyte was achieved using positive ion electrospray ionization(ESI)in the multiple reaction monitoring(MRM)mode.The MS/MS ion transitions monitored were m/z 370.2→m/z 288.2(cilostazol)and m/z 374.2→m/z 288.2(cilostazol-d4).The specificity,lower limit of quantitation and standard curve,precision and recovery rate and stability as well as matrix effect were investigated.Results The standard curve of cilostazol was Y=5.12×10^-3 X+4.86×10^-3(r=0.9954),and in 5.00-2000 ng·mL^-1 good linear relationship,the lowest concentration was 5.00 ng·mL^-1.The intra-and inter-batch precision were 3.2%-5.8%,and the relative deviation were-1.9%-6.6%.The matrix factor normalized by internal standard for cilostazol QC samples were(98.1±2.2)%,(98.2±1.3)%,(99.9±1.0)%,respectively.The recoveries in the three concentration cilostazol were(90.9±6.3)%,(90.9±9.0)%,(89.3±7.4)%,respectively.Conclusion The method was fast,accurate,sensitive and efficient,and it was suitable for the determination and pharmacokinetics of cilostazol in human plasma.