Pesticide Residue Analysis Webinar Series: Tips and Tricks for the Whole Workflow
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This webinar will provide pesticides residue analysts with valuable information on the development and optimization of chromatographic separations and mass spectrometry methods for the analysis of pesticide residues in food. The expert speakers will share their knowledge in understanding the critical aspects of the method, assisting analysts in optimizing their methods for the most challenging analyses.
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Pesticide Residue Analysis Webinar Series: Tips and Tricks for the Whole Workflow
- 1. Pesticide Residue Analysis Webinar Series PART TWO: Workflow Guide for the Use of LCMS Richard Fussell Vertical Marketing Manager, Food and Beverage Claudia Martins Applications Program Manager, Environmental and Food Safety
- 2. 2 Webinar Series Objectives • To provide pesticide residue analysts with valuable information on development and optimization of method workflows for the analysis of pesticide residues in food. • To understand the challenges, limitations and advantages of multi-residue workflows for pesticides residues in food • To review the latest developments in: • sample preparation • targeted and screening techniques using GC-MS/MS and LC-MS/MS • software advances to help with data interpretation and reporting.
- 3. 3 1. Sample Prep: March 24th 2. LC-MS Analysis: April 29th 3. GC-MS Analysis: June 17th 4. Data Processing/Analysis: July 15th Typical Pesticides Workflow Register at www.chromatographyonline.com/LCGCwebseminars
- 4. 4 Thermo ScientificTM TSQ QuantivaTM and TSQ EnduraTM Next generation Triple Quadrupole MS/MS LC-MS Timeline: Developments and Impact Electrospray QQQ-MS 1995 2015 APCI, Single quadrupole MS Karen A Barnes et al, Rapid Comunications in Mass Spectrometry (1995) 9, 1441-1445. Christel L. Hetherton et al, Rapid Communications in Mass Spectrometry, 2004, 18, 2443-2450. Quantification Limits QuEChERS 2002 -‐ 2011 Total # Analytical Results (x1000) Avg # pesticides analyzed per sample Total # residues detected (x10) # of different residues detected Thermo ScientificTM Q ExactiveTM Focus Routine HRAM MS/MS 2013 2000 2005 2010
- 5. 5 Pesticide Residues Analysis Laboratory Sample & Analyte Variability Number of Analytes High Throughput Cost Reduction Screening Identification Quantitation LC-MS & GC-MS Sensitivity
- 6. 6 Different Steps in Analytical Methodology Sample Preparation Chromatographic Separation Detection Data Processing & Reporting
- 7. Original QuEChERS Protocol* Weight 10 g of sample * Anastassiades et al. J AOAC Int., 86 (2) 412-431 Analysis – GC-MS and LC-MS Shake Vigorously 1 min Shake Vigorously 1 min Shake 30s + Centrifuge Shake vigorously 30s + Centrifuge Add 10 mL Acetonitrile Add ISTD - Solution Add 4 g MgSO4 + 1 g NaCl Take Aliquot and Mix with MgSO4 and PSA
- 8. Pros & Cons – Most-used Sample Prep Techniques QuEChERS + Standard Method + Effective + Simple - Contains manual steps - Difficulties when dealing with high fat content and carotinoides or chlorophyll rich samples Dilute & Shoot (Liquid Samples) + Simple - Dilution needed - Highly Sensitive and Robust methods needed - Regular maintenance of instrumentation needed Turbulent Flow Chromatography + Automated - Needs upgrade of standard configuration - Setup can be time-consuming OFFLINE ONLINE
- 9. 9 9 How Does Thermo Scientific TurboFlow Technology Work? • Big particle size + high flow rate turbulent flow • Small molecules diffuse into pores faster than large molecules • Components of interest (analytes) are retained • Matrix components are flushed through • A change in mobile phase composition elutes analytes of interest to the column (or an analytical detector) small moleculescolumn pores analytes analytes
- 10. 10 10 Examples of the Use of Turbulent Flow Chromatography Analytes Application Field Sample TFC column Loading Flow Rate Injection Volume Detection Sensitivity PFOS Environmental Analysis River Water 50 x 1.0 mm, 50µm C18 1 mL/min 1mL APPI-MS 5.35 ng/L (LOD) Anti-Infectives Environmental Analysis Waste Water 50 x 1.0 mm, 50µm C18 XL 3 mL/min 1mL ESI-MS/MS 15-53 ng/L (LOD) Enrofloxacin Ciprofloxacin Food Analysis Edible Tissues 50 x 1.0 mm, 50µm C18 Cyclone 5 mL/min 20µL ESI-MS/MS 25 µg/Kg (LOQ) Quinolones Food Analysis Honey 50 x 0.5 mm, 60µm Cyclone 1.5 mL/min 160µL ESI-MS/MS 5 µg/Kg (MLOQ) Veterinary drugs Food Analysis Milk 50 x 0.5 mm, 60µm Cyclone – Cyclone P (connected in tandem) 1.5 mL/min 50µL ESI-MS/MS 0.1-5.2 µg/L O.Núñez et al. J Chrom A 2012, 1228, 298-323.
- 11. 11 11 Vortex&Centrifugation 0.5 g sample + 990 µl MeOH + IS Squeeze Adjust pH 7 Application of Turbulent Flow Chromatography TFC-ESI-MS/MSanalysis
- 12. 12 12 Prior to Injection Time Savings - Comparison Online Clean Up Method Centrifugation/Filtration ~0.5 g sample in 1 mL solvent (shake briefly) TFC-ESI-MS/MS Analysis ~ 12 min/sample (+ 13 min online clean-up-LC analysis) Manual Method (QuEChERS) d-SPE clean-up (vortex) Supernatant recuperation Liquid-Liquid Extraction (Vortex) Centrifugation ~ 10 g sample in 10 mL solvent (shake briefly) Centrifugation, supernatant recuperation (filtration) ~ 30 min/sample ( + ~10 min LC analysis) LC-ESI-MS/MS Analysis Extraction of solid samples
- 13. 13 13 Configuration of the System Laszlo Hollosi et al. Chromatographia, 2012, 75:1377-1393.
- 14. 0 0 50 100 Polar Pesticides in Food Extracts Acephate in acetonitrile Acephate in acetonitrile-H2O Polar pesticides – dilution of QuEChERS extract necessary before injection to achieve good peak shape Alternatives: • Reduce injection volume • Increase tubing diameter between injector and column
- 15. Time (min) % H2O 5mM Ammonium Formate 0.1% FA % MeOH 5 mM Ammonium Formate 0.1% FA 0.0 98 2 0.5 98 2 2.0 60 40 20.0 5 95 22.0 5 95 22.1 98 2 25.0 98 2 Thermo Scientific™ Accucore™ aQ (100 x 2.1 mm, 2.6 µm) Chromatographic Separation of 440 Pesticides
- 16. 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 Time (min) 0 50 100 0.64 RelativeAbundance Glyphosate MeOH:Water Gradient Elution C18 column 0.2 0.6 1.0 1.4 1.8 2.2 2.6 3.0 3.4 Time (min) 0 50 100 0.80 RelativeAbundance Glyphosate ACN:Water Gradient Elution HILIC column Mix-‐mode: HILIC + Ion-‐Exchange ACN:0.1%HCOOH 600 µL/min AMPA Glyphosate 0 1 2 3 4 5 6 7 8 9 10 11 12 Time (min) RelativeAbundance 2.75 6.25 Chromatography is Important…. 0 50 100 0 50 100
- 17. 17 17 Impact on Maintenance After cleaning After 32 direct injections After 120 direct injections with TLX
- 18. 18 18 Matrix Effects Laszlo Hollosi et al. Chromatographia, 2012, 75:1377-1393.
- 19. 19 19 • Recoveries close to 100% most of compounds in range 80-110% • Small initial sample size <1 g homogenised sample • Multiresidual assay 48 pesticides of different classes - extendable • Minimal Matrix Effects • High Sensitivity all (except chlormequate) fullfills MRL criteria –below 10 ug/kg • Good Intermediate Precision below 25% • High speed total run time 13 min/sample (excl. extraction time) Method Validation Results Laszlo Hollosi et al. Chromatographia, 2012, 75:1377-1393.
- 20. 20 20 Adapted from Alexander Makarov’s presentation, 18th IMSC, Bremen The Ideal Mass Analyzer?
- 21. 21 What Workflow Should I Follow? SAMPLE Target Analysis Non-target Analysis Identification Screening Profiling Fingerprinting Differential Analysis Database & Spectrum Libraries Identification Quantitation
- 22. 22 The Industry’s Leading Portfolio of MS Solutions HRAM MS, MSn Applied Markets Research Markets Non-targeted Analysis Targeted Analysis Quantitative Qualitative • Biomarker Discovery • Proteomics • Metabolism • Metabolomics • Proteomics • Bioanalysis • Food Safety • Environmental • Clinical/Toxicology • Metabolomics • PTM Analysis • Lipidomics Transform Your Science Ion TrapsTriple Quads Tribrid OrbitrapExactives
- 23. 23 Thermo Scientific TSQ Quantiva and Thermo Scientific TSQ Endura Thermo Scientific™ TSQ Endura™ Triple Quadrupole Mass Spectrometer Thermo Scientific™ TSQ Quantiva™ Triple Quadrupole Mass Spectrometer TSQ Quantiva TSQ Endura Mass Range 10-1850 10-3400 Max SRM Number 30,000 SRMs 30,000 SRMs SRM/Sec 500 SRMs/sec 500 SRMs/sec Ion Optics Active Ion Management (AIM) • Ion Max NG source • Electrodynamic ion funnel • ion beam guide with neutral blocker • 6 mm HyperQuad quadrupoles with asymmetric RF drive S-LENS with Beam Blocker Technology Quadrupole Design 4mm Quadrupoles with Asymetric RF Reserpine Specification (RFQ) 100,000 : 1 S/N for 1 pg Reserpine 10,000 : 1 S/N for 1 pg Reserpine Extreme Quantitative Value • Designed for non-stop operation. • For scientists who need to run routine samples day in and day out. Extreme Quantitative Performance • Designed for the most challenging assays. • For scientists needing to stay at the forefront of analytical technology.
- 24. 24 What’s the Alternative to Optimization by Infusion? Select compounds from Thermo Scientific™ TraceFinderTM Compound Database to create instrument method Select Pre-configured TSQ method with LC and SRM conditions included - Load and Go! Edit existing pre-configured method to create new OR OR
- 25. 25 Typical Setup of a LC-QqQ-MS/MS Method 1. Compound Database 2. Master method 4. Data Review & Report 3. Acquisition
- 26. 26 Typical Triple Quad MS Technology in Action Q1 q2 Q3 408,4 408,2 256,2 256,3
- 27. 27 Why use Hyperbolic rods ? • Forms Pure Quadrupolar Fields • Reduces Fringing Field Effects • Significantly Improves Resolution • Improves Transmission • Improves Peak Shapes TSQ Quantum HyperQuads Technology
- 28. 28 The Impact of H-SRM Q1 Q2 Q3 408.8 408.4 0.7 Da 256.3 0.4 Da
- 29. 29 Advantages of H-SRM in the Analysis of Pesticides O.Núñez et al. J Chrom A ., 2012, 1249, 164-180. m/z • Remove background interferences • Smooth baselines • Improve % CV • Increase signal-to-noise
- 30. 30 T-SRM: Pictorial Representation 0 5 10 15 20 25 30 35 40 45 50 55 Time (min) 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 RelativeAbundance 29.85 15.15 29.68 27.16 39.01 34.40 26.28 40.72 51.6734.02 32.99 48.243.99 42.9315.53 36.34 30.18 7.93 55.3922.97 20.40 49.616.44 14.5310.61 47.98 52.102.83 NL: 3.59E6 TIC MS • Instead of continually monitoring all transitions across the entire chromatographic window, SRMs can be monitored in distinct time windows (based on the retention time of the analyte). • Increases number of transitions that can be monitored. • Increases dwell time for each transition monitored. • Therefore improved %CV.
- 31. 31 Overview of Identification Criteria LC-MS/MS http://dx.doi.org/10.1016/j.aca.2015.03.007
- 32. 32 The Use of QqQ for Pesticide Residue Analysis • Compatibility with simple sample preparation: QuEChERS, extract-n-shoot • Compatibility with UHPLC • High Sensitivity BUT… • Targeted analysis only • Limited screening capability of MS/MS (~ 200 compounds) • Data evaluation is time consuming • Multiple instruments to cover whole scope of method
- 33. 33 Where’s the Crossover Point? Kaufmann et al: Anal. Chim. Acta., 2010, 673, 60-72.
- 34. 34 Skip the Matrix: Dummy Transitions Concentrations “…the selectivity of LC-HRMS exceeds LC-MS/MS, if high resolution mass spectrometry (HRMS) data is recorded with a resolution of 50,000 full width at half maximum (FWHM) and a corresponding mass window…” Kaufmann et al: Anal. Chim. Acta., 2010, 673, 60-72.
- 35. 35 COMPANY CONFIDENTIAL Q Exactive & Q Exactive Plus • Orbitrap analyzer • Mass Range m/z 50 - 6000 • Mass Accuracy <1ppm • Max. Mass Resolution >140,000 • Scan speed up to 12Hz MS and MS/MS • Spectral Multiplexing Q Exactive HF • Ultra High Field Orbitrap analyzer • Mass Range m/z 50 - 6000 • Mass Accuracy <1ppm • Max. Mass Resolution >240,000 • Scan speed up to 18Hz MS and MS/MS Exactive Plus (EMR) • Orbitrap analyzer • Mass Range m/z 50 - 6000 • Mass Range EMR: 300 - 20,000 • Mass Accuracy: <1ppm • Mass Resolution >140,000 • Scan Speed up to 12 Hz Q Exactive Focus • Orbitrap analyzer • Mass Range m/z 50 - 2000 • Mass Accuracy <1ppm • Max. Mass Resolution >70,000 • Scan speed up to 12Hz MS and 12 Hz MS/MS • Polarity switching • Top 2 ddMS2 • No Multiplexing • Refined workflows that are optimal for routine laboratories VALUE PERFORMANCE What’s the Alternative to LC-QqQ-MS/MS?
- 36. 36 Typical LC-HRMS workflow QuEChERS LC-HRMS Identification Quantification Reporting Retention Time Exact Mass (± 5 ppm) Product ions Ion ratios Isotopic Pattern Library Matching Compound Inclusion List Screening Identification Reporting Generic Instrument Method
- 37. 37 Pesticides in Fruits and Vegetables by LC-HRMS • QuEChERS • 5 X Dilution Sample Treatment • Accucore aQ (13min) • FS/ddMS2 (Inclusion List) LC-HRMS Data presented at EPRW 2014
- 38. 38 Is Mass Resolving Power important? Data presented at EPRW 2014
- 39. 39 Add Sample Complexity… Data presented at EPRW 2014
- 40. 40 What Scan Mode is Right for Your Workflow? PRM/T-MS2 Full Scan MS Matrix Leek extract spiked with 0.1 ppb imazalil Precursorion@R=70,000Production@R=70,000 3.275 ppm Product Ion M.M.Gomez-Ramos et al. J Chrom A., 2013, 1287, 24-37.
- 41. 41 SANCO/12571/2013 • Method Validation & Quality Control Procedures for Pesticide Residues Analysis in Food & Feed • Implemented 1/1/2014 HRAM criteria ≥ 2 ions (pref. including quasi molecular ion) < 5 ppm mass accuracy At least one fragment ion Resolution typically >20000FWHM
- 42. 42 EU 2002/657/ EC SANCO 12571/2013 EU-RL- MB SOP Gerssen (2010) Mol (2012) Domènech (2014) Kumar (2014) Pitarch (2007) Analytes - Pesticides Lipophilic Toxins Lipophilic Toxins Pesticides Lipophilic Toxins Ronidazole Nitroimidazoles Priority organic micropollutants Matrix Food Food and Feed Molluscs Shellfish Vegetables &Fruits Mussels Muscle Water Technique HRMS HRMS LC-MS/MS LC-MS/MS LC-HRMS/MS LC-HRMS/MS LC-HRMS/MS GC-MS/MS Mass Accuracy - ˂ 5ppm - - ˂ 5ppm ˂ 5ppm ˂ 5ppm - Mass Resolving Power (FWHM) - ≥20,000 - - ≥20,000 ≥20,000 ≥70,000 - Retention Time (RT) Tolerance 2.5% 2.5% Not exceed 3% 5% 1% Mean ±3SD (not relative to time) ±1% Agreement - RT samples & standards Diagnostic Ions ≥ 2 ≥ 2 1 precursor 1 precursor ≥ 2 1 2 1 or 2 precursors Fragment Ions - At least one At least 2 precursor- product transitions 2 products At least one 1 At least 1 >20,000FWHM At least 2 precursor-product transitions Isotope Ions - - - - M+1 M+2 M+1 - - Ion Ratio Relative intensity (% at base peak) Relative intensity (% at base peak) Must be recorded As described 2002/657/EC Fragment Ion Ratio: Diagnostic/Fragment Isotope Ion Ratio: Diagnostic/M+1 (M +2) Fragment Ion Ratio: Diagnostic/ Fragment Isotope Ion Ratio: Diagnostic/M+1 At least one Ratio between quantitative and confirmation transition Fragment-Isotope Ion Ratio Tolerance 2 IPs for precursor ion 2.5 IPs for a product As described 2002/657/EC - As described 2002/657/EC Independent of relative intensity between ions: ±50% As described 2002/657/EC - As described 2002/657/EC Identification Criteria - Other ExamplesP. Lucci and C.P.B. Martins in Fast Liquid Chromatography – Mass Spectrometry Methods in Food and Environmental Analysis - World Scientific Publishing Company (March 2015)
- 43. 43 N. Cortés-Francisco, C. Flores, E. Moyano, J. Caixach. Anal. Bioanal. Chem., 2011 400(10):3595-606. Importance of Mass Accuracy on Unknown Screening 0 50 100 150 200 250 300 350 0.05 0.1 0.5 1 2 3 4 5 6 7 8 9 10 Mass Accuracy (ppm) NumberofPossibleCandidates CHO CHON CHONS CHONSF m/z 498.9302
- 44. Trends in pesticide residue analysis • IC-MS/MS for polar pesticides • Low flow separations (microflow) for highest sensitivity • Fast separation (< 15 min/run) • Matrix effects compensation by dilution (1:100-1000?) • The use of LC-HRMS as an alternative to LC-MS/MS • Multi-analyte screening (eg. pesticide & mycotoxin)
- 45. Conclusions • It is critical to adapt methodology for maximum performance • Sample preparation, chromatography, configuration, method set-up, etc • Different technologies and configurations complement each other to suit multiple needs
- 46. 46 Thermo Scientific Food and Environmental Communities: Resources • View application notes, on-demand webinars, product information, and many more resources on our Pesticides and Food Communities Libraries: www.thermoscientific.com/pesticides www.thermoscientific.com/foodandbeverage