同位素稀释液相色谱串联质谱法测定人血清尿酸

目的建立一种使用同位素稀释液相色谱串联质谱（ID-LC/MS/MS）测定人血清尿酸的候选参考方法。方法用1,3]1-5N2尿酸作内标,与一定量的血清充分混匀,加入等量乙腈沉淀血清蛋白并提取尿酸,氯仿纯化上清液后用液相色谱串联质谱（LC/MS/MS）分离测定。优化LC/MS/MS的色谱条件,评价该法的精密度、回收率和偏倚,并计算不确定度。结果对3个浓度水平的室间质量评价用质控血清测定3批,批内、批间和总变异系数（CV）的均值（范围）分别为0.54%（0.44%-0.69%）、0.66%（0.45%-0.79%）和0.86%（0.66%-1.02%）。加样回收试验回收率范围为98.38%-102.64%。美国国家标准与技术研究所（NIST）标准物质SRM 909b-Ⅰ、Ⅱ的测定均值与靶值的偏倚分别为0.04%和0.25%。3种质控血清的相对扩展不确定度分别为1.80%、2.19%和2.04%。结论建立的ID-LC/MS/MS测定血清尿酸的方法精密、准确,有望作为血清尿酸测定的参考方法。

Determination of serum uric acid by isotope dilution liquid chromatography tandem mass spectrometry

Objective To develop a candidate reference method for the measurement of serum uric acid by isotope dilution liquid chromatography tandem mass spectrometry (ID-LC/MS/MS). Methods An isotope labeled internal standard(IS) 1,3]-~(15)N_2 uric acid was weighed and spiked to serum samples. Acetonitrile was applied to precipitate serum proteins and extract uric acid and IS. After being treated with chloroform for further clean-up, the samples were analyzed by LC/MS/MS. Serum uric acid was quantified by a bracketing calibration. Results The within-run, between-run and total coefficients of variation(CV)of 3 batches of measurements of 3 levels of lyophilized sera used in external quality assessment ranged from 0.44% to 0.69%, 0.45 % to 0.79% and 0.66% to 1.02%, and the averages were 0.54%, 0.66% and 0.86%,respectively. The analytical recoveries ranged from 98.38% to 102.64% with an average of 100.55%. The results of analyzing the certified reference material SRM909b-Ⅰand Ⅱ showed 0.04% and 0.25% proportional bias compared with certified values. Evaluated uncertainty of measurement for the determination of (3 levels) of lyophilized sera and values of the relative expanded uncertainty were 1.80%,2.19% and 2.04%. Conclusions An ID-LC/MS/MS method for measuring serum uric acid has been developed. The method is highly precise and accurate and may be used as a candidate reference method for serum uric acid measurement.