Foodstuffs - Determination of phomopsin A in lupin seeds and lupin derived products by HPLC-MS/MS

This document describes a procedure for the determination of phomopsins in lupin seeds and lupin-derived products based on liquid chromatography with tandem mass spectrometry (LC-MS/MS). Several phomopsins exist, i.e. phomopsin A, B, C and D, but the method only deals with the quantitative measurement of phomopsin A due to lack of commercially available analytical reference standards for the other phomopsins.
The method has been validated for phomopsin A in naturally contaminated lupin seeds, lupin flour and crisp bread at levels ranging from approximately 5 µg/kg to 60 µg/kg.

Lebensmittel - Bestimmung von Phomopsin A in Lupinensamen und Lupinenerzeugnissen mit LC-MS/MS

Diese Europäische Norm beschreibt ein Verfahren zur Bestimmung von Phomopsinen in Lupinensamen und Lupinenerzeugnissen mit Flüssigchromatographie und Tandem-Massenspektrometrie (LC MS/MS). Es gibt mehrere Phomopsine, z. B. Phomopsin A, B, C und D, aber das Verfahren behandelt nur die quantitative Bestimmung von Phomopsin A, da Vergleichssubstanzen für die anderen Phomopsine nicht im Handel erhältlich sind.
Das Verfahren wurde für Phomopsin A in natürlich kontaminierten Lupinensamen, Lupinenmehl und Knäckebrot in Gehalten von etwa 5 μg/kg bis 60 μg/kg validiert.

Produits alimentaires - Détermination de la teneur en phomopsine A dans les graines de lupin et les produits dérivés du lupin par CL-SM/SM

Le présent document décrit un mode opératoire pour le dosage des phomopsines dans les graines de lupin et les produits dérivés du lupin par chromatographie liquide couplée à la spectrométrie de masse en tandem (CL-SM/SM). Il existe plusieurs phomopsines, à savoir la phomopsine A, B, C et D. Toutefois, en raison de l’absence d’étalons de référence analytiques disponibles dans le commerce pour les autres phomopsines, la méthode concerne uniquement le dosage quantitatif de la phomopsine A.
La méthode a été validée pour la phomopsine A dans les graines de lupin, la farine de lupin et le pain croustillant naturellement contaminés à des niveaux compris entre environ 5 µg/kg et 60 µg/kg.

Živila - Določevanje fomopsina A v semenih volčjega boba in predelanih proizvodih s HPLC-MS/MS

Ta dokument opisuje postopek za določevanje fomopsinov v semenih volčjega boba in predelanih proizvodih s tekočinsko kromatografijo s tandemsko masno spektrometrijo (LC-MS/MS). Obstaja več fomopsinov (fomopsin A, B, C in D), toda zaradi pomanjkanja komercialno razpoložljivih analitičnih referenčnih standardov za druge fomopsine metoda zajema le kvantitativno merjenje fomopsina A.
Metoda je bila validirana za fomopsin A v naravno kontaminiranih semenih volčjega boba, moki volčjega boba in hrustljavem kruhu v razponu od približno 5 do 60 µg/kg.

General Information

Status
Published
Public Enquiry End Date
19-Jul-2018
Publication Date
09-Feb-2020
Current Stage
6060 - National Implementation/Publication (Adopted Project)
Start Date
22-Jan-2020
Due Date
28-Mar-2020
Completion Date
10-Feb-2020

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SLOVENSKI STANDARD
SIST EN 17252:2020
01-marec-2020
Živila - Določevanje fomopsina A v semenih volčjega boba in predelanih
proizvodih s HPLC-MS/MS

Foodstuffs - Determination of phomopsin A in lupin seeds and lupin derived products by

HPLC-MS/MS
Lebensmittel - Bestimmung von Phomopsin A in Lupinensamen und
Lupinenerzeugnissen mit LC-MS/MS

Produits alimentaires - Détermination de la teneur en phomopsine A dans les graines de

lupin et les produits dérivés du lupin par CL-SM/SM
Ta slovenski standard je istoveten z: EN 17252:2020
ICS:
67.060 Žita, stročnice in proizvodi iz Cereals, pulses and derived
njih products
SIST EN 17252:2020 en,fr,de

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

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SIST EN 17252:2020
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SIST EN 17252:2020
EN 17252
EUROPEAN STANDARD
NORME EUROPÉENNE
January 2020
EUROPÄISCHE NORM
ICS 67.060
English Version
Foodstuffs - Determination of phomopsin A in lupin seeds
and lupin derived products by HPLC-MS/MS

Produits alimentaires - Détermination de la teneur en Lebensmittel - Bestimmung von Phomopsin A in

phomopsine A dans les graines de lupin et les produits Lupinensamen und Lupinenerzeugnissen mit

dérivés du lupin par CL-SM/SM LC-MS/MS
This European Standard was approved by CEN on 9 October 2019.

CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this

European Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references

concerning such national standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN

member.

This European Standard exists in three official versions (English, French, German). A version in any other language made by

translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management

Centre has the same status as the official versions.

CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,

Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,

Poland, Portugal, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and

United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATIO N
EUROPÄISCHES KOMITEE FÜR NORMUN G
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 17252:2020 E

worldwide for CEN national Members.
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SIST EN 17252:2020
EN 17252:2020 (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 and equipment ........................................................................................................................... 7

7 Procedure........................................................................................................................................................... 8

8 Calculation ...................................................................................................................................................... 10

9 Precision .......................................................................................................................................................... 10

10 Test report ...................................................................................................................................................... 11

Annex A (informative) Precision data ............................................................................................................... 12

Annex B (informative) Examples conditions for suitable LC-MS/MS systems with typical

chromatograms ............................................................................................................................................. 13

Bibliography ................................................................................................................................................................. 18

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SIST EN 17252:2020
EN 17252:2020 (E)
European foreword

This document (EN 17252:2020) has been prepared by Technical Committee CEN/TC 275 “Food

analysis - Horizontal methods”, the secretariat of which is held by DIN.

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 July 2020, and conflicting national standards shall be

withdrawn at the latest by July 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 mandate given to CEN by the European Commission and the

European Free Trade Association.

According to the CEN-CENELEC Internal Regulations, the national standards organisations of the

following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria,

Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland,

Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Republic of

North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the

United Kingdom.
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SIST EN 17252:2020
EN 17252:2020 (E)
Introduction

Phomopsins are mycotoxins produced by the fungus Diaporthe toxica. There are several phomopsins of

which phomopsin A is the major toxic congener. The main host of the fungus are lupins (Lupinus L.).

Lupin seeds are being used as food ingredients and therefore phomopsin A might occur in food

ingredients and food products containing lupin seeds or lupin flour.

WARNING 1 — Suitable precaution and protection measures need to be taken when carrying out

working steps with harmful chemicals. The latest version of the hazardous substances ordinance (EU)

1907/2006 [3] should be taken into account as well as appropriate national statements.

WARNING 2 — The use of this document can involve hazardous materials, operations and equipment.

This document does not purport to address all the safety problems associated with its use. It is the

responsibility of the user of this document to establish appropriate safety and health practices and

determine the applicability of regulatory limitations prior to use.
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SIST EN 17252:2020
EN 17252:2020 (E)
1 Scope

This document specifies a procedure for the determination of phomopsin A in lupin seeds and lupin-

derived products based on liquid chromatography with tandem mass spectrometry (LC-MS/MS).

Several phomopsins exist, i.e. phomopsin A, B, C and D, but the method only deals with the quantitative

measurement of phomopsin A due to lack of commercially available analytical reference standards for

the other phomopsins.

The method has been validated for phomopsin A in naturally contaminated lupin seeds, lupin flour and

crisp bread at levels ranging from approximately 5 µg/kg to 60 µg/kg.
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:

• IEC Electropedia: available at http://www.electropedia.org/
• ISO Online browsing platform: available at https://www.iso.org/obp
4 Principle

Phomopsin A is extracted from the homogenized sample material by shaking with a mixture of

acetonitrile/water/acetic acid (80+19+1, v+v+v). After centrifugation, an aliquot of the extract is

diluted with water, optionally filtered, and analysed by liquid chromatography coupled to tandem mass

spectrometry (LC-MS/MS). Phomopsin A is quantified by multi-level matrix-matched calibration.

5 Reagents

Use only reagents of recognized analytical grade and water complying with grade 1 of EN ISO 3696,

unless otherwise specified. Solutions shall be of quality for LC analysis, unless otherwise specified.

5.1 Water, deionised.
5.2 Water, LC-MS grade.
5.3 Acetonitrile, pro analysis (p.a.).
5.4 Methanol, LC-MS grade.
5.5 Acetic acid, purity greater than mass fraction w ≥ 98 %.
5.6 Ammonium formate, p.a.
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SIST EN 17252:2020
EN 17252:2020 (E)
5.7 Extraction solution acetonitrile/water/acetic acid, (80+19+1, v+v+v).

Mix 800 ml of acetonitrile (5.3), 190 ml of water (5.1 or 5.2) and 10 ml of acetic acid (5.5) in a bottle of

1 000 ml. This solution can be used for 3 months if stored at room temperature.

5.8 Phomopsin A, isolated from Phomopsis leptostromiformis, purity greater than w ≥ 98 %.

5.9 Phomopsin A stock solution (STD 1), mass concentration ρ = 500 mg/l.

Weigh 5 mg of the phomopsin A standard (5.8) to the nearest 0,1 mg into a 10 ml volumetric flask and

make up to the volume with methanol (5.4). Take into account the exact weight and the purity of the

standard. The solution can be used for 3 months if stored in an amber flask in the refrigerator at

approximately 4 °C.
5.10 Standard solution of phomopsin A (STD 2), ρ = 10 mg/l.

Pipette 100 µl of the standard solution (STD 1) (5.9) into a volumetric flask of 5 ml and make up to the

volume with methanol (5.4). The solution can be used for 3 months if stored in an amber vial in the

refrigerator at approximately 4 °C.
5.11 Standard solution of phomopsin A (STD 3), ρ = 250 µg/l.

Pipette 250 µl of the standard solution (STD 2) (5.10) into a volumetric flask of 10 ml and make up to

the volume with methanol (5.4).
5.12 Intermediate solutions for preparation of the matrix-matched standards.

To seven vials (6.9) add different volumes of the standard solution of phomopsin A (STD 3) (5.11) and

methanol (5.4) according to Table 1. Cap the vials and mix. Prepare these solutions freshly for each

batch of analysis.
Table 1 — Intermediate standard solutions of phomopsin A in methanol
Standard solution Methanol (5.4) Mass concentration
Intermediate solution
(STD 3) (5.11)
no µl µl µg/l
1 25 975 6,25
2 50 950 12,5
3 100 900 25
4 200 800 50
5 350 650 87,5
6 500 500 125
7 650 350 162,5
5.13 Matrix matched calibration solutions

Prepare matrix-matched calibration solutions in vials (6.9) according to Table 2.

The matrix matched calibration solutions may also be prepared directly in auto sampler vials with

insert or filter vials. In that case, proportionally reduce the volumes indicated in Table 2.

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SIST EN 17252:2020
EN 17252:2020 (E)

Table 2 — Matrix matched calibration solutions of phomopsin A in blank matrix extract

Calibration Mass Blank Intermediate Water Equivalent to mass
solution concentration extract solutions (5.2) fraction in sample
(7.2) (5.12) see
Table 1
no µg/l µl µl µl µg/kg
0 0 500 0 500 0
1 0,3125 450 50 µl no 1 500 2,5
2 0,625 450 50 µl no 2 500 5,0
3 1,25 450 50 µl no 3 500 10,0
4 2,5 450 50 µl no 4 500 20,0
5 4,375 450 50 µl no 5 500 35,0
6 6,25 450 50 µl no 6 500 50,0
7 8,125 450 50 µl no 7 500 65,0
6 Apparatus and equipment
Usual laboratory glassware and equipment, in particular, the following:
6.1 Conical polypropylene screw cap centrifuge tubes, 50 ml with caps.
6.2 Volumetric flasks, 5 ml and 10 ml.
6.3 Analytical balance, accuracy 0,1 mg.
6.4 Laboratory balance, accuracy 0,01 g.
6.5 Pipettes, e.g. 10 µl to 1 000 µl, for organic solutions.

6.6 Adjustable mechanical vertical or horizontal shaker or rotary tumbling machine.

6.7 Laboratory shaker.
6.8 Centrifuge, capable of generating a relative centrifugal force of 3 500 g.

6.9 Vials, 1,5 ml to 2 ml, used for intermediate solutions (5.12), made of glass or polypropylene, with

screw cap.

6.10 Syringe filter, 0,20 µm to 0,45 µm, nylon or polytetrafluoroethylene (PTFE) (for optional

filtration of final extracts).

6.11 Auto sampler vials, of appropriate size for the auto sampler in use, e.g. glass with insert vials, or

filter vials (PTFE 0,45 μm), with crimp cap or equivalent.
6.12 LC-MS/MS system with the following components:

6.12.1 LC pump, capable of delivering a binary gradient at flow rates appropriate for the analytical

column in use with sufficient accuracy.
---------------------- Page: 9 ----------------------
SIST EN 17252:2020
EN 17252:2020 (E)

6.12.2 Injection system, capable of injecting an appropriate volume of injection solution with

sufficient accuracy.

6.12.3 LC column, capable of retaining phomopsin A with a retention factor of at least two and that

ensures base line separation to distinguish peaks of phomopsin A from all other signals. Examples of

suitable columns are listed in Annex B.
6.12.4 Column oven, capable of maintaining a constant temperature.

6.12.5 Tandem mass spectrometer (MS/MS), capable of performing ionization of phomopsin A and

selected reaction monitoring (SRM), with a sufficiently wide dynamic
...

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.ODQLKLebensmittel - Bestimmung von Phomopsin A in Lupinensamen und Lupinenerzeugnissen mit LC-MS/MSProduits alimentaires - Détermination de la teneur en phomopsine A dans les graines de lupin et les produits dérivés du lupin par CL-SM/SMFoodstuffs - Determination of phomopsin A in lupin seeds and lupin derived products by LC-MS/MS67.060QMLKCereals, pulses and derived productsICS:Ta slovenski standard je istoveten z:prEN 17252oSIST prEN 17252:2018en,fr,de01-julij-2018oSIST prEN 17252:2018SLOVENSKI

STANDARD
oSIST prEN 17252:2018
EUROPEAN STANDARD NORME EUROPÉENNE EUROPÄISCHE NORM
DRAFT prEN 17252
May
t r s z ICS
x yä r x r English Version

Foodstuffs æ Determination of phomopsin A in lupin seeds Produits alimentaires æ Détermination de la teneur en phomopsine A dans les graines de lupin et les produits

Lebensmittel æ Bestimmung von Phomopsin A in Lupinensamen und Lupinenerzeugnissen mit LCæThis draft European Standard is submitted to CEN members for enquiryä It has been drawn up by the Technical Committee

If this draft becomes a European Standardá CEN members are bounwhich stipulate the conditions for giving this European Standard the status of a national standard without any alterationä

This draft European Standard was established by CEN in three ofer language made by translation under the responsibility of a CEN member into its own language and notified to the CENæCENELEC Management Centre has the same status as the official versionsä

CEN members are the national standards bodies of Austriaá Belgiumá Bulgariaá Croatiaá Cyprusá Czech Republicá Denmarká Estoniaá Finlandá Former Yugoslav Republic of Macedoniaá Franceá Germanyá Greeceá Hungaryá Icelandá Irelandá Italyá Latviaá Lithuaniaá Luxembourgá Maltaá Netherlandsá Norwayá Polandá Portugalá Romaniaá Serbiaá Slovakiaá Sloveniaá Spainá Swedená Switzerlandá Turkey and United Kingdomä

Recipients of this draft are invited to submitá with their commentsá notification of any relevant patent rights of which they are aware and to provide supporting documentationä

Warning ã This document is not a European Standardä It is distributed for review and commentsä It is subject to change without notice and shall not be referred to as a European Standardä

EUROPEAN COMMITTEE FOR STANDARDIZATION COMITÉ EUROPÉEN DE NORMALISATION EUROPÄISCHES KOMITEE FÜR NORMUNG

CEN-CENELEC Management Centre:
Rue de la Science 23,
B-1040 Brussels

t r s z CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Membersä Refä Noä prEN

s y t w tã t r s z EoSIST prEN 17252:2018
prEN 17252:2018 (E) 2 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 and equipment .............................................................................................................................. 7 7 Procedure .............................................................................................................................................................. 8 8 Calculation ......................................................................................................................................................... 10 9 Precision ............................................................................................................................................................. 10 10 Test report ......................................................................................................................................................... 11 Annex A (informative)

Precision data ............................................................................................................... 12 Annex B (informative)

Examples of extracted ion chromatograms........................................................ 13 Bibliography ................................................................................................................................................................. 18 oSIST prEN 17252:2018

prEN 17252:2018 (E) 3 European foreword This document (prEN 17252:2018) has been prepared by Technical Committee CEN/TC 275 “Food analysis - Horizontal methods”, the secretariat of which is held by DIN. 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.

oSIST prEN 17252:2018

prEN 17252:2018 (E) 4 Introduction Phomopsins are mycotoxins produced by the fungus Diaporthe toxica. There are several phomopsins of which phomopsin A is the major toxic congener. The main host of the fungus are lupins (Lupinus L.). Lupin seeds are being used as food ingredient and therefore phomopsin A might occur in food ingredients and food products containing lupin seeds or lupin flour. WARNING 1 — Suitable precaution and protection measures need to be taken when carrying out working steps with harmful chemicals. The latest version of the hazardous substances ordinance (EU) 1907/2006, [3] should be taken into account as well as appropriate National statements. WARNING 2 — The use of this document can involve hazardous materials, operations and equipment. This document does not purport to address all the safety problems associated with its use. It is the responsibility of the user of this document to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.

oSIST prEN 17252:2018

prEN 17252:2018 (E) 5 1 Scope This document describes a procedure for the determination of phomopsins in lupin seeds and lupin-derived products based on liquid chromatography with tandem mass spectrometry (LC-MS/MS). Several phomopsins exist, i.e. phomopsin A, B, C and D, but the method only deals with the quantitative measurement of phomopsin A due to lack of commercially available analytical reference standards for the other phomopsins. The method has been validated for phomopsin A in naturally contaminated lupin seeds, lupin flour and crisp bread at levels ranging from approximately 5 µg/kg to 60 µg/kg. 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:

IEC Electropedia: available at http://www.electropedia.org/

ISO Online browsing platform: available at http://www.iso.org/obp 4 Principle The phomopsins are extracted from the homogenized sample material by shaking with a mixture of acetonitrile/water/acetic acid (80+19+1, v+v+v). After centrifugation, an aliquot of the extract is diluted with water, optionally filtered, and analysed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Phomopsins are quantified by multi-level matrix-matched calibration. 5 Reagents Use only reagents of recognized analytical grade and water complying with grade 1 of EN ISO 3696, unless otherwise specified. Solvents shall be of quality for LC analysis, unless otherwise specified. 5.1 Water, deionised. 5.2 Water, LC-MS grade. 5.3 Acetonitrile, p.a. 5.4 Methanol, LC-MS grade. 5.5 Acetic acid, purity greater than w

· 98 %. 5.6 Ammonium formate, p.a. oSIST prEN 17252:2018

prEN 17252:2018 (E) 6 5.7 Extraction solution acetonitrile/water/acetic acid, (80+19+1, v+v+v). Mix 800 ml of acetonitrile (5.3), 190 ml of water (5.1 or 5.2) and 10 ml of acetic acid (5.4) in a bottle of 1000 ml. This solution is stable for 3 months if stored at room temperature. 5.8 Phomopsin A, isolated from Phomopsis leptostromiformis. 5.9 Phomopsin A stock solution (STD 1), mass concentration

= 500 mg/l. Accurately weigh between 5 mg and 6 mg of the phomopsin A standard (5.8) into an amber-coloured glass bottle of 30 ml. Add a volume of methanol (5.4) to produce a solution with a concentration of 500 mg/l. Take into account the weight and the purity of the standard. The solution is stable for 3 months if stored in the refrigerator at 4 °C. 5.10 Standard solution of phomopsin A (STD 2),

= 10 mg/l. Pipette 100 µl of the standard solution (STD 1) (5.9) into a calibrated volumetric flask of 5 ml and make up the volume with methanol (5.4). The solution is stable for 3 months if stored in the refrigerator at 4 °C. 5.11 Standard solution of phomopsin A (STD 3),

= 250 µg/l. Pipette 250 µl of the standard solution (STD 2) (5.10) into a calibrated volumetric flask of 10 ml and make up to the volume with methanol (5.4). 5.12 Intermediate solutions for preparation of the matrix-matched standards. To seven glass vials (6.9) add different volumes of the standard solution of phomopsin A (5.11) and methanol (5.4) according to Table 1. Close with screw cap and mix. Prepare these solutions freshly for each batch of analysis. Table 1 — Intermediate standard solutions of phomopsin A in methanol Intermediate solution Standard solution STD 3 (5.11) Methanol Mass concentration no µl µl µg/l 1 25 975 6,25 2 50 950 12,5 3 100 900 25 4 200 800 50 5 350 650 87,5 6 500 500 125 7 650 350 162,5 5.13 Matrix matched calibration solutions Prepare matrix-matched calibration solutions in vials (6.9) according to Table 2. The matrix matched calibration solutions may also be prepared directly in auto sampler vials with insert or filter vials. In that case, proportionally reduce the volumes indicated in Table 2. Once it has been shown that there is linearity, the number of levels may be adjusted to local needs and requirements. oSIST prEN 17252:2018

prEN 17252:2018 (E) 7 Table 2 — Matrix matched calibration solutions of phomopsin A in blank matrix extract Calibration solution Mass concentration Blank extract Phomopsin A intermediate solutions (5.12) see Table 1 Water (5.2) Equivalent to mass fraction in sample no µg/l µl µl µl µg/kg 0 0 500 0 500 0 1 0,3125 450 50 µl no 1 500 2,5 2 0,625 450 50 µl no 2 500 5,0 3 1,25 450 50 µl no 3 500 10,0 4 2,5 450 50 µl no 4 500 20,0 5 4,375 450 50 µl no 5 500 35,0 6 6,25 450 50 µl no 6 500 50,0 7 8,125 450 50 µl no 7 500 65,0 6 Apparatus and equipment Usual laboratory glassware and equipment, in particular, the following: 6.1 Conical polypropylene screw cap centrifuge tubes, 50 ml with caps. 6.2 Volumetric flasks, 5 ml and 10 ml. 6.3 Analytical balance, accuracy 0,1 mg. 6.4 Laboratory balance, accuracy 0,01 g. 6.5 Pipettes, e.g. 10 µl to 1000 µl, for organic solvents. 6.6 Adjustable mechanical vertical or horizontal shaker or rotary tumbling machine. 6.7 Laboratory shaker. 6.8 Centrifuge, capable of generating a relative centrifugal force of 3 500 g. 6.9 Vials, 1,5 ml to 2 ml, used for intermediate solutions (5.12), made of glass or polypropylene, with screw cap. 6.10 Syringe filter, 0,20 µm to 0,45 µm, nylon or PTFE (for optional filtration of final extracts). 6.11 Auto sampler vials, of appropriate size for the auto sampler in use, e.g. glass with insert vials, or filter vials (polytetrafluoroethylene (PTFE) 0,45

or equivalent. 6.12 LC-MS/MS system, with the following components: 6.12.1 LC pump, capable of deliv

...

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