4,5-Diaminophthalhy drazide as a highly sensitive chemiluminescence reagent for α-keto acids in liquid chromatography

4,5-Diaminophthalhydrazide dihydrochloride is studied as a highly sensitive and selective chemiluminescence derivatization reagent for α-keto acids in liquid chromatography (LC). The reagent reacts selectively with α-keto acids in dilute hydrochloric acid to give derivatives which produce chemiluminescence by reaction with hydrogen peroxide and potassium hexacyanoferrate(III). The derivatives in the reaction mixture of eight biologically important α-keto acids are separated within 50 min by reversed-phase LC with isocratic elution, followed by chemiluminescence detection. The detection limits for the acids are in the range 4–50 fmol for a 20-μl injection.