Determination of marker pteridines in urine by HPLC with fluorimetric detection and second-order multivariate calibration using MCR-ALS

MANCHA LLANOS; DE ZAN, MERCEDES; CULZONI M J; ESPINOSA MANSILLA, A; CAÑADA CAÑADA F; MUÑOZ DE LA PEÑA, A; GOICOECHEA, H C

ANALYTICAL AND BIOANALYTICAL CHEMISTRY

SPRINGER HEIDELBERG

Año: 2011 vol. 399 p. 2123 - 2135

1618-2642

A liquid chromatographic method has been developed,in combination with the multivariate curve resolutionalternatingleast squares algorithm (MCR-ALS), for thesimultaneous determination of marker pteridines in urinesamples. A central composite design has been applied tooptimize the factors influencing the separation (buffer concentration,buffer pH, flow rate, oven temperature, mobile-phasecomposition). A set of 15 calibration samples were randomlyprepared, in a concentration range of 0.5?10.5 ngmL−1 forneopterin, biopterin, and pterin; 4.0?8.0 ngmL−1 for xanthopterin;and 0.5?4.5 ngmL−1 for isoxanthopterin. Thevalidation was carried out with fortified urine samples fromhealthy adults. The optimized conditions were a mobile-phasecomposition of 10 mM citric buffer at pH 5.44 and acetonitrile(94.5/5.5, v/v), a flow rate of 1.0 mLmin−1, and an oventemperature of 25 °C. The detection systemconsisted of a fastscanningspectrofluorimeter, which allows obtaining ofsecond-order data matrices containing the fluorescence intensityas a function of retention time and emission wavelength. Inthis work, MCR-ALS was used to cope with coelutinginterferences, on account of the second-order advantageinherent to this algorithm which, in addition, is able to handledata sets deviating from trilinearity, like the high-performanceliquid chromatography data analyzed in the present report. Thedeveloped approach enabled us to determine five pteridines,some of them with overlapped profiles, reducing the experimentaltime and reagent consumption. Ratio values forpteridines/creatinine in urine, for infected children withdifferent pathologies, are reported in this work