HPLC-MS/MS 和 FPIA 法测定全血中环孢素浓度的比较方法学

 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 (2.1 mm × 150 mm ， 5 μm) ，以含有 10 mmol·L^-1 乙酸铵 - 0.1% 甲酸溶液 0.1 % 甲酸甲醇溶液为 10 ∶ 90 ，流速 0.25 mL·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 5 min 。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~ 1 000 μ g·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。

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

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，5 μm) 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.