HPLC Determination of α-keto Acids from Human Serum Using 2,3-Diamino-2,3-dimethylbutane as Derivatizing Reagent

Khalida Parveen Mahar; Muhammad Yar Khuhawar; Kulsoom Ubedullah Abbasi; Gulam Qadir Shar; Rafia Azmat; Jameel Ahmed Baig

doi:10.21743/

Authors

Khalida Parveen Mahar Department of Chemistry, Faculty of Science, Shah Abdul Latif University Khairpur, Pakistan Author
Muhammad Yar Khuhawar Institute of Advanced Research Studies in Chemical Science, University of Sindh, Jamshoro, Pakistan Author
Kulsoom Ubedullah Abbasi Institute of Advanced Research Studies in Chemical Science, University of Sindh, Jamshoro, Pakistan Author
Gulam Qadir Shar Department of Chemistry, Faculty of Science, Shah Abdul Latif University Khairpur, Pakistan Author
Rafia Azmat Department of Chemistry, University of Karachi, Pakistan Author
Jameel Ahmed Baig National Centre of Excellence in Analytical Chemistry, University of Sindh, Jamshoro 76080, Pakistan Author

DOI:

https://doi.org/10.21743/

Keywords:

α-keto acids; 2,3-diamino-2,3-dimethylbutane (DDB); serum; diabetic patients

Abstract

Seven α-keto acids, pyruvic acid (PYR), 2-oxobutyric acid (KB), 3-methyl-2-oxobutyric acid (MKBA), 3-methyl-2-oxovaleric acid (K3MVA), 4-methyl-2-oxovaleric acid (K4MVA), 2-oxoglutaric acid (KG) and Phenyl pyruvic acid (PPY) as derivatives of 2,3-diamino-2,3-dimethybutane (DDB) were separated by HPLC column Zorbax C–18 (4.6x150 mm-id). The compounds were eluted with methanol-water-acetonitrile (40:58:2 V/V/V) with flow rate 1 ml/min. UV detection was carried out by photodiode array at 255 nm. Linear calibration plots were obtained with 0.1 to 60 μg/ml with limits of detection (LoD) within 0.04-0.4 μg/ml. The method was applied for the analysis of α-keto acids from serum of diabetic patients with blood glucose level 430-458 mg/dl and healthy volunteers. The amounts of α-keto acids observed 3.24- 9.71 μg/ml with RSD 1.1-1.9% in diabetic patients were higher than healthy volunteer’s 0.11-1.3 μg/ml with RSD 0.9-2.6%.