FOOD AND NUTRITIONAL ANALYSIS / Additives 217

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

system, cleanups can be performed with little or no operator intervention, requiring only that the nal extracts be concentrated.
See also: Carbohydrates: Dietary Fiber Measured as Nonstarch Polysaccharides in Plant Foods. Extraction: Solvent Extraction Principles; Solid-Phase Extraction; Solid-Phase Microextraction. Food and Nutritional Analysis: Overview. Sampling: Theory; Practice. Sensory Evaluation.

Further Reading
AOAC International (1995) Ofcial Methods of Analysis, 16th edn. Gaithersburg, MD: AOAC International. Horwitz W (1988) Sampling and preparation of samples for chemical examination. Journal of the Association of Ofcial Analytical Chemists 71: 241245. Pomeranz Y and Meloan CE (1994) Food Analysis: Theory and Practice, 3rd edn, pp. 1129. New York: Chapman and Hall. Proctor A and Meullenet J-F (1998) Sampling and sample preparation. In: Nielsen SS (ed.) Food Analysis, 2nd edn, pp. 7182. Gaithersburg, MD: Aspen Publishers.

Additives
rdoba, Co rdoba, Spain M Gallego, University of Co lez, Instituto Canario de Investigaciones M Gonza Agrarias, La Laguna, Spain
& 2005, Elsevier Ltd. All Rights Reserved.

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

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

General Information about Food Additives

Definition of Additive

Among the various definitions of food additives that can be found in international legislation and accords is the one adopted by the European Union (EU) in its European Community Directive 89/107/EEC of the Council of 21 December 1988: any substance not normally consumed as a food in itself and not normally used as a characteristic ingredient of food whether or not it has nutritive value, the intentional addition of which to food for a technological purpose in the manufacture, processing, preparation,

218 FOOD AND NUTRITIONAL ANALYSIS / Additives

treatment, packaging, transport or storage of such food results, or may be reasonably expected to result, in it or its by-products becoming directly or indirectly a component of such foods. This definition emphasizes that additives are substances added intentionally to food to produce permanent functional effects and that they are generally not considered themselves to be foods. Another food additive definition is that proposed by the Codex Alimentarius Commission: any substance not normally consumed as a food itself and not used as a typical ingredient of the food, whether or not it has nutritive value, the intentional addition of which to food for a technological (including organoleptic) purpose in the manufacture, processing, preparation, treatment, packing, packaging, transport or holding of such food results, or may be reasonably expected to result (directly or indirectly), in it or its by-products becoming a component of or otherwise affecting the characteristics of such foods. The term does not include contaminants or substances added to food for maintaining or improving nutritional qualities. This definition indicates the difference between an additive that is added intentionally and a contaminant (e.g., microorganisms and their toxins, heavy metals) whose presence in a food is unintentional. These two examples illustrate that different food additive definitions tend to be very similar because a general consensus has been reached among the majority of countries as to what an additive is. It should also be pointed out that a special statute is normally reserved for dietetic products, nutritive substances added to food products (e.g., vitamins, amino acids, minerals, etc.), technological processing aids, and aromas.

Table 1 Comparison between the classications of food additives established by EU and Codex Alimentarius Improved characteristic Color Color Preservation Preservation Texture Texture Texture Texture Texture Flavor Flavor Flavor Texture Flavor Texture Texture Texture Texture Texture Texture Texture Texture Texture Texture Texture Texture EU legislation Color additives Preservatives Antioxidants Emulsiers Emulsifying salts Thickeners Gelling agents Stabilizers Flavor enhancers Acids Acidity regulators Anticaking agents Sweeteners Modied starch Raising agents Antifoaming agents Glazing agents and lubricants Firming agents Flour treatment agents Humectants Sequestrants Enzymes Bulking agents Propellants and packaging gases Codex Alimentarius Color additives Color stabilizers Preservatives Antioxidants Emulsiers Emulsifying salts Thickeners Gelling agents Stabilizers Flavor enhancers Acidity and pH regulators Anticaking agents Sweeteners Raising agents Antifoaming agents Foaming agents Lubricants Flour treatment agents Humectants Bulking agents Propellants

various categories. Table 1 shows a comparison between the two different classications. In practical terms, a more general classication could be created that includes the additive categories previously mentioned, separating them by the functions that they fulll in the foods: 1. additives that increase the shelf life or stability of a food, 2. additives that improve sensorial qualities without altering the nature or quality of the food in such a way that could deceive the consumer, 3. additives that improve the nutritional value of the foods, and 4. additives that aid in the manufacture, transformation, preparation, treatment, packaging, transportation, or storage of the foods.
Food Additive Regulations

Classication of Food Additives

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

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

FOOD AND NUTRITIONAL ANALYSIS / Additives 219

concern for humans and to achieve this end, our ancestors used salt to preserve meats and sh; preserved fruit with sugar; and pickled or canned vegetables with vinegar. During the nineteenth century and the beginning of the twentieth century, new chemical substances were discovered that could be used to preserve and color foods. In the twentieth century the need to combat commercial fraud and abuse led to the rst regulation of chemical food additives by France in 1912, giving rise to the concept of a positive list of chemicals allowed to be added to foods. For the rst time there was explicit prohibition to the addition of chemicals or colorants, which had not been previously approved by law, to foods. The concept of positive lists of additives has lasted until today and is still in use in modern international legislation. Regulations in Europe and in the United States Since 1988, the EU, using safety evaluations done by its Scientific Committee for Food (SCF), has harmonized the national legislation of its Member States through European Community Directive 89/107/ EEC, concerning additives that can be used in products destined for human consumption. The role of the SCF has grown in importance since the adoption of directives on sweeteners (Directive 94/35/EC), colorants (Directive 94/36/EC), and other additives (Directive 95/2/EC) in 1994 and 1995. The committee, created in 1974 by the European Commission, has dedicated itself to evaluating the possible risks of food additives and elaborating guidelines for their safe use. Meanwhile, the European Commission has established concrete criteria on the purity of additives, the general or specific categories of foods with which additives may be used, and the maximum amounts that can be added to them. In the United States, the regulation of additives is done by the Food and Drug Administration (FDA). As a response to the widespread use of many substances not approved by the FDA, in 1958 the agency created the GRAS lists (Generally Recognized As Safe), comprising substances that were unanimously recognized as safe. These lists, the US equivalent to the positive lists in European legislation, included only substances that had been proven by scientific practice and evidenced to be innocuous to human beings. In the same year, the Food Additive Amendment, to the Federal Food, Drug and Cosmetic Act (FD&C Act) was passed, requiring the explicit authorization of an additive before it could be added to a food. It also obligates the manufacturer to prove that the additive is safe in all of the forms in which it will be used. This amendment excluded two groups of substances from the additive regulation process:

those that the FDA or the United States Department of Agriculture (USDA) had deemed safe for use in foods previous to the 1958 amendment and those on the GRAS lists. In 1960, the US Congress approved the Color Additive Amendments to the FD&C Act, which state that all color additives to be used in foods, cosmetics, and drugs (including externally applied drugs and cosmetics) must be approved by the FDA before being sold on the market. Since 1958, the FDA and USDA have continued to monitor all prior sanctioned and GRAS substances in response to new scientific information and evidence on the safety of these substances. International organizations Every country needs to have access to reliable evaluations of the risks associated with the chemical substances used in foods, but relatively few have the technical capability and the economic means to do individual evaluations on the risks posed by the huge number of existing additives. The work done by the Joint FAO/OMS Expert Committee on Food Additives (JECFA) and the Codex Committee on Food Additives and Contaminants (CCFAC) is extremely important in this regard. Both committees were jointly created by the World Health Organization (WHO) and the Food and Agriculture Organization of the United Nations (FAO) and are dedicated to studying additives with the objective of producing international regulations for them. The JECFA works as the scientific advisor to the FAO, the WHO, the member governments of the FAO, and the WHO and the Codex Alimentarius Commission. The JECFA establishes guidelines to evaluate the safety of additives, makes toxicological evaluations, determines the ADI of each of the additives, prepares their purity specications, and evaluates the frequency of consumption of foods that contain additives. The CCFAC advises the Codex Alimentarius Commission on food additives, contaminants, and naturally present toxic substances; examines the purity characteristics and the identity of the additives; establishes the maximum level of contaminants in foods; and advises the Products Committee of the Codex Alimentarius in which foods an additive can be used and the maximum amounts that can be added to them. Labeling and identication of food additives In addition to the strict criteria applied to the evaluation of risk and the obligation of having a demonstrable use, the regulations also require that additives appear on the labels of the packaging of the foods and drinks that contain them. These labels must explain the function of the additive and its name or assigned number.

220 FOOD AND NUTRITIONAL ANALYSIS / Additives

The identication of food additives using a number is an alternative to stating the specific name of the additive, which is normally quite long and has a complex chemical structure. In 1960, the EU assigned conventional numbers to food additives to better identify them. It was decided that each substance would be identied with a number (of three or four digits) preceded by the letter E. It was thought that the additives could be separated into nine categories and that each category would have 100 numbers: the rst 100 numbers was assigned to color additives (100199); the second to preservatives (200299); the third to antioxidants (300399); and the fourth to emulsiers, thickeners, and gelling agents (400499). The initial letter E indicates that the additive has been certied by the SCF as safe for use in the entire EU. In the framework of the Codex Alimentarius, in 1989 the CCFAC created the International Numbering System (INS). This numbering system was created to reach an international agreement on the identication number of additives and establish a numerically ordered list of additives that included the number of each additive and its technological function. The European Commission and that of the Codex Alimentarius have agreed to adopt the same identication numbers, although the INS system does not include the E prex, which is reserved for the EU.
Criteria Used to Establish the Maximum Food Additive Dose

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

Legal Procedures for the Authorization of New Food Additives

To establish the maximum food additive dose that can be used, international authorities consider two basic criteria: technology and toxicology. On the one hand, they assess the amount of the additive that is technologically required to satisfy the objective need to include the additive in the food; on the other hand, they evaluate the innocuousness of the additive. The technologically useful dose of the additive is that which accomplishes the desired effect on the food. The tests that determine the technologically useful dose must follow the guidelines established in the Good Manufacturing Practice used by the food industry. The ADI is established by international organizations like the JECFA and the SCF after completing toxicological evaluations. The toxicological evaluation of additives is done using as reference the maximum dose that does not have a toxic effect on animals or the maximum dose that in short- and longterm toxicological studies has not had any signicant toxic effects. The ADI for man is dened as the additive dose that, when consumed daily during a lifetime, does not cause a noticeable risk and is

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

SCF

Committee of experts from the Member State

European Commission

Council of Ministers

European Parliament

European Council of Ministers

National authorization for 2 years

EC directive
Figure 1 Legislative procedures to authorize additives in the EU.

FOOD AND NUTRITIONAL ANALYSIS / Additives 221

Table 2 European Community Directives (updated on December 2003) EC Directive General legislation Directive 89/107/CEE Sweeteners Directive 94/35/CE Directive 95/31/CE Color additives Directive 94/36/CE Directive 95/45/CE Directive 99/75/CE Directive 2001/50/CE Food additives other than colors or sweeteners Directive 95/2/CE Directive 96/85/CE Directive 98/72/CE Directive 2001/5/CE Directive 96/77/CE Directive 98/86/CE Directive 2000/63/CE Directive 2001/30/CE Content Additives that can be used in products destined for human consumption Sweeteners Criteria on the purity of sweeteners Color additives Criteria on the purity of color additives Modication of the Directive 95/45/CE Modication of the Directive 95/45/CE On food additives other than colors or sweeteners Modication of the Directive 95/2/CE Modication of the Directive 95/2/CE Modication of the Directive 95/2/CE Criteria on the purity of food additives other than colors or sweeteners Modication of the Directive 96/77/CE Modication of the Directive 96/77/CE Modication of the Directive 96/77/CE

In the US, a request to authorize a new food additive is presented to the FDA. A petition for an additive or a colorant must include data pertaining to the toxicity of the additive accompanied by toxicological tests on animals and even on humans. To evaluate whether an additive should be approved, the agency rst considers the composition and attributes of the substance, the probable amount that will be ingested, the possible long-term effects on consumers and various additional safety factors. If the additive is approved, the FDA publishes regulations that may include the types of foods in which it can be used, the maximum quantities that can be added, and how it should be identied in the food labels.
Food Additive Legislation

Table 3 US additive legislation Part 7082 170173 174178 180 Content Regulation of color additives General regulation of food additives Regulation of indirect food additives Regulation of food additives permitted in food or in contact with food on an interim basis pending additional study Regulation of the substances afrmed as generally recognized as safe Regulation of the substances prohibited from use in human food

182186 189

Analytical Methods for the Determination of Food Additives

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

European legislation is compiled in European Community Directives that are published in the Bulletin of the EU. European Community Directives affect the entire EU, setting a time limit for their integration in the legislation of each Member State, after which they are required to be enforced in the entire EU. Table 2 is a catalog of European Community Directives, updated in December 2003, that regulate positive lists of additives, standards for the purity of additives, and the maximum amounts of additives that can be added to different categories of foods. The US additive legislation is incorporated in the Code of Federal Regulations, Title 21, Chapter 1. The content of the US legislation is organized in parts, shown in Table 3.

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

222 FOOD AND NUTRITIONAL ANALYSIS / Additives

technique employed for the determination of the additive. Often, very simple samples do not require prior treatment or at the most need uncomplicated pretreatments, but most foods need to be treated before the determination of the additive. Frequently, the rst step is to use liquidliquid extraction (LLE) to eliminate interferences. LLE can be simple or repetitive (with the same solvent and then combining all the extracts). The extractant and the extraction method used depend fundamentally on the kind of sample being analyzed. The extraction can be done with a single solvent, principally n-hexane, water or acidied water, acetonitrile, dichloromethane, ethanol or methanol, diethylic or petroleum ether, acetone, and chloroform, or with a binary or even ternary mixture of the solvents, containing a very polar solvent, like water or methanol, and other less polar solvents. However, other matrix compounds are extracted together with the analytes, making it necessary to include an extract-cleanup step. The thoroughness of this step depends on the detection system: if the detector being used is selective enough, an exceedingly thorough cleanup of the extract is not necessary; but if the detector is not very selective, this step must eliminate as many as possible of the coextracts that can interfere in the determination. Moreover, because the additive concentration in this extract is not sufcient for the detection of legal limits, it is necessary to preconcentrate the analytes. LLE is commonly used to separate additives from their coextracts. Nevertheless, the recent trend is to replace LLE with solid-phase extraction (SPE). It is possible to use SPE as a single cleanup/preconcentration step or as an additional step after LLE. The use of SPE has become popular because of the great variety of polar and nonpolar sorbents and ionic exchange resins that exist. The sorbents most often used to preconcentrate food additives are: RP-C18, silica gel, sorbents with amino groups, XAD-2 polymeric sorbent, polyamide and wool. Supercritical uid extraction is another technique used to extract food additives. Its main advantages are that it saves both time and solvents; the solvent most often employed is carbon dioxide, for its moderate critical temperature (311C) and pressure (73 atm). In addition, carbon dioxide is not inammable, toxic, or very expensive. Methanol is sometimes used as a modier to adjust the dissolution power of this uid. Quite recently, other extraction techniques have been developed and are being employed for the determination of food additives, but are not yet widely used. These techniques include countercurrent chromatography, gel permeation chromatography,

distillation, membrane separation, dialysis and microdialysis, microwave-assisted extraction, solvent extraction assisted by ultrasound, solvent microextraction, solid-phase microextraction, and stir bar sorptive extraction. It is important to emphasize that specific difculties arise when extracting antioxidants and preservatives from fatty foods. Most of the permitted antioxidants and preservatives are fat soluble, so it may rst be necessary to extract the fat from the food and then extract the antioxidants from the fat. As a result, the determination of these additives implies the removal of triglycerides; the techniques most frequently used to remove triglycerides are saponication of the extracts, esterication of the extracts in the presence of lipase, and the separation of the analytes by preparative liquid chromatography.
Determination, Separation, and Detection of Food Additives

For the determination of food additives several analytical methods are used based on spectroscopic, electrochemical, and separation techniques, the latter with detectors included. For the determination of individual additives several spectroscopic techniques are normally employed: ultravioletvisible spectrophotometry, spectrouorimetry, luminescence, and photoacoustic spectrometry. Sometimes, to increase method selectivity, a combination of spectroscopic techniques is used. Another alternative to determine individual additives are electrochemical techniques: voltammetry, polarography, amperometry, and potentiometry. However, additives are normally combined to complement and promote their activity; as a result, it is necessary to develop analytical methods for the determination of additive mixtures. Although some spectroscopic and chemical methods are used, it is preferable to use separation methods for this purpose. Most analytical methods used to determine food additives are based on chromatographic techniques, although several recent papers have demonstrated the usefulness of electrophoresis for the analysis of food colors, sweeteners, antioxidants, and/or preservatives. The separation of food colors has received most attention, with a number of articles published on both capillary zone electrophoresis and micellar electrokinetic chromatography. Thin-layer chromatography, in its normal and high resolution forms, is used for the separation and quantication of additives with minimal sample manipulation. The most common detection technique

FOOD AND NUTRITIONAL ANALYSIS / Additives 223

is ultravioletvisible spectrophotometry, although other techniques such as spectrouorimetry and mass spectrometry are also used.

Liquid chromatography is the preferred technique to determine food additives, which in general are not very volatile and sometimes are thermolabile.

Table 4 Ofcial AOAC methods of analysis applicable to antioxidants AOAC method 948.26 942.11 942.13 952.09 952.10 954.06 965.28 967.21 968.17 971.30 975.43 983.15 984.26 Antioxidant a-Tocopheryl acetate Quaternary ammonium compounds Quaternary ammonium compounds Propyl gallate Quaternary ammonium compounds Quaternary ammonium compounds Antioxidants Ascorbic acid Butylated hydroxyanisole, butylated hydroxytoluene a-Tocopherol, a-tocopheryl acetate a-Tocopherol Antioxidants Ascorbic acid Food Foods Milk Foods, beverages Foods Milk Milk Foods Juices Cereals Foods Foods Oils, fats Foods Analytical technique Colorimetry Qualitative test Bromophenol blue method Colorimetry Optical crystallographic properties of the Reineckates Eosin yellowish method Qualitative test Titrimetry Gas chromatography Colorimetry Polarimetry Liquid chromatography Semiautomated method uorimetry

Table 5 Ofcial AOAC methods of analysis applicable to preservatives AOAC method 892.02 910.02 931.08 935.34 950.24 953.12 953.13 959.09 960.27 960.38 961.09 962.16 963.19 963.20 964.13 967.15 968.16 969.26 970.33 970.34 971.15 972.19 974.08 974.10 975.10 975.26 975.30 975.31 975.32 980.17 983.16 987.04 990.28 990.29 990.30 990.31 993.03 Preservative Sulfurous acid free Benzoic acid Formaldehyde Benzoic acid Benzoate, caffeine, saccharin Dehydroacetic acid Dehydroacetic acid Boric acid Preservatives Benzoic acid Sultes Sulfurous acid total Benzoic acid Sulfurous acid total Nitrites Benzoic acid Fumaric acid Boric acid Boric acid, borates Boric acid Sorbic acid Boric acid Sorbic acid Sorbic acid Sorbic acid Boric acid Salicylic aid Salicylic acid Sulfurous acid Preservatives Benzoic acid, sorbic acid Sultes total Sultes Sultes total Sultes free Sultes Nitrates Food Meat Foods Foods Flour Soda beverages Cheese Cheese Meat Milk Non-solid foods, beverages Meat Foods Foods Dried fruit Curing preparations Foods Foods Caviar Foods Foods Cheeses Foods Wines Dairy products Wines Foods Foods, beverages Foods, beverages Foods Ground beef Foods Foods Foods Foods, beverages Wines Foods, beverage Baby foods Analytical technique Titrimetry Qualitative test Qualitative test Photometry Liquid chromatography Qualitative test Spectrophotometry Semiquantitative test Qualitative test Spectrophotometry Qualitative test Modied MonierWilliams method Titrimetry Colorimetry Titrimetry Thin-layer chromatography Polarography Spectrophotometry Qualitative test Titrimetry Oxidation method Atomic absorption spectrometry Spectrophotometry Spectrophotometry Colorimetry Emission spectroscopy Qualitative test Colorimetry Qualitative test Spectrophotometry Gas chromatography Differential pulse polarography Optimized MonierWilliams method Flow injection method spectrophotometry Flow injection method spectrophotometry Ion exclusion chromatography Spectrophotometry

224 FOOD AND NUTRITIONAL ANALYSIS / Additives

In most of the methods that have been developed, the stationary phase used for the additive separation is a polygosyl-bonded silica reversed-phase sorbent with octadecylsilane functional groups (RP-C18), although other stationary phases such as reverse RP-C30 and ionic-exchange phases have also been used. The detection of additives, after their separation by liquid

chromatography, has been done using ultraviolet visible, uorescence, electrochemical, and mass spectrometry detectors. Gas chromatography, with or without derivatization, is also used to selectively determine individual or mixtures of food additives. The additive separation is done using nonpolar stationary phases consisting

Table 6 Ofcial AOAC methods of analysis applicable to sweeteners AOAC method 934.04 941.10 947.10 950.24 957.09 957.10 957.11 969.27 969.28 973.28 973.29 980.18 Sweetener Saccharin Saccharin Saccharin Benzoate, caffeine, saccharin Cyclamate salts Cyclamate salts Dulcin Nonnutritive sweeteners Cyclamate salts Sorbitol Saccharin Saccharin Food Nonalcoholic beverages Foods Foods Soda beverages Nonalcoholic beverages Nonalcoholic beverages Fruits Nonalcoholic beverages Canned fruits Foods Foods Foods Analytical technique Semiquantitative method Qualitative test Sublimation Liquid chromatography Sodium nitrite test Gravimetry Qualitative test spectrophotometry Thin-layer chromatography Colorimetry Gas chromatography Gravimetry Differential pulse polarography

Table 7 Ofcial AOAC methods of analysis applicable to color additives AOAC method 900.01 920.114 920.119 920.209 925.28 930.17 930.38 938.04 942.19 948.07 955.18 966.21 970.65 981.15 985.31 988.13 Color additive Color additives Color additives Color additives Natural color additives Color additives Articial color additives Synthetic organic color additives Carotenoids Synthetic color additives Caramel Natural and articial organic and water-soluble color additives Oil-soluble color additives Riboavin Riboavin Riboavin FD&C color additives Food Milk Cream Butter fat Foods Ice cream, frozen desserts Distilled liquors Foods Macaroni products Oils, fats Wines Distilled liquors Foods Foods Foods Ready-to-feed milk-based infant formula Foods Analytical technique Qualitative test Qualitative test Qualitative test Qualitative test Qualitative test Qualitative test Column chromatography Colorimetry Spectrophotometry Qualitative test Spectrophotometry Column chromatography Fluorimetry Flow injection method uorimetry Fluorimetry Spectrophotometry thin-layer chromatography

Table 8 Ofcial AOAC methods of analysis applicable to emulsifying agents AOAC method 920.106 935.61 945.51 959.06 960.33 960.34 963.25 968.18 974.11 991.30 Emulsifying agent Gelatin Gums Gelatin Alginates Gums Alginates Alginates Sodium lauryl sulfate Polysorbate 60 Polydimethylsiloxane Food Milk, milk products Salad dressings Ice cream, frozen desserts Chocolate products Ice creams, frozen desserts Chocolate frozen desserts Food dressings Egg white Shortening, oils, dressings Pineapple juice Analytical technique Qualitative test Qualitative test Qualitative test Qualitative test Infrared spectroscopy Qualitative test Colorimetry Colorimetry Gravimetry Atomic absorption spectrometry

FOOD AND NUTRITIONAL ANALYSIS / Antioxidants and Preservatives 225

of 100% poly(dimethylsiloxane), intermediate polarity phases consisting of 5% diphenyl95% dimethylsiloxane or 50% diphenyl50% dimethylsiloxane, and polar phases consisting of 100% poly(ethyleneglycol). The detection of these analytes is usually done using a ame ionization detector, which is able to detect compounds with CH groups, or universal detectors like a mass spectrometry detector.
Identication of Food Additives

The Association of Ofcial Analytical Chemists (AOAC) publishes reference methods for the analysis of additives in foods. The following Tables 48 summarize the AOAC Ofcial Methods of Analysis applicable to antioxidants (Table 4), preservatives (Table 5), sweeteners (Table 6), color additives (Table 7), and emulsifying agents (Table 8).
See also: Food and Nutritional Analysis: Overview; Antioxidants and Preservatives. Gas Chromatography: Overview. Liquid Chromatography: Instrumentation. Sweeteners.

When analyzing food additives it is frequently necessary to identify the additives that have been determined quantitatively using the above cited detectors that, while supplying high sensitivity (essential for measuring the established legal limits), are not able to identify these compounds. To conrm the obtained results two strategies are used: on the one hand, the use of detectors that are able to unmistakably identify the analytes; on the other hand, the use of two different detectors to compare their responses against the same mixture of additives. Mass spectrometry is an excellent tool that can be used coupled to either a liquid or a gas chromatograph to conrm the identity of the additives present in foods. A complete mass spectrum of an additive provides structural information that is unique to the additive. Most of the studies that have been done use the ionization technique for electronic impact to a certain energy (normally 70 eV). Detection by a diode array detector can be used to conrm the identity of an additive. This detector is usually coupled with a liquid chromatograph to record the ultravioletvisible spectra for each of the chromatographic peaks so that they can be compared with the spectra of the pure compounds. Moreover, the purity of each of the chromatographic peaks can be checked. The use of two connected detectors, in succession or in parallel, is widely employed, coupling them to either a liquid or gas chromatograph.

Further Reading
Abidi SL (2000) Chromatographic analysis of tocol derived lipid antioxidants. Journal of Chromatography A 881: 197216. Belitz HD and Grosch W (eds.) (1999) Food Chemistry, 2nd edn. Berlin: Springer. Boyce MC (2001) Determination of additives in foods by capillary electrophoresis. Electrophoresis 22: 14471459. Burdock GA (ed.) (1996) Encyclopedia of Food and Color Additives. Boca Raton, FL: CRC Press. Clydesdale FM (ed.) (1996) Food additives: Toxicology, Regulation and Properties. Boca Raton, FL: CRC Press. Code of Federal Regulations Title 21, Chapter 1, Parts 7082, 170189. Washington, DC: US Government Printing Ofce, 2003; http://www.access.gpo.gov/cgi-bin/ cfrassemble.cgi?title 200321. Horwitz W (ed.) (2000) Ofcial Methods of Analysis of the Association of Ofcial Analytical Chemists, 17th edn. Arlington: AOAC International. Karovicova J and Simko P (2000) Determination of synthetic phenolic antioxidants in food by high performance liquid chromatography. Journal of Chromatography A 882: 271281. Macrae R, Robinson RK, and Sadler MJ (eds.) (1993) Encyclopaedia of Food Science, Food Technology and Nutrition, vols. I and II. San Diego: Academic Press. Pokorny J, Yanishlieva N, and Gordon MH (eds.) (2001) Antioxidants in Food: Practical Applications. Boca Raton, FL: CRC Press.

Antioxidants and Preservatives

M H Gordon, The University of Reading, Reading, UK
& 2005, Elsevier Ltd. All Rights Reserved.

Introduction
Antioxidants and preservatives are added to foods to retard their deterioration. Antioxidants retard

chemical oxidation of the lipids whereas preservatives retard microbiological deterioration. In the UK, the permitted antioxidants and preservatives are listed in the Miscellaneous Food Additives Regulations (1995). Common allowed antioxidants and preservatives are listed in Tables 1 and 2. The allowed additives vary from country to country with