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 (greater than 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.
Garibotto
G
G
Department of Internal Medicine, University of Genova, Italy.
Ancarani
P
P
Russo
R
R
Sala
M R
MR
Fiorini
F
F
Paoletti
E
E
D001692:Biological Transport; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D006835:Hydrazones / Q000737:chemistry*; D007536:Isomerism; D007651:Keto Acids / Q000097:blood*; D013056:Spectrophotometry, Ultraviolet; D013696:Temperature
D006835:Hydrazones; D007651:Keto Acids