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NBS SPECIAL PUBLICATION 635

U.S. DEPARTMENT OF COMMERCE/National Bureau of Standards

Reference

for Organic Nutrient

Measurement

c. 2
NATIONAL BUREAU OF STANDARDS

The National Bureau of Standards' was established by an act of Congress on March 3, 1901. The Bureau's overall goal is to strengthen and advance the Nation's science and technology

and facilitate their effective application for public benefit. To this end, the Bureau conducts

research and provides: (1) a basis for the Nation's physical measurement system, (2) scientific and technological services for industry and government, (3) a technical basis for equity in trade, and (4) technical services to promote public safety. The Bureau's technical work is per- formed by the National Measurement Laboratory, the National Engineering Laboratory, and

the Institute for Computer Sciences and Technology.

THE NATIONAL MEASUREMENT LABORATORY provides the national system of

physical and chemical and materials measurement; coordinates the system with measurement

systems of other nations and furnishes essential services leading to accurate and uniform

physical and chemical measurement throughout the Nation's scientific community, industry,

and commerce; conducts materials research leading to improved methods of measurement,

standards, and data on the properties of materials needed by industry, commerce, educational

institutions, and Government; provides advisory and research services to other Government

agencies; develops, produces, and distributes Standard Reference Materials; and provides

calibration services. The Laboratory consists of the following centers:

Absolute Physical Quantities 2 — Radiation Research — Chemical Physics —

Analytical Chemistry — Materials Science

THE NATIONAL ENGINEERING LABORATORY provides technology and technical ser- vices to the public and private sectors to address national needs and to solve national problems; conducts research in engineering and applied science in support of these efforts; builds and maintains competence in the necessary disciplines required to carry out this research and technical service; develops engineering data and measurement capabilities;

provides engineering measurement traceability services; develops test methods and proposes

engineering standards and code changes; develops and proposes new engineering practices;

and develops and improves mechanisms to transfer results of its research to the ultimate user. The Laboratory consists of the following centers:

Applied Mathematics — Electronics and Electrical Engineering2 — Manufacturing

Engineering — Building Technology — Fire Research — Chemical Engineering2

THE INSTITUTE FOR COMPUTER SCIENCES AND TECHNOLOGY conducts

research and provides scientific and technical services to aid Federal agencies in the selection, acquisition, application, and use of computer technology to improve effectiveness and

economy in Government operations in accordance with Public Law 89-306 (40 U.S.C. 759), relevant Executive Orders, and other directives; carries out this mission by managing the Federal Information Processing Standards Program, developing Federal ADP standards

guidelines, and managing Federal participation in ADP voluntary standardization activities; provides scientific and technological advisory services and assistance to Federal agencies; and

provides the technical foundation for computer-related policies of the Federal Government.

The Institute consists of the following centers:

Programming Science and Technology — Computer Systems Engineering.

'Headquarters and Laboratories at Gaithersburg, MD, unless otherwise noted; mailing address Washington, DC 20234. 'Some divisions within the center are located at Boulder, CO 80303.
Reference Materials

for Organic Nutrient Measurement

H K

Proceedings of a Workshop held at the National Bureau of Standards,

Gaithersburg, Maryland, October 23, 1980

Edited by Sam A. Margolis

National Measurement Laboratory

Center for Analytical Chemistry

National Bureau of Standards

Washington, DC 20234

Sponsored by:

National Bureau of Standards

Food and Drug Administration

Department of Agriculture

National Food Processors Association

U.S. DEPARTMENT OF COMMERCE, Malcolm Baldrige, Secretary

NATIONAL BUREAU OF STANDARDS, Ernest Ambler, Director

Issued August 1982
Library of Congress Catalog Card Number: 82-600575

National Bureau of Standards Special Publication 635

Natl. Bur. Stand. (U.S.), Spec. Publ. 635, 51 pages (Aug. 1982)

CODEN: XNBSAV

U.S. GOVERNMENT PRINTING OFFICE

WASHINGTON: 1982

For sale by the Superintendent of Documents, U.S. Government Printing Office, Washington, DC 20402

Price (Add 25 percent for other than U.S. mailing)
TABLE OF CONTENTS

PAGE

PREFACE iv

ABSTRACT v

WELCOMING REMARKS vi

D. R. Johnson

A GENERAL OUTLINE OF THE PROGRAM FOR THE NBS WORKSHOP ON MEASURING NUTRIENTS IN FOOD .... vii

S. A. Margolis

SESSION I

THE ROLE OF STANDARD REFERENCE MATERIALS IN THE DEVELOPMENT OF A SOUND DATA BASE FOR THE ASSESSMENT OF HUMAN NUTRITION 1 James T. Tanner and Jean A. T. Pennington

THE PROCESS AND REQUIREMENTS FOR THE DEVELOPMENT OF A STANDARD REFERENCE MATERIAL. ... 5 George A. Uriano

AN ASSESSMENT OF THE ACCURACY AND PRECISION OF THE METHODS USED FOR THE MEASUREMENT

OF ORGANIC NUTRIENTS IN CEREAL AND GRAIN PRODUCTS 8 David C. Egberg

SESSION II

ACCURACY AND PRECISION OF NUTRIENT METHODOLOGY 13

E. R. Elkins

PROBLEMS IN THE MEASUREMENT OF ORGANIC NUTRIENTS IN FOOD PRODUCTS: AN OVERVIEW 18

Kent K. Stewart

LONG TERM STABILITY OF ORGANIC NUTRIENTS IN FOODS 25

Wayne Thornburg

THE LONG-TERM STABILITY OF ORGANIC NUTRIENTS IN INFANT AND ADULT DIETARY SUPPLEMENTS . . 30

Steven Barnett

WORKSHOP SESSION

GENERAL OUTLINE FOR DISCUSSION DURING WORKSHOP SESSIONS 31

EXECUTIVE SUMMARY OF WORKSHOP SESSIONS . 32

S. A. Margolis

REPORT OF WORKSHOPS 36

A. Water-Sol uble Vitamins 36

J. R. Kirk and J. Pennington

B. Carbohydrate Analysis 37

W. J. Hurst and R. Schaffer

C. Fat-Soluble Vitamins, Cholesterol, and Fat 38

D. C. Egberg and H. T. Slover

Summary of Workshop Proposals 40

S.A. Margolis

LIST OF ATTENDEES 41

iii
PREFACE

In 1980, President Carter signed into law a bill (Public Law 96-

359) regulating the minimal nutritional content of infant formula.

Furthermore, the FDA regulation promulgated in 1973 mandated full

nutritional labeling on products whose merchandising includes any

nutritional claims or information. The accuracy and precision of some

of the methods used in measuring nutrient content are inadequate and

high-quality standard materials in representative food matrices, are

not always available for use in standardizing analytical procedures.

The objectives of this Workshop were to assess the state-of-theart of the measurement of organic nutrients in food matrices for the

purpose of developing a sound rationale for preparing suitable

Standard Reference Materials (SRM's). These proposed SRM's will

complement the SRM's which are already certified for inorganic

nutrients in food matrices. The aim of this meeting was to bring

together scientists from the industrial, research, and regulatory

communities to discuss the problems and propose strategies for the

development of SRM's including accurate and precise methods of

measurement. A priority list of organic nutrients and suitable

matrices to be certified as SRM's, was developed by the workshop

participants, who made a very important contribution to the knowledge

and understanding of the stability and measurement of organic

nutrients in foods.

iv
ABSTRACT

This publication is the formal report of the Workshop on Reference Materials for Organic Nutrient Measurement held at

the National Bureau of Standards on October 23, 1980. There

were seven formal presentations which provided the framework

for three workshop sessions. Each workshop session focused on one of three groups of nutrients: (1) cholesterol, fat, and

fat-soluble vitamins; (2) water-soluble vitamins; or (3) sugars.

Each workshop session reported on the state-of-the-art in

measurement techniques, suggested matrices which were appropriate

for Standard Reference Materials (SRM's), and indicated areas where

there were problems in measurement methodology. These recommendations are included in this publication.

Key Words: food matrices; methods of measurement; nutrients;

SRM's; stability; vitamins.

Disclaimer

Certain commercial equipment, instruments, or materials are identified in this report to specify adequately the experimental

procedure. Such identification does not imply recommendation or endorsement by the National Bureau of Standards, nor does it

imply that the materials or equipment identified are necessarily

the best available for the purpose.

v
WELCOMING REMARKS

I welcome you on behalf of the National Bureau of Standards to this Workshop on Reference Materials for Organic Nutrient

Measurements. This workshop is a cooperative effort between NBS and

the Food and Drug Administration, Department of Agriculture, and the

National Food Processors Association. We are very pleased to see the

turnout this morning, which I think is a bit more than we anticipated.

I would like to call your attention to the brochure which states rather

explicitly the purpose for this meeting. Let me articulate that

purpose in my own words. This meeting has been called to develop

rationalizations for a series of Standard Reference Materials for the

measurement of nutrients. I would like to point out to you that NBS

has been involved for many years in problems of measurement

standardization in various areas of the health care industry. Our work

in clinical chemistry, dental materials, and certain surgical implant

materials is well known. We also play the leadership role in

developing reference materials in many of these areas and, in fact, we now offer Standard Reference Materials for trace elements in food

matrices, such as spinach and flour. We recognize that there is a growing interest in standardization and measurement of nutrients in

other matrices and the exploration of this area of interest is the

purpose of this meeting today. According to your organizers during the

next few hours you will assess the state-of-the-art in the measurement

of organic nutrients in various matrices, examine nutrient stability

in the matrices, develop priority lists of nutrients for

standardization (That is something that is exceedingly important to

us, particularly for the people operating our Standard Reference

Materials program.) and develop a list of most appropriate matrices for

each group of nutrients. This is a big job and is going to require a lot of deliberation in your working groups this afternoon. I think

your steering committee has done an excellent job in arranging speakers

for you this morning. I wish you the best of success in the

deliberations over the next day and a half and I hope that you will

provide a very useful list of priorities for us. Thank you.

D. R. Johnson

Deputy Director

National Measurement Laboratory

vi
A GENERAL OUTLINE OF THE PROGRAM FOR THE NBS WORKSHOP ON MEASURING NUTRIENTS IN FOOD

The major stimuli for this workshop came from three directions: the recent legal requirement

to measure accurately the nutritional content of infant formula, the lack of availability of suitable

standard materials for the measurement of organic nutrients in food matrices, and the wide industrial

and governmental ' acceptance of NBS Standard Reference Materials (SRM's) for the measurement of trace

elements in foods and the measurement of alcohol in wine. With these points in mind the first paper

of the first session of the workshop examined the industrial need for SRM's in the development of an accurate data base for assessing human nutrition with respect to the levels of organic nutrients and

trace elements. SRM's are available from NBS for the measurement of trace elements in foods. How- ever there is no single source of Certified Reference Materials for the measurement of organic

nutrients.

Some reference materials for specific development or evaluation of methodologies are intermittently provided by the National Food Processors Association and the Association of Official

Analytical Chemists. There is no traceable, continuous source of SRM's for the measurement of

organic nutrients in food matrices which are comparable to the NBS SRM's for the measurement of

trace elements. The second paper outlined the process of development of the SRM's proposed for

organic nutrients and the possible cooperative role of NBS and industry in the development of such

standards.

The remaining formal presentations examined the current state of the accuracy and precision of

the measurements of organic nutrients in foods and identified the major problem areas in the

measurement of these nutrients. We will try also to delineate some of the conditions required for

long-term stability of organic nutrients in foods.

The afternoon workshop session composed of three parallel meetings dealing with separate classes

of nutrients was charged with examining the current methods of measurement and recommending the most

accurate ones for use in the analysis of potential SRM's. Each workshop group was also instructed to recommend a priority list of organic nutrients and appropriate matrices for development as SRM's.

These efforts will provide the guidelines for the NBS effort to prepare organic nutrient SRM's to meet the need of the industrial community.

Sam A. Margol is

Organic Analytical Research Division

Center for Analytical Chemistry

vii

National Bureau of Standards Special Publication 635. Proceedings of the Workshop on Reference

Materials for Organic Nutrient Measurement held at NBS, Gaithersburg, Maryland, October 23, 1980.

THE ROLE OF STANDARD REFERENCE MATERIALS IN THE DEVELOPMENT OF A SOUND DATA BASE FOR THE ASSESSMENT OF HUMAN NUTRITION

James T. Tanner and Jean A. T. Pennington

Division of Nutrition

Food and Drug Administration

Washington, D. C. 20204

1. Introduction

Food composition data bases are basic tools for nutrition researchers, clinical nutritionists,

dieticians, and food scientists. Such data bases are essential for developing menus for groups of

people and formulating specific therapeutic diets for individuals. They are necessary for assessing

the nutrient adequacy of the dietary intake records of groups or individuals. Establishment of the

relationship of foods to health requires use of data bases listing the various organic and inorganic

nutrients and contaminants in specific foods. Likewise, the challenge of improving the nutritional

quality of foods, meeting standards of food identity and nutrient fortification, and formulating new

food products also requires extensive data on food composition.

Food composition data are of great importance to the Food and Drug Administration in carrying

out its responsibilities of safeguarding the public health by insuring the safety and nutritional

quality of the food supply. Such a task requires surveillance of individual food comsumption;

conversion of food consumption data to nutrient consumption data (with food composition data bases);

and research activities on the use of nutrient consumption as a predictor of health status. Surveillance of food consumption allows FDA to continue assessing the nutritional effects of the

food supply on population groups.

The development of food composition data bases is a highly complex task. Data, old and new, are available from many sources - private laboratories, food industry, academic institutions, and

government agencies. The means, ranges, and standard deviations of nutrient values obtained in

various laboratories by various methodologies need to be evaluated and integrated. Standard

reference materials (SRM's) are going to be extremely valuable in the integration of nutrient values

from various laboratories so that the quality of nutrient composition data bases can be improved.

These reference materials of certified quality serve as points of common reference and allow data

from different laboratories determined by different methods to be compared, evaluated and

integrated. SRM's will promote the development of new methods of nutrient analysis which will be

comparable with current methods. SRM's will play a valuable role in nutrition labeling of foods. It

is anticipated that future additional nutrition labeling will be based on nutrient data banks. Use

of data banks for nutrition labeling of foods is desirable to prevent severe economic burdens on

small food companies who do not have the resources for extensive analytical work. It is essential

that the data from the nutrient banks be accurate to fulfill the purposes of nutrition labeling.

2. Food Label i ng

The concepts of nutrition labeling were initiated at the White House Conference on Food Nutrition and Health in 1969. These concepts were later incorporated in an FDA regulation proposed in

March 1972, and promulgated as a final regulation in 1973, entitled "Food, Nutrition Labeling". This

regulation provides for voluntary declaration of nutrition information, but regulates the substance

of the information, and specifies a mandatory format for presenting that information. For food

products which contain added nutrients or for products whose merchandising includes any nutrition

claims or information, full nutrition labeling is mandatory. Fresh fruits and vegetables have been

exempted from the provisions of these nutrition labeling regulations.

1
The nutrition information on the label includes: serving size; servings per container;

calories; protein, carbohydrate and fat in grams; and eight mandatory nutrients expressed as a percentage of the U.S. Recommended Daily Allowance (U.S. RDA). These eight mandatory nutrients are:

protein riboflavin

vitamin A niacin

vitamin C calcium

thiamin iron

Optional nutrients (vitamin D, vitamin E, vitamin B-6, folic acid, vitamin B-12, phosphorus,

iodine, magnesium, zinc, copper, biotin, and pantothenic acid) may be listed on food labels and must

be listed when they are added to a food. Currently the amount of sodium may be declared without

requiring full nutrition information. Nutrient quantities are expressed in terms of an average

serving. If the nutrients occur in amounts less than 2 percent of the U.S. RDA per serving, this is

indicated by a zero or the statement "contains less than 2 percent of the U.S. RDA for this

nutrient"

.

Since the 1973 regulation, many consumers have advocated more label information, whereas

industry spokespeople have questioned whether labeling has reached the saturation point both for

label space and for the consumer's capacity to utilize the information. In 1978, FDA joined with

USDA and the Federal Trade Commission (FTC) to review the total food label and consider updating the

existing food labeling laws and implementing regulations. USDA is responsible for the laws and

labeling of meat and poultry products, FDA is responsible for all other foods, and FTC is responsible

for the regulation of food advertising.

This was the first joint effort of these three agencies to review the entire field of food

labeling laws and regulations. They developed issue papers on various labeling topics and published

these papers along with an announcement of public hearings which were held in five cities during the

fall of 1978. Consumers were also invited to submit written comments regarding these papers. The

topics included: ingredient labeling; nutrition labeling and other dietary information; open date

labeling; food fortification; imitation and other substitute foods; safe and suitable ingredients;

and the total food label as a communications device.

Over 9000 comments were received, more than 2800 people attended the hearings, and 452 individuals testified. After careful review, a task force of FDA, USDA, and FTC members prepared a report

entitled "Food Labeling Report on the Analysis of Comments".

This report plus the results of a Consumer Food Labeling Survey of a statistically representative sample of the population were used to formulate tentative positions on changes in food

labeling. These tentative positions are summarized in the December 21, 1979 Federal Register which

lays out the strategy that FDA and other agencies plan to use in carrying out the first comprehensive revision of food labeling. This revision has a high priority in FDA and is expected to take 3-4 years to accomplish. The major concerns relative to nutrient labeling are: voluntary vs. mandatory requirement for nutrition labeling; information furnished by the label; and format of

the information on the label.

The following regulatory initiatives are being pursued:

1) FDA and USDA will seek legislative authority to make nutrition labeling mandatory or to require

nutrition labeling at their discretion. Many foods are already labeled nutritionally. An FDA survey

based on 1976 sales showed that on a dollar basis, approximately 40 percent of all packaged processed

foods and 24 percent of all foods in supermarkets carried nutrition labeling and that 60 percent of

foods with nutrition declarations were labeled voluntarily. The three agencies have formed a task

group to develop criteria for determining which additional foods should carry nutrition labeling.

This group has delayed action pending legislation in Congress. The criteria will primarily reflect

the significance of the food in the diet, the potential for misleading the public without labeling,

and other matters of public health significance. FDA seeks authority to require nutrition labeling

on many food categories which contribute to the diet in a significant way. To help relieve industry

of the task of nutrient analysis, the agencies have formalized a policy pertaining to the use of a composite data base for labeling those foods to which no nutrients have been added.

2) The agencies have agreed to conduct research to determine which nutritional labeling format consumers find more useful and convenient. Industry will be encouraged to experiment with formats

consistent with the principle of the current quantitative system.

3) FDA will propose regulations to establish uniform serving sizes for foods.

2
4) The Agencies plan to make sodium and potassium labeling a mandatory part of nutrition labeling

format and will ask for legislative authority to require such labeling on appropriate foods.

5) The Agencies will also require that cholesterol and fatty acids be linked in nutrition labeling

so that, if a claim is made for either, or if the manufacturer elects to include one on the label

then the other would also be required.

6) The Agencies plan to make labeling of total sugars a mandatory part of nutrition labeling format

and will ask for legislative authority to require such labeling on appropriate foods.

7) No change in fiber labeling is proposed, but FDA will intensify research efforts relative to methodology for fiber and its value in the diet.

Nutritional labeling is an effective educational tool. Its principal purpose is to provide

consumers with meaningful nutrition information and allow for product comparisons. It likewise

allows the consumer, measured assurance of nutrient content through government regulations. Any

product which carries nutrition information is subject to FDA laboratory analyses for all nutrients

claimed to be in the packaged product. To be in comformance with regulations, labeled products

must meet three criteria:

1. For essential nutrients which have been added to the product, FDA's analytical values for a composite sample from 12 packages of the product must equal or exceed label claims.

2. For essential nutrients that are indigenous to the product or its food ingredients, or for claimed

polyunsaturated fat, the analytical values must equal or exceed 80 percent of the label claims.

3. Analytical values for calories, carbohydrate, fat, saturated fat, cholesterol, and sodium must

not exceed 120 percent of the label claims. Products not meeting these criteria may be declared

misbranded.

3. Infant Formula

One specific type of food which requires mandatory declaration of nutrients is infant formula.

The President recently signed a bill which specifies the nutrient composition for infant formula.

These nutrient specifications are based on the recommendations of the Committee on Nutrition of the

American Academy of Pediatrics (CON/AAP). In addition to the responsibility of the manufacturing

company for the information provided on the nutrition label, a large number of independent, university, and government laboratories are performing additional analyses on infant formula collected from

store shelves. For many of these laboratories this is the first time they have analyzed infant

formula. As a result, FDA receives many complaints that Infant Formula Brand X is deficient in

certain nutrients. When these complaints are received, samples are collected and analyzed, not only

for the suspected deficient nutrients but for the other nutrients as well. The result has been that

most of the reported deficiencies are not really deficiencies at all but are errors in the analyses

of the products. The availability and use of a Nutrient Standard Reference Material would not guarantee accurate analyses but would at least provide the laboratory with a benchmark to gauge the

accuracy of their analytical results.

4. Association of Official Analytical Chemists

The Association of Official Analytical Chemists (AOAC) is a unique, non-profit scientific

organization whose primary purpose is to serve the needs of government, regulatory and research

agencies for analytical methods. The goal of the AOAC is to provide methods which will perform with

the necessary accuracy and precision under usual laboratory conditions. Since its founding in 1884

the AOAC has provided a mechanism to select methods of analysis from published literature or develop

new methods, collaboratively test them through interlaboratory studies, approve them, and publish

the approved methods. Only methods validated by collaborative tests receive approved status and

only approved methods are included in the AOAC compendium, "Official Methods of Analysis."

Although "Official Methods of Analysis" contains many analytical methods, numerous methods in

widespread use are not currently included. These methods are not included because the collaborative

studies necessary for acceptance of the methods have not been completed. Perhaps the primary reason

for this situation is that laboratories are unwilling to participate in these studies. Therefore,

although the AOAC Book of Methods may be regarded as unique and fulfilling a valuable function by

compiling tested, approved methods, it must also be recognized as being incomplete because it does

not contain all currently used analytical methods for nutrients.

3
Perhaps the ideal analytical results would be produced by a capable analyst, using proven AOAC

methodology and analyzing National Bureau of Standards (NBS) SRM's along with the samples to provide

a quality control check. Problems arise because sometimes all three are not available. There are certainly many capable analysts and the AOAC Book of Methods contains many analytical methods.

There are even several SRM's available; however, these are certified only for trace and major

elements. There are currently none for organic nutrients. A primary purpose of this workshop is to investigate the development of one or more SRM's certified for organic nutrients.

5*. Summary

In summary it can be said that:

1. Nutrition labeling currently is a voluntary regulation, but it is in widespread use. Pending legislation would provide USDA and FDA with authority to make nutrition labeling mandatory.

2. Data banks have been used to provide nutritional information for foods which contain only

indigenous nutrients, and use of data banks is expected to increase as mandatory labeling requirements

are issued. The information put into the data banks must be of the highest quality to ensure accurate nutritional information.

3. New and/or improved analytical methodology will be developed which is rapid and perhaps

automated in order to provide continuous quality control and to further enhance the data banks.

4. Availability of Standard Reference Materials is vital to the integration of nutrient data,

laboratory quality control, and analytical methods development. It is hoped that this workshop will

provide the basic information necessary to the development of SRM's for organic nutrients and will

provide a framework whereby future SRM's may be selected and developed.

4

1
National Bureau of Standards Special Publication 635. Proceedings of the Workshop on Reference

Materials for Organic Nutrient Measurement held at NBS, Gaithersburg, Maryland, October 23, 1980.

THE PROCESS AND REQUIREMENTS FOR THE DEVELOPMENT OF A STANDARD REFERENCE MATERIAL

(Copy of Notes Used During Presentation)

George A. Uriano

National Bureau of Standards

Washington, D. C. 20234

1. Reference Materials

Standard Reference Materials are wel 1 -characterized stable materials that are produced in

quantity and have one or more physical or chemical properties measured and certified by NBS. Today

I'd like to talk about the technical and programmatic requirements for the development of an SRM -

with particular emphasis on potential SRM's for organic nutrient analysis. I'll also comment briefly

on what I perceive is the role of SRM's in helping to assure accurate and compatible measurements on

a national scale. NBS now has available for sale nearly 1,000 different SRM's for use in four major

areas:

(1) Industrial Quality Control

(2) Environmental Analysis and Monitoring

(3) Clinical Analysis and Health Related Measurements

including Botanical/Biological

(4) Basic Metrological Applications.

During fiscal year 1980, NBS distributed over 40,000 SRM units to approximately 10,000 users throughout the world.

2. Guide for Requesting Development of SRM's

Generally the three criteria shown here should be satisfied before NBS will undertake development of a new SRM.

1. A technical need - usually associated with a critical measurement problem - must be

identified, i.e. , improving the accuracy of an important measurement method.

2. The production of the SRM at an organization other than NBS should not be technically or economically feasible or appropriate.

3. The SRM should address an industry-wide or community wide need rather than one of a particular manufacturer or individual laboratory.

In a few cases, the SRM's are mandated for use by federal regulations - many of these address public

health and safety problems such as in Air Pollution monitoring.

NBS would appreciate obtaining information which justifies the technical need for particular

SRM's for organic nutrient analysis in terms of economic impact and/or impact on public health and

safety. Some consideration should also be given to estimating future demand for the particular SRM. For your information, the average compositional SRM sells about 100 units per year. NBS would also

like to be made aware of laboratories that are willing to participate in the preparation or analysis

of the materials. Evidence of outside laboratory cooperation considerably strengthens justification

for producing the SRM.

5
3. NBS SRM Certification Philosophy

Three principal modes are used by NBS to certify SRM's.

1. Measurement by a so-called definitive method of known and demonstrated accuracy having

essentially no systematic error - an example is the Cholesterol in Serum method, based on isotope

dilution mass spectrometry.

2. Measurement by two or more independent - so-called reference methods - having inaccuracies

(including systematic errors) that are small relative to certification requirements. Example - the

two independent gas chromatography and liquid chromatography techniques used to certify the anticonvulsant drug in serum SRM - the first SRM ever certified by NBS for trace organic constituents.

3. The third certification mode involves interlaboratory testing and is generally used only

for renewals of previously issued SRM's or to obtain supplemental data.

The objective of the SRM Program is to achieve interlaboratory compatibility and accuracy on a national scale.

The total uncertainty assigned to the certified value of an SRM generally includes three

components:

1. The first is due to method precision and is amenable to standard procedures for statistical

analysi s.

2. The second is due to systematic error of the method or bias among methods. In many cases

the value assigned to this component is based on experience and/or judgment of the

analyst.

3. If significant , material variability or inhomogeneity accounts for the third component.

With regard to SRM's certified for chemical composition, three of the most important criteria

to be met during certification are:

1. The material must be homogeneous with regard to the constituents of interest. A testing

protocol must be developed to quantitatively assess homogeneity within or between bottles

and/or samples.

2. Long term stability of the material must be assured usually through the use of special

processing procedures or treatment of the material. Special storage requirements may also

be required.

3. Another necessary requirement to be met is the availability of one or more certification

methods, which have been well characterized with regard to precision and accuracy.

4. Homogeneity Testing

Normally preliminary homogeneity testing is performed on a limited number of key constituents

to detect trends or patterns in the variation of the levels of constituents, which could cause

problems.

If inhomogeneity is shown to be small, we normally proceed directly to carry out a certification

program.

If inhomogeneity is very large - causing the total uncertainty to be large relative to end-use

requirements - the material must be either rejected or reprocessed.

If inhomogeneity is of the same magnitude as the estimated errors of certification methods and

the total estimated uncertainty is small relative to end-use requirements, then one proceeds to quantify the inhomogeneity for inclusion in the uncertainty. Certain restrictions such as requiring

the use of minimum sample size for analysis may be imposed to assure proper level of homogeneity.

6
5. Stability of Food SRM'

The stability of materials is undoubtedly going to cause more serious problems than inhomogeneity

with regard to developing organic nutrient SRM's. Stability of constituents and the matrix itself

may be affected by microbial attack, and/or chemical reactions which in turn are sensitive to environmental conditions such as temperature, radiation, or presence of oxygen or moisture.

The following questions are pertinent to this aspect of SRM development.

(1) What materials could be used that would minimize stability problems? What steps might be

taken during processing and/or storage to assure adequate stability?

(2) Are there simple and accurate methods for monitoring the long term stability of the material

without having to essentially duplicate the certification procedure?

(3) What constraints do you have to impose on the user to minimize stability problems?

NBS has not had much experience with developing SRM's for organic nutrient analysis. We have

certified over the last 10 years, a series of agricultural products and plant tissues for a number

of elements at the trace, and minor constituent levels. The first in this series was orchard leaves

and the last, oyster tissue.

A number of different processes have been used to assure adequate stability of these materials.

These include freeze and air drying and Co-60 irradiation. Some of these techniques may not be

useful for stabilizing organic nutrient SRM's. For example, the use of Co-60 radiation would

probably damage both the matrix and many organic constituents of interest.

We probably will have to develop special processes to stabilize SRM's for organic nutrient

analysis. To be a useful SRM, the material should be stable for at least 4 or 5 years and preferably 10 years or more.

Sometimes, it is necessary to take a number of different steps to assure adequate stability in a material. A case in point is the soon-to-be-issued wine SRM 1

, which is being certified for its

ethanol content. At the recommendation of oenologists at the University of California, Davis and a committee of experts from the wine industry, a number of separate special processing steps were undertaken to stabilize the wine. These included ion exchange of K for Na, increasing the alcohol

content, and addition of Cu and Fe. I won't elaborate on why steps were necessary to stabilize the

wine. Suffice it to say that to obtain requisite stability - i.e., 5 years or more - some unusual

steps may have to be taken during the processing of material intended for use as an SRM.

6. Summary

In summary, certification of organic constituents in a food SRM will depend on (1) assuring the

homogeneity and long-term stability of both constituents and matrix, and (2) the availability of

certification methods of requisite precision and accuracy. Subsequent speakers will address these

questions. Particular attention must be paid to the question of stability.

Editor's Note: This material is available from NBS as SRM 1590, Stabilized Wine.

7
National Bureau of Standards Special Publication 635. Proceedings of the Workshop on Reference

Materials for Organic Nutrient Measurement held at NBS, Gaithersburg, Maryland, October 23, 1980.

AN ASSESSMENT OF THE ACCURACY AND PRECISION OF THE METHODS USED FOR THE MEASUREMENT OF ORGANIC NUTRIENTS IN CEREAL AND GRAIN PRODUCTS

David C. Egberg

General Mills, Inc.

9000 Plymouth

Minneapolis, Minnesota 55427

A sincere concern for the nutritional quality of the U.S. food supply on the part of industry

and government has given rise to nutritional labeling and nutrient fortification of foods. Under the

authority of the 1938 FD & C Act, the FDA formulated in 1941 requirements for Minimum Daily Requirement labeling and in 1942 standards for nutrient enriched flour became effective. Since that time,

these programs have been credited with the elimination of certain nutrient deficiency diseases in the

United States.

In 1973, the FDA published "voluntary" food labeling regulations with nutritional labeling

expressed in terms of the percent of the National Academy of Sciences' Recommended Daily Allowance of

nutrients on a per serving basis. Also, in January of this year the FDA published a food fortification policy. Clearly, the implication of all this interest and regulatory activity for the food

chemist, in the public or private sector, is an increased need for accurate nutritional analysis of

foods - the subject of this presentation.

The Code of Federal Regulations states that "... food composites shall be analyzed by AOAC

methods where available and when no AOAC method is available, by reliable and appropriate analytical

procedures. Alternative methods of analysis may be submitted to the FDA to determine their acceptability". This means that AOAC methods are used to check compliance by the FDA and hence, in a legal sense, are correct or accurate by definition.

It is generally recognized that due to the lengthy, and I might add necessary, collaborative

process required for official status, AOAC methods frequently are not the most current methods of

choice. Industrial chemists who are concerned with diminishing analytical costs may use any technology at their disposal provided there is sufficient correlation with AOAC methodology to guarantee

compliance upon FDA sampling. This striving for efficiency has lead to a proliferation of analytical

methods for nutrient analysis in foods. The objective of this workshop is to help the NBS provide

reference materials for organic nutrients. These reference materials may then be used to assess the

accuracy of analytical methods, as well as laboratory performance. It is worth repeating, however,

that for nutritional labeling, comparison with AOAC methods used by the FDA and state agencies is

currently the most fundamental criterion. No amount of checking with reference materials will change

a non-compliance situation.

In discussing the precision and accuracy of methods used for organic nutrient analysis, I will

focus primarily on the five vitamins required when a food product is labeled. Although protein,

carbohydrate, and fat analyses are important for labeling and calorie calculation, the methods used

are quite standardized for cereals and the CFR allows a 20 percent tolerance in calories, carbohydrate, and fat labeling.

In discussing the precision and accuracy of methods used for riboflavin, thiamin, niacin, vitamin A and vitamin C, I will review some sources of error associated with extraction, method interference, and method execution. Some reproducibility, repeatability, recovery and method comparison

will be presented. I will be dealing almost exclusively with instrumental and chemical methods

leaving microbiological assay to someone with more experience.

Between- laboratory reproducibility of nutrient methods may be assessed by collaborative study.

Within-laboratory repeatability is less of a concern and is usually controlled by individual laboratories to within five percent relative error. Accuracy or "the true value" is most important and more elusive; it is assessed operationally by recovery data, between-method comparative data and of course with reference materials which we anticipate will be forthcoming through the efforts of this

workshop.

8
1. Riboflavin

If protected from light, our laboratory has observed no loss of riboflavin in ground cereal

samples (2-8 percent moisture) at room temperature over 90 days. Riboflavin exists in foods as the

phosphate ester of the carbohydrate moiety as well as the adenine dinucleotide. Because of differences in fluorescence response and extraction efficiency, acid and/or enzymatic hydrolysis is

required when other than added riboflavin is analyzed. In the presence of elemental iron and hot

acid, riboflavin is reduced; this is usually not a problem with cereals but is a concern with con- centrates. If enzymes are used for hydrolysis, a correction is necessary for riboflavin added with

the enzyme.

Potential sources of error in the fluorometric method are as follows: (1) filter paper adsorption of riboflavin (glass filter recommended); (2) high iron; and (3) sulfur formation with

dithionite addition.

Any fluorescing material interferes which is not oxidized by permanganate and is reduced by

dithionite. These exist in certain foods and adsorption/desorption using zeolite, magnesium

silicate, and Fuller's earth is recommended for certain foods. Comparison of the fluorometric

method with an High Performance Liquid Chromatograph (HPLC) method in our lab for ready to eat (RTE)

cereals has demonstrated a bias of about 15 percent (1.5 mg/100 g) from the fluorometric even though

both methods show excellent recoveries. Within laboratory repeatability is usually about 4-6 percent

relative error and typical data are shown in Table 1. The recovery is from an automated method which

also shows better repeatability. Between laboratory variability is shown in Table 2 under controlled

method conditions and conditions using different methods. The reproducibility about doubles when

many different methods are used. The standard deviation of 25 percent with outliers removed graphically demonstrates the need for standard reference materials.

Table 1. Recovery and Repeatability of AOAC Fluorometric Riboflavin.

Twelve RTE Cereals (Corn, Oat, Wheat)

Average 3.35 mg/100 g (56% RDA)

Within Laboratory Standard Deviation 6.9%

(11 degrees of freedom)

Average Recovery

Automated 99 ± 4.6%

Table 2. Reproducibility of Riboflavin.

Sixteen laboratories AOAC fluorometric

Wheat Flour

Bread

Oat RTE Corn-Oat

Average 0. 50 mg/100 g Average 0.13 mg/100 g

Average 0.43 mg/100 g

Average 1.95 mg/100 g Average 2.45 mg/100 g

Relative

Standard Deviation

9.6%

18.3%

11.9%

10.9%

11.0%

American Association of Cereal Chemists (AACC)

flour check sample

Thirteen laboratories any method

Hard Wheat Flour Average 0.43 mg/100 g

Association of Vitamin Chemists (AVC) mixture

Nineteen laboratories any method

Fabricated Average 1.05 mg/100 g

19.0%

25.0%

To summarize, fluorometric and chromatographic methods provide reasonably precise and accurate

riboflavin results. The between laboratory reproducibility needs improvement.

9
2. Thiamin

Over a 90-day period, our laboratory has observed no loss of thiamin in ground cereal samples

(2-8 percent moisture) stored at room temperature.

Thiamin exists in foods as free thiamin and the phosphate ester which must be cleaved by enzyme

hydrolysis for isobutanol extraction in the standard thiochrome method. Like riboflavin, acid/

elemental iron reduction is a source of error in vitamin concentrates and the thiamin in an enzyme

used must be subtracted.

Potential sources of error in the fluorometric thiochrome method are as follows: (1) thiamin

as disulfide; (2) non-standard Decalso column recovery; (3) overloading Decalso (>10 ug/g).

Liquid chromatographic methods have been reported but with limited comparison data. Typical

within laboratory repeatability of the thiochrome method is 4 to 6 percent with good recoveries as shown in Table 3. The AACC flour sample and the AVC check sample show a between laboratory reproducibility of about 15 percent relative (Table 4) with outliers removed.

Table 3. Recovery and Repeatability of AOAC Thiochrome Method.

Ten RTE cereals (Corn, Oat, Wheat)

Average 3.10 mg/100 g (57% RDA)

Within Laboratory Standard Deviation 4-6.5%

(18 degrees of freedom)

Average Recovery

Automated 98 ± 3.8%

Table 4. Reproducibility of Thiamin.

Relative

Standard Deviation

AACC flour check sample

Fourteen laboratories any method

Hard Wheat Average 0.83 mg/100 g 19%

Average C.30 mg/100 g 13%

AVC mixture

Eighteen laboratories all thiochrome assay

Fabricated Average 1.34 mg/100 g 15%

3. Niacin

Over a 90-day period, our laboratory has observed no loss of niacin in ground cereal samples

(2-8 percent at room temperature).

Niacin exists in foods as free acid, amide, and amide derivatives, all of which are converted

to nicotinic acid by acid or base hydrolysis prior to analysis. Chemical analyses with cyanogen

bromide and microbiological methods are commonly used. Liquid chromatographic methods have been

reported but with little comparative data.

For cereal products the AOAC microbiological method using Lactobacillus plantarum- compares

favorably with the AOAC colorimetric method as shown in Table 5 giving some of the data from an AACC collaborative study using ten products and seven laboratories. The excellent reproducibility

of 11 percent or less is not typical when different methods are used under routine conditions as evidence by a 17 and 15 percent between laboratory reproducibility in two recent AVC collaborative

studies (6.61 and 2.66 mg/100 g, respectively).

10

i
Table 5. Reproducibility of Niacin.

Microbiological Chemical

AACC collaborative study over seven laboratories

Soy Flour Average d. ing/ 1 UU g

oL mg/ I UU g Standard Deviation 2 4% 11 0%

k 1 1 cerea

Average 1 / QO mg/ i uu g

1 Q1 3 4 mg/ i uu g Standard Devi ati on 4

r\0/ 5

00/

Flour Average 1 4 mg/100 g 1 3 mg/100 g Standard Deviation 7 1% 6 9%

Bread Average 7 03 mg/100 g 7 17 mg/100 g Standard Deviation 6 3% 5 4%

Rice Average 1. 23 mg/100 g 1 26 mg/100 g Standard Deviation 8 1% 10 0%

4. Vitamin A

Vitamin A activity exists in cereal products as pre-vitamin A carotenoids and as retinol esters

added as fortification. Over a 90-day period, our laboratory has observed no loss of retinol palmitate in ground cereal samples (2-8 percent moisture) stored at room temperature.

Liquid chromatographic, f 1 uorometri c , and Carr-Price colorimetric methods are commonly used

for vitamin A analysis. Potential sources of error in the popular AOAC Carr-Price method are:

(1) potential destruction during saponification; (2) incomplete partitioning into petroleum ether;

(3) incomplete recovery from alumina column; (4) contamination of the Carr-Price reagent, and

(5) improper timing of the antimony trichloride reaction. Most of these problems are eliminated

using liquid chromatography, but care must be taken to ensure complete extraction with encapsulated

vitamin A and to combine the all-trans and 13-cis peaks if the chromatographic conditions used,

separate these isomers.

An HPLC method used in our laboratory has a within relative standard deviation of about 4.0

percent, 95 percent recovery, and compares favorably with Carr-Price. In a recent AVC collaborative

study, however, 14 laboratories ranged from 0 to 11,880 vitamin A Ill's with a mean of 8,313 IU. Clearly, many of these labs could use a reference material.

Error associated with pre-vitamin A determination is related to destruction during saponification and the inability to separate pre-vitamin A carotenoids from other carotenoids prior to quantitation. To a large extent, these difficulties are eliminated by omitting saponification and

using HPLC, but it is still very difficult to separate a from 0 carotene.

5. Vitamin C

Ascorbic acid exists in cereal products due to fortification. It is a reactive vitamin and care must be exercised in storing the sample and extract. In our laboratory a ground cereal product

sample (2 percent moisture) showed no vitamin C destruction after 90 days, yet another product with

as little as 8.3 percent moisture showed 53 percent loss after 30 days.

Upon extraction of the vitamin C, rapid analysis within 30 minutes is recommended, especially

in the presence of solid material.

The ascorbic acid is reversibly oxidized to dehydroascorbic acid which is hydrolyzed to biologically unavailable diketogulonic acid. Only trace levels of dehydroascorbic acid are present

in cereal products. Of the many methods available for ascorbic acid determination, the most common

are the indophenol titration, microf 1 uorometric , osazone, and HPLC. The indophenol method is subject to great error due to reducing substances (sulfhydryl, reductones, etc.) other than ascorbic

acid and it doesn't measure the dehydroascorbic acid. The osazone technique is subject to interference by the gulonic acid although not a major concern. The HPLC method omits the dehydro form

unless it is reduced to ascorbic acid prior to analysis. The fluorometric method is comparatively

free of problems except when large amounts of browning products are present. Some typical perfor- mance specifications for the fluorometric, indophenol, and HPLC methods used in our laboratory are

11
shown in Table 6. Even though excellent repeatability and recovery data can be achieved, reproducibility data, from recent AVC collaborative study are very poor ranging from 3.8 to 293 mg/100 g 01 laboratories, fabricated food).

Table 6. Vitamin C.

HPLC

30 samples (corn, oat, wheat)

Within Laboratory Standard Deviation 4.5%

Recovery 98.4 ± 2.5%

No bias from fluorometric method

Indophenol

16 samples (corn, oat, wheat)

Within Laboratory Standard Deviation 4.0%

No bias from fluorometric method

Fl uorometric

34 samples (corn, oat, wheat)

Within Laboratory Standard Deviation 3.0%

Recovery 100.5 ± 2.0%

To summarize, numerous instrumental, chemical, and microbiological methods exist for the

analyses of organic nutrients in foods. Many of these are sufficiently accurate and repeatable if

applied properly by competent workers. The major difficulty exists in between laboratory reproducibility and poor method execution. I feel that Standard Reference Materials will help improve the

situation.

12
Proceedings of the Workshop on Reference

Gaithersburg, Maryland, October 23, 1980.

ACCURACY AND PRECISION OF NUTRIENT METHODOLOGY

E. R. Elkins

National Food Processors Association

1133 20th Street, N.W. Washington, DC 22036

Since 1964, the National Food Processors Association, formerly the National Canners Association,

Committee of Canning Industry Analytical Chemists (CCIAC), has been obtaining data on i nterl aboratory

variability of food composition analytical methods.

This paper will describe the CCIAC and discuss i nterl aboratory variability in nutrition methodology projects which the committee has cooperatively studied. We term these projects cooperative

studies because they are not strictly controlled like an AOAC collaborative study. AOAC methods are mostly used, however.

The members of the CCIAC are analytical chemists from the laboratories of companies belonging

to the NFPA. These companies are listed in Table 1.

Table 1. Committee of Canning Industry Analytical Chemists.

National Food Processors Association Members

American Home Foods

Baker/Beech-Nut, Inc. California Canners & Growers

Campbell Soup Company

Carnation Company

Del Monte Corporation

Environmental Science Associates

General Mills, Inc. Gerber Products Company

Green Giant Company

H. J. Heinz Company

Hunt-Wesson Foods, Inc. Libby, McNeill & Libby

National Food Processors Association

Ocean Spray Cranberries, Inc. Ralston Purina Company

Stokely-Van Camp, Inc. Van Camp Seafood Company

Westgate-Cal ifornia Foods, Inc.

Recently, participation in projects involving testing for nutrient quality and heavy metals has

been opened to interested commercial and government laboratories listed in Table 2.

National Bureau of Standards Special Publication 635.

Materials for Organic Nutrient Measurement held at NBS,

13
Table 2. Committee of Canning Industry Analytical Chemists.

Government and Commercial Laboratories

ABS Laboratories, Inc. Anresco

Columbia Laboratories

Food and Drug Administration

Nutrition International, Inc. Shankman Laboratories, Inc.

H. V. Shuster, Inc. Stoner Laboratories

United States Department of Agriculture

Warf Institute

Westreco

The committee was organized in 1964 to discuss problems in the field of pesticide residue

analysis. Since that time, its activities have broadened to include cooperative studies on environmental contaminants and nutrient testing. The committee meets twice a year to discuss the results

of the cooperative studies. Approximately 100 samples have been studied over the past 16 years.

The advent of nutrition labeling made it necessary for the canned foods industry to carry out

studies on currently available nutrition methodology; a typical nutritional labeling format is shown

in Table 3. The part of the nutrition labeling regulation which deals with method variability states

that no regulatory action will be based on the determination of a nutrient value which falls below

80 percent of the declared value by a factor less than the variability generally recognized for the

analytical method used in that food at the level involved.

Table 3. Sweet Peas Nutrition Information, Serving Size One Cup.

Calories 130 Carbohydrate 25 g

Protein 8 g Fat 1 g

Percent of U.S. Recommended Daily Allowance

Protein 10 Riboflavin 8

Vitamin A 15 Niacin 10

Vitamin C 40 Calcium 2

Thiamin 15 Iron 10

This variability has yet to be established. The processed foods industry through the CCIAC is actively engaged in obtaining data on interlaboratory variability for nutrition methods in a variety

of foods.

This workshop is designed to answer questions concerning the current state-of-the-art in organic

nutrient methodology. This paper will attempt to answer those questions concerning interlaboratory

variability for organic nutrients that must be measured if a manufacturer chooses to nutritionally

label their products. Since there are no products available in which exact concentrations of these

nutrients are known, we can only guess at the accuracy of the methods. We do feel that once a material has been tested by a large number of laboratories, the resulting mean is the best estimate

of the concentration of a particular analyte if the standard deviation is reasonably small at the

level being tested.

Many samples have been cooperatively studied by the CCIAC since 1973. Some of these are listed

in Table 4. For purposes of this paper, we have elected to discuss interlaboratory variability in

pureed peaches, green beans, a vegetable and liver mixture and a dry pet food.

14
Table 4. Committee of Canning Industry Analytical Chemists.

Nutrition Projects

Project Number

1 Tnniat'n 1 UIMCl LU Uliiir^ U 1 LC

9c Doac redo

"3 OUT II 4 jp 1 1 ICIL 1

1

t;J Fn y»mi 1 1 a T T r u i in lj i ci 11

6 Corn

7 L 1 VCI

QO

q rui hi u la li 10 Moat n a i \/onot ahl o anH Po^fla 1 ricd L , Da l i y , vcycidU I c dliu Lei cd I

1

1

r cdLl Icj

1 ? Di cdU L, i UlllUb 13

14 ^wppf Pnt^tnPQ

15 Tit una ma Fi^h i loll "in mi Oil \J \ i 16 Brsn

17 TnmAtn i unia f*at<^nn bd touu

l ft Hpp i e oauce

19 Bran

20 Mixed Vegetables and Meat

21 Corn

22 Vegetables and Liver

Table 5 shows data developed by the CCIAC on protein and fat in these products. The method

used for protein was the AOAC method, 1980 Edition, section 2.057-2.058; the factor of 6.25 was used

to calculate percent (%) protein from Kjeldahl nitrogen value. For fat, the AOAC method, 1980

Edition, section 18.043 of acid hydrolysis was used by the committee. Interlaboratory variation for

protein values at the level tested is considered good. The interlaboratory variation for fat analysis at the highest level is acceptable; variation at low fat levels is high, but we would expect it to be.

Table 6 shows interlaboratory variation for carotene, ascorbic acid and niacin content in green

beans, peaches, vegetable and liver mixture and a dry pet food. The dry pet food did not contain

carotene or ascorbic acid.

Methods of analysis for carotene in yellow fruits and vegetables are poor and this is reflected

in the high degree of variation in Table 6. The presence of xanthophylls makes the analysis difficult. HPLC methods for this determination looks promising.

Two methods are currently being used for ascorbic acid determinations. These are:

1. AOAC, 1980 Edition, section 43. 056-2-6 dichlorindophenol titration;

2. AOAC, 1980 Edition, section 43. 061-microf luorometric method.

The interlaboratory variability for this nutrient at higher levels would appear to be acceptable.

Results for niacin in these products at the levels tested, are also good.

15
Table 5. Inter laboratory Variability in Nutrient Analysis.

Product No. Mean Standard Deviation Coefficient of Variation

Protein Percent

Vegetables and Liver 14 2. 7 0. 10 4

Dry Pet Food 19 21. 4 0.46 2 Green Beans 18 1 , 6 0. 15 9 Peaches 23 0. 4 0. 10 25

Fat Percent

Vegetables and Liver 11 0. 46 0.05 12 Dry Pet Food 12 10. 66 0.60 6 Green Beans 15 0. 26 0.11 42

23 0. 22 0. 18 83

Table 6. Interlaboratory Variability in Nutrient Analysis.

Product No. Mean Standard Deviation Coefficient of

Carotene (u/100 g)

Green Beans 16 247 62 25

Peaches 17 516 200 39

Vegetables and Liver 12 591 135 23

Ascorbic Acid (mg/100 g)

Vegetables and Liver 12 2. 7 0.8 30

Green Beans 16 3. 5 1.7 49

Peaches 19 22. 2 7.8 8

Niacin (mg/100 g)

Peaches 18 0. 49 0. 12 24

Vegetables and Liver 12 1. 45 0. 11 8

Green Beans 18 0. 53 0. 15 28

Dry Pet Food 16 11. 21 1.18 11

In Table 7 are data on interlaboratory variability for thiamin and riboflavin analyses. Again,

at the levels tested, these data are considered satisfactory.

Some observations that can be made from these data are:

1. In many cases, but not all, the precision of the methods depends on the concentration

of the nutrient being tested. Interlaboratory variability i ncreases as concentration

decreases.

Interlaboratory variability also depends on the food matrix. Some foods

difficult to analyze, for example, carotene in yellow foods vs. green foods. are more

Extractions are easier in cooked foods.

Members of the NFPA-CCIAC have for some time used excess sample from a particular study as an inhouse reference material. The indication is that they will continue to do so. So far, these

samples have proved to be an excellent quality assurance program. However, the industry does need

Standard Reference Materials that have been certified by the National Bureau of Standards.

16
Table 7. Inter! aboratory Variability in Nutrient Analysis.

Product No. Mean Standard Deviation Coefficient of Variation

Thiamin (mg/100 g)

Peaches 18 Vegetables and Liver 12 Green Beans 17 Dry Pet Food 17

Peaches 19 Vegetables and Liver 13 Green Beans 17 Dry Pet Food 17

0.01

0.02

0.033

1.17

0.003

0.008

0. 008

0.21

Riboflavin (mg/100 g)

0.033

0. 29

0. 09

0.95

0.013

0.05

0.01

0.22

29

42

24

18

39

17

1123

17
National Bureau of Standards Special Publication 635. Proceedings of the Workshop on Reference

Materials for Organic Nutrient Measurement held at NBS, Gaithersburg, Maryland, October 23, 1980.

PROBLEMS IN THE MEASUREMENT OF ORGANIC NUTRIENTS IN FOOD PRODUCTS: AN OVERVIEW

Kent K. Stewart

Nutrient Composition Laboratory

Room 225, Bldg. 308

U.S. Department of Agriculture

Beltsville, Maryland 20705

The accurate and precise measurements of organic nutrients in foods are the underpinning of our understanding of the human requirements for those nutrients and our understanding of the nutrient

contents of foods so that we may meet these requirements. We would prefer to utilize methods which

contain the elements shown in figure 1. Of particular interest to this workshop are those features

of the method which are concerned with quality control. The availability of pure standards and

adequately documented standard reference materials are critical to adequate quality control for

analytical determinations.

FOOD SAMPLE

H0M0GENIZATI0N

AND SUBSAMPLING

•STANDARDS

i

SRM

i

EXTRACTION

r \ t SEPARATION

i f DETECTION AND IDENTIFICATION

r r r r CALCULATION OF RESULTS

AND REPORT GENERATION

Figure 1. Flow chart for an ideal analytical method for nutrient

analysis of food.

REPORT ON SRM

REPORT ON STANDARDS

REPORT ON SPIKED SAMPLES

REPORT ON UNKNOWNS

VALIDATED RESULTS

18
Before discussing this topic further, it is useful to briefly evaluate the state-of-the-art for

organic nutrient determinations in foods (see Table 1). If asked "If a qualified analyst uses current methods under the appropriate conditions, for which nutrients are the results of the determinations in food matrices likely to have sufficient accuracy and precision?", the answer must

be, for most foods, only those nutrients for which the methods are classified as "sufficient" or "substantial". Methods for those labeled "tentative" will probably give acceptable results. Those

determinations which are classified "conflicting" yield different results with different methods.

No one knows which, if any, of these results are accurate. Those nutrient methods listed as "fragmentory" have had so little study that the accuracy is unknown. As shown in Table 1, there are a large number of organic nutrients for which there are not adequate methods for determination of the

nutrient levels in foods.

Table 1. State of Development of Methodology for the Analysis of Organic Nutrients

in Foods as of 1980.

Sufficient Substantial

Amino Acids (Most)

Choi esterol

Fatty Acids

Individual Sugars

Niaci n

Ri bof 1 avi n Thiamin

Vitamin C

Tentatively Acceptable

Amino Acids (Some)

Fat (Total)

Trans-Fatty Acids

Conf 1 icting

Fiber

Fol aci n Pantothenic Acid

Protein (Total

)

b

Starch

Sterols (Others)

Vitamins A

B "

Fragmentary

Bi oti

n

Choi i ne Heme-Iron

Vitamin K

'12

New methods look very promising.

5 Total nitrogen methods would have a ranking of sufficient.

It is worthwhile to examine some of the background of this state of affairs. Basically "nutrients" are a group of compounds which elicit given biological responses. When too low a level of a "nutrient" is consumed, one set of biological responses - "a deficiency" - is observed in the test

animal. When a adequate level is consumed, another set of biological responses - "normal" - is observed. For most nutrients, several isomers of a given compound will elicit qualitatively (though

not always quantitatively) similar biological responses. For example, in the case of the vitamins,

niacin and vitamin C have two isomeric forms which elicit similar biological responses; riboflavin,

thiamin, and vitamin E have three; vitamin D has four; vitamin B 12 has five; vitamin B 6 has six;

and vitamin A and folacin each have greater than 10. Often the relative biological responses of the

nutrient isomers is species specific, thus nutrient activities determined using one species (i.e.,

rats) cannot be directly transposed to another species (i.e. , man). Many traditional organic nutrient

assay methods depend either upon feeding studies with a test species (usually microorganisms, rats,

or chicks) or upon single chemical reactions for which a number of isomers give similar responses.

While the originator of the assay usually did the necessary development and quality control for the

determination of a given nutrient in a given food matrix, these techniques have often been utilized

for the determination of that nutrient in different matrices without revalidating the methods.

Often these traditional methods do not provide sufficiently accurate and precise results for all food matrices. It is desirable to have assays which separate the biologically active isomers from

each other as well as from the bulk of the food matrix, to quantitate each isomer separately, to assign a biological activity to each isomer and then to sum the biological activities to obtain the

total nutrient activity for the food. These desired techniques should have built in quality control

systems which include internal standards, pure standards of each nutrient isomer and standard

reference materials of similar matrices to the food sample. Many believe that it is desirable to do the quality control operations in each set of determinations. Thus, it will be necessary to have pure standards for each isomer of each nutrient of interest and to have standard reference

materials representative of the important food matrices for these nutrients. These pure standards

and standard reference materials are needed for within method quality control and for development

and validation of new or untested methods. This is a large assignment and some sense of priorities

19
problems in the United States (see Table 3). From the information presented in Tables 2-3 it is

possible to set priorities for research on new methods (see Table 4) and for routine determinations

(see Table 5).

Table 2. Organic Label Nutrients.

Biotin

Carbohydrate

Cholesterol

Fat

Fatty Acids

saturates

polyunsaturates

Folacin

Niacin

Panthothenic Acid

Riboflavin

Thiamin

Vitamin A

B

6 B 12

C

D

E

Table 3. Inadequate and/or Excessive Organic Nutrient Intakes and Their

Contribution to Existing U.S. Public Health Problems.

No Known Contribution

to Existing Problems

Biotin

Choi i ne

Gl ucose

Mai tose

Pantothenic Acid

Starch

Suspected to be Contributing

to Existing Problems

Amino Acids 3

Fatty Acids

Fructose

Niaci n Other Sterols

Total Protein3

Trans-Fatty Acids 3

Vitamin E Vitamin K

Accepted as Contributing

to Existing Problems

Cholesterol

Folacin

Lactose

Nutrient Fiber

Riboflavin

Sucrose

Thiamin

Total Fat

Vitamin A

B,.

12

It is unlikely that increased information on the nutrient composition of foods

for these nutrients will significantly help in combating in public health problems associated with these nutrients.

20
Table 4. Suggested Priorities for Research on New or Improved Analytical

Methodologies for Organic Nutrients in Foods.

First Priority

Folacin

Second Priority

Third Priority

Niacin

Total Fat

Nutrient Fiber

Sugars

Riboflavin

Sterol

s

Vitamin A

Vitamin B,

Trans-Fatty Acids

Vitamin

Vitamin D Vitamin E

'12

Vitamin K

These priorities were determined by an assessment of the severity of the

U.S. public health problems and the adequacy of the analytical methods.

Table 5. Suggested Organic Nutrient Priorities for Routine Analysis of Foods.

First Priority

Cholesterol

Fatty Acids (including Trans)

Second Priority

Niacin

Thiamin

Riboflavin

Neutral Detergent Fiber

Sugars

Vitamin E

Total Fat

Vitamin C

These nutrient priorities were determined by an assessment of the relationship

of the nutrient to U.S. public health, state of the data for the nutrients, and

the adequacy of the analytical methods available for assay.

Pure standards are needed to develop adequate methods for determination of given nutrients

(see figure 2). A great deal of work has been done in this area and often either the U.S. Pharmacopoeia or the National Bureau of Standards or both have developed pure standards of many of the

major organic nutrients. This is particularly true of those isomeric forms which are used as supplements. However, there are a number of nutrient isomers for which pure standards are not

available. This is especially the case for those forms which are relatively unstable.

21
PREP AND STORAGE

OF PRIMARY PURE STANDARDS

SEPARATION AND QUANTITATIVE DETECTION

OF STANDARDS, EXTRACTS AND SPIKED EXTRACTS

VERIFICATION

OF IDENTIFICATION

AUTOMATION

FOOD EXTRACTION

METHODS AND

CLEAN UP

AUTOMATION

PREP AND STORAGE

OF PRIMARY

FOOD STANDARDS

r

SAMPLE

HOMOGEM I ZAT I ON AND SUBSAMPLING

AUTOMATION

L_.

QUALITY CONTROL

VALIDATION OF RESULTS

REPORT GENERATION

AUTOMATION

Figure 2. Flow chart for the development of an ideal analytical method.

The Standard Reference Materials for organic nutrients are much less well developed. Table 6 lists the Food Standard Reference Materials available as of 1978 with their certified nutrients.

When this is compared with the priorities for routine analysis (Table 5) it is apparent that much

remains to be done. The development of food standard reference materials fortified with those forms

of the nutrients most commonly added to foods would be an important first step. These SRM's would

be useful to the food industry, the regulatory laboratories and the research laboratories. Development of SRM's certified for the other isomeric nutrient contents could then follow.

22
Table 6. Food Standard Reference Materials Certified for Nutrient Content.

Food Category

Cereal Grain

Meat

Fish

Poul try

Vegetables, Leafy

Root, Vine

Fruit

Legumes

Eggs and Egg Products

Dairy Products

Fats and Oils

Beverages

NBS Available

Wheat Flour-SRM 1567

Rice Flour-SRM 1568

Bovine Liver-SRM 1577

Tuna-RM 50

Spinach-SRM 1570a

Nutrients Certified

Some Inorganic

Some Inorganic

Some Inorganic

Information Values

Some Inorganic

Some Inorganic

SRM Needed

Wheat Bran

Beef Muscle

Pork Muscle

Fish Meat

Chi cken

Lettuce

Potato, Tomato

Apple, Orange

Bean, Pea

Egg

Cheese

Cooking Oil

Soft Drink, Milk

Editor's Note: SRM 1570 is no longer available. Other plant tissue SRM's are available

which may be useful for these analyses.

Nutrient Composition Laboratory; Nutrition Institute; USDA, SEA-FR, NER, BARC; Beltsville,

Maryland 20705; 1978.

Summary

The state-of-the-art for determining the organic nutrients in biological materials and particularly foods needs improvement. The development (where necessary) of pure standards for isomeric

forms of the nutrients used in food fortification and Standard Reference Materials certified for

these nutrients would be an important first step in the development of the needed organic nutrients

reference materials.

Suggested Readings

Nutrient Analysis: Analytical Needs and Problems

Stewart, K. K. Statement before the Select Committee on Nutrition and Human Needs of the United

States Senate, Ninety-Fifth Congress; 1977. 21-27, 73-82.

Stewart, K. K. Nutrient Analyses of Foods: A Reexamination. Hertz, H. S. , and Chesler, S. N. , eds. Proceedings of the 9th Materials Research Symposium; 1978 April 10-13; Nat. Bur. Stand.

(U.S.) Spec. Publ. 519; 1979. 249-255.

Stewart, K. K. Nutrient Analysis of Foods: The State of the Art for Routine Analysis. Stewart,

K. K. , ed. Proceedings of Symposium entitled "Nutrient Analysis of Foods: The State of the

Art for Routine Analysis". Presented at the 93rd Annual Meeting of the Association of Official

Analytical Chemists. Washington, D.C.; October 1979. 1-19.

Wolf, W. R. Nutrient Analysis of Foods: Analytical Needs and Problems. Anal. Chem. 50: 190A-194A;

1978.

23
Standard Reference Materials and Meaningful Measurements. Seward, R. W. , ed. Proceedings of the

6th Materials Research Symposium 1973; Nat. Bur. Stand. (U.S.) Spec. Publ . 408; 1975.

Accuracy in Trace Analysis: Sampling, Sample Handling, Analysis. LaFleur, P. D. , ed. Proceedings

of the 7th Materials Research Symposium 1974; Nat. Bur. Stand. (U.S.) Spec. Publ. 422; 1976.

Report of Working Group on Cardiovascular Priorities in Food Composition Analysis. National Heart,

Lung and Blood Institute, National Institutes of Health, HEW, in press.

Nutrients and Public Health

Ten-State Nutrition Survey 1968-1970. U.S. Department of Health, Education and Welfare; DHEW Publ.

No. (HSM) 72-8134; 1972.

Recommended Dietary Allowances 9th Revised Edition. Food and Nutrition Board, National Council,

National Academy of Sciences; 1980.

Nutrition Reviews' Present Knowledge in Nutrition, 4th Edition. The Nutrition Foundation, Inc.,

New York, Washington; 1976.

Nutrition, A National Priority. A Report by Nutrition Canada to the Department of National Health

and Welfare; 1973.

Knowledge of Nutrient Composition

Watt, B. K. ; Merrill, A. L. Composition of Foods - Raw, Processed, and Prepared. Agriculture

Handbook No. 8; 1963.

U.S. Department of Agriculture. Composition of Foods - Raw, Processed, and Prepared.

a. Handbook No. 8- 1. b. Handbook No. 8- 2. c. Handbook No. 8- 3, d. Handbook No. 8- 4.

e. Handbook No. 8- 5.

f

Handbook No. 8- 6.

g- Handbook No. 8- 7.

Watt, B. K. ; Murphy, E. W

50-60; 1970.

fables of Food Composition, Scope and Needed Research. Food Tech. 24:

American Medical Association, Nutrients in Processed Foods, Fat and Carbohydrates. White, P. H. , Fletcher, D. C. , and Ellis, M. , eds. Acton, Massachusetts: Publishing Sciences Group, Inc.;

1975.

American Medical Association, Nutrients in Processed Foods, Proteins. White, D. H. , and Fletcher,

D. C. , eds. Acton, Massachusetts: Publishing Sciences Group; 1974.

American Medical Association, Nutrients in Processed Foods, Vitamins and Minerals. White, P. L. , and Fletcher, D. C. , eds. Acton, Massachusetts: Publishing Sciences Group; 1974.

Paul, A. A.; Southgate, D. A. T. McCane and Widdowson's - The Composition of Foods. New York,

New York: El sevier/North-Hol land Inc. Biomedical Press; 1978.

24
National Bureau of Standards Special Publication 635. Proceedings of the Workshop on Reference

Materials for Organic Nutrient Measurement held at NBS, Gaithersburg, Maryland, October 23, 1980.

LONG TERM STABILITY OF ORGANIC NUTRIENTS IN FOODS

Wayne Thornburg

Del Monte Corporation

205 N. Wiget Lane Walnut Creek, California 94598

Today we are going to discuss the long term stability of natural and fortified organic nutrients

in food products.

The way to avoid the loss of organic nutrients in food is to pick it fresh from the garden,

rinse it, and eat it. However, in modern society this is rarely possible. Most of our fruits and

vegetables are transported fresh in refrigerated and/or controlled atmosphere storage or processed

into frozen or canned foods which may be stored for long periods.

My talk will be principally about fruits and vegetables since most of our work has been with

these foods.

Hurt, in 1979, stated that the primary objective of any form of food preservation is to modify

basic raw agricultural commodities into a stable physical form that will ensure a safe and wholesome

food supply. In this regard, nutrition becomes only one of the factors in the equation of successful

food processing. Processed foods must have the necessary organoleptic qualities of desirable flavor,

odor, texture, and appearance, as well as be priced right before nutritional quality has any meaning.

People today eat foods not nutrients.

Two major methods of food preservation are canning and freezing.

Table 1 shows the changes in vegetable consumption between 1940 - 1975 and shows a slow increase

in the consumption of canned and frozen vegetables.

Table 1. Changes in Vegetable Consumption per Person, 1940 - 1975

a

.

Total Consumption Percentage Consumed As: Year (lb) Fresh Canned Frozen

1940 116 78 21 1

1955 118 71 23 6

1965 115 69 23 8

1975 123 64 24 10

a Source: USDA (1977).

When I searched the literature on the stability of vitamins in processed foods to compare it

with our experimental data I was surprised to find a great divergency in published data.

Table 2 compares Del Monte experimental data with data published by Lund in 1975. It shows the

percent loss of four vitamins in four foods caused by the canning process.

25
Table 2. Loss of Nutrients in the Canning Process.

Percent Losses of Nutrients

Vitamin A Thiami n Riboflavin Vitami n c Product Del Monte Lund

a Del Monte Lund Del Monte Lund Del Monte Lund

Green Beans 1.9 51 .7 20.0 62.5 0 63.6 59.7 78.9

Corn 22.4 32.5 33.3 80.0 0 58.3 20.0 58.3

Green Peas 45.4 29.7 PiR OJ . nU 74/ *T • Cm9 41 fi4 ~\ 25.9 66.7

Spinach 0 32. 1 66.6 80.0 22.2 50.0 68.2 72.5

a 1975.

Table 3 lists some of the reasons that there is such a variation in data.

Table 3. Factors Affecting the Apparent Percent of Loss

of Vitamins in Processed Food.

1. Variety - growing conditions

2. Handling after harvest

3. Type and temperature of blanch

4. Processing time and temperature

5. Oxygen level in the can

6. Storage after processing

7. Analytical techniques

Causes for Variation in Data

1. Variety - Vitamin levels vary in different varieties of food. Growing conditions may also

affect vitamin levels and influence loss of vitamins during processing.

2. Handling after Harvesting - poor handling will reduce levels of vitamin A and C in foods.

Under proper refrigerated storage, vitamins are stable until senescence at which time there is rapid

loss of vitamins.

3. Type and Temperature of Blanch - Both frozen and canned vegetables are blanched prior to processing. Either hot water or steam blanching can be used. There is a trend to steam blanching

because it is more energy efficient. Blanching removes water soluble nutrients (vitamins and

minerals) but not fat soluble vitamins. Up to this point the processing of canned and frozen food

is similar. Frozen food after freezing is generally held at -18 °C.

Canned Foods

4. Processing Time and Temperatures - The magnitude of the losses in canned foods obviously

reflect the sensitivity of the nutrients to the microenvi ronment in the food and the severity of the

thermal process. Canned tomatoes would receive a thermal process no higher than 100 °C while corn would receive a "hot" cook at temperatures near or in excess of 120 °C.

5. Oxygen Level Remaining in the Can - Oxygen levels will affect vitamin A and C levels. In years past, exhaust boxes were used to remove the air from cans. However, they are slow and very

energy wasteful. Now most cans are filled with brine or syrup and closed with a steam flow closer

where the air is displaced by steam and the can closed in a steam atmosphere. However, more air

remains in the can using a steam flow closer.

26
6. Storage After Processing - Higher storage temperatures may lead to loss of vitamins. This

is more pronounced in frozen than in canned food.

7. Analytical Technigues - We will discuss this at some length. Before you can measure the

retention or loss of vitamins in foods you must be able to analyze for them accurately.

Analytically, attempts to measure the loss of vitamins leads to a surprising amount of

di ff iculties.

For example, in 1977 J. D. Selman at the University of Technology in Great Britain reviewed the

vitamin C losses from peas during blanching in water. He concluded that no conclusions could be

drawn due to variations between experiments and the poor control of important parameters.

In our laboratory we found the same problem when we tried to compare the vitamin levels in

fresh, frozen, and canned foods. We now analyze the samples for moisture content and express the

nutrients on an egui-solids basis and thus levels can be compared.

With water insoluble vitamins the problem is much more complicated. When vegetables and fruits

are blanched, water soluble constituents are removed, but not water insoluble vitamins. Then if you

analyze the blanched samples you have increased the apparent fat soluble vitamins such as p-carotene.

This is very obvious if you compare raw and blanched vegetables on an egual solids basis. After

considerable number of experiments we found that the water insoluble solids (WIS) could be used to compare water insoluble vitamins.

Analysis of vitamins on fresh foods is further complicated by the presence of enzymes. Enzymes

in plant tissue destroy vitamins, especially vitamins A and C. This is important in two ways. For

the analyst it is important to inactivate the enzymes in fresh tissue. If you blend fresh spinach

in a Waring blender for 2 minutes, half of the ascorbic acid is lost before blending is completed.

If the blendate is allowed to stand for 30 minutes all the ascorbic acid is lost. Therefore, when

you analyze fresh vegetables for vitamin C it is necessary to blend the intact tissue with acetic

acid/meta phosphoric acid stabilizing solution.

We have found that beta-carotene is rapidly destroyed when fresh plant tissue is blended. We

use a procedure developed by Van de Weerdhof where the plant tissue is dropped into boiling

methanolic 0.5 mol/L potassium hydroxide solution. This procedure has two advantages:

The enzymes are destroyed and the sample is saponified. After suitable purification

the beta-carotene can be quantitated by the AOAC procedure or by high performance

liquid chromatography.

We have found that repeated freezing-thawing destroys vitamin C and beta-carotene in frozen

blended samples. Commercially both canned and frozen foods are blanched prior to freezing or canning. This operation inactivates the enzymes and softens the food so that it can be put in the can

or package.

Stability of Nutrients in Processed Foods

Canned Foods - As previously noted blanching removes water soluble nutrients which includes

water soluble vitamins. Nutrients in canned foods are quite stable for as long as the integrity of

the can remains intact. In canned foods light and air are excluded and enzymes are destroyed. Some

canned vegetables are lower in thiamin; all of this loss being apparently due to the sterilization

process.

People frequently ask us how long vitamins are stable in canned foods. Recently we were given

a set of canned foods packed in 1939. We believe they were stored in relatively cool storage in a basement. Let me hasten to inform you that the cans used at that time were hot dip tin plate and

lasted much longer than would the currently used electrolytic differential plate. We put these

canned foods through a complete nutritional analysis and were pleased to find that most of the

vitamins were retained and compared favorably with vitamins found in currently marketed foods.

Table 4 compares the vitamin levels found in some of these foods compared with the same food available

in the markets.

27
Table 4. Effect of Storage on Canned Food.

(Per 100 Grams)

Food Storage

Vitamin A

(I.U.

)

Thiamin

(mg)

Riboflavin

(mg)

Niacin

(mg)

Vitamin

(ma)

call O 1 c \* U i 1

1

tuAC) Yoarc rear a

-a en 0. 01 0. 05 1

.

2

oo Cream Style Corn Current 144 u

.

fid u

.

ne r,u

.

6

PrUIv-> 1/l\ ailu anH Roane Dcallb *+U leal b l ^n 0. 02 0. 04 0. 6 Pork and Beans Current 130 0. 08 0. 03 0. 6

Fruit Cocktail 40 Years 220 0. 02 0. 01 0. 7 3.1

Fruit Cocktail Current 233 0. 03 0. 02 0. 5 2.2

Green Peas 40 Years 328 0. 07 0. 06 0. 9 Green Peas Current 442 0. 09 0. 07 0. 7

Frozen Foods - Storage temperature has a special significance for frozen foods because they are defined as foods stored at temperatures below -18 °C. Even at such low temperatures certain enzymatic

and non-enzymatic changes continue, but at a much slower rate, to limit storage life of frozen

foods.

The nutrient that has been most thoroughly researched is ascorbic acid. The destruction of

ascorbic acid during storage is influenced by the amount of blanching (to inactivate enzymes) rate of

freezing and type of packaging. Generally, we have found that the quality deteriorates and vitamin

A and C levels decrease in frozen foods after 18 month's storage. Table 5 demonstrates this loss for

frozen green beans, Table 6 for frozen corn, Table 7 for frozen spinach, and Table 8 for frozen

green peas.

Table 5. Frozen Green Beans (18 Month's Storage) Nutritional Data on a 7.2 Percent Solids Basis.

(Per 100 Grams)

Storage

Vitamin A (I.U.)Thi ami n (mg)

Ri bof 1 avi n (mg)Niaci n (mg)

Vitamin C (mg)

Initial 503 0.06 0.07 0.46 2.8

6 Months 377 0.06 0.08 0.40 2.1

12 Months 400 0.06 0.07 0.40 1.0

18 Months 396 0.05 0.08 0.45 1.1

Table 6. Frozen Corn

25 Percent

(18 Month's

Solids Basis.

Storage) Nutritional Data on a

(Per 100 Grams)

Storage

Vitamin A (I.U.)Thiamin

(mg)

Riboflavin

(mg)

Niacin

(mg)

Vitamin C (mg)

Initial 728 0.09 0.09 1.16 12.3

6 Months 537 0. 11 0.09 1.21 11.4

12 Months 432 0. 10 0.08 1.06 11.3

18 Months 340 0.11 0.07 1.15 9.7

28
Table 7. Frozen Spinach (18 Month's Storage) Nutritional Data on a 7.2 Percent Solids Basis.

(Per 100 Grams)

Storage

Vitamin A (I.U.)Thiamin

(nig)

Riboflavin

(mg)

Niacin

(mg)

Vitamin C (mg)

Initial 6367 0.06 0.14 0.40 24

8 Months 6250 0.07 0. 15 0.39 15

18 Months 4752 0.07 0. 13 0.43 8

Table 8. Frozen

on a 15 Green Peas (18 Month's Storage)

Percent Solids Basis.

Nutritional Data

(Per 100 Grams)

Storage

\/i t ami n A V 1 Lam 1 [1 n (I.U.)

Thiamin

(mg)

Riboflavin

(mg)

n i ac l n (mg)V 1 UalU 1 n u (mg)

Initial 678 0.17 0.08 1.03 17

6 Months 898 0.15 0.10 1.11 13

12 Months 611 0.16 0.08 1.15 11

18 Months 566 0.17 0.07 1.00 10

Naturally Occurring vs. Added Nutrients

The question has been raised whether naturally-occurring nutrients are inherently more stable

than added nutrients. Some literature seems to indicate that under some conditions added nutrients

are inherently less stable than those presumably incorporated in a different way in the natural

state.

In summary we can conclude:

1. Vitamins are stable in cool stored fresh vegetables until senescence begins and

the food yellows when vitamin loss becomes rapid.

2. If fresh green foods are blended there is a rapid loss of vitamins C and A due to enzyme action and probably oxidation.

3. Both frozen and canned foods are blanched either in steam or hot water to destroy

enzymes. Water soluble nutrients (minerals and vitamins) are lost by this

operation.

4. Frozen foods begin to lose vitamins after about 18 month's storage.

5. Thiamin is lost in canned food probably due to the high temperatures used to sterilize non acid foods.

6. There is good reason to believe that damage to nutrient levels in both canned and

frozen foods occurs to the greatest degree during handling and preparation of the

foods in the home.

29
National Bureau of Standards Special Publication 635. Proceedings of the Workshop on Reference

Materials for Organic Nutrient Measurement held at NBS, Gaithersburg, Maryland, October 23, 1980.

THE LONG-TERM STABILITY OF ORGANIC NUTRIENTS IN INFANT AND ADULT DIETARY SUPPLEMENTS

Steven Barnett

Mead Johnson and Company

2404 Pennsylvania Avenue

Evansville Indiana 47712

Many factors affect the long-term stability of organic nutrients. These include raw material

composition, processing losses, nutrient-matrix interactions, product-container interactions, product

age, and storage temperature. The ability to measure the fat-soluble vitamins accurately is a difficult task. This is due to the inaccuracies in the methods, the occurrence of multiple forms of

the vitamins and their natural precursors, and the lack of purity of the reference materials which

are used as standards.

In April of 1980, we published a High-Performance Liquid Chromatographic procedure for the

determination of vitamins A, D, E, and K from a single sample preparation [l] 1

. This method is

unique in that an enzyme, Lipase, is used to hydrolyze the lipid material. This more controlled

hydrolysis minimizes vitamin degradation and facilitates vitamin extraction.

In using this method, we have observed that vitamin A reference materials contain variable

amounts of cis and trans isomers and vitamin K standards contain from 3 to 25 percent of the biological inactive cis isomer. Materials of similar purity are used to fortify foods; therefore, the

analytical procedure must quantify all the nutritionally added forms in addition to the naturally

occurring vitamin forms. This is evident in the variation of vitamin E isomers, contributed by

nutritional seed oils, which are added to fortify infant and adult dietary food products. The distribution of these isomers is so characteristic that identification of a particular oil, in a product, is possible.

Determination of the fat-soluble vitamins in our products, after 2 1/2 years of storage,

indicates that vitamin A undergoes facile isomerization to the 13 cis form and other isomers. All

have lesser degrees of biological activity and in addition, these isomers are not resolved from

the 13 cis form. Extraneous peaks are observed in the E alcohol region and there is evidence of some reduction in the amount of E acetate. The total vitamin K content does not appear to change;

however, current procedures do not separate cis and trans isomers, consequently, biological activity

is not unequi vocably defined. Vitamin D appears to be structurally quite stable in these near- neutral products, in the absence of light, in an atmosphere containing an anti -oxidant, such as a-tocopherol

The results of these studies demonstrate the critical need for suitable reference materials for

the multiple forms of the fat-soluble vitamins. The analytical methodology is improving to the point

where more suitable standards of matrices are required to carry out accurate measurements.

REFERENCE

[1] Barnett, S. A.; Frick, L. W. ; Baine, H. M. Anal. Chem. 52: 610-614; 1980.

1 Figure in brackets indicate the literature reference at the end of this paper.

30
National Bureau of Standards Special Publication 635. Proceedings of the Workshop on Reference

Materials for Organic Nutrient Measurement held at NBS, Gaithersburg, Maryland, October 23, 1980.

GENERAL OUTLINE FOR DISCUSSION DURING WORKSHOP SESSIONS

Collaborative studies, summarized by Stewart 1

, indicate that the measurement of many of the

vitamins are neither accurate nor precise. With this in mind as a starting point, we would like to examine the following questions during each of the three workshops.

Are there new methods which are more accurate than the accepted methods?

Which are the two most accurate and precise chemical methods for measurement of the nutrients

listed below in (a) formulated foods? (b) commodity foods?

What are the problems in extracting these nutrients from (a) commodity foods? (b) formulated

foods?

Do extraction methods need further study? What is the nature of the needed studies?

What are the five most useful matrices for standards for formulated foods, e.g., infant formula,

powdered milk, margarine?

What are the five most useful matrices for standards for commodity foods, e.g., puree peas,

wheat flour, frozen dried beef?

What are the conditions under which the analytes cited are stable for 1, 3, or 5 years in these

matrices?

Priority List for: (1) Analytes to be standardized

(2) Matrices to be evaluated

PROPOSED LIST OF NUTRIENTS FOR DISCUSSION

The following list of analytes which have special nutritional significance with respect to industrial measurements might serve as a guide for discussion.

Fat Soluble Water Soluble Sugars

Group I

Vitamin A Cholesterol

Group II

D

E

Niacin

Vitamin C

Thiamin

Ribof 1 avi n Pyridoxine and Analogs

Monosaccharides

01 igosaccharides

Stewart, K. K. , Nutrient Analysis of Foods : A Reexamination , in NBS Special Publication 519, Trace

Organic Analysis: A New Frontier in Analytical Chemistry, U.S. Govt. Printing Office, Washington,

D. C. (1979).

31
National Bureau of Standards Special Publication 635. Proceedings of the Workshop on Reference

Materials for Organic Nutrient Measurement held at NBS, Gaithersburg, Maryland, October 23, 1980.

EXECUTIVE SUMMARY OF WORKSHOP SESSIONS

S.A. Margolis, NBS

1. Introduction

The development of standards for the vitamins fall into three categories: those for vitamins

which are believed to present few or no analytical problems, those for vitamins which present some analytical problems but the preparation of standards appears feasible, and those for vitamins for

which current methods of analysis are neither accurate nor precise.

A major problem for the workshop was the alternative development of standards based on fortified

vitamin levels as opposed to standards in which the vitamins occur naturally. The analysis of

fortified standards presents fewer problems in extraction and measurement since the fortified vitamin

is often added to the matrix as a single species.

2. Workshop on Water-Soluble Vitamins

Most of the naturally occurring vitamins in the water-soluble class, with the exception of

ascorbic acid, occur in forms other than those which would be used for fortifying standards. For instance, niacin occurs naturally as NAD, NADH, and related compounds; riboflavin occurs naturally as FAD, FMN, and related compounds; and thiamin is naturally bound to proteins. Similarly B 6 , biotin,

and pantothenic acid occur naturally bound to macromolecules. Furthermore, the extraction procedures for some of these vitamins or the procedures used to release the vitamins from the bound form

may cause extreme rearrangement of the vitamin moiety. This occurs, for example, when niacin or nicotinamide is released from NAD and NADH or NADP and NADPH. The alkaline hydrolysis results in the

conversion of the nicotinamide ring of NAD and NADP to 2-hydroxynicotinaldehyde, and other products

as well. On the other hand, acid hydrolysis of NADH and NADPH results in the formation of products

in which the carbon at C-6 of the nicotinamide ring is hydroxylated and then condenses with the

,2' carbon of the ribose ring to yield 0

2

', 6-B-cyclotetrahydronicotinamide adenine dinucleotide.

This acid instability of one form and basic instability of another form may lead to the inability to adequately recover bound vitamins from natural materials. Because of this type of problem, the

workshop discussion centered primarily on the development of standards of the class of fortified

materials. High priorities were given to the most stable vitamins for which current FDA labeling

regulations require that measurements be made to support labeling statements.

Participants in the water-soluble vitamins workshop quickly established a general priority

listing. The high-priority group included thiamin, niacin, ascorbic acid, and riboflavin followed

by B 6 , pantothenic acid, folacin, biotin, and B 12 - Of the B vitamins, niacin appears to present the

fewest problems. It is extremely stable as nicotinamide, easy to extract, and easy to measure. Workshop participants believed that it can be measured quite accurately by the cyanogen bromide

method using an autoanalyzer as well as by microbiological techniques.

Thiamin was listed as the most stable compound. It exists in nature as enzyme-bound thiamin

pyrophosphate. It is heat sensitive, but can be released by hydrolysis with appropriate enzyme

preparations. The binding of thiamin pyrophosphate to the enzyme is through a phospho-ester linkage

possibly to serine, and the release of thiamin from the enzyme requires hydrolysis of this bond.

Another problem with the assay of thiamin is the occasional unavailability of the ion-exchange

material used for the final purification step. An alternate material, Bio-Rex 70, appears to be

satisfactory for this separation. The use of thiamin- fortified material would eliminate the need for

the hydrolysis and the ion-exchange chromatography. Without these steps the measurement of thiamin

is believed to be relatively problem free.

The measurement of riboflavin still represents a significant problem. Riboflavin is unstable

above pH 6 and is light sensitive. The microbiological method has not yet been approved as an AOAC

official method, and the official method which utilizes permanganate oxidation is of limited accuracy

and precision.

In the case of unfortified materials, the major problem is in the extraction procedure.

Presumably, the extraction procedures for riboflavin present the same problems as those for bound

niacin; and, in general, it is difficult to assess the completeness of the release of riboflavin from

the nucleotide forms. Because of the difficulties in measurement and extraction, riboflavin is given

32
a relatively low priority in the first category of compounds for which standards need to be made.

Ascorbic acid, on the other hand, which occurs as the free vitamin, is relatively easy to extract. However, it is somewhat unstable; and in the presence of oxygen, is oxidized to dehydroascorbi c acid,

a reaction which is speeded up by the presence of moisture and trace amounts of metals. However,

vitamin C can be stabilized at pH's in the range of 2-4 and by the use of matrices which have a low

water activity. It was pointed out in this workshop that the water content of the matrix is not as crucial as the water activity.

The next group of vitamins which have significant public health impact, but which have problems

either in the methods of analysis or isolation, include fol acin, Bg , pantothenic acid, and B^2- The official methods for the analysis of these vitamins are microbiological techniques, and suitable

assays need to be developed for the chemical assay of these vitamins. Folacin was only briefly

considered in the workshop because it exists in multiple forms which are not easy to interconvert to

a single form. B 12 also exists in several forms, both adenylo- and cyano-forms. It is measured by

radioimmunoassay techniques. At this time, both the researchers and the nutritionists need a wellcharacterized pure standard to use in the development of better assay techniques. In the case of

pantothenic acid, the major problem is the ability to hydrolyze the phosphoseri ne bond or phosphodiester bond with the AMP. At the moment, pantothenic acid is released by enzymatic digestion; and

the resulting material is measured by microbiological assay.

Of the four vitamins in this class which have significant public health impact, B 6 has the

highest priority. There is currently an urgent need for reference materials containing the six

vitamers of B 6

: the free and phosphoryl ated forms of pyridoxine, of pyridoxal , and of pyridoxami ne. The availability of these vitamers in pure form and also in a simple matrix would facilitate the

development of methods of analysis for this group of substances. In the case of plant food materials,

the incomplete extraction of vitamin B 6 , particularly that which is covalently bound to carbohydrate,

still requires further evaluation. The pure vitamers would aid in the evaluation of the completeness

of the hydrolysis of the vitamers from food substances and in the analysis of the multiple peaks

found by HPLC analysis of food extracts.

At the moment, the analysis and measurement of biotin is not considered important for public

health reasons; therefore, it was recommended that a biotin SRM would be less urgent than others and

would be necessary only to check the process of extraction.

3. Workshop on Analysis of Carbohydrates

Carbohydrate analysis was divided into two parts: a formal discussion of methods of analysis of

carbohydrate content and then a discussion of the types of materials needed as Standard Reference

Materials for carbohydrate analysis. The first formal presentation by Dr. Kent Stewart consisted of

a discussion of the automated analysis of carbohydrate content in food samples. Dr. Stewart pointed

out that this method of analysis is not suitable as a primary method but is desirable when a large

number of samples must be analyzed. The method involves the use of an autoanalyzer and consists of

hydrolyzing the carbohydrate samples with sulfuric acid, and then reacting the hydrolysate with

di nitrophenyl hydrazi ne to form a colored derivative that is measured spectrophotometri cal ly. The

problem with this method is that different sugars have different molar response factors. Glucose,

fructose, and invert sugar have similar response factors, but the factor for maltose is 30 percent

and for lactose is 23 percent of the others. For sucrose, the response factor is double the others

since it is hydrolyzed into fructose and glucose before the hydrazone is formed.

The second presentation by Dr. Betty Li was a consideration of methods of analysis of carbohydrates by gas chromatography which requires the conversion of carbohydrates into derivatives that are volatile. Four types of derivatives were discussed: trimethyl si lyl ethers, trimethyl si lyl ated

oximes, products of the reaction with N-methyl imidazol e followed by reaction with nitroacetate , and

products of reaction with sodium borohydride and acetic anhydride. The trimethyl si lyl ated oxime

method is preferred since it avoids anomerization and permits distinction between glucose and

fructose. It has a high degree of sensitivity, gives good resolution, and can detect as little as

1 percent of any sugar in the presence of other sugars. It permits both identification and quantitation of the sugars, and requires a relatively short period of time for analysis. It cannot be used

for sugars more complex than tetrasaccharides. The solvent for the deri vati zati on is 80 percent

methanol which prevents enzymatic hydrolysis of the polysaccharides. With automated samplers, the

standard deviation of the analysis for glucose was 2 percent. In the case of maltose and lactose,

the standard deviation was 5 percent. For food extracts, the precision was approximately 7 percent.

Furthermore, this method can be used to analyze sugar alcohols, sugar amines, or sugar phosphates;

and it is appropriate for analysis of sugar syrups.

33
Two presentations dealt with the high-performance liquid chromatographic analysis (HPLC) of

sugars. The HPLC methods do not require the deri vati zation of the sugars. Thus, samples can be

analyzed directly. However, HPLC methods are not as sensitive as the gas chromatographic methods.

The first of these methods (presented by M. J. Gray) dealt with the use of ion-exchange resins

for carbohydrate separation. The resins were polystyrene-di vinylbenzene copolymers which were either

polysul fonated or aminated. Calcium, lead, or silver ions are added to the solvent in the use of the

polysul fonated resins and are required for the separations. The basis for the separation is not clearly understood. It may be ion exclusion, molecular size exclusion, or ion exchange, or some combination of the three. The method requires the use of a precolumn to remove strong anions or cations

depending on the choice of resin and mobile phase. These resins are durable under aqueous conditions

and stable up to a temperature of 80 °C. The major disadvantages of this method are that di sac- charides cannot be resolved and the resolution is dependent upon the degree of cross-linking and upon

the counter ion. Thus, a resin with 6 percent cross- 1 i nki ng in the presence of silver will resolve

oligosaccharides up to hexasaccharides ; whereas with 4 percent cross-linking in the presence of

silver, oligomers as large as nonasaccharides can be resolved. Sugar alcohols can be resolved, by

increasing the concentration of acetoni tri 1 e.

For the resolution of disaccharides such as maltose and cellobiose, lead is used as the cation.

Solutions containing 1 percent saccharides can easily be analyzed. Anion and cation exchange resins

bonded to silica can also be used; and for these, the use of metal ions are not required. The solvent

commonly used is a mixture of water and acetonitrile, and efficient separations can be achieved

through the control of solvent composition and flow rate.

The second presentation by K. Ivie dealt with separations of carbohydrates on aminosilane

columns. In this case, the solvent is again water and acetonitrile. The separation can be achieved

at ambient temperature, and for the simple sugars appears to give better resolution than the other

liquid chromatographic methods. This column has been used to quantify sugars in foods; the example

cited was corn syrup. A Corasil m guard column was used to protect the analytical column. This

system resolved sucrose, glucose, and maltose; analyses with a CV of 1 to 2 percent and a relative

standard deviation of 1/10 of 1 percent were obtained using an automatic injection mode for more than

100 analyses. Maltose and maltotriose were also resolved by this system.

An alternate method of analysis uses a silica column and a solvent which contains water, acetonitrile, and a small amount of an amine. By controlling the concentration of the amine, one can control

the resolution of the column. However, this method does not resolve glucose and galactose. Alphaand beta-anomers can be resolved by reverse-phase chromatography. The amine modified silicas are adequate for analyzing sugar acids, sugar amines, sugar alcohols, as well as the sugars themselves.

The discussion which followed focused on the sugars most important to both the nutritionist and

the industrialist. These included sucrose, lactose, maltose, glucose, fructose, galactose, and

malotriose. The participants noted that corn syrup is widely used as a sweetening agent. A reference

material for use in determining the degree of adulteration of various corn syrup products is needed.

Adulteration is detected by measuring the amount of maltotriose present. The characteristics for a food matrix for a sugar SRM were then discussed. The conclusions were that the matrix must be

constant in its moisture content, must be free of fatty acid oxidation, must be free from bacteria,

must have little or no enzymatic activity, and should be a homogeneous, easily weighed material. A number of food materials were discussed; the dry products were preferred over the non-dry products

because hydrolysis of sucrose occurs in materials such as fruit and vegetable purees. Some of the

matrices which were discussed were carob powder, pasturized honey, milk chocolate, corn syrup, invert

syrup, dried skim milk, and a powdered beverage mix.

Three general industrial groups were cited as having needs for reference materials: the dairy

industry, the cereal and grain industry, and the processed fruit and vegetable industry. Based upon

the kinds of industries that would use such Standard Reference Materials, the following series were proposed at the end of the workshop: corn syrup, a ready- to-eat cereal made with whey, a milk

chocolate preparation made with skim milk, and a fortified skim milk. Of this list the proposed corn syrup SRM was considered by the food industry to have the greatest priority.

4. Workshop on Fat-Soluble Vitamins and Cholesterol

The fat-soluble analytes discussed were vitamin A, beta-carotenes, cholesterol, and tocopherols. Two methods are currently in use for the analysis of vitamin A: an HPLC method and the

Carr-Price method (an AOAC method). These often differ by 5 percent or more in their assessment of

vitamin A content. The forms of vitamin A which are commonly used for fortifying food materials or

as analytical standards are the alcohol, the palmitate ester, or acetate ester. The major problem

34
with vitamin A samples is that they contain a mixture of the thirteen cis and the thirteen trans

isomers. A vitamin A palmitate preparation can consist of anywhere from 5 to 20 percent of the

thirteen cis isomer. If the pure thirteen trans vitamin A palmitate is not available, then USP grade

vitamin A acetate, which contains essentially no thirteen cis isomer is suitable for use as a reference material. The current method for vitamin A analysis involves a preliminary saponification

of the vitamin A followed by ether extraction and chromatographic analysis. The major problems with

vitamin A are its relatively easy oxidation and its sensitivity to light. Hence, the ideal storage

conditions include low temperature, nitrogen environment, and storage in a low actinic glass in the

presence of antioxidants such as tetrabutyl hydroqui none or BHA. Two matrices for vitamin A believed

to be feasible for certification and useful for nutritional and industrial measurements, are hydrogenated oils or enriched flour, the former being the matrix of choice.

An HPLC method and the AOAC method are being used for the combined analysis of both the alphaand beta-carotenes. The availability of a primary standard material was questioned. Furthermore,

workshop participants pointed out that useful methods for the separation of the alpha- and betacarotenes were needed, as well as the separation of the carotenes from the xanthenes. The major

current problem is the extractions of carotenes from plant material, not their extraction from oil.

For cholesterol, gas chromatography appears to be the best method of analysis. The availability

of SRM Cholesterol (SRM 911a) eliminates the need to find a suitable reference material; thus, the

discussion focused on the type of matrix which would be most appropriate for a reference material for

cholesterol measurement in foods. A hydrogenated vegetable oil or some other stable oil that was not readily susceptible to oxidation was proposed as a matrix. If the NBS GC/IDMS method is used to establish the cholesterol content, then the participants felt there would be no need to free the oil

from possible contaminating sterols. Therefore, they proposed that a soybean oil or some other

vegetable oil be used as a matrix. Little time was spent on a discussion of the tocopherols,

although it was noted that GC and HPLC methods are available for tocopherol analysis and that their

levels in the foods usually run about 50 micrograms per 100 milligrams of material.

The question of how to package the fat-soluble material was of considerable interest. The group

generally concluded that the gelatin-type capsule used by the USP is the most convenient method of

distributing fat-soluble materials because the capsules can be weighed and dissolved relatively

easily. However, they do exhibit some degree of permeability to oxygen. This is significant and,

therefore, care must be taken to avoid oxidation. Consequently, the capsules have to be stored in some closed system, preferably low actinic glass and in a nitrogen environment. The workshop

participants recommend evaluating both the use of gelatin capsules and some sort of glass container

noting that the gelatin capsules are much more convenient for the analysts.

The discussion on appropriate matrices led to the posing of four questions. How applicable is

the oil matrix to the general needs of the analyst? How measurements of vitamins in an oil matrix

relate to the measurements on real samples? How does extraction of oil matrix samples relate to the

problems of extraction of real samples? Can a representative food material be selected? With these

points in mind, two additional matrices were proposed: dry milk powder and dog food. These two food

matrices can be spiked with reasonable confidence in obtaining a homogeneous product and the

reference material can contain more than one analyte.

The levels of the various analytes in standards should be equivalent to 30 to 50 percent of the

recommended daily allowance. Therefore, the workshop participants were asked to send us their ideas

on recommended spiking levels in the report of the workshop leaders. The minimum requirement by the users for accuracy of measurement using a given standard should be ±5 percent at the 95 percent

confidence limits. Finally, a relatively high analyte content in the standard was proposed to permit

the standard to be used for the assessment of accuracy of analysis by all methods.

35
National Bureau of Standards Special Publication 635. Proceedings of the Workshop on Reference

Materials for Organic Nutrient Measurement held at NBS, Gaithersburg, Maryland, October 23, 1980.

REPORT OF WORKSHOPS

A. Water-Soluble Vitamins

J. R. Kirk and J. Pennington

Materials with Methodologies Acceptable to Workshop Participants

Nutrient

Vitamin C

Thiamin

Riboflavin

Niacin

Extraction Method

Solvent Extraction

Solvent Extraction

HPLC

Ion-Exchange Bio-Rad 70

Solvent Extraction

Analytical Method (Horwitz, Chap. 43)

Fluorometric with o-phenylenedi amine

Redox titration with 2,4-dichloroindophenol

Spectrophotometry or refractive index

Fluorometric as thiochrome

Microbiological

Fluorometric

Spectrophotometry cyanogen bromide

and barbituric acid derivative

Microbiological

Horwitz, W. , Official Methods of Analysis of the Association of Official Analytical Chemists , 13th ed. , Association of Official Analytical Chemists, Washington, D. C. (1980).

Priority Rating of Some Water Soluble Vitamins

b Vitamin

Vitamin C B-l

B-2

Niacin

Method Adequacy

2

3

3

1

Stability

3

2

2

1

Public Health Need

1

2

2

3

The numbers indicate the relative position of each vitamin in each category

for determination of natural levels as determined by the participants.

Nutrients and Matrices for Proposed SRM's

A. For B-l , B-2, Niacin:

Non-Fat Dry Milk - B-l, B-2, and Niacin

Non-Fat Dry Mi 1 k + B-l

Non-Fat Dry Milk + B-2

Non-Fat Dry Milk + Niacin

B. For Vitamin C: Freeze-Dried Potato Granules

Freeze-Dried Tomato Juice

Dried Drink Mix with Sugar and

Vitamin C added

Vitamins with Methodological Problems

For B-6, B-12, Folacin, Pantothenic Acid, and Biotin:

B-6 and Folacin - of public health significance

Biotin - method adequate but of no public health significance

Folacin - methodology problems

B-6 and B-12 - need for reference materials for method development

B-6 - suggest to use bovine liver (SRM) with added B-6

B-12 - of lower priority than B-6

36
B. Carbohydrate Analysis

W. J. Hurst and R. Schaffer

Materials with Methodologies Acceptable to Workshop Participants

Methods:

Supplement/complement current Association of Official Analytical Chemists (AOAC)

methods (Horwitz, Chap. 31, pp. 506-532) by use of:

a) High-Performance Liquid Chromatography

b) Gas Chromatography

c) Enzymes - not AOAC approved but certainly a potentially

viable method

The materials should provide analysts with experience in extracting the

carbohydrates since there are not any inherent problems in analysis.

Analytes:

Phase I Common analytes of interest

(1) Sucrose

(2) Glucose

(3) Lactose

(4) Fructose

Phase II Galactose

Maltotriose''

Maltose

Deiry industry interest

The primary interest is for identifying corn syrup

related products (which contain maltotriose) as adulterants of "pure" products

Horwitz, W. , Official Methods of Analysi

of the Association of Official

Analytical Chemi sts , 13th ed. , Association of Official Analytical

Chemists, Washington, D. C. (1980).

'Need pure reference material for use by NBS.

Matrices for Proposed SRM's

Corn Syrup - Simple commercially available product, 100 mL units,

presenting no major analytical problems

- wide number of users

- possible suppliers: Staley, CPC

- HPLC is readily accepted as method for analysis

- certify for glucose, maltose, maltotriose, moisture

Ready-to-eat cereal with skim - 10 g sample size needed for analysis, 250 g units,

milk fortification 100 units/year (est. sales)

- low fat matrix

- medium extraction difficulty

- good number of probable users

- Fructose (1.6%), Glucose (1.6%), Sucrose (3.2%), Lactose (?)

- possible suppliers: Kellogg, General Mills, General Foods

Milk Chocolate with skim milk - 10 g sample size, 250 g units, 100 units/year (est. sales)

- high fat matrix

- medium extraction difficulty

- good number of probable users

- Sucrose (45%), Lactose (7+%), Moisture (?)

- Possible suppliers: Hershey, Nestle, M&M/Mars, Cadbury

- chip form

37
C. Fat-Soluble Vitamins, Cholesterol, and Fat

D. C. Egberg and H. T. Slover

Materials with Methodologies Acceptable to Workshop Participants

The following nutrients are listed in decreasing order of priority and increasing order of

analytical difficulty. The currently preferred method of analysis and additional pertinent information are also tabulated.

Nutrient

Choi esterol

Vitamin A Palmitate

(al 1-trans)

p-Carotene

or Tocopherol

Fat

(sol id matrix only)

Method of Analysis

GC

GC/MS

HPLC

Carr-Price

HPLC

AOAC

GC

A0AC b or best method

Suggested Source

NBS

Hof fmann-LaRoche

or USP

Eastman Kodak

Eastman Kodak

Durkee, Anderson,

Clayton, or Kraft

Key Contact

Additional Possible Fat Analyses:

Trans-unsaturates GC or infrared (IR) (in fat)

Polyunsaturates GC or Lipoxidase (in fat)

H. Slover

J. DeVries (Gen. Mills)

Stewart (U. of Florida)

or

S. Reeder (California)

H. Slover

H. Lento

L. L. Diosady (U. of Toronto)

A. Sheppard

A. Sheppard

Horwitz, W. , Official Methods of Analysis of the Association of Official Analytical Chemi sts , Chapt. 43, 13th ed. , Association of Official Analytical Chemists, Washington, D. C. (1980).

5 Met'iod is matrix dependent, see footnote a, Chapter 13-18.

Matrices for Proposed SRM's

Two SRM mixtures were proposed for fat, cholesterol, and fat-soluble vitamins: (1) a partially

hydrogenated vegetable oil containing cholesterol and the fat-soluble vitamins, and (2) this mixture

dispersed on fat-free dry milk powder.

Partially hydrogenated vegetable oil was selected to maximize the stability of the mixture.

This matrix is proposed rather than a totally hydrogenated oil because of the high melting point of

the latter. Furthermore, a partially hydrogenated oil (same sol id- fat index as a common margarine)

may serve also as a reference material for trans-saturated fats.

Storage Conditions

The oil (previously degassed to remove 0 2 ) should be put into low-actinic gelatin capsules and

stored at -20 °C in a nitrogen atmosphere. An antioxidant (tertiary butyl hydroqui none) should be

added to the oil at 200 ppm. The fat dispersed on dry milk powder should also be stored in sealed

containers under nitrogen at -20 °C.

38
Loads of Nutrients in Fat

Nutrient Hydrogenated Oil, amount 100 gm Non-Fat Dry Milk, amount 100 gm

Cholesterol 100 mg 25 mg

Vitamin A Palmitate 2500 IU 2500 IU Tocophenol 15 IU 15 IU Carotene 2500 IU 2500 IU

Suppliers of Raw Materials and Possible Processors

Non-Fat Dry Milk - Land-0- Lakes , Carnation

To Mix Nutrients - Producers of Dry Infant Formulas - Ross Laboratories,

Mead-Johnson

To Fill Capsules - G. D. Searle, Nutrilite Products, Inc.

39
Summary of Workshop Proposals

S. A. Margolis

The participants in each workshop session established a reference material priority list for the

nutrients discussed and the types of matrices to be used. Their recommendations are summarized in

the attached reports.

Several general observations were evident upon review of the workshop sessions:

1. With the exception of vitamin B 12 and folacin, there appears to be minimal need

for reference materials of higher purity than USP quality reference materials.

2. There is an immediate need for Standard Reference Materials (SRM's) consisting of

food matrices with specific nutrients at certified concentrations.

3. There was some conflict about the nature of the proposed SRM materials. Should

they be matrices certified for fortified concentrations of the nutrients or, rather, matrices certified for the natural content of the nutrient? The consensus was to certify for the content of native nutrient, only when the

nutrient was present in a free form, e.g., vitamin C. In other cases, the

initial effort should be directed toward the preparation of a nutrient

fortified food material, e.g., non-fat dry milk.

The levels of the certified nutrients should be between 25 percent and 50 percent of U.S. recommended daily allowance (U.S. RDA). The major reason for not attempting certification of bound

nutrients, particularly the B vitamins, is the instability of these nutrients under extraction

conditions.

The SRM priority lists proposed by the workshop participants fall into three categories, each

with several subcategories. The subcategory assignments do not indicate a preference for order of

certification.

1. Standards which are needed and which can be prepared with reasonable hope of success.

a. Corn syrup certified for glucose, maltose, maltotriose, and moisture: This

standard appears to be widely needed; existing isotope dilution mass spectrometry

(IDMS) methods can be used for glucose determinations.

b. Partially hydrogenated vegetable oil certified for cholesterol: Cholesterol

can be accurately measured by IDMS methods.

c. Non-fat dry milk certified for fortified amounts of niacin, thiamin, and

riboflavin.

d. Either non-fat dry milk or a sugar-containing dry drink mix certified

for vitamin C.

e. Finally, of lower priority, a non-fat dry milk certified for galactose and

1 actose.

2. Standards which are needed but present purity or stability problems.

Vitamin A palmitate and p-carotene in partially hydrogenated vegetable oil.

3. Other nutrients which present major problems of stability, purity, and/or selection of appropriate vitamers.

These are listed with their respective problems in the workshop reports.

40
LIST OF ATTENDEES

R. L. Alvarez

National Bureau of Standards

Washington, DC 20234

Jorg Augustin

University of Idaho

Food Research Center

Moscow, ID 83843

Steve Barnett

Mead Johnson Co. 2404 Penn. Ave. Evansville, IN 47712

Ofelia U. Barretto

Nutrilite Products, Inc. 5600 Beach Blvd.

Buena Park, CA 90620

Mart P. Bueno

Food & Drug Administration

200 C St. , S.W. Washington, DC 20204

Stephen N. Chesler

National Bureau of Standards

Washington, DC 20234

Bruce Coxon

National Bureau of Standards

Washington, DC 20234

Chester E. Daugherty

Campbell Soup Company

Line St. & Memorial Ave. Camden, NJ 08101

Phillip H. Davis

Ross Laboratories

625 Cleveland Ave. Columbus, OH 43216

L. L. Diosady

University of Toronto

Toronto, Ontario M5S 1A4

George J. Durany

Hunt-Wesson Foods R&D

1645 W. Valencia

Fullerton, CA 90631

David C. Egberg

General Mills

9000 Plymouth

Minneapolis, MN 55427

Edgar Elkins

National Food Processors Assoc.

1133 20th St., N.W. Washington, DC 22036

J. Ass is F. Faria

Food Science Dept.

512 Hamiltons St. Somerset, NJ 08873

Fengdi Feng

National Bureau of Standards

Washington, DC 20234

Tom Gills

National Bureau of Standards

Washington, DC 20234

Michael J. Gray

Bio-Rad Labs

2200 Wright Ave. Richmond, CA 94804

Lucy B. Hagan

National Bureau of Standards

Bldg. 222, Rm. # A309

Washington, DC 20234

Sally H. Harrison

National Bureau of Standards

Bldg. 235, Rm. # B]08

Washington, DC 20234

David B. Haytowitz

USDA-SEA-HN-CNC

6505 Bel crest Rd. Hyattsville, MD 20782

Mary Heckman

Ralston Purina Company

3RS, Checkerboard Square

St. Louis, M0 63188

Frank N. Hepburn

USDA-HN-CNC-SEA

6505 Belcrest Rd. Hyattsville, MD 20782

Harry S. Hertz

National Bureau of Standards

Washington, DC 20234

Joanne M. Hoi den

Nutrient Composition Laboratory

Center Rd.

Bldg. 308, Rm. # 101

Beltsville, MD 20705

Barbara F. Howell

National Bureau of Standards

Washington, DC 20234

Karen Huether

Planters Peanut Company

200 Johnson Ave. Suffolk, VA 23434

W. Jeffrey Hurst

Hershey Foods Technical Center

1025 Reese Ave. Hershey, PA 17033

John L. Iverson

Food & Drug Administration

Washington, DC 20204
Kert I vie

Waters Associates

24 Maple St. Mil ford, MA 01757

Robert G. Johansen

ITT-Continental Baking Co. P.O. Box 731 Rye, NY 10580

Nadine Jutzi

Beatrice Foods Co. 1526 So. State St. Chicago, IL 60605

James R. Kirk

Dept. Food Science & Human Nutrition

Univ. of Florida

Gainesville, FL 32605

Barbara P. Klein

Univ. of Illinois

274 Bevier Hall

Urbana, IL 61801

Walter F. Kline

National Bureau of Standards

Washington, DC 20234

Elaine Lanza

Nutrient Composition Lab.

BARC-E, Bldg. 264

Beltsville, MD 20705

Harry G. Lento

Campbell Soup Company

Line St. & Memorial Ave. Camden, NJ 08101

Betty W. Li Nutrient Composition Lab.

BARC-East

Beltsville, MD 20705

Sam A. Margol is National Bureau of Standards

Washington, DC 20234

Robert A. Martin, Jr. Hershey Foods Corporation

Technical Ctr., 1025 Reese Ave. Hershey, PA 17033

Ruth H. Matthews

USDA, SEA, HN

Rm. 314, Federal Bldg.

Hyattsville, MD 20782

Robert Maxwell

ERRC, SEA, USDA

600 E. Mermaid Lane

Philadelphia, PA 19118

Willie E. May

National Bureau of Standards

Washington, DC 20234

David McKay

Kellogg Co. 235 Porter

Battle Creek, MN 49016

Roger Mossberg

Tecator AB

P.O. Box 70

Hoganaes, Sweden S-26301

Ronald R. Oetgen

Dept. of Chemistry

Roanoke College

Salem, VA 24153

Xiurong Pan National Bureau of Standards

Bldg. 222, Rm. # A349

Washington, DC 20234

Nicholas Parris

ERRC, SEA, USDA

600 E. Mermaid Lane

Philadelphia, PA 19118

Jean Pennington

FDA

200 C. Street

Washington, DC 20234

Harry Radloff

Oscar Mayer & Co. P.O. Box 7188

Madison, WI 53707

S. D. Rasberry

National Bureau of Standards

Washington, DC 20234

W. P. Reed

National Bureau of Standards

Washington, DC 20234

Dennis J. Reeder

National Bureau of Standards

Washington, DC 20234

Curt W. Reimann

National Bureau of Standards

Bldg. 222, Rm. # A309

Washington, DC 20234

Robert D. Reynolds

Beltsville Human Nutrition Res. Ctr.

Rm. 217, Bldg. 307

Beltsville, MD 20705

Cecil e Robitaille

Health Protection Branch

1001 St-Laurent West Blvd.

Long-ueui 1 -Quebec J4K 1C7

CANADA

E. K. Ryu

National Bureau of Standards

Washington, DC 20234

42
Jacob M. Scheiner

Hoffman-La Rouche

340 Kingsland St. Nutley, NJ 07110

Priscilla J. Schuhmann

Nutrient Composition Lab.

Bldg. 308, Rm. 318

Beltsville, MD 20705

Katherine G. Sloman

General Foods

Technical Center

White Plains, NY 10625

Hal T. Slover

NCL, BHNRD

Beltsville Agri. Res. Center

Beltsville, MD 20705

Lorna T. Sniegoski

National Bureau of Standards

Washington, DC 20234

Roza Somkaite

CPC International, Div.. of Best Foods

1120 Commerce Ave.

P.O. 1534

Union, NJ 07083

Kent K. Steward

Nutrient Composition Lab.

Rm. 225, Bldg. 308

Beltsville, MD 20705

James Tanner

FDA, HFF-266

200 C. St. Washington, DC 20234

John K. Taylor

National Bureau of Standards

Bldg. 222, Rm. # A309

Washington, DC 20234

R. H. Thompson

Nutrient Composition Lab.

BARC-E, Bldg. 264

Beltsville, MD 20705

Wayne Thornburg

Del Monte Corp.

205 N. Wiget Ln. Walnut Creek, CA 94598

Richard A. Thornhi 1

Paniplus Co. 100 Paniplus Roadway

Olathe, KS 66061

Thomas N. Tweeten

Hewlett-Packard Co.

Rt. 41 Avondale, PA 19311

George A. Uriano

National "Bureau of Standards

Washington, DC 20234

Joseph Vandersiice

USDA-SEA-HN-Nuti rent Composition Lab.

Rm. 209, Bldg. 308

Beltsville, MD 20705

Ranee A. Velapoldi

National Bureau of Standards

Bldg. 222, Rm. # A309

Washington, DC 20234

John L. Weihrauch

Consumer Nutrition Center, SEA, USDA

7721 Fontaine St. Rochville, MD 20854

Stephen A. Wise

National Bureau of Standards

Washington, DC 20234

Wayne R. Wolf

Nutrient Composition Lab.

Bldg. 307, Rm. 226

Beltsville, MD 20705

H. T. Yolken

National Bureau of Standards

Bldg. 221 , Rm. # B318

Washington, DC 20234

Mary E. Zabik

Dept. Food Science and Human

Nutrition, M.S.U.

Michigan State Univ.

East Lansing, MI 48824

* U.S. GOVERNMENT PRINTING OFFICE: 1982 360-997/2200

43
NBS-114A (REV. 2-8C) U.S. DEPT. OF COMM. l i—1 1 i r~> i I /— A T i f~\ h i /~\ D

1. PUBLICATION OK 2. Performing Organ. Report No. 3. Publication Date

I RIRI inPRflPUIP nATfl DlDLIUuitHrmL UM 1 M REPORT NO.

SHEET (See instructions) NBS SP-635 August 1982

4. TITLE AND SUBTITLE

Reference Materials for Organic Nutrient Measurement Proceedings of a Workshop

held at the National Bureau of Standards, Gaithersburg, Maryland, October 23, 1980

5. AUTHOR(S)

Sam A. Margolis, Editor

6. PERFORMING ORGANIZATION (If joint or other than NBS. see instructions)

NATIONAL BUREAU OF STANDARDS

DEPARTMENT OF COMMERCE

WASHINGTON, D.C 20234

7. Contract/Grant No.

8. Type of Report & Period Covered

Final

9. SPONSORING ORGANIZATION NAME AND COMPLETE ADDRESS (Street. City, State. ZIP)

National Bureau of Standards

Food and Drug Administration

Department of Agriculture

National Food Processors Association

10. SUPPLEMENTARY NOTES

Library of Congress Catalog Card Number: 82-600575

^] Document describes a computer program; SF-185, FlPS Software Summary, is attached.

11. ABSTRACT ( A 200-word or less factual summary of most significant information. If document includes a significant

bi bliography or literature survey, mention it here) This publication is the formal report of the Workshop on Reference Materials

for Organic Nutrient Measurement held at the National Bureau of Standards on October 23, 1980. There were seven formal presentations which provided the

framework for three workshop sessions. Each workshop session focused on one

of three groups of nutrients: (1) cholesterol, fat, and fat-soluble vitamins;

(2) water-soluble vitamins; or (3) sugars. Each workshop session reported on the

state-of-the-art in measurement techniques, suggested matrices which were appropriate

for Standard Reference Materials (SRM's), and indicated areas where there were

problems in measurement methodology. These recommendations are included in this

publ i cat i on

12. KEY WORDS (Six to twelve entries; alphabetical order; capitalize only proper names; and separate key words by semicolons)

food matrices; methods of measurement; nutrients; SRM's; stability; vitamins

13. AVAILABILITY

f_X] Unlimited For Official Distribution. Do Not Release to NTIS

| "~y1 Order From Superintendent of Documents, U.S. Government Printing Office, Washington, D.C. 20402.

Order From National Technical Information Service (NTIS), Springfield, VA. 22161

14. NO. OF

PRINTED PAGES

51

15. Price

USCOMM-DC 6043-P80
NBS TECHNICAL PUBLICATIONS

PERIODICALS

JOURNAL OF RESEARCH—The Journal of Research of the National Bureau of Standards reports NBS research and develop- ment in those disciplines of the physical and engineering sciences in which the Bureau is active. These include physics, chemistry,

engineering, mathematics, and computer sciences. Papers cover a broad range of subjects, with major emphasis on measurement

methodology and the basic technology underlying standardization. Also included from time to time are survey articles on topics

closely related to the Bureau's technical and scientific programs. As a special service to subscribers each issue contains complete

citations to all recent Bureau publications in both NBS and non- NBS media. Issued six times a year. Annual subscription: domestic

$18: foreign $22.50. Single copy. $4.25 domestic; $5.35 foreign.

NONPERIODICALS

Monographs—Major contributions to the technical literature on

various subjects related to the Bureau's scientific and technical activities. Handbooks—Recommended codes of engineering and industrial practice (including safety codes) developed in cooperation with in- terested industries, professional organizations, and regulatory

bodies. Special Publications—Include proceedings of conferences sponsored by NBS, NBS annual reports, and other special publications

appropriate to this grouping such as wall charts, pocket cards, and

bibliographies. Applied Mathematics Series—Mathematical tables, manuals, and

studies of special interest to physicists, engineers, chemists,

biologists, mathematicians, computer programmers, and others engaged in scientific and technical work.

National Standard Reference Data Series—Provides quantitative data on the physical and chemical properties of materials, compiled from the world's literature and critically evaluated. Developed under a worldwide program coordinated by NBS under

the authority of the National Standard Data Act (Public Law

90-396).

NOTE: The principal publication outlet for the foregoing data is the Journal of Physical and Chemical Reference Data (JPCRD)

published quarterly for NBS by the American Chemical Society

(ACS) and the American Institute of Physics (AIP). Subscriptions,

reprints, and supplements available from ACS, 1 155 Sixteenth St., NW, Washington, DC 20056.

Building Science Series—Disseminates technical information developed at the Bureau on building materials, components,

systems, and whole structures. The series presents research results,

test methods, and performance criteria related to the structural and

environmental functions and the durability and safety characteristics of building elements and systems.

Technical Notes—Studies or reports which are complete in themselves but restrictive in their treatment of a subject. Analogous to monographs but not so comprehensive in scope or definitive in treatment of the subject area. Often serve as a vehicle for final reports of work performed at NBS under the sponsorship of other government agencies.

Voluntary Product Standards—Developed under procedures

published by the Department of Commerce in Part 10, Title 15, of

the Code of Federal Regulations. The standards establish nationally recognized requirements for products, and provide all concerned interests with a basis for common understanding of the

characteristics of the products. NBS administers this program as a supplement to the activities of the private sector standardizing

organizations. Consumer Information Series—Practical information, based on N BS research and experience, covering areas of interest to the con- sumer. Easily understandable language and illustrations provide

useful background knowledge for shopping in today's technological marketplace. Order the above NBS publications from: Superintendent of Documents. Government Printing OJfice. Washington, DC 20402. Order the following NBS publications—FIPS and NBSlR's—from

the National Technical Information Services. Springjield. VA 22161

.

Federal Information Processing Standards Publications (FIPS

PUB)—Publications in this series collectively constitute the Federal Information Processing Standards Register. The Register

serves as the official source of information in the Federal Government regarding standards issued by NBS pursuant to the Federal Property and Administrative Services Act of 1949 as amended.

Public Law 89-306 (79 Stat. 1127), and as implemented by Executive Order 1 1717 (38 FR 12315, dated May II, 1 973) and Part 6 of Title 15 CFR (Code of Federal Regulations).

NBS Interagency Reports (NBSIR)—A special series of interim or

final reports on work performed by NBS for outside sponsors

(both government and non-government). In general, initial dis- tribution is handled by the sponsor; public distribution is by the National Technical Information Services, Springfield, VA 22161,

in paper copy or microfiche form.
U.S. DEPARTMENT OF COMMERCE

National Bureau of Standards

Washington, DC 20234

OFFICIAL BUSINESS

Penalty for Private Use. $300

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