串联质谱技术检测孕妇胆汁酸谱的性能评估及其参考区间建立

Performance evaluation of tandem mass spectrometry for detecting bile acid profile in pregnant women and establishment of its reference interval

目的对基于串联质谱(LC-MS/MS)技术检测胆汁酸谱的方法进行性能评估并建立适合于南京地区孕妇的15种胆汁酸亚型参考区间。方法采用LC-MS/MS技术检测孕中期孕妇血清15种胆汁酸亚型,根据美国临床和实验室标准协会(CLSI)C62-A和美国食品药品监督管理局的生物分析方法验证标准,从定量检出限、准确度、精密度、线性、携带污染率等几个方面进行性能评估;根据CLSI EP28-A3c推荐的方法和程序建立南京地区孕妇孕中期15种胆汁酸亚型的参考区间,用小样本验证法对新建立的20例样本参考区间进行验证。结果LC-MS/MS技术可同时检测孕妇血清中15种胆汁酸亚组分,最低定量限为标准曲线最低浓度点(S1)浓度,最低检出限为1/4 S1;线性:各种亚型均呈现良好的线性关系,r^(2)均大于0.99;准确度:各种亚型加标回收试验回收率均为80%~120%,偏倚<15%;批内精密度和批间精密度变异系数均小于15%;携带污染率:小于最低定量限的25%;孕中期孕妇参考区间:以石胆酸为例,新的参考区间为0.07~13.46 ng/mL,95%的验证结果落于新的参考区间内。结论LC-MC/MS技术可以精准、快速地检测胆汁酸谱15种亚型,评估结果符合各项性能验证要求;新建立的胆汁酸谱15种亚型的参考区间适合于南京地区孕中期孕妇。

Objective To establish a method based on tandem mass spectrometry(LC-MS/MS)to detect bile acid spectrum and evaluate its performance,and to establish the reference intervals of 15 bile acid subtypes for pregnant women in Nanjing.Methods LC-MS/MS technology was used to detect 15 bile acids in the serum of pregnant women in the second trimester of pregnancy.According to Clinical and Laboratory Standards Institute(CLSI)C62-A and FDA guidelines,the performance was evaluated from several aspects,including the lower limit of measuring interval,the limit of detection,accuracy,precision,linear and carryover,etc.As recommended by the CLSI EP28-A3c,the reference intervals of 15 bile acids in the second trimester of pregnancy were established.Small sample validation method(20 cases)was used to verify the new reference interval.Results LC-MS/MS technology could simultaneously detect 15 bile acids in serum,the Limit of quantitation was the lowest concentration(S1)at the point of the standard curve.The limit of detection was 1/4 S1.Linear:all subtypes showed a good linear relationship,and r^(2) was more than 0.99 in all subtypes.Accuracy:the recoveries of each subtype were between 80%-120%,and the bias were less than 15%.The coefficien of variation of within-and between-batch were less than 15%.The carryover was less than 25%of the limit of detection.Reference intervals of pregnancy:taking LCA as an example,the new reference interval of the second trimester was 0.07-13.46 ng/mL,and 95%of the validation results fell in the new reference interval.Conclusion LC-MS/MS technology could accurately and rapidly detect 15 subtypes of bile acid spectrum,and the evaluation results meet the performance verification requirements.The newly established reference intervals of 15 subtypes of bile acid spectrum are suitable for pregnant women in the second trimester of pregnancy in Nanjing.The established LC-MS/MS technology can effectively separate and accurately detect 15 kinds of bile acids in serum and the evaluation results are conformed to all standards.The newly established reference intervals of 15 subtypes of bile acids are suitable for pregnant women in the second trimester of pregnancy in this region.