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

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that may hold food, efforts are being made to reduce

the possible contact to any tin that may be in the

items. The primary target is the food can, and some

efforts are included.

Food Cans

0034 Early food cans were made of tin because of their

permanent shiny appearance and the ease with

which they could be manufactured. However, the

lack of strength to maintain their shape led to the

use of steel, which was much sturdier and less expen-

sive. However, steel corrodes easily when exposed

to air.

0035 The findings that levels of tin that were at the levels

of those originally eluted by contact with food in the

tin cans has effects on the levels of copper, iron, and

zinc, which can affect health, has made the coating of

steel for food cans a very important issue. This led to

various means to prevent corrosion, which occurred

inside the cans with contents of juices of various

foodstuffs. The early method used to prevent this

corrosion (rusting) was to plate the surface with tin.

However, any small deformations or pinholes would

permit rusting of the steel. This led to the use of

various plastics.

0036 The literature is full of procedures for protecting

steel, and most are coatings, for which patents de-

scribe in detail the ingredients and the procedure for

preparing for them. An early simple procedure was to

plate the outer coat with tin and apply an inner

thermoplastic layer with particles of tin in them.

The contents of the can would then be exposed to

only a small amount of tin, and the outer surface of

the can would be protected by the tin coating.

0037 One Japanese patent describes a corrosion-resist-

ant metal–polyester laminate formed by laminating a

hot metal sheet with a polyester film coated with a

thin aluminum layer. A number of layered coatings

without plastic have also been patented. One such

example describes a steel strip flash-electroplated

with nickel, electroplated with tin, rinsed and dried,

and then chromated.

0038 Electric-resistance seam-welded cans are painted

internally and have a tin coating with double chro-

mate layers of chromium oxide layer. They then have

a final coating of epoxy-phenolic paint and are baked

at 205



C. The patents all state that the resulting steel

is suitable for food cans.

Summary

0039 Long-term exposure of weanling rats to levels of tin

expected in foods contained in tin cans has provided

quantitative data on their effects. Copper and zinc

levels were the primary targets of exposure through

the diet. Since these elements are a vital part of many

functions, tin has an indirect effect.

0040General exposure to tin is in the inorganic form in

foods contained within cans containing tin. Origin-

ally, this exposure was through the use of tin cans,

because tin seemed to be inert to general exposure

and was an easy metal to mold into cans. However,

the relative softness resulted in many dents in hand-

ling the cans, and many cans used to be rejected

because of the dents. For this reason, the industry

turned to the use of steel, a less expensive and much

sturdier material.

0041However, the surface of steel is easily oxidized

(rust), forming an unpleasant appearance. Initially,

the steel was plated with tin to coat both the inside

and outside surfaces. The tin coat did not provide

complete protection, because small holes would

allow exposure to the air, resulting in rust spots.

What happened inside the can was not of general

concern, because the amounts of soluble tin were

not considered to be harmful. The first solution to

maintain the external appearance was to coat the

external surface with some type of plastic.

0042There have been many patents on the use of various

plastics to coat the steel, and many elements including

tin are included in the plastics. Many of the resultant

steel sheets are satisfactorily electrowelded. The

patents all state that the steel sheets are suitable for

food cans. This interest is good evidence that the

health of the consumers of canned goods is very im-

portant to showing the concern to produce safe

materials for canning.

See also: Canning: Principles; Cans and their

Manufacture; Recent Developments in Can Design;

Chromatography: Thin-layer Chromatography; High-

performance Liquid Chromatography; Copper:

Physiology; Mercury: Properties and Determination;

Zinc: Deficiency

Further Reading

Boyer IJ (1989) Review paper. Toxicity of dibutyltin and

other organotin compounds to humans and experimen-

tal animals. Toxicology 55: 253–298.

Cappon CJ (1987) GLC speciation of selected trace elem-

ents. LG-GC 5: 400–418.

Cappon CJ (1990) Speciation of selected trace elements in

edible seafood. In: Nriagu JE and Simons MS (eds) Food

Contamination from Environmental Sources. New

York: Wiley.

Chang LW (1985) Neuropathological effects of toxic metal

ions. In: Gabay S, Harris J and Ho BT (eds) Metal Ions in

Neurology and Psychiatry, vol. 15, pp. 207–230. New

York: Alan R. Liss.

5788 TIN

Donaldson JD and Grimes SM (1989) The inorganic chem-

istry of tin. In: Harrison PG (ed.) Chemistry of Tin,

pp. 118–144. New York: Chapman & Hall.

Evans CJ (1989) Industrial uses of tin chemicals. In: Harri-

son PG (ed.) Chemistry of Tin, pp. 421–453. New York:

Chapman & Hall.

Furman NH (ed) (1962) Scott’s Standard Methods of

Chemical Analysis, vol. I, pp. 1073–1093. Princeton,

NJ: Van Nostrand.

Lickiss PD (1989) Organometallic compounds of bivalent

tin. In: Harrison PG (ed.) Chemistry of Tin, pp. 221–

244. New York: Chapman & Hall.

Molloy KC (1989) Organometallic compounds of tetrava-

lent tin. In: Harrison PG (ed.) Chemistry of Tin, pp. 187–

220. New York: Chapman & Hall.

Nielson FH (1976) Newer trace elements and possible ap-

plication in man. In: Prasad A (ed.) Trace Elements in

Human Health and Disease, II. Essential and Toxic

Elements, p. 395. New York: Academic Press.

Rader JI (1991) Anti-nutritive effects of dietary tin.

Advances in Experimental Medicine & Biology 289:

509–524

Selwyn MJ (1989) Biological chemistry of tin. In: Harrison

PG (ed.) Chemistry of Tin, pp. 359–396. New York:

Chapman & Hall.

Toribara TY (1985) Preparation of CH

203

HgCl of high

specific activity. International Journal of Applied

Radiation and Isotopes 36: 903–904.

Toribara TY, Jackson DA, French WR, Thompson AC and

Jaklevic JM (1982) Nondestructive X-ray fluorescence

spectrometry for determination of trace elements along

a single strand of hair. Analytical Chemistry 54:

1844–1849.

Underwood EJ (1977) Trace Elements in Human and

Animal Nutrition, 4th edn. pp. 449–458. New York:

Academic Press.

Walker JQ, Jackson MT Jr. and Maynard JB (1977) Chro-

matographic Systems. Maintenance and Troubleshoot-

ing, 2nd edn. New York: Academic Press.

Willard HH and Toribara TY (1942) A volumetric method

for determining tin based on the formation of a dioxa-

latothiometastannate. Industrial and Engineering

Chemistry Analytical Edition 14: 716–721.

Willard HH and Toribara TY (1942) I. The preparation and

properties of potassium oxalatostannate. II. A study of

complex dioxalatothiometastannates. Journal of the

American Chemical Society 64: 1759–1765.

TOCOPHEROLS

Contents

Properties and Determination

Physiology

Properties and Determination

D C Woollard, AgriQuality NZ, Auckland 1,

New Zealand

H E Indyk, Anchor Products, NZWP, Waitoa,

New Zealand

Physical Properties

0001 The generic term ’vitamin E’ is used to describe a

group of eight structurally related tocopherol and

tocotrienol compounds (Figure 1). The parent form

is commonly referred to as a-tocopherol and system-

atically named 2,5,7,8-tetramethyl-2-(4

,12

-tri-

methyltridecyl)chroman-6-ol, as a derivative of the

benzene-pyran ring structure.

0002 The tocopherols are insoluble in water, freely

soluble in fats and oils, and soluble in most organic

solvents. They exist as pale yellow viscous oils in

purified form but can be crystallized at subzero tem-

peratures, the melting point of a-tocopherol being

2.5–3.5



C. Each tocopherol contains three asymmet-

ric carbon atoms, making possible a total of eight

optical isomers, although the epimeric configuration

at carbon-2 dominates biological activity. Table 1

lists the major physical properties of the naturally

occurring RRR-tocopherols, as well as the synthetic

all-rac-a-tocopheryl acetate. Spectral properties and

specific optical rotations are highly dependent on the

solvent used during measurement.

0003All vitamin E congeners show weak UV absorp-

tion (290–300 nm) and unusually narrow band-

widths. However, they exhibit strong native

fluorescence, emitting radiation at 324–330 nm, the

practical use of which has increased with advances

in detector design. The esters of vitamin E have

TOCOPHEROLS/Properties and Determination 5789

bathochromically modified spectral properties as a

consequence of electronic interactions with the chro-

manol chromophore, and reduced extinction coeffi-

cients and quantum yields relative to the parent

alcohols. The tocopherols and tocotrienols show a

strong infrared band at 8.6 mm and O–HandC–H

stretching frequencies at 2.8–3.0 and 3.4–3.5 mm,

respectively.

Chemical Properties

0004The tocopherols and tocotrienols share a common

chemical structure based upon tocol. Tocol consists

of a chromanol nucleus with substitution at the 2- and

6-positions. A methyl group and a phytyl side-chain

are bonded to C-2, while the hydroxy functionality

on C-6 is available for esterification. Tocol itself is not

tbl0001 Table 1 Physical properties of the tocopherols

a-T b-T g-T d-T a-T acetate

Formula C

Molecular weight 430.69 416.66 416.66 402.62 472.73

Boiling point (



C, 10 kPa (0.1 atm)) 210–220 200–210 200–210 184

Optical rotation (a

546

in ethanol, RRR-isomers) þ0.32



þ2.9



þ3.2



þ3.4



þ3.2



max

(nm) in ethanol 294 297 298 298 285.5

Extinction coefficient, E

1cm

, in ethanol 71.0 86.4 92.8 91.2 43.6

See text for the abbreviated names used.

Tocopherols

Tocotrienols

α-Tocopherol and α-Tocotrienol

β-Tocopherol and β-Tocotrienol

γ-Tocopherol and γ-Tocotrienol

δ-Tocopherol and δ-Tocotrienol

fig0001 Figure 1 Structure of the tocopherols and tocotrienols.

5790 TOCOPHEROLS/Properties and Determination

widely regarded as an E-vitamin but shares some of

the properties of the group, particularly regarding its

antioxidant role, and occurs naturally in low concen-

tration.

0005 The tocopherols are derived from tocol by the

addition of methyl groups on the three available

carbons (5,7,8) of the benzene ring. Trimethylation

produces a-tocopherol, the most active E-vitamin as

defined by rat fertility and other biological activity

studies. In general, any structural modifications from

a-tocopherol markedly reduces biological activity,

including side-chain unsaturation, side-chain length,

oxidation of the chromanol ring, and loss of any

methyl group. While C-8 methylation is essential, b-

and g-tocopherol isomers (5,8- and 7,8-dimethyltocol,

respectively) have reduced bioactivities of *30% and

*12%, and d-tocopherol (8-methyltocol) exhibits

only 1% biopotency relative to a-tocopherol. Other

mono- and dimethyltocols are also known to occur, as

either minor natural components or synthetic bypro-

ducts, but are not classified as E-vitamins owing to

their lack of any measurable physiological response.

0006 Each of the four tocopherols has a related toco-

trienol (abbreviated T3) through phytyl side-chain

unsaturation at C-3

and 11

, resulting in reduced

biopotency: a-T3 (25%), b-T3 (5%), g-T3 (< 1%)

and d-T3 (< 1%) relative to a-tocopherol.

0007 Optical asymmetry at C-2 yields stereoisomers, of

which the d-form is biologically more active by a

factor of 2.38 compared with the l-isomer. Thus,

naturally occurring a-tocopherol, which exists exclu-

sively as the d-isomer has a higher efficacy than the

synthetic racemic equivalent.

0008 The tocopherols have two additional chiral carbon

atoms (4

and 8

), and naturally occurring d-a-toco-

pherol is more correctly described as 2R,4

R,8

R-

a-tocopherol (abbreviated RRR-a-tocopherol). The

stereochemistry of the phytyl ’tail’ is known to govern

biological properties, believed to be a consequence

of in vivo interaction with chiral biological

membranes. Thus, the natural RRR-a-T isomer is

approximately three times more effective than its

stereochemically inverted relative SRR-a-T (referred

to as 2-epi-a-tocopherol). While tocotrienols possess

no side-chain asymmetric centers, geometrical isom-

erism becomes a possibility, although only the trans-

isomers have been reported. The influence of cis

geometry on biopotency is therefore unknown.

0009 The tocopherols are vulnerable to aggressive forms

of oxygen, leading to the formation of biologically

inactive monomeric and dimeric quinones. These

reactions are accelerated by UV light, heat, high

pH, and certain transition elements, whereas in the

absence of oxygen, the E-vitamins are relatively stable.

Lability is decreased by esterification at the 6-hydroxy

substituent, presumably through stabilization of the

phenolic ring structure. Consequently, most nutri-

tional uses of vitamin E involve a-tocopherol esters.

0010To account for the variation in biological activities

of the isomers, the concentration of vitamin E is often

defined in international units rather than by weight.

The international unit (IU) is expressed relative to

synthetic all-rac-a-tocopheryl acetate, where 1 IU is

equivalent to 1 mg, based upon fetal resorption after

oral administration to rats. The information agrees

fairly well with rat hemolytic anemia studies. On this

basis, 1 unit is provided by 0.91 mg of all-rac-a-

tocopherol and 0.67 mg of RRR-a-tocopherol. The

bioactivities of other tocopherols and tocotrienols

are then calculated against these data by applying

the appropriate conversion factors.

0011As a consequence of their phenolic structure, the

tocopherols and tocotrienols are potent antioxidants,

conferring protection to vulnerable lipids in bio-

logical tissues and foods. Such antioxidant behavior

is presumed to depend on pyran ring cleavage to

form the hydroquinone intermediate, followed by

oxidation to tocopherol quinone. These and other

oxidation products (tocopheroxides, epoxides, and

polymers) are receiving increased attention.

Occurrence and Forms in Food

0012The dietary intake of vitamin E in the general popu-

lation appears to be sufficient to meet the US Recom-

mended Daily Allowances of 3 mg (infants) to 12 mg

(adult males). Avitaminosis E is comparatively rare

in well-nourished communities because of large

physiological reserves in tissues and organs, although

neuromuscular and other disorders have been associ-

ated with dietary deficiencies, particularly in chil-

dren. (See Cells; Dietary Requirements of Adults.)

0013The tocopherols are well distributed in nature, the

richest and most varied isomeric sources being vege-

table oils and products derived from them, such as

margarine. It has been common practice with these

products to speciate the various vitamers during

analysis (Table 2). Other valuable sources of vitamin

E include nuts, cereals, green vegetables, fruits, and

foods of animal origin such as eggs, dairy products,

and meats (Table 3). In practice, the contribution of

the non-a vitamers in most foods is sufficiently small

after biopotency factors are applied that they are

regularly excluded from estimations of total vitamin

E status. Animal tissues contain vitamin E almost

exclusively as a-tocopherol, with the exception of

egg yolk, where g-tocopherol is found in significant

concentrations. There is a fairly wide disparity in

literature data, probably as a consequence of genetic,

geographical, seasonal, and processing factors as well

TOCOPHEROLS/Properties and Determination 5791

as variations in analytical techniques. Refer to

individual foods.

0014 Tocopherols and tocotrienols are labile materials,

and their stability during food processing and storage

has been subject to intensive investigations. Storage

of foods under anaerobic conditions and the incorp-

oration of other synthetic or natural antioxidants are

common strategies to optimize the preservation of

their vitamin E status.

Role as Antioxidants

0015 Oxidation of polyunsaturated lipids operates through

a free-radical pathway that not only facilitates food

spoilage but creates substances of potential toxi-

cological significance that can have damaging

consequences upon ingestion. The tocopherols and

tocotrienols combat this process both in vitro and

in vivo by trapping the hydroperoxide intermediates

and preventing their propagation. The fate of the

stable vitamin E phenoxy radicals so produced is

variable, with formation of monomeric quinone,

which can react further with a secondary radical

forming a dimer. Under certain conditions, ascorbate

is considered capable of synergistically repairing the

phenoxy radical and regenerating the tocopherols.

(See Antioxidants: Natural Antioxidants; Oxidation

of Food Components.)

0016It has been confirmed that antioxidant activity of

the tocopherols follows the same order as their

biological potencies (a>b>g>d) and that this may

be rationalized on a stereoelectronic basis. Thus, a

higher resonance stabilization of the phenoxy radical

is achieved in a-tocopherol where ring planarity is

optimal relative to the other congeners.

0017Vegetable oils and margarines are particularly

prone to oxidation, owing to their high content of

polyunsaturated fatty acids but fortuitously also

possess substantial native vitamin E, which functions

protectively. However, additional stabilization is

often considered necessary, particularly for refined

oils where the tocopherols become depleted during

the harsh manufacturing conditions. Although animal

fats and foods rich in them are predominantly satur-

ated and less sensitive to oxidative processes, the

addition of tocopherols is usually of benefit since

natural vitamin E contents are low. (See Margarine:

Methods of Manufacture; Vegetable Oils: Types and

Properties.)

0018The ratio of tocopherol to polyunsaturated fat is an

important criterion in determining optimum oxida-

tive protection offered by vitamin E and also the

toxicological safety of the fat. Excessive concentra-

tions of vitamin E are reported to accelerate lipid

oxidation, although there is debate about the detail

tbl0003 Table 3 Vitamin E content of selected foods

Food

Tocopherol (mg per 100 g)

a-T

b-T g-T d-T

Peanut 7.0–8.3 8.0

Spinach 1.8–2.5 0.1 1.0

Lettuce 0.2–0.4 0.04 0.1

Parsley 0.8–1.2 0.1

Celery 0.3–0.5 0.1 0.4

Peas 0.2–0.5 0.05 0.2

Carrot 0.4–0.5 0.02 0.01

Tomato 0.4–0.9 0.03 0.1

Potato 0.05–0.1

Apple 0.3–0.6 0.02 0.07

Blackberry 3.0–3.5 4.7 4.5

Banana 0.2–0.3

Wheat flour 0.7–0.9 0.6

Beef, raw 0.4–0.8 0.02

Chicken, cooked 0.2–0.4 Trace

Halibut 0.4–1.3 Trace Trace

Milk 0.05–0.13 0.01

Butter 1.0–2.0 0.05

Margarine 10.0–15.0 25.0 3.0

Egg white, raw 0.07–1.95 0.04 0.2 0.01

a-Tocopherol values are expressed as typical ranges, whereas

representative values are reported for the less biologically active

congeners. Blanks indicate a lack of data or the absence of traceable

levels. Tocotrienol contributions are not reported as they are

predominantly absent from these foods.

To convert to IU per 100 g, multiply by 1.49 (as RRR-a-tocopherol).

tbl0002 Table 2 Vitamin E content of selected vegetable oils

Oil

Tocopherol (mg per100 g) Tocotrienol (mgper100 g)

a-T b-T g-T d-T a-T3 b-T3 g-T3 d-T3 Total

Coconut 0–2 0–1 0–1 0–1 0–1 0–4 1–5

Soya bean 1–35 0–4 40–240 15–95 60–330

Corn 2–55 0–30 25–250 2–8 0–24 0–5 0–40 0–2 30–340

Wheat germ 120–150 40–70 0–25 0–25 0–2 6–18 150–200

Sunflower 40–80 1–4 0–5 0–1 45–90

Palm 0–20 1–4 0–30 0–3 4–70 0–10 10–130

Olive 10–25 Trace 10–27

Safflower 20–45 3–10 25–50

Blanks indicate a lack of reported data, implying concentration below limits of detection.

5792 TOCOPHEROLS/Properties and Determination

and extent of this process. As expected, antioxidant

and prooxidant properties differ between the various

tocopherols and tocotrienols. Vitamin E acetate

cannot be used as an antioxidant because this prop-

erty is lost upon esterification.

Use in Food Fortification

0019 As indicated previously, the tocopherols are among

the most important fat-soluble antioxidants used in

the prevention of food spoilage. However, vitamin E

has other recognized biological functions and conse-

quently is added to a variety of foods for the manage-

ment of community health. This has particular

relevance for infant nutrition and for sections of the

population where inadequate nutrition is suspected.

To maintain vitamin efficacy, the free phenols are not

used during food fortification unless an antioxidant

function is also required. (See Food Fortification.)

0020 The most common nutritional additive is synthetic

all-rac-a-tocopheryl acetate owing largely to its

greatly enhanced oxidative stability. Technological

problems during the fortification process can be

overcome by utilizing the oily vitamin in a water-

dispersible ’beadlet’ form, contained within a gelatin

coating. The vitamin can then be blended as an emul-

sion into liquid foods, usually prior to spray drying.

Milk powders, dietetic, and infant formulae are often

supplemented in this way, facilitating a uniform dis-

tribution of the additive. In other products, the ingre-

dients may be mixed in the dry state, either because

the nature of the food is not conducive to wet-

blending procedures or because the vitamin E has a

diminished stability owing to low fat content. Cereals

and skim-milk powder products are regularly supple-

mented in this manner. The major disadvantage with

this protocol is the potential for vitamin hetero-

geneity. Particle-size control and correct mixing,

packaging, and storage conditions are therefore ne-

cessary to minimize uneven distribution of the vita-

min beadlets.

0021 Vitamin E succinate can be used if severe hetero-

geneity problems are encountered since this ester is a

solid at room temperature and more easily dry-

blended into foods and pharmaceuticals. A disadvan-

tage with the use of the succinate ester is its lower

bioavailability. The third commercially available

vitamin E ester, tocopheryl nicotinate, is used in

cosmetics rather than as a food additive.

Isolation and Analysis

0022 Vitamin E poses analytical challenges because of its

multiplicity of forms and its tendency to oxidize.

Traditional bioassays (e.g., resorption gestation,

encephalomalacia, myopathy, and erythrocyte hem-

olysis tests) do not discriminate between the various

vitamers (vitamin isomers) in foods. They are tedi-

ous and relatively imprecise techniques, although

they do exhibit true, host-specific, activity data.

Lipid peroxidation tests for the accumulation of

hydroperoxides or aldehydes in animals have also

been used to indicate vitamin E status. (See Vitamins:

Determination.)

0023Physicochemical tests have become more popular

because of their ease of use, speed, and precision

compared with bioassays. The traditional chemical

test has been the Emmerie–Engel reaction, which

detects the presence of fat-soluble reducing agents

through conversion of trivalent iron to the red ferrous

bipyridyl complex. The accuracy of this and related

colorimetric procedures is, however, compromised by

spectral interferences of other antioxidant materials

such as biologically inactive tocols, retinoids, carote-

noids, nonvitamin chromanol compounds, and syn-

thetic food antioxidants. The success of the method is

therefore dependent upon the degree of purity of the

vitamin E analytes prior to colorimetry. The problem

remains that the eight E-vitamins are not differenti-

ated and will be quantified as a single analyte. These

same difficulties remain, albeit to different degrees,

whether detection is by UV absorption, fluorescence,

or electrochemical techniques. In each case, a chro-

matographic step is required in the analytical scheme

in order to remove interferences and, ideally, to sep-

arate the vitamers. Traditional column or thin-layer

chromatographic (TLC) techniques have been used

extensively for this purpose. (See Chromatography:

Principles; Thin-layer Chromatography; Spectro-

scopy: Visible Spectroscopy and Colorimetry.)

0024Instrumental chromatographic techniques have

now become the methods of choice, with separation

and detection being performed concurrently. Gas–

liquid chromatography (GLC) using various packed-

or open-tubular capillary columns, coupled with flame

ionization detection, allows a quantitative analysis of

individual tocopherols and tocotrienols. A unique at-

tribute of capillary GLC is its recent success in separat-

ing the diastereoisomers of all-rac-a-tocopherol and

the potential of stereoactive chiral columns to resolve

all eight stereoisomers. (See Chromatography: Gas

Chromatography.)

0025The free phenols can be analyzed by GLC, where

elution occurs in order of increasing methylation

(d < g < b < a), although it is preferable to derivatize

the analytes to their more volatile trimethylsilyl ethers

or alkyl esters to improve the chromatographic qual-

ity. However, coelution of the b-andg-isomers is

often a problem unless very long capillary columns

are used. In the analysis of foods, a more significant

TOCOPHEROLS/Properties and Determination 5793

problem exists from interferences by other lipid con-

stituents, predominantly sterols. It is therefore usual

to fractionate the crude extracts by TLC or column

chromatography prior to the assay in order to facili-

tate resolution and interpretation.

0026 High-performance liquid chromatography (HPLC)

offers several advantages over GLC by avoiding the

need for multistage clean-up or derivatization tech-

niques. Either normal- or reversed-phase modes can

be used to achieve a successful separation. Important

advantages of normal-phase chromatography are its

ability to separate the b- and g-isomers and its solvent

compatibility with oil-rich vitamin E extracts. Never-

theless, reversed-phase analysis is technically more

robust and remains in common use, as illustrated in

Figure 2 for an infant formula analysis. It is also

amenable to gradient techniques, allowing concur-

rent determination of tocopherols and tocopheryl

acetate as well as degradation products such as to-

copherol quinone. Recently, variants of micellar

capillary electrophoresis (CE) have been applied to

fat-soluble vitamin separations. Although represent-

ing an alternative to HPLC techniques, the applica-

tion of CE to the hydrophobic vitamins is currently in

its infancy. (See Chromatography: High-performance

Liquid Chromatography.)

0027Direct UV detection is commonly employed for

samples with reasonable vitamin E concentrations.

The difficulty with this mode of detection is the

potential for other materials to interfere and also the

low extinction coefficients of the tocopherols and

tocotrienols. There is consequently a regular need to

increase the sophistication of sample preparation,

which can cause problems with such labile analytes.

Time (min)

fig0003Figure 3 Normal-phase HPLC chromatogram of a saponified

dietary formulation. Waters Radial-PAK 5-mm silica column with a

mobile phase of 10% diisopropylether in hexane. Flow, 2.0 ml

min

1

. Detection by fluorescence at 290 nm (excitation) and

325 nm (emission). Peak identification: 1 ¼a-tocopherol; 2 ¼

a-tocotrienol; 3 ¼b-tocopherol; 4 ¼g tocopherol; 5 ¼b-toco-

trienol; 6 ¼d-tocopherol; ¼elution position of g-tocotrienol;

þ¼elution position of d-tocotrienol.

Time (min)

fig0002 Figure 2 Reversed-phase HPLC chromatogram of a saponified

fully oil-filled infant formulation. Waters Radial-PAK 5-mmC

column with a mobile phase of methanol (100%). Flow rate,

1.0 ml min

1

. Detection by fluorescence at 295 nm (excitation)

and 330 nm (emission). Peak identification: 1 ¼d-tocopherol;

2 ¼b- and g-tocopherol; 3 ¼a-tocopherol.

5794 TOCOPHEROLS/Properties and Determination

0028 Through the use of more specific detection modes,

sample preparation can be further simplified together

with substantial increases in sensitivity. Fluorescence

detection at 292 nm (excitation) and 325 nm (emis-

sion) is a simple procedure with modern detectors,

and encounters very few interference problems pro-

viding the separation is optimized. Excitation can be

performed at 210 nm, but this is less common, except

where spectral overlaps in the detector are unavoid-

able, as with filter-based instruments. Oxidative

electrochemical detection (at 0.7–0.8 V) provides a

further order of magnitude improvement in sensitiv-

ity compared with fluorescence but is less commonly

used owing to operational difficulties and the need for

a supporting electrolyte, incompatible with normal-

phase separations. Similarly, recently reported

LC-MS techniques have been restricted to research

laboratories. (See Spectroscopy: Fluorescence.)

0029For most food products, it is generally necessary to

precede any analysis, whether traditional or instru-

mental, with a clean-up stage aimed largely at remov-

ing bulk lipids, rupturing lipoprotein interactions and

releasing supplemental vitamin E. Alkaline digestion

(saponification), followed by solvent extraction of

unsaponifiable material, is the usual technique

employed where the free tocopherols are measured.

If necessary, the vitamin-rich extract can then be

concentrated by evaporation and subjected to further

purification techniques. Figure 3 illustrates a complex

food product analyzed under these conditions, using

normal-phase HPLC. Six of the eight vitamin E con-

geners are visible, the elution order being a-T < a-T3

< b-T < g-T < b-T3 < g-T3 < d-T < d-T3.

0030Some simpler matrices, particularly oils, can alter-

natively be assayed by sample dilution, clarification

and direct injection, whereas other high-fat products

(e.g., dairy products) may lend themselves to simpli-

fied fat-extraction procedures without the need for a

saponification step (Figure 4). This approach has the

advantage of leaving any supplemental esters intact

and eliminating most of the oxidative difficulties

associated with the parent alcohols. Unfortunately,

a-tocopheryl acetate has poorer UV absorption

than a-tocopherol and greatly reduced fluorescence

properties, whereas normal-phase chromatography is

necessary in order to tolerate the high triglyceride

loading that would quickly foul C

columns. The

alternative vitamin E additive, a-tocopheryl succin-

ate, has no known fluorescence and is susceptible to

peak distortion, both of which encumber chromato-

graphic analysis, unless the sample is subjected to ion-

suppression techniques or measured as a-tocopherol

after saponification.

See also: Antioxidants: Natural Antioxidants; Cells;

Chromatography: Principles; Thin-layer

Chromatography; High-performance Liquid

Chromatography; Gas Chromatography; Dietary

Requirements of Adults; Food Fortification;

Margarine: Methods of Manufacture; Oxidation of Food

Components; Spectroscopy: Fluorescence; Visible

Spectroscopy and Colorimetry; Vegetable Oils: Types

and Properties; Vitamins: Determination

Further Reading

Ball GFM (1988) Fat-soluble Vitamin Assays in Food

Analyses. London: Elsevier.

Bates CJ (2000) Vitamins: fat and water soluble: analysis.

In: Meyers RA (ed.) Encyclopedia of Analytical Chemis-

try, pp. 1–35. Chichester, UK: Wiley.

Diplock AT (1985) Vitamin E. In: The Fat-soluble Vita-

mins, pp. 154–224. London: Heinemann.

01020

Time (min)

α-Tocopheryl

acetate

Lipids

fig0004 Figure 4 Normal-phase HPLC chromatogram of a fortified UHT

milk without the use of saponification. Waters Radial-PAK 5-mm

silica column with a mobile phase of 0.08% isopropanol in

hexane. Flow rate of 2.0 ml min

1

is programed to 8 ml min

1

after 15 min to elute lipid components. Detection by UV at

280 nm at 0.01 aufs. The identification of a-tocopherol acetate is

indicated. The retinyl esters (endogenous and supplemental) are

also visible (*).

TOCOPHEROLS/Properties and Determination 5795

Eitenmiller RR and Landen WO (1999) Vitamin Analyses

for the Health and Food Sciences, pp. 109–148. Boca

Raton: CRC Press.

Frankel EN (1998) Lipid Oxidation. Dundee: The Oily

Press.

Friedrich W (1988) Vitamin E. In: Vitamins, pp. 217–283.

New York: Walter de Gruyter.

Machlin LJ (1991) Vitamin E. In: Machlin LJ (ed.) Hand-

book of Vitamins, 2nd edn, pp. 99–144. New York:

Marcel Dekker.

Nelis HJ, De Bevere VORC and De Leenheer AP (1985)

Vitamin E tocopherols and tocotrienols. In: De Leenheer

AP, Lambert WE and De Ruyter MGM (eds) Modern

Chromatographic Analysis of the Vitamins, pp. 129–

200. New York: Marcel Dekker.

Sheppard AJ, Prosser AR and Hubbard WD (1972) Gas

chromatography of the fat-soluble vitamins: A review.

Journal of the American Oil Chemists Society 49:

619–633.

Wagner AF and Folkers K (1975) Vitamins and Coenzymes,

pp. 363–388. New York: Robert E. Krieger.

Physiology

W A Pryor, Louisiana State University, Baton Rouge,

LA, USA

This article is reproduced from Encyclopaedia of Food Science,

Background

0001 Vitamin E is the most important fat-soluble antioxi-

dant present in human tissues. It is found in the lipid-

rich areas of cells, such as cell membranes and fat

depots. Its role is thought to be the protection of

polyunsaturated fatty acids (PUFAs) such as linoleic,

linolenic, and arachidonic acids, which are present in

lipids, against the types of oxidative damage to which

these highly unsaturated fatty acids are so readily

susceptible. (See Antioxidants: Natural Antioxidants;

Fatty Acids: Properties.)

History

0002 In 1923, Evans and Bishop reported that rats failed

to reproduce if given a diet containing rancid lard,

unless it was supplemented with a factor, called

factor X, found in lettuce or whole wheat. Grad-

ually, over the next 20 years, it became clear that

factor X was associated with more than just repro-

ductive failure in the rat. For example, a series of

observations were reported linking this factor to

encephalomalacia in the chick and nutritional mus-

cular dystrophy in guinea-pigs and rabbits. In 1937,

Olcott and Emerson reported that the tocopherols

are antioxidants.

0003The first 15 years of the study of vitamin E were

made difficult by the absence of a concentrated source

of the vitamin or a knowledge of its structure and

chemical make-up. However, in 1936, Evans and his

group reported the isolation from wheatgerm of an

’alcohol’ which had biological activity consistent

with that of a tocopherol, and they suggested the

correct chemical empirical formula for a-tocopherol

(The free, unesterified tocopherols con-

tinue to be referred to as ’alcohols,’ whereas they are

actually phenols; in fact, alcohols are not, in general,

antioxidants, whereas phenols are.) In 1938, the

structure of a-tocopherol was deduced by Fernholz.

The need for a pure, concentrated, and available

source of the vitamin for nutritional studies was satis-

fied in 1939 when Karrer, of the Basel (Switzerland)

laboratories of Hoffmann–La Roche, and then Smith

and his group in America, reported the synthesis of

the vitamin. (See Phenolic Compounds.)

0004The first international symposium on vitamin E

was held in London in April 1939, and 15 papers

were presented. In the current era, there have been

major international symposia on the vitamin in

various countries almost every year, each with 50 or

100 papers that appreciably add to our expanding

knowledge of this fascinating vitamin.

Natural and Synthetic Forms of

Tocopherol

0005The side-chain of the vitamin (see Figure 1) contains

three carbons that are asymmetric; therefore, there

are eight possible isomers of each of the tocopherols.

Several forms of a-tocopherol are sold. One is

a material that is derived from natural sources,

called RRR-a-tocopherol, which has the stereo-

chemical arrangement of methyl groups shown in

Figure 1; another is the synthetic vitamin called all-

rac-a-tocopherol, which is a mixture of all eight

stereoisomers. Both the naturally derived material

and all-rac-tocopherol are marketed as the free alco-

hol, the acetate ester, and the somewhat less bioavail-

able succinate ester. Some vegetable sources of

tocopherols contain as much or more g-tocopherol

as a-tocopherol. The relative biopotencies of the

various forms of tocopherol are shown in Table 1.

Amounts of the tocopherols are expressed as

either international units (IU) or equivalents of

a-tocopherol (a -TE). The relationship between these

terms is as follows: 1 mg of d-a-tocopherol (the nat-

urally derived material) equals 1 a-TE; this is the unit

used in Recommended Dietary Allowances (10

edn.) by the Food and Nutrition Board of the Na-

tional Research Council (National Academy Press,

1989). The IU is used in commerce for labeling

5796 TOCOPHEROLS/Physiology

vitamin preparations and food, and is defined as

follows: 1 mg of d-a-tocopherol equals 1.46 IU. (See

Dietary Requirements of Adults.)

Vitamin E Requirements

0006 A recommended dietary allowance (RDA) for various

food factors was first recognized in the USA with the

publication of the first edition of the book on RDA

requirements in 1946. A specific RDA for vitamin E

was first recommended in the sixth edition (1964), in

which the concept of the RDA was broadened and

redefined from the 1946 idea – enough ’to insure

good nutrition’–to an amount necessary ’to permit

full realization of . . . potential.’ Thus, in the 1964

edition, the concept was expressed that vitamins

might have a pharmacological use beyond that neces-

sary merely to prevent vitamin deficiency diseases.

The 1997 edition sets the need for vitamin E as 10

a-TE per day for males and 8 mg for females, which is

equal to 10 mg of the natural RRR-a-tocopherol (see

Table 1).

0007The vitamin E requirement for humans increases as

their diet includes more PUFAs because of the suscep-

tibility of PUFAs to undergo autoxidation. A value of

0.4 for the ratio of the intake of RRR-a-tocopherol

(in mg) to the intake of PUFA (in g) in the diet has

been suggested to be adequate for adult humans.

Thus, if a person eating a 2000-cal per day diet con-

sumed 35% of their calories as fat, of which 40% was

unsaturated (typical of today’s American diet), they

would require a daily intake of about 13 mg of

a-tocopherol.

0008The intake of vitamin E for Americans on typical

diets has been estimated to vary between 5 and 20

a-TE. The requirement of 10 a-TE for men and 8

a-TE for women (who are generally smaller) was

established as the RDA because it is believed that

very few Americans have overt vitamin E deficiency

symptoms, and the amount ingested in a ’normal’

diet must therefore be sufficient. This rather unsatis-

factory, backward and ad hoc reasoning is thought

to be the only approach possible for establishing an

RDA for vitamin E, for which deficiency symptoms

are only obvious in humans who have severe mal-

absorption. There is an increasing body of evidence,

however, that vitamin E may provide benefits (such

as protection from chemically induced cancers, cat-

aracts, and ischemic heart disease) that require

higher daily intakes; scientific studies of the useful-

ness of vitamin E in these contexts generally use

amounts of vitamin E up to about 800 mg per day,

which is generally agreed to be safe for humans.

(See Cancer: Diet in Cancer Prevention.)

Metabolism

0009As more tocopherol is ingested, a smaller fraction is

absorbed, such that the uptake of tocopherol by

tissues varies with the logarithm of the tocopherol

intake. Thus, ingesting four times the amount of

the vitamin will raise tissue levels by only twofold.

Triglycerides enhance tocopherol absorption, and

PUFAs inhibit it. If the acetate or succinate is ingested,

it is hydrolyzed prior to absorption through the gut

wall. Since the hydrogen atom of the alcohol group is

intimately involved in the antioxidant properties of

the vitamin, it is not unexpected that only the free

alcohol is biologically active. For high doses, a higher

blood level is achieved using the free alcohol than the

acetate. Absorption of vitamin E is maximal in the

median portion of the small intestine, and none is

absorbed in the large intestine. Vitamin E is thought

to be absorbed through the gut epithelial cells as

a lipid–bile micelle, together with fatty acids,

tbl0001 Table 1 Biopotency of various forms of tocopherols

IU mg

1

a-TE mg

1

RRR-a-Tocopherol 1.46 1.00

RRR-a-Tocopherol acetate 1.36 0.91

RRR-a-Tocopherol succinate 1.21 0.81

All-rac-a-Tocopherol 1.10 0.74

All-rac-a-tocopherol acetate 1.00 0.67

All-rac-a-tocopherol succinate 0.89 0.59

RRR-b-Tocopherol 0.30 0.20

RRR-g-Tocopherol 0.15 0.10

RRR-d-Tocopherol 0.01 0.01

Adapted from Machlin LJ (ed.) (1991) Handbook of Vitamins. New York:

Marcel Dekker.

R''

fig0001 Figure 1 Structures and stereochemistry of the four

tocopherols. Reproduced from Tocopherols: Physiology, Encyclo-

paedia of Food Science, Food Technology and Nutrition, Macrae R,

Robinson RK and Sadler MJ (eds), 1993, Academic Press.

tbl0003 Table 3

Tocopherol Substitutionpattern Stereochemistry

a CH

2R,4

R,8

b CH

HCH

2R,4

R,8

g HCH

2R,4

R,8

d HHCH

2R,4

R,8

TOCOPHEROLS/Physiology 5797