oSIST prEN 17504:2020

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 - Določevanje gosipola v bombažnem semenu in krmi z LC-MS/MS

General Information

• Status: Not Published
• Public Enquiry End Date: 19-Jun-2020
• ICS: 65.120 - Animal feeding stuffs
• Technical Committee: KŽP - Agricultural food products
• Current Stage: 4020 - Public enquire (PE) (Adopted Project)
• Start Date: 22-Apr-2020
• Due Date: 09-Sep-2020
• Completion Date: 09-Jul-2020
• Directive: 882/2004 - Regulation (EC) No 882/2004 of the European Parliament and of the Council of 29 April 2004 onofficial controls performed to ensure the verification of compliance with feed and food law, animal health and animal welfare rules
• Mandate: M/523 - Mandate for standardisation addressed to CEN for methods of analysis in the field of animal nutrition - Part III
• Ref Project: prEN 17504 - Animal feeding stuffs: Methods of sampling and analysis - Determination of gossypol in cotton seed and feeding stuff by LC-MS/MS

Standards Content (sample)

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
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 (

...