Pesticide Screening Method with UPLC-MS/MS
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Pesticide Screening Method with UPLC-MS/MS Emma Eriksson Degree Project in Engineering Chemistry, 30 hp Report passed: June 2015 Supervisors: Daniel Jansson, FOI Anders Östin, FOI Richard Lindberg, Umeå University Emma Eriksson June 3, 2015 . Emma Eriksson June 3, 2015 Sammanfattning Pesticider används till stor del i jordbruk runt om i världen för att skydda grödor från skadedjur men på grund av deras toxicitet och strukturella likheter med nervgaser så kan pesticider potentiellt också användas vid avsiktlig förgiftning eller som kemiska vapen. När man misstänker att kemiska vapen eller toxiska föreningar har använts så behöver man en snabb identifiering av ämnet för att kunna varna allmänheten och förhindra vidare spridning. Av den anledningen utvecklades en snabb multi-metod för screening av 233 pesticider med ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). En generisk extraktionsmetod med acetonitril användes för extraktion av matriserna vatten, mjölk, apelsinjuice, barnmat, sand, jord och serum. Extraktionsmetodens prestanda demonstrerades genom att analysera spikade prover med koncentrationen 0,625 µg/ml eller 0,625 µg/g. Utbyten på 70 % eller bättre och relativ standard avvikelse på 20 % eller längre uppnåddes för 79 % av alla pesticider. Undersökning av kemisk nedbrytning av phorate, utfördes i vattenprover under normala, sura, basiska och oxidativa förhållanden. Phorate valdes ut på grund av dess toxicitet och strukturella likheter med nervgaser. Resultatet visade att phorate sulfoxide och phorate sulfone var de två väsentliga nedbrytningsprodukterna som bildades under oxidativa förhållanden och att pH inte påverkar nedbrytningen. Ytterligare en topp med m/z på 277 hittades med låga intensiteter i standard av phorate sulfone och oxidering av phorate och phorate sulfoxide, denna kunde dock inte identifieras. Alla prover analyserades igen efter 20 dagar i rumstemperatur och då påträffades två nya toppar med m/z 111 och 163. Det kunde inte fastställas att dessa två toppar kom från nedbrytningsprodukter av phorate och båda lämnades oidentifierade. Den utvecklade metoden är väl anpassad för snabb identifiering av pesticider i de vanligaste typer av prover som samlas in i situationer där man misstänker användning av kemiska vapen eller vid förgiftningar. Nyckelord: UPLC-MS/MS, multi-metod, pesticider, generisk extraktionsmetod i Emma Eriksson June 3, 2015 Summary Pesticides are widely used in the agriculture all around the world for protection of crops from pests but because of their toxicity and structural similarities to nerve gases, pesticides can potentially also be used for intentional poisoning and as chemical weapons. When chemical weapons or toxic compounds are suspected to have been used, there is a need of quick identification in order to be able to warn the public and to prevent further spreading. For this purpose, a fast multi-method for screening of 233 pesticides in environmental, biological and food samples was developed using ultra performance liquid chromatography coupled to tandem mass spectrometry (UPLC- MS/MS). A generic single step solvent extraction method with acetonitrile was used for extraction of water, milk, orange juice, baby food, sand, soil and serum samples. The performance of the extraction method was demonstrated by analysis of spiked samples at the pesticide concentration 0.625 µg/mL or 0.625 µg/g. Extraction recoveries of 70 % or higher and relative standard deviation of 20 % or lower was achieved for 79 % of the pesticides. Chemical degradation of phorate was evaluated in water samples under normal, acidic, basic and oxidative condition. Phorate was chosen because of its high toxicity and structural similarities to nerve gases. Phorate sulfoxide and phorate sulfone were found to be the major degradation products under oxidative conditions and pH did not affect the degradation. One additionally peak with m/z of 277 was found at low intensities in the standard of phorate sulfone and when phorate and phorate sulfoxide was oxidized. The peak could however not be identified. After 20 days at room temperature, all samples were analyzed again which showed two more peaks with m/z 111 and 163, respectively. It could not be concluded that these peaks were degradation products of phorate and both were left unidentified. It was concluded that the developed method is well suited for fast pesticide identification in the most common collected sample types in situations where use of chemical weapons or intentional poisoning are suspected. Keywords: UPLC-MS/MS, multi-method, pesticides, generic extraction method ii Emma Eriksson June 3, 2015 Table of contents 1 Introduction 1 1.1 Pesticide classification ...................................................................... 2 1.2 Analytical methods ............................................................................ 3 1.3 Purpose ............................................................................................. 5 2 Materials and methods 6 2.1 Reagents, standards and matrices ................................................... 6 2.2 Pre-study ........................................................................................... 8 2.3 Multi MRM method ............................................................................ 9 2.3.1 Optimization of MS/MS transition and retention time ................... 9 2.4 Sample preparations ....................................................................... 16 2.4.1 Extraction .................................................................................... 16 2.4.2 Extraction recoveries .................................................................. 16 2.5 Degradation of phorate ................................................................... 17 3 Result and discussion 18 3.1 Pre-study ......................................................................................... 18 3.2 Multi MRM method .......................................................................... 20 3.3 Screening of non-spiked matrices ................................................... 21 3.4 Extraction recoveries ...................................................................... 21 3.5 Degradation of phorate ................................................................... 28 4 Conclusion 35 5 Acknowledgments 36 6 References 37 Appendix 1 iii Emma Eriksson June 3, 2015 iv Emma Eriksson June 3, 2015 1 Introduction Pesticides are used to prevent, repel, or mitigate pests (insects, fungus, animals, weeds, microorganism, etc.) in order to protect and increase crop yields and also to inhibit negative effect on human health [1]. Pesticides have an important role to play in controlling vector-borne diseases since diseases from vectors, such as insects and rodents, are a significant health problem and amount to 17 % of the infectious diseases in the world. Pesticides are often mixed with inert ingredients to improve the efficacy and ease the spreading of the active ingredient and are therefore not only found as technical grade but also as powders, granules, emulsions and tablets [2]. More than 1000 active ingredients are used in different formulations [3] and the worldwide consumption of pesticides is approximately 2.5 million ton per year [4]. The toxicity to humans and the environment depends on the pesticide and the exposure. Some effects on humans are skin irritation, headache, nausea, cancerogenic and disturbance on the nervous, endocrine or hormone systems. Humans are exposed to pesticides through dermal, inhalation and/or ingestion exposure [1]. Pesticides are present in the environment from agricultural use by direct application on the soil to obstruct microorganisms and from excessive and inappropriate use on crops. The frequent use of pesticides in the agriculture is resulting in more persistent compounds being present in the soil due to rupture of the natural degradation which in turn can damage the soil to the extent that further growing of crops are reduced [5]. The problems of persistent pesticides are highlighted in the Stockholm Convention on persistent organic pollutants (POPs) where nine of the twelve original POPs are pesticides [6]. Moreover, the pesticides have the ability to spread from the soil to water, air or other environmental systems and since they are toxic to pest, other living organisms may also be affected [7]. In the 1930’s, a German scientist developed highly toxic organophosphates to be used as pesticides (see 1.1 Pesticide classification). These molecules where further developed into chemical weapons that were produced during World War II (WWII). In the end of WWII German had developed and produced the compounds tabun, soman and sarin which are known as the classical nerve gases. Germany was unaware of the fact that they were the only country in possess of nerve gases and because of the fear of a counter attack, they were never used in the war. The research of nerve gases continued after the war and USA found even more toxic compounds and they began producing a new nerve gas, VX in 1961. The structures of the nerve gases are very similar to the organophosphates that are used as pesticides (see Figure 1) and they all target the nervous system by inhibiting the enzyme acetylcholinesterase [8]. Muscles and nerve fibers are controlled by sending stimulated or inhibited signals between the synapses in the nervous system. A stimulating signal is transported by the neurotransmitter acetylcholine and when acetylcholinesterase breaks down