Unified Gas Chromatographic-mass Spectrometric Method for Quantitating Tyrosine Metabolites in Urine and Plasma

Tyrosine and many of its catabolites play significant roles in the in the toxicity associated with acquired and congenital forms of hypertyrosinemia. We now report a specific and sensitive GC/MS method for the simultaneous determination of tyrosine metabolites maleylacetone (MA), fumarylacetone (FA), succinylacetone (SA), fumarate and acetoacetate in urine and plasma. Tyrosine metabolites and an internal standard, 2-oxohexanoic acid (OHA), in urine or plasma samples were derivatized to their methyl esters with a 12% boron trifluoride-methanol complex (12%BF3-MeOH). The reaction mixture was extracted with methylene chloride and analyzed by GC/MS, using a selected ion monitoring (SIM) mode. The detection limits were in the range of 0.08-0.4 ng and the quantitation limits were 0.2-2 ng. Most of the intraday and interday coefficients of variation for three concentrations (low, medium and high) of the analytes were below 10%. Sensitivity and selectivity are superior to existing HPLC or enzymatic methods and derivatization of samples is simpler than the traditional silylation of organic acids used for analysis by GC/MS or derivatization to oximes, followed by silylation in the case of the ketoacids, such as SA. Furthermore, the current procedure can be performed in aqueous solution, which results in a high percentage yield without appreciable analyte degradation or formation of side products. Thus far, the method has been successfully applied in the analysis of over 5000 urine and plasma samples from humans and rodents.