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Digital Re-print - May | June 2012 LC-MS/MS: The New Reference Method

for Mycotoxin Analysis

www.gfmt.co.uk

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 agricultur-al commodities and products.

Approximately 300 to 400 substances are recognised as mycotoxins, comprising a broad variety of chemical structures pro-duced 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 analysisThe most important target analytes are

aflatoxins, trichothecenes, zearalenone and its derivatives, fumonisins, ochratoxins, ergot alkaloids, and patulin (1). Various mycotox-ins 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

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 deriva-tisation that can be time-consuming and prone to error, so that nowadays GC methods are used less frequently.

HPLC can be cou-pled with a variety of detectors, e.g. spectro-photometric (UV-Vis, diode array) detectors, refractometers (RI), fluo-rescence (FLD) detec-tors, electrochemical detectors, radioac-tivity detectors and mass spectrometers. Particularly the coupling of liquid chro-matography (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 spectrometryThe technology of liquid chromatogra-

phy-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 spectrom-eter detectors, can be used to measure a wide range of potential analytes. It has no molecular mass limitations, a very straightfor-ward 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 determina-tion and validation of 39 mycotoxins in wheat and maize was used for analys-ing A- and B-type trichothecenes and their metabolites, zearalenone and deriva-tives, fumonisins, enniatins, ergot alkaloids,

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

Mass spectrometry

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

by Dr Eva-Maria Binder Chief Scientific Officer, Erber Group, Austria

Grain&feed millinG technoloGy10 | may - June 2012

FEATURE

GFMT12.03.indd 10 22/06/2012 08:48

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 agricultur-al commodities and products.

Approximately 300 to 400 substances are recognised as mycotoxins, comprising a broad variety of chemical structures pro-duced 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 analysisThe most important target analytes are

aflatoxins, trichothecenes, zearalenone and its derivatives, fumonisins, ochratoxins, ergot alkaloids, and patulin (1). Various mycotox-ins 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

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 deriva-tisation that can be time-consuming and prone to error, so that nowadays GC methods are used less frequently.

HPLC can be cou-pled with a variety of detectors, e.g. spectro-photometric (UV-Vis, diode array) detectors, refractometers (RI), fluo-rescence (FLD) detec-tors, electrochemical detectors, radioac-tivity detectors and mass spectrometers. Particularly the coupling of liquid chro-matography (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 spectrometryThe technology of liquid chromatogra-

phy-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 spectrom-eter detectors, can be used to measure a wide range of potential analytes. It has no molecular mass limitations, a very straightfor-ward 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 determina-tion and validation of 39 mycotoxins in wheat and maize was used for analys-ing A- and B-type trichothecenes and their metabolites, zearalenone and deriva-tives, fumonisins, enniatins, ergot alkaloids,

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

Mass spectrometry

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

by Dr Eva-Maria Binder Chief Scientific Officer, Erber Group, Austria

Grain&feed millinG technoloGy10 | may - June 2012

FEATURE

GFMT12.03.indd 10 22/06/2012 08:48

orchratoxins, aflatoxin, and moniliformin (1).

A multi-mycotoxin method for food and feed matrices based on liquid chromatog-raphy/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 ana-lytes 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 quantita-tive results may be obtained without any clean-up and with very little chromatographic separation.

Unfortunately, co-eluting matrix compo-nents influence the ionization efficiency of the analyte positively or negatively, impairing the repeatability and accuracy of the ana-lytical method (1). As a consequence, only a few approaches describe the successful injec-tion 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 par-ticular the use of Mycosep® columns proved straightfor-ward and efficient (4,5,6,7,8,9).

Stable Isotope Dilution Assay

In order to overcome matrix effects and related quantification problems, external matrix calibration for each com-modity tested was recommended. This is extremely time-consuming and proved to be very impracti-cal 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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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. 3421-3425.

3 Schneweis, I., Meyer, K., Engelhardt, G., Bauer, J. 2002. Occurrence of zearalenone-4-�-D-glucopyranoside 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. 251-2585 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-of-flight 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, 8904-8910.

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 isotope-substituted (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.

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).

ConclusionsDirect coupling between a liquid phase

separation technique such as liquid chroma-tography and mass spectrometry has been recognised as a powerful tool for analysis of highly complex mixtures.

The main advantages include low detection lim-its, the ability to generate structural information, the requirement of minimal sam-ple treatment and the pos-sibility 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 rug-gedness and accuracy and pose a potential source of systematic errors.

Stable isotope labelled internal stand-ards 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 myco-toxins have been developed and published in recent years, however so far only a few were based on stable isotope labeled ana-lytes, mainly due to their limited availability and quality.

Only recently calibrants of thoroughly [13C]-labeled mycotoxins have been intro-duced thus opening a broad field of applica-tions and improvement in mycotoxin analy-sis. 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.

References: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.

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 sub-stances, i.e. their molecular structure should be as close as possible to the target analyte, while the molecular weight has to be differ-ent. 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 proper-ties of such substances, and especially their ionization potential is very similar to or nearly the same as of their naturally occurring target

analytes, but because of their higher molecular weight (due to the incorporated isotopes) dis-tinction 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 espe-cially 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 distribu-tion 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.

Contaminants, 22 (3), pp. 251-2585 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

include low detection lim-its, the ability to generate structural information, the requirement of minimal sam-ple treatment and the pos-sibility to cover a wide range

Despite their high sensitivity and selectivity, LC/

“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”

Grain&feed millinG technoloGy12 | may - June 2012

FEATURE

GFMT12.03.indd 12 22/06/2012 08:48GFMT12.03.indd 2 22/06/2012 08:48

Grain&feed millinG technoloGy may - June 2012 | 13

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