IC-MS Multi-residue pesticide methods, fantasy or reality?

Dr Stuart Adams stuart.adams@fera.co.uk

Presentation by Mike Dickinson

Overview

• Introduction to Fera

• Our current pesticide multi residue method approaches

• Single residue methods focussing on polar ionic pesticides

• Is there a new approach for PIPs?

• Summary

Fera Science Ltd.

• A leading supplier of scientific solutions, evidence and advice across the Agri-Food supply chain in Europe

• Approximately 150 chemists on site

• Turnover 50,000 samples for pesticide, veterinary medicines, mycotoxins and many other types of analysis

• www.fera.co.uk

Monitoring of pesticide residues in the UK by targeted analyses

• Risk-based monitoring targets the foodstuffs most likely to contain residues • Last year approx 3,600 samples were analysed at

Fera using QuEChERs in combination with LC-MS/MS (250) and GC-MS/MS (140) and other selected/single residue methods (10)

• Residues are often detected , often PIPs • Methods should be suitable for screening,

quantification and identification: often MS approaches used in complementary fashion.

QuEChERS method for pesticides Quick, Easy Cheap, Effective, Rugged, Safe

GC-MS/MS

LC-MS/MS

Analysis of QuEChERS extracts by LC-MS/MS with pos/neg switching Pear matrix spiked with 228 pesticides @ 5 µg/kg

Problems with multi-residue methods: peak shape of early eluters

Diluted extract 9:1 v/v aqueous/MeCN Neat MeCN as inj solvent

> Tubing diameter from inj to column : 0.17 to 0.25 mm i.d.

Single residue methods

• Not all ionic pesticides can be included in QuEChERS extraction methods and analysed by reversed phase chromatography methods

• Small polar ionic molecules • Difficult to separate on conventional chemistry columns • Often low detection limits required (1-4 µg/L in drinking

water)

• E.g chlormequat, mepiquat, glyphosate ,ethephon

Polar/Ionic pesticides of interest

Anions Zwitterions Polar Compounds

Cations Others

Chlorate Perchlorate Ethephon Ethephon-hydroxy 3-MPPA

Fluoroacetate

Glufosinate Glufosinate

-N-acetyl Glyphosate

AMPA Daminozide

ETU PTU

Cyromazine Fosetyl Al

Chlormequat Mepiquat Amitrole

Clopyralid Aminopyralid

Trinexapac-ethyl free acid

• Chromatographic options including HILIC, reversed phase chromatography and ion chromatography See QuPPe Version 8, March 2015 for an overview of methods

proposed, (Anastassiades, et, al 2013)

EURL Workshop and Training October 2014 Freiburg / Stuttgart

QuPPe Version 8 (ES Negative Mode)

M 1.1 M 1.2 M 1.3

M 1.4

M 2

Separation principle Anion Exchange

Anion Exchange Carbon Carbon HILIC

Column type AS-11 AS11-HC Hypercarb Hypercarb Obelisc-R

Ethephon HEPA Glufosinate N-Acetyl-Glufosinate MPPA Glyphosate AMPA Phosphonic acid N-Acetyl-AMPA Fosetyl-Al Maleic hydazide Perchlorate

Chlorate

Bialphos

Cyanuric acid

EURL Workshop and Training October 2014 Freiburg / Stuttgart

QuPPe Version 8 (ES Positive) M1.3 M 3 M4.1 M4.2 M 5 M 6 M 7 M8

Separation principle HILIC HILIC HILIC HILIC HILIC HILIC Carbon

Column type Obelisc-R Obelisc-R BEH-Amide PFP Obelisc-R Trinity P1 Hypercar

b Amitrole ETU PTU Cyromazine Trimesium Daminozide Chlormequat Mepiquat Difenzoquat Promaocarb Melamine Diquat Paraquat N,N-Dimethylhydrazine

Nereistoxin Streptomycin Kasugamycin Morpholine () () Diethanolamine () Triethanolamine () 1,2,4-Triazole Triazole-alanine Triazole-acetic acid Triazole-latic acid

Using QuPPe multi-residue extraction method

• Extraction of 10 grams of sample or 5 grams of dry sample (adjusted for water content to give 10 ml of water)

• Add 10 mL of 1% Formic acid in methanol and shake for 20 minutes

• Filter (cellulose based, 0.45 µm pore size) and analyse by IC-MS/MS

• Is it really that simple?

Quick Method for the Analysis of Residues of numerous Highly Polar

Pesticides in Foods of Plant Origin involving Simultaneous Extraction with Methanol and LC-MS/MS Determination. (Anastassiades, et, al 2013)

No, it’s not that simple:

IC-MS method 1 - anions

• Thermo Scientific™ Dionex™ ICS-3000 system using Thermo Scientific™ Dionex™ IonPac™ AS19 (250 x 2mm , with guard)

• Flow rate: 0.3 ml/min KOH with membrane electrolytic suppressor.

• 100 µl loop injection, online “trapping” previously evaluated and not used due to low pH of extracts.

IC-MS/MS methods – the MS

• Initially for method development purposes:

• But a higher spec MS system required for multi-residue PIPs method.

IC-MS/MS multi-residue: cereal matrix standard @ 10 µg/kg

2x10

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1

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Cpd 3: Glufosinate-N-acetyl (NAG): -ESI MRM Frag=100.0V CID@50.0 (222.0000 -> 63.0000) 43774_test004.d

11.162

10.866

1x10

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5

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Cpd 6: Glufosinate: -ESI MRM Frag=100.0V CID@16.0 (180.0000 -> 95.0000) 43774_test004.d

10.910

13.134

2x10

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1

1.

2

2.

3

3.

4

4.

Cpd 9: 3-Methylphosphinicopropionic acid (3-MPPA): -ESI MRM Frag=105.0V CID@36.0 (151.0000 -> 63.0000) 43774_test004.d

11.513

12.037

Counts vs. Acquisition Time (min)

10.4 10.6 10.8 11 11.2 11.4 11.6 11.8 12 12.2 12.4 12.6 12.8 13 13.2 13.4 13.6 13.8 14 14.2

Glufosinate and metabolites @ 5 µg/kg in Cereals

3-MPPA, 151 > 63

Glufosinate, 180 > 95

Glufosinate-N-acetyl, 222 > 63

Glyphosate (@ 100 µg/kg ) and AMPA (@ 10 µg/kg ) in cereals

2x10

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Cpd 8: Glyphosate: -ESI MRM Frag=85.0V CID@24.0 (168.0000 -> 63.0000) 43774_test004.d

12.570

23

Counts vs. Acquisition Time (min)

9.6 9.8 10 10.2 10.4 10.6 10.8 11 11.2 11.4 11.6 11.8 12 12.2 12.4 12.6 12.8 13 13.2 13.4

AMPA, 110> 63

Glyphosate, 168>63

Perchlorate and chlorate @ 10 µg/kg in cereals

2x10

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Cpd 15: Chlorate: -ESI MRM Frag=45.0V CID@24.0 (83.0000 -> 67.0000) 43774_data007.d

10.694

3 34

4x10

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Cpd 18: Perchlorate: -ESI MRM Frag=145.0V CID@30.0 (99.0000 -> 83.0000) 43774_data007.d

18.576

19.961

3 34

Counts vs. Acquisition Time (min)

10.5 11 11.5 12 12.5 13 13.5 14 14.5 15 15.5 16 16.5 17 17.5 18 18.5 19 19.5 20 20.5 21 21.5

Chlorate, 83 > 67

Perchlorate, 99 > 83

Fosetyl aluminium and associated metabolite @ 10 µg/kg in cereals

3x10

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Cpd 1: Fosetyl-aluminium: -ESI MRM Frag=70.0V CID@12.0 (109.0000 -> 81.0000) 43774_test004.d

7.597

6.908

23

2x10

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Cpd 16: Phosphonic acid: -ESI MRM Frag=50.0V CID@16.0 (81.0000 -> 79.0000) 43774_test004.d

11.605

23

Counts vs. Acquisition Time (min)

6.4 6.6 6.8 7 7.2 7.4 7.6 7.8 8 8.2 8.4 8.6 8.8 9 9.2 9.4 9.6 9.8 10 10.2 10.4 10.6 10.8 11 11.2 11.4 11.6 11.8 12 12.2 12.4 12.6 12.8

Fosetyl Al, 109 > 81

Phosphonic acid, 81 >79

Validation criteria for pesticide analysis

• Pesticide analysis for official control purposes: Document

No. SANCO/12571/2013 • Recoveries required are between 70 – 120%, n = 5 and at

two separate concentrations

• Relative Standard Deviations (RSD) of 20% or less

• Retention time (RT) • Minimum acceptable retention factor, x2 the RT corresponding to the

void volume of the column

Validation of method in cereals - 1

Compound Concentration ( (µg/kg)

Mean Rec

(n=5)

Mean % RSD

Chlorate (IStd used) 50 94 13

100 89 16

Perchlorate (IStd used) 50 90 8

100 96 18

Ethephon 50 89 10

100 60 15

Ethephon-hydroxy 10 103 28

50 75 5

Fosetyl-Al 10 82 21

50 50 3

Phosphonic acid 50 77 19

100 67 16

Validation of method in cereals - 2

Compound Concentration (µg/kg)

Mean Rec

(n=5)

Mean % RSD

Glyphosate 100 88 13

500 91 9

AMPA 50 81 13

100 81 19

Glufosinate 25 157 5

50 138 6

Glufosinate-N-acetyl 25 79 12

50 61 16

3-MPPA 5 113 27

25 74 11

All analytes in above table had internal standard correction

Internal standards (cereal matrix)

3x10

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Cpd 14: Perchlorate-IS: -ESI MRM Frag=140.0V CID@34.0 (107.0000 -> 89.0000) 43774_test003.d

18.571

3x10

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Cpd 15: Perchlorate: -ESI MRM Frag=145.0V CID@30.0 (99.0000 -> 83.0000) 43774_test003.d

18.627

20.676

Counts vs. Acquisition Time (min)

16.6 16.8 17 17.2 17.4 17.6 17.8 18 18.2 18.4 18.6 18.8 19 19.2 19.4 19.6 19.8 20 20.2 20.4 20.6 20.8 21 21.2

18O4-Perchlorate – labelled internal

standard

Perchlorate – native standard

3x10

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Cpd 9: Ethephon-IS: -ESI MRM Frag=55.0V CID@4.0 (147.0000 -> 111.0000) 43774_test003.d

11.299

2x10

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Cpd 10: Ethephon: -ESI MRM Frag=55.0V CID@4.0 (143.0000 -> 107.0000) 43774_test003.d

11.664

14.522

Counts vs. Acquisition Time (min)

9 9.2 9.4 9.6 9.8 10 10.2 10.4 10.6 10.8 11 11.2 11.4 11.6 11.8 12 12.2 12.4 12.6 12.8 13 13.2 13.4 13.6 13.8 14 14.2 14.4 14.6

Ethephon – native standard

d4-Ethephon – labelled internal

standard, could not be used

Interferent peak, MS dependant

Validation of method in cereals – 3 (improved method)

Compound Concentration (µg/kg)

Mean Rec

(n=5)

Mean % RSD

Glufosinate (IStd used) 10 68 19

50 90 9

Glufosinate-N-acetyl 10 98 17

(IStd used)

50 90 6

3-MPPA (IStd used) 10 101 9

50 93 8

Glyphosate (IStd used) 100 95 6

500 91 3

Ethephon (IStd used) 10 86 14

50 80 6

Clopyralid 50 65 10

500 57 5

Aminopyralid 50 68 6

500 63 3

Glyphosate in more detail

Weedkiller 'probably' causes cancer, says WHO study

Glyphosate is a 'probably carcinogenic' pesticide. Why do cities still use it? Cities use glyphosate to control weeds in parks and along verges. Now that the WHO says the pesticide is ‘probably carcinogenic to humans’, is it time to stop? Tuesday 21st April 2015

Tuesday 21st March 2015

IC-MS/MS Analysis of Cereal Based Baby Food

glyphosate (10 µg/kg) glufosinate (50 µg/kg) AMPA (100 µg/kg)

• Quantification and Identification at 10 µg/kg • Excellent linearity, sensitivity precision for glyphosate

Glyphosate Residues in Oats: IC-MS/MS vs GC-MS (derivatisation)

y = 1.2427x - 0.0057 R² = 0.9313

0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

1.60

1.80

0.00 0.50 1.00 1.50

Pesticide contamination of organic oats Source: Soil Assosiation UK 2008-12-1 Tags:Pesticide,contamination,chlormequat,glyphosate Some batches of oats being sold in the UK as organic have been found to contain one or both of the pesticides Chlormequat and Glyphosate…………….

www.farmersguardian.com/organic-oats-test

Organic oats test positive for herbicide and PGR use November 28th 2008

THE Soil Association has pledged to increase the amount of random testing…………

Incurred residues 0.3 – 1.5 mg/kg

Perchlorate in more detail (1)

Oranges @ 5 µg/kg 18O4 Perchlorate 107> 89

Quant transition 99> 83

99> 67

Perchlorate in more detail (2)

18O4 Perchlorate 107> 89

Quant transition 99> 83

99> 67

Mixed salad @ 10 µg/kg

Sodium Fluoroacetate

• Recent threats to New Zealand dairy sector from environmental activists

Same QuPPe extraction , without the formic acid Spike @ 10 µg/kg in infant formula IC-MS, m/z 77

Summary

• Detection of 12 different anion/zwitterionic pesticides is possible using IC-MS

• Generic extraction method a likely possibility

• Requires the use of labelled internal standards • Not perfect for quantitation purposes - poor peak

shapes, future plan to use IC x IC – MS/MS?

Acknowledgements

• The Fera LC-MS team, especially Chris Brown, Matt Emission, Jonathan Guest and Patrick Hough

• Dr Simon Hird and Richard Fussell

• Colleagues at Thermo Fisher Scientific

Thank you for your attention!