EN 16924:2017

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.Lebensmittel - Bestimmung von Zearalenon in pflanzlichen Speiseölen mit LC-FLD oder LC-MS/MSDenrées alimentaires - Dosage de la zéaralénone dans les huiles végétales alimentaires par CL-FLD ou CL-SM/SMFoodstuffs - Determination of zearalenone in edible vegetable oils by LC-FLD or LC-MS/MS67.200.10Animal and vegetable fats and oilsICS:Ta slovenski standard je istoveten z:EN 16924:2017SIST EN 16924:2017en,fr,de01-september-2017SIST EN 16924:2017SLOVENSKI
STANDARD
EUROPEAN STANDARD NORME EUROPÉENNE EUROPÄISCHE NORM
EN 16924
May
t r s y ICS
x yä t r rä s r English Version
Foodstuffs æ Determination of zearalenone in edible Produits alimentaires æ Dosage de la zéaralénone dans les huiles végétales alimentaires par CLæFLD ou CLæ Lebensmittel æ Bestimmung von Zearalenon in This European Standard was approved by CEN on
t y February
t r s yä
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
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t r s y CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Membersä Refä Noä EN
s x { t vã t r s y ESIST EN 16924:2017

Typical chromatograms . 16 Annex B (informative)
Example conditions for suitable LC-MS/MS systems . 22 Annex C (informative)
Precision data . 27 Bibliography . 30
= 10 g/l. Weigh in 1 g of ammonium hydrogen carbonate (4.4) into a 100 ml volumetric flask and fill up to the mark with water. Prepare a fresh solution each day of analysis. SIST EN 16924:2017

= 100 µg/ml. Weigh in 10 mg of crystalline zearalenone (4.7) to the nearest 0,1 mg into a 100 ml volumetric flask and fill up to the mark with acetonitrile (4.3). The stock solution is stable for at least 4 weeks to 12 weeks if stored at <
°C [7]. This step can be omitted when using the certified standard solution. The certified standard solution then serves as stock solution. 4.9 Standard solution of zearalenone,
= 10 µg/ml. Pipette 1 ml of the stock solution of zearalenone (4.8) into a 10 ml volumetric flask and fill up to the mark with acetonitrile (4.3). Determine the exact concentration of zearalenone in this standard solution spectrometrically. For this purpose, record the absorption curve of the standard solution from 190 nm to 350 nm against acetonitrile. Calculate the mass concentration of zearalenone in the standard solution st in µg/ml according to Formula (1): max100stEMρδε⋅⋅=⋅=(1)=where=Emax is the maximum extinction value determined from the absorption curve (here: 274 nm); M is the molar mass of zearalenone = 318,4 g/mol;
is the layer thickness of the cuvette in cm;
is the molar extinction coefficient in acetonitrile: 1262 m2/mol. 4.10 Reagents, mobile phases and calibration solutions for LC-FLD analysis: 4.10.1 Glacial acetic acid for LC-FLD, volume fraction
(CH3COOH) approximately 99 %. 4.10.2 Mobile phase A for LC-FLD: Acetonitrile/water/glacial acetic acid (47+52+1, v+v+v). Mix 47 volumes of acetonitrile (4.3) with 52 volumes of water and 1 volume of glacial acetic acid (4.10.1). For the stability of the chromatographic separation it is necessary to acidify the mobile phase. 4.10.3 Mobile phase B for LC-FLD: Acetonitrile (100 %) (4.3). 4.10.4 Calibration solutions of zearalenone for LC-FLD. Prepare a series of calibration solutions from the standard solution (4.9) of zearalenone. According to the pipetting scheme in Table 1, pipette the corresponding volumes of standard solution of zearalenone into volumetric flasks, evaporate the solvent with nitrogen at approximately 40 °C to dryness and dissolve the residue in mobile phase A (4.10.2). Dissolve by means of laboratory shaker (5.4) or ultrasonic bath (5.3) at room temperature. Then fill up to the mark. The calibration range shall be SIST EN 16924:2017

µl Mass concentration of calibration solution
ng/ml 1 10 25 25 2 10 50 50 3 10 100 100 4 10 150 150 5 10 200 200 6 10 250 250 4.11 Reagents, mobile phases, and additional standards and standard solutions for LC-MS/MS analysis: 4.11.1 Methanol, LC quality. 4.11.2 Ammonium hydrogen carbonate (NH4HCO3), for LC-MS, molecular weight: 79 g/mol. 4.11.3 Mobile phase A for LC-MS/MS: 1 mmol/l NH4HCO3 in water/methanol (95+5, v+v). Dissolve 79 mg of ammonium hydrogen carbonate (4.11.2) in 950 ml of water and mix with 50 ml of methanol (4.11.1). 4.11.4 Mobile phase B for LC-MS/MS: 1 mmol/l NH4HCO3 in water/methanol (5+95, v+v). Dissolve 79 mg of ammonium hydrogen carbonate (4.11.2) in 50 ml of water and mix with 950 ml of methanol (4.11.1). 4.11.5 Working solution of zearalenone for calibration,
= 200 ng/ml. Transfer 0,5 ml of the standard solution (4.9) into a 25 ml volumetric flask and fill up to the calibration mark with methanol (4.11.1). 4.11.6 Zearalanone, internal standard (ISTD 1) for LC-MS/MS, e.g. crystalline or as certified standard solution with 10 µg/ml. 4.11.7 Zearalenone, isotopic labelled internal Standard (ISTD 2) for LC-MS/MS, as optional internal standard, e.g. zearalenone [13C18]-labelled. 4.11.8 Stock solution of zearalanone (ISTD 1),
= 100 µg/ml. Weigh in 10 mg of zearalanone (ISTD 1) (4.11.6) to the nearest 0,1 mg into a 100 ml volumetric flask and fill up to the mark with acetonitrile (4.3). The ISTD 1 stock solution is stable for at least 4 weeks to 12 weeks if stored at <
°C [7]. If the certified standard solution is used, the preparation of this stock solution of zearalanone (ISTD 1) is not necessary. SIST EN 16924:2017

= 10 µg/ml. Transfer 1 ml of the stock solution of zearalanone (4.11.8) into a 10 ml volumetric flask and fill up to the calibration mark with methanol (4.11.1). This step can be omitted when using the certified standard solution of zearalanone. The certified standard solution then serves as the internal standard solution of zearalanone (ISTD 1). 4.11.10 Working solution of internal standard zearalanone (ISTD 1) for calibration,
= 200 ng/ml. Transfer 0,5 ml of the internal standard solution of zearalanone (ISTD 1) (4.11.9) into a 25 ml volumetric flask and fill up to the calibration mark with methanol (4.11.1). However, it is also possible to use isotopic labelled zearalenone (ISTD 2) (4.11.7) as internal standard instead of zearalanone (ISTD 1) (4.11.6), which is sufficient in most cases. 4.11.11 Calibration solutions with zearalenone and zearalanone (ISTD 1) for LC-MS/MS. Prepare a series of calibration solutions from the working solutions of zearalenone (4.11.5) and zearalanone (ISTD 1) (4.11.10). For this purpose, pipette the corresponding volumes of the working solutions into an LC vial (5.11), evaporate the solvent with nitrogen at approximately 40 °C to dryness and dissolve the residue in the alkaline methanol extraction solution (4.6). The mass concentration of the ISTDs and the calibration range shall be adapted to the desired working range. Taking into account the dilution steps, the calibration can be carried out as described in Table 2. The calibration solutions can be used for approximately 1 month if stored in the refrigerator at 4 °C. Table 2 — Examples for suitable calibration solutions for LC-MS/MS Calibration solution Volume of working solutions
(200 ng/ml) Final volume
ml Mass concentration of
calibration solution ng/ml Zearalenone (4.11.5) µl ISTD 1 Zearalanone (4.11.10) µl Zearalenone ISTD 1 Zearalanone 1 25 100 1 5 20 2 50 100 1 10 20 3 100 100 1 20 20 4 150 100 1 30 20 5 200 100 1 40 20 6 250 100 1 50 20 Control possible reciprocal interferences of zearalenone and zearalanone (ISTD 1) at the chosen measurement conditions, by using one calibration standard with the highest level of zearalenone only and one calibration standard with zearalanone (ISTD 1) with the highest level only. 5 Apparatus and equipment Usual laboratory apparatus and, in particular, the following: 5.1 Laboratory balance, accuracy: 0,01 g. SIST EN 16924:2017

1) g = 9,81 m „ sF2. 2) LiChroCART® 250-4 filled with LiChrospher® are trade names of products supplied by Merck, XBridge® is a trade name of a product supplied by Waters. Gemini® is a trade name of a product supplied by Phenomenex. 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. SIST EN 16924:2017
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