HPLC-MS/MS测定人血浆中盐酸左旋多巴甲酯浓度的不确定度评价

Uncertainty Evaluation for the Determination of Levodopa Methyl Ester Hydrochloride Concentration in Human Plasma by HPLC-MS/MS

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 analyze the measurement uncertainty in determination of levodopa methyl ester hydrochloride in plasma by LC-MS/MS. METHODS Thermo BDS HYPERSIL C18 column（4.6 mm×100 mm, 2.4 μm） was used, the mobile phase consisted of methanol and ammonium acetate（10 mmol·L-1, p H=5）, using gradient elution, and the flow rate was 0.4 m L·min-1. Mass spectrometry was performed in multiple reaction monitoring（MRM） mode, with m/z 212.4→152.3 for levodopa methyl ester hydrochloride and m/z of 166.4→148.3 for internal standard. A mathematical model was established to analyze the source of uncertainty. The various factors influencing the uncertainty in the whole process of determination, including precision, weighing, solution preparation, plasma containing drug preparation, plasma extraction, the apparatus and calibration curves fitting, were all analyzed and estimated. The uncertainty and synthetic uncertainty of each variable were calculated. The expanded uncertainty was analyzed with all the components. RESULTS When the confidence probability P=95%, the expanded uncertainty for low, medium and high（0.4, 4, 32 ng·m L-1） concentration of zidovudine were 0.10, 0.23, 1.62 ng·m L-1,respectively. CONCLUSION This method is suitable for the uncertainty evaluation for the determination of levodopa methyl ester hydrochloride concentration in human plasma by LC-MS/MS, and provides reference for uncertainty evaluation of complex biological matrix analysis.