Volume 6 • Issue 7 • 1000464 J Food Process Technol ISSN: 2157-7110 JFPT, an open access journal Open Access Research Article Kafouris et al., J Food Process Technol 2015, 6:7 DOI: 10.4172/2157-7110.1000464 *Corresonding author: Demetris Kafouris, State General Laboratory, Ministry of Health, Kimonos 44, 1451 Nicosia, Cyprus, Tel: +357 22 809167; Fax: +357 22 316434; E-mail: dkafouris@sgl.moh.gov.cy Received April 15, 2015; Accepted May 14, 2015; Published May 21, 2015 Citation: Kafouris D, Christofdou M, Christodoulou M, Christou E, Ioannou-Kakouri E (2015) A Validated UPLC-MS/MS Multi-mycotoxin Method for Nuts and Cereals for the Offcial Control in Cyprus within the EU Requirements. J Food Process Technol 6: 464. doi:10.4172/2157-7110.1000464 Copyright: © 2015 Kafouris D, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Abstract The validation of a rapid, reliable and sensitive method for the simultaneous determination of Afatoxins (AFB 1 , AFB 2 , AFG 1 , AFG 2 ), Ochratoxin A, Zearalenone, Deoxynivalenol, Fumonisins B 1 and B 2 , T-2 and HT-2 toxins, in nuts (peanuts, pistachio and almonds) and cereals (maize and wheat) is reported. The method was developed and validated to fulfl the requirements of the offcial control of mycotoxins according to EU legislation. This method is based on a single extraction step using an acetonitrile/ water mixture followed by the analysis of the diluted crude extract using ultra high performance liquid chromatography with tandem mass spectrometry (UPLC-MS/MS). The MS/MS detection was carried out using an electro spray-ionization interface (ESI) in positive ion mode. Matrix-matched calibration was used for the quantifcation of the mycotoxins, because of the absence of further clean up steps that reduce suppression/enhancement matrix effects. The method performance characteristics were determined after spiking blank samples on multiple levels. The mean recoveries of mycotoxins in spiked nuts ranged from 74.4% to 131.7%, while in cereals ranged from 52.8% to 113.9%. Relative standard deviations were lower than 20.4% for all target mycotoxins. Limits of detection and quantifcation for nuts and cereals ranged 0.08-30.0 and 0.25-99.0 μg/Κg, respectively. A Validated UPLC-MS/MS Multi-mycotoxin Method for Nuts and Cereals for the Official Control in Cyprus within the EU Requirements Demetris Kafouris*, Maria Christofdou, Markela Christodoulou, Eftychia Christou and Eleni Ioannou-Kakouri State General Laboratory, Ministry of Health, Kimonos 44, 1451 Nicosia, Cyprus Keywords: Multiycotoxin analysis; UPLC-MS/MS; Nuts; Cereals; Afatoxins; Ochratoxin A, Zearalenone; Deoxynivalenol; Fumonisins; T-2 toxin; HT-2 Toxin Introduction Mycotoxins are natural chemical contaminants produced as toxic secondary metabolites by some fungal species such as Aspergillus, Penicillium and Fusarium. Mycotoxin formation is afected by several biological factors, by harvesting, storage and processing conditions, insect damage in agricultural crops and fnally by climate changes (temperature, humidity) [1,2]. Consumption of food and feed contaminated with mycotoxins can cause severe efects on animal and human health, from allergic responses to cancer and death. Due to their high occurrence, the most common and widely investigated mycotoxins in food and feed, regulated by the European Union legislation [3,4] are: Afatoxins (AFB 1 , AFB 2 , AFG 1 , AFG 2 ), Ochratoxin A (OTA), trichothecenes [Deoxynivalenol (DON), T-2 and HT-2 toxins], Fumonisins (FB 1 , FB 2 ) and Zearalenone (ZON) [5-7]. AFs are primarily produced by Aspergillus favus and Aspergillus parasiticus and are among the most carcinogenic substances known. AFs are signifcant in nuts, maize and cereal grains [1,8]. OTA has been found in cereals (wheat, barley, oats and maize), spices and dried fruits and is primarily nephrotoxic and considered to be a possible human carcinogen. OTA is produced by Aspergillus ochraceus, Penicillium verrucosum and Penicillium viridicatum. Fumonisins, trichothecenes and ZON are produced by Fusarium species. Tese mycotoxins are mostly found in cereals, especially in maize [1,8]. Fumonisins have cancer promoting activity and mostly responsible for esophageal carcinoma. ZON can cause oestreogenic efects such as infertility, abortion and cervical cancer. Trichothecenes are related with fatal and chronic toxicoses. Tey inhibit DNA and protein synthesis, and they are responsible for outbreaks of acute diseases of the digestive system such as nausea, vomiting, diarrhea, dizziness and headache [1,7]. Several analytical (chemical and biochemical) methods have been developed for the determination of individual mycotoxins afer immunoafnity column clean-up [9-15]. Chromatographic methods commonly used for the quantitative determination of mycotoxins in foodstufs includes thin layer chromatography (TLC) [16], high performance liquid chromatography (HPLC) coupled with ultraviolet (UV), photo diode array (PDA), fuorescence detectors (FLD) or mass spectrometry (MS), and gas chromatography (GC) coupled with electron capture (ECD), fame ionization (FID) or MS detectors [17]. Due to the high toxicity and occurrence of mycotoxins, rapid and reliable screening methods need to be developed for their identifcation and quantifcation in foodstuf in order to ensure safety and compliance with the legislation. For the past years, multi-target methods for the simultaneous detection and quantifcation of diferent, co-occurring mycotoxins have been developed to replace the single analyte methods. Most of these methods are based on the combination of high- or ultra-high- performance liquid chromatography with tandem [18-21] or high- resolution [22,23] mass spectrometry. A major advantage of LC-MS- based multi-target methods is the elimination of the need for sample derivatization, in addition with the high selectivity and sensitivity. Furthermore, such multi-target methods are suitable for the analysis of highly variable mycotoxin concentrations. Mycotoxins present a great diversity in their physicochemical properties, therefore the optimization of an efective and efcient extraction procedure is of great importance. Mixtures of water with high amounts of methanol or acetonitrile (>70 %) are appropriate as extraction solvents for most mycotoxins. However, for fumonisins, higher extraction recoveries are achieved when the water proportion is increased and/or the pH of the solvent is decreased [19]. For the cleanup, multi-target methods use simple “dilute-and-shoot” approaches [19-20,24,25], solid phase extraction [21,26,27], immunoafnity columns [28,29] and, more recently QuEChERS methodology (Quick, Easy, Cheap, Efective, Journal of Food Processing & Technology J o u r n a l o f F o o d P r o c e s s i n g & T e c h n o l o g y ISSN: 2157-7110