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目的评价液-质联用法测定人血浆中左旋多巴甲酯浓度的不确定度。方法选用Thermo BDS HYPERSIL C18(4.6 mm×100 mm,2.4μm)色谱柱;流动相由甲醇-水(含10 mmol·L?1乙酸铵,p H=5.0)梯度洗脱;流速:0.4 m L·min?1。质谱条件:选用电喷雾(ESI)离子源,在正离子电离模式下,采用多反应监测(MRM)的质谱扫描方式,测定左旋多巴甲酯和内标的检测离子分别为:m/z 212.4→152.3、m/z 166.4→148.3。测定左旋多巴甲酯含量后,建立数学模型,分析过程中各分量引起的不确定度,包括测定精密度、称量、标准溶液的配制、含药血浆的配制、血浆提取、仪器、标准曲线拟合等,计算各变量的不确定度和合成不确定度,最终计算扩展不确定度。结果置信概率P为95%时,血浆低、中、高(0.4,4,32 ng·m L?1)浓度左旋多巴甲酯的扩展不确定度分别为0.10,0.23,1.62 ng·m L?1。结论该方法适用于LC-MS/MS测定人血浆中左旋多巴甲酯浓度的不确定度评价,为复杂生物基质分析过程的不确定度评价提供了参考依据。
Objective To evaluate the uncertainty of determination of levodopa methyl ester concentration in human plasma by liquid chromatography / mass spectrometry. Methods The column of Thermo BDS HYPERSIL C18 (4.6 mm × 100 mm, 2.4 μm) was used as the mobile phase. The mobile phase was eluted with a gradient of methanol-water (containing 10 mmol·L -1 ammonium acetate, p H = 5.0) · Min? 1. Mass spectrometry conditions: Electrospray ionization (ESI) ion source was used to determine the detected ions of levodopa methyl ester and internal standard by mass reaction monitoring (MRM) in positive ionization mode. The detection ions were: m / z 212.4 → 152.3, m / z 166.4 → 148.3. After determining the content of levodopa methyl ester, a mathematical model was established to analyze the uncertainty caused by each component in the process, including the determination of precision, weighing, preparation of standard solution, preparation of medicated plasma, plasma extraction, apparatus, standard curve Fitting and so on, calculates the uncertainty of each variable and the synthesis uncertainty, finally calculates the expansion uncertainty. Results When the confidence probability P was 95%, the expanded uncertainty of the low, middle and high concentrations of levodopa methyl ester at concentrations of 0.4, 4, 32 ng · m L -1 were 0.10, 0.23, 1.62 ng · m L ?1. Conclusion The method is suitable for the LC-MS / MS determination of uncertainty in the determination of levodopa methyl ester in human plasma, which provides a reference for the evaluation of the uncertainty of complex bio-matrix analysis.