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LC-MS/MS: The New Reference Method for Mycotoxin Analysis


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The 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.

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LC-MS/MS: The New Reference Method for Mycotoxin Analysis

  1. 1. Digital Re-print - May | June 2012 LC-MS/MS: The New Reference Method for Mycotoxin AnalysisGrain & 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 ofinformation published.©Copyright 2010 Perendale Publishers Ltd. All rights reserved. No part of this publication may be reproduced in any formor by any means without prior permission of the copyright owner. Printed by Perendale Publishers Ltd. ISSN: 1466-3872
  2. 2. FEATURE LC-MS/MS: The New Reference Method for Mycotoxin Analysisby Dr Eva-Maria Binder Chief Scientific Officer, Erber Group, AustriaT he analysis of mycotoxins has single mycotoxins or mycotoxin classes, thus Mass spectrometry become an issue of global interest, including a limited number of chemically The technology of liquid chromatogra- in particular because most related target analytes only. But as additive phy-mass spectrometry (LC/MS) opens thecountries already set up regulative limits and synergistic effects have been observed perspective of efficient spectrometric assaysor guideline levels for the tolerance concerning the health hazards posed by for routine laboratory settings, with highof such contaminants in agricultur- mycotoxins, efforts have been increased sample throughput. This technique, whichal commodities and products. to search for multi-toxin methods for the in many cases utilises multi-mass spectrom- simultaneous screening of different classes eter detectors, can be used to measure a Approximately 300 to 400 substances of mycotoxins. wide range of potential analytes. It has noare recognised as mycotoxins, comprising High performance liquid chromatography molecular mass limitations, a very straightfor-a broad variety of chemical structures pro- (HPLC) and gas chromatography (GC) have ward sample preparation, does not requireduced by various mould species on many traditionally been the favored choices for the chemical derivatisation and has, due to theagricultural commodities and processed food analyst when sensitive, reliable results are rugged instrumentation, limited maintenanceand feed. Globalisation of the trade of required with minimum variability. The major needs. Therefore, liquid chromatography/agricultural products contributed significantly disadvantage of mycotoxinto the discussion about potential hazards analysis using GC is basedinvolved and increased the awareness of on the necessity of deriva-mycotoxins. Safety awareness in food and tisation that can be time-feed production has also risen due to the consuming and prone tosimple fact that methods for testing residues error, so that nowadaysand undesirable substances have become GC methods are used lessnoticeably more sophisticated and available all points of the supply chain. HPLC can be cou- pled with a variety ofModern mycotoxin analysis detectors, e.g. spectro- The most important target analytes are photometric (UV-Vis,aflatoxins, trichothecenes, zearalenone and diode array) detectors,its derivatives, fumonisins, ochratoxins, ergot refractometers (RI), fluo-alkaloids, and patulin (1). Various mycotox- rescence (FLD) detec-ins may occur simultaneously, depending tors, electrochemical detectors, radioac- mass spectrometry (LC/MS) and particularlyon environmental and substrate conditions. tivity detectors and mass spectrometers. LC coupled to tandem mass spectrometryConsidering this coincident production, it Particularly the coupling of liquid chro- (LC/MS/MS) have become very popular inis very likely, that humans and animals matography (LC) and mass spectrometry mycotoxin analysis.are exposed to mixtures rather than to (MS) provided a great potential for the A liquid chromatography/tandem massindividual compounds. Recently, the natural analysis of mycotoxins, as the need for spectrometric method for the determina-occurrence of masked mycotoxins, where pre- or post-column sample derivatisation tion and validation of 39 mycotoxins inthe toxin is conjugated, has been reported, was eliminated. Thus, no other technique wheat and maize was used for analys-requiring even more selective and sensitive in the area of instrumental analysis of ing A- and B-type trichothecenes anddetection principles (1,2,3). environmental toxins developed so rapidly their metabolites, zearalenone and deriva- So far most analytical methods deal with during the past 10 years. tives, fumonisins, enniatins, ergot alkaloids,10 | may - June 2012 Grain &feed millinG technoloGy
  3. 3. BIN LEVELSorchratoxins, aflatoxin, and moniliformin cedure, and in par-(1). ticular the use of A multi-mycotoxin method for food and Mycosep® columnsfeed matrices based on liquid chromatog- proved straightfor-raphy/electrospray ionization-tandem massspectrometry (HPLC/ESI-MS/MS) covered ward and efficient (4,5,6,7,8,9). without climbing! smartBob and eBob softwarethe analysis of 186 fungal and bacterialmetabolites. The method is based on a single Stable Isotopeextraction step using an acidified acetonitrile/ Dilution Assaywater mixture followed by analysis of the In order todiluted crude extract (13). overcome matrix The development of LC/MS methods for effects and relatedmycotoxin determination is impeded to some quantificationextent by the chemical diversity of the ana- problems, externallytes and compromises that have to be made matrix calibrationon the conditions of sample preparation (1). for each com- Considering the wide range of polaritiesof the analytes the seemingly high selective modity tested was recommended. affordable. reliable. safe. sMS/MS detection could lead incorrectly to This is extremely Inventory management systemsthe perception that matrix interferences time-consuming and bin level indicatorscould be eliminated effectively and quantita- and proved totive results may be obtained without any be very impracti-clean-up and with very little chromatographic cal under routineseparation. conditions, where Unfortunately, co-eluting matrix compo- one is confrontednents influence the ionization efficiency of with a variety ofthe analyte positively or negatively, impairing matrices every day.the repeatability and accuracy of the ana- As an alternativelytical method (1). As a consequence, only a approach, the use Rotary Pressure Switch Vibrating Rod Capacitance Probefew approaches describe the successful injec- of [stable] isotopetion of crude extracts, and the majority ofpublications depict a sample clean-up prior labelled internal standards has been Binmaster level controlsto liquid chromatography with solid-phase introduced recently • www.binmaster.comextraction (SPE) as the most efficient pro- (10). These sub- © 2012 BinMaster, Lincoln, Nebraska uSa NEW! AgraStrip® + AgraVisionTM Quantitative Strip Tests for ß Aflatoxins ß Deoxynivalenol (DON) ß Fumonisins & ß GMOs The AgraVision™ is a new robust handheld reader with printer! Tel +43 2272 6153310 (AT) Tel +44 845 5192010 (UK) E-Mail www.romerlabs.comGrain &feed millinG technoloGy may - June 2012 | 11
  4. 4. FEATURE stances are not present in real world samples The same analyses without considering the 2 Berthiller, F., Dall’Asta, C., Schuhmacher, R., but have identical properties to the analytes. internal standard resulted in R2=0.9974 and Lemmens, M., Adam, G., Krska, A.R. 2005. Masked Internal standards are substances which a recovery rate of 76 percent +/- 1.9 percent mycotoxins: Determination of a deoxynivalenol are highly similar to the analytical target sub- , underlining the successful compensation glucoside in artificially and naturally contaminated stances, i.e. their molecular structure should for losses due to sample preparation and wheat by liquid chromatography-tandem mass be as close as possible to the target analyte, ion suppression effects by isotope labeled spectrometry. J. Agr. Food Chem. 53, 9, pp. 3421- while the molecular weight has to be differ- internal standards (10,11). 3425. ent. Within the analytical process, internal 3 Schneweis, I., Meyer, K., Engelhardt, G., Bauer, standards are added to both, the calibration Conclusions J. 2002. Occurrence of zearalenone-4-�-D- solutions and analytical samples, and by Direct coupling between a liquid phase glucopyranoside in wheat. J. Agric. Food Chem. 50 comparing the peak area ratio of internal separation technique such as liquid chroma- (6), pp. 1736-1738. standard and analyte, the concentration of tography and mass spectrometry has been 4 Biancardi, A., Gasparini, M., Dall’Asta, C., Marchelli, the analyte can be determined. recognised as a powerful tool for analysis of R. 2005. A rapid multiresidual determination of Ideal internal standards are isotope-marked highly complex mixtures. type A and type B trichothecenes in wheat molecules of a respective target analyte, which The main advantages flour by HPLC-ESI-MS. Food Additives and are usually prepared via organic synthesis by include low detection lim- Contaminants, 22 (3), pp. 251-258 exchanging some of the hydrogen atoms by its, the ability to generate 5 Berthiller, F., Schuhmacher, R., Buttinger, deuterium, or by exchanging carbon [12C] structural information, the G., Krska, R. 2005b. Rapid simultaneous atoms by [13C]. Physico-chemical proper- requirement of minimal sam- determination of major type A- and ties of such substances, and especially their ple treatment and the pos- B-trichothecenes as well as zearalenone ionization potential is very similar to or nearly sibility to cover a wide range in maize by high performance liquid the same as of their naturally occurring target of analytes differing in chromatography-tandem mass their polarities. spectrometry. J. Chromatog. A, 1062, 2, Depending on pp. 209-216.“Direct coupling between the applied interface 6 Biselli, S., Hummert, C. 2005. technique a wide Development of a multicomponent methoda liquid phase separation range of organic for Fusarium toxins using LC-MS/MS and itstechnique such as liquid compounds can be application during a survey for the content of detected and flows T-2 toxin and deoxynivalenol in various feedchromatography and mass up to 1.5ml/min can and food samples. Food Add. Contam. 22 be handled (12).spectrometry has been Despite their high (8), pp. 752-760. 7 Tanaka, H., Takino, M., Sugita-Konishi,recognised as a powerful sensitivity and selectivity, LC/ Y., Tanaka, T. 2006. Development MS/MS instruments are limited to of a liquid chromatography/time-of-tool for analysis of highly some extent due to matrix-induced flight mass spectrometric method for thecomplex mixtures” differences in ionization efficiencies and signal simultaneous determination of trichothecenes, intensities between calibrants and analytes. zearalenone and aflatoxins in foodstuffs. Ion suppression/enhancement due to matrix Rapid Commun. Mass Spectrom. 20 (9), pp. analytes, but because of their higher molecular compounds entering the mass spectrometer 1422-1428. weight (due to the incorporated isotopes) dis- together with the analytes limit also rug- 8 Milanez, T.V., Valente-Soares, L.M. 2006. tinction between internal standard and target gedness and accuracy and pose a potential Gas chromatography - Mass spectrometry analyte is possible. source of systematic errors. determination of trichothecene mycotoxins in Variations during sample preparation and Stable isotope labelled internal stand- commercial corn harvested in the State of São clean-up as well as during ionization are ards have been proven to overcome these Paulo, Brazil. Journal of the Brazilian Chemical compensated so that methods with espe- problems as well as to compensate also Society, 17 (2), pp. 412-416. cially high analytical accuracy and precision for fluctuations in sample preparation, e.g. 9 Klötzel, M., Gutsche, B., Lauber, U., Humpf, can be developed. Optimally, these isotope extraction and clean-up. Numerous LC/MS/ H.-U. 2005. Determination of 12 Type labeled analogues must have a large enough MS methods for the determination of myco- A and B Trichothecenes in Cereals by Liquid mass difference to nullify the effect of natural toxins have been developed and published Chromatography- Electrospray Ionization Tandem abundance heavy isotopes in the analyte. in recent years, however so far only a few Mass Spectrometry. J. Chromatog. 53, 8904- This mass difference will depend generally were based on stable isotope labeled ana- 8910. on the molecular weight of the analyte itself, lytes, mainly due to their limited availability 10 Häubl, G., Berthiller, F., Krska, R., Schuhmacher, in case of molecules with a molecular weight and quality. R. 2005. Sitability of a 13C isotope labeled range of 200 to 500, a minimum of three Only recently calibrants of thoroughly internal standard for the determination of the extra mass units might be required. [13C]-labeled mycotoxins have been intro- mycotoxin Deoxynivalenol by LC-MS/MS without Isotope labelled standards supplied by duced thus opening a broad field of applica- clean-up. Anal. Bioanal. Chem. 384 (3), pp. Biopure are fully labelled thus providing tions and improvement in mycotoxin analy- 692-696. an optimum mass unit difference between sis. Thus in particular the development of 11 Häubl, G., Berthiller, F., Rechthaler, J., Jaunecker, labeled standard and target analyte. For unified multi-toxin methods being suitable G., Binder, E.M., Krska, R., Schuhmacher, R. 2006. example, the [13C15]-DON standard, which for the determination of many types of Characterisation and application of isotope- is available as liquid calibrant (25mgl-1) was analyte/matrix combinations poses a great substituted (13C15)-deoxynivalenol (DON) as an thoroughly characterised by Häubl et al.(9) challenge for the future. internal standard for the determination of DON. with regard to purity and isotope distribu- Food Add. Contam. In print. tion and substitution, the latter being close References: 12 Sakairi, M., Kato, Y. 1998. Multi-atmospheric pressure to 99 percent. Fortification experiments 1 Sulyok, M., Berthiller, F., Krska., R., Schuhmacher, R. 2006. ionization interface for liquid chromatography-mass with maize proved the excellent suitability of Development and validation of a liquid chromatography/ spectrometry. J. Chromatography A, 794, 391-406. [13C15]-DON as internal standard indicating tandem mass spectrometric method for the determination 13 Vishwanath, V., Sulyhok, M., Labuda, R., Bicker, W., a correlation coefficient (R2) of 0.9977 and a of 39 mycotoxins in wheat and maize. Rapid Commun. Krska, R. (2009) Anal. Bioanal. Chem. 395:1355– recovery rate of 101 percent +/- 2.4 percent. Mass Spectrom. 20, 2649-2659. 1372. 12 | may - June 2012 Grain &feed millinG technoloGy
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  6. 6. This digital Re-print is part of the May | June 2012 edition of Grain & Feed Milling Technology magazine. Content from the magazine is available to view free-of-charge, both as a full LINKS online magazine on our website, and as an archive of individual features on the docstoc website. Please click here to view our other publications on May - June 2012 • See the full issue • LC-MS/MS: The New Reference Method for Mycotoxin Analysis • Visit the GFMT website • Contact the GFMT Team • Mould control in grain and feed preservation In this issue: • NIR in practice • Rice and • Fast, reliable contract • Subscribe to GFMT and flexible: terms the world of modern bulk weighing • New weighing software for UK co-operative A subscription magazine for the global flour & feed milling industries - first published in 1891 GFMT12.03.indd 1 22/06/2012 08:48 To purchase a paper copy of the magazine, or to subscribe to the paper edi- tion please contact our Circulation and Subscriptions Manager on the link adove. INFORMATION FOR ADVERTISERS - CLICK HERE Article reprints All Grain & Feed Milling Tecchnology feature articles can be re-printed as a 4 or 8 page booklets (these have been used as point of sale materials, promotional materials for shows and exhibitions etc). If you are interested in getting this article re-printed please contact the GFMT team for more informa- tion on - Tel: +44 1242 267707 - Email: or visit