Analysis of Carbamate and Organophosphate …...Analysis of Carbamate and Organophosphate Pesticides...
Transcript of Analysis of Carbamate and Organophosphate …...Analysis of Carbamate and Organophosphate Pesticides...
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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Pea
k A
rea
Rat
io
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First Stability Study Results
MalathionRefrigerator (4C)
MalathionRoomTemperature(25 C)
MalathionOven (40 C)
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0 5 10 15 20 25 30
Pea
k A
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Rat
io
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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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