Solatenol™ (SYN545192) SYN545192 – Analytical Method

Solatenol™ (SYN545192)

SYN545192 – Analytical Method GRM042.03A for the

Determination of SYN545192 and its Metabolite SYN546039 in Crops

Analytical Method

DATA REQUIREMENT(S): OECD ENV/JM/MONO(2007)17 EPA OPPTS 860.1340 (1996) SANCO/3029/99 rev. 4 (2000) SANCO/825/00 rev. 7 (2004) AUTHOR(S): Stephanie Braid Kaijun Lin STUDY COMPLETION DATE: August 4, 2011 PERFORMING LABORATORY: Syngenta Jealott’s Hill International Research Centre Bracknell, Berkshire, RG42 6EY, UK LABORATORY PROJECT ID: Report Number: GRM042.03A Task Number: T001407-08 SUBMITTER/SPONSOR: Syngenta Crop Protection, LLC 410 Swing Road Post Office Box 18300 Greensboro, NC 27419-8300 USA VOLUME 1 OF 1 OF STUDY PAGE 1 OF 92

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GRM042.03A Page 4 of 92

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Abbreviations and Symbols Abbreviation Definition

oC degrees Celsius or Centigrade CAS Chemical Abstract Services cm centimetre

EPA Environmental Protection Agency (U.S.) EC European Commission EU European Union g gram

GRM Global Residue Method HPLC high performance liquid chromatography

i.d. internal diameter IUPAC International Union of Pure and Applied Chemistry

kg kilogram L litre

LC-MS/MS tandem liquid chromatography/mass spectrometry/mass spectrometry LOD limit of detection LOQ limit of quantification

m metre MeCN acetonitrile MeOH methanol

µg microgram µL microlitre µm micrometre mg milligram mL millilitre mm millimetre

mmol millimole min minute mol mole ms millisecond

MS/MS tandem mass spectrometry mV millivolt MW molecular weight m/z mass to charge ratio


Abbreviation Definition N/A not applicable

ND or nd not detectable (below limit of detection) ng nanogram No. number OES Occupational Exposure Standards

OECD Organisation for Economic Co-operation and Development OPPTS Office of Prevention, Pesticides and Toxic Substances

pg picogram ppb parts per billion or micrograms per kilogram or micrograms per litre ppm parts per million or milligrams per kilogram or milligrams per litre

R (or r) correlation coefficient R2 (or r2) square of correlation coefficient

RSD relative standard deviation Rt retention time s second

SD standard deviation SHC Syngenta Hazard Category SPE Solid Phase Extraction UPW ultra pure water

V volt vol volume


TABLE OF CONTENTS

THE TITLE PAGE 1

STATEMENT OF DATA CONFIDENTIALITY CLAIMS 2

GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT 3

TABLE OF CONTENTS 7

1.0 INTRODUCTION 13

1.1 Scope of the Method ................................................................................. 13

1.2 Method Summary ...................................................................................... 13

2.0 MATERIALS AND APPARATUS 13

2.1 Apparatus .................................................................................................. 13

2.2 Reagents .................................................................................................... 14

2.3 Preparation of Analytical Standard Solutions ........................................... 14

2.3.1 Stock Solutions.......................................................................................... 14

2.3.2 Fortification Solutions ............................................................................... 15

2.3.3 Preparation of Calibration Standards for LC-MS/MS .............................. 15

2.3.4 Standard Solution Storage and Expiration ................................................ 15

2.4 Safety Precautions and Hazards ................................................................ 15

3.0 ANALYTICAL PROCEDURE 16

3.1 Sample Preparation ................................................................................... 16

3.2 Sample Fortification .................................................................................. 16

3.3 Extraction for High Water Content Crops ................................................ 17

3.4 Extraction for Dry Crops e.g. Straw.......................................................... 17

3.5 Liquid-Liquid Partition ............................................................................. 18

3.6 Acid Hydrolysis ........................................................................................ 19

3.7 Solid Phase Extraction Procedure ............................................................. 20

3.8 Experimental Precautions .......................................................................... 21

3.9 Time Required for Analysis ...................................................................... 22

3.10 Method Stopping Points ............................................................................ 22

4.0 FINAL DETERMINATION 22

4.1 Instrument Description .............................................................................. 22

4.2 Chromatography Conditions for SYN545192 and SYN546039 ............... 22

4.3 Mass Spectrometer Conditions for SYN545192 and SYN546039 ........... 23

4.4 Confirmatory Procedures for SYN545192 and SYN546039 .................... 24

5.0 CALCULATION OF RESULTS 24

5.1 Multi Point Calibration Procedure ............................................................ 24


5.2 Single Point Calibration Procedure ........................................................... 25

6.0 CONTROL AND RECOVERY SAMPLES 26

7.0 SPECIFICITY 26

7.1 Matrix ........................................................................................................ 26

7.2 Reagent and Solvent Interference ............................................................. 27

7.3 Labware Interference ................................................................................ 27

8.0 METHOD VALIDATION 27

8.1 Extractability ............................................................................................. 27

8.2 Recovery Data and Repeatability .............................................................. 27

8.3 Limit of Quantification (LOQ) .................................................................. 27

8.4 Limit of Detection (LOD) ......................................................................... 27

8.5 Matrix Effects ............................................................................................ 28

8.6 Detector Linearity ..................................................................................... 28

8.7 Extract Stability ......................................................................................... 28

9.0 LIMITATIONS 28

10.0 CONCLUSIONS 28

11.0 REFERENCES 30

TABLES 31

TABLE 1 Recovery Data - SYN545192 m/z 396→91 .............................................. 32

TABLE 2 Recovery Data - SYN546039 m/z 412→91 .............................................. 34

TABLE 3 Recovery Data - SYN545192 m/z 396→368 ............................................ 36

TABLE 4 Recovery Data - SYN546039 m/z 412→340 ............................................ 38

TABLE 5 LC-MS/MS Matrix Effects ....................................................................... 40

TABLE 6 Stability of SYN545192 in 50/50 Acetonitrile/Ultra Pure Water Stored at 4oC for 9 – 102 Days ................................................................................. 41

TABLE 7 Stability of SYN546039 in 50/50 Acetonitrile/Ultra Pure Water Stored at 4o C for 9 – 102 Days................................................................................. 42

FIGURES 44

CHEMICAL STRUCTURES ................................................................................................ 45

Figure 1: SYN545192 ............................................................................................... 45

Figure 2: SYN546039 ............................................................................................... 46

REPRESENTATIVE CHROMATOGRAMS ....................................................................... 47

Figure 3: 0.05 ng/mL SYN545192 Standard in 50/50 (v/v) Acetonitrile/Ultra Pure Water. SYN545192 Primary Transition m/z = 396 91 ........................ 47

Figure 4: 0.05 ng/mL SYN545192 Standard in 50/50 (v/v) Acetonitrile/Ultra Pure Water. SYN545192 Confirmatory Transition m/z = 396 91 ............... 47

Figure 5: Reagent Blank. SYN545192 Primary Transition m/z = 396 91 .......... 48


Figure 6: Reagent Blank. SYN545192 Confirmatory Transition m/z = 396 368 48

Figure 7: Untreated Wheat Forage. SYN545192 Primary Transition m/z = 396 91 Residue < LOQ ......................................................................................... 49

Figure 8: Untreated Wheat Forage. SYN545192 Confirmatory Transition m/z = 396 368 Residue < LOQ ............................................................................. 49

Figure 9: Untreated Wheat Forage Fortified with 0.01 mg/kg SYN545192. SYN545192 Primary Transition m/z = 396 91 Recovery = 77% ......... 50

Figure 10: Untreated Wheat Forage Fortified with 0.01 mg/kg SYN545192. SYN545192 Confirmatory Transition m/z = 396 368 Recovery = 91%50


Figure 12: Untreated Wheat Forage Fortified with 0.5 mg/kg SYN545192. SYN545192 Confirmatory Transition m/z = 396 368 Recovery = 102% ................................................................................................................... 51

Figure 13: Untreated Wheat Hay. SYN545192 Primary Transition m/z = 396 91 Residue < LOQ ......................................................................................... 52

Figure 14: Untreated Wheat Hay. SYN545192 Confirmatory Transition m/z = 396 368 Residue < LOQ .................................................................................. 52

Figure 15: Untreated Wheat Hay Fortified with 0.01 mg/kg SYN545192. SYN545192 Primary Transition m/z = 396 91 Recovery = 88% .............................. 53

Figure 16: Untreated Wheat Hay Fortified with 0.01 mg/kg SYN545192. SYN545192 Confirmatory Transition m/z = 396 368 Recovery = 83% ................... 53



Figure 19: Untreated Wheat Grain. SYN545192 Primary Transition m/z = 396 91 Residue < LOQ ......................................................................................... 55

Figure 20: Untreated Wheat Grain. SYN545192 Confirmatory Transition m/z = 396 368 Residue < LOQ .................................................................................. 55

Figure 21: Untreated Wheat Grain Fortified with 0.01 mg/kg SYN545192. SYN545192 Primary Transition m/z = 396 91 Recovery = 98% ......... 56

Figure 22: Untreated Wheat Grain Fortified with 0.01 mg/kg SYN545192. SYN545192 Confirmatory Transition m/z = 396 368 Recovery = 91%56

Figure 23: Untreated Wheat Grain Fortified with 0.1 mg/kg SYN545192. SYN545192 Primary Transition m/z = 396 91 Recovery = 97% .............................. 57

Figure 24: Untreated Wheat Grain Fortified with 0.1 mg/kg SYN545192. SYN545192 Confirmatory Transition m/z = 396 368 Recovery = 99% ................... 57

Figure 25: Untreated Orange Juice. SYN545192 Primary Transition m/z = 396 91 Residue < LOQ ......................................................................................... 58

Figure 26: Untreated Orange Juice. SYN545192 Confirmatory Transition m/z = 396 368 Residue < LOQ ............................................................................. 58


Figure 27: Untreated Orange Juice Fortified with 0.01 mg/kg SYN545192. SYN545192 Primary Transition m/z = 396 91 Recovery = 86% ......... 59

Figure 28: Untreated Orange Juice Fortified with 0.01 mg/kg SYN545192. SYN545192 Confirmatory Transition m/z = 396 368 Recovery = 84%59

Figure 29: Untreated Orange Juice Fortified with 0.1 mg/kg SYN545192. SYN545192 Primary Transition m/z = 396 91 Recovery = 86% .............................. 60

Figure 30: Untreated Orange Juice Fortified with 0.1 mg/kg SYN545192. SYN545192 Confirmatory Transition m/z = 396 368 Recovery = 88% ................... 60

Figure 31: Untreated Coffee Bean. SYN545192 Primary Transition m/z = 396 91 Residue < LOQ ......................................................................................... 61

Figure 32: Untreated Coffee Bean. SYN545192 Confirmatory Transition m/z = 396 368 Residue < LOQ .................................................................................. 61

Figure 33: Untreated Coffee Bean Fortified with 0.01 mg/kg SYN545192. SYN545192 Primary Transition m/z = 396 91 Recovery = 95% ......... 62

Figure 34: Untreated Coffee Bean Fortified with 0.01 mg/kg SYN545192. SYN545192 Confirmatory Transition m/z = 396 368 Recovery = 83%62

Figure 35: Untreated Coffee Bean Fortified with 0.1 mg/kg SYN545192. SYN545192 Primary Transition m/z = 396 91 Recovery = 74% .............................. 63

Figure 36: Untreated Coffee Bean Fortified with 0.1 mg/kg SYN545192. SYN545192 Confirmatory Transition m/z = 396 368 Recovery = 78% ................... 63

Figure 37: 0.05 ng/mL SYN546039 Standard in 50/50 (v/v) Acetonitrile/Ultra Pure Water. SYN545192 Primary Transition m/z = 412 91 ........................ 64

Figure 38: 0.05 ng/mL SYN546039 Standard in 50/50 (v/v) Acetonitrile/Ultra Pure Water. SYN545192 Confirmatory Transition m/z = 412 340 ............. 64

Figure 39: Reagent Blank. SYN546039 Primary Transition m/z = 412 91 .......... 65

Figure 40: Reagent Blank. SYN546039 Confirmatory Transition m/z = 412 340 65

Figure 41: Untreated Wheat Forage. SYN546039 Primary Transition m/z = 412 91 Residue < LOQ ......................................................................................... 66

Figure 42: Untreated Wheat Forage. SYN546039 Confirmatory Transition m/z = 412 340 Residue < LOQ ............................................................................. 66



Figure 45: Untreated Wheat Forage Fortified with 0.5 mg/kg SYN546039. SYN546039 Primary Transition m/z = 412 91 Recovery = 101% ....... 68


Figure 47: Untreated Wheat Hay. SYN546039 Primary Transition m/z = 412 91 Residue < LOQ ......................................................................................... 69

Figure 48: Untreated Wheat Hay. SYN546039 Confirmatory Transition m/z = 412 340 Residue < LOQ .................................................................................. 69






Figure 53: Untreated Wheat Grain. SYN546039 Primary Transition m/z = 412 91 Residue < LOQ ......................................................................................... 72

Figure 54: Untreated Wheat Grain. SYN546039 Confirmatory Transition m/z = 412 340 Residue < LOQ .................................................................................. 72

Figure 55: Untreated Wheat Grain Fortified with 0.01 mg/kg SYN546039. SYN546039 Primary Transition m/z = 412 91 Recovery = 94% ......... 73

Figure 56: Untreated Wheat Grain Fortified with 0.01 mg/kg SYN546039. SYN546039 Confirmatory Transition m/z = 412 340 Recovery = 95%73

Figure 57: Untreated Wheat Grain Fortified with 0.1 mg/kg SYN546039. SYN546039 Primary Transition m/z = 412 91 Recovery = 92% .............................. 74

Figure 58: Untreated Wheat Grain Fortified with 0.1 mg/kg SYN546039. SYN546039 Confirmatory Transition m/z = 412 340 Recovery = 93% ................... 74

Figure 59: Untreated Orange Juice. SYN546039 Primary Transition m/z = 412 91 Residue < LOQ ......................................................................................... 75

Figure 60: Untreated Orange Juice. SYN546039 Confirmatory Transition m/z = 412 340 Residue < LOQ ............................................................................. 75

Figure 61: Untreated Orange Juice Fortified with 0.01 mg/kg SYN546039. SYN546039 Primary Transition m/z = 412 91 Recovery = 81% ......... 76

Figure 62: Untreated Orange Juice Fortified with 0.01 mg/kg SYN546039. SYN546039 Confirmatory Transition m/z = 412 340 Recovery = 86%76

Figure 63: Untreated Orange Juice Fortified with 0.1 mg/kg SYN546039. SYN546039 Primary Transition m/z = 412 91 Recovery = 87% ............................... 77

Figure 64: Untreated Orange Juice Fortified with 0.1 mg/kg SYN546039. SYN546039 Confirmatory Transition m/z = 412 340 Recovery = 86% ................... 77

Figure 65: Untreated Coffee Bean. SYN546039 Primary Transition m/z = 412 91 Residue < LOQ ......................................................................................... 78

Figure 66: Untreated Coffee Bean. SYN546039 Confirmatory Transition m/z = 412 340 Residue < LOQ .................................................................................. 78

Figure 67: Untreated Coffee Bean Fortified with 0.01 mg/kg SYN546039. SYN546039 Primary Transition m/z = 412 91 Recovery = 82% ......... 79

Figure 68: Untreated Coffee Bean Fortified with 0.01 mg/kg SYN546039. SYN546039 Confirmatory Transition m/z = 412 340 Recovery = 82%79

Figure 69: Untreated Coffee Bean Fortified with 0.1 mg/kg SYN546039. SYN546039 Primary Transition m/z = 412 91 Recovery = 75% .............................. 80


Figure 70: Untreated Coffee Bean Fortified with 0.1 mg/kg SYN546039. SYN545192 Confirmatory Transition m/z = 412 340 Recovery = 71% ................... 80

DETECTOR LINEARITY GRAPHS .................................................................................... 81

Figure 71: LC-MS/MS Calibration Graph for SYN545192 Standards in 50/50 (v/v) Acetonitrile/Ultra Pure Water. Primary Transition m/z = 396 91 (no intercept set) .............................................................................................. 81

Figure 72: LC-MS/MS Calibration Graph for SYN545192 Standards in 50/50 (v/v) Acetonitrile/Ultra Pure Water. Confirmatory Transition m/z = 396 368 (no intercept set) ........................................................................................ 82

Figure 73: LC-MS/MS Calibration Graph for SYN546039 Standards in 50/50 (v/v) Acetonitrile/Ultra Pure Water. Primary Transition m/z = 412 91 (no intercept set) .............................................................................................. 83

Figure 74: LC-MS/MS Calibration Graph for SYN546039 Standards in 50/50 (v/v) Acetonitrile/Ultra Pure Water. Confirmatory Transition m/z = 412 340 (no intercept set) ........................................................................................ 84

MS/MS PRODUCT ION SPECTRA ..................................................................................... 85

Figure 75: Initial Product Ion Scan for SYN545192 (Negative Ionisation Mode). ... 85

Figure 76: Initial Product Ion Scan for SYN546039 (Negative Ionisation Mode). .... 86

APPENDICES 87

APPENDIX 1 APPARATUS ........................................................................................... 88

APPENDIX 2 REAGENTS .............................................................................................. 89

APPENDIX 3 LC-MS/MS TUNING PROCEDURE....................................................... 90

APPENDIX 4 METHOD FLOW CHART FOR HIGH WATER CONTENT CROPS .. 91

APPENDIX 5 METHOD FLOW CHART FOR DRY CROPS ....................................... 92


1.0 INTRODUCTION

1.1 Scope of the Method

Analytical method GRM042.03A is suitable for the determination of SYN545192 (Figure 1) and its metabolite SYN546039 (Figure 2) in crops. The limit of quantification (LOQ) of the method has been established at 0.01 mg/kg (or 0.01 ppm, 10 ppb) for both analytes. This method satisfies OECD Guidance Document ENV/JM/MONO(2007)17, US EPA guidelines EPA OPPTS 860.1340 and EC Guidance Documents SANCO/3029/99 rev 4 and SANCO/825/00 rev 7.

1.2 Method Summary

For high water content crops, 10 g sub samples of crop are extracted by homogenisation in 80/20 (v/v) acetonitrile/ultra pure water. For dry matrices e.g. straw, 5 g sub samples are pre-soaked with ultra pure water prior to homogenisation with 80/20 (v/v) acetonitrile/ultra pure water. 0.2 g aliquots of the crop extracts are evaporated under a stream of air or nitrogen to remove the acetonitrile. 2 mL of aqueous 1 M HCl is added prior to a liquid/liquid partition with iso-hexane (3 x 2 mL or 3 x 4 mL for dry matrices) to separate residues of SYN545192. The combined iso-hexane fractions are evaporated to dryness and dissolved in 50/50 (v/v) acetonitrile/ultra pure water for analysis of SYN545192 directly. Alternatively the iso-hexane fraction may be stored refrigerated, then combined with the post-hydrolysis extract should a sample clean-up be required. The remaining acidic, aqueous sample is transferred into a glass vial and heated at 100 oC for 6 hours in a water bath with agitation to hydrolyse conjugates of SYN546039. The samples are cooled and taken through a solid phase extraction (SPE) procedure. SYN546039 (and SYN545192 if included at this stage) is eluted from the cartridge with 50/50 (v/v) acetonitrile/ultra pure water. Samples are diluted with 50/50 (v/v) ultra pure water/acetonitrile as required then analysed by high performance liquid chromatography with triple quadrupole mass spectrometric detection (LC-MS/MS). The limit of quantification of the method is 0.01 mg/kg (0.01 ppm, 10 ppb) for SYN545192, and SYN546039.

2.0 MATERIALS AND APPARATUS

2.1 Apparatus

The recommended equipment and apparatus are listed in Appendix 1. Equipment with equivalent performance specifications may be substituted.


2.2 Reagents

All solvents and other reagents must be of high purity, e.g. glass distilled/HPLC grade solvents and analytical grade reagents. Particular care must be taken to avoid contamination of the reagents used. Reagents of comparable purity may be substituted as long as acceptable performance is demonstrated. A list of reagents used in this method along with details of preparation of solutions is included in Appendix 2.

2.3 Preparation of Analytical Standard Solutions

It is recommended that the following precautions should be taken when weighing the analytical materials.

1. Ensure good ventilation. 2. Wear gloves and laboratory coat. 3. Prevent inhalation and contact with mouth. 4. Wash any contaminated area immediately.

2.3.1 Stock Solutions

Prepare a 200 µg/mL stock solution for each of SYN545192 and SYN546039 by one of the following methods.

Weigh out accurately, using a five figure balance, sufficient SYN545192 and SYN546039 analytical standard into separate amber “Class A” volumetric flasks (50 mL). Dilute to the mark with acetonitrile to give individual 200 µg/mL stock solutions of SYN545192 and SYN546039.

Alternatively, the appropriate volume of acetonitrile to add to a known amount of standard material may be determined using the equation below. The standard concentration is corrected for its chemical purity.

1000××

=C

PWV

P = Standard purity in decimal form (P(%)/100) V = Volume of acetonitrile required W = Weight, in mg, of the solid analytical standard C = Desired concentration of the final solution, (µg/mL) 1000 = Unit conversion factor In this case, the standard material is weighed directly into an appropriate storage vessel.


2.3.2 Fortification Solutions

Sample fortification solutions of SYN545192 and SYN546039 should be prepared by serial dilution in acetonitrile. It is recommended that the following solutions are prepared: 10.0 µg/mL, 1.0 µg/mL and 0.1 µg/mL.

2.3.3 Preparation of Calibration Standards for LC-MS/MS

The matrix effects for SYN545192 and SYN546039 observed during method validation were generally not significant and non-matrix calibration standards should normally be used for quantification. Matrix-matched standards were used to quantify residues of SYN546039 in coffee beans in the method validation. Matrix effects may be compensated for by use of matrix matched standards at the discretion of the study director. Alternatively, further dilution of the final sample with 50/50 (v/v) acetonitrile/ultra pure water to reduce effects may be carried out should instrument sensitivity permit.

Matrix-matched standards should be prepared from further aliquots taken from the control extracts at the appropriate stage in the analytical procedure so that the matrix composition and concentration matches that of the samples to be analysed.

2.3.4 Standard Solution Storage and Expiration

All stock solutions should be stored in a refrigerator or freezer when not in use to prevent decomposition and/or concentration of the standard. Standard solutions should be allowed to equilibrate to room temperature prior to use.

Standard solutions of SYN545192 and SYN546039 at 0.1 µg/mL in acetonitrile have been shown to be stable on storage refrigerated at 4oC for at least 236 days over the course of the method validation. When tested at 1 µg/mL however, SYN546039 in acetonitrile stored under the same conditions appeared to show some degradation. An expiration date of six months for SYN545192 is recommended, but solutions of SYN546039 should be tested on a monthly basis to confirm stability.

2.4 Safety Precautions and Hazards

The following information is included as an indication to the analyst of the nature and hazards of the reagents used in this procedure. If in any doubt, consult the appropriate MSDS or a monograph such as ‘Hazards in the Chemical Laboratory’, edited by S G Luxon, The Chemical Society, London (Reference 1).


Solvent and Reagent hazards

Acetonitrile Hydrochloric acid

Methanol Acetic acid

Harmful Vapour

Highly Flammable

Harmful by Skin Absorption

Irritant to respiratory system and eyes

Causes severe burns

Syngenta Hazard Category (SHC) SHC-C, S N/A SHC-C, S SHC-C, S

OES Short Term (mg/m3) 105 7 310 37

OES Long Term (mg/m3) 70 N/A 260 25

N/A not known

At present there are insufficient data available to assign a Syngenta Hazard Classification for SYN545192 and SYN546039. They should be treated as category SHC-D compounds until further information indicates otherwise. The Syngenta Hazard Category scale rates highly toxic chemicals as category SHC-E and non-toxic chemicals as category SHC-A. An additional hazard category of S indicates the compound is a severe skin and eye irritant. In all cases avoid breathing vapour. Avoid contact with eyes and skin.

3.0 ANALYTICAL PROCEDURE

3.1 Sample Preparation

All samples should be prepared using an approved method of preparation to obtain a homogeneous sample prior to analysis. Composite crop samples should be homogenised using a suitable mincer or bowl chopper. Dry ice may be used during preparation if required.

3.2 Sample Fortification

In order to verify method performance and allow recovery corrections to be made (if appropriate), fortified control samples should be included with each sample set. Prior to extraction, recovery samples should be fortified with SYN545192 only. Fortification with SYN546039 is inappropriate at this stage as metabolism studies indicate that residues of SYN546039 are mainly conjugated.

During the acid hydrolysis step described in Section 3.6, residues of free SYN546039 are generated from conjugates present in crop commodities. To demonstrate that these free residues can be successfully taken through the subsequent SPE procedure, it is recommended that procedural recoveries are prepared by fortification of recovery samples with SYN546039 immediately prior to the SPE step described in Section 3.7.


To each pre-weighed control crop sample, add the appropriate amount of standard solution containing SYN545192 in acetonitrile. Let each sample stand for at least five minutes after fortification to allow the spiking solution to soak into the matrix before proceeding with the extraction procedure. At least one untreated control and two fortified control samples should be analysed with each sample set. For dry crops, fortification of recovery samples should be performed after pre-soaking the crop with ultra pure water.

3.3 Extraction for High Water Content Crops

A summary of the method for high water content crops is included in flow-chart form in Appendix 4. a) Weigh representative amounts of crop (10 g) into plastic centrifuge bottles

(250 mL size). At least one untreated control and two control samples fortified with known amounts of SYN545192 in acetonitrile (not more than 0.5 mL) should be analysed with each batch of samples using the same procedure to demonstrate acceptable method performance.

b) Add 80/20 (v/v) acetonitrile/ultra pure water (100 mL minus the water content of the samples). Homogenise at high speed for 3 – 5 minutes. Note: Estimate the percentage water content in each matrix type and hence the total volume of water in the 10 g sub-sample. E.g. for a 10 g sub-sample with 90% natural water content add 100 mL – (10 x 90/100) mL = 91 mL extraction solution. It is sufficient to round the natural water content to the nearest ten percent value. Any volume contraction due to mixing organic solvents with water and evaporation loss during extraction is considered to be negligible. The water content of matrices can be obtained from published sources or the relevant information can be obtained from the following USDA web site: http://www.nal.usda.gov/fnic/cgi-bin/nut_search.pl Alternatively, where information is not available from such sources, it may be necessary to determine the moisture content experimentally, following a suitable moisture content determination procedure.

c) Centrifuge samples at 3500 rpm (or at a speed that visibly separates the supernatant from the solid sample) for 5 minutes. The sample concentration is 0.1 g/mL.

d) Where instrument sensitivity permits, samples may be diluted with ultra pure water (2-fold) and analysed for SYN545192 residues by LC-MS/MS, or diluted still further with 50/50 (v/v) acetonitrile/ultra pure water.

3.4 Extraction for Dry Crops e.g. Straw

A summary of the method for dry crops is included in flow-chart form in Appendix 5.


a) Weigh representative amounts of dry crop sample (5 g) into plastic centrifuge bottles

(250 mL size).

b) Prior to fortification and extraction, samples should be wetted by addition of ultra pure water (25 mL). Leave samples to stand at room temperature for 30 minutes.

c) At least one untreated control and two control samples fortified with known amounts of SYN545192 in acetonitrile (not more than 0.5 mL) should be analysed with each batch of samples using the same procedure to demonstrate acceptable method performance.

d) Add 80/20 (v/v) acetonitrile/ultra pure water, (100 mL, so that the total volume is 125 mL), and homogenise at high speed for 3 – 5 minutes.

e) Centrifuge samples at 3500 rpm (or at a speed that visibly separates the supernatant from the solid sample) for 5 minutes. The final sample concentration 0.04 g/mL.

f) Where instrument sensitivity permits, samples may be diluted with ultra pure water (2-fold) and analysed for SYN545192 residues by LC-MS/MS, or diluted still further with 50/50 (v/v) acetonitrile/ultra pure water.

3.5 Liquid-Liquid Partition

Under the hydrolysis conditions specified in the following Section 3.6, degradation of SYN545192 was observed in the 14C metabolism study (Reference 2). It is important therefore that all the SYN545192 residues are separated from the sample using the liquid-liquid partition scheme described in this Section prior to hydrolysis. a) Transfer a 0.2 g aliquot of extract (2 mL for wet crops and 5 mL for dry crops) into a

15 mL plastic, graduated centrifuge tube. Place in a sample concentrator set at 50 oC under a steam of air or nitrogen and evaporate the sample to < 0.2 mL for wet crops and < 1 mL for dry crops. Should the sample be evaporated to dryness, the sample should be ultrasonicated thoroughly after addition of 1 M HCl and iso-hexane in the following step to prevent losses of SYN545192 due to adsorption. It is essential that all the acetonitrile is removed at this point. Any acetonitrile remaining in the sample will have an adverse effect on the recovery of SYN545192, with some SYN545192 remaining in the aqueous fraction after partition with iso-hexane. Add 1 M HCl (2 mL) to the evaporated sample and ultrasonicate briefly to mix thoroughly. Add iso-hexane (2 mL, or 4 mL for dry matrices), cap the tube securely and shake vigorously for 30 seconds. Centrifuge at a speed to separate the upper organic and lower aqueous phases e.g. 4500 rpm for 5 minutes.


b) Carefully transfer the upper iso-hexane layer into a clean, disposable glass test tube using e.g. a plastic disposable pipette, taking care not to transfer any of the lower aqueous layer. Retain both fractions.

c) Add a second 2 mL portion of iso-hexane (4 mL for dry matrices) to the acidic aqueous phase and repeat the above partition.

d) Combine the upper iso-hexane layer with the previous iso-hexane fraction. Repeat the process one more time to give a combined iso-hexane volume of 6 mL (12 mL for dry matrices).

e) Either: Evaporate the combined iso-hexane fractions just to dryness in a sample concentrator set at 30 oC under a stream of air or nitrogen. Add 50/50 (v/v) acetonitrile/ultra pure water (2 mL) and ultrasonicate briefly to mix thoroughly.

f) Transfer to a suitable autosampler vial for analysis of SYN545192 by LC-MS/MS. The final sample concentration is 0.1 g/mL. Further dilutions of the final sample with 50/50 (v/v) acetonitrile/ultra pure water may be performed to reduce any matrix effects, depending on instrument sensitivity.

g) Or: If there is insufficient instrument sensitivity or the samples are too dirty, refrigerate the combined iso-hexane fractions containing SYN545192 for a later SPE clean-up. Continue the procedure from Section 3.6 (d).

3.6 Acid Hydrolysis

The metabolite SYN546039 in crops is mainly conjugated (Reference 2). To release the free metabolites from conjugates, an acid hydrolysis step is required. The following hydrolysis conditions have been developed after extensive testing. It is important that the hydrolysis conditions are not changed as this may result in either incomplete deconjugation or degradation of free residues of SYN546039. a) Adjust the volume of the acidic, aqueous sample at Section 3.5 (d) to 4 mL with ultra

pure water so that the acid concentration is approximately 0.5 M HCl. Carefully transfer the contents into a glass, screw-top vial (15 mL size) and replace the lid securely.

b) Place in a suitable water bath capable of heating to 100 oC with sample agitation or shaking. Heat at 100 oC for 6 hours with shaking (approximately 140 - 150 strokes/minute).

c) Carefully remove the sample from the water bath wearing suitable protective equipment to avoid burns by scalding. Allow the sample to cool to room temperature before proceeding to Section 3.6 (e).

d) If an SPE clean-up for SYN545192 and SYN546039 is required, add methanol (0.5 mL) to the combined iso-hexane fractions from Section 3.5 (d) and evaporate to 0.1 - 0.2 mL in a sample concentrator set at 40 oC under a stream of air or nitrogen.


Evaporation to dryness for extended periods of time may have an adverse effect on the recovery of SYN545192 and SYN546039.

e) Transfer the cooled, hydrolyzed sample from Section 3.6 (c) into the glass tube from 3.6 (d) above after evaporation of the iso-hexane and ultrasonicate the mixture for 5 - 10 minutes before proceeding to the SPE step in Section 3.7.

3.7 Solid Phase Extraction Procedure

a) Take one Waters Oasis HLB SPE cartridge (60 mg, 3 mL size) for each sample to be analysed and place on a suitable vacuum manifold (e.g. IST Vacmaster). Add acetonitrile (2 mL) and allow to percolate through under gravity or draw through under vacuum to the level of the top frit at a rate of approximately 1 mL/min, discarding the column eluate. Do not allow the cartridges to become dry. Add ultra pure water (2 mL) to the top of each cartridge and allow to percolate through under gravity or draw through under vacuum to the level of the top frit at the same rate, again discarding the column eluate. Do not allow the cartridges to become dry.

b) Fortify the recovery samples from 3.6 (e) with SYN546039 at an appropriate level e.g. to fortify at the LOQ, add 20 µL of 0.1 µg/mL SYN546039 in acetonitrile.

c) Load the sample from Section 3.6 (e) or 3.7 (b) onto the SPE cartridges and allow to percolate through under gravity or under low vacuum, at a rate of approximately 1 - 2 mL/min, to the level of the top frit. Do not allow cartridges to become dry.

d) Add 80/20 (v/v) ultra pure water/methanol (3 mL) to the empty tube and ultrasonicate for 5 minutes.

e) Add the rinse to the top of the SPE cartridge and allow to percolate through under gravity or draw through under vacuum to the level of the top frit at the same rate, discarding the column eluate.

f) Add 50/50 (v/v) methanol/ultra pure water (3 mL) to the empty tube and ultrasonicate for 5 minutes. Add the rinse to the top of the SPE cartridge and draw through as before. Discard the eluate and do not allow cartridges to become dry.

g) Add 50/50 (v/v) methanol/ultra pure water (3 mL) to top of the SPE cartridge and draw through as before.

h) Add 35/65 (v/v) acetonitrile/ultra pure water (1 mL) to the SPE cartridge and allow to percolate through under gravity or low vacuum and discard the eluate. Do not allow cartridges to become dry.

i) Place a suitable disposable, graduated, plastic centrifuge tube under each port (e.g. 15 mL size), as required, in the manifold rack. Add 50/50 (v/v) acetonitrile/ultra pure water (2 x 3 mL) to the top of each cartridge and allow to percolate through under gravity. SYN545192 (if included at this stage) and SYN546039 are eluted from the cartridge in this step. Collect the cartridge eluate.


j) Remove the collection tubes from the manifold rack and adjust the final volume to 20 mL (or as appropriate depending on instrument sensitivity) with 50/50 (v/v). acetonitrile/ultra pure water. Further dilutions with 50/50 (v/v) acetonitrile/ultra pure water may be made to reduce any matrix effects where instrument sensitivity permits.

k) Transfer an aliquot of the sample into a suitable autosampler vial ready for final determination by LC-MS/MS. The final sample concentration is 0.01 g/mL.

3.8 Experimental Precautions

a) The SPE procedure above has been developed using cartridges from the stated manufacturer. Similar cartridges from other manufacturers may be used however in all cases, it is strongly recommended that the elution profile of the chosen batch of cartridges is checked prior to commencing analysis to assess any variation in manufacturers’ products and between batches.

b) In order to verify the performance of the SPE step, it is recommended that the recovery samples are fortified with known amounts of SYN546039 prior to loading onto the SPE cartridge. See Section 3.2 for further details.

c) Bottled HPLC grade ultra pure water is used to prepare the LC mobile phase, which produces a lower background noise in the MS/MS chromatograms than water taken from a laboratory water purification system.

d) To prevent contamination of the instrument and to minimise possible carry-over issues, it is recommended that high level recoveries (>0.1 mg/kg) and samples with expected residues greater than 0.1 mg/kg should be diluted so that the final analyte concentration does not exceed 0.005 µg/mL. It may also be useful to include blank injections of 50/50 (v/v) acetonitrile/ultra pure water after high level samples to clear any observed carry-over greater than 10% of the LOQ.

e) The hydrolysis conditions have been extensively tested to ensure efficient hydrolysis of the conjugated metabolites without degradation of SYN546039. It is essential that these conditions are not modified in any way. The acid concentration, temperature and length of time are all critical parameters. Use of a water bath with agitation or shaking has been found to be more effective than using a static heating block for the hydrolysis.

f) SYN545192 has been observed to adsorb to glass surfaces over a period of time in solutions with < 50% organic solvent. Storing samples containing SYN545192 in < 50% organic solutions must be avoided.

g) If the samples are not processed immediately following extraction, the supernatant should be decanted from the remaining crop after centrifugation and transferred into a clear plastic vessel for storage in a refrigerator. Separation of the extract into two phases has been observed in soybean seed and straw extracts on storage in a refrigerator. The extracts should be allowed to warm to room temperature and shaken


to combine the phases before aliquots are taken. Centrifugation may be necessary if particulate material is present.

3.9 Time Required for Analysis

The methodology is normally performed with a batch of 12 samples. One person can complete the analysis of 12 samples over 1.5 working days (8 hour working period).

3.10 Method Stopping Points

The analytical procedure can be stopped at various points for overnight and weekend breaks unless otherwise specified in the analytical procedure. Acceptable method recoveries will validate any work flow interruptions. Samples should be stored refrigerated in sealed containers where the analysis cannot be completed in a single day. See note 3.8 (g) above for storage of extracts.

4.0 FINAL DETERMINATION The method has been developed for use on an Applied Biosystems API4000. The following instrumentation and conditions have been found to be suitable for this analysis. Other instrumentation can also be used, though optimisation may be required to achieve the desired separation and sensitivity. The operating manuals for the instruments should always be consulted to ensure safe and optimum use.

4.1 Instrument Description Waters H Class UPLC system

: Quaternary pump, autosampler and column oven

Detector : Applied Biosystems API 4000 triple quadrupole mass spectrometer with Analyst™ software version 1.4.2

Autosampler : CTC HTS PAL Gas Supply : Peak Scientific NM20ZA gas station

4.2 Chromatography Conditions for SYN545192 and SYN546039

Column : Waters Atlantis T3 100 mm x 3 mm, 5µm Column Oven Temperature : 40°C Injection volume : 40 µL Stop Time : 11.5 minutes Injection protocol : Analyse calibration standard after 3 to 4 samples

injections Mobile phase : Solvent 1 = methanol

Solvent 2 = 0.2% (v/v) acetic acid in ultra pure water


Mobile Phase Composition

Time (mins) % solvent 1 % solvent 2 Flow rate (mL/min)

0.0 50 50 0.5 2.5 50 50 0.5 4.5 90 10 0.5 8.9 90 10 0.5 9.0 50 50 0.5 11.5 50 50 0.5

Notes : The column eluate is diverted to waste for the first 1 minute to prevent ionic material from the sample contaminating the mass spectrometer front plate. A secondary pump providing flow of mobile phase to the mass spectrometer when the column eluate is switched to waste has been found to be unnecessary. Under these conditions the retention time of SYN546039 is 5.2 minutes and SYN545192 is 6.2 minutes.

4.3 Mass Spectrometer Conditions for SYN545192 and SYN546039

Interface : TurboIonSpray

Polarity : Negative

Curtain gas (CUR) : Nitrogen set at 30 (arbitrary units)

Temperature (TEM) : 550°C

Ionspray voltage : -3500V

Collision gas setting (CAD) : Nitrogen set at 8 (arbitrary units)

Gas 1 (GS1) : Air set at 40 (arbitrary units)

Gas 2 (GS2) : Air set at 50 (arbitrary units)

Interface heater (ihe) : On

Scan type : MRM


MRM Conditions SYN545192

primary transition

SYN545192 confirmatory

transition

SYN546039 Primary

transition

SYN546039 confirmatory

transition

Q1 m/z : 396 396 412 412

Q3 m/z : 91 368 91 340

Dwell time : 150 ms 150 ms 150 ms 150 ms

Resolution Q1 : Unit Unit Unit Unit

Resolution Q3 : Unit Unit Unit Unit

Declustering potential (DP) : -95 V -95 V -85 V -85 V

Entrance potential (EP) : -10 V -10 V -10 V -10 V

Collision energy (CE) : -64 V -32 V -64 V -28 V

Collision cell exit potential (CXP) : -5 V -11 V -3 V -9 V

4.4 Confirmatory Procedures for SYN545192 and SYN546039 Final determination by LC-MS/MS with two transitions is considered to be highly specific; hence no further confirmatory conditions are included. Either transition may be used for quantification.

5.0 CALCULATION OF RESULTS

5.1 Multi Point Calibration Procedure SYN545192 and SYN546039 residues may be calculated in mg/kg for each sample as follows. a) Prepare standard solutions over a concentration range (e.g. 0.02 ng/mL to 0.5 ng/mL)

appropriate to the expected residues in the samples (for example, 50% LOQ to 10 x LOQ). An appropriate number of different concentrations within this range should be prepared (at least five).

b) Make an injection of each sample solution and measure the areas of the peaks

corresponding to SYN545192 and SYN546039. Calibration standard solutions should be interspersed throughout the analysis, after a maximum of four injections of sample solutions.

c) Generate calibration curve parameters using an appropriate regression package. d) The following equation can be rearranged and used to calculate residues as follows:

cmxy +=


Where y is the instrument response value, x is the standard concentration, m is the gradient of the line of best fit (“X-variable 1” in MS Excel) and c is the intercept value. An example of this equation generated using the experimental values of m and c should be included in the raw data, as should the “R-Squared” value for the regression.

Re-arrangement for x gives

mcyx −

=

e) Calculate the SYN545192 and SYN546039 residues in the sample, expressed as

mg/kg, as follows

)(g/mL conc. Sample)g/mL( found Analyte )(mg/kg Residue µ

=

Where analyte found (µg/mL) is calculated from the standard calibration curve and sample conc. is the final sample concentration in g/mL.

If residues need to be corrected for average percentage recovery e.g. for storage stability studies, then the equation below should be used.

(mg/kg) Recovery percentage Average

100 Residue = Residue Corrected ×

5.2 Single Point Calibration Procedure

SYN545192 and SYN546039 residues may be calculated in mg/kg for each sample using a mean standard response from each of the injections bracketing the sample as follows. a) Make repeated injections of a standard containing SYN545192 and SYN546039 at an

appropriate concentration into the LC-MS/MS operated under conditions as described in Section 4. When a consistent response is obtained, measure the peak areas obtained for SYN545192 and SYN546039.

b) Make an injection of each sample solution and measure the areas of the peaks

corresponding to SYN545192 and SYN546039. c) Re-inject the standard solution after a maximum of four injections of sample

solutions. d) Calculate the SYN545192 and SYN546039 residues in the sample, expressed as

mg/kg using a mean standard response from each of the injections bracketing the sample as follows.


Conc. SampleConc. Standard

(STD) areaPK (SA) areaPK )(mg/kg Residue ×=

PK area (SA) = Peak response for sample PK area (STD) = Average peak response for bracketing standards Standard Conc. = Concentration of standard (µg/mL) Sample Conc. = Sample concentration (g/mL)

If residues need to be corrected for average percentage recovery e.g. for storage stability studies, then the equation below should be used.

(mg/kg) Recovery percentage Average

100Residue = Residue Corrected ×

Although single point calibration may be used to quantify residues it is recommended that a calibration curve is generated with each analytical run to demonstrate the linearity of instrument response (Reference 3).

6.0 CONTROL AND RECOVERY SAMPLES

Control samples should be analysed with each set of samples to verify that the sample used to prepare recovery samples is free from contamination. A minimum of one control should be analysed with each batch of samples. At least two recovery samples (control samples accurately fortified with known amounts of SYN545192 in acetonitrile) should also be analysed alongside each set of samples. Provided the recovery values are acceptable they may be used to correct any residues found. The fortification levels should be appropriate to the residue levels expected. Recovery efficiency is generally considered acceptable when the mean values are between 70% and 110% and with a relative standard deviation of <20%. Where the method is used for monitoring purposes, control and recovery samples are not required where suitable control samples are not available. 7.0 SPECIFICITY

It is recommended that reagent blank samples be included in a sample set if contamination is suspected. 7.1 Matrix

LC-MS/MS is a highly specific detection technique. Interference arising from the matrices tested has not been observed.


7.2 Reagent and Solvent Interference

Using high purity solvents and reagents no interference has been found. 7.3 Labware Interference

This method uses mainly disposable labware. All reusable glassware should be detergent washed and then rinsed with HPLC grade methanol, acetone or acetonitrile prior to use. 8.0 METHOD VALIDATION

8.1 Extractability

SYN545192 and SYN546039 have been shown to be efficiently extracted in a radiolabelled metabolism study using the conditions described in Section 3 of the analytical procedure (Reference 2).

8.2 Recovery Data and Repeatability

Method validation has been carried out on the procedures described in Sections 3 and 4. The method validation data are reported in Syngenta report TK0002511 (Reference 4) and a summary is included in Tables 1 - 4. 8.3 Limit of Quantification (LOQ)

The limit of quantification of the method is defined as the lowest analyte concentration in a sample at which the methodology has been validated and a mean recovery of 70-110% with a relative standard deviation of ≤ 20% has been obtained. Generally, for accurate quantification, the response for an analyte peak should be no lower than four times the mean amplitude of the background noise in an untreated sample at the corresponding retention time. The limit of quantification has been set at 0.01 mg/kg (0.01 ppm, 10 ppb). 8.4 Limit of Detection (LOD)

The limit of detection of the method is defined as the lowest analyte concentration detectable above the mean amplitude of the background noise in an untreated sample at the corresponding retention time. An estimate of the LOD can be taken as three times background noise. Note that the LOD may vary between runs and from instrument to instrument. The LOD for SYN545192 and SYN546039 for both primary and confirmatory transitions was determined as approximately 1 pg injected on column, equivalent to 0.01 ng/mL when using a 40 µL injection volume in the method validation.


8.5 Matrix Effects Generally no significant matrix effects were observed in the crops types tested during method validation and non- matrix standards were used for quantification, with the exception of SYN546039 in coffee beans. In this case, significant matrix suppression was observed and matrix-matched standards were used to compensate for these effects. A summary of the matrix effects is included in Table 5. 8.6 Detector Linearity For accurate quantification of residue concentrations, analyses should be carried out within the linear range of the detector. For multi point calibration, detector range and linearity will be demonstrated within each sample set. The linearity of the LC-MS/MS detector response for SYN545192 and SYN546039 was tested in the range from 0.8 pg to 20 pg injected on column (equivalent to 0.02 ng/mL to 0.5 ng/mL standards when using a 40 µL injection volume) and was found to be linear. If a residue beyond the tested concentration range is expected, dilute the sample appropriately to bring it within the tested linear range prior to quantification. Standards of 6 different concentration levels were injected and the response plotted against the standard concentration, using Microsoft Excel for both primary and confirmatory transitions for SYN545192 and SYN546039. A plot of the response factor versus the analyte concentration for all calibration points is also included. Detector linearity graphs are presented in Figures 71 – 74.

8.7 Extract Stability Final crop extracts in 50/50 (v/v) acetonitrile/ultra pure water retained in vials and stored at a temperature of approximately 4oC were suitable for SYN545192 and SYN546039 residue analysis, for storage periods of up to 7 days. A summary of the stability data is presented in Tables 6 and 7.

9.0 LIMITATIONS The method has been tested on representative crop matrices. It can reasonably be assumed that the method can be applied to other crop matrices not tested in this method provided successful recovery tests at the relevant levels validate the suitability of the method.

10.0 CONCLUSIONS This procedure has been demonstrated to be a reliable and accurate procedure for the determination of SYN545192 and SYN546039 residues in crop commodities. Only commercially available laboratory equipment and reagents are required. The analysis of 12 crop samples for SYN545192 and SYN546039 can be completed by one person in 1.5 days


(8 working hour period). Untreated and fortified samples should be analysed with each set of samples to demonstrate absence of any interference and adequate recovery, if possible. The limit of quantification of the method is 0.01 mg/kg (0.01 ppm, 10 ppb). This method satisfies OECD Guidance Document ENV/JM/MONO(2007)17, US EPA guidelines EPA OPPTS 860.1340 and EC Guidance Documents SANCO/3029/99 rev 4 and SANCO/825/00 rev 7.


11.0 REFERENCES

1. Luxon S G (1992): Hazards in the Chemical Laboratory 5th Edition. The Royal Society of Chemistry. Thomas Graham House, The Science Park, Cambridge CB4 4WF, UK. ISBN 0-85186-229-2.

2. Mark Simmonds (2011): SYN545192 – Metabolism in Spring Wheat. Battelle UK Report Number NC/08/039 (yet to be issued)

3. Cardone M J, Palermo P J and Sybrand L B: Potential error in single point ratio calculations based on linear calibration curves with a significant intercept. Anal Chem., 52 pp 1187-1191, 1980

4. Kaijun Lin (2011): SYN545192 - Validation of Analytical Methods GRM042.03A for the Determination of SYN545192 and Its Metabolite SYN546039 in Crops and GRM042.04A for the Determination of SYN545192 and Its Metabolite SYN546039 in Soybean Commodities and SYN545720 in Soybean Seed Only. Syngenta Report number TK0002511.


TABLES


TABLE 1 Recovery Data - SYN545192 m/z 396→91

Matrix Fortification Level

(mg/kg)

Recovery (%)** n Mean (%)

RSD (%)

Range (%)

Wheat Forage 0.01* 77, 100, 89, 97, 91 5 91 9.8 77 - 100 0.5 94, 93, 95, 105, 97 5 97 5.0 93 - 105 Overall 10 94 7.9 77 - 105 Wheat Hay 0.01* 87, 79, 80, 82, 77 5 81 4.7 77 - 97 0.5 74, 82, 86, 77, 79 5 80 5.8 74 - 86 Overall 10 81 7.9 74 - 97 Wheat Grain 0.01* 98, 90, 95, 91, 91 5 93 3.7 90 - 98 0.1 97, 100, 92, 92, 94 5 95 3.7 92 - 100 Overall 10 94 3.6 90 - 100 Wheat Flour 0.01* 95, 90, 90, 99, 94 5 94 4.0 90 - 99 0.1 88, 96, 90, 98, 90 5 92 4.7 88 - 98 Overall 10 93 4.2 88 - 99 Spinach Leaves 0.01* 99, 94, 99, 88, 98 5 96 4.9 88 - 99 0.1 95, 94, 94, 98, 99 5 96 2.4 94 - 99 Overall 10 96 3.8 88 - 99 Carrot Root 0.01* 92, 94, 88, 91, 90 5 91 2.5 88 - 94 0.1 99, 99, 92, 94, 91 5 95 4.0 91 - 99 Overall 10 93 3.9 88 - 99 Apple Fruit 0.01* 91, 96, 90, 90, 87 5 91 3.6 87 - 96 0.1 94, 83, 90, 90, 89 5 89 4.4 83 - 94 Overall 10 90 3.9 83 - 96 Grape 0.01* 79, 85, 88, 81, 78 5 82 5.1 78 - 88 0.1 90, 88, 90, 91, 92 5 90 1.6 88 - 92 Overall 10 86 6.0 78 - 92 Orange Juice 0.01* 86, 86, 84, 88, 84 5 86 2.0 84 - 88 0.1 86, 90, 86, 85, 93 5 88 3.9 85 - 93 Overall 10 87 3.2 84 - 93 Sugarcane 0.01* 87, 96, 88, 86, 81 5 88 6.2 81 - 96 0.1 95, 82, 93, 83, 82 5 87 7.4 82 - 95 Overall 10 87 6.4 81 - 96


TABLE 1 Recovery Data - SYN545192 m/z 396→91 (continued) Matrix Fortification

Level (mg/kg)


RSD (%)

Range (%)

Peanut Meat 0.01* 93, 83, 86, 84, 90 5 87 4.8 83 - 93 0.1 85, 92, 87, 84, 90 5 88 3.8 84 - 92 Overall 10 87 4.1 83 - 93 Coffee Bean 0.01* 95, 91, 87, 86, 86 5 89 4.4 86 - 95 0.1 74, 91, 80, 93, 91 5 86 9.7 74 - 93 Overall 10 87 7.3 74 - 95

Note: (1) LOQ is defined by the lowest fortification level, i.e. 0.01 ppm (mg/kg) in this validation study; (2) Residues in control samples and reagent blanks were less than 30% of the LOQ; (3) Mean recoveries, standard deviations, and relative standard deviations were calculated using Microsoft Office Excel 2003. Results were rounded for reporting purposes but not during calculations




(mg/kg)


RSD (%)

Range (%)

Wheat Forage 0.01* 84, 104, 95, 96, 102 5 96 8.1 84 - 104 0.5 101, 94, 97, 88, 101 5 96 5.7 88 - 101 Overall 10 96 6.6 84 - 104Wheat Hay 0.01* 90, 96, 102, 109, 102 5 100 7.2 90 - 109 0.5 102, 93, 88, 100, 73 5 91 12.7 73 - 102 Overall 10 96 10.6 73 - 109Wheat Grain 0.01* 94, 83, 105, 81, 86 5 90 11.0 81 - 105 0.1 92, 89, 75, 82, 86 5 85 7.8 75 - 92 Overall 10 87 9.5 75 - 105Wheat Flour 0.01* 86, 98, 86, 89, 92 5 90 5.6 86 - 98 0.1 87, 91, 90, 95, 94 5 91 3.5 87 - 95 Overall 10 93 4.2 86 - 98 Spinach Leaves 0.01* 85, 73, 81, 88, 104 5 86 13.3 73 - 104 0.1 91, 82, 81, 78, 88 5 84 6.4 78 - 91 Overall 10 85 10.0 73 - 104Carrot Root 0.01* 80, 95, 94, 95, 97 5 92 7.5 80 - 97 0.1 96, 92, 98, 94, 99 5 96 3.0 92 -99 Overall 10 93 3.9 80 - 99 Apple Fruit 0.01* 87, 80, 101, 95, 71 5 87 13.7 71 - 101 0.1 90, 95, 81, 75, 81 5 84 9.5 75 - 95 Overall 10 86 11.2 71 - 101Grape 0.01* 89, 88, 96, 92, 89 5 91 3.6 88 - 96 0.1 88, 82, 82, 72, 94 5 84 9.8 72 - 94 Overall 10 87 8.0 72 - 96 Orange Juice 0.01* 81, 80, 93, 81, 74 5 82 8.4 74 - 93 0.1 98, 94, 87, 81, 77 5 87 10.0 77 - 98 Overall 10 85 9.4 74 - 98 Sugarcane 0.01* 88, 89, 92, 74, 83 5 85 8.3 74 - 92 0.1 91, 91, 88, 76, 89 5 87 7.2 76 - 91 Overall 10 86 7.4 74 - 92



Level (mg/kg)


RSD (%)

Range (%)






(mg/kg)


RSD (%)

Range (%)

Wheat Forage 0.01* 81, 98, 91, 98, 97 5 93 7.9 81 - 98 0.5 102, 100, 97, 107, 99 5 101 3.8 97 - 107 Overall 10 97 7.1 81 - 107 Wheat Hay 0.01* 83, 76, 78, 81, 81 5 80 3.5 76 - 83 0.5 87, 74, 85, 80, 87 5 83 6.8 74 - 87 Overall 10 81 5.4 74 - 87 Wheat Grain 0.01* 91, 89, 93, 94, 95 5 92 2.6 89 - 95 0.1 99, 102, 96, 94, 100 5 98 3.3 94 - 102 Overall 10 95 4.3 89 - 102 Wheat Flour 0.01* 92, 88, 91, 96, 90 5 91 3.3 88 - 96 0.1 86, 89, 92, 95, 91 5 91 3.7 86 - 95 Overall 10 91 3.3 86 - 96 Spinach Leaves 0.01* 93, 95, 87, 95, 101 5 94 5.3 87 - 101 0.1 88, 93, 98, 98, 97 5 95 4.6 88 - 98 Overall 10 95 4.7 87 - 101 Carrot Root 0.01* 87, 103, 90, 91, 91 5 92 6.7 87 - 103 0.1 105, 102, 95, 101, 97 5 100 4.0 95 - 105 Overall 10 96 6.6 87 -105 Apple Fruit 0.01* 85, 87, 86, 86, 91 5 87 2.7 85 - 91 0.1 81, 79, 91, 84, 87 5 84 5.7 79 - 91 Overall 10 86 4.4 79 - 91 Grape 0.01* 84, 88, 91, 84, 91 5 88 4.0 84 - 91 0.1 93, 96, 94, 91, 97 5 94 2.5 91 - 97 Overall 10 91 4.9 84 - 97 Orange Juice 0.01* 84, 90, 89, 92, 81 5 87 5.2 84 - 92 0.1 88, 93, 84, 83, 92 5 88 5.2 83 - 93 Overall 10 88 4.9 83 - 93 Sugarcane 0.01* 92, 92, 84, 88, 82 5 88 5.2 82 - 92 0.1 86, 83, 87, 84, 76 5 83 5.2 76 - 87 Overall 10 85 5.6 76 - 92



Level (mg/kg)


RSD (%)

Range (%)






(mg/kg)


RSD (%)

Range (%)

Wheat Forage 0.01* 83, 105, 102, 105, 104 5 100 9.5 83 - 105 0.5 108, 106, 97, 76, 109 5 99 13.9 76 - 109 Overall 10 100 11.2 76 - 109 Wheat Hay 0.01* 99, 110, 95, 86, 101 5 98 8.9 86 - 110 0.5 110, 102, 105, 94, 96 5 101 6.5 94 - 110 Overall 10 100 7.5 86 - 110 Wheat Grain 0.01* 95, 97, 97, 90, 96 5 95 3.1 90 - 97 0.1 93, 88, 76, 89, 79 5 85 8.4 76 - 93 Overall 10 90 8.2 76 - 97 Wheat Flour 0.01* 100, 98, 92, 94, 95 5 96 3.3 92 - 100 0.1 98, 90, 84, 93, 94 5 92 5.7 84 - 98 Overall 10 94 4.9 84 - 100 Spinach Leaves 0.01* 87, 82, 89, 96, 90 5 89 5.7 82 - 96 0.1 86, 89, 80, 89, 91 5 87 4.9 80 - 91 Overall 10 88 5.2 80 - 96 Carrot Root 0.01* 94, 95, 93, 93, 96 5 94 1.4 93 - 96 0.1 85, 84, 88, 84, 78 5 84 4.3 78 - 88 Overall 10 89 6.8 78 - 96 Apple Fruit 0.01* 92, 81, 95, 89, 85 5 88 6.3 81 - 95 0.1 78, 84, 77, 92, 86 5 83 7.4 77 - 92 Overall 10 86 7.1 77 - 95 Grape 0.01* 92, 95, 95, 92, 86 5 92 4.0 86 - 95 0.1 83, 81, 83, 84, 85 5 83 1.8 81 - 85 Overall 10 88 6.1 81 - 95 Orange Juice 0.01* 86, 76, 89, 89, 87 5 85 6.3 76 - 89 0.1 86, 75, 74, 77, 77 5 78 6.1 74 - 86 Overall 10 82 7.7 74 - 89 Sugarcane 0.01* 85, 78, 84, 77, 87 5 82 5.4 77 - 87 0.1 87, 89, 91, 87, 81 5 87 4.3 81 - 91 Overall 10 85 5.5 77 - 91



Level (mg/kg)


RSD (%)

Range (%)




Determination of LC-MS/MS Matrix Effects

The effect of crop matrices on the LC-MS/MS signal was assessed by preparing standards in the presence of crop matrix and comparing the peak areas of SYN545192 and SYN546039 against non-matrix standards at an equivalent concentration. See Section 2.3.3 for details of preparation of matrix-matched calibration standards.

Matrix effects during method validation were calculated using the following equation:

%100solventin standard of areapeak

solventin standard of areapeak -matrix in standard of areapeak EffectsMatrix x=

TABLE 5 LC-MS/MS Matrix Effects

MATRIX EFFECT SYN545192 SYN546192 SYN546039 SYN546039 m/z 396→91 m/z 396→368 m/z 412→91 m/z 412→340

Wheat Forage -1% -4% -4% -3% Wheat Hay +1% +3% -16% -6% Wheat Grain +2 % 0% -11% -10% Wheat Flour +6% +4% -6% -7% Spinach Leaves -1% 0% -14% -12% Carrot Root +4% +3% -13% -8% Apple Fruit 0% 0% -6% -4% Grape +13% +5% -6% +1% Sugarcane -4% +1% -8% -3% Peanut Nutmeat -1% +2% -15% -11% Orange Juice -5% -8% -8% -7% Coffee Bean -10% -9% -35% -38%

Figures rounded to nearest integer.

The magnitude of the matrix effects were generally considered not to be significant (<20%) for SYN545192 or SYN546039 in the method validation, and non-matrix standards were used for calibration with the following exception:

Significant matrix suppression (>20%) was observed for SYN546039 in the coffee beans tested and matrix-matched standards were used for calibration in method validation.

Matrix matched standards may be used to compensate for any significant effects, at the discretion of the study director. Alternatively, where instrument sensitivity permits, samples may be diluted further with 50/50 (v/v) acetonitrile/ultra pure water.


TABLE 6 Stability of SYN545192 in 50/50 Acetonitrile/Ultra Pure Water Stored at 4oC for 9 – 102 Days

Crop Commodities

Fortification Level ppm

(mg/kg)

Storage* (days)

Recovery (%)**

N

Average (%)

Relative Standard Deviation

(%) Wheat Forage 0.01 0 77,100, 89, 97, 91 5 91 10 Wheat Forage 0.01 16 75, 85, 87, 75, 74 5 79 8

Wheat Hay 0.01 0 87, 79, 80, 82, 77 5 81 5 Wheat Hay 0.01 19 66, 70, 67, 63, 62 5 66 5

Wheat Grain 0.01 0 98, 90, 95, 91, 91 5 93 4 Wheat Grain 0.01 65 92, 84, 87, 92, 94 5 90 5

Wheat Flour 0.01 0 95, 90, 90, 99, 94 5 94 4 Wheat Flour 0.01 96 87, 81, 101, 98, 83 5 90 10

Apple Fruit 0.01 0 91, 96, 90, 90, 87 5 91 4 Apple Fruit 0.01 60 75, 87, 85, 84, 90 5 84 7

Orange Juice 0.01 0 86, 86, 84, 88, 84 5 86 2 Orange Juice 0.01 50 87, 86, 84, 87, 89 5 87 2

Grape 0.01 0 79, 85, 88, 81, 78 82 5 Grape 0.01 48 81, 80, 87, 86, 91 85 5

Peanut Nutmeat 0.01 0 93, 83, 86, 84, 90 5 87 5 Peanut Nutmeat 0.01 41 80, 79, 82, 90, 88 5 84 6

Coffee Bean 0.01 0 95, 91, 87, 86, 86 5 89 4 Coffee Bean 0.01 14 81, 94, 86, 91, 91 5 89 6

Carrot Root 0.01 0 92, 94, 88, 91, 90 5 91 2 Carrot Root 0.01 73 89, 83, 85, 91, 79 5 85 6 Sugarcane 0.01 0 87,96, 88, 86, 81 5 88 6 Sugarcane 0.01 60 78, 80, 78, 76, 74 5 77 3

Spinach Leaves 0.01 0 99, 94, 99, 88, 98 5 96 5 Spinach Leaves 0.01 102 86, 84, 91, 83, 90 5 87 4

Note: Mean recoveries, standard deviations, and relative standard deviations were calculated using Microsoft Office Excel 2003. Results were rounded for reporting purposes but not during calculations. The zero date is defined as the date when the recovery samples were injected for analysis. The storage date is defined as the date when the same samples were re-injected after storing refrigerated for at least 7 days, analyzing against freshly prepared calibration standards, using the primary transition only.


TABLE 7 Stability of SYN546039 in 50/50 Acetonitrile/Ultra Pure Water Stored at 4o C for 9 – 102 Days

Crop Commodities

Fortification Level

ppm (mg/kg)

Storage (days)* Recovery (%)** N Average

(%)

Relative Standard Deviation

(%) Wheat Forage 0.01 0 84,104, 95, 96, 102 5 96 8 Wheat Forage 0.01 19 80, 87, 80, 86, 91 5 85 6

Wheat Hay 0.01 0 90, 96, 102, 109, 102 5 100 7 Wheat Hay 0.01 16 78, 97, 88, 95, 97 5 91 9

Wheat Grain 0.01 0 94, 83, 105, 81,86 5 90 11 Wheat Grain 0.01 65 80, 91, 102, 96, 96 5 93 9

Wheat Flour 0.01 0 86, 98, 86, 89, 92 5 90 6 Wheat Flour 0.01 96 89, 97, 106, 97, 83 5 94 9

Apple Fruit 0.01 0 87, 80, 101, 95, 71 5 87 14 Apple Fruit 0.01 60 92, 96, 81, 80, 86 5 87 8

Orange Juice 0.01 0 81, 80, 93, 81, 74 5 82 8 Orange Juice 0.01 50 77, 75, 86, 86, 75 5 80 7

Grape 0.01 0 89, 88, 96, 92, 89 5 91 4 Grape 0.01 48 74, 87, 93, 92, 94 5 88 9

Peanut Nutmeat 0.01 0 80, 90, 80, 80, 79 5 82 6 Peanut Nutmeat 0.01 41 85, 76, 90, 87, 80 5 84 7

Coffee Bean 0.01 0 82, 82, 97, 87, 88 5 87 7 Coffee Bean 0.01 14 87, 92, 92, 87, 84 5 90 6

Carrot Root 0.01 0 80, 95, 94, 95, 97 5 92 7 Carrot Root 0.01 73 93, 93, 101, 93, 88 5 94 5 Sugarcane 0.01 0 88, 89, 92, 74, 83 5 85 8 Sugarcane 0.01 60 81, 85, 82, 75, 1% 5 81 5

Spinach Leaves 0.01 0 85, 73, 81, 88, 104 5 86 13 Spinach Leaves 0.01 102 82, 88, 95, 88, 79 5 86 7

Note: Mean recoveries, standard deviations, and relative standard deviations were calculated using Microsoft Office Excel 2003. Results were rounded for reporting purposes but not during calculations. The zero date is defined as the date when the recovery samples were injected for analysis. The storage date is defined as the date when the same samples were re-injected after storing refrigerated for at least 7 days, analyzing against freshly prepared calibration standards, using the primary transition only.


Final crop extracts in 50/50 (v/v) acetonitrile/ultra pure water retained in vials and stored at a temperature of approximately 4oC were suitable for SYN545192 and SYN546039 residue analysis, for storage periods of at least 7 days.


FIGURES


CHEMICAL STRUCTURES

Figure 1: SYN545192

Alternative compound code number

: CSCD064398

CAS Number : 1072957-71-1 IUPAC Name : N-[(1RS,4SR)-9-(Dichloromethylidene)-1,2,3,4-

tetrahydro-1,4-methanonaphthalen-5-yl]-3-(difluoromethyl)-1-methyl-1H-pyrazole-4-carboxamide

Molecular Formula : C18H15Cl2F2N3O Molecular Weight : 398.2

NN

F

F

O

NH

ClCl


Figure 2: SYN546039

Alternative compound code number

: CSCD695908

CAS Number : Not in registry IUPAC Name : 3-Difluoromethyl-1-methyl-1H-pyrazole-4-carboxylic

acid ((1S,2R,4R)-9-dichloromethylene-2-hydroxy-1,2,3,4-tetrahydro-1,4-methano-naphthalen-5-yl)-amide

Molecular Formula : C18H15Cl2F2N3O2 Molecular Weight : 414

NN

F

F

O

NH

ClCl

OH


REPRESENTATIVE CHROMATOGRAMS

Figure 3: 0.05 ng/mL SYN545192 Standard in 50/50 (v/v) Acetonitrile/Ultra Pure Water. SYN545192 Primary Transition m/z = 396 91

Figure 4: 0.05 ng/mL SYN545192 Standard in 50/50 (v/v) Acetonitrile/Ultra Pure Water. SYN545192 Confirmatory Transition m/z = 396 91


Figure 5: Reagent Blank. SYN545192 Primary Transition m/z = 396 91

Figure 6: Reagent Blank. SYN545192 Confirmatory Transition m/z = 396 368


Figure 7: Untreated Wheat Forage. SYN545192 Primary Transition m/z = 396 91 Residue < LOQ

Figure 8: Untreated Wheat Forage.

SYN545192 Confirmatory Transition m/z = 396 368 Residue < LOQ


Figure 9: Untreated Wheat Forage Fortified with 0.01 mg/kg SYN545192. SYN545192 Primary Transition m/z = 396 91 Recovery = 77%

Figure 10: Untreated Wheat Forage Fortified with 0.01 mg/kg SYN545192. SYN545192 Confirmatory Transition m/z = 396 368 Recovery = 91%





Figure 13: Untreated Wheat Hay. SYN545192 Primary Transition m/z = 396 91 Residue < LOQ

Figure 14: Untreated Wheat Hay. SYN545192 Confirmatory Transition m/z = 396 368 Residue < LOQ


Figure 15: Untreated Wheat Hay Fortified with 0.01 mg/kg SYN545192. SYN545192 Primary Transition m/z = 396 91 Recovery = 88%

Figure 16: Untreated Wheat Hay Fortified with 0.01 mg/kg SYN545192. SYN545192 Confirmatory Transition m/z = 396 368 Recovery = 83%





Figure 19: Untreated Wheat Grain. SYN545192 Primary Transition m/z = 396 91 Residue < LOQ

Figure 20: Untreated Wheat Grain.



Figure 21: Untreated Wheat Grain Fortified with 0.01 mg/kg SYN545192. SYN545192 Primary Transition m/z = 396 91 Recovery = 98%

Figure 22: Untreated Wheat Grain Fortified with 0.01 mg/kg SYN545192. SYN545192 Confirmatory Transition m/z = 396 368 Recovery = 91%





Figure 25: Untreated Orange Juice. SYN545192 Primary Transition m/z = 396 91 Residue < LOQ

Figure 26: Untreated Orange Juice.



Figure 27: Untreated Orange Juice Fortified with 0.01 mg/kg SYN545192. SYN545192 Primary Transition m/z = 396 91 Recovery = 86%

Figure 28: Untreated Orange Juice Fortified with 0.01 mg/kg SYN545192. SYN545192 Confirmatory Transition m/z = 396 368 Recovery = 84%





Figure 31: Untreated Coffee Bean. SYN545192 Primary Transition m/z = 396 91 Residue < LOQ

Figure 32: Untreated Coffee Bean. SYN545192 Confirmatory Transition m/z = 396 368 Residue < LOQ


Figure 33: Untreated Coffee Bean Fortified with 0.01 mg/kg SYN545192. SYN545192 Primary Transition m/z = 396 91 Recovery = 95%

Figure 34: Untreated Coffee Bean Fortified with 0.01 mg/kg SYN545192. SYN545192 Confirmatory Transition m/z = 396 368 Recovery = 83%





Figure 37: 0.05 ng/mL SYN546039 Standard in 50/50 (v/v) Acetonitrile/Ultra Pure Water. SYN545192 Primary Transition m/z = 412 91

Figure 38: 0.05 ng/mL SYN546039 Standard in 50/50 (v/v) Acetonitrile/Ultra Pure Water. SYN545192 Confirmatory Transition m/z = 412 340


Figure 39: Reagent Blank. SYN546039 Primary Transition m/z = 412 91

Figure 40: Reagent Blank. SYN546039 Confirmatory Transition m/z = 412 340


Figure 41: Untreated Wheat Forage. SYN546039 Primary Transition m/z = 412 91 Residue < LOQ

Figure 42: Untreated Wheat Forage.









Figure 47: Untreated Wheat Hay. SYN546039 Primary Transition m/z = 412 91 Residue < LOQ

Figure 48: Untreated Wheat Hay. SYN546039 Confirmatory Transition m/z = 412 340 Residue < LOQ








Figure 53: Untreated Wheat Grain. SYN546039 Primary Transition m/z = 412 91 Residue < LOQ

Figure 54: Untreated Wheat Grain.









Figure 59: Untreated Orange Juice. SYN546039 Primary Transition m/z = 412 91 Residue < LOQ

Figure 60: Untreated Orange Juice.









Figure 65: Untreated Coffee Bean. SYN546039 Primary Transition m/z = 412 91 Residue < LOQ

Figure 66: Untreated Coffee Bean. SYN546039 Confirmatory Transition m/z = 412 340 Residue < LOQ








DETECTOR LINEARITY GRAPHS

Figure 71: LC-MS/MS Calibration Graph for SYN545192 Standards in 50/50 (v/v) Acetonitrile/Ultra Pure Water. Primary Transition m/z = 396 91 (no intercept set)

Amount SYN545192

Injected * (pg)

Standard Concentration

(ng/mL)

Peak Area (cps)

0.8 0.02 1130

2 0.05 3090

3.2 0.08 5310

4 0.1 6460

8 0.2 13300

20 0.5 33400

*40 µL injection volume


Figure 72: LC-MS/MS Calibration Graph for SYN545192 Standards in 50/50 (v/v) Acetonitrile/Ultra Pure Water. Confirmatory Transition m/z = 396 368 (no intercept set)

Amount SYN545192 Injected *

(pg)


(ng/mL)

Peak Area (cps)

0.8 0.02 1850

2 0.05 4110

3.2 0.08 7240

4 0.1 8810

8 0.2 18000

20 0.5 43900



Figure 73: LC-MS/MS Calibration Graph for SYN546039 Standards in 50/50 (v/v) Acetonitrile/Ultra Pure Water. Primary Transition m/z = 412 91 (no intercept set)

Amount SYN546039

Injected * (pg)


(ng/mL)

Peak Area (cps)

0.8 0.02 386

2 0.05 721

3.2 0.08 1200

4 0.1 1590

8 0.2 2750

20 0.5 7390



Figure 74: LC-MS/MS Calibration Graph for SYN546039 Standards in 50/50 (v/v) Acetonitrile/Ultra Pure Water. Confirmatory Transition m/z = 412 340 (no intercept set)

Amount SYN546039 Injected *

(pg)


(ng/mL)

Peak Area (cps)

0.8 0.02 798

2 0.05 2250

3.2 0.08 3740

4 0.1 4790

8 0.2 9590

20 0.5 24600



MS/MS PRODUCT ION SPECTRA

Figure 75: Initial Product Ion Scan for SYN545192

(Negative Ionisation Mode).

This scan was not produced as part of the validation study


Figure 76: Initial Product Ion Scan for SYN546039 (Negative Ionisation Mode).

This scan was not produced as part of the validation study


APPENDICES


APPENDIX 1 APPARATUS

Recommended Suppliers

Equipment Description Supplier General glassware General glassware www.thermofisher.com/global/en/home.asp Plastic centrifuge bottles 250 mL HDPE www.thermofisher.com/global/en/home.asp Macerator/homogeniser Ultra Turrax T 50 basic

4,000rpm to 10,000rpm speed range

www.thermofisher.com/global/en/home.asp

Screw capped glass vials 15 mL size www.thermofisher.com/global/en/home.asp Sample processing station/Vacuum manifold

Isolute® Vacmaster-20® sample processing station

www.Biotage.com

Solid Phase Extraction cartridges

Oasis HLB, 60 mg, 3 mL size www.waters.com

Plastic centrifuge tubes Polypropylene,15 mL size www.thermofisher.com/global/en/home.asp Autosampler vials Crimp cap, 2 mL size www.Agilent.com LC-MS/MS system API 4000 equipped with a

TurboIonSpray source www.AppliedBiosytems.com

HPLC system Waters H Class UPLC system equipped with quaternary pump, vacuum degasser and column oven compartment

www.waters.com

HPLC column Waters Atlantis T3 3 x 100 mm 5 µm. Part number 186003739

www.phenomenex.com

Nitrogen generator Peak Scientific NM20ZA gas station

www.peakscientific.com


APPENDIX 2 REAGENTS

Recommended suppliers

Reagent Description Supplier Ultra pure water HPLC grade www.thermofisher.com/global/en/home.asp Acetonitrile HPLC grade www.thermofisher.com/global/en/home.asp Methanol HPLC grade www.thermofisher.com/global/en/home.asp Hydrochloric acid Analytical grade www.sigmaaldrich.com Acetic acid Analytical grade www.sigmaaldrich.com SYN545192 and SYN546039 analytical standards

GLP certified GLP Testing Facility, Syngenta, CH-4333, Munchweilen, Switzerland or Syngenta Crop Protection, Inc., P.O. Box 18300, Greensboro, NC 27419-8300.

Preparation of Reagents

a) 50/50 (v/v) acetonitrile/ultra pure water: Measure 500 mL acetonitrile into a 1 L volumetric flask. Add 500 mL ultra pure water. Stopper flask securely and shake to mix thoroughly.

b) 80/20 (v/v) acetonitrile/ultra pure water: Measure 800 mL acetonitrile into a 1 L volumetric flask. Add 200 mL ultra pure water. Stopper flask securely and shake to mix thoroughly.

c) 1 M hydrochloric acid: Add 83.3 mL concentrated hydrochloric acid to ultra pure water in a 1 L volumetric flask. Adjust the volume to the 1 L mark with ultra pure water. Stopper the flask securely and shake to mix thoroughly.

d) 80/20 (v/v) ultra pure water/methanol: Add 200 mL methanol to 800 mL ultra pure water in a 1 L volumetric flask. Stopper the flask securely and shake to mix thoroughly.

e) 50/50 (v/v) ultra pure water/methanol: Measure 500 mL acetonitrile into a 1 L volumetric flask. Add 500 mL ultra pure water. Stopper flask securely and shake to mix thoroughly.

f) 65/35 (v/v) ultra pure water/acetonitrile: Measure 65 mL ultra pure water into a 100 mL volumetric flask. Add 35 mL acetonitrile. Stopper flask securely and shake to mix thoroughly.

g) 0.2% (v/v) acetic acid in ultra pure water: Add concentrated acetic acid (2 mL) to ultra pure water in a 1L volumetric flask. Adjust volume to the 1 L mark with ultra pure water. Stopper flask securely and shake to mix thoroughly.


APPENDIX 3 LC-MS/MS TUNING PROCEDURE

Calibration of Instrument The instrument must be mass calibrated on a regular basis using polypropylene glycol (PPG) solutions according to the manufacturer’s instructions. Calibrate both mass resolving quadrupoles (Q1 and Q3).

Tuning Instrument for SYN545192 and SYN546039 Note: SYN545192 and SYN546039 for this method have 2 chlorine atoms. Chlorine has two isotopic forms 35Cl and 37Cl and therefore the mass spectrum shows ions at M (35Cl x 2), M+2 (35Cl + 37Cl) and M+4 (37Cl x2). For all analytes on the instruments tested, the most abundant ions were for M (i.e.35Cl x 2).

Infuse a standard solutions of SYN545192 and SYN546039 (0.01 to 1.0 µg/mL) in mobile phase (see section 4) directly into the mass spectrometer interface at a rate at of approximately 10-20 µL/min. Roughly adjust interface parameters (sprayer position, spray, heater/auxiliary gas flows, as well as voltages of spray, orifice, and focusing ring) for a sufficiently high parent ion signal at m/z 396 for SYN545192 and m/z 412 for SYN546039 negative ionisation mode. Using the Analyst software quantitative optimisation routine, tune the instrument for SYN545192 and SYN546039, ensuring that the correct ion is selected. If desired, manual tuning of the ion optics and collision energy can be carried out to ensure maximum sensitivity. Finally, connect the LC-pump via the autosampler directly to the MS/MS instrument. Perform repetitive flow injection of SYN545192 and SYN546039 standards using mobile phase at the flow rate to be used. Tune the interface parameters (sprayer position, spray and heater gas flows, spray, orifice, and focusing ring voltages) and the collision gas flow for maximum sensitivity. For SYN545192, in negative ionisation mode, the deprotonated molecular ion generated in the ion source (m/z 396) is selected and subjected to further fragmentation by collisional activation. The two most sensitive daughter ions (m/z 368 and m/z 91) are then selected and used for quantitative analysis. The fragment m/z = 368 corresponds to loss of H2C=CH2 from the deprotonated molecular ion and m/z = 91 corresponds to the deprotonated 1,3 dimethyl pyrazole fragment. For SYN546039, in negative ionisation mode, the deprotonated molecular ion generated in the ion source (m/z 412) is selected and subjected to further fragmentation by collisional activation. The two most sensitive daughter ions (m/z 340 and m/z 91) are then selected and used for quantitative analysis. The fragments m/z = 376 corresponds to loss of 1 x HCl from the deprotonated molecular ion and m/z = 91 corresponds to the deprotonated 1,3 dimethyl pyrazole fragment.


APPENDIX 4 METHOD FLOW CHART FOR HIGH WATER CONTENT CROPS

Homogenise 10 g crop sample with 80/20 (v/v) MeCN/UPW (100 mL – natural water

content of crop sample) for 3 – 5 minutes ↓

Centrifuge* ↓

Evaporate 2 mL (0.2 g) aliquots in plastic graduated centrifuge tube to < 0.2 mL to remove MeCN ↓

Add 2 mL 1 M HCl and partition with 3 x 2 mL iso-hexane to remove SYN545192. Retain for later use**.

↓ Adjust aqueous fraction to 4 mL with UPW and transfer to 15 mL screw-top vial. Heat with

shaking at 100oC for 6 hours in a water bath ↓

Allow the aqueous, acidic hydrolysate to cool to room temperature ↓

Evaporate iso-hexane and either dissolve in 50/50 (v/v) MeCN/UPW for analysis of SYN545192 or evaporate and combine with aqueous post hydrolysis sample for SPE

clean-up ↓

Take sample through an Oasis HLB SPE procedure (fortify recoveries with SYN546039 as appropriate).

↓ Wash with 80/20 (v/v) UPW/MeOH (3 mL) followed by 50/50 (v/v) MeOH/UPW (2 x 3 mL)

↓ Wash with 35/65 (v/v) MeCN/UPW (1 mL)

↓ Elute with 50/50 (v/v) MeCN/UPW (2 x 3 mL)

↓ Adjust final volume to 20 mL (or other suitable volume, depending on instrument sensitivity)

with 50/50 (v/v) MeCN/UPW and shake to mix thoroughly ↓

Analyse by LC-MS/MS for SYN545192** and SYN546039

*Samples may be analysed by direct injection for SYN545192 depending on instrument sensitivity **Unless previously determined by direct injection of extract, or post iso-hexane partition and evaporation.


APPENDIX 5 METHOD FLOW CHART FOR DRY CROPS

Pre-soak a 5 g sample of straw or hay with UPW (25 mL) for 30 minutes

↓ Add 80/20 (v/v) MeCN/UPW (100 mL) and homogenise for 3 – 5 minutes

↓ Centrifuge*

↓ Evaporate 5 mL (0.2 g) aliquots in plastic graduated centrifuge tube to < 1 mL to remove

MeCN ↓

Add 2 mL 1 M HCl and partition with 3 x 4 mL iso-hexane to remove SYN545192. Retain for later use**.

↓ Adjust aqueous fraction to 4 mL with UPW and transfer to 15 mL screw-top vial. Heat with

shaking at 100oC for 6 hours in a water bath ↓

Evaporate iso-hexane and either dissolve in 50/50 (v/v) MeCN/UPW for analysis of SYN545192 or evaporate and combine with aqueous post hydrolysis sample for SPE clean-

up ↓

Take sample through an Oasis HLB SPE procedure (fortify recoveries with SYN546039 as appropriate).

↓ Wash with 80/20 (v/v) UPW/MeOH (3 mL) followed by 50/50 (v/v) MeOH/UPW (2 x 3 mL)

↓ Wash with 35/65 (v/v) MeCN/UPW (1 mL)

↓ Elute with 50/50 (v/v) MeCN/UPW (5 mL)

↓ Adjust final volume to 20 mL (or other suitable volume, depending on instrument sensitivity)

with 50/50 (v/v) MeCN/UPW and shake to mix thoroughly ↓

Analyse by LC-MS/MS for SYN545192** and SYN546039

*Samples may be analysed by direct injection for SYN545192 depending on instrument sensitivity **Unless previously determined by direct injection of extract, or post iso-hexane partition and evaporation.