Detection and Quantification of Organophosphate Pesticides in Hum

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Detection and Quantification of OrganophosphatePesticides in Human SerumPeter KuklenyikGeorgia State University

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I

DETECTION AND QUANTIFICATION ORGANOPHOSPHATE PESTICIDES IN

HUMAN SERUM

by

PETER KUKLENYIK

Under the Direction of Dr. Gabor Patonay

ABSTRACT

The United States Environmental Agency permits the use of 39 organophosphate pesticides.

Many of these pesticides are acutely toxic and have lasting effect on human health.

Organophosphates quickly metabolize in the body, therefore currently human exposure is studied

by measuring the metabolic products in urine.

In this work a suite of analytical methods was developed to determine the presence of un-

metabolized organophosphate pesticides in human serum. First mass spectroscopic detection

methods were evaluated. Gas chromatograph coupled tandem mass spectrometer was used to

compare the detection limits using chemical and electron impact ionization. Positive chemical

ionization was selected, because it provided better detection limits for this set of analytes.

Liquid chromatograph coupled tandem mass spectrometry was also evaluated and was found

advantageous over the gas chromatographic method for approximately 50% of the compounds.

Positive atmospheric pressure chemical ionization was chosen for this group of compounds.

Once the analytes were separated by detection methods, analytical separation methods were

compared: column and eluent was selected for liquid chromatography, column alone was

selected for gas chromatography.

Last step of the method development was to produce a suitable sample cleanup process.

Solid phase extraction was not suitable because the very wide range of solubility characteristics

and hydrolytic stability of the analytes. Lyophilization, liquid-liquid extraction methods were

tested and compared. A multi step cleanup method was produced, which starts with liquid-liquid

extraction using high pressure ethyl acetate in accelerated solvent extractor, solvent exchange

and a lipid removal step. The concentrated extract then injected in a HPLC-MS-MS system then

the same extract either directly injected in GC-MS-MS or further purified using headspace solid

phase micro extraction before the GC-NS-MS step.

The method was used with good results for analyzing samples collected from farm workers

using OP pesticides.

IV


HUMAN SERUM

by

PETER KUKLENYIK

A Dissertation Submitted in Partial Fulfillment of the Requirements for the Degree of

Doctor of Philosophy

In the College of Arts and Sciences

Georgia State University

Copyright by

Peter Kuklenyik

2008


HUMAN SERUM

by

PETER KUKLENYIK

Committee Chair: Dr. Gabor Patonay

Committee: Dr. Dana Barr

Dr Shahab Shamsi

Dr. Lucjan Strekowski

Electronic Version Approved:

Office of Graduate Studies

College of Arts and Sciences

Georgia State University

August 2008

IV

ACKNOWLEDGEMENTS

5

TABLE OF CONTENTS

CHAPTER 1: INTRODUCTION AND GOALS ...................................................................... 1

Toxicity .................................................................................................................................. 3

Inhibition of Acetyl Cholinesterase: Acute Cholinergic Syndrome .................................. 3

Intermediate Syndrome ...................................................................................................... 5

Organophosphate Induced Delayed Polyneurophaty (OPIDP) .......................................... 5

Organophosphorus Ester-Induced Chronic Neurotoxicity (OPICN) [2] ........................... 5

Exposure [11] ......................................................................................................................... 7

Oral Exposure..................................................................................................................... 7

Dermal Exposure ................................................................................................................ 7

Inhalation ........................................................................................................................... 7

Distribution ........................................................................................................................ 8

OP Metabolism [11] ............................................................................................................... 9

A-esterases ....................................................................................................................... 10

Carboxylesterases ............................................................................................................. 11

Amidases .......................................................................................................................... 12

Microsomal Oxigenases ................................................................................................... 12

Determining OP pesticide exposure ..................................................................................... 14

Non – assay methods. ....................................................................................................... 14

Urinary Assay of OP Metabolites .................................................................................... 14

Detection of OP Pesticides in Blood/Serum/Plasma ........................................................ 15

Goals and Objectives ............................................................................................................... 17

6

METHOD DEVELOPMENT .................................................................................................. 29

General development strategy .............................................................................................. 29

CHAPTER 2: GAS CHROMATOGRAPHY AND TANDEM MASS SPECTROMETRY

31

Process to optimize mass spectrometric parameters for GC-MS-MS systems. ................... 31

Determining Ionization type and ion selection .................................................................... 32

Chapter 2 Dicussion....................................................................................................... 38

Appendix A CI Positive, negative and EI full scans ......................................................... 48

CHAPTER 3: HIGH PERFORMANCE LIQUID CHROMATOGRAPHY – MASS

SPECTROMETRY .................................................................................................................. 87

Liquid Chromatography: HPLC ........................................................................................... 90

Selection of eluents. ......................................................................................................... 92

Alternative to the one column HPLC separation ................................................................. 99

CHAPTER 3 APPENDIX ............................................................................................. 101

Appendix 3A APCI Source Temperature Studies ...................................................... 101

Appendix 3B : Applied Solvent Gradient curves for testing HPLC columns and ......... 123

Appendix 3C: ESI and APCI positive and negative full scan spectra ........................... 134

CHAPTER 4: SAMPLE CLEANUP AND METHOD APPLICATION .......................... 174

Liquid-Liquid Extraction on Solid Backing ....................................................................... 175

Accelerated Solvent Extraction (ASE)............................................................................... 181

Second step cleanup: normal phase chromatography on SPE cartridges. .......................... 184

Serum Extraction ................................................................................................................ 193

Determination of Instrument Detection Limits LC-MS-MS and GC-MS-MS groups ...... 198

7

Effect of drying the samples. ............................................................................................. 200

Construction of calibration curves ..................................................................................... 203

LOD-s for the LC group ..................................................................................................... 205

Precision and accuracy of the method ................................................................................ 205

The problem with azinphos methyl and the power of tandem mass spectroscopy ............ 213

Chapter 4 Discussion ................................................................................................... 216

Solid phase extraction .................................................................................................... 217

Lyophilization ................................................................................................................ 217

Liquid- Liquid extraction ............................................................................................... 217

Novelty and significance of the method......................................................................... 220

CHAPTER 4 APPENDIX ............................................................................................. 227

Appendix 4A: Suggested fragmentation of OP pesticides .......................................... 227

Appendix 4B: Determination of recovery using internal standard ............................. 232

Appendix 4C: Determination of limit of detection (LOD) ........................................ 234

REFERENCES ....................................................................................................................... 236

8

LIST OF FIGURES

Figure 1 General Structure of Organophosphate Pesticides ................................................... 1

Figure 2 Esterase Inhibition by Organophosphate R: Ethyl or methyl, X: Specific group ..... 4

Figure 3 Non Persistent Pesticide: Concentration Change in Blood and in Urine [12] ........... 9

Figure 4 Paraoxon Hydrolysis to Diethyl phosphate ............................................................. 11

Figure 5 Hydrolysis of the Carboxyl Ester Side Chain of Malathion .................................... 11

Figure 6 Amidase Cleavage of Carboxylamide R: Methyl, Ethyl ...................................... 12

Figure 7 Oxidative Desulphuration of Parathion ................................................................... 12

Figure 8 Metabolism of Chlorpyrifos .................................................................................... 13

Figure 9 Simple temperature gradient to determine GC properties of OP compounds ........ 34

Figure 10 Transition selection for chlorpyrifos methyl ......................................................... 35

Figure 13 Retention times for OP compounds on 30m .25mm X 25µm DB-5 MS column,

linear temperature gradient. .......................................................................................................... 37

Figure 14 Polymer structure of phenyl-methyl (DB-5 MS) and cyanopropyl phenyl

(HP1701) stationary phases .......................................................................................................... 39

Figure 15 Major fragments of malathion in EI mode ............................................................ 40

Figure 16 Major fragments of malathion in CI positive mode............................................... 41

Figure 17 Major fragments of malathion in CI negative mode .............................................. 41

Figure 18 GC retention times as the function of vapor pressure data (logarithmic scale) ..... 42

Figure 19 Correlation factor analysis ..................................................................................... 44

Figure 20 Predicting OP retention times in GC ...................................................................... 45

Figure 21 Predicting OP retention times in GC – outliers (Oxydemethon methyl,

dicrotophos, tetrachlorvinphos, trichlorfon, all LC group members) taken out: ......................... 46

9

Figure 22 Acephate - Positive CI, Negative CI, EI spectra ................................................... 49

Figure 23 Azinphos Methyl - Positive CI, Negative CI, EI spectra ....................................... 50

Figure 24 Cadusafos - Positive CI, Negative CI, EI spectra .................................................. 51

Figure 25 Chlorethoxyfos - Positive CI, Negative CI, EI spectra .......................................... 52

Figure 26 Chlorpirifos - Positive CI, Negative CI, EI spectra ............................................... 53

Figure 27 Chlorpyrofos Methyl- Positive CI, Negative CI, EI spectra .................................. 54

Figure 28 Coumaphos - Positive CI, Negative CI, EI spectra ............................................... 55

Figure 29 Diazinon - Positive CI, Negative CI, EI spectra .................................................... 56

Figure 30 Dichlorvos - Positive CI, Negative CI, EI spectra ................................................ 57

Figure 31 Dichrotophos - Positive CI, Negative CI, EI spectra ............................................. 58

Figure 32 Dimethoate - Positive CI, Negative CI, EI spectra ................................................ 59

Figure 33 Disulfoton - Positive CI, Negative CI, EI spectra ................................................. 60

Figure 34 Ethion - Positive CI, Negative CI, EI spectra ........................................................ 61

Figure 35 Ethoprop - Positive CI, Negative CI, EI spectra .................................................. 62

Figure 36 Ethyl Parathion - Positive CI, Negative CI, EI spectra .......................................... 63

Figure 37 Fenamiphos - Positive CI, Negative CI, EI spectra ............................................... 64

Figure 38 Fenitrothion - Positive CI, Negative CI, EI spectra ............................................... 65

Figure 39 Fenthion - Positive CI, Negative CI, EI spectra .................................................... 66

Figure 40 Fonofos- Positive CI, Negative CI, EI spectra ...................................................... 67

Figure 41 Malathion - Positive CI, Negative CI, EI spectra .................................................. 68

Figure 42 Methamidophos- Positive CI, Negative CI, EI spectra .......................................... 69

Figure 43 Methidathion - Positive CI, Negative CI, EI spectra ............................................. 70

Figure 44 Methyl parathion - Positive CI, Negative CI, EI spectra ....................................... 71

10

Figure 45 Mevinphos - Positive CI, Negative CI, EI spectra................................................... 72

Figure 46 Naled - Positive CI, Negative CI, EI spectra ........................................................... 73

Figure 47 Oxydemethon methyl - Positive CI, Negative CI, EI spectra ................................ 74

Figure 48 Phorate - Positive CI, Negative CI, EI spectra ...................................................... 75

Figure 49 Phosalone - Positive CI, Negative CI, EI spectra .................................................. 76

Figure 50 Phosmet - Positive CI, Negative CI, EI spectra ..................................................... 77

Figure 51 Phostebupirim - Positive CI, Negative CI, EI spectra ........................................... 78

Figure 52 Pirimphos methyl - Positive CI, Negative CI, EI spectra ...................................... 79

Figure 53 Profenofos - Positive CI, Negative CI, EI spectra ................................................. 80

Figure 54 Propetamphos - Positive CI, Negative CI, EI spectra ............................................ 81

Figure 55 Sulfotepp - Positive CI, Negative CI, EI spectra ................................................... 82

Figure 56 Terbufos - Positive CI, Negative CI, EI spectra .................................................... 83

Figure 57 Tetrachlorvinphos - Positive CI, Negative CI, EI spectra ..................................... 84

Figure 58 Tribufos - Positive CI, Negative CI, EI spectra ..................................................... 85

Figure 59 Trichlorfon - Positive CI, Negative CI, EI spectra ................................................ 86

Figure 60 Viscosity profiles of Methanol - Water and Acetonitrile - Water blends, according

to Thompson [34] .......................................................................................................................... 93

Figure 61 Betasi C18 and Betasil Phenyl retention profile using a linear water methanol

gradient ......................................................................................................................................... 97

Figure 62 Aquasil C18 retention profile using a linear water methanol gradient and Betasil

Phenyl using the final, non lina=ear solvent gradient ................................................................... 98

11

Figure 63 Two column HPLC system. 1: Column A separates the first analyte group. 2: The

group of unresolved analytes pass column A and are loaded onto column B. 3: This second

group of analytes are gradient eluted and separated on column B. ............................................ 100

Figure 64 Acephate ESI positive, ESI negative, APCI positive, APCI negative full scan

spectra ......................................................................................................................................... 134

Figure 65 Azinphos Methyl ESI positive, ESI negative, APCI positive, APCI negative full

scan spectra ................................................................................................................................. 135

Figure 66 Bensulide ESI positive, ESI negative, APCI positive, APCI negative full scan

spectra ......................................................................................................................................... 136

Figure 67 Cadusafos ESI positive, ESI negative, APCI positive, APCI negative full scan

spectra ......................................................................................................................................... 137

Figure 68 Chlorethoxyfos ESI positive, ESI negative, APCI positive, APCI negative full

scan spectra ................................................................................................................................. 138

Figure 69 Chlorpyrifos ESI positive (weak), ESI negative (weak), APCI positive, APCI

negative full scan spectra ............................................................................................................ 139

Figure 70 Chlorpyrifos Methyl ESI positive, ESI negative, APCI positive, APCI negative

full scan spectra........................................................................................................................... 140

Figure 71 Coumaphos ESI positive, ESI negative (weak), APCI positive, APCI negative full

scan spectra ................................................................................................................................. 141

Figure 72 Diazinon ESI positive, ESI negative, APCI positive, APCI negative full scan

spectra ......................................................................................................................................... 142

Figure 73 Dichlorvos ESI positive, ESI negative, APCI positive, APCI negative full scan

spectra ......................................................................................................................................... 143

12

Figure 74 Dicrotophos ESI positive, ESI negative, APCI positive, APCI negative full scan

spectra ......................................................................................................................................... 144

Figure 75 Dimethoate ESI positive, ESI negative, APCI positive, APCI negative full scan

spectra ......................................................................................................................................... 145

Figure 76 Disulfoton ESI positive, ESI negative (weak), APCI positive, APCI negative full

scan spectra ................................................................................................................................. 146

Figure 77 Ethion ESI positive, ESI negative, APCI positive, APCI negative full scan spectra

..................................................................................................................................................... 147

Figure 78 Ethoprop ESI positive, ESI negative (weak), APCI positive, APCI negative full

scan spectra ................................................................................................................................. 148

Figure 79 Ethyl parathion ESI positive (weak), ESI negative (weak), APCI positive, APCI


Figure 80 Fenamiphos ESI positive (weak),, ESI negative (weak), APCI positive, APCI


Figure 81 Fenitrothion ESI positive (weak),, ESI negative, APCI positive, APCI negative

full scan spectra........................................................................................................................... 151

Figure 82 Fenthion ESI positive, ESI negative, APCI positive, APCI negative full scan

spectra ......................................................................................................................................... 152

Figure 83 Fonofos ESI positive, ESI negative, APCI positive, APCI negative full scan

spectra ......................................................................................................................................... 153

Figure 84 Malathion ESI positive, ESI negative, APCI positive, APCI negative full scan

spectra ......................................................................................................................................... 154

13

Figure 85 Methamidophos ESI positive, ESI negative, APCI positive, APCI negative

(weak)full scan spectra ............................................................................................................... 155

Figure 86 Methidathion ESI positive, ESI negative, APCI positive, APCI negative full scan

spectra ......................................................................................................................................... 156

Figure 87 Methyl parathion ESI positive (weak), ESI negative, APCI positive, APCI


Figure 88 Mevinphos ESI positive, ESI negative, APCI positive, APCI negative full scan

spectra ......................................................................................................................................... 158

Figure 89 Naled ESI positive, ESI negative, APCI positive, APCI negative full scan spectra

..................................................................................................................................................... 159

Figure 90 Oxydemethon methyl ESI positive, ESI negative (weak), APCI positive, APCI


Figure 91 Phorate ESI positive, ESI negative (weak), APCI positive, APCI negative full scan

spectra ......................................................................................................................................... 161

Figure 92 Phosalone ESI positive (weak), ESI negative, APCI positive, APCI negative full

scan spectra ................................................................................................................................. 162

Figure 93 Phosmet ESI positive, ESI negative (weak), APCI positive, APCI negative full

scan spectra ................................................................................................................................. 163

Figure 94 Phostebupirim ESI positive, ESI negative, APCI positive, APCI negative full scan

spectra ......................................................................................................................................... 164

Figure 95 Pirimphos methyl ESI positive, ESI negative, APCI positive, APCI negative

(weak) full scan spectra ........................................................................................................ 165

14

Figure 96 Profenofos ESI positive, ESI negative, APCI positive, APCI negative full scan

spectra ......................................................................................................................................... 166

Figure 97 Propetamphos ESI positive, ESI negative, APCI positive, APCI negative full scan

spectra ......................................................................................................................................... 167

Figure 98 Sulfotepp ESI positive, ESI negative, APCI positive, APCI negative full scan

spectra ......................................................................................................................................... 168

Figure 99 Temephos ESI positive (weak), ESI negative, APCI positive, APCI negative full

scan spectra ................................................................................................................................. 169

Figure 100 Terbufos ESI positive (weak), ESI negative, APCI positive, APCI negative full

scan spectra ................................................................................................................................. 170

Figure 101 Tetrachlorvinphos ESI positive, ESI negative, APCI positive, APCI negative full

scan spectra ................................................................................................................................. 171

Figure 102 Tribufos ESI positive, ESI negative, APCI positive, APCI negative full scan

spectra ......................................................................................................................................... 172

Figure 103 Trichlorfon ESI positive, ESI negative, APCI positive, APCI negative full scan

spectra ......................................................................................................................................... 173

Figure 104 ChemElut LLE experiment with combination of solvents .............................. 180

Figure 105 ASE temperature effect on recoveries ............................................................... 183

Figure 106 ASE dwell time and pressure effect on recoveries ............................................ 183

Figure 107 Structure of various sorbents ............................................................................. 185

Figure 108 Comparison of various sorbents for removing contaminants from serum extract

..................................................................................................................................................... 187

Figure 109 Elution of OP pesticides over different sorbents ............................................... 188

15

Figure 110 Two step cleanup: recoveries of LC group OP pesticides ................................. 192

Figure 111 Two step cleanup: recoveries of GC group OP pesticides ................................ 192

Figure 112 Sample dispersion process prior ASE extraction............................................... 193

Figure 113 Accelerated solvent extractor ............................................................................. 195

Figure 114 Comprehensive cleanup diagram ........................................................................ 197

Figure 115 Recoveries of LC compound group as the function of drying time .................. 201

Figure 116 Recoveries of LC compound group as the function of drying time, 2µL dodecane

added ........................................................................................................................................... 202

Figure 118 Decomposition of OP pesticides in serum ......................................................... 209

Figure 119 Decomposition of OP pesticides in serum, 100µL 50 mmolar EDTA solution is

added ........................................................................................................................................... 210

Figure 120 Azinphos methyl chromatography, monitoring all four major transitions for

azinphos methyl and phoseth ...................................................................................................... 214

Figure 121 Phosmet chromatography, monitoring all four major transitions for azinphos

methyl and phosmet .................................................................................................................... 214

Figure 122 Transition 318 -> 77 signal from azinphos methyl and phosmet ...................... 215

Figure 123 Dependence of ASE recovery on the number of nitrogen atoms in the OP

molecule. Emphasis on the halogenated species. ........................................................................ 219

Figure 124 Metabolism of Chlorpyrifos .............................................................................. 226

Figure 125 Environmental degradation of chlorpyrifos ...................................................... 226

Figure 126 Flow chart of determining recoveries of a given cleanup process..................... 233

Figure 127 Determination of limits of detection .................................................................. 235

16

LIST OF TABLES

Table 1 Groups of Organophosphates R, R1, R2, R3 : Ethyl, methyl. X: Specific Group .. 2

Table 2 Non-Specific Metabolites of OP Pesticides and their Structures .............................. 10

Table 3 OP pesticides of interest ............................................................................................ 19

Table 4 Specific OP Pesticide Metabolites ........................................................................... 23

Table 5 Specific and non specific metabolites of OP pesticides ............................................ 24

Table 6 Assay of OP Pesticides In Serum, Plasma or Whole Blood - Gas Chromatography 25

Table 7 Assay of OP Pesticides In Serum, Plasma or Whole Blood - Liquid

Chromatography ........................................................................................................................... 26

Table 8 Assay of OP esters In Serum, Plasma or Whole Blood ............................................ 27

Table 9 Available Native OP pesticides and their Labeled Reference Compounds [32] ...... 28

Table 10 Non optimized MS parameters ............................................................................... 32

Table 11 Compounds of line B (GC) ...................................................................................... 43

Table 12 GC predicted and actual retention times ................................................................. 47

Table 13 Table of LC-MS ions in APCI and ESI mode, positive or negative ionization

Bolded ions ara the most aboundant (Cont. next page) ................................................................ 88

Table 14 HPLC columns tested ............................................................................................. 91

17

Betasil Betasil Betasil C18 C18 Phenyl Width: 2.1mm 4.6mm 4.6mm 4.6mm No. OP Length: 100mm 100mm MeOH ACN

1 Acephate 8.1 7.94 6.15 5.62 2 Azinphos-methyl 21.83 19.35 20.53 17.11 3 Bensulide 24.2 21.4 21.72 19.08 4 Cadusafos* 25.64 22.39 21.05 17.59 5 Chlorethoxyfos 26.88 - N/F N/F E/B 6 Chlorpyrifos 27.41 23.5 22.53 28.06 E/B 7 Chlorpyrifos methyl 25.86 22.57 21.67 27.61 E/B 8 Coumaphos 24.85 21.88 22.24 19.15 9 Diazinon 24.8 21.83 21.18 17.92

10 Dichlorvos (DDVP) 19.08 16.72 15.67 N/F E/B 11 Dicrotophos 14.25 12.13 13.68 9.94 12 Dimethoate 15.71 14.29 14.16 11.68 13 Disulfoton 28.94 22.43 21.66 N/F 14 Ethion 26.97 23.19 23.1 20.58 15 Ethoprop 23.72 21.08 19.86 16.28 16 Ethyl parathion 24.46 21.62 21.09 N/F E/B 17 Fenamiphos 24.19 21.42 20.77 16.5 18 Fenitrothion 23.36 20.76 20.64 N/F E/B 19 Fenthion 24.83 21.9 21.82 N/F E/B 20 Fonofos** 24.91 21.98 21.25 18.85 21 Malathion 22.97 20.41 20.57 17.89 22 Methamidophos 4.08 6.43 3.67 3.47 23 Methidathion 21.66 19.18 19.75 16.86 24 Methyl parathion 22.46 19.98 19.66 N/F E/B 25 Mevinphos* 16.97 15.14 15.18 11.72 26 Naled 21.52 18.96 18.47 N/F 27 Oxydemeton methyl 12.79 10.67 12.34 10.22 split 28 Phorate 25.4 22.27 21.29 N/F E/B 29 Phosalone* 25.26 22.17 22.12 N/F E/B 30 Phosmet 29.03 N/F N/F N/F E/B 31 Phostebupirim 26.79 23.13 22.04 19.27 32 Pirimiphos methyl 24.72 21.71 20.69 17.56 33 Profenofos 26.4 22.86 22 N/F E/B 34 Propetamphos 23.21 20.64 19.87 17.68 35 Sulfotepp 24.56 21.67 21.31 19.11 36 Temephos 26.73 22.98 24.04 N/F E/B 37 Terbufos 26.75 23.09 22.15 N/F 38 Tetrachlorvinphos 24.28 21.51 21.03 17.53 39 Tribufos (DEF) 28.58 24.31 23.17 20.08 40 Trichlorfon 19.09 16.73 15.64 N/F E/B

Table 15 Retenetion times of OP pesticides on Betasil C18 and Phenyl Columns (N/F: not

found, E/B: extreme peak broadening) ......................................................................................... 94

18

Aquasil C18 Chromolith RP 18e Zorbax SBC3 Width: 4.6mm 4.6mm 4.6mm No. OP Length: 100 mm 100 mm 100 mm

1 Acephate 8.35 7.33 7.53 poor peakshape

2 Azinphos-methyl 20.12 18.42 19.43 3 Bensulide 21.56 21.19 21.19 4 Cadusafos* 22.76 22.51 22.19 5 Chlorethoxyfos 20.1 Poor peak N/F N/F 6 Chlorpyrifos 23.63 23.79 22.65 7 Chlorpyrifos methyl 22.77 Poor peak 22.61 21.63 8 Coumaphos 22.57 21.8 22.29 9 Diazinon 22.32 21.67 21.75

10 Dichlorvos (DDVP) 16.77 15.79 16.24 11 Dicrotophos 13.8 11.73 14.13 12 Dimethoate 14.12 12.42 14 13 Disulfoton 22.47 Bad tailing 22.39 Smearing 21.4 14 Ethion 23.55 23.59 22.58 15 Ethoprop 21.45 20.39 20.63 16 Ethyl parathion 21.86 21.25 20.97 17 Fenamiphos 21.79 21.05 20.93 18 Fenitrothion 21.04 19.91 20.15 19 Fenthion 22.23 21.66 21.01 20 Fonofos** 22.1 21.65 20.92 21 Malathion 20.45 19.52 20.01 22 Methamidophos 6.84 4.24 Excessive

Peak broadening

4.03

23 Methidathion 19.26 18.08 18.86 24 Methyl parathion 20.06 18.88 19.21 25 Mevinphos* 15.26 13.96 15.91 26 Naled * 18.06 N/F 27 Oxydemeton methyl 12.79 10.39 12.38 28 Phorate 22.35 22.14 21.13 29 Phosalone* 22.41 22.17 21.64 30 Phosmet N/F N/F N/F 31 Phostebupirim 23.26 23.38 22.48 32 Pirimiphos methyl 21.87 20.85 20.77 33 Profenofos 23.31 23.14 22.29 34 Propetamphos 20.64 19.79 20.07 35 Sulfotepp 21.71 21.43 20.86 36 Temephos 23.47 23.55 22.78 37 Terbufos 23.17 23.33 22.23 38 Tetrachlorvinphos 21.88 21.15 20.9 39 Tribufos (DEF) 24.31 24.51 23.6 40 Trichlorfon 15.27 14.17 15.88

Table 16 Retenetion times of OP pesticides on Aquasil C18 and Chromolit RP 18e, Zorbax

19

SBC3 ............................................................................................................................................. 95

20

Discovery HS F5 3µm Hypercarb Varian C18

Width: 4.6mm 4.6mm 4.6mm No. OP Length: 100 mm 100 mm 250mm

1 Acephate 7.61 3.22 12.74 2 Azinphos-methyl 20.61 N/F E/B 23.99 3 Bensulide 22.21 22.62 tailing 24.65 4 Cadusafos* 21.7 18.73 26.17 5 Chlorethoxyfos N/F N/F E/B N/F 6 Chlorpyrifos 22.85 N/F E/B 27.53 7 Chlorpyrifos methyl 22.12 N/F E/B N/F E/B 8 Coumaphos 23.83 N/F E/B 25.62 9 Diazinon 22.4 21.05 25.77

10 Dichlorvos (DDVP) 16.8 N/F E/B 21.64 11 Dicrotophos 13.39 9.33 16.69 12 Dimethoate 14.86 10.46 18.43 13 Disulfoton N/F N/F 26.21 14 Ethion 23.31 25.26 tailing 26.8 15 Ethoprop 19.98 15.63 24.98 16 Ethyl parathion 22.75 N/F E/B 25.28 17 Fenamiphos 20.97 N/F E/B 24.88 18 Fenitrothion 22.1 N/F E/B 24.7 19 Fenthion 21.99 N/F E/B 25.77 20 Fonofos** 22.02 8.23 25.92 21 Malathion 21.27 18.7 24.23 22 Methamidophos 4.28 1.43 12 23 Methidathion 20.4 22.03 23.74 24 Methyl parathion 21.57 N/F E/B 24.13 25 Mevinphos* 15.59 N/F E/B 19.41 26 Naled 18.55 N/F N/F 27 Oxydemeton methyl 11.57 8.23 15.35 28 Phorate 22.02 tailing N/F 26.9 29 Phosalone* 22.57 N/F 25.66 30 Phosmet N/F N/F E/B N/F 31 Phostebupirim 22.73 20.45 27 32 Pirimiphos methyl 26.26 N/F E/B 25.89 33 Profenofos 22.25 N/F E/B 26.72 34 Propetamphos 20.6 17.76 24.26 35 Sulfotepp 21.92 17.32 25.21 36 Temephos 23.59 N/F E/B 26.44 37 Terbufos 22.96 N/F 26.87 38 Tetrachlorvinphos 21.19 N/F E/B 25.09 39 Tribufos (DEF) 23.01 N/F 28.5 40 Trichlorfon 16.82 N/F E/B 19.38

Table 17 Retenetion times of OP pesticides on Discovery HS F5, Hyper carb and Varian C18

columns ......................................................................................................................................... 96

21

Table 18 List of elution curves applied while optimizing LC separations .......................... 124

Table 19 Recoveries for lyophilization extraction ............................................................... 177

Table 20 Recoveries for extraction on ChemElut Cartridges .............................................. 178

Table 21 ChemElut LLE experiment with combination of solvents................................... 179

Table 22 Effects on recoveries of varying ASE extraction parameters ............................... 182

Table 23 Two step cleanup: recoveries of GC group OP pesticides. Concentrations are

500ng/mL .................................................................................................................................... 191

Table 24 Two step cleanup: recoveries of LC group OP pesticides Concentrations are

500ng/mL .................................................................................................................................... 191

Table 25 Effect of drying on recoveries of LC compound group, with and without addition

of dodecane "catching solvent" ................................................................................................... 201

Table 26 Available isotope labeled internal standards ......................................................... 204

Table 27 LC group compound detection limits in pure form............................................... 205

Table 28 Accuracy of the LC group assay, 1 mL serum, n=5 ............................................. 206

Table 29 Relative standard deviations of three concentration of analytes - HPLC group .. 211

Table 30 Limits of detection of the LC group compounds, serum samples ......................... 212

Table 31 Major transitions and optimum collision energies for azinphos methyl and phosmet

..................................................................................................................................................... 213

Table 32 Summary of OP assays for human exposure .......................................................... 222

22

LIST OF ACRONYMS

AChE Acetyl Cholinesterase Enzyme

APCI Atmospheric Pressure Chemical Ionization

CI Chemical Ionization

DEDTP O,O-Diethyl Dithiophosphate

DEP Diethyl Phosphate

DETP O,O-Diethyl Dithiophosphate

DMDTP O,O-Dimethyl Dithiophosphate

DMP Dimethyl Phosphate

DMTP O,O-Dimethyl Thiophosphate

EI Electron Impact Ionization

EPA Environmental Protection Agency

GC Gas Chromatography

HPLC High Pressure Liquid Chromatography

LC Liquid Chromatography

LOD Limit of Detection

OP Organophosphate Pesticide

OPICN Organophosphorus Ester-Induced Chronic Neurotoxicity

OPIDP Organophosphate Induced Delayed Polyneurophaty

1

CHAPTER 1: INTRODUCTION AND GOALS

Organophosphates (OP) are a very effective and widely used class of pesticides.

Presently there are 39 organophosphate type pesticides registered with the Environmental

Protection Agency (EPA) [1]

OP pesticides became popular after the persistent organochlorine pesticides were banned in

the 1970s. Their wide availability relatively low price, short half life in the environment and low

susceptibility to pest resistance made them an attractive alternative.

The total insecticide use in the United States has been declining: it has dropped approximately

45% from 1980 to 2001, according to the latest available statistics from the EPA. The same time

the share of OP pesticides went up from 58 t 70% of the total use.

R: Methyl or Ethyl

Y: O or S

X: Specific Organic Group

Figure 1 General Structure of Organophosphate Pesticides

Using the above general structure, the organophosphate pesticides of interest can be grouped

as seen in Error! Reference source not found..

R1YP

R2YX

Y

2

Type of Phosphorous Group General Structure Pesticide

Phosphate

P OX

OR1O

R2O

Dichlorvos, Chlorfenvinphos, Heptenophos

Phosphonate

P OX

OR1O

R2

Trichlorfon

Phosphinate

P OX

OR1

R2

Glufosinate

(herbicide, not in the 40

compounds of interest)

S-alkyl phosphorothioate

RSP

ROOX

O

Phosphoramidate

R1

R1OO

P NR2

O

Phosphorotriamidate

R2NP NH2

OR1N

Phosphorothioamidate

R2SR1O

P NR2

O

O-alkyl phoshorothioate

R1OR1O P SX

O Omethoate

S-alkyl phosphorodithioate

RS PRO

OX

S

Phosphorodithioate

R1O

R1OP SXS

Phosphonothioate

RP

ROOX

S

Table 1 Groups of Organophosphates R, R1, R2, R3 : Ethyl, methyl. X: Specific Group

3

Toxicity

Inhibition of Acetyl Cholinesterase: Acute Cholinergic Syndrome

The primary reason for OP toxicity is its inhibition of the acetyl cholinesterase (AChE)

enzyme:

Acetyl cholinesterase is found in many tissues but its role mostly is understood in the nervous

system. It hydrolyzes the neurotransmitter acetylcholine, terminating its action. It is essentially

the “off switch” of the neuroimpulse. If the AChE is inhibited it leaves the synapse in the “on”

state hence the paralytic effect of the OP.

During inhibition the OP mimics the acetyl cholinesterase phosphorylation of the active site.

Hydrolysis of the phosphoryl-AChE is very slow so the enzyme’s catalytic potential is lost.

Inhibition of the AChE enzyme becomes irreversible when the methoxy or ethoxy groups

hydrolytically removed from the absorbed portion of the organophosphate pesticide. This process

is called aging Phosphorylated AChE undergoes two reactions at a very slow rate: recovery and

aging. Recovery is the hydrolytic removal of the phosphate moiety from the AChE enzyme. The

half life of the OP-AChE adduct depends on R1 and R2. In case of pesticides it takes place in

order of hours.

The other important process is aging. Aging is the dealkylation of the OP moiety in the OP-

AChE complex. Once aging takes place, the inhibited AChE can not be reactivated.

- Symptoms of acute OP poisoning are:

- Nausea, vomiting, abdominal cramps, diarrhea;

- Excessive salivation, rhinorrhea;

- Headache, vertigo;

4

- Fixed pinpoint pupils, blurred vision ocular pain;

- Muscle twitches, especially of face, tongue and neck;

- Difficulty in breathing, primarily due to excessive secretion and bronchoconstriction

- Random jerky movements, convulsions;

- Respiratory paralysis, death.

Depending on the dose, type/toxicity of the OP pesticide and quality of health facilities, the

reported mortality rates are between 4 % and 29 %.

Recovery of inhibited AChE in the nervous system is about 1 week in experimental animals

and it is believed to be the same in people. In untreated patients it takes 5-7 weeks for the AChE

activity to return to normal.

OHENZYME + X

O

PRORO

OHENZYME X

O

PRORO

OENZYME X

O

PRORO

O-

OENZYME ORX

O

P

Reactivation

OPEsterase Michealis Complex

Phosphorylation

Aging

Figure 2 Esterase Inhibition by Organophosphate R: Ethyl or methyl, X: Specific group

5

Intermediate Syndrome

The intermediate syndrome is a set of symptoms that may develop in patients after recovery

from acute OP pesticide poisoning. It too is caused by esterase inhibition and manifests itself

through muscle weakness tremors and respiratory distress. Since it develops after the acute

cholinergic syndrome but before the “OP induced delayed neuropathy, it was coined as the

“Intermediate syndrome”

Organophosphate Induced Delayed Polyneurophaty (OPIDP)

OPIDP is a set of symptoms caused by OP poisoning but with an onset of 1-3 weeks after the

initial acute poisoning. Its manifestations are: muscle weakness, cramps in the arms and legs.

Recovery is slow and usually incomplete.

The cause of OPIDP is the OP induced inhibition of the neuropathy target esterase (NTE)

Organophosphorus Ester-Induced Chronic Neurotoxicity (OPICN) [2]

OPICN is produced by acute or multiple subclinical doses of organophosphorus compounds.

Clinical signs, ranging from weeks to years after exposure, consist of neurological and

neurobehavioral abnormalities. These abnormalities continue for a prolonged time, and are

caused by damage to the central and peripheral nervous system.

Most examples in the literature are observed after acute OP poisoning. The OP exposure

causes lesions on the brain which results in neurological symptoms: increased incidence of

depression, irritability, confusion, and social isolation. Also there is observable decreased verbal

attention, visual memory, motoricity, and affectivity.

There is more information on acute exposure induced OPICN, probably because the cause is

well defined and the dose is quantifiable.

6

For our studies however, effects of repeated small doses – which do not cause acute poisoning

– is more relevant.

Professional pesticide applicators and farmers exposed to organophosphate pesticides show

the following symptoms: anxiety, diminished vigilance and reduced concentration. Kaplan et

al.[3] observed long-term cognitive problems in concentration, word finding, and short-term

memory in individuals exposed to low subclinical levels of chlorpyrifos. Sheep dippers exposed

to OP pesticides experienced significant neuropsychological problems.[4, 5] Male fruit farmers

who were chronically exposed to OP insecticides showed significant increase of their reaction

time.[6] The same symptoms were observed in female pesticide applicators, with the addition of

reduced motor steadiness, and increased muscle tension, depression, and fatigue . Workers

exposed to the OP insecticide quinalphos during manufacturing, exhibited changes in central

nervous system function: memory loss, impaired vigilance, slow learning and motor deficits. The

AChE activity of these workers was normal..[7]

Many rescue workers and some people who did not develop any acute symptoms during the

Tokyo nerve gas attack later developed chronic, persistent memory loss three years and nine

months after the attack.[8, 9] Pilkington et al.[10] reported strong correlation between chronic

low-level exposure to OP pesticide concentrates in sheep dips and neurological symptoms in

workers

7

Exposure [11]

The main routes of exposure are oral, dermal or inhalation.

Oral Exposure

The main route of the exposure of the general population to OP pesticides is ingestion via

food or drinking water. Regulatory controls of pesticide use is designed to keep the resulting

pesticide levels insufficient to cause adverse health effects.

Ingestion of substantial amounts can occur either deliberately (suicide attempts) or

accidentally as a result of mistaken identity or poor handling techniques.

Dermal Exposure

The main cause of dermal exposure is handling of OP pesticides, during manufacturing or

application. Dermal penetration is varying, depending the carrier solvent: usage of solvents

which can penetrate the skin – such as acetone – cause higher uptake than water based

suspensions. Absorption of OP pesticidesis also dependent on the hydratation and temperature of

skin.

Inhalation

Inhalation of OP pesticides is usually the result of use of sprays, mists and powders.

Absorption is fast and almost complete. The pesticide enters in the circulatory system without

passing through the liver. Exposure through inhalation is usually combined with exposure of

eyes and mucous membranes.

8

Distribution

In rhesus monkeys, 40% of intravenously administered radiolabelled diazinon, was excreted

in 24 hrs. (urinary excretion) This was followed by a slow and almost constant 5% / day

excretion for 3 – 7 days. Dermal exposure of humans followed a similar pattern.

9

OP Metabolism [11]

Many tissues like skin, gut lung and kidneys contain enzymes that can metabolize Ops, the

main sites are the liver and blood. Mammalian OP metabolism involves many enzymes and

possible pathways. The dominant metabolic process will depend on the type of OP, the exposure

route, and the dose. These are some of the possible routes, grouped by the participating enzyme:

Figure 3 Non Persistent Pesticide: Concentration Change in Blood and in Urine [12]

10

Table 2 Non-Specific Metabolites of OP Pesticides and their Structures

Chemical Name Abbreviation Structure

Dimethyl phosphate DMP

MeOP

MeOOH

O

O,O-Dimethyl

Thiophosphate DMTP

MeOP

MeOOH

S

O,O-Dimethyl

Dithiophosphate DMDTP

MeOP

MeOSH

S

Diethyl phosphate DEP

EtOP

EtOOH

O

O,O-Dimethyl

Thiophosphate DETP

EtOP

EtOOH

S

O,O-Dimethyl

Dithiophosphate DEDTP

EtOP

EtOSH

S

A-esterases

The A- esterases are a group of enzymes which are able to hydrolyze a broad range of

substrates, including OP pesticides. They are present in a number of tissues: in the liver, the

11

intestine and in serum. Most studied example is the hydrolysis of paraoxon to diethyl phosphate.

(Figure 4) Many similar OP hydrolyze to the respective dialkyl phosphate, following a similar

pattern.

The products of the hydrolysis are excreted in the urine. This hydrolytic process results in the

removal of the “leaving group”, which subsequently can be analyzed and used as a biomarker for

that specific OP. These removed leaving groups are also known as “specific metabolites”

Parathion and other thion compounds are not substrates for A-esterases.

NO2EtO P

OEt

O

O EtO P

OEt

O

OH

Figure 4 Paraoxon Hydrolysis to diethyl phosphate

Carboxylesterases

Carboxylesterases (CaEs) are present in mammalian plasma and in many tissues including

liver, kidney, brain, intestines, muscles, etc. Their normal physiological role is the metabolism of

lipids. They also contribute to the detoxification of OP pesticidesthrough two mechanisms:

1. Hydrolysis of carboxyl ester side chains.(Figure 5)

2. Irreversible binding of the OP without hydrolysis

OC2H5

MeO P

OMe

S

S CH

C

O

OC2H5

CH2 C

O

OC2H5

MeO P

OMe

S

S CH

C

O

OH

CH2 C

O

Figure 5 Hydrolysis of the Carboxyl Ester Side Chain of Malathion

12

Amidases

Amidases catalyse the hydrolytic cleavage of carboxylamide. (Figure 6)

R CO

NH CH3 R CO

OH

Figure 6 Amidase Cleavage of Carboxylamide R: Methyl, Ethyl

NO2EtO P

OEt

S

O NO2EtO P

OEt

O

O

Figure 7 Oxidative Desulphuration of Parathion

Microsomal Oxigenases

Microsomal oxigenases are a family of enzymes that contain cytochrome P450 and/or flavin.

These enzymes are found in most tissues but their predominant location is the liver. Microsomal

oxigenases catalyze several reactions:

- Oxidative desulfuration of OP pesticidessuch as conversion of parathion to paraoxon

(Figure 7). The resulting oxon is much more toxic.

- Oxidative O and N-dealkylation and dearylation

- Thioether oxidation

- Side chain oxidation

13

An example of OP metabolism is shown on Figure 8: Metabolism of Chlorpyrifos

Cl

Cl

Cl

N

EtO P

OEt

S

O

Cl

Cl

Cl

N

EtO P

OEt

O

O

EtO P

OEt

S

OH

Cl

Cl

Cl

N

OH

Diethyl thiophosphate

3,5,6-Trichloropyridinol

Chlorpyrifos

Chlorpyrifos Oxon

EtO P

OEt

O

OH

Diethyl phosphate

Cl

Cl

Cl

N

OH

3,5,6-Trichloropiridinol

OHOH

OH

HOOC

O

Cl

Cl

Cl

N

O + O-

O

O

Cl

Cl

Cl

N

S

3,5,6-Trichloropiridinol glucuonide 3,5,6-Trichloropiridinol sulfate

Cholinesterase Inhibition

Figure 8 Metabolism of Chlorpyrifos

14

Determining OP pesticide exposure

Non – assay methods.

Exposure to pesticides may be estimated by using empirical mathematical formulas based on

usage, time of exposure, exposed skin area and other factors. [4, 5]. Of course these methods are

only estimates, not actual measurements.

Urinary Assay of OP Metabolites

Most modern pesticides and all of the organophosphates of interest are “non persistent”,

which means that they quickly metabolized, the intact pesticide is only present in the

bloodstream – in any measurable amount - for a few hours to a few days

Traditionally most of the studies are carried out using urine samples analyzing for OP

metabolites. The two main reasons are that urine samples are much easier to obtain than blood

and that the metabolite concentration – time function gives a wider window of delectability.

Examples of urinary assays of organophosphate metabolites:

Hardt Et. Al. [13]used liquid-liquid extraction followed by centrifugation and drying under

vacuum. After derivatization overnight with 2,3,4,5,6 pentafluorobenzyl bromide. After a second

liquid-liquid extraction the reconstituted sample was separated with gas-chromatography (GC).

The detector was mass spectrometer.

Bravo et al. [14] greatly simplified the above method by first lyophilizing the urine samples

then using liquid extraction with a blend of acetonitrile and ethyl ether. Just like in the previous

example, the OP metabolites being highly polar compounds, had to be derivativized to make

them suitable for GC separation. In this case the derivatizing agent was 1-Chloro-3-iodopropane.

Using metabolites in urine as biomarker has drawbacks however; metabolites are much less

15

specific. Most OP pesticidesmetabolize to one of the seven dialkyl phosphates:

Second source of error is that the OP pesticidespath of breakdown is often similar to the

metabolic pathway. As a result a person may ingest a breakdown product, which is also the target

compound of the assay. Nonetheless today the preferred method of determining OP pesticide

exposure is the urinary assay of metabolites.

Detection of OP Pesticides in Blood/Serum/Plasma

Being able to detect the unchanged pesticide solves the above mentioned, but because the

concentrations in blood drop rapidly, (Figure 3) it requires a more sensitive detector. On the

other hand the unmetabolized form of these compounds is more lipophilic than the metabolite,

which should make sample cleanup and separation somewhat easier.

Measuring the parent compounds in blood or blood products has other advantage; it is a

regulated fluid. It means that blood volume is constant, unlike the urine volume in the human

body. Therefore no dilution-correction needed when using serum, plasma or whole blood.[15]

Two review articles [16, 17] summarize the most frequently used methods in OP analysis in

whole blood, plasma or serum matrix. This review also uses independent searches of “ISI web of

science”, focusing on assays OP compounds, blood / serum / plasma as matrix and –for the most

part – mass spectrometer as a detector. The results are tabulated in Table 6 - Table 8, grouped by

methods of separation

The first group of methods, listed in Table 6 list recent works based on GC.

The most recent work by Mushoff et. Al. [18] used solid phase microextraction, (SPME), GC

and mass spectrometric detection with electron impact ionization and single ion monitoring to

determine 22 OP pesticides. Detection limits were reported between 10 and 300 ng/mL. SPME is

16

a clean and simple technique the method is hampered though with low recoveries.

The lowest limits of detection was achieved by Barr and her coworkers [19] They analyzed 29

contemporary pesticides in blood, 9 of them OP-s. using GC coupled with high resolution mass

spectrometer they achieved 0.5 to 2 ppt detection limits This is probably the most important and

applicable method four our purposes; the LOD-s are likely low enough to study background

levels of exposure.

17

Goals and Objectives

To develop an analytical method to measure the non metabolized form of all 39 EPA

registered organophosphate pesticides in human serum as matrix.

Test various cleanup methods such as solid phase extraction, liquid-liquid extraction,

lyophilization, and their automated variants for feasibility. Determine recoveries for each

analyte. While high recovery is important, focus of the cleanup has to be removal of

interfering contaminants.

Test HPLC and GC for analytical separation methods, optimize separation parameters.

Determine Limits of Detection, Limits of Quantitation.

Study the robustness of the method. (Sensitivity to parameter variations.)

Study the stability of OP pesticides at different temperatures.

Study the stability of organophosphorus pesticides id blood and means to stabilize it.

In order to achieve these goals, the project has to be broken down to the following blocks:

18

Development Step

Experimental Choices Considerations

Cleanup First Choice: For all sample cleanup / pre-concentration steps the main considerations are: 1. Recovery 2. Purity (not to extract compounds interfering with further steps of the analysis) 3. Feasibility for automation / Sample throughput

SPE LLE

Analytical Separation

GC Will work for the intact OP pesticides

HPLC Probably preferred for the metabolites or heat labile OP-s. Interfacing with HRMS may be a problem

Detection HRMS - First Choice, MS-MS - Second choice, but should give good

results with the newer instruments. (Quantum, Sciex 4000)

Stability Study This is like a project in a project: it may run parallel with the above outlined

parts on a limited number of OP pesticides or: subsequently on all or most of the OP pesticides.

19

MeO P

SMe

O

NH C

CH3

O Acephate

Me

i-Pr

EtO P

OEt

S

O N

N

Diazinon

MeO P

OMe

S

S CH2 N

NN

O

Azinphos-methyl

Cl

ClMeO P

OMe

O

O C

CH3

C

Dichlorvos (DDVP)

i-Pr

i-Pr

O

O

CH2O P

O

S

S CH2 NH S

Bensulide

MeO P

OMe

O

O C

CH3

CH

CH3

NC

O CH3

Dicrotophos

EtO P

O

S s-Bu

S s-Bu Cadusafos

CH3NHMeO P

OMe

S

S CH2 C

O

Dimethoate

Cl

Cl

Cl

EtO P

OEt

S

O CH C

Cl Chlorethoxyphos

EtEtO P

OEt

S

S SCH2CH2

Disulfoton

Cl

Cl

Cl

N

EtO P

OEt

S

O

Chlorpyrifos

EtO P

OEt

S

S CH2 OEtP

OEt

S

S

Ethion

Table 3 OP pesticides of interest

20

Cl

Cl

Cl

N

MeO P

OMe

S

O

Chlorpyrifos methyl

n-Pr

n-PrEtO P

S

O

S

Ethoprop

CH3

Cl

O

EtO P

OEt

S

O

O

Coumaphos

NO2EtO P

OEt

S

O

Ethyl Parathion

CH3

CH3

i-Pr NH P

OEt

O

O

Fenamiphos

MeO P

OMe

O

O C

CH3

CH CH3C

O

Mevinphos

CH3

NO2MeO P

OMe

S

O

Fenitrothion

Cl

Cl

Br

MeO P

OMe

O

O CH

Br

C

Naled

CH3

CH3

SMeO P

OMe

S

O

Fenthion

CH3MeO P

OMe

O

S CH2 CH2 CH2S

O

Oxydemeton methyl

Et P

OEt

S

S

Fonofos

EtO P

OEt

S

S EtSCH2

Phorate

Table 3 (cont.) OP pesticides of interest

21

OEt

MeO P

OMe

S

S CHCO

OEt

CH2 CO

Malathion

Cl

O

NEtO P

OEt

S

S O

Phosalone

MeO P

SMe

O

NH2

Methamidophos

CH2MeO P

OMe

S

S

O

O

N

Phosmet

O

CH2MeO P

OMe

S

S N

C

N

S

C O CH3

Methidathion

CH3

CH3

CH3

C

i-Pr

CH2EtO P

O

S

O

N

N Phostebupirim (tebupirimfos)

NO2MeO P

OMe

S

O

Methyl parathion

MeO P

OMe

S

O S OMeP

OMe

S

O

Temephos CH3

Et

Et

N

MeO P

OMe

S

O N

N

Pirimiphos methyl

EtO P

OEt

S

S CH2 S t-Bu

Terbufos


22

Cl

BrEtO P

S

O

O

n-Pr

Profenofos

H

Cl

ClC

MeO P

OMe

O

O C

Cl

Cl

Tetrachlorvinphos

OCH3

MeO P

NH

S

O C

Et

CH

C O i-Pr

Propetamphos

n-Pr

n-Pr S P

S

O

S n-Pr

Tribufos (DEF)

EtO P

OEt

S

OEtP

OEt

S

O

Sulfotepp

OH

CHMeO P

OMe

O

O C

Cl

Cl

Cl Trichlorfon


23

Abbreviation Chemical Name BTA 1,2,3-benzotriazin-4(3H)-one CIT 5-chloro-1-isopropyl-(1H)-1,2,4-triazol-3-ol CMHC 3-chloro-7-hydroxy-4-methyl-2H-chromen-2-ol DEAMPY 2-(diethylamino)-6-methylpyrimidin-4-ol DEDTP O,O-Diethyl Dithiophosphate DMDTP O,O-Dimethyl Dithiophosphate IMPY 2-isopropyl-4-methyl-6-hydroxypyrimidine MSMB Methysulfonyl methylbenzazimide

Table 4 Specific OP Pesticide Metabolites

24

Pesticides Non Specific metabolites Specific

DMP

DMTP

DMDTP

DEP

DETP

DEDTP metabolites Acephate — — — — — — Acephate, methamidophos Azinphos-methyl X X X — — — BTA, MSMB Bensulide — — — — — — — Cadusafos — — — — — — — Chlorethoxyphos — — — X X — — Chlorpyrifos — — — X X — 3,5,6-TCPY Chlorpyrifos-methyl X X — — — — 3,5,6-TCPY Coumaphos — — — X X — CMHC Diazinon — — — X X — IMPY Dichlorvos (DDVP) X — — — — — — Dicrotophos X — — — — — — Dimethoate X X X — — — — Disulfoton — — — X X X — Ethion — — — X X X — Ethoprop — — — — — — — Ethyl parathion — — — X X — p-Nitrophenol Fenamiphos — — — — — — — Fenitrothion X X — — — — — Fenthion X X — — — — — Malathion X X X — — — Malathion dicarboxylic acid, mono-carboxylic acid Methamidophos — — — — — — Methamidophos Methidathion X X X — — — — Methyl parathion X X — — — — p-Nitrophenol Mevinphos X — — — — — — Naled X — — — — — — Oxydemeton-methyl X X — — — — — Phorate — — — X X X — Phosalone — — — X X X — Phosmet X X X — — — — Phostebupirim (tebupirimphos) — — — — — — — Pirimiphos-methyl X X — — — — DEAMPY Profenofos — — — — — — — Propetamphos — — — — — — — Sulfotepp — — — X X — — Temephos X X — — — — — Terbufos X X X X Tetrachlorvinphos X — — — — — — Tribufos — — — X X — — Trichlorfon X — — — — — —

Table 5 Specific and non specific metabolites of OP pesticides

Abbreviations: —: not applicable, X: exposure to the pesticide listed, DMP, DMTP,DMDTP, DEP, DETP, DEDTP see; BTA, CIT, CMHC, DEAMPY; DEDTP, DMDTP, IMPY MSMB: see Table 4

25

Ref First Year Matrix Type

Sample Cleanup

Analytical Separation MS Results

No Author Publ. Amount Type Recovery [%]

Type Column Ioniz. MS Type #of OP-s (Ops of Interest)

LOD

[18] Mushoff 2002 Whole Blood 0.5 g

Headspace SPME

0.1-19.6 GC HP5-MS 30mx.25mmx.25μm

EI HP5972 22 (13)

10-30 ng/mL

[19] Barr 2002 Serum, Plasma, 4g

SPE Oasis HLB

14-27 GC DB1701 (J&W) 30mX.25mmX.25μm

EI High Res. Finnigan MAT-900

9 (9)

0.5 –2 ppt

[20] Hernandez 2001 Whole Blood 0.5

HS-SPME GC HP Ultra 2 30mX.32mmX.50μm

EI Ms-MS Ion trap

3 (3)

0.02-0.7 ng/mL

[21] Tarbah 2001 Blood, Plasma 0.7g

LLE

50-133 GC HP5-MS 30mX.25mmX.25μm

EI

SIM HP MSD 5970

23 (10)

- stability studies

[22] Lacassie 2001 Whole Blood

SPE (HLB, C18)

41.2-107.9

GC Supelco PTE5 30mX.32mmX.25μm

EI

SIM Shm QP-5000

29

5-25 ng/mL

[23] Lacassie

2001

Serum 1 mL

SPE (HLB)

40-99% ? not detailed

GC

Supelco PTE5 30mX.25mmX.25μm

EI

SIM Shm QP-5000

29 (21)

5 ng/mL

[24] Liu

2001 Serum6 mL

LLE, hexane 2X

100%

GC

RTX CLPesticides 30mX.25mmX.25μm

EI

SIM 5971A

4 2-5 ng/mL

[25] López

2000

Serum (.5 mL, dil.)

SPME of 3 mL of dil. sample

60-100%. relative to water

GC Brand? 30mX .32mmX .5µm

FPD det.

7 (7)

1-15 ng/mL

Table 6 Assay of OP Pesticides In Serum, Plasma or Whole Blood - Gas Chromatography

26


Sample Cleanup



Type Column Ioniz.

MS Type # of OP-s (Ops of Interest)

LOD

[26] Sancho

2000 Serum 0.5 mL

0.5mL ser.+ .75mL ACN 10µL inj 2

87-113 col switching LC

Columns: #1: Discovery C18 #2: Supelco ABZ

ESI Micromass Quattro Zspray Ms-ms

Chlor-pyriphos & metab

1.5 ng/mL

[27] Abu-Qare

2001 (Rat) Plasma /0.5ml

SPE C18 (Sep-Pack)

80.1-83.9 LC

C18 UV detection

2 + metab

20-50 ng/mL

Table 7 Assay of OP Pesticides In Serum, Plasma or Whole Blood - Liquid Chromatography

27


Sample Cleanup



Type Column Ioniz. MS Type No of OP-s (Ops of Interest)

LOD

[28] Amini

2003

Plasma /.5ml

RAM (restricted access materials)

60-92 LC C18 ESI & APCI

ms-ms MM, Quattro

9 OP esters

0.2 –1.8 ng/mL

[29] Jonsson

2003 Plasma 5g

Micro-porous membrane

Stir bar assisted 32-92 % Counter Current 23-78 %

GC DB5 MS 30m X .25mm X .1µm

NPD 8 OP esters

0.2 – 0.9 ng/g

[30] Jonsson

2001 Plasma 5g

Membrane Extraction Device

72-83 % GC DB5 MS 30m X .32mm X .1µm

NPD 9 OP esters

0.06-4 ng/g

[31] Jonsson

2003 Plasma 50 µL !

Hollow Fiber Extraction Device

40-80 % GC DB17 MS 30mX.25mmX .15µm

EI

TSQ 700

8 OP esters

0.06-4 ng/g

Table 8 Assay of OP esters In Serum, Plasma or Whole Blood

28

No. OP Native Labeled 1 Acephate X X, D6 2 Azinphos-methyl X ─ 3 Bensulide X X, D14 4 Cadusafos* X ─ 5 Chlorethoxyfos X ─ 6 Chlorpyrifos X X, D10 7 Chlorpyrifos methyl X X, D6 8 Coumaphos X X, D10 9 Diazinon X ─

10 Dichlorvos (DDVP) X ─ 11 Dicrotophos X ─ 12 Dimethoate X X, D6 13 Disulfoton X X, D10 14 Ethion X ─ 15 Ethoprop X ─ 16 Ethyl parathion X X, D10 17 Fenamiphos X ─ 18 Fenitrothion X ─ 19 Fenthion X X, D6 20 Fonofos** X X, 13C6 21 Malathion X X, D10 22 Methamidophos X X, D6 23 Methidathion X ─ 24 Methyl parathion X ─ 25 Mevinphos* X ─ 26 Naled X ─ 27 Oxydemeton methyl X X, D6 28 Phorate X X, 13C4 29 Phosalone* X ─ 30 Phosmet X X, D6 31 Phostebupirim X ─ 32 Pirimiphos methyl X ─ 33 Profenofos X ─ 34 Propetamphos X ─ 35 Sulfotepp X X, D20 36 Temephos X ─ 37 Terbufos X X, 13C4 38 Tetrachlorvinphos X ─ 39 Tribufos (DEF) X ─ 40 Trichlorfon X ─

Table 9 Available Native OP pesticides and their Labeled Reference Compounds [32] Abbreviations: X: compound is available; ─: compound is not available, Dn: n number of Hydrogen Atoms Replaced with Deuterium (Labeled Compound); 13Cn n number of carbons replaced with 13C isotope.(Labeled Compound)

29

METHOD DEVELOPMENT

General development strategy

Our goal was as stated in the introduction, to develop a method to detect and measure

trace concentration of the EPA approved OP pesticides. The difficulty of the project lies

in the fact that these compounds encompass a very wide range of concentrations many of

them are labile and all of them are subject of enzymatic degradation while in the matrix.

Possible matrices – other than whole blood – are serum and plasma.

We choose serum as matrix of preference, mainly because of easier cleanup and

availability.

The development process started with determining the correct mass spectrometric

detection method. Detection has to be developed in conjunction with the separation

techniques applied. Analytical separation techniques such as choice of solvents, effect

decisions made at the detection part of the project, at the same time one needs a detection

method to develop any analytical separation.

• First, GC-MS was evaluated electron impact ionization was compared with

chemical ionization.

Chemical ionization positive polarization was chosen.

• Tandem mass spectrometry: MS-MS fragmentation was optimized and ions

chosen

30

• LC/MS method was chosen (APCI positive mode) then MS-MS transitions were

selected , parameters optimized.

• HPLC method was finalized

Sample Cleanup section

Cleanup methods evaluated:

• SPE was not considered because previous experience and the range of compounds

involved with varying physical and chemical properties.

• Freeze drying followed with solvent extraction was tried – not chosen

• Liquid-liquid extraction on solid carrier (ChemElut) was tested with better results

• Based on the promising results of the ChemElut method, accelerated solvent

extraction (ASE)was tried and proved very successful.

• ASE was optimized proved to be successful

• A “normal phase chromatography” like additional cleanup step was developed

using the “Rapid Trace” automated SPE instrument. It is to further purify the

serum extract and make it usable. In a GC application

• As an alternative to the second step cleanup headspace solid phase

microextraction

• Checked for accuracy and precision

• Applied the method to a study of farm workers, potentially exposed to pesticides

31

CHAPTER 2: GAS CHROMATOGRAPHY AND TANDEM MASS

SPECTROMETRY

First step in the development process was to establish a method of detection: without

detection one can not study separations. In our laboratory the most sensitive detectors are

the mass spectrometers. The available instruments were GC coupled TSQ-7000 triple

quadropole mass spectrometers and the newer LC coupled TSQ-Quantum Ultras, also of

the triple quadropole type. We also have a MAT-95 high resolution sector instrument, but

decided against using it mainly because of the limited availability and reliability issues at

the time.

The initial task was to determine the preferred ionization form for both: liquid

chromatography (LC) and gas chromatography (GC) coupled systems. The selection

process started with the GC-MS-MS system. After initial experiments it became apparent

that chemical ionization – being a soft ionization mode – yield a higher proportion of

molecular ions than electron impact ionization. For that reason chemical ionization (CI)

was chosen. Later in this chapter actual limits of detections will be compared and the

choice of CI quantitatively justified.

Process to optimize mass spectrometric parameters for GC-MS-MS systems.

When deciding on a detection method for a group of compounds, first the followings

have to be decided:

32

1. Type of ionization: The main modes are electron impact ionization(EI) and

chemical ionization.(CI) The polarity of ionization is always positive in EI, in

CI however it can be either positive or negative.

2. Ion selection: Two transitions have to be selected: one for quantitation, one for

confirmation.

3. Once the transitions are selected, the collision energy needs to be optimized.

Some of the parameters were not optimized but used at or close to their default value.

CI EI

Filament Current 300 mA 1300 mA

Source temperature 150°C 180°C

Reagent Gas Methane @ 1500 mT

Table 10 Non optimized MS parameters

Increasing filament current may increase signal strength but greatly decreases service

life. Elevating source temperature will increase reaction rate, but also cause

decomposition of thermally labile molecules. Lower source temperatures also cause

increased fouling of the CI source and accelerate signal loss.

Determining Ionization type and ion selection

First individual solutions of the analytes were prepared at 2 µg/mL concentration in

toluene. Based on prior literature, [18, 21-24] and availability at the time, W&J-s DB-5

MS column was selected for initial testing. The column is 30m in length, has 0.25mm

internal diameter and 25µm coating thickness. 1 µL of the pure analyte solution was

injected and the developing chromatogram observed. Once the analyte peak was detected,

33

retention time recorded and one of more dominant ion selected. Preferably one of the ions

of interest a molecular ion, but OP pesticides being often labile, it is not always possible.

After one or more potential parent ion was selected, a second injection was made. This

time the MS was set to product ion scan: quadropole one selects the parent ion,

quadropole two fragments the parent ion and quadropole three was scanning the

fragmentation products. Observing the product ions of each parent ion, two or three

promising transition was selected. For each of these transitions (parent ion -> product

ion) a selected reaction monitoring (SRM) table was set up with different collision

energies. Collision energy is the voltage difference between the last lens before

quadropole 2 (Lens 2-3) and the first lens after quadropole two (Lens 3-1). This is the

potential difference, that accelerate the parent ion (select in quadropole one) down on

quadropole two. Quadropole two is filled wit low pressure ( ~2 mTorr) argon. The parent

ions hitting the argon atoms break up, producing the product ions. At a set argon

(collision gas) pressure, the pattern of fragmentation and the amount of product ions

depend on the collision energy. For each transition of interest, the collision energy had to

be optimized. The before mentioned SRM table is the instrument of collision energy

optimization. A third injection was made in SRM mode, covering all transitions and

collision energies of interest. After the GC-MS-MS run, for each transition and collision

energy setting the chromatographic curve was plotted and the respective peaks integrated.

For each transition the area count – collision energy curve was plotted and the optimum

collision energy determined. This process is shown in Figure 9-Figure 12.

34

This process was repeated for each analyte in positive chemical ionization. For

electron impact and negative chemical ionization, only the full scan (parent ion selection)

and the product ion scan were performed.

0

50

100

150

200

250

300

350

0 2 4 6 8 10 12

Time [min]

Tem

pera

ture

[°C]

Figure 9 Simple temperature gradient to determine GC properties of OP compounds

35

F:\OldGCWork\041229_07 12/29/2004 12:14:38 PM Chlorpyrifos MeOP 1:10 dil stock Sol in ACN in ACN Q1 fs POS CIRT: 0.00 - 13.02

0 1 2 3 4 5 6 7 8 9 10 11 12 13Time (min)

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e Ab

unda

nce

8.97

3.15 10.043.63 10.809.373.91 7.864.75 11.20 12.27 12.6511.927.666.115.47 6.87 8.38

NL:8.90E8TIC MS 041229_07

041229_07 #686 RT: 8.97 AV: 1 NL: 1.82E8T: + c CI Q1MS [50.00-500.00]

50 100 150 200 250 300 350 400 450m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e Ab

unda

nce

323.69

325.67

349.73285.67

287.68124.66353.71289.66 327.67

365.72293.6292.70 110.69 142.6178.72 447.69196.62 213.65181.6262.70 228.62 269.67 431.64395.82 490.77464.76

Cl

Cl

Cl

N

MeO P

OMe

S

OFull Scan(quad 1)

Product Ion Scan

Cl

Cl

Cl37

N

P+

OMe

S

O

F:\NewGC\060630\060816_msmsFS08 8/21/2006 5:19:53 PM Chlorpyrifps MethylChlorpyrifps Methyl, 2ppm in toluene msmsFSRT: 0.00 - 13.01

0 1 2 3 4 5 6 7 8 9 10 11 12 13Time (min)

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e Ab

unda

nce

9.38

8.678.13 9.999.316.58 12.797.817.27 10.35 11.106.125.29 11.63 12.325.415.12

NL:2.91E6TIC MS 060816_msmsFS08

060816_msmsFS08 #1106-1108 RT: 9.38-9.39 AV: 3 SB: 43 9.28-9.35 , 9.43-9.52 NL: 8.03E5T: + c CI ms2 [email protected] [50.00-400.00]

60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e Ab

unda

nce

291.90

125.00

213.94289.90109.24 211.91

324.0679.38 180.19 277.17192.60126.97102.68 239.61143.0262.38 112.78 162.09 292.61

MeOP

+

OMe

S

Figure 10 Transition selection for chlorpyrifos methyl

36

RT: 0.00 - 13.01

0 1 2 3 4 5 6 7 8 9 10 11 12 13Time (min)

0

50

1000

50

1000

50

1000

50

1000

50

100

Rel

ativ

e A

bund

ance 0

50

1000

50

1000

50

100AA: 1317554

AA: 1897489

AA: 1704100

AA: 1176298

AA: 594232

AA: 253427

AA: 82282

AA: 29149

NL: 1.01E6TIC F: + c CI SRM ms2 [email protected] [289.70-290.30] MS ICIS 060816_msmsFS09








Figure 11 Area counts for clorpyrofos methyl 322 -> 290 transition at different collision energies

Figure 12 Otimization of collision energies for clorpyrofos methyl 322 -> 290 transition

0

500000

1000000

1500000

2000000

2500000

10 15 20 25 30

Collision Energy [V]

Are

a C

ount

37

4 5 6 7 8 9 10 11 12 13

Oxydemeton methylDichlorvos (DDVP)

NaledMethamidophos

MevinphosAcephate

ChlorethoxyfosEthoprop

DicrotophosSulfotepp

CadusafosPhosmetPhorate

DimethoatePropetamphos

TerbufosDiazinon

Fonofos**Disulfoton

PhostebupirimChlorpyrifos methyl

Methyl parathionPirimiphos methyl

FenitrothionMalathionFenthion

ChlorpyrifosEthyl parathion

MethidathionTetrachlorvinphos

FenamiphosTrichlorfonProfenofos

Tribufos (DEF)Ethion

PhosaloneCoumaphos

Figure 13 Retention times for OP compounds on 30m .25mm X 25µm DB-5 MS column, linear temperature gradient.

38

Chapter 2 Dicussion

When developing the GC-MS-MS part, there were two questions to consider.

The first one is the selection of ionization mode. Using MS and MS-MS, the available

ionization modes are EI, CI positive and negative polarity. Most of the available literature

uses EI. [8]-[14]. The reason for this is probably twofold: EI is more economical in the

sense that it produces multiple fragments in the ion source. As a result a compound can

be identified from the main ions or fragments and their ratios. One does not need tandem

mass spectrometer to produce fragments. The second reason is probably more practical:

EI ions form in a partially open ion source. It remains clean longer than CI sources. CI

sources or “ion volumes” are closed to the outside, save the few holes needed for material

transport. Inside is the end of the GC column, and the reagent gas – most often methane –

bombarded by the electron beam. As a result, contaminants from the sample leaving the

GC column and their decomposition products tend to coat the inside of the ion volume.

Once the inside of the source becomes less conductive it also less effective, resulting in

loss of sensitivity.

Chemical ionization on the other hand has the advantage of the more controlled ion

formation. To maximize sensitivity, our focus was to generate as much as possible

molecular ion or a close fragment where unavoidable. Tandem mass spectrometers were

available, these ions could be fragmented at a later stage.

Using CI the choice is between positive and negative ionization. All things being equal,

analyzing biological samples, negative ionization sometimes has an advantage. It is easier

to ionize a compound in positive mode because it is easier to add a charge – usually a

proton – to a molecule. In negative ionization, the removable proton (removable proton)

39

has to be there the first place. So if the molecule of interest ionizes in negative mode, the

background is likely to be lower.

OP pesticides however pose a particular problem: they tend to break apart in negative

ionization. When this happens, often the non-specific part (phosphate-methyl, ethyl ester,

thio-ester) is dominant.

These issues can be demonstrated on the example of malathion, a very common

pesticide. Figure 15 shows the many fragments produced by EI. CI, positive ionization

(Figure 16 ) produces mainly the molecular ion (331), an adduct (C2H5+) and one more

ion which created with a loss of an ethoxy. Negative chemical ionization chiefly produces

the ion with the m/z of 157. As it shown on Figure 17, it is a very non specific fragment.

In a crowded separation it may come from many different compounds, such as Fonofos

and Dimethoate. For this reason, CI positive was chosen as preferred ionization mode.

For gas chromatography portion of the analytical separations part, most authors used

either DB-5 / DB-5MS (5% phenyl, methyl polysiloxane) type columns or HP1701

((14%-Cyanopropyl-phenyl)-methylpolysiloxane). (Figure 14)

nm CH3CH3

CH3

OSi O Si

nm CH2CH3

CH3

OSi O Si

C N3

Figure 14 Polymer structure of phenyl-methyl (DB-5 MS) and cyanopropyl phenyl (HP1701) stationary phases

40

OEt

MeO P

OMe

S

S CHCO

OEt

CH2 CO

MeO P

OMe

S

S CHCO

OEt

CH2 C+

O

OEt

CH2

CO

OEt

CH+

CO

m/z

285

m/z

173

OH

CH2

CO

OEt

CH+

CO

MeO P

OMe

SH2+

MeO P+

OMe

S

m/z

127

m/z

145

m/z

125

Figure 15 Major fragments of malathion in EI mode

41

OEt

MeO P

OMe

S

S CHCO

OEt

CH2 CO

MeO P

S

SH+

MeO P

OMe

SH+

S CHCO

OEt

CH2 CHO

OEt

MeO P

OMe

SH+

S CHCO

OEt

CH2 CO

OEt

MeO P

OMe

S

S CHCO

OEt

CH2 CO

CH2+

CH3

m/z

359

m/z

331

m/z

285m/z

127

Figure 16 Major fragments of malathion in CI positive mode

OEt

MeO P

OMe

S

S CHCO

OEt

CH2 CO

MeO P

OMe

S

S-

m/z

157

Figure 17 Major fragments of malathion in CI negative mode

42

When looking for correlations in the GC section, the first approach is to plot retention

time as a function of published[33] vapor pressure data. Since vapor pressure covers a

wide range of values, logarithmic scale is useful. Figure 18 shows these vapor pressure

values. It is tempting to find two groups, represented by lines A and B, but examining

line B and the compounds it corresponds to, there is no structural commonality. (Table 11

)

y = -0.4671Ln(x) + 9.414

0.00

2.00

4.00

6.00

8.00

10.00

12.00

14.00

0.001 0.01 0.1 1 10 100 1000 10000

Vapor Pressure [mPa]

Ret

entio

n Ti

me

[min

]

Acephate

Dimethoate

Methyl Parathion

Methidathion

TrichlorfonMethidathion

A

B

Figure 18 GC retention times as the function of vapor pressure data (logarithmic scale)

43

H

Cl

ClC

MeO P

OMe

O

O C

Cl

Cl

1 Tetrachlorvinphos

CH2MeO P

OMe

S

S

O

O

N

2 Phosmet

MeO P

SMe

O

NH C

CH3

O 3 Acephate

MeO P

SMe

O

NH2

4 Methamidophos

CH3MeO P

OMe

O

S CH2 CH2 CH2S

O

5 Oxydemeton methyl

Table 11 Compounds of line B (GC)

Next step is to look at the outliers at an area where the vapor pressure is the same but

the retention time is markedly different. Such is the boxed area on Figure 18. The two

most extreme outliers are acephate and methamidophos. Both are very water soluble, so it

is useful to factor in hydrophilic – hydrophobic properties in the investigation. Kow

values, describe a compound hydrophobicity.

Next, a retention factor “R” was proposed to predict an OP retention time (DB-5

column, linear temperature gradient) along with the equation R = log(Kow) - a ∗ log(P).

The correlation factor for Retention time –R factor arrays was calculated for several “a”

parameters and charted. A=1.5 was approximated to be the optimum. (Figure 19)

44

0.5

0.55

0.6

0.65

0.7

0.75

0.8

0.85

0.9

0 1 2 3 4

"a" parameter

corre

latio

n fa

ctor

Figure 19 Correlation factor analysis

Calculating the R value for an OP using the R = log(Kow) - a ∗ log(P )equation, the

retention time of an OP may be easily predicted.

45

TR = 0.55R + 7.4

4.00

5.00

6.00

7.00

8.00

9.00

10.00

11.00

12.00

13.00

-4 -2 0 2 4 6 8

"R" factor

Ret

entio

n tim

e [m

in]

R = log(Kow) - 1.5 x log(P)

Figure 20 Predicting OP retention times in GC

46

TR = 0.61R + 7.3

4.00

5.00

6.00

7.00

8.00

9.00

10.00

11.00

12.00

13.00

-4 -2 0 2 4 6 8

"R" factor

Ret

entio

n tim

e [m

in]

R = log(Kow) - 1.5 x log(P)

Figure 21 Predicting OP retention times in GC – outliers (Oxydemethon methyl, dicrotophos, tetrachlorvinphos, trichlorfon, all LC group members) taken out:

47

Retention Time

[min] predicted actual Cadusafos 7.8 8.0 Chlorethoxyfos 8.2 7.6 Chlorpyrofos Me 9.8 9.4 Diazinon 8.3 8.5 Disulfoton 8.9 8.6 Et Parathion 9.7 9.4 Fenamiphos 10.2 10.1 Fenitrothion 8.2 9.2 Fenthion 10.4 9.4 Fonofos 8.4 8.5 Malathion 8.3 9.3 Me Parathion 9.8 9.0 Mevinphos 6.3 6.6 Naled 5.9 5.5 Phorate 7.9 8.0 Phosalone 10.9 11.7 Profenofos 10.8 10.2 Sulfotepp 8.7 7.9 Terbufos 7.6 8.4

Table 12 GC predicted and actual retention times

48

CHAPTER 2 APPENDIX

Appendix A CI Positive, negative and EI full scans

These MS full scan experiments were necessary for the development of the GC-MS-

MS par t of the method. Unlike the LC –MS portion, few automated tools are available.

Acetonitrile based stock solution was diluted with toluene to produce 1 µg/mL samples.2

µL of these were injected on DB-5 MS column, using a linear temperature gradient.

Source conditions were the recommended starting values:

Source Temperature

[°C]

Reagent Gas Pressure [mTorr]

Filament Current

[µA]

Electron Energy

[eV]

CI positive 150 1500 300 -200

CI negative 150 1500 300 -200

EI 150 N/A 1300 -70

49

041229_01 #446-458 RT: 6.89-6.99 AV: 13 SB: 177 4.83-5.59 , 6.28-7.02 NL: 4.82E6T: + c CI Q1MS [50.00-500.00]

50 100 150 200 250 300 350 400 450m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Relative Abu

ndan

ce

142.66

183.70

135.70

170.71124.7360.80 211.81 282.95242.89 406.87330.97 374.30 431.26 492.95

041229N_01 #440-458 RT: 6.82-6.98 AV: 19 SB: 177 4.84-5.59 , 6.27-7.00 NL: 5.85E4T: - c CI Q1MS [50.00-500.00]

50 100 150 200 250 300 350 400 450m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Relative Abu

ndan

ce

167.84

146.88218.03 287.94105.05 421.9378.80 352.09 479.68251.18 391.20

041227_01 #328-357 RT: 5.84-6.09 AV: 30 SB: 177 4.81-5.57 , 6.24-6.97 NL: 2.89E5T: + c EI Q1MS [50.00-500.00]

50 100 150 200 250 300 350 400 450m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Relative Abu

ndan

ce

141.75

126.68

300.87 428.93110.95 341.85210.41 263.9661.76 369.15176.23 463.62 493.66

Figure 22 Acephate - Positive CI, Negative CI, EI spectra

50

041229_02 #356 RT: 6.11 AV: 1 SB: 26 5.95-6.08 , 6.24-6.32 NL: 1.45E5T: + c CI Q1MS [50.00-500.00]

50 100 150 200 250 300 350 400 450m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100Relative Abu

ndan

ce332.81

316.78

360.96

55.80

94.79 174.80 373.01123.92 211.50 293.05 425.50248.98 483.36

N/F


50 100 150 200 250 300 350 400 450m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Relative Abu

ndan

ce

316.82

331.96

192.78268.93136.90 345.82165.95 218.1990.86 405.03 488.31431.02366.00

Figure 23 Azinphos Methyl - Positive CI, Negative CI, EI spectra

51

041229_04 #577-579 RT: 8.04-8.06 AV: 3 NL: 2.79E6T: + c CI Q1MS [50.00-500.00]

50 100 150 200 250 300 350 400 450m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Relative Abu

ndan

ce

270.83

214.76

56.81242.79

180.74

158.6592.85 298.89310.89

124.75391.09357.02 414.04 470.87

041229N_04 #576-579 RT: 7.96-7.98 AV: 4 NL: 2.02E7T: - c CI Q1MS [50.00-500.00]

50 100 150 200 250 300 350 400 450m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Relative Abu

ndan

ce

212.95

126.74 180.9294.72 240.9962.75 427.02285.83 390.13366.89 446.98313.10 498


50 100 150 200 250 300 350 400 450m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Relative Abund

ance

158.70

269.86

126.7287.78

212.8096.7056.85

181.7960.78

236.88 363.00 452.01338.96278.91 419.60 4

Figure 24 Cadusafos - Positive CI, Negative CI, EI spectra

52

041229_05 #530-533 RT: 7.62-7.65 AV: 4 NL: 1.19E8T: + c CI Q1MS [50.00-500.00]

50 100 150 200 250 300 350 400 450m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Relative Abundance

152.69

336.67298.67

364.69264.67

170.71 376.7096.63 488.73142.65 198.75 236.6464.78 416.75 446.67


50 100 150 200 250 300 350 400 450m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Relative Abundance

269.80

306.80

168.86

69.69197.82 234.79

71.79

158.8073.78 370.84205.80126.74 336.86 420.96 471.63


50 100 150 200 250 300 350 400 450m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Relative Abu

ndan

ce

152.73

96.67

298.71

124.71

165.60

262.73272.65244.62

335.6664.84206.65

488.88370.29 420.76 446.44

Figure 25 Chlorethoxyfos - Positive CI, Negative CI, EI spectra

53


50 100 150 200 250 300 350 400 450m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Relative Abu

ndan

ce

349.71

313.71

379.76152.69

389.76197.63287.6696.63 257.62124.67 170.6464.76 437.69 469.85


50 100 150 200 250 300 350 400 450m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Relative Abu

ndan

ce

312.82

211.81

168.85

94.70285.79

139.8278.73 192.80 248.84 385.89350.92 472.87408.80


50 100 150 200 250 300 350 400 450m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Relative Abu

ndan

ce

313.74196.66

257.6696.67

285.70

207.71

212.69124.70

348.70243.67168.66

64.85160.6892.74

429.91400.91 463.88 494.02

Figure 26 Chlorpirifos - Positive CI, Negative CI, EI spectra

54


50 100 150 200 250 300 350 400 450m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Relative Abu

ndan

ce

323.69

349.73

285.67

124.66

363.72

92.71 142.6278.72 447.69196.62 228.63 269.67 395.83 490.76


50 100 150 200 250 300 350 400 450m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Relative Abu

ndan

ce

211.81

140.73

284.7894.77

270.8278.81320.86192.8096.75 248.8770.70 355.80 469.78391.08 447.68


50 100 150 200 250 300 350 400 450m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Relative Abu

ndan

ce

285.71

124.70

322.6778.74196.67

167.63 270.68227.69 375.27 447.78418.41 470.23

Figure 27 Chlorpyrofos Methyl- Positive CI, Negative CI, EI spectra

55


50 100 150 200 250 300 350 400 450m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Relative Abu

ndance

362.80

390.85

402.85328.81206.74 225.71138.72108.68 280.7864.79 180.73 429.10 450.46 493.16

041229N_08 #1110 RT: 12.49 AV: 1 SB: 24 9.00-9.09 , 9.36-9.46 NL: 2.23E7T: - c CI Q1MS [50.00-500.00]

50 100 150 200 250 300 350 400 450m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Relative Abu

ndan

ce

361.88

224.79

168.86328.02190.90126.75 297.97 377.06239.77 495.28451.7194.63 408.2776.69

041227_08 #1108 RT: 12.47 AV: 1 SB: 47 12.03-12.26 , 12.61-12.76 NL: 1.10E6T: + c EI Q1MS [50.00-500.00]

50 100 150 200 250 300 350 400 450m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Relative Abu

ndan

ce

361.79

225.76

209.78

108.83

96.78 333.76124.85 305.74

136.86 181.8188.85 288.73240.97394.07 432.33 463.12 495.9

Figure 28 Coumaphos - Positive CI, Negative CI, EI spectra

56


50 100 150 200 250 300 350 400 450m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Relative Abu

ndan

ce

304.83

332.93

258.79344.91

288.83178.80

151.78 247.76198.7692.72 456.96350.85 424.98 485.05


50 100 150 200 250 300 350 400 450m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Relative Abu

ndan

ce

168.84

126.73 275.01177.9794.75 303.09242.99 338.99 473.1278.65 362.01 451.90416.79


50 100 150 200 250 300 350 400 450m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Relative Abu

ndan

ce

303.86

178.88

136.79

151.82 198.80

275.82226.85

92.7796.67

65.79

428.92354.87 376.77 475.02

Figure 29 Diazinon - Positive CI, Negative CI, EI spectra

57


50 100 150 200 250 300 350 400 450m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Relative Abu

ndan

ce

220.69

184.66108.67

248.72126.68 260.72 442.6978.71 328.71150.70 348.76 406.90 463.25


50 100 150 200 250 300 350 400 450m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Relative Abu

ndan

ce

124.85

133.72

169.81

78.79 204.85147.75110.70 390.28262.09 437.28358.01285.95


50 100 150 200 250 300 350 400 450m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Relative Abu

ndan

ce

108.73

184.71

219.74144.7278.86

112.6792.72173.66 328.85234.85 266.79 384.13 437.15 478.00

Figure 30 Dichlorvos - Positive CI, Negative CI, EI spectra

58


50 100 150 200 250 300 350 400 450m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100Relative Abu

ndan

ce237.79

192.71111.75

265.84

277.84139.79

474.9771.77 154.71 348.90197.73 429.89308.86 391.99 479.32


50 100 150 200 250 300 350 400 450m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Relative Abu

ndan

ce

124.84

177.89210.71146.8078.73 251.91 360.10305.10 430.29390.30 459.47 484.1


50 100 150 200 250 300 350 400 450m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Relative Abu

ndan

ce

126.74

236.85192.78

66.80

110.8082.83 163.78 219.89 418.85268.27 326.78 450.12 478.90360.90

Figure 31 Dichrotophos - Positive CI, Negative CI, EI spectra

59


50 100 150 200 250 300 350 400 450m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100Relative Abu

ndan

ce198.65

229.72

257.7687.71

86.71269.76142.63115.71 170.6673.78 458.81316.80 426.75399.77353.29 479.33


50 100 150 200 250 300 350 400 450m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Relative Abundance

156.72

126.74 385.93196.87102.72 355.88215.90 263.03 286.82 447.08 467.18


50 100 150 200 250 300 350 400 450m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Relative Abu

ndan

ce

86.77

124.71

92.74

228.77

141.6757.91

156.71197.76

263.70 328.08 405.09 451.03348.51 487.90

Figure 32 Dimethoate - Positive CI, Negative CI, EI spectra

60


50 100 150 200 250 300 350 400 450m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Relative Abu

ndan

ce

88.74

362.82

87.73 212.72 273.75184.6589.75

228.70152.68244.7096.64 124.6560.75 458.80 486.85426.80314.81 394.87


50 100 150 200 250 300 350 400 450m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Relative Abu

ndan

ce

184.76

126.73 206.94 244.89155.8294.7378.73 425.83375.90271.88 327.70 490.69449.54


50 100 150 200 250 300 350 400 450m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Relative Abundance

87.77

273.78

88.78

185.72

141.6996.72

59.80124.70

244.75211.78

311.97 408.18361.29 429.86 461.51

Figure 33 Disulfoton - Positive CI, Negative CI, EI spectra

61


50 100 150 200 250 300 350 400 450m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100Relative Abu

ndan

ce198.69

384.78

230.70

338.74152.69

412.81170.65120.7096.65 366.81260.73 292.71 458.82 478.92


50 100 150 200 250 300 350 400 450m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Relative Abu

ndance

184.77

126.73 354.89152.86 308.94 425.9194.78 198.91 229.8378.77 285.88 397.79 450.02 479.93


50 100 150 200 250 300 350 400 450m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Relative Abu

ndan

ce

230.75

383.77

152.73

96.67 124.70

198.7464.85260.76 337.78292.73 404.84 449.97 480.72

Figure 34 Ethion - Positive CI, Negative CI, EI spectra

62


50 100 150 200 250 300 350 400 450m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Relative Abu

ndan

ce

242.78

166.71

270.83

282.83 484.94

408.86196.71138.68

124.6696.64 438.86380.83334.81288.83


50 100 150 200 250 300 350 400 450m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Relative Abu

ndan

ce

198.90

126.75 166.8994.74 212.9262.69 336.98304.92247.90 399.04 453.99 480.98


50 100 150 200 250 300 350 400 450m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Relative Abu

ndan

ce

157.69

241.83199.76

125.7196.68

92.7373.79 167.76

213.80 354.85334.90 408.84280.87 441.01 464.01

Figure 35 Ethoprop - Positive CI, Negative CI, EI spectra

63


50 100 150 200 250 300 350 400 450m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Relative Abu

ndan

ce

291.78

319.83

263.73

331.83

136.71 235.70108.68 185.6664.75 427.84355.92 399.88 461.81 498.0


50 100 150 200 250 300 350 400 450m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Relative Abu

ndan

ce

290.90

153.75

168.85137.83 261.93 319.02 460.06232.00107.80 181.82 430.04383.0678.66 492.23


50 100 150 200 250 300 350 400 450m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Relative Abu

ndan

ce

290.82

108.73

96.69 136.76154.73

185.73 234.75262.77

217.7564.79

308.06 408.27336.04 451.04 480.74

Figure 36 Ethyl Parathion - Positive CI, Negative CI, EI spectra

64


50 100 150 200 250 300 350 400 450m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100Relative Abu

ndan

ce303.84

331.90

343.90287.80261.78153.72 194.79121.7279.79 452.98360.92 390.98 498.0


50 100 150 200 250 300 350 400 450m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Relative Abu

ndan

ce

126.74

288.03

165.89

302.06

147.76 273.8678.72

315.03 390.00192.80 229.83119.95 445.19367.08 423.11 480.08


50 100 150 200 250 300 350 400 450m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Relative Abu

ndan

ce

302.89

153.78

287.86

216.78

259.83194.87129.77

242.8179.85 179.82108.77 310.19 422.51374.97 488.00464.39

Figure 37 Fenamiphos - Positive CI, Negative CI, EI spectra

65


50 100 150 200 250 300 350 400 450m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100Relative Abu

ndan

ce277.73

305.80

317.80247.76

124.65 231.7592.70 169.6978.70 385.78355.86 426.87 490.72


50 100 150 200 250 300 350 400 450m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Relative Abu

ndan

ce

276.90

167.77

140.72

261.92124.83 290.98 417.98230.94182.91 317.1893.84 477.18397.9957.60


50 100 150 200 250 300 350 400 450m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Relative Abu

ndan

ce

276.79

259.80124.71

108.73

78.77213.79149.77

181.77 291.96 369.02 417.72 448.98 491.90330.38

Figure 38 Fenitrothion - Positive CI, Negative CI, EI spectra

66


50 100 150 200 250 300 350 400 450m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100Relative Abu

ndan

ce

278.74

306.80

318.80

246.72124.65 168.6992.7178.72 214.73 402.78370.82 446.82324.84 492.85


50 100 150 200 250 300 350 400 450m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Relative Abu

ndan

ce

140.83

126.74

262.95

152.88 276.8794.78 190.89 303.99229.9878.70 336.10 372.39 425.28


50 100 150 200 250 300 350 400 450m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Relative Abu

ndan

ce

277.78

124.70 168.74

152.74244.8292.7478.77

214.78 292.95 402.94369.24338.64 446.14 478.87

Figure 39 Fenthion - Positive CI, Negative CI, EI spectra

67


50 100 150 200 250 300 350 400 450m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Relative Abu

ndan

ce

278.74

306.80

318.88246.8192.8268.84 138.80 201.91 359.21 404.59 434.78 466.89


50 100 150 200 250 300 350 400 450m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Relative Abu

ndan

ce

168.77

108.74

245.95136.85 216.9191.81 354.94 393.70309.89 472.20


50 100 150 200 250 300 350 400 450m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Relative Abu

ndan

ce

245.82

108.72

136.76

173.7080.80 201.78170.68

429.03354.78 382.88281.93 326.95 450.02 491.80

Figure 40 Fonofos- Positive CI, Negative CI, EI spectra

68


50 100 150 200 250 300 350 400 450m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Relative Abu

ndan

ce

284.73

330.79

126.71

358.82

172.75

200.79 370.85298.76

158.65 256.7292.70 456.86216.8062.70 422.82 486.75384.84


50 100 150 200 250 300 350 400 450m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Relative Abu

ndan

ce

156.72

171.93126.74 242.94 314.99276.9397.81 199.95 369.80 389.9878.70 441.68 464.09


50 100 150 200 250 300 350 400 450m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Relative Abu

ndan

ce

172.83

126.78

157.70

92.74

284.7998.75255.78

210.7578.76237.73183.73 329.86 422.87358.91 449.91 496.0

Figure 41 Malathion - Positive CI, Negative CI, EI spectra

69


50 100 150 200 250 300 350 400 450m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100Relative Abu

ndan

ce

141.67

93.67

181.72124.6592.73 282.74234.76195.84 314.99 415.01350.38 435.22 471.46


50 100 150 200 250 300 350 400 450m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Relative Abu

ndan

ce

171.93

126.74287.98 344.28243.99 413.19168.00 180.8974.81 370.0090.89 452.05


50 100 150 200 250 300 350 400 450m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Relative Abu

ndan

ce

93.75

140.76

94.81

63.83

125.8078.78

300.85 428.92340.89280.93192.84146.88 404.93224.92 491.94

Figure 42 Methamidophos- Positive CI, Negative CI, EI spectra

70


50 100 150 200 250 300 350 400 450m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Relative Abu

ndan

ce

144.67

302.69

330.7384.73 446.75270.67242.71124.65 170.6357.77 210.69 342.75 386.77 426.69 472.62


50 100 150 200 250 300 350 400 450m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Relative Abu

ndan

ce

144.67

302.69

330.7384.73 446.75270.67242.71124.65 170.6357.77 210.69 342.75 386.77 426.69 472.62


50 100 150 200 250 300 350 400 450m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Relative Abu

ndan

ce

144.75

84.79

301.76124.71

57.83156.6994.68 206.85 270.72 314.62 340.97 388.81 465.13408.09 493.8

Figure 43 Methidathion - Positive CI, Negative CI, EI spectra

71


50 100 150 200 250 300 350 400 450m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100Relative Abu

ndan

ce263.72

291.79

303.78233.74

108.67217.72155.6878.71 371.78 496.8341.81 398.81 462.85


50 100 150 200 250 300 350 400 450m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Relative Abu

ndan

ce

262.95

153.76

140.78

247.87126.74 276.95215.89 403.97178.9393.75 345.07 452.02 476.01


50 100 150 200 250 300 350 400 450m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Relative Abu

ndan

ce

262.80

108.74

124.71

78.75245.79199.77136.75

167.76 280.93 316.74 374.96 451.13407.04 482.97

Figure 44 Methyl parathion - Positive CI, Negative CI, EI spectra

72


50 100 150 200 250 300 350 400 450m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Relative Abu

ndan

ce

192.70

224.76

126.70252.80

98.72 264.80448.89154.71 350.8094.72 416.84322.85 489.07


50 100 150 200 250 300 350 400 450m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Relative Abu

ndan

ce

124.83

177.89138.90 218.90 348.9998.7978.77 250.97 285.94 390.18 417.69 466.28


50 100 150 200 250 300 350 400 450m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Relative Abu

ndan

ce

126.75

191.78

108.73

163.7666.80 223.83

78.77 140.79234.91 374.95342.81285.36 404.99 461.20 481.09

Figure 45 Mevinphos - Positive CI, Negative CI, EI spectra

73


50 100 150 200 250 300 350 400 450m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100Relative Abu

ndan

ce78.77

80.76

250.80

204.83159.70

115.71

124.84 364.73169.79 344.72262.86 460.72218.8197.70 414.71 485.03


50 100 150 200 250 300 350 400 450m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Relative Abu

ndan

ce

108.74 144.73

300.67

188.74

78.86

156.75128.6894.74

59.86 218.78 254.68 313.06 405.98344.82 450.96 480.95


50 100 150 200 250 300 350 400 450m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Relative Abu

ndan

ce

220.67

300.59

126.68

380.55144.64

344.55154.71

108.66266.61

184.64

188.61 248.6982.67 408.57328.66 442.67 486.68

Figure 46 Naled - Positive CI, Negative CI, EI spectra

74


50 100 150 200 250 300 350 400 450m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100Relative Abu

ndan

ce

168.67

124.66196.72

142.66108.67 208.7466.78 336.79292.82259.04 391.11 479.28429.17


50 100 150 200 250 300 350 400 450m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Relative Abu

ndan

ce

63.82

126.7565.80

250.70 285.87152.6990.65 210.81 338.09 390.17


50 100 150 200 250 300 350 400 450m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Relative Abu

ndan

ce

109.74

167.76

141.74

78.77

92.84

356.11266.86 391.40196.99 324.35 452.91241.90 478.17

Figure 47 Oxydemethon methyl - Positive CI, Negative CI, EI spectra

75

041229_28 #584-586 RT: 8.02-8.03 AV: 3 NL: 1.75E8T: + c CI Q1MS [50.00-500.00]

50 100 150 200 250 300 350 400 450m/z

0

10

20

30

40

50

60

70

80

90

100Rel

ative

Abu

ndan

ce74.75

260.75

198.69

334.79

152.67214.70

230.6976.74 300.78120.70 170.64 412.80 458.8096.63 380.82288.79 320.75 477.79360.71


50 100 150 200 250 300 350 400 450m/z

0

10

20

30

40

50

60

70

80

90

100

Relative Abu

ndan

ce

184.76

126.74 192.89 230.88152.85110.7176.80 286.03 425.88324.85 390.29 451.99355.71 485.48

041227_28 #584-586 RT: 8.02-8.03 AV: 3 NL: 6.20E7T: + c EI Q1MS [50.00-500.00]

50 100 150 200 250 300 350 400 450m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e Abu

ndan

ce

74.79

259.81

120.76

230.76

96.72

261.80124.71 152.76154.78 186.75

262.81 380.93334.95292.73 412.73 462.80 478.99

Figure 48 Phorate - Positive CI, Negative CI, EI spectra

76

041229_29 #1013 RT: 11.68 AV: 1 NL: 4.02E7T: + c CI Q1MS [50.00-500.00]

50 100 150 200 250 300 350 400 450m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ative

Abu

ndan

ce

367.77

181.69

369.76

183.68

366.78321.74 395.79198.70

152.68

209.73 370.78 397.80323.74371.76211.74169.67 307.75 409.79120.7174.85 221.74 339.6896.66 137.69 372.78280.80268.78 441.77 487.84453.05

041229N_29 #1015 RT: 11.68 AV: 1 NL: 5.98E7T: - c CI Q1MS [50.00-500.00]

50 100 150 200 250 300 350 400 450m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Relative Abu

ndan

ce

184.77

186.74

187.76 366.97184.09 302.94126.73 213.91152.80 401.99337.94110.6878.62 432.59242.09 457.00259.00

041227_29 #1014-1017 RT: 11.67-11.69 AV: 4 NL: 3.07E6T: + c EI Q1MS [50.00-500.00]

50 100 150 200 250 300 350 400 450m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ative

Abu

ndan

ce

181.76

366.80

183.74120.77

153.76368.80

152.7596.76

198.7564.86 369.82280.83213.81 321.76252.88 340.83 404.98 428.72 464.99 489.2

Figure 49 Phosalone - Positive CI, Negative CI, EI spectra

77


50 100 150 200 250 300 350 400 450m/z

0

10

20

30

40

50

60

70

80

90

100R

elat

ive

Abu

ndan

ce159.71

317.75

161.75318.77285.73124.65 357.76162.76 201.74104.81 271.94 476.93390.90 423.35


50 100 150 200 250 300 350 400 450m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bund

ance

156.82

158.81

146.87 192.89 284.99 316.87 369.57252.7294.86 451.9875.70 388.09


50 100 150 200 250 300 350 400 450m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bund

ance

159.78

160.81 192.77132.77103.75193.80 260.08 477.54351.87 371.12 419.00325.52

Figure 50 Phosmet - Positive CI, Negative CI, EI spectra

78


50 100 150 200 250 300 350 400 450m/z

0

10

20

30

40

50

60

70

80

90

100R

elat

ive

Abu

ndan

ce318.87

276.80

304.85

319.89346.91

358.90275.77166.69 233.72151.7796.6856.80 484.96442.84414.02


50 100 150 200 250 300 350 400 450m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bund

ance

156.72

158.80

146.87 192.91 284.97 317.03238.82110.64 369.71 390.2878.98


50 100 150 200 250 300 350 400 450m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bund

ance

317.91

260.83233.80

275.84151.83

302.89136.80 318.92214.79198.80109.77 319.9156.85

321.91 359.66 422.95 442.84 475.76

Figure 51 Phostebupirim - Positive CI, Negative CI, EI spectra

79

041229_32 #718-723 RT: 9.15-9.20 AV: 6 NL: 1.44E8T: + c CI Q1MS [50.00-500.00]

50 100 150 200 250 300 350 400 450m/z

0

10

20

30

40

50

60

70

80

90

100Rel

ative

Abu

ndan

ce305.84

333.90

273.80345.88

163.78 261.77232.74124.6492.7154.79 195.81 429.86397.92359.91 468.02 484.9


50 100 150 200 250 300 350 400 450m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ative

Abu

ndan

ce

140.83

126.74

199.97 291.05146.83107.7792.82 275.08244.83 324.38 360.99 420.29 454.28378.31

041227_32 #719-723 RT: 9.16-9.19 AV: 5 NL: 3.65E7T: + c EI Q1MS [50.00-500.00]

50 100 150 200 250 300 350 400 450m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ative

Abu

ndan

ce

304.89289.88

275.84

232.82

261.85179.88

124.72

162.86243.8492.75 150.7971.86 211.82

317.93 341.04 429.89 451.04400.91 479.05

Figure 52 Pirimphos methyl - Positive CI, Negative CI, EI spectra

80

041229_33 #836-840 RT: 10.16-10.19 AV: 5 NL: 1.15E8T: + c CI Q1MS [50.00-500.00]

50 100 150 200 250 300 350 400 450m/z

0

10

20

30

40

50

60

70

80

90

100Rel

ative

Abu

ndan

ce374.71

166.69402.75

346.65294.74 414.75

332.63138.67 207.61 266.6596.63 231.6662.78 460.90421.09 498.7


50 100 150 200 250 300 350 400 450m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e Abu

ndan

ce

266.74

78.76

307.79

337.83

182.89 295.78250.89 344.87 373.92139.81 408.88116.67 452.8578.13 213.88165.84 470.81

041227_33 #836-839 RT: 10.16-10.18 AV: 4 NL: 1.16E7T: + c EI Q1MS [50.00-500.00]

50 100 150 200 250 300 350 400 450m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e Abu

ndan

ce

338.77

207.69

373.75138.74

96.68296.72268.67

124.71

166.77 308.73221.73

62.80

98.7174.82 251.69 344.71 422.29388.76 450.94 480.81

Figure 53 Profenofos - Positive CI, Negative CI, EI spectra

81

041229_34 #626-629 RT: 8.37-8.40 AV: 4 NL: 1.31E8T: + c CI Q1MS [50.00-500.00]

50 100 150 200 250 300 350 400 450m/z

0

10

20

30

40

50

60

70

80

90

100Rel

ativ

e Abu

ndan

ce221.76155.70

309.87

197.76137.70

281.82

239.76

194.67321.87

126.75 183.72418.88 436.88267.7984.72 478.95391.91347.84


50 100 150 200 250 300 350 400 450m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ative

Abu

ndan

ce

153.78

434.98193.86126.77 281.0199.79 308.98220.9783.82 264.96 407.81370.98336.94 451.81 498.8

041227_34 #625-628 RT: 8.37-8.39 AV: 4 NL: 3.33E7T: + c EI Q1MS [50.00-500.00]

50 100 150 200 250 300 350 400 450m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ative

Abu

ndan

ce

137.75

193.76 235.82

221.84

121.76 155.76

105.76

63.76 165.73280.87 324.97 369.21 408.07 451.05 479.14

Figure 54 Propetamphos - Positive CI, Negative CI, EI spectra

82

041229_35 #571-575 RT: 7.91-7.94 AV: 5 NL: 1.68E8T: + c CI Q1MS [50.00-500.00]

50 100 150 200 250 300 350 400 450m/z

0

10

20

30

40

50

60

70

80

90

100R

elat

ive

Abu

ndan

ce322.79

350.82

294.74 362.83276.71

201.68170.70 244.7396.67 120.7164.80 442.88 474.89384.79 416.78


50 100 150 200 250 300 350 400 450m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e Abu

ndan

ce

168.88292.96

94.78

126.75

146.79277.83

78.75 231.93192.95 262.92 363.78110.59 452.78390.17

041227_35 #574 RT: 7.93 AV: 1 NL: 9.59E7T: + c EI Q1MS [50.00-500.00]

50 100 150 200 250 300 350 400 450m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ative

Abu

ndan

ce

321.82

201.74

265.73237.71

173.68293.7896.67 216.76120.75

64.85 144.6980.71 353.86 384.97 475.07407.26 442.28

Figure 55 Sulfotepp - Positive CI, Negative CI, EI spectra

83


50 100 150 200 250 300 350 400 450m/z

0

10

20

30

40

50

60

70

80

90

100R

elat

ive

Abu

ndan

ce102.73

232.70 288.79

56.81 390.87

334.78186.65198.68 260.74

152.6874.74 124.65 362.82 440.82306.73 486.85408.85


50 100 150 200 250 300 350 400 450m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ative

Abu

ndan

ce

184.77

126.74 152.87 258.94110.70 192.9076.72 288.75 425.90375.89310.81 452.99357.99


50 100 150 200 250 300 350 400 450m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ative

Abu

ndan

ce

230.76

56.86

287.83

152.74102.78

185.7396.72124.70 202.71

64.81 174.6478.72 272.89 334.93 409.01385.02300.91 493.09432.84

Figure 56 Terbufos - Positive CI, Negative CI, EI spectra

84


50 100 150 200 250 300 350 400 450m/z

0

10

20

30

40

50

60

70

80

90

100Rel

ativ

e Abu

ndan

ce366.66

126.67

330.66

394.68240.61

406.68154.71108.66 206.63 268.64 492.7178.70 166.67 300.64 358.74 474.75419.07


50 100 150 200 250 300 350 400 450m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e Abu

ndan

ce

124.77

241.84221.87110.78 138.86 277.87 490.92329.91 350.8678.72 187.84 380.87 451.88418.19


50 100 150 200 250 300 350 400 450m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e Abu

ndan

ce

328.71

108.71

206.71 239.69203.6878.80 228.75 365.71156.69132.72 313.73265.82 450.81402.08 479.01

Figure 57 Tetrachlorvinphos - Positive CI, Negative CI, EI spectra

85


50 100 150 200 250 300 350 400 450m/z

0

10

20

30

40

50

60

70

80

90

100R

elat

ive

Abu

ndan

ce224.76 314.83

342.89

168.65

354.88298.82201.65 257.74144.7856.81

270.76102.7288.73 482.88176.74 370.92 416.94 450.91


50 100 150 200 250 300 350 400 450m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e Abu

ndan

ce

256.90

224.97167.82135.83 270.98110.68 200.8278.73 392.98293.84 425.05336.79 451.90 470.68361.09


50 100 150 200 250 300 350 400 450m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ative

Abu

ndan

ce

168.72

201.73

56.86

313.88

257.82

225.84146.66

112.64

136.7588.79

86.77

57.89 176.81 284.92 422.20376.90326.87 448.92 475.16

Figure 58 Tribufos - Positive CI, Negative CI, EI spectra

86


50 100 150 200 250 300 350 400 450m/z

0

10

20

30

40

50

60

70

80

90

100R

elat

ive

Abu

ndan

ce314.83

224.76

312.83 342.89225.77168.66

298.82 354.8856.81 257.74201.66144.79102.71 391.03 437.87 483.05


50 100 150 200 250 300 350 400 450m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bund

ance

256.87

258.97

259.86224.97167.85126.7678.76 401.49336.69 451.79


50 100 150 200 250 300 350 400 450m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bund

ance

168.74201.74

56.86

313.89146.66 257.82225.85

112.65

88.8986.76

259.97 314.9157.85422.48 452.09356.20 474.90

Figure 59 Trichlorfon - Positive CI, Negative CI, EI spectra

87

CHAPTER 3: HIGH PERFORMANCE LIQUID CHROMATOGRAPHY – MASS SPECTROMETRY

Many of the analytes are thermally labile, highly polar or otherwise unsuitable for GC

separations. Examples are methamidophos, acephate, and azinphos methyl. These

compounds are the candidates for HPLC separations and form the core of the LC group

of compounds. Other analytes were added because after preliminary experiments it was

determined that they likely have a better LOD in an LC-MS-MS method.

In the HPLC-MS-MS system two ionization modes were available: electrospray

ionization (ESI) and atmospheric pressure chemical ionization (APCI), positive or

negative polarity for each. The feasibility of ionization modes and polarizations were

tested by injecting concentrated (approximately 5 μg/mL samples into a rapid HPLC

eluent gradient and observing full scan spectra, using only a single quadropole of the

mass spectrometer. This process was repeated for all four combinations of ionization

modes in both polarities. All forty of the analytes were tested, because at this point it was

unclear weather or not all OP pesticides can be analyzed solely on a HPLC-MS-MS

system.

As it is apparent by looking at the ionization tables (Table 13) that more compounds

ionize in APCI mode than in ESI mode. The reason is that ESI ionization takes place in

the solution phase, while the APCI ionization happens in the plasma surrounding the

charged needle. When one has to choose between positive and negative ionization, -

especially when studying biological, dirty samples – negative ionization has an

advantage. If the analyte of interest is ionizing in the negative mode, the background is

often lower. The reason is that in the majority of cases positive ionization requires

88

addition of a proton, while negative ionization needs the removal of one. It is easier to

add a proton from some external source than find an already existing proton to lose.

Mono Ions (m/z), nominal amu isotopic ESI APCI No. OP Mass Positive Negative Positive Negative

1 Acephate 183.012 184 182 184 168 2 Azinphos-methyl 317.006 318 318 157 3 Bensulide 397.061 398 396 442 356, 398 213 4 Cadusafos* 270.088 271, 541 426 271, 541 213

5 Chlorethoxyfos 333.892 305, 307, 309

6 Chlorpyrifos 348.926 350 313, 315, 317

7 Chlorpyrifos

methyl 320.895 115, 143, 306 308 321 212, 214 216

8 Coumaphos 362.014 363 363 225, 208, 333

9 Diazinon 304.101 305 169 215 305 169, 215

10 Dichlorvos

(DDVP) 219.946 262 264 205 207 262, 253 205, 207 11 Dicrotophos 237.077 475 223 291 709 270, 283 171 12 Dimethoate 230.000 230, 459 274 230 157 13 Disulfoton 274.028 305 185 363 185 14 Ethion 383.988 385 385 185 15 Ethoprop 242.560 485 213 243, 485 199

16 Ethyl parathion 291.033 173, 243, 305 262 262, 154, 138

17 Fenamiphos 271.134 304, 363, 607 247

18 Fenitrothion 277.017 305 115 262 383 124, 165,

248, 289 168 19 Fenthion 278.020 342 263 279, 311, 320 263 20 Fonofos** 246.030 247, 173 137 247 169 21 Malathion 330.036 331 315 331 157

Table 13 Table of LC-MS ions in APCI and ESI mode, positive or negative ionization Bolded ions ara the most aboundant (Cont. next page)

89

Mono Ion Masses isotopic ESI APCI No. OP Mass Positive Negative Positive Negative

22 Methamidophos 141.001 142, 283 141 187 283, 174, 142 23 Methidathion 301.962 303 366 248 287 303 157

24 Methyl parathion 263.002 115 277 138 248

325 151, 234, 275 248, 154

25 Mevinphos* 224.045 225 449 209 255 225, 257 171, 125, 237

26 Naled 377.783 420 422 424 363 365 367 411, 413, 426 363, 365, 367

27 Oxydemeton

methyl 246.015 247 231 247 141 28 Phorate 260.013 335 115 143 185 231 355 261, 335 185 29 Phosalone* 366.986 368 167 368 185 30 Phosmet 316.995 318 157 31 Phostebupirim 318.117 319 319 275

32 Pirimiphos methyl 305.096 318, 381 290 306 141, 290 187

33 Profenofos 371.935 306 313 315, 317

343 345 347 373 405, 407 343, 345, 347

34 Propetamphos 281.085 282 282…327 154, 280, 200

35 Sulfotepp 322.023 323 169, 293, 155

36 Temephos 465.990 451 467 451, 141 37 Terbufos 288.044 115 143 185 245, 289 185

38 Tetrachlorvinphos 363.899365

406,408,410 349 351

353… 373 365,

397..399… 171, 125 39 Tribufos (DEF) 314.096 315 315, 347, 629 257

40 Trichlorfon 255.923 257… 515 301 303 305143, 152,

221, 257, 289

Table 13 Table of LC-MS ions in ESI and APCI mode, positive or negative Bolded ions ara the most aboundant (Weak) ionization (cont. from previous page)

90

Liquid Chromatography: HPLC

After deciding an ionization mode the next step is to develop the analytical separation

part of the method

In the literature the commonly used column type for the analytical separation of OP

pesticides is the reversed phase C-18. [26-28] For this reason we started with variants of

C-18 columns: betasil C18, Aquasil C18. Hydrophilic – hydrophobic properties of our

compounds of interest vary widely as it was observed in the introduction. Compounds

like acephate and methamidophos are extremely water soluble, do not retain very well on

most packing. Very lipophilic, oily OP pesticides, such as Tribufos on the other hand;

have very good retention.

91

Name Manufacturer Length Diam. Particle size Carbon

Pore Size End-

[mm] [mm] [µm] Load cap

Aquasil C18 Thermo 100 4.6 5 12% 100 A Yes High purity,base deactivated C18

Betasil C8 Thermo 100 4.6 5 12% 100 A Yes High purity,base deactivated C8

Betasil C18 Thermo 100 4.6, & 2.1 5 20% 100 A Yes High purity,base deactivated C18

Betasil Phenyl Thermo 100 4.6 5 11% 100 A Yes High purity,base deactivated

Merck Chromolit RP-e

Merck Life Sciences 100 4.6 5 17% 120 A Yes

High purity silica gel C18

Discovery HS F5 Supelco 100 4.6 3 12% 120 A Yes

silica gel, high purity, spherical particle platform

Pentafluorophenyl propyl

Fluorphase RP Thermo 50 2.1 5 10% 100 A Yes High purity,base deactivated perfluorinated C6

Prism RP Thermo 50 2.1 5 12% 100 A Yes High purity,base deactivated Urea+ C12 chain

Zorbax SB C3 Agilent 100 4.6 5 4% 80 A No Diisopropyl propyl

Table 14 HPLC columns tested

92

Using the C-18 type columns , it became apparent that when applying linear – or close

to linear – elution curves, that the compounds separate into two groups: early and late

eluting compounds. Many of the experiments listed in tables 15-17 were attempts to

further resolve these two elution groups to their components. While it is not absolutely

necessary when using tandem mass spectrometer as detector, resolving peaks can help by

eliminating possible interferents and, ion competition. If many analytes elute very

closely, it can complicate the instrument method setup. Since the MS has to spend a finite

amount of time on each transition, in the presence of many overlapping peaks the number

of data points for each peak may be limited.

An other aspect of the overlapping peaks is the signal to noise ratio. The detector

works in ion counting mode, so when the time to collect ion counts is limited –

background being the same- the signal to noise ratio worsens. So even in with the

resolving power of tandem mass spectrometry, good chromatographic resolution provides

an advantage.

Selection of eluents.

Before developing the analytical separations part of the method, the detection was

decided to be APCI, positive ionization. The selection of ionization mode has a major

influence on the on the selection of solvents, sizing of the column and picking the right

flow rate. The two most often used combination of eluents are: water-methanol and

water-acetonitrile.

93

Figure 60 Viscosity profiles of Methanol - Water and Acetonitrile - Water blends, according to Thompson [34]

One of the practical differences between the two pairs is that the water - methanol

gradient has a viscosity maximum while the water – acetonitrile mixture does not.

Acetonitrile and methanol gives different retention times.

Another difference between the two solvents is the propensity to form adducts. From

previous work we saw that both solvents tend to form adducts with many of the analytes.

Polarity of ionization makes a difference: acetonitrile is more likely to form adducts in

positive ionization while methanol adducts are favored in negative ionization.

The TSQ Quantum-s APCI probe can be operated between .2 and 2 mL/min, the

recommended optimum being approximately 1mL/min. This flow rate will work well

with a 4.6 mm internal diameter column. Smaller diameter columns may be – and were –

tried, but the increased pressure leads to leaks and sometimes clogging. In this method

real life samples, with varying degree of purity have to be run reliably. The 4.6mm

internal diameter, 100mm length combined with 1.0 mL/min flow rate proved to be the

most useful combination.

94

Betasil Betasil Betasil C18 C18 Phenyl Width: 2.1mm 4.6mm 4.6mm 4.6mm No. OP Length: 100mm 100mm MeOH ACN

1 Acephate 8.1 7.94 6.15 5.62 2 Azinphos-methyl 21.83 19.35 20.53 17.11 3 Bensulide 24.2 21.4 21.72 19.08 4 Cadusafos* 25.64 22.39 21.05 17.59 5 Chlorethoxyfos 26.88 - N/F N/F E/B 6 Chlorpyrifos 27.41 23.5 22.53 28.06 E/B 7 Chlorpyrifos methyl 25.86 22.57 21.67 27.61 E/B 8 Coumaphos 24.85 21.88 22.24 19.15 9 Diazinon 24.8 21.83 21.18 17.92

10 Dichlorvos (DDVP) 19.08 16.72 15.67 N/F E/B 11 Dicrotophos 14.25 12.13 13.68 9.94 12 Dimethoate 15.71 14.29 14.16 11.68 13 Disulfoton 28.94 22.43 21.66 N/F 14 Ethion 26.97 23.19 23.1 20.58 15 Ethoprop 23.72 21.08 19.86 16.28 16 Ethyl parathion 24.46 21.62 21.09 N/F E/B 17 Fenamiphos 24.19 21.42 20.77 16.5 18 Fenitrothion 23.36 20.76 20.64 N/F E/B 19 Fenthion 24.83 21.9 21.82 N/F E/B 20 Fonofos** 24.91 21.98 21.25 18.85 21 Malathion 22.97 20.41 20.57 17.89 22 Methamidophos 4.08 6.43 3.67 3.47 23 Methidathion 21.66 19.18 19.75 16.86 24 Methyl parathion 22.46 19.98 19.66 N/F E/B 25 Mevinphos* 16.97 15.14 15.18 11.72 26 Naled 21.52 18.96 18.47 N/F 27 Oxydemeton methyl 12.79 10.67 12.34 10.22 split 28 Phorate 25.4 22.27 21.29 N/F E/B 29 Phosalone* 25.26 22.17 22.12 N/F E/B 30 Phosmet 29.03 N/F N/F N/F E/B 31 Phostebupirim 26.79 23.13 22.04 19.27 32 Pirimiphos methyl 24.72 21.71 20.69 17.56 33 Profenofos 26.4 22.86 22 N/F E/B 34 Propetamphos 23.21 20.64 19.87 17.68 35 Sulfotepp 24.56 21.67 21.31 19.11 36 Temephos 26.73 22.98 24.04 N/F E/B 37 Terbufos 26.75 23.09 22.15 N/F 38 Tetrachlorvinphos 24.28 21.51 21.03 17.53 39 Tribufos (DEF) 28.58 24.31 23.17 20.08 40 Trichlorfon 19.09 16.73 15.64 N/F E/B

Table 15 Retenetion times of OP pesticides on Betasil C18 and Phenyl Columns (N/F: not found, E/B: extreme peak broadening)

95

Aquasil C18 Chromolith RP 18e Zorbax SBC3 Width: 4.6mm 4.6mm 4.6mm No. OP Length: 100 mm 100 mm 100 mm

1 Acephate 8.35 7.33 7.53 poor peakshape

2 Azinphos-methyl 20.12 18.42 19.43 3 Bensulide 21.56 21.19 21.19 4 Cadusafos* 22.76 22.51 22.19 5 Chlorethoxyfos 20.1 Poor peak N/F N/F 6 Chlorpyrifos 23.63 23.79 22.65 7 Chlorpyrifos methyl 22.77 Poor peak 22.61 21.63 8 Coumaphos 22.57 21.8 22.29 9 Diazinon 22.32 21.67 21.75

10 Dichlorvos (DDVP) 16.77 15.79 16.24 11 Dicrotophos 13.8 11.73 14.13 12 Dimethoate 14.12 12.42 14 13 Disulfoton 22.47 Bad tailing 22.39 Smearing 21.4 14 Ethion 23.55 23.59 22.58 15 Ethoprop 21.45 20.39 20.63 16 Ethyl parathion 21.86 21.25 20.97 17 Fenamiphos 21.79 21.05 20.93 18 Fenitrothion 21.04 19.91 20.15 19 Fenthion 22.23 21.66 21.01 20 Fonofos** 22.1 21.65 20.92 21 Malathion 20.45 19.52 20.01 22 Methamidophos 6.84 4.24 Excessive

Peak broadening

4.03

23 Methidathion 19.26 18.08 18.86 24 Methyl parathion 20.06 18.88 19.21 25 Mevinphos* 15.26 13.96 15.91 26 Naled * 18.06 N/F 27 Oxydemeton methyl 12.79 10.39 12.38 28 Phorate 22.35 22.14 21.13 29 Phosalone* 22.41 22.17 21.64 30 Phosmet N/F N/F N/F 31 Phostebupirim 23.26 23.38 22.48 32 Pirimiphos methyl 21.87 20.85 20.77 33 Profenofos 23.31 23.14 22.29 34 Propetamphos 20.64 19.79 20.07 35 Sulfotepp 21.71 21.43 20.86 36 Temephos 23.47 23.55 22.78 37 Terbufos 23.17 23.33 22.23 38 Tetrachlorvinphos 21.88 21.15 20.9 39 Tribufos (DEF) 24.31 24.51 23.6 40 Trichlorfon 15.27 14.17 15.88

Table 16 Retenetion times of OP pesticides on Aquasil C18 and Chromolit RP 18e, Zorbax SBC3

96

Discovery HS F5 3µm Hypercarb Varian C18

Width: 4.6mm 4.6mm 4.6mm No. OP Length: 100 mm 100 mm 250mm

1 Acephate 7.61 3.22 12.74 2 Azinphos-methyl 20.61 N/F E/B 23.99 3 Bensulide 22.21 22.62 tailing 24.65 4 Cadusafos* 21.7 18.73 26.17 5 Chlorethoxyfos N/F N/F E/B N/F 6 Chlorpyrifos 22.85 N/F E/B 27.53 7 Chlorpyrifos methyl 22.12 N/F E/B N/F E/B 8 Coumaphos 23.83 N/F E/B 25.62 9 Diazinon 22.4 21.05 25.77

10 Dichlorvos (DDVP) 16.8 N/F E/B 21.64 11 Dicrotophos 13.39 9.33 16.69 12 Dimethoate 14.86 10.46 18.43 13 Disulfoton N/F N/F 26.21 14 Ethion 23.31 25.26 tailing 26.8 15 Ethoprop 19.98 15.63 24.98 16 Ethyl parathion 22.75 N/F E/B 25.28 17 Fenamiphos 20.97 N/F E/B 24.88 18 Fenitrothion 22.1 N/F E/B 24.7 19 Fenthion 21.99 N/F E/B 25.77 20 Fonofos** 22.02 8.23 25.92 21 Malathion 21.27 18.7 24.23 22 Methamidophos 4.28 1.43 12 23 Methidathion 20.4 22.03 23.74 24 Methyl parathion 21.57 N/F E/B 24.13 25 Mevinphos* 15.59 N/F E/B 19.41 26 Naled 18.55 N/F N/F 27 Oxydemeton methyl 11.57 8.23 15.35 28 Phorate 22.02 tailing N/F 26.9 29 Phosalone* 22.57 N/F 25.66 30 Phosmet N/F N/F E/B N/F 31 Phostebupirim 22.73 20.45 27 32 Pirimiphos methyl 26.26 N/F E/B 25.89 33 Profenofos 22.25 N/F E/B 26.72 34 Propetamphos 20.6 17.76 24.26 35 Sulfotepp 21.92 17.32 25.21 36 Temephos 23.59 N/F E/B 26.44 37 Terbufos 22.96 N/F 26.87 38 Tetrachlorvinphos 21.19 N/F E/B 25.09 39 Tribufos (DEF) 23.01 N/F 28.5 40 Trichlorfon 16.82 N/F E/B 19.38

Table 17 Retenetion times of OP pesticides on Discovery HS F5, Hyper carb and Varian C18 columns

97

0 5 10 15 20 25 30

MethamidophosAcephate

Oxydemeton methylDicrotophosDimethoateMevinphos*Trichlorfon

Dichlorvos (DDVP)Naled

Methyl parathionMethidathion

EthopropPropetamphos

Azinphos-methylMalathion

FenitrothionPirimiphos methyl

FenamiphosTetrachlorvinphos

Cadusafos*Ethyl parathion

DiazinonFonofos**

PhorateSulfotepp

DisulfotonChlorpyrifos methyl

BensulideFenthion

ProfenofosPhostebupirim

Phosalone*Terbufos

CoumaphosChlorpyrifos

EthionTribufos (DEF)

Temephos

Betasil C18 0 5 10 15 20 25 30



Dichlorvos (DDVP)Methidathion

Methyl parathionChlorethoxyfos

Azinphos-methylMalathion

PropetamphosFenitrothion

EthopropBensulideSulfotepp

FenamiphosEthyl parathion

Pirimiphos methylTetrachlorvinphos

Fonofos**FenthionDiazinonPhorate

Phosalone*Disulfoton

CoumaphosCadusafos*

Chlorpyrifos methylTerbufos

PhostebupirimProfenofosTemephos

EthionChlorpyrifos

Tribufos (DEF)

Betasil Phenyl

Figure 61 Betasi C18 and Betasil Phenyl retention profile using a linear water methanol gradient

98

0 5 10 15 20 25 30




MethidathionAzinphos-methylMethyl parathion

MalathionPropetamphos

FenitrothionEthoprop

Pirimiphos methylFenamiphos

TetrachlorvinphosBensulide

Ethyl parathionSulfoteppFonofos**

FenthionDiazinon

CoumaphosPhorate

Phosalone*Disulfoton

Cadusafos*Chlorpyrifos methyl

ProfenofosTerbufos

PhostebupirimTemephos

EthionChlorpyrifos

Tribufos (DEF)

Aquasil C18

0 5 10 15 20 25 30


Oxydemeton methylDicrotophosDimethoate

Mevinphos*Trichlorfon


Methyl parathionMethidathion

PropetamphosEthoprop

Azinphos-methylPhosmet

MalathionPirimiphos methyl

FenitrothionFenamiphos

TetrachlorvinphosEthyl parathion

DiazinonFonofos**

PhorateCadusafos*

SulfoteppChlorpyrifos methyl

DisulfotonFenthion

BensulideProfenofos

PhostebupirimPhosalone*

TerbufosCoumaphosChlorpyrifos

EthionTribufos (DEF)

Temephos

Betasil Phenyl, final gradient (GC group is grayed out)

Figure 62 Aquasil C18 retention profile using a linear water methanol gradient and Betasil Phenyl using the final, non lina=ear solvent gradient

99

Alternative to the one column HPLC separation

For sake of simplicity, as mentioned earlier, a single column, the 4.6X100mm Betasil

Phenyl column was selected. This column did not resolve all the compounds in the late

eluting group, but with the modified gradient gave sufficient separation. The reason for

this is the same as the core difficulty of our problem: we have to deal with a very large

group of compounds. It is difficult if not impossible to find one single column that will

separate all the analytes in an even manner.

An alternative is to use two columns and create a “two dimensional system”, where

one column separates the early eluting group but not the late eluting ones. This late group

then directed on a different type of column, where it is separated with a more organic rich

gradient. It would be recommended to use Aquasil C18 or Betasil Phenyl for first column

and Discovery HF S5 as a second column.

This system can be achieved with two properly timed six port valves. As it is

illustrated in Figure 63

100

Figure 63 Two column HPLC system. 1: Column A separates the first analyte group. 2: The group of unresolved analytes pass column A and are loaded onto column B. 3: This second group of analytes are gradient eluted and separated on column B.

101

CHAPTER 3 APPENDIX

Appendix 3A APCI Source Temperature Studies

Because many of the OP pesticides known to be thermally labile, an investigation was

undertaken to study the effect of source temperature on on signal strength.

In APCI, source temperature has two components:

• Evaporator temperature is the temperature of the porcelain tube where the LC

solvent stream enters in the source. This is where the liquid eluent starts to

evaporate and forms an aerosol with the nitrogen sheet gas. Evaporator

temperature may be raised up to 600°C.

• Capillary or “ion transfer tube” temperature may be adjusted between 0 and 400°.

The capillary is the narrow ion guide that separates the atmospheric part of the

APCI source from the first stage of vacuum. Its temperature is important

optimization factor because this is where the last traces of water is dried off, or

where the dehydratation of the ions is completed.

Too low temperature will result in incomplete drying, too high temperature causes

thermal decomposition.

While both temperatures may be adjusted, neither one of them is suitable to change

during a run. A pair of compromise temperatures have to be found to accommodate

all the analytes.

102

The following set of charts illustrates the effect of these temperature combinations for

all analytes. 350 °C for evaporator temperature and 250°C capillary temperature was

found to be the best combination for the analytes selected for HPLC MS-MS.

103

12Area Count/1000 150 250 400

VAP 250 1.4 1.5 0.3Vaporizer VAP 350 2.2 1.6 0.3

VAP 450 2 1.4 0.19

Vaporizer - Heated Capillary Temperature Effect

13Area Count/1000 150 250 400

VAP 250 0.015 0.014 0Vaporizer VAP 350 0.045 0 0

VAP 450 0.11 0 0

Dimethoate Capillary

Disulfoton Capillary

150250

400

VAP 250

VAP 350

VAP 450

0

0.5

1

1.5

2

2.5

Are

a C

ount

/100

0

Ion Tranfer Tube Temp.[C]

Vaporizer Temp. [C]

150250

400

VAP 250

VAP 350

VAP 450

0

0.02

0.04

0.06

0.08

0.1

0.12

Are

a C

ount

/100

0


Vaporizer Temp. [C]

104

3Area Count/1000 150 250 400

VAP 250 0.74 0.12 0Vaporizer VAP 350 0.85 0.07 0

VAP 450 0.45 0.01 0

4Area Count/1000 150 250 400


VAP 450 3.2 1.1 0

Bensulide Capillary

Cadusafos Capillary

150250

400

VAP 250

VAP 350

VAP 450

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

Are

a C

ount

/100

0


Vaporizer Temp. [C]

150250

400

VAP 250

VAP 350

VAP 450

0

0.5

1

1.5

2

2.5

3

3.5

Are

a C

ount

/100

0


Vaporizer Temp. [C]

105

5Area Count/1000 150 250 400

VAP 250 0 0 0Vaporizer VAP 350 0 0 0.001327

VAP 450 0 0 0.007349

6Area Count/1000 150 250 400


VAP 450 0.11 0.72 0.25

Clorethoxyfos Capillary

Clorpyrifos Capillary

150250

400

VAP 250

VAP 350

VAP 450

0

0.001

0.002

0.003

0.004

0.005

0.006

0.007

0.008

Are

a C

ount

/100

0


Vaporizer Temp. [C]

150250

400

VAP 250

VAP 350

VAP 450

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

Are

a C

ount

/100

0


Vaporizer Temp. [C]

106

7Area Count/1000 150 250 400

VAP 250 0 0.002 0.062Vaporizer VAP 350 0 0.03 0.11

VAP 450 0 0.068 0.093

8Area Count/1000 150 250 400

VAP 250 0.92 1.4 1.4Vaporizer VAP 350 1.6 2 1.9

VAP 450 2.1 2.3 1.9

Clorpyrifos -Me Capillary

Coumaphos Capillary

150250

400

VAP 250

VAP 350

VAP 450

0

0.02

0.04

0.06

0.08

0.1

0.12

Are

a C

ount

/100

0


Vaporizer Temp. [C]

150250

400

VAP 250

VAP 350

VAP 450

0

0.5

1

1.5

2

2.5

Are

a C

ount

/100

0


Vaporizer Temp. [C]

107

9Area Count/1000 150 250 400


VAP 450 3 2.5 1.1

10Area Count/1000 150 250 400


VAP 450 0.12 0.097 0.071

Diazinon Capillary

Dichlorvos Capillary

150250

400

VAP 250

VAP 350

VAP 450

0

0.5

1

1.5

2

2.5

3

Are

a C

ount

/100

0


Vaporizer Temp. [C]

150250

400

VAP 250

VAP 350

VAP 450

0

0.02

0.04

0.06

0.08

0.1

0.12

Are

a C

ount

/100

0


Vaporizer Temp. [C]

108

11Area Count/1000 150 250 400


VAP 450 0.19 0.19 0.044

12Area Count/1000 150 250 400


VAP 450 2 1.4 0.19

Dichrotophos Capillary

Dimethoate Capillary

150250

400

VAP 250

VAP 350

VAP 450

0

0.05

0.1

0.15

0.2

0.25

Are

a C

ount

/100

0


Vaporizer Temp. [C]

150250

400

VAP 250

VAP 350

VAP 450

0

0.5

1

1.5

2

2.5

Are

a C

ount

/100

0


Vaporizer Temp. [C]

109

13Area Count/1000 150 250 400


VAP 450 0.11 0 0

14Area Count/1000 150 250 400

VAP 250 0.19 0.51 0.06Vaporizer VAP 350 0.66 0.62 0

VAP 450 0.91 0.39 0

Disulfoton Capillary

Ethion Capillary

150250

400

VAP 250

VAP 350

VAP 450

0

0.02

0.04

0.06

0.08

0.1

0.12

Are

a C

ount

/100

0


Vaporizer Temp. [C]

150250

400

VAP 250

VAP 350

VAP 450

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

Are

a C

ount

/100

0


Vaporizer Temp. [C]

110

15Area Count/1000 150 250 400


VAP 450 0.86 0.4 0.09

16Area Count/1000 150 250 400

VAP 250 0.16 0.16 0.026Vaporizer VAP 350 0.24 0.1 0

VAP 450 0.18 0.05 0

Ethoprop Capillary

Ethyl Parathion Capillary

150250

400

VAP 250

VAP 350

VAP 450

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

Are

a C

ount

/100

0


Vaporizer Temp. [C]

150250

400

VAP 250

VAP 350

VAP 450

0

0.05

0.1

0.15

0.2

0.25

Are

a C

ount

/100

0


Vaporizer Temp. [C]

111

17Area Count/1000 150 250 400


VAP 450 0.38 0.23 0

18Area Count/1000 150 250 400


VAP 450 0.051 0.026 0

Fenamiphos Capillary

Fenitrothion Capillary

150250

400

VAP 250

VAP 350

VAP 450

00.050.1

0.150.2

0.250.3

0.350.4

Are

a C

ount

/100

0


Vaporizer Temp. [C]

150250

400

VAP 250

VAP 350

VAP 450

00.010.020.030.040.050.060.070.080.09

Are

a C

ount

/100

0


Vaporizer Temp. [C]

112

19Area Count/1000 150 250 400

VAP 250 0 0.058 0.049Vaporizer VAP 350 0.005 0.17 0.024

VAP 450 0.007 0.45 0

20Area Count/1000 150 250 400

VAP 250 0.82 0.6Vaporizer VAP 350 0.96

VAP 450 0.75

Fenthion Capillary

Fonofos Capillary

150250

400

VAP 250

VAP 350

VAP 450

00.050.1

0.150.2

0.250.3

0.350.4

0.45

Are

a C

ount

/100

0


Vaporizer Temp. [C]

150250

400

VAP 250

VAP 350

VAP 450

0

0.2

0.4

0.6

0.8

1

Are

a C

ount

/100

0


Vaporizer Temp. [C]

113

21Area Count/1000 150 250 400


VAP 450 1.61 0.25 0

22Area Count/1000 150 250 400


VAP 450 1.5 0.76 0.03

Malathion Capillary

MMP Capillary

150250

400

VAP 250

VAP 350

VAP 450

00.20.40.60.8

11.21.41.61.8

Are

a C

ount

/100

0


Vaporizer Temp. [C]

150250

400

VAP 250

VAP 350

VAP 450

00.20.40.60.8

11.21.41.6

Are

a C

ount

/100

0


Vaporizer Temp. [C]

114

23Area Count/1000 150 250 400


VAP 450 0.47 0.016 0

24Area Count/1000 150 250 400


VAP 450 0.4 0.17 0.027

Methidathion Capillary

Me-Parathion Capillary

150250

400

VAP 250

VAP 350

VAP 450

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0.5

Are

a C

ount

/100

0


Vaporizer Temp. [C]

150250

400

VAP 250

VAP 350

VAP 450

0

0.1

0.2

0.3

0.4

0.5

0.6

Are

a C

ount

/100

0


Vaporizer Temp. [C]

115

Area Count/1000 150 250 400VAP 250 0 0 0

Vaporizer VAP 350 0 0 0VAP 450 0 0 0


31Area Count/1000 150 250 400


VAP 450 1.3 0.5 0.008

Phostebupyrim Capillary

150250

400

VAP 250VAP 350

VAP 4500

0.20.4

0.6

0.8

1A

rea

Cou

nt/1

000


Vaporizer Temp. [C]

150250

400

VAP 250VAP 350

VAP 4500

0.5

1

1.5

Are

a C

ount

/100

0


Vaporizer Temp. [C]

116

27Area Count/1000 150 250 400


VAP 450 2 0.61 0

28Area Count/1000 150 250 400

VAP 250 0 0.0029 0Vaporizer VAP 350 0 0.0007 0

VAP 450 0 0.006 0

Oxydemethon-Me Capillary

Phorate Capillary

150250

400

VAP 250

VAP 350

VAP 450

0

0.5

1

1.5

2

2.5

Are

a C

ount

/100

0


Vaporizer Temp. [C]

150250

400

VAP 250

VAP 350

VAP 450

0

0.001

0.002

0.003

0.004

0.005

0.006

Are

a C

ount

/100

0


Vaporizer Temp. [C]

117

29Area Count/1000 150 250 400


VAP 450 0.26 0.19 0

30Area Count/1000 150 250 400

VAP 250 0 0 0Vaporizer VAP 350 0 0 0

VAP 450 0 0 0

Phosalone Capillary

Phosmet Capillary

150250

400

VAP 250VAP 350

VAP 4500

0.050.1

0.150.2

0.250.3

Are

a C

ount

/100

0


Vaporizer Temp. [C]

150250

400

VAP 250VAP 350

VAP 4500

0.2

0.4

0.6

0.8

1

Are

a C

ount

/100

0


Vaporizer Temp. [C]

118

31Area Count/1000 150 250 400


VAP 450 1.3 0.5 0.008

32Area Count/1000 150 250 400


VAP 450 0.079 0.045 0.021

Phostebupyrim Capillary

Pirimiphos - Me Capillary

150250

400

VAP 250VAP 350

VAP 4500

0.5

1

1.5

Are

a C

ount

/100

0


Vaporizer Temp. [C]

150250

400

VAP 250VAP 350

VAP 4500

0.02

0.04

0.06

0.08

Are

a C

ount

/100

0


Vaporizer Temp. [C]

119

38Area Count/1000 150 250 400


VAP 450 0.34 0.24 0


39Area Count/1000 150 250 400


VAP 450 0.014 0.0046 0

Tetrachlorvinphos Capillary

Tribufos Capillary

150250

400

VAP 250

VAP 350

VAP 450

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

Are

a C

ount

/100

0


Vaporizer Temp. [C]

150250

400

VAP 250

VAP 350

VAP 450

0

0.002

0.004

0.006

0.008

0.01

0.012

0.014

Are

a C

ount

/100

0


Vaporizer Temp. [C]

120

35Area Count/1000 150 250 400


VAP 450 1.6 0.77 0.099

36Area Count/1000 150 250 400


VAP 450 0.025 0 0

Sulfotepp Capillary

Temephos Capillary

150250

400

VAP 250

VAP 350

VAP 450

00.20.40.60.8

11.21.41.6

Are

a C

ount

/100

0


Vaporizer Temp. [C]

150250

400

VAP 250

VAP 350

VAP 450

00.010.020.030.040.050.060.070.080.09

Are

a C

ount

/100

0


Vaporizer Temp. [C]

121

37Area Count/1000 150 250 400

VAP 250 0 0.023 0Vaporizer VAP 350 0.013 0.011 0

VAP 450 0.036 0 0

38Area Count/1000 150 250 400


VAP 450 0.34 0.24 0

Terbufos Capillary

Tetrachlorvinphos Capillary

150250

400

VAP 250

VAP 350

VAP 450

0

0.005

0.01

0.015

0.02

0.025

0.03

0.035

0.04

Are

a C

ount

/100

0


Vaporizer Temp. [C]

150250

400

VAP 250

VAP 350

VAP 450

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

Are

a C

ount

/100

0


Vaporizer Temp. [C]

122

39Area Count/1000 150 250 400


VAP 450 0.014 0.0046 0

40Area Count/1000 150 250 400


VAP 450 0.47 0 0

Tribufos Capillary

Trichlorfon Capillary

150250

400

VAP 250

VAP 350

VAP 450

0

0.002

0.004

0.006

0.008

0.01

0.012

0.014

Are

a C

ount

/100

0


Vaporizer Temp. [C]

150250

400

VAP 250

VAP 350

VAP 450

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

Are

a C

ount

/100

0


Vaporizer Temp. [C]

123

Appendix 3B : Applied Solvent Gradient curves for testing HPLC columns and

This section lists the solvent gradients and their reference codes used while developing

the HPLC portion of the analytical separations.

124

code Gradient Graph 11_1 1 mL/min 0.1% formic acid in both

Time [min]

MeOH [%V/V]

0 23 2

49 10055 10056 260 2

0

50

100

0 20 40 60

Time [min]

MeO

H [

%]

11_2, 11_3 1 ,mL/min 0.1% formic acid in both

Time [min]

MeOH [%V/V]

0 23 2

24.5 10027.5 100

28 230 2

0

50

100

0 10 20 30

Time [min]

MeO

H [

%]

11_4 0.2mL/min 0.1% formic acid in both

Time [min]

MeOH [%V/V]

0 105 10

24.5 10027.5 100

28 1030 10

0

50

100

0 10 20 30

Time [min]

MeO

H [

%]

11_5 0.2mL/min 0.1% formic acid in both

Time [min]

MeOH [%V/V]

0 54 55 30

15 3020 10023 10024 530 5

0

50

100

0 10 20 30

Time [min]

MeO

H [

%]

Table 18 List of elution curves applied while optimizing LC separations

125


Time [min]

MeOH [%V/V]

0 54 55 50

15 5020 10023 10024 530 5

0

50

100

0 10 20 30

Time [min]

MeO

H [

%]

11_7 1 mL/min 0.1% formic acid in both

Time [min]

MeOH [%V/V]

0 53 54 30

15 7017 10023 10024 530 5

0

50

100

0 10 20 30

Time [min]

MeO

H [

%]


Time [min]

MeOH [%V/V]

0 203 20

23 10025 100

25.5 2030 20

0

50

100

0 10 20 30

Time [min]

MeO

H [

%]


Time [min]

MeOH [%V/V]

0 202 205 75

10 9015 9520 10025 10026 2030 20

0

50

100

0 10 20 30

Time [min]

MeO

H [

%]

Table Table 18 (cont) List of elution curves applied while optimizing LC separations

126

code Gradient Graph 11-10 1 mL/min 0.1% formic acid in both

Time [min]

MeOH [%V/V]

0 201 202 60

10 7515 8520 10025 10026 2030 20

0

50

100

0 10 20 30

Time [min]

MeO

H [

%]


Time [min]

MeOH [%V/V]

0 201 202 60

10 6515 7020 10025 10026 2030 20

0

50

100

0 10 20 30

Time [min]

MeO

H [

%]


Time [min]

MeOH [%V/V]

0 5025 10028 10029 5030 50

0

50

100

0 10 20 30

Time [min]

MeO

H [

%]


Time [min]

MeOH [%V/V]

0 7525 90

25.5 10028 10029 5030 50

0

50

100

0 10 20 30

Time [min]MeO

H [

%]


127


Time [min]

MeOH [%V/V]

0 6525 75

25.5 10027.5 100

28 5030 50

0

50

100

0 10 20 30

Time [min]

MeO

H [

%]


Time [min]

MeOH [%V/V]

0 53 5

25 10027.5 100

28 530 5

0

50

100

0 10 20 30

Time [min]

MeO

H [

%]


Time [min]

MeOH [%V/V]

0 23 2

24.5 10027.5 100

28 230 2

0

50

100

0 10 20 30

Time [min]

MeO

H [

%]


Time [min]

MeOH [%V/V]

0 4025 10028 10029 4030 40

0

50

100

0 10 20 30

Time [min]

MeO

H [

%]


128


Time [min]

MeOH [%V/V]

0 6025 10028 10029 6030 60

0

50

100

0 10 20 30

Time [min]MeO

H [

%]


Time [min]

MeOH [%V/V]

0 605 80

25 10028 100

28.5 6030 60

0

50

100

0 10 20 30

Time [min]

MeO

H [

%]


Time [min]

MeOH [%V/V]

0 603 60

24 9025 10028 100

28.5 6030 60

0

50

100

0 10 20 30

Time [min]

MeO

H [

%]


Time [min]

MeOH [%V/V]

0 5025 50

25.5 10028 100

28.5 5030 50

0

50

100

0 10 20 30

Time [min]

MeO

H [

%]


129


Time [min]

MeOH [%V/V]

0 6025 60

25.5 10028 100

28.5 6030 60

0

50

100

0 10 20 30

Time [min]

MeO

H [

%]


Time [min]

MeOH [%V/V]

0 7025 70

25.5 10028 100

28.5 7030 70

0

50

100

0 10 20 30

Time [min]

MeO

H [

%]


Time [min]

MeOH [%V/V]

0 8025 80

25.5 10028 100

28.5 8030 80

0

50

100

0 10 20 30

Time [min]

MeO

H [

%]


Time [min]

MeOH [%V/V]

0 9025 90

25.5 10028 100

28.5 9030 90

0

50

100

0 10 20 30

Time [min]

MeO

H [

%]


130

code Gradient Graph 12 1 mL/min 0.1% formic acid in both

Time [min]

MeOH [%V/V]

0 23 28 25

10 7515 9525 10027 100

27.5 230 2

0

50

100

0 10 20 30

Time [min]

MeO

H [

%]

13 1 mL/min 0.1% formic acid in both

Time [min]

MeOH [%V/V]

0 53 58 20

10 6025 10027 100

27.5 530 5

0

50

100

0 10 20 30

Time [min]

MeO

H [

%]


Time [min]

MeOH [%V/V]

0 53 57 20

10 8024 10026 100

26.5 530 5

0

50

100

0 10 20 30

Time [min]

MeO

H [

%]


Time [min]

MeOH [%V/V]

0 53 57 208 70

20 7522 10026 100

26.5 530 5

0

50

100

0 10 20 30

Time [min]MeO

H [

%]


131

code Gradient Graph 16 1 mL/min 0.1% formic acid in both

Time [min]

MeOH [%V/V]

0 44 47 208 65

22 6524 10026 100

26.5 430 4

0

50

100

0 10 20 30

Time [min]MeO

H [

%]


Time [min]

MeOH [%V/V]

0 100.5 10

3 6043 8045 10053 10055 1060 10

0

50

100

0 20 40 60

Time [min]

MeO

H [

%]


Time [min]

MeOH [%V/V]

0 100.5 10

3 7043 8045 10053 10055 1060 10

0

50

100

0 20 40 60

Time [min]

MeO

H [

%]


Time [min]

MeOH [%V/V]

0 100.5 10

3 8043 8045 10053 10055 1060 10

0

50

100

0 20 40 60

Time [min]

MeO

H [

%]


132


Time [min]

MeOH [%V/V]

0 503 50

25 10030 10035 10036 5040 50

0

50

100

0 10 20 30 40

Time [min]

MeO

H [

%]


Time [min]

MeOH [%V/V]

0 703 70

25 10030 10035 10036 7040 70

0

50

100

0 10 20 30 40

Time [min]

MeO

H [

%]


Time [min]

MeOH [%V/V]

0 803 80

20 10030 10035 10036 8040 80

0

50

100

0 10 20 30 40

Time [min]

MeO

H [

%]


Time [min]

MeOH [%V/V]

0 803 80

25 9526 10035 10036 8040 80

0

50

100

0 10 20 30 40

Time [min]

MeO

H [

%]


133


Time [min]

MeOH [%V/V]

0 803 90

20 9021 10035 10036 8040 80

0

50

100

0 10 20 30 40

Time [min]MeO

H [

%]

18_6 3-21min: .75mL/min Else: 1 mL/min 0.1% formic acid in both

Time [min]

MeOH [%V/V]

0 803 90

20 9021 10035 10036 8040 80

0

50

100

0 10 20 30 40

Time [min]M

eOH

[%]

18_7 3-15min: Increasing .75 to 1.0 mL/min else: 1 mL/min 0.1% formic acid in both

Time [min]

MeOH [%V/V]

0 803 80

15 8321 10035 10036 8040 80

0

50

100

0 10 20 30 40

Time [min]MeO

H [

%]

Final 1 mL/min 0.1% formic acid in both

Time [min]

MeOH

[%V/V]0 23 24 60

18 6525 90

25.5 10027.5 100

28 230 2

0

50

100

0 10 20 30

Time [min]

MeO

H [

%]


134

Appendix 3C: ESI and APCI positive and negative full scan spectra

050920ESIfs_01 #20 RT: 0.33 AV: 1 NL: 2.71E8T: + c Q1MS [100.00-600.00]

100 150 200 250 300 350 400 450 500 550 600m/z

0

20

40

60

80

100

Rel

ativ

e A

bund

ance

184.02

367.04

388.99

201.04143.01 294.51 389.98 477.56247.02 587.96351.03 491.51

050920ESI_Nfs_01 #22 RT: 0.37 AV: 1 NL: 3.34E7T: - c Q1MS [100.00-1000.00]

100 150 200 250 300 350 400 450 500 550 600m/z

0

20

40

60

80

100

Rel

ativ

e A

bund

ance

181.93

227.92364.85

182.97136.93 386.82273.91 426.81295.84 591.74454.84 522.77

050922APCI_P_fs01 #7 RT: 0.11 AV: 1 NL: 2.50E8T: + c Q1MS [100.00-1000.00]

100 150 200 250 300 350 400 450 500 550 600m/z

0

20

40

60

80

100

Rel

ativ

e A

bund

ance

184.02

366.99224.97

310.00388.98227.01

324.99168.04 389.97115.08 552.94522.93480.00 591.32

050922APCI_N_fs01 #6 RT: 0.09 AV: 1 NL: 5.12E7T: - c Q1MS [100.00-1000.00]

100 150 200 250 300 350 400 450 500 550 600m/z

0

20

40

60

80

100

Rel

ativ

e Ab

unda

nce

167.92

252.91186.89

124.92 331.78279.86222.86 350.83 416.84 495.69 580.64550.69

Figure 64 Acephate ESI positive, ESI negative, APCI positive, APCI negative full scan spectra

135


100 150 200 250 300 350 400 450 500 550 600m/z

0

20

40

60

80

100R

elat

ive

Abun

danc

e318.00

160.08 318.98132.04 359.00260.95 495.56184.02 247.04 417.09 442.97 587.00523.02283.10

N/F 050922APCI_P_fs02 #6-11 RT: 0.09-0.18 AV: 6 SB: 12 0.01-0.06 , 0.39-0.51 NL: 2.13E8T: + c Q1MS [100.00-1000.00]

100 150 200 250 300 350 400 450 500 550 600m/z

0

20

40

60

80

100

Rel

ativ

e Ab

unda

nce

317.96

164.06

160.04 175.08318.97260.95132.03 451.04358.99289.97205.07 349.99 537.82469.04 599.7

050922APCI_N_fs02 #6-9 RT: 0.09-0.15 AV: 4 SB: 12 0.01-0.06 , 0.39-0.51 NL: 9.24E7T: - c Q1MS [100.00-1000.00]

100 150 200 250 300 350 400 450 500 550 600m/z

0

20

40

60

80

100

Rel

ativ

e Ab

unda

nce

156.86

336.72158.85224.83 301.84 338.73 481.72117.96 422.65 534.57 598.4273.84

Figure 65 Azinphos Methyl ESI positive, ESI negative, APCI positive, APCI negative full scan spectra

136


100 150 200 250 300 350 400 450 500 550 600m/z

0

20

40

60

80

100R

elat

ive

Abun

danc

e398.05

399.04355.99420.03313.96 358.00184.04 218.03 497.18 581.03443.08151.08 254.90 533.02

050920ESI_Nfs_03 #22 RT: 0.37 AV: 1 SB: 23 0.06-0.25 , 0.46-0.63 NL: 5.31E6T: - c Q1MS [100.00-1000.00]

100 150 200 250 300 350 400 450 500 550 600m/z

0

20

40

60

80

100

Rel

ativ

e Ab

unda

nce

441.88

395.86

212.89442.95

396.89 458.85 487.85214.96 311.01 398.93 578.79254.98 326.03196.91107.94

050922APCI_P_fs03 #7-12 RT: 0.11-0.20 AV: 6 NL: 2.53E8T: + c Q1MS [100.00-1000.00]

100 150 200 250 300 350 400 450 500 550 600m/z

0

20

40

60

80

100

Rel

ativ

e Ab

unda

nce

355.98

398.01

399.02345.96 357.97419.99313.90 596.9200.00115.07 441.05 554.96519.09222.07184.02


100 150 200 250 300 350 400 450 500 550 600m/z

0

20

40

60

80

100

Rel

ativ

e Ab

unda

nce

212.91

214.90353.84140.89 537.86168.92 589.81293.80215.91 404.83 517.79443.74

Figure 66 Bensulide ESI positive, ESI negative, APCI positive, APCI negative full scan spectra

137


100 150 200 250 300 350 400 450 500 550 600m/z

0

20

40

60

80

100R

elat

ive

Abun

danc

e541.19

542.19271.10

563.13334.09312.10 564.12200.02 256.04 370.19 476.15 527.19172.00 422.22105.05


100 150 200 250 300 350 400 450 500 550 600m/z

0

20

40

60

80

100

Rel

ativ

e Ab

unda

nce

426.75

247.07135.02 441.85240.98

395.91299.00 372.94136.92 442.91181.92 526.78 578.68


100 150 200 250 300 350 400 450 500 550 600m/z

0

20

40

60

80

100

Rel

ativ

e Ab

unda

nce

541.13271.05

303.07

542.12

304.06 544.09247.01190.99 527.10471.01370.15162.97 442.95


100 150 200 250 300 350 400 450 500 550 600m/z

0

20

40

60

80

100

Rel

ativ

e Ab

unda

nce

212.93

154.90

258.87200.90 390.87140.88 376.76280.87 448.83 554.69510.70 596.7

Figure 67 Cadusafos ESI positive, ESI negative, APCI positive, APCI negative full scan spectra

138

N/F N/F N/F 050922APCI_N_fs05 #17-22 RT: 0.29-0.37 AV: 6 SB: 12 0.01-0.06 , 0.39-0.51 NL: 1.55E7T: - c Q1MS [100.00-1000.00]

100 150 200 250 300 350 400 450 500 550 600m/z

0

20

40

60

80

100

Rel

ativ

e Ab

unda

nce

306.68

304.71

308.70234.78

236.78154.90197.83 310.69140.88 292.73 352.70 564.57414.68 482.64 538.54

Figure 68 Chlorethoxyfos ESI positive, ESI negative, APCI positive, APCI negative full scan spectra

139

N/F N/F 050922APCI_P_fs06 #22-28 RT: 0.37-0.48 AV: 7 NL: 2.28E8T: + c Q1MS [100.00-1000.00]

100 150 200 250 300 350 400 450 500 550 600m/z

0

20

40

60

80

100

Rel

ativ

e Ab

unda

nce

351.89

353.89

126.02 355.88162.01 217.03 315.94 405.88254.99 457.96 481.77 551.87 571.81

050922APCI_N_fs06 #21-29 RT: 0.36-0.50 AV: 9 NL: 2.23E7T: - c Q1MS [100.00-1000.00]

100 150 200 250 300 350 400 450 500 550 600m/z

0

20

40

60

80

100

Rel

ativ

e Ab

unda

nce

312.79

314.79197.80168.91

213.76214.90 315.80

285.78 316.78215.81154.90 347.78245.78 380.76 490.71 533.63432.58 558.71

Figure 69 Chlorpyrifos ESI positive (weak), ESI negative (weak), APCI positive, APCI negative full scan spectra

140


100 150 200 250 300 350 400 450 500 550 600m/z

0

20

40

60

80

100R

elat

ive

Abun

danc

e143.09

271.09312.29 334.11

247.04173.09 368.16370.20272.16 454.30 522.59240.15 541.20


100 150 200 250 300 350 400 450 500 550 600m/z

0

20

40

60

80

100

Rel

ativ

e Ab

unda

nce

307.73

195.81

426.79197.82309.71

243.81351.62247.04

427.77 472.72355.02140.89 526.84 568.94

050922APCI_P_fs07 #14-16 RT: 0.23-0.27 AV: 3 SB: 8 0.15-0.20 , 0.32-0.37 NL: 2.78E7T: + c Q1MS [100.00-1000.00]

100 150 200 250 300 350 400 450 500 550 600m/z

0

20

40

60

80

100

Rel

ativ

e Ab

unda

nce

126.03

321.87

129.99 162.01

325.88164.01287.93196.01 271.07 327.91 377.91 427.91 452.89 486.76 534.07 580.06


100 150 200 250 300 350 400 450 500 550 600m/z

0

20

40

60

80

100

Rel

ativ

e Ab

unda

nce

140.88213.77

197.79

186.88305.73215.76

284.77241.79 309.69142.88 317.76 361.68126.88 416.64 436.43 524.54 596.5

Figure 70 Chlorpyrifos Methyl ESI positive, ESI negative, APCI positive, APCI negative full scan spectra

141


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e363.01

365.01

404.02 426.03115.11 143.10 303.65271.08184.04 467.10329.08 522.56 550.60


100 150 200 250 300 350 400 450 500 550 600m/z

0

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nce

136.94

369.86332.81 415.87208.91137.95 254.86 304.86 472.48 579.57500.79


100 150 200 250 300 350 400 450 500 550 600m/z

0

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60

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Rel

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nce

362.98

177.04 364.97209.05 395.01

396.99329.01151.05539.02218.05 427.98243.02 513.03301.04 572.97137.03


100 150 200 250 300 350 400 450 500 550 600m/z

0

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Rel

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224.85

208.89

332.81226.85

418.83168.92 334.81254.87318.80 400.78256.87 421.84 542.77335.82161.98 510.73 564.70

Figure 71 Coumaphos ESI positive, ESI negative (weak), APCI positive, APCI negative full scan spectra

142


100 150 200 250 300 350 400 450 500 550 600m/z

0

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Abun

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e305.10

306.10

307.08

327.07173.10 488.11359.15206.05 299.18 404.20 510.14457.12152.15 575.25


100 150 200 250 300 350 400 450 500 550 600m/z

0

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40

60

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Rel

ativ

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unda

nce

136.92

168.92

214.88

299.00 345.02274.94181.92 372.99 416.99 461.08 504.97 570.67


100 150 200 250 300 350 400 450 500 550 600m/z

0

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60

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Rel

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nce

306.07

307.06

319.08153.08 181.10 359.08291.06 457.14233.01 395.01 505.05 561.63


100 150 200 250 300 350 400 450 500 550 600m/z

0

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Rel

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168.91

214.90

154.90 200.89 274.91236.88 346.84112.90 414.80 510.78482.79 564.75

Figure 72 Diazinon ESI positive, ESI negative, APCI positive, APCI negative full scan spectra

143


100 150 200 250 300 350 400 450 500 550 600m/z

0

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e261.97

263.98 305.09

252.97 442.86265.98 444.90105.05 220.96 306.08182.09 525.05360.96 571.85407.23 464.88


100 150 200 250 300 350 400 450 500 550 600m/z

0

20

40

60

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Rel

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e Ab

unda

nce

204.79

250.81252.80

170.91 254.77 334.71 370.65208.81150.94 412.64 434.66 466.68 538.56 568.51

050922APCI_P_fs10 #7-10 RT: 0.11-0.16 AV: 4 SB: 4 0.02-0.08 NL: 1.15E8T: + c Q1MS [100.00-1000.00]

100 150 200 250 300 350 400 450 500 550 600m/z

0

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Rel

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nce

261.95

252.96263.95182.05

442.86115.07 173.05265.95 360.94233.01 332.94 446.86402.92 513.83 538.75 581.64


100 150 200 250 300 350 400 450 500 550 600m/z

0

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204.81

206.82

250.81170.90124.91 254.79 434.63216.85 410.66302.76 352.76 502.67 582.61530.57

Figure 73 Dichlorvos ESI positive, ESI negative, APCI positive, APCI negative full scan spectra

144


100 150 200 250 300 350 400 450 500 550 600m/z

0

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Abun

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e475.16

270.09

497.14238.07

375.62271.10 498.13193.02 305.07 389.57130.08 533.11 583.11443.18


100 150 200 250 300 350 400 450 500 550 600m/z

0

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Rel

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unda

nce

136.91

290.89222.92468.85307.87 334.90213.88 267.81181.95 368.80 496.83 530.72427.83 574.73


100 150 200 250 300 350 400 450 500 550 600m/z

0

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60

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Rel

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nce

270.08

283.11

475.12

284.09238.06 349.11112.05 476.12144.08 364.05285.09 408.01193.01 555.98 579.0


100 150 200 250 300 350 400 450 500 550 600m/z

0

20

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60

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Rel

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e Ab

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170.90

124.92236.86

250.93221.92

156.89 272.89 420.83216.91 340.85 384.74 568.76517.91452.60

Figure 74 Dicrotophos ESI positive, ESI negative, APCI positive, APCI negative full scan spectra

145


100 150 200 250 300 350 400 450 500 550 600m/z

0

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40

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Abun

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e230.00

458.99

293.00 480.97251.96198.98 592.57482.95305.08 363.46134.07 413.02 534.10


100 150 200 250 300 350 400 450 500 550 600m/z

0

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Rel

ativ

e Ab

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nce

273.87

319.83213.86317.89197.92134.92 252.87 502.76369.79 426.84 527.79485.90 581.75


100 150 200 250 300 350 400 450 500 550 600m/z

0

20

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60

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Rel

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nce

229.98

458.95

271.01231.97

198.95 460.95292.98171.00 335.01115.06 386.01 437.99 544.10 595.5


100 150 200 250 300 350 400 450 500 550 600m/z

0

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60

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Rel

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nce

156.87

213.86

158.86 385.78224.83140.87 369.79273.89 442.81 484.72 538.90 598.4

Figure 75 Dimethoate ESI positive, ESI negative, APCI positive, APCI negative full scan spectra

146


100 150 200 250 300 350 400 450 500 550 600m/z

0

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40

60

80

100R

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Abun

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e305.11

173.08115.09 306.10

230.00 270.12184.03 368.14312.30 444.04 525.20482.52 550.67


100 150 200 250 300 350 400 450 500 550 600m/z

0

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Rel

ativ

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unda

nce

136.94

184.88213.91 273.85137.91 368.78 491.52345.07 432.73 577.11527.66


100 150 200 250 300 350 400 450 500 550 600m/z

0

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40

60

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Rel

ativ

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nce

363.04

365.02

213.00 366.02275.01184.99106.06 487.01289.03217.00 458.97 522.90 584.73


100 150 200 250 300 350 400 450 500 550 600m/z

0

20

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60

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Rel

ativ

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unda

nce

184.90

186.86154.87 252.84200.90 348.83320.79 378.76 430.78 452.76 512.63 540.77 580.83

Figure 76 Disulfoton ESI positive, ESI negative (weak), APCI positive, APCI negative full scan spectra

147


100 150 200 250 300 350 400 450 500 550 600m/z

0

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80

100R

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Abun

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e384.98

173.10 386.99305.10105.05 198.99 448.01273.53 359.24 484.10 554.12 596.0

N/F 050922APCI_P_fs14 #21-28 RT: 0.36-0.48 AV: 8 SB: 4 0.02-0.08 NL: 3.18E8T: + c Q1MS [100.00-1000.00]

100 150 200 250 300 350 400 450 500 550 600m/z

0

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100

Rel

ativ

e Ab

unda

nce

384.95

386.95230.99

198.98398.94185.02 232.98 338.95 438.94 491.00153.06 582.93281.03 556.86


100 150 200 250 300 350 400 450 500 550 600m/z

0

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Rel

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nce

184.89

186.89354.78156.85 252.84 392.78334.78 430.79 562.71478.72 526.73

Figure 77 Ethion ESI positive, ESI negative, APCI positive, APCI negative full scan spectra

148


100 150 200 250 300 350 400 450 500 550 600m/z

0

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Abun

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e485.09

243.08486.08284.08 306.04

244.04 508.07308.08 455.11115.07 213.06 547.15347.11173.11 425.14

050920ESI_Nfs_15 #16-26 RT: 0.27-0.44 AV: 11 SB: 18 0.02-0.15 , 0.56-0.72 NL: 4.70E5T: - c Q1MS [100.00-1000.00]

100 150 200 250 300 350 400 450 500 550 600m/z

0

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Rel

ativ

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nce

136.93

212.93 265.04299.04198.93 345.04258.89

181.94 327.09 349.13 383.00 441.87 580.96523.09468.14


100 150 200 250 300 350 400 450 500 550 600m/z

0

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Rel

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nce

485.08

243.03

275.05

284.05 486.08

285.05 488.06455.08233.02 330.09146.05 425.11203.03 557.06 576.9


100 150 200 250 300 350 400 450 500 550 600m/z

0

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Rel

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nce

198.90

200.89 244.88170.91124.92 376.82310.82 414.74 444.78 510.79 554.73 582.78

Figure 78 Ethoprop ESI positive, ESI negative (weak), APCI positive, APCI negative full scan spectra

149


100 150 200 250 300 350 400 450 500 550 600m/z

0

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100R

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Abun

danc

e243.08

173.09

115.09305.09

306.07143.13284.09 384.97

270.11 323.07 359.19230.04 386.00 485.14454.29 554.03 569.41

050920ESI_Nfs_16 #22 RT: 0.37 AV: 1 SB: 11 0.01-0.18 NL: 4.03E5T: - c Q1MS [100.00-1000.00]

100 150 200 250 300 350 400 450 500 550 600m/z

0

20

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60

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Rel

ativ

e Ab

unda

nce

261.84354.85

103.89137.93 345.03183.97

299.04 373.04 400.78237.68 301.01 441.83181.92 461.12 540.49 581.22


100 150 200 250 300 350 400 450 500 550 600m/z

0

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Rel

ativ

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nce

262.04

151.07

142.06 323.00263.03

230.04152.06 324.00 432.05371.09 584.03523.09493.97


100 150 200 250 300 350 400 450 500 550 600m/z

0

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60

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Rel

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nce

261.86

153.90

137.93

276.90247.85168.91 329.82546.77245.87 400.84 524.82453.80330.82 575.84

Figure 79 Ethyl parathion ESI positive (weak), ESI negative (weak), APCI positive, APCI negative full scan spectra

150

N/F 050920ESI_Nfs_17 #18 RT: 0.30 AV: 1 SB: 11 0.01-0.18 NL: 5.76E5T: - c Q1MS [100.00-1000.00]

100 150 200 250 300 350 400 450 500 550 600m/z

0

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60

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100

Rel

ativ

e Ab

unda

nce

347.94

107.89 136.93

368.94181.90 273.89213.77

306.99 369.73147.97 415.01 548.78456.84 586.02487.15


100 150 200 250 300 350 400 450 500 550 600m/z

0

20

40

60

80

100

Rel

ativ

e Ab

unda

nce

363.14

304.07

364.13336.09

365.13512.20250.13209.12 548.08101.10 485.16409.11 596.0146.07


100 150 200 250 300 350 400 450 500 550 600m/z

0

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60

80

100

Rel

ativ

e Ab

unda

nce

246.89

292.90314.86

136.93 382.82156.89 232.89 319.91 516.84409.91 584.80494.80

Figure 80 Fenamiphos ESI positive (weak),, ESI negative (weak), APCI positive, APCI negative full scan spectra

151

050920ESIfs_18 #22 RT: 0.37 AV: 1 SB: 24 0.06-0.23 , 0.63-0.83 NL: 1.11E7T: + c Q1MS [100.00-600.00]

100 150 200 250 300 350 400 450 500 550 600m/z

0

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Abun

danc

e115.09

305.10

143.11

367.12304.11261.08146.07 211.15 345.16274.37 368.14 454.29 488.37 522.43 567.01

050920ESI_Nfs_17 #16-20 RT: 0.27-0.34 AV: 5 SB: 11 0.01-0.18 NL: 4.37E5T: - c Q1MS [100.00-1000.00]

100 150 200 250 300 350 400 450 500 550 600m/z

0

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60

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100

Rel

ativ

e Ab

unda

nce

347.93

107.91

368.90134.93 273.88213.84181.90

393.90306.92147.90 414.88 504.97 586.15565.33


100 150 200 250 300 350 400 450 500 550 600m/z

0

20

40

60

80

100

Rel

ativ

e Ab

unda

nce

165.09

124.05248.04

156.09289.05

262.02179.07 294.04216.06 371.09 401.04 495.07 525.02431.07 555.19


100 150 200 250 300 350 400 450 500 550 600m/z

0

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Rel

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unda

nce

167.92

151.94293.87

186.89276.85208.86 305.93135.95 414.86344.86 546.81493.70436.86 586.73

Figure 81 Fenitrothion ESI positive (weak),, ESI negative, APCI positive, APCI negative full scan spectra

152


100 150 200 250 300 350 400 450 500 550 600m/z

0

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100R

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Abun

danc

e342.03

115.10557.04

320.04279.03 579.02143.10

343.04173.09 512.16261.50 378.14 451.05208.05


100 150 200 250 300 350 400 450 500 550 600m/z

0

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60

80

100

Rel

ativ

e Ab

unda

nce

262.86

308.86299.04 382.87345.06107.91 213.84181.93 523.17417.03 555.03460.81


100 150 200 250 300 350 400 450 500 550 600m/z

0

20

40

60

80

100

Rel

ativ

e Ab

unda

nce

279.00

311.03

247.04320.04

106.05 325.00154.04 385.01 557.01233.15 429.08 491.07 579.0


100 150 200 250 300 350 400 450 500 550 600m/z

0

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40

60

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Rel

ativ

e Ab

unda

nce

262.87

263.86186.89140.91 330.84308.83208.86 398.82 548.76 596.6466.85 520.56

Figure 82 Fenthion ESI positive, ESI negative, APCI positive, APCI negative full scan spectra

153


100 150 200 250 300 350 400 450 500 550 600m/z

0

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80

100R

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Abun

danc

e115.10

173.10247.03

143.09 305.10

299.18368.16245.54 312.29248.05 524.01454.28369.17 538.71


100 150 200 250 300 350 400 450 500 550 600m/z

0

20

40

60

80

100

Rel

ativ

e Ab

unda

nce

324.87

299.04 345.04373.01260.89198.89 416.95152.89

461.06247.04 374.06 581.25561.03505.06


100 150 200 250 300 350 400 450 500 550 600m/z

0

20

40

60

80

100

Rel

ativ

e Ab

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nce

247.01

248.02

261.02169.03 215.04106.06 353.05310.11 383.10 459.09 503.12 547.25 595.0


100 150 200 250 300 350 400 450 500 550 600m/z

0

20

40

60

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Rel

ativ

e Ab

unda

nce

168.90

138.92184.92 216.87 284.85 304.84 352.78 408.83 446.68 484.96 514.63 572.91

Figure 83 Fonofos ESI positive, ESI negative, APCI positive, APCI negative full scan spectra

154


100 150 200 250 300 350 400 450 500 550 600m/z

0

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40

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80

100R

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Abun

danc

e331.04

332.04285.00 348.05 394.04115.10 430.13257.03 515.11159.06 217.13 488.08 558.19 586.0


100 150 200 250 300 350 400 450 500 550 600m/z

0

20

40

60

80

100

Rel

ativ

e Ab

unda

nce

314.87

315.86156.87 360.85233.04 299.00 317.86213.83 416.99 437.48 543.68494.83 568.88


100 150 200 250 300 350 400 450 500 550 600m/z

0

20

40

60

80

100

Rel

ativ

e Ab

unda

nce

331.01

332.01284.97

352.99247.03173.06115.06 394.02 550.86437.06 486.96 587.03

050922APCI_N_fs21 #9 RT: 0.15 AV: 1 SB: 11 0.03-0.09 , 0.27-0.36 NL: 6.47E7T: - c Q1MS [100.00-1000.00]

100 150 200 250 300 350 400 450 500 550 600m/z

0

20

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60

80

100

Rel

ativ

e Ab

unda

nce

156.87

314.87158.87 224.84 336.71142.88 292.80 424.60 494.79159.86 536.57 598.5

Figure 84 Malathion ESI positive, ESI negative, APCI positive, APCI negative full scan spectra

155


100 150 200 250 300 350 400 450 500 550 600m/z

0

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80

100R

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Abun

danc

e283.00

142.02

183.04 285.02205.01 372.50267.07 443.03126.05 479.34 549.30 587.66

050920ESI_Nfs_22 #22 RT: 0.37 AV: 1 SB: 11 0.01-0.18 NL: 5.07E6T: - c Q1MS [100.00-1000.00]

100 150 200 250 300 350 400 450 500 550 600m/z

0

20

40

60

80

100

Rel

ativ

e Ab

unda

nce

186.90140.91

232.86

110.88 263.83156.92 278.83188.93 369.82 385.66 486.51 530.38 580.79461.24


100 150 200 250 300 350 400 450 500 550 600m/z

0

20

40

60

80

100

Rel

ativ

e Ab

unda

nce

282.98

183.03

142.01284.98

266.99205.00 297.02126.00 329.01 390.00 445.09 530.97 578.85468.80

N/F

Figure 85 Methamidophos ESI positive, ESI negative, APCI positive, APCI negative (weak)full scan spectra

156


100 150 200 250 300 350 400 450 500 550 600m/z

0

20

40

60

80

100R

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ive

Abun

danc

e302.95

365.98304.94115.10 473.00145.01 367.99324.94183.04 474.88218.08 402.07252.99 548.07 595.2


100 150 200 250 300 350 400 450 500 550 600m/z

0

20

40

60

80

100

Rel

ativ

e Ab

unda

nce

286.81

270.83156.85 416.87288.82 332.77213.85417.89 460.93374.03 504.94 548.87 581.09


100 150 200 250 300 350 400 450 500 550 600m/z

0

20

40

60

80

100

Rel

ativ

e Ab

unda

nce

302.94

304.94177.03 365.93115.07 319.96218.04 522.82446.92366.92286.99 586.69

050922APCI_N_fs23 #6-11 RT: 0.09-0.18 AV: 6 SB: 4 0.01-0.06 NL: 6.80E7T: - c Q1MS [100.00-1000.00]

100 150 200 250 300 350 400 450 500 550 600m/z

0

20

40

60

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100

Rel

ativ

e Ab

unda

nce

156.87

286.79 336.73158.87224.83 466.71142.86 422.66 534.60354.76 598.5242.77

Figure 86 Methidathion ESI positive, ESI negative, APCI positive, APCI negative full scan spectra

157


100 150 200 250 300 350 400 450 500 550 600m/z

0

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40

60

80

100R

elat

ive

Abun

danc

e115.11

143.12198.05 254.09 312.28 368.17 394.11 455.45 495.08 528.41 580.18


100 150 200 250 300 350 400 450 500 550 600m/z

0

20

40

60

80

100

Rel

ativ

e Ab

unda

nce

276.92

137.93247.85 325.04

183.94 338.94

311.02 386.83231.88138.95 464.00387.88 479.07 595.1553.70


100 150 200 250 300 350 400 450 500 550 600m/z

0

20

40

60

80

100

Rel

ativ

e Ab

unda

nce

151.07

234.02110.03

275.03149.05

152.08 192.12 316.06243.06 343.06 391.25 467.02 497.03 536.00 564.14


100 150 200 250 300 350 400 450 500 550 600m/z

0

20

40

60

80

100

Rel

ativ

e Ab

unda

nce

247.86153.91

137.92276.92

279.86

183.93386.82315.82205.90 401.81 518.73463.79 586.69

Figure 87 Methyl parathion ESI positive (weak), ESI negative, APCI positive, APCI negative full scan spectra

158


100 150 200 250 300 350 400 450 500 550 600m/z

0

20

40

60

80

100R

elat

ive

Abun

danc

e225.07 449.08

471.05193.06 288.04257.05

168.05 472.03 580.11289.03 356.09 505.03127.02 427.13


100 150 200 250 300 350 400 450 500 550 600m/z

0

20

40

60

80

100

Rel

ativ

e Ab

unda

nce

107.92

208.91

254.90290.88

213.85148.92296.92 334.88 368.78 468.89420.71 530.72 558.79


100 150 200 250 300 350 400 450 500 550 600m/z

0

20

40

60

80

100

Rel

ativ

e Ab

unda

nce

225.02257.05

193.01351.03258.04 449.05159.02

289.05 352.01 471.02106.05 394.98 528.99 590.98


100 150 200 250 300 350 400 450 500 550 600m/z

0

20

40

60

80

100

Rel

ativ

e Ab

unda

nce

170.91

124.91

236.88

156.91 250.88208.91 272.86 420.80384.80356.82 568.77504.83438.64

Figure 88 Mevinphos ESI positive, ESI negative, APCI positive, APCI negative full scan spectra

159


100 150 200 250 300 350 400 450 500 550 600m/z

0

20

40

60

80

100R

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ive

Abun

danc

e423.78

115.08

425.78298.04419.79257.08168.06 412.82 453.82146.07 380.75 491.01252.12183.06 334.25 508.82 592.69


100 150 200 250 300 350 400 450 500 550 600m/z

0

20

40

60

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100

Rel

ativ

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unda

nce

364.59366.59

362.60 410.59574.41

408.60 414.57 578.3254.73 458.56170.91 370.60218.86124.91 570.42334.72271.72 490.54


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412.79

252.96115.06 267.02

421.78168.03

208.98 410.77182.04

425.78298.94141.05 235.01 391.21 506.76346.96 547.68 598.6


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364.59366.58

174.79204.81 362.59172.82 250.75 368.58

206.80218.81 410.59252.76 286.64 468.49 572.39538.49159.74

Figure 89 Naled ESI positive, ESI negative, APCI positive, APCI negative full scan spectra

160


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e247.02

493.03

249.02 515.02169.02 389.02 530.99115.07 310.03229.05 339.10 417.72 484.09 586.56


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134.95

230.86335.84

334.85351.84

318.88156.92 200.92 276.82

364.80 400.75 520.78455.45 597.1


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247.00

249.00201.02

168.99 492.98 514.97268.98210.02139.01 310.02 338.95 416.95 446.93 545.14


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140.89

186.89

230.86 304.78170.92124.91 282.81208.85 394.77306.80 468.73 490.67 558.67 578.6

Figure 90 Oxydemethon methyl ESI positive, ESI negative (weak), APCI positive, APCI negative full scan spectra

161


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e335.03115.10

143.08

337.03

339.02173.08 310.13269.01247.01 423.23 510.93462.28 582.12


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184.88

354.81230.87

292.85345.03 416.91373.01134.96

250.02214.91 432.72384.68 461.00 583.19556.58


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260.99

335.01

338.98

262.98244.97 340.97198.98106.05 159.02 508.89277.03 413.00 444.98 572.86


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184.89

186.88170.88 230.86140.88 392.78 478.71348.77320.78 438.77 542.64 588.63

Figure 91 Phorate ESI positive, ESI negative (weak), APCI positive, APCI negative full scan spectra

162


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Abun

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e367.98115.08 430.99

242.26 433.00369.99143.12 415.03352.03

571.61389.99265.06 334.10 433.96183.07 467.10 562.51208.05


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167.89

213.88321.81 411.78337.79134.94 184.91 215.87 413.78 457.82339.82257.88 490.70 534.71 580.18


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367.95

369.95110.03

188.03 477.01142.07 389.95 510.98190.04 421.97246.01 260.99 334.01 554.98 580.03


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184.89

167.89 186.88 337.77252.84 392.79303.83156.88 551.78531.68478.73430.74 587.59

Figure 92 Phosalone ESI positive (weak), ESI negative, APCI positive, APCI negative full scan spectra

163


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e317.97

380.99

115.10

495.55381.99319.00143.13 260.48192.07 529.15448.06417.06 562.83


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301.83

156.89

134.95302.83 347.78200.92 413.82 435.62268.86 534.53481.42 593.69


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317.97

476.99

318.96 477.99115.07 224.06192.05 380.97166.07 537.83451.02 479.99301.99233.09 583.00


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156.86

158.86 336.74224.82 301.81 481.73422.66 536.58142.85 352.71 598.5

Figure 93 Phosmet ESI positive, ESI negative (weak), APCI positive, APCI negative full scan spectra

164


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Abun

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e319.11

320.10321.11 360.13173.07 277.08105.03 247.01 502.16454.28 543.19 595.1432.10


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274.93

134.93

288.94

250.00 299.08 345.03233.04154.93 580.93426.76 506.95372.96327.08213.89 460.91 569.17


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319.08

320.09

321.08153.08 305.08277.04

334.10154.08 385.03106.04 241.09 597.0471.23 515.32 568.97427.27


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274.92

275.92260.91572.91550.93182.92 276.92 342.89196.99151.00 574.92426.97 480.89398.87

Figure 94 Phostebupirim ESI positive, ESI negative, APCI positive, APCI negative full scan spectra

165


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Abun

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e306.09

307.12

308.12

309.06173.08105.04 347.12196.11 552.10266.12 489.11404.61 454.29 576.04


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289.93

136.92 290.92 335.92250.01 434.76373.02181.91 581.09460.90 518.88


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306.08

307.07

182.11 308.06

320.06183.11153.09 360.06277.06 487.19 509.17416.04 562.16


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140.88

289.92

186.88

290.93208.84142.90 357.91276.78124.91 434.71412.79 453.77 531.14 587.96

Figure 95 Pirimphos methyl ESI positive, ESI negative, APCI positive, APCI negative (weak) full scan spectra

166


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e306.08

307.12415.96374.93 437.93115.09 309.08 478.93224.13 515.91183.08 254.11 556.98


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344.73

314.74

312.77

182.92346.74228.91

198.93 252.78152.94 390.72298.96 412.72 520.58496.83 548.59 581.0


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406.92

404.92

372.90

408.91306.07

415.92319.07182.11 295.00153.08 254.99 447.96 509.92 528.89 572.94


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124.84

344.73330.72264.76328.73182.92

250.80 294.78 346.72228.91372.75170.90 398.69 432.60 470.60 514.64 550.62

Figure 96 Profenofos ESI positive, ESI negative, APCI positive, APCI negative full scan spectra

167

050920ESIfs_34 #23-27 RT: 0.39-0.46 AV: 5 NL: 7.97E7T: + c Q1MS [100.00-600.00]

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e282.08 306.10

314.11563.17222.04

197.05156.03 240.04 394.14 416.09 564.17345.09 457.18115.10 516.79


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252.91391.97

325.93

437.96298.99 364.94107.93 392.99

250.01213.86 438.99393.97 483.97 579.98134.92 534.00


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327.12

299.08

328.11254.03222.00198.05170.00 329.10 391.05144.08 419.06 535.10 563.12479.11


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279.94153.91

199.91

280.93221.87155.92 347.89 392.87 434.90140.89 283.90265.92 511.80481.72 560.89

Figure 97 Propetamphos ESI positive, ESI negative, APCI positive, APCI negative full scan spectra

168

050920ESIfs_35 #21-26 RT: 0.35-0.44 AV: 6 NL: 9.31E7T: + c Q1MS [100.00-600.00]

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e323.03

306.10

324.00115.10 282.08173.08 386.02 447.00 503.09263.08 570.97


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292.85

168.92416.79

214.91

103.92345.04

250.02107.92 364.72184.90 418.82327.08 540.70 581.15461.25


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322.99

323.99

446.96337.01115.07 321.99 386.00217.05185.03 240.07 468.95 522.97 554.93


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292.82154.91

200.90214.89

140.90294.84 332.83222.86126.87 470.80 510.76400.83 538.85 592.46

Figure 98 Sulfotepp ESI positive, ESI negative, APCI positive, APCI negative full scan spectra

169


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e115.08

323.03

306.10143.07

263.09 340.07173.09 386.07 499.02242.48 484.00263.92 531.92 589.27


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450.79

136.92 451.82 496.80497.84453.76178.95 340.86200.93 562.83386.80273.84 430.74


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466.96

467.96

498.98

499.97436.96218.01173.06 556.91319.09 356.03 592.9389.03143.03 251.12


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450.78

186.88140.89

451.81356.83262.90 518.79208.84 453.81142.87 276.82 586.70124.92 424.79

Figure 99 Temephos ESI positive (weak), ESI negative, APCI positive, APCI negative full scan spectra

170


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e319.13

306.09391.09

115.08 289.04 320.13173.09 244.97 393.08321.10143.12 466.98277.09 569.03208.05 539.01


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184.89

134.96 345.05186.92 299.05258.89149.88 450.81 491.61349.06 581.23214.88 393.76 529.13


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244.95

289.01

391.08246.96

335.00232.96 393.07103.03 536.91198.98 248.95 337.00 466.96439.03 598.9


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184.89

186.87170.88252.85 392.82364.78 480.69140.92 450.75334.81 558.67 588.7512.72

Figure 100 Terbufos ESI positive (weak), ESI negative, APCI positive, APCI negative full scan spectra

171


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e407.92

405.91

409.92398.92

366.90306.09 429.90

431.89319.11105.04 466.00141.14 589.49505.95230.20 288.29


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373.01

417.02350.72348.72134.94328.99 461.04

374.02200.93156.92418.04 505.06250.00173.91 549.04298.93 434.78 593.05484.96


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398.88396.89

400.88

407.88366.86

115.06 411.88173.04 506.89322.99 465.96271.04244.05 536.82 563.78


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170.91

124.92

350.72192.88 216.90156.94 354.70 396.68260.83 328.77 564.87498.65441.03

Figure 101 Tetrachlorvinphos ESI positive, ESI negative, APCI positive, APCI negative full scan spectra

172


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e315.09

316.09356.11 414.21306.10 566.25520.21466.17179.09 259.04115.09 228.17

N/F 050922APCI_P_fs39 #35-46 RT: 0.60-0.79 AV: 12 SB: 3 0.02-0.06 NL: 2.89E8T: + c Q1MS [100.00-1000.00]

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315.07

347.08

348.07414.16291.04233.04 571.09501.03450.07201.05


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256.90

258.89224.93324.86270.93 392.83 420.80140.88 186.89 488.78 536.81 568.85

Figure 102 Tribufos ESI positive, ESI negative, APCI positive, APCI negative full scan spectra

173


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e256.92

297.95299.95

514.83

288.92 516.82319.92 512.86518.83105.03 152.07 323.93 368.96 534.82221.00 480.91439.89 556.70

050920ESI_Nfs_40 #17-22 RT: 0.29-0.37 AV: 6 SB: 25 0.08-0.29 , 0.60-0.79 NL: 6.45E6T: - c Q1MS [100.00-1000.00]

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302.77

304.77292.77 346.78

134.93 319.75 350.78204.83 240.77 558.64512.66428.74 460.63 580.6


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152.03

143.03

297.92221.02 288.93115.06 166.05 368.91301.94

514.82382.93 437.88 550.84 572.98


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140.91

242.80190.89

204.81176.88

244.78128.86290.75 350.76

354.75250.81 498.61388.69 462.63 526.61 570.58

Figure 103 Trichlorfon ESI positive, ESI negative, APCI positive, APCI negative full scan spectra

174

CHAPTER 4: SAMPLE CLEANUP AND METHOD APPLICATION

When analyzing biological samples, almost always the first step is cleanup, followed

by sample concentration. The preferred first step is solid phase extraction (SPE) because

it is relatively inexpensive, easy to automatise, and a quick method.

It was clear from previous [35] work, that SPE unlikely to be successful because the

analytes of interests encompass a very wide range of physical properties. Particularly the

highly hydrophilic analytes, like acephate and methamidophos are very difficult to retain

together with hydrophobic OP pesticides.

Also based on experiences in [35] the better cleanup method is lyophilization or freeze

drying. When working with urine as matrix, freeze drying yielded very good result. It is

not as easy to automatise as SPE, but the actual manual preparation is limited, and small

amounts of solvent can remove most of the analytes. Reasons against lyophilization are

that it requires specialized equipment, and it is a long process, creating a bottleneck in the

cleanup procedure.

Should lyophilization fail, there are number of options, essentially variants of liquid-

liquid extraction (LLE):

• Manual liquid-liquid extraction. It can yield good information on feasibility for

a certain solvent, but uses a lot of it and practically impossible to automatise.

• Cartridge based LLE, such as Varian’s ChemElut. It is a form of LLE, but

because the hydrophilic carrier increases surface area, it can have good results

with less solvent.

175

• Accelerated solvent extraction (ASE) is a LLE carried out under pressure and

potentially elevated temperatures. While it uses more solvent than the

ChemElut system does, it is an automated process

Liquid-Liquid Extraction on Solid Backing

Since ChemElut cartridges perform similarly to LLE in a flask, it is a good starting

point for investigating LLE feasibility. ChemElut cartridges are made by Varian, Inc.,

and they serve as an alternative to the conventional liquid-liquid extraction. These

cartridges are ultrapure polypropylene tubes filled with Hydromatrix. Hydromatrix is a

purified and of controlled particle size diatomaceous earth.

When performing liquid-liquid extraction with a ChemElut cartridge, the aqueous

sample first added to the tube. The Hydromatrix adsorbs the water on the surface of the

particles and generates a very large potential contact surface wit the extraction solvent.

After an approximately 3-5 minutes waiting, the eluent is added and led gravity-flow

through the system. The stationary Hydromatrix does not let the aqueous sample to

escape from the cartridge. The organic solvent surrounds the immobilized sample and the

extraction process takes place. ChemElut cartridges are a good alternative to manual

liquid/liquid extraction. In the experiments testing the ChemElut cartridges, 1ml serum

samples were used spiked with 500 ng/mL OP pesticides. Recoveries were calculated

according to the method described in Appendix 4B.

The results of the experiments are summarized in Table 20 and Table 21. First (Table

20), serum samples were tested on 3mL ChemElut cartridges with ethyl ether, ethyl

acetate, dichloromethane, 1-chlorobutane, toluene and hexane as extraction solvent

176

candidates. In the second round of experiments (Table 21), ethyl acetate,

dichloromethane and hexane were tested by themselves and in combination. In the first

screening 5 mL solvent was used. In the second group the amount of solvent was

increased to 2 x 5mL. From the tested neat and combination solvents, ethyl acetate was

the most promising throughout the range of the analytes.

177

Analyte Solvent

Diethyl Ether Ethyl Acetate Dichloro methane

1-Chloro butane

Toluene n-Hexane

MMP 2.3% 17.8% 4.2% 1.0% 0.9% 0.1%Acephate 0.7% 8.7% 1.8% 0.1% 0.1% 0.0%Oxydemethon Methyl 3.6% 9.9% 2.9% 1.0% 0.9% 0.6%Dimethoate 3.5% 9.5% 2.3% 1.3% 0.8% 0.3%Trichlorfon 4.3% 13.9% 4.2% 3.7% 10.0% 2.7%Dichlorvos 8.7% 2.4% 3.1% 5.3% 3.1% 0.2%Methidathion 28.6% 12.4% 20.2% 10.9% 8.7% 8.5%Propetamphos 0.0% 0.0% 33.2% 14.3% 19.0% 9.1%Ethoprop 27.3% 23.4% 17.2% 8.4% 9.6% 3.8%Azinphos Me 0.0% 0.0% 28.7% 16.8% 14.1% 41.3%Pirimphos Me 0.1% 14.6% 9.0% 7.3% 6.8% 13.5%Tetrachlorvinphos 11.7% 21.9% 0.0% 6.0% 7.1% 5.8%Fonofos 0.0% 0.0% 12.4% 7.7% 8.2% 6.2%Bensulide 0.0% 0.0% 0.0% 25.1% 26.8% 4.1%Coumaphos 0.0% 6.4% 0.0% 37.4% 33.9% 15.3%Chlorpyrifos 0.0% 0.0% 3.5% 8.0% 7.6% 0.4%Ethion 0.0% 0.0% 0.0% 39.0% 12.1% 11.6%Tribufos 22.4% 26.8% 0.0% 33.0% 34.4% 29.8%Temephos 0.0% 14.0% 0.0% 36.2% 31.4% 46.0%

Table 19 Recoveries for lyophilization extraction

178

Analyte Solvent

Diethyl Ether Ethyl Acetate Dichloro methane

1-Chloro butane

Toluene n-Hexane

MMP 16.3% 42.7% 35.7% 0.6% 0.6% 0.2% Acephate 3.5% 23.8% 17.3% 0.0% 0.0% 0.0% Oxydemethon Methyl 4.3% 33.3% 87.5% 0.2% 1.1% 0.3% Dimethoate 81.2% 84.8% 87.7% 74.6% 78.4% 0.1% Trichlorfon 80.9% 38.8% 40.4% 71.3% 93.9% 3.8% Dichlorvos 75.4% 16.2% 9.0% 10.3% 25.3% 3.6% Methidathion 23.7% 23.6% 19.1% 25.7% 27.6% 27.8% Propetamphos 12.7% 13.1% 10.9% 16.7% 14.2% 18.5% Ethoprop 38.0% 30.6% 22.4% 30.9% 37.6% 26.6% Azinphos Me 22.8% 18.0% 16.8% 16.6% 17.1% 11.5% Pirimphos Me 13.0% 13.5% 10.0% 14.9% 15.9% 21.5% Tetrachlorvinphos 16.7% 14.5% 16.6% 18.8% 19.8% 10.3% Fonofos 14.3% 11.1% 12.9% 20.5% 20.3% 26.9% Bensulide 11.8% 10.9% 11.5% 20.1% 21.6% 0.0% Coumaphos 13.3% 12.0% 5.8% 16.2% 14.2% 16.4% Chlorpyrifos 8.8% 9.4% 4.8% 10.5% 8.7% 19.2% Ethion 8.0% 8.6% 3.1% 6.5% 6.3% 18.2% Tribufos 9.4% 10.5% 4.8% 10.7% 9.5% 17.2% Temephos 0.5% 5.9% 1.5% 2.4% 2.2% 10.6% Table 20 Recoveries for extraction on ChemElut Cartridges

179

Analyte

Solvent Ethyl Acetate Dichloro

methane n-Hexane Blend of

Ethyil Acetate Dichloromethan

e n-Hexane

1 Ethyl Acetate2 Dichloro- methane

1 Ethyl Acetate2 n-Hexane

1 Dichloro methane

2 n-hexane

MMP 45.7% 24.2% 0.3% 12.4% 38.3% 20.7% 0.2% Acephate 44.3% 36.1% 0.2% 19.7% 45.4% 19.6% 0.1% Oxydemethon Methyl 43.5% 71.8% 0.5% 45.6% 60.6% 26.0% 0.1% Dimethoate 75.6% 74.7% 9.2% 68.4% 63.9% 54.5% 0.1% Trichlorfon 52.7% 22.6% 8.3% 23.2% 41.9% 33.0% 0.2% Dichlorvos 36.1% 1.2% 0.4% 0.7% 17.0% 5.7% 0.3% Methidathion 38.1% 18.8% 46.9% 56.5% 31.4% 45.9% 35.9% Propetamphos 18.0% 9.5% 36.1% 34.2% 14.5% 32.3% 22.0% Ethoprop 40.7% 15.1% 28.5% 37.6% 32.2% 39.0% 9.6% Azinphos Me 31.3% 13.4% 31.5% 52.7% 25.2% 36.1% 20.9% Pirimphos Me 16.9% 5.7% 41.0% 33.4% 14.1% 31.8% 32.2% Tetrachlorvinphos 22.0% 9.0% 25.9% 45.6% 18.2% 30.8% 15.6% Fonofos 18.6% 6.2% 38.2% 31.6% 15.6% 25.1% 17.9% bensulide 14.2% 6.9% 5.4% 38.3% 12.7% 30.4% 0.1% Coumaphos 15.6% 4.2% 33.4% 35.1% 14.7% 29.0% 24.8% Chlorpyrifos 13.4% 3.3% 33.7% 17.4% 12.5% 21.5% 23.4% Ethion 12.8% 3.8% 30.6% 11.5% 11.7% 18.9% 27.5% Tribufos 16.7% 8.0% 29.2% 20.9% 14.6% 25.9% 26.6% Temephos 10.3% 2.2% 17.5% 6.2% 9.2% 12.3% 17.1%

Table 21 ChemElut LLE experiment with combination of solvents

180

0.0%

10.0%

20.0%

30.0%

40.0%

50.0%

60.0%

70.0%

80.0%

MMP

Acephate

Oxydem

ethon M

ethyl

Dimeth

oate

Trichlorfo

n

Dichlorvo

s

Methidath

ion

Propeta

mphos

Ethoprop

Azinphos M

e

Pirimphos M

e

Tetrac

hlorvinphos

Fonofos

bensu

lide

Coumaphos

Chlorpyri

fos

Ethion

Tribufos

temep

hos

Analytes in HPLC elution order

Rec

over

y

Ethyl AcetateDichloromethanen-Hexanemix of EA, DCM and hexaneEA, then DCMEA then HexaneDCM then Hexane

Figure 104 ChemElut LLE experiment with combination of solvents

181

Accelerated Solvent Extraction (ASE)

Accelerated solvent extraction is a variant of solid backed LLE. The most common

carrier is Hydromatrix, a purified form diatomaceous earth marketed by Varian Inc. Just

like in the case of LLE cartridges, the particles of the solid backing provide a large

surface area and increase the contact area between the matrix and the eluting solvent.

ASE extraction is carried out in pressurizable stainless steel cartridges. The application of

pressure further increases relative surface by pressing the matrix inside the solid particles.

Applying pressure allows for the use of elevated temperatures, where the pressure

prevents the solvent from boiling. Using hot solvents for extraction can accelerate the

process because the lowered viscosities and increased diffusion.

The solvent of choice for OP pesticides was ethyl acetate. This selection was made based

on the results using ChemElut cartridges (Table 20, Table 21). Once the extraction

solvent was selected, extraction temperature, static time and pressure was varied and

optimized. The results are tabulated in Table 22 and charted in Figure 105 and Figure

106. Increasing temperatures, static(extraction) time and pressure has a positive effect on

most compounds, but has a dramatic decrease of recovery on trichlorfon, dichlorvos and

tetrachlorvinphos. Fro that reason the parameters selected were 5 minutes static time, at

room temperature (20C) and 1500 PSI nitrogen pressure.

182

Compound Temperature (Pressure = 1500 PSI, 5 min Static time)

Temp = 20°C

20°C 40°C 75°C 100°C 10 min static time

2500 PSI

MMP 79.9% 81.9% 84.9% 77.4% 79.2% 71.9% Acephate 49.5% 59.4% 88.4% 84.6% 51.5% 44.3% Oxydem. Me 60.4% 81.6% 94.4% 81.7% 72.3% 73.6% Dimethoate 98.5% 92.9% 95.5% 83.8% 93.5% 87.6% Trichlorfon 81.2% 64.9% 31.7% 13.9% 59.9% 27.8% Dichlorvos 91.2% 34.5% 1.4% 0.1% 34.7% 0.3% Methidathion 84.5% 84.3% 89.1% 76.1% 88.8% 79.8% Propetamphos 88.7% 91.4% 99.2% 94.3% 85.4% 89.4% Ethoprop 81.4% 83.6% 94.9% 93.3% 87.1% 86.7% Azinphos Me 87.5% 88.3% 90.6% 72.4% 36.2% 18.1% Pirimphos Me 67.0% 71.6% 66.9% 45.1% 71.5% 59.7% Tetrachlorvinphos 69.0% 54.7% 27.4% 3.2% 47.0% 18.0% Fonofos 81.3% 89.8% 102.1% 97.8% 79.4% 82.4% Bensulide 66.6% 81.4% 102.2% 91.6% 81.1% 83.2% Coumaphos 74.0% 82.1% 102.2% 99.0% 69.3% 66.1% Chlorpyrifos 62.9% 76.8% 93.1% 84.7% 70.3% 70.3% Ethion 62.4% 76.8% 100.9% 97.8% 67.5% 70.3% Tribufos 64.4% 75.8% 93.0% 90.7% 79.3% 83.3% Temephos 56.2% 73.8% 103.2% 98.7% 58.8% 59.9%

Table 22 Effects on recoveries of varying ASE extraction parameters

183

MMPAcephate

Oxydem. MeDimethoate

TrichlorfonDichlorvos

MethidathionPropetamphos

EthopropAzinphos Me

Pirimphos MeTetrachlorvinphos

FonofosBensulide


EthionTribufos

Temephos

20°40°

75°

100°

0.00%

50.00%

100.00%

Rec

over

y

Analytes in the order of ElutionCell Temp.

20°40°75°100°

Figure 105 ASE temperature effect on recoveries

MMP Acephate

Oxydem Me Dimethoate

TrichlorfonDichlorvos

MthidathionEthoprop

PropetamphosAzinphos Me

Pirimphos MeTetrachlorvinphos

FonofosBensulide


EthionTribufos

Temephos

10 min static time

2500 PSI0.0%

50.0%

100.0%

Rec

over

y

Analytes in the order of elution

Figure 106 ASE dwell time and pressure effect on recoveries

184

Second step cleanup: normal phase chromatography on SPE cartridges.

After ASE extraction with ethyl acetate, the samples are clean enough to – after the lipid

rich portion of the serum. If these samples are injected without further cleanup, the lipids

will first concentrate in the column, causing retention time shifts and peak shape changes.

Next the lipids migrating into the MS, contaminate the ion source and will cause rapid

signal degradation. For this reason a second step of the cleanup process was developed.

The approach to further purify the ASE extract was to transfer it to a sorbent filled

cartridge and wash it with a polar solvent. The elution process was to be fractionated and

tested for the analytes as well for contaminants.

The process was carried out as follows:

The instrument used was Rapid Trace (originally from Zymark corporation, now Caliper

Life Sciences Hopkinton, MA) 3mL SPE cartridges were filled with 200mg sorbents

each. The ASE extract (0.35 mL) was then transferred to the Rapid Trace. The automated

system placed the extract on top of pre-wetted sorbent. Then the cartridge was washed

with 8mL of ACN. 1 mL fractions were collected and analyzed for all the LC-group

compounds. Half of the fractions were also dried down (1hr 105°C) to check solids

content.

185

Silica Surface

OHOHOHOH

SiO

SiO

Si O SiH O Si

Supelco PSA Structure

OSi

O O NHNH2

Varian HLB Sorbent

O

O N

N

Figure 107 Structure of various sorbents

186

The following sorbents were tested:

- Precipitated silica (Varian, Inc. Palo Alto, CA ) (Figure 107)

- Alumina (Varian, Inc. Palo Alto, CA)

- LRA or Lipid removal agent, a synthetic calcium silicate from (Advanced

Minerals, Santa Barbara, CA) [36]

- PSA or primary, secondary amine. It is a surface treated silica from Supelco,

primarily used for foodstuff analysis (Figure 107)

- Varian HLB (Figure 107)

The cumulative solids were plotted on Figure 108.

Next, the cumulative amount of analytes were plotted for each sorbent and the

cumulative solids/contaminants curve superimposed on each. Figure 109

All the conventional sorbents (silica, alumina) showed varying affinity to the different

OP pesticides. LRA did not show a different retention for OP pesticides and

contaminants.

The choice came down between PSA and Varian HLB. PSA worked excellent, except

for its strong retention of acephate. Because acephate is one of our important compounds,

the choice became the Varian HLB bulk sorbent.

In the extraction method, the first 1.5mL is saved, concentrated and used for HPLC

and GC both. As it seen on Figure 109, this cut allows close to 100% of the analytes and

less than 10% of the contaminants.

There is a possibility of carryover of trace water. Ethyl acetate can contain up to 7%

water at room temperature. And the repeated solvent exchanges may not remove all of it.

Trace of water can damage GC columns over time. To remove residual water, a

187

combination of 200 mg bulk HLB and the same amount of anhydrous sodium sulfate was

tried. Dichlorvos. Tetrachlorvinphos and phosmet showed decreased recoveries (Table

23, Table 24) so this step is not applied in the final method.

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

1 2 3 4 5 6 7 8mL ACN eluent

mg

Solid

s

PSASilicaAluminaHLBHLB +Na2SO4LRA

Figure 108 Comparison of various sorbents for removing contaminants from serum extract

188

Silica

0.00%

20.00%

40.00%

60.00%

80.00%

100.00%

120.00%

140.00%

160.00%

0.0 1.0 2.0 3.0 4.0 5.0Eluent Volume [mL]

Rec

over

y

0

0.2

0.4

0.6

0.8

1

1.2

Sol

ids

[mg/

mL]

MMPAcephateOxydemethon MethylDimethoateTrichlorfonDichlorvosMethidathionPropetamphosEthopropAzinphos MePirimphos MeTetrachlorvinphosFonofosBensulideCoumaphosChlorpyrifosEthionTribufosTemephosSolidsPoly. (Solids)

Aluminum Oxide

0.00%20.00%40.00%60.00%80.00%

100.00%120.00%140.00%160.00%

0.0 1.0 2.0 3.0 4.0 5.0Eluent Volume [mL]

Rec

over

y

0

0.2

0.4

0.6

0.8

1

1.2

Sol

ids

[mg/

mL]

MMPAcephateOxydemethon MethylDimethoateTrichlorfonDichlorvosMethidathionPropetamphosEthopropAzinphos MePirimphos MeTetrachlorvinphosFonofosBensulideCoumaphosChlorpyrifosEthionTribufosTemephosPoly. (Solids)

Figure 109 Elution of OP pesticides over different sorbents

189

PSAPrimary Secondary Amine

0.00%

20.00%

40.00%

60.00%

80.00%

100.00%

120.00%

140.00%

160.00%

0.0 1.0 2.0 3.0 4.0 5.0Eluent Volume [mL]

Rec

over

y

0

0.2

0.4

0.6

0.8

1

1.2

Sol

ids

[mg/

mL]


LRALipid removal Agent

0.00%

20.00%

40.00%

60.00%

80.00%

100.00%

120.00%

140.00%

160.00%

0.0 1.0 2.0 3.0 4.0 5.0

Eluent Volume [mL]

Rec

over

y

0

0.2

0.4

0.6

0.8

1

1.2

Sol

ids

[mg/

mL]


Figure 109, continued: Elution of OP pesticides over different sorbents

190

Oasis HLB

0.00%

20.00%

40.00%

60.00%

80.00%

100.00%

120.00%

140.00%

160.00%

0.0 1.0 2.0 3.0 4.0 5.0Eluent Volume [mL]

Rec

over

y

0

0.2

0.4

0.6

0.8

1

1.2

Sol

ids

[mg/

mL]


Figure 109, continued: Elution of OP pesticides over different sorbents

191

Average Recoveries Standard Deviations

200 mg HLB

200 mg HLB

+ 200 mg Na2SO4

200 mg HLB

200 mg HLB + 200 mg Na2SO4

Mevinphos 69.2% 36.8% 7.0% 17.2% Chlorethoxyfos 55.8% 67.9% 4.4% 9.0% Phorate 59.7% 75.9% 3.3% 7.7% Cadusafos 82.7% 81.9% 6.3% 12.7% Dicrotophos 82.2% 73.7% 12.0% 12.1% Sulfotepp 62.4% 50.1% 2.4% 10.7% Diazinon 71.6% 91.5% 16.8% 58.3% Terbufos 57.1% 69.5% 13.2% 38.0% Fonofos 72.2% 94.1% 16.7% 62.1% Phostebupirim 62.5% 73.3% 9.3% 20.1% Chlorpirifos Me 50.3% 46.9% 6.0% 1.7% Me parathion 45.9% 64.6% 14.1% 3.4% Malathion 64.9% 37.4% 12.1% 9.5% Fenthion 55.2% 58.1% 9.7% 4.4% Et Parathion 47.0% 70.6% 21.3% 26.2% Fenamiphos 56.7% 69.5% 13.7% 3.0% Profenofos 31.7% 12.6% 11.5% 5.9% Phosmet 30.1% 2.2% 21.1% 0.8% Phosalone 62.6% 74.7% 13.0% 9.4%

Table 23 Two step cleanup: recoveries of GC group OP pesticides. Concentrations are 500ng/mL

Average Recoveries Standard Deviations

200 mg HLB

200 mg HLB

+ 200 mg Na2SO4

200 mg HLB 200 mg HLB

+ 200 mg Na2SO4

MMP 65.9% 66.0% 1.0% 2.3% Acephate 43.9% 50.7% 2.5% 14.7% Oxydem Me 65.5% 63.7% 8.7% 9.0% Dimethoate 80.2% 80.9% 9.8% 5.6% Trichlorfon 53.6% 27.5% 11.2% 12.3% Dichlorvos 47.2% 18.3% 40.1% 1.5% Methidathion 77.9% 74.6% 8.0% 5.7% Ethoprop 75.6% 82.3% 2.7% 11.2% Propetamphos 69.9% 77.4% 1.7% 14.2% Azinphos_Me 69.3% 66.2% 8.5% 5.9% PirimphosMe 60.6% 51.4% 9.2% 5.1% Tetrachlorvinphos 48.4% 14.2% 14.7% 11.2% Fonofos 68.1% 74.8% 0.4% 7.2% Bensulide 63.9% 73.3% 4.1% 8.3% Coumaphos 64.8% 72.6% 6.2% 15.1% Chlorpyrifos 62.2% 69.1% 4.7% 10.8% Ethion 58.9% 73.0% 2.5% 13.5% Tribufos 63.9% 76.9% 0.6% 11.5% Temephos 52.1% 65.3% 3.9% 16.1%

Table 24 Two step cleanup: recoveries of LC group OP pesticides Concentrations are 500ng/mL

192

MMP

Acephate

Oxydem Me

Dimethoate

Trichlorfon

Dichlorvos

Methidathion

Ethoprop Propetamphos

Azinphos_Me

PirimphosMe

Tetrachlorvinphos

Fonofos Bensulide

Coumaphos

Chlorpyrifos Ethion

Tribufos Temephos

200 mg HLB


0.0%

50.0%

100.0%

Rec

over

y

Analytes in the Order of Elution

Figure 110 Two step cleanup: recoveries of LC group OP pesticides

MevinphosChlorethoxyfos

PhorateCadusafosDicrotophosSulfoteppDiazinonTerbufosFonofosPhostebupirim

Chlorpirifos MeMe parathion

MalathionFenthionEt ParathionFenamiphosProfenofosPhosmetPhosalone

200 mg HLB


0.0%

50.0%

100.0%

Rec

over

y

Analytes in the Order of Detection

Figure 111 Two step cleanup: recoveries of GC group OP pesticides

193

Serum Extraction

Vortex Mixer

Sample dispersed in 10mL

Hydromatrix

~ 10 mL dry Hydromatrix

22 mL ASE cell

~ 10 mL dry Hydromatrix

1 mL SerumSpiked with 10uL Internal Standard

Vortex Mixing

To ASE

22 mLFlat bottom glass vial

Filter

Cap5 mLEthyl acete

Figure 112 Sample dispersion process prior ASE extraction

Frozen unknown and blank serum samples were first thawed out. 1 mL of each was

aliquoted into 15 mL conical bottom centrifuge tubes. All samples were spiked with

10µL internal standard solution. Each centrifuge tubes were mixed with vortex mixer for

ten seconds. Next, the standard series were spiked with 10µL standard solutions. The

centrifuge tubes were vortex mixed once more for ten seconds.

In equal number as the centrifuge tubes, ASE cartridges were prepared as follows:

Cartridge body and cap were hand tightened, and then one fiberglass based filter disc

was placed inside. 10 mL of Hydromatrix absorbent was placed in the cartridges.

194

The same number of 20 mL flat bottomed vials was also filled with 10 mL Hydromatrix.

Each serum sample was then pipetted on top of the corresponding glass vial, where the

Hydromatrix promptly absorbed it. Each vial then was vortex mixed at high speed to

disperse the serum in the Hydromatrix. The absorbent/serum mix was then transferred in

a previously “half filled” cartridge.

A second filter disk was placed on top of the cartridge; the top cap was put in place

and hand tightened. The cartridges were placed on the ASE rack, the receptacle vials

were put in place and labeled. The extraction solvent reservoir was filled with fresh ethyl

acetate and 1500 PSI nitrogen pressure applied.

The extraction process consisted of two extraction cycles of ten minutes duration at

1500 PSI and room temperature. Machine purge (automatic flush between samples) was

turned on to prevent cross contamination.

195

Pressure Cell CarusselPurge Valve

Solvent Reservoir

Nitrogen

High Pressure Pump

Receptacle Vial Carussel

Oven

Static Valve

Extract

Figure 113 Accelerated solvent extractor

After the extraction the receptacle vials were collected, and the ethyl acetate extract

was transferred into 50 ML Turbovap II tubes (with 0.5 mL tips). The sensors of the

Turbovap II evaporator were customized to signal endpoint at 350µL instead the regular

0.5 mL. Applied nitrogen pressure was 13 PSI, the water bath temperature was adjusted

to 35°C.

The concentration took place in thee steps:

First the ethyl acetate was blown off until the sensor stopped the process, or

approximately .35 mL. Next 5 mL acetonitrile was added to each tube and the process

repeated. Finally an other 5 mL of acetonitrile was added to each tube, but this time the

196

evaporator was programmed, to stop the drying process at 6 minutes after sensors detect

the meniscus. This program resulted in approximately 100 µL final extract volume in

acetonitrile.

The extract samples were filled in HPLC vials, and refrigerated until the next step:

HPLC-MS-MS.

197

Extract from ASEIn ethyl acetate

Dry to 0.5 mL

Add 5 mL acetonitrile

Dry to 0.5 mL

Add 5 mL acetonitrile

Rapid TraceExtraction on HLB

Eluent: ACN

200 mg HLB OasisSorbent

First 2 mL captured

Is GC-CINeeded?

Yes

Dry to ~0.1 mL

Dry to ~0.1 mL

No

Figure 114 Comprehensive cleanup diagram

198

Determination of Instrument Detection Limits LC-MS-MS and GC-MS-MS groups

LODs were determined by using a series of neat standard solutions. The starting

concentrations were 1.00, 2.50, 5.00, 10.00, 25.00, 50.00, 100, 250 and 500 ng/mL,

identical concentrations for each analyte. These standards were previously prepared in

acetonitrile.

The internal standard stock solution was of the concentration of 2.5 µg/mL in acetonitrile.

This solution was diluted twenty times with acetonitrile. The resulting diluted internal

standard solution had the concentration of 125 ng/mL for each isotope labeled standard.

To make the standard series for the “LC analyte group”, 10 µL of the native standard

solution was pipetted into a LC vial, 10 µL of diluted internal standard solution added,

and then followed with 20 µL of acetonitrile. This process was repeated for each

concentration levels. The resulting series had concentrations of 0.250, 0.625, 1.25, 2.50,

6.25, 12.5, 25.0, 62.5, 125 ng/mL in serum €for native compounds and 31.3 ng/mL for

isotope labeled standards. The standard series samples were injected at 1µL each, using

the HPLC-MS-MS method described previously.

For the “GC analyte group” the same standard series was used as a starting point, but

with a different preparation: 20µL of the original standard series, then 20 µL diluted

internal standard was pipetted into a 15 mL conical bottom centrifuge tube. The tube then

was placed in a Turbovap LV evaporator. Temperature was 40°C, nitrogen pressure was

set to 15 PSI. The drying process was carefully monitored and stopped just before

complete dryness. 80 µL toluene was added to the centrifuge tube, mixed thoroughly on a

vortex mixer, then transferred in a GC vial and capped.

199

The 1 µL of the prepared samples were injected in the GC-MS-MS system.

For the LC and GC experiment both, three injections were made.

The LC-MS-MS experiment was only conducted in positive APCI mode because – as

it was shown earlier – not all OP pesticides ionize in ESI mode.

Since not all OP pesticides ionize in negative mode, positive ionization was selected.

LOD-s were determined as follows:

For each compound a linear calibration plot was established by using linear regression

and 1/concentration weigthing. The vertical axis of the calibration curve was the area

ratio (area of analyte peak divided by the internal standard peak area). The horizontal axis

was the concentration. Once the calibration curve was defined, for each concentration and

each analyte a “calculated concentration” was determined, based on the slope and

intercept of the calibration curve and the response factor for the particular measurement.

Next, for each concentration of the standard series the standard deviation of the

“calculated concentrations” was determined and plotted over the actual concentrations.

Limit of detection was determined by establishing a “standard deviation – concentration”

relationship trend line and extrapolating it to zero concentration. LOD is defined as three

times the expected value of the standard deviations of the calculated concentrations at

zero concentration.

200

Effect of drying the samples.

During the cleanup process the samples are dried in the presence of acetonitrile to

remove water and ethyl acetate. To determine the effect of the drying process, and to

determine if complete dryness causes sample loss, the following experiment was carried

out:

A stock solution was prepared which contained all the OP analytes at 500 ng/mL:

concentration, but no internal standard. 0.5 mL of this solution was added to Turbovap II

tubes started to dry the samples at 40°C and 12 PSI nitrogen pressure. Drying was

stopped at 10 and 20 minutes, the samples reconstituted to 0.5 mL and internal standard

added. The experiment was done in multiples of 3.

A second set of experiments was also completed with one difference: at the beginning

approximately 5 µL dodecane was added, the experiment was similarly stopped at 10 and

20 minutes. Everything was carried identically to the first set of experiment.

The reconstituted samples were analyzed with the HPLC-MS-MS system; response

factors were averaged for identical samples then compared

201

Start 10 min 20 min start

10 min, 2µL dodecane

20 min, 2µL dodecane

MMP 100.00% 45.20% 21.11% 100.00% 61.92% 41.76% Acephate 100.00% 86.57% 66.91% 100.00% 94.46% 79.76% Oxydemethon Methyl 100.00% 92.93% 101.83% 100.00% 92.46% 98.10% Dimethoate 100.00% 85.47% 76.35% 100.00% 87.99% 84.31% Trichlorfon 100.00% 28.00% 15.10% 100.00% 52.35% 22.05% Dichlorvos 100.00% 6.67% 3.37% 100.00% 22.47% 3.09% Methidathion 100.00% 94.91% 91.72% 100.00% 99.42% 98.68% Propetamphos 100.00% 86.07% 51.42% 100.00% 97.18% 76.52% Ethoprop 100.00% 35.29% 11.90% 100.00% 76.42% 19.36% Azinphos Me 100.00% 99.95% 108.24% 100.00% 97.25% 102.03% Pirimphos Me 100.00% 77.67% 54.49% 100.00% 84.65% 70.65% Tetrachlorvinphos 100.00% 120.74% 125.45% 100.00% 121.15% 146.78% Fonofos 100.00% 38.89% 5.64% 100.00% 76.56% 20.09% Bensulide 100.00% 104.90% 111.46% 100.00% 100.20% 107.56% Coumaphos 100.00% 103.83% 108.55% 100.00% 101.26% 105.96% Chlorpyrifos 100.00% 74.77% 44.84% 100.00% 87.31% 66.36% Ethion 100.00% 99.13% 95.45% 100.00% 98.24% 103.38% Tribufos 100.00% 99.19% 84.62% 100.00% 97.97% 92.87% Temephos 100.00% 110.98% 118.84% 100.00% 111.10% 111.43%

Table 25 Effect of drying on recoveries of LC compound group, with and without addition of dodecane "catching solvent"

From Table 25 and figures

Analyte Loss During Turbovap Drying

0.0%

50.0%

100.0%

150.0%

0 10 20Drying Time [min]

Ana

lyte

Pre

sent

MMPAcephateOxydemethon MethylDimethoateTrichlorfonDichlorvosMethidathionPropetamphosEthopropAzinphos MePirimphos MeTetrachlorvinphosFonofosBensulideCoumaphosChlorpyrifosEthionTribufos

Figure 115 Recoveries of LC compound group as the function of drying time

To dryness:

~6-7.5 min

202

Analyte Loss During Turbovap Drying2 uL Dodecane added

0.0%

50.0%

100.0%

150.0%

0 10 20

Drying Time [min]

Ana

lyte

Pre

sent

MMPAcephateOxydemethon MethylDimethoateTrichlorfonDichlorvosMethidathionPropetamphosEthopropAzinphos MePirimphos MeTetrachlorvinphosFonofosBensulideCoumaphosChlorpyrifosEthionTribufosTemephos

Figure 116 Recoveries of LC compound group as the function of drying time, 2µL dodecane added

To dryness:

~6-7.5 min

203

Construction of calibration curves

First a series of stock solutions was prepared for each analyte. The stock solutions

were 1000 µg/mL concentration in acetonitrile. By blending the forty stock solutions at

equal volume, a 25 µg/mL blended stock was made. Using the blended stock as a starting

solution, the following standard concentrations were developed by gradual dilution:

1.00, 2.50, 5.00, 10.0, 25.0, 50.0, 100, 250, 500, 1000 ng/mL

10µL of above standards was added to 1 mL serum resulting in the following

concentration series:

0.010, 0.025, 0.050, 0.100, 0.250, 0.500, 1.00 ng/mL analyte in serum

Calibration curves were constructed following the isotope dilution method. For the

forty compounds of interest we obtained seventeen isotopically labeled variants for using

them as internal standards. A mixture of internal standards was dissolved in acetonitrile at

a concentration of approximately 0.25 ng/mL. This level was selected because all the

reference compounds are well detectable at this concentration. Increasing the amount of

reference compounds may causes problems because they contain small amounts of the

non labeled variant. This can introduce an “offset amount of” analyte, not coming from

the matrix.

For each analyte a corresponding internal standard was selected. After the analysis

was completed the date files were collected and evaluated using Thermo Finnigan’s

Xcalibur software. For all calibration curves the linear model was chosen with 1/x

weighing. 1/x weighing puts more emphasis to the lower concentration points. This is

more desirable for trace analysis.

204

MMP_QY = 0.0183435+0.578204*X R^2 = 0.9995 W: 1/X

0 2 4 6 8 100.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5

6.0A

rea

Rat

io

Name Type of Label Location of the Label Acephate D6 Deuterated dimethyl Bensulide D14 Deuterated diisopropyl Chlorpyrifos D10 Deuterated diethyl Chlorpyrifos methyl D6 Deuterated dimethyl Coumaphos D10 Deuterated diethyl Dimethoate D6 Deuterated dimethyl Disulfoton D10 Deuterated diethyl Ethyl parathion D10 Deuterated diethyl Fenthion D6 Deuterated dimethyl Fonofos** 13C6 Malathion D10 Deuterated diethyl Methamidophos D6 Deuterated dimethyl Oxydemeton methyl D6 Deuterated dimethyl Phorate 13C4 Phosmet D6 Deuterated dimethyl Sulfotepp D20 Deuterated tetraethyl Terbufos 13C4

Table 26 Available isotope labeled internal standards

Figure 117 Methamidophos Calibration Plot

205

LOD-s for the LC group

Instrument LOD-s were determined by injecting a series of neat standards dissolved in

acetonitrile, thus not performing the actual cleanup. Final LOD-s were calculated from

triplicates as described above:

OP name LOD [pg]: MMP 0.29Acephate 0.26Oxydemethon Methyl 1.19Dimethoate 0.27Trichlorfon 1.13Dichlorvos 8.99Methidathion 0.16Propetamphos 1.82Ethoprop 0.97Azinphos Me 0.52Pirimphos Me 0.18Tetrachlorvinphos 3.32Fonofos 0.44Bensulide 0.75Coumaphos 1.38Chlorpyrifos 0.95Ethion 0.25Tribufos 0.92Temephos 6.07

Table 27 LC group compound detection limits in pure form

Precision and accuracy of the method

Precision of the method is described by the standard deviation of measurements at a

certain concentration. Accuracy is the quality that compares the measured values with the

expected concentrations. Both, accuracy and precision are displayed as percentages: the

206

mean of measured concentration/expected concentration ratio and the relative standard

deviation, respectively.

We used two concentrations, 0.25 ng/mL and 5 ng/mL to show accuracy and

precision of the method

Standard Deviation Accuracy OP concentration 0.25 5 0.25 5 in serum [ng/mL] [ng/mL] [ng/mL] [ng/mL]

Analyte MMP 5.5% 1.8% 97% 100% Acephate 16% 5.2% 94% 98% Oxydemethon Me 0.2% 10% 124% 95% Dimethoate 10% 6.4% 93% 99% Trichlorfon 8.3% 8.3% 81% 99% Dichlorvos 14% 8.1% 154% 97% Propetamphos 8.7% 6.4% 102% 97% Methidathion 20% 4.9% 93% 94% Ethoprop 9.8% 5.3% 103% 97% Azinphos Methyl 20% 4.4% 98% 97% Pirimphos Methyl 4.7% 3.2% 102% 96% Tetrachlorvinphos 14% 3.5% 93% 96% Fonofos 9.4% 4.9% 100% 99% Bensulide 12% 4.6% 83% 95% Coumaphos 6.2% 4.6% 99% 98% Chlorpyrifos 6.8% 4.6% 95% 99% Ethion 11% 3.7% 102% 97% Tribufos 17% 4.4% 96% 97% Temephos 21% 7.1% 89% 102%

Table 28 Accuracy of the LC group assay, 1 mL serum, n=5

207

Standard Deviation Accuracy

OP concentration 0.25 5 0.25 5 in serum [ng/mL] [ng/mL] [ng/mL] [ng/mL]

Analyte Mevinphos 9.1% 7.9% 94% 107%Chlorethoxyfos 3.1% 5.3% 88% 100%Dicrotophos 19% 1.4% 93% 119%Sulfotepp 5.9% 5.3% 76% 105%Cadusafos 6.7% 5.1% 90% 109%Phorate 5.1% 17% 99% 100%Diazinon 0.7% 1.6% 78% 110%Fonofos 19% 3.4% 97% 102%Phostebupirim 19% 6.8% 103% 99%Chlorpyrofos Me 13% 2.8% 91% 99%Me Parathion 20% 8.7% 110% 101%Malathion 5.0% 13% 138% 101%Fenthion 18% 7.8% 85% 106%Et Parathion 21% 6.2% 95% 102%Fenamiphos 10% 19% 81% 118%Phosalone 5.2% 3.7% 76% 108%

Standard Deviation Accuracy

OP concentration 0.25 5 0.25 5 in serum [ng/mL] [ng/mL] [ng/mL] [ng/mL]

Analyte Mevinphos 18% 2.4% 118% 97%Chlorethoxyfos 10% 11% 75% 104%Dicrotophos 19% 8.6% 107% 101%Sulfotepp 5.1% 2.2% 105% 103%Cadusafos 15% 3.0% 102% 101%Phorate 2.8% 1.2% 95% 99%Diazinon 23% 4.0% 96% 112%Fonofos 5.1% 4.1% 93% 98%Phostebupirim 5.6% 2.2% 106% 97%Chlorpyrofos Me 44% 8.0% 109% 103%Me Parathion 43% 7.2% 167% 100%Malathion 45% 17.6% 99% 107%Fenthion 46% 3.8% 126% 100%Et Parathion 2.3% 22% 98% 116%

To to test the precision of the method, serum samples of three concentrations were

prepared: .250 ng/mL, 1 ng/mL and 5 ng/mL. These samples were prepared, spiked and

208

extracted individually, then the concentrations were determined and plotted. At this point

was it determined that was it determined that length of extraction time plays an important

role when extracting larger number of samples on ASE. The ASE is a carousel system,

where the assembled cells with serum inside are waiting for their turn to be extracted.

While the cells wait at room temperature, enzymatic and hydrolytic decomposition is

taking place. This theory was tested by keeping spiked serum at room temperature for up

to 24 hrs and testing recoveries at different times. As it shows in

Figure 118, about half of the compounds lose more than 50% of their original

concentration. If, - as in the original method – we use two five minutes static cycles when

all the other time adds up a 24 cartridge carousel takes over six hours to process. This

time delay while working with full carousels, can and do effect detection limits and the

precision of the measurement. The main problem is, that not all compounds have their

own isotope labeled equivalent as internal standard. Different compounds hydrolyze at a

different rate and this will effect response factors and calculated concentrations.

The first attempt to solve the decomposition problem was to add 100µL 50 mM EDTA

solution. The idea was that EDTA will chelate the metal ions in the A-esterases and

denature these enzymes. Comparing Figure 118 and Figure 119 it is clear that the EDTA

did not have the desired effect.

The second approach was to fine-tune the first part of the sample preparation. First,

the ASE pressurized dwell time was reduced from 2x5 to 2x2 minutes, but using the same

amount of ethyl acetate. This alone reduced the total run time to 3 hrs.

Second, the sample dispersion method was altered. Instead of adding the serum directly

to the filled cartridge, it was separately pre-dispersed in 10mL of Hydromatrix. Lastly, 5

209

mL of ethyl acetate was added to the cartridge before closing, to denature any enzyme

and to begin the extraction process while waiting in the line. The results were tabulated in

Table 29. At the end of the table, averages were calculated for the range of OP pesticides

covered (HPLC group).

0

1

0 10 20 30

Time [hrs]

Nor

mal

ized

resp

onse

Fac

tor


Figure 118 Decomposition of OP pesticides in serum

210

0

1

0 10 20 30

Time [hrs]

Nor

mal

ized

resp

onse

Fac

tor


Figure 119 Decomposition of OP pesticides in serum, 100µL 50 mmolar EDTA solution is added

211

Relative standard deviations of calculated

concentrations

no vortex, no ethyl acetate 5 mL ethyl acetate but no vortex mixing

5 mL ethyl acetate, vortex mixing

Concentration [ng/mL] Analyte 0.25 1 5 0.25 1 5 0.25 1 5 MMP 6.9% 3.2% 3.8% 4.5% 6.1% 2.5% 7.4% 8.6% 1.6%Acephate 30.4% 9.9% 3.8% 25.2% 14.7% 4.2% 10.2% 11.0% 2.9%Oxydem Me 20.8% 44.3% 26.1% 26.9% 30.3% 11.7% N/F 16.8% 11.3%Dimethoate 11.2% 9.2% 8.1% 13.3% 6.1% 8.7% 11.0% 11.8% 4.0%Trichlorfon 100.0% 108.3% 75.5% 19.8% 12.7% 12.2% 9.0% 11.9% 4.7%Dichlorvos N/F N/F N/F N/F 19.2% 38.9% 15.0% 26.3% 24.3%Methidathion 25.0% 10.3% 5.9% 28.3% 7.4% 12.2% 12.0% 10.1% 7.3%Ethoprop 18.9% 9.4% 5.7% 24.3% 11.5% 13.8% 18.5% 7.2% 7.1%Propetamphos 38.3% 37.9% 12.0% 60.2% 31.2% 26.6% 13.0% 11.1% 10.1%Azinphos Me 69.3% 20.7% 9.2% 21.1% 18.9% 9.8% 26.4% 5.5% 10.7%Pirimphos Me 37.2% 32.8% 21.3% 16.8% 10.4% 32.5% 5.7% 9.9% 6.5%Tetrachlorvinphos 160.4% 123.0% 95.6% 49.0% 18.4% 19.7% 22.2% 10.5% 8.4%Fonofos 23.6% 8.1% 4.2% 7.0% 9.5% 6.4% 18.2% 6.2% 5.3%Bensulide 56.2% 15.8% 18.8% 47.9% 28.8% 18.1% 15.6% 13.0% 9.2%Coumaphos 19.2% 14.5% 10.9% 15.7% 5.1% 12.7% 8.5% 9.6% 6.4%Chlorpyrifos 23.2% 20.8% 12.1% 31.1% 3.5% 8.7% 22.2% 15.1% 10.7%Ethion 15.3% 7.6% 7.1% 8.8% 6.3% 20.3% 5.5% 9.8% 5.1%Tribufos 34.7% 12.5% 13.7% 23.2% 25.8% 41.9% 15.4% 14.2% 4.2%Temephos 39.5% 39.3% 27.5% 53.7% 36.9% 24.5% 26.6% 13.2% 4.1%Average 40.6% 29.3% 20.1% 26.5% 15.9% 17.1% 14.6% 11.7% 7.6%

Table 29 Relative standard deviations of three concentration of analytes - HPLC group

212

Detection limits for the cleaned up serum samples are sown in Table 30

LOD [ppt] MMP 33Acephate 22Oxydemethon Methyl 29Dimethoate 69Trichlorfon Dichlorvos Methidathion 5.2Propetamphos 38Ethoprop 85Azinphos Me 41Pirimphos Me 22Tetrachlorvinphos Fonofos 245Bensulide 1.0Coumaphos 17Chlorpyrifos 61Ethion 7.5Tribufos 27Temephos 16

Table 30 Limits of detection of the LC group compounds, serum samples

213

The problem with azinphos methyl and the power of tandem mass spectroscopy

Analyzing real samples from the field, several clear “hits” were detected for azinphos

methyl. The peaks were clear, well defined, but the concentrations determined by using

the “quantitation ion” (318->132) were substantially different from those indicated by the

conformation ion. (318->77). QC results were within the expected range for both

transitions.

Further investigation has shown that phosmet co-elutes (not fully resolved) with

azinphos methyl and has the same molecular mass, 317. This usually isn’t a problem

using tandem MS, but in this case one of the fragments, 77 is the same. Phosmet is

analyzed in the GC-MS-MS part of the method, so this coincidence only became apparent

while comparing ion ratios of the transitions 318->132 and 318-77 in standards, QC-s

and actual samples.

This problem was solved by changing confirmation ion from 318-77 to 318-> 104.

Table 31 Major transitions and optimum collision energies for azinphos methyl and phosmet

Analyte Parent Ion m/Z

Fragment m/Z

Optimum Collision Energy

[V]

Azinphos Methyl 318

77 51 132 21 104 26 125 24

Phosmet 318

160 33 133 49 77 52

105 56

214

Figure 120 Azinphos methyl chromatography, monitoring all four major transitions for azinphos methyl and phoseth

Figure 121 Phosmet chromatography, monitoring all four major transitions for azinphos methyl and phosmet

RT: 0.00 - 30.01

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RT: 25.91AA: 4663 RT: 27.47

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NL: 8.86E3TIC F: + c APCI sid=10.00 SRM ms2 [email protected] [76.500-77.500] MS ICIS 0801010AzMe05_081015185749








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NL: 2.86E4TIC F: + c APCI sid=10.00 SRM ms2 [email protected] [76.500-77.500] MS ICIS 0801010azme06_081015183815


NL: 6.58E2TIC F: + c APCI sid=10.00 SRM ms2 [email protected] [103.500-104.500] MS 0801010azme06_081015183815






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318 -> 133

215

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Figure 122 Transition 318 -> 77 signal from azinphos methyl and phosmet

216

Chapter 4 Discussion

The firs step of the method is the cleanup and it was to be developed last. Detection

methods have to be established first, so the effectiveness of different approaches to the

cleanup may be evaluated.

The purpose of the cleanup procedure is twofold: First to remove those contaminants

from the sample that interfere with the detection or analytical separation. In serum these

contaminants are mainly salts, proteins and lipids.[37] .

It is also important to concentrate the sample. The available serum sample is usually 1-

5 mL. LC and GC methods only inject a few micro liters of sample. The solution

containing the analytes, have to be concentrated.

The most important cleanup consideration in our work is that it has to be universal.

The serum samples are usually part of studies, supplies are limited and most often they

came from an anonymous individual. The cleanup and analytical methods are destructive.

Once a sample is processed that amount of serum is used up, it can not be replenished.

Considering availability of instrumentation and materials, the usual approach to

cleanup is as follows:

- Solid phase extraction (SPE)

- Lyophilization followed by solid-liquid extraction

- Various forms of liquid-liquid extraction (LLE)

217

Solid phase extraction

Solid phase extraction is the usual first and preferred approach. It is specific, uses less

solvent than the varieties of LLE methods. It is also highly automatable. Methods

developed for individual tubes are easily transferred to 96 well plates and automated

liquid handlers. The problem with SPE in this application is the range of compounds and

consequently the range of their properties. For example: some are extremely water

soluble (methamidophos (MMP), acephate) others are very lipophilic. Some are ionizable

in solution, most are not. This problem was confronted in a previous work [35], where

SPE had to be abandoned. (The matrix was urine.) The reason was that the highly

hydrophilic compounds could not be retained sufficiently on any sorbent and washed.

Acephate and MMP always eluted with wash step. Eventually freeze drying and liquid –

solid extraction with dichloromethane had to be applied.

Lyophilization

Lyophilization or freeze drying was tried as described in the experimental section, but

if did not bring the same results as in urine matrix. The most likely explanation is that

when serum dries, it forms a hard crust leaving limited area for the solvent to interact

with. Urine on the other hand, - when freeze dried – turns into a crumbling powdery

material which has a very large surface are.

Liquid- Liquid extraction

The method was developed with the intention to run many samples in the future, so

LLE in a separatory funnel was never considered. LLE was first tested using ChemElut

LLE cartridges. Using these cartridges shoved promise, but we did not have the

218

equipment to automate. For this reason and because of the need for better control the

LLE portion of the method was transferred to ASE, where timing, temperature and

pressure is controlled. As described previously, ethyl acetate solvent and 1500 PSI

(~10,000 kPa) at room temperature was chosen as operating parameters. While the many

of the more lipophilic (late eluting in HPLC) compounds improve their recovery at higher

pressures and temperatures, three analytes: trichlorfon, dichlorvos and tetrachlorvinphos

show abnormally low recoveries at alleviated pressure temperature or dwell time. The

commonality of these three compounds is that they have chlorine near the ester bond,

possibly weakening it and predisposing it to thermal breakdown. Interestingly, these three

compounds are also among those five compounds in the GC-MS-MS section that were

shown unpredictable retention behavior.

Different structural properties of OP pesticides were examined and correlated with

ASE extraction data. Only the LC group was used because – as it was pointed out earlier

in this chapter- after ASE extraction the samples are not clean enough for GC-MS-MS. It

was found that the strongest correlation is between the number of nitrogen atoms in the

molecule and the recovery percentage. Interestingly, the Correlation factor between

log(Kow) and recovery is only 0.23, while the same between the number of nitrogen

atoms and the recovery is -0.83.

219

Figure 123 Dependence of ASE recovery on the number of nitrogen atoms in the OP molecule. Emphasis on the halogenated species.

y = -0.1122x + 0.9539

0%

20%

40%

60%

80%

100%

0 1 2 3

H

Cl

Cl

MeO P

OMe

O

O

Cl

Cl

Tetrachlorvinphos

Cl

ClMeO P

OMe

O

O

CH3

Dichlorvos

OH

MeO P

MeO

O

O

Cl

Cl

Cl

Trichlorfon

Cl

Cl

Cl

N

EtO P

OEt

S

O

Chlorpyrifos CH3

Cl

O

EtO P

OEt

S

O

OCoumaphos

220

Novelty and significance of the method

Currently there are three different approaches to analyze OP exposure. These methods

can be grouped as indirect and direct (parent compound) methods.

- Urine Analysis of OP metabolites [13]

Most large studies use urinary analysis of OP metabolites. This method gives a

window to OP exposure of few days. It is also a cumulative indicator of OP exposure.

Advantage of the urinary assay is that urine samples are easy and non-invasive to

obtain and clean up is relatively easier than that of blood products. Some OP pesticides

like chlorpyrifos, coumaphos, diazinon, parathion, malathion have specific metabolites

detectable in urinary assay.

While urinary metabolite assay is the most common analysis of OP exposure, it has

two shortcomings. Many OP pesticides do not have easily detectable specific metabolites,

so the source of non specific metabolites has to be estimated from external data, such as

usage statistics. The second and more general shortcoming is, that – as shown later on the

chlorpyrifos example – the source of metabolites may be environmental degradation prior

exposure. The degradation products are not cholinesterase inhibitors, not known to be

toxic. This results in the underestimation of the health outcome – exposure correlation.

- Butyryl Cholinesterase adduct assay [38, 39]

Butyryl cholinesterase is a scavenger of anti-cholinesterase compounds and it is

abundantly available in serum. The OP compounds of interest form adducts at the active

221

sites, lose their specific group. These adducts may be detected for a few months after

exposure.

Comprehensive OP assay for OP adducts is currently under implementation at CDC. It

will provide an alternative to the urinary assay.

It also will inherit one of the shortcomings of the above method: non-specificity. On

the other hand, environmental degradation products do not form serum cholinesterase

adducts.

2 Parent compound methods

Direct analysis of the parent compounds in serum

This is the method developed in this dissertation.

As shown in the graph, after exposure the first opportunity for detection is in the

blood. While the window of detection is short, hours to few days depending the type of

OP, sensitivity and metabolic conditions, direct analysis has significant advantages.

- It is a specific method. The compounds of interests are directly analyzed,

exposure is determined, not deducted from secondary markers. (metabolites)

- It is a non-cumulative method, like a snapshot of state of OP exposure of a

group in question.

- It relies on a regulated fluid: blood, so subsequent concentration correction is

not necessary.

- It eliminates the otherwise always present question of the source of the

secondary biomarker: is it exposure to OP or exposure to environmental

degradants.

222

A practical significance of the method described in the thesis is that it is a sensitive

method, with sub ng/mL limits of detection, useful for exposure studies, that covers all 39

EPA approved OP compounds.

Assay Detected Compound

Specificity Cumulative? Window of exp.

Urinary Metabolites

Nonspecific metabolites: Alkyl phosphate and thiophosphate metabolates

Few specific metabolates

Only for the OP pesticides that have detectable specific metabolites

Yes, days

Butyryl Cholinesterase Adducts

Digested protein segment with OP remaining grouop still attached

Non specific Yes, months

Serum OP (Dissertation

method)

Original, non metabolized OP pesticides

Highly specific No, Hours to days

Table 32 Summary of OP assays for human exposure

The Chlorpyrifos example.

Chlorpyrifos is a broad-spectrum, chlorinated organophosphate insecticide, acaricide

and nematicide. It was first introduced in 1965, to control insects on food and animal feed

crops. It is one of the most commonly used insecticide. It is still used for food crops, golf

courses turf, non-structural wood treatment for termite control.

Once ingested, chlorpyrifos metabolizes according the mechanism illustrated in Figure

124.

223

In water and soil chlorpyrifos also degrades in a similar pattern. (

Figure 125) Both degradation pathway includes the oxon formation step. Environmental

decomposition does not include the glucoronization step. Non specific metabolites are

deithylphosphate and diethyl thiophosphate, while specific metabolites are 3,5,6

trichloropiridinol and 3,5,6 trichloropiridinol glucoronide.

While specific metabolites may provide a way to specify the OP in question, the

source of the specific metabolite may be environmental degradation, prior ingestion and

not metabolism of the original OP. This alternate pathway clouds the speciation. It also

makes it difficult to assign health effects to OP exposure, since the metabolic pathway

and the environmental degradation partially overlap.

The same is true for the non specific metabolites. Urinary assay of non specific

metabolites is used to track cumulative exposure to OP pesticides. To interpret the

results, one needs to have knowledge of the composition and types of the pesticides the

population is exposed to. But even with this information it is not certain if the non

specific metabolites detected in the urine were results of actual OP exposure or prior

environmental degradation.[40]

Clarification of the OP exposure may be approached in several ways.

One is the above mentioned determination of the composition of likely exposure from

agricultural usage and environmental data.

Second approach is – which was developed in this thesis – is to use the presence of

unmetabolized parent compounds in blood as biomarker of OP exposure.

224

Why is the method unique?

The method developed is unique for two reasons:

1. The range of compounds it detects.

This method covers 35 of the 39 EPA registered OP pesticides. Before, the highest

number was 21. [23].

Serum methods covering a limited number of OP pesticides existed before, but they

concentrated on poisoning cases where the possible toxicants are already known and the

concentrations are relatively high. These forensic cases don’t need high sensitivity or

selectivity.

2. Limits of detection

Detection limits are 1-2 orders of magnitude lower than previous methods, covering a

larger number of OP pesticides.

The reasons for this increased limits of detection is three fold:

- The ASE / ethyl acetate extraction provides recovery through the range of OP

pesticides.

- For the “GC group compounds” The secondary, normal phase cleanup (with

HLB sorbent) eliminated much of the lipid contaminants, allowing for

225

chemical ionization for serum sample extracts. This purer extract also helped

lowering the chemical background.

- Tandem mass spectrometry was used throughout the method. Tandem MS

adds an other layer of selectivity and increases LOD

This lower detection limit, combined with the wide range of detectable compounds

makes the method developed in this dissertation a uniquely applicable assay for the

“snapshot detection” of OP pesticide exposure.

The cleanup and sample concentration part of the method may be used to detect of low

levels of similar, ester type analytes. (ref to Angela’s poster)

226

Cl

Cl

Cl

N

EtO P

OEt

S

O

Cl

Cl

Cl

N

EtO P

OEt

O

O

EtO P

OEt

S

OH

Cl

Cl

Cl

N

OH

Diethylthiophosphate


Chlorpyrifos

Chlorpyrifos Oxon

EtO P

OEt

O

OH

Diethylphosphate

Cl

Cl

Cl

N

OH


OHOH

OH

HOOC

O

Cl

Cl

Cl

N

O + O-

O

O

Cl

Cl

Cl

N

S

3,5,6-Trichloropiridinol glucuonide 3,5,6-Trichloropiridinol sulfate

Cholinesterase Inhibition

Figure 124 Metabolism of Chlorpyrifos

Cl

Cl

Cl

N

EtO P

OEt

S

O

Cl

Cl

Cl

N

EtO P

OEt

O

O

EtO P

OEt

S

OH

Diethylthiophosphate

Chlorpyrifos

Chlorpyrifos Oxon

EtO P

OEt

O

OH

Diethylphosphate

Cl

Cl

Cl

N

OH


Figure 125 Environmental degradation of chlorpyrifos

227

CHAPTER 4 APPENDIX

Appendix 4A: Suggested fragmentation of OP pesticides

Pesticide name Chemical Structure

MeO P

SMe

O

NH C

CH3

O184+H

125 143

Acephate

Me

i-Pr

EtO P

OEt

S

O N

N

+H 305

153

169 Diazinon

MeO P

OMe

S

S CH2 N

NN

O

+H 318

104

132

Azinphos-methyl

Cl

ClMeO P

OMe

O

O C

CH3

C

79

109

+H 221 Dichlorvos (DDVP)

i-Pr

i-Pr

O

O

CH2O P

O

S

S CH2 NH S

398+H

158 141

Bensulide

MeO P

OMe

O

O C

CH3

CH

CH3

NC

O CH3

+H 238

72

127

Dicrotophos

EtO P

O

S s-Bu

S

s-Bu+H 271

97

131

Cadusafos

CH3NHMeO P

OMe

S

S CH2 C

O

+H 230125

171

Dimethoate

228

Cl

Cl

Cl

EtO P

OEt

S

O CH C

Cl

+H 335

115143

Chlorethoxyphos

EtEtO P

OEt

S

S SCH2CH2

+H

88

274

Disulfoton

Cl

Cl

Cl

N

EtO P

OEt

S

O

350+H

198

97

Chlorpyrifos

EtO P

OEt

S

S CH2 OEtP

OEt

S

S

143 171

+H 385

Ethion

Cl

Cl

Cl

N

MeO P

OMe

S

O

+H 322, 324

290, 292

Chlorpyrifos methyl

n-Pr

n-PrEtO P

S

O

S

243+H

97

131

Ethoprop

CH3

Cl

O

EtO P

OEt

S

O

O

+H 363

- 2 Et307

227

Coumaphos

NO2EtO P

OEt

S

O

+H 292

236

110

Ethyl Parathion

CH3

i-Pr NH P

OEt

O

O S Et

318

217

+H

202

Fenamiphos

MeO P

OMe

O

O C

CH3

CH C

O

O CH3

+H 22512767

Mevinphos

229

CH3

NO2MeO P

OMe

S

O

+H 278109

79

Fenitrothion

Cl

Cl

Br

MeO P

OMe

O

O CH

Br

C

-2Br+H 221, 223109

Naled

CH3

CH3

SMeO P

OMe

S

O

+H 279

169105

Fenthion

CH3MeO P

OMe

O

S CH2 CH2 CH2S

O

+H 247

109169

Oxydemeton methyl

Et P

OEt

S

S

137

109

+H 247

Fonofos

EtO P

OEt

S

S EtSCH2

+H 261

75

199

97

Phorate

OEt

MeO P

OMe

S

S CHCO

OEt

CH2 C

O

+H 331

99

Malathion

Cl

O

N O

SPO

O

S

Et

Et

+H 368

182128

Phosalone

MeO P

SMe

O

NH2

+H 142125

94

Methamidophos

CH2MeO P

OMe

S

S

O

O

N

+H 318

16077

230

Phosmet

O

CH2MeO P

OMe

S

S N

C

N

S

C O CH3

145

+H 303

85

Methidathion

CH3

CH3

CH3

C

N

N

i-Pr

EtO P

O

S

O

+H 319

153

277

Phostebupirim (tebupirimfos)

NO2MeO P

OMe

S

O

+H 264

109

127

Methyl parathion

MeO P

OMe

S

O S OMeP

OMe

S

O

467+H

341

419

Temephos

CH3

Et

Et

N

MeO P

OMe

S

O N

N

+H 306

164

108

Pirimiphos methyl

EtO P

OEt

S

S CH2 S t-Bu

+H 289

233

97

Terbufos6

Cl

BrEtO P

S

O

O

n-Pr+H 373, 375

303, 305

Profenofos

H

Cl

ClC

MeO P

OMe

O

O C

Cl

Cl

+H 365204 (-Cl)

127

Tetrachlorvinphos

231

OCH3

MeO P

NH

S

O C

Et

CH

C O i-Pr

+H 282156

138

Propetamphos

n-Bu

n-Bu S P

S

O

S n-Bu

+H 315

169 113

Tribufos (DEF)

+H 323

97

EtEtOO

EtO P

S

OEtP

S

O

115

Sulfotepp

MeO P

OMe

O OH

CH C

Cl

Cl

Cl

+H 257109

127

Trichlorfon

232

Appendix 4B: Determination of recovery using internal standard

Recoveries are the fractions of the analytes extracted after a clean up step, or after the

entire cleanup process. To determine recoveries one need two samples of the matrix, A

and B. Sample A is spiked with a predetermined amount of analyte and homogenized.

Sample B is not spiked. Next both of the samples are put through the clean up process.

After completing the cleanup, the same amount of analyte added to B as it was added

sample A before. Next internal standard is added to both A and B and homogenized.

Both extracted and concentrated samples A and B is analyzed for all compounds of

interest and response factor is determined for all in A and B.

The recovery for each analyte is calculated as follows:

B

A

ffrec =

Equation 1 Calculation of recoveries fA and fB are the response factors for the particular analyte in samples A and B

Finally recovery calculations are done on multiple sample pairs, average recoveries

and standard deviations are calculated. The experimental process is illustrated on

Figure 126. This approach of determining recoveries eliminates the matrix effect,

because the cleanup sample (A) and the control (B) both contain extracted matrix.

233

Cleanup

BlankA

Cleanup

BlankB

Analyte Spike

Internal Standard

Analyte Spike

Analysis Analysis

fA fB

Internal Standard

A B

Figure 126 Flow chart of determining recoveries of a given cleanup process

234

Appendix 4C: Determination of limit of detection (LOD)

Limits of detections were calculated as described by Taylor [41] and illustrated on

Figure 127. Multiple calibration series were plotted and for each known concentration

(“actual concentration” on Figure 127 ) the response factor was determined. From the

response factor, “calculated concentrations” were computed, using the calibration curve.

As shown in the second part of Figure 127, the standard deviations of the calculated

concentrations ware plotted against the actual concentrations. By fitting a line to the

standard deviation series of data points, the intercept was determined. The intercept is the

standard deviation of the calculated concentration at zero concentration, or S0. Three

times S0 is defined as the limit of detection.

235

Concentration

Res

pons

e fa

ctor

CalculatedConcentrations

ActualConcentration

Concentration

Sta

ndar

d D

evia

tion

of

Cal

cula

ted

Con

cent

ratio

ns

Intercept

LOD

=3

x In

terc

ept

Figure 127 Determination of limits of detection

236

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