Foodstuffs - Determination of ergot alkaloids in cereals and cereal products by dSPE clean-up and LC-MS/MS

This document describes a method for the determination of the sum total of six ergot alkaloids (ergocornine, ergometrine, ergocristine, ergotamine, ergosine and ergocryptine) and their  inine epimer pairs by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) after clean-up by dispersive solid phase extraction (SPE).
The method has been validated for cereals and cereal-based food products.
The method has been validated in the range 13,2 µg/kg to 168 µg/kg for the sum of the twelve ergot alkaloids, in rye flour, rye bread and cereal products (breakfast cereal, infant breakfast cereal, and crispbread) that contained rye as an ingredient, as well as seeded wholemeal flour and a barley and rye flour mixture.
Method performance was satisfactory in the range 24,1 µg/kg to 168 µg/kg, however at lower concentrations RSDR values were greater than 44 %, and HorRat values exceeded 2,0, indicating the method may not be fully suitable at concentrations below 24 µg/kg for sum of ergot alkaloids, although it is suitable for screening at these concentrations. Method performance may be improved by inclusion of an isotopically labelled internal standard, but this was not available at the time of the method validation study.

Lebensmittel - Bestimmung von Ergotalkaloiden in Getreiden und Getreideerzeugnissen mit dSPE-Reinigung und LC-MS/MS

Dieses Dokument beschreibt ein Verfahren für die Bestimmung der Summe von sechs Ergotalkaloiden (Ergocornin, Ergometrin, Ergocristin, Ergotamin, Ergosin und Ergocryptin) und ihrer -inin-Epimerenpaare, wobei die Flüssigchromatographie mit Tandem-Massenspektrometrie (LC-MS/MS) nach Reinigung durch dispersive Festphasenextraktion (dSPE) zum Einsatz kommt.
Dieses Verfahren wurde für die Summe von zwölf Ergotalkaloiden im Bereich von 13,2 μg/kg bis 168 μg/kg in Roggenmehl, Roggenbrot und Getreideerzeugnissen (Frühstückscerealien, Frühstückscerealien für Kleinkinder und Knäckebrot), die Roggen als Inhaltsstoff enthielten, wie auch in Vollkornmehl und einer Mehlmischung aus Roggen und Gerste validiert.
Das Verfahren erbrachte zufriedenstellende Ergebnisse im Bereich von 24,1 μg/kg bis 168 μg/kg, allerdings betrugen die RSDR-Werte bei den niedrigeren Konzentrationen mehr als 44 % und die HorRat-Werte überschritten 2,0, was darauf hinweist, dass das Verfahren möglicherweise als nicht zufriedenstellend bei Konzentrationen unter 24 μg/kg für die Summe der Ergotalkaloide zu bewerten ist. Dennoch ist das Verfahren bei diesen Konzentrationen zum Screening geeignet.

Produits alimentaires - Dosage des alcaloïdes de l’ergot dans les céréales et les produits céréaliers par purification par dSPE et CL-SM/SM

Le présent document décrit une méthode pour le dosage de la somme des six alcaloïdes de l’ergot (ergocornine, ergométrine, ergocristine, ergotamine, ergosine et ergocryptine) et de leurs épimères ( inines) par chromatographie liquide couplée à la spectrométrie de masse en tandem (CL-SM/SM) après purification par extraction sur phase solide dispersée (dSPE).
La méthode a été validée dans la gamme de 13,2 µg/kg à 168 µg/kg pour la somme des douze alcaloïdes de l’ergot, dans la farine de seigle, le pain de seigle et les produits céréaliers (céréales pour petit-déjeuner, céréales pour nourrissons et biscottes) contenant du seigle, ainsi que dans la farine complète aux graines et un mélange de farines de seigle et d’orge.
La performance de la méthode s’est révélée satisfaisante dans la gamme de 24,1 µg/kg à 168 µg/kg, cependant à des concentrations inférieures, les valeurs de RSDR étaient supérieures à 44 % et les valeurs de HorRat dépassaient 2,0, ce qui indique que la méthode peut ne pas être parfaitement adaptée à des concentrations de moins de 24 µg/kg pour la somme des alcaloïdes de l’ergot, bien qu’elle soit adaptée au criblage à ces concentrations.

Živila - Določevanje alkaloidov rženih rožičkov (ergot) v žitu in žitnih proizvodih s čiščenjem dSPE in LC-MS/MS

General Information

Status
Published
Public Enquiry End Date
19-Oct-2019
Publication Date
18-May-2021
Current Stage
6060 - National Implementation/Publication (Adopted Project)
Start Date
02-Mar-2021
Due Date
07-May-2021
Completion Date
19-May-2021

Buy Standard

Standard
SIST EN 17425:2021
English language
44 pages
sale 10% off
Preview
sale 10% off
Preview

e-Library read for
1 day

Standards Content (sample)

SLOVENSKI STANDARD
SIST EN 17425:2021
01-julij-2021

Živila - Določevanje alkaloidov rženih rožičkov (ergot) v žitu in žitnih proizvodih s

čiščenjem dSPE in LC-MS/MS

Foodstuffs - Determination of ergot alkaloids in cereals and cereal products by dSPE

clean-up and LC-MS/MS

Lebensmittel - Bestimmung von Ergotalkaloiden in Getreiden und Getreideerzeugnissen

mit dSPE-Reinigung und LC-MS/MS

Produits alimentaires - Dosage des alcaloïdes de l’ergot dans les céréales et les produits

céréaliers par purification par dSPE et CL-SM/SM
Ta slovenski standard je istoveten z: EN 17425:2021
ICS:
67.050 Splošne preskusne in General methods of tests and
analizne metode za živilske analysis for food products
proizvode
67.060 Žita, stročnice in proizvodi iz Cereals, pulses and derived
njih products
SIST EN 17425:2021 en,fr,de

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

---------------------- Page: 1 ----------------------
SIST EN 17425:2021
---------------------- Page: 2 ----------------------
SIST EN 17425:2021
EN 17425
EUROPEAN STANDARD
NORME EUROPÉENNE
April 2021
EUROPÄISCHE NORM
ICS 67.050; 67.060
English Version
Foodstuffs - Determination of ergot alkaloids in cereals
and cereal products by dSPE clean-up and HPLC-MS/MS

Produits alimentaires - Dosage des alcaloïdes de l'ergot Lebensmittel - Bestimmung von Ergotalkaloiden in

dans les céréales et les produits céréaliers par Getreiden und Getreideerzeugnissen mit dSPE-

purification par dSPE et CL-SM/SM Reinigung und LC-MS/MS
This European Standard was approved by CEN on 21 September 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

© 2021 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN 17425:2021 E

worldwide for CEN national Members.
---------------------- Page: 3 ----------------------
SIST EN 17425:2021
EN 17425:2021 (E)
Contents Page

European foreword ...................................................................................................................................................... 4

Introduction .................................................................................................................................................................... 5

1 Scope .................................................................................................................................................................... 6

2 Normative references .................................................................................................................................... 6

3 Terms and definitions ................................................................................................................................... 6

4 Principle ............................................................................................................................................................. 6

5 Reagents ............................................................................................................................................................. 6

6 Apparatus and equipment ........................................................................................................................... 8

7 Procedure .......................................................................................................................................................... 9

7.1 Preparation of the test sample ................................................................................................................... 9

7.2 Extraction of ergot alkaloids..................................................................................................................... 10

7.2.1 Precautions ..................................................................................................................................................... 10

7.2.2 Test sample ..................................................................................................................................................... 10

7.2.3 Spiking procedure ........................................................................................................................................ 10

7.2.4 Sample extraction ......................................................................................................................................... 10

7.2.5 Clean-up............................................................................................................................................................ 10

8 LC-MS/MS analysis ........................................................................................................................................ 10

8.1 General.............................................................................................................................................................. 10

8.2 Batch composition and analytical sequence ....................................................................................... 11

8.3 Identification .................................................................................................................................................. 11

8.4 Calibration ....................................................................................................................................................... 11

9 Calculation ....................................................................................................................................................... 11

9.1 Calculation of individual ergot alkaloid concentration .................................................................. 11

9.2 Calculation of ergot alkaloid sum concentration .............................................................................. 12

10 Precision .......................................................................................................................................................... 12

10.1 General.............................................................................................................................................................. 12

10.2 Repeatability .................................................................................................................................................. 12

10.3 Reproducibility .............................................................................................................................................. 12

11 Test report ....................................................................................................................................................... 13

Annex A (informative) Example conditions for suitable LC-MS/MS systems ....................................... 14

® ®

A.1 System settings for Waters Xevo TQ-S Mass Spectrometer....................................................... 14

A.1.1 Settings for chromatography .................................................................................................................... 14

A.1.2 Detector parameters.................................................................................................................................... 15

A.2 System settings for Micromass Quattro Ultima Pt Mass Spectrometer .................................. 17

A.2.1 Settings for chromatography .................................................................................................................... 17

A.2.2 Detector parameters.................................................................................................................................... 18

A.3 System settings for SCIEX API 5500 Mass Spectrometer ................................................................ 19

A.3.1 Settings for chromatography .................................................................................................................... 19

---------------------- Page: 4 ----------------------
SIST EN 17425:2021
EN 17425:2021 (E)

A.3.2 Detector parameters ................................................................................................................................... 20

A.4 Other LC conditions ..................................................................................................................................... 22

Annex B (informative) Typical chromatograms ............................................................................................. 23

Annex C (informative) Precision data ................................................................................................................ 29

Annex D (informative) Data comparison .......................................................................................................... 43

Bibliography ................................................................................................................................................................. 44

---------------------- Page: 5 ----------------------
SIST EN 17425:2021
EN 17425:2021 (E)
European foreword

This document (EN 17425:2021) 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 October 2021, and conflicting national standards shall

be withdrawn at the latest by October 2021.

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.
---------------------- Page: 6 ----------------------
SIST EN 17425:2021
EN 17425:2021 (E)
Introduction

Ergot alkaloids are a group of mycotoxins produced by several species of Claviceps fungi growing on

cereals and forage grass. These toxins are a risk for consumers as they can enter the food chain. All

ergot alkaloids share a common structure, the ergoline system, and are divided into several classes,

based on the presence of functional groups. The chiral carbon atom C-8 is responsible for the

epimerization.

The isomers of each of these compounds are nominally known as ‘ines’ and the ‘inines’. And besides,

ergocryptine and ergocryptinine can both occur as α- and β-forms.

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, Regulation (EC) No 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.

WARNING 3 — Ergot alkaloids can cause vasoconstrictive, neurotoxic, reproductive and

developmental adverse effects, and can be acutely and chronically toxic [4].
---------------------- Page: 7 ----------------------
SIST EN 17425:2021
EN 17425:2021 (E)
1 Scope

This document describes a method for the determination of the sum of six ergot alkaloids (ergocornine,

ergometrine, ergocristine, ergotamine, ergosine and ergocryptine) and their -inine epimer pairs by

liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) after clean-up by

dispersive solid phase extraction (dSPE).

The method has been validated in the range 13,2 µg/kg to 168 µg/kg for the sum of the twelve ergot

alkaloids, in rye flour, rye bread and cereal products (breakfast cereal, infant breakfast cereal, and

crispbread) that contained rye as an ingredient, as well as seeded wholemeal flour and a barley and rye

flour mixture.

Method performance was satisfactory in the range 24,1 µg/kg to 168 µg/kg, however at lower

concentrations RSD values were greater than 44 %, and HorRat values exceeded 2,0, indicating the

method may not be fully suitable at concentrations below 24 µg/kg for sum of ergot alkaloids, although

it is suitable for screening at these concentrations.
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

Ergot alkaloids are extracted from cereals and cereal-based foods with a buffer at pH 8,9 and cleaned up

with a dispersive solid phase material prior to filtering. Ergot alkaloids are detected and quantified by

liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS).
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.

NOTE Ergometrine and ergotamine are listed as Category 1 scheduled substances in Regulation (EC)

No 273/2004 [5] on drug precursors. It is a requirement to have an appropriate licence in order to purchase and

store these compounds (and their related -inine epimers).
5.1 Ergocornine, e.g. crystalline, as a film or as certified standard solution.

5.2 Ergocorninine, e.g. crystalline, as a film or as certified standard solution.

5.3 Ergocristine, e.g. crystalline, as a film or as certified standard solution.
---------------------- Page: 8 ----------------------
SIST EN 17425:2021
EN 17425:2021 (E)

5.4 Ergocristinine, e.g. crystalline, as a film or as certified standard solution.

5.5 α-Ergocryptine, e.g. crystalline, as a film or as certified standard solution.

5.6 α-Ergocryptinine, e.g. crystalline, as a film or as certified standard solution.

5.7 Ergometrine (maleate), e.g. crystalline, as a film or as certified standard solution.

5.8 Ergometrinine, e.g. crystalline, as a film or as certified standard solution.

5.9 Ergosine, e.g. crystalline, as a film or as certified standard solution.
5.10 Ergosinine, e.g. crystalline, as a film or as certified standard solution.

5.11 Ergotamine (tartrate), e.g. crystalline, as a film or as certified standard solution.

5.12 Ergotaminine, e.g. crystalline, as a film or as certified standard solution.

5.13 Acetonitrile, LC-MS grade.
5.14 Methanol, LC grade.
5.15 Water, suitable for LC-MS/MS.

5.16 Solid phase extraction material, primary secondary amine (PSA) bulk sorbent, 40 µm, 10 g; e.g.

TM, 1
Bondesil .
5.17 Sodium hydroxide (NaOH), analytical reagent grade.
5.18 Sodium hydroxide solution, molar concentration c(NaOH) = 1,0 mol/l.
Dissolve 4 g NaOH (5.17) in water (5.15) to a final volume of 100 ml.

5.19 Hydrochloric acid (HCl), analytical reagent grade, volume fraction φ(HCl) = 37 % (acidimetric),

density: 1,18 g/cm (20 °C).
5.20 Hydrochloric acid solution, molar concentration c(HCl) = 1,0 mol/l.
Dilute 8,3 ml HCl (5.19) in water (5.15) to a final volume of 100 ml.
5.21 Ammonium carbonate ((NH ) CO ), LC-MS grade.
4 2 3

5.22 Ammonium carbonate solution, mass concentration ρ((NH ) CO ) = 200 mg/l; pH = 8,9 ± 0,3.

4 2 3

Weigh 200 mg of ammonium carbonate (5.21) to the nearest 1 mg and transfer into a 1 l glass flask. Add

500 ml of water (5.15). Shake the flask vigorously to ensure all solid has been dissolved.

After dissolution adjust the pH 8,9 ± 0,3 with sodium hydroxide solution (5.18) or hydrochloric acid

solution (5.20) as appropriate, then fill up to the mark with water (5.15).
This solution can be stored at room temperature for 3 days.

Bondesil ™ is a trade name of a product commercially available from various suppliers. This information is given

for the convenience of users of this European standard and does not constitute an endorsement by CEN of the

products named. Equivalent products may be used if they can be shown to lead to the same results.

---------------------- Page: 9 ----------------------
SIST EN 17425:2021
EN 17425:2021 (E)
5.23 Extraction solvent.

Mix acetonitrile (5.13) and ammonium carbonate solution (5.22) (84 + 16, V + V). Shake vigorously.

5.24 Individual stock solutions, ρ = 100 µg/ml, in acetonitrile.

Follow any specific manufacturers’ instructions to re-dissolve the films of individual ergot alkaloids (5.1

to 5.12). If crystalline material is used, weigh 5 mg to the nearest 0,2 mg, of each of the solid standards

(5.1 to 5.12) individually into glass weighing boats and transfer quantitatively into individual 50 ml

volumetric flasks, then fill up to the mark with acetonitrile (5.13). For ergometrine (5.7) and

ergometrinine (5.8) a mixture of acetonitrile (5.13) and water (5.15) (90 + 10, V + V) is recommended

to ensure complete dissolution. Shake vigorously.
5.25 Mixed standard solution, ρ = 0,5 µg/ml.

Using a pipette (6.5), transfer 50 µl of each of the individual stock solutions (5.24) into a 10 ml

volumetric flask and make up to volume with acetonitrile (5.13).

When using an individual stock solution with a mass concentration other than ρ = 100 µg/ml calculate

the appropriate volume required to prepare the 0,5 µg/ml standard solution mixture.

5.26 Calibration solutions.

Prepare e.g. the following calibration solutions as outlined in Table 1. Dispense volumes of mixed

standard solution (5.25) into volumetric flasks and fill up to the mark with acetonitrile (5.13).

Table 1 — Examples of suitable calibration solutions
Calibration Mixed standard Final volume Mass Equivalent to
solution solution (5.25) concentration mass fraction of
of alkaloids alkaloids
µl ml ng/ml µg/kg
1 10 50 0,1 0,5
2 20 50 0,2 1
3 10 5 1 5
4 20 5 2 10
5 40 5 4 20
6 100 5 10 50
6 Apparatus and equipment

Usual laboratory apparatus and, in particular, the following. Unless otherwise stated volumetric

glassware shall be of grade ‘A’ quality. Ergot alkaloids are sensitive to epimerization by light and amber

glass shall be used where possible.
6.1 Laboratory balance, accuracy of 0,01 g.
6.2 Analytical balance, accuracy of 0,1 mg.
6.3 Single or multiple grinding mill.

6.4 Extraction flasks with cap, of sufficient volume to contain 50 ml extraction solvent and 10 g

sample, e.g. 100 ml.
---------------------- Page: 10 ----------------------
SIST EN 17425:2021
EN 17425:2021 (E)

6.5 Pipette, adjustable, e.g. 25 µl, 50 µl, 250 µl, 1 000 µl, suitable for organic solvents, with

disposable tips.
6.6 Laboratory shaker, for solvent extraction with suitable 100 ml flasks.
6.7 Folded filter paper, diameter 12,5 cm, hardened, grade 54.
6.8 Vials with caps, 40 ml amber vials.
6.9 Vials with caps, 4,0 ml amber vials.
6.10 Plastic Luer-lock syringe, 1 ml.
6.11 Polytetrafluoroethylene (PTFE) plastic syringe filters, 13 mm × 0,22 µm.
6.12 Vials with caps, 2,0 ml amber vials.
6.13 Autosampler vials suitable for LC-MS/MS analysis, e.g. 200 µl.
6.14 LC-MS/MS system with the following components:

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

column in use with sufficient accuracy.

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

sufficient accuracy.

6.14.3 LC column, capable of retaining the ergot alkaloids, preferably with a retention factor of at least

two. The column shall be suitable for use with a mobile phase at pH > 7.
6.14.4 Column filter, in-line filter suitable for the LC column used (6.14.3).
6.14.5 Column oven, capable of maintaining a constant temperature.

6.14.6 Tandem mass spectrometer (MS/MS), capable of ionization of the ergot alkaloids (resulting in

positive ions) and selected reaction monitoring (SRM) with a sufficiently wide dynamic range.

Any ionization source providing sufficient yield may be used.
6.14.7 Data evaluation system.
7 Procedure
7.1 Preparation of the test sample

Grind and homogenize the sample with a grinding mill with a mesh or sieve size of 0,5 mm or smaller

(6.3) before analysis.

Depending on the starting material (ground or unground material), it is advisable to first grind the

sample through a sieve of 1 mm to prevent excessive heat formation during milling, which could lead to

partial decomposition of the analytes. Then grind the sample through a sieve of 0,5 mm.

Mix samples well before taking a test portion for analysis. Store the samples at room temperature.

---------------------- Page: 11 ----------------------
SIST EN 17425:2021
EN 17425:2021 (E)
7.2 Extraction of ergot alkaloids
7.2.1 Precautions

Ergot alkaloids are sensitive to epimerization by light and amber glass shall be used where possible.

Analysis should take place immediately after extraction. Only if absolutely necessary, extracts may be

stored overnight at 4 °C.
7.2.2 Test sample
Weigh 10 g of the sample to the nearest 0,05 g into a flask (6.4).

A larger test sample size may be used. In that case the amount of extraction solvent shall be adjusted

accordingly (7.2.4).
7.2.3 Spiking procedure

Add 200 µl of the mixed standard solution (5.25) to 10 g ± 0,05 g of a ‘blank’ sample. Allow the spiked

samples to dry. Extract the samples as described in 7.2.4. If a larger test sample size has been used

adjust the spiking volume accordingly.
7.2.4 Sample extraction

Add 50 ml of the extraction solvent (5.23) to the flask (6.4) using a measuring cylinder.

Place the sample flasks in the laboratory shaker (6.6). Shake the samples for approximately 30 min at a

moderate speed.

Whilst the samples are shaking, prepare sufficient glass funnels containing folded filter paper (6.7)

ready to filter the samples into 40 ml amber vials (6.8).

When the samples have finished shaking, shake each flask individually by hand for approximately 10 s

prior to pouring through funnels and filter paper (6.7) into 40 ml amber vials (6.8).

7.2.5 Clean-up

Transfer 1 ml of sample filtrate into a 4 ml amber vial (6.9) containing 50 mg ± 5 mg of the solid phase

extraction material (5.16).

Shake each tightly sealed sample vial with a laboratory shaker (6.6) at high speed for approximately

45 s.

Using a plastic Luer-lock syringe (6.10), take up as much as possible of the sample. Fit a 13 mm

PTFE 0,22 µm syringe filter (6.11) and holding the syringe vertically, allow any solid phase material to

rest on the bottom of the syringe. Push the liquid through the filter into a 2 ml amber vial (6.12).

8 LC-MS/MS analysis
8.1 General

Optimize analytical parameters (i.e. selection of masses of precursor and product ions, cone voltages

and collision energies) by infusion and injection of standard solutions of individual ergot alkaloids. Use

a tandem mass spectrometer or equivalent in positive electrospray ionization (ESI ) mode. Set the

acquisition mode to multiple reaction monitoring (MRM), monitoring at least two product ions [6].

Examples are given in Table A.3.

Satisfactory separation of ergot alkaloid epimers can be achieved using a mobile phase with a pH > 7.

The use of a mobile phase with pH > 7, requires an analytical column that contains a stationary phase

that is resistant to high pH.
---------------------- Page: 12 ----------------------
SIST EN 17425:2021
EN 17425:2021 (E)

Examples of measurement conditions and transitions are given in Annex A. Examples of typical

chromatograms are shown in Annex B.
8.2 Batch composition and analytical sequence

An example is as follows: The first injection of every run sequence (following any test or priming

injections) is usually an aliquot of sample extraction solvent (5.23). Then inject the calibration

solutions, followed by an extraction solvent to check for possible carry over. Subsequently inject the

sample test solutions, inject one calibration solution at periodic intervals, e.g. one calibration solution

for every 5 to 10 sample test solutions injected. At the end of the batch, re-inject the calibration

solutions.
8.3 Identification

Identify each ergot alkaloid by comparing the retention times of the calibration solutions with that of

the sample test solution. Identify the analyte on the basis of at least two mass transitions. In addition

the retention times (peaks in both mass traces) and the area ratio of the two peaks shall match that of

the standard substance [6].

Under the conditions given (Annex A) it is possible that a naturally contaminated sample shows

separate α- and β-ergocryptine and α- and β-ergocryptinine peaks in the chromatogram. As long as

standards for β-ergocryptine and β-ergocryptinine are not available, use standards for α-ergocryptine

and α-ergocryptinine to quantify both the α- and β-ergocryptine and the α- and β-ergocryptinine

respectively. Report a sum for α- and β-ergocryptine and α- and β-ergocryptinine. The separation of the

α- and β- pairs is not critical for the application of the method as results are reported as total

ergocryptine and total ergocryptinine.
8.4 Calibration

For each ergot alkaloid, plot the peak areas of the quantifier ion (y-axis) of all individual calibration

solutions (5.26, calibration solutions 1 to 6) against the corresponding mass fraction (µg/kg) (x-axis).

The quantifier is the transition which overall gives the best S/N (signal/noise) ratio. Construct a

calibration curve using (possibly weighted) regression with all individual data points obtained,

compute the slope and possible intercept of each of the calibration curves.
9 Calculation
9.1 Calculation of individual ergot alkaloid concentration

Using the calibration graph for each analyte (8.4), calculate the mass fraction w of each ergot alkaloid in

the sample, expressed in μg/kg, according to Formula (1):
Ab −
w= (1)
where
A is the peak area from the chromatogram of the test solution;
a is the value of the slope of the linear function;
b is the value where the calibration function intercepts the y-axis.

Rate after recovery: Calculate the spike recovery RR for each analyte, expressed in %, according to

Formula (2):
ww −
RR × 100 (2)
---------------------- Page: 13 ----------------------
SIST EN 17425:2021
EN 17425:2021 (E)
w is the measured mass fraction of the spiked sample, in µg/kg;
w is the mass fraction of the unspiked sample, in µg/kg;
w is the mass fraction added to the spiked sample, in µg/kg (here: 10 µg/kg).

If required, calculate the recovery corrected mass fraction for each analyte, w in µg/kg; using

Formula (3):
w× 100
w = (3)
9.2 Calculation of ergot alkaloid sum concentration

To obtain the sum value of ergot alkaloids in the test sample add the mass fraction of each individual

ergot alkaloid obtained from the calibration graphs. Where an analyte result is less than the limit of

quantitation (LOQ) [7] of the method, use a value of zero for that analyte in the calculation of the sum

concentration.
10 Precision
10.1 General

Details of the interlaboratory test of the precision of the method are summarized in Annex C. The values

derived from the interlaboratory test may not be applicable to analyte concentration ranges and

matrices other than given in Annex C.
10.2 Repeatability

The absolute difference between two single test results found on identical test material by one operator

using the same apparatus within the s
...

Questions, Comments and Discussion

Ask us and Technical Secretary will try to provide an answer. You can facilitate discussion about the standard in here.