A-316 A quantitative gas chromatography tandem mass spectrometry method for metabolic profiling of urine organic acids and acylglycines

Abstract Background The analysis of urine organic acids (UOA) and acylglycines (UAG) are an essential investigation for the diagnosis inborn errors of metabolism (IEMs). The vast majority of methods for these analytes use gas chromatography mass spectrometry (GC-MS) and are only qualitative. Gas chromatography tandem mass spectrometry (GC-MS/MS) offers greater sensitivity and selectivity and enables the combination of qualitative screening with quantitative analysis. We describe the development and validation results of a quantitative GC-MS/MS method. Methods Urine creatinine concentrations were measured (CobasPro, Roche Diagnostics) and normalized to 1.25 mmol/L. After internal standard addition, each urine specimen, calibration standard and QC were incubated with a hydroxylamine solution followed by a liquid-liquid extraction. Further sample concentration permitted the isolated UOA and UAG to be derivatized using N,O-bis(trimethylsilyl)trifluoroacetamine (BSTFA) with 1% chlorotrimethylsilane (TMCS) prior to injection. GC-MS/MS analysis was performed using a GC-MS-TQ8050 triple quadrupole system (Shimadzu) with a 30 m DB-5MS (Agilent J&W) capillary column (0.25 mm, ID of 0.25 µm). Split injection mode was deployed with an initial column temperature of 70°C. The mass spectrometer was operated in electron ionization (EI) and multiple reaction monitoring (MRM) modes. Results The optimal temperature gradient for high-resolution UOA and UAG separations was derived and their respective retention times determined. MS/MS detection parameters were optimized to permit the identification of precursor and production ions for all (N=36) derivatized analytes. Total analysis time was 33.8 min per injection. This method permits the measurement of 11 samples in one batch and is highly linear for all analytes (R^2 >0.99 and average slope 1.04). The limit of quantification (LOQ) was established and ranged from <0.05 to 2.1 µmol/mmol creatinine. Intra- and inter-day imprecision (CV%) was determined using matrix-matched abnormal and normal QC. Intra-day imprecision (N=10) ranged from 1.3% (2-ketoglutaric acid) to 9.7% (3-hydroxybutyric acid). Inter-day imprecision (N=30 over 15 days) ranged from 2.2% (ethylmalonic acid and methylmalonic acid) to 20.5% (3-hydroxy-3-methylglutaric acid). The method had an average accuracy of 113±14% for analytes included in the ERNDiM quantitative organic acids (urine) external quality assurance program. For analytes not included in this proficiency testing, the method showed good agreement when compared to a national reference laboratory, with average Deming correlation coefficients and regression slopes being 0.9944 and 1.1, respectively. No interferences were detected when monitoring the analyte quantifier and qualifier ion ratios in patient urine. Conclusions The devised GC-MS/MS protocol permits the unbiased identification and quantification of UOA and UAG used to investigate IEMs. The method has acceptable sensitivity, specificity, precision and accuracy for clinical routine clinical testing.