B-201 Development of a Quantitative Gas Chromatography Tandem Mass Spectrometry Method for Metabolic Profiling of Urine Organic Acids

Abstract Background Urine organic acids (UOA) analysis is an essential investigation for inborn errors of metabolism (IEMs). The vast majority methods use gas chromatography mass spectrometry (GC-MS) and are only qualitative. Gas tandem (GC-MS/MS) offers greater sensitivity selectivity, enables the combination qualitative screening with quantitative analysis. We describe development preliminary validation results a GC-MS/MS method. Methods creatinine concentrations were measured (CobasPro, Roche Diagnostics) normalized to 1.25 mmol/L. After internal standard addition, each urine specimen, calibration QC incubated hydroxylamine solution followed by liquid-liquid extraction. Further sample concentration permitted isolated UOA be derivatized using N,O-bis(trimethylsilyl)trifluoroacetamine (BSTFA) 1% chlorotrimethylsilane (TMCS) prior injection. was performed GC-MS-TQ8050 triple quadrupole system (Shimadzu) 30 m DB-5MS (Agilent J&W) capillary column (0.25 mm, ID 0.25 µm). Split injection mode deployed initial temperature 70°C. spectrometer operated in electron ionization (EI) multiple reaction monitoring (MRM) modes. linear ranges targeted metabolites within devised protocol determined. Results optimal gradient high-resolution separations derived their respective retention times MS/MS detection parameters optimized permit identification precursor production ions all (N = 38) UOA. Total time 33.8 min per achieved metabolite respectively Linear correlation coefficients representative UOA: 3-hydroxyisovaleric acid (range 9 708 µmol/L, R2 0.9874); lactic 15 751 0.9936); 3-hydroxybutyric 0.9991); 2-methyl-3-hydroxybutyric 10 501 0.9996). remaining ranged from 0.9458 (4-hydroxyphenylpyruvic acid) 0.9998 (3-methylglutanoic ethylmalonic acid). Conclusion permits unbiased quantification used investigate IEMs. analytes determined appropriate these markers inherited enzyme deficiencies. analytical performance this method must further validated its diagnostic selected IEMs characterized.