Midwest AOAC, St. Paul, MN, June 10, 2003 Development of a Multi-Residue Method for Mycotoxin Analysis in Feeds and Grains --Multi-residue mycotoxin separation and detection by HPLC and Post-Column Derivatization --although Aspergillus and generally associated with peanuts these fungi and those that produce otehr toxins are not host selective and so can cross plant species. The situation is complicated by microscopic mold may not be visible to the naked eye also, when infected grains are processed, and visible mold is lost but the toxic metabolites carry over into the finished products. multi-residue analytical screens for toxins in grain and finished goods are a wiser choice than single-family protocols 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 -Current methods either use a separate sample prep and detection method or ELISA for screening (not very sensitive, can lead to false positives) --Pickering has developed a rugged method for the separation and detection of the mycotoxins --A cleanup with immunoaffinity columns has been well-demonstrated by Vicam and ATF on beer, wine and sake --Pickering’s goal was to take this method and adapt a sample prep for grains and feeds -Many feeds, foods can contain several mycotoxins because of mixing of plant species and resultant combination of mycotoxins -Samples can range from wheat to beer, from corn to feeds, from rice to sake -Have several options as to how to prep. the samples. Can use silica clean-up column, or immunoaffinity column Multi-residue method needed for ease of analyzing several mycotoxins in one run and increased sensitivity Two basic methods can screen for or quantitate 5 classes of toxins The ruggedness of separation and detection of the mycotoxins has been well-established Produce a reliable and affordable sample cleanup for feeds, grains Easy to use with current post-column system Many different types of samples can be run on the same method

2 Methods to Analyze 5 classes of Mycotoxins Trichothecene (DON) Aflatoxins (incl. M1 & M2) Zearalenone Ochratoxin A Method 2: Trichothecene (DON) Aflatoxins (incl. M1 & M2) Fumonisins (FB1, FB2, FB3) –alumina eats ochratoxin and fumonisins (not 100%)

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 -- Duplex system with 2 OPA panels is ideal so there is no contamination between the Iodine reagent and the OPA reagent -However, simplex system will work fine, but requires excessive flushing to remove Iodine from panel for OPA method and vice-versa --Also, an Aflatoxin system should work fine. This contains a 1.4 ml reactor. The smaller reactor should work with no problem. Most likely the 70°C listed in the literature will not be sufficient, however 90°C should probably work well. -- PCX Control software connected to PCX via serial cable to facilitate pump control

Flow Paths of Post-Column Systems Method 1 Method 2

Concentration (animal feed) Concentration (humans) Maximum Levels Analyte Species of Fungus Concentration (animal feed) Concentration (humans) DON Fusarium 5 ppm, 10 ppm for chickens 1 ppm Fumonisin 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 N/A --Can easily detect these values with post-column method --Took lowest values listed on FDA’s Guidance for Industry “Fumonisin Levels in Human Foods and Animal Feeds”, November 2001 --Took lowest values listed on USDA (GIPSA)’s Fact Sheet on Vomitoxin in wheat, October 1996 --Took lowest Aflatoxin values for humans from EC’s Official Journal of the European Communities “Commission Regulation (EC) No 1525/98, July 1998 --Took lowest Ochratoxin A values for humans from EC’s Official Journal of the European Communities “Commission Regulation (EC) No 472/2002, March 2002 --N/A values for Zearalenone are per Darsa Siantar, ATTB, no established limits

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 -- Ochratoxin A eaten up by alumina, but immunoaffinity column works well. -- Take chromatograms of Darsa’s samples with Vicam’s column -- Make a solution of 84:16 ACN:Water -- Add ACN/H2O to 25 g of sample and shake for one hour to extract mycotoxins -- For fatty samples (like feeds) freeze the sample so that any fats coagulate. --Centrifuge to separate fat --Pass the extract through the column --Then inject --After passing the extract through the C18/Alumina column, evaporate and reconstitute in MeOH --Evaporate and reconstitute in MeOH --This will result in a larger response from DON --But does not decrease response for other mycotoxins --The MeOH is not strong enough to wash mycotoxins from Al/C18 column with the same recoveries as ACN/H2O (SEE RECOVERY NOTES ) --If it is know that there is no DON in the sample, this step can be skipped

Method 1 Conditions Mobile 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 Detection

Standard, method 1 Note that the pump is turned off at 30 minutes, but drop in absorbance takes time before you notice it on the detector DON is collected using the UV detector, and is on a different channel

Corn, m1 -- Large B1 goes off scale on spiked sample -- Only spikes reconstituted in MeOH. Natural samples not reconstituted. SAMPLE 2

Wheat, m1 SAMPLE 6

Pig Feed, m1 SAMPLE 1

Feed sample, m1 SAMPLE 4 (Range land cake)

Corn 2, m1 SAMPLE 8

Hay, m1 ** low density sample = light weight sample, used 4x more extraction solution. Sample was not concentrated after cleanup (prior to injection) SAMPLE 9

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 -- ACN has low response for fumonisin and DON -- Reconstituting in straight MeOH could help increase DON and fumonisin response, but hasn’t been tried yet --to increase the response even more, reconstitute in a lower volume

Method 2 Conditions Mobile 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 --Phosphate buffer: NaOH (0.025 M) in phosphoric acid, pH 3.3

Method 2 Detection

Standard for Method 2 -Note increase in background – due to OPA -Separation has been optimized so that Fumonisins elute after the baseline has stabilized

Corn Sample Expanded, m2 SAMPLE 2

Corn Samples SAMPLE 8 AND 5 --Corn contaminate with FB 3 --Sample spiked with FB1 and FB2, but not FB3

Feed Samples, m2 --Pig feed labeled as simply feeds – one is pig, one is “feed” --Bottom chromatogram is pig feed SAMPLE 3 (on top) SAMPLE 1 (on bottom)

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 - Fumonisin extraction can always be achieved using immunoaffinity columns, or with strong anion exchange (SAX) and C18

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