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Method Abstract 208

MA 208 / MULTI-RESIDUE MYCOTOXIN ANALYSIS OF DRY DISTILLERS GRAINS

SINGLE RUN ANALYSIS OF AFLATOXINS , OCHRATOXIN A, ZEARALENONE AND FUMONISINS BY HPLC AND POST-COLUMN DERIVATIZATION

Distillers grains (DG) are the still residues after the ethanol has been collected. Approximately 90 % of US production is used in domestic animal feed. Any Mycotoxins present in the fresh corn can be concentrated by a factor of three. Contamination can also occur during storage. This raises concern about the potential animal and human health hazards from the use of Mycotoxin contaminated distillers grains. Corn entering the ethanol processing plant as well as distillers grains should be routinely tested for Mycotoxin contaminations to ensure compliance with guidelines set by FDA.

We present a single screen method to cover 4 families of toxins that could be present in dry distillers grains (DDG).

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 OchratoxinA 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 and combined with eluant from AOZ column. 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
 
 

chart-m-208a
 

HPLC PROGRAM

MYCOTOXIN

SPIKE CONC, ppb

NATURAL CONTAMINATION LEVEL, ppb

RECOVERIES %

RSD N=4, %

Aflatoxin B1

10.0

 

65

7.6

Aflatoxin B2

3.4

 

79

6.3

Aflatoxin G1

10.2

 

75

9.4

Aflatoxin G2

4.4

 

82

9.1

Ochratoxin A

203

 

89

7.1
Zearalenone 1057 231 75 8.8
Fumonisin B1 1042 801 109 5.8
Fumonisin B2 1379 223 104 6.8

REFERENCES:
Ofitserova, M., Nerkar, S., Pickering, M., Torma, L., Thex, 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-208b

5-POINT CALIBRATION CURVES 

MYCOTOXIN

CONCENTRATION RANGE

CORRELATION

Aflatoxin B1

0.23 – 113.1 ppb

0.99926

Aflatoxin B2

0.2 – 39.7 ppb

0.99966

Aflatoxin G1

0.5 – 58.2 ppb

0.99933

Aflatoxin G2

0.2 – 24.7 ppb

0.99941

Ochratoxin A

9.2 – 1155 ppb

0.99926

Zearalenone 0.024 – 12.01 ppm 0.99908
Fumonisin FB1 0.024 – 11.84 ppm 0.99987
Fumonisin FB2 0.031 – 7.84 ppm 0.99993