New Reference Method for Mycotoxin Analysis

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

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