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From: Food Quality & Safety magazine, June/July 2014

LC/MS/MS in Detecting and Quantifying Mycotoxins

Control of these potentially life threatening fungai toxins in food and animal feed is vitally important

by Thomas Glauner, PhD

Testing

Mycotoxins are produced primarily by Aspergillus, Penicillium, and Fusarium fungi growing on a variety of agricultural commodities worldwide. They pose a major threat to human and animal health, as they have been implicated as causes of cancer and mutagenicity, as well as estrogenic, gastrointestinal, urogenital, vascular, kidney, and nervous disorders. Some may also impair resistance to infectious disease by compromising the immune system. Their impact on human health, animal productivity, and international trade results in significant economic losses.

The mycotoxins that pose the biggest threat to food safety include the aflatoxins, ochratoxin A, and toxins produced by Fusarium molds, including fumonisins, trichothecenes, and zearalenone. Aflatoxins (B1, B2, G1, G2, and M1), are the most toxic, including damage to DNA that can cause cancer in animals. In fact, AFB1 and mixtures of AFB1, AFG1, and AFM1 are proven human carcinogens, and AFM1 and AFB2 are designated as probable human carcinogens by the International Agency for Research on Cancer (IARC). They contaminate many crops grown in hot and humid regions of the world, including peanuts, corn, cottonseed, and pistachios.

Ochratoxin A is produced by several Penicillium and Aspergillus fungal strains, and it occurs in a large variety of foods. It is classified by the IARC as a probable human carcinogen and is also implicated in kidney damage, birth defects, and immune deficiency.

Fumonisins are the result of fungal infection of maize, tomatoes, asparagus, and garlic, but maize-containing foods are the major food safety concern for fumonisin contamination. There are at least 15 related fumonisin compounds, and fumonisin B1 can cause necrotic lesions in the cerebrum in horses, and pulmonary edema in swine. The fumonisins are weak carcinogens in rodents and probable human carcinogens that have been associated with esophageal cancer in South Africa and China. The level of fumonisin contamination in corn was relatively high in the U.S. between 1988 and 1991, but has been low in recent years.

Only a few of the nearly 200 trichothecenes occur at concentrations high enough to pose significant threats to human health. The most prevalent of these is deoxynivalenol (DON), also known as vomitoxin. DON occurs predominantly in grains such as wheat, barley, oats, rye, and maize, and it is immunotoxic in animal models. It is not a known carcinogen and its major symptom in animals is reduced feed intake. Large amounts of grain with vomitoxin would have to be consumed to pose a health risk to humans. Type A trichothecenes like T-2 toxin or HT-2 toxin are more toxic to mammals than type B trichothecenes such as DON, but fortunately often occur in lower concentrations. Oats are the most prone cereals for contamination by trichothecenes, followed by barley and maize.

Zearalenone is an estrogenic compound found almost entirely in grains that has received recent focus due to concerns that environmental estrogens can disrupt sex steroid hormone functions. In fact, occasional outbreaks of zearalenone mycotoxicosis in livestock have caused infertility. Zearalenone has also been reported to have genotoxic activity.

hromatogram of a calibration sample containing all 11 EU regulated mycotoxins illustrating the separation efficiency of the UHPLC method run on an Agilent 1290 Infinity LC system and an Agilent 6490 Triple Quadrupole LC/MS.
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Figure 1: Chromatogram of a calibration sample containing all 11 EU regulated mycotoxins illustrating the separation efficiency of the UHPLC method run on an Agilent 1290 Infinity LC system and an Agilent 6490 Triple Quadrupole LC/MS.

Regulating Levels in Food and Feed

Limiting mycotoxin exposure to humans and agricultural animals is paramount, and more than 100 countries regulate levels of mycotoxins in foods and feed because of their public health significance and commercial impact. The U.S. FDA has established advisory levels for DON and fumonisins and action levels for aflatoxin, but regulatory limits have not been established in the U.S. for mycotoxins. China, Brazil, and Mexico have the most comprehensive legislation on aflatoxin. China and Russia have established limits for ochratoxins in cereals and other products. Several countries, including India and Japan, have maximum limits for DON. However, in the international markets, no maximum limits for fuminisins exist in several countries, including Russia, Canada, and many Latin American countries. Several countries do have maximum limits for zearalenone.

The European Union (EU) has comprehensive regulations that are referenced by several other countries for establishment of their own limits. Commission Regulation (EC) No. 1881/2006 and its amendments set out specific rules in relation to mycotoxins and other contaminants. It includes specific maximum levels for 11  mycotoxins, including aflatoxins, ochratoxin A, type A and B trichothecenes, fumonisins, and zearalenone. This regulation applies to all food business operators involved, for example, in the import, production, processing, storage, distribution, and sale of food.

Extracted quantifier ion peak, qualifier to quantifier ion ratios, triggered spectra library matching (upper panel) and calibration curve and structure for aflatoxin M1 (lower panel), using the UHPLC/MS/MS method for infant formula. An Agilent 1290 Infinity LC system and ­Agilent 6490 Triple Quadrupole LC/MS with triggered MRM was used.
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Figure 2: Extracted quantifier ion peak, qualifier to quantifier ion ratios, triggered spectra library matching (upper panel) and calibration curve and structure for aflatoxin M1 (lower panel), using the UHPLC/MS/MS method for infant formula. An Agilent 1290 Infinity LC system and ­Agilent 6490 Triple Quadrupole LC/MS with triggered MRM was used.

Efficient Testing

Most traditional methods for the determination of mycotoxins in food or feed have been single-analyte methods, and few of them used liquid chromatography coupled to tandem mass spectrometry (LC/MS/MS) until a few years ago. However, tandem mass spectrometry is a powerful tool capable of accurately detecting and quantitating the levels of mycotoxins that are dictated by the regulations. Several LC/MS/MS methods have been developed that enable high throughput analysis of food products for accurate and reproducible quantitation of very low levels of several mycotoxins at once. A few are presented here.

Accurate quantitation in complex food matrices can be hampered by suppression or enhancement of the analyte signal due to matrix effects during the mass spectrometry ionization process. Differences in the degree of matrix effects cannot only be expected between different commodities but, to a lesser extent, also between individual samples of one matrix type.

There are different strategies to compensate for matrix effects such as the dilution of the sample, matrix-matched calibrations, standard addition, or the use of internal standards. For busy routine testing laboratories, the use of internal standards which behave exactly like the target compounds but are still distinctive, is most attractive. In the past, internal standards have often been analogs of a single compound or group of compounds. However, this has limited value when the intention is to compensate for matrix effects, since such effects are retention time dependent and target compounds rarely elute concurrent with such analogs.

Stable isotopically-labeled compounds are ideally suited as internal standards since they share the same physicochemical properties (meaning they elute together with the target compound) but are still distinguishable by MS due to their different molecular mass. In addition, they are not present in naturally contaminated samples. Since the naturally abundant isotopic distribution of the analyte is diluted by the addition of stable isotopically labeled compounds, this procedure is often referred to as stable isotope dilution assay (SIDA).

A SIDA LC/MS/MS assay has been developed for the analysis of the 11 mycotoxins regulated by the EU in maize. To assure accurate quantitation, a uniformly (13C)-labeled homolog for each target analyte was used as the internal standard (Figure 1). A two-step extraction without further cleanup was combined with ultra high performance liquid chromatography (UHPLC) separation and highly sensitive MS/MS detection using Dynamic Multiple Reaction Monitoring (dMRM). This method was successfully validated for maize based on method performance parameters including linearity of response, the limit of quantitation (LOQ) based on the signal-to-noise (S/N) ratio, and repeatability. The accuracy and reliability of the method were proven by analyzing several test materials with well-characterized concentrations. The key benefits of this method are the simple and complete extraction, the improved accuracy for a wide variety of matrices enabled by efficient compensation of all matrix effects, and high sensitivity.

Forty analytes could be identified in different kinds of nut samples. The chart shows the number of each kind of nut sample that contained the given fungal compound. An Agilent 1290 Infinity LC system and Agilent 6460 Triple Quadrupole LC/MS was used.
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Figure 3: Forty analytes could be identified in different kinds of nut samples. The chart shows the number of each kind of nut sample that contained the given fungal compound. An Agilent 1290 Infinity LC system and Agilent 6460 Triple Quadrupole LC/MS was used.

Providing feed to cows that is contaminated with mycotoxins can result in the contamination of products processed from their milk, including infant formula. The EU regulation for the presence of mycotoxins in formula is quite stringent, limiting the maximum concentrations of aflatoxin M1, aflatoxin B1 and ochratoxin A, for example, to 0.025, 0.1, and 0.5 microgram/kilogram, respectively. Most current methods for this analysis involve labor intensive and time consuming sample purification and concentration steps required to achieve these detection levels using liquid chromatography with fluorescence detection or LC/MS.

A UHPLC/MS/MS assay for the EU regulated mycotoxins in baby formula has been developed that uses a simple extraction without a concentration step to attain the sub-part per billion detection limits required by the regulation. This method utilizes triggered MRM acquisition (tMRM) for ultimate confidence in the identification of the mycotoxins. Pre-selected MRM transitions trigger the collection of additional MS/MS transitions, each with optimized collision energy and maximized dwell time to enable the highest sensitivity. The collected ions are formulated into a spectrum, which is compared to a triggered MRM library spectrum for confirmation. This method enables the detection of the regulated mycotoxins in infant formula at levels below the maximum allowable limits, as is demonstrated by the results for aflatoxin M1, which is typically associated with mycotoxin contamination of milk (Figure 2). In addition to the ideal sensitivity and precision of the method, its key benefit is the high confidence in the result due to the availability of high quality spectra down to very low concentration levels, which is only possible with triggered MRM.

Expanding Detection Capabilities

A method for the analysis of mycotoxins in nuts exploits the power of UHPLC and tandem mass spectrometry by enabling the detection and semi-quantitation of 191 mycotoxins and other fungal metabolites, in just two chromatographic runs per sample. UHPLC allows better separation of the analytes from the matrix, when compared to other LC/MS/MS methods, and the overall repeatability is superior to other published methods. This method features fast and easy sample preparation that includes only a single extraction step before injection of the diluted raw extract into the UHPLC/MS/MS. The multiplex analysis capability of the method enables a throughput of 25 samples per day.

This method has been utilized to survey 53 different nut samples for the presence of the 191 fungal compounds (Figure 3). The importance of using multi-mycotoxin methods was demonstrated by the detection of 40 different analytes in the nut samples. The key benefit of this method is the ability to detect mycotoxins in unlikely matrices. By applying comprehensive screening methods, the availability of occurrence data is greatly improved. In addition, this method is a good repository of MRM transitions for method extension of, for example, one of the two methods mentioned previously.

Although aflatoxins are the only mycotoxins regulated in nuts in the EU, these results suggested that other toxins may also be relevant. Major mycotoxins found in more than 50 percent of the samples were beauvericin, enniatin B, macrosporin, 3-nitropropionic acid, emodin, and alternariol methyl ether. These results also confirmed for the first time the presence of HT-2 and T-2 toxins in hazelnuts. Analysis of such a large number of fungal toxins might be useful in the future since possible toxic effects on humans are still not fully evaluated and additive or synergistic effects of such toxins are largely unknown.


Dr. Glauner is a senior LC/MS applications scientist for Agilent Technologies, Inc.,Waldbronn, Germany. Reach him at thomas_glauner@agilent.com.

References Furnished Upon Request

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