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ORS-Office of Regulatory Science

Document Number:

MAN-000063

Revision #: 03

Revised: 

31 Oct 2023

Title: 

ORA Lab Manual Vol. IV Section 10 - Food Standards and Additives (IV-10)

Page 1 of 85

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Sections in This Document 

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

Scope ....................................................................................................................................... 1

Responsibility............................................................................................................................ 1

Procedure ................................................................................................................................. 2

4.1. Preliminary documents for review.................................................................................. 2

4.2. Food Standards.............................................................................................................. 3

4.2.1. Food Standards Exercises............................................................................... 6

4.3. Food Additives ............................................................................................................... 8

4.3.1. Food Additive Exercises ................................................................................13

4.4. Color Additives.............................................................................................................23

4.4.1. Color Additives Exercises ..............................................................................29

Document History ...................................................................................................................34

Change History.......................................................................................................................34

Attachments............................................................................................................................35

Attachment A - Color Additive Training Samples....................................................................36

Attachment B - Answer Key....................................................................................................37

Attachment C - Quantitative Determination of FD&C Colors (Graichen Method)...................52

Attachment D - Newburger’s Chapter 19................................................................................78

Purpose

This chapter provides preliminary training to new analysts in sample 

preparation, analytical techniques, instrumentation, labeling, and other 

requirements for food standards, food additives, and color additives. The 

exercises in this chapter should be used to supplement in-house training.

Scope

This document applies to trainees and other analysts who analyze FDA 

regulated products for food standards, additives, and colors.

Responsibility

A. It is the responsibility of laboratory management and quality 

management staff to ensure that all analysts are properly trained and 

have the necessary knowledge and skills to analyze FDA-regulated 
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Document Number:

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31 Oct 2023

Title: 

ORA Lab Manual Vol. IV Section 10 - Food Standards and Additives (IV-10)

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products to meet regulatory requirements of the agency, and that all 

training is documented.

B. This chapter is divided into three sections: Food Standards, Food 

Additives, and Color Additives. For each assigned training exercise, the 

trainee is required to read the background information, applicable 

methodology, instrument/equipment SOPs or work instructions, and 

relevant Safety Data Sheets before starting any sample analysis. 

The trainee will complete a worksheet for each assigned exercise. 

C. The trainer will address any questions the trainee has prior to starting 

an exercise. Once the exercise is completed, the trainer will review the 

analysis, worksheet, and exercise questions with the trainee.

Procedure

4.1. Preliminary documents for review

The trainee should review and become familiar with the following documents:

A. Definition of Terms and Explanatory Notes, Official Methods of Analysis 

of AOAC (current edition), Gaithersburg MD.

B. General Provisions and Requirements Applying to Specifications, Tests, 

and Assays of the Food Chemicals Codex (starting with General 

Specifications and Statements), Food Chemicals Codex, (current 

edition) United States Pharmacopeia, Rockville, MD.

C. ORA Laboratory Manual:

1. MAN-000037 Volume II: Methods, Method Verification and 

Validation (ORA-LAB.5.4.5). 

2. MAN-000038 Volume II: Estimation of Measurement Uncertainty 

(ORA-LAB.5.4.6).

3. MAN-000043 Volume II: Ensuring the Quality of Test Results (ORALAB.5.9).

4. MAN-000048 Volume III: Basic Statistics and Data Presentation (III04). 

D. Local SOPs/Work Instructions:

1. Laboratory Safety Procedures.

2. Chemical Management and Hygiene.

3. Hazardous Waste.
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4. Physical Reference Standards and Reference Materials.

E. Labeling:

1. Food, Drug, and Cosmetic Act, Section 201(k), (l), and (m). 

Washington DC: Library of Congress.

2. Code of Federal Regulations, Title 21, Pt. 101 – Food Labeling. 

Washington DC: Office of the Federal Register National Archives 

and Records Administration.

4.2. Food Standards

Planned work in the Food Standards Program area is infrequent. Therefore, 

the laboratory has the option of not completing this training section. However, 

it is recommended that trainee reviews this information to gain a basic 

understanding of food standards and related regulations.

A. Background

1. “Food Standards" is FDA’s common designation for Standards of 

Identity, Standards of Quality, and Standards of Fill of Container 

enacted under Section 401 of the FD&C Act. Section 401 

requires regulations be promulgated “fixing and establishing for 

any food, under its common or usual name so far as practicable, 

a reasonable definition and standard of identity, a reasonable 

standard of quality or reasonable standards of fill of container”. 

The purpose of food standards is to maintain the integrity of food 

products, so consumers get what they reasonably expect. 

2. Food standards were published and incorporated into law to 

prevent or mitigate the harm from economically motived 

adulteration. They are formulated in precise and definite terms, 

so they are defensible in court when challenged. 

3. Economically motivated adulteration is a type of food fraud that 

has existed since man first started trading food products. It 

comprises a wide array of dishonest acts that are designed to 

deceive consumers about the content or true value of a food 

product. 

a. Early forms of economic adulteration included diluting alcohol 

or milk with water, increasing the density of flour by adding 

chalk powder, and substituting or mixing expensive imported 

spices with less expensive spices. 

b. An entirely new level of economic motivated adulteration 

came into existence with the Industrial Revolution. Less 

expensive synthetic ingredients replaced natural ingredients 
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in processed foods. Manufacturers used deceptive packaging 

practices and containers to hide the foods’ quality or quantity. 

c. Economically motived adulteration increased as foods 

became more complex. It became easier for unscrupulous 

manufacturers to add, substitute, or omit ingredients. 

d. The impact of economically motivated adulteration affects 

more than just the economy. Depending on what was added, 

substituted, or removed, it can lead to health issues, and in 

some cases, death. Economically motivated adulteration 

continues to be a growing concern of the food industry, 

consumer advocacy groups and regulatory authorities. 

4. Standards of Identity legally define what a food is. They establish 

the common or usual name and define the nature and essential 

characteristics of a food and the ingredients that must be used or 

may be used. Ingredients not recognized in the standard are not 

permitted.

a. The standards of identity may specify minimum levels of 

valuable constituents and maximum levels for fillers. For 

example, oleomargarine must not have less than 80 percent 

fat as determined by the method specified in the Standard. 

b. They may designate a method of production or formulation if 

it is relevant to the identity of the finished food. 

c. They also describe the labeling requirements. 

d. Foods that have a legal standard of identity are considered 

“standardized” foods. 

5. Not all foods have a Standard of Identity. For example, the Act 

does not require a standard of identity be established for fresh 

and dried fruits and vegetables. 

a. These foods are referred to as “non-standardized foods”. 

b. 21 CFR Part 102 lists some non-standardized foods that have 

no Standard of Identify per se but are subject to specific 

regulatory requirements. 

c. Non-standardized foods without a specific naming 

requirement must be labeled using the common or usual 

name of the food. 

6. Quality Standards establish minimum specifications for 

characteristics such as freedom from defects, tenderness, and 
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color along with objective methods for measuring these 

characteristics. 

a. These standards are crucial for foods packaged in opaque 

containers where such characteristics would not be readily 

apparent to the consumer. For example, green beans that are 

highly fibrous do not have good eating quality. 

b. The quality standard for canned green beans sets a limit for 

fiber and prescribes the method to be followed to determine 

the fiber content. 

c. Section 403(h)(1) of the Act requires any food with an 

established quality standard whose quality falls below that of 

the standard to bear the statement that it falls below the 

quality standard on the label in a prescribed size and type or 

it is considered misbranded.

7. Fill of Container standards define how much product must be in 

the container. This standard is particularly important for foods 

packed in liquid and sealed in opaque containers. 

a. This standard has been promulgated only for certain fruits, 

vegetables, fish, shellfish, and nuts that are canned, packed 

in glass, or packed in semi-rigid containers. 

b. Fill standards vary widely, from those without methods (e.g. 

canned peaches) to those where both method and apparatus 

are specified in detail (e.g. press weight for tuna). 

c. Some standards (e.g. canned applesauce) prescribe 

"sampling and acceptance" procedures, which state the 

number of units to be examined and the acceptable number 

of defective units permitted for a specified size lot. A lot is 

considered to fall below the standard of fill when the number 

of defective units exceeds the acceptable number.

d. Section 403(h)(2) of the Act requires any food with a 

prescribed fill of container standard whose contents fall below 

that standard to bear a statement that it falls below the fill of 

container standard on the label in a prescribed size and type 

or it is considered misbranded.

B. Questions

1. What consumer protection was presumed in the promulgation of 

food standards?

2. Differentiate between misbranding and adulteration.
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3. What are the requirements for the fill of a container?

4. When is the packing liquid included in net weight calculations?

5. Define edible portion.

6. Can the net contents be satisfactory and fill of container fail?

7. What, if any, is the relationship between the vignette and the 

contents?

8. What is the maximum amount (in %) of peanuts allowed in mixed 

nuts?

4.2.1. Food Standards Exercises

This section is intended to acquaint the trainee with food standards, the law 

authorizing such standards, their basis, the procedures, techniques used in 

determining compliance with the standards, and the significance of the results. 

The trainee should carefully read sections 401, 403(g) and (h), and 701(e)(1) 

of the Act and review 21 CFR Parts 130-169 to become familiar with foods that 

have been standardized.

The analyst should be aware of the law in examining any product for 

standards.

NOTE: AOAC methods referenced in the exercises are the updated version of 

the methods listed in the CFR.

NOTE: Training samples may be used for more than one training exercise.

4.2.1.1. Cheese

A. The trainer will provide one or more types of cheese. Perform the 

following tests in duplicate.

1. Determine moisture using AOAC Method 926.08 “Loss on Drying 

(Moisture) in Cheese Method I”.

2. Determine fat content using – AOAC Method 933.05 “Fat in 

Cheese IDF-ISO-AOAC Method, Codex-Adopted –AOAC Method”.

3. Compare results with established standards.

B. References

1. Code of Federal Regulations. Title 21, Pt. 133-Cheeses, and 

Related Cheese Products. Washington DC: Office of the Federal 

Register National Archives and Records Administration. 

2. Official Methods of Analysis of AOAC International, AOAC 

International, (current ed.) Gaithersburg MD.
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3. General Provisions and Requirements Applying to Specifications, 

Tests, and Assays of the Food Chemicals Codex – Analytical 

Sample, Water and Loss on Drying, and Weighing Practices, 

Foods Chemical Codex (FCC), (current ed.). Rockville MD

C. Questions

1. Why is speed essential in sample preparation?

2. Differentiate between a cheese spread and cheese food.

3. List two kinds of cheese in which sorbic acid is permitted and 

include the conditions under which it might be used. List two kinds 

of cheese for which sorbic acid is not permitted.

4. What is a processed cheese?

5. In the AOAC procedure, is the moisture content directly 

determined?

6. In the cheese fat extraction, what other substances may be found?

7. Why is petroleum ether used for the extraction?

4.2.1.2. Jams and Jellies

Jams and jellies are highly susceptible to adulteration for economic gain. An 

economic cheat involves the substitution of a cheap, easily obtainable 

ingredient, such as water, for a more expensive one, such as fruit. Tests have 

been devised to check certain indices or ratios of indices related to product 

composition to determine their compliance with standards.

A. The trainer will provide one or more types of jams or jellies. Make the 

following determinations:

1. Net contents.

2. Soluble solids by refractometry – AOAC Method 932.12 “Solids 

(Soluble) in Fruits and Fruit Products: Refractometer Method”.

3. Compare results with established standards.

B. References

1. Code of Federal Regulations. Title 21, Pt. 150-Fruit Butters, 

Jellies, Preserves, and Related Products. Washington DC: Office 

of the Federal Register National Archives and Records 

Administration.

2. Official Methods of Analysis of AOAC International, AOAC 

International, (current ed.) Gaithersburg MD.
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C. Questions

1. What is jam?

2. What is an imitation jelly?

3. Define “degrees Brix”.

4.2.1.3. Egg Noodles

Egg noodles are also susceptible to adulteration for economic gain.

A. The trainer will provide one or more types of noodles. Make the 

following determinations:

1. Prepare a well-mixed composite using AOAC Method 926.06 

“Macaroni Products Preparation of Samples”.

2. Determine total solids and moisture in duplicate using AOAC 

Method 926.07 “Solids (Total) and Loss on Drying (Moisture) in 

Macaroni Products”.

3. Compare the results to the standards in 21 CFR 139.150 Noodle 

products.

B. References

1. Code of Federal Regulations. Title 21, Pt. 139 - Macaroni and 

Noodle Products. Washington DC: Office of Federal Register 

National Archives and Records Administration. 

2. Official Methods of Analysis of AOAC International, AOAC 

International, (current ed.) Gaithersburg MD

C. Questions

1. Does the label have an ingredient statement? Why?

2. Do all noodle products contain eggs?

3. Compare the two drying methods. Which one is to be used to 

determine compliance with the standard for total solids?

4. What is the difference between macaroni and noodles?

4.3. Food Additives

A. Background

1. Section 201(s) of the FD&C Act defines a food additive as “any 

substance the intended use of which results or may reasonably be 

expected to result, directly or indirectly , in its becoming a 

component or otherwise affecting the characteristic of any food 
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(including any substance intended for use in producing, 

manufacturing, packing, processing, preparing, treating, packaging, 

transporting or holding food; and including any source of radiation 

intended for any such use), if such substance is not generally 

recognized, among experts qualified by scientific training and 

experience to evaluate its safety, as having been adequately 

shown through scientific procedures (or, in the case of a substance 

used in food prior to January 1, 1958, through either scientific 

procedures or experience based on common use in food) to be 

safe under the conditions of its intended use...” 

2. Food additives are used to modify chemical, biological, sensory, or 

physical characteristics of food and drink. They are added as 

preservatives, antioxidants, acid regulators, thickeners, stabilizers, 

emulsifiers, anti-caking agents, flavors, and flavor enhancers.

3. Food additives have been used for centuries to enhance the flavor 

of food and drink and preserve the quality of food. To prevent 

spoilage, salt was added to meat. Sugar, vinegar, and alcohol were 

used to preserve fruits and vegetables. 

4. With the Industrial Revolution, food production moved from the 

farm to food factories as the population shifted from rural areas to 

the cities. New chemical preservatives such as borax and boric 

acid, sulfites, salicylic acid, benzoic acid, and formaldehyde were 

liberally added to food to increase food production, prevent 

spoilage, and increase profits. 

5. As the food processing industry grew, new artificial thickeners, 

emulsifiers, and flavors were synthesized and used. The safety of 

these new additives was unknown, and regulations did not exist to 

prevent manufacturers from adding whatever they liked to their 

products and using deceptive labeling to sell them.

6. Department of Agriculture Chief Chemist, Dr. Harvey Wiley began 

researching the safety of chemical preservatives and published his 

assessment in a report to Congress in 1889. He acknowledged that 

it was probably not wise to absolutely prohibit the use of 

preservatives in foods, but it was “imperative” that any food 

containing a preservative should clearly state so on the label. 

7. In 1902, Congress appropriated Dr. Wiley $5,000 to study the 

effects of chemical preservatives on health. Dr. Wiley established 

the “hygienic table trails” and assembled a squad of young men 

volunteers who would consume steadily increasing amounts of 

various preservatives carefully tracking the effects they had on 
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their bodies. This group of young men became known as the 

“Poison Squad”. Of the substances studied, only benzoate of soda 

and sulfites were sanctioned for continued use in food. The use of 

sulfite was limited, and the use of benzoate had to be indicated on 

the label.

8. The first Pure Food and Drug Act was passed by Congress in 

1906, prohibiting interstate commerce of adulterated food, drink, 

and drugs. This ACT advanced food safety but also had several 

shortcomings. Provisions were left out which would have 

established similar standards for foods as those established for 

drugs. The law did not include a scientific definition of harmfulness 

by which to judge substances used in foods as either safe or 

“poisonous or deleterious”, and it only applied to a product once it 

was marketed. 

9. Over the next several decades, USDA parameters were 

established for additives through court hearings and regulations. In 

1938, the Pure Food and Drug Act was replaced by The Federal 

Food, Drug, and Cosmetic Act. Section 402(a)(1) considers a food 

that contains a poisonous or deleterious substance that was not 

added, not to be adulterated if the quantity of the substance is not 

injurious to health. 

10.Under 402(a)(2)(C)(i), a food is deemed adulterated if it contains 

an unsafe food additive whose use is avoidable unless its use 

conforms to the terms of an exemption or regulation prescribing the 

conditions under which the additive can be safely used. 

11.Section 406 allowed FDA to set tolerances for poisonous or 

deleterious substances required to be used during production that 

cannot be avoided. Any quantity used that exceeds the established 

limit will cause the food to be adulterated.

12.The processed food industry saw a dramatic growth surge after 

World War II. More processed foods became available in grocery 

stores and these foods used new chemical ingredients and 

packaging. By 1947, over 500 different chemical entities had been 

proposed for use in foods.

13.The Food Additives Amendment was passed in 1958 in response 

to concerns about the safety of additives used in food. It became 

Section 409 of the Act. This amendment and associated regulation 

established the requirement for a premarket review and approval 

by FDA, for any substance added to food unless the substance is 
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Generally Recognized as Safe (GRAS) or was sanctioned or 

approved by FDA or USDA prior to 1958. 

14.A substance can be classified as GRAS if qualified experts have a 

reasonable certainty, it is not harmful under the conditions of its 

intended use based on appropriate scientific analyses, or its 

common use in food has demonstrated with reasonable certainty 

the substance is safe based on a substantial history of 

consumption by a significant number of people. 

15.By definition, GRAS substances are not food additives and do not 

require premarket approval by FDA. This means that the 

determination of an ingredient as GRAS can be made by qualified 

experts outside of FDA. With the implementation of the GRAS 

Notification Program in 1997, a company can independently 

conclude an ingredient is GRAS, and they can voluntarily inform 

FDA of their conclusion. 

16.The company is still required to ensure the ingredient meets the 

safety requirements and required regulations under the law. It is 

important to note whether a substance is deemed to be GRAS or 

approved as a food additive, safety determination is always limited 

to its intended conditions of use and not to the substance itself. 

17.Incorporated within Section 409 is what is known as the “Delaney 

Clause”, which restricts FDA from approving food additives that 

have been found to cause cancer when ingested by man or animal 

or if it is found after appropriate tests to evaluate the safety of the 

additive, to induce cancer in people or animals

18.A stipulation to this clause allows for use of a carcinogen as a feed 

ingredient for animals raised for food production provided the 

additive will not adversely affect the animals under conditions of 

use and no residue of the additive can be found by methods 

prescribed or approved by regulation, in any edible portion of the 

animal after slaughter. 

19.Additional amendments were made to Section 409 under the Food 

and Drug Administration Modernization Act (FDAMA) in 1977 when 

subsection (h) was added. This subsection established the 

requirement for premarket notification and approval of food contact 

substances. Subsection (k), food additives intended for use in 

animal food was added in 2018.
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20.Any substance added to a food for a specific purpose in that food is 

referred to as a direct food additive and must be declared as an 

ingredient on the label. 

21.Substances added to a food during processing are referred to as a 

secondary direct food additive. They have no technical effect in the 

finished food and do not have to be declared as an ingredient. 

22.Indirect food additives are substances that come into contact with 

the food but are not to become a component of or have a technical 

effect in or on the food. Packaging, coatings, and adhesives are 

examples of indirect food additives. Indirect food additives are also 

not required to be listed on the label. 

23.Both prior-sanctioned and GRAS substances are required to be 

listed as an ingredient on the product label.

B. References

1. Code of Federal Regulations, Title 21, Washington DC: Office of 

Federal Register National Archives and Records Administration 

a. Part 170 – Food Additives. 

b. Part 172- Food Additives Permitted for Direct Addition to Food 

for Human Consumption.

c. Part 173 – Secondary Direct Food Additives Permitted in Food 

for Human Consumption.

d. Part 174 – Indirect Food Additives: General.

e. Part 175 – Indirect Food Additives: Adhesives and Components 

of Coatings.

f. Part 176 – Indirect Food Additives: Paper and Paperboard 

Component. 

g. Part 177 – Indirect Food Additives: Polymers. 

h. Part 178 – Indirect Food Additives: Adjuvants, Production Aids, 

and Sanitizers.

i. Part 180 – Food Additives Permitted in Food or in Contact with 

Food on an Interim Basis Pending Additional Study.

j. Part 181 – Prior-Sanctioned Food Ingredients.

k. Part 182 – Substances Generally Recognized as Safe. 

l. Part 184 – Direct Food Substances Affirmed as Generally 

Recognized as Safe.
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m. Part 185 - Indirect Food Substances Affirmed as Generally 

Recognized as Safe.

n. Part 189 – Substances Prohibited from Use in Human Food.

o. Part 101 – Food Labeling. 

2. US Food & Drug Administration, Compliance Program 7309.006 –

Domestic and Import Food Additives and Color Additives (current 

edition). 

3. US Food & Drug Administration, Center for Food Safety and 

Applied Nutrition, Food Additive Status List. 

4. US Food & Drug Administration, Center for Food Safety and 

Applied Nutrition, Substances Added to Food (formerly EAFUS).

5. US Food & Drug Administration, Center for Food Safety and 

Applied Nutrition, GRAS Notices.

6. US Food & Drug Administration, Center for Food Safety and 

Applied Nutrition, Food Ingredient & Packaging Inventories.

7. US Food & Drug Administration, Center for Food Safety and 

Applied Nutrition, A Food Labeling Guide, Guide for Industry, 

January 2013.

C. Questions

1. What are the three conditions that are to be met for a preservative 

to be used according to good manufacturing practices?

2. List 10 standardized foods for which the addition of chemical 

preservatives has been permitted by regulation.

3. Are all substances added to food required to be declared on the 

product label? How should they be declared?

4. What is the term “Do.” an abbreviation for?

4.3.1. Food Additive Exercises

The exercises in this section will introduce the trainee to food additive analysis 

and associated regulations. The exercises do not need to be performed in 

order and can be abbreviated or omitted if the laboratory does not perform the 

analysis.
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4.3.1.1. Water Phase Salt and Sodium Nitrite in Smoked Fish

This exercise has been developed to introduce the principles of analysis for 

common chemical preservatives such as sodium chloride and sodium nitrite, 

and related regulations.

A. The trainer will provide a sample of smoked fish. Determine water 

phase salt and nitrites as described.

1. Sample Preparation

Prepare each subsample separately for analysis according to AOAC 

937.07(e), except take the loin muscle as defined in 21 CFR 

161.190(a)(3) for the sample portion. i.e. “the longitudinal quarter of 

the great lateral muscle (loin muscle) freed from skin, scales, visible 

blood clots, bones, gills and viscera and from the nonstriated part of 

such muscle." Grind the loin muscle according to AOAC 937.07(a), 

paragraph 2.

2. Water Phase Salt

a. Analyze each of the prepared subs by AOAC 952.08A for 

moisture content (total solids) and AOAC 937.09 for salt content. 

(See 21 CFR 172.177.)

b. Calculate salt content in the water phase of the loin muscle 

according to the formula:

% Salt in water phase of loin muscle = [% Salt / (%Salt + % 

moisture)] x 100

3. Sodium Nitrite

Analyze each of the prepared subs for nitrite using AOAC 973.31. 

Report nitrite as ppm sodium nitrite in loin muscle.

B. References

1. Code of Federal Regulations. Title 21, Pt. 110-Current Good 

Manufacturing Practice in Manufacturing, Packing, or Holding 

Human Food, and Sections 172.170 - Sodium Nitrate and 172.177-

Sodium Nitrite Used in Processing Smoked Chub. Washington DC: 

Office of Federal Register National Archives and Records 

Administration 

2. Official Methods of Analysis of AOAC International, AOAC 

International, (current ed.). Gaithersburg MD

3. Fish and Fishery Product Hazards and Controls Guidance (current 

ed.), US Food and Drug Administration.
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4. FDA CPGM 7303.842 Seafood Processor Inspection ProgramDomestic and Foreign Facilities (current ed.)

5. FDA CPGM 7303.844 Import Seafood Products (current ed.)

6. Appendix I: Apparatus for Tests and Assays – Volumetric Apparatus, 

Food Chemicals Codex (current edition). United States 

Pharmacopeia, Rockville, MD.

7. <1857> Ultraviolet-Visible Spectroscopy – Theory and Practice, 

USP-NF General Chapters, General Information (current edition).

C. Questions

1. Which fish or fish products may legally contain nitrates or nitrites? 

List the maximum legal level for each product and describe how the 

retail container must be labeled.

2. Where is "water phase salt" defined? How does water phase salt 

vary with moisture content? Write the equation used for calculating 

the salt concentration in the water phase.

3. Why is water added to the portion taken for analysis by AOAC 

952.08A?

4. How would one show that all the volatile components had been 

evaporated from the tissue?

5. Show the reactions involved in the titrimetric analysis for total 

chloride.

6. Why is the HNO3 solution boiled before the indicator is added?

7. AOAC 937.09B(b) states “With 10 g sample each ml 0.1 N AgNO3 = 

0.058% NaCl.” How is this factor obtained?

8. What is the structure of the colored product formed during the nitrite 

analysis? Show the reactions involved.

9. How has the order of addition of reagents been shown to affect this 

type of analysis?

10.What is the correlation between pH and color stability?

4.3.1.2. Nitrate and Nitrite in Bottled Water

A. Background

1. Nitrogen is an essential nutrient for all life and exits in several 

forms in nature. Bacteria in the soil convert nitrogen into ammonia 

and nitrate which are assimilated by plants. Nitrogenous 
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compounds not taken up by plants or converted to nitrous oxide by 

bacteria is converted to nitrate. Nitrate is readily soluble in water, 

passing through the soil into groundwater. 

2. Man acquires nitrates and nitrites from consuming vegetables, 

processed meat, seafood, and water. When nitrate and nitrite are 

consumed, a little over half of the nitrate is absorbed and rapidly 

excreted as urea and ammonia in the urine. The remaining nitrate 

is converted by bacteria and stomach acid into nitrite and nitric 

oxide. Consuming high levels of these compounds has been 

shown to cause methemoglobinemia. 

3. Methemoglobin is formed when nitrite oxidizes ferrous iron in 

hemoglobin to the ferric form. Methemoglobin is unable to bind and 

transport oxygen through the body causing weakness, cyanosis, 

coma, and in some cases death. In young children and infants, 

Methemoglobinemia (referred to as “blue baby syndrome”) can be 

caused by drinking water that is high in nitrates. 

4. FDA is responsible for regulating the safety of bottled drinking 

water. 21CFR 165.110 defines bottled water as “water that is 

intended for human consumption and that is sealed in bottles or 

other containers with no added ingredients except that it may 

optionally contain safe and suitable antimicrobial agents”. 

5. Flavored and nutrient-added water beverages are also required to 

meet bottled water regulations if it is declared as “water” on the 

label. 

6. Artificially carbonated waters like soda water (club soda), tonic 

water, and seltzer water are not regulated as bottled water. 

7. The Safe Drinking Water Act was passed in 1974 establishing the 

EPA drinking water standards. FDA regulations for bottled water 

are based on the EPA tap water standards. 

B. The trainer will provide a sample of bottled water. Determine the 

amount of nitrate and nitrite in the water as described:

1. Prepare a composite as outlined in CPGM 7309.006 Part IV, 

Methodology, Food Additives.

2. Determine the amount of nitrate and nitrite using EPA method 

300.1 - “The Determination of Inorganic Anions in Water by Ion 

Chromatography”.

3. Compare the results with 21 CFR165.110(b)(4)(iii)(A).

C. References
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1. Code of Federal Regulations, Title 21, Part 165.110 - Bottled 

Water. Washington DC: Office of Federal Register National 

Archives and Records Administration

2. US Food & Drug Administration, Compliance Program 

7309.006 – Domestic and Import Food Additives and Color 

Additives (current edition).

3. Method 300.1, Environmental Monitoring Systems Laboratory 

Office of Research and Development. US Environmental 

Protection Agency (current revision), Cincinnati, Ohio.

4. <1065> Ion Chromatography, USP-NF General Chapters, 

General Information (current edition).

D. Questions

1. What is the stability and storage conditions for the stock 

standards? Working standards? 

2. What factors can affect trace analysis by IC?

3. How does diluting the eluate affect the analysis?

4. What does the analyst need to be aware of when using a 

pretreatment cartridge to eliminate certain types of matrix 

interferences?

4.3.1.3. Sulfites

A. Background

1. Sulfiting agents have been used as a food additive for centuries 

and are considered GRAS by FDA based on its extensive 

history of use. 

2. Sulfites are added to many different types of foods for several 

technical purposes. 

a. Sulfur dioxide, sodium and potassium bisulfites, and 

metabisulfites are used in foods as antioxidants and 

bleaching agents. 

b. Sulfites are commonly used as preservatives in dried fruits, 

as a bleaching agent in the processing of maraschino 

cherries, on shrimp and lobster to prevent melanosis (“black 

spot”), to retain the color of cooked octopus, and prevent 

conch meat from darkening. 
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3. Sulfites were used for years on fresh fruit and vegetables to 

preserve the color and crispness but their use on fresh produce, 

except for potatoes, was prohibited in 1986, based on an 

increase of reported allergic-type reactions in individuals who 

are sensitive to sulfites. 

4. To further protect these individuals, the labeling regulation 

requiring the presence of sulfites to be declared on any food 

containing at least 10 ppm sulfites was enacted in 1987. 

5. This labeling requirement applies to foods containing added 

sulfites, not to foods with naturally occurring sulfur-containing 

compounds such as vegetables in the Brassica and Allium 

families. 

B. The trainer will provide one or more samples of dried fruit preserved 

with sulfites. Determine the amount of sulfite, calculated as sulfur 

dioxide present in the food using both the AOAC and CAM methods.

1. Prepare a composite as outlined in CPGM 7309.006 Part IV, 

Methodology, Food Additives.

2. Determine the amount of sulfite using AOAC 990.28 “Sulfites in 

Foods Optimized Monier- Williams Method”. Report both the 

titrimetric and gravimetric results.

3. Determine the amount of sulfite using CAM Method C-004 

“Determination of Sulfites in Food using Liquid 

Chromatography-Tandem Mass Spectrometry (LC-MS/MS)” 

(Current version). 

4. Compare your results to the regulations.

C. References

1. Code of Federal Regulations. Title 21, Part. 130.9 - Sulfites in 

Standardized Foods; Part 182, Subpart D – Chemical 

Preservatives. Washington DC: Office of Federal Register 

National Archives and Records Administration. 

2. Official Methods of Analysis of AOAC International, AOAC 

International, (current ed.). Gaithersburg MD.

3. US Food & Drug Administration, Center for Food Safety and 

Applied Nutrition FDA Foods Program Compendium of 

Analytical Laboratory Methods: Chemical Analytical Manual 

(CAM).
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4. US Food & Drug Administration, Compliance Program 

7309.006 – Domestic and Import Food Additives and Color 

Additives (current edition).

5. <1738> Applications of Mass Spectrometry, USP-NF General 

Chapters, General Information (current edition).

6. Appendix 1: Apparatus for Tests and Assays – Volumetric 

Apparatus, Foods Chemical Codex (FCC), (current ed.). 

Rockville MD

D. Questions

1) Monier-Williams 

1. Why doesn't the HCl distill into the sulfur dioxide absorber? 

What is an azeotrope?

2. What reaction occurs in the distilling flask? In the sulfur dioxide 

absorber?

3. What is the purpose of the gravimetric determination?

4. What purpose does the pyrogallol/KOH gas wash serve?

2) LC/MS/MS Sulfite Method 

1. What is the purpose of the addition of 0.2% formaldehyde for 

sample extraction?

2. What volume of extracting solvent is used for the first extraction 

for the three different solid procedures (basic protocol, low 

moisture solids, and high moisture solids) 

3. Why is the sample run through a C18 solid phase extraction 

(SPE) clean-up?

4. The method uses a HILIC column for addition separation of 

matrix constituents prior to MS/MS detection. What is a HILIC 

column?

5. Which type of calibration curve is used? 

6. What are the monitored transitions?

7. What is the calculation to convert the concentration in vial from 

the calibration curve to concentration in sample (ppm)?

8. Can sulfites be declared using advisory statement such as “may 

contain sulfites”?
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4.3.1.4. Sweeteners

A. Background

1. Artificial sweeteners have been used by the food industry since the 

1800’s. Saccharin was discovered in 1879 and by the early 1900s, 

was widely used in processed foods to replace sugar. By the 1970s, 

concerns were growing about saccharin's safety. Based on 

laboratory test results, FDA revoked its GRAS classification and 

imposed restrictions for the use of saccharin in foods. In 1977, 

Canada banned saccharin based on the Canadian Health Protection 

Branch Study. In the US, the Saccharin Study and Labeling Act of 

1977 was passed. This ACT established criteria for the scientific 

study into the safety of saccharin and its impurities and required any 

food containing saccharin to carry a warning statement on its label. 

The requirement for warning statement was repealed in 2001.

2. Dulcin was discovered in 1883, but never gained the popularity 

saccharin had. Dulcin was removed from the market in 1954 due to 

studies linking it with cancer. Cyclamate was discovered in 1937 and 

by the 1960s, had become the sweetener of choice since it had less 

of a bitter aftertaste then saccharin. Cyclamate was banned for use 

in 1970 by FDA due to studies linking the sweetener to bladder 

cancer.

3. The development and use of sweeteners has increased dramatically 

since the 1970s. FDA approved six new synthetic sweeteners as 

food additives., Some plant- and fruit-based sweeteners, sugar 

alcohols, and sugars that are metabolized differently than sucrose 

have been classified as GRAS.

B. The trainer will provide at least one sample containing sweeteners. 

Identify any sweeteners present in the sample.

1. Prepare a composite as outlined in CPGM 7309.006 Part IV, 

Methodology, Food Additives.

2. Analyze the sample using the method designated by the trainer. If 

a validated method is not available, use AOAC Method 969.27, 

“Non-Nutritive Sweeteners in Nonalcoholic Beverages, Qualitative 

Thin-Layer Chromatographic Method”.

3. Compare your results to the regulations.

C. References

1. Code of Federal Regulations. Title 21, Sections 172.800 -

Acesulfame potassium; 172.803 - Advantame; 172.804 -
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Aspartame; 172.829 - Neotame; 172.831 - Sucralose; 180.37 -

Saccharin, Ammonium Saccharin, Calcium Saccharin, and Sodium 

Saccharin; 189.135 - Cyclamate and its Derivatives; 189.145 -

Dulcin; 172.395 - Xylitol; 180.25 - Mannitol; 184.1835 Sorbitol. 

Washington DC: Office of Federal Register National Archives and 

Records Administration. 

2. US Food & Drug Administration, Center for Food Safety and 

Applied Nutrition, GRAS Notices.

3. Official Methods of Analysis of AOAC International, AOAC 

International, (current ed.). Gaithersburg MD.

4. US Food & Drug Administration, Compliance Program 7309.006 –

Domestic and Import Food Additives and Color Additives (current 

edition).

5. Physical Tests and Determinations – Thin-Layer Chromatography, 

Food Chemicals Codex Appendix II (current edition).

D. Questions

1. You have been given a dried fruit to analyze for cyclamates using 

AOAC method 969.27. Is this method suitable for the analysis? 

What should you do?

2. In terms of the TLC extraction procedure, what is the purpose of 

adding 5 ml of 50% NaOH solution?

3. Why do foods that contain sugar alcohols require a warning on the 

label? What is the warning?

4.3.1.5. Benzoates

A. Background

1. Sodium benzoate was the first preservative approved by FDA. 

Sodium benzoate is the salt of benzoic acid and unlike benzoic 

acid, is soluble in water. It is widely used in acidic foods to inhibit 

the growth of bacteria, yeast, and mold. It is also used as a 

flavoring agent and adjuvant. When used under acidic conditions, 

sodium benzoate is converted to benzoic acid. There is concern 

that benzoate can be transformed by decarboxylation into benzene 

when used in combination with vitamin C, so some manufacturers 

have replaced sodium benzoate with a different preservative.

2. This ingredient is used in food at levels not to exceed good 

manufacturing practice. Current usage results in a maximum level 

of 0.1 percent in food. 
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B. The trainer will provide at least one beverage sample. Determine if the 

sample contains benzoate. If present, determine the amount of 

benzoate the product contains.

1. Prepare a composite as outlined in CPGM 7309.006 Part IV, 

Methodology, Food Additives.

2. Analyze the sample using the method designated by the trainer. If a 

validated method is not available, use AOAC Method 979.08, 

“Benzoate, Caffeine, and Saccharin in Carbonated Beverages”

3. Compare your results to the regulations.

C. References

1. Code of Federal Regulations. Title 21, Sections 582.3733 - Sodium 

Benzoate. Washington DC: Office of Federal Register National 

Archives and Records Administration.

2. Official Methods of Analysis of AOAC International, AOAC 

International, (current ed.). Gaithersburg MD.

3. US Food & Drug Administration, Compliance Program 7309.006 –

Domestic and Import Food Additives and Color Additives (current 

edition).

4. <621> Chromatography, USP-NF General Chapters, General Tests 

and Assays, (current edition).

D. Questions

1. The lab has a large supply of Silica Gel G plates that are used in 

several different analyses in the lab, including colors. Can these TLC 

plates be used for this analysis?

2. Is the pH of the mobile phase used in AOAC 979.08 critical? What 

affect can pH have on retention times?

3. What formula should be used to calculate % benzoate in the 

sample?

4. What is the salt of benzoic acid? What pH does sodium benzoate 

optimally function? Which is more soluble, sodium benzoate or 

benzoic acid?

5. In terms of HPLC, what may occur if there is a change in the 

composition of the mobile phase?

6. In terms of HPLC how critical is the detection wavelength?
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4.4. Color Additives

A. Background

1. Section 201(t) of the FD&C Act defines a color additive as “a dye, 

pigment, or other substance made by a process of synthesis or 

similar artifice, or extracted, isolated, or otherwise derived, with or 

without intermediate, or final change of identity, from a vegetable, 

animal, mineral or other source, when added or applied to a food, 

drug or cosmetic, or the human body or any part thereof, is capable 

(alone or through reaction with other substance) of imparting color 

thereto”. The term “color” includes black, white, and shades of gray. 

2. Colors are used to enhance a person’s appearance, offset color loss

due to light, air, temperature extremes, moisture, and storage 

conditions, correct natural variations in color, decorate baked goods, 

and in certain products such as candy, can be associated with 

flavors. Unscrupulous companies will sometimes use color additives 

to deceive consumers by concealing damaged or inferior foods. 

3. The earliest color additives were natural pigments from plants and 

minerals. This changed in 1885 when the first synthetic organic dye 

called Mauve was accidently discovered by William Henry Perkins 

while trying to synthesize quinine from aniline isolated from coal tar. 

This led to the discovery of other synthetic organic dyes produced 

from the by-product of coal processing. These colors became known 

as “coal-tar dyes”. By the 1900’s, most food, drugs, and cosmetics 

were artificially colored. Reported injuries and deaths associated 

with their use resulted in congress appropriating funds to examine 

these dyes. Subsequent testing found many of them contained 

dangerous and poisonous materials such as lead, arsenic, and 

mercury. 

4. The Federal Food and Drugs Act of 1906 also known as the Pure 

Food and Drug Act was the start of federal color regulation. The 

1906 Act prohibited the use of poisonous or deleterious colors in 

confectionary products and the staining or coloring of food to 

conceal inferior or damaged foods. 

5. In 1907, USDA (FDA did not exist at this time) published the first list 

of synthetic organic dyes considered to be safe for use in foods. Out 

of the 80 dyes tested, seven were found to be safe and placed on 

this list. These were the first “approved” colors.

6. Over time, it was recognized the Pure Food and Drug Act of 1906 

did not go far enough to protect the public from misbranded, 
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adulterated, and dangerous products. Marketed cosmetics contained 

hazardous ingredients such as arsenic, radium, mercury, and 

thallium. 

7. In 1933, users of “Lash Lure” a new permanent mascara containing 

p-phenylenediamine, an oxidizing coloring agent, were injured and, 

in some cases, blinded. This incident has been linked to the passing 

of the first cosmetic regulations in the Food, Drug and Cosmetic Act 

of 1938. The 1938 Act increased government oversight of colors. 

“Harmless” coal-tar colors, other than those used in hair dye, were 

required to be listed in the recently established Code of Federal 

Regulations. Voluntary certification of listed color additive batches 

became mandatory and certification fees were established. 

Misbranding and adulteration provisions for colors were added. 

8. In the same year, FDA developed labeling and record keeping 

provisions, established procedures for requesting color certification 

and adding new colors to the permitted list, and diluents that could 

be added to colors were identified. 

9. A new nomenclature was formulated for certified coal-tar dyes 

consisting of a prefix (FD&C - Food, Drug and Cosmetic, D&C –

Drug and Cosmetic, or Ext. D&C – External Drug and Cosmetic) 

followed by the specific color, a number, and the term “Lake” if 

applicable. A lake is an insoluble pigment formed by chemically 

reacting a straight dye with precipitants and substrata. Color additive 

lakes are provisionally listed under 21 CFR 81.1 and 21 CFR part 

82. 

10.Color regulations were further enhanced with the passing of the 

Color Amendments of 1960. The term “color additive” was defined. 

Factors to determine the safety of the color based on its use were 

established and specific conditions for their safe use had to be 

included in the regulations. The “Delany Clause” which prohibits the 

listing of a color additive that has been shown to be a carcinogen 

was added. The purity of the dye and an acceptable level of 

impurities had to be determined through scientific analysis. 

11.A process for premarket approval of a new color additive or 

amendment for an existing color was created. Around 200 listed 

color additives, except those used to color hair, were moved to a 

provisional list until they were shown to be safe and permanently 

listed or their use was terminated. About half of these colors were 

approved and listed. Coal-tar colors used to color hair are still 

excluded from the regulations. 
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12.The passing of the Nutrition Labeling and Education Act (NLEA) in 

1990 required all certified color additives to be declared on the 

product label with a few exceptions. CFR 101.22(k)(3) states color 

additives added to butter, cheese, and ice cream do not have to be 

declared as an ingredient unless it is required by regulation in parts 

73 or 74 of the CFR.

13.Approved (listed) color additives are divided into two categories; 

those that are subject to batch certification and those that are 

exempt from batch certification. Color additives subject to batch 

certification are synthetic organic dyes, pigments, or lakes that are 

now derived from petroleum products instead of by-products of coal 

manufacturing. They are still referred to as “coal-tar colors”. They 

are classified into five main groups based on their chemical 

structure: azo, triphenylmethane, xanthene, indigoid, and quinoline. 

14.Only a few synthetic organic dyes are permanently listed for use in 

foods; FD&C Yellow No. 5, FD&C Yellow No. 6, FD&C Blue No. 1, 

FD&C Blue No. 2, FD&C Red No. 3, FD&C Red No. 40, and FD&C 

Green No. 3. Except for FD&C Red No. 3, their aluminum lakes are 

permitted to be used in foods. Citrus Red No. 2 and Orange B are 

also listed for use in foods, but their use is restricted to specific 

foods. 

15.Additional synthetic organic dyes are permanently listed for use in 

drugs and cosmetics. Lakes prepared from D&C colors are also 

permitted. Except for Ext. D&C Yellow No 7, lakes prepared from 

Ext. D&C colors are not permitted. Any use restrictions for D&C and 

Ext. D&C colors are specified in the CFR. 

16.Color additives that are exempt from batch certification are obtained 

from natural sources such as plants and minerals. Cochineal extract 

(Carmine) is the only approved color derived from an insect. 

Certification exempt color additives must be approved by FDA 

before they can be used. They are required to comply with the 

identity, purity, and use restrictions listed in the CFR. 

17.CFR 101.22(k)(1) requires certified color additives to be declared by 

their listed name in the CFR. For example, “FD&C Yellow No. 5” or 

“FD&C Blue No. 1 Lake” or abbreviated to “Yellow 5” or “Blue 1 

Lake”. 

18.CFR 101.22(k)(2) requires color additives not subject to certification 

to be declared by the common or usual name listed in the CFR. 

These colors may also be declared as “Artificial Color”. “Artificial 

Color Added”, “Color Added”, or “Colored with ______”, or “_______ 
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color” with the blanks filled in with the name of the color additive. 

CFR 73.100(d)(2) requires food products that contain cochineal 

extract or carmine, including butter, cheese, and ice cream, declare 

its presence as an ingredient using its common or usual name; 

“Cochineal Extract” or “Carmine”. Labeling of color additives not 

subject to certification can be challenging. For example, the color 

additive Turmeric cannot be declared as Curcumin which is a yellow 

pigment found in Turmeric. Also, only the carotenoids listed in part 

73 of the CFR are approved color additives and must be declared by 

the listed name in the CFR. Vegetable Juice is a permitted color 

additive while Black Carrot juice is not. 

19.Labeling of colors in cosmetics is subject to additional regulations 

that are not applicable to foods. CFR 701.3(f)(3) permits color 

additives to be grouped together on the label without respect to 

order of predominance. CFR 701.3(g) states color additives can be 

declared using the phrase “may contain” if the same declaration of 

ingredients is used for other products similar in composition and all 

products that share the common declaration of ingredients are sold 

under a common trade name or brand designation. As an alternative 

to listing color additives on each product sold in a cosmetic 

assortment, CFR 701.3(h) allows color additives to be declared in a 

single composite list provided it is not misleading to the consumer 

and the list pertains to all the products in the assortment. 

20.Other color designations such as the common name of the color, 

Colour Index Number (C.I. number), European Number (E code), or 

International Numbering System (INS) are used in other countries 

and can also be declared in parentheses along with the US 

approved name in the ingredients. INS numbers generally 

correspond with the E code for the same compound. For example, 

the common name for FD&C Yellow 5 is Tartrazine, C.I. number is 

C.I. 19140, E code is E102, and the INS number is INS102. 

Improperly declared color additives imply the color used may not be 

from a certified batch.

21.The analysis of colors has both a subjective and objective 

component. Three common attributes used to visually identify a 

color are hue, lightness, and chroma. Hue is the attribute of a color 

that allows it to be discerned as red, yellow, green, etc. and is 

dependent on its dominant wavelength. Lightness is the quality that 

distinguishes a lighter color from a darker one. Chroma is the purity 

or intensity of a color. Because of these attributes, colors cannot be 

solely described in spectrophotometric terms.
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22.Color molecules contain a visible chromophore that is used to 

objectively analyze colors. A chromophore is a group of one or more 

unsaturated bonds that absorb light in the ultraviolet and visible 

region. Those that absorb light in the visible region impart color to 

the molecule. In most cases, they are covalent unsaturated bonds 

such as C=C, C=O, C=NH, CH=N, N=N, and aromatic rings. 

23.An auxochrome is a group such as -OH, -NH2, -SO3H, and -COOH 

which by itself does not act as a chromophore, but when attached to 

a chromophore increases the absorption intensity and shifts the 

absorption wavelength. 

24.A shift to a longer wavelength is a bathochromic effect (red shift). A 

shift to a shorter wavelength is a hypsochromic effect (blue shift). An 

increase in absorption is a hyperchromic effect and a decrease in 

absorption is a hypochromic effect.

B. References:

1. Code of Federal Regulations, Title 21, Washington DC: Office of 

Federal Register National Archives and Records Administration.

a. Part 70 – Color Additives

b. Part 73- Listing of Color Additives Exempt from Certification

c. Part 74 – Listing of Color Additives Subject to Certification

d. Part 81- General Specifications and General Restrictions for 

Provisional Color Additives for Use in Foods, Drugs, and 

Cosmetics 

e. Part 82 – Listing of Certified Provisionally Listed Color and 

Specifications

f. Part 101 – Food Labeling 

g. Part 701 – Cosmetic Labeling

2. US Food & Drug Administration, Compliance Program 7309.006 –

Domestic and Import Food Additives and Color Additives (current 

edition). 

3. US Food & Drug Administration, Center for Food Safety and Applied 

Nutrition, Summary of Color Additives for Use in the United States in 

Foods, Drugs, Cosmetics, and Medical Devices. 

C. Questions:

1. What is a color Additive?
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2. Define these terms: straight color, lake, diluent.

3. What are “certified” colors?

4. Under what conditions may color additives be used in the area of the 

eye, In injections, or in surgical sutures?

5. List the restrictions, if any, for the following colors:

a. Citrus Red No. 2

b. Orange B

c. Titanium Dioxide

d. FD&C Blue No. 1

e. FD&C Red No. 3

f. FD&C Red No. 40

g. FD&C Yellow No. 5

6. Give one example of a triphenylmethane color.

7. List the Colour Index Number (C.I. Number) and common name for:

a. FD&C Red No. 1

b. FD&C Red No. 3

c. D&C Orange No. 3 

d. FD&C Green No. 3

e. D&C Yellow No. 7

f. D&C Yellow No. 9

g. FD&C Blue No. 2

h. FD&C Violet No. 1

8. A label of a food product declares “...E123...” as an ingredient. Is 

this product permitted in the U.S.?

9. A label of a food product declares “...Sunset Yellow FCF, 

Erythrosine Lake, Allura Red AC”. Is this product correctly labeled? 

If not, why? If the importer relabels the product, would it be permitted 

in the U.S.?

10.In terms of solubility, state whether the following color additives are 

water soluble or oil soluble.

a. “Natural” colors such as riboflavin or saffron
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b. Tartrazine

c. Sudan 1

d. D&C Red 19 or D&C Red 28

4.4.1. Color Additives Exercises

The purpose of the exercises in this section is to acquaint the trainee to 

analytical methods, techniques, and technologies used by FDA to test for 

colors in foods and cosmetics and familiarize the trainee with color additive 

regulations. 

These exercises do not need to be performed in order and can be abbreviated 

or omitted if the laboratory does not perform the analysis. 

The trainee must read and have a theoretical knowledge about the methods 

listed below. Attachment A contains a list of suggested color training samples. 

4.4.1.1. Quantitative Determination of FD&C Colors in Foods (Graichen 

Method)

A. Background

This method was developed by Charles Graichen and John Molitor for 

analyzing colors in foods. The method was published in the Journal of 

Association of Agricultural Chemists (JAOAC) which later became the 

Journal of the Association of Official Analytical Chemists in 1963. The 

method was revised by Charles Graichen in 1973 and can be found in 

Attachment C.

In general, an extraction flows as follows:

1. A food is finely ground with an aqueous solution and Celite 

(diatomaceous earth) and packed into a chromatographic column. 

2. Chloroform is added to remove oils/fats and natural colors.

3. An anion exchange resin solution is used to extract the colors. 

4. Weak basic solutions are used to extract the colors from the resin. 

5. All eluates collected are scanned by ultraviolet/visible (UV-VIS) 

spectroscopy to obtain an initial indication of color.

6. Eluates requiring further analysis are neutralized for further cleanup 

and isolation by SPE column using AOAC method 988.13. 

7. Colors are identified by ultraviolet/visible (UV-Vis) spectroscopy.

B. The trainer will provide two or more samples to analyze for colors.
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1. Extract and identify colors present in the sample using the Graichen 

method.

2. Determine if the color(s) detected meets labeling and color 

regulations.

C. References

1. Graichen and Molitor (1963). Quantitative Determination of FD&C 

Colors in Foods, Journal of the Association of Agricultural Chemists, 

Vol. 46, No. 2, pages 1022 – 1029. Revised in 1973. Attachment B 

contains a method copy including revisions.

2. Physical Tests and Determinations – Column Chromatography, 

Food Chemicals Codex Appendix II (current edition).

3. <1857> Ultraviolet-Visible Spectroscopy – Theory and Practice, 

USP-NF General Chapters, General Information (current edition).

D. Questions

1. What is the difference between Procedure I, II, and III?

2. A sample is prepared as per the method following Procedure I. In 

which eluate would the following colors be seen:

a. Amaranth

b. Green 3

c. Beta Carotene

4.4.1.2. FD&C Color Additives in Foods: Rapid Cleanup for 

Spectrophotometric and Thin-Layer Chromatographic 

Identification (AOAC Method 988.13)

A. Background

1. This method was developed by Mary Young and published in the 

AOAC in 1988. It is a quick screening method that uses a reverse 

phase C-18 cartridge and different concentrations of isopropyl 

alcohol (the method includes an elution scheme to use as a guide) 

to separate, isolate, and concentrate colors present in the sample. 

The colors are then identified by visible spectrometry and confirmed 

by thin layer chromatography. 

2. This method requires little expenditure of solvents and is capable of 

separating colors that are exempt from certification (i.e. “natural” 

colors) from synthetic colors and permitted colors from non-
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permitted colors. It can be applied to a variety of products except 

high fat products such as baked goods.

B. The trainer will provide two or more samples to analyze for colors.

1. Extract and identify the colors present in the sample using AOAC 

Method 988.13.

2. Determine if the color(s) detected meets labeling and color 

regulations

C. References

1. Official Methods of Analysis of AOAC International, AOAC 

International, (current ed.). Gaithersburg MD

2. Physical Tests and Determinations – Thin-Layer Chromatography, 

Food Chemicals Codex Appendix II (current edition).

3. <1857> Ultraviolet-Visible Spectroscopy – Theory and Practice, 

USP-NF General Chapters, General Information (current edition).

D. Questions

1. How does the C18 sep-pak isolate and concentrate colors?

2. Yellow No. 5 is not retained as well as the other colors on the sep-pak. 

How can you improve its retention?

3. What are chromophores and auxochromes? Identify the chromophores 

and auxochromes in two of the following colors

a. Citrus Red 2

b. Orange B

c. Titanium Dioxide

d. FD&C Blue No. 1

e. FD&C Red No. 3

f. FD&C Red No. 40

g. FD&C Yellow No. 5

4. What is hypsochromic shift? Give the effect of hypochromic shift for two 

of the colors in the question above.

4.4.1.3. Color Additive Analysis in Foods and Cosmetics using UPLC 

with Extended Photo-Diode Array Detection (LIB 4643)

A. Background
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1. This method developed by Susan Clark, et al. describes a simple and 

efficient extraction and clean-up process applicable to a wide variety of 

different food and cosmetic sample types. 

2. Colors are extracted/purified using three types of solid-phase extraction 

columns (cation-exchange (CBX), silica gel (SiOH), and hydrophiliclipophilic balanced (HLB)) and identified by UPLC – PDA (ultraperformance liquid chromatography with extended photo-diode array 

detector). 

3. This method has high sensitivity with minimal solvent use. Instrument 

run time is approximately 17 minutes. It utilizes a large spectral library 

and is capable of identifying over 200 color additives. 

B. The trainer will provide two or more samples to analyze for colors.

1. Extract and identify the colors present in the sample using LIB 4643.

2. Determine if the color(s) detected meets labeling and color 

regulations

C. References

1. Laboratory Information Bulletins (2000 – present), ORA Office of 

Regulatory Science SharePoint site.

2. <621> Chromatography, USP-NF General Chapters, General Tests 

and Assays, (current edition).

D. Questions

1. How versatile is this method? 

2. Describe how to determine the appropriate method section to use for 

a sample.

3. When is an HLB SPE column used? 

4. Is it critical to filter both mobile phases? 

4.4.1.4. Determination of Colors in Cosmetics

A. Background

1. Chapter 19 in the Newburgers Manual of Cosmetic Analysis is a 

collection of methods for isolating and identifying colors in a variety 

of cosmetics. The methods use TLC, solvent-solvent extraction, and 

column chromatography to isolate and cleanup the colors, which are 

then identified by UV/VIS spectroscopy. 

2. This manual was last published in 1977. Attachment D contains a 

copy of Chapter 19 from Newburgers Manual of Cosmetic Analysis.
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NOTE: The TLC solvents used in method 19.1 “Lipstick - Tube (Thin Layer 

Chromatographic Method)” are also used to analyze other powdered and 

liquid makeup. For the determination of colors, methylene chloride is used 

to move waxes and oils to the top of the plate and isolate oil-soluble colors. 

Water-soluble colors are scraped from the plate, dissolved in a suitable 

solvent, and analyzed using AOAC Method 988.13.

B. The trainer will provide at least two different types of cosmetics to 

analyze for colors.

1. Extract and identify the colors present in the sample using the 

methods in Newburgers Chapter 19.

2. Determine if the color(s) detected meets labeling and color 

regulations

C. References

1. (1977). Newburgers manual of cosmetic analysis (2nd ed., chap. 

19). Arlington, VA: Association of Official Analytical Chemists.

2. Physical Tests and Determinations – Column Chromatography, 

Food Chemicals Codex Appendix II (current edition).

3. Physical Tests and Determinations – Thin-Layer Chromatography, 

Food Chemicals Codex Appendix II (current edition).

4. <1857> Ultraviolet-Visible Spectroscopy – Theory and Practice, 

USP-NF General Chapters, General Information (current edition).

D. Questions

1. How many synthetic colors are approved for use in cosmetics? 

Which of these colors are permitted in eye area cosmetics?

2. What colors or pigments are approved for use in tattoos? What 

about henna?

3. A lipstick sample was analyzed using Newburgers, 19.1. One bright 

pink band of color, exhibiting orange fluorescence, was detected. 

Using Table 12 in Newburgers (page 119), what color(s) was(were) 

detected? Are these possible color(s) permitted for use?
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Document History

Revision

#

Status*

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

1.3 R 06/06/2008 LMEB LMEB

1.4 R 02/02/2010 LMEB LMEB

1.5 R 02/06/2012 LMEB LMEB

1.6 R 02/14/2013 LMEB LMEB

1.7 R 05/02/2014 LMEB LMEB

02 R 11/12/2020 LMEB LMEB

03 R See QMiS

COL, FAD, and DEC 

Operation Program 

Managers

LMEB

* - D: Draft, I: Initial, R: Revision

Change History

Revision 

# Change

1.3 Answer Key 10.2.3, 2., straight color definition changed and mixture definition added

1.4

10.1.2.1 C., 10.1.2.2 C., 10.1.2.3 C., 10.1.2.4 C., 10.1.2.5 C., 10.2.2.1 C., 10.2.2.2 

C, 10.2.2.3 C.,

10.2.2.4 C., 10.2.2.5 C. – References updated or deleted

10.2.2.2 B. – “sulfides” changed to “sulfites”

10.2.3 B – revised

10.2.3. D. – “Neuberger” changed to

“Newberger” Footer – web link 

updated

1.5 10.2.2.2 B. – updated method

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

1.7

10.2.2.2 D. 1. & 2. – deleted U tube

10.3 Answer Key 10.2.2.2. 1. & 2. – deleted U tube
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Revision 

# Change

02

Reformatted entire document which led to change in the numbering and bullets

Revised the exercises and questions for Food Additives 

Updated answers to colors questions

Removed obsolete links

03

Entire document was extensively revised with input obtained from COL, FAD, and DEC 

Operation committee members to capture current processes and references and must be read 

in its entirety. Document has been transferred to new TEMPLATE-000054 for formatting.

Attachments

List of Attachments

Attachment A - Color Additive Training Samples ........................................................36

Attachment B - Answer Key ........................................................................................37

Attachment C - Quantitative Determination of FD&C Colors (Graichen Method)........52

Attachment D - Newburger’s Chapter 19 ....................................................................78
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Attachment A - Color Additive Training Samples

Foods

• Bakery items (cookie, cake, bread)

• Candy (hard, soft)

• Gelatin

• Cheese

• Chocolate (candy, cocoa, powdered drink mix)

• Dried fruits and vegetables

• Spices

• Mukhwas (mouth freshener/digestive aid consisting of various seeds like 

fennel, anise, coriander, etc.) 

• Gum 

 

Cosmetics

• Lipstick, lip gloss or lip tint

• Powdered makeup (eyeshadow, blush, powder)

• Soap (liquid, bar)

• Liquid eyeliner

• Mascara

• Temporary hair color sprays and colored dry shampoo

• Lotions

• Liquid cosmetics (cologne, aftershave)

• Temporary tattoos
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Attachment B - Answer Key

4.2. Food Standards

1. What consumer protection was presumed in the promulgation of food standards? 

Consumers are protected from adulterated and misbranded foods by use of 

standards. 

2. Differentiate between misbranding and adulteration.

A food shall be deemed to be “adulterated” if: 

a. It bears or contains any poisonous or deleterious substance which may render 

it injurious to health. 

b. It bears or contains any added poisonous or added deleterious substance 

(other than a substance that is a pesticide chemical in or on a raw agricultural 

commodity; or processed food, a food additive, a color additive, or a new 

animal drug) that is unsafe within the meaning of section 406 of The Act. 

c. It consists in whole or in part of any filthy, putrid, or decomposed substance, 

or if it is otherwise unfit for food. 

d. It has been prepared, packed, or held under unsanitary conditions whereby it 

may have become contaminated with filth, or whereby it may have been 

rendered injurious to health. 

A food shall be deemed to be “misbranded” if: 

a. Its labeling is false or misleading in any particular, or in the case of a food to 

which section 411 of the Act applies, its advertising is false or misleading in a 

material respect or its labeling is in violation of section 411(b)(2). 

b. It is offered for sale under the name of another food. 

c. Its container is so made, formed, or filled as to be misleading. 

d. In the case of nuts with misleading vignettes, misrepresentation of product, 

and 

e. incorrect net weight, misbranding charges apply. 

3. What are the requirements for the fill of container? 

a. Food containers must be so made, formed, or filled so they are not misleading 

(FD&C Act, Sec. 403(d)). 

b. Fill of container is the minimum quantity of the solid food in the container after 

processing: not less than 90 percent of the total capacity of the container. 

4. When is the packing liquid included in net weight calculations? 
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The packing liquid is included in the net weight calculations when it is also used by 

the consumer. 

5. Define edible portion. 

Edible portion is any portion of a food product that is fit to be eaten. 

6. Can the net contents be satisfactory and fill of container fail? 

When the volume of product settles during shipment the correct net contents may 

no longer properly fill the container. When this is expected, the manufacturer may 

include a comment on the label to that effect. 

7. What, if any, is the relationship between the vignette and the contents? 

If the label bears any pictorial representation (vignette) of the mixture of nuts, it 

must depict the relative proportions of the nut ingredients contained in the finished 

food. 

8. What is the maximum amount (in %) of peanuts allowed in mixed nuts? 

In mixed nuts, each nut ingredient shall be present in a quantity not less than 2 

percent and not more than 80 percent by weight of the finished food.

4.2.1.1 Cheese

1. Why is speed essential in sample preparation? 

Speed is essential in sample preparation to prevent moisture loss, which would 

affect the results in the fat calculations of cheeses. 

2. Differentiate between a cheese spread and cheese food. 

A cheese food is the food prepared by comminuting and mixing, without the aid of 

heat, one or more optional ingredients into a homogeneous mass. 

A cheese spread is the food prepared by comminuting and mixing, with the aid of 

heat, one or more optional ingredients into a homogeneous mass. 

3. List two kinds of cheese in which sorbic acid is permitted and include the 

conditions under which it might be used. List two kinds of cheese for which sorbic 

acid is not permitted. 

a. Sorbic Acid is permitted for use in cold pack cheeses in an amount not to 

exceed 0.3 percent by weight and in pasteurized process cheeses in an 

amount not to exceed 0.2 percent by weight. 

b. Sorbic Acid is not permitted for use in Nuworld and Roquefort cheeses. 

4. What is a processed cheese? 
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A processed cheese is the food prepared by comminuting and mixing, with the aid 

of heat, one or more of the optional cheese ingredients, into a homogeneous mass 

that is spreadable.

5. In the AOAC procedure, is the moisture content directly determined? 

The AOAC procedure uses an indirect method and moisture loss is determined, 

not content. 

6. In the cheese fat extraction, what other substances may be found? 

Other product ingredients, such as oil-soluble natural colors, may be extracted if 

present. 

7. Why is petroleum ether used for the extraction? 

Petroleum ether is used because fats and oils are readily soluble in this solvent, 

and it is easily evaporated. 

4.2.1.2 Jams and Jelly

1. What is jam? 

A viscous or semi solid food, from a mixture of one or more fruits, mixed with or 

without water, and concentrated with or without heat. The soluble solids content of 

the finished product should not be less than 65 per cent. The standards of identity 

for jams and jellies (21 CFR 150) require that these products be prepared by mixing 

not less than 45 parts by weight of certain specified fruits (or fruit juice in the case 

of jelly), and 47 parts by weight of other designated fruits, to each 55 parts by 

weight of sugar or other optional nutritive carbohydrate sweetening ingredient. 

2. What is an imitation jelly? 

A food is an imitation if it is a substitute for, and resembles another food, but is 

nutritionally inferior to that food (21 CFR 101.3(e)). Nutritional inferiority includes 

any reduction in the content of an essential nutrient that is present in a measurable 

amount. 

3. Define "degrees Brix." 

A unit of measure on the Brix scale for measuring the density or concentration of 

sugar (sucrose) in solution, determined as percent by weight. It is used to measure 

the density of the packing medium of fruits and vegetables, or strength of juices.

4.2.1.3 Egg Noodles 

1. Does the label have an ingredient statement? Why? 

The label of an egg noodle must have an ingredient statement when optional 

ingredients such as onions, garlic salt, gum gluten, or glyceryl monostearate are 

added. 
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2. Do all noodle products contain eggs? 

As defined in the standard, all noodle products should contain egg; in noodles, the 

total solids of egg or egg yolk are not less than 5.5 percent. 

3. Compare the two drying methods. Which must be used to determine compliance 

with the standard for total solids? 

a. One method uses a vacuum oven with drying to a constant weight at temp 98-

100 oC (~ 5 hrs.) and the second uses an air oven at 130 oC for 1 hour. 

b. CFR 21.139.150(a)4 specifies the vacuum oven method. 

4. What is the difference between macaroni and noodles? 

The difference between macaroni and noodles is the egg content. In macaroni the 

total solids from egg white, frozen egg white, or dried egg white is not less than 0.5 

and not more than 2.0 percent. In noodles, the total solids of egg or egg yolk are 

not less than 5.5 percent. 

4.3 Food Additives

1. What are the three requirements that must be met for a preservative to be used 

according to good manufacturing practices? The three requirements for 

preservative use are the following: generally recognized as safe for such use (a 

food additive is covered by food additive regulations prescribing conditions of safe 

use); not used in such a way as to conceal damage or inferiority or to make the food 

appear better or of greater value than it is; and proper declaration on the food label 

in which used.

2. List 10 standardized foods for which the addition of chemical preservatives has 

been permitted by regulation. Milk and milk products, cereal, fish, macaroni, 

cheeses, canned vegetables, nuts, fruits bakery goods, and cacao. 

3. Are all substances added to food required to be declared on the product label? Any 

substance directly added to food must be declared on the label. Substances added 

during processing that have no technical effect on the product and indirect food 

additives are not required to be declared. Substances added to foods should be 

declared as an ingredient by its common or usual name in descending order of 

predominance in the finished food. Ingredients cannot be listed “collectively” unless 

excepted from the requirement for using its specific name. 

4. What is “Do.”? “Do.” Is the abbreviation for ditto.

4.3.1.1 Water Phase Salt and Sodium Nitrite in Smoked Fish

1. Which fish or fish products may legally contain nitrates or nitrites? List the maximum 

legal level for each product and describe how the retail container must be labeled. 
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The fish or fish products that may contain nitrates or nitrites are smoked cured 

sablefish, salmon, shad, and chubs at levels not to exceed (NTE) 200 ppm sodium 

nitrite and NTE 500 ppm sodium nitrate. In smoked tuna fish products, the level of 

sodium nitrite is NTE 10 ppm. The container label must bear the name and 

concentration of the additive.

2. Where is "water phase salt" defined? 

Water phase salt (WPS) is defined as the salt content of the water phase portion of 

the edible portion of the finished smoked product, as measured in the loin muscle 

(21 CFR 172.177). Generally, WPS should not be less than 3.5%. However, consult 

Compliance Programs 7303.844 and 7303.842 Domestic/Imported Fish and 

Fisheries Products for specific requirements, or the HACCP program (Fish and 

Fisheries Products Hazards & Controls Guide). 

How does water phase salt vary with moisture content? 

The water phase salt content is inversely proportional to the moisture content. 

Write the equation used for calculating the salt concentration in the water phase.

% salt in water phase = [(% salt / (% salt + %water)] x 100.

3. Why is water added to the portion taken for analysis by AOAC method 952.08A?

Water is added for the purpose of mixing and to help uniformly distribute the 

sample portion across the surface of the drying pan. A smooth layer of sample 

promotes even drying.

4. How would one show that all the volatile components had been evaporated from the 

tissue? 

If no significant change in weight is observed after an additional period of drying, 

then all volatiles have been evaporated.

5. Show the reactions involved in the titrimetric analysis for total chloride.

The procedure uses a modification of the old Volhard Titration.

For fish and fish products, the organic matter is oxidized by heating with HNO3; the 

Cl- is precipitated by the addition of Ag+. This results in an excess of Ag+.

Ag+ + Cl-  AgCl ppt + excess Ag+

The excess Ag+ is then titrated with SCN- (thiocyanate). 

Ag+ + SCN-  AgSCN ppt

One drop past the end point there is an excess of SCN-

. This excess SCN- then 

reacts with the Fe+3 to produce a colored ferric thiocyanate complex:

Fe+3 + SCN-  FeSCN2+ (reddish-brown color)
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So, the total amount of Ag+ is known, and the excess Ag+ after AgCl formation is

known from the titration. Subtracting the moles of each yields the amount of Ag+ 

that reacts with the Cl-, which in turn tells us the amount of Cl-. The Fe+3 does not 

do anything until there is an excess of SCN-; then it reacts and produces the 

colored FeSCN2+ complex which is used for the end point. The reddish-brown 

FeSCN2+ actually has a more complex formula. Bromide, iodide, and cyanide are 

interferences. Also, if one does not do the titration quickly, then there will be an 

interference from the AgCl (Ksp =1x10E-10) reacting with the SNC- since AgSCN 

has a Ksp = 1x10E-12). The analyst should get an RSD of < 5% and a relative error 

of < 2%.

6. Why is the HNO3 solution to be boiled before the indicator is added? 

The HNO3 solution is used to digest the protein and liberate the chloride in solution 

before the indicator is added.

7. AOAC method 937.09B(b) states "With 10g sample each mL 0.1N AgNO3 = 0.058% 

NaCl." How is this factor obtained?

Assuming a 10.000 gram sample is used:

100% XX 58.44 mmmm NNNNNNNN

mmmmmm

XX 0.1 NN SS −

10,000 mmmm ss ss = 0.058%

8. What is the structure of the colored product formed during the nitrite analysis? 

Show the reactions involved. 

The nitrosation of sulfanilic acid form the diazonium salt of sulfanilic acid which then 

couples with 1-naphthylamine to form a pink azo compound.

9. How has the order of addition of reagents been shown to affect this type of 

analysis? 

Nitrite was added to sulfanilic acid, and the mixture was allowed to stand 20 

minutes before 1-naphthylamine was added. The resulting amount of azo dye was 
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the same as that formed when nitrite was added to a mixture of sulfanilic acid and 

1-naphthylamine.

10.What is the correlation between pH and color stability? 

Use of more acid and substitution of acids were investigated and the amount of dye 

compound formed was not affected.

4.3.1.2 Nitrate and Nitrite in Bottled Water

1. What is the stability and storage conditions for the stock standards? Working 

standards?

The nitrate stock standard is stable for 6 months if stored at 4 °C. The nitrite stock 

standard is stable for 1 month if stored at 4 °C. Nitrite in water readily transforms 

into nitrate, so the working standards must be prepared fresh daily. 

2. What factors can affect trace analysis by IC? 

Contamination control is important. The samples and solutions must be stored in a 

container made of polytetrafluoroethylene (PFTS), polypropylene (PP) or 

polystyrene. Glass has ion exchange properties and should be avoided. Deionized 

water used to prepare standards, eluents, and used to dilute samples must be the 

highest quality possible (< 0.1 μS·cm-1). Contamination can also come from the 

apparatus used to process the sample.

3. How does diluting the eluate affect the analysis? 

Diluting the eluate to separate co-eluting peaks results in a reduction in the overall 

response of the anion. Late eluting anions will be retained longer, lengthening the 

run time. Peaks will be shorter and broadened which will adversely affect the 

Method Detection Limit (MDL) of the anion. The QC must be repeated to show the 

dilution does not negatively impact the analysis. 

4. What does the analyst need to be aware of when using a pretreatment cartridge to 

eliminate certain types of matrix interference? 

All method calibration standards must be treated in the same manner as the 

sample. Caution should also be exercised when using pretreatment cartridges 

because artifacts can be leached from the cartridge which can damage the guard 

and analytical columns causing a loss of column capacity resulting in shortened 

retention times and irreproducible results. 

4.3.1.3 Sulfites

Monier-Williams Determination

1. Why doesn't the HCl distill into the sulfur dioxide absorber? What is an azeotrope?

The risk of interfering substances reaching the hydrogen peroxide trap (sulfur 

dioxide absorber) by aerosolization, co-distillation, or steam distillation has been 
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reduced by condenser coolant temperatures, reflux rates, and nitrogen flow. The 

HCl is trapped in the reflux column. 

An azeotrope is a mixture of liquids that has a constant boiling point and cannot be 

separated by distillation.

2. What reaction occurs in the distilling flask? In the sulfur dioxide absorber? 

An oxidation reaction: SO2  SO3. 

3. What is the purpose of the gravimetric determination? 

The gravimetric determination serves as a confirmatory test specific for sulfites.

4. What purpose does the pyrogallol/KOH gas wash serve? 

The pyrogallol/KOH wash serves as an oxygen scrubbing solution.

LC-MS/MS Determination

1. What is the purpose of the addition of 0.2% formaldehyde for sample extraction?

It converts free sulfite to a more stable adduct  hydroxymethylsulfonate (HMS).

2. What volume of extracting solvent is used for the first extraction for the three 

different solid procedures (basic protocol, low moisture solids, and high moisture 

solids)?

Basic – 20 mL, low moisture – 30 mL, high moisture – 15 mL

3. Why is the sample run through a C18 solid phase extraction (SPE) clean-up? 

To remove all lipophilic matrix components

4. The method uses a HILIC column for addition separation of matrix constituents 

prior to MS/MS detection. What is a HILIC column?

Hydrophilic Interaction Liquid Chromatography. Typically used when retention time 

in reverse phase chromatography is insufficient. Typically involves more polar or 

ionizable analytes.

5. Which type of calibration curve is used? 

Quadratic with 1/x2 weighting

6. What are the monitored transitions?

a. HMS: 111-81 and 111-80

b. Internal Std HMS (34S): 113-83 and 113-82

7. What is the calculation to convert the concentration in vial from the calibration curve 

to concentration in sample (ppm)?
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iiii ss ss = �

xx μμ NNNN2SSSS3

mmmm vv � ∗ �

1 mmmm vv

vv � ∗ �

dddd

mm � ∗ � 64 mmmm −1 SSSS2126 mmmm −1NNNN2SSSS3

�

Where x is the concentration (ug/mL) in vial from the calibration curve, df is the 

dilution factor for the sample, m is the mass (g) of sample analyzed, v is the 

volume (mL) of the extract added to the LC vial. The final term of the equation is 

used to convert from concentration Na2SO3 to SO2. All values were reported as μg 

SO2/g food sample.

8. Can sulfites be declared using an advisory statement such as “may contain 

sulfites”? 

No. Sulfiting agents is a food intolerance substance, not an allergen. The sulfiting 

agent used must be declared on the label as an ingredient.

4.3.1.4 Sweeteners

1. You have been given a dried fruit to analyze for cyclamates using AOAC method 

969.27. Is this method suitable for the analysis? What should you do? 

No. A method extension or modification is needed prior to analyzing this sample.

2. In terms of the TLC extraction procedure, what is the purpose of adding 5 ml of 

50% NaOH solution? 

Modification of the nature of the aqueous phase to basic changes the solubility of 

the 4 analytes. This will promote their extracting into the ethyl acetate organic 

phase.

3. Why do foods that contain sugar alcohols require a warning statement on the 

label? What is the warning? 

Sugar alcohols can product abdominal gas, bloating and diarrhea in some 

individuals. 

Labels must include the warning “excess consumption may have a laxative effect.”

4.3.1.5 Benzoates

1. The lab has a large supply of Silica Gel G plates that are used in different 

analyses in the lab, including colors. Can these TLC plates be used for this 

analysis? 

No. Plates specified in LIB 3385 should be used.

2. Is the pH of the mobile phase used in AOAC 979.08 critical? What affect can 

pH have on retention times? 

Yes. Adjustments to pH can affect the retention time of certain analytes. 

3. What formula should be used to calculate % benzoate in the sample?
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% Benzoates = C1 x (H/H1) x (V1/V) x 0.1

4. What is the salt of benzoic acid? What pH does sodium benzoate optimally 

function? Which is more soluble, sodium benzoate or benzoic acid?

a. Sodium or potassium benzoate. 

b. pH 2.5 – 4.0 

c. Sodium benzoate is 180 times more soluble than benzoic acid. 

5. In terms of HPLC, what may occur if there is a change in the composition of 

the mobile phase? 

The chromatographic elution pattern will change and possibly the peak shape; 

the resolution between peaks may not meet the performance criteria, 

components may elute in the void volume or greatly increase elution time 

resulting in poor chromatography and efficiency.

6. In terms of HPLC how critical is the detection wavelength? 

The wavelength at which the analyte of interest absorbs is critical information. 

When using HPLC with a UV/VIS fixed detector and attempting to 

chromatograph several analytes at once, a wavelength that is common for the 

group of analytes should be selected. However, modern instrumentation allows 

for different wavelengths to be simultaneously or sequentially monitored 

throughout the course of a chromatographic run.

4.4 Color Additives

1. What is a color additive? 

A color additive is defined as a dye, pigment, or other substance - whether 

synthetic or derived from a vegetable, animal, mineral, or other source - which 

is added to impart color to a food, drug, cosmetic, applied to the human body 

or medical device.

2. Define these terms: straight color, lake, and diluent. 

a. A straight color is a color additive that has not been mixed or chemically 

reacted with any other substance. Straight colors subject to certification are 

listed in 21 CFR Part 74. Straight colors exempt from certification are listed 

in 21 CFR Part 73. In addition, 21 CFR Section 81.10 and 81.30 identify 

straight colors whose listings or certain uses have been terminated or 

whose certificates have been cancelled. 

b. Lakes are pigments prepared by precipitating a soluble dye onto an 

insoluble reactive or adsorptive substratum or diluent. They are waterinsoluble and more stable than straight dyes and are ideal for product in 
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which leaching of the color is undesirable (coated tablets and hard candies, 

for example). 

c. A diluent is any component of a color additive mixture that is not of itself a 

color additive and has been added to facilitate the use of the mixture as a 

coloring agent. The term mixture means a color additive made by mixing 

two or more straight colors, or one or more straight colors and one or more 

diluents.

3. What are "certified" colors? 

Certified colors are listed color additives in Code of Federal Regulations (21 

CFR Part 74) from certified batches analyzed by FDA's Color Certification 

Branch, CCB, to assure the listed specifications are met. These certifiable 

color additives are synthetic organic dyes, pigments, or lakes of a dye. In order 

to be used in foods, drugs, cosmetics, and medical devices manufactured or 

sold in the United States, the color additives must meet the following criteria: 

come from a certified batch, be allowed in the respective product, and the color 

additive name must be declared in the ingredients label.

4. Under what conditions may color additives be used in the area of the eye, in 

injections, or in surgical sutures? 

Color additives may be used in the area of eye, injections, or in surgical 

sutures only if the color additives are specifically listed for such use.

5. List the restrictions, if any, for the following colors:

a. Citrus Red No.2 - Permanently listed for use only in coloring of skins of 

mature oranges. Limit of 2 ppm calculated on basis of whole fruit 

weight.

b. Orange B - Permanently listed for use in coloring surfaces and casings 

of frankfurters or sausages. Limit of 150 ppm by wt. of finished product.

c. Titanium dioxide - Limit: of 1.0% by wt. of food.

d. FD&C Blue No. 1 - For food, drug, and cosmetic use, including drugs 

and cosmetics for eye area - GMP - 74.101, 74.1101, 74.2101, 82.101.

e. FD&C Red No. 3 - For food and ingested drugs - GMP - 74.303, 4.1303. 

Lake use terminated 2-1-90; All cosmetic uses terminated 2-1-90.

f. FD&C Red No. 40 - For food, drug, and cosmetic use, including drugs 

and cosmetics for eye area - GMP - 74.340, 74.1340, 74.2340.

g. FD&C Yellow No. 5 - For food, drug, and cosmetics, including drugs 

and cosmetics for eye area - GMP - 74.705, 74.1705, 74.2705, 82.705.

6. Give one example of a triphenylmethane color. 
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Brilliant Blue FCF (CI 42090, certifiable as FD&C Blue No.1) is an example of 

a triphenylmethane color. [Other triphenylmethane dyes, such as malachite 

green or gentian violet, are sometimes used as fungicides in the food industry.] 

Triphenylmethane colors consist of a common moiety of three phenyl groups 

attached to a central carbon (hence "methane"). Other functional groups are 

then attached to the phenyls, and these determine the different colors.

7. List the Color Index Number (C.I. No.) and common name for FD&C Red No. l, 

FD&C Red No. 3, D&C Orange No. 3, FD&C Green No. 3. D&C Yellow No. 7, 

D&C Yellow No. 9, FD&C Blue No. 2, and FD&C Violet No. 1.

Color C.I. No. Common 

FD&C Red No.1 CI 16155 Ponceau 3R

FD&C Red No.3 CI 45430 Erythrosine

D&C Orange No.3 CI 16230 Orange G

FD&C Green No.3 CI Fast Green

D&C Yellow No. 7 CI Fluorescein

D&C Yellow No. 9 CI Uranine K

FD&C Blue No.2 CI 73015 Indigotine

FD&C Violet No.1 CI 42640 Wool Violet 

5BN

8. A label of a food product declares "...E123..." as an ingredient. Is this product 

permitted in the U.S.? 

E123 is the European code designation for Amaranth (a.k.a. CI 16185, former 

FD&C Red No.2) which is not permitted for use in the U.S. No E numbers are 

permitted in the US since we do not know that they meet the specifications 

listed in the CFR.

9. A label of a food product declares "...Sunset Yellow FCF, Eythrosine Lake, 

Allura Red AC.” Is this product correctly labeled? If not, why? If the importer 

re-labels the product, would it be permitted in the U.S.? 

Artificial colors listed in 21 CFR part 74 must be declared on the labels by 

listing the color additive name (i.e. Red 40) among the list of ingredients (21 

CFR 101.22(k)(1)). When colors are declared using their common names (i.e. 

Allura Red AC), it is assumed that the colors used were not from a certified lot 

and the product can be detained based on adulteration. 

Use of Erythrosine Lake is not permitted in the U.S. and the product would not 

be permitted to be sold even if relabeled.

10.In terms of solubility, state whether the following color additives are water 

soluble or oil soluble: 
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a. “Natural” colors such as riboflavin or saffron (water soluble)

b. Tartrazine (water soluble)

c. Sudan 1 (oil soluble)

d. D&C Red 19 or DC Red 28 (water soluble)

4.4.1.1 Quantitative Determination of FD&C Colors in Foods (Graichen Method)

1. What is the difference between Procedure I, II, and III? 

All use chloroform to remove FD&C Red 3, fats, and “natural” colors (i.e. 

carotenoids, chlorophyll, etc.). However, 

Procedure I uses 20% acetic acid as the aqueous phase and separately 

extracts azo colors from triphenylmethane colors. 

Procedure II uses pH 7.5 buffer as the aqueous phase and is required for the 

extraction of FD&C Blue 2. 

Procedure III uses 0.1 N Hydrochloric Acid and is required if the colors in the 

product were added as lakes.

2. A sample is prepared as per the method following Procedure I. In which eluate 

would the following colors be seen: 

a. Amaranth will elute in resin-in-hexane.

b. Green 3 will elute resin-in-butanol.

c. Beta Carotene will elute in chloroform.

4.4.1.2 FD&C Color Additives in Foods: Rapid Cleanup for Spectrophotometric and ThinLayer Chromatographic Identification (AOAC Method 988.13)

1. How does the C18 sep-pak isolate and concentrate colors? 

The C18 sep-pak is a small reverse phase liquid chromatography column. 

Colors are adsorbed onto the C18 while more polar compounds (sugars, flavors, 

etc.) are not retained. The colors separate into bands based on their polarity. 

2. Yellow No. 5 is not retained as well as the other colors on the sep-pak. How 

can you improve its retention? 

The C18 sep-pak is flushed with 5 ml of acetic acid to improve adsorption of the 

colors on the sep-pak. Yellow 5 is more polar and has a lower affinity for the 

sep-pak. Acidifying the sample solution will strengthen its adsorption on the 

sep-pak.

3. What are chromophores and auxochromes? Identify the chromophores and 

auxochromes in two of the following colors: Citrus Red 2, Orange B, Titanium 
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Dioxide, FD&C Blue No. 1, FD&C Red No. 3, FD&C Red No. 40, and FD&C 

Yellow No. 5. 

Chromophores are groups of one or more unsaturated bonds that absorb light 

(in the ultraviolet or visible region) and thus produce colored substances. They 

are, in most cases, covalent unsaturated groups such as C=C, C=O, C=NH,

CH=N, and N=N. 

Examples are FD&C Yellow No. 5 (N=N) and FD&C Blue No. 1 (N=CH). 

Auxochromes are functional groups that do not confer color to a substance 

themselves but increase the coloring power of a chromophore. They contain 

functional groups, such as –OH, –NH2, –SO3 H, –COOH, that don’t exhibit 

absorption in visible wavelengths (however, auxochromes may absorb strongly 

in the far-ultraviolet region). When an auxochrome is combined with a 

chromophore in the same molecule, the chromophore absorption will typically 

shift to a longer wavelength (a bathochromic shift) and show an increase in 

intensity (a hyperchromic shift). Auxochromes provide an unshared pair of 

elections in conjunction to the chromophore. 

Examples are FD&C Blue No. 1 (–SO3) and Citrus Red 2 (–OH).

4. What is hypsochromic shift? Give the effect of hypochromic shift for two of the 

colors in the question above.

Hypsochromic shift is a change in wavelength maxima to a shorter wavelength, 

and thus change in color. 

For FD&C Red No. 40 and Citrus Red 2, the addition of base produces a 

change in color from red to orange and a wavelength maximum shift of 

approximately 50 nanometers.

4.4.1.3 Color Additive Analysis in Foods and Cosmetics using UPLC with Extended 

Photo-Diode Array Detection (LIB 4643)

1. How versatile is this method? 

LIB 4643 was designed to be a suite of techniques to analyze a broad spectrum 

of synthetic and natural colors in a vast variety of foods, cosmetics, and other 

marketed products. This method can analyze water and some oil-soluble colors. 

The extraction and cleanup is fast and simple, and the colors are identified by 

UPLC-PDA analysis. Over 150 different synthetic dyes and natural pigments 

have been characterized and stored in a spectral library on the instrument to 

assist in identifying undeclared colors. 

2. Describe how to determine the appropriate method section to use for a sample.
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Method selection is based on whether the sample is a liquid or solid, water or 

oil-based, or considered a “crossover” product. A method selection flowchart is 

included in the LIB. 

3. When is an HLB SPE column used? 

The HLB column is a secondary column that can retain most synthetic dyes or 

natural pigments not bound by the CBX. If color is observed passing through the 

CBX SPE when the sample is loaded onto the column, the eluate can be 

collected for further analysis following the HLB column protocol. 

4. Is it critical to filter both mobile phases? 

No, only mobile phase A must be filtered. Mobile phase B is a mix of high-grade 

organic solvents and does not require filtering.

4.4.1.4 Determination of Colors in Cosmetics

1. How many synthetic colors are approved for use in cosmetics? Which of these 

colors are permitted in eye area cosmetics? 

Presently there are 36 synthetic colors approved for use in cosmetics, of which 

only six are permitted in eye area cosmetics: FD&C Yellow No.5, FD&C Red No. 

40, FD&C Blue No.1, D&C Green No.5, D&C Black No. 2, and D&C Black No. 3.

2. What colors or pigments are approved for use in tattoos? What about henna?

Color additives are not approved for tattoos since tattoos require the color to be 

injected under the skin. No color additives are approved for direct injection. 

The color additive henna is not permitted to be applied to the skin and is only 

approved for coloring hair.

3. A lipstick sample was analyzed using Newburgers, 19.1. One bright pink band of 

color, exhibiting orange fluorescence, was detected. Using Table 12 in 

Newburgers (page 119), what color(s) was(were) detected? Are these possible 

colors permitted for use? 

A bright pink band of color exhibiting orange fluorescence under UV light can be 

due to either D&C Red No. 27, which is permitted for use in cosmetics, or it can 

be due to D&C Red No. 19, which is not permitted for use in the U.S.
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Attachment C - Quantitative Determination of FD&C Colors (Graichen Method)
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Attachment D - Newburger’s Chapter 19
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