Wageningen Academic World Mycotoxin Journal, February 2014; 7 (1): 3-33 Publishers

Developments in mycotoxin analysis: an update for 2012-2013

F. Berthiller1, P.A. Burdaspal2, C. Crews3, M.H. Iha4, R. Krska1, V.M.T. Lattanzio5, S. MacDonald3, R.J. Malone6, C. Maragos7, M. Solfrizzo5, J. Stroka8 and T.B. Whitaker9

1University of Natural Resources and Life Sciences, Vienna, Department for Agrobiotechnology (IFA-Tulln), Christian Doppler Laboratory for Mycotoxin Metabolism and Center for Analytical Chemistry, Konrad Lorenz Str. 20, 3430 Tulln, Austria; 2National Centre for Food, Spanish Food Safety and Nutrition Agency, Carretera de Majadahonda a Pozuelo km 5, 228220 Majadahonda, Spain; 3The Food and Environment Research Agency, Sand Hutton, York YO41 1LZ, United Kingdom; 4Instituto Adolfo Lutz, Laboratrio I de Ribeiro Preto, Av Dr Arnaldo 355, CEP 14085-410, Ribeiro Preto SP, Brazil; 5Institute of Sciences of Food Production, National Research Council, Via Amendola 122/o, Bari 700126, Italy; 6Trilogy Analytical Laboratory, 870 Vossbrink Drive, Washington, MO 63090, USA; 7USDA, ARS National Center for Agricultural Utilization Research, 1815 N. University St., Peoria, IL 61604, USA; 8Institute for Reference Materials and Measurements (IRMM), European Commission Joint Research Centre, Retieseweg 111, 2440 Geel, Belgium; 9Biological and Agricultural Engineering Department, N.C. State University, P.O. Box 7625, Raleigh, NC 27695-7625, USA; [email protected]

Received: 12 September 2013 / Accepted: 2 December 2013 © 2014 Wageningen Academic Publishers REVIEW ARTICLE Abstract

This review highlights developments in mycotoxin analysis and sampling over a period between mid-2012 and mid-2013. It covers the major mycotoxins: aflatoxins, Alternaria toxins, ergot alkaloids, fumonisins, ochratoxins, patulin, trichothecenes and zearalenone. A wide range of analytical methods for mycotoxin determination in food and feed were developed last year, in particular immunochemical methods and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS)-based methods. After a section on sampling and sample preparation, due to the rapid spread and developments in the field of LC-MS/MS multimycotoxin methods, a separate section has been devoted to this area of research. It is followed by a section on mycotoxins in botanicals and spices, before continuing with the format of previous reviews in this series with dedicated sections on method developments for the individual mycotoxins.

Keywords: aflatoxin, Alternaria toxins, ergot alkaloids, fumonisin, ochratoxin, patulin, trichothecene, zearalenone, sampling, multimycotoxin, botanicals, method development

1. Introduction and spices (section 4), aflatoxins (section 5), Alternaria toxins (section 6), ergot alkaloids (section 7), fumonisins This review continues from a previous paper that covers (section 8), ochratoxins (section 9), patulin (PAT, section analytical developments in mycotoxin determination over 10), trichothecenes (section 11) and zearalenone (ZEA, the period mid-2011 to mid-2012 (Shephard et al., 2013). section 12). As in the previous paper, this review emphasises new methodology published over the title period, although A number of more general articles on mycotoxin some natural occurrence data have been included, especially developments have been published lately. Tabata (2012) where it demonstrates the applicability of new methods. summarised the development of analytical methods Also some toxicological findings are addressed at the in particular for the determination of aflatoxins, PAT, discretion of the authors. Topics covered are sampling ochratoxins and citrinin including the research for and sample preparation (section 2), multimycotoxin food safety from a Japanese perspective. Moreover, the liquid chromatography-tandem mass spectrometry (LC- current status and prospects for advanced hyphenated MS/MS) methods (section 3), mycotoxins in botanicals chromatography-mass spectrometry in mycotoxin

ISSN 1875-0710 print, ISSN 1875-0796 online, DOI 10.3920/WMJ2013.1637 3 F. Berthiller et al.

of variation (CV) associated with the sampling step for determination has been reviewed extensively by Li et manual and automatic sample selection methods was al. (2013a). A comprehensive overview of the current 8.9 and 6.4%, respectively. The CVs associated with the knowledge on plant metabolites of mycotoxins, also called sample preparation and analytical steps were about the same masked mycotoxins, was recently published (Berthiller (approximately 3%) for both sample selection methods. et al., 2013). The decontamination of mycotoxins, in particular by microbes or their enzymes, has been subject Lippolis et al. (2012) studied the variability and distribution to a book chapter (Juodeikiene et al., 2012) and a review among 100 test portions used to measure deoxynivalenol (McCormick, 2013). Finally, a book on the improvement of (DON) and ochratoxin A (OTA) concentrations when two public health through mycotoxin control has been released 10 kg laboratory wheat samples (each naturally contaminated by the International Agency for Research on Cancer with DON and OTA) were prepared using dry comminution (edited by Pitt et al., 2012). Dedicated chapters deal with and water-slurry mixing. The DON distribution among mycotoxin producing fungi, physicochemical attributes the 100 test portions could be represented by a normal of mycotoxins, sampling, analysis, effects of mycotoxins distribution for both dry comminution and water-slurry in animals and humans, risk assessment, economics and mixing. The variability, represented by the CVs, among practical approaches to control mycotoxins. the 100 test portions for DON measurements were 4.6 and 6.4% for water-slurry and dry comminution, respectively. 2. Sampling and sample preparation The small CVs suggest that the variability associated with both sample preparation methods was negligible and was Improvements in sampling and sample preparation methods mostly due to the analytical step. The OTA distribution used to detect mycotoxins and other quality attributes in among the 100 test portions for dry comminution was food and feed products continues to be a high priority positively skewed while the OTA distribution among the among regulatory agencies, international organisations 100 test portions for water-slurry mixing could be described and commodity industries worldwide. As in recent years, by the normal distribution. The variability among the 100 more studies are investigating the effect of sample selection test portions for OTA measurements was 4 and 75% for methods on the additional variability added to the sampling water-slurry and dry comminution, respectively. Results step by various selection techniques. For instance, the clearly indicated that slurry mixing continued to reduce Codex Committee on Contaminants in Foods (CCCF) particle size and reduce sample preparation variability met 26-30 March 2012 to discuss harmonised aflatoxin specifically for OTA, but not DON. maximum levels (ML) and aflatoxin sampling plans for dried figs traded in the international markets. CCCF 3. Multimycotoxin LC-MS(/MS) methods delegates agreed on a ML of 10 µg/kg total aflatoxins and a sampling plan that required three 10 kg samples of dried A survey of the available literature relevant to the period figs to all test below the ML of 10 µg/kg total aflatoxins 2012-2013 shows that sample preparation continues to (3×10 kg <10 µg/kg) to accept a lot for trade. The ML be the most challenging task in multimycotoxin analysis. and sampling plan were sent forward for adoption by the Strategies for sample preparation based on either traditional Codex Alimentarius Commission (CAC). The CAC met clean-up devices or innovative systems were evaluated for 2-7 July 2012 and adopted the aflatoxin ML and sampling their contribution in satisfactory method performances, plan for dried figs as the official Codex Standards (Codex including acceptable matrix effects. Alimentarius Committee, 2012). An LC-MS/MS method based on a combination of Mallmann et al. (2013) studied the efficiency of two sample direct injection (after dilution) and extract purification selection methods to detect fumonisins in maize. The first using MultiSep®226 (Romer, Union, MO, USA), has been sample selection method was a manual system using a developed to monitor the co-occurrence of DON, ZEA, sampling probe or spear to select whole grain samples OTA, FB1, FB2, T-2 (T-2) and HT-2 (HT-2) toxins in animal from a lot. The second sample selection method was a feeds (Åberg et al., 2013). Method apparent recovery continuous flow automatic system to select milled maize was higher than 86% for all the mycotoxins and limits of samples from a lot. Ten samples, one kg each (10×1 kg) quantification (LOQs) ranged from 2.5 to 115 μg/kg. Two were taken from each of 11 lots by each sample selection examples of the use of accelerated solvent extraction (ASE) method. Using a nested design, the total variance associated coupled with solid phase extraction (SPE) clean-up for with measuring fumonisins B1+B2 (FB1+FB2) among the multimycotoxin extraction have been reported. An LC-MS/ 10×1 kg samples was partitioned into sampling, sample MS method with a pressurised liquid extraction followed preparation and analytical variances. For both the manual by clean-up through polymeric SPE cartridges (Oasis HLB, and automatic sample selection methods, the sampling Waters, Milford, MA, USA) has been developed for the step was the largest source of error accounting for 71 and simultaneous determination of aflatoxins and OTA in a 40% of the total variation, respectively. The coefficient range of animal derived foods (Chen et al., 2012). A similar

4 World Mycotoxin Journal 7 (1) Developments in mycotoxin analysis: an update for 2012-2013

approach, which involved ASE and homemade clean-up was that LC-MS/MS analysis of samples without clean-up cartridges consisting of silica gel, has been proposed for is adequate for screening, but when using LC-MS/MS for the extraction and purification of 35 mycotoxins in various definitive measurements (e.g. for food regulatory control traditional Chinese medicine matrixes (Han et al., 2012a) purposes) IAC clean-up remains essential. for subsequent analysis by ultra high performance liquid chromatography (UHPLC)-MS/MS. Both methods showed On the other side, the direct injection of crude extracts limits of detection (LODs) in the sub µg/kg range and coupled with highly sensitive instrumentation has been used satisfactory recoveries higher than 68%. to develop relatively rapid methods. A UHPLC-MS/MS method based on direct injection of diluted extracts has The use of TurboFlow™ technology, a new automated on been proposed for the determination of 18 mycotoxins line sample clean-up system, directly coupled to an LC-high in different food matrices (Beltrán et al., 2013). Sample resolution mass spectrometry (HRMS) equipment, has preparation consisted of extraction with acidified MeCN/ been described by Ates et al. (2013) for the simultaneous water. A defatting step with MeCN/hexane was introduced determination of Fusarium toxins (DON, T-2, HT-2, ZEA, for oil samples. The dilution of the sample extract was FB1 and FB2) in maize, wheat and animal feed. Toxins were applied to minimise matrix effects. As stated by the authors, extracted with an acidified water-acetonitrile (MeCN) this was feasible due to the high sensitivity of the modern mixture. Average recoveries, relative standard deviations instrumentation used. Extraction recoveries between 70 (RSDs) and intermediate precision values were 71-120%, and 120% and RSDs lower than 20% were obtained for the 1-19% and 4-19%, respectively. The main perspective of this majority of analyte-matrix combinations, whereas LOQs technology, coupled with full scan analysis by HRMS, is the were ≤1 μg/kg for most of the toxins. A similar approach was inclusion of masked mycotoxins and/or the identification developed to detect 26 mycotoxins (aflatoxins, ochratoxins, of other metabolites by retrospective analysis. fumonisins, trichothecenes and ergot alkaloids) in maize, rice, wheat, almond, peanut and pistachio products by Also this year the increasing interest in evaluating the use LC-MS/MS (Liao et al., 2013). Homogenised grain or nut of HRMS for multimycotoxin analysis in comparison with products were extracted with MeCN:water (85:15, v/v), triple quadrupole-based methodologies was confirmed. followed by high-speed centrifugation and dilution with Rubert et al. (2013) compared two mass spectrometry water. Mean recoveries were in the range 84-104%, with strategies for multiple mycotoxin analysis in European RSDs lower than 16%. LOQs ranged from 0.2 to 12.8 μg/kg, beers. LC-HRMS (based on Orbitrap technology) was used depending on the mycotoxin. for unambiguous identification of 18 target mycotoxins, as well as for screening non-target mycotoxins, such as Based on the number of publications (more than 10) in the enniatins, fusaproliferin and DON-3-glucoside (DON-3G), period 2012-2013, the use of QuEChERS (quick, easy, cheap, whereas mycotoxin quantification was carried out using effective, rugged and safe) as generic sample pre-treatment a triple quadrupole (LC-QqQ) instrument. The authors is becoming a popular alternative to the dilute-and-shoot concluded that the LC-QqQ was most satisfactory for approach. The effectiveness of four extraction methods quantification purposes, whereas LC-HRMS improved (modified QuEChERS, matrix solid-phase dispersion unambiguous identification and screening capabilities. (MSPD), solid-liquid extraction and SPE clean-up) was evaluated for simultaneous determination of 32 mycotoxins Apparently not in line with the trend of simplifying the by UHPLC-HRMS (Rubert et al., 2012). QuEChERS clean- sample preparation and of joining targeted and untargeted up was fast and yielded satisfactory recoveries (from 64- screening, selective clean-up based on multi-antibody 93%) for the widest range of mycotoxins. A QuEChERS- immunoaffinity columns (IAC) continues to represent based extraction procedure has also been used to develop a valuable tool to develop robust methodologies and to an LC-MS/MS method for the determination of multiple achieve low detection limits. The use of multi-antibody mycotoxins in highly pigmented spice matrices, namely red IAC, Myco6in1™ (Vicam, Watertown, MA, USA), has been chilli (Capsicum annum ssp.), black and white pepper (Piper re-proposed prior to UHPLC-MS/MS for determination of nigrum ssp.) (Yogendrarajah et al., 2013). Significant matrix regulated mycotoxins, namely aflatoxins, fumonisins, DON, effects were observed but compensated using matrix- ZEA, OTA, T-2 and HT-2, in grains (Tang et al., 2013). The matched calibration curves. Satisfactory recoveries (from average recovery rates in rice, maize, wheat and peanut were 75-117%) and intra-day reproducibility (from 4-22%) were mostly between 70 and 120%, with RSDs less than 15%. The obtained for most of the mycotoxins. The LOQs ranged LOQs ranged between 0.5 and 20 µg/kg. An interesting from 2.3 to 146 μg/kg. A QuEChERS-like procedure critical comparison between the use of IAC and direct prior to UHPLC-MS/MS analysis was developed for the analysis of crude extracts has been published by Senyuva determination of 34 mycotoxins in dietary supplements et al. (2012). LC-MS/MS ion ratios and peak profiles, containing green coffee bean extracts (Vaclavik et al., repeatability and LOQs were used as the basis for a detailed 2013). Average recoveries of the analytes were in the comparison of the two approaches. The overall conclusion range of 75-110% with RSDs below 12%. LOQs ranged

World Mycotoxin Journal 7 (1) 5 F. Berthiller et al.

from 2.5 to 100 μg/kg. Also in this case, significant matrix effects whereas external calibration gave reliable results effects were observed and corrected by standard addition by injecting <10 mg of matrix equivalent amounts. It is method. Finally, the use of QuEChERS in combination worth mentioning that unacceptable high recovery and with dispersive liquid-liquid microextraction (DLLME) high variability of fumonisin results were obtained by the has been proposed for the UHPLC-MS/MS determination majority of laboratories for spiked maize. Results of an of 15 mycotoxins in milk thistle (Silybum marianum), inter-laboratory study concerning relative and absolute including aflatoxins, fumonisins, trichothecenes, OTA, matrix effects in multimycotoxin determination have been citrinin, sterigmatocystin and ZEA (Arroyo-Manzaranes et reported by Malachova et al. (2013). The applicability of al., 2013). The method was validated in extract and seeds commonly used strategies in matrix effect reduction was of milk thistle, obtaining LOQs lower than those usually tested in the quantitative determination of nivalenol (NIV), required by legislation in food matrices, with precisions DON, FB1, FB2 and ZEA in complex feed matrices. The lower than 10%. Recoveries were between 62 and 99%, study showed that the use of any purification technique except for ZEA and citrinin. helped to improve absolute matrix effects for some analytes, whereas other factors such as changes in LC conditions or Being now routinely used to compensate matrix effects, switching of ion source polarity had a minor impact. These different approaches for fully isotope-labelled mycotoxins as studies provide a great deal of information on currently internal standard have been studied. A novel normalisation used methodologies enabling a deeper understanding on approach of the signal response of 32 mycotoxins was the performances of different LC-MS-based approaches investigated using the isotopically labelled versions of only for multimycotoxins analysis in real food matrices. three mycotoxins, i.e. DON, aflatoxin B1 (AFB1) and ZEA, as both true isotopologues and as structurally different internal The increasing availability of LC-MS(/MS) methods standards. Mycotoxins were extracted via a single-step for multimycotoxin determination provides data about procedure using a mixture of MeCN/water/formic acid and the co-occurrence of multiple mycotoxins in the same the extracts were directly analysed by UHPLC-MS/MS. This sample, including a wide array of less known or ‘emerging’ approach reduced both the number of standard compounds mycotoxins and other metabolites. LC-MS/MS has been needed for quantification and therefore cost of analysis used to investigate the co-occurrence of beauvericin per sample (Jackson et al., 2012a). Zhang et al. (2013b) and enniatins (Blesa et al., 2012; Juan et al., 2013a) described a procedure for quantitative determination of also in combination with other Fusarium toxins such 12 mycotoxins (aflatoxins, OTA and Fusarium toxins) in as trichothecenes and zearalenones (Juan et al., 2013b; milk-based infant formula and foods using the relative Serrano et al., 2012), or fusaproliferin (Serrano et al., 2013) response factors of the 13C-uniformly labelled internal in cereals and cereal products. Further surveys provided standards and target mycotoxins. data on co-occurrence of major mycotoxins (aflatoxins, fumonisins, OTA, trichothecenes and ZEA) in a wide range Still far from harmonisation, efforts are being made of food matrices, such as Serbian wheat flours (Škrbić et for method comparison and deeper understanding of al., 2012), peanut cakes from Nigerian markets (Ezekiel performances of available LC-MS/MS methodologies et al., 2012), compound feeds from South Africa (Njobeh for multimycotoxin analysis. Within the EU network of et al., 2012), raw cereals and cereal foods from Tunisian Excellence MoniQA (www.MoniQA.eu) a proficiency test markets (Oueslati et al., 2012), staple food commodities was conducted to benchmark laboratories using LC-MS/ from Cameroon (Abia et al., 2013). Alternaria, Penicillium (MS) for multimycotoxins analysis and to obtain information and Aspergillus toxins were surveyed in wine, cider and their on currently used methodologies and related method cork stoppers from eight countries (Scussel et al., 2013). performances (De Girolamo et al., 2013; Solfrizzo et al., LC-MS/MS screening for a broader range of mycotoxins and 2013a). The study involved 41 laboratories from 14 countries other fungal metabolites showed the presence of up to 69 and was conducted for the simultaneous determination metabolites in a single feed sample (Streit et al., 2013; Warth of up to 11 mycotoxins (aflatoxins, OTA, FB1, FB2, ZEA, et al., 2012a) emphasising the great variety of potential DON, T-2 and HT-2) in spiked and naturally contaminated mycotoxin co-exposure. maize. Only two laboratories obtained acceptable results of z-score for all mycotoxins. In general, extraction mixtures of With use of advanced LC-MS(/MS) methods, low water with MeCN, methanol or both provided good results concentrations of mycotoxins and relevant metabolites for quantitative extraction of mycotoxins from maize. can be quantified simultaneously in biological fluids Laboratories using extract clean-up reported acceptable and tissues and be used as biomarkers. An overview of results for the majority of mycotoxins. Good results were published LC-MS/MS-based multibiomarker approaches also obtained by laboratories that analysed crude extracts for the assessment of human exposure to mycotoxin has although a high variability of results was observed for all been published by Warth et al. (2013). Furthermore, a tested mycotoxins. Matrix-matched calibration or isotope- recent comparison of single and multi-analyte LC-MS/MS labelled internal standards efficiently compensated matrix methodologies for mycotoxin biomarker determination in

6 World Mycotoxin Journal 7 (1) Developments in mycotoxin analysis: an update for 2012-2013

human urine provided insights in performances of methods approach is the most commonly used when developing based on direct injection or sample clean-up (Solfrizzo et multi-contaminant extraction, either in combination al., 2013b). In the last year, multi-analyte methods have with HRMS based on TOF (time-of-flight) or Orbitrap been developed for the identification and quantification of mass analysers or with last generation QqQ enabling fast several toxicological important mycotoxins in human urine, detection of multiple analytes. QuEChERS-like methods pig plasma and pig urine. Njumbe Ediage et al. (2012a) have been developed and optimised for the simultaneous developed an LC-MS/MS method for the determination of determination of 22 carbamate insecticides and 17 seven mycotoxins and their metabolites (including DON- mycotoxins in cereals (Zhang et al., 2013b) by UHPLC- glucuronide (DON-GlcA)) and ZEA-glucuronide (ZEA- MS/MS, for simultaneous analysis of veterinary drugs and GlcA)) in human urine. Urine samples were extracted with mycotoxins in hen eggs by LC-MS/MS (Capriotti et al., ethyl acetate/formic acid followed by SPE clean-up. For pig 2012b) and for purifying bakery raw materials and finished plasma, two LC-MS/MS methods have been developed products to detect pesticides, mycotoxins and veterinary for two sets of mycotoxins, namely for DON, deepoxy- drugs by UHPLC-HRMS (De Dominicis et al., 2012). On the deoxynivalenol (DOM-1), T-2, HT-2, ZEA, α-zearalenol other side, some authors proposed commodity-dedicated (α-ZOL), β-zearalenol (β-ZOL), α-zearalanol (α-ZAL), methods for the determination of a restricted number of β-zearalanol (β-ZAL), OTA, FB1 and AFB1 (Devreese et mycotoxins together with other contaminants of interest al., 2012) and for beauvericin and enniatins (Devreese et in specific matrices. Mornar et al.(2013) developed a al., 2013). In both cases, sample clean-up consisted of a method for simultaneous determination of citrinin and deproteinisation step using MeCN, followed by evaporation cholesterol-lowering compounds present in red fermented of the supernatant and re-suspension of the dry residue in rice using connected diode array and fluorescence (FLD) water/methanol mixture. However for toxicokinetic studies and MS detectors, whereas Song et al. (2013b) reported with individual mycotoxins, highly sensitive analyte-specific an LC-MS method for the simultaneous determination LC-MS/MS methods were developed within the same study. of AFB1, OTA and PAT together with bisphenol A for For pig urine, Song et al.(2013a) developed an LC-MS/ regulatory purposes, in beverages and food products. A MS method for simultaneous analysis of AFB1, DON, screening method for the detection and identification of FB1, OTA, ZEA, T-2 and their metabolites. A salting-out undesirable organic compounds, including antibiotics, assisted liquid-liquid extraction (LLE) procedure was used pesticides and mycotoxins in aquaculture products based for sample preparation. The developed method was also on direct injection of MeCN/water extracts in the LC- validated for human urine as an extension of its application. HRMS system, has been reported by Nácher-Mestre et al. Finally, Veršilovskis et al. (2012) reported on the use of (2013). Retrospective analysis of accurate mass full spectra LC-MS/MS for the simultaneous determination of masked provided by QTOF-MS enabled detection and tentative forms of DON and ZEA after oral dosing in rats. A total identification of other undesirable organic compounds of 21 metabolites were quantified in rat organs giving different from those included in the validated list. insights in metabolisms of masked mycotoxins. A multi- biomarker LC-MS/MS method was used to validate urinary 4. Mycotoxins in botanicals and spices biomarkers of five mycotoxins (DON, FB1, ZEA, OTA and AFB1) in piglets fed with boluses contaminated with There was an increase in published methods for mycotoxin increasing levels of the toxins (Gambacorta et al., 2013). determination in botanicals and spices this past year compared to previous years. This increase was mainly due A current trend in chemical food safety control is to the multiple LC-MS/MS methods recently published. represented by an effort to integrate analyses of various In addition, there were several new methods published groups of food contaminants/toxicants into a single, high- along with several surveillance studies using HPLC-FLD. throughput method. Again, the power of modern LC- There were also a group of publications that described MS(/MS) instrumentation to screen for a large number of methodologies specifically for the analysis of mycotoxins contaminants is employed. Increasing attention has been in Chinese herbal medicines. given to the choice of optimal sample preparation procedures that represents one of the key steps to achieve satisfactory Six published methods for mycotoxins in botanicals and performance characteristics. As an example, Lacina et spices used HPLC-MS/MS. Two of the methods described al. (2012) reported results of a study dedicated to test the analysis of aflatoxins and OTA in liquorice roots. The different sample preparation procedures: aqueous MeCN first method was developed for analysing liquorice roots extraction followed by partition (QuEChERS-like method), and fritillary bulbs for the simultaneous determination of aqueous MeCN extraction and pure MeCN extraction AFB1 and OTA using LC-MS/MS (Wang et al., 2013b). for the analysis of a list of target analytes, including 288 The samples were concentrated and purified using a low pesticides together with 38 mycotoxins. The QuEChERS- cost SPE clean-up prior to HPLC analysis. The precision like method showed the best performance in terms of co- of the method was excellent (RSD <2.8%) with analytical extraction of focused analytes. Indeed the QuEChERS recoveries ranging from 93-105%. The LOD and LOQ

World Mycotoxin Journal 7 (1) 7 F. Berthiller et al.

were 0.02 and 0.09 µg/kg, respectively. The second for the determination of the AFB1, AFB2, AFG1 and AFG2 method (Wei et al., 2013) described the analysis of AFB1, in peanuts, rice and chilli was developed (Khayoon et aflatoxin B2 (AFB2), aflatoxin G1 (AFG1), aflatoxin G2 al., 2012). The sample was extracted with MeCN:water (AFG2) and OTA in Glycyrrhiza uralnensis, one of the (90:10, v/v) and then purified using SPE. After a pre-column main sources of liquorice in China. This method used derivatisation, the analytes were separated within 3.7 min sonication extraction using methanol:water (80:20, v/v) using a RP-18 monolithic HPLC column. The recoveries followed by an IAC extract purification with HPLC-MS/MS of aflatoxins that were spiked into the samples were 86- detection. This purification method was used due to the 105% and the RSDs were <4.4%. Another method utilising complexity and high coloration of the liquorice root matrix. UHPLC-FLD was developed for the determination of OTA The LOQs of AFB1, AFB2, AFG1, AFG2 and OTA were in ginger using molecularly imprinted polymer (MIP)- 0.02, 0.02, 0.01, 0.05 and 0.01 µg/kg, respectively. The SPE clean-up (Cao et al., 2013). The recoveries of OTA results of analysing a total of 15 samples of G. uralnensis from spiked ginger samples ranged from 87-94%. After showed that almost all the samples were contaminated a regeneration procedure, this clean-up column could be with aflatoxins and OTA. This was the first report of the reused at least forty-one times to obtain more than 80% co-occurrence of aflatoxins and OTA in G. uralnensis in recovery of OTA from ginger samples. Other surveillance China. A multimycotoxin method validation was published studies from Turkey, Malaysia and Pakistan investigated the (Škrbić et al., 2013) utilising UHPLC-MS/MS for the incidence of aflatoxins and OTA in spices using HPLC-FLD analysis of crude extracts of red and black pepper. This and IAC (Iqbal et al., 2013; Jalili and Jinap, 2012; Ozbey validation was performed to establish a high-throughput and Kabak, 2012). multi-matrix procedure for the simultaneous determination of mycotoxins included in the EU regulations for spices. A number of articles were published this past year The data obtained on red pepper samples fortified with specifically describing methodologies for the analysis of AFB1, AFB2, AFG1, AFG2 and OTA were in compliance mycotoxins in Chinese medicinal plants. One published with these regulations. However, validation parameters article reviewed the advancement of detection methods for analysis of some mycotoxins (recoveries and LODs for mycotoxins in Chinese herbal medicines (Xu et al., for AFG1, AFG2 and OTA) did not comply. Another 2012). The natural occurrence of citrinin in red yeast publication evaluated different extraction procedures for rice, medicinal plants and their related products was the simultaneous determination of pesticide residues and investigated for the first time (Li et al., 2012d). The mycotoxins in fruits, cereals, spices and oil seeds employing samples were extracted by methanol/water, purified with UHPLC-MS/MS (Lacina et al., 2012). Acceptable recoveries IAC for citrinin and quantified by HPLC-FLD. The mean (70-120%) for most of the analytes were obtained by the recoveries on spiked samples ranged from 73-93% with analysis of spiked matrices. However, during the analysis RSDs of 1.4-7.9%. Out of a total of 109 samples, citrinin was of dry samples with incurred analytes, pure MeCN did detected in 31 of the red yeast rice matrices ranging from not efficiently extract some common contaminants. The 16.6 to 5,253 µg/kg. A gas chromatography (GC)-electron development and validation of an HPLC-MS/MS method capture detection (ECD) method was developed for the for the simultaneous quantification of AFB1, AFB2, AFG1 simultaneous determination of T-2 and HT-2 in Chinese and AFG2 in lotus seeds was published (Liu et al., 2013a). herbal medicines and related products (Kong et al., 2012). The samples were extracted with methanol:water solution The method used IAC for sample extract purification and (80:20, v/v) and then purified using IAC. Recoveries for a pre-column derivatisation with N-heptafluoro-butyryl samples of spiked lotus seeds were all above 66% with the imidazole. The LODs for T-2 and HT-2 were 1.9 and RSDs all below 15%. Nineteen out of twenty samples of 0.5 µg/kg, and the recoveries for different herbal matrices lotus seeds collected from different stores and markets in ranged from 89 to 99%. T-2 and HT-2 were not detected China were found to be contaminated with aflatoxins at in all 89 Chinese herbal medicines analysed using this different levels ranging from 0.02 to 688.4 µg/kg. methodology.

Several methods were published utilising HPLC-FLD for 5. Aflatoxins the analysis of mycotoxins in spices and botanicals. A collaborative study was performed to validate an analytical Aflatoxins remain of interest, either as model substances method for the determination of OTA in liquorice and to prove the concept of new immunoassays or sensors or liquorice extracts (Lerda et al., 2013). The samples were as target analytes in a number of multimycotoxin methods. extracted with a mixture of methanol and aqueous sodium Certainly the current situation of an apparent increased bicarbonate, purified with IAC and quantified by HPLC- aflatoxin occurrence in agricultural products will promote FLD. The mean recoveries were 87% for liquorice root and methods for early as well high throughput screening. 86% for liquorice extracts. The RSDs for reproducibility ranged from 10-17% and from 11-22% in liquorice extracts Taking note of the rapid progress of immunochemical and liquorice roots, respectively. An HPLC-FLD method methods, 133 publications mainly of the last 3 years have

8 World Mycotoxin Journal 7 (1) Developments in mycotoxin analysis: an update for 2012-2013

been reviewed (Li et al., 2012a) for current development of Degola et al. (2012) described a fluorometry method for the microfluidic immunosensing approaches for mycotoxins determination of aflatoxins in culture media. The method including aflatoxins. Another review (Li et al., 2012c) reads the induced fluorescence of the culture media and summarises the developments in surface plasmon resonance allows the evaluation of the aflatoxins progression in the (SPR) biosensors for mycotoxins, including aflatoxins. The media. The method was compared with an LC method, and review covers 28 publications of the years 1999-2011. A a good correlation between the observed fluorescence and few new immunoassays and sensors have been described the determined aflatoxins was achieved. The addition of by various authors (Abhijith and Thakur, 2012; Dinçkaya et cyclodextrins further facilitated the procedure. The authors al., 2012; He et al., 2012; Jiang et al., 2013; Li et al., 2012b, showed the usefulness of the method for monitoring 2013b; Linting et al., 2012; Masoomi et al., 2013; Nachi the production or inhibition of aflatoxins in cultures. Rossi et al., 2012; Wang et al., 2013d; Xu et al., 2013; Yu Unfortunately, the authors did not address to what degree et al., 2013; Zhang et al., 2012) and a number of method their procedure is of value for other fungal metabolites that parameters that are specific to the test system applied are also exhibit fluorescence like kojic acid (after reaction with given by the authors. However, as method performance peroxidase from the seed). Therefore, some knowledge of depends on the availability of the antibodies used, which are the metabolic products of the cell cultures tested seem to either not readily available and often raised in the laboratory be important. Chen and Zhang (2013) reported the use of the authors, arrive as a gift, or are only temporarily of ASE for aflatoxin-fortified grains. Various extraction available in case of polyclonal antibodies, these methods parameters (temperature, solvent, time) were investigated are not further reviewed for their performance and the and some indicated satisfactory performance comparable claims made, but are mentioned for completeness. to conventional extraction and clean-up procedures. The authors (like others before) did unfortunately not address The inhibition of acetylcholinesterase by AFB1 was the issue of the possibility of analyte migration into the used (Pohanka, 2013) for its determination in a matrix, rather than the assumption of matrix extraction. spectrophotometric assay. The author describes the system This is, however, an important aspect as similar procedures with respect to many variables for neat AFB1 in solution. (parboiling) are used to promote the migration of potential Ma et al. (2013a) developed a new immunoaffinity column analytes (niacin or calcium) into the matrix (rice), rather linking the antibodies to silicagel as carrier. They report than extracting them. Jinap et al. (2012) described an in- on various loading, washing and elution parameters of line IAC clean-up coupled to HPLC. The method offers the developed product, which have been described in the advantage that the IAC (mounted via a six port valve) the past having the potential of being reusable. Authors can be used for a number of cycles and facilitates aflatoxin considered the aspect of reusing their columns but also concentration and purification by direct transfer onto the mention the restrictions that apply. Oasis HLB® columns chromatographic column. The method was tested for a (Waters) have been proposed (Wang et al., 2012b) for the number of performance parameters (recovery, repeatability purification of milk and milk powder as alternative for and intermediate precision) that all met current provisions IAC. The method was tested on various dairy products for methods in official control laboratories. Furthermore, and the authors reported recovery rates of approx. 80- the authors demonstrated the specificity of their IAC used 90% with repeatabilities of less than 5%. The supplied and lack of cross-reactivity to other potential interfering chromatograms show, however, that the purification by analytes. immunoaffinity is superior, while the one by Oasis HLB appears to be sufficient for the described purpose. A similar A clean-up procedure for aflatoxins in edible oil that is based approach was followed by Tamura et al. (2012) who used on IAC clean-up with further manipulation by DLLME an Oasis HLB column and a MycoSep® column (Romer) has been described (Afzali et al., 2012). The proposed for clean-up for the determination of aflatoxins next to 10 method was extensively studied for various parameters other mycotoxins in wine. After validation, the method was and validated, however, it lacked the demonstration that used to test red and white wines. Only fumonisins and OTA it is superior to conventional IAC clean-up followed by could be identified in a few cases. Bertuzzi et al. (2012) evaporation, especially as no interferences were reported. compared currently used methanol- and acetone-based Interesting is that the authors used an elution solvent based extractants at various ratios and identified that a ratio of on diluted MeCN with an aqueous NaNO3 solution. This 6+4 (acetone:water) favours better extraction yields for solution allowed a direct injection of a larger aliquot of the total aflatoxins. Other ratios favour AFB1, while one key IAC eluate onto an HPLC system. A method for a cluster of observation was that the used IAC was found to be robust almost 40 analytes ranging from insecticides to mycotoxins for all aflatoxins up to a concentration of 8.5% acetone in cereals has been reported by Zhang et al. (2013b). Next after 10-fold extract dilution. The method was applied to to some Fusarium toxins and OTA, aflatoxins were subject maize and resulted in recoveries above 90% with acceptable of investigation. The method is based on QuEChERS clean- repeatability figures. up followed by LC-MS. Recovery values and precision data were generated over a range of 5-50 µg/kg for rice,

World Mycotoxin Journal 7 (1) 9 F. Berthiller et al.

wheat and maize. Recovery values for all aflatoxins were products for the simultaneous purification of AFB1 and T-2 above 85%, with repeatability values of less than 15% and toxin in plasma, the authors used a self-prepared clean-up slightly higher levels for intermediate precision. Song et column on the basis of silica gel and florisil after testing al. (2013b) described an LC-MS method with SPE clean- the performance of this clean-up in comparison with other up for the determination of AFB1, next to OTA, PAT and alternative clean-up materials, such as charcoal, aluminium bisphenol A, a rather heterogeneous combination for a oxide and kieselguhr. LOQs of 0.05 ng/ml, recoveries of 71- variety of matrices, i.e. peanut butter, cereals and fruit 108% and RSDs below 14% were achieved for both toxins juices. Even though the methodological components have in plasma and tissue samples. A multitoxin LC-MS method been described before, the authors managed to tune the has been described for a variety of mycotoxins in the method for satisfactory performance for each matrix. In botanical Silybum marianum (Arroyo-Manzanares et al., particular, aflatoxin recoveries ranged from 70-93% for dry 2013). The method combines QuEChERS clean-up followed foods, while all precision experiments demonstrated figures by DLLME prior to identification and quantification by below 15% (typically below 10%). The authors found that LC-MS. The authors compared the effectiveness of both matrix-matched calibration is key for the performance, clean-up steps with respect to ion suppression. Precision but only demonstrated the performance on solvent-spiked figures over different levels were below 10% (typically materials. This might be critical in that respect that solvent- around 5-7%) for all tested mycotoxins, including AFB1. spiked analytes (e.g. AFB1) might have different extraction The authors analysed commercial samples and confirmed behaviour depending on the matrix (e.g. peanut butter) and the presence of mycotoxins, but not of AFB1. The method the way they are incurred and extracted. Wang et al. (2013b) performance figures are comparable with those reported described a method for the determination of AFB1 and OTA by Zhang et al. (2013b) and Afzali et al. (2012) who used in liquorice roots and fritillary bulbs based on an SPE clean- similar methodologies for clean-up. However, it should up with C18 material followed by LC-MS. They reported be taken into account these authors covered a different precision data of less than 3% for both analytes with a range of mycotoxins and other matrices. Song et al. (2013a) recovery rate above 90%. Moreover, the LOQs for AFB1 developed a method for the assessment of a number of and OTA were 0.024 and 0.095 µg/kg, respectively. The mycotoxins, including AFB1 and aflatoxin M1 (AFM1), authors used the method to analyse more than 20 samples, as biomarkers in urine. The method made use of LLE half of which were liquorice. As a result, the co-occurrence followed by LC-MS. Recovery was assessed to range from of both analytes was investigated. Different from previous 70-108% for all analytes with an intermediate precision of studies, they could only identify OTA in a single sample less than 25% for most analytes when tested at different despite a rather low LOQ, while other studies indicated concentrations. The method confirmed the presence that the majority of tested samples were contaminated with of AFB1 next to other mycotoxins in pig urine samples. OTA. This is confirmed by one of the authors who, despite Abia et al. (2013) described an LC-MS-based multitoxin substantial efforts, was unable to identify blank liquorice method that includes aflatoxins and some of its precursors roots for OTA during a collaborative method validation, in the metabolic pathway (averufin and versicolorins). It unless pealed and treated. These different findings in the was reported to be suitable for nuts, cereals and products occurrence of OTA (and possibly AFB1) using different thereof (e.g. beer). The method required no clean-up and methodologies are surely an interesting aspect of further confirmed a high abundance of aflatoxins in local products. investigation to clarify which factors affect the presence of OTA in liquorice. An clean-up for the determination of AFM1 as an alternative to IAC has been proposed by Biancardi et al. (2013). The Devreese et al. (2012) developed and validated a multi- authors used liquid-liquid extraction of defatted milk with mycotoxin method for animal blood sera, including OTA. ethyl acetate and subsequent measurement by LC-MS. The method was benchmarked for comparison with single The method was compared with the official ISO method analyte methods and found sufficient for the purpose, 14501 and proposed as an alternative one. The method despite slightly higher LOQs. The method is considerably did not use any proprietary products, which is an asset simple, covering 11 analytes. In practice, the sample is for analysts in infrastructural or economically challenged deproteinised using MeCN and evaporated. The dried regions. Unfortunately, the authors only applied this new residue is taken up in aqeous methanol and injected into an concept to LC-MS, leaving the question unanswered LC-MS system. The working range is 5-1000 µg/l with an whether their novel clean-up procedure is also suitable for LOD of 0.05 µg/l for AFB1. The recovery was determined LC-FLD. The IAC purification drawbacks highlighted by the at 99%. Acceptable precision limits were calculated for authors appear a crucial driver for the search of a clean-up different levels according to Horwitz (1982), while a alternative. This highlights how diverse the experiences of goodness-of-fit coefficient for the calibration was reported different research groups are with respect to IACs. While with a magnitude of 4.1%. Han et al. (2012b) studied the some scientists further implement the application of toxicokinetics of AFB1 and T-2 toxin using a stable isotope IACs in methods for improving the performance of their mass spectroscopic method. Instead of specific clean-up methods, others put effort to find alternatives for the same

10 World Mycotoxin Journal 7 (1) Developments in mycotoxin analysis: an update for 2012-2013

reason. As a result, an in-depth consideration of pro and 6. Alternaria toxins contra arguments is key to identify suitable methods for different scenarios that apply to individual needs. Chen The stable isotope dilution assay (SIDA) approach was used et al. (2012) described a method based on pressurised by Liu and Rychlik (2013) to develop an LC-MS/MS method liquid extraction with SPE clean-up followed by LC-MS for for the determination of tentoxin (TTX), dihydrotentoxin the determination of a number of aflatoxins and OTA in (DHT) and isotentoxin (isoTTX) in different food products. animal products, including organs, muscle tissue, egg, milk Triply deuterated internal standards were prepared via total and fat. The method was evaluated for precision (<17% in synthesis. Samples were extracted with MeCN-water (84:16, all cases), recovery at 0.25 and 1 µg/kg (68-106%) as well v/v) and purified on a C18-phenyl SPE column providing as other important performance provisions according to a more selective binding of these phenyl-containing cyclic Commission Decision 657/2002/EC, e.g. CCα and CCβ, peptides. For the three toxins LODs ranged from 0.10 to identification points for MS, selectivity and linearity (EC, 0.99 µg/kg, recoveries ranged between 98 and 115% and 2002). Among the 18 tested samples, AFB1 was detected in method precision was below 9%. The method was applied 8 samples, while a residue level of around 1 μg/kg of AFG1 to 103 food samples and 85% were found contaminated was detected in swine liver and kidneys. with TTX at levels up to 52 µg/kg, 55% with DHT at levels up to 36 µg/kg whereas isoTTX was not quantifiable. The In the review period, only two methods have been SIDA approach was also used by Lohrey et al. (2013) who published that concerned the inter-laboratory validation developed an LC-MS/MS method for determination of of analytical methods, a classical approach to validate tenuazonic acid (TeA) in tomato and pepper products. 13 methods for performance parameters providing evidence Isotopically labelled internal standard C2-TeA was for implementation of the method in laboratories beyond synthesised and added to samples before extraction. the laboratory of the method developer. Bao et al. (2012) Samples were slurried with water, extracted with MeCN- studied a method using a classical concept (IAC with HPLC- formic acid (10:1.1, v/v) and sample extracts were purified FLD) in a collaborative trial study for olive, peanut and by using a QuEChERS approach. Values of LOD and LOQ sesame oils. The levels tested ranged from 2 to 4 µg/kg were 0.9 and 2.9 µg/kg, respectively. Acceptable values of (total aflatoxins) and resulted in HorRat values of <0.5 for recoveries (91-99%) and precision (1-6%) were reported. total toxins as well as AFB1. Recovery rates were 91-93%. The application of this method to samples from a German As a result, the method demonstrated suitable performance market revealed the presence of TeA in all 30 analysed characteristics meeting the requirements by AOAC samples at levels ranging from 3 to 2,330 µg/kg. International following the IUPAC protocol for the design, conduct and interpretation of method performance studies A comparison of the performances of two LC-MS/MS (Horwitz, 1995) and was accepted as an official method of methods for the determination of TeA and its analogues analysis. Bailey and Davis (2012) described the validation was performed on sorghum-based infant food (Asam according to AOAC International provisions of a lateral flow et al., 2013). One method involved derivatisation with kit that allows the quantification of total aflatoxins and is 2,4-dinitrophenylhydrazine and the other no derivatisation. designed for a working range of 0-100 µg/kg. Interestingly, A good chromatographic separation of TeA and its four the authors tested the kit over a temperature range of 18- analogues was obtained whereas the separation of TeA 30 °C and found it was stable over this temperature range. and TeA analogues dinitrophenylhydrazones was only marginal, and the isobaric dinitrophenylhydrazones of TeA Two publications deserve to be mentioned, although their and its analogue derived from leucine (LeuTeA) had to be main topic is not related to the development of analytical differentiated by mass spectrometric means. LODs were methods. Burdaspal and Legarda (2013) conducted a survey between 1-3 µg/kg (with derivatisation) and 50-80 µg/kg on aflatoxins in beer and calculated the contribution to (without derivatisation). Analogues of TeA derived from daily intake. Exposure data generated from a large valine (ValTeA) and LeuTeA were detected in sorghum- number of findings below LOQ (or LOD) bears the risk based infant food highly contaminated with TeA. Prelle of questionable results, provided that the LOQ is higher et al. (2013) developed an LC-MS/MS method for the than what is acceptable for proper exposure estimation. simultaneous determination of alternariol, alternariol Therefore, the authors discussed upper bound and lower monomethyl ether, altenuene, TTX and TeA in apple juices, bound approaches and adviced correctly to cautious beers, tomato sauces, olives and dried basil. For sample interpretation of survey and exposure data with respect extract clean-up, four different SPE columns were selected to method performance of the analytical methods used. to optimise the purification step for each food matrix. Durakovic et al. (2012) described the effect of AFM1 LODs and LOQs were in the range 0.2-12 and 0.5-41 µg/kg, removal by dehydroacetic acid analogues in yoghurt. In respectively. Recoveries were generally above 70% and experiments with 5-bromo-4-hydroxy-3-(p-toluoyl)-6- precision in the range of 1-38%. Thirty out of 70 samples (p-tolyl)-2H-pyrane-2-one, the concentration of AFM1 from an Italian market were contaminated with at least was reduced by 20-100% after 28 days. one Alternaria toxin at levels up to 35 µg/kg.

World Mycotoxin Journal 7 (1) 11 F. Berthiller et al.

Within the mandate M/520, the European Committee for significantly between the grain types with up to 80 to 90% Standardization (CEN, 2013) has recently launched a call signal suppression of ergometrine in barley and oats, and for tender for the development of a standardised method 50% suppression of ergocryptinine and ergocristinine for the analysis of Alternaria toxins in food. Probably due to in oats and barley. There was almost no suppression or the increasing interest for Alternaria toxins, several papers enhancement of the later-eluting compounds ergocryptinine were published over the last year on the toxicity of these and ergocristinine in rye, wheat and triticale. There were toxins (Fleck et al., 2012; Schwarz et al., 2012a,b; Solhaug also differences in matrix effects between three varieties et al., 2012, 2013; Tiessen et al., 2013). of rye. These results emphasise the need for a careful and correct choice of blank matrix for the preparation of matrix- 7. Ergot alkaloids matched standards. Sample preparation procedures strongly affected the matrix effects, the highest signal suppression The European Food Safety Authority (EFSA) has delivered being observed for ergometrine and ergometrinine. Samples a Scientific Opinion on ergot alkaloids in food and feed cleaned up using MycoSep® multifunctional columns (EFSA, 2012). The opinion considered over 20,000 analytical eliminated matrix interferences better than other SPE results for ergot alkaloid levels in 1,716 food, 496 feed and techniques for the majority of ergot alkaloids, although 67 unprocessed grain samples, and made a risk assessment recovery of ergometrine was poor. Atmospheric pressure based on the major ergot alkaloids identified in sclerotia chemical ionization gave a tremendous signal enhancement from Claviceps purpurea. The EFSA Panel on Contaminants for most of the ergot alkaloids compared to electrospray in the Food Chain (CONTAM Panel) considered the ionisation (ESI), leading to an overestimation of the levels main C. purpurea alkaloids (ergometrine, ergotamine, of ergot alkaloids in some cereal samples. Overall, a method ergosine, ergocristine, ergocryptine, ergocornine and their based on liquid-liquid extraction combined with UHPLC- corresponding -inine epimers) as the most significant. MS/MS in ESI+ mode was recommended. Considering estimates of chronic and acute exposure for various age groups in European countries, a group Malysheva et al. (2013b) also developed a rapid LC-MS/MS acute reference dose of 1 μg/kg body weight (b.w.) and method to determine ergot alkaloids in buffered solutions a group tolerable daily intake of 0.6 μg/kg b.w. were from in vitro studies. Analytes were obtained using liquid- established. The opinion gives a comprehensive overview liquid extraction under alkaline conditions and determined of analytical methods. The CONTAM Panel recommended by LC-MS/MS. Recoveries of the six major alkaloids and that efforts should continue to collect analytical data on their epimers were good (91-123%). The method was occurrence of ergot alkaloids in relevant food and feed applied to a study of the efficacy of a clay-based mycotoxin commodities. The alkaloids monitored should include binder towards ergometrine, ergosine and ergocornine. The at least the C. purpurea compounds listed above, along samples were adjusted to pH 10 and the ergot alkaloids with monitoring of dihydroergosine and agroclavine from extracted with ethyl acetate. The extract was evaporated Claviceps africana and Claviceps fusiformis. The need for to dryness, reconstituted in a methanol:water:MeCN commercially available reference standards was reiterated mixture and analysed by LC-MS/MS in positive ESI (the most important are now available), and in particular the mode. Two selected reaction monitoring transitions were need for isotope-labelled internal standards and certified selected for each of the analytes. LOQ values between reference materials, which are not so freely accessible. 0.19 to 1.45 μg/l and recoveries above 70% were achieved. The method showed the clay to have a binding efficacy The most important analytical contribution over the above 93% for the peptide ergot alkaloids, and 24% was current period was a comprehensive study of the variability observed for ergometrine. Liao et al. (2013) measured six of matrix effects encountered in LC-MS/MS analysis ergot alkaloids as part of a simple and rapid LC-MS/MS of ergot alkaloids in cereals (Malysheva et al., 2013a). method to detect 26 mycotoxins (aflatoxins, ochratoxins, In a systematic investigation of matrix effects, several fumonisins, trichothecenes, and ergot alkaloids) in a maize, approaches to the elimination or minimisation of matrix rice, wheat and three nut products. The analytes were effects were investigated for rye, wheat, triticale, oat and extracted with MeCN:water (85:15, v/v), centrifuged and barley. These included variation of ionisation techniques, diluted with water. Separation and identification of epimers chromatography and sample preparation for a range of was not included in the method. The extraction procedure different grain types and varieties. Signal suppression or was optimised for the maximum number of analytes but the enhancement could not be eliminated but could be reduced acid conditions could have led to degradation of the ergot by the use of UHPLC methods and choice of sample alkaloids. Recoveries of the ergot alkaloids were typically preparation procedure. Sample preparation conditions (pH above 75%, lower in pistachio spiked at 10 µg/kg and higher and extraction buffer concentration, shaking time, drying (>100%) in peanuts. LOQs were above 1 µg/kg. temperature and extraction volumes) did not affect the recoveries of ergot alkaloids significantly, but subsequent In a typical clinical application, Nakamichi et al. (2012) clean-up and detection methods did. Matrix effects differed measured the postnatal uterotonic drug methylergometrine

12 World Mycotoxin Journal 7 (1) Developments in mycotoxin analysis: an update for 2012-2013

in human breast milk using solid-phase extraction and Wakimoto et al. (2013) offered the first report of the HPLC-FLD. The analysis was complete in 8.5 min, isolation of an ergopeptine from a marine creature, the performed well and had an LOD of about 50 ng/l. sea slug Pleurobranchus forskalii. An ethanolic extract of the frozen slug was evaporated and partitioned between Two significant alternative approaches have been published. water and chloroform, and the chloroform-soluble material Lenain et al. (2012) gave the first report of the use of a subjected to flash chromatography on a silica gel using a MIP that recognised six ergot alkaloids and their epimers. gradient of methanol (0-20%) in chloroform. The collected Metergoline was used as a template for the production fractions were analysed by HPLC with diode array ultra of suspended polymerised beads that acted as a selective violet (UV) detection at 210 and 311 nm. The major peak sorbent in a solid-phase extraction column. was identified as ergosinine by means of both time of flight mass spectrometry in ESI mode and 1H-NMR. Rouah-Martin et al. (2012) produced the first report of the production and characterisation of aptamers for ergot Ergot alkaloid formation pathways via the intermediate alkaloids for use in a biosensor. Aptamers for the ergot chanoclavine-I were studied in the non-toxin-producing alkaloids metergoline and lysergamine were produced Aspergillus nidulans innoculated with fragments of using the iterative selection procedure SELEX (Systematic genomic DNA from the ergot alkaloid producer Aspergillus Evolution of Ligands by EXponential enrichment). The fumigatus (Ryan et al., 2013). New pathway intermediates aptamers were isolated, amplified and sequenced before and an ergot alkaloid-like compound were detected but being characterised by SPR. The selected aptamers not identified. Precursors of ergot alkaloids were extracted had a high sensitivity for lysergamine. A colorimetric from A. nidulans colonies by washing with methanol, reaction could be achieved with an aptamer linked to gold centrifugation and evaporation, prior to HPLC with nanoparticles producing a specific colour change in the fluorescence detection. Ergot alkaloids were determined presence of lysergamine and small ergot alkaloids. The by LC-MS using positive ESI and detected by scanning for system was considered suitable for development with regard ions with a m/z between 200-400. to producing a lateral flow-based dipstick test. 8. Fumonisins A complete procedure for detecting ergot bodies in cereals based on near-infrared (NIR) hyperspectral imaging Various aspects of fumonisins, including occurrence, was developed by Vermeulen et al. (2013). NIR spectral toxicity, analysis and chemoprotection of plant extracts characteristics were used to build relevant decision rules were recently reviewed (Waskiewicz et al., 2012; Yang et based on chemometric tools and on the morphological al., 2012), and sampling plans for maize (Mallmann et al., information obtained from the NIR images. The procedure 2013) and workplace air (Jargot and Melin, 2013) were was transferred from a pilot online system to the industrial recently described. The absence of a good chromophore level. The discrimination between cereals and ergot was in the fumonisin structure influences the methods used dependent on the different fat and starch contents of cereal for their detection. Widely used methods for these toxins kernels and ergot bodies. Validation experiments carried generally fit into one of three groups: those based upon out using ergot and wheat, rye, rapeseed, straw, barley physical properties, such as mass/charge ratio (i.e. various straw and cardboard showed the method to be quite stable forms of MS), those based upon derivatisation with more and repeatable, with good correlation with the reference easily detectable labels (such as fluorescence) and those method based on stereomicroscopy. No false positives were based upon indirect detection. The later usually involve produced. It could detect an ergot body concentration of the interaction with selective binding materials, such as 0.02%, less than the EC limit for intervention cereals of antibodies, or more recently synthetic materials, such as 0.05%. aptamers. Research effort in these areas has changed over time and the literature is not evenly distributed among Merkel et al. (2012) studied the fate of ergot alkaloids on these groups. Over the past several years, there has baking cookies made from contaminated rye flour and been a pronounced shift towards the use of LC-MS/MS also monitored the action of in vitro digestion. Baking of for fumonisins detection, particularly in the context of the cookies caused a 2-30% degradation of ergot alkaloids multitoxin methods, while the number of literature reports and formation of an increased proportion of (S)-epimers. developing new LC-FLD methods has declined. The indirect The digestion model showed a selective increase in the assays, such as immunoassays and biosensors, continue to proportion of toxic (R)-epimers in the case of ergotamine- be well represented. As with the other technologies, there type alkaloids (ergotamine and ergosine). Ergotoxine type has been a trend towards multitoxin quantification. alkaloids (ergocornine, alpha- and beta-ergocryptine and ergocristine) were predominantly transformed into their By far the greatest research effort, as reflected in the biologically inactive (S)-epimers. published literature, has been towards the development of MS-based assays. The large number of papers dictates that,

World Mycotoxin Journal 7 (1) 13 F. Berthiller et al.

with few exceptions, they can be only briefly summarised vary according to the clean-up desired and the detection here. Because most of the MS methods have LODs well technique used. Most of the LC-MS/MS methods that below current regulatory limits, in general the LOD for these include fumonisins extract using mixtures of MeCN:water, methods will not be provided here. The MS methods fall which is often acidified (Åberg et al., 2013; Abia et al., 2013; into two categories: those for which the fumonisins are the Ates et al., 2013; Liao et al., 2013; Nácher-Mestre et al., sole analytes and those for which fumonisins are one class 2013; Soleimany et al., 2012; Varga et al., 2013a; Warth et al., of analytes among many tested (multi-analyte assays). The 2012a). While most of the multitoxin methods that include distinction is important, because the extraction conditions fumonisins were developed for cereals or cereal-based feeds, that are optimal for the fumonisins may not be the same several of them dealt with other commodities such as nuts as the extraction conditions that are optimal for multi- (Abia et al., 2013; Liao et al., 2013; Varga et al., 2013a), fish analyte assays. Multiple mycotoxin detection using MS was feeds (Nácher-Mestre et al., 2013), and wines and beers recently reviewed (Capriotti et al., 2012a). In work focused (Al-Taher et al., 2013; Pérez-Ortega et al., 2012; Rubert upon fumonisins, Marschik et al. (2013) tested extraction et al., 2013; Tamura et al., 2012). Solfrizzo et al. (2013a) solutions previously described for multimycotoxin reported results of a proficiency test for a multimycotoxin LC-MS/MS methods in their own LC-MS/MS method and method that included fumonisins. From the 41 laboratories compared the results to a reference method (EN 14352:2004; involved in the performance test, 30 results from spiked CEN, 2004) that combined methanol:MeCN:water maize were reported for FB1 and 29 results were reported (25:25:50, v/v/v) extraction and LC-FLD. Extraction for FB2. For contaminated maize, 32 results were reported solutions examined included: (a) methanol:MeCN:water for FB1 and 31 for FB2. The percentage with satisfactory (1:1:2); (b) MeCN:water:formic acid (80:19.9:0.1, v/v/v) z scores (those with an absolute value less than or equal to followed by MeCN:water:formic acid (20:79.9:0.1, v/v/v); 2) in spiked maize were: 30% (for FB1) and 38% (for FB2). (c) MeCN:water (75:25, v/v) containing 50 mM formic For contaminated maize the results were: 50% (FB1) and acid; (d) MeCN:water:acetic acid (79:20:1, v/v/v); and (e) 52% (FB2). Poor performance (z scores with an absolute MeCN:water (80:20, v/v). Overall, the most appropriate value above 3) were seen for 31% (FB1) and 32% (FB2) of extractant for spiked or naturally contaminated maize the results when testing the contaminated maize, and 63% was methanol:MeCN:water (1:1:2). Other LC-MS(/MS) (FB1) and 52% (FB2) for spiked maize. Mean recovery results methods focused on fumonisins in matrices such as red were acceptable for all the toxins tested, except for the cargo rice (Tansakul et al., 2012), wheat having black point fumonisins, which were unacceptably high (159% for FB1, disease (Busman et al., 2012), culture media and mouldy 163% for FB2). This suggests that many of the multi-analyte maize (Falavigna et al., 2012b), and maize and wheat-based assays are likely compromised when it comes to fumonisin products (Bryła et al., 2013). The method described by quantification. Falavigna et al. (2012b) also included fumonisins beyond the commonly tested for FB1, FB2 and fumonisin B3 (FB3), As described earlier, fumonisins can also be detected specifically the fumonisin A and C series. The method by derivatising them to yield fluorescent products. described by Bryła et al. (2013) is notable because of the Methods based upon LC with either pre- or post-column use of a MIP-SPE to isolate FB1, FB2 and FB3 from cereal derivatisation are still widely used. Several derivatisation products. The MIP-SPE was a commercial product that reagents have been previously described and investigations showed almost equal selectivity for FB1, FB2 and FB3. For on improving fluorescence detection continue. Recently these three fumonisins, the recoveries from spiked samples four reagents were compared for pre-column derivatisation averaged from 95-106%, with repeatabilities less than 17%. of FB1, FB2 and FB3 and separation by UHPLC (Wang et al., 2013a). The reagents were: o-phthaldialdehyde (OPA), Most of the published methods for fumonisins involve their naphthalene-2,3-dicarboxaldehyde, danysl chloride and inclusion among other analytes in multi-analyte assays. As 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC). noted previously, methods for detecting multiple groups From among the four, OPA was selected for development of analytes employ either compromise solvent mixtures to of a method. The LOQs in maize were 25, 14 and 17 µg/kg extract analytes of widely different polarities, or involve for FB1, FB2 and FB3, respectively (Wang et al., 2013a). The multi-step extractions with different polarity mixtures of reagent AQC was also used in a pre-column derivatisation solvents. Extraction techniques continue to evolve and method for measuring FB1 and FB2 in Chinese rice wine (Ma recent trends in the use of MSPD for clean-up have been et al., 2013b). The AQC derivatives were stable for 5 days reviewed (Capriotti et al., 2013). Several of the multi-analyte and the LOD for FB1 and FB2 standards was 6 µg/l. Observed papers use forms of QuEChERS for analysis of various recoveries were in the range of 88-95%. A reference LC- matrices including milk thistle (Arroyo-Manzanares et al., FLD method (EN 16006; CEN, 2011), based upon pre- or 2013), dietary supplements (Vaclavik et al., 2013), spices post-column derivatisation, was used to determine the (Yogendrarajah et al., 2013) and cereals (Zhang et al., extent to which analytical performance was affected by the 2013b). The multitoxin assays have generally been applied addition of mycotoxin binders in the formulation of animal to multiple commodities or foods. Extraction conditions feed (Kolosova and Stroka, 2012). Twenty commercial

14 World Mycotoxin Journal 7 (1) Developments in mycotoxin analysis: an update for 2012-2013

binders were evaluated. The incorporation of binders into glycol diamine (Oswald et al., 2013). A substrate yielding a the feed did not significantly affect the levels of FB1 or FB2 chemiluminescent product was used for detection. Assays that were found. Methods based on LC-FLD were also took 19 min. The response to FB1 was linear from 168 to used in comparisons of extraction and clean-up of FBs 2,215 µg/kg, with an IC50 of 645±59 µg/kg. Chips could be from maize (Chilaka et al., 2012; Marschik et al., 2013). regenerated 50 times. Recoveries of 55-80% were observed As described above, one of these involved a comparison from spiked oat, wheat, rye and maize-polenta. Another of extraction solutions with detection by LC-MS/MS assay format involved FRET between aptamers tagged with (Marschik et al., 2013). A single-laboratory comparison fluorescent (donor) unconversion nanoparticles (UCNPs) was also conducted of six methods for detecting fumonisins and a graphene oxide (GO) acceptor (Wu et al., 2012b). in naturally contaminated maize (Chilaka et al., 2012). The This very novel format used aptamers that, in the absence methods tested used a variety of clean-up and detection of toxin, bound to GO that quenched the fluorescence of strategies. These included: strong anion exchange (SAX) the UCNP. In the presence of toxin, the aptamer-UCNPs clean-up followed by thin layer chromatography or LC-FLD; did not bind to the GO and were not quenched. In this IAC clean-up followed by either LC, direct testing with a fashion, the signal from the aptasensor was proportional to fluorometer, or commercial enzyme-linked immunosorbent toxin level. The sensor had a linear range of 0.1 to 500 µg/l assay (ELISA) and lateral flow device (LFD) methods. The for FB1 and an LOD of 0.1 µg/l. Recoveries of FB1 were contamination levels of total fumonisins found using the between 84-124%. Results from the multiplexed FRET SAX clean-up combined with HPLC and those found using were correlated to those of a commercial ELISA for 15 IAC clean-up were significantly lower than levels found naturally contaminated samples. Several other formats for using ELISA and LFD. Interestingly, this came despite multiplexing immunoassays used microspheres of various the lower recoveries seen with the ELISA method (50%) forms (Czeh et al., 2012; Deng et al., 2013; Peters et al., relative to the two LC methods (72 and 92%). The higher 2013). The first of these used three different colours of contamination shown by ELISA and LFD methods could be silica photonic crystal microspheres (SPCMs), each coated attributed to cross-reactivities with related compounds as with a different toxin-BSA conjugate. The immobilised well as matrix interferences, since no clean-up procedures toxin competed with toxin from samples for fluorescein were applied in these methods. isothiocyanate-labelled antibody. After washing, the SPCMs were applied to a slide surface. The SPCMs were categorised The third group of fumonisin assays involve indirect by reflectance spectrometry and the fluorescence (inversely detection mediated by a binding event, such as with proportional to toxin content) was determined. The LOD antibodies in immunoassays. While antibodies against for FB1 was 1 ng/l, with a dynamic range of 0.001 to 10 µg/l. the fumonisins have existed for many years, improving Spiked over the range from 1 to 10,000 pg/g, the recoveries immunoassays using a variety of formats (ELISAs, LFDs, ranged from 87-117% (peanuts), 88-124% (maize) and 79- biosensors) continues to be an active area of research. 119% (wheat). Another way that microspheres have been While sensitive monoclonal antibodies and ELISAs for used is in formats based upon flow cytometers, e.g. Luminex fumonisins continue to be developed (Sheng et al., 2012), (Austin, TX, USA) or similar instruments (Czeh et al., 2012; much of the literature now concerns either novel fumonisin Peters et al., 2013). One such assay, termed a competitive binding elements, or multiplexed assays. A novel ELISA fluorescent microsphere immunoassay (CFIA), used colour- based upon a mimotope peptide was reported and applied coded polystyrene microspheres coated with anti-toxin to cereals (Liu et al., 2013b). The mimotope peptide, which antibodies (Czeh et al., 2012). Competition was between bound to a fumonisin antibody (analogous to FB1), was phycoerythrin-labeled (fluorescent) toxin and unlabelled attached to bovine serum albumin (BSA) and used as a toxin for binding to the microspheres. The multiplex assay solid phase antigen for the ELISA (e.g. instead of a FB1- was used to detect six groups of mycotoxins, including FB1, protein conjugate). The LOD was 1.2 µg/l, with an IC50 of using a single extraction (with MeCN:water, 84:16, v/v). 6.1 µg/l. The assay compared well to a commercial ELISA Interested readers are referred to the original manuscript and an LC method for 32 cereal samples (R2 of 0.95 and 0.90 for the recoveries from spiked wheat or peas, as the data respectively). Interestingly, in competitive assays 1,289 µM were presented in the form of bar graphs rather than tabular of the free peptide (CT-452) was equivalent to 1 µM of FB1 form. Another type of microsphere assay used paramagnetic (Liu et al., 2013b). Fumonisins were among the analytes colour-coded beads coated with anti-toxin antibody (Peters measured in multimycotoxin assays in formats that included et al., 2013). Competition for binding to the microspheres an immunochromatographic strip (Wang et al., 2013e), a was between phycoerythrin-labelled toxin and unlabelled flow-through sepharose gel (Njumbe Ediage et al., 2012b), a toxin (i.e. from samples). The beads were isolated and the chip-based microarray (Oswald et al., 2013), a fluorescence fluorescence (indirectly proportional to toxin content) was energy transfer (FRET) apatasensor (Wu et al., 2012a), detected. Two Luminex flow cytometers were evaluated and and several microsphere-based assays. The chip-based the assays were compared to an LC-MS/MS method. When microarray was essentially an antigen-immobilised ELISA, applied to naturally contaminated wheat, maize or feed, the conducted on glass slides functionalised with polyethylene assay worked well for certain of the mycotoxins, but severely

World Mycotoxin Journal 7 (1) 15 F. Berthiller et al.

overestimated fumonisin content. Despite this fact, it is 9. Ochratoxins clear from the aforementioned papers that the detection of fumonisins in the context of multiplexed immunoassays is Apparently, LC-FLD with IAC clean-up continued being the making significant progress, with several of the assays able most widely used technique in routine analytical activities to quantify fumonisins in relevant matrices. for the determination of OTA in food and feed along the last year. As an example, in a proficiency test (PT) conducted on Increased interest in fumonisin metabolism and potential OTA in cocoa, 29 out of 36 participants used IAC clean-up hidden or ‘masked’ mycotoxins has lead to several reports and only 2 participants used SPE, 27 out of 31 participants of fumonisin biomarkers and masked fumonisins in foods. declared the use of LC-FLD with only 2 participants using Application of chromatographic and immunochemical MS/MS detectors and 2 participants using UV detection methods for detecting masked mycotoxins were recently (FAPAS, 2012). Nevertheless, some PT organisers (FAPAS) reviewed (Cirlini et al., 2012; Goryacheva and DeSaeger, have launched also specific rounds for users of ELISA, 2012). The masked fumonisins were further subdivided into what may be an indication about the increasing number of two categories: ‘hidden’ or ‘bound’ (Falavigna et al., 2012a). laboratories using this type of screening technique in the The authors of that work suggested the distinction be made routine analysis of OTA. based upon whether the attachment was covalent (e.g. bound) or non-covalent (e.g. hidden). The terminology gets Regarding procedures based on LC, Lerda et al. (2013) entangled because the ‘hidden’ group is described as ‘bound published the results of a collaborative study for the non-covalently’, which is contradictory to the definition. determination of OTA in liquorice and liquorice extracts Despite problems with terminology, the importance of by LC-FLD with IAC clean-up. The test portion was detecting such materials is well recognised. Both hidden extracted with a mixture of methanol and aqueous sodium and bound fumonisins were found to co-occur in thermally bicarbonate solution; the extract was filtered and diluted processed maize-based products (Falavigna et al., 2012a), with phosphate-buffered saline and purified by IAC. suggesting further study is needed to determine the extent Twenty laboratories, all from different countries, took part to which these are found in commodities and foods. In in this study. The tested contents of OTA ranged from related topics, the fate of fumonisins following extrusion 7.7 to 141 µg/kg OTA in both types of matrices. Mean of maize and biomarkers of fungal contamination were recoveries were 87% for liquorice root and 84-88% for also reviewed (Jackson et al., 2012b; Turner et al., 2012). liquorice extracts; reproducibility values ranged from Several methods were developed to detect fumonisins in 10-17% and from 11-22% in liquorice extracts and root, biological fluids, such as plasma (Devreese et al., 2012) respectively. A novel approach for microextraction of OTA and urine (Song et al., 2013a; Njumbe Ediage et al., 2012a). from cereal matrixes was reported by Campone et al. (2012). There are also several recent reports of finding fumonisins The procedure used the so-termed pH-controlled DLLME, in commodities in which they were not commonly expected, which was based on two successive steps conducted at including pineapples (Stępień et al., 2013), onion bulbs opposite pH values. One ml of the raw methanolic extract (Varga et al., 2012) and banana beer (Shale et al., 2012). was re-extracted with 400 µl CCl4 at pH 8 in order to eliminate the hydrophobic matrix interferences. Further, The methods described in this section have all involved the pH of the aqueous phase was adjusted to pH 2 and methods requiring extraction of fumonisins for detection. the toxin was extracted with 150 µl C2H4Br2. The final Methods have also been explored that avoid extraction by determination of OTA was accomplished by conventional measuring spectroscopic properties of solids. One such LC-FLD. The LOQ estimated was 0.06 µg/kg and recoveries method used data from Fourier transform NIR to develop were in the range of 81-90%. Among procedures based on a regression model with 15 principal components. The LC-MS/MS and dealing specifically with OTA, Duarte model was used to predict FB1+FB2 content of maize meal et al. (2013) presented a new analytical methodology for (Gaspardo et al., 2012). A set of 143 maize meal samples was determination of OTA in pork. The method used acidified used to define the principle components. The developed methanol, ultrasound treatment and centrifugation before model was then used to predict fumonisin contamination of the IAC clean-up, followed by LC-ESI-MS/MS in positive an additional 25 samples that were also tested by a reference mode using SRM scanning. The LOQ was estimated LC-FLD method. Content of the samples as measured by 0.2 µg/kg with recoveries varying between 99 and 101%. The the reference method were correlated with values predicted validation study was carried out according the guidelines by the model, suggesting the technology may have value from the European Commission Decision 2002/657/EC for screening maize meal. (EC, 2002) for this type of products.

Various innovative approaches were developed for the analysis of OTA in wine, specifically. Arroyo-Manzanares et al. (2012) proposed the use of DLLME and capillary LC coupled to laser-induced fluorescence detection allowing

16 World Mycotoxin Journal 7 (1) Developments in mycotoxin analysis: an update for 2012-2013

the quantification of OTA at the level of 0.2 µg/l with an investigated the possibility to apply QuEChERS extraction intermediate precision of 4.9% and recoveries >90% when for OTA determination in bread samples by checking the applied to white, rose and red wines. The method was performance of a number of different types of QuEChERS. based on the treatment of an aliquot of 5 ml wine by a After establishing the optimal parameters in terms of mixture of chloroform as extraction solvent and MeCN extraction time, volume of extraction solvent and sample as the disperser solvent. Other feature was the addition of mass, the authors concluded that salt mixtures from three an anionic surfactant (0.2 M SDS) to the isocratic mobile different suppliers were adequate for OTA extraction from phase for increasing the fluorescence intensity. Mao et al. bread samples. The use of that type of QuEChERS for the (2013) reported the quantification of OTA in red wines by analysis of OTA by LC-FLD in bread samples obtained conventional LC-FLD using a commercial column packed recoveries ranging from 95-97%, repeatabilities ranging with 2.6 µm core-shell particles. Core-shell particles are between 1.0 and 10% with an LOQ at the level of 0.05 µg/kg. different from the totally porous particles in that they Sirhan et al. (2012) also described the use of the QuEChERS have a solid core surrounded by a thin porous shell. This method combined with LC-FLD in the analysis for OTA type of columns can provide similar speed and efficiency in cereal and cereal products with recoveries >85% and a while maintaining low back pressure, thus allowing their chromatographic separation of the toxin performed within use on conventional LC instruments. Under optimised 4 min by using a reversed-phase C18 column at 30 °C under conditions, the authors were able to analyse extracts from isocratic conditions. Llorent-Martínez et al. (2013) reported red wines after IAC clean-up, with recovery rates between the use of this extraction approach in a sequential injection 81 and 83% and a retention time for OTA of less than 5 min. analysis coupled to two different flow-through optosensors Longobardi et al. (2013) described a new method in red for comparison between them. One of the sensors was wine that used a double extract-clean-up and fluorometric based on fluorescence detection whereas the other one detection. The prepared sample was first cleaned-up by used terbium-sensitised luminescence (TSL) detection. IAC and the eluted extract was further purified through The instrumentation was formed by an automatic analyser a commercial aminopropyl silica SPE cartridge. The OTA unit in line with a luminescence spectrometer. In each case, in the final extract was measured fluorometrically and the flow-cell was filled with the appropriate solid support: quantified after deconvolution of fluorescence spectra. Sephadex QAE A-25 (GE Healthcare, Little Chalfont, UK) This mathematical treatment allowed significant recovery in the fluorescence detection and C18 silica gel in the TSL improvement by removing the contributions arising from sensor. Taking into account the better sensitivity and sample interfering substances eluting with OTA. As an example, throughput, the TSL system was chosen to perform the the recovery went down from 118 to 95% in samples spiked analysis of some cereals and pet feed with recoveries in the at the level of 2.0 µg/l. The repeatability values ranged from 86-112% range at spiking levels around 250 µg/kg. 4-15% at spiking levels between 0.5 and 3.0 µg/l. Some attention also deserves the use of immunochemical In spite of the availability of commercial MIPs for selective methods for OTA detection. Anfossi et al. (2013) adapted SPE of OTA in foodstuffs, very few articles on its use were a lateral flow immunoassay previously developed for OTA published along the last year. Cao et al. (2013) proposed detection in cereals for measuring OTA in wine and grape a method based on the use of MIP as an alternative to must (Anfossi et al., 2011). Red wines and grape juice needed IAC for the determination of OTA in ginger by UHPLC- special treatment in order to avoid the matrix interference FLD. The method was optimised after checking the with red-purple lines arisen from gold nanoparticles used influence of several parameters, such as the pH value of in most common LFDs. Wines were diluted 1:2:2 with loading solution, the volume and composition of washing NaHCO3 (0.15 M, pH 9.0) and a water solution of PEG (4%, solution and volume of eluting solution: pH 1.0, 5 ml 0.1 M w/v) and pipetted into the sample well. Grapes musts were HCl:MeCN (60:40, v/v) and 2 ml of methanol:acetic acid treated in the same way, except for the fact that 12% (v/v) (98:2, v/v) respectively, were found the best conditions. The of absolute ethanol was added before dilution. Optimal recoveries ranged from 88-95% in ginger spiked at 5, 15 concentration of reagents for the test and control lines and 25 µg/kg with an inter-day precision ranging from 2.2- and gold nanoparticles-labelled antibodies for detection 3.7%. More interferences were observed in chromatograms was redefined to achieve the best sensitivity. The LOD of after clean-up with MIP compared to IAC, although they the assay was 1.0 µg/l. Despite that intra- and inter-day had no adverse effect on the separation of OTA. It is worth precision were found to be high comprising between 20 mentioning, that MIP columns were found to be reusable and 40%, the authors proposed this method for the reliable up to forty-one consecutive times after a single wash step first level monitoring of OTA. Lippolis et al. (2013) gave an with 10 ml methanol. account of the development of a fluorescence polarisation immunoassay applicable to the determination of OTA in The use of QuEChERS methods for the extraction of wheat samples. The authors described the synthesis of a mycotoxins is starting to appear in an increasing number fluorescent-labelled OTA tracer and studied its binding of articles in the scientific literature. Paíga et al. (2012) response with three monoclonal antibodies tested, showing

World Mycotoxin Journal 7 (1) 17 F. Berthiller et al.

an IC50 value of 0.5 µg/l in the most sensitive case with the linking oligonucleotide and then measured after being negligible cross-reactivity for ochratoxin B (1.7%) and no separated with an external magnetic field. The method was cross-reactivity for other mycotoxins that may be frequent applied to the determination of OTA in wheat samples in wheat. Samples were extracted and purified by SPE using spiked at 1.0, 3.7 and 9.2 µg/kg with recoveries >97% and an aminopropyl column prior to the assay. The authors RSDs <4.8%. Another innovative approach was followed claimed an overall time of analysis of 20 min, the LOQ by Zhang et al. (2013a) who incorporated the use of Tb3+ was estimated at 2.0 µg/kg with average recoveries of 87% and magnetic beads in the aptasensor. Some biotinylated in spiked samples (3 to 10 µg/kg) and RSDs lower than 6%. anti-OTA aptamer was captured onto streptavidin-coated magnetic beads and two single-stranded complementary Also aptamers have been used by some research groups. oligonucleotides were added in order to allow their Thus, Wu et al. (2012a) reported the development of a hybridisation to the aptamer. The resulting magnetic beads single-strand aptamer functionalised electrochemical were magnetically separated and the resulting supernatant aptasensor able to detect the presence of OTA at levels of liquid contained no single-stranded oligonucleotides and less than 0.01 ng/l. The preparation of the electrochemical could not increase the emission of Tb3+ in the absence aptasensor was described after optimisation of some key of OTA. In the presence of OTA, the OTA-aptamer parameters, such as the density of the aptamer on the surface binding was produced while releasing the single-stranded of the electrode, the reaction time between the aptamer and oligonucleotides, which finally were present in the OTA, and the effect of metal ions including Mg2+ and Ca2+. supernatant and could enhance the fluorescence intensity The aptasensor was also applied for detection of OTA in of Tb3+. This aptasensor was used for determination of OTA wine samples spiked at different levels (1 to 950 ng/l) and in spiked wheat samples in order to evaluate its practical dilution with phosphate buffered saline as the only sample applicability and results were found to be consistent with pre-treatment. Recoveries were always >94% and results those obtained with ELISA. compared well with those obtained by LC analysis conducted on the same samples. Ma et al. (2013c) also described the As a final remark, a new call for tenders for project leaders development of another ultrasensitive aptasensor specific for the development of standardised methods for the analysis to OTA allowing its detection at femtogram measurable of mycotoxins in food (Mandate M/520) was issued very levels. The whole procedure was accomplished in a single recently by the European Committee for Standardization PCR tube. A single-strand OTA-specific biotin-labelled (CEN, 2013) in the framework of the Regulation (EC) aptamer and its complementary DNA fragment were 882/2004 (EC, 2004). The execution of the mandate was incubated forming a double-strand DNA immobilised on divided into eleven projects. Two of them dealt with OTA, the surface of a previously streptavidin-coated PCR tube. namely: determination of OTA in liquorice and spices (for In the presence of OTA, the aptamer had a conformational which an EU ML has been established) and in cocoa and change induced by the target binding and the single- cocoa products and determination of OTA in meat, meat stranded DNA complementary to the aptamer was released. products and edible offal. In both cases, the methods need The OTA concentration was measured by the change in to be interlaboratory validated, in accordance with ISO 5725 the amplification signal generated in real-time quantitative (ISO, 1994) or with AOAC International guidelines. The PCR. The assay proved to be highly specific when replacing techniques to be used could be either fluorimetric or MS OTA by four mycotoxins (ZEA, DON, AFB1 and FB1) detection. In principle, it was recognised that there were as target molecules, which were not recognised by the methods based on LC-FLD, which could serve as good biotin-labelled aptamer. The application of this assay to the basic methods, suitable for interlaboratory validation of analysis of red wines required just a simple pre-treatment the missing matrices, i.e. OTA in chilli and paprika, and to remove the wine sediments; recoveries in the range of OTA in pig and poultry serum and blood derivatives. Those 99-112% were obtained at seven spiking levels ranging two projects may be considered as an indication about from 5×10-6 to 5 µg/l, demonstrating the potential to the what is nowadays demanded to analytically support the analysis of trace amounts of the toxin. Hun et al. (2013) food legislation. reported a method based on the combination of cleavage of nicking endonuclease with target-induced strand release. 10. Patulin The OTA aptamer and OTA aptamer complementary oligonucleotide were loaded onto a magnetic bead. When New and improved methods for PAT determination using OTA was present, the aptamer preferentially bound it and a range of analytical techniques continue to be published. caused the release of some strands from complementary Xiao and Fu (2012) developed and validated a GC-MS oligonucleotide that acted as the primer for a subsequent method for the detection of PAT in apple juice. The method polymerisation and nicking endonuclease scission process, used NaHCO3 during LLE and acetic acid into the organic producing a large amount of short single-stranded linker extract with 3-nitrobenzyl alcohol as internal standard. oligonucleotides. A chemoluminescence nanoparticle probe The LOD and LOQ were 2 and 5 µg/l, respectively. The previously prepared was captured by hybridisation with extraction efficiency was 86% with an RSD of 14%; for

18 World Mycotoxin Journal 7 (1) Developments in mycotoxin analysis: an update for 2012-2013

precision the RSD was 6.5-10%. Kharandi et al. (2013) binary solvents-based DLLME method was developed by developed a GC-MS method using a QuEChERS procedure Maham et al. (2013) for the determination of PAT in apple for the detection and determination of PAT in apple juice. juice followed by LC quantification. The method involved This procedure includes an initial extraction with MeCN, the use of an appropriate solvent to form fine droplets of a partitioning with addition of magnesium sulphate, extractant in a sample solution. The authors optimised sodium carbonate and sodium chloride and a clean-up the parameters that affect the extraction efficiency: (1) the using dispersive solid-phase extraction by the addition of type and volume of high-density extraction solvent; (2) the a mixture of magnesium sulphate and primary secondary volume of ethyl acetate; (3) the kind and volume of disperser amine sorbent. N,O-bis(trimethylsilyl)trifluoroacetamide solvent; and (4) salt addition. The LOD and LOQ were 2.0 was used to derivatise PAT prior to GC-MS in order and 10.0 µg/l, respectively. The RSD for five measurements to increase the sensitivity of the method. The recovery of 25 µg/l of PAT was 3.8% and the relative recoveries at ranged from 80-88% with an LOD of 0.4 µg/l and an LOQ spiking levels of 25, 50 and 75 µg/l ranged from 91-95%. of 1.3 µg/l, with an RSD lower than 9.5%. Shirasawa et al. (2013) used a cyclodextrin-based polymer for clean-up of PAT from apple juice and subsequent HPLC- Song et al. (2013b) reported a method for the simultaneous UV analysis. After optimising the procedure, recoveries of determination of bisphenol A, AFB1, OTA and PAT in food the method were 67-78% with RSDs of 3-20%, while the matrices by LC-MS/MS. Analytes were extracted from food LOQ was 10 µg/l. matrices, such as cereal, peanut butter, cereal-based baby formula and fruit juices, and were enriched by SPE before There were several surveys of PAT in food published this LC-MS analysis using negative ESI with selected reaction past year from different countries. In Italy, Pattonoa et al. monitoring and matrix-matched external calibration for (2013) reported a survey of the presence of PAT and OTA in quantitation. The LOQs for PAT were 1 µg/l in juices and traditional hand-made, semi-hard cheeses. OTA was present 0.8-1.5 µg/kg in solid matrices, while the recoveries were 95- in both the rind and the inner part of the cheeses, while 127%. Malysheva et al. (2012) demonstrated the formation PAT was mainly detected in the rind and the inner part of and use of a methanol adduct of PAT in ESI+. A study of the only one cheese sample. The observed PAT concentration fragmentation pathway confirmed the authenticity of the ranged from 15 to 460 μg/kg, while the observed OTA PAT adduct, while the use of an ion trap and high resolution concentration ranged from 1 to 262 μg/kg in the rind and Orbitrap mass spectrometry allowed reliable assignment from 18 to 146 μg/kg in the cheese interior. In Tunisia, a of the PAT fragment ions. Exploiting the formation of the total of 85 apple juice, baby food and mixed juice samples methanol adduct, PAT has been successfully included in a collected during 2011 from major supermarkets and stores single run multimycotoxin LC-MS/MS method in support located in Tunisia were analysed (Zaied et al., 2013). Results of ex vivo-in vitro biomedical studies. showed that the incidence of PAT contamination was 35%. The levels of contamination determined in the total Güray et al. (2013a) developed and validated a method samples ranged to 167 μg/l with a mean value of 20 μg/l for determining PAT in apple juice using capillary zone and a median of 13 μg/l. 18% of the total juice samples electrophoresis. The run buffer was 25 mM of sodium (apple juices and mixed juices) and 28% of the baby food tetraborate and 10% MeCN at pH 10. The optimal samples exceeded the EU maximum levels, which are 50 conditions included an applied voltage of 25 kV (normal and 10 μg/l, respectively. A study of the occurrence of PAT polarity), a temperature of 25 °C and an injection time of in organic and conventional apple-based food marketed in 10 s at 50 mbar. The signal of PAT and phenobarbital as Catalonia and an exposure assessment was reported (Piqué internal standard were detected at 276 nm. The LOQ was et al., 2013). A total of 93 apple-based products marketed 18 µg/l, while a recovery of 68% was reached. This method in Catalonia were analysed, 49 of which were derived from required fewer chemicals and less time than other methods conventional farming and 44 from organic farming. The for the determination of PAT. results showed a higher incidence of positive samples and a higher concentration of PAT in organic apple purees as Chanique et al. (2013) developed a method based on square compared with conventional ones. In the case of juices, voltammetry for the quantitative determination of PAT in significant differences were found between conventional both fresh and commercial apple juices in the presence of and organic samples, but applying a multivariate analysis 5-hydroxymethylfurfural. The electro-reduction of PAT the type of agriculture did not seem to have a relevant and 5-hydroxymethylfurfural at glassy carbon electrodes contribution to PAT occurrence, cloudiness being the main in MeCN, tetrabutylammonium perchlorate, with and factor involved. without different aliquots of trifluoroacetic acid, was also reported. The electrochemical techniques were cyclic and square wave voltammetry and controlled potential bulk electrolysis. Ultraviolet, visible and infrared spectroscopy was used to identify PAT electro-reduction products. A

World Mycotoxin Journal 7 (1) 19 F. Berthiller et al.

11. Trichothecenes et al., 2013a). The mycotoxins were extracted from the samples with MeCN and the extracts were cleaned up In the past year, there have been a significant number using the QuEChERS method. A novel approach was of publications about trichothecenes, with the focus to produce derivatives of the target components with increasingly on LC-MS(/MS) methods. In March 2013, trifluoracetic anhydride (TFAA) simultaneously with the European Commission published Recommendation additional purification and pre-concentration of the extract 2013/165/EU about T-2 and HT-2. This requested member by DLLME. This led to a much reduced analysis time of states to collect data on T-2 and HT-2 occurrence, as well as less than 1 h, giving a major time efficiency saving for this co-occurrence with other Fusarium toxins where possible, analysis. including the masked forms of these toxins, in particular the mono and di-glycosides of T-2 and HT-2 (EC, 2013). Rapid test kits for T-2 and HT-2 and DON have been evaluated (Aamot et al., 2012, 2013). The studies were There was little novel in the way of HPLC methods for carried out to evaluate rapid test kits performance and DON. However, two papers were of note, as both could applicability. For HT-2 and T-2, the combined toxins were detect DON with other trichothecenes without using analysed in naturally contaminated oat samples using three MS. An HPLC method with UV detection was used to test kits: two ELISA kits and a LFD. Mycotoxin analysis separate and quantify DON, 15-acetylDON (15-ADON) by LC-MS/MS was used as a reference method. The LFD and 3-acetylDON (3-ADON) in cereals. The analytes offered the best reliability, with the greatest agreement were separated on a reversed-phase C18 column using with the reference method. In addition, it also showed gradient elution and detection at 224 nm. 15- and 3-ADON cross-reaction of 100% with both T-2 and HT-2, whereas showed effective baseline separation. The method gave for the ELISA kits, cross-reactions were below 100% with good linearity and excellent precision for inter- and HT-2. Similarly in the DON study, oats and wheat were intra-day determinations. Average recovery rates for tested by three rapid kits. Again, the LFD showed the best the tested matrices ranged from 71-92%. The LODs and reliability, achieving detection that was stable across toxin LOQs ranged from 16 to 25 μg/kg and 48 to 60 μg/kg, levels in both matrices, while the ELISA method achieved respectively (Yang et al., 2013). In addition, Muscarella et the highest accuracy at a DON level of 1,750 μg/kg in oats. al. (2012) reported an analytical method for the quantitative For both toxins, it was noted that ELISA is more applicable determination of DON and NIV in cereals. The method, for laboratory use as it allows multiple samples to be tested, based on HPLC-FLD, used automated 2 channel post- while the LFD formats are well-suited for on-site detection. column derivatisation. Separation was achieved using a C18 Goryacheva and De Saeger (2012) reviewed recent literature column with isocratic elution, with FLD using excitation and on possible applications of antibody-based techniques emission wavelengths of 360 nm and 470 nm, respectively. for masked mycotoxins. They concluded that for many Complete separation of DON and NIV was obtained in less immunochemical methods cross-reactivity of the antibodies than 20 min, LODs were as low as 14 μg/kg. The method to masked mycotoxins is unknown or poorly characterised. was validated and the analytical performances of linearity, This can lead to overestimation of target mycotoxins, selectivity, precision and recovery were determined. An however, the methods may be able to measure the sum of interlaboratory study to evaluate LC methods with UV or the target compounds with co-existing analogues, which MS detection for simultaneous analysis of DON and NIV can be useful for risk assessment studies. However, they did in wheat was conducted (Aoyama et al., 2012). DON and also note that progress in immunochemical methods for NIV were purified using a multifunctional column and the masked mycotoxins is hindered due to a lack of analytical concentrations determined using LC-UV or LC-MS(/MS). standards. No internal standards were used. Three fortified wheat samples (0.1, 0.5 and 1 mg/kg), one naturally contaminated Two papers that could help improve analysis focussed on wheat sample and one blank wheat sample were used. Eleven different aspects of method performance. Köppen et al. laboratories produced recovery data in the range from (2013) described the preparation and certification of a 90-110% for DON and from 76-83% for NIV. Regardless reference material for Fusarium mycotoxins in wheat flour. of sample and detector, the HorRat values for DON and The material is the first to be certified for the determination NIV ranged from 0.4 to 1.4. The study demonstrated that of DON and NIV as well as ZEA. The certified values and both LC-UV and LC-MS(/MS) methods can be considered corresponding expanded uncertainties were assigned in suitable for use as official methods. compliance with the requirements of ISO Guide 35 (ISO, 2006). The material should be of great benefit for internal Very few papers using GC were published, with little in quality control in addition to facilitating the validation of terms of novel developments. A method of note for the new analytical methods. Asam and Rychlik (2012) reviewed simultaneous determination of trichothecene mycotoxins the synthetic routes to stable isotope-labelled 3-ADON T-2, HT-2, DON and NIV in grain and mixed feed by gas- and 15-ADON that can be used as internal standards in liquid chromatography with ECD was reported (Amelin SIDAs. They concluded that the benefits of SIDAs in terms

20 World Mycotoxin Journal 7 (1) Developments in mycotoxin analysis: an update for 2012-2013

of precision and reliability are great making the effort to this mycotoxin at levels up to 842 μg/kg. Yoshinari et al. produce labelled standards worthwhile. (2012) reported a method based on LC-MS/MS for the identification of DON, DON-3G, 3-ADON and 15-ADON. The bulk of analytical method papers have focussed on the Comparison of five multifunction columns revealed that use of LC-MS(/MS), with increasing numbers of authors DON and its acetylated derivatives were recovered well reporting the use of HRMS or Orbitrap technologies. The (96-120%) by all columns, but DON-3G was recovered use of LC-MS(/MS) lends itself to the detection of many adequately (94%) by only one column, InertSep VRA-3 (GL different types of mycotoxins in one analysis, which has Sciences, Torrance, CA, USA). Samples of maize grits and resulted in many papers being published as multitoxin maize flour were analysed. DON, DON-3G and 15-ADON methods. These are reviewed in their own section. However, were the major contaminants in the samples harvested in many authors report methods for Fusarium toxins as a group 2009, but only DON was detected in the samples harvested or include ZEA with trichothecenes due to their common in 2010. A sensitive UHPLC-MS/MS method for type A co-occurrence. Therefore, some of the papers here also and type B trichothecenes in rice with LOQs from 0.5 to include ZEA due to their relevance to this review. The use 10 μg/kg was described (Wang et al., 2012a). The method of HRMS methods has also led to another increasing trend, used Oasis HLB cartridges for clean-up and gave recoveries which is the rise in the number of papers reporting methods of 71-103%. A new LC-MS/MS method for the simultaneous for masked mycotoxins, including some novel masked analysis of T-2 and HT-2 in cereals was reported by Tölgyesi forms that were previously unreported. A comparison was and Kunsági (2013). Extracted samples were cleaned-up on made between IAC clean-up and no clean-up for animal Strata-XL solid-phase extraction cartridges and analysed feed analysis by LC-MS/MS. It was reported that, while with LC-MS/MS equipped with multimode ion source. IAC improved repeatability and reduced interference, The key point of note was that the HPLC separation was there was little difference in quantification. Nonetheless, tested with both fully porous (Gemini C18; Phenomenex, the authors concluded that analysis without clean-up is Torrance, CA, USA) and core-shell type (Kinetex XB suitable for screening, but for regulatory purposes the C18; Phenomenex) columns, the core-shell column gave use of IAC is recommended (Senyuva et al., 2012). An enhanced peak shapes for both toxins. LC-MS/MS method was developed and validated for the simultaneous determination of DON, ZEA, T-2, HT-2 and A novel water-based slow injection ultrasound-assisted metabolites, including 3-ADON, 15-ADON, DON-3G, emulsification microextraction (SI-USAEME) method α-ZOL, β-ZOL, ZEA-14-glucoside (ZEA-14G), α-ZOL- followed by UHPLC-MS/MS analysis was developed 14-glucoside, β-ZOL-14-glucoside and ZEA-14-sulphate for the rapid pre-treatment and determination of DON in maize, wheat, oats, cornflakes and bread (De Boevre et and its metabolite DOM-1, in maize and pork samples. al., 2012). A sample clean-up procedure was not included A study comparing IAC clean-up, SPE clean-up and the because of the low recoveries of free and masked mycotoxins developed SI-USAEME method was presented. In addition and their differences in polarity. The method was validated to providing comparable concentration and purification for several parameters such as linearity, apparent recovery, efficiency to the IAC and SPE methods, the SI-USAEME LOD, LOQ, precision, expanded measurement uncertainty method was simple and considerably lower in cost than and specificity. The LODs varied from 5 to 13 μg/kg, and the the IAC method, demonstrating that it could become a LOQs from 10 to 26 μg/kg. The method was subsequently simple, low-cost alternative method. The method was applied to collect information on human exposure to these successfully applied to the analysis of commercial maize mycotoxins in Belgium (De Boevre et al., 2013). and pork products (Cui et al., 2013).

There were many publications reporting the use of LC-MS/ In addition to foodstuffs, LC-MS/MS methods have been MS for the analysis of various trichothecene mycotoxins developed to analyse trichothecenes and their metabolites in a variety of cereal matrices. Many authors applied the in biological systems. An assessment of DON exposure methods to obtain large amounts of occurrence data. using an LC-MS/MS biomarker method was reported by Malachova et al. (2012) reported the first triple quadrupole Warth et al. (2012b). The method was used for a pilot survey LC-MS/MS method for the determination of DON, DON- to investigate the level of DON exposure in Austrian adults 3G and 3-ADON in beer. The method was applied to by measurement of DON and its glucuronide conjugates, conduct a survey of 374 samples of beer. While DON and as biomarkers of exposure, in first morning urine. DON- DON-3G were detected at a high frequency, concentrations GlcAs were directly quantified by LC-MS/MS and the were generally low and it was concluded that beer is not a results were compared with indirect quantification after major risk factor to moderate beer drinkers (Varga et al., enzymatic hydrolysis that confirmed the suitability of the 2013b). Dall’Asta et al. (2013) reported a method for DON direct method. The in vivo metabolism of DON in humans and DON-3G in durum wheat that was used to analyse was studied and two closely eluting DON-GlcAs in naturally 150 samples from Italy. A high incidence of DON-3G was contaminated urine samples were determined for the first recorded, with 85% of the analysed samples containing time. In contrast to previous findings, DON-15-glucuronide

World Mycotoxin Journal 7 (1) 21 F. Berthiller et al.

was tentatively identified as a major DON metabolite in that accurate mass targeted screening using current single- human urine based on the analysis of these samples. stage HRMS analytical instrumentation can be used for screening for multiple classes of contaminants in both The use of HRMS has increased, as the technique offers bakery raw materials and finished products. many advantages, not least the ability to analyse for both known compounds for which standards are not available, 12. Zearalenone but also to identify unknown compounds. The method was used to good effect to identify masked mycotoxins derived Already the majority of last year´s publications describe from type A trichothecenes. These novel glucosides were the concurrent detection and quantification of ZEA in identified as T-2-glucoside and HT-2-glucoside on the food and feed among other mycotoxins by LC-MS(/MS)- basis of accurate mass measurements of characteristic ions based multitoxin methods, which are covered in another and fragmentation patterns using LC-HRMS (Orbitrap) section of this review. Still, dedicated chromatographic analysis. Although their absolute structures were not methods for ZEA and its metabolites are continuously clarified, 3-OH-glucosylation appeared to be the most developed. For instance, Rodriguez-Carrasco et al. probable route for T-2 (Nakagawa et al., 2012). In addition, (2012) developed a method for the determination of ten the existence of a di-glucosylated derivative of T-2 in mycotoxins including ZEA in wheat semolina using a plant (maize powder) was confirmed for the first time QuEChERS-based purification and GC-MS measurement. in addition to that of HT-2. These masked mycotoxins Derivatisation was performed with a mixture of N,O- were identified as T-2-di-glucoside and HT-2-di-glucoside bis(trimethylsilyl)acetamide, trimethylchlorosilane and based on accurate mass measurements of characteristic N-trimethylsilylimidazole to silylate all hydroxyl groups. ions and fragmentation patterns using LC-Orbitrap MS For ZEA, recoveries of 60-67% have been gained. The analysis. In addition, 15-monoacetoxyscirpenol-glucoside intra- and inter-day precision was at around 10 and 20% was also detected in the same sample (Nakagawa et al., RSD, which is still very much acceptable despite a rather 2013). Veprikova et al. (2012) also reported a new analytical poor linearity of the calibration (r2=0.93). An LOQ of procedure for monitoring T-2/HT-2 conjugates in cereal around 10 μg/kg was calculated in wheat semolina. A samples. They used dedicated immunoaffinity cartridges similar method was developed for the determination of for the selective isolation and pre-concentration of the OTA, T-2, DON and ZEA using GC-ECD (Amelin et al., target conjugated analytes. The samples were analysed 2013b). This method is applicable to various grains, feed by UHPLC-HRMS that allowed the confirmation of the mixes and also meat. Again, mycotoxins were extracted presence of conjugated T-2 and HT-2. In addition to mono- with MeCN and purified by QuEChERS. Trifluoroacetic glycosylated forms of T-2 and HT-2 detected in naturally anhydride was used as derivatising agent, introducing highly contaminated barley, wheat and oats, diglucosides of HT-2 electronegative fluorine atoms into the analytes. RSDs below were found in barley. Suman et al. (2013) reported the 8% were gained and the LOQ for ZEA was around 50 μg/kg. development of an LC-linear ion trap-MS method capable of Capillary zone electrophoresis coupled with UV detection determining DON-3G in different processed cereal-derived was developed and used to determine ZEA in cereals and products. Chromatographic separation was performed feed (Güray et al., 2013b). A sodium tetraborate buffer using a core-shell C18 column. An LC-HRMS method was with MeCN was used and 15 kV of potential difference was used to carry out untargeted screening for the detection of applied to the capillary. Phenobarbital was found to be a biotransformation products of DON in plants using stable suitable internal standard and increased the repeatability isotopic labelling (Kluger et al., 2013). Flowering ears of of the overall method. LOQs of 25 µg/l have been achieved wheat were treated with a mixture of labelled and non- in solution. HPLC-FLD was used to determine ZEA in beer labelled DON. In addition to DON and DON-3G, eight after DLLME using chloroform as extraction solvent and further DON bio-transformation products were found MeCN as dispersive solvent (Antep and Merdivan, 2012). by the untargeted screening approach, including DON- This way, ZEA could be enriched by a factor of 40 while glutathione conjugate, which was reported in wheat for the extraction recovery was 83%. Sub-μg/kg LOQs were the first time. This demonstrates what a powerful tool the achieved for the quantification of ZEA in beer, which was LC-HRMS approach can be, particularly in metabolomics detected in 11 out of 13 tested samples. Another HPLC- studies, and no doubt the area of untargeted analysis will UV-FLD method for the determination of DON and ZEA continue to develop in the mycotoxin field. has been developed (Sebaei et al., 2012). Cereal samples were extracted with water and MeCN and the extracts were The final example of the usefulness of LC-HRMS was purified by QuEChERS and in the case of DON additionally reported by De Dominicis et al. (2012). A fast method by SPE. At a spiking level of 100 µg/kg, 85, 93 and 91% of for extracting and purifying bakery matrices for analytes ZEA was recovered in maize, rice and wheat, respectively. including pesticides, aflatoxins, trichothecene toxins and RSDs of 7% were calculated, while LOQs of 20 μg/kg were veterinary drugs was developed using UHPLC coupled to determined for all tested matrices. a Orbitrap Exactive HRMS. Method performance showed

22 World Mycotoxin Journal 7 (1) Developments in mycotoxin analysis: an update for 2012-2013

An alternative to the determination of ZEA in food and 70-117% on the Orbitrap, and excellent LODs below feed is the use of LC-MS-based biomarker methods 0.1 µg/l for all analytes were achieved on both instruments. to quantify ZEA or its metabolites in biological fluids Repeatabilities were in the range of 3-22% on the QqQ and like urine or plasma. In the last years, a variety of such from 1-15% on the Orbitrap. Besides ZEA, α-ZOL and methods has been developed and published. For instance, β-ZOL, ZEA-GlcA was also detected in pig urine using the an LC-MS/MS method for the quantification of seven HRMS. The same group also published a multibiomarker mycotoxins (and their metabolites), including ZEA, α-ZOL, method that was very similar in setup (and performance β-ZOL and ZEA-GlcA, in human urine was developed regarding ZEA and its metabolites) to the previous method, (Njumbe Ediage et al., 2012a). Samples were extracted but also contained DON, T-2, HT-2, OTA, FB1 and AFB1 with acidified ethyl acetate. This extract was combined (Devreese et al., 2012). with the acidified aqueous fraction after SAX-SPE clean-up before LC-MS/MS measurement in positive electrospray Besides chromatographic methods, also a wealth of mode. LOQs of around 2 µg/l (or 7 µg/l for ZEA-GlcA) immunoanalytical methods were developed and employed could be reached, with intra-day precision of 8-13%. for the determination of ZEA in cereals and other matrices. ZEA was detected in 4 out of 40 analysed samples. Three For instance, a group-specific monoclonal antibody against samples were contaminated with β-ZOL, while no α-ZOL ZEA was produced and applied in two direct competitive was detected. In a follow-up paper by the same group, a ELISA systems (Cha et al., 2012). Cross-reactivities method for the determination of 6 mycotoxins and their ranged from 101-152% for α-ZOL, β-ZOL, α-ZAL and metabolites (ignoring potential glucuronidation) in pig β-ZAL, rendering the system suitable for the concurrent urine was developed (Song et al., 2013a). A salting-out quantification of ZEA and its metabolites. LODs of assisted LLE procedure was used for sample preparation 10 µg/kg could be achieved with recoveries of 106-123% before LC-MS/MS analysis. Therefore, urine was treated and RSDs below 10%. Another ZEA ELISA was developed with MgSO4 and consecutively extracted with ethyl acetate by Pei et al. (2013). Their raised antibody showed cross- and MeCN. Compared to dilute-and-shoot techniques, the reactivities of 24% for β-ZOL and 189% for α-ZOL in the used extraction technique resulted in less matrix effects assay, recovery rates of 80-128% from spiked maize samples, and better method sensitivity. LOQs of around 1 µg/l were and an LOQ of 0.5 µg/kg. Wang et al. (2013g) developed a achieved for both pig and human urine for ZEA, α-ZOL chemiluminescence immunoassay to detect ZEA in food and β-ZOL. Recoveries reached from 75-121% depending using biotinylated ZEA conjugates and gold nanoparticles on the analyte, matrix and spiking level, while the intra- labelled with streptavidin-horseradish peroxidase for signal day RSDs were below 18%. Matrix-matched calibration amplification. Low cross-reactivities below 32% were shown was used for quantification. Another LC-MS/MS method for ZEA metabolites. Including extraction and dilution, the was developed for the determination of ZEA, α-ZOL, linear working range was 0.4-10 µg/kg. Very good recoveries β-ZOL, zearalanone (ZAN), α-ZAL and β-ZAL in bovine (97-117%) and repeatabilities (<10%) were achieved. The and porcine urine (Matraszek-Zuchowska et al., 2013). same group also developed an electrochemical ELISA for After glucuronidase treatment, LLE and two SPE stages, ZEA determination in grains (Wang et al., 2013h). Here, samples were injected into an LC-MS/MS system, operating the enzymatic conversion of the substrate is detected by a in negative electrospray mode. Recoveries ranged from microplate reader and the signal subsequently converted 76-116% with repeatabilities below 20%. As LODs were into an electric signal. The detection range of this unrealistically low, detection capabilities were calculated immunoassay is wide (0.1-240 µg/kg) and the test showed being 0.3 µg/l for ZEA and even lower for all other analytes. excellent recoveries (92-113%) and RSDs (<8%). Quantum Nine hormones, including ZEA, α-ZAL and β-ZAL can be dot-based rapid tests for ZEA determination have been detected with a newly developed method using a UHPLC- developed (Beloglazova et al., 2012). CdSe/ZnS core/shell quadrupole-orbitrap hybrid system (Kumar et al., 2013). quantum dots were used as the label in a fluorescent- Urine was treated with β-glucuronidase and subjected labelled immunosorbent assay. The application of this label to SPE and LLE. Extraction recoveries for the three ZEA resulted in better IC50 values and LOQs (about 1 µg/kg) metabolites ranged from 79-83% and detection capabilities compared with classical ELISA, but in a small linear range from 0.5-0.8 μg/l. At or above 2 µg/l, repeatabilities below (factor of 5). A non-enzymatic electrochemical biosensor 9% were obtained. Animal blood plasma is also a suitable for ZEA determination was developed (Feng et al., 2013). matrix for ZEA biomarker methods. De Baere et al. (2012) The biosensor was prepared by immobilising nitrogen- developed two LC-MS/MS-based methods to quantify doped graphene sheets capturing the primary antibodies ZEA, α-ZOL, β-ZOL, ZAN, α-ZAL and β-ZAL in chicken on a glassy carbon electrode. Extracted ZEA binds to the and pig plasma using an LC-ESI--QqQ and a UHPLC-ESI-- primary antibodies and is further captured by secondary Orbitrap MS. After protein precipitation with MeCN, urine antibodies, labelled with nanoporous bimetallic PtCo alloy, samples were injected and separated within 10 min. For which can reduce H2O2. A linear range of 0.005-25 µg/l was quantification, matrix-matched calibration was applied. achieved for standard solutions of ZEA. A suspension array Recoveries ranged from 82-96% on the QqQ and from for quantifying ZEA, AFB1, DON and T-2 in maize and

World Mycotoxin Journal 7 (1) 23 F. Berthiller et al.

peanuts was introduced (Wang et al., 2013c). Using 1-ethyl- in line with a study showing that masked mycotoxins can 3-(3-dimethylaminopropyl) carbodiimide hydrochloride, be hydrolysed by human colonic microbiota (Dall’Erta et mycotoxin antigens were attached to microspheres. al., 2013). Both ZEA-14-sulphate and -glucoside released Mycotoxins were then indirect competitively quantified. ZEA already after 30 min of intestinal fermentation. Linear working ranges of about three orders of magnitude and sub-μg/kg LOQs were achieved for both matrices. Disclaimer 80-116% of ZEA was recovered in the two matrices, with intra-assay RSDs <15%. The mention of trade names or commercial products in this publication is solely for the purpose of providing Easy-to-use LFDs become more and more popular for specific information and does not imply recommendation mycotoxin detection and several ones have been developed or endorsement by the authors of this article or any of for the determination of ZEA along with other mycotoxins. their institutions. The institutions of all authors are equal Within the last 12 months alone, test strips for the opportunity providers and employers. quantification of ZEA and DON (Huang et al., 2012), ZEA and FB1 (Wang et al., 2013f), ZEA, OTA and AFB1 (Li et References al., 2013c), ZEA, DON, OTA and AFB1 (Njumbe Ediage et al., 2012b) as well as ZEA, T-2, HT-2, DON and fumonisins Aamot, H.U., Hofgaard, I.S., Brodal, G., Elen, O., Holen, B. and (Lattanzio et al., 2013) were developed. All these tests are Klemsdal, S.S., 2013. Evaluation of rapid test kits for quantification suited for the analysis of various cereals and the cut-off of HT-2 and T-2 toxins in naturally contaminated oats. World levels for ZEA were in the range of 10-175 µg/kg. Mycotoxin Journal 6: 31-41. Aamot, H.U., Hofgaard, I.S., Brodal, G., Elen, O., Jestoi, M. and The application of aptamers instead of antibodies also gains Klemsdal, S.S., 2012. Evaluation of rapid test kits for quantification attention in mycotoxin determination. Chen et al. (2013) of DON in naturally contaminated oats and wheat. World Mycotoxin isolated and identified single-stranded DNA aptamers Journal 5: 339-350. recognising ZEA. After repeated selection, 12 representative Åberg, A.T., Solyakov, A. and Bondesson, U., 2013. Development and sequences were assayed for their affinity and specificity in-house validation of an LC-MS/MS method for the quantification towards ZEA. The best aptamer was then applied in the of the mycotoxins deoxynivalenol, zearalenone, T-2 and HT-2 toxin, specific detection of ZEA in real samples. The method ochratoxin A and fumonisin B1 and B2 in vegetable animal feed. recovered 85-105% of spiked ZEA in beer and showed a Food Additives and Contaminants Part A 30: 541-549. linear range of 4 orders of magnitude from 1 to 10 mg/l. Abhijith, K.S. and Thakur, M.S., 2012. Application of green synthesis

of gold nanoparticles for sensitive detection of aflatoxin B1 based Finally, the determination of conjugated or masked forms of on metal enhanced fluorescence. Analytical Methods 4: 4250-4256. ZEA also improved significantly. So far, a main disadvantage Abia, W.A., Warth, B., Sulyok, M., Krska, R., Tchana, A.N., Njobeh, was the non-availability of analytical standards. This has P.B., Dutton, M.F. and Moundipa, P.F., 2013. Determination of been largely overcome as (E)-6-(1-alkenyl)-substituted multi-mycotoxin occurrence in cereals, nuts and their products in β-resorcylic acid esters have been synthesised as ZEA Cameroon by liquid chromatography tandem mass spectrometry. mimics (Mikula et al., 2013a) and used to optimise the Food Control 31: 438-453. synthesis of several ZEA conjugates. In this context, ZEA- Afzali, D., Ghanbarian M., Mostafavi A., Shamspur T. and 14-β,D-glucuronide, which is a major metabolite of ZEA Ghaseminezhad S., 2012. A novel method for high preconcentration and occurring in urine of various animals, was synthesised of ultra trace amounts of B1, B2, G1 and G2 aflatoxins in edible oils (Mikula et al., 2012). The same group was successful to by dispersive liquid-liquid microextraction after immunoaffinity produce the Fusarium metabolite ZEA-14-sulphate as column clean-up. Journal of Chromatography A 1247: 35-41. ammonium salt (Mikula et al., 2013b) and glucosides and Al-Taher, F., Banaszewski, K., Jackson, L., Zweigenbaum, J., Ryu, D. glucuronides of α-ZOL and β-ZOL (Mikula et al., 2013c). and Cappozzo, J., 2013. Rapid method for the determination of ZEA-14G detection techniques based on acidic or enzymatic multiple mycotoxins in wines and beers by LC-MS/MS using a hydrolysis to ZEA and immunochemical determination of stable isotope dilution assay. Journal of Agricultural and Food total ZEA were developed (Beloglazova et al., 2013). While Chemistry 61: 2378-2384. trifluoromethanesulfonic acid yielded about 70% of cleavage Amelin, V.G., Karaseva, N.M. and Tretyakov, A.V., 2013a. to ZEA, several enzymes – including β-glucosidase from Combination of the QuEChERS method with dispersive liquid- Aspergillus niger – performed even better, cleaving around liquid microextraction and derivatization in the determination of 85% of the masked mycotoxin in cereal samples. Direct mycotoxins in grain and mixed feed by gas-liquid chromatography LC-MS/MS methods for the determination of ZEA and with an electron-capture detector. Journal of Analytical Chemistry DON conjugates have also been developed and used to 68: 552-557. verify their stability after oral dosing of rats (Versilovskis et al., 2012). Already after 55 min, ZEA was found in the digestive tract, proving its release from ZEA-14G. This is

24 World Mycotoxin Journal 7 (1) Developments in mycotoxin analysis: an update for 2012-2013

Amelin, V.G., Karaseva, N.M. and Tretyakov, A.V., 2013b. Simultaneous Beloglazova, N.V., Speranskaya, E.S., De Saeger, S., Hens, Z., Abe, determination of trichothecene mycotoxins, ochratoxin A and S. and Goryacheva, I.Y., 2012. Quantum dot based rapid tests for zearalenone in grain and products of its processing, feed premixes zearalenone detection. Analytical and Bioanalytical Chemistry and meat by gas chromatography. Journal of Analytical Chemistry 403: 3013-3024. 68: 61-67. Beltrán, E., Ibáñez, M., Portolés, T., Ripollés, C., Sancho, J.V., Yusà, V., Anfossi, L., D´Arco, G., Baggiani, C., Giovanoli, C. and Giraudi, G., Marín, S. and Hernández, F., 2013. Development of sensitive and 2011. A lateral flow immunoassay for measuring ochratoxin A: rapid analytical methodology for food analysis of 18 mycotoxins development of a single system for maize, wheat and durum wheat. included in a total diet study. Analytica Chimica Acta 783: 39-48. Food Control 22: 1965-1970. Berthiller, F., Crews, C., Dall’Asta, C., De Saeger, S., Haesaert, G., Anfossi, L., Giovanoli, C., Giraudi, G., Biagioli, F., Passini, C. and Karlovsky, P., Oswald, I.P., Seefelder, W., Speijers, G. and Stroka, Baggiani, C., 2013. A lateral flow immunoassay for the rapid J., 2013. Masked mycotoxins: a review. Molecular Nutrition and detection of ochratoxin A in wine and grape must. Journal of Food Research 57: 165-186. Agricultural and Food Chemistry 60: 11491-11497. Bertuzzi, T., Rastelli, S., Mulazzi, A. and Pietri, A., 2012. Evaluation and

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