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.
Keywords: amino acid; liquid chromatography-tandem mass spectrometry; organic acid; random urine; vitamin B12.