Metabolite profiling methods are important tools for measurement of metabolite pools in biological systems. While most metabolite profiling methods report relative intensities or depend on a few internal standards representing all metabolites, the ultimate requirement for a quantitative description of the metabolite pool in biological cells and fluids is absolute concentration determination. We report here a high-throughput and sensitive gas chromatography/tandem mass spectrometry (GC/MS/MS) targeted metabolite profiling method enabling absolute quantification of all detected metabolites. The method is based on methyl chloroformate derivatization and quantification by spiking samples with metabolite standards separately derivatized with deuterated derivatization reagents. The traditional electron impact ionization is replaced with positive chemical ionization since the latter to a much larger extent preserve the molecular ion and other high molecular weight fragments. This made it easier to select unique MS/MS transitions among the many coeluting metabolites. Currently, the novel GC/MS/MS method comprises 67 common primary metabolites of which most belong to the groups of amino and nonamino organic acids. We show the applicability of the method on urine and serum samples. The method is a significant improvement of present methodology for quantitative GC/MS metabolite profiling of amino acids and nonamino organic acids.
