SIST EN 15637:2009
(Main)Foods of plant origin - Determination of pesticide residues using LC-MS/MS following methanol extraction and clean-up using diatomaceous earth
Foods of plant origin - Determination of pesticide residues using LC-MS/MS following methanol extraction and clean-up using diatomaceous earth
This European Standard describes a method for the analysis of pesticide residues in foods of plant origin, such as fruits, vegetables, cereals, nuts as well as processed products including dried fruits. The method has been collaboratively studied on a large number of commodity/pesticide combinations.
Pflanzliche Lebensmittel - LC-MS/MS-Verfahren zur Bestimmung von Pestizidrückständen mit Methanolextraktion und Reinigung an Diatomeenerde
Diese Europäische Norm beschreibt ein Verfahren für die Analyse von Pestizidrückständen in pflanzlichen Lebens¬mitteln, wie Früchten, Gemüse, Getreide, Nüssen, als auch verarbeiteten Erzeugnissen, wie beispiels-weise getrockneten Früchten. Das Verfahren wurde gemeinsam mit einer großen Anzahl an Ernte-güter/Pestizid-Kombinationen überprüft.
Aliments d'origine végétale - Détermination des pesticides par LC-MS/MS après extraction méthanolique et purification sur terre de diatomées
La présente Norme européenne spécifie les exigences auxquelles doivent satisfaire les produits manufacturés en polystyrène expansé, avec ou sans parements ou revêtements, utilisés pour l'isolation thermique des bâtiments. Les produits sont fabriqués sous forme de panneaux ou de rouleaux ou toute autre présentation préformée.
La présente norme européenne décrit les caractéristiques du produit et contient les procédures d’essai, d’évaluation de la conformité, de marquage et d’étiquetage.
Les produits relevant du domaine d'application de la présente norme européenne sont également utilisés pour l’isolation acoustique dans des systèmes d'isolation thermique et des panneaux composites préfabriqués ; la performance des systèmes dans lesquels entrent ces produits n'est pas définie.
La présente norme européenne ne spécifie pas, pour une propriété donnée, le niveau exigé que doit atteindre un produit pour démontrer son aptitude à l’emploi dans une application particulière. Les classes et niveaux requis pour une application donnée figurent dans les réglementations ou normes non conflictuelles.
Les produits dont la résistance thermique déclarée à une température de 10 °C est inférieure à 0,25 m2.K/W ou dont la conductivité thermique déclarée à une température de 10°C est supérieure à 0,060 W/(m.K) ne sont pas du domaine d'application de la présente norme européenne.
Hrana rastlinskega izvora - Določanje ostankov pesticidov z uporabo LC-MS/MS za ekstrakcijo z metanolom in čiščenjem z diatomejsko zemljo
General Information
Standards Content (Sample)
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.Pflanzliche Lebensmittel - LC-MS/MS-Verfahren zur Bestimmung von Pestizidrückständen mit Methanolextraktion und Reinigung an DiatomeenerdeAliments d'origine végétale - Détermination des pesticides par LC-MS/MS après extraction méthanolique et purification sur terre de diatoméesFoods of plant origin - Determination of pesticide residues using LC-MS/MS following methanol extraction and clean-up using diatomaceous earth67.050Splošne preskusne in analizne metode za živilske proizvodeGeneral methods of tests and analysis for food productsICS:Ta slovenski standard je istoveten z:EN 15637:2008SIST EN 15637:2009en,fr,de01-april-2009SIST EN 15637:2009SLOVENSKI
STANDARD
SIST EN 15637:2009
EUROPEAN STANDARDNORME EUROPÉENNEEUROPÄISCHE NORMEN 15637November 2008ICS 67.050 English VersionFoods of plant origin - Determination of pesticide residues usingLC-MS/MS following methanol extraction and clean-up usingdiatomaceous earthAliments d'origine végétale - Détermination des résidus despesticides par LC-MS/MS après extraction méthanolique etpurification sur terre de diatoméesPflanzliche Lebensmittel - LC-MS/MS-Verfahren zurBestimmung von Pestizidrückständen mitMethanolextraktion und Reinigung an DiatomeenerdeThis 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 15637:2008: ESIST EN 15637:2009
EN 15637:2008 (E) 2 Contents Page Foreword.3 1 Scope.4 2 Principle.4 3 Reagents.4 4 Apparatus.6 5 Procedure.7 6 Evaluation of results.12 7 Confirmatory tests.16 8 Precision.16 9 Test report.16 Annex A (informative)
Example for appropriate experimental conditions.17 Annex B (informative)
Precision data.21 Bibliography.63
SIST EN 15637:2009
EN 15637:2008 (E) 3 Foreword This document (EN 15637:2008) 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 May 2009, and conflicting national standards shall be withdrawn at the latest by May 2009. 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. WARNING — The application of this standard may involve hazardous materials, operations and equipment. It does not claim to address all safety issues associated with its use. The user of this standard is responsible for establishing appropriate safety and health practices and determining 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, 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 the United Kingdom. SIST EN 15637:2009
EN 15637:2008 (E) 4
1 Scope This European Standard describes a method for the analysis of pesticide residues in foods of plant origin, such as fruits, vegetables, cereals, nuts as well as processed products including dried fruits. The method has been collaboratively studied on a large number of commodity/pesticide combinations. 2 Principle The sample is extracted with methanol after addition of some water. After partition into dichloromethane the organic phase is evaporated and the residue is reconstituted with methanol. Quantification of pesticide residues is performed by liquid chromatography with tandem mass spectrometric detection, using electrospray ionisation. To achieve the required selectivity the mass spectrometer is operated in the selected reaction monitoring mode (SRM). 3 Reagents 3.1 General and safety considerations Unless otherwise specified, use reagents of recognised analytical grade. Take every precaution to avoid possible contamination of water, solvents, inorganic salts, etc.
3.2 Ammonium formate
3.3 Sodium chloride 3.4 Water, HPLC quality 3.5 Dichloromethane, for residue analysis
3.6 Methanol, HPLC quality 3.7 Internal Standard (ISTD) solutions in methanol,
= 10 µg/ml to 50 µg/ml1)
Table 1 shows a list of potential internal standards that may be used in this method. The concentrations listed refer to the ISTD solutions that should be added at the first extraction step (5.2) and to standard solutions.
Table 1: Potential internal standards (ISTDs) or quality control (QC) standards Name of the compound Log P (octanol-water partion coefficient) Chlorine atoms Concentration C ISTD µg/ml Triphenyl phosphate 4,59 - 20 Tris-(1,3-dichlorisopropyl)-phosphate
3,65 6 50 Bis-nitrophenyl urea (nicarbazin) 3,76 - 10
1)
ρ = mass concentration SIST EN 15637:2009
EN 15637:2008 (E) 5
3.8 Pesticide stock solutions Prepare individual stock solutions of analytical standards at concentrations that are sufficiently high to allow the preparation of complex pesticide mixtures. The solvent used should not negatively influence the stability of the pesticides employed.
NOTE Usually, store stock solutions at ≤ -18 °C. Check the stability of stock solutions during storage regularly. In some cases the addition of acids or bases can be helpful to enhance stability and extend the acceptable storage period. 3.9 Pesticide mixtures Because of the broad applicability of this method and due to the partly divergent pH-stability of pesticides, analyte mixtures of different composition can be needed. These are prepared by mixing together defined volumes of the required analyte stock solutions (3.8) and appropriately diluting them with methanol. The analyte concentrations in this mixture should be sufficient to allow the preparation of the required matrix matched standards (see 3.10.3) with moderate dilution of the blank sample extract (e.g. less than 20 %). Usually, store pesticide mixtures at ≤ -18 °C. Since the stability of the pesticides in the mixture may be lower than in stock solutions, stability has to be checked regularly. In some cases the addition of acids or bases can be helpful to enhance stability and extend acceptable storage times. 3.10 Standard solutions 3.10.1 Standard solutions prepared in pure solvent (solvent-based standards)
Solvent-based standards are prepared by mixing a certain volume of methanol with known amounts of pesticide mixtures (3.9). The preparation of multiple standards of different pesticide concentration is useful to cover a broad concentration range.
NOTE An analyte concentration of 1 µg/ml correlates to a residue level of 0,4 mg/kg when a 10 g sample is employed (e.g. samples with water content > 30 %) or 0,8 mg/kg when a 5 g sample is employed (e.g. cereals). 3.10.2 Standard solutions with internal standard prepared in pure solvent Solvent-based standards with ISTD are prepared by mixing a certain volume of methanol with known amounts of pesticide mixtures (3.9) and a fixed volume of internal standard solution (3.7). The volume used shall result in that concentration of ISTD which is obtained in the final extracts after sample extraction and clean-up (see 5.2 and 5.3). The concentration of internal standard in the final extract (CsampleISTD ) can be calculated using Equation (1). The preparation of multiple standards of different pesticide concentration but with constant ISTD concentration is useful to cover a broad concentration range. endexISTDISTDsampleISTDVVVVVVCVC×××−××=2312)( (1) where: V ISTD is the volume of internal standard solution (3.7) added to the test portion; C ISTD is the concentration of internal standard solution (3.7); V1 is the volume of NaCl solution (2,5 ml); V2 is the volume of measuring flask used in 5.2 (10 ml); SIST EN 15637:2009
EN 15637:2008 (E) 6 V3 is the volume used for solid supported liquid/liquid extraction (5 ml); Vex is the total volume of extraction solvents and natural water (30 ml); Vend is the final volume of extract obtained after clean-up (0,5 ml). NOTE The internal standard may correct for deviations from the correct extraction volume, a wrong estimate of water content of samples, losses of methanol during preparation of the final extract and fluctuations of instrument sensitivity during a batch of measurements. However, validation results in Annex B were obtained without internal standards.
3.10.3 Standard solutions prepared in blank matrix extracts (matrix-matched standards)
Prepare matrix-matched standards in the same way as the solvent-based standards, however, instead of pure methanol use extracts of blank samples (prepared as described in 5.2, 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, carrots for carrot samples, etc.).
The stability of pesticide in matrix-matched standards may be lower than that of standards in pure acetonitrile and has to be checked more thoroughly. 3.11 5 ml cartridge for solid supported liquid/liquid extraction, 5 ml sample volume, diatomaceous earth, for example ChemElut CE 10052)
3.12 20 ml cartridge for solid supported liquid/liquid extraction, 20 ml sample volume, diatomaceous earth, for example ChemElut CE 10202)
4 Apparatus Usual laboratory apparatus and, in particular, the following: 4.1 Carving board and knife, for chopping up food samples for analysis 4.2 Homogenizer or high speed blender, fitted with jar 4.3 Laboratory balance 4.4 Measuring flasks, 10 ml and 20 ml 4.5 Ultrasonic bath 4.6 Centrifuge tubes, 80 ml 4.7 Centrifuge, capable of producing a relative centrifugal force (RCF) of at least 3000 g (at the bottom of the tube) 4.8 Round bottom flasks, 50 ml and 250 ml 4.9 Glass syringe, minimum volume 2 ml
2) ChemElut is a product supplied by Varian, Inc. (Palo Alto, CA, USA). This information is given for the convenience of users of this European Standard and does not constitute an endorsement by CEN of the product named. Equivalent products may be used if they can be shown to lead to the same results. SIST EN 15637:2009
EN 15637:2008 (E) 7 4.10 Microliter syringes, for sample fortification 4.11 Rotary evaporator, with temperature-controlled water bath 4.12 Syringe filters, 0,45 µm pore size, 4 mm diameter, polytetrafluoroethylene (PTFE) membrane 4.13 Glass vials and caps, 1,8 ml volume, suitable for an autosampler 4.14 LC-MS/MS system, triple quadrupole mass spectrometer with electrospray interface 5 Procedure 5.1 Preparation and storage of the samples 5.1.1 General Sample processing and storage procedures should be demonstrated to have no significant effect on the residues present in the test sample (sometimes also called “analytical sample”). Processing should also ensure that the test sample is homogeneous enough so that sub-sampling variability is acceptable. If a single analytical portion is unlikely to be representative of the test sample, larger or replicate portions shall be analysed, to provide a better estimate of the true value. The degree of comminution supports a quantitative residue extraction. 5.1.2 Laboratory sample
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 shall 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 [1]. 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 SIST EN 15637:2009
EN 15637:2008 (E) 8 pesticides does not occur to a significant extent. Comminution of samples in a frozen state can significantly reduce losses of chemically labile analytes and usually results in smaller particle sizes and thus achieve 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. 5.1.5 Test portion
Individual test portions each sufficient for one analysis are taken from the comminuted test sample. These test portions should be analysed immediately. If test portions cannot be analysed directly, the test sample or the test portions shall be frozen until required. If test portions are taken from test samples after being stored frozen, the test samples shall be mixed before taking test portions to ensure that homogeneity has been re-established. 5.2 Extraction Transfer a representative test portion of mA = 10 g into a centrifuge tube (4.6). For dry sample materials like cereal products, weigh a homogenised portion of 5 g (mA) into the centrifuge tube. Add sufficient water, that a total volume (added and natural) of 10 ml water is obtained. For typical water contents of crops and cereals, see Table 2. In the case of dry sample materials wait 10 min after addition of water. Add 20 ml of methanol (3.6) to the mixture and homogenise for 2 min using the high speed blender (4.2). Take at least 10 ml of the resulting extract of 30 ml (= Vex) and centrifuge at approximately 3000 g. Pipette 2,5 ml of NaCl solution (20 %, w/w) (= V1) into a 10 ml measuring flask (= V2) (4.4), fill up to the mark with supernatant of centrifugation and mix. As an option an internal standard can be used additionally. In that case add a small volume (<1 % of Vex) of internal standard solution (=V ISTD) to the test portion after addition of 20 ml of methanol. SIST EN 15637:2009
EN 15637:2008 (E) 9
Table 2 — Water content of selected foods and amount of water, which have to be added Food group Food Typical water content g/100 g Amount of water added to 10 g of test portion g Amount of water added to 5 g of test portion g Fruits Citrus fruits citrus juices 90 1,0
grapefruit 90 1,0
lemon 90 1,0
orange 85 1,5
orange peel 75 2,5
tangerine 90 1,0
Pome fruit apple 85 1,5
apple, dried 30
8,5
apple sauce 80 2,0
apple juice 90 1,0
pear 85 1,5
quince 85 1,5
Stone fruit apricot 85 1,5
apricot, dried 30
8,5
apricot nectar 85 1,5
cherry 85 1,5
mirabelle 80 2,0
nectarine 85 1,5
peach 90 1,0
peach, dried 20
9,0
plum 85 1,5
plum, dried 20
9,0 Soft and small fruits blackberry 85 1,5
blueberry 85 1,5
currant 85 1,5
elderberry 80 2,0
gooseberry 90 1,0
grapes 80 2,0
raspberry 85 1,5
raisin 20
9,0
strawberry 90 1,0
Other fruits pineapple 85 1,5
banana 75 2,5
SIST EN 15637:2009
EN 15637:2008 (E) 10
Table 2 (continued) Food group Food Typical water content g/100 g Amount of water added to 10 g of test portion g Amount of water added to 5 g of test portion g
fig, dried 20
9,0
kiwi 85 1,5
mango 80 2,0
papaya 90 1,0
Vegetables Root and tuber
beetroot 90 1,0
vegetables carrot 90 1,0
celeriac 90 1,0
horseradish 75 2,5
parsley root 90 1,0
radish 95 0,5
scorzonera (black salsify) 80 2,0
shallot 80 2,0
Onions garlic 60
7,0
onion 90 1,0
Fruiting vegetables aubergine 90 1,0
cucumber 95 0,5
melon 90 1,0
pepper, sweet 90 1,0
pumpkin 95 0,5
tomato 95 0,5
zucchini (courgette) 95 0,5
Cabbage broccoli 90 1,0
Brussels sprouts 85 1,5
cauliflower 90 1,0
Chinese cabbage 95 0,5
kale 90 1,0
kohlrabi 90 1,0
red cabbage 90 1,0
savoy cabbage 90 1,0
white cabbage 90 1,0
SIST EN 15637:2009
EN 15637:2008 (E) 11 Table 2 (continued) Food group Food Typical water content g/100 g Amount of water added to 10 g of test portion g Amount of water added to 5 g of test portion g Leafy vegetables and
butterhead lettuce 95 0,5
herbs chive 85 1,5
cress 90 1,0
endive 95 0,5
iceberg lettuce 95 0,5
lamb’s lettuce 85 1,5
parsley 80 2,0
spinach 90 1,0
witloof chicory 95 0,5
Stem vegetables artichokes 85 1,5
asparagus 95 0,5
celery 95 0,5
leek 85 1,5
rhubarb 95 0,5
Beans, peas (fresh) beans 90 1,0
peas with pods 80 2,0
Beans, peas (dried) beans, peas, lentil 10
9,5 Other
beer 90 1,0
cereals (grain, flour, etc.) 10
9,5
coffee (raw) 10
9,5
mushrooms 90 1,0
must (grape) 90 1,0
potato 80 2,0
tea 10
9,5
wine 90 1,0
5.3 Clean-up Apply 5 ml of the diluted centrifugate (= V3) from 5.2 to a 5 ml cartridge (3.11). After 5 minutes, elute into a 50 ml round bottom flask (4.8), using 12,5 ml of dichloromethane (3.5). Repeat the elution with another 12,5 ml of dichloromethane. Reduce the combined eluates almost to dryness using the rotary evaporator (4.11). Remaining dichloromethane should be removed with a stream of nitrogen. SIST EN 15637:2009
EN 15637:2008 (E) 12 Add 500 µl methanol (3.6) to the round bottom flask and weigh with stopper. Carefully dissolve the residue by swirling the flask in the ultrasonic bath (4.5), but avoid losses of methanol. If losses of methanol occur (re-weighing), add methanol to obtain the previous total weight. Filter the obtained sample test solution of 0,5 ml (= Vend) through a PTFE-filter (4.12) into a sample vial (4.13) for injection. To obtain a larger amount of sample test solution for the preparation of matrix-matched standards (3.10.3) a 20 ml cartridge (3.12) can be used. In that case, 400 % of all volumes mentioned above have to be used.
NOTE The sample test solution contains the extractable components of 2,5 g sample per millilitre final extract (or 1,25 g/0,5 ml). 5.4 Determination The sample test solutions (5.3) and calibration solutions (3.10.1, 3.10.2 or 3.10.3) are injected into the LC-MS/MS instrument in an appropriate sequence. This can involve bracketing of the sample extracts with the calibration solutions. In the injector needle of the HPLC system the sample test solution should be diluted with eluent A. The LC-MS/MS instrument shall be operated in the selected reaction monitoring (SRM) mode with transitions selective for the pesticides under investigation. For suitable experimental conditions see CEN/TR 15641 [4]. Nevertheless, individual tuning of the compounds on the instrument that is used for measurement usually provides better sensitivities. The measurement can be performed using various instruments, instrument parameters and columns. Some instrument parameters and columns are listed in Annex A. These conditions have been shown to provide satisfactory results.
NOTE Most validation results listed in Annex B have been obtained after mixing of sample test solution with water in a LC vial and not in the injector of the HPLC system. In that case a ratio between methanolic extract and water of 1:4 (V/V) was used. Also, standard solutions were diluted with water in the volume ratio 1:4 (V/V). Most samples contained small amounts of co-extracted components that are not soluble in the resulting methanol/water mixture. As a result a turbid emulsion (or suspension) was obtained. It was recognized that recovery of some less polar pesticides was reduced, if such emulsions have formed.
5.5 Test for interference and recovery Prepare reagent blanks and carry out spiked recovery tests at levels appropriate to the maximum residue level. The chromatogram of the reagent blank should not show any significant peak (e. g. 10 % of relevant MRL) at the retention time of the analytes. 6 Evaluation of results 6.1 Identification and quantification To identify analytes compare the retention times obtained from the sample test solution with those obtained from the calibration solutions. Positive findings are confirmed by comparing the peak intensity ratios of the first and second compound specific m/z transition with the peak intensity ratios found in standards. If the peak ratio of a residue peak differs more than 20 % from the expected response ratio, check the EU-quality control guidelines described in the SANCO/2007/3131 document [2]. A different LC column, another eluent or an additional m/z transition may be used, if additional measures are necessary. Use standard solutions (3.10.1 or 3.10.2) or matrix-matched standards (3.10.3) to check linearity and to determine the calibration functions for each active substance by plotting the peak areas or heights (if ISTDs are not used) or peak ratios (if ISTDs are used) of one SRM transition against the analyte concentration [ng/ml] of the standard solution.
For a first estimate of the residue level of pesticides in the food or to show their absence, the standard solutions (3.10.1 or 3.10.2) in pure methanol 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 SIST EN 15637:2009
EN 15637:2008 (E) 13 results obtained. As soon as relevant residue concentrations are detected (e.g. suspected MRL violations), a more precise determination using matrix-matched standards (3.10.3) or the standard addition method should be preferred. 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 can be necessary to construct more than one calibration graph from the results of calibration measurements. When using ISTDs it is important to know that any shift in the ISTD signal will directly influence the calculated concentration of the analytes. Ideally, the ISTD signal should only shift due to volume differences and thus improve the accuracy of measurement. However, there are also other, non-desirable, factors that may also affect the signals of the ISTD thus introducing errors in the analyte quantification. Losses of the ISTD during clean-up will result in an overestimation of analyte concentration. Such losses should thus be minimal. A specific suppression of the ISTD signal, potentially occurring in LC-MS applications due to co-eluting matrix components, will also result in analyte overestimations. Matrix effects will depend on whether the commodity extract contains specific components that will co-elute with the ISTD and affect its ionisation process.
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 are mandatory. 6.2 Calculation of residue concentrations without standard addition If standard addition method is not used, the residue level wR of a pesticide in the food sample is calculated from the obtained peak area (or height) using Equation 2: ××−×××−=kgmg1000)(3122VVVVVmVbcAwendaexR (2) where: A is the peak area, peak height or peak ratio for one SRM transition measured, in arbitrary units (a.u) or without dimension; c is the intercept of the corresponding calibration graph, in a.u. or without dimension; b is the slope of the corresponding calibration graph, in a.u. × ml/ng (without ISTD) or ml/ng (with ISTD); Vex is the total volume of extraction solvents and natural water (30 ml); ma is the initial sample weight, in grams; V1 is the volume of NaCl solution (2,5 ml); V2 is the volume of measuring flask used in 5.2 (10 ml); V3 is the volume used for solid supported liquid/liquid extraction (5 ml); Vend is the final volume of extract obtained after clean-up (0,5 ml); 1000 is the conversion factor. SIST EN 15637:2009
EN 15637:2008 (E) 14 If the results indicate that the amount of residue approaches or exceeds the maximum residue level, at least one further test portion shall be analysed. 6.3 Calculation of residue concentrations with standard addition In case of suspected violative residues, or for compounds which are known to be strongly affected by matrix-induced enhancement or suppression phenomena, the procedure of standard additions is recommended provided that the function between response and concentrations at the concentration range in question is linear. In that case several aliquots of the final sample extract are fortified with increasing known amounts of the analyte of intere
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