Caballero B. (ed.) Encyclopaedia of Food Science, Food Technology and Nutrition. Ten-Volume Set

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International Standards

0012 The Food and Agriculture Organization (FAO), the

World Health Organization (WHO) and the Joint

Codex Alimentarius Commission are concerned

with the safety of food and food ingredients. The

aims of the Codex Alimentarius include protecting

the health of the consumer and insuring fair practices

in the food trade, coordination of all food standards

work, determining priorities and initiating, guiding,

and finalizing the preparation of food standards.

There are published recommended international

standards for individual foods (sugars, edible oils,

canned fruits and vegetables, processed meat, fish

products, cocoa products, etc.), as well as provisions

with respect to food hygiene, food additives, contam-

inants, and labeling. The key role of the Codex Ali-

mentarius in developing international food standards

was recognized when the World Trade Organization

(WTO) was set up in 1995. The WTO agreements

accept the Codex Alimentarius standards and this

enhanced status has raised the profile of the activities

of the Codex. However, the impact of WTO on the

Codex is not yet clear and fears have been expressed

that the body may lose its ability to take decisions

based strictly on science.

0013 All of the standards are detailed specifications,

most of which require analytical methods for their

realization. This task is backed by other standardiz-

ing organizations such as the International Union of

Pure and Applied Chemistry (IUPAC), the Inter-

national Standards Organization (ISO), and the As-

sociation of Official Analytical Chemists (AOAC),

which are involved in the development of methods

of sampling and analysis. These organizations consult

with the various international bodies representing

individual commodity interests, such as the Inter-

national Dairy Federation (IDF), the International

Association for Cereal Chemistry (ICC), the Inter-

national Olive Oil Council (IOOC), the International

Office of Cocoa and Chocolate (IOCC), and others.

European Union Laws

0014 In the European Union (EU), a huge market with a

population of 320 million, a programme of food law

measures has been designed to create a single Euro-

pean market in foodstuffs. The laws of the commu-

nity are expressed in the form of regulations and

directives. Regulations, made under Article 43 of the

Treaty of Rome, are binding on all member states and

are directly applicable. The directive has no force of

law in a member state until it is incorporated into the

legislation of that country. Dates are prescribed in

directives to insure compliance within the time stated.

Some of the rules and specifications of the EU direct-

ives rely on Codex Alimentarius or other inter-

national guidelines. The Community is also laying

down tougher rules for food analysis which

laboratories must adopt. Bringing a multitude of na-

tional laws into line with a common objective is a

difficult task but food legislation at EU level is

expected to deal successfully with questions such as

protection of public health, consumer information,

and fair-trading. The member states of the EU have

speeded up harmonization, since their governments

are aware that outdated legislation can no longer act

as a basis for competition in this large market. Coun-

cil directives 89/397/EEC and 93/99/EEC laid down

uniform general rules for the official control of food-

stuffs and also for laboratories entrusted by the

member states to carry out food inspection and an-

alysis. Since 1993 coordinated food inspection pro-

grams among the responsible authorities in member

states were also set up to establish compliance with

food legislation and insure that food is fit for

consumption. In July 1996, the so-called Black

List Anti-Fraud Regulation came into effect in the

EU. This regulation aims to reduce fraud within

export refund and intervention measures by intensi-

fying inspection. Those found to have committed a

fraud are excluded from EU funds for at least 6

months.

Regulating the Safety of Food in the USA

0015In the USA there were no food regulation laws prior

to the 1900s. At that time food companies could add

anything to their products. In 1902, the Director of

the Bureau of Chemistry in the US Department of

Agriculture (USDA), Harvey Wiley, who was the

leader of a campaign against food adulteration, set

up a group of male volunteers to evaluate the safety of

common food preservatives and ingredients. This

group became known as the Poison Squad. At about

the same time the Poison Squad was formed, Upton

Sinclair wrote a book, The Jungle, dealing with the

unsanitary conditions of the Chicago meat-packing

industry. This crusade resulted in the passage of the

Pure Food and Drug Act in 1906: this Act defined

food adulteration and made the distribution and sale

of adulterated foods and drugs illegal. These early

efforts of the Poison Squad marked the beginning of

food toxicology and provided impetus for the regula-

tion of substances added to foods.

0016In 1931, the Food and Drug Administration (FDA)

was formed as a separate unit of the USDA to admin-

ister the law. This agency regulates food under two

general criteria – adulteration and misbranding. The

FDA was ineffective until 1938, at which time the

44 ADULTERATION OF FOODS/History and Occurrence

passage of the Federal Food, Drug and Cosmetic Act

of 1938 updated and tightened the definition of

adulteration. This gave the agency the power to fine

violators of the laws.

0017 After 20 years of application, interpretation, and

enforcement of this Act, a significant revision took

place in 1958. The Food Additives Amendment of

1958 led to a major change in the FDA’s approach

to its activities. Congress applied the term ‘safe’

as the criterion for action, supplementing the term

‘adulteration.’

0018 Over the years, with advances in science and ana-

lytical methods, the FDA has become more confident

in its evaluation of food safety and US courts have

given general endorsement to the agency’s decisions.

In the fake food fight, the Agency has sought

and won convictions against companies and individ-

uals engaged in selling bogus fruit juices, honey,

maple sugar, cream, olive oil, seafood, and many

others.

0019 The FDA has major responsibilities for food supply

in the USA, but other agencies also exert significant

regulatory control over foods and beverages. The

major agencies so far involved are the Department

of Health and Human Services, the US Department of

Agriculture Inspection Services, the US Department

of Commerce, the Environmental Protection Agency

and Centers for Disease Control and Prevention

(CDC). The FDA relies heavily on industry and con-

sumers to help identify instances of economic fraud.

The detective work of FDA inspectors in the premises

of suspect companies is also valuable. Cooperation

with other foreign government bodies prevents adul-

terated foods banned in one country being shipped

to another. Some adulterants recently reported in

FDA files are: beet sugar or corn syrup in orange

juice; canola oil in olive oil; sugar, water, hydrolyzed

inulin syrup in apple juice; corn oil in dairy cream;

corn syrup in maple and sorghum syrups or honey;

water and sodium tripolyphosphate in scallops;

potato starch in horseradish; salt and water in milk

and sawdust in ginseng dietary supplement. The Safe

Food Act of 1997, by which the food safety and inspec-

tion functions of 12 agencies have been consolidated

into a single independent agency, is expected to combat

even more effectively dishonest or fraudulent practices

not only in a federal but also in an international con-

text. (See Food and Drug Administration.)

Toxicological Implications of Economic

Adulteration of Food

0020 In the past, the deliberate adulteration of food with

untested chemicals or poisonous substances was

widespread. In the 20th century, developments in

the analytical field and improvement of legislation

have brought many of these illegal and unethical

practices under control, but cases of food adulter-

ation with toxicological consequences have not com-

pletely disappeared.

0021One serious case of malpractice in the food indus-

try is the use of denatured rapeseed oil for edible

purposes. This caused the outbreak referred to as

Spanish toxic oil syndrome. In 1981, a disease broke

out in Spain which rapidly took on epidemic propor-

tions. The reported symptoms were respiratory dis-

tress, nausea, fever, vomiting, headaches, myalgias,

abdominal pain, and skin eruptions. Approximately

20 000 people were affected and more than 400

deaths were recorded. For many years, scientists all

over the world tried hard to find an explanation and

identify the toxic compounds causing the illness.

Today, toxicologists and chemists are convinced that

the toxic oil syndrome should be attributed to aniline

used for denaturation, but there are different opinions

concerning the nature of the toxic compounds.

Phenylalamine, originating from the glucosinolates

present in rapeseed oil, was for many years believed

to be the etiological agent in toxic oil syndrome. The

course of events, however, in the two factories

processing the aniline containing rapeseed oil and

simulating studies conducted in the UK do not

confirm this hypothesis.

0022Another explanation may be the formation of N-

phenyl-amino propane diol esters of fatty esters under

conditions favoring dehydration. This suggests that

deodorization sludges containing free fatty acids (and

aniline) were catalytically treated with glycerol to

obtain reconstruction of triacylglycerols. The prob-

lem will remain unresolved unless experts visit Seville

again, study in more depth the conditions of the

episode, and implement simulation processes that

include esterification of fatty acids with glycerol in

the presence of aniline.

Cases of Economic Adulteration

of Food

0023Cases of adulteration frequently reported in the

literature concern not only the basic foods (oils and

fats, milk and dairy products, meat, fish, and cereals)

but also fruit juices, alcoholic beverages and wine,

honey, chocolate, coffee and spices, essential oils,

and flavorings.

0024Some cases of the last decades with serious eco-

nomic and other implications are briefly discussed

below.

ADULTERATION OF FOODS/History and Occurrence 45

Wines

0025 Some Austrian, German, and Italian wines were

found in 1985 to have been illegally supplemented

with diethylene glycol (DEG), not a permitted food

additive, to improve body and sweetness. Soon after

the scandal, a 30% drop in demand for wines was

observed in Austria. In 1987 some Japanese wines

were also reported to contain the same adulterant.

(See Wines: Types of Table Wine.)

Fruit Juices

0026 One case which involved economic fraud and cover-

up efforts is the adulterated apple juice for babies

scandal. For many years, Beech-nut Nutrition Cor-

poration purchased and used concentrated apple juice

which contained cheap ingredients such as corn

syrup, invert sugar, malic acid, caramel, and imitation

flavor. Although it was the director of research and

development who discovered the use of these adulter-

ants, he was forced to resign. The FDA took action

and in 1988 the company was heavily fined.

0027 Citrus juices are frequently adulterated in many

ways. The price of grapefruit juice is always lower

than that of orange juice, so their admixture can

be expected. Misbranding of fruit juices made

from imported concentrates is a common problem

in Florida.

Vanilla Extracts

0028 The adulteration of vanilla extracts with inexpensive

synthetic vanillin represents a loss of millions of

dollars to the vanilla extract industry. To obtain

equal strength of flavor, either 28 g of synthetic vanil-

lin or 4.5 l of single-fold vanilla extract is required.

The ratio of cost is approximately 1:120, and this

significant difference is a strong stimulus for adulter-

ation.

0029 Nonvanilla vanillin can be prepared from lignin,

eugenol, or guaiacol. Synthetic products have a char-

acteristic vanilla-like note but they are inferior in

organoleptic properties because the quality of natural

vanilla derives not only from the concentration of

vanillin but also from the presence of other valuable

volatile flavor compounds.

Olive Oil

0030 Olive oil, a food staple in the warmer regions around

the Mediterranean, is now becoming more popular

throughout Europe, the USA, Japan, and Australia.

Olive oil is often adulterated with seed oils and olive

residue oil (olive pomace, olive kernel, or olive husk

oil). Esterified oil, which is prepared by re-esterifying

low-grade olive oil or olive oil soapstock recovered

from alkali refining, has also been used to adulterate

olive oil products. A survey in 1982 in the USA dem-

onstrated that many olive oil products (imported or

locally produced) contained undeclared olive husk

oil, esterified oils, and seed oils. The European legis-

lation for olive oil characteristics issued in 1991 (and

amended many times since then) is an important

tool against sophisticated frauds. The most recently

reported cases of virgin olive oil frauds are those

where lower-grade olive oil (desterolized or mildly

deodorized), or other oils having a similar compos-

ition (hazelnut oil, high oleic-sunflower oil) have been

used. (See Olive Oil.)

Fish, Meat and their Products

0031Meat and fish commercial products, being the main

food protein sources for human consumption, are

most frequently adulterated with other proteins of

inferior-quality fish species or of nonmeat origin

(e.g., undeclared soya protein in pork sausages).

Authentication of species is important because all

meats are not safe for consumers (e.g., meat from

cows which had bovine spongiform encephalopathy

(BSE)) or meats which should not be used (endan-

gered or overexploited resources). Identification is

more complex, especially when the analysis has to

be carried out on cooked products. Fish traded in

fillets or as fish fingers is the most frequent case of

economic adulteration. Surimi, which was used in the

past as a substitute for crab sticks, is no longer a

cheap raw material because of overexploitation.

Characteristic incidences of substitution of cheaper

materials for more expensive ones include the fraudu-

lent replacement of salmon, trout, plaice or lemon

sole, and caviar. DNA analysis seems to be the answer

to speciation problems related to fish trade. (See Fish:

Fish as Food; Meat: Eating Quality.)

Milk and Dairy Products

0032The addition of water is still the most frequent case of

milk adulteration. False labeling of the animal origin

of milk, the use of reconstituted powdered milk, and

the addition of partially hydrogenated vegetable oils

are currently a matter of investigation for milk and

dairy products. Roquefort in France, manchego in

Spain, pecorino in Italy and feta in Greece are cheeses

of large economic importance which are frequently

adulterated or misbranded. The manufacture of these

products is strictly regulated in these countries and

within the EU. Consumption of products containing

undeclared milk has been associated with certain

food allergies. (See Milk: Liquid Milk for the Con-

sumer; Cheeses: Chemistry and Microbiology of

Maturation.)

46 ADULTERATION OF FOODS/History and Occurrence

Wheat and Other Cereals

0033 The main cases of adulteration reported and ad-

dressed analytically are the admixture of wheat and

barley cultivars, the fraud of durum wheat flour with

common wheat (mainly in pasta products), and the

misbranding of various commercial types (cargo,

milled, broken grains, parboiled) or the origin of

Basmati rice. (See Cereals: Bulk Storage of Grain.)

Genetically Modified Food

0034 The rapid pace of development of genetically modi-

fied organisms (GMOs), especially food plants, and

the commercialization of novel foods derived from

GMOs have caused intense debate and consumer

concern. Although there is no evidence of actual or

potential harm of GM food, nothing can be certain in

a field of rapid scientific and technological progress.

The present EU legislation (regulations 258/97/EC

and 1139/98) recognizes that a genuine choice of

non-GM foods should be available to the public and

that food containing identifiable GM material should

be properly labeled. Against this background numer-

ous questions have emerged concerning analytical

aspects, ranging from sampling procedures to robust-

ness of methods. (See Genetically Modified Foods.)

See also: Cereals: Bulk Storage of Grain; Cheeses:

Chemistry and Microbiology of Maturation;

Contamination of Food; Fish: Fish as Food; Food

Additives: Safety; Food and Drug Administration;

Genetically Modified Foods; Legislation: International

Standards; Additives; Contaminants and Adulterants;

Codex; Meat: Eating Quality; Milk: Liquid Milk for the

Consumer; Olive Oil; Wines: Types of Table Wine

Further Reading

Ashurst PR and Dennis MJ (eds) (1996) Food Authentica-

tion. London: Blackie Academic and Professional.

Barai BK, Nayak RR, Singhal RS and Kulkarni PR (1992)

Approaches to the detection of meat adulteration.

Trends in Food Science and Technology 3: 69–73.

Farrer KT (1997) Dr AH Hassal and food technology. Food

Science and Technology Today 11: 81–87.

Fennema OR (ed.) (1996) Food Chemistry, 3rd edn,

pp. 1–15. New York: Marcel Dekker.

Musgrove S (1998) The enforcement of food authenticity

issues in the UK: practical problems in the European

context. Food Science and Technology Today 12: 84–94.

Robinson C (1997) Genetically modified foods and con-

sumer choice. Trends in Food Science and Technology

8: 84–88.

Widmer WW, Cancalon PF and Nagy S (1992) Methods for

determining the adulteration of citrus juices. Trends in

Food Science and Technology 3: 278–286.

Wrolstad RE (1991) Ethical issues concerning food adulter-

ation. Food Technology 45: 108–114.

Detection

M Tsimidou and D Boskou, Aristotle University of

Thessaloniki, Thessaloniki, Greece

Introduction

0001Methods of detecting food adulteration are based on

physical, chemical, biochemical, and other tech-

niques. All these methods, which have replaced the

early organoleptic and other empirical tests, are

continuously updated because food adulteration is

unceasing, and new problems are always arising. A

number of analytical procedures are usually available

for most cases of adulteration so that the analyst

has the flexibility to choose the appropriate one

(number of samples to be analyzed, sensitivity re-

quired, etc).

0002Proximate analysis of a foodstuff indicates the

extent of abstraction for the main compositional

components – moisture, fat, protein, carbohydrates,

fibre, and ash. However, it cannot answer problems

of authenticity or speciation. Very often, adulteration

can be detected from the presence of minor compon-

ents that occur in the adulterant and not in the food

itself. In such cases, trace chemical analysis is neces-

sary to assess the purity. Data banks of authentic

values that account for seasonal, regional or other

variation may support authenticity testing in some

cases (fruit juices).

0003This article outlines the main applications of ana-

lytical techniques that are routinely used in laborator-

ies authorized to check the purity of foods and drinks,

and also presents in brief the state of the art in

authenticity testing.

Sensory Methods

0004Sensory methods have not been incorporated yet into

legislation for the detection of food adulteration, al-

though routine analysis of a sample always includes

the examination of its organoleptic characteristics.

International organizations such as the International

Standards Organization (ISO), American Society

for Testing Materials (ASTM), and others have

developed and recommended sensory methods for

foods in general and for particular commodities.

Such methods are useful in trade (e.g., grading of

goods) and the management of resources. Moreover,

the US Food and Drug Administration (FDA) has

accepted results of sensory panel tests as prima facie

evidence of product efficacy, and in some cases, legal

ADULTERATION OF FOODS/Detection 47

decisions in the USA have relied exclusively upon

sensory evaluation data.

0005 The limitations concerning the application of

sensory methods for the detection of adulteration

are fewer now that multivariate statistical techniques

are employed. With these methods, sensory data are

better evaluated, and more reliable conclusions can

be drawn. For example, the results of Free-choice

Profiling tests can be analyzed by the Generalized

Procrustes method. The latter adjusts for several

types of variation in the way each panelist describes

samples and then manipulates the data to combine

terms that appear to measure the same characteris-

tics. Such an approach has been meaningfully applied

to classify coffee, tea, and alcoholic beverages.

Separation Techniques

Gas Chromatography (GC)

0006 GC has proved to be essential for the analysis of lipids

and, therefore, is widely used in the detection of

adulteration of edible fats. Adulteration of fats is

usually detected by GC analysis of fatty acid methyl

esters (FAMEs), triacylglycerols, sterols, and their

dehydration products.

0007 Fatty acid composition Separation of FAMEs,

based on different molecular weights and degrees of

unsaturation, is usually achieved on capillary

columns. Positional and geometrical isomers of the

FAMEs are also analyzed under the same conditions.

Standard methods are available for the analysis of

FAMEs. Edible fats have characteristic fatty acid

compositions and can be grouped into various cat-

egories. The percentage values for each fatty acid are

compared with those recommended by national and

international organizations. An increase in the levels

of certain fatty acids indicates the presence of certain

adulterants (e.g., lauric acid, (12:0), content >0.1

indicates the presence of lauric oils to sunflower oil

margarine). An increase in the Trans isomers, natur-

ally found in low levels in animal fats, is an indication

of treatments such as hydrogenation.

0008 Triacylglycerol composition Triacylglycerols are

separated according to carbon number. This is im-

portant for the detection of cocoa butter equivalents

(CBEs) in cocoa products. The addition of 5%

noncocoa butter apart from milk fat is permitted

in many countries, and it is a common practice in

the industry. Fincke, and also Padley and Timms,

proposed a methodology based on the well-docu-

mented linear relationship between C

and C

species for all cocoa butters. All CBEs contain fewer

triacylglycerols compared with cocoa butter, and

this difference causes a significant deviation from the

above relationship. Levels of POP, POS, and SOS

(P¼palmitic, O¼oleic, S¼ stearic acid) for natural

cocoa butters of different origins were used to con-

struct a ternary diagram to discriminate them from

synthetic ones.

0009Analysis of sterols and other minor components Ide-

ntification and determination of sterols by GC are

mainly used to reveal the presence of vegetable fats

in animal fats or admixtures of vegetable fats. The

principle of recommended procedures involves sa-

ponification of the sample, isolation of the sterols

from the nonsaponifiable matter on thin-layer plates,

and GC analysis of sterols or their derivatives. The

separation is usually with SE-30, OV-17 or JXR at

elevated temperatures (230–240



C). Qualitative and

quantitative differences among sterol fractions are

well established for a great variety of vegetable fats.

Sterol dehydration products formed during refining

have been successfully used to identify cheaper edible

oils in virgin olive oil or vegetable oils in chocolate.

Various groups of researchers developed excellent

procedures for the determination of steroidal hydro-

carbons, which are useful to verify the thermal treat-

ment of oils and to demonstrate the presence of

desterolized oils in an admixture. A minor compon-

ent of olive residue oil, erythrodiol, a triterpenic diol,

is used as an indication of adulteration of genuine

olive oil with olive residue oil. The detection is

based on GC of the combined sterol and triterpenic

diol fraction after silanization. The presence of

wax esters with 40–60 carbon atoms indicates the

presence of solvent extracted or refined oils in virgin

olive oils.

0010Recent applications GC–mass spectroscopy (GC–

MS, GC–MS–MS) techniques have been proved un-

equivocal in identifying the presence of undesirable

components and in revealing adulteration. For

example, a GC–MS method was used to identify the

illegal addition of diethylene glycol in wines or ethyl-

ene oxide in spices. Improvements in column technol-

ogy required new sample application techniques. A

variety of injection modes compatible with capillary

columns have emerged. Headspace sampling tech-

niques are ideal for trapping very volatile components

prior to GC analysis. They can also be employed to

explore the volatiles of samples that contain large

amounts of nonvolatile components. This is the case

in fermented beverages such as wine, beer, and dis-

tilled liquors. The profile of the aromatic compounds

of fermented beverages chromatographed by head-

space techniques is used to check misbranding of

48 ADULTERATION OF FOODS/Detection

these heavily taxed commodities. The test involves a

comparative GC analysis of the suspected sample and

a standard for the particular brand.

High-performance Liquid Chromatography (HPLC)

0011 HPLC techniques are increasingly being used in rou-

tine analysis and incorporated into the legislation of

many countries. Today, well-established procedures

for many classes of organic compounds or individual

food components are available; many of these pro-

cedures can be used to check food adulteration.

0012 Carbohydrate composition Carbohydrate analysis

by HPLC is usually performed on amino-bonded

reversed-phase columns. A 70–80% aqueous aceto-

nitrile solution is suitable to elute low-molecular-

weight sugars, isocratically, within 20 min; for the

detection of the eluate, refractive index detectors are

necessary. The sample injected (10–100 ml) must be

free from particulate material; careful extraction and

clean-up are, thus, essential. Simultaneous determin-

ation of glucose, fructose, and sucrose is careful to

detect the illegal addition of inverted sugar in manu-

factured products. Nutritional claims may also be

checked by HPLC profiling of carbohydrates. For

example, lactose content extrapolates the fat level of

skim milk powder; raffinose and stachyose determin-

ation is an indicator of the additon of texturized soya

in meat products, etc. Oligosaccharide analysis is

carried out by similar elution systems to those used

for low-molecular-weight sugars (40–60% aqueous

acetonitrile, 30 min) or by anion exchange

chromatography and pulsed amperometric detection

(potassium hydroxide-based mobile phase). Molecu-

lar-weight carbohydrate analysis is necessary for the

control of the addition of undeclared starch syrups in

honey, fruit juices, and other valuable natural prod-

ucts. The authenticity of instant coffee, using meas-

urements of total glucose (to detect maltodextrins or

caramel), total xylose, and free mannitol (to detect

husks and skins) and free fructose (to detect chicory),

is assisted by the use of pulsed amperometric detec-

tion, which provides an increased sensitivity over the

refractive index detection.

0013 Triacylglycerol composition The HPLC analysis

of triacylglycerols is achieved on reversed-phase

columns, isocratically or by gradient nonaqueous elu-

tion. The separation is based on the concept of

equivalent carbon number (ECN): ECN¼CN2DB,

where CN is the carbon number of the triacylglycerol

molecule, and DB is the double bond number. Tri-

acylglycerols with the same equivalent carbon

number are referred to in the literature as ‘critical

pairs’. The complete resolution of critical pairs is

not necessary for detection of adulterated oils and

fats or fat speciation, as long as the separation is

characteristic and reproducible. The International

Union of Pure and Applied Chemistry recommends

a simple isocratic procedure using acetone–acetoni-

trile mixtures and refractive index detection to deter-

mine the nature and the relative percentages of

triacylglycerols in fat. Based on such simple proced-

ures, the EU has proposed a maximum level of 0.5%

trilinolein for olive oil. The distribution of saturated

(S) and unsaturated (U) fatty acids in the 1, 2, 3

positions of triacylglycerol molecules is used as a

basis for differentiating pork from other fats. The

addition of pork to beef would increase the SSU:SUS

ratio, thus permitting its detection even at low

levels (2%).

0014Amino acid composition The search for an equally

powerful technique for amino acid analysis may

explain the fact that the first reported application of

HPLC to food and beverage analysis was the deter-

mination of free amino acids in cocoa beans in 1980.

Precolumn derivatization of amino acids with various

reagents (phenyl isothiocyanate, dansyl chloride,

2,4-dinitrofluorobenzene or o-phthaldialdehyde), re-

versed-phase columns, gradient elution with mixtures

of acetonitrile (or methanol) and a buffer and a suit-

able detector (UV-Vis or fluorescence) are the usual

chromatographic parameters. Ion-exchange chroma-

tography is followed by postcolumn derivatization

with the ninhydrin reaction and detection in the vis-

ible region. Among the most important applications

are the determination of authenticity and type of

juices, and the detection of the origin of honeys. The

concentration and relative proportions of amino

acids are two of the best criteria for identification of

the type of juices and for estimation of the content of

juice in a beverage or syrup. This evaluation of results

is based on the small variability of relative quantities

of l-amino acids in juices, which cannot be easily

manipulated. Besides, commercial amino acid mix-

tures are expensive adulterants. A stepwise regression

analysis performed on amino acid data produced by

HPLC is very useful in solving complex problems of

adulteration such as the presence of more than one

adulterant, or the addition of acidulants and coloring

agents.

0015Other applications HPLC is a very promising tech-

nique for the determination of minor components and

metabolites. For example, illegal addition of coloring

matter in juices and wine can be detected by HPLC

analysis of pigments; diacetyl determination in butter,

flavonoids in apple and pear juice, and naringin and

prunin in grapefruit are also used to detect aroma

ADULTERATION OF FOODS/Detection 49

supplementation or the type of fruit. HPLC analysis

of minor components usually involves careful extrac-

tion and clean-up of the determinant. The widespread

application of solid-phase extraction with commer-

cial cartridges facilitates these steps.

Electrophoresis and Related Techniques

0016 In recent years, electrophoretic techniques have been

used to reveal adulteration of many food products

rich in protein. Among the most important applica-

tions reported are the detection of: the addition of

soya protein to meat products (polyacrylamide gel

electrophoresis (PAGE) ); the substitution of cow’s

milk for goats milk in goat dairy products (PAGE,

isoelectric focusing (IEF) ); the origin (season, area,

and strain) of wheat flour (PAGE) and the partial

replacement of wheat flour by other cereal flours

(PAGE). Electrophoretic techniques are used almost

exclusively for the species identification of fishery

products. The large amount of fish species consumed,

the close relations among some of them, and the

removal of the external features due to processing

are the major difficulties in the identification of fish

species. The latter is achieved using, as determinants,

sarcoplasmic and myofibrillar proteins under well-

established standard procedures. Reference samples

are considered necessary for identification. Today,

preparations of species-specific proteins are commer-

cially available. Information is also available for the

characteristic electrophoretic protein patterns of a

great number of fish species. PAGE with an anionic

detergent (sodium dodecyl sulfate, SDS) and a

reducing agent (mercaptoethanol) used to separate

subunits by size is of great value for the identification

of salmon, rainbow trout or brown trout eggs. Elec-

trofocusing is suitable because as an equilibrium tech-

nique, it is easily standardized. It is the preferred

procedure for the identification of raw species.

Enzymic staining instead of amido black is used

with IEF to differentiate some species having similar

protein profiles (e.g., mutton from goat meat using

phosphogluconate dehydrogenase). Two-dimensional

electrophoresis (IEF and SDS–PAGE) is a powerful

technique able to resolve 1000 proteins in a mixture.

Conjunction of electrophoretic systems with image

analysis equipment is a means of quantitative estima-

tion of the various species present in a mixture.

Simplified speeded up PAGE procedures (e.g., the

Micrograd system) are applied for discrimination of

the most relevant wheat varieties. HPLC is sometimes

used to complement the PAGE data and vice versa.

0017 Capillary zone electrophoresis The potential of ca-

pillary zone electrophoresis will be tested in a routine

analysis of various analytes in the near future. At the

moment, it is not considered as an alternative method

for protein speciation.

Spectrometry–Spectroscopy

0018Ultraviolet-visible techniques are frequently used for

the assessment of adulteration of citrus juices, coffee

and tea, virgin olive oils, and colored food products.

Minerals in foods are almost exclusively determined

using atomic absorption and emission spectroscopy.

Infrared radiation is the basis of automated equip-

ment for the quality control of milk, grains, or fats.

More sophisticated techniques are employed, where

necessary (nuclear magnetic resonance, mass spec-

trometry).

UV-Vis Spectrophotometry

0019Citrus products The approximation of orange juice

and pulpwash content in citrus products is based on

equations derived from absorption data of the alco-

holic extract in the visible region (465, 443, and

425 nm; carotenes) and the ultraviolet (325, 280,

and 254 nm; polyphenols, flavonoids, and ascorbic

acid) regions. The ratio A

273 nm

326 nm

is found to

be constant for lemon juice (A ¼ absorbance). Any

deviation from this constant value indicates the add-

ition of grapefruit, orange, or apple juices. Lumines-

cence spectrophotometry is also useful for the

characterization of mixtures of citrus fruit juices.

0020Tea and coffee The caffeine content of these highly

priced commodities and their products is a significant

quality criterion. The method, which is based on

measurement of the absorbance at 276 nm, is sensi-

tive enough to test decaffeinated green or roasted

coffee samples.

0021Oils and fats The state of autoxidation is indicated

by the specific extinction values at 232 nm (conju-

gated dienes) and 268 nm (conjugated trienes). The

specific extinction at 268 nm is also suitable to check

admixing of virgin olive oil with residue or refined

olive oil because conjugated trienes are present in the

processed oils.

0022Colored foods Ultraviolet-visible spectra of permit-

ted and nonpermitted pigments are important for the

identification of added coloring matter in food. Care-

ful extraction and isolation of the dyes are necessary

prior to analysis. Lists for permitted colors (FDA, EU)

and spectra for food colors are available for compari-

son (German Research Society).

50 ADULTERATION OF FOODS/Detection

Atomic Absorption and Emission Spectroscopy

0023 Atomic absorption has a high sensitivity but does not

permit multicomponent analysis. The latter is feasible

when inductively coupled plasma–atomic emission

spectrometry (ICP) is coupled with MS. ICP–atomic

emission spectrometry coupled with a pattern recog-

nition program can provide information for the

presence of pulp wash in juices on the basis of the

increased levels of calcium, silicon, and sodium.

Barium, rubidium, and copper were the most import-

ant elements for the geographical differentiation of

orange juices using a similar approach. Sample diges-

tion is a crucial step in this sensitive analytical tech-

nique. Wines can be classified according to origin and

grape cultivar on the basis of simultaneous determin-

ation of a considerable number of minerals and multi-

variate analysis of data. It is worth noting that for

different regions, different elements were found useful

for the discrimination. It seems that grape varieties

selectively take up certain elements from the soil.

Near-infrared Reflectance or Transmission

Spectroscopy (NIR/NIT) (0.7–2.5 mm) and mid-

infrared Spectroscopy (2.5–15 mm)

0024 NIR, or NIT, spectroscopy has been used in the

analysis of a wide array of raw materials and food

products. Many of its industrial applications are

designed for specific products and production lines.

Quality control of products includes determination of

moisture, protein, fat, carbohydrates/starch, and ash

content. Milk and dairy products (whole, skim,

powdered milk, cheese, caseinates, whey concen-

trates, etc.), meat and products, grains, vegetables,

baked goods, and snacks are the most frequently

analyzed materials. The combination of NIR technol-

ogy with multivariate analysis software enables pro-

cessing of all recorded absorption data.

0025 Mid-IR spectroscopy was extensively used in the

dairy industry in the proximate analysis of milk and

soon became a standard method (AOAC, 1980).

Infrared Milk Analyzers evolved as the tools for

producer payment and efficient, rapid, cheap routine

quality control (one milk sample per 20 s). Fourier

transform IR spectroscopy and attenuated total re-

flectance technology are powerful potential methods

in the field of proximate analysis of food components.

Isotopic Analysis

0026 The isotopic composition of various constituents has

been used since the late 1970s as a means of deter-

mining the authenticity of foods of plant origin.

activity analysis and stable isotope ratio analysis

(SIRA), utilizing an isotope ratio mass spectrometer

(IRMS) or a gas chromatography combined with

IRMS, are the most frequently used techniques. The

ratios are usually expressed as ‘d’ units, where



isotope ratio

R sample  R reference standard

R reference standard



 10

0027

C activity measurement The use of

C analysis

in the food industry for determining the origin

of ethanol in wines and other fermented beverages

dates back to 1952. Since then,

C-activity meas-

urement has been extended to other natural prod-

ucts, e.g., spices and flavors. Atmospheric carbon

dioxide through photosynthesis provides plants

with the unstable isotope

C. The age of a certain

plant or plant material is approximated from the

measurement of its

C content. Hence, inexpensive

artificial flavorings from petrochemicals, materials

free of

C activity, can be easily detected. The tech-

nique is used in the USA to check the purity of cham-

pagne from the addition of artificially carbonated

drinks. In a similar way, the vintage of a wine can

be effectively determined because the

C activity of

agricultural products has been well documented for

the last four decades. However, this approach fails in

detecting semisynthetic molecules derived from nat-

ural precursors or manipulated levels of

C to resem-

ble expected levels.

0028Stable isotope ratio analysis Photosynthesis initiates

isotopic fractionation of

C and

C. Most plants

cultivated for food fix carbon dioxide by Calvin syn-

thesis (or the C

photosynthetic mode). Exceptions to

this rule are sugar cane and corn syrup, which follow

the Hatch–Slack (C

) pathway. C

plant material is

more abundant in

C, and thus

C ratios are

higher than in C

plants. Therefore, the

C ratios

are used to detect the low-cost sugar cane and high-

fructose corn syrups (HFCS) in valuable natural

products such as honey and most juices (except for

pineapple, which follows the Crassulacian acid me-

tabolism). No other analysis could identify the above

adulterants in these products. The Association of Of-

ficial Analytical Chemists has adopted a method for

honey, which involves complete burning of a sample

to carbon dioxide and water, purification of carbon

dioxide, and measurement of the

C ratio in an

isotope ratio mass spectrometer.

0029A very important application of the

C ratio is

the detection of synthetic vanillin in vanilla extract.

Vanilla plants follow an intermediate biosynthetic

path compared with C

and C

modes, and the ex-

tracts have very characteristic

C ratio limits. In

this way, natural vanillin can be differentiated from

its synthetic counterpart. Sophisticate fraud involves

enrichment of the plant material with

C to escape

ADULTERATION OF FOODS/Detection 51

MS detection. The

C ratio is also useful for

ensuring the authenticity of corn oil and can provide

a good indication of the presence of corn oil in other

oils. An atypical carbon d value is also indicative

of the addition of cane sugar spirit to brandy.

The combination of IRMS and site-specific isotopic

fractionation (SNIF) (site-specific natural isotopic

fractionation)–NMR appears to be useful to over-

come technical limitations in certain cases.

0030 Another very important application is the discrim-

ination between natural citrus juices and reconsti-

tuted juices (water addition). Plant water, compared

with ground- or rain-water, is richer in

O due to

losses in the lighter

O isotope during leaf evapo-

transpiration.

O values are also used to check

wine appellation claims as well as the addition of beet

sugar syrups prepared with tap-water.

0031 To establish isotopic methods for the identification

of origin, it is necessary to construct the natural vari-

ability of the different bioelement’s stable isotope

ratios within the area of interest. Such data banks

have been installed for wine-producing areas within

the European Union since the beginning of the 1990s.

0032 The distribution of

H in water is influenced by

similar factors as in the case of oxygen isotopes.

Deuterium concentrations are used to detect the add-

ition of groundwater to orange juice (fresh or recon-

stituted) or the addition of beet invert sugar to orange

juice. Analytical difficulties due to the exchange-

able oxygen-bound hydrogen atoms are overcome

through the formation of nitrate esters (the level of

carbon-bound

H is determined).

0033 Site-specific isotopic fractionation–nuclear magnetic

resonance spectroscopy (SNIF–NMR) SNIF–NMR

is a sophisticated technique for detecting the presence

of added sugars in fruit juices and characterizing the

origin of wines. It takes into account the nonrandom

distribution of deuterium within organic molecules, in

particular ethanol molecules. The methodology in-

volves fermentation of the fruit juice to ethanol and

deduction of the botanical origin of the sugars from

the

H ratio at the methyl and methylene site of the

molecule. The methyl site isotope ratio is much lower

in ethanol from beet sugar than that from grape or

citrus fruit fermentation. Addition of 1% beet ethanol

induces a

H shift of 0.88 p.p.m. The analytical

requirements are great (equipment, sample size, data

banks), and the sensitivity of the method is extremely

low in the case of commercial orange juice blends.

Microscopic Methods

0034 Standard microscopic methods have been developed

for the authentication of herbs, spices, flours, and

honey. These examinations are simple in principle,

but the evaluation of results requires considerable

experience of the microscopist. Herbs and spices are

ground, and a slide is prepared and examined under

different power objectives. For comparison, ISO

standards describing histological characteristics of a

great number of spices and condiments can be used.

The potential of microscopic examination of pollen

for the recognition of geographical origin of honey

has been extensively studied. The principle of the

method is the identification of pollen from character-

istic and preferably indigenous plant species of a

region. The visualization of proteins in cereal prod-

ucts, cheese, or chocolate is carried out using fluores-

cence microscopy with specific stains. Microscopy is

used as a screening method in the detection of adul-

teration or misdescription of meat and meat products.

Staining or polarization microscopy enhances the

potential of this examination.

Biospecific Methods

Enzymatic Methods

0035Enzymatic methods are applied to check the purity of

fruit juices and starch and to characterize various

sugars in foodstuffs and formulae.

0036Fruit juices Among the various parameters required

in the evaluation of fruit juice quality, the determin-

ation of sugars and organic acids is extremely import-

ant. Enzymatic determination of sucrose and invert

sugars is more accurate and specific compared with

chemical methods. By applying enzymatic techniques,

more reliable sugar/invert sugar ratios can be calcu-

lated.

0037Determination of organic acids is also useful in

detecting adulteration or checking the type of pro-

cessing and the raw materials used. The addition of

synthetic d,l-malic acid is revealed by a combination

of enzymatic determination of l-malic acid (the nat-

ural existing isomer) and HPLC. Enzyme kits are also

available to catalyse the oxidation of d-malic acid to

pyruvate. The concentration of the formed NADH

monitored spectrophotometrically depends on the

original concentration of d-malic acid. Deviation

from the narrow range of the citric/isocitric acid

ratio in orange juice indicates adulteration with

sugar solutions and citric acid. Other organic acids

such as ascorbic, dehydroascorbic, lactic, and formic

acids are also easily determined enzymatically.

0038Starches There is a need for a reliable and reprodu-

cible technique to determine starch purity. The latter,

for example, is extremely important for the EU

52 ADULTERATION OF FOODS/Detection

refunds for food-grade starches. A multienzyme

regime consisting of a-amylase, amyloglucosidase,

and a debranching enzyme, pullalanase, is utilized to

overcome the problem of incomplete starch hydroly-

sis (EC Regulation 4154/87).

0039 Other applications Enzymatic determination of glu-

cose, sucrose, and starch or glucose, maltose, sucrose,

and partially hydrolyzed starch is recommended by

various bodies for the characterization of sugars

added to meat products, sausages, baby foods, etc.

These determinations of complex mixtures are readily

carried out with commercial multienzyme test kits.

0040 Very sensitive enzymatic methods are also available

for the determination of ethanol. Such methods are

particularly useful in the quality control of wine, beer,

other alcoholic beverages, alcohol-free products, and

alcohol-containing chocolate products.

Immunochemical Methods

0041 Immunochemical procedures were initially developed

for clinical analysis, but today, they find many appli-

cations in food quality control. Immunodiffusion and

newer techniques such as the various forms of

enzyme-linked immunosorbent assay (ELISA) and

radioimmunoassay (RIA) are the techniques most

widely used in food analysis.

0042 Speciation of meat and meat products Identification

of meat species is important not only for economic

but also for religious reasons (e.g., kosher food). The

speciation of fresh, unheated frozen or mildly cooked

meat is carried out by immunodiffusion and ELISA

tests. Immunodiffusion procedures are used as

screening techniques because they are influenced by

the antigen content, which varies from sample to

sample. ELISA tests are more suitable for quantifi-

cation.

0043 Identification of thoroughly cooked meat species

involves tedious steps for both sample (extraction and

protein recovery) and sera preparation. Double-diffu-

sion techniques are capable of detecting 5–10% meat

species in cooked beef sausages by employing anti-

gens resistant to thermal denaturation (e.g., adrenal

antigens). Recently, multiple species-specific thermo-

stable antigens have been used to detect various

species with ELISA systems. In these cases, cross-

reactivity has been reported for very closely related

systems (beef/lamb and turkey/chicken). Combin-

ations of immunoassays and electrofocusing in order

to increase sensitivity have also been used to solve