EN 15637:2008

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
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

Example for appropriate experimental conditions.17 Annex B (informative)
Precision data.21 Bibliography.63
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

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

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

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

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

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
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
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

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

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

from preliminary analysis.
The standard solutions used for standard addition shall have nearly identical solvent composition compared to the sample test solution from 5.3. Assuming a sample (used sample amount 10 g) with an estimated residue level of wR = 0,8 mg/kg, the following pipetting scheme may be appropriate. (In the case of other residue levels wR an adjusted concentration of the analyte standard solution and/or more appropriate volumes of analyte standard solution and solvent are needed.) The amount of analyte in the sample is calculated using a graphical presentation of resulting response data as shown in Figure 1 via linear regression.
(20 µg/ml) 0 µl 5 µl
10 µl
15 µl
Resulting mass of analyte added 0 µg 0,1 µg 0,2 µg 0,3 µg Volume of solvent 15 µl 10 µl 5 µl 0 µl Final volume 115 µl 115 µl 115 µl 115 µl
Key: Y Peak area Analyte X Added absolute amount of analyte in µg |x| Absolute amount of analyte in the sample extract (in µg) before standard addition (y = 0)
)()(bcurvetheofslopecterceptinyx−=
Figure 1 — Internal calibration using the procedure of standard additions, schematically SIST EN 15637:2009

b is the slope of the calibration graph of the analyte in question (a.u./µg);
Vex is the total volume of extraction solvents and natural water (30 ml); ma is the initial sample weight (5 g or 10 g); 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); Valiq is the aliquot of final volume of extract obtained after clean-up (0,1 ml). 7 Confirmatory tests A confirmation of quantity involves analysis of a second sample portion and is to be performed if the first analysis indicates a suspected volative residue. For more information about the confirmation of identity refer to the EU- quality control guidelines described in the SANCO/2007/3131 document [2].
8 Precision Details of the inter-laboratory test of the precision of the method according to ISO 5725-1 and ISO 5725-2 [3] are summarised in Annex B. The values derived from the inter-laboratory test may not be applicable to analyte concentration ranges and matrices other than given in Annex B. 9 Test report The test report shall contain at least the following:  all information necessary for the identification of the sample;  reference to this European Standard;  date and type of sampling procedure (if possible);  date of receipt of sample in the laboratory;  date of test;  results and the units in which the results have been expressed;  any particular points observed in the course of the test;  any operations not specified in the method or regarded as optional which might have affected the results. SIST EN 15637:2009

Example for appropriate experimental conditions The following LC-MS/MS operating conditions have been proven to be satisfactory. A.1 HPLC system 1 For most LC-amenable compounds: HPLC pump HP1100 Binary Pump (G1312A) Autosampler HP1100 (G1313A) Injector programme
draw 3 µl eluent A draw 2 µl sample
wash needle with methanol draw 2 µl eluant A draw 2 µl sample
wash needle with methanol draw 2 µl eluant A
draw 2 µl sample
wash needle with methanol draw 2 µl eluant A
draw 2 µl sample
wash needle with methanol draw 3 µl eluent A Column Phenomenex Aqua 5µ C18 125 Å, 50 mm × 2 mm Mobile phase A Methanol/water 2+8 (V/V) with 5 mmol/l ammonium formate (3.2) Mobile phase B Methanol/water 9+1 (V/V) with 5 mmol/l ammonium formate Column temperature 20 °C Table A.1 — Flow rate and elution gradient Time
min Flow rate
µl/min Mobile phase A % Mobile phase B % 0 200 100 0 11 200 0 100 23 200 0 100 25 200 100 0 33 200 100 0
Zorbax XDB C18, length 150 mm, inner diameter 2,1 mm, particle size 3,5 µm
Mobile phase A Ammonium formate solution in water, c = 5 mmol/l
Mobile phase B Ammonium formate solution in methanol, c = 5 mmol/l
Column temperature
40 °C
Injection volume
5 µl Table A.2 — Flow rate and elution gradient Time
min Flow rate
µl/min Mobile phase A % Mobile phase B % 0 300 50 50 20 300 0 100 25 300 0 100 26 300 50 50 30 300 50 50 A.3 HPLC-System 3 For polar compounds that show low retention at reversed-phased columns: Column
Phenomenex Aqua, length 150 mm, inner diameter 2 mm, filled with 125 A C18-material, particle size 3 µm
Mobile phase A Ammonium formate solution in water, c = 5 mmol/l
Mobile phase B Ammonium formate solution in methanol, c = 5 mmol/l
Column temperature
40 °C
Injection volume
3 µl, automatically diluted with 3 µl of mobile phase A during injection procedure Table A.3 — Flow rate and elution gradient:
Time
min Flow rate
µl/min Mobile phase A % Mobile phase B % 0 100 100 0 3 100 30 70 6 300 15 85 9 300 10 90 20,5 300 10 90 21 300 100 0 32 300 100 0
NOTE Should the possibility for an automated dilution of the solutions in the instrument injector not exist, these should be manually diluted with mobile phase A (1 : 1), and 6 µl thereof should be injected.
Zorbax XDB C18, length 150 mm, inner diameter 2,1 mm, particle size 3,5 µm
Mobile phase A Acetic acid solution in water, 1 = 0,1 ml glacial acetic acid /l Mobile phase B Acetic acid solution in acetonitrile, 1 = 0,1 ml glacial acetic acid /l Column temperature
40 °C
Injection volume 5 µl Table A.4 — Flow rate and elution gradient:
Time
min Flow rate µl/min Mobile phase A % Mobile phase B % 0 300 80 20 20 300 0 100 22 300 0 100 22,1 300 80 20 30 300 80 20
A.5 MS/MS system 1 MS/MS instrument Applied Biosystems API 2000 Ion source Turbo Ion Spray (ESI) Table A.5 — Ion source and general parameters Curtain gas nitrogen, 35 psi Gas 2 temperature 400 °C Collision gas nitrogen, 2 units Resolution MS 1 unit Ion spray voltage 5500 V Resolution MS 2 unit Gas 1 nitrogen, 60 psi Dwell time 25 ms Gas 2 nitrogen, 60 psi Focusing potential 360 V
MS1 LM Resolution 14,7 Desolvation gas flow
nitrogen, 552 l/h
MS1 HM Resolution 14,7 Desolvation temp.
350 °C MS2 LM Resolution 14,7 Capillary voltage 3500 V MS2 HM Resolution 14,7 Gas cell
9,2 x 10-4 mbar
Precision data In accordance with ISO 5725-1 and ISO 5725-2, the parameters given in Table B.1 have been defined in an inter-laboratory test. The precision data listed in Table B.2 are summarized from single laboratory validation trails. An updated version of validation data can be found on the website www.crl-pesticides-datapool.eu, which is run by the EU Community Reference Laboratories for Pesticides.
Table B.1 — Results of validation study of the German working group „Unterarbeitsgruppe Analytik der Bund-Länder-Arbeitsgruppe Pflanzenschutz- und Schädlingsbekämpfungsmittel“ (n approximately 12 000) Recovery a
Pesticide Matrix type Spiked amount mg/kg X
% V % n Number of labs 3,4,5-Trimethacarb water containing 0,100 97
35 7 3,4,5-Trimethacarb water containing 0,010 95
40 8 3,4,5-Trimethacarb acidic 0,100 87
40 8 3,4,5-Trimethacarb acidic 0,010 88
40 8 3,4,5-Trimethacarb cereal (dry) 0,100 82
25 5 3,4,5-Trimethacarb cereal (dry) 0,010 73
25 5 3,4,5-Trimethacarb fatty 0,100 79
25 5 3,4,5-Trimethacarb fatty 0,010 81
20 4
Acephate water containing 0,100 87
35 7 Acephate water containing 0,010 85
40 8 Acephate acidic 0,100 81
40 8 Acephate acidic 0,010 88
40 8 Acephate cereal (dry) 0,100 85
25 5 Acephate cereal (dry) 0,010 74
25 5 Acephate fatty 0,100 85
25 5 Acephate fatty 0,010 86
20 4
Aldicarb water containing 0,100 85
30 6 Aldicarb water containing 0,010 82
35 7 Aldicarb acidic 0,100 79
35 7 Aldicarb acidic 0,010 74
30 6 Aldicarb cereal (dry) 0,100 92
25 5 Aldicarb cereal (dry) 0,010 89
25 5 Aldicarb fatty 0,100 97
25 5 SIST EN 15637:2009
Pesticide Matrix type Spiked amount mg/kg X
% V % n Number of labs Aldicarb fatty 0,010 88
20 4
Azoxystrobin water containing 0,100 92
35 7 Azoxystrobin water containing 0,010 97
35 7 Azoxystrobin acidic 0,100 72
40 8 Azoxystrobin acidic 0,010 70
40 8 Azoxystrobin cereal (dry) 0,100 72
25 5 Azoxystrobin cereal (dry) 0,010 69
25 5 Azoxystrobin fatty 0,100 78
25 5 Azoxystrobin fatty 0,010 81
20 4
Bendiocarb water containing 0,100 101
30 6 Bendiocarb water containing 0,010 98
35 7 Bendiocarb acidic 0,100 92
35 7 Bendiocarb acidic 0,010 95
35 7 Bendiocarb cereal (dry) 0,100 84
20 4 Bendiocarb cereal (dry) 0,010 91
25 5 Bendiocarb fatty 0,100 95
25 5 Bendiocarb fatty 0,010 95
20 4
Butocarboxim water containing 0,100 61
30 6 Butocarboxim water containing 0,010 70
35 7 Butocarboxim acidic 0,100 62
35 7 Butocarboxim acidic 0,010 60
25 5 Butocarboxim cereal (dry) 0,100 88
20 4 Butocarboxim cereal (dry) 0,010 81
20 4 Butocarboxim fatty 0,100 91
20 4 Butocarboxim fatty 0,010 76
15 3
Carbaryl water containing 0,100 99
35 7 Carbaryl water containing 0,010 96
40 8 Carbaryl acidic 0,100 91
40 8 Carbaryl acidic 0,010 84
40 8 Carbaryl cereal (dry) 0,100 90
25 5 Carbaryl cereal (dry) 0,010 78
25 5 SIST EN 15637:2009
Pesticide Matrix type Spiked amount mg/kg X
% V % n Number of labs Carbaryl fatty 0,100 81
25 5 Carbaryl fatty 0,010 101
20 4
Carbendazim water containing 0,100 67
35 7 Carbendazim water containing 0,010 69
40 8 Carbendazim acidic 0,100 84
40 8 Carbendazim acidic 0,010 88
35 7 Carbendazim cereal (dry) 0,100 80
25 5 Carbendazim cereal (dry) 0,010 66
25 5 Carbendazim fatty 0,100 63
25 5 Carbendazim fatty 0,010 71
20 4
Carbofuran water containing 0,100 104
35 7 Carbofuran water containing 0,010 108
40 8 Carbofuran acidic 0,100 102
40 8 Carbofuran acidic 0,010 105
40 8 Carbofuran cereal (dry) 0,100 104
25 5 Carbofuran cereal (dry) 0,010 89
25 5 Carbofuran fatty 0,100 100
25 5 Carbofuran fatty 0,010 104
20 4
Cinosulfuron water containing 0,100 83
35 7 Cinosulfuron water containing 0,010 86
40 8 Cinosulfuron acidic 0,100 84
40 8 Cinosulfuron acidic 0,010 92
40 8 Cinosulfuron cereal (dry) 0,100 95
25 5 Cinosulfuron cereal (dry) 0,010 80
25 5 Cinosulfuron fatty 0,100 84
25 5 Cinosulfuron fatty 0,010 94
20 4
Cyprodinil water containing 0,100 77
30 6 Cyprodinil water containing 0,010 75
35 7 Cyprodinil acidic 0,100 41
40 8 Cyprodinil acidic 0,010 45
40 8 Cyprodinil cereal (dry) 0,100 90
25 5 SIST EN 15637:2009
Pesticide Matrix type Spiked amount mg/kg X
% V % n Number of labs Cyprodinil cereal (dry) 0,010 67
20 4 Cyprodinil fatty 0,100 11
25 5 Cyprodinil fatty 0,010 49
10 2
Dimethoat water containing 0,100 107
35 7 Dimethoat water containing 0,010 104
35 7 Dimethoat acidic 0,100 101
40 8 Dimethoat acidic 0,010 108
40 8 Dimethoat cereal (dry) 0,100 104
25 5 Dimethoat cereal (dry) 0,010 92
25 5 Dimethoat fatty 0,100 109
25 5 Dimethoat fatty 0,010 108
20 4
Ethiofencarb water containing 0,100 33
30 6 Ethiofencarb water containing 0,010 22
35 7 Ethiofencarb acidic 0,100 56
40 8 Ethiofencarb acidic 0,010 47
40 8 Ethiofencarb cereal (dry) 0,100 54
25 5 Ethiofencarb cereal (dry) 0,010 49
25 5 Ethiofencarb fatty 0,100 72
25 5 Ethiofencarb fatty 0,010 87
20 4
Fenhexamid water containing 0,100 87
35 7 Fenhexamid water containing 0,010 75
35 7 Fenhexamid acidic 0,100 69
40 8 Fenhexamid acidic 0,010 69
35 7 Fenhexamid cereal (dry) 0,100 86
25 5 Fenhexamid cereal (dry) 0,010 81
20 4 Fenhexamid fatty 0,100 78
25 5 Fenhexamid fatty 0,010 70
15 3
Fenoxycarb water containing 0,100 75
35 7 Fenoxycarb water containing 0,010 75
40 8 Fenoxycarb acidic 0,100 47
40 8 Fenoxycarb acidic 0,010 51
35 7 SIST EN 15637:2009
Pesticide Matrix type Spiked amount mg/kg X
% V % n Number of labs Fenoxycarb cereal (dry) 0,100 56
25 5 Fenoxycarb cereal (dry) 0,010 58
20 4 Fenoxycarb fatty 0,100 32
25 5 Fenoxycarb fatty 0,010 40
15 3
Fenpropimorph water containing 0,100 72
35 7 Fenpropimorph water containing 0,010 59
40 8 Fenpropimorph acidic 0,100 72
40 8 Fenpropimorph acidic 0,010 74
40 8 Fenpropimorph cereal (dry) 0,100 50
25 5 Fenpropimorph cereal (dry) 0,010 42
25 5 Fenpropimorph fatty 0,100 13
20 4 Fenpropimorph fatty 0,010 26
15 3
Flufenoxuron water containing 0,100 65
30 6 Flufenoxuron water containing 0,010 64
40 8 Flufenoxuron acidic 0,100 29
30 6 Flufenoxuron acidic 0,010 18
40 8 Flufenoxuron cereal (dry) 0,100 45
25 5 Flufenoxuron cereal (dry) 0,010 38
20 4 Flufenoxuron fatty 0,100 18
15 3 Flufenoxuron fatty 0,010 25
10 2
Imazalil water containing 0,100 74
25 5 Imazalil water containing 0,010 94
25 5 Imazalil acidic 0,100 78
30 6 Imazalil acidic 0,010 80
30 6 Imazalil cereal (dry) 0,100 57
15 3 Imazalil cereal (dry) 0,010 31
20 4 Imazalil fatty 0,100 54
15 3 Imazalil fatty 0,010 74
10 2
Imidacloprid water containing 0,100 99
35 7 Imidacloprid water containing 0,010 102
40 8 Imidacloprid acidic 0,100 95
40 8 SIST EN 15637:2009
Pesticide Matrix type Spiked amount mg/kg X
% V % n Number of labs Imidacloprid acidic 0,010 103
40 8 Imidacloprid cereal (dry) 0,100 96
25 5 Imidacloprid cereal (dry) 0,010 86
25 5 Imidacloprid fatty 0,100 98
25 5 Imidacloprid fatty 0,010 96
20 4
Indoxacarb water containing 0,100 71
35 7 Indoxacarb water containing 0,010 62
35 7 Indoxacarb acidic 0,100 28
40 8 Indoxacarb acidic 0,010 44
35 7 Indoxacarb cereal (dry) 0,100 50
25 5 Indoxacarb cereal (dry) 0,010 44
15 3 Indoxacarb fatty 0,1
...