Analytica Chimica Acta 705 (2011) 24–29 Contents lists available at ScienceDirect Analytica Chimica Acta jo u rn al hom epa ge: www.elsevier.com/locate/aca A liquid–liquid extraction procedure followed by a low temperature purification step for the analysis of macrocyclic lactones in milk by liquid chromatography–tandem mass spectrometry and fluorescence detection Gabriel Rübensam a,b,∗ , Fabiano Barreto b , Rodrigo Barcellos Hoff b , Tarso Ledur Kist a , Tânia Mara Pizzolato c a Programa de Pós-Graduac ¸ ão em Biologia Celular e Molecular, Centro de Biotecnologia do Estado do Rio Grande do Sul, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil b Ministério da Agricultura, Pecuária e Abastecimento, Laboratório Nacional Agropecuário - LANAGRO/RS, Porto Alegre, RS, Brazil c Instituto de Química, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil a r t i c l e i n f o Article history: Received 1 October 2010 Received in revised form 12 February 2011 Accepted 17 February 2011 Available online 24 February 2011 Keywords: Avermectins Low temperature purification Extraction Liquid chromatography–tandem mass spectrometry Liquid chromatography–fluorescence detection a b s t r a c t In this work a method is proposed and demonstrated for the analysis of the macrocyclic lactones abamectin, doramectin, eprinomectin, ivermectin and moxidectin in bovine milk by liquid chromatogra- phy coupled to mass spectrometry (LC–MS/MS) and liquid chromatography with fluorescence detection (LC–FL). The method is based on liquid–liquid extraction followed by a low temperature purification (LLE–LTP) step. Moreover, the proposed method was validated according to the Commission Decision 2002/657/EC, using LC–MS/MS and LC–FL for confirmatory and quantitative analysis, respectively. For LC–MS/MS the recovery rates observed ranged from 101.2 to 141.6% with coefficient of variation from 2.6 to 19.8%. For LC–FL the recovery rates observed ranged from 100.2 to 105% and coefficient of varia- tions from 2.9 to 8.8%. Matrix effects were negligible due to the low temperature purification step. The quantification limits were far below the maximum limits established by regulations of all countries con- sulted. The proposed method proved to be simple, easy, and adequate for high-throughput analysis of a large number of samples per day at low cost. © 2011 Elsevier B.V. All rights reserved. 1. Introduction The avermectins abamectin (ABA), doramectin (DOR), epri- nomectin (EPR), ivermectin (IVR), and the milbemycin moxidectin (MOX) are macrocyclic lactones (ML) widely used for veterinary purposes due to their antiparasitary properties at low doses [1]. These chemicals are slowly eliminated from the cattle body [2] and their extensive or extra-label use may result in the presence of their residues in milk, liver, and muscle tissue. Residues of vet- erinary drugs in food are monitored by regulatory agencies using established maximum residue limits (MRL). Thus, formulations containing ABA, DOR, or IVR are not authorized for use in milk- ing cows in the European Union (EU) [3]. The same regulation is applied for DOR and IVR, in the United States [4]. For the CODEX ALIMETARIUS, no MRL was set for MOX. ABA is considered a pesti- cide with MRL of 5 ng mL -1 in milk [5]. In Brazil, which is the world’s ∗ Corresponding author at: Ministério da Agricultura, Pecuária e Abastecimento, Laboratório Nacional Agropecuário – LANAGRO/RS, Estrada da Ponta Grossa, 3036, CEP 91780-580, Porto Alegre, RS, Brazil. Tel.: +55 51 3248 2133. E-mail address: grubensam@yahoo.com (G. Rübensam). sixth largest milk producer [6], the avermectins are monitored by the National Residue Control Plan (NRCP), adopting the MRL val- ues suggested by CODEX, Food and Drug Administration, and EU, respectively. In the Brazilian NRCP, the MRL values for ABA, IVR, MOX in milk were established as 10 ng mL -1 . For DOR and EPR, the MRL was set as 15 and 20 ng mL -1 , respectively [7]. Residual levels of ML in milk samples have been analyzed by multiresidue methods based LC–FL after derivatization with acetic anhydride [1] or trifluoroacetic anhydride [8–10], and by liquid chromatography–tandem mass spectrometry (LC–MS/MS). Solid phase extraction (SPE) is widely used for macrocyclic lac- tones extraction in milk [1,8,9,11–13]. Other sample preparation procedures were developed as an alternative to SPE, such as QuECh- ERS (Quick Easy Cheap Effective Rugged and Safe) [14,15], protein precipitation [10], protein precipitation followed by ultrafiltration [16], and liquid–liquid extraction (LLE) [17]. LLE are simple and con- venient extraction techniques. However, intense matrix effects are observed when the extracts are analyzed by LC–MS/MS with elec- trospray ionization (ESI) [14]. Therefore, a previous cleanup step is necessary to remove interfering compounds. The low-temperature purification (LTP) method was introduced by Lentza-Rizos et al. [18] for the analysis of organophosphorous 0003-2670/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2011.02.041