Analysis of Carbamate and Organophosphate Pesticides in Animal

Poisoning Cases by GC/MS

Jessica M Greene, BSBarry Logan, PhD, Stephen Donovan, PhD, Karen Scott, PhD, Alana Balogh, BFA

03/02/17

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Background

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Africa

• Pesticides are readily available in Africa for legitimate use

• Pesticides are thus available for use as poisons

• Conflicts between farmers and wildlife

• Poachers attempting to hide illicit activities

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The Poisonings Effect

• Lions

• Vultures

• Elephants

• Hyenas

• Humans

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Organophosphate & Carbamate Pesticides

• Acetylcholinesterase inhibitors

• CNS effects can be varied but poisoning ultimately will lead to muscle contractions followed by paralysis and death due to respiratory failure

• Absorption occurs through all routes

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Figure 1: Organophosphate Basic Structure

Figure 2: Carbamate Basic Structure

Aims

• Develop and optimize a method for OP and carbamates

• Create an extraction procedure for the pesticides

• Receive authentic samples

• Provide reliable and authoritative results to wildlife advocates in Africa

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Method Development

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Original Method

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Finalization of Method

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GC/MS Parameters

GC Agilent 7890A

Column HP-4MS Ultra Inert

Column Length 30 m

Carrier Gas Hydrogen

Injection Type/Volume Splitless/ 1 µL

Inlet Temperature 250 ⁰C

Oven Program 40 ⁰C for 1 minute15 ⁰C/min to 170 ⁰C, hold for 5 minutes25 ⁰C/min to 350 ⁰C, hold for 3 minutes

Flow 1.1 mL/min

MS Agilent 5975C

MS Scan Range 35-500 amu

Test Mix on Final Method

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Extraction Optimization

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In-Field Extraction

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Add acetone to biological material and mix

Pour mixture through a funnel using a paper towel as a filter

Pour filtrate on clean paper towel and allow to dry

Place dry paper towel in plastic bag and follow shipping procedures

In-Field Extraction

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In-Lab Extraction Method

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Place paper towel in tube with 2 mL ethyl acetate, rotate (x2)

Wash ethyl acetate with deionized water

Wash ethyl acetate with 10% sodium bicarbonate

Wash ethyl acetate with saturated sodium chloride in deionized water

Add ~0.5 g sodium sulfate

Dry down under nitrogen gas and reconstitute in ethyl acetate

Remove aqueous layer

Remove aqueous layer

Remove aqueous layer

Decant ethyl acetate

Decant ethyl acetate

Method Validation

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Limit of Detection

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LOD (ng on column) Compounds

1 Aldicarb nitrile, propoxur

10 Methomyl, 3-hydroxycarbofuran, thionazin, sulfotep, phorate, dimethoate, carbofuran, disulfoton, methyl parathion, carbaryl, methiocarb, parathion, famphur

25 Triethyl thiophosphate, oxamyl

Recovery

Beta-Naphthol Carbaryl

Recovery from In-Field Extraction

8.85% 8.48%

Recovery from Lab Extraction

73.57% 70.64%

Recovery from Both Extractions

6.51% 5.99%

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0.5

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1.5

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2.5

0 5 10 15 20 25 30 35

Pea

k A

rea

Rat

io

Days

First Stability Study Results

MalathionRefrigerator (4C)

MalathionRoomTemperature(25 C)

MalathionOven (40 C)

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0

2

4

6

8

10

12

0 5 10 15 20 25 30

Pea

k A

rea

Rat

io

Days

Second Stability Study Results

MalathionFridge (4 C)

MalathionRoom Temp(25 C)

Carbaryl Fridge(4 C)

Carbaryl RoomTemp (25 C)

Authentic Samples

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Extraction Video

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Authentic Samples

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Elephants at Rubbish Dump in Zimbabwe

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Blank Stomach Extract

Stomach contents from suspected poisoning case

Future Work

• Continue receiving samples from Africa for testing

• Build a database to track poisoning cases

• Developing LC/MS/MS method if more sensitivity is required

• Evaluating the stability in the refrigerator (4⁰C) and determine reason for spike

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Acknowledgements

• Mandi Mohr, Melissa Friscia and Alex Krotulski

• Arcadia University Class of 2017

• The Center for Forensic Science Research and Education

• Arcadia University

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References

1. Quinn LP, B, de J, Fernandes-Whaley M, Roos C, Bouwman H, et al. Pesticide Use in South Africa: One of the Largest Importers of Pesticides in Africa [Internet]. In: Pesticides in the Modern World - Pesticides Use and Management. InTech, 2011 [cited 2016 Aug 10]; Available from: http://www.intechopen.com/books/pesticides-in-the-modern-world-pesticides-use-and-management/pesticide-use-in-south-africa-one-of-the-largest-importers-of-pesticides-in-africa2. Richards N. Carbofuran and Wildlife Poisonings [Internet]. Chichester, UK: John Wiley & Sons, Ltd, 2011 [cited 2016 Aug 10]; Available from: http://doi.wiley.com/10.1002/9781119998532.fmatter3. Ogada D, Shaw P, Beyers RL, Buij R, Murn C, Thiollay JM, et al. Another Continental Vulture Crisis: Africa’s Vultures Collapsing toward Extinction. 4. Chung SWC, Chan BTP. Validation and use of a fast sample preparation method and liquid chromatography-tandem mass spectrometry in analysis of ultra-trace levels of 98 organophosphorus pesticide and carbamate residues in a total diet study involving diversified food types. J Chromatogr A [Internet] 2010;1217(29):4815–24. Available from: http://dx.doi.org/10.1016/j.chroma.2010.05.0435. King AM, Aaron CK. Organophosphate and Carbamate Poisoning. Emerg Med Clin North Am [Internet] 2015;33(1):133–51. Available from: http://www.ncbi.nlm.nih.gov/pubmed/254556666. Papoutsis I, Mendonis M, Nikolaou P, Athanaselis S, Pistos C, Maravelias C, et al. Development and Validation of a Simple GC-MS Method for the Simultaneous Determination of 11 Anticholinesterase Pesticides in Blood-Clinical and Forensic Toxicology Applications. J Forensic Sci 2012;7. Luzardo OP, Ruiz-su??rez N, Valer??n PF, Camacho M, Zumbado M, Henr??quez-hern??ndez LA, et al. Methodology for the identification of 117 pesticides commonly involved in the poisoning of wildlife using gc-ms-ms and lc-ms-ms. J Anal Toxicol 2014;38(3):155–63. 8. SEMIVOLATILE ORGANIC COMPOUNDS BY GAS CHROMATOGRAPHY/MASS SPECTROMETRY (GC/MS). 1996;9. Trehy ML, Yost RA, McCreary JJ. Determination of aldicarb, aldicarb oxime, and aldicarb nitrile in water by gas chromatography/mass spectrometry. Anal Chem [Internet] 1984 [cited 2017 Mar 17];56(8):1281–5. Available from: http://pubs.acs.org/doi/abs/10.1021/ac00272a020

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Questions?jgreene_01@arcadia.edu

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