J. Sep. Sci. 2006, 29 A. Espinosa-Mansilla et al. Anunciación Espinosa- Mansilla Arsenio Muæoz de la Peæa David Gonzµlez Gómez Florentina Caæada-Caæada Department of Analytical Chemistry, Faculty of Sciences, University of Extremadura, Campus Universitario, Badajoz, Spain Original Paper HPLC determination of ciprofloxacin, cloxacillin, and ibuprofen drugs in human urine samples This paper reports, for the first time, a liquid chromatographic method for the simultaneous determination of three frequently co-administered active principles, two antibiotics, ciprofloxacin (CIPRO) and cloxacillin (CLOXA) belonging to the fluo- roquinolones and b-lactam families, respectively, and ibuprofen (IBU), a non-stero- idal anti-inflammatory drug. The chromatographic separation was performed on a C-18 analytical column, using isocratic elution with methanol-acetonitrile-pH 3 for- mate buffer (C T = 0.1 M) (15 : 12 : 73, v/v/v) for 3 min and, after that, a linear gradient with methanol-acetonitrile (88 : 12, v/v) for 8 min. Several absorption spectra were obtained for each peak using a DAD detector. Chromatograms at the maximum absorption wavelength for each analyte, 220 nm for both IBU and CLOXA, and 280 nm for CIPRO were selected as the most suitable. The proposed chromatogra- phic method requires about 15 min per sample. The presence of a urine background was tested and no interference was found. The method was satisfactorily applied to the determination of CIPRO, CLOXA, and IBU, in fortified urine, and in urine samp- les from a patient undergoing treatment with these three active principles, among others. Limits of quantification in urine were 1.00, 1.70, and 2.87 lg/mL for CIPRO, CLOXA, and IBU, respectively. Keywords: Ciprofloxacin / Cloxacillin / HPLC / Ibuprofen / Urine / Received: March 21, 2006; revised: April 7, 2006; accepted: April 7, 2006 DOI 10.1002/jssc.200600126 1 Introduction Ciprofloxacin {1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxo- 7-(1-piperazinyl)-3-quinoline carboxylic acid; CIPRO} is the most powerful quinolone against Gram-negative bac- teria. It is particularly active against Pseudomonas aerugi- nosa. CIPRO is less active than some of the more recently developed quinolones against Gram-positive bacteria. This quinolone is well absorbed after oral administra- tion. Approximately, 60% of unchanged CIPRO and 15% of metabolites are recovered from the urine 24 h after administration. Although CIPRO is primarily excreted by the kidneys, over 40% can be excreted in the bile if the renal route is not available [1]. Cloxacillin {(2S,5R,6R)-6-[[[3-(2-chlorophenyl)-5-methyl-4- isoxazolyl]carbonyl]amino]-3,3-dimethyl-7-oxo-4-thia-1- azabicyclo[3.2.0]heptane-2-carboxylic acid, CLOXA}, is a b-lactam antibiotic, of broad spectrum against Gram- positive and negative bacteria. It is widely used for the treatment of several bacterial infections in humans and animals [2]. Ibuprofen (IBU), a propionic acid derivative {(l)-(R,S)-2-(4- isobutylphenyl)propionic acid} is a non-steroidal anti- inflammatory drug, used in the treatment of rheuma- toid arthritis and other inflammatory conditions [3]. This compound was the first non-steroidal anti-inflam- matory drug (NSAIDs) to be commonly used for the treat- ment of pain and inflammation [4]. The structures of the studied analytes are shown in Fig. 1. In human diseases, bacterial infection usually leads to a painful inflammatory process. Hence, co-administration of antibiotics and anti-inflammatory drugs is commonly used to overcome these effects. Moreover, when the infec- tion is caused by unknown bacteria, mixtures of antibio- tics belonging to different groups are frequently admi- nistered. In the literature, there are no reports of these three com- pounds being simultaneously determined. Nor have papers been published in which only two of these com- pounds are determined in the presence of urine. How- ever, the literature abounds in papers about the resolu- Correspondence: Dr. Florentina Caæada-Caæada, Departamento de Quimica Analitica, Facultad de Ciencias, Avda. Elvas s/n, 06071 Badajoz, Spain. E-mail: flori@unex.es Fax: +34-924-289-375 Abbreviations: CIPRO, ciprofloxacin; CLOXA, cloxacillin; DAD, diode array detector; IBU, ibuprofen i 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com