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MAN-000060

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Title: 

ORA Lab Manual Vol. IV Section 7 - Mycotoxin Analysis (IV-07) Page 1 of 24

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Sections in This Document

1. Purpose ................................................................................................................................... 1

2. Scope....................................................................................................................................... 2

3. Responsibility ......................................................................................................................... 2

4. Background............................................................................................................................. 3

5. References .............................................................................................................................. 4

6. Procedure................................................................................................................................ 6

6.1. Safety Precautions....................................................................................................... 6

6.2. Sample Preparation ..................................................................................................... 7

6.3. Exercises ...................................................................................................................... 8

6.3.1. Aflatoxins B1, B2, G1, and G2 ......................................................................... 9

6.3.2. Aflatoxin M1 ..................................................................................................10

6.3.3. Ochratoxin A ................................................................................................11

6.3.4. Zearalenone..................................................................................................12

6.3.5. Trichothecenes ............................................................................................12

6.3.6. Patulin...........................................................................................................13

6.3.7. Fumonisins...................................................................................................14

6.3.8. Multi-Mycotoxins Method............................................................................15

7. Glossary/Definitions.............................................................................................................16

8. Records ................................................................................................................................. 16

9. Supporting Documents........................................................................................................16

10. Document History ...................................................................................................................18

11. Change History.......................................................................................................................19

12. Attachments............................................................................................................................19

Attachment A - Answer Key....................................................................................................20

1. Purpose

The mycotoxin analysis training program provides background and training in 

the analysis of mycotoxins in foods and feeds. The Office of Training, 

Education and Development (OTED) provides the New Hire Laboratory 

Analyst Training curriculum (Bingo card) which specifies sections of the ORA 

Lab Manual as part of the New Hire Curriculum. This section describes 

mycotoxin analyses for new hire training and provides suggested on-the-job 

training of selected techniques.
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The Center for Food Safety and Applied Nutrition (CFSAN) and the Center for 

Veterinary Medicine (CVM) are responsible for establishing the inspectional 

and analytical guidelines associated with the FDA mycotoxin programs. The 

two mycotoxin Compliance Programs, 7307.001 Mycotoxins in Domestic and 

Imported Foods and 7371.003 Feed Contaminants Program, are found in the 

Compliance Program Guidance Manual (CPGM).

This training chapter introduces the trainee to regulatory analyses of 

mycotoxins in the FDA, safety precautions, sample preparation, analytical

separation techniques and instrumentation. The trainee should discuss each 

exercise in advance with the trainer. If a laboratory does not perform analyses 

in that area, that portion of the training can be abbreviated or eliminated. 

When the trainee has completed each exercise, the analysis, worksheet 

preparation, and answers to the questions should be discussed thoroughly. 

This provides the analyst the opportunity to demonstrate that they are 

proficient in the technique and instrumentation. 

2. Scope

The incumbent is a regulatory scientist in an FDA/ORA field laboratory and is 

expected to bring full professional competence in their discipline to bear in 

carrying out analyses and interpreting the significance of test results. The 

incumbent is assigned scientific analyses on a wide range of samples 

including those that are difficult, complex, or unusual.

These responsibilities support the FDA and its mission to support public health 

through the regulation of mycotoxins in foods and feeds under the jurisdiction 

of the Federal Food, Drug, and Cosmetic Act (FD&C Act) and related Acts.

3. Responsibility

A. Trainer

1. Coordinates training with Supervisor(s) and Trainee.

2. Works with trainees to ensure completion of all training needed to 

meet their regulatory responsibilities.

B. Trainee

1. Completes required training within specified timeframes.

2. Reports training received and submits documentation for training to 

supervisor.
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A. Supervisor

3. Implements and reviews training records.

C. Quality Management or designee

1. Maintains employee competency records for staff.

4. Background

Mycotoxins are natural poisons produced by fungi as secondary metabolites. 

Foods and animal feeds may become contaminated with mycotoxins because 

of mold growth during harvest or storage. Three genera are responsible for 

most of the mycotoxins with which FDA is concerned: Aspergillus, Penicillium, 

and Fusarium. Of the numerous mycotoxins elaborated by these fungi, FDA is 

actively concerned with aflatoxins, fumonisins, trichothecenes (i.e., DON, HT2, T-2), ochratoxin A, patulin, and zearalenone. The potential for a product to 

contain a naturally incurred mycotoxin depends on whether the product 

contains and supports the growth of a mycotoxin-producing mold species, and 

whether the optimum temperature and humidity are present.

In addition to giving instructions to the FDA field (the laboratory, investigation, 

and compliance branches) on how to accomplish their mycotoxin assignments, 

the FDA mycotoxin compliance programs give introductory information about 

these mycotoxins and the products which are susceptible to contamination by 

them (see Section 5 References A-B). The compliance programs also list the 

analytical methods for each mycotoxin. For more information on the 

occurrence, health impacts and sources of mycotoxins, see Section 5 

References C-K.

An extensive amount of information and guidance about mycotoxins is 

available on the internet. One example, CFSAN’s Bad Bug Book, can be found 

in Section 5 Reference L and provides many details about aflatoxins. There is 

a corresponding FDA Compliance Policy Guide (CPG) for each of the 

mycotoxins that have an established action level. For example, CPG Sec. 

510.150 Apple Juice, Apple Juice Concentrates, and Apple Juice Products – 

Adulteration with Patulin (Section 5 Reference M) establishes the action level 

for patulin in apple juice, apple juice concentrate, and apple juice products. 

CPGs give guidance for recommending legal action, such as detention of 

imports. They specify criteria for recommending action, the wording of the legal 

charge, and which analyses are needed. In addition to regulatory action levels, 

the FDA has set guidance and advisory levels for other mycotoxins in order to 

protect food and feed safety and to guide food and feed industries. These 

additional regulatory limits can be found in the mycotoxin compliance 
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programs or in FDA guidance for industry documents (Section 5 References 

N-O).

5. References 

A. U.S. Food & Drug Administration, Center for Food Safety and Applied 

Nutrition. Compliance Program Guidance Manual. Compliance Program

7307.001 Mycotoxins in Domestic and Import Foods. 

B. U.S. Food & Drug Administration, Center for Veterinary Medicine. 

Compliance Program Guidance Manual. Compliance Program 7371.003 

Feed Contaminants Program. 

C. Bennett, J. W., & Klich, M. (2003). Mycotoxins. Clin. Microbiol. Rev., 16, 

497–516.

D. Marin, S., Ramos, A. J., Cano-Sancho, G., & Sanchis, V. (2013). 

Mycotoxins: occurrence, toxicology, and exposure assessment. Food 

Chem. Toxicol., 60, 218–237.

E. Lee, H. J, & Ryu, D. (2017). Worldwide Occurrence of Mycotoxins in 

Cereals and Cereal-Derived Food Products: Public Health Perspectives 

of Their Co-occurrence. J. Agric. Food Chem., 65, 7034-7051.

F. Fink‐Gremmels, J., (1999). Mycotoxins: their implications for human and 

animal health. Vet. Quart., 21, 115–120.

G. Liu Y., & Wu, F. (2010). Global Burden of Aflatoxin-Induced 

Hepatocellular Carcinoma: A Risk Assessment. Envir. Health 

Perspectives, 118, 818-824.

H. Omotayo, O. P., Omotayo, A. O., Mwanza, M., & Babalola, O. O. 

(2019). Prevalence of Mycotoxins and Their Consequences on Human 

Health. Toxicol. Res., 35, 1–7.

I. Ponce-García, N., Serna-Saldivar, S. O., & Garcia-Lara, S. (2018). 

Fumonisins and their analogues in contaminated corn and its processed 

foods - a review. Food Addit. Contam. Part A, 35, 2183-2203.

J. Munkvold, G. P. (2017). Fusarium Species and Their Associated 

Mycotoxins. Methods Mol. Biol., 1542, 51-106.

K. Perrone, G., & Gallo, A. (2017). Aspergillus Species and Their 

Associated Mycotoxins. Methods Mol. Biol., 1542, 33-49.

L. U.S. Food & Drug Administration, Center for Food Safety and Applied 

Nutrition. Bad Bug Book: Handbook of Foodborne Pathogenic 
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Microorganisms and Natural Toxins. Second Edition. (2012). Retrieve 

from: http://www.fda.gov/media/83271/download 

M. U.S. Food & Drug Administration, Office of Compliance. (2001, October 

22). Compliance Policy Guide. CPG Sec. 510.150 Apple Juice, Apple 

Juice Concentrates, and Apple Juice Products - Adulteration with 

Patulin. Retrieve from: https://www.fda.gov/regulatoryinformation/search-fda-guidance-documents/cpg-sec-510150-applejuice-apple-juice-concentrates-and-apple-juice-products-adulterationpatulin 

N. Guidance for Industry: Fumonisin Levels in Human Foods and Animal 

Feeds (2001). Retrieve from: 

https://www.fda.gov/RegulatoryInformation/Guidances/ucm109231.htm 

O. Guidance for Industry and FDA: Advisory Levels for Deoxynivalenol 

(DON) in Finished Wheat Products for Human Consumption and Grains 

and Grain By-Products used for Animal Feed (2010). Retrieve from: 

https://www.fda.gov/regulatory-information/search-fda-guidancedocuments/guidance-industry-and-fda-advisory-levels-deoxynivalenoldon-finished-wheat-products-human 

P. AOAC Official Methods of Analysis (OMA), Online or Current Edition. 

AOAC International. Retrieve from: http://www.eoma.aoac.org/ 

Q. Stoloff, L., Campbell, A. D., Beckwith, A.C., Nesheim, S., Winbush, J.S., 

Jr., & Fordham, O. M., Jr. (1969). Sample preparation for aflatoxin 

assay: The nature of the problem and approaches to a solution. J. Am. 

Oil Chem.’ Soc., 46, 678-684.

R. Stoloff, L. & Dantzman, J. (1972). Preparation of lot samples of nut 

meats for mycotoxin assay. J. Am. Oil Chem.’ Soc., 49, 264-266.

S. Francis, O.J., Jr. (1979). Sample Preparation of Some Shelled Treenuts 

and Peanuts in a Vertical Cutter-Mixer for Mycotoxin Analysis. J. AOAC 

Int., 62, 1182-1185.

T. Stinson, E. E., Osman, S. F., Huhtanen, C. N., & Bills, D. D. (1978). 

Disappearance of patulin during alcoholic fermentation of apple juice. 

Appl. Environ. Microbiol., 36 (4), 620-622.

U. U.S. Food & Drug Administration, Office of Regulatory Affairs, (current 

edition). Investigations Operations Manual (Chap. 4, Chart 6, Mycotoxin 

Sample Sizes). Retrieve from: 

https://www.fda.gov/media/75243/download 
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V. Vega, V., Young, M., & Todd, S. (2016). Laboratory Information Bulletin: 

Quantitation of Aflatoxin M1 in Bovine Milk by Liquid Chromatography 

with Fluorescence Detection. J. AOAC Int., 99, 174-179.

W. Trucksess, M. W., Giler, J., Young, K., White, D., & Page, S. W. (1999). 

Determination and Survey of Ochratoxin A in Wheat, Barley, and 

Coffee—1997. J. AOAC Int., 82, 85-89.

X. Carter, L., Jr., Young M.B., & Williams A.D. (2011). US FDA Laboratory 

Information Bulletin (LIB) No. 4488 Analysis and Confirmation of 

Zearalenone in Grain Feeds by Liquid Chromatography with 

Fluorescence and Mass Spectrometry Detection. 1-14.

Y. Trucksess, M. W., Ready, D. E., Pender, M. K., Ligmond, C. A., Wood, 

G. E., & Page, S. W. (1996). Determination and Survey of 

Deoxynivalenol in White Flour, Whole Wheat Flour, and Bran. J. AOAC 

Int., 79, 883-887.

Z. Vega, V. A., Young, M., & Dorsey, L. (2015). A Quick Assay for the 

Quantitation of Deoxynivalenol in Grain Samples by Liquid 

Chromatography with UV Detection. J. AOAC Int., 98, 806–809.

AA. FDA Foods Program Compendium of Analytical Laboratory Method 

#C-003 for Mycotoxins: Determination of Mycotoxins in Corn, Peanut 

Butter, and Wheat Flour Using Stable Isotope Dilution Assay (SIDA) 

and Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS). 

BB. Rupp, H.S. and Turnipseed, S.B. (2000). Confirmation of Patulin and 

5-hydroxy-methylfurfural in Apple Juice by Gas Chromatography/Mass 

Spectroscopy. J. AOAC Int., 83: 612-620.

6. Procedure

6.1. Safety Precautions

General laboratory safety precautions concerning toxic substances are 

addressed in the ORA Laboratory Manual (Volume III, Section 2, 

Environmental Health and Safety) and safety considerations regarding 

mycotoxins should be understood before any laboratory work is conducted. 

Aflatoxins are carcinogenic to humans and are highly toxic, especially aflatoxin 

B1. Trichothecenes are potent dermal irritants and orally toxic. Never dispose 

of these or any other mycotoxins by pouring their aqueous solutions down the 

drain or their organic solutions into a waste container. Chemical inactivation of 

all mycotoxins should be performed using a sodium hypochlorite solution of the 
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appropriate strength and volume in accordance with the laboratory's chemical 

hygiene plan for disposal and treatment of spills.

Weighing and transferring mycotoxins in dry form should be avoided. The 

electrostatic nature of several mycotoxins in dry form results in a tendency for 

them to be easily dispersed in the working area and to be attracted to exposed 

skin and clothes. If mycotoxin standards are received in a dry form, they 

should be dissolved in a solvent while in the original container using the label 

statement of weight as a guide, and the concentrations should be determined 

spectrophotometrically. Containers of mycotoxin standard solutions should be 

tightly capped, and their weights should be recorded for future reference 

before wrapping them in foil and storing them in a freezer (see Section 5 

Reference P, Chapter 49, 971.22E). Keeping records of the weights of stored 

solutions will provide a means of determining if the solutions have become 

concentrated during storage. To store the material as a film, the solvent should 

be removed at a moderately elevated temperature under a stream of nitrogen, 

or by rotary evaporation before freezer storage.

6.2. Sample Preparation

The objective of sample preparation in trace analysis is to obtain a sample

of convenient size (usually 1-100 g) whose composition accurately reflects the

composition of the bulk sample from which it was taken (usually 2-25 kg). That 

sample composite needs to be representative of the entire sample collected for 

analysis. The nature of mycotoxin contamination makes this objective more

difficult to attain than for most other types of trace analyses. Because the

molds that produce mycotoxins do not grow uniformly on the substrate,

contamination of the commodity is usually far from uniform (See Section 5 

Reference P, Chapter 49, Method 977.16). However, when food is prepared

from that commodity, the mycotoxin present in perhaps only a small portion of 

the commodity may be spread throughout the entire lot of food. 

Sample preparation can be divided into three steps: sample size reduction, 

particle size reduction, and mixing. Sample size reduction is dividing or 

splitting the sample while maintaining a random distribution between the split 

portions in order to ensure these portions remain representative of the entire 

sample. One of the acceptable methods for sample size reduction is using a 

sample splitter such as a Jones riffle divider/splitter. Particle size reduction can 

be accomplished using a vertical cutter/mixer (VCM), cutting mill, rotor mill, 

meat grinder, or other equivalent equipment. Mixing can be accomplished 

using a VCM, polytron homogenizer, laboratory blender, or other equivalent 

equipment. All three steps are not carried out for all samples and if so, not 

necessarily in the order given. In some cases, the grinding equipment 

combines the particle size reduction with the mixing step. In other cases, the 
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steps must be performed separately because the grinding operation does not 

thoroughly mix the sample composite. Choose the proper equipment for each 

sample in order to obtain a properly ground and mixed sample, called a 

“composite”. Following compositing of regulatory samples, two equally 

representative portions of the composite should be placed in two containers –

one for an “Original” analysis, and one for a “Check” analysis. An additional 

representative portion of composite may also be saved as a “Reserve” portion. 

The remainder of the sample composite is disposed. In the case of a violative 

sample, the remaining original and check portions, as well as any 702(b) 

portion, should be sealed and returned to the Sample Custodian as the 

reserve sample.

The samples themselves may be grouped in two categories: those that are 

moist or relatively high in oil content and yield a paste when ground (e.g., 

peanuts, tree nuts, dates), and those that are dry and yield a powder or dry 

particles when ground (e.g., corn, small grains). For contextual guidance on 

the preparation of nut samples, see Section 5 References Q-S. Dry samples 

present a different set of problems that come from two sources. The kernel or 

grain of a dry commodity is often not the same hardness and density 

throughout, and the mold may grow or produce mycotoxin preferentially on one 

part of the kernel or grain. When the sample is ground, the softer portion of the 

kernel or grain is often ground to a smaller particle size than the harder 

portion. These smaller particles migrate to the bottom of the sample container 

when the sample is handled. Consequently, the sample withdrawn for analysis 

may no longer be representative, and the analytical result may be inaccurate. 

To minimize these problems, one needs to mix the sample thoroughly and 

withdraw the portion for analytical determination as soon as practical after 

mixing. The trainer will further discuss the selection and proper use of the 

sample preparation equipment in the laboratory.

Proper storage of sample composites is critical for mycotoxins analysis to 

ensure sample integrity. Mycotoxin sample composites should not be stored in 

conditions of high temperature or humidity and should be frozen for extended 

storage to prevent mold growth or any additional spoilage. Aspergillus species 

will not grow under refrigeration or freezing, however some Penicillium and 

Fusarium species can grow under refrigeration and just below freezing. 

Sample composites for patulin analysis should always be stored frozen to 

prevent any possible fermentation caused by spoilage as fermentation can 

destroy patulin in juices (see Section 5 Reference T).

6.3. Exercises

The purpose of these exercises is to familiarize the analyst with the equipment 

and procedures used to obtain a representative composite and the 
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methodology used for the isolation, determination, and confirmation of the 

most encountered mycotoxins.

The trainer will instruct each trainee in the proper sample preparation 

procedure for each commodity. The trainer should obtain bulk samples of 2-25 

kg each of whole-kernel corn, shelled peanuts, and wheat or barley. Enough of 

each prepared commodity should be given to each trainee for use in the 

following exercises.

Note: Training on all the various mycotoxin methods is not required if the 

laboratory does not perform those analyses. Even though the official method 

for confirmation of identity of aflatoxin B1 is by chemical derivative, 

confirmation of identity for all mycotoxins is performed by Liquid 

Chromatography-Tandem Mass Spectrometry (LC-MS/MS) for violative 

samples when MS instrumentation is available.

6.3.1. Aflatoxins B1, B2, G1, and G2

Aflatoxins are metabolic products of the molds Aspergillus flavus and 

Aspergillus parasiticus and may occur in food because of mold growth in a 

number of susceptible commodities, including peanuts, corn, Brazil nuts, 

pistachio nuts, pumpkin seeds, cotton seeds, and watermelon seeds. Other 

nuts, grains, and seeds are susceptible but less prone to contamination with 

aflatoxins. Because aflatoxins are known carcinogens to humans and animals, 

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 the aflatoxin is in the edible 

portion of the product, and none is in the shell. For nuts, this is called a 

nutmeat basis. The units are μg/kg or ppb.

Liquid Chromatography (LC-Fluorescence), Immunoaffinity Method:

A. Assignments

1. Prepare standard solutions of aflatoxins B1, B2, G1, and G2 as 

described in AOAC 991.31D (see Section 5 Reference P).

2. Weigh four 25 g portions of an assigned sample composite into 

blender cups with covers. Two of these samples are to be spiked at 

the same level with aflatoxins by the trainer. Analyze all four 

samples as described in AOAC 991.31 (see Section 5 Reference P). 

Identify and calculate the quantities of the aflatoxins found. Be 

prepared to discuss the results and any problems encountered.
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3. Confirm the identity of the aflatoxins B1, B2, G1, and G2 found in one 

of the spiked samples by using LC-MS/MS.

Note: Trainer may use other accepted methods for aflatoxins B1, B2, G1, 

and G2 analysis.

B. Questions

1. What are aflatoxins? How do they affect humans and animals?

2. Why is the preparation of a representative or homogeneous sample 

so critical in this determination?

3. In what order do these four aflatoxins (B1, B2, G1, and G2) show up 

on the chromatogram using this method (reversed-phase 

chromatography)?

4. Why is the dilution of the sample critical before placing the sample 

solution on the immunoaffinity column?

5. Explain the principle of reversed phase HPLC.

6. Describe another way to confirm aflatoxin B1 or G1 found in a 

sample other than by TFA derivative formation.

7. How many grams of ground in-shell pistachios should be weighed to 

obtain 25 g of nutmeat for analysis?

8. What references would be consulted first for information on new or 

improved techniques for the determination of aflatoxins or other 

mycotoxins?

6.3.2. Aflatoxin M1

Aflatoxin M1 is produced by lactating animals consuming aflatoxin 

contaminated feed. Aflatoxin B1 is metabolized into aflatoxin M1. Monitoring 

aflatoxin M1 is an important part of the food safety program because milk is an 

important basic food, particularly for infants and children.

Liquid Chromatography (LC-Fluorescence), Immunoaffinity Method:

A. Assignments

1. Prepare standard solutions of aflatoxin M1 as described in J. AOAC 

Int. 99, 2016, 174-179 (see Section 5 Reference V).

2. Weigh four 10 g portions of an assigned milk sample composite into 

an appropriate container. Two of these samples are to be spiked at 

the same level by the trainer. Analyze all four samples as described 

in J. AOAC Int. 99, 2016, 174-179 (see Section 5 Reference V).
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3. Confirm the identity of the aflatoxins M1 found in one of the spiked 

samples by using LC-MS/MS.

Note: Trainer may use other accepted methods for the analysis of 

aflatoxin M1.

B. Questions

1. What is the acceptable level of aflatoxin M1 in milk?

2. What is the purpose of centrifugation at low temperatures?

3. Why are sodium chloride (NaCl) and magnesium sulfate (MgSO4) 

used in this method?

4. In using the immunoaffinity method are antibodies on the 

immunoaffinity column (IAC) monoclonal or polyclonal?

6.3.3. Ochratoxin A

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 can be 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.

Liquid Chromatography (LC-Fluorescence), Immunoaffinity Method:

A. Assignments

1. Prepare standard solutions of ochratoxin A as described in AOAC 

2000.03 (see Section 5 Reference P) or J. AOAC Int. 82, 1999, 85-

89 (see Section 5 Reference W).

2. Weigh four 25 g portions of an assigned sample composite into 

blender cups with covers. Two of the samples are to be spiked at the 

same level by the trainer. Analyze all four samples by AOAC 

2000.03 or J. AOAC Int. 82, 1999, 85-89 (see Section 5 References 

P or W).

3. Confirm the identity of the ochratoxin A found in one of the spiked 

samples by using LC-MS/MS.

Note: Trainer may use other accepted methods for ochratoxin A 

analysis.

B. Questions
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1. What is an advantage of using an immunoaffinity column method?

2. Why is a buffer solution of pH 7.4 needed for this method?

6.3.4. Zearalenone

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 also found in other 

important crops such as wheat, barley, sorghum, and rye.

Liquid Chromatography (LC-Fluorescence), Immunoaffinity Method:

A. Assignments

1. Prepare standards of zearalenone as described in LIB 4488 (see 

Section 5 Reference X).

2. Weigh four 25 g portions of an assigned sample composite into 

blender cups with covers. Two of these samples are to be spiked at 

the same level by the trainer. Analyze all four samples as described 

in LIB 4488 (see Section 5 Reference X).

3. Confirm the identity of zearalenone found in both spiked samples as 

described in LIB 4488 (see Section 5 Reference X).

Note: Trainer may use other accepted methods for zearalenone 

analysis.

B. Questions

1. What is the method detection level (MDL) and limit of quantitation 

(LOQ) for this method?

2. What other mycotoxins can be separated using this method and 

how?

6.3.5. Trichothecenes

The trichothecene mycotoxins (i.e., DON, HT-2, T-2) 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 (DON/Vomitoxin), LC-UV (SPE or Immunoaffinity Method):

A. Assignments
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1. Prepare standards of deoxynivalenol (DON) as described in J. 

AOAC Int. 79, 1996, 883-888 or J. AOAC Int. 98, 2015, 806-809 

(see Section 5 References Y-Z).

2. Weigh four 25 g portions of an assigned sample composite into 

blender cups with covers or other appropriate containers. Two of 

these samples are to be spiked at the same level by the trainer. 

Analyze all four samples for DON as described in J. AOAC Int. 79, 

1996, 883-888 or J. AOAC Int. 98, 2015, 806-809 (see Section 5 

References Y-Z).

3. Confirm the identity of DON found in one of the spiked samples by 

using LC-MS.

Note: Trainer may use other accepted methods for DON analysis.

B. Questions

1. Can conclusive identification be achieved by equivalent HPLC 

retention times (RT) between a sample peak and the DON standard 

peak alone? 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 SPE cleanup column packing used in 

J. AOAC Int. 79, 1996, 883-888 (see Section 5 References Y)?

3. Why is a combination of isocratic and step gradient conditions used 

in J. AOAC Int. 79, 1996, 883-888 (see Section 5 References Y)?

4. Is DON stable during most processing procedures including baking?

6.3.6. Patulin

Patulin is a mycotoxin that is produced by certain species of Penicillium,

Aspergillus, and Byssochlamys molds that may grow on variety of foods

including fruit, grains, and cheese. Patulin has been found to occur in

several 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. 

Samples of frozen concentrate or bulk concentrate should be diluted either

as per the labeling instructions for dilution or to a Brix value of 11.5° (single 

strength) before analysis. See Section 5 Reference P, Chapter 44, AOAC

932.14C, Solids in Syrup.

Liquid Chromatography (LC-UV):

A. Assignments
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1. Prepare standard solutions of patulin as described in AOAC 

995.10C (see Section 5 Reference P) at the time of analysis.

2. Measure 5 mL of an assigned apple juice sample composite into 

four 20 x 150 mm glass culture tubes or other appropriate 

containers. Two of these samples are to be spiked at the same level 

by the trainer. Analyze all four samples as described in AOAC 

995.10 (see Section 5 Reference P). 

3. Confirm the identity of patulin found in one of the spiked samples by 

GC/MS (see Section 5 Reference BB) or equivalent.

Note: Trainer may use other accepted methods for patulin analysis.

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 instruments can be used for determining the Brix value of a 

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 kinds of products are

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?

6.3.7. Fumonisins

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) 
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and fumonisin B3 (FB3) are the major fumonisins of concern produced in 

nature.

Liquid Chromatography (LC-Fluorescence):

A. Assignments

1. Prepare standard solutions of fumonisin B1, B2, and B3 as described 

in AOAC 995.15 (see Section 5 Reference P).

2. Weigh four 25 g portions of an assigned sample composite into 

blender cups with covers or other appropriate containers. Two of 

these samples are to be spiked at the same level by the trainer. 

Analyze all four samples as described in AOAC 995.15 (see Section 

5 Reference P). 

Note: Trainer may use other accepted methods for the analysis of 

fumonisins B1, B2, and B3.

B. Questions

1. Why does the OPA reaction not work with fumonisins A1 and A2?

2. Why is there no screening procedure for fumonisins A1 and A2?

3. What condition occurs in horses upon ingesting high levels of 

fumonisin contaminated feed?

4. What are the recommended maximum levels of fumonisins in human 

foods and in animal feeds established by FDA?

6.3.8. Multi-Mycotoxins Method

The multi-mycotoxins method uses stable isotope dilution assay (SIDA) and 

liquid chromatography–tandem mass spectrometry (LC-MS/MS) for the 

simultaneous determination of aflatoxins B1, B2, G1, and G2; deoxynivalenol; 

fumonisins B1, B2, and B3; ochratoxin A; HT-2 toxin; T-2 toxin; and 

zearalenone in foods (see Section 5 Reference AA). 

A. Assignments

1. Prepare standard solutions containing aflatoxins B1, B2, G1, G2; 

deoxynivalenol, fumonisins B1, B2, and B3, ochratoxin A, T-2, HT-2, 

zearalenone, and their corresponding isotopically labeled internal 

standards as described in the LC-MS/MS multi-mycotoxins method 

(see Section 5 Reference AA).

2. Weigh four equal portions of an assigned product that has been preprocessed as described in Section 5 Reference AA into 15 mL 

polypropylene centrifuge tubes or other appropriate containers. Two 
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of these samples are to be spiked at the same level with mycotoxins 

by the trainer. Analyze all four samples as described in the LCMS/MS multi-mycotoxins method. Identify and calculate the 

quantities of the compounds found. Be prepared to discuss the 

results and any problems encountered.

B. Questions

1. What is the molecular weight of each native mycotoxin analyzed for 

in this method according to Section 5 Reference AA?

2. Explain how LC-MS/MS definitively confirms the identity of a 

substance and why, even though liquid chromatography on its own 

is non-specific, it becomes specific when paired with MS/MS.

3. Describe how you would analyze a sample with a high level of one 

mycotoxin.

7. Glossary/Definitions

None

8. Records

None

9. Supporting Documents

A. Chapter IV of the FD&C Act. Retrieve from: 

https://www.fda.gov/regulatoryinformation/lawsenforcedbyfda/federalfoo

ddrugandcosmeticactfdcact/default.htm 

B. U.S. Food & Drug Administration, Center for Food Safety and Applied 

Nutrition. Compliance Program Guidance Manual. Compliance Program

7307.001 Mycotoxins in Domestic and Import Foods. Retrieve from:

http://inside.fda.gov:9003/downloads/ProgramsInitiatives/Food/FieldProg

rams/ucm019307.pdf

C. U.S. Food & Drug Administration, Center for Veterinary Medicine. 

Compliance Program Guidance Manual. Compliance Program 7371.003 

Feed Contaminants Program. Retrieve from: 

https://www.fda.gov/media/74766/download 
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D. U.S. Food & Drug Administration, Office of Compliance. (2001, October 

22). Compliance Policy Guide. CPG Sec. 510.150 Apple Juice, Apple 

Juice Concentrates, and Apple Juice Products - Adulteration with 

Patulin. Retrieve from: https://www.fda.gov/regulatoryinformation/search-fda-guidance-documents/cpg-sec-510150-applejuice-apple-juice-concentrates-and-apple-juice-products-adulterationpatulin 

E. U.S. Food & Drug Administration, Office of Compliance. (1980, October 

01). Compliance Policy Guide. CPG Sec. 527.400 Whole Milk, Low fat 

Milk, Skim Milk – Aflatoxin M1. Retrieve from: 

https://www.fda.gov/regulatory-information/search-fda-guidancedocuments/cpg-sec-527400-whole-milk-lowfat-milk-skim-milk-aflatoxinm1 

F. U.S. Food & Drug Administration, Office of Compliance. (1980, October 

01). Compliance Policy Guide. CPG Sec. 555.400 Foods - Adulteration 

with Aflatoxin. Retrieve from: https://www.fda.gov/regulatoryinformation/search-fda-guidance-documents/cpg-sec-555400-foodsadulteration-aflatoxin 

G. U.S. Food & Drug Administration, Office of Compliance. (1983, August 

01). Compliance Policy Guide. CPG Sec. 570.200 Brazil Nuts - 

Adulteration with Aflatoxin. Retrieve from: 

https://www.fda.gov/media/72053/download 

H. U.S. Food & Drug Administration, Office of Compliance. (1980, October 

01). Compliance Policy Guide. CPG Sec. 570.375 Aflatoxin in Peanuts 

and Peanut Products. Retrieve from: 

https://www.fda.gov/media/72073/download 

I. U.S. Food & Drug Administration, Office of Compliance. (1980, October 

01). Compliance Policy Guide. CPG Sec. 570.500 Pistachio Nuts – 

Aflatoxin Adulteration. Retrieve from: 

https://www.fda.gov/media/72084/download 

J. U.S. Food & Drug Administration, Office of Regulatory Affairs, (current 

edition). Investigations Operations Manual (Chap. 4, Chart 6, Mycotoxin 

Sample Sizes). Retrieve from: 

https://www.fda.gov/media/75243/download
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10. Document History

Revision

#

Status*

(D, I, R) Date Author Name and Title Approving Official Name and Title

1.3 R 09/01/2005 LMEB LMEB

1.4 R 02/02/2010 LMEB LMEB

1.5 R 02/06/2012 LMEB LMEB

1.6 R 02/13/2014 LMEB LMEB

02 R 05/27/2020 LMEB LMEB

03 R REFER TO 

QMIS 

MYCOTOXIN HARMONIZATION 

COMMITTEE

DR. SEAN LINDER, ASSOC.

DIRECTOR, ORS

* - D: Draft, I: Initial, R: Revision
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11. Change History

Revision 

# Change

1.3 Table of Contents – 7.6 title change; 7.7 added.

Section 7.2 revised first sentence; 7.4.3 revised last sentence

1.4

NOTE: - revised last sentence

7.4.2 Aflatoxin M1 II. A. - revised

7.4.2 Aflatoxin M1 II. C. – added 4.

7.4.4 A. – added NOTE:

7.4.5 A. – changed Reference 35 to 27; added NOTE:

7.4.7 B. – deleted 1.

7.5 – updated web links

7.6 7.4.2 Aflatoxin M1 – added 4.

7.6 7.4.7 – deleted 1. 

Footer – updated web link

1.5

7.5 – 1. Compliance Program reference 7307.001 title updated; 

3. removed compliance Program 7307.002

7.6 – 7.4.1 6. question and answer revised

1.6 Header – Division of Field Science to Office of Regulatory Science

7.5 15. – removed Division name

02

Revised into new template: 

Created new sections (1-3 & 9), changed formatting and revised contents throughout.

Changes made to all sections, including extensive updating of methods, removal of 

outdated methods and updating of references. 

Added section 6.3.8 Multi-Mycotoxins Method.

03

Section 5. updated link for reference A., updated reference AA to FDA Compendium instead 

of Journal. Section 6.3 updated technology name, and removed questions related to 

surveillance sample size. Section 6.3.8.A #2 removed specific sample weights. Attachment A 

removed related answers to removed questions. Updated Instruction 6.3.8.3 in Attachment A 

– Answer Key.

12. Attachments

List of Attachments

Attachment A - Answer Key ........................................................................................20
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Attachment A - Answer Key

6.3.1. Aflatoxins B1, B2, G1, and G2

1. What are aflatoxins? How do they affect humans and animals? Aflatoxins are 

a group of toxins produced by the molds, Aspergillus flavus and Aspergillus 

parasiticus as metabolic products. The toxins consist primarily of aflatoxin B1, B2, 

G1, and G2. M1 is produced from B1 in cow’s milk. In humans and animals, 

aflatoxins cause liver cancer, suppress the immune system, are mutagenic, and 

decrease the production of milk and eggs in animals. Aflatoxin B1 is the most 

potent carcinogen.

2. Why is the preparation of a representative or homogeneous sample so 

critical in this determination? Both sampling and laboratory preparation of a 

representative/homogeneous sample composite are important to obtain a 

laboratory result that reflects the level of toxin present in the lot of product. Since 

aflatoxins are produced by mold growth on the product, the contamination is not 

evenly distributed but occurs in pockets of high contamination. Sample 

preparation results in a finely ground, well mixed sample composite.

3. In what order do these four aflatoxins (B1, B2, G1, and G2) show up on the 

chromatogram using this method (reverse phase chromatography)? The four 

aflatoxins appear in the following order: G2, G1, B2, and B1.

4. Why is the dilution of the sample critical before placing the sample solution 

on the immunoaffinity column? To reduce the methanol concentration from the 

70% of the extraction solvent to about 23%. Methanol is finally used as an elution 

solvent to remove the aflatoxin from the antibodies in the column. The 

immunoaffinity for aflatoxin is low in high concentrations of methanol.

5. Explain the principle of reversed phase HPLC. Liquid chromatography is based 

on using the competing affinities of compounds between a stationary phase and a 

mobile phase to create separation of the compounds for analysis. Reversedphase liquid chromatography uses a non-polar stationary phase and a polar 

mobile phase, which is the opposite set of conditions from the classical mode of 

normal-phase liquid chromatography. The non-polar stationary phase, which 

usually consists of hydrocarbon chains bonded to an inert material, has a stronger 

affinity for less polar or hydrophobic compounds. Therefore, more polar or 

hydrophilic compounds will elute earlier in reversed phase HPLC. The polar 

mobile phase usually consists of an aqueous blend of water with a miscible, polar 

organic solvent, such as acetonitrile or methanol. The most common reversedphase HPLC columns, C18 columns, use an octadecyl carbon chain (C18)-

bonded silica packing as the stationary phase.

6. Describe another way to confirm aflatoxin B1 or G1 found in a sample other 

than by TFA derivative formation. Confirmation of identity by liquid 

chromatography-mass spectrometry (LC-MS).
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7. How many grams of ground in-shell pistachios should be weighed to obtain 

25 grams of nutmeat for analysis? 50 grams should be weighed. Note: In-shell 

pistachios are 50% nut meat by weight. For different ratios, an equation can be 

set up such as: 0.50X = 25 g, where X = the amount of sample to be weighed.

8. What references would be consulted first for information on new or 

improved techniques for the determination of aflatoxins or other 

mycotoxins? The Journal of the Association of Analytical Chemists, the Journal 

of Agricultural and Food Chemistry, Food Additives and Contaminants (found on 

the internet through Medline), or Science Direct are good sources.

6.3.2 Aflatoxin M1

1. What is the acceptable level of aflatoxin M1 in milk? Aflatoxin M1 level in milk 

samples should be less than 0.5 ppb.

2. What is the purpose of centrifugation at low temperatures? Centrifugation at 

low temperatures allows the sample to completely precipitate thereby producing 

a clean extract. 

3. Why are sodium chloride (NaCl) and magnesium sulfate (MgSO4) used in 

this method? NaCl and MgSO4 are used as drying agents to remove the water 

from the organic layer.

4. In using the immunoaffinity method, are antibodies on the immunoaffinity 

column (IAC) monoclonal or polyclonal? Monoclonal antibodies since they 

bind to a specific antigen (aflatoxin M1).

6.3.3 Ochratoxin A

1. What is an advantage of using immunoaffinity column method? The 

immunoaffinity column uses a very selective antibody that binds only with 

ochratoxin A, which acts as the antigen. Other components of the matrix in the 

sample extract are washed from the column prior to the elution of ochratoxin A, 

therefore eliminating sources of potential interference or contamination.

2. Why is a buffer solution of pH 7.4 needed for this method? The antibody in 

the immunoaffinity column generally works best around a pH of 7. Any changes 

in pH will affect its binding capacity for capturing the antigen.

6.3.4 Zearalenone

1. What is the method detection level (MDL) and limit of quantitation (LOQ) for 

this method? The method detection level (MDL) is about 24 ng/g and the limit of 

quantitation (LOQ) is approximately 50 ng/g (about 2.1 times the MDL).

2. What other mycotoxin can be separated using this method and how? 

Aflatoxins B1, B2, G1, and G2 can also be separated using this method by using 

the same extraction procedure but separate immunoaffinity columns.

6.3.5 Trichothecenes
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1. Can conclusive identification be achieved by equivalent HPLC retention 

times (RT) between a sample peak and the DON standard peak alone? What 

other method or instrument could be used for confirmation of identity of the 

compound found in the sample extract? No, it is possible for other compounds 

extracted from the sample to have the same retention time as DON. Confirmation 

of identity can be proven by comparing the compound in the sample extract with 

DON standard using mass spectrometry.

2. Does DON have affinity to the SPE cleanup column packing used in J. AOAC 

Int. 79, 1996, 883-888 (see Section 5 References Y)? No, DON passes through 

the charcoal/alumina/celite column. Interferences are mostly adsorbed on the 

column packing.

3. Why is a combination of isocratic and step gradient conditions used in J. 

AOAC Int. 79, 1996, 883-888 (see Section 5 References Y)? The isocratic 

condition is used to elute the DON peak. The use of a wash solvent or step 

gradient elution is to elute compounds after the elution of DON that may interfere 

with the next run and then re-equilibrate the column in the isocratic condition.

4. Is DON stable during most processing procedures including baking? Yes

6.3.6 Patulin

1. Is there another way that the extraction could have been carried out? 

Discuss the advantages of the proposed way and the official method. 

Separatory funnels instead of test tubes could be used to carry out the extraction. 

The advantage of this modified procedure is that it is easier to more separate the 

layers of solvents during the extraction without carrying over aqueous phase into 

the ethyl acetate as when using a disposable pipet. The advantage of the official 

method procedure is that it is easier to separate the layers of solvents without any

loss of the ethyl acetate layer.

2. What instruments can be used for determining the Brix value of a 

concentrate of apple juice? A refractometer or a hydrometer is used for 

determining the Brix value of apple juice concentrate.

3. Why is the combined ethyl acetate sample extract washed with 1.5% 

Na2CO3? Sodium carbonate solution removes acidic interferences from the 

sample.

4. What factors affect the stability of patulin standards and patulin sample 

extracts? Heat and pH are factors that can affect patulin stability. Therefore, the 

method calls for diluting the standards and reconstituting the sample extracts with 

pH 4 water.

5. What is the regulatory guidance for recommending legal actions against 

products collected for patulin analysis? The FDA Compliance Policy Guide for 

patulin adulteration of apple juice, apple juice concentrates, and apple juice 

products gives the following criteria to be considered: The sample is analyzed in 

accordance with applicable methods of the AOAC, original and check analysis 
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show patulin at or above 50 ppb in single strength juice, identity of patulin is 

confirmed by LC-MS.

6. If the HPLC hydroxymethylfurfural (HMF) peak is close to the patulin peak, 

how may the separation between the two peaks be increased to obtain 

better resolution? What kinds of products are more likely to have a 

significant HMF peak present in the chromatogram? If using acetonitrile, 

decrease the amount of acetonitrile in the mobile phase (i.e., 5% to 4%). If using 

water as the mobile phase, change to another column. From experience it has 

been found that processed apple juice products such as pasteurized apple juice, 

and especially apple juice concentrate tend to have more HMF. Fresh apple juice 

and sweet apple cider have little or no HMF.

7. Why is it important to use anhydrous sodium sulfate to dry the ethyl acetate 

extract? The main reason is given in AOAC method 995.10 which contains a note 

stating that patulin may be destroyed when wet ethyl acetate extract is evaporated 

to dryness. Anhydrous sodium sulfate removes water.

6.3.7 Fumonisins

1. Why does the OPA reaction not work with fumonisins A1 and A2? Fumonisin 

A1 and A2 are secondary amines.

2. Why is there no screening procedure for fumonisins A1 and A2? Fumonisin 

A1 and A2 are not as toxic as fumonisin B1, B2, and B3.

3. What condition occurs in horses upon ingesting high levels of fumonisin 

contaminated feed? Horses develop a disease that causes a softening of the 

white matter in the brains. This disease is called equine leukoencephalomalacia 

(ELEM).

4. What are the recommended maximum levels of fumonisins in human foods 

and animal feeds established by FDA? The recommended maximum level of 

fumonisins in human foods is from 2-4 ppm based upon the particular corn-based 

product. The recommended maximum level of fumonisins in animal feed is from 1-

100 ppm depending on the animals that the feed is intended for. (See Section 5 

Reference A-B).

6.3.8 Multi-Mycotoxins Method

1. What is the molecular weight of each native mycotoxin analyzed for in this 

method according to Section 5 Reference AA? Aflatoxin B1 = 312.1; Aflatoxin 

B2 = 314.1; Aflatoxin G1 = 328.1; Aflatoxin G2 = 330.1; Deoxynivalenol = 296.1; 

Fumonisin B1 = 721.4; Fumonisin B2 = 705.4; Fumonisin B3 = 705.4; HT-2 toxin = 

424.2; Ochratoxin A = 403.1; T-2 toxin = 466.2; Zearalenone = 318.1

2. Explain how LC-MS/MS definitively confirms the identity of a substance and 

why, even though liquid chromatography on its own is non-specific, it 

becomes specific when paired with MS/MS. Mass spectrometric specificity is 

based on the structure and abundance of ions or fragments. Ion abundance is, in 
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turn, dependent on the structure and stability of the ions formed. Masses should 

match and ion abundances (or abundance ratios) should closely match those of a 

standard or a reference. Although chromatographic retention time is in itself nonspecific, for identification the chromatographic retention time must match that of 

the compound whose mass spectrum is obtained.

3. Describe how you would analyze a sample with a high level of one 

mycotoxin. If the level of the mycotoxin is above the regulatory limit, which 

renders the sample to be potentially violative, a check analysis is needed, and will 

be conducted by a second analyst on the reserved “Check” sample portion. 

Appropriate dilution of the sample extract may be needed to ensure the level of 

the mycotoxin of interest in the sample falls within the method calibration curve 

range. The check analysis should meet all quality assurance requirements 

specified by applicable ISO 17025, ORA Laboratory Manual, and ORS policies 

and procedures.