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