CEN/TS 15754:2008

SLOVENSKI STANDARD
01-december-2008
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Animal feeding stuffs - Determination of sugar content - High performance exchange
chromatographic method (HPAEC-PAD)
Futtermittel - Bestimmung des Zuckergehalts - Hochleistungs-Anionenaustausch-
Chromatographieverfahren (HPAEC-PAD)
Aliments des animaux - Détermination de la teneur en sucre - Chromatographie
d'échange d'anions haute performance couplée à la détection par ampérométrie pulsée
(HPAEC-PAD)
Ta slovenski standard je istoveten z: CEN/TS 15754:2008
ICS:
65.120 Krmila Animal feeding stuffs
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

TECHNICAL SPECIFICATION
CEN/TS 15754
SPÉCIFICATION TECHNIQUE
TECHNISCHE SPEZIFIKATION
September 2008
ICS 65.120
English Version
Animal feeding stuffs - Determination of sugar content - High
performance exchange chromatographic method (HPAEC-PAD)
Aliments des animaux - Détermination de la teneur en Futtermittel - Bestimmung des Zuckergehalts -
sucre - Chromatographie d'échange d'anions haute Hochleistungs-Anionenaustausch-
performance couplée à la détection par ampérométrie Chromatographieverfahren (HPAEC-PAD)
pulsée (HPAEC-PAD)
This Technical Specification (CEN/TS) was approved by CEN on 17 May 2008 for provisional application.
The period of validity of this CEN/TS is limited initially to three years. After two years the members of CEN will be requested to submit their
comments, particularly on the question whether the CEN/TS can be converted into a European Standard.
CEN members are required to announce the existence of this CEN/TS in the same way as for an EN and to make the CEN/TS available
promptly at national level in an appropriate form. It is permissible to keep conflicting national standards in force (in parallel to the CEN/TS)
until the final decision about the possible conversion of the CEN/TS into an EN is reached.
CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Cyprus, Czech Republic, Denmark, Estonia, Finland,
France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal,
Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
Management Centre: rue de Stassart, 36  B-1050 Brussels
© 2008 CEN All rights of exploitation in any form and by any means reserved Ref. No. CEN/TS 15754:2008: E
worldwide for CEN national Members.

Contents Page
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 Preparation of the test sample .8
9 Procedure .8
10 Calculation and expression of the results .11
11 Precision.12
12 Test report .12
Annex A (informative) Results of an interlaboratory test for each individual sugar.13
Bibliography .20

Foreword
This document (CEN/TS 15754:2008) 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.
Attention is drawn to the possibility that some of the elements of this document may be the subject of patent
rights. CEN [and/or CENELEC] shall not be held responsible for identifying any or all such patent rights.
This document has been prepared under a mandate given to CEN by the European Commission and the
European Free Trade Association, and supports essential requirements of EU Directive(s).
According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following
countries are bound to announce this CEN Technical Specification: Austria, Belgium, Bulgaria, Cyprus, Czech
Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia,
Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain,
Sweden, Switzerland and United Kingdom.
Introduction
This Technical Specification describes an application of the High Performance Anion Exchange
Chromatography with Pulsed Amperometric Detection (HPAEC-PAD) for the determination of different
individual sugars, both mono- and disaccharides, in dry animal feeding stuffs.
HPAEC-PAD is a modern liquid chromatographic technique with high selectivity and sensitivity for especially
carbohydrates. Due to this high selectivity and sensitivity of HPAEC-PAD for carbohydrates just a minimum of
sample clean-up is required in most carbohydrate applications in food and feed matrices.
This technical specification is meant as an introduction of a new method for the determination of the content of
the individual sugars present in animal feeding stuffs. The document should be used to get experienced with
this new powerful technique and with the described method for the quantification of the individual sugars in
animal feeding stuffs.
Currently the HPAEC-PAD equipment is mainly used in research environments and not wide spread in use in
the feed and control laboratories. Therefore, the method is at this time not yet suitable for official control
applications of the sugar content in feeding stuffs. It is however expected that within several years more and
more laboratories in the field will have this instrumentation at their disposal.
1 Scope
This Technical Specification describes the quantitative determination of specific sugars (glucose, fructose,
galactose, sucrose, maltose, and lactose) in dry animal feeding stuffs at the g/kg level by a sophisticated high
performance anion exchange chromatography in combination with pulsed amperometric detection (HPAEC-
PAD).
2 Normative references
The following referenced documents are indispensable for the application 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 3696, Water for analytical laboratory use - Specification and test methods (ISO 3696:1987)
ISO 6498:1998, Animal feeding stuffs — Preparation of test samples
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
total sugar content
sum of the content of the individual sugars, glucose, fructose, galactose, maltose, sucrose and lactose,
expressed as g/kg in the sample as such
4 Principle
The sugars present in the test potion of the animal feeding stuff sample are extracted with water. The clean-up
of the aqueous extract is done by either a C-18 SPE procedure, or by a deproteination with acetonitrile. After
the clean-up, the sample solution is diluted and the sugars present are separated by high performance anion
exchange chromatography and detected by pulsed amperometric detection. Quantitation of the sugars
present is done by comparison of the peak areas and/or heights with those of an external calibration graph
prepared with standard solutions.
5 Reagents
Use only reagents of recognized analytical grade, unless otherwise specified.
5.1 Water, complying with EN ISO 3696, grade 3.
5.2 Acetonitrile
5.3 Dimethyl Sulfoxide (DMSO)
5.4 Sodium hydroxide solution, mass fraction, w(NaOH) = 50 % in water
The reagent should contain the minimum amount of carbonate and mercury. Do not shake or stir the solution
before use.
5.5 Sodium acetate trihydrate (CH COONa·3H O)
3 2
5.6 Eluent 1 (E1), aqueous solution of 500 mmol/l sodium hydroxide (NaOH)
Add to a 1 000 ml volumetric flask about 800 ml degassed water (eluent 3, 5.8) followed by 40,0 g of sodium
hydroxide 50 % (m/m) (5.4). Then dilute the aqueous solution to the mark with degassed demineralised water
(5.8).
5.7 Eluent 2 (E2), aqueous solution of 500 mmol/l sodium acetate
Add to a 1 000 ml volumetric flask about 800 ml degassed demineralised water (eluent 3, 5.8) followed by
68,0 g sodium acetate trihydrate (CH COONa·3H O) (5.5). Then dilute the aqueous solution to the mark with
3 2
degassed demineralised water (5.8).
5.8 Eluent 3 (E3), degassed demineralized water
Filter the demineralized water through a 0,2 membrane filter. Degas by sparging with helium for at least
30 min.
5.9 Sugar standard solutions
5.9.1 Preparation stock solution
Prepare daily fresh aqueous solutions of glucose, fructose, galactose, maltose, sucrose, and lactose. Weigh
approximately 40 mg of each sugar to the nearest 0,1 mg into separate 100 ml volumetric flasks (6.6) and
dilute to the mark with water (stock standard solutions of 400 mg/l).
NOTE 1 Mixed standard solutions can also be prepared once the retention time of the individual sugars is known under
the prevailing chromatographic conditions.
NOTE 2 The standard solutions can be further diluted to reach sugar concentrations similar to those found in the
sample solutions.
5.9.2 Working standard solutions for calibration
Prepare dilutions of the sugar standard solutions as specified in Table 1. Dilute 0,25 ml of each obtained
standard solution with 1,25 ml DMSO (5.3) in an HPLC vial. Close the vial and mix well.
Table 1 — Preparation aqueous DMSO sugar standard solutions
Standard solution Volume sugar Volume water Sugar concentration after
calibration graph standard stock dilution with DMSO
solution
ml
a
µg/ml
ml
Standard 1 0,00 10,00 0
Standard 2 0,10 9,90 0,67
Standard 3 0,30 9,70 2,00
Standard 4 0,70 9,30 4,67
Standard 5 1,40 8,60 9,33
Standard 6 2,00 8,00 13,33
a
Based upon a sugar content of exactly 400 mg/l in the aqueous stock solution.

These aqueous DMSO sugar standard solutions will be used for preparing the calibration graphs.
NOTE Aqueous DMSO is a good solvent for carbohydrates and the use of DMSO prevent microbial deterioration of
the carbohydrates in the solutions.
6 Apparatus
Usual laboratory equipment and, in particular the following:
6.1 Analytical balance, capable of weighting to an accuracy of ± 0,1 mg.
6.2 Glass centrifugation tubes, of a capacity of 50 ml.
6.3 Homogenizer
6.4 Swing-out centrifuge, adjusted to a centrifugation force of about 2 800 g.
6.5 Screw cap closed centrifugation tubes, with screw caps with a polytetrafluoroethylene (PTFE) inlay,
of a capacity of about 10 ml.
6.6 One mark volumetric flasks, of a capacity of 100 ml and 1 000 ml.
6.7 Dispensors, resistant to organic solvents and adjusted to 2,5 ml and 20 ml.
6.8 Disposable C-18 SPE cartridges, to be used according to the manufacturer’s recommendations.

An IKA Ultra turrax with apprropriate probe is an example of a suitable commercially available homogenizer.
6.9 Metal free liquid chromatographic system , applicable for a ternair gradient elution profile.
6.10 Column oven, adjusted at 30 °C.
6.11 High performance anion exchange column , filled with pellicularpolystyrene-divinylbenzene resin
and pre- column (guard column), mounted in the column oven (6.10).
6.12 Pulsed amperometric detector , with a gold electrode, situated together with the columns in the
column oven at 30 °C (6.10).
Detector settings are as follows:
 0 s – 0,40 s potential working electrode + 0,1 V;
 0,41 s – 0,60 s potential working electrode + 0,7 V;
 0,61 s – 1,00 s potential working electrode – 0,1 V;
 integration window 0,20 s – 0,40 s.
6.13 Integrator chromatography data system
7 Sampling
It is important that the laboratory receives a sample which is truly representative and has not been damaged
or changed during transport and/or storage.
Sampling is not a part of the method specified in this Technical Specification. A recommended sampling
method is given in EN-ISO 6497 [1].
8 Preparation of the test sample
Prepare the test sampl
...