EN 17992:2024

SLOVENSKI STANDARD
01-januar-2025
Pristnost živil - Določanje vsote 16-O-metilkafestola, 16-O-metilkaveola in njunih
derivatov v praženi kavi z metodo ¹H-qNMR
Food authenticity - Determination of the sum of 16-O-methylcafestol, 16-O-
Methylkahweol and their derivatives in roasted coffee by ¹H-qNMR
Lebensmittelauthentizität - Bestimmung des Gehalts von 16-O-Methylcafestol, 16-O-
Methylkahweol und deren Derivaten als Summenparameter in Röstkaffee mittels ¹H-
qNMR
Authenticité alimentaire du café - Détermination de la teneur en 16-O-méthylcafestol -
Méthode par NMR
Ta slovenski standard je istoveten z: EN 17992:2024
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 17992
EUROPEAN STANDARD
NORME EUROPÉENNE
November 2024
EUROPÄISCHE NORM
ICS 67.140.20
English Version
Food authenticity - Determination of the sum of 16-O-
Methylcafestol, 16-O-Methylkahweol and their derivatives
in roasted coffee by ¹H-qNMR
Authenticité des aliments - Détermination par Lebensmittelauthentizität - Bestimmung des Gehalts
résonance magnétique nucléaire quantitative du von 16-O-Methylcafestol, 16-O-Methylkahweol und
proton (RMN-¹H) de la somme des teneurs en 16-O- deren Derivaten als Summenparameter in Röstkaffee
méthylcafestol, 16-O-méthylkahwéol et leurs dérivés mittels ¹H-qNMR
dans le café torréfié
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 17992:2024 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 . 8
5 Reagents . 9
6 Apparatus . 10
7 Measurement and test methods . 11
7.1 General. 11
7.2 Sampling . 11
7.3 Sample preparation . 11
7.3.1 General. 11
7.3.2 Sample preparation of coffee beans . 11
7.3.3 Sample preparation of ground coffee . 11
7.3.4 Extraction and preparation of the measuring solution . 12
7.4 NMR experiments . 12
7.4.1 General. 12
7.4.2 Fundamental criteria for the NMR Spectrum . 12
7.4.3 Valid exemplary measuring conditions/ parameters for the 1D experiment . 13
7.4.4 Acquisition parameters for the 1D experiment . 14
7.4.5 Processing parameters for the 1D experiment . 14
7.5 Quality control monitoring . 15
7.5.1 Checking the acquisition quality of the spectra . 15
7.5.2 Checking the qNMR calibration and sample preparation process . 15
7.6 Determination of the PULCON factor . 16
7.7 Calculation of the analyte mass fractions in coffee. 16
7.8 List of analytes and relevant parameters . 17
8 Accuracy . 18
8.1 Ring test / Interlaboratory comparison test . 18
8.2 Repeatability . 18
8.3 Reproducibility . 18
8.4 Limit of quantification (LOQ) . 18
9 Test report . 18
Annex A (informative) Summary of the statistical evaluation of the method validation study
“16-OMC in coffee” . 19
Annex B (informative) Common collaborative study of this NMR method and the HPLC
method (EN 18003:2024) . 23
Bibliography . 24

European foreword
This document (EN 17992:2024) 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 standardization 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 roasted coffee samples the claim “100 %
arabica”, “pure arabica” can be authenticated by analysing the mass fraction of 16-O-Methylcafestol and
16-O-Methylkahweol and their derivatives (e.g. fatty acid esters). Whereas arabica coffees contain no
detectable or only very small amounts of these analytes (less than 20 mg/kg), the mass fractions in
1, [1]-[7]
robusta coffees are significantly higher in the approximate range of 800 to 2 500 mg/kg .
[8]
Metrologically, quantitative NMR (qNMR) is a proven primary method of measurement . This means
that the quantity determination by NMR is directly related to the definition of the unit of measurement
and/ or basic fundamental constants. Thus, the measurement process is physically fully understood and
a complete error budget and its physical causation can be described. NMR does not need substance
specific calibration.
Three approaches to qNMR are common and regularly used:
— use of an internal standard (adding an exact quantity of a reference substance to the sample);
— projection of a concentration-calibrated artificial signal into the sample’s spectrum (called ERETIC
[9]
method) ;
— calibration of the spectrometer’s response (under comparable acquisition conditions) with an
[10]
external standard of exactly known concentrations (called PULCON method or ERETIC2) .
This analysis method uses the PULCON method to quantify analytes with NMR.

These published values are quoted as an orientation only and not intended as strict threshold recommendations.
1 Scope
This document specifies a method for the determination of soluble 16-O-Methylcafestol and 16-O-
Methylkahweol content (the sum of free forms and derivatives, e.g. fatty acid esters, henceforth
abbreviated as 16-OMD = “diterpenes”) in roasted coffee (beans or ground), using quantitative proton
nuclear magnetic resonance spectroscopy ( H-qNMR).
If complying with the experimental parameters described below, this test procedure has been proven for
the following mass fraction range:
w : 20 mg/kg to 2 000 mg/kg.
16-OMD
The mass fraction range can be expanded by suitable changes of the experimental parameters, e.g. a
different weighed portion of ground coffee or the accumulation of more NMR-transients.
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.
EN ISO 1042, Laboratory glassware — One-mark volumetric flasks (ISO 1042)
ISO 3310-1, Test sieves — Technical requirements and testing — Part 1: Test sieves of metal wire cloth
ISO 3509, Coffee and coffee products — Vocabulary
3 Terms and definitions
For the purposes of this document, the terms and definitions given in ISO 3509 and the following 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-OMD content
amount of 16-OMC and 16-OMK (both are 16-O-Methylditerpenes) and their derivatives in coffee
Note 1 to entry: It is extractable with chloroform, determined according to this test procedure and usually quoted
as a mass fraction w in mg/kg coffee, calculated as free 16-OMC.
Note 2 to entry: An additional fraction of the total 16-OMD content (ca. 25 %) can only be extracted by vigorous
chemical processing.
Note 3 to entry: Approximately a mass fraction of 90 % of the 16-OMD is represented by 16-O-Methylcafestol, and
16-O-Methylkahweol represent the remaining mass fraction of 10 % of the mixture.
Note 4 to entry: The relative error due to differences in molar weight is 0,6 %.
[1]
[SOURCE: bibliography ]
3.2
16-OMC
16-O-Methylcafestol ®
(CAS RN : 108214-28-4), M = 330,5 g/mol
Note 1 to entry: The substance appears in its free form or esterified, see Figure 1.

Key
R residue (H or fatty acid)
Figure 1 — 16-OMC
3.3
16-OMK
16-O-Methylkahweol ®
(CAS RN : not yet available), M = 328,5 g/mol
Note 1 to entry: The substance appears in its free form or esterified, see Figure 2.

Key
R residue (H or fatty acid)
Figure 2 — 16-OMK
3.4
FID
free induction decay
time-domain NMR signal that results from the precession of the nuclear magnetization vector inside the
probe coil after application of an excitation RF (radio frequency) pulse to a sample in a static magnetic
field
[SOURCE: ISO 24583:2022, 3.6, modified – RF explanation added]
3.5
ERETIC
Electronic REferencing To In-vivo Concentrations
quantitative NMR method using a synthetic signal with an integral calibrated against an exactly known
concentration of protons
Note 1 to entry: By adding this signal to a sample’s real spectrum, this spectrum has an artificial signal that can be
used for concentration determination like the signal of an internal standard.
Note 2 to entry: The advantage of the ERETIC signal is that it can be set in an empty region of the sample spectrum,
the disadvantage is that an extra signal generating electronic circuitry is needed in the spectrometer.
Note 3 to entry: ERETIC2 is a commercial word mark for the PULCON method.
3.6
FWHM
full width at half maximum
width of a line shape at half the maximum signal intensity
Note 1 to entry: It is expressed in Hz.
[SOURCE: ISO 24583:2022, 3.9 – modified: Note 2 to entry deleted]
3.7
NMR
nuclear magnetic resonance spectroscopy
method based on the selective absorption of high frequency radio waves by atomic nuclei subjected to a
stationary magnetic field
Note 1 to entry: NMR provides chemical and structural properties of molecules.
[SOURCE: ISO 23118:2021, 3.10]
3.8
ppm
part per million
pseudo-unit for the chemical shift (δ) of a resonance signal
Note 1 to entry: The frequency difference of a signal’s resonance f from a reference compound’s resonance
signal
f (both frequencies in Hz, cycles per second) is divided by the frequency of the reference compound’s
referene
resonance and multiplied with 10 . This yields a normalized shift measure that is independent of the spectrometer’s
field strength. See the following formula for explanation:
f − f
signal reference
δ = ×10
signal
f
reference
Note 2 to entry: ppm is not a (SI) unit but a symbol for an order of magnitude (10–6), like the % symbol (10–2).
3.9
PULCON
PUlse-Length-based CONcentration determination
quantitative NMR method with external standard factoring in the duration of the 90° excitation pulse into
the calculation of concentrations
Note 1 to entry: ERETIC2 is a commercial word mark for the PULCON method.
3.10
QuantRef
Quantification Reference
external standard to determine the spectrometer’s response (signal integral per amount of NMR-active,
measured nuclei)
Note 1 to entry: The QuantRef shall be a stable solution of one or more certified reference substances showing at
least one intensive and baseline-separated resonance in the NMR-spectrum:
— Preferably substances with short T transversal relaxation time should be used, thus enabling the use of shorter
recycle delays between FIDs.
— The concentration(s) should be high enough to yield an SNR > 150 (reducing the uncertainty due to noise under
1 %) under applied NMR acquisition parameters (typical concentrations should be between 10 to 50 mmol/L).
— Reference substances intended for use in a QuantRef shall have a proven long-lasting stability in this mixture
(ambient light, normal room temperature)
— Signals of the reference substances should not be close to the residual non-deuterated solvent signal or a trace
water signal.
— Care shall be taken to ensure signals are separated by a clear, flat baseline without signal overlap, e.g. from C
satellites.
3.11
sample temperature
calibrated temperature of the sample during the NMR experiment(s)
3.12
SNR
signal to noise ratio
quotient of a selected signal’s intensity by the root mean square value of the noise (measured in an empty
region of the spectrum)
4 Principle
A well homogenized sample of ground coffee is weighed and mixed with a defined amount of deuterated
chloroform. After thorough extraction the suspension is separated, filtered and filled into an NMR tube
for measurement. The analytes are quantified by integration of specific resonances using the PULCON
qNMR method. Alternatively, an internal standard may be added to the coffee extract for quantification.
5 Reagents
Only certified and traceable substances of highest purity shall be used as reference substances. These
reference substances shall be stored separately from the samples.
®2
5.1 Deuterated Chloroform (CDCl ), minimum degree of deuteration 99,8 % (at.), CAS RN : 865-49-
Deuterated solvent for the extraction and the NMR measurement of lipophilic substances in coffee. ®
5.2 Tetramethylsilane (TMS), CAS RN : 75-76-3
Internal standard used for the calibration of the ppm-scale δ(TMS) = 0,00 ppm (def). It may be omitted if
the solvent’s residual non-deuterated signal is used for calibration of the chemical shift scale.
5.3 Deuterated Chloroform with TMS
TMS is added to CDCl to achieve a TMS content between 0,03 % and 0,1 %. The solution shall be stored
in the dark, preferably in a brown glass bottle. Some cm of silver tape should be added to hinder the
formation of aggressive compounds (phosgene, hydrogen chloride) that can subsequently deteriorate the
samples. The addition of molecular sieves (3 Å or 4 Å) can help to absorb any residual water. ®
5.4 1,2,4,5-Tetrachloro-3-nitrobenzene (TCNB), CAS RN : 117-18-0, M = 260,89 g/mol
Exemplary reference substance for the QuantRef solution, δ (in CDCl ) = 7,74 ppm, s, 1 H; very stable in
CDCl , but long T transversal relaxation time.
3 1 ®
5.5 Ethylbenzene, CAS RN : 100-41-4, M = 106,17 g/mol
Exemplary reference substance for the QuantRef solution, δ (in CDCl ) = 7,30 ppm to 7,26 ppm, m, 2 H;
7,23 ppm to 7,15 ppm, m, 2 H; 7,23 ppm to 7,15 ppm, m, 1 H; 2,65 ppm, q (7,6 Hz), 2 H and 1,24 ppm, t
(7,6 Hz), 3 H. ®
5.6 1,3,5-Trimethoxybenzene, CAS RN : 621-23-8, M = 168,19 g/mol
Exemplary reference substance for the QuantRef solution, δ (in CDCl ) = 6,1 ppm, s, 3 H and 3,8 ppm, s,
9 H. ®
5.7 p-Xylene, CAS RN : 106-42-3, M = 106,2 g/mol
Exemplary reference substance for the QuantRef solution, δ (in CDCl ) = 7,06 ppm, s, 4 H and 2,31 ppm,
s, 6 H. ®
5.8 Dimethylsulfon, CAS RN : 67-71-0, M = 94,13 g/mol
Exemplary reference substance for the QuantRef solution, δ (in CDCl ) = 2,31 ppm, s, 6 H.
® 4
5.9 Cyclohexane, CAS RN : 110-82-7, M = 84,16 g/mol

2 ® ®
CAS Registry Number (CAS RN ) is the trademark of a product supplied by CAS (Chemical Abstracts Service).
This information is given for the convenience of users of this document and does not constitute an endorsement by
CEN of the product named. Equivalent products may be used if they can be shown to lead to the same results.
1 Å = 10⁻¹⁰ m, molecular sieves of different pore sizes are generally offered in Ångström units.
Chloroform can slowly deteriorate the coffee extract and is a hazardous solvent. Cyclohexane has been proven
(elsewhere) to extract the 16-OMD as well as chloroform, thus it can be used as a substitute: Prepare a 95/5 mixture
of non-deuterated/deuterated cyclohexane, use this in the extraction step, then centrifuge the suspension
(centrifugation works well with cyclohexane, but not with chloroform). From the clear supernatant take a filtered
600 µL aliquot for NMR measurement. Adapt the NMR experiment to suppress the cyclohexane resonance
Exemplary reference substance for the QuantRef solution, δ (in CDCl ) = 1,43 ppm, s, 12 H.
NOTE Caution: Cyclehexane’s resonance is close to the signal from water traces in CDCl .
5.10 QuantRef solution
In a 5 mL volumetric flask at least one appropriate (see 3.10) reference substance (e.g. 5.4 to 5.9) is/ are
dissolved in CDCl to achieve concentrations between 5 to 10 mg/mL. The exact weighed portions shall
be documented (see 7.3.1). After thorough mixing, fill 0,6 mL of this QuantRef solution into an NMR tube
that subsequently is fused shut. By repeated weighing the tube shall be checked against leakage, the
proven tube shall be labelled with a distinct ID and the date of preparation.
In general, other reference substances and concentrations may be used for the QuantRef solution as long
as the conditions stated in 3.10 are fulfilled. By using more than one substance, a higher reliability of the
QuantRef solution can be reached.
5.11 Quality control (QC) sample for monitoring repeatability and reproducibility
To supervise the whole process regularly, an appropriately sized sample of real coffee with a substantial
16-OMD content shall be stored in a tightly closed vessel in the dark. From this quality control sample,
one aliquot shall be prepared, measured and evaluated with each new sample series, see 7.5.2 for details.
6 Apparatus
Normal laboratory equipment shall be used, furthermore the instruments stated in 6.1 to 6.12.
Volumetric equipment shall comply to class A according to EN ISO 1042.
Any vessel or tool coming into direct contact with the sample or the solutions shall be reliably clean to
prevent contamination and to minimize the blank values.
6.1 High-field NMR spectrometer, Typically, a 400 MHz FT-NMR with a 5 mm proton probe adapted
for the analysis of solutions and temperature stabilization of ±0,2 K, or an instrument with at least
equivalent performance, is used.
6.2 NMR tubes, 5 mm outer diameter, suitable for high-field NMR measurements, less than ±1 %
variation of the inner diameter to ensure a low variance of stimulated volume from sample to sample and
(e.g. PTFE). The same type of tubes shall be used for the
with tight-fitting cap plug suitable for CDCl3
QuantRef and all samples.
6.3 Spinner for 5-mm-NMR-tubes
6.4 Gauge for the correct depth-adjustment of the NMR tube in the probe
6.5 Analytical balance, suitable for weighing with a precision of at least ±0,000 1 g.
6.6 Coffee- or laboratory mill, suitable for the milling of roasted coffee beans. The mill shall be
calibrated to deliver a homogenous particle size, as used for the preparation of filtered coffee. At least
2/3 of the ground coffee particles shall be finer than 0,63 mm. The mill should have a minimal dead
volume and be easy to clean.
6.7 Laboratory sieves, Wire sieves with 0,63 mm mesh and 0,25 mm mesh fulfilling the requirements
according to ISO 3310-1.
(1,41 ppm, s), then proceed as usual. Cyclohexane’s extraction capacity for other substances (HMF, furfuryl alcohol)
can be unsatisfying. This modification would need individual validation.
6.8 Disposable syringes 1 mL or 2 mL, preferably non-siliconised and with Luer lock.
6.9 Syringe filter, with chloroform-resistant membrane (e.g. PTFE), porosity (Por.) of 0,45 µm.
NOTE A combination filter (glass fibre / PTFE, Por. 1 µm / 0,45 µm) can ease the filtration.
6.10 Centrifuge filters (optional), with chloroform-resistant membrane (e.g. PTFE), Por. ≤ 0,45 µm,
capacity ≥ 800 µL.
6.11 Positive displacement pipette, adjustable up to 1 000 µL or 2 000 µL.
To minimize the volume error of the added CDCl3, preferably a positive displacement pipette should be
to exactly measure the true amount added
used, alternatively weigh the sample after addition of the CDCl3
(taking into account the ρ = 1,500 g/mL).
CDCl3, 25 °C
6.12 Cyclic shaker (vortexer), capable of > 500 min- (revolutions per minute).
7 Measurement and test methods
7.1 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 ground coffee sample should be analysed in duplicate. If the difference between the results is larger
than 5 %, the analysis shall be repeated from the sieved mid-fraction (0,63 mm > x > 0,25 mm particle
size) of the coffee powder.
7.2 Sampling
The sampling method is not regulated by this document. Sampling should be done as agreed on by the
stakeholders. Care shall be taken to ensure that samples are not damaged or adulterated during transport
or interim storage.
7.3 Sample preparation
7.3.1 General
All numerical values used to calculate the result(s), e.g. weighed portions or liquid volumes, shall be
measured and documented with at least three, better four significant numbers.
The roasted coffee beans (or ground coffee) shall be homogenized thoroughly to ensure the sub-sample
is representative. The sub-sample should weigh at least 200 g (approximately 1 000 beans).
7.3.2 Sample preparation of coffee beans
Roasted coffee beans shall be ground in a mill (6.6). The milling grade shall be adjusted to yield between
30 % and 50 % of the ground coffee as the mid-fraction (between 0,25 mm and 0,63 mm particle size). At
least 2/3 of the ground coffee
...