analytical strategies to evaluate antioxidants in food: a...

Trends in Food Science & Technology 21 (2010) 229e246

Review

* Corresponding author.

0924-2244/$ - see front matter � 2010 Elsevier Ltd. All rights reserved.doi:10.1016/j.tifs.2009.12.003

Analytical strategies

to evaluate

antioxidants in food:

a review

C. Andréa, I. Castanheiraa,

J.M. Cruzb, P. Paseirob and

A. Sanches-Silvaa,*aNational Institute of Health Dr. Ricardo Jorge, Av.

Padre Cruz, 1600 Lisbon, Portugal (National Institute

of Health Dr. Ricardo Jorge, Food and Nutrition, Av.

Padre Cruz, 1600 Lisbon, Portugal. Tel./fax: D351

938380493; e-mail: [email protected])bDepartment of Analytical Chemistry, Nutrition and

Food Science, Pharmacy Faculty, University of

Santiago de Compostela, Campus Sur, 15782 Santiagode Compostela, Spain

Antioxidants are an important group of food additives that

have the ability to protect against detrimental change of oxi-

dizable nutrients and consequently they extend shelf-life of

foods.

The present paper is an updated review on the analysis of

ten antioxidants (both synthetic and natural). Three types of

gallates, propyl (PG), octyl (OG) and dodecyl (DG), tert-Butyl-

hydroquinone (TBHQ), Butylated Hydroxytoluene (BHT), tert-

Butylhydroxyanisole (BHA), a-tocopherol, ascorbic acid,

erythorbic acid and sodium D-isoascorbate were the antioxi-

dants selected. The physico-chemical properties, sample prep-

aration procedures and analytical methods for their

determination are compared and discussed.

IntroductionNowadays, antioxidants receive remarkable attention in

the literature, due to the ability to preserve foodstuffs byretarding deterioration, rancidity and/or discoloration

caused by oxidation. Detrimental effects include undesir-able chemical compounds formation like aldehydes, ke-tones and organic acids than yield off-flavours (Saadet al., 2007).

In biological systems, antioxidants protect against oxi-dative damage and help to prevent cardiovascular, neuro-logical and/or carcinogenic diseases (Lanina, Toledo,Sampels, Kamal-Eldin, & Jastrebova, 2007).

Following the recommendations of international organi-zations, the use of synthetic antioxidants like PG, OG,BHA, BHT and TBHQ is regulated by legal authorities ina limited number of foods, with maximum limits in eachcase or association. There are several documents legislatingfood additives such as European Union Directives and Reg-ulations (Directive 95/2/EC, 1995; EC Regulation No 1333/2008, 2008), FDA in USA (FDA, 2001a, 2001b), FoodStandards Australia New Zealand for Australia and NewZealand (The Australia New Zealand Food StandardsCode, 2009), The Joint FAO/WHO Expert Committee onFood Additives (JECFA, 2003) and Codex Alimentarius(CGSFA, 1995).

The use of antioxidants (AO) is not restricted to food-stuffs. They can also be used to prevent the degradationof food packaging during processing and storage, improv-ing the end-use application. Nevertheless, they or their deg-radation products can migrate into foodstuffs. EUlegislation imposes migration limits of substances thatcan be used in the manufacture of food packaging.

Antioxidants can be classified as primary or long-termantioxidants and as secondary or processing antioxidants.Primary antioxidants include hindered phenols and second-ary aryl amines and secondary antioxidants include orga-nophosphites and thioesters (Dopico-Garcı́a, Lopez-Vilariño, & González-Rodrı́guez, 2003). The primaryare active radical scavengers or hydrogen donors or chainreaction breakers while secondary are peroxidedecomposers.

Most of the methods described in the literature for thequantitative analysis of antioxidants or antioxidant mixturespresent interference problems, low resolution and are time-consuming. The establishment of analytical methods tomonitor the use of prohibited antioxidants and/or the exces-sive use of permitted AO and their degenerative products isa real need. In 2000, Karovi�cová & Šimko (2000) have car-ried out a review regarding the determination of syntheticphenolic antioxidants in food by high-performance liquid

mailto:[email protected]

Table 1. Chemical and physical information of the antioxidants.

Migrants CAS No Formula/Elementalanalysis

MW/Exactmass

Mp (�C) Bp/Fp (�C) Density (g/cm3) Logkow Enthalpy of vap. Solubility

Propyl gallate, Benzoicacid, 3,4,5-trihydroxy-,propyl ester (9CI)

121-79-9 C10H12O5 212.20a 145e146�Ca

CASFp: 181.3�20.8�C,bp: 448.6�40.0�C,Press: 760Torra

1.363�0.06g/cm3,Temp: 20�C,Press: 760Torra

2.600�0.333,Temp: 25�Ca

73.43�3.0kJ/mol,Press: 760Torra

Slightly soluble inwater; freelysoluble in ethanol,ether and propane-1,2-diolc

Octyl gallate, Benzoicacid, 3,4,5-trihydroxy-,octyl ester (9CI)

1034-01-1 C15H22O5 282.33a 101e104�Ca

91e92�Cabp: 482.9�40.0�C,Press: 760Torr,Fp:177.1�20.8�Ca


5.256�0.333,Temp: 25�Ca


Insoluble in water,freely soluble inethanol, ether andpropane-1,2-diolc

Lauryl gallate, Benzoicacid, 3,4,5-trihydroxy-,dodecyl ester (9CI)

1166-52-5 C19H30O5 338.44 94e96�Ca bp:521.7�45.0�C,

Press: 760Torr,Fp:180.3�22.2�Ca


7.382�0.333,Temp: 25�Ca


Insoluble in water;freely soluble inethanol and etherc

Erythorbic acid,D-erythro-Hex-2-enonicacid, g-lactone (8CI, 9CI)

89-65-6 C6H8O6 176.12 167e174�Ca bp: 552.7�50.0�C,

Press: 760Torr, Fp:238.2�23.6�Ca

1.954�0.06g/cm3,Temp: 20�C,Press: 760Torr (calc),1.654g/cm3 (exp)a

–2.410�0.450,Temp: 25�Ca


Freely soluble inwater, soluble inethanolc

Sodium, D-isoascorbatemonohydrate D-erythro-Hex-2-enonic acid,g-lactone, monosodiumsalt, monohydrate (9CI)

63524-04-9 C6H8O6,H2O, Na

216.12b 165�C (dec.)(lit.)b

Ascorbic acid,L-Ascorbic acid (8CI, 9CI)

50-81-7 C6H8O6 176.12 190e194�Cb Fp: 238.2�23.6�C, bp:

552.7�50.0�C, Press:760Torr (calc)a

1.954�0.06g/cm3,Temp: 20�C (calc)a

–2.410�0.450,Temp: 25�C(calc)a

95.78�6.0kJ/mol,Press: 760Torr(calc)a

Freely soluble inwater; sparinglysoluble in ethanol;insoluble in etherc

a-Tocopherol, 2H-1-Benzopyran-6-ol,3,4-dihydro-2,5,7,8-tetramethyl-2-(4,8,12-trimethyltridecyl)-(9CI)

10191-41-0e

C29H50O2 430.71 bp:485.9�0.0�C,Press: 760Torr (calc),Fp: 210.2�24.4�C(calc)a

0.930�0.06g/cm3,Temp: 20�C,Press: 760Torr (calc),a

0.950g/mL at 20�C(lit.)b

11.902�0.267,Temp: 25�C(calc)a

78.00�3.0kJ/mol,Press: 760Torr (calc)a

Insoluble in water,freely soluble inethanol, misciblewith etherc

tert-Butylhydroquinone,1,4-Benzenediol, 2-(1,1-dimethylethyl)-(9CI),TBHQ

1948-33-0 C10H14O2 166.22 126.5e129�C

(exp)abp: 291.3�20.0�C,Press: 760Torr, Fp:138.7�16.4�Ca

1.086�0.06g/cm3, Temp:20�C, Press: 760Torra

2.333�0.225,Temp: 25�Ca


Practicallyinsoluble in water;soluble in ethanolc

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231C. André et al. / Trends in Food Science & Technology 21 (2010) 229e246

chromatography. Robbins (2003) has also carried out a re-view concerning the analytical methodologies to determinephenolic acids in foods.

This review aims to carry out an updated evaluation ofa wide range of analytical methods used to separate, iden-tify and quantify antioxidants, phenolic and other, in food-stuffs and food packaging.

Chemical and physical information of antioxidantsCurrently, food industry requires simple and rapid ana-

lytical assays for the control of synthetic phenolic antioxi-dants added to food (Morales, González, Reviejo, &Pingarrón, 2005; Morales, González, Serra, Reviejo, & Pin-garrón, 2005).

Many studies have been dedicated to the effects of anti-oxidants (EFSA, 2004a, 2004b, 2008; Fidler, Davidsson,Zeder, & Hurrell, 2004; Georgantelis, Blekas, Katikou,Ambrosiadis, & Fletouris, 2007; Gharavi & El-Kadi,2005; Hirose et al., 1999; Kuwabara, Watanabe, Adachi,Nakanishi, & Matsuno, 2003; Sauberlich, Tamura, Craig,Freeberg, & Liu, 1996; Zurita et al., 2007). In fact, recently,the safety of AO has been questioned (Yang, Lin, &Choong, 2002). Studies demonstrate that excess use ofPG, BHA, BHT and TBHQ, synthetic antioxidants addedto foodstuff, may cause a loss of sustenance and even pro-duce toxic substances to harm people’s health (Guan, Chu,Fu, Wu, & Ye, 2006). The use of high concentrations ofBHA and BHT in rats was associated with cancer develop-ment. Therefore, although they unequivocal advantages,they have been subject of controversy (Saad et al., 2007).

Limits are established according to the toxicity of sub-stances. The Acceptable Daily Intake (ADI) recommendedby the Joint FAO/WHO Expert Committee on Food Addi-tives (JECFA, 2003) is 0e1.4mg of additive/kg bodyweight for Octyl gallate, Dodecyl gallate, BHA, BHT andTBHQ, while the Scientific Committee for Food (SCF) es-tablished a 0.5mg/kg value. Erythorbic acid is allocateda numerical ADI of 6mg/kg bw by the SCF. In the particu-lar case of BHT, the ADI was established at 0e0.3mg/kgbody weight but did not include BHT in the drinking watercontaminants list (JECFA, 2003).

In 2003, SCF set a tolerable upper intake of vitamin E,as a-tocopherol, for adults of 300mg of a-tocopherol equiv-alents per day while JECFA established an ADI of 0.15e2mg/kg of body weight per day calculated as a-tocopherol(JECFA, 2003).

Table 1 is summarizes chemical and physical informa-tion of 10 antioxidants and Table 2 resumes the methodsused to determine antioxidants in foodstuffs (according tothe type of food), packaging and pharmaceutical or cos-metic products.

Sample preparationThe present review has focused the analysis of antioxi-

dants in foodstuffs. However, a few papers that have beenfound also extract antioxidants from food packaging

http://www.fao.comhttp://www.Sigma Aldrich.com

Table 2. Methods used to determine antioxidants in foodstuffs (according to the type of food), packaging and pharmaceutical or cosmeticproducts.

Type of food Antioxidants Analytical methods References

Fats, oils and theirproducts

BHA, BHT, PG, OG, DG HPLCePDA Sin et al., 2006GCeFIDTLC

BHA, BHT, TBHQ, PG, OG RPeHPLCeUVevis Perrin & Meyer, 2002BHA, BHT, PG, OG, DG RPeHPLCeUVeVis Aparicio et al., 2000BHA, BHT, TBHQ, PG RPeHPLCeUV Saad et al., 2007BHA, BHT, TBHQ HPLCeUV Maziero et al., 2001Tocopherols LCeMSeAPCI Lanina et al., 2007

LCeMSeESIBHA, BHT, TBHQ, PG LCeMS & GCeMS Tsuji, Nakanoi, Terada, Tamura, &

Tonogai, 2005BHA, BHT, TBHQ GCeFID Yang et al., 2002BHA, BHT, TBHQ GCeFID Suh et al., 2005TBHQ, BHA, BHT, PG MECCeED Guan et al., 2006BHA, BHT, DG MECC-UV Delgado-Zamarreño, González-Maza,

Sánchez-Pérez, & Martı́nez, 2007BHA, BHT, TBHQ VT Raymundo, Paula, Franco, & Fett, 2007Tocopherols HPLCeFl Gliszczyńska-Świg1o, & Sikorska, 2004

Cereals and cerealsproducts (e.g.Bakery products)

Tocopherols NPeHPLCeUVevis Devi, Jayalekshmy, & Arumughan, 2007BHA, BHT, PG, OG, DG LCeUV Rafecas et al., 1998

Vegetables andvegetablesproducts

Total AA HPLCeUV & MECC Thompson & Trenerry, 1995AA Enzymatic/Spectrophotometric,

RPeHPLCeFluRaghu, Platel, & Srinivasan, 2007

Tocopherol HPLCeDAD & HPLCeFlu Gómez-Coronado, Ibañez, Rupérez, &Barbas, 2004

TBHQ, BHA, BHT, PG MECCeED Guan et al., 2006LeAA RPeHPLCeFlu Burini, 2007

Fruit and fruitproducts

AA RPeHPLCeFlu Raghu et al., 2007Total AA HPLCeUV & MECC Thompson & Trenerry, 1995LeAA HPLCePDA & HPLCeDADeESIeMS Shui & Leong, 2004AA & EA LCeHILICeUV Tai & Gohda, 2007AA & EA CEeUVeVIS Versari, Mattioli, Parpinello, & Galassi,

2004AA CE e ED Wu, Guan, & Ye, 2007LeAA RPeHPLCeFlu Burini, 2007

Milk and milkproducts (e.g.cheese and cheeseproducts)

Total AA RPeHPLCeFlu Burini, 2007Tocopherol RPeHPLCeUV & RPeHPLCeMSeAPCI

& RPeHPLCeMSeESILanina et al., 2007

Tocopherol NPeHPLCeFl Rodrigo, Alegrı́a, Barberá, & Farré, 2002BHA, TBHQ, PG, OG, DG HPLCeDAD Noguera-Ortı́ et al., 2000BHA, BHT, TBHQ, PG RPeHPLCeUV Saad et al., 2007BHA, BHT, TBHQ GCeFID Yang et al., 2002

Meat and meatproducts

BHA, BHT, TBHQ, PG, DG, OG HPLCeUVePAD Pinho, Ferreira, Oliveira, & Ferreira,2000

Fish, fish productsand sea food

Tocopherol HPLCeDAD López-Cervantes et al., 2006TBHQ, BHA, BHT, PG MECCeED Guan et al., 2006AA HPLCeUV and HPLCeFl Quirós, Lopez-Hernandez, & Simal-

Lozano, 2001

Mixed foods BHA, BHT, TBHQ, PG LCeMS & GCeMS Tsuji et al., 2005PG, OG, DG CE-Microchip MEC Amperometric

determinationDing et al., 2006

Spice and otheringredients

BHA, BHT, TBHQ, PG LCeMSeGCeMS Tsuji et al., 2005

Dry foods (e.g.Sugar, honey andproducts thereof)

BHA, BHT, TBHQ, PG, OG RPeHPLCePDA Perrin & Meyer, 2002BHA, BHT, TBHQ, PG LCeMSeGCeMS Tsuji et al., 2005

Table 2 (continued )

Food for specialnutrition use

Total AA RPeHPLCeUV Fontannaz, Kilinç, & Heudi, 2006Tocopherol NPeHPLCeFlu Rodrigo et al., 2002BHA, TBHQ, PG, OG, DG MLCeDAD Noguera-Ortı́ et al., 2000AA & EA LCeHILICeUV Tai & Gohda, 2007

Other foods BHA, BHT, TBHQ GCeFID Suh et al., 2005BHA, PG (fatty foods) Flow injection solid-phase spectrometry Capitán-Vallvey, Valencia, & Nicolás,

2003

Food simulants BHA, BHT HPLCePDA Dopico-Garcı́a, López-Vilariño, &González-Rodrı́guez, 2005

BHA, BHT RPeHPLCeDAD Dopico-Garcı́a et al., 2003BHA, BHT RPeHPLCeDAD Dopico-Garcı́a, Vilariño, & Gonzalez-

Rodriguez, 2007

Plastic products a-Tocopherol HPLCeFlu & DRIFT-IR Graciano-Verdugo et al., 2006BHT GCeMS Hill et al., 2003

Beverages AA & EA HPLCeUV Tai & Gohda, 2007BHT GCeMS Tombesi & Freije, 2002AA CE conventional microchip Law, Kubán, Zhao, Li, & Hauser, 2005

Chewing gum BHT, BHA, PG, TBHQ HPLCeDAD Ruiz, Garcı́a-Moreno, Barbas, &Pingarrón, 1999

Pharmaceutical&/or Cosmeticproducts

AA HPLCeUVevis Maia et al., 2007AA Iodometric titration/NIR Blanco & Alcalá, 2006Tocopherols RPeHPTLC Pyka & Sliwiok, 2001

NPeHPLCeUVRPeHPLCeUVGC


(Graciano-Verdugo, Peralta, González-Rı́os, & Soto-Val-dez, 2006; Hill, Shaw, & Wu, 2003) or pharmaceutical&/or cosmetic products (Blanco & Alcalá, 2006; Maiaet al., 2007; Pyka & Sliwiok, 2001).

Most of the methods found in the literature, to determineAO in foodstuffs, were tested in fats, oils and their prod-ucts, as it can be seen in Table 4. HPLC with UV wasthe most used technique (Aparicio, Andrés, & Vera,2000; Maziero, Baunwart, & Toledo, 2001; Perrin &Meyer, 2002; Sin, Wong, Mak, Sze, & Yao, 2006; Saadet al., 2007).

The quantification of antioxidants in foodstuffs presentsseveral difficulties in what regards to incomplete extractionof antioxidants and co-extraction with interfering sub-stances (Perrin & Meyer, 2002).

Table 3 resumes some of the extraction techniques foundin the literature to determine antioxidants from foodstuffs.The literature reports several extraction solvents due tothe wide range of polarity of antioxidants. Some studies in-clude comparison of several solvents for the extraction ofantioxidants (Perrin & Meyer, 2002) and it has concludedthat methanol was the most suitable for the antioxidantsextraction.

The most common method to extract these antioxidantsis Solvent extraction (SE), which can be either LiquideLiq-uid Extraction (LLE) or liquidesolid extraction (LSE). Insome matrices are also described Solid-Phase Extraction(SPE), SolideLiquid Extraction, SolideGas Extraction

and Solid-Phase Micro Extraction (SPME) (Tombesi &Freije, 2002). SolideLiquid Extraction and SolideGasExtraction are described in order to analyse a-tocopheroland BHT in plastics (Graciano-Verdugo et al., 2006; Hillet al., 2003).

Direct sample injection in a chromatograph is not a com-mon practise, although it has been used (Noguera-Ortı́,Villanueva-Camañas, & Ramis-Ramos, 2000).

Solid-Phase Extraction is more rapid and environmen-tally friendly (due to the solvents save) than LLE.

A lot of different solvents were used to extract BHA,BHT, TBHQ, PG, OG and DG from different matrices.For instance, to extract BHA, BHT, PG, OG and DG inoil samples Sin et al. (2006) used n-hexane, AcN anda methanolic ascorbic acid solution while Aparicio et al.(2000) used only hexane and AcN for BHT and petroleumether and ethanol for the others. The gallates were ex-tracted with methanol and AcN with ammonium acetatesolution from food samples by Ding, Mora, and Garcia(2006).

Regarding the analysis of vitamin C, the official methodinvolves the titration of vitamin C (AA and iso AA) bya mild oxidizing agent (2,6-dichlorophenol-indophenol)(AOAC, 1995).

For the analysis of a-tocopherol, saponification is oftenrequired to obtain full extraction of lipids (Lanina et al.,2007; López-Cervantes, Sánchez-Machado, & Rios-Vaz-quez, 2006).

Table 3. Extraction techniques used to determine antioxidants.

Extraction technique Analyte determineda Sample Solvent used in the extractionprocedure

References

LiquideLiquid Phenolicantioxidants

BHA, BHT, PG, OG, DG Oil n-Hex, ACN Sin et al., 2006BHA, BHT, PG, OG, DG Oil sample For BHT: Hex, ACN Aparicio et al., 2000

For others: petroleum ether,EtOH (72%, v/v)

PG, TBHQ, OG, DG, BHA, BHT Oils and fats MeOH IUPAC, 1992AOAC, 1995AOCS, 1997

BHA, BHT, PG, OG, DG Bakery products ACN-isopropanol (1:1, v/v) Rafecas et al., 1998BHA, BHT, TBHQ Cooking oil, fish oil, butter, margarine,

cheese, mayonnaise, salad dressingDiethyl ether Yang et al., 2002

BHA, BHT, TBHQ Mayonnaise MeOH Raymundo et al., 2007BHA BHT TBHQ PG Vegetable oil, butter, margarine, cheese MeOH/ACN (1:1, v/v) Saad et al., 2007BHA BHT TBHQ PG OG Gravies, dehydrated soups, bouillons,

dehydrated meat, dry pet foodMeOH; Hex/2-propanol Perrin & Meyer, 2002

BHA BHT TBHQ PG DG OG Liver pâtés n-Hex saturated with ACN,ACN saturated with n-Hex

Pinho et al., 2000

BHA BHT LDPE n-Hex Dopico-Garcı́a et al., 2003BHA BHT DG Sunflower oil, olive oil MeOH, ACN Delgado-Zamarreño et al., 2007PG OG DG Food sample (gravy mix) MeOH Ding et al., 2006

Vitamin C Total AA Fortified food products TCEP$HCl, trichloroacetic acid.For starch containingsamples, taka-diastase wasalso used.

Fontannaz et al., 2006

AA & EA (1) Tea drinks, (2) Nutritionalsupplementfood, (3)dried fruit

For (1) and (2): ACNe66.7mMammonium acetatesolution (85:15, v/v)

Tai & Gohda, 2007

For (3): acetonitrileewater(30:70, v/v)

AA & EA Apricot Juices Dilution 1:1, v/v with 10%Metaphosphoric acid

Versari et al., 2004

Total AA Fruit vegetables 3% Metaphosphoric acid, Thompson & Trenerry, 1995AA Star fruit 50% aqueous acetone Shui & Leong, 2004AA Emblica officinalis, guava fruits,

chilli fruitsand amaranth leaves

2% m-phosphoric acid Raghu et al., 2007

AA Grapefruit EtOH 99.7% Wu et al., 2007Vitamin E a-tocopherol Shrimp waste n-Hex López-Cervantes et al., 2006

a-tocopherol Olive, grapeseed, corn rapeseed,peanut,sunflower, soybean oils

2-propanol Gliszczyńska-Świg1o, & Sikorska,2004

tocopherol (1) Sunflower oil, (2) Milk For (1):Saponification with KOHin ethanol. Extraction with Hex

Lanina et al., 2007

For (2): Saponification withaqueous KOH. Extraction with Hex

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ew.


Analytical methods to determine antioxidantsA large variety of methods were found in the literature.

Antioxidants analysis of food and plastic samples aremainly performed by chromatographic methods as it canbe confirmed by Table 4 which resumes the separation tech-niques and conditions used for the determination of theantioxidants.

Method performanceAntioxidants have a wide polarity range. Therefore,

when analysing them simultaneously a low recovery canbe achieved for some of these compounds (Rafecas, Guar-diola, Illera, Codony, & Boatella, 1998). As shown in Table5 the difference methods have different behaviour concern-ing the property and kind of food. These antioxidants aremost use in fats, oils and their products.

TBHQ was determined in many samples but only foundin fats and fish and fish products. All recoveries are good,being in the range 88.7e108.3%. There are no limits ofquantification in the references and the higher limit of de-tection was achieved in fats, oils and their products andin other foods.

BHA was found in more foods than TBHQ and also inplastics. In vegetables, fish, milk, food for special nutri-tional use and other foods BHA was not found. The limitsof detection found were lower than TBHQ. The worst re-covery was in oil and butter (81.80%).

BHT is very similar to BHA but it was also found in bev-erages. The recoveries are not so good, the lowest wasfound in cereals (57.8%).

The most used gallate is PG. It was determined in fats,oils, vegetables and mixed foods. OG and DG were notfound in any kind of foods. The limit of detection (LD) isvery similar being the higher in other foods. The worst re-covery found was for DG in cereals and cereals productsfrom 94.5 to 95.8%.

From the selected food, tocopherol was not found in ce-reals and cereals products and presents the worst recoveryin this kind of food. The LD was higher in the plasticproducts.

Total vitamin C, was just checked in food for special nu-tritional use. The recoveries were around 100%.

Ascorbic acid is used in different kind of food and alsoin cosmetic and pharmaceutical products. The only valuenot found was for food for special nutritional use. Thehigher limit of detection determined was for beverages. Re-coveries were tested with 2 different solvents which presentsignificant different values.

Erythorbic acid was just found in beverages with a limitof detection lower than the one found in fruit. Recoverywas tested with 2 different methods of extraction, givinga good recovery for the first method and worst for thesecond.

For sodium D-isoascorbate there are no values in thereferences.

Table 4. Separation techniques and conditions used for the determination of selected antioxidants.

Technique Detector Matrix Analytes Conditions References

HPLC DAD (l¼280nm) Oil BHA, BHT, PG,OG, DG

Column: C18 Hypersil ODS (150mm�4.6mm, 5mm) Sin et al., 2006Mobile phase solvents: A e acetic acid 5%;B e methanol/AcN 1:1 (Gradient elution)

DAD (l¼280nm;220e320nm)

Dry food PG, OG BHA,BHT, TBHQ

Column: Supelcosil LC-18 30�C (150mm�4.6mm, 5mm) Perrin & Meyer,2002Mobile phase solvents: A e Water pH3.0 acidified with

phosphoric acid 1% (v/v)B e MeOH-AcN 1:1 (v/v) (gradient elution)

DAD (l¼276nm) Aqueous foodsimulants

BHA, BHT Column: Nova Pack C18 (150mm�3.9mm, 4mm) Dopico-Garcı́aet al., 2005Mobile phase solvents: MeOH/Water

DAD Aqueous foodsimulants

BHA, BHT Column: Symmetry (150mm�3.0mm, 3.5mm) Dopico-Garcı́aet al., 2007Mobile phase solvents: MeOH/Water (gradient elution)

DAD Fatty foodsimulants

BHA, BHT Column: Symmetry (150mm�3.0mm, 3.5mm) Dopico-Garcı́aet al., 2007Mobile phase solvents: MeOH/Acn/THF/Water (gradient

elution)DAD (l¼280nm)HPLCeDADeESIeMS-ESIeMS 250�C4.5kV

Star fruit AA Column: Shim-Pack VPeODS (250mm�4.6mm) Shui & Leong,2004Column temperature: 27�C

Mobile phase solvents: A e 0.1% formic acid in water;B e MeOH

DAD (l¼208nm) Shrimp waste a-Tocopherol Column: SS Exil ODS (250m�4.6mm, 5mm) López-Cervanteset al., 2006Column temperature: 36�C

Mobile phase solvents: MeOH:ACN:water (68:28:4, v/v/v)DAD (l¼276nm) Food simulant BHA, BHT Column: Nova-Pak C18 (150mm�3.9mm, 4mm) Dopico-Garcı́a

et al., 2003Column temperature: 30�CMobile phase solvents: A e MeOH; B e Water (gradientelution)

DAD (l¼295nm)Fl (lex¼295nm,lem¼350nm)

Fresh leaves Tocopherol Column: Discovery C18 (250mm�4.6mm, 5mm) Gómez-Coronadoet al., 2004Column temperature: 35�C

Mobile phase solvents: A e Purified-for-HPLC water; B eAcNemethanol (70:30, v/v) with 0.1% (v/v) of acetic acidadded (gradient elution)

UV DAD(l¼280nm)

Liver pâtés TBHQ, BHA, BHT,PG, DG, OG

Column: Spherisorb C18 (250mm�4.6mm, 5mm) Pinho et al., 2000Mobile phase solvents: A e 5% acetic acid in water; B eAcN with 5% acetic acid

UV (l¼254nm) Fruits andvegetables

AA, EA Column: C18 NOVAPAK Radial-PAK (100mm�80mm,4mm) Thompson &Trenerry, 1995Mobile phase solvent: 0.2% (v/v) aqueous orthophosphoric

acidUV (l¼280nm) Vegetable oil,

hydrogenatedvegetable fat,margarine,vegetable cream,halvarina

TBHQ, BHA, BHT Column: C18 (ODS) Vydac 201-TP 54 (250mm�4.6mm,5mm)

Maziero et al.,2001

Mobile phase solvents: AcN: methanol:5% acetic acidTBHQ and BHA: (25:25:5 0 v/v/v)BHT:(42.5:42. 5:15 v/v/v)

UV (l¼280nm) Vegetable oil,butter, cheesemargarine,

PG, TBHQ, BHA,BHT

Column: LiChrospher RP (250mm�4.0mm, 5mm) Saad et al., 2007Mobile phase solvents: A e AcN; B e water with 1% aceticacid (gradient elution)

UVeVis(l¼290nm)

Oil sample BHA, BHT, PG,OG, DG

Column: Lichrosorb RP-18 (150mm�3.9mm, 10mm) Aparicio et al.,2000Mobile phase solvents: SDS/H3PO4/n-propanol 20e50% v/v

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CTAB/H3PO4/n-propanol 20e50% v/vUV (l¼265nm) Fortified food

productsTotal vitamin C Column: LiChrospher RP-C18 (250mm x4.6mm, 5mm) Fontannaz et al.,

2006Mobile phase solvents: decylamine, AcN, sodium acetatesolution; distilled water (pH¼5.4 phosphoric acid 85%);TCEP.HCl (isocratic elu n)

UV (l¼280nm) Bakery products PG, OG, DG, BHT,BHA

Column: C18 Extrasil O S2 (250mm�4.6mm, 5mm) Rafecas et al.,1998Mobile phase solvents: e acetic acid-MeOH (5:95,v/v); B

e acetic acid-water (5 v/v) (gradient elution)UV (l¼260nm) Tea drinks, dried

fruit, nutritionalsupplement

AA, EA Column: GL Sciences 0mm x4.6mm, 5mm) Tai & Gohda, 2007Mobile phase solvents: cN:water with 66.7mMammonium acetate (85 ,v/v)

UVeVis (l¼�297nm)

Deffated rice bran Tocopherol Column: Shim-pack (2 m�4.6mm) Devi et al., 2007Mobile phase solvents: Hex/isopropanol (96:4); v/v

NPeUV(l¼290nm)

Mix of tocopherolsin ethanol

Tocopherol Column: Hypersil Silic 250mm�4.6mm, 5mm) Pyka & Sliwiok,2001Mobile phase solvents: ex:amyl alcohol (99.5:0.5, v/v)

RPeHPTLCeUV(l¼254nm)

Mix of tocopherolsin chloroform

Tocopherol Column: RPeHPTLC (1 m �10cm) Pyka & Sliwiok,2001Solvents: EtOH:water v me proportions 10:0; 9.5:0.5; 9:1

RPeUV(l¼290nm)

Mix of tocopherolsin ethanol

Tocopherol Column: RP C18 (150m �4mm, 5mm) Pyka & Sliwiok,2001Mobile phase solvents: eOH-water (9:1, v/v)

UV (l¼292nm)MS-APCI(þ)(�)MS-ESI(þ)(�)

Sunflower oil milk Tocopherol Column: Fluophase PF 200mm�4.6mm, 5mm) Lanina et al., 2007Column temperature: 2 CMobile phase solvents: eOH:water 95:5,v/v or AcN:water78:22, v/v

UVevis(l¼254nm)

Semisolidpharmaceutical/cosmeticormulations

AA Column: Phenomenex hrospher 100-RP18(250mm�4.6mm, 5mm

Maia et al., 2007

Mobile phase solvents: 2% metaphosphoric acid/MeOH/AcN (90:8:2, v/v/v)

UV (l¼245nm) Sea urchin AA Column: Kromasil 100 50mm�4mm, 5mm), Teknokroma Quirós et al., 2001Mobile phase solvents: illi-Q Water adjusted to pH 2.2with metaphosphoric a

Fl (lex¼350nm,lem¼430nm)

Sea urchin AA Column: Kromasil 100 50mm�4mm, 5mm), Teknokroma Quirós et al., 2001Mobile phase solvents ethanol/0.1% metaphosphoricacid (7:1)


Infant formulas tocopherol ColumnNova-Pak: (150 m�3.9mm, 5mm) Rodrigo et al.,2002Column temperature: r m temp

Mobile phase solvents ex-ethyl acetate (98:2, v/v)Fl (lex¼290nm,lem¼330nm)DRIFT-IR

Plastic LDPE a-tocopherol Column: Omnispher C column (150mm�4.6mm, 5mm);Microsorb

Graciano-Verdugoet al., 2006

Mobile phase solvents eOH:water (98:2) (isocraticelution)


Olive, peanut,grapeseed,rapeseed,sunflower,corn,soybean oils

tocopherols Column: Symmetry C1 50mm�3.9mm, 5mm) Gliszczyńska-Świg1o, & Sikorska,2004

Mobile phase solvents: cN/MeOH (1:1) (elution gradient)

Fl (lex¼355nm,lem¼350nm)425nm emission

Various food, milk AA Column: Nova-Pak C18 50mm�3.9mm, 4mm) Burini, 2007Mobile phase solvents: osphate pH¼7.8containing16%MeOH v) (isocratic elution)

Fl (lex¼350nm, Emblica fruits, AA Column: SS Excil C18 0mm�4.6mm, 10mm) Raghu et al., 2007(continued on next page)

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tioD

A:95(25A:15

50mn-

a (H0columM

P (3�

M

LiC)0.

(2Mcid(2

: M

moo: H18

: M

8 (1A

(1ph(v/(25

Table 4 (continued).

Technique Detector Matrix Analytes Conditions References

lem¼430nm) guava, chilli fruits,amaranth leaves

Mobile phase solvents: Me :water (55:45) (isocraticelution)

Amperometricdetection

Chewing gum AA Nickel phthalocyanine pol er-coated glassy carbonelectrodes

Ruiz et al., 1999

GC MS m/z 35e500V¼70eV

Drinking water BHT Column: HP5 column (30m 0.25mm) Tombesi & Freije,2002Oven ramp: 40�C 5min; to 50�C 20�C/min 5min

MS m/z 10e55070eV

PVC tubes BHT Column: Supelcowax 10(3 �0.25mm, thickeness0.25mm)

Hill et al., 2003

Injector temperature: 250�

Oven ramp: 35�C held 2m increased at 15�C/min to250�C

FID Cooking oil, fishoil, butter,margarine, cheese,mayonnaise, saladdressing

BHA, BHT, TBHQ Column: ChromPack CP-Si CB (30m�0.53mm, 1.5mm) Yang et al., 2002Injector temperature: 250� Detector temperature: 290�COven ramp:150�C held 1m , increased to 200�C at 7�C/min, increased to 300�C a 0�C/min, held 5min at 300�CCarrier gas: H2 (4ml/min)

FID Soybean oil, cornoil, other vegetableoil, shortening,margarine,seasoned driedfish, dried fish,salted fisheryproduct and frozenfishery product(combined),chewing gum,mayonnaise andbreakfast cereal

BHA, BHT, TBHQ Suh et al., 2005

FID Oil BHA, BHT, PG,OG, DG

Column: AT-5 (30m�0.53m ) Sin et al., 2006Injector temperature: and d ector temperature: 250�CColumn oven: 125�C held min; increased to 250�C at20�C/minCarrier gas. H2 30mL/min 300mL/min

Electroforesis CEElectrochemicaldetection

Grapefruit AA Column: Fused-silica capil y (75cm�25mm i.d. and360mm o.d.)

Wu et al., 2007

Electrochemical cell (WE: 0mm diameter carbon disk þplatimium electrode þ SCE n combination with BAS LC-4C AD

CE Capillary zonemode (CZE) UVeVis

Fruit juice AA and EA Uncoated fused-silica capi ry (total length¼50cm;effective length to the dete r¼42.4cm; i.d.¼75mm)conditioned with 0.1M Na (3min); CE-grade water(5min) rinsed with running ffer 50mM tricine at pH 8.8(5min) sample injection ca ed out at the anode for 5s.T¼30�C; V¼11kV across th capillary applied for 15min.

Versari et al., 2004

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OH

ym

�2

0m

Cin,

l 8C;in

t 4

met20

Air

lar

30) i

llactoOHbu

rrie

MECC l¼254nm Fruit andvegetables

AA and EA Fused-silica capillary columns (65cm�7.5mm i.d., effectivelength to the detector¼40cm for fruit; 75cm�75mm id.effective length to the detector¼50cm for vegetables)

Thompson &Trenerry, 1995

Buffer 0.05M sodium deoxycholate, 0.01M sodium borateand 0.01M potassium dihydrogen orthophosphate.V¼25kV; T¼28�C; Buffer for 2min between runs.

MECC-UV l¼200,214, 254, 280nm

Edible oils BHA, BHT, DG Column: Fused-silica capillary (57cm�75mm i.d.) Delgado-Zamarreño et al.,2007

Separation solution: 20% AcN, 20mM boric-borate buffer(pH¼9.2) and 20mM bis-(2-ethylhexyl))sodiumsulfosuccinate surfactant

MECCelectrochemicaldetection

Stock solutions BHA, BHT, PG,TBHQ

Column: fused-silica capillary (75cm�25mm i.d. and360mm o.d.)

Guan et al., 2006

Injector: V¼18kV for 6s; T¼25�CElectrochemical cell (WE: 300mm diameter carbondiscþplatinium electrodeþSCE in combination with BASLC-4C AD

Microchip MEC Food sample PG, OG, DG Detection electrode: 25mm diameter, 99.95% gold wire Ding et al., 2006T¼22�2�C

TLC Edible oil BHA, BHT, PG,OG, DG

TLC plate: silica gel 60, 20�20cm2, Merck Sin et al., 2006Mobile phases: Petroleum ether(40:60):toluene:aceticacid:AcN (4:4:1:0.4 by volume)Spraying 0.5% Gibb’s reagent; dried at 105�C for 15minExposed to ammonia vapour

NIR Pharmaceuticalproducts

AA 1100e2498nm, 32 scans/spectrum Blanco & Alcalá,2006

DRIFT-IR Plastic LDPE A-Tocopherol 4000e400cm�1 Graciano-Verdugoet al., 2006

Voltametrictechniques

Mayonnaise BHA, TBHQ, BHT WE: glassy carbon disk (GC, 0.950cm2) and Pt disk (Pt,0.502cm2). Reference: Hg/Hg2Cl2/KCl (sat.) electrodes andhome made plate auxiliary electrode

Raymundo et al.,2007

Cyclic voltammetry: yb¼50mVs�1Differential pulse voltammetry: DE¼25mV; yb¼50mVs�1Square wave voltammetry: DE¼25mV; f¼60Hz

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Table 5. Method performance of analytical methods for determination of antioxidants.

Compound Food group Range LOD Recovery References

TBHQ Meat and meatproducts

n.d. e 93.6% at 30.3mg/mLPinho et al., 2000

Fish and fishproducts

7.35�103 mg/kg 0.80 � 10�6mol/l 95.40% Guan et al., 2006

Cheese and cheeseproducts

n.d. 0.1mg/ml 97.3e108.4% Yang et al., 2002n.d. 0.5mg/l Cheese 85.3e93.1%Saad et al., 2007

Milk and cheeseproducts

e 0.12 ng 98e105% Noguera-Ortı́ et al.,2000


n.d.e 20.2 mg/kg 0.5mg/l Oil: 103.9e104.0%Bread Spread: 95.0e108.3%

Saad et al., 2007

n.d.e70.3mg/kg 0.1mg/ml 97.3e108.4% Yang et al., 2002n.d.e 143 mg/kg 3.0mg/kg 103e108% Maziero et al., 2001e 5mg/kg 95.40% Suh et al., 200556.7e73.8 mg/kg e e Raymundo et al., 2007n.d. 0.80 � 10�6mol/l 95.80% Guan et al., 2006

Other foods(mushroom cream)

n.d. 0.80 � 10�6mol/l 99.70% Guan et al., 2006

Several foods(shortening,seasoned dried fish,dried fish, saltedfishery product andfrozen fisheryproduct, chewinggum, mayonnaise,breakfast cereal, etc)

e 5mg/kg 88.70% Suh et al., 2005e 2mg/kg e Perrin & Meyer, 2002

Food for specialnutritional use

e 0.12 ng 94.8e100.8% Noguera-Ortı́ et al.,2000

BHA Meat and meatproducts

92.4 mg/kg e 97.80% Pinho et al., 2000


n.d. 1.0�10�6mol/l 102.30% Guan et al., 2006


n.d. 0.5mg/l 97.4e101.2% Saad et al., 20075.4e12.4 mg/kg 0.1mg/ml Yang et al., 2002


e 0.3 ng 92e102% Noguera-Ortı́ et al.,2000

Bakery (doughnuts,biscuits, varioustypes of cakes)

Sample:3.9e10.7 mg/kgFat fraction: 17.5e55.6 mg/kg

e 94.5e95.8% Rafecas et al., 1998


n.d. 1.0�10�6mol/l 97.40% Guan et al., 2006


n.d.e103.9 mg/kg 0.5mg/l Oil-96.7e97.6%Bread spread-97.5e97.7%

Saad et al., 2007

e 0.41-0.67pg 96.2e105.7% Aparicio et al., 2000n.d.-135.4 mg/kg 0.1mg/ml 101.2e103.8% Yang et al., 2002n.d. 0.45ppm Found 0.75g/Kg

adding 0.8g/Kg(MeOH)0.79g/kg adding0.8g/Kg (sunfloweroil)0.70g/Kg adding0.8g/Kg

Delgado-Zamarreño etal., 2007

21.3e58.1mg/kg e e Raymundo et al., 2007164mg/kg 2e4mg/g 97.60% Sin et al., 2006e 3mg/kg 81.80% Suh et al., 2005n.d. 0.6mg/kg 87e98% Maziero et al., 2001n.d. 1.0�10�6mol/l 96.50% Guan et al., 2006e 0.6mg/kg e Maziero et al., 2001


e 0.3 ng 93.4e99.7% Noguera-Ortı́ et al.,2000

240 C. André et al. / Trends in Food Science & Technology 21 (2010) 229e246

(continued on next page)

Table 5 (continued )

Several foods(shortening,seasoned dried fish,dried fish, saltedfishery product andfrozen fisheryproduct, chewinggum, mayonnaise,breakfast cereal, etc)

e 3mg/kg 89.20% Suh et al., 2005

Dry foods e 95e104% Perrin & Meyer, 2002Chewing gum n.d. 5.5ng e Ruiz et al., 1999Food simulant e 0.052mgL�1 104% Dopico-Garcı́a et al.,

20054.2�10�3mgdm�2(aqueous simulant)

Dopico-Garcı́a et al.,2005

4.4�10�3mgdm�2(simulant D-olive oil)

Plastic products 16�10�3e30�10�3g/l 6.5mgl�1 88% Dopico-Garcı́a et al.,2003

BHT Meat and meatproducts

e e 99.90% Pinho et al., 2000


n.d. 2.7�10�6mol/l 95.90% Guan et al., 2006


56.7e87.1mg/kg 0.1mg/ml Yang et al., 2002n.d. 0.5mgl�1 Cheese e 89.5e

91.4%Saad et al., 2007


n.d. 2.7�10�6mol/l 94.30% Guan et al., 2006

Bakery products Sample: 3.0e17.0mg/kg e 85.7e89.4% Rafecas et al., 1998Fat fraction: 14.9e90.0mg/kg


e 3mg/kg 94.50% Suh et al., 200517.0e319mg/kg 0.1mg/ml 92.5e97.6% Yang et al., 2002n.d. e 154.2mg/kg 0.5mgl�1 Oil e 88.2e94.6% Saad et al., 2007

Bread spread e73.9e80.2%

46.5mg/kg 2e4mg/g/10mg/g 75.80% Sin et al., 2006e 1.42e5.12pg 83.0e85.5% Aparicio et al., 200017.6e26.7mg/kg e e Raymundo et al., 2007n.d. 1.47ppm Adding 0.8g/kg

found 0.064g/kg(MeOH) 0.99g/kg(Sunflower oil)0.64g/kg (olive oil)

Delgado-Zamarreñoet al., 2007

n.d. e 197mg/kg 2.7mg/kg 78e98% Maziero et al., 2001n.d. 2.7�10�6mol/l 92.10% Guan et al., 2006

Other foods e 2mg/kg e Perrin & Meyer 2002e 3mg/kg 92.60% Suh et al., 2005

Beverages 0e38mg/L 4.2ml�1 84%e119% Tombesi & Freije, 2002Chewing gum 51�3mgg�1 30ng 97�4% Ruiz et al., 1999Food simulant e 0.045mgl�1 82% Dopico-Garcı́a et al.,

2005n.d. 5.1�10�3 mgdm�2

(aqueous simulant) 7.7�10�3mgdm�2 (simulant D)

Dopico-Garcı́a et al.,2007

Plastic products 5.1e5.6�10�10mol 0.13nmol e Hill et al., 200316e30mg/l/100mL simulant 5.6mgl�1 70% Dopico-Garcı́a et al.,

2003

PG Meat and meatproducts

n.d. e 78.00% Pinho et al., 2000

Fish and fish n.d. 0.29�10�6mol/l 98.70% Guan et al., 2006


productsFats, oils and theirproducts

n.d. 0.3mgl�1 Oil e 103.5e108.3%

Saad et al., 2007

n.d. Bread spread e103.6e107.3%

e 2e4mg/g Spike 0.01 e 7.6% Sin et al., 2006e 0.14e0.23pg 95.3e100.9% Aparicio et al., 2000103mg/kg 0.29�10�6mol/l 99.10% Guan et al., 2006


n.d. 0.3mgl�1 93.3e99.5% Saad et al., 2007


e 0.05ng 96e103% Noguera-Ortı́ et al.,2000


217mg/kg 0.29�10�6mol/l 101.80% Guan et al., 2006

Bakery products n.d. e 88.9e89.5% Rafecas et al., 1998Food for specialnutritional use

e 0.05ng 100.6e101.3% Noguera-Ortı́ et al.,2000

Mixed foods 45mg/kg 2.2mM 95�6% Ding et al., 2006Chewing gum n.d. 7.5ng e Ruiz et al., 1999Other foods e 85e106% Perrin & Meyer, 2002

OG Meat and meatproducts

n.d. e 82.10% Pinho et al., 2000

Bakery products n.d. e 77.9e79.7% Rafecas et al., 1998Milk and cheeseproducts

e 0.2ng 99e100.1% Noguera-Ortı́, et al.,2000


e 0.31e0.40pg 92.5e105.0% Aparicio et al., 2000

e 2e4mg/g 95.80% Sin et al., 2006Mixed foods e 2.3mM 95�6% Ding et al., 2006Food for specialnutritional use

e 0.2ng 100e103% Noguera-Ortı́ et al.,2000

Other foods e 83e85% Perrin & Meyer, 2002

DG Meat and meatproducts

n.d. e 94.30% Pinho et al., 2000

Bakery products n.d. e 39.8e40.7% Rafecas et al., 1998Fats, oils and theirproducts

e 2e4mg/g 97.60% Sin et al., 2006e 0.30e0.33pg 95.7e105.7% Aparicio et al., 2000n.d. 0.27ppm Adding 0.8g/kg,

found 0.77g/kg(MeOH), 0.75g/kg(sunflower oil,0.82g/kg (olive oil)

Delgado-Zamarreñoet al., 2007

Mixed foods e 4.1mM 95�6% Ding et al., 2006

Tocopherol Fish and fishproducts

351mg/kg 80ng/mL 100.80% López-Cervantes et al.,2006


100.16e203.78mg/kg 28ng/mL (a-tocopherol) Gliszczyńska-Świg1o, &Sikorska, 20048ng/mL (g and

d tocopherol)393mg/kg APCI(-): 2.5e3ng/mL 102e112% Lanina et al., 2007

ESI(-): 5e20ng/mLVegetables andvegetable products

13e1322mg/kg 0.05mg/100g a e 87% Gómez-Coronado et al.,2004g e 57e60%

Cereals and cerealproducts

51e347ppm (alltocopherols)

e 19.9%e47.7%based on crudeextraction

Devi et al., 2007

Milk and milkproducts

0.939mg/kg APCI(�): 3.0ng/mL 104e109% Lanina et al., 2007ESI(�): 20ng/mL


275mg/kg 1.60ng/50mL inj volume e Rodrigo et al., 2002

Plastic product (LPPEfilm)

0.26e1.59% (HPLC)/0.21e1.42% (DRIFT-IR)

0.25mg/mL (HPLC)/1300mg/g (DRIFT-IR)

71.3e86.0% (HPLC)Graciano-Verdugo et al.,2006


Compound Food group Range LOD Recovery References


Fruits 979.17�10�6g/mL(grapefruit juice)/500�10�6g/g grapefruit peel

1�10�6g/mL 95% Wu et al., 2007

Total vitamin C Food for specialnutritional use

6.2e208.5mg/100g 0.1mg/100g 93e105% Fontannaz et al., 2006

Ascorbic acid Beverages 30mg/L 3mg/L (conventional CE) e Law, Kubán et al., 200510mg/L (microship E)

31.9e267.5mg/l 0.3mg/ml AcN/water 30/70using dithiothreitol:92%

Tai & Gohda, 2007

AcN/water 30/70:38%

Wu et al., 2007


500�10�6g/g (grapefruitpeel)/979.17�10�6g/g(grapefruit juice)

1.0�10�6g/mL 95%

45e243mg/l 1.6mg/l e Versari et al., 200458.6e236.2mg/100g(HPLC)

e e Raghu et al., 2007

52.4e213.5 (Enzymatic)71.1e206.8 (indophenol-xylene)55.0e468.8 (2,4-DNPH)2e38mg/100g 0.27mg/mL e Burini, 20072e101mg/100g e e Thompson & Trenerry,

1995n.d. 0.3mg/ml AcN/water 30/70

using dithiothreitol:92%

Tai & Gohda, 2007

AcN/water 30/70:38%

Vegetables andvegetable products

41.1mg/100g (HPLC) e e Raghu et al., 200736.8mg/100g (Enzymatic)60.4mg/100g (indophenol-xylene)62.3mg/100g (2,4-DNPH)2e200mg/100g e e Thompson & Trenerry,

19952.8e7.0mg/100g 0.27mg/mL e Burini, 2007

Milk and milkproducts

1.15e1.44mg/kg 0.27mg/mL e Burini, 2007

Sea Food HPLCeFl: Fresh 26.57mg/100g

HPLCeFl: 0.19mg/ml,0.082mg/ml

98.12% (HPLCeFl) Quirós et al., 2001

Canned 14.25mg/100gPharmaceutical/cosmetic products

1.909e10.154mg/ml 0.05mg/mL�1 95.46e101.54% Maia et al., 20070.17mg/mL�1

2.5% wt. e e Blanco & Alcalá 2006

Erythorbic acid Beverages 23.4mg/ml 0.3mg/ml AcN/water 30/70using dithiothreitol:92%

Tai & Gohda, 2007

Food for specialnutrition use

n.d. AcN/water 30/70:38%


n.d.


e 1.1mg/l e Versari et al., 2004




ConclusionsThe use of antioxidants has been growing in the past few

years due to the advantages offered in the preservation of food.The most common method to analyse these antioxidants

is reversed phase HPLC with UV detection. This UV detec-tion can be a simple UVevis detector or DAD. Althoughtocopherol can be detected by RPeHPLC there aremethods in normal phase. Various solvents can be used,but usually is selected an organic phase, AcN or methanoland an aqueous phase, water acidified or not.

The present legislation is being very strict in the concen-tration limits of antioxidants that can be use in variousfoods. The analytical methods reviewed for this articlepresents limits of detection below the maximum permittedby legislation. In this way the methods used are suitable fordetect and quantify the antioxidants in study.

AcknowledgementsAuthors are grateful to the ‘‘Fundaç~ao para a Ciência e

Tecnologia’’, Portugal, for the Postdoctoral contract ofAna Sanches Silva in the frame of the Program ‘‘Science2007’’ and for the Bilateral Cooperation between Portugaland Italy. The authors are also grateful to ‘‘Instituto Nacio-nal de Saúde Doutor Ricardo Jorge’’ for the PhD scholar-ship of Catarina André (BID 04/2006-I).

Abbreviations

AA: ascorbic acidAcN: acetonitrileAD: amperometric detectionAPCI: atmosphere pressure chemical ionizationCE: capillary electrophoresisDAD: diode array detectorDDTD: dynamic thermal desorptionDG¼LG: dodecyl gallate or lauryl gallateDRIFT: diffuse reflectance Fourier transform infraredEA: erythorbic acid (D-isoascorbic)ED: electrochemical detectionESI: electrospray ionizationEtOH: ethanolFID: flame ionization detectorFlu: fluorescence detectorGC: gas chromatographyHex: hexaneHILIC: hydrophilic interaction chromatographyHPLC: high-performance liquid chromatographyIR: infraredLC: liquid chromatographyMECC: micellar electrokinetic capillary chromatographyMeOH: methanolMLC: micellar liquid chromatographyMS: mass spectrometryNIRS: near infrared spectroscopyNP: normal phase

PDA: photodiode arrayRP: reverse phaseSCE: saturated calomel electrodeSFE: supercritical fluid extractionSPME: solid-phase microextractionTCEP$HCl: tris-(2-carboxyethyl)-phosphine hydro-chlorideTHF: tetrahydrofuranTLC: thin layer chromatographyUVevis: ultraviolet and visible detectorVT: voltametric techniquesWE: working electrodeWt.: weight

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Analytical strategies to evaluate antioxidants in food: a reviewIntroductionChemical and physical information of antioxidantsSample preparationAnalytical methods to determine antioxidantsAnalytical methods to determine antioxidantsMethod performanceConclusionsConclusionsConclusionsAcknowledgementsAbbreviationsReferences

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