SIST EN 17504:2022
(Main)Animal feeding stuffs: Methods of sampling and analysis - Determination of gossypol in cotton seed and feeding stuff by LC-MS/MS
Animal feeding stuffs: Methods of sampling and analysis - Determination of gossypol in cotton seed and feeding stuff by LC-MS/MS
This document describes a method for the determination of free gossypol, extractable by acidified
acetonitrile/water, in cottonseeds, cottonseed cake and complete feed by high performance liquid
chromatography (HPLC) coupled with tandem mass spectrometry (MS/MS)
This method has been in-house validated in the range 20-6000 mg/kg.
Futtermittel: Probenahme- und Untersuchungsverfahren - Bestimmung von Gossypol in Baumwollsamen und Futtermitteln mittels LC-MS/MS
Dieses Dokument beschreibt ein Verfahren zur Bestimmung von freiem Gossypol, das mittels angesäuertem Acetonitril/Wasser extrahierbar ist, in Baumwollsamen, aus Baumwollsamen hergestellten Produkten und Alleinfuttermitteln durch Flüssigchromatographie mit Tandem-Massenspektrometrie (LC-MS/MS).
Durch einen Ringversuch mit den nachfolgend angegebenen Matrices ist dieses Dokument für den Bereich von 69 mg/kg bis 5 950 mg/kg erfolgreich validiert worden: Baumwollsamen, Baumwollsamenprodukte (Kuchen/Mehl, Schalen) und Alleinfuttermittel für Rinder, Schweine und Geflügel.
Aliments des animaux - Méthodes d’échantillonnage et d’analyse - Dosage du gossypol dans les graines de coton et les aliments pour animaux par CL-SM/SM
La présente norme décrit une méthode pour le dosage du gossypol libre, extractible par un mélange acétonitrile acidifié/eau dans des graines de coton, des produits issus de graines de coton et des aliments complets pour animaux par chromatographie liquide couplée à une spectrométrie de masse en tandem (CL-SM/SM).
Le présent document a été appliqué avec succès dans la plage allant de 69 mg/kg à 5 950 mg/kg lors d’un essai interlaboratoires réalisé sur les matrices suivantes : graines de coton, produits issus de graines de coton (tourteau/farine, coques) et aliments complets pour bovins, porcs et volailles.
Krma: metode vzorčenja in analize - Ugotavljanje gosipola v bombažnem semenu in krmi z LC-MS/MS
Ta dokument opisuje metodo za ugotavljanje prostega gosipola, ki ga je mogoče pridobiti z nakisanim acetonitrilom/vodo iz bombažnega semena, bombažne moke in krme z visoko zmogljivo tekočinsko kromatografijo (HPLC) v kombinaciji s tandemsko masno spektrometrijo (MS/MS).
Ta metoda je bila interno potrjena v območju od 20 do 6000 mg/kg.
General Information
Standards Content (Sample)
SLOVENSKI STANDARD
SIST EN 17504:2022
01-junij-2022
Krma: metode vzorčenja in analize - Ugotavljanje gosipola v bombažnem semenu
in krmi z LC-MS/MS
Animal feeding stuffs: Methods of sampling and analysis - Determination of gossypol in
cotton seed and feeding stuff by LC-MS/MS
Futtermittel: Probenahme- und Untersuchungsverfahren - Bestimmung von Gossypol in
Baumwollsamen und Futtermitteln mittels LC-MS/MS
Aliments des animaux - Méthodes d’échantillonnage et d’analyse - Dosage du gossypol
dans les graines de coton et les aliments pour animaux par CL-SM/SM
Ta slovenski standard je istoveten z: EN 17504:2022
ICS:
65.120 Krmila Animal feeding stuffs
SIST EN 17504:2022 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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SIST EN 17504:2022
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SIST EN 17504:2022
EN 17504
EUROPEAN STANDARD
NORME EUROPÉENNE
March 2022
EUROPÄISCHE NORM
ICS 65.120
English Version
Animal feeding stuffs: Methods of sampling and analysis -
Determination of gossypol in cotton seed and feeding stuff
by LC-MS/MS
Aliments des animaux - Méthodes d'échantillonnage et Futtermittel: Probenahme- und
d'analyse - Dosage du gossypol dans les graines de Untersuchungsverfahren - Bestimmung von Gossypol
coton et les aliments pour animaux par CL-SM/SM in Baumwollsamen und Futtermitteln mittels LC-
MS/MS
This European Standard was approved by CEN on 10 January 2022.
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
© 2022 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN 17504:2022 E
worldwide for CEN national Members.
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EN 17504:2022 (E)
Contents Page
European foreword . 3
Introduction . 4
1 Scope . 5
2 Normative references . 5
3 Terms and definitions . 5
4 Principle . 5
5 Reagents . 5
6 Apparatus . 7
7 Procedure. 8
7.1 General . 8
7.2 Sample pre-treatment . 8
7.3 Test portion . 9
7.4 Extraction . 9
8 LC-MS/MS analysis . 10
8.1 General . 10
8.2 Analysis sequence . 11
9 Results . 11
9.1 Identification . 11
9.2 Quantification . 12
10 Precision . 14
10.1 General . 14
10.2 Repeatability . 14
10.3 Reproducibility . 14
11 Test report . 15
Annex A (informative) Precision data. 16
Annex B (informative) Example of LC-MS/MS conditions . 18
B.1 General . 18
B.2 HPLC conditions . 18
B.3 MS conditions . 19
Annex C (informative) Example chromatogram gossypol in compound feed . 20
Bibliography . 21
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EN 17504:2022 (E)
European foreword
This document (EN 17504:2022) 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 September 2022, and conflicting national standards shall
be withdrawn at the latest by September 2022.
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.
Any feedback and questions on this document should be directed to the users’ national standards body.
A complete listing of these bodies can be found on the CEN website.
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
Gossypol is a polyphenolic plant toxin produced by species of the genus Gossypium (cotton plant). The
plants are primarily grown for fibre and oil production. The seeds and processed seed materials are also
used as animal feeding stuffs. Gossypol is present in the seeds in two forms: free gossypol and bound
gossypol (mostly to proteins). The measured content of free gossypol depends on the method of
extraction and the specificity of the subsequent method of analysis used ([1], [2]). Consequently, this
method might not be directly comparable to existing standards based on spectrophotometric
measurement (e.g. [3], [4]).
WARNING — 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 protection
measures and to ensure that regulatory and legal requirements are complied with.
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1 Scope
This document specifies a method for the determination of free gossypol, extractable by acidified
acetonitrile/water, in cottonseed, cottonseed products and compound feeds by liquid chromatography
with tandem mass spectrometry (LC-MS/MS).
The method described in this document has been successfully validated in the range of 69 mg/kg to
5 950 mg/kg by collaborative trial in the following matrices: cottonseed, cottonseed products
(cake/meal, hulls) and compound feeds for bovine, porcine and poultry.
NOTE It is possible to reach quantification limits of approximatively 5 mg/kg in compound feeds. The method
might be applicable at lower and at higher concentrations than the concentration range validated in the
collaborative trial. However, this needs to be assessed by in-house validation.
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)
3 Terms and definitions
No terms and definitions are listed in this document.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https://www.iso.org/obp
— IEC Electropedia: available at https://www.electropedia.org/
4 Principle
Free gossypol is extracted by mixing an homogenized sample of 0,4 g of cottonseed or 1,0 g of cottonseed
product or compound feed with 40 ml of 0,1 % phosphoric acid in acetonitrile:water (80:20) (V:V). The
mixture is shaken for 1 h. After centrifugation a portion of the supernatant is diluted with extraction
solvent in a vial. The final extract is analysed by reversed phase liquid chromatography with tandem mass
spectrometry (LC-MS/MS). Quantification of gossypol in cottonseed and cottonseed products is based on
multi-level calibration using standards in extraction solvent. Quantification of gossypol in compound
feeds is based on multi-level calibration using standards in extraction solvent and corrected for the
apparent recovery.
5 Reagents
WARNING — Gossypol can be hazardous to health. It has a strong toxic effect on the reproductive organs.
Depending on the level of exposure, both acute and chronic effects are possible.
5.1 Gossypol, ≥ 95 %, as gossypol base
Gossypol is a racemic mixture of (+)- and (-)-gossypol. In this method the enantiomers of gossypol are
not separated. A standard containing a mixture of both enantiomers may be used.
Gossypol is also available in the form of a 1:1 complex with acetic acid. In this method either a standard
of gossypol or gossypol-acetic acid may be used.
NOTE Gossypol is sensitive to light.
5
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5.2 Acetonitrile, LC-MS or HPLC quality
3
5.3 Phosphoric acid 85 % w/w, d = 1,71 g/cm , p.a. quality
5.4 Formic acid, ≥ 98 %, p.a. quality
5.5 Water
Water of LC-MS grade, double-distilled or water of grade 1 as defined in EN ISO 3696.
5.6 Extraction solvent: 0,1 % phosphoric acid in acetonitrile:water (80:20)
Mix 800 ml acetonitrile (5.2) and 200 ml water (5.5) in a bottle of 1 000 ml. Add 1,0 ml phosphoric acid
(5.3) and mix. This solution is stored at room temperature and may be used for 1 month.
5.7 Mobile phase A: 0,5 % formic acid in acetonitrile:water (5:95)
Mix 50 ml acetonitrile (5.2) and 950 ml water (5.5) in a bottle of 1 000 ml. Add 5 ml formic acid (5.4) and
mix. This solution is stored at room temperature and may be used for 1 month.
5.8 Mobile phase B: 0,5 % formic acid in acetonitrile:water (95:5)
Mix 950 ml acetonitrile (5.2) and 50 ml water (5.5) in a bottle of 1 000 ml. Add 5 ml formic acid (5.4) and
mix. This solution is stored at room temperature and may be used for 1 month.
5.9 Gossypol stock solution, 1 mg/ml
Weigh (6.1) 20 mg gossypol (5.1) and transfer into a 20 ml amber volumetric flask. Take into account the
mass, the purity and the chemical form of the standard. Add extraction solvent (5.6), dissolve by mixing
and make up to the mark with extraction solvent (5.6). Transfer to an amber glass bottle (6.7) and store
the stock solution at < −18 °C. Under these conditions the solution may be used for 6 months.
5.10 Gossypol standard solution, 10 µg/ml
Transfer 100 µl of gossypol stock solution (5.9) into a 10 ml amber volumetric flask (6.7) using a pipette.
Fill up to the mark with extraction solvent (5.6) and mix. Transfer to an amber glass bottle (6.7). Store
the standard solution at < −18 °C. Under these conditions the solution can be used for 2 months. Use this
standard solution for preparation of calibration curves.
5.11 Gossypol standard solution, 1 µg/ml
Transfer 1,00 ml of gossypol standard solution (5.10) into a 10 ml amber volumetric flask (6.7) using a
pipette. Fill up to the mark with extraction solvent (5.6) and mix. Transfer to an amber glass bottle (6.7).
Store the standard solution at < −18 °C. Under these conditions the solution can be used for 2 months.
Use this standard solution for preparation of calibration curves.
5.12 Calibration standards
Prepare calibration standards using the standard solutions (5.10) and (5.11) and extraction solvent (5.6)
according to Table 1 (See NOTE). Pipette directly in HPLC vials (6.8).
NOTE The number of calibration points required depends on the concentrations expected in the samples (7.4)
and the dynamic range of the mass spectrometer. It is advised to use all calibration standards, but at least calibration
standards Cal 1, 2, 4 to 6 and 8.
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Table 1 — Preparation of calibration standards
Concentration Standard solution of Standard solution of Extraction solvent
1 µg/ml (5.11) 10 µg/ml (5.10) (5.6)
µg/ml
µl µl µl
Cal 1 0,00 0 0 1 000
Cal 2 0,025 25 0 975
Cal 3 0,05 50 0 950
Cal 4 0,10 100 0 900
Cal 5 0,25 0 25 975
Cal 6 0,50 0 50 950
Cal 7 0,75 0 75 925
Cal 8 1,00 0 100 900
6 Apparatus
Usual laboratory equipment and, in particular, the following items:
6.1 Analytical balance, with an accuracy of 0,1 mg or better
6.2 Laboratory balance, with an accuracy of 0,01 g or better
6.3 Pipettors, adjustable, suitable for organic solvents, properly calibrated, with appropriate tips
6.4 Filter disc units, polytetrafluoroethylene (PTFE) with a pore size of 0,45 µm or less
6.5 Centrifuge tubes, polypropylene, 50 ml with screw cap
6.6 Centrifuge, suitable for 50 ml centrifuge tubes (6.5)
6.7 Glass bottles and volumetric flasks, amber coloured, different sizes
6.8 HPLC autosampler vials, amber glass 1,5 ml with caps
6.9 Minishaker or vortex mixer
6.10 Ultrasonic bath
6.11 Vertical or horizontal shaker, adjustable
6.12 Laboratory mill
6.13 HPLC system, consisting of:
6.13.1 Autosampler, thermostated
As an example, an autosampler capable of maintaining a temperature of 10 °C ± 1 °C is suitable.
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6.13.2 Binary pump system
Capable of delivering a binary gradient at flow rates appropriate for the analytical column in use with
sufficient accuracy.
6.13.3 Column oven, thermostated
As an example, a column oven capable of maintaining a temperature of 40 °C ± 1 °C is suitable.
6.13.4 Analytical column
Containing C18 reversed phase packing material, capable of the base-line separation of the analyte from
compounds with identical molecular mass.
6.13.5 Pre-column, optional
With the same stationary phase material as the analytical column and with appropriate dimensions.
6.14 Tandem mass spectrometer
Capable of performing multiple selected reaction monitoring in negative ionization mode, with a
sufficiently wide dynamic range and capable of unit mass separation and equipped with a computer-
based data processing system. Any ionization source giving sufficient yield may be employed.
7 Procedure
7.1 General
Animal feed is a complex matrix containing a wide range of ingredients in varying amounts. Sample and
instrument dependent matrix effects (suppression/enhancement) can occur that may affect the
quantification of the analyte in the sample (see NOTE). To correct for these matrix effects a recovery
sample (7.4.3) is included.
Depending on the sensitivity and linear range of the mass spectrometric instrument, it can be necessary
to dilute the sample extracts (7.4) by an appropriate factor with extraction solvent (5.6), taking into
account the expected concentration of the analyte in the sample, to keep the response of the detector
within the dynamic range of the mass spectrometer. Alternatively, the injection volume of the sample
may be reduced, within the calibrated range of the injection system.
NOTE At the conditions described in this document, matrix effects (ion suppression or enhancement) were not
significant for cottonseed and cottonseed products. For compound feed matrix effects were observed.
7.2 Sample pre-treatment
Laboratory samples should be taken and prepared in accordance with European legislation [3] where
applicable or, in any other case with EN ISO 6498.
Homogenize samples in a laboratory mill (6.12) to < 1 mm.
Cottonseeds can contain high amounts of gossypol. Carefully clean all parts of the grinder between
samples to avoid significant carry-over. When cottonseed samples are analysed together with cottonseed
products or compound feed materials, the cottonseed products and the compound feed samples should
be ground prior to the cottonseed samples.
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7.3 Test portion
7.3.1 Cottonseed
The amount of homogenized cottonseed material examined is 0,4 g ± 0,01 g.
A larger test sample size may be used by the laboratory in order to improve the representativeness of the
sample. The amount of extraction solvent shall in that case be adjusted accordingly (7.4).
7.3.2 Cottonseed products and compound feed
The amount of homogenized cottonseed product or compound feed material examined is 1,0 g ± 0,05 g.
A larger test sample size may be used by the laboratory in order to improve the representativeness of the
sample. The amount of extraction solvent shall in that case be adjusted accordingly (7.4).
7.4 Extraction
7.4.1 Cottonseed
Weigh (6.2) a test portion of 0,4 g ± 0,01 g homogenized sample (7.3) into a centrifuge tube of 50 ml (6.5).
Add 40,0 ml of extraction solvent (5.6) and mix for 15 s using a vortex mixer (6.9). Place the tube in an
ultrasonic bath (6.10) for 5 min and subsequently shake the tube for 1 h (6.11).
-1
Centrifuge the tube for 5 min at 3 000 min at room temperature (6.6). Transfer 10 µl of the extract into
a HPLC vial (6.8) and add 990 µl extraction solvent (5.6) and mix (see NOTE).
NOTE Cottonseeds can contain high concentrations of gossypol (ranging from 2 000 mg/kg to 8 000 mg/kg).
The expected gossypol concentration in undiluted cottonseed extracts is in the range of 20 µg/ml to 80 µg/ml. After
dilution of the sample extract the expected concentration is in the range of 0,2 µg/ml to 0,8 µg/ml.
7.4.2 Cottonseed products and compound feed
Weigh (6.2) a test portion of 1,0 g ± 0,05 g homogenized sample (7.3) into a centrifuge tube of 50 ml (6.5).
Add 40,0 ml of extraction solvent (5.6) and mix for 15 s using a vortex mixer (6.9). Place the tube in an
ultrasonic bath (6.10) for 5 min and subsequently shake the tube for 1 h (6.11).
-1
Centrifuge the tube for 5 min at 3 000 min at room temperature (6.6). Transfer 200 µl of the extract
(see NOTE 1) into a HPLC vial (6.8) and add 800 µl extraction solvent (5.6) and mix (see NOTE 2).
When a sample contains gossypol in a concentration that exceeds the range of the calibration curve,
reanalysis is required. For this purpose, the sample extract is diluted with extraction solvent (5.6) in an
appropriate ratio, to produce a concentration in the sample extract that is within the range of the
calibration curve.
In case of turbid extracts, 1 ml of the extract may first be filtered through a 0,45 µm PTFE membrane filter
(6.4) into a new HPLC vial. Take 200 µl of this extract to dilute with 800 µl extraction solvent.
NOTE 1 The expected range of gossypol in cottonseed products or compound feeds is from 10 mg/kg to
200 mg/kg. The gossypol concentration in undiluted extracts prepared from cottonseed products or compound feed
is expected to be in the range of 0,25 µg/ml to 5 µg/ml. After dilution of the sample extract the concentration is
expected to range from 0,05 µg/ml to 1 µg/ml.
NOTE 2 In specific products higher concentrations of gossypol can be present, in the range of 200 mg/kg to
1 000 mg/kg. For these products the gossypol concentration in undiluted extracts is expected to range from 5 µg/ml
to 25 µg/ml and in the diluted extracts from 1 µg/ml to 5 µg/ml. By applying an additional fivefold dilution step, the
gossypol concentration in the sample extracts is expected to range from 0,2 µg/ml to 1,0 µg/ml.
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7.4.3 Recovery sample for compound feed
For preparation of the recovery sample take a representative blank feed sample (see NOTE).
Weigh (6.2) a test portion of 1,0 g ± 0,05 g homogenized sample into a centrifuge tube of 50 ml (6.5). Add
100 µl of gossypol stock solution of 1 mg/ml (5.9). This is equivalent to 100 mg/kg. Vortex (6.9) the
spiked sample for 15 s and let soak for 30 min. Add 40,0 ml of extraction solvent (5.6) and mix for 15 s
using a vortex mixer (6.9). Place the tube in an ultrasonic bath (6.10) for 5 min and subsequently shake
the tube for 1 h (6.11).
-1
Centrifuge the tube for 5 min at 3 000 min at room temperature (6.6). Filter 1 ml of the extract through
a 0,45 µm membrane filter (6.4) into a HPLC vial (6.8). Pipette 200 µl of this extract into a new HPLC vial
(6.8), add 800 µl extraction solvent (5.6) and mix.
NOTE A blank sample is a sample shown by a preceding analysis not to contain gossypol in a concentration
above the limit of detection.
8 LC-MS/MS analysis
8.1 General
Chromatographic and mass spectrometric conditions can be chosen freely. The optimal measuring
conditions strongly depend on the instrumentation used. Important criteria and parameters with respect
to the chromatographic separation and detection of the analyte are:
— The chosen column dimensions and chromatographic conditions should be appropriate to obtain
base line separation of the analyte from other substances with the same molecular mass-to-charge
ratio.
— The chosen mass spectrometric conditions should be appropriate to measure the analyte with
sufficient sensitivity and specificity. Preferably the deprotonated molecular parent ion should be
selected as precursor ion and at least two product ions specific for the compound should be selected.
The chromatographic peak should be composed of at least 10 data points.
— The analytical series should not be started before it is verified, by injecting calibration standard Cal
4 (5.12) at least three times, that the system produces stable analyte retention times and that the
sensitivity of the detector is sufficient and stable.
— It should be checked whether carry-over effects are occurring by injecting a high gossypol
concentration in solvent or matrix extract standard followed by a solvent blank or blank extract. The
amount of carry-over should be reduced as much as possible by selection of an adequate washing
procedure of the injector system. The use of a wash solvent containing a high proportion of organic
solvent in combination with an organic acid (e.g. mobile phase B (5.8)) can reduce carry-over
significantly. It can be necessary to inject one or more extraction solvent blanks between the
individual sample extracts.
— Depending on the sensitivity and linear dynamic range of the mass spectrometric instrument it can
be necessary to dilute the calibration standards and sample extracts by an additional factor with
extraction solvent (5.6).
An example of suitable measuring conditions is provided in Annex B and an example chromatogram is
given in Annex C. Other instrumentation with appropriate conditions can also be suited.
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8.2 Analysis sequence
An appropriate analysis sequence for cottonseed and cottonseed product samples:
— Calibration standard 4 of 0,1 µg/ml (5.12), injected at least three times or until stable measurement
conditions are obtained;
— Calibration standards range (5.12);
— Extraction solvent (5.6);
— Sample extracts (7.4.1 and/or 7.4.2);
— Extraction solvent (5.6);
— Calibration standards range (5.12).
Depending on the number of samples, additional calibration standards can be included in between the
samples (e.g. every 20 samples) to correct for deviations of the analyte response during the sequence.
An appropriate analysis sequence for compound feed samples:
— Calibration standard 4 of 0,1 µg/ml (5.12), injected at least three times or until stable measurement
conditions are obtained;
— Calibration standards range (5.12);
— Extraction solvent (5.6);
— Recovery sample (7.4.3);
— Extraction solvent (5.6);
— Sample extracts (7.4.2);
— Extraction solvent (5.6);
— Calibration standards range (5.12).
Depending on the number of samples, additional calibration standards can be included in between the
samples (e.g. every 20 samples) to correct for deviations of the analyte response during the sequence.
9 Results
9.1 Identification
The detection and identification of gossypol in the sample is based on the presence of co-eluting
chromatographic peaks for the transitions measured for the analyte. Retention time and relative
abundance of the transitions should match with those in the calibration standards. The deviation of the
retention time of gossypol in the sample should not exceed 0,2 min in comparison to the average
retention time of gossypol in the calibration standards 3 to 8 (Table 1). The relative intensities of the
transitions in the sample should not deviate more than 30 % in comparison to the average of the relative
intensities of the transitions in the calibration standards 3 to 8 (Table 1).
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9.2 Quantification
9.2.1 General
For cottonseed and cottonseed products quantification of gossypol is based on multi-level calibration in
extraction solvent (5.6). For compound feeds quantification is based on multi-level calibration in
extractio
...
SLOVENSKI STANDARD
oSIST prEN 17504:2020
01-junij-2020
Krma: metode vzorčenja in analize - Določevanje gosipola v bombažnem semenu
in krmi z LC-MS/MS
Animal feeding stuffs: Methods of sampling and analysis - Determination of gossypol in
cotton seed and feeding stuff by LC-MS/MS
Futtermittel - Probenahme- und Untersuchungsverfahren - Bestimmung von Gossypol in
Baumwollsamen und Futtermitteln mittels LC-MS/MS
Aliments des animaux - Méthodes d’échantillonnage et d’analyse - Dosage du gossypol
dans les graines de coton et les aliments pour animaux par CL-SM/SM
Ta slovenski standard je istoveten z: prEN 17504
ICS:
65.120 Krmila Animal feeding stuffs
oSIST prEN 17504:2020 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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oSIST prEN 17504:2020
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oSIST prEN 17504:2020
DRAFT
EUROPEAN STANDARD
prEN 17504
NORME EUROPÉENNE
EUROPÄISCHE NORM
April 2020
ICS 65.120
English Version
Animal feeding stuffs: Methods of sampling and analysis -
Determination of gossypol in cotton seed and feeding stuff
by LC-MS/MS
Aliments des animaux - Méthodes d'échantillonnage et Futtermittel - Probenahme- und
d'analyse - Dosage du gossypol dans les graines de Untersuchungsverfahren - Bestimmung von Gossypol
coton et les aliments pour animaux par CL-SM/SM in Baumwollsamen und Futtermitteln mittels LC-
MS/MS
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, 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.
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
© 2020 CEN All rights of exploitation in any form and by any means reserved Ref. No. prEN 17504:2020 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 . 5
4 Principle . 5
5 Reagents . 5
6 Apparatus . 7
7 Procedure. 8
7.1 General . 8
7.2 Sample pre-treatment . 8
7.3 Test portion . 9
7.4 Extraction . 9
8 LC-MS/MS analysis . 10
8.1 General . 10
8.2 Analysis sequence . 11
9 Results . 11
9.1 Identification . 11
9.2 Quantification . 12
10 Precision . 14
10.1 Repeatability . 14
10.2 Reproducibility . 14
11 Test report . 15
Annex A (informative) Precision data . 16
Annex B (informative) Example of LC-MS/MS conditions . 18
B.1 General . 18
B.2 HPLC conditions . 18
B.3 MS conditions . 18
Annex C (informative) Example chromatogram gossypol in compound feed . 20
Bibliography . 21
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European foreword
This document (prEN 17504:2020) 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 standardization request given to CEN by the European
Commission and the European Free Trade Association.
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Introduction
Gossypol is a polyphenolic plant toxin produced by species of the genus Gossypium (cotton plant). The
plants are primarily grown for fibre and oil production. The seeds and processed seed materials are also
used as animal feeding stuffs. Gossypol is present in the seeds in two forms: free gossypol and bound
gossypol (mostly to proteins). The measured content of free gossypol depends on the method of
extraction and the specificity of the subsequent method of analysis used ([1], [2]). Consequently, this
method may not be directly comparable to existing standards based on spectrophotometric
measurement (e.g. [3], [4]).
WARNING — 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
protection measures and to ensure that regulatory and legal requirements are complied with.
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1 Scope
This document describes a method for the determination of free gossypol, extractable by acidified
acetonitrile/water, in cottonseed, cottonseed products and complete feed by liquid chromatography with
tandem mass spectrometry (LC-MS/MS).
This document has been successfully validated in the range of 69 mg/kg to 5 950 mg/kg by collaborative
trial in the following matrices: cottonseed, cottonseed products (cake/meal, hulls) and complete feed for
bovine, porcine and poultry.
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)
3 Terms and definitions
No terms and definitions are listed in this document.
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
4 Principle
Free gossypol is extracted by mixing a sample of 0,4 g of cottonseed or 1,0 g of cottonseed product or
compound feed with 40 ml acetonitrile:water:phosphoric acid (80:20:0,1). The mixture is shaken for 1 h.
After centrifugation a portion of the supernatant is diluted with extraction solvent in a vial. The final
extract is analysed by reversed phase liquid chromatography with tandem mass spectrometry (LC-
MS/MS). Quantification of gossypol in cottonseed and cottonseed products is based on multi-level
calibration using standards in extraction solvent. Quantification of gossypol in compound feeds is based
on multi-level calibration using standards in extraction solvent and corrected for the apparent recovery.
5 Reagents
WARNING — Gossypol may be hazardous to health. It has a strong toxic effect on the reproductive organs.
Depending on the level of exposure, both acute and chronic effects are possible.
5.1 Gossypol, ≥ 95 %, HPLC grade
NOTE 1 Gossypol is a racemic mixture of (+)- and (-)-gossypol. In this method the enantiomers of gossypol are
not separated.
NOTE 2 Gossypol is also available in the form of a 1:1 complex with acetic acid. In this method either a standard
of gossypol or gossypol-acetic acid can be used.
NOTE 3 Gossypol is sensitive to light.
5.2 Acetonitrile, LC-MS or HPLC quality
5.3 Phosphoric acid 85 %
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5.4 Formic acid
5.5 Water
Water of LC-MS grade, double-distilled or water of grade 1 as defined in EN ISO 3696.
5.6 Extraction solvent: 0,1 % phosphoric acid in acetonitrile:water (80:20)
Mix 800 ml acetonitrile (5.2) and 200 ml water (5.5) in a bottle of 1 000 ml. Add 1,0 ml phosphoric acid
(5.3) and mix. This solution is stored at room temperature and can be used for 1 month.
5.7 Mobile phase A: 0,5 % formic acid in acetonitrile:water (5:95)
Mix 50 ml acetonitrile (5.2) and 950 ml water (5.5) in a bottle of 1 000 ml. Add 5 ml formic acid (5.4) and
mix. This solution is stored at room temperature and can be used for 1 month.
5.8 Mobile phase B: 0,5 % formic acid in acetonitrile:water (95:5)
Mix 950 ml acetonitrile (5.2) and 50 ml water (5.5) in a bottle of 1 000 ml and mix. Add 5 ml formic acid
(5.4) and mix. This solution is stored at room temperature and can be used for 1 month.
5.9 Gossypol stock solution, 1 mg/ml
Weigh (6.1) 20 mg gossypol (5.1) and transfer into a 20 ml volumetric flask. Take into account the weight,
the purity and the appearance form of the standard. Add extraction solvent (5.6), dissolve by mixing and
make up to the mark with extraction solution (5.6). Transfer to an amber glass bottle (6.7) and store the
stock solution at < −18 °C. Under these conditions the solution can be used for 6 months.
5.10 Gossypol standard solution, 10 µg/ml
Transfer 100 µl of gossypol stock solution (5.9) into an amber glass bottle (6.7) using a pipette. Add
9,90 ml extraction solvent (5.6) and mix. Store the standard solution at < 18 °C. Under these conditions
the solution can be used for 2 months. Use this standard solution for preparation of calibration curves.
5.11 Gossypol standard solution, 1 µg/ml
Transfer 1,00 ml of gossypol standard solution (5.10) into an amber glass bottle (6.7) using a pipette. Add
9,00 ml extraction solvent (5.6) and mix. Store the standard solution at < 18 °C. Under these conditions
the solution can be used for 2 months. Use this standard solution for preparation of calibration curves.
5.12 Calibration solutions
Prepare calibration solutions using the standard solutions (5.10) and (5.11) and extraction solvent (5.6)
according to Table 1. Pipette directly in HPLC vials (6.8).
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Table 1 — Preparation of calibration standards
Concentration Standard solution of Standard solution of Extraction solvent
1 µg/ml (5.11) 10 µg/ml (5.10) (5.6)
µg/ml
µl µl µl
Cal 1 0,00 0 0 1 000
Cal 2 0,025 25 0 975
Cal 3 0,05 50 0 950
Cal 4 0,10 100 0 900
Cal 5 0,25 0 25 975
Cal 6 0,50 0 50 950
Cal 7 0,75 0 75 925
Cal 8 1,00 0 100 900
6 Apparatus
Usual laboratory equipment and, in particular, the following items.
6.1 Analytical balance, with an accuracy of 0,1 mg or better
6.2 Laboratory balance, with an accuracy of 0,01 g or better
6.3 Pipettors
Adjustable, suitable for organic solvents, properly calibrated, with appropriate tips.
6.4 Filter disc units, polytetrafluoroethylene (PTFE) with a pore size of 0,45 µm or less
6.5 Centrifuge tubes, polypropylene, 50 ml with screw cap
6.6 Centrifuge, suitable for 50 ml centrifuge tubes (6.5)
6.7 Glass bottles, amber coloured, different sizes
6.8 HPLC autosampler vials, amber glass 1,5 ml with caps
6.9 Minishaker or vortex mixer
6.10 Ultrasonic bath
6.11 Vertical or horizontal shaker, adjustable
6.12 Laboratory mill
6.13 HPLC system, consisting of:
6.13.1 Autosampler, thermostated
Capable of maintaining a temperature of 10 °C ± 1 °C.
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6.13.2 Binary pump system
Capable of delivering a binary gradient at flow rates appropriate for the analytical column in use with
sufficient accuracy.
6.13.3 Column oven, thermostated
Capable of maintaining a temperature of at least 40 °C ± 1 °C.
6.13.4 Analytical column
Containing C18 reversed phase packing material, capable of the base-line separation of the analyte from
compounds with identical molecular mass.
6.13.5 Pre-column, optional
With the same stationary phase material as the analytical column and with appropriate dimensions.
6.14 Tandem mass spectrometer
Capable of performing multiple selected reaction monitoring in negative ionization mode, with a
sufficiently wide dynamic range and capable of unit mass separation and equipped with a computer based
data processing system. Any ionization source giving sufficient yield may be employed.
7 Procedure
7.1 General
Animal feed is a complex matrix containing a wide range of ingredients in varying amounts. Sample and
instrument dependent matrix effects (suppression/enhancement) can occur that may affect the
quantification of the analyte in the sample (see NOTE). To correct for these matrix effects a recovery
sample (7.4.3) is included.
Depending on the sensitivity and linear range of the mass spectrometric instrument, it may be necessary
to dilute the sample extracts (7.4) by an appropriate factor with extraction solvent (5.6), taking into
account the expected concentration of the analyte in the sample, to keep the response of the detector
within the dynamic range of the mass spectrometer. Alternatively, the injection volume of the sample
may be reduced, within the calibrated range of the injection system.
NOTE At the conditions described in this document, matrix effects (ion suppression or enhancement) were not
significant for cottonseed and cottonseed products. For compound feed matrix effects were observed.
7.2 Sample pre-treatment
Laboratory samples should be taken and prepared in accordance with European legislation [2] where
applicable or, in any other case with EN ISO 6498.
Homogenize samples in a laboratory mill (6.12) to < 1 mm.
Cottonseeds may contain high amounts of gossypol. Carefully clean all parts of the grinder between
samples to avoid significant carry-over. When cottonseed samples are analysed together with cottonseed
products or compound feed materials, the cottonseed products and the compound feed samples should
be ground prior to the cottonseed samples.
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7.3 Test portion
7.3.1 Cottonseed
The amount of homogenized cottonseed material examined is 0,4 g ± 0,01 g.
A larger test sample size may be used by the laboratory in order to improve the representativeness of the
sample. The amount of extraction solvent shall in that case be adjusted accordingly (7.4).
7.3.2 Cottonseed products and compound feed
The amount of homogenized cottonseed product or compound feed material examined is 1,0 g ± 0,05 g.
A larger test sample size may be used by the laboratory in order to improve the representativeness of the
sample. The amount of extraction solvent shall in that case be adjusted accordingly (7.4).
7.4 Extraction
7.4.1 Cottonseed
Weigh (6.2) a test portion of 0,4 g ± 0,01 g homogenized sample (7.3) into a centrifuge tube of 50 ml (6.5).
Add 40,0 ml of extraction solvent (5.6) and mix for 15 s using a vortex mixer (6.9). Place the tube in an
ultrasonic bath (6.10) for 5 min and subsequently shake the tube for 1 h (6.11).
Centrifuge the tube for 5 min at 3 000 rpm at room temperature (6.6). Transfer 10 µl of the extract into a
HPLC vial (6.8) and add 990 µl extraction solvent (5.6) and mix (see NOTE).
NOTE Cottonseeds can contain high concentrations of gossypol (ranging from 2 000 mg/kg to 8 000 mg/kg).
The expected gossypol concentration in undiluted cottonseed extracts is in the range of 20 µg/ml to 80 µg/ml. After
dilution of the sample extract the expected concentration is in the range of 0,2 µg/ml to 0,8 µg/ml.
7.4.2 Cottonseed products and compound feed
Weigh (6.2) a test portion of 1,0 g ± 0,05 g homogenized sample (7.3) into a centrifuge tube of 50 ml (6.5).
Add 40,0 ml of extraction solvent (5.6) and mix for 15 s using a vortex mixer (6.9). Place the tube in an
ultrasonic bath (6.10) for 5 min and subsequently shake the tube for 1 h (6.11).
Centrifuge the tube for 5 min at 3 000 rpm at room temperature (6.6). Transfer 200 µl of the extract (
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