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Keywords:

  • mycotoxins;
  • GC/MS;
  • LC/MS

Abstract

  1. Top of page
  2. Abstract
  3. I. INTRODUCTION
  4. II. PATULIN
  5. III. AFLATOXINS
  6. IV. OCHRATOXIN A
  7. V. TRICHOTHECENES
  8. VI. ZEARALENONE
  9. VII. FUMONISINS
  10. VIII. CONCLUSIONS
  11. REFERENCES
  12. Biographical Information

Mycotoxins are fungal toxins produced by molds, which occur universally in food and feed derivatives, and are produced under certain environmental conditions in the field before harvest, post-harvest, during storage, processing, and feeding. Mycotoxin contamination is one of the most relevant and worrisome problem concerning food and feed safety because it can cause a variety of toxic acute and chronic effects in human and animals. In this review we report the use of mass spectrometry in connection with chromatographic techniques for mycotoxin determination by considering separately the most diffuse class of mycotoxins: patulin, aflatoxins, ochratoxin A, zearalenone, trichothecenes, and fumonisins. Although the selectivity of mass spectrometry is unchallenged if compared to common GC and LC detection methods, accuracy, precision, and sensitivity may be extremely variable concerning the different mycotoxins, matrices, and instruments. The sensitivity issue may be a real problem in the case of LC/MS, where the response can be very different for the different ionization techniques (ESI, APCI, APPI). Therefore, when other detection methods (such as fluorescence or UV absorbance) can be used for the quantitative determination, LC/MS appears to be only an outstanding confirmatory technique. In contrast, when the toxins are not volatile and do not bear suitable chromophores or fluorophores, LC/MS appears to be the unique method to perform quantitative and qualitative analyses without requiring any derivatization procedure. The problem of exact quantitative determination in GC/MS and LC/MS methods is particularly important for mycotoxin determination in food, given the high variability of the matrices, and can be solved only by the use of isotopically labeled internal standards or by the use of ionization interfaces able to lower matrix effects and ion suppressions. When the problems linked to inconstant ionization and matrix effects will be solved, only MS detectors will allow to simplify more and more the sample preparation procedures and to avoid clean-up procedures, making feasible low-cost, high-throughput determination of mycotoxins in many different food matrices. © 2005 Wiley Periodicals, Inc.


I. INTRODUCTION

  1. Top of page
  2. Abstract
  3. I. INTRODUCTION
  4. II. PATULIN
  5. III. AFLATOXINS
  6. IV. OCHRATOXIN A
  7. V. TRICHOTHECENES
  8. VI. ZEARALENONE
  9. VII. FUMONISINS
  10. VIII. CONCLUSIONS
  11. REFERENCES
  12. Biographical Information

Mycotoxins are fungal toxins produced by molds, which occur universally in food and feed derivatives, and are produced under certain environmental conditions: proper moisture, sufficient oxygen, suitable temperature, physical damage to the commodity, and presence of the fungal spores. Production of mycotoxins can occur in the field before harvest, post-harvest, during storage, processing, and feeding. Mold growth and the occurrence of mycotoxins are often related to extremes in weather conditions, which can cause plant stress or hydration of feedstuffs, poor storage practices, which affect feedstuff quality and feeding conditions.

Molds of the genus Aspergillus, Fusarium, and Penicillium are the most important in producing mycotoxins (Council for Agricultural Science and Technology (CAST), 1989). Mycotoxin contamination is one of the most relevant and worrisome problem concerning food and feed safety, since they cause a variety of toxic effects in humans and animals, due to their different chemical structures. Acute effects are generally produced by high amounts of toxins present in food or feed, so that fatal incidents are usually restricted to the less developed areas of the world, where resources for control are limited, or to livestock. Chronic effects are also of concern for the long-term health of the human population and must not be underestimated since many toxins are present in low amounts in daily intaken food. Actually, some mycotoxins are carcinogenic, genotoxic or may affect the kidneys, liver, or immune system (DeVries & Trucksess, 2002).

It is a priority of the European Food Safety Authority (EFSA) to establish maximum allowed limits in food and feed and a duty of the producers to comply with them. Thus, the availability of sound analytical methods for monitoring the presence of mycotoxins along the food chain is of the utmost importance for keeping the contamination under control.

Immunological techniques for mycotoxin determination based on specific monoclonal and polyclonal antibodies produced against several toxins are commercially available and are essentially of two types: immunoaffinity column-based analyses and ELISA tests (Trucksess & Poland, 2001). Although these methods are generally good for rapid qualitative screenings, they fell short in providing a definitive confirmation of the toxin and an accurate quantitative determination. Better suited to the purpose are the common analytical techniques such as gas chromatography (GC) and high pressure liquid chromatography (HPLC), given their good performances in terms of accuracy, precision, sensitivity, and reproducibility (Trucksess & Poland, 2001).

In this review, we will report the use of mass spectrometry (MS) in connection with chromatographic techniques for mycotoxin determination. Although the selectivity of MS is unchallenged if compared to common GC and LC detection methods and accuracy and precision are generally high, other factors must often be taken into account for evaluating the performances of MS detectors and the necessity of their use, such as the sensitivity, which may be different in different instruments and can be much lower than with fluorescence detectors, and the price of the instrument, which is generally higher than that of other detectors. The sensitivity issue may be a real problem especially in the case of LC-MS, in which the choice of the right interface for every given toxin is fundamental and the response can be very different for the different ionization techniques. To comprehensively understand the problems connected with every single toxin determination, a comparison with other common detectors will be made.

The problem of sample preparation, which is of primary importance in mycotoxin determination, will not be thoroughly approached, since it affects every chromatographic determination with any detector. When necessary, since it may affect recovery and sensitivity (due to matrix interference), a few comments on the pre-analysis preparation of the samples will be given.

We will consider separately the most widely spread classes of mycotoxins: patulin (produced from Penicillium, Aspergillus, and Byssochlamys molds), aflatoxins (produced from several species of Aspergillus molds), ochratoxin A (produced from Penicillium and Aspergillus molds), zearalenone (ZEN), trichothecenes, and fumonisins (all produced from Fusarium molds).

The literature has been surveyed until the first months of 2004.

II. PATULIN

  1. Top of page
  2. Abstract
  3. I. INTRODUCTION
  4. II. PATULIN
  5. III. AFLATOXINS
  6. IV. OCHRATOXIN A
  7. V. TRICHOTHECENES
  8. VI. ZEARALENONE
  9. VII. FUMONISINS
  10. VIII. CONCLUSIONS
  11. REFERENCES
  12. Biographical Information

Patulin (Fig. 1) is a toxic metabolite produced by several species of Penicillium and Aspergillus (Lovett & Thompson, 1978; Notholt, van Egmond, & Paulsch, 1978).

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Figure 1. Patulin structure.

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The most commonly occurring fungus is Penicillium expansum, the apple blue mold rot. Apple juices are the most important source of patulin in the human diet (Jelinek, Poland, & Wood, 1989), although the toxin has also been detected in berries, bread, and meat products. Toxicological studies on patulin demonstrated that it is acutely toxic (Burghardt, 1992) and able to produce tumors in rats (Becci et al., 1981). The presence and the extent of patulin contamination in apple products can serve as a marker for the quality of the product. The EU adopted a maximum allowed level for patulin in various foods (mainly apple derivatives) ranging from 10 to 50 μg/kg. Analogously, FDA recommends a maximum level of 50 μg/kg in apple juices and apple derivatives.

Liquid–liquid extraction and solid phase extraction (SPE) are the traditional methods of sample preparation for analysis of patulin in food samples (AOAC, 1997). In view of the recognized adverse effects caused by this mycotoxin and the need for regulatory controls, several analytical methods have been proposed, mainly concerning GC and LC with UV detection. Recently, MS has been associated with both LC and GC, allowing for a conclusive confirmation of the toxin. MS-MS methods provide additional selectivity and increased sensitivity.

A. Gas Chromatography/Mass Spectrometry Determination of Patulin

Quantitative GC/MS determinations of patulin are usually based on previous extraction and derivatization, either as silyl or acetyl derivatives, and require isotopically labeled patulin as internal standard, which is not commercially available.

An earlier example was presented by Price (1979), who used a stable isotope dilution GC/MS method by using patulin labeled with (2H9)-trimethylsilane ((2H9)-TMS) as internal standard. Unfortunately, the derivatization of patulin as (1H9)-TMS derivative in the presence of the (2H9)-TMS internal standard led to exchange of the “light” and “heavy” sylanol groups between the analyte and the standard itself, thus preventing the exact quantification.

Rychlik & Schieberle (1999) developed both HRGC/HRMS and LC/MS methods for the determination of patulin in foodstuffs, by previously extracting and concentrating the toxin in ethyl acetate and using 13C2-Patulin as internal standard. The GC method was based on previous derivatization with bis(trimethylsilyl)trifluoroacetamide (BSTFA) and GC/HRMS analysis (EI source) by monitoring the 12C2 and 13C2 mass peaks of the trimethylsilylderivatives. Outstanding results were obtained: LOD of 12 ppt and LOQ of 35 ppt were determined by adding patulin to a patulin-free apple matrix and using a high-resolution mass spectrometer. However, when used in the low-resolution mode, which is close to the commercially available benchtop instruments, the limits of detection were higher (LOQ of 500 ppt). In any case, very accurate results in apple juices were obtained with this method, also because of the high recovery (96%) of the extraction procedure. The authors suggested that the very low LOD and LOQ achievable with the HRGC/HRMS method might allow for patulin determination in metabolic studies.

A method essentially analogous to the previous one (but with no labeled internal standard) was presented by Rupp & Turnipseed (2000) for the qualitative confirmation of patulin and 5-hydroxymethylfurfural in apple juice by GC/MS with an EI source and selective ion monitoring (SIM) of the corresponding ions.

Sheu & Shyu (1999) reported a sensitive GC/MS method using a direct acetylation procedure before extraction to increase the stability and the recovery of the patulin as acetate and subsequent determination with the SIM mode, obtaining a limit of quantification of 10 μg/L. To improve the sample clean-up and concentration, a new technique combining diphasic dialysis extraction (DDE) (Dominguez et al., 1992) and in situ derivatization with acetic anhydride and GC/MS confirmation of the patulin acetate was utilized in food analysis with nitrobenzene as internal standard (LOD of 1 ppb and LOQ of 10 ppb). However, the method was affected by quite low recovery (<80%) in the real samples when patulin was present at levels close to the LOQ, and it also required a long (>1 day) sample preparation. Actually, the derivatization steps may result in sample loss for incomplete reaction or decomposition before analysis.

Some methods were developed to detect the underivatized patulin, which can be used for the qualitative identification of the molecule. Gas chromatography/mass spectrometry of underivatized patulin has been utilized mainly for confirmatory assays.

A GC/MS technique for the underivatized patulin, based on direct “on-column” injection (to avoid patulin thermal decomposition) with an instrument equipped with an electronic pressure control (EPC) to improve sample transfer to the head of the GC column, was proposed as a confirmatory qualitative technique after positive LC/UV analyses (Llovera et al., 1999). The acquisition was carried out in the SIM mode (EI source) with hexachlorobenzene as internal standard. Unfortunately, the ratio between the base mass peak of patulin (110 m/z) and that of hexachlorobenzene (284 m/z) was shown to undergo a great variation in two consecutive days, hampering the exact quantitative determination. In any case, the authors suggested the use of this technique for qualitative confirmation and estimated an LOD ranging from 4 to 10 ppb, according to the extraction procedure adopted.

Finally, a comparative study on the determination of underivatized patulin by GC/MS was presented by Roach et al. (2000). The authors reported about their experience in a FDA laboratory in the period 1993–2000 using GC/MS as confirmatory technique for samples previously turned out to be positive in the LC/MS analysis. During that period, different capillary columns and several mass analyzers (quadrupole, ion trap, magnetic sector) were used. Quite interestingly, methane chemical ionization (CI) in the negative ion mode was found to be the most sensitive ionization technique for underivatized patulin, given the intense signal of the deprotonated molecule and the fewer signals due to coextracted compounds than full scan EI data. Positive ion CI spectra showed less fragmentation than EI spectra and negative ion CI even lesser. The reduced fragmentation increased the sensitivity of the assay because the total signal for the analyte was contained in a smaller set of ions. Similar negative ion CI spectra were obtained with an ion trap analyzer. By GC/MS, it was possible to confirm presumptive findings of patulin in apple juices in the range 68–3,700 ppb. The same group also presented the same approach in a subsequent study (Roach et al., 2002).

A summary of the main recent GC/MS methods for patulin determination is reported in Table 1.

Table 1. Main recent GC/MS methods for patulin determinationThumbnail image of
  • aThese values can be achieved only when the mass spectrometer is operated in the high resolution mode.

  • bThis method was affected by low recovery (<80%).

  • cThe LOD was not accurately calculated, this value being merely the lowest detected amount in naturally contaminated or spiked samples.

  • The better sensitivity of new MS instruments, such as those equipped with ion trap analyzer, in combination with better GC columns, may allow to apply the GC/MS technique to significantly low patulin levels. However, the necessity of derivatization makes exact quantification more suitable for LC rather than GC/MS, unless a stable isotope-labeled patulin is available as internal standard.

    B. Liquid Chromatography/Mass Spectrometry Determination of Patulin

    Reversed phase HPLC coupled with UV detection is most suitable since the molecule is polar and exhibits a strong absorption spectrum. However, the shortfall of UV detection arises when other interfering substances are present, especially 5-hydroxymethyl-2-furaldehyde (5-HMF). Quantitative LC/MS determinations of patulin are performed on the underivatized molecule, after extraction from the matrices. LC/MS methods are usually more robust and more reproducible than the corresponding GC/MS methods, although in many cases less sensitive. However, given the unavailability of a commercial labeled internal standard for GC/MS, LC/MS at the moment has to be considered the method of choice for exact quantification and confirmation of patulin.

    The LC/MS method developed by Rychlik & Schieberle (1999) in the above cited study was based on the ESI determination of the deprotonated molecular ions in the negative ion mode, using 13C2-labeled patulin as internal standard. The ratio of patulin and its isotopomer was determined by monitoring the quasimolecular ions at m/z 153 and 155, respectively. The response of the patulin in the ESI interface allowed for a LOD of 20 ppb and for a LOQ of 63 ppb. Addition of ammonium acetate did not increase the ionization, whereas addition of formic acid increased the sensitivity in the positive ion mode, not more than that achieved in the negative ion mode. Thus the negative ion mode, mainly on account of the cleaner background produced, seems to be more appropriate. The APCI technique in the negative ion mode showed a response similar to the ESI technique. A comparison of the single isotope dilution assay (SIDA) with the commonly used LC/UV analysis showed that the latter technique allowed to obtain better LOD and LOQ than the LC/MS, respectively of 1.3 and 4 ppb. Actually, as far as patulin routine determinations in food derivatives are concerned, the LC-UV method seems to be fully adequate. However, when the selectivity is compared, both GC/HRMS and LC/MS methods are more specific than UV detection.

    Sewram et al. (2002) used an LC/APCI-MS/MS method with an ion trap analyzer (negative ion mode) for the analysis of patulin in apple juices. Negative ion mode was found to be more sensitive than the positive ion mode, and both ammonium acetate and ammonium hydroxide did not improve the signal. By monitoring the fragments at m/z 139 (loss of water), 125 (loss of CO), and 109 (loss of acetaldehyde or CO2) with the optimized parameters, a good linearity in the 10–135 ppb range was obtained, using spiked patulin-free apple juices, with a LOD of 4 and a LOQ of 10 ppb. Quite interestingly, the vaporizer temperature in the APCI source had to be set at 450°C, since lower temperatures resulted in consistent LC peak tailing associated to retarded ion formation. To verify possible errors in quantification due to the use of an ion trap analyzer, the authors calculated the patulin concentrations also by a standard LC/UV method, obtaining a good correlation coefficient (r2 = 0.99) between the data calculated by LC/UV and those calculated by LC/APCI-MS/MS.

    Very recently a comparative study between APCI and the atmospheric pressure photoionization (APPI) technique for the determination of patulin in apple juices was carried out by Takino, Daishima, & Nakahara (2003). In the APPI mass spectrum with or without acetone as dopant, in the negative ion mode, the deprotonated ion [M−H] at m/z 153 was observed as the predominant peak with no other significant ions. In contrast, in the APCI mass spectrum, the radical anion [M]·− at m/z 154 was observed as the most relevant ion, although [M−H] was also observed. Thus, ionization of patulin by APCI in the negative ion mode proceeds simultaneously via electron capture and deprotonation. The APPI parameters (capillary voltage, vaporizer temperature, presence of dopant) were carefully optimized: the dopant was found not to be necessary, the optimum capillary voltage was 1,000 V, and the optimum temperature 350°C. Also chromatographic conditions were optimized: the best eluent was 10 mM ammonium acetate and methanol with an optimal flow of 0.3 mL/min. Linearity, LOD and precision were compared between the LC/APPI-MS and LC/APCI-MS methods, using a standard solution of patulin and a SIM acquisition mode on the deprotonated ion. The apple juice samples were injected without any previous extraction since the authors developed an online purification method based on precolumn switching. The results were very similar for APCI and APPI, with a good linearity in both cases (r2 > 0.999), LOD of 0.13 and 0.20 ppb in standard solutions, respectively, and precision of 2.1% and 6.5%, respectively. The only difference was a cleaner chromatogram with the APPI detection. Moreover, the authors developed an online extraction technique and studied the performances of the two sources for the routine determination of patulin in apple juice matrices. Again, the chromatogram obtained by APPI detection was cleaner, indicating a higher selectivity and a lower matrix effect than APCI. After 1 day of analysis, the relative intensity of the peak observed in the last analysis compared to the first was 90% for APPI detection, whereas it was 51% for APCI detection. Finally, the LC/APPI-MS method was fully validated by spiking patulin-free apple juices: the recovery ranged from 94% to 103%, the precision from 2.3% to 6.7% and the LOD in matrices was found in the 1.05–1.50 ppb range. According to these data, HPLC-APPI-MS seems to be convenient for routine analysis of patulin residues in apple juices at trace levels.

    A summary of the main recent LC/MS methods for patulin determination is reported in Table 2.

    Table 2. Main recent LC/MS methods for patulin determinationThumbnail image of

    III. AFLATOXINS

    1. Top of page
    2. Abstract
    3. I. INTRODUCTION
    4. II. PATULIN
    5. III. AFLATOXINS
    6. IV. OCHRATOXIN A
    7. V. TRICHOTHECENES
    8. VI. ZEARALENONE
    9. VII. FUMONISINS
    10. VIII. CONCLUSIONS
    11. REFERENCES
    12. Biographical Information

    Aflatoxins are a group of closely related metabolites produced by the genus Aspergillus, in particular A. flavus and A. parasiticus. The main aflatoxins are the B1 (AFB1), B2 (AFB2), G1 (AFG1), and G2 (AFG2) together with their metabolites, among which the most important is the aflatoxin M1 (AFM1). The structures of aflatoxins are reported in Figure 2.

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    Figure 2. Aflatoxin structures.

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    Aflatoxins have been found in a variety of agricultural and food products; peanuts, corn, tree nuts, and spices are the main commodities with high aflatoxin contamination (Bhatnagar, Yu, & Ehrlich, 2002). AFM1 is the main contaminant in cow's milk (Veldman et al., 1992).

    Based on epidemiological evidence, aflatoxins have been classified as human liver carcinogens (Krabyll & Shapiro, 1969). The AFB1 is listed as group 1 carcinogen by the International Agency for Research on Cancer (IARC), an organism of the World Health Organization (1987). Acute toxic effects have also been reported (Krishnamachari et al., 1975). The FDA allows for a maximum of 20 ppb of total aflatoxin in food for human consumption in the USA (0.5 ppb of AFM1 in milk), whereas the EU established maximum acceptable levels ranging from 2 to 5 ppb of AFB1 and from 4 to 10 ppb of total aflatoxins in various foods (but for infant foods, the maximum level is 0.025 ppb of AFM1 and 0.1 ppb of AFB1).

    The chromatographic method of choice for aflatoxin detection is HPLC-FLD, with the very efficient, low noise, and quantitative robust fluorescence detection. However, aflatoxins have a weak native fluorescence, which can be enhanced by pre- or post-column derivatization. Addition of the cyclodextrin to the LC eluent has been proved to highly enhance cyclodextrin fluorescence (Chiavaro et al., 2001). The method usually requires extraction and clean-up procedures, which enhance sensitivity and specificity. The published LC/MS methods are quite scarce in the literature, on account of the relatively high limits of detection of single stage analyzers.

    A first approach to the analysis of aflatoxins using a thermospray LC/MS interface was introduced by Hurst, Martin, & Vestal (1991). After extraction of peanut samples by an SPE method, they used HPLC coupled to a thermospray MS detector for the determination of aflatoxins B1, B2, G1, and G2.

    Cappiello, Famiglini, & Tirillini (1995) reported the use of a reversed phase capillary packed column coupled to a modified particle beam interface for the determination of aflatoxins in peanuts. The particle beam interface (PB-MS) provided library searchable electron impact (EI) mass spectra for unambiguous identification of compounds. Selected ion monitoring (SIM) detection allowed to identify and quantify the substance on the base of the characteristic fragmentation. In particular, three characteristic ions were monitored for each aflatoxin. Although good linearities in the calibration curves were obtained with standards, no application to real samples was reported and, most important, the estimated detection limits appeared to be too high (>10 ppb) for the current regulatory limits.

    In the last few years, the new generations of tandem MS/MS instruments allowed to reach detection limits comparable to those obtained with fluorescence detectors, stimulating the development of LC/MS methods for confirmation of samples already turned out to be positive with fluorescence detection. It is important to bear in mind that the specificity advantage given by MS detectors is often not essential in aflatoxin determination, these compounds usually being the main fluorescent molecules in food matrices. Moreover, when using mass spectrometric detection, although the specificity increases (which is good for qualitative confirmation), problems due to matrix effects seemed to affect the robustness of the quantitative determinations more than with fluorescence detection (Mallet, Lu, & Mazzeo, 2004).

    A good example of the problems usually encountered when using LC/MS/MS for aflatoxin determination is given by Vahl & Jorgensen (1998). By using an ESI interface in the positive ion mode, they monitored one daughter ion for each aflatoxin in multiple reaction monitoring (MRM) experiments for the LC/MS quantification of aflatoxins in various foods (peanuts, chili, pepper, pistachios) by using aflatoxin M1 as internal standard. A comparison with the fluorescence detection was also carried out. Although MS detection limits were fully satisfactory for confirmatory analyses (0.1 ppb), the quantification was totally unsatisfactory, due to interferences in the mass chromatograms. Moreover, the percentage recovery of aflatoxins from spiked extracts varied from 40% to 280% for spices, with high variabilities being also observed even in the same samples. Quantitative data obtained by MS/MS in real samples were generally inconsistent with those obtained by fluorescence detection. The authors suggested that samples must undergo extensive clean-up before LC/MS/MS analysis and the use of isotope-labeled aflatoxins as internal standards (not commercially available) is mandatory to get acceptable quantitative results.

    The use of MS detectors in confirmatory LC methods has been also reported by Trucksess et al. (2002), who used an LC/APCI-MS/MS method to confirm aflatoxin contaminations in samples of Incaparina, a mixture of corn and cottonseed flour with added vitamins, minerals, and a preservative. It is marketed as a high-protein food supplement in Guatemala to help reducing malnutrition by providing a lower cost alternative to cow's milk. The authors, as a further example of the quantitative problems often encountered when using LC/MS with aflatoxins, reported an average recovery of 80% with an average standard deviation (SD) of 26%.

    A recent direct comparison of FLD, MS detection, and ELISA test was reported by Blesa et al. (2003), who applied a new extraction procedure (matrix solid phase dispersion, MSPD) before the LC determination of aflatoxins in peanuts. This study represents a striking example of the improvement in the S/N ratio that can be obtained by eliminating matrix interferences. The MS detection (single quadrupole analyzer) was performed in the positive ion mode by monitoring protonated and sodiated molecular ions. The outstanding clean-up of the matrix obtained by the MSPD method allowed for a significant improvement of the ESI performance: after the MSPD procedure, the limits obtained by FLD and MS detection were comparable (0.04–0.75 ng/g with FLD and 0.07–0.2 ng/g with MS). Unfortunately, the extensive clean-up also resulted in incomplete recoveries (78–86%). These results demonstrate that for aflatoxin analysis of peanuts, ELISA can be used as test for initial screening followed by MSPD and LC. Being LC/FLD chromatograms free of interferents, LC/FLD was used for quantification and LC/MS was used only as a confirmatory technique.

    Very recently, Takino et al. (2004) reported an optimized LC/APPI-MS method (single quadrupole analyzer) for the detection of aflatoxins in various foods (corn, peanuts, nuts, pepper). Again, analogously to the results obtained by the same group for patulin determination, this technique seems to be less affected by matrix interferences than other atmospheric pressure ionization techniques, such as ESI and APCI, resulting in more stable signals and better limits of detection. Using a water–methanol eluent containing ammonium acetate, a capillary voltage of 1,500 V, a vaporizer temperature of 350°C, and a flow of 0.5 mL/min, the protonated molecular ions [MH]+ were monitored and no other ions were observed. Quite interestingly, the use of acetonitrile in the eluent, although better performing from a chromatographic point of view, was found to unfavor the formation of protonated ions. Thus, when acetonitrile was replaced with methanol, a significant signal increase was observed and the S/N ratio also increased. Probably, aflatoxins are ionized by photons to radical cations [M]·+, which can capture a H· atom from methanol to produce the stable [MH]+. Experiments carried out with standard solutions showed that the APPI interface was better performing than the ESI interface in terms of linearity, reproducibility, and limits of detection (0.04–0.18 ng/mL against 0.18–0.53 ng/mL obtained with ESI on standard solutions). The application to real food matrices showed the practical absence of matrix effects (only a 5% signal variation was observed between standard solutions and spiked toxin-free food samples containing the same amount of toxins), good precision, recovery, repeatability, and reproducibility. Being the instrument equipped with a single quadrupole analyzer, quite outstanding LODs ranging from 0.11 to 0.5 ppb in spiked food matrices were obtained, probably on account of the reduced matrix-derived noise. Although naturally contaminated samples have not yet been tested, the method seems to be convenient for analysis of aflatoxins in food matrices, on account of its improved selectivity relative to the chemical background of the system.

    A summary of the main recent LC/MS methods for aflatoxin determination is reported in Table 3.

    Table 3. Main recent LC/MS methods for aflatoxin determinationThumbnail image of
  • aRecovery percentage varied from 40% to 280%.

  • bThese methods were used only as a qualitative confirmation. Quantitation was carried out by LC/FLD.

  • cRecovery percentage varied from 78% to 86%.

  • IV. OCHRATOXIN A

    1. Top of page
    2. Abstract
    3. I. INTRODUCTION
    4. II. PATULIN
    5. III. AFLATOXINS
    6. IV. OCHRATOXIN A
    7. V. TRICHOTHECENES
    8. VI. ZEARALENONE
    9. VII. FUMONISINS
    10. VIII. CONCLUSIONS
    11. REFERENCES
    12. Biographical Information

    Ochratoxin A (OTA, Fig. 3) is produced by fungi of the genus Aspergillus (mainly A. alutaceus, formerly ochraceus) and Penicillium (mainly P. verrucosum) (Marquardt & Frohlich, 1992). The dechloro-analog, known as ochratoxin B (OTB), although not toxic, and the ethyl ester analog (ochratoxin C) are also known fungal metabolites.

    thumbnail image

    Figure 3. Ochratoxin A structure.

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    OTA has been demonstrated to be a potent kidney carcinogen and an immunosuppressant in rodents, to be the cause of mycotoxicoses in farm animals and is associated with chronic human kidney diseases (Kuiper-Goodman & Scott, 1989; Creppy, Castegnaro, & Dirheimer, 1993).

    OTA has been found in many different foods, such as cereals, beans, coffee, beer, wine, grape juices, spices (Pohland, Nesheim, & Friedman, 1992; Pittet, 1998). The EU regulations have established maximum levels of OTA ranging from 3 to 10 ppb, according to the different food products (but for infant foods, the maximum level is 0.5 ppb).

    LC is the technique of choice for OTA analysis, and MS detectors have long been used to confirm and quantitate ochratoxin in foods with high selectivity. However, as it will be discussed in the following text, LC using reversed phase columns and fluorescence detection appears to be fully adequate to the task, with lower LODs than MS/MS methods, being chromatograms usually clean enough to ensure a clear identification of the peak. Therefore, OTA detection and quantification is another case, such as for aflatoxins, where fluorescence is more satisfying than MS in terms of sensitivity, not to mention the easiness of operation and the lower cost of the instrument. The higher selectivity displayed by MS detectors is not necessary for many food matrices, if a suitable sample preparation is performed. The best application for MS detectors is the clear-cut confirmation of ochratoxin in samples already turned out to be positive by LC/FLD analysis. On the other side, the use of MS detection, together with isotopically labeled standards, is fundamental to overcome problems encountered in the clean-up of the samples. It should be noted in any case that even with very specific MS/MS methods, problems due to matrix interference are often encountered in OTA determination in food matrices. One study discussing OTA determination, after derivatization, by GC/MS has also been reported (Soleas, Yan, & Goldberg, 2001).

    A. Liquid Chromatography/Mass Spectrometry Determination of Ochratoxin A

    Earlier LC/MS methods made use of the first generation of interfaces, such as thermospray (Rajakyla, Laasasenaho, & Sakkers, 1987) or a direct liquid introduction (Abramson, 1987), with the known disadvantages in terms of robustness, sensitivity, and ease of operation.

    The first work with modern instrumentation can be attributed to Becker et al. (1998), who used an LC/ESI-MS/MS method for the quantification of OTA in wheat, coffee, and beer. The molecule was analyzed in the positive ion mode, where the fragmentation of the protonated molecular ion (m/z 404) yielded fragments corresponding to the loss of water (m/z 386), water/carbon monoxide (m/z 358), phenylalanine (m/z 239), and water/ammonia/carbon monoxide (m/z 341). The fragmentation of the deprotonated molecular ion (m/z 402) in the negative mode yielded only the fragment corresponding to the loss of carbon dioxide (m/z 358): being this loss not very characteristic, the positive ion mode was chosen for the selected reaction monitoring (SRM) analysis, by monitoring the fragments at m/z 358 and 239 generated by the protonated molecular ion. An LOD of 20 pg (as absolute amount injected) was obtained, almost comparable to that obtained with fluorescence detectors. In food samples, after an SPE and a concentration procedure with good recoveries, this meant an LOD of 0.01 ppb. Quantification was performed with standard solutions with external calibration (LOQ 60 pg or 0.03 ppb). Thus, the method can be a good confirmatory test for samples already turned out to be positive with fluorescence detection. The same procedure was applied by the same group (Degelmann et al., 1999) for the detection of OTA in beer. Again, the LC/MS/MS method was used mainly for confirmatory purposes, after a positive sample was detected by LC/FLD. In this case, the LODs were higher if compared to the previous study (0.1 ppb). Quite interestingly, dark beer could not be analyzed by LC/MS/MS due to matrix interferences.

    A very interesting LC/MS/MS method for the analysis and confirmation of OTA in foods, after derivatization to the methyl ester and using the isotopically labeled OTA methyl(D3) ester as internal standard, was reported by Jorgensen & Vahl (1999). The deuterated methyl derivative was synthesized by the authors starting from the methanol(D4)-boron trifluoride complex and a standard solution of OTA. The same reaction with “light” methanol was then used to derivatize OTA in the food matrices, followed by mixing with the “heavy” internal standard OTA methyl(D3) ester. The MS/MS detection for both isotopes was performed by ESI in the positive ion mode through a MRM experiment by monitoring three daughter ions for the “light” methyl ester derivative (m/z 239, 221, and 193) and one for the “heavy” internal standard methyl ester derivative (m/z 239). However, quantification was done by using only the daughter ion m/z 239 also for the “light” labeled analyte, and an LOD of 0.02 ppb was reported. The samples were extracted and concentrated before derivatization. The precision of the method was around 10% although the mean recovery was quite high (104–121%). This could be due to the fact that the pseudomolecular ion at m/z 421 (the protonated molecular ion for the “heavy” standard) was present with discrete intensity also in the spectrum of the “light” analyte. As a consequence, the transition from 421 to 239 cannot be considered specific only for the standard, since it can be detected also for the analyte, thus interfering with an accurate determination of the real OTA content.

    A comparison of two different LC/MS/MS methods, the former based on ESI interface and the latter on APCI interface, was reported by Lau et al. (2000). In the positive ion mode, the ESI interface showed much higher sensitivity than the APCI interface. A sensitive LC/ESI-MS/MS method was extensively studied for the detection of OTA in human plasma and was also applied to the analysis of contaminated coffee. The fragmentation patterns were extensively discussed and the transitions to be monitored to achieve maximum sensitivity were carefully optimized: the monitored transitions were those from m/z 404 to 239, 404 to 358 (see above), and also the unusual 426 to 261, i.e., the transitions from the sodiated molecular ion to the sodiated 239 ion. Since a sodiated ion was also taken into account for quantification, the effect of the presence of alkali metal ions in the sample was also studied and found to be negligible. OTB was used as internal standard in plasma, although its use in foods is not envisaged, since it may be present as contaminant. The LOD obtained with the optimized ESI-MS/MS detection was 5 pg (as absolute amount injected). Finally, by using LC/FLD to confirm the quantitative results, three different methods of quantification were compared: external standard, internal standard, and standard addition methods. The results were compared with those from the conventional LC/FLD method: all methods were in reasonable agreement (< ± 20% deviation from the average). The authors suggested that a suitably isotopically labeled OTA would be the best internal standard.

    Two different columns were used by Zoellner et al. (2000a) to determine OTA in wines by LC/ESI-MS/MS in the MRM mode. The wines were extracted and concentrated by a SPE procedure. The monolithic column was found to give comparable results to the standard particle-based column, while enabling higher flow rates, thus reducing the analysis time. The usual transitions from 404 to 239 and to 358 and also the very specific transition from 406 (minor isotope of the protonated molecular ion) to 241 were monitored in the positive ion mode. Good linearity range, precision, and LOD (0.025 ppb) were achieved by using the mycotoxin ZEN as internal standard. Very interestingly, this method was compared in a subsequent study from the same group with widely used LC/FLD determinations of OTA in 18 naturally contaminated wine samples (Leitner et al., 2002). The simpler sample preparation by SPE allowed by the high selectivity of the MS method led to higher recoveries than liquid–liquid extraction and comparable with those obtained by IAC column purification. When the same sample preparation was used, the results of the MS method were highly consistent with those obtained by fluorescence detection. The major and evident drawback was in the LOD (and subsequently in the limit of quantification), which was five times higher (0.05 ppb against 0.01 ppb) by using MS detection. As a consequence, samples in which quantification was possible by LC/FLD were under the limit of quantification or even under the limit of detection when using LC/MS/MS. Although MS chromatograms were usually cleaner, OTA in all wine samples could be confidently identified and quantified by LC/FLD.

    Another recent study reported the use of LC/ESI-MS/MS (MRM mode), with procedures essentially analog to those previously reported, for the confirmation of OTA in South African wines previously turned out to be positive by LC/FLD analysis (Shepard et al., 2003). Recently, we showed that by changing the pH of the eluent (from 5.5 to 9.8) it is possible to detect OTA by directly injecting wine in HPLC/FLD with LOD of 0.05 ng/mL (Dall' Asta et al., 2004a). Actually, the presence of OTA in wine is a widespread problem in the world and further studies are needed to investigate its natural occurrence in wines and for developing methods of prevention and decontamination.

    An LC/FLD method with confirmation of positive samples by LC/ESI-MS (single quadrupole analyzer, positive ion mode, single ion recording of m/z 404) was also reported for the quantification of OTA in coffee (Ventura et al., 2003). The LOD of the fluorescence detector was reported to be 0.1 ppb, and the LOD of the MS detector was claimed to be the same, although data were not reported. The importance of the study mainly consists in the claim that a single quadrupole MS is sufficient for confirming by LC/MS the samples positive by LC/FLD.

    A very interesting stable isotope dilution assay was recently reported by Lindenmeier, Schieberle, & Rychlik (2004), who elegantly synthesized the (2H5)-OTA by hydrolyzing the phenylalanyl moiety from standard OTA and coupling the isocoumarin derivative with (2H5)-phenylalanine. The labeled internal standard was mixed with the matrix and OTA was extracted by an SPE technique or by immunoaffinity clean-up of wine and coffee samples. The ESI-MS/MS method was based on a SRM experiment by monitoring the specific transition from 404 to 358 for OTA and from 409 to 363 for labeled OTA. Both extraction procedures worked well for the wine samples, whereas for the coffee samples only the immunoaffinity clean-up yielded interferent-free MS chromatograms and was used as the preparative method of choice. The LOD was calculated at 0.5 ppb in wheat flour, with good recovery and precision. Finally, many different food samples were tested for their OTA content with the developed method.

    Since Aspergillus is known to produce different mycotoxins, in particular ochratoxin and aflatoxins, under certain conditions, there is a need to develop methods which allow the simultaneous detection of multiple residues. A fundamental problem is the co-extraction of toxins with different structures and different polarities. The simultaneous detection of ochratoxin A together with other toxins produced by Aspergillus fungi, including aflatoxins, was reported by Tuomi et al. (2001) by using an ion trap analyzer in an LC/ESI-MS/MS. In the positive ion mode, the sodiated molecular ion of OTA, which was more intense than the protonated ion, was used as parent ion, and the fragment at m/z 279 (loss of phenylalanine residue, sodiated) and 261 (loss of a water molecule from the previous one) were used as daughter ions for the quantification experiments. Although the method presented turned out to be good for a preliminary screening, given the fast and easy extraction procedure, with a LOQ of 200 ng for all compounds, it suffered evident drawbacks for an accurate quantification, including low recoveries (28–99%, although OTA was extracted for the 99%), high errors (6–30% without internal standard and 11–53% with reserpine as internal standard), and low accuracy (65–190%). Although this method is very important since it approaches a multiresidual determination, its low performance can be attributed partly to the use of an ion trap as analyzer, which clearly affects accuracy and precision, and partly to the use of a unified method for the extraction of different molecules having different polarities and different solubility properties.

    A summary of the main recent LC/MS methods for ochratoxin determination is reported in Table 4.

    Table 4. Main recent LC/MS methods for ochratoxin A determinationThumbnail image of
  • aThese values are those found for wine. In beer, the LOD was higher than one order of magnitude (0.1 ppb).

  • bThis value is somewhat higher than others because of the different matrix, since it has been calculated in human plasma.

  • cThe method, although proposed for aflatoxins, OTA and other toxins, showed good recovery (99%) only for OTA. Moreover, the method also showed low precision and low accuracy. Again, OTA gave best results (5–27% RSD and 69–110% accuracy).

  • B. Gas Chromatography/Mass Spectrometry Determinations of Ochratoxin A

    A GC/MS method for the detection and quantification of OTA in wines was reported by Soleas, Yan, & Goldberg (2001). The samples were extracted in dichloromethane, dried and derivatized with bis(trimethylsilyl)trifluoroacetamide (BSTFA) to yield the trimethylsilyl derivative of OTA (MW = 619), which was analyzed by GC/MS in the SIM mode by monitoring eight specific ions. The LOD of the method was 0.1 ppb, higher than that obtained by an LC/PDA method presented in the same study (0.05 ppb). Moreover, also recovery and precision were much worse in the GC/MS method, which is to be considered suitable for confirmation, due to its high selectivity, but not for routine detection of OTA in wines.

    V. TRICHOTHECENES

    1. Top of page
    2. Abstract
    3. I. INTRODUCTION
    4. II. PATULIN
    5. III. AFLATOXINS
    6. IV. OCHRATOXIN A
    7. V. TRICHOTHECENES
    8. VI. ZEARALENONE
    9. VII. FUMONISINS
    10. VIII. CONCLUSIONS
    11. REFERENCES
    12. Biographical Information

    Trichothecenes are a group of mycotoxins produced by a variety of different Fusarium species, such as F. culmorum, F. equiseti, F. graminearum, F. moniliforme, F. sporotrichioides, and F. proliferatum. In particular, F. graminearum, a plant pathogen of gramineous plants, especially wheat, is known to produce deoxynivalenol (DON), nivalenol (NIV), and ZEN. It is the most widely distributed toxinogenic Fusarium species (Miller & Trenholm, 1997).

    The structure of trichothecenes presents a tetracyclic sesquiterpene skeleton, which (although trichothecenes are a class of very diverse chemical compounds) usually includes a six-membered oxane ring, an extremely stable epoxide group in the 12,13 position and a 9,10 olefinic bond. The main naturally occurring trichothecenes in grains are divided in two different groups: the more polar substances, with a keto group at C-8 (type B trichothecenes) and the less polar toxins, which do not contain the keto function at C-8 and also usually bear fewer free hydroxyl groups (type A trichothecenes). The chemical structures of the main type A and type B trichothecenes are reported in Figure 4.

    thumbnail image

    Figure 4. Chemical structures of the main type A and type B trichothecenes.

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    DON, the most important type B trichothecene, occurs predominantly in grains such as wheat, barley, and maize and less often in oats, rice, rye, sorghum, and triticale. After short-term administration, DON induces decrease in feed consumption (anorexia) and emesis (vomiting in animals). DON has been detected not only in cereals (wheat, barley, rice, rye, sorghum, oats), but also in some processed food products in Europe and North America. In addition, it has been reported in barley products, such as malt and beer (Hussein & Brasel, 2001; Creppy, 2002). Under natural conditions, DON is found in wheat and barley in association with several strictly related compounds, such as 3-acetyl-deoxynivalenol (3-ADON), 15-acetyl-deoxynivalenol (15-ADON), 3,4-diacetyl-nivalenol (3,4-DA-NIV), and 3,15-diacetyl-deoxynivalenol (3,15-DA-DON). The same situation occurs for T-2 toxin (T2) and diacetoxyscirpenol (DAS), which are often found together with closely related toxins contributing to the overall toxicity. Generally, the same Fusarium strains are also producers of ZEN, a mycoestrogen, which occurs along with DON in maize, barley, and wheat (see below).

    Trichotechenes are not regulated by FDA, nor by the EU, although the latter organism is introducing a new legislation proposing legal limits for trichothecenes around 500 ppb.

    The most common methods for the determination of trichothecenes involve the use of a gas-chromatographic system with an FID, ECD or mass spectrometric detector or an HPLC system with UV or fluorescence detection (after derivatization of the molecules). The GC methods allow to get detection limits in the range 1–10 ppb for type A and 10–30 ppb for type B trichothecenes, whereas the LODs obtained by HPLC-UV methods are higher (100–200 ppb). Likely, the HPLC would be better suited for the trichothecene determination from a chromatographic point of view, but the lower sensitivities obtained by using UV detectors have always hampered the spreading of the technique. Since trichothecenes are not fluorescent, the LC/FLD can be used only after derivatization of the trichothecene with suitable fluorophores (Dall'Asta et al., 2004b). Therefore, the use of GC for trichothecene analysis is due to the necessity to reach sensitivities in the range of low ppbs, although it requires derivatization steps prior to the analysis, introducing points of uncertainty. Samples can be readily confirmed by GC/MS. As far as HPLC is concerned, MS detection is essential to selectively detect underivatized trichothecenes at a fair sensitivity (Langseth & Rundberget, 1998).

    A. Gas Chromatography/Mass Spectrometry Determination of Trichothecenes

    GC analysis is mostly used for the determination of type A trichothecenes, since they are not fluorescent and do not strongly adsorb in the UV-Vis range. GC is also widely employed for the determination of type B trichothecenes, although the conjugation of the carbonyl group with the double bond makes them more suited for UV detection and therefore HPLC analysis. The GC determination is mostly based on derivatization of the hydroxyl groups by trimethylsilylation or fluoroacylation to trifluoroacetyl (TF), pentafluoropropionyl (PFP), or heptafluorobutiryl (HFB) derivatives. FID detectors are commonly utilized or alternatively, ECD for the fluoroacyl derivatives. Although several GC/MS methods have been proposed as alternatives to GC/ECD or GC/FID, none seems to allow for a good quantitative determination. At the moment, no isotope dilution approach has been developed. Moreover, only few GC/MS methods allow the simultaneous detection of type A and type B trichothecenes. The GC/MS approach seems to give the best results for confirmatory analyses and also for obtaining structural information on several derivatives of the main trichothecenes, which is fundamental for toxicological and metabolic studies. The widespread possibility of using GC/MS for the development of mass spectral databases inspired some recent studies.

    A GC/MS method used for the analysis of DON and derivatives (3-ADON, 15-ADON, NIV, 7-ADON) in wheat and barley, described by Mirocha et al. (1998), is based on selected ion monitoring (SIM) detection of the mycotoxin as trimethylsilyl-ethers (TMS). The analysis involved electron impact ionization (EI) of the derivatives and monitoring of the diagnostic ions (from four to seven according to the different toxins). The sensitivity varied according to the column, the conditions of the filament, as well as the instrument tuning. Moreover, ionization turned out to be matrix dependent, so that internal calibration was often required, in particular with complex matrices such as feeds. Quantification was performed by external calibration with good precision (around 5% SD on mean values) and good recovery (around 90%), although LOD and LOQ were not reported.

    Onjy et al. (1998) suggested that the derivatization step of trichothecenes before the GC/MS analysis might cause a lack of structural informations for the identification of compounds. As a possible approach, they proposed a direct analytical method to determine the native mycotoxins extracted from several cereals without any derivatization procedure, using an improved cold on-column injection equipped with a laboratory-made inlet liner directly connected to the GC capillary column. Prior to GC/MS analysis, sample clean-up procedures (zinc acetate treatment and Florisil columns) for separation of toxins from fats are indispensable. The chromatographic separation, performed for DON, 3-ADON, FUSX, DAS, scirpentriol (SCT), 15-monoacetoxyscirpenol (15-MAS), T-2, and ZEN, allowed to achieve good peak resolutions, with the EI spectra showing the molecular ions of the analytes and the specific fragmentation for each toxin. However, the direct analysis of NIV was not possible, because of the degradation of this compound into several fragments after injection. Moreover, although average recovery was 93%, two toxins (FUSX and SCT) showed a recovery of 49% and 42%, respectively, again for likely degradation of these compounds. The LODs of the method, measured for barley sample extracts, ranged from 100 to 500 ppb using an SIM mode.

    A GC/MS method for the determination of eight trichothecenes of type A and B developed by Schoellenberg et al. (1998) involved the use of an ion trap mass spectrometer operating in chemical ionization mode, using isobutane as reactant gas. Also in this case, it was necessary to apply different clean-up steps to remove the co-extracted matrix components. The authors suggested that the critical point of the whole procedure, which was applied to complex matrices including heavily molded cereals, cereal foods, and feed, was the derivatization step to obtain TF derivatives, so that an internal standard was added to control the reaction. Since the ion trap system allowed to obtain the typical fragmentation for each toxin, the quantification was based both on the main fragment ion and on a secondary characteristic ion, which allowed to correctly identify the analyte. The reproducibility of the fragmentation was good, with differences between the two selected ions below 10%. The LODs and LOQs of the whole method, measured by injection of standard solutions, ranged from 2 to 12 ppb and from 6 to 38 ppb, respectively. The highest detection limit was obtained for NIV, consistently with the lower ionization properties of this compound.

    Quite often GC/MS methods allow the simultaneous detection of trichothecenes and ZEN, which is also produced by Fusarium molds (see below). Recently, Tanaka et al. (2000) proposed a GC/MS method, essentially analog to that used by Mirocha (see above), for the simultaneous detection of seven trichothecenes (DON, NIV, FUSX, 3-ADON, T-2, neosolaniol (NEO), DAS), and ZEN as trimethylsilyl derivatives by single ion monitoring (SIM) of the main ion of each compound. The authors decided to detect trichothecenes as TMS derivatives according to the mass range limitation (600–700 m/z) of their bench-top type GC/MS, obtaining detection limits in cereals ranging from 5 to 10 ppb. The recovery ranged from 81% to 94% for the different toxins in the different matrices.

    Although not intended for food analysis, Nielsen & Thrane (2001) proposed a useful GC/MS/MS data bank for the characteristic fragmentation of 21 trichothecenes from Fusarium and Stachybotrys fungi as PFP derivatives. The use of PFP anhydride as derivatizing agent allowed to achieve the highest sensitivity by GC/MS analysis, if a mass spectrometer with a range higher than 800 amu is available. The disadvantage of this reagent, as mentioned by the authors, was the isomerization of type B trichothecenes during the reaction. Moreover, the derivatization reaction itself was also very slow. The method used an ion trap detector for MS/MS analysis. Indeed, MS/MS spectra were much more reproducible than simple MS spectra, where a concentration dependence of the intensity was observed, a typical problem of the instrumentation utilized. In general, trichothecenes were detected by using electron impact (EI) ionization in a positive ion mode, although in some cases, a chemical ionization (CI) in the negative ion mode with methane as ionizing gas was used for peak confirmation.

    A database obtained by GC/MS analysis (EI ionization) of the TMS-derivatized trichothecenes was also recently proposed (Rodrigues-Fo et al., 2002). The authors studied the spectral behavior of DON and several DON-related compounds isolated from moulds (NIV, 7-ADON, 15-ADON, 3-ADON, 3-lactyl-DON, 4,15-DA-ADON, 3-ANIV). The 3-acetyl-nivalenol was identified for the first time. Structures of the main ions at m/z 103, 117, 147, and 191 were elucidated by high resolution MS (HRMS) experiments and a general fragmentation scheme for the DON-derivatives was suggested. The relative abundances of the assigned ions in the mass spectra of trichothecenes allowed a fast structural investigation in the analysis of complex matrices and could be used for database identification of the investigated compounds.

    A summary of the main recent GC/MS methods for trichothecene determination is reported in Table 5. The last two methods are not reported in this table since they are intended neither for food nor for feed.

    Table 5. Main recent GC/MS methods for trichothecene determinationThumbnail image of
  • aRecovery range: 42–108%.

  • bCold on-column injection.

  • cExtracted chromatograms corresponding to the molecular ions and to the main fragments were used for the quantification.

  • B. Liquid Chromatography/Mass Spectrometry Determination of Trichothecenes

    LC/MS analysis is becoming the method of choice for the determination of trichothecenes, which do not contain characteristic chromophores or fluorophores, thus avoiding derivatization steps and achieving more robust and reproducible results. Moreover, the LC/MS analysis also allows to reduce the sample preparation step: different studies showed that the direct analysis of the sample extract can be performed without the need of the immunoaffinity purification. The injection of raw extracts requires, however, an extremely careful calibration: it was widely demonstrated that the matrix effect on ionization is particularly significant for trichothecenes. Thus, the most important limitation, at the moment, is the difficulty to find a proper internal standard, being the isotope-labeled compounds not commercially available or very expensive.

    Another important area of research concerns the development of multiresidual methods, able to detect a great number of trichothecene toxins in many different matrices: in this case, not only the ionization but also the extraction method needs to be carefully studied and completely validated. Quite interestingly, almost all published methods make use of the less common APCI interface, rather than of the more common ESI. Several reasons probably justify this preference: the ionization yield in the ESI interface is likely to be more affected from matrix effects, it usually does not allow the simultaneous detection of type A and type B trichothecenes with the same ionization mode and generally both classes of toxins show a poor ESI response.

    An interesting LC/MS method based on ion trap MS for the determination of nine trichothecenes in wheat using LC-APCI-MSn in positive ionization mode was described by Berger, Oehme, & Kuhn (1999). Clean-up procedures with Mycosep columns were performed allowing for fair recovery (≥80%). The method allowed the separation of the trichothecenes (NIV, DON, FUSX, NEO, 3-ADON, 15-ADON, DAS, HT-2, and T-2) in less than 12 min using a MRM detection mode, with LODs of 10 ppb for NIV and 15-ADON, 6 ppb for DON, 4 ppb for NEO, 3 ppb for FUSX, 3-ADON, and T-2, and 1 ppb for DAS and HT-2. The method did not allow for the separation of the two acetylated DON-derivatives, but they could be distinguished by the specific fragmentation performed in an ion trap mass spectrometer. The quantification was based on the addition of an internal standard (verrucarol, a semisynthethic trichothecene) to the sample before the extraction and was carried out using the [M + H]+ ions or the main fragment ions. Moreover, the authors presented a scheme to unequivocally identify trichothecenes by online MS/MS and ion adduct formation with ammonium acetate. For the ion adduct formation experiments, ammonium acetate was added connecting a T-tube between the HPLC column and the ionization chamber. The addition of ammonium acetate resulted in increased sensitivity for the type A trichothecenes as [M + NH4]+ adducts and for type B toxins as [M + CH3COO] ions. MS2–MS6 experiments were performed to study the fragmentation in the ion trap. Compared to the type A trichothecenes, type B easily lost the side chain C15, likely resulting in a quinoic structure followed by the rearrangement into a stable aromatic alcohol. Online HPLC/MS/MS experiments provided information about the substituents present. Since the hydroxyl groups were cleaved off as water and the ester as their corresponding acids, every lost substituent generated a double bond equivalent. Type B trichothecenes with three or four substituents usually generated fragments of 200 m/z, while a higher energy was required to break the 3-ring backbone generating a fingerprint profile typical of both type A and type B trichothecenes. The LODs and the LOQs of the whole method ranged from 1 to 10 ppb and from 10 to 100 ppb, respectively.

    An isocratic HPLC method combined with negative APCI-MS detection for the determination of NIV and DON in wheat was published by Razzazi-Fazeli, Bohm, & Luf (1999). The instrument employed was a single quadrupole mass spectrometer and the detection of each compound was based on the molecular ion. All the ionization parameters were optimized for the analysis, to obtain the best sensitivity. The best flow was found to be 1 mL/min, without splitting. The effect of the nebulizing temperature on the sensitivity and on the fragmentation pattern was studied, showing the need of high temperatures (>300°C) to allow high ionization yields. The cone voltage was found to have a strong effect on fragmentation: the intensity of the deprotonated ion [M−H] and of the adducts decreased by increasing the cone voltage to values higher than 20 V. For both molecules, by using a vaporizer temperature of 400°C, the loss of 30 mass units, together with other fragments, was observed, which was explained by the authors as the cleavage of the epoxy group yielding fragments [M−H-CH2O] at m/z 265 (DON) and 281 (NIV). The authors also studied the effect of buffer addition on the ionization of DON and NIV. In particular, a decrease in the signal-to-noise ratio was observed by the use of modifiers to the mobile phase such as ammonium acetate or acetic acid, whereas addition of ammonia resulted in no visible effect. With the optimized method and using a SIM mode of acquisition on the deprotonated molecular ions, LOQs (LOD not reported) of 40 ppb for DON and of 50 ppb for NIV (in matrix) with fair recoveries (70–86%) were obtained.

    More recently, the same group (Razzazi-Fazeli et al., 2003) extended the proposed negative ion mode APCI-MS method to the detection of the acetylated derivatives of DON (3-ADON and 15-ADON) and to the de-epoxy metabolite of DON called DOM-1. This metabolite is formed from DON by gut and rumen bacteria through reduction of the epoxide group of DON, and it was found in some mammalian tissues and in cow's milk. DOM-1 was proposed as a marker for the exposition to DON in animals. The type B trichothecenes separation involved the use of a linear gradient from H2O-CH3CN-CH3OH 82:9:9 to H2O-CH3CN 40:60 as mobile phase and a Polar-RP standard bore column (150 mm × 4.6 mm, 4 μm). The quantification was performed using the [M−H] ion, while the identification was based on the main fragments. This is the first method, which allows the separation of 3-ADON and 15-ADON without changing the flow rate, by choosing a more polar column than the commonly used C18. The detection limits for type B trichothecenes were 70 ppb for NIV, 50 ppb for DON, 3-ADON, FUS-X, and 150 ppb for 15-ADON.

    The same authors also published a study for the LC/APCI-MS determination of type A trichothecenes T-2, HT-2, Acetyl-T-2, DAS, 15-acetoxyscirpenol (15-AS), and NEO (Razzazi-Fazeli et al., 2002). A C18 narrowbore column (200 mm × 2.1 mm, 5 μm) and a linear gradient consisting of 1 mM aqueous ammonium acetate–acetonitrile mobile phase was used. The MS experiment was performed in the APCI positive ion mode. The quantitative determination of each compound was based on the molecular protonated ions, on the ammonium adducts, or on the main fragments. In contrast to type B trichothecenes, the response in positive ion mode for all the investigated type A toxins was found to be distinctly more sensitive than in the negative ion mode. The mass spectra of the compounds often showed a [M + NH4]+ adduct more intense than the [M + H]+ ion. The loss of the acetyl group could be observed for all the tested molecules, while the formation of very intense ammonium adducts was typical of those with ester functions at the C15 or C4 positions. Although optimal flow was found to be around 1 mL/min, the use of a narrow bore column required a flow of 0.3 mL/min. LODs were not reported, but LOQs in matrix ranged from 50 to 85 ppb. As internal standard deuterated T2 toxin (D3-T2) was used. Nevertheless, the precision was nevertheless not very high (up to 25% of interday variability).

    As a good example of methods using an LC/ESI-MS, Plattner (1999) reported the analysis of DON in food matrices in the negative ion mode with a single quadrupole analyzer. The sensitivity was typically low (LOD >2 ppm in matrices without clean-up treatment), although comparable with UV detection. However, the author stated that the ESI interface was more rugged and forgiving than the APCI when dirty samples were analyzed and thus more suited for routine determinations.

    In a very recent study, the analysis of four type B trichothecenes (NIV, DON, FUSX, 3-ADON) was performed in maize by an LC/MS/MS technique, comparing the commonly used Atmospheric Pressure Chemical Ionization (APCI) interface to the TurboIonSpray (TISP) interface in both negative and positive ion modes (Laganà et al., 2003). The most valuable spectra for each trichothecene were obtained using the turbo ion spray TISP-ESI system in the negative ion mode, gradient elution from water/methanol/acetonitrile 90:3:7 to water/methanol/acetonitrile 20:24:56, with no modifier. The APCI responses were definitely lower than those obtained for ESI. Moreover, the ESI interface was again found to be more rugged, maintaining sensitivity after many injections of dirty samples and requiring less maintenance than the APCI interface. In the negative ion mode, the type B trichothecenes showed a very specific fragmentation, although the [M−H] ion was the base peak for all the tested compounds. The MRM quantification was based on the [M−30] ion, assigned by the authors to the cleavage of the epoxy group. The best results were found by TISP in the negative ion mode and in the MRM mode for the quantification, obtaining detection limits ranging between 1.5 and 10 ppb.

    Very recently, we succeeded in developing a method for the simultaneous determination of several type A (NEO, DAS, T-2, HT-2) and several type B trichothecenes (DON, 15-Ac-DON, NIV, FUSX) at good sensitivity with an ESI interface and a single quadrupole analyzer. The determination was carried out for all trichothecenes in the positive ion mode by using a water/methanol 35:65 mixture as isocratic eluent. To enhance the naturally poor response of the trichothecenes in the ESI interface, we started from the observation that this class of molecules forms very stable adducts with the sodium ion. The addition of sodium chloride to the eluent (100 μM) not only simplified the spectra, which only showed the sodiated molecular ions, but also enhanced the response for all trichothecenes of about one order of magnitude. The LODs obtained with this method by monitoring the sodiated molecular ion by a SIM acquisition ranged from 20 to 50 ppb, as determined in matrices, extracted, and analyzed without any clean-up treatment. The method was tested by determining DON in a certified reference material, giving very consistent results (Dall'Asta et al., 2004c).

    A summary of the main recent LC/MS methods for trichothecene determination is reported in Table 6.

    Table 6. Main recent LC/MS methods for trichothecene determinationThumbnail image of
  • aThe LOD without Mycosep clean-up was 2 ppm.

  • bNaCl was added as cationization agent to the HPLC eluent.

  • VI. ZEARALENONE

    1. Top of page
    2. Abstract
    3. I. INTRODUCTION
    4. II. PATULIN
    5. III. AFLATOXINS
    6. IV. OCHRATOXIN A
    7. V. TRICHOTHECENES
    8. VI. ZEARALENONE
    9. VII. FUMONISINS
    10. VIII. CONCLUSIONS
    11. REFERENCES
    12. Biographical Information

    ZEN is a secondary metabolite produced by several species of Fusarium fungi, mainly by F.graminearum and F. culmorum, which grow on several commodities, especially cereals such as maize, barley, oats, wheat, and sorghum (Betina, 1989). Depending on climatic, harvest, and storage conditions, the levels of ZEN found in cereals are between 1 and 2,900 ppb (Kuiper-Goodman, Scott, & Watanabe, 1987).

    The structure of ZEN consists of a resorcinol moiety fused to a 14-membered macrocyclic lactone ring, which includes a trans double bond, a ketone, and a methyl side group (Fig. 5).

    thumbnail image

    Figure 5. The structure of Zearalenone.

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    The structure is flexible enough to allow ZEN to adopt a conformation able to bind to the mammalian estrogen receptor, although with lower affinity than the natural estrogen 17-β-estradiol, thus resulting in severe effects on the reproductive system in several animal species, in particular pigs. The reduction of the C-6′ ketone in the liver of animals and humans gives two different diastereomers, namely α- and β-zearalenol (ZOL). The estrogenic effect of α-ZOL is approximately three times stronger than that of ZEN and β-ZOL. Only α-ZOL has been observed to occur in naturally contaminated cereal grains (Mirocha, 1971; Hussein & Brasel, 2001). The carcinogenic properties of ZEN are not yet well defined and are under study.

    Due to its metabolic activity and its frequent occurrence in cereals, several countries have established guidelines or maximum tolerable levels for ZEN, ranging from 0 (sic!) in the Netherlands and in Hungary to 1,000 ppb in Russia. Also the European Union will provide legal limits for the Member Countries, in particular to regulate the presence of this mycoestrogen for cereal-based foods for human consumption and for baby foods (Creppy, 2002).

    Usually, extraction and clean-up are the critical points of the procedures and are performed in different ways: (a) SPE; (b) liquid–liquid partitioning; (c) immunoaffinity columns (Schumacher et al., 1998). One of the most promising sample preparation techniques is the pressurized liquid extraction (PLE), which allows for an automated sample extraction step (Pallaroni & von Holst, 2003). However, only ZEN was extracted with this method, whereas nothing is reported for its metabolites.

    Consistent with the fluorescent properties of ZEN, the most commonly used method for its analytical determination in food is based on HPLC with fluorescence detection, although extensive clean-up of the sample is required. Excitation is usually carried out at 275–280 nm and fluorescence emission is measured at 450–470 nm, allowing limit of detections down to 2 ng/g.

    A. Gas Chromatography/Mass Spectrometry Determination of Zearalenone

    Very few methods involving the gas-chromatographic detection of ZEN have been reported, given its polarity and its fluorescent properties, which make it more suited for HPLC-FLD analysis. The possible simultaneous occurrence of several trichothecenes, in particular DON, and ZEN induced some authors to extend the GC/MS method for the trichothecenes detection also to ZEN, but none of these was developed specifically for this compound (Onjy et al., 1998; Tanaka et al., 2000).

    B. Liquid Chromatography/Mass Spectrometry Determination of Zearalenone

    Several LC methods based on mass spectrometric detection were developed in the last few years for the simultaneous determination of ZEN and its metabolites in food commodities and in animal tissues (Krska & Josephs, 2001). At the moment, the most important problem for the analysis of ZEN and its derivatives using LC/MS techniques, given the complex food matrices, seems to be the availability of an opportune internal standard. Generally, LC/MS methods make use of the saturated zearalanone (ZAN) as internal standard, according to its properties closely related to ZEN. However, this choice is not suitable for mass spectrometric methods based on single quadrupole instruments or for monitoring only the molecular ions, since ZAN is isobaric with α- and β-ZOLs and it coelutes with ZEN if a common C18 column is used, thus it could be recognized only on account of its specific fragmentation. Recently 1′,2′-dideuterated ZEN (D2-ZEN) was opportunely synthesized and used as internal standard for detecting ZEN and its metabolites in urine, muscle tissue, and liver samples of pigs fed with mycotoxin-contaminated oats (Zoellner et al., 2002).

    Many methods have been developed in the last 5 years based on LC/MS techniques, most of them interfaced by APCI. The high selectivity of MS as compared to other detection techniques allows to simplify clean-up procedures even for complex matrices as feeds and animal tissues, suitable also for toxicokinetic analyses. Moreover, ESI and APCI techniques offer the clear advantages of robustness and easy handling, important requirements for routine analyses with high sample throughput.

    The first developed LC/APCI-MS method for the determination of ZEN in food and feed was proposed by Rosenberg et al. (1998). Several parameters (ionization mode, nebulizer gas flow, vaporizer temperature, fragment and capillary voltages, corona current) were evaluated to achieve the best ionization of ZEN. In particular, although it was noticed that the best response for ZEN was obtained by negative ionization mode because of the presence of two phenolic groups in the ZEN chemical structure, and the most abundant ion was the quasi-molecular ion [M−H], the positive ion mode was chosen of the stability for the signal and the better baseline. In a working range of 2,000–4,000 V, the response was independent from the capillary voltage, while already at low fragmentor voltage a significant fragmentation of the quasi-molecular ion occurred. The method was successfully applied to the quantification of ZEN in cereal samples, obtaining a detection limit of 2.5 ng/ml extract. This corresponds to a limit of detection of 0.12 μg/kg corn, approximately 50 times lower than that reported for ZEN the by HPLC/FLD method.

    Recently, Zoellner, Jodlbauer, & Lindner (1999) proposed a robust LC-APCI-MS/MS method for the determination of ZEN in grains, using ZAN as internal standard, which does not occur in nature but it was found in tissues and microbial cultures as a ZEN metabolite. The method involved purification on an RP-C18 SPE column or IAC clean-up and an MRM quantification. Both positive and negative ionization modes were evaluated, achieving the best results with the latter, in contrast with the previous data (Rosenberg et al., 1998). For MRM analysis, the transition of the [M−H] ion (m/z 317) to the characteristic fragment at m/z 175 was used for ZEN quantification, while for ZAN the transition from 319 m/z ([M−H] ion) to m/z 205 was selected. In the reported conditions, a limit of detection of 0.5 μg/kg in maize was achieved. The quantitative data for ZEN obtained with or without internal standard were evaluated in different grain samples, showing a strong matrix effect, in particular if a simplified sample clean-up was used. This result suggested the need of a deuterated analyte as internal standard to achieve a higher accuracy, even if ZAN exhibits a quite similar mass spectrometric behavior as ZEN. The method was successfully validated by an interlaboratory test on grain certified material.

    The described method was then applied to the analysis of ZEN and its metabolites α- and β-ZOL in beer samples (Zoellner et al., 2000b). Also in this case, an MRM quantification in the negative ion mode was performed, which was found to be by a factor of 10 more sensitive than the positive ion mode. It was based on the specific transition of ZEN (from 317 to 175 m/z) and ZOLs (from 319 to 275 m/z), using ZAN as internal standard. The main problem of MS detection of these compounds was the formation of identical molecular ions in SIM mode or identical ion pairs in MRM mode, on account of the closely related chemical structure of these compounds. Actually, ZAN, α- and β-ZOL, contribute to the same 319 to 275 m/z fragmentation pathway, therefore an improved chromatographic separation was necessary for such multi-residual determination. A sufficient resolution was achieved using a “shielded” RP-8 column. The limits of detection (LOD) in beer were 0.03 ppb for ZEN and 0.06 ppb for both ZOLs; limits of quantification (LOQs) were 0.06 μg/L for ZEN and 0.15 μg/L for both ZOLs. The performance of the method was excellent when applied to a particular beer brand, but it was not possible to use a general calibration curve for all beer brands, since calibration curves varied considerably from brand to brand in relation to matrix effects. Other potential limitations for the LC/MS/MS method include selectivity problems of the detection system when similar compounds have to be analyzed and the yield of the selected ions in the presence of coeluting matrix compounds. Therefore, a more accurate sample clean-up and/or more selective chromatographic methods prior to MS analysis may be required.

    Finally, the range of application of the proposed method was opened to the analysis of urine, muscle, and liver samples of pigs fed with contaminated oats, to investigate the toxicological fate of ZEN and the incorporation of its metabolites in animal tissues, which are intended to be used for human nutrition purposes (Zoellner et al., 2002). In particular, the analysis allowed the simultaneous detection of ZEN, α- and β-zearalenol (α- and β-ZOL), α- and β-zearalanol (α- and β-ZAL), and ZAN. In this case, ZAN could not be used as internal standard, since it occurs in animal tissues; thus the quantification was performed with 1′,2′-dideuterated ZEN (MW 322) opportunely synthesized as internal standard. For the MRM quantification, the deprotonated ions of ZEN (m/z 317), α- and β-ZOLs and ZAN (m/z 319), α- and β-ZALs, and D2-ZEN (m/z 321) were selected, while the quantification was based on the following product ions: m/z 277/303 for α- and β-ZALs, m/z 207 for D2-ZEN, m/z 160/174 for α- and β-ZOLs, m/z 205/275 for ZAN, and m/z 131/175 for ZEN. The method allowed to achieve limits of detection of 0.1 ppb for ZEN, α-ZOL, α-ZAL and ZAN, 0.3 ppb for β-ZOL, and 1 ppb for β-ZAL in pig liver.

    Although an LC/ESI-MS method for the determination of six resorcyclic acid lactones ZEN-related in bovine tissues using α-ZAL-d4 and β-ZAL-d4 as internal standard has also been reported (van Bennekom et al., 2002), at our knowledge only one method based on an electrospray interface was published allowing the quantification of ZEN in grain samples (Pallaroni & von Holst, 2003). Ideally, a deuterated analog should always be used as internal standard, but these compounds are still not readily available and, if commercially available, too expensive for routine analyses. The method developed by Pallaroni & von Holst (2003) for the determination of ZEN in wheat and corn is based on the already mentioned PLE and LC/MS detection equipped with an electrospray ionization interface without performing any clean-up step. Quantification of ZEN was carried out by using matrix-matched standard curves to compensate for matrix related adverse effects and ZAN was used as internal standard. The method involved an isocratic RP-elution; the mass spectra were recorded in the full scan mode (200–550 m/z) using the negative ionization mode, while the quantification was based on the extract ion chromatograms of the deprotonated molecular ions [M−H] at m/z 317 for ZEN and m/z 319 for ZAN, obtaining a LOD of 4 ppb in corn and 5 ppb in wheat and a LOQ of 15 ppb in corn and 12 ppb in wheat. Although other methods showed lower detection limits, this approach has the main advantage of reducing the sample preparation procedure without loosing accuracy; moreover, the use of a less expensive single quadrupole mass spectrometer as detector offers a simple tools, which could be applied also in laboratories for routine analyses.

    A summary of the main recent LC/MS methods for ZEN determination is reported in Table 7.

    Table 7. Main recent LC/MS methods for zearalenone determinationThumbnail image of

    VII. FUMONISINS

    1. Top of page
    2. Abstract
    3. I. INTRODUCTION
    4. II. PATULIN
    5. III. AFLATOXINS
    6. IV. OCHRATOXIN A
    7. V. TRICHOTHECENES
    8. VI. ZEARALENONE
    9. VII. FUMONISINS
    10. VIII. CONCLUSIONS
    11. REFERENCES
    12. Biographical Information

    Fumonisins are a structurally related group of Fusarium mycotoxins produced mainly by F. verticillioides (formerly monoliforme) and F. proliferatum, which are characterized by a 19 or 20 carbon aminopolyhydroxy-alkyl chain diesterified with propane-1,2,3-tricarboxylic acid (tricarballylic acid, TCA) and also bear an amino or a 3-hydroxypyridinium (3HP) group (Fig. 6). Fumonisin B1 (FB1) is the most abundant natural contaminant in corn-based foods and feeds among the several structurally related homologs (Nelson, Desjardins, & Plattner, 1993).

    thumbnail image

    Figure 6. The structure of fumonisins.

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    The most abundant fumonisins found in naturally contaminated corn are fumonisins B1, B2, and B3, although over 14 other fumonisins are produced, including N-acetylated fumonisins (“A” series), C1 des-methylated fumonisins (“C” series), and hydroxypyridinium containing fumonisins (“P” series). The fully hydrolyzed forms of the B series, such as HFB1, are not naturally occurring and have only been found in processed foods (Musser, Eppley, & Trucksess, 2002).

    The toxicity of fumonisins can cause a variety of diseases in animals, such as Equine Leukoencephalomalacia (ELEM) in horses and Porcine Pulmonary Edema in swines. Fumonisins are also hepatocarcinogenic, hepatotoxic, and nephrotoxic and show cytotoxic effects in various mammalian cell lines (Hussein & Brasel, 2001). The high incidence of human esophageal cancer in South Africa and China has been associated with FB1. For these reasons, fumonisin B1 has been declared by IARC as a class 2B carcinogen, which means a possible human carcinogen (IARC, 1993). At the moment, there is no wide-scale introduction of limits for fumonisins. Limits have been established in some countries to protect susceptible animals and temporary guidelines have been introduced in a few countries to limit the exposure for humans: for example, Switzerland has set a limit for the total fumonisin B1 and B2 at 1,000 ppb in maize intended for human consumption. Recently, the European Commission established a new safety limit for fumonisin in foods of 500 ppb.

    The widespread prevalence of fumonisins and the toxicity associated with the ingestion have resulted in the development of a number of analytical methods for determining the amount of fumonisins present in foods. Among the most common methods utilized are liquid chromatographic (LC) separation with fluorescence detection with a pre-column derivatization with o-phthaldialdheyde (OPA) and LC/MS.

    While yielding reliable results and good limits of detection of total fumonisin B1, B2, and B3 at levels ≥0.8 μg/g (Sydenham et al., 1996), the OPA derivatives of fumonisin decompose very rapidly, thus leading to underestimation of the level of contamination. Moreover, OPA may react with other amino group containing compounds, leading to a good deal of interference. Enzyme-linked immunosorbent assays (ELISA) have also been developed providing a good sensitivity with a limit of detection of 5–10 ng/g. However, they can lead to both over- and underestimation of fumonisin in food, due to a combination of several factors. The LC/MS provides quantitative analyses as well as confirmation of identity of the fumonisins without any need of derivatization. Moreover, the commercial availability of the isotopically labeled D6-FB1 allowed the development of LC/MS methods based on the isotopic dilution approach, which are usually characterized by high recoveries and accuracy. Although some GC/MS methods have been reported several years ago, in a recent review concerning the determination of the fumonisins using chromatographic methods (Shephard, 1998), the author underlined a shifting trend away from the GC/MS technique, which requires multiple sample handling steps (sample hydrolysis, clean-up, and derivatization) before the analysis, towards LC/MS approaches. According to this trend, in the past 5 years, no innovative GC/MS methods were developed, whereas several LC/MS methods were proposed.

    A. Liquid Chromatography/Mass Spectrometry Analysis of Fumonisins

    ESI-MS is an ideal way to detect and measure fumonisins, which are ionic compounds and therefore produce abundant signals in both positive and negative ion modes. In the positive ion mode analysis of the most common fumonisin B1, the protonated ion [M + H]+ = 722 is the base peak in the mass spectrum, and in general, only a low fragmentation is observed. In the negative ion mode, the base peak in the mass spectrum is the [M−H] ion at m/z = 720. Although fumonisins can be observed in both ionization modes, the most common methods make use of the positive ion mode. Compared to the other LC separation techniques, the major advantage of the LC/ESI/MS detection of fumonisins is that no derivatization or special sample preparation are required and, during the ionization, a low fragmentation is produced: for these reasons, it is possible to directly analyze culture materials and food products by simply extracting, filtering, and injecting the sample on the LC column. Moreover, the LC/MS/MS experiments allow to enhance the sensitivity of the method using a MRM determination and to identify the fumonisins by characteristic sequential losses of the TCA side chains, as recently reviewed by Musser, Eppley, & Trucksess (2002).

    The first LC/ESI-MS study about the characterization of fumonisins using an ion trap and triple quadrupole MS was published by Josephs (1996). Multi-stage tandem mass spectrometry (MSn) and LC/tandem mass spectrometry (LC/MS/MS) of FB1 and of 15 synthetically prepared methyl esters of FB1 allowed the investigation of the fragmentation pathways of this class of compounds. The FB1 was analyzed by flow injection analysis, the full scan MS spectrum showing a protonated molecule at m/z 722, with low formation of sodium and potassium adducts and no fragmentation or solvent adduction. The CID fragmentation patterns were acquired by using the triple quadrupole and the ion trap mass analyzers. Several specific fragmentation pathways were proposed to explain the CID spectra obtained on a triple quadrupole instrument and to easily design precursor-scan experiments. In particular, the author hinted that the ability to perform data-dependent experiments reduces the number of LC/MS/MS analysis for the structural identification of unknown fumonisin derivatives and impurities.

    The first LC/ESI-MS/MS validated method for the simultaneous detection of FB1 and FB2 was proposed by Lukacs et al. (1996). In particular, in this work, an isotope dilution approach was performed, using D6-FB1 obtained by adding D3-methionine to a liquid culture medium before inoculation with Fusarium monoliforme. The separation was performed on a C18 column using a gradient elution with a water–methanol–TFA mobile phase by using a positive ion mode for the mass spectrometric detection. Essentially, only the protonated [M + H]+ ions were observed in the ESI-MS experiments, while under collision-induced dissociation of the protonated fumonisins a characteristic spectrum was obtained for both FB1 and FB2, due to the loss of the TCA side-chains. For the qualitative confirmation of FB1 and FB2, a SRM experiment was performed, choosing the specific transition from [M + H]+ to [M + H-2TCA-H2O]+ for both compounds. Since quantitative analysis in the SRM mode was affected by fluctuations in the skimmer pressure in the ESI interface and changes of the collision gas pressure, the quantification of FB1 was performed by SIM experiments, by monitoring the [M + H]+ ions. Since the D6-FB1 standard was 90% pure, a calibration curve in the range of 1–50 ng of FB1 was calculated as a response factor due to the area ratio between FB1 and D6-FB1. The limit of detection as signal to noise ratio 5:1 was 10.4 ppb for FB1.

    An LC/MS method for the FB1 detection was reported by Plattner (1999), in the study already cited in the trichotechene paragraph, concerning the simultaneous determination of DON and FB1 by using an ESI interface (single quadrupole analyzer) and a reversed phase separation on a C18 column. On account of the ionic structure of the FB1, the author suggested that the separation on C18 columns is based on a mixture of reverse phase and ion exchange mechanisms. For this reason, the selectivities of different commercially available columns for B1 are different and a buffered solvent system is required. Actually, the author proposed an acidic elution of FB1, involving a gradient with a water–methanol–acetic acid mobile phase. Under these conditions, a separation of FB1, FB2, FB3, FB4, and the hydrolyzed HFB1 was achieved, as well as of their A series and P series analogs. The method allowed the quantification of the main fumonisins by an external calibration with detection limits below an injected amount of 0.1 ng. For the “B” fumonisins, both the free amino group and the carboxyl groups on the side chains are protonated, so that the response under acidic conditions is similar. Although the “A” series of fumonisins showed a simple ESI+ mass spectrum with both [M + H]+ and [M + Na]+ ions, the LC/MS analysis was complicated by the presence of rearrangement products due to the transfer of the acetyl group from the amino to the adjacent hydroxyl group.

    The LC/ESI-MS detection of FB1 and of its hydrolyzed form HFB1 in processed corn-based food was also achieved by Hartl and Humpf using an isotope dilution approach (Hartl & Humpf, 1999). In particular, using D6-labeled FB1 as internal standard, the detection limits in corn were 5 ppb for FB1 and 8 ppb for HFB1. The method involved a gradient elution using a methanol-water mobile phase with TFA as acidic modifier. According to their ESI-MS/MS experiments, the authors suggested that a MRM analysis was not more specific than a SIM detection: the product ion scans for HFB1 and FB1 showed the presence of characteristic fragments due to the loss of TCA side chains for FB1 and FB2 and the presence of unspecific signals due to the loss of one to four molecules of water for HFB1. Whereas the D6-FB1 showed a response factor close to 1 for FB1, the resulting peak areas for HFB1 were always smaller than expected, causing a response factor for the ratio HFB1/D6-FB1 curve of 1.25. Two factors were suggested as possible explanations: the HFB1 was more strongly discriminated during sample workup as compared to the isotopically labeled D6-FB1 and its ionization in the ESI source was lower than that of D6-FB1, in the presence of matrix components.

    The same group also proposed this method for the determination of N-(carboxymethyl)-fumonisin B1 (NCM-FB1), the degradation product formed by reaction of fumonisin B1 with reducing sugars, in corn products (Seefelder, Hartl, & Humpf, 2001). By using D6-FB1 as internal standard, they obtained a LOD of 10 ppb by monitoring the protonated ion of NCM-FB1. The response factor of the NCM-FB1 was almost six times lower than deuterated FB1, a characteristic attributed to the blockage of the amino group. In model compounds, NCM-FB1 was shown to be present, although at a low extent, only when corn was heated with reducing sugars. With sucrose, only heating at 180°C produced some NCM-FB1.

    Recently, the same group again proposed the use of D6-FB1 as isotopically labeled internal standard for the determination of fumonisins B1 in asparagus and garlic, obtaining good results, a recovery close to 100% and an accurate quantification. Also fumonisin B2 and B3 were detected, but the corresponding labeled standards not being available, they were not quantified (Seefelder, Gossmann, & Humpf, 2002).

    A summary of the main recent LC/MS methods for fumonisin determination is reported in Table 8.

    Table 8. Main recent LC/MS methods for fumonisin determinationThumbnail image of

    VIII. CONCLUSIONS

    1. Top of page
    2. Abstract
    3. I. INTRODUCTION
    4. II. PATULIN
    5. III. AFLATOXINS
    6. IV. OCHRATOXIN A
    7. V. TRICHOTHECENES
    8. VI. ZEARALENONE
    9. VII. FUMONISINS
    10. VIII. CONCLUSIONS
    11. REFERENCES
    12. Biographical Information

    When analyzing complex mixtures, such as food or beverages, the main goal is to keep the sample preparation as simplest as possible, since many steps introduce many uncertainties in the accurate determination of the analytes. This also means that the detector should be very specific.

    Many examples of micotoxin determination in food matrices have been reported which made use of chromatography with mass spectrometric detection. Actually, when using a mass spectrometer as a detector for LC or GC, the first and clear advantage over other detection methods is specificity, which makes LC/MS or GC/MS methods outstanding confirmatory techniques also when other detection methods are available. This is clear the case of patulin, aflatoxins, ochratoxin A, and zearalenone, which are usually extracted, analyzed, and quantified with good accuracy with chromatographic techniques with other detectors than mass spectrometer. For these classes of toxins, MS detection is not strictly necessary, with the exception of complex matrices containing close interferents, unless it is mandatory to confirm the exact identity of the analyte. It is always important also to bear in mind that GC/FID and LC/FLD methods, when they can be used, usually attain sensitivities which are hardly reached only by multiple stage MS detectors, with a clear disadvantage from the point of view of cost/benefit ratio. In contrast, when the toxins are not volatile and/or do not bear suitable chromophores or fluorophores, LC/MS is the unique method to perform specific analyses without requiring any derivatization procedure. For trichothecenes and fumonisins, LC/MS methods are unrivalled for the quantitative and qualitative determination, the latter being particularly important since many similar toxins are present.

    If the specificity of identification is the strength of the MS detectors, the exact quantitative determination is often their Achilles' heel. Almost all the GC/MS methods and LC/MS methods suffer from the fact that the ionization of a given amount of a given molecule is hardly a constant factor, but it is strongly influenced by instrumental parameters, on one side, and from matrix effects, on the other side. The latter parameter is particularly important for mycotoxin determination in food, given the high variability of the matrices. Many approaches can be successfully used to overcome this problem, although the only one which ensures a very high accuracy is the use of isotopically labeled internal standards. However, since labeled standards are not available for many analytes or are very costly, the use of systems able to lower matrix and ion suppressions effects is particularly important. From this point of view, the APPI interface seems to be one of the most promising, although the available data on its use for mycotoxin determinations in food are still quite scarce in the literature.

    The poor ESI response of many toxins is confirmed by many studies presenting the use of APCI as LC interface for analyzing these compounds in foods. Although the response for several toxins is definitely better in the APCI interface, it should always be remembered that there are several limitations that strongly hamper its use for routine determination of trace compounds in complex matrices, such as the necessity of high flows and, therefore, the propensity of the source to be easily contaminated, both affecting sensitivity and reproducibility.

    Given the increasing attention to the problem of mycotoxin determination, it is our opinion that most of the future researches will focus on the ionization problems, since this represent a kind of rate limiting step for the diffusion of the MS technique. When the troubles linked to inconstant ionization and matrix effects will be solved, only MS detectors will allow to simplify more and more the sample preparation and to avoid clean-up procedures, making feasible low-cost high-throughput determination of mycotoxins in many different food matrices.

    ABBREVIATIONS
    ADON

    acetyl-deoxynivalenol

    AFB1

    aflatoxin B1

    AFB2

    aflatoxin B2

    AFG1

    aflatoxin G1

    AFG2

    aflatoxin G2

    AFM1

    aflatoxin M1

    ANIV

    acetyl-nivalenol

    AOAC

    Association of Analytical Communities

    APCI

    atmospheric pressure chemical ionization

    APPI

    atmospheric pressure photeochemical ionization

    BSTFA

    bis(trimethylsilyl)trifluoroacetamide

    CAST

    Council for Agricultural Science and Technology

    CI

    chemical ionization

    CID

    collisionally induced dissociation

    DADON

    diacetyl-deoxynivalenol

    DANIV

    diacetyl-nivalenol

    DAS

    diacetoxyscirpenol

    DDE

    diphasic dialysis extraction

    DON

    deoxynivalenol

    ECD

    electron capture detector

    EFSA

    European Food Safety Authority

    ELISA

    enzyme-linked immunosorbent assay

    EI

    electron impact

    EPC

    electronic pressure control

    ESI

    electrospray ionization

    EU

    European Union

    FB1

    fumonisin B1

    FB2

    fumonisin B2

    FB3

    fumonisin B3

    FB4

    fumonisin B4

    FDA

    Food and Drug Administration

    FID

    flame ionization detector

    FLD

    fluorescence detection

    FUSX

    fusarenone X

    GC

    gas chromatography

    HFB

    heptafluorobutyryl

    HFB1

    hydrolized fumonisin B1

    HMF

    hydroxymethyl-2-furaldehyde

    HP

    hydroxypyridnum

    HPLC

    high pressure liquid chromatography

    HR

    high resolution

    IAC

    immunoaffinity clean-up

    IARC

    International Agency for Research on Cancer

    LC

    liquid chromatography

    LOD

    limit of detection

    LOQ

    limit of quantification

    MAS

    monoacetoxyscirpenol

    MRM

    multiple reaction monitoring

    MS

    mass spectrometry

    MSPD

    matrix solid phase dispersion

    NCM

    N-carboxymethyl

    NEO

    neosolaniol

    OPA

    orto-phthaldialdehyde

    OTA

    ochratoxin A

    OTB

    ochratoxin B

    PB

    particle beam

    PDA

    photodiode array

    PFP

    pentafluoropropionyl

    PLE

    pressurized liquid extraction

    SCT

    scirpentriol

    SD

    standard deviation

    SIDA

    single isotope dilution assay

    SIM

    single ion monitoring

    SPE

    solid phase extraction

    SRM

    selected reaction monitoring

    TCA

    tricarballylic acid

    TF

    trifluoroacetyl

    TFA

    trifluoroacetic acid

    TISP

    turbo ion spray

    TMS

    trimethylsilyl

    ZAL

    zearalanol

    ZAN

    zearalanone

    ZEN

    zearalenone

    ZOL

    zearalenol

    REFERENCES

    1. Top of page
    2. Abstract
    3. I. INTRODUCTION
    4. II. PATULIN
    5. III. AFLATOXINS
    6. IV. OCHRATOXIN A
    7. V. TRICHOTHECENES
    8. VI. ZEARALENONE
    9. VII. FUMONISINS
    10. VIII. CONCLUSIONS
    11. REFERENCES
    12. Biographical Information
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    Biographical Information

    1. Top of page
    2. Abstract
    3. I. INTRODUCTION
    4. II. PATULIN
    5. III. AFLATOXINS
    6. IV. OCHRATOXIN A
    7. V. TRICHOTHECENES
    8. VI. ZEARALENONE
    9. VII. FUMONISINS
    10. VIII. CONCLUSIONS
    11. REFERENCES
    12. Biographical Information

    Stefano Sforza graduated in Chemistry in 1993 at the University of Parma and received his PhD in Chemical Sciences in 1998 from the same University presenting a dissertation on Peptide Nucleic Acids (PNAs), modified oligonucleotides with a pseudopeptidic backbone. The subject of his researches, which he also performed at the Oersted Institut and at the Panum Institut of the University of Copenhagen (Denmark), were the development of new synthetic methods and the study of the recognition properties of chiral PNAs. As a post-doc, he used his experience on peptide-like molecules by developing innovative ESI approaches in order to study the presence of peptides in food matrices, such as ham and cheese. The experience gained by using mass spectrometry applied to complex food matrices was lately applied to the development of new MS techniques for detecting mycotoxins in foods. He is currently Research Associate at the Department of Organic and Industrial Chemistry of the University of Parma, and his research interests cover the synthesis and study of chiral PNAs, determination of peptides and mycotoxins in foods, and development of mass spectrometric methods for studying biomolecules.

    Chiara Dall'Asta graduated in Chemistry at the University of Parma in 2000 and earned her PhD in Food Science and Technology at the same University in 2004 with a dissertation concerning the development of innovative LC-MS and LC-FLD methods for the determination of mycotoxins in food. After her PhD, she spent 6 months at the IFA-Tulln Institute, University of Natural Resources and Applied Life Sciences, Vienna, where she carried out researches on the identification and characterization of masked-mycotoxins by LC-MS/MS. At the moment, she is attending a PostDoctoral Fellowship at the University of Parma, directing her studies to the use of mass spectrometry for the analysis of mycotoxin metabolites.

    Rosangela Marchelli graduated in Chemistry at the University of Pavia in 1965. She was a post-doctoral fellow at the National Research Council of Canada in Halifax (Canada) from 1967 to 1969. Since 1970 she has been working at the University of Parma, where she became Full Professor of Organic Chemistry in 1986. Since 1993 she has been the Dean of the Faculty of Agriculture. Her scientific interests started from the chemistry of natural products, biosynthesis of mold metabolites, covered amino acids and peptides, and lately mycotoxins. A large part of her work was dedicated to the study of the mechanisms of chiral discrimination and to the development of new methods for chiral separations. More recently, she became involved in the study of chiral PNAs, as a mean to perform molecular recognition of DNA. The PNA probes have been successfully applied to the recognition of GMOs and allergens in food. Mass spectrometry has been a constant interest through the different research pathways of her life.