EN 18003:2024

Food authenticity - Determination of 16-O-Methylcafestol content of green and roasted coffee - HPLC-method

This document specifies a high-performance liquid chromatography (HPLC) method for determining the 16-O-Methylcafestol content in green and roasted coffee.
The method is suitable for a content of 40 mg/kg to 1 600 mg/kg of 16-O-Methylcafestol of green and roasted coffee, respectively. The collaborative study has shown that mass fractions also between 20 mg/kg to 40 mg/kg can be successfully analysed depending on the laboratory equipment.
The compliance assessment process is not part of this document.

Lebensmittelauthentizität - Bestimmung des Gehaltes an 16‑O‑Methylcafestol in Roh- und Röstkaffee - HPLC-Verfahren

Dieses Dokument legt ein Hochleistungsflüssigchromatographie-(HPLC-)Verfahren zur Bestimmung des Gehaltes an 16-O-Methylcafestol in Roh- und Röstkaffee fest.
Das Verfahren ist für einen Gehalt von 40 mg/kg bis 1 600 mg/kg 16-O-Methylcafestol in Roh- bzw. Röstkaffee geeignet. Der Ringversuch hat gezeigt, dass je nach Laborausrüstung auch Massenanteile zwischen 20 mg/kg und 40 mg/kg erfolgreich analysiert werden können.
Der Prozess der Konformitätsbewertung ist nicht Teil dieses Dokuments.

Authenticité des aliments - Détermination de la teneur en 16-O-méthylcafestol du café vert et torréfié - Méthode CLHP

Le présent document spécifie une méthode de chromatographie en phase liquide à haute performance (CLHP) permettant de déterminer la teneur en 16-O-méthylcafestol dans le café vert et le café torréfié.
La méthode est adaptée pour quantifier une teneur en 16-O-méthylcafestol comprise entre 40 mg/kg et 1 600 mg/kg pour le café vert et le café torréfié respectivement. L’étude interlaboratoires a montré que la méthode permet aussi de quantifier des fractions massiques comprises entre 20 mg/kg et 40 mg/kg, en fonction de l’équipement de laboratoire.
Le présent document ne couvre pas le processus d’évaluation de la conformité.

Pristnost živil - Določanje vsebnosti 16-O-metilkafestola v surovi in praženi kavi - Metoda HPLC

Ta dokument določa metodo tekočinske kromatografije z visoko ločljivostjo (HPLC) za določanje vsebnosti 16-O-metilkafestola v surovi in praženi kavi.
Metoda je primerna za vsebnost od 40 mg/kg do 1600 mg/kg 16-O-metilkafestola v zeleni oziroma praženi kavi. V sodelovalni študiji je bilo ugotovljeno, da je mogoče uspešno analizirati tudi masne deleže med 20 mg/kg in 40 mg/kg, kar je odvisno od laboratorijske opreme.
Postopek ocenjevanja skladnosti ni del tega dokumenta.

General Information

• Status: Published
• Publication Date: 05-Nov-2024
• ICS: 67.140.20 - Coffee and coffee substitutes
• Technical Committee: CEN/TC 460 - Food Authenticity
• Drafting Committee: CEN/TC 460/WG 3 - Coffee and coffee products
• Current Stage: 6060 - Definitive text made available (DAV) - Publishing
• Start Date: 06-Nov-2024
• Due Date: 06-Mar-2025
• Completion Date: 06-Nov-2024
• Ref Project: SIST EN 18003:2025 - Food Authenticity - Determination of 16-O-methylcafestol content of green and roasted coffee - HPLC-method

Overview - EN 18003:2024 (Food authenticity, HPLC method)

EN 18003:2024 is a CEN standard that specifies a validated high-performance liquid chromatography (HPLC) procedure to determine the 16-O‑Methylcafestol (16‑OMC) content of green and roasted coffee. The method supports food authenticity testing and laboratory quality control by measuring 16‑OMC mass fractions in the range 40 mg/kg to 1 600 mg/kg (with collaborative study evidence that 20–40 mg/kg can be analysed depending on equipment). The standard covers sample preparation, reagents, apparatus, HPLC analysis, calculation and reporting. The compliance assessment process is explicitly excluded.

Key topics and technical requirements

• Analyte and units: Mass fraction of 16‑O‑Methylcafestol expressed in mg/kg.
• Scope and limits: Method validated for 40–1 600 mg/kg; lower-range analysis down to ~20 mg/kg may be achievable.
• Analytical principle: Hydrolysis of an aliquot with ethanolic potassium hydroxide, isolation of the unsaponifiable fraction by repeated extraction with tert‑butylmethylether (tBME), solvent evaporation, reconstitution in HPLC mobile phase, and quantification by HPLC with UV detection (224 nm) using an external calibration (peak-area) method.
• Calibration: Defined five-point calibration sets for both high and low ranges; stock and working standard preparation from a ≥95 % purity 16‑OMC reference.
• Reagents and solvents: Includes acetonitrile, methanol, ethanol, ethanolic KOH (freshly prepared), tBME, sodium chloride solution, and ascorbic acid. Water quality per ISO 3696.
• Apparatus essentials: Coffee mills (for green and roasted beans), sieves, ultrasonic bath, rotary evaporator or solvent stripping device, HPLC system with reversed-phase C18 column, UV detector at 224 nm, membrane filtration (0.2 µm).
• Documentation: Test report requirements, annexed HPLC chromatograms, precision/statistics and collaborative study results.

Practical applications and users

• Food authenticity laboratories verifying “100% arabica” claims by detecting 16‑OMC (arabica typically <20 mg/kg; robusta commonly much higher - orientation values only).
• Industry quality control in coffee roasting, trading and blends to detect robusta adulteration or verify varietal composition.
• Official food control authorities implementing standardized methods for enforcement and surveillance.
• Analytical contract labs offering coffee authenticity testing services.

Related standards and references

• EN 17992:2024 (NMR method for coffee authenticity) - a collaborative study is referenced.
• ISO 3696 (water for analytical laboratory use) and ISO 3310‑1 (test sieve requirements) are normative references in EN 18003:2024.

Keywords: EN 18003:2024, 16‑O‑Methylcafestol, 16‑OMC, HPLC method, food authenticity, green coffee, roasted coffee, coffee authentication, CEN.

Standards Content (Sample)

SLOVENSKI STANDARD
01-januar-2025
Pristnost živil - Določanje vsebnosti 16-O-metilkafestola v surovi 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: EN 18003:2024
ICS:
67.140.20 Kava in kavni nadomestki Coffee and coffee substitutes
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EN 18003
EUROPEAN STANDARD
NORME EUROPÉENNE
November 2024
EUROPÄISCHE NORM
ICS 67.140.20
English Version
Food authenticity - Determination of 16-O-Methylcafestol
content of green and roasted coffee - HPLC-method
Authenticité des aliments - Détermination de la teneur Lebensmittelauthentizität - Bestimmung des Gehaltes
en 16-O-méthylcafestol du café vert et torréfié - an 16-O-Methylcafestol in Roh- und Röstkaffee - HPLC-
Méthode CLHP Verfahren
This European Standard was approved by CEN on 2 September 2024.

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. Up-to-date lists and bibliographical references
concerning such national standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN
member.
This European Standard exists 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.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION

EUROPÄISCHES KOMITEE FÜR NORMUNG

CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2024 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN 18003:2024 E
worldwide for CEN national Members.

Contents Page
European foreword . 3
Introduction . 4
1 Scope . 5
2 Normative references . 5
3 Terms and definitions . 5
4 Principle . 5
5 Reagents . 5
6 Apparatus . 7
7 Sampling . 8
8 Test method . 8
8.1 General. 8
8.2 Sample preparation of roasted coffee . 9
8.2.1 Roasted whole beans . 9
8.2.2 Commercial ground roasted coffee . 9
8.3 Sample preparation of green coffee . 9
8.3.1 Green whole coffee beans . 9
8.4 Isolation of the unsaponifiable matter. 9
8.4.1 Hydrolysis . 9
8.4.2 Extraction . 9
8.4.3 Subsequent washing . 10
8.4.4 Preparation of test solution . 10
8.4.5 High-performance liquid chromatography (HPLC) . 10
9 Evaluation . 10
9.1 General. 10
9.2 Calculation . 10
10 Test report . 11
11 Collaborative study . 11
Annex A (informative) Typical HPLC chromatograms. 12
Annex B (informative) Statistical parameters and precision data . 14
Annex C (informative) Common collaborative study of this HPLC method and the NMR method
(EN 17992:2024) regarding roasted coffees . 17
C.1 Background of the collaborative study . 17
C.2 Explanation of systematic differences between the results of the NMR method and
HPLC method . 17
Bibliography . 18

European foreword
This document (EN 18003:2024) has been prepared by Technical Committee CEN/TC 460 “Food
authenticity”, the secretariat of which is held by DIN.
This European Standard shall be given the status of a national standard, either by publication of an
identical text or by endorsement, at the latest by May 2025, and conflicting national standards shall be
withdrawn at the latest by May 2025.
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. CEN shall not be held responsible for identifying any or all such patent rights.
Any feedback and questions on this document should be directed to the users’ national standards body.
A complete listing of these bodies can be found on the CEN website.
According to the CEN-CENELEC Internal Regulations, the national standards organisations of the
following countries are bound to implement this European Standard: 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 the United
Kingdom.
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. For green and roasted coffee samples, the claim
“100 % arabica”, “pure arabica” can be authenticated by analysing the mass fraction of 16-O-
Methylcafestol (16-OMC). Whereas arabica coffees contain no detectable or only very small amounts of
16-OMC (<20 mg/kg), the mass fractions in robusta coffees are significantly higher in the approximate
range of 800 mg/kg to 2 500 mg/kg.
NOTE These published values are quoted as an orientation only and not intended as strict threshold
recommendations.
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 is suitable for a content of 40 mg/kg to 1 600 mg/kg of 16-O-Methylcafestol of green and
roasted coffee, respectively. The collaborative study has shown that mass fractions also between
20 mg/kg to 40 mg/kg can be successfully analysed depending on the laboratory equipment.
The compliance assessment process is not part of this document.
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
mass fraction of 16-O-Methylcafestol in green coffee or roasted coffee
Note 1 to entry: 16-O-Methylcafestol content is expressed in mg/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 against 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).
The mixing ratio should be adjusted depending on the analysis system used.
5.5 Potassium hydroxide pellets, p.a. (quality grade).
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 only be used for a maximum of two days.
5.7 Methanol, HPLC grade.
5.8 Sodium chloride, p.a. (quality grade).
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)
Dilute the stock solution (5.12.2) 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 γ 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).
5.12.4 Calibration solutions for low calibration range (16 mg/kg to 120 mg/kg)
Dilute the stock solution (5.12.2) 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 γ 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 × 10 mm.

1 ®
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.
2 ®
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.

6.8 Heating device for stripping solvents .
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 bot
...