微量随机尿液中多种氨基酸和有机酸定量方法及其在维生素B12营养状况及相关代谢的初步应用

目的 建立一种高效液相色谱-串联质谱同时定量微量随机尿液中多种氨基酸和有机酸的方法。方法 10μl随机尿液样本经乙腈沉淀蛋白后使用饱和的盐酸正丁醇进行衍生化，衍生后的样本经ACE Excel 2 AQ(50×2.1 mm, 2μm)色谱柱分离，采用电喷雾离子源，在正离子扫描和多反应监测模式下检测。参照现有指南，从灵敏度、特异性、准确度、精密度、回收率、基质效应、稳定性等方面完成系统方法学确证。并检测70例健康儿童、27例维生素B12可疑缺乏儿童及3例甲基丙二酸血症cblC型患儿尿液中的19种目标待测物。结果 19种目标待测物定量下限为0.01～1.00μmol/L,标准曲线线性良好，R²均>0.990。准确度良好(相对误差均<±15%),日内、日间精密度均<15%。分析时间为8 min,除精氨酸外，其余待测物未见明显基质效应，加标回收率为80.20%～114.97%,室温8 h和3个冻融循环稳定性良好。健康儿童尿液中目标待测物测定值在Labcorp实验室公布的儿童参考区间内，维生素B12可疑缺乏儿童尿液中甲基丙二酸(P=0.030)和同型半胱氨酸...

A Method for Quantitative Determination of Amino Acids and Organic Acids in Trace Random Urine and Preliminary Application in Research on Vitamin B12 Nutritional Status and Related Metabolism

Objective To develop a method for the quantification of amino acids and organic acids in trace urine by high performance liquid chromatography-tandem mass spectrometry. Methods Random urine samples(10 μl each)were precipitated by acetonitrile and underwent derivatization with 3 mol/L HCl in n-butanol.The analytes were separated by ACE Excel 2 AQ column(50×2.1 mm,2 μm).Electrospray ionization in positive ion mode was carried out and the analytes were detected in multiple reaction monitoring mode.According to existing guidelines,the method was systematically evaluated in terms of sensitivity,specificity,accuracy,precision,recovery,matrix effect,and stability.Then,the established method was employed to detect 19 target compounds in urine samples from 70 healthy children,27 children with suspected vitamin B12 deficiency,and 3 children with cblC type methylmalonic acidemia. Results The lower limit of quantification of the method for the 19 compounds ranged from 0.01 μmol/L to 1.00 μmol/L,and the calibration curves were linear,R²>0.990.The method showed good accuracy with relative error less than ±15% and the intra-day and intra-day precision less than 15%.The run time was 8 min.No obvious matrix effect was detected except for arginine,and the recovery ranged from 80.20% to 114.97%.The samples were stable after 8 h at room temperature and 3 freeze-thaw cycles.The measured values of the compounds in the urine of healthy children were within the children’s reference intervals published by Labcorp.The levels of methylmalonic acid(P=0.030)and homocysteine(P<0.001)in the urine samples of children with suspected vitamin B12 deficiency were higher than those in healthy children.The levels of methylmalonic acid,methylcitric acid,and homocysteine in the urine samples of children with cblC type methylmalonic acidemia were 5.14-76.52 times higher than the median levels of healthy children. Conclusions The method established in this study has small sample demand and short run time,which can accurately quantify the levels of amino acids and metabolites in the urine of children.Moreover,it can provide data support for related studies about the metabolic characteristics of urine amino acids and their metabolites in children with vitamin B12 deficiency.