Analysis of Carbamate and Organophosphate Pesticides in Animal Poisoning Cases by GC/MS Jessica M Greene, BS Barry Logan, PhD, Stephen Donovan, PhD, Karen Scott, PhD, Alana Balogh, BFA 03/02/17 1 Background 2 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 3 The Poisonings Effect • Lions • Vultures • Elephants • Hyenas • Humans 4 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 Figure 1: Organophosphate Figure 2: Carbamate Basic Structure Basic Structure 5 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 6 Method Development 7 Original Method 8 Finalization of Method 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 minute 15 ⁰C/min to 170 ⁰C, hold for 5 minutes 25 ⁰C/min to 350 ⁰C, hold for 3 minutes Flow 1.1 mL/min MS Agilent 5975C MS Scan Range 35-500 amu 9 Test Mix on Final Method 10 Extraction Optimization 11 In-Field Extraction 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 12 In-Field Extraction 13 In-Lab Extraction Method Place paper towel in tube with 2 mL ethyl acetate, rotate (x2) Decant ethyl acetate Wash ethyl acetate with deionized water Remove aqueous layer Wash ethyl acetate with 10% sodium bicarbonate Remove aqueous layer Wash ethyl acetate with saturated sodium chloride in deionized water Remove aqueous layer Add ~0.5 g sodium sulfate Decant ethyl acetate Dry down under nitrogen gas and reconstitute in ethyl acetate 14 Method Validation 15 Limit of Detection 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 16 Recovery Beta-Naphthol Carbaryl Recovery from In-Field 8.85% 8.48% Extraction Recovery from Lab 73.57% 70.64% Extraction Recovery from Both 6.51% 5.99% Extractions 17 First Stability Study Results 2.5 Malathion Refrigerator (4 2 C) 1.5 Malathion Room Temperature Peak Area Ratio 1 (25 C) Malathion 0.5 Oven (40 C) 0 0 5 10 15 20 25 30 35 Days 18 Second Stability Study Results 12 Malathion Fridge (4 C) 10 8 Malathion Room Temp (25 C) 6 Carbaryl Fridge Peak Area Ratio (4 C) 4 Carbaryl Room 2 Temp (25 C) 0 0 5 10 15 20 25 30 Days 19 Authentic Samples 20 Extraction Video 21 Authentic Samples 22 Elephants at Rubbish Dump in Zimbabwe 23 Blank Stomach Extract Stomach contents from suspected poisoning case 24 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 25 Acknowledgements • Mandi Mohr, Melissa Friscia and Alex Krotulski • Arcadia University Class of 2017 • The Center for Forensic Science Research and Education • Arcadia University 26 References 1. 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