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Method Abstract 203.1

MA 203.1 / MULTI-RESIDUE MYCOTOXIN ANALYSIS

Single Run Analysis of Deox ynivalenol , Aflatoxins , Ochratoxin A, Zearalenone and Fumonisin by HPLC and Post-column Derivatization

Maria Ofitserova, PhD, Sareeta Nerkar, PhD, Michael Pickering, PhD

Although Aspergillus (Aflatoxins, Ochratoxin A) are generally associated with peanuts and Fusarium (Deoxynivalenol, Zearalenone) with wheat, these fungi and those that produce other toxins are not host-selective and so can cross plant species. This situation is complicated by the fact that the microscopic mold may not be visible to the naked eye. Also, when infected grains are processed, any visible mold is lost but the toxic metabolites carry over into the finished products. Thus, multi-residue analytical screens for toxins in grain and finished goods are a wiser choice than single-family protocols. We present a single screen to cover five families of toxins. This method is suitable for analyzing beverages, grains and feeds.

Sample Extraction and Clean-up

25 g of finely grounded sample is extracted with 150 mL of water/Methanol mixture (30/70). 20 mL of filtered extract is diluted with 70 mL of Phosphate Buffered Saline (PBS) solution . Aflatoxins, Zearealenone and Ochratoxin A are isolated using AOZ Immunoaffinity column (Vicam, USA) according to the procedure from the column manufacture. Toxins are eluted with 2 x 2 mL of Methanol. Fumonisins are isolated using FumoniTest Immunoaffinity column (Vicam, USA) according to the procedure from the column manufacture. Toxins are eluted with 2 x 1.5 mL of Methanol.

To isolate DON 3 mL of filtered extract is mixed with 6 mL of Acetonitrile and cleaned with MycoSep 277 column (Romer Labs, USA) according to the manufacture’s instructions. The cleaned solution is filtered and 5.5 mL of it is combined with eluants from AOZ and FumoniTest columns. The solution is evaporated to 0.5 mL and final volume is adjusted to 1 mL with Methanol.
 

METHOD

Analytical Conditions

Column: MYCOTOX™ reversed-phase C18, 4.6×250 mm Catalog No. 1612124
Temperature: 40 ºC
Flow Rate: 1 mL/min
Mobile Phase: Sodium Phosphate buffer, pH 3.5
                     Catalog No 1700-1108/MeOH/ACN

Post-column Conditions

Post-column System: Pinnacle PCX
Reactor Volume: 1.4 mL
Temperature: 60 ˚C
Reagent: OPA, Thiofluor, Brij 35® in OD104
Photochemical Reactor: UVE™

Detection:

Fluorescence
Aflatoxins (photochemical derivatization)
λex = 365 nm; λem = 455 nm

Fumonisins (post-column derivatization with OPA)
λex = 330 nm; λem = 465 nm

Ochratoxin A
λex = 335 nm; λem = 455 nm

Zearalenone
λex = 275 nm; λem = 455 nm

UV/Vis
Deoxynivalenol
λ=218 nm
  
HPLC PROGRAM

TIME (Min)

1700-1108 ,%

METHANOL, %

ACETONITRILE, %

0.0

85

0

15

5.0

85

0

15

5.1

57

28

15

20.0

57

28

15

23.0

40

60

0

40.0 40 60 0
50.0 20 0 80
60.0 20 0 80

REFERENCES
Ofitserova, M., Nerkar, S., Pickering, M., Torma, L., Thiex, N., Multiresidue Mycotoxin Analysis in Corn Grain by Column High-performance Liquid Chromatography with Post-column Photochemical and Chemical Derivatization: Single-Laboratory Validation., (2009), J AOAC Int., 92, 15-25

chart-m-203a

Chromatogram of a standard solution of mycotoxins. Concentrations of toxins (ng/mL): deoxynivalenol (DON) 930, aflatoxin B1 4.5, aflatoxin B2 1.6, aflatoxin G1 4.7, aflatoxin G2 2, ochratoxin A 92, zearalenone 481, fumonisin B1 474, and fumonisin B2 627.

chart-m-203b

Chromatogram of a corn grain sample naturally contaminated with fumonisins FB1 and FB2 and spiked with DON; aflatoxins B1, B2, G1, and G2; ochratoxin A; and zearalenone. Concentrations of toxins in the sample (ng/g): deoxynivalenol 930, aflatoxin B1 5.0, aflatoxin B2 1.7, aflatoxin G1 5.1, aflatoxin G2 2.2, ochratoxin A 102, zearalenone 529, fumonisin B1 1838, and fumonisin B2 1107.