Reversed-phase high-performance liquid chromatographic analysis of branched-chain keto acid hydrazone derivatives: optimization of techniques and application to branched-chain keto acid balance studies across the forearm

A sensitive method of quantifying branched-chain keto acids in plasma and whole blood samples is described. It is based on the separation by ion-pair reversed-phase liquid chromatography of 2,4-dinitrophenylhydrazine derivatives with ultraviolet detection. The sample clean-up steps that are usually required for reversed-phase high-performance liquid chromatography are eliminated. A reduction in ketoisocaproate isomer formation is obtained by incubation of derivatives in ice. The method is reproducible (coefficient of variation 2%, n = 5, at the 200-pmol level) and the ultraviolet response is linearly related to branched-chain keto acid concentration. Recoveries are high (>95%). Other keto acids do not co-elute with branched-chain keto acids. Because of its sensitivity and precision, this method can be proposed for whole blood branched-chain keto acid balance studies across organs.