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
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i
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
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
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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
1
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 acetylcholine, this signal is inhibited. Consequently, acetylcholinesterase is terminating signals so next impulse can be transmitted. When acetylcholinesterase is inhibited by a nerve gas or organophosphate pesticide, the nerve signals are not stopped causing symptoms from weakness, headache, vomiting to diarrhea, muscular tremors, breathing difficulty, and death [9].
Figure 1: Structure of the most common nerve gases and the general formula for organophosphates.
P O (or S)
X R2 N R1
S P O
O VX
P O
F O
Sarin
P O O
N N Tabun
O
F O P
Soman Organophosphates
Chemical weapons are today most likely to be used in local conflicts against guerrillas or in civil war [8]. Syria is a recent example, where an armed conflict between the national government and rebel groups led to the use of chemical weapons in the Ghouta area of Damascus. Analysis of both environmental and biological samples confirmed the use of sarin at the militant parties but also against civilians [10]. Terrorism is an increasing concern when it comes to use of chemical weapons and the sarin attack in the Tokyo subway by the group Aum Shinrikyo in 1995 is an example of this [11]. Attacks on the food industry with intentional contamination of food are another concern in the security and safety field. Production, transportation and storage of food are relatively easy targets and an attack of can cause casualties, shortage of provisions and huge economic losses [12]. The effects of a terror attack have been illustrated by a model simulating the spread of contaminated milk in California where it was shown that 100,000 persons could easily be poisoned in a very short time [13].
When the Chemical Weapon Convention (CWC) came into effect in 1997 and banned chemical weapons, the threat level of non-classical chemical weapons increased. Because of the similarity to nerve gases, pesticides are likely to be used as warfare agents since they are found in large quantities all over the world and are therefore more easily accessible. They also have a reduced risk of being detected for inappropriate use since they are not included in the CWC inspections. Furthermore, pesticides have already been used for intentional poisoning in the food industry. For example, a food plant worker in China poisoned dumplings with the pesticide methamidophos to make a statement of the low wages [14] and in Japan malathion was used to poison frozen food which sicken about 2 800 people [15, 16]. Intentional poisoning with pesticides comprehend about one third of all suicides in the world [17] which also manifest the lethality of pesticides.
1.1 Pesticide classification
The only common denominator among pesticides is that they control pests. The chemical and physical structures and properties vary greatly among the pesticides but they can anyhow be classified by chemical structure, the pest they control or hazard. The chemical classification is divided into organophosphates, carbamates, organochlorines and pyrethroids. Organophosphates consist of over 100 compounds which are mostly used in the agriculture but also in preparation of pharmaceuticals. Humans are often exposed to organophosphates through skin absorption, inhalation or food ingestion and they affect the nervous system by inhibiting the enzyme acetylcholinesterase [18]. Degradation products can be even more toxic, persistent and travel long distances in the environment [19]. The general formula of organophosphates is shown in Figure 1.
Carbamates have been used in large scale for the last 40 years, both in agriculture and as biocides in industrial products. There are more than 50 known compounds and all are derived from carbamic acid. Carbamate exposure to human occur through skin absorption or inhalation and as for organophosphates, they target the nervous system by inhibiting acetylcholinesterase. In contrary to the organophosphates, carbamates are more rapidly metabolized and the symptoms (described above) can disappear after a few hours.
Carbamates are not stable under aquatic conditions and the toxicity is overall low in the environment, but some can bioaccumulate in fish because of the slow metabolism and the microflora can be affected at high dose exposure [20]. Figure 2 shows the general formula of carbamates.
Figure 2: General formula for carbamates.
C OR 2 R 1 NH
O
3
Organochlorines are the oldest synthetic and most toxic pesticides. They are affecting the nervous system by damaging the ion channels and thereby interrupting the transport of ions such as calcium, chloride, sodium, and potassium, to and from nerve cells. For example, DDT affects the sodium channel and cyclodienes alter the chloride current [21].
Exposure from organochlorines comes from inhalation or ingestion (mostly form fish) and additional symptoms are cancer, Parkinson’s and Alzimer’s disease. Many organochlorines are persistent in the environment and have high bioaccumulation. People still have traces of organochlorines in their body today, despite many were banned in the 1970-1980s [22]. A general structure does not exist for organochlorines, the only common part of the chemical structure are carbon-chlorine bonds [23].
Pyrethroids are a synthetic analogous of natural occurring pyrethrins from chrysanthemum flowers. Pyrethrin I & II, Cinerin I & II and Jasmolin I & II are the six compounds referred to as pyrethrins and some examples of pyrethroids are cypermethrin, tau-fluvalinate, resmethrin and tetramethrin [24]. Human exposure through skin adsorption causes mild effects but ingestion gives symptoms like muscular twitching, convulsion and coma that can takes days or month to recover from [25]. Pyrethroids are affecting the nervous system by interactions with the sodium channels [26] and in the environment, they can accumulate in sediments and are therefore especially toxic to aquatic organisms [27].
General structure for pyrethroids can be seen in Figure 3.
Figure 3: General structure for pyrethroids.
Pesticide classification by the pest they control includes for example, acaricides (kills ticks and mites), attractants (attract pests), fungicides (kills fungi), fumigants (produce gas or vapor to kill pests), herbicides (kills weeds), insecticides (kills insects) and nematicides (kills nematodes) [1].
The World Health Organization (WHO) recommends classification according to the hazard of the pesticide. Each pesticide is divided into six groups between “Extremely hazardous” and “Unlikely to present acute hazard” based on oral and/or dermal LD50- values for rat [28].
1.2 Analytical methods
The European Union (EU) has strict regulations of pesticide use and residue-levels in the food. Each active ingredient needs to be evaluated and approved prior to use in the agriculture and maximum residue level (MRL) are set for every pesticide to make sure that the food is safe for human consumption (children, pregnant and vegetarian are included) [29]. Each member state in EU is obligated to have a control program that monitor residue levels in food and make sure that no prohibited pesticides are used on the crops. The Swedish Food Agency is responsible for this in Sweden and analysis 1500-2000 random food samples every year [30]. The regulation has resulted in development and optimization of multi-residue analysis that can screen for and identify a large number of pesticides in different food stuff.
Gas chromatography mass spectrometry (GC-MS) has been the first choice in analysis of pesticide residue for a long time but more recently liquid chromatography mass spectrometry (LC-MS) has become more attractive and is now the most used technique.
The change depends on the development of tandem mass spectrometry and new ionization
R2
O R1
O
sources such as electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) [31]. The revolutionary with ESI and APCI is the ionization efficiency that are 103-104 times better than a reduced-pressure source due to the atmospheric pressure in the source, the fact that multiple charged ions can be obtained and that these ion sources can easily interface with HPLC [32, pp. 33-47]. A common tandem MS (MS/MS) instrument is the triple quadrupole (TQ or QqQ) where two quadrupoles are used as analyzers and connected in line with a reaction chamber (q) for fragmentation.
This results in a versatile instrument with higher selectivity and sensitivity since both the precursor ion and its fragments can be analyzed in one run. In comparison to a single MS, MS/MS obtain more structural information on a single ion, enable observations of ion- molecule reactions, ease the determination of fragmentation mechanisms, and gives better selectivity and sensitivity. The determination of fragmentation mechanisms are possible due to the second quadrupole and the ion-molecule reactions can be observed since the gas introduced in the collision cell (reaction chamber) can give raise to gas-phase reactions.
[32, pp. 133-154, 33, pp. 86-93].
The development of ultra performance liquid chromatography (UPLC) has improved the analysis on LC even more. The small particles (diameter < 2 µm) in the column together with an instrument that can handle the high pressures, minimize band spreading and enhances resolution, sensitivity and peak capacity at the same time as the speed of analysis is improved [34]. In the content of a multi-residue analysis, this becomes very important in order to separate large numbers of compounds in a sample [35]. Studies has also proven that LC-MS/MS is superior to GC-MS for this type of application in means of sensitivity, selectivity, limit of quantification (LOQ) and number of pesticides covered in one run [36, 37].
In the literature several multi residue LC/MS/MS methods with 7-341 pesticides in each has been reported for the following foods; green pepper, tomatoes, oranges, mineral waters, lettuce, cucumber, grape, apple, lemon, fruit juice, egg, milk, baby food, maize flour, soil, sediment, human serum, blood and tissue with high extraction recoveries [5, 31, 35, 36, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48]. Two review articles summarize MS/MS- transitions for many of the existing pesticides and come to the conclusion that LC-MS/MS is superior technique for analysis of pesticides [37, 3]. General extraction methods with acetone, ethyl acetate or acetonitrile followed by clean-up steps with for example PSA or dispersive SPE are well-known and efficient for food samples [3, 42]. Acetonitrile has also been used for extraction of biological samples (urine, tissue, blood and serum) [39, 48, 47]
and of soil samples [41, 5, 40, 49]. Extraction of soil samples are reported to be difficult due to the complex composition of organic and inorganic compounds which can interact with the pesticides. The soils particles themselves, in contrast to food samples, bond to the pesticides and the degree of interactions (adsorption, leaching and degradation) depends on the pesticide and the properties of the soil (pH, organic content and texture and mineral fraction). A hydration step before the extraction weakens these interactions since the addition of water makes the pores in the soil more reachable by the extraction solvent [49].
The extraction methods are often time consuming because of the extensive clean-up steps but on the other hand they are well optimized to give high recoveries for one or similar food. In situations that require screening for pesticides in many different matrices it is more suitable to use a generic extraction method. Studies have shown that a single step solvent extraction can be used for this purpose. Mol et. al. (2008) compared acetonitrile, methanol and acetone as extraction solvent with three existing and commonly used extraction methods for pesticides (QuEChERS Method, Ethyl Acetate Method and Modified Luke Method). It was found that all three single step solvent extractions were more generic than the already existing methods and that acetonitrile was the overall best extraction solvent [46]. A more extensive study with 19 different food and beverages, and acetonitrile as extraction solvent showed that the method meets the requirements of a fast, simple and generic extraction with high recoveries (75 % of the compounds had recoveries higher than 70 %). Ion suppression from matrix effects was found to be at an acceptable
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level and it was noted that this kind of method can cause more dirty extracts since no clean-up steps are included [50].
1.3 Purpose
During war, terror attacks, deliberate food contaminations or other scenarios where chemical weapons or toxic compounds are suspected to have been used, there is a need of fast identification in order to be able to warn the public and to prevent further spreading.
Since the likelihood of pesticides being used as potential threat substances has increased, these compounds are in need of a fast standardized analysis method. With this in view, the aim of the project was to develop a fast multi-residue method for screening of pesticides with UPLC-MS/MS. The priority was to create a qualitative analytical method that could establish if pesticides are present in environmental, biological and food samples. As an extension to the method, the goal was to follow the chemical degradation of one pesticide (phorate) over time in different samples.
Initially a pre-study with 5 pesticides (acephate, cypermethrin, fenarimol, imidacloprid and phorate) was performed. The pesticides were chosen based on reported retention times in order to cover the retention time span of all pesticides in the multi-method and the purpose of the pre-study was to gain knowledge of the analysis instrumentation and to investigate extraction recoveries and LC-MS/MS conditions such as gradients and mobile phases.
2 Materials and methods
2.1 Reagents, standards and matrices
Methanol (MeOH) of gradient grade (eluent) and of LC grade (dissolution of standard solutions), acetonitrile of gradient grade (eluent) and of LC grade (extraction solvent) were purchased from LiChrosolv, pro analysis grade ammonium acetate and acetic acid was from Merck, pro analysis grade formic acid and ammonium bicarbonate from Sigma- Aldrich and reagent grade DMSO was bought from Scharlau. Ultra-pure water (Milli-Q) was prepared in the laboratory by using Q-POD® Ultrapure Water Remote Dispenser from Merck Millipore.
Twelve standards, each containing 19-25 pesticides dissolved in MeOH with concentration of 25 µg/mL (28-177 µM) were gifts from the National Swedish Food Agency together with documentation of their multi-method. A list of the pesticides is found in Table 1 and the complete summary with classification, mass, molecular formula and structure are listed in Appendix 1. Two working solutions were prepared by diluting the standards 100 times with 50 % acetonitrile, 0.1 % formic acid for identification of MS/MS transitions and 2 times with 50 % MeOH for use as spiking solution in the recovery studies.
Acephate, cypermethrin (isomers), fenarimol, imidacloprid, phorate, phorate sulfone and phorate sulfoxide were of analytical standard grade and were bought from Sigma-Aldrich.
Stock solution of each pesticide (except phorate sulfone and sulfoxide) at a concentration of 10 mM was prepared in dimethyl sulfoxide (DMSO). Working solutions at 10 and 100 µM was prepared from the stock solution by diluting in 50 % MeOH, 0.1 % formic acid except for cypermethrin, which was diluted in 50 % acetonitrile. Stock solutions of phorate, phorate sulfone and phorate sulfoxide for investigation of degradation products was prepared at 10 mM in MeOH. Working solutions at 5 µM and 200 µM was constructed by diluting the stock solutions with MeOH.
The environmental, biological and food samples listed in Table 2 were used as matrices to which the pesticides were spiked in the recovery study.
Table 1: List of pesticides.
Abamectin Demeton-S-methyl Fenthion sulfoxide Methidathion Pyrethrin II Acephate Demeton-S-methyl sulfone Fluazifop-P-butyl Methiocarb Pyridaphenthion Acetamiprid Demeton-S-methyl sulfoxide Flucythrinate Methiocarb sulfone Pyrifenox (E & Z) Acibenzolar-S-methyl Desmetryn Fludioxonil Methiocarb sulfoxide Pyriproxyfen
Aldicarb Dialifos Flumetralin Methomyl Quinoxyfen
Aldicarb sulfoxide Diazinon Fluopyram Methoprene Quizalofop-ethyl
Aldicarb-sulfone Dichlorvos Fluquinconazole Methoxyfenozide Resmethrin
Aminocarb Dicrotophos Flusilazole Metobromuron Rotenone
Amisulbrom Diethofencarb Fonofos Monocrotophos Simazine
Aspon Difenoconazole Formetanate Myclobutanil Spinosyn A
Atrazine Dimethoate Fuberidazole Napropamide Spinosyn D
Atrazine-desethyl Dimethomorph (E & Z) Furalaxyl Nitenpyram Spirodiclofen Atrazine-desisopropyl Dimoxystrobin Furathiocarb Novaluron Spiroxamine
Azadirachtin Diniconazole Haloxyfop Ofurace Sulfentrazone
Azoxystrobin Diphenamide Haloxyfop ethoxyethyl Omethoate Tau-fluvalinate
Benalaxyl Disulfoton Haloxyfop-methyl Oxamyl Tebuconazole
Bendiocarb Disulfoton sulfone Heptenophos Oxamyl-oxime Tebufenozide
Benfuracarb Disulfoton sulfoxide Hexaconazole Paclobutrazole Tebufenpyrad
Bitertanol DMF Hexazinone Paraoxon-ethyl TEPP
Boscalid DMSA Hexythiazox Paraoxon-methyl Tepraloxydim
Bupirimate DMST Imazalil Penconazole Terbufos
Buprofezin Dodine Imidacloprid Pencycuron Terbufos sulfone
Butocarboxim Epoxiconazole Indoxacarb Phenmedipham Terbufos sulfoxide
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Butocarboxim sulfoxide Ethiofencarb Iprovalicarb Phenothrin Terbuthylazine
Butoxycarboxim Ethiofencarb sulfone Isazofos Phorate Terbutryn
Butralin Ethiofencarb sulfoxide Isocarbophos Phorate sulfone Tetrachlorvinphos
Carbaryl Ethion Isofenphos Phorate sulfoxide Tetraconazole
Carbendazim Ethirimol Isofenphos methyl Phosphamidon (E & Z) Tetramethrin
Carbofuran Ethofenprox Isoprocarb Phoxim Thiabendazole
Carbofuran-3-OH Ethofumesate Isopropalin Picoxystrobin Thiacloprid
Carbophenthion Ethoprophos Isoprothiolane Piperonyl butoxide Thiamethoxam
Carbosulfan Etrimfos Isoproturon Pirimicarb Thiodicarb
Carboxin Famoxadone Isoxaben Pirimicarb desmethyl Thiometon
Carfentrazone-ethyl Fenamidone Jasmolin I Pirimicarb-desmethyl- Thiophanate methyl
Chlorantraniliprole Fenamiphos Jasmolin II formamido Tralomethrin
Chlorbromuron Fenamiphos sulfone Krexoxim-methyl Prochloraz Triadimefon Chlorfenvinphos (E & Z) Fenamiphos sulfoxide Linuron Promecarb Triadimenol (R & S)
Cinerin I Fenarimol Malathion Prometryn Tribenuron-methyl
Cinerin II Fenazaquin Malaoxon Propamocarb Trichlorfon
Clofentezine Fenbuconazole Mandipropamid Propanil Tricyclazole
Clomazone Fenhexamide Mecarbam Propaquizafop Trifloxystrobin
Clopyralid Fenoxycarb Mepanipyrim Propetamphos (E & Z) Triflumuron
Clothianidin Fenpiclonil Mepanipyrim-2- Propiconazole Triforin
Coumaphos Fenpropidin hydroxypropyl Propoxur Trimetarcarb-3,4,5
Cyanazine Fenpropimorph Mephosfolan Prosulfocarb Trimethacarb-2,3,5
Cyazofamid Fenpyroximate Metaflumizone Prothioconazole-desthio Trinexapac ethyl
Cymoxanil Fensulfothion oxon Metalaxyl Prothiofos Triticonazole
Cypermethrin (E & Z) Fensulfothion oxon sulfone Metconazole Pymetrozine Vamidothion Cyproconazole (R & S) Fensulfothion sulfone Methabenzthiazuron Pyraclostrobin Vamidothion
Danifos Fenthion Methacriphos Pyrazophos sulfoxide
Demeton-S Fenthion sulfone Methamidophos Pyrethrin I Zoxamide
Table 2: Description of the sample matrices included in the recovery study.
Matrix Origin and comments
Water Milli-Q from Q-POD® Ultrapure Water Remote Dispenser (Merck Millipore)
Milk Norrmejerier milk, 3 % fat, Norrmejerier AB Orange juice Rynkeby, no pulp, Rynkeby HB
Baby food NaturNes baby food, potato, tomato, beef, Nestlé Sweden AB Sand Hörnefors, Sweden. Pelagia Miljökonsult AB
Soil Hörnefors, Sweden. Pelagia Miljökonsult AB 66.66 % Sand, 16,67 % Clay, 16,67 % Humus Rat serum Sigma-Aldrich. Product number: R9759
2.2 Pre-study
An Agilent Infinity 1200 liquid chromatography coupled to a Micromass Quattro Micro triple quadrupole mass spectrometer with an electrospray ionization source was used. The pesticides were analyzed in positive ionization mode with capillary voltage set to 3.5 kV, source temperature 150 °C and desolvation gas temperature 500 °C. Desolvation gas flow and cone gas flow were 500 L/h and 50 L/h, respectively. Nitrogen was used as desolvation gas and cone gas and argon was used as collision gas. The column was kept at 60 °C and the injection volume was 10 µL. Eluent A consisted of 10 mM ammonium acetate and eluent B was MeOH. The flow rate was set to 0.225 mL/min and the linear gradient was: 0-1 min, 2 % B (v/v); 1-5 min, 1-98 % B; 5-6 min, 98 % B; 6-7 min; 98-2 % B; 7-15 min, 2 % B. MassLynx 4.1 and Agilent Chemstation for LC systems software’s were used to control the LC-MS/MS instrument.
The working solutions of the five pesticides (acephate, cypermethrin, fenarimol, imidacloprid and phorate) at a concentration of 10 µM were used to optimize the MS- parameters. Precursor ion, product ion and optimal cone voltage and collision energy was manually determined from MS and MS/MS spectra acquired from infusion of the working solutions directly into the mass spectrometer. Precursor ion was identified in MS Scan mode and optimal cone voltage was determined by varying the voltage between 10 - 40 V in 4 steps. The product ion was established by fragmenting the precursor ion in Product Scan mode and the optimal collision energy was determined in the same way as for the cone voltage. A multiple reaction monitoring (MRM) method was built in the software with these MS/MS - transitions. Retention times were found by analyzing each standard solution with the instrument settings noted above together with the created MRM-method.
Chromatograms from each MRM-transition were evaluated and the retention time for each peak was noted. Optimized MS/MS parameters and retention times for the five pesticides are shown in Table 3.
Table 3: Retention times and MS/MS parameters for the pesticides included in the pre-study.
Compound tR (min) Precursor ion (m/z)
Product ion (m/z)
CV (V)
CE (eV)
Acephate 5.2 184.2 143.0 10 10
Cypermethrina 7.3/7.6 433.3 191.2 20 20
Fenarimol 7.7 331.2 268.2 30 30
Imidacloprid 6.3 256.2 209.1 20 20
Phorate 8.2 261.2 74.9 20 15
a Detected as NH4+ adduct
Spiked samples were created by adding 5 µL of each pesticide working solution (100 µM) to 100 µL water, milk, orange juice and serum, respectively or to 0.1 g (w.w) baby food, sand and soil, respectively, followed by mixing on a vortex mixer. 300 µL acetonitrile was added and the tube was centrifuged for 10 minutes at 12 000 rpm in a Microfuge® 18 centrifuge from Beckman Coulter. The solvent phase was then collected and diluted 1:1 with water in a 200 µL HPLC vial (90 µL solvent phase, 90 µL water). The final concentration in the samples was 5 µM. 100 µL or 0.1 g blank matrix samples were extracted in the same way as the spiked samples and 5 µL of each pesticide working solution were spiked to the blank extracts after extraction. All samples were made in triplicates and the average peak area of the spiked extract was divided with the average peak area from the blank matrix extract spiked after extraction in order to establish the extraction recoveries.
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The same matrices described in Table 2 were used apart from mouse serum being used instead of rat serum. The same spiked samples water, milk, orange juice and baby food were also analyzed on a Waters Acquity UPLC coupled to a Waters Xevo TQ MS.
2.3 Multi MRM method
A Waters Acquity UPLC coupled to a Waters Xevo triple quadrupole mass spectrometer with an electrospray ionization source was used. The pesticides were analyzed in positive ionization mode with capillary voltage set to 3.5 kV, source temperature 150 °C and desolvation gas temperature 500 °C. Desolvation gas flow and cone gas flow were 500 L/h and 50 L/h, respectively. Nitrogen was used as desolvation gas and cone gas and argon was used as collision gas. The column was kept at 60 °C and the injection volume was 10 µL. The two eluents were 10 mM ammonium acetate, 0.1 % formic acid in water (A) and methanol (B) and the flow rate was 0.4 mL/minute. A linear gradient was used to elute the compounds: 0-1 min, 5% B (v/v); 1-5 min, 5-42% B; 5-11 min, 42-70% B; 11-12 min, 70- 98% B; 12-14 min, 98 % B; 14-14.1 min, 98-5% B; 14.1-16 min, 5% B. MassLynx 4.1 software was used to control the instruments and for data acquisition and processing.
2.3.1 Optimization of MS/MS transition and retention time
Optimization of MS/MS transitions for each pesticide was done with QuanOptimize in MassLynx 4.1. One (of twelve) working solution was optimized at a time and for every pesticide, two injection of 5 µL passed a peek-tube (no column was used) into the mass spectrometer at a flow rate of 0.15 mL/minute. The mobile phase consisted of 0.1 % formic acid in water (30 %) and 0.1 % formic acid in acetonitrile (70%). Each injection was scanned for one minute.
A list of the pesticides in every working solution and their masses were infilled in the software (Figure 4) and the noted masses were used by the software to automatically search for each precursor ion. Precursor ion and optimal cone voltage were identified in the first injection and the second injection was used to identify product ions (two product ions for each pesticide) and optimize the collision energy. The cone voltage and collision energy was varied between 10-40 V with 5 and 10 steps, respectively and a cycle time of 0.25 second (see Method Editor in Figure 4).
Figure 4: Sample list and settings in QuanOptimize for optimization of MS/MS-transitions.
From the result, a MRM-method consisting of the precursor ion, the two product ions, optimal cone voltage and collision energy was created in the software. This method was used to determine the retention times for each pesticide by using the LC method described above. The most intense product ion of each pesticide was identified from the chromatograms and it was used in the MRM-method for the later experiments. The need of 2-3 product ions for quantification and/or verifying identification is known but since this is a screening method, one product ion was considered to be enough.
The final multi MRM-method (parameters in Table 4) was created with ten LC retention time segments to decrease the cycle time and gain enough data points over each peak. The time segments consisted of 15-30 MRM transitions in each segment and were partially overlapping in order to detect pesticides eluting at the front and end of each segment and to manage possible drift in retention times. Figure 5 shows the MRM-method and which pesticide belonging to which MRM segment is seen in Table 4. Distribution of the time windows for each segment in the entire analysis (0-16 minutes) is visually presented by the green boxes in the column “Time” to the right in the figure.
Figure 5: The MRM-method and its time window.
11
Table 4: Optimized MS/MS-transitions and retention times for the pesticides.
MRM segment no.
Compound tR (min) Pre
(m/z)
Pro (m/z)
CV (V)
CE (eV)
1 Methamidophos 1.8 142.0 94.0 20 10
1 Acephate 2.5 183.7 142.4 15 10
1 Oxamyl-oxime 3.0 163.1 90.0 40 15
1 Omethoate 3.0 213.8 124.7 20 20
1 Butocarboxim sulfoxide 3.1 206.8 131.4 20 10
1 Propamocarb 3.1 189.0 101.9 15 15
1 Aldicarb sulfoxide 3.3 207.0 88.8 20 20
1 Butoxycarboxim 3.4 223.1 105.7 15 10
1 Aldicarb-sulfone 3.5 222.8 85.6 25 20
1 Pymetrozine 3.6 217.7 104.1 30 30
1 Oxamyla 3.7 237.2 72.0 30 5
1 Vamidothion sulfoxide 3.77/3.86 303.9 124.8 40 30
1 Nitenpyram 3.8 271.1 125.9 40 30
1 Methomyl 4.0 162.7 105.6 10 10
1 Demeton-S-methyl
sulfoxide 4.0 247.2 108.7 25 30
1 Thiamethoxam 4.0 293.2 210.9 20 20
1 Aminocarb 4.0 208.8 151.7 25 20
1 Demeton-S-methyl
sulphone 4.1 262.9 108.8 25 30
1 Monocrotophos 4.3 224.0 193.0 15 5
1 Atrazine-desisopropyl 4.5 174.0 68.0 40 30
1 Clothianidin 4.7 250.2 132.0 40 15
1 Ethiofencarb sulfonea 4.7 274.9 106.6 15 30
1 Methiocarb sulfone 4.7 258.2 106.7 25 20
1 Imidacloprid 4.7 256.1 209.0 40 10
1 Dicrotophos 4.7 237.7 126.7 15 20
1 Carbendazim 4.8 191.7 159.7 25 20
1 Methacriphos 4.8 241.4 198.2 20 10
2 Dimethoate 5.1 229.8 124.7 25 20
2 Trichlorfon 5.2 257.0 109.2 20 15
2 Acetamiprid 5.2 223.3 126.7 20 20
2 Carbofuran-3-OH 5.3 237.8 163.0 15 20
2 Vamidothion 5.3 288.0 145.5 40 20
2 Thiabendazole 5.4 202.2 174.6 20 30
2 Thiacloprid 5.6 253.0 126.0 35 20
2 Fuberidazole 5.6 184.7 156.7 30 20
2 Atrazine-desethyl 5.6 188.1 146.0 40 15
2 Pirimicarb desmethyl 5.9 224.8 167.7 25 20
2 Tricyclazole 6.0 189.8 135.7 40 30
2 Butocarboxima 6.1 213.1 75.0 40 10
2 DMSAa 6.1 200.7 91.7 20 20
2 Aldicarba 6.1 208.0 116.0 35 5
2 Fensulfothion oxon 6.2 292.8 139.7 35 40
2 Paraoxon-methyl 6.3 248.0 90.0 30 25
2 Fensulfothion oxon sulfone 6.4 308.8 252.7 15 20
2 DMF 6.4 150.0 107.2 35 20
2 Phosphamidon (E & Z) 6.75/6.92 301.2 126.6 40 20
2 Cyanazine 6.8 241.2 213.7 20 20
2 Dichlorvos 6.9 221.0 108.9 35 15
4 Thiophanate methyl 7.0 343.4 151.2 15 20
4 Bendiocarb 7.1 224.0 108.8 20 20
4 Mephosfolan 7.1 270.2 139.6 40 30
4 Simazine 7.1 202.1 131.9 25 15
4 Propoxur 7.2 209.9 110.7 10 20
4 Hexazinone 7.2 252.9 170.7 30 20
4 DMST 7.2 214.8 105.8 15 20
4 Formetanate 7.2 221.8 122.8 20 20
4 Carbofuran 7.3 221.8 122.8 25 20
4 Demeton-S-methyl 7.3 231.1 88.5 25 20
4 Ofurace 7.3 282.3 147.9 20 20
4 Ethirimol 7.4 209.8 97.8 30 30
4 Fenamiphos sulfoxidea 7.5 337.2 320.1 25 5
4 Carbaryl 7.5 202.0 127.0 30 25
4 Malaoxon 7.5 315.0 127.0 20 10
4 Fenthion sulfoxide 7.5 294.9 108.7 15 30
4 Sulfentrazone 7.6 387.0 145.8 35 50
4 Carboxin 7.6 236.0 142.6 20 20
4 Fenamiphos sulfone 7.6 336.3 187.8 20 30
4 Fenthion sulfone 7.7 311.2 124.9 35 20
4 Ethiofencarb 7.9 226.0 107.0 25 10
4 Pirimicarb 8.0 238.8 72.1 30 20
3 Thiodicarb 8.0 355.1 87.4 25 20
3 Metobromuron 8.1 260.6 171.9 20 20
3 Desmetryn 8.1 214.3 171.7 40 20
3 Disulfoton sulfoxide 8.2 291.2 96.6 20 30
3 Phorate sulfoxide 8.2 276.9 96.5 35 40
3 Methabenzthiazuron 8.2 222.0 164.6 10 20
3 Disulfoton sulfone 8.3 306.9 96.4 30 30
3 Isoprocarb 8.3 194.0 95.0 15 10
3 Phorate sulfone 8.4 293.1 247.0 40 5
3 Carbophenthion 8.4 344.0 120.6 25 20
3 Paraoxon-ethyl 8.4 275.8 93.6 15 30
3 Atrazine 8.5 216.3 173.6 25 20
3 Imazalil 8.6 298.4 160.5 15 20
3 Isoproturon 8.6 206.8 164.5 30 20
3 Azadirachtinb 8.6 743.3 725.4 20 30
3 Mepanipyrim-2-
hydroxypropyl 8.7 244.0 225.3 35 20
3 Trimethacarb-2.3.5 8.7 194.0 137.0 20 10
3 Trimetarcarb-3.4.5 8.7 194.1 122.0 25 25
3 Trinexapac ethyl 8.8 253.1 69.0 25 20
3 Heptenophos 8.8 251.1 126.8 30 20
3 Isocarbophosb 8.8 312.0 269.9 15 10
6 Fensulfothion sulfone 8.9 325.1 269.1 30 20
6 Fenthion 8.9 279.8 159.8 25 30
6 Metalaxyl 8.9 279.9 191.8 25 20
6 Methidathion 8.9 302.9 84.9 25 20
6 Fenpropidin 9.0 274.4 146.8 40 30
6 Fenpiclonila 9.0 254.0 202.0 25 25
6 Diphenamide 9.1 240.4 133.8 10 20
6 Phenmedipham 9.2 300.9 136.0 35 20
6 Triforin (R & S) 9.21/9.52 436.2 391.3 20 20
6 Clomazone 9.4 240.0 125.0 25 20
6 Linuron 9.4 250.4 161.7 25 20
6 Pirimicarb-desmethyl-
formamido 9.4 252.9 163.5 30 20
13
6 Chlorantraniliprole 9.5 483.7 452.5 15 20
6 Demeton-S 9.5 258.9 88.6 10 10
6 Spiroxamine 9.60/9.73 298.6 99.7 20 40
6 Methiocarb 9.6 226.0 121.0 25 15
6 Fludioxonila 9.7 266.0 158.0 40 35
6 Ethofumesate 9.7 287.0 121.0 30 10
6 Terbufos sulfone 9.8 321.0 114.9 35 25
6 Terbufos sulfoxide 9.8 305.3 96.7 25 40
6 Chlorbromuron 9.8 294.4 206.0 25 20
6 Diethofencarb 9.8 267.9 123.8 20 40
6 Azoxystrobin 9.9 403.9 371.9 25 20
6 Furalaxyl 9.9 301.9 94.6 15 30
5 Promecarb 10.0 207.8 108.7 15 20
5 Dimethomorph (E & Z) 10.03/10.38 389.2 302.5 20 20
5 Terbuthylazine 10.0 230.2 173.7 30 20
5 Fenamidone 10.1 312.0 91.8 25 30
5 Boscalid 10.2 344.2 271.9 30 30
5 Paclobutrazole 10.3 294.3 70.1 35 15
5 Isoxaben 10.4 332.9 164.8 10 30
5 Isoprothiolane 10.4 291.3 188.8 30 20
5 Mandipropamid 10.4 413.3 329.6 25 20
5 Malathion 10.4 331.2 127.2 15 10
5 Cyproconazole (R & S) 10.40/10.65 292.2 125.0 25 30
5 Myclobutanil 10.4 289.0 125.0 20 40
5 Triadimefon 10.5 295.2 198.4 20 20
5 Pyridaphenthion 10.6 341.0 91.6 25 40
5 Triadimenol (R & S) 10.66/10.78 296.0 70.0 25 10 5 Pyrifenox (E & Z) 10.68/11.10 296.6 92.6 30 20
5 Mepanipyrim 10.7 223.8 105.9 25 30
5 Methoxyfenozide 10.8 369.0 148.7 10 20
5 Prometryn 10.8 242.2 157.7 40 30
5 Fluquinconazole 10.8 377.3 107.6 35 40
5 Fenarimol 10.9 332.6 80.6 35 30
5 Tepraloxydim 10.9 342.2 250.1 15 15
5 Terbutryn 11.0 241.9 185.8 25 20
5 Isazofos 11.0 315.1 96.7 25 30
5 Fenhexamide 11.0 302.0 96.9 25 25
5 Fluopyram 11.0 398.2 172.7 25 30
5 Tetraconazole 11.0 373.6 160.1 35 30
5 Triticonazole 11.1 318.2 70.1 40 15
5 Mecarbam 11.1 330.1 170.5 10 20
5 Fenpropimorph 11.1 305.0 147.3 20 30
7 Prothioconazole-desthio 11.1 313.4 124.7 35 30
7 Epoxiconazole 11.2 330.1 121.1 15 25
7 Iprovalicarb 11.2 321.0 118.9 25 30
7 Ethoprophos 11.2 243.0 96.5 15 30
7 Haloxyfop 11.2 362.0 91.0 40 32
7 Napropamide 11.2 271.9 128.7 20 20
7 Fenbuconazole 11.2 337.0 125.0 15 40
7 & 9 Propetamphos (E & Z) 11.38/12.55 281.9 90.7 35 5
7 Flusilazole 11.4 316.4 164.7 30 30
7 Fenoxycarb 11.4 301.9 87.6 20 20
7 Rotenone 11.5 394.9 212.8 25 20
7 Fenamiphos 11.5 303.9 216.7 40 20
7 Bupirimate 11.5 317.4 165.8 10 30
7 Picoxystrobin 11.7 367.9 144.9 10 20
7 Tetrachlorvinphos 11.7 366.5 205.7 20 40
7 Dimoxystrobin 11.7 326.9 115.7 10 30
7 Penconazole 11.7 285.6 160.7 15 30
7 Tebufenozide 11.7 353.0 132.8 15 20
7 Dodine 11.8 228.0 57.0 25 20
7 Krexoxim-methyl 11.8 314.2 130.5 15 20
7 Jasmolin I 11.8 331.1 131.2 10 20
7 Tebuconazole 11.8 308.2 70.1 35 20
7 Etrimfos 11.9 292.8 124.6 30 30
7 Carfentrazone-ethyl 11.9 413.3 347.9 25 30
7 Fonofos 11.9 247.0 109.1 25 15
7 Zoxamide 12.0 336.1 187.1 15 20
7 Danifos 12.0 327.0 157.0 35 5
7 Isophenphos methyl 12.0 332.0 230.9 10 20
10 Propiconazole 12.0 342.1 159.2 40 25
10 Hexaconazole 12.0 314.0 70.1 25 20
10 Coumaphos 12.1 364.2 228.6 30 20
10 Famoxadonea 12.1 392.2 238.0 40 15
10 Benalaxyl 12.1 326.0 90.5 30 30
10 Chlorfenvinphos (E & Z) 12.1 360.9 98.7 25 30
10 Metconazole 12.1 320.2 70.1 40 25
10 Diazinon 12.1 305.3 152.6 10 20
10 Phorate 12.1 261.1 75.1 10 10
10 Prochloraz 12.2 377.3 268.1 15 20
10 Triflumuron 12.2 359.3 139.1 35 40
10 Bitertanol 12.2 338.0 268.8 15 10
10 Pyraclostrobin 12.2 389.3 163.4 20 20
10 Clofentezine 12.2 303.0 102.0 20 40
10 Diniconazole 12.2 326.1 70.0 35 30
10 Pyrazophos 12.3 374.1 193.8 30 30
10 Pencycuron 12.3 329.2 125.0 25 25
10 Difenoconazole 12.3 407.4 252.7 25 30
10 Spinosyn A 12.3 732.4 142.0 35 30
10 Isofenphos 12.3 346.0 120.7 10 40
10 Cinerin II 12.4 361.2 148.9 20 20
10 Indoxacarb 12.4 528.0 203.0 30 40
10 Haloxyfop-methyl 12.4 377.2 90.5 35 30
9 Trifloxystrobin 12.4 408.9 185.8 15 30
9 Pyrethrin II 12.4 373.0 160.9 20 10
9 Spinosyn D 12.4 747.4 142.0 35 30
9 Prosulfocarb 12.5 252.1 90.7 25 20
9 Metaflumizone 12.5 506.9 177.8 25 20
9 Quizalofop-ethyl 12.5 373.1 299.0 35 15
9 Tetramethrin 12.6 332.1 163.5 20 20
9 Haloxyfop ethoxyethyl 12.6 434.0 91.0 15 40
9 Jasmolin II 12.6 375.2 107.0 20 20
9 Fluazifop-P-butyl 12.6 384.0 90.6 40 40
9 Tebufenpyrad 12.6 334.0 117.0 40 40
9 Furathiocarb 12.6 383.0 194.7 35 20
9 Propaquizafop 12.6 445.3 99.7 20 20
9 Buprofezin 12.6 306.0 116.1 30 10
9 Ethion 12.6 385.4 142.7 15 30
9 Pyriproxyfen 12.7 321.9 95.7 35 20
9 Piperonyl butoxidea 12.7 356.1 176.8 25 20
9 Quinoxyfen 12.7 308.0 197.0 30 35
9 Hexythiazox 12.7 353.0 168.0 15 20
15
9 Aspon 12.8 379.1 114.6 30 30
9 Pyrethrin I 12.8 329.1 160.7 10 10
9 Cinerin I 12.8 317.2 107.0 40 20
8 Butralin 12.8 296.0 240.1 15 20
8 Fenpyroximate 12.8 422.0 365.9 10 20
8 Cypermethrina 12.8 433.3 190.7 15 20
8 Isopropalin 12.9 310.3 206.1 15 15
8 Spirodiclofen 12.9 412.2 71.1 25 15
8 Tau-fluvalinate 12.9 503.1 180.9 15 35
8 Prothiofos 12.9 346.3 242.2 15 20
8 Fenazaquin 12.9 307.0 57.0 30 25
8 Abamectina 13.0 890.6 305.5 10 20
8 Resmethrin 13.0 339.2 170.9 20 20
8 Ethiofencarb sulfoxide 13.0 242.2 185.0 25 10
8 Phenothrin 13.0 351.0 182.8 15 20
8 Methoprene 13.0 311.1 190.8 10 10
8 Dialifos 13.1 395.0 106.4 15 40
8 Ethofenproxa 13.1 394.0 177.0 15 10
Acibenzolar-S-methylc Amisulbromc
Benfuracarbc Carbosulfanc Clopyralidc Cyazofamidc Cymoxanilc Disulfotonc Flucythrinatec Flumetralinc
Methiocarb sulfoxidec Novaluronc
Phoximc Propanilc TEPPc Terbufosc Thiometonc Tralomethrinc Tribenuron-methylc tR: Retention time
Pre: Precursor ion Pro: Product ion CV: Cone voltage CE: Collision energy
a Detected as NH4+ adduct
b Detected as Na+ adduct
c Not found in MS/MS optimization
2.4 Sample preparations
2.4.1 Extraction
100 µL of water, milk, orange juice and rat serum, respectively or 0.1 g baby food was placed in a 2 mL Eppendorf tube. 300 µL acetonitrile was added and the tube was centrifuged for 10 minutes at 12 000 rpm in a Microfuge® 18 centrifuge from Beckman Coulter. The solvent phase was then collected and diluted 1:1 with water in a 200 µL HPLC vial (90 µL solvent phase, 90 µL water).
For sand and soil, an extra hydration step was included before the addition of acetonitrile.
0.1 g sand or soil sample was placed in a 2 mL Eppendorf tube. 200 µL water was added to the sample, rigorously mixed on a Vortex-Genie 2 from Scientific Industries and left on a HulaMixer® from Life technologies for one hour with 90° as reciprocal degree. Finally, the samples were mixed once again on the Vortex-Genie 2 before addition of 300 µL acetonitrile. The sample was centrifuged for 10 minutes at 12 000 rpm, the solvent phase was collected and diluted 4.8:1 with water in a 200 µL vial (150 µL solvent phase, 31 µL water). Schematic view of the workflow can be seen in Figure 6.
Figure 6: Scheme over the extraction.
2.4.2 Extraction recoveries
Spiked samples were created by adding 5 µL of pesticide working solution to 100 µL or 0.1 g blank matrices followed by mixing on a vortex mixer. The samples were extracted as described above. The final concentration in water, milk, orange juice and rat serum were 0.625 µg/mL and 0.625 µg/g in baby food, sand, and soil.
100 µL or 0.1 g (w.w) blank matrix samples were extracted in the same way as the spiked samples. 5 µL of pesticide working solution were spiked to the blank extracts after extraction. All samples were made in triplicates and the average peak area of the spiked extract was divided with the average peak area from the blank matrix extract spiked after extraction in order to establish the extraction recoveries. One working solution was spiked at the time for each matrix.
17
2.5 Degradation of phorate
In addition to the screening method, chemical degradation of one pesticide was investigated to give a better insight of pesticide degradation over time. FOI is already involved in an EU-project called GIFT (Generic Integrated Forensic Toolbox) with the goal of developing forensic analysis (screening, degradation in biological samples and profiling). Phorate is included in that study since it is very toxic, has known metabolites and has very similar structure compared to nerve gases. For that reason, phorate was also chosen in these degradation experiments.
Chemical degradation of phorate was investigated in water under normal, acidic, basic and oxidative conditions. Working solutions of phorate, phorate sulfoxide and phorate sulfone (structure in Figure 7) at a concentration of 5 µM were analyzed in full scan mode with both positive and negative electrospray ionization. Cone voltage was set to 10 V with the mass range of 50-500 m/z and the instrument settings (MS settings, gradient and mobile phase) used is described in 2.3 Multi MRM method.
Degradation of the pesticides under normal conditions was evaluated by separately spiking 5 µL of the three pesticide working solutions with a concentration of 200 µM to 100 µL water and left for one hour. The samples were extracted with the extraction method in 2.4.1 Extraction. The same methodology was used for acidic and basic conditions but with 1 % acetic acid (pH = 2.89) and 10 mM ammonium bicarbonate (pH = 8.75) instead of water, respectively. The samples was oxidized by spiking the pesticides to 90 µL water and 10 µL 30 % hydrogen peroxide (H2O2) followed by the same method described above.
All samples were analyzed with the same settings as the working solutions.
The same samples were left at room temperature and analyzed again after 20 days.
Figure 7: Chemical structure of phorate, phorate sulfoxide and phorate sulfone.
P S O
O
S S P
S O
O
S S
O
P S O
O
S S
O
O
Phorate Phorate sulfoxide Phorate sulfone
3 Result and discussion
Developing multi residue LC-MS/MS methods are difficult due to the high number of compounds with a wide range of chemical and physical properties. When the range of compounds varies from very polar to non-polar combined with high diversity in structure and properties, each compound cannot be analyzed under optimal conditions. Instead, parameters like mobile phase, gradient, column and instrument settings have to be adjusted to be acceptable for most of the compounds resulting in a method that is a compromise between all the compounds included. In this study, different mobile phases and gradients were tested and evaluated to give the best possible method seen to all pesticides included.
The column, a Waters Acquity UPLC HSS T3 (1.8 µm, 2.1x100 mm) is developed for separations of polar and non-polar compounds [51] which was considered to be satisfactory for the wide range of polarity among the tested pesticides.
3.1 Pre-study
The pre-study with five pesticides was performed in order to get to know the analysis instrument and to start the development and evaluation of the multi residue MRM screening method (both extraction and LC-MS/MS method). The amount of available pesticide standard solutions was limited, which also favored a pre-study to establish basic chromatographic conditions. The compounds in the pre-study were chosen among the pesticides (Appendix 1) based on the retention times reported by the Swedish Food Agency. The goal was to include pesticides with a big retention time span, so that the rest of the pesticides could be fitted into the developed LC method.
Cypermethrin was diluted with 50 % acetonitrile due to solubility problem with 50 % MeOH. Ammonium acetate was added to the working solution to be able to ionize cypermethrin in ESI mode. The LC-method was established by testing several gradients with the aim of well separated peaks. A longer gradient than necessary was used since the same gradient was intended for the multi-method with all pesticides. Two different eluent systems were compared. The first consisted of 1 % formic acid in water and acetonitrile and the second eluent combination used 10 mM ammonium acetate as eluent A and MeOH as eluent B. In the former, cypermethrin could not be ionized, not even when ammonium acetate was added to the working solution. However, with the ammonium acetate/methanol eluent system, cypermethrin could be detected as an ammonium adduct.
Chromatograms of the five pesticides are seen in Figure 8.
Working solutions with concentration of 10 µM was first used as spiking solution for determination of extraction recoveries which resulted in a final pesticide concentration of 0.5 µM in the samples prior to the extraction. This concentration gave too low peak intensities for all compounds in all matrices except for cypermethrin in water. Ten times higher peak intensities were desired and the concentration of the spiking solution was increased to 100 µM which gave a final pesticide concentration of 5 µM in the samples.
This gave satisfactory results for all five pesticides. In order to avoid retention time shifts due to high concentration of organic solvent, dilution of the solvent phase from the extraction with water was investigated. The solvent phase was diluted 1:1, 1:2 and 1:5 with water after the centrifugation step in the extraction method. No difference in retention times was found but the early-eluting pesticide, acephate, gave broad and split peaks at higher concentration of acetonitrile. Even though the poor peak shapes were overcome by diluting the extracts with more water, it was still possible to integrate the broad peaks and therefore the 1:1 dilution was selected in order to maintain as high concentration of the pesticides as possible in the samples.
19
Extraction recoveries for the five pesticides in all seven matrices are presented in Table 2.
The average extraction recovery of the individual compounds was 92 % and varied between 60 % (phorate in soil) to 141 % (cypermethrin in serum). Recoveries in soil were lower compared to the other matrices but this was expected due the complexity of soil composition and was not examined further.
Figure 8: Chromatogram from analysis of the five pesticides included in the pre-study on Agilent Infinity 1200 LC coupled to a Micromass Quattro Micro TQ MS. Cypermethrin consisted of two isomers (E & Z) which explains the two peaks in the chromatogram.
Table 5: Extraction recoveries (%) for the compounds included in the pre-study.
Compound Water Milk Juice Baby food
Sand Soil Serum
Acephate 90 94 81 86 98 64 77
Cypermethrin 111 73 131 92 96 113 141
Fenarimol 78 91 84 88 88 76 113
Imidacloprid 107 110 89 80 93 68 114
Phorate 85 87 86 80 81 60 127
The same samples of spiked water, milk, orange juice and baby food were analyzed on two different instruments and the results showed significant difference in sensitivity between the two. The Waters Xevo TQ MS had around 100 times higher peak intensity compared to the Micromass Quattro Micro TQ MS. Different flow rates were used on the two systems, which explain the differences in retention times (Table 3). Chromatogram from Waters instrument is shown in Figure 9.
Figure 9: Chromatogram from analysis of the five pesticides included in the pre-study Waters Acquity UPLC coupled to Waters Xevo TQ MS.
3.2 Multi MRM method
The used standard solutions were expired reference solutions from the national pesticide control program at the Swedish Food Agency (SLV) and the solutions were kind gifts from SLV. The total number of pesticides in the standard solutions added up to 252 compounds, 19 of these were not found during the optimization of the MS/MS parameters (Table 4) and were therefore not included in the final method. It was possible that these pesticides were unstable and had degraded. For example, terbufos and disulfoton were not found in the standards solutions but their breakdown products terbufos sulfoxide and disulfoton sulfoxide were identified. Since the overall condition of the pesticide solutions were not known, no further experiments were done to find the missing pesticides.
The two pesticides carbofuran and formetanate (Table 6) were optimized to the same MS/MS-transitions and retention time. The pesticides could not be separately identified but they were still included in the method since they most likely can be separated with an identification analysis containing three or more product ions or the whole fragmentation chromatogram.
Table 6: Mass, molecular formula and structure for carbofuran and formetanate.
Standard solution
Compound Mass (Da) Molecular formula
Structure
LC-E Carbofuran 221.252 C12H15NO3
LC-N Formetanate 221.256 C11H15N3O2
O N
O
O
O O
N N
N
21
The gradient used in the pre-study showed that the majority of the compounds eluted between 7-10 minutes. Several new gradients were tested on the standard solution LC-F to better separate the peaks in this region. A new eluent A with 10 mM ammonium acetate and 0.1 % formic acid was also tested. The addition of formic acid improved the ionization and gave higher peak intensities for the most of the pesticides and was then chosen over the eluent without formic acid that was used in the pre-study. The final chromatogram with all 233 pesticides included is shown in Figure 10.
Figure 10: Chromatogram of the 233 pesticides found in optimization of MS/MS-transitions. Each color represents one standard solution.
Butocarboxim, fenpiclonil and tau-fluvalinate did only have one product ion with detectable signal when run through the column. These compounds were included in the method anyway since the final MRM-method only consisted of one product ion.
Reproducibility of the retention times in and between matrices was tested on the Waters instrument by analyzing seven replicates of each matrix spiked with the standard solution LC-D. Variation in the retention times was ± 0.1 minute. The time windows in the MRM- method were set to overlap with 0.2-0.3 minutes to include some margin for variation of the retention times.
3.3 Screening of non-spiked matrices
Possible pesticide residues in the matrices used in the study were determined by analysis of blank extracts. No pesticides were found in any of the matrices.
3.4 Extraction recoveries
Extraction recoveries and relative standard deviation for all pesticides in the different matrices are presented in Table 7. Due to insufficient amount of pesticide standard solutions, recoveries for all pesticides in all matrices was not analyzed. The number of pesticides (n) for each matrix included in the recovery study are noted below and in Table 7. Recoveries of 70 % or higher and standard deviation of 20 % or lower was found for 79
% of all compounds. The average extraction recovery was 84 % and varied between 10 % (hepentophos and furathiocarb in serum) and 235 % (pyrethrin II in orange juice). The average recovery in water was 92 % (n = 204) and ranged from 30 % (furathiocarb) to 167
% (haloxyfop ethoxyethyl) and in milk, the recovery interval was 29 % (propetamphos) to 165 % (demeton-S-methyl) with the average 88 % (n = 203). For orange juice the average was 89 % (n = 184) and varied from 33 % (furathiocarb) to 235 % (pyrethrin II) and for baby food the average was 76 % (n = 149) and varied from 13 % (ethion) to 138 % (ofurace). Sand had the average recovery 80 % (n = 128) ranging from 21 % (ethion) to 149 % (dimoxystrobin) and serum varied between 10 % (hepentophos and furathiocarb) and 141 % (hexaconazole) with the average 80 % (n = 142). The distribution of compounds in the extraction recovery range for each matrix is presented in Figure 11.
Figure 11: Distribution of compounds in the extraction recovery range for each matrix.
Twelve pesticides were not detected at sufficiency high intensities in any matrix, neither in the spiked extracts nor the blank extracts spiked after extraction, to be able to determine the recoveries (Table 7). These pesticides already showed low intensities when the standard solutions were analyzed and were probably diluted too much by the extraction method to be detected in the samples. Because of limited amounts of standard solutions, a more concentrated standard could not be spiked to the samples. The pesticides were nevertheless included in the method since they were detected in the standard solutions at higher concentration. Tetramethrin, thiophanate methyl, thiodicarb and phenmedipham could not be extracted from serum and spirodiclofen could not be extracted from milk and orange juice. Overall, the pesticides eluting later than twelve minutes in the chromatogram showed lower recoveries and higher relative standard deviation than the earlier eluting pesticides. This might be explained by the fact that these compounds are fatty and non- polar and they may be more difficult to extract with the proposed extraction method.
These results were not seen as a problem and the extraction method was still considered as sufficient for its purpose. The alternative was to use DMSO as extraction solvent which
