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LC-MS/MS and GC-MS/MS Multi Residue Pesticide Analysis in Fruit and Vegetable Extracts on a Single Tandem Quadrupole Mass Spectrometer

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LC-MS/MS and GC-MS/MS Multi Residue Pesticide Analysis in Fruit and Vegetable Extracts on a Single Tandem Quadrupole Mass Spectrometer 36 May / June 2017 LC-MS/MS and GC-MS/MS Multi Residue Pesticide Analysis in Fruit and Vegetable Extracts on a Single Tandem Quadrupole Mass Spectrometer by Kari Organtini1, Gareth Cleland1, Eimear McCall2, and Simon Hird2 1Waters Corporation, Milford, MA, USA 2Waters Corporation, Wilmslow, UK Hundreds of pesticides are commercially available and are approved for use on various fruit and vegetable plants to prevent pest infestation and improve shelf-life of fresh produce. Maximum Residue Levels (MRLs) are set at the highest level of pesticide that the relevant regulatory body would expect to find in that crop when it has been treated in accordance with good agricultural practice. In the EU, if a pesticide is not explicitly mentioned in the MRL legislation, a default MRL is used for enforcement. This default value is set to be equal to the limit of quantification (LOQ) achievable with the analytical methods used for analysis. National authorities control and enforce MRLs by testing samples for pesticide residue levels using analytical surveillance programs. These programs check for compliance with MRLs, assess dietary exposure, and check for use of unauthorised pesticides. The food industry also carries out its own due diligence analyses. Mass spectrometry (MS) coupled with fragile or easily fragmented compounds. in this study (listed in appendix tables) were both gas chromatography (GC) and liquid APGC ionisation can occur using two chosen to cover a wide range of different chromatography (LC) is needed to provide mechanisms; proton transfer (wet source) pesticide classes and chemistries. The multi comprehensive analysis of a wide range of or charge transfer (dry source). In proton residue MS/MS methods were generated pesticide residues with sufficient sensitivity transfer ionisation, [M+H]+ ions are formed, using the Quanpedia™ database, with to meet global MRL regulations. The use whereas in charge transfer ionisation, M+· separate databases utilised for generation of Quick, Easy, Cheap, Efficient, Rugged ions are formed. of the LC and GC methods. Each database and Safe (QuEChERS) sample extraction contains MRMs and retention time In this work, a single workflow for the and clean up has streamlined analytical information for each compound. When multi residue analysis of pesticides is efficiencies for multi residue analyses [1]. the MS method is generated the MRM demonstrated on a variety of fruit and The advantage of ultra high performance function windows are automatically set for vegetable samples. Utilising the universal liquid chromatography (UHPLC) coupled each compound. For the LC method, a source of Waters Xevo® TQ-S micro mass with tandem quadrupole mass spectrometry window of 1 minute was placed around each spectrometer allows for LC (electrospray (MS/MS) for multi residue pesticide analysis compound’s expected retention time. For ionisation) and GC (atmospheric pressure is widely reported [2]. More recently the use the GC method, a window of 30 seconds ionisation) analyses to be completed of GC-MS/MS utilising atmospheric pressure was used due to the narrower peak widths on the same tandem quadrupole MS ionisation (APGC) has been shown to offer exhibited in GC analysis. In addition to the instrument, with less than 30 minutes significant improvements in performance MS methods, the TargetLynx™ software data needed to switch between chromatographic over EI for challenging pesticides, in terms processing methods and LC inlet method inlets. The performance of the method of selectivity, specificity, and speed of were also generated through the Quanpedia will be highlighted in terms of sensitivity, analysis [3,4]. database. repeatability, and linearity for both LC The APGC source ionises compounds and GC in compliance with the SANTE using a corona discharge at atmospheric guidelines (11945/2015) for pesticide Sample Extraction and Cleanup pressure in an APCI-like manner. Therefore, analysis [5]. this ionisation mechanism is a much softer Celery, lemon, corn, and kale samples technique than classic electron impact (EI) were purchased at a local grocery store. ionisation and produces larger amounts of Methods Samples were chosen to be representative of different types of matrix complexity from intact parent ions, especially in the case of The LC and GC suites of pesticides analysed 37 different commodity groups, including high water content (celery and kale), high acid content (lemon), and high starch/ protein with low water content (corn). Samples were immediately homogenised in a food processer and frozen until sample preparation was performed. QuEChERS extraction was performed according to the official AOAC method 2007.01 using Waters DisQuE™ Dispersive Solid Phase Extraction (d-SPE) product [6]. Figure 1 highlights the sample extraction. Table 1: dSPE clean up conditions used for each sample matrix. Sample MgSO4 PSA GCB Volume Celery 150 mg 25 mg 7.5 mg 1 mL Lemon 150 mg 25 mg - 1 mL Corn 150 mg 25 mg - 1 mL Kale 900 mg 150 mg 150 mg 6 mL Figure 1: DisQuE sample extraction method. Experimental LC-MS/MS Conditions GC-MS/MS Conditions Results and Discussion LC system: ACQUITY UPLC H-Class GC System: 7890A Method Management Using the Column: ACQUITY UPLC BEH C18 Autosampler: CTC PAL RTC Quanpedia Database Column: 30 m x 0.25 mm x 1.7 μm, 2.1 x 100 mm Working with methods involving large 0.25 μm Rxi-5MS Column temp.: 45°C numbers of compounds can be time Carrier gas: Helium Injection volume: 5 μl consuming when done manually and is Flow rate: 2.0 mL/min Flow rate: 0.45 mL/min prone to errors when setting up time Mobile phase A: Water + 10 mM Injection: Splitless segmented acquisition. Quanpedia is ammonium acetate Injector temp: 280°C a compound centric database typically Mobile phase B: Methanol + 10 mM Injection volume: 1 μl used for method generation, but it can ammonium acetate Makeup gas: Nitrogen at 250 mL/min also function as a method management Gradient: Transfer line temp.: 320°C tool. Initial methods for this analysis were Oven program: Time (min) % A % B generated using existing LC and APGC databases (Figure 2). Retention time changes 0.00 98 2 Rate (°C/min) Temp. (°C) Hold (min) resulting from further method development or method changes were updated in the 0.25 98 2 - 80 1.00 database. This allowed for immediate and 12.25 1 99 25 150 0.00 automatic updates to be made in the MS processing methods by re-generating the 13.00 1 99 8 270 0.00 methods with three simple clicks. 13.01 98 2 20 320 4.10 17.00 98 2 MS system: Xevo TQ-S micro Robust and Rapid Ionisation mode: APGC+ Data Acquisition MS System: Xevo TQ-S micro Ionisation For the successful analysis of large Ionisation mode: ESI+ mechanism: Proton transfer numbers of pesticides and their metabolites, Capillary voltage: 1 kV (3 vials of uncapped it is important that the mass spectrometer Desolvation temp.: 500°C water in source) can maintain sufficient sensitivity while Desolvation Corona current: 20 μA for first 3.5 min acquiring MRM transitions with a fast scan gas flow: 1000 L/hr 3.0 μA for rest of run speed in order to provide enough data Source temp.: 150°C Cone gas flow: 0 L/hr points across each chromatographic peak Auxiliary gas flow: 250 L/hr (e.g. minimum of 12 points per peak). Source temp.: 150°C 38 May / June 2017 Figure 2: Quanpedia databases used to manage the methods used for both LC and GC analyses demonstrating the three click workflow of method generation The fast scanning speeds of the Xevo TQ-S micro provide this robust and rapid data acquisition while maintaining large retention time windows to accommodate any shift in retention time due to column maintenance (GC) or chromatography changes caused by the different matrices [6]. Figure 3 highlights one of the busiest sections of the APGC MS method. In this example, flutolanil is just one of approximately 30 pesticides (set across 30 channels, each acquiring at least two transitions per compound) eluting in a 1.5 minute time window. Figure 3: Demonstration of the rapid scanning capabilities of the Xevo TQ-S micro showing the retention of peak quality at a fast scan time. Figure 4: Overlay of a selection of pesticides at 0.010 mg/kg analysed in a celery extract on A. APGC, and B. UHPLC. 39 Figure 5: Matrix matched calibration curves and chromatograms for standards at 0.001 mg/kg for peaks from: A. GC analysis of leptophos in celery and lemon, and B. LC analysis of carbofuran in corn and kale. Figure 6: The percentage of pesticides detected in the 0.01 mg/kg standard for each matrix using both GC and LC. The dwell time calculated for this compound using the autodwell function was 0.006 s. The resulting chromatogram of three replicate injections of 0.010 mg/kg of flutolanil in a celery matrix can be seen in Figure 3. Even with the fast scanning speed, 19 points were collected across the peak and the RSD of three consecutive injections in matrix was 5.2%. The same is true for the LC method used for this analysis. Pesticides in Matrix Matrix matched standards were prepared in celery, lemon, corn, and kale over a range of 0.001 to 0.050 mg/kg, and replicate injections made using the LC and GC methods. A TIC overlay for a selection of pesticides is shown in Figure 4, with 0.010 mg/kg in celery extract from both the A. APGC, and B. UHPLC analyses. The data were fitted with the best fit calibration: for the UHPLC data, the response was shown to be linear, whereas the APGC response over the range investigated was non-linear and Figure 7: Percentage of compounds detected at 0.01 mg/kg in each matrix and associated RSDs.
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