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Pesticide Residues Screening and Quantitation Analysis in Olive Oil Using an Orbitrap Exploris 240 HRMS

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Pesticide Residues Screening and Quantitation Analysis in Olive Oil Using an Orbitrap Exploris 240 HRMS thermo scientific APPLICATION NOTE 65901 Pesticide residues screening and quantitation analysis in olive oil using an Orbitrap Exploris 240 HRMS Authors: Francesca Barbetti1, Charles Yang 2, Debora D’Addonna3, Christian Klaas4 1ISVEA S.r.L., Siena, Italy 2Thermo Fisher Scientific, San Jose, CA 3Thermo Fisher Scientific, Radano, Italy 4Thermo Fisher Scientific, Bremen, Germany Keywords: Pesticide Explorer, QuEChERS, Orbitrap Exploris 240 Mass Spectrometer, Orbitrap Analyzer, quantitation, unknown screening, high-resolution accurate mass Introduction (HRAM), olive oil, TraceFinder The demand for quick and simple analysis of large numbers of samples in agriculture applications is growing Goal yearly. Throughout the world, pesticides are used to control To develop and test a multi-residue instrument method pests that are harmful to crops, humans, and animals. that can be applied for high-throughput quantitation and These substances can pose a significant health threat, unknown screening of pesticide residues in food matrices and therefore need to be accurately detected at the levels at or below the current legislative requirements. A high- required by governmental authorities. Typically, maximum resolution, accurate-mass (HRAM) mass spectrometer residue levels for pesticides in different products of plant was operated in Full Scan-Data-Independent Acquisition and animal origin are set at low part-per-billion (ppb) (DIA) mode and using a Thermo Scientific™ AcquireX™ levels. The current regulations present significant analytical Background Exclusion workflow, providing an option for challenges with respect to the low limits of quantification full spectrum filtering, retrospective analysis, and multi- and high number of target analytes. parameter-based compound identification. The method was tested on over 400 target pesticides, in positive and negative mode, with an option for future extension to a larger number of analytes. Most routine LC-based methods usually employ triple Sample preparation Table 1. Option A for content table appearance quadrupole mass spectrometry. In recent years, Thermo Olive oil was purchased from a local market and analyzed Scientific™ Orbitrap™ mass analyzers have become forDolor background sit amet levels ofNiminci pesticides. Approximately 5 g available, providing higher confidence in compound ofRun olive time oil was weighed30 into min a expected 50 mL conical (Fast 96-well) tube. Then, identification and confirmation along with quantitative 10Resolution mL of acetonitrile, 61.5-fold mL of water,changes and for 100singleplex µL of reactioninternal capabilities comparable to triple quadrupole MS/MS. Mass standardExcitation were source added toOptiFlex the tube.™ System This withmixture halogen was lamp capped accuracy (typically below 5 ppm) minimizes interferences andSection shaken separator vigorously for 5 minutes. A Thermo Scientific™ Detection channels*™ Decoupled—6 emission, 6 excitation from co-eluting analytes and matrix co-extractives, and QuEChERS salt slim pack (4000 mg MgSO4, 1000 mg • Optional software module A thus reduces the potential for false positive and negative NaCl,21 CFR 500 p11 mg Na2 citrate and 1000 mg Na3 citrate), which results. This work describes the method performance includescompliance all themodule salts required• Optional for softwarethe extraction module process, B parameters using the latest benchtop LC-Orbitrap wasStoreiciis added. aut The mixtureQuiat was volore, shaken comnis for about aut explaturerum 1 minute instrument, the Thermo Scientific™ Orbitrap Exploris™ and* Asterisked then text centrifugedor other footnote at >3300 rpm for 15 minutes. 240 mass spectrometer and its application to screening A 2 mL aliquot of the supernatant was aspirated and hundreds of target analytes at or below legislative levels filtered through a 0.45 µm filter into a vial. The recovered Table 2. Option B for content table appearance (maximum residue levels – MRLs) in olive oil matrix. supernatant was passed through a lipid removal cartridge 96-well, fast 96-well, 384-well intoBlock an HPLC vial and spiked(runs fast with or standard),the necessary TaqMan calibration Experimental levels.configurations A 1 μL injection Arraywas madeMicrofluidic with the Thermo ™ ™ 30 min expected Reagents, all Fisher Scientific ScientificRun time Vanquish Flex Binary UHPLC system equipped (Fast 96-well); • Acetonitrile, uHPLC-MS grade (P/N A956-1) with a Thermo Scientific™ Accucore™ aQ reversed phase Resolution 1.5-fold changes for singleplex reaction column. • Ammonium formate >99% (P/N A115-50) Excitation source OptiFlex™ System with halogen lamp Detection channels* Decoupled—6 emission, 6 excitation Calibration standard preparation • Methanol, uHPLC-MS grade (P/N A458-1) 21 CFR p11 compliance Optional software module Amodule MegaMix high stock standard was prepared by taking • Formic acid, extra pure for HPLC (P/N 28905) 10* Asterisked µL from text or othereach footnote vial (~1000 μg/mL) and adding them together. For calibration, a 5-level calibration set was • Water, uHPLC-MS grade (P/N W8-1) prepared by post-spiking the control olive oil extract with Table 3. Option C for content table appearance the mixture at concentrations ranging from 0.5 to 500 Consumables (0.5,Dolor 1.0, sit 5, amet* 10, 500)Niminci ppb. All calibration levelsLuptas were injected • Thermo Scientific™ HPLC Vial (P/N A4954-010) sixExample times. 1 Result A Result B Example 2 Result A Result B • Thermo Scientific™ National™ 10 mm Wide Opening InstrumentExample 3 analysisResult A Result B Screw Caps and Septa (P/N C4010-60A) SampleExample analysis 4 wasResult carried A out on a VanquishResult B Flex Binary • Thermo Scientific™ Accucore™ aQ column, 100 × 2.1 mm, UHPLCExample system 5 coupledResult to Aan Orbitrap ExplorisResult B 240 mass 2.6 μm (P/N 17326-102130) spectrometer.Example 6 Result A Result B * Asterisked text or other footnote Standards Pesticides were purchased from Ultra Scientific. See results Table 4. Option D for content table appearance, with optional tables for the identity of all pesticides investigated for paragraph style for content table title that has a lot of text, causing it to be more than two lines. Use Figure Caption paragraph style, as seen targeted analysis. here. Use on all content tables within the same tactic. Dolor sit amet* Niminci Luptas Volup Example 1 Result A Result B Result C Example 2 Result A Result B Result C Example 3 Result A Result B Result C Example 4 Result A Result B Result C Example 5 Result A Result B Result C Example 6 Result A Result B Result C * Asterisked text or other footnote HPLC parameters Data acquisition and processing Data were acquired and processed using Thermo Column 30 ºC Scientific™ TraceFinder™ software to ensure full automation temperature from instrument setup to raw data collection, processing, Flow rate 300 µL/min and reporting. Data acquired from DIA were analyzed with an extraction mass tolerance of ≤5 ppm for both precursor Total run time 15 min and product ions. Analytes were quantified based on full Injection volume 1 μL scan data. In addition, confirmation of target pesticides Column Accucore aQ, 100 × 2.1 mm, 2.6 µm was performed using DIA fragment matching against a curated, high-resolution spectral library. The Thermo A: Water with 5 mM ammonium Scientific™ AcquireX™ Background Exclusion workflow was formate, 0.1% formic acid Mobile phases applied, and the data was extracted with a mass tolerance B: Methanol with 5 mM ammonium of ≤5 ppm for both precursor and product ions. Analytes formate, 0.1% formic acid were first confirmed using a targeted list of pesticides from Gradient Table 1 a compound database and matched with the spectral library. All unknown pesticides were automatically passed to the unknown workflow in TraceFinder software to Table 1. HPLC gradient run program search online compound databases as well as other local Flow rate databases. Time [min] A% B% Curve [mL/min] 0.0 0.300 98 2 5 Results and discussion 1.0 0.300 98 2 5 Simplified in-house validation for screening and semi- 2.0 0.300 50 50 5 quantitative methods was carried out on targeted 9.0 0.300 2 98 5 pesticides. The linearity of the calibration curves was 12.0 0.300 2 98 5 assessed over the range from 0.5 to 500 ng/mL to 12.1 0.300 98 2 5 demonstrate the potential of the method for quantitative 15.0 0.300 98 2 5 analysis. Method selectivity was evaluated by comparison of blank olive oil and spiked samples (n=6). The evaluation was based on accurate precursor m/z value of the analyte Orbitrap Exploris 240 MS settings at the specified retention time window (±0.1 min). Full scan quantitation based upon mono-isotopic match, Spray voltage 3.5 kV Pos / 2.5 kV Neg presence of fragment ions (FI), and high-resolution curated Sheath gas 30 arb pesticide spectral library search (LS) was additionally applied for confirmation according to References 1 and 2. Aux gas 6 arb Acceptance values were set to less than or equal to 5 ppm Sweep gas 1 arb for mass accuracy (FS and MS2), ± 0.1 min for retention Capillary temperature 290 ºC time, reproducibility at LOQ ≤15%, at least one fragment ion present, and for passing LS matching a report out Vaporizer temperature 350 ºC of greater than or equal to 60% and a r2 ≥ 0.9000. The Ion polarity Pos / Neg switching established values are shown in Table 2. Full scan mass range 110 – 1100 m/z Full scan resolution 45,000 Data-Independent 30,000 Acquisition (DIA) resolution HCD collision energy Stepped 18, 35, 60 The methodology showed excellent reproducibility in terms a chromatogram of a 10 ppb matrix-matched standard of a) consistent peak shapes and robust column life, with (MMS) for a method containing over 500 pesticides over 250 injections and b) consistent peak response over with positive and negative polarity switching occurring time. Figure 1 shows select pesticides across the retention throughout the run. Enough data points were obtained time range of the method (0 – 8 minutes), for 250 injections across each peak for accurate quantitation.
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