Digital Re-print - May | June 2012

LC-MS/MS: The New Reference Method for Mycotoxin Analysis
Grain & Feed Milling Technology is published six times a year by Perendale Publishers Ltd of the United Kingdom. All data is published in good faith, based on information received, and while every care is taken to prevent inaccuracies, the publishers accept no liability for any errors or omissions or for the consequences of action taken on the basis of information published. ©Copyright 2010 Perendale Publishers Ltd. All rights reserved. No part of this publication may be reproduced in any form or by any means without prior permission of the copyright owner. Printed by Perendale Publishers Ltd. ISSN: 1466-3872



The New Reference Method for Mycotoxin Analysis

by Dr Eva-Maria Binder Chief Scientific Officer, Erber Group, Austria
he analysis of mycotoxins has become an issue of global interest, in particular because most countries already set up regulative limits or guideline levels for the tolerance of such contaminants in agricultural commodities and products. Approximately 300 to 400 substances are recognised as mycotoxins, comprising a broad variety of chemical structures produced by various mould species on many agricultural commodities and processed food and feed. Globalisation of the trade of agricultural products contributed significantly to the discussion about potential hazards involved and increased the awareness of mycotoxins. Safety awareness in food and feed production has also risen due to the simple fact that methods for testing residues and undesirable substances have become noticeably more sophisticated and available at all points of the supply chain.


Modern mycotoxin analysis
The most important target analytes are aflatoxins, trichothecenes, zearalenone and its derivatives, fumonisins, ochratoxins, ergot alkaloids, and patulin (1). Various mycotoxins may occur simultaneously, depending on environmental and substrate conditions. Considering this coincident production, it is very likely, that humans and animals are exposed to mixtures rather than to individual compounds. Recently, the natural occurrence of masked mycotoxins, where the toxin is conjugated, has been reported, requiring even more selective and sensitive detection principles (1,2,3). So far most analytical methods deal with
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single mycotoxins or mycotoxin classes, thus including a limited number of chemically related target analytes only. But as additive and synergistic effects have been observed concerning the health hazards posed by mycotoxins, efforts have been increased to search for multi-toxin methods for the simultaneous screening of different classes of mycotoxins. High performance liquid chromatography (HPLC) and gas chromatography (GC) have traditionally been the favored choices for the analyst when sensitive, reliable results are required with minimum variability. The major disadvantage of mycotoxin analysis using GC is based on the necessity of derivatisation that can be timeconsuming and prone to error, so that nowadays GC methods are used less frequently. HPLC can be coupled with a variety of detectors, e.g. spectrophotometric (UV-Vis, diode array) detectors, refractometers (RI), fluorescence (FLD) detectors, electrochemical detectors, radioactivity detectors and mass spectrometers. Particularly the coupling of liquid chromatography (LC) and mass spectrometry (MS) provided a great potential for the analysis of mycotoxins, as the need for pre- or post-column sample derivatisation was eliminated. Thus, no other technique in the area of instrumental analysis of environmental toxins developed so rapidly during the past 10 years.

Mass spectrometry
The technology of liquid chromatography-mass spectrometry (LC/MS) opens the perspective of efficient spectrometric assays for routine laboratory settings, with high sample throughput. This technique, which in many cases utilises multi-mass spectrometer detectors, can be used to measure a wide range of potential analytes. It has no molecular mass limitations, a very straightforward sample preparation, does not require chemical derivatisation and has, due to the rugged instrumentation, limited maintenance needs. Therefore, liquid chromatography/

mass spectrometry (LC/MS) and particularly LC coupled to tandem mass spectrometry (LC/MS/MS) have become very popular in mycotoxin analysis. A liquid chromatography/tandem mass spectrometric method for the determination and validation of 39 mycotoxins in wheat and maize was used for analysing A- and B-type trichothecenes and their metabolites, zearalenone and derivatives, fumonisins, enniatins, ergot alkaloids,

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orchratoxins, aflatoxin, and moniliformin (1). A multi-mycotoxin method for food and feed matrices based on liquid chromatography/electrospray ionization-tandem mass spectrometry (HPLC/ESI-MS/MS) covered the analysis of 186 fungal and bacterial metabolites. The method is based on a single extraction step using an acidified acetonitrile/ water mixture followed by analysis of the diluted crude extract (13). The development of LC/MS methods for mycotoxin determination is impeded to some extent by the chemical diversity of the analytes and compromises that have to be made on the conditions of sample preparation (1). Considering the wide range of polarities of the analytes the seemingly high selective MS/MS detection could lead incorrectly to the perception that matrix interferences could be eliminated effectively and quantitative results may be obtained without any clean-up and with very little chromatographic separation. Unfortunately, co-eluting matrix components influence the ionization efficiency of the analyte positively or negatively, impairing the repeatability and accuracy of the analytical method (1). As a consequence, only a few approaches describe the successful injection of crude extracts, and the majority of publications depict a sample clean-up prior to liquid chromatography with solid-phase extraction (SPE) as the most efficient pro-

cedure, and in particular the use of Mycosep® columns proved straightforward and efficient (4,5,6,7,8,9).

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In order to overcome matrix effects and related quantification problems, external matrix calibration for each commodity tested was recommended. This is extremely time-consuming and proved to be very impractical under routine conditions, where one is confronted with a variety of matrices every day. As an alternative approach, the use of [stable] isotope labelled internal standards has been introduced recently (10). These sub-

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FEATURE stances are not present in real world samples but have identical properties to the analytes. Internal standards are substances which are highly similar to the analytical target substances, i.e. their molecular structure should be as close as possible to the target analyte, while the molecular weight has to be different. Within the analytical process, internal standards are added to both, the calibration solutions and analytical samples, and by comparing the peak area ratio of internal standard and analyte, the concentration of the analyte can be determined. Ideal internal standards are isotope-marked molecules of a respective target analyte, which are usually prepared via organic synthesis by exchanging some of the hydrogen atoms by deuterium, or by exchanging carbon [12C] atoms by [13C]. Physico-chemical properties of such substances, and especially their ionization potential is very similar to or nearly the same as of their naturally occurring target The same analyses without considering the internal standard resulted in R2=0.9974 and a recovery rate of 76 percent +/- 1.9 percent , underlining the successful compensation for losses due to sample preparation and ion suppression effects by isotope labeled internal standards (10,11).
2 Berthiller, F., Dall’Asta, C., Schuhmacher, R., Lemmens, M., Adam, G., Krska, A.R. 2005. Masked mycotoxins: Determination of a deoxynivalenol glucoside in artificially and naturally contaminated wheat by liquid chromatography-tandem mass spectrometry. J. Agr. Food Chem. 53, 9, pp. 34213425. 3 Schneweis, I., Meyer, K., Engelhardt, G., Bauer, J. 2002. Occurrence of zearalenone-4-�-Dglucopyranoside in wheat. J. Agric. Food Chem. 50 (6), pp. 1736-1738. 4 Biancardi, A., Gasparini, M., Dall’Asta, C., Marchelli, R. 2005. A rapid multiresidual determination of type A and type B trichothecenes in wheat flour by HPLC-ESI-MS. Food Additives and Contaminants, 22 (3), pp. 251-258 5 Berthiller, F., Schuhmacher, R., Buttinger, G., Krska, R. 2005b. Rapid simultaneous determination of major type A- and B-trichothecenes as well as zearalenone in maize by high performance liquid chromatography-tandem mass spectrometry. J. Chromatog. A, 1062, 2, pp. 209-216. 6 Biselli, S., Hummert, C. 2005. Development of a multicomponent method for Fusarium toxins using LC-MS/MS and its application during a survey for the content of T-2 toxin and deoxynivalenol in various feed and food samples. Food Add. Contam. 22 (8), pp. 752-760. 7 Tanaka, H., Takino, M., Sugita-Konishi, Y., Tanaka, T. 2006. Development of a liquid chromatography/time-offlight mass spectrometric method for the simultaneous determination of trichothecenes, zearalenone and aflatoxins in foodstuffs. Rapid Commun. Mass Spectrom. 20 (9), pp. 1422-1428. 8 Milanez, T.V., Valente-Soares, L.M. 2006. Gas chromatography - Mass spectrometry determination of trichothecene mycotoxins in commercial corn harvested in the State of São Paulo, Brazil. Journal of the Brazilian Chemical Society, 17 (2), pp. 412-416. 9 Klötzel, M., Gutsche, B., Lauber, U., Humpf, H.-U. 2005. Determination of 12 Type A and B Trichothecenes in Cereals by Liquid Chromatography- Electrospray Ionization Tandem Mass Spectrometry. J. Chromatog. 53, 89048910. 10 Häubl, G., Berthiller, F., Krska, R., Schuhmacher, R. 2005. Sitability of a 13C isotope labeled internal standard for the determination of the mycotoxin Deoxynivalenol by LC-MS/MS without clean-up. Anal. Bioanal. Chem. 384 (3), pp. 692-696. 11 Häubl, G., Berthiller, F., Rechthaler, J., Jaunecker, G., Binder, E.M., Krska, R., Schuhmacher, R. 2006. Characterisation and application of isotopesubstituted (13C15)-deoxynivalenol (DON) as an internal standard for the determination of DON. Food Add. Contam. In print. 12 Sakairi, M., Kato, Y. 1998. Multi-atmospheric pressure ionization interface for liquid chromatography-mass spectrometry. J. Chromatography A, 794, 391-406. 13 Vishwanath, V., Sulyhok, M., Labuda, R., Bicker, W., Krska, R. (2009) Anal. Bioanal. Chem. 395:1355– 1372.

Direct coupling between a liquid phase separation technique such as liquid chromatography and mass spectrometry has been recognised as a powerful tool for analysis of highly complex mixtures. The main advantages include low detection limits, the ability to generate structural information, the requirement of minimal sample treatment and the possibility to cover a wide range of analytes differing in their polarities. Depending on the applied interface technique a wide range of organic compounds can be detected and flows up to 1.5ml/min can be handled (12). Despite their high sensitivity and selectivity, LC/ MS/MS instruments are limited to some extent due to matrix-induced differences in ionization efficiencies and signal intensities between calibrants and analytes. Ion suppression/enhancement due to matrix compounds entering the mass spectrometer together with the analytes limit also ruggedness and accuracy and pose a potential source of systematic errors. Stable isotope labelled internal standards have been proven to overcome these problems as well as to compensate also for fluctuations in sample preparation, e.g. extraction and clean-up. Numerous LC/MS/ MS methods for the determination of mycotoxins have been developed and published in recent years, however so far only a few were based on stable isotope labeled analytes, mainly due to their limited availability and quality. Only recently calibrants of thoroughly [13C]-labeled mycotoxins have been introduced thus opening a broad field of applications and improvement in mycotoxin analysis. Thus in particular the development of unified multi-toxin methods being suitable for the determination of many types of analyte/matrix combinations poses a great challenge for the future.

“Direct coupling between a liquid phase separation technique such as liquid chromatography and mass spectrometry has been recognised as a powerful tool for analysis of highly complex mixtures”
analytes, but because of their higher molecular weight (due to the incorporated isotopes) distinction between internal standard and target analyte is possible. Variations during sample preparation and clean-up as well as during ionization are compensated so that methods with especially high analytical accuracy and precision can be developed. Optimally, these isotope labeled analogues must have a large enough mass difference to nullify the effect of natural abundance heavy isotopes in the analyte. This mass difference will depend generally on the molecular weight of the analyte itself, in case of molecules with a molecular weight range of 200 to 500, a minimum of three extra mass units might be required. Isotope labelled standards supplied by Biopure are fully labelled thus providing an optimum mass unit difference between labeled standard and target analyte. For example, the [13C15]-DON standard, which is available as liquid calibrant (25mgl-1) was thoroughly characterised by Häubl et al.(9) with regard to purity and isotope distribution and substitution, the latter being close to 99 percent. Fortification experiments with maize proved the excellent suitability of [13C15]-DON as internal standard indicating a correlation coefficient (R2) of 0.9977 and a recovery rate of 101 percent +/- 2.4 percent.
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1 Sulyok, M., Berthiller, F., Krska., R., Schuhmacher, R. 2006. Development and validation of a liquid chromatography/ tandem mass spectrometric method for the determination of 39 mycotoxins in wheat and maize. Rapid Commun. Mass Spectrom. 20, 2649-2659.

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