Aflatoxins are metabolic products of the molds, Apergillus flavus and Apergillus parasiticus, and may occur in food as a result of mold growth in a number of susceptible commodities, including peanuts, corn, Brazil nuts, pistachio nuts, pumpkin seeds, and watermelon seeds. Other nuts, grains, and seeds are susceptible but less prone to contamination with aflatoxins. Because aflatoxins are known carcinogens to laboratory animals and presumably man, the presence of aflatoxins in foods should be restricted to the minimum levels practically attainable using processing techniques.

Results for aflatoxins found in in-shell nuts and seeds are calculated and reported on an edible basis, assuming that all of the aflatoxin is in the edible portion of the product and none is in the shell. For nuts, it is called a nutmeat basis. The units are µg/kg or ppb.

I. Thin Layer Chromatography

A.  Assignments 

1. Prepare standard solutions of aflatoxins B1, B2, G1, and G2 for thin layer chromatography (TLC) as described in AOAC 971.22. 
2. Weigh four 50 g portions of the shelled peanut sample prepared earlier into 500 mL glass-stoppered Erlenmeyer flasks. Two of these samples are to be spiked with aflatoxins added at the same level by the trainer, and two will be analyzed as blanks. Analyze all four samples as described in AOAC 968.22. Identify and calculate the quantities of the aflatoxins found. Be prepared to discuss the results and any problems encountered. Reserve a second 50 mL portion of the extract from one of the blank samples. Evaporate the solvent over a steam bath and determine the volume of fat in the sample. Retain the final extracts for use in the next step. 
3. Confirm aflatoxins found in the samples (step 2) by derivative formation as described in AOAC 975.37. Obtain and read Section 7.5 References 24-29.

B. Questions 

1. What are aflatoxins? How do they affect humans and animals? 
2. Why is the preparation of a homogeneous sample so critical in this determination? 
3. How do the names of these four aflatoxins (B1, B2, G1, and G2) correlate with their appearance and chromatographic pattern on a TLC plate? 
4. Several AOAC and Laboratory Information Bulletins (LIB) methods are used for the determination of aflatoxins in a number of commodities. Given the constraints on an analytical chemist working for a regulatory agency, which of the methods would one select to analyze a sample of peanuts for aflatoxins? For corn or for animal feed? Why? 
5. What is the derivative that is prepared in the confirmation method in AOAC 975.37? What is the reaction? Which of these four aflatoxins can be derivatized using trifluoroacetic acid (TFA)? Why? How does this method differ from the derivative formed in AOAC 970.47? Which procedure would one use? Why? 
6. Two samples of shelled filberts are received, one sample of in-shell pecans, and a sample of shelled walnuts. The samples are to be analyzed as described in AOAC 968.22. What details peculiar to each of these samples requires attention in order to perform an accurate analysis?  
7. What reference would be consulted first for information on new or improved techniques for the determination of aflatoxins? For other mycotoxins?

II. Liquid Chromatography

A.  Assignments

1. Prepare standard solutions of aflatoxins B1, B2, G1, and G2 for high pressure liquid chromatography (HPLC) as described in AOAC 991.31D (g). 
2. Weigh four 25 g portions of prepared shelled peanut in blender with cover. Two of these samples are to be spiked with aflatoxins added at the same level by the trainer, and two will be analyzed as blanks. Analyze all four samples as described in 991.31. Identify and calculate the quantities of the aflatoxins found. Be prepared to discuss the results and any problems encountered. Refer to Volume IV, Mycotoxin Analysis, Section 7.4.1 Part I-Thin Layer Chromatography, and No.3 (above) for confirmation of aflatoxins.

B. Questions

1. In what order do these four aflatoxins (B1, B2, G2, and G2) show up on the chromatogram using this method (reverse phase chromatography)?  
2. Why is the dilution of the sample critical before placing the sample solution on the immunoaffinity column?
3. AOAC method 991.31 is for corn, raw peanuts, and peanut butter. Describe the methodology used for other commodities.
4. For AIAC method 991.31, what are levels of quantitation (LOQ) for aflatoxins B1, B2, G1, and G2? How does this compare with the LOQs using thin layer chromatography?
5. Explain the principles of reverse phase HPLC. 
6. Describe another way to confirm aflatoxin B1 or G1 found in a sample by TFA derivative formation, other than by the TLC method specified in I. A No. 3 above.
7. How many grams of ground in-shell pistachios should be weighed to obtain 25 g of nutmeat for analysis?
8. Does the method found in Section 7.5 Reference 30 always give accurate results? Discuss.
9. (If not doing the TLC unit) Answer questions 1, 2, 4, 5, and 7 under Volume IV, Mycotoxin Analysis, Section 7.4.1, I. Thin Layer Chromatography, above.

Aflatoxin M1 is produced by lactating animals consuming aflatoxin contaminated feed. Aflatoxin B1 is metabolized into aflatoxin M1.

I.  Thin Layer Chromatography

A. Assignments 

1. Prepare aflatoxin M1, standard solution as described in 980.21B (f). 
2. Transfer four 50 mL aliquots of a milk sample obtained from the trainer into 250 mL Erlenmeyer flasks. Two of these samples are to be spiked by the trainer. Analyze all four samples as described in 980.21A-E. 
3. Confirm the identity of the aflatoxin M1, in one of the spiked samples as described in 980.21E.

B. Questions

What is the minimum detectable quantity (MDQ) of aflatoxin M1, by this method? How does this compare with the MDQ for the other aflatoxins? 

1. Is there a regulatory guideline for aflatoxin M1, in milk? If so, what is it? 

II. Liquid Chromatography

A. Assignments

1. Prepare aflatoxin M1, standard solution as described in AOAC 986.16B (e).
2. Transfer four 20 mL aliquots of a milk sample obtained from the trainer into graduated cylinders containing 20 mL hot (ca 80 °C) water. Two of these samples are to be spiked by the trainer. Analyze all four samples as described in AOAC 986.16A-E.

B. Questions

1. What reaction occurs when aflatoxin M1 is derivatized using trifloroacetic acid (TFA)?
2. What is the purpose for using the C-18 cartridge and silica gel column in this method?
3. What is the principle of the procedure found in Section 7.5 References 31, and what are the advantages of using this method? Would this method, alone, be used for analyzing a violative sample?

Ochratoxin A is a naturally occurring nephrotoxic fungal metabolite produced by certain species of the genera Aspergillus and Penicillium. It is mainly a contaminant of cereals (corn, barley, wheat, and oats), and has been found in edible animal tissues as well as in human sera and milk. It has also been found in raisins, currants, and green coffee. Studies indicate that this toxin is carcinogenic in mice and rats. It is destroyed during the processing and cooking of food, therefore the implication of health risk to human health and safety is considered less than that of aflatoxins.

I. SPE Method, HPLC

A. Assignments

1. Prepare ochratoxin A standard stock solution of ca 24 µg/mL in toluene-acetic acid (99+1) as described in the European Committee for Standardization method (see Section 7.5 References 32-33), a modification of AOAC 991.44C(f), determining the concentration spectrophotometrically. Dilute the stock solution to obtain a working standard solution of 4 µg/ml. Alternately, prepare 5 mL of a working standard solution of 0.4 µg/mL in toluene-acetic acid (99+1). This will result in HPLC injection standards of 1/10 the concentration described in the method. These injection standards will be closer in concentration to those of the immunoaffinity method.
2. Weigh four 50 g portions of wheat or barley into blenders with covers. Two of these samples are to be spiked with added ochratoxin A by the trainer. Analyze all four samples as described in (see Section 7.5 References 32).   
3. Confirm the identity of the ochratoxin A found in one of the spiked samples as described in AOAC 991.44J or reference (see Section 7.5 References 32, 34). 

B. Questions 

1. The principle of separation of aflatoxins on silica gel is adsorption chromatography. What is the principle of separation of ochratoxin A in AOAC 991.44? 
2. Using standard chemical structure notation, describe the reaction of ochratoxin A with BF3 in methanol. 
3. If ochratoxins are found in a sample, what other mycotoxins  might be found? 

II. Immunoaffinity Method, HPLC

A. Assignments

1. Prepare standard ochratoxin A to concentrations in (see Section 7.5 References 34). In this method, 5 mg of standard is weighed.
2. Weigh four 25 g portions of wheat or barley in blenders with covers. Two of the samples are to be spiked with added ochratoxin A by the trainer. Analyze all four samples by Section 7.5 References 34. 
3. Confirm the identity of the ochratoxin A found in one of the spiked samples as described in the method.   

B. Questions

1. What are the advantages of using the immunoaffinity method over the SPE method?
2. Why is a buffer solution of pH 7.4  needed for this method?

Zearalenone is an estrogenic mycotoxin produced by fungus Fusarium graminearum. Zearalenone can interfere with conception, ovulation, implantation, fetal development, and the viability of newborn animals. This toxin can occur in corn and corn based feeds. It is found also in other important crops such as wheat, barley, sorghum, and rye.

A. Assignments 

1. Prepare standard solution of zearalenone as described in AOAC 985.18C (a). 
2. Weigh four 50 g portions into 500 mL glass-stoppered extraction flask. Two of these samples are to be spiked by the trainer. Analyze all four samples as described in AOAC 985.18A-G. 
3. Confirm the identity of zearalenone found in both spiked samples as described in AOAC 985.18H. 

B. Questions 

1. Describe the confirmation step in this method. How reliable is this confirmation method? 
2. If zearalenone is found in a sample of wheat or corn, what other mycotoxins might be found? What other mycotoxins probably would not be found? 

The trichothecene mycotoxins (i.e. T-2, DON) are a group of closely related, secondary metabolites produced by various strains of Fusarium, Trichoderma, Myrothecium, and some other fungi. Some of the most common toxicological effects caused by these toxins are necrosis, diarrhea, and vomiting. These toxins occur in corn, wheat, barley, oats, rice, rye, and other crops.

Deoxynivalenol (DO N, Vomitoxin)

A. Assignments 

1. Prepare fresh deoxynivalenol (DON) stock solution as described in AOAC 986.17 (see Section 7.5 References 35). Follow the journal method (see Section 7.5 References 35) to prepare a more dilute stock of 105 ug per 2100 uL of methanol-water (1+1). Finally prepare a 5 ng DON/uL working standard as per the method.
2. Weigh four 25 g portions of wheat or corn into 250 mL Erlenmeyer flasks or blenders with covers. Two of these samples are to be spiked with added DON by the trainer. Analyze all four samples for DON as described in (see Section 7.5 References 35). 

B. Questions 

1. Is the HPLC retention time (RT) of a chromatographic sample injection peak being equal to the DON standard peak’s RT sufficient to provide conclusive identification? What other method or instrument could be used for confirmation of identity of the compound found in the sample extract?
2. Does DON have affinity to the cleanup column packing?
3. Why is a combination of isocratic and step gradient elution used in the HPLC?
4. Is DON stable during most processing procedures including baking?

Patulin is a mycotoxin that is produced by certain species of Penicillium, Apergillus, and Byssochylamys molds that may grow on variety of foods including fruit, grains, and cheese. Patulin has been found to occur in a number of foods including apple juice, apples, and pears. Patulin contamination is primarily associated with damaged and rotting fruits and fruit juices made from poor quality fruits.  

A. Assignments 

1. Prepare a standard stock solution of patulin for HPLC as described in AOAC 995.10C (e) calculating the patulin concentration as in AOAC 974.18C (d). Prepare patulin standard working solutions as in AOAC 995.10C (f) at the time of analysis. 
2. Measure 5 mL apple juice or diluted concentrate into four 20 x 150 mm glass culture tubes. Two of these samples are to be spiked with patulin added at the same level by the trainer, and two will be analyzed as blanks. Analyze all four samples as described in AOAC 995.10. Identify and calculate the quantities of the patulin found. Be prepared to discuss the results and any problems encountered. 

Note:  As per the CPs, samples of frozen concentrate or bulk concentrate should be diluted either as per recommendation for dilution or to a Brix value of 11.5° (single strength) before analysis. See Section 7.5 References 19, Chapter 44, AOAC  932.14C, Solids in Syrup.

B. Questions

1. Is there another way that the extraction could have been carried out? Discuss the advantages of  the proposed way and the official method.
2. What instrument is used for determining Brix value of a frozen concentrate of apple juice?
3. Why is the combined ethyl acetate sample extract washed with 1.5 % Na2CO3?
4. What factors affect the stability of patulin standards and patulin sample extracts?
5. What is today’s regulatory guidance for recommending legal actions against products collected for patulin analysis?
6. If the HPLC hydroxymethylfurfural (HMF) peak is close to the patulin peak, how may the separation of the two peaks be increased to obtain better resolution? Which kind of products is more likely to have a significant HMF peak present in the chromatogram?
7. Why is it important to use anhydrous sodium sulfate to dry the ethyl acetate extract?

Fumonisins are natural toxins produced by Fusarium moniliforme, and other Fusarium species; these molds are common natural contaminants of corn. Fumonisins have been linked to fatalities in horses and swine. Recent studies have demonstrated the presence of fumonisins in human foods, including corn meal and breakfast cereals. More than ten types of fumonisins have been isolated and characterized. Of these, fumonisin B1 (FB1), fumonisin B2 (FB2) and fumonisin B3 (FB3) are the major fumonisins produced in nature.

A. Assignments

1. Prepare fresh fumonisin B1, B2, and B3 standards by the AOAC 995.15 procedure.
2. Weigh four 50 g portions of ground corn into 250 mL plastic centrifuge bottles. Two of these samples are to be spiked with added fumonisin by the trainer. Analyze by above procedure.

B. Questions

1. Describe the OPA reaction with fumonisin B1, B2 and B3.
2. Why does reaction not work with fumonisins A1 and A2?
3. Why is there no screening procedure for fumonisins A1 and A2?
4. What condition occurs in horses upon ingesting high levels of fumonisin contaminated feed?
5. What are the recommended maximum levels of fumonisins in human foods and in animal feeds established by FDA?

Note on the Preparation of Standard Mycotoxin Solutions for Training:

Mycotoxin standards are toxic, hazardous to handle, expensive, and distributed only in small quantities. The method exercises include the complete methods starting from the preparation of concentrated standard stock solutions. The trainers should use judgment in deciding the starting point for trainees. The beginning point could be pure standards, concentrated stock solutions, or intermediate stock solutions. It may be impractical to train large numbers of analysts starting from pure standards, which are toxic and should be handled by experienced analysts.