Development of a QuEChERS-Based Method for the Simultaneous

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Development of a QuEChERS-based method for the simultaneous determination of acidic pesticides, their esters and conjugates following alkaline hydrolysis Angelika Steinborn, Lutz Alder, Madeleine Spitzke, Daniela Doerk, and Michelangelo Anastassiades J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b05407 • Publication Date (Web): 18 Jan 2017 Downloaded from http://pubs.acs.org on January 23, 2017

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Journal of Agricultural and Food Chemistry

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Development of a QuEChERS-based method for the simultaneous

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determination of acidic pesticides, their esters and conjugates following

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alkaline hydrolysis

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Angelika Steinborna*, Lutz Aldera, Madeleine Spitzkeb, Daniela Dörkc and

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Michelangelo Anastassiadesc

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a

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Dohrn-Str. 8-10, 10589 Berlin, Germany

Federal Institute for Risk Assessment, Department of Pesticides Safety, Max-

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b

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Laboratories for Pesticide Residues, Diedersdorfer Weg 1, 12277 Berlin, Germany

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c

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Methods (EURL-SRM), hosted at the Chemisches und Veterinäruntersuchungsamt

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Stuttgart, Schaflandstr. 3/2, 70736 Fellbach, Stuttgart, Germany

Federal Office of Consumer Protection and Food Safety (BVL), National Reference

EU-Reference Laboratory for Residues of Pesticides Requiring Single Residue

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*Corresponding author.

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Phone: +4930-18412-4792; e-mail: [email protected]

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Abstract

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An analytical procedure was developed allowing the simultaneous determination of

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acidic pesticides and their conjugates by addition of an alkaline hydrolysis step into

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the EU version of the QuEChERS method. The procedure resulted additionally in

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hydrolysis of most esters of phenoxy acids. Based on information from metabolism

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studies and the hydrolytic conditions employed in supervised field trials as well as

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results on the influence of physical and chemical parameters (temperature, time, type

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of solvent, type of matrix) alkaline hydrolysis for 30 min at 40 °C was deemed as a

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good compromise for the determination of residues of 2,4-D, dichlorprop, fluazifop,

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haloxyfop, MCPA and MCPB.

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The applicability of the proposed method was tested by analyzing food samples with

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incurred residues in 6 German labs not involved in method development. Up to 6

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times higher residues are measured by using the QuEChERS extraction procedure

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with the new developed alkaline hydrolysis step.

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Keywords

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Acidic pesticides, QuEChERS method, alkaline hydrolysis, LC-MS/MS, EU reference

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method

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1

Introduction

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For several pesticides, the European Union (EU) residue definitions for monitoring

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established within the Regulation on maximum residue levels (MRLs) of pesticides in

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or on food and feed of plant and animal origin1 include esters and conjugates of

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acids. At present, this applies amongst others to 2,4-D, 2,4-DB,

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acibenzolar-S-methyl, dichlorprop(-P), fluazifop-P-butyl, fluroxypyr, haloxyfop(-R),

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MCPA and MCPB. The consideration of conjugates for setting of MRLs follows the

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Guidance Document on the Definition of Residue 2 prepared by the Organization for

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Economic Co-operation and Development (OECD). Inclusion of conjugates in the

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MRL residue definition is recommended when they contribute significantly to the total

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radioactive residue in metabolism studies in plants, livestock, or rotational crops and

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is justified “I due to their potential to be converted back to a biologically active

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compound following hydrolysis in the mammalian digestive system.”

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Conjugation in plants may take place with a multitude of natural compounds (e.g.

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sugars, polysaccharides, amino acids, proteins and plant cuticles containing epoxy

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groups.3,4,5 The chemical structures of these conjugates are thus often not fully

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identified in metabolism studies. In such cases, the MRL regulation refers to

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conjugates in general rather than to specific compounds with defined structures (e.g.

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glucosides or glucuronides). In addition, conjugation with endogenous

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macromolecules may result in the formation of non-extractable residues.6

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Nevertheless, based on the OECD Guidance Document on Pesticide Residue

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Analytical Methods7 analytical methods for pesticide residues should be able to

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measure all components included in the residue definition. If the residue definition

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includes conjugated residues, the method must include appropriate techniques for

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releasing the conjugated moiety. Appropriate techniques to release conjugated

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residues are mentioned in the Residue Chemistry Test Guidelines of the United ACS Paragon Plus Environment

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States Environmental Protection Agency (U.S. EPA).8 “Whenever there are

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indications of the formation of bound components which may not be recovered by the

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extracting solvent but are of toxicological concern, modifications should be made in

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the procedure that will free and recover the liberated components. One such

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modification would be the initial hydrolysis of the treated crop under acidic, alkaline,

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or enzymatic conditions to free the components.”

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The aim of this study was to develop an analytical method for the liberation of acidic

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pesticides from their esters and conjugates. Analytical methods used within the

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framework of residue trials should also conform to methods used for MRL

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enforcement to ensure the correct (i.e. uniform) quantification of residues.

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Unfortunately, metabolism and residue studies are rarely published in the open

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literature by manufacturers of pesticides. Publications on the nature of the conjugates

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formed during metabolism of acidic pesticides in plants are also rare. The most

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extensive information available is on 2,4-D. The most important conjugates of 2,4-D

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in monocotyledonous crops like grasses and cereals are glycoside esters of the

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parent compound 9,10,11 or glycosides of hydroxylated metabolites.5,12 Susceptible

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dicots such as broad-leaf weeds, soybean, sunflower, and tobacco also possess

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these same detoxification mechanisms but to a lesser degree.5,13 These plants

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primarily conjugate 2,4-D with amino acids.13 Both, dichlorprop and its 4-hydroxy

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derivatives undergo conjugation with diglucoses via the carboxyl group and the

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hydroxyl group, respectively.14 Similar to 2,4-D, amino acid conjugates of dichlorprop

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are not observed in cereals and grass. 15,16 Fluazifop-butyl partly degrades to the free

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acid fluazifop 17, 18, which is also conjugated in the plant.19 Condensed information

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on the metabolism of 2,4-D 20, haloxyfop 21, dicamba 22 and MCPA 23 is available

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from reports of the WHO/FAO Joint Meeting on Pesticide residues (JMPR). The

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JMPR evaluations of 2,4-D, haloxyfop and MCPA mention the fast conversion of ACS Paragon Plus Environment

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esters into free acids after application and emphasize the importance of conjugates

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(typically glycosides), which release the free acids after hydrolysis. The liberation of

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conjugated acids is possible by alkaline, acidic or enzymatic hydrolysis or

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combinations thereof. One of the first studies was conducted with incurred residues

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of MCPA in wheat. 24 The authors noticed a higher level of MCPA after alkaline

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hydrolysis compared to hydrolysis under acidic conditions. A second early study on

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incurred 2,4-D residues in potatoes tested acidic hydrolysis only and noticed a

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significant enhancement of free 2,4-D. 25 Non-extractable residues of 2,4-D in wheat

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straw and forage mainly released 2,4-D (free acid) by strong alkaline hydrolysis. 11 A

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combination of acidic and enzymatic hydrolysis was found most appropriate by Løkke

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in his investigation on residues of dichlorprop and 2,4-D in cereals. 26 Chkanikov et

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al. noticed similar recovery of 2,4-D from its glycosides under alkaline and acidic

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conditions.10 By contrast, amino acid conjugates which are minor metabolites in

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cereals, were hydrolysed by acids only. The JMPR report for haloxyfop 21 mentioned

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the necessity of ‘mild’ alkaline hydrolysis to release the free acid from glycosides or

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triglycerides. Glycosidic conjugates of diclorprop were hydrolysed with the enzyme

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cellulose.15, 16 A summary of these and many subsequent studies on the hydrolysis of

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conjugates is given in Table 1, in which it can be observed that most authors

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preferred alkaline hydrolysis, which was mainly applied before extraction with organic

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solvents. Only one author compared hydrolysis before and after the extraction step

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and found fewer problems and better performance with hydrolysis after extraction.27

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Several of the methods presented in Table 1 were used for samples with incurred

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residues and results are compared to extractions without hydrolysis. After applying

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acidic hydrolysis to immature cereal plants, 2,4-D levels increased 3-fold compared

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to the levels determined without hydrolysis.10 In potato tubers the increase following

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acidic hydrolysis was up to 6.8-fold. 25 Following alkaline hydrolysis of citrus fruit the ACS Paragon Plus Environment

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2,4-D levels were reported to increase 1.6- to 2.1-fold in oranges 28, 2.4- to 6.3-fold in

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grapefruit and 2.8- to 5.7-fold in lemons.29 In immature wheat plants MCPA levels

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were reported to increase 6-fold 24 after alkaline hydrolysis. In whole wheat grain

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which was treated with MCPA in the field and with mecoprop post-harvest, the

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increase of MCPA upon alkaline hydrolysis was 7-fold compared to only 1.4-fold in

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the case of Mecoprop.30 A QuEChERS-based method entailing an alkaline hydrolysis

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directly after water-addition to the sample, was employed in these cases. The

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detected residue of dichlorprop in wheat grain from residue studies increased from

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2.3 mg/kg to 7.0 mg/kg (approx. 3-fold) after alkaline hydrolysis.31 In peanut butter,

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sunflower seeds, white bread, cornbread and oranges an increased level of residues

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of 2,4-D and clopyralid was detected after alkaline hydrolysis.32

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Today, the analysis of acidic pesticides is possible by liquid-chromatography-tandem

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mass spectrometry (LC-MS/MS) and no longer requires derivatization. An excellent

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overview on the LC-MS/MS methods used for acidic pesticides was presented by

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Sack et al..32 Extraction and cleanup procedures based on the well-known

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QuEChERS method 33, 34, 35 may be used if the partitioning into acetonitrile from the

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water phase is supported by a lower pH value. 36, 37 Further, the typical cleanup by

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dispersive SPE with primary secondary amine sorbent (PSA) should not be used in

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order to avoid losses of free acids. 32 Due to the high popularity and broad use of the

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QuEChERS method, it was decided that the reference method developed here

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should follow this procedure as closely as possible. Conjugates of acidic pesticides

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are unfortunately not available as analytical standards or reference compounds and

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could thus not be used for method development. Therefore, it was decided to use

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esters of the acidic pesticides for hydrolysis experiments. From prior experience we

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expected that the saponification of esters requires harsher conditions than the

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hydrolysis of conjugates. ACS Paragon Plus Environment

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2

Materials and Methods

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2.1

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Pesticide standards used in this work (purity > 96%) were obtained from Dr.

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Ehrenstorfer (Augsburg, Germany), Sigma-Aldrich Chemie (Seelze, Germany), High

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Purity Compounds (Cunnersdorf, Germany) or Honeywell Speciality Chemicals

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(Seelze, Germany) and listed in Supporting Information, Table S1.

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Deuterium labelled internal standard of pirimicab-d6 (purity 99.8%) and nicarbazin

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(1,3-Bis(4-nitrophenyl)urea- 4,6-dimethyl-2(1H)-pyrimidinone (1:1) (purity 99.4%)

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used as internal standards and QuEChERS extraction salts (4 mg magnesium

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sulfate, 1 g sodium chloride, 1 g trisodium dihydrate, 0.5 g disodium hydrogencitrate

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sesquihydrate, cat. no. 55227-U were purchased from Sigma-Aldrich Chemie

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(Seelze, Germany).

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Deuterium labelled internal standard of 2,4-D-d3 (purity 99.0%) and TRIS

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(tris(hydroxymethyl)aminomethane) (purity 97.0%) used as internal standard were

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purchased from Dr. Ehrenstorfer (Augsburg, Germany). Hydrochloric acid (HCl) 1 M,

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acetic acid 1 M, sodium hydroxide (NaOH) 5 mol/L, 15 mol/L, potassium hydroxide

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(KOH) solution 1 mol/L, sulfuric acid (H2SO4) 2.5 mol/L (5 N), 5 mol/L (10 N) and

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Phenolphthalein (3,3-Bis(4-hydroxyphenyl)-1(3H)-isobenzofuranone) indicator

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solution (ACS, Reg. Ph Eur., 0.1% in ethanol) were purchased from Merck KGaA

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(Darmstadt, Germany).

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The following solvents and chemicals were used: acetonitrile (ULC/MS grade,

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Biosolve BV, Valkenswaard, NL), acetic acid glacial (HPLC grade, Fisher Scientific,

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UK), formic acid (99%, ULC/MS grade, Biosolve BV, Valkenswaard, NL), water

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(deionized, from Milli-Q Advantage A10 System, Millipore).

Chemicals

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2.2

Preparation of standard solutions

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Individual stock solutions of pesticide esters and acids were prepared at 1 mg/mL or

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0.1 mg/L. Working solutions of pesticide esters were prepared from a mixed stock

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solution at 2.5 µg/mL by dilution with acetonitrile/water (1:1 v/v) or blank matrix

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extract resulting in final concentrations from 5 to 250 ng/mL for each pesticide ester.

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Working solutions of pesticide acids were prepared from a mixed stock solution at

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1.25 µg/mL by dilution with acetonitrile/water (1:1, v/v) or blank matrix extract

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resulting in final concentrations from 12.5 to 400 ng/mL.

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2.3

Estimation of the lowest amount of hydroxide required to maintain pH ≥ 12 (tests on sodium hydroxide consumption)

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Food samples from a local supermarket were homogenized at low temperature and

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the pH value of the samples was measured. 5.0 ±0.1 g of the homogenized sample

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were weighed in an Erlenmeyer flask. The samples were tempered for at least 15

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min in a water bath. 10.0 mL of 1 M NaOH solution were added to the sample

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homogenate. The samples were shaken for 2 min and let to stand for 10 min.

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Phenolphthalein indicator solution in ethanol was added. The amount of base was

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titrated with 1 M HCl until the colour of the indicator turned to pink.

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2.4

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Hydrolysis of phenoxy acid esters and fluroxypyr-meptyl in acetonitrile/water solutions

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The experiments were performed in 5 separate screw cap vials. 1.0 mL of a mixed

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pesticide ester working solution (250 ng/mL of each pesticide ester in

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acetonitrile/water, 1/1, v/v) and 10.0 µL internal standard solution (TRIS and

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pirimicarb-d6, 1 µg/mL each) were added to the vials and vortexed for 10 s. To each

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vial 100 µL 1 M KOH were added and the solutions were vortexed for 10 s. The

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reaction was stopped after 60 s, 5 min, 10 min, 30 min or 60 min by addition of

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125 µL acetic acid (1 mol/L). The slightly higher quantity of acetic acid for ACS Paragon Plus Environment

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neutralization ensured a pH < 6 in the final solution. Immediately after stopping the

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reaction the solutions were again vortexed for 10 s. The experiments at 40 °C were

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performed in a water bath. The pH-value of the reaction mixture after neutralization

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was checked by appropriate pH indicator strips. An aliquot of the reaction mixture

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was filtered and transferred into a HPLC vial.

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2.5

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Preliminary tests of QuEChERS extraction combined with alkaline hydrolysis of esters of phenoxy acids

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Organic food samples from a local supermarket were extracted according to the

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following procedure. 10.0 g ± 0.1 g (cucumber, grapefruit); 5.0 g ± 0.05 g (whole

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wheat flour, lentils) and 2.0 g ± 0.02 g (black tea) homogenized sample material was

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weighed in 50 mL disposable screw capped polypropylene centrifuge bottles. For

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whole wheat flour and lentils 10.0 mL cold distilled water were added, immediately

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vortexed and soaked for 15 min. At this stage samples were fortified with mixed

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pesticide ester standard solution resulting in a final level of 0.25 mg/kg pesticide

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ester each and internal standard mixture. To sample homogenates of wheat flour,

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lentils, cucumber and tea 10.0 mL acetonitrile and 0.7 mL 5 N NaOH were added for

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simultaneous extraction and hydrolysis. For the grapefruit sample, 10.0 mL

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acetonitrile and 0.7 mL 15 N NaOH were added. The samples were mixed intensively

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for 10 s and shaken for 30 min in a water bath showing a temperature of 40 ± 0.5 °C.

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The reaction was stopped by addition of 0.35 mL 5 M H2SO4 except for grapefruit

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where 0.7 mL 5 M H2SO4 was added. The pH-value was checked after neutralization.

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After addition of magnesium sulfate, sodium chloride, and buffering citrate salts, the

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mixture was shaken intensively and centrifuged for phase separation. Aliquots of the

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organic phases for all samples except cucumber and tea were stored for 2 h in a

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freezer (-25 °C). After additional centrifugation, the clear supernatants were

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standards were prepared according to the same procedure without fortification. One

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portion of each of the five commodities was fortified with the pesticide ester standard

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mixture and extracted without addition of sodium hydroxide and subsequent

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neutralization with sulfuric acid to control the recovery of the fortified pesticide esters

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and to check whether some free acids are formed when applying the normal

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QuEChERS procedure.

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2.6

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QuEChERS extraction of samples with incurred residues (final reference method)

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Food samples with incurred residues of acidic pesticides were provided by Eurofins

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SOFIA GmbH, Berlin, Germany and Eurofins Dr. Specht Laboratories, Hamburg,

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Germany. Some fresh samples previously identified by the CVUA Stuttgart to contain

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residues of acidic pesticides were also tested. Dry samples (cereal grain, rape seed,

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lentils, dried tomatoes and dried orange) were stored at room temperature in the

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dark. Fresh samples were stored at -25 °C in a freezer.

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10.0 g ± 0.1 g (Brussels sprouts, grapefruit, lime), 5.0 g ± 0.05 g (wheat grain, beans,

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lentils, peas, soybeans, rape seed) or 2.0 g ± 0.02 g (orange dried, tomato dried)

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homogenized sample material were weighed in 50 mL disposable screw capped

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polypropylene centrifuge bottles. For dry samples (lentils, beans, wheat, peas, and

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dried orange, dried tomato), 6.0 mL or 10.0 mL cold distilled water were added and

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soaked for 15 min. 0.1 mL internal standard mixture were added to the samples. For

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extraction and simultaneous hydrolysis 10.0 mL acetonitrile and 1 mL 5 N NaOH

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(wheat grain, beans, lentils, peas, and dried orange) were added. For grapefruit

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samples, 2 mL 5 N NaOH were added. The samples were mixed 10 s intensively and

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shaken for 30 min in a preheated water bath at a temperature of 40 ± 0.5 °C. The

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reaction was stopped by addition of 1 mL 2.5 M H2SO4 except for grapefruit (1.4 mL

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2.5 M H2SO4). After addition of magnesium sulfate, sodium chloride, and buffering ACS Paragon Plus Environment

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citrate salts, the mixture was shaken intensively and centrifuged for phase

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separation. Aliquots of the organic phases for all samples were stored for 2 h in a

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freezer (-25 °C). After additional centrifugation, the clear supernatants were

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transferred to HPLC vials and were ready for analysis. Non-hydrolysed sample

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extracts were prepared in the same way omitting addition of sodium hydroxide and

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subsequent neutralization. Instead of shaking for 30 min at 40 °C samples were

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shaken mechanically for 15 min at room temperature.

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2.7

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Liquid chromatography was achieved using Agilent 1190 system as well as Agilent

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Infinity 1290 system. Each system consisted of a binary pump, a vacuum solvent

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degasser unit, a column oven and a temperature controlled automated liquid sampler

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(Agilent Technologies Deutschland GmbH, Waldbronn, Germany). The Agilent 1190

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system was interfaced to AB SCIEX API 4000 triple Quad (AB SCIEX Instruments)

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mass spectrometer and controlled by Analyst 1.5.1 software.

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The Agilent Infinity 1290 system was interfaced to AB SCIEX API 5500 QTrap (AB

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SCIEX Instruments) and controlled by Analyst 1.6.2 software. Both mass

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spectrometers were equipped with a TUBO V Ion source. The separation was

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performed using Synergi Fusion-RP columns, 150 mm x 2 mm internal diameter, 4

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µm or 2.5 µm particle sizes (Phenomenex, Aschaffenburg, Germany) coupled with

278

the corresponding pre-column (4 mm x 2 mm). The flow rate was 0.2 mL/min or 0.15

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mL/min. The injection volume was 20 µL. Mobile phase A was methanol/water (1/4,

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v/v) and mobile phase B was methanol/water (9/1, v/v), both of which contained 5

281

mM ammonium formate. Directly after injection, elution was performed with 100%

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eluent A for 8 min. The gradient was raised from 100% eluent A to 100% eluent B

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within 3 min. For a further 12 min the elution was performed with 100% eluent B.

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Within 2 min the solvent was changed to 100% A. The total run time was 25 min.

LC-MS/MS analysis


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Electrospray ionization (ESI) in positive or negative mode for analytes was used. For

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the API 4000 triple Quad system separate runs for analytes with ESI in positive mode

287

and negative mode were made. For the API 5500 QTRAP, the scheduled MRM

288

mode was used (MRM detection window 120 s, target scan time 0.6 s). Gas flow, gas

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temperature, and voltages were set as needed for the particular instrument to obtain

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optimum sensitivity (see Table S2 of the Supporting Information for details).

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2.8

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To determine the concentrations of the acids and the residual esters, calibration lines

293

were calculated from the peak areas of calibration solutions. The concentrations of

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the calibration solutions of the esters ranged from 5 to 250 ng/mL and for acids from

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12.5 to 300 ng/mL. The peak area ratio of the standard and the internal standard was

296

used for calculation. The correlation coefficients of the corresponding regression

297

lines were always ≥0.99. Standard solutions in (hydrolysed) blank matrix extracts

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were used to prepare matrix-matched calibration standards.

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3

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3.1 Conditions of hydrolysis and selection of most relevant pesticides

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There are two diverging concepts for the hydrolysis of conjugates and bound

302

residues. In the first case, an attempt is made to hydrolyse all conjugates and bound

303

residues. This requires a stepwise hydrolysis to avoid degradation of liberated

304

compounds, which may occur if too harsh conditions are used from the beginning.

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The second concept is to hydrolyse the main part of conjugates (and bound residues)

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in a milder one-step procedure. As the aim was to develop a procedure for the

307

hydrolysis of esters and conjugates which is widely applicable and acceptable

308

several factors were taken into account. Previous experiments with conjugates have

309

shown that they are relatively quickly hydrolyzed and do not require strong hydrolysis

310

conditions. Applying longer extraction times and higher temperatures did not increase

Validation of the method

Results and Discussion


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the determined residue levels of free acids. The hydrolysis yield of various, especially

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sterically hindered, esters was an important aspect to check. Published data can be

313

found in Table 1. Summarising the results, it becomes clear that alkaline hydrolysis

314

was used in nearly all cases. There was no clear preference with respect to the

315

temperature of the hydrolysis... Since hydrolytic treatments of crop samples widely

316

differed by pesticide, we decided that the proposed reference method should focus

317

on average hydrolysis conditions applied for the most relevant acidic pesticides with

318

regard to residue findings.

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The selection of the most relevant pesticides was done by using data from the

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German Food Monitoring. For detailed explanation and graphical results please refer

321

to figure S1 of the Supporting Information. The pesticide most often found was 2,4-D.

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Fluazifop and haloxyfop followed on rank two and three with 343 and 83 positive

323

findings, respectively. MCPA/MCPB, dichlorprop and fluroxypyr were found in

324

≤0.02% of investigated samples. 2,4-DB was never reported.

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3.2

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Hydrolysis of conjugates and esters depending on temperature, matrix and solvent

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Lowest amount of hydroxide needed for hydrolysis

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Different kinds of fruits and vegetables were treated with sodium hydroxide to

329

estimate the lowest amount of base required to maintain pH ≥ 12 used in field

330

studies. The remaining amount of hydroxide after 30 min treatment at 40 °C was

331

measured by titration with 1 mol/L aqueous HCl. Sample materials like apple,

332

avocado, carrot, cauliflower, cucumber, grapes, kohlrabi, iceberg lettuce, onions,

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oregano and potato “consumed” between 0.03 and 0.2 mmol/g sample. Only highly

334

acidic fruits like lime and lemons consumed more than 0.2 mmol/g. The highest

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amount (0.66 mmol) of NaOH was consumed by one gram of limes. All measured

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sample pH values and the individual NaOH consumption data are listed in Table S4 ACS Paragon Plus Environment

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of the Supporting Information. The pH value of the homogenized samples is not a

338

sufficient indicator for NaOH consumption. Summarizing the treatment with sodium

339

hydroxide, our data show that a maximum of 2 mmol NaOH per g sample are

340

required to maintain a pH ≥ 12 for hydrolysis of conjugates (and esters) in one gram

341

sample material.

342

Timing of hydrolysis step (before or after addition of acetonitrile)

343

Experimental tests of the hydrolysis of conjugates were conducted with cereal grains

344

containing incurred residues of MCPA at a level of 0.284 mg/kg. The sample material

345

was produced in 2007 for the EU proficiency test C1-SRM2.30 The second test was

346

conducted with peas containing residues of fluazifop at a level of 0.362 mg/kg

347

(without hydrolysis). Samples were analysed by three different methods: a) by the

348

standard QuEChERS method without a hydrolysis33, b) by a modified QuEChERS

349

version entailing an alkaline hydrolysis module, which includes the addition of 2 mmol

350

NaOH/g sample directly after addition of water, i.e. prior to the addition of acetonitrile,

351

and c) by a modified QuEChERS version entailing an alkaline hydrolysis module,

352

which includes the addition of 2 mmol NaOH/g sample after the addition of water and

353

acetonitrile. Each of the three methods was conducted at three different

354

extraction/hydrolysis temperatures (room temperature, 40 °C and 60 °C). The results

355

presented in Figure 1 obtained with both sample materials indicate the advantage of

356

conducting the hydrolysis step after the addition of acetonitrile. Interestingly, the

357

obtained amounts of free phenoxy acids were higher at 40 °C (or lower temperature).

358

This is most likely related to the fact that hydrolysis at higher temperatures results in

359

more extensive coagulation of the material causing parts of the material to be

360

inaccessible to hydroxide. When conducting the hydrolysis after the addition of

361

acetonitrile, coagulation was clearly less pronounced. We assume that the strong

362

coagulation at 60 °C when acetonitrile was added after hydrolysis resulted in an ACS Paragon Plus Environment

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363

incomplete wetting of wheat grain with NaOH solution causing the reduced liberation

364

of free MCPA. The results indicated that an amount of 0.5 – 2 mmol NaOH/g sample

365

and a temperature of 40 °C are sufficient to liberate conjugated phenoxy acids.

366

Hydrolysis of esters in the absence of sample matrix

367

Herbicide esters are reported to quickly hydrolyze into their free acids within the

368

plants or the soil and to further form metabolites and conjugates within the plants.

369

There are however bulky esters which are more resistant to hydrolysis in plants and

370

which can thus still be detected in the harvested products (e.g. fluazifop-butyl). For

371

many of the herbicides there are numerous esters in the market. To avoid additional

372

measurements of numerous esters, their nearly quantitative conversion to the

373

respective acids would reduce the need of additional measurements of these esters.

374

Unfortunately, our tests have shown that especially bulky esters of acidic pesticides

375

require quite harsh conditions for hydrolysis. Nevertheless, detection of esters in food

376

samples analysed for MRL compliance or in residue trials is rarely because of fast

377

degradation of esters to the free acids.20, 21As demonstrated in Figure 1, the

378

conjugates are typically easily hydrolysed with the yield of the released free acids

379

reaching a plateau already at relatively mild hydrolysis conditions. Esters of the

380

pesticides can thus be regarded as “worst-case” model compounds for tests of

381

hydrolytic conditions. Three sets of experiments were performed including (1)

382

hydrolysis of pesticide esters in acetonitrile/water depending on temperature, time

383

and the added amount of base, (2) investigations of the influence of plant matrix on

384

reaction rate, and (3) application of the final procedure to samples with incurred

385

residues. In set (1) the degradation of pesticide esters and the concurrent formation

386

of acids in alkaline acetonitrile/water were measured from 1 min to 60 min (a) at room

387

temperature and (b) at 40 °C. In the absence of sample matrix, the added amount of

388

0.1 mmol NaOH/ mL solvent was sufficient to obtain a pH >12. Reaction mixtures ACS Paragon Plus Environment

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389

were measured separately for pesticide esters and acids by LC-MS/MS without

390

cleanup or salting-out steps. Figure 2 shows the degradation of esters and

391

simultaneous appearance of acids at room temperature. After 10 min the residual

392

quantities of esters were below 5% with the exception of fluazifop-butyl, fluroxypyr-

393

meptyl and MCPB-methyl. For these esters, the hydrolysis rate was much slower and

394

60 min were needed to convert ≥90% of the esters to the respective free acid.

395

To investigate the impact of higher temperature on hydrolysis rate, the hydrolysis

396

experiments were repeated at 40 °C. Degradation of esters and formation of acids

397

was much faster (see Figure 3). After 30 min, residual esters were no longer

398

detectable. A hydrolysis time of 30 min seems to be sufficient in absence of matrix.

399

The mean recoveries expressed as sum of detected esters and acids were higher

400

than 90% for all pesticides in these experiments.

401

Hydrolysis of esters in the presence of sample matrix

402

The influence of sample matrix on the hydrolysis was tested by spiking different food

403

samples with esters and hydrolysing them at 40 °C for 30 min as described above. It

404

was expected that high water content commodities, e.g. cucumber, represent the

405

simplest matrix. The samples used for testing the alkaline hydrolysis during

406

QuEChERS extraction were chosen according to the proposed commodity groups

407

stated in the OECD guidance document.7 The selected samples represented the

408

commodity groups of high water content (cucumber), high protein content (lentils),

409

high starch content (wheat flour), and high acid content (grapefruit). Black tea was

410

the representative sample for commodities that are difficult to analyse. The added

411

amount of sodium hydroxide was 0.35 mmol/g sample for cucumber. Because of the

412

half sample weight in combination with an unchanged volume of sodium hydroxide,

413

the added alkali was raised to 0.7 mmol hydroxide/g sample for lentils and wheat

414

flour. For grapefruit, a higher amount of alkali was added (1.1 mmol hydroxide/g) to ACS Paragon Plus Environment

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415

account for the higher acidity of the sample. The detected amounts of residual esters

416

demonstrated a nearly complete hydrolysis for most investigated compounds for

417

cucumber only. For grapefruit, relatively low amounts of residual esters between only

418

23% and 33% of the starting value were detected for fluroxypyr-meptyl and MCPB-

419

methyl. The results in Figure 4 indicated that a) the type of analyte and b) the type of

420

matrix have an influence on the yield of the hydrolysis step.

421

In an additional experiment we have compared the recovery of remaining intact

422

esters after fortification to cucumber when applying hydrolysis before and after

423

addition of acetonitrile. As can be seen in Table 2, this experiment confirmed that

424

hydrolysis after addition of acetonitrile is much more effective with hydrolysis yields

425

being virtually quantitative after 30 min at 40 °C.

426

3.3

427

The final extraction procedure for different sample types is given in Figure 5. The

428

figure shows that compared to QuEChERS without hydrolysis in the worst case

429

(acidic samples with high water content) 3.4 mL additional water, i.e. 2 mL with

430

NaOH and 1.4 mL with H2SO4, are added to acidic samples with high water content.

431

In all other cases, hydrolysis results in a surplus of ≤ 2 mL of water. Our results show

432

that this additional water amount does not prevent the complete phase separation

433

between water and acetonitrile after addition of QuEChERS salts (magnesium sulfate

434

and sodium chloride).

435

The applicability of the proposed reference method for the release of conjugates was

436

tested by analysing 20 different food samples with incurred residues of acidic

437

pesticides. It should be noted that six of these samples contained multiple residues.

438

The analytical results for the pesticides determined after QuEChERS extraction and

439

after application of the reference method are given in Table 3. The highest increase

440

was detected in citrus fruits (grapefruit, oranges and lime). For cereals and dry

Results of samples with incurred residues


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441

legumes, the results were more variable. It is interesting that even in samples of the

442

same matrix (e.g. wheat grain) a difference of up to 115% was observed. A possible

443

reason for this variability might be that the amount of conjugated bound residues

444

depends on the time between application and harvest, the so-called pre harvest

445

interval, the type of formulation employed (salt, ester, acid) and the time elapsing

446

between harvest and sampling/analysis.

447

Additionally, the developed reference method was tested in an interlaboratory trial.

448

Five samples (dried lentils, mungo beans, linseed, yellow peas and rape seed)

449

containing incurred residues were sent to 6 German food monitoring laboratories not

450

involved in the method development as well as to two laboratories involved in the

451

method development. The laboratories analysed the samples by the QuEChERS

452

multiresidue method with and without the hydrolysis module. The measured residue

453

for a pesticide without hydrolysis is set as 100%. The detected residues of 2,4-D in

454

dried lentils were 108 ± 10% after hydrolysis with 10% being the standard deviation.

455

For haloxyfop in mungo beans, the mean residues compared to non-hydrolysed

456

samples raised to 119 ± 22%. The corresponding data for fluazifop were 88 ± 21% in

457

rape seed, 127 ± 18% in peas, 129 ± 42% in mungo beans, 132 ± 36% in dried lentils

458

and 729 ± 170% in linseed, respectively.

459

Summarizing the results, the proposed extension of the widely accepted QuEChERS

460

method by an alkaline hydrolysis modules is applicable for the determination of

461

residues of 2,4-D, dichlorprop(-P), fluazifop(-P), fluroxypyr, haloxyfop(-R), MCPA and

462

MCPB. It was developed considering the results of metabolism studies with these

463

pesticides. As hydrolysis is conducted in presence of the matrix bulk, the method

464

allows the liberation of residues conjugated to non-extractable endogenous

465

macromolecules, which is also done in most residue studies. The early addition of

466

the extraction solvent (acetonitrile) before hydrolysis reduces the time needed for ACS Paragon Plus Environment

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467

hydrolysis and allows the application of lower temperatures. Esters of phenoxy

468

pesticides have been chosen as suitable surrogates for the investigations since

469

defined conjugates were not available as reference materials. Results obtained with

470

samples containing only esters of phenoxy pesticides have shown that the chosen

471

hydrolysis conditions allow at least their detection and often also their quantification

472

as free phenoxy acids. Since the proposed hydrolysis conditions of the new method

473

reflect conditions used in analysis of samples from residue studies quite well, a better

474

matching of MRL setting and MRL enforcement can be reached. The developed

475

method is proposed as reference method for alkaline hydrolysis in order to further

476

harmonize the analysis of residues of acidic pesticides in plant materials in the EU.

477

4

478

The authors wish to thank Ms. Melanie Hoffmann (German National Reference

479

Laboratory on Pesticide Residues located in the Federal Office of Consumer

480

Protection and Food Safety) for thorough laboratory studies during method

481

development and analysis of incurred samples and Mr. Rainer Binner (Federal Office

482

of Consumer Protection and Food Safety) for extracting the relevant German

483

Monitoring data from the National Database. The teams of Eurofins NDSC Food

484

Testing Germany GmbH in Hamburg (Eurofins Dr. Specht Laboratorium, Mr. Manfred

485

Linkerhägner) and Berlin (Eurofins SOFIA GmbH) provided a large number of

486

samples with incurred residues. We would also like to kindly thank the official

487

laboratories from the German Federal States that participated in the interlaboratory

488

exercises.

489

Supporting information available:

490

Tables S1-S4 included information on pesticide standards used in this work (list of

491

pesticide standards, structural formulas, molecular weights and suppliers), MS-MS

492

parameters including transitions, retention times of pesticides and internal standards,

Acknowledgment


Seite 19


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493

gas and source parameters for AB Sciex 5500 Qtrap as well as experimental results

494

on hydroxide consumption by various food commodities.

495

Table S5 listed hydrolysis conditions used in residue studies conducted for MRL

496

setting including references and explanation of content of table.

497

Figure S1 included a description of analysis of data from German food monitoring

498

data including graphical presentation of results, figure S2 and S3 give additional

499

results for samples with incurred residues.

500

Figure S4 included a description of determination of non-volatile coextracted matrix

501

including graphical presentation of results.

502

This material is available free of charge via the Internet at http://pubs.acs.org.

503 504

References

505

(1)

506

lex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:L:2005:070:0001:0016:en:PDF;

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509

and Safety Publications, Series on Testing and Assessment No. 63 and Series on

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Pesticides No. 31, ENV/JM/MONO(2009)30.

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Potential controlled-release herbicides from 2,4-D esters of starches. Weed Science

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plant cuticles. Arch. Environ. Con. Tox. 1986, 15, 97-105.

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Riederer, M.; Schönherr, J. Covalent binding of chlorophenoxyacetic acids to

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dichlorophenoxyacetic acid. 10. Identification of metabolites in rice root callus tissue

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V. P.; Klimov, O. V. Variety of 2,4-D metabolic pathways in plants; its significance in

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N. D.; Smith, G. A. Comparative degradation of [14C]-2,4-dichlorophenoxyacetic acid

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US EPA Residue Chemistry Test Guideline OPPTS 860.1340, Residue

Feung, C.-S.; Hamilton, R. H.; Mumma, R. O. Metabolism of 2,4-

Chkanikov, D. I.; Makeyev, A. M.; Pavlova, N. N.; Grygoryeva, L. V.; Dubovoi

Hamburg, A.; Puvanesarajah, V.; Burnett, T. J.; Barnekow, D. E.; Premkumar,

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549

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(2,4-Dichlorophenoxy)-propionic acid (dichlorprop) in barley (hordeum vulgare). Z.

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and Field Grass. Arch. Environ. Con. Tox. 1988, 17, 81-85.

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H. R. Zum Rückstandsverhalten und Metabolismus von Dichlorprop in Getreide.

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Nahrung 1985, 29, 357-367.

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digitaria sanguinalis resistant to the herbicide fluazifop-P-butyl. Pesticide Biochem.

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Phys. 1997, 57, 137–146.

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fluazifop-butyl in soybean plants. Weed Res. 1992, 32, 143–147.

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Bärenwald, G.; Schneider, B.; Schütte, H.-R. Metabolism of the herbicide 2-

Goedicke, H. J.; Banasiak, U. Fate and Metabolism of Dichlorprop in Cereals

Banasiak, U.; Binner, R.; Franke, G.; Goedicke, H. J.; Gruendel, D.; Schuette,

Hidayat, I.; Preston, P. Enhanced metabolism of fluazifop acid in a biotype of

Balinova, A. M.; Lalova, M. P. Translocation, metabolism and residues of

Clegg, B. S. Gas Chromatographic analysis of fluazifop-butyl (Fusilade) in

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pesticides in fruits and vegetables using SFE – a contribution to reduce analytical

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deficits. 2001, Thesis, University of Hohenheim, Shaker Verlag Aachen (Germany);

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ISBN 3-8265-9618-8 (in German).


Chow, C.; Montgomery, M. L.; Yu, Te C. Methodology and analysis for

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Report on proficiency test on incurred and spiked pesticides in wheat (EUPT-

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C1-SRM2 2007), http://www.crl-

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pesticides.eu/userfiles/file/EurlSRM/EUPT_C1SRM2_FinalReport.pdf, accessed

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Chlorphenoxycarbonsaeure-Herbiciden in Weizenmehl, Z. Lebensm. Unters. For.

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1982, 175, 327-332.

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herbicides using modified QuEChERS with fast switching ESI+/ESI− LC-MS/MS. J.

606

Agric. Food Chem. 2015, 63, 9657-9665.

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easy multiresidue method employing acetonitrile extraction/partitioning and

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dispersive solid-phase extraction for the determination of pesticide residues in

610

produce. J. AOAC Int. 2003, 86, 412-431.

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MS (/MS) following acetonitrile extraction/partitioning and cleanup by dispersive SPE

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- QuEChERS method, 2009, Brussels, Belgium, European Committee for

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Standardization.

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Extraction and Partitioning with Magnesium Sulfate, Gas Chromatography Mass

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Spectrometry and Liquid Chromatography Tandem Mass Spectrometry, First Action

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2007.

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determination of pesticide residues and mycotoxins in fruits, cereals, spices and oil

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seeds employing ultra-high performance liquid chromatography−tandem mass

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Sack, S.; Vonderbrink, J.; Smoker, M.; Smith, R.E. Determination of acid

Anastassiades, M.; Lehotay, S. J.; Stajnbaher, D.; Schenck, F.J. Fast and

EN 15662:2008 Determination of pesticide residues using GC-MS and/or LC-

AOAC Official Method 2007.01 Pesticide Residues in Foods by Acetonitrile

Lacina, O.; Zachariasova, M.; Urbanova, J.; Vaclavikova, M.; Cajka, T.;


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Hua; Shao; Maojun, Jin; Jing; Wang. Simultaneous determination of five plant growth

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regulators in fruits by modified quick, easy, cheap, effective, rugged, and safe

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(QuEChERS) extraction and liquid chromatography–tandem mass spectrometry. J.

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Agric. Food Chem. 2012, 60, 60–65.

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dichlorophenoxyacetic acid (2,4-D) in bean plants. J. Agric. Food Chem. 1964, 12, 3-

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Dichlorophenoxy)acetic acid and bromoxynil from wheat. J. Agric. Food Chem. 1980,

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Rückständen an Pflanzenbehandlungsmitteln nach Clean-up über Gel-

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Chromatographie und Mini-Kieselgel-Säulen-Chromatographie 4. Mitteilung Gas-

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chromatographische Bestimmung von 11 herbiciden Phenoxyalkancarbonsäuren und

639

ihren Estern in Pflanzenmaterial. Fresen. Z. Anal. Chem. 1981, 307, 257-264.

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and hydrolyzable residues of 2,4-dichlorophenoxyacetic acid and 2,4-dichlorophenol

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in potatoes. J. Agric. Food Chem. 1982, 30, 137–144.

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simple method for the determination of five chlorophenoxyalkane carboxylic acids in

645

cereals. Fresen. Z. Anal. Chem. 1989, 334, 133-135.

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thiophanate methyl and 2,4-dichlorophenoxyacetic acid in fruits and vegetables after

648

supercritical fluid extraction. J. Chromatogr. A 1998, 825, 45−54.

Xiaomei, Shi; Fen, Jin; Yuting, Huang; Xinwei, Du; Chunmei, Li; Miao, Wang;

Crosby, D. G. Metabolism of herbicides, metabolites of 2,4-

Cessna, A. J. Simultaneous extraction and detection of residues of (2,4-

Specht, W.; Tillkes, M. Gas-chromatographische Bestimmung von

Bristol, D. W.; Cook, L. W.; Koterba, M. T.; Nelson, D. C. Determination of free

Steinwandter, H. Contributions to the analysis of chlorophenoxy acids - A

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650

acid herbicides in vegetation and soil by liquid chromatography/electrospray-tandem

651

mass spectrometry. J. AOAC Int. 2007, 90, 1402-1410.

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pesticides in cereals with liquid chromatography-tandem mass Spectrometry, master

654

thesis, 2009, http://www.diva-

655

portal.org/smash/record.jsf?pid=diva2%3A302672&dswid=-9324, accessed

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acidic herbicides in cereals by QuEChERS extraction and LC/MS/MS. J. Environ.

659

Sci. Heal. B 2011, 46, 535-543.

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CF), http://www.crl-

662

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version.pdf, accessed 02/08/2016.

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EU Reference Laboratory for Pesticides in Cereals and Feedingstuffs (EURL-


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664

Figure captions

665

Figure 1. Relative amount of MCPA detected in EU proficiency test sample C1-SRM2

666

(wheat) (A); and fluazifop in peas (B) depending on conditions used for extraction

667

and hydrolysis.

668 669

Figure 2. Alkaline hydrolysis of pesticide esters in acetonitrile/water at room

670

temperature. Left: degradation of esters. Right: formation of acids.

671 672

Figure 3. Alkaline hydrolysis of pesticide esters in acetonitrile/water at 40 °C. Left:

673

degradation of esters. Right: formation of acids.

674 675

Figure 4. Percentage of detected residual esters measured by LC-MS/MS after

676

alkaline hydrolysis for 30 min at 40 °C and QuEChERS extraction of different plant

677

commodities spiked with pesticide esters (0.25 mg/kg).

678 679

Figure 5. Flow chart of the proposed reference method.

680 681 682 683 684 685 686 687 688


Seite 27


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Table 1. Conditions for Hydrolysis used in Published Methods for Acidic Pesticides Regulated with Esters and Conjugates point of addition of extraction solvent after hydrolysis

author/ reference

12 mL diethylether

after hydrolysis

Chow et al. 24

Not stated

after hydrolysis

Nelson et al.

3 mL diethylether/ petroleum ether

after hydrolysis

Løkke 26

30 min / 80 °C

22.5 mL diethylether

after hydrolysis

Cessna 39

0.6 – 3.0 mmol NaOH

120 min / 100 °C

12 – 60 mL acetone/ dichlorometha ne (2/1, v/v)

after hydrolysis

Specht et al.40

2,4 D

2.1 mmol H2SO4

60 min / 100 °C

1.5 mL benzene

after hydrolysis

Bristol et al.41

2,4 D, 2,4 DB, dichlorprop, MCPA, MCPB

1.25 mmol NaOH

60 min / 90 °C

10 mL dichlorometha ne

after hydrolysis

Gilsbach et al. 31

fluazifopbutyl

0.3 – 1 mmol NaOH

90 min / 60 °C

0.5 – 1.6 mL dichlorometha ne

after hydrolysis

Clegg 19

2,4 D, 2,4 DB, dichlorprop

4 mmol NaOH

45 min / 100 °C

75 mL acetone/ dichlorometha ne (4/3; v/v)

after hydrolysis

Steinwandter

2,4 D

0.075 mmol K2CO3

20 min / 100 °C

9 mL acetone/ cyclohexane/ ethyl acetate (5/2/2; v/v/v)

after hydrolysis

Anastassiade s et al. 28, 43

2,4 D, 2,4 DB, clopyralid, dicamba, dichlorprop,

1.0 mmol NaOH

>10 h / room temperature

15 mL trichlorometha ne

after hydrolysis

Schaner et al.

pesticide(s)

amount of reagent added (per g sample)

time & temperatur e for hydrolysis

extraction solvent (per g sample)

2,4 D

H2SO4

100 °C

diethylether

MCPA

0.6 mmol NaOH

120 min / 100 °C

2,4 D

6 N H2SO4

100 °C

2,4 D, dichlorprop

enzymatic hydrolysis

120 min / 50 °C

2,4 D

1.25 mmol NaOH

2,4 D, 2,4 DB, dichlorprop, MCPA, MCPB


Crosby 38

25

42

44

Seite 28

Page 29 of 38


MCPA 2,4 D, dicamba, dichlorprop, fluazifop, Fluroxypyr, Haloxyfop, MCPA

0.3 mmol NaOH

30 min / 80 °C

2 mL ethyl acetate + 0.5% acetic acid

after hydrolysis

Östlund 45

2,4 D, Dichlorprop, Fluazifop, Fluroxypyr, MCPA

0.3 mmol NaOH

30 min / room temp.

2 mL acetonitrile

after hydrolysis

Santilio et al.

(not specified)

0.3 mmol NaOH

30 min / room temperature

2 mL acetonitrile

after hydrolysis

EU reference laboratory for pesticides in cereals and feeding stuffs

46

47

2,4 D, 2,4 DB, Clopyralid, Dicamba, Dichlorprop, Fluroxypyr, Haloxyfop, MCPA, MCPB

0.3 mmol NaOH

30 min / room temperature

5 mL acetonitrile + 1% formic acid


after hydrolysis

Sack et al. 32

Seite 29


Page 30 of 38

Table 2. Impact of Alkaline Hydrolysis at various Temperatures and Extraction Times on various Esters Spiked on Cucumber with Hydrolysis being Applied (a) prior to the Addition of Acetonitrile and (b) after the Addition of Acetonitrile at a Spiking Level of 0.2 mg/kg 30 min, 40 60 Min, 40 30 Min, 30 Min, 40 60 Min, 40 °C °C 80 °C °C °C Hydrolysed Hydrolysed before acetonitrile addition, % of after acetonitrile addition, starting value % of starting value 2,4-Dichlorprop0 ethylhexyl 104 65 11 6 0 Fluazifop-butyl 22 12 0 0 0 Fluroxypyr-meptyl 70 29 3 0 0 Haloxyfop-ethoxyethyl 11 6 0 0 0 MCPA butoxyethyl 3 2 0 0 0 Mecoprop-1-octyl 110 70 12 0


Seite 30

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Table 3. Results of Determination of Acidic Pesticides in Samples with Incurred Residues sampl e no.

commodity

sample type

pesticide

1

Lentils, sample 1

1

Lentils, sample 1

2

Lentils, sample 2

2

Lentils, sample 2

3

Lentils, sample 3

4

Mungo bean seed, sample 1 Mungo bean seed, sample 1 Beans dried, sample 1

dry, high starch content dry, high starch content dry, high starch content dry, high starch content dry, high starch content dry, high starch content dry, high starch content dry, high starch content dry, high starch content dry, high starch content dry, high starch content dry, high starch content dry, high starch content dry, high starch content dry, high starch content dry, high starch content dry

4

5

5

Beans dried, sample 1

6

Wheat grain, sample 1

7


8


9

Yellow peas, sample 1

9


10


11


12

Tomato dried

fluazifop

amount (QuEChERS extraction), mg/kg 0.087

amount (reference method), mg/kg 0.112

129

2,4-D

0.10

0.14

138

haloxyfop

0.051

0.053

104

2,4-D

0.042

0.041

98

2,4-D

0.078

0.077

99

haloxyfop

0.613

0.665

108

fluazifop

0.042

0.057

136

haloxyfop

0.403

0.455

113

fluazifop

0.024

0.033

138

2,4-D

0.059

0.127

215

2,4-D

0.164

0.212

129

2,4-D

0.075

0.068

91

2,4-D

0.034

0.037

109

fluazifop

0.053

0.071

134

2,4-D

0.107

0.104

97

fluazifop

0.362

0.482

133

fluazifop

0.036

0.041

114


Seite 31

ratio (QuEChERS = 100%)


13

Soybeans

14

Rape seed, sample 1 Rape seed sample 1 Oranges dried Oranges dried Grapefruit, Grapefruit Lime, fresh Brussels sprouts

14 15 16 17 18 19 20

high fat content high fat content high fat content acidic

Page 32 of 38

fluazifop

0.43

0.52

121

fluazifop

0.49

0.49

100

2,4-D

0.006

0.02

333

2,4-D

0.063

0.089

141

acidic

2,4-D

0.015

0.029

193

acidic acidic acidic High water content

2,4-D 2,4-D 2,4-D fluazifop

0.06 0.078 0.398 0.027

0.41 0.188 0.657 0.027

683 241 165 100


Seite 32

Page 33 of 38


Figure 1


Seite 33


120% Dichlorprop-methyl Fluazifop-butyl Fluroxypyr-meptyl Haloxyfop-methyl MCPA-ethylhexyl MCPB-methyl

100% 80% 60% 40% 20% 0% 0

10

20

30

40

50

60

Amount (% of theoretical value at 100% yield)

Amount (% of initial value)

120%

Page 34 of 38

100% 80% Dichlorprop Fluazifop Fluroxypyr Haloxyfop MCPA MCPB 2,4-D

60% 40% 20% 0%

Time (min)

0

10

20 30 40 Time (min)

Figure 2


Seite 34

50

60


120%

120% 100%

Amount (% of theoretical value at 100% yield)

Amount (% of initial value)

Page 35 of 38

100%

Dichlorprop-methyl Fluazifop-butyl Fluroxypyr-meptyl Haloxyfop-methyl MCPA-ethylhexyl MCPB-methyl

80% 60% 40% 20% 0% 0

10

20 30 40 Time (min)

50

80%

Dichlorprop Fluazifop Fluroxypyr Haloxyfop MCPA MCPB 2,4-D

60% 40% 20% 0%

60

0

10

20 30 40 Time (min)

Figure 3


Seite 35

50

60


Page 36 of 38

Remaining esters (% of initial value)

100% 90%

Dichlorprop-methyl

80%

Fluazifop-butyl Fluroxypyr-meptyl

70%

Haloxyfop-methyl

60%

MCPA-ethylhexyl

50%

MCPB-methyl

40% 30% 20% 10% 0% Lentils

Grapefruit

Cucumber

Tea

Wheat flour

Sampe Type

Figure 4


Seite 36

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Plant material with high water content (≥80%)

Plant material with low water content (

Development of a QuEChERS-Based Method for the Simultaneous

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