A Simple and Sensitive Liquid Chromatography-Mass Spectrometry Confirmatory Method for Analyzing Sulfonamide Antibacterials in Milk and Egg CHIARA CAVALIERE,ROBERTA CURINI,ANTONIO DI CORCIA,* MANUELA NAZZARI, AND ROBERTO SAMPERI Dipartimento d Chimica, Universita ` “La Sapienza”, Piazza Aldo Moro 5, 00185 Roma, Italy A simple and specific method able to identify and quantify traces of 14 sulfonamide antibacterials (SAs) in milk and eggs is presented. This method uses a single solid-phase extraction (SPE) cartridge for simultaneous extraction and purification of SAs in the above matrices. Milk and egg samples are passed through a Carbograph 4 sorption cartridge. After analyte desorption, an aliquot of the final extract is injected into a liquid chromatography-mass spectrometry (LC-MS) instrument equipped with an electrospray ion source (ESI) and a single quadrupole. MS data acquisition is performed in the positive-ion mode and by a time-scheduled multiple-ion selected ion monitoring program. Compared to two published methods, the present protocol extracted larger amounts of SAs from both milk and egg and decreased the analysis time by a factor of 3 with milk samples and by a factor of 2 with egg samples. Recovery of SAs in milk at the 5 ppb level ranged between 76 and 112% with relative standard deviations (RSDs) of E13%. Recovery of SAs in egg at the 50 ppb level ranged between 68 and 106% with RSDs of e12%. Estimated limits of quantification (S/N ) 10) of the method were 1-6 ppb of SAs in whole milk and 5-13 ppb of SAs in eggs. Analyses of eggs from three layer chickens treated with sulfoquinoxaline revealed this antibacterial was still present at the 150 ppb level 1 week after withdrawal. KEYWORDS: Sulfonamides; milk; egg; SPE extraction; LC-MS INTRODUCTION Sulfonamides (SAs) comprise a large number of synthetic bacteriostatic compounds. They act by competing with p- aminobenzoic acid in the enzymatic synthesis of dihydrofolic acid. This leads to a decreased availability of the reduced folates that are essential in the synthesis of nucleic acids. No fewer than 10 SAs are routinely used in veterinary medicine to treat a variety of bacterial and protozoan infections in cattle, swine, and poultry. Analysis of SAs in foodstuffs is of particular concern because of the potential carcinogenic character (1, 2). To ensure the safety of food for consumers, Regulation 281/96 of the EU Commission has laid down maximum residue limits of 100 ppb of SAs as a total in milk. Although several SAs are approved for medicinal purposes in chickens, no SAs is approved for use in laying hens. Violative residues in eggs could result from giving SAs intended for broilers to laying hens. Public health agencies in many countries rely on detection by mass spectrometry (MS) for unambiguous confirmation of xenobiotics in foodstuffs. EU Commission Decision 93/256/ EEC states that “Methods based only on chromatographic analysis without the use of molecular spectrometric detection are not suitable for use as confirmatory methods”. Liquid chromatography (LC)-MS is thus the ideal technique to determine nonvolatile, polar compounds such as sulfonamide antibacterials. At present, three LC-MS methods are quoted in the literature for determining SA residues in milk (3-5). One of these methods (3) is based on the use of the thermospray interface that is no longer commercially available. Doerge et al. (4) demonstrated the practicality of using a benchtop single- quadrupole LC-MS instrumentation for sensitive detection of SAs in milk. Protonated molecules were generated by an atmospheric pressure chemical ionization (APCI) ion source, whereas fragment (product) ions were obtained by in-source collision-induced dissociation (CID) reactions in the first part of the ion transmission region. Volmer (5) elaborated a method based on LC-tandem MS with an electrospray (ESI) ion source for detecting and quantifying 21 SAs in milk at levels of <1 ppb. A drawback of this method is that it is time-consuming, as one of the steps of the sample treatment involves evaporation of ∼15 mL of water. Despite the fact that SAs are widely used for poultry, little attention has been given in the past to elaborate LC-MS confirmatory methods of these antibacterials in egg. Tarbin et al. (6) elaborated an LC-APCI-MS method for determining parts per billion levels of 16 SAs in whole egg. Identification and * Author to whom correspondence should be addressed (fax +39-06- 490631; e-mail antonio.dicorcia@uniroma1.it). 558 J. Agric. Food Chem. 2003, 51, 558-566 10.1021/jf020834w CCC: $25.00 © 2003 American Chemical Society Published on Web 12/18/2002