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
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
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
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
standards has been
Binmaster level controls
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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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