Foods of plant origin - Multimethod for the determination of pesticide residues in vegetable oils by LC-MS/MS (QuOil)

This Technical Specification describes a method for the analysis of pesticide residues in fatty oils of plant origin (essential oils are excluded). It has been validated in an interlaboratory test with olive oil. However, laboratory experiences have shown that this method is also applicable to other kinds of oils such as sunflower seed oil, sesame oil, flax seed oil, rape seed oil, grape seed oil, thistle oil and pumpkin seed oil.

Pflanzliche Lebensmittel - Multiverfahren zur Bestimmung von Pestizidrückständen in pflanzlichen Ölen mit LC-MS/MS (QuOil)

Diese Technische Spezifikation beschreibt ein Verfahren zur Bestimmung von Pflanzenschutzmittel-rückständen in fettigen pflanzlichen Ölen (ätherische Öle werden nicht berücksichtigt). Das Verfahren wurde in einem Ringversuch mit Olivenöl validiert. Laborerfahrungen haben jedoch gezeigt, dass dieses Verfahren auch bei anderen Ölsorten wie Sonnenblumenkernöl, Sesamöl, Leinöl, Rapsöl, Traubenkernöl, Distelöl und Kürbiskernöl anwendbar ist.

Aliments d’origine végétale - Multiméthode de détermination des résidus de pesticides dans les huiles végétales par CL-SM/SM (QuOil)

La présente Spécification technique décrit une méthode d’analyse des résidus de pesticides dans les huiles grasses d’origine végétale (les huiles essentielles sont exclues). Elle a été validée lors d’un essai interlaboratoires avec de l’huile d’olive. Toutefois, les expériences menées en laboratoire montrent que cette méthode est également applicable à d’autres types d’huiles, telles que l’huile de tournesol, l’huile de sésame, l’huile de lin, l’huile de colza, l’huile de pépins de raisin, l’huile de chardon et l’huile de pépins de courge.

Hrana rastlinskega izvora - Multirezidualna metoda za določanje ostankov pesticidov v rastlinskih oljih z LC-MS/MS (QuOil)

Ta tehnična specifikacija opisuje metodo za analizo ostankov pesticidov v maščobnih oljih rastlinskega izvora (eterična olja niso zajeta). Potrjena je bila v okviru medlaboratorijskega preskusa z olivnim oljem. Rezultati laboratorijskih preskusov pa so pokazali, da se lahko ta metoda uporablja tudi za druge vrste olj, kot so olje iz sončničnih semen, sezamovo olje, olje iz lanenih semen, olje iz oljne repice, olje grozdnih pečk, olje iz semen žafranike in bučno olje.

General Information

Status
Published
Publication Date
03-Sep-2019
Current Stage
6060 - Definitive text made available (DAV) - Publishing
Due Date
04-Sep-2019
Completion Date
04-Sep-2019

RELATIONS

Buy Standard

Technical specification
-TS CEN/TS 17062:2019
English language
28 pages
sale 10% off
Preview
sale 10% off
Preview

e-Library read for
1 day
Technical specification
-TS FprCEN/TS 17062:2019
English language
28 pages
sale 10% off
Preview
sale 10% off
Preview

e-Library read for
1 day

Standards Content (sample)

SLOVENSKI STANDARD
SIST-TS CEN/TS 17062:2019
01-november-2019
Nadomešča:
SIST-TS CEN/TS 17062:2017
Hrana rastlinskega izvora - Multirezidualna metoda za določanje ostankov
pesticidov v rastlinskih oljih z LC-MS/MS (QuOil)

Foods of plant origin - Multimethod for the determination of pesticide residues in

vegetable oils by LC-MS/MS (QuOil)

Pflanzliche Lebensmittel - Multiverfahren zur Bestimmung von Pestizidrückständen in

pflanzlichen Ölen mit LC-MS/MS (QuOil)

Aliments d’origine végétale - Multiméthode de détermination des résidus de pesticides

dans les huiles végétales par CL-SM/SM (QuOil)
Ta slovenski standard je istoveten z: CEN/TS 17062:2019
ICS:
67.050 Splošne preskusne in General methods of tests and
analizne metode za živilske analysis for food products
proizvode
67.200.10 Rastlinske in živalske Animal and vegetable fats
maščobe in olja and oils
SIST-TS CEN/TS 17062:2019 en,fr,de

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

---------------------- Page: 1 ----------------------
SIST-TS CEN/TS 17062:2019
---------------------- Page: 2 ----------------------
SIST-TS CEN/TS 17062:2019
CEN/TS 17062
TECHNICAL SPECIFICATION
SPÉCIFICATION TECHNIQUE
September 2019
TECHNISCHE SPEZIFIKATION
ICS 67.050
English Version
Foods of plant origin - Multimethod for the determination
of pesticide residues in vegetable oils by LC-MS/MS
(QuOil)

Aliments d'origine végétale - Multiméthode de Pflanzliche Lebensmittel - Multiverfahren zur

détermination des résidus de pesticides dans les huiles Bestimmung von Pestizidrückständen in pflanzlichen

végétales par CL-SM/SM (QuOil) Ölen mit LC-MS/MS (QuOil)

This Technical Specification (CEN/TS) was approved by CEN on 14 July 2019 for provisional application.

The period of validity of this CEN/TS is limited initially to three years. After two years the members of CEN will be requested to

submit their comments, particularly on the question whether the CEN/TS can be converted into a European Standard.

CEN members are required to announce the existence of this CEN/TS in the same way as for an EN and to make the CEN/TS

available promptly at national level in an appropriate form. It is permissible to keep conflicting national standards in force (in

parallel to the CEN/TS) until the final decision about the possible conversion of the CEN/TS into an EN is reached.

CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,

Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,

Poland, Portugal, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and

United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels

© 2019 CEN All rights of exploitation in any form and by any means reserved Ref. No. CEN/TS 17062:2019 E

worldwide for CEN national Members.
---------------------- Page: 3 ----------------------
SIST-TS CEN/TS 17062:2019
CEN/TS 17062:2019 (E)
Contents Page

European foreword ....................................................................................................................................................... 3

1 Scope .................................................................................................................................................................... 4

2 Normative references .................................................................................................................................... 4

3 Terms and definitions ................................................................................................................................... 4

4 Principle ............................................................................................................................................................. 4

5 Reagents ............................................................................................................................................................. 4

6 Apparatus ........................................................................................................................................................... 7

7 Procedure........................................................................................................................................................... 8

7.1 Extraction ........................................................................................................................................................... 8

7.2 Clean-up .............................................................................................................................................................. 8

7.3 Determination by liquid chromatography with tandem mass spectrometry (LC-

MS/MS) ................................................................................................................................................................ 9

8 Evaluation of results ...................................................................................................................................... 9

8.1 Identification and quantification .............................................................................................................. 9

8.2 Calculation of residue concentrations using the internal standard .......................................... 10

8.3 Calculation of residue concentrations without internal standards ........................................... 10

8.4 Calculation of residue concentration using the standard additions approach ..................... 10

9 Precision .......................................................................................................................................................... 10

10 Test report ...................................................................................................................................................... 11

Annex A (informative) Examples of experimental conditions .................................................................. 12

Annex B (informative) Precision data and recovery .................................................................................... 15

Annex C (informative) Scheme of procedure (for 2 g of sample) ............................................................. 24

Annex D (informative) Recovery studies .......................................................................................................... 25

Annex E (informative) Abbreviations ................................................................................................................ 26

Bibliography ................................................................................................................................................................. 28

---------------------- Page: 4 ----------------------
SIST-TS CEN/TS 17062:2019
CEN/TS 17062:2019 (E)
European foreword

This document (CEN/TS 17062:2019) has been prepared by Technical Committee CEN/TC 275 “Food

analysis - Horizontal methods”, the secretariat of which is held by DIN.

Attention is drawn to the possibility that some of the elements of this document may be the subject of

patent rights. CEN shall not be held responsible for identifying any or all such patent rights.

This document will supersede CEN/TS 17062:2017.
Compared to CEN/TS 17062:2017, the following changes have been made:
— Annex E (informative) containing a list of abbreviations was added;
— The document has been editorially revised.
— Annex E (informative) contains a list of abbreviations.

WARNING — The application of this Technical Specification may involve hazardous materials,

operations and equipment. This Technical Specification does not claim to address all the safety

problems associated with its use. It is the responsibility of the user of this Technical

Specification to establish appropriate safety and health practices and to determine the

applicability of regulatory limitations prior to use.

According to the CEN/CENELEC Internal Regulations, the national standards organisations of the

following countries are bound to announce this Technical Specification: Austria, Belgium, Bulgaria,

Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland,

Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Republic of

North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the

United Kingdom.
---------------------- Page: 5 ----------------------
SIST-TS CEN/TS 17062:2019
CEN/TS 17062:2019 (E)
1 Scope

This Technical Specification describes a method for the analysis of pesticide residues in fatty oils of

plant origin (essential oils are excluded). It has been validated in an interlaboratory test with olive oil.

However, laboratory experiences have shown that this method is also applicable to other kinds of oils

such as sunflower seed oil, sesame oil, flax seed oil, rape seed oil, grape seed oil, thistle oil and pumpkin

seed oil.
2 Normative references

The following documents are referred to in the text in such a way that some or all of their content

constitutes requirements of this document. For dated references, only the edition cited applies. For

undated references, the latest edition of the referenced document (including any amendments) applies.

CEN/TS 17061:2019, Foodstuffs — Guideline for the calibration and quantitative determination of

chromatographic methods for the determination of pesticide residues and organic contaminants

3 Terms and definitions
No terms and definitions are listed in this document.

ISO and IEC maintain terminological databases for use in standardization at the following addresses:

• IEC Electropedia: available at http://www.electropedia.org/
• ISO Online browsing platform: available at https://www.iso.org/obp
4 Principle

The homogeneous sample is extracted with acetonitrile. After centrifugation, an aliquot of the organic

phase is cleaned-up by dispersive solid phase extraction (D-SPE; sorbents PSA and C18). To separate

co-extracted fat a freeze-out step of the acetonitrile phase can be applied. After clean up an additional

centrifugation step is performed. The 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 used

for LC-MS/MS analysis. A scheme of the procedure is given in Annex C.

NOTE In contrast to the method described in EN 15662 [1], this procedure does not include any addition of

water.
5 Reagents

Unless otherwise specified, use reagents of recognized analytical grade. Take every precaution to avoid

possible contamination of water, solvents, sorbents, inorganic salts, etc.
5.1 Water, HPLC quality.
5.2 Acetonitrile, HPLC quality.
5.3 Methanol, HPLC quality.
5.4 Acetic acid.
5.5 Ammonium formate.

5.6 Formic acid solution in acetonitrile, volume concentration σ = 5 ml formic acid/100 ml :

---------------------- Page: 6 ----------------------
SIST-TS CEN/TS 17062:2019
CEN/TS 17062:2019 (E)

Dilute 5 ml of formic acid (mass fraction w ≥ 95 %) to 100 ml with acetonitrile (5.2).

® 1)

5.7 Primary secondary amin sorbent (PSA), e.g. Bondesil-PSA 40 µm Agilent No. 12213023 .

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.
5.8 C-18-sorbent (Octadecyl-silyl-modified silica gel), Bulk material 50 µm.

5.9 Internal standard and quality control standard solutions in acetonitrile, mass concentration

ρ = 10 µg/ml to 100 µg/ml.

Table 1 shows a list of potential internal standards (ISTDs) and quality control (QC) standards that may

be used in this method.
Table 1 — Potential internal standards (ISTDs) or quality control (QC) standards
Compound Log P Suggested MS/MS MS/MS
(octanol-water concentration ESI (+) ESI (-)
partition C
ISTD
coefficient)
[µg/ml]
Tris-(1,3-dichlorisopropyl)- 3,65 10 +++ +
phosphate
Linuron-D6 3,00 10 ++ -
Carbofuran-D3 1,80 10 ++ -
Chlorpyrifos-D10 4,70 10 +++ -
Bis-nitrophenyl urea 3,76 10 - +++
(Nicarbazin)
+++ very good detectable
++ good detectable
+ poor detectable
- not detectable
5.10 Primary pesticide standards
Use standards of known purity, only.

Bondesil-PSA is a product supplied by Agilent. This information is given for the convenience of users of this

European Technical Specification 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.

---------------------- Page: 7 ----------------------
SIST-TS CEN/TS 17062:2019
CEN/TS 17062:2019 (E)
5.11 Pesticide stock solutions

Prepare individual stock solutions of analytical standards at concentrations that are sufficient to allow

the preparation of complex pesticide working solutions 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 storage period. Before withdrawing any aliquot from this solution redissolve any

precipitation that may have occurred.
5.12 Pesticide working solutions

Because of the broad applicability of this method and due to the partly divergent pH-stability of

pesticides, more than one working solution each containing one or more pesticides can be needed to

cover the entire pesticide spectrum of interest. These are prepared by mixing together defined volumes

of the required pesticide stock solutions (5.11) and appropriately diluting them with acetonitrile. The

pesticide concentrations in these mixtures should be sufficient to allow the preparation of the required

matrix matched standards (5.13.2) with moderate dilution of the blank sample extract (e.g. less than

20 %).

Usually, pesticide working solutions should be stored at low temperature in the dark. Check the stability

of pesticides contained in these mixtures during storage regularly [2] and adapt the storing conditions

accordingly. In some cases the addition of acids or bases can be helpful to enhance stability and extend

acceptable storage times.
5.13 Standard solutions (calibration mixtures)
5.13.1 Solvent-based standards

Prepare solvent-based standards by mixing known volumes of the pesticide working solutions (5.12)

and make up to volume with acetonitrile. The preparation of solvent based calibration mixtures with

cal cal

different analyte concentrations (ρ ) and identical internal standard concentrations (ρ ) is

A ISTD
necessary to create a calibration graph.
cal

The concentration of the internal standards in the calibration mixtures ( ) shall be equivalent to

ISTD

the concentration of the internal standard in the sample extracts, as the internal standards are added

cal cal

after extraction. The quotient V /V from the volume (V ) of the internal standard (5.9) and the

ISTD Std ISTD

final volume of the calibration standards (V ) shall be equivalent to the quotient V /V (see

Std ISTD Aliquot

7.1). If 60 µl ISTD solution (5.9) are added to 6 ml of aliquot of the centrifugate, 6 ml of standard

solution shall be spiked with 60 µl of ISTD solution. If other volumes of calibration standards are used,

the addition of ISTD solution shall be adjusted.

NOTE A pesticide concentration of 1 µg/ml correlates to a residue level of 5 mg/kg when a 2 g test portion is

employed.
5.13.2 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 (samples, where no pesticides have been found with this

method). The extract is prepared as described in Clause 7 (but without ISTD addition). To minimize

errors caused by matrix induced effects during chromatography, it is best to choose similar

commodities (e.g. olive oil for olive oil samples etc.).
---------------------- Page: 8 ----------------------
SIST-TS CEN/TS 17062:2019
CEN/TS 17062:2019 (E)

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.
5.14 Mobile phase A1

Ammonium formate solution in water for HPLC, ρ = 0,315 g ammonium formate / 1 000 ml, substance

concentration c = 5 mmol/l.
5.15 Mobile phase B1

Ammonium formate solution in methanol for HPLC, ρ = 0,315 g ammonium formate / 1 000 ml,

c = 5 mmol/l.
5.16 Mobile phase A2

Acetic acid solution in water, add 0,1 ml of glacial acetic acid to 1 000 ml of water.

5.17 Mobile phase B2

Acetic acid solution in acetonitrile, add 0,1 ml of glacial acetic acid to 1 000 ml of acetonitrile.

5.18 Mobile phase A3

Methanol/water 2+8 (V/V) with 5 mmol/l ammonium formate, ρ = 0,315 g ammonium formate /

1 000 ml.
5.19 Mobile phase B3

Methanol/water 9+1 (V/V) with 5 mmol/l ammonium formate, ρ = 0,315 g ammonium formate /

1 000 ml.
5.20 Cotton wool.
6 Apparatus
Usual laboratory apparatus and, in particular, the following:

6.1 Automatic pipettes, suitable for handling volumes of 10 µl to 100 µl, 200 µl to 1 000 µl and 2 ml

to 10 ml.

NOTE Instead of the latter, 10 ml graduated glass pipettes can be used alternatively.

6.2 Single use 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.
6.3 Polypropylene-single use tubes with screw caps, 10 ml or 12 ml

6.4 Centrifuges, suitable for the centrifuge tubes employed in the procedure (7.2.2 and 7.2.3) and

capable of achieving at least 1 000 g.

6.5 10 ml solvent-dispenser for acetonitrile, for use with the acetonitrile reservoir bottle.

6.6 Injection vials, 1,5 ml, suitable for LC autosampler, if necessary with micro-inserts.

---------------------- Page: 9 ----------------------
SIST-TS CEN/TS 17062:2019
CEN/TS 17062:2019 (E)
6.7 Vibration device, e.g. Vortex (used for recovery studies).
6.8 Freezer, > 60 l, ≤ –18 °C.

6.9 LC-MS/MS system, equipped with electrospray ionization (ESI) interface (see Annex A).

7 Procedure
7.1 Extraction

Transfer a representative test portion of 2 g (m ) of the homogenous sample into a 50 ml

Sample

centrifuge tube (6.2) [4]. Add 10 ml of acetonitrile (5.2) (V ). Close the tube and shake vigorously for

1 min. Centrifuge for 5 min with at least 1 000 g for better separation of the phases.

Transfer an aliquot of the acetonitrile phase V (e.g. 6 ml extract) into a tube with screw cap (6.3).

Aliquot

Add a defined volume (V ) of the ISTD solution (5.9). The volume corresponds to 1 % of the aliquot

ISTD
volume (e.g. 60 µl ISTD solution to 6 ml acetonitrile phase).
7.2 Clean-up
7.2.1 General

The two different clean-up methods described in 7.2.2 and 7.2.3 were successfully validated and may be

used alternatively.
7.2.2 Clean-up with amino-sorbent and silica-based reversed phase sorbent

Transfer an aliquot of 4 ml of the acetonitrile phase (7.1) into a Polypropylene-single use tube (6.3)

already containing 100 mg of PSA (5.7) and 100 mg of C18 sorbent (5.8). Close the tube, shake

vigorously for 30 s and centrifuge (5 min at ≥ 1 000 g). Immediately isolate and acidify the clear extract

as described in 7.2.4.

In case residues with acetic groups (e.g. phenoxy carboxylic acids) shall be determined, a second aliquot

of the centrifuged extract from 7.1 is filled into an injection vial and analysed directly with LC-MS/MS to

avoid losses of acidic groups by PSA clean-up.
25 mg PSA and 25 mg C18 sorbent are needed per ml of extract.
7.2.3 Freezing-out of co-extracted fat and clean-up with amino-sorbent

Store an aliquot of the extract from 7.1 containing the internal standard for at least 1,5 h at ≤ – 18 °C to

freeze out most of the fat in the extract. For separation of the latter filter the extract over cotton wool

(5.20). Take 4 ml from the cold and fat separated solution for dispersive SPE.

Transfer an aliquot of 4 ml of the acetonitrile phase into a Polypropylene-single use tube (6.3) already

containing 100 mg of PSA (5.7). Close the tube, shake vigorously for 30 s and centrifuge (5 min

at ≥ 1 000 g). Immediately isolate and acidify the clear extract as described in 7.2.4.

If residues with acetic groups shall be determined, transfer a second aliquot into an injection vial and

analyse directly with LC-MS/MS to avoid losses of acidic groups with PSA clean-up.

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. 25 mg PSA sorbent are needed per ml of extract.

7.2.4 Extract stabilization

Transfer an aliquot of 3 ml of the cleaned-up extract from 7.2.2 or 7.2.3 into a screw cap storage vial

(6.3), taking care to avoid sorbent particles of being carried over, and slightly acidify by adding 30 µl of

---------------------- Page: 10 ----------------------
SIST-TS CEN/TS 17062:2019
CEN/TS 17062:2019 (E)

a 5 % formic acid solution in acetonitrile (5.6). Transfer the pH-adjusted extract into auto-sampler vials

and use it for 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 (5.6) are necessary.
7.3 Determination by liquid chromatography with tandem mass spectrometry (LC-
MS/MS)

Inject the sample extracts derived from 7.2.2 to 7.2.4 and standard solutions (5.13) into the LC

instruments in an appropriate sequence. This may involve bracketing of the sample extracts with the

calibration solutions.

The measurement may 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, but are provided as examples, only.

For some gradient/column combinations it is necessary to mix the extract with water or the aqueous

mobile phase to achieve a sufficient separation of the analytes.

NOTE If extracts are diluted with water or aqueous mobile phases it is important to avoid that non-polar

parts of the extract precipitate or emulsions occur. This could lead to losses of lipophilic analytes. In this case an

injection applying an injector programme can be helpful (see A.4).

The chromatographic conditions as outlined in Annex A have been shown to be satisfactory.

Suitable experimental conditions of LC-MS/MS measurements are outlined in CEN/TR 15641 [3].

Nevertheless, individual tuning of the compounds on the instrument that is used for measurement

usually provides better sensitivities.
8 Evaluation of results
8.1 Identification and quantification

For the identification of residues in the final extract, use relative retention time ratio against the ISTD

(Rt /Rt ) obtained from the same run. Check positive results by comparing the intensity ratios

(A) (ISTD)

between the SIM masses (m/z) or SRM transitions of the analyte. The expected intensity ratios can be

determined with the standard solutions. If the ratios of the samples and the standards have a variation

of more than 30 %, the rules of EU Quality Control Procedures will be followed [2]. According to these

procedures positive results shall be ensured by using additional measures, e.g. additional SIM masses or

SRM transitions or other chromatographic conditions (column, eluents).

For calibration and for checking the linearity of detection of each substance, plot the peak area ratio or

cal cal

peak height ratio of pesticide and internal standard y / y (if an internal standard is used) versus

A ISTD
cal cal

the concentration ratio of the analyte against the ISTD (ρ / ρ ) in the standard solution (5.13). If

A ISTD
cal

no internal standards are used, plot the peak areas or peak height y against the concentration of the

cal
analyte ρ .

The calibration area shall be adapted to the residue concentration and should not exceed a decimal

power. Possibly more calibration graphs shall be established using the standard solution. The

calibration function is selected according to CEN/TS 17061:2019, 6.2.1.

For a first estimation of the residue level or for the verification of absence of residues, solvent based

standards (5.13.1) can be used. They can also be used for quantification, if it was shown that no

enhancement or suppression of the analyte signal through matrix occurs. If relevant residue levels are

---------------------- Page: 11 ----------------------
SIST-TS CEN/TS 17062:2019
CEN/TS 17062:2019 (E)

observed (e.g. with possible MRL violation) matrix matched standard shall be preferred for exact

quantification.
8.2 Calculation of residue concentrations using the internal standard

The determination of the concentration of the analyte ρ in the final extract is performed by using the

measured peak area ratio or peak height ratio from pesticide and internal standard y /y in the

A ISTD

sample as described in CEN/TS 17061. Calculate the mass fraction w of the analyte in the sample, in

milligram per kilogram with Formula (1):
ρ ×V
A ex
w = (1)
Sample
where

is the mass concentration of the analyte in the final extract, in microgram per millilitre;

V is the volume of acetonitrile used in 7.1, in millilitre;
m is the mass of test portion in 7.1, in gram.
Sample
8.3 Calculation of residue concentrations without internal standards

Determine the concentration of the analyte ρ in the final extract by using the measured peak area or

peak height from pesticide y in the sample as described in CEN/TS 17061:2019, 6.4.2 to 6.4.5.

Calculate the mass fraction w of the analyte in the sample by using Formula (1).
8.4 Calculation of residue concentration using the standard additions approach

In case of suspected violative residues, or for compounds which are known to be strongly affected by

matrix-induced enhancement or suppression phenomena, standard additions are recommended

provided that the function between response and concentrations at the concentration range in question

is linear.

In case of the standard addition to the final extract, determine the concentration of the analyte ρ in

the final extract using a linear regression graph of peak areas or peak height versus spiked

concentrations and the volume of the applied aliquot of the final extract as described in

CEN/TS 17061:2019, 6.6.1. Calculate the mass fraction w of the analyte in the sample by using

Formula (1).

In case of standard addition to the sample, determine the mass of the analyte in the weighted sample

using a linear regression graph of peak areas or peak height versus spiked analyte masses as described

in CEN/TS 17061:2019, 6.6.2. The mass fraction of the analyte in the sample is the quotient of the mass

of the analyte m in the weighted sample and the weighted sample m .
A Sample

NOTE With the standard addition approach, the sought analyte concentration is determined using linear

extrapolation. Therefore, it is important that the analyte has linear detection properties in the investigated

calibration range. It can be necessary to dilute the extract to achieve the calibration range using LC-MS(/MS).

9 Precision

The method was validated in two interlaboratory tests with representative analytes. The results for LC-

MS/MS validation and ongoing verification are shown in Annex B. An updated and detailed list of

validation results can be found in the internet www.eurl-pesticides-datapool.eu operated by the EU

---------------------- Page: 12 ----------------------
SIST-TS CEN/TS 17062:2019
CEN/TS 17062:2019 (E)

reference laboratories for pesticides residues. A summary of the validation data are given in Table B.1

in Annex B. Data for compounds are given there if these were available from at least two laboratories

and if the number of individual results was at least five (in total). Further information on how to

conduct recovery studies is given in Annex D.

Tested matrices were vegetable oils such as olive oil, sunflower seed oil, sesame oil, flax seed oil, rape

seed, oil, grape seed oil, thistle oil and pumpkin seed oil, see www.eurl-pesticides-datapool.eu.

However, it has been noted that coconut oils containing a high amount of short chain fatty acids can

cause problems as they behave in a different way to the other oils.

With addition of the levels 0,01 mg/kg to 0,10 mg/kg the recoveries obtained were usually between

70 % and 110 %.

The detection limits are depending on the analyte of interest and the sensitivity of the equipment. In

general, pesticide residues of 0,01 mg/kg (lowest maximum residue level in most cases) can be

analysed with modern systems.
10 Test report
The test report shall contain at least the following:
— all information necessary for the identification of the sample;
— a reference to this Technical Specification;
— the results and the units in which the results have been expressed;
— the date and type of sampling procedure (if possible);
— the date of receipt of sample in the laboratory;
— the date of test;
— any particular observations made in the course of the test;

— any operations not specified in the method or regarded as optional which might have affected the

results.
-----------
...

SLOVENSKI STANDARD
kSIST-TS FprCEN/TS 17062:2019
01-maj-2019
+UDQDUDVWOLQVNHJDL]YRUD9HþHOHPHQWQDPHWRGD]DGRORþDQMHRVWDQNRY
SHVWLFLGRYYUDVWOLQVNLKROMLK]/&0606 4X2LO

Foods of plant origin - Multimethod for the determination of pesticide residues in

vegetable oils by LC-MS/MS (QuOil)

Pflanzliche Lebensmittel - Multiverfahren zur Bestimmung von Pestizidrückständen in

pflanzlichen Ölen mit LC-MS/MS (QuOil)

Aliments d’origine végétale - Multiméthode de détermination des résidus de pesticides

dans les huiles végétales par CL-SM/SM (QuOil)
Ta slovenski standard je istoveten z: FprCEN/TS 17062
ICS:
67.050 Splošne preskusne in General methods of tests and
analizne metode za živilske analysis for food products
proizvode
67.200.10 5DVWOLQVNHLQåLYDOVNH Animal and vegetable fats
PDãþREHLQROMD and oils
kSIST-TS FprCEN/TS 17062:2019 en,fr,de

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

---------------------- Page: 1 ----------------------
kSIST-TS FprCEN/TS 17062:2019
---------------------- Page: 2 ----------------------
kSIST-TS FprCEN/TS 17062:2019
FINAL DRAFT
TECHNICAL SPECIFICATION
FprCEN/TS 17062
SPÉCIFICATION TECHNIQUE
TECHNISCHE SPEZIFIKATION
March 2019
ICS 67.050 Will supersede CEN/TS 17062:2017
English Version
Foods of plant origin - Multimethod for the determination
of pesticide residues in vegetable oils by LC-MS/MS
(QuOil)

Aliments d'origine végétale - Multiméthode de Pflanzliche Lebensmittel - Multiverfahren zur

détermination des résidus de pesticides dans les huiles Bestimmung von Pestizidrückständen in pflanzlichen

végétales par CL-SM/SM (QuOil) Ölen mit LC-MS/MS (QuOil)

This draft Technical Specification is submitted to CEN members for Vote. It has been drawn up by the Technical Committee

CEN/TC 275.

CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,

Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania,

Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland,

Turkey and United Kingdom.

Recipients of this draft are invited to submit, with their comments, notification of any relevant patent rights of which they are

aware and to provide supporting documentation.

Warning : This document is not a Technical Specification. It is distributed for review and comments. It is subject to change

without notice and shall not be referred to as a Technical Specification.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels

© 2019 CEN All rights of exploitation in any form and by any means reserved Ref. No. FprCEN/TS 17062:2019 E

worldwide for CEN national Members.
---------------------- Page: 3 ----------------------
kSIST-TS FprCEN/TS 17062:2019
FprCEN/TS 17062:2019 (E)
Contents Page

European foreword ....................................................................................................................................................... 3

1 Scope .................................................................................................................................................................... 4

2 Normative references .................................................................................................................................... 4

3 Terms and definitions ................................................................................................................................... 4

4 Principle ............................................................................................................................................................. 4

5 Reagents ............................................................................................................................................................. 4

6 Apparatus ........................................................................................................................................................... 7

7 Procedure........................................................................................................................................................... 8

8 Evaluation of results ...................................................................................................................................... 9

9 Precision .......................................................................................................................................................... 10

10 Test report ...................................................................................................................................................... 11

Annex A (informative) Examples of experimental conditions .................................................................. 12

A.1 HPLC-System 1............................................................................................................................................... 12

A.2 HPLC-System 2............................................................................................................................................... 12

A.3 HPLC-System 3............................................................................................................................................... 13

A.4 HPLC System 4 ............................................................................................................................................... 14

Annex B (informative) Precision data and recovery .................................................................................... 15

Annex C (informative) Scheme of procedure (for 2 g of sample) ............................................................. 24

Annex D (informative) Recovery studies .......................................................................................................... 25

Annex E (informative) Abbreviations ................................................................................................................ 26

Bibliography ................................................................................................................................................................. 28

---------------------- Page: 4 ----------------------
kSIST-TS FprCEN/TS 17062:2019
FprCEN/TS 17062:2019 (E)
European foreword

This document (FprCEN/TS 17062:2019) has been prepared by Technical Committee CEN/TC 275

“Food analysis - Horizontal methods”, the secretariat of which is held by DIN.
This document is currently submitted to the Vote on TS.
This document will supersede CEN/TS 17062:2017.
Compared to CEN/TS 17062:2017, the following changes have been made:
— Annex E (informative) containing a list of abbreviations was added;
— The document has been editorially revised.
Annex E (informative) contains a list of abbreviations.
---------------------- Page: 5 ----------------------
kSIST-TS FprCEN/TS 17062:2019
FprCEN/TS 17062:2019 (E)

WARNING — The application of this Technical Specification may involve hazardous materials,

operations and equipment. This Technical Specification does not claim to address all the safety

problems associated with its use. It is the responsibility of the user of this Technical

Specification to establish appropriate safety and health practices and to determine the

applicability of regulatory limitations prior to use.
1 Scope

This Technical Specification describes a method for the analysis of pesticide residues in fatty oils of

plant origin (essential oils are excluded). It has been validated in an interlaboratory test with olive oil.

However, laboratory experiences have shown that this method is also applicable to other kinds of oils

such as sunflower seed oil, sesame oil, flax seed oil, rape seed oil, grape seed oil, thistle oil and pumpkin

seed oil.
2 Normative references

The following documents are referred to in the text in such a way that some or all of their content

constitutes requirements of this document. For dated references, only the edition cited applies. For

undated references, the latest edition of the referenced document (including any amendments) applies.

FprCEN/TS 17061:2019, Foodstuffs — Guideline for the calibration and quantitative determination of

chromatographic methods for the determination of pesticide residues and organic contaminants

3 Terms and definitions
No terms and definitions are listed in this document.

ISO and IEC maintain terminological databases for use in standardization at the following addresses:

• IEC Electropedia: available at http://www.electropedia.org/
• ISO Online browsing platform: available at http://www.iso.org/obp
4 Principle

The homogeneous sample is extracted with acetonitrile. After centrifugation, an aliquot of the organic

phase is cleaned-up by dispersive solid phase extraction (D-SPE; sorbents PSA and C18). To separate

co-extracted fat a freeze-out step of the acetonitrile phase can be applied. After clean up an additional

centrifugation step is performed. The 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 used

for LC-MS/MS analysis. A scheme of the procedure is given in Annex C.

NOTE In contrast to the method described in EN 15662 [1], this procedure does not include any addition of

water.
5 Reagents

Unless otherwise specified, use reagents of recognized analytical grade. Take every precaution to avoid

possible contamination of water, solvents, sorbents, inorganic salts, etc.
5.1 Water, HPLC quality.
5.2 Acetonitrile, HPLC quality.
5.3 Methanol, HPLC quality.
---------------------- Page: 6 ----------------------
kSIST-TS FprCEN/TS 17062:2019
FprCEN/TS 17062:2019 (E)
5.4 Acetic acid.
5.5 Ammonium formate.

5.6 Formic acid solution in acetonitrile, volume concentration σ = 5 ml formic acid/100 ml :

Dilute 5 ml of formic acid (mass fraction w ≥ 95 %) to 100 ml with acetonitrile (5.2).

® 1)

5.7 Primary secondary amin sorbent (PSA), e.g. Bondesil-PSA 40 µm Agilent No. 12213023 .

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.
5.8 C-18-sorbent (Octadecyl-silyl-modified silica gel), Bulk material 50 µm.

5.9 Internal standard and quality control standard solutions in acetonitrile, mass concentration

ρ = 10 µg/ml to 100 µg/ml.

Table 1 shows a list of potential internal standards (ISTDs) and quality control (QC) standards that may

be used in this method.
Table 1 — Potential internal standards (ISTDs) or quality control (QC) standards
Compound Log P Suggested MS/MS MS/MS
(octanol-water concentration ESI (+) ESI (-)
partition C
ISTD
coefficient)
[µg/ml]
Tris-(1,3-dichlorisopropyl)- 3,65 10 +++ +
phosphate
Linuron-D6 3,00 10 ++ -
Carbofuran-D3 1,80 10 ++ -
Chlorpyrifos-D10 4,70 10 +++ -
Bis-nitrophenyl urea 3,76 10 - +++
(Nicarbazin)
+++ very good detectable
++ good detectable
+ poor detectable
- not detectable
5.10 Primary pesticide standards
Use standards of known purity, only.

Bondesil-PSA is a product supplied by Agilent. This information is given for the convenience of users of this

European Technical Specification 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.

---------------------- Page: 7 ----------------------
kSIST-TS FprCEN/TS 17062:2019
FprCEN/TS 17062:2019 (E)
5.11 Pesticide stock solutions

Prepare individual stock solutions of analytical standards at concentrations that are sufficient to allow

the preparation of complex pesticide working solutions 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 storage period. Before withdrawing any aliquot from this solution redissolve any

precipitation that may have occurred.
5.12 Pesticide working solutions

Because of the broad applicability of this method and due to the partly divergent pH-stability of

pesticides, more than one working solution each containing one or more pesticides can be needed to

cover the entire pesticide spectrum of interest. These are prepared by mixing together defined volumes

of the required pesticide stock solutions (5.11) and appropriately diluting them with acetonitrile. The

pesticide concentrations in these mixtures should be sufficient to allow the preparation of the required

matrix matched standards (5.13.2) with moderate dilution of the blank sample extract (e.g. less than

20 %).

Usually, pesticide working solutions should be stored at low temperature in the dark. Check the stability

of pesticides contained in these mixtures during storage regularly [2] and adapt the storing conditions

accordingly. In some cases the addition of acids or bases can be helpful to enhance stability and extend

acceptable storage times.
5.13 Standard solutions (calibration mixtures)
5.13.1 Solvent-based standards

Prepare solvent-based standards by mixing known volumes of the pesticide working solutions (5.12)

and make up to volume with acetonitrile. The preparation of solvent based calibration mixtures with

cal cal

different analyte concentrations (ρ ) and identical internal standard concentrations (ρ ) is

A ISTD
necessary to create a calibration graph.
cal

The concentration of the internal standards in the calibration mixtures (ρ ) shall be equivalent to

ISTD

the concentration of the internal standard in the sample extracts, as the internal standards are added

cal cal

after extraction. The quotient V /V from the volume (V ) of the internal standard (5.9) and the

Std
ISTD ISTD

final volume of the calibration standards (V ) shall be equivalent to the quotient V /V (see

Std ISTD Aliquot

7.1). If 60 µl ISTD solution (5.9) are added to 6 ml of aliquot of the centrifugate, 6 ml of standard

solution shall be spiked with 60 µl of ISTD solution. If other volumes of calibration standards are used,

the addition of ISTD solution shall be adjusted.

NOTE A pesticide concentration of 1 µg/ml correlates to a residue level of 5 mg/kg when a 2 g test portion is

employed.
5.13.2 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 (samples, where no pesticides have been found with this

method). The extract is prepared as described in Clause 7 (but without ISTD addition). To minimize

errors caused by matrix induced effects during chromatography, it is best to choose similar

commodities (e.g. olive oil for olive oil samples etc.).
---------------------- Page: 8 ----------------------
kSIST-TS FprCEN/TS 17062:2019
FprCEN/TS 17062:2019 (E)

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.
5.14 Mobile phase A1

Ammonium formate solution in water for HPLC, ρ = 0,315 g ammonium formate / 1 000 ml, substance

concentration c = 5 mmol/l.
5.15 Mobile phase B1

Ammonium formate solution in methanol for HPLC, ρ = 0,315 g ammonium formate / 1 000 ml,

c = 5 mmol/l.
5.16 Mobile phase A2

Acetic acid solution in water, add 0,1 ml of glacial acetic acid to 1 000 ml of water.

5.17 Mobile phase B2

Acetic acid solution in acetonitrile, add 0,1 ml of glacial acetic acid to 1 000 ml of acetonitrile.

5.18 Mobile phase A3

Methanol/water 2+8 (V/V) with 5 mmol/l ammonium formate, ρ = 0,315 g ammonium formate /

1 000 ml.
5.19 Mobile phase B3

Methanol/water 9+1 (V/V) with 5 mmol/l ammonium formate, ρ = 0,315 g ammonium formate /

1 000 ml.
5.20 Cotton wool.
6 Apparatus
Usual laboratory apparatus and, in particular, the following:

6.1 Automatic pipettes, suitable for handling volumes of 10 µl to 100 µl, 200 µl to 1 000 µl and 2 ml

to 10 ml.

NOTE Instead of the latter, 10 ml graduated glass pipettes can be used alternatively.

6.2 Single use 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.
6.3 Polypropylene-single use tubes with screw caps, 10 ml or 12 ml

6.4 Centrifuges, suitable for the centrifuge tubes employed in the procedure (7.2.2 and 7.2.3) and

capable of achieving at least 1 000 g.

6.5 10 ml solvent-dispenser for acetonitrile, for use with the acetonitrile reservoir bottle.

6.6 Injection vials, 1,5 ml, suitable for LC autosampler, if necessary with micro-inserts.

---------------------- Page: 9 ----------------------
kSIST-TS FprCEN/TS 17062:2019
FprCEN/TS 17062:2019 (E)
6.7 Vibration device, e.g. Vortex (used for recovery studies).
6.8 Freezer, > 60 l, ≤ –18 °C.

6.9 LC-MS/MS system, equipped with electrospray ionization (ESI) interface (see Annex A).

7 Procedure
7.1 Extraction

Transfer a representative test portion of 2 g (m ) of the homogenous sample into a 50 ml

Sample

centrifuge tube (6.2) [4]. Add 10 ml of acetonitrile (5.2) (V ). Close the tube and shake vigorously for

1 min. Centrifuge for 5 min with at least 1 000 g for better separation of the phases.

Transfer an aliquot of the acetonitrile phase V (e.g. 6 ml extract) into a tube with screw cap (6.3).

Aliquot

Add a defined volume (V ) of the ISTD solution (5.9). The volume corresponds to 1 % of the aliquot

ISTD
volume (e.g. 60 µl ISTD solution to 6 ml acetonitrile phase).
7.2 Clean-up
7.2.1 General

The two different clean-up methods described in 7.2.2 and 7.2.3 were successfully validated and may be

used alternatively.
7.2.2 Clean-up with amino-sorbent and silica-based reversed phase sorbent

Transfer an aliquot of 4 ml of the acetonitrile phase (7.1) into a Polypropylene-single use tube (6.3)

already containing 100 mg of PSA (5.7) and 100 mg of C18 sorbent (5.8). Close the tube, shake

vigorously for 30 s and centrifuge (5 min at ≥ 1 000 g). Immediately isolate and acidify the clear extract

as described in 7.2.4.

In case residues with acetic groups (e.g. phenoxy carboxylic acids) shall be determined, a second aliquot

of the centrifuged extract from 7.1 is filled into an injection vial and analysed directly with LC-MS/MS to

avoid losses of acidic groups by PSA clean-up.
25 mg PSA and 25 mg C18 sorbent are needed per ml of extract.
7.2.3 Freezing-out of co-extracted fat and clean-up with amino-sorbent

Store an aliquot of the extract from 7.1 containing the internal standard for at least 1,5 h at ≤ – 18 °C to

freeze out most of the fat in the extract. For separation of the latter filter the extract over cotton wool

(5.20). Take 4 ml from the cold and fat separated solution for dispersive SPE.

Transfer an aliquot of 4 ml of the acetonitrile phase into a Polypropylene-single use tube (6.3) already

containing 100 mg of PSA (5.7). Close the tube, shake vigorously for 30 s and centrifuge (5 min

at ≥ 1 000 g). Immediately isolate and acidify the clear extract as described in 7.2.4.

If residues with acetic groups shall be determined, transfer a second aliquot into an injection vial and

analyse directly with LC-MS/MS to avoid losses of acidic groups with PSA clean-up.

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. 25 mg PSA sorbent are needed per ml of extract.

7.2.4 Extract stabilization

Transfer an aliquot of 3 ml of the cleaned-up extract from 7.2.2 or 7.2.3 into a screw cap storage vial

(6.3), taking care to avoid sorbent particles of being carried over, and slightly acidify by adding 30 µl of

---------------------- Page: 10 ----------------------
kSIST-TS FprCEN/TS 17062:2019
FprCEN/TS 17062:2019 (E)

a 5 % formic acid solution in acetonitrile (5.6). Transfer the pH-adjusted extract into auto-sampler vials

and use it for 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 (5.6) are necessary.
7.3 Determination by liquid chromatography with tandem mass spectrometry (LC-
MS/MS)

Inject the sample extracts derived from 7.2.2 to 7.2.4 and standard solutions (5.13) into the LC

instruments in an appropriate sequence. This may involve bracketing of the sample extracts with the

calibration solutions.

The measurement may 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, but are provided as examples, only.

For some gradient/column combinations it is necessary to mix the extract with water or the aqueous

mobile phase to achieve a sufficient separation of the analytes.

NOTE If extracts are diluted with water or aqueous mobile phases it is important to avoid that non-polar

parts of the extract precipitate or emulsions occur. This could lead to losses of lipophilic analytes. In this case an

injection applying an injector programme can be helpful (see A.4).

The chromatographic conditions as outlined in Annex A have been shown to be satisfactory.

Suitable experimental conditions of LC-MS/MS measurements are outlined in CEN/TR 15641 [3].

Nevertheless, individual tuning of the compounds on the instrument that is used for measurement

usually provides better sensitivities.
8 Evaluation of results
8.1 Identification and quantification

For the identification of residues in the final extract, use relative retention time ratio against the ISTD

(Rt /Rt ) obtained from the same run. Check positive results by comparing the intensity ratios

(A) (ISTD)

between the SIM masses (m/z) or SRM transitions of the analyte. The expected intensity ratios can be

determined with the standard solutions. If the ratios of the samples and the standards have a variation

of more than 30 %, the rules of EU Quality Control Procedures will be followed [2]. According to these

procedures positive results shall be ensured by using additional measures, e.g. additional SIM masses or

SRM transitions or other chromatographic conditions (column, eluents).

For calibration and for checking the linearity of detection of each substance, plot the peak area ratio or

cal cal

peak height ratio of pesticide and internal standard y / y (if an internal standard is used) versus

A ISTD
cal cal

the concentration ratio of the analyte against the ISTD ( / ) in the standard solution (5.13). If

ρ ρ
A ISTD
cal

no internal standards are used, plot the peak areas or peak height y against the concentration of the

cal
analyte ρ .

The calibration area shall be adapted to the residue concentration and should not exceed a decimal

power. Possibly more calibration graphs shall be established using the standard solution. The

calibration function is selected according to FprCEN/TS 17061:2019, 6.2.1.

For a first estimation of the residue level or for the verification of absence of residues, solvent based

standards (5.13.1) can be used. They can also be used for quantification, if it was shown that no

enhancement or suppression of the analyte signal through matrix occurs. If relevant residue levels are

---------------------- Page: 11 ----------------------
kSIST-TS FprCEN/TS 17062:2019
FprCEN/TS 17062:2019 (E)

observed (e.g. with possible MRL violation) matrix matched standard shall be preferred for exact

quantification.
8.2 Calculation of residue concentrations using the internal standard

The determination of the concentration of the analyte ρ in the final extract is performed by using the

measured peak area ratio or peak height ratio from pesticide and internal standard y /y in the

A ISTD

sample as described in FprCEN/TS 17061. Calculate the mass fraction w of the analyte in the sample,

in milligram per kilogram with Formula (1):
ρ ×V
A ex
w = (1)
Sample
where

is the mass concentration of the analyte in the final extract, in microgram per millilitre;

V is the volume of acetonitrile used in 7.1, in millilitre;
m is the mass of test portion in 7.1, in gram.
Sample
8.3 Calculation of residue concentrations without internal standards

Determine the concentration of the analyte ρ in the final extract by using the measured peak area or

peak height from pesticide y in the sample as described in FprCEN/TS 17061:2019, 6.4.2 to 6.4.5.

Calculate the mass fraction w of the analyte in the sample by using Formula (1).
8.4 Calculation of residue concentration using the standard additions approach

In case of suspected violative residues, or for compounds which are known to be strongly affected by

matrix-induced enhancement or suppression phenomena, standard additions are recommended

provided that the function between response and concentrations at the concentration range in question

is linear.

In case of the standard addition to the final extract, determine the concentration of the analyte ρ in

the final extract using a linear regression graph of peak areas or peak height versus spiked

concentrations and the volume of the applied aliquot of the final extract as described in

FprCEN/TS 17061:2019, 6.6.1. Calculate the mass fraction w of the analyte in the sample by using

Formula (1).

In case of standard addition to the sample, determine the mass of the analyte in the weighted sample

using a linear regression graph of peak areas or peak height versus spiked analyte masses as described

in FprCEN/TS 17061:2019, 6.6.2. The mass fraction of the analyte in the sample is the quotient of the

mass of the analyte m in the weighted sample and the weighted sample m .
A Sample

NOTE With the standard addition approach, the sought analyte concentration is determined using linear

extrapolation. Therefore, it is important that the analyte has linear detection properties in the investigated

calibration range. It can be necessary to dilute the extract to achieve the calibration range using LC-MS(/MS).

9 Precision

The method was validated in two interlaboratory tests with representative analytes. The results for LC-

MS/MS validation and ongoing verification are shown in Annex B. An updated and detailed list of

validation results can be found in the internet www.eurl-pesticides-datapool.eu operated by the EU

---------------------- Page: 12 ----------------------
kSIST-TS FprCEN/TS 17062:2019
FprCEN/TS 17062:2019 (E)

reference laboratories for pesticides residues. A summary of the validation data are given in Table B.1

in Annex B. Data for compounds are given there if these were available from at least two laboratories

and if the number of individual results was at least five (in total). Further information on how to

conduct recovery studies is given in Annex D.

Tested matrices were vegetable oils such as olive oil, sunflower seed oil, sesame oil, flax seed oil, rape

seed, oil, grape seed oil, thistle oil and pumpkin seed oil, see www.eurl-pesticides-datapool.eu.

However, it has been noted that coconut oils containing a high amount of short chain fatty acids can

cause problems as they behave in a different way to the other oils.

With addition of the levels 0,01 mg/kg to 0,10 mg/kg the recoveries obtained were usually between

70 % and 110 %.

The detection limits are depending on the analyte of interest and the sensitivity of the equipment. In

general, pesticide residues of 0,01 mg/kg (lowest maximum residue level in most cases) can be

analysed with modern systems.
10 Test report
The test report shall contain at least the following:
— all information necessary for the identification of the sample;
— a reference to this Technical Specification;
— the results and the units in which the results have been expressed;
— the date and type of sampling procedure (if possible);
— the date of receipt of sample in the laboratory;
— the date of test;
— any particular observations made in the course of the test;

— any operations not specified in the method or regarded as optional which might have affected the

results.
---------------------- Page: 13 ----------------------
kSIST-TS FprCEN/TS 17062:2019
FprCEN/TS 17062:2019 (E)
Annex A
(informative)
Examples of experimental conditions
The following LC-MS operating conditions have been shown to be satisfactory.
A.1 HPLC-System 1
For most LC-amenable compounds:
Column ZORBAX™ Eclipse XDB-C18, length 150 mm, inner diameter 2,1 mm, particle
size 3,5 µm
Mobile phase A1 (5.14) Ammonium formate solution in water, c = 5 mmol/l
Mobile phase B1 (5.15) Ammonium formate solution in methanol, c = 5 mmol/l
Column temperature 40 °C
Injection volume 3 µl
Table A.1 — Flow rate and elution gradient
Time Flow Mobile phase A1 Mobile phase B1
min rate % %
µl/min
0 300 50 50
20 300 0 100
25 300 0 100
26 300 50 50
30 300 50 50
A.2 HPLC-System 2

For polar compounds (e.g. with log P < 0,5) 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 A1 (5.14) Ammonium formate solution in water, c = 5 mmol/l
ZORBAX™ Eclipse XDB-C1
...

Questions, Comments and Discussion

Ask us and Technical Secretary will try to provide an answer. You can facilitate discussion about the standard in here.