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 ultra-violet 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 and compound feed and for benzoic acid in water. This method determines the total extractable concentration of these organic acids and their salts.
The working range of the method must be 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. A working range between 5 mg/l and 100 mg/l should be 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 weigh and extraction volume, limits of quantification (LOQ), as calculated (Table 1) on the basis of a wavelength of 230 nm, should 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 per¬formance liquid chromatography with ultraviolet detection) fest.
Das Verfahren ist für die Verwendung zur Bestimmung von Benzoesäure und Sorbinsäure als Wirkstoffe in Futtermittelzusatzstoffen, 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.
Tabelle 1 — Bestimmungsgrenzen (LOQ) bei 230 nm
Organische Säure   LOQ
(mg/kg)
Benzoesäure   200
Sorbinsäure   200

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 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
Public Enquiry End Date
24-Oct-2018
Publication Date
01-Sep-2019
Current Stage
6060 - National Implementation/Publication (Adopted Project)
Start Date
19-Aug-2019
Due Date
24-Oct-2019
Completion Date
02-Sep-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.

---------------------- Page: 1 ----------------------
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.

---------------------- Page: 3 ----------------------
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
2

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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.
3

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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.
4

---------------------- Page: 6 ----------------------
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]]
5

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SIST EN 17298:2019
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.
6

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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)
S
V
where
C is the experimental concentration of the organic acid in the standard stock solution, in mg/ml;
S
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
...

SLOVENSKI STANDARD
oSIST prEN 17298:2018
01-oktober-2018
.UPDPHWRGHY]RUþHQMDLQDQDOL]H'RORþHYDQMHEHQ]RMVNHLQVRUELQVNHNLVOLQHV
WHNRþLQVNRNURPDWRJUDILMRYLVRNHORþOMLYRVWL +3/&
Animal feeding stuffs: Methods of sampling and analysis - Determination of benzoic and
sorbic acid by High Pressure 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: prEN 17298
ICS:
65.120 Krmila Animal feeding stuffs
71.040.40 Kemijska analiza Chemical analysis
oSIST prEN 17298:2018 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

---------------------- Page: 1 ----------------------
oSIST prEN 17298:2018

---------------------- Page: 2 ----------------------
oSIST prEN 17298:2018


DRAFT
EUROPEAN STANDARD
prEN 17298
NORME EUROPÉENNE

EUROPÄISCHE NORM

August 2018
ICS 65.120; 71.040.40
English Version

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

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 17298:2018 E
worldwide for CEN national Members.

---------------------- Page: 3 ----------------------
oSIST prEN 17298:2018
prEN 17298:2018 (E)
Contents
Page
European foreword . 4
Introduction . 5
1 Scope . 6
2 Normative references . 6
3 Term and definitions . 6
4 Principle . 7
5 Reagents . 7
5.1 Water, complying with grade 2 in accordance with EN ISO 3696:1995 . 7
5.2 Benzoic acid,p.a., minimum 99 % purity . 7
5.3 Sorbic acid, p.a., minimum 99 % purity . 7
5.4 Acetic acid, p.a., ω = 100 % . 7
5.5 Methanol, gradient grade, ω ≥ 99,8 % . 7
5.6 Sodium acetate trihydrate, p.a . 7
5.7 Sodium acetate solution, c = 1 mol/l . 7
5.8 Acetic acid solution, c = 1 mol/l . 7
5.9 Acetate buffer, c = 0,1 mol/l . 7
5.10 HPLC Mobile phase . 7
5.11 Extraction solution . 8
5.12 Standard stock solution, app. 500 mg/l . 8
6 Apparatus . 8
7 Sampling . 9
8 Preparation of test sample . 9
9 Procedure . 9
9.1 Extraction . 9
9.2 Calibration . 10
9.3 Measurement of the calibration standards and sample solutions . 11
9.4 Confirmation of identity . 11
10 Calculation and expression of results . 11
11 Precision . 12
11.1 Interlaboratory test . 12
11.2 Repeatability . 12
11.3 Reproducibility . 12
2

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oSIST prEN 17298:2018
prEN 17298:2018 (E)
12 Test report . 13
Annex A (informative) Precision data - Results of the validation study . 14
Annex B (informative) Flow chart . 17
Bibliography . 18
3

---------------------- Page: 5 ----------------------
oSIST prEN 17298:2018
prEN 17298:2018 (E)
European foreword
This document (prEN 17298:2018) 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 mandate given to CEN by the European Commission and the
European Free Trade Association.
4

---------------------- Page: 6 ----------------------
oSIST prEN 17298:2018
prEN 17298:2018 (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 the animal
feeding by improving the animals’ performance and decrease 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.
5

---------------------- Page: 7 ----------------------
oSIST prEN 17298:2018
prEN 17298:2018 (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 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 weigh 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 3696:1995, Water for analytical laboratory use - Specification and test methods (ISO 3696:1987)
EN ISO 6498, Animal feeding stuffs - Guidelines for sample preparation (ISO 6498)
3 Term 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]]
6

---------------------- Page: 8 ----------------------
oSIST prEN 17298:2018
prEN 17298:2018 (E)
4 Principle
The sample is extracted with a mixture of sodium acetate buffer and methanol. The extract is filtrated 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, complying with grade 2 in accordance with EN ISO 3696:1995
5.2 Benzoic acid,p.a., minimum 99 % purity
5.3 Sorbic acid, p.a., minimum 99 % purity
5.4 Acetic acid, p.a., ω = 100 %
5.5 Methanol, gradient grade, ω ≥ 99,8 %
5.6 Sodium acetate trihydrate, p.a
5.7 Sodium acetate solution, c = 1 mol/l
Dissolve 13,6 g sodium acetate (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
By means of pipette add 50,0 ml sodium acetate solution (5.7) and 50,0 ml acetic acid solution (5.8) in a
1000 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.
7

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oSIST prEN 17298:2018
prEN 17298:2018 (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.
EXAMPLE Example of dilution:
Determine the actual concentration of the stock solution using the reference standard purity value
provided by the supplier according to Fomula (1).
mP×
C = (
...

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