Determination of marker pteridins in urine by hplc with fluorimetric detection and second-order multivariate calibration using MCR-ALS.

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

Granada

Congreso; VII Coloquium Chemiometricum Mediterraneum; 2010

     A liquid chromatographic method has been developed, in combination with the multivariate curve resolution alternating least squares (MCR-ALS) algorithm, for the simultaneous determination of marker pteridins in urine samples. A central composite design was applied to optimize the factors influencing the separation (buffer concentration, buffer pH, flow rate, mobile phase composition). A set of 15 calibration samples were randomly prepared, in a concentration range of 0.5-10.5 ng mL-1 for neopterin (NEO), biopterin (BIO) and pterin (PT); 4.0-8.0 ng mL-1 for xanthopterin (XAN) and 0.5-4.5 ng mL-1 for isoxanthopterin (ISO). The validation was carried out with urine samples from healthy adults (dilution 1:250). The optimized conditions were a mobile phase composition of citric buffer 10 mM at pH=5.44-acetonitrile (94.5/5.5, v/v). The flow rate was 1.0 mL min-1 and the oven temperature was maintained at 25 ºC. The detection system consisted of a fast-scanning spectrofluorimeter, which allows one to obtain second-order data matrices containing the fluorescence intensity as a function of retention time and emission wavelength. In this work, MCR-ALS was used to cope with coeluting interferences, on account of the second order advantage inherent to this algorithm, which, in addition, is able to handle data sets deviating from trilinearity, like the HPLC-data analyzed in the present report. The developed approach enabled us to determine five pteridins, some of them with overlapped profiles, reducing the experimental time and reagent consumption. Ratio values for pteridins/CREA in urine, for infected children with different pathologies, are reported in this communication.