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Dott. Luca Gerardo IC/SP Application Specialist, Thermo Fisher Scientific, Rodano (MI)

Ion Chromatography Coupled to MS, a Powerful Approach for Polar Pesticides Determination

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

H2N P

O

OHOH

• In 2015, the World Health Organization's International Agency for Research on Cancer (IARC) classified glyphosate as "probably carcinogenic to humans." • International scientists found there was a particularly strong connection to non-Hodgkins

lymphoma.

• 2015: The European Food Safety Authority and the European Chemicals Agency have said glyphosate is NOT likely to be carcinogenic to humans. • Under scrutiny for research conducted by chemical companies

• 2016: The World Health Organization/ United Nations Joint Meeting on Pesticide Residues also cleared glyphosate as unlikely to pose a risk to humans, • tarnished by conflicts of interest regarding ties to the International Life Sciences Institute, a

food industry front group

• March, 2017: California Environmental Protection Agency's Office of

Environmental Health Hazard Assessment confirmed that it would add glyphosate to list of cancer causing chemicals.

.

Glyphosate – Health Concerns Timeline

• 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).

• FDA found glyphosate in US honey at double the levels allowed in the EU.

• More than 50 lawsuits against glyphosate producers are pending in US District Court in San Francisco.

• Several hundred similar actions are pending in

state courts.

Glyphosate in the News

http://www.anh-usa.org/http://www.anh-usa.org/https://usrtk.org/our-investigations/fda-finds-monsantos-weed-killer-in-u-s-honey/

HPLC methods: • Typically require extensive sample preparation

• SPE • QuePP method

• Preparation of buffers/acidic mobile phases

• Non retentive on C18 columns • Pre or post column derivatisation • Speciality columns – poor resolving power • Fluorescence detection

• Matrix effects and interferences

Analysis of Glyphosate

Time and additional source of errors

Robustness issues

QuPPe-PO v 9.1-Negative Mode Compounds

• Method lists a total of 42 different (pos and neg mode) analytes.

http://www.crl-pesticides.eu/userfiles/file/EurlSRM/meth_QuPPe-PO_EurlSRM.pdf

http://www.crl-pesticides.eu/userfiles/file/EurlSRM/meth_QuPPe-PO_EurlSRM.pdf

Why Couple Ion Chromatography (IC) to MS?

- IC columns provide great specificity and selectivity for ionic compounds.

- Metal-free flow path reduces fouling of ion-exchange columns.

- MS and conductivity detection in series

Possibility to determine high concentration contaminants (Anions / Cations) and trace contaminants (Pesticides/Disinfection byproducts) in the same chromatographic run.

- Eluent suppression to Water

Enable rapid switching from anion to cation analysis sharing MS device.

- Just add water to prepare eluents for anions and cations analysis.

Principles of RFIC - Eluent Generation

Vent

Pump

[KOH] α Current

Flow rate

H2

Degas unit

Cation exchange connector

Thermo Scientific™ Dionex ™ CR-ATC anion trap

KOH + H2

KOH electrolytic chamber

Thermo Scientific™ EluGen™ KOH

cartridge

Pt anode

[ + ]

K+

OH–

KOH

K-reservoir

H2O

Thermo Scientific™ RFIC technology - Reagent-free IC without manual eluent preparation

(H2O 2H+ + ½O2 + 2e–)

(2H2O + 2e– 2OH– + H2)

KOH [ - ] Pt cathode

NaOH, H2

Waste/Vent Waste/Vent Anode

To Detector

H+ + O2 H2 + OH-

H2O H2O H+ , X- in H2O

H2O H2O

H2O, O2

Cation- Exchange

membranes

OH-

Na+, X- in NaOH eluent Cathode

H2O 2H+ + ½ O2 + 2e

- 2H2O + 2e- 2OH- + H2

X- H+

Na+ OH-

Chemistry and Ion Movement … … in an Thermo Scientific™ Dionex™ ERS™ electrolytically regenerated suppressor

HPLC Separation … Thermo Scientific™ Hypercarb™ and Silica C18 columns

• 10 µg/kg spike in grapes (Fosetyl & Phosphonic acid @ 100 µg/kg)

IC-MS/MS Pesticide Multi Residue Ion Chromatogram

IC Separations - Polar Pesticides …

Glyphosate

Glufosinate

Ethephon

Phosphonic Acid

Fosethyl Al

Maleic Hydrazide

Bromide

AMPA

… on a Thermo Scientific™ Dionex™ IonPac™ AS19 4 µm column

AS19 4 um Gradient

Ion Chromatography Selectivity

• 0- 12 minutes - Matrix interferences in conductivity trace can be sent to waste to preserve the ion source.

• 12 minutes +/- analytes of interest directed to MS

Polar_mix_20ppb_AS19_161006104310 10/06/16 10:43:10

RT: 0.00 - 31.93

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30Time (min)

0

1000

1000

1000

100R

ela

tive

Ab

un

da

nce

0

1000

10025.36

25.67 26.91 30.7128.5424.3920.47 22.2310.916.0914.92

15.1915.94 16.91 25.7518.07 26.1620.56 23.72 29.64 31.4321.2213.433.54 9.50 11.832.53 4.9916.63

17.12 18.036.27 20.44 22.24 24.56 25.388.15 28.43 30.273.81 5.51 10.55 11.558.80 15.7314.902.6411.92

12.64 13.47 15.25 16.89 18.34 23.48 31.5028.04 30.1424.27 27.1520.85 22.355.61 9.336.64 8.21 11.494.753.422.681.7024.69

25.0317.03 25.8016.78 27.8217.90 28.98 30.6015.166.42 13.56 18.595.53 23.2412.29 21.0310.787.63 9.314.412.2331.08

30.5724.87 29.0027.7222.5419.85 21.3817.25 19.1013.625.54 16.4812.686.78 8.15 8.73 10.863.682.12

NL: 8.15E5Base Peak m/z= 168.0059-168.0075 F: FTMS - p ESI Full ms [50.0000-200.0000] MS Polar_mix_20ppb_AS19_161006104310

NL: 2.60E5Base Peak m/z= 110.0006-110.0018 F: FTMS - p ESI Full ms [50.0000-200.0000] MS Polar_mix_20ppb_AS19_161006104310

NL: 3.75E6Base Peak m/z= 80.9743-80.9751 F: FTMS - p ESI Full ms [50.0000-200.0000] MS Polar_mix_20ppb_AS19_161006104310

NL: 4.04E6Base Peak m/z= 82.9537-82.9545 F: FTMS - p ESI Full ms [50.0000-200.0000] MS Polar_mix_20ppb_AS19_161006104310

NL: 1.85E5Base Peak m/z= 128.0096-128.0108 F: FTMS - p ESI Full ms [50.0000-200.0000] MS Polar_mix_20ppb_AS19_161006104310

NL: 1.81E6Base Peak m/z= 98.9486-98.9496 F: FTMS - p ESI Full ms [50.0000-200.0000] MS Polar_mix_20ppb_AS19_161006104310

Polar_mix_20ppb_AS19_161006104310 #4117 RT: 18.35 AV: 1 NL: 1.89E7T: FTMS - p ESI Full ms [50.0000-200.0000]

50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200m/z

0

10

20

30

40

50

60

70

80

90

100

Re

lative

Ab

un

da

nce

59.9850

94.9809

60.992896.9602

76.9880

61.9881 183.0123

79.9574 194.9277184.991659.0135 98.9560 172.9915116.928780.9652 140.9673111.947389.0245180.9967

155.988963.9621 75.0087 134.8949 147.0300129.0559 161.0457

um Matrix Interferences Removed

TIC showing 20ppb: *Glyphosate *AMPA *Phosphonic acid *Chlorate *Cyanuric acid *Perchlorate

Flow Diagram— IC-HR/AM MS System

Conductivity Detector

EG IC column Suppressor IC pump 25 µL/min

H2O

Methanol

Low dead volume

mixing tee

Waste AXP-MS pump 10 µL/min

High pressure ion chromatography system

Injection from autosampler

To assist desolvation

Thermo Scientific™ Q Exactive™ Orbitrap™

mass spectrometer

Grounding union

H2O

AXP pump 40 µL/min

Trap Guard

40 µL/min

Degas

Wang, J., Christison, T., et al. Submitted to Analytical Chemistry

Data Management

Ion Chromatography Coupled with Orbitrap HRMS (Q Exactive Orbitrap MS)

HRMS for IC and HPLC

Comprehensive solution for pesticide analysis

AMPA Linearity from 20 to 2000 ppt in Drinking Water

AMPA Repeatability at 30 ppt in Drinking Water

Picco Area

1 112109

2 110436

3 111219

4 114398

5 111643

Media 111961

StDev 1494.84

CV% 1.3

μS

Time (min)

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

1.

2.

5. 4.

3.

Fosetyl-Al Glyphosate Clopyralid AMPA Glufosinate

Conductivity Traces of Anions Present in Different Matrices

20 ppt AMPA Spiked in Water Containing Cl- (5 to 50 ppm)

Glyphosate Lineatity from 20 to 2000 ppt

Glyphosate Repeatability at 10 ppt in Drinking Water

Picco Area 1 199496

2 190406

3 197228

4 195575

5 183081

Media 193157.2

StDev 6551.6

CV% 3.4

2 – 5 – 10 ppt of Glyphosate in Drinking Water

Grafico1

35886

105416

195575

Sheet1

PiccoArea

1199496

2190406

3197228

4195575

5183081

Media193157.2

StDev6551.6498456493

CV%3.3918745176

235886

5105416

10195575

Sheet1

Repeatability of Glyphosate 500 ppt on Lettuce Extract

Picco Area

1 565481

2 609915

3 543858

4 595630

5 609373

Media 584851.4

StDev 29160.51

CV% 4.99

Cation Polar Pesticides

Note: Triazole-lactic acid missing Courtesy of Dr. Richard Fussell

Cation Polar Pesticides on Dionex IonPac CS17 Column

Courtesy of Dr. Richard Fussell

Paraquat – Mepiquat – Chlormequat - Diquat

Clormequat

Mepiquat

Paraquat

Diquat

Dionex IonPac CS17 column full scan determination of cation polar pesticides

• Determination of several analytes in a single anionic run: • Glyphosate, AMPA, Glufosinate, anionic polar pesticides • Haloacetic Acid HAA9 • Bromate (at ppt level) • Perclorate (at ppt level) • Organic ccids • Anions

• Determination of several analytes in a single cationic run: • Diquat, Paraquat, Chlormequat, cationic polar pesticides • Amines • Cations

IC-MS Advantages

Do you have any questions?

Do you have additional questions or do you want to talk to an expert from Thermo Fisher Scientific? Please send an E-Mail to analyze.eu@thermofisher.com and we will get back to you.

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Ion Chromatography Coupled to MS, a Powerful Approach for Polar Pesticides DeterminationWhat is Glyphosate?Glyphosate – Health Concerns TimelineGlyphosate in the NewsAnalysis of GlyphosateQuPPe-PO v 9.1-Negative Mode CompoundsWhy Couple Ion Chromatography (IC) to MS?Principles of RFIC - Eluent GenerationChemistry and Ion Movement …HPLC SeparationIC-MS/MS Pesticide Multi Residue Ion ChromatogramIC Separations - Polar Pesticides …Ion Chromatography SelectivityMatrix Interferences Removed Flow Diagram— IC-HR/AM MS SystemIon Chromatography Coupled�with Orbitrap HRMS �(Q Exactive Orbitrap MS)HRMS for IC and HPLCAMPA Linearity from 20 to 2000 ppt in Drinking WaterAMPA Repeatability at 30 ppt in Drinking WaterConductivity Traces of Anions Present in Different Matrices20 ppt AMPA Spiked in Water Containing Cl- (5 to 50 ppm)Glyphosate Lineatity from 20 to 2000 pptGlyphosate Repeatability at 10 ppt in Drinking Water2 – 5 – 10 ppt of Glyphosate in Drinking WaterRepeatability of Glyphosate 500 ppt on Lettuce ExtractCation Polar PesticidesCation Polar Pesticides on Dionex IonPac CS17 ColumnParaquat – Mepiquat – Chlormequat - DiquatIC-MS AdvantagesDo you have any questions?