Vibrational characterization and surface- enhanced Raman scattering detection of probenecid doping drug † Irene Izquierdo-Lorenzo, José Vicente García-Ramos and Santiago Sanchez-Cortes* Probenecid (PB), a drug employed to reduce the excretion of other drugs in urine and also employed as masking agent in sport doping, is characterized for the first time in this work by vibrational spectroscopy. Previously, FTIR and FT-Raman spectra were assigned on the basis of density functional theory calculations. Afterwards, a detection protocol of PB was developed on the basis of surface-enhanced Raman scattering (SERS) spectroscopy. To accomplish this, the experimental conditions of SERS measurement were previously optimized by varying parameters such as the metal, its reduction protocol to fabricate the nanoparticles and the pH. The adsorption isotherms of PB on the metal nanoparticles were obtained, and the adsorption mechanism of this drug on Ag nanoparticles was deduced from the analysis of SERS spectra. Finally, a quantitative SERS study was carried out on the basis of the calibration curve deduced from the adsorption isotherms. The results shown in this study demonstrate that PB can be detected by SERS at concentrations as low as 1.2 mg/ml. Copyright © 2013 John Wiley & Sons, Ltd. Keywords: Probenecid; antidoping; SERS; adsorption Introduction Surface-enhanced Raman spectroscopy (SERS) has largely proved in recent years its potential as analytical technique, [1] as it provides both qualitative and quantitative information on a sample. Although it is usually highlighted the ultrasensitive character of SERS, reaching to obtain spectra of single molecules, [2] it is necessary to also point out its selective character given by the qualitative infor- mation deducible from a SERS spectrum, which is often neglected. In fact, SERS spectra are often registered without a deep analysis of the vibrational bands. But to take full advantage of the SERS phenomenon for analytical purposes, the structural information should be withdrawn, and for that purpose, primary vibrational stud- ies of the molecules of interest must be performed beforehand. [1,3] In this work, we have applied SERS to the study of a pharmaceutical drug: probenecid (p-(dipropylsulphamyl) benzoic acid, PB, Fig. 1). Although originally developed for reducing renal excretion of antibi- otics, it is most used in the treatment of gout because of its uricosuric properties. [4] Apart from antibiotics, excretion of certain substances other than antibiotics in urine is diminished, such as beta-adrenergic agonists and other drugs abused in sport doping. [5] After PB consumption, these substances remain inside the body for a longer period, and their presence is not detected in antidoping tests. For this reason, PB itself was included in the World Anti-Doping Agency Prohibited list. [6] For doping tests requiring both univocal identi fica- tion of the consumed substance and a high sensitivity, SERS could be regarded as a potential technique to be applied in antidoping tests. Under the structural point of view, PB contains an aromatic moiety with a carboxylic substituent and an aliphatic moiety constituted by two propyl groups, joined by a sulphonamide group. Surprisingly, no vibrational studies of this molecule prior to this work were found in the literature. On the one hand, the structural information of a SERS spectrum must be directly related to that of the conventional Raman spectrum of the molecule to extract information regarding the influence of the surface on the analyte structure. For this reason, prior to the application of SERS with analytical purposes, the analysis of the vibrational spectra of PB is a necessary step. On the other hand, trace level detection by SERS requires an optimization process to obtain the highest sensitivity, selectivity and reproducibility. This optimization involved a study of the effectiveness of Ag and Au nanoparticles (NPs) prepared by different reduction protocols and a study at different pH. Once the optimal conditions were determined, a quantitative detection of PB was addressed by performing the adsorption isotherm and the subsequent calculation of the affinity constant, the maximum SERS intensity and the limit of detection. Experimental section Probenecid was acquired from Sigma-Aldrich. A 0.1 M stock solution was prepared in ethanol, from which further dilutions were * Correspondence to: Santiago Sanchez-Cortes, Instituto de Estructura de la Materia, IEM-CSIC, Serrano 121, 28006 Madrid, Spain E-mail: s.sanchez.cortes@csic.es † This article is from the GISR part of the joint special issue on the European Conference on Nonlinear Optical Spectroscopy (ECONOS 2012) with Guest Editors Johannes Kiefer and Peter Radi and the II Congresso Nationale di Spettroscopie Raman ed Effetti Ottici Non Lineari (GISR 2012) with Guest Editor Maria Grazia Giorgini. Instituto de Estructura de la Materia, IEM-CSIC, Serrano 121, 28006 Madrid, Spain J. Raman Spectrosc. (2013) Copyright © 2013 John Wiley & Sons, Ltd. Research article Received: 9 November 2012 Revised: 22 December 2012 Accepted: 22 January 2013 Published online in Wiley Online Library (wileyonlinelibrary.com) DOI 10.1002/jrs.4284