The fluorometric determination of malic acid.

A sensitive and specific chemical method for the estimation of malic acid in biological materials has long been desired. The condensation of malic acid with orcinol in the presence of concentrated sulfuric acid to form the highly fluorescent homoumbelliferone (7-hydroxyd-methylcoumarin) was reported years ago (1). Although this reaction is relatively specific for malic acid, the quantitative precipitation of calcium malate by alcohol as a means of fractionation from biological materials gives it additional specificity. If, in conjunction with this treatment, a small amount of 2,4-dinitrophenylhydrazine is added, most of the simple carbohydrate impurities which give an interfering amber color with orcinol become soluble in alcohol and thus may be removed.