HPLC法测定人血浆中咖啡因含量的不确定度评定

摘要

目的 对高效液相色谱法测定血浆中咖啡因的不确定度进行分析,找出影响因素,对不确定度进行评估,如实反映测量的置信度和准确度.方法 采用ACE 5 C18-AR柱(150 mm×4.6 mm,5μm),流动相:甲醇-0.2％醋酸溶液(17∶83),检测波长:273 nm,流速:1.0 mL/min,测定咖啡因含量后,建立数学模型,分析过程中各分量引起的不确定度,采用A类评定程序评价随机效应引起的不确定度,B类评定程序评价其他因素引起的不确定度.结果 置信概率P为95％时,血浆中咖啡因可表示为:低浓度(1 058.08±29.24) ng/mL,中浓度(3 489.53±90.21) ng/mL,高浓度(8 453.32±115.92) ng/mL.结论 该方法适用于HPLC法测定血浆中咖啡因浓度的不确定度评定,能为复杂生物样品分析过程的不确定度评定提供一定参考.%Objective To analyze the measurement uncertainty in determination of caffeine in plasma by HPLC,and identify the influencing factors to assess the uncertainty.Methods The ACE 5 C18-AR (150 mm × 4.6 mm,5 μm) was used,the mobile phase consisted of methanol-0.2 ％ acetic acid (17∶ 83),the flow rate was 1.0 rnL/min,and the detective wavelength was 273 nm.The mathematical model was established to analyze the source of uncertainty.The uncertainty caused by various factors in the whole process of determination was evaluated.The uncertainty caused by random effects was evaluated with type A and others were evaluated with type B.The combined uncertainty was then finally obtained by synthesizing the uncertainties of various component variables.Results The expanded uncertainty for low,medium and high level of caffeine can be expressed (1 058.08 ± 29.24) ng/mL,(3 489.53 ± 90.21)ng/mL and(8 453.32 ± 115.92)ng/mL,respectively (P =95 ％).Conclusion The method is applicable for the uncertainty evaluation of caffeine determination in plasma.It provides an important reference for the uncertainty evaluation for the determination in complex biological sample.