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

Подождите немного. Документ загружается.

Rico AG (ed.) (1986) Drug Residues in Animals. London:

Academic Press.

Woodward KN (1991) The licensing of veterinary medi-

cinal products in the United Kingdom – the work of

the Veterinary Medicines Directorate. Biologist 38:

105–108.

Residue Determination

A A Bergwerff and J Schloesser, Utrecht University,

Utrecht, The Netherlands

Residue Determination

0001 Farming of husbandry animals, as well as aquatic

animals, is associated with veterinary application

of pharmaceuticals, with the aim of influencing

growth, weight gain, fat/protein distribution, milk

production, stress, microbial infections, inflamma-

tion reactions etc. (See Antibiotics and Drugs:

Uses in Food Production). This administration of

pharmaceuticals results in so-called residues in the

treated animal, including its edible parts, and prod-

ucts such as muscle (meat), organs, eggs, honey,

and milk.

0002 In some cases, residues are persistent, and concen-

trations may be high enough to affect the health or

well-being of the consumer. In the case of direct car-

cinogenic substances, in theory, a single molecule may

cause malignancies in the consumer. The veterinary

use of such substances, when xenobiotic, is of course

unacceptable. Veterinary medicinal products that

have the ability to accumulate in the animal are

banned world-wide as well. As well as the risk to

public health, the production or quality of food may

be affected by residues, for example by inhibition of

fermentation processes during cheese production.

0003 This phenomenon of negative effects of lagging

drugs is known as the ‘residue problem.’ Controlling

this problem through residue determination in the

food chain from ‘stable to table’ is necessary to

guarantee food quality and safety and to secure

the consumer’s trust.

Test at Maximum Residue Limit

0004 In the case of registered substances, and thus sub-

stances allowed for veterinary use, international, na-

tional, and supranational legal bodies have agreed on

so-called ‘maximum residue limits’ (MRL) for each

edible product, and for each husbandry species. The

consumer may be exposed to a nonacceptable health

risk when this MRL is offended, i.e., the concentra-

tion of the indicator molecule is in excess of this

concentration. This makes statutory and nonstatu-

tory control of residues in food of major importance.

MRL values may differ from one (inter)national

authority to another, as consensus is not always

reached on quantitative public health risks.

0005Authorities have also introduced a definition

for residues of veterinary medicinal drugs, which,

according to EU council regulation 2377/90, is:

All pharmacologically active substances whether active

principles, excipients or degradation products, and their

metabolites which remain in food stuffs obtained from

animals to which the veterinary medicinal product in

question has been administered.

This description clearly does not include contamin-

ation arising from environmental substances, phyto-,

phyco-, or mycotoxins, or as a result of food hand-

ling, processing, and preparation. The description

highlights the environmental influences on the

original molecules administered to the animal. The

chemical appearance of a residue in muscle (ante

mortem) or in meat (post mortem) is not static. In

addition to biochemical processes in the animal, stor-

age, food processing, and food preparation, which

may include fermentation and heating, frequently

initiate reactions converting the original pharmacon

into derivatives. The residue analyst has to account

for these processes, since they may mask the presence

of residues.

0006Relatively soon after administration, furazolidone,

for example, is metabolized completely and occurs

as a so-called bound residue, which is a covalent

linkage of the residue molecule with predominantly

proteins. Bound residues are, by definition, not ex-

tractable. The remaining and nonreacting 3-ami-

no-2-oxazolidone fragment is extractable and is used

for furazolidone residue determination. The cepha-

losporin ceftiofur is converted into a reactive des-

furoyl ceftiofur species, which reacts with itself,

proteins, and other biomolecules. The residue is re-

leased chemically and stabilized prior to residue an-

alysis. Another example is that of the antibiotic

sulfadimidine, which may occur in the presence of

relatively high concentrations of glucose, as in saus-

ages, as an extractable sulfadiminyl glucose residue.

In the living animal, many veterinary drugs are sul-

fated or conjugated to glucuronic acid in order to

detoxify and excrete these substances. Incubation

with sulfatases and glucuronidases prior to residue

determination may be necessary to obtain reliable

results.

254 ANTIBIOTICS AND DRUGS/Residue Determination

0007 Authorities have to decide on the indicator residue

molecule for analysis. The MRL value of this indica-

tor molecule should correspond with the total

concentration of residue components, including

derivatives of ante- and post-mortem reactions.

Analysis Strategies

0008 Before going into a detailed description of residue

analysis techniques, it should be noted that two dif-

ferent situations occur, which dramatically influence

the desired degree of reliability of an analytical

method.

0009 In the situation of registration of pharmaceuticals,

methods are designed and validated in well-defined

experiments. A defined number of healthy animals

are treated, allowing statistical analysis and aver-

aging of errors. Target tissues may be sampled, and

these usually contain easily detectable amounts of

residues. In contrast, in an inspection or a monitoring

situation, a juridically useful judgment should be

made on the basis of a single sample for which the

history is generally unknown and the concentrations

are usually relatively low. In most cases, meat is

sampled, which is mostly not the ideal matrix to

trace the (illegal) use of pharmaceuticals. Further-

more, in contrast to the ‘registration methods’, time

pressure is relatively high in the field of ‘forensics’

especially in the case of inspection of carcasses in the

slaughter line. Swine are slaughtered at a rate of up to

12 carcasses per minute, whereas chickens are slaugh-

tered at a rate of up to 270 exemplars each minute.

The storage capacity becomes critical when a decision

on condemnation of a carcass has to be made. The

quality criteria for analytical methods in these

different situations may therefore vary considerably.

0010 The situation and analysis strategy therefore deter-

mine the characteristics of the assay of choice. The

ideal method, which is inexpensive, fast, specific,

selective, sensitive and allows a high throughput,

does not exist. Compromises have to be made. A

low-cost screening method with a high throughput

may be followed by a usually more expensive, more

selective, more sensitive, and specific confirmatory

method. In arbitrage situations, a reconfirmation

may be needed through so-called reference methods

involving expensive instrumental techniques requir-

ing highly skilled personnel.

Inventory Expected Residue Levels

0011 Of the enormous collection of veterinary drugs, anti-

biotics are used most frequently and in the largest

amounts compared with the other groups listed in

Table 1. In the EU, a considerable part of antibiotic

use is still for growth promotion. From the point of

view of residue analysis strategies, it is important to

consider the use of a drug before developing or intro-

ducing a new method in the residue laboratory. The

different levels at which veterinary drugs are applied

will result usually in proportional concentrations in

edible tissues. For that reason, legal antibiotic growth

promoters do not result in high levels of residues.

Tracing the use of illegal antibiotic growth promoters

in animal products may be more worthwhile, but will

require sensitive tests amenable for detecting rela-

tively low concentrations. Therapeutic use of antibi-

otics, however, may yield residues at concentrations

of milligrams per kilogram, when withdrawal times

are not obeyed, or as high as grams per kilogram at

the injection site. The withdrawal time is the pre-

scribed interval between administration and allow-

ance to bring products of a treated animal to the

market.

0012Besides antibiotics, the occurrence of substances

with hormonal activity is attracting much attention,

at least in the EU. Since steroid hormones are effective

at very low levels, tracing residues in meat needs

ultrasensitive assays for detecting concentrations as

low as the subnanogram per kilogram level. These

methods also may need to be able to differentiate

between endogenous and administered, exogenous

hormones, as in the case of nortestosterone of 17b-

estradiol, for example. In these situations, it is diffi-

cult to answer the questions ‘when is a residue a

residue, and when is a residue not a residue?’

0013Monitoring and inspection programs should be

aware of the use of ‘cocktails’ of illegal hormonal

compounds. The mixture will exert its biological

effect, but the individual components may not be

detected as a result of low concentrations per com-

ponent. The illegal use of b-agonists may co-occur, for

example, with corticoids and thyreostats. Residue

analysis strategies may therefore focus on the most

easily extractable and detectable residue in first

screening.

Sampling

0014In setting up a method, a few steps can be recognized

(Figure 1). Each step from sampling, extraction, and

concentration to (instrumental) analysis is essential

and should be carried out appropriately for successful

analysis. The statement: ‘collect the wrong sample, or

collect the right sample incorrectly, and you will trivi-

alize all that follows, rendering your data worthless’

emphasizes the importance of the first step, namely

sampling. The selection of the laboratory sample for

analysis, its transport, and storage should always

be carefully evaluated (cf. Table 2). The storage of a

ANTIBIOTICS AND DRUGS/Residue Determination 255

laboratory sample of homogenated or intact tissue

may influence the final determined residue content

dramatically. Furthermore, the test sample should

reflect the laboratory sample, which usually means

thorough homogenization of the latter. The labora-

tory sample should reflect the tissue, body fluid, or

total animal that is sampled. At the same time, the

result of an individual sample should not be over-

valued. Unfortunately, many mistakes have been

made at this level, sometimes with huge political

impact.

0015 When tracking the illegal use of prohibited sub-

stances, the choice for the most suitable matrix for

analysis is obvious, since the MRL in an edible prod-

uct does not have to be tested. In such cases, urine

(anabolics), feces (steroid hormones), bile (antibiot-

ics), retina (b-agonists), or hair (b-agonists), instead

of meat, may be most suitable.

Extraction and Clean-up

0016 Milk and diluted honey, but also urine, serum used to

trace contamination of the animal, may be analyzed

in some cases without extensive sample processing

(Figure 1). Solid matrices, including fat, hair, meat,

kidney, retina, and skin, however necessitate extrac-

tion of residues prior to analysis. Specific solvents are

mixed with the homogenized matrix to facilitate solu-

bilization of the residues. As an exception, the collec-

tion of meat drip through heating or compressing, or

of pre-urine from the kidney cortex may be sufficient

for microbiological growth-inhibition assays (see

below).

0017Matrices are complex, and it should be noted that

the residue analyst looks for a single particle in a heap

of a million to billion other potentially interfering

particles. These particles are also for a part solubil-

ized and extracted from the matrix. Clean-up of

samples is therefore necessary in most cases to over-

come any interference of the analysis method.

0018A good knowledge of the chemical nature of the

analyte in terms of stability, pK

value(s), polarity,

and functional groups will support the design of a

selective, effective, and appropriate clean-up proced-

ure. Before the availability of sorbent materials in

the mid-1980s, sample clean-up was carried out by

tbl0001 Table 1 Classes and examples of veterinary drugs

Activity/class Subclass Examples

Antibiotics, including

growth-promoters

and chemotherapeutics

Nitrofurans Furazolidine, nitrofurazone, furaltadone

(Fluoro)quinolones Enrofloxacin, oxolinic acid, flumequine

Tetracyclines Oxytetracycline, doxycycline, chlortetracycline

Penicillins and cephalosporins Amoxiciline, cloxacillin, penicillin G, ampicillin, ceftiofur

Aminoglycosides Gentamicin, lincomycin, streptomycin

Sulfonamides Sulfadimidine, sulphaquinoxaline

Macrolides Erythromycin A, spiramycin, tylosine

Peptides Virginiamycin, bacitracin

Miscellaneous Chloramphenicol, carbadox, salinomycin

Anti-ectoparasitica Phosphoesters, carbamates,

pyrethroids a.o.

Dichlorvos, carbaryl, deltamethrine, lufenurone, cyromazin, lindane

Anthelmintics Imidazothiazoles Levamisole, dexamisol

Benzimidazoles Mebendazole, fenbendazole, lobendazole

Tetrahydropyrimides Pyrantel

Avermectins Ivermectin, abamectin, moxidectine

Salicylanilides Closantel, rafoxanide, niclosamide

Coccidiostats

amprolium, dimitridazole, monensin,

Antimycotica nystatine, griseofulvine, enilconazole

Hormones Glucocorticosteroids Dexamethasone, cortisone (hydrocortisone)

Androgenic and estrogenic

sex steroids

17b-Estradiol, methyltestosteron, trenbolone

Progastegens Megestrol, medroxyprogesteronacetate

Resorcylic acid lactones Zeranol, taleranol

Stilbenes Diethylstilboestrol, hexoestrol, dienoestrol

b-agonists Clenbuterol, salbutamol, bromobuterol

Thyreostats Tapazole, thiouracil

Proteins Somatropins

Tranquilizers Azaperone, carazolol, xylazine

This table is a summary and not a complete list of compounds and classes of compounds used in veterinary medicine.

Some of these compounds are registered veterinary drugs. The EU-accepted MRL values in food from bees, cattle, deer, horses, pigs, poultry,

salmonidae,sheep,etc.arepublishedontheinternetathttp://eudrams1.is.eudra.org

Many antibiotics are used as coccidiostats as well.

256 ANTIBIOTICS AND DRUGS/Residue Determination

liquid/liquid partition and/or atmospheric pressure

chromatography. Sample concentration was obtained

through rotary evaporation. A major improvement

in this laborious work was obtained by the launch

of commercially available, and thus standardized,

solid-phase extraction (SPE) cartridges. The cart-

ridges enabled selectivity and concentration in a

single step with minimal sample and solvent con-

sumption. The choice of stationary phases and sup-

portive material in SPE is comparable with that used

in high-performance liquid chromatography (HPLC),

including normal- and reversed-phase, partition and

ion-exchange chromatography. Combinations of

phases within a single cartridge are available as

well. For example, so-called ‘restricted access mater-

ials’, such as alkyl-diol-silica particles, combine the

ability of gel filtration and solid-phase extraction

within one material. These special SPE materials

allow the extraction of aqueous extracts or liquid

samples like milk or urine without prior deproteina-

tion. With the advent of chelating agents and the

availability of antibodies and receptors, it is now

possible to develop cartridges facilitating affinity

extraction with a high selectivity.

0019More recently, disks consisting of membranes of

polymers with high, reversed and ion-exchange

phase capacity have been introduced. These disks

are used as filters, allow fast-flow processing, purify

and concentrate analytes like SPE cartridges with

small to large sample volumes. Interfaces are avail-

able, enabling the coupling of such disks to HPLC

columns.

0020Another sorbent extraction approach is matrix

solid-phase dispersion, which allows homogenization

and extraction by mixing of sorbent material in a

single run omitting liquid extraction of the homogen-

ate as a separate step. Columns prepared with the

mixture are eluted in a similar way to SPE cartridges.

Alternatives for these extraction procedures are

supercritical fluid extraction and accelerated solvent

extraction. The approaches are slowly gaining popu-

larity but are still used in niche applications.

0021After extraction, the analyte-containing solution

often has to be concentrated by removing excess solv-

ent through evaporation or sublimation by applying a

vaccum or a flow of an inert gas (nitrogen) sometimes

in combination with heat. Loss of a part of the

amount of the analyte can occur if unsuitable glass-

or plastic ware is used. Many analytes, like penicillins

and avermectins, tend to become adsorbed by free

silanol groups of glass surfaces. Deactivation of

free silanol groups through silanization of glassware

Eggs

Laboratory

sample

Milk, urine,

serum

Homogenization

Tissue, feces,

feed

Grinding and

homogenization

Test

sample

Centrifugation or filtration

Liquid extraction

Extraction, clean-up and concentration: SPE

(evaporation of solvent)

Test portion

Analysis of analyte: high-performance liquid

chromatography, gas chromatography, mass-spectrometry

fig0001 Figure 1 Typical scheme for sample processing involving SPE

and determination of residues of veterinary drugs in different

matrices.

tbl0002 Table 2 Stability of veterinary drug residues in different matrices upon cold storage and heating

Pharmaceutical Storage at 18



CStorageatþ4



C Heated (Percentage activityrecovered)

Penicillin Stable in muscle for 14 days Stable in muscle for 14 days 10% in pork at 80



C for 10 min

Degraded in kidney Degraded in kidney 0% in sterilized sausages at 125



C for 65 min

Furazolidone Unstable Unstable

Oxytetracycline Stable for 6 months Stable for 6 months No recoverable activity in pork at 100



C for 10 min

Sulfadimidine

Degraded in liver Degraded in liver 100% in well-done steak at 80



C for 30 min

Chloramphenicol Degraded in liver Degraded in liver and kidney 37% in pork at 80



C for 10 min

Spectinomycin Degraded in liver and kidney Degraded in liver and kidney

Sulfadimidine reacts with nitrite and glucose in sausages.

ANTIBIOTICS AND DRUGS/Residue Determination 257

using chlorsilane often enhances the recovery in such

situations.

Analysis of Analyte

0022 Overviewing all available methods for separation and

detection of residues in the nonprocessed test sample

or in the obtained extract, an enormous gamma of

(immuno)chemical and physicochemical approaches

can be disentangled (Table 3). Thin-layer chromatog-

raphy (TLC) was applied frequently in the early days

of residue analyses and was closely followed in time

by gas chromatography (GC). Analytes on TLC plates

were visualized by generating chromophores or fluor-

escents by applying (through spraying or dipping)

derivatizing molecules. Thermal conductivity, flame

ionization, and, in certain applications, electron-

capture and nitrogen phosphorus (NPD), detectors

were popular in GC analysis. In current residue GC

methods, the universality, selectivity and specificity

of the mass spectrometer (MS) in combination with

electron-impact ionization (EI) is by far preferred.

0023 HPLC, in combination with a range of different

detectors, became popular in the 1980s and 1990s.

The stationary phases used predominantly are

reversed-phase materials, especially phenyl-hexyl,

octyl- or dodecyl-modified silica gels, but also, parti-

tion and ion-exchange chromatography is found in

residue-determination protocols. Highly end-capped

silica gels or polymeric stationary phases may be

necessary to prevent tailing of amphoteric residues,

such as tetracyclines. Polymeric phases also allow

elution under alkaline conditions without solving

the supporting silica material.

0024 In addition to adequate extraction, clean-up and

chromatography, the choice of detection system is

an important parameter to achieve selectivity and

specificity. Analytes, which are not detected by light

absorption, refractive index, or fluorescence at the

level they are expected to occur in animal-derived

matrices, may require chemical modifications

rendering them as fluorescents, chromophores, or

UV-light absorbing compounds. For example, specti-

nomycin is converted postcolumn into a fluorescent

derivative following oxidation and reaction of two

o-phthaldialdehyde molecules with the two primary

amines into conjugated iso-indoles. In such cases,

chemical modification may also introduce a tremen-

dous improvement in the selectivity of the method.

0025Alternative detection systems compatible with LC

that have been used in residue analyses comprise

evaporative laser-scattering and electrochemical de-

tection, including (pulsed) amperometric detection.

0026In electrochromatography, electroosmotic flow is

used for mobile phase delivery. Packed and open

tubular columns are used in these systems, giving

very small band widths, since extremely small par-

ticles can be used as the stationary phase. The devel-

opment of heat is, however, a major drawback of this

separation technique. Instead of chromatography,

capillary electrophoresis with a high resolving power

may be considered as well. However, the maximum

sample size, which is very small, limits its use in

residue analysis. To obtain namely dissolved extracts,

relatively large solvent volumes are required, which

reduce residue concentrations.

0027The identification of analytes in TLC, GC, and LC

separation systems is carried out by comparison of

the migration or elution positions with those of stand-

ard substances under identical chromatographic and

detection conditions. However, comigration or coelu-

tion is usually not considered as a proof of identity of

the analyte, especially if juridical consequences are

involved. For this reason, combination of chromatog-

raphy with spectrometric (MS) or spectroscopic de-

tection techniques (NMR, IR) may provide data with

tbl0003 Table 3 Examples of residue analysis approaches

Analyte Matrix type Analyticalmethod Sample pretreatment

Penicillins

(group-specific

method)

Meat GC-NPD or GC-EI-MS Acetonitrile/buffer extraction, multiple liquid–

liquid partition ion-exchange-SPE, elution

with sodium chloride, liquid/liquid partition,

derivatization

Penicillins Milk HPLC, photochemical postcolumn

derivatization, UV detection at 300 nm

or electrochemical detection

Centrifugation and ultrafiltration, on-line SPE

(restricted-access material)

Avermectins Liver HPLC, fluorescence detection

: 365 nm l

470 nm)

Acetone/water extraction, liquid–liquid partition,

SPE, concentration, precolumn derivatization

Quinolones Poultry meat HPLC-APCl-MS/MS Phosphate-buffer extraction, SPE

Tetracyclines Kidney HPLC-ESI-MS/MS Mcllvain-buffer extraction, on-line SPE

Sulfamethazine,

Sulfdaizine

Meat, milk Biosensor (surface plasmon resonance) Homogenization with buffer, centrifugation

258 ANTIBIOTICS AND DRUGS/Residue Determination

sufficient evidence. Of the spectroscopic approaches,

nuclear magnetic resonance (NMR) and Fourier-

transform infrared (FTIR) spectroscopy are most suit-

able, although certainly not in routine settings. The

so-called hyphenated techniques, like in TLC-MS,

GC-MS, LC-MS, LC-NMR, and GC-FTIR, have

proved very powerful techniques for separation and

molecular identification of the residue.

0028 The evolution, and sometimes revolution, of mass

selectors, ionization techniques, and their interfacing,

has made the mass spectrometer standard equipment

in a residue analysis laboratory. Mass selectors com-

prise sector, quadropole, ion-trap, time-of-flight

instruments, and combinations thereof. In particular,

mild-ionization techniques, such as atmospheric pres-

sure chemical ionization (APCI) and electrospray

ionization (ESI), facilitate MS analysis of small to

relatively large and hydrophobic to hydrophilic mol-

ecules. APCI and ESI are therefore very suitable for

residue determination. Using mass spectrometer-

induced molecular fragmentation, it is possible to

elucidate the chemical structure of the residue. A

major pitfall is matrix-caused interference, which

may reduce the response of the analyte to nil.

0037 Besides physicochemical methods, the use of

microbiological growth-inhibition assays to test

meat and milk for the presence of antibiotics is popu-

lar over a long period of time. In terms of the number

of analyses performed each year, this type of residue

determination is most important. In addition to the

low cost, this may be a result of an early implementa-

tion of such tests in national legislation. Examples of

such methods are the EU Four Plate Test, the New

Dutch Kidney Test, and the Delvotest SP, which use

very antibiotic-sensitive bacterial reporter strains,

such as Bacillus subtilis and Bacillus stearothermo-

philus var. calidolactis. These bacteria are inoculated

under optimal conditions with and without sample.

After culturing, results are read from visible inhib-

ition zones or from the color change of the bacterial

suspension in agar gels.

0029 The availability of relatively large amounts of

immunoglobulins in the 1980s, of which the pro-

duction could be assured, enabled the development

of immunoassays, like radio-immunoassays, enzyme-

immunoassays, enzyme-linked immunosorbent

assays, and later strip- and dip stick-based immuno-

assays. Specific enzymes and receptors have been used

in a similar way in such analysis approaches, which are

referred to as receptor assays, such as the commercially

available CHARM Test II and Penzyme III. The major

advantage of immunoglobulins and receptors is their

high specificity, which reduces the sample clean-up

to a minimum. However, their high specificity is a

disadvantage in multiresidue methods.

0030In immuno- and receptor assays, the binding of

small-molecular-weight compounds to their corres-

ponding receptors or immunoglobulins is not directly

detectable. These assays are therefore mostly com-

petitive assays in which the analyte competes with a

radio-isotope- or enzyme-labeled reporter compound

for a limited number of binding sites. In contrast to

conventional immuno- and receptor assays, advanced

biosensor techniques do not require this labeling in

most cases. From the point of view of signal transduc-

tion, biosensors fall into four categories, namely

mass, optical, electrical, and thermal. Of the few

applications so far, the ‘surface plasmon resonance’

optical biosensor has shown benefits in screening

residues, including ciprofloxacin, clenbuterol, enro-

floxacin, gentamycin, streptamycin, sulfadiazine, and

sulfamethazine.

0031The ability of tetracyclines to fluoresce and to form

complexes with alkali metals has revealed its presence

in many cases (Figure 2). The article entitled ‘The dog

with the luminous bone’ in the Daily Telegraph sev-

eral years ago is certainly an example of a tetracycline

contamination of bone tissue.

Quality Assurance

0032The validation of a developed method, but also that

of an existing method newly introduced in a lab, is of

absolute importance to assure reliable outcomes. In

fact, international trading requiring certification of

foodstuffs dictates that the performance of analytical

methods be supported by validation data. The topics

that have to be assessed are limit of detection

and quantification, specificity, selectivity, accuracy,

Figure 2 Eggs examined under UV light. The white/yellow

fluorescence of the eggs in the middle is a result of tetracycline

residues complexed with calcium in the eggshell. Positive eggs

were collected from tetracycline treated laying hens. Courtesy of

Dr. Michael Petz and Dr. Georg Zurhelle, University of Wuppertal,

Germany.

ANTIBIOTICS AND DRUGS/Residue Determination 259

precision, reproducibility, repeatability, etc. The ro-

bustness of the method should also attract attention.

Critical points that may influence the measurement

like minor changes in pH, temperature, SPE flow rate,

the use of different SPE batches or of different pro-

ducers, etc. therefore have to be evaluated as well.

0033 In analysis strategies, the predictive value of the

method should be considered. The false-negative

rate of validated screening methods should be less

than 5% (b-error) at the level of interest, whereas

up to 20% false positive outcomes are not appreci-

ated but are generally accepted. In contrast, false-

positive results are not accepted in confirmatory

methods, which usually carry a higher risk with sys-

temic errors and interference. Criteria for the positive

identification and quantification of residues are

increasingly gaining attention. The suitability of con-

firmatory and/or reference methods for registered

veterinary drugs, as proposed, is given in Table 4.In

the case of banned substances, methods have to be

based on molecular spectrometry, and the number of

so-called ‘identification points’ (Table 5) should be at

least four for positive identification.

0034Besides validation of methods, quality-assurance

systems, such as good laboratory practice (GLP) are

also essential for residue laboratories. Such systems

not only ensure clarity on the performance of the

method, but also increase the traceability of the

results. Furthermore, GLP or similar systems assure

appropriate laboratory management and properly

trained analysts.

Outlook in Residue Analysis

0035‘Multianalyte’, i.e., testing various residues in a single

run with a single instrument, is a magical word in the

field of residue analysis. It requires generic extraction

methods and highly resolving and robust analytical

techniques. The fulfillment of this ideal still seems to

be far away. Computerization and miniaturization

and the introduction of SPE cartridges already have

revolutionized residue analysis in the past. Advances

in computerization and miniaturization will certainly

increase sensitivity and selectivity further. This and

further technical improvements in instrumental tech-

niques will make multianalyte analyses possible to an

increasing degree. Automation and analysis within

minutes and even seconds, but also the type of matri-

ces (body fluids and excreta), will transform future

residue analysis labs into clinical chemistry labs.

0036Recent scandals have shown that residue determin-

ation at the slaughter-line stage is not effectual in

protecting public health. The need for high analysis

frequencies to guarantee consumers’ trust is obvious.

However, it is impossible to test every product for all

possible contaminations. Analysis should be per-

formed in the food chain, starting in the stable and

at the feed mill. Laboratory samples should be col-

lected not only from animals, but also from the feed

and drinking water in order to trace cross- or other

accidental contamination. Furthermore, inspection

systems will not focus on the individual animal any-

more, but on the complete herd or flock. Analysis of a

number easily collectable samples will need to reflect

the complete herd and predict the level of contamin-

ation of the animals, so that its products can be

processed and placed on the market accordingly. At

the same time, future veterinary control systems are

being developed that will monitor residue levels to be

in compliance with withdrawal times and MRL

values. The residue lab may therefore be (at least in

part) situated in the slaughterhouse or located in a

vehicle visiting farms, e.g., for certification purposes.

In particular, biosensor systems are attracting consid-

erable attention for such targets. (See Antibiotics and

Drugs: Uses in Food Production.)

tbl0004 Table 4 Suitability of methods for confirmatory analysis of

nonbanned veterinary medicinal products

Technique Criterion

LC or GC in combination with MS Suitable (at least three

identification points)

LC-diode array detector Suitable

Two-dimensional

TLC-spectrophotometry

Suitable

GC-electron capture detector Suitable, only if combined

with other methods

LC-immunogram Suitable, in combination

with other techniques

LC-UV/VIS (single wavelength) Suitable, in combination

with other techniques

See Table 5 for clarification of identification points. Adapted from the

pending revised EU commission Decision 93/256/EC.

tbl0005 Table 5 Awarding mass-spectrometric approaches for

confirmatory analysis of banned veterinary products with so-

called identification points

MS technique Identification points

awarded per ion

Low-resolution (LR) MS 1.0

LR-MS

precursor ion

1.0

LR-MS

transition products 1.5

High resolution (HR) MS 2.0

HR-MS

precursor ion 2.0

HR-MS

transition products 2.5

At least four identification points are necessary for confirmation of

banned veterinary drugs. Adapted from the pending revised EU

Commission Decision 93/256/EC.

spectra are generated with tandem MS machines (MS

) or with

ion-trap mass selectors.

260 ANTIBIOTICS AND DRUGS/Residue Determination

See also: Antibiotics and Drugs: Uses in Food

Production; Chromatography: Thin-layer

Chromatography; High-performance Liquid

Chromatography; Gas Chromatography; Combined

Chromatography and Mass Spectrometry;

Contamination of Food; Quality Assurance and

Quality Control; Spectroscopy: Overview; Infrared and

Raman; Near-infrared; Fluorescence; Atomic Emission

and Absorption; Nuclear Magnetic Resonance

Further Reading

Commission of the European Communities, Directorate

General SANCO (2002) Commission Decision of Imple-

menting Council Directive 96/23/EC concerning the per-

formance of analytical methods and the interpretation of

results. Draft document SANCO/1085/2000 Rev. 7.

Funk W, Dammann V and Donnevert G (1995) Quality

Assurance in Analytical Chemistry. Weinheim: Wiley-

VCH.

Haagsma N and Ruiter A (eds) (1996) Residues of Veterin-

ary Drugs in Food. Proceedings of the EuroResidue III

Conference, Veldhoven, May 3–6. Utrecht: Utrecht Uni-

versity.

Horwitz W (ed.) (2000) Official Methods of Analysis of

AOAC INTERNATIONAL, 17th edn. Arlington, VA:

AOAC International.

Nollet LML (ed.) (1996) Residues and other food com-

ponent analysis. In: Handbook of Food Analysis (vol. 2)

New York: Marcel Dekker.

Oka H, Nakazawa H, Harada K and MacNeil JD (eds)

(1995) Chemical Analysis of Antibiotics Used in Agri-

culture, p. 452. Arlington, VA: AOAC International.

O’Keeffe M (ed.) (2000) Residue Analysis in Food – Prin-

ciples and Applications. Singapore: Harwood Academic

Publishers.

Stephany RW (1989) Molecular spectroscopy in forensic

residue analysis – a general overview of variability, ap-

plicability and cost-effectiveness. Journal of Chromatog-

raphy 489: 3–9.

Van Ginkel LA and Ruiter A (eds) (2000) Residues of

Veterinary Drugs in Food. Proceedings of the Euro-

Residue IV Conference, Veldhoven, May 8–10. Biltho-

ven: National Institute of Public Health and the

Environment.

ANTIOXIDANTS

Contents

Natural Antioxidants

Synthetic Antioxidants

Synthetic Antioxidants, Characterization and Analysis

Role of Antioxidant Nutrients in Defense Systems

Natural Antioxidants

M H Gordon, The University of Reading, Reading, UK

Background

0001 Natural antioxidants have been of interest for many

years because of their ability to retard the develop-

ment of off-flavors in foods. However, an upsurge in

interest in these components has occurred in recent

years because of their importance for the prevention

of diseases mediated by free radical reactions in vivo.

The onset of a variety of major health problems,

including cancer, atherosclerosis, rheumatoid arth-

ritis, inflammatory bowel disease, immune system

decline, brain dysfunction, cataracts, and malaria

may be delayed by natural antioxidants.

0002 Natural antioxidants are primarily plant phenolic

compounds which may occur in all parts of the

plant. Nonphenolics including carotenoids and

phospholipids may also show antioxidant activity

under some conditions. Plant phenolics are multi-

functional. They can act as radical scavengers, metal

chelators, singlet oxygen quenchers, or reducing

agents. Though they are considered to be safe at

normal levels of consumption they have not been

toxicologically tested in many cases and therefore

the possibility of mutagenic or carcinogenic activity

cannot be discounted for some natural phenolics at

unnaturally high levels of consumption. Some anti-

oxidants add color, aftertaste, or off-flavor to a prod-

uct and this may restrict their use in some types of

foods.

0003Natural antioxidants can be found in a wide range

of food raw materials. Tocopherols and related com-

pounds occur at levels of about 50–1000 mg kg

1

vegetable oils. Animal fats contain very low levels

(e.g., butter oil 20–50 mg kg

1

), but cereal germ oils

are very rich sources (1500–5000 mg kg

1

ANTIOXIDANTS/Natural Antioxidants 261

0004 Tocopherols are added to some foods as anti-

oxidants. They occur with various degrees of

methylation of the dihydrochromanol ring as a, b, g

and d-tocopherol. They have the chemical structure

shown in Figure 1.

0004 The activity of the tocopherols depends on their

concentration, the food system, and the presence of

heavy metals since they can act as prooxidants with

iron or copper. Tocopherols are strongly effective as

antioxidants in emulsions, but they are less effective

in oils. Their antioxidant activity under common test

conditions in oils decreases from d-tocopherol>

g-tocopherol > b-tocopherol > a-tocopherol. They are

most effective in the presence of a synergist like as-

corbic or citric acid. In biological systems, however,

a-tocopherol or vitamin E is the most active anti-

oxidant with a strong antioxidant effect in biological

membranes. Tocopherols are commercially produced

and they are used as food additives. (See Tocopherols:

Properties and Determination.)

0009 About 50 carotenoids can be found in the human

diet as minor components of fruit and vegetables.

Carotenoids, including b-carotene (Figure 2), demon-

strate antioxidant properties under certain condi-

tions. Due to their multiple conjugated double-bond

system, the carotenoids act as singlet oxygen

quenchers during photosynthesis. They are also able

to scavenge free radicals and act as antioxidants at

low partial pressure of oxygen.

0010 There is considerable interest in the nutritional

properties of carotenoids. Besides the provitamin A

property of some of the carotenoids, the antioxidant

properties may also be of nutritional significance.

Epidemiological studies have found that consumption

of fruit and vegetables, which contain carotenoids, is

associated with a reduced risk for several chronic

diseases, including cardiovascular and photosensitiv-

ity diseases, cataracts, and some cancers. However,

the results from large controlled trials involving

b-carotene supplementation do not always support

these beneficial associations, providing in some

cases evidence for adverse effects, including an

increase in the incidence of lung cancer and overall

mortality in smokers.

0011It has been suggested that other carotenoids,

including a-carotene, lycopene, lutein, and zea-

xanthin, may show a higher potency than b-carotene

in the suppression of the development of various

types of cancer, including lung, breast, and stomach

cancer. Dietary consumption of tomato products con-

taining lycopene is associated with reduced risk of

cancer and coronary heart disease. Although the anti-

oxidant properties of lycopene may be responsible for

its beneficial properties, other mechanisms, including

effects on intercellular gap junction communication,

or on the hormonal or immune system, may be partly

responsible. (See Carotenoids: Occurrence, Proper-

ties, and Determination.)

0012Flavonoidsareanimportantclassofnaturalantioxi-

dants that occur in a wide variety of fruit, vegetables,

leaves, and flowers. They are found mainly as glyco-

sides and methylated derivatives. The most important

subgroups are the colorless catechins, the red-purple-

colored anthocyanidins, the yellow flavonols and fla-

vones, as well as the colorless proanthocyanidins.

0013The flavonoids share a common C

-C

skel-

eton. The flavonoid aglycone normally consists of

an aromatic ring (A) condensed with a six-membered

oxygen-containing ring (C) which carries a phenyl

substituent in the 2-position (B). The six-membered

ring condensed with the benzene ring is either a

g-pyrone ring (flavonols and flavones) or its dihydro

derivatives (flavanols and flavanones).

Flavonoid Structure(Figure3)

0014The position of the attachment of the B-ring to

the central C-ring divides the flavonoid class into

5,7,8-Trimethyltocol (α-tocopherol)

7,8-Dimethyltocol (β-tocopherol)

5,8-Dimethyltocol (γ-tocopherol)

8-Methyltocol (δ-tocopherol)

H, R

, R

H, R

4' 8'

fig0001 Figure 1 Tocopherol structure.

fig0002 Figure 2 Structure of b-carotene.

6'2

fig0003Figure 3 Flavonoid structure.

262 ANTIOXIDANTS/Natural Antioxidants

flavonoids (2-position) and isoflavonoids (3-pos-

ition). Flavonols including quercetin and myricetin

are important natural antioxidants. Quercetin occurs

as the glycoside quercetrin in onions, apple skins, and

black tea. Anthocyanidins differ from the other fla-

vonoids in having a charged oxygen in the C-ring.

The C-ring is open in the chalcones. Flavonoids are

often hydroxylated in position C-3,5,7,3

or 5

Flavonol and Flavone Structure(Figure4)

0019 Attention was focused on the nutritional significance

of dietary flavonols and flavones by the Zutphen

Elderly Study in The Netherlands. The study started

in 1960 with 878 men aged 40–59 years and con-

tinued until 1985 with 555 of the original subjects

and 250 replacements aged 65–84 years. Nutritional

intake was studied by cross-checked dietary history

for 5 years. The mean baseline of flavonoid intake

was 25.9 mg day

1

(as aglycones). The major flavo-

noids in the food were quercetin, myricetin, kaemp-

ferol, apigenin, and luteolin. Their sources of intake

were mainly tea (61%), onions (13%), and apples

(10%).

002 0 After adjustment for age, body mass index,

smoking, serum total and high-density lipoprotein

cholesterol, blood pressure, physical activity, coffee

consumption, and intake of other antioxidants and

fiber, the flavonoid intake was significantly inversely

associated with mortality from coronary heart disease

and showed an inverse relationship with the incidence

of myocardial infarction.

002 1 In Mediterranean countries, wine is an important

source of flavonoids (10–20 mg l

1

combined flavo-

noids). This may partly contribute to the low risk of

coronary heart disease of wine drinkers who consume

a high level of saturated fats in France, the so-called

French paradox.

002 2 Tea catechins are very strong antioxidants. For

the tea catechins, stronger activity than BHA or

a-tocopherol in lard autooxidation systems was

shown with activity increasing in the order:

( )-epicatechin < ()-epicatechin gallate< ()-epi-

gallocatechin < ()-epigallocatechin gallate (Figures

5–8).

OOH

quercetin R

OH, R

myricetin R

quercitrin R

OH, R

luteolin R

OH, R

Flavonol aglycone: X OH

Flavonol: X glucose

Flavone aglycone: X H

fig0004 Figure 4 Flavonol and flavone structure.

fig0005Figure 5 Epicatechin.

fig0008Figure 8 Epigallocatechin gallate.

fig0007Figure 7 Epigallocatechin.

fig0006Figure 6 Epicatechin gallate.

ANTIOXIDANTS/Natural Antioxidants 263