EN CASO DE INGESTIÓN DE PRODUCTOS QUÍMICOS Formaldehído (formol)

Nitrates and nitrites Xylenol Meat — 27 Nitrate and nitrite Spectrophotometric Meat products — 28 Nitrate and nitrite Ion-exchange

chromatography

Meat products Nitrite >40 29

a _{For meanings of these abbreviations see Table 5.1.}

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determination of benzoic acid and sorbic acid in foods involves extraction with ether and successive partitionings into aqueous NaOH and CH2Cl2, derivatization to a trimethylsilyl

ester, and fl ame ionization detection (FID). A GC-mass spectrometric technique has been reported for the simultaneous determination of sorbic acid, benzoic acid, and parabens in foods.37 _{A new application of pyrolytic methylation has been developed to determine}

benzoic acid in soft drinks by GC without any pretreatment and using a special pyrolyzer. Samples containing benzoic acid could be accurately determined by direct-injection GC on the medium polar stationary phase column.38

Specifi c GC determination of benzoates in meat products has been reported.39,40

Simultaneous GC determination of preservatives (sorbic acid, benzoic acid, and their esters) in fatty foods (pâté) without derivatization has been described.41 _{Sample pretreatment}

includes solvent extraction and SPE. GC-FID for quantifi cation and mass spectrometry has been used in the conventional electron impact mode for identifi cation. Sorbates, benzoates, and parabens were simultaneously analyzed in cured meat products by GC-FID.42

High performance liquid chromatographic methods. Determination of preservatives by HPLC

off ers high specifi city with minimal preparation and does not require derivatization. Numerous HPLC methods for simultaneous determination of benzoic acid, sorbic acid, and parabens have been reported as applicable to selected foodstuff s.4,5,43 _{In general, the}

extraction system of the applicable procedures varies according to the complexity and composition of the foodstuff s, and may include clarifi cation/purifi cation, which is essential for eliminating high–molecular-mass matrix interferences (e.g., proteins, fats, and poly- saccharides). Sample pretreatment prior to HPLC analysis involves solvent extraction, fi ltration, centrifugation, and SPE. Separation is done essentially by reversed-phase HPLC with UV detection at the wavelengths of maximum absorption of the compounds. Diff erent eluents have been used, including phosphate buff er, methanol, tetrahydrofuran, acetate buff er, and acetonitrile.4,43–46

Nowadays HPLC is the most common analytical procedure for the detection and quantifi cation of these preservatives in foods, although there are very few published analytical methods that are specifi cally applicable to meat systems. Ali47 _{reported that}

benzoic acid, sorbic acid, and parabens were extracted from meat with 70% ethanol. After fi ltration, extracts were analyzed using reversed-phase liquid chromatography. An analytical procedure has been developed for the analysis of benzoic acid; p-hydroxybenzoic acid; and methyl-, ethyl-, propyl-, isopropyl-, and butyl esters of p-hydroxybenzoic acid by micellar liquid chromatography in food samples, including chicken spread.48

Capillary electrophoretic methods. CE has recently been employed as an effi cient tool for preservative determination in food due to its many advantages, which include high separation effi ciency, excellent resolution, and short analysis time. Various CE methods, such as capillary zone electrophoresis, micellar electrokinetic chromatography (MEKC), and micro- emulsion electrokinetic chromatography (MEEKC), have been reported for determination of preservatives in foods. Th ese methods have generally been used on carbohydrate-rich matrices (soft drinks, wine, jam, soy sauce, etc.). In most cases, real samples cannot be injected directly into CE systems, and an extraction cleanup process is necessary. Th is stage can be even more necessary in complex matrices (protein, fat, etc.), like meat derivatives, for which no published methods have been found.

Kuo and Hsieh9 _{described a CE method for the simultaneous separation of nine}

preservatives, including benzoic acid, sorbic acid, p-hydroxybenzoic acid, and six alkyl esters of

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Additives: Preservatives 䡲 97

p- hydroxybenzoic acid in plum preserves, bean curd, and soy sauce. MEKC has been

successfully used to simultaneously analyze p-hydroxybenzoic acid methyl ester, p-hydroxy- benzoic acid ethyl ester, benzoic acid, and sorbic acid.49 _{Huang et al.}11 _{used the MEEKC}

method to separate parabens (methyl, ethyl, propyl, and butyl), sorbic acid, and benzoic acid in various food products (soft drinks, soy sauces, and wines). Th e separation and detection of benzoate and sorbate in soft drinks by both conventional CE and microchip electrophoresis with capacitively coupled contactless conductivity detection has been reported.50 _Capillary

electrochromatography, a hybrid separation technique that combines the features of HPLC and CE, has been used to analyze sorbic acid, benzoic acid, and parabens in some products (cold syrups, lotions, soy sauces, and wines).10

Enzymatic determination. A method for the determination of sorbic acid based on spectro-

photometric measurement of sorbyl coenzyme A at 300 nm has been reported.51 _{Th e method}

has been tested for various food matrices (wine, alcoholic and nonalcoholic beverages, fruit preserves, and tomato ketchup).

5.4 Sulfi tes

Sulfur dioxide and sulfi tes comprise the group of compounds known collectively as sulfi tes (Table 5.1). Th ese sulfi ting agents, or S(IV) oxoanion compounds, are considered relatively strong preservatives, because of their strong antimicrobial activity. Moreover, even a small amount of sulfi te in meat imparts a bright red color. Th ese compounds are not permitted for use in meat in the United States. In the European Community, the maximum permitted amount of added sulfi ting agents in the various diff erent meat products is 450 mg/kg, expressed as SO2 (Table 5.2).

Sulfi tes have been associated with allergic reactions and food intolerance symptoms. Th ey are known to degrade thiamine (vitamin B1), of which meat is a good source. Th e ADI for sulfi te

(expressed as SO2) is 0.7 mg/kg body weight.52

When added to a food matrix, some of the sulfi ting agents bind to diff erent components of food. Th e portion of sulfi ting agent that does not combine with the food is called free sulfi te. Bound sulfi te can be categorized as reversibly or irreversibly bound sulfi te. Th e relative presence of each one varies according to the reactivity of sulfur dioxide in meat systems; this in turn is associated with factors involved with composition or with processing and storage conditions. For instance, following incorporation of additives to meat products, there can be irreversible losses of as much as 50%, depending on these factors.53,54 _{Cooking meat products also causes sulfi te}

reduction.55 _{Th en again, sulfi te ions may cleave disulfi de bonds in meat proteins.}54 _{Th e analytical}

determination of sulfi te, then, does not refl ect the preservatives that were initially added.

5.4.1 Analytical Methods

Many analytical methods have been reported for sulfi te determination in foods and beverages. Th ese methods include titrimetry, spectrophotometry, enzymatic analysis, chromatography, fl ow injection analysis (FIA), and electroanalysis.5,56,57 _{However, not all of them are equally suitable}

for the determination of sulfi tes in solid, complex protein matrices such as processed meats, where sulfi te-binding problems may arise from interaction with other food components or entrapment within food particles.56,58

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Extraction procedure. Analytical determination requires some means of removing and recovering

the sulfur dioxide (free and reversibly bound) and then quantifying the level found. Sample preparation and analysis should be as rapid as possible to avoid loss of labile forms of sulfi te.

Numerous procedures utilize the Monier-Williams distillation process for sulfi te separation by means of distillation from a suspension or solution of the food in hot HCl. In some cases, Monier-Williams distillation has been used as a preparatory tool for obtaining free and bound sulfi te fractions to accommodate more selective quantitation techniques, whereas in others extraction has been used instead of distillation.56 _{Other procedures that do not utilize}

distillation or vapor phase transfer require the conversion of sulfi te to a stabilized sulfi te derivative compound to take advantage of some property that serves for quantitation.

Titrimetry (Monier-Williams) method. Th is procedure, derived from the classic studies of Monier-Williams, measures free sulfi te plus the reproducible portion of bound sulfi te. It is based on acid distillation followed by vapor phase transfer of the SO2, facilitated by a

carrier gas stream, to an oxide-trapping solution. Th e sulfur dioxide is oxidized to sulfuric acid for quantitation by titration or determination of sulfi te by precipitation with barium. Th e sulfuric acid is stoichiometrically related to the sulfur dioxide distilled from the test solution (modifi ed Monier-Williams method)19 _{(Table 5.3). An optimized Monier-Williams}

method20 _{for the analysis of sulfi tes in foods has been collaboratively tested and accepted as}

an offi cial method (Table 5.3). Very few foods not treated with sulfi te give a false positive even at levels below 10 ppm; Allium and Brassica vegetables and isolated soy protein are important exceptions to this rule. As a comparative procedure, this methodology has been assayed in diff erent muscle foods including beef, pork, and chicken meat products.53,55

Monier- Williams methods are the ones most commonly employed by food control labora- tories for meat product analysis (Table 5.4). A method based on distilling of sample in an acidic medium followed by iodometric titration has also been also assayed (Table 5.4), although its use is limited to products with high levels of sulfi te.56

Spectrophotometric methods. Various spectrophotometric procedures have been reported for

determination of sulfi ting agents in foods. An AOAC method12 _{(Table 5.3) for sulfi te}

Table 5.4 Examples of the Analytical Methods Used for Meat Product Preservative Determination in Evaluation of Profi ciency Testing of Analytical Laboratories (Interlaboratory Comparisons)

Sulfi tea _{in Pork Sausage}

(Number of Laboratories Participating: 75)

Nitriteb _{in Gammon Steak}

(Number of Laboratories Participating: 101) Analytical Methods Used by Laboratories (%) Analytical Methods Used by Laboratories (%)

Modifi ed Monier-Williams 43.9 Colorimetry 52.2 Optimized Monier-Williams 28.1 IC 21.7 Distillation and titration

with iodine

15.8 HPLC 18.5 Spectrophotometry 8.8 FIA 4.4

IC 1.8 Enzymatic 2.2

Others 1.8 CE 1.1

a _{Food Analysis Performance Assessment Scheme (FAPAS), Profi ciency Test 2046. August– October}

2006.

b _{FAPAS, Profi ciency Test 1547. January–February 2007.}

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Additives: Preservatives 䡲 99

determination in ground beef is based on a colorimetric reaction with p-rosaniline after reaction with mercuric extractant. Another method often used for determination of sulfur dioxide in meat products is based on distillation/spectrophotometric analysis using 5,5′ di-thiobis-(2-nitrobenzoic acid) (DTNB). It has been used in fresh sausages,53 _{and commi-}

nuted pork meat.54 _{Determination of sulfi tes has been performed in commercial sausages}

by direct extraction and spectrophotometric methods based on a step reaction using the reagent DTNB.58

Diff erential pulse polarographic method. A method based on diff erential pulse polarography

(DPP) and applicable to the determination of sulfi tes (total) in foods has been proposed23

(Table 5.3). Th e method, based on a collaborative study,59 _{was tested on a number of muscle}

foods including shrimp. It measures SO2, which is purged with N2 from acidifi ed test sus-

pension, collected in electrolyte-trapping solution, and then determined by DPP. Modifi ed Monier-Williams distillation followed by DPP has also been used to determine sulfi ting agents in foods.60

Chromatographic methods. Anion exclusion chromatography has proven a useful technique

for determining sulfi tes in foods. An electrochemical detection system is the most commonly used,55,61–63 _{although conductivity detection}64,65 _{and direct UV detection have been also}

reported.66 _{In the AOAC chromatographic method}25 _{(Table 5.3), SO}

2 is released by direct

alkali extraction, followed by anion exclusion chromatographic separation and electro- chemical (amperometric) detection.62,67 _{Th e method was tested on diff erent food matrices,}

including a muscle food (dehydrated seafood). Improvements in amperometric detection of sulfi te in food matrices have been reported.68

Although chromatographic methods may be appropriate for use on meat products, very little has been published in the literature in that respect. Free and total sulfi te have been determined in fresh sausages by HPLC;63 _{the method includes extraction of both free and}

total sulfi te by dissolution of the sample in a suitable solvent and determination by HPLC (anion exclusion column) using electrochemical detection. Th is procedure was also used to determine sulfi te content in fresh and cooked (beef, pork, and chicken) burgers.55

Enzymatic methods. Enzymatic methods have been developed for sulfi te analysis in food. In

these procedures, sulfi te is usually oxidized to sulfate; this is catalyzed by sulfi te oxidase to release hydrogen peroxide, and the hydrogen peroxide is measured by linking it to the oxi- dation of reduced nicotinamide adenine dinucleotide (NADH) in the presence of NADH peroxidase. Hydrogen peroxide was measured by spectrophotometry.22,56,69 _{Various sulfi te}

oxidase biosensors have also been reported70,71 _{(Table 5.3). Enzymatic methods have been}

used to determine SO2 in muscle foods (shrimp).72

FIA. Th is method off ers the advantages of simplicity and precision with a high analytical sampling rate, while requiring only low-cost equipment, reducing the need for large volumes of toxic reagents, and requiring little analysis time. Several FIA methods have been used for sulfi te determination in food and beverages,4,72,73 _{but few published reports can be found}

dealing with their application to muscle foods, and most of these refer to shrimp.72,74 _With

the support of an interlaboratory study,74 _{an AOAC method}24 _{has been proposed for FIA}

sulfi te determination (Table 5.3) based on sulfi te reaction with malachite green. A test solution is made to react with NaOH to release aldehyde-bound sulfi te; then, the test stream is acidifi ed to produce SO2 gas, which diff uses across a Tefl on membrane in the gas diff usion

cell into a fl owing stream of malachite green, which is discolored. With this procedure it is possible to assay samples containing ingredients from liliaceae (garlic, onions, leeks) for which the Monier-Williams reference method is not suitable.

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Capillary electrophoresis. Sulfi te content in foods and beverages can be determined by CE. Th e sulfi te is converted to sulfur dioxide and fi nally to sulfate by Monier-Williams distillation. Th e sulfate is then determined by CE. Th e results for sulfi te content of seafood agree very well with those determined by titrimetry.75

5.5 Nitrite and Nitrate

Sodium and potassium nitrates and sodium and potassium nitrites (Table 5.1) are used in meat curing because they stabilize red meat color, inhibit some spoilage and food poisoning anaerobic microorganisms, delay the development of oxidative rancidity, and contribute to fl avor development. Depending on the type of processed meat, processing conditions, presence of sodium ascorbate, and other factors, the added nitrite reacts with many components in the matrix (myoglobin, nonheme proteins, lipids), so that the analytical detection of the nitrite or nitrate content does not refl ect the preservative initially added. Analytical methods therefore usually determine the residual nitrate/nitrite, which can reach only about 10–20% of the original nitrite amount added.76,77 _To

detect bad practice and use of high nitrite levels, it is more eff ective to control nitrite at input.77

Nitrite levels in meat products are important because nitrite can react with secondary amines to form nitrosamines, which are recognized as having carcinogenic eff ects. In the EU, potassium and sodium nitrite and nitrate are authorized for use in diff erent meat products, and maximum ingoing amounts (150 mg/kg for nitrite and 300 mg/kg for nitrate) are established for all products, as well as maximum residual levels for some of them (Table 5.2). Current regulations on use of nitrite and nitrate in the United States vary depending on the curing method used and the product that is cured. For comminuted products, the maximum ingoing nitrite and nitrate limits are 156 mg/kg and 1718 mg/kg, respectively. For immersion-cured and massaged or pumped products, those limits are 200 mg/kg and 700 mg/kg, respectively. In dry-cured products nitrite is limited to 625 ppm and nitrate to 2187 ppm.78 _{Th e ADI for nitrites, as nitrite ion, is 0.07 mg/kg body}

weight, and 3.7 mg/kg for nitrate, as nitrate ion.79,80

5.5.1 Analytical Methods

Several methods have been reported for quantitative determination of nitrate and nitrite in foods, including spectrophotometry, chromatography, electrochemical detection (potentiometry, amperometry, polarography), CE, and others.4,5,81,82 _{However, not all of them are equally suitable}

for use on highly heterogeneous solid matrices like processed meats and poultry.

Extraction procedure. Nitrite and nitrate determination requires an extraction stage, generally

involving dispersion in hot water. Meat products contain various compounds (ascorbic acid, fat, protein, sodium chloride, etc.) that can interfere in nitrite and nitrate measurement, and so a number of procedures have been tried to clean up the extracts prior to determination. Th ese include clarifi cation stages (fat and protein precipitation, fi ltration, etc.) using diff erent compounds or solvents (Carrez or borax reagents, zinc sulfate or potassium ferrocyanide, acetonitrile, and others) or pretreatment cartridges to remove sample matrix interferences.3,83

Another possibility is to separate the fat by centrifugation and rapid cooling followed by in-line dialysis to remove protein and remaining fat. Obviously the choice of a specifi c

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Additives: Preservatives 䡲 101

procedure to clean up an extract prior to determination will depend on the analytical method used.

Spectrometric methods. Of the methods available for quantitative determination of residual

nitrite in meat products, the most commonly used are based on colorimetric determination using Griess diazotization, which involves the formation of azo dye produced by coupling a diazonium salt with an aromatic amine or phenol. Th e diazo compound is formed when nitrite (aqueous extract from meat) reacts with sulfanilamide and the coupling agent

N-(1-naphtyl) ethylenediamine-2HCl.3,4,81 _{Th e color that develops is measured spectrophoto-}

metrically (540 nm). Th e same reaction can be used to determine nitrate. To do this, the relatively inert nitrate is reduced to nitrite, which can then be determined by Griess diazotization. Nitrate can be reduced by chemical and enzymatic procedures. A variety of agents have been investigated for chemical reduction,81 _{the most common arrangement}

being a spongy cadmium column, which can achieve effi ciencies of nitrate-to-nitrite conver- sion approaching 100%. Enzymatic reduction of nitrate to nitrite has been accomplished with nicotinamide adenosine dinucleotide phosphate in the presence of the enzyme nitrate reductase.28 _{Spectrophotometric methods involving the reduction of nitrate to nitrite and}

subsequent colorimetric determination of nitrite with a diazo coupling reaction have been adopted26,28 _{for meat products (Table 5.3). Nitrite and nitrate have also been determined in}

meat products using m-xylenol27 _{(Table 5.3).}

Other colorimetric reactions have been used to determine nitrites and nitrates in meat products. A number of these are based on the reduction of phosphomolybdic acid to phosphomolybdenum blue complex by sodium sulfi de, which is oxidized by the addi- tion of nitrite, causing a reduction in the intensity of the blue color and a reduction in the absorbance measured at 814 nm;84 _{others are based on the catalytic eff ect of nitrite}

on the oxidation of methyl red by bromate, and the absorbance is measured at 520 nm.85

A spectrofl uorimetric method has been developed for nitrite determination in meat systems.86

Enzymatic methods. Procedures based on enzymatic reduction coupled with spectrophotometric

detection can be used to determine nitrite and nitrate in meat samples. Nitrite is measured enzymatically through its reaction with nitrite reductase coupled with NADH, and the reaction is measured spectrophotometrically.87

FIA. Several FIA-based methodologies for the determination of nitrite and nitrate in meat

products have been reported. Most of the FIA methods that are used to simultaneously determine nitrates and nitrites in meat products are based on a diazotization/coupling reaction.88–91 _{Th ere have also been other applications of FIA, based on the reduction of nitrite}

and nitrate to nitric oxide followed by reaction with iron (II) and thiocyanate in an acid medium to form FeSCNNO+ chromophore, which is measured at 460 nm;82 _{based on the}

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