Keto acids, DNPHs

The dinitrophenylhydrazine (DNPH) assay detects a-keto acids in the urine [1, 4]. 

Substances with low solubility in acid can interfere with the DNPH assay. Mandel-amine (methenamine man delate), an antibacterial medication, and radiopaque contrast material will form a precipitate immediately upon addition of the DNPH reagent. The color and immediacy of formation of this precipitate distinguishes it from the yellowish precipitate of a-keto acid hydrazones. It is very important to have information on patient medications prior to evaluation of the DNPH test. 

Separation of a number of keto acid hydrazones may be accomplished as their free hydrazones [37], as sodium salts [38] or as ammonium salts [39]. For TLC separation of the sodium salts a plate (20 X 20 cm) is coated with a 0.25-mm layer of a mixture of silica gel and 0.1 N sodium bicarbonate (1 2 w/v). The plate is activated by heating at 110 °C for 40 min and is then cooled and kept in a desiccator until required. The solvent systems are ethyl acetate (saturated with 0.1 N sodium bicarbonate)-methanol (5 1) and butanol-ethanol-0.1 N sodium bicarbonate (10 3 10) (upper layer). The plates are developed for 2.5 h at room temperature. For quantitation, the spots may be removed from the plate and dissolved in 2.07V sodium hydroxide for color development and determination in solution. Treatment of the plate directly with base (as a spray) should also be possible for quantitation in situ. The wavelengths of the absorption maxima of a number of DNPH-keto acids in aqueous base are listed in Table 4.6... 

Dinitrophenylhydrazones (DNPHs) were applied to the GC analysis of keto acids. As with carbonyl compounds, they are prepared by reaction with 2,4-dinitrophenylhydrazine and are also used mainly for the preliminary isolation of keto acids. They can be isolated from a dilute aqueous sample by adsorption on activated carbon and selective desorption [178] hydrazones of aldehydes with a methyl formate-dichloromethane mixture and hydrazones of keto acids with a pyridine-water azeotropic mixture. Hydrazones of acids are released from their pyridine salts with methanol containing hydrogen chloride. After... 

Various keto acids in tobacco seeds were identified from their 2,4-DNPH derivatives. 

Examination of 2,4-DNPH derivatives from tobacco and smoke revealed presence of several keto acids. 

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