Study Of The Application Of The GC-MS Quantitative Method In Diagnosis Of Inborn Error Of Metabolism

Objective1. To establish a quantitative method for measurement of urinary abnormal metabolites by gas chromatography-mass spectrometry(GC-MS);2. To examine identification and quantitation of urinary metabolites by different pre-treatment methods. ]Methods1. To quantify 12 urinary organic acids, which is key diagnostic metabolites by GC-MS2. To establish calibration curves.Quantification is by comparison to calibration of pure standard compounds in ratio to an internal standard.3. Validation data, including detection limit, accuracy, precision, recovery have been studied and linearity has been evaluated for 12 organic acids.4. Measurement of 12 urinary organic acids in normal children.5. 49 positive urine specimens including organic aciduria, amino acid metabolism disorder, NICCD patient and 9 positive controls from ERNDIM were prepared prior to GC/MS analysis.6. To analyse all urine by organic solvent extraction and urease-pretreatment prior to GC-MS.7. To identify each compound in the urine by comparing with the standard spectrum in the NIST library.8. To quantify 12 urinary organic acids by different pre-treatment method and to analyse detection limit, accuracy, precision, recovery, etc.9. To compare urease pretreatment and organic solvent extraction for analyzing indentified metabolites qualitatively and quantitatively by t-test, if P<0.05, there is statistical significance.Results1. After urease pre-treatment, derivatization, the retention time of 3-hydroxypropionic acid, methylmalonic acid, 2-hydroxyisocaproic acid, uracil, glutaric acid, adipic acid, 2-hydroxyphenylacetic acid, 3-hydroxy-3-methylglutaric acid, suberic acid, phenypyruvic acid, orotic acid, sebacic acid is 8.23min, 9.75min, 10.18min, 12.07min, 13.15min, 14.79min, 15.78min, 16.35min, 17.60min, 17.79min, 18.38min, 20.16min.Except phenypyruvic acid, correlation coefficients of all the other organic acids are 0.981-0.999, intra- and inter-assay imprecisions are less than 15%, recoveries range from 85%-111%. After the extraction and derivatization, correlation coefficient of pheypyruvic acid is 0.998, intra- and inter-assay imprecision is less than 15%, recovery ranges from 92%-104%, which to fit the bioannalytical method validation.2. Hexanoylglycine and phenypropionylglycine can't be detected by GC-MS prior to urease pretreatment. Galactose, galactitol, galactonic acid and amino acid can't be detected by organic solvent extraction.3. After urease pretreatment or organic solvent extraction, the intra- assay imprecision of methylmalonic acid, glutaric acid, adipic acid, suberic acid, sebacic acid, 2-hydroxyphenylactic acid range from 3%~`10%, inter-assay imprecision is 9%~15%, recovery is 90%~105%, there is no statistical significance.4. After urease pretreatment, intra-assay imprecision of 3-hydroxypropionic acid, uracil acid, orotic acid and 3-hydroxy-3-methylglutaric acid range from 2%~13%, inter-assay imprecisions below 15%, recoveries range from 90%~105%; After organic solvent extraction, intra- and inter- assay imprecisions are above 15%, and recoveries are below 90% or above 105%, there is statistical significance.Conclusions1. Establish a sensitive and accurate method of quantify 12 urinary organic acids, which is key diagnostic metabolites by GC-MS.2. Hexanoylglycine and phenypropionylglycine can't be detected by GC-MS prior to urease pretreatment. Fatty acid disorder can't be diagnosed by urease pretreatment.3. To quantify the polar organic acids, for example 3-hydroxypropionic acid, orotic acid, uracil acid, etc. there is statistical significance between urease pre-treatment and organic solvent extraction. After urease pre-treatment, there is a better liner correlation, imprecision and recovery.