高效液相色谱串联质谱法测定人血清中同型半胱氨酸的方法学研究

The Methodological Study of Determining Homocysteine in Human Serum by HPLC-MS/MS

目的评价使用高效液相色谱串联质谱法(HPLC-MS/MS)对血清同型半胱氨酸的测定。方法本实验收集76例需要测定同型半胱氨酸水平的血清样本,分别使用HPLC-MS/MS法和循环酶法(ECA)测定,评价HPLC-MS/MS和ECA测定结果的相关性,并使用低、中、高值质控对HPLC-MS/MS法进行精密度和准确度的评价。结果 76例血清样本中同型半胱氨酸水平分布范围为1.50~62.64μmol/L,2组测定结果方程式为YHPLC-MS/MS=1.369XECA(r=0.928,n=76),相关性良好;同型半胱氨酸在HPLC-MS/MS方法中的日内精密度低值质控的变异系数(CV)是2.19%,中值质控的CV是3.43%,高值质控的CV是0.82%;日间精密度低值质控的CV是3.08%,中值质控的CV是4.02%,高值质控的CV是1.37%;本实验通过回收率表示准确度,测得的低值平均回收率是101.48%,中值平均回收率是98.81%,高值平均回收率是100.30%,表示准确度良好。结论通过HPLC-MS/MS和ECA测定血清样本中同型半胱氨酸的水平,表明实验结果相关性较好,且经过实验验证HPLC-MS/MS的精密度和准确度均符合临床实验室标准。所以HPLC-MS/MS法测定人血清中同型半胱氨酸可以在临床中应用。

Objective To evaluate the measurement of serum homocysteine by HPLC-MS/MS.Methods Seventy-six serum samples were collected to determine the concentration of homocysteine.HPLC-MS/MS method and cyclic enzyme method(ECA)were used to evaluate the correlation between results of HPLC-MS/MS and ECA,and the precision and accuracy of HPLC-MS/MS method were evaluated by low,medium and high value quality control.Results The distribution range of homocysteine concentration in 76 serum samples was 1.50-62.64(μmol/L).The achieved correlation equation of the two groups was YHPLC-MS/MS=1.369XECA(r=0.928,n=76),suggesting a good performance.In HPLC-MS/MS method,the intraday low-value quality control CV of homocysteine was 2.19%,the median quality control CV was 3.43%,and the high-value quality control CV was 0.82%.The CV of low-value quality control for daytime precision was 3.08%,the CV of mid-value quality control was 4.02%,and the CV of high-value quality control was 1.37%.In this experiment,the accuracy was represented by the mean recovery rate.We observed that the low mean recovery rate was 101.48%,the median mean recovery rate was 98.81%,and the high mean recovery rate was 100.30%.Conclusion In the current study,HPLC-MS/MS and ECA were used to determine the concentration of homocysteine in serum of the samples,which indicated that the experimental results were well correlated.The accuracy and precision of HPLC-MS/MS were verified and meet the clinical laboratory standards.Therefore,HPLC-MS/MS can be used to determine homocysteine in human serum.