Triazine residues in raw milk and infant formulas from Spanish northwest, by a diphasic dialysis extraction M. Ángeles García ⇑ , M. Santaeufemia, M. Julia Melgar Department of Toxicology, University of Santiago de Compostela, 27002 Lugo, Spain article info Article history: Received 21 July 2011 Accepted 15 November 2011 Available online 25 November 2011 Keywords: Atrazine Simazine Terbuthylazine Milk Diphasic dialysis abstract Residue levels of herbicides (triazines) were determined in milk by a new and sensitive analytical method. A total of 312 samples were collected (70 infant formulas and 242 raw milk) biweekly during a 24-month period. A method is reported for the analysis of triazine with extraction by diphasic dialysis and determination by gas chromatography with nitrogen–phosphorus detection (CG/NPD) and by liquid chromatography/mass spectrometry (LC/MS) to confirm positive results. Detection limits (LODs) were 0.01 mg/l for terbuthylazine, 0.07 mg/l for simazine and 0.04 mg/l for atrazine by CG/NPD. Optimal extraction was obtained with dichloromethane at 34 °C, and stirring at 200 rpm for 4 h. This extraction method improves recovery by up to 89% for terbuthylazine, 75% for simazine and 116% for atrazine. The frequency of total samples containing triazine residues was 12.50%; considering, separately, raw milk and infant formulas, this percentage was 16.11% and 5.71%, respectively. Finally, because one sample con- tained triazine levels slightly higher than established MRLs and owing to the known effects of these con- taminants on infants health and development, urgent actions are needed for monitoring and controlling of the level of contamination in infant formula which not exceeds tolerable limit recommended by Euro- pean and Spanish legislation. Ó 2011 Elsevier Ltd. All rights reserved. 1. Introduction The triazines are popular herbicides introduced about 30 years ago and applied to a variety of cultures as well as to pasture and hayfields to control perennial weeds. As a result, triazine residues may contaminate crops and pastures, and thus enter the food chain. Triazines and triazine residues show marked chemical stability and persistence in the environment, and can be transferred to milk from food or from well water (Lagana et al., 1995). Human milk is considered to be the best source of nutrition for the infant, but in the absence of breast milk, the infant milk formu- las serve as substitutes for human milk and play particular role in the diets of infants, because they are the major source of nutrients and should be of good quality and free of pesticide residues whose levels should not pose an unacceptable risk to consumers (Chao et al., 2004; Kazi et al., 2009; Melø et al., 2008; Riu et al., 2008; Opydo-Szymaczek and Opydo, 2011; European Commission, 2008; Directive 91/414/EEC; Regulation (EC) No 396/2005). Several authors have reported low levels of herbicides, includ- ing triazines, in milk (Gajduskova, 1983; Tekel et al., 1988; Bush- way et al., 1992; Balduini et al., 2003; Baranowska et al., 2005; Nougadère et al., 2011). Recent EU decisions (Decision 2004/247/CE and Decision 2004/ 248/CE) have prohibited the inclusion of atrazine and simazine in herbicide compounds. To conform to legislation prescribing acceptable limits for these residues, sensitive, rapid and easy-to- use analytical methods are required. Single-residue and multiresi- due methods have been developed for determining triazine resi- dues in water and other matrices (Moltó et al., 1991); however, little attention has been paid to analyzing herbicide residues in materials of animal origin like milk. For the analysis of herbicide residues in milk, the following steps are necessary (Lagana et al., 1995; Hajslova et al., 1990; Mel- gar et al., 2010): extraction of analytes from the sample matrix, clean-up to remove interfering coextractants, and separation of analytes from each other and any remaining interfering sub- stances. One of the most popular extraction methods is solid phase-extraction (Picó et al., 1995; Baynes and Bowen, 1995). In contrast, liquid extraction is more usually used for the analysis of triazines in water (Carabias-Martínez et al., 2005; Papadopoulus et al., 2007). The diphasic dialysis technique enables direct extraction of ana- lytes from organic matrices, with no need for complementary puri- fication steps. It is a new liquid–liquid extraction technique based on a cellulose membrane, with equilibrium reached between the amount of analyte inside the extraction membrane and in the extraction solvent in which the membrane is introduced. Conditions for optimal extraction must be properly selected: the extraction 0278-6915/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2011.11.019 ⇑ Corresponding author. Tel.: +34 982 822204; fax: +34 982 822001. E-mail address: mangeles.garcia@usc.es (M. Ángeles García). Food and Chemical Toxicology 50 (2012) 503–510 Contents lists available at SciVerse ScienceDirect Food and Chemical Toxicology journal homepage: www.elsevier.com/locate/foodchemtox