High Performance Liquid Chromatographic Determination Of α-keto Acids From A Pharmaceutical Preparation (Tablet) Using 4-Nitro-1, 2-Phenylene Diamine As A Derivatizing Reagent

A rapid, sensitive and selective HPLC separation with photodiode array detector was developed for the determination of the αketo acids from pharmaceutical tablet. α-keto acids (1) 3-methyl 2-oxobutyric acid (MKBA), (2) 3-methyl 2-oxovaleric acid (K3MVA), (3) 4-methyl-2-oxovaleric acid (K4MVA) and (4) Phenyl pyruvic acid (PPY) as derivatives with 4-dinitro 1,2- phenylenediamine (NPD) eluted and separated from HPLC column Zorbax C–18 and the UV detection was carried out by photodiode array at 255 nm at pH 3. The isocratic elution was with methanol-water-acetonitrile (42:56:2V/V/V) with flow rate 0.9 ml/min. The standard curves were linear from 0.45-100 µg/ml and detection limits were within 0.1-0.266 µg/ml of each acid. The elution and separation of four keto acids was rapid within 10 min. The method was repeatable with relative standard deviation (RSD) within 0.1-2.9% for each of the α-keto acids. The method was applied for determination of α-keto acids from a pharmaceutical preparation. The result was further confirmed by standard addition technique.