Foodstuffs - Determination of T-2 toxin and HT-2 toxin in cereals and cereal products for infants and young children by LC-MS/MS after SPE cleanup

This European Standard describes a method for the determination of the content of T-2 toxin and HT-2 toxin in cereals and cereal based products e.g. oats, intended for nutrition of infants and young children by high performance liquid chromatography (HPLC) coupled with tandem mass spectrometry (MS/MS) after cleanup by solid phase extraction (SPE) [5].
The method has been validated for HT-2 toxin in oat flour at levels of 9,3 µg/kg and 28,1 µg/kg, oat flakes at levels of 16,5 µg/kg and 21,4 µg/kg, and breakfast cereals (containing oat flakes) at a level of 8,1 µg/kg and for T-2 toxin in oat flour at levels of 4,4 µg/kg and 8,3 µg/kg, oat flakes at levels of 4,9 µg/kg and 6,6 µg/kg and breakfast cereals (containing oat flakes) at a level of 3,5 µg/kg.
Laboratory experiences [6] have shown that the method is also applicable to highly swelling materials (dry cereal based porridges and modified starches), but these were not examined in the method validation study. Details are outlined in 6.3.
The method can also be applied to oat-by-products at higher levels of T-2- and HT-2 toxin. In this case, the dilution steps need to be considered [6].

Lebensmittel - Bestimmung von T-2-Toxin und HT-2-Toxin in Getreide und Säuglings- und Kleinkindernahrung auf Getreidebasis mit LC-MS/MS nach SPE-Reinigung

Diese Europäische Norm legt ein Verfahren zur Bestimmung des Gehaltes von T-2-Toxin und HT 2 Toxin durch Hochleistungsflüssigchromatographie (HPLC) in Kopplung mit der Tandem-Massenspektrometrie (MS/MS) nach Festphasenreinigung (SPE, en: solid phase extraction) [5] in Getreide und getreidebasierten Produkten, z. B. Haferflocken, fest, die für die Ernährung von Säuglingen und Kleinkindern vorgesehen sind.
Das Verfahren wurde für HT 2 Toxin in Hafermehl bei Konzentrationen von 9,3 µg/kg und 28,1 µg/kg, in Haferflocken bei Konzentrationen von 16,5 µg/kg und 21,4 µg/kg und bei Frühstückscerealien (die Hafer-flocken enthalten) bei einer Konzentration von 8,1 µg/kg und für T 2 Toxin in Hafermehl bei Konzentrationen von 4,4 µg/kg und 8,3 µg/kg, in Haferflocken bei Konzentrationen von 4,9 µg/kg und 6,6 µg/kg und bei Frühstückscerealien (die Haferflocken enthalten) bei einer Konzentration von 3,5 µg/kg validiert.
Laborerfahrungen [6] haben gezeigt, dass das Verfahren auch für hochgradig aufquellende Materialien (auf trockenem Getreide basierendem Porridge und modifizierte Stärken) anwendbar ist, jedoch wurden diese in der Validierungsuntersuchung des Verfahrens nicht überprüft. Einzelheiten hierzu sind in 6.3 enthalten.
Das Verfahren kann ebenso bei Nebenprodukten von Hafer bei höheren Konzentrationen von T-2-Toxin und HT 2 Toxin angewendet werden. In diesem Fall müssen die Verdünnungsschritte berücksichtigt werden [6].

Produits alimentaires - Dosage des toxines T-2 et HT-2 dans les céréales et les produits céréaliers pour nourrissons et enfants en bas âge par CL-SM/SM après purification par SPE

La présente Norme européenne décrit une méthode de détermination de la teneur en toxines T-2 et HT-2 dans les céréales et les produits céréaliers, par exemple l’avoine, destinés aux nourrissons et aux jeunes enfants par chromatographie liquide à haute performance (CLHP) couplée à une spectrométrie de masse en tandem (SM/SM) après purification par extraction en phase solide (SPE) [3].
La méthode a été validée pour la toxine HT-2 présente dans la farine d’avoine à des niveaux de 9,3 µg/kg et 28,1 µg/kg, dans les flocons d’avoine à des niveaux de 16,5 µg/kg et 21,4 µg/kg et dans les céréales de petit-déjeuner (contenant des flocons d’avoine) à un niveau de 8,1 µg/kg, et pour la toxine T-2 présente dans la farine d’avoine à des niveaux de 4,4 µg/kg et 8,3 µg/kg, dans les flocons d’avoine à des niveaux de 4,9 µg/kg et 6,6 µg/kg et dans les céréales de petit-déjeuner (contenant des flocons d’avoine) à un niveau de 3,5 µg/kg.
Les expériences menées en laboratoire [4] ont démontré que la méthode est également applicable aux matières qui gonflent beaucoup (bouillies à base de céréales sèches et amidons modifiés), mais ces matières n’ont pas été examinées lors de l’étude de validation de la méthode. Le paragraphe 6.3 fournit plus d’informations.
La méthode peut également être appliquée aux sous-produits de l’avoine à des teneurs en toxines T-2 et HT-2 plus élevées. Dans ce cas, les étapes de dilution doivent être prises en compte [4].

Živila - Določevanje toksinov T-2 in HT-2 v žitu in žitnih proizvodih za dojenčke in majhne otroke z LC-MS/MS po čiščenju s SPE

Ta evropski standard opisuje metodo za določanje vsebnosti toksinov T-2 in HT-2 v žitu in žitnih proizvodih za dojenčke in majhne otroke s tekočinsko kromatografijo visoke ločljivosti (HPLC) skupaj s tandemsko masno spektrometrijo (MS/MS) po čiščenju z ekstrakcijo na trdni fazi (SPE) [5].
Metoda je potrjena za toksin HT-2 v ovseni moki pri koncentracijah 9,3 μg/kg in 28,1 μg/kg, ovsenih kosmičih pri koncentracijah 16,5 μg/kg in 21,4 μg/kg ter žitih za zajtrk (ki vsebujejo ovsene kosmiče) pri koncentracijah 8,1 μg/kg, ter za toksin T-2 v ovseni moki pri koncentracijah 4,4 μg/kg in 8,3 μg/kg, ovsenih kosmičih pri koncentracijah 4,9 μg/kg in 6,6 μg/kg ter žitih za zajtrk (ki vsebujejo ovsene kosmiče) pri koncentraciji 3,5 μg/kg.
Laboratorijske izkušnje [6] kažejo, da se metoda uporablja tudi za materiale, ki močno nabreknejo (kaše iz suhih žit in modificiranih škrobov), vendar ti materiali niso bili raziskani v študiji validativnosti metode. Podrobnosti so opredeljene v točki 6.3.
Metoda se lahko uporabi tudi za stranske proizvode iz ovsa z višjimi ravnmi toksinov T-2 in HT-2. V tem primeru je treba upoštevati korake za redčenje [6].

General Information

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

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2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.Lebensmittel - Bestimmung von T-2-Toxin und HT-2-Toxin in Getreide und Säuglings- und Kleinkindernahrung auf Getreidebasis mit LC-MS/MS nach SPE-ReinigungProduits alimentaires - Dosage des toxines T-2 et HT-2 dans les céréales et les produits céréaliers pour nourrissons et enfants en bas âge par CL-SM/SM après purification par SPEFoodstuffs - Determination of T-2 toxin and HT-2 toxin in cereals and cereal products for infants and young children by LC-MS/MS after SPE cleanup67.230Predpakirana in pripravljena hranaPrepackaged and prepared foods67.060QMLKCereals, pulses and derived productsICS:Ta slovenski standard je istoveten z:EN 16923:2017SIST EN 16923:2017en,fr,de01-september-2017SIST EN 16923:2017SLOVENSKI

STANDARD
SIST EN 16923:2017
EUROPEAN STANDARD NORME EUROPÉENNE EUROPÄISCHE NORM
EN 16923
May
t r s y ICS
x yä r x râ
x yä t u r English Version

Foodstuffs æ Determination of Tæ t toxin and HTæ t toxin in cereals and cereal products for infants and young children Produits alimentaires æ Dosage des toxines Tæ t et HTæ t dans les céréales et les produits céréaliers pour purification par SPE

Lebensmittel æ Bestimmung von Tæ tæToxin und HTæ tæToxin in Getreide und Säuglingsæ und nach SPEæReinigung 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 uã t r s y ESIST EN 16923:2017

EN 16923:2017 (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 ........................................................................................................................... 7 6 Procedure........................................................................................................................................................... 9 7 Calculation ...................................................................................................................................................... 10 8 Precision .......................................................................................................................................................... 11 9 Test report ...................................................................................................................................................... 12 Annex A (informative)

Example chromatograms (API 4000™) ................................................................ 13 Annex B (informative)

Example conditions for suitable LC-MS/MS systems ...................................... 17 Annex C (informative)

Precision data ................................................................................................................ 24 Bibliography ................................................................................................................................................................. 26

SIST EN 16923:2017

EN 16923:2017 (E) 3 European foreword This document (EN 16923:2017) 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 16923:2017

EN 16923:2017 (E) 4 Introduction The mycotoxin T-2 toxin and its metabolite HT-2 toxin belong to the group of trichothecenes which are produced by various Fusarium species. Cereals like maize, wheat, barley, oats, and rye are most likely to be affected. WARNING 1 — Suitable precaution and protection measures need to be taken when carrying out working steps with harmful chemicals. The latest version of the hazardous substances ordinance, Regulation (EC) No 1907/2006 [3], should be taken into account as well as 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 — T-2 toxin and its metabolite HT-2 toxin are known to have carcinogenic effects. SIST EN 16923:2017

EN 16923:2017 (E) 5 1 Scope This European Standard describes a method for the determination of T-2 toxin and HT-2 toxin in cereals and cereal based products e.g. oats, intended for nutrition of infants and young children by high performance liquid chromatography (HPLC) coupled with tandem mass spectrometry (MS/MS) after cleanup by solid phase extraction (SPE) [5]. The method has been validated for HT-2 toxin in oat flour at levels of 9,3 µg/kg and 28,1 µg/kg, oat flakes at levels of 16,5 µg/kg and 21,4 µg/kg, and breakfast cereals (containing oat flakes) at a level of 8,1 µg/kg and for T-2 toxin in oat flour at levels of 4,4 µg/kg and 8,3 µg/kg, oat flakes at levels of 4,9 µg/kg and 6,6 µg/kg and breakfast cereals (containing oat flakes) at a level of 3,5 µg/kg. Laboratory experiences [6] have shown that the method is also applicable to highly swelling materials (dry cereal based porridges and modified starches), but these were not examined in the method validation study. Details are outlined in 6.3. The method can also be applied to oat-by-products at higher levels of T-2- and HT-2 toxin. In this case, the dilution steps need to be considered [6]. The method can also be applied to cereals and cereal products for infants and young children based on e.g. wheat, barley, and rice. In this case, the method needs to be in-house-validated for each material. At the time of the interlaboratory study, planned range was 10 µg/kg to 100 µg/kg, and it is known from the pre-study that the method works well in the whole range, although final validation was only done in the range from 3,5 µg/kg to 28,1 µg/kg. 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 T-2 toxin and HT-2 toxin are extracted with acetonitrile-water mixture and by shaking manually or with a laboratory blender. A solid phase extraction column or a pass through column is used to clean up and concentrate the filtered and diluted extract, see also [7]. The toxins are determined by HPLC coupled with tandem mass spectrometry. 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 HPLC analysis, unless otherwise specified. 4.1 Acetonitrile, HPLC grade. 4.2 Methanol, HPLC grade. 4.3 Solvent mixture. Mix 20 parts of acetonitrile (4.1) and 80 parts of water (20+80, v+v). 4.4 Extraction mixture. Mix 84 parts of acetonitrile (4.1) and 16 parts of water (84+16, v+v). SIST EN 16923:2017

EN 16923:2017 (E) 6 4.5

Eluent for LC-MS/MS. Examples of eluents suitable for LC-MS/MS systems are given in Annex B. Filter the solution through a membrane filter (5.18). 4.6

Nitrogen, purity of at least 99,9 %. 4.7

Activated charcoal for column chromatography (particle size: 63 µm to 200 µm). 4.8

Aluminium oxide (neutral, for liquid chromatography). 4.9

Finely ground/pulverized diatomaceous earth (diatomite, kieselgur), e.g. Celite® 545. 4.10

Siliconization reagent, e.g. SurfaSil™1) (optional). 4.11
Cyclohexane, analytical quality, (optional). 4.12

Preparation of the diluted siliconization reagent, (optional). Add e.g. 50 ml of a siliconization reagent (4.10) to 950 ml cyclohexane (4.11). 4.13

Formic acid, HPLC quality. 4.14

Ammonia solution, substance concentration c(NH3) = 13,4 mol/l or mass concentration (NH3) = 250 g/l. 4.15

Ammonium acetate (CH3CO2NH4), LC-MS/MS quality. 4.16 Anti-clogging material, such as washed sea sand, glass beads, or polyethylene beads, (optional). 4.17 Stock solution of T-2 toxin, mass concentration

= 100
4.18 Stock solution of HT-2 toxin,
= 100
4.19 Internal standard solution of [13C24]-T-2 toxin,
= 25

Other suitable isotopic labelled standards of T-2 toxin than the [13C24]-T-2 toxin may be used. 4.20 Internal standard solution of [13C22]-HT-2 toxin,

= 25

Other suitable isotopic labelled standards of HT-2 toxin than the [13C22]-HT-2 toxin may be used. 4.21 Mixed standard solution,

= 500 ng/ml. Pipette 25 µl of each T-2 toxin and HT-2 toxin stock solution (4.17 and 4.18), respectively, into a 5 ml volumetric flask, and dilute up to the mark with solvent mixture (4.3). This solution can be stored at

« s z °C for 12 months.

1) Surfasil ™ is a trade name of a product commercially available from various suppliers. This information is given for the convenience of users of this European Standard and does not constitute an endorsement by CEN of the products named. Equivalent products may be used if they can be shown to lead to the same results. SIST EN 16923:2017

EN 16923:2017 (E) 7 4.22 Mixed internal standard solution,

= 1000 ng/ml Dilute 200 µl of the internal standard solutions (4.19 and 4.20) with solvent mixture (4.3) in a 5 ml volumetric flask. This solution can be stored at – 18 °C for 6 months. 4.23 Calibration solutions. For the calibration of the measuring system, prepare calibration solutions within a range from 5 ng/ml to 100 ng/ml. Prepare e.g. the following calibration solutions as outlined in Table 1: Table 1 — Examples of suitable calibration solutions Calibration solution Mass concentration per analyte Mass concentration per isotope labelled analyte Mixed standard solution (4.21) Mixed internal standard solution (4.22) Solvent mixture (4.3)

ng/ml ng/ml µl µl µl IS-Blank 0 50 – 50 950 1 5 50 10 50 940 2 10 50 20 50 930 3 20 50 40 50 910 4 40 50 80 50 870 5 60 50 120 50 830 6 80 50 160 50 790 7 100 50 200 50 750 5 Apparatus and equipment Usual laboratory apparatus and, in particular, the following. 5.1 Laboratory balance, accuracy of 0,01 g. 5.2 Analytical balance, accuracy of 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 5 ml. 5.6 Dispenser, suitable for 20 ml. 5.7 250 ml-Erlenmeyer flasks with stoppers, or 250 ml-centrifuge tubes. SIST EN 16923:2017

EN 16923:2017 (E) 8 5.8

Syringe filters (0,45 µm), or centrifugal filters (e.g. Durapore® PVDF (0,45 µm), or Millipore Ultrafree-MC® 0,5 ml), fitting with centrifuge for reaction vessels, e.g. Eppendorf® vessels2). 5.9 Folded filter, pore size 4 µm to 7 µm, diameter 100 mm. 5.10

Laboratory centrifuge. 5.11

Cartridges (6 ml), made from polypropylene (PP) and corresponding frits from polyethylene (PE), or commercially available SPE columns, e.g. CHROMABOND® Carbon/Alox/Celite®2), 6 ml, 500 mg. 5.12

SPE vacuum/elution station. 5.13
Laboratory shaker, e.g. overhead shaker. 5.14

Laboratory blender, e.g. Ultra Turrax®2). 5.15 Test tubes, suitable for a volume up to 10,0 ml. 5.16 Siliconized test tubes (optional). After thorough cleaning of the test tubes (5.15), fill up to the top with the diluted siliconization reagent (4.12) and allow them to stand for 1 min. Then, pouring out the reagent solution, make sure to collect it for repeated usage. Afterwards rinse the tubes with cyclohexane (4.11) and acetonitrile (4.1) or methanol (4.2) successively in this order. The rinsing solutions may be used again. Finally rinse the tubes twice with double-distilled water and allow them to dry. WARNING — Surfasil™2) being a chloride silane solvent, readily reacts with water by forming hydrochloric acid vapour. Therefore never rinse tubes with water directly after derivatization. Tubes that are not siliconized, such as made from polypropylene, may be used, if formally proved suitable. 5.17 Concentration evaporator workstation, e.g. TurboVap®2) Zymark, or similar. 5.18 Membrane filters for aqueous solutions (pore size 0,45 µm). 5.19 LC-MS/MS system with the following components: 5.19.1 HPLC pump. 5.19.2 Injection system. 5.19.3 HPLC column, e.g. octadecylsilane (ODS), that ensures base line separation to distinguish peaks of the T-2 toxin and HT-2 toxin from all other signals, 150 mm length, 2,00 mm inner diameter, particle size 5 µm, suitable reversed-phase pre-column.

2) Durapore® PVDF, Millipore Ultrafree-MC®, Ultra Turrax ®, TurboVap®LV Zymark and Surfasil are trade names of products commercially available from various suppliers. Eppendorf® vessel is an example of a product commercially available from Eppendorf, Chromabond® is the trade name of a product, commercially available from by Macherey-Nagel. 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 16923:2017

EN 16923:2017 (E) 9 Columns of different dimensions may also be used. 5.19.4 Column thermostat. 5.19.5 Tandem mass spectrometer (MS/MS). 5.19.6 Data evaluation system. 6 Procedure 6.1 Preparation of the test sample Grind and homogenize the sample to particle sizes less than 1 mm before analysis. 6.2 Preparation of the solid phase column Mix 42 g of activated charcoal (4.7) with 30 g of neutral Al2O3 (4.8) and 18 g of Celite 545 (4.9) in a glass vessel (500 ml) and homogenize with a shaker (5.13) for 1 h (ratio 7:5:3 activated charcoal/neutral Al2O3/Celite 545; m/m/m). Place the homogenized mixture, 0,5 g respectively, in empty 6 ml cartridges provided with three PE frits (2 frits below, and one on top for covering). Alternatively, commercially available SPE-columns may be used. For this reason, clean up procedure shall be checked for recovery and shall be optimized if necessary. [7] 6.3 Extraction of T-2 toxin and HT-2 toxin Weigh 25,0 g of the homogenized and finely ground sample (6.1) with an accuracy of 0,1 g into a 250 ml beaker/Erlenmeyer flask, or into a 250 ml centrifuge tube (5.7), add 100 ml of the extraction mixture (4.4) and close the vessel. Shake the mixture manually or with a shaker (5.13) for 1 h at room temperature. Alternatively, use a laboratory blender (5.14) for extraction. In this case, homogenize the mixture for 3 min at a great speed. After extraction, pass slightly more than 10 ml extract through a folded filter (5.9) into a glass vessel. Centrifuge this portion at 2 500 × g at room temperature for 10 min, Remove 10 ml of the upper solution of the centrifugate. If highly swelling food matrices are analysed, increase the water content in the extraction medium up to 200 % or alternatively reduce the weight of the sample amount down to 50 % of the described amount. To prevent clogging of the swelling material, add the same amount of e.g. sea sand (4.16) as the sample weight. Take volume and/or weight adjustments into account in the final calculation. 6.4 Clean-up by solid phase filtration Plug the prepared column containing 0,5 g of activated charcoal/Al2O3/Celite (6.2) on the SPE station (5.12), and place a test tube (5.15) beneath to collect the eluate. Pass 5,0 ml of the extract (6.3) through the SPE-column and collect the eluate. Apply a low vacuum in order to obtain an elution speed of 1 drop to 2 drops per s. Rinse the cartridge again with 5 ml of extraction mixture (4.4), and collect that eluate also. Add 25 µl of mixed internal standard solution (4.22) to the combined eluates, and evaporate to dryness with nitrogen (4.6) using a concentration evaporator workstation (at 45 °C for 30 min, and 10 psi gas pressure). Re-dissolve the residue in 500 µl of solvent mixture (4.3) by shaking turbulently (5.4) for 60 s, and, if necessary, apply an ultrasonic bath (5.3) for 5 min at room

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