HPLC Determination of α-keto Acids in Human Serum and Urine after Derivatization with 4-Nitro-1,2-phenylenediamine

The determination of α-keto acids has clinical importance, because these are intermediates in a number of biochemical processes. This work reports the development of an HPLC procedure for the analysis α-keto acids in blood and urine samples after derivatization with 4-nitro-1,2-phenylenediamine (NPD). Nine α-keto acids: glyoxylic acid (GA), pyruvic acid (PYR), 2-oxobutyric acid (KB), 3-methyl-2-oxobutyric acid (MKBA), 3-methyl-2-oxovaleric acid (K3MVA), 2-oxoglutaric acid (KG), 4-methyl-2-oxovaleric acid (K4MVA), 2-oxohexanoic acid (KHA) and phenylpyruvic acid (PPY) were derivatized with (NPD) at pH 3 and separated on a Zorbax 300 SB-C18 HPLC column (4.6x150mm id) and photodiode array detection at 255 nm. The isocratic elution was performed with methanol: water: acetonitrile (42: 56:2, v/ v/ v) with a flow rate 0.9 mL/min. The keto acids separated within 14 min. The method was repeatable with a relative standard deviation (RSD) of 0.1-2.9% for each of the α-keto acids. The limits of detection and quantitation were obtained within the range 0.05-0.26 µg/ mL and 0.15-0.8 µg/ mL respectively. The method was applied for determination of α-keto acids from a pharmaceutical preparation, human serum and urine samples of healthy volunteers and diabetic patients. The results were further confirmed by standard addition technique. The method is rapid and simple and is suitable for the separation and determination of α-keto acids from clinical samples