28 February / March 2018 Utilisation of LC/MSMS (QTRAP) and Polarity Switching for the Quantitative Analysis of Over 300 Pesticides in Crude QuEChERS Extracts from Various Fruit and Vegetable Matrices by Laura M. Melton, Michael J. Taylor, Emily E. Flynn, Kirsty B. Reid, Katie J. Viezens, Devanshi S. Vyas, Andrew T. Wilson Science and Advice for Scottish Agriculture (SASA), Roddinglaw Road, Edinburgh, UK, EH12 9FJ Science and Advice for Scottish Agriculture (SASA) is an Official UK laboratory that on behalf of the Scottish Government, participates in statutory UK and EU annual surveillance programmes that monitor UK and imported food & drink for residues of pesticides and their metabolites. LC/MSMS has been a front-line tool in the laboratory for many years but with older instrumentation it was not possible to generate reliable data for all pesticides in a single run. A highly sensitive LC/MSMS instrument has been carry out this statutory work. several analytical proficiency tests each year. commissioned. The instrument is capable of Gas Chromatography coupled with Tandem In this work, a highly sensitive Sciex 6500 simultaneous acquisition of hundreds of MRMs Mass Spectrometry (GC/MSMS) and Liquid (QTRAP) LC/MSMS instrument was used incorporating fast polarity switching without Chromatography coupled with Tandem Mass to improve the workflow. The instrument compromising data or generation of sufficient Spectrometry (LC/MSMS) have been front- is capable of simultaneous acquisition of data points across each peak for quantitation. line tools in official pesticide laboratories hundreds of Multiple Reaction Monitoring However, due to the excellent robustness of for many years. Tandem mass spectrometry (MRM) experiments incorporating fast polarity the system, it has been possible to modify the provides the required specificity to detect switching without compromising data and generic QuEChERS procedure and omit the and quantify hundreds of pesticides in ensuring generation of sufficient data points clean-up step. Consequently, we directly carry complex matrices. As well as the specificity, across each peak for quantitation. The out quantitative analysis of crude extracts. the instrumentation must also have adequate instrument has excellent robustness allowing The above features are demonstrated with sensitivity to comply with the regulations. modification of the QuEChERS procedure, in examples of quantitative surveillance data, order to directly analyse crude extracts without Official control laboratories have implemented UK and EU Proficiency Test data and system the need for a clean-up step. various multi-residue sample preparation performance data. approaches to include many pesticides with The above features will be demonstrated with different physicochemical properties such examples of quantitative surveillance data, Introduction as the Quick, Easy, Cheap, Efficient, Rugged UK and EU Proficiency Test data and system and Safe (QuEChERS) extraction technique performance data. EU legislation requires member states to carry [3] followed by GC and/or LC coupled with out post-approval surveillance monitoring MS. Methods must comply with Method of pesticides in food and feed to ensure that Validation and Quality Control Procedures for Experimental residues do not exceed Maximum Residue Pesticide Residues Analysis in Food and Feed Sample preparation Levels (MRLs) [1]. A MRL is the highest level SANTE guidelines [4]. The aim for laboratories Fruit and vegetable samples submitted as of a pesticide that is legally tolerated in or on is to generate SANTE compliant data for all part of the 2016 UK monitoring programme food and feed. MRLs are not safety limits but pesticides in as few analytical runs as possible. were cryogenically milled using solid carbon the highest level expected when pesticides With older instrumentation it was not possible dioxide and a R23 vertical cutter mixer are used in accordance with good agricultural to generate reliable data for all pesticides in a from RobotCoupe (UK), Middlesex, UK and practice. The regulation covers pesticides single run. Samples often had to be analysed stored at -20°C until required for analysis. currently and previously used in agriculture in using several different approaches in order 10 g of sample were extracted using citrate or out with the EU. For pesticides not listed, a to achieve the required detection limits of 10 QuEChERS extraction method [5]. Q-sep default MRL of 0.01 mg/kg is applied. The co- parts per billion (ppb), to cover all pesticides QuEChERS salts were purchased from ordinated EU multi-annual control programme and to provide sufficient identification points to Restek (Thames-Restek), High Wycombe, sets out for each member state the minimum confirm positive residues. Testing Laboratories UK. The dispersive SPE clean-up step was number of samples to test and which specific must be accredited to international standards omitted. Sample extracts in acetonitrile with pesticide and crop combinations to test [2]. (ISO 17025:2005) and as part of this must equivalent to 1 gml-1 matrix were filtered Member states appoint official laboratories to demonstrate proficiency by participating in 29 through 0.45 µm PTFE syringe filters into Gradient elution: 200 ppb these were diluted into the linear vials for subsequent analysis by LC/MSMS. range of the instrument. Time (min) % Eluent A %Eluent B Pesticides to be detected by LC/MSMS were The position of the probe in the Turbo VTM 0.1 75 25 purchased as neat reference standards of source can greatly affect the sensitivity of purity ≥ 98% from either QMX Laboratories 0.7 40 60 the analysis. It is desirable to ensure that (Thaxted, UK), LGC standards (Teddington, UK) 11.8 2 98 for typical LC flow rates the position of the or Greyhound Chromatography (Birkenhead, 13.8 2 98 electrode is not too close to the orifice of UK). Single stock solutions of each pesticide 14.0 75 25 the mass spectrometer to avoid undesirable were prepared in-house in methanol at components fouling the system. In this concentrations of approximately 400 µg/ml. MS Set-up work the probe vertical micrometer position Newly prepared single stock solutions were was set to 3 mm and the probe horizontal Ionisation compared against old stock solutions as per micrometer position was set to 6 mm. mode: Electrospray in positive SANTE guidelines. Single stock solutions and negative ion mode The use of the advanced Scheduled MRMTM were combined into mixed standards at with polarity switching. Pro algorithm in Analyst software aided the approximately 5 µg/ml for most pesticides Multiple Reaction acquisition of the large number of MRMs and stored at 4-7°C until required for analysis. Monitoring (MRM). whilst still ensuring that there were sufficient Pesticides were grouped into two sets of Ion Source data points across each peak (>10 points mixtures (A and B) to safeguard that pesticides Temperature: 425°C required for quantitation) even in congested known to convert to one another were in Collision (CAD) gas: High areas of the acquisition method. separate mixtures, for example thiophanate- Ionspray voltage: switching between methyl and carbendazim, ensuring that Figure 1a shows the total ion current (TIC) +4500 and -4500V quantitative results could be achieved in positive ion mode (red) and negative ion Curtain gas: 35 psi for positive samples without the need for mode (blue) of a strawberry matrix standard Ionspray gas 1: 60 psi analysis with separate standards. HPLC grade at 10 ppb. Figure 1b shows the TIC of a 2016 Ionspray gas 2: 50 psi methanol and acetonitrile were supplied strawberry sample that contained 16 pesticide Entrance potential: 10 V (positive ion) and by Rathburn Chemicals (Walkerburn, UK). residues in the range 10 – 300 ppb. 15 of -10 V (negative ion) Ammonium acetate was supplied by Fisher these pesticides were acquired in positive Scientific UK, Loughborough, UK. Matrix- ion mode (red) and one was acquired in matched calibration standards at 5 levels Results and Discussion negative ion mode (blue). The extracted ion were prepared in appropriate (organic) fruit chromatograms (XIC) presented Figure 1c There are many considerations when or vegetable matrix that had been extracted show boscalid (+MRM) quantifier and qualifier deciding upon a workflow for pesticide using the citrate QuEChERS extraction transitions for the strawberry matrix standard residue analysis. Hundreds of pesticides method. A mixture of internal standards at 10 ppb and 2016 strawberry sample and their metabolites must be screened containing carbendazim D4, methomyl D3 and containing 16 pesticide residues including for, in complex matrices down to low levels pendimethalin D5 was added to each sample boscalid (+MRM) at 80 ppb. XIC of fludioxonil and positive residues must be quantified and standard as an injection internal standard. (-MRM) quantifier and qualifier transitions for and confirmed with sufficient identification strawberry matrix standard at 10ppb and 2016 A Shimadzu Nexera UHPLC system points in accordance with SANTE guidelines. strawberry sample containing 16 pesticide (Shimadzu, Milton Keynes, UK) was coupled Detection limits are typically 10 ppb for most residues including fludioxonil (-MRM) at 100 with a Sciex 6500 QTRAP mass spectrometer pesticides although some pesticides need ppb are displayed in Figure 1d. (Sciex, Warrington, UK). A Genius 3031 to have lower detection limits since they The figure is typical of the good peak integrity nitrogen generator from Peak Scientific,