title: Determination of marker pteridins and biopterin reduced forms, tetrahydrobiopterin and dihydrobiopterin, in human urine, using a post-column photoinduced fluorescence liquid chromatographic derivatization method.
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 microg mL(-1) for CREA. Ratio values for NEO/CREA, PT/CREA, BH4/CREA, BH2/CREA, NEO/BIO and BIO(total)/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 (Fukushimas method), has been performed, in urine samples belonging to healthy volunteers. The values obtained were BH4/CREA: 0.41, BH2/CREA: 0.31 and BIO(total)/CREA: 0.73, by the proposed method, and BH4/CREA: 0.35, BH2/CREA: 0.20 and BIO(total)/CREA: 0.48, by iodine oxidation method.
authors.affiliation: Department of Analytical Chemistry, University of Extremadura, 06071 Badajoz, Spain. floricanada@gmail.com
authors.forename: Florentina
authors.initials: F
authors.lastname: Cañada-Cañada
authors.forename: Anunciación
authors.initials: A
authors.lastname: Espinosa-Mansilla
authors.forename: Arsenio
authors.initials: A
authors.lastname: Muñoz de la Peña
authors.forename: Alicia
authors.initials: A
authors.lastname: Mancha de Llanos
mesh_terms: D000328:Adult; D015415:Biomarkers / Q000652:urine; D001708:Biopterins / Q000031:analogs & derivatives* / Q000652:urine*; D002648:Child; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005260:Female; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D007244:Infectious Mononucleosis / Q000652:urine; D008297:Male; D010661:Phenylketonurias / Q000652:urine; D011621:Pteridines / Q000652:urine*
chemical_list: D015415:Biomarkers; D005456:Fluorescent Dyes; D011621:Pteridines; D001708:Biopterins; C017226:7,8-dihydrobiopterin; C003402:sapropterin