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Determination of selected α-keto acids in dried blood samples using HPLC with fluorescence detection
R. Kanďár, M. Kopčil, L. Laštovičková
Language English Country Great Britain
Document type Journal Article
- MeSH
- Amino Acids * MeSH
- Indicators and Reagents MeSH
- Keto Acids * MeSH
- Chromatography, High Pressure Liquid methods MeSH
- Publication type
- Journal Article MeSH
The determination of α-keto acids derived from amino acids is currently the most reliable approach for the diagnosis of some congenital metabolic diseases. An HPLC method for the simultaneous measurement of selected α-keto acids in dried blood samples has been developed and evaluated. Blood spot samples from a group of healthy blood donors were collected onto #903 Specimen Collection Paper. Prior the separation, the α-keto acids were derivatized with 1,2-diamino-4,5-dimethoxybenzene to the corresponding 3-substituted-6,7-dimethoxy-2(1 H)-quinoxalinol derivatives. For the separation, a reverse-phase column LichroCart 125-4, Purospher RP-18e, 5 μm, was used. The mixture of 25% ACN in deionized water (mobile phase A) and 100% ACN (mobile phase B) were used for a gradient elution of α-keto acids derivatives. Analytical performance of this method is satisfactory for all α-keto acids. The intra-assay and inter-assay coefficients were below 10% and recoveries were close to 100%. We have developed relatively simple, rapid, selective and sufficiently sensitive HPLC method with fluorescence detection for the determination of selected α-keto acids in dried blood samples. The presented method is suitable for clinical testing purposes.
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- $a The determination of α-keto acids derived from amino acids is currently the most reliable approach for the diagnosis of some congenital metabolic diseases. An HPLC method for the simultaneous measurement of selected α-keto acids in dried blood samples has been developed and evaluated. Blood spot samples from a group of healthy blood donors were collected onto #903 Specimen Collection Paper. Prior the separation, the α-keto acids were derivatized with 1,2-diamino-4,5-dimethoxybenzene to the corresponding 3-substituted-6,7-dimethoxy-2(1 H)-quinoxalinol derivatives. For the separation, a reverse-phase column LichroCart 125-4, Purospher RP-18e, 5 μm, was used. The mixture of 25% ACN in deionized water (mobile phase A) and 100% ACN (mobile phase B) were used for a gradient elution of α-keto acids derivatives. Analytical performance of this method is satisfactory for all α-keto acids. The intra-assay and inter-assay coefficients were below 10% and recoveries were close to 100%. We have developed relatively simple, rapid, selective and sufficiently sensitive HPLC method with fluorescence detection for the determination of selected α-keto acids in dried blood samples. The presented method is suitable for clinical testing purposes.
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