Fast Analysis of Polar Pesticides in Water by IC-MS/MS Michal Godula, Ph.D. Thermo Fisher Scientific The world leader in serving science
What is Glyphosate • Glyphosate ( N -(phosphonomethyl)glycine) is a broad spectrum systemic herbicide commonly used as weed control. • It is rapidly degraded to aminomethylphosphonic acid ( AMPA ) metabolite frequently found in plants, water and soil. • Very polar, zwitterionic compound • Difficult to retain on C18 LC column 2
Glyphosate in the News • In 2016 • The Munich Environmental Institute group - found glyphosate in 14 of Germany’s most popular beers (0.46 – 29.74 µg/L) • Alliance for Natural Health USA tested 24 popular breakfast foods, 10 of 24 goods had detectable levels of glyphosate (86 – 1,327 µg/kg) (www.anh-usa.org) 3
Polar Pesticides Story : An Imperfect Compromise Food area – ppb levels Water area – ppt levels • QuPPe method with generic extraction using acidified • Derivatization needed (FMOC) to reach 10 ng/l levels with methanol- no partition, no clean-up, LC based separation LC/MS/MS (Hypercarb) • Only Glyphosate/AMPA and Glufosinate analyzed • The QuPPe method developed by EURL-SRM is not perfect – • Derivatization the main obstacle though it can be automated... early eluting compounds, strong matrix effects – compromised LOQs • What about other compounds? Dalapon, Ethephon etc.. • Discussion started at EU level to change the concept 4
Options for the analysis • HPLC, GC-MS/MS after derivatization, not used much nowadays • HPLC-MS/MS (or HRAM) – reversed phase After FMOC derivatization, standard RPC18 column used, can be automated but mostly offline, only for Gly/AMPA/Glufosinate • HPLC-MS/MS (or HRAM) – alternative LC column phase Separation on HILIC, Hypercarb, anion exchange (IC like), Thermo Scientific TM Acclaim TM Trinity, Obelisc etc.. Presented in QuPPe method (food) – not very robust, LOQ in ppb range – not suited for water analysis • IC-MS/MS or HRAM better separation, less matrix effects, good peak shapes, sensitivity matching requirements 5
LC-MS analysis after FMOC derivatization LOD LOQ LOD LOQ Compound (fg on column) (fg on column) ng/L ng/L 1-2.5 1-2.5 AMPA-FMOC 100-250 100-250 1 2.5 Glyphosate – FMOC 100 250 • Column Synchronis Phenyl 50 x 2.1 mm, 1.7 µm • Eluent C: 2 mM Ammonium Carbonate (pH 9) D: Methanol •Injection volume 100 µL •Column Temperature 30 ºC •Flow 300 µL/min 6
Scheme of IC-MS/MS High-Pressure Non-Metallic Makeup Pump AERS regeneration Pump Eluent Generator (OH – or H + ) CR-TC Separation Column Electrolytic Conductivity AS 24 Waste Eluent Detector Suppressor Sample Inject (Autosampler) 0.31 µS Data Management Triple Quadrupole MS/MS 7
Introduction to TSQ Altis and TSQ Quantis TSQ Altis Performance: Sensitivity, Selectivity (H-SRM) Thermo Scientific TM Thermo Scientific TM TSQ Altis TM TSQ Quantis TM High-end Mid-tier Mass Range 5-2000 5-3000 SRM/sec 600 600 Selectivity (H-SRM) 0.2 Da FWHM 0.4 Da FWHM TSQ Quantis Sensitivity 500,000:1 150,000:1 (HESI Reserpine 1 pg) Omics, Research, Pharma/Biopharma, Environmental and Food Safety, Clinical Targeted Market Clinical Research and Forensic Research, and Forensic Toxicology Toxicology Robustness, Reproducibility, Speed, Ease-of-Use, Flexibility 8
TSQ Altis: Sensitivity with Robustness, No Compromises Active collision cell with axial DC field facilitates up to 600 SRMs/sec Ion beam guide with neutral blocker Reduces chemical background High capacity Ion transfer tube (HCTT) Increases ion flux Segmented Quadrupoles with hyperbolic surface for enhanced performance with both SRM and H-SRM (0.2 FWHM) NEW! Electrodynamic ion funnel (EDIF) Increases ion flux OptaMax TM NG Enhanced dual-mode electron multiplier detector NEW! NEW! APCI ready Ensures excellent linearity and dynamic range 9
Figure 2 . Signal improvement using different make-up solvents; Measured at 500 ng/l concentration levels Influence of make-up solvent ACN MeOH IPA 250 Peak area [counts*sec] 200 150 100 50 0 Fosetyl Glufosinate AMPA Clopyralid Glyphosate 10
IC-MS/MS System used for analysis Thermo Scientific TM Dionex Integrion TM Ion Chromatograph: Thermo Scientific TM TSQ Quantis TM Mass Spectrometer: Column: AS24 (2 x 250 mm) Guard Column: AG24 (2 x 50 mm) Eluent: KOH Injection volume: 100 µL Column Temperature: 21 ºC Flow rate: 0.3 ml/min Make-up flow: 0.1 ml/min Make-up solvent: CH 3 OH Time (min) Concentration of KOH in eluent (mM) 0 25 0.2 25 11 80 11.1 100 12.5 100 12.6 25 17.0 25 11
Calibration surface water * Levels 5, 10, 25, 50, 100, 500, 12 1000 ppt, 1 injections each
SRM transitions in surface water, spiked at 50 ng/l level 13
Calibration curves of glyphosate obtained in different matrices Glyphosate Clopyralid 5500 16000 Surface water Surface water 5000 Deionized water Deionized water 14000 Bottled water Bottled water 4500 Drinking water Drinking water 12000 4000 Artificial matrix Artificial matrix 3500 10000 Area Area 3000 8000 2500 6000 2000 4000 1500 1000 2000 500 0 0 200 400 600 800 1000 0 0 200 400 600 800 1000 c (ng/l) c (ng/l) 14
Possible suppression sources: anions present in different matrices 1. Artificial matrix Cl � 2. Drinking water 1. 3. Surface water 4. Bottled water 5. Deionized water μ S �� SO � 2. AMPA Clopyralid Fosetyl-Al Glufosinate Glyphosate � 3. NO � 4. 5. Time (min) 15
Bottled mineral water performance LOD LOQ RSD % Name [ppt] [ppt] (10 ppt) level Fosetyl-Al 1 2.5 5 Clopyralid 10 50 9 AMPA 2 5 9 Glyphosate 5 10 15 Glufosinate 2 5 4 16
Expected signal suppression and LOQ in heavy matrix Artificial water* Surface Water Analyte LOD (ng/l) LOQ (ng/l) LOD (ng/l) LOQ (ng/l) Fosetyl-Al 25 50 2.5 5 Glufosinate 5 10 5 10 AMPA 25 50 5 10 Clopyralid 250 500 10 50 Glyphosate 10 25 5 10 *Consisted of: 250 mg/l Cl - and SO 4 2- 150 mg/l HCO 3- 20 mg/l NO 3- 17
Validation results Component Matrix LOD [ppt]] LOQ [ppt] REC [%] RSD [%] 10 ppt 20 ppt 50 ppt 10 ppt 100 ppt 1000 ppt Drinking 133 122 132 9.0 1.3 0.7 2.5 5 Fosetyl Bottled 121 123 128 2.0 0.9 1.1 1 2.5 Surface 105 105 104 2.0 1.1 0.6 2.5 5 Drinking 133 121 95 9.3 2.4 0.9 5 10 Glufosinate Bottled 56 112 96 4.1 3.4 0.8 5 10 Surface 124 111 93 2.2 1.6 1.2 5 10 Drinking 5 10 91 93 83 9.1 2.3 0.8 AMPA Bottled 109 107 95 8.7 2.1 0.8 5 10 Surface 91 100 98 4.5 3.7 1.0 5 10 Drinking 110 88 90 11.9 1.4 1.0 10 50 Clopyralid Bottled 53 89 86 9.1 1.4 0.9 5 10 Surface 5 10 109 114 140 10.0 1.4 0.6 Drinking 89 106 83 6.3 2.3 1.3 10 50 Glyphosate Bottled 66 108 105 14.4 1.7 3.4 10 50 Surface 82 101 90 5.0 10.1 1.8 10 50 18
IC vs. LC arguments Ion Chromatography Liquid Chromatography Matrix Food Water Food Water Sample preparation Extraction None Extraction (QuPPe) FMOC* Derivatization (QuPPe) (filtration) (optional FMOC* (SPE cleanup) derivatization) (automation possible) Separation quality Excellent Excellent Poor Very good (Hypercarb) (Reversed Phase) LOQs 1-10 ppb 5 -10 ppt 1 – 10 ppb 5 – 10 ppt MS/MS **TSQ Endura **TSQ Quantiva **TSQ Quantiva **TSQ Quantiva (Endura not suitable) Matrix tolerance Very good Very good Limited Good Time consumption Low Low Low - medium Medium – High (automation possible) *FMOC derivatization applicable only for Gly, AMPA, Glu! **Thermo Scientific™ TSQ™ Endura, Thermo Scientific™ TSQ™ Quantiva™ 19
Food Analysis; chromatogram in lettuce at 10 ug/kg Fosetyl-Al HEPA N-acetyl-AMPA Phosphonic Glyphosate acid N-acetyl-Glufosinate MPPA Ethephon Glufosinate AMPA 20
Calibration curves – lettuce – range: 0 – 20 ppb II. Glufosinate Glyphosate MPPA N-acetyl-AMPA 21
Repeatability and recovery in lettuce matrix Level 1 - 1 ppb Level 2 - 5 ppb Level 3 - 10 ppb Compound RSD % REC % RSD % REC % RSD % REC % Phosphonic acid 14 86 5 102 3 102 Fosetyl 4 85 1 91 2 93 AMPA 15 82 3 92 2 94 Glufosinate 10 79 2 92 3 93 N-acetyl-glufosinate 5 81 2 92 2 95 Ethephon-hydroxy 3 78 4 87 2 92 N-acetyl-AMPA 5 88 2 90 2 92 MPPA 5 81 3 86 2 91 Ethephon 10 86 3 88 3 91 Glyphosate 2 72 4 92 2 95 22
Conclusions • Current IC/LC-MS/MS portfolio allows determination of polar pesticides in both food and environmental samples well below regulatory limits • Simple sample preparation for IC separation – no FMOC needed! • Good separation efficiency of IC makes it a suitable method for most polar pesticides • TSQ Quantis is the recommended MS/MS for water and food analysis @ ppt levels • Full application support is provided 23
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