PROANALYSIS LABORATORY

Document No .: Date of publication: Rev: 00 / Date: Page.: 1/6

U.7.2.111

DETERMINATION OF CAFFEİNE-ALL FOODS

1. PURPOSE AND SCOPE

This method detects the active ingredients of caffeine by HPLC UV aims.

2. CHEMICALS
Caffeine standard (≥98)
Methanol (HPLC Grade)
Acetonitrile (HPLC Grade)
Water (Ultra pure water)
Phosphoric acid (85%)
Potassium dihydrogen phosphate
Potassium hexacyanoferrate(II)
Zincsulphate
Ammonium acetate
Magnesium oxide

2.1 Preparation of Necessary Chemicals

2.1.1 Standard and Calibration Solutions

Taking into account the impurity, 1000 µg/ml main stock solution is prepared with the
help of the formula below.

Amount weighed ( mg )
Volume ( ml )= x 1000
concentration ( mg ) x impurity

An intermediate stock solution of 10 µg/ml and 1 µg/ml is prepared from the prepared
main stock solution and measurement solutions are prepared with the help of the table
below. Prepared solutions are given to the device at least twice. The R 2 value of the
calibration curve drawn with the help of the device software should be at least 0.995. If
there are values deviating from the curve, they are removed from the curve, provided
that the lowest point remains and the number of levels does not fall below 5.

Caffeine
Standard Concentration Master Stock Absorbed Final Volume ml
µg/ml µg/ml Volume µl
0,1 1 0,1 1
0,25 1 0,25 1
0,5 1 0,5 1
1 10 0,1 1
2,5 10 0,25 1
5 10 0,5 1
10 1000 0,01 1
25 1000 0,025 1
50 1000 0,05 1
100 1000 0,1 1

2.1.2 Carrez I solution: 15g of potassium hexacyanoferrate(II) is dissolved in 100ml of

water.( It is prepared for 3 months.)
2.1.3 Carez II solution: 20 g of zinc sulfate is dissolved in 100 ml of water. (It is prepared for
3 months.)
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U.7.2.111

DETERMINATION OF CAFFEİNE-ALL FOODS

2.1.4 5% phosphoric acid: 3 ml of 85% orthophosphoric acid is taken and diluted to 25 ml.
(Prepared weekly.)
2.1.5 Phosphate Buffer: 20mM (2.72g) Potassium dihydrogen phosphate is dissolved in
1000ml water, pH: adjusted to 3.5 (2.1.4.) The membrane is filtered through the filter.
(Prepared weekly.)
2.1.6 Ammonium Acetate Buffer: Dissolve 20 mm of ammonium acetate in 1000 ml of
distilled water. pH:4.8 is adjusted with 85% orthophosphoric acid. The membrane is
filtered through the filter. (Prepared weekly.)
2.1.7 Mobile phase:
I. Phosphate Buffer (2.1.5) + Acetonitrile (90:10 v/v)
II. Ammonium Acetate Buffer(2.1.6) + Acetonitrile(90:10 v/v)
III. Water + Methanol (70:30 v/v)
Note: One of them is used.
3. TOOLS AND EQUIPMENT
Precision balance
Homogenizer
Vortex
Mixer
pH meter
Measure (100,500,1000ml)
Automatic pipettes; 10-100µl, 100-1000µl, 1000-5000µl
Vial (1,20,40ml)
Balloons (10, 25, 50 and 100ml)
Injector, 10 ml
0.45 µm filter(PTFE-L)
Membrane filter
Centrifugal
Watman No:4
Falcon
Funnel
Ultrasonic water bath
HPLC UV detector
C18 column:4.6x150mm, 5µm or similar column

4. Process
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DETERMINATION OF CAFFEİNE-ALL FOODS

4.1 Preparation of Samples

Carbonated drinks are kept in an ultrasonic water bath. If solid and semi-solid products are
not homogeneous, they can be made homogeneous by adding water in the grinder or mixer
when necessary. The amount of water used in the homogenization stage is recorded and taken
into account during weighing. For example, if homogenization is performed by adding 200
ml of water to a 200 g sample, 10 g of the sample, which should be weighed 5 g according to
the method, is weighed.
4.2 Extraction
For soft drinks and beverages
- 5 grams of sample is taken into a 50mL flask.
- It is completed with water up to the volume line.
- If the solution is not dense or clear, 50 ml of water is added to the flask and 2 ml of Carrez I
and Carez II solutions are added, and the flask is completed to the volume line.
- Centrifuged, filtered through watman.
- The solution is passed through a filter (PTFE) and taken into a vial.
Coffee, tea and products
-0.25 grams of sample is weighed into a 100 ml bottle.
-If necessary, some magnesium oxide is added.
-Approximately 50 ml of water is added to the balloon.
-It is heated on the stove until it boils.
-After boiling, it is expected to cool down and the volume of the balloon is completed.
-It is filtered through the filter, taken into a bottle and given to the device.
- Note: the amount of sample taken may vary depending on the amount of active
substance. The dilution rate is adjusted according to the amount of active substance in
the sample.
4.3 HPLC Analysis
-Pump Flow Rate: 1 ml/min
-Wavelength: 272 nm
-Injection Volume: 20 µl
-Column Temperature:30ºC
5. CALCULATION AND REPORTING
Result (mg/kg)= 𝐴 × DC ± 𝑈(𝑋)
A: The amount of active substance found by using the calibration curve (µg/ml)
DC: Dilution coefficient
U(X): Extended Uncertainty x Result
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DETERMINATION OF CAFFEİNE-ALL FOODS

Extended Uncertainty = Standard Uncertainty x k

k=2(expansion coefficient for 95% confidence interval)
Dilution coefficient = (𝑚 𝑣𝑒𝑦𝑎 𝑉s ) ⁄ 𝑉F
m: Amount of sample weighed (g)
VS: Volume from the sample (ml)
VF: Final volume (ml)
6. QUALITY CONTROL
-The two levels determined in the measurement (calibration) curve are given to the device,
one before the samples and the other after the samples, and the curve is controlled.
- Acceptance values of these two points are evaluated on the basis of AOAC recovery
acceptance values
- Control standards are prepared fresh each time. If the obtained values exceed the control
value, the process is repeated. If necessary, the measurement run is repeated.
- Measurements (calibration) are renewed every three months
- Quality control work is carried out with each sample lot that comes to the laboratory for
analysis and is recorded on the quality control card created for the method. In case the sample
quantity is low, quality control work is carried out once a year.
- The interpretation of the value entered on the quality control card is done in the following
ways.
There are three possible states at the end of the operand value
I. The method is under control.
If the value is within the warning limits, the result is reported
If the value is between the warning and action limits, and the two previous control values are
within the warning limit, the result is reported. However, in this case, the analysis can be
repeated if desired.
II. The method is under control, but it can be considered statistically out of control even if the
results are within the warning limits and if the following conditions are present:
If 7 consecutive control values gradually increase or decrease
If 10 of the 11 consecutive control values are on the same side of the center line In these cases,
the employee can report the analysis results but a problem may develop. In order to avoid
future problems, trends should be determined and precautions should be taken.
III. The method is out of control.
If the value is outside the action limits, the results are never reported.
If the value is between the warning and action limits, and at least one of the two previous
control values is between the warning and action limits, the result is not reported.
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DETERMINATION OF CAFFEİNE-ALL FOODS

- After the nonconformity is eliminated, the quality control study and sample studies are
renewed.
- In quality control studies, it is obligatory to have all employees authorized in the analysis.
An employee can perform a maximum of four consecutive quality control exercises. At the
end of four consecutive studies, the other employee or employees authorized in the analysis
must perform at least one quality control study.
Thus, all employees authorized in the analysis are included in the system.
7. REFERENCE
-ISO 10727
-Journal of AOAC Vol-76
-TS ISO 10095-1999
-Fast, Low Pressure Analysis Of Food And Beverage Additives Using A Superficially Porous
Application Note
8. DISTRIBUTION
Controlled copies of this analysis method are distributed to the relevant Analysts

9. REVISIONS

Revision Number Date Revised Article Reason for Revision Revision By Person

ИЗРАБОТИЛ ОДОБРИЛ
Одговорен на оддел за хемиска анализа Раководител на лабораторија
 PROANALYSIS LABORATORY
Document No .: Date of publication: Rev: 00 / Date: Page.: 6/6
U.7.2.111

DETERMINATION OF CAFFEİNE-ALL FOODS