Gas Chromatography In Mycotoxin Analysis

Introduction

The multi-exposure to various mycotoxins may lead to additive, synergic or antagonist toxic effects (Ficheux et al, 2012) to humans and animals. This may cause chronic and acute diseases thereby calling for food safety measures. Many countries have setup strict regulations for mycotoxin control in food and feed and established legislation to control their possible contamination (Juan et al., 2012). The most frequently studied mycotoxins on which there are more data are TRC (Trichothecenes), FMs (Fumonisins), ZEN (Zearalenone), AFs (Aflatoxins) and OTA (Ochratoxin A), with more emerging mycotoxins like FUS (fusaproliferin), BEA (Beauvericin), ENs (Enniatins), MON (moniliformin) being reported in raw and processed cereals. Mycotoxins are produced by fungi species belonging to genera of Aspergillus, Penicillium and Fusarium.

Most mycotoxins are lowly volatile or not at all, are present at low levels, and are highly polar hence their matrices are often very complex. Hence their analysis is challenging. However, despite the challenges, efforts to develop analytical methodologies for effective determination of mycotoxins have been undertaken, particularly for multi-mycotoxin methods.

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Chromatographic techniques have the ability to separate a great number of analytes of different chemical structure. Techniques such as liquid chromatography (LC), gas chromatography (GC), LC/mass spectrometry (MS), and gas chromatography (GC) coupled to mass spectrometry (MS) detector, GC-MS, and other novel techniques are used for separation and quantification of mycotoxins.

Chromatographic analytical methodology includes; define the problem, obtain a representative sample, prepare the sample for analysis and performing necessary chemical separation, performing the measurement, and calculate the results and presenting the date. This review features an overview of the gas chromatographic techniques reported on mycotoxin analysis of cereal and cereal products, focusing on the popularity, utility, advantages, drawbacks and future potential of this technique.

Gas chromatography

Gas chromatography is applied in three broad methods; gas liquid chromatography-using a packed column with a liquid stationary phase coated onto inert support particles, Capillary column GC- where open tubular columns are used with the liquid or solid stationary phase coated onto the inner walls of the column tubing referred to as wall coated, porous layer and surface coated open tubular columns, and Gas-solid chromatography (GSC)- using a packed column with the solid surface of the particles forming the stationary phase, for example alumina or a cross-linked polymer. A GC instrumentation system consists of six sections; Carrier gas supply and controls- gives gaseous mobile phase ( carrier gas), Sample introduction/injector system, Chromatographic column and oven- contains the stationary phase, Detector- generate a minute signal current that require an amplifier for output to produce the chromatogram, Amplifier and signal processing and control electronics, Integrator and chromatogram printout

Gas chromatography in combination with superior separation on the capillary columns based on flame ionization detector, electron capture detector and mass spectrometry detection are largely used for quantification of mycotoxins especially trichothecenes in cereals and cereal based products. GC-MS is the most popular because it allows the simultaneous identification and quantification of the compounds.

Gas Chromatography analysis can be done using a single capillary column or a dual column (Seeley, 2012). The dual column has an advantage over a single column in that it enables a multidimensional separation. Hence this providing an enhanced separation power of the analytes. In an application of a single capillary column, Rodriques-Carrasco et al., (2014) using an analytical protocol based on QuEChERS and gas chromatography–tandem mass spectrometry (GC–MS/MS), successfully determined trichothecenes, patulin and zearalenone in 182 milled grain-based samples.

However, the LOQs were lower than 10µg/kg for the selected mycotoxins. Similarly, in the study by Ferreira et al (2012) on GC/MS method determination for multi-mycotoxins (deoxynivalenol, nivalenol, 15-acetyl-deoxynivalenol, fusarenon X and zearalenone) in 30 unpopped and popped popcorn samples. Although one sample was positive for nivalenol and zearalenone, at concentrations below the maximum permissible level; however low levels of detection and quantification lower than 65µg/kg and 196µg/kg were reported.

In an application of a dual column, Cunha and Fernandes (2010) reported successful application in the determination of DON, ZEN (Zearalenone), FUS-X (fusarenon-X), 15-AcDON (15-acetyldeoxynivalenol) and NIV (nivalenol) in breakfast cereals and flours (maize, wheat and cassava). In conclusion, gas chromatography gives a poor performance for analysis of mycotoxins for instance trichothecenes in terms of accuracy, recovery, and precision. This may be due to matrix interference.

Mycotoxins are lowly volatile or not volatile at all, highly polar; this makes them unsuitable for GC analysis. Compounds containing functional groups with active hydrogens such as -SH, -OH, -NH and - COOH are of primary concern because of the tendency of these functional groups to form intermolecular hydrogen bonds (Zaikin and Halket, 2003). These intermolecular hydrogen bonds affect the inherent volatility of compounds containing them, their tendency to interact with column packingmaterials and their thermal stability (Sobolevsky et al., 2003). Hence to ensure suitability, modification of the functional group of the mycotoxin molecule by derivatisation during sample preparation is required. Derivatisation will serve the following functions;

• Suitability; compound of interest to be volatile with respect to gas chromatographic analysis conditions, as compared to liquid chromatography where the compound of interest should be soluble in the mobile phase.
• Efficiency; derivatisation of analyte molecules reduce interaction between the compound themselves and also between the compounds and the GC column which may otherwise reduce separation efficiency of many compounds and mixtures (Knapp, 1979).
• Detectability; given the low levels of mycotoxins, derivatisation may increase the amount of materials to a point at which they can be detected in GC. This may be achieved either by increasing the bulk of the compound or by introducing onto the analyte compound, atoms or functional groups that interact strongly with the detector and hence improve signal identification. Hence improve detector response, peak separations and peak symmetry. For example the addition of halogen atoms to analyte molecules for electron capture detectors and the formation of trimethylsilyl ether derivatives to produce readily identifiable fragmentation patterns and mass ions (Knapp, 1979).

Derivatisation reactions used for GC are alkylation which is generally esterification, acylation and silylation. For derivatisation of mycotoxins, silylation and acylation are generally employed to obtain a volatile material.

Ochratoxin A (OTA) is not volatile, hence cannot be directly determined by GC. Therefore, to enable OTA detection, they are meant to be derivatised. Silylating agents have been used prior to GC/MS analysis (Montes et al., 2012) for trichothecenes determination in breakfast cereals. Acylation reactions have been used (Ibanez-Vea et al., 2011) in the determination of trichothecenes in barley samples.

Due to matrix interference, derivatisation step is a nearly always requirement to convert analytes to a suitable state to enable analysis by gas chromatography. Liquid chromatography methods on the other hand don’t require derivatisation, and are able to simultaneously identify and quantify almost all the mycotoxins at low levels. With improvements of the conventional liquid chromatography to ultra performance liquid chromatography separation techniques, enhancement in speed, resolution and sensitivity of analysis have been achieved.

For mycotoxin determination, use of LC-MS, use of electrospray ionization (ESI) and ionization techniques (API) have enabled their success. ESI is well suited for analysis of polar compounds while API is highly effective for the analysis of medium and low polar compounds (Boyd et al., 2008). Hence this gives LC-MS techniques a great advantage over GC methods.

Conclusion

Gas chromatography has been successfully applied in the analysis of mycotoxins in cereals. However, the need to derivatise all non volatile and polar samples prior to analysis by gas chromatography is a shortcoming compared with liquid chromatographic techniques may be a potential source of error. Further, thermal stability is a problem because heating sometimes degrades the samples. In the future, it is expected that mycotoxins determination in foodstuffs will continue to be mainly performed with LC-MS/MS equipments allowing multi-toxin detection and accurate quantification and identification.

References

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