Foodstuffs - Determination of citrinin in food by HPLC-MS/MS

This document specifies a procedure for the determination of the citrinin content in food (cereals, red yeast rice (RYR)), herbs and food supplements by liquid chromatography tandem mass spectrometry (LC-MS/MS).
This method has been validated for citrinin in red yeast rice and in the formulated food supplements in the range of 2,5 µg/kg to 3000 µg/kg and in wheat flour in the range of 2,5 µg/kg to 100 µg/kg.
Laboratory experiences have shown that this method is also applicable to white rice, herbs such as a powder of ginkgo biloba leaves and the formulated food supplements in the range of 2,5 µg/kg to 50 µg/kg.

Lebensmittel - Bestimmung von Citrinin in Lebensmitteln mit HPLC-MS/MS

Dieses Dokument beschreibt ein Verfahren zur Bestimmung des Citriningehalts in Lebensmitteln (Getreide, rotem Reis [RYR, en: red yeast rice]), Kräutern und Nahrungsergänzungsmitteln mittels Flüssigchromatographie und Tandem-Massenspektrometrie (LC-MS/MS).
Dieses Verfahren wurde für Citrinin in rotem Reis und in entsprechend zusammengesetzten Nahrungsergänzungsmitteln im Bereich von 2,5 μg/kg bis 3 000 μg/kg sowie für Citrinin in Weizenmehl im Bereich von 2,5 μg/kg bis 100 μg/kg validiert.
Laborerfahrungen haben gezeigt, dass dieses Verfahren auch auf weißen Reis, auf Kräuter wie Ginkgo-Biloba-Blätter in Pulverform und entsprechend zusammengesetzte Nahrungsergänzungsmittel im Bereich von 2,5 μg/kg bis 50 μg/kg anwendbar ist.

Produits alimentaires - Dosage de la citrinine dans les produits alimentaires par CLHP-SM/SM

Le présent document décrit un mode opératoire de détermination de la teneur en citrinine dans les aliments [céréales, levure de riz rouge (LRR)], les herbes aromatiques et les compléments alimentaires par chromatographie liquide couplée à une spectrométrie de masse en tandem (CL-SM/SM).
Cette méthode a été validée pour la citrinine dans la levure de riz rouge et dans les compléments alimentaires à base de levure de riz rouge dans la gamme de concentration de 2,5 μg/kg à 3 000 μg/kg et dans la farine de blé dans la gamme de concentration de 2,5 μg/kg à 100 μg/kg.
Les expériences en laboratoire ont montré que cette méthode est également applicable au riz blanc, aux herbes aromatiques telles que les feuilles de Ginkgo biloba en poudre et aux compléments alimentaires formulés dans la gamme de concentration de 2,5 μg/kg à 50 μg/kg.

Živila - Določevanje citrinina v živilih z metodo HPLC-MS/MS

General Information

Status
Published
Public Enquiry End Date
19-Dec-2019
Publication Date
28-Jun-2021
Current Stage
6060 - National Implementation/Publication (Adopted Project)
Start Date
14-Jun-2021
Due Date
19-Aug-2021
Completion Date
29-Jun-2021

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SLOVENSKI STANDARD
SIST EN 17203:2021
01-september-2021
Nadomešča:
SIST EN 17203:2019
Živila - Določevanje citrinina v živilih z metodo HPLC-MS/MS
Foodstuffs - Determination of citrinin in food by HPLC-MS/MS
Lebensmittel - Bestimmung von Citrinin in Lebensmitteln mit HPLC-MS/MS

Produits alimentaires - Dosage de la citrinine dans les produits alimentaires par CLHP-

SM/SM
Ta slovenski standard je istoveten z: EN 17203:2021
ICS:
67.050 Splošne preskusne in General methods of tests and
analizne metode za živilske analysis for food products
proizvode
SIST EN 17203:2021 en,fr,de

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

---------------------- Page: 1 ----------------------
SIST EN 17203:2021
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SIST EN 17203:2021
EN 17203
EUROPEAN STANDARD
NORME EUROPÉENNE
May 2021
EUROPÄISCHE NORM
ICS 67.050 Supersedes EN 17203:2018
English Version
Foodstuffs - Determination of citrinin in food by HPLC-
MS/MS

Produits alimentaires - Dosage de la citrinine dans les Lebensmittel - Bestimmung von Citrinin in

produits alimentaires par CLHP-SM/SM Lebensmitteln mit HPLC-MS/MS
This European Standard was approved by CEN on 19 March 2021.

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 17203:2021 E

worldwide for CEN national Members.
---------------------- Page: 3 ----------------------
SIST EN 17203:2021
EN 17203:2021 (E)
Contents Page

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

Introduction .................................................................................................................................................................... 4

1 Scope .................................................................................................................................................................... 5

2 Normative references .................................................................................................................................... 5

3 Terms and definitions ................................................................................................................................... 5

4 Principle ............................................................................................................................................................. 5

5 Reagents ............................................................................................................................................................. 5

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

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

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

7.2 Extraction of citrinin ...................................................................................................................................... 9

7.3 Spiking procedure ........................................................................................................................................ 10

7.4 Preparation of sample test solutions .................................................................................................... 10

7.5 LC-MS/MS analysis ....................................................................................................................................... 10

7.5.1 General ............................................................................................................................................................. 10

7.5.2 LC-MS/MS operating conditions ............................................................................................................. 11

7.6 Identification ................................................................................................................................................. 12

8 Calculation ...................................................................................................................................................... 12

8.1 General ............................................................................................................................................................. 12

8.2 Calculation with the internal standard ................................................................................................ 12

9 Precision .......................................................................................................................................................... 14

9.1 General ............................................................................................................................................................. 14

9.2 Repeatability .................................................................................................................................................. 14

9.3 Reproducibility ............................................................................................................................................. 14

10 Test report ...................................................................................................................................................... 14

Annex A (informative) Example conditions for suitable LC-MS/MS systems ...................................... 15

Annex B (informative) Typical chromatograms ............................................................................................ 17

Annex C (informative) Precision data ................................................................................................................ 20

Bibliography ................................................................................................................................................................. 23

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SIST EN 17203:2021
EN 17203:2021 (E)
European foreword

This document (EN 17203:2021) 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 November 2021, and conflicting national standards shall

be withdrawn at the latest by November 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 supersedes EN 17203:2018.

This document has been prepared under a standardization request given to CEN by the European

Commission and the European Free Trade Association.
The alterations to the version of 2018 are as follows:

— 5.24 The necessity to prepare calibration solutions freshly every day was deleted.

— 6.14.2 The requirement for cross contamination below 1 % was deleted.
— 7.5.1 First sentence was re-worded in better language.

— 7.5.2 The last part “when negative ion mode is used”of second para before Table 2, starting with

“When an adduct ion is used as precursor ion” was deleted as not applicable for this method.

— 7.5.2 Table 2, the last column for the 2nd qualifier was re-introduced again.
— 7.6 Para 2 line 1 was re-worded in a clearer way.
— 8.1 Para 3 was aligned with other standards of CEN/TC 275/WG 5.

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: 5 ----------------------
SIST EN 17203:2021
EN 17203:2021 (E)
Introduction

The mycotoxin citrinin is a polyketide secondary metabolite produced mainly post-harvest in food and

feed by several fungi of the genera Penicillium (e.g. P. citrinum), Aspergillus (e.g. A. candidus), and

Monascus (e.g. M. purpureus). Citrinin occurs mainly in stored grains like rice, maize, wheat, barley, oats,

and rye. Citrinin can be found as a contaminant in red fermented rice with Monascus purpureus and its

formulated dietary supplements.

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 [5] 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 — Citrinin is known to have nephrotoxic properties, damaging the proximal tubules of the

kidney [6].
---------------------- Page: 6 ----------------------
SIST EN 17203:2021
EN 17203:2021 (E)
1 Scope

This document describes a procedure for the determination of the citrinin content in food (cereals,

red yeast rice (RYR)), herbs and food supplements by liquid chromatography tandem mass spectrometry

(LC-MS/MS).

This method has been validated for citrinin in red yeast rice and in the formulated food supplements in

the range of 2,5 µg/kg to 3 000 µg/kg and in wheat flour in the range of 2,5 µg/kg to 100 µg/kg.

Laboratory experiences have shown that this method is also applicable to white rice, herbs such as a

powder of ginkgo biloba leaves and the formulated food supplements in the range of 2,5 µg/kg to

50 µg/kg.
2 Normative references

The following documents are referred to in the text in such a way that some or all of their content

constitutes requirements of this document. For dated references, only the edition cited applies. For

undated references, the latest edition of the referenced document (including any amendments) applies.

EN ISO 3696, Water for analytical laboratory use - Specification and test methods (ISO 3696)

3 Terms and definitions
No terms and definitions are listed in this document.

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

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

A test portion is humidified with a hydrochloric acid aqueous solution and extracted with ethyl

acetate/acetonitrile/glacial acetic acid mixture for 60 min. Magnesium sulfate and sodium chloride are

added to the extract, agitated and centrifuged in order to expel water and allow phase separation from

the mixture. An aliquot of supernatant is collected, filtered, internal standard (ISTD) solution is added

and analysed by reversed phase LC-MS/MS. Quantification is based on matching citrinin/citrinin- C

ratios and citrinin concentrations.
5 Reagents

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

unless otherwise specified. Commercially available solutions with equivalent properties to those listed

may also be used.
5.1 Ethyl acetate, analytical grade or higher.
5.2 Acetonitrile, LC-MS grade.
5.3 Glacial acetic acid (CH COOH), analytical grade or higher.
5.4 Glacial acetic acid (CH COOH), LC-MS grade.
5.5 Magnesium sulfate; anhydrous (MgSO ), analytical grade or higher.
---------------------- Page: 7 ----------------------
SIST EN 17203:2021
EN 17203:2021 (E)
5.6 Sodium chloride (NaCl), analytical grade or higher.

5.7 Hydrochloric acid solution (HCl), analytical grade or higher, volume fraction φ(HCl) = 37 %

(acidimetric).
5.8 Water (H O), deionised (Ultrapure).
5.9 Water (H O), LC-MS grade.
5.10 Methanol (MeOH), LC-MS grade.
5.11 Ammonium acetate (CH COONH ), LC-MS grade.
3 4
5.12 Extraction solution 1.

Add 10 ml of glacial acetic acid (5.3) to 990 ml of water (5.8) and mix (water + glacial acetic acid, 99+1,

v+v). Dissolve 100 g of sodium chloride (5.6) in 1 l of this mixture and add 16 ml of hydrochloric acid

solution (5.7). This solution can be used for 1 month if stored at room temperature.

5.13 Extraction solution 2.

Mix 240 ml of acetonitrile (5.2) with 750 ml of ethyl acetate (5.1) and 10 ml of glacial acetic acid (5.3).

This solution (ethyl acetate + acetonitrile + glacial acetic acid, 75+24+1, v+v+v) can be used for 1 month

if stored at room temperature.
5.14 Dilution solution.

Mix 80 ml of methanol (5.10), 18 ml of water (5.9) and 2 ml of glacial acetic acid (5.4). This solution

(methanol + water + glacial acetic acid, 80+18+2, v+v+v) can be used for 1 month if stored at room

temperature.
5.15 Ammonium acetate/glacial acetic acid in water.

Dissolve 9,5 g of ammonium acetate (5.11) in 12,5 ml of water (5.9), then add 12,5 ml of glacial acetic acid

(5.4) and mix thoroughly. This solution can be used for 12 months if stored at < −18 °C.

5.16 Mobile phase A: ammonium acetate/glacial acetic acid in water, molar concentration c = 5 mmol/l.

Add 1 ml of ammonium acetate/glacial acetic acid in water (5.15) to 999 ml of water (5.9) and mix

thoroughly.

5.17 Mobile phase B: ammonium acetate/glacial acetic acid in methanol, c = 5 mmol/l.

Add 1 ml of ammonium acetate/glacial acetic acid in water (5.15) to 999 ml of methanol (5.10) and mix

thoroughly.

5.18 Citrinin, analytical standard > 99 %, e.g. crystalline or as certified standard solution.

5.19 Citrinin stock solution, mass concentration ρ = 500 µg/ml.

Weigh 5 mg of crystalline citrinin to the nearest 0,1 mg into a 10 ml volumetric flask and dissolve with

acetonitrile (5.2) by filling up to the mark. The mass concentration of this stock solution shall be checked.

This can be achieved via LC-MS/MS analysis against the certified standard solution (5.18) or by a

photometric determination of concentration using the molar extinction coefficient [8].

The certified standard solution (5.18) can alternatively be used as stock solution.

---------------------- Page: 8 ----------------------
SIST EN 17203:2021
EN 17203:2021 (E)
5.20 Citrinin working solution, ρ = 100 µg/ml.

Pipette 1 ml of the stock solution (5.19) into 4 ml of the dilution solution (5.14) and homogenize the

mixture.

A correction factor (see 5.19) shall be determined and used to correct the exact concentration of this

working solution (ρ = 100 µg/ml). This step may be omitted when using the certified standard solution

(5.18).

The stock and working solutions can be used for 12 months if stored at < −18 °C and protected against

light and moisture.
5.21 Citrinin intermediate solution, ρ = 1 µg/ml.

Pipette 50 µl of the citrinin working solution (5.20) into 4 950 µl of the dilution solution (5.14) which

results in 5 ml and homogenize the mixture.

Use this intermediate solution as spiking solution for recovery experiments. This solution can be used for

3 months if stored in amber flasks (6.13) at < −18 °C.

5.22 Citrinin- C stock solution, as standard solution at a given mass concentration, e.g. ρ = 100 µg/ml.

After opening, the stock solution can usually be used for 12 months if stored at < −18 °C in amber flasks

(see its expiry date in the Certificate of Analysis).

If obtained as crystalline, dissolve the citrinin- C in a suitable amount of acetonitrile (5.2) to obtain a

stock solution at a suitable mass concentration (e.g. ρ = 100 µg/ml). This stock solution can also usually

be used for 12 months if stored at < −18°C.
5.23 Citrinin- C intermediate solution, ρ = 100 ng/ml.

Dilute the citrinin- C stock solution (5.22) with dilution solution (5.14) and mix well to obtain the

citrinin- C intermediate solution at a concentration of ρ = 100 ng/ml. Use this solution as internal

standard (ISTD) and add it to each of the sample extracts, the calibration solutions (5.24) and citrinin

control solution (5.25).

This solution can be used for 6 months if stored in amber flasks at < −18 °C. Check the stability of the

solution when using after 6 months.
5.24 Calibration solutions.

Prepare e.g. the following calibration solutions (within a range from 0,25 ng/ml to 50 ng/ml) as outlined

in Table 1 using the citrinin intermediate solution (5.21) and the dilution solution (5.14). Homogenize

the mixtures.

Start with calibration solution 6 which is then used to prepare calibration solutions 1, 2 and 3 (see

Table 1).
---------------------- Page: 9 ----------------------
SIST EN 17203:2021
EN 17203:2021 (E)
Table 1 — Examples of suitable calibration solutions
Calibration Mass Total volume Citrinin Calibration Dilution
solution concentration intermediate solution 6 solution
solution (5.14)
(5.21)
ng/ml µl µl µl µl
1 0,25 1 000 5 995
2 0,5 1 000 10 990
3 1 1 000 20 980
4 10 1 000 10 990
5 20 1 000 20 980
6 50 1 000 50 950

Take 45 µl from every calibration solution (1 to 6), add 5 µl of the citrinin- C intermediate solution

(5.23) and mix.

It is also possible to increase the volumes of the calibration and intermediate solutions in proportion to

one another.
5.25 Citrinin control solution, ρ = 25 ng/ml.

Prepare a citrinin control solution from the certified standard solution (5.18). Alternatively, prepare a

citrinin control solution from a suitable independent citrinin stock solution that has been checked

photometrically or has been checked via a certified standard solution (5.18). Prepare the control solution

(ρ = 25 ng/ml) by diluting the stock solution with dilution solution (5.14).

Add 5 µl of the citrinin- C intermediate solution (5.23) to 45 µl of this control solution before analysis.

This control solution can be used for 3 months if stored in amber flasks at < −18 °C.

6 Apparatus and equipment
Usual laboratory glassware and equipment, in particular, the following.

For glassware, use preferably amber coloured to reduce light effect and degradation of citrinin.

6.1 Laboratory balance, accuracy of 0,01 g.
6.2 Analytical balance, accuracy of 0,1 mg.
6.3 Laboratory shaker.
6.4 Centrifuge, suitable for 50 ml centrifuge tubes.
6.5 Centrifuge, suitable for 1,5 ml centrifuge tubes.

6.6 Pipettes, adjustable e.g. 1 µl to 1 000 µl, suitable for organic solvents, with appropriate tips.

6.7 Centrifuge tubes, 50 ml.
---------------------- Page: 10 ----------------------
SIST EN 17203:2021
EN 17203:2021 (E)
6.8 Centrifuge tubes, polypropylene, (e.g. Eppendorf tube), 1,5 ml.
6.9 Syringe, 2 ml.

6.10 Filters for syringe, polytetrafluoroethylene (PTFE), with 0,2 µm pore diameter.

6.11 Amber vials for injection, with screw cap or equivalent, 1,5 ml.
6.12 Glass inserts.
6.13 Amber flasks.
6.14 LC-MS/MS system, comprising the following.
6.14.1 LC pump, suitable for gradient elution.

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

accuracy.

6.14.3 LC column, e.g. HSST3 C18 column, 1,8 µm, 2,1 mm × 100 mm and corresponding pre-filter or

pre-column.

Columns of different dimensions may also be used that ensure base line separation to distinguish peaks

of the citrinin from all other signals.
6.14.4 Column thermostat.

6.14.5 Tandem mass spectrometer (MS/MS), capable of performing ionization of citrinin and multiple

reaction monitoring (MRM) with a sufficiently wide dynamic range.
6.14.6 Data evaluation system.
7 Procedure
7.1 Preparation of the test sample

Finely grind the laboratory sample, homogenize and store in the dark before taking a test portion for

analysis.
7.2 Extraction of citrinin

Weigh a test portion of 4,00 g of the homogenized laboratory sample to the nearest 0,02 g into a 50 ml

centrifugation tube (6.7).

Add 10 ml of the extraction solution 1 (5.12) followed immediately by 20 ml (V ) of extraction solution 2

(5.13) and cap the tube.

Shake the mixture for approximately 1 h using a laboratory shaker (6.3) at room temperature.

Thereafter, add 6,0 g of magnesium sulfate (5.5) and 1,5 g of sodium chloride (5.6) to the mixture and

immediately shake vigorously for 30 s to avoid aggregation of the salts.

Eppendorf is a trade name of a centrifugation tube supplied by Eppendorf International. This information is given

for the convenience of users of this European
...

SLOVENSKI STANDARD
oSIST prEN 17203:2019
01-december-2019
Živila - Določevanje citrinina v živilih s HPLC-MS/MS
Foodstuffs - Determination of citrinin in food by HPLC-MS/MS
Lebensmittel - Bestimmung von Citrinin in Lebensmitteln mit HPLC-MS/MS

Produits alimentaires - Dosage de la citrinine dans les produits alimentaires par CLHP-

SM/SM
Ta slovenski standard je istoveten z: prEN 17203
ICS:
67.050 Splošne preskusne in General methods of tests and
analizne metode za živilske analysis for food products
proizvode
oSIST prEN 17203:2019 en,fr,de

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

---------------------- Page: 1 ----------------------
oSIST prEN 17203:2019
---------------------- Page: 2 ----------------------
oSIST prEN 17203:2019
DRAFT
EUROPEAN STANDARD
prEN 17203
NORME EUROPÉENNE
EUROPÄISCHE NORM
October 2019
ICS Will supersede EN 17203:2018
English Version
Foodstuffs - Determination of citrinin in food by HPLC-
MS/MS

Produits alimentaires - Dosage de la citrinine dans les Lebensmittel - Bestimmung von Citrinin in

produits alimentaires par CLHP-SM/SM Lebensmitteln mit HPLC-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 17203:2019 E

worldwide for CEN national Members.
---------------------- Page: 3 ----------------------
oSIST prEN 17203:2019
prEN 17203:2019 (E)
Contents Page

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

Introduction .................................................................................................................................................................... 4

1 Scope .................................................................................................................................................................... 5

2 Normative references .................................................................................................................................... 5

3 Terms and definitions ................................................................................................................................... 5

4 Principle ............................................................................................................................................................. 5

5 Reagents ............................................................................................................................................................. 5

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

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

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

7.2 Extraction of citrinin ...................................................................................................................................... 9

7.3 Spiking procedure ........................................................................................................................................ 10

7.4 Preparation of sample test solutions .................................................................................................... 10

7.5 LC-MS/MS analysis ....................................................................................................................................... 10

7.5.1 General ............................................................................................................................................................. 10

7.5.2 LC-MS/MS operating conditions ............................................................................................................. 10

7.6 Identification ................................................................................................................................................. 12

8 Calculation ...................................................................................................................................................... 12

8.1 General ............................................................................................................................................................. 12

8.2 Calculation with the internal standard ................................................................................................ 12

9 Precision .......................................................................................................................................................... 14

9.1 General ............................................................................................................................................................. 14

9.2 Repeatability .................................................................................................................................................. 14

9.3 Reproducibility ............................................................................................................................................. 14

10 Test report ...................................................................................................................................................... 14

Annex A (informative) Example conditions for suitable LC-MS/MS systems ...................................... 15

A.1 Settings for chromatography for WATERS Acquity H-class coupled to a Xevo® TQ-S ........ 15

A.2 Mass spectrometric detection settings ................................................................................................. 16

Annex B (informative) Typical chromatograms ............................................................................................ 17

Annex C (informative) Precision data ................................................................................................................ 20

Bibliography ................................................................................................................................................................. 23

---------------------- Page: 4 ----------------------
oSIST prEN 17203:2019
prEN 17203:2019 (E)
European foreword

This document (prEN 17203:2019) 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 will supersede EN 17203:2018.

This document has been prepared under a standardization request given to CEN by the European

Commission and the European Free Trade Association.
The alterations to the version of 2018 are as follows:

— 5.24 The necessity to prepare calibration solutions freshly every day was deleted.

— 6.14.2 The requirement for cross contamination below 1 % was deleted.
— 7.5.1 First sentence was re-worded in better language.

— 7.5.2 The last part "when negative ion mode is used"of second para before Table 2, starting with

"When an adduct ion is used as precursor ion" was deleted as not applicable for this method.

— 7.5.2 Table 2, The last column for the 2 qualifier was re-introduced again.
— 7.6 Para 2 line 1 was re-worded in a clearer way.
— 8.1 Para 3 was aligned with other standards of CEN/TC 275/WG 5.
---------------------- Page: 5 ----------------------
oSIST prEN 17203:2019
prEN 17203:2019 (E)
Introduction

The mycotoxin citrinin is a polyketide secondary metabolite produced mainly post-harvest in food and

feed by several fungi of the genera Penicillium (e.g. P. citrinum), Aspergillus (e.g. A. candidus), and

Monascus (e.g. M. purpureus). Citrinin occurs mainly in stored grains like rice, maize, wheat, barley, oats,

and rye. Citrinin can be found as a contaminant in red fermented rice with Monascus purpureus and its

formulated dietary supplements.

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 [5] 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 — Citrinin is known to have nephrotoxic properties, damaging the proximal tubules

of the kidney [6].
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1 Scope

This document describes a procedure for the determination of the citrinin content in food (cereals, red

yeast rice (RYR)), herbs and food supplements by liquid chromatography tandem mass spectrometry

(LC-MS/MS).

This method has been validated for citrinin in red yeast rice and in the formulated food supplements in

the range of 2,5 µg/kg to 3000 µg/kg and in wheat flour in the range of 2,5 µg/kg to 100 µg/kg.

Laboratory experiences have shown that this method is also applicable to white rice, herbs such as a

powder of ginkgo biloba leaves and the formulated food supplements in the range of 2,5 µg/kg to

50 µg/kg.
2 Normative references

The following documents are referred to in the text in such a way that some or all of their content

constitutes requirements of this document. For dated references, only the edition cited applies. For

undated references, the latest edition of the referenced document (including any amendments) applies.

EN ISO 3696, Water for analytical laboratory use — Specification and test methods (ISO 3696)

3 Terms and definitions
No terms and definitions are listed in this document.

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

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

A test portion is humidified with a hydrochloric acid aqueous solution and extracted with ethyl

acetate/acetonitrile/glacial acetic acid mixture for 60 min. Magnesium sulfate and sodium chloride are

added to the extract, agitated and centrifuged in order to expel water and allow phase separation from

the mixture. An aliquot of supernatant is collected, filtered, isotopic labelled internal standard (ISTD)

solution is added and analysed by reversed phase LC-MS/MS. Quantification is based on matching

citrinin/citrinin- C ratios and citrinin concentrations.
5 Reagents

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

unless otherwise specified. Commercially available solutions with equivalent properties to those listed

may also be used.
5.1 Ethyl acetate, analytical grade or higher.
5.2 Acetonitrile, LC-MS grade. .
5.3 Glacial acetic acid (CH COOH), analytical grade or higher.
5.4 Glacial acetic acid (CH COOH), LC-MS grade.
5.5 Magnesium sulfate; anhydrous (MgSO ), analytical grade or higher.
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5.6 Sodium chloride (NaCl), analytical grade or higher.

5.7 Hydrochloric acid solution (HCl), analytical grade or higher, volume fraction φ(HCl) = 37 %

(acidimetric).
5.8 Water (H O), deionised (Ultrapure).
5.9 Water (H O), LC-MS grade.
5.10 Methanol (MeOH), LC-MS grade.
5.11 Ammonium acetate (CH COONH ), LC-MS grade.
3 4
5.12 Extraction solution 1.

Add 10 ml of glacial acetic acid (5.3) to 990 ml of water (5.8) and mix (water + glacial acetic acid, 99+1,

v+v). Dissolve 100 g of sodium chloride (5.6) in 1 l of this mixture and add 16 ml of hydrochloric acid

solution (5.7). This solution can be used for 1 month if stored at room temperature.

5.13 Extraction solution 2.

Mix 240 ml of acetonitrile (5.2) with 750 ml of ethyl acetate (5.1) and 10 ml of glacial acetic acid (5.3).

This solution (ethyl acetate + acetonitrile + glacial acetic acid, 75+24+1, v+v+v) can be used for 1 month

if stored at room temperature.
5.14 Dilution solution.

Mix 80 ml of methanol (5.10), 18 ml of water (5.9) and 2 ml of glacial acetic acid (5.4). This solution

(methanol + water + glacial acetic acid, 80+18+2, v+v+v) can be used for 1 month if stored at room

temperature.
5.15 Ammonium acetate/glacial acetic acid in water.

Dissolve 9,5 g of ammonium acetate (5.11) in 12,5 ml of water (5.9), then add 12,5 ml of glacial acetic

acid (5.4) and mix thoroughly. This solution can be used for 12 months if stored at < −18 °C.

5.16 Mobile phase A: ammonium acetate/glacial acetic acid in water, c = 5 mmol/l.

Add 1 ml of ammonium acetate/glacial acetic acid in water (5.15) to 999 ml of water (5.9) and mix

thoroughly.

5.17 Mobile phase B: ammonium acetate/glacial acetic acid in methanol, c = 5 mmol/l.

Add 1 ml of ammonium acetate/glacial acetic acid in water (5.15) to 999 ml of methanol (5.10) and mix

thoroughly.

5.18 Citrinin, analytical standard > 99 %, e.g. crystalline or as certified standard solution.

5.19 Citrinin stock solution, mass concentration ρ = 500 µg/ml.

Weigh 5 mg of crystalline citrinin to the nearest 0,1 mg into a 10 ml volumetric flask and dissolve with

acetonitrile (5.2) by filling up to the mark. The mass concentration of this stock solution shall be

checked. This can be achieved via LC-MS/MS analysis against the certified standard solution (5.18) or

by a photometric determination of concentration using the molar extinction coefficient [8].

The certified standard solution (5.18) can alternatively be used as stock solution.

5.20 Citrinin working solution, ρ = 100 µg/ml.

Pipette 1 ml of the stock solution (5.19) into 4 ml of the dilution solution (5.14) and homogenize the

mixture.
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A correction factor (see 5.19) shall be determined and used to correct the exact concentration of this

working solution (mass concentration ρ = 100 µg/ml). This step may be omitted when using the

certified standard solution (5.18).

The stock and working solutions can be used for 12 months if stored at < −18 °C and protected against

light and moisture.
5.21 Citrinin intermediate solution, ρ = 1 µg/ml.

Pipette 50 µl of the citrinin working solution (5.20) into 4950 µl of the dilution solution (5.14) which

results in 5 ml and homogenize the mixture.

Use this intermediate solution as spiking solution for recovery experiments. This solution can be used

for 3 months if stored in amber flasks (6.13) at < −18 °C.

5.22 Citrinin- C stock solution, as standard solution at a given mass concentration, e.g.

ρ = 100 µg/ml.

After opening, the stock solution can usually be used for 12 months if stored at < −18 °C in amber flasks

(see its expiry date in the Certificate of Analysis).

If obtained as crystalline, dissolve the citrinin- C in a suitable amount of acetonitrile (5.2) to obtain a

stock solution at a suitable mass concentration (e.g. ρ = 100 µg/ml). This stock solution can also usually

be used for 12 months if stored at < −18°C
5.23 Citrinin- C intermediate solution, ρ = 100 ng/ml.

Dilute the citrinin- C stock solution (5.22) with dilution solution (5.14) and mix well to obtain the

citrinin- C intermediate solution at a concentration of ρ = 100 ng/ml. Use this solution as internal

standard (ISTD) and add it to each of the sample extracts, the calibration solutions (5.24) and citrinin

control solution (5.25).

This solution can be used for 6 months if stored in amber flasks at < −18 °C. Check the stability of the

solution when using after 6 months.
5.24 Calibration solutions.

Prepare e.g. the following calibration solutions (within a range from 0,25 ng/ml to 50 ng/ml) as

outlined in Table 1 using the citrinin intermediate solution (5.21) and the dilution solution (5.14).

Homogenize the mixtures.

Start with calibration solution 6 which is then used to prepare calibration solutions 1, 2 and 3 (see

Table 1).
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Table 1 — Examples of suitable calibration solutions
Calibration Mass Total volume Citrinin Calibration Dilution
solution concentration intermediate solution 6 solution
solution (5.14)
(5.21)
ng/ml µl µl µl µl
1 0,25 1000 5 995
2 0,5 1000 10 990
3 1 1000 20 980
4 10 1000 10 990
5 20 1000 20 980
6 50 1000 50 950

Take 45 µl from every calibration solution (1 to 6), add 5 µl of the citrinin- C intermediate solution

(5.23) and mix.

It is also possible to increase the volumes of the calibration and intermediate solutions in proportion to

one another.
5.25 Citrinin control solution, ρ = 25 ng/ml.

Prepare a citrinin control solution from the certified standard solution (5.18). Alternatively, prepare a

citrinin control solution from a suitable independent citrinin stock solution that has been checked

photometrically or has been checked via a certified standard solution (5.18). Prepare the control

solution (ρ = 25 ng/ml) by diluting the stock solution with dilution solution (5.14).

Add 5 µl of the citrinin- C intermediate solution (5.23) to 45 µl of this control solution before analysis.

This control solution can be used for 3 months if stored in amber flasks at < −18 °C.

6 Apparatus and equipment
Usual laboratory glassware and equipment, in particular, the following.

For glassware, use preferably amber coloured to reduce light effect and degradation of citrinin.

6.1 Laboratory balance, accuracy of 0,01 g.
6.2 Analytical balance, accuracy of 0,1 mg.
6.3 Laboratory shaker.
6.4 Centrifuge, suitable for 50 ml centrifuge tubes.
6.5 Centrifuge, suitable for 1,5 ml centrifuge tubes.

6.6 Pipettes, adjustable e.g. 1 µl to 1000 µl, suitable for organic solvents, with appropriate tips.

6.7 Centrifuge tubes, 50 ml.
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6.8 Centrifuge tubes, polypropylene, (e.g. Eppendorf tube), 1,5 ml.
6.9 Syringe, 2 ml.

6.10 Filters for syringe, polytetrafluoroethylene (PTFE), with 0,2 µm pore diameter.

6.11 Amber vials for injection (1,5 ml), with screw cap or equivalent.
6.12 Glass inserts.
6.13 Amber flasks.
6.14 LC-MS/MS system, comprising the following:
6.14.1 LC pump, suitable for gradient elution.

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

sufficient accuracy.

6.14.3 LC column, e.g. HSST3 C18 column, 1,8 µm, 2,1 mm ⨯100 mm and corresponding pre-filter or

pre-column.

Columns of different dimensions may also be used that ensure base line separation to distinguish peaks

of the citrinin from all other signals.
6.14.4 Column thermostat.

6.14.5 Tandem mass spectrometer (MS/MS), capable of performing ionization of citrinin and

multiple reaction monitoring (MRM) with a sufficiently wide dynamic range.
6.14.6 Data evaluation system.
7 Procedure
7.1 Preparation of the test sample

Finely grind the laboratory sample, homogenize and store in the dark before taking a test portion for

analysis.
7.2 Extraction of citrinin

Weigh a test portion of 4,00 g of the homogenized laboratory sample to the nearest 0,02 g into a 50-ml

centrifugation tube (6.7).

Add 10 ml of the extraction solution 1 (5.12) followed immediately by 20 ml (V ) of extraction

solution 2 (5.13) and cap the tube.

Shake the mixture for approximately 1 h using a laboratory shaker (6.3) at room temperature.

Thereafter, add 6,0 g of magnesium sulfate (5.5) and 1,5 g of sodium chloride (5.6) to the mixture and

immediately shake vigorously for 30 s to avoid aggregation of the salts.

NOTE This step is exothermic and results in moderate but noticeable warming of the tube and its contents.

Eppendorf is a trade name of a centrifugation tube supplied by Eppendorf International. This information is

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

the product named. Equivalent tube
...

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