SIST EN 16930:2017

SLOVENSKI STANDARD
oSIST prEN 16930:2016
01-januar-2016
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Animal feeding stuffs: Methods of sampling and analysis - Determination of carbadox
and olaquindox by high performance liquid chromatography - UV detection (HPLC/UV)
Futtermittel - Probenahme- und Untersuchungsverfahren - Bestimmung von Carbadox
und Olaquindox mittels Hochleistungsflüssigkeitschromatographie mit UV-Detektion
(HPLC/UV)
Aliments des animaux - Méthodes d'échantillonnage et d'analyse - Détermination des
teneurs en carbadox et olaquindox par chromatographique liquide à haute performance
avec détection UV (CLHP/UV)
Ta slovenski standard je istoveten z: prEN 16930
ICS:
65.120 Krmila Animal feeding stuffs
71.040.50 Fizikalnokemijske analitske Physicochemical methods of
metode analysis
oSIST prEN 16930:2016 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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oSIST prEN 16930:2016

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oSIST prEN 16930:2016


DRAFT
EUROPEAN STANDARD
prEN 16930
NORME EUROPÉENNE

EUROPÄISCHE NORM

November 2015
ICS 65.120
English Version

Animal feeding stuffs: Methods of sampling and analysis -
Determination of carbadox and olaquindox by high
performance liquid chromatography - UV detection
(HPLC/UV)
 Bestimmung von Carbadox und Olaquindox in
Konzentrationen unterhalb von Zusatzstoffen in
Mischfuttermitteln mittels HPLC-UV
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, 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: Avenue Marnix 17, B-1000 Brussels
© 2015 CEN All rights of exploitation in any form and by any means reserved Ref. No. prEN 16930:2015 E
worldwide for CEN national Members.

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Contents Page
European foreword . 3
1 Scope . 4
2 Normative references . 4
3 Principle . 4
4 Reagents and materials . 4
5 Apparatus . 6
6 Sampling . 7
7 Preparation of test sample . 8
7.1 General . 8
7.2 Laboratory sample . 8
7.3 Test sample . 8
7.4 Test portion . 8
8 Procedure. 8
-1 -1
8.1 Extraction of feeding stuffs containing 0,5 mg kg to 100 mg kg of growth
promoters . 8
8.2 Filtration . 8
8.3 Purification . 8
8.3.1 Open glass clean up . 8
8.3.2 SPE clean up . 8
8.4 HPLC analysis . 9
8.4.1 Analytical conditions . 9
8.4.2 External calibration curve . 9
8.4.3 Sample extracts . 9
8.5 System suitability – Confirmation . 9
8.5.1 Co-chromatography . 9
8.5.2 DAD . 10
8.5.3 Spiking procedures for analytical recovery determination . 11
9 Calculation . 11
10 Precision . 12
10.1 Collaborative study . 12
10.2 Repeatability . 12
10.3 Reproducibility . 12
11 Test report . 12
Annex A (informative) Results of the collaborative study . 14
A.1 Procedure. 14
A.2 Materials . 14
A.3 Statistical analysis of results . 16
A.4 Results and interpretation . 16
A.5 Example chromatogram . 23
Bibliography . 24

2

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European foreword
This document (prEN 16930:2015) 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.
WARNING — The use of this protocol involves hazardous materials, operations and equipment.
This protocol does not purport to address all the safety problems associated with its use. It is the
responsibility of the user of this protocol to establish appropriate safety and health practices
and determine the applicability of regulatory limitations prior to use.
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1 Scope
This European Standard specifies a high performance liquid chromatographic – UV detection (HPLC-
UV) method for the simultaneous determination of two growth promoters Carbadox and Olaquindox
contents in compound feeds and raw materials at levels ranging from the limit of quantification to
100 mg kg-1.
The limit of quantification of the method has been demonstrated to be better than 3 mg kg-1 for
olaquindox and 4 mg kg-1 for carbadox.
2 Normative references
The following documents, in whole or in part, are normatively referenced in this document and are
indispensable for its application. For dated references, only the edition cited applies. For undated
references, the latest edition of the referenced document (including any amendments) applies.
ISO 6498, Animal feeding stuffs — Guidelines for sample preparation
3 Principle
The two growth promoters are extracted from the sample with a mixture of methanol and water 1:1,
v:v. The extract of animal feeds is purified through a short open glass aluminium oxide column or by an
alumina Solid Phase Extraction (SPE).
The final extract is analysed by reversed phase HPLC with UV detection at a wavelength of 375 nm.
Alternatively, for confirmation purposes, a Diode Array Detector (DAD) can be used.
The presence of furazolidone can interfere with the determination of carbadox.
If a DAD is used, many other veterinary drugs or feed additives can be detected (ronidazole,
meticlorpindol, nitrofurazone, dimetridazol, furaltadon, sulphonamides.), but their signals do not
interfere with the target ones.
In some special feeds, matrix interfering peaks may be present, very close to the carbadox peak.
4 Reagents and materials
WARNING
a) This method requires the use of solutions of Carbadox and Olaquindox. These substances were
used as growth-promoting feed additives for piglets. Carbadox and Olaquindox are
chemotherapeutics belonging to the quinoxaline group. They are suspected to be teratogen and
mutagen. Avoid inhalation of and exposure to the toxic standard materials and solutions.
b) Use adequate protection of materials: wear safety glasses, protective clothing and avoid skin
contact.
c) These 2 growth promoters are subject to light degradation. Protect analytical work adequately
from daylight, and keep standard solutions protected from light by using amber glassware, amber
vials or aluminium foil.
Use only reagents recognized as analytical grade at least unless otherwise stated.
4.1 Water, demineralized or deionized or at least equivalent
4.2 Methanol
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4.3 Acetonitrile
4.4 Glacial acetic acid, minimum purity 96 %
4.5 Ammonium acetate water free salt, CH CO NH
3 2 4
4.6 Extraction solvent: methanol water mixture (1:1 v:v)
Combine equal volumes of methanol (4.2) and water (4.1). Mix well.
NOTE Only for 8.1 purposes, it is possible to combine equal volume of technical methanol (4.14) and water
(4.1). Mix well.
4.7 Ammonium acetate buffer, 25 mM, pH = 4,35
Weigh 2,0 ± 0,1 g of ammonium acetate (4.5) into a 1 000 ml volumetric flask. Dissolve in 900 ml of
water (4.1). Add 3,0 ml of glacial acetic acid (4.4). Adjust (5.1) the pH to pH = 4,35 with acetic acid (4.4),
if necessary. Dilute to the mark with water (4.1) and mix well.
4.8 Mobile phase for HPLC: acetonitrile:buffer mixture (10:90; v:v)
Transfer 100 ml of acetonitrile (4.3) into a 1 000 ml volumetric flask. Dilute to the mark with
ammonium acetate buffer (4.7). Filter the eluent through a 0,45 µm cellulose acetate membrane filter
(5.9) using a solvent filtration system (5.2). If necessary, perform degassing for 5 min in an ultrasonic
bath (5.3).
NOTE This mobile phase is suitable for the three recommended columns in 5.11.8. If another column is used,
optimization of the mobile phase composition may be necessary before performing real analysis (see 8.4.1).
4.9 Carbadox dissolving solvent: methanol:acetonitrile mixture (1:1; v:v)
Combine equal volumes of methanol (4.2) and acetonitrile (4.3). Mix well.
4.10 Reference standards
Guaranteed purity is required for each lot of reference standard.
4.10.1 Carbadox, 3-(2-quinoxalinyl methylene) carbazic acid methyl ester N,N’-dioxide
4.10.2 Olaquindox, (2-[N-2'-(hydroxyethyl) carbamoyl] 3–methyl quinoxaline) N,N’ dioxide)
4.11 Standard solutions
Protect all standard solutions from daily light.
−1
4.11.1 Carbadox stock standard solution ca. 100 µg ml
Weigh 25 mg of carbadox (4.10.1), to the nearest 0,1 mg, into a 250 ml amber volumetric flask. Dissolve
in 200 ml of mixture (4.9). Mix well and place the flask in an ultrasonic bath (5.3) until total dissolution.
Allow to cool down to room temperature, dilute to the mark with mixture (4.9) and mix well. Calculate
the accurate concentration taking into account the purity of the reference standard (4.10.1).
Prepare fresh every month. Store in the dark at 0 °C to 8 °C.
−1
4.11.2 Olaquindox stock standard solution ca. 250 µg ml
Weigh 50 mg of olaquindox (4.10.2), to the nearest 0,1 mg, into a 200 ml amber volumetric flask.
Dissolve in about 190 ml of water (4.1). Mix well and place the flask in an ultrasonic bath (5.3) until
total dissolution. Allow to cool down to room temperature. Dilute to the mark with water (4.1) and mix
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well. Calculate the accurate concentration taking into account the purity of the reference standard
(4.10.2).
Prepare fresh every month. Store in the dark at 0 °C to 8 °C.
4.11.3 Calibrations solutions
−1
4.11.3.1 Carbadox/olaquindox calibration solution ca. 10 µg ml
Pipette 5 ml of the carbadox stock standard solution (4.11.1) and 2 ml of the olaquindox stock standard
solution (4.11.2) in a 50 ml volumetric flask. Dilute to the mark with the extraction solvent (4.5) and
mix well.
Prepare fresh for each series of samples.
−1
4.11.3.2 Carbadox/olaquindox calibration solution ca. 5 µg ml
Pipette 5 ml of the carbadox stock standard solution (4.11.1) and 2 ml of the olaquindox stock standard
solution (4.11.2) in a 100 ml volumetric flask. Dilute to the mark with the extraction solvent (4.5) and
mix well.
Prepare fresh for each series of samples.
−1
4.11.3.3 Carbadox/olaquindox calibration solution ca. 2,5 µg ml
Pipette 5 ml of the carbadox stock standard solution (4.11.1) and 2 ml of the olaquindox stock standard
solution (4.11.2) in a 200 ml volumetric flask. Dilute to the mark with the extraction solvent (4.5) and
mix well.
Prepare fresh for each series of samples.
−1
4.11.3.4 Carbadox/olaquindox calibration solution ca. 1 µg ml
−1
Pipette 5 ml of the Carbadox/olaquindox calibration solution ca. 10 µg ml (4.11.3.1) in a 50 ml
volumetric flask. Dilute to the mark with the extraction solvent (4.5) and mix well.
Prepare fresh for each series of samples.
−1
4.11.3.5 Carbadox/olaquindox calibration solution ca. 0,5 µg ml
−1
Pipette 5 ml of the Carbadox/olaquindox calibration solution ca. 5 µg ml (4.11.3.2) in a 50 ml
volumetric flask. Dilute to the mark with the extraction solvent (4.5) and mix well.
Prepare fresh for each series of samples.
−1
4.11.3.6 Carbadox/olaquindox calibration solution ca. 0,25 µg ml
−1
Pipette 5 ml of the Carbadox/olaquindox calibration solution ca. 2,5 µg ml (4.11.3.3) in a 50 ml
volumetric flask. Dilute to the mark with the extraction solvent (4.5) and mix well.
Prepare fresh for each series of samples.
4.12 Neutral aluminium oxide, Brockmann activity I
3
4.13 Neutral aluminium oxide SPE cartridge, 2 cm , 1 850 mg
4.14 Technical methanol
5 Apparatus
Usual laboratory apparatus and, in particular, the following:
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5.1 pH meter
5.2 Solvent filtration system, suitable for 0,45 µm membrane filters
5.3 Ultrasonic bath
5.4 Rotary on mechanical shaker
5.5 Centrifuge
5.6 Glass wool
5.7 Glass column for chromatography, length 200 nm to 400 nm; internal diameter 10 mm,
restricted at the end and fitted with a wad of glass wool (5.6) or equivalent column
5.8 Filter papers or glass microfibre filter
5.9 Cellulose acetate membrane filters of pore size 0,45 µm
5.10 PVDF syringe filters of pore size 0,45 µm and adaptable syringes
5.11 HPLC system
−1 −1
5.11.1 pump, capable of maintaining a volume flow rate of 0,4 ml min to 1,5 ml min
5.11.2 Column heater set at 30 °C
5.11.3 Injection system, with a loop suitable for 10 µl to 50 µl injections
5.11.4 UV detector, suitable for measurements at a wavelength of 375 nm
5.11.5 Diode array detector for confirmation purposes
5.11.6 Recorder or data acquisition system
5.11.7 Guard column, silica bounded C
8
5.11.8 LC analytical column, 250 × 3 mm, 5 µm pore size, packed with Lichrosorb C or Lichrospher
8
C or Lichrospher C or equivalent column.
8 18
NOTE During the method validation, these recommended LC columns have proved to be fit for purpose,
especially with samples containing interfering peaks close to the carbadox zone.
5.11.9 Refrigerated autosampler set between 0 °C and 8 °C
6 Sampling
It is important that the laboratory receives a sample that 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 Commission Regulation (EU) No 691/2013 [1].
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7 Preparation of test sample
7.1 General
Prepare the test sample in accordance with EN ISO 6498.
7.2 Laboratory sample
Grind the laboratory sample (usually 50 g) so that is passes completely through a sieve with 1 mm
apertures. Mix thoroughly.
7.3 Test sample
The test sample consists of a representative and homogenized aliquot of the ground laboratory sample
of at least 20 g.
7.4 Test portion
Accurately weigh 10,0 g to the nearest 0,1 g of the thoroughly mixed test sample into a 250 ml conical
flask. Note down the mass expressed in g. Submit it to the analysis procedure (8).
8 Procedure
-1 -1
8.1 Extraction of feeding stuffs containing 0,5 mg kg to 100 mg kg of growth
promoters
Add 100,0 ml of extraction solvent (4.5) to the test portion (7.4), stopper and shake vigorously for 1 h
or 2 h on the rotary shaker (5.4).
8.2 Filtration
Filter the solution obtained in 8.1 through a paper or glass micro fibre filter (5.8).
Alternatively centrifugation (5.5) is possible, if filtration is too long, for example.
Use the filtrate or the supernatant for the purification step.
8.3 Purification
Apply 8.3.1 or 8.3.2.
8.3.1 Open glass clean up
For each sample extract, dry-pack a glass column (5.7), fitted at the end with a plug of glass wool (5.6)
with 5 g of aluminium oxide (4.12). Load 20 ml of extract (8.2) on the column and discard the first 5 ml
of eluate. Collect the next 5 ml. Filtrate these 5 ml through a PVDF syringe filter (5.10). Use this filtrate
for HPLC determination.
8.3.2 SPE clean up
Load 8 ml of extract (8.2), on a SPE cartridge (4.13) and discard the first 1,5 ml of eluate. Collect the
next 2 ml. Filtrate these 2 ml through a PVDF syringe filter (5.10). Use this filtrate for HPLC
determination.
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8.4 HPLC analysis
8.4.1 Analytical conditions
The following conditions are provided for guidance. Other conditions may be used provided they
yield to equivalent results.
8.4.1.1 HPLC column: as in 5.11.8.
8.4.1.2 guard column: as in 5.11.7.
−1
8.4.1.3 mobile phase: as in 4.8, flow rate: 0,5 ml min .
8.4.1.4 injection volume: 20 µl-50 µl.
8.4.1.5 column temperature: 30 °C.
8.4.1.6 detection wavelength: 375 nm.
Using these conditions the retention times should approximately be 4,0 min to 6,0 min and 18,0 min to
22,0 min for olaquindox and carbadox respectively with respective capacity factors of 3 and 13
8.4.1.7 system set up
Check the stability of the HPLC system by injecting several times one of the calibration solutions
(4.11.3.1; 4.11.3.2; 4.11.3.3) until constant peak heights and retention times are achieved.
NOTE Furazoline has approximately the same retention time than carbadox. In case of doubt, confirm the
result. See 8.5.1 or 8.5.2.
In few particular feeds, a matrix interfering peak is present and has approximately the same retention
time than carbadox. Using the described LC parameters, the interfering peak is eluted just before the
carbadox peak. Apex should however be sufficiently separated. In case of doubt, confirm the result. See
8.5.1 or 8.5.2.
8.4.2 External calibration curve
Inject each calibration solution (4.11.3) at the beginning and at the end of a sample series.
Plot each of the individual measured peak height or peak area (y-axis) versus the respective
−1
concentration of carbadox or olaquindox (x-axis) in µg ml .
8.4.3 Sample extracts
Inject several times (the determined area or peak height should be stable) the sample extract obtained
in (8.3). The injection volume should be the same than the one selected for the calibration solutions.
Determine the mean peak height (or peak area) for the carbadox and/or olaquindox signals.
8.5 System suitability – Confirmation
The identity of the target analyte(s) can be confirmed by co-chromatography or by using a DAD.
8.5.1 Co-chromatography
Prepare a spiked sample extract by adding an appropriate amount of one of the carbadox/olaquindox
calibration solutions (4.11.3) to the sample extract. The amount of carbadox or olaquindox added shall
be approximately equal to the estimated amount of the corresponding growth promoter detected in the
sample extract.
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Inject the spiked sample extract. If the signal detected in the chromatogram obtained in 8.4.3 was linked
to the detection of carbadox, only this signal intensity should increase. The related peak height increase
should be proportional to the spiking level of carbadox and the related peak width at half height should
increase by no more than ± 10 % of the original width.
If the signal detected in the chromatogram obtained in 8.4.3 was linked to the detection of olaquindox,
only this signal intensity should increase. The related peak height increase should be proportional to
the spiking level of olaquindox and the related peak width at half height should increase by no more
than ± 10 % of the original width.
If the signals detected in the chromatogram obtained in 8.4.3 were linked to the detection of carbadox
and olaquindox, only these signals intensities should increase. The respective related peak heights
increase should be proportional to the spiking level of carbadox and olaquindox respectively and the
respective related peak widths at half height should increase by no more than ± 10 % of the respective
original widths.
8.5.2 DAD
8.5.2.1 Analytical parameters
If a DAD is used instead of a UV detector, conditions to be used are:
a) Sample wavelength: 375 nm
b) Sample bandwidth: 4 nm
c) Spectrum range: 220 nm to 400 nm
d) Spectrum: baseline, apex, up-slope and down slope inflection points
8.5.2.2 Evaluation
Plot the normalized difference spectra (sample – baseline) of the sample peak, recorded both at the
apex and at the up-slope and down-slope inflection points.
Plot the normalized spectra of the sample peak and of the carbadox or olaquindox corresponding
calibration solution peak, both recorded at the apex.
8.5.2.3 Confirmation criteria
The identity of the analyte is confirmed if the following three criteria are satisfied:
a) The retention time of the sample peak shall be equal (±5 %) to the retention time of the standard
peak. In case of doubt, standard addition (standard material added to the sample) shall be
performed.
b) Assess the purity of the sample peak on the basis of the conformity of the difference spectra,
recorded at the apex and at up-slope and down-slope inflection points. For those parts of the
spectra with a relative absorption of at least 10 % (between 10 % and 100 % in a normalized
spectrum), at each wavelength the relative absorption shall be equal (within 15 % for contents
−1 −1
higher than 20 mg kg and 25 % for contents lower than 20 mg kg ) for all spectra.
c) The difference spectra of the sample and standard peaks recorded at the peak apex shall not be
visually different for those parts of the spectra with a relative absorption of at least 10 % (between
10 % and 100 % in a normalized spectrum). This criterion is met when:
— the same maxima are present within a margin determined by the resolution of the detection
system (4 nm).
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— at those particular maxima wavelengths, the relative deviation, in comparison with the
absorbance of the standard analyte, between the two spectra (sample - standard) not
−1
exceed 15 % for contents higher than 20 mg kg and 25 % for contents lower than
−1
20 mg kg .
8.5.3 Spiking procedures for analytical recovery determination
For the determination of the analytical recovery, the spiking procedure shall be carried out using the
carbadox stock standard solution (4.11.1) and the olaquindox stock standard solution (4.11.2). The
spiking level shall be within the calibration range (preferably at the mean value). Attention shall be paid
that not more than 3 ml of the spiking solvent are added and that the subsequent evaporation takes
place in the dark and should last 0,25 h-0,5 h.
−1
For example, for a spiking level of 10 mg kg , pipette 0,4 ml of the olaquindox stock standard solution
(4.11.2) and 1 ml of the carbadox stock standard solution (4.11.1) and add to 10,0 g ± 0,1 g of sample
test portion. Shake slowly and manually. Allow penetration for at least 15 min, in the dark before
submitting to analysis (Clause 8).
The analytical recovery should be comprised between 75 % and 125 % for olaquindox and 70 % and
130 % for carbadox.
9 Calculation
Determine the content of carbadox and / or olaquindox in the sample feed using Formula (1):
( or A − b
( )
V
(1)
W ×
am
where
−1
W is the carbadox or olaquindox content in mg kg of feed sample;
( is the mean peak height calculated for carbadox or olaquindox in the feed sample extract (8.4.3);
A is the mean peak area calculated for carbadox or olaquindox in the feed sample extract (8.4.3);
b is the intercept of the calibration curve obtained from the standard solutions (8.4.2);
a is the slope coefficient of the calibration curve obtained from the standar
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