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

  • Khalida Parveen Mahar Shah Abdul Latif University Khairpur Pakistan
  • Kulsiim Ubedullah Abbasi Institute of Advanced Research Studies in Chemical Sciences, University of Sindh, Jamshoro, Pakistan
  • Mohammad Yaar Khuhawar Institute of Advanced Research Studies in Chemical Sciences, University of Sindh, Jamshoro, Pakistan
  • Gulam Murtaza Mastoi Institute of Advanced Research Studies in Chemical Sciences, University of Sindh, Jamshoro, Pakistan
  • Adbul Hmeed Channer Institute of Advanced Research Studies in Chemical Sciences, University of Sindh, Jamshoro, Pakistan
  • Abdul Jabbar Kandhro Institute of Advanced Research Studies in Chemical Sciences, University of Sindh, Jamshoro, Pakistan
  • Shamroz Bano sahito Shah Abdul Latif University Khairpur Pakistan

Abstract

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-1,2phenylenediamine (NPD). Nine α-keto acids: glyoxylic acid (GA), pyruvic acid (PYR), 2oxobutyric 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.

Published
Dec 2, 2013
How to Cite
MAHAR, Khalida Parveen et al. HPLC Determination of α-keto Acids in Human Serum and Urine after Derivatization with 4-Nitro-1,2-phenylenediamine. Pakistan Journal of Analytical & Environmental Chemistry, [S.l.], v. 14, n. 1, p. 10, dec. 2013. ISSN 2221-5255. Available at: <http://pjaec.pk/index.php/pjaec/article/view/231>. Date accessed: 17 dec. 2017.