Animal feeding stuffs: Methods of sampling and analysis - Determination of pentachlorophenol (PCP) in feed materials and compound feed by LC-MS/MS

This document specifies a liquid chromatographic method with triple-quadrupole mass spectrometry (MS/MS) detection for the determination of pentachlorophenol (PCP) in feed materials and animal feed.
The limit of quantitation (LOQ) for the PCP determination in guar gum, fatty acid distillates (FAD) and animal feed is 10 µg/kg. Individual laboratories are responsible for ensuring that the equipment that they use will achieve this limit of quantification.
The method is validated in an international collaborative trial for pentachlorophenol in compound feed, guar gum and fatty acid distillate in the range between 9 µg/kg and 22 µg/kg.
The results of the collaborative trial, in which 16 laboratories participated, have shown that the method is applicable for the determination of PCP in compound feed, guar gum and FAD at the desired limit of 10 µg/kg. Satisfactory results were obtained for one compound feed sample, guar gum and the two FAD samples (HorRat <2), while for the second compound feed sample a HorRat value of 2,2 was obtained.

Futtermittel: Probenahme- und Untersuchungsverfahren - Bestimmung von Pentachlorphenol (PCP) in Futtermittel und Mischfuttermittel mittels LC-MS/MS

Dieses Dokument legt ein flüssigkeitschromatographisches Verfahren mit Nachweis mittels Triple-Quadrupol Massenspektrometrie (MS/MS) für die Bestimmung von Pentachlorphenol (PCP) in Einzelfutter  und Mischfuttermitteln fest.
Die Bestimmungsgrenze (LOQ) für die PCP Bestimmung in Guargummi, Fettsäuredestillaten (FAD) und Futtermitteln liegt bei 10 µg/kg. Die einzelnen Laboratorien sind selbst dafür verantwortlich sicherzustellen, dass die von ihm verwendeten Geräte diese Bestimmungsgrenze auch erreichen.
Das Verfahren ist in einem internationalen Ringversuch zur Pentachlorphenol Bestimmung in Mischfutter, Guargummi und Fettsäuredestillat im Bereich zwischen 9 µg/kg und 22 µg/kg validiert worden.
Die Ergebnisse des Ringversuchs, an dem 16 Laboratorien teilnahmen, haben gezeigt, dass das Verfahren für die Bestimmung von PCP in Mischfutter, Guargummi und FAD beim gewünschten Grenzwert von 10 µg/kg anwendbar ist. Zufriedenstellende Ergebnisse (HorRat Wert < 2) wurden für eine Mischfutter Probe, Guargummi und die beiden FAD Proben erhalten, während bei der zweiten Mischfutter Probe ein HorRat Wert von 2,2 erhalten wurde.

Aliments pour animaux : Méthodes d’échantillonnage et d’analyse - Détermination de la teneur en pentachlorophénol (PCP) dans les matières premières pour aliments des animaux et les aliments composés pour animaux par CL-SM/SM

Le présent document spécifie une méthode de chromatographie liquide couplée à la détection par spectrométrie de masse triple quadripôle (SM/SM) pour la détermination de la teneur en pentachlorophénol (PCP) dans les matières premières pour aliments des animaux et les aliments pour animaux.
La limite de quantification (LOQ) applicable à la détermination de la teneur en PCP dans la gomme de guar, les distillats d’acides gras (FAD) et les aliments pour animaux est de 10 µg/kg. Chaque laboratoire est tenu de s’assurer que l’équipement qu’il utilise atteindra cette limite de quantification.
La méthode a été validée lors d’un essai interlaboratoires international portant sur le pentachlorophénol présent dans les aliments composés pour animaux, la gomme de guar et les distillats d’acides gras dans la gamme allant de 9 µg/kg à 22 µg/kg.
Les résultats de l’essai interlaboratoires, auquel ont participé 16 laboratoires, ont montré que la méthode est applicable à la détermination de la teneur en PCP dans les aliments composés pour animaux, la gomme de guar et les FAD à la limite souhaitée de 10 µg/kg. Des résultats satisfaisants ont été obtenus pour un échantillon d’aliment composé, la gomme de guar et les deux échantillons de FAD (HorRat < 2), alors qu’une valeur de HorRat de 2,2 a été obtenue pour le deuxième échantillon d’aliment composé.

Krma: metode vzorčenja in analize - Določevanje pentaklorofenola (PCP) v sestavinah krme in krmni mešanici z LC-MS/MS

General Information

Status
Published
Public Enquiry End Date
24-Apr-2019
Publication Date
18-Jun-2020
Current Stage
6060 - National Implementation/Publication (Adopted Project)
Start Date
19-May-2020
Due Date
24-Jul-2020
Completion Date
19-Jun-2020

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SLOVENSKI STANDARD
SIST EN 17362:2020
01-julij-2020
Krma: metode vzorčenja in analize - Določevanje pentaklorofenola (PCP) v
sestavinah krme in krmni mešanici z LC-MS/MS
Animal feeding stuffs: Methods of sampling and analysis - Determination of
pentachlorophenol (PCP) in feed materials and compound feed by LC-MS/MS
Futtermittel: Probenahme- und Untersuchungsverfahren - Bestimmung von
Pentachlorphenol (PCP) in Futtermittel und Mischfuttermittel mittels LC-MS/MS

Aliments pour animaux : Méthodes d’échantillonnage et d’analyse - Détermination de la

teneur en pentachlorophénol (PCP) dans les matières premières pour aliments des
animaux et les aliments composés pour animaux par CL-SM/SM
Ta slovenski standard je istoveten z: EN 17362:2020
ICS:
65.120 Krmila Animal feeding stuffs
SIST EN 17362:2020 en,fr,de

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

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SIST EN 17362:2020
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SIST EN 17362:2020
EN 17362
EUROPEAN STANDARD
NORME EUROPÉENNE
April 2020
EUROPÄISCHE NORM
ICS 65.120
English Version
Animal feeding stuffs: Methods of sampling and analysis -
Determination of pentachlorophenol (PCP) in feed
materials and compound feed by LC-MS/MS

Aliments pour animaux : Méthodes d'échantillonnage Futtermittel: Probenahme- und

et d'analyse - Détermination de la teneur en Untersuchungsverfahren - Bestimmung von

pentachlorophénol (PCP) dans les matières premières Pentachlorphenol (PCP) in Futtermittel und

pour aliments des animaux et les aliments composés Mischfuttermittel mittels LC-MS/MS

pour animaux par CL-SM/SM
This European Standard was approved by CEN on 7 March 2020.

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

© 2020 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN 17362:2020 E

worldwide for CEN national Members.
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SIST EN 17362:2020
EN 17362:2020 (E)
Contents Page

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

1 Scope .................................................................................................................................................................... 4

2 Normative references .................................................................................................................................... 4

3 Terms and definitions ................................................................................................................................... 4

4 Principle ............................................................................................................................................................. 7

5 Reagents and materials ................................................................................................................................. 7

5.1 General ................................................................................................................................................................ 7

5.2 Chemicals ........................................................................................................................................................... 7

6 Apparatus ........................................................................................................................................................ 10

6.1 Analytical balance, accuracy to 0,1 mg ................................................................................................. 10

6.2 Centrifuge ........................................................................................................................................................ 10

6.3 Water bath ...................................................................................................................................................... 10

6.4 Liquid chromatograph connected to a triple-quadrupole mass spectrometer ..................... 10

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

8 Preparation of the test sample ................................................................................................................ 12

9 Procedure........................................................................................................................................................ 12

9.1 General ............................................................................................................................................................. 12

9.2 Test portions and extraction .................................................................................................................... 12

9.3 LC-MS/MS ........................................................................................................................................................ 13

10 Calculation and expression of results ................................................................................................... 13

10.1 General ............................................................................................................................................................. 13

10.2 Calibration criteria ...................................................................................................................................... 14

10.3 Identification and confirmation.............................................................................................................. 14

10.4 Calculation ...................................................................................................................................................... 14

10.5 Recovery .......................................................................................................................................................... 15

11 Precision .......................................................................................................................................................... 15

11.1 Collaborative test ......................................................................................................................................... 15

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

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

12 Test report ...................................................................................................................................................... 16

Annex A (informative) Collaborative trial........................................................................................................ 17

Bibliography ................................................................................................................................................................. 19

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SIST EN 17362:2020
EN 17362:2020 (E)
European foreword

This document (EN 17362:2020) 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 shall be given the status of a national standard, either by publication of an identical text

or by endorsement, at the latest by October 2020, and conflicting national standards shall be withdrawn

at the latest by October 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 document: 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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SIST EN 17362:2020
EN 17362:2020 (E)
1 Scope

This document specifies a liquid chromatographic method with triple-quadrupole mass spectrometry

(MS/MS) detection for the determination of pentachlorophenol (PCP) in feed materials and compound

feed.

The limit of quantitation (LOQ) for the PCP determination in guar gum, fatty acid distillates (FAD) and

compound feed is 10 µg/kg. Individual laboratories are responsible for ensuring that the equipment that

they use will achieve this limit of quantification.

The method is validated in an international collaborative trial for pentachlorophenol in compound feed,

guar gum and fatty acid distillate in the range between 9 µg/kg and 22 µg/kg.

The results of the collaborative trial, in which 16 laboratories participated, have shown that the method

is applicable for the determination of PCP in compound feed, guar gum and FAD at the desired limit of

10 µg/kg. Satisfactory results were obtained for one compound feed sample, guar gum and the two FAD

samples (HorRat < 2), while for the second compound feed sample a HorRat value of 2,2 was obtained.

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 6498, Animal feeding stuffs — Guidelines for sample preparation (ISO 6498)

3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.

ISO and IEC maintain terminological databases for use in standardization at the following addresses:

— IEC Electropedia: available at http://www.electropedia.org/
— ISO Online browsing platform: available at https://www.iso.org/obp
3.1
calibration

complete set of operations which estimates under specified conditions the calibration function from

observations of the response variable obtained on reference states
[SOURCE: ISO 3534-2:2006, 3.5.13 [1]]
3.2
collaborative trial
interlaboratory comparisons

organization, performance and evaluation of measurements or tests on the same or similar items by two

or more laboratories in accordance with predetermined conditions
[SOURCE: EN ISO/IEC 17043:2010, 3.4 [2]]
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3.3
feed material

products of vegetable or animal origin, whose principal purpose is to meet animals’ nutritional needs, in

their natural state, fresh or preserved, and products derived from the industrial processing thereof, and

organic or inorganic substances, whether or not containing feed additives, which are intended for use in

oral animal-feeding either directly as such, or after processing, or in the preparation of compound feed,

or as carrier of premixtures
[SOURCE: Regulation (EC) No 767/2009 (Article 3(2)(g)) [3]]
3.4
HorRat

ratio of the reproducibility relative standard deviation to that calculated from the Horwitz equation

–0,15
Note 1 to entry: Predicted relative standard deviation PRSD = 2 C
HorRatR = RSDR / PRSDR (1)
HorRatr = RSDr / PRSDr (2)

Note 2 to entry: If applied to within-laboratory studies, the normal range of HorRat(r) is 0,30 to 1,30.

Note 3 to entry: To check proper calculation of PRSDR, a C of 10 should give a PRSDR of 16 %. C is concentration

expressed as a mass fraction (both numerator and denominator expressed in the same units). The HorRat is

indicative of method performance for a large majority of methods in chemistry. Normal values lie between 0,50 and

2,00.
[SOURCE: ISO 16577:2016, 3.75 [4]]
3.5
limit of quantitation

measured quantity value, obtained by a given measurement procedure, which is the lowest concentration

of a measurand that can be determined with an acceptable level of repeatability precision and trueness

Note 1 to entry: The “limit of quantitation” is not a concept defined in ISO/IEC Guide 99:2007 [5] but has been

defined as described above.
Note 2 to entry: The abbreviation LOQ is sometimes used.
3.6
precision

closeness of agreement between indications or measured quantity values obtained by replicate

measurements on the same or similar objects under specified conditions

Note 1 to entry: Measurement precision is usually expressed numerically by measures of imprecision, such as

standard deviation, variance, or coefficient of variation under the specified conditions of measurement.

Note 2 to entry: The ‘specified conditions’ can be, for example, repeatability conditions of measurement,

intermediate precision conditions of measurement, or reproducibility conditions of measurement (see ISO 5725-1

[6]).

Note 3 to entry: Measurement precision is used to define measurement repeatability, intermediate measurement

precision, and measurement reproducibility.

Note 4 to entry: Sometimes 'measurement precision' is erroneously used to mean 'measurement accuracy'.

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EN 17362:2020 (E)
[SOURCE: ISO IEC Guide 99:2007, 2.15 [5]]
3.7
repeatability
precision under repeatability conditions

Note 1 to entry: Repeatability can be expressed quantitatively in terms of the dispersion characteristics of the

results.
[SOURCE: ISO 3534-2:2006, 3.3.5 [1]]
3.8
repeatability limit
repeatability critical difference for a specified probability of 95 %
[SOURCE: ISO 3534-2:2006, 3.3.9 [1]]
3.9
repeatability standard deviation

standard deviation of test results or measurement results obtained under repeatability conditions

Note 1 to entry: It is a measure of the dispersion of the distribution of test or measurement results under

repeatability conditions.

Note 2 to entry: Similarly, “repeatability variance” and “repeatability coefficient of variation” can be defined and

used as measures of the dispersion of test or measurement results under repeatability conditions.

[SOURCE: ISO 3534-2:2006, 3.3.7 [1]]
3.10
reproducibility
precision under reproducibility conditions

Note 1 to entry: Reproducibility can be expressed quantitatively in terms of the dispersion characteristics of the

results.
Note 2 to entry: Results are usually understood to be corrected results.
[SOURCE: ISO 3534-2:2006, 3.3.10 [1]]
3.11
reproducibility limit
reproducibility critical difference for a specified probability of 95 %
[SOURCE: ISO 3534-2:2006, 3.3.14 [1]]
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SIST EN 17362:2020
EN 17362:2020 (E)
3.12
reproducibility standard deviation

standard deviation of test results or measurement results obtained under reproducibility conditions

Note 1 to entry: It is a measure of the dispersion of the distribution of test or measurement results under

reproducibility conditions.

Note 2 to entry: Similarly, “reproducibility variance” and “reproducibility coefficient of variation” can be defined

and used as measures of the dispersion of test or measurement results under reproducibility conditions.

[SOURCE: ISO 3534-2:2006, 3.3.12 [1]]
4 Principle

In order to check for the presence of PCP, a test portion of sample material is fortified with internal

standards ( C-PCP). The test portion is extracted using a QuEChERS approach for compound feed and

FAD.

A ‘reversed’ QuEChERS approach is used for guar gum. For FAD samples, lipids are removed prior to

QuEChERS extraction by the addition of sulfuric acid.

Final extracts from all matrices are analysed by liquid chromatography coupled to triple quadrupole mass

spectrometry, operated in negative electrospray ionization mode.

Identification is done on the basis of retention time and mass-to-charge ratio. Quantification is done using

the internal standard method.
5 Reagents and materials
5.1 General

Use only reagents of recognized analytical grade and with a purity suitable for organochlorine residue

analysis. Check the purity of the reagents by performing a blank test under the same conditions as used

in the method. The chromatogram should not show any interfering impurity at the retention time of

compounds of interest.
5.2 Chemicals
5.2.1 Diethylamine (DEA)
5.2.2 Acetonitrile (ACN)
5.2.3 Methanol
5.2.4 Acetone
5.2.5 n-Hexane
5.2.6 Sulphuric acid (concentrated, 95 % – 98 %)
5.2.7 Deionized water
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SIST EN 17362:2020
EN 17362:2020 (E)
5.2.8 Sodium hydroxide
5.2.8.1 Sodium hydroxide solution (10 M)

Weigh 4 g of sodium hydroxide (5.2.8) and add 10 ml deionized water (5.2.7) to achieve a concentration

of 10 M. Store at room temperature in a closed glass bottle with a plastic cap or stopper.

5.2.9 Magnesium sulphate
5.2.10 Sodium chloride
5.2.11 Formic acid
5.2.12 Ammonium formate
5.2.12.1 Ammonium formate solution (1 M)

Weigh 6,3 g ammonium formate (5.2.12) and add 100 ml deionized water (5.2.7) to achieve a

concentration of 1 M. Store the solution at room temperature. The solution is stable under these

conditions for at least 1 month.
5.2.13 Mobile phase A

Take 1 ml ammonium formate solution (1 M) (5.2.12.1) and add 999 ml deionized water (5.2.7) and 20 µL

formic acid (5.2.11). Mix well. Store the solution at room temperature. The solution is stable under these

conditions for at least 1 month.
5.2.14 Mobile phase B

Take 1 ml ammonium formate solution (1 M) (5.2.12.1) and add 50 ml deionized water (5.2.7), 949 ml

methanol (5.2.3) and 20 µl formic acid (5.2.11) and mix well. Store the solution at room temperature. The

solution is stable under these conditions for at least 1 month.
5.2.15 Pentachlorophenol (PCP sodium salt, 95 % purity)
5.2.15.1 PCP Stock solution 1 (2 000 µg/ml)

Weigh 25 mg (± 0,01 mg) of PCP (5.2.15) (taking impurities into consideration) and add 12,5 ml

deionized water (5.2.7) to achieve a concentration of 2 000 µg/ml. Store the solution in a refrigerator at

4 °C (± 3 °C). The solution is stable under these conditions for at least 12 months.

5.2.15.2 PCP Stock solution 2 (1 ng/µl)

Dilute 50 µl of PCP Stock solution 1 (5.2.15.1) to 100 ml deionized water (5.2.7) in a volumetric flask

(5.2.20). Store the solutio
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