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

Abstract

A liquid chromatographic method has been developed, in combination with the multivariate curve resolution-alternating least squares algorithm (MCR-ALS), for the simultaneous determination of marker pteridines in urine samples. A central composite design has been applied to optimize the factors influencing the separation (buffer concentration, buffer pH, flow rate, oven temperature, 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, biopterin, and pterin; 4.0-8.0 ng mL(-1) for xanthopterin; and 0.5-4.5 ng mL(-1) for isoxanthopterin. The validation was carried out with fortified urine samples from healthy adults. The optimized conditions were a mobile-phase composition of 10 mM citric buffer at pH 5.44 and acetonitrile (94.5/5.5, v/v), a flow rate of 1.0 mL min(-1), and an oven temperature of 25 °C. The detection system consisted of a fast-scanning spectrofluorimeter, which allows obtaining of 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 high-performance liquid chromatography data analyzed in the present report. The developed approach enabled us to determine five pteridines, some of them with overlapped profiles, reducing the experimental time and reagent consumption. Ratio values for pteridines/creatinine in urine, for infected children with different pathologies, are reported in this work.

Second- and higher-order data generation and calibration: A tutorial

“…As an example, we consider the development of an HPLC-FSFD method for the 664 simultaneous determination of five marker pteridines in urine samples: neopterin 665 (NEO), biopterin (BIO), pterin (PT), xanthopterin (XAN) and isoxanthopterin (ISO) 666 [72]. Figure 12 shows a chromatogram registered at  exc = 272 nm and  em = 445 nm 667 and elution times between 2.0 and 6.5 min for a urine sample, after spiking it with the 668 five analytes.…”

Pteridine determination in human serum with special emphasis on HPLC methods with fluorimetric detection

“…First, an approach to determine five of the most important pteridine markers (BIO, NEO, ISO, XAN and PT) by means of a chromatographic method linked to a second-order multivariate procedure has been reported. The developed method enables the determination of the listed pteridines in urine, some of them with overlapping profiles, in a short analysis time and with the consequent reduction of reagent consumption [67,68]. A multivariate curve resolution-alternating least squares (MCR-ALS) algorithm gives good results for the investigated system, where significantly overlapped signal are detected.…”

Development of an HPLC Assay Methodology for a Desonide Cream with Chemometrics Assisted Optimization

“…Several strategies for obtaining acceptable resolution in chromatograms with overlapped peaks using chemometrics have been proposed (Duarte and Capelo 2006;Li et al 2006). Partial least squares regression (PLSR) and multivariate curve resolution (MCR) have proven to be valuable chemometrics resources in overcoming the problem of overlapping peaks de Llanos et al 2011;Donmez et al 2011;Gonzalez-Garcia et al 2008;Mas et al 2011;Rodriguez-Cuesta et al 2005) and matrix effects (Galera et al 2010;Garcia et al 2009) in chromatographic determinations.…”