Foodstuffs - Determination of citrinin in food by liquid chromatography tandem mass spectrometry (LC-MS/MS)

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

Lebensmittel - Bestimmung von Citrinin in Lebensmitteln mit Flüssigchromatographie und Tandem-Massenspektrometrie (LC-MS/MS)

Diese Europäische Norm beschreibt ein Verfahren zur Bestimmung des Citriningehalts in Lebensmitteln (Getreide, rotem Reis [RYR, en: red yeast rice)], Kräutern und Nahrungsergänzungsmitteln mit Flüssigchromatographie und Tandem-Massenspektrometrie (LC-MS/MS).
Dieses Verfahren wurde für Citrinin in rotem Reis und in entsprechend zusammengesetzten Nahrungs-ergänzungsmitteln im Bereich von 2,5 μg/kg bis 3000 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 chromatographie liquide couplée à une spectrométrie de masse en tandem (CL-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 s tekočinsko kromatografijo s tandemsko masno spektrometrijo (LC-MS/MS)

Ta evropski standard opisuje postopek za določevanje vsebnosti citrinina v živilih (žito, rdeči riž), zeliščih in prehranskih dopolnilih s tekočinsko kromatografijo s tandemsko masno spektrometrijo (LC-MS/MS).
Ta metoda je potrjena za rdeči kvasni riž v razponu od 2,5 do 3000 μg/kg in pšenično moko v razponu od 2,5 do 100 μg/kg.
Laboratorijske izkušnje kažejo, da se metoda uporablja tudi za beli riž, zelišča, kot so listi dvokrpega ginka (Ginkgo biloba) v prahu, in že pripravljena prehranska dopolnila.

General Information

Status
Withdrawn
Public Enquiry End Date
14-Feb-2018
Publication Date
28-Jan-2019
Withdrawal Date
13-Jun-2021
Current Stage
9900 - Withdrawal (Adopted Project)
Start Date
14-Jun-2021
Due Date
07-Jul-2021
Completion Date
14-Jun-2021

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2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.Lebensmittel - Bestimmung von Citrinin in Lebensmitteln mit Flüssigchromatographie und Tandem-Massenspektrometrie (LC-MS/MS)Produits alimentaires - Dosage de la citrinine dans les produits alimentaires par chromatographie liquide couplée à une spectrométrie de masse en tandem (CL-SM/SM)Foodstuffs - Determination of citrinin in food by liquid chromatography tandem mass spectrometry (LC-MS/MS)67.050Splošne preskusne in analizne metode za živilske proizvodeGeneral methods of tests and analysis for food productsICS:Ta slovenski standard je istoveten z:EN 17203:2018SIST EN 17203:2019en,fr,de01-marec-2019SIST EN 17203:2019SLOVENSKI
STANDARD



SIST EN 17203:2019



EUROPEAN STANDARD NORME EUROPÉENNE EUROPÄISCHE NORM
EN 17203
December
t r s z ICS
x yä r w r English Version
Foodstuffs æ Determination of citrinin in food by liquid Produits alimentaires æ Dosage de la citrinine dans les produits alimentaires par chromatographie liquide
Lebensmittel æ Bestimmung von Citrinin in Lebensmitteln mit Flüssigchromatographie und TandemæMassenspektroThis European Standard was approved by CEN on
{ November
t r s zä
egulations which stipulate the conditions for giving this European Standard the status of a national standard without any alterationä Upætoædate lists and bibliographical references concerning such national standards may be obtained on application to the CENæCENELEC Management Centre or to any CEN memberä
translation under the responsibility of a CEN member into its own language and notified to the CENæCENELEC Management Centre has the same status as the official versionsä
CEN members are the national standards bodies of Austriaá Belgiumá Bulgariaá Croatiaá Cyprusá Czech Republicá Denmarká Estoniaá Finlandá Former Yugoslav Republic of Macedoniaá Franceá Germanyá Greeceá Hungaryá Icelandá Irelandá Italyá Latviaá Lithuaniaá Luxembourgá Maltaá Netherlandsá Norwayá Polandá Portugalá Romaniaá Serbiaá Slovakiaá Sloveniaá Spainá Swedená Switzerlandá Turkey and United Kingdomä
EUROPEAN COMMITTEE FOR STANDARDIZATION COMITÉ EUROPÉEN DE NORMALISATION EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre:
Rue de la Science 23,
B-1040 Brussels
9
t r s z CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Membersä Refä Noä EN
s y t r uã t r s z ESIST EN 17203:2019



EN 17203:2018 (E) 2 Content
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 7.6 Identification . 12 8 Calculation . 12 8.1 General . 12 8.2 Calculation with the internal standard citrinin (ISTD) . 12 9 Precision . 14 9.1 General . 14 9.2 Repeatability . 14 9.3 Reproducibility . 14 10 Test report . 15 Annex A (informative)
Example conditions for suitable LC-MS/MS systems . 16 A.1 Settings for chromatography for WATERS Acquity H-class coupled to a Xevo® TQ-S . 16 A.2 Mass spectrometric detection settings . 17 Annex B (informative)
Typical chromatograms . 18 Annex C (informative)
Precision data . 21 Bibliography . 24
SIST EN 17203:2019



EN 17203:2018 (E) 3 European foreword This document (EN 17203:2018) 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 June 2019, and conflicting national standards shall be withdrawn at the latest by June 2019. 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, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom. SIST EN 17203:2019



EN 17203:2018 (E) 4 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]. SIST EN 17203:2019



EN 17203:2018 (E) 5 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-13C 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 (CH3COOH), analytical grade or higher. 5.4 Glacial acetic acid (CH3COOH), LC-MS grade. 5.5 Magnesium sulfate; anhydrous (MgSO4), analytical grade or higher. SIST EN 17203:2019



EN 17203:2018 (E) 6 5.6 Sodium chloride (NaCl), analytical grade or higher. 5.7 Hydrochloric acid solution (HCl), analytical grade or higher, volume fraction (HCl) = 37 % (acidimetric). 5.8 Water (H2O), deionised (Ultrapure). 5.9 Water (H2O), LC-MS grade. 5.10 Methanol (MeOH), LC-MS grade. 5.11 Ammonium acetate (CH3COONH4), LC-MS grade. 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 <
« s z °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
= 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. SIST EN 17203:2019



EN 17203:2018 (E) 7 5.20 Citrinin working solution,
= 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 (mass concentration
= 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 <
« s z °C and protected against light and moisture. 5.21 Citrinin intermediate solution,
= 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 <
« s z °C. 5.22 Citrinin-13C stock solution, as standard solution at a given mass concentration, e.g.
= 100 µg/ml. After opening, the stock solution can usually be used for 12 months if stored at <
« s z °C in amber flasks (see its expiry date in the Certificate of Analysis). If obtained as crystalline, dissolve the citrinin-13C in a suitable amount of acetonitrile (5.2) to obtain a stock solution at a suitable mass concentration (e.g.
= 100 µg/ml). The stock solution can usually be used for 12 months if stored at <
« s z °C. 5.23 Citrinin-13C intermediate solution,
= 100 ng/ml. Dilute the citrinin-13C stock solution (5.22) with dilution solution (5.14) and mix well to obtain the citrinin-13C intermediate solution at a concentration of
= 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 <
« s z °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. These calibration solutions shall be freshly prepared on the day of the analysis. Start with calibration solution 6 which is then used to prepare calibration solutions 1, 2 and 3 (see Table 1). SIST EN 17203:2019



EN 17203:2018 (E) 8 Table 1 — Examples of suitable calibration solutions Calibration solution Mass concentration Total volume Citrinin intermediate solution (5.21) Calibration solution 6 Dilution solution (5.14)
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-13C 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,
= 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 ( = 25 ng/ml) by diluting the stock solution with dilution solution (5.14). Add 5 µl of the citrinin-13C 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 <
« s z °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. SIST EN 17203:2019



EN 17203:2018 (E) 9 6.8 Centrifuge tubes, polypropylene, (e.g. Eppendorf®1 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, and cross-contamination below 1 %. 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 (Vex) 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.
1 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 tubes may be used if they can be shown to lead to the same results. SIST EN 17203:2019



EN 17203:2018 (E) 10 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. Shake the mixture again with a
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