Foodstuffs - Determination of zearalenone in edible vegetable oils by LC-FLD or LC-MS/MS

This European Standard describes a procedure for the determination of the zearalenone content in edible vegetable oils specifically maize germ oil by either of the following techniques: High performance liquid chromatography with fluorescence detection (LC-FLD) or high performance liquid chromatography with tandem mass spectrometry (LC-MS/MS) after basic extraction of the diluted oil.
The method has been validated for zearalenone in naturally contaminated maize germ oil at levels of 61,2 µg/kg to 515 µg/kg [5].
Laboratory experiences [6] have shown that this method is also applicable to vegetable oils such as wheat germ oil (n = 4), sunflower oil (n = 5), pumpkin seed oil (n = 1), soybean oil (n = 5), hemp seed oil (n = 5), rape seed oil (n = 11), and mixed oils including maize germ oils (n = 3). However occasionally, samples can result in interferences in the FLD-chromatograms. In this case, the detection with MS/MS is recommended.

Lebensmittel - Bestimmung von Zearalenon in pflanzlichen Speiseölen mit LC-FLD oder LC-MS/MS

Diese Europäische Norm beschreibt ein Verfahren für die Bestimmung des Zearalenongehalts in pflanzlichen Speiseölen, insbesondere in Maiskeimöl, mit einem der beiden folgenden Verfahren: Hochleistungsflüssig¬chromatographie mit Fluoreszenzdetektion (LC-FLD) oder Hochleistungsflüssigchromatographie mit Tandem-Massenspektrometrie (LC-MS/MS) nach basischer Extraktion des verdünnten Öles.
Das Verfahren wurde für Zearalenon in natürlich kontaminiertem Maiskeimöl bei Konzentrationen von 61,2 µg/kg bis 515 µg/kg validiert [5].
Laboruntersuchungen [6] haben gezeigt, dass das Verfahren auch für weitere pflanzliche Öle, wie z. B. Weizenkeimöl (n = 4), Sonnenblumenöl (n = 5), Kürbiskernöl (n = 1), Sojaöl (n = 5), Hanfsamenöl (n = 5), Rapsöl (n = 11) sowie Pflanzenölmischungen (n = 3), die Maiskeimöle enthalten, anwendbar ist. Jedoch können Proben gelegentlich zu Interferenzen in den FLD-Chromatogrammen führen. In diesem Fall wird die Detektion mit MS/MS empfohlen.

Denrées alimentaires - Dosage de la zéaralénone dans les huiles végétales alimentaires par CL-FLD ou CL-SM/SM

La présente Norme européenne décrit un mode opératoire de détermination de la teneur en zéaralénone dans les huiles végétales alimentaires, notamment l’huile de germes de maïs, par l’une des techniques suivantes : chromatographie liquide à haute performance avec détection de fluorescence (CL-FLD) ou chromatographie liquide à haute performance couplée à une spectrométrie de masse en tandem (CL-SM/SM) après extraction basique de l’huile diluée.
La méthode a été validée pour la zéaralénone présente dans l’huile de germes de maïs naturellement contaminée à des niveaux de 61,2 µg/kg à 515 µg/kg [3].
Les expériences menées en laboratoire [4] ont démontré que cette méthode est également applicable aux huiles végétales telles que l’huile de germes de blé (n = 4), l’huile de tournesol (n = 5), l’huile de pépins de courge (n = 1), l’huile de soja (n = 5), l’huile de chanvre (n = 5), l’huile de colza (n = 11) et les mélanges d’huiles tels que les huiles de germes de maïs (n = 3). Toutefois, les échantillons peuvent parfois provoquer des interférences dans les chromatogrammes FLD. Dans ce cas, il est recommandé d’utiliser la détection par SM/SM.

Živila - Določevanje zearalenona v jedilnih rastlinskih oljih z LC-FLD ali LC-MS/MS

Ta evropski standard opisuje postopek za določanje vsebnosti zearalenona v jedilnih rastlinskih oljih, zlasti v olju iz koruznih kalčkov, z eno od naslednjih tehnik: tekočinska kromatografija visoke ločljivosti s fluorescenčno detekcijo (LC-FLD) ali tekočinska kromatografija visoke ločljivosti s tandemsko masno spektrometrijo (LC-MS/MS) po osnovni ekstrakciji razredčenega olja.
Ta metoda je bila potrjena za zearalenon v naravno kontaminiranem olju iz koruznih kalčkov pri koncentracijah 61,2–515 μg/kg [5].
Laboratorijske izkušnje [6] kažejo, da se ta metoda uporablja tudi za rastlinska olja, kot so olje pšeničnih kalčkov (n = 4), sončnično olje (n = 5), bučno olje (n = 1), sojino olje (n = 5), konopljino olje (n = 5), olje oljne ogrščice (n = 11) in mešana olja, vključno z olji iz koruznih kalčkov (n = 3). Vendar pa vzorci lahko občasno povzročijo motnje v kromatogramih FLD. V tem primeru je priporočena detekcija z MS/MS.

General Information

Status
Published
Public Enquiry End Date
29-Dec-2015
Publication Date
10-Aug-2017
Current Stage
6060 - National Implementation/Publication (Adopted Project)
Start Date
02-Aug-2017
Due Date
07-Oct-2017
Completion Date
11-Aug-2017

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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
SIST EN 16924:2017
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

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:
Avenue Marnix 17,
B-1000 Brussels

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

EN 16924:2017 (E) 2 Contents Page European foreword ....................................................................................................................................................... 3 Introduction .................................................................................................................................................................... 4 1 Scope .................................................................................................................................................................... 5 2 Normative references .................................................................................................................................... 5 3 Principle ............................................................................................................................................................. 5 4 Reagents ............................................................................................................................................................. 5 5 Apparatus and equipment ........................................................................................................................... 8 6 Procedure........................................................................................................................................................ 10 7 Calculation ...................................................................................................................................................... 12 8 Precision .......................................................................................................................................................... 14 9 Test report ...................................................................................................................................................... 15 Annex A (informative)

Typical chromatograms ............................................................................................ 16 Annex B (informative)

Example conditions for suitable LC-MS/MS systems ...................................... 22 Annex C (informative)

Precision data ................................................................................................................ 27 Bibliography ................................................................................................................................................................. 30

SIST EN 16924:2017

EN 16924:2017 (E) 3 European foreword This document (EN 16924:2017) 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 2017, and conflicting national standards shall be withdrawn at the latest by November 2017. 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 organizations 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 16924:2017

EN 16924:2017 (E) 4 Introduction The mycotoxin zearalenone is a resorcylic acid derivative, which is produced by several species of the fungi genus Fusarium, in particular by Fusarium roseum var. graminearum. Especially cereals like maize and wheat are affected, so that zearalenone can also be detected in the oils produced from them. WARNING 1 — Suitable precaution and protection measures need to be taken when carrying out working steps with harmful chemicals. The hazardous substances ordinance, Regulation (EC) No 1907/2006 [3], should be taken into account as well as appropriate National statements e.g. such as in [4]. 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 — Zearalenone is known to have strong oestrogenic effects. SIST EN 16924:2017

EN 16924:2017 (E) 5 1 Scope This European Standard describes a procedure for the determination of the zearalenone content in edible vegetable oils specifically maize germ oil by either of the following techniques: High performance liquid chromatography with fluorescence detection (LC-FLD) or high performance liquid chromatography with tandem mass spectrometry (LC-MS/MS) after basic extraction of the diluted oil. The method has been validated for zearalenone in naturally contaminated maize germ oil at levels of 61,2 µg/kg to 515 µg/kg [5]. Laboratory experiences [6] have shown that this method is also applicable to other vegetable oils such as wheat germ oil (n = 4), sunflower oil (n = 5), pumpkin seed oil (n = 1), soybean oil (n = 5), hemp seed oil (n = 5), rape seed oil (n = 11), and mixed oils including maize germ oil (n = 3). However occasionally, samples can result in interferences in the FLD-chromatograms. In this case, the detection with MS/MS is recommended. 2 Normative references The following documents, in whole or in part, are normatively referenced in this document and are indispensable for its application. 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 Principle After diluting the edible vegetable oil, zearalenone is extracted by shaking with an alkaline methanol - ammonium hydrogen carbonate mixture. For the determination by LC-FLD, an aliquot of the centrifuged methanolic-alkaline extract is evaporated to dryness, then the residue is diluted in acidified LC-eluent and the zearalenone content is determined by LC-FLD. For the determination by LC-MS/MS, an aliquot of the centrifuged methanolic-alkaline extract is used directly for analysis. 4 Reagents Use only reagents of recognized analytical grade and water complying with grade 1 of EN ISO 3696, unless otherwise specified. Solvents shall be of quality for LC analysis, unless otherwise specified. 4.1 Methanol, p. a. (pro analysis) for extraction. 4.2 Dilution solvent, (defatting solvent), n-hexane or, alternatively, n-heptane, p. a. n-Heptane may be used instead of n-hexane, however, only n-hexane was used in the interlaboratory test. 4.3 Acetonitrile, LC quality. 4.4 Ammonium hydrogen carbonate (NH4HCO3). 4.5 Ammonium hydrogen carbonate solution, mass concentration

= 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

EN 16924:2017 (E) 6 4.6 Alkaline methanol extraction solution. Mix 9 volumes of methanol (4.1) with 1 volume of ammonium hydrogen carbonate solution (4.5). Prepare a fresh alkaline methanol extraction solution each day of analysis. 4.7 Zearalenone, e.g. crystalline, purity greater than 98 % mass fraction, or as certified standard solution with 100 µg/ml. 4.8 Stock solution of zearalenone, mass concentration

= 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

EN 16924:2017 (E) 7 adapted to the desired working range. The calibration solutions can be used for approximately 1 month if stored in the refrigerator at 4 °C. Table 1 — Examples of suitable calibration solutions for LC-FLD Calibration solution Volume of volumetric flask ml Volume of standard solution (4.9)

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

EN 16924:2017 (E) 8 4.11.9 Internal standard solution of zearalanone (ISTD 1),

= 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

EN 16924:2017 (E) 9 5.2 Analytical balance, accuracy: 0,1 mg. 5.3 Ultrasonic bath. 5.4 Laboratory shaker for test tubes. 5.5 Manual dispensers, microlitre syringes or microlitre pipettes for 10 µl to 2 ml. 5.6 Dispenser, suitable for 20 ml. 5.7 Solvent evaporator with heating module. 5.8 Centrifuge, suitable for 3 000 g1). 5.9 Microcentrifuge, suitable for 14 000 g. 5.10 Centrifugation tubes, 50 ml, made of polypropylene with screw cap. 5.11 LC vials. 5.12 UV-spectrometer with quartz cuvettes. 5.13 LC-FLD system with the following components: 5.13.1 LC pump, suitable for gradient elution. 5.13.2 Injection system. 5.13.3 LC column, e.g. LiChroCART® 250-4 filled with LiChrospher®2) 100 RP-18 250 mm x 4 mm, particle size 5 µm and corresponding pre-column. 5.13.4 Column thermostat. 5.13.5 Fluorescence detector, with variable wavelengths. 5.13.6 Data evaluation system. 5.14 LC-MS/MS system with the following components: 5.14.1 LC pump, suitable for gradient elution. 5.14.2 Injection system. 5.14.3 LC column, suitable for chromatography under alkaline conditions, e.g. XBridge®2) C18 or Shield RP18 100 mm × 3 mm; particle size 3,5 µm, or Gemini®2) C18 150 mm × 3 mm; particle size 5 µm and corresponding pre-column. 5.14.4 Column thermostat.

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

EN 16924:2017 (E) 10 5.14.5 Tandem mass spectrometer (MS/MS): Interface: electrospray ionization (ESI); acquisition mode: multiple reaction monitoring (MRM); Ion mode: negative ion mode. 5.14.6 Data evaluation system. 6 Procedure 6.1 Extraction of zearalenone from the sample 6.1.1 General Weigh in 2 g of the sample into a 50 ml centrifugation tube (5.10) to the nearest 0,01 g, add 2,0 ml of dilution solvent (4.2) and 20,0 ml of alkaline methanol extraction solution (Ve) (4.6). If the sample is measured by LC-MS/MS, add 40 µl of zearalanone internal standard solution (ISTD 1) (4.11.9) to the sample prior to extraction (corresponds to a mass fraction of 200 µg/kg zearalanone in the sample). To determine the recovery rate, add 40 µl of the standard solution of zearalenone (4.9) to the sample prior to extraction (corresponds to a mass fraction of 200 µg/kg zearalenone in the sample) and leave for approximately 5 min at room temperature. Extract the mixture for approximately 20 min using a laboratory shaker (5.4). To separate the phases, centrifuge for 10 min at approximately 3 000 g (5.8). 6.1.2 Preparation for LC-FLD Evaporate 5,0 ml of the clear, alkaline methanol supernatant of the extract to dryness at approximately 40 °C and dissolve in 1,0 ml of mobile phase A (4.10.2). Dissolve the residue using an ultrasonic bath (5.3). If necessary, centrifuge the sample solution again in a microcentrifuge (5.9) for 2 min at 14 000 g and transfer into an LC vial (5.11). 6.1.3 Preparation for LC-MS/MS Transfer a part of the clear, alkaline methanol supernatant into an LC vial (5.11). When isotopic labelled zearalenone (ISTD 2) (4.11.7) is used to identify the matrix effect, dilute this supernatant. In this case, the methanol content in this diluted supernatant shall still be 90%. 6.2 LC-FLD analysis Inject equal suitable volumes of the sample test solution and of each calibration solution (4.10.4) into the LC-FLD system. SIST EN 16924:2017

EN 16924:2017 (E) 11 When using the column specified under 5.13.3 and the mobile phases specified in 4.10, the following parameters and those in Table 3 have been shown to be applicable for LC: Flow: 0,9 ml/min Injection volume: 20 µl to 50 µl Column temperature: 30 °C Excitation wavelength: 274 nm Emission wavelength: 446 nm Gradient: see Table 3 Table 3 — Example of a gradient programme Time Percentage of mobile phase A (4.10.2) Percentage of mobile phase B (4.10.3) min % %

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