Pesticide Screening Method with UPLC-MS/MS826306/... · 2015. 6. 25. · Pesticide Screening Method...

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

.


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


ii

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


iii

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


iv


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

R1NS

P

O

O

VX

P

O

F

O

Sarin

P

O

O

N

N

Tabun

O

F

OP

Soman Organophosphates


2

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 OR2

R1NH

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

OR1

O


4

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


5

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.


6

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


7

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


8

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.


9

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.


10

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


12

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


14

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


16

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

SS

P

S

O

O

SS

OP

S

O

O

SS

O

O

Phorate Phorate sulfoxide Phorate sulfone


18

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.


20

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

ON

O

O

O

O

NNN


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


22

% 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


23

requires an extra evaporation step and the goal of a fast and generic extraction method

would then not be achieved.

The compounds with extraction recovery exceeding 120 % in general had a very high

standard deviation of the three blank extracts that were spiked after the extraction but

neither this or the overall variation in extraction recoveries and relative standard deviations

among the pesticides could be fully resolved. No pesticide residues were found in the

blank matrices (3.3 Screening of non-spiked matrices) and variation due to pipetting errors

and sample injection on the instrument were ruled out since the standard deviations varied

among the pesticides in the same standard solution. Matrix effects, solubility problems and

degradation are however some possible explanations but these were not investigated. As

mentioned above, a multi-method is a compromise rather than an optimized method for

every compound and therefore some variation in the results are acceptable. If the screening

method confirms presence of a compound, further analysis has to be done to confirm the

results. The extraction method can then be optimized for that particularly compound and

the LC-MS/MS method can be improved by addition of heavily labeled internal standard

and quantifier and qualifier ions.

Table 7: Extraction recoveries (%) for all pesticides included in the study.

Standard

solution Compound

Water

(n = 204)

Milk

(n = 203)

Orange

juice

(n = 184)

Baby

food

(n = 149)

Sand

(n = 128)

Serum

(n = 142)

LC-B Abamectin 102 (57) 68 (28) N/A N/A N/A N/A LC-A Acephate 87 (23) 68 (15) 69 (19) 59 (24) 72 (12) 68 (15)

LC-G Acetamiprid 96 (11) 111 (9) 105 (9) 92 (3) 98 (6) 97 (7)

LC-C Aldicarba

LC-C Aldicarb sulfone 99 (3) 95 (11) 89 (2) 82 (5) 92 (4) 97 (5)

LC-D Aldicarb sulfoxide 95 (13) 71 (10) 80 (13) 74 (5) 88 (10) 77 (9)

LC-J Aminocarb 110 (5) 88 (11) 99 (11) 77 (3) 80 (2) 86 (15)

LC-J Aspon 97 (24) 92 (71) 69 (54) 97 (43) 42 (55) 108 (12)

LC-A Atrazine 84 (17) 75 (4) 70 (7) 64 (9) 75 (9) 79 (7)

LC-K Atrazine-desethyl N/A N/A N/A N/A N/A N/A LC-J Atrazine-desisopropyl 130 (16) 83 (18) 92 (13) 79 (18) 92 (9) 73 (9)

LC-B Azadirachtin 110 (0) 81 (7) N/A N/A N/A N/A LC-C Azoxystrobin 102 (4) 92 (5) 86 (12) 81 (4) 90 (0) 100 (8)

LC-J Benalaxyl 116 (22) 100 (27) 92 (20) 52 (26) 67 (17) 64 (18)

LC-E Bendiocarb 102 (3) 96 (5) 107 (3) N/A N/A N/A LC-C Bitertanol 68 (10) 80 (31) 80 (11) 70 (15) 81 (30) 128 (12)

LC-H Boscalid 104 (14) 90 (4) 101 (7) N/A N/A 91 (10)

LC-G Bupirimate 60 (19) 77 (13) 72 (13) 59 (13) 73 (12) 75 (7)

LC-J Buprofezin 114 (17) 64 (15) 60 (17) 121 (28) 64 (40) 56 (17)

LC-F Butocarboxim sulfoxide 92 (11) 97 (1) 94 (9) 76 (5) 104 (11) 87 (3)

LC-E Butocarboxima

LC-E Butoxycarboxim 104 (7) 92 (1) 107 (3) N/A N/A N/A LC-I Butralin 57 (3) 133 (50) 60 (19) 52 (43) N/A N/A LC-B Carbaryl 102 (26) 92 (24) N/A N/A N/A N/A LC-B Carbendazim 116 (5) 84 (5) N/A N/A N/A N/A LC-E Carbofuran 104 (7) 90 (7) 105 (4) N/A N/A N/A LC-E Carbofuran-3-OH 105 (3) 101 (9) 111 (6) N/A N/A N/A LC-K Carbophenthion N/A N/A N/A N/A N/A N/A LC-F Carboxin 104 (4) 106 (2) 105 (4) 93 (7) 119 (2) 97 (4)

LC-H Carfentrazone-ethyl 93 (45) 105 (19) 145 (18) N/A N/A 28 (30) LC-B Chlorantraniliprole 114 (1) 85 (5) N/A N/A N/A N/A LC-F Chlorbromuron 102 (5) 107 (1) 109 (3) 90 (7) 107 (5) 90 (6)


24

LC-A Chlorfenvinphos (E & Z) 67 (6) 57 (17) 47 (7) 45 (12) 51 (13) 50 (5)

LC-D Cinerin Ia

LC-D Cinerin IIa

LC-E Clofentezine 66 (40) 72 (24) 109 (34) N/A N/A N/A LC-I Clomazone 78 (7) 81 (3) 77 (3) 70 (6) N/A N/A LC-K Clothianidin N/A N/A N/A N/A N/A N/A LC-I Coumaphos 77 (35) 60 (24) 47 (19) 124 (23) N/A N/A LC-C Cyanazine 94 (3) 101 (8) 91 (13) 85 (8) 98 (3) 107 (4)

LC-B Cypermethrin 114 (7) 80 (19) N/A N/A N/A N/A LC-K Cyproconazole (R & S) N/A N/A N/A N/A N/A N/A LC-A Danifos 55 (60) 44 (46) 46 (43) 29 (43) 39 (21) 34 (15)

LC-H Demeton-S 95 (14) 91 (5) 100 (5) N/A N/A 84 (8)

LC-C Demeton-S-methyl 95 (44) 165 (5) 146 (55) 97 (25) 80 (8) 75 (44)

LC-D

Demeton-S-methyl

sulfoxide 103 (10) 70 (11) 86 (15) 75 (3) 86 (7) 72 (13)

LC-C

Demeton-S-methyl

sulphone 101 (7) 93 (15) 94 (5) 86 (8) 93 (4) 87 (9)

LC-J Desmetryn 108 (9) 86 (8) 102 (7) 81 (7) 99 (5) 84 (14)

LC-J Dialifos 125 (10) 85 (16) 80 (12) 71 (17) 40 (18) 78 (23)

LC-A Diazinon 62 (7) 47 (40) 35 (17) 36 (17) 40 (6) 47 (3)

LC-A Dichlorvos 81 (24) 73 (18) 62 (30) 58 (37) 69 (5) 31 (35)

LC-J Dicrotophos 105 (4) 88 (6) 107 (7) 74 (6) 97 (1) 79 (11)

LC-J Diethofencarb 109 (10) 85 (9) 102 (13) 82 (5) 87 (4) 86 (17)

LC-C Difenoconazole 82 (11) 91 (22) 82 (25) 103 (2) 54 (22) 122 (10)

LC-A Dimethoate 95 (11) 77 (5) 72 (13) 68 (18) 75 (2) 75 (4)

LC-F Dimethomorph (E & Z) 108 (3) 113 (2) 109 (4) 90 (4) 108 (11) 96 (5)

LC-F Dimoxystrobin 121 (20) 97 (16) 103 (14) 89 (16) 149 (2) 76 (14)

LC-N Diniconazole 105 (46) 93 (19) 121 (25) 71 (39) 101 (26) 67 (8)

LC-I Diphenamide 86 (3) 83 (4) 85 (4) 80 (4) N/A N/A LC-C Disulfoton sulfone 99 (2) 97 (7) 89 (14) 79 (4) 95 (2) 100 (10)

LC-D Disulfoton sulfoxide 98 (17) 80 (10) 92 (10) 72 (11) 91 (10) 78 (12)

LC-F DMF 96 (4) 102 (5) 100 (8) 85 (5) 111 (5) 86 (2)

LC-H DMSA 97 (17) 94 (4) 97 (3) N/A N/A 87 (11)

LC-H DMST 94 (16) 94 (4) 93 (7) N/A N/A 90 (11)

LC-N Dodine 94 (26) 102 (8) 90 (22) 63 (13) 39 (36) 82 (18)

LC-K Epoxiconazole N/A N/A N/A N/A N/A N/A LC-E Ethiofencarb 106 (6) 100 (7) 106 (4) N/A N/A N/A LC-E Ethiofencarb sulfone 107 (9) 92 (6) 109 (6) N/A N/A N/A LC-F Ethiofencarb sulfoxidea

LC-A Ethion 71 (40) 76 (21) 33 (36) 13 (33) 21 (22) 30 (4)

LC-N Ethirimol 101 (1) 84 (9) 91 (8) 61 (13) 81 (5) 85 (4)

LC-J Ethofenprox 104 (15) 79 (16) 79 (15) 68 (26) 40 (9) 80 (17)

LC-J Ethofumesate 114 (6) 89 (12) 107 (9) 81 (2) 93 (8) 77 (12)

LC-G Ethoprophos 68 (21) 79 (9) 80 (15) 76 (17) 75 (13) 72 (9)

LC-J Etrimfos 106 (7) 81 (8) 101 (14) 85 (2) 76 (11) 92 (14)

LC-C Famoxadonea

LC-N Fenamidone 93 (12) 88 (2) 100 (8) 70 (11) 97 (6) 91 (5)

LC-N Fenamiphos 110 (10) 72 (27) 118 (6) 63 (14) 81 (13) 58 (15)

LC-H Fenamiphos sulfone 102 (10) 96 (4) 112 (5) N/A N/A 52 (4)

LC-K Fenamiphos sulfoxide N/A N/A N/A N/A N/A N/A LC-A Fenarimol 76 (14) 69 (7) 58 (14) 63 (13) 61 (3) 58 (7)

LC-J Fenazaquin 107 (9) 81 (12) 110 (25) 81 (5) 56 (5) 75 (14)

LC-C Fenbuconazole 93 (7) 97 (5) 87 (6) 79 (7) 89 (6) 106 (8)

LC-D Fenhexamide 84 (15) 92 (11) 92 (10) 85 (9) 108 (10) 87 (3)

LC-E Fenoxycarb 112 (6) 75 (6) 117 (3) N/A N/A N/A LC-F Fenpiclonila


25

LC-C Fenpropidin 97 (9) 93 (6) 87 (16) 77 (5) 35 (36) 97 (6)

LC-D Fenpropimorph 110 (64) 99 (8) 89 (8) 65 (4) 37 (31) 63 (15)

LC-H Fenpyroximate 56 (15) 75 (13) 148 (35) N/A N/A 84 (10)

LC-H Fensulfothion oxon 99 (14) 94 (2) 99 (2) N/A N/A 74 (10)

LC-G

Fensulfothion oxon

sulfone 85 (9) 96 (14) 98 (7) 86 (6) 98 (8) 32 (14)

LC-G Fensulfothion sulfone 67 (15) 86 (7) 83 (12) 74 (8) 78 (7) 86 (9)

LC-A Fenthion 96 (2) 72 (2) 71 (6) 61 (11) 73 (8) 76 (6)

LC-A Fenthion sulfone 81 (10) 65 (18) 58 (13) 54 (13) 62 (8) 68 (9)

LC-B Fenthion sulfoxide 124 (6) 87 (3) N/A N/A N/A N/A LC-I Fluazifop-P-butyl 89 (64) 97 (37) 39 (17) 61 (36) N/A N/A LC-K Fludioxonil N/A N/A N/A N/A N/A N/A LC-N Fluopyram 97 (12) 90 (2) 97 (8) 68 (11) 91 (14) 90 (4)

LC-J Fluquinconazole 119 (20) 79 (8) 95 (25) 91 (18) 115 (21) 83 (37)

LC-C Flusilazole 83 (10) 113 (6) 90 (11) 100 (7) 98 (6) 125 (13)

LC-K Fonofos N/A N/A N/A N/A N/A N/A LC-N Formetanate 96 (9) 81 (8) 104 (9) 63 (18) 103 (10) 101 (10)

LC-C Fuberidazole 99 (4) 94 (6) 91 (9) 81 (5) 71 (10) 105 (8)

LC-J Furalaxyl 113 (6) 88 (11) 99 (12) 77 (4) 89 (3) 82 (14)

LC-G Furathiocarb 30 (28) 47 (43) 33 (7) 39 (19) 37 (6) 10 (11)

LC-G Haloxyfop 75 (30) 91 (5) 109 (18) 83 (5) 92 (6) 77 (19)

LC-H Haloxyfop ethoxyethyl 167 (10) 101 (14) 86 (18) N/A N/A 35 (40)

LC-H Haloxyfop-methyl 71 (17) 112 (15) 103 (44) N/A N/A 57 (72)

LC-J Heptenophos 114 (10) 87 (6) 105 (10) 78 (5) 81 (9) 10 (31)

LC-C Hexaconazole 91 (33) 95 (13) 91 (19) 89 (27) 123 (11) 141 (18)

LC-G Hexazinone 72 (9) 93 (17) 85 (7) 81 (8) 94 (4) 94 (8)

LC-E Hexythiazox 88 (64) 155 (43) 61 (28) N/A N/A N/A LC-C Imazalil 114 (5) 92 (8) 90 (12) 73 (7) 49 (24) 93 (10)

LC-C Imidacloprid 80 (20) 89 (22) 70 (15) 78 (16) 79 (23) 98 (20)

LC-H Indoxacarb 49 (21) 97 (15) 88 (9) N/A N/A 72 (26)

LC-F Iprovalicarb 102 (7) 105 (1) 107 (4) 95 (7) 112 (4) 96 (5)

LC-F Isazofos 107 (10) 110 (5) 104 (13) 105 (16) 100 (5) 94 (9)

LC-H Isocarbophosa

LC-J Isofenphos 83 (19) 122 (22) 75 (46) 64 (26) 83 (24) 75 (28)

LC-G Isophenphos methyl 55 (15) 59 (9) 58 (25) 52 (14) 61 (12) 60 (19)

LC-E Isoprocarb 100 (7) 85 (9) 111 (3) N/A N/A N/A LC-I Isopropalin 67 (81) 86 (43) 52 (47) 100 (43) N/A N/A LC-E Isoprothiolane 104 (3) 94 (8) 116 (6) N/A N/A N/A LC-F Isoproturon 96 (7) 108 (10) 108 (3) 90 (6) 109 (8) 92 (3)

LC-C Isoxaben 97 (1) 99 (7) 89 (12) 87 (4) 88 (6) 101 (13)

LC-D Jasmolin I 113 (49) 94 (6) 116 (55) 99 (20) 40 (25) 37 (10)

LC-D Jasmolin IIa

LC-D Krexoxim-methyl 112 (19) 100 (16) 121 (25) 102 (25) 94 (10) 21 (9)

LC-B Linuron 116 (6) 83 (11) N/A N/A N/A N/A LC-K Malaoxon N/A N/A N/A N/A N/A N/A LC-B Malathion 110 (3) 94 (10) N/A N/A N/A N/A LC-N Mandipropamid 85 (17) 92 (5) 102 (10) 74 (11) 97 (10) 91 (10)

LC-J Mecarbama

LC-H Mepanipyrim 92 (10) 92 (4) 97 (3) N/A N/A 96 (9)

LC-N

Mepanipyrim-2-

hydroxypropyl 105 (2) 84 (7) 89 (1) 69 (23) 88 (15) 87 (3)

LC-J Mephosfolan 113 (1) 79 (11) 108 (10) 83 (7) 101 (9) 82 (11)

LC-N Metaflumizone 79 (45) 81 (34) 95 (48) 83 (13) 80 (20) 94 (6)

LC-A Metalaxyl 94 (2) 74 (6) 71 (8) 60 (13) 73 (9) 76 (2)

LC-N Metconazole 84 (29) 105 (14) 98 (33) 68 (34) 82 (24) 134 (19)

LC-F Methabenzthiazuron 103 (3) 106 (3) 107 (5) 88 (3) 97 (8) 90 (3)


26

LC-D Methacriphos 84 (5) 72 (11) 83 (14) 61 (19) 87 (11) 78 (12)

LC-B Methamidophos 110 (5) 85 (4) N/A N/A N/A N/A LC-A Methidathion 67 (6) 58 (14) 50 (4) 44 (14) 47 (9) 54 (9)

LC-E Methiocarb 98 (3) 103 (6) 100 (3) N/A N/A N/A LC-E Methiocarb sulfone 106 (7) 100 (7) 105 (1) N/A N/A N/A LC-B Methomyl 117 (5) 91 (2) N/A N/A N/A N/A LC-B Methoprene 75 (24) 89 (16) N/A N/A N/A N/A LC-F Methoxyfenozide 107 (4) 108 (3) 115 (2) 89 (7) 97 (4) 102 (4)

LC-N Metobromuron 92 (12) 81 (9) 89 (7) 78 (10) 102 (6) 89 (9)

LC-G Monocrotophos 85 (2) 95 (12) 96 (8) 81 (8) 101 (10) 93 (12)

LC-J Myclobutanil 108 (10) 83 (11) 110 (13) 86 (21) 98 (7) 84 (16)

LC-I Napropamide 75 (8) 77 (6) 85 (5) 75 (7) N/A N/A LC-N Nitenpyrama

LC-G Ofurace 83 (11) 101 (7) 100 (20) 138 (2) 92 (8) 94 (6)

LC-A Omethoate 95 (11) 73 (8) 72 (20) 62 (19) 77 (3) 58 (14)

LC-F Oxamyl 77 (9) 100 (12) 87 (12) 88 (16) 99 (6) 92 (26)

LC-E Oxamyl-oxime 97 (14) 77 (18) 108 (11) N/A N/A N/A LC-G Paclobutrazole 91 (15) 94 (22) 94 (14) 94 (9) 93 (3) 100 (9)

LC-K Paraoxon-ethyl N/A N/A N/A N/A N/A N/A LC-A Paraoxon-methyl 78 (58) 55 (53) 53 (58) 48 (53) 57 (51) 46 (52)

LC-D Penconazole 113 (18) 95 (8) 91 (17) 71 (13) 84 (15) 87 (26)

LC-I Pencycuron 85 (42) 50 (23) 68 (4) 64 (41) N/A N/A LC-G Phenmedipham 71 (23) 94 (21) 102 (22) 112 (19) 95 (10)

LC-K Phenothrin N/A N/A N/A N/A N/A N/A LC-H Phorate 70 (47) 76 (24) 111 (32) N/A N/A 85 (83)

LC-H Phorate sulfone 98 (14) 107 (3) 101 (9) N/A N/A 86 (6)

LC-G Phorate sulfoxide 78 (7) 96 (15) 94 (19) 92 (1) 90 (13) 89 (10)

LC-K Phosphamidon (E & Z) N/A N/A N/A N/A N/A N/A LC-G Picoxystrobin 47 (18) 63 (12) 52 (7) 49 (4) 54 (2) 55 (10)

LC-B Piperonyl butoxide 68 (43) 79 (28) N/A N/A N/A N/A LC-B Pirimicarb 112 (3) 89 (5) N/A N/A N/A N/A LC-D Pirimicarb desmethyl 95 (11) 75 (5) 89 (10) 83 (3) 76 (13) 75 (7)

LC-B

Pirimicarb-desmethyl-

formamido 127 (13) 84 (12) N/A N/A N/A N/A LC-A Prochloraz 83 (13) 53 (3) 58 (15) 54 (12) 55 (3) 65 (7)

LC-E Promecarb 102 (5) 94 (9) 111 (3) N/A N/A N/A LC-A Prometryn 76 (3) 64 (8) 56 (5) 53 (8) 58 (3) 65 (8)

LC-F Propamocarb 95 (3) 92 (2) 88 (6) 72 (6) 38 (13) 85 (3)

LC-I Propaquizafop 60 (10) 80 (13) 66 (32) 93 (30) N/A N/A LC-I Propetamphos (E & Z) 59 (53) 29 (64) 41 (61) 85 (42) N/A N/A LC-A Propiconazole 70 (10) 63 (14) 55 (6) 51 (13) 58 (2) 60 (8)

LC-F Propoxur 96 (5) 105 (2) 113 (3) 78 (6) 112 (6) 87 (7)

LC-D Prosulfocarb 67 (11) 77 (5) 77 (18) 86 (34) 68 (34) 95 (43)

LC-I Prothioconazole-desthio 77 (6) 79 (2) 89 (3) 77 (9) N/A N/A LC-I Prothiofos 78 (6) 92 (22) 43 (88) 62 (74) N/A N/A LC-H Pymetrozine 101 (11) 88 (5) 91 (4) N/A N/A 80 (11)

LC-D Pyraclostrobin 82 (5) 79 (2) 97 (8) 80 (17) 68 (8) 113 (83)

LC-B Pyrazophos 144 (19) 99 (28) N/A N/A N/A N/A LC-D Pyrethrin I 74 (84) 97 (22) 54 (62) 57 (10) 61 (31) 75 (20)

LC-D Pyrethrin II 120 (21) 126 (56) 235 (51) 102 (78) 44 (46) 88 (23)

LC-I Pyridaphenthion 76 (8) 76 (3) 70 (1) 66 (8) N/A N/A LC-J Pyrifenox (E & Z) 104 (6) 84 (8) 103 (14) 81 (3) 87 (5) 84 (14)

LC-D Pyriproxyfen 58 (12) 78 (13) 83 (22) 53 (13) 64 (60) 39 (21)

LC-I Quinoxyfen 85 (45) 94 (19) 45 (30) 48 (32) N/A N/A LC-I Quizalofop-ethyl 73 (16) 73 (30) 37 (37) 86 (30) N/A N/A LC-E Resmethrin 61 (11) 118 (40) 53 (15) N/A N/A N/A


27

LC-K Rotenone N/A N/A N/A N/A N/A N/A LC-B Simazine 107 (10) 86 (11) N/A N/A N/A N/A LC-I Spinosyn A 70 (36) 59 (16) 48 (23) 62 (33) N/A N/A LC-I Spinosyn D 61 (46) 137 (26) 53 (21) 73 (43) N/A N/A LC-N Spirodiclofen 125 (39) 58 (40) 70 (35) 93 (50)

LC-D Spiroxamine 92 (24) 82 (5) 89 (9) 79 (1) 38 (29) 74 (12)

LC-H Sulfentrazone 98 (14) 98 (2) 104 (5) N/A N/A 93 (11)

LC-K Tau-fluvalinate N/A N/A N/A N/A N/A N/A LC-A Tebuconazole 88 (9) 70 (5) 65 (8) 62 (9) 69 (11) 79 (9)

LC-F Tebufenozide 130 (32) 80 (29) 98 (12) 113 (17) 103 (19) 96 (30)

LC-K Tebufenpyrad N/A N/A N/A N/A N/A N/A LC-I Tepraloxydim 96 (4) 94 (4) 94 (6) 79 (7) N/A N/A LC-G Terbufos sulfone 50 (22) 77 (19) 70 (3) 64 (29) 73 (18) 63 (23)

LC-H Terbufos sulfoxide 80 (21) 87 (14) 93 (5) N/A N/A 98 (5)

LC-K Terbuthylazine N/A N/A N/A N/A N/A N/A LC-G Terbutryn 62 (11) 78 (17) 73 (8) 66 (8) 70 (6) 70 (5)

LC-K Tetrachlorvinphos N/A N/A N/A N/A N/A N/A LC-G Tetraconazole 71 (13) 96 (15) 84 (11) 79 (3) 85 (6) 77 (4)

LC-N Tetramethrin 78 (21) 114 (27) 117 (35) 85 (9) 78 (25)

LC-D Thiabendazole 91 (15) 64 (7) 97 (13) 84 (6) 60 (24) 73 (21)

LC-G Thiacloprid 86 (9) 100 (12) 97 (10) 90 (4) 96 (7) 97 (6)

LC-I Thiamethoxam 98 (8) 92 (3) 99 (3) 84 (4) N/A N/A LC-D Thiodicarb 87 (16) 67 (8) 86 (3) 76 (11) 91 (17)

LC-D Thiophanate methyl 106 (23) 107 (14) 92 (11) 91 (3) 77 (10)

LC-A Triadimefon 74 (5) 62 (8) 61 (9) 54 (13) 58 (9) 65 (17)

LC-B Triadimenol (R & S) 110 (8) 85 (10) N/A N/A N/A N/A LC-K Trichlorfon N/A N/A N/A N/A N/A N/A LC-E Tricyclazole 104 (5) 91 (7) 108 (8) N/A N/A N/A LC-I Trifloxystrobin 55 (20) 55 (17) 82 (25) 91 (41) N/A N/A LC-B Triflumuron 95 (15) 104 (25) N/A N/A N/A N/A LC-N Triforin (R & S) 101 (6) 99 (9) 101 (8) 73 (14) 96 (12) 98 (2)

LC-E Trimetarcarb-3,4,5 103 (6) 92 (4) 102 (3) N/A N/A N/A LC-F Trimethacarb-2,3,5 99 (5) 105 (3) 112 (1) 89 (3) 119 (5) 90 (1)

LC-D Trinexapac ethyl 90 (17) 72 (9) 91 (13) 75 (1) 85 (11) 72 (22)

LC-I Triticonazole 85 (8) 91 (11) 90 (9) 78 (10) N/A N/A LC-H Vamidothion 109 (14) 83 (8) 93 (2) N/A N/A 76 (20)

LC-F Vamidothion sulfoxide 94 (5) 97 (1) 109 (12) 83 (6) 94 (3) 86 (3)

LC-C Zoxamide 62 (18) 109 (12) 69 (27) 111 (6) 106 (27) 128 (33)

a Not detected at sufficiency high intensities

The first analysis showed lower extraction recoveries in sand and soil than expected from

the pre-study. This led us to investigate adding a hydration step in the extraction method to

weaken the interaction between the soil and the pesticides. Extraction recoveries from

addition of 50 µL and 200 µL water and 200 µL, 50 mM ammonium acetate (AmAc) prior

to the extraction of sand and soil were evaluated and compared with the original extraction

method (Table 8). Addition of 200 µL water was the only change in the method that gave

higher recoveries for soil in comparison with the original extraction method. For sand, the

recoveries increased for all types of hydration steps, with the best result for 200 µL AmAc.

Since the extraction method was aiming to be generic for all matrices, a hydration step

with 200 µL water was chosen to be added to the extraction method, but only for the

environmental samples (sand and soil).

The hydration step was tested with both mixing on the Vortex-Genie 2 and the HulaMixer

(as described in 2.4.1 Extraction) and with no mixing at all (only left to settle for 1 hour).


28

The samples that were rigorously mixed had in average 28 % lower recovery than the

samples that were not mixed at all during the hydration step. It was concluded that the

pesticides stayed in the water phase when no mixing was applied, but thorough mixing

resulted in pesticides sticking to the sand/soil. These interactions make it more difficult to

extract the pesticides from the soil which explains the lower extraction recoveries found

when mixing was utilized. Rigorously mixing was included to the hydration step to better

simulate extractions of real soil samples where these interactions are likely to occur. A

change in dilution of the solvent phase after the centrifugation step for the environmental

samples was implemented as a compensation for the extra addition of water. The pesticide

concentration and water content are the same in the samples to be analyzed regardless of

which matrix extracted.

Table 8: Extraction recoveries (%) for sand and soil with different hydration steps added to the original extraction method.

Original 50 µL water 200 µL water 200 µL AmAc

Sand Soil Sand Soil Sand Soil Sand Soil

45 68 66 49 66 79 98 34

The absence of additional clean-up steps in the extraction methods makes the extracts

dirty. Especially soil samples made both the column and the instrument dirty, resulting in

higher back pressure and lower sensitivity. Due to the low extraction recoveries and

instrumental problems together with the fact that a standard operating procedure for

extraction of soil samples already exists at FOI, soil was excluded from the rest of the

study. Usage of FOI’s extraction method will however be more complicated and might

require some modifications to be suited for LC-MS/MS analysis. As a result of the dirty

column and higher back pressure, the retention times shifted so the time segments in the

MRM-method had to be increased.

3.5 Degradation of phorate

The results from chemical degradation of phorate were seen as a good starting point for

later experiments. Phorate was subjected to oxidative, acidic and basic conditions in order

to simulate environmental conditions. Phorate sulfoxide and phorate sulfone were included

in the reference solutions since they were already known degradation products of phorate.

Pesticides or degradation products were detected in positive electrospray ionization mode,

but none were detected in negative electrospray ionization mode. Analysis of the working

solutions of phorate, phorate sulfoxide and phorate sulfone gave distinct peaks in the

chromatogram. An unidentified peak with m/z 277 was observed at 6.07 minutes in the

chromatogram of phorate sulfone. Phorate sulfoxide (C7H17O3PS3, Mw= 276 Da) was

dismissed as an alternative to this peak even though the m/z-ratio was a match because of

the difference in retention time. Chromatograms are presented in Figure 12.


29

Figure 12: Extracted mass chromatogram from full scan of working solutions for phorate, phorate sulfoxide and phorate sulfone

In the degradation experiments under acidic and basic condition, pH was selected with

regards to the most likely pH extremes of environmental, biological and food samples.

Therefore, a pH lower than 2.5 and higher than 9 were considered as unrealistic. No new

products or significant change of the origin compounds were found under acidic or basic

conditions and it was therefore concluded that degradation of phorate is not influenced by

pH.

Neither phorate, phorate sulfoxide or phorate sulfone showed any short-time degradation

in water but when the compounds were oxidized with hydrogen peroxide, new products

were formed. Phorate subjected to hydrogen peroxide was completely oxidized and could

no longer be found in the sample, whereas phorate sulfoxide and phorate sulfone were

formed and identified in the sample (Figure 13). When hydrogen peroxide was added to

phorate sulfoxide, low levels of the sulfoxide was still found but phorate sulfone had been

formed (Figure 14). Phorate sulfone was the only compound that did not react with

hydrogen peroxide to form any detectable product (Figure 15). This indicates that phorate

sulfoxide and phorate sulfone are the main degradation products of phorate and phorate

sulfone is the most stable degradation product since phorate sulfoxide can, to some extent,

be further oxidized to phorate sulfone. All chromatograms below are extracted mass

chromatograms from the identified peaks in the full scan chromatogram to ease the

interpretation.

P

S

O

O

SS

P

S

O

O

SS

OP

S

O

O

SS

O

O


P

S

O

O

SS

P

S

O

O

SS

OP

S

O

O

SS

O

O


P

S

O

O

SS

P

S

O

O

SS

OP

S

O

O

SS

O

O



30

Figure 13: Extracted mass chromatogram from full scan of phorate in water (top) and phorate oxidized with hydrogen peroxide (bottom).

P

S

O

O

SS

P

S

O

O

SS

OP

S

O

O

SS

O

O


P

S

O

O

SS

P

S

O

O

SS

OP

S

O

O

SS

O

O


P

S

O

O

SS

P

S

O

O

SS

OP

S

O

O

SS

O

O



31

Figure 14: Extracted mass chromatogram from full scan of phorate sulfoxide in water (top) and

phorate sulfoxide oxidized with hydrogen peroxide (bottom).

P

S

O

O

SS

P

S

O

O

SS

OP

S

O

O

SS

O

O


P

S

O

O

SS

P

S

O

O

SS

OP

S

O

O

SS

O

O


P

S

O

O

SS

P

S

O

O

SS

OP

S

O

O

SS

O

O



32

Figure 15: Extracted mass chromatogram from full scan of phorate sulfone in water (top) and phorate

sulfone oxidized with hydrogen peroxide (bottom).

The unidentified compound eluting at 6 minutes in the working solution of phorate sulfone

was also found at low intensity when phorate sulfoxide was oxidized with hydrogen

peroxide and at even lower intensities when phorate was oxidized. Mass chromatograms

of m/z 277 extracted from the full scan analysis of these three samples are shown in Figure

17. The peak at 8.8 minutes is phorate sulfoxide. Since the unidentified compound was

formed during oxidation of both phorate and phorate sulfoxide, it was suspected to be a

degradation product of the sulfoxide and a possible candidate is the phorate oxygen

sulfone (Figure 16) with the mass of 276.3 Da. Product scan of phorate sulfoxide, phorate

sulfone and the unidentified compound was acquired in the mass range of 50-500 m/z with

collision energy of 20 eV as an attempt to identify the compound by the fragmentation

pattern (Figure 18). The fragmentation of phorate sulfoxide and sulfone are very similar

with only fragment extra for phorate sulfone but the fragmentation pattern for the

unidentified compound was too different in comparison to phorate sulfoxide and sulfone to

be able to positively identify it as the phorate oxygen sulfone analog.

Hydrogen peroxide provides the most extreme conditions possible for degradation in the

matrices included in this study and because the two major degradation products found

were already known, long-time test for investigation of chemical degradation in the rest of

matrices was not carried out.

Figure 16: Chemical structure of phorate oxygen analog sulfone.

P

O

O

O

SS

O

O

Phorate oxygen analog sulfone

P

S

O

O

SS

P

S

O

O

SS

OP

S

O

O

SS

O

O


P

S

O

O

SS

P

S

O

O

SS

OP

S

O

O

SS

O

O



33

Figure 17: Mass chromatogram of m/z 277 from full scan analysis of phorate sulfone working solution (top), phorate sulfoxide oxidized with hydrogen peroxide (middle) and phorate oxidized with hydrogen

peroxide (bottom).

Figure 18: Spectra from product scan of phorate sulfone (precursor ion: 292), phorate sulfoxide pPrecursor ion: 277) and the unidentified peak (precursor ion: 277).

P

O

O

O

SS

O

O


P

O

O

O

SS

O

O


P

O

O

O

SS

O

O


?

?

?

P

S

O

O

SS

P

S

O

O

SS

OP

S

O

O

SS

O

O


P

O

O

O

SS

O

O


?

P

S

O

O

SS

P

S

O

O

SS

OP

S

O

O

SS

O

O


Phorate sulfone standard

Oxidation of phorate sulfoxide

Oxidation of phorate

P

S

O

O

SS

P

S

O

O

SS

OP

S

O

O

SS

O

O


P

S

O

O

SS

P

S

O

O

SS

OP

S

O

O

SS

O

O


P

S

O

O

SS

P

S

O

O

SS

OP

S

O

O

SS

O

O


P

S

O

O

SS

P

S

O

O

SS

OP

S

O

O

SS

O

O


P

S

O

O

SS

P

S

O

O

SS

OP

S

O

O

SS

O

O



34

After 20 days at room temperature, the same samples were run again in full scan mode

with positive electrospray. All samples (standards and water samples under normal, acidic,

basic and oxidative conditions) contained two new peaks. One at the retention time 5.84

minutes and m/z 111 and the other at the retention time 6.81 minutes and m/z 163.

Fragmentation of both masses were investigated and compared to fragments of phorate but

the peaks could not be identified or even concluded to be degradation products of phorate.

The unidentified peak from the first analysis (m/z 277, tR = 6.07 min) was at day 20 found

in all samples and at the same time phorate, phorate sulfoxide and phorate sulfone had

decreased peak intensities or completely disappeared. The degradation of phorate can be

followed in Figure 19. The top chromatogram is from analysis of phorate at in water

(normal conditions) at day 0, the middle chromatogram is from analysis phorate oxidized

with hydrogen peroxide at day 0 and the bottom chromatogram is from analysis of the

same sample as the middle one but after 20 days.

Figure 19: Extracted mass chromatogram from full scan of phorate at day 0 in water (top), day 0

(middle) and day 20 (bottom) oxidized with hydrogen peroxide.

Water Day 0

Hydrogen peroxide Day 0

Hydrogen peroxide Day 20

Phorate 12.34

P

S

O

O

SS

P

S

O

O

SS

OP

S

O

O

SS

O

O


Phorate sulfone 8.98

Phorate sulfoxide 8.78

Unidentified (m/z 277)

6.07

P

S

O

O

SS

P

S

O

O

SS

OP

S

O

O

SS

O

O


P

S

O

O

SS

P

S

O

O

SS

OP

S

O

O

SS

O

O



6.81


5.84


6.07


35

4 Conclusion The developed multi-residue LC-MS/MS method allows quantitative determination of 233

pesticides with high variation in both chemical and physical properties. Together with the

simple generic extraction method based on acetonitrile, analysis of spiked samples in

different matrices was possible. The extraction method was shown to be applicable to a

wide range of different environmental, biological and food samples. The results showed

good recoveries (≥ 70 %) and precision (RSD ≤ 20 %) for the majority of the pesticides,

regardless of sample type. Most of the pesticides could be detected at the concentration

0.625 µg/mL or 0.625 µg/g in all matrices. Soil samples were too difficult for the

extraction method due to dirty extract and low recoveries. Five pesticides displayed

extraction difficulties in one or two matrices and late eluting compounds gave in general

lower extraction recoveries and higher standard deviations than earlier eluting compounds,

possible due to their non-polar properties.

During chemical degradation of phorate in water samples under oxidative conditions,

phorate sulfoxide and phorate sulfone were found as the major degradation products. For

normal, acidic and basic condition no degradation occurred and it was concluded that pH

was not a contributing factor to the degradation. One additional degradation product was

found at low intensities at m/z 277 but could not be identified. After 20 days at room

temperature, analysis of the same samples showed two new peaks with m/z 111 and 163. It

could not be concluded that these peaks came from degradation products of phorate and

both were left unidentified.

The quick, easy and generic extraction method in combination with the short analysis time

demonstrates that the requirements of a fast and standardized screening method are

fulfilled. Environmental, biological and food samples are the most common collected

sample types in situations where use of chemical weapons or intentional poisoning are

suspected which makes this method well adapted for threat or emergency situations. After

identification with the screening method, the next step in the analysis chain is target

conformational analysis for gathering forensic evidence such as chemical profiling in

order to identify the source, production method or link the sample to a confiscated

material.

Moreover, addition of new pesticides or degradation products into the method is possible

without any need of modifications since the method works satisfactory for a wide range of

structurally different compounds.


36

5 Acknowledgments The kind gifts of pesticide standard solutions from Tuija Pihlström at the Swedish Food

Agency are gratefully acknowledged.


37

6 References

[1] United States Environmental Protection Agency (EPA), ”Pesticides,” 05 08 2014.

[Online]. Available: http://www.epa.gov/pesticides/. [Använd 29 01 2015].

[2] World Healt Organization - WHO Pesticide evaluation scheme, ”Pesticides and their

application, sixth edition,” World Health Organization, 2006.

[3] A. . R. Fernández-Alba och J. F. García-Reyes, ”Large-scale multi-residue methods

for pesticides and their degradation products in food by advanced LC-MS,” Trends in

Analytical Chemistry, vol. 27, nr 11, pp. 973-990, 2008.

[4] C. R. Michael och P. H. Alavanja, ”Pesticides Use and Exposure Extensive

Worldwide,” Reviews on Environmental Health, vol. 24, nr 4, p. 303–309, 2009.

[5] G. L. Martins, C. A. Friggi, O. D. Prestes, M. C. Vicari, D. A. Friggi, M. B. Adaime

och R. Zanella, ”Simultaneous LC-MS/MS Determination of Imidazolinone

Herbicides Together with Other Multiclass Pesticide Residues in Soil,” Clean - Soil,

Air, Water, vol. 42, nr 10, pp. 1441-1449, 2014.

[6] Stockholm Convention, ”Stockholm Convention Protecting human healt and the

environment from persistent organic pollutants,” Stockholm Convention, [Online].

Available: http://chm.pops.int. [Använd 06 02 15].

[7] Toxics Action Center, ”The problem with pesticides,” Toxics Action Center,

[Online]. Available: http://www.toxicsaction.org/problems-and-solutions/pesticides.

[Använd 15 03 2015].

[8] FOI orienterar om Kemiska vapen - hot, verkan och skydd, Stockholm: FOI, 2002.

[9] Extonext - Extension Toxicology Network, ”CHOLINESTERASE INHIBITION,”

Extonext - Extension Toxicology Network, 09 1993. [Online]. Available:

http://extoxnet.orst.edu/tibs/cholines.htm. [Använd 13 03 2015].

[10] Å. Sellström, ”Report on the Alleged Use of Chemical Weapons in the Ghouta Area

of Damascus on 21 August 2013,” United Nations, The Hague, 2013.

[11] R. Danzig, M. Sageman, T. Leighton, L. Hough, H. Yuki, R. Kotani och Z. M.

Hosford, ”Aum Shinrikyo - Insights Into How Terrorists Develop Biological and

Chemical Weapons,” Center for a New American Security, Washington DC, 2012.

[12] Office of Inspector General, ”The Department of Homeland Security's Role in Food

Defense and Critical Infrastructure Protection,” U.S Departmend of Homeland

Security, Washington DC, 2007.

[13] L. M. Wein och Y. Liu, ”Analyzing a bioterror attack on the food supply: The case of

botulinum toxin in milk,” PNAS, vol. 102, nr 28, pp. 9984-9989, 2005.

[14] Daily News, ”Daily News,” 20 01 2014. [Online]. Available:

http://www.nydailynews.com/news/world/insecticide-poisoned-frozen-dumplings-

means-life-jail-chinese-worker-article-1.1585225. [Använd 17 02 2015].

[15] F&D Buzz, ”F&D Buzz,” 27 01 2014. [Online]. Available:

http://www.foodanddrinkbuzz.com/food-news/japanese-foods-pesticide-news.html.

[Använd 17 02 2015].

[16] The Japan Times News, ”The Japan Times News,” 08 08 2014. [Online]. Available:

http://www.japantimes.co.jp/news/2014/08/08/national/crime-legal/factory-worker-


38

sentenced-for-lacing-seafood-with-malathion/#.VONOs9pOXcs. [Använd 17 02

2015].

[17] J. M. Bertolote, A. Fleischmann, M. Eddleston och D. Gunnell, ”Deaths from

pesticide poisoning: a global response,” The British Journal of Phsychiatry, vol. 189,

nr 3, pp. 201-203, 2006.

[18] W. A. Temple, N. A. Smith och M. Ruse, ”InChem,” Internationl Programme on

Chemical Saftey (IPCS), 03 1998. [Online]. Available:

http://www.inchem.org/documents/pims/chemical/pimg001.htm. [Använd 16 02

2015].

[19] PAN North America, ”PAN North America,” Pesticide Action Network, [Online].

Available: http://www.panna.org/resources/organophosphates. [Använd 16 02 2015].

[20] United Nations Environment Programme, International Labour Organisation, World

Health Organization, ”InChem,” International Programme on Chemical Saftey

(IPCS), 1986. [Online]. Available:

http://www.inchem.org/documents/ehc/ehc/ehc64.htm. [Använd 16 02 2015].

[21] M. C. Keifer och J. Firestone, ”Neurotoxicity of Pesticides,” Journal of

Agromedicine, vol. 12, nr 1, pp. 17-25, 2007.

[22] PAN North America, ”PAN North America,” Pesticide Action Network, [Online].

Available: http://www.panna.org/resources/organochlorines. [Använd 16 02 2015].

[23] PAN North America, ”Pesticide Action Network Pesticide Database,” Pesticide

Action Network, [Online]. Available:

http://www.pesticideinfo.org/Docs/ref_general3.html#Organochlorines. [Använd 17

02 2015].

[24] U.S Environmental Protection Agency (EPA), ”Pyrethroids and Pyrethrins,” U.S

Environmental Protection Agency (EPA), 05 08 2014. [Online]. Available:

http://www.epa.gov/oppsrrd1/reevaluation/pyrethroids-pyrethrins.html. [Använd 28

04 2015].

[25] National Poisons Information Service, ”InChem,” International Programme on

Chemical Saftey (IPCS), 1998. [Online]. Available:

http://www.inchem.org/documents/ukpids/ukpids/ukpid75.htm. [Använd 16 02

2015].

[26] D. E. Ray och J. R. Fry, ”A reassessment of the neurotoxicity of pyrethroid

insecticides,” Pharmacology & Therapeutics, vol. 111, p. 174 – 193, 2006.

[27] Environmental Protection Agency United States, ”Pesticides: Regulating Pesticides,”

Environmental Protection Agency United States, 05 08 2014. [Online]. Available:

http://www.epa.gov/oppsrrd1/reevaluation/pyrethroids-pyrethrins.html. [Använd 16

02 2015].

[28] World Health Organization, ”The WHO Recommended Classification of Pesticides

by Hazard and Guidelines to Classification 2009,” World Health Organization,

Stuttgart, 2009.

[29] European commisson, ”Pesticides,” European Union, 09 03 2015. [Online].

Available: http://ec.europa.eu/food/plant/pesticides/index_en.htm. [Använd 20 04

2015].

[30] Livsmedelsverket, ”Kontroll av bekämpningsmedelsrester,” Livsmedelsverket, 10 02 2015. [Online]. Available: http://www.livsmedelsverket.se/produktion-handel--

kontroll/livsmedelskontroll/offentlig-kontroll/livsmedelsverkets-kontroll-av-


39

livsmedel/bekampningsmedelsrester---kontroll/. [Använd 20 04 2015].

[31] T. Kovalzcuk, O. Lacina, M. Jech, J. Poustka och J. Hajšlová, ”Novel approach to

fast determination of multiple pesticide residues using ultra-performance liquid

chromatography-tandem mass spectrometry (UPLC-MS/MS),” Food Additives and

Contaminants, vol. 25, nr 4, pp. 444-457, 2008.

[32] E. de Hoffman och V. Stroobant, Mass Spectrometry 2nd edition, Chichester: John

Wiley & sons, Ltd, 2001.

[33] B. M. Ham, Even Electon Mass Spectrometry with Biomolecule Applications, New

Jersey: John Wiley & Sons, 2008.

[34] Waters, ”Beginners Guide to UPLC,” Waters, [Online]. Available:

http://www.waters.com/waters/en_SE/UPLC---Ultra-Performance-Liquid-

Chromatography-Beginner%27s-Guide/nav.htm?cid=134803622&locale=en_SE.

[Använd 20 04 2015].

[35] I. Ferrer, M. E. Thurman och J. A. Zweigenbaum, ”Screening and confirmation of

100 pesticides in food samples by liquid chromatography/tandem mass

spectrometry,” Rapid communication in mass spectrometry, vol. 21, p. 3869–3882,

2007.

[36] M. Hiemstra och A. de Kok, ”Comprehensive multi-residue method for the target

analysis of pesticides in crops using liquid chromatography–tandem mass

spectrometry,” Journal of Chromatography A, vol. 1154, pp. 3-25, 2007.

[37] L. Alder, K. Greulich, G. Kempe och B. Vieth, ”Residue analysis of 500 high

priority pesticides: better by GC-MS or LC-MS/MS?,” Mass Spectrometry Reviews,

vol. 25, pp. 838-865, 2006.

[38] K. Greulich och L. Alder, ”Fast multiresidue screening of 300 pesticides in water for

human consumption by LC-MS/MS,” Analytical and Bioanalytical Chemistry, vol.

391, pp. 183-197, 2008.

[39] S. Inoue, T. Saito, H. Mase, Y. Suzuki, K. Takazawa, I. Yamamotoa och S. Inokuchi,

”Rapid simultaneous determination for organophosphorus pesticides in human serum

by LC–MS,” Journal of Pharmaceutical and Biomedical Analysis, vol. 44, p. 258–

264, 2007.

[40] M. Kvíčalová, P. Doubravová, R. Jobánek, M. Jokešová, V. Očenášková, H.

Süssenbeková och A. Svobodová, ”Application of Different Extraction Methods for

the Determination of Selected Pesticide Residues in Sediments,” Bulletin of

Environmental Contamination and Toxicology, vol. 89, pp. 21-26, 2002.

[41] C. Lsuerur, M. Gartner, A. Mentler och M. Fuerhacker, ”Comparison of four

extraction methods for the analysis of 24 pesticides in soil samples with gas

chromatography-mass spectrometry an dliquid chromatography-ion trap-mass

spectrometry,” Talanta, vol. 75, pp. 284-293, 2008.

[42] H. G. J. Mol, A. Rooseboom, R. van Dam, M. Roding, K. Arondeus och S. Sunarto,

”Modification and re-validation of the ethyl acetate-based multi-residue method for

pesticides in produce,” Analytical Bioanalytical Chemistry, vol. 389, p. 1715–1754,

2007.

[43] T. Pihlström, G. Blomkvist, P. Friman, U. Pagard och B.-G. Österdahl, ”Analysis of

pesticide residues in fruit and vegetables with ethyl acetate extraction using gas and liquid chromatography with tandem mass spectrometric detection,” Analytical and

Bioanalytical Chemistry, vol. 389, pp. 1773-1789, 2007.


40

[44] R. Romero-González, A. Garrido Frenich och J. L. Martínez Vidal, ”Multiresidue

method for fast determination of pesticides in fruit juices by ultra performance liquid

chromatography coupled to tandem mass spectrometry,” Talanta, vol. 76, p. 211–

225, 2008.

[45] J. Wang och D. Leung, ”Applications of Ultra-performance Liquid Chromatography

Electrospray Ionization Quadrupole Time-of-Flight Mass Spectrometry on Analysis

of 138 Pesticides in Fruit- and Vegetable-Based Infant Foods,” Journal of

agriculture and food chemistry, vol. 57, pp. 2162-2173, 2009.

[46] H. G. Mol, P. Plaza-Bolaños, P. Zomer, T. C. de Rijk, A. A. Stolker och P. P.

Mulder, ”Toward a Generic Extraction Method for Simultaneous Determination of

Pesticides, Mycotoxins, Plant Toxins, and Veterinary Drugs in Mycotoxins, Plant

Toxins, and Veterinary Drugs in,” Analytical Chemistry, vol. 80, nr 24, pp. 9450-

9459, 2008.

[47] P. D. DeArmond, M. K. Brittain, G. E. Platoff, Jr och D. T. Yeung, ”QuEChERS-

based approach toward the analysis of two insecticides, methomyl and aldicarb, in

blood and brain tissue,” Analytical Methods, vol. 7, pp. 321-328, 2015.

[48] D. Vudathala, M. Cummings och L. Murphy, ”Analysis of Multiple Anticoagulant

Rodenticides in Animal Blood and Liver Tissue Using Principles of QuEChERS

Method,” Journal of Analytical Toxicology, vol. 34, pp. 273-279, 2010.

[49] J. Vera, L. Cirreis-Sá, P. Paíga, I. Braganca, V. C. Fernandes, V. F. Domingues och

C. Delerue-Matos, ”QuEChERS and soil analyis. An Overview.,” Sample Preparation, vol. 1, pp. 54-77, 2013.

[50] A. Herrmann, J. Rosén, D. Jansson och K.-E. Hellenäs, ”Evaluation of a generic

multi-analyte method for detection of >100 representative compounds correlated to

emergency events in 19 food types by ultrahigh-pressure liquid chromatography-

tandem mass spectrometry,” Journal of Chromatography A, vol. 1235, pp. 115-124,

2012.

[51] Waters, ”ACQUITY UPLC HSS T3 Column,” Waters, [Online]. Available:

http://www.waters.com/waters/partDetail.htm?partNumber=186003539. [Använd 23

04 2015].


I

Appendix 1

Vial Compound CAS Classification

Average

mass (Da)

Monoisotopic

mass (Da) Molecular formula Structure

LC-B Abamectin 65195-55-3 Acaricide, anthelminthic drug 873.077 872.492188 C48H72O14

LC-A Acephate 30560-19-1 Insecticide 183.166 183.011902 C4H10NO3PS

LC-G Acetamiprid 135410-20-7 Insecticide 222.674 222.067230 C10H11ClN4

LC-H Acibenzolar-S-methyl 135158-54-2 Plant growth regulator 210.276 209.992157 C8H6N2OS2

LC-C Aldicarb 116-06-3

Insecticide, acaricide,

fumigant, nematicide 190.263 190.077591 C7H14N2O2S

LC-D Aldicarb sulfoxide 1646-87-3 Insecticide, acaricide 206.263 206.072510

C13H13BrFN5O4S2

LC-C Aldicarb-sulfone

1646-88-4


nematicide 222.262 222.067429

C7H14N2O4S

LC-J Aminocarb 2032-59-9 Insecticide, acaricide 208.257 208.121185 C11H16N2O2


II

LC-N Amisulbrom 348635-87-0 Fungicide 466.306 464.957642

C7H14N2O3S

LC-J Aspon 3244-90-4 Insecticide, acaricide 378.425 378.085327 C12H28O5P2S2

LC-A Atrazine 1912-24-9 Herbicide 215.683 215.093781 C8H14ClN5

LC-K Atrazine-desethyl 6190-65-4 Herbicide 187.630 187.062469

C6H10ClN5

LC-J Atrazine-desisopropyl 1007-28-9 Herbicide 173.604 173.046829

C5H8ClN5

LC-B Azadirachtin 11141-17-6 Insecticide, antimalarial 720.714 720.262939 C35H44O16

LC-C Azoxystrobin 131860-33-8 Fungicide 403.388 403.116821 C22H17N3O5

LC-J Benalaxyl 71626-11-4 Fungicide 325.401 325.167786

C20H23NO3

LC-E Bendiocarb 22781-23-3 Insectidice 223.225 223.084457 C11H13NO4

LC-N Benfuracarb 82560-54-1 Insecticide 410.53 408.208282 C20H30N2O5S


III

LC-C Bitertanol

55179-31-2 Fungicide, antibacterial drug 337.415 337.179016

C20H23N3O2

LC-H Boscalid 188425-85-6 Herbicide 343.207 342.032654

C18H12Cl2N2O

LC-G Bupirimate 41483-43-6 Fungicide, antibacterial drug 316.420 316.156921 C13H24N4O3S

LC-J Buprofezin 69327-76-0 Insecticide, acaricide 305.438 305.156189

C16H23N3OS

LC-E Butocarboxim 34681-10-2 Insecticide 190.263 190.077591

C7H14N2O2S

LC-F Butocarboxim sulfoxide 34681-24-8 Herbicide 206.263 206.072510

C7H14N2O3S

LC-E Butoxycarboxim 34681-23-7 Insectidice 222.262 222.067429

C7H14N2O4S

LC-I Butralin 33629-47-9

Herbicide, plant growth

regulator 295.334 295.153198

C14H21N3O4

LC-B Carbaryl 63-25-2

Acaricide, plant growth

regulator 201.221 201.078979 C12H11NO2

LC-B Carbendazim 10605-21-7

Fungicide, nematicide,

antibacterial drug 191.187 191.069473 C9H9N3O2


IV

LC-E Carbofuran 1563-66-2

Insecticide, acaricide, avicide,

nematicide 221.252 221.105194 C12H15NO3

LC-E Carbofuran-3-OH 16655-82-6 Insecticide 237.252 237.100113 C12H15NO4

LC-K Carbophenthion 786-19-6 Insecticide, acaricide 342.865 341.973846

C11H16ClO2PS3

LC-G Carbosulfan 55285-14-8


nematicide 380.545 380.213348

C20H32N2O3S

LC-F Carboxin 5234-68-4 Fungicide, antibacterial drug 235.302 235.066696

C12H13NO2S

LC-H Carfentrazone-ethyl 128639-02-1 Herbicide 412.191 411.036438

C15H14Cl2F3N3O3

LC-B

Chlorantraniliprole 500008-45-7 Insecticide 483.146 480.970795 C18H14BrCl2N5O2

LC-F Chlorbromuron 13360-45-7 Herbicide 293.545 291.961395

C9H10BrClN2O

LC-A

Chlorfenvinphos (E &

Z) 470-90-6 Insecticide, acaricide 359.570 357.969513 C12H14Cl3O4P

LC-D Cinerin I 25402-06-6 Insecticide 316.435 316.203857 C20H28O3


V

LC-D Cinerin II 121-20-0 Insecticide 360.444 360.193665 C21H28O5

LC-E Clofentezine 74115-24-5 Insecticide, acaricide 303.146 302.012604

C14H8Cl2N4

LC-I Clomazone 81777-89-1 Herbicide 239.698 239.071304 C12H14ClNO2

LC-N Clopyralid 1702-17-6 Herbicide 191.999 190.954086 C6H3Cl2NO2

LC-K Clothianidin 210880-92-5 Insecticide 249.678 249.008728

C6H8ClN5O2S

LC-I Coumaphos 56-72-4

Insecticide, avicide,

nematicide, anthelminthic

drug 362.766 362.014465 C14H16ClO5PS

LC-C Cyanazine

21725-46-2 Herbicide 240.693 240.089020

C9H13ClN6

LC-E Cyazofamid 120116-88-3 Fungicide 324.786 324.044769

C13H13ClN4O2S

LC-N Cymoxanil 57966-95-7 Fungicide 198.179 198.075287 C7H10N4O3

LC-B Cypermethrin (E & Z) 52315-07-8 Insecticide 416.297 415.074188 C22H19Cl2NO3


VI

LC-K Cyproconazole (R & S) 94361-06-5 Fungicide 291.776 291.113831

C15H18ClN3O

LC-A Danifos 7173-84-4 Insecticide, acaricide 326.800 325.996704 C11H16ClO3PS2

LC-H Demeton-S 126-75-0 Insecticide 258.338 258.051331 C8H19O3PS2

LC-C Demeton-S-methyl 919-86-8 Insecticide, acaricide 230.285 230.020020 C6H15O3PS2

LC-D

Demeton-S-methyl

sulfoxide

301-12-2

Insecticide, acaricide 246.285 246.014938

C6H15O4PS2

LC-C

Demeton-S-methyl

sulphone 17040-19-6 Insecticide, acaricide 262.284 262.009857 C6H15O5PS2

LC-J Desmetryn 1014-69-3 Herbicide 213.303 213.104813

C8H15N5S

LC-J Dialifos 10311-84-9 Insecticide, acaricide 393.846 393.002502

C14H17ClNO4PS2

LC-A Diazinon 333-41-5


nematicide 304.345 304.101044 C12H21N2O3PS

LC-A Dichlorvos 62-73-7


fungicide, anthelminthic drug 220.976 219.945908 C4H7Cl2O4P


VII

LC-J Dicrotophos 141-66-2 Insecticide, acaricide, avicide 237.190 237.076614 C8H16NO5P

LC-J Diethofencarb 87130-20-9 Fungicide, antibacterial drug 267.321 267.147064

C14H21NO4

LC-C Difenoconazole


C19H17Cl2N3O3

LC-A Dimethoate 60-51-5 Insecticide 229.257 228.999619 C5H12NO3PS2

LC-F Dimethomorph (E & Z) 110488-70-5 Fungicide 387.857 387.123749 C21H22ClNO4

LC-F Dimoxystrobin 149961-52-4 Fungicide 326.390 326.163055

C19H22N2O3

LC-N Diniconazole 83657-24-3 Fungicide, antibacterial drug 326.221 325.074860 C15H17Cl2N3O

LC-I Diphenamide 957-51-7 Herbicide 239.312 239.131012 C16H17NO

LC-C Disulfoton 298-04-4 Insecticide, acaricide 274.404 274.028473 C8H19O2PS3

LC-C Disulfoton sulfone

2497-06-05 Insecticide 306.403 306.018311

C8H19O4PS3


VIII

LC-D Disulfoton sulfoxide

301-12-2 Insecticide, acaricide 290.403 290.023376

C8H19O3PS3

LC-F DMF 60397-77-5 Breakdown product 149.190 149.084061 C9H11NO

LC-H DMSA 110-61-2

Herbicide, plant growth

regulator 182.218 181.970749 C4H6O4S2

LC-H DMST 66840-71-9 Breakdown product 214.285 214.077591

C9H14N2O2S

LC-N Dodine 112-65-2 Fungicide, antibacterial drug 227.389 227.236145 C13H29N3

LC-K Epoxiconazole 133855-98-8 Fungicide 329.756 329.073120 C17H13ClFN3O

LC-E Ethiofencarb 29973-13-5 Insecticide 225.307 225.082352 C11H15NO2S

LC-E Ethiofencarb sulfone 53380-23-7 Insecticide 257.306 257.072174

C11H15NO4S

LC-F Ethiofencarb sulfoxide 53380-22-6 Insecticide 241.307 241.077271

C11H15NO3S

LC-A Ethion 563-12-2 Insecticide, acaricide 384.476 383.987610 C9H22O4P2S4


IX

LC-N Ethirimol 23947-60-6 Fungicide, antibacterial drug 209.288 209.152817

C11H19N3O

LC-J Ethofenprox 80844-07-1 Insecticide, acaricide 376.488 376.203857 C25H28O3

LC-J Ethofumesate 26225-79-6 Herbicide 286.344 286.087494

C13H18O5S

LC-G Ethoprophos 13194-48-4

Insecticide, fungicide,

fumigant, nematicide 242.339 242.056412 C8H19O2PS2

LC-J Etrimfos 38260-54-7 Insecticide 292.292 292.064667

C10H17N2O4PS

LC-C Famoxadone 131807-57-3 Fungicide 374.389 374.126648 C22H18N2O4

LC-N Fenamidone 161326-34-7 Fungicide 311.401 311.109222 C17H17N3OS

LC-N Fenamiphos 22224-92-6


nematicide 303.357 303.105804 C13H22NO3PS

LC-H Fenamiphos sulfone 31972-44-8 Insecticide 335.356 335.095642

C13H22NO5PS

LC-K Fenamiphos sulfoxide 31972-43-7 Insecticide 319.357 319.100708

C13H22NO4PS


X

LC-A Fenarimol 60168-88-9 Fungicide, antibacterial drug 331.196 330.032654 C17H12Cl2N2O

LC-J Fenazaquin 120928-09-8 Insecticide, acaricide 306.401 306.173218

C20H22N2O

LC-C Fenbuconazole


C19H17ClN4

LC-D Fenhexamide 126833-17-8 Fungicide 302.196 301.063629 C14H17Cl2NO2

LC-E Fenoxycarb 72490-01-8 Insecticide 301.337 301.131409 C17H19NO4

LC-F Fenpiclonil 74738-17-3 Fungicide, antibacterial drug 237.085 235.990799

C11H6Cl2N2

LC-C Fenpropidin

67306-00-7

Fungicide, antibacterial drug 273.456 273.245636

C19H31N

LC-D Fenpropimorph 67564-91-4 Fungicide, antibacterial drug 303.482 303.256226 C20H33NO

LC-H Fenpyroximate 134098-61-6 Acaricide 421.489 421.200165

C24H27N3O4

LC-H Fensulfothion oxon 6552-21-2 Insecticide 292.288 292.053436 C11H17O5PS


XI

LC-G

Fensulfothion oxon

sulfone 6132-17-8 Insecticide 308.288 308.048340

C11H17O6PS

LC-G Fensulfothion sulfone 14255-72-2 Insecticide 324.353 324.025513 C11H17O5PS2

LC-A Fenthion 55-38-9

Acaricide, avicide, insect

attractant 278.328 278.020020 C10H15O3PS2

LC-A Fenthion sulfone 3763-41-0 Insceticide 310.327 310.009857

C10H15O5PS2

LC-B Fenthion sulfoxide

3761-41-9 Insecticide 294.328 294.014923

C10H15O4PS2

LC-I Fluazifop-P-butyl 79241-46-6 Herbicide 383.362 383.134430

C19H20F3NO4

LC-F Flucythrinate 70124-77-5 Insecticide, acaricide 451.462 451.159515 C26H23F2NO4

LC-K Fludioxonil 131341-86-1 Fungicide 248.185 248.039734

C12H6F2N2O2

LC-I Flumetralin 62924-70-3 Plant growth regulator 421.731 421.045258

C16H12ClF4N3O4

LC-N Fluopyram 658066-35-4 Fungicide 396.715 396.046417

C16H11ClF6N2O


XII

LC-J Fluquinconazole 136426-54-5 Fungicide 376.172 375.009003

C16H8Cl2FN5O

LC-C Flusilazole 85509-19-9 Fungicide, antibacterial drug 315.393 315.100342 C16H15F2N3Si

LC-K Fonofos 944-22-9 Insecticide 246.329 246.030197 C10H15OPS2

LC-N Formetanate 22259-30-9 Insecticide, acaricide 221.256 221.116425 C11H15N3O2

LC-C Fuberidazole 3878-19-1 Fungicide, antibacterial drug 184.194 184.063660 C11H8N2O

LC-J Furalaxyl 57646-30-7 Fungicide, antibacterial drug 301.337 301.131409

C17H19NO4

LC-G Furathiocarb 65907-30-4 Insecticide 382.474 382.156250

C18H26N2O5S

LC-G Haloxyfop 69806-34-4 Insecticide, herbicide 361.700 361.032867 C15H11ClF3NO4

LC-H Haloxyfop ethoxyethyl 87237-48-7 Herbicide 433.806 433.090393

C19H19ClF3NO5

LC-H Haloxyfop-methyl 72619-32-0 Herbicide 375.727 375.048523

C16H13ClF3NO4


XIII

LC-J Heptenophos 23560-59-0 Insecticide, acaricide 250.616 250.016174

C9H12ClO4P

LC-C Hexaconazole 79983-71-4 Fungicide, antibacterial drug 314.210 313.074860 C14H17Cl2N3O

LC-G Hexazinone 51235-04-2 Herbicide 252.313 252.158630 C12H20N4O2

LC-E Hexythiazox 78587-05-0 Insect growth regulator 352.879 352.101227

C17H21ClN2O2S

LC-C Imazalil 35554-44-0

Fungicide, antibacterial drug,

antifungal agent 297.180 296.048309 C14H14Cl2N2O

LC-C Imidacloprid 138261-41-3 Insecticide 255.661 255.052307 C9H10ClN5O2

LC-H Indoxacarb 173584-44-6 Insecticide 527.834 527.070740 C22H17ClF3N3O7

LC-F Iprovalicarb 140923-17-7 Fungicide 320.427 320.209991 C18H28N2O3

LC-F Isazofos 42509-80-8


antibacterial drug, nematicide 313.741 313.041687

C9H17ClN3O3PS

LC-H Isocarbophos 24353-61-5 Insecticide, acaricide 289.288 289.053772

C11H16NO4PS


XIV

LC-J Isofenphos 25311-71-1 Insecticide 345.394 345.116364 C15H24NO4PS

LC-G Isofenphos methyl 99675-03-3 Insecticide 331.367 331.100708

C14H22NO4PS

LC-E Isoprocarb 2631-40-5 Insecticide 193.242 193.110275

C11H15NO2

LC-I Isopropalin 33820-53-0 Herbicide 309.361 309.168854

C15H23N3O4

LC-E Isoprothiolane 50512-35-1


antibacterial drug 290.399 290.064636 C12H18O4S2

LC-F Isoproturon 34123-59-6 Herbicide 206.284 206.141907

C12H18N2O

LC-C Isoxaben 82558-50-7 Herbicide 332.394 332.173615 C18H24N2O4

LC-D Jasmolin I 4466-14-2 Insecticide 330.461 330.219482 C21H30O3

LC-D Jasmolin II 1172-63-0 Insecticide 374.471 374.209320 C22H30O5

LC-D Krexoxim-methyl 143390-89-0 Fungicide, antifungal agent 313.348 313.131409 C18H19NO4


XV

LC-B Linuron 330-55-2 Herbicide 249.094 248.011932 C9H10Cl2N2O2

LC-B Malathion 121-75-5 Insecticide, acaricide 330.358 330.036072 C10H19O6PS2

LC-K Malaoxon 1634-78-2 Insecticide 314.292 314.058899 C10H19O7PS

LC-N Mandipropamid 374726-62-2 Fungicide 411.878 411.123749

C23H22ClNO4

LC-J Mecarbam 2595-54-2 Insecticide 329.373 329.052063

C10H20NO5PS2

LC-H Mepanipyrim 110235-47-7 Fungicide, antibacterial drug 223.273 223.110947

C14H13N3

LC-N

Mepanipyrim-2-

hydroxypropyl 204571-52-8 Fungicide 243.30 243.137161 C14H17N3O

LC-J Mephosfolan 950-10-7 Insecticice, acaricide 269.321 269.030914

C8H16NO3PS2

LC-N Metaflumizone 139968-49-3 Insecticide 506.400 506.117737 C24H16F6N4O2

LC-A Metalaxyl 57837-19-1 Fungicide, antibacterial drug 279.332 279.147064 C15H21NO4


XVI

LC-N Metconazole 125116-23-6 Fungicide 319.829 319.145142

C17H22ClN3O

LC-F Methabenzthiazuron 18691-97-9 Herbicide 221.279 221.062286

C10H11N3OS

LC-D Methacriphos

62610-77-9 Insecticide, acaricide 240.214 240.022125

C7H13O5PS

LC-B Methamidophos 10265-92-6 Insecticide, acaricide, avicide 141.129 141.001328 C2H8NO2PS

LC-A Methidathion 950-37-8 Insecticide, acaricide 302.331 301.961853 C6H11N2O4PS3

LC-E Methiocarb 2032-65-7 Insecticied, acaricide, avicide 225.307 225.082352 C11H15NO2S

LC-E Methiocarb sulfone 2179-25-1 Insecticide 257.306 257.072174 C11H15NO4S

LC-F Methiocarb sulfoxide 2635-10-01 Breakdown product 241.307 241.077271

C11H15NO3S

LC-B Methomyl 16752-77-5 Insecticide 162.210 162.046295 C5H10N2O2S

LC-B Methoprene 40596-69-8 Insecticide 310.471 310.250793 C19H34O3


XVII

LC-F Methoxyfenozide 161050-58-4 Insecticide 368.469 368.209991

C22H28N2O3

LC-N Metobromuron 3060-89-7 Herbicide 259.100 258.000397

C9H11BrN2O2

LC-G Monocrotophos 6923-22-4 Insecticide, acaricide, avicide 223.163 223.060959 C7H14NO5P

LC-J Myclobutanil 88671-89-0 Fungicide, antibacterial drug 288.775 288.114166 C15H17ClN4

LC-I Napropamide 15299-99-7 Herbicide 271.354 271.157227 C17H21NO2

LC-N Nitenpyram 150824-47-8 Insecticide 270.715 270.088348 C11H15ClN4O2

LC-N Novaluron 116714-46-6 Insecticide 492.705 492.012299

C17H9ClF8N2O4

LC-G Ofurace 58810-48-3 Fungicide, antibacterial drug 281.735 281.081879

C14H16ClNO3

LC-A Omethoate 1113-02-6 Insecticide 213.192 213.022461 C5H12NO4PS

LC-F Oxamyl 23135-22-0


nematicide 219.261 219.067764 C7H13N3O3S


XVIII

LC-E Oxamyl-oxime 30558-43-1 Breakdown product 162.210 162.046295 C5H10N2O2S

LC-G Paclobutrazole 76738-62-0 Plant growth regulator 293.792 293.129486

C15H20ClN3O

LC-K Paraoxon-ethyl 311-45-5 Insecticide 275.195 275.055878 C10H14NO6P

LC-A Paraoxon-methyl 950-35-6 Insecticide 247.142 247.024567

C8H10NO6P

LC-D Penconazole 66246-88-6 Fungicide, antibacterial drug 284.184 283.064301

C13H15Cl2N3

LC-I Pencycuron 66063-05-6 Fungicide, antibacterial drug 328.836 328.134247 C19H21ClN2O

LC-G Phenmedipham 13684-63-4 Herbicide 300.309 300.110992

C16H16N2O4

LC-K Phenothrin 26002-80-2 Insecticide 350.451 350.188202 C23H26O3

LC-H Phorate 298-02-2 Insecticide, acaricide 260.377 260.012817 C7H17O2PS3

LC-H Phorate sulfone 2588-04-07 Breakdown product 292.376 292.002655

C7H17O4PS3


XIX

LC-G Phorate sulfoxide 2588-03-06 Insecticide 276.377 276.007751 C7H17O3PS3

LC-K Phosphamidon (E & Z) 13171-21-6 Insecticide, acaricide 299.688 299.068939 C10H19ClNO5P

LC-N Phoxim 14816-18-3 Insecticide 298.298 298.054108 C12H15N2O3PS

LC-G Picoxystrobin 117428-22-5 Fungicide 367.319 367.103149 C18H16F3NO4

LC-B Piperonyl butoxide 51-03-6 Insecticide, insect attractant 338.439 338.209320 C19H30O5

LC-B Pirimicarb 23103-98-2 Insecticide 238.286 238.142975 C11H18N4O2

LC-D Pirimicarb desmethyl 30614-22-3 Insecticides 224.260 224.127319

C10H16N4O2

LC-B

Pirimicarb-desmethyl-

formamido

27218-04-8 Insecticide 252.270 252.122238

C11H16N4O3

LC-A Prochloraz 67747-09-5 Fungicide 376.665 375.030823 C15H16Cl3N3O2


XX

LC-E Promecarb 2631-37-0 Insecticide, acaricide 207.269 207.125931 C12H17NO2

LC-A Prometryn 7287-19-6 Herbicide 241.356 241.136108 C10H19N5S

LC-F Propamocarb 24579-73-5 Fungicide 188.267 188.152481 C9H20N2O2

LC-K Propanil 709-98-8 Herbicide, nematicide 218.080 217.006119 C9H9Cl2NO

LC-I Propaquizafop 111479-05-1 Herbicide 443.880 443.124786

C22H22ClN3O5

LC-I Propetamphos (E & Z) 31218-83-4 Insecticide 281.309 281.085052

C10H20NO4PS

LC-A Propiconazole 60207-90-1 Fungicide, antibacterial drug 342.220 341.069794 C15H17Cl2N3O2

LC-F Propoxur 114-26-1 Insecticide, acaricide 209.242 209.105194 C11H15NO3

LC-D Prosulfocarb 52888-80-9 Herbicide 251.388 251.134384

C14H21NOS

LC-I Prothioconazole-desthio 120983-64-4 Fungicide 312.194 311.059204

C14H15Cl2N3O


XXI

LC-I Prothiofos 34643-46-4 Insecticide 345.245 343.962799

C11H15Cl2O2PS2

LC-H Pymetrozine 123312-89-0 Insecticide 217.227 217.096359

C10H11N5O

LC-D Pyraclostrobin 175013-18-0 Fungicide 387.817 387.098572 C19H18ClN3O4

LC-B Pyrazophos 13457-18-6


antibacterial drug 373.365 373.086121 C14H20N3O5PS

LC-D Pyrethrin I 121-21-1 Insecticide 328.445 328.203857 C21H28O3

LC-D Pyrethrin II 121-29-9 Insecticide 372.455 372.193665 C22H28O5

LC-I Pyridaphenthion 119-12-0 Insecticide 340.335 340.064667

C14H17N2O4PS

LC-J Pyrifenox (E & Z) 88283-41-4 Fungicide, antibacterial drug 295.164 294.032654

C14H12Cl2N2O

LC-D Pyriproxyfen 95737-68-1 Insecticide 321.370 321.136505 C20H19NO3

LC-I Quinoxyfen 124495-18-7 Fungicide 308.134 306.996704

C15H8Cl2FNO


XXII

LC-I Quizalofop-ethyl 76578-14-8 Herbicide 372.802 372.087677

C19H17ClN2O4

LC-E Resmethrin 10453-86-8 Insecticide 338.440 338.188202 C22H26O3

LC-K Rotenone 83-79-4 Insecticide, acaricide 394.417 394.141632 C23H22O6

LC-B Simazine 122-34-9 Herbicide 201.657 201.078125 C7H12ClN5

LC-I Spinosyn A 131929-60-7 Insecticide 731.956 731.460876 C41H65NO10

LC-I Spinosyn D 131929-63-0 Insecticide 745.982 745.476501 C42H67NO10

LC-N Spirodiclofen 148477-71-8 Insecticide 411.319 410.105164

C21H24Cl2O4

LC-D Spiroxamine 118134-30-8 Fungicide 297.476 297.266785

C18H35NO2

LC-H Sulfentrazone 122836-35-5 Herbicide 387.190 385.981873 C11H10Cl2F2N4O3S

LC-K Tau-fluvalinate 102851-06-9 Insecticide, acaricide 502.913 502.127106 C26H22ClF3N2O3


XXIII

LC-A Tebuconazole 107534-96-3 Fungicide, antibacterial drug 307.818 307.145142 C16H22ClN3O

LC-F Tebufenozide 112410-23-8 Insecticide 352.470 352.215088 C22H28N2O2

LC-K Tebufenpyrad 119168-77-3 Insecticide, acaricide 333.856 333.160797 C18H24ClN3O

LC-K TEPP 107-49-3 Insecticide, acaricide 290.188 290.068420 C8H20O7P2

LC-I Tepraloxydim 149979-41-9 Herbicide 341.830 341.139374

C17H24ClNO4

LC-G Terbufos 13071-79-9 Insecticide, nematicide 288.431 288.044128 C9H21O2PS3

LC-G Terbufos sulfone 56070-16-7 Breakdown product 320.429 320.033966

C9H21O4PS3

LC-H Terbufos sulfoxide 10548-10-4 Insecticide 304.430 304.039032

C9H21O3PS3

LC-K Terbuthylazine 5915-41-3 Herbicide 229.710 229.109421 C9H16ClN5

LC-G Terbutryn 886-50-0 Herbicide 241.356 241.136108

C10H19N5S


XXIV

LC-K Tetrachlorvinphos 22248-79-9 Insecticide 365.962 363.899261 C10H9Cl4O4P

LC-G Tetraconazole 112281-77-3 Fungicide 372.146 371.021515

C13H11Cl2F4N3O

LC-N Tetramethrin 7696-12-0 Insecticide 331.406 331.178345 C19H25NO4

LC-D Thiabendazole 148-79-8 Fungicide, nematicide 201.248 201.036072 C10H7N3S

LC-G Thiacloprid 111988-49-9 Insecticide 252.723 252.023651 C10H9ClN4S

LC-I Thiamethoxam 153719-23-4 Insecticide 291.715 291.019287 C8H10ClN5O3S

LC-D Thiodicarb 59669-26-0 Insecticide 354.469 354.049011

C10H18N4O4S3

LC-D Thiometon 640-15-3 Insecticide, acaricide 246.351 245.997177 C6H15O2PS3

LC-D Thiophanate methyl 23564-05-8 Fungicide, antibacterial drug 342.394 342.045654

C12H14N4O4S2

LC-B Tralomethrin 66841-25-6 Insecticide 665.007 660.809814 C22H19Br4NO3


XXV

LC-A Triadimefon 43121-43-3 Fungicide, antibacterial drug 293.749 293.093109 C14H16ClN3O2

LC-B Triadimenol (R & S)


C14H18ClN3O2

LC-D Tribenuron-methyl 101200-48-0 Herbicide 395.390 395.089966 C15H17N5O6S

LC-K Trichlorfon 52-68-6

Insecticide, anthelminthic

drug 257.437 255.922577 C4H8Cl3O4P

LC-E Tricyclazole 41814-78-2

Fungicide, antibacterial drug,

antifungal agent 189.237 189.036072 C9H7N3S

LC-I Trifloxystrobin 141517-21-7 Fungicide 408.371 408.129700

C20H19F3N2O4

LC-B Triflumuron

64628-44-0 Insecticide 358.700 358.033203

C15H10ClF3N2O3

LC-N Triforin 26644-46-2 Fungicide, antibacterial drug 434.962 431.924805

C10H14Cl6N4O2

LC-E Trimetarcarb-3,4,5 12407-86-2 Insecticide 193.242 193.110275 C11H15NO2

LC-F Trimethacarb-2,3,5 002655-15-4 Insecticide 193.242 193.110275 C11H15NO2


XXVI

LC-D Trinexapac ethyl 95266-40-3 Plant growth regulator 252.263 252.099777

C13H16O5

LC-I Triticonazole 131983-72-7 Fungicide 317.813 317.129486

C17H20ClN3O

LC-H Vamidothion 2275-23-2


fungicide 287.337 287.041473

C8H18NO4PS2

LC-F Vamidothion sulfoxide 20300-00-9 Insecticide 303.336 303.036407

C8H18NO5PS2

LC-C Zoxamide

156052-68-5 Fungicide 336.641 335.024658

C14H16Cl3NO2

Department of Chemistry

S-901 87 Umeå, Sweden

Telephone +46 90 786 50 00

Text telephone +46 90 786 59 00

www.umu.se

Pesticide Screening Method with UPLC-MS/MS826306/... · 2015. 6. 25. · Pesticide Screening Method...

Documents

Transcript of Pesticide Screening Method with UPLC-MS/MS826306/... · 2015. 6. 25. · Pesticide Screening Method...