Branched-chain alpha-keto acid analysis in biological fluids: preparative clean-up by anion-exchange and analysis by...

A method is given for the quantitative analysis of the alpha-keto derivatives of the branched-chain amino acids in physiological fluids. A sample containing alpha-ketovalerate and alpha-ketocaproate as internal standards is passed through a weak anion-exchange resin at neutral pH. After washing the resin with distilled water, the alpha-keto acids are eluted with 4 M hydrochloric acid--ethanol (50:50). Quinoxalinol derivatives are prepared directly in the eluent, extracted with methylene chloride, and trimethylsilylated. Separation of the derivatives is by capillary gas chromatography on a 30 m fused-silica SE-30 column. Chromatographic separation is superior to that reported for packed column methods, thereby permitting the use of alpha-ketovalerate and alpha-ketocaproate as internal standards.