FprEN 18003

SLOVENSKI STANDARD
oSIST prEN 18003:2023
01-december-2023
Pristnost živil - Določanje vsebnosti 16-O-metilkafestola v zeleni in praženi kavi -
Metoda HPLC
Food Authenticity — Determination of 16-O-methylcafestol content of green and roasted
coffee — HPLC-method
Lebensmittelauthentizität - Bestimmung des Gehaltes an 16-O-Methylcafestol in Roh-
und Röstkaffee - HPLC-Verfahren
Authenticité des aliments - Détermination de la teneur en 16-O-méthylcafestol du café
vert et torréfié - Méthode CLHP
Ta slovenski standard je istoveten z: prEN 18003
ICS:
67.140.20 Kava in kavni nadomestki Coffee and coffee substitutes
oSIST prEN 18003:2023 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

oSIST prEN 18003:2023
oSIST prEN 18003:2023
DRAFT
EUROPEAN STANDARD
prEN 18003
NORME EUROPÉENNE
EUROPÄISCHE NORM
August 2023
ICS
English Version
Food Authenticity - Determination of 16-O-methylcafestol
content of green and roasted coffee - HPLC-method
Authenticité alimentaire du café et des produits á base Lebensmittelauthentizität - Bestimmung des Gehaltes
de café ? Détermination de la teneur en 16-O- an 16-O-Methylcafestol in Roh- und Röstkaffee - HPLC-
méthylcafestol dans le café vert et le café torréfié ? Verfahren
Méthode par HPLC
This draft European Standard is submitted to CEN members for enquiry. It has been drawn up by the Technical Committee
CEN/TC 460.
If this draft becomes a European Standard, CEN members are bound to comply with the CEN/CENELEC Internal Regulations
which stipulate the conditions for giving this European Standard the status of a national standard without any alteration.

This draft European Standard was established by CEN in three official versions (English, French, German). A version in any other
language made by translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC
Management Centre has the same status as the official versions.

CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,
Poland, Portugal, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Türkiye and
United Kingdom.
Recipients of this draft are invited to submit, with their comments, notification of any relevant patent rights of which they are
aware and to provide supporting documentation.

Warning : This document is not a European Standard. It is distributed for review and comments. It is subject to change without
notice and shall not be referred to as a European Standard.

EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION

EUROPÄISCHES KOMITEE FÜR NORMUNG

CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2023 CEN All rights of exploitation in any form and by any means reserved Ref. No. prEN 18003:2023 E
worldwide for CEN national Members.

oSIST prEN 18003:2023
prEN 18003:2023 (E)
Contents Page
European foreword . Error! Bookmark not defined.
Introduction . Error! Bookmark not defined.
8.1 General. Error! Bookmark not defined.
8.2 Sample preparation of roasted coffee . Error! Bookmark not defined.
8.3 Sample preparation of green coffee . Error! Bookmark not defined.
8.4 Isolation of the unsaponifiable matter. Error! Bookmark not defined.
9.1 General. Error! Bookmark not defined.
9.2 Calculation . Error! Bookmark not defined.
Annex A (informative) Typical HPLC chromatograms . Error! Bookmark not defined.
Annex B (informative) Statistical parameters and precision data . Error! Bookmark not defined.
Annex C (informative) Common collaborative study of the HPLC and NMR methods
(prEN XXXX:2023 and prEN 17992:2023) regarding roasted coffeesError! Bookmark not defined.
C.1 Background of the collaborative study . Error! Bookmark not defined.
C.2 Explanation of systematic differences between the results of the NMR and HPLC
methods . Error! Bookmark not defined.
Bibliography . Error! Bookmark not defined.
oSIST prEN 18003:2023
prEN 18003:2023 (E)
European foreword
This document (prEN 18003:2023) has been prepared by Technical Committee CEN/TC 460 “Food
authenticity”, the secretariat of which is held by DIN.
This document is currently submitted to the CEN Enquiry.
oSIST prEN 18003:2023
prEN 18003:2023 (E)
Introduction
This document was developed in response to demand for an efficient and reliable test method allowing
the confirmation of coffee authenticity both for commercial quality control and for official food control.
The coffee species with the greatest commercial importance are Coffea arabica and Coffea canephora
var. robusta, commonly known as “arabica” and “robusta”. Within these species, arabica coffees have a
significantly higher market value than robusta coffees. In unblended coffee the botanical origin of coffee
can be determined unambiguously by analysing the concentration of 16-O-methylcafestol because this
marker substance is present in arabica coffee at very low concentrations (typically < 20 mg/kg). In
robusta coffees the concentration levels are significantly higher in the approximate range of 800 to
2 500 mg/kg.
NOTE These published values are quoted as an orientation only and not intended as strict threshold
recommendations.
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1 Scope
This document specifies a high-performance liquid chromatography (HPLC) method for determining
the 16-O-methylcafestol content in green and roasted coffee.
The method described is suitable with a reasonable precision for a content of 40 mg to 1 600 mg of 16-
O-methylcafestol per kg of green and roasted coffee respectively. The collaborative study has shown
that concentrations also between 20 to 40 mg/kg can be successfully analysed depending on the
laboratory equipment.
2 Normative references
The following documents are referred to in the text in such a way that some or all of their content
constitutes requirements of this document. For dated references, only the edition cited applies. For
undated references, the latest edition of the referenced document (including any amendments) applies.
ISO 3310-1, Test sieves — Technical requirements and testing — Part 1: Test sieves of metal wire cloth
ISO 3696, Water for analytical laboratory use — Specification and test methods
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply
ISO and IEC maintain terminology databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https://www.iso.org/obp/
— IEC Electropedia: available at https://www.electropedia.org/
3.1
16-O-methylcafestol content
concentration of 16-O-methylcafestol in green coffee or roasted coffee, expressed in mg per kg
4 Principle
An aliquot of the ground and well-mixed sample is hydrolysed using ethanolic potassium hydroxide
solution. Then, repeated shaking with tert-butylmethylether (tBME) isolates the unhydrolysable
portion. The combined ether-extract phases are reduced to dryness after being washed. The residue is
taken up in HPLC mobile phase, and the 16-O-methylcafestol content is examined by HPLC.
The substance is identified by comparison of its retention time with that of the standard substance, and
quantification is carried out by the external standard method based on peak areas.
5 Reagents
Unless otherwise stated, analytical grade reagents shall be used. The water used shall be of quality
grade 1, as described in ISO 3696.
5.1 Acetonitrile, HPLC grade.
5.2 L(+)-Ascorbic acid, 99,5 %, suitable for analytical purposes.
5.3 Ethanol, HPLC grade.
5.4 Mobile phase, acetonitrile/aqua bidest or methanol/aqua bidest (see 8.4.5).
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NOTE The mixing ratio may be adjusted depending on the analysis system used.
5.5 Potassium hydroxide pellets.
5.6 Ethanolic potassium hydroxide solution, prepared by dissolving 10 g of potassium hydroxide
in 10 ml of aqua bidest and making up to 100 ml with ethanol; the solution shall always be freshly
prepared and may be used for a maximum of two days.
5.7 Methanol, HPLC grade.
5.8 Sodium chloride.
5.9 Sodium chloride solution, prepared by dissolving 2 g of sodium chloride in a 100 ml volumetric
flask filled with water and making up to the calibration mark.
5.10 tert-Butylmethylether (tBME), HPLC grade.
5.11 liquid nitrogen or dry ice (optional), for cooling.
5.12 Standard 16-O-methylcafestol solutions.
5.12.1 Standard substance
A 16-O-methylcafestol standard shall be used that has a minimum purity of 95 %.
5.12.2 Stock solution
Weigh about 10 mg of 16-O-methylcafestol into a 50 ml volumetric flask (approximately 200 μg/ml).
Dissolve the substance in acetonitrile (5.1) using an ultrasonic bath, and then make up the flask to the
mark with acetonitrile (5.1).
NOTE The stock solution will remain stable for at least 12 weeks if refrigerated.
5.12.3 Calibration solutions for high calibration range (160 mg/kg to 1 600 mg/kg)
The stock solution (5.11.2) is diluted with acetonitrile (5.1) to produce five different dilute solutions for
creating the five-point calibration and calculating the 16-O-methylcafestol content, shown in Table 1.
NOTE The calibration solutions will remain stable for at least three weeks if refrigerated or stored at ambient
temperature (20 °C) away from direct sunlight.
Table 1 — Standard solutions for high calibration range (160 mg/kg to 1 600 mg/kg)
Standard solution V C W
16-OMC 16-OMC 16-OMC
ml µg/ml mg/kg
H1 2,0 80 1 600
H2 1,5 60 1 200
H3 1,0 40 800
H4 0,5 20 400
H5 0,2 8 160
Pipette the specified volumes into a 5 ml volumetric flask and fill up with acetonitrile (5.1).
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prEN 18003:2023 (E)
5.12.4 Calibration solutions for low calibration range (16 mg/kg to 120 mg/kg)
First the stock solution (5.12.2) is diluted with acetonitrile (5.1) 1:10 to produce a standard solution
with 20 µg/ml (10 ml). Thereof prepare five different dilute solutions for creating the five-point
calibration and calculating the 16-O-methylcafestol content, shown in Table 2.
Table 2 — Standard solutions for low calibration range (16 mg kg to 120 mg/kg)
Standard solution V C W
16-OMC 16-OMC 16-OMC
ml µg/ml mg/kg
L1 1,5 6,0 120
L2 1,2 4,8 96
L3 0,8 3,2 64
L4 0,4 1,6 32
L5 0,2 0,8 16
Pipette the specified volumes into a 5 ml volumetric flask and fill up with acetonitrile (5.1).
If the content of the coffee measurement solution exceeds the highest value of the calibration curve, the
measurement solution shall be diluted 1:5 or 1:10. The linearity of the calibration curves shall be
checked individually.
6 Apparatus
Standard laboratory equipment shall be used, as well as the devices listed in 6.1 to 6.20.
6.1 Coffee mill, cold resistant and therefore suitable for grinding green coffee beans.
NOTE For example, cryogenic mills have been found to be suitable for grinding green coffee beans.
6.2 Mill, suitable for grinding roasted coffee beans.
NOTE For example, standard mills have been found to be suitable for grinding roasted coffee beans.
6.3 Sieves, with wire screens of mesh sizes 0,63 mm and 0,25 mm, which meet the requirements of
ISO 3310-1.
6.4 Glycerine bath or heating block.
6.5 Heating plate with integrated agitator.
1 2
6.6 Screw capped tubes and caps for screw capped tubes (for hydrolysis).
6.7 Magnetic rod, triangular, 16 mm x 10 mm.
6.8 Heating device for stripping solvents .

DURAN culture tubes (Z620262), 30 ml volume (180 mm × 18 mm, height × diameter) made of borosilicate glass
are an example for commercially available screw capped tubes. This information is given for the convenience of
users of this document and does not constitute an endorsement by CEN of this product.
PBT cap for DURAN (2924011), temperature-stable up to 180 °C, size GL 18 is an example for commercially
available caps for screw capped tubes. This information is given for the convenience of users of this document and
does not constitute an endorsement by CEN of this product.
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6.9 Ultrasonic bath, room temperature.
6.10 Graduated cylinder, 10 ml.
6.11 Volumetric flask, different sizes (5 ml, 10 ml, 50 ml, 100 ml).
6.12 Pasteur pipettes.
6.13 Screw top vials with cap, 60 ml volume (height x diameter 140 mm x 28 mm).
6.14 Round bottom flask, 50 ml.
6.15 Vacuum rotary evaporator or similar laboratory equipment.
6.16 Membrane filter, regenerated cellulose, pore size of 0,2 μm or similar membrane.
6.17 HPLC Vials 2 ml, clear glass or brown glass.
6.18 High-performance liquid chromatograph with UV detector, measurements at 224 nm, with
evaluation system, preferably with a column oven.
6.19 Analytical column, Reversed Phase C
18 .
6.20 vessel, stainless-steel, 600 ml.
7 Sampling
It is assumed that the sample will be representative.
8 Test method
8.1 General
WARNING — The use of this document can involve hazardous materials, operations and equipment. It
does not purport to address all of the safety or environmental problems associated with its use.
The coffee sample (ground powder) shall be thoroughly homog
...