oSIST prEN 17425:2019

SLOVENSKI STANDARD
oSIST prEN 17425:2019
01-oktober-2019

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

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

Lebensmittel - Bestimmung von Ergotalkaloiden in Getreiden und Getreideerzeugnissen

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

This draft European Standard is submitted to CEN members for enquiry. It has been drawn up by the Technical Committee

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

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

Recipients of this draft are invited to submit, with their comments, notification of any relevant patent rights of which they are

Warning : This document is not a European Standard. It is distributed for review and comments. It is subject to change without

© 2019 CEN All rights of exploitation in any form and by any means reserved Ref. No. prEN 17425:2019 E

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

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

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

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

This document (prEN 17425:2019) has been prepared by Technical Committee CEN/TC 275 “Food

This document has been prepared under a mandate given to CEN by the European Commission and the

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

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

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

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

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

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. Method performance may be improved by inclusion

of an isotopically labelled internal standard, but this was not available at the time of the method

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)

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

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

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

5.2 Ergocorninine, 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.

5.11 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.

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

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

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

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

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.

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

Alternatively, a ready to use ammonium carbonate solution (c((NH ) CO ) = 3,03 mmol/l) of the correct

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

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

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

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.

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).

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

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

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

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

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

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

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

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.

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

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.

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

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

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

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

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).

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

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

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

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).

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].

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

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

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

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

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.

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

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

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

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

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

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.