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...
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National Centre of Excellence in Analytical Chemsitry
2013-06-01
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doaj-107465e288ae4e26acf885d90828e3582020-11-24T22:40:43ZengNational Centre of Excellence in Analytical ChemsitryPakistan Journal of Analytical & Environmental Chemistry1996-918X2221-52552013-06-0114011625HPLC Determination of α-keto Acids in Human Serum and Urine after Derivatization with 4-Nitro-1,2-phenylenediamineShamroz Bano SahitoAbdul Hameed ChannerGulam Murtaza MastoiMuhammad Yar KhuhawarKhalida Parveen MaharKulsoom Ubedullah AbbasiAbdul Jabbar KandhroThe 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.http://www.ceacsu.edu.pk/PDF%20file/Volume%2014%20No%201/16-25-PJAEC-02052013-013.pdfα-Keto acids4-nitro-12-phenylenediamineHPLCSerumUrine. |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Shamroz Bano Sahito Abdul Hameed Channer Gulam Murtaza Mastoi Muhammad Yar Khuhawar Khalida Parveen Mahar Kulsoom Ubedullah Abbasi Abdul Jabbar Kandhro |
spellingShingle |
Shamroz Bano Sahito Abdul Hameed Channer Gulam Murtaza Mastoi Muhammad Yar Khuhawar Khalida Parveen Mahar Kulsoom Ubedullah Abbasi Abdul Jabbar Kandhro 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 α-Keto acids 4-nitro-1 2-phenylenediamine HPLC Serum Urine. |
author_facet |
Shamroz Bano Sahito Abdul Hameed Channer Gulam Murtaza Mastoi Muhammad Yar Khuhawar Khalida Parveen Mahar Kulsoom Ubedullah Abbasi Abdul Jabbar Kandhro |
author_sort |
Shamroz Bano Sahito |
title |
HPLC Determination of α-keto Acids in Human Serum and Urine after Derivatization with 4-Nitro-1,2-phenylenediamine |
title_short |
HPLC Determination of α-keto Acids in Human Serum and Urine after Derivatization with 4-Nitro-1,2-phenylenediamine |
title_full |
HPLC Determination of α-keto Acids in Human Serum and Urine after Derivatization with 4-Nitro-1,2-phenylenediamine |
title_fullStr |
HPLC Determination of α-keto Acids in Human Serum and Urine after Derivatization with 4-Nitro-1,2-phenylenediamine |
title_full_unstemmed |
HPLC Determination of α-keto Acids in Human Serum and Urine after Derivatization with 4-Nitro-1,2-phenylenediamine |
title_sort |
hplc determination of α-keto acids in human serum and urine after derivatization with 4-nitro-1,2-phenylenediamine |
publisher |
National Centre of Excellence in Analytical Chemsitry |
series |
Pakistan Journal of Analytical & Environmental Chemistry |
issn |
1996-918X 2221-5255 |
publishDate |
2013-06-01 |
description |
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. |
topic |
α-Keto acids 4-nitro-1 2-phenylenediamine HPLC Serum Urine. |
url |
http://www.ceacsu.edu.pk/PDF%20file/Volume%2014%20No%201/16-25-PJAEC-02052013-013.pdf |
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