Talanta 72 (2007) 932–940 Square wave adsorptive stripping voltametric determination of the mixture of nalidixic acid and its main metabolite (7-hydroxymethylnalidixic acid) by multivariate methods and artificial neural network A. Guiberteau Cabanillas ∗ , M.I. Rodr´ ıguez C´ aceres, M.A. Mart´ ınez Ca ˜ nas, J.M. Ortiz Burguillos, T. Galeano D´ ıaz Department of Analytical Chemistry, University of Extremadura, 06071 Badajoz, Spain Received 25 January 2006; received in revised form 4 December 2006; accepted 14 December 2006 Available online 5 January 2007 Abstract Nalidixic acid (NA) and its main metabolite, 7-hydroximethylnalidixic acid (OHNA), are quinolones antibacterial used as agents used for the treatment of urinary tract infection. For both compounds an adsorption process on a hanging mercury electrode (HMDE). On this basis, a square wave adsorptive stripping voltammetry (SWadSV) method has been developed for the individual and simultaneous determination of NA and OHNA. The variables that affect to accumulation process, such as concentration of perchloric acid, accumulation potential and accumulation time have been optimised by using an experimental design (concretely a Box–Behnken design with three levels) together with the response surface methodology (RSM). Calibration curves were linear in the range (0–1.38) × 10 -7 mol L -1 for NA and (0–3.23) × 10 -8 mol L -1 for OHNA, in the optimized conditions, with detection limits of 9.48 × 10 -9 mol L -1 and 8.06 × 10 -10 mol L -1 for NA and OHNA, respectively. The method was applied to urine samples containing only one of the analytes with satisfactory recoveries. As the voltammetric signals of these compounds show a high overlapping, different chemometric methods, such as classical least squares (CLS), partial least squares (PLS), principal component regression (PCR) and artificial neural network (ANN) have been used for the resolution of the mixture. The analysis of these compounds in urine samples were carried out using the different chemometric tools and the best recoveries were obtained by using ANN. No pre-treatment of the sample was necessary. © 2007 Elsevier B.V. All rights reserved. Keywords: Nalidixic acid; 7-Hydroxymethylnalidixic acid; Square wave adsorptive stripping voltammetry; Artificial neural network 1. Introduction Nalidixic acid (NA) (Fig. 1A) is an antibacterial agent which has been used extensively in the treatment of gram-negative uri- nary tract infections [1]. It was synthesized by Lesher et al. [2] in the 1960s of 20th century. Its major metabolite is the 7- hydroxymethyl analogue (OHNA) (Fig. 1B) which exhibits at least in vitro anti-bacterial activity identical in spectrum and potency to that of the parent compound [3]. Besides its human use, NA is also used in fish farms. Fish farming, and particu- larly freshwater fish farming, has spectacularly grown during the last few decades. Because of fish concentration and poor water quality, bacterial diseases become an important problem ∗ Corresponding author. E-mail address: aguibert@unex.es (A.G. Cabanillas). and antibacterial agents are consequently used in order to keep good productivity. A survey of the bibliography shows that nalidixic acid has been widely studied by several techniques, such as, spectropho- tometry [4,5], fluorescence [6,7], phosphorescence [8], HPLC with several detectors [9–11] and capillary electrophoresis [12]. Also, a spectrofluorimetric method has been found for the deter- mination of OHNA [13], in which OHNA form a complex with gamma-cyclodextrin in aqueous solution. There are several papers in which the mixture NA + OHNA is analyzed. Thus, HPLC has been used for plasma samples with a dynamic anion-exchange system [14]. Also, HPLC with fluorescence and ultra-violet detection in series has been used [15]. A micellar electrokinetic capillary electrophoresis method has been developed for the separation and determination of NA, OHNA and carboxynalidixic acid in serum and urine samples [16]. 0039-9140/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.talanta.2006.12.035