Animal feeding stuffs: Methods of sampling and analysis - Determination of theobromine in feed materials and compound feed, including cocoa derived ingredients, by liquid chromatography

This document method is applicable for the determination of theobromine in compound feed by liquid chromatography with UV detection in the tested range of 27 to 307 mg/kg. This method has been validated using complementary compound feed for adult dogs and complementary compound feedstuff for horses. The actual working range may extend beyond the tested range. Alternative chromatography conditions using liquid chromatography tandem mass spectrometry (LC-MS/MS) are also provided for the validated range of 49 to 307 mg/kg. This method has also been shown to be fit for purpose for the determination of theobromine in baking chocolate by both HPLC-UV and LC-MS/MS.

Futtermittel: Probenahme- und Untersuchungsverfahren - Bestimmung von Theobromin in Einzel- und Mischfuttermitteln, einschließlich aus Kakao gewonnenen Bestandteilen, mittels Flüssigchromatographie

Das Verfahren in diesem Dokument gilt für die Bestimmung von Theobromin in Mischfutter mittels Flüssigchromatographie mit UV-Detektion im Prüfbereich von 27 mg/kg bis 307 mg/kg. Dieses Verfahren wurde unter Verwendung von Ergänzungsmischfuttermittel für erwachsene Hunde und Ergänzungs-mischfuttermittel für Pferde validiert. Der tatsächliche Arbeitsbereich kann größer sein als der geprüfte Bereich. Es werden auch alternative Chromatographiebedingungen unter Verwendung von Flüssigkeits-chromatographie mit Tandem-Massenspektrometrie (LC-MS/MS) für den validierten Bereich von 49 mg/kg bis 307 mg/kg bereitgestellt. Dieses Verfahren hat sich ebenfalls als gebrauchstauglich für die Bestimmung von Theobromin in Blockschokolade mittels HPLC-UV oder LC-MS/MS erwiesen.

Aliments des animaux : Méthodes d'échantillonnage et d'analyse — Détermination par chromatographie en phase liquide de la teneur en théobromine dans les matières premières destinées aux aliments des animaux et dans les aliments composés pour animaux, y compris les ingrédients issus du cacao

La méthode de la présente Norme européenne est applicable à la détermination de la teneur en théobromine dans les aliments composés par chromatographie en phase liquide avec détection UV dans la gamme d’essai de 27 mg/kg à 307 mg/kg. Cette méthode a été validée avec des aliments composés complémentaires pour chiens adultes et des aliments composés complémentaires pour chevaux. La gamme de travail réelle peut dépasser la gamme d’essai. D’autres conditions chromatographiques utilisant la chromatographie en phase liquide couplée à une spectrométrie de masse en tandem (CL‐SM/SM) sont également proposées pour la gamme validée de 49 mg/kg à 307 mg/kg. Cette méthode s’est également révélée adaptée pour déterminer la teneur en théobromine dans le chocolat pâtissier par CLHP‐UV et CL‐SM/SM.

Krma: metode vzorčenja in analize - Določevanje teobromina v sestavinah krme in krmnih mešanicah, vključno s pridobljenimi sestavinami iz kakava, s tekočinsko kromatografijo

Metoda v tem dokumentu je uporabna za določanje teobromina v krmni mešanici s tekočinsko kromatografijo z UV-odkrivanjem v preskusnem obsegu 27–307 mg/kg. Ta metoda je bila potrjena z dopolnilno krmno mešanico za odrasle pse in dopolnilno krmno mešanico za konje. Dejanski delovni obseg lahko presega preskušeni obseg. Alternativni pogoji kromatografije s tandemsko masno spektrometrijo s tekočinsko kromatografijo (LC-MS/MS) so prav tako zagotovljeni za potrjeni obseg 49–307 mg/kg. Pokazalo se je tudi, da je ta metoda primerna za določanje teobromina v čokoladi za peko s HPLC-UV in LC-MS/MS.

General Information

Status
Published
Public Enquiry End Date
24-Oct-2018
Publication Date
09-Dec-2019
Current Stage
6060 - National Implementation/Publication (Adopted Project)
Start Date
27-Nov-2019
Due Date
01-Feb-2020
Completion Date
10-Dec-2019

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Standards Content (Sample)

SLOVENSKI STANDARD
SIST EN 17270:2020
01-januar-2020
Krma: metode vzorčenja in analize - Določevanje teobromina v sestavinah krme in
krmnih mešanicah, vključno s pridobljenimi sestavinami iz kakava, s tekočinsko
kromatografijo
Animal feeding stuffs: Methods of sampling and analysis - Determination of theobromine
in feed materials and compound feed, including cocoa derived ingredients, by liquid
chromatography
Futtermittel: Probenahme- und Untersuchungsverfahren - Bestimmung von Theobromin
in Einzel- und Mischfuttermitteln, einschließlich aus Kakao gewonnenen Bestandteilen,
mittels Flüssigchromatographie
Aliments des animaux : Méthodes d'échantillonnage et d'analyse — Détermination par
chromatographie en phase liquide de la teneur en théobromine dans les matières
premières destinées aux aliments des animaux et dans les aliments composés pour
animaux, y compris les ingrédients issus du cacao
Ta slovenski standard je istoveten z: EN 17270:2019
ICS:
65.120 Krmila Animal feeding stuffs
71.040.50 Fizikalnokemijske analitske Physicochemical methods of
metode analysis
SIST EN 17270:2020 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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SIST EN 17270:2020

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SIST EN 17270:2020


EN 17270
EUROPEAN STANDARD

NORME EUROPÉENNE

October 2019
EUROPÄISCHE NORM
ICS 65.120
English Version

Animal feeding stuffs: Methods of sampling and analysis -
Determination of theobromine in feed materials and
compound feed, including cocoa derived ingredients, by
liquid chromatography
Aliments des animaux : Méthodes d'échantillonnage et Futtermittel: Probenahme- und
d'analyse - Détermination par chromatographie en Untersuchungsverfahren - Bestimmung von
phase liquide de la teneur en théobromine dans les Theobromin in Einzelfuttermitteln, vor allem aus
matières premières destinées aux aliments des Kakao gewonnen sowie in Mischfuttermitteln mittels
animaux et dans les aliments composés pour animaux, Flüssigchromatographie
y compris les ingrédients issus du cacao
This European Standard was approved by CEN on 28 July 2019.

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, Turkey 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
© 2019 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN 17270:2019 E
worldwide for CEN national Members.

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SIST EN 17270:2020
EN 17270:2019 (E)
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 Procedure. 8
8 Chromatographic analysis . 9
9 Calculation of results . 9
10 Accuracy . 10
11 Test report . 10
Annex A (informative) Example HPLC-UV conditions . 11
Annex B (informative) Example LC-MS/MS conditions . 14
Annex C (informative) Method performance data obtained during single laboratory
validation . 20
Annex D (informative) Method performance data obtained during collaborative trial . 21
Bibliography . 23
2

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SIST EN 17270:2020
EN 17270:2019 (E)
European foreword
This document (EN 17270:2019) has been prepared by Technical Committee CEN/TC 327 “Animal
feeding stuffs: Methods of sampling and analysis”, the secretariat of which is held by NEN.
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 April 2020, and conflicting national standards shall be
withdrawn at the latest by April 2020.
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.
This document has been prepared under a standardization request given to CEN by the European
Commission and the European Free Trade Association.
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, Turkey and the
United Kingdom.

3

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SIST EN 17270:2020
EN 17270:2019 (E)
Introduction
Theobromine is naturally present in the cacao tree and its seeds and consequently in cocoa product and
by-products. Cocoa bean shells, cocoa bean meal, cocoa germs and discarded confectionery are used for
feed purposes in Europe. Maximum levels of theobromine in feeding stuffs are controlled by EU
regulations.
WARNING — the use of this protocol involves hazardous materials, operations and equipment.
This protocol does not purport to address all the safety problems associated with its use. It is the
responsibility of the user of this protocol to establish appropriate health and safety practices
and determine the compatibility with regulatory limitations prior to use.

4

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SIST EN 17270:2020
EN 17270:2019 (E)
1 Scope
This document specifies a test method for the determination of theobromine in feed material or
compound feed in the working range 27 mg/kg to 307 mg/kg using liquid chromatography coupled to a
UV detector (HPLC-UV) or in the working range 49 mg/kg to 307 mg/kg using liquid chromatography
with tandem mass spectrometry (LC-MS/MS).
This method has been fully validated using complementary compound feed for adult dogs and
complementary compound feed for horses.
This method is also considered applicable for determining theobromine in baking chocolate using either
HPLC-UV or LC-MS/MS systems.
The working range can be extended provided the extended range is validated.
2 Normative references
There are no normative references in this document.
3 Terms and definitions
No terms and definitions are listed in this document.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
• IEC Electropedia: available at http://www.electropedia.org/
• ISO Online browsing platform: available at https://www.iso.org/obp
4 Principle
A test portion of finely ground and homogeneous material is defatted with hexane, an internal standard
added and the theobromine extracted into ammonium acetate buffer. The extract is cleaned with the
addition of Carrez reagents, filtered and the extract analysed by liquid chromatography with UV
detection. Alternatively, the theobromine content can be determined by liquid chromatography tandem
mass spectrometry (LC-MS/MS) providing it can be demonstrated that there is no interference from the
sample matrix.
5 Reagents
Use only reagents of recognized analytical grade unless otherwise specified. Commercially available
solutions with equivalent properties to those listed may be used. References to products or vendors are
for information only and do not preclude the use of products of similar quality from alternative
suppliers.
For reagents specific to the analysis of the extracts by HPLC-UV see Annex A. For reagents specific to the
analysis of the extracts by LC-MS/MS see Annex B.
WARNING — Dispose of waste solvents according to applicable environmental rules and regulations.
5.1 Ammonium acetate, analytical reagent grade
5.2 Glacial acetic acid, 99,5 %
5.3 Acetic acid, 1 mol/l
5

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SIST EN 17270:2020
EN 17270:2019 (E)
Add 5,7 ml of glacial acetic acid (5.2) to 60 ml high-purity water (5.4) in a 100 ml volumetric flask, mix
thoroughly and dilute to the mark with water (5.4). Mix the flask contents thoroughly again by
inversion before use.
5.4 Water, LC-MS grade or of comparable purity, e.g. resistance of 18,2 MΩ cm or conductivity of
55 nS/cm at 20 °C
5.5 2,5 mol/l Ammonium acetate buffer, pH 5,5
Weigh 192,7 g ± 0,1 g ammonium acetate (5.1) into a 1 l beaker and dissolve in approximately 600 ml of
high-purity water (5.4). Using a calibrated pH meter (6.13), adjust the pH of the buffer using initially
glacial acetic acid (5.2) and then 1 mol/l acetic acid (5.3) until it is in the range of pH 5,4 to 5,6.
Quantitatively transfer the pH-adjusted buffer from the beaker to a 1 l volumetric flask and then dilute
to volume with water (5.4). Mix thoroughly by inversion prior to use. This solution is stable for up to
1 month when stored at room temperature.
5.6 Hexane, reagent grade
5.7 Zinc acetate dihydrate, reagent grade
5.8 Potassium ferrocyanide trihydrate, reagent grade
5.9 Carrez reagent I
Weigh 219 g ± 1 g zinc acetate dihydrate (5.7) into a 1 l beaker, add 30 ml glacial acetic acid (5.2) and
approximately 800 ml water (5.4). Mix thoroughly until dissolved, transfer to a 1 l volumetric flask and
dilute to volume with water (5.4). Mix well before use. This solution is stable for up to 3 months when
stored at room temperature.
5.10 Carrez reagent II
Weigh 106 g ± 1 g potassium ferrocyanide trihydrate (5.8) into a 1 l beaker and add approximately
800 ml water (5.4). Mix thoroughly until dissolved, transfer to a 1 l volumetric flask and dilute to
volume with water (5.4). Mix well before use. This solution is stable for up to 3 months when stored at
room temperature.
5.11 Theobromine, ≥ 98,5 %
5.12 Theobromine stock solution, 125 µg/ml
Weigh 62,5 mg ± 1 mg theobromine (5.11) into a 500 ml volumetric flask and add approximately 400 ml
water (5.4). Place in an ultrasonic bath until the theobromine has completely dissolved then dilute to
volume with water (5.4). Mix well before use. The exact weight of theobromine taken should be
recorded and the concentration of the solution calculated. This solution is stable for up to 1 month
when stored at 2 °C to 8 °C.
5.13 7-(β-Hydroxyethyl)theophylline, ≥ 98 %
5.14 7-(β-Hydroxyethyl)theophylline internal standard stock solution, 1 mg/ml
Weigh 100 mg ± 1 mg 7-(β-Hydroxyethyl)theophylline (5.13) into a 100 ml volumetric flask and add
approximately 80 ml water (5.4). Shake well to dissolve then dilute to volume with water (5.4). Mix well
before use. This solution is stable for up to 1 month when stored at 2 °C to 8 °C.
6

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SIST EN 17270:2020
EN 17270:2019 (E)
5.15 7-(β-Hydroxyethyl)theophylline internal standard solution, 100 µg/ml
Pipette 1 ml 7-(β-Hydroxyethyl)theophylline internal standard stock solution, 1 mg/ml (5.14) into a
10 ml volumetric flask and dilute to volume with water (5.4). Mix well before use. This solution is stable
for up to 1 month when stored at 2 °C to 8 °C.
5.16 Calibration standards
Add by pipette 7-(β-Hydroxyethyl)theophylline solution, 100 µg/ml (5.15) and different volumes of
theobromine stock solution, 125 µg/ml (5.12), into six 10 ml volumetric flasks such that six calibration
standards across the calibration range are obtained. The solutions should be diluted to volume with
water (5.4). These solutions are stable for up to 1 week when stored at room temperature.
Table 1 provides examples of volumes to be taken to obtain calibration standards at 0 µg/ml, 0,5 µg/ml,
1, 5 µg/ml, 10 µg/ml and 15 µg/ml theobromine. If the extraction procedure is followed, the calibration
standards are equivalent to 0 mg/kg, 20 mg/kg, 40 mg/kg, 200 mg/kg, 400 mg/kg and 600 mg/kg in
the sample. The exact concentration of the calibration standards should be calculated from the weight
of theobromine used to prepare the stock standard solution (5.12).
Table 1 — Suggested Calibration Standards
Calibration Standard (nominal concentration µg/ml) 0 0,5 1 5 10 15
Volume (µl) of
0 40 80 400 800 1200
125 µg/ml theobromine (5.12)
Volume (µl) of
100 µg/ml 200 200 200 200 200 200
7-(β-Hydroxyethyl)theophylline (5.15)
5.17 Quality control material
It is recommended that a suitable quality control material be analysed in every batch, for example NIST
SRM 2384 Baking chocolate, certified value for theobromine (11 600 ± 1 100) mg/kg. For NIST
SRM 2384 a sample weight of 1,0 g ± 0,1 g should be used, 2 ml of 1 mg/ml 7-(β-
Hydroxyethyl)theophylline internal standard stock solution (5.14) should be added and the extract
should be diluted, for example, by a factor of 10 prior to analysis by HPLC-UV or LC-MS/MS.
6 Apparatus
Standard laboratory glassware and equipment including the following:
6.1 Mill, single mill or multiple mills, capable of comminuting test materials to particle sizes
of < 500 μm
6.2 Sieve, 500 µm
6.3 Mixer, capable of sufficiently homogenizing the comminuted test materials
6.4 Conical polypropylene (PP) screw-cap centrifuge tubes, with screw cap, 50 ml or similar
6.5 Balance, with a mass resolution of 0,001 g or better
6.6 Centrifuge, capable of generating a relative centrifugal force (rcf) of 3000 g
7

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SIST EN 17270:2020
EN 17270:2019 (E)
6.7 Glass vials for use in the autosampler (usually approximately 1,5 ml capacity) and screw caps or
equivalent
6.8 Nylon syringe filter, small internal volume, pore size: 0,45 μm
6.9 General purpose filter papers
6.10 Vortex mixer
6.11 Water bath, capable of maintaining 40 °C ± 5 °C.
6.12 Ultrasonic bath
6.13 Equipment for measuring pH, calibrated and sufficiently accurate for the purposes of this
European Standard
7 Procedure
7.1 Sample preparation
It is important that the laboratory receives a sample which is truly representative and has not been
damaged or altered during transport or storage. Laboratory samples should be taken and prepared in
accordance with European legislation where applicable [1]. The laboratory sample should be finely
ground and thoroughly mixed using a mill (6.1) and a mixer (6.3) or another process for which
adequate homogenization has been demonstrated, before a test portion is taken for analysis.
It is important that the test portion is taken from a subsample which is sufficiently homogenous with a
particle size of ≤ 500 μm. Care should be taken not to overheat the sample during this process.
7.2 Extraction
Weigh 2,5 g ± 0,1 g homogenized sample into a polypropylene screw-cap tube (6.4). Add 6 ml hexane
(5.6) and mix thoroughly by vortex (6.10) then centrifuge at 2 700 g for 5 min (6.6). Discard the top
hexane layer.
Repeat the hexane extraction two more times, each with 3 ml hexane.
Dry the sample with a stream of nitrogen to remove the last traces of hexane then add 0,2 ml of
1 mg/ml 7-(β-Hydroxyethyl)theophylline internal standard (5.14).
Add 25 ml 2,5 mol/l ammonium acetate, pH 5,5 (5.5), shake vigorously then place in a water bath (6.11)
set at 40 °C ± 1 °C for (15 ± 2) min. Vortex to mix thoroughly ensuring that no solid material remains
adhered to the bottom of the tube, then place in an ultrasonic bath (6.12) for (20 ± 2) min.
Transfer the sample and extract to a 100 ml volumetric flask, using water (5.4) to rinse the
polypropylene tube.
Add 5 ml Carrez I (5.9) and mix well by hand. Add 5 ml Carrez II (5.10) and mix well by hand.
Dilute to volume with water (5.4) and mix well then filter through a general purpose filter paper (6.9).
Pass an aliquot of the filtrate through a 0,45 µm syringe filter (6.8).
8

----------------------
...

SLOVENSKI STANDARD
oSIST prEN 17270:2018
01-oktober-2018
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NUPQLKPHãDQLFDKSUHGYVHPL]NDNDYDSULGREOMHQLKVHVWDYLQVWHNRþLQVNR
NURPDWRJUDILMR.RPSOHPHQWDUQLHOHPHQW
Animal feeding stuffs: Methods of sampling and analysis - Determination of theobromine
in feed materials and compound feed, including cocoa derived ingredients, by liquid
chromatography - Complementary element
Futtermittel: Probenahme- und Untersuchungsverfahren - Bestimmung von Theobromin
in Einzelfuttermitteln, vor allem aus Kakao gewonnen sowie in Mischfuttermitteln mittels
Flüssigchromatographie
Aliments des animaux : Méthodes d'échantillonnage et d'analyse - Détermination par
chromatographie en phase liquide de la teneur en théobromine dans les matières
premières destinées aux aliments des animaux et dans les aliments composés pour
animaux, y compris les ingrédients issus du cacao
Ta slovenski standard je istoveten z: prEN 17270
ICS:
65.120 Krmila Animal feeding stuffs
71.040.50 Fizikalnokemijske analitske Physicochemical methods of
metode analysis
oSIST prEN 17270:2018 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

---------------------- Page: 1 ----------------------
oSIST prEN 17270:2018

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oSIST prEN 17270:2018


DRAFT
EUROPEAN STANDARD
prEN 17270
NORME EUROPÉENNE

EUROPÄISCHE NORM

August 2018
ICS 65.120
English Version

Animal feeding stuffs: Methods of sampling and analysis -
Determination of theobromine in feed materials and
compound feed, including cocoa derived ingredients, by
liquid chromatography - Complementary element
Aliments des animaux : Méthodes d'échantillonnage et Futtermittel: Probenahme- und
d'analyse - Détermination par chromatographie en Untersuchungsverfahren - Bestimmung von
phase liquide de la teneur en théobromine dans les Theobromin in Einzelfuttermitteln, vor allem aus
matières premières destinées aux aliments des Kakao gewonnen sowie in Mischfuttermitteln mittels
animaux et dans les aliments composés pour animaux, Flüssigchromatographie
y compris les ingrédients issus du cacao
This draft European Standard is submitted to CEN members for enquiry. It has been drawn up by the Technical Committee
CEN/TC 327.

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, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania,
Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland,
Turkey 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
© 2018 CEN All rights of exploitation in any form and by any means reserved Ref. No. prEN 17270:2018 E
worldwide for CEN national Members.

---------------------- Page: 3 ----------------------
oSIST prEN 17270:2018
prEN 17270:2018 (E)
Contents
Page
European foreword . 4
Introduction . 5
1 Scope . 6
2 Normative references . 6
3 Terms and definitions . 6
4 Principle . 6
5 Reagents . 6
5.1 Ammonium Acetate . 6
5.2 Glacial Acetic Acid, 99,5 % w/v . 6
5.3 Acetic acid, 1 M . 6
5.4 Water, LC-MS grade or of comparable purity, e.g. resistance of 18,2 MΩ cm or
conductivity of 55 nS/cm at 20 °C. 7
5.5 Ammonium acetate. 7
5.6 2,5 M Ammonium acetate buffer, pH 5,5. 7
5.7 Hexane . 7
5.8 Zinc acetate dihydrate . 7
5.9 Potassium ferrocyanide trihydrate . 7
5.10 Carrez reagent I . 7
5.11 Carrez reagent II . 7
5.12 Theobromine, ≥ 98,5 % . 7
5.13 Theobromine stock solution, 125 µg/ml . 7
5.14 7-(β-Hydroxyethyl)theophylline, ≥ 98 % . 7
5.15 7-(β-Hydroxyethyl)theophylline stock solution, 1 mg/ml . 7
5.16 7-(β-Hydroxyethyl)theophylline solution, 100 µg/ml . 8
5.17 Calibration standards . 8
5.18 Quality control material . 8
6 Apparatus . 8
7 Procedure. 9
7.1 Sample preparation . 9
7.2 Extraction . 9
8 Chromatographic analysis . 9
9 Calculation of results . 10
10 Accuracy . 11
11 Test report . 11
Annex A (informative)  Example HPLC-UV conditions . 12
A.1 HPLC-UV analysis . 12
A.2 Reagents . 12
A.3 Apparatus . 13
2

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oSIST prEN 17270:2018
prEN 17270:2018 (E)
A.4 HPLC conditions . 13
A.5 Example chromatogram . 14
Annex B (informative) Example LC-MS/MS conditions . 15
B.1 General . 15
B.2 LC-MS/MS analysis . 15
B.3 Reagents . 16
B.4 Apparatus . 16
B.5 HPLC conditions . 17
B.6 MS conditions . 17
B.7 Example chromatograms . 18
Annex C (informative) Method performance data obtained during single laboratory
validation . 22
Annex D (informative) Method performance data obtained during collaborative trial . 23
Bibliography . 25

3

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oSIST prEN 17270:2018
prEN 17270:2018 (E)
European foreword
This document (prEN 17270:2018) has been prepared by Technical Committee CEN/TC 327 “Animal
feeding stuffs: Methods of sampling and analysis”, the secretariat of which is held by NEN.
This document is currently submitted to the CEN Enquiry.
This document has been prepared under a standardization request given to CEN by the European
Commission and the European Free Trade Association.

4

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oSIST prEN 17270:2018
prEN 17270:2018 (E)
Introduction
Theobromine is naturally present in the cacao tree and its seeds and consequently in cocoa product and
by-products. Cocoa bean shells, cocoa bean meal, cocoa germs and discarded confectionery are used for
feed purposes in Europe. Maximum levels of theobromine in feeding stuffs are controlled by EU
regulations.
WARNING — the use of this protocol involves hazardous materials, operations and equipment.
This protocol does not purport to address all the safety problems associated with its use. It is the
responsibility of the user of this protocol to establish appropriate health and safety practices
and determine the compatibility with regulatory limitations prior to use.

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1 Scope
This document method is applicable for the determination of theobromine in compound feed by liquid
chromatography with UV detection in the tested range of 27 mg/kg to 307 mg/kg. This method has
been validated using complementary compound feed for adult dogs and complementary compound
feedstuff for horses. The actual working range can extend beyond the tested range. Alternative
chromatography conditions using liquid chromatography tandem mass spectrometry (LC-MS/MS) are
also provided for the validated range of 49 mg/kg to 307 mg/kg. This method has also been shown to
be fit for purpose for the determination of theobromine in baking chocolate by both HPLC-UV and LC-
MS/MS.
2 Normative references
There are no normative references in this document.
3 Terms and definitions
No terms and definitions are listed in this document.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
• IEC Electropedia: available at http://www.electropedia.org/
• ISO Online browsing platform: available at http://www.iso.org/obp
4 Principle
A test portion of finely ground and homogeneous material is defatted with hexane, an internal standard
added and the theobromine extracted into ammonium acetate buffer. The extract is cleaned with the
addition of Carrez reagents, filtered and the extract analysed by liquid chromatography with UV
detection. Alternatively, the theobromine content can be determined by liquid chromatography tandem
mass spectrometry (LC-MS/MS) providing it can be demonstrated that there is no interference from the
sample matrix.
5 Reagents
Use only reagents of recognized analytical grade unless otherwise specified. Commercially available
solutions with equivalent properties to those listed may be used. References to products or vendors are
for information only and do not preclude the use of products of similar quality from alternative
suppliers.
For reagents specific to the analysis of the extracts by HPLC-UV see Annex A. For reagents specific to the
analysis of the extracts by LC-MS/MS see Annex B.
WARNING — Dispose of waste solvents according to applicable environmental rules and regulations.
5.1 Ammonium Acetate
5.2 Glacial Acetic Acid, 99,5 % w/v
5.3 Acetic acid, 1 M
Add 5,7 ml of glacial acetic acid (5.2) to 60 ml high-purity water (5.4) in a 100 ml volumetric flask, mix
thoroughly and dilute to the mark with water. Mix the flask contents thoroughly again by inversion
before use.
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5.4 Water, LC-MS grade or of comparable purity, e.g. resistance of 18,2 MΩ cm or
conductivity of 55 nS/cm at 20 °C
5.5 Ammonium acetate
5.6 2,5 M Ammonium acetate buffer, pH 5,5
Weigh 192,7 ± 0,1 g ammonium acetate (5.5) into a 1 l beaker and dissolve in approximately 600 ml of
high-purity water (5.4). Using a calibrated pH meter, adjust the pH of the buffer using initially glacial
acetic acid (5.2) and then 1 M acetic acid (5.3) until it is in the range of pH 5,4 to 5,6.
Quantitatively transfer the pH-adjusted buffer from the beaker to a 1 l volumetric flask and then dilute
to volume with water. Mix thoroughly by inversion prior to use. This solution is stable for up to 1 month
when stored at room temperature.
5.7 Hexane
5.8 Zinc acetate dihydrate
5.9 Potassium ferrocyanide trihydrate
5.10 Carrez reagent I
Weigh 219 ± 1 g zinc acetate dihydrate (5.8) into a 1 l beaker, add 30 ml glacial acetic acid (5.2) and
approximately 800 ml water (5.4). Mix thoroughly until dissolved, transfer to a 1 l volumetric flask and
dilute to volume with water. Mix well before use. This solution is stable for up to 3 months when stored
at room temperature.
5.11 Carrez reagent II
Weigh 106 ± 1 g potassium ferrocyanide trihydrate (5.9) into a 1 l beaker and add approximately
800 ml water (5.4). Mix thoroughly until dissolved, transfer to a 1 l volumetric flask and dilute to
volume with water. Mix well before use. This solution is stable for up to 3 months when stored at room
temperature.
5.12 Theobromine, ≥ 98,5 %
5.13 Theobromine stock solution, 125 µg/ml
Weigh 62,5 ± 1 mg theobromine (5.12) into a 500 ml volumetric flask and add approximately 400 ml
water (5.4). Place in an ultrasonic bath until the theobromine has completely dissolved then dilute to
volume with water. Mix well before use. The exact weight of theobromine taken should be recorded and
the concentration of the solution calculated. This solution is stable for up to 1 month when stored at 2 –
8 °C.
5.14 7-(β-Hydroxyethyl)theophylline, ≥ 98 %
5.15 7-(β-Hydroxyethyl)theophylline stock solution, 1 mg/ml
Weigh 100 ± 1 mg 7-(β-Hydroxyethyl)theophylline (5.14) into a 100 ml volumetric flask and add
approximately 80 ml water (5.4). Shake well to dissolve then dilute to volume with water. Mix well
before use. This solution is stable for up to 1 month when stored at 2 – 8 °C.
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5.16 7-(β-Hydroxyethyl)theophylline solution, 100 µg/ml
Pipette 1 ml 7-(β-Hydroxyethyl)theophylline stock solution, 1 mg/ml (5.15) into a 10 ml volumetric
flask and dilute to volume with water (5.4). Mix well before use. This solution is stable for up to 1 month
when stored at 2 – 8 °C.
5.17 Calibration standards
Add by pipette 7-(β-Hydroxyethyl)theophylline solution, 100 µg/ml (5.16) and different volumes of
theobromine stock solution, 125 µg/ml (5.13), into 6 10 ml volumetric flasks such that 6 calibration
standards across the calibration range are obtained. The solutions should be diluted to volume with
water. These solutions are stable for up to 1 week when stored at room temperature.
Table 1 provides examples of volumes to be taken to obtain calibration standards at 0, 0,5, 1, 5, 10 and
15 µg/ml theobromine. If the extraction procedure is followed, the calibration standards are equivalent
to 0 mg/kg, 20 mg/kg, 40 mg/kg, 200 mg/kg, 400 mg/kg and 600 mg/kg in the sample. The exact
concentration of the calibration standards should be calculated from the weight of theobromine used to
prepare the stock standard solution (5.13).
Table 1 — Suggested Calibration Standards
Calibration Standard
(nominal 0 0,5 1 5 10 15
concentration µg/ml)
Volume (µl) of
125 µg/ml 0 40 80 400 800 1200
theobromine
Volume (µl) of
100 µg/ml
200 200 200 200 200 200
7-(β-Hydroxyethyl)
theophylline
5.18 Quality control material
It is recommended that a suitable quality control material be analysed in every batch, for example NIST
SRM 2384 Baking chocolate, certified value for theobromine (11 600 ± 1 100) mg/kg. For NIST
SRM 2384 a sample weight of 1,0 ± 0,1 g should be used, 2 ml of 1 mg/ml 7-(β-
Hydroxyethyl)theophylline internal standard (5.15) should be added and the extract should be diluted,
for example, by a factor of 10 prior to analysis by HPLC-UV or LC-MS/MS.
6 Apparatus
Standard laboratory glassware and equipment including the following:
6.1 Mill, single mill or multiple mills, capable of comminuting test materials to particle sizes
of < 500 μm
6.2 Sieve, 500 µm
6.3 Mixer, capable of sufficiently homogenizing the comminuted test materials
6.4 Conical polypropylene (PP) screw-cap centrifuge tubes, with screw cap, 50 ml or similar
6.5 Balance, with a mass resolution of 0,001 g or better
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6.6 Centrifuge, capa
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