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Separation and determination of 11 marker pteridines in human urine by liquid chromatography and fluorimetric detection
Author(s) -
de Llanos Alicia M.,
EspinosaMansilla Anunciación,
CañadaCañada Florentina,
de la Peña Arsenio M.
Publication year - 2011
Publication title -
journal of separation science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.72
H-Index - 102
eISSN - 1615-9314
pISSN - 1615-9306
DOI - 10.1002/jssc.201000900
Subject(s) - chemistry , chromatography , detection limit , urine , analyte , buffer solution , column chromatography , iodide , tris , analytical chemistry (journal) , inorganic chemistry , biochemistry
A simple liquid chromatographic method has been developed to achieve the complete separation and determination of a wide range of pteridinic compounds and creatinine (CREA) in urine samples, in just one run. The influences of mobile phase composition and buffer pH have been studied. The optimized mobile phase was composed of a Tris‐HCl buffer (15 mmol/L) at pH 6.10 solution (eluent A) and a Tris‐HCl buffer (15 mmol/L) at pH 6.40 solution (eluent B), in gradient mode. Analytes were determined by fluorimetric detection, exciting at 272 nm, and measuring the fluorescence emission at three wavelengths, 410, 445 and 465 nm. CREA, as a reference of metabolites excretion in urine, was determined by photometric detection at 230 nm. Pteridines detection limits varied from 0.2 to 6.1 ng/mL, and 0.2 g/mL for CREA. Calculated precision values expressed as RSD (%) varied from 1.1 to 5.9. Two different oxidation procedures for urine samples were optimized. The neopterin/biopterin ratios found were 0.98 and 0.86 for adults and children, respectively, by means of the alkaline iodide/iodine oxidation and 0.45 and 0.57 using neutral KMnO 4 oxidation.