Food analysis Additives

Various cleanup techniques can be used to remove

these contaminants, but they are time-consuming,
may remove target compounds, and cannot remove
all interfering compounds. One of the primary class-
es of problem compounds is lipid in food samples.
GPC cleanup is a technique that separates target
compounds from a sample based on their molecular
size. Since lipids are very large molecules compared
to the target compounds in these methods, they are
effectively removed from the extract prior to analy-
sis. The packing gel used in GPC cleanup is porous
and is characterized by the range or uniformity of
that pore size. GPC operates on the principle of
loading all components in an extract on the gel bed,
and then selectively removing the components of
larger molecular size. This procedure provides for the
efﬁcient separation of typical semivolatile and pesti-
cide components from various higher-molecular
weight compounds. This also improves method
performance and extends GC column life, leading
to more efﬁcient analyses. With autosampling and
automatic fraction collection as part of the GPC

system, cleanups can be performed with little or no
operator intervention, requiring only that the ﬁnal
extracts be concentrated.

See also: Carbohydrates: Dietary Fiber Measured as Non-
starch Polysaccharides in Plant Foods. Extraction: Solvent
Extraction Principles; Solid-Phase Extraction; Solid-Phase
Microextraction. Food and Nutritional Analysis: Over-
view. Sampling: Theory; Practice. Sensory Evaluation.

Further Reading

AOAC International (1995) Ofﬁcial Methods of Analysis,

16th edn. Gaithersburg, MD: AOAC International.

Horwitz W (1988) Sampling and preparation of samples

for chemical examination. Journal of the Association of
Ofﬁcial Analytical Chemists 71: 241–245.

Pomeranz Y and Meloan CE (1994) Food Analysis: Theory

and Practice, 3rd edn, pp. 11–29. New York: Chapman
and Hall.

Proctor A and Meullenet J-F (1998) Sampling and sample

preparation. In: Nielsen SS (ed.) Food Analysis, 2nd edn,
pp. 71–82. Gaithersburg, MD: Aspen Publishers.

Additives

M Gallego

, University of Co´rdoba, Co´rdoba, Spain

M Gonza´lez

, Instituto Canario de Investigaciones

Agrarias, La Laguna, Spain

Introduction

Foods naturally provide a series of substances that
maintain the vital processes and normal development
of the body, but sometimes it is desirable to add
foreign substances to foods in order to modify some
aspect of its properties; these substances are called
food additives. The advantages of food additives are
undeniable and so no country prohibits them en-
tirely. Currently, the establishment of international
positive lists and the acceptable daily intake (ADI)
instituted by international organizations ensure that
these additives, used at a normal dosage, will not
have toxic effects on the human body. Analytical
methods for the determination of additives have been
developed so that the stability of these substances
until the food’s consumption can be monitored and
also to ensure that the additives are within the limits
established by legislation. The sample treatment

methods (which consume more than 70% of the an-
alytical process) and the determinative and disc-
riminative methodologies for the determination of
additives in foods have gone through remarkable
changes in the last few years as a consequence of the
spectacular advances in analytical instrumentation.
However, in this regard it has to be pointed out that
these modern methodologies (with sophisticated
instrumentation) are uncommon in many food
industries.

General Information about
Food Additives

Definition of Additive

Among the various definitions of food additives that
can be found in international legislation and accords
is the one adopted by the European Union (EU) in its
European Community Directive 89/107/EEC of the
Council of 21 December 1988: ‘‘any substance not
normally consumed as a food in itself and not nor-
mally used as a characteristic ingredient of food
whether or not it has nutritive value, the intentional
addition of which to food for a technological pur-
pose in the manufacture, processing, preparation,

FOOD AND NUTRITIONAL ANALYSIS

/ Additives

217

treatment, packaging, transport or storage of such
food results, or may be reasonably expected to result,
in it or its by-products becoming directly or indi-
rectly a component of such foods.’’ This definition
emphasizes that additives are substances added in-
tentionally to food to produce permanent functional
effects and that they are generally not considered
themselves to be foods.

Another food additive definition is that proposed

by the Codex Alimentarius Commission: ‘‘any sub-
stance not normally consumed as a food itself and
not used as a typical ingredient of the food, whether
or not it has nutritive value, the intentional addition
of which to food for a technological (including
organoleptic) purpose in the manufacture, process-
ing, preparation, treatment, packing, packaging,
transport or holding of such food results, or may
be reasonably expected to result (directly or indi-
rectly), in it or its by-products becoming a compo-
nent of or otherwise affecting the characteristics of
such foods. The term does not include ‘contami-
nants’ or substances added to food for maintaining
or improving nutritional qualities.’’ This definition
indicates the difference between an additive that is
added intentionally and a contaminant (e.g., micro-
organisms and their toxins, heavy metals) whose
presence in a food is unintentional.

These two examples illustrate that different food

additive definitions tend to be very similar because a
general consensus has been reached among the ma-
jority of countries as to what an additive is. It should
also be pointed out that a special statute is normally
reserved for dietetic products, nutritive substances
added to food products (e.g., vitamins, amino acids,
minerals, etc.), technological processing aids, and
aromas.

Classiﬁcation of Food Additives

There are many classiﬁcation methods for additives:
alphabetical order, numeration of each substance
within a collection of chemicals, or classiﬁcation by
chemical function. The most common method is to
group them by functional categories, that is, by an-
ticipated use. This is the method used by the EU,
which classiﬁes additives in 24 categories, and by the
Codex Alimentarius Commission, which uses 21
categories. The juxtaposition between the types
of additives classiﬁed by the EU and the Codex
Alimentarius Commission is not perfect, given that
the number of categories is different and some
categories that exist in one do not exist in the oth-
er. Furthermore, there is a large amount of multi-
functional additives that could be classiﬁed in

various categories. Table 1 shows a comparison be-
tween the two different classiﬁcations.

In practical terms, a more general classiﬁcation

could be created that includes the additive categories
previously mentioned, separating them by the func-
tions that they fulﬁll in the foods:

1. additives that increase the shelf life or stability of a

food,

2. additives that improve sensorial qualities without

altering the nature or quality of the food in such a
way that could deceive the consumer,

3. additives that improve the nutritional value of the

foods, and

4. additives that aid in the manufacture, transfor-

mation, preparation, treatment, packaging, trans-
portation, or storage of the foods.

Food Additive Regulations

Although additives are generally associated with the
modern food industry, in reality they have been used
for centuries. Preserving food has always been a

Table 1

Comparison between the classiﬁcations of food add-

itives established by EU and Codex Alimentarius

Improved

characteristic

EU legislation

Codex Alimentarius

Color

Color additives

Color

–

Color stabilizers

Preservation

Preservatives

Preservation

Antioxidants

Texture

Emulsiﬁers

Texture

Emulsifying salts

Texture

Thickeners

Texture

Gelling agents

Texture

Stabilizers

Flavor

Flavor enhancers

Flavor

Acids

–

Flavor

Acidity regulators

Acidity and pH

regulators

Texture

Anticaking agents

Flavor

Sweeteners

Texture

Modiﬁed starch

–

Texture

Raising agents

Texture

Antifoaming agents

Texture

–

Foaming agents

Texture

Glazing agents and

lubricants

Lubricants

Texture

Firming agents

–

Texture

Flour treatment

agents

Flour treatment

agents

Texture

Humectants

Texture

Sequestrants

–

Texture

Enzymes

–

Texture

Bulking agents

Texture

Propellants and

packaging gases

Propellants

218

FOOD AND NUTRITIONAL ANALYSIS

/ Additives

concern for humans and to achieve this end, our an-
cestors used salt to preserve meats and ﬁsh; preserved
fruit with sugar; and pickled or canned vegetables
with vinegar. During the nineteenth century and the
beginning of the twentieth century, new chemical
substances were discovered that could be used to
preserve and color foods. In the twentieth century the
need to combat commercial fraud and abuse led to
the ﬁrst regulation of chemical food additives by
France in 1912, giving rise to the concept of a ‘po-
sitive list’ of chemicals allowed to be added to foods.
For the ﬁrst time there was explicit prohibition to the
addition of chemicals or colorants, which had not
been previously approved by law, to foods. The con-
cept of positive lists of additives has lasted until to-
day and is still in use in modern international
legislation.

Regulations in Europe and in the United States

Since 1988, the EU, using safety evaluations done by
its Scientific Committee for Food (SCF), has harmo-
nized the national legislation of its Member States
through European Community Directive 89/107/
EEC, concerning additives that can be used in prod-
ucts destined for human consumption. The role of
the SCF has grown in importance since the adoption
of directives on sweeteners (Directive 94/35/EC),
colorants (Directive 94/36/EC), and other additives
(Directive 95/2/EC) in 1994 and 1995. The commit-
tee, created in 1974 by the European Commission,
has dedicated itself to evaluating the possible risks of
food additives and elaborating guidelines for their
safe use. Meanwhile, the European Commission has
established concrete criteria on the purity of add-
itives, the general or specific categories of foods with
which additives may be used, and the maximum
amounts that can be added to them.

In the United States, the regulation of additives is

done by the Food and Drug Administration (FDA).
As a response to the widespread use of many sub-
stances not approved by the FDA, in 1958 the agency
created the ‘GRAS lists’ (Generally Recognized As
Safe), comprising substances that were unanimously
recognized as safe. These lists, the US equivalent to
the ‘positive lists’ in European legislation, included
only substances that had been proven by scientific
practice and evidenced to be innocuous to human
beings. In the same year, the ‘Food Additive Amend-
ment, to the Federal Food, Drug and Cosmetic Act
(FD&C Act) was passed, requiring the explicit au-
thorization of an additive before it could be added to
a food. It also obligates the manufacturer to prove
that the additive is safe in all of the forms in which it
will be used. This amendment excluded two groups
of substances from the additive regulation process:

those that the FDA or the United States Department
of Agriculture (USDA) had deemed safe for use in
foods previous to the 1958 amendment and those on
the GRAS lists. In 1960, the US Congress approved
the ‘Color Additive Amendments’ to the FD&C Act,
which state that all color additives to be used in
foods, cosmetics, and drugs (including externally ap-
plied drugs and cosmetics) must be approved by the
FDA before being sold on the market. Since 1958,
the FDA and USDA have continued to monitor all
prior sanctioned and GRAS substances in response to
new scientific information and evidence on the safety
of these substances.

International organizations

Every country needs to

have access to reliable evaluations of the risks asso-
ciated with the chemical substances used in foods,
but relatively few have the technical capability and
the economic means to do individual evaluations on
the risks posed by the huge number of existing add-
itives. The work done by the Joint FAO/OMS Expert
Committee on Food Additives (JECFA) and the
Codex Committee on Food Additives and Contam-
inants (CCFAC) is extremely important in this
regard. Both committees were jointly created by the
World Health Organization (WHO) and the Food
and Agriculture Organization of the United Nations
(FAO) and are dedicated to studying additives with
the objective of producing international regulations
for them. The JECFA works as the scientific advisor
to the FAO, the WHO, the member governments of
the FAO, and the WHO and the Codex Alimentarius
Commission. The JECFA establishes guidelines to
evaluate the safety of additives, makes toxicological
evaluations, determines the ADI of each of the add-
itives, prepares their purity speciﬁcations, and evalu-
ates the frequency of consumption of foods that
contain additives. The CCFAC advises the Codex
Alimentarius Commission on food additives, conta-
minants, and naturally present toxic substances; ex-
amines the purity characteristics and the identity of
the additives; establishes the maximum level of con-
taminants in foods; and advises the Products Com-
mittee of the Codex Alimentarius in which foods an
additive can be used and the maximum amounts that
can be added to them.

Labeling and identiﬁcation of food additives

In ad-

dition to the strict criteria applied to the evaluation
of risk and the obligation of having a demonstrable
use, the regulations also require that additives appear
on the labels of the packaging of the foods and drinks
that contain them. These labels must explain the
function of the additive and its name or assigned
number.

FOOD AND NUTRITIONAL ANALYSIS

/ Additives

219

The identiﬁcation of food additives using a

number is an alternative to stating the specific name
of the additive, which is normally quite long and has
a complex chemical structure. In 1960, the EU
assigned conventional numbers to food additives to
better identify them. It was decided that each sub-
stance would be identiﬁed with a number (of three or
four digits) preceded by the letter E. It was thought
that the additives could be separated into nine
categories and that each category would have 100
numbers: the ﬁrst 100 numbers was assigned to color
additives (100–199); the second to preservatives
(200–299); the third to antioxidants (300–399);
and the fourth to emulsiﬁers, thickeners, and gelling
agents (400–499). The initial letter E indicates that
the additive has been certiﬁed by the SCF as safe for
use in the entire EU. In the framework of the Codex
Alimentarius, in 1989 the CCFAC created the Inter-
national Numbering System (INS). This numbering
system was created to reach an international agre-
ement on the identiﬁcation number of additives and
establish a numerically ordered list of additives that
included the number of each additive and its tech-
nological function. The European Commission and
that of the Codex Alimentarius have agreed to adopt
the same identiﬁcation numbers, although the INS
system does not include the E preﬁx, which is re-
served for the EU.

Criteria Used to Establish the Maximum Food
Additive Dose

To establish the maximum food additive dose that
can be used, international authorities consider two
basic criteria: technology and toxicology. On the one
hand, they assess the amount of the additive that is
technologically required to satisfy the objective need
to include the additive in the food; on the other hand,
they evaluate the innocuousness of the additive.

The technologically useful dose of the additive is

that which accomplishes the desired effect on the
food. The tests that determine the technologically
useful dose must follow the guidelines established in
the Good Manufacturing Practice used by the food
industry.

The ADI is established by international organizat-

ions like the JECFA and the SCF after completing
toxicological evaluations. The toxicological evaluat-
ion of additives is done using as reference the max-
imum dose that does not have a toxic effect on
animals or the maximum dose that in short- and long-
term toxicological studies has not had any signiﬁcant
toxic effects. The ADI for man is deﬁned as the add-
itive dose that, when consumed daily during a life-
time, does not cause a noticeable risk and is

expressed in milligrams of additive per kilogram of
body weight per day.

Legal Procedures for the Authorization of
New Food Additives

The EU has established a legislative structure for the
authorization of new additives proposed by either
the EU or any of the individual Member States. This
authorization requires a safety evaluation by the SCF,
which evaluates the information provided by the
manufacturers of the new food additive, including
toxicity data and the function of the new additive. If
the additive passes the safety standards, the commit-
tee establishes its ADI. The European Commission
then develops legislation based on the committee’s
recommendations and then presents it to the Euro-
pean Council of Ministers and the European Parlia-
ment. The ﬁnal result is published in a European
Community Directive that obligates each Member
State to make the necessary changes in their national
legislation to include the new additive. Alternatively,
the manufacturers can ask one of the Member States
for a provisional national authorization while the EU
deliberates on whether or not to legalize the additive.
In this case, a committee of experts from the Member
State evaluates the request, following procedures
similar to those of the SCF. If the Member State es-
tablishes an ADI, the additive can be approved for
use in that country for a period of 2 years, while the
authorization request is studied in the EU. If the SCF
authorizes the additive it is incorporated in a Euro-
pean Community Directive. If the SCF denies the
authorization request, the additive must immediately
be banned in all of the Member States. Figure 1
summarizes the legislative procedures required to
authorize additives in the EU.

EC directive

Food additive

EU evaluation

SCF

European Commission

European

Parliament

European Council

of Ministers

National authorization

for 2 years

Council of Ministers

Committee of experts

from the Member State

Provisional national

authorization

Figure 1

Legislative procedures to authorize additives in

the EU.

220

FOOD AND NUTRITIONAL ANALYSIS

/ Additives

In the US, a request to authorize a new food add-

itive is presented to the FDA. A petition for an add-
itive or a colorant must include data pertaining to the
toxicity of the additive accompanied by toxicological
tests on animals and even on humans. To evaluate
whether an additive should be approved, the agency
ﬁrst considers the composition and attributes of the
substance, the probable amount that will be ingested,
the possible long-term effects on consumers and var-
ious additional safety factors. If the additive is ap-
proved, the FDA publishes regulations that may
include the types of foods in which it can be used, the
maximum quantities that can be added, and how it
should be identiﬁed in the food labels.

Food Additive Legislation

European legislation is compiled in European Com-
munity Directives that are published in the Bulletin
of the EU. European Community Directives affect
the entire EU, setting a time limit for their integration
in the legislation of each Member State, after which
they are required to be enforced in the entire EU.
Table 2 is a catalog of European Community Di-
rectives, updated in December 2003, that regulate
positive lists of additives, standards for the purity of
additives, and the maximum amounts of additives
that can be added to different categories of foods.

The US additive legislation is incorporated in the

Code of Federal Regulations, Title 21, Chapter 1.
The content of the US legislation is organized in
parts, shown in Table 3.

Analytical Methods for the
Determination of Food Additives

The development of analytical methods for the de-
termination of food additives responds to the need to
check the stability of the additives during processing
and storage or, primarily, to legal requirements such
as: (1) establishing whether there are additives
present in foods and whether they are permitted,
(2) determining their concentration to ensure that
they are within the legally established limits, and (3)
conﬁrming the absence of illegal additives in foods.

Sample Pretreatment: Extraction and Cleanup

The pretreatment of foods before additive determi-
nation varies greatly depending on the type of sample
(liquid or solid foods, samples with high content of
lipids, proteins or carbohydrates) or the analytical

Table 2

European Community Directives (updated on December 2003)

EC Directive

Content

General legislation
Directive 89/107/CEE

Additives that can be used in products destined for human consumption

Sweeteners
Directive 94/35/CE

Sweeteners

Directive 95/31/CE

Criteria on the purity of sweeteners

Color additives
Directive 94/36/CE

Color additives

Directive 95/45/CE

Criteria on the purity of color additives

Directive 99/75/CE

Modiﬁcation of the Directive 95/45/CE

Directive 2001/50/CE

Modiﬁcation of the Directive 95/45/CE

Food additives other than colors or sweeteners
Directive 95/2/CE

On food additives other than colors or sweeteners

Directive 96/85/CE

Modiﬁcation of the Directive 95/2/CE

Directive 98/72/CE

Modiﬁcation of the Directive 95/2/CE

Directive 2001/5/CE

Modiﬁcation of the Directive 95/2/CE

Directive 96/77/CE

Criteria on the purity of food additives other than colors or sweeteners

Directive 98/86/CE

Modiﬁcation of the Directive 96/77/CE

Directive 2000/63/CE

Modiﬁcation of the Directive 96/77/CE

Directive 2001/30/CE

Modiﬁcation of the Directive 96/77/CE

Table 3

US additive legislation

Part

Content

70–82

Regulation of color additives

170–173

General regulation of food additives

174–178

Regulation of indirect food additives

180

Regulation of food additives permitted in food or in

contact with food on an interim basis pending
additional study

182–186

Regulation of the substances afﬁrmed as generally

recognized as safe

189

Regulation of the substances prohibited from use in

human food

FOOD AND NUTRITIONAL ANALYSIS

/ Additives

221

technique employed for the determination of the
additive. Often, very simple samples do not require
prior treatment or at the most need uncomplicated
pretreatments, but most foods need to be treated be-
fore the determination of the additive. Frequently,
the ﬁrst step is to use liquid–liquid extraction (LLE)
to eliminate interferences. LLE can be simple or re-
petitive (with the same solvent and then combining
all the extracts). The extractant and the extraction
method used depend fundamentally on the kind of
sample being analyzed. The extraction can be done
with a single solvent, principally n-hexane, water
or acidiﬁed water, acetonitrile, dichloromethane,
ethanol or methanol, diethylic or petroleum ether,
acetone, and chloroform, or with a binary or even
ternary mixture of the solvents, containing a very
polar solvent, like water or methanol, and other less
polar solvents.

However, other matrix compounds are extracted

together with the analytes, making it necessary to
include an extract-cleanup step. The thoroughness of
this step depends on the detection system: if the de-
tector being used is selective enough, an exceedingly
thorough cleanup of the extract is not necessary; but
if the detector is not very selective, this step must
eliminate as many as possible of the coextracts that
can interfere in the determination.

Moreover, because the additive concentration in

this extract is not sufﬁcient for the detection of legal
limits, it is necessary to preconcentrate the analytes.
LLE is commonly used to separate additives from
their coextracts. Nevertheless, the recent trend is to
replace LLE with solid-phase extraction (SPE). It is
possible to use SPE as a single cleanup/preconcen-
tration step or as an additional step after LLE. The
use of SPE has become popular because of the great
variety of polar and nonpolar sorbents and ionic ex-
change resins that exist. The sorbents most often
used to preconcentrate food additives are: RP-C

silica gel, sorbents with amino groups, XAD-2 poly-
meric sorbent, polyamide and wool.

Supercritical ﬂuid extraction is another technique

used to extract food additives. Its main advantages
are that it saves both time and solvents; the solvent
most often employed is carbon dioxide, for its mod-
erate critical temperature (31

1C) and pressure (73

atm). In addition, carbon dioxide is not inﬂammable,
toxic, or very expensive. Methanol is sometimes used
as a modiﬁer to adjust the dissolution power of this
ﬂuid.

Quite recently, other extraction techniques have

been developed and are being employed for the de-
termination of food additives, but are not yet widely
used.

These

techniques

include

countercurrent

chromatography, gel permeation chromatography,

distillation, membrane separation, dialysis and
microdialysis, microwave-assisted extraction, solvent
extraction assisted by ultrasound, solvent microex-
traction, solid-phase microextraction, and stir bar
sorptive extraction.

It is important to emphasize that specific difﬁcul-

ties arise when extracting antioxidants and pre-
servatives from fatty foods. Most of the permitted
antioxidants and preservatives are fat soluble, so it
may ﬁrst be necessary to extract the fat from the food
and then extract the antioxidants from the fat.
As a result, the determination of these additives
implies the removal of triglycerides; the techniques
most frequently used to remove triglycerides are
saponiﬁcation of the extracts, esteriﬁcation of
the extracts in the presence of lipase, and the
separation of the analytes by preparative liquid
chromatography.

Determination, Separation, and Detection of
Food Additives

For the determination of food additives several an-
alytical methods are used based on spectroscopic,
electrochemical, and separation techniques, the latter
with detectors included.

For the determination of individual additives

several spectroscopic techniques are normally em-
ployed: ultraviolet–visible spectrophotometry, spec-
troﬂuorimetry, luminescence, and photoacoustic
spectrometry. Sometimes, to increase method selec-
tivity, a combination of spectroscopic techniques is
used. Another alternative to determine individual
additives are electrochemical techniques: voltamme-
try, polarography, amperometry, and potentiometry.

However, additives are normally combined to

complement and promote their activity; as a result,
it is necessary to develop analytical methods for the
determination of additive mixtures. Although some
spectroscopic and chemical methods are used, it is
preferable to use separation methods for this pur-
pose. Most analytical methods used to determine
food additives are based on chromatographic
techniques, although several recent papers have
demonstrated the usefulness of electrophoresis
for the analysis of food colors, sweeteners, antioxi-
dants, and/or preservatives. The separation of
food colors has received most attention, with a
number of articles published on both capillary
zone electrophoresis and micellar electrokinetic
chromatography.

Thin-layer chromatography, in its normal and high

resolution forms, is used for the separation and
quantiﬁcation of additives with minimal sample
manipulation. The most common detection technique

222

FOOD AND NUTRITIONAL ANALYSIS

/ Additives

is ultraviolet–visible spectrophotometry, although
other techniques such as spectroﬂuorimetry and mass
spectrometry are also used.

Liquid chromatography is the preferred technique

to determine food additives, which in general are
not very volatile and sometimes are thermolabile.

Table 4

Ofﬁcial AOAC methods of analysis applicable to antioxidants

AOAC method

Antioxidant

Food

Analytical technique

948.26

-Tocopheryl acetate

Foods

Colorimetry

942.11

Quaternary ammonium compounds

Milk

Qualitative test

942.13

Quaternary ammonium compounds

Foods, beverages

Bromophenol blue method

952.09

Propyl gallate

Foods

Colorimetry

952.10

Quaternary ammonium compounds

Milk

Optical crystallographic properties of

the Reineckates

954.06

Quaternary ammonium compounds

Milk

Eosin yellowish method

965.28

Antioxidants

Foods

Qualitative test

967.21

Ascorbic acid

Juices

Titrimetry

968.17

Butylated hydroxyanisole, butylated

hydroxytoluene

Cereals

Gas chromatography

971.30

a-Tocopherol, a-tocopheryl acetate

Foods

Colorimetry

975.43

a-Tocopherol

Foods

Polarimetry

983.15

Antioxidants

Oils, fats

Liquid chromatography

984.26

Ascorbic acid

Foods

Semiautomated method – ﬂuorimetry

Table 5

Ofﬁcial AOAC methods of analysis applicable to preservatives

AOAC method

Preservative

Food

Analytical technique

892.02

Sulfurous acid free

Meat

Titrimetry

910.02

Benzoic acid

Foods

Qualitative test

931.08

Formaldehyde

Foods

Qualitative test

935.34

Benzoic acid

Flour

Photometry

950.24

Benzoate, caffeine, saccharin

Soda beverages

Liquid chromatography

953.12

Dehydroacetic acid

Cheese

Qualitative test

953.13

Dehydroacetic acid

Cheese

Spectrophotometry

959.09

Boric acid

Meat

Semiquantitative test

960.27

Preservatives

Milk

Qualitative test

960.38

Benzoic acid

Non-solid foods, beverages

Spectrophotometry

961.09

Sulﬁtes

Meat

Qualitative test

962.16

Sulfurous acid total

Foods

Modiﬁed Monier–Williams method

963.19

Benzoic acid

Foods

Titrimetry

963.20

Sulfurous acid total

Dried fruit

Colorimetry

964.13

Nitrites

Curing preparations

Titrimetry

967.15

Benzoic acid

Foods

Thin-layer chromatography

968.16

Fumaric acid

Foods

Polarography

969.26

Boric acid

Caviar

Spectrophotometry

970.33

Boric acid, borates

Foods

Qualitative test

970.34

Boric acid

Foods

Titrimetry

971.15

Sorbic acid

Cheeses

Oxidation method

972.19

Boric acid

Foods

Atomic absorption spectrometry

974.08

Sorbic acid

Wines

Spectrophotometry

974.10

Sorbic acid

Dairy products

Spectrophotometry

975.10

Sorbic acid

Wines

Colorimetry

975.26

Boric acid

Foods

Emission spectroscopy

975.30

Salicylic aid

Foods, beverages

Qualitative test

975.31

Salicylic acid

Foods, beverages

Colorimetry

975.32

Sulfurous acid

Foods

Qualitative test

980.17

Preservatives

Ground beef

Spectrophotometry

983.16

Benzoic acid, sorbic acid

Foods

Gas chromatography

987.04

Sulﬁtes total

Foods

Differential pulse polarography

990.28

Sulﬁtes

Foods

Optimized Monier–Williams method

990.29

Sulﬁtes total

Foods, beverages

Flow injection method – spectrophotometry

990.30

Sulﬁtes free

Wines

Flow injection method – spectrophotometry

990.31

Sulﬁtes

Foods, beverage

Ion exclusion chromatography

993.03

Nitrates

Baby foods

Spectrophotometry

FOOD AND NUTRITIONAL ANALYSIS

/ Additives

223

In most of the methods that have been developed, the
stationary phase used for the additive separation is a
polygosyl-bonded silica reversed-phase sorbent with
octadecylsilane functional groups (RP-C

), although

other stationary phases such as reverse RP-C

and

ionic-exchange phases have also been used. The de-
tection of additives, after their separation by liquid

chromatography, has been done using ultraviolet–
visible, ﬂuorescence, electrochemical, and mass
spectrometry detectors.

Gas chromatography, with or without derivatiza-

tion, is also used to selectively determine individual or
mixtures of food additives. The additive separation is
done using nonpolar stationary phases consisting

Table 6

Ofﬁcial AOAC methods of analysis applicable to sweeteners

AOAC method

Sweetener

Food

Analytical technique

934.04

Saccharin

Nonalcoholic beverages

Semiquantitative method

941.10

Saccharin

Foods

Qualitative test

947.10

Saccharin

Foods

Sublimation

950.24

Benzoate, caffeine, saccharin

Soda beverages

Liquid chromatography

957.09

Cyclamate salts

Nonalcoholic beverages

Sodium nitrite test

957.10

Cyclamate salts

Nonalcoholic beverages

Gravimetry

957.11

Dulcin

Fruits

Qualitative test – spectrophotometry

969.27

Nonnutritive sweeteners

Nonalcoholic beverages

Thin-layer chromatography

969.28

Cyclamate salts

Canned fruits

Colorimetry

973.28

Sorbitol

Foods

Gas chromatography

973.29

Saccharin

Foods

Gravimetry

980.18

Saccharin

Foods

Differential pulse polarography

Table 7

Ofﬁcial AOAC methods of analysis applicable to color additives

AOAC method

Color additive

Food

Analytical technique

900.01

Color additives

Milk

Qualitative test

920.114

Color additives

Cream

Qualitative test

920.119

Color additives

Butter fat

Qualitative test

920.209

Natural color additives

Foods

Qualitative test

925.28

Color additives

Ice cream, frozen desserts

Qualitative test

930.17

Artiﬁcial color additives

Distilled liquors

Qualitative test

930.38

Synthetic organic color additives

Foods

Column chromatography

938.04

Carotenoids

Macaroni products

Colorimetry

942.19

Synthetic color additives

Oils, fats

Spectrophotometry

948.07

Caramel

Wines

Qualitative test

955.18

Natural and artiﬁcial organic and

water-soluble color additives

Distilled liquors

Spectrophotometry

966.21

Oil-soluble color additives

Foods

Column chromatography

970.65

Riboﬂavin

Foods

Fluorimetry

981.15

Riboﬂavin

Foods

Flow injection method – ﬂuorimetry

985.31

Riboﬂavin

Ready-to-feed milk-based infant

formula

Fluorimetry

988.13

FD&C color additives

Foods

Spectrophotometry – thin-layer

chromatography

Table 8

Ofﬁcial AOAC methods of analysis applicable to emulsifying agents

AOAC method

Emulsifying agent

Food

Analytical technique

920.106

Gelatin

Milk, milk products

Qualitative test

935.61

Gums

Salad dressings

Qualitative test

945.51

Gelatin

Ice cream, frozen desserts

Qualitative test

959.06

Alginates

Chocolate products

Qualitative test

960.33

Gums

Ice creams, frozen desserts

Infrared spectroscopy

960.34

Alginates

Chocolate frozen desserts

Qualitative test

963.25

Alginates

Food dressings

Colorimetry

968.18

Sodium lauryl sulfate

Egg white

Colorimetry

974.11

Polysorbate 60

Shortening, oils, dressings

Gravimetry

991.30

Polydimethylsiloxane

Pineapple juice

Atomic absorption spectrometry

224

FOOD AND NUTRITIONAL ANALYSIS

/ Additives

100%

poly(dimethylsiloxane),

intermediate

polarity phases consisting of 5% diphenyl–95%
dimethylsiloxane or 50% diphenyl–50% dime-
thylsiloxane, and polar phases consisting of 100%
poly(ethyleneglycol). The detection of these analytes
is usually done using a ﬂame ionization detector,
which is able to detect compounds with –CH–
groups, or universal detectors like a mass spectrome-
try detector.

Identiﬁcation of Food Additives

When analyzing food additives it is frequently nec-
essary to identify the additives that have been deter-
mined quantitatively using the above cited detectors
that, while supplying high sensitivity (essential for
measuring the established legal limits), are not able
to identify these compounds. To conﬁrm the ob-
tained results two strategies are used: on the one
hand, the use of detectors that are able to unmistak-
ably identify the analytes; on the other hand, the use
of two different detectors to compare their responses
against the same mixture of additives.

Mass spectrometry is an excellent tool that can be

used coupled to either a liquid or a gas chro-
matograph to conﬁrm the identity of the additives
present in foods. A complete mass spectrum of an
additive provides structural information that is
unique to the additive. Most of the studies that have
been done use the ionization technique for electronic
impact to a certain energy (normally 70 eV).

Detection by a diode array detector can be used to

conﬁrm the identity of an additive. This detector is
usually coupled with a liquid chromatograph to
record the ultraviolet–visible spectra for each of the
chromatographic peaks so that they can be compared
with the spectra of the pure compounds. Moreover,
the purity of each of the chromatographic peaks can
be checked.

The use of two connected detectors, in succession

or in parallel, is widely employed, coupling them to
either a liquid or gas chromatograph.

The Association of Ofﬁcial Analytical Chemists

(AOAC) publishes reference methods for the analysis
of additives in foods. The following Tables 4–8
summarize the AOAC Ofﬁcial Methods of Analysis
applicable to antioxidants (Table 4), preserva-
tives (Table 5), sweeteners (Table 6), color additives
(Table 7), and emulsifying agents (Table 8).

See also: Food and Nutritional Analysis: Overview;
Antioxidants and Preservatives. Gas Chromatography:
Overview. Liquid Chromatography: Instrumentation.
Sweeteners.