Fast Analysis of Polar Pesticides in Water by IC-MS/MS Michal - - PowerPoint PPT Presentation

The world leader in serving science

Michal Godula, Ph.D. Thermo Fisher Scientific

Fast Analysis of Polar Pesticides in Water by IC-MS/MS

2

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

3

• 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)

Glyphosate in the News

4

Water area – ppt levels

• Derivatization needed (FMOC) to reach 10 ng/l levels with

LC/MS/MS

• Only Glyphosate/AMPA and Glufosinate analyzed
• Derivatization the main obstacle though it can be automated...
• What about other compounds? Dalapon, Ethephon etc..

Polar Pesticides Story : An Imperfect Compromise

Food area – ppb levels

• QuPPe method with generic extraction using acidified

methanol- no partition, no clean-up, LC based separation (Hypercarb)

• The QuPPe method developed by EURL-SRM is not perfect –

early eluting compounds, strong matrix effects – compromised LOQs

• Discussion started at EU level to change the concept

5

• 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 ScientificTM AcclaimTM 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

Options for the analysis

6

LC-MS analysis after FMOC derivatization

Compound LOD (fg on column) LOQ (fg on column) LOD ng/L LOQ ng/L

AMPA-FMOC 100-250 100-250

1-2.5 1-2.5

Glyphosate – FMOC 100 250

1 2.5

• 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

7

Scheme of IC-MS/MS

High-Pressure Non-Metallic Pump Eluent Generator (OH– or H+) Waste Sample Inject (Autosampler) CR-TC Electrolytic Eluent Suppressor Separation Column AS 24 Triple Quadrupole MS/MS Conductivity Detector 0.31 µS Data Management Makeup Pump AERS regeneration

8

Introduction to TSQ Altis and TSQ Quantis

TSQ Altis TSQ Quantis

Performance: Sensitivity, Selectivity (H-SRM) Robustness, Reproducibility, Speed, Ease-of-Use, Flexibility

Thermo ScientificTM TSQ AltisTM

High-end

Thermo ScientificTM TSQ QuantisTM

Mid-tier

Mass Range 5-2000 5-3000 SRM/sec 600 600 Selectivity (H-SRM) 0.2 Da FWHM 0.4 Da FWHM Sensitivity (HESI Reserpine 1 pg) 500,000:1 150,000:1 Targeted Market Omics, Research, Pharma/Biopharma, Clinical Research and Forensic Toxicology Environmental and Food Safety, Clinical Research, and Forensic Toxicology

9

TSQ Altis: Sensitivity with Robustness, No Compromises

Segmented Quadrupoles with hyperbolic surface for enhanced performance with both SRM and H-SRM (0.2 FWHM) Enhanced dual-mode electron multiplier detector Ensures excellent linearity and dynamic range Electrodynamic ion funnel (EDIF) Increases ion flux High capacity Ion transfer tube (HCTT) Increases ion flux Ion beam guide with neutral blocker Reduces chemical background Active collision cell with axial DC field facilitates up to 600 SRMs/sec OptaMaxTM NG APCI ready NEW! NEW! NEW!

10

50 100 150 200 250

Fosetyl Glufosinate AMPA Clopyralid Glyphosate

Peak area [counts*sec]

ACN MeOH IPA

Figure 2.Signal improvement using different make-up solvents; Measured at 500 ng/l concentration levels

Influence of make-up solvent

11

IC-MS/MS System used for analysis

Ion Chromatograph: Thermo ScientificTM Dionex IntegrionTM Mass Spectrometer: Thermo ScientificTM TSQ QuantisTM

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: CH3OH

Time (min) Concentration of KOH in eluent (mM) 25 0.2 25 11 80 11.1 100 12.5 100 12.6 25 17.0 25

12

Calibration surface water

* Levels 5, 10, 25, 50, 100, 500, 1000 ppt, 1 injections each

13

SRM transitions in surface water, spiked at 50 ng/l level

14

500 1000 1500 2000 2500 3000 3500 4000 4500 5000 5500 200 400 600 800 1000 Area c (ng/l)

Glyphosate

Surface water Deionized water Bottled water Drinking water Artificial matrix

Calibration curves of glyphosate obtained in different matrices

2000 4000 6000 8000 10000 12000 14000 16000 200 400 600 800 1000 Area c (ng/l)

Clopyralid

Surface water Deionized water Bottled water Drinking water Artificial matrix

15 μS Time (min)

1. Artificial matrix 2. Drinking water 3. Surface water 4. Bottled water 5. Deionized water

1.

2.

5. 4. 3.

Cl SO

• NO
• Fosetyl-Al

Glyphosate Clopyralid AMPA Glufosinate

Possible suppression sources: anions present in different matrices

16

Bottled mineral water performance

Name

LOD [ppt] LOQ [ppt] RSD % (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

17

Expected signal suppression and LOQ in heavy matrix

Analyte Artificial water* Surface Water 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 SO4

2-

150 mg/l HCO3- 20 mg/l NO3-

18

Component Matrix LOD [ppt]] LOQ [ppt] REC [%] RSD [%] 10 ppt 20 ppt 50 ppt 10 ppt 100 ppt 1000 ppt Fosetyl Drinking 2.5 5 133 122 132 9.0 1.3 0.7 Bottled 1 2.5 121 123 128 2.0 0.9 1.1 Surface 2.5 5 105 105 104 2.0 1.1 0.6 Glufosinate Drinking 5 10 133 121 95 9.3 2.4 0.9 Bottled 5 10 56 112 96 4.1 3.4 0.8 Surface 5 10 124 111 93 2.2 1.6 1.2 AMPA Drinking 5 10 91 93 83 9.1 2.3 0.8 Bottled 5 10 109 107 95 8.7 2.1 0.8 Surface 5 10 91 100 98 4.5 3.7 1.0 Clopyralid Drinking 10 50 110 88 90 11.9 1.4 1.0 Bottled 5 10 53 89 86 9.1 1.4 0.9 Surface 5 10 109 114 140 10.0 1.4 0.6 Glyphosate Drinking 10 50 89 106 83 6.3 2.3 1.3 Bottled 10 50 66 108 105 14.4 1.7 3.4 Surface 10 50 82 101 90 5.0 10.1 1.8

Validation results

19

Ion Chromatography Liquid Chromatography

Matrix Food Water Food Water Sample preparation Extraction (QuPPe) None (filtration) Extraction (QuPPe) (optional FMOC* derivatization) FMOC* Derivatization (SPE cleanup) (automation possible) Separation quality Excellent Excellent Poor (Hypercarb) Very good (Reversed Phase) LOQs 1-10 ppb 5 -10 ppt 1 – 10 ppb 5 – 10 ppt MS/MS **TSQ Endura **TSQ Quantiva **TSQ Quantiva (Endura not suitable) **TSQ Quantiva Matrix tolerance Very good Very good Limited Good Time consumption Low Low Low - medium Medium – High (automation possible)

IC vs. LC arguments

*FMOC derivatization applicable only for Gly, AMPA, Glu!

**Thermo Scientific™ TSQ™ Endura, Thermo Scientific™ TSQ™ Quantiva™

20

Food Analysis; chromatogram in lettuce at 10 ug/kg

Phosphonic acid Fosetyl-Al Glyphosate Ethephon MPPA HEPA N-acetyl-AMPA Glufosinate N-acetyl-Glufosinate AMPA

21

Calibration curves – lettuce – range: 0 – 20 ppb II.

Glufosinate Glyphosate MPPA N-acetyl-AMPA

22

Repeatability and recovery in lettuce matrix

Compound Level 1 - 1 ppb Level 2 - 5 ppb Level 3 - 10 ppb 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

23

• 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

Conclusions