Animal feeding stuffs - Methods of sampling and analysis - Determination of organic acids by Ion Chromatography with Conductivity Detection (IC-CD)

This document specifies a method for the determination of organic acids in animal feeding stuffs by Ion Chromatography with conductivity detection (IC-CD).
The method is intended to be used for the determination of formic acid, lactic acid, propionic acid, citric acid, fumaric acid and malic acid as active substances in feed additives, premixtures, feed materials, compound feed and water and for acetic acid in a limited manner in the same matrices. This method determines the total extractable concentration of the above mentioned organic acids and their salts.
It is advisable that the user of this standard determines the working range of the method for each organic acid. The lower limit of the working range depends on the matrix and the interferences encountered. It is advisable that a working range between 10 mg/l and 100 mg/l is achievable.
The method was successfully tested in an inter-laboratory study in concentrations between 0,02 % up to 27 % of the above mentioned organic acids.
NOTE   Limitation occurs during simultaneous determination of high concentration of lactic acid and low concentration of acetic acid. If the ratio of concentration of lactic acid to acetic acid exceeds factor 20, the determination of acetic acid is not guaranteed.
On the basis of the referred working range, sample weight and extraction volume, limits of quantification (LOQ), as calculated (Table 1) can be achievable.

Futtermittel - Probenahme- und Untersuchungsverfahren - Bestimmung organischer Säuren mittels Ionenchromatographie mit Leitfähigkeitsdetektion (IC-CD)

Dieses Dokument legt ein Verfahren zur Bestimmung organischer Säuren in Futtermitteln mittels Ionen-chromatographie mit Leitfähigkeitsdetektion (en: IC-CD, ion chromatography with conductivity detection) fest.
Das Verfahren ist für die Verwendung zur Bestimmung von Ameisensäure, Milchsäure, Propionsäure, Zitronensäure, Fumarsäure und Apfelsäure als Wirkstoffe in Futtermittelzusatzstoffen, Vormischungen, Futtermittel-Ausgangserzeugnissen, Mischfuttermitteln und Wasser sowie für Essigsäure in begrenztem Maße in denselben Matrices vorgesehen. Dieses Verfahren ermittelt die extrahierbare Gesamtkonzentration der vorstehend aufgeführten 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 10 mg/l und 100 mg/l eingehalten wird.
Das Verfahren wurde im Rahmen einer Ringversuchsstudie bei Konzentrationen von 0,02 % bis zu 27 % der vorgenannten organischen Säuren erfolgreich geprüft.
ANMERKUNG Bei der gleichzeitigen Bestimmung einer hohen Konzentration von Milchsäure und einer niedrigen Konzentration von Essigsäure tritt eine Einschränkung auf. Wenn das Verhältnis der Konzentration der Milchsäure zu der von Essigsäure den Faktor 20 überschreitet, ist die Bestimmung der Essigsäure nicht sichergestellt.
Auf der Grundlage des zugehörigen Arbeitsbereichs, der Probenmasse und des Extraktionsvolumens können Bestimmungsgrenzen (en: LOQ, limits of quantification) wie berechnet (Tabelle 1) erreichbar sein.

Aliments des animaux - Méthodes d’échantillonnage et d’analyse - Dosage des acides organiques par chromatographie ionique avec détection conductimétrique (CI-DC)

Le présent document spécifie une méthode de dosage des acides organiques dans les aliments pour animaux par chromatographie ionique avec détection conductimétrique (CI-DC).
La méthode est destinée à permettre le dosage de l’acide formique, de l’acide lactique, de l’acide propionique, de l’acide citrique, de l’acide fumarique et de l’acide malique présents comme substances actives dans les additifs pour l’alimentation animale, les prémélanges, les matières premières pour aliments des animaux, les aliments composés pour animaux, ainsi que l’eau et l’acide acétique de manière limitée dans les mêmes matrices. Cette méthode détermine la concentration totale extractible des acides organiques susmentionnés 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 est recommandé de pouvoir atteindre un domaine de mesure compris entre 10 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 27 % pour les acides organiques susmentionnés.
NOTE   La détermination simultanée d’une concentration élevée d’acide lactique et d’une faible concentration d’acide acétique présente des limites. Si le rapport de la concentration d’acide lactique à la concentration d’acide acétique dépasse un facteur de 20, le dosage de l’acide acétique n’est pas garanti.
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).

Krma - Metode vzorčenja in analize - Določevanje organskih kislin z ionsko kromatografijo in detekcijo na osnovi prevodnosti (IC-CD)

Ta dokument določa metodo za določevanje organskih kislin v krmi z ionsko kromatografijo in detekcijo na osnovi prevodnosti (IC-CD).
Ta metoda je predvidena za določevanje mravljične kisline, mlečne kisline, propionske kisline, citronske kisline, fumarne kisline in jabolčne kisline kot aktivnih snovi v dodatkih za krmo, premiksih, krmnih mešanicah in vodi ter za varnostne preglede ocetne kisline pri istih matrikah. Ta metoda določa skupno koncentracijo omenjenih 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 10 [mg/l] in 100 [mg/l] naj bi bil dosegljiv.
Metoda je bila uspešno preskušena v medlaboratorijski študiji pri koncentracijah od 0,02 % do 27 % omenjenih organskih kislin.
OPOMBA:   Omejitev se pojavi pri hkratnem določevanju velike koncentracije mlečne kisline in majhne koncentracije ocetne kisline. Če razmerje med koncentracijo mlečne in ocetne kisline presega faktor 20, potem določevanje ocetne kisline ni zagotovljeno.
Na podlagi omenjenega delovnega razpona, teže vzorca in volumna ekstrakta naj bi bile mejne vrednosti kvantifikacije (LOQ) dosegljive, kot so izračunane (preglednica 1).

General Information

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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 17294:2019
01-oktober-2019
Krma - Metode vzorčenja in analize - Določevanje organskih kislin z ionsko
kromatografijo in detekcijo na osnovi prevodnosti (IC-CD)

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

acids by Ion Chromatography with Conductivity Detection (IC-CD)
Futtermittel - Probenahme- und Untersuchungsverfahren - Bestimmung organischer
Säuren mittels Ionenchromatographie mit Leitfähigkeitsdetektion (IC-CD)

Aliments des animaux - Méthodes d’échantillonnage et d’analyse - Dosage des acides

organiques par chromatographie ionique avec détection conductimétrique (CI-DC)
Ta slovenski standard je istoveten z: EN 17294:2019
ICS:
65.120 Krmila Animal feeding stuffs
71.040.40 Kemijska analiza Chemical analysis
SIST EN 17294:2019 en,fr,de

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

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SIST EN 17294:2019
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SIST EN 17294:2019
EN 17294
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 organic acids by Ion Chromatography
with Conductivity Detection (IC-CD)

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

d'analyse - Dosage des acides organiques par Untersuchungsverfahren - Bestimmung organischer

chromatographie ionique avec détection Säuren mittels Ionenchromatographie mit
conductimétrique (CI-DC) Leitfähigkeitsdetektion (IC-CD)
This European Standard was approved by CEN on 24 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 17294:2019 E

worldwide for CEN national Members.
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Contents Page

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

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

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

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

3 Terms and definitions ................................................................................................................................... 6

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

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

6 Apparatus ........................................................................................................................................................... 8

7 Sampling .......................................................................................................................................................... 11

8 Preparation of test sample ....................................................................................................................... 11

9 Procedure........................................................................................................................................................ 11

9.1 General ............................................................................................................................................................. 11

9.2 Calibration ...................................................................................................................................................... 13

9.3 Measurement of the calibration standards and sample solutions ............................................. 13

9.4 Confirmation of identity ............................................................................................................................ 13

10 Calculation and expression of results ................................................................................................... 14

11 Precision .......................................................................................................................................................... 14

11.1 Interlaboratory test ..................................................................................................................................... 14

11.2 Repeatability .................................................................................................................................................. 15

11.3 Reproducibility ............................................................................................................................................. 15

12 Test report ...................................................................................................................................................... 19

Annex A (normative) Flow Chart ......................................................................................................................... 20

Annex B (informative) Examples of measuring equipment and appropriate eluents ..................... 21

Annex C (informative) Precision data ................................................................................................................ 25

Bibliography ................................................................................................................................................................. 33

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European foreword

This document (EN 17294:2019) 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 Feburary 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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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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1 Scope

This document specifies a method for the determination of organic acids in animal feeding stuffs by Ion

Chromatography with conductivity detection (IC-CD).

The method is intended to be used for the determination of formic acid, lactic acid, propionic acid, citric

acid, fumaric acid and malic acid as active substances in feed additives, premixtures, feed materials,

compound feed and water and for acetic acid in a limited manner in the same matrices. This method

determines the total extractable concentration of the above mentioned organic acids and their salts.

It is advisable that the user of this standard determines the working range of the method for each organic

acid. The lower limit of the working range depends on the matrix and the interferences encountered. It is

advisable that a working range between 10 mg/l and 100 mg/l is achievable.

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

27 % of the above mentioned organic acids.

NOTE Limitation occurs during simultaneous determination of high concentration of lactic acid and low

concentration of acetic acid. If the ratio of concentration of lactic acid to acetic acid exceeds factor 20, the

determination of acetic acid is not guaranteed.

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

quantification (LOQ), as calculated (Table 1) can be achievable.
Table 1 — Limits of quantification (LOQ)
Organic acid LOQ
mg/kg
Formic acid 200
Lactic acid 200
Propionic acid 200
Citric acid 200
Fumaric acid 200
Acetic acid 200
Malic 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)

EN ISO 10304-1, Water quality — Determination of dissolved anions by liquid chromatography of ions —

Part 1: Determination of bromide, chloride, fluoride, nitrate, nitrite, phosphate and sulfate (ISO 10304-1)

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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]]
4 Principle

The sample is extracted with water. The extract is filtered or centrifuged and – if necessary – diluted. The

amount of organic acids extracted from the sample is determined with ion chromatography (IC) in

conjunction with conductivity detection (CD) using external calibration.

When using CD it is essential that the eluent shows a sufficiently low conductivity. For this reason, CD is

usually combined with a suppressor device (cation exchanger), which will reduce the conductivity of the

eluent and transform the sample acids into their respective salts.

The method description follows a proven approach where the chromatographic resolution R shall be

checked to ensure that it complies with the required separation conditions in accordance with

EN ISO 10304-1.

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
Use only reagents of recognized analytical grade, unless otherwise specified.
5.1 Water, grade 1 in accordance with EN ISO 3696

5.2 Formic acids, lactic acid, propionic acid, citric acid, acetic acid, malic acid standard solution,

c = 1 000 [mg/l]

Single acids standard solutions with adequate and required specification are commercially available

(ready-to-use solutions).
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5.3 Alternative preparation of stock solution based single standard substances
NOTE Differentiation between the enantiomers (D, L, DL) is not of interest.
5.3.1 Citric acid, minimum 99 % purity.
5.3.2 Malic acid, minimum 99 % purity.
5.3.3 Formic Acid, minimum 98 % purity.
5.3.4 Lactic acid, minimum 85 % purity.
NOTE Lithium Lactate, minimum 95 % purity, can also be used.
5.3.5 Acetic acid, minimum 99 % purity.
5.3.6 Fumaric acid, minimum 99 % purity.
5.3.7 Propionic acid, minimum 99 % purity.
5.3.8 Single standard stock solutions, app. 2 500 [mg/l].

Weigh 250 mg acid (5.3.1 to 5.3.7) each into a 100 ml volumetric flask. Dissolve with approximately 80 ml

water (5.1), mix and fill up to the mark with water (5.1).

For fumaric acid weigh 250 mg (5.3.6) into a 100 ml volumetric flask. Add 80 ml water (5.1) and dissolve

10 min in an ultrasonic bath at 60 °C. After cooling to room temperature fill up to the mark with water.

The maximum storage time is 6 month at 4 °C.

NOTE Addition of 1 % Isopropanol has a positive effect on the stability of this solution.

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 in percent divided by 100;

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.
5.4 Mixed standard solution, (app.) 100 [mg/l]

Accurately pipette 4,0 ml of each single standard stock solution (5.3.8) into a 100 ml volumetric flask and

fill up to the mark with water (5.1). The exact concentration of each organic acid shall be calculated

according to the real concentration of the single standard stock solutions.

In the case of ready-to-use solutions (5.2) pipette 10,0 ml each into a 100 ml volumetric flask and fill up

to the mark with water (5.1).
The maximum storage time is 2 months at 4 °C.
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5.5 IC Mobile phase
Degas all water used for eluent preparation.

The choice of eluent depends on the chosen column and detector (examples in Annex B, Table B.1). The

chosen combination of ion-chromatography column (IC-column) and eluent shall meet the resolution

requirements stated in 6.11.
A selection of reagents for common eluents is given in Annex B.
6 Apparatus
Usual laboratory apparatus, 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 Magnteic stirrer, with Polytetrafluoroethylene (PTFE)-coated stirring bar.
6.4 Ultrasonic bath
6.5 Pipettes (electronic or manual), in the range 100 μl to 5 000 μl.
6.6 Centrifuge, approx. 5 000 × g.
6.7 Folded filter, pore size 4 µm to 7 µm (ash free paper filter).

6.8 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).
6.9 Reversed phase solid phase extract (RP SPE)
Optional for protecting the IC column, e.g. OnGuard II RP (Dionex).

6.10 Ion chromatograph (pump, autosampler) with suppressed conductivity detection

6.10.1 IC column, with specified separation performance (6.11).
6.10.2 Precolumn
6.11 Quality requirements for the separator column

In chromatograms of samples and standard solutions (see Figure 1), the peak resolution, R, between the

acid of interest and its nearest peak, shall not fall below 1,3 [see Formula (2) or Formula (3) and Figure 2].

Separation conditions shall be such that possible interfering organic acids or substances will not interfere

with the organic acids of interest.
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Key
X = retention time t (s) 1 to 10 = peak 1 to 10
Y = signal
Figure 1 — Example chromatogram of organic acids with sufficient peak resolution
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Key
X retention time tR (s) 1 peak 1
Y signal 2 peak 2
W peak width (s)

Figure 2 — Graphical representation of the parameters to calculate the peak resolution R

Base peak widths, w and w , are obtained by constructing isosceles triangles over the Gaussian peaks.

1 2
Calculate the peak resolution R using Formula (2).
2×()tt−
R2 R1
R =
(2)
2,1
w + w
where
R is the peak resolution;
2,1
t is the retention time of the first peak, in s;
tR2 is the retention time of the second peak, in s;
W is the peak width on the time axis of the first peak, in s;
W is the peak width on the time axis of the second peak, in s.
NOTE Mostly the chromatography software can calculated R by itself.
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If R shall be calculated manually follow the described procedure: The width at the base of a peak is the

segment of the peak base intercept by the tangents drawn to the inflection points on either side of the

peak. Alternatively the peaks width can be measured at half the peak height using Formula (3).

18,(× tt− )
R2 R1
R =
2,1
ww+
h2 h1
(3)
where
R is the peak resolution;
2,1
t is the retention time of the first peak, in s;
t is the retention time of the second peak, in s;

W is the peak width on the time axis of the first peak at half the peak height, in s;

W is the peak width on the time axis of the second peak at half the peak height, in s.

7 Sampling

It is important that the laboratory receives a sample which is truly representative and has not been

damaged or changed during transport or storage.

Sampling is not part of the method specified in this European Standard. A recommended sampling

method is given in EN ISO 6497 [3].

Store the sample in such a way that deterioration and change in its composition are prevented.

8 Preparation of test sample
Prepare the test sample in accordance with EN ISO 6498.

Grinding (≤ 1 mm) shall be done in such conditions that the substance is not appreciably heated.

The whole ground product is placed in a flask made of e.g. polypropylene, which can be stoppered and

stored in such way to prevent any change in composition.

Before any weighing is carried out for the analysis, the whole test sample shall be thoroughly mixed for

reasons of homogeneity.
9 Procedure
9.1 General
An overview of the procedure is given in Annex A.
9.1.1 Extraction of formic, lactic, propionic, citric, acetic and malic acid

Accurately weigh 5,00 g ± 10 mg of prepared sample into a 250 ml conical flask. Add 100,0 ml water (5.1)

and mix for 60 min on a magnetic stirrer at ambient temperature (20 °C to 25 °C).

NOTE Samples can also be extracted 30 min by ultrasonic bath at ambient temperature (20 °C to 25 °C).

Let the particles settle down. Filter through folded filter (6.7), discarding the first 3 ml of the filtrate, and

collect the rest. Alternatively centrifuge the sample extract for 3 min (appr. 5 000 × g).

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Dilute the filtered/centrifuged solution with water (5.1) to a final concentration according to the working

range of the calibration (10 µg/ml to 100 µg/ml) – see below for a dilution example. For different

concentration levels of organic acids more than one dilution can be done.
Before IC analysis filter through a 0,45 µm membrane filter (6.8).
9.1.2 Extraction of fumaric acid

Accurately weigh 5,00 g ± 10 mg of prepared sample into a 250 ml conical flask. Add 100,0 ml water (5.1),

stopper the flask, heat up to 60 °C for complete solution and mix for 60 min on a magnetic stirrer.

NOTE Samples can also be extracted 30 min by ultrasonic bath at 60 °C.
The flask should be stoppered in order to avoid evaporation of extract solution.

Cool to room temperature and let the particles settle down. Filter through folded filter (6.7), discarding

the first 3 ml of the filtrate, and collect the rest. Alternatively centrifuge the sample extract for 3 min

(appr. 5 000 × g).

Dilute the filtered/centrifuged solution with water (5.1) to a final concentration according to the working

range of the calibration (10 µg/ml to 100 µg/ml) – see below for a dilution example.

Before IC analysis filter through a 0,45 µm membrane filter (6.8).
9.1.3 Extraction of coated acids

Accurately weigh 5,00 g ± 10 mg of prepared sample into a 250 ml conical flask. Add 100,0 ml water (5.1),

stopper the flask and extract 30 min by ultrasonic bath at 60 °C.
The flask shall be stoppered in order to avoid evaporation of extract solution

Cool to room temperature and let the particles settle down. Filter through folded filter (6.7), discarding

the first 3 ml of the filtrate, and collect the rest. Alternatively centrifuge the sample extract for 3 min

(appr. 5 000 × g).

Dilute the filtered/centrifuged solution with water (5.1) to a final concentration according to the working

range of the calibration (10 µg/ml to 100 µg/ml) – see below for a dilution example.

Before IC analysis filter through a 0,45 µm membrane filter (6.8).

For protecting the IC column the sample solution can be purified through an RP SPE (6.9). Follow the

description of the supplier. Blank solution and recoveries of organic acids shall be checked.

EXAMPLE Calculation of dilution:
Expected concentration of the sample 0,8 % (8 000 µg/g) formic acid.
Desired final concentration: 80 µg/ml.
5 g sample weight dissolved in 100 ml.
5g× 8000µg / g
Dilution factor 5
100ml× 80µg / ml

Therefore the sample extract solution shall be diluted 1:5 to meet the calibration range.

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9.2 Calibration
The following calibration solutions are recommended.

Prepare the calibration standards with water (5.1) according to Table 2. The exact concentration of each

organic acid shall be calculated according to the concentration of the mixed standard solution (5.4).

Table 2 — Preparation of calibration standards
Concentration of organic
ml standard
acids each in the calibration
Calibration standard solution (5.4)
solution in µg/ml
in 20 ml
(working range)
1 2 10
2 5 25
3 10 50
4 15 75
5 20 100

Level 1: pipette 2,0 ml mixed standard solution (5.4) in a 20 ml volumetric flask and

fill to the mark with water (5.1)

Level 2: pipette 5,0 ml mixed standard solution (5.4) in a 20 ml volumetric flask and

fill to the mark with water (5.1)

Level 3: pipette 10,0 ml mixed standard solution (5.4) in a 20 ml volumetric flask

and fill to the mark with water (5.1)

Level 4: pipette 15,0 ml mixed standard solution (5.4) in a 20 ml volumetric flask

and fill to the mark with water (5.1)

Level 5: pipette 20,0 ml mixed standard solution (5.4) in a 20 ml volumetric flask

and fill to the mark with water (5.1)
The actual concentration shall be calculated.
The maximum storage time is 2 months at 4 °C.
9.3 Measurement of the calibration standards and sample solutions

Conditions in Annex B (informative) have been shown to provide examples for an adequate separation.

Due to the different laboratory equipment for ion chromatography the described specifications are given

for guidance. Other parameters should be applicable if they lead to comparable results.

Inject twice at least, randomly the calibration standards 1 to 5 prepared according to 9.2 into the

IC system (6.10) to calculate a linear standard calibration curve (R2 should be 0,99 at a minimum).

Inject the sample extract solution prepared according to 9.1. If the concentration of the analyte exceeds

the calibration range, dilute the sample according to 9.1 or establish a separate calibration function for a

higher working range and re-analyse it.
9.4 Confirmation of identity
The identity of the target analyte(s) can be confirmed by co-chromatography.

Prepare a spiked sample extract by adding an appropriate amount of calibration solution of the organic

acid in question to the sample extract. The amount of organic acid added shall be approximately equal to

the estimated amount of the corresponding organic acid detected in the sample extract. Inject the spiked

sample extract. If the signal detected in the chromatogram obtained in (9.3) was linked to the detection

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of the organic acid, only this signal intensity should increase. The related peak height increase should be

proportional to the spiking level of the organic acid and the related peak width at half height should

increase by no more than ±10 % of the original width.
10 Calculation and expression of results

In general the calibration curve and the concentration of the test solution are calculated by the

measurement system itself. The quantification is carried out on the basis of the peak areas determined

according 9.2 by the calibration regression line (calibration curve) by the method of external standards.

y = bx+a

The concentration of organic acids in the sample, expressed in mg/kg, is determined using Formula (4):

C ××VD
Chr E
(4)
C =
where

C is the concentration of the organic acid in the sample, in mg/kg with 3 significant digits;

C is the concentration of the organic acid in the measurement solution, in µg/ml;

Chr
V is the extraction volume, in ml;
D is the dilution factor according 9.1 (extraction volume x dilution);
m is the mass of the sample, in g.

Especially the critical pair of peaks regarding the peak resolution R should be taken into account. If the

ratio of concentration of lactic acid to acetic acid exceeds factor 20, the determination of acetic acid is not

guaranteed.

If the acid is to be expressed as the corresponding salt, Formula (5) should be applied:

C C×
(5)
s A
where

C is the concentration of the salt of the organic acid in the sample, in mg/kg with 3 significant

digits;
C is the concentration of the organic acid in the sample, in mg/kg;
MW is the molecular weight of the salt of the organic acid;
MW is the molecular weight of the organic acid.
11 Precision
11.1 Interlaboratory test

An overview of an interlaboratory study on the precision of the method is shown in Table 3 to Table 9.

Details are shown in Annex C.

The values for performance characteristics derived from this interlaboratory study may not be applicable

to concentration ranges and/or matrices other than those given.
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11.2 Repeatability

The absolute difference between two independent single test results or the ratio of the higher to the lower

of the two test results on the normal scale, obtained using the same method on identical test material in

the same laboratory by the same operator using the same apparatus within the shortest feasible time

...

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