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Development and Validation of Ion Chromatography Tandem Mass Spectrometry Based Method for the MultiResidue Determination of Polar Ionic Pesticides in Food Stuart Adams, Jonathan Guest, Michael Dickinson, Richard J Fussell, Jonathan Beck, and Frans Schoutsen J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b00476 • Publication Date (Web): 07 Apr 2017 Downloaded from http://pubs.acs.org on April 8, 2017

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Journal of Agricultural and Food Chemistry

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Development and Validation of Ion Chromatography Tandem Mass Spectrometry Based Method for the Multi-Residue Determination of Polar Ionic Pesticides in Food

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Stuart Adams, 1 Jonathan Guest, 1 Michael Dickinson, 1 Richard J. Fussell, 2 Jonathan Beck, 3 and Frans Schoutsen4

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Fera Science Ltd, Sand Hutton, York, YO41 1LZ, UK; 2Thermo Fisher Scientific, Hemel Hempstead, UK; 3Thermo Fisher Scientific, San Jose, CA, USA; 4Thermo Fisher Scientific, Special Solutions Center, Dreieich, Germany. [email protected] 1

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ACS Paragon Plus Environment


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Abstract

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An extraction method using acidified methanol based on the Quick Polar Pesticide (QuPPe) method

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using suppressed ion chromatography coupled to mass spectrometry for determination was

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developed and validated for the direct analysis polar pesticides, without the need for derivatisation

13

or ion paring, in cereals and grapes. The method was robust and results for glyphosate, AMPA, N-

14

acetyl-AMPA, glufosinate, 3-MPPA, N-acetyl glufosinate, ethephon, chlorate, perchlorate, fosetyl

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aluminium and phosphonic acid at 3 concentration levels (typically 0.01, 0.05 and 0.1 mg/kg) were

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compliant with SANTE/11945/2015 guideline method performance criteria. Cereal based infant food

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proved to be a more challenging matrix and validated only for glyphosate, chlorate and perchlorate

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at 0.005, 0.01 and 0.05 mg/kg. The developed method enables the multi-residue analysis of 11 ionic

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pesticides and relevant metabolites in a single analysis. Until now, the analysis of these compounds

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required several different single residue methods using different chromatographic conditions. This

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multi-residue approach offers the possibility of more cost effective and more efficient monitoring of

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polar ionic pesticides and contaminants that are of concern to food regulation bodies and

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consumers.

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Keywords: glyphosate, chlorate, perchlorate, ion chromatography, mass spectrometry

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Introduction

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The group of polar ionic pesticides include some of the most frequently used pesticides worldwide.

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Although these compounds result in residues in food and have been the subject of recent

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controversy, they have been infrequently monitored in food testing programs. In the U.S. for

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example, a report by the Government Audit Office (1) criticised the responsible government

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agencies (Environmental Protection Agency (EPA), Food and Drug Administration (FDA) and United

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States Department of Agriculture (UDSA)) with respect to the lack of testing for glyphosate residues

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in food. The lack of testing is simply because of the analytical difficulties and higher costs associated

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with the single residue methods that have been available until recently. Historically, pesticides such

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as glyphosate, glufosinate, fosetyl, and alike were analysed individually using specialist methods

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involving derivatisation or ion pairing to overcome unwanted interactions during extraction and

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chromatographic separation.

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Methods for the analysis of glyphosate ([N-(phosphono,metyl) glycine] and its main metabolite

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aminomethyl phosphonic acid (AMPA) include the use of derivatisation with heptafluorobutanol and

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trifluoroacetic anhydride, followed by ion-exchange clean-up prior to GC-MS (2); using 9-fluoroenyl

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methyl chloroformate (FMOC) prior to liquid chromatography mass spectrometry (LC-MS) (3,4,5).

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More recently Montra Piriyapittaya et al reported the microscale membrane extraction of

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glyphosate and AMPA followed by determination using LC- -Fluorescence detection with post

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column derivatisation using 0-pthaldehyde (OPA) reagent (6). Recent advances in mixed mode

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columns have enabled methods based on cation exchange chromatography, for the analysis of

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glyphosate in cereal based crops (7). A review of methods for difficult pesticides in food was

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published by R Raina-Fulton in 2014 (8): including methods for the measurement of glyphosate,

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glufosinate, AMPA

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chromatography) or ion-pair separation prior to LC-MS (Liquid chromatography mass spectrometry)

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determination.

and

3-MPPA

by

derivatisation, and

HILIC


(Hydrophilic

interaction


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Analysis of glyphosate and AMPA based on derivatisation is more successful for water, but such

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methods are limited to glyphosate and glufosinate and some of their metabolites, and issues with

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matrix interferences in water have been reported by M. Ibanez et al (9) and I Freuze et al (10). In our

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experience derivatisation methods are not robust for analysis across a wide range of food matrices.

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Hernandez et al (11) determined residues of fosetyl-aluminium in vegetables by LC-MS after addition

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of tetra butyl ammonium acetate as ion-pairing agent. Although derivatisation and ion-pairing can

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provide some benefits the methods are limited to 1 or 2 analytes and have not always proved to be

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robust for the analysis of food extracts as control of pH is a critical factor.

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Other polar/ionic compounds that are coming under closer scrutiny are chlorate and perchlorate,

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which have been detected at high frequency in food in recent years. Chlorate and perchlorate and

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predominately appear as by-products of biocides used for cleaning food preparation facilities (12,

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13). Chlorate is still currently considered as a pesticide (registered as sodium chlorate New EU

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maximum residue levels (MRLs) have been set for both compounds with challenging limits of

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detection as low as 0.01 mg/kg for some commodities (14). Table I lists the current EU MRLs for

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polar compounds/pesticides included in this study. There are other potential options for the direct

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analysis of these compounds, such as HILIC (15), but suppressed ion chromatography has been our

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technique of choice.

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A new multi-residue approach for the extraction of polar analytes is the Quick Polar Pesticides

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Method (QuPPe) developed by the European Reference Laboratory for Single Residue Methods (16).

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The method is based on acidic methanol extraction without clean-up. Although the method is

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capable of extracting a wide range of polar analytes, the extracts can contain high concentrations of

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matrix-co-extractives that contaminate the instruments. The overall approach also requires the use

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of several different chromatographic separations (including HILIC and non-suppressed ion exchange

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chromatography) to determine all of the anionic analytes listed in the method.


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In this work we have explored the use of Ion-chromatography with post column suppression of the

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eluent to analyse a higher number of anionic analytes in a single analysis of extracts prepared using

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the QuPPe method. Granby et al (17) and Andersen et al (18) used ion chromatography with post

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column suppression coupled to MS for the analysis of glyphosate. In this project we further explored

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the possibilities for the analysis of multiple polar analytes using a modern ion chromatography

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system. Compared to these reports we were able to use KOH mobile phase instead of carbonate

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mobile phases, electrolytic eluent generation and electrolytic post column suppressors in place of

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hollow membrane suppressors. All advancements that provide greater capacity for high

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concentration of matrix and thus greater retention of analytes, improved robustness and stability of

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retention times, improved peak shape and resolution. In addition advancements in MS technology

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provided higher sensitivity especially at low m/z values

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The aim of this work was to investigate the suitability of using suppressed ion chromatography

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coupled to tandem mass spectrometry (IC-MS/MS) for the analysis of polar pesticides including

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glyphosate, AMPA, N-acetyl-AMPA, glufosinate, N-acetyl glufosinate, 3-MPPA, ethephon, chlorate,

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perchlorate, fosetyl aluminium, phosphonic acid, clopyralid, bialaphos and cyanuric acid in a single

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injection and in diverse

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illustrated in Figure I. The extraction method used was the QuPPe-PO (QuPPe method for products

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of plant origin) method, version 9.2 published by the European Union Reference Laboratory for

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single residue methods (CVUA Stuttgart), but with slight operational modifications for the matrices

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used.

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Materials and Methods

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Reagents and Chemicals

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An Elga Purelab® Ultra (Veolia Water Technologies UK, High Wycombe, UK) was used to provide

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deionised water with a purity of 18.2 MΩ. Formic acid (98-100% Certified AR), methanol (HPLC

matrices. The chemical structures of the compounds of interest are



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grade) acetonitrile (Optima UHPLC/MS Grade) and Kinesis Mixed Cellulose syringe filters, (30mm,

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0.22 µm) were purchased from Fisher Scientific (Loughborough, UK).

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Glufosinate ammonium, N-acetyl-glufosinate, 3-methylphosphinicopropionic acid (3-MPPA),

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glyphosate, aminomethyl phosphonic acid (AMPA), phosphonic acid (neat reference standard

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materials) and, ethephon-D4 (solution, 100 ng/µL) were purchased from QMx (Essex, UK). Ethephon,

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fosetyl aluminium, cyanuric acid, clopyralid and cyanuric acid-13C3 were purchased from Sigma-

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Aldrich (Dorset, UK) as neat materials with chlorate and perchlorate as stock solutions at 1000

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µg/mL. Bialaphos, glufosinate-D3 hydrochloride, N-acetyl-glufosinate-D3 disodium salt, 3-

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methylphosphinicopropionic acid-D3 sodium salt and

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Toronto Research Chemicals (Toronto, Canada). N-acetyl-AMPA was purchased from Carbosynth

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Limited (Compton, UK).

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All standards were prepared in deionised water at an approximate stock concentration of 1 mg/mL

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and given a 1 month shelf life. Mixed standards were prepared (excluding fosetyl aluminium and

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phosphonic acid) at 100, 10 and 1 µg/mL. Single solutions of phosphonic acid and fosetyl aluminium

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were prepared at 100, 10 and 1 µg/mL. Separate single standards were prepared due to fosetyl

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aluminium degrading to phosphonic acid.

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Commodities Selected:

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Three different types of matrices were selected for the validation of the method. Oat flour to

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represent group 5 (High starch and/or protein content and low water and fat content) of the

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SANTE/11945/2015 document, grapes to represent group 2 (High acid and high water content).

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Infant food (creamy porridge) represents group 6 (“Difficult or unique commodities”).

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Sample Preparation

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Samples of organic oat flour, grapes and infant food (creamy porridge) were purchased from a retail

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outlet. No further preparation work was carried out on the oat flour which was stored at room

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C215N glyphosate were purchased from


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temperature. The grape sample was cryogenically milled and stored at -20°C until required. The

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creamy porridge was reconstituted, using deionised water, in the proportions recommended on the

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label instructions and stored at -20°C until required.

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QuPPe Sample Extraction

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For all three sample types investigated the QuPPe extraction method (15) was used, but with

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additional steps to reduce the particulate matter in the extracts.

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Homogenised cereal (oat flour) samples (5 ± 0.05 g) were each weighed into 50 mL polypropylene

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centrifuge tubes. Samples were spiked with internal standards and native standards as appropriate

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and left to stand (10 minutes). Deionised water (9.5 mL) was added, followed by acidified methanol

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1% formic acid (10 mL). The extracts were mixed using a rotary shaker for 20 minutes. Samples were

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then were placed in a freezer at -20°C for 10 minutes on a bed of dry ice. Afterwards the samples

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were centrifuged at 4,500 rpm for 5 minutes at 4 °C. Once centrifuged the falcon tubes containing

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the samples were placed on a bed of dry ice to keep the supernatant cold with the supernatant

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filtered through a mixed cellulose syringe filter (0.22 µm) as soon as possible after centrifugation.

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The final extract was diluted 10-fold with deionised water in a plastic 2 mL vial ready for

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determination using IC-MS/MS.

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Matrix-matched calibration standards were prepared by preparing the highest concentration

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calibration standard in matrix blank (spiked with internal standard after extraction) followed by

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serial dilution with the same matrix blank that had been spiked with internal standards after

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extraction. Matrix-matched calibration standards were prepared at, 0.2, 0.1, 0.05, 0.025, 0.01 and

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0.005 mg/kg (0.05, 0.025, 0.0125, 0.00625, 0.0025 and 0.00125 µg/mL before 1/10 dilution), for all

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compounds, except phosphonic acid was at the following concentrations 4, 2, 1, 0.5, 0.2 and 0.1

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mg/kg (equivalent to 1, 0.5, 0.25, 0.125, 0.05 and 0.025 µg/mL before 1/10 dilution) and fosetyl



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aluminium. Matrix-matched standards for fosetyl aluminium were prepared separately at, 4, 2, 1,

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0.5, 0.2 and 0.1 mg/kg.

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Homogenised grape samples (10 ± 0.1 g) were weighed into a 50 mL polypropylene centrifuge tube.

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Samples were spiked with internal standard and native standards as appropriate and left to stand for

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10 minutes. Deionised water (2 mL) was added, followed by 10 mL of acidified methanol (1% formic

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acid). The sample was then placed on a rotary shaker for 20 minutes. Afterwards the samples were

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centrifuged at 4,500 rpm for 5 minutes. Supernatant was then filtered through a mixed cellulose

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syringe filter (0.22 µm). The final extract was diluted 10-fold with deionised water and an aliquot

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transferred to a plastic 2 mL vial ready for IC-MS/MS analysis. Plastic ware was used throughout to

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avoid adsorption of the analytes on to glass surfaces.

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Matrix matched calibration standards were prepared by making the top calibration standard in the

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matrix blank (spiked with internal standard after extraction) and then serial dilution with blank that

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had been spiked with internal standards after extraction. The following concentrations were used to

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calibrate the IC-MS system, 0.2, 0.1, 0.05, 0.025, 0.01 and 0.005 mg/kg (0.1, 0. 05, 0.025, 0.0125,

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0.005 and 0.0025 µg/mL before 1/10 dilution), where phosphonic acid was at the following

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concentrations 1, 0.5, 0.25, 0.125, 0.05 and 0.025 mg/kg (0.5, 0.25, 0.125, 0.00625, 0.0025 and

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0.0125 µg/mL before 1/10 dilution). Separate matrix matched standards for fosetyl aluminium only,

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were prepared at the following concentrations, 4, 2, 1, 0.5, 0.2 and 0.1 mg/kg, and by serial dilution

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of the top calibration standard prepared in matrix.

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For infant food samples the same method was followed as for grapes with the exception that only 1

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mL of deionised water was added. Matrix matched calibration standards were prepared at the

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following levels 0.1, 0.05, 0.025, 0.0.1, 0.005, 0.0025 mg/kg (0.05, 0.025, 0.0125, 0.005, 0.0025 and

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0.00125 µg/mL before 1/10 dilution). Matrix matched standards for fosetyl aluminium only, were

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prepared separately at 0.1, 0.05, 0.025, 0.01, 0.005 and 0.0025 mg/kg.


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IC-MS/MS Analysis

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The IC-MS/MS analysis was performed using a Thermo Scientific ™ Dionex™ ICS-5000+ Reagent

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Free™ HPIC™ System (Sunnyvale, CA, US) coupled to a Thermo Scientific™ TSQ Quantiva™ Triple

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Quadrupole Mass Spectrometer (San Jose, CA, US). The ion chromatography separation column

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system was a Thermo Scientific ™ Dionex™ IonPac™ AS19-4µm (2 x 250 mm, 4µm particle size) with

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a guard column Dionex IonPac™ AG19-4µm (2 x 50 mm) maintained at 30 °C. The eluent flow rate

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was 0.35 mL/min with a gradient from 5 mM KOH (aq) to 20 mM KOH (aq) at 8 minutes, then to 60

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mM KOH (aq) at 12 minutes, held at 60 mM KOH (aq) until 22 minutes and back to 5 mM KOH (aq) at

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22.1 minutes, with a cycle time of 26 minutes. The KOH eluent was neutralised using a Dionex AERS

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500e 2mm electrolytically regenerated suppressor (Thermo Scientific, Sunnyvale CA, US). The

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injection volume was 100 µL of the extract diluted 10-fold with water. Figure II shows the system

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configuration.

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The TSQ Quantiva™ was tuned using an extended mass range solution (Thermo Fisher Scientific P/N

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88340) with the following ions in the negative mode, m/z 69, 113, 302, 602, 1033, 2233 and 2833.

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Multiple-reaction monitoring (MRM) acquisition was conducted in the ESI negative mode. Table II

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lists the details for the MS method. The vaporizer temperature was set to 250 °C and the ion transfer

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tube temperature to 350 °C. The following recommended values were set for gas flows, Sheath Gas,

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42, Auxiliary Gas, 12 and the Sweep Gas, 1 with the spray voltage set to -3,000 V. Thermo Scientific

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™ TraceFinder™ 3.2 software was used for instrument control and data acquisition.

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Data Analysis

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Data analysis was done using TraceFinder 4.0 software. Glyphosate, glufosinate, 3-MPPA, N-acetyl-

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glufosinate, chlorate, perchlorate, ethephon and cyanuric acid were all internally standardised using

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labelled parent compounds. AMPA and N-acetyl-AMPA were internally standardised against N-



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acetyl-glufosinate-D3 as this eluted closely to these compounds in the chromatographic run.

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Bialaphos, phosphonic acid and fosetyl aluminium were not internally standardised. The validation

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results are presented with recoveries calculated using internally standardised external calibration,.

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Calibration criteria were set at the R2 value ≥ 0.95 and residuals for the calibration graph within ±

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20%. Data generated from calibration graphs that do not meet these criteria are identified in the

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relevant tables. Matrix effects were calculated as per equation 1 for all validation batches using data

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not subject to internal standard correction.

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Equation 1.

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Single suppression/enhancement (SSE) (%) = gradient matrix matched standards/gradient neat

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solvent standards x 100

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The SANTE/11945/2015 Guidance document on analytical quality control and method validation

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procedures for pesticides residues in food and feed (19) was used to verify that the method was fit

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for purpose.

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Results and Discussion

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Ion Chromatography Optimisation

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A series of IC separation methods were investigated using the AS19-4 µm column with the starting

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method based on an already established method used at Fera (20). The starting conditions were

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altered to increase the retention of fosetyl aluminium on the system with the method selected

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giving sufficient retention of fosetyl aluminium (at just over 2 column void volumes) and allowing for

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the partial separation of analytes during the 10 - 12 minute region of the chromatogram. To

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maintain acceptable chromatography it was necessary to dilute the extracts 10-fold with water prior

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to the injection of 100 µL. This approach caused less distortion of the peak shape for glufosinate

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compared to an injection of 10 µL of extract without dilution as shown in Figure III


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Optimisation of Mass Spectrometer Method Settings

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The analytes were infused individually into the TSQ Quantiva triple quadrupole MS system. The

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parent ions to product ion transitions were optimised using the automatic optimisation function in

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the TraceFinder software. At least 2 Transitions were selected for each analyte to allow identification

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of analytes of the analytes across the concentration range used in the validation study.

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The addition of an organic solvent modifier, after the conductivity detector (inline after the

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suppressor) and before the MS, was investigated as a possible aid desolvation within the ion source.

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The use of methanol was quickly discounted due to the high viscosity (0.55 cp at 20 °C) which would

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significantly increase the backpressure on the IC electrolytic suppressor. Acetonitrile proved to be

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the most suitable solvent due to its lower viscosity, (0.36 cp at 20 °C). The optimum flowrate was

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found to be 0.2 mL/min, giving a total flow into the source of 0.55 mL/min which is acceptable. The

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analyte responses were improved by a factor of 2.8 – 6.3 as shown in Table III which clearly

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demonstrate the benefits of using a post suppressor modifier. This additional flow of acetonitrile

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was sufficiently low, to ensure the backpressure on the suppressor was below 150 psi.

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The optimum temperatures for the vaporisation and ion transfer tube were investigated and 300°C

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and 250°C gave the best response for the analytes of interest. The optimum electrospray spray

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voltage was -3,000 s.

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Validation Results: Cereal (Oat Flour)

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The cereal matrix was validated at three concentrations 0.01, 0.05 and 0.1 mg/kg (fosetyl aluminium

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and phosphonic acid were validated at 0.2, 1 and 2 mg/kg to reflect the significantly higher EU MRL).

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Sample chromatograms for the analytes in the method are displayed in Figure IV and show that the

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method gives acceptable peak shapes for the analytes included in the method.

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The results of the cereal validation are listed in Table IV. All the calibration graphs that were

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internally standardised pass the calibration criteria, but without the use of internal standard



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correction the following analytes calibration graphs fail the either on residuals tolerance or R2 value

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was less than 0.95 when externally calibrated, AMPA, ethephon, glyphosate, N-acetyl-AMPA, N-

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acetyl-glufosinate and perchlorate. For bialaphos a suitable internal standard was not identified and

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the external calibration failed on residual values exceeding 20%.

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The non-corrected recovery of glyphosate was approximately 50% while the relative (IS corrected)

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recoveries were approximately 90% with acceptable RSDs, all below 10%. The internal standard also

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brings the calibration residuals within the acceptable range. Glyphosate passes the both ion ratio

248

and retention time identification criteria. Glyphosate demonstrated acceptable and stable retention

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as shown in Figure V. AMPA and N-acetyl-AMPA gave both acceptable non-corrected and relative

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recoveries with the exception of the 0.01 mg/kg spike concentration for AMPA and both analytes

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had acceptable RSDs. All identification criteria passed including retention time stability compared to

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the matrix standards.

253

The non-corrected recovery for both chlorate and perchlorate were lower than 70%, however the

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relative recoveries were both approximately 90% with acceptable RSDs, all below 10% for both sets

255

of results. Both compounds pass all the identification criteria for both ion ratio and retention time

256

identification. They are both well retained using this method and there was acceptable retention

257

time stability of chlorate and perchlorate, which is demonstrated in Figure V.

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In oats ethephon non-corrected recovery is lower than 70% at the 0.01 mg/kg spike concentration

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but was acceptable for the 0.05 and 0.1 mg/kg spike concentrations. The relative recoveries were

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between 85% - 104% with acceptable RSDs for both sets of data. At 0.01 mg/kg concentration only

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the quantifier ion is detected, but at the 0.05 and 0.1 mg/kg spike concentrations the ion ratio

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identification criteria pass with identification by retention time passing at all spike concentrations.

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Glufosinate and the two relevant metabolites, 3-MPPA and N-acetyl-glufosinate gave acceptable

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results for the relative recoveries with all 3 analytes giving recoveries greater than 80% and all the


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RSDs were 10% or less. Similar results were observed for the non-corrected recoveries except for

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glufosinate at the 0.01 mg/kg spike concentration level where the recovery average was 42% with a

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very high RSD of 31%. All identification criteria passed for both ion ratios and retention time. All

268

analytes demonstrated acceptable retention on column.

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Validation Results: Grapes

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The grape matrix was validated at three concentrations 0.01, 0.05 and 0.1 mg/kg (fosetyl aluminium

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and phosphonic acid were validated at 0.1, 0.5 and 1 mg/kg to reflect the significantly higher EU

272

MRLs).The results of the validation are listed in Table IV. All the calibration graphs that were

273

internally and externally standardised pass the calibration criteria.

274

For all analytes both the non-corrected and corrected results show acceptable recoveries across all

275

concentrations when both internally and externally standardised. RSDs for the results were all below

276

20%. with the exception of AMPA at the 0.01 mg/kg which gave recoveries outside specification

277

when corrected and when non-corrected gave a high RSD of 22%.

278

For the grape validation all analytes passed the identification criteria in both ion ratio comparisons

279

with the matrix standards and retention time. The retention time stability was similar to those

280

observed in the oats validation batch. Figure VI shows acceptable retention time stability for

281

chlorate, glyphosate and perchlorate in the grapes validation.

282

Validation Results: Infant Food (creamy porridge)

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The infant food matrix was validated at three concentrations 0.005, 0.01 and 0.05 mg/kg .The results

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of the infant food validation are listed in Table V. All the calibration graphs for glyphosate and

285

perchlorate that were internally and externally standardised pass the calibration criteria. Many of

286

the analytes of interest were not detected using this approach. Some analytes were readily

287

detectable with sufficient signal to noise in the matrix matched calibration standards but not in the

288

recovery samples indicating that the analytes were not extracted using the QuPPe method. The



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decision to apply the standard QuPPe method and not evaluate the QuPPe-AO (QuPPe for products

290

of Animal Origin) (21) was based on problems observed at Fera in the past using the QuPPe AO to

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determine glyphosate in milk products, where the glyphosate was not recovered following the clean-

292

up listed in the method (dSPE using ODS octadecylsilane).

293

The results for glyphosate show a similar pattern to those obtained for cereal with low non-

294

corrected recoveries but acceptable relative recoveries with both sets of results giving RSDs of 12%

295

or less. Glyphosate passes all the identification criteria for both ion ratio and retention time

296

identification with Figure VII showing acceptable retention time stability throughout the validation.

297

The results for perchlorate have been corrected to account for a natural incurred residue of

298

perchlorate at the 0.002 mg/kg level, calculated using standard addition (hence the concentration

299

levels have been listed as 0.007, 0.012 and 0.052 mg/kg). Perchlorate passes all the identification

300

criteria for both ion ratio and retention time identification with Figure VII showing acceptable

301

retention time stability throughout the validation.

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Chlorate was also readily detected in all spikes but there was a high incurred residue present in the

303

sample, 0.038 mg/kg (calculated by standard addition) which is significantly higher than the current

304

MRL of 0.01 mg/kg. This highlights one of the problems for the analytes in this method in that

305

organic food should be free from pesticides but analytes such as chlorate and perchlorate should be

306

more correctly considered as processing contaminants. Chlorate displayed acceptable retention time

307

stability as show in Figure VII similar to the previous two validation batches.

308

Method Performance

309

Across the two batches there were problems determining AMPA at the 0.01 mg/kg spike

310

concentration but this analyte gave acceptable results at the 0.05 and 0.1 mg/kg spike

311

concentrations. Cyanuric acid failed the validation in cereals and bialaphos failed in both cereals and

312

grapes. Overall the combination of QuPPe method with determination by IC-MS/MS can extract and


Page 14 of 29

Page 15 of 29


313

accurately quantify a variety of compounds that have weak or strong anionic properties, in both

314

cereals and grapes. In both validation batches stable retention times were observed and all analytes

315

could be confirmed by both ion ratio and retention time when compared to matrix standards. The

316

method can be used for analysis of glufosinate (as the sum of the 3 MRL components) and achieve a

317

LOQ under the assigned MRL and if the glyphosate MRL was to include other components this

318

method would be able to analyse for AMPA and N-acetyl-AMPA.

319

The method performance in infant food was poor due to extraction problems, as many analytes

320

were detected in the matrix standards but not spikes indicating an extraction efficiency problem.

321

Both glyphosate and perchlorate were successfully validated using this method meeting all

322

identification criteria and demonstrating stable retention times. Chlorate was detected in the

323

organic blank sample at a considerable concentration which excludes the use of the data to support

324

method validation using this method.

325

Matrix suppression was calculated for all the three validation batches with the results presented in

326

Table VI. Overall there is no suppression for the majority of the analytes across the three validation

327

batches, the exceptions being AMPA and ethephon in grapes where significant suppression was

328

observed. The low effects of suppression are most likely attributable to the 10 fold dilution of the

329

extracts before analysis.

330

The results presented in this research paper support the use of an alternative approach to the

331

determination approach of polar pesticides. There are many methods that are able to analyse a

332

limited number of polar compounds successfully such as glyphosate and AMPA, (2), glyphosate,

333

glufosinate and AMPA (8) or perchlorate (15). The scope of all of these methods is too narrow. By

334

contrast the IC-MS/MS approach is able to analyse all of these compounds and more in a single

335

analysis without the need for derivatisation or ion-pairing. The ion-chromatography columns have

336

higher capacity than HILIC columns so can withstand a high matrix loading and still provide

337

reproducible chromatography and retention times. Also, the columns are very robust and can be



338

cleaned multiple times , even with strong acids, without loss of resolution. All characteristics which

339

are beneficial in routine analysis. The IC-MS/MS system consists of more components (suppressor,

340

conductivity detector, additional pump for the addition of organic modifier) compared to the single

341

residue methods, but the system is integrated and automated and easy to use. Such a multiple

342

residue method can potentially enable laboratories to provide the additional monitoring results that

343

meet MRLs, comply with the SANTE method performance criteria and are required by regulators to

344

make more informed risk assessments. This is especially important for heavily used pesticides such

345

as glyphosate.

346 347

Overall the results support the application of IC-MS/MS as a multi-residue detection method with

348

successful validations for 12 analytes in cereal matrix, 13 analytes in grape matrix and 2 important

349

analytes (glyphosate, perchlorate) in infant food with the potential for a 3rd if a suitable blank sample

350

is identified. All the listed MRLs in Table I are achievable using this method, including those of

351

glufosinate that have a multi-component listing, and if the MRL for glyphosate should incorporate

352

metabolites the potential for this method to include them has been established.

353 354

Abbreviations Used

355

3-MPPA, 3-methylphosphinicopropionic acid

356

AMPA, aminomethyl phosphonic acid

357

dSPE, dispersive Solid Phase Extraction

358

EPA, Environmental Protection Agency

359

EURL, European Union Reference Laboratory

360

FDA, Food and Drug Administration

361

HILIC, hydrophilic interaction chromatograph

362

IC-MS/MS, ion chromatography tandem mass spectrometry


Page 16 of 29

Page 17 of 29


363

IC, ion chromatography

364

LC-MS, Liquid chromatography mass spectrometry

365

MRL, maximum residue level

366

QuPPe, Quick Polar Pesticide Extraction Method

367

QuPPe-AO, QuPPe for products of Animal Origin

368

QuPPe-PO, QuPPe for products of plant origin

369

USDA, United States Department of Agriculture

370

Acknowledgment

371

The authors would like to thank Dr Sadat Nawaz, pesticide residues team leader at Fera for his

372

support in supplying blank samples for use in validation batches and useful discussions regarding

373

pesticide MRLs.

374

References

375 376 377

1.GAO-15-38, Report to the Ranking Member, Subcommittee on Environment and the Economy, Committee on Energy and Commerce, House of Representatives, URL (http://www.gao.gov/assets/670/666408.pdf) (accessed 17/01/2017)

378 379 380 381 382 383 384

2. Alferness, P.;Wiebe. L. Determination of Glyphosate and Aminomethylphosphonic Acid in Crops by Capillary Gas Chromatography with Mass-Selective Detection: Collaborative Study. J. AOAC Int, 2001, 84, 3, 823-846 3. Poyer, A.; Beguin, S.; Tabet, J.C., Hulot, S.; Reding, M.A.; Communal, P.Y. Determination of glyphosate and aminomethylphosphonic acid residues in water by gas chromatography with tandem mass spectrometry after exchange ion resin purification and dervatization. Application on vegetable matrixes. Anal. Chem. 2000, 72, 16, 3826-3832

385 386 387 388

4.Sancho, J.V.; Hernandez, F.; Lopez, F.J.; Hogendoorn, E.A., Dijkman, E. Rapid determination of glufosinate, glyphosate and aminomethylphosphonic acid in environmental water samples using precolumn fluorogenic labeling and coupled-column liquid chromatography. J. Chrom A, 1996, 737, 1, 75-83

389 390 391

5. Ibanez, M.; Pozo, O.J.; Sancho, J.V.; Lopez, F.J.; Hernandez, F.; Reside determination of glyphosate, glufosinate and aminomethylphosphonic acid in water and soil samples by liquid chromatography coupled to electrospray tandem mass spectrometry. J. Chrom A, 2005, 1081, 2, 145-155

392 393 394 395

6. Piriyapittaya, M.; Jayanta, S.; Mitra, S., Leepipatpiboon, N.; Micro-scale membrane extraction of glyphosate and aminomethylphosphonic acid in water followed by high-performance liquid chromatography and post-column derivatization with fluorescence detector. J. Chrom A, 2008, 1189, 1-2, 483-492



396 397 398

7.Chamkasem, N.; Harmon, T.; Direct determination of glyphosate, glufosinate, and AMPA in soybean and corn by liquid chromatography/tandem mass spectrometry. Anal Bioanal Chem. 2016, 408, 18, 4995-5004

399 400 401 402 403 404 405

8 Raina-Fulton, R; A Review of Methods for the Analysis of Orphan and Difficult Pesticides: Glyphosate, Glufosinate, Quaternary Ammonium and Phenoxy Acid Herbicides and Dithiocarbamate and Phthalimide Fungicides. J. AOAC Int, 2014, 97,4, 965-977

406 407 408

10. Freuze, I.; Jadas-Hecart, A.; Royer, A.; Communal, P.; influence of complexation phenomena with multivalent cations on the analysis of glyphosate and aminomethyl phosphonic acid in water. J. Chrom A, 2007, 1175, 2, 197-206

409 410 411

11. Hernandez, F.; Sancho.; J.V.; Pozo., O.J.; Grimalt, S.; Rapid Determination of Fosetyl-Aluminium Residues in Lettuce by Liquid Chromatography/Electrospray Tandem Mass. J. AOAC Int, 2003, 86, 4, 832-838

412 413 414

12. Kettlitz, B.; Kemendi, G.; Thorgrimsson, N.; Cattor, N.; Verzegnassi, L.; Bail-Collet, Y.L.; Maphosa, F.; Perrichet, A.; Christall, B.; Stadler, R.H.; Food Additives and Contaminants, Part A, 2016, 33, 6, 968-982

415 416

13. Scientific Opinion on the risks to public health related to the presence of perchlorate in food, in particular fruits and vegetables. EFSA Journal, 2014; 12, 10, 3869

417

14. Liaison MRL database, https://secure.fera.defra.gov.uk/liaison/secure/ (accessed 17/01/2017)

418 419

15. Chen, L.; Chen, H.; Shen, M.; Zhou, Z.; Ma. A. Analysis of perchlorate in milk powder and milk by hydrophilic interaction. J. Agric. Food Chem., 2010, 58, 6, 3736–3740

420 421 422 423 424

16. Anastassiades, M.; Kolberg, D.I.; Benkenstein, A.; Eichhorn, E.; Zechmann, S.; Mack, D.; Wildgrube, C.; Sigalov, I.; Dork, D.; Barth, A. Quick Method for the Analysis of numerous Highly Polar Pesticides in Foods of Plant origin via LC-MS/MS involving Simultaneous Extraction with Methanol (QuPPe-Method), version 9.2, http://www.eurl-pesticides.eu/userfiles/file/EurlSRM/meth_QuPPePO_EurlSRM.pdf (accessed 17/01/2017)

425 426 427

17. Granby, K.; Johannesen, S.; Gabrielsen, M.V. Analysis of glyphosate residues in cereals using liquid chromatography mass-spectrometry (LC-MS/MS., Food Addit Contam, Part A, 2003, 20, 8, 692698

428 429 430

18. Andersen, J.H.; Bille, R.L.L.; Granby, K. An intercomparison study of the determination of glyphosate, chlormequat and mepiquat residues in wheat. Food Additives and Contaminants, Part A, 2007, 24, 2, 140-148

431 432

19. Guidance document on analytical quality control and method validation procedures for pesticides residues analysis in food and feed, SANTE/11945/2015,

9. Ibanez, M.; Pozo, O.J.; Sancho, J.V.; Lopez, F.J.; Hernandez, F.; Re-evaluation of glyphosate determination in water by liquid chromatography coupled to electrospray tandem mass spectrometry. J. Chrom A, 2006, 1134, 1-2, 51-55


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433 434

(https://ec.europa.eu/food/sites/food/files/plant/docs/pesticides_mrl_guidelines_wrkdoc_11945.p df) (accessed 17/01/2017)

435 436 437 438

20. PS2538, Improved methodology for the determination of ionic pesticides in dried food commodities, (http://randd.defra.gov.uk/Default.aspx?Menu=Menu&Module=More&Location=None&Completed =0&ProjectID=15441) (accessed 17/01/2017)

439 440 441 442 443

21. Anastassiades, M.; Kolberg, D.I.; Benkenstein, A.; Zechmann, S.; Mack, D.; Barth, A,; Wildgrube, C.; Dork, D.Quick Method for the Analysis of numerous Highly Polar Pesticides in Foods commodities involving Simultaneous Extraction with Methanol and Determination via LC-MS/MS (QuPPe-AOMethod), II Food of Animal Origin, version 2, (http://www.eurlpesticides.eu/userfiles/file/EurlSRM/meth_QuPPe_AO.pdf) (accessed 17/01/2017)

444 445

Figure captions

446

Tables

447

Table I. MRL of selected pesticides in selected commodities Compounds in residue definition Clopyralid Ethephon Fosetyl Al

Cereal mg/kg

Grapes (Table) mg/kg

Infant Food mg/kg

2 1/0.05/1, Barley, Oat, Wheat

0.5 1

0.01 0.01

2

100

0.01

0.1

0.15 (Wine grapes, 0.15 in force as of 14/01/2017)

0.01

20/20/10, Barley, Oat, Wheat

0.5

0.01

Fosetyl-Al † Phosphonic acid

Glufosinate

Glufosinate-ammonium ♦ N-Acetyl-Glufosinateɑ

Glyphosate

3-MPPAɑ AMPA N-Aectyl-AMPA

Perchlorate

448

0.1

Chlorate 0.01 (proposed 0.04) †sum of fosetyl, phosphonic acid and their salts, expressed as fosetyl

449

♦

450

ɑ

0.1

0.02

0.01 (proposed 0.015)

0.01

sum of glufosinate and its salts MPP and NAG expressed as glufosinate equivalents

451 452

Table II. Information on MS/MS transitions (quantification transition in bold) Compound

Retention Time (min)

Fosetyl Al

5.8

Precursor (m/z) 109.1 109.1


Product (m/z) 62.9 78.9

Collision Energy (V) 32 24


Clopyralid

8.7

Chlorate

9.8

IS-Chlorate

9.8

Bialaphos

10.5

Glufosinate

12.2

IS-Glufosinate

12.2

AMPA

11.7

Page 20 of 29

109.1 190 192

80.9 145.8 147.9

13 10 10

83.1 83.1 85.1

66.9 50.9 68.9

22 33 22

89.1 322.2 322.2

70.9 88 94

22 30 31

322.2 180.1

133.9 62.9

31 39

180.1 183.1 110.1

136 62.9 80.9

18 39 13

110.1 152.09

78.9 62.9

31 31

N-acetyl-AMPA

11.7

152.09 152.09 222.2

78.9 110 62.9

33 11 49

N-acetyl-glufosinate

11.7

IS-N-acetyl-glufosinate

11.7

3-MPPA

12.2

IS-3-MPPA

12.2

Phosphonic acid

12.4

222.2 222.2 225.2 151.1 151.1 154.1 81.1 81.1

133.9 136 62.9 62.9 132.9 62.9 62.9 78.8

21 23 49 35 12 35 31 12

Ethephon

12.6

IS-Ethephon

12.6

Glyphosate

15.1

IS-Glyphosate

15.1

Cyanuric acid

15.5

IS-Cyanuric acid

15.5

Perchlorate

19.3

IS-Perchlorate

20.0

143.1 143.1 147.1 168 168 171 128 128 131 99 99 99 101 107

106.9 78.9 110.9 62.9 78.9 63 42 85 43 82.9 66.9 50.9 84.9 88.9

10 21 10 25 40 25 28 13 28 27 39 47 28 27

453


Page 21 of 29

454 455


Table III. Organic modifier (post suppressor) effect on polar pesticide response. All evaluations were undertaken using 20 µL injection volume of a 0.1 µg/mL solvent standard.

Compound

Average Peak Area (n=4) without post suppressor MeCN

Average Peak Area (n=4) with post suppressor MeCN

Response Increase (MeCN/no MeCH)

Glyphosate

307,755

1,294,126

4.2

AMPA N-Acetyl-AMPA

96,480 497,987

463,516 1,854,753

4.8 3.7

Glufosinate 3-MPPA

92,423 726,123

337,068 2,837,665

3.6 3.9

N-AcetylGlufosinate

118,348

426,449

3.6

Perchlorate Chlorate

3,435,072 727,635

14,781,008 3,333,338

4.3 4.6

Ethephon Clopyralid

195,581 458,844

803,967 1,304,068

4.1 2.8

Fosetyl Al Phosphonic acid

414,228 336,819

2,594,673 1,401,104

6.3 4.2

Cyanuric acid

13,645

68,229

5.0

456 457

Table IV. Validation results for polar pesticides in cereal and grapes Cereal (flour) Internally Standardised

Compound

Glyphosate

N-AcetylAMPA †

Glufosinate

Externally Standardised

Concn (mg/kg)

Mean Recovery (n=5)

Mean % RSD

Mean Recovery (n=5)

Mean % RSD

Concn (mg/kg)

Mean Recovery (n=5)

Mean % RSD

Mean Recovery (n=5)

Mean % RSD

0.01

104

3

56

9

0.01

112

15

96

1

0.05

89

4

48

4

0.05

108

12

92

2

0.1

92

2

48

3

0.1

111

7

94

1

11

85

4

0.01

121

10

92

22

10

0.05

111

16

98

13 3

0.01 AMPA †

Grape Internally Standardised

Externally Standardised

69

0.05

89

12

85

100

89

4

80

3

0.1

108

8

97

0.01

89

5

97

6

0.01

100

2

100

3

0.05

80

11

75

9

0.05

93

2

88

2

0.1

84

2

74

1

0.1

99

2

96

2

0.01

120

5

42

31

0.01

100

16

108

2

0.05

88

3

81

6

0.05

109

11

90

2

0.1

91

8

104

9

0.1

109

8

94

2



0.01 3-MPPA

N-AcetylGlufosinate

Perchlorate

Chlorate

Ethephon

94

5

5

0.01

106

17

90

2

0.05

83

2

79

3

0.05

108

13

91

2

0.1

88

2

80

4

0.1

111

7

96

2

0.01

88

7

98

7

0.01

104

15

92

4

0.05

86

10

80

7

0.05

109

13

93

2

0.1

88

2

78

3

0.1

110

7

98

2

0.01

107

1

66

2

0.01

110

17

94

11

0.05

91

6

57

6

0.05

110

12

91

2

0.1

93

3

56

5

0.1

113

6

96

1

0.01

91

5

74

3

0.01

112

19

92

6

0.05

84

1

67

3

0.05

111

12

96

2

0.1

88

1

67

2

0.1

115

6

100

2

0.01

104

10

66

9

0.01

114

17

104

7

0.05

85

7

82

5

0.05

95

14

92

5

0.1

87

7

95

6

0.1

102

10

92

5

67

11

0.01

90

2

76

2

0.05

91

2

81

5

0.1

97

2

60

4

0.1

98

3

92

2

90

2

102

2

97

7

0.01 Clopyralid

95

Page 22 of 29

0.05 0.1

No internally standardised results

0.2 Fosetyl Al

1 2 0.2

Phosphonic acid

1



2

71

4

0.5

72

2

1

106

5

0.1

94

4

0.5




97

2

2

103

2

0.05

75

37

75

37

0.05

116

12

93

4

0.1

74

8

74

8

0.1

113

8

97

3

Cyanuric acid 0.01 Bialaphos

0.05


0.1

89

16

118

13

135

5

Not Detected

458

†Internally standardised with IS-N-Acetyl-Glufosinate

459

Table V. Validation results for glyphosate and perchlorate in infant food Infant Food (creamy porridge) Internally Standardised Externally Standardised Compound

Glyphosate

Perchlorate

Concn. (mg/kg)

Mean Recovery (n=5)

Mean % RSD

Mean Recovery (n=5)

Mean % RSD

0.005

110

5

51

6

0.01 0.05

120 102

12 4

66 58

10 2

0.007 0.012

116 115

2 4

87 89

3 4

0.052

104

1

82

3

460


Page 23 of 29

461


Table VI Matrix effects on mass spectrometer response for polar pesticides SSE % Grapes

Compound

Cereals

3-MPPA AMPA

110.1 89.1

108.1 48.5

Bialaphos Chlorate

127.9 100.8

nm 105.8

Clopyralid Cyanuric acid

113.5 81.8

125.9 71.1

Ethephon Glufosinate

77.6 128.2

15.5 73.0

Glyphosate N-Acetyl-AMPA

135.5 94.5

96.8 101.9

N-Acetyl-Glufosinate Perchlorate

117.5 108.3

105.5 88.3

Phosphonic acid Fosetyl Aluminium

110.7 74.7

124.1 126.9

Infant Food

462

nm = not measured due to interference peak in blank matrix

463

Figure graphics

464

Figure I Chemical structures of polar pesticides

94.5

102.6

89.3

465 466



467

Figure II Ion chromatograph tandem mass spectrometer configuration

468 469 470

Figure III Effect of solvent composition on the peak shape of glufosinate

471 472

Figure IV Chromatograms of polar pesticides in cereal


Page 24 of 29

Page 25 of 29


473

474 475 476



Page 26 of 29

477 478 479

Figure V Retention time stability of polar pesticides in cereals

(

21.5 R e 19.5 t e n 17.5 t m i i 15.5 o n n s 13.5

IS-Chlorate IS-Glyphosate

)

IS-Perchlorate T i m e

11.5 9.5 7.5 2

7

12

17

22

27

Injection Number

480 481

Figure VI Retention time stability of polar pesticides in grapes


32

Page 27 of 29


(

21.5 R e 19.5 t e 17.5 n m t i i 15.5 n o i n s 13.5

IS-Chlorate IS-Glyphosate IS-Perchlorate

) T i m e

11.5 9.5 7.5 2

7

12

482 483

17 22 Injection Number

27

32

Figure VII Retention time stability of polar pesticides in infant food

484

(

21.5 R e 19.5 t e n 17.5 t m i i 15.5 o n n s 13.5

IS-Chlorate IS-Glyphosate

)

IS-Perchlorate T i m e

11.5 9.5 7.5 2

7

12

17

22

27

Injection Number

485 486


32


487

Graphic for table of content

488 489


Page 28 of 29

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