prEN 16923
(Main)Foodstuffs - Determination of T-2 toxin and HT-2 toxin in cereals and cereal products for infants and young children by SPE clean up and HPLC-MS/MS
Foodstuffs - Determination of T-2 toxin and HT-2 toxin in cereals and cereal products for infants and young children by SPE clean up and HPLC-MS/MS
This document 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 7.3.
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.
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
Dieses Dokument beschreibt ein Verfahren zur Bestimmung von T-2-Toxin und HT-2-Toxin durch Hochleistungsflüssigchromatographie (HPLC, en: high performance liquid chromatography) in Kopplung mit der Tandem-Massenspektrometrie (MS/MS) nach Festphasenreinigung (SPE, en: solid phase extraction) [5] in Getreide und getreidebasierten Produkten, z. B. Haferflocken, 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 Haferflocken enthalten) bei einer Konzentration von 8,1 µg/kg sowie 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 stark 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 7.3 enthalten.
Das Verfahren kann ebenso bei Hafer-Nebenprodukten bei höheren Konzentrationen von T-2-Toxin und HT-2-Toxin angewendet werden. In diesem Fall müssen die Verdünnungsschritte berücksichtigt werden [6].
Das Verfahren kann auch auf Getreide und Getreideerzeugnisse auf der Basis von Weizen, Gerste und Reis für Säuglinge und Kleinkinder angewendet werden. In diesem Falle ist es erforderlich, das Verfahren im Labor für jedes Material zu validieren (In-house-Validation). Zum Zeitpunkt des Ringversuches war ein Bereich von 10 µg/kg bis 100 µg/kg zur Validierung vorgesehen, und die Vorstudien haben gezeigt, dass das Verfahren diesen gesamten Bereich gut abdeckt, obwohl das Verfahren letztendlich nur für den Bereich von 3,5 µg/kg bis 28,1 µg/kg validiert wurde.
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
Le présent document décrit une méthode de dosage des toxines T-2 et HT-2 dans les céréales et les produits céréaliers, par exemple l’avoine, destinés à l’alimentation des nourrissons et des enfants en bas âge 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) [5].
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 pour 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 pour petit-déjeuner (contenant des flocons d’avoine) à un niveau de 3,5 µg/kg.
Les expériences menées en laboratoire [6] ont démontré que la méthode est également applicable aux matériaux qui gonflent beaucoup (bouillies à base de céréales sèches et amidons modifiés), mais ces matériaux n’ont pas été examinés lors de l’étude de validation de la méthode. Le paragraphe 7.3 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 [6].
La méthode peut également être appliquée aux céréales et aux produits céréaliers pour nourrissons et enfants en bas âge à base par exemple de blé, d’orge et de riz. Dans ce cas, la méthode doit être validée en interne pour chaque matrice. Lors de l’étude interlaboratoires, la gamme prévue allait de 10 µg/kg à 100 µg/kg et il est connu, grâce à l’étude préalable, que la méthode fonctionne bien sur toute l’étendue de la gamme, bien que la validation finale ait seulement été faite pour la gamme de concentrations de 3,5 µg/kg à 28,1 µg/kg.
Živila - Določevanje toksinov T-2 in HT-2 v žitu in žitnih proizvodih za dojenčke in majhne otroke z HPLC-MS/MS po čiščenju s SPE
General Information
RELATIONS
Standards Content (sample)
SLOVENSKI STANDARD
oSIST prEN 16923:2020
01-oktober-2020
Živila - Določevanje toksinov T-2 in HT-2 v žitu in žitnih proizvodih za dojenčke in
majhne otroke z HPLC-MS/MS po čiščenju s SPEFoodstuffs - Determination of T-2 toxin and HT-2 toxin in cereals and cereal products for
infants and young children by SPE clean up and HPLC-MS/MSLebensmittel - 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
SPETa slovenski standard je istoveten z: prEN 16923
ICS:
67.060 Žita, stročnice in proizvodi iz Cereals, pulses and derived
njih products
67.230 Predpakirana in pripravljena Prepackaged and prepared
hrana foods
oSIST prEN 16923:2020 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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DRAFT
EUROPEAN STANDARD
prEN 16923
NORME EUROPÉENNE
EUROPÄISCHE NORM
August 2020
ICS 67.060; 67.230 Will supersede EN 16923:2017
English Version
Foodstuffs - Determination of T-2 toxin and HT-2 toxin in
cereals and cereal products for infants and young children
by SPE clean up and HPLC-MS/MS
Produits alimentaires - Dosage des toxines T-2 et HT-2 Lebensmittel - Bestimmung von T-2-Toxin und HT-2-
dans les céréales et les produits céréaliers pour Toxin in Getreide und Säuglings- und
nourrissons et enfants en bas âge par CL-SM/SM après Kleinkindernahrung auf Getreidebasis mit LC-MS/MS
purification par SPE nach SPE ReinigungThis 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
© 2020 CEN All rights of exploitation in any form and by any means reserved Ref. No. prEN 16923:2020 E
worldwide for CEN national Members.---------------------- Page: 3 ----------------------
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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 ........................................................................................................................... 7
7 Procedure........................................................................................................................................................... 9
8 Calculation ...................................................................................................................................................... 10
9 Precision .......................................................................................................................................................... 12
10 Test report ...................................................................................................................................................... 13
Annex A (informative) Example chromatograms (API 4000™) ................................................................ 14
Annex B (informative) Example conditions for suitable LC-MS/MS systems ...................................... 18
Annex C (informative) Precision data ................................................................................................................ 25
Bibliography ................................................................................................................................................................. 27
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European foreword
This document (prEN 16923:2020) 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 16923:2017.
Alterations to the version of 2017 are as follows:
— The second elution step in the solid phase extraction in 7.4 is more clearly described.
This document has been prepared under a standardization request given to CEN by the European
Commission and the European Free Trade Association.---------------------- Page: 5 ----------------------
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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.
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1 Scope
This document 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 7.3.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 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 definitionsNo terms and definitions are listed in this document.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
• ISO Online browsing platform: available at https://www.iso.org/obp/ui• IEC Electropedia: available at http://www.electropedia.org/
4 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.5 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.
5.1 Acetonitrile, HPLC grade.---------------------- Page: 7 ----------------------
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5.2 Methanol, HPLC grade.
5.3 Solvent mixture.
Mix 20 parts of acetonitrile (5.1) and 80 parts of water (20+80, v+v).
5.4 Extraction mixture.
Mix 84 parts of acetonitrile (5.1) and 16 parts of water (84+16, v+v).
5.5 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 (6.18).5.6 Nitrogen, purity of at least 99,9 %.
5.7 Activated charcoal for column chromatography (particle size: 63 µm to 200 µm).
5.8 Aluminium oxide (neutral, for liquid chromatography).5.9 Finely ground/pulverized diatomaceous earth (diatomite, kieselgur), e.g. Celite® 545.
5.10 Siliconization reagent, e.g. Surfasil™ (optional).5.11 Cyclohexane, analytical quality, (optional).
5.12 Preparation of the diluted siliconization reagent, (optional).
Add e.g. 50 ml of a siliconization reagent (5.10) to 950 ml cyclohexane (5.11).
5.13 Formic acid, HPLC quality.
5.14 Ammonia solution, substance concentration c(NH ) = 13,4 mol/l or mass concentration
ρ(NH ) = 250 g/l.5.15 Ammonium acetate (CH CO NH ), LC-MS/MS quality.
3 2 4
5.16 Anti-clogging material, such as washed sea sand, glass beads, or polyethylene beads, (optional).
5.17 Stock solution of T-2 toxin, mass concentration ρ = 100 μg/ml, in acetonitrile.
5.18 Stock solution of HT-2 toxin, ρ = 100 μg/ml, in acetonitrile.5.19 Internal standard solution of [ C ]-T-2 toxin, ρ = 25 μg/ml, in acetonitrile.
Other suitable isotopic labelled standards of T-2 toxin than the [ C ]-T-2 toxin may be used.
5.20 Internal standard solution of [ C ]-HT-2 toxin, ρ = 25 μg/ml, in acetonitrile.
Other suitable isotopic labelled standards of HT-2 toxin than the [ C ]-HT-2 toxin may be used.
Surfasil™ is a trade name of a product commercially available from various suppliers. This information is given for the
convenience of users of this document 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.
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5.21 Mixed standard solution, ρ = 500 ng/ml.
Pipette 25 µl of each T-2 toxin and HT-2 toxin stock solution (5.17 and 5.18), respectively, into a 5 ml
volumetric flask, and dilute up to the mark with solvent mixture (5.3).This solution can be stored at −18 °C for 12 months.
5.22 Mixed internal standard solution, ρ = 1000 ng/ml
Dilute 200 µl of the internal standard solutions (5.19 and 5.20) with solvent mixture (5.3) in a 5 ml
volumetric flask.This solution can be stored at –18 °C for 6 months.
5.23 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
Mixed
Mass Mixed
Mass internal Solvent
Calibration concentration standard
concentration standard mixture
solution per isotope solution
per analyte solution (5.3)
labelled analyte (5.21)
(5.22)
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
6 Apparatus and equipment
Usual laboratory apparatus and, in particular, the following.
6.1 Laboratory balance, accuracy of 0,01 g.
6.2 Analytical balance, accuracy of 0,1 mg.
6.3 Ultrasonic bath.
6.4 Laboratory shaker for test tubes.
6.5 Manual dispensers, microlitre syringes or microlitre pipettes for 10 µl to 5 ml.
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6.6 Dispenser, suitable for 20 ml.
6.7 250 ml-Erlenmeyer flasks with stoppers, or 250 ml-centrifuge tubes.
6.8 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® vessels.
6.9 Folded filter, pore size 4 µm to 7 µm, diameter 100 mm.6.10 Laboratory centrifuge.
6.11 Cartridges (6 ml), made from polypropylene (PP) and corresponding frits from polyethylene
(PE), or commercially available SPE columns, e.g. CHROMABOND® Carbon/Alox/Celite® , 6 ml,
500 mg.6.12 SPE vacuum/elution station.
6.13 Laboratory shaker, e.g. overhead shaker.
6.14 Laboratory blender, e.g. Ultra Turrax® .
6.15 Test tubes, suitable for a volume up to 10,0 ml.
6.16 Siliconized test tubes (optional).
After thorough cleaning of the test tubes (6.15), fill up to the top with the diluted siliconization reagent
(5.12) 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 (5.11) and acetonitrile (5.1) or
methanol (5.2) 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™, 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 those made from polypropylene, may be used, if formally proved
suitable.6.17 Concentration evaporator workstation, e.g. TurboVap® Zymark , or similar.
6.18 Membrane filters for aqueous solutions (pore size 0,45 µm).
6.19 LC-MS/MS system with the following components:
6.19.1 HPLC pump.
6.19.2 Injection system.
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 document 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.
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6.19.3 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.Columns of different dimensions may also be used.
6.19.4 Column thermostat.
6.19.5 Tandem mass spectrometer (MS/MS).
6.19.6 Data evaluation system.
7 Procedure
7.1 Preparation of the test sample
Grind and homogenize the sample to particle sizes less than 1 mm before analysis.
7.2 Preparation of the solid phase columnMix 42 g of activated charcoal (5.7) with 30 g of neutral Al O (5.8) and 18 g of Celite 545 (5.9) in a glass
2 3vessel (500 ml) and homogenize with a shaker (6.13) for 1 h (ratio 7:5:3 activated charcoal/neutral
Al O /Celite 545; m/m/m). Place the homogenized mixture, 0,5 g respectively, in empty 6 ml cartridges
2 3provided 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]7.3 Extraction of T-2 toxin and HT-2 toxin
Weigh 25,0 g of the homogenized and finely ground sample (7.1) with an accuracy of 0,1 g into a 250 ml
beaker/Erlenmeyer flask, or into a 250 ml centrifuge tube (6.7), add 100 ml of the extraction mixture
(5.4) and close the vessel. Shake the mixture manually or with a shaker (6.13) for 1 h at room
temperature.Alternatively, use a laboratory blender (6.14) 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 (6.9) 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 (5.16) as the sample
weight.Take volume and/or weight adjustments into account in the final calculation.
7.4 Clean-up by solid phase filtration
Plug the prepared column containing 0,5 g of activated charcoal/Al O /Celite (7.2) on the SPE station
2 3(6.12), and place a test tube (6.15) beneath to collect the eluate. Pass 5,0 ml of the extract (7.3) 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 two times with 5 ml of extraction mixture (5.4), and collect the
eluates also in the same test tube.---------------------- Page: 11 ----------------------
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Add 25 µl of mixed internal standard solution (5.22) to the combined eluates, and evaporate to dryness
with nitrogen (5.6) 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 (5.3) by shaking turbulently (6.4) for 60 s, and, if
necessary, apply an ultrasonic bath (6.3) for 5 min at room tem...
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