Analytica Chimica Acta 654 (2009) 127–132 Contents lists available at ScienceDirect Analytica Chimica Acta journal homepage: www.elsevier.com/locate/aca A multidimensional high performance liquid chromatography method coupled with amperometric detection using a boron-doped diamond electrode for the simultaneous determination of sulfamethoxazole and trimethoprim in bovine milk Leonardo S. Andrade, Marcela C. de Moraes, Romeu C. Rocha-Filho, Orlando Fatibello-Filho, Quezia B. Cass ∗ Departamento de Química, Universidade Federal de São Carlos, C.P. 676, 13560-970 São Carlos - SP, Brazil article info Article history: Received 15 July 2009 Received in revised form 18 September 2009 Accepted 23 September 2009 Available online 27 September 2009 Keywords: Multidimensional HPLC Restricted-access media (RAM) Bovine milk Sulfonamides determination Electrochemical detection Boron-doped diamond electrode abstract The development and validation of a multidimensional HPLC method using an on-line clean-up column coupled with amperometric detection employing a boron-doped diamond (BDD) electrode for the simul- taneous determination of sulfamethoxazole (SMX) and trimethoprim (TMP) in bovine milk are presented. Aliquots of pre-prepared skim-milk samples were directly injected into a RAM octyl-BSA column in order to remove proteins that otherwise would interfere with milk analysis. After exclusion of the milk pro- teins, SMX and TMP were transferred to the analytical column (an octyl column) and the separation of the compounds from one another and from other endogenous milk components was achieved. SMX and TMP were detected amperometrically at 1.25 V vs. Ag/AgCl (3.0 mol L -1 KCl). Results with good linearity in the concentration ranges 50–800 and 25–400 gL -1 for SMX and TMP, respectively, were obtained and no fouling of the BDD electrode was observed within the experimental period of several hours. The intra- and inter-assay coefficients of variation were less than 10% for both drugs and the obtained LOD values for SMX and TMP were 25.0 and 15.0 gL -1 , respectively. © 2009 Elsevier B.V. All rights reserved. 1. Introduction A series of synthetic bacteriostatic compounds is used in dairy farming for the treatment of diseases evolving from bacterial infections, such as mastitis, pneumocystis pneumonia, chronic bronchitis, meningococcal meningitis, acute otitis, and toxoplas- mosis [1]. Sulfonamides have been used for several decades in the treatment of bacterial infection. They competitively inhibit the bacterial enzyme dihydropholate synthetase [2,3]. In veterinary medicine, sulfonamides are commonly used to treat or prevent livestock diseases, such as respiratory and gastrointestinal tract infections [4]. The corresponding pharmaceutical products usually consist of a sulfonamide mixed with another drug that increases its antibacterial activity (in a 5:1 ratio sulfonamide:potentiator), as for example the sulfamethoxazole (SMX) and trimethoprim (TMP) mixture, which is commonly known as SMX–TMP (for their chemi- cal structures, see de Paula et al. [5]). However, the use of SMX–TMP may lead to residues in milk, which subsequently may induce aller- gic reactions in humans. In addition, they can give rise to an increase ∗ Corresponding author. Tel.: +55 16 3351 8087; fax: +55 16 3351 8350. E-mail address: quezia@pesquisador.cnpq.br (Q.B. Cass). in the resistance of pathogenic bacteria to them, which may result in health problems [1]. To protect consumer’s health, the maximum residue limit (MRL) adopted by the US Food and Drug Administra- tion (FDA) for sulfonamides in milk is 100 g kg -1 [6]. In the same way, the European Medicines Agency (EMEA) has set the MRL of trimethoprim in meat and milk as 50 g kg -1 [7]. Therefore, the search for analytical methods that can detect these individual sub- stances at low concentration levels is quite relevant. Commonly, the monitoring of sulfonamides in milk is carried out using microbial methods, but the use of instrumental meth- ods of analysis based on LC with UV or fluorescence becomes necessary when more conclusive results are required [2]. How- ever, problems involving the direct injection of milk samples into chromatographic phases can limit their use for practical purposes, since proteins present in this type of biofluid promptly clog the chromatographic column, leading to a significant loss of chromato- graphic efficiency [8]. Aiming to avoid any pretreatment before the chromatographic analysis, the interest in having on-line techniques for the handling of untreated biological samples has been increased. The main advantage of on-line techniques is their non-requirement of further handling of the samples; thus they are highly suitable to become fully automated techniques that can be used on site [9]. In this sense, HPLC methods incorporating restricted-access media 0003-2670/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2009.09.035