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Article
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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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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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
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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
19
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
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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
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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.
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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
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identification. They are both well retained using this method and there was acceptable retention
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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
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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
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internally and externally standardised pass the calibration criteria.
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For all analytes both the non-corrected and corrected results show acceptable recoveries across all
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concentrations when both internally and externally standardised. RSDs for the results were all below
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20%. with the exception of AMPA at the 0.01 mg/kg which gave recoveries outside specification
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when corrected and when non-corrected gave a high RSD of 22%.
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For the grape validation all analytes passed the identification criteria in both ion ratio comparisons
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with the matrix standards and retention time. The retention time stability was similar to those
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observed in the oats validation batch. Figure VI shows acceptable retention time stability for
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chlorate, glyphosate and perchlorate in the grapes validation.
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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
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perchlorate that were internally and externally standardised pass the calibration criteria. Many of
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the analytes of interest were not detected using this approach. Some analytes were readily
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detectable with sufficient signal to noise in the matrix matched calibration standards but not in the
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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
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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).
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The results for glyphosate show a similar pattern to those obtained for cereal with low non-
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corrected recoveries but acceptable relative recoveries with both sets of results giving RSDs of 12%
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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.
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The results for perchlorate have been corrected to account for a natural incurred residue of
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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
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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
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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
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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
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IC, ion chromatography
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LC-MS, Liquid chromatography mass spectrometry
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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
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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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(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
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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
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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
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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
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0.05 0.1
No internally standardised results
0.2 Fosetyl Al
1 2 0.2
Phosphonic acid
1
No internally standardised results
No internally standardised results
2
71
4
0.5
72
2
1
106
5
0.1
94
4
0.5
No internally standardised results
No internally standardised results
No internally standardised results
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
No internally standardised results
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
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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
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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
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474 475 476
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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
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(
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
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Graphic for table of content
488 489
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