Food analysis - Determination of acrylamide in food by liquid chromatography tandem mass spectrometry (LC-ESI-MS/MS)

This European Standard specifies a method for the determination of acrylamide in bakery ware such as bread, toasted bread, crisp bread, butter cookies, and biscuits, as well as potato products such as potato chips, potato crisps, and potato pan cake and roasted coffee, by liquid chromatography in combination with electrospray ionization and tandem mass spectrometry (LC-ESI-MS/MS). This method has been validated in an interlaboratory study via the analysis of both naturally contaminated and spiked samples, ranging from 14,3 µg/kg to 9 083 µg/kg. It was developed at the Swedish National Food Administration and validated in a study organized by the Directorate General Joint Research Centre (DG JRC), Swedish National Food Administration and the Nordic Committee on Food Analysis (NMKL), see [1] and [2].
The limit of quantification (LOQ) depends on the type of instrument used and on the actual performance of the instrument. The majority of the laboratories participating in the validation study were able to determine acrylamide in a butter cookie sample at a level of 14,3 µg/kg. Thus, the validation by interlaboratory study showed that LOQ can be expected to be in the range between below 15 µg/kg and 30 µg/kg.

Lebensmittelanalytik - Bestimmung von Acrylamid in Lebensmitteln mit Flüssigchromatographie und Tandem-Massenspektrometrie (LC-ESI-MS/MS)

Diese Europäische Norm legt ein Verfahren zur Bestimmung von Acrylamid in Backwaren, wie z. B. Brot, getoastetem (geröstetem) Brot, Knäckebrot, Butterkeksen und Keksen, sowie in Kartoffelprodukten, wie z. B. Pommes frites, Kartoffelchips und Kartoffelpuffern, und Röstkaffee durch Flüssigchromatographie in Kombination mit Elektrospray-Ionisation und Tandem-Massenspektrometrie (LC-ESI-MS/MS) fest. Dieses Verfahren wurde in einem Ringversuch mit sowohl natürlich kontaminierten als auch aufgestockten Proben im Bereich von 14,3 µg/kg bis 9 083 µg/kg validiert. Es wurde von der Schwedischen Behörde für Lebensmittelsicherheit (Swedish National Food Administration) entwickelt und in einer Studie validiert, die von der Generaldirektion Gemeinsame Forschungsstelle (Directorate General Joint Research Centre, DG JRC), der Schwedischen Behörde für Lebensmittelsicherheit und dem Nordischen Komitee für Lebensmittelanalytik (Nordic Committee on Food Analysis, NMKL) organisiert wurde, siehe [1] und [2].
Die Bestimmungsgrenze (en: LOQ, limit of quantification) hängt von der Art des eingesetzten Gerätes und dessen tatsächlichem Leistungsvermögen ab. Die Mehrheit der an der Validierungsstudie teilnehmenden Laboratorien war in der Lage, Acrylamid in einer Butterkeksprobe bei einer Konzentration von 14,3 µg/kg zu bestimmen. Die Validierung durch den Ringversuch zeigte daher, dass die Bestimmungsgrenze im Bereich von weniger als 15 µg/kg bis 30 µg/kg erwartet werden kann.

Analyse des produits alimentaires - Dosage de l'acrylamide dans les produits alimentaires par chromatographie en phase liquide couplée à la spectrométrie de masse en tandem (CL-ESI-SM-SM)

Le présent document spécifie une méthode de dosage, par chromatographie en phase liquide en mode ionisation par électrovaporisation couplée à la spectrométrie de masse en tandem (CL-ESI-SM/SM), de l’acrylamide dans les produits de boulangerie tels que le pain, le pain grillé, le pain suédois, les biscuits au beurre et les biscuits épicés, ainsi que dans les produits à base de pomme de terre tels que les frites, les chips et les galettes de pomme de terre et le café torréfié. Cette méthode a été validée lors d’un essai interlaboratoires impliquant l’analyse d’échantillons naturellement contaminés et d’échantillons enrichis, sur une plage de 14,3 µg/kg à 9 083 µg/kg. Elle a été développée au sein de l’Agence suédoise des aliments et validée lors d’un essai organisé par le Centre commun de recherche de la Direction générale (DG JRC), l’Agence suédoise des aliments et le Comité nordique sur l’analyse des aliments (NMKL), voir [1] et [2].La limite de quantification (LOQ) dépend du type d’instrument utilisé et de la performance réelle de l’instrument. La majorité des laboratoires participant à l’essai de validation a pu doser l’acrylamide dans un échantillon de biscuit au beurre à un niveau de 14,3 µg/kg. La validation effectuée lors de l’essai interlaboratoires a montré que ce niveau devait se situer entre une valeur inférieure à 15 µg/kg et 30 µg/kg.

Analize živil - Določevanje akrilamida v živilih s tekočinsko kromatografijo s tandemsko masno spektrometrijo (LC-ESI-MS-MS)

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2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.Lebensmittelanalytik - Bestimmung von Acrylamid in Lebensmitteln mit Flüssigkeitschromatographie und Tandem-Massenspektrometrie (LC-ESI-MS/MS)Analyse des produits alimentaires - Dosage de l'acrylamide dans les produits alimentaires par chromatographie en phase liquide couplée à la spectrométrie de masse en tandem (CL-ESI-SM-SM)Food analysis - Determination of acrylamide in food by liquid chromatography tandem mass spectrometry (LC-ESI-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 16618:2015SIST EN 16618:2015en,fr,de01-julij-2015SIST EN 16618:2015SLOVENSKI

STANDARD
SIST EN 16618:2015
EUROPEAN STANDARD NORME EUROPÉENNE EUROPÄISCHE NORM
EN 16618
April 2015 ICS 67.050; 67.060; 67.080.20; 67.140.20 English Version

Food analysis - Determination of acrylamide in food by liquid chromatography tandem mass spectrometry (LC-ESI-MS/MS)

Analyse des produits alimentaires - Dosage de l'acrylamide dans les produits alimentaires par chromatographie en phase liquide couplée à la spectrométrie de masse en tandem (CL-ESI-SM-SM)

Lebensmittelanalytik - Bestimmung von Acrylamid in Lebensmitteln mit Flüssigchromatographie und Tandem-Massenspektrometrie (LC-ESI-MS/MS) This European Standard was approved by CEN on 7 February 2015.

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. Up-to-date lists and bibliographical references concerning such national standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN member.

This European Standard exists 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, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and United Kingdom.

EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre:
Avenue Marnix 17,

B-1000 Brussels © 2015 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN 16618:2015 ESIST EN 16618:2015

EN 16618:2015 (E) 2 Contents Page Foreword ..............................................................................................................................................................3 1 Scope ......................................................................................................................................................4 2 Normative references ............................................................................................................................4 3 Principle ..................................................................................................................................................4 4 Reagents .................................................................................................................................................4 5 Apparatus ...............................................................................................................................................7 6 Sample preparation ...............................................................................................................................8 7 Measurement ....................................................................................................................................... 10 8 Determination of concentrations ...................................................................................................... 13 9 Precision .............................................................................................................................................. 15 10 Test report ........................................................................................................................................... 17 Annex A (informative)

Typical chromatograms ............................................................................................ 18 Annex B (informative)

Precision data ............................................................................................................. 20 Bibliography ..................................................................................................................................................... 24

SIST EN 16618:2015

EN 16618:2015 (E) 3 Foreword This document (EN 16618:2015) 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 October 2015 and conflicting national standards shall be withdrawn at the latest by October 2015. Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. CEN [and/or CENELEC] 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. WARNING — 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. 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, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom. SIST EN 16618:2015

EN 16618:2015 (E) 4 1 Scope This European Standard specifies a method for the determination of acrylamide in bakery ware such as bread, toasted bread, crisp bread, butter cookies, and biscuits, as well as potato products such as potato chips, potato crisps, and potato pan cake and roasted coffee, by liquid chromatography in combination with electrospray ionization and tandem mass spectrometry (LC-ESI-MS/MS). This method has been validated in an interlaboratory study via the analysis of both naturally contaminated and spiked samples, ranging from 14,3 µg/kg to 9 083 µg/kg. It was developed at the Swedish National Food Administration and validated in a study organized by the Directorate General Joint Research Centre (DG JRC), Swedish National Food Administration and the Nordic Committee on Food Analysis (NMKL), see [1] and [2]. The limit of quantification (LOQ) depends on the type of instrument used and on the actual performance of the instrument. The majority of the laboratories participating in the validation study were able to determine acrylamide in a butter cookie sample at a level of 14,3 µg/kg. Thus, the validation by interlaboratory study showed that LOQ can be expected to be in the range between below 15 µg/kg and 30 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 1042:1999, Laboratory glassware - One-mark volumetric flasks (ISO 1042:1998) EN ISO 3696:1995, Water for analytical laboratory use - Specification and test methods (ISO 3696:1987) 3 Principle Acrylamide is extracted with water and isotopic labelled acrylamide is added. The extract is centrifuged and the supernatant is cleaned up with two solid phase extraction (SPE) columns. The first SPE column contains silica based C18 groups as well as anion and cation exchangers, and since acrylamide is not retained by the column, the extract is just passed and collected. The reason for using this column is to retain as many matrix components as possible (non-polar compounds as well as anions and cations) without retaining acrylamide, i.e. this first SPE column is used as a chemical filter. The second SPE column contains a polymer based phase with a relatively high capacity to bind acrylamide. The extract is loaded onto the column, the column is washed with water and finally eluted with a mixture of 60 parts per volume of methanol and 40 parts per volume of water. The purpose of this step, apart from further cleaning of the extract, is to concentrate the extract and to obtain low limits of quantification. After evaporation of the methanol, the extract is analysed by LC-MS/MS. For this purpose an HPLC column with graphitized carbon as stationary phase is used, since the retention factor (k) is relatively high (k = 4 when no organic solvent is added in the mobile phase) compared to other commercially available columns. 4 Reagents Use only reagents of recognized analytical grade and water complying with grade 1 of EN ISO 3696:1995, unless otherwise specified. Solvents shall be of quality for HPLC analysis. 4.1 Acrylamide (CAS 79-06-1), purity not less than 99,9 % mass fraction. SIST EN 16618:2015

EN 16618:2015 (E) 5 The chemical structure is:

Figure 1 — Acrylamide WARNING — Acrylamide has been classified by the International Agency for Research on Cancer (IARC) as probably carcinogenic to humans. Protective equipment as laboratory coat, disposable gloves and safety glasses shall be used. All handlings of acrylamide and organic solvents shall be performed in a fume cupboard with adequate air flow. 4.2 Deuterium-labelled acrylamide – acrylamide-2,3,3-D3 (CAS 122775-19-3). The chemical structure is:

Figure 2 — Deuterium-labelled acrylamide Alternatively, 13C-labelled acrylamide (acrylamide-13C3, CAS 287399-26-2) may be used. 4.3 Methanol (CAS 67-56-1). 4.4 Glacial acetic acid (CAS 64-19-7). 4.5 n-Hexane (CAS 110-54-3). Alternatively, cyclohexane (CAS 110-82-7) may be used. 4.6 Eluent for SPE column 2 (5.2.3) Mix 60 parts per volume of methanol (4.3) with 40 parts per volume of water. 4.7 HPLC mobile phase Mix 1 part per volume of glacial acetic acid (4.4) with 1 000 parts per volume of water. 4.8 Stock solutions of acrylamide and acrylamide-2,3,3-D3, mass concentration

= 1 000

Weigh, to the nearest 0,05 mg, approximately 100 mg of acrylamide and acrylamide-2,3,3-D3 respectively into separate 100 ml volumetric flasks, dissolve in water and dilute to 100 ml. Solutions can be stored at 4 °C for at least 3 months. 4.9 Internal standard solution 1,

= 10

Transfer 1 000 -2,3,3-D3 (4.8) to a 100 ml volumetric flask and dilute to the mark with water. SIST EN 16618:2015

EN 16618:2015 (E) 6 4.10 Internal standard solution 2,
= 1 000 ng/ml. Transfer 5 000

internal standard solution 1 (4.9) to a 50 ml volumetric flask and dilute to the mark with water. 4.11 Acrylamide standard solution 1,

= 100

Transfer 5 000 4.8) to a 50 ml volumetric flask and dilute to the mark with water. 4.12 Acrylamide standard solution 2,

= 10
Transfer 5 000

1 (4.11) to a 50 ml volumetric flask and dilute to the mark with water. 4.13 Acrylamide standard solution 3,

= 100 ng/ml. Transfer 1 000

2 (4.12) to a 100 ml volumetric flask and dilute to the mark with water. 4.14 LC-MS calibration solutions Dilute aliquots from standard solutions (4.9), (4.11), (4.12) and (4.13) with water to give calibration solutions of e.g. 0 ng/ml, 5 ng/ml, 10 ng/ml, 20 ng/ml, 50 ng/ml, 100 ng/ml, 250 ng/ml, 500 ng/ml, 1 000 ng/ml, 2 000 ng/ml, 5 000 ng/ml and 10 000 ng/ml respectively of acrylamide, all containing 400 ng/ml of acrylamide-2,3,3-D3. Examples for the preparation of calibration solutions are given in Table 1. Table 2 indicates the relation between calibration solution concentrations and acrylamide contents of food samples. Calibration shall be performed on at least six concentration levels distributed properly over the working range. The analysis of an even higher number of calibration solutions should be analysed if such a broad range of concentrations (0

000

Table 1 — Preparation of LC-MS calibration solutions Calibration solution ng/ml Volumetric flask ml Internal standard solution (4.9)

Acrylamide standard solution

0 100 4 000 0 5 100 4 000 5 000 of (4.13) 10 100 4 000 10 000 of (4.13) 20 100 4 000 200 of (4.12) 50 100 4 000 500 of (4.12) 100 100 4 000 1 000 of (4.12) 250 100 4 000 2 500 of (4.12) 500 100 4 000 5 000 of (4.12) 1 000 100 4 000 1 000 of (4.11) 2 000 100 4 000 2 000 of (4.11) 5 000 100 4 000 5 000 of (4.11) 10 000 50 4 000 5 000 of (4.11) SIST EN 16618:2015

EN 16618:2015 (E) 7 Table 2 — Relation between acrylamide contents of calibration solutions and contents in food Calibration solution ng/ml Bakery and potato products

Roasted coffee

10 10 50 5 Apparatus Usual laboratory glassware and equipment and, in particular, the following: 5.1 LC-MS/MS system 5.1.1 HPLC apparatus, comprising the following: 5.1.1.1 Thermostated column compartment. 5.1.1.2 Injection system, capable of injecting 10

5.1.1.3 Mobile phase pump, capable of maintaining a mobile phase flow of 0,4 ml/min. 5.1.2 HPLC column The stationary phase of the column is graphitized carbon1a), particle size 5

mm x 2,1 mm with a guard column1a), particle size 5

mm x 2 mm. Alternative columns/stationary phases may be applied provided that similar performance to the graphitized carbon column can be demonstrated. 5.1.3 Mass spectrometer Triple quadrupole mass spectrometer operating in positive electrospray and, selected reaction monitoring mode (SRM), set to obtain unit resolution. 5.1.4 Data acquisition and analysis system Suitable data collection and evaluation software. 5.1.5 Divert valve (optional) HPLC valve installed between HPLC column and mass spectrometer in order to direct the HPLC effluent either to waste or to the mass spectrometer, see 7.1.1. 5.2 Solid phase extraction system 5.2.1 Vacuum manifold for solid phase extraction

1) a) Hypercarb™ column, Thermo Hypersil-Keystone® column, b) ISOLUTE ® Multimode SPE column and c) ISOLUTE ® ENV+ SPE column from Biotage® are examples of suitable products available commercially. This information is given for the convenience of users of this European Standard and does not constitute an endorsement by CEN of these products. Equivalent products may be used if they can be shown to lead to the same results. SIST EN 16618:2015

EN 16618:2015 (E) 8 5.2.2 SPE column 1 Multimode SPE column comprising non-polar, strong anion exchange, and strong cation exchange properties1b), 1 000 mg/6 ml. 5.2.3 SPE column 2 Crosslinked polystyrene-divinylbenzene copolymer for the extraction of polar analytes from aqueous samples1c), 500 mg/6 ml. 5.3 Analytical balance, accuracy to the nearest 0,01 mg. 5.4 Laboratory balance, accuracy to the nearest 0,01 g. 5.5 Calibrated precision microlitre pipettes, of 200

000
000
000

5.6 Centrifuge tubes, volume of 50 ml, polypropylene, disposable. 5.7 Mechanical shaker, e.g. wrist arm shaker, allowing well mixing of different phases, capable of holding 50 ml centrifuge tubes. 5.8 Vortex mixer. 5.9 Cooled centrifuge, capable of a centrifugal force of 3 600 g for 50 ml centrifuge tubes. 5.10 Volumetric flasks, volume of 50 ml, 100 ml, etc. according to EN ISO 1042:1999. 5.11 Glass vials, volume of at least 4 ml, suitable for the evaporation equipment. 5.12 Amber glass autosampler vials, suitable for the HPLC autosampler. 5.13 Evaporation equipment, based on vacuum or a stream of inert gas. The evaporation temperature shall not exceed 40 °C. 6 Sample preparation 6.1 General Residues of acrylamide have sometimes been found in laboratory ware as e.g. filters. Make sure the laboratory ware does not contain any measureable amounts of acrylamide, and include procedural blank samples as controls in each series of samples. Acrylamide has been found to be formed as an artefact in some analytical procedures for acrylamide, e.g. during extraction or in the injection port of GC instruments. Even if this is not a problem for HPLC analysis, make sure to never exceed 40 °C during extraction or the work-up process. It has been proven that acrylamide is efficiently extracted from various types of food by shaking with water if the particles of the samples are small enough. Make sure that the particles are < 1 mm before extraction and use, if necessary, a mechanical device for preparation of homogenous slurry2).

2) Ultra Turrax® and Waring blender® are examples of suitable products available commercially. This information is given for the convenience of the users of this European Standard and does not constitute an endorsement by CEN of these products. Equivalent products may be used if they can be shown to lead to the same results. SIST EN 16618:2015

EN 16618:2015 (E) 9 Sample extracts can sometimes cause problems by e.g. clogging the SPE columns. The amount of extract loaded on the SPE columns can be reduced provided that the abundances of the peaks of both acrylamide and internal standard are large enough to comply with quantification criteria. NOTE Complementary information regarding sample preparation and chromatographic separation of acrylamide is given by Petersson et al. [3] and Rosén et al. [1]. 6.2 Extraction 6.2.1 Extraction procedure for bakery ware and potato product samples Weigh, to the nearest 0,01 g, a 2,0 g test portion into a 50 ml centrifuge tube (5.6). Add 40 ml of water. Add 400

2, c = 1 000 ng/ml (4.10). Shake intensively for 15 s to 30 s by hand and 10 s to 15 s with a vortex mixer (5.8), and then for 60 min on a mechanical shaker (5.7) adjusted to maximum sample-extractant agitation. Centrifuge in a cooled centrifuge (5.9) at 10 °C, 3 600 x g for 20 min and take off 10 ml of the aqueous phase to a clean test tube. Avoid to transfer parts of the fat layer that will be formed and found on the top, depending of the fat content of the sample. Take care that a homogenous slurry is formed and that the whole sample is in contact with the extractant. If the described procedure is for any reason not sufficient to produce a homogenous slurry, additional mechanical forces shall be applied by e.g. application of a device for preparation of homogenous slurry. 6.2.2 Extraction procedure for coffee samples Weigh, to the nearest 0,01 g, a 2,0 g test portion into a 50 ml centrifuge tube (5.6). Add 5 ml of n-hexane (alternatively cyclohexane). Add 40 ml of water. Add 400

2, c = 1 000 ng/ml (4.10). Shake intensively for 15 s to 30 s by hand and 10 s to 15 s with a vortex mixer (5.8), and then for 60 min on a mechanical shaker (5.7), adjusted to maximum sample-extractant agitation. Centrifuge in a cooled centrifuge (5.9) at 10 °C, 3 600 x g for 20 min. Check for proper phase separation of n-hexane (or cyclohexane), aqueous and solid phase. Remove and discard the organic solvent phase (n-hexane or cyclohexane), and transfer 10 ml of the aqueous phase to a clean test tube. Take care that a homogenous slurry is formed and that the whole sample is in contact with the extractant. If the described procedure is for any reason not sufficient to produce a homogenous slurry, additional mechanical forces shall be applied by, e.g. application of a device for preparation of homogenous slurry. NOTE Cyclohexane is a suitable alternative for n-hexane. 6.3 Cleanup 6.3.1 Cleanup for bakery and potato product sample For all steps adjust the flow of the SPE columns to let the liquid elute drop wise (about 30 drops per min). Check the completeness of elution of acrylamide from the SPE column 2 (5.2.3) by recording the elution profile, at least for each new batch of columns. Fit SPE column 1 (5.2.2) to the vacuum manifold (5.2.1). Condition the column with 3 ml of methanol and 2 times with 6 ml of water. Pass 10 ml of the aqueous extract (6.2.1) through the column and collect the eluate. Fit SPE column 2 (5.2.3) to the vacuum manifold (5.2.1). Condition the column with 5 ml of methanol and 5 ml of water. Load the extract (approximately 10 ml) from the previous column and discard the eluate. Rinse the column once with 4 ml of water and discard the rinsing solvent. Assure that no eluate is left in the valves or flow channels of the vacuum manifold by e.g. placing the column on another (dry) position of the vacuum manifold. Rinsing solvent that is left in the valves could contain co-extracts that could interfere with the internal standard peak. After rinsing, elute acrylamide with 2 ml of 60 % methanol in water (4.6). Collect the elution SIST EN 16618:2015

EN 16618:2015 (E) 10 solvent and the residual solvent in the column (by applying a slight vacuum or a slight pressure) in a glass vial of at least 4 ml, which is compatible with the evaporation equipment (5.11). Evaporate the methanol from the extract, never exceeding 40 °C. This can be achieved by means of e.g. a vortex evaporator under vacuum for approximately 30 min at 40 °C, or by a gentle stream of inert gas, heating the glass vial to a maximum of 40 °C. Do not reduce the volume to less than approximately 500 Transfer the sample to a suitable autosampler vial and perform LC-MS/MS analysis. 6.3.2 Cleanup for coffee samples For all steps adjust the flow of the SPE columns to let the liquid elute drop wise (about 30 drops per min). Check the completeness of elution of acrylamide from the SPE column 2 (5.2.3) by recording the elution profile, at least for each new batch of columns. Fit SPE column 1 (5.2.2) to the vacuum manifold (5.2.1). Condition the column with 3 ml of methanol and 2 times with 6 ml of water. Pass 2 ml of the aqueous phase (6.2.2) through the column, followed by 3 ml of water, and collect the combined eluate, consequently approximately 5 ml in total. Fit SPE column 2 (5.2.3) to the vacuum manifold (5.2.1). Condition the column with 5 ml of methanol and 5 ml of water. Load the extract (approximately 5 ml) from the previous column and discard the eluate. Rinse the column once with 4 ml of water and discard the rinsing solvent. Assure that no eluate is left in the valves or flow channels of the vacuum manifold by e.g. placing the column on another (dry) position of the vacuum manifold. Rinsing solvent that is left in the valves could contain co-extracts that could interfere with the internal standard peak. After rinsing, elute acrylamide with 2 ml of 60 % methanol in water

(4.6). Collect the elution solvent and the residual solvent in the column (by applying a slight vacuum or a slight pressure) in a glass vial of at least 4 ml, which is compatible with the evaporation equipment (5.11). Evaporate the methanol from the extract, never exceeding 40 °C. This can be achieved by means of e.g. a vortex evaporator under vacuum for approximately 30 min at 40 °C, or by a gentle stream of inert gas, heating the glass vial to a maximum of 40 °C. Do not reduce the volume to less than approximately 500 Transfer the sample to a suitable autosampler vial and perform LC-MS/MS analysis. 7 Measurement 7.1 LC-MS/MS determination 7.1.1 LC-MS/MS conditions For a successful analysis it is of paramount importance that the instrument is in good condition and that all instrumental parameters are optimized. Use the HPLC column (5.1.2) and the mobile phase (4.7) at a flow rate of 400 room temperature (22 °C ± 2 °C). The injection volume is 10 phy columns may be used, provided system suitability provisions (see 7.1.2) are fulfilled. Use electrospray ionization, Selected Reaction Monitoring (SRM) and unit resolution. Optimize all parameters as different temperatures, gas flows, voltages and probe position for the detection of acrylamide at the flow rate of the mobile phase. Optimize collision energy individually for each of the following transitions: m/z 72 > 55, 72 > 54, 72 > 44 and 75 > 58. Use optimal settings of dwell time and inter-channel delay to obtain the best sensitivity, avoiding any crosstalk and to obtain chromatograms with at least 15 data points per channel over the peak. Detect acrylamide and the internal standard with the transitions m/z 72 > 55 and 75 > 58 for quantitative purposes, and m/z 72 > 55, 72 > 54 and 72 > 44 for confirmation of the identity of acrylamide. Depending on the type of LC-MS/MS instrument used, other transitions can be available for peak qualification too. SIST EN 16618:2015

EN 16618:2015 (E) 11 NOTE Definition of selected reaction monitoring: data acquired from one or more specific product ions corresponding to m/z selected precursor ions recorded via two or more stages of mass spectrometry. The following parameter setting were successfully applied3): Table 3 — LC-MS/MS conditions HPLC parameters HPLC column Hypercarb™ column (50 mm x 2,1 mm) equipped with a Hypercarb™ pre-column (10 mm x 2,1 mm) Column temperature Room temperature (22 °C ± 2 °C) Injection volume 10

Mobile phase 0,1 % acetic acid in water Mobile phase flow 400

Total run time 8 min MS parameters Desolvation gas N2, 600 l/h Desolvation temperature 400 °C Nebulising gas N2, fully open Cone gas N2, 200 l/h Collision gas Argon, 2,3 × 10−3 mbar Ion source temperature 125 °C Capillary voltage 2 kV Cone voltage 20 V Hexapole voltage 10 V Collision energies:

72 > 55 and 75 > 58 9 eV 72 > 44 20 eV 72 > 54 16 eV Dwell time 0,15 s Inter channel delay 0,03 s Inject the extract of each sample preferably twice. Use a run time of 8 min for samples to allow matrix components to elute from the column. If necessary, wash the column with 80 % of acetonitrile in water, as described in 7.1.3, in between the set of samples, or after the end of the batch. The application of a divert valve (optional) is recommended in order to increase instrument stability and times between maintenance interventions. By applying a divert valve, the effluent from the chromatographic column

3) A Quattro Ultima PT from Micromass ® was used for the measurements. This is an example of a suitable product available commercially. This information is given for the convenience of users of this European Standard and does not constitute an endorsement by CEN of these products. Equivalent products may be used if they can be shown to lead to the same results. SIST EN 16618:2015

EN 16618:2015 (E) 12 is directed to the mass spectrometer only between expected retention time of acrylamide minus 0,5 min and expected retention time of acrylamide plus 1,5 min, and signals are recorded only between these two times. 7.1.2 System suitability The response of the LC-MS can vary from day to day or over longer periods. Also the HPLC column can deteriorate after having been used several times, or just once, depending on the number of injections and type of samples analysed. Therefore, the system should be checked prior to each series of analysis in the following way. Equilibrate the column with mobile phase and the mass spectrometer for, e.g. 30 min. Inject at least three times one of the standard solutions to check the response of the LC-MS equipment as well as retention time, peak shape and peak width of the acrylamide and internal standard peaks. The response should be similar as after the optimization. If not, the interface needs to be cleaned and/or the mass spectrometer needs to be re-optimized. The retention time of acrylamide should be on the Hypercarb™ column (5.1.2) above 1,7 min (at a flow of 0,4 ml/min), and the peak width at half height should be below 0,2 min. The retention factor (k) should be above 3,5. Tailing occurs even for fresh columns, but the distance from peak maximum to tailing edge of peak (measured at 10 % height) should not be more than twice the distance from peak maximum to the leading edge of the peak. For better illustration, a typical chromatogram for potato products is shown in Annex A. Columns not fulfilling these requirements could be regenerated by washing (see 7.1.3). If regeneration was not successful, the column shall be exchanged. Inject pure water to check for possible contamination of the system. No traces of acrylamide should be detected. 7.1.3 Regeneration of HPLC column If the performance of the HPLC column (5.1.2) is significantly worse than expected or required (see 7.1.2) it may be restored by regeneration of the column. Regeneration of columns shall be performed according to the recommendations given by the column supplier. For graphitized carbon columns the following procedures are recommended. The column can be flushed with 80 % acetonitrile in water at 0,4 ml/min for 30 min in line with the LC-MS. This may be done routinely after each day or even between each set of injections. Make sure to give time for equilibration with the mobile phase. For more severe cases, the following washing procedure, preferably performed off-line the LC-MS, can be used. Dispose of the guard column. Change the flow direction and flush the column at room temperature at 0,2 ml/min in a consecutive order for: a) two h with a mixture of 50 % tetrahydrofuran (THF), 10 % ammonia and 40 % water. Consider that some polymers used for HPLC tubings are not resistant against THF; b) 30 min with pure methano

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