紫外-可见分光光度法测定b型流感嗜血杆菌结合疫苗中多糖含量的不确定度评估

Uncertainty evaluation of determination of polysaccharide content in Haemophilus influenzae type b conjugate vaccine by UV spectrophotometry

摘要

目的: 对紫外—可见分光光度法测定 b 型流感嗜血杆菌结合疫苗中多糖含量的不确定度进行评定分析。方法: 依据现行版《中华人民共和国药典》通则3421对b型流感嗜血杆菌结合疫苗中多糖含量进行测定。根据《测量不确定度评定与表示》《化学分析中不确定度的评估指南》及实验过程,分析测量过程的各个不确定度来源,计算其大小并合成。结果: 多糖含量的不确定度来源主要有标准溶液配制 u（P）、试样处理 u（S）、标准曲线拟合 u（C）、测量重复性 u（rep）、紫外分光光度计 u（I）及方法偏倚 u（B）;合成标准不确定度 u_c 为 1.1090μg⋅ mL^-1,扩展不确定度 U 为 2.2180μg⋅mL^-1 (k=2,置信概率为 95%)。结论: 测定结果的不确定度主要来自标准曲线拟合、试样处理和方法偏倚。控制标准曲线拟合降低不确定度分量,效果最为明显;试样处理引入的不确定度主要来源于移液器操作,需计量检定,试样处理1 mL 及以上样品时用移液管,1 mL 以下用移液器配制标准曲线效率高;方法偏倚引入的不确定度需严格依照标准检验,把控好实验细节;控制实验环境温度,对仪器定期检定与维护;本研究可为基于紫外-分光光度法的药品质量控制的不确定度评估提供参考。

Abstract

Objective: To evaluate the uncertainty of the determination of polysaccharide content in Haemophilus influenzae type b（Hib）conjugate vaccine by ultraviolet-visible（UV）spectrophotometry. Methods: According to the General Rule 3421 of the Chinese Pharmacopoeia,the polysaccharide content in Haemophilus influenzae type b conjugate vaccine was determined.Based on the experimental process,the Evaluation and Representation of Measurement Uncertainty,and the Guidelines for the Evaluation of Uncertainty in Chemical Analysis, the uncertainties of the measurement results were systematically evaluated. Results: The sources of uncertainty mainly included the preparation of standard solution u(P),sample processing u(S),standard curve fitting u(C), measurement repeatability u（rep）,UV spectrophotometry u(I),and method bias u(B).The combined standard uncertainty（u_c）was 1.1090μg⋅mL^-1,and the expanded uncertainty（U）was 2.2180μ" g⋅mL^-1（coverage factor k=2,95% confidence probability）. Conclusion: The dominant contributors to measurement uncertainty were standard curve fitting,sample processing,and method bias.The fitting of the control standard curve significantly reduces measurement uncertainty,with this effect being particularly pronounced.The uncertainty introduced during sample processing primarily stems from pipette operations,which necessitate rigorous metrological verification. While standard curve preparation is time-consuming,optimized workflow efficiency can be achieved by employing pipettes for sample processing volumes of 1 mL or greater and utilizing micropipettes for standard curve preparation when the volumes are 1 mL or less.Method bias-related uncertainty requires strict compliance with standardized testing protocols,with particular attention to meticulous control of experimental parameters.Furthermore,maintaining appropriate laboratory temperature conditions,implementing regular instrument calibration,and performing systematic equipment maintenance all contribute substantially to uncertainty mitigation.This study can provide references for uncertainty assessment of drug quality control based on UV spectrophotometry.

关键词

紫外-可见分光光度法 / b型流感嗜血杆菌结合疫苗 / 多糖含量 / 不确定度 / 评估

Key words

ultraviolet-visible spectrophotometry / Haemophilus influenzae type b conjugate vaccine / polysaccharide content / measurement uncertainty / uncertainty evaluation

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