SIST EN 17425:2021
(Main)Foodstuffs - Determination of ergot alkaloids in cereals and cereal products by dSPE clean-up and LC-MS/MS
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
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.
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SIST EN 17425:2021
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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.
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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
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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
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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.
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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].
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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
R
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.
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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),
3
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.
1
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.
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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.
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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.
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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.
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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)
a
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 −
su
RR × 100 (2)
w
L
11
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w is the measured mass fraction of the spiked sample, in µg/kg;
s
w is the mass fraction of the unspiked sample, in µg/kg;
u
w is the mass fraction added to the spiked sample, in µg/kg (here: 10 µg/kg).
L
If required, calculate the recovery corrected mass fraction for each analyte, w in µg/kg; using
c
Formula (3):
w× 100
w = (3)
c
RR
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
...
SLOVENSKI STANDARD
oSIST prEN 17425:2019
01-oktober-2019
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
Ta slovenski standard je istoveten z: prEN 17425
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
oSIST prEN 17425:2019 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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DRAFT
EUROPEAN STANDARD
prEN 17425
NORME EUROPÉENNE
EUROPÄISCHE NORM
August 2019
ICS 67.050; 67.060
English Version
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
This draft European Standard is submitted to CEN members for enquiry. It has been drawn up by the Technical Committee
CEN/TC 275.
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
© 2019 CEN All rights of exploitation in any form and by any means reserved Ref. No. prEN 17425:2019 E
worldwide for CEN national Members.
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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 . 10
7.1 Preparation of the test sample . 10
7.2 Extraction of ergot alkaloids. 10
7.2.1 Precautions . 10
7.2.2 Test sample . 10
7.2.3 Sample extraction . 10
7.2.4 Spiking procedure . 10
7.2.5 Clean-up. 10
8 LC-MS/MS analysis . 11
8.1 General. 11
8.2 Batch composition and analytical sequence . 11
8.3 Identification . 11
8.4 Calibration . 11
9 Calculation . 12
9.1 Calculation of individual ergot alkaloid concentration . 12
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. 17
5
A.3 System settings for SCIEX API 5500 Mass Spectrometer . 19
A.3.1 Settings for chromatography . 19
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A.3.2 Detector parameters . 20
A.4 Other LC conditions . 21
Annex B (informative) Typical chromatograms . 23
Annex C (informative) Precision data . 29
Annex D (informative) Data comparison . 43
Bibliography . 44
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European foreword
This document (prEN 17425:2019) 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.
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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 the ‘inines’.
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].
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1 Scope
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 RSD values were greater than 44 %, and HorRat values exceeded 2,0, indicating the
R
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.
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
Ergot alkaloids are extracted from cereals and cereal-based foods with buffer at pH 8,9 and cleaned up
with a dispersive solid phase material prior to filtering. The ergot alkaloids are 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.
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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.
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, substance 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),
3
density: 1,18 g/cm (20 °C).
5.20 Hydrochloric acid solution, 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 2 mg and transfer into a 1 l glass
laboratory screw top flask. Add 500 ml of water (5.15). Shake the flask vigorously to ensure all solid has
been dissolved.
1
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.
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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 months.
Alternatively, a ready to use ammonium carbonate solution (c((NH ) CO ) = 3,03 mmol/l) of the correct
4 2 3
analytical grade may be used ensuring the pH 8,9 ± 0,3.
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).
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 epimerisation 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.
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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.
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.
NOTE Under the conditions given it can be possible to separate α- and β-ergocryptine. This is not critical for
the application of the method as results are reported as total ergocryptine using α-ergocryptine for quantification.
If it is not possible to separate α- and β-ergocryptinine using these conditions, a single peak will be measured that
should be quantified with α-ergocryptinine standard.
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.
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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.
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.
Analyse the samples 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 (7.2.3).
7.2.3 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 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).
7.2.4 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. At least one blank and a spike of the appropriate sample shall be included with each
batch. Extract the samples as described in 7.2.3. If a larger test sample size has been used adjust the
spiking volume accordingly.
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).
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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.
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 mycotoxin 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].
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 α- and β-
ergocryptinine and report a sum for α- and β-ergocryptine and α- and β-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, estimate
the slope and possible intercept of each of the calibration curves. If higher deviations or nonlinearity is
observed, identify the cause and, if necessary, re-run the analyses.
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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)
a
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.
Calculate the spike recovery RR, expressed in %, according to Formula (2):
wc −w
s u
(2)
RR × 100
w
L
w is the measured mass fraction of the spiked sample, in µg/kg;
s
w is the mass fraction of the unspiked sample, in µg/kg;
u
w is the mass fraction added to the spiked sample, in µg/kg (here: 10 µg/kg).
L
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 shortest feasible time interval will exceed the repeatability limit r
in Table 2 in not more than 5 % of the cases.
10.3 Reproducibility
The absolute difference between two single test results found on identical test material reported by two
laboratories will exceed the reproducibility limit R in Table 2 in not more than 5 % of the cases.
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Table 2 — Validation data for sum (12) ergot alkaloids
Sample x̅ r R
µg/kg µg/kg µg/kg
rye flour 150 44,9 84,0
rye flour 168 82,4 111
b
...
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