ENHANCED MCR-ALS MODELING OF HPLC WITH FAST SCAN FLUORIMETRIC DETECTION SECOND ORDER DATA FOR QUANTITATION OF METABOLIC DISORDER MARKER PTERIDINES IN URINE

MANCHA LLANOS; A MUÑOZ DE LA PEÑA; ESPINOSA MANSILLA, A; CAÑADA CAÑADA F; CULZONI M J; DE ZAN, MM; GOICOECHEA, HC

Barcelona

Congreso; Jornada de Quimica Analitica Instrumental 2011; 2011

Pteridines are very important cofactors in several cell metabolisms and, in consequence, the amounts of these pteridines in biological fluids have significant importance in clinical diagnostic.The pteridines are excreted in urine and its levels are related with activation of the cellular immune system due to certain diseases such as cancer, viral infections and renal disease.A significant increase in the urinary excretion of xantopterin (XAN), neopterin (NEO) and pterin (PT), as well as a significant decrease in isoxantopterin (ISO) and an NEO/BIO increment, was found in cancer patients. In mononucleosis infected children an increase of the NEO amount was also observed.Furthermore, tetrahydrobiopterin (BH4) is the major unconjugated pteridine present in human biological fluids. It is known that alterations on the metabolic mechanism of BH4 give rise to different diseases, named hyperphenylalaninaemia (HPAs). The differential diagnostic of these HPAs is important to establish the adequate therapeutic protocol and is based in the measurement of pteridines and other metabolites excretion. Hence, the classical phenylketonuria produces increments in the urinary levels of NEO and biopterin (BIO), while the BH4 deficit produces a decrease in the NEO and BIO levels.It is, in consequence, of interest the development of methods of determination of the most important metabolic disorder marker pteridines.High performance liquid chromatography with fluorimetric detection is the most used analytical method for its determination in biological fluids (1). A recent review reports on different strategies to enhance chromatographic methodologies by the use of second order multivariate calibration with the object of resolving overlapped peaks (2), and a recent Editorial and a special issue of Talanta have been dedicated to this subject (3).The problem has been addressed using fast scan fluorimetry as chromatographic detection, allowing the obtaining of second order matrices containing fluorescence intensity in function of retention time and of emission wavelength. The multivariate curve resolution-alternating least squares (MCR-ALA) method was used to model the system in the presence of co-eluted interferents, in base to the second order advantage inherent to this algorithm. Two different strategies were followed for the mixture resolution, in which three of the analytes, NEO, BIO and PT, present identical emission spectra. In the first one (4), data were split in five different regions, building five MCR-ALS models with augmented matrices in the spectral axis, while in the second one an unique MCR-ALS model was build, composed of augmented matrices in the spectral axis, it is to say, the temporal profiles remain invariant while the emission spectra may vary from a sample to another. The obtained results with the unique MCR-ALS model were compared with the ones obtained with the strategy in which five regions were used, found better results of prediction for NEO and PT with the new strategy, as well as a considerable reduction in the time of analysis.