EN 17270:2019

SLOVENSKI STANDARD
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
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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.

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
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.
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.

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
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.
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 stabl
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