A New Method For Quantitative Determination Of Various Amino Acids And Organic Acids In Trace Random Midstream Urine And Preliminary Application

Objective:1.To develop a new method for the quantification of various amino acids and organic acids in trace random midstream urine by liquid chromatography-tandem mass spectrometry(LC-MS/MS).Amino acids include:L-Homocysteine(HCY),L-Methionine(MET),L-Cystathionine(CYSTA),L-Alanine(ALA),L-Arginine(ARG),L-Citrulline(CIT),Glycine(GLY),L-Histidine(HIS),L-Isoleucine(ILE),L-Leucine(LEU),L-Phenylalanine(PHE),L-Proline(PRO),L-Serine(SER),L-Threonine(THR),L-Tryptophan(TRP),L-Tyrosine(TYR),L-Valine(VAL);and organic acids include:Methylmalonic acid(MMA)and Methylcitric acid(MCA).2.To establish the biological reference intervals of various amino acids and organic acids in random midstream urine of children aged 1-7 years in Beijing area.Methods:1.Sample pretreatment and adjustment of LC-MS/MS analysis conditions and parameters:10μL random midstream urine samples were precipitated by acetonitrile and derivated with 3M HCL in n-butatol.The analytes were separated by ACE Excel 2 AQ(50×2.1mm,2μm)column.Electrospray ionization(ESI)in positive ion mode was used and the analytes were detected in multiple reaction monitoring(MRM)mode.A series of standard curves were prepared from standards and synthetic urine and the stable isotope internal standard method was used to quantification.2.Verification for the method:Synthetic urine samples with different concentrations of standards at low,medium and high concentrations were tested in 3 run batches,and 5 parallel samples were set for each batch at each concentration level to investigate the accuracy and precision of the method.At the same time,the selectivity,recovery,matrix effect,stability of the method were systematically validated according to existing guidelines.3.Verify the practicability and reliability of the method:The urine levels of amino acids and organic acids were detected in 28 healthy children and 3 children of cbl C type methylmalonic acidemia(methylmalonic acidemia combined with homocysteinemia)and corrected by creatinine to further verify the practicality and reliability of the method.4.The establishment of reference intervals:Urine of 388 healthy children aged 1-7years from Beijing area was collected.The levels of amino acids and organic acids in urine were detected by the new method,and the concentration were corrected by creatinine.Excel 2016 and SPSS 26.0 software were used for data statistical analysis and reference interval calculation.Firstly,the Dixon method was used to initially eliminate the outliers,and then the groups were initially grouped according to the physiological development stage.The Harris&Boyd method was used to confirm the groups,and the Tukey test was used to further eliminate the outliers of each group after confirming the grouping.Finally,95%reference intervals of each group were calculated using non-parametric method and 90%confidence intervals of upper and lower limits were calculated using Bootstrap.Another 40 healthy children at the same age were collected for reference interval validation.Results:1.The method established in this study can simultaneously quantify the levels of various amino acids and organic acids in urine,and the run time was 8 minutes,with good selectivity.The accuracy of RE<±15%,intra-day and inter-day precision RSD<15%.The lower limit of quantification for 19 compounds ranged from0.01μmol/L to 1.00μmol/L,and the calibration curves were linear,R~2>0.990.No obvious matrix effect was found except arginine,and recoveries ranged from 80.20%to114.97%.The samples were stable after 8h at room temperature and 3 freeze-thaw cycles.2.The measured values of 28 healthy children were consistent with previous studies,and the levels of MMA,MCA and HCY in 3 children of cbl C type methylmalonic acidemia were 3.99-93.52 times higher than the median level of healthy children.3.The reference intervals of most amino acids and organic acids in urine of children aged 1-7 in Beijing were basically consistent with those in foreign countries,and the upper limits of HCY,MMA and TYR reference intervals were slightly higher than those in foreign countries.The results showed that there were no gender differences in urine amino acids and organic acids between 1 and 7 years old,and the levels of ALA,LEU,MET,PHE,PRO,SER,TRP,TYR,VAL and MCA in early childhood(1 to 3 years old)were higher than those in preschool age(>3～7 years old).There was no difference in other amino acids and organic acids in early childhood and preschool age.Conclusion:In this study,a new LC-MS/MS method was developed for the simultaneous quantification of 17 amino acids(HCY、MET、CYSTA、ALA、ARG、CIT、GLY、HIS、ILE、LEU、PHE、PRO、SER、THR、TRP、TYR、VAL)and 2 organic acids(MMA and MCA)in random midstream urine.The method has the advantages of small sample consumption,low cost,short analysis time,high sensitivity and good specificity,and can accurately reflect the level of components to be measured in urine of children,providing a useful tool for the study of amino acid metabolism disorders.The reference intervals of urine amino acids and organic acids in healthy children aged from1 to 7 years in Beijing were established by this method,which provided the basis for clinical diagnosis,treatment and research.