Quantitation of 2-ketoacids in biological fluids by gas chromatography chemical ionization mass spectrometry of O-trimethylsilyl-quinoxalinol derivatives.

A very sensitive and specific method of 2-ketoacid determination in various biological fluids using gas chromatography chemical ionization mass spectrometry of O-trimethylsilyl-quinoxalinol derivatives is described. After derivatization with o-phenylenediamine in acidic medium and extraction, the 2-ketoacids, and the 2-ketovaleric acid used as internal standard, are silylated and resolved on a capillary column and their concentrations automatically determined by monitoring the m/z values corresponding to their respective protonated molecular ions, using ammonia as reactant gas. The detection limit is below 50 ng ml-1 of each 2-ketoacid. The recoveries of 2-ketoacids from urine and plasma were between 97 and 104%. Problems encountered with interfering substances were tested for and discussed. This method has been applied successfully to various metabolic disorders characterized by an accumulation of aliphatic 2-ketoacids. The metabolic interrelationship of branched chain 2-ketoacids and other compounds, especially pyruvate, is discussed.