Preliminary Study Of The Application Of The Two-step Derivztive Reaction Unite GC-MS Method In Diagnosis Of Phenylktonuria

Objective 1. To preliminary establish the application of the two-step derivatine reaction unite gas chromatography-mass spectrometry method in diagnosis of phenylketonuria through analysis the urine of phenylketonuria and the normal, and compared with the Literature; 2.To assessment the methodology validation of the two-step derivatine reaction; 3. To establish the normal reference range of biomarkers.Methods 1. Collecting urine samples of 60 healthy children(control group) and 20 untreated phenylketonuria(PKU group), after treatment with two step chemical derivative method and analysis by gas chromatography-mass spectrometry, record the concentration difference obvious biomarkers, make qualitative analysis and compared with the Literature; 2. using creatinine as internal standard to set up the standard curve of phenylpyruvic acid, evaluating the precision and accuracy of the two step chemical derivative method; 3. Collecting urine samples of 120 healthy children, analysis the biomarkers,establish the normal reference range of biomarkers;Results 1.After treatment with the two-step derivatine reaction and analysis with GC-MS, we can synchronous detection 14 kinds of biomarkers in the urine of PKU group, That is phenylacetylglutamine, phenylacetic acid, mandelic acid, phenylalanine,o–hydroxyphenylacetic acid, phenyllactic acid, p- hydroxyphenylacetic acid, phenylpyruvic acid, N- acetylphenylalanine, vanilmandelic acid, p-hydroxyphenyllactic acid, p-hydroxyphenylpyruvic acid, 5-hydroxyindolepropionic acid and 5-Hydroxyindoleacetic acid. the Retention Time(RT) of the biomarker is steady;2.Compared with PKU group and control group, we find phenylacetylglutamine, phenylacetic acid, mandelic acid, phenylalanine,o–hydroxyphenylacetic acid, phenyllactic acid, phenylpyruvic acid, N- acetylphenylalanine, vanilmandelic acid, p-hydroxyphenyllactic acid, p-hydroxyphenylpyruvic acid, 5-hydroxyindolepropionic acid and 5-Hydroxyindoleacetic acid has statistical significance in two groups, which can be used as examination indicator, consistent with the literature reported; phydroxyphenylacetic acid was no statistical significance in two groups; 3.After two-step derivatine reaction pretreatment, the standar curve of phenylpyruvic acid is y=0.1042x-0.0147, correlation coefficient is r2=0.996, linear relationship is good. intra-day and inter-day relative standard deviation of three different concentration standard solution are below 5%, recovery is above 85%. Precision and accurate methed is good;4.we can conclude reference range of biomarker by the 120 healthy children(biomarkers/creatinine): phenylacetylglutamine 0, phenylacetic acid 0,mandelic acid 0~0.5387, o–hydroxyphenylacetic acid 0~0.1740,phenyllactic acid0, phenylpyruvic acid 0, N- acetylphenylalanine 0, vanilmandelic acid 0~1.1336, p- hydroxyphenylpyuvic acid 0~0.8574,p-hydroxyphenylpyruvic acid 0~0.0703, 5- hydroxyindolepropionic acid 0, 5-Hydroxyindoleacetic acid 0~0.8415.Conclusion 1. The two-step derivatine reaction can synchronously detect 14 kinds of metabolic substances in PKU urine. Except phydroxyphenylacetic acid, the other 13 kinds of metabolic substances between the two groups show significant difference, consistent with the literature reported, can be used as urinary biomarkers of PKU screening; 3. The method shows small difference in intra-day and inter-day, stable retention time, good precision, high recovery rate, small RSD and high accuracy, conform to all the basic requirements of a biological sample analysing method.The test is non-invasive,with easy specimen collection and preservation, suitable for PKU screening and monitoring,and ready for clinical promotion; 2. we conform the reference range of biomarkers in urine with two-step chemical derivative method combined GC- MS...