1145 Research Article Received: 6 February 2008 Revised: 3 December 2008 Accepted: 26 January 2009 Published online in Wiley Interscience: 16 March 2009 (www.interscience.wiley.com) DOI 10.1002/jsfa.3564 Multitoxin extraction and detection of trichothecenes in cereals: an improved LC-MS/MS approach Antonello Santini, a * Rosalia Ferracane, a Maria Carmela Somma, a Alejandro Arag ´ on b and Alberto Ritieni a Abstract BACKGROUND: Many multiresidual methods to evaluate natural occurrence of Fusarium toxins are already reported in the scientific literature but a new rapid, reliable, cost-efficient and high-sensitivity method for the simultaneous determination of several fusariotoxins is always welcome. Nivalenol (NIV), deoxynivalenol, fusarenon-X (FUS-X), 3-acetyldeoxynivalenol, diacetoxyscirpenol (DAS), HT-2 toxin, T-2 toxin, neosolaniol (NEO), zearalanone and zearalenone (ZON) belong to the most common mycotoxins in food matrix grains, e.g., wheat and maize. The proposed method is a multitoxin analytical method that combines high-performance liquid chromatography (HPLC), atmospheric pressure chemical ionization (APCI), triple- quadrupole tandem mass spectrometry (LC-MS/MS) under the selected reaction monitoring (SRM) mode, and it is focused on the optimization of the sample preparation without the need for any cleanup. RESULTS: Three different methods for sample preparation and for the simultaneous extractions of the above-mentioned fusariotoxins were tested: two of these were followed by a different cleanup step for comparison, while the extraction method proposed in this work, which uses an 84% (v/v) acetonitrile aqueous solution, sample homogenization and subsequent filtration, was validated without any further cleanup step. CONCLUSION: Calibration curves for all analytes are linear, except DAS, HT-2 and ZON, over the working range of 10 – 1000 μg kg -1 . The calibration curve of DAS was linear between 10 and 500 μg kg -1 , although the curves of HT-2 and ZON were linear in the range 10 – 250 μg kg -1 . Squared correlation coefficients (R 2 ) were in the range 0.995 – 0.998 for the all point calibration curves. The lowest limits of detection (LOD) were found for DON and ZAN with 0.5 and 0.2 μg kg -1 , respectively, while the highest LODs were obtained for NIV, FUS-X and NEO, with 3.3 μg kg -1 for each toxin. c  2009 Society of Chemical Industry Supporting information may be found in the online version of this article. Keywords: Fusarium mycotoxins; trichothecenes; zearalenone; cereals; LC-MS/MS; simultaneous extraction; simultaneous determination INTRODUCTION Mycotoxins are toxic secondary metabolites produced by various fungi growing on agricultural commodities in the field or during storage. The Fusarium genus is the most frequent fungal contaminant of a broad range of hosts, including barley, maize, millet, oat, rice, rye and wheat, 1 especially in temperate regions. 2,3 Trichothecenes and zearalenone are major Fusarium mycotoxins. 4 The trichothecenes are polycyclic sesquiterpenoids, possessing a C9–C10 double bond, an epoxide between carbons atoms C12 and C13, plus a variable number of hydroxyl and acetoxy groups in the molecule. They are commonly classified into four types, namely A, B, C, D, according to the functional groups present in the molecule, and only a few of them have been encountered as natural contaminants of cereal products. Figure 1 illustrates the chemical structures of trichothecenes and the characteristic functional groups present in their molecules. Type A trichothecenes include HT-2 toxin (HT-2), T-2 toxin (T-2), diacetoxyscirpenol (DAS) and neosolaniol (NEO) and differ from type-B trichothecenes, such as deoxynivalenol (DON, also known as vomitoxin), nivalenol (NIV) and their acetyl derivatives, respectively 3-acetyldeoxynivalenol (3AcDON) and fusarenon-X (FUS-X), by the absence of a carbonyl group at the C(8) position. 5 DON and NIV, the most commonly found trichothecenes, followed only by T-2, HT-2 and by the DON-acetyl derivates, 5 have been investigated in this work. Dietary exposure to fusariotoxins can induce a variety of adverse health effects in farm animals and humans, including toxic effects on skin and mucosal surfaces, feed refusal, weight loss, vomiting, immunosuppressive and haemorrhaging effects. 5 ∗ Correspondence to: Antonello Santini, Dipartimento di Scienza degli Alimenti, Universit` a di Napoli ‘Federico II’, Via Universit` a 100, 80055 Portici, Naples, Italy. E-mail: asantini@unina.it a Dipartimento di Scienza degli Alimenti, Universit` a di Napoli ‘Federico II’, Via Universit` a 100, 80055 Portici, Naples, Italy b Facultad de Ciencias Agrarias y Forestales, Universidad de La Plata, Calle 60 y 119, 1900 La Plata, Buenos Aires, Argentina J Sci Food Agric 2009; 89: 1145–1153 www.soci.org c  2009 Society of Chemical Industry