Development and validation of a liquid chromatographic method for the determination of hydroxymethylfurfural and alpha-ketoglutaric acid in human plasma |
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Authors: | Michail K Juan H Maier A Matzi V Greilberger J Wintersteiger R |
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Affiliation: | a Institute of Pharmaceutical Sciences, University of Graz, Austria b Faculty of Pharmacy, University of Alexandria, Egypt c Department of Biomedical Research, Medical University of Graz, Austria d Department of Thoracic and Hyperbaric Surgery, Medical University of Graz, Austria e Institute of Physiological Chemistry, Medical University of Graz, Austria |
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Abstract: | Hydroxymethylfurfural (HMF) and alpha-ketoglutaric acid (KG) have been recently investigated as potential cancer cell damaging agents. We herein report for the first time a validated quantitative assay for their simultaneous determination in human plasma which is amenable to be applied in the future screening of the target compounds in human probands in order to properly design a targeted chemotherapeutic regimen for certain types of malignant tumors.A simple liquid chromatographic method in conjunction to derivatization after a two-step optimized solid phase clean-up procedure is described. The method is based on the reaction of HMF and KG with 2-nitrophenylhydrazine or 2,4-dinitrophenylhydrazine in an aqueous environment. Reaction conditions were studied with respect to pH, reagent volume, reaction temperature and time. Exact testing of such parameters beside careful selection of the mobile phase composition rendered feasible the quantification of the chemically significantly differing analytes along a single chromatographic run. The formed derivatives could be separated isocratically by reversed-phase LC on a C8-column. Detection in the UV and in the visible range is possible. Results showed good recovery and reproducibility with detection limits (S/N = 3) down to 2 picomoles analyte on column. Resolution of the syn and anti geometric isomers of the HMF and KG derivatives is possible. The isomeric ratio in relation to the reaction pH is discussed. |
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Keywords: | High-performance liquid chromatography (HPLC) Derivatization Hydroxymethylfurfural and alpha-ketoglutaric acid 2-Nitrophenylhydrazine 2,4-Dinitrophenylhydrazine Plasma |
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