ORIGINAL PAPER Jose´ Manuel Amigo Æ Jordi Coello Æ Santiago Maspoch Three-way partial least-squares regression for the simultaneous kinetic-enzymatic determination of xanthine and hypoxanthine in human urine Received: 22 February 2005 / Accepted: 22 April 2005 / Published online: 7 June 2005 Ó Springer-Verlag 2005 Abstract The performance of three-way principal com- ponent analysis and three-way partial least-squares regression when applied to a complex kinetic-enzymatic system is studied, in order to investigate the analytical potential of the combined use of these chemometric technologies for non-selective enzymatic systems. A enzymatic-kinetic procedure for the simultaneous determination of hypoxanthine and xanthine in spiked samples of human urine is proposed. The chemical sys- tem involves two consecutive reactions catalyzed by xanthine oxidase (EC 1.17.3.2). This enzyme catalyzes the oxidation of hypoxanthine, first to xanthine and then to uric acid, a competitive inhibitor of the reactions. The influence of uric acid during quantitative determination was considered in the design of the calibration set. The sample and enzyme solution were mixed in a stopped- flow module and the reaction was monitored using a diode array spectrophotometer. The recorded data have an intrinsical three-component structure (samples, time and wavelength). This data array was studied via three- way principal component analysis and was modeled for quantitative purposes using a three-way partial least- squares calibration procedure. Results are compared with those obtained by applying classical bilinear PLS to the previously unfolded data matrix. Keywords Oxipurines Æ Urine analysis Æ Three-way principal component analysis Æ Three-way partial least- squares regression Æ Simultaneous kinetic determination Introduction While enzymes are usually visualized as highly selective catalysts and are used with the aim of obtaining a specific product, there are a number of them that, in fact, show group selectivity. The development of multivariate cali- bration procedures and their application to kinetics [1–4] has renewed analytical interest in non-selective enzymes, since they can potentially be applied to the simultaneous and rapid determination of several analytes using rela- tively simple instrumentation. The use of enzymes showing group selectivity becomes an analytical choice when applied to the kinetic resolution of similar com- pounds with the same functional groups. The potential for this kind of resolution has already been proved dur- ing the simultaneous determination of ethanol and methanol using an alcohol oxidase [5]. Since the same product is formed from both compounds, discrimination is only possible via kinetic information, and this non- linear system was solved by applying an artificial neural network to the data recorded at a single wavelength. The system studied in this work is more complex in the sense that it consists of two consecutive reactions (A fi B fi C) catalyzed by the same enzyme, where A and B are the analytes and C, the product, acts as an inhibitor [6]. However, although the kinetic system is complex, there is a large amount of information avail- able since all three compounds absorb in the ultraviolet region, although with highly overlapped absorption bands. Therefore, the spectral and kinetic data from the system could contribute useful information that may be used to discriminate the responses of the analytes, and therefore to resolve the mixtures. The oxipurine (hypoxanthine and xanthine) and uric acid levels in blood, plasma and urine may provide sensitive indicators of certain pathologic states, includ- ing xanthinuria, gout, renal failure, toxaemia during pregnancy, and other diseases [7]. These compounds have similar chemical structures and UV–Vis spectra (Fig. 1a), which has prompted the use of analytical separation techniques such as high performance liquid chromatography (HPLC) [8–10] or high performance capillary electrophoresis (HPCE) [11] for their simulta- neous analysis. In addition, several electrochemical methods have been proposed [12, 13]. Until now, the J. M. Amigo Æ J. Coello (&) Æ S. Maspoch Universitat Auto´noma de Barcelona, Bellaterra, 08193 Barcelona, Spain E-mail: jordi.coello@uab.es Tel.: +34-93-5812122 Fax: +34-93-5812379 Anal Bioanal Chem (2005) 382: 1380–1388 DOI 10.1007/s00216-005-3275-4