HPLC-MS／MS和FPIA法测定全血中环孢素浓度的比较方法学

Determination of Cyclosporin A in Human Blood With HPLC-MS/MS and Its Comparison with FPIA Methods

目的建立液质联用(HPLC-MS/MS)法同时测定人血中环孢素(CsA),并与荧光偏振免疫法(FPIA)进行比较。方法液质联用法:Waters Quattro Premier串联质谱检测器,采用Symmetry C18色谱柱(2.1mm×150mm,5μm),以含有10mmol·L-1乙酸铵-0.1%甲酸溶液0.1%甲酸甲醇溶液为10∶90,流速0.25mL·min-1,采用多反应监测进行定量,血样处理:采用乙腈沉淀蛋白,分析时间为5min。并收集162人份临床使用CsA的重症肌无力患者的血样分别用HPLC-MS/MS及FPIA法测定并进行评价。结果HPLC-MS/MS:CsA在人血中50～1000μg·L-1内线性良好,相对回收率在98.67%～103.56%之间,日内及日间精密度(RSD%)均小于10%。2种方法测定患者血样,HPLC-MS/MS结果大多数低于FPIA法。结论2种测定方法的相关性较差,串联质谱法方法用于监测CsA血药浓度特异性好,灵敏度高,经济,可适性强。与FPIA法相比,对CsA的母药有高度的选择性,并能够提高分析的灵敏度。

OBJECTIVE To develop and validate A HPLC-MS/MS method for determining cyclosporine A in human blood and compare with the fluorescence polarization immunoassay（FPIA） method. METHODS The separation was performed on a symmetry C18 （2.1 mm× 150 mm, 5μm） column with a mobile phase comsisting of water （containing 10 mmol.L-1 ammonium acetate and 0.1% formic acid ） and methanol （containing 0.1% formic acid）. Multi-reaction monitoring mode was employed for the quantitative determination. Proteins were precipitated by acetonitrile. Cycle time was 5 min. 162 Samples from patients with myasthenia gravis were determined by the HPLC-MS/MS and FPIA methods respectively. RESULTS For the HPLC-MS/MS assay, the standard curve of cyclosporine was linear over the range of 25-1 000 μg.L-1. The relative recovery was among the range of 98.67%-103.56%. The RSDs of intra- and inter-day accuracy results were less than 10%. For comparisons, 162 blood samples were determined by the HPLC-MS/MS and FPIA methods respectivelY. The HPLC-MS/MS gave significantly lower values than the FPIA. CONCLUSION There is bad correlation between HPLC/MS/MS and FPIA methods. HPLC-MS/MS will provide a flexible, robust, and cost-effective solution for the monitoring of CsA. The HPLC-MS/MS assay has some advantages over the FPIA method, namely, better specificity for the parent compound, improved assay sensitivity.