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From: Food Quality & Safety magazine, February/March 2013

New Reference Method for Mycotoxin Analysis

by Eva-Maria Binder

LC/MS/MS have become valuable tools in the analysis of mycotoxins. The use of internal standards (inset) is a method for overcoming inherent limitations.

The analysis of mycotoxins has become a global issue, and most countries have already set up regulatory limits or guideline levels for the tolerance of such contaminants in agricultural commodities and products. Approximately 300 to 400 substances are recognized as mycotoxins, comprising a broad variety of chemical structures produced by various mold species on many commodities and processed food and feed.

Globalization of the agricultural product trade has contributed significantly to the discussion about potential hazards and increased awareness of mycotoxins, at the same time as knowledge of safety in food and feed production has 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.

Internal standards are substances that are highly similar to the analytical target substances: Their molecular structure should be as close as possible to the target analyte.

Requirements of 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, humans and animals are likely exposed to mixtures rather than to individual compounds. Recently, the natural occurrence of masked mycotoxins, in which 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, including a limited number of chemically related target analytes. But as additive and synergistic effects have been observed concerning the health hazards posed by mycotoxins, the need for multi-toxin methods for the simultaneous screening of different classes of mycotoxins has risen.

High-performance liquid chromatography and gas chromatography have traditionally been the favored choices for the analyst when sensitive, reliable results with minimum variability are required. The major disadvantage of mycotoxin analysis using GC is the necessity of derivatization, which can be time-consuming and prone to error; as a result, GC methods are used less frequently.

HPLC can be coupled with a variety of detectors, including spectrophotometric detectors (UV-Vis, diode array), refractometers, fluorescence detectors, electrochemical detectors, radioactivity detectors, and mass spectrometers. The coupling of liquid chromatography and mass spectrometry, which eliminates the need for pre- or post-column sample derivatization, provides great potential for the analysis of mycotoxins. No other technique in the area of instrumental analysis of environmental toxins has developed so rapidly during the past 10 years.

Analysis is performed on LC/MS/MS equipment at Romer Labs in Union, Mo.
Analysis is performed on LC/MS/MS equipment at Romer Labs in Union, Mo.
By compensating for variations in sample preparation and cleanup, internal standards can help achieve high analytical accuracy and precision.
By compensating for variations in sample preparation and cleanup, internal standards can help achieve high analytical accuracy and precision.

Liquid Chromatography/Mass Spectrometry

Liquid chromatography/mass spectrometry technology enables efficient spectrometric assays in routine laboratory settings with high sample throughput. This technique, which in many cases utilizes multi-mass spectrometer detectors, can be used for the measurement of a wide range of potential analytes, faces no limitations by molecular mass, offers a very straightforward sample preparation, does not require chemical derivatization, and requires only limited maintenance due to rugged instrumentation. The method has become very popular in mycotoxin analysis, particularly when LC is coupled to tandem mass spectrometry.

Recently, an LC/MS/MS method for the determination and validation of 39 mycotoxins in wheat and maize was published. The analytes determined were A- and B-type trichothecenes and their metabolites; zearalenone and derivatives; fumonisins; enniatins; ergot alkaloids; orchratoxins; aflatoxin; and moniliformin.1

The development of LC/MS methods for mycotoxin determination is impeded to some extent by the chemical diversity of the analytes and the compromises that have to be made on the conditions of sample preparation.1 Considering the wide range of polarities of the analytes, the seemingly highly selective MS/MS detection could lead to the misperception that matrix interferences could be eliminated effectively and quantitative results be obtained without any cleanup 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

Consequently, few approaches describe the successful injection of crude extracts, and the majority of publications describe a sample cleanup prior to liquid chromatography with solid-phase extraction as the most efficient procedure; in particular, the use of Mycosep columns proved straightforward and efficient. 4,5,6,7,8,9

Stable Isotope Dilution Assay

To overcome matrix effects and related quantification problems, external matrix calibration for each commodity tested has so far been recommended. This process is extremely time consuming and impractical under routine conditions, in which a variety of matrices are present every day. An alternative approach, the use of stable isotope-labeled internal standards, has recently been introduced.10

These substances are not present in real-world samples but have properties identical to the analytes. Internal standards are substances that are highly similar to the analytical target substances: Their molecular structure should be as close as possible to the target analyte, while the molecular weight is different. Within the analytical process, internal standards are added to both the calibration solutions and analytical samples. By comparing the peak area ratio of an internal standard and the 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 using organic synthesis by exchanging some of the hydrogen atoms with deuterium or exchanging carbon-12 with carbon-13 atoms. Physicochemical properties of such substances, especially ionization potential, are very similar to or nearly the same as their naturally occurring target analytes, but because of their higher molecular weight (due to the incorporated isotopes), distinction between the internal standard and target analyte is possible.

Considering the wide range of polarities of the analytes, the seemingly highly selective MS/MS detection could lead to the misperception that matrix interferences could be eliminated effectively and quantitative results be obtained without any cleanup and with very little chromatographic separation.

Variations during sample preparation and cleanup, as well as during ionization, are compensated for 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 naturally abundant heavy isotopes in the analyte. This mass difference will generally depend on the molecular weight of the analyte itself; in the case of molecules with a molecular weight range of 200 to 500, a minimum of three extra mass units might be required.

Isotope-labeled standards supplied by Biopure are fully labeled, providing an optimum mass unit difference between the labeled standard and target analyte. For example, the [13C15]-DON standard, which is available as a liquid calibrant (25mgl-1), was thoroughly characterized by Häubl and colleagues with regard to purity and isotope distribution and substitution, the latter being close to 99%.9 Fortification experiments with maize confirmed the excellent suitability of [13C15]-DON as an internal standard, indicating a correlation coefficient of 0.9977 and a recovery rate of 101% +/- 2.4%. When the same analyses were run without considering the internal standard, the correlation coefficient was 0.9974 and the recovery rate was 76% +/- 1.9%, underlining the successful compensation for losses due to sample preparation and ion suppression effects by the isotope-labeled internal standards.10,11

Conclusions

Direct coupling of a liquid phase separation technique such as liquid chromatography and mass spectrometry has been recognized 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 with different polarities.

Depending on the applied interface technique, a wide range of organic compounds can be detected and flows up to 1.5 ml/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 also limit ruggedness and accuracy and pose a potential source of systematic errors.

Stable isotope-labeled internal standards have been proven to overcome these problems and compensate for fluctuations in sample preparation, such as extraction and cleanup. Numerous LC/MS/MS methods for detecting mycotoxins have been developed and published in recent years; however, only a few so far have been based on stable isotope-labeled analytes, mainly due to their limited availability and quality. Only recently have calibrants of thoroughly [13C]-labeled mycotoxins been introduced, thus opening a broad field of applications and improvement in mycotoxin analysis. The challenge for the industry is to accelerate the development of unified multi-toxin methods suitable for many types of analyte/matrix combinations.


Dr. Eva-Maria Binder is chief scientific officer at the Erber Group. She can be reached at eva.binder@erber-group.net.

References

  1. Sulyok M, Berthiller F, Krska R, Schuhmacher R. 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. 2006;20(18):2649-2659.
  2. Berthiller F, Dall'Asta C, Schuhmacher R, Lemmens M, Adam G, Krska R. Masked mycotoxins: determination of a deoxynivalenol glucoside in artificially and naturally contaminated wheat by liquid chromatography-tandem mass spectrometry. J Agric Food Chem. 2005;53(9):3421-3425.
  3. Schneweis I, Meyer K, Engelhardt G, Bauer J. Occurrence of zearalenone-4-β-D-glucopyranoside in wheat. J Agric Food Chem. 2002;50(6):1736-1738.
  4. Biancardi A, Gasparini M, Dall'Asta C, Marchelli R. A rapid multiresidual determination of type A and type B trichothecenes in wheat flour by HPLC-ESI-MS. Food Addit Contam. 2005;22(3):251-258.
  5. Berthiller F, Schuhmacher R, Buttinger G, Krska R. 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 Chromatogr A. 2005;1062(2):209-216.
  6. Biselli S, Hummert C. 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 Addit Contam. 2005;22(8):752-760.
  7. Tanaka H, Takino M, Sugita-Konishi Y, Tanaka T. 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. 2006;20(9):1422-1428.
  8. Milanez TV, Valente-Soares LM. Gas chromatography: mass spectrometry determination of trichothecene mycotoxins in commercial corn harvested in the state of São Paulo, Brazil. J Braz Chem Soc. 2006;17(2):412-416.
  9. Klötzel M, Gutsche B, Lauber U, Humpf HU. Determination of 12 type A and B trichothecenes in cereals by liquid chromatography-electrospray ionization tandem mass spectrometry. J Agric Food Chem. 2005;53(23):8904-8910.
  10. Häubl G, Berthiller F, Krska R, Schuhmacher R. Suitability of a 13C isotope labeled internal standard for the determination of the mycotoxin deoxynivalenol by LC-MS/MS without clean up. Anal Bioanal Chem. 2006;384(3):692-696.
  11. Häubl G, Berthiller F, Rechthaler J, et al. Characterization and application of isotope-substituted (13C15)-deoxynivalenol (DON) as an internal standard for the determination of DON. Food Addit Contam. 2006;23(11):1187-1193.
  12. Sakairi M, Kato Y. Multi-atmospheric pressure ionization interface for liquid chromatography-mass spectrometry. J Chromatogry A. 1998;794(1-2):391-406.

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