SOME ANALYTICALMETHODS OF FOOD ADDITIVES AND RELATED
SUBSTANCES BY CHROMATOGRAPHIC TEHNIQUES AT EUROFINS
SAC KY HAI DANG COMPANY
Nguyen Huynh Tuan Anh, DinhThi Ha Long, Do Vu Phuong Thao, Vuong Quang
Huy, Nguyen Vu Khanh, Pham Thi Anh, 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 the decision No 3742/2001/QD-BYT of August
31/2011, setting the list of food additives allowed in food. Under this decision,
Food additives can be divided into several groups(Table 1).
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Table 1:Group of food additives
No.
Group
Functions
Some compounds in group
1.
Acidity regulators
Acidity regulators are used to
change or otherwise control the
acidity and alkalinity of foods.
41 compounds such as:potassium
acetate, citric acid, orthophosphoric
acid…
2.
Flavor enhancers
Flavor enhancers enhance food's
existing flavors.
8 compounds such as:glutamic acid,
monosodium glutamate, guanilic acid,
inosinic acid…
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 prevent or inhibit
spoilage of food due
to fungi, bacteria and
other microorganisms.
29 compounds such as:sorbic acid,
sodium sorbate, potassium bisulfite…
Anticaking agents keep powders
such as milk powder from caking or
sticking.
14 compounds such as:trimagnesium
orthophosphate, ammonium citrate…
Antioxidants prevent foods from
being oxidized, or going rancid.
15 compounds such as: ascorbic acid, a
lpha tocopherol, TBHQ, BHA….
Antifoaming agents reduce or
prevent foaming in foods.
4 compounds such as: polypropylene
glycol, polyethyleneglycol,
polyoxyethylene(20), sorbitan,
monooleate,polydimethylsiloxane
Bulking agents such as starch are
additives that increase the bulk of a
food without affecting its taste.
3 compounds such as microcrystalline
cellulose, carnauba wax, sodium
alginate.
Sweeteners are added to foods for
flavoring. Sweeteners other
than sugar are added to keep
the food energy (calories) low, or
because they have beneficial effects
for diabetes mellitus, tooth
decay and diarrhea.
7 compounds such as: mannitol,
acesulfame potassium, saccharin, ….
Modified starches are used
practically in all starch applications,
such as in food products as
a thickening
agent, stabilizer or emulsifier
19 compounds such as: dextrins,
roasted starch white and yellow, acidtreated starch
Enzymes are catalysts in food
processing
6 compounds such as: amylase,
protease, glucose oxydase
3.
Stabilizers
4.
Preservatives
5.
Anti Caking agents
6.
Antioxidants
7.
8.
9.
10.
11.
Antifoaming agents
Bulking agents
Artificial Sweeteners
Modified starches
Enzyms
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Propellants help propelling food from
a container.
2 compounds such as: nitrogen,
nitrogen dioxide
Glazing
Glazing improves appearance and
able to protect food.
6 compounds such as: beeswax,
mineral oil (food grade),…
14.
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,…
15.
Humectants
Humectants keep foods moist.
2 compoundssuch ass: glycerol, calcium
dihydrogendiphosphate
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, calcium sulfate,…
Emulsifiers allow water and oils to
remain mixed together in
an emulsion, as in mayonnaise, ice
cream, and homogenized milk.
24 compounds such as: mono-and
diglycerides of fatty acids, lactic and
fatty acid esters of glycerol,…
12.
Propellants
13.
16.
Firming agents
17.
Emulsifiers
18.
Coloring agents
Coloring agents enhance or add
colour.
24 synthetic coloring agents and 11
natural coloring agents such as:
curcumin, tartrazine, quinoline,…
19.
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 disodiumetylendiamintetraacetate, sodium polyphosphate,
sodium gluconate,…
20.
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 improve
baking quality.
1 compound: azodicarbonamide
Flavouring agents improve the taste
or flavour of the foods
60 compouds including natural and
synthetic flavouring agents
21.
22.
Flour treatment agents
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
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 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:
sweeteners
National technical regulation on food additive– Artificial
 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.
Quantify food additive
There are several advantages and disadvantages in food additive analysis.
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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 complex food matrices
2. SOME ANALYTICAL METHODS OF FOOD ADDITIVES AND RELATED SUBSTANCES
BY CHROMATOGRAPHY TEHNIQUES AT 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-diaminoazobenzene were extracted with 1 % acetic acid in
acetonitrile. The extracted solution was cleaned by Primary Secondary Amine
(PSA) and C18 sorbent, filtered through a 0.45 µm filter and analyzed by HPLCUV.
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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, shaken for 1
min and centrifuge at 5000 rpm for 5 min.
Step 4: Transfer supernatant solution into new centrifuge tube and add mixture 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:Redissolve the 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
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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
excellent correlation coefficients (0.9999 – 1.000)
Table 2: Linear equations representing area versus concentrations of orange II and 2,4diaminoazobenzene
Equation
Compound
Range of concentration
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
Between 1 and 20 mg/L
y =437899x-24054
1.000
2,4-iaminoazobenzene
Figure 1: 0.5 mg/L standards of 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.
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Table 3: LOD of orange II and 2, 4-diaminoazobenzen in chicken and roast pork
The matrices
Chicken
Roast pork
Compound
(mg/Kg)
Orange II
0.045
2,4-diaminoazobenzene
0.046
Orange II
0.042
2,4-diaminoazobenzene
0.044
0.62 mg/Kg of 2,4-diaminoazobenzene
in chicken
LOD
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
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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 solution into a C18 column after being conditioned
with 10 mL of acetonitrile), discard the first 3 mL 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
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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.
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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.
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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 sonicate for 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: Repeat step 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.
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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
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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 potassium and saccharin in candy, jam and
beverage were respectively 20 ppm and 10 ppm.
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2.4. Sodium benzoate and potassium sorbate in grilled chopped fish and beef
meatball
2.4.1. Principle
Sodium benzoate and potassium sorbate were extracted with distilled water,
filtered through a 0.45 µm filter and analyzed by HPLC-UV.
2.4.2. Molecular formula
Sodium benzoate Potassiumsorbate
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 diethyl ether 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: Repeat step 3, 4.
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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 formate buffer (15:85)
Flow rate: 0.8 mL/min
Injection volume: 20 µL
2.4.6. Calibration range
The calibration curves represented area versus concentrations.
4500000
4000000
y = 81548x + 2385.5
3500000
2
R =1
3000000
2500000
2000000
1500000
1000000
500000
0
0
10
20
30
40
50
60
Figure 9: Standard curve of sodium benzoate from concentrations of 0.5 to 50 mg/L
14000000
y = 233013x + 20520
12000000
2
R =1
10000000
8000000
6000000
4000000
2000000
0
0
10
20
30
40
50
60
Figure 10: Standard curves of potassium sorbate from concentrations of 0.5 to 50 mg/L
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Figure 11:Chromatogram of 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
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Figure 13: Grilled chopped fish containing 18.3 mg/Kg of sodium benzoate and 11 mg/kg of
potassium sorbate.
3.
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.
4.
REFERENCES
[1]. TCVN 8471 - Foods – Analysis of Acesulfame potassium, Aspartame and
Saccharin by HPLC (2010).
[2]. TCVN 8472 - Foods – Analysis of cylamate 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 Science 30 (14), 194-196 (2009).
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[5] Wang X.,Song G., Wu W., Zhao J., Hu Y - Determination of the Food colorant,
Chrysoidine, in Fish by GC/MS, Chromatographia 68, 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
Chem 122, 1230-1234 (2010).
[7]. QCVN 4 to QCVN 12 – National Technical Regulations concerning Food
Additives and Preservatives (2010).
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