Some Analyticalmethods of Food Additives and Related Substances by Chromatographic Tehniquesat

SOME ANALYTICALMETHODS OF FOOD ADDITIVES AND RELATED SUBSTANCES BY CHROMATOGRAPHIC TEHNIQUESAT EUROFINS SAC KY HAI DANG COMPANY

Nguyen Huynh Tuan Anh,DinhThi Ha Long, Do Vu Phuong Thao, VuongQuangHuy, Nguyen Vu Khanh, Pham ThiAnh, Diep Ngoc Suong, Chu Pham Ngoc Son.

1. INTRODUCTION

1.1. Food additives

A food additive is any chemical substance that is added to food during the preparation or storage and either becomes a part of the food or affects its characteristics for the purpose of achieving a particular technical effect.

Substances that are used in food to maintain its nutritive quality, enhance its keeping quality, and make it attractive or to aid in its processing, packaging or storage are all considered to be food additives. However, some substances that aid in the processing of food, under certain conditions, are considered to be food processing aids, not food additives.

Examples of food additives include colouring agents that give foods an appetizing appearance, preservatives that prevent or delay undesirable spoilage in food, and certain sweeteners that are used to sweeten foods without appreciably adding to the caloric value of the foods.

1.2 Categories

The Ministry of Health has issued thedecision No 3742/2001/QD-BYT of August 31/2011, setting the list of food additives allowed in food. Under this decision, Food additivescan be divided into severalgroups(Table 1).

Table 1:Group of food additives

No. / Group / Functions / Some compounds in group
Acidity regulators / Acidity regulatorsare used to change or otherwise control the acidity andalkalinityof foods. / 41 compounds such as:potassium acetate, citric acid, orthophosphoric acid…
Flavor enhancers / Flavor enhancers enhance food's existing flavors. / 8 compounds such as:glutamic acid, monosodium glutamate, guanilic acid, inosinic acid…
Stabilizers / Stabilizers, thickeners and gelling agents, like agar or pectin (used in jam for example) give foods a firmer texture / 13 compounds such as:polivinylpyrrolidone, Calcium carbonate, potassium chloride…
Preservatives / Preservativesprevent or inhibit spoilage of food due tofungi,bacteriaand othermicroorganisms. / 29 compounds such as:sorbic acid, sodium sorbate, potassiumbisulfite…
Anti Caking agents / Anticaking agentskeep powders such as milk powder from caking or sticking. / 14 compounds such as:trimagnesiumorthophosphate, ammonium citrate…
Antioxidants / Antioxidants prevent foods from being oxidized, or going rancid. / 15 compounds such as: ascorbic acid, a lpha tocopherol, TBHQ, BHA….
Antifoaming agents / Antifoaming agentsreduce or prevent foaming in foods. / 4 compounds such as: polypropylene glycol, polyethyleneglycol, polyoxyethylene(20),sorbitan, monooleate,polydimethylsiloxane
Bulking agents / Bulking agents such asstarchare additives that increase the bulk of a food without affecting its taste. / 3 compounds such asmicrocrystallinecellulose, carnauba wax, sodium alginate.
Artificial Sweeteners / Sweetenersare added to foods for flavoring. Sweeteners other thansugarare added to keep thefood energy(calories) low, or because they have beneficial effects fordiabetes mellitus,tooth decayanddiarrhea. / 7 compounds such as: mannitol, acesulfame potassium, saccharin, ….
Modified starches / Modified starches are used practically in all starch applications, such as in food products as athickeningagent,stabilizeroremulsifier / 19 compounds such as: dextrins, roasted starch white and yellow, acid-treated starch
Enzyms / Enzymes are catalysts in food processing / 6 compounds such as: amylase, protease, glucose oxydase
Propellants / Propellants help propelling food from a container. / 2 compounds such as: nitrogen, nitrogen dioxide
Glazing / Glazing improves appearance andable toprotect food. / 6 compounds such as: beeswax, mineral oil (food grade),…
Thickeners / Thickening agents are substances which, when added to the mixture, increase its viscosity without substantially modifying its other properties. / 20 compounds such as:alginic acid, carrageenan,…
Humectants / Humectants keep foods moist. / 2 compoundssuch ass: glycerol, calcium dihydrogendiphosphate
Firming agents / Firming agents are food additives added in order to precipitate residual pectin, thus strengthening the structure of the food and preventing its collapse during processing. / 8 compounds such as: calcium citrate, calcium chloride, calciumsulfate,…
Emulsifiers / Emulsifiersallow water and oils to remain mixed together in anemulsion, as inmayonnaise,ice cream, and homogenizedmilk. / 24 compounds such as: mono-and diglyceridesof fatty acids, lactic and fatty acid esters of glycerol,…
Coloring agents / Coloring agents enhance or add colour. / 24 synthetic coloring agents and 11 natural coloring agents such as: curcumin, tartrazine, quinoline,…
Sequestrants / Sequestrants improve the quality and stability of the food products.
Sequestrants form chelate complexes with polyvalent metal ions. / 14 compounds such as: Isopropylcitrate, calcium disodiumetylendiamin-tetraacetate, sodium polyphosphate, sodium gluconate,…
Raising agents / Raising agents increase the volume of food through the use of gases / 2 compounds such as: ammonium carbonate, sodium carbonate
Flour treatment agents / Flour treatment agents improve baking quality. / 1 compound: azodicarbonamide
Flavouring agents / Flavouring agents improve the taste or flavour of the foods / 60 compouds including natural and synthetic flavouring agents

1.3 National technical regulation on food additive

National technical regulation on food additive follow:

QCVN 4-1/2010: National technical regulation on food additive – Flavour Enhancer

QCVN 4-2/2010: National technical regulation on food additive– Humectants

QCVN 4-3/2010: National technical regulation on food additive– Raising agents

QCVN 4-4/2010: National technical regulation on food additive– Anticaking agents

QCVN 4-5/2010: National technical regulation on food additive– Colour retention agent

QCVN 4-6/2010: National technical regulation on food additive– Antioxidant agents

QCVN 4-7/2010: National technical regulation on food additive– Antifoaming Agent

QCVN 4-8/2010: National technical regulation on food additive– Artificial sweeteners

QCVN 4-9/2010: National technical regulation on food additive– Firming agents

QCVN 4-10/2010 National technical regulation on food additive– Colours

QCVN 4-11/2010: National technical regulation on food additive– Acidity regulators

QCVN 4-12/2010: National technical regulation on food additive– Preservatives

QCVN 4-13/2010: National technical regulation on food additive– Stabilizers

QCVN 4-14/2010: National technical regulation on food additive– Sequestrants

QCVN 4-15/2010: National technical regulation on food additive– Flour treatment agents

QCVN 4-16/2010: National technical regulation on food additive– Bulking agents

QCVN 4-17/2010: National technical regulation on food additive– Propellants

QCVN 4-18/2011: National technical regulation on food additive– Modified starches

QCVN 4-19/2011: National technical regulation on food additive– Enzyme

QCVN 4-20/2011: National technical regulation on food additive– Glazing agent

QCVN 4-21/2011: National technical regulation on food additive– Thickeners

QCVN 4-22/2011: National technical regulation on food additive– Emulsifier

QCVN 4-23/201: National technical regulation on food additive– Foaming agent

1.4 Purpose of food additive analysis

Assess that food additive is in the lists of permitted food additives

Assess food additive quality

Control the abuse of non-permitted food additives.

Quantifyfood additive

There are several advantages and disadvantages in food additive analysis.

1.5 Advantages

Most of analytical methods for food additives and related subtances in food are available.

Equipments for food additive analysis such as: HPLC, IC, GC, AAS, Spectrophotometers….are also available in our company

1.6 Disadvantages

High matrix effect in food additive analysis.

Numerous and complexfood matrices

2. SOME ANALYTICALMETHODS OF FOOD ADDITIVES AND RELATED SUBSTANCES BY CHROMATOGRAPHY TEHNIQUESAT EUROFINS SAC KY HAI DANG COMPANY

2.1.Orange II and 2,4-diaminoazobenzene in chicken, roast pork

2.1.1.Principle

Orange II and 2,4-diaminoazobenzenewereextracted with 1 % acetic acid in acetonitrile. The extracted solution wascleaned by Primary Secondary Amine (PSA) and C18 sorbent, filtered through a 0.45 µm filter and analyzed by HPLC-UV.

2.1.2.Molecular formula

Orange II 2,4-diaminoazobenzene

2.1.3.Equipment

HPLC-UV with C18 column (250mm × 4.6 mm × 5µm).

2.1.4.Sample preparation

Step 1: Weigh 5 g of sample into 50 mL centrifuge tube.

Step 2: Add 15 mL of 1 % acid acetic in acetonitrile solution and vortex for 5 min.

Step 3: Add mixture I of 1.5 g of CH3COONa and 6.5 g of MgSO4, shakenfor 1 minand centrifugeat 5000 rpm for 5 min.

Step 4: Transfer supernatant solution into new centrifuge tube and addmixture II of 0.15 g PSA and 0.15 g C18, shaken for 1 min and centrifuge at 5000 rpm for 5 min.

Step 5: Pipet 5 mL of supernatant solution into 50 mL round bottom flask and then evaporate the solvent

Step 6:Redissolvethe residue in 1 mL of acetonitrile;filter the solution through a 0.45 µm filter before injection into HPLC-UV equipment.

2.1.5.HPLC operating conditions

Wavelength: λOrangeII = 492 nm λ2,4-diaminoazobenzene = 437 nm

Eluent: Acetonitrile : pH 4.8 buffer acetate (20:80)

Flow rate: 1.0 mL/min

Injection volume: 10 µL

2.1.6.Calibration range

The calibration curves represented area versus concentrations. Table 2 showed the corresponding linear equations for orange II and 2,4-diaminoazobenzene with excellentcorrelation coefficients (0.9999 – 1.000)

Table 2: Linear equations representing area versus concentrations of orange II and 2,4-diaminoazobenzene

Compound / Range of concentration / Equation / Equation coefficient
(R2)
Orange II / Between 0.01 and 1 mg/L / y=406763x-271.44 / 0.9999
2,4-diaminoazobenzene / Between 0.01 and 1 mg/L / y=4109696x-1586.8 / 1.000
Orange II / Between 1 and 20 mg/L / y= 413961x-12529 / 1.000
2,4-iaminoazobenzene / Between 1 and 20 mg/L / y =437899x-24054 / 1.000

Figure 1:0.5 mg/L standardsof orange II and 2,4-diaminoazobenzene

2.1.7.Limit of Detection (LOD)

The limits of detection of 2, 4-diaminoazobenzene and orange II in chicken and roast pork were shown in table 3.

Table 3: LOD of orange II and 2, 4-diaminoazobenzen in chicken and roast pork

The matrices / Compound / LOD
(mg/Kg)
Chicken / Orange II / 0.045
2,4-diaminoazobenzene / 0.046
Roast pork / Orange II / 0.042
2,4-diaminoazobenzene / 0.044
0.62 mg/Kg of 2,4-diaminoazobenzene in chicken / 8.35 mg/Kg of orange II in roast pork

Figure 2:Analytical results of orange II and 2,4-diaminoazobenzene

2.2.Cyclamate in fried fish

2.2.1.Principle

Cyclamate was extracted with acetonitrile : water (2:8) solution. The extracted solution was cleaned on C18 SPE, filtered through a 0.45 µm filter and analyzed by ion chromatography (IC).

2.2.2.Molecular formula

Sodium cyclamate

2.2.3.Equipment

Metrohm 850 Professional IC with Metrosep A Supp7 250/4.0

2.2.4.Sample preparation

Step 1: Weigh 0.5 g of sample into 50 mL centrifuge tube.

Step 2: Add 10 mL of acetonitrile: water (2:8) solution, vortex for 1 min, sonicate for 10 min.

Step 3: Centrifuge at 5000 rpm for 5 min.

Step 4: Transfer supernatant solution into 50 mL volumetric flask

Step 5: Repeat step 2, 3, 4 two times and make up to 50 mL with bi-distilled water.

Step 6: Transfer extracted solutioninto a C18 column after being conditioned with 10 mL of acetonitrile), discard thefirst 3mL of solution.

Step 7: Then collect 7 mL of solution and filter through a 0.45 µm filter before injection into IC equipment.

2.2.5.IC operating conditions

Injection Volume: 20 µl

Flow rate: 0.7 mL/min

Detector: conductivity

Suppression: H2SO4 0.1 M

Eluent: Na2CO3 3.6mM

2.2.6.Calibration range

The calibration curves represented area versus concentrations.

Figure 3:Standard curve of cyclamate from concentrations of 0.5 to 50 mg/L

Figure 4: Chromatogram of cyclamate

2.2.7.Limit of Detection (LOD)

The limit of detection of cyclamate in dried fish is 10 mg/Kg.

Figure 5: Chromatogram of dried fish containing cyclamate

2.3.Acesulfame potassium and Saccharin in candy, jam and beverage

2.3.1.Principle

Acesulfame potassium and saccharin were extracted with distilled water, filtered through a 0.45 µm filter and analyzed by HPLC-UV.

2.3.2.Molecular formula

Acesulfame potassium Saccharin

2.3.3.Equipment

HPLC-UV with C18 column (250 mm × 4.6 mm × 5 µm).

2.3.4.Sample preparation

Step 1: Weigh 5 g of sample into 50 mL centrifuge tube.

Step 2: Add 30 mL of distilled water, vortex and sonicatefor 10 min. Add 5 mL of 20 % Zn(CH3COO)2 and 5 mLof Na2HPO4saturated solution

Step 3: Vortex for 5 min, centrifuge for 10 min at 5000 rpm.

Step 4: Transfer the supernatant into 100 mL volumetric flask.

Step 5: Repeatstep 3, 4.

Step 6: Make up to 100 mLwith distilledwater and filter through a 0.45 µm filter into a vial and inject into the HPLC-UV.

2.3.5.HPLC operating conditions

Wavelength: 220 nm

Eluent: ACN: pH 3.4 phosphate buffer (10:90)

Flow rate: 1.0 mL/min

Injection volume: 10 µL

2.3.6.Calibration range

The calibration curves represented area versus concentrations.

Standard curves of Acesulfame potassium
Between 0 and 250 mg/L / Between 0 and 15 mg/L
Equation : y = 11004 x + 13346 / Equation : y = 11168 x + 1049

Figure 6:Standard curves of Acesulfame potassium

Standard curves of saccharin
Between 0 and 250 mg/L / Between 0 and 15 mg/L
Equation : y = 188370 x + 24412 / Equation : y = 19185 x + 729,1

Figure 7: Standard curves of Saccharin

Figure 8:Chromatogram of 10 mg/L of Acesulfame potassium and Saccharin solution

2.3.7.Limit of Detection (LOD)

The limits of detection of acesulfame potassiumand saccharin in candy, jam and beverage were respectively 20 ppm and 10 ppm.

2.4.Sodium benzoate and potassium sorbate in grilled chopped fish and beef meatball

2.4.1.Principle

Sodium benzoate and potassiumsorbatewere extracted with distilled water, filtered through a 0.45 µm filter and analyzed by HPLC-UV.

2.4.2.Molecular formula

Sodium benzoatePotassiumsorbate

2.4.3.Equipment

HPLC-UV with C18 column (150 mm × 4.6 mm × 5 µm).

2.4.4.Sample preparation

Step 1: Weigh 5 g of sample into 50 mL centrifuge tube.

Step 2: Add 1 mL of HCl (1M) and 10 mL of distilled water, vortex and sonicate for 15 min.

Step 3: Add 20 mL of diethylether into sample, vortex for 5 min, centrifuge for 5 min at 5000 rpm.

Step 4: Transfer the supernatant into 100 mL round bottom flask.

Step 5: Repeatstep 3, 4.

Step 6: Evaporate the solvent.

Step 6: Redissolve the residue in 1 mL of acetonitrile; filter through a 0.45 µm filter before inject into HPLC-UV equipment.

2.4.5.HPLC operating conditions

Wavelength:λsodium benzoate= 225 nm λpotassiumsorbate = 250 nm

Eluent: ACN: pH 3.4 formatebuffer (15:85)

Flow rate: 0.8 mL/min

Injection volume: 20 µL

2.4.6.Calibration range

The calibration curves represented area versus concentrations.

Figure 9: Standard curve of sodium benzoate from concentrations of 0.5 to 50 mg/L

Figure 10: Standard curves of potassium sorbate from concentrations of 0.5 to 50 mg/L

Figure 11:Chromatogramof sodium benzoate and potassiumsorbate

2.4.7.Limit of Detection (LOD)

The limit of detection of sodium benzoate and potassium sorbate in grilled chopped fish and beef meatball was 10 mg/Kg

Figure 12:Beef meatball containing 26.3 mg/Kg of sodium benzoate

Figure 13: Grilled chopped fish containing 18.3 mg/Kg of sodium benzoate and 11 mg/kgof potassium sorbate.

1. CONCLUSION

Food additive analysis is very important in assessing food quality.As different methods are today available, choice of appropriate one depends on food matrices and the range of concentrations to be determined.

1. REFERENCES

[1]. TCVN 8471 - Foods – Analysis ofAcesulfame potassium, Aspartame and Saccharin by HPLC (2010).

[2]. TCVN 8472 - Foods – Analysis ofcylamate by HPLC (2010).

[3] AOAC official method 2007.01 - Pesticide residues in food by acetonitrile extraction and partitioning magnesium sulfate, Gas chromatography/Mass spectrometry and Liquid chromatography/Tanden mass spectrometry.

[4] QinL., Xiao-Yan Z., Shu-Kun .H et al - Simultaneous High Performance Liquid Chromatographic Determination of Chrysoidine, Auramine O and Safranine T in Food.Food Science30 (14), 194-196 (2009).

[5] Wang X.,Song G., Wu W., Zhao J., Hu Y - Determination of the Food colorant, Chrysoidine, in Fish by GC/MS, Chromatographia68,659-662 (2008).

[6] Guui W., Xu Y., Shou L., Zhu G., Ren Y - Liquid chromatography- tandem mass spectrometry for the determination of chrysoidine in yellow-fin tuna, Food Chem122, 1230-1234 (2010).

[7]. QCVN 4 to QCVN 12 –National Technical Regulations concerning Food Additives and Preservatives (2010).

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