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

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even in acid medium. Orthophosphoric acid can be

used to assist in caramelization (FDA 73.85), as a

neutralizing agent in cacao (FDA 163.110), and as

an acidifying agent in cheese manufacture (FDA

133.123, 124). Several sodium and potassium ortho-

phosphates and pyrophosphates can also be used as

emulsifying agents to process cheese (FDA 133.169,

173). The acid is also added to soft drinks and has

miscellaneous industrial applications.

Ethylenediaminetetraacetates

004 4 In an alkaline medium, the dibasic anion of the ethy-

lenediaminetetraacetic acid (EDTA) forms very stable

complexes with most divalent and trivalent metal

ions. Thus, citric acid and EDTA salts are the most

common chelating agents used as antioxidant syner-

gists in food. As a consequence of the concurrence

with hydrogen ions, the sequestrant capability of

EDTA decreases as the pH is lowered; however, the

EDTA complexes of iron, copper, zinc, aluminum,

and many other metal ions are still very stable in

neutral and slightly acid media. EDTA also lowers

the redox potential of the Fe

3 þ

/Fe

2 þ

pair. The use of

calcium disodium EDTA, rather than disodium

EDTA, prevents the calcium-depleting action of the

ligand in the body. Calcium disodium EDTA can be

added to margarine (0.0075%, FDA 166.110), and is

also permitted whether alone or in combination with

disodium EDTA in a variety of canned vegetables,

emulsions, and dressings to preserve color and flavor

(FDA 172.120, 169.150).

Thiodipropionic Acid and its Fatty Esters

004 5 Thiodipropionic acid and the dilauryl and distearyl

thiodipropionates are weak reducing substances, but

they are very effective in the destruction of hydro-

peroxides, which are reduced to alcohols. The sulfur

atom is eventually oxidized to sulfate, but the reduced

sulfur species originated as reaction intermediates

catalyze the decomposition of more hydroperoxide

molecules. Up to 20 moles of hydroperoxide can be

decomposed by a mole of thiodipropionate. Thiodi-

propionates display strong synergism with BHT and

other phenol derivatives, which also terminate free

radical chain reactions through a rather different

mechanism.

0046 Thiodipropionic acid and its esters are GRAS for

use in food when the total content of antioxidants is

not over 0.02% of fat or oil content of the food (FDA

182.3109, 3280). They are used to retain color in

dried bananas, beans, peas, canned clams and shrimp,

and frozen potatoes. They improve flavor retention

in canned carbonated beverages, dressings, mayon-

naise, margarine, and sauces. They are also used as

antioxidants in soap products and lubricants, and in

the manufacture of a variety of polymers (including

adhesives). When used in packaging materials, they

should not migrate to food in amounts exceeding a

concentration of 0.005% in the food (FDA 178.2010,

181.24).

Stannous Chloride

0047Stannous chloride is a very strong reducing agent that

absorbs oxygen from the air. The colorless crystalline

solid or the white powder is severely irritating and

corrosive, and reacts with alcohols, amines, alkalies,

and oxidizers, sometimes with violence; the reaction

with hydrogen peroxide is strongly exothermic, even

in solution. As a food preservative, it can be added to

canned asparagus packed in glass containers in a

quantity not exceeding 15 mgg

1

calculated as tin

(Sn), but up to 20 mgg

1

can be present if the con-

tainer lid is lined with an inert material (FDA

155.200). It is also used as a stabilizer for colors and

perfumes in soaps, and as a reducing agent in many

other industrial applications.

Sodium thiosulfate

0048The addition of sulfur to sodium sulfite produces

sodium thiosulfate, a salt that contains an S–S bond.

Its antioxidant properties are due to the presence of a

reduced sulfur atom, but it also forms strong com-

plexes with iron, copper, and other metal ions. It can

be added to alcoholic beverages (<0.00005%) and

table salt (< 0.1%) (FDA 184.1807). It is also used

as a reducing (e.g., to remove residual chlorine from

solutions), bleaching, and complexing agent in a

variety of industrial applications.

4-Hexylresorcinol

00494-Hexylresorcinol is a pale yellow, viscous liquid (be-

coming solid on standing at room temperature) with a

pungent faint fatty odor and a sharp astringent taste.

It is an irritant, and its concentrated solutions can

cause burns on the skin and mucous membranes.

The Canadian Food Inspection Agency allows the

addition of up to 1 mgg

1

hexylresorcinol to frozen

and fresh crustacean (http://inspection.gc.ca). It is

used in medicine as a topical and urinary antiseptic,

anthelmintic, cleanser for skin wounds, and anes-

thetic for throat pain.

See also: Antioxidants: Natural Antioxidants; Synthetic

Antioxidants, Characterization and Analysis; Role of

Antioxidant Nutrients in Defense Systems;

Atherosclerosis; Cancer: Carcinogens in the Food

Chain; Diet in Cancer Prevention; Diet in Cancer

Treatment; Coronary Heart Disease: Antioxidant Status;

Legislation: Additives; Mutagens; Oxidation of Food

274 ANTIOXIDANTS/Synthetic Antioxidants

Components; Phenolic Compounds; Storage

Stability:ParametersAffectingStorageStability;

Tannins and Polyphenols; Tocopherols:Properties

andDetermination; Vitamins:Overview

Further Reading

Adegoke GO, Kumar MV, Krishna AGG et al. (1998) Anti-

oxidants and lipid oxidation in foods. A critical ap-

praisal. Journal of Food Science and Technology

(Mysore) 35: 283–298.

Bauerova K and Bezek S (1999) Role of reactive oxygen and

nitrogen species in etiopathogenesis of rheumatoid arth-

ritis. General Physiology and Biophysics 18(special

issue) 15–20.

Botterweck AAM, Verhagen H, Goldbohm RA, Kleinjans J

and vanden-Brandt PA (2000) Intake of butylated hydro-

xyanisole and butylated hydroxytoluene and stomach

cancer risk: Results from analyses in The Netherlands

cohorts study. Food Chemistry and Toxicology 38:

599–605.

Budavary S (ed.) (1996) The Merck Index, 12th edn.

Rahway, NJ: Merck & Co.

Coupland JN and McClements DJ (1996) Lipid oxidation

in food emulsions. Trends in Food Science and Tech-

nology 7: 83–91.

Dangles O, Dufour C and Fargeix G (2000) Inhibition of

lipid peroxidation by quercetin and quercetin deriva-

tives: antioxidant and prooxidant effects. Journal of

the Chemistry Society Perkin Transactions 6:

1215–1222.

Fukumoto LR and Mazza G (2000) Assessing antioxidant

and prooxidant activities of phenolic compounds. Jour-

nal of Agricultural and Food Chemistry 48: 3597–3604.

Gertz C, Klostermann S and Kochhar SP (2000) Testing and

comparing oxidative stability of vegetable oils and fats

at frying temperature. European Journal of Lipid Sci-

ence and Technology 102: 543–551.

Gower JD (1988) A role for dietary lipids and antioxidants

in the activation of carcinogens. Free Radical Biology

and Medicine 5: 95–111.

Hras AR, Hadolin M, Knez Z and Bauman D (2000) Com-

parison of antioxidative and synergistic effects of rose-

mary extract with alphatocopherol, ascorbyl palmitate

and citric acids in sunflower oil. Food Chemistry 71:

229–233.

Iuliano L, Pedersen JZ, Camastra C et al. (1999) Protection

of low density lipoprotein oxidation by the antioxidant

agent IRFI005, a new synthetic hydrophilic vitamin E

analogue. Free Radical Biology and Medicine 26:

858–868.

Kirk R and Othmer DF (eds) (1978–1984) Encyclopedia of

Chemical Technology, 3rd edn. New York: Wiley.

Kirk R and Othmer DF (eds) (1991–1995) Encyclopedia of

Chemical Technology, 4th edn. New York: Wiley.

Morsel JT and Meusel D (1990) Fortschrittsbericht. Lipid-

peroxydation. 2. Mitt. Sekundare Reaktionen [Progress

report. Lipid peroxidation. Part 2. Secondary reactions].

Nahrung 34: 13–27.

Moure A, Cruz JM, Franco D et al. (2001) Natural anti-

oxidants from residual sources. Food Chemistry 72:

145–171.

Schubert MA, Wiggins MJ, DeFife KM, Hiltner A and

Anderson JM (1996) Vitamin E as an antioxidant for

poly(etherurethane urea): in vivo studies. Journal of

Biomedical Materials Research 32: 493–504.

Ueda T, Ueda T and Armstrong D (1996) Preventive effect

of natural and synthetic antioxidants on lipid peroxida-

tion in the mammalian eye. Ophthalmic Research 28:

184–192.

Valenzuela A and Nieto S (1996) Synthetic and natural

antioxidants: Food quality protectors. Grasas y aceites

47: 186–196.

Weisburger JH (1999) Mechanisms of action of antioxi-

dants as exemplified in vegetables, tomatoes and tea.

Food Chemistry and Toxicology 37: 943–948.

Code of Federal Regulations, USA http://www.access.gpo.-

gov/nara/cfr/index.html

FAO/WHO Joint Expert Committee on Food Additives

(JECFA). Monographs and Evaluations http://

www.inchem.org/jecfa.html

International Numbering System (INS) for Food Addi-

tives ftp://ftp.fao.org/codex/standard/volumela /en/

Cxxot04e.pdf

Miscellaneous databases on chemical products: http://

www.chemfinder.com, http://www.nutritionfocus.com,

http://www.sigma-aldrich.com

National Toxicology Program, National Institute of Envir-

onmental Health Sciences, Research Triangle Park, NC

http://ntp-server.niehs.nih.gov

School of Food Biosciences, The University of Reading, UK

http://www.fst.rdg.ac.uk/foodlaw

US Food and Drug Administration, Center for Food Safety

& Applied Nutrition, Office of Premarket Approval,

EAFUS: A Food Additive Database http://vm.cfsan.fda.-

gov/*dms/eafus.html

Synthetic Antioxidants,

Characterization and Analysis

G Ramis-Ramos, Universidad de Valencia, Burjassot,

Spain

Phenolic Antioxidants

Reversed-phase High-performance Liquid

Chromatography

0001The determination of phenolic antioxidants and

ethoxyquin (EQ) in food is most frequently accom-

plished by reversed-phase high-performance liquid

chromatography (RP-HPLC), while both HPLC and

thin-layer chromatography (TLC) are normally used

ANTIOXIDANTS/Synthetic Antioxidants, Characterization and Analysis 275

for the screening of antioxidant residues in food prod-

ucts. Usually, sample preparation for RP-HPLC in-

volves dilution or suspension with hexane, extraction

with a polar solvent (most frequently acetonitrile

mixed with some water), preconcentration by evap-

oration under reduced pressure, reconstitution (e.g.,

with isopropanol), and, if necessary, clean-up with

unmixable solvents or solid phases. Extraction with

supercritical CO

has been used for capillary electro-

separations. RP-HPLC separations are normally per-

formed on C18 columns using gradient elution with

aqueous phases containing methanol or acetonitrile

and acetic acid, followed by spectrophotometric

detection at 280–285 nm. Pentachlorophenol and

2,4,6-trimethylphenol have been employed as in-

ternal standards in the analysis of fats and oils and

bakery products.

0002 A modification of the AOAC official method

applied to butter oil has yielded recoveries higher

than 95% for alkyl gallates, t-butylhydroxyquinone

(TBHQ), butylated hydroxyanisole (BHA), nordihy-

droguaiaretic acid (NDGA), 2,4,5-trihydroxybutyro-

phenone, and 2,6-di-t-butyl-4-hydroxymethylphenol,

and lower for butylated hydroxytoluene (BHT)

(79%). Other reported data indicate recoveries higher

than 90% for BHA and BHT in vegetable oil and

propyl gallate (PG) in lard, and for dodecyl gallate

(DG), BHA, BHT, TBHQ, and NDGA in liver pa

but the less hydrophobic PG and octyl gallate (OG)

gave recoveries around 80% in this sample. Using

methanol, recoveries between 85 and 95% have

been reported for PG, OG, DG, 2- and 3-BHA,

NDGA and TBHQ, but 3,5-di-t-butylcresol gave

72%. The intralab relative standard deviations

(RSDs) range typically between 3 and 7%, but values

lower than 2% have also been obtained. Reported

interlab RSDs range from 7% for DG to 26% for

TBHQ. Low or rather disperse recoveries can be due

to oxidation during sample storage and preparation,

which easily occurs with TBHQ.

0003 In one procedure for dry food, the sample was

homogenized with hexane, water, and acetonitrile

(25:5:75) and the combined acetonitrile extracts con-

centrated by evaporation, injected, and detected at

280 nm. Separation was performed on a C18 column

with gradient elution using water–acetonitrile mobile

phases containing 5% acetic acid. The detection

limits (DLs) for PG, BHA, BHT, t-butylquinol,

2,4,5-trihydroxybutyrophenone, 3,5-di-t-butyl-4-

hydroxymethylphenol, and NDGA ranged from 0.4

to 2.7 mgml

1

. For a typical 20-ml injection loop,

these data roughly match with other DLs reported

as absolute values: 25 (OG, DG), 35 (PG) and

100 ng (NDGA). In the determination of nine phen-

olic antioxidants in corn oil, butter oil, butter, niboshi

(dried sardines), and frozen shrimps, the DLs were

1.0 mg per gram of sample (85–102% recoveries).

0004Lower DLs can be achieved with fluorometric,

chemoluminometric, and amperometric detection.

For BHA, BHT, and TBHQ, excitation at 280 nm

yields maximum emission at c. 310 nm. Procedures

based on the chemiluminescence of luminol are

mainly used to measure antioxidant activity, and

these are discussed in detail later in the corresponding

section. t-Butylquinol, 2,6-di-t-butyl-p-cresol, and

2- and 3-t-butyl-4-methoxyphenol have been deter-

mined in oils by HPLC with amperometric detection

on a glassy carbon electrode. With a previous modifi-

cation of the carbon electrode surface by electropoly-

merization of nickel phthalocyanine, DLs ranging

from 0.1 to 0.6 mgml

1

have been achieved. When

used in a flow-injection nonchromatographic system,

the modified electrode provides a DL of 2.7 ng ml

1

with a 1.8% RSD (at 0.5 mgml

1

) for BHA in bis-

cuits. Using 0.3- and 0.7-mm microbore HPLC with

electrochemical detection, DLs as low as 0.1 and

0.6 fmol l

1

have been obtained for BHA, BHT,

and PG.

HPLC with Ion-pairing and Micellar Mobile Phases

0005The selectivity of the HPLC separations can be modi-

fied by ion-pairing agents, such as alkyl sulfates and

carboxylates, and alkyl ammonium salts. These re-

agents form micelles when at least one of the alkyl

chains has 12 or more carbon atoms; however, mi-

celles are disrupted in the presence of a large organic

solvent concentration, thus leading to an ion-pairing

rather than a micellar separation mechanism. PG,

OG, DG, BHA, and BHT can be determined in spiked

olive oil with a mobile phase containing sodium

dodecyl sulfate (SDS) and 30% propanol in 18 min.

0006An aqueous micellar mobile phase, which may

also contain a lower percentage of an organic solvent

(usually 5–15% of a short-chain alcohol), is used in

micellar liquid chromatography (MLC). In MLC,

physiological fluids and tissue extracts can be injected

without deproteinization, because proteins are solved

in the micellar phase and elute with the solvent front.

Moreover, hydrophobic samples can be simply di-

luted with a compatible solvent and injected. Thus,

several oils and fats diluted with n-pentanol (5–30%

solutions) can be injected on a C18 column and eluted

with an SDS/n-propanol mobile phase; PG, OG,

TBHQ, and BHA have been quantified in c. 15 min

with absolute DLs ranging from 0.05 to 0.3 ng.

Avoiding a previous two-phase partitioning step sim-

plifies the procedure and may help in obtaining high

recoveries and in reducing random and systematic

errors, particularly when easily oxidizable solutes

such as TBHQ are quantified.

276 ANTIOXIDANTS/Synthetic Antioxidants, Characterization and Analysis

0007 Further, since retention time in MLC is linearly

proportional to solute hydrophobicity, isocratic elu-

tion is always suitable to separate solutes with large

hydrophobicity differences, whereas gradient elution

is required in most cases with conventional hydroor-

ganic phases, owing to the exponential dependence of

the retention time with solute hydrophobicity. Also,

in comparison to hydroorganic phases, micellar

phases have a lower toxicity and produce a milder

environmental impact, and owing to the lower vapor

pressure of its components, a small volume of phase

can be recirculated and reused many times without

any perceivable alteration of the chromatograms. To

prevent clogging by precipitation of the surfactant, a

simple cleaning protocol with water and methanol

should be followed before stopping the pumps or

storing a used column.

0008 Another advantage of MLC is the excellent repro-

ducibility of the chromatograms and, frequently, the

almost perfect Gaussian shape of the peaks, which

facilitates modeling and prediction. Thus, the chro-

matographic retention of seven antioxidants has been

described, with errors below 3%, by the equation

1=k ¼ c

þ c

 þ c

’ þ c

’

where k is the relative retention (capacity factor), m

and ’ are the concentrations of SDS and organic

solvent (n-propanol in this study), and c

to c

are

regression coefficients, which are calculated for each

solute from five chromatograms obtained with differ-

ent m and ’ values. Modeling of retention speeds up

the optimization of chromatographic procedures, i.e.,

the resolution between all the peak pairs of a group of

solutes can be maximized, and the interference of

matrix peaks can be avoided, by using the model

predictions. This procedure can be used to optimize

the MLC separation of a mixture of antioxidants in a

short elution time.

Capillary Electroseparations

0009 Capillary electroseparations are rapid, versatile, and

very efficient, solutions in solvent mixtures of all

types with the sole requirement of supporting some

conductivity can be used to fill up the capillary, and

most hydrophilic and hydrophobic samples can be

injected without pretreatment. Further, only the

anions or the cations present in a sample can be

selectively introduced in the capillary by using elec-

trokinetic injection. Antioxidants can be separated in

at least two modes: (1) capillary zone electrophoresis

(CZE), which is based on the different electrophoretic

mobilities of the charged solutes, and (2) micellar

electrokinetic capillary chromatography (MECC), in

which differential migration results from the different

strength of the association of uncharged solutes with

charged discriminant agents such as ionic surfactants

(both as individual ions or as micelles), vesicles and

derivatized (e.g., sulfated) cyclodextrines. In mixed

MECC, charged solutes are separated by the joint

action of their own electrophoretic mobilities and

the different strength of the solute–discriminant

agent interactions.

0010Using a CZE method with spectrophotometric

detection, flavonoids and phenolic antioxidants

have been determined in red wine and olive leaves.

In one experiment, the wine samples were directly

injected, and the leaf extracts were prepared by diges-

tion with boiling water or aqueous methanol, Soxhlet

extraction with acetone/CH

, and supercritical

fluid extraction with CO

. Triphenyl acetic and

benzoic acids were used as migration time markers,

to assist in the identification of the analytes. The

absolute DL was 3 pmol.

0011In a mixed MECC procedure, a buffer containing

SDS was used to separate 11 phenolic antioxidants

with DLs of 2.8–8.6 mgml

1

. Vesicles of bis(2-

ethylhexyl)sodium sulfosuccinate have been used to

separate six food antioxidants in 15 min. Sodium

cholate and SDS have been used to separate nine

phenolic antioxidants and ascorbic acid with DLs in

the range of 1–10 mgml

1

(spectrophotometric detec-

tion at 214 nm), and the procedure has been applied

to wine and sesame seed oil.

0012In a previous study, CZE, MECC, and HPLC were

compared, using a sodium borate buffer of pH 9.5 for

CZE, adding SDS to this buffer for MECC, and

performing RP-HPLC separations on a C18 column

with isopropanol/water/acetonitrile (2:17:31). The

resolution was poor with CZE and excellent with

MECC. The antioxidants were separated in 6 and

25 min by MECC and HPLC, with absolute DLs in

the picomole and nanomole ranges, respectively. The

reader should not be misled by the reported absolute

DLs, which are usually extremely low due to the very

small injection volumes (in the picoliter range).

Owing to the short optical path length (limited to

the capillary inner diameter), the relative DLs are in

fact higher than in HPLC with spectrophotometric

detection. The DLs of capillary electroseparations

can be reduced by a factor of five to 10 by using

capillaries with specially extended path lengths, or

more effectively by implementing fluorimetric or

mass-spectrometric detection.

Gas Chromatography

0013BHA, BHT, TBHQ, a-tocopherol, and a-tocopherol

acetate have been determined using a gas chromato-

graph coupled to a solid-phase extractor device,

extracting powdered soup with n-hexane in the pres-

ence of 2-t-butyl-4-methylphenol and 5-a-cholestane

ANTIOXIDANTS/Synthetic Antioxidants, Characterization and Analysis 277

as internal standards, and washing the extract with

0.2 M NaCl to remove any water-soluble compounds.

Similarly, oil and margarine samples were spiked with

the internal standards and diluted with n-hexane. The

antioxidants were retained on Amberlite XAD-7 resin

and eluted with isopropanol, and portions of the

eluate were injected. Using a coated fused-silica

column and a flame ionization detector (FID), the

DLs ranged from 2 to 8 mg l

1

, and the RSDs for

50 mg l

1

were 2.7–3.5%. The recoveries of 125 mg

of antioxidants per kilogram from spiked sunflower

seed oil were 93.6–101.4%. Synthetic antioxidants

have also been determined by GC in soya-bean oil

and sesame oil.

Thin-layer Chromatography

0014 To screen PG, OG, DG, BHA, and BHT by TLC, oil

samples have been diluted with hexane and extracted

with 80% ethanol and acetonitrile. The combined

extracts were evaporated under reduced pressure,

and the concentrate was spotted on the TLC plates.

After development, the solutes were revealed by

oxidative coupling with N,N-dimethyl-p-phenylene-

diamine in the presence of hexacyanoferrate (III). The

values and the color of the spots obtained with

several mobile phases were then tabulated.

Nonchromatographic Techniques

0015 Rapidity at the cost of a lower selectivity can be

gained by using nonchromatographic techniques.

Selectivity can be improved by using kinetic ap-

proaches and other masking techniques, and, as

detailed below, if the signals given by several solutes

overlap, the required qualitative and quantitative in-

formation can be retrieved by using multivariate data

treatment techniques, including principal-component

regression (PCR) and partial least-squares regression

when all solutes exhibit linear signal–concentration

relationships, and artificial neural network (ANN)

algorithms in case of nonlinearity. Thus, in a differen-

tial kinetic determination, the oxidation of several

antioxidants with Fe(III) has been monitored with

2,2

-dipyridyl, and the spectrophotometric-time data

treated with an ANN algorithm.

0016 In comparison to ordinary spectrophotometry,

selectivity can be improved by using derivative spec-

trophotometry. This technique has been used to de-

termine BHA and BHT in lard, diluting the sample

with hexane, extracting with dimethyl sulfoxide, and

measuring the second derivatives of the spectrum at

311.5 and 278.5 nm for BHA and BHT, respectively

(with recoveries of 99.5–109.3% and 90.6–108.1%).

0017 Sensitization of the luminescence of Tb(III) in a

micellar medium has been used to determine PG by

a stopped-flow initial-rate kinetic method. Dilution

of the oil samples with hexane, followed by extrac-

tion in an ammonium acetate aqueous solution,

mixing with the Tb(III) reagent and data collection

over 1 s has yielded recoveries of 90.1–108.2%, a

RSD of * 2%, and a DL of 0.02 mgml

1

. Mixtures

of PG and BHA have also been determined with a

stopped-flow setup, the former being evaluated by the

initial rate method, by monitoring the luminescence

of Tb(III) at 0.1 s, and the latter being determined

from the equilibrium signal of its reaction product

with the oxidized form of Nile Blue, measured at

1 s. Both systems are excited at 310 nm, and the emis-

sion wavelengths are 545 nm for PG and 665 nm for

BHA. Recoveries from food samples have been found

to range between 94 and 103%, with RSDs of ap-

proximately 2% and DLs of 0.03 and 0.09 mgml

1

for PG and BHA, respectively.

0018Phenolic antioxidants present well-defined oxida-

tion waves, with peak potentials of 629, 818, 599,

and 501 mV, with linear concentration ranges of

1–15, 0.5–15, 0.5–8, and 1–15 mg l

1

, for PG, BHA,

BHT, and TBHQ, respectively. Square-wave voltam-

metry on carbon-fiber disk ultramicroelectrodes has

been used to determine BHA and BHT in acetonitrile

extracts of vegetable oils and lard, yielding DLs of a

few micromoles per liter and RSDs of about 6–7%.

To determine BHA in chewing gum, samples are

shaken with ethyl acetate and the polymer compon-

ents precipitated by freezing; the supernatant is then

diluted with a 1% solution of the polymeric nonionic

surfactant Pluronic F-68 and buffered. Differential

pulsed voltammetry at a glassy carbon electrode has

yielded DLs below 1 mmol l

1

0019Problems arising from overlapping oxidation

waves in mixtures can be overcome by improving

the electrode kinetics through modification of the

electrode surface. Thus, PG, BHA, TBHQ, and BHT

can be simultaneously determined in mixtures by

differential-pulse voltammetry on a carbon-fiber elec-

trode coated with a poly-(3-methylthiophene) film. In

micellar solutions and in an oil-in-water emulsion, a

DL for PG of 0.8 mmol l

1

and a RSD for 50 mMof

1.3% have been found. The method has been applied

to dehydrated soups. Cyclic voltammetry with modi-

fied Pt electrodes has been used to determine PG in

cornflakes and potato flakes, extraction with 1:1

water/methanol leading to quantitative recoveries

and a DL of 1.5 mgml

1

0020As indicated above, overlapped signals can be dis-

cerned by using multivariate data treatment tech-

niques, and thus PCR and PLS have been applied to

voltammetric data. Differential pulse voltammetry

with PLS calibration has been used to determine PG,

BHA, and BHT in dehydrated soup, with DLs of 0.15,

278 ANTIOXIDANTS/Synthetic Antioxidants, Characterization and Analysis

0.7, and 7 mmol l

1

, respectively, and recoveries

ranging from 77 to 101%.

0021 Finally, an amperometric enzyme biosensor (BAS

VC-2 tyrosinase electrode) has been used to deter-

mine BHA in biscuits, extracting the samples with

ethyl acetate, concentrating the extracts, and diluting

with a medium of dioctyl sulfosuccinate reversed

micelles. Under flow conditions, the DL has been

found to be 30 mmol l

1

Characterization and Determination of Anoxomer

0022 Anoxomer is a nonabsorbable polymeric phenol

obtained by condensation polymerization of m- and

p-divinylbenzene with TBHQ, t-butylphenol, hydro-

xyanisole, p-cresol and 4,4

-isopropylidenediphenol.

Two types of analytical problems should be addressed

in relation to anoxomer: (1) to verify regulations

concerning its composition and purity and (2) to

detect and determine anoxomer in food products

and wrapping materials. According to the US Code

of Federal Regulations (CFR 21, 172.105), the

specifications to be met are: (1) the purity should

not be lower than 98.0%, (2) the total amount of

monomers, dimers, and trimers below a molecular

mass of 500 should not be more than 1%, (3) the

phenol content should range from 3.2 to 3.8 meq g

1

and (4) traces of Pb, As, and Hg should not exceed 10,

3, and 1 mgg

1

, respectively. Assays to check specifi-

cations (1), (2), and (3) are available from the Center

for Food Safety and Applied Nutrition and other US

Administration agencies.

0023 Several qualitative and quantitative procedures for

anoxomer have been investigated, but no methods for

real samples have been published. A linear calibration

graph over the range of 5–50 mgml

1

can be con-

structed by heating the product with nitric acid in a

medium containing acetic acid, adjusting the pH to

8 with ammonia, and measuring the absorbance at

420 nm. A linear calibration graph over the range

1–10 mgml

1

has been obtained by measuring the

native fluorescence of anoxomer in diethylene oxide.

A lower sensitivity has been obtained by measuring

the infrared absorption of anoxomer in tetrachloro-

ethylene solutions at 3525 cm

1

(-OH stretching;

linearity range: 150–580 mgml

1

4-Hexylresorcinol

0024 4-Hexylresorcinol has been determined in shrimps by

extraction with methanol and RP-HPLC with fluori-

metric detection (excitation at 280 nm, emission at

310 nm), with a recovery of * 82%, RSD of 2.1%

within the range 1.5–2.5 mg kg

1

, and a DL of

1.98 ng ml

1

in the extract and 80 mgkg

1

in shrimp.

In another method for shrimps, the ethyl acetate

extracts have been cleaned up by solid-phase extrac-

tion, followed by gradient elution on a C18 column

with trifluoroacetic acid/acetonitrile/methanol/water,

and detection at 214 nm (DL ¼6 ng, 94% recovery).

In a method for crab meat, extraction with acetoni-

trile, cleaning up by solid-phase extraction, and

HPLC with diode array detection has been used

(RSD ¼ 7.1%, 86–104% recovery).

Nonphenolic Synthetic Antioxidants

Ethoxyquin

0025EQ can be determined by extracting the samples with

hexane, followed by HPLC with spectrophotometric

(absorption bands at 230 and 365 nm), fluorimetric

(emission at 435 nm with excitation at 365 nm),

or electrochemical (oxidation wave at þ0.45 V) de-

tection, or by GC with a FID or with thermionic

detection, or using TLC on fluorescent plates. In a

comparative study, significant differences among the

RDSs were not found for EQ in fish meals using

different techniques, which was attributed to the

large variability in the extraction process.

0026EQ is decomposed by light, which should be taken

into account in storing samples. It has been found that

EQ is stable for at least 9 days by keeping the apple

extracts in deep freeze, and for at least 20 days by

storing the pieces of apple in hexane in the dark.

Further, its degradation products are found in

significant concentrations in the food samples. EQ

and up to 12 of its degradation products have been

resolved by HPLC. Also, EQ and its two major oxi-

dation products, 1,8

-di-1,2-dihydro-6-ethoxy-2,2,4-

trimethylquinoline (DM) and 2,6-dihydro-2,2,4-tri-

methyl-6-quinolone (QI), have been determined

in fish meals by HPLC (DL ¼5mgkg

1

for EQ

and DM, and 0.5 mg kg

1

for QI) and gas chro-

matography (GC), and the changes in the EQ, DM,

and QI concentrations during storage have been

studied.

0027HPLC procedures for EQ in meat meals and ex-

truded pet foods (with fluorimetric detection), apples,

skins of apples, pears, citrus fruit, and bananas, chilli

powder, and paprika have been developed. For pap-

rika, and using spectrophotometric detection, the DL

has been found to be 2 mgml

1

, and the RSDs for 20

and 40 mgml

1

, 2.4 and 1.9%, respectively; however,

with fluorimetric detection, the DL has been found to

be 0.2 mgml

1

, and the RSD for 0.5 mgml

1

, 3.2%.

EQ in apples has been also determined using electro-

chemical detection (DL ¼0.03 ng) [69]. The levels of

EQ residues in apple varieties stored under different

conditions have been tabulated (with spectrophoto-

metric detection, DL ¼0.25 mgml

1

in the extract

ANTIOXIDANTS/Synthetic Antioxidants, Characterization and Analysis 279

and 0.2 mg kg

1

in the fruit). EQ has also been deter-

mined in apples by GC (DL ¼ 50 mgkg

1

, * 74%

recovery). The use of guaiacol as an internal standard

in the GC determination of phenolic antioxidants and

EQ has been described.

Thiodipropionic and Thiodiglycolic Acids and their

Fatty Esters

0028 3,3

-Thiodipropionic acid, thiodiglycolic acid (or

2,2

-thiodiacetic acid) and their dialkyl esters have

been separated and identified by HPLC on a C18

column with a water–acetonitrile mobile phase

of pH 7.95 and spectrophotometric detection at

254 nm. The determination of thiodipropionic acid

esters of fatty alcohols in polyethylene for food pack-

aging has been performed by Soxhlet extraction with

CHCl

, followed by saponification with methanolic

KOH and capillary GC of the liberated fatty alcohols.

The distearyl thiodipropionate has also been deter-

mined by GC analysis of the compounds evolved by

pyrolysis of the polymers used for packaging.

Selection of the Analytical Method for

Phenols and EQ

0037 For screening purposes, TLC on high-performance

microplates has the advantages of simplicity and rap-

idity, while HPLC with both hydroorganic or micellar

mobile phases should be recommended for screening

and evaluation. Generally, gradient elution is re-

quired with hydroorganic phases, whereas solutes

with large hydrophobicity differences can be separ-

ated in a short time, with good resolution and excel-

lent reproducibility, by isocratic MLC. In relation to

spectrophotometry, the DLs of phenols and EQ can

be reduced by several decades using fluorimetric or

voltammetric detection.

0038 Rapid and extremely selective separations can be

achieved by capillary electroseparation techniques, as

CZE and MECC; however, the DLs are larger than in

HPLC. To reduce the DLs, laser-induced fluorescence

(LIF) can be used. Both the selectivity and sensitivity

in HPLC and capillary electroseparation can be

largely expanded using MS detection. Moreover, MS

is excellent for structural characterization studies at

the molecular level, and can be further powered by

inserting an ion trap (ITMS) between the mass filter

and the ion sink.

0039 GC separations are rapid and very efficient, and the

antioxidants are sensibly detected with a FID; how-

ever, before injection the hydroxyl and carboxyl

groups should be derivatized. Superior selectivity

and sensitivity can be achieved with MS detection.

Further, many mass spectra within the elution time of

a single peak can be obtained with a time-of-flight

mass spectrometric detector (TOFMS). The commer-

cial availability of portable gas chromatographs, also

including miniaturized ITMS and TOFMS detectors,

have increased the interest of GC.

0040Nonchromatographic kinetic and cyclic voltam-

metric approaches, although useful to resolve simple

mixtures, have a limited selectivity. High selectivity

and rapidity, almost without sample preparation,

can be achieved with computer-aided near-infrared-

spectroscopy (NIR). The poor chemical selectivity is

compensated by powerful multivariant data treat-

ment techniques. This approach is also applied to

sensor-array- and MS-based electronic noses (EN),

but in this case, only the volatile fraction of the

sample can be analyzed. An advantage of NIR and

EN is the capability of direct evaluation of ‘funda-

mental’ variables or key factors, such as specific

flavors or off-flavors (including rancidity), while

skipping the tedious and sometimes impracticable

evaluation of all the chemical species on which a

given flavor could rely.

Evaluation of the Prooxidant/Antioxidant

Activity

0029The evaluation of the primary prooxidant/anti-

oxidant activity in food is important to compare the

performance of antioxidants in different food prod-

ucts under diverse conditions. In these studies, alkyl

gallates, BHT, BHA and a-tocopherol are frequently

used as references, to evaluate the antioxidant activity

of other compounds, including vegetal extracts, but

most of the procedures mentioned below can also be

used to determine the antioxidants.

0030The prooxidant/antioxidant activity can be meas-

ured with a wide variety of methods, including the

evaluation of sensory quality (color and smell) by a

panel of ‘rancidity’-trained experts, the spectrophoto-

metric monitoring of the 2,2-diphenyl-1-picrylhydra-

zyl (DPPH), 2,2

-azinobis(3-ethylbenzothiazoline)-6-

sulfonic acid, and N,N-dimethyl-p-phenylenediamine

radicals, the bleaching of b-carotene, the inhibition of

the chemiluminescence of luminol, the GC evaluation

of the C18:2/C16:0 ratio and free fatty acid (particu-

larly, linoleic acid) contents, the iodine, peroxide, and

thiobarbituric acid values, and other methods which

are discussed below. In spite of their promising cap-

abilities, little work has been still published concern-

ing the use of electronic noses in detecting and

monitoring rancidification processes.

0031In a fast TLC test for the evaluation of the total

free-radical scavenger capacity of foodstuffs, silica-

gel plates were stained with a methanolic solution of

the DPPH radical; this test is sufficiently sensitive

280 ANTIOXIDANTS/Synthetic Antioxidants, Characterization and Analysis

for water- and methanol-soluble products, but not for

lipid-soluble compounds. In a method for the screen-

ing of complex mixtures for radical scavenging com-

ponents, postcolumn mixing of the HPLC eluate with

the DPPH radical was implemented, the bleaching

induced by the presence of antioxidants in the eluate

being detected as negative peaks at 517 nm; this

simple, rapid, and quantitative method is suitable

for both isocratic and gradient HPLC runs, with

mobile-phase compositions ranging from 10 to 90%

organic solvent, with DLs within the 0.33–94 mgml

1

range, depending on the compound tested.

0032 The antioxidant activity has been evaluated in

edible plant material by the inhibition capacity on

the hemoglobin-catalyzed peroxidation of linoleic

acid, which can be monitored spectrophotometri-

cally. For eight synthetic antioxidants, the antioxi-

dant activity has been shown to be linear with

respect to the logarithm of their concentrations.

Hydrophilic and hydrophobic antioxidants can be

assayed with this method within 15 min.

0033 Inhibition of luminol chemiluminescence has

been used to measure the antioxidant capacity of

lipid-soluble antioxidants in the lipid phase of

systems as blood plasma and tissue homogenates

(measuring range for a-tocopherol: 0.1–3 nmol). In a

postcolumn HPLC procedure, radical scavengers

were detected in vegetal extracts by mixing the eluate

with luminol and hydrogen peroxide in the presence

of peroxidase at pH 10; by monitoring the quenching

of the luminiscent signal, the antioxidants were

detected at the nanogram level.

0034 The antioxidant activity of olive oils and wines has

been measured with a flow-injection system with

electrochemical detection. Using the ‘Rancimat’

instrument (Metrohm, Herisau, Switzerland) the

sample is heated (under computer control), the

volatile organic acids produced by lipid oxidation

are collected in water, and the resulting conductivity

increase is monitored. The rancidification induction

time and stability index of the product are automatic-

ally obtained. Reported applications include cereals,

cookies and biscuits, lard, vegetal oils, butter and

margarine, walnut oil, and olive oil. The antioxidant

activities of red grape marc (skin and seeds), BHA,

BHT, and PG when used in protecting olive oil have

been compared. Similarly, the antioxidant activity in

soybean oil of extracts from grape bagasse has been

evaluated.

0035 The formation of volatile peroxides during lipid

oxidation can be followed by static headspace GC.

Using this technique, the ability of BHT, BHA and the

natural polyphenol antioxidants from olive oil to

inhibit lipid oxidation in tuna can be compared. GC

with mass spectrometric detection can also be used.

In addition, lipid oxidation can be monitored by

headspace GC of hexanal. Capillary GC has been

used to determine the oxidized fatty acids.

0036Finally, the free radicals generated during lipid oxi-

dation can be monitored directly by electron spin

resonance (ESR) spectroscopy. The free radicals also

induce a strong ESR signal in the exposed proteins

and amino acids, and simultaneously the fluorescence

of the proteins increases, which has been attributed to

cross-linking. When added to the proteins prior to

incubation with oxidized lipids, antioxidants, includ-

ing BHT, BHA, ascorbic acid and a-tocopherol,

inhibit both the ESR signal and the increase in fluor-

escence. The ESR of free radicals has been used to

compare the efficiency of OG, BHA, and plant ex-

tracts (tea, coffee, rosemary, and others) in protecting

dehydrated chicken meat.

See also: Antioxidants: Natural Antioxidants; Synthetic

Antioxidants; Ascorbic Acid: Properties and

Determination; Chromatography: Principles; Thin-layer

Chromatography; High-performance Liquid

Chromatography; Gas Chromatography; Supercritical

Fluid Chromatography; Combined Chromatography and

Mass Spectrometry; Fats: Classification; Food

Additives: Safety; Legislation: Contaminants and

Adulterants; Preservation of Food; Preservatives:

Analysis; Tocopherols: Properties and Determination