Comprehensive two-dimensional gas chromatography (GC x GC) time-of-flight mass spectrometry (ToFMS) was applied to the analysis of urinary organic acids from patients with inborn errors of metabolism. Abnormal profiles were obtained from all five patients studied. Methylmalonic academia and deficiencies of 3-methylcrotonyl-CoA carboxylase and medium chain acyl-CoA dehydrogenase gave diagnostic profiles while deficiencies of very long chain acyl-CoA dehydrogenase and mitochondrial 3-hydroxy-3-methylglutaryl CoA synthase gave profiles with significant increases in dicarboxylic acids suggestive of these disorders. The Superior resolving power of CC x CC with ToFMS detection was useful in separating isomeric organic acids that were not resolved using one-dimensional GC: A novel urinary metabolite, crotonyl glycine, was also discovered in the mitochondrial 3-hydroxy-3-methylglutaryl CoA synthase sample which may be a useful specific diagnostic marker for this disorder. The quantitative aspects of CC x CC were investigated using stable isotope dilution analyses of glutaric, glyceric, orotic, 4-hydroxybutyric acids and 3-methylcrotonylglycine. Correlation coefficients for linear calibrations of the analytes ranged from 0.9805 to 0.9993 (R-2) and analytical recoveries from 77 to 99 . This study illustrates the potential of GC x GC-ToFMS for the diagnosis of organic acidurias and detailed analysis of the complex profiles that are often associated with these disorders. (C) 2009 Elsevier B.V. All rights reserved.