Animal feeding stuffs - Methods of sampling and analysis - Determination of benzoic and sorbic acid by High Performance Liquid Chromatography (HPLC)

This document specifies a method for the determination of benzoic acid and sorbic acid in animal feeding stuffs by high-performance liquid chromatography method with ultraviolet detection (HPLC-UV).
The method is intended to be used for the determination of benzoic acid and sorbic acid as active substances in feed additives, premixtures, feed materials and compound feed and for benzoic acid in water. This method determines the total extractable concentration of these organic acids and their salts.
It is advisable that the working range of the method is determined for each organic acid by the user of this standard. The lower limit of the working range depends on the matrix and the interferences encountered. It is advisable that a working range between 5 mg/l and 100 mg/l is accessible.
The method was successfully tested in an inter-laboratory study in concentrations between 0,02 % up to 9,0 %.
On the basis of the referred working range, sample weight and extraction volume, limits of quantification (LOQ), as calculated (Table 1) on the basis of a wavelength of 230 nm, can be achievable.

Futtermittel - Probenahme- und Untersuchungsverfahren - Bestimmung von Benzoesäure und Sorbinsäure mittels Hochleistungs-Flüssigchromatographie (HPLC)

Dieses Dokument legt ein Verfahren zur Bestimmung von Benzoesäure und Sorbinsäure in Futtermitteln mittels Hochleistungs-Flüssigchromatographie mit Ultraviolett Detektion (HPLC UV, en: high performance liquid chromatography with ultraviolet detection) fest.
Das Verfahren ist für die Verwendung zur Bestimmung von Benzoesäure und Sorbinsäure als Wirkstoffe in Futtermittelzusatzstoffen, Futtermittel Ausgangserzeugnissen, Vormischungen, Mischfuttermitteln und für Benzoesäure in Wasser vorgesehen. Dieses Verfahren ermittelt die extrahierbare Gesamtkonzentration dieser organischen Säuren und ihrer Salze.
Dem Anwender dieser Norm wird empfohlen, den Arbeitsbereich des Verfahrens für jede organische Säure zu ermitteln. Der untere Grenzwert des Arbeitsbereiches hängt von der Matrix und den aufgetretenen Störungen ab. Es wird empfohlen, dass ein Arbeitsbereich zwischen 5 mg/l und 100 mg/l eingehalten wird.
Das Verfahren wurde im Rahmen einer Ringversuchsstudie bei Konzentrationen von 0,02 % bis 9,0 % erfolg¬reich geprüft.
Auf der Grundlage des zugehörigen Arbeitsbereichs, der Probenmasse und des Extraktionsvolumens können Bestimmungsgrenzen (LOQ) (en: limits of quantification) wie auf der Grundlage einer Wellen¬länge von 230 nm berechnet (Tabelle 1) erreichbar sein.

Aliments des animaux - Méthodes d’échantillonnage et d’analyse - Dosage de l’acide benzoïque et de l’acide sorbique par chromatographie liquide à haute pression (CLHP)

Le présent document spécifie une méthode de dosage de l’acide benzoïque et de l’acide sorbique dans les aliments pour animaux, par chromatographie liquide à haute performance avec détecteur UV (CLHP UV).
La méthode est destinée à permettre le dosage de l’acide benzoïque et de l’acide sorbique présents comme substances actives dans les additifs pour l’alimentation animale, les prémélanges, les matières premières pour aliments et les aliments composés pour animaux, ainsi que le dosage de l’acide benzoïque dans l’eau. Cette méthode détermine la concentration totale extractible de ces acides organiques et de leurs sels.
Il est recommandé à l’utilisateur de la présente norme de déterminer le domaine de mesure de la méthode pour chaque acide organique. La limite inférieure du domaine de mesure dépend de la matrice et des interférences rencontrées. Il convient de pouvoir atteindre un domaine de mesure compris entre 5 mg/l et 100 mg/l.
La méthode a été soumise à essai avec succès lors d’une étude interlaboratoires à des concentrations allant de 0,02 % à 9,0 %.
En s’appuyant sur le domaine de mesure mentionné, le poids de l’échantillon et le volume d’extraction, il est possible d’atteindre les limites de quantification (LQ), telles que calculées (Tableau 1) pour une longueur d’onde de 230 nm.
Tableau 1 - Limites de quantification (LQ) à 230 nm
Acide organique   LQ (mg/kg)
Acide benzoïque   200
Acide sorbique   200

Krma - Metode vzorčenja in analize - Določevanje benzojske in sorbinske kisline s tekočinsko kromatografijo visoke ločljivosti (HPLC)

Ta dokument določa metodo za določevanje benzojske kisline in sorbinske kisline v krmi s tekočinsko kromatografijo visoke ločljivosti z UV-detekcijo (HPLC-UV).
Ta metoda je predvidena za določevanje benzojske kisline in sorbinske kisline kot aktivnih snovi v dodatkih za krmo, premiksih, krmnih mešanicah ter za benzojsko kislino v vodi. Ta metoda določa skupno koncentracijo teh organskih kislin in njihovih soli, ki jo je mogoče ekstrahirati.
Uporabnik tega standarda mora določiti delovni razpon metode za vsako organsko kislino. Spodnja meja delovnega razpona je odvisna od matrice in motenj, ki se pojavijo. Delovni razpon med 5 mg/l in 100 mg/l naj bi bil dosegljiv.
Metoda je bila uspešno preskušena v medlaboratorijski študiji pri koncentracijah od 0,02 % do 9 %.
Na podlagi omenjenega delovnega razpona, teže vzorca in volumna ekstrakta naj bi bile mejne vrednosti kvantifikacije (LOQ) dosegljive, kot so izračunane na podlagi valovne dolžine 230 nm (preglednica 1).

General Information

Status
Published
Publication Date
06-Aug-2019
Current Stage
6060 - Definitive text made available (DAV) - Publishing
Due Date
07-Aug-2019
Completion Date
07-Aug-2019

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SLOVENSKI STANDARD
SIST EN 17298:2019
01-oktober-2019

Krma - Metode vzorčenja in analize - Določevanje benzojske in sorbinske kisline s

tekočinsko kromatografijo visoke ločljivosti (HPLC)

Animal feeding stuffs - Methods of sampling and analysis - Determination of benzoic and

sorbic acid by High Performance Liquid Chromatography (HPLC)
Futtermittel - Probenahme- und Untersuchungsverfahren - Bestimmung von
Benzoesäure und Sorbinsäure mittels Hochleistungs-Flüssigchromatographie (HPLC)

Aliments des animaux - Méthodes d’échantillonnage et d’analyse - Dosage de l’acide

benzoïque et de l’acide sorbique par chromatographie liquide à haute pression (CLHP)

Ta slovenski standard je istoveten z: EN 17298:2019
ICS:
65.120 Krmila Animal feeding stuffs
71.040.40 Kemijska analiza Chemical analysis
SIST EN 17298:2019 en,fr,de

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

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SIST EN 17298:2019
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SIST EN 17298:2019
EN 17298
EUROPEAN STANDARD
NORME EUROPÉENNE
August 2019
EUROPÄISCHE NORM
ICS 65.120; 71.040.40
English Version
Animal feeding stuffs - Methods of sampling and analysis -
Determination of benzoic and sorbic acid by High
Performance Liquid Chromatography (HPLC)

Aliments des animaux - Méthodes d'échantillonnage et Futtermittel - Probenahme- und

d'analyse - Dosage de l'acide benzoïque et de l'acide Untersuchungsverfahren - Bestimmung von

sorbique par chromatographie liquide à haute pression Benzoesäure und Sorbinsäure mittels Hochleistungs-

(CLHP) Flüssigchromatographie (HPLC)
This European Standard was approved by CEN on 17 June 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 17298:2019 E

worldwide for CEN national Members.
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SIST EN 17298:2019
EN 17298:2019 (E)
Contents Page

European foreword ....................................................................................................................................................... 3

Introduction .................................................................................................................................................................... 4

1 Scope .................................................................................................................................................................... 5

2 Normative references .................................................................................................................................... 5

3 Terms and definitions ................................................................................................................................... 5

4 Principle ............................................................................................................................................................. 6

5 Reagents ............................................................................................................................................................. 6

6 Apparatus ........................................................................................................................................................... 7

7 Sampling ............................................................................................................................................................. 8

8 Preparation of test sample .......................................................................................................................... 8

9 Procedure........................................................................................................................................................... 8

9.1 General ................................................................................................................................................................ 8

9.2 Extraction ........................................................................................................................................................... 8

9.3 Calibration ......................................................................................................................................................... 9

9.4 Measurement of the calibration standards and sample solutions ............................................. 10

9.5 Confirmation of identity ............................................................................................................................ 10

10 Calculation and expression of results ................................................................................................... 10

11 Precision .......................................................................................................................................................... 11

11.1 Interlaboratory test ..................................................................................................................................... 11

11.2 Repeatability .................................................................................................................................................. 12

11.3 Reproducibility ............................................................................................................................................. 12

12 Test report ...................................................................................................................................................... 12

Annex A (normative) Flow chart............................................................................................................................ 13

Annex B (informative) Precision data — Results of the validation study ............................................... 14

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SIST EN 17298:2019
EN 17298:2019 (E)
European foreword

This document (EN 17298:2019) 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 February 2020, and conflicting national standards shall

be withdrawn at the latest by February 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.
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SIST EN 17298:2019
EN 17298:2019 (E)
Introduction

Organic acids and their salts such as citric acid, formic acid, lactic acid, acetic acid, propionic acid, fumaric

acid, benzoic acid and sorbic acid are animal feed additives which play an important role in animal feeding

by improving the animals’ performance and decreasing the development of pathogenic microorganisms

in the intestine especially in the pig production. Concerning the feed legislation the substances can be

used for different purposes depending on its functions and properties. According to their functional

principle or their function, the relevant organic acid could be allocated within one or more of the

functional groups mentioned in Annex I of Regulation (EC) no. 1831/2003 as preservative, acidity

regulators, flavouring compounds, silage additives or other zoo-technical additives.

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SIST EN 17298:2019
EN 17298:2019 (E)
1 Scope

This document specifies a method for the determination of benzoic acid and sorbic acid in animal feeding

stuffs by high-performance liquid chromatography method with ultraviolet detection (HPLC-UV).

The method is intended to be used for the determination of benzoic acid and sorbic acid as active

substances in feed additives, premixtures, feed materials and compound feed and for benzoic acid in

water. This method determines the total extractable concentration of these organic acids and their salts.

It is advisable that the working range of the method is determined for each organic acid by the user of

this standard. The lower limit of the working range depends on the matrix and the interferences

encountered. It is advisable that a working range between 5 mg/l and 100 mg/l is accessible.

The method was successfully tested in an inter-laboratory study in concentrations between 0,02 % up to

9,0 %.

On the basis of the referred working range, sample weight and extraction volume, limits of quantification

(LOQ), as calculated (Table 1) on the basis of a wavelength of 230 nm, can be achievable.

Table 1 — Limits of quantification (LOQ) at 230 nm
Organic acid LOQ (mg/kg)
Benzoic acid 200
Sorbic acid 200
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 3696, Water for analytical laboratory use — Specification and test methods (ISO 3696)

EN ISO 6498, Animal feeding stuffs — Guidelines for sample preparation (ISO 6498)

3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.

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
3.1
feed additives

substances, micro-organisms or preparations, other than feed material and premixtures, which are

intentionally added to feed or water
[SOURCE: Regulation (EC) No 1831/2003/Article 2/2 a [1]]
3.2
animal feeding stuffs

any substance or product, including additives, whether processed, partially processed or unprocessed,

intended to be used for oral feeding to animals
[SOURCE: Regulation (EC) No 178/2002/Article 2/4 [2]]
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EN 17298:2019 (E)
4 Principle

The sample is extracted with a mixture of sodium acetate buffer and methanol. The extract is filtered or

centrifuged and – if necessary – diluted. The amount of organic acids extracted from the sample is

determined with high-performance liquid chromatography (HPLC), applying a RP 18 column, in

conjunction with diode array detection (DAD) or ultraviolet detection (UV). The quantification is carried

out by an external calibration.

WARNING — The use of this European Standard can involve hazardous materials, operations and

equipment. This standard does not purport to address all the safety problems associated with its use. It

is the responsibility of the user of this European Standard to establish appropriate safety and health

practices and determine the applicability of regulatory limitations prior to use.

5 Reagents
5.1 Water, grade 2 in accordance with EN ISO 3696.
5.2 Benzoic acid, p.a., minimum 99 % purity.
5.3 Sorbic acid, p.a., minimum 99 % purity.
5.4 Acetic acid, p.a., w = 100 %.
5.5 Methanol, gradient grade, w ≥ 99,8 %.
5.6 Sodium acetate trihydrate, p.a.
5.7 Sodium acetate solution, c = 1 mol/l.

Dissolve 13,6 g sodium acetate trihydrate (5.6) with approximately 80 ml water (5.1) in a 100 ml

volumetric flask, mix and fill up to the mark with water (5.1).
The maximum storage time is 3 months at room temperature.
5.8 Acetic acid solution, c = 1 mol/l.

Dissolve 6,00 g acetic acid (5.4) with approximately 80 ml water (5.1) in a 100 ml volumetric flask, mix

and fill up to the mark with water (5.1).
The maximum storage time is 3 months at room temperature.
5.9 Acetate buffer, c = 0,1 mol/l.

Mix 50,0 ml sodium acetate solution (5.7) and 50,0 ml acetic acid solution (5.8) in a 1 000 ml volumetric

flask and add approximately 700 ml water (5.1). Adjust the pH with acetic acid solution (5.8) to 4,6. Fill

up to the mark with water (5.1).
The maximum storage time is 6 months at room temperature.

NOTE A pH of 4,6 is the best range for an optimal HPLC separation regarding the referred conditions.

5.10 HPLC Mobile phase

Mix acetate buffer (5.9) and methanol (5.5) in proportion of 75:25 (v/v) by means of graduated cylinder.

Filter for HPLC use.
The maximum storage time is 6 months at room temperature.
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SIST EN 17298:2019
EN 17298:2019 (E)
5.11 Extraction solution

Mix acetate buffer (5.9) and methanol (5.5) in proportion of 60:40 (v/v) by means of graduated cylinder.

The maximum storage time is 6 months at room temperature.
5.12 Standard stock solution, app. 500 mg/l.

Weigh 125 mg benzoic acid (5.2) and 125 mg sorbic acid (5.3) into the same 250 ml volumetric flask.

Dissolve with approximately 150 ml extraction solution (5.11), dissolve 5 min in an ultrasonic bath and

fill to the mark with extraction solution (5.11).
The maximum storage time is 2 months at 4 °C.

NOTE 1 The shelf life of the stock solution is limited because of esterification reactions.

NOTE 2 If available, single acid standard solutions with adequate and required specifications can be used.

Determine the exact concentration of the stock solution using the reference standard purity value

provided by the supplier according to Formula (1).
mP×
C = (1)
where

C is the experimental concentration of the organic acid in the standard stock solution, in mg/ml;

P is the purity of the organic acid standard given by the supplier divided by 100, in %;

NOTE For example 0,98.
m is the weighed mass of the organic acid, in mg;
V is the volume of the volumetric flask, in ml.
6 Apparatus
Usual laboratory equipment and, in particular, the following:

6.1 Laboratory grinder, capable of grinding to a particle size of less than or equal to 1,0 mm.

6.2 Analytical balance, suitable to accurately weigh between 0 g and 10 g with an accuracy of 0,1 mg.

6.3 Ultrasonic bath
6.4 pH meter capable of being read to at least one place of decimals.
6.5 Pipettes (electronic or manual) in the range 100 μl to 5 000 μl.
6.6 Centrifuge, approx. 5 000 × g.

6.7 Membrane filter for HPLC use, 0,45 µm (for example: Ready-to-use filter unit with a hydrophilic,

low protein-binding membrane made of regenerated cellulose).
6.8 Folded filter, pore size 4 µm to 7 µm (ash free paper filter).
6.9 HPLC system (pump, autosampler, column oven) with D
...

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