Determination of marker pteridins and biopterin reduced forms, tetrahydrobiopterin and dihydrobiopterin, in human urine, using a post-column photoinduced fluorescence liquid chromatographic derivatization method |
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Authors: | Florentina Cañ ada-Cañ ada,Anunciació n Espinosa-Mansilla,Arsenio Muñ oz de la Peñ a,Alicia Mancha de Llanos |
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Affiliation: | Department of Analytical Chemistry, University of Extremadura, 06071 Badajoz, Spain |
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Abstract: | A liquid chromatographic method for the simultaneous analysis of marker pteridins and biopterin reduced forms, in urine samples is proposed. A Zorbax Eclipse XDB-C18 column was used for the chromatographic separation, using a 98/2 (v/v), citrate buffer (pH 5.5)-acetonitrile mobile phase, in isocratic mode. A post-column photoderivatization was carried out with an on-line photoreactor, located between a diode array detector (DAD) and a fast scanning fluorescence detector (FSFD). Neopterin (NEO), biopterin (BIO), pterin (PT) and dihydrobiopterin (BH2) were determined by measuring native fluorescence, using the photoreactor in OFF-mode, and tetrahydrobiopterin (BH4) was determined by measuring of the induced fluorescence of the generated photoproducts, using the photoreactor in ON-mode. In addition, Creatinine (CREA), as a reference of metabolites excrection in urine, was simultaneously determined using the DAD detector. Detection limits were 0.2, 13.0, 0.3, 0.3 and 3.5 ng mL−1, for NEO, BH2, BIO, PT and BH4, respectively, and 0.4 μg mL−1 for CREA. Ratio values for NEO/CREA, PT/CREA, BH4/CREA, BH2/CREA, NEO/BIO and BIOtotal/CREA, in urine samples, of healthy children and adults, phenylketonuric children and infected mononucleosis children, are reported. A comparative study, about the mean values obtained for each of the compounds, by the present procedure and by the classical iodine oxidation method (Fukushimás method), has been performed, in urine samples belonging to healthy volunteers. The values obtained were BH4/CREA: 0.41, BH2/CREA: 0.31 and BIOtotal/CREA: 0.73, by the proposed method, and BH4/CREA: 0.35, BH2/CREA: 0.20 and BIOtotal/CREA: 0.48, by iodine oxidation method. |
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Keywords: | BH4, tetrahydrobiopterin GTP, guanosine triphosphate PAH, phenylalanine-4-hydroxylase TH, tyrosine-3-hydroxylase TPH, tryptophan-5-hydroxylase BH2, dihydrobiopterin DHPR, dihydropteridine reductase NEO, neopterin BIO, biopterin ISO, isoxanthopterin PT, pterin HPA, hyperphenylalaninaemia PKU, phenylketonuria 6-PTS, 6-pyruvoyltetrahydropterin synthase DTE, dithioerythritol DTT, dithiothreitol DAD, diode array detector FSFD, fast scanning fluorescence detector CREA, creatinine HPAs, hyperphenylalaninaemias |
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