LC-MS/MS: The New Reference Method for Mycotoxin Analysis

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					Digital Re-print - May | June 2012
  LC-MS/MS: The New Reference Method
         for Mycotoxin Analysis

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   LC-MS/MS:                                                                                        The New
                                                                                                   Reference Method
                                                                                                   for Mycotoxin

by Dr Eva-Maria Binder Chief Scientific Officer, Erber Group, Austria

      he analysis of mycotoxins has               single mycotoxins or mycotoxin classes, thus     Mass spectrometry
      become an issue of global interest,         including a limited number of chemically            The technology of liquid chromatogra-
      in particular because most                  related target analytes only. But as additive    phy-mass spectrometry (LC/MS) opens the
countries already set up regulative limits        and synergistic effects have been observed       perspective of efficient spectrometric assays
or guideline levels for the tolerance             concerning the health hazards posed by           for routine laboratory settings, with high
of such contaminants in agricultur-               mycotoxins, efforts have been increased          sample throughput. This technique, which
al commodities and products.                      to search for multi-toxin methods for the        in many cases utilises multi-mass spectrom-
                                                  simultaneous screening of different classes      eter detectors, can be used to measure a
    Approximately 300 to 400 substances           of mycotoxins.                                   wide range of potential analytes. It has no
are recognised as mycotoxins, comprising              High performance liquid chromatography       molecular mass limitations, a very straightfor-
a broad variety of chemical structures pro-       (HPLC) and gas chromatography (GC) have          ward sample preparation, does not require
duced by various mould species on many            traditionally been the favored choices for the   chemical derivatisation and has, due to the
agricultural commodities and processed food       analyst when sensitive, reliable results are     rugged instrumentation, limited maintenance
and feed. Globalisation of the trade of           required with minimum variability. The major     needs. Therefore, liquid chromatography/
agricultural products contributed significantly   disadvantage of mycotoxin
to the discussion about potential hazards         analysis using GC is based
involved and increased the awareness of           on the necessity of deriva-
mycotoxins. Safety awareness in food and          tisation that can be time-
feed production has also risen due to the         consuming and prone to
simple fact that methods for testing residues     error, so that nowadays
and undesirable substances have become            GC methods are used less
noticeably more sophisticated and available       frequently.
at all points of the supply chain.                    HPLC can be cou-
                                                  pled with a variety of
Modern mycotoxin analysis                         detectors, e.g. spectro-
    The most important target analytes are        photometric (UV-Vis,
aflatoxins, trichothecenes, zearalenone and       diode array) detectors,
its derivatives, fumonisins, ochratoxins, ergot   refractometers (RI), fluo-
alkaloids, and patulin (1). Various mycotox-      rescence (FLD) detec-
ins may occur simultaneously, depending           tors, electrochemical detectors, radioac-        mass spectrometry (LC/MS) and particularly
on environmental and substrate conditions.        tivity detectors and mass spectrometers.         LC coupled to tandem mass spectrometry
Considering this coincident production, it        Particularly the coupling of liquid chro-        (LC/MS/MS) have become very popular in
is very likely, that humans and animals           matography (LC) and mass spectrometry            mycotoxin analysis.
are exposed to mixtures rather than to            (MS) provided a great potential for the              A liquid chromatography/tandem mass
individual compounds. Recently, the natural       analysis of mycotoxins, as the need for          spectrometric method for the determina-
occurrence of masked mycotoxins, where            pre- or post-column sample derivatisation        tion and validation of 39 mycotoxins in
the toxin is conjugated, has been reported,       was eliminated. Thus, no other technique         wheat and maize was used for analys-
requiring even more selective and sensitive       in the area of instrumental analysis of          ing A- and B-type trichothecenes and
detection principles (1,2,3).                     environmental toxins developed so rapidly        their metabolites, zearalenone and deriva-
    So far most analytical methods deal with      during the past 10 years.                        tives, fumonisins, enniatins, ergot alkaloids,

10 | may - June 2012                                                                                         Grain &feed millinG technoloGy
                                                                             BIN LEVELS
orchratoxins, aflatoxin, and moniliformin          cedure, and in par-
(1).                                               ticular the use of
    A multi-mycotoxin method for food and          Mycosep® columns
feed matrices based on liquid chromatog-           proved straightfor-
raphy/electrospray ionization-tandem mass
spectrometry (HPLC/ESI-MS/MS) covered
                                                   ward and efficient
                                                                                   without climbing!
                                                                            smartBob and eBob software
the analysis of 186 fungal and bacterial
metabolites. The method is based on a single       Stable Isotope
extraction step using an acidified acetonitrile/   Dilution Assay
water mixture followed by analysis of the              In order to
diluted crude extract (13).                        overcome matrix
    The development of LC/MS methods for           effects and related
mycotoxin determination is impeded to some         quantification
extent by the chemical diversity of the ana-       problems, external
lytes and compromises that have to be made         matrix calibration
on the conditions of sample preparation (1).       for each com-
    Considering the wide range of polarities
of the analytes the seemingly high selective
                                                   modity tested was
                                                                                                        affordable. reliable. safe.
MS/MS detection could lead incorrectly to          This is extremely                                    Inventory management systems
the perception that matrix interferences           time-consuming                                       and bin level indicators
could be eliminated effectively and quantita-      and proved to
tive results may be obtained without any           be very impracti-
clean-up and with very little chromatographic      cal under routine
separation.                                        conditions, where
    Unfortunately, co-eluting matrix compo-        one is confronted
nents influence the ionization efficiency of       with a variety of
the analyte positively or negatively, impairing    matrices every day.
the repeatability and accuracy of the ana-         As an alternative
lytical method (1). As a consequence, only a       approach, the use            Rotary       Pressure Switch    Vibrating Rod      Capacitance Probe
few approaches describe the successful injec-      of [stable] isotope
tion of crude extracts, and the majority of
publications depict a sample clean-up prior
                                                   labelled internal
                                                   standards has been
                                                                            Binmaster level controls
to liquid chromatography with solid-phase          introduced recently •
extraction (SPE) as the most efficient pro-        (10). These sub-                      © 2012 BinMaster, Lincoln, Nebraska uSa

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   stances are not present in real world samples       The same analyses without considering the                         2 Berthiller, F., Dall’Asta, C., Schuhmacher, R.,
   but have identical properties to the analytes.      internal standard resulted in R2=0.9974 and                         Lemmens, M., Adam, G., Krska, A.R. 2005. Masked
       Internal standards are substances which         a recovery rate of 76 percent +/- 1.9 percent                       mycotoxins: Determination of a deoxynivalenol
   are highly similar to the analytical target sub-    , underlining the successful compensation                           glucoside in artificially and naturally contaminated
   stances, i.e. their molecular structure should      for losses due to sample preparation and                            wheat by liquid chromatography-tandem mass
   be as close as possible to the target analyte,      ion suppression effects by isotope labeled                          spectrometry. J. Agr. Food Chem. 53, 9, pp. 3421-
   while the molecular weight has to be differ-        internal standards (10,11).                                         3425.
   ent. Within the analytical process, internal                                                                          3 Schneweis, I., Meyer, K., Engelhardt, G., Bauer,
   standards are added to both, the calibration        Conclusions                                                         J. 2002. Occurrence of zearalenone-4-�-D-
   solutions and analytical samples, and by                Direct coupling between a liquid phase                          glucopyranoside in wheat. J. Agric. Food Chem. 50
   comparing the peak area ratio of internal           separation technique such as liquid chroma-                         (6), pp. 1736-1738.
   standard and analyte, the concentration of          tography and mass spectrometry has been                           4 Biancardi, A., Gasparini, M., Dall’Asta, C., Marchelli,
   the analyte can be determined.                      recognised as a powerful tool for analysis of                       R. 2005. A rapid multiresidual determination of
       Ideal internal standards are isotope-marked     highly complex mixtures.                                                    type A and type B trichothecenes in wheat
   molecules of a respective target analyte, which         The main advantages                                                        flour by HPLC-ESI-MS. Food Additives and
   are usually prepared via organic synthesis by       include low detection lim-                                                      Contaminants, 22 (3), pp. 251-258
   exchanging some of the hydrogen atoms by            its, the ability to generate                                                     5 Berthiller, F., Schuhmacher, R., Buttinger,
   deuterium, or by exchanging carbon [12C]            structural information, the                                                      G., Krska, R. 2005b. Rapid simultaneous
   atoms by [13C]. Physico-chemical proper-            requirement of minimal sam-                                                       determination of major type A- and
   ties of such substances, and especially their       ple treatment and the pos-                                                         B-trichothecenes as well as zearalenone
   ionization potential is very similar to or nearly   sibility to cover a wide range                                                     in maize by high performance liquid
   the same as of their naturally occurring target     of analytes differing in                                                           chromatography-tandem               mass
                                                       their polarities.                                                                spectrometry. J. Chromatog. A, 1062, 2,
                                                           Depending on                                                              pp. 209-216.
“Direct coupling between                               the applied interface                                                         6 Biselli, S., Hummert, C. 2005.
                                                       technique a wide                                                             Development of a multicomponent method
a liquid phase separation                              range of organic                                                             for Fusarium toxins using LC-MS/MS and its
technique such as liquid                               compounds can be                                                             application during a survey for the content of
                                                       detected and flows                                                           T-2 toxin and deoxynivalenol in various feed
chromatography and mass                                up to 1.5ml/min can                                                         and food samples. Food Add. Contam. 22
                                                       be handled (12).
spectrometry has been                                      Despite their high
                                                                                                                                    (8), pp. 752-760.
                                                                                                                                    7 Tanaka, H., Takino, M., Sugita-Konishi,
recognised as a powerful                               sensitivity and selectivity, LC/                                             Y., Tanaka, T. 2006. Development
                                                       MS/MS instruments are limited to                                            of a liquid chromatography/time-of-
tool for analysis of highly                            some extent due to matrix-induced                                      flight mass spectrometric method for the
complex mixtures”                                      differences in ionization efficiencies and signal                   simultaneous determination of trichothecenes,
                                                       intensities between calibrants and analytes.                        zearalenone and aflatoxins in foodstuffs.
                                                       Ion suppression/enhancement due to matrix                           Rapid Commun. Mass Spectrom. 20 (9), pp.
   analytes, but because of their higher molecular     compounds entering the mass spectrometer                            1422-1428.
   weight (due to the incorporated isotopes) dis-      together with the analytes limit also rug-                        8 Milanez, T.V., Valente-Soares, L.M. 2006.
   tinction between internal standard and target       gedness and accuracy and pose a potential                           Gas chromatography - Mass spectrometry
   analyte is possible.                                source of systematic errors.                                        determination of trichothecene mycotoxins in
       Variations during sample preparation and            Stable isotope labelled internal stand-                         commercial corn harvested in the State of São
   clean-up as well as during ionization are           ards have been proven to overcome these                             Paulo, Brazil. Journal of the Brazilian Chemical
   compensated so that methods with espe-              problems as well as to compensate also                              Society, 17 (2), pp. 412-416.
   cially high analytical accuracy and precision       for fluctuations in sample preparation, e.g.                      9 Klötzel, M., Gutsche, B., Lauber, U., Humpf,
   can be developed. Optimally, these isotope          extraction and clean-up. Numerous LC/MS/                            H.-U. 2005. Determination of 12 Type
   labeled analogues must have a large enough          MS methods for the determination of myco-                           A and B Trichothecenes in Cereals by Liquid
   mass difference to nullify the effect of natural    toxins have been developed and published                            Chromatography- Electrospray Ionization Tandem
   abundance heavy isotopes in the analyte.            in recent years, however so far only a few                          Mass Spectrometry. J. Chromatog. 53, 8904-
   This mass difference will depend generally          were based on stable isotope labeled ana-                           8910.
   on the molecular weight of the analyte itself,      lytes, mainly due to their limited availability                   10 Häubl, G., Berthiller, F., Krska, R., Schuhmacher,
   in case of molecules with a molecular weight        and quality.                                                        R. 2005. Sitability of a 13C isotope labeled
   range of 200 to 500, a minimum of three                 Only recently calibrants of thoroughly                          internal standard for the determination of the
   extra mass units might be required.                 [13C]-labeled mycotoxins have been intro-                           mycotoxin Deoxynivalenol by LC-MS/MS without
       Isotope labelled standards supplied by          duced thus opening a broad field of applica-                        clean-up. Anal. Bioanal. Chem. 384 (3), pp.
   Biopure are fully labelled thus providing           tions and improvement in mycotoxin analy-                           692-696.
   an optimum mass unit difference between             sis. Thus in particular the development of                        11 Häubl, G., Berthiller, F., Rechthaler, J., Jaunecker,
   labeled standard and target analyte. For            unified multi-toxin methods being suitable                          G., Binder, E.M., Krska, R., Schuhmacher, R. 2006.
   example, the [13C15]-DON standard, which            for the determination of many types of                              Characterisation and application of isotope-
   is available as liquid calibrant (25mgl-1) was      analyte/matrix combinations poses a great                           substituted (13C15)-deoxynivalenol (DON) as an
   thoroughly characterised by Häubl et al.(9)         challenge for the future.                                           internal standard for the determination of DON.
   with regard to purity and isotope distribu-                                                                             Food Add. Contam. In print.
   tion and substitution, the latter being close       References:                                                       12 Sakairi, M., Kato, Y. 1998. Multi-atmospheric pressure
   to 99 percent. Fortification experiments            1 Sulyok, M., Berthiller, F., Krska., R., Schuhmacher, R. 2006.     ionization interface for liquid chromatography-mass
   with maize proved the excellent suitability of        Development and validation of a liquid chromatography/            spectrometry. J. Chromatography A, 794, 391-406.
   [13C15]-DON as internal standard indicating           tandem mass spectrometric method for the determination          13 Vishwanath, V., Sulyhok, M., Labuda, R., Bicker, W.,
   a correlation coefficient (R2) of 0.9977 and a        of 39 mycotoxins in wheat and maize. Rapid Commun.                Krska, R. (2009) Anal. Bioanal. Chem. 395:1355–
   recovery rate of 101 percent +/- 2.4 percent.         Mass Spectrom. 20, 2649-2659.                                     1372.

  12 | may - June 2012                                                                                                                Grain   &feed millinG technoloGy
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Grain&feed millinG technoloGy                                                                may - June 2012 | 13
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Description: The analysis of mycotoxins has become an issue of global interest, in particular because most countries already set up regulative limits or guideline levels for the tolerance of such contaminants in agricultural commodities and products.