Development of a Multi-Residue Method for

Mycotoxin Analysis in Feeds and Grains

Midwest AOAC, St. Paul, MN, June 10, 2003

Introduction and background

Two basic methods can screen for or quantitate 5 classes of toxins

Multi-residue method needed for convenience and increased sensitivity

The ruggedness of separation and detection of the mycotoxins has been well-established

Easy to use with current post-column system

Uses a reliable and affordable sample cleanup for feeds, grains

2 Methods to Analyze 5 classes of Mycotoxins Method 1:

Trichothecene (DON)

Aflatoxins (incl. M1 & M2)

Zearalenone Ochratoxin A

Method 2: Trichothecene

(DON) Aflatoxins

(incl. M1 & M2) Fumonisins (FB1, FB2, FB3)

Post Column Set Up

PCX 5200 with duplex pump, 2 ml reactor Simplex pump will also be sufficient,

but requires more conversion time PCX Control software Photochemical reactor (Needed for

Aflatoxins Only)

UV detector Fluorescence detector

Flow Paths of Post-Column Systems

Method 1 Method 2

Maximum LevelsAnalyte Species of

FungusConcentration (animal feed)

Concentration (humans)

DON Fusarium 5 ppm, 10 ppm for chickens

1 ppm

Fumonisin Fusarium 5 ppm – horses 2 ppm (FB1+FB2+FB3)

Aflatoxin Aspergilllus 50 ppb – dairy feed

2 ppb for B1

4 ppb for B1+B2+G1+G2

Ochratoxin A

Aspergillus 3 ppb

Zearalenone

Fusarium N/A N/A

Sample Preparation and Cleanup Method 1

Aflatoxin, DON, Zearalenone ACN : H2O (84:16) 100 mL Add 25 g of ground sample C18 : Alumina (1:1) Fill column with 1.5g Filter 6 mL of extract Inject

Note: for increased DON response, evaporate and reconstitute in MeOH

Method 1 ConditionsMobile Phase H2O, ACN, MeOH, Phosphate

Buffer (pH3.3):MeOH (90:10)

HPLC Flow Rate 1.0 mL/min

Column Reverse-phase C18, 4.6x250mm

Column Temperature 40o C

Post-column Reagent Iodine (100mg/L)

Reagent Flow Rate 0.3 mL/min

Reactor Volume 2.0 mL

Reactor Temperature 90o C

Method 1 DetectionMethod 1 - Analytes

Analyte DON Aflatoxins Ochratoxins Zearalenone

Detection Post-column derivatization with Iodine

Detector DAD FLD FLD FLD

Notes = 218 nm

ex = 365 nm

em = 455 nm

ex = 335 nm

em = 455 nm

ex = 275 nm

em = 455 nm

Method 1 – Time Program

Pump Status Time (min)

ON 0

OFF 21

OFF 35

Standard, method 1

Corn, m1

Wheat, m1

Pig Feed, m1

Feed sample, m1

Corn 2, m1

Hay, m1

Conclusions for Method 1 Sample prep works well for most

samples Certain matrices have interferences

with portions of the chromatogram Good sensitivity on the detection.

Can see well below recommended amounts

Method can easily match the allowed limits

Sample Preparation and Cleanup, Method 2 DON, Aflatoxins, Fumonisins

MeOH : H2O (80:20) 100 mL Add 50 g of ground sample C18 only Fill column with 1.5 g Filter 6 mL of extract

Note: No alumina – the Fumonisins stick to the alumina

Method 2 ConditionsMobile Phase H2O, ACN, MeOH, Phosphate

Buffer (pH 3.3): MeOH (90:10)

HPLC Flow Rate 1.0 mL/min

Column Type Reverse-phase C18, 4.6x250 mm

Column Temperature 40° C

Photochemical

Reactor (Needed for Aflatoxins only)

ambient temp

Post-column Reagent OPA, Thiofluor in GA104

Reagent Flow Rate 0.3 mL/min

Reactor Volume 2.0 mL

Reactor Temperature 60° C

Method 2 DetectionMethod 2 - Analytes

Analyte DON Aflatoxins Fumonisins

Detection Photochemical derivatization

Post-column derivatization with OPA/Thiofluor,

Detector DAD FLD FLD

Notes = 218 nm

ex = 365 nm

em = 455 nm

ex = 330 nm

em = 465 nm

Method 2 – Time Program

Pump Status Time (min)

OFF 0

ON 24

ON 40

Standard for Method 2

Corn Sample Expanded, m2

Corn Samples

Feed Samples, m2

Conclusions for Method 2 Reliable extraction for Fumonisins

requires more investigation Some interferences with Aflatoxins

as with Method 1 Can easily detect below allowed

limits

Future work

Improve sample preparation and cleanup for Fumonisins

Examine interferences in some samples

Try reconstituting in MeOH Investigate recoveries

Acknowledgements

Nancy Thiex, South Dakota State University

Beth Tacke, North Dakota State University

Maria Ofitserova, Ph.D., Pickering Laboratories, Inc.

Darsa Siantar, Ph.D., ATTB

Questions and Discussion

Sareeta Nerkar & Maria Ofitserova