Determination of selected α-keto acids in dried blood samples using HPLC with fluorescence detection

• Published in: Journal of pharmaceutical and biomedical analysis 2022-05, Vol.214, p.114738-114738, Article 114738
• Main Authors: Kanďár, Roman, Kopčil, Michal, Laštovičková, Lenka
• Format: Article
• Language: English
• Subjects: 1,2-Diamino-4,5-dimethoxybenzene; Dried blood spot; HPLC with fluorescence detection; α-Keto acids
• ISSN: 0731-7085; 1873-264X
• DOI: 10.1016/j.jpba.2022.114738

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. •The first reported HPLC-FLD method capable of quantifying phenylpyruvate in dried blood spot samples.•The limit of detection values are in fmol.•Method is suitable for screening examination.•The method only required about 20 μL of blood for accurate analysis.