EN 15662:2008

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.4X(&K(56Pflanzliche Lebensmittel - Bestimmung von Pestizidrückständen mit GC-MS und/oder LC-MS/MS nach Acetonitril-Extraktion/Verteilung und Reinigung mit dispersiver SPE - QuEChERS-VerfahrenAliments d'origine végétale - Méthode polyvalente de détermination des résidus des pesticides par CG-SM et CL/SM(/SM) avec extraction/partition avec de l'acétonitrile et nettoyage par SPE dispersée - Méthode QuEChERSFoods of plant origin - Determination of pesticide residues using GC-MS and/or LC-MS/MS following acetonitrile extraction/partitioning and clean-up by dispersive SPE - QuEChERS-method67.050Splošne preskusne in analizne metode za živilske proizvodeGeneral methods of tests and analysis for food productsICS:Ta slovenski standard je istoveten z:EN 15662:2008SIST EN 15662:2009en,fr,de01-januar-2009SIST EN 15662:2009SLOVENSKI
STANDARD
EUROPEAN STANDARDNORME EUROPÉENNEEUROPÄISCHE NORMEN 15662November 2008ICS 67.050 English VersionFoods of plant origin - Determination of pesticide residues usingGC-MS and/or LC-MS/MS following acetonitrileextraction/partitioning and clean-up by dispersive SPE -QuEChERS-methodAliments d'origine végétale - Méthode polyvalente dedétermination des résidus des pesticides par CG-SM etSL/SM(SM) avec extraction/partition avec de l'acétonitrile etnettoyage par SPE dispersés - Méthode QuEchERSPflanzliche Lebensmittel - Bestimmung vonPestizidrückständen mit GC-MS und/oder LC-MS/MS nachAcetonitril-Extraktion/Verteilung und Reinigung mitdispersiver SPE - QuEChERS-VerfahrenThis European Standard was approved by CEN on 13 September 2008.CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this EuropeanStandard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such nationalstandards may be obtained on application to the CEN 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 translationunder the responsibility of a CEN member into its own language and notified to the CEN Management Centre has the same status as theofficial versions.CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Cyprus, Czech Republic, Denmark, Estonia, Finland,France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal,Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.EUROPEAN COMMITTEE FOR STANDARDIZATIONCOMITÉ EUROPÉEN DE NORMALISATIONEUROPÄISCHES KOMITEE FÜR NORMUNGManagement Centre: rue de Stassart, 36
B-1050 Brussels© 2008 CENAll rights of exploitation in any form and by any means reservedworldwide for CEN national Members.Ref. No. EN 15662:2008: ESIST EN 15662:2009

Examples of experimental conditions.22 Annex B (informative)
Precision data.25 Annex C (informative)
Procedure schematically (for 10 g sample).75 Annex D (informative)
Complementary information.76 Bibliography.81
1 Scope This European Standard describes a method for the analysis of pesticide residues in foods of plant origin, such as fruits (including dried fruits), vegetables, cereals and processed products thereof. The method has been collaboratively studied on a large number of commodity/pesticide combinations. 2 Principle The homogeneous sample is extracted with the help of acetonitrile. Samples with low water content (< 80 %) require the addition of water before the initial extraction to get a total of approximately 10 g of water. After addition of magnesium sulfate, sodium chloride and buffering citrate salts, the mixture is shaken intensively and centrifuged for phase separation. An aliquot of the organic phase is cleaned-up by dispersive solid phase extraction (D-SPE) employing bulk sorbents as well as magnesium sulfate for the removal of residual water. Following clean-up with amino-sorbents (e.g. primary secondary amin sorbent, PSA) extracts are acidified by adding a small amount of formic acid, to improve the storage stability of certain base-sensitive pesticides. The final extract can be directly employed for GC- and LC-based determinative analysis. Quantification is performed using an internal standard, which is added to the extract after the initial addition of acetonitrile. A brief overview of the method is shown in the flowchart in Annex C. 3 Reagents 3.1 General and safety aspects Unless otherwise specified, use reagents of recognized analytical grade. Take every precaution to avoid possible contamination of water, solvents, sorbents, inorganic salts, etc.
DISCLAIMER — This standard refers to several trade names products and instruments which are commercially available and suitable for the described procedure. 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 equivalent results. 3.2 Water, HPLC quality 3.3 Acetonitrile, HPLC quality 3.4 Methanol, HPLC quality 3.5 Ammonium formate
3.6 Magnesium sulfate, anhydrous, grit, e.g. Fluka No. 63135
Phthalates may be removed in a muffle furnace by heating to 550 °C (e.g. overnight).
3.7 Magnesium sulfate, anhydrous, fine powder
Phthalates may be removed in a muffle furnace by heating to 550 °C (e.g. overnight). 3.8 Sodium chloride 3.9 Disodium hydrogencitrate sesquihydrate SIST EN 15662:2009

3.11 Sodium hydroxide solution, substance concentration c = 5 mol/l Dissolve 2 g of sodium hydroxide in approximately 5 ml of water and dilute to 10 ml. 3.12 Buffer-salt-mixture for second extraction and partitioning:
Weigh 4 g ± 0,2 g of magnesium sulfate anhydrous (3.6), 1 g ± 0,05 g of sodium chloride, 1 g ± 0,05 g of trisodium citrate dihydrate and 0,5 g ± 0,03 g of disodium hydrogencitrate sesquihydrate into a cup (4.11). These amounts refer to approximately 10 ml water in the sample. For highly acidic samples (with pH < 3) the pH-value achieved after the addition of buffering salts is usually below 5. To better protect acid labile compounds the pH-value can be elevated by adding 5 mol/l sodium hydroxide solution (3.11): For lemons, limes and currants add 600 µl and for raspberry 200 µl of sodium hydroxide solution directly to the salt mixture.
NOTE It is advisable to prepare a sufficient number of buffer-salt-mixtures in advance so that extraction series can be performed quickly without interruption. The preparation of the salt mixtures can be enormously facilitated using a sample divider (4.12). The amounts of salts given above are to be used for sample portions containing approximately 10 g water. 3.13 Formic acid solution in acetonitrile, volume fraction 3 = 5 ml formic acid/100 ml
Dilute 0,5 ml of formic acid (mass fraction w = > 95 %) to 10 ml with acetonitrile (3.3).
3.14 Primary secondary amin sorbent For example, Bondesil-PSA® 40 µm Varian No. 122130231). Other amino sorbents may be used, but investigations may be necessary to prove equivalency especially regarding analyte losses and pH value of the end extracts.
3.15 Graphitised Carbon Black sorbent (GCB), e.g. Supelco Supelclean Envi-Carb® 1) SPE Bulk Packing, No. 57210U Other graphitised carbon sorbents may be used, but investigations will be necessary to prove equivalency especially regarding analyte losses.
3.16 Sorption mixture 1: GCB (3.15)/ magnesium sulfate anhydrous fine powder (3.7)-mixture, 1 + 59 mass portions
Mix the two components intensively to form a visually homogeneous mixture.
3.17 Sorption mixture 2: GCB (3.15)/ magnesium sulfate anhydrous fine powder (3.7)-mixture, 1 + 19 mass portions
Mix the two components intensively to form a visually homogeneous mixture. NOTE It is highly advisable to prepare the sorption mixtures 1 (3.16) and 2 (3.17) in advance and store them in sealable vessels. For the extract clean-up according to 5.4.3 the pre-mixed sorption mixtures 1 or 2 are weighed into the centrifuge tubes (4.4).
1) Bondesil-PSA® is a product supplied by Varian, Inc. (Palo Alto, CA, USA). Envi-Carb is a product supplied by Supelco. 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 15662:2009

= 10 µg/ml to 50 µg/ml Table 1 shows a list of potential internal standards (ISTDs) and quality control (QC) standards that may be used in this method. The suggested concentration values (CISTD) listed refers to the ISTD solutions that should be added at the first extraction step (5.2). An appropriate dilution of this solution (mixcalISTDC) should be prepared to be used for the preparation of the standard solutions. For more details see 3.22. Table 1 — Potential internal standards (ISTDs) or quality control (QC) standards GC LC Name of the compound Log P (octanol-water partition coefficient) Chlorine atoms Sugges-ted concen-trationCISTD [µg/ml]a ECDNPDMSD EI (+) MSD
CI (-) MS/MS
ESI (+) MS/MS ESI (-) Potential Internal Standards PCB 18
5,55 3 50 +++ - ++ +++ - - PCB 28
5,62 3 50 +++ - ++ +++ - - PCB 52 6,09 4 50 +++ - ++ +++ - - Triphenyl phosphate 4,59 - 20 - +++ +++ - +++ - Tris-(1,3-dichlorisopropyl)-phosphate
3,65 6 50 +++ +++ +++ +++ +++ + Triphenylmethane 5,37 - 10 - - +++ - - - Bis-nitrophenyl urea (nicarbazin) 3,76 - 10 - - - - - +++ Potential Quality Control Standards (may be contained in the same mixture as the other ISTDs used or added at a different stage of analysis to detect and localize sources of error) PCB 138b 6,83 6 50 +++ - ++ +++ - - PCB 153b 7,75 6 50 +++ - ++ +++ - - Anthracene (or its d10 analogue)c 4,45 - 100 - - ++ - - - a
Exemplary concentrations of the ISTD solutions to be added to the test samples in 5.2, use acetonitrile as solvent. b
Recoveries of PCB 138 and 153 drop as lipid amount in the sample increases, recoveries of those two compounds exceeding 70 % indicate that no unacceptable partitioning losses have occurred even for the most lipophilic pesticides. c
Recoveries of anthracene exceeding 70 % will indicate that no unacceptable losses of pesticides with high carbon affinity have occurred during dispersive SPE with GCB.
3.20 Pesticide stock solutions Prepare individual stock solutions of analytical standards at concentrations that are sufficient to allow the preparation of complex pesticide working solutions (3.21) that are used for the preparation of standard solutions.
Usually, store stock solutions at ≤ -18 °C. Check the stability of stock solutions during storage regularly [2]. In some cases the addition of acids or bases can be helpful to enhance stability and extend the acceptable SIST EN 15662:2009

The volume of the ISTD solution to be employed (mixcalISTDV) will depend on the volume of the standard solution to be prepared (mixcalV) and should be such to ensure an ISTD concentration similar to that in the sample test solutions (5.3, 5.4).
EXAMPLE If 1 ml solvent-based standard is prepared the volume of ISTD solution to be added should contain a mass of ISTD (mixcalISTDmixcalISTDmixcalISTDVxCm=) which is 10-fold smaller than the mass of ISTD added to the test portions in 5.2.3, where 10 ml of acetonitrile are used for extraction. It is thus indicated to appropriately dilute the concentration of internal standard solution (in this case mixcalISTDC = 0,1 × C ISTD). Then the same pipette volume can be used to add ISTDs to spike test samples and for the preparation of standard solutions. Table 2 shows exemplarily the ratio of the ISTD mass that should be added to the test portions (5.2.3) and the standard solutions (3.22). The preparation of multiple standard solutions covering a broad concentration range will allow the construction of a calibration curve (see 6.2).
NOTE A pesticide concentration of 1 µg/ml correlates to a residue level of 1 mg/kg when a 10 g sample is employed (e.g. samples with water content > 30 %) or 2 mg/kg when 5 g sample is employed (e.g. cereals). 3.22.2 Matrix-matched standards Prepare matrix-matched standards in the same way as solvent-based standards, however, instead of pure acetonitrile use extracts of blank samples (prepared as described in 5.1 to 5.4, but without ISTD addition). To minimize errors caused by matrix induced effects during chromatography, it is best to choose similar commodities (e.g. apple for apple samples, carrot for carrot samples, etc.). Should the dilution of the blank sample extract upon addition of the pesticide working solutions exceed 20 %, a volume adjustment may be necessary to avoid errors caused by differences in the matrix-induced enhancement effect between sample extract and matrix-matched standard.
The stability of pesticides in matrix-matched standards can be lower than that of standards in pure acetonitrile and has to be checked more thoroughly. SIST EN 15662:2009

Volume of standard solution V cal mix ml mixcalISTDsampleISTDmm = calmixISTDmix calISTD sampleISTDISTDx V C x V C 1 10 2 5 5 2 10 1 NOTE The values given in this table refer to sample extract volumes of ca. 10 ml (i.e. following addition of 10 ml acetonitrile in 5.2.3). The blank sample employed to prepare the matrix-matched standard should be extracted in the same way as the sample.
3.23 Cold water (< 4 °C) 3.24 Dry ice
3.25 Mobile phase A1: Ammonium formate solution in water, c = 5 mmol/l
3.26 Mobile phase B1: Ammonium formate solution in methanol, c = 5 mmol/l
3.27 Mobile phase A2: Acetic acid solution in water, 3 = 0,1 ml glacial acetic acid /l 3.28 Mobile phase B2: Acetic acid solution in acetonitrile, 3= 0,1 ml glacial acetic acid /l 3.29 Mobile phase A3: Methanol/water 2+8 (V/V) with 5 mmol/l ammonium formate 3.30 Mobile phase B3: Methanol/water 9+1 (V/V) with 5 mmol/l ammonium formate
4 Apparatus Usual laboratory apparatus and, in particular, the following: 4.1 Sample processing equipment, e. g. Stephan UM 5 universal
4.2 High speed dispersing device
Diameter of the dispersing elements should fit the openings of the centrifuge tubes (4.4) used. 4.3 Automatic pipettes, suitable for handling volumes of 10 µl to 100 µl, 200
µl to 1 000 µl and 1 ml to 10 ml.
NOTE Instead of the latter, 10 ml graduated glass pipettes may be used alternatively.
4.4 Centrifuge tubes with screw caps, 50 ml
EXAMPLES a) 50 ml centrifuge tubes made of poly-tetrafluoroethylene with screw caps, or
b) disposable 50 ml polypropylene centrifuge tubes with screw caps SIST EN 15662:2009

4.9 Injection vials, 1,5 ml, suitable for GC and LC autosampler, if necessary with micro-inserts
4.10 Screw capped glass vials, e.g. 20 ml, for the storage of excessive amounts of the final extract, if necessary 4.11 Plastic cups (stackable), 25 ml, used for the storage of buffer-salt mixture portions (3.12). 4.12 Sample divider, to automatically portion salts and sorbents For example from Retsch/Haan, PT 100 or Fritsch/Idar-Oberstein, Laborette 27 or Bürkle/Lörrach, Repro high-precision sample divider2). Their use is optional but highly recommended when dealing with high numbers of samples. NOTE The first two are better for portioning the buffer-salt-mixture (3.12) while the Bürkle Repro is designed for smaller amounts of solids and is much more suitable for portioning the PSA (3.14) / magnesium sulfate (3.6) mixture needed for „dispersive SPE” (5.4.2). The 10 ml polypropylene tubes from Simport Canada, 17 mm x 84 mm, article-no. T550-10AT2) (4.5) perfectly fit the Bürkle Repro.
4.13 Vibration device, e.g. Vortex (used for recovery studies)
4.14 LC-MS/MS system equipped with electrospray ionisation (ESI) interface (see Annex A) 4.15 GC-MS system, equipped with appropriate detectors e.g. MS, MS/MS, TOF and with PTV-injector with solvent vent mode (see GC-MS equipment described in Annex A)
2) PT 100, Laborette 27, Repro high-precision sample divider and T550-10AT 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.
A laboratory sample that is wholly or extensively spoiled or degraded should not be analysed. When possible, prepare laboratory samples immediately after arrival and in any event, before any significant physical or chemical changes have taken place. If a laboratory sample cannot be prepared without delay, it should be stored under appropriate conditions to keep it fresh and to avoid deterioration. Generally, laboratory samples should not be stored longer than 3 days before preparation. Dried or similarly processed samples should be analysed within their stated shelf life.
5.1.3 Partly-prepared test sample
For preparation of the partly-prepared test sample take only the portion of the laboratory sample to which the maximum residue level applies. No further plant-parts may be removed.
The reduction of the laboratory sample shall be carried out in such a way that representative portions are obtained (e. g. by sub-division into four and selection of opposite quarters). For samples of small units (e. g. small fruits such as berries, legumes, cereals), the sample must be thoroughly mixed before weighing out the partly-prepared test sample. When the samples are made up of larger units, take wedge-shaped sections (e.g. melons) or cross sections (e. g. cucumbers) that include the skin (outer surface) from each unit [2]. 5.1.4 Test sample
From each partly-prepared test sample, any parts that would cause difficulties with the homogenisation process should be removed. In the case of stone fruits, the stones shall be removed. A record of the plant-parts that have been removed shall be kept. Precautions should be taken to avoid any losses of juice or flesh. This is the test sample. Calculation of the residue shall be based on the mass of the original test sample (including the stones). Where the homogeneity of the test sample is not sufficient or the extraction of residues may be significantly compromised due to large particle sizes, intensive comminution should be performed using appropriate means. This is possible at ambient temperature, if separation of flesh and juice or degradation of target pesticides does not occur to a significant extent. Comminution of samples in a frozen state can significantly reduce losses of chemically labile pesticides and usually results in smaller particle sizes and a higher degree of homogeneity. Cutting the samples coarsely (e. g. 3 cm x 3 cm) with a knife and putting them into the freezer (e. g. -18 °C overnight) prior to comminution facilitates processing. Processing can be also assisted and improved by cryogenic milling (using dry ice or liquid nitrogen) by keeping the temperature below 0 °C. Especially in the case of fruits and vegetables, cryogenic milling is much more effective at homogenising commodities that have tough skins (e.g. tomatoes or grapes) compared to milling at ambient temperature. Given the fact that non-systemic pesticides often predominantly occur on the skin, cryogenic milling significantly reduces sub-sampling variability. When processing test samples at low temperatures, condensation caused by high humidity should be avoided. Residual carbon dioxide should be allowed to sufficiently dissipate so that its contribution to weigh of the sample will be negligible. SIST EN 15662:2009

5.2.2 Water addition For samples having water content below 80 % add sufficient cold water (3.23), leading to a total water content in the tube of approximately 10 g. See Table 3 for typical water content and the amount of water to be added to the corresponding test portions. NOTE The homogenates derived from 5.1.6 do not need additional water. 5.2.3 Solvent and ISTD addition Add 10 ml of acetonitrile and a defined small volume of the ISTD solution (sampleISTDV e.g. 100 µl) containing one or several of the compounds listed in Table 1 at the concentrations exemplary given (CISTD).
5.2.4 Extraction Close the tube and shake vigorously for 1 min. If the sample’s degree of comminution is insufficient or the residues do not readily extract from the matrix, the extraction time may be prolonged (e.g. to 20 min using a mechanical shaker) or assisted by a high-speed disperser (e.g. Ultra-Turrax). The dispersing element is immersed into the sample/acetonitrile mixture and comminution is performed for about 2 min to 5 min at high speed. In either case ensure that no significant degradation of the target pesticides occurs. As the ISTD solution has already been added, no rinsing of the dispersing element is necessary. Nevertheless, it still has to be cleaned thoroughly before being used for the next sample to avoid cross-contamination.
Make sure to employ dispersing elements that can pass through the opening of the centrifuge tubes (4.4).
Samples should be extracted frozen or while in the process of thawing (except dry samples with water content < 20 %). If samples are employed for extraction at ambient temperature, it shall be ensured that no significant degradation of the target pesticides occurs. SIST EN 15662:2009

grapefruit 90
lemon/lime 85
orange 85
orange peel 75 2,5
Citrus fruits tangerine 90
Add 600 µl 5 mol/l NaOH-solution to buffer salts as stated in 3.12 (applies only to lemon/lime). Optionally perform freeze out step to remove waxes; see 5.4.1 (applies to all citrus fruits). apple 85
apple, dried 30
8,5 (see 5.1.6)
apple sauce 80
apple juice 90
pear 85
Pome fruit quince 85
apricot 85
apricot, dried 30
8,5 (see 5.1.6)
apricot nectar 85
cherry 85
mirabelle 80
nectarine 85
peach 90
peach, dried 20
8,5 (see 5.1.6)
plum 85
Stone fruit plum, dried 20
8,5 (see 5.1.6)
blackberry 85
blueberry 85
currant 85
Add 600 µl 5 mol/l NaOH-solution to buffer salts as stated in 3.12. elderberry 80
gooseberry 90
grapes 80
raspberry 85
Add 200 µl 5 mol/l NaOH-solution to buffer salts as stated in 3.12. raisin 20
8,5 (see 5.1.6)
Soft and small fruits strawberry 90
banana 75 2,5
fig, dried 20
8,5 (see 5.1.6)
kiwi 85
mango 80
Use GCB in dispersive SPE as stated in 3.16 and 5.4.3 (mixture 1).
Other fruits papaya 90
Vegetables beetroot 90
carrot 90
Use GCB in dispersive SPE asstated in 3.16 and 5.4.3 (mixture 1). celeriac 90
horseradish 75 2,5
parsley root 90
radish 95
black salsify 80
Root and tuber
vegetables potato 80
garlic 60
7,0
onion 90
leek 85
shallot 80
Leek plants chive 85
Use GCB in dispersive SPE as stated in 3.17 and 5.4.3 (mixture 2). aubergine 90
cucumber 95
melon 90
pepper, sweet 90
For red sweet pepper use GCB in dispersive SPE as stated in 3.17 and 5.4.3 (mixture 2). pumpkin 95
For strongly coloured varieties use GCB in dispersive SPE as stated in 3.16 and 5.4.3 (mixture 1). tomato 95
Fruiting vegetables zucchini (courgette) 95
broccoli 90
brussels sprouts 85
cauliflower 90
chinese cabbage 95
kale 90
kohlrabi 90
Cabbage red cabbage 90
white cabbage 90
lettuce varieties 95
endive 95
For strongly coloured varieties use GCB in dispersive SPE as stated in 3.16 and 5.4.3 (mixture 1). cress 90
lamb’s lettuce 85
parsley 80
rucola 85
Leafy vegetables and herbs spinach 90
Use GCB in dispersive SPE as stated in 3.17 and 5.4.3 (mixture 2). asparagus 95
celery 95
leek 85
rhubarb 95
Stem vegetables artichokes 85
beans, peas, lentils (dried) <10
Legumes
beans (fresh) 75 2.5
Other Cereals cereals
(grain, flour, etc.) 10
10 Optionally perform freeze out step or add 25 mg C18 sorbent per ml extract at the dispersive SPE step to remove lipids (see 5.4.1). coffee beans
<10
tea <10
10 (use 2 g sample if fermented) Add 75 mg PSA sorbent per ml extract at the dispersive SPE step if fermented. dry herbs and spices <10
10 (use 2 g sample if extract-rich)
mushrooms 90
wine 90
5.3 Second extraction step and partitioning Add the prepared buffer-salt mixture (3.12) to the suspension from 5.2. Close the tube and immediately shake vigorously for 1 min and centrifuge for 5 min at > 3 000 g.
NOTE When dealing with highly acidic commodities, such as lemons, limes, currants and raspberries, use buffer-salt mixtures to which NaOH was added as stated in 3.12. In the presence of water, magnesium sulfate tends to form lumps, which can harden rapidly. This can be avoided, if immediately after the addition of the salt mixture the centrifuge tube is shaken vigorously for few seconds. The 1 min extraction of the entire batch may be performed in parallel after the salts have been added to all the samples. Pesticides with acidic groups (e.g. phenoxyalcanoic acids) interact with amino-sorbents such as PSA. Thus, if such pesticides are within the scope of analysis, their determinative analysis (preferably via LC-MS/MS ESI SIST EN 15662:2009

5.4 Clean-up 5.4.1 Removal of co-extracted fat, wax, sugars (e.g. for cereals, citrus fruits)
Transfer an aliquot of 8 ml of the acetonitrile phase (5.3) into a centrifuge tube (4.5) and store overnight in a freezer (for flour, 2 h are sufficient), wherewith the major part of fat and waxes solidify and precipitate. Following a short centrifugation (where necessary), 6 ml of the still cold extract is taken for dispersive SPE according to 5.4.2.
NOTE Freezing out also helps to partly remove some additional sample co-extractives with limited solubility in acetonitrile such as sugars. Co-extracted fat and waxes, which may negatively affect the ruggedness of GC analysis can be also effectively removed using silica-based reversed phase sorbents (ODS-, C18-type). For this, 25 mg ODS together with 25 mg PSA and 150 mg magnesium sulfate per millilitre extract is employed in the dispersive SPE step. Pesticides and the proposed internal and QC-standards (see Table 1) are not affected by these treatments. 5.4.2 Clean-up with amino-sorbent („Dispersive SPE“ with PSA) An aliquot of 6 ml of the acetonitrile phase from 5.3 or 5.4.1 is transferred into a PP-single use centrifuge tube (4.5) already containing 150 mg PSA (3.14) and 900 mg of magnesium sulfate (3.6). Close the tube, shake vigorously for 30 s and centrifuge (for 5 min at > 3000 g). Immediately isolate and acidify the clear extract as described in 5.4.4.
For 1 ml of extract 150 mg magnesium sulfate and 25 mg PSA are necessary.
NOTE It is helpful to load the centrifuge tubes with the dispersive SPE sorbents before beginning the extraction procedure needed for one batch of samples. The use of a sample divider (4.12) substantially facilitates this procedure. 5.4.3 Clean-up with a mixture of amino-sorbent + GCB („Dispersive SPE“ with PSA + GCB)
For samples, with a high content of carotinoides (e.g. red sweet pepper, carrots) or chlorophyll (e.g. spinach, lamb’s lettuce, rucola, curly kale, vine leaves und Lactuca varieties except iceberg lettuce and lettuce hearts), dispersive SPE is performed using a combination of PSA and GCB. Transfer an aliquot of 6 ml of the acetonitrile phase from 5.3 into a centrifuge tube (4.5), which already contains 150 mg PSA (3.14) and for extracts of carrots, Lactuca varieties 900 mg of sorption mixture 1 (3.16), and for extracts of red sweet pepper, spinach, lamb’s lettuce or rucola 900 mg of sorption mixture 2 (3.17). Close the tube, shake vigorously for 2 min and centrifuge (e.g. for 5 min at > 3 000 g). Immediately isolate and acidify the clear extract as described in 5.4.4. For 1 ml extract 25 mg PSA and, depending on the sample, 150 mg sorption mixture 1 or 2 are necessary.
NOTE It is helpful to load the centrifuge tubes with the dispersive SPE sorbents before beginning the extraction procedure needed for one batch of samples. The use of a sample divider (4.12) substantially facilitates this procedure. 5.4.4 Extract stabilisation
Transfer 5 ml aliquot of the cleaned-up extract from 5.4.2 or 5.4.3 into a screw cup storage vial (4.10), taking care to avoid sorbent particles of being carried over, and slightly acidify by adding 50 µl of a 5 % formic acid solution in acetonitrile (3.13). Fill the pH-adjusted extract into auto-sampler vials and use it for gas- and liquid chromatographic analysis. Store the residual extract in a refrigerator to be used if necessary. For 1 ml extract 10 µl of the formic acid solution (3.13) are necessary.
For suitable experimental conditions of LC-MS/MS measurements, see CEN/TR 15641 [5]. For suitable experimental conditions of GC-MS measurement, refer to [3]. Nevertheless, individual tuning of the compounds on the instrument that is used for measurement usually provides better sensitivities.
5.6 Test for interference and recovery Prepare reagent blanks and carry out spiked recovery tests at various appropriate levels. The chromatogram of the reagent blank should not show any significantly interfering peak at the retention time of the analytes. 6 Evaluation of results 6.1 Identification and quantification A number of parameters can be employed to determine the identity of an analyte present in the sample extract. This includes:
1) retention time of the analyte in question (RT) or, even better, the retention time ratio against the ISTD (Rt(A)/Rt(ISTD)) obtained from the same run;
2) peak shape of the analyte; and
3) in case of MS or MS/MS detection, the relative abundance of the recorded masses (in general 2 SRM transitions are required in MS/MS and 3 ions in MS applications).
The parameters obtained for the analyte to be identified in the sample extract are compared with those obtained for the pesticides in the calibration solution(s). Should a higher degree of certainty be required for the confirmation of the analyte identity, additional measures may be necessary, such as the use of different chromatographic separation conditions or the evaluation of additional m/z or SRM-transitions. For more information about the required confirmation criteria refer to the EU-quality control guidelines described in the SANCO/2007/3131 document [1]. Table 1 gives a list of the ISTDs that can be employed. The use of more than one ISTD will provide some backup information.
Use standard solutions (3.22.1 or 3.22.2) to check linearity and to determine the calibration functions for each active substance as described in 6.2. The use of matrix-matched standards is to be preferred, however, for a first estimate of the residue level of pesticides in the food or to show their absence, the standard solutions in pure solvent (3.22.1) can be used. They can be also used for quantification if preliminary experiments indicate that any suppression or enhancement effects experienced do not significantly affect the results obtained. As soon as relevant residue concentrations are detected (e.g. suspected MRL violations), a more precise determination using matrix-matched standards (3.22.2) or the standard addition method (6.3) should be used. NOTE 1 Matrix effects influence the response of target analytes in sample extracts compared to the response of standard solutions in pure solvent.
NOTE 2 The calibration range should be appropriate to the residue concentrations to be quantified. Thus, it may be necessary to construct more than one calibration graph from the results of calibration measurements. This standard prescribes the use of an internal standard for quantification and identification. Nevertheless, it is still possible to quantify without ISTD. Without ISTD, the volume of the acetonitrile phase (5.3) is assumed to be identical to the volume of acetonitrile added to the sample in 5.2.3 (10 ml). SIST EN 15662:2009

In any case it is always crucial to introduce quality control measures to ensure that any error introduced by the ISTD remains insignificant. Quality control measures may include the use of backup ISTDs and quality control standards that may be added at other stages of the analytical procedure (e.g. to the final extract) and that may help to identify any non-volume related shifts of the ISTD signal. Very helpful for quality control is the observation of the signal intensity of the ISTD in every sample within a sequence. Should a significant signal shift occur, quantification should be performed using a backup ISTD or without using ISTD. In the latter case exact liquid transfers and equalisation of the volumes of the standard solutions and the sample extracts is mandatory.
6.2 Calculation of residue concentration without standard addition
Variables used:  Concentration of internal standard in ISTD solution C ISTD µg/ml  Concentration of pesticide in calibration mixture mixcalpestC µg/ml  Concentration of internal standard in calibration mixture mixcalISTDC µg/ml  Concentration of pesticide in the final extract samplepestC µg/ml  Concentration of internal standard in the final extract sampleISTDC µg/ml  Mass of test portion m a g  Volume of ISTD added to the test portion sampleISTDV ml  Peak area of pesticide obtained from calibration mixture mixcalpestA (counts)  Peak area of ISTD obtained from calibration mixture mixcalISTDA (counts)  Peak area of pesticide obtained from the final extract samplepestA (counts)  Peak area of ISTD obtained from the final extract sampleISTDA (counts)  Peak ratio obtained from calibration mixture PR cal mix
(dimensionless) SIST EN 15662:2009

(dimensionless)  Slope of calibration graph a cal
(dimensionless)  Bias of calibration graph b cal
(dimensionless)  Mass fraction of pesticide in the sample w R
mg/kg Furthermore in the text:  Mass of the internal standard(s) to be employed for preparation of standard solution mixcalISTDm  Resulting mass of pesticide added to each vial (for standard addition) addstdpestm  Volume of the standard solution (solvent based or matrix-matched standard) mixcalV  Volume of the internal standard solution to be employed for preparation of standard solution mixcalISTDV  Volume of the pesticide working solutions used to prepare the calibration mixtures mixcalpestV  Added volume of an appropriate dilution of pesticide stock solution (for standard addition) addstdpestV
Determine the calibration functions for each active substance by plotting the peak ratio PR cal mix (mixcalpestA/ mixcalISTDA) of each calibration level against the dimensionless concentration ratio (mixcalpestC/
mixcalISTDC)
of the standard solution. From the corresponding calibration graph described by the following formula: calmixcalISTDmixcalpestcalmixcalbCCaPR+×= (1) each expected concentration ratio (mixcalpestC/
mixcalISTDC) can be calculated as follows:
calcalmixcalmixcalISTDmixc
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