Animal Health Research Institute

Alex food inspection lab

Determination of Additives in FOOD

Code No. Test Procedure ( )

Version No. 1
Issue Date 16/01/2022
No. of Pages 8

Preparation Revision Approval

Name Dr\ahmed Dr\mohamed Dr\ahmed aiad
ABOUKHASHABA elsendiony
Position ANALYST Quality manger General manager
Sign
Date

) ‫( جدول التعديالت‬

‫اعتمادات الوثيقة‬ ‫التاريخ‬ ‫رقم الصفحة‬ ‫بيان التعديل‬ ‫مراجعة‬ ‫إصدار‬

\ ‫إصدار‬
‫مراجعة و اعتماد‬ ‫إعداد‬ ‫مراجعة‬

CONTENTS

1. Purpose:

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2. Scope:
3. Applicability.
4. Environment.
5. Equipment And Media.
6. Precautions.
Procedure.
Retention And Disposal.
9. Method Validation.
7. Test Report
11 Assuring the test result
12. Appendices.
13 Distribution List.

1. Purpose:
A food additive is defined as a substance or mixture of substances, which are generally added to processed
foods for a specific purpose such as prevention of spoilage, conservation or fortification of color, flavor,
texture, or control of pH, moisture, crispness etc. However, the excessive use of food additives could lead to
adverse effects such as metabolic acidosis, convulsions and hyperpnoea in human.
2. Scope:

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Alex food inspection lab

The method involves preservatives and artificial sweeteners. Ascorbic acid, Acesulfame potassium,
saccharine, caffeine, Aspartame, Sorbic acid and benzoic acid. According to Knauer Separation of Additives in
soft drink, Method VFD0017J , Knauer Fast analyses of additives in soft drinks minimal sample preparation-
application note and Food control 13(2002)117-123, Determination of benzoic acid and Sorbic acid in Brazilian.

3. Applicability.
This procedure shall be applied by all personnel who authorized to
Carry out this test.
4. Environment.
 No air currents
 No dusty atmosphere
 Atmospheric confederation .22±2 and 45-50% humidity
5. Equipments and Media.
5.1. Equipments
Hplc set up:-

Column: PerfectSIL 120 C8 5µm, 150 x 4.0 mm ID

Phase: PerfectSIL 120 C8
Conditions: Eluent: 20 mM KH2PO4 (pH 3) / Acetonitrile (5:1)
Gradient: isocratic
Flow rate: 1.2 ml/min
Temperature: 30 °C
Volume: 10 μl
Detection: UV at 220 nm
Substances: Ascorbic acid, Acesulfame potassium, saccharine, caffeine, Aspartame, Sorbic acid
and benzoic acid.

Usual laboratory apparatus and, in particular, the following

1 Laboratory mill or explosion proof high speed blender:
2 . Necessary for the production and extraction of pastes from peanuts, pistachios, with
suitable blender jar.
3 Adjustable vertical or horizontal shaker
4 Paper filter: e.g. 24 cm diameter, prefolded
5 Conical flask: with screw top or glass stopper
6 Glass microfiber filter paper: retention size 1.6 μm or smaller
7 Volumetric glassware: flasks of e.g. 5 ml,10 ml and 20 ml, with an accuracy of at least
0.5%
8 HPLC system, consisting of:
1 HPLC Low Pressure gradient pump, (AZURA® P 6.1L) SN: FBI 1200600002, suitable
for flow rate at 1.2 ml/min.

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2 Autosampler (AZURA® AS 6.1L) SN: FZB194000003, capable for Partial loop

injection. A valve with a 20 μl loop is recommended.
3 RP-HPLC column. PerfectSIL 120 C8 5µm, 150 x 4.0 mm ID
4 The DAD 2.1L SN: FOU 200600005
4.1 UV/VIS detector with 8-variable UV measuring channels From 190-750 nm and
for this method a wavelength of λ = 220 nm.
10 Disposable filter unit:
7.1 of pore size 0.45 μm
11 Pipettes, 2 ml and 10 ml capacity, with an accuracy of at least 0.5%
12 Analytical balance, capable of weighing to 0.1 mg; Explorer Pro OHAUS; Calibrated
by NIS; Model: EP2142; SN: 124112514039
13 Laboratory balance, capable of weighing to 0.01 g; AND GX 2000;
Calibrated by NIS; SN: 14526089.
14 ISO lab pipette 20-200 μl; SN: 031434 with calibration certificate.
14.1 ISO lab pipette 100-1000 μl; SN: 2130132 with calibration certificate.
15 acuum manifold, optional

6-Chemicals and Reagents:

General 1
Use only reagents of recognized analytical grade and deionized water complying with
grade 3 of EN ISO 3696, unless otherwise specified.
2 Acetonitrile, HPLC grade
3 Methanol, HPLC grade
4 Mobile phase, 20 mM KH2po4(pH 3) / Acetonitrile (5 : 1 )
4.1 Prepare 20 mM KH2po4(pH 3) by weight 1.3601 g of KH 2po4 make up to 500
ml by dist. Water adjust pH to 3 by phosphoric acid 85 %
4.2 Degas the solution before use.
Preparation of Ascorbic acid, Acesulfame potassium, saccharine, caffeine, Aspartame, 5
.Sorbic acid and benzoic acid Mixture standards

5.1 All standard solution were prepared with 100% buffer

5.2 Prepare the stock solution by weighting out 100 mg of Ascorbic acid, Acesulfame potassium,
saccharine, caffeine, Aspartame, Sorbic acid and benzoic acid. In vol.flask 100ml complete to
mark by buffer.

5.3 We obtain of mixture stock solution of 1000 µg/ml of ascorbic acid, caffeine, Sorbic acid and
benzoic acid.

5.4 Prepare working solution as follow on table in 10 ml vol.flask

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Alex food inspection lab

Mass concentration of standard solution , in µg/ml

Taken from
stoc
k
Standard solut
Acesulfam
solution ion
e
(8.5. sacchari
Ascorbic po caffein Sorbic Benzoic
2) (in n Aspartame
acid tas acid acid
μl) e
siu
m

1 100 10 10 10 10 10 10 10
2 200 20 20 20 20 20 20 20
3 400 40 40 40 40 40 40 40
4 800 80 80 80 80 80 80 80
5 1000 100 100 100 100 100 100 100

5.5 Complete the volume taken from stock solution up to 10 ml by buffer

5.6 Wrap the flask tightly in aluminum foil and store it at 4°C.

6 Potassium dihydrogen phosphate analytical grade

7 Phosphoric acid 85%

7 Diethyl ether analytical grade

8 Sodium hydroxide analytical grade

9 Ethanol analytical grade

10 Caffeine anhydrous 98% extra pure-LOBA Chemie.

11 Sorbic Acid 99% extra pure - LOBA Chemie.

12 Ascorbic acid – alpha.

7. Procedure:
7.1 Soft drink
200 ml of samples, degas the sample by placing it in a vacuum flask and

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Connecting the flask to a vacuum pump or water aspirator. Keep it under vacuum
Until no more bubbles appear in the soda sample. (If no vacuum is available, allow
The soda to stand open overnight or place in a sonicator bath).
7.1.1 Instant powder beverages, prepare as indicated on the package for a
normal serving using the methanol/water solution instead of just water. Any
insoluble material will be removed in the filtering step.
7.1.2 Filter the beverage sample through a 0.45μm syringe filter with a 5mL
syringe. Dispense the initial 1mL into waste. Dispense the remaining volume into
the sample vial.
7.1.3 Rinse the syringe filter with three 2-3 mL aliquots of the methanol/water
solution between each use.

7.1.4 Beverages with a high Preservatives content may need to be diluted. If the area of the Preservatives
peak for a beverage sample is greater than the area of the peak for the 200 ppm standard, dilute the
beverage sample with the methanol/water solution and run again. Start with a 1:1 dilution.

7.2 Fruit juice

Sample were centrifuged (2000 rpm) for 10 min and 1 ml of supernatant was diluted with water.

7.3 Margarine
5 gm of sample dissolve in 50 ml diethyl ether and extracted twice with 10 ml of 0.1 M sodium hydroxide in a
separating funnel the basic aqueous extracts was acidified with 1 ml of 2.5M sulphuric acid in vol. flask
and diluted to the volume with water.

7.4 Yoghurt and petit Suisse

The extraction procedure weight A 5 gm of sample and 15 ml 2% H3PO4 ( ortho-phosphoric acid ), placed in
tube with screw shake for 1 min then filtrate with whatman=0 no.1 and diluted with water.

7.5 Cheese

Wt.10 gm of sample and 40 ml of ethanol, shake for 30 min. then filtrate with whatman no.1filter paper and
diluted with water.
Reporting:
1 The results reported as µg/g for Ascorbic acid, Acesulfame potassium, saccharine, caffeine, Aspartame,
Sorbic acid and benzoic acid.

.2 Adding measuring uncertainty in case of critical results (the results very close to the limits stated in the
Health decision No.204/2015)

8 Method Validation .
According to - Knauer Separation of Additives in soft drink, Method VFD0017J
- Knauer Fast analyses of additives in soft drinks minimal sample preparation-application note.
-Food control 13(2002)117-123, Determination of benzoic acid and Sorbic acid in Brazilian.

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Alex food inspection lab

.
9 Test Report .
The final laboratory report has a ( form 20-1)
7. Assuring the quality of test results
The laboratory applied the quality control program to insure the quality
7.1 Internal quality control :
 Spiked samples
 Repeatability
 Duplicate test

11. Distribution List :

 Technical Manger specialty
 Staff of HPLC unit

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Alex food inspection lab

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