Detection and Quantification of Organophosphate Pesticides in Human Serum

Home , Bensulide, Chlorethoxyfos, Diisopropyl paraoxon

Georgia State University ScholarWorks @ Georgia State University

Chemistry Dissertations Department of Chemistry

7-15-2009

Peter Kuklenyik Georgia State University

Follow this and additional works at: https://scholarworks.gsu.edu/chemistry_diss

Part of the Chemistry Commons

Recommended Citation Kuklenyik, Peter, "Detection and Quantification of Organophosphate Pesticides in Human Serum." Dissertation, Georgia State University, 2009. https://scholarworks.gsu.edu/chemistry_diss/45

This Dissertation is brought to you for free and open access by the Department of Chemistry at ScholarWorks @ Georgia State University. It has been accepted for inclusion in Chemistry Dissertations by an authorized administrator of ScholarWorks @ Georgia State University. For more information, please contact [email protected]. I

DETECTION AND QUANTIFICATION ORGANOPHOSPHATE PESTICIDES IN

HUMAN SERUM

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 unmetabolized 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.

DETECTION AND QUANTIFICATION ORGANOPHOSPHATE PESTICIDES IN

HUMAN SERUM

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

Peter Kuklenyik

2008

DETECTION AND QUANTIFICATION ORGANOPHOSPHATE PESTICIDES IN

HUMAN SERUM

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

ACKNOWLEDGEMENTS

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

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

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 negative full scan spectra ...... 149

Figure 80 Fenamiphos ESI positive (weak),, ESI negative (weak), APCI positive, APCI negative full scan spectra ...... 150

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 negative full scan spectra ...... 157

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 negative full scan spectra ...... 160

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

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 poor 1 Acephate 8.35 7.33 7.53 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 4.03 Peak broadening 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

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 R Y Y 1 Y: O or S P X X: Specific Organic Group R2Y

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.. 2

Type of Phosphorous Group General Structure Pesticide

Phosphate O Dichlorvos, R1O Chlorfenvinphos, POX R2O Heptenophos Phosphonate O Trichlorfon R1O POX R2 Phosphinate O Glufosinate R1 (herbicide, not in the 40 POX R 2 compounds of interest) S-alkyl phosphorothioate O RS P OX RO Phosphoramidate O R1O PNR2 R1O Phosphorotriamidate O R1N PNH2 R2N Phosphorothioamidate O R1O PNR2 R2S O-alkyl phoshorothioate O Omethoate R O 1 P SX R1O S-alkyl phosphorodithioate S RS P OX RO Phosphorodithioate R1O S PSX R1O Phosphonothioate S R P OX RO 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.

O O OR OR ENZYME OH + X P ENZYME OH X P OR OR EsteraseOP Michealis Complex

Phosphorylation

Reactivation O OR ENZYME O X P OR

Aging

ENZYME O X P OR - O

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

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

O MeO P OH Dimethyl phosphate DMP MeO

S MeO O,O-Dimethyl P OH DMTP MeO Thiophosphate

S MeO O,O-Dimethyl P SH DMDTP MeO Dithiophosphate

O EtO P OH Diethyl phosphate DEP EtO

S EtO O,O-Dimethyl P OH DETP EtO Thiophosphate

S EtO O,O-Dimethyl P SH DEDTP EtO Dithiophosphate

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.

O O

EtO P O NO2 EtO P OH

OEt OEt

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

O O S S C OH C OC2H5

MeO P SCH MeO P SCH

CH C OC H OMe CH2 C OC2H5 OMe 2 2 5

O O

Figure 5 Hydrolysis of the Carboxyl Ester Side Chain of Malathion 12

Amidases

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

O O RCNH CH RCOH 3

Figure 6 Amidase Cleavage of Carboxylamide R: Methyl, Ethyl

S O

EtO P O NO2 EtO P O NO2

OEt OEt

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

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

S N

EtO P O Cl

OEt Cl S Chlorpyrifos EtO P OH

OEt Diethyl thiophosphate Cl Cl

O N N

EtO P O Cl HO Cl

OEt

Cl Cl Cl Chlorpyrifos Oxon 3,5,6-Trichloropyridinol N

HO Cl O

EtO P OH Cl

OEt 3,5,6-Trichloropiridinol Diethyl phosphate

Cholinesterase Inhibition

Cl Cl HOOC O N O N - HO O Cl + O S Cl O

HO OH Cl Cl

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

Figure 8 Metabolism of Chlorpyrifos

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:

Development Experimental Choices Considerations Step Cleanup First Choice: SPE For all sample cleanup / pre- LLE 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

Analytical GC Will work for the intact OP pesticides Separation 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.

i-Pr O CH3 S N

MeO P NH C EtO P ON

OEt SMe O Me Acephate Diazinon O O S Cl MeO P OCC MeO P S CH2 N N N Cl OMe OMe CH Azinphos-methyl 3 Dichlorvos (DDVP)

S O O O CH3 OCCH C N i-Pr OPS CH2 CH2 NH S MeO P CH OMe 3 CH3 O i-Pr O Dicrotophos Bensulide

O S O

MeO P SCHC NH CH EtO P S s-Bu 2 3 S s-Bu OMe Cadusafos Dimethoate

S Cl S

EtO P OCHCCl EtO P S CH2 CH2 S Et

Cl Cl OEt OEt Chlorethoxyphos Disulfoton Cl S S N S

EtO P O Cl EtO P SCH2 S P OEt OEt OEt OEt Cl Ethion Chlorpyrifos Table 3 OP pesticides of interest

Cl O S N

MeO P O Cl EtO P S n-Pr

OMe S n-Pr Cl Ethoprop Chlorpyrifos methyl

O S O

S Cl EtO P O NO2 EtO P O OEt OEt CH3 Ethyl Parathion Coumaphos

CH3 O O O

MeO P OCCHC CH3 i-Pr NH P O CH3 OMe CH OEt 3 Fenamiphos Mevinphos S O Cl MeO P O NO 2 MeO P OCHC Cl

OMe OMe Br Br CH 3 Naled Fenitrothion S O O

MeO P O S CH3 MeO P SCH2 CH2 S CH2 CH3 OMe OMe CH3 Fenthion Oxydemeton methyl S S

Et P S EtO P S CH2 S Et

OEt OEt Fonofos Phorate Table 3 (cont.) OP pesticides of interest 21

S O

S O EtO P SON C OEt MeO P SCH O OEt CH C OEt OMe 2 Malathion Cl Phosalone

O S O

MeO P NH2 MeO P S CH2 N

SMe OMe O Methamidophos Phosmet O S C S CH S N 3 MeO P SNCH 2 EtO P O CH2 C CH3 C O CH N 3 N OMe O i-Pr CH3 Phostebupirim (tebupirimfos) Methidathion S S S

MeO P O NO2 MeO P O S O P OMe

OMe OMe OMe Methyl parathion Temephos

CH3 S S

MeO P O N EtO P SCH2 S t-Bu

OMe N OEt

N Et Terbufos Et Pirimiphos methyl Table 3 (cont.) OP pesticides of interest

O H Cl Cl O C EtO P O Br MeO P OC Cl

S n-Pr OMe Cl Cl Profenofos Tetrachlorvinphos

S O H MeO P O C C C O i-Pr n-Pr SPS n-Pr

HN Et CH3 O S n-Pr Propetamphos Tribufos (DEF) S S O OH Cl

EtO P O P OEt MeO P OCCH Cl

OEt OEt OMe Cl Sulfotepp Trichlorfon Table 3 (cont.) OP pesticides of interest

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

Non Specific metabolites Specific Pesticides 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 Sample Analytical Separation MS Results Type Cleanup No Author Publ. Amount Type Recovery Type Column Ioniz. MS Type #of OP-s LOD [%] (Ops of Interest) [18] Mushoff 2002 Whole Headspace 0.1-19.6 GC HP5-MS EI HP5972 22 10-30 Blood SPME 30mx.25mmx.25μm (13) ng/mL 0.5 g [19] Barr 2002 Serum, SPE 14-27 GC DB1701 (J&W) EI High Res. 9 0.5 –2 Plasma, Oasis 30mX.25mmX.25μm Finnigan (9) ppt 4g HLB MAT-900 [20] Hernandez 2001 Whole HS-SPME GC HP Ultra 2 EI Ms-MS 3 0.02-0.7 Blood 30mX.32mmX.50μm Ion trap (3) ng/mL 0.5 [21] Tarbah 2001 Blood, LLE 50-133 GC HP5-MS EI SIM 23 - Plasma 30mX.25mmX.25μm HP MSD (10) stability 0.7g 5970 studies [22] Lacassie 2001 Whole SPE 41.2-107.9 GC Supelco PTE5 EI SIM 29 5-25 Blood (HLB, 30mX.32mmX.25μm Shm QP- ng/mL C18) 5000 [23] Lacassie 2001 Serum SPE 40-99% ? GC Supelco PTE5 EI SIM 29 5 ng/mL 1 mL (HLB) not 30mX.25mmX.25μm Shm QP- (21) detailed 5000 [24] Liu 2001 Serum6 LLE, 100% GC RTX CLPesticides EI SIM 4 2-5 mL hexane 2X 30mX.25mmX.25μm 5971A ng/mL

[25] López 2000 Serum SPME of 3 60-100%. GC Brand? FPD 7 1-15 (.5 mL, dil.) mL of dil. relative 30mX .32mmX .5µm det. (7) ng/mL sample to water Table 6 Assay of OP Pesticides In Serum, Plasma or Whole Blood - Gas Chromatography

Ref First Year Matrix Sample Analytical Separation MS Results Type Cleanup No Author Publ. Amount Type Recovery Type Column Ioniz MS Type # of OP-s LOD [%] . (Ops of Interest) [26] Sancho 2000 Serum 0.5mL 87-113 col Columns: ESI Micromass Chlor- 1.5 0.5 mL ser.+ switching #1: Quattro pyriphos ng/mL .75mL LC Discovery Zspray & metab ACN C18 Ms-ms 10µL inj #2: Supelco 2 ABZ [27] Abu-Qare 2001 (Rat) SPE C18 80.1-83.9 LC C18 2 + 20-50 Plasma (Sep- UV detection metab ng/mL /0.5ml Pack)

Table 7 Assay of OP Pesticides In Serum, Plasma or Whole Blood - Liquid Chromatography 27

Ref First Year Matrix Sample Analytical Separation MS Results Type Cleanup No Author Publ. Amount Type Recovery Type Column Ioniz. MS Type No of OP-s LOD [%] (Ops of Interest) [28] Amini 2003 Plasma RAM 60-92 LC C18 ESI & ms-ms 9 OP 0.2 –1.8 /.5ml (restricted APCI MM, esters ng/mL access Quattro materials) [29] Jonsson 2003 Plasma Micro- Stir bar GC DB5 MS NPD 8 OP 0.2 – 0.9 5g porous assisted 30m X .25mm esters ng/g membrane 32-92 % X .1µm Counter Current 23-78 % [30] Jonsson 2001 Plasma Membrane 72-83 % GC DB5 MS NPD 9 OP 0.06-4 5g Extraction 30m X .32mm esters ng/g Device X .1µm

[31] Jonsson 2003 Plasma Hollow 40-80 % GC DB17 MS EI TSQ 700 8 OP 0.06-4 50 µL ! Fiber 30mX.25mmX esters ng/g Extraction .15µm Device

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

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.

350

300

250

200

150

Temperature [°C] 100

0 024681012 Time [min]

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 Me OP 1:10 dil stock Sol in ACN in ACN Q1 fs POS CI RT: 0.00 - 13.02 8.97 NL: 100 8.90E8 TIC MS 90 Cl 041229_07 80 S N Full Scan 70 MeO P O Cl (quad 1) 60 OMe 50 Cl 40

Relative Abundance Relative 30

10 3.15 10.04 3.63 3.91 4.75 5.476.11 6.877.66 7.86 8.38 9.37 10.80 11.20 11.92 12.27 12.65 0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 Time (min)

041229_07 #686 RT: 8.97 AV: 1 NL: 1.82E8 T: + c CI Q1MS [50.00-500.00] 323.69 100

50 325.67

Relative Abundance Relative 349.73 30 285.67 287.68 20 124.66 289.66 353.71 10 327.67 62.70 78.72 92.70 110.69 142.61 181.62 196.62 213.65 228.62 269.67293.62 365.72 395.82 431.64 447.69 464.76 490.77 0 50 100 150 200 250 300 350 400 450 m/z

F:\NewGC\060630\060816_msmsFS08 8/21/2006 5:19:53 PM Chlorpyrifps Methyl Chlorpyrifps Methyl, 2ppm in toluene msmsFS RT: 0.00 - 13.01 9.38 NL: 100 2.91E6 TIC MS 90 Product Ion 060816_ms msFS08 80 Scan 70

Relative Abundance Relative 30

10 8.138.67 9.31 9.99 5.12 5.29 5.41 6.12 6.58 7.277.81 10.35 11.10 11.63 12.32 12.79 0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 Time (min)

060816_msmsFS08 #1106-1108 RT: 9.38-9.39 AV: 3 SB: 43 9.28-9.35 , 9.43-9.52 NL: 8.03E5 T: + c CI ms2 [email protected] [50.00-400.00] 291.90 100 37 90 S Cl + S 80 N P + 70 MeO OMe P O Cl

60 OMe

50 Cl 125.00 40

Relative Abundance Relative 30

20 213.94 109.24 211.91 289.90 10 324.06 79.38 180.19 62.38102.68 112.78 126.97 143.02 162.09 192.60 239.61 277.17 292.61 0 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 m/z

Figure 10 Transition selection for chlorpyrifos methyl 36

RT: 0.00 - 13.01 AA: 1317554 NL: 1.01E6 100 TIC F: + c CI SRM ms2 [email protected] 50 [289.70-290.30] MS ICIS 060816_msmsFS09 0 AA: 1897489 NL: 1.44E6 100 TIC F: + c CI SRM ms2 [email protected] 50 [289.70-290.30] MS ICIS 060816_msmsFS09 0 AA: 1704100 NL: 1.29E6 100 TIC F: + c CI SRM ms2 [email protected] 50 [289.70-290.30] MS ICIS 060816_msmsFS09 0 AA: 1176298 NL: 8.70E5 100 TIC F: + c CI SRM ms2 [email protected] 50 [289.70-290.30] MS ICIS 060816_msmsFS09 0 AA: 594232 NL: 4.19E5

Relative Abundance 100 TIC F: + c CI SRM ms2 [email protected] 50 [289.70-290.30] MS ICIS 060816_msmsFS09 0 AA: 253427 NL: 1.73E5 100 TIC F: + c CI SRM ms2 [email protected] 50 [289.70-290.30] MS ICIS 060816_msmsFS09 0 AA: 82282 NL: 5.20E4 100 TIC F: + c CI SRM ms2 [email protected] 50 [289.70-290.30] MS ICIS 060816_msmsFS09 0 AA: 29149 NL: 1.29E4 100 TIC F: + c CI SRM ms2 [email protected] 50 [289.70-290.30] MS ICIS 060816_msmsFS09 0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 Time (min) Figure 11 Area counts for clorpyrofos methyl 322 -> 290 transition at different collision energies

2500000

2000000

1500000

1000000 Area Count 500000

0 10 15 20 25 30 Collision Energy [V]

Figure 12 Otimization of collision energies for clorpyrofos methyl 322 -> 290 transition 37

45678910111213

Oxydemeton methyl Dichlorvos (DDVP) Naled Methamidophos Mevinphos Acephate Chlorethoxyfos Ethoprop Dicrotophos Sulfotepp Cadusafos Phosmet Phorate Dimethoate Propetamphos Terbufos Diazinon Fonofos** Disulfoton Phostebupirim Chlorpyrifos methyl Methyl parathion Pirimiphos methyl Fenitrothion Malathion Fenthion Chlorpyrifos Ethyl parathion Methidathion Tetrachlorvinphos Fenamiphos Trichlorfon Profenofos Tribufos (DEF) Ethion Phosalone Coumaphos

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)

CH3 CH3

Si O Si O Si O Si O

CH CH CH 3 m CH3 n 3 m 2 n 3 C N

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

S O C OEt MeO P SCH O m/z + O CH C S OMe 2 285 C OEt MeO P SCH O CH C OEt OMe 2

O C OEt m/z H C 2 O + 173 CH C OEt

O C OEt m/z H C 2 O + 145 CH C OH

+ S SH2 + MeO P m/z MeO P m/z 125 127 OMe OMe

Figure 15 Major fragments of malathion in EI mode 41

+ S H2C CH3 O O S C OEt C OEt MeO P SCH m/z MeO P SCH O O CH2 C OEt 359 CH C OEt OMe OMe 2

+ SH O C OEt MeO P SCH m/z O CH C OEt 331 OMe 2

S + SH O + MeO P SH C OEt MeO P SCH m/z O CH CH 285 m/z OMe 2 127

Figure 16 Major fragments of malathion in CI positive mode

S O C OEt MeO P SCH O CH C OEt OMe 2

S - MeO P S

OMe m/z 157

Figure 17 Major fragments of malathion in CI negative mode

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

)

14.00

12.00

Methidathion Trichlorfon 10.00 Methidathion y = -0.4671Ln(x) + 9.414 Methyl Parathion 8.00 Dimethoate Acephate A 6.00 Retention Time [min] Time Retention 4.00 B

2.00

0.00 0.001 0.01 0.1 1 10 100 1000 10000 Vapor Pressure [mPa]

Figure 18 GC retention times as the function of vapor pressure data (logarithmic scale) 43

H Cl Cl O C S O

MeO P OC Cl MeO P S CH2 N

OMe OMe O Cl 2 Phosmet 1 Tetrachlorvinphos O CH3 O

MeO P NH C MeO P NH2

O SMe SMe 4 Methamidophos 3 Acephate O O

MeO P SCH2 CH2 S CH2 CH3

OMe 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

0.9

0.85

0.8

0.75

0.7

0.65 correlation factor 0.6

0.55

0.5 01234 "a" parameter

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

13.00

12.00

TR = 0.55R + 7.4 11.00

10.00

9.00

8.00

7.00 Retention time [min]

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

5.00

4.00 -4 -2 0 2 4 6 8 "R" factor

Figure 20 Predicting OP retention times in GC

13.00

12.00

TR = 0.61R + 7.3 11.00

10.00

9.00

8.00

7.00 Retention time [min]

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

5.00

4.00 -4 -2 0 2 4 6 8 "R" factor

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 Reagent Gas Filament Electron Temperature Pressure Current Energy [mTorr] [°C] [µA] [eV]

CI positive 150 1500 300 -200

CI negative 150 1500 300 -200

EI 150 N/A 1300 -70

041229_01 #446-458 RT: 6.89-6.99 AV: 13 SB: 177 4.83-5.59 , 6.28-7.02 NL: 4.82E6 T: + c CI Q1MS [50.00-500.00] 142.66 100 95 90 85 80 75 70 65 60 55

bundance 50 45

el ati ve A 40 R 35 30 25 20 183.70 15 135.70 10 170.71 5 60.80 124.73 211.81 242.89 282.95 330.97 374.30406.87 431.26 492.95 0 50 100 150 200 250 300 350 400 450 m/z

041229N_01 #440-458 RT: 6.82-6.98 AV: 19 SB: 177 4.84-5.59 , 6.27-7.00 NL: 5.85E4 T: - c CI Q1MS [50.00-500.00] 167.84 100 95 90 85 80 75 70 65 60 55

bundance 50 45

el ati ve A 40 R 35 30 25 20 15 10 5 146.88 78.80 105.05 218.03 251.18287.94 352.09 391.20 421.93 479.68 0 50 100 150 200 250 300 350 400 450 m/z

041227_01 #328-357 RT: 5.84-6.09 AV: 30 SB: 177 4.81-5.57 , 6.24-6.97 NL: 2.89E5 T: + c EI Q1MS [50.00-500.00] 141.75 100 95 90 85 80 75 70 65 60 55

bundance 50 45

el ati ve A 40 R 35 30 25 126.68 20 15 10 5 61.76 110.95 176.23210.41 263.96 300.87 341.85 369.15 428.93 463.62 493.66 0 50 100 150 200 250 300 350 400 450 m/z

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: 1.45E5 T: + c CI Q1MS [50.00-500.00] 332.81 100 95 90 85 80 75 70 65 60 55

bundance 50 316.78 45

el ati ve A 40 R 35 30 360.96 25 20 55.80 15 10 94.79 123.92 174.80 373.01 5 211.50 248.98 293.05 425.50 483.36 0 50 100 150 200 250 300 350 400 450 m/z

N/F

041227_02 #358-360 RT: 6.10-6.12 AV: 3 SB: 26 5.93-6.06 , 6.21-6.29 NL: 3.00E4 T: + c EI Q1MS [50.00-500.00] 316.82 100 95 90 85 80 75 70 65 60 55

bundance 50 45

el ati ve A 40 R 35 30 25 331.96 20 15 10 192.78 268.93 136.90 5 90.86 165.95 218.19 345.82 366.00 405.03 431.02 488.31 0 50 100 150 200 250 300 350 400 450 m/z

Figure 23 Azinphos Methyl - Positive CI, Negative CI, EI spectra 51

041229_04 #577-579 RT: 8.04-8.06 AV: 3 NL: 2.79E6 T: + c CI Q1MS [50.00-500.00] 270.83 100 95 90 85 80 214.76 75 70 65 60 nce a

d 55 un b 50 45 lative A lative

e 40 R 35 30 56.81 242.79 25 20 180.74 15 158.65 92.85 298.89 10 310.89 5 124.75 357.02391.09 414.04 470.87 0 50 100 150 200 250 300 350 400 450 m/z 041229N_04 #576-579 RT: 7.96-7.98 AV: 4 NL: 2.02E7 T: - c CI Q1MS [50.00-500.00] 212.95 100 95 90 85 80 75 70 65

ce 60 n a

d 55 n u b 50 A

tive 45 la e 40 R 35 30 25 20 15 10 5 62.75 94.72126.74 180.92 240.99 285.83313.10 366.89 390.13 427.02 446.98 498 0 50 100 150 200 250 300 350 400 450 m/z

041227_04 #576-578 RT: 7.95-7.97 AV: 3 SB: 26 5.93-6.06 , 6.21-6.29 NL: 5.87E7 T: + c EI Q1MS [50.00-500.00] 158.70 100 95 90 85 80 75 70 65 e

c 60 n a

d 55 n u b 50 A e

tiv 45 la

e 40 R 269.86 35 30 126.72 25 87.78 20 96.70 212.80 56.85 15 10 181.79 60.78 5 236.88 278.91 338.96 363.00419.60 452.01 4 0 50 100 150 200 250 300 350 400 450 m/z

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

041229_05 #530-533 RT: 7.62-7.65 AV: 4 NL: 1.19E8 T: + c CI Q1MS [50.00-500.00] 152.69 100 95 90 85 336.67 80 298.67 75 70 65 e

c 60 n a d 55 n u b 50 A e

tiv 45 la e 40 R 35 30 25 20 15 364.69 10 264.67 170.71 376.70 5 96.63 488.73 142.65 198.75 236.64 64.78 416.75 446.67 0 50 100 150 200 250 300 350 400 450 m/z

041229N_05 #536-540 RT: 7.61-7.65 AV: 5 NL: 4.79E6 T: - c CI Q1MS [50.00-500.00] 269.80 100 95 90 85 80 75 70 65 e

c 60 n a d

n 55 u b 50 A e 306.80 tiv 45 la e 40 R 35

30 168.86 25 69.69 20 197.82 234.79 15 71.79 10 158.80 5 73.78 205.80 370.84 126.74 336.86 420.96 471.63 0 50 100 150 200 250 300 350 400 450 m/z

041227_05 #538-540 RT: 7.63-7.64 AV: 3 SB: 14 7.51-7.56 , 7.70-7.75 NL: 3.02E7 T: + c EI Q1MS [50.00-500.00] 152.73 100 95 90 85 80 75 70 65 e

c 60 n a

d 55 n u b 50 96.67 A e

tiv 45 la e 40 298.71 R 35 124.71 30 25 165.60 20 262.73 15 244.62 272.65 10 64.84 335.66 5 206.65 370.29 420.76 446.44 488.88 0 50 100 150 200 250 300 350 400 450 m/z

Figure 25 Chlorethoxyfos - Positive CI, Negative CI, EI spectra 53

041229_06 #736-738 RT: 9.37-9.39 AV: 3 SB: 24 9.05-9.14 , 9.41-9.52 NL: 1.75E8 T: + c CI Q1MS [50.00-500.00] 349.71 100 95 90 85 80 75 70 65

ce 60 n a d 55 n u b 50 A

tive 45 la

e 40 R 35 30 25 313.71 20 379.76 152.69 15 10 197.63 389.76 5 96.63 257.62 287.66 64.76 124.67 170.64 437.69 469.85 0 50 100 150 200 250 300 350 400 450 m/z

041229N_06 #742-745 RT: 9.37-9.39 AV: 4 SB: 24 9.00-9.09 , 9.36-9.46 NL: 3.00E7 T: - c CI Q1MS [50.00-500.00] 312.82 100 95 90 85 80 75 70 65 60 nce a

d 55 un b 50 45 lative A lative

e 40 R 35 30 211.81 25

20 168.85 15

10 94.70 5 285.79 78.73 139.82 192.80 248.84 350.92385.89 408.80 472.87 0 50 100 150 200 250 300 350 400 450 m/z

041227_06 #743-746 RT: 9.37-9.40 AV: 4 SB: 24 9.00-9.08 , 9.35-9.45 NL: 1.68E7 T: + c EI Q1MS [50.00-500.00] 313.74 100 196.66 95 90 85 80 75 70 65

ce 60 n a

d 55 n u b 50 257.66 A 96.67

tive 45 285.70 la

e 40 R 207.71 35 30 25 212.69 20 124.70 15 348.70 168.66 243.67 10 64.85 5 92.74 160.68 400.91 429.91 463.88 494.02 0 50 100 150 200 250 300 350 400 450 m/z

Figure 26 Chlorpirifos - Positive CI, Negative CI, EI spectra 54

041229_07 #686 RT: 8.97 AV: 1 SB: 24 9.08-9.17 , 9.45-9.55 NL: 1.82E8 T: + c CI Q1MS [50.00-500.00] 323.69 100 95 90 85 80 75 70 65

ce 60 n a d 55 n u b 50 A

tive 45 la

e 40 R 35 30 349.73 285.67 25 20 124.66 15 10 363.72 5 78.72 92.71 142.62 196.62 228.63 269.67 395.83447.69 490.76 0 50 100 150 200 250 300 350 400 450 m/z

041229N_07 #694-698 RT: 8.95-8.99 AV: 5 SB: 24 8.99-9.08 , 9.35-9.45 NL: 2.10E7 T: - c CI Q1MS [50.00-500.00] 211.81 100 95 90 85 80 75 70 140.73 65

ce 60 n a

d 55 n u b 50 A

tive 45 la

e 40 R 35 30 25 20 284.78 94.77 15 10 78.81 270.82 5 70.70 96.75 192.80 248.87 320.86 355.80391.08 447.68 469.78 0 50 100 150 200 250 300 350 400 450 m/z

041227_07 #694-697 RT: 8.95-8.98 AV: 4 SB: 24 9.00-9.08 , 9.35-9.46 NL: 2.59E7 T: + c EI Q1MS [50.00-500.00] 285.71 100 95 90 85 80 75 70 65

ce 60 n a d 55 n u b 50 A

tive 45 la e 40 R 35 30 124.70 25 20 15 10 78.74 322.67 5 196.67 167.63 227.69 270.68 375.27418.41 447.78 470.23 0 50 100 150 200 250 300 350 400 450 m/z

Figure 27 Chlorpyrofos Methyl- Positive CI, Negative CI, EI spectra 55

041229_08 #1095 RT: 12.49 AV: 1 SB: 24 9.06-9.15 , 9.42-9.53 NL: 5.34E7 T: + c CI Q1MS [50.00-500.00] 362.80 100 95 90 85 80 75 70 65 e

c 60 n a

d 55 n u b 50 A e

tiv 45 la e 40 R 35 30 25

20 390.85 15 10 402.85 5 328.81 64.79108.68 138.72 180.73 206.74 225.71 280.78 429.10 450.46 493.16 0 50 100 150 200 250 300 350 400 450 m/z

041229N_08 #1110 RT: 12.49 AV: 1 SB: 24 9.00-9.09 , 9.36-9.46 NL: 2.23E7 T: - c CI Q1MS [50.00-500.00] 361.88 100 95 90 85 80 75 70 65

ce 60 n a

d 55 n u b 50 A 45 224.79 lative

e 40 R 35 30 25 20 15 10 5 168.86 190.90 328.02 76.69 94.63 126.75239.77 297.97 377.06 408.27 451.71 495.28 0 50 100 150 200 250 300 350 400 450 m/z

041227_08 #1108 RT: 12.47 AV: 1 SB: 47 12.03-12.26 , 12.61-12.76 NL: 1.10E6 T: + c EI Q1MS [50.00-500.00] 361.79 100 95 90 85 80 75 70 65 60 nce a 55 nd

bu 50 A 45

el ati ve 40 225.76 R 35 30 209.78 25 20 108.83 96.78 15 333.76 10 124.85 305.74 136.86 181.81 5 88.85 240.97 288.73 394.07 432.33 463.12 495.9 0 50 100 150 200 250 300 350 400 450 m/z

Figure 28 Coumaphos - Positive CI, Negative CI, EI spectra 56

041229_09 #634 RT: 8.48 AV: 1 SB: 94 7.82-8.28 , 8.65-8.98 NL: 2.35E8 T: + c CI Q1MS [50.00-500.00] 304.83 100 95 90 85 80 75 70 332.93 65

ce 60 n a d

n 55 u b 50 A

tive 45 la

e 40 R 35 30 25 20 258.79 344.91 15 288.83 10 178.80 151.78 5 198.76 247.76 92.72 350.85 424.98456.96 485.05 0 50 100 150 200 250 300 350 400 450 m/z

041229N_09 #636-638 RT: 8.46-8.48 AV: 3 SB: 94 7.79-8.24 , 8.61-8.94 NL: 3.65E7 T: - c CI Q1MS [50.00-500.00] 168.84 100 95 90 85 80 75 70 65 e

c 60 n a d 55 n u b 50 A

tive 45 la

e 40 R 35 30 25 20 15 10 5 78.65 94.75126.73 177.97 242.99 275.01 303.09 338.99 362.01 416.79 451.90 473.12 0 50 100 150 200 250 300 350 400 450 m/z

041227_09 #637 RT: 8.47 AV: 1 SB: 94 7.79-8.24 , 8.60-8.94 NL: 5.99E7 T: + c EI Q1MS [50.00-500.00] 303.86 100 95 90 85 178.88 80 75 70 65

ce 60 n a

d 55 n u b 50 A 136.79

tive 45 la e 40 151.82 R 198.80 35 30 275.82 25 226.85 20 15 92.77 10 96.67 65.79 5 354.87 376.77 428.92 475.02 0 50 100 150 200 250 300 350 400 450 m/z

Figure 29 Diazinon - Positive CI, Negative CI, EI spectra 57

041229_10 #286 RT: 5.50 AV: 1 SB: 94 7.81-8.27 , 8.64-8.97 NL: 1.93E8 T: + c CI Q1MS [50.00-500.00] 220.69 100 95 90 85 80 75 70 65 60 ance 55

bund 50 45

el ati ve A 40 R 35 30 25 20 184.66 15 108.67 10 248.72 5 126.68 260.72 78.71 150.70 328.71 348.76 406.90442.69 463.25 0 50 100 150 200 250 300 350 400 450 m/z

041229N_10 #284 RT: 5.47 AV: 1 SB: 94 7.79-8.24 , 8.61-8.94 NL: 1.41E6 T: - c CI Q1MS [50.00-500.00] 124.85 100 95 90 85 80 75 70 65 e

c 60 n a d 55 n u b 50 A

tive 45 la

e 40 R 35 30 133.72 25 20 15 169.81 10 5 78.79 204.85 147.75 110.70 262.09285.95 358.01 390.28 437.28 0 50 100 150 200 250 300 350 400 450 m/z

041227_10 #286 RT: 5.49 AV: 1 SB: 94 7.79-8.24 , 8.61-8.94 NL: 4.39E7 T: + c EI Q1MS [50.00-500.00] 108.73 100 95 90 85 80 75 70 65

ce 60 n

a 184.71 d 55 n u b 50 A 45 lative

e 40 R 35 30 25 20 144.72 219.74 15 78.86 10

5 92.72 112.67 173.66 234.85 266.79 328.85 384.13 437.15 478.00 0 50 100 150 200 250 300 350 400 450 m/z

Figure 30 Dichlorvos - Positive CI, Negative CI, EI spectra 58

041229_11 #562-564 RT: 7.87-7.89 AV: 3 SB: 94 7.83-8.29 , 8.66-9.00 NL: 1.86E8 T: + c CI Q1MS [50.00-500.00] 237.79 100 95 90 85 80 75 70 65

ce 60 n a d 55 n u b 50 A

tive 45 la

e 40 R 35 111.75 192.71 30 25 20 265.84 15 10 277.84 139.79 5 71.77 474.97 154.71 197.73 308.86 348.90 391.99429.89 479.32 0 50 100 150 200 250 300 350 400 450 m/z

041229N_11 #566-569 RT: 7.87-7.89 AV: 4 SB: 94 7.79-8.24 , 8.61-8.94 NL: 1.94E5 T: - c CI Q1MS [50.00-500.00] 124.84 100 95 90 85 80 75 70 65

ce 60 n a

d 55 n u b 50 A e

tiv 45 la e 40 R 35 30 25 20 15 10 5 177.89 78.73 146.80 210.71 251.91 305.10360.10 390.30 430.29 459.47 484.1 0 50 100 150 200 250 300 350 400 450 m/z

041227_11 #566-569 RT: 7.87-7.89 AV: 4 SB: 94 7.79-8.24 , 8.60-8.93 NL: 9.23E6 T: + c EI Q1MS [50.00-500.00] 126.74 100 95 90 85 80 75 70 65

ce 60 n a

d 55 n u b 50 A

tive 45 la

e 40 R 35 30

25 236.85 20 192.78 15 66.80 10 110.80 5 82.83 163.78 219.89 268.27 326.78360.90 418.85 450.12 478.90 0 50 100 150 200 250 300 350 400 450 m/z

Figure 31 Dichrotophos - Positive CI, Negative CI, EI spectra 59

041229_12 #601-605 RT: 8.24-8.27 AV: 5 SB: 94 7.86-8.31 , 8.69-9.02 NL: 7.25E7 T: + c CI Q1MS [50.00-500.00] 198.65 100 95 90 85 80 75 229.72 70 65

ce 60 n a d 55 n u b 50 A

tive 45 la

e 40 R 35 30

25 257.76 20 87.71 15

10 86.71 269.76 5 115.71 142.63 170.66 73.78 316.80353.29399.77 426.75 458.81 479.33 0 50 100 150 200 250 300 350 400 450 m/z

041229N_12 #608-614 RT: 8.23-8.28 AV: 7 SB: 94 7.79-8.24 , 8.61-8.94 NL: 2.37E7 T: - c CI Q1MS [50.00-500.00] 156.72 100 95 90 85 80 75 70 65 e

c 60 n a

d 55 n u b 50 A e

tiv 45 la e 40 R 35 30 25 20 15 10 5 102.72 126.74 196.87 215.90 263.03 286.82 355.88 385.93 447.08 467.18 0 50 100 150 200 250 300 350 400 450 m/z

041227_12 #610-614 RT: 8.24-8.27 AV: 5 SB: 94 7.79-8.24 , 8.61-8.94 NL: 1.66E6 T: + c EI Q1MS [50.00-500.00] 86.77 100 95 90 85 80 75 70 65

e 124.71 c 60 n a d 55 92.74 n u b 50 A

tive 45 la

e 40 R 35 228.77 30 25 20 141.67 15 57.91 10 156.71 5 197.76 263.70 328.08348.51 405.09 451.03 487.90 0 50 100 150 200 250 300 350 400 450 m/z

Figure 32 Dimethoate - Positive CI, Negative CI, EI spectra 60

041229_13 #639-643 RT: 8.55-8.59 AV: 5 SB: 94 7.85-8.30 , 8.67-9.01 NL: 1.66E8 T: + c CI Q1MS [50.00-500.00] 88.74 100 95 90 85 80 75 70 65 e

c 60 n a

d 55 n u b 50 A e

tiv 45 la

e 40 R 35 30 25 362.82 20 15 87.73 212.72 273.75 184.65 10 89.75 152.68 228.70 5 244.70 60.75 96.64 124.65 314.81 394.87 426.80 458.80 486.85 0 50 100 150 200 250 300 350 400 450 m/z

041229N_13 #647-650 RT: 8.56-8.59 AV: 4 SB: 94 7.80-8.25 , 8.61-8.94 NL: 1.30E7 T: - c CI Q1MS [50.00-500.00] 184.76 100 95 90 85 80 75 70 65

ce 60 n a

d 55 n u b 50 A

tive 45 la

e 40 R 35 30 25 20 15 10 5 78.73 94.73 126.73 155.82 206.94 244.89 271.88 327.70375.90 425.83 449.54 490.69 0 50 100 150 200 250 300 350 400 450 m/z

041227_13 #646-650 RT: 8.55-8.58 AV: 5 SB: 94 7.79-8.24 , 8.61-8.94 NL: 3.13E7 T: + c EI Q1MS [50.00-500.00] 87.77 100 95 90 85 80 75 70 65 273.78 e

c 60 n a

d 55 n u b 50 A e

tiv 45 la

e 40 R 88.78 35 30 185.72 25 141.69 96.72 20 59.80 124.70 15 10 244.75 5 211.78 311.97361.29 408.18 429.86 461.51 0 50 100 150 200 250 300 350 400 450 m/z

Figure 33 Disulfoton - Positive CI, Negative CI, EI spectra 61

041229_14 #871-875 RT: 10.55-10.59 AV: 5 SB: 94 7.84-8.30 , 8.67-9.01 NL: 1.75E8 T: + c CI Q1MS [50.00-500.00] 198.69 100 95 90 85 384.78 80 75 70 65

ce 60 n a

d 55 n u b 50 A e

tiv 45 la e 40 R 35 230.70 30 25 20 338.74 15 152.69 10 412.81 5 170.65 96.65 120.70 366.81 260.73 292.71 458.82 478.92 0 50 100 150 200 250 300 350 400 450 m/z

041229N_14 #881-887 RT: 10.55-10.60 AV: 7 SB: 94 7.79-8.24 , 8.61-8.94 NL: 5.29E7 T: - c CI Q1MS [50.00-500.00] 184.77 100 95 90 85 80 75 70 65 e

c 60 n a

d 55 n u b 50 A e

tiv 45 la e 40 R 35 30 25 20 15 10 5 78.77 94.78126.73152.86 198.91 229.83 285.88 308.94 354.89 397.79 425.91 450.02 479.93 0 50 100 150 200 250 300 350 400 450 m/z

041227_14 #883-885 RT: 10.56-10.58 AV: 3 SB: 94 7.79-8.24 , 8.61-8.94 NL: 8.62E7 T: + c EI Q1MS [50.00-500.00] 230.75 100 95 90 85 80 75 70 65

ce 60 n a d

n 55 u b 50 A

tive 45

la 383.77 e 40 R 152.73 35 30 25 96.67 20 124.70 15 10 64.85 198.74 5 260.76 337.78 292.73 404.84 449.97 480.72 0 50 100 150 200 250 300 350 400 450 m/z Figure 34 Ethion - Positive CI, Negative CI, EI spectra 62

041229_15 #541-544 RT: 7.69-7.71 AV: 4 SB: 94 7.82-8.28 , 8.64-8.98 NL: 2.12E8 T: + c CI Q1MS [50.00-500.00] 242.78 100 95 90 85 80 75 70 65

ce 60 166.71 n a d

n 55 u b 50 A

tive 45 270.83 la

e 40 R 35 30 25 20 15 282.83 484.94 408.86 10 138.68 196.71 5 96.64 124.66 288.83 334.81 380.83 438.86 0 50 100 150 200 250 300 350 400 450 m/z

041229N_15 #544-548 RT: 7.68-7.72 AV: 5 SB: 94 7.79-8.24 , 8.61-8.94 NL: 2.48E7 T: - c CI Q1MS [50.00-500.00] 198.90 100 95 90 85 80 75 70 65 60 55

bundance 50 45

el ati ve A 40 R 35 30 25 20 15 10 5 62.69 94.74126.75 166.89 212.92 247.90 304.92 336.98 399.04 453.99 480.98 0 50 100 150 200 250 300 350 400 450 m/z

041227_15 #545-547 RT: 7.69-7.71 AV: 3 SB: 94 7.79-8.24 , 8.61-8.94 NL: 5.49E7 T: + c EI Q1MS [50.00-500.00] 157.69 100 95 90 85 80 75 70 65 60 55

bundance 50 241.83 45 199.76

el ati ve A 40 R 125.71 35 96.68 30 25

20 92.73 15 73.79 167.76 10 5 213.80 280.87334.90 354.85 408.84 441.01 464.01 0 50 100 150 200 250 300 350 400 450 m/z

Figure 35 Ethoprop - Positive CI, Negative CI, EI spectra 63

041229_16 #738-742 RT: 9.39-9.43 AV: 5 SB: 94 7.83-8.29 , 8.66-8.99 NL: 1.81E8 T: + c CI Q1MS [50.00-500.00] 291.78 100 95 90 85 80 75 70 65 60 55

bundance 50

45 319.83

el ati ve A 40 R 35 30 25 263.73 20 15 331.83 10 5 108.68 136.71 235.70 64.75 185.66 355.92 399.88 427.84 461.81 498.0 0 50 100 150 200 250 300 350 400 450 m/z

041229N_16 #744-749 RT: 9.38-9.42 AV: 6 SB: 94 7.79-8.24 , 8.61-8.94 NL: 4.71E7 T: - c CI Q1MS [50.00-500.00] 290.90 100 95 90 85 80 75 70 65

ce 60 n a

d 55 n u b 50 A

tive 45 la

e 40 R 35 153.75 30 25 20 15 10 5 168.85 78.66 107.80 137.83 181.82 232.00 261.93 319.02383.06 430.04 460.06 492.23 0 50 100 150 200 250 300 350 400 450 m/z

041227_16 #745-749 RT: 9.38-9.42 AV: 5 SB: 94 7.79-8.24 , 8.60-8.93 NL: 2.95E7 T: + c EI Q1MS [50.00-500.00] 290.82 100 95 90 85 80 75 70 65 60 55

bundance 50 45

el ati ve A 40 R 35 108.73 30

25 96.69 136.76 20 154.73 15 185.73 234.75 10 262.77 217.75 64.79 5 308.06 336.04 408.27 451.04 480.74 0 50 100 150 200 250 300 350 400 450 m/z

Figure 36 Ethyl Parathion - Positive CI, Negative CI, EI spectra 64

041229_17 #813 RT: 10.06 AV: 1 SB: 172 8.93-9.79 , 10.52-11.12 NL: 1.96E8 T: + c CI Q1MS [50.00-500.00] 303.84 100 95 90 85 80 75 70 65

ce 60 n a

d 55 n u b 50 A

tive 45 la

e 40 R 35 331.90 30 25 20 15 10 343.90 5 261.78 287.80 79.79 121.72 153.72 194.79 360.92 390.98 452.98 498.0 0 50 100 150 200 250 300 350 400 450 m/z

041229N_17 #826-828 RT: 10.07-10.09 AV: 3 SB: 23 9.88-10.02 , 10.10-10.14 NL: 4.83E3 T: - c CI Q1MS [50.00-500.00] 126.74 100 288.03 95 90 85 80 75 70 65

ce 60 n a d

n 55 u b 50 A

tive 45 la

e 40 R 35 165.89 30 25 302.06 20 15 147.76 273.86 10 78.72 192.80 229.83 315.03 390.00 5 119.95 367.08 445.19 423.11 480.08 0 50 100 150 200 250 300 350 400 450 m/z

041227_17 #827 RT: 10.08 AV: 1 SB: 172 8.85-9.70 , 10.41-11.01 NL: 8.94E7 T: + c EI Q1MS [50.00-500.00] 302.89 100 95 90 85 80 75 70 65 e

c 60 n a d 55 n u b 50 A

tive 45 la

e 40 R 35 153.78 30 25 287.86 20 216.78 15 259.83 194.87 10 129.77 79.85 179.82 242.81 5 108.77 310.19 374.97422.51 464.39 488.00 0 50 100 150 200 250 300 350 400 450 m/z

Figure 37 Fenamiphos - Positive CI, Negative CI, EI spectra 65

041229_18 #716-719 RT: 9.21-9.23 AV: 4 SB: 23 9.96-10.10 , 10.18-10.23 NL: 2.15E8 T: + c CI Q1MS [50.00-500.00] 277.73 100 95 90 85 80 75 70 65

ce 60 n a d 55 n u b 50 A

tive 45

la 305.80 e 40 R 35 30 25 20 15 317.80 10 247.76 5 124.65 78.70 92.70 169.69 231.75 355.86 385.78 426.87 490.72 0 50 100 150 200 250 300 350 400 450 m/z

041229N_18 #722-726 RT: 9.20-9.23 AV: 5 SB: 23 9.88-10.02 , 10.10-10.15 NL: 5.30E7 T: - c CI Q1MS [50.00-500.00] 276.90 100 95 90 85 80 75 70 65 60

nce 167.77 a 55 nd

bu 50 A 45

el ati ve 40 R 35 30 25 20

15 140.72 10 5 57.60 93.84 124.83 182.91 230.94 261.92 290.98 317.18 397.99 417.98 477.18 0 50 100 150 200 250 300 350 400 450 m/z

041227_18 #723-726 RT: 9.20-9.23 AV: 4 SB: 23 9.88-10.01 , 10.10-10.14 NL: 2.31E7 T: + c EI Q1MS [50.00-500.00] 276.79 100 95 90 85 80 75 70 65

ce 60 n a

d 55 n u b 50 A 259.80

tive 45

la 124.71 e 40 R 35 108.73 30 25 20 15 10 78.77 5 149.77 213.79 181.77 291.96 330.38 369.02 417.72 448.98 491.90 0 50 100 150 200 250 300 350 400 450 m/z

Figure 38 Fenitrothion - Positive CI, Negative CI, EI spectra 66

041229_19 #734-739 RT: 9.35-9.40 AV: 6 SB: 23 9.95-10.09 , 10.17-10.22 NL: 1.69E8 T: + c CI Q1MS [50.00-500.00] 278.74 100 95 90 85 80 75 70 65

ce 60 n a d 55 n

u 306.80 b 50 A

tive 45 la

e 40 R 35 30 25 20 15 318.80 10 5 246.72 124.65 78.72 92.71 168.69 214.73 324.84 370.82402.78 446.82 492.85 0 50 100 150 200 250 300 350 400 450 m/z

041229N_19 #741-746 RT: 9.36-9.40 AV: 6 SB: 16 9.28-9.33 , 9.43-9.50 NL: 4.12E4 T: - c CI Q1MS [50.00-500.00] 140.83 100 95 90 85 80 75 70 126.74 65

ce 60 an

d 55 n

bu 50

tive A tive 45 la

e 40 R 35 30 262.95 25 20 15 10 5 152.88 94.78 276.87 78.70 190.89 229.98 303.99 336.10 372.39 425.28 0 50 100 150 200 250 300 350 400 450 m/z 041227_19 #742-745 RT: 9.36-9.38 AV: 4 SB: 23 9.87-10.01 , 10.09-10.14 NL: 9.17E7 T: + c EI Q1MS [50.00-500.00] 277.78 100 95 90 85 80 75 70 65

ce 60 n a

d 55 n u b 50 A

tive 45 la

e 40 R 35 30 25 20 15 124.70 168.74 10 152.74 5 78.77 92.74 244.82 214.78 292.95 338.64 369.24 402.94 446.14 478.87 0 50 100 150 200 250 300 350 400 450 m/z Figure 39 Fenthion - Positive CI, Negative CI, EI spectra 67

041229_20 #735-739 RT: 9.34-9.37 AV: 5 SB: 16 9.31-9.36 , 9.46-9.53 NL: 5.32E5 T: + c CI Q1MS [50.00-500.00] 278.74 100 95 90 85 80 75 70 65 60 nce a 55 nd

bu 50 45

el ati ve A 40 R 35 30 306.80 25 20 15 10 318.88 5 246.81 68.84 92.82 138.80 201.91 359.21 404.59 434.78 466.89 0 50 100 150 200 250 300 350 400 450 m/z 041229N_20 #638 RT: 8.48 AV: 1 SB: 16 9.28-9.33 , 9.43-9.50 NL: 5.61E7 T: - c CI Q1MS [50.00-500.00] 168.77 100 95 90 85 80 75 70 65 60 55

bundance 50 45 108.74

el ati ve A 40 R 35 30 25 20 15 10 5 91.81136.85 216.91 245.95 309.89 354.94 393.70 472.20 0 50 100 150 200 250 300 350 400 450 m/z 041227_20 #637 RT: 8.46 AV: 1 SB: 16 9.27-9.32 , 9.42-9.49 NL: 3.69E7 T: + c EI Q1MS [50.00-500.00] 245.82 100 95 90 85 80 75 70 65 60 108.72 55

bundance 50

45 136.76

el ati ve A 40 R 35 30 25 20 15

10 173.70 80.80 5 170.68 201.78 281.93 326.95 354.78 382.88 429.03 450.02 491.80 0 50 100 150 200 250 300 350 400 450 m/z Figure 40 Fonofos- Positive CI, Negative CI, EI spectra 68

041229_21 #723-727 RT: 9.24-9.27 AV: 5 SB: 16 9.32-9.37 , 9.47-9.54 NL: 1.34E8 T: + c CI Q1MS [50.00-500.00] 284.73 100 95 90 85 80 75 70 330.79 65 60 55 126.71 bundance 50 45

el ati ve A 40 358.82 R 35 30 25 172.75 20 15 200.79 298.76 370.85 10 5 158.65 92.70 256.72 62.70 216.80 384.84 422.82456.86 486.75 0 50 100 150 200 250 300 350 400 450 m/z

041229N_21 #727-731 RT: 9.24-9.27 AV: 5 SB: 16 9.28-9.33 , 9.43-9.50 NL: 4.17E7 T: - c CI Q1MS [50.00-500.00] 156.72 100 95 90 85 80 75 70 65

ce 60 n a

d 55 n u b 50 A

tive 45 la

e 40 R 35 30 25 20 15 10 5 78.70 97.81 126.74 171.93 199.95 242.94276.93 314.99 369.80 389.98 441.68 464.09 0 50 100 150 200 250 300 350 400 450 m/z

041227_21 #729 RT: 9.25 AV: 1 SB: 16 9.28-9.33 , 9.43-9.50 NL: 3.35E7 T: + c EI Q1MS [50.00-500.00] 172.83 100 95 90 85 80 75 70 65

ce 60 n a d

n 55 u b 50 A 126.78

tive 45 la

e 40 R 35 157.70 30 92.74 25 20 15 98.75 284.79 10 255.78 210.75 78.76 5 237.73 183.73 329.86 358.91 422.87 449.91 496.0 0 50 100 150 200 250 300 350 400 450 m/z

Figure 41 Malathion - Positive CI, Negative CI, EI spectra 69

041229_22 #298-311 RT: 5.60-5.71 AV: 14 SB: 16 9.31-9.36 , 9.47-9.53 NL: 4.24E7 T: + c CI Q1MS [50.00-500.00] 141.67 100 95 90 85 80 75 70 65 60 ance 55

bund 50 A

tive 45

el a 40 R 35 30 25 20 15 93.67 10 181.72 5 124.65 92.73 195.84 234.76 282.74 314.99350.38 415.01 435.22 471.46 0 50 100 150 200 250 300 350 400 450 m/z

041229N_22 #228-240 RT: 5.00-5.10 AV: 13 SB: 79 4.56-4.85 , 5.26-5.62 NL: 2.17E4 T: - c CI Q1MS [50.00-500.00] 171.93 100 95 90 85 80 75 70 65 60 55

bundance 50 45

el ati ve A 40 R 35 30 25 20 15 10 126.74 5 74.8190.89 168.00 180.89 243.99287.98 344.28 370.00 413.19 452.05 0 50 100 150 200 250 300 350 400 450 m/z

041227_22 #297-336 RT: 5.58-5.91 AV: 40 SB: 79 4.56-4.85 , 5.26-5.63 NL: 8.24E5 T: + c EI Q1MS [50.00-500.00] 93.75 100 95 90 85 140.76 80 75 70 94.81 65 60 55

bundance 50 45

el ati ve A 40 R 35 30 25 20 63.83 15 125.80 78.78 10 5 300.85 146.88 192.84 224.92 280.93 340.89 404.93 428.92 491.94 0 50 100 150 200 250 300 350 400 450 m/z

Figure 42 Methamidophos- Positive CI, Negative CI, EI spectra 70

041229_23 #804-807 RT: 9.92-9.95 AV: 4 SB: 79 4.57-4.86 , 5.27-5.63 NL: 1.37E8 T: + c CI Q1MS [50.00-500.00] 144.67 100 95 90 85 80 75 70 65

ce 60 n a

d 55 n u b 50 A

tive 45 la

e 40 R 35 30 25 20 15 10 302.69 330.73 5 84.73 124.65 242.71 270.67 446.75 57.77 170.63 210.69 342.75 386.77 426.69 472.62 0 50 100 150 200 250 300 350 400 450 m/z

041229_23 #804-807 RT: 9.92-9.95 AV: 4 SB: 79 4.57-4.86 , 5.27-5.63 NL: 1.37E8 T: + c CI Q1MS [50.00-500.00] 144.67 100 95 90 85 80 75 70 65 60 nce a 55 nd u b 50 A 45

el ati ve 40 R 35 30 25 20 15 10 302.69 5 84.73 242.71 270.67 330.73 446.75 124.65 170.63 57.77 210.69 342.75 386.77 426.69 472.62 0 50 100 150 200 250 300 350 400 450 m/z

041227_23 #807-810 RT: 9.91-9.93 AV: 4 SB: 79 4.56-4.85 , 5.26-5.62 NL: 7.36E6 T: + c EI Q1MS [50.00-500.00] 144.75 100 95 90 85 80 75 70 65 60 55

bundance 50 45

el ati ve A 40 R 84.79 35 30 25 20 301.76 15 124.71 10 57.83 5 156.69 94.68 206.85 270.72 314.62 340.97 388.81408.09 465.13 493.8 0 50 100 150 200 250 300 350 400 450 m/z

Figure 43 Methidathion - Positive CI, Negative CI, EI spectra 71

041229_24 #694-698 RT: 8.97-9.01 AV: 5 SB: 79 4.57-4.86 , 5.26-5.63 NL: 1.84E8 T: + c CI Q1MS [50.00-500.00] 263.72 100 95 90 85 80 75 70 65

ce 60 n a d 55 n u b 50 A

tive 45 la

e 40 R 291.79 35 30 25 20 15 303.78 10 233.74 5 108.67 78.71 155.68 217.72 341.81 371.78 398.81 462.85 496.8 0 50 100 150 200 250 300 350 400 450 m/z

041229N_24 #694-700 RT: 8.96-9.01 AV: 7 SB: 79 4.57-4.86 , 5.26-5.63 NL: 3.07E7 T: - c CI Q1MS [50.00-500.00] 262.95 100 95 90 85 80 153.76 75 70 65 60 nce a 55 nd u b 50 A 45

el ati ve 40 R 35 30 25 20 15

10 140.78 5 93.75 126.74 178.93 215.89 247.87 276.95 345.07403.97 452.02 476.01 0 50 100 150 200 250 300 350 400 450 m/z

041227_24 #697-700 RT: 8.97-9.00 AV: 4 SB: 79 4.56-4.85 , 5.25-5.62 NL: 2.48E7 T: + c EI Q1MS [50.00-500.00] 262.80 100 95 90 85 80 75 70 65 60 ance 55 und b 50 A 108.74 45 124.71 el ati ve 40 R 35 30 25 20 15 10 78.75 199.77 245.79 5 136.75 167.76 280.93 316.74 374.96407.04 451.13 482.97 0 50 100 150 200 250 300 350 400 450 m/z

Figure 44 Methyl parathion - Positive CI, Negative CI, EI spectra 72

041229_25 #411-415 RT: 6.56-6.59 AV: 5 SB: 79 4.57-4.86 , 5.27-5.63 NL: 1.75E8 T: + c CI Q1MS [50.00-500.00] 192.70 100 95 90 85 80 75 70 65 224.76

ce 60 n a

d 55 n u b 50 A

tive 45 la

e 40 R 35 30 126.70 25 252.80 20 15 10 98.72 264.80 5 154.71 350.80 448.89 94.72 322.85 416.84 489.07 0 50 100 150 200 250 300 350 400 450 m/z 041229N_25 #412-414 RT: 6.56-6.58 AV: 3 SB: 79 4.57-4.86 , 5.26-5.63 NL: 3.27E7 T: - c CI Q1MS [50.00-500.00] 124.83 100 95 90 85 80 75 70 65 60 55

bundance 50 45

el ati ve A 40 R 35 30 25 20 15 10 5 78.77 98.79 138.90 177.89 218.90250.97 285.94 348.99 390.18 417.69 466.28 0 50 100 150 200 250 300 350 400 450 m/z

041227_25 #412-415 RT: 6.56-6.58 AV: 4 SB: 79 4.57-4.85 , 5.26-5.63 NL: 4.67E7 T: + c EI Q1MS [50.00-500.00] 126.75 100 95 90 85 80 75 70 65 60 55 191.78

bundance 50 45

el ati ve A 40 R 35 30 25

20 108.73 15 163.76 10 66.80 223.83 5 78.77 140.79 234.91 285.36342.81 374.95 404.99 461.20 481.09 0 50 100 150 200 250 300 350 400 450 m/z Figure 45 Mevinphos - Positive CI, Negative CI, EI spectra 73

041229N_26 #562-566 RT: 7.84-7.87 AV: 5 SB: 79 4.56-4.85 , 5.26-5.63 NL: 7.05E6 T: - c CI Q1MS [50.00-500.00] 78.77 100 80.76 95 90 85 80 75 70 65 60 nce a 55 nd

bu 50 250.80 A 45

el ati ve 40 R 35 30 204.83 25 159.70 20 115.71 15 10

5 124.84 364.73 97.70 169.79 218.81 262.86 344.72 414.71460.72 485.03 0 50 100 150 200 250 300 350 400 450 m/z

041227_26 #563-566 RT: 7.83-7.86 AV: 4 SB: 79 4.56-4.85 , 5.25-5.62 NL: 1.52E6 T: + c EI Q1MS [50.00-500.00] 108.74 100 144.73 95 90 85 80 75 70 65 60 55

bundance 50 300.67 45

el ati ve A 40 R 35 30 25 188.74 20

15 78.86 10 156.75 94.74 128.68 5 59.86 218.78 254.68 313.06 344.82 405.98 450.96 480.95 0 50 100 150 200 250 300 350 400 450 m/z

041229_26 #562-565 RT: 7.84-7.86 AV: 4 SB: 79 4.56-4.85 , 5.26-5.62 NL: 2.08E7 T: + c CI Q1MS [50.00-500.00] 220.67 100 95 300.59 90 85 80 75 126.68 70 65 60 55

bundance 50 45

el ati ve A 40 380.55 R 144.64 35 30 25 344.55 154.71 20 108.66 15 266.61 184.64 10 188.61 248.69 5 82.67 408.57 328.66 442.67 486.68 0 50 100 150 200 250 300 350 400 450 m/z

Figure 46 Naled - Positive CI, Negative CI, EI spectra 74

041229_27 #206 RT: 4.81 AV: 1 SB: 79 4.57-4.86 , 5.26-5.63 NL: 2.94E7 T: + c CI Q1MS [50.00-500.00] 168.67 100 95 90 85 80 75 70 65 60 55

bundance 50 45

el ati ve A 40 R 35 30 25 20 15 124.66 10 196.72

5 108.67 142.66 208.74 66.78 259.04292.82 336.79 391.11429.17 479.28 0 50 100 150 200 250 300 350 400 450 m/z

041229N_27 #193-203 RT: 4.70-4.78 AV: 11 SB: 79 4.56-4.85 , 5.26-5.62 NL: 1.76E4 T: - c CI Q1MS [50.00-500.00] 63.82 100 95 90 85 80 75 70 65 60 55

bundance 50 45

el ati ve A 40 R 35 30 25 20 15 10 65.80 126.75 5 90.65152.69 210.81 250.70 285.87 338.09 390.17 0 50 100 150 200 250 300 350 400 450 m/z

041227_27 #204 RT: 4.79 AV: 1 SB: 79 4.56-4.85 , 5.26-5.62 NL: 1.98E6 T: + c EI Q1MS [50.00-500.00] 109.74 100 95

90 167.76 85 80 75 70 65 60 55

bundance 50 141.74 45

el ati ve A 40 78.77 R 35 30 25 92.84 20 15 10 5 196.99241.90 266.86 324.35 356.11 391.40 452.91 478.17 0 50 100 150 200 250 300 350 400 450 m/z Figure 47 Oxydemethon methyl - Positive CI, Negative CI, EI spectra 75

041229_28 #584-586 RT: 8.02-8.03 AV: 3 NL: 1.75E8 T: + c CI Q1MS [50.00-500.00] 74.75 100 260.75 90

70 198.69

334.79 60

40 Relative Abundance

20 152.67 214.70 10 230.69 76.74 300.78 120.70 170.64 412.80 458.80 96.63 288.79 320.75 360.71 380.82 477.79 0 50 100 150 200 250 300 350 400 450 m/z

041229N_28 #583-586 RT: 8.01-8.04 AV: 4 NL: 2.54E7 T: - c CI Q1MS [50.00-500.00] 184.76 100

40 Relative Abundance

76.80 110.71 126.74 152.85 192.89 230.88 286.03324.85355.71 390.29 425.88 451.99 485.48 0 50 100 150 200 250 300 350 400 450 m/z

041227_28 #584-586 RT: 8.02-8.03 AV: 3 NL: 6.20E7 T: + c EI Q1MS [50.00-500.00] 74.79 100

90 259.81 80

60 120.76

40 Relative Abundance Relative

230.76 30

96.72 20

124.71 261.80 10 152.76 154.78 186.75 262.81 292.73 334.95 380.93 412.73 462.80 478.99 0 50 100 150 200 250 300 350 400 450 m/z Figure 48 Phorate - Positive CI, Negative CI, EI spectra 76

041229_29 #1013 RT: 11.68 AV: 1 NL: 4.02E7 T: + c CI Q1MS [50.00-500.00] 367.77 100

60 181.69 55

45 369.76 Relative Abundance 40

35 183.68 30

20 366.78 321.74 15 198.70 395.79 152.68 10 209.73 323.74 370.78 397.80 5 169.67 211.74 307.75 371.76 74.85 120.71 221.74 409.79 96.66 137.69 268.78 280.80 339.68 372.78 441.77453.05 487.84 0 50 100 150 200 250 300 350 400 450 m/z

041229N_29 #1015 RT: 11.68 AV: 1 NL: 5.98E7 T: - c CI Q1MS [50.00-500.00] 184.77 100

45 Rel ative Abundance 40

15 186.74 10

5 78.62 110.68 126.73152.80 184.09 187.76 213.91 242.09 259.00 302.94 337.94 366.97 401.99 432.59 457.00 0 50 100 150 200 250 300 350 400 450 m/z

041227_29 #1014-1017 RT: 11.67-11.69 AV: 4 NL: 3.07E6 T: + c EI Q1MS [50.00-500.00] 181.76 100

366.80 50

40 Relative Abundance 183.74 120.77 30 153.76 368.80 20 96.76 152.75 10 64.86 198.75 369.82 213.81 252.88 280.83 321.76 340.83 404.98 428.72 464.99 489.2 0 50 100 150 200 250 300 350 400 450 m/z

Figure 49 Phosalone - Positive CI, Negative CI, EI spectra 77

041229_30 #974 RT: 11.34 AV: 1 SB: 162 10.41-11.23 , 11.49-12.04 NL: 9.72E6 T: + c CI Q1MS [50.00-500.00] 159.71 100 90 80 70 60 50 40 317.75 30 Relative Abundance Relative 20 161.75 10 124.65 285.73 318.77 104.81 162.76 201.74 271.94 357.76 390.90 423.35 476.93 0 50 100 150 200 250 300 350 400 450 m/z 041229N_30 #975 RT: 11.34 AV: 1 SB: 165 10.41-11.23 , 11.49-12.05 NL: 2.40E7 T: - c CI Q1MS [50.00-500.00] 156.82 100 90 80 70 60 50 40 30 Relative Abundance Relative 20 158.81 10 75.70 94.86 146.87 192.89 252.72 284.99 316.87 369.57 388.09 451.98 0 50 100 150 200 250 300 350 400 450 m/z 041227_30 #583 RT: 8.01 AV: 1 SB: 165 10.41-11.23 , 11.49-12.05 NL: 1.11E7 T: + c EI Q1MS [50.00-500.00] 159.78 100 90 80 70 60 50 40 30 Relative Abundance Relative 20 160.81 10 103.75 132.77 192.77 193.80 260.08325.52 351.87 371.12 419.00 477.54 0 50 100 150 200 250 300 350 400 450 m/z

Figure 50 Phosmet - Positive CI, Negative CI, EI spectra 78

041229_31 #659 RT: 8.65 AV: 1 SB: 164 10.40-11.23 , 11.48-12.05 NL: 1.30E8 T: + c CI Q1MS [50.00-500.00] 318.87 100 90 276.80 80 70 60 50 40 30 304.85 Relative Abundance Relative 20 319.89 346.91 10 275.77 358.90 56.80 96.68 151.77 166.69 233.72 414.02 442.84 484.96 0 50 100 150 200 250 300 350 400 450 m/z 041229N_30 #974 RT: 11.33 AV: 1 SB: 165 10.41-11.23 , 11.49-12.05 NL: 1.32E7 T: - c CI Q1MS [50.00-500.00] 156.72 100 90 80 70 60 50 40 30 Relative Abundance Relative 20 158.80 10 78.98 110.64 146.87 192.91 238.82 284.97 317.03 369.71 390.28 0 50 100 150 200 250 300 350 400 450 m/z 041227_31 #660 RT: 8.66 AV: 1 SB: 165 10.40-11.24 , 11.49-12.04 NL: 5.87E7 T: + c EI Q1MS [50.00-500.00] 317.91 100 90 80 70 260.83 60 233.80 50 275.84 40 151.83 30 Relative Abundance Relative 20 136.80 302.89 318.92 214.79 10 56.85 109.77 198.80 319.91 321.91 359.66 422.95 442.84 475.76 0 50 100 150 200 250 300 350 400 450 m/z

Figure 51 Phostebupirim - Positive CI, Negative CI, EI spectra 79

041229_32 #718-723 RT: 9.15-9.20 AV: 6 NL: 1.44E8 T: + c CI Q1MS [50.00-500.00] 305.84 100

40 333.90 Relative Abundance

20 273.80 10 345.88 163.78 54.79 92.71 124.64 195.81 232.74 261.77 359.91 397.92 429.86 468.02 484.9 0 50 100 150 200 250 300 350 400 450 m/z

041229N_32 #718-723 RT: 9.16-9.21 AV: 6 NL: 3.27E5 T: - c CI Q1MS [50.00-500.00] 140.83 100

40 Relative Abundance

126.74 10

92.82 107.77 146.83 199.97244.83 275.08 291.05 324.38 360.99378.31 420.29 454.28 0 50 100 150 200 250 300 350 400 450 m/z

041227_32 #719-723 RT: 9.16-9.19 AV: 5 NL: 3.65E7 T: + c EI Q1MS [50.00-500.00] 304.89 100 289.88

80 275.84 70

40 Relative Abundance

30 232.82 179.88 261.85 20 124.72

162.86 10 92.75 243.84 71.86 150.79 211.82 317.93 341.04 400.91429.89 451.04 479.05 0 50 100 150 200 250 300 350 400 450 m/z

Figure 52 Pirimphos methyl - Positive CI, Negative CI, EI spectra 80

041229_33 #836-840 RT: 10.16-10.19 AV: 5 NL: 1.15E8 T: + c CI Q1MS [50.00-500.00] 374.71 100

40 Relative Abundance

30 166.69 20 402.75

346.65 10 294.74 414.75 138.67 207.61 332.63 62.78 96.63 231.66 266.65 421.09 460.90 498.7 0 50 100 150 200 250 300 350 400 450 m/z

041229N_33 #834-840 RT: 10.15-10.20 AV: 7 NL: 8.67E6 T: - c CI Q1MS [50.00-500.00] 266.74 100

60 78.76

50 307.79

40 Relative Abundance Relative 337.83 30

10 182.89 295.78 250.89 344.87 78.13 116.67 139.81 165.84 213.88 373.92 408.88 452.85 470.81 0 50 100 150 200 250 300 350 400 450 m/z 041227_33 #836-839 RT: 10.16-10.18 AV: 4 NL: 1.16E7 T: + c EI Q1MS [50.00-500.00] 338.77 100

90 207.69

70 373.75 138.74 60

96.68 40 296.72

Relative Abundance 268.67

30 124.71

20 166.77 308.73 221.73 62.80 10 98.71 74.82 251.69 344.71 388.76 422.29 450.94 480.81 0 50 100 150 200 250 300 350 400 450 m/z Figure 53 Profenofos - Positive CI, Negative CI, EI spectra 81

041229_34 #626-629 RT: 8.37-8.40 AV: 4 NL: 1.31E8 T: + c CI Q1MS [50.00-500.00] 221.76 100 155.70

60 309.87

50 137.70 197.76

40 281.82 Relative Abundance

30 239.76

20 194.67 321.87

10 126.75 183.72 267.79 418.88 436.88 84.72 347.84 391.91 478.95 0 50 100 150 200 250 300 350 400 450 m/z

041229N_34 #624-628 RT: 8.36-8.40 AV: 5 NL: 4.82E7 T: - c CI Q1MS [50.00-500.00] 153.78 100

40 Relative Abundance

10 434.98 83.82 99.79126.77193.86 220.97 264.96 281.01 308.98 336.94370.98 407.81 451.81 498.8 0 50 100 150 200 250 300 350 400 450 m/z

041227_34 #625-628 RT: 8.37-8.39 AV: 4 NL: 3.33E7 T: + c EI Q1MS [50.00-500.00] 137.75 100

80 193.76 235.82

40 221.84 Relative Abundance

121.76 20 155.76

105.76 10 63.76 165.73 280.87 324.97 369.21 408.07 451.05 479.14 0 50 100 150 200 250 300 350 400 450 m/z

Figure 54 Propetamphos - Positive CI, Negative CI, EI spectra 82

041229_35 #571-575 RT: 7.91-7.94 AV: 5 NL: 1.68E8 T: + c CI Q1MS [50.00-500.00] 322.79 100

40 Relative Abundance

30 350.82

294.74 10 362.83 276.71 64.80 96.67 120.71 170.70201.68 244.73 384.79 416.78 442.88 474.89 0 50 100 150 200 250 300 350 400 450 m/z 041229N_35 #572-573 RT: 7.92-7.93 AV: 2 SB: 13 7.83-7.89 , 7.96-7.99 NL: 2.27E4 T: - c CI Q1MS [50.00-500.00] 168.88 100 292.96

40 94.78 Relative Abundance

30 126.75

146.79 10 277.83 78.75 231.93 110.59 192.95 262.92 363.78 390.17 452.78 0 50 100 150 200 250 300 350 400 450 m/z

041227_35 #574 RT: 7.93 AV: 1 NL: 9.59E7 T: + c EI Q1MS [50.00-500.00] 321.82 100

Relative Abundance 201.74

30 265.73 237.71 20 173.68 96.67 216.76 293.78 120.75 10 64.85 144.69 80.71 353.86 384.97407.26 442.28 475.07 0 50 100 150 200 250 300 350 400 450 m/z

Figure 55 Sulfotepp - Positive CI, Negative CI, EI spectra 83

041229_37 #629-633 RT: 8.40-8.43 AV: 5 SB: 13 7.82-7.88 , 7.95-7.98 NL: 1.49E8 T: + c CI Q1MS [50.00-500.00] 102.73 100

232.70 288.79 40 Relative Abundance

20 56.81 390.87 186.65 334.78 10 198.68 260.74 74.74 152.68 124.65 306.73362.82 408.85 440.82 486.85 0 50 100 150 200 250 300 350 400 450 m/z

041229N_37 #628-632 RT: 8.40-8.43 AV: 5 SB: 13 7.83-7.89 , 7.95-7.99 NL: 1.78E7 T: - c CI Q1MS [50.00-500.00] 184.77 100

40 Relative Abundance

76.72 110.70 126.74 152.87 192.90 258.94 288.75310.81 357.99 375.89 425.90 452.99 0 50 100 150 200 250 300 350 400 450 m/z

041227_37 #630-633 RT: 8.41-8.43 AV: 4 SB: 13 7.82-7.88 , 7.95-7.98 NL: 9.57E7 T: + c EI Q1MS [50.00-500.00] 230.76 100

60 56.86

40 Relative Abundance

30 287.83

20 102.78 152.74

96.72 185.73 10 124.70 202.71 64.81 174.64 78.72 272.89300.91 334.93385.02 409.01 432.84 493.09 0 50 100 150 200 250 300 350 400 450 m/z

Figure 56 Terbufos - Positive CI, Negative CI, EI spectra 84

041229_38 #813-815 RT: 9.96-9.98 AV: 3 SB: 13 7.83-7.88 , 7.95-7.99 NL: 3.80E7 T: + c CI Q1MS [50.00-500.00] 366.66 100

90 126.67 80

40 Relative Abundance

30 330.66 20 394.68 10 240.61 406.68 108.66 154.71 206.63 268.64 78.70 166.67 300.64 358.74 419.07 474.75 492.71 0 50 100 150 200 250 300 350 400 450 m/z 041229N_38 #811-816 RT: 9.95-9.99 AV: 6 SB: 13 7.83-7.88 , 7.95-7.99 NL: 4.05E7 T: - c CI Q1MS [50.00-500.00] 124.77 100

40 Relative Abundance

78.72110.78 138.86 187.84 221.87 241.84 277.87329.91 350.86 380.87 418.19 451.88 490.92 0 50 100 150 200 250 300 350 400 450 m/z 041227_38 #813-815 RT: 9.96-9.98 AV: 3 SB: 13 7.82-7.88 , 7.95-7.99 NL: 3.53E6 T: + c EI Q1MS [50.00-500.00] 328.71 100

Relative Abundance 108.71

10 206.71 239.69 78.80 203.68 132.72 156.69 228.75 365.71 265.82 313.73 402.08450.81 479.01 0 50 100 150 200 250 300 350 400 450 m/z

Figure 57 Tetrachlorvinphos - Positive CI, Negative CI, EI spectra 85

041229_39 #836-840 RT: 10.16-10.20 AV: 5 SB: 13 7.83-7.89 , 7.95-7.99 NL: 1.60E8 T: + c CI Q1MS [50.00-500.00] 224.76 100 314.83

40 Relative Abundance

30 342.89

20 168.65

354.88 10 201.65 257.74 298.82 56.81 144.78 270.76 88.73 102.72 176.74 370.92 416.94 450.91 482.88 0 50 100 150 200 250 300 350 400 450 m/z 041229N_39 #834-840 RT: 10.15-10.20 AV: 7 SB: 13 7.83-7.89 , 7.96-7.99 NL: 6.26E7 T: - c CI Q1MS [50.00-500.00] 256.90 100

40 Relative Abundance

78.73 110.68 135.83167.82 200.82 224.97 270.98 293.84 336.79361.09 392.98 425.05 451.90 470.68 0 50 100 150 200 250 300 350 400 450 m/z

041227_39 #837-840 RT: 10.17-10.19 AV: 4 SB: 13 7.82-7.88 , 7.95-7.98 NL: 2.70E7 T: + c EI Q1MS [50.00-500.00] 168.72 100 201.73

80 56.86

60 313.88 50 257.82 225.84 40 146.66 Relative Abundance

30 112.64

136.75 20 88.79 86.77 10 57.89 176.81 284.92 326.87 376.90 422.20 448.92 475.16 0 50 100 150 200 250 300 350 400 450 m/z

Figure 58 Tribufos - Positive CI, Negative CI, EI spectra 86

041229_40 #836-837 RT: 10.16-10.16 AV: 2 SB: 73 7.22-7.47 , 7.86-8.21 NL: 1.07E7 T: + c CI Q1MS [50.00-500.00] 314.83 100 224.76 90 80 70 60 50 40 30 Relative Abundance Relative 20 312.83 342.89 225.77 168.66 10 56.81 201.66 257.74 298.82 354.88 102.71 144.79 391.03 437.87 483.05 0 50 100 150 200 250 300 350 400 450 m/z 041229N_40 #835 RT: 10.16 AV: 1 SB: 74 7.22-7.47 , 7.85-8.21 NL: 4.03E7 T: - c CI Q1MS [50.00-500.00] 256.87 100 90 80 70 60 50 40 30 Relative AbundanceRelative 20 258.97 10 78.76 126.76 167.85 224.97 259.86 336.69401.49 451.79 0 50 100 150 200 250 300 350 400 450 m/z 041227_40 #837 RT: 10.16 AV: 1 SB: 73 7.22-7.47 , 7.86-8.21 NL: 1.49E6 T: + c EI Q1MS [50.00-500.00] 168.74 100 201.74 90 56.86 80 70 60 50 313.89 146.66 225.85 257.82 40 30 112.65 Relative Abundance Relative 20 88.89 86.76 10 57.85 259.97 314.91 356.20 422.48 452.09 474.90 0 50 100 150 200 250 300 350 400 450 m/z

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, 541 213 305, 307, 5 Chlorethoxyfos 333.892 309 313, 315, 6 Chlorpyrifos 348.926 350 317 Chlorpyrifos 212, 214 7 methyl 320.895 115, 143, 306 308 321 216 225, 208, 8 Coumaphos 362.014 363 363 333 9 Diazinon 304.101 305 169 215 305 169, 215 Dichlorvos 10 (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, 485 199 262, 154, 16 Ethyl parathion 291.033 173, 243, 305 262 138 17 Fenamiphos 271.134 304, 363, 607 247 124, 165, 18 Fenitrothion 277.017 305 115 262 383 248, 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)

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 301.962 303 366 248 287 303 157 277 138 248 24 Methyl parathion 263.002 115 325 151, 234, 275 248, 154 171, 125, 25 Mevinphos* 224.045 225 449 209 255 225, 257 237 363, 365, 26 Naled 377.783 420 422 424 363 365 367 411, 413, 426 367 Oxydemeton 27 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 318.117 319 319 275 141, 290 32 Pirimiphos methyl 305.096 318, 381 290 306 187 313 315, 317 343, 345, 33 Profenofos 371.935 306 343 345 347 373 405, 407 347 154, 280, 34 Propetamphos 281.085 282 282…327 200 169, 293, 35 Sulfotepp 322.023 323 155 36 Temephos 465.990 451 467 451, 141 37 Terbufos 288.044 115 143 185 245, 289 185 365 349 351 365, 38 Tetrachlorvinphos 363.899 406,408,410 353… 373 397..399… 171, 125 39 Tribufos (DEF) 314.096 315 315, 347, 629 257 143, 152, 40 Trichlorfon 255.923 257… 515 301 303 305 221, 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

Particle Pore Name Manufacturer Length Diam. size Carbon Size End- [mm] [mm] [µm] Load cap High purity,base Aquasil C18 Thermo 100 4.6 5 12% 100 A Yes deactivated C18 High purity,base Betasil C8 Thermo 100 4.6 5 12% 100 A Yes deactivated C8 High purity,base Betasil C18 Thermo 100 4.6, & 2.1 5 20% 100 A Yes deactivated C18 High purity,base Betasil Phenyl Thermo 100 4.6 5 11% 100 A Yes deactivated Merck Chromolit Merck Life High purity silica RP-e Sciences 100 4.6 5 17% 120 A Yes gel C18 silica gel, high purity, spherical Pentafluorophenyl propyl Discovery HS F5 Supelco 100 4.6 3 12% 120 A Yes particle platform High purity,base Fluorphase RP Thermo 50 2.1 5 10% 100 A Yes deactivated perfluorinated C6 High purity,base Prism RP Thermo 50 2.1 5 12% 100 A Yes deactivated Urea+ C12 chain Zorbax SB C3 Agilent 100 4.6 5 4% 80 A No Diisopropyl propyl Table 14 HPLC columns tested

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.

Table 15 Retenetion times of OP pesticides on Betasil C18 and Phenyl Columns (N/F: not found, E/B: extreme peak broadening) 95

Table 16 Retenetion times of OP pesticides on Aquasil C18 and Chromolit RP 18e, Zorbax SBC3 96

Discovery HS F5 3µm Hypercarb Varian C18

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

97 Temephos Tribufos (DEF) Tribufos (DEF) Chlorpyrifos Ethion Ethion Chlorpyrifos Temephos Coumaphos Profenofos Terbufos Phostebupirim Phosalone* Terbufos Phostebupirim Chlorpyrifos methyl Profenofos Cadusafos* Fenthion Coumaphos Bensulide Disulfoton Chlorpyrifos methyl Phosalone* Disulfoton Phorate Sulfotepp Diazinon Phorate Fenthion Fonofos** Fonofos** Diazinon Tetrachlorvinphos Ethyl parathion Pirimiphos methyl Cadusafos* Ethyl parathion Tetrachlorvinphos Fenamiphos Fenamiphos Sulfotepp Pirimiphos methyl Bensulide Fenitrothion Ethoprop Malathion Fenitrothion Azinphos-methyl Propetamphos Propetamphos Malathion Ethoprop Azinphos-methyl Methidathion Chlorethoxyfos Methyl parathion Methyl parathion Naled Methidathion Dichlorvos (DDVP) Dichlorvos (DDVP) Trichlorfon Trichlorfon Mevinphos* Mevinphos* Dimethoate Dimethoate Dicrotophos Dicrotophos Oxydemeton methyl Oxydemeton methyl Acephate Acephate Methamidophos Methamidophos

0 5 10 15 20 25 30 0 5 10 15 20 25 30 Betasil C18 Betasil Phenyl

Figure 61 Betasi C18 and Betasil Phenyl retention profile using a linear water methanol gradient Tribuf os (DEF) Temephos 98 Chlorpyrifos Tribuf os (DEF) Ethion Ethion Temephos Chlorpyrif os Phostebupirim Coumaphos Terbufos Terbuf os Profenofos Phosalone* Chlorpyrifos methyl Phostebupirim Cadusafos* Profenofos Disulfoton Bensulide Phosalone* Fenthion Phor ate Disulf oton Coumaphos Chlorpyrifos methyl Diazinon Sulfotepp Fenthion Cadusaf os * Phor ate Fonofos** Fonof os** Sulfotepp Diazinon Ethyl parathion Ethy l par athion Bensulide Tetrachlorvinphos Tetrachlorvinphos Fenamiphos Fenamiphos Fenitrothion Pirimiphos methyl Pirimiphos methyl Ethoprop Malathion Fenitrothion Phos met Propetamphos Azinphos-methyl Malathion Ethoprop Methyl parathion Propetamphos Azinphos-methyl Methidathion Methidathion Methyl parathion Naled Naled Dichlorvos (DDVP) Dichlorvos (DDVP) Trichlorfon Trichlorf on Mevinphos* Mevinphos* Dimethoate Dimethoate Dicrotophos Dicrotophos Oxydemeton methyl Oxydemeton methyl Acephate Acephate Methamidophos Methamidophos 0 5 10 15 20 25 30 0 5 10 15 20 25 30

Aquasil C18 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

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

12 Dimethoate Capillary Area Count/1000 150 250 400 VAP 250 1.4 1.5 0.3 Vaporizer VAP 350 2.2 1.6 0.3 VAP 450 2 1.4 0.19

2.5

1.5

Area Count/1000 Area 0.5 VAP 450 0 VAP 350

150 VAP 250 Vaporizer Temp. [C] 250 400 Ion Tranfer Tube Temp.[C]

Vaporizer - Heated Capillary Temperature Effect

13 Disulfoton Capillary Area Count/1000 150 250 400 VAP 250 0.015 0.014 0 Vaporizer VAP 350 0.045 0 0 VAP 450 0.11 0 0

0.12

0.1

0.08 0.06 0.04

Area Count/1000 Area 0.02 VAP 450 0 VAP 350

150 VAP 250 Vaporizer Temp. [C] 250 400 Ion Tranfer Tube Temp.[C] 104

3 Bensulide Capillary Area Count/1000 150 250 400 VAP 250 0.74 0.12 0 Vaporizer VAP 350 0.85 0.07 0 VAP 450 0.45 0.01 0

0.9

0.8

0.7

0.6 1000 0.5 ount/ 0.4

Area C Area 0.3

0.2 VAP 450 0.1

0 VAP 350

150 Vaporizer Temp. [C] VAP 250 250 400 Ion Tranfer Tube Temp.[C]

4 Cadusafos Capillary Area Count/1000 150 250 400 VAP 250 3.4 2.5 0 Vaporizer VAP 350 3.4 1.7 0 VAP 450 3.2 1.1 0

3.5

2.5 1000 2 ount/ 1.5

Area C Area 1

0.5 VAP 450

0 VAP 350

150 Vaporizer Temp. [C] VAP 250 250 400 Ion Tranfer Tube Temp.[C] 105

5 Clorethoxyfos Capillary Area Count/1000 150 250 400 VAP 250 000 Vaporizer VAP 350 0 0 0.001327 VAP 450 0 0 0.007349

0.008

0.007

0.006

0.005 1000

0.004 ount/

0.003 Area C Area 0.002 VAP 450 0.001

0 VAP 350

150 Vaporizer Temp. [C] VAP 250 250 400 Ion Tranfer Tube Temp.[C]

6 Clorpyrifos Capillary Area Count/1000 150 250 400 VAP 250 0.003 0.26 0.29 Vaporizer VAP 350 0.022 0.58 0.34 VAP 450 0.11 0.72 0.25

0.8

0.7

0.6

0.5 1000

0.4 ount/

0.3 Area C Area 0.2 VAP 450 0.1

0 VAP 350

150 Vaporizer Temp. [C] VAP 250 250 400 Ion Tranfer Tube Temp.[C] 106

7 Clorpyrifos -Me Capillary Area Count/1000 150 250 400 VAP 250 0 0.002 0.062 Vaporizer VAP 350 0 0.03 0.11 VAP 450 0 0.068 0.093

0.12

0.1

0.08 1000

0.06 ount/

Area C Area 0.04

0.02 VAP 450

0 VAP 350

150 Vaporizer Temp. [C] VAP 250 250 400 Ion Tranfer Tube Temp.[C]

8 Coumaphos Capillary Area Count/1000 150 250 400 VAP 250 0.92 1.4 1.4 Vaporizer VAP 350 1.6 2 1.9 VAP 450 2.1 2.3 1.9

2.5

1000 1.5 ount/

1 Area C Area

0.5 VAP 450

0 VAP 350

150 Vaporizer Temp. [C] VAP 250 250 400 Ion Tranfer Tube Temp.[C] 107

9 Diazinon Capillary Area Count/1000 150 250 400 VAP 250 2.5 2.2 1.2 Vaporizer VAP 350 2.9 2.8 1.2 VAP 450 32.51.1

2.5

2 1000

ount/ 1.5

Area C Area 1

0.5 VAP 450

0 VAP 350

150 Vaporizer Temp. [C] VAP 250 250 400 Ion Tranfer Tube Temp.[C]

10 Dichlorvos Capillary Area Count/1000 150 250 400 VAP 250 0.043 0.088 0.11 Vaporizer VAP 350 0.087 0.12 0.084 VAP 450 0.12 0.097 0.071

0.12

0.1

0.08 1000

0.06 ount/

Area C Area 0.04

0.02 VAP 450

0 VAP 350

150 Vaporizer Temp. [C] VAP 250 250 400 Ion Tranfer Tube Temp.[C] 108

11 Dichrotophos Capillary Area Count/1000 150 250 400 VAP 250 0.13 0.14 0.03 Vaporizer VAP 350 0.23 0.18 0.043 VAP 450 0.19 0.19 0.044

0.25

0.2

1000 0.15 ount/ 0.1 Area C Area

0.05 VAP 450

0 VAP 350

150 Vaporizer Temp. [C] VAP 250 250 400 Ion Tranfer Tube Temp.[C]

12 Dimethoate Capillary Area Count/1000 150 250 400 VAP 250 1.4 1.5 0.3 Vaporizer VAP 350 2.2 1.6 0.3 VAP 450 2 1.4 0.19

2.5

1000 1.5 ount/

1 Area C Area

0.5 VAP 450

0 VAP 350

150 Vaporizer Temp. [C] VAP 250 250 400 Ion Tranfer Tube Temp.[C] 109

13 Disulfoton Capillary Area Count/1000 150 250 400 VAP 250 0.015 0.014 0 Vaporizer VAP 350 0.045 0 0 VAP 450 0.11 0 0

0.12

0.1

0.08 1000

0.06 ount/

Area C Area 0.04

0.02 VAP 450

0 VAP 350

150 Vaporizer Temp. [C] VAP 250 250 400 Ion Tranfer Tube Temp.[C]

14 Ethion Capillary Area Count/1000 150 250 400 VAP 250 0.19 0.51 0.06 Vaporizer VAP 350 0.66 0.62 0 VAP 450 0.91 0.39 0

0.9

0.8

0.7

1000 0.6

0.5 ount/ 0.4

Area C Area 0.3 0.2 VAP 450 0.1 0 VAP 350

150 Vaporizer Temp. [C] VAP 250 250 400 Ion Tranfer Tube Temp.[C] 110

15 Ethoprop Capillary Area Count/1000 150 250 400 VAP 250 0.74 0.6 0.15 Vaporizer VAP 350 0.81 0.46 0.11 VAP 450 0.86 0.4 0.09

0.9

0.8

0.7

0.6 1000 0.5 ount/ 0.4

Area C Area 0.3

0.2 VAP 450 0.1

0 VAP 350

150 Vaporizer Temp. [C] VAP 250 250 400 Ion Tranfer Tube Temp.[C]

16 Ethyl Parathion Capillary Area Count/1000 150 250 400 VAP 250 0.16 0.16 0.026 Vaporizer VAP 350 0.24 0.1 0 VAP 450 0.18 0.05 0

0.25

0.2

1000 0.15 ount/ 0.1 Area C Area

0.05 VAP 450

0 VAP 350

150 Vaporizer Temp. [C] VAP 250 250 400 Ion Tranfer Tube Temp.[C] 111

17 Fenamiphos Capillary Area Count/1000 150 250 400 VAP 250 0.24 0.21 0.014 Vaporizer VAP 350 0.34 0.23 0.007 VAP 450 0.38 0.23 0

0.4 0.35 0.3 0.25 0.2 0.15 0.1 Area Count/1000 Area 0.05 VAP 450 0 VAP 350

150 VAP 250 Vaporizer Temp. [C] 250 400 Ion Tranfer Tube Temp.[C]

18 Fenitrothion Capillary Area Count/1000 150 250 400 VAP 250 0.069 0.059 0.022 Vaporizer VAP 350 0.089 0.06 0.0087 VAP 450 0.051 0.026 0

0.09 0.08 0.07 0.06 0.05 0.04 0.03 0.02 Area Count/1000 Area VAP 450 0.01 0 VAP 350

150 VAP 250 Vaporizer Temp. [C] 250 400 Ion Tranfer Tube Temp.[C] 112

19 Fenthion Capillary Area Count/1000 150 250 400 VAP 250 0 0.058 0.049 Vaporizer VAP 350 0.005 0.17 0.024 VAP 450 0.007 0.45 0

0.45 0.4 0.35 0.3 0.25 0.2 0.15 0.1 Area Count/1000 Area VAP 450 0.05 0 VAP 350

150 VAP 250 Vaporizer Temp. [C] 250 400 Ion Tranfer Tube Temp.[C]

20 Fonofos Capillary Area Count/1000 150 250 400 VAP 250 0.82 0.6 Vaporizer VAP 350 0.96 VAP 450 0.75

0.8

0.6

0.4

Area Count/1000 Area 0.2 VAP 450 0 VAP 350

150 VAP 250 Vaporizer Temp. [C] 250 400 Ion Tranfer Tube Temp.[C] 113

21 Malathion Capillary Area Count/1000 150 250 400 VAP 250 1.48 0.73 0 Vaporizer VAP 350 1.68 0.57 0 VAP 450 1.61 0.25 0

1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 Area Count/1000 Area VAP 450 0.2 0 VAP 350

150 VAP 250 Vaporizer Temp. [C] 250 400 Ion Tranfer Tube Temp.[C]

22 MMP Capillary Area Count/1000 150 250 400 VAP 250 1.2 1.5 0.084 Vaporizer VAP 350 1.3 1.1 0.05 VAP 450 1.5 0.76 0.03

1.6 1.4 1.2 1 0.8 0.6 0.4 Area Count/1000 Area 0.2 VAP 450 0 VAP 350

150 VAP 250 Vaporizer Temp. [C] 250 400 Ion Tranfer Tube Temp.[C] 114

23 Methidathion Capillary Area Count/1000 150 250 400 VAP 250 0.21 0.17 0 Vaporizer VAP 350 0.42 0.097 0 VAP 450 0.47 0.016 0

0.5

0.45

0.4

0.35

1000 0.3

ount/ 0.25 0.2

Area C Area 0.15 0.1 VAP 450 0.05 0 VAP 350

150 Vaporizer Temp. [C] VAP 250 250 400 Ion Tranfer Tube Temp.[C]

24 Me-Parathion Capillary Area Count/1000 150 250 400 VAP 250 0.4 0.43 0.11 Vaporizer VAP 350 0.54 0.35 0.12 VAP 450 0.4 0.17 0.027

0.6

0.5

0.4 1000

ount/ 0.3

Area C Area 0.2

0.1 VAP 450

0 VAP 350

150 Vaporizer Temp. [C] VAP 250 250 400 Ion Tranfer Tube Temp.[C] 115

Area Count/1000 150 250 400 VAP 250 000 Vaporizer VAP 350 000 VAP 450 000

1 0.8 0.6 0.4 0.2 VAP 450

Area Count/1000 Area 0 VAP 350 150 VAP 250 Vaporizer Temp. 250 400 [C] Ion Tranfer Tube Temp.[C]

Vaporizer - Heated Capillary Temperature Effect

31 Phostebupyrim Capillary Area Count/1000 150 250 400 VAP 250 1.5 1.11 0.021 Vaporizer VAP 350 1.5 0.87 0.0092 VAP 450 1.3 0.5 0.008

1.5

0.5 VAP 450 Area Count/1000 0 VAP 350 150 VAP 250 Vaporizer Temp. 250 400 [C] Ion Tranfer Tube Temp.[C] 116

27 Oxydemethon-Me Capillary Area Count/1000 150 250 400 VAP 250 2.2 1.15 0.022 Vaporizer VAP 350 2.5 0.97 0.014 VAP 450 20.610

2.5

1000 1.5 ount/

1 Area C Area

0.5 VAP 450

0 VAP 350

150 Vaporizer Temp. [C] VAP 250 250 400 Ion Tranfer Tube Temp.[C]

28 Phorate Capillary Area Count/1000 150 250 400 VAP 250 0 0.0029 0 Vaporizer VAP 350 0 0.0007 0 VAP 450 0 0.006 0

0.006

0.005

0.004 1000

0.003 ount/

Area C Area 0.002

0.001 VAP 450

0 VAP 350

150 Vaporizer Temp. [C] VAP 250 250 400 Ion Tranfer Tube Temp.[C] 117

29 Phosalone Capillary Area Count/1000 150 250 400 VAP 250 0.11 0.14 0 Vaporizer VAP 350 0.22 0.19 0 VAP 450 0.26 0.19 0

0.3 0.25 0.2 0.15 0.1 0.05 VAP 450 Area Count/1000 0 VAP 350 150 VAP 250 Vaporizer Temp. 250 400 [C] Ion Tranfer Tube Temp.[C]

30 Phosmet Capillary Area Count/1000 150 250 400 VAP 250 000 Vaporizer VAP 350 000 VAP 450 000

1 0.8 0.6 0.4 0.2 VAP 450 Area Count/1000 0 VAP 350 150 VAP 250 Vaporizer Temp. 250 400 [C] Ion Tranfer Tube Temp.[C] 118

31 Phostebupyrim Capillary Area Count/1000 150 250 400 VAP 250 1.5 1.11 0.021 Vaporizer VAP 350 1.5 0.87 0.0092 VAP 450 1.3 0.5 0.008

1.5

0.5 VAP 450 Area Count/1000 0 VAP 350 150 VAP 250 Vaporizer Temp. 250 400 [C] Ion Tranfer Tube Temp.[C]

32 Pirimiphos - Me Capillary Area Count/1000 150 250 400 VAP 250 0.066 0.04 0.022 Vaporizer VAP 350 0.07 0.055 0.019 VAP 450 0.079 0.045 0.021

0.08

0.06

0.04

0.02 VAP 450 Area Count/1000 0 VAP 350 150 VAP 250 Vaporizer Temp. 250 400 [C] Ion Tranfer Tube Temp.[C] 119

38 Tetrachlorvinphos Capillary Area Count/1000 150 250 400 VAP 250 0.13 0.21 0 Vaporizer VAP 350 0.2 0.25 0 VAP 450 0.34 0.24 0

0.35

0.3

0.25 1000 0.2

0.15

Area Count/ 0.1

0.05 VAP 450

0 VAP 350

150 Vaporizer Temp. [C] VAP 250 250 400 Ion Tranfer Tube Temp.[C]

Vaporizer - Heated Capillary Temperature Effect

39 Tribufos Capillary Area Count/1000 150 250 400 VAP 250 0.013 0.006 0 Vaporizer VAP 350 0.005 0.008 0 VAP 450 0.014 0.0046 0

0.014

0.012

0.01

0.008

0.006

Area Count/1000 0.004

0.002 VAP 450

0 VAP 350

150 Vaporizer Temp. [C] VAP 250 250 400 Ion Tranfer Tube Temp.[C] 120

35 Sulfotepp Capillary Area Count/1000 150 250 400 VAP 250 0.7 1.2 0.17 Vaporizer VAP 350 1.4 1.1 0.12 VAP 450 1.6 0.77 0.099

1.6 1.4 1.2 1 0.8 0.6 0.4 Area Count/1000 Area 0.2 VAP 450 0 VAP 350

150 VAP 250 Vaporizer Temp. [C] 250 400 Ion Tranfer Tube Temp.[C]

36 Temephos Capillary Area Count/1000 150 250 400 VAP 250 0.012 0.04 0 Vaporizer VAP 350 0.086 0.077 0 VAP 450 0.025 0 0

0.09 0.08 0.07 0.06 0.05 0.04 0.03 0.02 Area Count/1000 Area VAP 450 0.01 0 VAP 350

150 VAP 250 Vaporizer Temp. [C] 250 400 Ion Tranfer Tube Temp.[C] 121

37 Terbufos Capillary Area Count/1000 150 250 400 VAP 250 0 0.023 0 Vaporizer VAP 350 0.013 0.011 0 VAP 450 0.036 0 0

0.04

0.035

0.03

0.025 1000

0.02 ount/

0.015 Area C Area 0.01 VAP 450 0.005

0 VAP 350

150 Vaporizer Temp. [C] VAP 250 250 400 Ion Tranfer Tube Temp.[C]

38 Tetrachlorvinphos Capillary Area Count/1000 150 250 400 VAP 250 0.13 0.21 0 Vaporizer VAP 350 0.2 0.25 0 VAP 450 0.34 0.24 0

0.35

0.3

0.25

1000 0.2 ount/ 0.15

Area C Area 0.1

0.05 VAP 450

0 VAP 350

150 Vaporizer Temp. [C] VAP 250 250 400 Ion Tranfer Tube Temp.[C] 122

39 Tribufos Capillary Area Count/1000 150 250 400 VAP 250 0.013 0.006 0 Vaporizer VAP 350 0.005 0.008 0 VAP 450 0.014 0.0046 0

0.014

0.012

0.01

1000 0.008 ount/ 0.006

Area C Area 0.004

0.002 VAP 450

0 VAP 350

150 Vaporizer Temp. [C] VAP 250 250 400 Ion Tranfer Tube Temp.[C]

40 Trichlorfon Capillary Area Count/1000 150 250 400 VAP 250 0.61 0.17 0 Vaporizer VAP 350 0.64 0 0 VAP 450 0.47 0 0

0.7

0.6

0.5 1000 0.4 ount/ 0.3 Area C Area 0.2

0.1 VAP 450

0 VAP 350

150 Vaporizer Temp. [C] VAP 250 250 400 Ion Tranfer Tube 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 Time MeOH 1 mL/min [min] [%V/V] 100 0.1% 02 formic 32 49 100 acid in 50 both 55 100 56 2 MeOH [%] 60 2 Time [min]

0 0204060

Time MeOH 11_2, [min] [%V/V] 100 11_3 02 1 ,mL/min 32 24.5 100 50 27.5 100 0.1% [%] MeOH formic 28 2 30 2 Time [min] acid in 0 both 0102030 Time MeOH 11_4 [min] [%V/V] 100 0.2mL/min 010 510 0.1% 24.5 100 50 27.5 100

formic MeOH [%] acid in 28 10 30 10 Time [min] both 0 0 102030

Time MeOH 11_5 [min] [%V/V] 100 0.2mL/min 05 45 530 0.1% 50 formic 15 30 MeOH [%] acid in 20 100 23 100 both Time [min] 24 5 0 30 5 0 102030

Table 18 List of elution curves applied while optimizing LC separations 125

code Gradient Graph 11_6 Time MeOH 1 mL/min [min] [%V/V] 100 05 0.1% 45 550 formic 50 acid in 15 50 MeOH [%] both 20 100 23 100 Time [min] 24 5 0 30 5 0102030 11_7 Time MeOH [min] [%V/V] 100 1 mL/min 05 35 430 0.1% 50 formic 15 70 MeOH [%] acid in 17 100 23 100 both Time [min] 24 5 0 30 5 0102030 11_8 Time MeOH [min] [%V/V] 100 1 mL/min 020 320 23 100 0.1% 50 25 100 formic MeOH [%] acid in 25.5 20 30 20 both Time [min] 0 0 102030

11_9 Time MeOH 1 mL/min [min] [%V/V] 100 020 0.1% 220 575 formic 50 acid in 10 90 MeOH [%] both 15 95 20 100 25 100 Time [min] 0 26 20 30 20 0102030

Table Table 18 (cont) List of elution curves applied while optimizing LC separations

126

code Gradient Graph 11-10 Time MeOH 1 [min] [%V/V] 100 mL/min 020 120

260 MeOH [%] 0.1% 50 formic 10 75 acid in 15 85 20 100 both 25 100 Time [min] 0 26 20 30 20 0102030

11_11 Time MeOH 1 [min] [%V/V] 100 mL/min 020 120 260 0.1% 50 formic 10 65 acid in 15 70 MeOH [%] 20 100 both 25 100 Time [min] 26 20 0 30 20 0102030

11_12 Time MeOH 1 [min] [%V/V] 100 mL/min 050 25 100 28 100 0.1% 50 formic 29 50 30 50

acid in MeOH [%] both Time [min] 0 0102030

11_13 Time MeOH 1 [min] [%V/V] 100 mL/min 075 25 90 25.5 100 0.1% 50 formic 28 100 acid in 29 50 30 50 both MeOH [%] Time [min] 0 0102030

Table Table 18 (cont) List of elution curves applied while optimizing LC separations 127

code Gradient Graph 11_14 Time MeOH 1 mL/min [min] [%V/V] 100 065 0.1% 25 75 25.5 100 formic 50 acid in 27.5 100 both 28 50 30 50 MeOH [%] Time [min] 0 0102030 11_15 Time MeOH 1 mL/min [min] [%V/V] 100 05 0.1% 35 25 100 formic 50 acid in 27.5 100 MeOH [%] both 28 5 30 5 Time [min] 0 0102030 11_16 Time MeOH 1 mL/min [min] [%V/V] 100 02 0.1% 32 24.5 100 formic 50 acid in 27.5 100 28 2 both MeOH [%] 30 2 Time [min] 0 0102030 11_17 Time MeOH 1 mL/min [min] [%V/V] 100 040 0.1% 25 100 28 100 formic 50 acid in 29 40

30 40 MeOH [%] both Time [min] 0 0102030

Table Table 18 (cont) List of elution curves applied while optimizing LC separations

128

code Gradient Graph 11_18 Time MeOH 1 mL/min [min] [%V/V] 100 060 0.1% 25 100 28 100 formic 50 acid in 29 60 30 60 both

MeOH [%] Time [min] 0 0102030 11_19 Time MeOH 1 mL/min [min] [%V/V] 100 060 0.1% 580 25 100 formic 50 acid in 28 100 both 28.5 60

30 60 MeOH [%] Time [min] 0 0102030 11_20 Time MeOH 1 mL/min [min] [%V/V] 100 060 0.1% 360 24 90 formic 50 acid in 25 100 28 100

both MeOH [%] 28.5 60 30 60 Time [min] 0 0102030 11_21 Time MeOH 1 mL/min [min] [%V/V] 100 050 0.1% 25 50 25.5 100 formic 50 acid in 28 100 both 28.5 50

30 50 MeOH [%] Time [min] 0 0102030

Table Table 18 (cont) List of elution curves applied while optimizing LC separations 129

code Gradient Graph 11_22 Time MeOH 1 mL/min [min] [%V/V] 100 060 0.1% 25 60 25.5 100 formic acid 50 in both 28 100 28.5 60

30 60 MeOH [%] Time [min] 0 0102030 11_23 Time MeOH 1 mL/min [min] [%V/V] 100 070 0.1% 25 70 25.5 100 formic acid 50 in both 28 100 28.5 70

30 70 MeOH [%] Time [min] 0 0102030 11_24 Time MeOH 1 mL/min [min] [%V/V] 100 080 0.1% 25 80 25.5 100 formic acid 50 in both 28 100 28.5 80

30 80 MeOH [%] Time [min] 0 0102030 11_25 Time MeOH 1 mL/min [min] [%V/V] 100 090 0.1% 25 90 25.5 100 formic acid 50 in both 28 100 28.5 90

30 90 MeOH [%] Time [min] 0 0102030

Table Table 18 (cont) List of elution curves applied while optimizing LC separations 130

code Gradient Graph 12 Time MeOH 1 mL/min [min] [%V/V] 100 02 0.1% 32 825 formic 50 acid in 10 75 both 15 95 25 100 MeOH [%] 27 100 Time [min] 27.5 2 0 30 2 0102030

13 Time MeOH 1 mL/min [min] [%V/V] 100 05 0.1% 35 820 formic 50 acid in 10 60 25 100 both MeOH [%] 27 100 27.5 5 Time [min] 30 5 0

0102030 14 Time MeOH 1 mL/min [min] [%V/V] 100 05 0.1% 35 720 formic 50 acid in 10 80 24 100 both MeOH [%] 26 100 26.5 5 Time [min] 30 5 0

0102030 15 Time MeOH 1 mL/min [min] [%V/V] 100 05 0.1% 35 720 formic 50 acid in 870 both 20 75 22 100 MeOH [%] Time [min] 26 100 26.5 5 0 30 5 0102030

Table Table 18 (cont) List of elution curves applied while optimizing LC separations 131

code Gradient Graph 16 Time MeOH 1 mL/min [min] [%V/V] 100 04 0.1% 44 formic 720 50 acid in 865 both 22 65 24 100 26 100 MeOH [%] Time [min] 26.5 4 0 30 4 0102030

17_1 Time MeOH 1 mL/min [min] [%V/V] 100 010 0.1% 0.5 10 formic 360 50 acid in 43 80 45 100 both MeOH [%] 53 100 55 10 Time [min] 60 10 0

0204060 17_2 Time MeOH 1 mL/min [min] [%V/V] 100 010 0.1% 0.5 10 formic 370 50 acid in 43 80 45 100

both MeOH [%] 53 100 55 10 Time [min] 60 10 0

0204060 17_3 Time MeOH 1 mL/min [min] [%V/V] 100 010 0.1% 0.5 10 formic 380 50 acid in 43 80 both 45 100 53 100 MeOH [%] 55 10 Time [min] 60 10 0

0204060

Table Table 18 (cont) List of elution curves applied while optimizing LC separations 132

code Gradient Graph 18_1 Time MeOH 1 mL/min [min] [%V/V] 100 050 0.1% 350 formic 25 100 50 acid in 30 100 both 35 100 MeOH [%] 36 50 40 50 Time [min]

0 0 10203040 18_2 Time MeOH 1 mL/min [min] [%V/V] 100 070 0.1% 370 formic 25 100 50 acid in 30 100 both 35 100 36 70 MeOH [%] 40 70 Time [min]

0 0 10203040 18_3 Time MeOH 1 mL/min [min] [%V/V] 100 080 0.1% 380 formic 20 100 50 acid in 30 100 both 35 100 36 80 MeOH [%] 40 80 Time [min]

0 010203040 18_4 Time MeOH 1 mL/min [min] [%V/V] 100 080 0.1% 380 formic 25 95 50 acid in 26 100 both 35 100 36 80 MeOH [%] 40 80 Time [min]

0 010203040

Table Table 18 (cont) List of elution curves applied while optimizing LC separations 133

code Gradient Graph 18_5 Time MeOH 1 mL/min [min] [%V/V] 100 080 0.1% formic 390 acid in both 20 90 21 100 50 35 100 36 80 MeOH [%] 40 80 Time [min]

0 0 10203040 18_6 Time MeOH 3-21min: [min] [%V/V] 100 .75mL/min 080 390 20 90 Else: 50 1 mL/min 21 100 35 100

[%] MeOH 36 80 Time [min] 0.1% formic 40 80 acid in both 0 0 10203040 18_7 Time MeOH [min] [%V/V] 100 3-15min: 080 Increasing 380 15 83 .75 to 1.0 50 mL/min 21 100 35 100 36 80 MeOH [%] MeOH Time [min] else: 40 80 1 mL/min 0 0.1% formic 0 10203040 acid in both Final MeO Time H 100 1 mL/min [min] [%V/V] 0 2

3 2 MeOH [%] 0.1% formic 50 4 60 acid in both 18 65 25 90 Time [min] 25.5 100 0 27.5 100 0 102030 28 2 30 2

Table Table 18 (cont) List of elution curves applied while optimizing LC separations 134

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

050920ESIfs_01 #20 RT: 0.33 AV: 1 NL: 2.71E8 T: + c Q1MS [100.00-600.00] 184.02 100

80 367.04

40 388.99

20 143.01 201.04 Relative Abundance Relative 294.51 247.02 351.03 389.98 477.56 491.51 587.96 0 100 150 200 250 300 350 400 450 500 550 600 m/z 050920ESI_Nfs_01 #22 RT: 0.37 AV: 1 NL: 3.34E7 T: - c Q1MS [100.00-1000.00] 181.93 100

40 227.92

Relative Abundance 20 364.85 136.93 182.97 273.91 295.84 386.82 426.81 454.84 522.77 591.74 0 100 150 200 250 300 350 400 450 500 550 600 m/z 050922APCI_P_fs01 #7 RT: 0.11 AV: 1 NL: 2.50E8 T: + c Q1MS [100.00-1000.00] 184.02 100

60 366.99 40 224.97 310.00

Relative Abundance 20 227.01 388.98 324.99 115.08 168.04 389.97 480.00 522.93 552.94 591.32 0 100 150 200 250 300 350 400 450 500 550 600 m/z 050922APCI_N_fs01 #6 RT: 0.09 AV: 1 NL: 5.12E7 T: - c Q1MS [100.00-1000.00] 167.92 100

40 252.91 186.89

Relative Abundance Relative 20 124.92 222.86 279.86 331.78 350.83 416.84 495.69 550.69 580.64 0 100 150 200 250 300 350 400 450 500 550 600 m/z

Figure 64 Acephate ESI positive, ESI negative, APCI positive, APCI negative full scan spectra 135

050920ESIfs_02 #30 RT: 0.51 AV: 1 NL: 1.94E8 T: + c Q1MS [100.00-600.00] 318.00 100

20 160.08 318.98 Relative Abundance Relative 132.04 359.00 184.02 247.04 260.95 283.10 417.09 442.97 495.56 523.02 587.00 0 100 150 200 250 300 350 400 450 500 550 600 m/z

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: 2.13E8 T: + c Q1MS [100.00-1000.00] 317.96 100

60 164.06

40 160.04 175.08 20 132.03 260.95 318.97 Relative Abundance Relative 289.97 358.99 451.04 205.07 349.99 469.04 537.82 599.7 0 100 150 200 250 300 350 400 450 500 550 600 m/z

050922APCI_N_fs02 #6-9 RT: 0.09-0.15 AV: 4 SB: 12 0.01-0.06 , 0.39-0.51 NL: 9.24E7 T: - c Q1MS [100.00-1000.00] 156.86 100

20 336.72

Relative Abundance Relative 158.85 117.96 224.83273.84 301.84 338.73 422.65 481.72 534.57 598.4 0 100 150 200 250 300 350 400 450 500 550 600 m/z

Figure 65 Azinphos Methyl ESI positive, ESI negative, APCI positive, APCI negative full scan spectra 136

050920ESIfs_03 #49 RT: 0.83 AV: 1 NL: 2.85E8 T: + c Q1MS [100.00-600.00] 398.05 100

40 399.04 20 355.99

Relative Abundance Relative 313.96 420.03 151.08 184.04 218.03 254.90 358.00 443.08 497.18 533.02 581.03 0 100 150 200 250 300 350 400 450 500 550 600 m/z

050920ESI_Nfs_03 #22 RT: 0.37 AV: 1 SB: 23 0.06-0.25 , 0.46-0.63 NL: 5.31E6 T: - c Q1MS [100.00-1000.00] 441.88 100 395.86 80

40 212.89 442.95 20 396.89 458.85 Relative Abundance Relative 487.85 107.94 196.91 214.96 254.98 311.01326.03 398.93 578.79 0 100 150 200 250 300 350 400 450 500 550 600 m/z

050922APCI_P_fs03 #7-12 RT: 0.11-0.20 AV: 6 NL: 2.53E8 T: + c Q1MS [100.00-1000.00] 355.98 100 398.01 80

40 345.96 357.97 399.02 20

Relative Abundance Relative 313.90 419.99 115.07 184.02 200.00 222.07 441.05 519.09 554.96 596.9 0 100 150 200 250 300 350 400 450 500 550 600 m/z

050922APCI_N_fs03 #7-12 RT: 0.11-0.20 AV: 6 SB: 12 0.01-0.06 , 0.39-0.51 NL: 1.35E8 T: - c Q1MS [100.00-1000.00] 212.91 100

20 214.90 Relative Abundance Relative 353.84 140.89 168.92 215.91 293.80 404.83 443.74 517.79 537.86 589.81 0 100 150 200 250 300 350 400 450 500 550 600 m/z

Figure 66 Bensulide ESI positive, ESI negative, APCI positive, APCI negative full scan spectra 137

050920ESIfs_04 #27 RT: 0.46 AV: 1 NL: 2.81E8 T: + c Q1MS [100.00-600.00] 541.19 100

40 542.19 271.10 563.13 20 334.09 Relative Abundance Relative 312.10 105.05 172.00 200.02 256.04 370.19 422.22 476.15 527.19 564.12 0 100 150 200 250 300 350 400 450 500 550 600 m/z

050920ESI_Nfs_04 #24 RT: 0.41 AV: 1 SB: 23 0.06-0.25 , 0.46-0.63 NL: 1.91E6 T: - c Q1MS [100.00-1000.00] 426.75 100

40 247.07 135.02 240.98 441.85 20 395.91 136.92 299.00 372.94 Relative Abundance Relative 442.91 181.92 526.78 578.68 0 100 150 200 250 300 350 400 450 500 550 600 m/z

050922APCI_P_fs04 #11-14 RT: 0.18-0.23 AV: 4 NL: 3.11E8 T: + c Q1MS [100.00-1000.00] 541.13 100 271.05

60 303.07 542.12 40

Relative Abundance Relative 247.01 304.06 544.09 162.97 190.99 370.15 442.95 471.01 527.10 0 100 150 200 250 300 350 400 450 500 550 600 m/z

050922APCI_N_fs04 #11-15 RT: 0.18-0.25 AV: 5 SB: 12 0.01-0.06 , 0.39-0.51 NL: 4.27E7 T: - c Q1MS [100.00-1000.00] 212.93 100

40 154.90 20 258.87 Relative Abundance Relative 200.90 140.88 280.87 376.76 390.87 448.83 510.70 554.69 596.7 0 100 150 200 250 300 350 400 450 500 550 600 m/z

Figure 67 Cadusafos ESI positive, ESI negative, APCI positive, APCI negative full scan spectra 138

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: 1.55E7 T: - c Q1MS [100.00-1000.00] 306.68 100

80 304.71 60 308.70 234.78 40 154.90 236.78 197.83 20 310.69 Relative Abundance Relative 140.88 292.73 352.70 414.68 482.64 538.54 564.57 0 100 150 200 250 300 350 400 450 500 550 600 m/z

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

139

N/F

050922APCI_P_fs06 #22-28 RT: 0.37-0.48 AV: 7 NL: 2.28E8 T: + c Q1MS [100.00-1000.00] 351.89 100

40 353.89

Relative Abundance Relative 126.02 355.88 162.01 217.03 254.99 315.94 405.88 457.96 481.77 551.87 571.81 0 100 150 200 250 300 350 400 450 500 550 600 m/z

050922APCI_N_fs06 #21-29 RT: 0.36-0.50 AV: 9 NL: 2.23E7 T: - c Q1MS [100.00-1000.00] 312.79 100

80 314.79 168.91 197.80 60 213.76 40 214.90 315.80 285.78 316.78 20 154.90 215.81 Relative Abundance Relative 245.78 347.78 380.76 432.58 490.71 533.63 558.71 0 100 150 200 250 300 350 400 450 500 550 600 m/z

Figure 69 Chlorpyrifos ESI positive (weak), ESI negative (weak), APCI positive, APCI negative full scan spectra 140

050920ESIfs_07 #29 RT: 0.49 AV: 1 NL: 8.51E6 T: + c Q1MS [100.00-600.00] 143.09 100

60 271.09 312.29 334.11 40 247.04 173.09 368.16 20 240.15 272.16 370.20 454.30 522.59 Relative Abundance Relative 541.20 0 100 150 200 250 300 350 400 450 500 550 600 m/z

050920ESI_Nfs_07 #26 RT: 0.44 AV: 1 SB: 23 0.06-0.25 , 0.46-0.63 NL: 2.42E6 T: - c Q1MS [100.00-1000.00] 307.73 100

80 195.81 60 197.82 426.79 40 309.71 243.81 351.62 20 247.04

Relative Abundance Relative 355.02 427.77 472.72 140.89 526.84 568.94 0 100 150 200 250 300 350 400 450 500 550 600 m/z

050922APCI_P_fs07 #14-16 RT: 0.23-0.27 AV: 3 SB: 8 0.15-0.20 , 0.32-0.37 NL: 2.78E7 T: + c Q1MS [100.00-1000.00] 126.03 100 321.87 80

60 129.99 162.01

40 164.01 325.88 20 196.01 287.93 Relative Abundance Relative 271.07 327.91 377.91 427.91 452.89 486.76 534.07 580.06 0 100 150 200 250 300 350 400 450 500 550 600 m/z

050922APCI_N_fs07 #13-15 RT: 0.22-0.25 AV: 3 NL: 2.17E7 T: - c Q1MS [100.00-1000.00] 140.88 100 213.77

80 197.79

60 186.88 40 215.76 305.73 284.77 20 241.79 309.69

Relative Abundance Relative 142.88 126.88 317.76 361.68 416.64 436.43 524.54 596.5 0 100 150 200 250 300 350 400 450 500 550 600 m/z

Figure 70 Chlorpyrifos Methyl ESI positive, ESI negative, APCI positive, APCI negative full scan spectra 141

050920ESIfs_08 #27 RT: 0.46 AV: 1 NL: 7.40E7 T: + c Q1MS [100.00-600.00] 363.01 100

40 365.01

20 404.02 426.03 Relative Abundance Relative 115.11 143.10 184.04 271.08 303.65 329.08 467.10 522.56 550.60 0 100 150 200 250 300 350 400 450 500 550 600 m/z

050920ESI_Nfs_08 #20 RT: 0.34 AV: 1 SB: 23 0.06-0.25 , 0.46-0.64 NL: 2.15E6 T: - c Q1MS [100.00-1000.00] 136.94 100

40 369.86 20 332.81 415.87 Relative Abundance Relative 137.95 208.91 254.86 304.86 472.48 500.79 579.57 0 100 150 200 250 300 350 400 450 500 550 600 m/z

050922APCI_P_fs08 #9-14 RT: 0.15-0.23 AV: 6 SB: 8 0.15-0.20 , 0.32-0.37 NL: 1.12E8 T: + c Q1MS [100.00-1000.00] 362.98 100

60 177.04 364.97 40 209.05 395.01

20 151.05 329.01 396.99 Relative Abundance Relative 539.02 137.03 218.05 243.02 301.04 427.98 513.03 572.97 0 100 150 200 250 300 350 400 450 500 550 600 m/z

050922APCI_N_fs08 #7-12 RT: 0.11-0.20 AV: 6 NL: 5.09E7 T: - c Q1MS [100.00-1000.00] 224.85 100

80 208.89

60 332.81 40 226.85 418.83 20 168.92254.87 334.81

Relative Abundance Relative 256.87 318.80 400.78 161.98 335.82 421.84 510.73 542.77 564.70 0 100 150 200 250 300 350 400 450 500 550 600 m/z

Figure 71 Coumaphos ESI positive, ESI negative (weak), APCI positive, APCI negative full scan spectra 142

050920ESIfs_09 #49 RT: 0.83 AV: 1 NL: 3.19E8 T: + c Q1MS [100.00-600.00] 305.10 100

60 306.10 40 307.08 20 Relative Abundance Relative 152.15 173.10 206.05 299.18 327.07 359.15 404.20 457.12 488.11 510.14 575.25 0 100 150 200 250 300 350 400 450 500 550 600 m/z

050920ESI_Nfs_09 #24 RT: 0.41 AV: 1 SB: 23 0.06-0.25 , 0.46-0.63 NL: 2.84E6 T: - c Q1MS [100.00-1000.00] 136.92 100 168.92 80

60 214.88

20 274.94 299.00 345.02 Relative Abundance Relative 181.92 372.99 416.99 461.08 504.97 570.67 0 100 150 200 250 300 350 400 450 500 550 600 m/z

050922APCI_P_fs09 #8-16 RT: 0.13-0.27 AV: 9 SB: 8 0.15-0.20 , 0.32-0.37 NL: 4.76E7 T: + c Q1MS [100.00-1000.00] 306.07 100

40 307.06

Relative Abundance Relative 319.08 153.08 181.10 233.01 291.06 359.08 395.01 457.14 505.05 561.63 0 100 150 200 250 300 350 400 450 500 550 600 m/z

050922APCI_N_fs09 #8-11 RT: 0.13-0.18 AV: 4 NL: 5.78E7 T: - c Q1MS [100.00-1000.00] 168.91 100

60 214.90 40

20 154.90 Relative Abundance Relative 200.89 274.91 112.90 236.88 346.84 414.80 482.79 510.78 564.75 0 100 150 200 250 300 350 400 450 500 550 600 m/z

Figure 72 Diazinon ESI positive, ESI negative, APCI positive, APCI negative full scan spectra 143

050920ESIfs_10 #24 RT: 0.40 AV: 1 NL: 1.11E8 T: + c Q1MS [100.00-600.00] 261.97 100

60 263.98 305.09

40 252.97 442.86 20 105.05 220.96 265.98 444.90 Relative Abundance Relative 306.08 182.09 360.96 407.23 464.88 525.05 571.85 0 100 150 200 250 300 350 400 450 500 550 600 m/z

050920ESI_Nfs_10 #19 RT: 0.32 AV: 1 SB: 23 0.06-0.25 , 0.46-0.63 NL: 7.89E6 T: - c Q1MS [100.00-1000.00] 204.79 100

60 250.81 40 252.80

20 170.91 254.77 334.71 Relative Abundance Relative 150.94 208.81 370.65 412.64 434.66 466.68 538.56 568.51 0 100 150 200 250 300 350 400 450 500 550 600 m/z

050922APCI_P_fs10 #7-10 RT: 0.11-0.16 AV: 4 SB: 4 0.02-0.08 NL: 1.15E8 T: + c Q1MS [100.00-1000.00] 261.95 100

80 252.96 182.05 263.95 60

40 442.86 20 115.07 173.05 265.95 360.94 Relative Abundance Relative 233.01 332.94 402.92 446.86 513.83 538.75 581.64 0 100 150 200 250 300 350 400 450 500 550 600 m/z

050922APCI_N_fs10 #5-6 RT: 0.08-0.09 AV: 2 NL: 6.61E7 T: - c Q1MS [100.00-1000.00] 204.81 100

80 206.82 60

20 250.81

Relative Abundance Relative 170.90 124.91 216.85 254.79 302.76 352.76 410.66 434.63 502.67 530.57 582.61 0 100 150 200 250 300 350 400 450 500 550 600 m/z

Figure 73 Dichlorvos ESI positive, ESI negative, APCI positive, APCI negative full scan spectra 144

050920ESIfs_11 #18 RT: 0.30 AV: 1 NL: 2.80E8 T: + c Q1MS [100.00-600.00] 475.16 100 270.09 80

40 497.14 238.07 20

Relative Abundance Relative 271.10 375.62 498.13 130.08 193.02 305.07 389.57 443.18 533.11 583.11 0 100 150 200 250 300 350 400 450 500 550 600 m/z

050920ESI_Nfs_11 #20 RT: 0.34 AV: 1 SB: 23 0.06-0.25 , 0.46-0.63 NL: 2.55E6 T: - c Q1MS [100.00-1000.00] 136.91 100

40 222.92 290.89 20 468.85 Relative Abundance Relative 213.88 267.81 307.87 334.90 181.95 368.80 427.83 496.83 530.72 574.73 0 100 150 200 250 300 350 400 450 500 550 600 m/z

050922APCI_P_fs11 #6-10 RT: 0.09-0.16 AV: 5 SB: 4 0.02-0.08 NL: 2.64E8 T: + c Q1MS [100.00-1000.00] 270.08 100 283.11 80

60 475.12 40 284.09 20 112.05 238.06 349.11 Relative Abundance Relative 476.12 144.08 193.01 285.09 364.05 408.01 555.98 579.0 0 100 150 200 250 300 350 400 450 500 550 600 m/z

050922APCI_N_fs11 #6-8 RT: 0.09-0.13 AV: 3 NL: 2.56E7 T: - c Q1MS [100.00-1000.00] 170.90 100

60 124.92 236.86 40 250.93 221.92 20

Relative Abundance Relative 156.89 272.89 420.83 216.91 340.85 384.74 452.60 517.91 568.76 0 100 150 200 250 300 350 400 450 500 550 600 m/z

Figure 74 Dicrotophos ESI positive, ESI negative, APCI positive, APCI negative full scan spectra 145

050920ESIfs_12 #21 RT: 0.35 AV: 1 NL: 2.16E8 T: + c Q1MS [100.00-600.00] 230.00 100 458.99 80

40 293.00 480.97 20 251.96

Relative Abundance Relative 198.98 134.07 305.08 363.46 413.02 482.95 534.10 592.57 0 100 150 200 250 300 350 400 450 500 550 600 m/z

050920ESI_Nfs_12 #19 RT: 0.32 AV: 1 SB: 23 0.06-0.25 , 0.46-0.63 NL: 1.33E7 T: - c Q1MS [100.00-1000.00] 273.87 100

40 319.83 20 213.86

Relative Abundance Relative 197.92 317.89 134.92 252.87 369.79 426.84 485.90 502.76 527.79 581.75 0 100 150 200 250 300 350 400 450 500 550 600 m/z

050922APCI_P_fs12 #5-10 RT: 0.08-0.16 AV: 6 SB: 4 0.03-0.08 NL: 2.38E8 T: + c Q1MS [100.00-1000.00] 229.98 100

60 458.95 40 271.01 231.97 20 198.95 Relative Abundance Relative 292.98 460.95 115.06 171.00 335.01 386.01 437.99 544.10 595.5 0 100 150 200 250 300 350 400 450 500 550 600 m/z

050922APCI_N_fs12 #6-9 RT: 0.09-0.15 AV: 4 NL: 6.56E7 T: - c Q1MS [100.00-1000.00] 156.87 100

40 213.86 20

Relative Abundance Relative 158.86 385.78 140.87 224.83 273.89 369.79 442.81 484.72 538.90 598.4 0 100 150 200 250 300 350 400 450 500 550 600 m/z

Figure 75 Dimethoate ESI positive, ESI negative, APCI positive, APCI negative full scan spectra 146

050920ESIfs_13 #27 RT: 0.46 AV: 1 NL: 5.57E7 T: + c Q1MS [100.00-600.00] 305.11 100

40 173.08 20 115.09 306.10

Relative Abundance Relative 230.00 270.12 184.03 312.30 368.14 444.04 482.52525.20 550.67 0 100 150 200 250 300 350 400 450 500 550 600 m/z

050920ESI_Nfs_13 #19 RT: 0.32 AV: 1 SB: 23 0.06-0.25 , 0.46-0.63 NL: 2.97E6 T: - c Q1MS [100.00-1000.00] 136.94 100

20 184.88 Relative Abundance Relative 213.91 137.91 273.85 345.07 368.78 432.73 491.52 527.66 577.11 0 100 150 200 250 300 350 400 450 500 550 600 m/z

050922APCI_P_fs13 #10-18 RT: 0.16-0.30 AV: 9 SB: 4 0.03-0.08 NL: 2.67E8 T: + c Q1MS [100.00-1000.00] 363.04 100

60 365.02 40

20 213.00 Relative Abundance Relative 366.02 106.06 184.99 217.00 275.01 289.03 458.97 487.01 522.90 584.73 0 100 150 200 250 300 350 400 450 500 550 600 m/z

050922APCI_N_fs13 #10-16 RT: 0.16-0.27 AV: 7 NL: 5.41E7 T: - c Q1MS [100.00-1000.00] 184.90 100

20 186.86 Relative Abundance Relative 154.87 200.90 252.84 320.79 348.83 378.76 430.78 452.76 512.63 540.77 580.83 0 100 150 200 250 300 350 400 450 500 550 600 m/z

Figure 76 Disulfoton ESI positive, ESI negative (weak), APCI positive, APCI negative full scan spectra 147

050920ESIfs_14 #28 RT: 0.47 AV: 1 NL: 8.15E7 T: + c Q1MS [100.00-600.00] 384.98 100

40 173.10 386.99 20 305.10 Relative Abundance Relative 105.05 198.99 273.53 359.24 448.01 484.10 554.12 596.0 0 100 150 200 250 300 350 400 450 500 550 600 m/z

N/F

050922APCI_P_fs14 #21-28 RT: 0.36-0.48 AV: 8 SB: 4 0.02-0.08 NL: 3.18E8 T: + c Q1MS [100.00-1000.00] 384.95 100

60 230.99 386.95 40 198.98 20 398.94

Relative Abundance Relative 185.02 153.06 232.98 281.03 338.95 438.94 491.00 556.86 582.93 0 100 150 200 250 300 350 400 450 500 550 600 m/z

050922APCI_N_fs14 #19-27 RT: 0.32-0.46 AV: 9 NL: 1.23E8 T: - c Q1MS [100.00-1000.00] 184.89 100

20 186.89 Relative Abundance Relative 156.85 252.84 334.78 354.78 392.78 430.79478.72 526.73 562.71 0 100 150 200 250 300 350 400 450 500 550 600 m/z

Figure 77 Ethion ESI positive, ESI negative, APCI positive, APCI negative full scan spectra 148

050920ESIfs_15 #21 RT: 0.35 AV: 1 NL: 2.75E8 T: + c Q1MS [100.00-600.00] 485.09 100

40 243.08 486.08 20 284.08 306.04 Relative Abundance Relative 115.07 173.11 213.06244.04 308.08 347.11 425.14 455.11 508.07 547.15 0 100 150 200 250 300 350 400 450 500 550 600 m/z

050920ESI_Nfs_15 #16-26 RT: 0.27-0.44 AV: 11 SB: 18 0.02-0.15 , 0.56-0.72 NL: 4.70E5 T: - c Q1MS [100.00-1000.00] 136.93 100

60 212.93 265.04 40 299.04 198.93 258.89 345.04 20 181.94 327.09 Relative Abundance Relative 349.13 383.00 441.87 468.14 523.09 580.96 0 100 150 200 250 300 350 400 450 500 550 600 m/z

050922APCI_P_fs15 #7-13 RT: 0.11-0.22 AV: 7 SB: 4 0.02-0.08 NL: 3.04E8 T: + c Q1MS [100.00-1000.00] 485.08 100 243.03 80

60 275.05

40 284.05 486.08

Relative Abundance Relative 285.05 488.06 146.05203.03 233.02 330.09 425.11 455.08 557.06 576.9 0 100 150 200 250 300 350 400 450 500 550 600 m/z

050922APCI_N_fs15 #6-12 RT: 0.09-0.20 AV: 7 NL: 8.08E7 T: - c Q1MS [100.00-1000.00] 198.90 100

20 200.89 244.88 Relative Abundance Relative 124.92 170.91 310.82 376.82 414.74 444.78 510.79 554.73 582.78 0 100 150 200 250 300 350 400 450 500 550 600 m/z

Figure 78 Ethoprop ESI positive, ESI negative (weak), APCI positive, APCI negative full scan spectra 149

050920ESIfs_16 #26 RT: 0.44 AV: 1 NL: 1.74E7 T: + c Q1MS [100.00-600.00] 243.08 100 173.09 80

60 115.09 305.09 306.07 40 143.13 284.09 384.97 20 230.04 270.11 323.07 359.19 Relative Abundance Relative 386.00 485.14 454.29 554.03 569.41 0 100 150 200 250 300 350 400 450 500 550 600 m/z

050920ESI_Nfs_16 #22 RT: 0.37 AV: 1 SB: 11 0.01-0.18 NL: 4.03E5 T: - c Q1MS [100.00-1000.00] 261.84 100 354.85 80

60 103.89 137.93 183.97 345.03 40 299.04 373.04 400.78 237.68 301.01 20 441.83 Relative Abundance Relative 181.92 461.12 540.49 581.22 0 100 150 200 250 300 350 400 450 500 550 600 m/z

050922APCI_P_fs16 #8-14 RT: 0.13-0.23 AV: 7 SB: 4 0.03-0.08 NL: 1.75E8 T: + c Q1MS [100.00-1000.00] 262.04 100

60 151.07 40 142.06 323.00 20 263.03

Relative Abundance Relative 230.04 152.06 324.00 371.09 432.05 493.97 523.09 584.03 0 100 150 200 250 300 350 400 450 500 550 600 m/z

050922APCI_N_fs16 #6-12 RT: 0.09-0.20 AV: 7 NL: 4.08E7 T: - c Q1MS [100.00-1000.00] 261.86 100

80 153.90 60 137.93 40 276.90 247.85 20 168.91 329.82

Relative Abundance Relative 245.87 546.77 330.82 400.84 453.80 524.82 575.84 0 100 150 200 250 300 350 400 450 500 550 600 m/z

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: 5.76E5 T: - c Q1MS [100.00-1000.00] 347.94 100

80 107.89 136.93 60

40 368.94 181.90 20 213.77 273.89 Relative Abundance Relative 306.99 147.97 369.73 415.01 456.84 487.15 548.78 586.02 0 100 150 200 250 300 350 400 450 500 550 600 m/z

050922APCI_P_fs17 #7-14 RT: 0.11-0.23 AV: 8 SB: 4 0.03-0.08 NL: 2.68E8 T: + c Q1MS [100.00-1000.00] 363.14 100

60 304.07

40 336.09 364.13 20

Relative Abundance Relative 365.13 101.10 146.07 209.12 250.13 409.11 485.16 512.20 548.08 596.0 0 100 150 200 250 300 350 400 450 500 550 600 m/z

050922APCI_N_fs17 #6-11 RT: 0.09-0.18 AV: 6 SB: 6 0.03-0.04 , 0.27-0.32 NL: 4.30E6 T: - c Q1MS [100.00-1000.00] 246.89 100

40 292.90 314.86 20 136.93

Relative Abundance Relative 156.89 232.89 319.91 382.82 409.91 494.80 516.84 584.80 0 100 150 200 250 300 350 400 450 500 550 600 m/z

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: 1.11E7 T: + c Q1MS [100.00-600.00] 115.09 100 305.10 80 143.11 60

40 367.12 261.08 304.11 20 146.07 211.15 274.37 345.16 Relative Abundance Relative 368.14 454.29 488.37 522.43 567.01 0 100 150 200 250 300 350 400 450 500 550 600 m/z

050920ESI_Nfs_17 #16-20 RT: 0.27-0.34 AV: 5 SB: 11 0.01-0.18 NL: 4.37E5 T: - c Q1MS [100.00-1000.00] 347.93 100 107.91 80

40 368.90 134.93 273.88 20 181.90 213.84

Relative Abundance Relative 306.92 393.90 147.90 414.88 504.97 565.33 586.15 0 100 150 200 250 300 350 400 450 500 550 600 m/z

050922APCI_P_fs18 #7-11 RT: 0.11-0.18 AV: 5 SB: 4 0.02-0.08 NL: 1.22E8 T: + c Q1MS [100.00-1000.00] 165.09 100

80 124.05 248.04 60

40 156.09 289.05 20 179.07 262.02 Relative Abundance Relative 216.06 294.04 371.09 401.04 431.07 495.07 525.02 555.19 0 100 150 200 250 300 350 400 450 500 550 600 m/z

050922APCI_N_fs18 #7-15 RT: 0.11-0.25 AV: 9 SB: 6 0.02-0.04 , 0.27-0.32 NL: 3.99E7 T: - c Q1MS [100.00-1000.00] 167.92 100

40 151.94 293.87 20 186.89 Relative Abundance Relative 276.85 135.95 208.86 305.93 344.86 414.86 436.86 493.70 546.81 586.73 0 100 150 200 250 300 350 400 450 500 550 600 m/z

Figure 81 Fenitrothion ESI positive (weak),, ESI negative, APCI positive, APCI negative full scan spectra 152

050920ESIfs_19 #26 RT: 0.44 AV: 1 SB: 24 0.06-0.23 , 0.63-0.83 NL: 1.88E7 T: + c Q1MS [100.00-600.00] 342.03 100

80 115.10 60 557.04 320.04 40 279.03 579.02 143.10 343.04 20 173.09 512.16 Relative Abundance Relative 261.50 378.14 451.05 208.05 0 100 150 200 250 300 350 400 450 500 550 600 m/z

050920ESI_Nfs_19 #23-29 RT: 0.39-0.50 AV: 7 SB: 11 0.01-0.18 NL: 3.80E6 T: - c Q1MS [100.00-1000.00] 262.86 100

20 308.86 Relative Abundance Relative 107.91 181.93 213.84 299.04 345.06 382.87 417.03 460.81 523.17 555.03 0 100 150 200 250 300 350 400 450 500 550 600 m/z

050922APCI_P_fs19 #8-14 RT: 0.13-0.23 AV: 7 SB: 4 0.02-0.08 NL: 7.07E7 T: + c Q1MS [100.00-1000.00] 279.00 100 311.03 80

40 247.04 320.04 20 106.05 325.00 Relative Abundance Relative 154.04 385.01 233.15 429.08 491.07 557.01 579.0 0 100 150 200 250 300 350 400 450 500 550 600 m/z

050922APCI_N_fs19 #7-15 RT: 0.11-0.25 AV: 9 SB: 6 0.02-0.04 , 0.27-0.32 NL: 8.55E6 T: - c Q1MS [100.00-1000.00] 262.87 100

20 140.91 186.89 263.86 Relative Abundance Relative 330.84 208.86 308.83 398.82 466.85 520.56 548.76 596.6 0 100 150 200 250 300 350 400 450 500 550 600 m/z

Figure 82 Fenthion ESI positive, ESI negative, APCI positive, APCI negative full scan spectra 153

050920ESIfs_20 #23 RT: 0.39 AV: 1 SB: 26 0.06-0.23 , 0.59-0.83 NL: 1.20E7 T: + c Q1MS [100.00-600.00] 115.10 100 173.10 247.03 80 143.09 60 305.10

40 299.18 20 245.54 312.29 368.16

Relative Abundance Relative 248.05 369.17 454.28 524.01 538.71 0 100 150 200 250 300 350 400 450 500 550 600 m/z

050920ESI_Nfs_20 #20-23 RT: 0.34-0.39 AV: 4 SB: 11 0.01-0.18 NL: 7.29E5 T: - c Q1MS [100.00-1000.00] 324.87 100

40 299.04 345.04 373.01 198.89 260.89 20 152.89 416.95

Relative Abundance Relative 461.06 247.04 374.06 505.06 561.03 581.25 0 100 150 200 250 300 350 400 450 500 550 600 m/z

050922APCI_P_fs20 #8-17 RT: 0.13-0.29 AV: 10 SB: 4 0.03-0.08 NL: 2.90E8 T: + c Q1MS [100.00-1000.00] 247.01 100

40 248.02 20

Relative Abundance Relative 261.02 106.06 169.03 215.04 310.11353.05 383.10 459.09 503.12 547.25 595.0 0 100 150 200 250 300 350 400 450 500 550 600 m/z

050922APCI_N_fs20 #9-15 RT: 0.15-0.25 AV: 7 NL: 1.39E7 T: - c Q1MS [100.00-1000.00] 168.90 100

40 138.92 20 184.92 Relative Abundance Relative 216.87 284.85 304.84 352.78 408.83 446.68 484.96 514.63 572.91 0 100 150 200 250 300 350 400 450 500 550 600 m/z

Figure 83 Fonofos ESI positive, ESI negative, APCI positive, APCI negative full scan spectra 154

050920ESIfs_21 #19 RT: 0.32 AV: 1 SB: 26 0.06-0.23 , 0.59-0.83 NL: 2.00E8 T: + c Q1MS [100.00-600.00] 331.04 100

40 332.04 20

Relative Abundance Relative 285.00 348.05 115.10 159.06 217.13 257.03 394.04 430.13 488.08 515.11 558.19 586.0 0 100 150 200 250 300 350 400 450 500 550 600 m/z

050920ESI_Nfs_21 #20-25 RT: 0.34-0.43 AV: 6 SB: 11 0.01-0.18 NL: 6.64E6 T: - c Q1MS [100.00-1000.00] 314.87 100

20 315.86

Relative Abundance Relative 156.87 360.85 213.83 233.04 299.00 317.86 416.99 437.48 494.83 543.68 568.88 0 100 150 200 250 300 350 400 450 500 550 600 m/z

050922APCI_P_fs21 #7-13 RT: 0.11-0.22 AV: 7 SB: 4 0.03-0.08 NL: 2.39E8 T: + c Q1MS [100.00-1000.00] 331.01 100

40 332.01 284.97 20 352.99 Relative Abundance Relative 173.06 247.03 115.06 394.02 437.06 486.96550.86 587.03 0 100 150 200 250 300 350 400 450 500 550 600 m/z

050922APCI_N_fs21 #9 RT: 0.15 AV: 1 SB: 11 0.03-0.09 , 0.27-0.36 NL: 6.47E7 T: - c Q1MS [100.00-1000.00] 156.87 100

20 158.87 314.87 Relative Abundance Relative 224.84 336.71 142.88 159.86 292.80 424.60 494.79 536.57 598.5 0 100 150 200 250 300 350 400 450 500 550 600 m/z

Figure 84 Malathion ESI positive, ESI negative, APCI positive, APCI negative full scan spectra 155

050920ESIfs_22 #23 RT: 0.39 AV: 1 SB: 26 0.06-0.23 , 0.59-0.83 NL: 2.72E8 T: + c Q1MS [100.00-600.00] 283.00 100

40 142.02

20 183.04 285.02 Relative Abundance Relative 205.01 126.05 267.07 372.50 443.03 479.34 549.30 587.66 0 100 150 200 250 300 350 400 450 500 550 600 m/z

050920ESI_Nfs_22 #22 RT: 0.37 AV: 1 SB: 11 0.01-0.18 NL: 5.07E6 T: - c Q1MS [100.00-1000.00] 186.90 100 140.91

40 232.86 20 110.88 263.83 Relative Abundance Relative 156.92 188.93 278.83 369.82 385.66 461.24 486.51 530.38 580.79 0 100 150 200 250 300 350 400 450 500 550 600 m/z

050922APCI_P_fs22 #6-10 RT: 0.09-0.16 AV: 5 SB: 4 0.03-0.08 NL: 2.52E8 T: + c Q1MS [100.00-1000.00] 282.98 100

60 183.03 40 142.01 20 284.98

Relative Abundance Relative 266.99 126.00 205.00 297.02 329.01 390.00 445.09 468.80 530.97 578.85 0 100 150 200 250 300 350 400 450 500 550 600 m/z

N/F

Figure 85 Methamidophos ESI positive, ESI negative, APCI positive, APCI negative (weak)full scan spectra 156

050920ESIfs_23 #26 RT: 0.44 AV: 1 SB: 26 0.06-0.23 , 0.59-0.83 NL: 6.84E7 T: + c Q1MS [100.00-600.00] 302.95 100

40 365.98 20 115.10 304.94 473.00 Relative Abundance Relative 145.01 367.99 183.04 218.08 252.99 324.94 402.07 474.88 548.07 595.2 0 100 150 200 250 300 350 400 450 500 550 600 m/z

050920ESI_Nfs_23 #20-27 RT: 0.34-0.46 AV: 8 SB: 11 0.01-0.18 NL: 9.39E5 T: - c Q1MS [100.00-1000.00] 286.81 100

40 156.85 270.83 416.87 20 213.85 288.82 332.77 Relative Abundance Relative 374.03 417.89 460.93 504.94 548.87 581.09 0 100 150 200 250 300 350 400 450 500 550 600 m/z

050922APCI_P_fs23 #7-10 RT: 0.11-0.16 AV: 4 SB: 4 0.02-0.08 NL: 2.18E8 T: + c Q1MS [100.00-1000.00] 302.94 100

40 304.94 20 115.07 177.03 365.93 Relative Abundance Relative 218.04 319.96 286.99 366.92 446.92 522.82 586.69 0 100 150 200 250 300 350 400 450 500 550 600 m/z

050922APCI_N_fs23 #6-11 RT: 0.09-0.18 AV: 6 SB: 4 0.01-0.06 NL: 6.80E7 T: - c Q1MS [100.00-1000.00] 156.87 100

20 286.79

Relative Abundance Relative 158.87 336.73 142.86 224.83 242.77 354.76 422.66 466.71 534.60 598.5 0 100 150 200 250 300 350 400 450 500 550 600 m/z

Figure 86 Methidathion ESI positive, ESI negative, APCI positive, APCI negative full scan spectra 157

050920ESIfs_24 #27 RT: 0.46 AV: 1 SB: 26 0.06-0.23 , 0.59-0.83 NL: 1.38E7 T: + c Q1MS [100.00-600.00] 115.11 100

40 143.12 20 198.05 254.09 Relative Abundance Relative 312.28 368.17 394.11 455.45 495.08 528.41 580.18 0 100 150 200 250 300 350 400 450 500 550 600 m/z

050920ESI_Nfs_24 #19-29 RT: 0.32-0.50 AV: 11 SB: 11 0.01-0.18 NL: 3.72E6 T: - c Q1MS [100.00-1000.00] 276.92 100

80 137.93 247.85 325.04 60 183.94 40 338.94 311.02 386.83 20 231.88 Relative Abundance Relative 138.95 387.88 464.00 479.07 553.70 595.1 0 100 150 200 250 300 350 400 450 500 550 600 m/z

050922APCI_P_fs24 #5-9 RT: 0.08-0.15 AV: 5 NL: 1.56E8 T: + c Q1MS [100.00-1000.00] 151.07 100

60 234.02 40 110.03 275.03 149.05 20 152.08 Relative Abundance Relative 192.12 243.06 316.06 343.06 391.25 467.02 497.03 536.00 564.14 0 100 150 200 250 300 350 400 450 500 550 600 m/z

050922APCI_N_fs24 #6-11 RT: 0.09-0.18 AV: 6 SB: 4 0.01-0.06 NL: 3.65E7 T: - c Q1MS [100.00-1000.00] 247.86 100 153.91 80

60 137.92 276.92 40 279.86

20 183.93 Relative Abundance Relative 386.82 205.90 315.82 401.81 463.79518.73 586.69 0 100 150 200 250 300 350 400 450 500 550 600 m/z

Figure 87 Methyl parathion ESI positive (weak), ESI negative, APCI positive, APCI negative full scan spectra 158

050920ESIfs_25 #24 RT: 0.40 AV: 1 SB: 26 0.06-0.23 , 0.59-0.83 NL: 1.56E8 T: + c Q1MS [100.00-600.00] 225.07 100 449.08

40 471.05 193.06 288.04 20 257.05

Relative Abundance Relative 168.05 472.03 127.02 289.03 356.09 427.13 505.03 580.11 0 100 150 200 250 300 350 400 450 500 550 600 m/z

050920ESI_Nfs_25 #20-26 RT: 0.34-0.44 AV: 7 SB: 10 0.06-0.22 NL: 6.36E5 T: - c Q1MS [100.00-1000.00] 107.92 100

80 208.91 60 254.90 40 290.88 148.92 213.85 20 296.92 334.88 Relative Abundance Relative 368.78 420.71 468.89 530.72 558.79 0 100 150 200 250 300 350 400 450 500 550 600 m/z

050922APCI_P_fs25 #7-13 RT: 0.11-0.22 AV: 7 NL: 2.57E8 T: + c Q1MS [100.00-1000.00] 225.02 100 257.05 80

40 193.01 20 258.04 351.03 449.05

Relative Abundance Relative 159.02 106.05 289.05 352.01 394.98 471.02 528.99 590.98 0 100 150 200 250 300 350 400 450 500 550 600 m/z

050922APCI_N_fs25 #6-9 RT: 0.10-0.15 AV: 4 SB: 4 0.01-0.06 NL: 3.63E7 T: - c Q1MS [100.00-1000.00] 170.91 100

80 124.91

40 236.88

20 156.91 208.91 250.88 Relative Abundance Relative 272.86 356.82 384.80 420.80 438.64 504.83 568.77 0 100 150 200 250 300 350 400 450 500 550 600 m/z

Figure 88 Mevinphos ESI positive, ESI negative, APCI positive, APCI negative full scan spectra 159

050920ESIfs_26 #25 RT: 0.42 AV: 1 SB: 26 0.06-0.23 , 0.59-0.83 NL: 2.11E7 T: + c Q1MS [100.00-600.00] 423.78 100

80 115.08 60

40 298.04 425.78 419.79 168.06 257.08 20 412.82 453.82 Relative Abundance Relative 146.07 380.75 491.01 183.06 252.12 334.25 508.82 592.69 0 100 150 200 250 300 350 400 450 500 550 600 m/z

050920ESI_Nfs_26 #20-27 RT: 0.34-0.46 AV: 8 SB: 10 0.06-0.22 NL: 1.33E7 T: - c Q1MS [100.00-1000.00] 364.59 100 366.59 80

40 362.60 410.59 574.41 20 408.60 254.73 414.57 578.3 Relative Abundance Relative 458.56 124.91 170.91 218.86 271.72 334.72 370.60 490.54 570.42 0 100 150 200 250 300 350 400 450 500 550 600 m/z

050922APCI_P_fs26 #6-10 RT: 0.09-0.16 AV: 5 NL: 2.82E7 T: + c Q1MS [100.00-1000.00] 412.79 100 115.06 252.96 80 267.02 421.78 60 168.03 208.98 410.77 40 182.04 425.78 20 141.05 298.94 Relative Abundance Relative 235.01 346.96 391.21 506.76 547.68 598.6 0 100 150 200 250 300 350 400 450 500 550 600 m/z

050922APCI_N_fs26 #6-8 RT: 0.09-0.13 AV: 3 SB: 4 0.01-0.06 NL: 1.90E7 T: - c Q1MS [100.00-1000.00] 364.59 100 366.58 80

60 174.79 204.81 362.59 40 172.82 250.75 368.58 206.80 20 218.81 252.76 410.59 Relative Abundance Relative 286.64 159.74 468.49 538.49 572.39 0 100 150 200 250 300 350 400 450 500 550 600 m/z

Figure 89 Naled ESI positive, ESI negative, APCI positive, APCI negative full scan spectra 160

050920ESIfs_27 #20 RT: 0.33 AV: 1 SB: 26 0.06-0.23 , 0.59-0.83 NL: 2.49E8 T: + c Q1MS [100.00-600.00] 247.02 100

40 493.03

20 249.02 515.02 Relative Abundance Relative 115.07 169.02 229.05 310.03 339.10 389.02 417.72 484.09 530.99 586.56 0 100 150 200 250 300 350 400 450 500 550 600 m/z

050920ESI_Nfs_27 #18-24 RT: 0.30-0.41 AV: 7 SB: 10 0.06-0.22 NL: 6.57E5 T: - c Q1MS [100.00-1000.00] 134.95 100

80 230.86 335.84 60 334.85 351.84 40 318.88 156.92 200.92 276.82 20 364.80 Relative Abundance Relative 400.75 455.45 520.78 597.1 0 100 150 200 250 300 350 400 450 500 550 600 m/z

050922APCI_P_fs27 #5-14 RT: 0.08-0.23 AV: 10 NL: 2.18E8 T: + c Q1MS [100.00-1000.00] 247.00 100

40 249.00 20 201.02

Relative Abundance Relative 168.99 268.98 492.98 514.97 139.01 210.02 310.02 338.95 416.95 446.93 545.14 0 100 150 200 250 300 350 400 450 500 550 600 m/z

050922APCI_N_fs27 #5-8 RT: 0.08-0.13 AV: 4 SB: 4 0.01-0.06 NL: 3.47E7 T: - c Q1MS [100.00-1000.00] 140.89 100

60 186.89 40 230.86 20 170.92 304.78 Relative Abundance Relative 124.91 282.81 208.85 306.80 394.77 468.73 490.67 558.67 578.6 0 100 150 200 250 300 350 400 450 500 550 600 m/z

Figure 90 Oxydemethon methyl ESI positive, ESI negative (weak), APCI positive, APCI negative full scan spectra 161

050920ESIfs_28 #29 RT: 0.49 AV: 1 SB: 26 0.06-0.23 , 0.59-0.83 NL: 9.46E6 T: + c Q1MS [100.00-600.00] 335.03 100 115.10

60 143.08

40 337.03 339.02 20 173.08 310.13

Relative Abundance Relative 247.01 269.01 423.23 462.28 510.93 582.12 0 100 150 200 250 300 350 400 450 500 550 600 m/z

050920ESI_Nfs_28 #18-23 RT: 0.30-0.39 AV: 6 SB: 10 0.06-0.22 NL: 8.83E5 T: - c Q1MS [100.00-1000.00] 184.88 100

80 354.81 60 230.87 292.85 40 345.03 134.96 373.01 416.91 20 214.91 250.02 384.68 432.72 461.00 583.19 Relative Abundance Relative 556.58 0 100 150 200 250 300 350 400 450 500 550 600 m/z

050922APCI_P_fs28 #10-17 RT: 0.16-0.29 AV: 8 NL: 1.77E8 T: + c Q1MS [100.00-1000.00] 260.99 100 335.01 80

40 338.98

262.98 20 244.97 Relative Abundance Relative 106.05 159.02 198.98 277.03 340.97 413.00 444.98 508.89 572.86 0 100 150 200 250 300 350 400 450 500 550 600 m/z

050922APCI_N_fs28 #9-17 RT: 0.15-0.29 AV: 9 SB: 4 0.01-0.06 NL: 6.62E7 T: - c Q1MS [100.00-1000.00] 184.89 100

20 186.88 Relative Abundance Relative 170.88 140.88 230.86 320.78 348.77 392.78 438.77 478.71 542.64 588.63 0 100 150 200 250 300 350 400 450 500 550 600 m/z

Figure 91 Phorate ESI positive, ESI negative (weak), APCI positive, APCI negative full scan spectra 162

050920ESIfs_29 #27 RT: 0.46 AV: 1 SB: 26 0.06-0.23 , 0.59-0.83 NL: 6.99E6 T: + c Q1MS [100.00-600.00] 367.98 100 115.08 430.99

40 242.26 369.99 433.00 143.12 352.03 415.03 20 389.99 571.61 Relative Abundance Relative 183.07 265.06 334.10 433.96 208.05 467.10 562.51 0 100 150 200 250 300 350 400 450 500 550 600 m/z

050920ESI_Nfs_29 #18-25 RT: 0.30-0.43 AV: 8 SB: 10 0.04-0.20 NL: 1.10E6 T: - c Q1MS [100.00-1000.00] 167.89 100

40 213.88 321.81 337.79 411.78 20 134.94 184.91 215.87 413.78 457.82 Relative Abundance Relative 257.88 339.82 490.70 534.71 580.18 0 100 150 200 250 300 350 400 450 500 550 600 m/z

050922APCI_P_fs29 #10-16 RT: 0.16-0.27 AV: 7 NL: 1.31E8 T: + c Q1MS [100.00-1000.00] 367.95 100

40 369.95 110.03 188.03 477.01 20 142.07 389.95 Relative Abundance Relative 190.04 421.97 510.98 246.01 260.99 334.01 554.98 580.03 0 100 150 200 250 300 350 400 450 500 550 600 m/z

050922APCI_N_fs29 #9-16 RT: 0.15-0.27 AV: 8 SB: 4 0.01-0.06 NL: 1.25E8 T: - c Q1MS [100.00-1000.00] 184.89 100

20 167.89 186.88 Relative Abundance Relative 337.77 156.88 252.84 303.83 392.79 430.74478.73 531.68 551.78 587.59 0 100 150 200 250 300 350 400 450 500 550 600 m/z

Figure 92 Phosalone ESI positive (weak), ESI negative, APCI positive, APCI negative full scan spectra 163

050920ESIfs_30 #24 RT: 0.40 AV: 1 SB: 26 0.06-0.23 , 0.59-0.83 NL: 1.89E7 T: + c Q1MS [100.00-600.00] 317.97 100 380.99 80

60 115.10 40

20 319.00 381.99 495.55

Relative Abundance Relative 143.13 192.07 260.48 417.06 448.06 529.15 562.83 0 100 150 200 250 300 350 400 450 500 550 600 m/z

050920ESI_Nfs_30 #19-27 RT: 0.32-0.46 AV: 9 SB: 10 0.04-0.20 NL: 2.21E6 T: - c Q1MS [100.00-1000.00] 301.83 100

60 156.89 40 134.95 20 302.83 347.78 Relative Abundance Relative 200.92 268.86 413.82 435.62 481.42 534.53 593.69 0 100 150 200 250 300 350 400 450 500 550 600 m/z

050922APCI_P_fs30 #6-10 RT: 0.09-0.16 AV: 5 NL: 1.83E8 T: + c Q1MS [100.00-1000.00] 317.97 100

80 476.99 60

40 318.96 477.99 20 115.07 192.05 224.06

Relative Abundance Relative 380.97 166.07 233.09 301.99 451.02 479.99 537.83 583.00 0 100 150 200 250 300 350 400 450 500 550 600 m/z

050922APCI_N_fs30 #5-10 RT: 0.08-0.16 AV: 6 SB: 4 0.01-0.06 NL: 8.79E7 T: - c Q1MS [100.00-1000.00] 156.86 100

Relative Abundance Relative 158.86 336.74 142.85 224.82 301.81 352.71 422.66 481.73 536.58 598.5 0 100 150 200 250 300 350 400 450 500 550 600 m/z

Figure 93 Phosmet ESI positive, ESI negative (weak), APCI positive, APCI negative full scan spectra 164

050920ESIfs_31 #31 RT: 0.52 AV: 1 SB: 26 0.06-0.23 , 0.59-0.83 NL: 2.10E8 T: + c Q1MS [100.00-600.00] 319.11 100

40 320.10 20

Relative Abundance Relative 321.11 360.13 105.03 173.07 247.01 277.08 432.10 454.28 502.16 543.19 595.1 0 100 150 200 250 300 350 400 450 500 550 600 m/z

050920ESI_Nfs_31 #23-28 RT: 0.39-0.48 AV: 6 SB: 10 0.04-0.20 NL: 8.72E5 T: - c Q1MS [100.00-1000.00] 274.93 100

80 134.93

60 288.94 40 250.00 299.08 345.03 20 154.93 233.04 426.76 580.93

Relative Abundance Relative 327.08 372.96 506.95 213.89 460.91 569.17 0 100 150 200 250 300 350 400 450 500 550 600 m/z

050922APCI_P_fs31 #14-28 RT: 0.23-0.48 AV: 15 NL: 2.94E8 T: + c Q1MS [100.00-1000.00] 319.08 100

60 320.09 40 321.08 20 153.08 305.08

Relative Abundance Relative 277.04 106.04 154.08 241.09 334.10 385.03 427.27 471.23 515.32 568.97 597.0 0 100 150 200 250 300 350 400 450 500 550 600 m/z

050922APCI_N_fs31 #14-23 RT: 0.23-0.39 AV: 10 SB: 4 0.01-0.06 NL: 1.04E8 T: - c Q1MS [100.00-1000.00] 274.92 100

40 260.91 275.92 20 550.93 572.91 Relative Abundance Relative 182.92 276.92 342.89 151.00 196.99 398.87 426.97 480.89 574.92 0 100 150 200 250 300 350 400 450 500 550 600 m/z

Figure 94 Phostebupirim ESI positive, ESI negative, APCI positive, APCI negative full scan spectra 165

050920ESIfs_32 #29 RT: 0.49 AV: 1 SB: 26 0.06-0.23 , 0.59-0.83 NL: 2.38E8 T: + c Q1MS [100.00-600.00] 306.09 100

40 307.12

20 308.12 Relative Abundance Relative 105.04 173.08 196.11 266.12309.06 347.12 404.61 454.29 489.11 552.10 576.04 0 100 150 200 250 300 350 400 450 500 550 600 m/z

050920ESI_Nfs_32 #21-26 RT: 0.36-0.44 AV: 6 SB: 10 0.04-0.20 NL: 3.77E6 T: - c Q1MS [100.00-1000.00] 289.93 100

20 136.92 290.92

Relative Abundance Relative 250.01 335.92 181.91 373.02 434.76 460.90 518.88 581.09 0 100 150 200 250 300 350 400 450 500 550 600 m/z

050922APCI_P_fs32 #8-19 RT: 0.13-0.32 AV: 12 NL: 3.13E8 T: + c Q1MS [100.00-1000.00] 306.08 100

60 307.07 40 182.11 308.06 20 Relative Abundance Relative 153.09 183.11 277.06 320.06 360.06 416.04487.19 509.17 562.16 0 100 150 200 250 300 350 400 450 500 550 600 m/z

050922APCI_N_fs32 #9-13 RT: 0.15-0.22 AV: 5 SB: 4 0.01-0.06 NL: 1.09E7 T: - c Q1MS [100.00-1000.00] 140.88 100

80 289.92

60 186.88 40

20 290.93 Relative Abundance Relative 142.90 208.84 124.91 276.78 357.91 412.79 434.71 453.77 531.14 587.96 0 100 150 200 250 300 350 400 450 500 550 600 m/z

Figure 95 Pirimphos methyl ESI positive, ESI negative, APCI positive, APCI negative (weak) full scan spectra 166

050920ESIfs_33 #24 RT: 0.40 AV: 1 SB: 26 0.06-0.23 , 0.59-0.83 NL: 1.61E8 T: + c Q1MS [100.00-600.00] 306.08 100

20 307.12

Relative Abundance Relative 374.93 415.96 437.93 115.09 183.08224.13 254.11 309.08 478.93 515.91 556.98 0 100 150 200 250 300 350 400 450 500 550 600 m/z

050920ESI_Nfs_33 #22-30 RT: 0.37-0.51 AV: 9 SB: 11 0.03-0.20 NL: 3.11E6 T: - c Q1MS [100.00-1000.00] 344.73 100

80 314.74

60 312.77

40 182.92 228.91 346.74 20 198.93 Relative Abundance Relative 152.94 252.78 390.72 298.96 412.72 496.83 520.58 548.59 581.0 0 100 150 200 250 300 350 400 450 500 550 600 m/z

050922APCI_P_fs33 #13-21 RT: 0.22-0.36 AV: 9 NL: 1.99E8 T: + c Q1MS [100.00-1000.00] 406.92 100 404.92 80 372.90 60

40 408.91 306.07 20

Relative Abundance Relative 319.07 415.92 153.08 182.11 254.99 295.00 447.96 509.92 528.89 572.94 0 100 150 200 250 300 350 400 450 500 550 600 m/z

050922APCI_N_fs33 #12-21 RT: 0.20-0.36 AV: 10 SB: 4 0.01-0.06 NL: 5.79E6 T: - c Q1MS [100.00-1000.00] 124.84 100

80 344.73 264.76 330.72 60 182.92 328.73 40 250.80 294.78 228.91 346.72 20 372.75 Relative Abundance Relative 170.90 398.69 432.60 470.60 514.64 550.62 0 100 150 200 250 300 350 400 450 500 550 600 m/z

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: 7.97E7 T: + c Q1MS [100.00-600.00] 282.08 306.10 100

40 314.11 222.04 563.17 197.05 20 156.03 240.04 Relative Abundance Relative 394.14 416.09 564.17 115.10 345.09 457.18 516.79 0 100 150 200 250 300 350 400 450 500 550 600 m/z

050920ESI_Nfs_34 #19-25 RT: 0.32-0.43 AV: 7 SB: 11 0.03-0.20 NL: 1.51E6 T: - c Q1MS [100.00-1000.00] 252.91 100 391.97 80 325.93 60

40 437.96 298.99 364.94 107.93 392.99 20 250.01 Relative Abundance Relative 213.86 393.97 438.99 134.92 483.97 534.00 579.98 0 100 150 200 250 300 350 400 450 500 550 600 m/z

050922APCI_P_fs34 #7-13 RT: 0.11-0.22 AV: 7 SB: 3 0.02-0.06 NL: 1.70E8 T: + c Q1MS [100.00-1000.00] 327.12 100

60 299.08 40 222.00 254.03 328.11 20 170.00 198.05 329.10 Relative Abundance Relative 144.08 391.05 419.06 479.11 535.10 563.12 0 100 150 200 250 300 350 400 450 500 550 600 m/z

050922APCI_N_fs34 #6-11 RT: 0.09-0.18 AV: 6 SB: 4 0.01-0.06 NL: 6.58E7 T: - c Q1MS [100.00-1000.00] 279.94 100 153.91 80

60 199.91 40

20 280.93 Relative Abundance Relative 221.87 140.89 155.92 265.92 283.90 347.89 392.87 434.90 481.72 511.80 560.89 0 100 150 200 250 300 350 400 450 500 550 600 m/z

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: 9.31E7 T: + c Q1MS [100.00-600.00] 323.03 100

80 306.10 60

20 324.00 Relative Abundance Relative 115.10 173.08 282.08 386.02 263.08 447.00 503.09 570.97 0 100 150 200 250 300 350 400 450 500 550 600 m/z

050920ESI_Nfs_35 #18-25 RT: 0.30-0.43 AV: 8 SB: 10 0.04-0.20 NL: 1.09E6 T: - c Q1MS [100.00-1000.00] 292.85 100

80 168.92 416.79 60 214.91 40 103.92 345.04 20 107.92 250.02 364.72 Relative Abundance Relative 184.90 327.08 418.82 540.70 461.25 581.15 0 100 150 200 250 300 350 400 450 500 550 600 m/z

050922APCI_P_fs35 #7-17 RT: 0.11-0.29 AV: 11 SB: 3 0.03-0.06 NL: 2.86E8 T: + c Q1MS [100.00-1000.00] 322.99 100

40 323.99

20 337.01 446.96 Relative Abundance Relative 115.07 185.03 217.05 240.07 321.99 386.00 468.95 522.97 554.93 0 100 150 200 250 300 350 400 450 500 550 600 m/z

050922APCI_N_fs35 #9-12 RT: 0.15-0.20 AV: 4 SB: 4 0.01-0.06 NL: 1.39E7 T: - c Q1MS [100.00-1000.00] 292.82 100 154.91 80

60 200.90 40 214.89 140.90 20 294.84 Relative Abundance Relative 222.86 332.83 126.87 400.83 470.80 510.76 538.85 592.46 0 100 150 200 250 300 350 400 450 500 550 600 m/z

Figure 98 Sulfotepp ESI positive, ESI negative, APCI positive, APCI negative full scan spectra 169

050920ESIfs_36 #24 RT: 0.40 AV: 1 SB: 21 0.11-0.30 , 0.54-0.68 NL: 1.27E7 T: + c Q1MS [100.00-600.00] 115.08 100

60 323.03

40 306.10 143.07 20 173.09 263.09 340.07 Relative Abundance Relative 242.48 386.07 499.02 263.92 484.00 531.92 589.27 0 100 150 200 250 300 350 400 450 500 550 600 m/z

050920ESI_Nfs_36 #18-23 RT: 0.30-0.39 AV: 6 SB: 11 0.03-0.20 NL: 3.37E6 T: - c Q1MS [100.00-1000.00] 450.79 100

40 136.92 451.82 20 496.80 Relative Abundance Relative 178.95 200.93 273.84 340.86 386.80 430.74 453.76 497.84 562.83 0 100 150 200 250 300 350 400 450 500 550 600 m/z

050922APCI_P_fs36 #19-31 RT: 0.32-0.53 AV: 13 SB: 3 0.02-0.06 NL: 3.21E8 T: + c Q1MS [100.00-1000.00] 466.96 100

60 467.96 40 498.98 20

Relative Abundance Relative 499.97 143.03 173.06 218.01 251.12 319.09 356.03 389.03 436.96 556.91 592.9 0 100 150 200 250 300 350 400 450 500 550 600 m/z

050922APCI_N_fs36 #21-26 RT: 0.36-0.44 AV: 6 SB: 4 0.01-0.06 NL: 2.55E7 T: - c Q1MS [100.00-1000.00] 450.78 100

60 186.88 140.89 40

20 451.81

Relative Abundance Relative 356.83 124.92 142.87 208.84 262.90 276.82 424.79 453.81 518.79 586.70 0 100 150 200 250 300 350 400 450 500 550 600 m/z

Figure 99 Temephos ESI positive (weak), ESI negative, APCI positive, APCI negative full scan spectra 170

050920ESIfs_37 #30 RT: 0.51 AV: 1 SB: 26 0.06-0.23 , 0.59-0.83 NL: 2.63E7 T: + c Q1MS [100.00-600.00] 319.13 100

60 306.09 40 391.09 115.08 289.04 320.13 20 173.09 244.97

Relative Abundance Relative 321.10 393.08 143.12 208.05 277.09 466.98 539.01 569.03 0 100 150 200 250 300 350 400 450 500 550 600 m/z

050920ESI_Nfs_37 #22-29 RT: 0.37-0.50 AV: 8 SB: 10 0.04-0.20 NL: 1.81E6 T: - c Q1MS [100.00-1000.00] 184.89 100

20 134.96 186.92 258.89 299.05 345.05 Relative Abundance Relative 149.88 214.88 349.06 393.76 450.81 491.61 529.13 581.23 0 100 150 200 250 300 350 400 450 500 550 600 m/z

050922APCI_P_fs37 #16-21 RT: 0.27-0.36 AV: 6 SB: 3 0.02-0.06 NL: 2.50E8 T: + c Q1MS [100.00-1000.00] 244.95 100

80 289.01

40 391.08 246.96 20 232.96 335.00 393.07 Relative Abundance Relative 103.03 198.98 248.95 337.00 439.03 466.96 536.91 598.9 0 100 150 200 250 300 350 400 450 500 550 600 m/z

050922APCI_N_fs37 #16-23 RT: 0.27-0.39 AV: 8 SB: 4 0.01-0.06 NL: 5.71E7 T: - c Q1MS [100.00-1000.00] 184.89 100

Relative Abundance Relative 170.88 186.87 140.92 252.85 334.81 364.78 392.82 450.75 480.69 512.72 558.67 588.7 0 100 150 200 250 300 350 400 450 500 550 600 m/z

Figure 100 Terbufos ESI positive (weak), ESI negative, APCI positive, APCI negative full scan spectra 171

050920ESIfs_38 #27 RT: 0.46 AV: 1 SB: 26 0.06-0.23 , 0.59-0.83 NL: 3.50E7 T: + c Q1MS [100.00-600.00] 407.92 100 405.91 80

60 409.92 398.92 40 366.90 306.09 20 429.90

Relative Abundance Relative 319.11 431.89 105.04 141.14 230.20 288.29 466.00 505.95 589.49 0 100 150 200 250 300 350 400 450 500 550 600 m/z

050920ESI_Nfs_38 #20-27 RT: 0.34-0.46 AV: 8 SB: 10 0.04-0.20 NL: 1.50E6 T: - c Q1MS [100.00-1000.00] 373.01 100

80 417.02 60 350.72 134.94 348.72 40 328.99 461.04 156.92 200.93 374.02 20 418.04 505.06

Relative Abundance Relative 173.91 250.00 298.93 434.78 484.96 549.04 593.05 0 100 150 200 250 300 350 400 450 500 550 600 m/z

050922APCI_P_fs38 #8-13 RT: 0.13-0.22 AV: 6 SB: 3 0.02-0.06 NL: 2.33E8 T: + c Q1MS [100.00-1000.00] 398.88 100 396.89 80

60 400.88 40 366.86 407.88 20 Relative Abundance Relative 115.06 173.04 244.05 271.04 322.99 411.88 465.96 506.89 536.82 563.78 0 100 150 200 250 300 350 400 450 500 550 600 m/z

050922APCI_N_fs38 #6-11 RT: 0.09-0.18 AV: 6 SB: 4 0.01-0.06 NL: 4.25E7 T: - c Q1MS [100.00-1000.00] 170.91 100

80 124.92 60

20 350.72

Relative Abundance Relative 156.94 192.88 216.90 260.83 328.77 354.70 396.68 441.03 498.65 564.87 0 100 150 200 250 300 350 400 450 500 550 600 m/z

Figure 101 Tetrachlorvinphos ESI positive, ESI negative, APCI positive, APCI negative full scan spectra 172

050920ESIfs_39 #35 RT: 0.59 AV: 1 SB: 26 0.06-0.23 , 0.59-0.83 NL: 6.52E7 T: + c Q1MS [100.00-600.00] 315.09 100

20 316.09

Relative Abundance Relative 356.11 115.09 179.09228.17 259.04 306.10 414.21 466.17 520.21 566.25 0 100 150 200 250 300 350 400 450 500 550 600 m/z

N/F

050922APCI_P_fs39 #35-46 RT: 0.60-0.79 AV: 12 SB: 3 0.02-0.06 NL: 2.89E8 T: + c Q1MS [100.00-1000.00] 315.07 100

80 347.08 60

20 348.07 Relative Abundance Relative 201.05 233.04291.04 414.16 450.07 501.03 571.09 0 100 150 200 250 300 350 400 450 500 550 600 m/z

050922APCI_N_fs39 #32-45 RT: 0.55-0.77 AV: 14 SB: 4 0.01-0.06 NL: 1.80E8 T: - c Q1MS [100.00-1000.00] 256.90 100

20 224.93 258.89 Relative Abundance Relative 140.88 186.89270.93 324.86 392.83 420.80 488.78 536.81 568.85 0 100 150 200 250 300 350 400 450 500 550 600 m/z

Figure 102 Tribufos ESI positive, ESI negative, APCI positive, APCI negative full scan spectra 173

050920ESIfs_40 #19 RT: 0.32 AV: 1 SB: 26 0.06-0.23 , 0.59-0.83 NL: 7.20E7 T: + c Q1MS [100.00-600.00] 256.92 100

80 297.95 60 299.95 514.83 40 288.92 319.92 512.86 516.82 20 105.03 518.83

Relative Abundance Relative 152.07 323.93 368.96 534.82 221.00 439.89480.91 556.70 0 100 150 200 250 300 350 400 450 500 550 600 m/z

050920ESI_Nfs_40 #17-22 RT: 0.29-0.37 AV: 6 SB: 25 0.08-0.29 , 0.60-0.79 NL: 6.45E6 T: - c Q1MS [100.00-1000.00] 302.77 100

40 304.77 292.77 20 346.78

Relative Abundance Relative 134.93 319.75 204.83 240.77 350.78 428.74 460.63 512.66 558.64 580.6 0 100 150 200 250 300 350 400 450 500 550 600 m/z

050922APCI_P_fs40 #5-9 RT: 0.08-0.15 AV: 5 SB: 8 0.01-0.04 , 0.39-0.46 NL: 1.04E8 T: + c Q1MS [100.00-1000.00] 152.03 100

60 143.03

40 297.92 221.02 288.93 20 115.06 166.05

Relative Abundance Relative 301.94 368.91 382.93 437.88 514.82 550.84 572.98 0 100 150 200 250 300 350 400 450 500 550 600 m/z

050922APCI_N_fs40 #6-8 RT: 0.09-0.13 AV: 3 SB: 6 0.02-0.06 , 0.22-0.25 NL: 1.19E7 T: - c Q1MS [100.00-1000.00] 140.91 100 242.80 80 190.89 204.81 60 176.88

40 128.86 244.78 20 290.75 350.76

Relative Abundance Relative 250.81 354.75 388.69 462.63 498.61 526.61 570.58 0 100 150 200 250 300 350 400 450 500 550 600 m/z

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

Solvent Analyte Diethyl Ether Ethyl Acetate Dichloro 1-Chloro Toluene n-Hexane methane butane 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

Solvent Analyte Diethyl Ether Ethyl Acetate Dichloro 1-Chloro Toluene n-Hexane methane butane 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

Solvent Ethyl Acetate Dichloro n-Hexane Blend of 1 Ethyl Acetate 1 Ethyl Acetate 1 Dichloro Analyte methane Ethyil Acetate 2 Dichloro- 2 n-Hexane methane Dichloromethan methane 2 n-hexane e 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

80.0% Ethyl Acetate Dichloromethane 70.0% n-Hexane mix of EA, DCM and hexane EA, then DCM 60.0% EA then Hexane DCM then Hexane 50.0%

40.0% Recovery

30.0%

20.0%

10.0%

0.0%

P s e e n s te os o o os M a vos ion o rop os fos M thyl r p h h i e os M os M p thi h inp onofos E idath v F ma Tribuf Acephate ichlo Etho r bensulide TrichlorfonD imph temeph Dimetho eth Cou M Azinp ir chlo Chlorpyr Propetamph P Tetra Oxydemethon M Analytes in HPLC elution order

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 Temp = 20°C (Pressure = 1500 PSI, 5 min Static time) 20°C 40°C 75°C 100°C 10 min 2500 static PSI time 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

20° 40° 75° 100°

100.00% Temephos Tribufos Ethion Chlorpyrifos Coumaphos Bensulide Fonofos 50.00% Tetrachlorvinphos

Recovery Pirimphos Me Azinphos Me Ethoprop Propetamphos Methidathion Dichlorvos Trichlorfon 0.00% Dimethoate ° Oxydem. Me 20 0° Acephate 4 ° MMP Cell 75 0° Analytes in the order of Elution Temp. 10

Figure 105 ASE temperature effect on recoveries

Temephos 100.0% Tribufos Ethion Chlorpyrifos Coumaphos Bensulide Fonofos Tetrachlorvinphos Pirimphos Me Azinphos Me 50.0% Propetamphos Recovery Ethoprop Mthidathion Dichlorvos Trichlorfon Dimethoate Oxydem Me Analytes in the order of elution 0.0% Acephate ime MMP atic t I in st 0 PS 10 m 250

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

HO OH HO OH O O Si Si Si O Si Silica Surface O SiH

O Si NH2 Supelco O NH PSA Structure O

O N

Varian HLB Sorbent

O 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.

1.8

1.6

1.4

1.2

1.0

0.8 mg Solids Solids mg PSA 0.6 Silica 0.4 Alumina HLB 0.2 HLB +Na2SO4 LRA 0.0 12345678 mL ACN eluent

Figure 108 Comparison of various sorbents for removing contaminants from serum extract 188

MMP Silica Acephate 1.2 Oxydemethon Methyl 160.00% Dimethoate Trichlorfon 140.00% 1 Dichlorvos Methidathion 120.00% Propetamphos 0.8 Ethoprop y 100.00% Azinphos Me 0.6 Pirimphos Me 80.00% Tetrachlorvinphos Recover

Solids [mg/mL] Fonofos 60.00% 0.4 Bensulide 40.00% Coumaphos 0.2 Chlorpyrifos 20.00% Ethion Tribufos 0.00% 0 Temephos 0.01.02.03.04.05.0 Solids Eluent Volume [mL] Poly. (Solids)

MMP Aluminum Oxide Acephate Oxydemethon Methyl Dimethoate 1.2 160.00% Trichlorfon Dichlorvos 140.00% 1 Methidathion 120.00% Propetamphos 0.8 Ethoprop y 100.00% Azinphos Me 80.00% 0.6 Pirimphos Me

Recover Tetrachlorvinphos

60.00% Solids [mg/mL] 0.4 Fonofos 40.00% Bensulide 0.2 20.00% Coumaphos Chlorpyrifos 0.00% 0 Ethion 0.0 1.0 2.0 3.0 4.0 5.0 Tribufos Eluent Volume [mL] Temephos Poly. (Solids)

Figure 109 Elution of OP pesticides over different sorbents 189

MMP PSA Acephate Primary Secondary Amine 1.2 Oxydemethon Methyl 160.00% Dimethoate Trichlorfon 1 140.00% Dichlorvos Methidathion 120.00% 0.8 Propetamphos Ethoprop y 100.00% Azinphos Me 0.6 80.00% Pirimphos Me Recover Tetrachlorvinphos 60.00% Solids [mg/mL] 0.4 Fonofos Bensulide 40.00% Coumaphos 0.2 20.00% Chlorpyrifos Ethion 0.00% 0 Tribufos 0.0 1.0 2.0 3.0 4.0 5.0 Temephos Eluent Volume [mL] Poly. (Solids)

LRA MMP Lipid removal Agent Acephate Oxydemethon Methyl 1.2 Dimethoate 160.00% Trichlorfon 140.00% 1 Dichlorvos Methidathion 120.00% 0.8 Propetamphos Ethoprop y 100.00% Azinphos Me 0.6 80.00% Pirimphos Me

Recover Tetrachlorvinphos

60.00% 0.4 Solids [mg/mL] Fonofos Bensulide 40.00% Coumaphos 0.2 20.00% Chlorpyrifos Ethion 0.00% 0 Tribufos 0.01.02.03.04.05.0 Temephos Eluent Volume [mL] Poly. (Solids)

Figure 109, continued: Elution of OP pesticides over different sorbents 190

Oasis HLB MMP Acephate 1.2 Oxydemethon Methyl 160.00% Dimethoate Trichlorfon 140.00% 1 Dichlorvos 120.00% Methidathion 0.8 Propetamphos 100.00% Ethoprop 0.6 Azinphos Me 80.00% Pirimphos Me Recovery Tetrachlorvinphos 60.00% [mg/mL] Solids 0.4 Fonofos 40.00% Bensulide 0.2 Coumaphos 20.00% Chlorpyrifos Ethion 0.00% 0 Tribufos 0.0 1.0 2.0 3.0 4.0 5.0 Temephos Eluent Volume [mL] Poly. (Solids)

Figure 109, continued: Elution of OP pesticides over different sorbents 191

Average Recoveries Standard Deviations 200 mg 200 mg HLB HLB 200 mg 200 mg HLB + 200 mg + 200 mg HLB Na2SO4 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 200 mg HLB HLB 200 mg HLB 200 mg HLB + 200 mg + 200 mg Na2SO4 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

Te mephos Tribufos Ethion Chl orpyrifos Coumaph Be os nsulide 100.0% Fonofos Tetra chlorvinph Pirim os phosMe Azinp hos_Me Pr opetamph E os 50.0% thoprop Meth idathion Dichlorv Recovery os Tr ichlorfon Dim Analytes in the Order of Elution 0.0% ethoate B Oxydem M g HL e 0 m Ac 20 O4 ephate Na2S mg MMP 200 LB + mg H 200

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

M ev Ch inph P lore os ho tho Ca rate xyf D dus os icro afo Su top s lfot ho 100.0% Dia epp s T zin erb on F ufo ono s Ph fos ost Ch ebu lorp pir 50.0% Me irif im pa os M Ma rath e lath ion Fen ion

Recovery t E hion t Pa F rath 0.0% ena ion P mip HLB rof ho mg eno s 200 O4 Ph fos a2S osm mg N P et 200 hos LB + alon mg H e 200 Analytes in the Order of Detection

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

193

Serum Extraction

1 mL Serum Vortex Mixing Spiked with 10uL Internal Standard 5 mL Cap Ethyl acete

Filter

22 mL Sample dispersed in 10mL ASE cell Hydromatrix

~ 10 mL dry Hydromatrix ~ 10 mL dry Vortex Mixer Hydromatrix

22 mL Flat bottom glass vial To ASE

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

Oven

High Pressure Pump

Pressure Purge Valve Cell Carussel

Static Valve

Nitrogen Solvent Reservoir

Receptacle Extract Vial Carussel

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 ASE In ethyl acetate

Add 5 mL acetonitrile Dry to 0.5 mL

Add 5 mL acetonitrile

Is GC-CI No Needed?

Yes Dry to ~0.1 mL Dry to 0.5 mL

200 mg HLB Oasis Sorbent Rapid Trace Extraction on HLB Eluent: ACN

First 2 mL captured

Dry to ~0.1 mL

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

10 min, 20 min, 2µL 2µL Start 10 min 20 min start dodecane 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

MMP Analyte Loss During Turbovap Drying Acephate Oxydemethon Methyl 150.0% Dimethoate To dryness: Trichlorfon ~6-7.5 min Dichlorvos Methidathion 100.0% Propetamphos Ethoprop Azinphos Me Pirimphos Me 50.0% Analyte Present Tetrachlorvinphos Fonofos Bensulide Coumaphos 0.0% Chlorpyrifos 01020 Ethion Drying Time [min] Tribufos

Figure 115 Recoveries of LC compound group as the function of drying time 202

Analyte Loss During Turbovap Drying MMP 2 uL Dodecane added Acephate Oxydemethon Methyl 150.0% Dimethoate To dryness: Trichlorfon ~6-7.5 min Dichlorvos Methidathion 100.0% Propetamphos Ethoprop Azinphos Me Pirimphos Me Tetrachlorvinphos

Analyte Present 50.0% Fonofos Bensulide Coumaphos Chlorpyrifos 0.0% 01020Ethion Tribufos Drying Time [min] Temephos

Figure 116 Recoveries of LC compound group as the function of drying time, 2µL dodecane added 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_Q Y = 0.0183435+0.578204*X R^2 = 0.9995 W: 1/X

6.0 5.5 5.0 4.5 4.0 3.5 3.0

Area Ratio Area 2.5 2.0 1.5 1.0 0.5 0.0 0 2 4 6 8 10 Figure 117 Methamidophos Calibration Plot

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 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 0.29 Acephate 0.26 Oxydemethon Methyl 1.19 Dimethoate 0.27 Trichlorfon 1.13 Dichlorvos 8.99 Methidathion 0.16 Propetamphos 1.82 Ethoprop 0.97 Azinphos Me 0.52 Pirimphos Me 0.18 Tetrachlorvinphos 3.32 Fonofos 0.44 Bensulide 0.75 Coumaphos 1.38 Chlorpyrifos 0.95 Ethion 0.25 Tribufos 0.92 Temephos 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).

MMP 1 Acephate Oxydemethon Methyl Dimethoate Trichlorfon Dichlorvos Methidathion Propetamphos Ethoprop Azinphos Me Pirimphos Me Tetrachlorvinphos Fonofos Normalized responseFactor Normalized Bensulide Coumaphos Chlorpyrifos 0 Ethion 0 102030Tribufos Time [hrs] Temephos

Figure 118 Decomposition of OP pesticides in serum 210

MMP 1 Acephate Oxydemethon Methyl Dimethoate Trichlorfon Dichlorvos Methidathion Propetamphos Ethoprop Azinphos Me Pirimphos Me Tetrachlorvinphos Fonofos Normalized response Factor response Normalized Bensulide Coumaphos Chlorpyrifos 0 Ethion 0 102030Tribufos Time [hrs] Temephos

Figure 119 Decomposition of OP pesticides in serum, 100µL 50 mmolar EDTA solution is added 211

Relative standard 5 mL ethyl acetate but 5 mL ethyl acetate, no vortex, no ethyl acetate deviations of no vortex mixing vortex mixing calculated concentrations 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 33 Acephate 22 Oxydemethon Methyl 29 Dimethoate 69 Trichlorfon Dichlorvos Methidathion 5.2 Propetamphos 38 Ethoprop 85 Azinphos Me 41 Pirimphos Me 22 Tetrachlorvinphos Fonofos 245 Bensulide 1.0 Coumaphos 17 Chlorpyrifos 61 Ethion 7.5 Tribufos 27 Temephos 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.

Analyte Parent Fragment Optimum Collision Ion m/Z Energy m/Z [V] 77 51 Azinphos 132 21 318 Methyl 104 26 125 24 160 33 133 49 Phosmet 318 77 52 105 56

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

214

RT: 0.00 - 30.01 RT: 12.40 NL: 8.86E3 AA: 85086 TIC F: + c APCI sid=10.00 SRM ms2 100 [email protected] [76.500-77.500] MS 318 -> 77 RT: 27.79 50 RT: 23.46 RT: 25.89 ICIS 0801010AzMe05_081015185749 AA: 13354 AA: 7094 AA: 567 0 RT: 12.34 NL: 1.27E4 AA: 141687 TIC F: + c APCI sid=10.00 SRM ms2 100 [email protected] [131.500-132.500] MS ICIS 50 RT: 27.89 0801010AzMe05_081015185749 AA: 774 0 RT: 12.39 NL: 5.22E3 AA: 50454 TIC F: + c APCI sid=10.00 SRM ms2 100 [email protected] [103.500-104.500] 50 318 -> 132 MS ICIS 0801010AzMe05_081015185749 0 RT: 12.39 NL: 1.11E4 AA: 113779 TIC F: + c APCI sid=10.00 SRM ms2 100 [email protected] [124.500-125.500] 50 MS ICIS 0801010AzMe05_081015185749 0 NL: 1.72E3 TIC F: + c APCI sid=10.00 SRM ms2 100 RT: 27.91 AA: 1171 [email protected] [159.500-160.500] 50 RT: 2.28 318 -> 160 MS ICIS AA: 357 0801010AzMe05_081015185749 0 RT: 29.75 NL: 6.63E2 TIC F: + c APCI sid=10.00 SRM ms2 100 AA: 299 [email protected] [132.500-133.500] 50 MS ICIS 0801010AzMe05_081015185749 0 RT: 12.34 NL: 5.94E3 AA: 77495 TIC F: + c APCI sid=10.00 SRM ms2 100 318 -> 133RT: 25.94 RT: 28.03 [email protected] [76.500-77.500] MS 50 RT: 23.56 AA: 7331 AA: 5089 ICIS 0801010AzMe05_081015185749 AA: 1148 0 RT: 25.91 NL: 2.04E3 RT: 23.78 RT: 27.47 AA: 4663 TIC F: + c APCI sid=10.00 SRM ms2 100 AA: 590 AA: 2640 [email protected] [104.500-105.500] 50 MS ICIS 0801010AzMe05_081015185749 0 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 Time (min)

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

RT: 0.00 - 30.00 RT: 12.25 NL: 2.86E4 AA: 394375 TIC F: + c APCI sid=10.00 SRM ms2 100 318 -> 77 [email protected] [76.500-77.500] MS 50 ICIS 0801010azme06_081015183815

0 RT: 12.21 NL: 1.31E3 AA: 1670 TIC F: + c APCI sid=10.00 SRM ms2 100 [email protected] [131.500-132.500] RT: 12.25 MS ICIS 0801010azme06_081015183815 50 AA: 649 0 NL: 6.58E2 12.31 TIC F: + c APCI sid=10.00 SRM ms2 100 12.17 318 -> 132 [email protected] [103.500-104.500] 50 MS 0801010azme06_081015183815 0.86 2.275.14 5.79 7.50 8.33 9.90 11.19 13.60 16.09 17.09 0 NL: 1.17E3 TIC F: + c APCI sid=10.00 SRM ms2 100 [email protected] [124.500-125.500] RT: 12.24 MS ICIS 0801010azme06_081015183815 50 AA: 384 0 RT: 12.23 NL: 9.59E4 AA: 1211308 TIC F: + c APCI sid=10.00 SRM ms2 100 [email protected] [159.500-160.500] MS ICIS 0801010azme06_081015183815 50 RT: 12.63 318 -> 160 AA: 3963 0 RT: 12.25 NL: 1.63E4 AA: 190719 TIC F: + c APCI sid=10.00 SRM ms2 100 [email protected] [132.500-133.500] 50 MS ICIS 0801010azme06_081015183815

0 RT: 12.25 NL: 2.85E4 AA: 393438 TIC F: + c APCI sid=10.00 SRM ms2 100 318 -> 133 [email protected] [76.500-77.500] MS 50 ICIS 0801010azme06_081015183815

0 RT: 12.25 NL: 1.10E4 AA: 93788 TIC F: + c APCI sid=10.00 SRM ms2 100 [email protected] [104.500-105.500] 50 MS ICIS 0801010azme06_081015183815

0 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 Time (min)

Figure 121 Phosmet chromatography, monitoring all four major transitions for azinphos methyl and phosmet 215

30000

25000 Phosmet Azinphos Methyl 20000

15000 Count

10000

5000

0 10 11 12 13 14 15 Time [min]

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

Coumaphos O O S Cl EtO P O 100% OEt CH3 Chlorpyrifos Cl S N EtO P O Cl OEt Cl 80%

y = -0.1122x + 0.9539

60%

Trichlorfon O OH Cl MeO P O Cl MeO Cl 40% Tetrachlorvinphos H Cl Cl O

MeO P O Cl

OMe 20% Cl Dichlorvos O Cl MeO P O Cl OMe 0% CH3 0123

Figure 123 Dependence of ASE recovery on the number of nitrogen atoms in the OP molecule. Emphasis on the halogenated species. 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 Specificity Cumulative? Compound Window of exp. Urinary Nonspecific Only for the OP Yes, Metabolites metabolites: Alkyl pesticides that have phosphate and detectable specific days thiophosphate metabolites metabolates Few specific metabolates Butyryl Digested protein Non specific Yes, Cholinesterase segment with OP Adducts remaining grouop months still attached Serum OP Original, non Highly specific No, (Dissertation metabolized OP method) pesticides 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

S N Cl EtO P O Cl S N OEt Cl EtO P O Cl S Chlorpyrifos EtO P OH OEt Cl OEt S Diethylthiophosphate Chlorpyrifos Cl Cl EtO P OH O N N OEt EtO P O Cl HO Cl Diethylthiophosphate OEt Cl Cl Cl O N Cl Chlorpyrifos Oxon 3,5,6-Trichloropiridinol EtO P O Cl N

HO Cl OEt O Cl

EtO P OH Cl Cl Chlorpyrifos Oxon OEt 3,5,6-Trichloropiridinol N Diethylphosphate HO Cl O Cholinesterase Inhibition EtO P OH Cl

OEt 3,5,6-Trichloropiridinol Cl Cl Diethylphosphate HOOC O N O N - HO O Cl + O S Cl O HO OH Cl Cl 3,5,6-Trichloropiridinol sulfate 3,5,6-Trichloropiridinol glucuonide

Figure 124 Metabolism of Chlorpyrifos Figure 125 Environmental degradation of chlorpyrifos

227

CHAPTER 4 APPENDIX

Appendix 4A: Suggested fragmentation of OP pesticides

Pesticide name Chemical Structure

O 125 143 CH3 153 i-Pr S N MeO P NH C EtO P ON

SMe O +H 184 OEt Me +H 305 169 Acephate Diazinon

O 109 O S Cl MeO P OCC MeO P S CH2 N N N 104 79 Cl OMe OMe CH3 132 +H 318 +H 221 Azinphos-methyl Dichlorvos (DDVP)

72 S O O O CH 158 141 3 MeO P O C CH C N i-Pr OPS CH2 CH2 NH S CH3 OMe CH3 O i-Pr O 127 +H 398 +H 238

Bensulide Dicrotophos

O S 131 O

MeO P SCH2 C NH CH3 EtO P S s-Bu OMe 171 S 97 125 +H s-Bu 230 +H 271

Dimethoate Cadusafos 228

S 143 88 S Cl 115 EtO P S CH2 CH2 S Et EtO P O CH C Cl OEt OEt Cl Cl +H 274 +H 335

Chlorethoxyphos Disulfoton Cl S 198 S 143 S N 171 EtO P O Cl EtO P SCH2 S P OEt 97 OEt OEt OEt Cl +H 350 +H 385

Chlorpyrifos Ethion

Cl O 131 S N

MeO P O Cl EtO P S n-Pr

290, 292 OMe S 97 Cl n-Pr +H 243 +H 322, 324 Chlorpyrifos methyl Ethoprop S O 110

O EtO P O NO2 227 S Cl 236 OEt EtO P O +H 292 - 2 Et OEt CH3 307 +H 363

Coumaphos Ethyl Parathion

O 217 CH3 O O 202 MeO P O C CH C OCH i-Pr NH P O SEt OMe 3 CH3 67 127 +H 225 OEt +H 318 Fenamiphos Mevinphos 229

S O Cl 79 MeO P OCHC Cl MeO P O NO2

OMe OMe Br Br CH 109 3 109 +H 278 -2Br+H 221, 223

Fenitrothion Naled S O 169 109 O MeO P O S CH3 MeO P SCH2 CH2 S CH2 CH3 169 OMe 105 CH3 OMe +H 279 +H 247

Fenthion Oxydemeton methyl

109 S S 97 75

Et P S EtO P S CH2 S Et OEt 199 137 OEt +H 247 +H 261

Fonofos Phorate

S S O 128 99 O C OEt EtO P S 182 MeO P S HC O O NO CH C OEt OMe 2 Et +H 331

+H 368 Cl Malathion Phosalone O O 77 S 160 MeO P NH2 94

SMe MeO P S CH2 N +H 142 125 OMe O +H 318

Methamidophos 230

Phosmet

O 145 153 S 85 C S CH S N 3

MeO P SNCH2 EtO P O C CH3 C O CH3 N N OMe 277 O CH3 +H 303 i-Pr +H 319

Methidathion Phostebupirim (tebupirimfos)

341 S S S MeO P O NO2 109 MeO P O S O P OMe OMe +H 264 127 OMe OMe 419 +H 467 Temephos Methyl parathion

CH3 164 S S 97

MeO P O N EtO P SCH2 S t-Bu 108 233 OMe N OEt +H 289 N Et

Et +H 306 Terbufos6 Pirimiphos methyl O H Cl Cl O EtO P O Br 127 C MeO P OC Cl 303, 305 S Cl n-Pr OMe +H 373, 375 Cl 204 (-Cl) +H 365

Profenofos Tetrachlorvinphos 231

S O H 169 113 MeO P OCC C O i-Pr n-Bu SPS n-Bu

HN CH3 O S n-Bu Et 156 +H 282 +H 315 138

Propetamphos Tribufos (DEF)

S S 127 115 97 OOHCl EtO P O P OEt MeO P CH C Cl O O Et Et OMe Cl +H 323 +H 257 109 Sulfotepp 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:

f rec = A fB

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

Blank Blank A B

Analyte Spike

Cleanup Cleanup

Analyte Spike

Internal Standard Internal Standard

Analysis Analysis

fA fB 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 Response factor

Calculated Concentrations

Concentration

Actual Concentration Standard Deviation of Deviation Standard Calculated Concentrations Calculated LOD = Intercept 3 x Intercept

Concentration

Figure 127 Determination of limits of detection 236

REFERENCES

1. EPA. Pesticide Reregistration Status for Organophosphates. [Web Page] 2008 2008 [cited; Available from: http://www.epa.gov/pesticides/reregistration/status_op.htm. 2. Abou-Donia, M.B., Organophosphorus ester-induced chronic neurotoxicity. Archives of Environmental Health, 2003. 58(8): p. 484-497. 3. Kaplan, Sensory Neuropathy Associated with Dursban (Chlorpyrifos) Exposure (Vol 43, Pg 2139, 1993). Neurology, 1994. 44(2): p. 367-367. 4. Beach, J.R., et al., Abnormalities on neurological examination among sheep farmers exposed to organophosphorous pesticides. Occupational and Environmental Medicine, 1996. 53(8): p. 520-525. 5. London, L., et al. An investigation into neurologic and neurobehavioral effects of long-term agrichemical use among deciduous fruit farm workers in the Western Cape, South Africa. 1997: Academic Press Inc Jnl-Comp Subscriptions. 6. Fiedler, N., et al. The assessment of neurobehavioral toxicity: SGOMSEC Joint Report. 1996: Natl Inst Environ Health Sci. 7. Srivastava, A.K., et al., Clinical, biochemical and neurobehavioural studies of workers engaged in the manufacture of quinalphos. Food and Chemical Toxicology, 2000. 38(1): p. 65-69. 8. Miyaki, K., et al., Effects of sarin on the nervous system of subway workers seven years after the Tokyo subway sarin attack. Journal of Occupational Health, 2005. 47(4): p. 299-304. 9. Nishiwaki, Y., et al., Effects of sarin on the nervous system in rescue team staff members and police officers 3 years after the Tokyo subway sarin attack. Environmental Health Perspectives, 2001. 109(11): p. 1169-1173. 10. Pilkington, A., et al., An epidemiological study of the relations between exposure to organophosphate pesticides and indices of chronic peripheral neuropathy and neuropsychological abnormalities in sheep farmers and dippers. Occupational and Environmental Medicine, 2001. 58(11): p. 702-710. 11. Edwards, P., et al., Factors Influencing Organophosphate Toxicity in Humans, in Organophosphates and Health. 2001, Imperial College Press: London. p. 61-81. 12. Needham, L.L. and K. Sexton, Assessing children's exposure to hazardous environmental chemicals: an overview of selected research challenges and complexities - Introduction and overview. Journal of Exposure Analysis and Environmental Epidemiology, 2000. 10(6): p. 611-629. 13. Hardt, L. and J. Angerer, Determination of dialkyl phosphates in human urine using gas chromatography-mass spectrometry. Journal of Analytical Toxicology, 2000. 24(8): p. 678-684. 14. Bravo, R., et al., Measurement of dialkyl phosphate metabolites of organophosphorus pesticides in human urine using lyophilization with gas chromatography-tandem mass spectrometry and isotope dilution quantification. 237

Journal of Exposure Analysis and Environmental Epidemiology, 2004. 14(3): p. 249-259. 15. Barr, D.B., et al., Strategies for biological monitoring of exposure for contemporary-use pesticides. Toxicology and Industrial Health, 1999. 15(1-2): p. 168-179. 16. Aprea, C., et al., Biological monitoring of pesticide exposure: a review of analytical methods. Journal of Chromatography B-Analytical Technologies in the Biomedical and Life Sciences, 2002. 769(2): p. 191-219. 17. Barr, D.B. and L.L. Needham, Analytical methods for biological monitoring of exposure to pesticides: a review. Journal of Chromatography B-Analytical Technologies in the Biomedical and Life Sciences, 2002. 778(1-2): p. 5-29. 18. Musshoff, F., H. Junker, and B. Madea, Simple determination of 22 organophosphorous pesticides in human blood using headspace solid-phase microextraction and gas chromatography with mass spectrometric detection. Journal of Chromatographic Science, 2002. 40(1): p. 29-34. 19. Barr, D.B., et al., A multi-analyte method for the quantification of contemporary pesticides in human serum and plasma using high-resolution mass spectrometry. Journal of Chromatography B-Analytical Technologies in the Biomedical and Life Sciences, 2002. 778(1-2): p. 99-111. 20. Hernandez, F., et al., Headspace solid-phase microextraction in combination with gas chromatography and tandem mass spectrometry for the determination of organochlorine and organophosphorus pesticides in whole human blood. Journal of Chromatography B-Analytical Technologies in the Biomedical and Life Sciences, 2002. 769(1): p. 65-77. 21. Tarbah, F.A., et al., An analytical method for the rapid screening of organophosphate pesticides in human biological samples and foodstuffs. Forensic Science International, 2001. 121(1-2): p. 126-133. 22. Lacassie, E., et al., Multiresidue determination method for organophosphorus pesticides in serum and whole blood by gas chromatography-mass-selective detection. Journal of Chromatography B, 2001. 759(1): p. 109-116. 23. Lacassie, E., et al., Sensitive and specific multiresidue methods for the determination of pesticides of various classes in clinical and forensic toxicology. Forensic Science International, 2001. 121(1-2): p. 116-125. 24. Liu, S.M. and J.D. Pleil, Human blood and environmental media screening method for pesticides and polychlorinated biphenyl compounds using liquid extraction and gas chromatography-mass spectrometry analysis. Journal of Chromatography B-Analytical Technologies in the Biomedical and Life Sciences, 2002. 769(1): p. 155-167. 25. Lopez, F.J., et al., Gas chromatographic determination of organochlorine and organophosphorus pesticides in human fluids using solid phase microextraction. Analytica Chimica Acta, 2001. 433(2): p. 217-226. 26. Sancho, J.V., O.J. Pozo, and F. Hernandez, Direct determination of chlorpyrifos and its main metabolite 3,5,6-trichloro-2-pyridinol in human serum and urine by coupled-column liquid chromatography/electrospray-tandem mass spectrometry. Rapid Communications in Mass Spectrometry, 2000. 14(16): p. 1485-1490. 238

27. Abu-Qare, A.W. and M.B. Abou-Donia, Simultaneous determination of malathion, permethrin, DEET (N,N-diethyl-m-toluamide), and their metabolites in rat plasma and urine using high performance liquid chromatography. Journal of Pharmaceutical and Biomedical Analysis, 2001. 26(2): p. 291-299. 28. Amini, N. and C. Crescenzi, Feasibility of an on-line restricted access material/liquid chromatography/tandem mass spectrometry method in the rapid and sensitive determination of organophosphorus triesters in human blood plasma. Journal of Chromatography B-Analytical Technologies in the Biomedical and Life Sciences, 2003. 795(2): p. 245-256. 29. Jonsson, O.B. and U.L. Nilsson, Determination of organophosphate ester plasticisers in blood donor plasma using a new stir-bar assisted microporous membrane liquid-liquid extractor. Journal of Separation Science, 2003. 26(9-10): p. 886-892. 30. Jonsson, O.B., E. Dyremark, and U.L. Nilsson, Development of a microporous membrane liquid-liquid extractor for organophosphate esters in human blood plasma: identification of triphenyl phosphate and octyl diphenyl phosphate in donor plasma. Journal of Chromatography B, 2001. 755(1-2): p. 157-164. 31. Jonsson, O.B. and U.L. Nilsson, Miniaturized dynamic liquid-liquid extraction of organophosphate triesters from blood plasma using the hollow fibre-based XT- tube extractor. Analytical and Bioanalytical Chemistry, 2003. 377(1): p. 182-188. 32. Wessels, D., D.B. Barr, and P. Mendola, Use of biomarkers to indicate exposure of children to organophosphate pesticides: Implications for a longitudinal study of children's environmental health. Environmental Health Perspectives, 2003. 111(16): p. 1939-1946. 33. Tomlin, C., Pesticide Manual. 2003: BCPC Publications. 34. Thompson, J.W., T.J. Kaiser, and J.W. Jorgenson, Viscosity measurements of methanol–water and acetonitrile–water mixtures at pressures up to 3500 bar using a novel capillary time-of-flight viscometer. Journal of Chromatography A, 2006. 1134: p. 201–209. 35. Montesano, M.A., et al., Method for determination of acephate, methamidophos, omethoate, dimethoate, ethylenethiourea and propylenethiourea in human urine using high-performance liquid chromatography-atmospheric pressure chemical ionization tandem mass spectrometry. Journal of Exposure Science and Environmental Epidemiology 2006. 17: p. 321-330. 36. Aldrich, S., LRA Lipid Removal Agent. Advanced Minerals Corporation, 2003. 37. Simpson, N.J.K., Solid Phase Extraction. 2000, New York: Marcel Dekker, Inc. 38. Fidder, A., et al., Retrospective detection of exposure to organophosphorus anti- cholinesterases: Mass spectrometric analysis of phosphylated human butyrylcholinesterase. Chemical Research in Toxicology, 2002. 15(4): p. 582- 590. 39. Noort, D., et al., Verification of exposure to organophosphates: Generic mass spectrometric method for detection of human butyrylcholinesterase adducts. Analytical Chemistry, 2006. 78(18): p. 6640-6644. 40. Needham, L.L., et al., Uses of speciation techniques in biomonitoring for assessing human exposure to organic environmental chemicals. Analytical and Bioanalytical Chemistry, 2005. 381(2): p. 397-404. 239

41. Taylor, J.K., Quality Assurance of Analytical Measurements. 1987, Bocaraton, London, New York, Washingtom DC: Lewis Publishers, a CRC Press Company.