Quick Method for the Analysis of Highly Polar Pesticides in Food Involving

Extraction with Acidified Methanol and LC- or IC-MS/MS Measurement

I. Food of Plant Origin (QuPPe-PO-Method)
Version 12.1 (17.03.2023, Document History, see page 99)
Check for latest version of this Method under www.quppe.eu ; older versions: obsolete versions
Authors: M. Anastassiades; A.-K. Wachtler; D. I. Kolberg; E. Eichhorn; H. Marks; A. Benkenstein; S. Zechmann;
D. Mack; C. Wildgrube; A. Barth; I. Sigalov; S. Görlich; D. Dörk; G. Cerchia
EU Reference Laboratory for pesticides requiring Single Residue Methods (EURL-SRM)
Contact: CVUA Stuttgart, Schaflandstr. 3/2, DE-70736 Fellbach, Germany, www.eurl-pesticides.eu; [email protected]
Note: Changes from V12 to V12.1 are highlighted in yellow

1. Scope and Short Description ............................................................................................................................................................................................. 2

2. Apparatus and Consumables............................................................................................................................................................................................. 3
3. Chemicals ............................................................................................................................................................................................................................. 4
4. Disclaimer ............................................................................................................................................................................................................................. 7
5. Procedure ............................................................................................................................................................................................................................. 7
5.1. Sample preparation........................................................................................................................................................................................................... 7
5.2. Extraction / Freeze-Out / Centrifugation / Cleanup / Filtration .................................................................................................................................... 8
5.3. Preparation of blank extracts ......................................................................................................................................................................................... 13
5.4. Recovery experiments .................................................................................................................................................................................................... 13
5.5. Preparation of calibration standards ............................................................................................................................................................................. 14
Solvent-based calibration standards .................................................................................................................................................................... 14
Matrix-based and matrix-matched calibration standards................................................................................................................................... 14
Standard-Additions approach ............................................................................................................................................................................... 15
Procedural calibration standards .......................................................................................................................................................................... 16
5.6. LC-and IC-MS/MS analysis............................................................................................................................................................................................ 16
General hints on analytes to avoid pitfalls ........................................................................................................................................................... 22
Method 1.1 (M1.1): “Gly&Co. AS 11” ................................................................................................................................................................... 33
Method 1.2 (M1.2): “Gly&Co. AS 11-HC” ............................................................................................................................................................ 35
Method 1.3 (M1.3): “Gly&Co. Hypercarb”............................................................................................................................................................ 37
Method 1.4 (M1.4): “PerChloPhos” ...................................................................................................................................................................... 39
Method 1.5 (M1.5): “Gly&Co. on Trinity Q1” ....................................................................................................................................................... 41
Method 1.6 “Gly&Co. on Torus DEA; (M1.6a)” or “Gly&Co. on Anionic Polar Pesticide Column (APPC); (M1.6b)” ................................. 43
Method 1.7 “PerChloPhos on Torus DEA; (M1.7a)” or “PerChloPhos on Anionic Polar Pesticide Column (APPC); (M1.7b)” ................ 47
Method 1.8 (M1.8): “PerChloCyanMalein on Anionic Polar Pesticide Column (APPC)” ............................................................................... 49
Method 1.9 (M1.9): “Gly&Co. on Raptor Polar X” ............................................................................................................................................ 50
Method 1.10 (M1.10): “Gly&Co. on Obelisc N” ................................................................................................................................................. 52
Method 2 (M2): “Fosetyl and Maleic Hydrazide” .............................................................................................................................................. 53
Method 3 (M3): “Amitrole&Co.” ........................................................................................................................................................................... 55
Method 4.1 (M4.1): “Quats&Co. Obelisc R” ...................................................................................................................................................... 57
Method 4.2 (M4.2): “Quats&Co. BEH Amide” ................................................................................................................................................... 59
Method 5 (M5): “Quats&Co. MonoChrom MS” ................................................................................................................................................. 63
Method 6 (M6): “Streptomycin and Kasugamycin”........................................................................................................................................... 64
Method 7 (M7): “Morpholine, Diethanolamine and Triethanolamine” ............................................................................................................ 65
Method 8 (M8): “Triazole derivative metabolites (TDMs)”............................................................................................................................... 66
Method 9 (M9): “Difluoroacetic acid and Trifluoroacetic acid” ........................................................................................................................ 67
Method 10 (M10): “Triazole derivative metabolites (TDMs) on Torus DEA”................................................................................................. 68
Method 11 (M11): “Gly&Co. by IC on AS19” .................................................................................................................................................... 69
5.7. Calibration and Calculations .......................................................................................................................................................................................... 71
6. Stability and purity of standards ...................................................................................................................................................................................... 74
7. Performance Data ............................................................................................................................................................................................................. 76
8. References ......................................................................................................................................................................................................................... 87
9. ANNEX................................................................................................................................................................................................................................ 88

EU Reference Laboratory for pesticides requiring Single Residue Methods (EURL-SRM)

1. Scope and Short Description
A method is described for the residue analysis of very polar, non-QuEChERS-amenable, pesticides in foods of plant
origin such as fruits, vegetables, cereals, dry pulses, oily seeds and nuts as well as in honey.
Residues are extracted from the test portion following water adjustment and addition of acidified methanol. In the
case of cereals, pulses, nuts and oily seeds, EDTA is added for the complexation of metal ions, such as calcium and
magnesium, which can affect the analysis of certain compounds (e.g. glyphosate and AMPA). The mixture is centri-
fuged, filtered and directly analyzed by LC-MS/MS. Various LC- or IC-MS/MS methods allowing simultaneous analysis
of different combinations of pesticides are provided. Quantification is in most cases performed employing isotope
labeled analogues of the target analytes as internal standards (IL-ISs). So far available, these IL-ISs are added directly
to the test portion at the beginning of the procedure to compensate for any factors having an influence on the re-
covery-rates such as volume-deviations, analyte losses during sample preparation as well as matrix-effects during
measurement. Due to the simplicity of the procedure strong matrix-effects are frequently observed.

Shortcut-Links to useful information

 Flow Chart - QuPPe-PO-Method at a glance (procedure for most commodities)

 Flow Chart - QuPPe-PO-Method at a glance (procedure for cereals, pulses, nuts and oily seeds)
 Pipetting Scheme (exemplary) - Preparation of Calibration Standards
 Pipetting Scheme (exemplary) - Standard-Additions-Approach
 Scope Overview- LC-Methods covering ESI-pos. Analytes
 Scope Overview - LC-Methods covering ESI-neg Analytes
 General hints on analytes to avoid pitfalls
 Calibration and Calculations
 Analyte Stability
 Performance Data
 Conversion Factors (between purchased standards and target analytes)
 Analyte Stock and Working Solutions (exemplary)
 Exemplary Providers of IL-ISs (Isotopically Labelled Internal Standards)
 IL-IS-Working Solutions (exemplary)
 Water Addition Overview

How to Cite (proposal):

Quick Method for the Analysis of Highly Polar Pesticides in Food Involving Extraction with Acidified Methanol and LC- or IC-
MS/MS Measurement - I. Food of Plant Origin (QuPPe-PO-Method) – Version 12 (published on EURL-SRM website on July 23,
2021); M. Anastassiades; A.-K. Wachtler; D. I. Kolberg; E. Eichhorn; H. Marks; A. Benkenstein; S. Zechmann; D. Mack; C. Wild-
grube; A. Barth; I. Sigalov; S. Görlich; D. Dörk and G. Cerchia.
URL: https://www.eurl-pesticides.eu/docs/public/tmplt_article.asp?CntID=887&LabID=200&Lang=EN

EU Reference Laboratory for pesticides requiring Single Residue Methods (EURL-SRM) 2 of 102
2. Apparatus and Consumables

2.1. Powerful sample processing equipment,

for milling samples. For fruits and vegetables, e.g. Stephan UM 5 or Retsch200 by Retsch Grindomix GM 300 or Vor-
werk-Thermomix TM31-1. For dry commodities such as cereals, e.g. ZM 200 by Retsch equipped with a 0.5 mm sieve.

2.2. Plastic tub,

for filling-in liquid nitrogen to immerse the samples prior to milling. e.g. 20 to 40 L polypropylene or polyethylene
tub with handles. Styrofoam boxes are also suitable. Take precautions when working with liquid nitrogen.

2.3. 50 mL centrifuge tubes with screw caps,

e.g.: a) reusable 50 mL Teflon® centrifuge tubes with screw caps (e.g. Nalgene/Rochester, USA; Oak-ridge, article-no.
3114-0050) or b) disposable 50 mL centrifuge tubes (e.g. Sarstedt / Germany, 114x28 mm, PP, article-no. 62.548.004).

2.4. 10 mL centrifuge tubes with screw caps,

for the d-SPE step (5.2.5), e.g.: disposable 10 mL PP-tubes by Simport/Beloeil (Canada), article-no. T550-10AT.

2.5. Automatic pipettes,

suitable for handling volumes of 10 to 100 μL, 200 to 1000 μL and 1 to 10 mL.

2.6. 10 mL solvent-dispenser,
for the acidified methanol (3.6).

2.7. Mechanical shaker,

suitable for 50 mL-centrifuge tubes, e.g. Geno/Grinder® 2010; SPEX® SamplePrep.

2.8. Water Bath,

adjustable to at least 80°C and automatically shaking.

2.9. Centrifuge,
suitable for the centrifuge tubes employed in the procedure (2.3) and capable of achieving > 3,000 g. E.g. Rotanta
460 by Hettich, Tuttlingen/Germany. Centrifuges capable of achieving higher centrifugal forces and of refrigerating
the sample during centrifugation (e.g. Avanti JXN-26 by Beckman Coulter, Brea/USA) are to be preferred.
Notes: Higher relative centrifugal forces (e.g. RCFs > 10,000 g) and cooling during centrifugation (e.g. to -10°C) are beneficial by
causing increased precipitation of matrix components. Check centrifuge tubes for suitability for higher velocities.

2.10. Disposable syringes,

suitable to the filters used; e.g. 2 or 5 mL disposable polypropylene syringes with luer tip by Macherey-Nagel, Düren
/ Germany (Ref. 729100 and 729101 respectively). These are suitable for the syringe filters listed below (2.11).

2.11. Disposable syringe filters,

e.g. . Ø 25 mm CHROMAFIL® filters and 0.2 µm pore size filters of the following materials: Hydrophilized polytetra-
fluoroethylene (H-PTFE) or Cellulose Mixed Ester or Polyester (Ref. No. 729245, 729006 and 729021 respectively) all
by Macherey-Nagel, Düren / Germany. 0.45 µm pore size filters of the above types (Ref. No. 729246 H-PTFE) may be
attached in front of the 0.2 µm filters if the latter get clogged when used directly. In case of filter-clogging during
honey extracts filtration, filters with 5.0 µm pore sizes (e.g. Rotilab® PTFE filters by ROTH Ref. No. SE4M075I99) may
be used for pre-filtration (or even instead of the filters with smaller pore-sizes), as pollen grains are typically > 10 µm
in diameter.

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Note: Check filters for any contamination with analytes of interest. Significant levels of Perchlorate and Chlorate were detected
in the above mentioned polyester filters. For testing suitability consider the worst-case scenario, where filters are clogged quickly
(e.g. elute only 200 µL through each filter). Such severe clogging was for example observed with industrially milled cereals, pears
and pineapples.

2.12. Ultrafiltration filters,

5 or 10 kDa molecular weight cutoff filters suitable for centrifuges, e.g. Vivaspin® 6 mL 5 kDa entailing Polyethersul-
fone membranes OR Amicon® Ultra-15 10K entailing Ultracel® low binding regenerated cellulose.

2.13. Autosampler vials,

suitable for LC auto-samplers, e.g. Vials Screw top 2 mL Cat No. 9502S-PP-CLEAR, 12x32 mm MicroSolv Technology
Corporation (MTC), USA; Lids for plastic vials: Lid G9-L/Sil-CS Art.-No. 2.301398-Blau WE13989, Ziemer GmbH, Lang-
erwehe / Germany
Notes: The use of plastic vials is highly recommended as several of the compounds covered by this method (e.g. Phosphonate,
Nicotine, Paraquat, Diquat, Streptomycin and Glyphosate)1 tend to interact with glass-surfaces. Such interactions with glass
surfaces are typically more pronounced in solutions consisting of aprotic solvents (e.g. acetonitrile). Increasing water content
and/or acidity typically reduces such interactions. Percent losses due to such interactions are typically higher at low concentra-
tions.

2.14. Volumetric flask with stoppers,

for the preparation of stock and working solutions, e.g. 20 mL; 25 mL; 50 mL, 100 mL glass flasks.
Notes: The use of plastic flasks and stoppers is highly recommended as several of the compounds covered by this method tend
to interact with glass-surfaces (see examples under 2.13).

2.15. Screw-cap storage vessels,

for storage of sample extracts or storage of stock and working solutions, e.g. 20 mL.
Notes: The use of plastic flasks and stoppers is highly recommended as several of the compounds covered by this method tend
to interact with glass-surfaces (see examples under 2.13).

2.16. LC-MS/MS instrumentation,

equipped with ESI source and appropriate columns, see details in chapters 5.6.2 to 5.6.225.6.21.

2.17. IC-MS/MS instrumentation,

equipped with ESI source and appropriate columns, see details in chapters 5.6.22.

3. Chemicals
Unless otherwise specified, use reagents of recognized analytical grade. Take every precaution to avoid possible con-
tamination of water, solvents, sorbents, inorganic salts, etc.

3.1. Water (deionized),

for water additions to the samples

3.1. Water, ultrapure

e.g. prepared by a laboratory water purification system. Commercially available MS-quality water can be
used for LC-MS/MS mobile phases and IC-quality water for IC-MS/MS mobile phases.

1
The list of compounds requiring plastic vessels is not comprehensive (this remark applies to the entire document).
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 3.2. Methanol at least HPLC quality,

for the preparation of mobile phases preferably use MS-quality methanol.

3.3. Acetonitrile at least HPLC quality,

for the preparation of mobile phases preferably use MS-quality acetonitrile.

3.4. Formic acid (concentrated; > 98%),

for the preparation of mobile phases preferably use MS-quality formic acid.

3.5. Acetic Acid (concentrated; >98%),

for the preparation of mobile phases preferably use MS-quality acetic acid.

3.6. Acidified methanol,

for the extraction of the majority of samples, prepared by pipetting 10 mL formic acid (3.4) into a 1000 mL volumetric
flask and filling up to volume with methanol (3.1).

3.7. C-18 sorbent (ODS-sorbent),

e.g. Polygoprep 30-300 µm Macherey-Nagel GmbH & Co KG/Düren (Germany), article-no. 711720.100).

3.8. Citric acid-monohydrate (p.a.)

3.9. Dimethylamine,
e.g. 40 % by Fluka (article-no. 38940).

3.10. Ammonium formate (p.a.)

3.11. Ammonium citrate-tribasic, anhydrous (p.a.)

3.12. Sodium hydroxide (p.a.)

3.13. Di-Sodiumtetraborate-decahydrate (p.a.)

3.14. Ethylenediaminetetraacetic acid tetrasodium

e.g. tetrasodium dihydrate salt (CAS Number 10378-23-1): E6511 Sigma Aldrich (MW=416.20)
OR tetrasodium tetrahydrate salt (CAS No.: 13235-36-4): 34103-M EMD Millipore/Merck (MW=452.23)

3.15. 10% aqueous EDTA solution,

prepared by weighing 15.85 EDTA tetrasodium tetrahydrate (OR 14.59 g EDTA tetrasodium dihydrate) into a 100 mL
volumetric flask with stopper, dissolving it in 80 mL water and filling up to 100 mL with water. This solution contains
10 % (w/v) EDTA tetra-anion.

3.16. Dry ice,

technical grade can be used; periodically check that it does not contain compounds of interest at relevant levels.

3.17. Pesticide Standards,

of known purity.

3.18. Pesticide stock solutions,

e.g. 1 mg/mL solutions of pesticide standards (3.17) in a water miscible solvent. Suggestions of solvents suitable for
the preparation of stock solutions can be found in Table 43.
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Notes: Keep in mind that some standards are sold as salts or hydrates. Some exemplary conversion factors to be applied be-
tween typical standards and the analytes are shown in Table 42. Keep solutions in plastic vessels as several of the compounds
covered by this method tend to interact with glass-surfaces (see examples under 2.13).

3.19. Pesticide working solutions / mixtures,

prepared at appropriate concentrations by diluting pesticide stock solutions (3.18) of one or more pesticides with
water-miscible solvents as required for the spiking of samples in recovery experiments (5.4) or for the preparation
of calibration standards (5.5). Suggestions of solvents for preparing stock solutions can be found in Table 43.
Notes: Keep solutions in plastic vessels as several of the compounds covered by this method tend to interact with glass-surfaces
(see examples under 2.13).

3.20. Internal Standards (ISs),

Exemplary sources are shown in Table 44. Check whether the ISs contain native compounds at levels, which would
lead to false positives or quantification errors.

3.21. IS-stock solutions,

e.g. 1 mg/mL solutions of ISs (3.20) in a water miscible solvent (e.g. methanol, acetonitrile, water or mixtures
thereof). For solvent-suggestions see Table 43 in the ANNEX.
Notes: Keep solutions in plastic vessels as several of the compounds covered by this method tend to interact with glass-surfaces
(see examples under 2.13).
In general the absolute concentrations of the IL-IS-solutions are not important as long as the IL-IS-concentration in the final
extract is high enough to produce a well measurable signal that is not relevantly disturbed by co-eluting matrix components. An
IL-IS standard with a relatively low purity may be still acceptable as long as the content of native analytes (irrespective whether
they were initially present - as impurity - or whether they were formed during storage of working solutions) is low enough to
exclude false positive results and to ensure that any influence on quantification of positive results is negligible. Some examples
where care is needed to avoid formation of native analytes from IL-ISs are N-Acetyl-Glyphosate (acetyl D3) that may de-acetylate
into native Glyphosate, Fosetyl-D5 that tends to hydrolyse to native Phosphonic acid (see 5.6.3 under Hints on Method 1.2) and
Maleic Hydrazide D2 the standard of which typically contains a small, but relevant, fraction of the native compound as impurity
(see 5.6.9).
For quantification purposes it is of foremost importance that the ratio between the absolute IL-IS amount added to the sample
prior to extraction (or to the isolated aliquot of the sample extract) and the absolute amount of IL-IS added to the calibration
standard solutions is known as it is used in calculations.

3.22. IS-working solution I (IS-WSln-1) for spiking samples prior to extraction,

prepared at appropriate concentrations by diluting IS-stock solutions (3.21) of one or more ISs with water-miscible
solvents. Suggestions for solvents are shown in Table 43 and suggestions for the concentrations in Table 45.
Notes: Keep solutions in plastic vessels 2.15 as several of the compounds covered by this method tend to interact with glass-
surfaces (see examples under 2.13).
In presence of water and especially at high pH levels, Phosphonic acid 18O3 will gradually convert to 18O216O1 , 18O116O2 and even-
tually of 16O3 (native) Phosphonic acid. The 18O3 Phosphonic acid standard solution provided by the EURLs should be preferably
diluted in acetonitrile, where it was shown to be stable for long periods.

3.23. IS-working solution II (IS-WSln-2) for preparation of calibration standards,

prepared at appropriate concentrations by diluting IS-WSln-1 (3.22) with water-miscible solvents. Suggestions for
solvents are shown in Table 43 and for concentrations in Table 45.
Notes: Keep solutions in plastic vessels as several of the compounds covered by this method tend to interact with glass-surfaces
(see examples under 2.13).
For short term usage (e.g. up to one month) the IL-IS of Phosphonic acid can be diluted in acidified methanol (3.6).

3.24. LC-MS/MS mobile phases and other consumables,

see details in chapters 5.6.2 to 5.6.21.

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 3.25. IC-MS/MS mobile phases and other consumables,

see details in chapter 5.6.22.

4. Disclaimer
This method refers to several trade names of products and instruments which are commercially available and suita-
ble for the described procedure. This information is given for the convenience of the users of this method and does
not constitute an endorsement by the EURL of the products named. The application of this method may involve
hazardous materials, operations and equipment. It is the responsibility of the users of this method to establish ap-
propriate safety and health practices prior to use. Any consumables and chemicals used in the procedure should be
periodically checked, e.g. through reagent blank tests, for any relevant levels of the analytes of interest.

5. Procedure

5.1. Sample preparation

To obtain representative test-portions from the laboratory sample, proceed as required by the valid regulations and
guidelines.
Fruits and vegetables are preferably milled cryogenically (e.g. using dry ice). This is done to reduce analyte degrada-
tion and particle sizes, with the latter resulting in improved homogeneity and residue accessibility. One possibility
for cryogenic milling is to cut large units coarsely to ca 3x3 cm pieces, freeze them and then mill them for ca. 1-2
minutes with a powerful mill. Then add dry ice (ca. 150-200 g per 500 g sample) and continue milling until barely any
carbon dioxide fumes are observed. Alternatively fill a plastic or polystyrene container with a ca 5-10 cm thick layer
of liquid nitrogen and immerse the sample pieces into liquid nitrogen. When completely frozen transfer the material
into a powerful knife mill and grind at high speed until it gets a free flowing snow-like consistency. If necessary crush
large units with a hammer before milling. If the material starts defrosting during milling, add some more liquid nitro-
gen or dry ice and continue milling as described above.
Dry commodities (e.g. cereals, pulses) are intensively milled to reduce particle size and improve the accessibility of
residues enclosed in the interior of the materials. Particle sizes (e.g. <500 µm) are preferable. The larger the particles
are the longer the extraction times required to achieve quantitative extraction of systemically distributed com-
pounds. Ultra centrifugal mills with 500 µm sieves were found to be suitable for this purpose. Addition of dry ice
during milling (e.g. at a sample: dry ice ratio of 2:1) reduces heat. The use of knife mills is also possible but prolonged
milling times are needed to reduce the size of particles. Add some dry ice periodically to reduce heat formation.
Alternatively a two stage milling can be helpful. For this a representative portion of the first milling step is transferred
to a second smaller mill and homogenized further.
Dry and oily commodities (e.g. oily seeds and nuts) tend to form a thick paste that prevents proper milling and is
difficult to handle, when using knife mills at room temperature. Milling with ultra centrifugal mills typically leads to
a clogging of the filters. For such materials cryogenic grinding with a powerful knife mill is recommended. Precool
the mill with dry ice and then mill the material at a sample to dry ice ratio of ca 2:1 until a fine powder is obtained.
Keep temperature low to avoid that the material becomes clumpy and thus more difficult to handle. Alternatively
immerse the sample in a plastic or polystyrene container containning liquid nitrogen. When completely frozen trans-
fer it into a powerful knife mill and grind until a fine powder is obtained. Do not mill too long as the material with
thaw and become clumpy and thus more difficult to handle.
For dried fruits and similar commodities ( 1 ̴ 5 to ≤ 40% water content) the following procedure is proposed: Weigh
500 g of frozen dried fruits, add X g* of cold water (see
Table 1) and homogenize the mixture using a strong mixer (2.1), if possible with addition of dry ice to prevent or
slow-down any chemical and enzymatic reactions (3.13). Weigh Y g* of homogenate (see

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Table 1; corresponding to 5 g sample).

Table 1: Weight of dried fruits required for slurry homogenization and analztical portions of rehydratized homogenates to be
emplozed for analzsis
Moisture content of product Water amount added Weight of analytical portion
(X g) (Y g; corresponding to 5 g of original dry sample)
1
̴ 5 to <25 % 900 g 14 g
25 to <35 % 850 g 13.5 g
≥35 to 40 % 800 g 13 g

Alternatively, immerse the sample material in a plastic or polystyrol container containing liquid nitrogen. When com-
pletely frozen transfer it into a powerful knife mill and grind until a fine powder is obtained. Do not mill too long and
quickly transfer the frozen powder into a storage container and place it into the freezer to avoid that it becomes
clumpy and more difficult to handle.
For freeze-dried fruit and vegetables homogenize with a high speed knife mill preferably adding dry ice to keep the
sample cool. Thereof 2 g sample may be employed for analysis (as in the case of spices, herbs and other extract-rich
commodities).

5.2. Extraction / Freeze-Out / Centrifugation / Cleanup / Filtration

A flow chart of the analytical procedure is shown in Figure 1 (for most commodities) and in Figure 2 (for pulses, nuts
and oily seeds)

Weighing of analytical portions

Weigh a representative analytical portion (ma) of the sample homogenate (5.1) into a 50 mL centrifuge tube (2.3).
In case of fresh fruits and vegetables and juices weigh 10 g  0.1 g of the homogenized sample. In case of cereals,
dried pulses, oily seeds, nuts, dried fruits, dried vegetables, dried mushrooms and honey weigh 5 g  0.05 g of the
homogenates. In case of dry fruits rehydrated according to 5.1, weigh Y g (e.g. 13.5 g  0.1 g, see
Table 1) of the re-hydrated and homogenized material (corresponding to 5 g sample). Smaller analytical portions
may have to be used for extract-rich commodities, such as spices, herbs or fermented products, or commodities with
very high water-absorbing capacity not allowing proper extraction.

Adjustment of water content

For commodities with ≥ 80% of natural moisture, water adjustment to 10 mL is not essential and may be skipped
when appropriate ISs are employed before any aliquotation. If no IS is used, add water (3.1), as indicated in Table
46 to minimize volumetric errors. Continue with step 5.2.3.
For commodities with < 80% of natural moisture (except honey, chia seeds and lineseeds, see notes), add water
(3.1) to the analytical portion (5.2.1) to reach a total water content of approx. 10 g according to the indications in
Table 46. No further water adjustment is needed where re-hydrated commodities (see 5.1) are employed. Continue
with step 5.2.3.
Notes: Keep in mind that the water volume adjustments in Table 46 are approximate.
In the case of honey, it needs to be taken into account, that sugars completely dissolve in the methanol/water mixture and that
they contribute to the volume. Instead of adding 9 mL of water (to account for ~1 mL of water contained in honey with ~20%
moisture content), the amount of water to be added is 7.5 mL.
For oily seeds, nuts and pulses the water contained in the aqueous EDTA solution (added during the extraction step (5.2.3) is
also considered in the overall water content. Therefore 9 mL of water + 1 mL of aqueous EDTA solution are added in total. See
also Table 46.
In the case of chia seeds and lineseeds (flaxseeds) adding water directly to the samples leads to a soaking and the formation
of a gellike layer, which hinders the accessibility of residues. To suppress this phenomenon, change the order of solvent addi-

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tion as follows. First add 10 mL acidified methanol (3.6) and 100 µL formic acid (3.4), shake shortly, and then add the 9 mL wa-
ter and the 1 mL EDTA solution and continue with 15 min shaking as described under 5.2.3. Then continue with step 5.2.4-(2)
or (3) and further with step 5.2.5-(2).

Extraction
A) General procedure
(1) All commodities of plant origin except cereals, pulses, nuts and oily seeds: Add 10 mL acidified methanol
(3.6) and 100 µL (or another appropriate small volume) of the IS-WSln-1 (3.22) containing isotopically la-
beled analogues of the analytes of interest (added IS mass = mISsample). Close the tube and shake vigorously
for 1 to 15 min by hand or a mechanical shaker.
(2) Cereals, pulses, nuts and oily seeds: Add 10 mL acidified methanol (3.6) and 100 µL (or another appropriate
small volume) of the IS-WSln-1 (3.22) containing isotopically labeled analogues of the analytes of interest
(added IS mass = mISsample) and agitate shortly to distribute the ISs. Add an extra amount of 100 µL formic
acid (3.4). Close the tube and shake for a few seconds to distribute the acid and allow proteins to coagulate.
Add 1 mL 10% aqueous EDTA solution (3.15) and shake for 15 min by an automatic shaker. For chia seeds
and lineseeds please refer to the notes under 5.2.2. Where no automatic shaker is available, dry products
may be shaken for 1 min by hand followed by a soaking period of 15 min and a subsequent second 1 min
vigorous shaking by hand.
Notes: Where no IL-ISs are used the aim should be to reach a total volume of the liquid phase as close as possible to 20 mL.
This volume will mainly consist of the water naturally contained in the sample, the water added during the procedure (includ-
ing that of the EDTA solution), the extraction solvent added, the IS solution added as well as the extra formic acid added. A
volume contraction is also taking place and is partly matched by the IS and the formic acid. Further alternatives to avoid errors
due to volumetric deviations are calibrations that compensate for recovery, such as standard additions to sample portions and
procedural calibrations using a suitable blank matrix. The 20 mL volume of the extractant corresponds to 0.5 g / 0.25 g sample
per mL extract if 10 g / 5 g sample are used. Where the raw extract is diluted with acetonitrile for cleanup purposes (see 5.2.5-
(2) concerning pulses, oily seeds and nuts) the final concentration in the extract is reduced to 0.125 g/mL.
For screening purposes, the IS can be alternatively added to a sample extract aliquot (e.g. the 1 mL aliquot transferred to the
autosampler vial, see below), assuming that 1 mL extract entails exactly 0.5 or 0.25 or 0.125 g sample equivalents, see above.
This way, the added amount of IS per sample can be drastically reduced (e.g. 20-fold if added to 1 mL extract). The IS added at
this step will compensate for matrix effects including retention-time shifts but not for recovery and volume deviations during
extraction. The quantitative result should therefore be considered tentative. For more accuracy, samples should be re-ex-
tracted with the IS being added to the analytical portion before aliquotation.
Particle size plays an important role for dry products (e.g. cereals, pulses) as far as extractability is concerned. If a considerable
fraction of the particles exceed 500 µm, shaking or soaking times may have to be extended, otherwise the extraction will need
to involve additional breakup of the sample particles, e.g. by the use of a high speed dispenser (e.g. Ultra Turrax).
The addition of EDTA solution is highly recommended when targeting analytes showing poor recoveries in absence of EDTA.
Affected are compounds with a tendency to form complexes with metals, such as Glyphosate and metabolites, Glufosinate and
metabolites. If affected analytes are not targeted, EDTA addition may be skipped and 10 mL of water are added.

B) Procedure for Paraquat and Diquat

For the analysis of Paraquat and Diquat add 10 mL of a 1:1 mixture of methanol + aqueous HCl 0.1M to the water-
adjusted analytical portion (from 5.2.2), close the vessel and shake initially for 1 minute. Then place the extraction
vessel in a shaking water bath (2.8) at 80 °C for 15 minutes. Then shake again for 1 minute and wait for the sample
to cool down to room temperature or lower before centrifuging.
Notes: The above mentioned extraction conditions for paraquat and diquat are needed for the quantitative extraction of in-
curred residues2. Extractions with the normal QuPPe solvent (methanol containing 1% formic acid) at room temperature lead
to poorer extraction yields but are still well suitable for screening. Extractions with the normal QuPPe solvent involving thermal
treatment (80 °C for 15 minutes) were shown to provide quantitative extraction yields of incurred Diquat and Paraquat resi-
dues in wheat and potatoes but not in lentils.

2
Kolberg DI, Mack D, Anastassiades M, Hetmanski MT, Fussell RJ, Meijer T, Mol HG. Anal Bioanal Chem. 404(8):2465-74 (2012); Development and independent
laboratory validation of a simple method for the determination of paraquat and diquat in potato, cereals and pulses.
EU Reference Laboratory for pesticides requiring Single Residue Methods (EURL-SRM) 9 of 102
 Freeze-Out and Centrifugation
Depending on the available centrifugation equipment there is various options, e.g.:
(1) Ambient centrifugation: Centrifuge the extracts from 5.2.3 for 5 min at ≥3,000 g (the higher the centrifu-
gation force the better). This procedure is NOT recommended for extracts of commodities that pose diffi-
culties in filtration (e.g. finely milled cereals, pineapples, strawberry, asparagus, kaki, banana and pears).
For such commodities better use the following options (2) or (3).
(2) Ambient centrifugation following freeze-out: Place the extracts from 5.2.3 into a freezer (e.g. at ca. -80 °C
for 30 min or for > 120 min at ca. -20 °C) and centrifuge while still cold for 5 min at ≥ 3,000 g. Higher cen-
trifugation forces (e.g. ≥ 10,000 g) and cold centrifugation are preferred. This procedure is suitable for the
extracts of all samples and especially recommended for those posing difficulties in filtration.
(3) Refrigerated high-speed centrifugation: Centrifuge the extracts from 5.2.3 for > 20 min at high centrifuga-
tion speed (e.g. > 10,000 g) and low temperatures (e.g. lower than -5 °C). Centrifugation time may be re-
duced to 5 min if the extract is pre-frozen. This procedure is suitable for extracts of all samples and espe-
cially recommended for those posing difficulties in filtration.
Notes: Solid metal racks suitable for falcon tubes (e.g. VWR® Modular Blocks for Conical-Bottom 50 mL Centrifuge Tubes) may
be used to speed up freeze-out.
Low temperatures reduce the solubility of interfering matrix components resulting in increased precipitation, which considera-
bly facilitates the filtration step as well as the subsequent LC-MS/MS analysis by reducing matrix effects and increasing the
lifespan of columns. To avoid redissolvation of the matrix components in the cases (2) and (3), it is recommended to transfer
an aliquot of the cold supernatant into a sealable container for later use, or to proceed immediately with the next steps,
while the extract is still cold.

Removal of proteins and lipids

(1) Cereals and pulses: transfer 2 mL of the supernatant into a 10 mL centrifuge vial containing 2 mL of ace-
tonitrile (3.3) and shake for 1 min. Then centrifuge for 5 minutes at >3000 g (see 2.7).
(2) Nuts and oily seeds: transfer 2 mL of the supernatant into a 10 mL centrifuge vial containing 2 mL of ace-
tonitrile (3.3) and 100 mg of C-18 sorbent and shake for 1 min. Then centrifuge for 5 minutes at >3000 g
(see 2.7).
(3) Oily fruits (e.g. avocado): transfer 4 mL of the supernatant (from 5.2.4) into a 10 mL centrifuge vial con-
taining 200 mg of C-18 sorbent and shake for 1 min. Centrifuge for 5 min at >3000 g (see 2.7). This step may
be skipped if the sample was centrifuged frozen (5.2.4-(2) and 5.2.4-(3)), with the supernatant being re-
moved while still very cold.

Filtration

(1) All commodities of plant origin except cereals, pulses, nuts and oily seeds: Withdraw an aliquot (e.g. 2-3
mL) of the supernatant from 5.2.4 or 5.2.5 using a syringe (2.10) and filter it through a syringe filter (2.11)
either directly into an auto-sampler vial (2.13) or into a sealable storage vessel.
Notes: Where centrifugation with the available means results in extracts that are difficult to filter, a 2-step filtration
may be performed by connecting a 0.45 µm syringe filter on top of a 0.2 µm one (2.10).
Where a high lipid and low protein content commodity (e.g. avocado) was centrifuged frozen (under 5.2.4), and step
5.2.5 was skipped, filter the supernatant quickly to avoid that lipids redissolve.

(2) Cereals, pulses, nuts and oily seeds: Transfer a 3 mL aliquot of the supernatant from 5.2.5 into an ultrafil-
tration unit (2.12) and centrifuge at ca. 3,000 g until enough filtrate is accumulated in the reservoir (5 min
are typically enough). Transfer an aliquot of the filtrate into an autosampler vial for measurement.
Notes: Filtration of honey extracts: In case of clogging of the filters because of pollen or wax particles, use 5.0 µm pore size
syringe filters (2.11) for pre-filtration (or even instead of the filters with smaller pore-sizes), as pollen grains are typically > 10
µm in diameter. High-speed centrifugation (see 5.2.4 (3)) also helps to separate pollen.

EU Reference Laboratory for pesticides requiring Single Residue Methods (EURL-SRM) 10 of 102
 QuPPe-PO-Method at a glance (procedure for most commodities)
WEIGH sample homogenate into 50 mL centrifuge tube
Fresh fruits and vegetables (with high water content): 10 g 0.1 g
Previously re-hydrated dry fruit: e.g. 13.5 g 0.1 g (containing 5 g sample)
Dry commodities (e.g. herbs): 2 g 0.02 g

ADJUST WATER CONTENT of sample to 10 mL

(Mandatory for matrices w. <80% water. If no IL-IS used manadatory for ALL matrices)
e.g. +10 mL of water to 2g of dried mint;
+2 mL water to 10 g potato; + 3.5 mL water to 10 g garlic

Add 100 µL isotopically-labeled internal standard (IL-IS) mix

ADD EXTRACTION SOLVENT (10 mL methanol containing 1 % formic acid)

SHAKE thoroughly for 1 min to 15 min for dry commodities

Preferably FREEZE-OUT extract until completely frozen

e.g. >90 min at -18 C or ca. 30 min at -80 C

CENTRIFUGE (5 min at >3,000 g but preferably >10,000 g);

preferably cryogenic centrifugation (e.g. at -10 C)
(if centrifuge is not refrigerated, swiftly proceed with centrifugation and the following step
to avoid redissolvation of matrix)

dSPE to Remove Lipids for High Oil Content samples (e.g. avocado):
(this step may be skipped if sample was centrifuged frozen at ≤ -10 C and ≥ 20 min)
TRANSFER 4 mL raw extract into a tube containing 200 mg C18-sorbent,
SHAKE for 1 min and CENTRIFUGE (>3,000 g for 5 min)

WITHDRAW SUPERNATANT AND FILTER it into a plastic autosampler vial

(use syringe filter of 0.2 µm pore size; e.g. H-PTFE)
(plastic vials are recommended as some compounds tend to interact with glass)
(withdraw cold supernatant quickly after centrifugation to avoid that matrix components redissolve)

LC-MS/MS and IC-MS/MS analysis

Figure 1: QuPPe-PO-Method at a glance (general procedure for most commodities, not considering paraquat and diquat)

EU Reference Laboratory for pesticides requiring Single Residue Methods (EURL-SRM) 11 of 102
 QuPPe-PO-Method at a glance (procedure for cereals, pulses, nuts and oily seeds)

Weigh 5 0.05 g of sample homogenate into a 50 mL centrifuge tube

Add 9 mL of water

Add 100 µL isotopically-labelled internal standard (IL-IS) mix

Add 10 mL MeOH containing 1 % formic acid + extra 100 µL formic acid

close tube and shake

Add 1 mL 10% aqueous EDTA solution

Shake thoroughly for 15 min by a mechanical shaker

Option 1 Option2:
Freeze-out sample
e.g. 30 min at -80 C or >90 min at -20 C
Refrigerated High-Speed Centrifugation
Immediately Centrifuge e.g. >10,000 g at -10 C for ≥20 min
>3,000 g for 5 min (>10,000 g preferred)
(refrigerated centrifugation preferred)

Removal of proteins and lipids

Transfer 2 mL of raw extract into a tube containing …
a) Oily Seeds, Nuts: 2 mL acetonitrile and 100 mg C18-sorbent
b) Pulses and Cereals: 2 mL acetonitrile
Shake for 1 min and centrifuge at >3,000 g for 5 min

Filter aliquot of supernatant

Centrifugation assisted ultrafiltration through a 5 kDa cut-off filter
(e.g. polyethersulfone membrane)

LC-MS/MS and IC-MS/MS analysis

Figure 2: QuPPe-PO-Method at a glance; procedure for cereals, pulses, oily seeds and nuts

EU Reference Laboratory for pesticides requiring Single Residue Methods (EURL-SRM) 12 of 102
 QuPPe-PO-Method at a glance (procedure for honey)
Weigh sample homogenate into a 50 mL centrifuge tube
5 g 0,05 g

Adjust water content of sample to 10 mL (not mandatory if IL-IS is used)

+7.5 mL

Add 100 µL isotopically-labelled internal standard (IL-IS) mix

Add 10 mL MeOH containing 1 % formic acid,

close tube

Shake thoroughly for 5-15 min by a mechanical shaker

Option 1 Option 2
Freeze-out sample till completely frozen
e.g. 30 min at -80 C or >90 min at -20 C
Refrigerated High-Speed
Immediately Centrifuge Centrifugation
>3,000 g for 5 min (>10,000 g preferred) e.g. >10,000 g at -10 C
(refrigerated centrifugation preferred) for ≥20 min

WITHDRAW SUPERNATANT AND FILTER it into a plastic autosampler vial

(use syringe filter of 0.2 µm pore size; e.g. H-PTFE)
(plastic vials are recommended as some compounds tend to interact with glass)
(withdraw cold supernatant quickly after centrifugation to avoid that matrix components redissolve)
(in case of clogging of the filters because of pollen or wax particles,
use 5.0 µm pore size syringe filters for a pre-filtration;
Note: high-speed centrifugation (Option 2) also helps to separate pollen)

LC-MS/MS and IC-MS/MS analysis

Figure 3: QuPPe-PO-Method at a glance; procedure for honey

5.3. Preparation of blank extracts

Using homogenates of suitable blank commodities (not containing any relevant residues of the analytes of interest),
proceed sample preparation exactly as described in 5.2 but SKIP THE ADDITION OF ISs.

5.4. Recovery experiments

Weigh an appropriate portion (see 5.2.1) of a blank commodity homogenate into a 50 mL centrifuge tube (2.3) and
spike it with a suitable pesticide working solution (3.19 and Table 43). Spike directly to the matrix, prior to any water
or solvent addition. Use small volumes of pesticide working solutions (e.g. 50-300 µL), to avoid too strong dilution.
Conduct sample preparation as described in 5.2.
EU Reference Laboratory for pesticides requiring Single Residue Methods (EURL-SRM) 13 of 102
 5.5. Preparation of calibration standards

Solvent-based calibration standards

An exemplary pipetting scheme for preparing solvent-based calibration standards is shown in Table 2.
The calculation of the mass-fraction WR of the pesticide in the sample, when IS is used, is shown in 5.7.1. Where
solvent-based calibrations are used the use of IL-ISs for quantification is essential as the IS compensates for any
matrix-related signal suppressions / enhancements.
Notes: Though matrix-matched calibration is considered the best option, solvent-based calibrations can also produce accurate
results as IL-ISs can compensate for errors irrespective on whether the calibration is solvent-based, matrix-based or matrix-
matched. Nevertheless, in some cases the use of matrix-based calibrations are to be preferred over solvent-based calibrations
as the matrix present can decrease unwanted interactions with surfaces (e.g. in the injector area) thus leading to peak shapes
and retention times that are closer to those observed from sample extracts.

Matrix-based and matrix-matched calibration standards

Transfer suitable aliquots of a blank extract (5.3) to auto-sampler vials and proceed as shown in Table 2.
The calculation of the mass-fraction WR of the pesticide in the sample using matrix-matched calibration standards,
with and without the use of IL-IS, is shown in 5.7.1 and 5.7.2 respectively.

Table 2: Exemplary pipetting scheme for the preparation of calibration standards

Calibration standards
Solvent based (5.5.1) Matrix-matched (5.5.2)
4
using IL-IS Without IL-IS5 using IL-IS4

Calibr. levels in µg pesticide /mL OR

0.056 0.1 0.25 0.05 0.1 0.25 0.05 0.1 0.25
in µg pesticide/ “IL-IS-portion”1

Blank extract (5.3) - - - 850 µL 850 µL 850 µL 800 µL 800 µL 800 µL

1:1 (v/v) mix of water (3.1) and
850 µL 800 µL 850 µL 100 µL 50 µL 100 µL 50 µL - 50 µL
acidified methanol (3.6)

Pesticide working 1 µg/mL 50 µL 100 µL - 50 µL 100 µL - 50 µL 100 µL -

solutions (3.19) 2 5 µg/mL - - 50 µL - - 50 µL - - 50 µL
IS-WSln-2 (3.23)1,3 100 µL 100 µL 100 µL - - - 100 µL 100 µL 100 µL
Total volume 1000 µL 1000 µL 1000 µL 1000 µL 1000 µL 1000 µL 1000 µL 1000 µL 1000 µL
1
One IL-IS portion would correspond to the IL-IS mass contained in 100 µL IS-WSln-2 (which in the particular example is added
to each calibration standard).
2
The concentration of the pesticide working solution(s) should be sufficiently high to avoid excessive dilution of the blank ex-
tract, which would result in matrix effect deviations.
3
For calibration standards of 1 mL it is highly recommended to prepare the IS-WSln-2 (3.23) by diluting IS-WSln-1 (3.22) appro-
priately (e.g. 20-fold). The same volume and pipette as in 5.2.3 can be used for preparing the calibration standards.
4
When employing IL-ISs matrix-matching, volume adjustments are of less importance as the IL-IS compensates for any matrix-
related signal suppressions / enhancements. Also solvent-based calibrations can be used here. Important is that a) the mass
ratio of pesticide and IL-IS in the respective calibration standards and b) the ratio between the IL-IS mass added to the sample
(5.2.3) and the IL-IS mass added to the calibration standard(s) (5.5.1 and 5.5.2) is known and recorded. For convenience the
latter mass-ratio should be kept constant throughout all calibration levels (e.g. at 20:1 when preparing calibration standards of
1 mL).
4
Where IL-ISs are not available/employed, matrix-matched standards Table 2) or the standard additions approach (5.5.3) are
particularly important to compensate for matrix effects in measurement. In both cases the total volume of the sample raw
extracts is assumed to be exactly 20 mL. This translates into 0.5 g sample equivalents per mL when using 10 g test portions.
6
The calibration level of 0.05 µg/mL corresponds to 0.1 mg pesticide /kg sample, when using 10 g test portions, or to
0.2 mg/kg sample when using 5 g test portions. Where the raw extract is diluted further and when using 5 g test portions (e.g.
pulses, nuts and oily seeds), the 0.05 µg/mL calibration level corresponds to 0.4 mg pesticide /kg sample.

EU Reference Laboratory for pesticides requiring Single Residue Methods (EURL-SRM) 14 of 102
 Standard-Additions approach
Where no appropriate IL-ISs are available the method of standard additions is a very effective approach for compen-
sating matrix-induced enhancement or suppression phenomena. As this procedure involves a linear extrapolation it
is mandatory that pesticide concentrations and detection signals show a linear relationship throughout the relevant
concentration range. The procedure furthermore requires knowledge of the approximate (estimated) residue level
in the sample (wR(approx)). This info is derived from a preliminary analysis.
Prepare 4 equal portions of the final extract and spike 3 of them with increasing amounts of analyte. The amounts
to be added should be chosen in such a way to remain within the linear range. It should be avoided that the added
levels are too close to the expected analyte level to avoid that measurement variability will influence too much the
slope, which is used to calculate the analyte level. In case the concentrations are outside the linear range a dilution
of all 4 extracts with the extraction solvent is indicated.
Prepare a working solution (3.19) of the analyte at a concentration level where 50 or 100 µL of the solution contain
the lowest amount of analyte to be added.
Example A: Vial 1) no addition; vial 2) 0.5 x wR(approx), vial 3) 1 x wR(approx), and vial 4) 1.5 x wR(approx),
Example B: Vial 1) no addition; vial 2) 1 x wR(approx), vial 3) 2 x wR(approx), and vial 4) 3 x wR(approx).
Adjust the volume within all vials by adding the corresponding solvent amounts.
An exemplary pipetting scheme according to Example B in shown in Table 3. The calculation of the mass fraction of
the pesticide in the sample wR is shown in 5.7.2.

Table 3: Exemplary pipetting scheme of a standard additions to extract aliquots approach (for a sample extract containing 0.5 g
sample equivalents per mL and an estimated residue level (wR(approx)) of 0.5 mg/kg = 0.25 µg/1000 µl
Additions Vial 1 Vial 2 Vial 3 Vial 4
1000 µL 1000 µL 1000 µL 1000 µL
Volume of sample extract
(= 0.5 g sample) (= 0.5 g sample) (= 0.5 g sample) (= 0.5 g sample)
Internal Standard (IS) none none none none
Added volume of pesticide working solution
- 50 µL 100 µL 150 µL
containing 5 µg/mL (3.19)

m std
pest
add
- 0.25 µg 0.5 µg 0.75 µg
Mass of pesticide added to each vial ( )
Volume of solvent (for volume equalization) 150 µL 100 µL 50 µL -
Final volume 1150 µL 1150 µL 1150 µL 1150 µL

EU Reference Laboratory for pesticides requiring Single Residue Methods (EURL-SRM) 15 of 102
 Procedural calibration standards
Procedural calibration is most useful where numerous samples of the same commodity type are analyzed within the
same badge and can help to largely compensate for recovery and matrix effects. An ideal precondition is the availa-
bility of a blank matrix of exactly the same type as the samples to be analyzed. For this, prepare 4 analytical portions
of a suitable blank sample and spike three of them with increasing amounts of the pesticides of interest (as done in
recovery experiments, see also 5.4). The aim should be to cover the concentration range of the analytes expected in
the samples. These spiked samples are extracted as described above and the obtained extracts are used in the same
way as any other matrix-matched standards.

5.6. LC-and IC-MS/MS analysis

Any suitable LC- or IC-MS/MS conditions generating peaks that can be well integrated may be used. The use of IL-
ISs typically ensures a good method accuracy and robustness even when matrix components have a strong influ-
ence on signals or retention times. Some exemplary instrument measurement conditions are given below. An over-
view of LC- and IC-MS/MS conditions proposed within this document is given in Table 4 and following.

EU Reference Laboratory for pesticides requiring Single Residue Methods (EURL-SRM) 16 of 102
 Table 4: Scope of QuPPe-LC-Methods of analytes analyzed in the ESI-pos. mode (see legent under Table 7) Part.I
M 1.1 M 1.2 M 1.3 M 1.4 M 1.5 M 1.6a/b M 1.7 a/b M 1.10 M 1.11 M 1.12
QuPPe method code
(5.6.2) (5.6.3) (5.6.4) (5.6.5) (5.6.6) (5.6.7) (5.6.8) (5.6.9) (5.6.10) (5.6.11)
Separation principle Anion Ex. Anion Ex. Carbon Carbon HILIC HILIC HILIC HILIC HILIC HILIC
a: Torus DEA/ a: Torus DEA/ Raptor ObeliscN
Column type AS 11 AS 11-HC Hypercarb Hypercarb Trinity Q1 APPC
b: APPC b: APPC PolarX
ANALYTES COVERED BY LC-MS/MS IN THE ESI-POSITIVE MODE
Amitrole NT NT - NT NT NT NT NT NT NT
ETU NT NT  NT NT NT NT NT NT NT
PTU NT NT  NT NT NT NT NT NT NT
Cyromazine NT NT NT NT NT NT NT NT NT NT
Trimesium NT NT NT NT NT NT NT NT NT NT
Daminozide NT NT NT NT NT NT NT NT NT NT
Chlormequat NT NT  NT NT NT NT NT NT NT
Mepiquat NT NT  NT NT NT NT NT NT NT
Difenzoquat NT NT - NT NT NT NT NT NT NT
Propamocarb NT NT NT NT NT NT NT NT NT NT
Melamine NT NT NT NT NT NT NT NT NT NT
Diquat NT NT - NT NT NT NT NT NT NT
Paraquat NT NT - NT NT NT NT NT NT NT
N,N-Dimethylhydrazine NT NT - NT NT NT NT NT NT NT
Nereistoxin NT NT  NT NT NT NT NT NT NT
Streptomycin NT NT NT NT NT NT NT NT NT NT
Kasugamycin NT NT NT NT NT NT NT NT NT NT
Morpholine NT NT NT NT NT NT NT NT NT NT
Diethanolamine NT NT NT NT NT NT NT NT NT NT
Triethanolamine NT NT NT NT NT NT NT NT NT NT
1,2,4-Triazole NT NT NT NT NT NT NT NT NT NT
Triazole-alanine NT NT NT NT NT NT NT NT NT NT
Triazole-acetic acid NT NT NT NT NT NT NT NT NT NT
Triazole-lactic acid NT NT NT NT NT NT NT NT NT NT
Aminocyclopyrachlor NT NT NT NT NT NT NT NT NT NT
Chloridazon-desphenyl NT NT NT NT NT NT NT NT NT NT
Mepiquat-4-hydroxy NT NT NT NT NT NT NT NT NT NT
Propamocarb-N-desmethyl NT NT NT NT NT NT NT NT NT NT
Propamocarb-N-oxide NT NT NT NT NT NT NT NT NT NT
Maleic Hydrazide NT NT NT NT NT NT NT NT NT NT
Nicotine NT NT NT NT NT NT NT NT NT NT
Matrine NT NT NT NT NT NT NT NT NT NT
Oxymatrine NT NT NT NT NT NT NT NT NT NT

EU Reference Laboratory for pesticides requiring Single Residue Methods (EURL-SRM) 17 of 102
 Table 5: Scope of QuPPe-LC- and IC-Methods of analytes analyzed in the ESI-pos. mode (see legent under Table 7) Part.II
M2 M3 M 4.1 M 4.2 M5 M6 M7 M8 M9 M 10 M 11
QuPPe method code
(5.6.12) (5.6.13) (5.6.14) (5.6.15) (5.6.16) (5.6.17) (5.6.18) (5.6.19) (5.6.20) (5.6.21) (5.6.22)
HILIC Ion Chroma-
Separation principle HILIC HILIC HILIC HILIC HILIC HILIC HILIC Carbon HILIC
tography
Column type Obelisc-R Obelisc-R Obelisc-R BEH-Amide PFP Obelisc-R Trinity P1 Hypercarb Trinity P1 Torus DEA AS19
ANALYTES COVERED BY LC-and IC-MS/MS IN THE ESI-POSITIVE MODE
Amitrole NT  -  NT NT NT NT NT NT NT
ETU NT  -   NT NT NT NT NT NT
PTU NT  -   NT NT NT NT NT NT
Cyromazine NT    NT NT NT NT NT NT NT
Trimesium NT    NT NT NT NT NT NT NT
Daminozide NT    NT NT NT NT NT NT NT
Chlormequat NT     NT NT NT NT NT NT
Mepiquat NT     NT NT NT NT NT NT
Difenzoquat NT     NT NT NT NT NT NT
Propamocarb NT    NT NT NT NT NT NT NT
Melamine NT NT   NT NT NT NT NT NT NT
Diquat NT NT  ()**** NT NT NT NT NT NT NT
Paraquat NT NT  ()**** NT NT NT NT NT NT NT
N,N-Dimethylhydrazine NT NT  - NT NT NT NT NT NT NT
Nereistoxin NT NT   NT NT NT NT NT NT NT
Streptomycin NT NT NT NT NT  NT NT NT NT NT
Kasugamycin NT NT NT NT NT  NT NT NT NT NT
Morpholine NT NT () () NT NT  NT  NT NT
Diethanolamine NT NT () () NT NT  NT NT NT NT
Triethanolamine NT NT () () NT NT  NT NT NT NT
1,2,4-Triazole NT NT () - NT NT NT  NT () NT
Triazole-alanine NT NT () - NT NT NT  NT  NT
Triazole-acetic acid NT NT () - NT NT NT  NT  NT
Triazole-lactic acid NT NT NT - NT NT NT  NT  NT
Aminocyclopyrachlor NT NT NT  NT NT NT NT NT NT NT
Chloridazon-desphenyl NT NT NT  NT NT NT NT NT NT NT
Mepiquat-4-hydroxy NT NT NT  NT NT NT NT NT NT NT
Propamocarb-N-desmethyl NT NT NT  NT NT NT NT NT NT NT
Propamocarb-N-oxide NT NT NT  NT NT NT NT NT NT NT
Maleic Hydrazide NT NT NT  NT NT NT NT NT NT NT
Nicotine NT NT NT  NT NT NT NT NT NT NT
Matrine NT NT NT  NT NT NT NT NT NT NT
Oxymatrine NT NT NT  NT NT NT NT NT NT NT

EU Reference Laboratory for pesticides requiring Single Residue Methods (EURL-SRM) 18 of 102
 Table 6: Scope of QuPPe-LC-Methods of analytes analyzed in the ESI-neg. mode Part.I
M 1.1 M 1.2 M 1.3 M 1.4 M 1.5 M 1.6a/b M 1.7 a/b M 1.10 M 1.11 M 1.12
QuPPe method code
(5.6.2) (5.6.3) (5.6.4) (5.6.5) (5.6.6) (5.6.7) (5.6.8) (5.6.9) (5.6.10) (5.6.11)
Separation principle Anion Ex. Anion Ex. Carbon Carbon HILIC HILIC HILIC HILIC HILIC HILIC
a: Torus DEA/ a: Torus DEA/ Raptor ObeliscN
Column type AS 11 AS 11-HC Hypercarb Hypercarb Trinity Q1 APPC
b: APPC b: APPC PolarX
ANALYTES COVERED BY LC-MS/MS IN THE ESI-NEGATIVE MODE
Ethephon    NT   ()  
HEPA    NT   ()  
Glufosinate    NT   ()  
N-Acetyl-Glufosinate    NT   ()  
MPPA    NT   ()  
Glyphosate    NT   ()  
AMPA    NT   ()  ()
Phosphonic acid () ()       ()***
N-Acetyl-AMPA NT   NT   () NT NT
Fosetyl-Al -   NT   ()  ()***
Maleic Hydrazide - -  NT - - - () () ()
Perchlorate NT -    ()**    
Chlorate NT -    ()**   () 
Bialaphos NT NT  NT  NT NT  NT
Cyanuric acid NT NT  NT - - -  () ()
Bromide NT NT -   ()**   
Bromate NT NT ()  NT NT  NT NT
N-Acetyl-Glyphosate NT NT  NT ()**  () - 
Difluoroacetic acid NT NT NT NT NT NT NT  NT
Trifluoroacetic acid NT NT NT NT NT NT NT  
Thiocyanate NT NT ()**  NT NT NT  NT
Desmethyl-Dimethoate NT NT  - NT NT NT NT NT
 = satisfactory chomatography and detection sensitivity achieved,
NT = Not tested under the conditions shown in the respective sections,
() = possible but compromised due to matrix effects or lacking separation or limited sensitivity or limitations in the detection of qualifiers compromising identification.
“-“ analysis was tested and found to be poor under the described conditions,
* Using a gradient (98% B -> 60% B in 5 min, hold 2 min)
** Different LC-conditions required to improve peaks (see M1.7 or M1.9)
*** Compromised quantitation of Phosphonic acid due to co-elution of Phosphonic acid and Fosetyl, see also General Hints 5.6.1
**** Quality of analysis may strongly depend on instrument type and condition

EU Reference Laboratory for pesticides requiring Single Residue Methods (EURL-SRM) 19 of 102
 Table 7: Scope of QuPPe-LC- and IC-Methods of analytes analyzed in the ESI-neg. mode Part.II
M2 M3 M 4.1 M 4.2 M5 M6 M7 M8 M9 M 10 M 11
QuPPe method code
(5.6.12) (5.6.13) (5.6.14) (5.6.15) (5.6.16) (5.6.17) (5.6.18) (5.6.19) (5.6.20) (5.6.21) (5.6.22)
Ion Chroma-
Separation principle HILIC HILIC HILIC HILIC HILIC HILIC HILIC Carbon HILIC HILIC
tography
Column type Obelisc-R Obelisc-R Obelisc-R BEH-Amide PFP Obelisc-R Trinity P1 Hypercarb Trinity P1 Torus DEA AS19
ANALYTES COVERED BY LC-MS/MS IN THE ESI-NEGATIVE MODE
Ethephon NT NT NT NT NT NT - NT NT NT 
HEPA NT NT NT NT NT NT - NT NT NT 
Glufosinate NT NT NT NT NT NT - NT NT NT 
N-Acetyl-Glufosinate NT NT NT NT NT NT - NT NT NT 
MPPA NT NT NT NT NT NT - NT NT NT 
Glyphosate NT NT NT NT NT NT - NT NT NT 
AMPA NT NT NT NT NT NT - NT NT NT 
Phosphonic acid NT NT NT NT NT NT - NT NT NT 
N-Acetyl-AMPA NT NT NT NT NT NT - NT NT NT 
Fosetyl-Al  NT NT NT NT NT * NT NT NT 
Maleic Hydrazide  NT NT NT NT NT * NT NT NT ()
Perchlorate  NT NT NT NT NT * NT NT NT 
Chlorate NT NT NT NT NT NT * NT NT NT 
Bialaphos NT NT NT NT NT NT - NT NT NT -
Cyanuric acid NT NT NT NT NT NT * NT NT NT 
Bromide NT NT NT NT NT NT NT NT NT NT 
Bromate NT NT NT NT NT NT NT NT NT NT NT
N-Acetyl-Glyphosate NT NT NT NT NT NT NT NT NT NT 
Difluoroacetic acid NT NT NT NT NT NT NT NT  NT 
Trifluoroacetic acid NT NT NT NT NT NT NT NT  NT 
Thiocyanate NT NT NT NT NT NT NT NT NT NT 
Desmethyl-Dimethoate NT NT NT NT NT NT NT NT NT NT -
 = satisfactory chomatography and detection sensitivity achieved,
NT = Not tested under the conditions shown in the respective sections,
() = possible but compromised due to matrix effects or lacking separation or limited sensitivity or limitations in the detection of qualifiers compromising identification.
“-“ analysis was tested and found to be poor under the described conditions,
* Using a gradient (98% B -> 60% B in 5 min, hold 2 min)
** Different LC-conditions required to improve peaks (see M1.7 or M1.9)

EU Reference Laboratory for pesticides requiring Single Residue Methods (EURL-SRM) 20 of 102
Table 8 : Methods mainly used by CVUA Stuttgart
Method Special remarks on Substances LC-MS/MS Comments
Glyphosate
AMPA
N-Acetyl-AMPA
N-Acetyl-Glyphosate
Ethephon
HEPA
Evaluation via solvent calibration and
Glufosinate
IL-ISs except for Bialaphos and N-Ace-
N-Acetyl-Glufosinate 1290 Agilent In-
M 1.3: Glyphosate & Co. Hyper- tyl-AMPA (IL-IS not yet available)
MPPA finity II and Sciex
carb (see 5.6.4) M 1.5 and M 1.6 are currently being
Fosetyl-Al QTRAP 6500+
tested for their suitability to replace
Phosphonic acid (screening)
M 1.3
Maleic Hydrazide
Perchlorate (screening))
Chlorate (screening)
Cyanuric acid
Bialaphos
Desmethyl-Dimethoate
Perchlorate (quantitative) Mostly employed directly (option:
Chlorate (quantitative) screening by M 1.3, if postive -> M
1290 Agilent In-
M 1.4: Method 1.4 (M1.4): “Per- Phosphonic acid (quantitative) 1.4)
finity II and Sciex
ChloPhos” (see 5.6.5) Bromide (Screening, quantitative) Dilution 5-fold
QTRAP 6500+
Bromate (quantitative) Evaluation via solvent calibration and
Thiocyanate IL-ISs
1290 Agilent In- Analysis of specific relevant commod-
M 4.1: “Quats & Co Obelisc R Paraquat (for specific commodities)
finity II and Sciex ities. Evaluation via matrix-based cali-
(see 5.6.14) Diquat (for specific commodities)
QTRAP 5500 bration and IL-ISs
Amitrole
ETU
Chlormequat
Mepiquat
Daminozide
PTU
Cyromazine
Trimethylsulfonium
Nereistoxin
Difenzoquat Evaluation via matrix-based calibra-
Melamine 1290 Agilent In- tion and IL-ISs (except for Difen-
M 4.2: “Quats & Co BEH Amide”
Propamocarb finity II and Sciex zoquat, Aminocyclopyrachlor, Mepi-
(see 5.6.15)
Morpholine (1st screening) QTRAP 5500 quat-4-hydroxy, Propamocarb-N-
Diethanolamine (1st screening) desmethyl, Propamocarb-N-oxide)
Triethanolamine (1st screening)
Aminocyclopyrachlor
Chloridazon-desphenyl
Mepiquat-4-hydroxy
Propamocarb-N-desmethyl
Propamocarb-N-oxide
Nicotine
Matrine
Oxamatrine
Employed if screening by M 4.2 was
positive and in matrices where DEA
M 7: “Morpholine, Diethanola- Morpholine (quantitative) 1290 Agilent In-
tends to give false negative results in
mine and Triethanolamine” (see Diethanolamine (quantitative) finity II and Sciex
M 4.2 (e.g. in cereals, dried mush-
5.6.18) Triethanolamine (quantitative) QTRAP 5500
rooms). Quantification via solvent-
based calibration and IL-ISs

EU Reference Laboratory for pesticides requiring Single Residue Methods (EURL-SRM) 21 of 102
 General hints on analytes to avoid pitfalls
1. Mass spectrometric interferences:
a) AMPA and Fosetyl share the mass-transition 110/81. Chromatographic separation is thus needed.

b) Interference of Phosphonic acid by Phosphoric acid; take care when using M 1.4 (Hypercarb column):
When extracts containing high levels of Phosphoric acid (which is naturally contained at high concentra-
tions in many samples) are injected, the chromatographic separation of Phosphoric and Phosphonic acid
is compromised. This often results in a suppression of the Phosphonic acid signal and in some cases even
leads to false negative results. The most important qualifier mass-transition of Phosphonic acid (81/63)
also occurs as a minor transition of Phosphoric acid, but as the latter is often present at much higher levels
than Phosphonic acid its interference on this mass transition can still be significant, especially if these two
compounds elute in close vicinity (e.g. M 1.4 using Hypercarb column). Exemplary chormatograms demon-
strating this effect are shown in Table 10.
The chromatographic separation of Phosphoric and Phosphonic acid considerably improves following di-
lution of the extracts typically allowing proper detection, identification and quantification of Phosphonic
acid next to high levels of phosphoric acid. When using method M 1.4 it is thus beneficial to inject smaller
volumes of sample extract (e.g. 1-2 µL) or to dilute QuPPe extracts 5-10-fold before injection.
Fortunately, both Phosphoric and Phosphonic acid have at least 1 proper individual mass-transition (97/63
and 81/79 respectively, shown in Table 10), which in the case of Phosphonic acid can be used for quanti-
tation and to improve identification certainty. The elution time and peak shape of the Phosphonic acid IL-
IS can also be used to distinguish it from Phosphoric acid and to avoid false positives. Using signals on the
81/63 mass trace it was calculated that 20 mg/kg Phosphoric acid would simulate 0.1 mg/kg Phosphonic
acid if this mass transition was used for quantification. Different instrument settings may result in a dif-
ferent degree of interference.
In an experiment using Differential Mobility Separation (DMS) a separation of the phosphonate generated
in-source from Phosphate and the original Phosphonate (mass trace m/z 81/63) was achieved, possibly
due to a different molecular structure of the P(OH)3 species and the HPO(OH)2 species.

Tip: Using method M 1.7 (Torus DEA and APPC) and method M 1.8 (Raptor Polar X), sufficient chromato-
graphic separation was achieved without dilution of the sample extract. Exemplary chromatograms for
M1.7 are shown in Table 9

Table 9: Chromatograms of Phosphonate transition 81/63 (also common to Phosphate) at 0.01 µg phosphonate/mL in grape, onion
and infant formula using M 1.7. Both substances are separated sufficiently.
Grape Onion Infant Formula

From Phosphate
From Phosphate From Phosphate
Phosphonic
acid 81/63

EU Reference Laboratory for pesticides requiring Single Residue Methods (EURL-SRM) 22 of 102
Table 10: Chromatographic and mass-spectrometric separation of Phosphoric and Phosphonic acid using M 1.4.
Detected signals at mass trace of
81/63
Sample 97/63 81/79
common to Phosphoric and
unique to Phosphoric acid unique to Phosphonic acid
Phosphonic acid

12-fold
Pear approx. 160 mg/kg 13 mg/kg

White 32-fold
approx. 230 mg/kg 7.2 mg/kg
grapes 1

White 940-fold
approx. 150 mg/kg 0.16 mg/kg
grapes 2

Cucum- 540-fold
approx. 440 mg/kg 0.82 mg/kg
ber

Raisins
140-fold
approx. 470 mg/kg 3.3 mg/kg

c) Intereference of Phosphonic acid by Fosetyl and Fosetyl-D5:

Fosetyl and its D5-analogon tend to degrade to Phosphonic acid both in solutions and via in-source frag-
mentation in LC-MS/MS, see also below and 6. A good chromatographic separation between Fosetyl and
Phosphonic acid is thus necessary.
Table 11 shows an example of this in-source fragmentation using M 1.3 and M 1.4 (Hypercarb column).
Upon injection of 0.1 µg/mL Fosetyl, a peak showed up on the mass traces of Phosphonic acid at the re-
tention time of Fosetyl. The signal intensity of this peak corresponded to 0.04 µg/mL Phosphonic acid.
When injecting Fosetyl-D5 at 0.1 µg/kg the in-source fragmentation was less abundant (corresponding to
approx. 0.001 µg/mL Phosphonic acid) but Phosphonic acid as impurity showed up at its proper retention
time at a concentration corresponding to approx. 0.007 µg/mL.
Tip: To be on the safe side, Fosetyl-IL-IS should not be added to calibration solutions, sample test portions
or sample extracts intended to be used for the analysing native Phosphonic acid. Further, calibration so-
lutions used for the analysis of Phosphonic acid should better not contain any native Fosetyl.

EU Reference Laboratory for pesticides requiring Single Residue Methods (EURL-SRM) 23 of 102
Table 11: Chromatograms of Phosphonic acid, Fosetyl and Fosetyl-D5 (each at 0.1 µg/mL) using Method M 1.3 and M 1.4.
Detected signals at mass trace of …
Calibration standard injected Fosetyl 109/81 (top) or
Phosphonic acid 81/79
Fosetyl-D5 114/82 (bottom)

Phosphonic acid 0.1 µg/mL

Very slight degradation to

Fosetyl 0.1 µg/mL Phosphonate in solution

Phosphonate present in
the injected solution

Fosetyl-D5 0.1 µg/mL

Note: In addition to the proper mass-traces of Fosetyl and Fosetyl-D5 the mass trace of Phosphonic acid is also shown to
demonstrate the occurrence of in-source fragmentation of Fosetyl and Fosetyl-D5 towards Phosphonic acid as well as the pres-
ence of Phosphonic acid as an impurity of the Fosetyl-D5 standard solution.

d) Paraquat interfered by Diquat:

Tranistions of Paraquat ([M2+- H+]+ 185/#) are interfered by Diquat. Diquat and Paraquat produce several
parent ions within the ion-source, each one fragmenting to various product ions, see 3.a). MRMs of singly
charged protonated Paraquat ([M2+- H+]+ 185/#) tend to be interfered by Diquat, e.g. [M2+- H+]+ 185/170
and [M2+- H+]+ 185/169. Same applies to the respective MRM of Paraquat D8 ([M2+ - H+]+193/#) , which is
interfered by Diquat D8. Furthermore, those transitions are less sensitive.

e) Bromide; take care when using M 1.4:

High levels of Phosphoric acid (which is naturally contained at high concentrations in many samples) or
Phosphonic acid (that is used as fungicide) could affect the determination of bromide. Depending on the
condition of the column, the separation of these three compounds could be insufficient, resulting in com-
promised identification and quantification, especially when using M 1.4 (Hypercarb column).
Bromide is mainly composed of two naturally occurring stable isotopes, that are almost equally frequent
(79Br¯ and 81Br¯). For bromide (element-ion), no MS/MS fragmentation is possible so that MS/MS analysis
has to rely on “parent/parent” analysis. The mass trace m/z 81/81 is highly recommended for quantifica-
tions whereas m/z 79/79 can be used as a qualifier.
The mass trace m/z 81/81 is interfered by Phosphonic acid (m/z of [H2PO3]- = 81) whereas m/z 79/79 is
highly affected by Phosphoric acid due to in-source fragmentation (Table 12, the two columns declared as
“CE -5 V”). In practice the interference by Phosphoric acid is more critical as it is naturally contained at
high levels (e.g. 100-2000 mg/kg) in various samples.
Tip: A 50-fold dilution of QuPPe extracts typically allows better identification and quantification of bro-
mide next to high levels of Phosphoric and Phosphonic acid as chromatographic separation is improved
and matrix-effects reduced (when using M 1.4, Hypercarb).
To improve selectivity and increase quantification accuracy and identification certainty, the interferences

EU Reference Laboratory for pesticides requiring Single Residue Methods (EURL-SRM) 24 of 102
 caused by Phosphoric and Phosphonic acid can be further reduced by increasing the Collision Energy (CE)
for the m/z 81 and 79 (Table 12, the two columns declared as “CE -70 V”). While Bromide cannot be frag-
mented, the interfering quasi-molecular ion of Phosphonic acid (m/z 81) as well as the interfering in-
source fragments of Phosphoric and Phosphonic acid (m/z 79) are largely destroyed by increased collision
induced dissociation. While losing up to a 100-fold of absolute sensitivity, the interferences were largely
decreased resulting in satisfactory signal-to-noise ratio.

Table 12: Chromatograms of Bromide using non-optimized collision energies (CE -5 V) showing the interference by Phosphoric and
Phosphonic acid as well as optimized collision energies (CE -60 V and -70 V, the) showing reduced interferences using M 1.4.
m/z 81/81 m/z 79/79
CE -5 V CE -60 V CE -5 V CE -70 V
Cucumber
containing 1.7 mg/kg Bromide, 2.0 mg/kg Phosphonic acid & approx. 300 mg/kg Phosphoric acid

Bromide peak Phosphoric acid

Bromide peak Bromide peak
ca. 10-fold lower interference
ca. 100-fold lower
Slight interference sensitivity but
sensitivity but
higher selectivity Bromide peak higher selectivity

Mint leaves
containing 1.1 mg/kg Bromide, approx. 370 mg/kg Phosphoric acid; Phosphonic acid n. d.

Phosphoric acid
interference

Fennel
containing 2.2 mg/kg Bromide, 5.4 mg/kg Phosphonic acid & approx. 400 mg/kg Phosphoric acid

Phosphoric acid
interference

Sweet Corn
containing 1.1 mg/kg Bromide, approx. 350 mg/kg Phosphoric acid; Phosphonic acid n. d.

Phosphoric acid
interference

EU Reference Laboratory for pesticides requiring Single Residue Methods (EURL-SRM) 25 of 102
2. Degradation and Contamination:
a) Issues concerning the purity of N-Acetyl-Glufosinate D3:
There is two types of N-Acetyl-Glufosinate D3 standards on the market. Both contain the three deuterium
atoms on a methyl group, but the first one contains them on the methyl group of the acetyl moiety and
the other one on the methyl group that is attached to the phosphorus atom. In theory, the acetyl group
can be hydrolytically detached, so that native glufosinate may be formed in working solutions of N-Acetyl-
Glufosinate (acetyl-D3), leading to false positive results. Fortunately the degradation rate observed in the
water:acetonitrile 9:1 mixture (see Figure 37) was negligible. More important is the content of native
glufosinate in purchased N-Acetyl-Glufosinate (acetyl-D3) standards.
Tip: Before first use, the standards should be checked for the presence of native glufosinate impurities
and not used if the levels of native compound are considered critical. The levels of native glufosinate im-
purities depend on the manufacturer and the badge. Where e.g. 0.5 µg IL-IS is added to 1 g sample, the
presence of 2% native glufosinate (a level once encountered) can lead to glufosinate levels of 0.01 mg/kg.
See also chapter 6.

b) Possible contaminations by consumables:

Check the filters and other consumables used for sample preparation for any contamination of Perchlorate
and Chlorate. Cellulose mixed ester filters were found to be suitable for this application (see comments
under 2.11).

c) Stability of the Phosphonic acid IL-IS:

In presence of water and especially at high pH levels, Phosphonic acid 18O3 will gradually convert to
18
O216O1 , 18O116O2 and eventually of 16O3 (native) Phosphonic acid. The 18O3 Phosphonic acid standard so-
lution provided by the EURLs should be preferably diluted in acetonitrile, where it was shown to be
stable for long periods.

d) Degradation of Ethephon and Fosetyl to Phosphonic acid:

Fosetyl and Ethephon as well as their respective IL-IS’s degrade to Phosphonic acid. Table 13 shows a small
peak of Phosphonic acid (corresponding to 0.002 µg/mL) that showed up when Ethephon standard at 1
µg/mL was injected using M 1.4. This contamination is considered negligible. However Table 13 also shows
chromatograms of an unsuitable Ethephon-D4 standard containing only ca. 0.08 µg/mL instead of the ex-
pected 1 µg/mL Ethephon-D4 and ca. 0.8 µg/mL Phosphonic acid. The use of such an IL-IS would contami-
nate the sample with Phosphonic acid leading to false positive results.
Tip: To be on the safe side Fosetyl, Ethephon and their respective IL-IS’s should thus not be added to
calibration solutions or samples or sample extracts intended to be used for the analysis of native phos-
phonic acid. Furthermore calibration solutions used for the analysis of phosphonic acid should better not
contain any native Ethephon or Fosetyl.
See also “Intereference of Phosphonic acid by Fosetyl: Fosetyl and its D5-analogon tend to degrade to
Phosphonic acid both in solutions and via in-source fragmentation in LC-MS/MS” and Chapter 6.

EU Reference Laboratory for pesticides requiring Single Residue Methods (EURL-SRM) 26 of 102
Table 13: Chromatograms of Phosphonic acid, Ethephon and an unsuitable Ethephon-D4 standard (each at 1.0 µg/mL) using
Method M 1.3 and M 1.4 (Hypercarb column).
Detected signals at mass trace of …
Calibration standard injected 143/107 Ethephon (top) or
81/79 Phosphonic acid
147/111 Ethephon-D4 (bottom)

Set at 100%
Phosphonic acid 1.0 µg/mL

<0.002 µg/mL
Set at 100%
Ethephon 1.0 µg/mL

Phosphonic acid level higher than Approx. 8% of respective

what would be expected in case signal in native standard
Ethephon-D4 1.0 µg/mL of full conversion of Ethephon D4
(Example of an unsuitable commer- to phosphonic acid
cial standard!!)

Note: Whereas Phosphonic acid is only present at very low concentrations in the Ethephon standard the amount of Phosphonic
acid in the Ethephon-D4 standard is unacceptably high. That is caused by the Phosphonic acid having already been present at
high amounts in the purchased standard.

e) Contamination of Maleic hydrazide D2 with native Maleic hydrazide:

In the case of Maleic Hydrazide (MH), the amount of IL-IS added is comparably high due to the low detec-
tion sensitivity achieved for this compound. Assuming native MH being contained as impurity in D2-MH at
0.25 % (a typical level encountered) the resulting concentration of native MH following the addition of 20
µg D2-MH to 10 g sample will be at 0.005 mg /kg sample. This aspect is to be considered when setting the
Reporting Limits of MH as well as when judging residue levels in samples having low MRLs (e.g. baby food)
or organic food.

f) Chlorate can be a minor contaminant of Perchlorate solutions

Chlorate can be a minor contaminant of Perchlorate solutions and is also a minor in-source fragment of
Perchlorate. In the example below, Perchlorate standard at 0.2 µg/mL was injected resulting in two peaks
on the mass traces of Chlorate (see Table 14). The first one originating from Chlorate contained as impurity
in the Perchlorate solution (at approx. 0.35%) and the second one originating from in-source fragmenta-
tion at the retention time of Perchlorate, corresponding to a Chlorate amount of 0.001 µg/mL. This means
that calibration solutions containing both chlorate and perchlorate at the same level the chlorate signal
will be overestimated by approx. 0.5% which is negligible. Also samples containing perchlorate may fake
the presence of chlorate at very low levels, normally well below the reporting level of chlorate. When
chlorate IL-IS is co-injected misidentification is unlikely as the two compounds typically separate well chro-
matographically.

EU Reference Laboratory for pesticides requiring Single Residue Methods (EURL-SRM) 27 of 102
Table 14: Chromatograms of Chlorate and Perchlorate at 0.2 µg/mL and of a mixture of Chlorate-18O3 and Perchlorate-18O4,
containing approx. 0.2 µg/mL Chlorate 18O3 and approx. 0.02 µg/mL Perchlorate-18O4.
Calibration standard injected Detected signals at mass trace of …

Chlorate 0.2 µg/mL

Chlorate 83/67

Perchlorate 0.2 µg/mL

Chlorate 83/67 Perchlorate 99/83

RT slightly shifted
18 compared to injec-
Chlorate- O3 (approx. tion above
0.18 µg/mL) +
Perchlorate-18O4 (approx. 0.02
µg/mL)

Chlorate-18O3 89/71 Perchlorate-18O4 107/89

3. Miscellaneous
a) Diquat and Paraquat:
Diquat and Paraquat produce several parent ions within the ion-source, each one fragmenting to various
product ions. The most prominent parent ions observed are the doubly charged ones ([M]2+), the singly
charged protonated ones ([M2+ - H+]+) and the singly charged radical ones ([M]+•). The relative yields of the
various parent ions were shown to greatly depend on the co-eluting matrix, which gives an additional
dimension to the matrix-effects. Mass transitions originating from the same parent ion are simmilarly af-
fected by co-eluting matrix (this also applies to the IL-IS), unlike those originating from different parent
ions. For a proper equalization of matrix effects and correct quantitations, it is thus paramount to use
equivalent parent ions (or even better, equivalent mass-transitions) of native analyte and the corre-
sponsing IL-IS. Generally, measurements achieved when using transitions from doubly charged parent ions
([M]2+) were more robust and validation results fluctuating less.
Table 15 gives an overview of mass transitions of Diquat while Table 16 shows matrix effects for various
mass-transitions in infant formula powder.

EU Reference Laboratory for pesticides requiring Single Residue Methods (EURL-SRM) 28 of 102
Table 15: Individual transitions and MS/MS settings (Sciex API 5500) for Diquat and Paraquat and their respective IL-ISs on Sciex
5500 QTrap ESI(+). Transitions are grouped by parent type.
Suitable IL-IS Sensitivity
Q1 (m/z) Q3 (m/z) DP (V) CE (V) CXP (V)
transition Ranking*
Diquat [M]2+ 92/84 1 92 84.4 61 21 4
IL-IS Diquat D8
Diquat [M]2+ 92/157 3 92 157 61 19 12
[M]2+
Diquat [M]2+ 92/78 6 92 78 61 31 12
96/88
Diquat [M]2+ 92/130 5 92 130 61 25 8
Diquat [M2+ - H+]+ 183/157 2 183 157 161 31 10
IL-IS Diquat D8
Diquat [M2+ - H+]+ 183/130 4 183 130 161 43 8
[M2+- H+]+
Diquat [M2+ - H+]+ 183/168 5 183 168 161 37 10
191/165
Diquat [M2+ - H+]+ 183/78 4 183 78 161 51 12
Diquat [M] +• 184/128*** 4 184 128 60 55 8
Diquat [M] +• 184/106 5 184 106 60 23 8
Diquat [M+•184/78 IL-IS Diquat D8 5 184 78 60 65 12
Diquat [M] +• 184/156*** [M] +• 4 184 156 60 29 10
Diquat [M] +• 184/169 192/134 5 184 169 60 27 12
Diquat [M] +• 184/155 5 184 155 60 43 12
Diquat [M] +• 184/168 5 184 168 60 45 12
IL-IS Diquat D8 [M]2+ 96/88 - 96 88.4 61 21 4
IL-IS Diquat D8 [M2+- H+]+ 191/165 - 191 165 101 31 10
IL-IS Diquat D8 [M] +• 192/134 - 192 134 156 55 8

Paraquat [M]2+ 93/171 2 93 171 46 15 12

IL-IS Paraquat D8
Paraquat [M]2+ 93/77 3 93 77 46 31 12
[M]2+
Paraquat [M]2+ 93/155 4 93 155 46 25 10
97/179
Paraquat [M]2+ 93/144 4 93 144 46 17 8
Paraquat [M]+• 186/171 1 186 171 41 25 12
Paraquat [M]+• 186/77 IL-IS Paraquat D8 3 186 77 41 57 4
Paraquat [M]+• 186/155 [M]+• 4 186 155 41 55 8
Paraquat [M]+• 186/128 194/179 5 186 128 41 57 12
Paraquat [M]+• 186/103 4 186 103 41 49 12
Paraquat [M2+- H+]+ 185/170 ** 185 170 61 23 8
IL-IS Paraquat D8
Paraquat [M2+- H+]+ 185/169 ** 185 169 61 37 8
[M2+- H+]+
Paraquat [M2+- H+]+ 185/144 ** 185 144 61 29 8
193/178
Paraquat [M2+- H+]+ 185/115 ** 185 115 61 55 8
IL-IS Paraquat D8 [M]2+ 97/179 - 97 179 46 15 12
IL-IS Paraquat D8 [M] +• 194/179 - 194 179 71 27 10
IL-IS Paraquat D8 [M2+- H+]+ - 193 178 86 29 12
193/178
* The ranking in this table only refers to the signal to noise ratio. Further experiments are planned to study signal repeatability of various mass
transitions also in comparison with the transitions of the respective IL-IS.
** MRMs of singly charged protonated Paraquat ([M2+- H+]+ 185/#) are typically less sensitive and tend to show more variable signals than the
MRMs of the other two parent ions ([M]2+ and [M] +•). Furthermore these transitions tend to be interfered by Diquat. Same applies to the re-
spective MRM of Paraquat D8 ([M2+- H+]+193/#) , which is interfered by Diquat D8.
*** Removed from this Table as the signals showed more variability than the ones newly included

EU Reference Laboratory for pesticides requiring Single Residue Methods (EURL-SRM) 29 of 102
Table 16: Exemplary matrix effects of Diquat in infant formula powder considering mass transitions resulting from different parents
(the conc. of Diquat and Paraquat in the final extract was 0.015 µg/mL each)
Matrix Effect of parent (%) Matrix Effect of corresponding IL-IS D8 (%)
Analyte Type of parent ion
(MRM) (MRM)
[M]2+ +57 (92/84) +54 (96/88)
Diquat [M2+ - H+]+ -92 (183/157) -91 (191/165)
[M] +• -91 (184/128) -93 (192/134)
[M]2+ +111 (93/171) +112 (97/179)
Paraquat [M2+ - H+]+ -74 (185/170) -78 (193/178)
[M] +• -81 (186/171) -78 (194/179)

b) Carry-over:
Be aware that some analytes are prone to carry-over effects and regularly observe blank solvent or blank
matrix extract injections for the occurrence of any carry-over. In some cases signals deriving from carry-
over are more intensive when injecting blank matrix extracts compared to pure solvent.
Examples of analytes where carry-over has been observed: Diquat, Paraquat, Phosphonic acid, Chlorate,
Glyphosate.
c) Avoid glass containers for certain analytes:
Keep solutions in plastic vessels 2.15 as several of the compounds covered by this method tend to interact
with glass-surfaces (see examples under 2.12).

4. Handling of column, pre-column and pre-filters:

a) AS11 and AS11HC:

Priming and reconditioning of column: before first use, after long storage (e.g. >2 weeks), after injection
of 50-100 sample extracts):
- Flush column for 30 minutes with 100 mmol aqueous Borax solution (7.62 g di-sodium tetra-
borate decahydrate in 200 mL water) at 0.3 mL/min OR
- Flush for 1 hour with 30 mM NaOH (240 mg NaOH in 200 mL water) at 0.3 mL/min
- Flush column for 30 minutes with Eluent A (water) at 0.3 mL/minRun system 3-4 times with full
gradient (inject standards in matrix)
Note: When flushing NaOH or Borax solution through the column make sure that it will go directly into
waste and not to the MS ion source!.
Storage of column: If to be stored for short periods (<2 weeks), columns can be put aside after any normal
sequence/run (full gradient). Run system 3-4 times with full gradient to reactivate the column (inject
standards in matrix) before starting a sequence. If to be stored for longer periods (e.g. >2 months) recon-
dition the column as described above.
Pre-filters: If pre-filters are used exchange them as soon as backpressure increases significantly. For prac-
tical and convenience reasons it is highly recommended to exchange pre-filters when performing other
maintenance operations such as reconditioning or pre-column exchange. Losses of glyphosate, that could
be clearly linked to interactions with a dirty pre-filter, have been once observed.
Pre-columns (guard columns): The pre-column should be exchanged as soon as a clear deterioration of
the separation performance (worsening of peak-shape) is noticed. The pre-column of M 1.1. needs to be
exchanged more often than that of M 1.2 and M 1.3. If after pre-filter exchange (see above) the pressure
does not come back to normal levels, the frit of the pre-column should be exchanged.

For further information on the storage and cleanup of column, see: http://www.dionex.com/en-us/web-
docs/113497-Man-065463-03-IonPac-AS11-HC-4um-Nov12.pdf

EU Reference Laboratory for pesticides requiring Single Residue Methods (EURL-SRM) 30 of 102
 b) Hypercarb
Priming and reconditioning of column: Before first use, Hypercarb columns and pre-columns have to be
thoroughly primed to cover certain active sites on the surface. Priming with solutions containing planar
molecules such as chlorophyll and anthocyans accelerates the priming period. Priming may be performed
by multiple injection of a QuPPe extract of spinach or of a grape skin extract solution (prepared by dissolv-
ing 100 mg grape skin extract in 20 mL methanol + 1% FA-H2O 1:1). For a quick equilibration, the LC-
conditions shown in Table 17 may be used. 10-15 injections of spinach extracts are typically required for
the pre-column and ca. 50 injections for the column and pre-column combined. If possible inject 50 µL
each time. This masking of the active sites is temporary as the activity of the column gradually increases
with the injection of solvent or diluted extracts. Following a sequence of injections with low or no matrix
load will typically raise the need for intermediate conditioning with extracts to reobtain sufficient column
masking. The impact of priming on the chromatographic properties of the column is exemplary shown in
Figure 4, Figure 5 and Figure 6.

Table 17: Proposed LC-MS/MS conditions for priming and reconditioning of the Hypercarb column.
Instrument parameters Conditions
Ionisation mode ESI neg
Column/temperature Hypercarb 2.1 x 100 mm 5 µm (P/N 35005-102130); 40°C
Pre-column Hypercarb Guard 2.1 x 10 mm 5 µm (P/N 35005-102101)
Pre-filters e.g. Supelco column saver 2.0 µm Filter (optional)
Eluent A 1% acetic acid in water + 5% methanol
Eluent B 1% acetic acid in methanol
%A Flow [mL/min] Time [min]
100 0.3 0
Gradient
70 0.3 7
100 0.3 7.1
100 0.3 12
Injection volume 50 µL
MS-System If possible disconnect the MS-System to prevent contamination of the MS.

HEPA 125/63 T Ethephon 143/107 T N-Acetyl-AMPA 152/63 T Glyphosate 168/63 T

Figure 4: Chromatograms obtained using a new Hypercarb column, poor chromatographic behavior due to strong interactions of
analytes with active sites. Same behavior is observed when the pre-column is new.

HEPA 125/63 T Ethephon 143/107 T N-Acetyl-AMPA 152/63 T Glyphosate 168/63 T

Figure 5: Chromatograms following priming with 25 injections QuPPe extracts of spinach. Injection volume 50 µL per injection

EU Reference Laboratory for pesticides requiring Single Residue Methods (EURL-SRM) 31 of 102
 HEPA 125/63 T Ethephon 143/107 T N-Acetyl-AMPA 152/63 T Glyphosate 168/63 T

Figure 6: Chromatograms after additional injection of approximately 100 QuPPe-extracts of various fruit and vegetables during
normal routine use.

Pre-columns (guard columns): The pre-column should be exchanged as soon as a clear deterioration of
the separation performance (worsening of peak-shape) is noticed. The pre-column of M 1.3 needs to be
clearly less often exchanged compared to the pre-columns of M 1.1 and M 1.2. Any exchange of the pre-
column requires priming as described above. For this the pre-column does not have to be attached to the
column. Connecting several pre-columns in a row and priming them simultaneously is also an option.
Storage of columns: Following normal operation the column can be stored directly after any normal se-
quence/run (full gradient). Run system 3-4 times with full gradient to reactivate the column (inject stand-
ards in matrix) before starting the sequence. If to be stored for longer periods (e.g. >2 months) it is highly
recommended to recondition the column as described above.
Pre-filters: If pre-filters are used exchange them as soon as backpressure increases significantly. For prac-
tical and convenience reasons it is highly recommended to exchange pre-filters when performing other
maintenance operations such as reconditioning or pre-column exchange. If after pre-filter exchange (see
above) the pressure does not come back to normal levels, the frit of the pre-column may need to be ex-
changed.
Note: Losses of Glyphosate, that could be clearly linked to interactions with a dirty pre-filter, have been
once observed.

c) Torus DEA
Priming:
The Torus DEA column should be conditioned before use following the manufacturer’s Start-up Guide,
which foresees flushing the column with a 5 mmol/L solution of Na2EDTA. Afterwards it is important to
prime thoroughly.

d) Raptor Polar X
Priming:
The manufacturing company of this column recommends “passivating” the LC-system with a methanolic
solution of Methylenediphosphonic Acid (Medronic Acid) (1984-15-2).

EU Reference Laboratory for pesticides requiring Single Residue Methods (EURL-SRM) 32 of 102
 Method 1.1 (M1.1): “Gly&Co. AS 11”
Table 18: Proposed LC-MS/MS conditions for Ethephon, HEPA (Ethephon metabolite), Glyphosat, AMPA (Glyphosate metabolite),
Glufosinate, MPPA (Glufosinate metabolite), N-Acetyl-Glufosinate (Glufosinate metabolite), Phosphonic acid.
Instrument parameters Conditions
Ionization mode ESI neg
Column/temperature (see notes) Dionex IonPac AS 11 2 x 250 mm (P/N 44077); 40°C
Pre-column Dionex IonPac AG 11 2 x 50 mm (P/N 44079)
Pre-filters e.g. Supelco column saver 2.0 µm Filter (optional)
Eluent A Water (3.1)
1 mM citric acid in water adjusted to pH 11 with dimethylamine (DMA)
Eluent B Note: You will need approx 0.5 mL DMA solution for 500 mL 1 mM citric acid in water
Make sure your eluent filters can handle alkaline solvents (see notes)!!
%A Flow [mL/min] Time [min]
100 0.3 0
50 0.3 8
Gradient
50 0.3 15
100 0.3 15.1
100 0.3 23
10-20 µL
Injection volume
(Note: in case of analyzing only Ethephon 5 µL may be enough -depending on the instrument)
Calibration standards and levels e.g. 0.05 or 0.1 µg/IS-portion* + one level at the reporting limit
Compound Mass Transitions (m/z)
Glyphosate 168/63, 168/124, 168/150, 168/81
Glyphosate-13C2,15N1 (IL-IS) 171/63, 171/126
AMPA 110/63, 110/79, 110/81**
AMPA-13C115N1 (IL-IS) 112/63, 112/81
Ethephon 143/107, 143/79, 145/107
Ethephon-D4 (IL-IS) 147/111, 147/79 (optional, in case of interferences)
HEPA 125/79, 125/95, 125/63
Acquired mass transitions (m/z)
HEPA-D4 (IL-IS) 129/79, 129/97
Glufosinate 180/63, 180/136, 180/85, 180/95
Glufosinate-D3 (IL-IS) 183/63, 183/98
N-Acetyl-Glufosinate 222/63, 222/59, 222/136
N-Acetyl-Glufosinate-[acetyl]D3 (IL-IS) 225/63, 225/137
N-Acetyl-Glufosinate-[methyl]D3 (IL-IS) 225/63
MPPA 151/63, 151/107, 151/133
MPPA-D3 (IL-IS) 154/63, 154/136
AMPA: Aminomethylphosphonic acid;
MPPA: 3-Methylphosphinicopropionic acid;
HEPA: 2-Hydroxyethylphosphonic acid (= hydroxy-ethephon),
* One IS portion is the absolute IS-mass contained in the prepared calibration standard solution (see also Table 2).
** See also 5.6.1.

Hints on Method 1.1

1. pH-related precautions: As the pH of the mobile phase is quite high, it is recommendable to use alkali-compati-
ble components, e.g. metal frits instead of silica frits in the Eluent B reservoir; borosilicate 3.3 bottles instead of
glass bottles for eluent B; rotor-seals from alkali-persistent materials, such as PEEK (polyetherketone) or Tefzel,
rather than Vespel.
2. Handling of column, pre-column and pre-filters: See 5.6.1. point 4.a)
3. For general hints on analytes: See 5.6.1

EU Reference Laboratory for pesticides requiring Single Residue Methods (EURL-SRM) 33 of 102
 SRM problems with RT-shift / peak shape can
occur depending on the matrix!

Glyphosate AMPA Glufosinate MPPA Ethephon

168/63 110/63 180/63 150/63 143/107
Spargel mit 0.05 ug/IS bw. mL in M eOH+1%AS - Glyphosat 167.9 / 62.9 (Unknown) 167.9/62.9 amu - sample 25 of 34 from Validierun... Sp argel m it 0.05 u g/IS b w. m L in M eO H+1% AS - AM PA 110.0 / 62.8 ( Unknown) 110.0/62.8 am u - sam ple 25 o f 34 from Validierung Et... Spargel mit 0.05 ug/IS bw. m L in M eOH +1% AS - Glu fosinate 180.0 / 62.9 (U nknown ) 180.0/62.9 am u - samp le 25 of 34 fro m Validieru... Spargel m it 0.05 ug /IS b w. m L in MeOH +1% AS - MPPA 150.9 / 62.8 (U nknown ) 150.9/62.8 amu - sam ple 25 of 34 from Validierung Et... Spargel m it 0.05 ug/IS bw. m L in MeOH+1% AS - Ethephon 142.9 / 106.9 (U nknow n) 142.9/106.9 amu - sample 25 of 35 from Validieru...
Area: 2.12e+004 counts H eight: 3.43e+003 cps R T: 9.792 min A rea: 3.99e+004 cou nts Heigh t: 3.78e+003 cps RT: 7.341 m in Area: 4.42e+004 counts Heigh t: 7.09e+003 cps RT : 7.269 m in A rea: 1.27e+005 coun ts Heig ht: 2.01e+004 cps RT: 7.836 min Area: 4.77e+004 counts Height: 6.63e+003 cps R T: 9.096 m in

9. 79 7. 34 7 . 27 7. 84 9.1 0
34 00 2. 0 e4
65 00
3 6 00
32 00 6 50 0 1. 9 e4
3 4 00

0.1 ppm
1. 8 e4 60 00
30 00
3 2 00 6 00 0
1. 7 e4
28 00 55 00
3 0 00
5 50 0 1. 6 e4
26 00
2 8 00 1. 5 e4 50 00
24 00 5 00 0
2 6 00 1. 4 e4
45 00
22 00 2 4 00 4 50 0 1. 3 e4
I n t e n s it y , c p s
In t e n s i ty , c p s

I n te n s i ty , c p s
1. 2 e4 40 00

I n te n s i t y , c p s

I n te n s i ty , c p s
20 00 2 2 00

on asparagus
4 00 0
2 0 00 1. 1 e4
18 00 35 00
3 50 0 1. 0 e4
1 8 00
16 00
90 0 0 .0 30 00
1 6 00 3 00 0
14 00
80 0 0 .0
1 4 00 25 00
12 00 2 50 0 70 0 0 .0
1 2 00
10 00 60 0 0 .0 20 00
2 00 0
1 0 00
80 0 50 0 0 .0
800 7 .8 3 15 00
1 50 0
40 0 0 .0
60 0
600
30 0 0 .0 10 00
1 00 0
40 0 400
20 0 0 .0
20 0 200 5 00 50 0
10 0 0 .0
0 0 0. 0
1 2 3 4 5 6 7 8 9 10 11 1 2 3 4 5 6 7 8 9 10 11 0 0
1 2 3 4 5 6 7 8 9 10 11 1 2 3 4 5 6 7 8 9 10 11 1 2 3 4 5 6 7 8 9 10 11
T im e, m in T im e , m in T im e , m in
T im e , m in T im e , m in
K artoffel mit 0.05 ug/IS bw. mL in MeOH+1%AS - Glyphosat 167.9 / 62.9 (Unknown) 167.9/62.9 am u - sample 27 of 34 from Validieru... K artoffel mit 0.05 ug/IS bw. mL in M eOH +1% AS - A MPA 110.0 / 62.8 (U nkn own) 110.0/62.8 amu - sam ple 27 of 34 from Validieru ng E... Kartoffel mit 0.05 ug/IS bw. mL in MeOH+1%AS - Glufosinate 180.0 / 62.9 (Unknown) 180.0/62.9 amu - sample 27 of 34 from Validier... K artof fel m it 0.05 u g/IS b w. m L in M eO H+ 1% A S - M PPA 150.9 / 62.8 (U nkn own ) 150.9/62.8 am u - sam ple 27 of 34 from Valid ierun g E ... K artoffel mit 0.05 ug /IS bw. mL in M eOH +1%A S - Ethephon 142.9 / 106.9 (Unknown) 142.9/106.9 am u - sam ple 27 of 35 from Validier...
Area: 2.06e+004 counts Height: 3.38e+003 cps RT: 9.627 min Area: 4.11e+004 cou nts Height: 5.63e+003 cps RT: 7.559 min Area: 4.74e+004 counts Height: 7.01e+003 cps RT: 7.535 min A rea: 2.00e+ 005 cou nts H eigh t: 2.62e+004 cp s R T : 7.723 m in Area: 4.78e+004 counts Height: 7.27e+003 cps R T: 9.035 m in

9 .63 7 .5 6 7.53 7 .7 2 9 .04

7000 2. 6 e 4
5 50 0 70 00
32 00

0.1 ppm
6500 2. 4 e 4
65 00
30 00 5 00 0
6000 2. 2 e 4
28 00 60 00
4 50 0
26 00 5500 2. 0 e 4 55 00

24 00 4 00 0 5000
1. 8 e 4 50 00
22 00
3 50 0 4500 45 00

on potato
1. 6 e 4

In te n s ity , c p s
I n te n s i t y , c p s
I n t e n s it y , c p s

I n te n s i ty , c p s
20 00
I n te n s i ty , c p s

4000 40 00
18 00 3 00 0 1. 4 e 4

16 00
3500 35 00
1. 2 e 4
2 50 0
14 00 3000 30 00
1. 0 e 4
12 00 2 00 0 25 00
2500
10 00 80 0 0 . 0
1 50 0 2000 20 00
80 0
60 0 0 . 0
1500 15 00
60 0 1 00 0
40 0 0 . 0
1000 10 00
40 0
5 00
20 0 0 . 0 50 0
20 0 8 .1 5 500
8 .1 5
0 0 0. 0 0
1 2 3 4 5 6 7 8 9 10 11 1 2 3 4 5 6 7 8 9 10 11 0 1 2 3 4 5 6 7 8 9 10 11 1 2 3 4 5 6 7 8 9 10 11
T im e, m in T im e , m in
1 2 3 4 5 6 7 8 9 10 11
T im e , m in T im e , m in
Tim e, m in
Erdbeere mit 0.05 ug/IS bw. mL in MeOH+1%AS - Glyphosat 167.9 / 62.9 (Unknown) 167.9/62.9 amu - sample 28 of 34 from Validieru... Erdbeere mit 0.05 ug/IS bw. mL in M eOH +1%A S - AM PA 110.0 / 62.8 (Un known) 110.0/62.8 amu - sample 28 of 34 from Validierung E... Erdbeere mit 0.05 ug/IS bw. mL in MeOH+1%AS - Glufosinate 180.0 / 62.9 (Unknown) 180.0/62.9 amu - sample 28 of 34 from Validier... E rdbeere mit 0.05 ug/IS bw. mL in MeOH+1%AS - MPPA 150.9 / 62.8 (Unknown) 150.9/62.8 amu - sample 28 of 34 from Validierung E... Erdbeere mit 0.05 ug/IS bw. mL in MeOH+1%AS - Ethephon 142.9 / 106.9 (Unknown) 142.9/106.9 amu - sample 28 of 35 from Validier...
Area: 2.37e+004 counts Height: 3.90e+003 cps RT: 9.601 min Area: 8.87e+004 co unts Height: 9.32e+003 cps RT: 7.086 min Area: 8.30e+004 counts Height: 1.25e+004 cps RT: 7.032 min Area: 1.25e+ 005 counts Height: 1.96e+004 cps RT: 7.386 min Area: 5.16e+004 counts Height: 7.93e+003 cps RT: 9.060 min

9.60 7. 09 7.03 7.39 9.06

90 00 1.9e4
3800
1.20e4 7500
85 00 1.8e4
3600
80 00 1.7e4 7000
3400 1.10e4

0.1 ppm
75 00 1.6e4 6500
3200
1.00e4
70 00 1.5e4
3000 6000
65 00 1.4e4
2800 9000.00
5500
60 00
1.3e4
2600
8000.00 5000
2400 55 00
1.2e4
I n te n s i ty , c p s

I n t e n s i ty , c p s

In te n s ity , c p s
In t e n s i ty , c p s

I n te n s i ty , c p s
2200 1.1e4 4500
50 00 7000.00

on strawberry
2000 1.0e4 4000
45 00
6000.00 9000.0
1800 40 00 3500
1600 8000.0
35 00 5000.00
3000
1400 7000.0
30 00
1200 4000.00 6000.0 2500
25 00
1000 5000.0 2000
20 00 3000.00
800 4000.0
15 00
1500
600 2000.00 3000.0
10 00 1000
400 2000.0
50 0
1000.00
7.7 6 500 1.58
200 1000.0
0
0 1 2 3 4 5 6 7 8 9 10 11 0.00 0.0 0
1 2 3 4 5 6 7 8 9 10 11 1 2 3 4 5 6 7 8 9 10 11 1 2 3 4 5 6 7 8 9 10 11 1 2 3 4 5 6 7 8 9 10 11
T im e , m in
T im e, min Tim e, m in T im e, m in T im e, m in

Community Reference Laboratory

CRL-SRM for Pesticide Residues
EPRW 2008, June 1-5, Berlin HEPA in real samples problems with RT-shift / peak shape can using Single Residue Methods
Figure 7: Typical chromatograms of Glyphosate, AMPA, Glufosinate, MPPA and Ethephon spiked on blank-QuPPe extracts
occur depending on the matrix!

HEPA D4 (IS) HEPA HEPA HEPA

129/79 125/95 125/79 T 125/63

Oat
0.034 ppm

Figgs
0.12 ppm

Pineapple
0.009 ppm

Figure 8: Typical chromatograms of HEPA in real samples

Community Reference Laboratory
CRL-SRM
EPRW 2008, June 1-5, Berlin
for Pesticide Residues
using Single Residue Methods

EU Reference Laboratory for pesticides requiring Single Residue Methods (EURL-SRM) 34 of 102
 Method 1.2 (M1.2): “Gly&Co. AS 11-HC”
Table 19: Proposed LC-MS/MS conditions for Ethephon, HEPA (Ethephon metabolite), Glyphosat, AMPA (Glyphosate metabolite),
Glufosinate, MPPA (Glufosinate metabolite), N-Acetyl-Glufosinate (Glufosinate metabolite), Fosetyl-Al, N-Acetyl-AMPA and Phos-
phonic acid.
Instrument parameters Conditions
Ionization mode ESI neg
Dionex IonPac AS 11-HC 2 x 250 mm (P/N 052961); 40°C
Column/temperature
(see also notes below)
Pre-column Dionex IonPac AG11-HC 2 x 50 mm (P/N 052963)
Pre-filters e.g. Supelco column saver 2.0 µm Filter (optional)
Eluent A water (3.1)
Eluent B 1 mM tribasic Ammonium citrate in water
%A Flow [mL/min] Time [min]
100 0.3 0
0 0.3 8
Gradient
0 0.3 16
100 0.3 16.1
100 0.3 23
Injection volume 10 µL
Calibration standards and levels e.g. 0.05 or 0.1 µg/IS-portion* + one level at the reporting limit
Compound Mass Transitions (m/z)
Glyphosate 168/63, 168/124, 168/150, 168/81
Glyphosate-13C2,15N (IL-IS) 171/63, 171/126
AMPA 110/63, 110/79, 110/81**
AMPA-13C,15N (IL-IS) 112/63, 112/81
N-Acetyl-AMPA 152/63, 152/79, 152/110
Ethephon 143/107, 143/79, 145/107
Ethephon-D4 (IL-IS) 147/111, 147/79 (optional, in case of interferences)
HEPA 125/79, 125/95, 125/63
HEPA-D4 (IL-IS) 129/79, 129/97
Acquired mass transitions (m/z) Glufosinate 180/63, 180/136, 180/85, 180/95
Glufosinate-D3 (IL-IS) 183/63, 183/98
N-Acetyl-Glufosinate 222/63, 222/59, 222/136
N-Acetyl-Glufosinate-[acetyl]D3 (IL-IS) 225/63, 225/137
N-Acetyl-Glufosinate-[methyl]D3 (IL-IS) 225/63
MPPA 151/63, 151/107, 151/133
MPPA-D3 (IL-IS) 154/63, 154/136
Fosetyl-Al: 109/81, 109/63 (Fosetyl)
Fosetyl-Al-D15 (IL-IS): 114/82, 114/63 (Fosetyl-D5)
Phosphonic acid*** 81/79, 81/63
Phosphonic acid-18O3 (IL-IS) 87/85, 87/67
AMPA: Aminomethylphosphonic acid;
MPPA: 3-Methylphosphinicopropionic acid;
HEPA: 2-Hydroxyethylphosphonic acid (=hydroxy-ethephon)
* One IS portion is the absolute IS-mass contained in the prepared calibration standard solution (see also Table 2).
** See also 5.6.1
*** See also 5.6.1

EU Reference Laboratory for pesticides requiring Single Residue Methods (EURL-SRM) 35 of 102
Hints on Method 1.2
1. Handling of column, pre-column and pre-filters: See 5.6.1. point 4.a)
2. Peak splitting: Using this M 1.2 some compounds (e.g. Glyphosate) in some commodities tend to give two sharp
peaks. The corresponding IL-IS typically behaves equally, so that quantification with any of the two peaks remains
accurate
3. Intereference of Phosphonic acid by Fosetyl: See 5.6.1.
4. Intereference of Phosphonic acid by Phosphoric acid: See 5.6.1.
5. IL-IS of N-Acetyl-Glufosinate D3: See 6
6. For general hints on analytes: See 5.6.1

Ethephon 143/ 107 T MPPA 151/ 63 T Glyphosate 168/ 63 T Glufosinate 180/ 63 T

HEPA 125/ 79 T N-Acetyl-Glufosinate 222/ 63 T AMPA 110/ 63 T Fosetyl 109/ 81 T

N-Acetyl-AMPA 152/ 63 T Phosphonic acid 81/79

Figure 9: Typical chromatograms of Ethephon, HEPA, Glyphosat, AMPA, Glufosinate, MPPA, N-Acetyl-AMPA, N-Acetyl-Glufosinate,
Fosetyl-Al and Phosphonic acid at 0.1 mg/L in methanol with 1% formic acid.

EU Reference Laboratory for pesticides requiring Single Residue Methods (EURL-SRM) 36 of 102
 Method 1.3 (M1.3): “Gly&Co. Hypercarb”
Table 20: Proposed LC-MS/MS conditions for Ethephon, HEPA (Ethephon metabolite), Glyphosat, AMPA (Glyphosate metabolite),
N-Acetyl-Glyphosate (Glyphosate metabolite), N-Acetyl-AMPA (Glyphosate metabolite), Glufosinate, MPPA (Glufosinate metabo-
lite), N-Acetyl-Glufosinate (Glufosinate metabolite), Fosetyl-Al, Maleic Hydrazide, Cyanuric acid and Bialaphos.
Instrument parameters Conditions
Ionization mode ESI neg
Column/temperature Hypercarb 2.1 x 100 mm 5 µm (P/N 35005-102130); 40°C
Pre-column Hypercarb Guard 2.1 x 10 mm 5 µm (P/N 35005-102101)
Pre-filters e.g. Supelco column saver 2.0 µm Filter (optional)
Eluent A 1% acetic acid in water + 5% methanol
Eluent B 1% acetic acid in methanol
%A Flow [mL/min] Time [min]
100 0.2 0
70 0.2 10
70 0.4 11
Gradient
70 0.4 18
10 0.4 19
10 0.4 22
100 0.2 22.1
100 0.2 30
Injection volume 5 µL
Dilution Not regularly; in case of strong matrix interferences 5-10-fold (see also Hints 8.)
Calibration standards and levels e.g. 0.05 or 0.1 µg/IS-portion* + one level at the reporting limit
Compound Mass Transitions (m/z)
Glyphosate 168/63, 168/124, 168/150, 168/81
13 15
Glyphosate- C2, N (IL-IS) 171/63, 171/126
AMPA** 110/63, 110/79, 110/81**
AMPA-13C,15N (IL-IS) 112/63, 112/81
N-Acetyl-AMPA: 152/63, 152/79, 152/110
N-Acetyl-Glyphosate 210/63, 210/150, 210/79, 210/148
N-Acetyl-Glyphosate-D3 (IL-IS) 213/63, 213/153
Ethephon 143/107, 143/79, 145/107
Ethephon-D4 (IL-IS) 147/111, 147/79
HEPA 125/79, 125/95, 125/63
HEPA-D4 (IL-IS) 129/79, 129/97
Glufosinate 180/63, 180/136, 180/85, 180/95
Glufosinate-D3 (IL-IS) 183/63, 183/98
Acquired mass transitions (m/z)
N-Acetyl-Glufosinate: 222/63, 222/59, 222/136
N-Acetyl-Glufosinate-[acetyl]D3 (IL-IS) 225/63, 225/137
N-Acetyl-Glufosinate-[methyl]D3 (IL-IS) 225/63
MPPA 151/63, 151/107, 151/133
MPPA-D3 (IL-IS) 154/63, 154/136
Fosetyl-Al 109/81, 109/63 (detected as Fosetyl)
Fosetyl-Al-D15 (IL-IS) 114/82, 114/63 (detected as Fosetyl-D5)
Maleic Hydrazide 111/82, 111/42, 111/55, 111/83
Maleic Hydrazide-D2 (IL-IS) 113/42, 113/85
Maleic Hydrazide-13C4 (IL-IS) 115/87, 115/58
Cyanuric acid 128/42, 128/85
Cyanuric acid-13C3 131/43, 131/87
Bialaphos 322/88, 322/94, 322/134
Desmethyl-Dimethoate 214/104, 214/95, 214/136
* One IS portion is the absolute IS-mass contained in the prepared calibration standard solution (see also Table 2).
** See also 5.6.1
*** See also 5.6.1

EU Reference Laboratory for pesticides requiring Single Residue Methods (EURL-SRM) 37 of 102
Hints on Method 1.3
1. Handling of column, pre-column and pre-filters: See 5.6.1 point 4.a)4.b)
2. Mass spectrometric interference of AMPA and Fosetyl: see 5.6.1
3. Intereference of Phosphonic acid by Fosetyl: see 5.6.1
4. Degradation of Ethephon to Phosphonic acid: see 5.6.1
5. Dilution: For certain matrices it can be beneficial to dilute the sample extract 5-10-fold before injection or to inject
smaller volumes (1-2 µL). Dilution of the sample extract is highly recommended for matrices containing high
amounts of protein such as oily seeds, pulses and commodities of animal origin in general. In routine analysis there
is an option run undiluted extracts for screening and in case of a positive result repeat measurement with a diluted
extract (provided that there is no issues with false negatives, in non-diluted extracts are injected, see also 5.6.1).
6. IL-IS of N-Acetyl-Glufosinate D3 see 6
7. Reference: In case of the determination of Fosetyl and Phosphonic acid on the Hypercarb-column, we refer to the
patent of D. Rosati and C. Venet from Bayer CropScience (Patent-No. WO 2006079566 A1).
8. For general hints on analytes: See 5.6.1

Desmethyl-Dimethoate 214/104 Glyphosate 168/63 T AMPA 110/63 T N-Acetyl-AMPA 152/63 T

Ethephon 143/107 T N-Acetyl-Glyphosate 210/63 Glufosinate 180/63 T N-Acetyl-Glufosinate 222/63 T

MPPA 151/63 T Cyanuric acid 128/42 T Fosetyl 109/81 T Maleic Hydrazide 111/82 T

HEPA 125/63 T Bialaphos 322/88 T

Figure 10: Chromatograms of Glyphosate, AMPA, N-Acetyl-AMPA, N-Acetyl-Glyphosate, Ethephon, HEPA, Glufosinate, MPPA, N-
Acetyl-Glufosinate, Fosetyl, Maleic Hydrazide, Cyanuric acid and Bialaphos at 0.02 mg/kg on apple extract and Desmethyl-Dime-
thoate at 0.03 mg/kg on cherry extract.

EU Reference Laboratory for pesticides requiring Single Residue Methods (EURL-SRM) 38 of 102
 Method 1.4 (M1.4): “PerChloPhos”
Table 21: Proposed LC-MS/MS conditions for Phosphonic acid (Fosetyl metabolite), Perchlorate, Chlorate, Bromide and Bromate.
Instrument parameters Conditions
Ionisation mode ESI neg
Column/temperature Hypercarb 2.1 x 100 mm 5 µm (P/N 35005-102130); 40°C
Pre-column Hypercarb Guard 2.1 x 10 mm 5 µm (P/N 35005-102101)
Pre-filters e.g. Supelco column saver 2.0 µm Filter (optional)
Eluent A 1% acetic acid in water + 5% methanol
Eluent B 1% acetic acid in methanol
%A Flow [mL/min] Time [min]
100 0.4 0
Gradient
70 0.4 10
100 0.4 10.1
100 0.4 15
Injection volume 5 µL
5-fold dilution with methanol + 1% formic acid
Dilution
(1 µL sample extract + 4 µL methanol + 1% formic acid)
Calibration standards and levels e.g. 0.05 or 0.1 µg/IS portion* + one level at the reporting limit
Compound Mass Transitions (m/z)
Bromate 127/95, 129/113, 127/111, 129/97
Bromate-18O3 (IL-IS) 135/117
Bromide* 81/81, 79/79
Chlorate 83/67, 85/69
Acquired mass transitions Chlorate-18O3 (IL-IS) 89/71, 91/73
Perchlorate 99/83, 101/85
Perchlorate-18O4 (IL-IS) 107/89, 109/91
Phosphonic acid 81/79, 81/63
Phosphonic acid-18O3 (IL-IS) 87/85, 87/67
Thiocyanate 58/58
Thiocyanate 13C 15N 60/60
* A 5-fold dilution is used for Bromide screening. For quantification purposes where Bromide exceeds approx. 1 mg/kg, the sam-
ple extracts should be diluted e.g. 250-fold (50-fold manually and 5-fold by the HPLC).

Hints on Method 1.4

1. Handling of column, pre-column and pre-filters: see 5.6.1 point 4.a)4.b).
2. Cross-contamination and other issues on Perchlorate and Chlorate: See 5.6.1
3. Degradation of Ethephon and Fosetyl to Phosphonic acid: See 5.6.1.
4. Intereference of Phosphonic acid by Phosphoric acid and impact of dilution: See 5.6.1.
5. Improving selectivity of Bromide analysis: See 5.6.1.
6. For general hints on analytes: See 5.6.1

EU Reference Laboratory for pesticides requiring Single Residue Methods (EURL-SRM) 39 of 102
 Bromate 129/113 Chlorate 83/67 T Perchlorate 99/83 T Phosphonic acid 81/79 T

Bromate-18O3 135/117 Chlorate 18O3 IS 89/71 Perchlorate 18O4 IS 107/89 Phosphonic acid 18O3 IS 87/85

Bromide 81/81 T Thiocyanat 58/58

Figure 11: Chromatograms of Bromate (0.02 mg/kg) in currant extract, Bromide (1 mg/kg) in currant extract, Phosphonic acid
(0.05 mg/kg) in currant extract, Perchlorate (0.01 mg/kg) in currant extract, Chlorate (0.01 mg/kg) in currant extract and Thiocy-
anate (1 mg/kg) in porree extract.

EU Reference Laboratory for pesticides requiring Single Residue Methods (EURL-SRM) 40 of 102
 Method 1.5 (M1.5): “Gly&Co. on Trinity Q1”
Table 22: Proposed LC-MS/MS conditions for Glyphosate, AMPA, N-Acetyl-AMPA, N-Acetyl-glyphosate, Ethephon, HEPA,
Glufosinate, N-Acetyl-Glufosinate, MPPA and Fosetyl-Al, Maleic Hydrazide, Cyanuric acid, Bialaphos, Bromide, Chlorate, Perchlo-
rate, Phosphonic acid
Instrument parameters Conditions
Ionisation mode ESI neg
Column/temperature Acclaim Trinity Q1 100x2.1 mm; 3 µm (P/N 079717; Thermo Fisher Scientific); 30 °C
Pre-column Acclaim Trinity Q1 Guard Cartridge 2.1x10 mm, 5 µm (P/N 083244; Thermo Fisher Scientific)
Pre-filters e.g. Supelco column saver 2.0 µm Filter (optional)
50 mM Ammonium formate (pH 2.9) in water+acetonitrile 6+4
Eluent A
5 mL 5 M Ammoniumformate and 4.5 mL Formic acid ad 300 mL water, add 200 mL ACN
Eluent B Acetonitrile
Time [min] Flow [mL/min] %A
0 0.5 100
10 0.5 100
Gradient
10.1 0.5 18.2 (≙ 90 % acetonitrile)
13 0.5 18.2 (≙ 90 % acetonitrile)
13.1 0.5 100
18 0.5 100
Injection volume 10 µL
Dilution Not regularly; in case of many matrix interferences 5-10-fold
Calibration standards and levels e.g. 0.05 or 0.1 µg/IS portion* + one level at the reporting limit
Compound Mass Transitions (m/z)
Glyphosate 168/63, 168/124, 168/150, 168/81
Glyphosate-13C2,15N (IL-IS) 171/63, 171/126
AMPA** 110/63, 110/79, 110/81**
AMPA-13C,15N (IL-IS) 112/63, 112/81
N-Acetyl-AMPA: 152/63, 152/79, 152/110
N-Acetyl-Glyphosate 210/63, 210/150, 210/79, 210/148
N-Acetyl-Glyphosate-D3 (IL-IS) 213/63, 213/153
Ethephon 143/107, 143/79, 145/107
Ethephon-D4 (IL-IS) 147/111, 147/79 (optional, when interferences)
HEPA 125/79, 125/95, 125/63
HEPA-D4 (IL-IS) 129/79, 129/97
Glufosinate: 180/63, 180/136, 180/85, 180/95
Glufosinate-D3 (IL-IS): 183/63, 183/98
N-Acetyl-Glufosinate 222/63, 222/59, 222/136
N-Acetyl-Glufosinate-[acetyl]D3 (IL-IS) 225/63, 225/137
N-Acetyl-Glufosinate-[methyl]D3 (IL-IS) 225/63
Acquired mass transitions (m/z)
MPPA: 151/63, 151/107, 151/133
MPPA-D3 (IL-IS) 154/63, 154/136
Fosetyl-Al 109/81, 109/63 (each detected as Fosetyl)
Fosetyl-Al-D15 (IL-IS) 114/82, 114/63 (each detected as Fosetyl- D5)
Maleic Hydrazide 111/82, 111/42, 111/55, 111/83
Maleic Hydrazide-D2 (IL-IS) 113/42, 113/85
Maleic Hydrazide-13C4 (IL-IS) 115/87, 115/58
Cyanuric acid 128/42, 128/85
Cyanuric acid-13C3 131/43, 131/87
Bialaphos 322/88, 322/94, 322/134
Bromide* 81/81, 79/79
Chlorate 83/67, 85/69
Chlorate-18O3 (IL-IS) 89/71, 91/73
Perchlorate 99/83, 101/85
Perchlorate-18O4 (IL-IS) 107/89, 109/91
Phosphonic acid 81/79, 81/63
Phosphonic acid 18O3 (IL-IS) 87/85, 87/67
* It is recommended to use an optimized collision energy for Bromide as described in 5.6.1.

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Hints on Method 1.5
1. For general hints on analytes see 5.6.1

AMPA 110/63 T Bialaphos 322/216 T Ethephon 143/107 T Fosetyl 109/63 T

Glufosinate 180/63 T Glyphosate 168/63 T HEPA 125/63 MPPA 151/63 T

N-Acetyl-AMPA 152/63 T N-Acetyl-Glufosinate 222/136 T N-Acetyl-Glyphosate 210/63 Phosphonic Acid 81/79

Chlorate 83/67 Perchlorate 99/83 Bromide 81/81

Figure 12: Chromatograms of Glyphosate, AMPA, N-Acetyl-AMPA, N-Acetyl-Glyphosate, Ethephon, HEPA, Glufosinate, MPPA, N-
Acetyl-Glufosinate, Fosetyl, Maleic Hydrazide, Cyanuric acid, Bialaphos and Bromide at 0.02 mg/kg each, Phosphonic acid at 0.05
mg/kg, as well as Chlorate and Perchlorate at 0.005 mg/kg each, all in black currant extract.

EU Reference Laboratory for pesticides requiring Single Residue Methods (EURL-SRM) 42 of 102
 Method 1.6 “Gly&Co. on Torus DEA; (M1.6a)” or “Gly&Co. on Anionic Polar Pesticide Column (APPC);
(M1.6b)”

Table 23: Proposed LC-MS/MS conditions for Glyphosate, AMPA, N-Acetyl-AMPA, N-Acetyl-Glyphosate, Ethephon, HEPA,
Glufosinate, N-Acetyl-Glufosinate, MPPA, Fosetyl-Al, Trifluoroacetic acid, Phosphonic acid and Bromide.
Instrument parameters Conditions
Ionisation mode ESI neg
M1.6a: Waters Torus™DEA 2.1 mm x 100 mm; 1.7 µm; 50 °C
Column/temperature
M1.6b: Waters Anionic Polar Pesticide Column (APPC), 130Å, 5 µm, 2.1 mm x 100 mm; 50 °C
M1.6a: Waters Torus™DEA VanGuard™ 2.1 mm x 5 mm; 1.7 µm
Pre-column
M1.6b: Waters Anionic Polar Pesticide VanGuard Cartridge, 130Å, 5µm, 2.1 mm X 5 mm
Pre-filters Waters ACQUITY UPLC Column In-Line Filter Kit [205000343]
Eluent A 1.2% formic acid in water
Eluent B 0.5 % formic acid in Acetonitrile
%A Flow [mL/min] Time [min]
10 0.5 0
10 0.5 0.5
Gradient 80 0.5 1.5
90 0.5 4.5
90 0.5 17.5
10 0.5 17.6
10 0.5 23
Injection volume 10 µL
e.g. 0.05 or 0.1 µg/IS portion* + one level at the reporting limit;
Calibration standards and levels Standard solutions of Fosetyl and Ethephon (and their IL-ISs) may be contaminated with native
Phosphonic acid which may potentially lead to false positives or shifted calibration, see 5.6.1.
Compound Mass Transitions (m/z)
Glyphosate 168/63, 168/124, 168/150, 168/81
Glyphosate-13C2,15N (IL-IS) 171/63, 171/126
AMPA 110/63, 110/79, 110/81
AMPA-13C,15N (IL-IS) 112/63, 112/81
N-Acetyl-AMPA: 152/63, 152/79, 152/110
N-Acetyl-Glyphosate 210/63, 210/150, 210/79, 210/148
N-Acetyl-Glyphosate-D3 (IL-IS) 213/63, 213/153
Ethephon 143/107, 143/79, 145/107
Ethephon-D4 (IL-IS) 147/111, 147/79 (optional, when interferences)
HEPA 125/79, 125/95, 125/63
HEPA-D4 (IL-IS) 129/79, 129/97
Glufosinate 180/63, 180/136, 180/85, 180/95
Acquired mass transitions (m/z)
Glufosinate-D3 (IL-IS) 183/63, 183/98
N-Acetyl-Glufosinate 222/63, 222/59, 222/136
N-Acetyl-Glufosinate-[acetyl]D3 (IL-IS) 225/63, 225/137
N-Acetyl-Glufosinate-[methyl]D3 (IL-IS) 225/63
MPPA 151/63, 151/107, 151/133
MPPA-D3 (IL-IS) 154/63, 154/136
Fosetyl-Al 109/81, 109/63 (each detected as Fosetyl)
Fosetyl-Al-D15 (IL-IS) 114/82, 114/63 (each detected as Fosetyl- D5)
Trifluoroacetic acid (TFA) 113/69, 113/113
Trifluoroacetic acid -13C2 (IL-IS) 115/70
Phosphonic acid 81/79, 81/63
18
Phosphonic acid- O3 (IL-IS) 87/85, 87/67
Bromide 81/81, 79/79

EU Reference Laboratory for pesticides requiring Single Residue Methods (EURL-SRM) 43 of 102
Hints on Method 1.6a and Method 1.6b
1. Handling of column, pre-column and pre-filters: see 5.6.1 point 4.a)4.c)
2. Maleic hydrazide and Cyanuric acid on Torus DEA: The intention was to cover all analytes of M1.3 with M1.6.
During method development, however, it became clear that Maleic hydrazide and Cyanuric acid showed a very
poor retention on this column, with retention times close to the dead-time, heavy interefernces of matrix com-
ponents on peak shapes and intensities (signal suppression). Figure 15 shows exemplarily chromatograms ob-
tained upon injection of standards in solvent and in extracts of plum, broccoli, soy and onion at 0.1 µg/mL. Proper
evaluation of the peaks at low concentrations is often not possible. Fortunately Maleic Hydrazide can also be cov-
ered by M 4.2 (5.6.15), whereas Cyanuric acid is not regulated.
3. For general hints on analytes: See 5.6.1

AMPA 110/63 T Ethephon 143/107 T Fosetyl 109/81 T Glufosinate 180/63T

Area: 3.996e4 Area: 3.873e5 Area: 4.008e6 Area: 5.510e4

Glyphosate 168/63T HEPA 125/63 T MPPA 151/63T N-acetyl-AMPA 152/63T

Area: 2.718e5 Area: 8.807e4 Area: 2.755e5 Area: 6.585e5

N-Acetylglufosinate 222/63T N-Acetyl-Glyphosate 210/150T

Area: 2.806e5 Area: 4.252e5

Figure 13: Chromatograms of Glyphosate, AMPA, N-Acetyl-AMPA, N-Acetyl-Glyphosate, Ethephon, HEPA, Glufosinate, MPPA,
N-Acetyl-Glufosinate, Fosetyl at 0.04 mg/kg on cucumber extract using i: Waters Torus™DEA 2.1 mm x 100 mm .

EU Reference Laboratory for pesticides requiring Single Residue Methods (EURL-SRM) 44 of 102
Figure 14: Chromatograms of Glyphosate, AMPA, N-Acetyl-Glyphosate at 0.06 mg/kg, Glufosinate at 0.036 mg/kg, HEPA, MPPA,
N-Acetyl-Glufosinate at 0.024 mg/kg, Ethephon, Fosetyl at 0.012 mg/kg, all in strawberry extract and Phosphonic acid at
0.06 mg/kg, Bromide at 6 mg/kg both in lemon extract using ii: Waters Anionic Polar Pesticide Column, 130Å, 5 µm, 2.1 mm x 100
mm.

EU Reference Laboratory for pesticides requiring Single Residue Methods (EURL-SRM) 45 of 102
 Solvent Plum Broccoli Soy Onion

Cyanuric acid 13C3

131/43

Cyanuric acid 128/42

Cyanuric acid 128/85

Maleic hydrazide D2
113/42

Maleic hydrazide
111/42

Maleic hydrazide
111/82

Maleic hydrazide
111/83

Figure 15: Exemplary peak shapes of Cyanuric acid and Maleic hydrazide in solvent-based standards and in standards of plum,
broccoli, soy and onion extracts at 0.1 µg/mL (Please also read the note under “Hints on Method 1.6a and Method 1.6b” above)

EU Reference Laboratory for pesticides requiring Single Residue Methods (EURL-SRM) 46 of 102
 Method 1.7 “PerChloPhos on Torus DEA; (M1.7a)” or “PerChloPhos on Anionic Polar Pesticide Col-
umn (APPC); (M1.7b)”
Table 24: Proposed LC-MS/MS conditions for PerChlorate, Chlorate, Phosphonic acid and Bormide
Instrument parameters Conditions
Ionisation mode ESI neg
M1.7a M1.7b
Waters Torus™DEA 2.1 mm x 100 mm; 1.7 Waters Anionic Polar Pesticide Column, 130Å,
Column/temperature
µm; 50 °C 5 µm, 2.1 mm x 100 mm; 50 °C
Waters Torus™DEA VanGuard™ 2.1 mm x 5 Waters Anionic Polar Pesticide VanGuard Car-
Pre-column
mm; 1.7 µm tridge, 130Å, 5µm, 2.1 mm X 5 mm
Waters ACQUITY UPLC Column In-Line Filter Waters ACQUITY UPLC Column In-Line Filter
Pre-filters
Kit [205000343] Kit [205000343]
1.2% formic acid + 10 mmol ammonium for- 1.2% formic acid + 15 mmol ammonium for-
Eluent A
mate in water mate in water
Eluent B 0.5 % formic acid in Acetonitrile 0.5 % formic acid in Acetonitrile
Flow Flow
%A Time [min] %A Time [min]
[mL/min] [mL/min]
10 0.5 0 10 0.5 0
10 0.5 0.5 10 0.5 0.5
Gradient
80 0.5 1.5 80 0.5 1.5
90 0.5 4.5 90 0.5 4.5
90 0.5 17.5 90 0.5 13.5
10 0.5 17.6 10 0.5 13.6
10 0.5 23 10 0.5 23
Injection volume 10 µL
Calibration standards and levels e.g. 0.05 or 0.1 µg/IS portion* + one level at the reporting limit
Compound Mass Transitions (m/z)
Bromide 81/81, 79/79
Chlorate 83/67, 85/69
Chlorate-18O3 (IL-IS) 89/71, 91/73
Acquired mass transitions (m/z)
Perchlorate 99/83, 101/85
Perchlorate-18O4 (IL-IS) 107/89, 109/91
Phosphonic acid 81/79, 81/63
Phosphonic acid-18O3 (IL-IS) 87/85, 87/67

Hints on Method 1.7

1. Handling of column, pre-column and pre-filters: See 5.6.1 point 4.a)4.c)
2. Intereference of Phosphonic acid by Phosphoric acid: See 5.6.1
3. For general hints on analytes: See 5.6.1

EU Reference Laboratory for pesticides requiring Single Residue Methods (EURL-SRM) 47 of 102
Figure 16: Chromatograms of Chlorate at 0.03mg/kg, Perchlorate at 0.01 mg/kg, Phosphonic acid at 0.05 mg/kg and Bromide at
5.0 mg/kg, all in lemon extract using ii: Waters Anionic Polar Pesticide Column, 130Å, 5 µm, 2.1 mm x 100 mm; 50 °C.

Grape Onion

Phosphonic acid 81/79

Perchlorate 99/83

Chlorate 83/67

Bromide 81/81

Figure 17: Exemplary chromatograms of Phosphonic acid, Perchlorate, Chlorate and Bromide at 0.01 mg/kg on Garpe and Onion
using i: Waters Torus™DEA 2.1 mm x 100 mm; 1.7 µm; 50 °C.

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 Method 1.8 (M1.8): “PerChloCyanMalein on Anionic Polar Pesticide Column (APPC)”
Table 25: Proposed LC-MS/MS conditions for Perchlorate, Chlorate, Cyanuric acid and Maleic hydrazide
Instrument parameters Conditions
Ionisation mode ESI neg
Column/temperature Waters Anionic Polar Pesticide Column, 130Å, 5 µm, 2.1 mm x 100 mm; 50 °C
Pre-column Waters Anionic Polar Pesticide VanGuard Cartridge, 130Å, 5µm, 2.1 mm X 5 mm
Pre-filters Waters ACQUITY UPLC Column In-Line Filter Kit [205000343]
Eluent A 1.2% formic acid and 50mM NH4-formate in water
Eluent B 85 % ACN : 10 % MeOH : 5 % water
%A Flow [mL/min] Time [min]
0 0.5 0
0 0.5 1.5
Gradient
70 0.5 4.5
70 0.5 7.0
0 0.5 7.1
0 0.5 15
Injection volume 10 µL
Calibration standards and levels e.g. 0.05 or 0.1 µg/IS portion* + one level at the reporting limit
Compound Mass Transitions (m/z)
Cyanuric acid 128/42, 128/85
Cyanuric acid 13C3 (IL-IS) 131/43
Chlorate 83/67, 85/69, 83/51
Chlorate-18O3 (IL-IS) 89/71, 91/73
Acquired mass transitions (m/z)
Perchlorate 99/83, 101/85, 99/67
Perchlorate-18O4 (IL-IS) 107/89, 109/91
Maleic hydrazide 111/83, 111/55, 111/41
Maleic hydrazide D2 (IL-IS) 113/85
Maleic Hydrazide-13C4 (IL-IS) 115/87, 115/58
* One IS portion is the absolute IS-mass contained in the prepared calibration standard solution (see also Table 2).
** Depending on matrix; better use M 1.3

Hints on Method 1.8

1. For general hints on analytes: See 5.6.1

Figure 18: Chromatograms of Chlorate at 0.03mg/kg, Perchlorate at 0.01 mg/kg, Cyanuric acid and Maleic hydrazide at 0.05
mg/kg, all in lemon extract

EU Reference Laboratory for pesticides requiring Single Residue Methods (EURL-SRM) 49 of 102
 Method 1.9 (M1.9): “Gly&Co. on Raptor Polar X”
Table 26: Proposed LC-MS/MS conditions for Glyphosate, AMPA, Ethephon, HEPA, Glufosinate, MPPA, N-Acetyl-Glufosinate,
Fosetyl-Al, Bialaphos, Bromide, (Chlorate,) Perchlorate, Phosphonic acid
Instrument parameters Conditions
Ionisation mode ESI neg
Column/temperature Restek Raptor Polar X LC column, 90Å, 2,7 µm, 2.1 mm x 30 mm; 50 °C
Pre-column Restek Raptor Polar X LC column, 90Å, 2,7 µm, 2.1 mm x 5 mm; 50 °C
Eluent A 0.5% formic acid in water
Eluent B 0.5% formic acid in acetonitrile
%A Flow [mL/min] Time [min]
10 0.5 0.5
60 0.5 1.5
Gradient
90 0.5 11.5
90 0.5 14
10 0.5 14.1
10 0.5 17
Injection volume 10 µL
e.g. 0.05 or 0.1 µg/IS portion + one level at the reporting limit;
Calibration standards and levels Standard solutions of Fosetyl and Ethephon (and their IL-ISs) may be contaminated with native
Phosphonic acid which may potentially lead to false positives or shifted calibration, see 5.6.1.
Compound Mass Transitions (m/z)
Glyphosate 168/63, 168/124, 168/150, 168/81
Glyphosate-13C2,15N (IL-IS) 171/63, 171/126
AMPA 110/63, 110/79, 110/81
AMPA-13C,15N (IL-IS) 112/63, 112/81
Ethephon 143/107, 143/79, 145/107
Ethephon-D4 (IL-IS) 147/111, 147/79 (optional, when interferences)
HEPA 125/79, 125/95, 125/63
HEPA-D4 (IL-IS) 129/79, 129/97
Glufosinate 180/63, 180/136, 180/85, 180/95
Glufosinate-D3 (IL-IS) 183/63, 183/98
N-Acetyl-Glufosinate 222/63, 222/59, 222/136
N-Acetyl-Glufosinate-[acetyl]D3 (IL-IS) 225/63, 225/137
N-Acetyl-Glufosinate-[methyl]D3 (IL-IS) 225/63
Acquired mass transitions (m/z)
MPPA 151/63, 151/107, 151/133
MPPA-D3 (IL-IS) 154/63, 154/136
Fosetyl-Al 109/81, 109/63 (each detected as Fosetyl)
Fosetyl-Al-D15 (IL-IS) 114/82, 114/63 (each detected as Fosetyl- D5)
Trifluoroacetic acid (TFA) 113/69, 113/113
13
Trifluoroacetic acid - C2 (IL-IS) 115/70
Bialaphos 322/88, 322/94, 322/134
Bromide 81/81, 79/79
Chlorate 83/67, 85/69
Chlorate-18O3 (IL-IS) 89/71, 91/73
Perchlorate 99/83, 101/85
Perchlorate-18O4 (IL-IS) 107/89, 109/91
Phosphonic acid 81/79, 81/63
Phosphonic acid-18O3 (IL-IS) 87/85, 87/67

Hints on Method 1.9

1. Handling of column, pre-column and pre-filters: See 5.6.1 point 4.a)4.c)
2. For general hints on analytes: See 5.6.1

EU Reference Laboratory for pesticides requiring Single Residue Methods (EURL-SRM) 50 of 102
 AMPA 110/63 T Ethephon 143/107 T Fosetyl 109/81 T Glufosinate 180/63T

Glyphosate 168/63T HEPA 125/63 T MPPA 151/63T N-Acetyl-Glufosinate 222/63T

Perchlorate 99/83 Phosphonic acid 81/79 Trifluoracetic acid 113/69 Bromide 79/79

Chlorate 83/67*

Figure 19: Typical chromatograms in strawberry extracts spiked at 0.1 mg/kg.

*for Chlorate matrix dependent retention time shifts were observed

EU Reference Laboratory for pesticides requiring Single Residue Methods (EURL-SRM) 51 of 102
 Method 1.10 (M1.10): “Gly&Co. on Obelisc N”
Table 27: Proposed LC-MS/MS conditions for for Glyphosate, AMPA, N-Acetyl-Glyphosate, Ethephon, HEPA, Glufosinate, MPPA, N-
Acetyl-Glufosinate, Bromide, Chlorate, Perchlorate, (Fosetyl-Al and Phosphonic acid)
Instrument parameters Conditions
Ionisation mode ESI neg
Column/temperature Sielc Obelisc N, 100Å, 5 µm, 2.1 mm x 150 mm (SIELC; ON-21.150.0510), 50°C
Pre-column Sielc Obelisc N, 100Å, 5 µm, 2.1 mm x 10 mm (SIELC; ON-21.G.0510), 50°C
Pre-filters e.g. Supelco column saver 2.0 µm Filter
Eluent A 1% formic acid in water
Eluent B 0.5 % formic acid in Acetonitrile
%A Flow [mL/min] Time [min]
10 0.4 0.5
Gradient 80 0.4 2
90 0.4 9
10 0.4 9.1
10 0.4 13
Injection volume 10 µL
e.g. 0.05 or 0.1 µg/IS portion + one level at the reporting limit;
Calibration standards and levels Standard solutions of Fosetyl and Ethephon (and their IL-ISs) may be contaminated with native
Phosphonic acid which may potentially lead to false positives or shifted calibration, see 5.6.1.
Compound Mass Transitions (m/z)
Glyphosate 168/63, 168/124, 168/150, 168/81
Glyphosate-13C2,15N (IL-IS) 171/63, 171/126
AMPA 110/63, 110/79, 110/81
AMPA-13C,15N (IL-IS) 112/63, 112/81
N-Acetyl-Glyphosate 210/63, 210/150, 210/79, 210/148
N-Acetyl-Glyphosate-D3 (IL-IS) 213/63, 213/153
Ethephon 143/107, 143/79, 145/107
Ethephon-D4 (IL-IS) 147/111, 147/79
HEPA 125/79, 125/95, 125/63
HEPA-D4 (IL-IS) 129/79, 129/97
Glufosinate 180/63, 180/136, 180/85, 180/95
Glufosinate-D3 (IL-IS 183/63, 183/98
N-Acetyl-Glufosinate 222/63, 222/59, 222/136
N-Acetyl-Glufosinate-[acetyl]D3 (IL-IS) 225/63, 225/137
N-Acetyl-Glufosinate-[methyl]D3 (IL-IS) 225/63
MPPA 151/63, 151/107, 151/133
Acquired mass transitions (m/z)
MPPA-D3 (IL-IS) 154/63, 154/136
Fosetyl-Al 109/81, 109/63 (each detected as Fosetyl)
Fosetyl-Al-D15 (IL-IS) 114/82, 114/63 (each detected as Fosetyl- D5)
Trifluoroacetic acid (TFA) 113/69, 113/113
Trifluoroacetic acid -13C2 (IL-IS) 115/70
Maleic Hydrazide 111/82, 111/42, 111/55, 111/83
Maleic Hydrazide-D2 (IL-IS) 113/42, 113/85
Maleic Hydrazide-13C4 (IL-IS) 115/87, 115/58
Cyanuric acid 128/42, 128/85
Cyanuric acid-13C3 131/43, 131/87
Bromide: 81/81, 79/79
Chlorate 83/67, 85/69
Chlorate-18O3 (IL-IS) 89/71, 91/73
Perchlorate 99/83, 101/85
Perchlorate-18O4 (IL-IS) 107/89, 109/91
Phosphonic acid 81/79, 81/63
Phosphonic acid-18O3 (IL-IS) 87/85, 87/67
Hints on Method 1.9
1. For general hints on analytes: See 5.6.1

EU Reference Laboratory for pesticides requiring Single Residue Methods (EURL-SRM) 52 of 102
 Method 2 (M2): “Fosetyl and Maleic Hydrazide”
Table 28: Proposed LC-MS/MS conditions for Fosetyl-Al, Maleic Hydrazide and Perchlorate
Instrument parameters
Ionization mode ESI neg
Column/temperature Obelisc R 2.1 x 150 mm 5 µm 100 Å; (SIELC; OR-21.150.0510)
Pre-filters e.g. Supelco column saver 2.0 µm Filter
Obelisc R 2.1 x 10mm 5 µm
Pre-column
(SIELC; OR-21.G.0510)
50 mmol NH4-formate in water + 0.1 % formic acid
Eluent A
use brown glass bottles
Eluent B Acetonitrile
%A Flow [mL/min] Time [min]
3 0.3 0
10 0.3 6
Gradient
70 0.5 15
70 0.5 18
3 0.5 18.1
3 0.5 28
Injection volume 5 µL
e.g. 0.05 or 0.1 µg/IS portion*, + one level at the reporting limit
For Maleic Hydrazide (MH) an additional level at 1 or 2 µg/mL may be useful as well, due to high
Calibration standards and levels
residue levels; consider that MH is typically only relevant for potatoes and crops of the leek
family (onions etc.)
Compound Mass Transitions (m/z)
Fosetyl-Al 109/81, 109/63 (detected as fosetyl)
Fosetyl-Al-D15 (IL-IS) 114/82, 114/63 (detected as fosetyl-D5)
Maleic Hydrazide 111/82, 111/42, 111/55, 111/83
Acquired mass transitions
Maleic Hydrazide-D2 (IL-IS) 113/42, 113/85
Maleic Hydrazide-13C4 (IL-IS) 115/87, 115/58
Perchlorate 99/83, 101/85
Perchlorate-18O4 (IL-IS) 107/89, 109/91
* One IS portion is the absolute IS-mass contained in the prepared calibration standard solution (see also Table 2).

Hints on Method 2
1. Contamination of Maleic hydrazide D2 with native Maleic hydrazide: See 5.6.1
2. For Perchlorate better run Method 1.3 or 1.4 !
3. For general hints on analytes: See 5.6.1

EU Reference Laboratory for pesticides requiring Single Residue Methods (EURL-SRM) 53 of 102
 Wdf 1 Erdbeere 0,1 ppm 0,5g/IS 1%AS in MeOH - Fosetyl-Al 109.0 / 81.0 (Unknown) 109.0/81.0 amu - sample 32 of 38 from Validieru... Wdf 1 Erdbeere 0,1 ppm 0,5g/IS 1%AS in MeOH - Fosetyl-Al 109.0 / 63.0 (Unknown) 109.0/63.0 amu - sample 32 of 38 from Validieru... Wdf 1 Erdbeere 0,1 ppm 0,5g/IS 1%AS in MeOH - Fosetyl-Al D15(IS) (Unknown) 113.9/81.9 amu - sample 32 of 38 from Validierung_...
Area: 3.37e+004 counts Height: 6.03e+003 cps RT: 13.03 min Area: 1.18e+004 counts Height: 2.12e+003 cps RT: 13.03 min Area: 8.88e+003 counts Height: 1.52e+003 cps RT: 13.01 min

13.03 13.03 13.01

6000 2100 1500

2000 1400
5500
1900
1300

Recovery test on strawberry

5000 1800
1700 1200
4500 1600
1100
1500
4000 1000

0.1 mg/kg
1400
In te n s ity , c p s

In te n s ity , c p s
In te n s ity , c p s
1300 900
3500
1200
800
3000 1100
700

(corresponds to 0.05 µg/mL at 100%

1000
2500
900 600
800
2000 500
700
1500 600 400

1000
500
400
300
8.60
9.29
9.55
300

200
recovery)
500
200 100

0 100
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 0
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
Time, min 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 Time, min
Time, min

0.005 ug/IS in 1%AS in MeOH - Fosetyl-Al 109.0 / 81.0 (Unknown) 109.0/81.0 amu - sample 8 of 38 from Validierung_Malein_Fosetyl.... 0.005 ug/IS in 1%AS in MeOH - Fosetyl-Al 109.0 / 63.0 (Unknown) 109.0/63.0 amu - sample 8 of 38 from Validierung_Malein_Fosetyl.... 0.005 ug/IS in 1%AS in MeOH - Fosetyl-Al D15(IS) (Unknown) 113.9/81.9 amu - sample 8 of 38 from Validierung_Malein_Fosetyl.wiff
Area: 6.63e+003 counts Height: 1.12e+003 cps RT: 13.05 min Area: 2.40e+003 counts Height: 4.32e+002 cps RT: 13.04 min Area: 1.76e+004 counts Height: 2.88e+003 cps RT: 13.03 min

13.05 13.04 13.03

450 2800
1100
2600
1000 400
2400

900
350 2200

800 2000

300
1800
Solvent calib. 0.005 µg/mL
In te n s ity , c p s

In te n s ity , c p s
700
In te n s ity , c p s

1600
600 250
1400
500

400
200

150
1200

1000
(Corresponds to 0.01 mg/kg)
300 800

100 600
200
400
100 50
200

0 0.93 4.37
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 0
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
Time, min 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 Time, min
Time, min

0.05 ug/IS in 1%AS in MeOH - Fosetyl-Al 109.0 / 81.0 (Unknown) 109.0/81.0 amu - sample 11 of 38 from Validierung_Malein_Fosetyl.... 0.05 ug/IS in 1%AS in MeOH - Fosetyl-Al 109.0 / 63.0 (Unknown) 109.0/63.0 amu - sample 11 of 38 from Validierung_Malein_Fosetyl.... 0.05 ug/IS in 1%AS in MeOH - Fosetyl-Al D15(IS) (Unknown) 113.9/81.9 amu - sample 11 of 38 from Validierung_Malein_Fosetyl.wiff
Area: 7.83e+004 counts Height: 1.35e+004 cps RT: 13.03 min Area: 2.65e+004 counts Height: 4.57e+003 cps RT: 13.03 min Area: 2.40e+004 counts Height: 4.00e+003 cps RT: 13.01 min

13.03 13.03 13.01

1.3e4
3800

1.2e4 4000 3600

3400
1.1e4
3200
3500
1.0e4 3000
2800
9000.0

Solvent calib. 0.05 µg/mL

3000
2600
In te n s ity , c p s

In te n s ity , c p s

In te n s ity , c p s
8000.0 2400
2500 2200
7000.0
2000

Corresponds to 0.1 mg/kg

6000.0
2000 1800

5000.0 1600
1400
1500
4000.0
1200

3000.0 1000
1000
800
2000.0
600
1000.0 500 400
200
0.0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 0 0
Time, min 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
Time, min Time, min

Fosetyl-Al 109 / 81 Fosetyl-Al 109 / 63 Fosetyl-Al D15 114 / 82 (IS)

Figure 20: Typical chromatograms of Fosetyl-Al in strawberry extract and in solvent-based standards

Maleinsaeure hydrazid 0,1 ug/IS - Maleic hydrazid D2 113/42(IS) (Unknown) 113.0/41.9 amu - sample 4 of 12 from 080131Mc_Malein.... Maleinsaeure hydrazid 0,1 ug/IS - Maleic hydrazid 111 / 82 T T (Unknown) 111.0/81.8 amu - sample 4 of 12 from 080131Mc_Malein.... Maleinsaeure hydrazid 0,1 ug/IS - Maleic hydrazid 111 / 55 (Unknown) 111.0/55.0 amu - sample 4 of 12 from 080131Mc_Malein.wiff Maleinsaeure hydrazid 0,1 ug/IS - Maleic hydrazid 111 / 42 (Unknown) 111.0/41.9 amu - sample 4 of 12 from 080131Mc_Malein.wiff
Area: 5.84e+005 counts Height: 6.17e+004 cps RT: 8.715 min Area: 2.88e+005 counts Height: 3.17e+004 cps RT: 8.713 min Area: 1.23e+005 counts Height: 1.36e+004 cps RT: 8.715 min Area: 3.74e+004 counts Height: 4.55e+003 cps RT: 8.721 min

8.71 8.71 8.71 8.72

6.0e4
3.0e4 1.3e4
4500
5.5e4 2.8e4 1.2e4

2.6e4 4000
5.0e4 1.1e4

2.4e4
4.5e4 1.0e4 3500

Solvent calib. 0.1 mg/kg

2.2e4
9000.0
4.0e4
2.0e4
In te n s ity , c p s

3000
In te n s ity , c p s

In te n s ity , c p s
In te n s ity , c p s

8000.0
3.5e4 1.8e4
7000.0 2500
1.6e4
3.0e4

Corresponds to 0.05 µg/mL

1.4e4 6000.0
2000
2.5e4
1.2e4 5000.0

2.0e4 1.0e4 4000.0 1500

8000.0
1.5e4 3000.0
1000
6000.0
2000.0
1.0e4
4000.0
1000.0 500
5000.0 2000.0
0.0
0.0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Time, min 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
Time, min Time, min Time, min

2493 Zwiebel 0,5g/IS - Maleic hydrazid D2 113/42(IS) (Unknown) 113.0/41.9 amu - sample 8 of 12 from 080131Mc_Malein.wiff 2493 Zwiebel 0,5g/IS - Maleic hydrazid 111 / 82 T T (Unknown) 111.0/81.8 amu - sample 8 of 12 from 080131Mc_Malein.wiff 2493 Zwiebel 0,5g/IS - Maleic hydrazid 111 / 55 (Unknown) 111.0/55.0 amu - sample 8 of 12 from 080131Mc_Malein.wiff 2493 Zwiebel 0,5g/IS - Maleic hydrazid 111 / 42 (Unknown) 111.0/41.9 amu - sample 8 of 12 from 080131Mc_Malein.wiff
Area: 3.05e+005 counts Height: 3.21e+004 cps RT: 9.443 min Area: 2.19e+003 counts Height: 3.02e+002 cps RT: 9.448 min Area: 6.67e+002 counts Height: 1.57e+002 cps RT: 7.245 min Area: 6.00e+002 counts Height: 1.17e+002 cps RT: 7.167 min

9.44 2.04 2.36

3.2e4 420
14.48 4.2e4 5000

Extract of organic onion from the

3.0e4 400
4.0e4
380
2.8e4 3.8e4 4500

see note
360
2.6e4 3.6e4
340 2.35
3.4e4 4000
2.4e4 320 2.92
3.2e4
300

market: n.d.
2.2e4 3.0e4 3500
280 1.73
2.0e4 2.8e4
In te n s ity , c p s

In te n s ity , c p s
In te n s ity , c p s

260
In te n s ity , c p s

1.92
2.6e4 3000
1.8e4 240
2.4e4
1.6e4 220
2.2e4 2500
200

(0.5 g onion equivalents /mL)

1.4e4 2.0e4
180
1.8e4 2000
1.2e4 160
3.08
1.6e4
1.0e4 140
1.4e4 1500
120
8000.0 1.2e4
100
6000.0 1.0e4 1000
80
60 8000.0
4000.0
6000.0 500
40 6.10
2000.0
20 4000.0
4.81 5.13 7.30 7.63 8.77 10.06
0 2000.0 0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
Time, min 0.0 Time, min
Time, min 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
Time, min

5618 Zwiebel 0,5g/IS - Maleic hydrazid D2 113/42(IS) (Unknown) 113.0/41.9 amu - sample 8 of 11 from 080229_Malein.wiff 5618 Zwiebel 0,5g/IS - Maleic hydrazid 111 / 82 T (Unknown) 111.0/81.8 amu - sample 8 of 11 from 080229_Malein.wiff 5618 Zwiebel 0,5g/IS - Maleic hydrazid 111 / 55 (Unknown) 111.0/55.0 amu - sample 8 of 11 from 080229_Malein.wiff 5618 Zwiebel 0,5g/IS - Maleic hydrazid 111 / 42 (Unknown) 111.0/41.9 amu - sample 8 of 11 from 080229_Malein.wiff
Area: 1.46e+005 counts Height: 1.28e+004 cps RT: 9.345 min Area: 6.32e+004 counts Height: 5.18e+003 cps RT: 9.344 min Area: 3.18e+004 counts Height: 2.42e+003 cps RT: 9.353 min Area: 1.08e+004 counts Height: 9.44e+002 cps RT: 9.346 min

6.5e4

6.0e4
14.57

5000

4500
9.34

8000

7500
1.83

2.11
7500

7000

6500
2.37

Extract of onion from the market

7000
5.5e4

containing ca. 0.1 mg/kg Maleic

6000 2.20
4000 6500
5.0e4
6000 5500
1.99
4.5e4 3500 5000
5500
In te n s ity , c p s
In te n s ity , c p s

In te n s ity , c p s

5000 4500
In te n s ity , c p s

4.0e4 3000

Hydrazide.
4500 4000
3.5e4
2500
4000 3500
3.0e4
3500 3000
2000
2.5e4 3000 9.35 2500

(0.5 g onion equivalents /mL)

1500 2500 2000
2.0e4 2.73
2000 1500
1000 2.86 9.35
1.5e4
9.34 1500 1000 2.95

1.0e4 500 1000

3.48 500

500 0
5000.0 0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
0 Time, min
Time, min 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Time, min
Time, min

2419 Zwiebel 0,5g/IS - Maleic hydrazid D2 113/42(IS) (Unknown) 113.0/41.9 amu - sample 7 of 12 from 080131Mc_Malein.wiff 2419 Zwiebel 0,5g/IS - Maleic hydrazid 111 / 82 T T (Unknown) 111.0/81.8 amu - sample 7 of 12 from 080131Mc_Malein.wiff 2419 Zwiebel 0,5g/IS - Maleic hydrazid 111 / 55 (Unknown) 111.0/55.0 amu - sample 7 of 12 from 080131Mc_Malein.wiff 2419 Zwiebel 0,5g/IS - Maleic hydrazid 111 / 42 (Unknown) 111.0/41.9 amu - sample 7 of 12 from 080131Mc_Malein.wiff
Area: 2.80e+005 counts Height: 2.58e+004 cps RT: 9.409 min Area: 5.73e+006 counts Height: 5.32e+005 cps RT: 9.417 min Area: 2.50e+006 counts Height: 2.29e+005 cps RT: 9.423 min Area: 8.13e+005 counts Height: 7.41e+004 cps RT: 9.419 min

2.8e4

2.6e4
14.50

5.0e5

4.5e5
9.42

2.2e5

2.0e5
9.42

7.0e4

6.5e4
9.42

Extract of onion from the market

2.4e4
6.0e4

containing ca. 4 mg/kg Maleic Hy-

1.8e5
2.2e4 4.0e5 5.5e4

2.0e4 1.6e5 5.0e4

3.5e5
In te n s ity , c p s
In te n s ity , c p s

1.8e4 4.5e4
In te n s ity , c p s

1.4e5
In te n s ity , c p s

3.0e5
4.0e4

drazide.
1.6e4
1.2e5
2.5e5 3.5e4
1.4e4
1.0e5 3.0e4
1.2e4 2.0e5
2.5e4
8.0e4
1.0e4

(0.5 g onion equivalents /mL)

1.5e5 2.0e4
6.0e4 2.03
8000.0 1.5e4
1.0e5
6000.0 4.0e4 1.73 2.31 1.0e4
2.38
5.0e4 3.14
4000.0 5000.0 1.95 3.00
2.0e4 6.01
8.93
7.85 9.06 0.0
2000.0 0.0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 0.0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Time, min
Time, min
Time, min
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
Time, min

Maleic Hydrazide-D2 Maleic Hydrazide Maleic Hydrazide Maleic Hydrazide

113/42 (IS) 111 / 82 (target ion) 111 / 55 111 / 42
Figure 21: Typical chromatograms of Maleic Hydrazide in onion extracts and in solvent-based standards

EU Reference Laboratory for pesticides requiring Single Residue Methods (EURL-SRM) 54 of 102
 Method 3 (M3): “Amitrole&Co.”
Table 29: Proposed LC-MS/MS conditions for Amitrole, Chlormequat, Mepiquat, Daminozide, ETU, PTU, Trimesium, Difenzoquat
and Cyromazine.
Instrument parameters Conditions
Ionisation mode ESI pos
Column/temperature Obelisc R 2.1 x 150 mm 5 µm 100 Å (SIELC; OR-21.150.0510); 40°C
Obelisc R 2.1 x 10 mm 5 µm
Pre-column
(SIELC; OR-21.G.0510)
Pre-filters e.g. Supelco column saver 2.0 µm Filter
5 mmol NH4-formate in water
Eluent A
Use brown glass bottles
Eluent B 5 mmol NH4-formate acetonitrile/water 95 :5 (v/v)
%A Flow [mL/min] Time [min]
2 0.4 0
2 0.4 2.5
Gradient
80 0.4 5
80 0.4 11
2 0.4 11.1
2 0.4 18
Injection volume 5 µL
Calibration standards and levels e.g. 0.05 or 0.1 µg/IS portion* + one level at the reporting limit
Compound Mass Transitions (m/z)
Amitrole 85/43, 85/57, 85/58
15
Amitrole- N (IL-IS) 86/43
Amitrole-15N2,13C2 (IL-IS) 89/44
Chlormequat 122/58, 122/63, 124/58
Chlormequat-D4 (IL-IS) 126/58
Mepiquat 114/98, 114/58
Mepiquat-D3 (IL-IS) 117/101
Daminozide 161/143, 161/61, 161/101 , 161/115, 161/44
Daminozide-13C4 (IL-IS) 165/147, 165/44
Acquired mass transitions Daminozide-D6 (IL-IS) 167/149, 165/97
Cyromazine 167/68, 167/125, 167/85, 167/108,
Cyromazine-D4 (IL-IS) 171/86, 171/68
ETU (Ethylenethiourea) 103/44, 103/60, 103/86
ETU-D4 (IL-IS) 107/48
PTU - N,N′-(1,2-Propylene)thiourea)**: 117/100, 117/58, 117/60, 117/72
PTU-D6 - N,N′-(1,2-Propylene)thiourea –D6**: 123/64, 126/74
PTU-D6 - N,N′-(1,3-Propylene)thiourea -D6)** (123/64)
Trimethylsulfonium 77/62, 77/47
Trimethylsulfonium-D9 (IL-IS) 86/68, 86/50
Difenzoquat 249/77, 249/130, 249/193
No IL-IS currently available -
* One IS portion is the absolute IS-mass contained in the prepared calibration standard solution (see also Table 2).
** The acronym PTU, commonly used for the propineb degradant 4-Methyl-2-imidazolidinethione = N,N′-(1,2-Propylene)thiou-
rea = N,N′-iso-propylenethiourea (CAS No. 2055-46-1). The same accronym is, however, also used for N,N′-propylenethiourea =
N,N′-(1,3-Propylene)thiourea = N,N′-Trimethylenethiourea (CAS No.: 2122-19-2).

Hints on Method 3
1. For Paraquat, Diquat, Trimethylsulfonium and N,N-Dimethylhydrazine better run Method 4 (5.6.14)
2. For general hints on analytes: See 5.6.1

EU Reference Laboratory for pesticides requiring Single Residue Methods (EURL-SRM) 55 of 102
 Amitrole 85 /43 Mepiquat 114/98 Chlormequat 122/58 Daminozide 161 /143

Cyromazine 167/68 ETU 103/44 PTU 117/100

Figure 22: Typical chromatograms of Amitrole, Chlormequat, Mepiquat, Daminozide, ETU, PTU and Cyromazine in apple extract at
0.01 mg/kg

EU Reference Laboratory for pesticides requiring Single Residue Methods (EURL-SRM) 56 of 102
 Method 4.1 (M4.1): “Quats&Co. Obelisc R”
Table 30: Proposed LC-MS/MS conditions Diquat, Paraquat, Chlormequat, Mepiquat, Daminozide N,N-Dimethylhydrazine, Cyrom-
azine, Trimethylsulfonium, Nereistoxin, Difenzoquat, Melamine and Propamocarb.
Instrument parameters Conditions
Ionisation mode ESI pos
Column/temperature Obelisc R 2.1 x 150 mm 5 µm 100 Å (SIELC; OR-21.150.0510); 40°C
Pre-filters e.g. Supelco column saver 2.0 µm Filter
Pre-column Obelisc R 2.1 x 10 mm 5 µm (SIELC; OR-21.G.0510)
20 mmol NH4-formate in water (adjust to pH 3 with formic acid), for this mix 1.8 mL formic acid (3.4)
with 500 mL 20 mmol NH4-formate in water Use brown glass bottles!
Eluent A
Alternative eluent A: 50 mmol NH4formate in water (adjust to pH 3 with formic acid). This eluent com-
ponents is also used in M 4.2 “Quats & Co BEH Amide”
Eluent B Acetonitrile
%A Flow [mL/min] Time [min]
20 0.4 0
Gradient 80 0.4 4
80 0.4 12
20 0.4 12.1
20 0.4 20
Injection volume 10 µL
e.g. 0.05 or 0.1 µg/IS portion* + one level at the reporting limit
Calibration standards and levels
(use plastic vials if Paraquat and Diquat are within your scope!)
Compound Mass Transitions (m/z)
Diquat 92/84, 183/157, 92/157, 184/156, 184/128
Diquat-D4 (IL-IS) 188/160 (this IL-IS showed problems with stability)
Diquat-D8 (IL-IS) 96/88, 191/165
Paraquat 186/171, 93/171, 93/77, 171/77, 171/155
Paraquat-D8 (IL-IS) 194/179, 97/179
Chlormequat 122/58, 122/63, 124/58
Chlormequat-D4 (IL-IS) 126/58
Mepiquat 114/98, 114/58
Mepiquat-D3 (IL-IS) 117/101
Daminozide 161/143, 161/61, 161/101 , 161/115, 161/44
Daminozide-13C4 (IL-IS) 165/147
Daminozide-D6 (IL-IS) 167/149
Acquired mass transitions
N,N-Dimethylhydrazine 61/44, 61/45
N,N-Dimethylhydrazine-D6 (IL-IS) 67/49
Cyromazine 167/68, 167/125, 167/85, 167/108,
Cyromazine-D4 (IL-IS) 171/86
Trimethylsulfonium 77/62, 77/47
Trimethylsulfonium-D9 (IL-IS) 86/68
Nereistoxin 150/105, 150/61, 150/71
Nereistoxin-D6 (IL-IS) 156/105
Difenzoquat 249/77, 249/130, 249/193
No IL-IS currently available -
Melamine 127/85, 127/68, (127/60)
Melamine-15N3 (IL-IS) 130/87
Propamocarb 189/144, 189/102, 189/74
Propamocarb-D7 (IL-IS) 196/103
* One IS portion is the absolute IS-mass contained in the prepared calibration standard solution (see also Table 1).

Hints on Method 4.1

1. For Morpholin, DEA and TEA better run Method 7 (5.6.18). As DEA converts to Morpholine in the ion source,
chromatogr. separation is paramount. With Method 4.1 (5.6.14) these two peaks do not sufficiently separate.
2. Diquat and Paraquat require special extraction conditions (see 5.2.3-B)
3. For general hints on analytes: See 5.6.1

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 Diquat 183/157 Paraquat 186/171 Trimethylsulfonium 77/62 N, N-Dimethylhydrazine 61/45

Mepiquat 114/98 Chlormequat 122/58 Daminozide 161/143 Nereistoxin 150/105

Cyromazine 167/68 Difenzoquat 249/77 Melamine 127/85 Propamocarb 189/102

Figure 23: Typical chromatograms of Diquat, Paraquat, Chlormequat, Mepiquat, Daminozide, N,N-Dimethylhydrazine, Trimethyl-
sulfonium, Cyromazine, Nereistoxin, Difenzoquat, Melamine and Propamocarb in apple extract at 0.1 mg/kg.

Diquat [M]2+ 92/84 Diquat [M]2+ 92/157 Diquat [M2+ - H+]+ 183/157 Diquat [M2+ - H+]+ 183/130

Diquat [M]+• 184/128 Diquat [M]+• 184/106 Paraquat [M]2+ 93/171 Paraquat [M]2+ 93/77

Paraquat [M]2+ 93/144 Paraquat [M]2+ 93/155 Paraquat [M]+• 186/171 Paraquat [M]+• 186/155

Figure 24: Typical chromatograms of Diquat and Paraquat in rice 0.005 µg/mL final extract (corresponding to 0,04 mg/kg)

EU Reference Laboratory for pesticides requiring Single Residue Methods (EURL-SRM) 58 of 102
 Method 4.2 (M4.2): “Quats&Co. BEH Amide”
Table 31: Proposed LC-MS/MS conditions for ESI-pos. compounds listed in the table below.
Instrument parameters Conditions
Ionisation mode ESI pos.
Column/temperature BEH Amide 2.1 x 100mm 1.7 µm (P/N: 186004801); 40°C
Pre-column / Pre-filters BEH Amide 1.7 µm (P/N: 186004799) / e.g. Supelco column saver 2.0 µm Filter
Eluent A 50 mmol NH4-formate in water (adjust to pH 3 with formic acid) Use brown glass !
Eluent B Acetonitrile
%A Flow [mL/min] Time [min]
3 0.5 0
3 0.5 0.5
Gradient 30 0.5 4.0
60 0.5 5.0
60 0.5 6.0
3 0.5 6.1
3 0.5 10
Injection volume 2 µL (0.5 µL for Waters Xevo TQ-Sµ)
Calibration standards and levels e.g. one level at the reporting limit plus 0.05 or 0.1 µg/IS portion* +
Compound Mass Transitions (m/z)
Aminocyclopyrachlor 214/170, 214/168, 214/101, 214/68
Amitrole 85/43, 85/57, 85/58
Amitrole-15N (IL-IS) 86/43
Amitrole-15N2 13C2 (IL-IS) 89/44
Chlormequat 122/58, 124/58, 122/63, 122/59, 124/59
Chlormequat-D4 (IL-IS) 126/58; 126/59
Chloridazon-desphenyl 146/117, 146/101, 146/66, 148/119
Chloridazon-desphenyl-15N2 (IL-IS) 148/117, 148/102
Cyromazine 167/68, 167/125, 167/108, 167/85, 167/60
Cyromazine-D4 (IL-IS) 171/86, 171/68
Daminozide 161/143, 161/61, 161/101, 161/115, 161/44
Daminozide-13C4 (IL-IS) 165/147, 165/44
Daminozide-D6 (IL-IS) 167/149, 165/97
Diethanolamine (DEA) 106/88, 106/70, 106/45
Diethanolamine-D4 (IL-IS) 110/92
Difenzoquat 249/130, 249/77, 249/193,
Diquat:*** 92/84, 92/157, 183/157
Diquat D8 (IL-IS) 96/89, 191/165
ETU (Ethylenethiourea) 103/60, 103/44, 103/86
ETU-D4 (IS) 107/48
Melamine 127/85, 127/68, (127/60)
Melamine-15N3 (IL-IS) 130/87, 130/44
Maleic Hydrazide 113/67, 113/40
Maleic Hydrazide D2 115/69, 115/87
Acquired mass transitions
Maleic Hydrazide-13C4 (IL-IS) 115/87, 115/58
Matrine 249/148, 249/150, 249/110, 249/55
Matrine D3 252/148, 252/150, 252/96
Mepiquat 114/98, 114/58
Mepiquat-D3 (IL-IS) 117/101
Mepiquat-4-hydroxy 130/58, 130/96, 130/114
Morpholine 88/70, 88/45, 88/44
Morpholine-D8 (IL-IS) 96/78, 96/46
Nereistoxin: 150/105, 150/61, 150/71, 150/72
Nereistoxin-D6 (IL-IS): 156/105, 156/61
Nicotine 163/130, 163/132, 163/84, 163/106
Nicotine D4 167/84
Oxymatrine 265/247, 265/205, 265/148, 265/136
Oxymatrine D3 268/250, 268/208
Paraquat*** 93/171, 93/85, 185/170
Paraquat D8 (IL-IS) 97/179, 193/178
Propamocarb: 189/144, 189/74, 189/102
Propamocarb-D7 (IL-IS) 196/103, 196/75
Propamocarb-N-desmethyl 175/102, 175/144, 175/74, 175/116
Propamocarb-N-oxide: 205/102, 205/144, 205/74
PTU - N,N′-(1,2-Propylene)thiourea)** 117/100, 117/58, 117/60, 117/72, 117/41
PTU-D6 - N,N′-(1,2-Propylene)thiourea –D6** 123/64, 123/74
Triethanolamine (TEA) 150/132, 150/70, 150/88
Triethanolamine-D12 (IL-IS) 162/144
Trimethylsulfonium 77/62, 77/47
Trimethylsulfonium-D9 (IL-IS) 86/68, 86/50
* One IS portion is the absolute IS-mass contained in the prepared calibration standard solution (see also Table 2).
**See comments on PTU under M3 (5.6.13).
*** Diquat and paraquat were only measured on the Waters Xevo TQ-Sµ

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Hints on Method 4.2
1. As the signals of Morpholin, DEA and TEA better run Method 7 (5.6.18): are often strongly suppressed by matrix
using these LC-conditions. For DEA even false negative results are observed in some cases.This effect is reduced if
the extract is diluted e.g. 5/10 fold.
2. Diquat and Paraquat require special extraction conditions (see 5.2.3-B). The screening option for diquat was
removed as the diquat peak is very broad. Deprotonated diquat (which is formed, e.g. in methanolic standards)
gives an earlier eluting sharp peak, but this peak does not appear in fresh extracts of real samples and is thus
unsuited for screening
3. For general hints on analytes: See 5.6.1

Figure 25: Typical chromatograms of Diquat and Paraquat in sesame extracts spiked at 0.05 mg/kg using Waters Xevo TQ-Sµ.

Nicotine 163/130 Nicotine 163/132 Nicotine 163/84 Nicotine D4 167/84

Maleic hydrazide 113/67 Maleic hydrazide D2 115/69

Maleic hydrazide 113/40

Figure 26: Exemplary chromatograms of nicotine in flour (spelt, whole-grain) Maleic hydrazide in solvent. Nicotine at 0.01 mg/kg
(0.005 µg/mL); nicotine D4 at 0.1 mg/kg (0.05 µg/mL); Maleic hydrazide and Maleic hydrazide D 2 at 0.2 mg/kg (0.1 µg/mL).

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 Aminocyclopyrachlor 214/168 T Amitrole 85/43 T Chlormequat 122/58 T Chloridazon-desphenyl 146/117 T

Cyromazin 167/68 T Daminozide 161/143 T Diethanolamine 106/88 T Difenzoquat 249/77 T

ETU 103/60 T Melamine 127/85 T Mepiquat 114/98 T Mepiquat-4-Hydroxy 130/58

Morpholine 88/70 T Nereistoxin 150/105 T Propamocarb 189/144 T Propamocarb-N-desmethyl 175/102 T

Propamocarb-N-oxide 204/102 T PTU 117/60 T Triethanolamine 150/132 T Trimethylsulfonium 77/62 T

Matrine 249/148 T Oxymatrine 265/247 T

Figure 27: Typical chromatograms of Aminocyclopyrachlor, Amitrole, Chlormequat, Chloridazon-desphenyl, Cyromazine, Damino-
zide, Diethanolamine, Difenzoquat, ETU, Melamine, Mepiquat, Mepiquat-4-hydroxy, Morpholine, Nereistoxin, Propamocarb, Pro-
pamocarb-N-desmethyl, Propamocarb-N-oxide, PTU, Triethanolamine, Trimesium (Trimethylsulfonium) in tomato extracts spiked
at 0.05 mg/kg; additionally Matrine and Oxymatrine at 0.01mg/kg in grape extract.

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Figure 28: Typical chromatograms of Aminocyclopyrachlor, Amitrole, Chlormequat, Chloridazon-desphenyl, Cyromazine, Damino-
zide, Diethanolamine, Difenzoquat, ETU, Melamine, Mepiquat, Mepiquat-4-hydroxy, Morpholine, Nereistoxin, Propamocarb, Pro-
pamocarb-N-desmethyl, Propamocarb-N-oxide, PTU, Triethanolamine, Trimesium (Trimethylsulfonium) in tomato extracts spiked
at 0.06 mg/kg.

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 Method 5 (M5): “Quats&Co. MonoChrom MS”
Table 32: Proposed alternative LC-MS/MS conditions for Chlormequat and Mepiquat
Instrument parameters Conditions
Ionisation mode ESI pos
Column/temperature MonoChrom MS 100x2 mm; 5 µm (Varian); at 40°C
Eluent A 5 mmol/L NH4-acetate + 0.1% acetic acid in water
Eluent B Acetonitrile
%A Flow [mL/min] Time [min]
5 0.4 0
95 0.4 2
Gradient
95 0.4 5
5 0.4 5.1
5 0.4 15
Injection volume 5 µL
Calibration standards and levels e.g. 0.05 or 0.1 µg/IS portion*+ one level at the reporting limit
Compound Mass Transitions (m/z)
Chlormequat 122/58, 122/63, 124/58
Chlormequat-D4 (IL-IS) 126/58
Mepiquat 114/98, 114/58
Mepiquat-D3 (IL-IS) 117/101
Acquired mass transitions Difenzoquat: 249/77, 249/130, 249/193
No IS currently available -
ETU (Ethylenethiourea) 103/44, 103/60, 103/86
ETU-D4 (IL-IS) 107/48
PTU - N,N′-(1,2-Propylene)thiourea)** 117/100, 117/58, 117/60, 117/72
PTU-D6 - N,N′-(1,2-Propylene)thiourea –D6** 123/64, 123/74
* One IS portion is the absolute IS-mass contained in the prepared calibration standard solution (see also Table 2).
** See comments on PTU under M 3 (5.6.13).

Hints on Method 5
1. For general hints on analytes: See 5.6.1
2. For more information on method 5 please refer to the following document within the EURL homepage:
http://www.crl-pesticides.eu/library/docs/srm/meth_ChlormequatMepiquat_CrlSrm.pdf

EU Reference Laboratory for pesticides requiring Single Residue Methods (EURL-SRM) 63 of 102
 Method 6 (M6): “Streptomycin and Kasugamycin”
Table 33: Proposed LC-MS/MS conditions Streptomycin and Kasugamycin
Instrument parameters Conditions
Ionisation mode ESI pos
Obelisc R 2.1 x 150 mm 5µm 100 Å
Column
(SIELC; OR-21.150.0510); 40°C
Pre-filters e.g. Supelco column saver 2.0 µm Filter
Obelisc R 2.1 x 10 mm 5 µm
Pre-column
(SIELC; OR-21.G.0510)
Eluent A 0.1% formic acid in water
Eluent B 0.1% formic acid in acetonitrile
%A Flow [mL/min] Time [min]
20 0.3 0
Gradient 20 0.3 8
20 0.3 13
80 0.5 18
80 0.5 23
Injection volume 20 µL; dwell time increased to 200 ms
e.g. 0.05 or 0.1 µg/IS portion* one level at the reporting limit
Calibration standards and levels
(use plastic vials if Streptomycin is within your scope)
Compound Mass Transitions (m/z)
Streptomycin 582/263, 582/246, 582/ 221
Acquired mass transitions No IS currently available -
Dihydrostreptomycin (IS) 584/263
Kasugamycin 380/112, 380/200
No IS currently available -
* One IS portion is the absolute IS-mass contained in the prepared calibration standard solution (see also Table 2).

Hints on Method 6
1. For general hints on analytes: See 5.6.1
2. Dihydrostreptomycin is a veterinary drug itself. It may be used as IS for the quantification of strepromycin if
shown to be absent from the sample (and vice versa)

Streptomycin 582/263 T Kasugamycin 380/112 T Dihydrostreptomycin IS 584/263

Figure 29: Typical chromatograms of Streptomycin and Kasugamycin in apple extracts spiked at 0.01 mg/kg.

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 Method 7 (M7): “Morpholine, Diethanolamine and Triethanolamine”
Table 34: Proposed LC-MS/MS conditions Morpholine, Diethanolamine and Triethanolamine
Instrument parameters Conditions
Ionisation mode ESI pos
Column Dionex Acclaim Trinity P1 2.1 x 100 mm (3 m) (P/N 071389); 40°C
Pre-filters e.g. Supelco column saver 2.0 µm Filter
Pre-column Dionex Acclaim Trinity P1 2.1 x 10 mm (3 m) (P/N 071391)
50 mmol NH4-formate in water (adjust to pH 3 with formic acid)
Eluent A
Use brown glass bottles!
Eluent B Acetonitrile
%A Flow [mL/min] Time [min]
Gradient
10 0.4 0
10 0.4 10
Injection volume 5 µL
Calibration standards and levels e.g. 0.05 or 0.1 µg/IS portion+ one level at the reporting limit
Compound Mass Transitions (m/z)
Morpholine 88/70, 88/45, 88/44
Morpholine-D8 (IS) 96/78, 96/46
Acquired mass transitions
Diethanolamine (DEA) 106/88, 106/70, 106/45
Diethanolamine-D4 (IS) 110/92
Triethanolamine (TEA) 150/132, 150/70, 150/88
Triethanolamine-D12 (IS) 162/144
* One IS portion is the absolute IS-mass contained in the prepared calibration standard solution (see also Table 2).

Hints on Method 7
1. For general hints on analytes: See 5.6.1
2. Morpholin, DEA and TEA are not pesticides, they are additive of waxes used to coat crops (citrus, apples and
mangoes etc). They are included in this method for the sake of convenience and synergy. As these three com-
pounds can be analyzed very sensitively 5-10-fold dilution of the extracts before injection is recommendable
where possible, especially in absence of an IS requiring standard additions approach (5.5.3)

Morpholine-D8 Diethanolamine-D4 Triethanolamine-D12

Morpholine 88/70 T Diethanolamine 106/88 T Triethanolamine 150/132 T

Figure 30: Typical chromatograms of Morpholine, Diethanolamine and Triethanolamine in apple extracts at 0.05 mg/kg (extract
were diluted 10-fold before injection)

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 Method 8 (M8): “Triazole derivative metabolites (TDMs)”
Table 35: Proposed LC-MS/MS conditions 1,2,4-Triazole, Triazole-alanine, Triazole-acetic acid, Triazole-lactic acid and 1,2,3- Tria-
zole
Instrument parameters Conditions
Ionisation mode ESI pos
Column Hypercarb 2.1 x 100 mm 5 m (P/N 35005-102130); 40°C
Pre-column Hypercarb Guard 2.1 x 10 mm 5 m (P/N 35005-102101)
Pre-filter e.g. Supelco column saver 2.0 µm Filter (optional)
Eluent A 1% acetic acid in water + 5% methanol
Eluent B 1% acetic acid in methanol
%A Flow [mL/min] Time [min]
100 0.6 0
Gradient 10 0.6 5
10 0.6 6
100 0.6 6.1
100 0.6 10
Injection volume 2 µL
Calibration standards and levels e.g. 0.05 or 0.1 µg/IS portion* one level at the reporting limit
DMS-Settings
Compound Mass Transitions (m/z) (SelexIon Q-Trap® 5500) **
COV (V) SV (V)
1,2,4-Triazole#: 70/43, 70/70 -10 2600
1,2,4-Triazole-13C2,15N3 (IS) 75/46 -13.75 3000
Acquired mass transitions
Triazole-alanine: 157/70, 157/88, 157/42 -2.0 3000
Triazole-alanine-13C2,15N3 (IS) 162/75 -1.75 3100
Triazole-acetic acid: 128/70, 128/43, 128/73 -6.0 3100
Triazole-acetic acid-13C2,15N3 (IS) 133/75 -6.0 3500
Triazole-lactic acid: 158/70, 158/43, 158/112 -3.0 3300
Triazole-lactic acid-13C2,15N3 (IS) 163/75 -2.25 3500
* One IS portion is the absolute IS-mass contained in the prepared calibration standard solution (see also Table 2).
** Further parameters: DMS temp.: low; CUR 20, GS1 60, GS2 70, DMO -3.0; DMS condition differ to some extent from instru-
ment to instrument DMS condition differ to some extent from instrument to instrument

Hints on Method 8
1. For general hints on analytes: See 5.6.1
2. 1,2,4-Triazole is used as nitrification inhibitors in fertilizers
3. The following commercially available isotopically labelled components were not tested with this method: 1,2,4-
Triazole-D2, 1, 2, 4-Triazole-acetic acid-D2, 1, 2, 4-Triazole-alanine-D2, 1, 2, 4-Triazole-lactic acid-D2

1,2,4-Triazole 70/43 1,2,4-Triazole-acetic acid 128/70 1,2,4-Triazole-1yl-alanine 157/70 1,2,4-Triazole-lactic acid 158/70

Figure 31: Typical chromatograms of TDMs in avocado extracts spiked at 0.01 mg/kg.

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 Method 9 (M9): “Difluoroacetic acid and Trifluoroacetic acid”

Table 36: Proposed LC-MS/MS and SelexIon conditions Difluoroacetic and Trifluoroacetic acid
Instrument parameters Conditions
Ionisation mode ESI neg
Column Dionex/Thermo, Acclaim Trinity P1, 2.1 x 100 mm, (3 µm) (P/N 071389); 40°C
Pre-column Thermo Guard Cartrige Acclaim Trinity P1, 2.1 x 10 mm, (3 µm) (P/N 071391)
Pre-filter e.g. Supelco column saver 2.0 µm Filter (optional)
Eluent A 50 mmol NH4-formate, adjusted to pH 3 with formic acid
Eluent B Acetonitrile
%A Flow [mL/min] Time [min]
10 0.45 0
10 0.45 3.5
Gradient
50 0.45 4
50 0.45 6
10 0.45 6.1
10 0.45 10
Injection volume 2 µL
e.g. 0.05 or 0.1 µg/IS portion*, one level at the reporting limit. Always use matrix based calibrations
Calibration standards and levels
(e.g. blank tomato extract) instead of solvent based.
DMS-Settings
Compound Mass Transitions (m/z) (SelexIon Q-Trap® 5500) ***
COV (V) SV (V)
Acquired mass transitions
Difluoroacetic acid (DFA) 95/51, 95/95** -9.5 2500
Difluoroacetic acid -13C2 (IL-IS) 75/46 -12 3000
Trifluoroacetic acid (TFA) 113/69, 113/113** -5.6 2200
Trifluoroacetic acid -13C2 (IL-IS) 115/70 -5.5 2300
* One IS portion is the absolute IS-mass contained in the prepared calibration standard solution (see also Table 2).
** Despite not having a mass transition the DMS provides good selectivity
*** Further parameters: DMS temp.: medium; CUR 20, GS1 60, GS2 70, DMO -3.0; DMS condition differ to some extent from
instrument to instrument

Hints on Method 9
1. For general hints on analytes: See 5.6.1

DFA 95/51 DFA 95/95 TFA 113/69 TFA 113/113

Figure 32: Typical chromatograms of DFA and TFA in tomato extracts spiked at 0.05 mg/kg

EU Reference Laboratory for pesticides requiring Single Residue Methods (EURL-SRM) 67 of 102
 Method 10 (M10): “Triazole derivative metabolites (TDMs) on Torus DEA”
Table 37: Proposed LC-MS/MS conditions 1,2,4-Triazole, Triazole-alanine, Triazole-acetic acid, Triazole-lactic acid and 1,2,3- Tria-
zole
Instrument parameters Conditions
Ionisation mode ESI pos
Column Waters Torus™DEA 2.1 mm x 100 mm; 1.7 µm; 50 °C
Pre-column Waters Torus™DEA VanGuard™ 2.1 mm x 5 mm; 1.7 µm
Pre-filter Waters ACQUITY UPLC Column In-Line Filter Kit [205000343]
Eluent A 1.2% formic acid in water
Eluent B 0.5 % formic acid in Acetonitrile
%A Flow [mL/min] Time [min]
10 0.5 0
10 0.5 0.5
Gradient 80 0.5 1.5
90 0.5 4.5
90 0.5 5
10 0.5 5.5
10 0.5 10
Injection volume 2 µL
Calibration standards and levels e.g. 0.05 or 0.1 µg/IS portion* one level at the reporting limit

Compound Mass Transitions (m/z)

Triazole-alanine: 157/88, 157/70, 157/42

Triazole-alanine-13C2,15N3 (IS): 162/75
Acquired mass transitions Triazole-alanine D2: 159/42
Triazole-acetic acid: 128/70, 128/43, 128/73
Triazole-acetic acid-13C2,15N3 (IS): 133/75
Triazole-acetic acid D2: 130/72
Triazole-lactic acid: 158/70, 158/43, 158/112
Triazole-lactic acid-13C2,15N3 (IS): 163/75
Triazole-lactic acid D2: 160/72
* One IS portion is the absolute IS-mass contained in the prepared calibration standard solution (see also Table 2).

Hints on Method 10
1. For general hints on analytes: See 5.6.1
2. 1,2,4-Triazole is measured by M8: See 5.6.19

1,2,4-Triazole-acetic acid 128/70 1,2,4-Triazole-1yl-alanine 157/88 1,2,4-Triazole-lactic acid 158/70

Figure 33: Typical chromatograms of TDMs in strawberry extracts spiked at 0.05 mg/kg.

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 Method 11 (M11): “Gly&Co. by IC on AS19”3
Table 38: Proposed IC-MS/MS conditions for Glyphosate, AMPA, N-Acetyl-Glyphosate, Ethephon, HEPA, Glufosinate, MPPA,
Fosetyl-Al, Cyanuric acid, Bromide, Chlorate, Perchlorate, Phosphonic acid and TFA
Instrument parameters Conditions
Ionisation mode ESI neg
Column/temperature Thermo ScientificTM DionexTM IonPacTM AS19,2x250mm; 32°C
Pre-column Thermo ScientificTM DionexTM IonPacTM AG19,2x50mm
Eluent Thermo ScientificTM DionexTM EGC 500TM KOH eluent generator cartridge
c [KOH] Flow [mL/min] Time [min]
15 0.3 0
15 0.3 8
36 0.3 13
Gradient
36 0.3 21
70 0.3 21.5
70 0.3 25
15 0.3 25.5
15 0.3 30
Injection volume 5 µL of 5-fold diluted extracts in water (preferably ultrapure)
Flow Make-up Solvent before ion source 0.15 mL/min acetonitrile
e.g. 0.05 or 0.1 µg/IS portion + one level at the reporting limit;
Calibration standards and levels Standard solutions of Fosetyl and Ethephon (and their IL-ISs) may be contaminated with native
Phosphonic acid which may potentially lead to false positives or shifted calibration, see 5.6.1.
Compound Mass Transitions (m/z)
Glyphosate: 168/63, 168/124, 168/150, 168/81
Glyphosate-13C2,15N (IL-IS): 171/63, 171/126
AMPA: 110/63, 110/79, 110/81
AMPA-13C,15N (IL-IS): 112/63, 112/81
N-Acetyl-Glyphosate: 210/63, 210/150, 210/79, 210/148
N-Acetyl-Glyphosate-D3 (IL-IS): 213/63, 213/153
Ethephon: 143/107, 143/79, 145/107
Ethephon-D4 (IL-IS): 147/111, 147/79
HEPA: 125/79, 125/95, 125/63
HEPA-D4 (IL-IS): 129/79, 129/97
Glufosinate: 180/63, 180/136, 180/85, 180/95
Glufosinate-D3 (IL-IS): 183/63, 183/98
N-Acetyl-Glufosinate: 222/63, 222/59, 222/136
N-Acetyl-Glufosinate-[acetyl]D3 (IL-IS): 225/63, 225/137
Acquired mass transitions (m/z) N-Acetyl-Glufosinate-[methyl]D3 (IL-IS): 225/63
MPPA: 151/63, 151/107, 151/133
MPPA-D3 (IL-IS): 154/63, 154/136
Fosetyl-Al: 109/81, 109/63 (each detected as Fosetyl)
Fosetyl-Al-D15 (IL-IS): 114/82, 114/63 (each detected as Fosetyl- D5)
Trifluoroacetic acid (TFA) 113/69, 113/113
Trifluoroacetic acid -13C2 (IL-IS): 115/70
Cyanuric acid: 128/42, 128/85
Cyanuric acid-13C3: 131/43, 131/87
Bromide: 81/81, 79/79
Chlorate: 83/67, 85/69
18
Chlorate- O3 (IL-IS): 89/71, 91/73
Perchlorate: 99/83, 101/85
Perchlorate-18O4 (IL-IS): 107/89, 109/91
Phosphonic acid: 81/79, 81/63
Phosphonic acid-18O3 (IL-IS): 87/85, 87/67
Hints on Method 11
1. For general hints on analytes: See 5.6.1

3
https://www.eurl-pesticides.eu/userfiles/file/EurlSRM/EPRW%202020%20-%20PD87.pdf
EU Reference Laboratory for pesticides requiring Single Residue Methods (EURL-SRM) 69 of 102
 AMPA 110/63 T Ethephon 143/107 T Fosetyl 109/81 T Glufosinate 180/63T

Glyphosate 168/63T HEPA 125/63 T MPPA 151/63T N-Acetyl-Glufosinate 222/63T

N-Acetyl-Glyphosate 210/150T Cyanuric acid 128/42

Figure 34: Typical chromatograms in cucumber extracts spiked at 0.05 mg/kg.

Perchlorate 99/83 Chlorate 83/67 Phosphonic acid 81/79 Trifluoracetic acid 113/69

Bromide 79/79

Figure 35: Typical chromatograms in milk extracts spiked at 0.01 mg/kg (Perchlorate and Chlorate); at 0.05 mg/kg (Phosphonic
acid); 0.025 mg/kg (Trifluoracetic acid) and 5 mg/kg (Bromide).

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 5.7. Calibration and Calculations

Using IS
Where IS is added to the sample before any aliquotation:
The following calculation approach requires that the ratio of the IS masses added to the test portions (5.2.3) and to
the calibration standard(s) (145.5) (mISsample / mIScal mix) is known and constant. By keeping the IS constant throughout
the calibration levels the peak ratio PR cal mix (A pest cal mix/ A IS cal mix) of each calibration level can be plotted against the
absolute mass of the pesticide mpestcal mix rather than the ratio mpestcal mix / mIScal mix(the mIScal mix is set as 1).
The calibration graph (to be plotted for each pesticide separately) is described by the following formula:

PRcal mix  acal  m cal

pest
mix
 bcal
(1)

The mass fraction (wR) of a given pesticide in a given sample can be calculated as follows using the respective peak
ratio of pesticide and internal standard obtained from the sample extract (PR sample = A pest Sample / A IS Sample), the correc-
tion factor (mISsample / mIScal mix) as well as the weight of the test portion (ma).
( PR Sample  bcal ) 1 mISTD
Sample
 mg 
wR    cal mix  
acal ma mISTD  kg  (2)

Reasonably (but not necessarily) the calibration standards should be prepared in such a way that the ratio mISsample /
mIScal mix equals the assumed volume ratio of sample extract versus calibration standard (20 for most samples and 40
for cereals, pulses, nuts and oily seeds). The absolute masses of the IS-WS I and II do not need to be necessarily known
(see also the notes of Table 2).

Where IS is added to an aliquot of the extract

When adding the IS to an aliquot of the extract (e.g. 1 mL) the knowledge of the exact total volume of the sample
extract becomes important. Water adjustment is thus essential and if it is done as described in 5.2.2 and Table 35, the
total volume can be assumed to be exactly 20 mL. 1 mL sample extract will correspond to 1/20th of the test portion
(ma) in case of most samples (or to 1/40th. in case of cereals, pulses, nuts and oily seeds, where extracts are diluted 2-
fold during cleanup). The mass of the IS to be added to an aliquot (mISaliquot ) should be scaled according to the aliquot
volume used (Valiquot) with the IS mass ratio (mISaliquot / mIScal mix ) being important for the calculation. Reasonably (but
not necessarily) mISaliquot should be derived using the following formula mISaliquot = mISsample x Valiquot/20 (or mISaliquot =
mISsample x Valiquot/40 in case of cereals, pulses, nuts and oily seeds), with mISsample being the IS mass that would have
been added to the entire sample portion according to 5.2.2 and Table 35.
Following the above, the mass fraction (wR) of a given pesticide in a given sample can be calculated as follows using
the respective peak ratio of pesticide and internal standard obtained from the sample extract (PR sample = A pest sample /
A IS sample), the correction factor (mISaliquot / mIScal mix) as well as the weight of the sample equivalents in the aliquot
(maliquot= ma x Valiquot/20 or maliquot= ma x Valiquot/40 in case of a 2-fold dilution during cleanup).
( PR sample  bcal ) 1 maliquot  mg 
wR    ISTD
cal mix
 
acal maliquot mISTD  kg  (3)

Variables used
Mass of pesticide in calibration mixture m cal
pest
mix
µg

Mass of pesticide in final extract m sample

pest µg

cal mix
Mass of internal standard in calibration mixture mISTD µg

Mass of internal standard added to test portion (sample) sample µg

mISTD

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 aliquot
Mass of internal standard added to aliquot of sample extract m ISTD µg
Volume of sample extract aliquot used (5.7.1 and 5.5.3) to spike the IS or for
standard additions
V aliquot mL

Mass of test portion ma g

Mass of test portion represented in an aliquot m aliquot g

Mass fraction of pesticide in the sample wR mg/kg

cal mix
Peak area of pesticide obtained from calibration standard (mixture) Apest (counts)

cal mix
Peak area of IS obtained from calibration standard (mixture) AISTD (counts)

sample
Peak area of pesticide obtained from the injected extract A pest (counts)

sample
Peak area of IS obtained from the injected extract AISTD (counts)

Peak ratio of pesticide vs. IS obtained from calibration mixture PR cal mix (dimensionless)

Peak ratio of pesticide vs. IS obtained from injected extract PR sample (dimensionless)

Slope of calibration graph a cal (dimensionless)

Bias of calibration graph (intercept) b cal (dimensionless)

Not using IS
If no appropriate ISs are used it is of high importance to properly compensate for matrix effects. For the compensation
of matrix effects matrix-matched calibrations (5.5.2) and the standard additions approach (5.5.3) are recommended.
In both cases the assumption is made that the total volume of the sample extract is exactly 20 mL. Adjustment of the
water content (and extract volume) in the sample is thus paramount.

Calculations when employing matrix-matched calibration without IS

The calibration graph (to be plotted for each pesticide separately) is described by the following formula:
cal mix
Apest  acal  C pest
cal mix
 bcal
(1)

The mass fraction (wR) of a given pesticide in a given sample can be calculated as follows using the respective peak
sample
area of the pesticide obtained from the sample extract ( Apest ) and a correction factor (V) as well as the weight of
the test portion (ma).
Sample
( Apest  bcal ) 1  mg 
wR    Vend  
acal ma  kg  (2)

where Vend is the total volume of the sample extract (20 mL or 40 mL in case of a 2-fold dilution during cleanup).

All other variables are listed in 5.7.1.

Calculations when employing the standard additions approach

The standard additions approach is the method of choice where no appropriate IL-IS is available. This approach typically compen-
sates matrix effect better than the matrix-matched calibrations (5.5.2). The mass fraction of the pesticide in the sample (w R) is
calculated via linear regression using a graphical presentation as shown in Figure 36. The Y-intercept of the calibration graph will
indicate the pesticide mass contained in the non-fortified aliquot of the sample extract.

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Figure 36: Internal calibration using the procedure of standard additions, schematically

Key:
Y Peak area of analyte
X Added absolute mass of analyte m std
pest
add
in µg
|x| absolute amount of analyte in the sample extract (in µg) before standard addition (y = 0)

y  int ercept (b)

With x = (µg)
slope of the curve (a)

The calculation is performed as follows using the regression graph shown in

b V  mg 
wR   end  
a Val  ma  kg 
where:
b Y-intercept of the calibration graph of the analyte in question;
a Slope of the calibration graph of the analyte in question (1/µg);
Vend Volume of sample extract (mL) (should be 20 mL)
Val Volume of aliquots used for the standard additions approach (mL)
ma Weight of initial sample portion (g)

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6. Stability and purity of standards
A general overview regarding the stability of Glyphosate & Co. compounds in stock solutions is given in Table 39. For
the compounds of this method (Maleic Hydrazide and Cyanuric acid excluded) the use of water with 10 % acetonitrile
was shown to be a suitable solvent, see also Table 43.
In case of Ethephon (native compound or IL-IS), which is sensitive towards neutral and alkaline pH, acidifying the stock
solution with hydrochloric acid is recommended. The addition of 0,1 % (v/v) of concentrated HCl (37 %) is proposed.
This acid content will also sufficiently stabilize 100-fold diluted working solutions (of e.g. 10 µg/mL) without the need
of adding further acid. Other compounds of this method are not markedly compromised in their stability by this acid
content.
The previously recommended solvent of methanol/water+1 % formic acid 1/1 proved to be less suitable in the long
run with methylations, formylations as well as dehydrations being observed for some compounds, such as glyphosate.
To some extent degradation also takes place in QuPPe extracts (consisting of Water/Methanol+1 % Formic acid (1/1,
v/v)) with AMPA and N-Acetyl-Glyphosate being most affected. In general degradation is negligible if extracts stored
at room temperature are analyzed within 14 days . In any case such losses can be effectively corrected by the respec-
tive IL-ISs (if added at any stage prior to extract storage). The stability of compounds of the “Glyphosate & Co. group”
in water containin g 10% acetonitrile, over a period of 7 months in the refrigerator, is demonstrated in Figure 37. The
stability in stock solutions is generally better that in working solutions.

Table 39: Overview of experiments on long-term stability “Glyphosate & Co.” compounds dissolved in differently composed sol-
vents. Concentration of analytes in stored mixtures 10 µg/mL ; storage duration: 6 months; storage temperature: 6°C
Composition of storage solvent
Pure Water Water / MeOH Pure MeOH Water / ACN
(MeOH 25 and 50 %)* (ACN 25 and 50 %)*
Native Compound w/o acid 1% FA 1% AA 0.1% HCl** w/o acid 1% FA 1% AA w/o acid 1% FA w/o acid 1% FA 1% AA
AMPA NT NT NT NT  NT NT NT NT NT NT NT
Bialaphos NT NT NT NT  NT NT NT NT NT NT NT
Cyanuric acid NT NT NT NT  NT NT NT NT NT NT NT
Ethephon     NT NT NT NT  NT NT NT
Fosetyl-Al      NT NT NT NT NT NT NT
Glufosinate NT NT NT NT  NT NT NT NT NT NT NT
Glyphosate    NT    NT NT   
HEPA NT NT NT NT NT NT NT   NT NT NT
Maleic Hydrazide NT NT NT NT NT NT NT   NT NT NT
MPPA    NT    NT NT   
N-Acetyl-AMPA    NT    NT NT   
N-Acetyl-Glufosinate NT NT NT NT  NT NT NT NT NT NT NT
N-Acetyl-Glyphosate    NT    NT NT   
= sufficiently stable (deviating less than ±10 % from a freshly prepared standard of the same composition) ; =not stable
MeOH = Methanol; ACN = Acetonitrile
* Solutions of both 25 % and 50 % of organic solvent have been tested.
** 0.1% HCl-conc. (37%) in water (v/v)

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Figure 37: Deviations of the concentration of Glyphosate & Co. compounds in a working solution of 10 µg/mL water containing 10
% acetonitrile and 1‰ HCl conc. (v/v), following 7 months of storage at 6 °C. Compared against a freshly prepared standard of
same composition

Issues concerning the purity of N-Acetyl-Glufosinate D3: There is two types of N-Acetyl-Glufosinate D3 standards on
the market. Both contain the three deuterium atoms on a methyl group, but the first one contains them on the methyl
group of the acetyl moiety and the other one on the methyl group that is attached to the phosphorus atom. In theory
the acetyl group can be hydrolytically detached, native glufosinate may be formed in working solutions of N-Acetyl-
Glufosinate (acetyl-D3), leading to false positive results. Fortunately the degradation rate observed in the water:ace-
tonitrile 9:1 mixture (see Figure 37) was negligible. More important is the content of native glufosinate in purchased
N-Acetyl-Glufosinate (acetyl-D3) standards. Before first use the standards should be checked for the presence of native
glufosinate impurities and object the product if it does not meet the producer’s specifications. The levels of native
glufosinate impurities depend on the manufacturer and the badge. Where e.g. 0.5 µg IL-IS is added to 1 g sample, the
presence of 2% native glufosinate (a typical level encountered) can lead to glufosinate levels of 0.01 mg/kg.

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7. Performance Data
Validation data of the presented methods according to SANTE/11945/2015 guidance document are shown at the EURL
validation database at www.eurl-pesticides-datapool.eu. Exemplary LOQs of the presented methods are listed in Table
40.

Table 40: Overview of lowest successfully validated levels per matrix

Spiking Mean
Method Analyte Commodity Group Matrix Level n Recov. RSD
(mg/kg) %
AMPA High water content + acidic Grapes 0.02 12 110 9
AMPA Dry (cereals) Barley 0.02 5 101 14
AMPA Dry (pulses) Lentil 0.1 10 95 17
AMPA Dry (cereals) Wheat flour 0.1 5 119 6
AMPA High water content Apple 0.02 17 100 12
AMPA Dry (cereals)* Rice 0.1 5 99 6
AMPA Dry (pulses)* Soybean 0.1 5 106 6
AMPA High sugar and low water content Honey 0.02 5 101 2
Cyanuric Acid High water content Cucumber 0.02 3 106 13
Cyanuric Acid Dry (cereals)* Rice 0.1 5 105 5
Cyanuric Acid Dry (pulses)* Soybean 0.1 5 115 12
Ethephon Dry (cereals) Barley 0.02 5 110 2
Ethephon Dry (cereals) Wheat flour 0.1 5 85 6
Ethephon High water content Apple 0.02 7 105 11
Ethephon High water content Cucumber 0.02 3 101 11
Ethephon High water content + acidic Grapes 0.01 5 104 4
Ethephon Dry (cereals)* Rice 0.02 5 92 8
Ethephon Dry (pulses)* Soybean 0.02 5 107 11
Ethephon High sugar and low water content Honey 0.02 5 101 7
Fosetyl High water content + acidic Strawberry 0.1 6 94 4
Fosetyl Dry (cereals) Barley 0.02 5 106 7
Fosetyl High water content Apple 0.02 7 104 5
Fosetyl High water content Cucumber 0.02 3 103 5
M1.3
Fosetyl High water content + acidic Grapes 0.01 5 105 2
Fosetyl Dry (cereals)* Rice 0.02 5 91 3
Fosetyl Dry (pulses)* Soybean 0.02 5 96 4
Fosetyl High sugar and low water content Honey 0.02 5 109 4
Glufosinate High water content + acidic Grapes 0.05 5 96 10
Glufosinate Dry (cereals) Barley 0.02 5 101 13
Glufosinate Dry (cereals) Wheat flour 0.1 5 85 5
Glufosinate High water content Apple 0.02 7 106 8
Glufosinate High water content Cucumber 0.02 3 115 4
Glufosinate Dry (cereals)* Rice 0.06 5 96 5
Glufosinate Dry (pulses)* Soybean 0.06 5 105 8
Glufosinate High sugar and low water content Honey 0.02 5 96 2
Glyphosate High water content + acidic Grapes 0.02 12 112 8
Glyphosate High water content + acidic Grapes 0.02 5 102 6
Glyphosate Dry (cereals) Barley 0.02 5 105 8
Glyphosate Dry (pulses) Lentil 0.1 11 107 18
Glyphosate High oil content, dry (oily seeds, nuts) Bean, Soya 0.1 10 95 10
Glyphosate High water content Apple 0.02 16 93 12
Glyphosate High water content Cucumber 0.02 3 94 3
Glyphosate Dry (cereals)* Rice 0.1 5 91 6
Glyphosate Dry (pulses)* Soybean 0.06 5 99 5
Glyphosate High sugar and low water content Honey 0.02 5 105 4
HEPA Dry (cereals) Barley 0.02 5 106 17

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 Spiking Mean
Method Analyte Commodity Group Matrix Level n Recov. RSD
(mg/kg) %
HEPA High water content Apple 0.02 7 109 14
HEPA High water content Cucumber 0.02 3 104 6
HEPA Dry (cereals)* Rice 0.04 5 98 1
HEPA Dry (pulses)* Soybean 0.04 5 93 6
HEPA High sugar and low water content Honey 0.02 5 100 2
Maleic Hydrazide Dry (cereals) Barley 0.02 5 100 9
Maleic Hydrazide High water content Apple 0.02 7 110 9
Maleic Hydrazide High water content Cucumber 0.02 3 103 13
Maleic Hydrazide High water content, extract rich Onion 0.1 5 106 4
Maleic Hydrazide High water content + acidic Grapes 0.01 5 110 11
Maleic Hydrazide Dry (cereals)* Rice 0.08 5 96 8
Maleic Hydrazide Dry (pulses)* Soybean 0.08 5 97 14
MPPA Dry (cereals) Barley 0.02 5 106 10
MPPA Dry (cereals) Wheat flour 0.1 5 85 1
MPPA High water content Apple 0.02 7 88 11
MPPA High water content Cucumber 0.02 3 107 14
MPPA High water content + acidic Grapes 0.02 5 102 3
MPPA Dry (cereals)* Rice 0.04 5 97 3
MPPA Dry (pulses)* Soybean 0.04 5 101 3
MPPA High sugar and low water content Honey 0.02 5 99 2
N-Acetyl AMPA Dry (cereals) Barley 0.02 5 108 3
N-Acetyl AMPA High water content Apple 0.02 7 120 11
N-Acetyl AMPA High water content Cucumber 0.02 3 89 7
N-Acetyl Glufosinate Dry (cereals) Barley 0.02 5 103 5
N-Acetyl Glufosinate High water content Apple 0.02 7 112 9
N-Acetyl Glufosinate High water content Cucumber 0.02 3 101 3
N-Acetyl Glufosinate High water content + acidic Grapes 0.01 5 97 4
N-Acetyl Glufosinate Dry (cereals)* Rice 0.04 5 99 2
N-Acetyl Glufosinate Dry (pulses)* Soybean 0.04 5 98 3
N-Acetyl Glufosinate High sugar and low water content Honey 0.02 5 99 2
N-Acetyl Glyphosate High water content + acidic Grapes 0.01 10 109 8
N-Acetyl Glyphosate Dry (cereals) Corn flour 0.02 10 104 10
N-Acetyl Glyphosate Dry (pulses) Lentil 0.05 10 104 8
N-Acetyl Glyphosate High oil content, dry (oily seeds, nuts) Bean, Soya 0.05 10 102 7
N-Acetyl Glyphosate High water content Apple 0.01 10 109 8
N-Acetyl Glyphosate Dry (cereals)* Rice 0.1 5 94 2
N-Acetyl Glyphosate Dry (pulses)* Soybean 0.1 5 101 3
N-Acetyl Glyphosate High sugar and low water content Honey 0.02 5 100 3
Bromate High water content Lettuce varieties 0.02 5 103 6
Bromide (inorg.) High water content + acidic Currant 1 5 98 4
Bromide (inorg.) High water content Cauliflower 1 5 94 12
Chlorate High water content + acidic Currant 0.01 5 102 7
Chlorate Dry (cereals) Rice 0.02 5 108 2
Chlorate High water content Cauliflower 0.01 5 100 5
Perchlorate High water content + acidic Currant 0.01 5 100 4
Perchlorate Dry (cereals) Barley 0.01 5 106 2
M1.4
Perchlorate Dry (cereals) Rice 0.02 5 100 7
Perchlorate High water content Apple 0.01 5 108 3
Perchlorate High water content Cauliflower 0.01 5 97 3
Phosphonic Acid High water content + acidic Currant 0.1 5 102 3
Phosphonic Acid High water content + acidic Mandarine 0.1 5 99 10
Phosphonic Acid Dry (cereals) Rice 0.2 5 97 4
Phosphonic Acid High water content Apple 0.1 6 102 9
Phosphonic Acid High water content Cauliflower 0.1 5 87 2

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 Spiking Mean
Method Analyte Commodity Group Matrix Level n Recov. RSD
(mg/kg) %
Phosphonic Acid High water content Mango 0.1 5 99 9
AMPA High water content Apple 0.02 5 102 8
AMPA High water content + acidic Grape 0.02 5 112 8
AMPA Dry (oily seeds) Soy flour 0.1 5 92 7
AMPA Dry (pulses) Lentils 0.1 5 103 6
Ethephon High water content Apple 0.025 5 97 0
Ethephon High water content + acidic Grape 0.025 5 80 8
Ethephon Dry (oily seeds) Soy flour 0.05 5 92 7
Ethephon Dry (pulses) Lentils 0.05 5 97 4
Fosetyl High water content Apple 0.01 5 99 9
Fosetyl High water content + acidic Grape 0.01 5 100 3
Fosetyl Dry (oily seeds) Soy flour 0.1 5 98 3
Fosetyl Dry (pulses) Lentils 0.1 5 99 1
Glufosinate High water content Apple 0.02 5 94 2
Glufosinate High water content + acidic Grape 0.02 5 106 4
Glufosinate Dry (oily seeds) Soy flour 0.1 5 98 4
Glufosinate Dry (pulses) Lentils 0.1 5 101 5
Glyphosate High water content Apple 0.02 5 98 8
M1.5
Glyphosate High water content + acidic Grape 0.02 5 106 5
Glyphosate Dry (oily seeds) Soy flour 0.1 5 102 3
Glyphosate Dry (pulses) Lentils 0.1 5 102 4
HEPA High water content Apple 0.02 5 102 8
HEPA High water content + acidic Grape 0.02 5 100 6
HEPA Dry (oily seeds) Soy flour 0.1 5 96 4
HEPA Dry (pulses) Lentils 0.1 5 98 2
MPPA High water content Apple 0.02 5 97 4
MPPA High water content + acidic Grape 0.02 5 106 3
MPPA Dry (oily seeds) Soy flour 0.1 5 103 1
MPPA Dry (pulses) Lentils 0.1 5 103 2
N-Acetyl-Glufosinate High water content Apple 0.01 5 100 3
N-Acetyl-Glufosinate High water content + acidic Grape 0.01 5 103 3
N-Acetyl-Glufosinate Dry (oily seeds) Soy flour 0.05 5 99 3
N-Acetyl-Glufosinate Dry (pulses) Lentils 0.05 5 102 1
N-Acetyl-Glyphosate Dry (oily seeds) Soy flour 0.05 5 105 10
N-Acetyl-Glyphosate Dry (pulses) Lentils 0.05 5 99 12
AMPA High water content Cucumber 0.02 5 99 7
AMPA Dry (pulses)* Lentil 0.1 5 107 10
Ethephon High water content Cucumber 0.02 5 95 5
Ethephon Dry (pulses)* Lentil 0.1 5 89 12
Fosetyl High water content Cucumber 0.02 5 98 3
Fosetyl Dry (pulses)* Lentil 0.1 5 104 3
Glufosinat High water content Cucumber 0.02 5 95 4
Glufosinat Dry (pulses)* Lentil 0.1 5 82 8
Glyphosat High water content Cucumber 0.02 5 107 3
M1.6a: To-
Glyphosat Dry (pulses)* Lentil 0.1 5 115 9
rus DEA HEPA High water content Cucumber 0.02 5 108 8
HEPA Dry (pulses)* Lentil 0.1 5 100 5
MPPA High water content Cucumber 0.02 5 103 3
MPPA Dry (pulses)* Lentil 0.1 5 108 5
N-Acetyl-AMPA High water content Cucumber 0.02 5 104 1
N-Acetyl-Glufosinat High water content Cucumber 0.02 5 98 2
N-Acetyl-Glufosinat Dry (pulses)* Lentil 0.1 5 113 4
N-Acetyl-Glyphosat High water content Cucumber 0.02 5 98 2
N-Acetyl-Glyphosat Dry (pulses)* Lentil 0.1 5 101 8

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 Spiking Mean
Method Analyte Commodity Group Matrix Level n Recov. RSD
(mg/kg) %
AMPA High water content + acidic Strawberry 0.05 5 106 6
Bromide (inorg.) High water content + acidic Lemon 5 5 101 6
Ethephon High water content + acidic Strawberry 0.01 5 105 2
Ethephon Dry (oily seeds)* Soybean 0.02 5 110 10
Fosetyl High water content + acidic Strawberry 0.01 5 99 1
Fosetyl Dry (oily seeds)* Soybean 0.02 5 98 3
Glufosinate High water content + acidic Strawberry 0.03 5 98 10
Glufosinate Dry (oily seeds)* Soybean 0.06 5 96 6
Glyphosate High water content + acidic Strawberry 0.05 5 104 8
M1.6b: Glyphosate Dry (oily seeds)* Soybean 0.1 5 101 5
APPC HEPA High water content + acidic Strawberry 0.02 5 107 8
HEPA Dry (oily seeds)* Soybean 0.04 5 99 4
MPPA High water content + acidic Strawberry 0.02 5 96 13
MPPA Dry (oily seeds)* Soybean 0.04 5 92 13
N-Acetyl-Glufosinate High water content + acidic Strawberry 0.02 5 100 6
N-Acetyl-Glufosinate Dry (oily seeds)* Soybean 0.04 5 92 5
N-Acetyl-Glyphosate High water content + acidic Strawberry 0.05 5 97 1
N-Acetyl-Glyphosate Dry (oily seeds)* Soybean 0.1 5 95 5
Phosphonic Acid High water content + acidic Lemon 0.05 5 99 3
Phosphonic Acid Dry (oily seeds)* Sesame 0.2 5 99 7
Bromide (inorg.) High water content + acidic Lemon 5 5 99 2.1
M1.7a: To- Chlorate High water content + acidic Lemon 0.03 5 100 5.6
rus DEA Perchlorate High water content + acidic Lemon 0.01 5 112 2.6
Phosphonic acid High water content + acidic Lemon 0.05 5 100 4.6
Bromide (inorg.) High water content + acidic Lemon 5 5 100 3
Bromide (inorg.) Dry (oily seeds)* Sesame 20 5 81 1
Chlorate High water content + acidic Lemon 0.03 5 115 6
M1.7b:
Chlorate Dry (oily seeds)* Sesame 0.12 5 91 6
APPC Perchlorate High water content + acidic Lemon 0.01 5 104 14
Phosphonic acid High water content + acidic Lemon 0.05 5 100 8
Phosphonic acid Dry (oily seeds)* Sesame 0.2 5 102 18
Chlorate High water content + acidic Lemon 0.03 5 99 4
Chlorate Dry (oily seeds)* Sesame 0.12 5 102 6
Cyanuric Acid High water content + acidic Lemon 0.05 5 99 1
M1.8 Cyanuric Acid Dry (oily seeds)* Sesame 0.2 5 76 2
Maleic Hydrazide Dry (oily seeds)* Sesame 0.2 5 107 16
Perchlorate High water content + acidic Lemon 0.01 5 103 5
Perchlorate Dry (oily seeds)* Sesame 0.04 5 101 6
AMPA Dry (oily seeds)* Soybean 0.2 5 88 5.9
Ethephon Dry (oily seeds)* Soybean 0.02 5 111 11.7
Fosetyl Dry (oily seeds)* Soybean 0.02 5 108 3.9
Glufosinat Dry (oily seeds)* Soybean 0.06 5 102 4.8
Glyphosat Dry (oily seeds)* Soybean 0.1 5 103 2.0
HEPA Dry (oily seeds)* Soybean 0.04 5 99 5.3
M1.9
MPPA Dry (oily seeds)* Soybean 0.08 5 94 6.9
N-Acetyl-Glufosinat Dry (oily seeds)* Soybean 0.08 5 96 11.9
Bromide (inorg.) High water content + acidic Lemon 5 5 98 4.1
Chlorate High water content + acidic Lemon 0.03 5 100 1.3
Perchlorate High water content + acidic Lemon 0.02 5 100 14.1
Phosphonic acid High water content + acidic Lemon 0.05 5 95 2.2
Fosetyl High water content + acidic Strawberry 0.01 5 104 3.2
Glyphosat High water content + acidic Strawberry 0.1 5 101 4.3
M1.10
MPPA High water content + acidic Strawberry 0.04 5 97 2.7
N-Acetyl-Glufosinat High water content + acidic Strawberry 0.04 5 103 4.6

EU Reference Laboratory for pesticides requiring Single Residue Methods (EURL-SRM) 79 of 102
 Spiking Mean
Method Analyte Commodity Group Matrix Level n Recov. RSD
(mg/kg) %
N-Acetyl-Glyphosat High water content + acidic Strawberry 0.05 5 106 1.8
Chlorate High water content + acidic Lemon 0.03 5 98 8.1
Perchlorate High water content + acidic Lemon 0.01 5 106 8.3
Phosphonic acid*** High water content + acidic Lemon 0.05 5 101 5.3
Trifluoro-acetic acid High water content + acidic Lemon 0.025 5 102 9.3
Amitrole High water content + acidic Orange 0.01 6 107 5
Amitrole Dry (cereals) Barley 0.01 5 111 2
Amitrole High water content Apple 0.01 7 93 11
Amitrole High water content Cucumber 0.01 6 92 4
Chloridazon, Desphenyl- High water content + acidic Currant 0.02 5 99 4
Chloridazon, Desphenyl- Other Swine meat 0.02 5 94 4
Chloridazon, Desphenyl- High water content Lettuce varieties 0.02 5 97 3
Chlormequat High water content + acidic Grapes 0.01 6 93 10
Chlormequat High water content + acidic Grapes 0.2 5 102 1
Chlormequat Dry (cereals) Barley 0.01 5 97 5
Chlormequat Dry (cereals) Wheat flour 0.1 5 97 5
Chlormequat High oil content, wet (oily fruits) Avocado 0.01 7 103 8
Chlormequat High water content Apple 0.01 6 102 6
Chlormequat High water content Cucumber 0.01 6 103 4
Chlormequat High water content Potato 0.01 6 99 4
Cyromazine High water content + acidic Grapes 0.01 6 101 4
Cyromazine Dry (cereals) Barley 0.01 5 109 6
Cyromazine High oil content, wet (oily fruits) Avocado 0.01 7 107 2
Cyromazine High water content Apple 0.01 6 102 8
Cyromazine High water content Potato 0.01 6 103 8
Daminozide High water content + acidic Orange 0.01 3 113 1
Daminozide Dry (cereals) Barley 0.01 5 113 6
Daminozide High oil content, wet (oily fruits) Avocado 0.01 6 112 10
Daminozide High water content Apple 0.01 6 100 9
M4.1 Daminozide High water content Cucumber 0.01 6 93 12
Diethanolamine High water content + acidic Mandarine 0.1 5 103 1
Diethanolamine High water content Apple 0.1 5 103 3
Diethanolamine High water content Mango 0.1 6 101 14
Difenzoquat Dry (cereals) Barley 0.01 5 99 8
Difenzoquat High water content Apple 0.01 6 99 11
Diquat Dry (cereals) Barley 0.01 10 103 7
Diquat High water content Apple 0.01 5 107 4
ETU Dry (cereals) Barley 0.01 5 96 10
ETU High water content Apple 0.01 7 102 9
Melamine High water content + acidic Grapes 0.01 6 87 13
Melamine High oil content, dry (oily seeds, nuts) Bean, Soya 0.02 3 109 5
Melamine High oil content, wet (oily fruits) Avocado 0.01 7 108 6
Mepiquat High water content + acidic Grapes 0.01 6 95 5
Mepiquat High water content + acidic Orange 0.01 6 101 9
Mepiquat Dry (cereals) Barley 0.01 5 108 3
Mepiquat Dry (cereals) Wheat flour 0.1 5 102 5
Mepiquat High oil content, wet (oily fruits) Avocado 0.01 6 104 5
Mepiquat High water content Apple 0.01 6 98 7
Mepiquat High water content Cucumber 0.01 6 107 6
Mepiquat High water content Potato 0.01 6 99 3
Morpholine High water content + acidic Mandarine 0.1 5 95 7
Morpholine High water content Apple 0.1 5 94 3
Morpholine High water content Mango 0.1 5 95 2
Nereistoxin High water content + acidic Grapes 0.01 6 93 9

EU Reference Laboratory for pesticides requiring Single Residue Methods (EURL-SRM) 80 of 102
 Spiking Mean
Method Analyte Commodity Group Matrix Level n Recov. RSD
(mg/kg) %
Nereistoxin Dry (cereals) Barley 0.01 5 104 13
Nereistoxin High oil content, wet (oily fruits) Avocado 0.01 5 103 6
Nereistoxin High water content Apple 0.01 6 118 2
Nereistoxin High water content Potato 0.01 6 113 9
Paraquat Dry (cereals) Barley 0.01 10 106 15
Paraquat High oil content, wet (oily fruits) Avocado 0.05 5 83 10
Paraquat High water content Apple 0.01 5 106 5
Paraquat High water content Potato 0.01 10 103 13
PTU Dry (cereals) Barley 0.01 5 113 3
Triethanolamine High water content + acidic Mandarine 0.1 5 112 4
Triethanolamine High water content Apple 0.1 5 108 6
Triethanolamine High water content Mango 0.1 5 120 5
Triethanolamine High water content Pear 0.1 3 107 11
Trimesium High water content + acidic Grapes 0.01 6 93 7
Trimesium Dry (cereals) Barley 0.01 5 118 3
Trimesium Dry (cereals) Wheat flour 0.1 5 105 2
Trimesium High oil content, wet (oily fruits) Avocado 0.01 7 93 14
Trimesium High water content Potato 0.01 6 84 5
Aminocyclopyrachlor High water content Apple 0.01 5 110 5
Aminocyclopyrachlor Dry (cereals) Oat 0.02 5 106 7
Aminocyclopyrachlor High water content Cucumber 0.01 5 101 6
Aminocyclopyrachlor High water content + acidic Lemon 0.01 5 112 9
Aminocyclopyrachlor High water content Mint 0.01 5 108 7
Aminocyclopyrachlor High sugar and low water content Honey 0.05 5 97 7
Amitole High water content Apple 0.01 5 99 6
Amitole Dry (cereals) Oat 0.02 5 117 4
Amitole High water content + acidic Raspberry 0.01 5 120 5
Amitole High water content Cucumber 0.01 5 104 6
Amitole High water content + acidic Lemon 0.01 5 96 4
Amitrole High sugar and low water content Honey 0.02 5 113 8
Chlormequat High water content Cucumber 0.01 5 106 3
Chlormequat High water content + acidic Lemon 0.01 5 103 2
Chlormequat High water content Mint 0.01 5 102 1
Chlormequat High water content Apple 0.01 5 101 2
Chlormequat Dry (cereals) Oat 0.02 5 119 2
Chlormequat High sugar and low water content Honey 0.02 5 104 4
M4.2
Chloridazon-desphenyl High water content Cucumber 0.01 5 104 3
Chloridazon-desphenyl High water content + acidic Lemon 0.01 5 108 8
Chloridazon-desphenyl High water content Mint 0.01 5 108 10
Chloridazon-desphenyl High water content Apple 0.01 5 97 5
Chloridazon-desphenyl Dry (cereals) Oat 0.02 5 113 9
Cyromazine High water content Cucumber 0.01 5 101 5
Cyromazine High water content + acidic Lemon 0.01 5 95 3
Cyromazine High water content Mint 0.01 5 100 5
Cyromazine High water content Apple 0.01 5 98 3
Cyromazine Dry (cereals) Oat 0.02 5 114 4
Cyromazine High sugar and low water content Honey 0.02 5 107 3
Daminozide High water content Apple 0.01 5 101 2
Daminozide Dry (cereals) Oat 0.02 5 116 2
Daminozide High water content + acidic Raspberry 0.01 5 119 3
Daminozide High water content Cucumber 0.01 5 103 6
Daminozide High water content + acidic Lemon 0.01 5 102 1
Daminozide High water content Mint 0.01 5 104 3
Daminozide High sugar and low water content Honey 0.02 5 105 3

EU Reference Laboratory for pesticides requiring Single Residue Methods (EURL-SRM) 81 of 102
 Spiking Mean
Method Analyte Commodity Group Matrix Level n Recov. RSD
(mg/kg) %
Diethanolamin Dry (cereals) Oat 0.02 5 106 14
Difenzoquat High water content Cucumber 0.01 5 105 1
Difenzoquat High water content + acidic Lemon 0.01 5 105 3
Difenzoquat High water content Apple 0.01 5 105 4
Difenzoquat Dry (cereals) Oat 0.02 5 97 6
Difenzoquat High sugar and low water content Honey 0.02 5 98 1
ETU High water content Cucumber 0.01 5 87 10
ETU High water content + acidic Lemon 0.01 5 104 11
ETU Dry (cereals) Oat 0.02 5 103 14
ETU High water content + acidic Raspberry 0.01 5 109 5
ETU High sugar and low water content Honey 0.05 5 103 10
Matrine High sugar and low water content Honey 0.02 5 101 2
Melamine High water content Cucumber 0.01 5 90 13
Melamine High water content + acidic Lemon 0.01 5 91 11
Melamine High water content Mint 0.01 5 93 11
Melamine High water content Apple 0.01 5 97 8
Melamine Dry (cereals) Oat 0.02 5 117 8
Melamine High sugar and low water content Honey 0.02 5 100 8
Mepiquat High water content Cucumber 0.01 5 102 3
Mepiquat High water content + acidic Lemon 0.01 5 104 4
Mepiquat High water content Mint 0.01 5 96 3
Mepiquat High water content Apple 0.01 5 104 3
Mepiquat Dry (cereals) Oat 0.02 5 114 5
Mepiquat High sugar and low water content Honey 0.02 5 99 3
Mepiquat, 4-Hydroxy High water content Cucumber 0.01 5 108 2
Mepiquat, 4-Hydroxy High water content + acidic Lemon 0.01 5 107 4
Mepiquat, 4-Hydroxy High water content Mint 0.01 5 105 2
Mepiquat, 4-Hydroxy High water content Apple 0.01 5 110 2
Mepiquat, 4-Hydroxy Dry (cereals) Oat 0.02 5 112 3
Mepiquat, 4-Hydroxy High sugar and low water content Honey 0.02 5 101 2
Morpholine High water content Cucumber 0.01 5 97 10
Morpholine High water content + acidic Lemon 0.01 5 92 9
Morpholine High water content Apple 0.01 5 84 15
Morpholine High water content + acidic Raspberry 0.01 5 84 18
Morpholine High sugar and low water content Honey 0.02 5 101 4
Nereistoxin High water content Cucumber 0.01 5 94 8
Nereistoxin High water content + acidic Lemon 0.01 5 99 2
Nereistoxin High water content Mint 0.01 5 90 3
Nereistoxin High water content Apple 0.01 5 101 3
Nereistoxin Dry (cereals) Oat 0.02 5 113 2
Nereistoxin High water content + acidic Raspberry 0.01 5 114 2
Nereistoxin High sugar and low water content Honey 0.02 5 106 2
Nicotine High water content Apple 0.01 5 90 3
Nicotine High water content Lamb’s lettuce 0.01 5 95 8
Nicotine High water content + acidic Orange 0.01 5 104 4
Nicotine High water content + acidic Grape 0.01 5 99 2
Nicotine Dry (cereals) Whole flour (spelt) 0.01 5 101 4
Nicotine High sugar and low water content Honey 0.02 5 108 3
Oxymatrine High sugar and low water content Honey 0.02 5 106 6
Propamocarb High water content Cucumber 0.01 5 99 2
Propamocarb High water content + acidic Lemon 0.01 5 84 6
Propamocarb High water content Mint 0.01 5 102 2
Propamocarb High water content Apple 0.01 5 102 2

EU Reference Laboratory for pesticides requiring Single Residue Methods (EURL-SRM) 82 of 102
 Spiking Mean
Method Analyte Commodity Group Matrix Level n Recov. RSD
(mg/kg) %
Propamocarb Dry (cereals) Oat 0.02 5 113 3
Propamocarb High sugar and low water content Honey 0.02 5 102 3
Propamocarb-N-Desmethyl High water content Apple 0.01 5 113 3
Propamocarb-N-Desmethyl Dry (cereals) Oat 0.02 5 94 3
Propamocarb-N-Desmethyl High water content Cucumber 0.01 5 106 2
Propamocarb-N-Desmethyl High sugar and low water content Honey 0.02 5 105 3
Propamocarb-N-Oxide High water content Cucumber 0.01 5 102 2
Propamocarb-N-Oxide High water content + acidic Lemon 0.01 5 109 4
Propamocarb-N-Oxide High water content Mint 0.01 5 111 3
Propamocarb-N-Oxide High water content Apple 0.01 5 110 4
Propamocarb-N-Oxide High sugar and low water content Honey 0.02 5 101 3
PTU High water content Cucumber 0.01 5 97 4
PTU High water content + acidic Lemon 0.01 5 100 5
PTU Dry (cereals) Oat 0.02 5 113 6
PTU High water content + acidic Raspberry 0.01 5 115 6
PTU High sugar and low water content Honey 0.02 5 116 3
Triethanolamine High water content Apple 0.01 5 73 15
Triethanolamine High water content + acidic Raspberry 0.01 5 106 4
Trimethylsulfonium High water content Cucumber 0.01 5 119 3
Trimethylsulfonium High water content + acidic Lemon 0.01 5 110 2
Trimethylsulfonium High water content Mint 0.01 5 116 3
Trimethylsulfonium High water content Apple 0.01 5 119 2
Trimethylsulfonium High sugar and low water content Honey 0.02 5 103 4
Diquat** Dry (oily seeds)** Sesame 0.05 5 105 7
Paraquat** Dry (oily seeds)** Sesame 0.02 5 100 10
M5 See un der http://www.crl-pesticides.eu/library/docs/srm/meth_ChlormequatMepiquat_CrlSrm.pdf
Kasugamycin High water content Apple 0.01 5 98 4
M6
Streptomycin High water content Apple 0.01 10 106 9
Morpholine High water content Apple 0.1 5 94 3
Morpholine High water content Mango 0.1 5 95 2
Morpholine High water content + acidic Mandarin 0.1 5 95 7
Diethanolamine High water content Apple 0.1 5 103 3
M7 Diethanolamine High water content Mango 0.1 5 107 1
Diethanolamine High water content + acidic Mandarin 0.1 5 103 1
Triethanolamine High water content Apple 0.1 5 108 6
Triethanolamine High water content Mango 0.1 5 118 3
Triethanolamine High water content + acidic Mandarin 0.1 5 112 4
1,2,4-Triazole High water content Cucumber 0.1 5 85 12
1,2,4-Triazole High water content Potatoes 0.01 5 100 8
1,2,4-Triazole High acid content Orange 0.1 5 94 20
1,2,4-Triazole High acid content Grapes 0.01 5 90 10
1,2,4-Triazole Dry (cereals) Rice 0.2 5 86 3
1,2,4-Triazole Dry (cereals) Barley 0.1 5 104 6
1,2,4-Triazole Fatty, wet Avocado 0.01 5 94 10
Triazole-acetic acid High water content Cucumber 0.01 5 100 2
M8
Triazole-acetic acid High water content Potatoes 0.01 5 96 6
Triazole-acetic acid High acid content Orange 0.01 5 104 9
Triazole-acetic acid High acid content Grapes 0.01 5 95 4
Triazole-acetic acid Dry (cereals) Rice 0.02 5 74 5
Triazole-acetic acid Dry (cereals) Barley 0.01 5 109 5
Triazole-acetic acid Fatty, wet Avocado 0.01 5 97 2
Triazole-alanine High water content Cucumber 0.01 5 100 19
Triazole-alanine High water content Potatoes 0.01 5 102 18

EU Reference Laboratory for pesticides requiring Single Residue Methods (EURL-SRM) 83 of 102
 Spiking Mean
Method Analyte Commodity Group Matrix Level n Recov. RSD
(mg/kg) %
Triazole-alanine High acid content Orange 0.01 5 98 5
Triazole-alanine High acid content Grapes 0.01 5 95 11
Triazole-alanine Dry (cereals) Rice 0.02 5 88 4
Triazole-alanine Dry (cereals) Barley 0.02 5 119 9
Triazole-alanine Fatty, wet Avocado 0.01 5 91 13
Triazole-lactic acid High water content Cucumber 0.01 5 107 3
Triazole-lactic acid High water content Potatoes 0.01 5 102 6
Triazole-lactic acid High acid content Orange 0.01 5 111 12
Triazole-lactic acid High acid content Grapes 0.01 5 100 5
Triazole-lactic acid Dry (cereals) Rice 0.02 5 71 4
Triazole-lactic acid Dry (cereals) Barley 0.02 5 99 4
Triazole-lactic acid Fatty, wet Avocado 0.01 5 97 4
See also: http://www.eurl-pesticides.eu/userfiles/file/EurlSRM/EurlSrm_meth_TriazoleDerivativeMetabolites.pdf
Difluoroacetic acid High water content Apple 0.01 5 94 7
Difluoroacetic acid Fatty, wet (oily fruits) Avocado 0.02 5 103 8
Difluoroacetic acid High water content Cucumber 0.01 5 70 2
Difluoroacetic acid Dry (cereals) Flour 0.02 5 77 9
Difluoroacetic acid High acid content Grapes 0.01 5 80 5
Difluoroacetic acid High acid content Grapes 0.01 5 106 15
Difluoroacetic acid High acid content Orange 0.01 5 109 11
Difluoroacetic acid Dry (cereals) Rice 0.02 5 80 3
M9
Trifluoroacetic acid High water content Apple 0.01 5 93 6
Trifluoroacetic acid Fatty, wet (oily fruits) Avocado 0.04 5 77 4
Trifluoroacetic acid Dry (cereals) Flour 0.04 5 84 6
Trifluoroacetic acid High acid content Gooseberry 0.02 5 128 11
Trifluoroacetic acid High acid content Grapes 0.01 5 87 14
Trifluoroacetic acid High acid content Orange 0.01 5 107 3
Trifluoroacetic acid Dry (cereals) Rice 0.04 5 72 4
Trifluoroacetic acid High water content Tomato 0.02 5 76 15
M10
AMPA High water content Cucumber 0.02 5 97 5.9
Ethephon High water content Cucumber 0.02 5 90 5.2
Fosetyl High water content Cucumber 0.02 5 101 3.1
Glufosinat High water content Cucumber 0.02 5 99 4.6
Glyphosat High water content Cucumber 0.02 5 96 2.3
HEPA High water content Cucumber 0.02 5 101 14.5
MPPA High water content Cucumber 0.02 5 103 4.0
M11
N-Acetyl-Glufosinat High water content Cucumber 0.02 5 103 3.0
N-Acetyl-Glyphosat High water content Cucumber 0.02 5 104 2.7
Bromide (inorg.) High water content + acidic Lemon 10 5 110 11.3
Chlorate High water content + acidic Lemon 0.06 5 96 2.7
Perchlorate High water content + acidic Lemon 0.02 5 97 2.0
Phosphonic acid High water content + acidic Lemon 0.1 5 100 4.1
TFA High water content + acidic Lemon 0.05 5 100 1.5
*The extract was prepared according to QuPPe PO V10 or newer versions, so EDTA solution was used during extraction (for oily
seeds, nuts pulses and cereals). All other data was generated without using EDTA solution during extraction.
**analysed with Waters Xevo TQ-Sµ
*** spiked separately from co-eluting Fosetyl to avoid interference that affects quantification

EU Reference Laboratory for pesticides requiring Single Residue Methods (EURL-SRM) 84 of 102
Table 41: Validation data deriving from two QuPPe interlaboratory validation studies organized by the EURL-SRM, Round 1.
Solvent + IL-IS Calibration Matrix Matched Calibration Matrix + IL-IS Calibration
Level Mean Recovery RSD Mean Recovery RSD No. Mean Recovery RSD No.
Matrix (mg/kg) (%) (± %) No. Labs (%) (± %) Labs (%) (± %) Labs
Cyromazine
0.01 102 4 7 89 5 9 100 5 9
Potatoes 0.05 115 4 8 91 4 9 102 3 9
0.2 100 3 9 92 2 9 102 3 9
0.01 101 4 8 96 6 10 103 4 10
Grapes 0.05 99 3 8 96 3 9 103 3 10
0.2 97 2 8 96 3 10 102 2 10
0.01 119 7 6 85 13 7 102 9 7
Rye flour 0.05 104 6 8 85 5 9 100 7 9
0.2 97 4 8 86 4 9 98 3 9
0.01 100 4 6 92 4 8 104 4 7
Avocados 0.05 102 2 9 93 3 10 102 3 9
0.2 99 2 8 86 4 11 102 2 10
Daminozide
0.01 107 2 3 100 6 8 89 4 8
Potatoes 0.05 99 3 7 97 4 10 97 4 9
0.2 93 4 7 99 3 10 94 4 10
0.01 102 6 4 97 5 10 97 7 8
Grapes 0.05 97 2 8 97 3 11 97 4 11
0.2 97 2 8 97 3 11 97 4 11
0.01 107 11 5 105 6 5 97 5 5
Rye flour 0.05 90 5 7 113 5 7 106 4 8
0.2 89 4 7 109 3 7 101 4 8
0.01 110 2 5 100 7 7 95 6 6
Avocados 0.05 99 4 8 104 4 10 99 3 9
0.2 95 2 8 99 3 10 99 2 9
Chlormequat
0.01 99 3 9 98 4 10 101 3 10
Potatoes 0.05 99 3 9 98 3 10 102 3 10
0.2 105 4 10 101 3 10 102 4 10
0.01 97 3 10 99 4 10 104 3 10
Grapes 0.05 99 3 10 95 2 10 101 2 11
0.2 101 2 10 97 2 11 103 2 11
0.01 109 5 9 105 7 10 102 4 10
Rye flour 0.05 103 5 9 101 4 10 106 5 10
0.2 102 3 9 103 3 10 104 3 10
0.01 97 3 10 97 4 10 103 3 10
Avocados 0.05 99 3 9 96 3 10 102 3 10
0.2 101 2 10 91 3 11 103 2 10
Trimesium
0.01 87 5 9 98 4 10 104 4 10
Potatoes 0.05 92 3 7 97 4 10 104 4 10
0.2 93 2 8 97 3 10 101 3 10
0.01 93 3 10 95 3 11 101 2 11
Grapes 0.05 96 2 9 95 2 10 100 2 11
0.2 97 2 9 95 3 11 102 2 11
0.01 126 6 9 102 7 10 107 5 10
Rye flour 0.05 122 5 9 100 4 10 106 4 10
0.2 120 4 9 101 3 10 101 4 10
0.01 93 4 9 89 4 10 98 4 10
Avocados 0.05 92 3 9 91 4 10 101 3 10
0.2 93 3 10 88 4 10 99 3 9
Nereistoxin
Potatoes 0.01 128 6 5 91 8 6 105 9 6

EU Reference Laboratory for pesticides requiring Single Residue Methods (EURL-SRM) 85 of 102
 Solvent + IL-IS Calibration Matrix Matched Calibration Matrix + IL-IS Calibration
Level Mean Recovery RSD Mean Recovery RSD No. Mean Recovery RSD No.
Matrix (mg/kg) (%) (± %) No. Labs (%) (± %) Labs (%) (± %) Labs
0.05 110 7 5 91 5 8 98 5 7
0.2 111 3 8 94 3 9 100 3 9
0.01 109 7 4 94 7 9 97 6 9
Grapes 0.05 107 5 8 96 5 10 100 6 11
0.2 115 3 9 94 4 11 100 4 11
0.01 184 8 6 93 9 7 99 7 7
Rye flour 0.05 137 6 8 89 7 9 104 6 9
0.2 131 4 8 90 4 9 102 5 9
0.01 100 6 3 84 8 5 102 7 5
Avocados 0.05 108 2 7 84 4 7 102 3 8
0.2 108 3 7 80 4 9 106 5 9
Melamine
0.01 121 4 4 78 6 7 103 7 7
Potatoes 0.05 105 4 6 73 5 9 101 5 9
0.2 97 3 7 81 4 9 104 5 9
0.01 102 6 5 91 7 7 102 7 8
Grapes 0.05 95 5 8 94 3 9 101 4 10
0.2 99 3 9 96 4 10 102 2 10
0.01 196 5 5 71 7 6 148 13 7
Rye flour 0.05 109 5 7 63 14 8 115 8 8
0.2 106 5 8 60 7 9 105 5 9
0.01 120 3 4 88 6 5 109 4 4
0.05 102 6 7 90 4 8 101 3 9
Avocados
0.2 96 5 9 82 4 10 102 4 9
0.2 114 14 6 104 7 6
Perchlorate
0.01 99 9.3 11
Carrot 0.02 99 11.4 11
0.2 96 10.5 10
0.01 105 6.4 11
Lemon 0.02 104 7.4 11
0.2 102 5.4 12
0.01 100 8.6 11
Rye flour 0.02 102 8.7 12
0.2 101 9.8 14
0.01 104 11.5 10
Avocado 0.02 105 10.2 11
0.2 102 8.1 11
Chlorate
0.01 105 11.6 12
Carrot 0.02 103 8.5 11
0.2 100 4.9 10
0.01 99 13.4 12
Lemon 0.02 104 18.1 11
0.2 100 5.2 12
0.01 108 13.3 12
Rye flour 0.02 105 15.9 14
0.2 101 6.5 13
0.01 99 4.1 8
Avocado 0.02 102 5.5 8
0.2 106 8.7 11
Phosphonic acid
0.01 100 18.8 6
Carrot
0.02 106 7.2 7

EU Reference Laboratory for pesticides requiring Single Residue Methods (EURL-SRM) 86 of 102
 Solvent + IL-IS Calibration Matrix Matched Calibration Matrix + IL-IS Calibration
Level Mean Recovery RSD Mean Recovery RSD No. Mean Recovery RSD No.
Matrix (mg/kg) (%) (± %) No. Labs (%) (± %) Labs (%) (± %) Labs
0.2 108 14.3 6
0.01 104 8.0 5
Lemon 0.02 101 10.1 5
0.2 98 8.7 10
0.01 104 20.3 5
Rye flour 0.02 103 18.1 7
0.2 105 14.1 11
0.01 99 10.2 7
Avocado 0.02 105 12.1 8
0.2 102 7.9 9
Bromide
0.01 103 11.2 7
Carrot 0.02 106 8.7 8
0.2 115 17.0 7
0.01 99 10.5 10
Lemon 0.02 94 18.9 10
0.2 100 10.6 10
0.01 82 10.9 9
Rye flour 0.02 83 10.3 10
0.2 85 34.8 11
0.01 102 15.9 10
Avocado 0.02 102 10.5 11
0.2 106 9.6 11

8. References
Anastassiades, M and Mack, D (2008); New Developments in the Analysis of Pesticides Typically not Covered by Mul-
tiresidue Methods; European Pesticide Residue Workshop, EPRW 2008, Berlin, oral presentation O1, Book of Abstracts

Kolberg DI, Mack D, Anastassiades M, Hetmanski MT, Fussell RJ, Meijer T, Mol HG. Anal Bioanal Chem. 404(8):2465-
74 (2012); Development and independent laboratory validation of a simple method for the determination of paraquat
and diquat in potato, cereals and pulses

Alder L. and Startin J. R. (2005); Determination of Chlormequat and Mepiquat in Foods by Liquid Chromatog-
raphy/Mass Spectrometry or Liquid Chromatography/Tandem Mass Spectrometry: Interlaboratory Study; Journal of
AOAC International Vol. 88, No. 6: 1762-1776

Vahl, M. et al. (1998); Analysis of Chlormequat residues in grain using liquid chromatography-mass spectrometry (LC-
MS/MS); Fresenius J Anal Chem 361:817-820

EU Reference Laboratory for pesticides requiring Single Residue Methods (EURL-SRM) 87 of 102
9. ANNEX
Table 42: Conversion factors between typical purchased standards and target analytes (3.18).
MW Conv. Factor
Compound MW [g/mol] Compound as sold Inverse CF
[g/mol] (CF)
Bialaphos 323.3 Bialaphos-sodium 345.3 0.94 1.07
Bromate (anion) 127.9 Potassium bromate 167.0 0.77 1.31
Bromide (anion) 79.9 Potassium bromide 119.0 0.67 1.49
Chlorate (anion) 83.5 Chlorate-sodium 106.4 0.78 1.27
Chlormequat (cation)* 122.6 Chlormequat-chloride* 158.1 0.78 1.29
Chlormequat-D4 (cation) 126.6 Chlormequat-D4-chloride 162.1 0.78 1.28
Difenzoquat (cation) 249.3 Difenzoquat-methylsulfate 360.4 0.69 1.45
Difluoroacetic acid-13C2 96.0 Sodium difluoroacetate-13C2 120.0 0.80 1.25
Dihydrostreptomycin 583.6 Dihydrostreptomycin-sesquisulfate 730.7 0.80 1.25
Diquat (dication) 184.2 Diquat-dibromide-monohydrate 362.1 0.51 1.97
Diquat-D4 (dication) 188.2 Diquat-D4-dibromide-monohydrate 366.1 0.51 1.95
Fosetyl (neutral = protonated) 110.05x3 = 330.14** Fosetyl-Al 354.10 0.932 1.07
115.08x3=345.23** Fosetyl-Al D15 369.19 0.935 1.07
Fosetyl-D5 (neutral = protonated)
115.08 Fosetyl-D5-sodium 137.0 0.84 1.19
Glufosinate 181.1 Glufosinate-ammonium 198.2 0.91 1.09
Glufosinate-D3 184.1 Glufosinate-D3-hydrochloride 220.6 0.83 1.20
Kasugamycin 379.4 Kasugamycin-hydrochloride-monohydrate 433.8 0.87 1.14
Mepiquat (cation)* 114.2 Mepiquat-chloride* 149.7 0.76 1.31
Mepiquat-D3 (cation) 117.2 Mepiquat-D3-iodide 244.1 0.48 2.08
Mepiquat-4-hydroxy 130.2 Mepiquat-4-hydroxy-chloride 165.7 0.79 1.27
N,N’-Dimethylhydrazine-D6 66.1 Dimethylhydrazine-D6–hydrochloride 102.6 0.64 1.55
N-Acetyl-Glufosinate 223.2 N-Acetyl-Glufosinate-disodium 267.2 0.84 1.20
N-Acetyl-Glufosinate-D3 226.2 N-Acetyl-Glufosinate-D3-disodium 270.2 0.84 1.19
Nereistoxin 149.3 Nereistoxin-oxalate 239.3 0.62 1.60
Nereistoxin-D6 155.3 Nereistoxin-D6-oxalate 245.3 0.63 1.58
Nicotine 162.2 Nicotine hemisulfate 422.5*** 0.77 1.30
Paraquat (dication) 186.3 Paraquat-dichloride 257.2 0.72 1.38
Paraquat-D6 (dication) 192.3 Paraquat-D6-diiodide 446.1 0.43 2.32
Propamocarb-N-oxide 204.3 Propamocarb-N-oxide hydrochloride 240.7 0.85 1.17
Streptomycin 581.6 Streptomycin-sesquisulfate 728.7 0.80 1.25
Trifluoroacetic acid -13C2 114.0 Sodium trifluoroacetate-13C2 138.0 0.83 1.21
Trimethylsulfonium (cation) 77.2 Trimethylsulfonium-iodide 204.1 0.38 2.64
Trimethylsulfonium-D9 (cation) 86.2 Trimethylsulfonium-D9-iodide 213.1 0.40 2.47
* Attention: The EU – Maximum Residue Levels are now expressed as the respective chloride salts. Thus no conversion of the
chloride to the cation is needed.
** Taking into account that 1 mol fosetyl-Al (MW 354.10) contains 3 mols of fosetyl anion (MW 109.04x3=327,12) leading to 3
mols fosetyl acid (MW 110.05x3=330,14). For the ILIS the following numbers apply 1 mol fosetyl-Al D15 (MW 369.19) contains 3
mols of fosetyl D5 anion (MW 114.07x3=342.21) leading to 3 mols fosetyl D5 acid (MW 115.08*3=345.23)
*** MW refers to the following formula C10H14N2)2 · H2SO4 which entails two nicotine molecules

EU Reference Laboratory for pesticides requiring Single Residue Methods (EURL-SRM) 88 of 102
Table 43: Exemplary concentrations of pesticide stock and working solutions (3.18 and 3.19), (solvent proposals also apply to IL-IS,
see 3.21, 3.22, 3.23). Prefereably use plastic vials (e.g. PP) as many of the compounds tend to interact with glass surfaces.
Stock Solution (exemplary) Working Solutions including mixtures (exemplary)
Compound
Solvent used to prepare [mg/mL] Solvent used to prepare [µg/mL]
Aminocyclopyrachlor MeOH 1 MeOH 10 / 1 / 0.1
Amitrole MeOH 1 MeOH 10 / 1 / 0.1
AMPA 10 % ACN in water 1 10 % ACN in water 10 / 1 / 0.1
Bromate Water/MeOH (50:50) 1 MeOH 10 / 1 / 0.1 / 0.01
Bromide MeOH 1 MeOH 10 / 1 / 0.1 / 0.01
Chlorate 10 % ACN in water 1 MeOH 10 / 1 / 0.1 / 0.01
Chloridazon-desphenyl MeOH 1 MeOH 10 / 1 / 0.1
Chlormequat MeOH 1 MeOH 10 / 1 / 0.1
Cyanuric acid MeOH 1 10 % ACN in water 10 / 1 / 0.1
Cyromazine MeOH 1 MeOH 10 / 1 / 0.1
Daminozide MeOH 1 MeOH 10 / 1 / 0.1
Diethanolamine ACN 1 MeOH 10 / 1 / 0.1
Difenzoquat ACN 1 MeOH 10 / 1 / 0.1
Difluoroacetic acid ACN with 5% water 1 ACN with 5% water 10 / 1/ 0.1
Diquat** 10 % ACN in water 1 10 % ACN in water 10 / 1 / 0.1
Ethephon 10 % ACN in water + 0,1 % HCl 1 10 % ACN in water +0,1 % HCl 10 / 1 / 0.1
ETU MeOH 1 MeOH 10 / 1 / 0.1
Fosetyl 10 % ACN in water 0.1 10 % ACN in water 10 / 1 / 0.1
Glufosinate 10 % ACN in water 1 10 % ACN in water 10 / 1 / 0.1
Glyphosate* 10 % ACN in water 1 10 % ACN in water 10 / 1 / 0.1
HEPA 10 % ACN in water 1 10 % ACN in water 10 / 1 / 0.1
Kasugamycin MeOH 1 MeOH 10 / 1 / 0.1
Matrine ACN 1 ACN 10 / 1 / 0.1
Maleic Hydrazide MeOH 1 10 % ACN in water 10 / 1 / 0.1
Melamine MeOH:water (90:10) 1 MeOH 10 / 1 / 0.1
Mepiquat MeOH 1 MeOH 10 / 1 / 0.1
Mepiquat-4-hydroxy MeOH 1 MeOH 10 / 1 / 0.1
Morpholine MeOH 1 MeOH 10 / 1 / 0.1
MPPA 10 % ACN in water 1 10 % ACN in water 10 / 1 / 0.1
N,N-Dimethylhydrazine MeOH 1 MeOH 10 / 1 / 0.1
N-Acetyl- AMPA 10 % ACN in water 1 10 % ACN in water 10 / 1 / 0.1
N-Acetyl-Glufosinate 10 % ACN in water 1 10 % ACN in water 10 / 1 / 0.1
N-Acetyl-Glyphosate 10 % ACN in water 1 10 % ACN in water 10 / 1 / 0.1
Nereistoxin MeOH / water (3:1) 1 MeOH 10 / 1 / 0.1
Nicotine* ACN 1 ACN 1 / 0.1
Oxymatrine ACN 1 ACN 10 / 1 / 0.1
Paraquat** 10 % ACN in water 1 10 % ACN in water 10 / 1 / 0.1
Perchlorate 10 % ACN in water 1 MeOH 10 / 1 / 0.1 / 0.01
Phosphonic acid* 10 % ACN in water 1 ACN*** 10 / 1 / 0.1 / 0.01
Propamocarb ACN 1 MeOH 10 / 1 / 0.1
Propamocarb-N-desmethyl ACN:Acetone (1 mL acetone to initially dissolve) 1 MeOH 10 / 1 / 0.1
Propamocarb-N-oxide ACN 1 MeOH 10 / 1 / 0.1
PTU MeOH 1 MeOH 10 / 1 / 0.1
Streptomycin* Water / MeOH (1:1) 0,5 MeOH 10 / 1 / 0.1
Triazole MeOH 1 MeOH 10 / 1 / 0.1
Triazole-lactic acid MeOH 1 MeOH 10 / 1 / 0.1
Triazole-acetic acid MeOH 1 MeOH 10 / 1 / 0.1
Triazole-alanine MeOH/Water (1:3) 1 MeOH 10 / 1 / 0.1
Triethanolamine MeOH 1 MeOH 10 / 1 / 0.1
Trifluoroacetic acid ACN with 5% water 1 ACN with 5% water 10 / 1/ 0.1
Trimethylsulfonium MeOH 1 MeOH 10 / 1 / 0.1
* Use plastic vessels and stoppers for compounds that tend to interact with glass surfaces
** Use plastic vials and protect solutions from light exposure
*** Pure water (18O-H2O for the IL-IS) is also suitable for the working solution. 10% ACN will reduce growth of microorganisms
MeOH: Methanol; ACN: Acetonitrile; FA: Formic acid

EU Reference Laboratory for pesticides requiring Single Residue Methods (EURL-SRM) 89 of 102
Table 44: Exemplary providers of isotopically labelled internal standards 3.20.
Conc. Amount Prices in €-cent (see diclaimer)
Isotope labelled compound Source Article-No.
(µg/mL) per unit 1 unit 2 µg* 0.1 µg**
15N 1 XA10240100ME 100 1.1 mL 165 € 300 c 15 c
15N, 13C 1 XA10240110AL 100 1.1 mL 332 € 604 c 30 c
Amitrole 15N , 13C
7 A633382 10 mg 1.500 € 30 c 1.5 c
2 2
14 LBS9AZ3L3293 1000 1.1 mL 1.380 € 251 c 12.5 c
15N , 13C 8 C4313 10 mg
4 2

1 CIL-CDNLM-6786-1.2 100 1.2 mL 464 € 773 c 39 c

13C, 15N, D 5 CDNLM-6786-1.2 100 1.2 mL 464 € 773 c 39 c
2

10 CDNLM-6786-1.2 100 1.2 mL 465 € 775 c 39 c

AMPA
7 A617342 10 mg 1.690 € 34 c 1.7 c
13C, 15N 1 XA10205100WA 100 1.1 mL 332 € 604 c 30 c
14 LBS9AZ3L1603 1000 1.1 mL 1.380 € 251 c 12.5 c
Bromate-18O3 1 CIL-OLM-8283-18O-1.2 100 1.2 mL 406 € 677 c 34 c
7 C292762 No indication 1 mL 4.300 €
Chlorate-18O3
12*** - 200 5 mL 250 € 50 c 2.5 c
13 8399.4-10MG 10 mg 1.380 € 28 c 1.4 c
14 LBS9G3L3294 1000 1.1 mL 1.380 € 251 c 12.5 c
Chloridazon-desphenyl-15N2 3 679027 5 mg 790 € 31.6 c 1.6 c
11 sc-218161 1 mg 326 € 65.2 c 3.3 c
18 20273 10 mg 810 € 16.2 c 0.8 c
Chloridazon-methyl-desphenyl-D3 18 20229 10 mg 695 € 13.9 c 0.7 c
1 X 11340100DO 100 10 mL 286 € 57 c 2.9 c
1 XA11340100DO 100 1.1 mL 73 € 133 c 6.6 c
6 D3386 10 mg 756 € 15 c 0.8 c
1,1,2,2-D4
Chlormequat-chloride 1 CA11340100 5 mg 389 € 16 c 0.8 c
9 00291 5 mg 485 € 19 c 1.0 c
14 CRM9G3L1612 1000 1.1 mL 320 € 58 c 2.9 c
D9 3 673151 5 mg 320 € 13 c 0.6 c
7 C987717 5 mg 164 € 6.6 c 0.3 c
13C 9 32679 10 mg 470 € 9.4 c 0.5 c
3

Cyanuric acid 14 LBS9G3L1609 1000 1.1 mL 200 € 36 c 1.8 c

18O 3 673141 10 mg 299 € 6.0 c 0.3 c
3
13C 15N 15 S-O-C695-A-1.2ML 100 1.2 mL 378 € 630 c 31.4 c
3, 3

1 DRE-C11920010 10 mg 366 € 7.3 c 0.4 c

1 XA11920010EA 100 1.1 mL 118 € 215 c 11 c
Cyromazine-D4 7 C989302 10 mg 1.255 € 25.1 c 1.3 c
9 93101 5 mg 164 € 6.6 c 0.3 c
14 LBS9G3L1613 1000 1.1 mL 170 € 31 c 1.5 c
1 XA11960100AL 100 1.1 mL 87 € 158 c 7.9 c
D6
7 D416717 25 mg 647 € 5.2 c 0.3 c
Daminozide
14 LBS9G3L2291 1000 1.1 mL 320 € 58 c 2.9 c
D4
6 D45297 50 mg 441 € 1.8 c 0.09 c
4 D-5307 100 mg 432 € 0.9 c 0.04 c
D4
14 LBS9B3L3152 1000 1.1 mL 180 € 33 c 1.6 c
Diethanolamine
7 D441902 100 mg 1.100 € 2.2 c 0.1 c
D8
14 LBS9B3L3095 1000 1.1 mL 180 € 33 c 1.6 c
13
Difluoroacetic acid - C2 (Sodium salt) 2 friendly donation
sesquisulfate-hydrate 1 C 12635300 100 mg 29 € 0.1 c 0.003 c
Dihydrostreptomycin
sulfate 1 EPD1954000 25 mg 120 € 1.0 c 0.048
Diquat-D8 Dibromide (dipyridine-D8) 4 D-7990 10 mg
1 DRE-CA12960010 50 mg 315 € 1.3 c 0.06 c
1 XA12960010DO 100 1.1 mL 82 € 149 c 7.5 c
Diquat-D4-dibromide (ethylene-D4)*
4 D-3932 10 mg 144 € 2.9 c 0.1 c
*stability problems
6 D17071 50 mg 840 € 3.4 c 0.2 c
7 D492902 5 mg 117 € 4.7 c 0.2 c

EU Reference Laboratory for pesticides requiring Single Residue Methods (EURL-SRM) 90 of 102
 Conc. Amount Prices in €-cent (see diclaimer)
Isotope labelled compound Source Article-No.
(µg/mL) per unit 1 unit 2 µg* 0.1 µg**
9 3627 5 mg 152 € 6.1 c 0.3 c
10 B130022-10 10 mg 1.100 € 22 c 1.1 c
11 sc-218246 5 mg 234 € 9.4 c 0.5 c
14 LBS9AZ3L2482 1000 1.1 mL 240 € 44 c 2.2 c
XA13230100AC 100 1.1 mL 127 € 231 c 12 c
1
DRE-C13230100 10 mg 1.200 € 24 c 1.2 c
D4 6 D8328 5 mg 1.400 € 56 c 2.8 c
Ethephon
7 C366177 10 mg 1.120 € 22 c 1.1 c
14 LBS9BK3L1600 1000 1.1 mL 250 € 45.5 c 2.3 c
13C 7 C366178 0.25 mg 210 € 170 c 8c
2

1 C 13330100 50 mg 316 € 1.3 c 0.06 c

1 XA13330100AC 100 1.1 mL 127 € 231 c 12 c
Ethylenethiourea-D4 (ETU-D4) 6 D1965 100 mg 733 € 1.5 c 0.07 c
7 I367002 10 mg 98 € 2.0 c 0.1 c
14 LBS9G3L2293 1000 1.1 mL 150 € 27 c 1.4 c
1 CA13940010 10 mg 380 € 7.6 c 0.4 c
D15 (Aluminium salt)
Fosetyl 14 LBS2AZ3L1607 100 1.1 mL 178 € 324 c 16.2 c
D5 (Sodium salt) 8 C5607 10 mg 825 € 17 c 0.8 c
2 Friendly donation
D3 3 680888 100 1 mL 380 € 760 c 38 c
Glufosinate 14 CRM9AZ3L1604 1000 1.1 mL 1.380 € 251 c 12.5 c
3 681220 100 1 mL 380 € 760 c 38 c
D3-Chloride
7 G596952 10 mg 1.900 € 38 c 1.9 c
1 XA14050100WA 100 1.1 mL 304 € 553 c 28 c
CNLM-4666-1.2 100 1.2 mL 361 € 602 c 30 c
5
CNLM-4666-10X-1.2 1000 1.2 mL 1.170 € 196 c 9.8 c
1 CIL-CNLM-4666-1.2 100 1.2 mL 344 € 573 c 29 c
13C 15N
6 CN10570 5 mg 1.990€ 80 c 4.0 c
2,
7 G765002 10 mg 1.048 € 21 c 1.0 c
Glyphosate 11 sc-280758 1 mg 262 € 52 c 2.6 c
14 CRM17AZ3L1602 200 1.1 mL 470 € 427 c 21.4 c
15 S-FCN1104S-1.2ML 100 1.2 mL 393 € 655 c 32.7 c
17 R009984 10 mg 2.165 € 43.4 c 2.2 c
13C, 15N 9 90479 5 mg 536 € 21 c 1.1 c
13C
7 G765001 5 mg 210 € 8.4 c 0.4 c
9 606502 10 mg 785 € 16 c 0.9 c
1 CA13230200 10 mg 256 € 5.1 c 0.3 c
7 H939652 25 mg 1.125 € 9.0 c 0.5 c
HEPA (Hydroxy-Ethephon)-D4 2 Friendly donation
3 676639 100 1 mL 99 € 200 c 10 c
14 LBS9AZ3L1601 1000 1.1 mL 580 € 105.5 c 5.3 c
Matrine-D3 7 M197872 10 mg 820 € 16 c 0.8 c
1 C 14730100 10 mg 235 € 4.7 c 0.2 c
1 c (0.5
3 673799 10 mg 199 € 20c (10µg)
D2 µg)
Maleic Hydrazide
7 M124502 5 mg 141 € 5.6 c 0.3 c
14 LBS9G3L1608 1000 1.1 mL 270 € 49 c 2.5 c
13C 9 04311-10MG 10 mg 228 € 4.6 c 0.2 c
4
13C 15N
3, 3 1 CIL-CNLM-8150-10X-1.2 1000 1.2 mL 1.300 € 260 c 13 c
9 80038 10 mg 647 € 13 0.7 c
15N 3 673055 10 mg 289 € 5.8 c 0.3 c
3
Melamine
14 LBS9G3L1616 1000 1.1 mL 270 € 49 c 2.5 c
13C
3 679703 10 mg 480 € 9.6 c 0.5 c
3
1 B-MYC8020-1.2 100 1.2 mL 528 8.8 € 440 c c

EU Reference Laboratory for pesticides requiring Single Residue Methods (EURL-SRM) 91 of 102
 Conc. Amount Prices in €-cent (see diclaimer)
Isotope labelled compound Source Article-No.
(µg/mL) per unit 1 unit 2 µg* 0.1 µg**
6 D14539 50 mg 1.350 € 5.4 c 0.3 c
D16 –chloride 9 52485 5 mg 214 € 8.6 c 0.4 c
14 CRM9G3L2292 1000 1.1 mL 300 € 54.5 c 2.7 c
1 X 14880100DO 100 10 mL 378 € 76 c 3.8 c
Mepiquat
1 XA14880100DO 100 1.1 mL 68 € 124 c 6.2 c
D3 (methyl-D3) -iodide 9 78278 10 mg 379 € 7.6 c 0.4 c
3 677008 10 mg 320 € 6.4 c 0.3 c
14 LBS9G3L1531 1000 1.1 mL 290 € 53 c 2.6 c
0.94 c 0.05c
4 D-1895/0.5 500 mg 468 €
(10µg) (0.5µg)
D8
Morpholine 7 M723728 25 mg 131 € 1.1 c 0.05 c
14 LBS9G3L3094 1000 1.1 mL 200 € 36 c 1.8 c
13C 7 M723727 1 mg 131 € 26 c 1.3 c
4

D3 (methyl-D3) 2 Friendly donation

7 A178237 5 mg 141 € 5.6 c 0.3 c
9 05567 5 mg 97.50 € 3.9 c 0.2 c
N-Acetyl-Glufosinate 3 680264 100 1 mL 280 € 560 c 28 c
D3 (Acetylamino-D3)
14 LBS9AZ3L1606 1000 1.1 mL 180 € 33 c 1.6 c
18 20053 20 mg 240 € 2.4 c 0.12 c
11 sc-479498 5 mg 230 € 9.2 c 0.46 c
7 A178248 25 mg 1.153 € 9.2 c 0.5 c
D3 (methyl-D3)
14 LBS9AZ3L2868 1000 1.1 mL 580 € 105.5 c 5.3 c
N-Acetyl-Glyphosate
13C 15N
7 A178247 10 mg 1.326 € 26.5 c 1.3 c
2,
17 R052712 10 mg 2.982 € 59.6 c 3.0 c
1 C 15502010 10 mg 245 € 5c 0.3 c
Nereistoxin-oxalate-D6
14 LBS9AR3L1615 1000 1.1 mL 270 € 49 c 2.5 c
4 D-5098 100 mg 400 € 0.8 c 0.04 c
Nicotine-D4
14 LBS9B3L3297 1000 1.1 mL 420 € 76 c 3.8 c
Oxymatrine-D3 7 O876302 5 mg 600 € 24 c 1.2 c
2 Friendly donation
7 M326162 10 mg 1.921 € 38 c 1.9 c
MPPA-D3
3 680891 100 1mL 380 € 760 c 38 c
14 LBS9AZ3L1605 1000 1.1 mL 1.800 € 327 c 16.4 c
1 C 15870200 50 mg 256 € 1.0 c 0.05 c
D6-diiodide
14 CRM9AZ3L1611 1000 1.1 mL 180 € 33 c 1.6 c
D6-dichloride (dime-
Paraquat 1 DRE-C15870050 50 mg 390 € 1.6 c 0.08 c
thyl D6)
1 DRE-CA15870100 50 mg 390 € 1.6 c 0.08 c
D8-dichloride
7 P191902 25 mg 920 € 7.3 c 0.4 c
5 OLM-7310-1.2 100 1.2 mL 326 € 272 c 14 c
Perchlorate-18O4 12*** 40 5 mL 250 € 125 c 6.3 c
9 631981 10 mg 4.500 € 90 c 4.5 c
0.3 c
Phosphonic acid-18O3 12 2000 1 mL 125 6.3 c

D6 7 P758462 10 mg 1050 € 21 c 1.1 c

4 DER-XA16390100AC 100 1.1 mL 82 € 149 c 7.5 c
Propamocarb
D7 9 80757 5 mg 230 € 9.2 c 0.5 c
14 LBS9G3L3296 1000 1.1 mL 320 € 58 c 2.9 c
6 D535 (not available) 100 mg 756 € 1.5 c 0.1 c
D6
7 P836802**** 10 mg 1.100 € 22 c 1.1 c
PTU
9 07359 5 mg 205 € 8.2 c 0.4 c
D3
14 LBS9G3L3151 1000 1.1 mL 220 € 40 c 2.0 c
13C 15N
2 Friendly donation
2, 3
1, 2, 4-Triazole 16 3201 5 mg 1.854 $ 74.2 c 3.7 c
D2 19 RCG-401 10 mg 350.61 € 7c 0,35 c

EU Reference Laboratory for pesticides requiring Single Residue Methods (EURL-SRM) 92 of 102
 Conc. Amount Prices in €-cent (see diclaimer)
Isotope labelled compound Source Article-No.
(µg/mL) per unit 1 unit 2 µg* 0.1 µg**
13C 15N
2 Friendly donation
1, 2, 4-Triazole-acetic 2, 3
16 15297 1 mg 420 $ 84 c 4.2 c
acid
D2 19 RCG-398 10 mg 350.61 € 7c 0,35 c
13C , 15N 2 Friendly donation
2 3
1, 2, 4-Triazole-alanine
D2 19 RCG-399 10 mg 350.61 € 7c 0,35 c
13C 15N
2 Friendly donation
1, 2, 4-Triazole-lactic 2, 3
16 15295 1 mg 420 $ 84 c 4.2 c
acid
D2 19 RCG-400 10 mg 350.61 € 7c 0,35 c
“D15“ (in reality D12) 1 CIL-DLM-7663 1 mg 153 € 31 c 1.5 c
Triethanolamine 7 T775582 10 mg 141 € 2.8 c 0.15 c
D12
14 LBS9G3L3096 1000 1.1 mL 180 € 33 c 1.6 c
Trifluoroacetic acid -13C2 (Sodium acetate) 7 S673752 10 mg 2.670 € 53 c 2.7 c
6 D2677 100 mg 730 € 0.7 c 0.04 c
6 D2677 10 mg 270 € 2.6 c 0.13 c
Trimethylsulfonium- D9
4 D-6093 500mg 430 € 0.2 c 0.009 c
(iodide)
14 LBS9G3L1614 1000 1.1 mL 605.71 € 110 c 5.5 c
D3 3 684243 10 mg 100 € 2c 0.1 c
Providers of compounds:
1: LGC Stndards 11: Santa Cruz biotechnology. Inc.
2: Bayer Crop Science 12: EURL-SRM (hosted at CVUA Stuttgart)
3: HPC (High Purity Compounds) 13: Campro Scientific / Chiron AS
4: CDN Isotopes (distributed in Germany by EQ Laboratories GmbH) 14: Lab Instruments
5: Cambridge Isotope Lab. Inc. 15: Chem Service Inc.
6: Medical isotopes 16: IsoSciences
7: Toronto Research Chemicals 17: MuseChem
8: ALSACHIM 18: ASCA GmbH
9: Sigma-Aldrich-Supelco (Merck) 19: ReseaChem GmbH
10: Cerilliant (by Sigma Aldrich)
(Disclaimer: The use of trade names is for the information and convenience of the reader. Such use does not constitute an official endorsement
or approval by the EURL of any product to the exclusion of others. Market prices and currency exchange rates may be subject to changes. Shipping
costs are not included in the pricing.
* 2 µg IS are typically employed to samples (typically 10 g) at the beginning of the procedure
** 0.1 µg are typically added to 1 mL aliquots of sample extracts (typically corresponding to 0.5 g sample), in this case only ma-
trix-effects are compensated
*** Due to manufacturing process the stock solution of 18O3-Chlorate is accompanied by ca 20% 18O4-Perchlorate.. As perchlorate
typically exhibits a ca. 5-fold higher LC-MS/MS-sensitivity compared to chlorate the signal intensities of the two are end up within
the same range.
**** The PTU-D6 offered by (7) used to be the non-branched 1,3 propylene variant. This product did not exactly co-elute with the
target analyte and thus not compensating mtrix effects. It now seems to be the right product N,N’-(1,2-Propylene)thiourea-D6

EU Reference Laboratory for pesticides requiring Single Residue Methods (EURL-SRM) 93 of 102
Table 45: Exemplary concentrations of Internal Standard Working Solutions (IS-WS) (3.22)
IS –Addition to samples (5.2.3) IS-Addition to calibration standard(s) (5.5)
Absolute mass of IS Expected approx.. IS-
Absolute mass of IS
Suggested con- spiked to calibration concentration in sample ex-
Suggested concentration spiked to sample (100
Internal Standard (IS)* centration of IS- standard tracts (~20 mL) and calibra-
of IS-WSln1 (3.22) µL IS-WSln1)
WSln2 (3.23) ** (100 µL IS-WSln2) tion standards (~1 mL)
(mISsample)
(mIScal mix)
µg/mL µg µg/mL µg µg/mL
Amitrole-(15N)/ (15N2,13C2) 20 2 1 0.1 0.1
AMPA-13C.15N 20 2 1 0.1 0.1
Bromate-18O3 200 20 10 1 1
Chlorate-18O3 20 2 1 0.1 0.1
Chloridazon-desphenyl-15N2 (IL-IS) 40 2 2 0.2 0.2
Chlormequat-D4 10 1 0.5 0.05 0.05
Cyromazine-D4 20 2 1 0.1 0.1
Daminozid-D6 10 1 0.5 0.05 0.05
Diethanolamine-D6 20 2 1 0.1 0.1
Difluoroacetic acid -13C2 10 1 1 0.05 0.05
Dihydrostreptomycin**** 20 2 1 0.1 0.1
Diquat-D4 40 4 2 0.2 0.2
Ethephon-D4 20 2 1 0.1 0.1
ETU-D4 20 2 1 0.1 0.1
Fosetyl-D5
20 2 1 0.1 0.1
(from fosetyl-aluminium-D15)
Glufosinate-D3 20 2 1 0.1 0.1
Glyphosate-13C2.15N 20 2 1 0.1 0.1
HEPA-D4 20 2 1 0.1 0.1
Maleic Hydrazide-D2 20 2 1 0.1 0.1
Melamine-15N3 20 2 1 0.1 0.1
Mepiquat-D3 10 1 0.5 0.05 0.05
Morpholine-D8 20 2 1 0.1 0.1
MPPA-D3 20 2 1 0.1 0.1
N-Acetyl-Glufosinate-D3 20 2 1 0.1 0.1
N-Acetyl-glyphosate-13C2.15N 20 2 1 0.1 0.1
Nereistoxin-D4 10 1 0.5 0.05 0.05
Nicotine-D4 10 1 0.5 0.05 0.05
Paraquat-D6 40 4 2 0.2 0.2
Perchlorate-18°4 20 2 1 0.1 0.1
Phosphonic acid-18O3 20 2 1 0.1 0.1
Propamocarb-D7 2 0.2 0.1 0.01 0.01
PTU-D6 10 1 0.5 0.05 0.05
Triethanolamine-D12 10 1 0.5 0.05 0.05
Trifluoroacetic acid -13C2 10 1 1 0.05 0.05
Trimethylsulfonium-D10 10 1 0.5 0.05 0.05
* The concentration of the IL-IS should be high enough to ensure good detection with little influence of signal noise (S/N>20 is
typically fine). It should be kept in mind. However. That isotopically labeled ISs (IL-ISs) sometimes contain small amounts of the
non-labeled analogues. To minimize the risk of false positives the amount of IL-IS added to the samples should thus not be
higher than necessary. Quantification of the parent is typically not affected to a great extend as the cross-contamination is typi-
cally at low levels and as similar concentrations of the native pesticide originating from the IL-IS will also be present in the cali-
bration standards and thus subtracted via the intercept. In the case of Maleic Hydrazide. Where the IL-IS is added at higher con-
centrations to the samples special attention is necessary (see also comments under 5.6.3).
** a 20-fold dilution of the IS working solution used to spike samples in step 5.2.3 .
*** Dihydrostreptomycin is not isotopically labeled but still suitable for compensation of matrix effects on Streptomycin, if LC
conditions are adjusted to ensure exact co-elution and thus equivalent matrix-effects.
NOTE: If detections of a compound are rather seldom and the IS expensive it is advisable to add the IL-IS to the 1 mL aliquot
transferred to the auto-sampler vial (see Table 44). Alternatively. It can be even skipped entirely in the first screening analysis
and only added in a second analysis in case the first one was positive. The first approach is to be preferred especially where the
retention times of a compound tends to shift. By comparing the retention time between the IS and the suspected peak as well as
the peak shape the certainty of identification significantly improves.

EU Reference Laboratory for pesticides requiring Single Residue Methods (EURL-SRM) 94 of 102
Table 46: Water content of selected foods and water amount to be added to test portions prior to extraction (5.2.2) depending on
the analytical approach
Typical natural Water addition may be
Water to be
Commodity group Commodity Sample weight water content skipped if suitable IS is Remarks
added
g/100 g used before aliquotation
Fruits
Citrus fruit Citrus juices 10 g 90 1 Yes
Grapefruit 10 g 90 1 Yes
Lemon/lime 10 g 85 1.5 Yes
Orange 10 g 85 1.5 Yes
Tangerine 10 g 90 1 Yes
Pome fruit Apple 10 g 85 1.5 Yes
Apple sauce 10 g 80 2 Yes
Apple juice 10 g 90 1 Yes
Pear 10 g 85 1.5 Yes
Quince 10 g 85 1.5 Yes
Stone fruit Apricot 10 g 85 1.5 Yes
Apricot nectar 10 g 85 1.5 Yes
Cherry 10 g 85 1.5 Yes
Mirabelle 10 g 80 2 Yes
Nectarine 10 g 85 1.5 Yes
Peach 10 g 90 1 Yes
Plum 10 g 85 1.5 Yes
Soft and small fruit Blackberry 10 g 85 1.5 Yes
Blueberry 10 g 85 1.5 Yes
Currant 10 g 85 1.5 Yes
Elderberry 10 g 80 2 Yes
Gooseberry 10 g 90 1 Yes
Grapes 10 g 80 2 Yes
Raspberry 10 g 85 1.5 Yes
Strawberry 10 g 90 1 Yes
Pineapple 10 g 85 1.5 Yes
Other fruits Banana 10 g 75 2.5 No
Fig 10 g 80 2 Yes
Kiwi 10 g 85 1.5 Yes
Mango 10 g 80 2 Yes
Papaya 10 g 90 1 Yes
Kaki/persimmon 10 g 90 1 Yes
Dried fruit Apple, dried 5g 20 9 No
Apricot, dried 5g 20 9 No Weigh 14 g rehydra-
Figs, dried 5g 20 9 No tized homogenate
Prunes (dried plums) 5g 20 9 No (500 g +900 g water)
Raisins 5g 15 9 No
Apricot, dried soft 5g 30-35 8,5 No Weigh 13.5 g rehy-
dratized homogenate
Figs, dried soft 5g 30-35 8,5 No
(500 g +850 g water)
Weigh 13 g rehydra-
Prunes, soft 5g 35-40 8 No tized homogenate
(500 g +800 g water)
Vegetables
Root and tuber vege- Beetroot 10 g 90 1 Yes
tables Carrot 10 g 90 1 Yes
Celeriac 10 g 90 1 Yes
Horseradish 10 g 75 2.5 No
Parsley root 10 g 90 1 Yes
Radish 10 g 95 0.5 Yes
Black salsify 10 g 80 2 Yes
Potato 10 g 80 2 Yes

Garlic 10 g 65 3.5 No

EU Reference Laboratory for pesticides requiring Single Residue Methods (EURL-SRM) 95 of 102
 Typical natural Water addition may be
Water to be
Commodity group Commodity Sample weight water content skipped if suitable IS is Remarks
added
g/100 g used before aliquotation
Leek plants Onion 10 g 90 1 Yes
Leek 10 g 85 1.5 Yes
Shallot 10 g 80 2 Yes

Chives 10 g 85 1.5 Yes

Fruiting vegetables Aubergine 10 g 90 1 Yes

Cucumber 10 g 95 0.5 Yes
Melon 10 g 90 1 Yes
Pepper. Sweet 10 g 90 1 Yes
Pumpkin 10 g 95 0.5 Yes
Tomato 10 g 95 0.5 Yes
Zucchini 10 g 95 0.5 Yes
Broccoli 10 g 90 1 Yes
Cabbage Brussel sprouts 10 g 85 1.5 Yes
Cauliflower 10 g 90 1 Yes
Chinese cabbage 10 g 95 0.5 Yes
Kale 10 g 90 1 Yes
Kohlrabi 10 g 90 1 Yes
Red cabbage 10 g 90 1 Yes
Savoy cabbage 10 g 90 1 Yes
White cabbage 10 g 90 1 Yes
Lettuce varieties 10 g 95 0.5 Yes
Endive 10 g 95 0.5 Yes
Leafy vegetables and Cress 10 g 90 1 Yes
herbs Lamb’s lettuce 10 g 85 1.5 Yes
Parsley 10 g 80 2 Yes
Rucola 10 g 85 1.5 Yes

Spinach 10 g 90 1 Yes

Stem Asparagus 10 g 95 0.5 Yes

vegetables Celery 10 g 95 0.5 Yes
Leek 10 g 85 1.5 Yes
Rhubarb 10 g 95 0.5 Yes
Artichokes 10 g 85 1.5 Yes
Legumes / Pulses Sample amount may
9 mL water
Pulses (dried Beans, need to be reduced if
5g <10 and 1 mL EDTA No
Peas, Lentils) material strongly ab-
solution
sorbs water
Fresh Peas 10 g 75 2.5 No
Green Beans 10 g 90 1 Yes
Cereals Sample amount may
9 mL water
need to be reduced if
Grain. Flour etc. 5g 10 and 1 mL EDTA No
material strongly ab-
solution*
sorbs water
Oily seeds Peanuts, Poppy seeds,
9 mL water
Pumpkin seeds, Ses-
5g <10 and 1 mL EDTA No
ame seeds, Soyabeans,
solution*
Sunflower seeds
To reduce slime for-
mation, which hin-
9 mL water ders residue accesi-
Linseeds, Chiaseeds 5g <10 and 1 mL EDTA No bility, change se-
solution* quence! First add
acidified methanol
and then EDTA/water

EU Reference Laboratory for pesticides requiring Single Residue Methods (EURL-SRM) 96 of 102
 Typical natural Water addition may be
Water to be
Commodity group Commodity Sample weight water content skipped if suitable IS is Remarks
added
g/100 g used before aliquotation
Nuts Almonds, Cashew nuts,
Dried coconuts, Hazel- 9 mL water
nuts, Macadamias, Pe- 5g <10 and 1 mL EDTA No
cans, Pistachios, Wal- solution*
nuts
Miscellaneous
Extract-rich (“diffi- 9 mL water
cult”) commodities Coffee beans 2g <10 and 1 mL EDTA No Different sample
solution* amounts may be
Tea 2g <10 10 No used depending on
extract-richness
Dry herbs and spices 2g <10 10 No
Miscellaneous Other Mushrooms fresh 10 g 90 1 Yes
Mushrooms dried 2g <10 10 No
Wine 10 g 90 1 Yes
Honey 5g 20 7.5 No
Avocado 10 g 70 3 No
Fat melting needed,
Coconut copra 5g <10 0 see QuPPe-AO (ani-
mal fat)
Olives 10 g 70 3 No
* The addition of EDTA solution is highly recommended when targeting analytes showing poor recoveries in absence of EDTA.
Affected are compounds with a tendency to form complexes with metals, such as Glyphosate and metabolites, Glufosinate and
metabolites. If affected analytes are not targeted, EDTA addition may be skipped and 10 mL of water are added.

Table 47: Exemplary LC-MS/MS parameters for Sciex Qtrap 5500

Methods
1.1/1.2/1.5/ Method Method Method Method Method Method Method Method
Parameters Method 10 Method 11
1.6/ 1.3 1.4 2 3/ 4.1/ 5 4.2 6 7 8/9
1.7/1.9/1.10
Ion source
(ESI. Turbo pos. / neg.
negative negative negative negative positive positive positive positive positive negative
Ion Spray) SelexIonTM
Mode
Curtain gas 30 psi 40 psi 40 psi 30 psi 30 psi 30 psi 30 psi 40 psi 20 psi 20 psi 40 psi
(N2) (2.07 bar) (2.76 bar) (2.76 bar) (2.07 bar) (2.07 bar) (2.07 bar) (2.07 bar) (2.76 bar) (1.38 bar) (1.38 bar) (2.76 bar)

Collision gas medium high

Ion spray 5500 / -

-4500 -4500 -4500 -4500 1500 5000 5500 1500 5500 -4500
voltage 5500
Gas 1
50 psi 60 psi 60 psi 50 psi 50 psi 60 psi 50 psi 60 psi 60 psi 60 psi 60 psi
(Zero Grade
(3.45 bar) (4.14 bar) (4.14 bar) (3.45 bar) (3.45 bar) (4.14 bar) (3.45 bar) (4.14 bar) (4.14 bar) (4.14 bar) (4.14 bar)
Air or N2)
Gas 2
60 psi 60 psi 70 psi 60 psi 60 psi 50 psi 60 psi 70 psi 70 psi 70 psi 60 psi
(Zero Grade
(4.14 bar) (4.14 bar) (4.83 bar) (4.14 bar) (4.14 bar) (3.45 bar) (4.14 bar) (4.83 bar) (4.83 bar) (4.83 bar) (4.14 bar)
Air or N2)

Tempera-
600°C 550°C 550°C 500°C 500°C 500°C 550°C 500°C 550°C 550°C 600°C
ture of Gas 2

Resolution
unit (approx.. 0.7 amu FWHM*)
MS 1
Resolution
unit (approx.. 0.7 amu FWHM)
MS 2

Dwell time 20 20 20 50 20 10 50 20 20 / 40 20 20
*FWHM = full width at half maximum

EU Reference Laboratory for pesticides requiring Single Residue Methods (EURL-SRM) 97 of 102
Table 48: Exemplary LC-MS/MS parameters for Waters Xevo TQ-Sµ

Method 1 Method
Parameters
M1.6b/M1.7b/M1.8 M4.2

Ion source (ESI) negative Positive

Source Temperature 150 °C 150 °C

Desolvation Temperature 600 °C 600 °C

Cone Gas Flow 50 L/h 150 L/h

Desolvation Gas Flow 1000 L/h 1000 L/h

Capillary 0.5 kV 0.5 kV

Resolution MS unit unit

EU Reference Laboratory for pesticides requiring Single Residue Methods (EURL-SRM) 98 of 102
Table 49: Document History
Action When? Version
Development of Method by the CRL-SRM 2006-2008
-
Presentation of method at the EPRW in Berlin (oral presentation plus poster) June 2008
Drafting of V1 Nov.-Dec. 2008
V1
Placing of V1 in CRL-Website Jan. 2009
Update of Table 1.
Expected concentrations of Iss were calculated with a wrong dilution factor in previous version. Arithmet-
Aug. 2009 V2
ical errors were corrected.
Introduction of measurement conditions for HEPA within the “Glyphosate & Co.” method
Introduction of measurement conditions for the screening of diquat and paraquat within the “Quats & Co.
method”
Introduction of measurement conditions for Amitrole. Chlormequat. Mepiquat and daminozide “Amitrole Nov 2009 V3
& Co.” method
Extensive text revisions
Introduction of measurement conditions for Streptomycin Kasugamycin
Introduction of measurement conditions for the screening of Perchlorate ion May 2010 V4
Extensive text revisions
Extensive text revisions and restructuring of document
Introduction of measurement conditions for ETU. ETU D4. PTU. PTU D6. Cyromazine. Cyromazine D4. N- Nov 2010 V5
Acetyl-Glufosinate. N-Acetyl-Glufosinate D3. Glufosinate D3. MPPA D3. Morpholin. Morpholin D8
Introduction of an acronym for the method (QuPPe)
Advice to use plastic vessels and stoppers for Glyphosate
Minor modification and additional instructions in Method 1 (M1)
Modification of mobile phase of M3 to improve analysis of ETU and PTU
Introd. Of measurement cond. For Amitrole15N13C and Amitrole15N in M3
Introd. Of measurement cond. For Nereistoxin and Nereistoxin D6 in M4
New method (M7) for the analysis of Morpholin/Morpholin D8; Diethanonamine/diethanolanmine D6; Tri- July 2011 V6
ethanolamine/Triethanolamine D12 (M7)
Removal of Morpholin from M4 as it does not separate from the interfering diethanolamine
Introduction of ETU and PTU and their corresponding IL-ISs in Method 5
Correction of dimension of stock solutions conc. In Table 12 (to mg/mL)
Text and Table revisions
Extensive revision of table concerning possible sources of purchase of Iss
Some additions in “Apparatus and Consumables” chapter
Clarifications in chapter concerning standard additions
Overview table concerning the scope of the methods 1.1. 1.2. 1.3 and 2
Addition of Phosphonic acid in Method 1.1 (“Glyphosate & Co.”)
New LC-method (Method 1.2) for “Glyphosate & Co.” using a Dionex ionPac AS11-HC column and an Eluent
with near to neutral pH; additionallycovering Fosetyl
New LC-method (Method 1.3) for “Glyphosate & Co.” using a Hypercarb column and an acidic Eluent cov-
Dec. 2012 V7
ering all analytes covered by Method 1.1. Method 1.2 and Method 2 (including perchlorate).
Update of practical considerations for methods 1.1-1.3
Update of table with performance data
Table with exemplary recovery data was deleted (recovery figures can be obtained in the EURL-DataPool
Update of table with LOQs
Update of table with providers of IL-ISs
Elimination of errors in text
Addition of Chlorate in Method 1.3
Update of practical considerations for methods 1.1-1.3 (Column C) Nov. 2013 V7.1
Update of table with performance data

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Action When? Version
Update of table with LOQs
Introduction of Trimethylsulfonium-D9 and N.N-Dimethylhydrazine-D6 in Method 4
Thorough revision of text and elimination of errors
Practical advices on the choice of filter materials
New Table 15: Conversion factors between standard materials and analytes
Advices as regards the use of IL-ISs
Update of Table 5.6: LC-MS/MS measurement conditions
New chapters “Hints on Method 1.1 – 1.4” and replacement of the section “Practical care and use consid-
erations concerning the columns of methods 1.1-1.3. This includes information on various potential sources
of errors such as in-source fragmentations of Fosetyl and Ethephon to Phosphonic acid and of Perchlorate
to Chlorate as well as degradation of compounds in solution.
Introduction of Cyanuric acid and Bialaphos in M1.3
Correction of a typing error concerning the mass-transitions of Phosphonic acid (81/79 instead of 81/81)
Mar. 2015 V8
Introduction of the IL-IS of Phosphonic acid and chlorate in M1.3 and 1.4
New LC Method (1.4) for “PerChloPhos” using a Hypercarb column and an acidic Eluent optimized for chlo-
rate. Perchlorate. Phosphonic acid compared to Method 1.3
Change of name of former M4 to M4.1
Introduction of Melamine and Propamocarb as well as the corresponding IL-ISs in M4.1
New LC Method (M4.2) employing a Hilic-Type BEH Amide column allowing the simultaneous analysis of
many polar pesticides
Reduction of injection volume and increase of dwell-time in method M6
New LC-method (M8) for the analysis of triazole derivative metabolite (TDMs) and their corresponsing IL-
ISs
Update of Table 17: Providers of isotopically labeled internal standards
5.1 Sample preparation: note to importance of having small particle sizes
5.2.4 notes to extraction time for dry products and the influence of particle size
May 2015 V8.1
5.6 information on the methods currently routinely used at CVUA Stuttgart
Update Table 20: Exemplary LC-MS/MS parameters for Sciex QTRAP 5500
Update of Chapter 5: Procedure including the extraction procedure at a glance
Update of Table 4: Overview and scope of the methods proposed within this document for the QuPPe
method
Update of Table 8: Methods mainly used by CVUA Stuttgart
Update of Chapter 5.7.3.1.: Hints on Method 1.3
Update of Method 1.4: Introduction of measurement conditions for the measurement of Bromate and Bro-
mide ion
Update of Chapter 5.7.4.1.: Hints on Method 1.4
Update of Method 4.2 : “Quats & Co BEH Amide” including Aminocyclopyrachlor. Chloridazon-desphenyl.
Mepiquat-4-hydroxy. Propamocarb-N-desmethyl. Propamocarb-N-oxide
Mar. 2016 V9
Update of Method 6 : “Streptomycin and Kasugamycin”. Change of gradient and new chromatograms
Update of Method 8 (M8): “Triazole derivative metabolites (TDMs)” new DMS parameters
Update of Table 40: Overview of approximate limits of quantification (LOQs)*
Update of Table 42: Conversion factors between typical purchased standards and target analytes (3.18):
Update of Table 43: Exemplary concentrations of pesticide stock and working solutions
Update of

Table 44: Providers of isotopically labeled internal standards

Update of Table 45: Exemplary concentrations of IS working solutions
Elimination of an error in method 1.4 (Change in dilution procedure) May. 2016 V9.1
Inclusion of N-Acetyl-Glyphosate in Table 3: Overview and scope of the methods proposed within this doc-
October 2016 V9.2
ument for the QuPPe method:

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Action When? Version
Inclusion of N-Acetyl-Glyphosate in Table 4: Practical Information: Mainly used methods used at CVUA
Stuttgart
Addition of a further Ethephon-IL-IS mass trace and inclusion of N-Acetyl-Glyphosate in Table 7: Proposed
LC-MS/MS conditions for Ethephon. HEPA (Ethephon metabolite). Glyphosat. AMPA (Glyphosate metabo-
lite). N-Acetyl-Glyphosate (Glyphosate metabolite). N-Acetyl-AMPA (Glyphosate metabolite). Glufosinate.
MPPA (Glufosinate metabolite). N-Acetyl-Glufosinate (Glufosinate metabolite). Fosetyl-Al. Maleic Hydra-
zide. Cyanuric acid and Bialaphos.
Update of Figure 4: Chromatograms of Ethephon. HEPA. Glyphosat. AMPA. Glufosinate. MPPA. N-Acetyl-
AMPA. N-Acetyl-Glufosinate. Fosetyl-Al. Maleic Hydrazide. Cyanuric acid.Bialaphos and N-Acetyl-Glypho-
sate at 0.1 mg/kg on almond extract.
Inclusion of N-Acetyl-Glyphosate in Table 18: Overview of approximate limits of quantification (LOQs)
Update of Table 19: Conversion factors between typical purchased standards and target analytes (3.15)
Update of Table 20: Exemplary concentrations of pesticide stock and working solutions (3.15 and 3.16).
solvent proposals also apply to IL-ISs (see 3.18. 3.19 and 3.20).
Inclusion of N-Acetyl-Glyphosate in Table 21: Exemplary providers of isotopically labeled internal standards
3.17.
Update of Table 22: Exemplary concentrations of IS working solutions (3.19)
New Method: (Method 9 “Difluoroacetic acid and Trifluoroacetic acid”), see 5.6.20
Proposed volume of IS-WS II changed to match with volume of IS-WS I (see Table 2)
Update of Table 4: data on M 9 were included
Hints on stability of standard solutions added in 0, including Table 39
Overview of lowest successfully validated levels (Table 40)
April 2017 V9.3
Update of Table 42 : DFA and TFA added
Update of Table 43: DFA and TFA added; solvents for Ethephon, Fosetyl and Maleic Hydrazide changed
Update of Table 48
Update of Table 45: DFA and TFA added
Table 47 : data on M 9 were included
Extensive general revision of text, tables and figures
Addition of nuts and oily seeds to the scope of the method
Update of centrifuge information under 2.5
Update of syringe filters information under 2.6
Revision of sample preparation conditions section (5.1) to include milling of oily seeds and nuts and more
details on how to accomplish cryogenic milling using carbon dioxide and liquid nitrogen
Revision of the chapter concerning centrifugation (5.2). Inclusion of pre-centrifugation freeze-out and cry-
ogenic centrifugation as an option to improve the subsequent filtration behaviour
Revision of Figure 1 QuPPe-PO-Method at a glance
Splitting of Table 3 (Overview and scope of methods) and splitting into Table 3 and 4 Dec 2018 V10
New method M 1.5 (Glyphosate&Co. using Trinity Q1)
New Method M 1.6 (Glyphosate&Co. using DEA Torus)
Inclusion of Nicotine under Method 4.2
Introduction of Chapter 6 on Analyte Stability
Extention of Table 22 (Overview of lowest successfully validated levels per matrix)
Addition of Table 23 (Validation data deriving from Interlab validation studies)
Update of Table 24 (Conversion factors between typical purchased standards and target analytes)
Update of Table 25 (Exemplary concentrations of pesticide stock and working solutions)
Update of Table 26 (Exemplary providers of isotopically labeled internal standards)
Change of the wording of the document title
Update of method for pulses, oily seeds and nuts. Method now involves addition of EDTA during the ex-
traction step for complexation metals that may interfere with analysis of certain analytes April 2019 V10.1
Update of cleanup procedure for the removal of lipids and proteins
Addition of Method 1.7 for phosphonate, bromide, chlorate and perchlorate

EU Reference Laboratory for pesticides requiring Single Residue Methods (EURL-SRM) 101 of 102
Action When? Version
Maleic hydrazide added to Methode 4.2
Introduction of a list with shortcut-links
Update of Table 4 (Overview of scope)
Update of Table 5 (Overview of main methods)
Update of method for cereals. It now involves addition of EDTA during the extraction step for complexation
metals that may interfere with analysis of certain analytes
Feb 2020 V11
Update of Method M 4.1 (Quats & Co. Obelisc R), new IL-IS and additional MRMs for Diquat
Update of Table 31 (Exemplary providers of isotopically labeled internal standards)
Update of Table 33 (Water contents of selected commodities)
Update of Chapter 6 (information on purity of N-acetyl-glufosinate D3 standards)
Inclusion of Thiocyanate (M1.3 and M1.4) and Desmethyl-Dimethoate (M1.3) to the scope
Inclusion of Matrine and Oxymatrine (M 4.2) to the scope March 2021 V11.1
Restructuring of document to improve clarity (e.g. Hints and comments applying to more than one method
are merged)
Introduction of a Chapter containing collected hints (5.6.1: Hints on analytes to avoid pitfalls)
Thorough revision of text and elimination of errors
Additional differentiation in solvent grades
Extension of Apparatus list
Revision of text for dried fruits
Introduction of Method M1.6b, M1.7b, M 1.8, M 1.9, M 1.10, M 10, M 11
July 2021 V12
Update of Table 40: Additional validation data
Update of Table 45: data on Melamine, Matrine, Oxymatrine, Triazole, Triazole-lactic acid,
Triazole-acetic acid, Triazole-alanine included
Update of Table 46: dried fruits; olives, coconut copra, dried mushrooms, kaki included; garlic updated
New ILISs: Maleic Hydrazide 13C4, 1, 2, 4-Triazole-D2, 1, 2, 4-Triazole-acetic acid-D2, 1, 2, 4-Triazole-alanine-
D2, 1, 2, 4-Triazole-lactic acid-D2
Table 47 : data on M 1.10, M 10, M 11
Introduction of Table 48: MS Parameters for Waters Xevo TQ-Sµ
Revision of procedure for honey including water adjustment for honey, hints on how to use syringe / par-
March 2023 V12.1
ticle filters. New figure showing the procedure at a glance for honey, and validation data

EU Reference Laboratory for pesticides requiring Single Residue Methods (EURL-SRM) 102 of 102