DOCUMENT NUMBER JSM0269. TEST GUIDELINE EPA OPPTS Guideline AUTHOR Stephen Brewin. STUDY COMPLETION DATE October 4, 2012

Transcription

1 STUDY TITLE IKI-3106 AND NK-1375: VALIDATION OF METHODOLOGY FOR THE DETERMINATION OF RESIDUES OF IKI-3106 AND NK-1375 IN GRAPE, WINE, PEACHES, OILSEED RAPE SEEDS AND DRY BEANS DOCUMENT NUMBER TEST GUIDELINE EPA OPPTS Guideline AUTHOR Stephen Brewin STUDY COMPLETION DATE October 4, 2012 PERFORMING LABORATORY STUDY SPONSOR Huntingdon Life Sciences Ltd Ishihara Sangyo Kaisha Ltd Eye Research Centre 3-15 Edobori 1-Chome Eye, Suffolk Nishi-ku IP23 7PX Osaka ENGLAND JAPAN SUBMITTED BY ISK BIOSCIENCES CORPORATION 7470 Auburn Road, Suite A Concord, Ohio Page 1 of 107 pages with 3 front pages

2 STATEMENT OF NO CLAIM OF CONFIDENTIALITY1 No claim of confidentiality, on any basis whatsoever, is made for any information contained in this document. I acknowledge that information not designated as within the scope of FIFRA sec. 10(d)(1)(A), (B), or (C) and which pertains to a registered or previously registered pesticide is not entitled to confidential treatment and may be released to the public, subject to the provisions regarding disclosure to multinational entities under FIFRA 10(g). COMPANY: ISHIHARA SANGYO KAISHA, LTD. COMPANY AGENT: (Submitter) Gregory A. Leyes Typed Name Agent for ISHIHARA SANGYO KAISHA, LTD. Vice President, Regulatory Affairs ISK Biosciences Corporation Title ^tjr~uaju^ 13-1 )ate / V 1 We submitted this material to the United States Environmental Protection Agency specifically under provisions contained in FIFRA as amended, and thereby consent to use and disclosure o f this material by EPA according to FIFRA. Notwithstanding the wording of our marking (either as "Property of ISK BIOSCIENCES CORPORATION" or "Property of ISHIHARA SANGYO KAISHA, LTD."), this marking by itself conveys no supplemental claims o f confidentiality under FIFRA Sections 10(a) or 10(b). In submitting this material to the EPA according to the method and format requirements contained in PR Notice 86-5 and PR Notice , we do not waive any protection or right involving this material that would have been claimed by ISK BIOSCIENCES CORPORATION or by ISHIHARA SANGYO KAISHA, LTD. or agents o f these companies anywhere else in the world, if this material had not been submitted to the EPA. IB

3 COMPLIANCE STATEMENT ISK Biosciences Corporation This study was conducted and reported in compliance with Good Laboratory Practice Regulations set forth in Title 40, Part 160 of the Code of Federal Regulations of the United States of America. Leyei ^ice President, Regulatory Affairs ISK BIOSCIENCES CORPORATION Agent for STUDY SPONSOR ISHIHARA SANGYO KAISHA, LTD. Date Mark Gelin Manager, International Registrations Regulatory Affairs ISK BIOSCIENCES CORPORATION, APPLICANT Date iqi ioiy REFERENCE TO COMPLIANCE STATEMENT WITHIN THE REPORT A GOOD LABORATORY PRACTICES compliance statement with the signature of the STUDY DIRECTOR found on page 2 of the report. 1C

4 Working for a better future Final Report IKI-3106 AND NK-1375: VALIDATION OF METHODOLOGY FOR THE DETERMINATION OF RESIDUES OF IKI-3106 AND NK-1375 IN GRAPE, WINE, PEACHES, OILSEED RAPE SEEDS AND DRY BEANS Huntingdon Life Sciences Project Identity Version ID Final Study completion date 4 th October 2012 Author Stephen Brewin Sponsor Ishihara Sangyo Kaisha, Ltd. 3-15, Edobori 1-Chome Nishi-ku, Osaka, JAPAN Performing Laboratory Huntingdon Life Sciences Ltd Eye Research Centre Eye Suffolk IP23 7PX ENGLAND Page 1 of 107

5

6

7 Contributing Scientists IKI-3106 and NK-1375: Validation of Methodology for the Determination of Residues of IKI-3106 and NK-1375 in Grape, Wine, Peaches, Oilseed Rape Seeds and Dry Beans The following staff member has reviewed this report. D Airs, BSc (Hons) Senior Study Manager Environmental Analysis The following staff members were responsible for the conduct of the work and reporting of the results. S Brewin, BSc (Hons), MRSC, CChem Senior Study Manager Environmental Analysis C Miller Senior Study Analyst Environmental Analysis 4

8 Table of Contents Compliance with Good Laboratory Practice... 2 Quality Assurance Statement... 3 Contributing Scientists... 4 Table of Contents... 5 Summary Introduction Materials Analytical Standard 1 IKI Analytical Standard 2 NK Control matrices Methods Validation Final extract stability Matrix effects Analytical method Fortification/calibration solutions Calculation of results for validation samples Results Calibration (linearity) Method validation (accuracy/precision) Limit of quantitation (LOQ) Limit of detection (LOD) Specificity/confirmation of residues Final extract stability Matrix effects Conclusion List of Tables Table 1 Table 2 Table 3 Table 4 Typical standard calibration for IKI-3106 by LC-MS/MS (quantitation transition monitored)...20 Typical standard calibration for IKI-3106 by LC-MS/MS (confirmation transition monitored)...20 Typical standard calibration for NK-1375 by LC-MS/MS (quantitation transition monitored)...21 Typical standard calibration for NK-1375 by LC-MS/MS (confirmation transition monitored)

9 Table 5 Table 6 Validation data IKI-3106 in grapes (quantitation transition monitored)...22 Validation data IKI-3106 in grapes (confirmation transition monitored)...23 Table 7 Validation data NK-1375 in grapes (quantitation transition monitored)...24 Table 8 Validation data NK-1375 in grapes (confirmation transition monitored)...25 Table 9 Validation data IKI-3106 in wine (quantitation transition monitored)...26 Table 10 Validation data IKI-3106 in wine (confirmation transition monitored)...27 Table 11 Table 12 Validation data NK-1375 in wine (quantitation transition monitored)...28 Validation data NK-1375 in wine (confirmation transition monitored)...29 Table 13 Validation data IKI-3106 in peaches (quantitation transition monitored)...30 Table 14 Validation data IKI-3106 in peaches (confirmation transition monitored)...31 Table 15 Table 16 Table 17 Table 18 Table 19 Table 20 Validation data NK-1375 in peaches (quantitation transition monitored)...32 Validation data NK-1375 in peaches (confirmation transition monitored)...33 Validation data IKI-3106 in oilseed rape seeds (quantitation transition monitored)...34 Validation data IKI-3106 in oilseed rape seeds (confirmation transition monitored)...35 Validation data NK-1375 in oilseed rape seeds (quantitation transition monitored)...36 Validation data NK-1375 in oilseed rape seeds (confirmation transition monitored)...37 Table 21 Validation data IKI-3106 in dry beans (quantitation transition monitored)..38 Table 22 Validation data IKI-3106 in dry beans (confirmation transition monitored).39 Table 23 Table 24 Table 25 Validation data NK-1375 in dry beans (quantitation transition monitored)..40 Validation data NK-1375 in dry beans (confirmation transition monitored).41 Final extract stability data

10 List of Figures Figure 1 Figure 2 Figure 3 Figure 4 Typical standard calibration for IKI-3106 by LC-MS/MS (quantitation transition monitored)...43 Typical standard calibration for IKI-3106 by LC-MS/MS (confirmation transition monitored)...44 Typical standard calibration for NK-1375 by LC-MS/MS (quantitation transition monitored)...45 Typical standard calibration for NK-1375 by LC-MS/MS (confirmation transition monitored)...46 Figure 5 Mass spectrum daughter ion scan of m/z 602 (IKI-3106)...47 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Figure 14 Figure 15 Figure 16 Figure 17 Figure 18 Mass spectrum daughter ion scan of m/z 566 (NK-1375)...48 Typical chromatogram of a 10 ng/ml calibration standard (quantitation transition monitored)...49 Typical chromatogram of a 10 ng/ml calibration standard (confirmation transition monitored)...50 Typical chromatogram of a 1 ng/ml calibration standard (quantitation transition monitored)...51 Typical chromatogram of a 1 ng/ml calibration standard (confirmation transition monitored)...52 Typical chromatogram of a ng/ml calibration standard (quantitation transition monitored)...53 Typical chromatogram of a ng/ml calibration standard (confirmation transition monitored)...54 Typical chromatogram of an extract of control grapes (quantitation transition monitored)...55 Typical chromatogram of an extract of control grapes (confirmation transition monitored)...56 Typical chromatogram of an extract of control grapes fortified at 0.01 mg/kg (quantitation transition monitored)...57 Typical chromatogram of an extract of control grapes fortified at 0.01 mg/kg (confirmation transition monitored)...58 Typical chromatogram of an extract of control grapes fortified at 0.5 mg/kg (quantitation transition monitored)...59 Typical chromatogram of an extract of control grapes fortified at 0.5 mg/kg (confirmation transition monitored)

11 Figure 19 Figure 20 Figure 21 Figure 22 Figure 23 Figure 24 Figure 25 Figure 26 Figure 27 Figure 28 Figure 29 Figure 30 Figure 31 Figure 32 Figure 33 Figure 34 Figure 35 Figure 36 Typical chromatogram of an extract of control wine (quantitation transition monitored)...61 Typical chromatogram of an extract of control wine (confirmation transition monitored)...62 Typical chromatogram of an extract of control wine fortified at 0.01 mg/kg (quantitation transition monitored)...63 Typical chromatogram of an extract of control wine fortified at 0.01 mg/kg (confirmation transition monitored)...64 Typical chromatogram of an extract of control wine fortified at 0.5 mg/kg (quantitation transition monitored)...65 Typical chromatogram of an extract of control wine fortified at 0.5 mg/kg (confirmation transition monitored)...66 Typical chromatogram of an extract of control peaches (quantitation transition monitored)...67 Typical chromatogram of an extract of control peaches (confirmation transition monitored)...68 Typical chromatogram of an extract of control peaches fortified at 0.01 mg/kg (quantitation transition monitored)...69 Typical chromatogram of an extract of control peaches fortified at 0.01 mg/kg (confirmation transition monitored)...70 Typical chromatogram of an extract of control peaches fortified at 0.5 mg/kg (quantitation transition monitored)...71 Typical chromatogram of an extract of control peaches fortified at 0.5 mg/kg (confirmation transition monitored)...72 Typical chromatogram of an extract of control oilseed rape seeds (quantitation transition monitored)...73 Typical chromatogram of an extract of control oilseed rape seeds (confirmation transition monitored)...74 Typical chromatogram of an extract of control oilseed rape seeds fortified at 0.01 mg/kg (quantitation transition monitored)...75 Typical chromatogram of an extract of control oilseed rape seeds fortified at 0.01 mg/kg (confirmation transition monitored)...76 Typical chromatogram of an extract of control oilseed rape seeds fortified at 0.5 mg/kg (quantitation transition monitored)...77 Typical chromatogram of an extract of control oilseed rape seeds fortified at 0.5 mg/kg (confirmation transition monitored)

12 Figure 37 Figure 38 Figure 39 Figure 40 Figure 41 Figure 42 Typical chromatogram of an extract of control dry beans (quantitation transition monitored)...79 Typical chromatogram of an extract of control dry beans (confirmation transition monitored)...80 Typical chromatogram of an extract of control dry beans fortified at 0.01 mg/kg (quantitation transition monitored)...81 Typical chromatogram of an extract of control dry beans fortified at 0.01 mg/kg (confirmation transition monitored)...82 Typical chromatogram of an extract of control dry beans fortified at 0.5 mg/kg (quantitation transition monitored)...83 Typical chromatogram of an extract of control dry beans fortified at 0.5 mg/kg (confirmation transition monitored)...84 List of Appendices Appendix 1 Certificates of Analysis...85 Appendix 2 Primary Data Tables...87 Appendix 3 Analytical Method Appendix 4 Eye Research Centre GLP Compliance Statements 2011 and

13 Summary IKI-3106 and NK-1375: Validation of Methodology for the Determination of Residues of IKI-3106 and NK-1375 in Grape, Wine, Peaches, Oilseed Rape Seeds and Dry Beans A method, based on previous work performed by the sponsor, was validated for the determination of IKI-3106 and its metabolite NK-1375 in five matrix types (grape, wine, peaches, oilseed rape seeds and dry beans). Samples were extracted with acetonitrile prior to solid phase extraction (SPE) cleanup. Quantitation was performed using liquid chromatography with tandem mass spectrometric detection (LC-MS/MS). Method validation Control (untreated) samples of each sample type were analysed using the analytical method. There was no apparent response (i.e. < 30% of the LOQ) in the region in the chromatograms corresponding to the retention time of IKI-3106 or NK The method was validated at 0.01 and 0.5 mg/kg for the detection of both analytes in the matrix samples tested. The quantitation was performed by the use of LC-MS/MS. The accuracy and precision data are summarised in the following table. Fortification Recovery range Mean recovery Analyte Matrix CV (%) level (mg/kg) (%) (%) Grape Wine IKI-3106 Peach Oilseed rape seed Dry bean Grape Wine NK-1375 Peach Oilseed rape seed Dry bean The validation of the methodology for the determination of IKI-3106 or NK-1375 in the five matrix types demonstrated that they could be accurately determined at 0.01 and 0.5 mg/kg. The additional analysis of the validation samples using an alternative MS/MS ion transition for each analyte demonstrated a suitable confirmatory technique. 10

14 1. Introduction The objective of this study was to validate methodology for the determination of residues of IKI-3106 and NK-1375 in grape, wine, peaches, oilseed rape seeds and dry beans, and to demonstrate a suitable confirmatory technique. To determine the validity of the analytical method, it was necessary to determine: - recovery / accuracy - precision - linearity - specificity - limit of detection - limit of quantitation This study was based upon fulfilling the requirements of the following regulatory guidelines: Guidance for Generating and Reporting Methods of Analysis in Support of Residue Data Requirements for Annex II (part A, Section 4), and Annex III (part A, Section 5) of Directive 91/414, SANCO/3029/99 rev. 4. SANCO/825/00 rev.8.1 of 16 November 2010 : Guidance document on residue analytical methods; to fulfil the post-registration monitoring data requirements for Annex II (part A, Section 4), and Annex III (part A, Section 5) of Council Directive 91/414/EEC as amended by Commission Directive 96/46/EC. Study organisation The sample analysis took place at: Huntingdon Life Sciences, Ltd. Eye Research Centre Eye Suffolk IP23 7PX England Relevant study dates Study initiation : 1 February 2012 Analytical phase commenced : 19 March 2012 Analytical phase completed : 20 June

15 2. Materials 2.1 Analytical Standard 1 IKI-3106 Chemical name: Structural formula: 3-Bromo-N-[2-bromo-4-chloro-6-[[(1- cyclopropylethyl)amino]carbonyl]phenyl]-1-(3-chloro-2- pyridinyl)-1h-pyrazole-5-carboxamide (CA) Br Br H N N N Cl O N Cl O N H Purity: 99.18% Expiry date: 24 January 2016 Batch number: Physical state: Solid Storage conditions: Freezer (approximately -20 ºC, in the dark) 12

16 2.2 Analytical Standard 2 NK-1375 Chemical name: 3-Bromo-2-((2-bromo-4H-pyrazolo[1,5-d]pyrido[3,2- b][1,4]oxazin-4-ylidene)amino)-5-chloro-n-(1- cyclopropylethyl)benzamide Structural formula: O NH Br N N N Cl Br O N Purity: 98.6% Expiry date: 25 July 2015 Batch number: Physical state: Solid Storage conditions: Freezer (approximately -20 ºC, in the dark) Certificates of Analysis are presented in Appendix Control matrices The control matrices were obtained from previous studies where excess untreated control material was available. These samples were assigned unique identification numbers and stored at approximately 20 C prior to use. With the exception of wine, the samples were transferred individually from freezer storage and processed from frozen by homogenisation with dry ice using a Robot Coupe blender. The samples were returned to freezer storage after processing and left to allow dissipation of the dry ice prior to use. The wine sample was analysed directly without any processing, after allowing to thaw. 13

17 3. Methods 3.1 Validation Sub-samples of each of the five matrix types were fortified with known concentrations of the two test substances and analysed according to the following regime: 2 sub-samples of untreated sample matrix 5 sub-samples of untreated sample matrix fortified at the LOQ (0.01 mg/kg) 5 sub-samples of untreated sample matrix fortified at 0.5 mg/kg These samples were then analysed using the analytical methodology, with each sample injected onto the chromatographic system once. 3.2 Final extract stability An experiment was set up to demonstrate the stability of the two analytes under the typical storage conditions of the final extracts if they are not quantified immediately after preparation. Processed control extracts, fortified with IKI-3106 and NK-1375 were stored at approximately -20 ºC in the dark (i.e. in a freezer). Aliquots of each of the control sample extracts were fortified with IKI-3106 and NK-1375 at a concentration of 5 ng analyte/ml of final extract. The concentration of analytes in the stored extracts was determined at day 0 and after 7 days. The concentration of the analytes in freshly fortified control extracts was also determined at that time. 3.3 Matrix effects Any possible sample matrix effects were investigated by the comparison of the instrument response to the analytes in the fortified final extract samples with the response of the analytes in solvent based calibration standard solutions prepared at the same time. 3.4 Analytical method Samples were extracted with acetonitrile prior to solid phase extraction (SPE) cleanup. Quantitation was performed using liquid chromatography with tandem mass spectrometric detection (LC-MS/MS). The analytical method used in the laboratory is presented in Appendix 3. 14

18 3.5 Fortification/calibration solutions Individual stock standard solutions of IKI-3106 and NK-1375 were prepared by dissolving an accurately weighed amount of each material in a suitable volume of acetonitrile, correcting for purity as appropriate. These stock solutions were further diluted with acetonitrile to produce mixed fortification solutions at 100 µg/ml, 10 µg/ml and 1 µg/ml concentrations. The instrument calibration solutions, over the concentration range ng/ml to 10 ng/ml, were prepared by serial dilution of the mixed fortification solution in acetonitrile, as detailed below: Standard solution used (ng/ml) Volume taken (ml) Final volume (ml) Nominal concentration (ng/ml)

19 3.6 Calculation of results for validation samples Test samples were quantified using the following equation: Residue found (mg/kg) = 1 x D M Where x (residue concentration in final solution) was calculated using the linear regression y = m x + c where x (concentration in ng/ml) = c = intercept m = slope y = peak area of sample M = matrix concentration (g/ml) D = dilution factor y c m Example calculation of IKI-3106 detected in dry beans fortified at 0.5 mg/kg (analytical identification 11/00/2988 F0.5 A, analysis batch 6). The primary data for this sample is presented in Table 26, Appendix 2. Linear regression y = m x + c where y = e4 m = c = e4 = x Therefore, concentration of IKI-3106 (x) = Matrix concentration = 0.01 g matrix/ml final extract Dilution factor = e = ng/ml IKI-3106 detected (mg/kg) = ng/ml 1 = ng/g = mg/kg 0.01 g/ml Recovery (%) = mg/kg mg/kg = 89% 16

20 4. Results 4.1 Calibration (linearity) The response of the LC-MS/MS system to standard solutions of IKI-3106 and NK-1375 was linear over the range to 10 ng/ml (equivalent to to 1 mg/kg in sample matrix). Typical calibration data are presented in Tables 1 to 4 and Figures 1 to 4. Typical chromatograms of calibration standards are presented in Figures 7 to Method validation (accuracy/precision) The method was validated at 0.01 and 0.5 mg/kg for the detection of both analytes in the five matrix types. The quantification was performed by the use of LC-MS/MS. The accuracy and precision data are summarised in the following table. Fortification Recovery range Mean recovery Analyte Matrix CV (%) level (mg/kg) (%) (%) Grape Wine IKI-3106 Peach Oilseed rape seed Dry bean Grape Wine NK-1375 Peach Oilseed rape seed Dry bean The validation of the methodology demonstrated that both analytes could be accurately determined at 0.01 and 0.5 mg/kg in the five matrix types. Example chromatograms of the fortified samples are presented in Figures 13 to Limit of quantitation (LOQ) The LOQ was defined as the lowest fortification level at which acceptable recovery data was obtained. The validation of the methodology demonstrated that both analytes can be accurately determined at a LOQ of 0.01 mg/kg in the five matrix types tested. 4.4 Limit of detection (LOD) The LOD of the method was defined as the concentration of the lowest calibration standard chromatographed that gave rise to a measurable chromatographic response. For this study the 17

21 LOD of the method was shown to be ng/ml (equivalent to mg/kg in sample matrix). 4.5 Specificity/confirmation of residues Control (untreated) samples of each of the five matrices were analysed. There was no apparent response (i.e. < 30% of the LOQ) in the region in the chromatograms corresponding to the retention time of IKI-3106 and NK Typical chromatograms of untreated extracts are presented in Figures 13, 14, 19, 20, 25, 26, 31, 32, 37 and 38. The LC-MS/MS quantitation was performed by the instrument in the SRM (selected reaction monitoring) MS/MS mode. In this mode, the selected parent ions (in this case assigned as the protonated molecular ion of each of the analytes) undergoes collision-inducted dissociation (CID) to product characteristic daughter ions. The resulting mass chromatograms are based on the instrument monitoring only the selected ion transitions and quantitation was performed on the peak areas of the resulting chromatographic peaks. The ion transitions monitored were m/z 602>284 (IKI-3106) and m/z 566>498 (NK-1375), which were monitored for quantitation purposes. To demonstrate possible confirmation of residues, alternative ion transitions, m/z 602>177 (IKI-3106) and m/z 566>266 (NK-1375) were also monitored. The analysis of the validation samples using these confirmatory transitions produced data comparable to that acquired monitoring the quantitation ion transitions. Typical mass spectra showing the observed daughter ions subsequently used for SRM quantitation are presented in Figures 5 and 6. A summary of the validation data obtained by the instrument monitoring the quantitation and confirmation ion transitions are presented in Tables 5 to 24. Example chromatograms of control and fortified samples, monitoring the confirmatory ion transitions, are shown in Figures 13 to 42. As the chosen detection principle (LC-MS/MS with 2 transitions for each analyte) is highly specific this method also addresses the confirmatory requirement and thus no separate confirmatory method is required. 4.6 Final extract stability The results from this test demonstrate that the analytes are stable in the final extracts when stored at approximately -20 C. The storage period tested was 7 days. The storage period tested was sufficient to cover the maximum storage period of extracts from this study and also demonstrates that the samples can be stored for short periods prior to instrument analysis. The results are summarised in Table 25 with the primary data presented in Appendix Matrix effects No significant enhancement or suppression of response was observed for the analytes in the final sample extracts. The primary data are presented in Appendix 2 (Tables 46 to 53). 18

22 5. Conclusion The analytical method for the determination of IKI-3106 and NK-1375 has been fully validated at 0.01 and 0.5 mg/kg in five matrix types (grape, wine, peaches, oilseed rape seeds and dry beans) using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The limit of quantitation (LOQ) of 0.01 mg/kg of the residue method in these matrix types was determined as the lowest level validated. Recovery data at both validated levels demonstrated acceptable precision and accuracy of the analytical method. A suitable confirmatory technique was also demonstrated by the quantitation of the validation samples using an alternative MS/MS ion transition for each analyte. 19

23 Table 1 Typical standard calibration for IKI-3106 by LC-MS/MS (quantitation transition monitored) Standard Equivalent concentration concentration (ng/ml) in matrix (mg/kg) Peak area E E E E E E E E E+04 Linear regression: y = x (zero value included, 1/x weighting) r = Where x = concentration (ng/ml) y = peak area r = correlation coefficient Table 2 Typical standard calibration for IKI-3106 by LC-MS/MS (confirmation transition monitored) Standard Equivalent concentration concentration (ng/ml) in matrix (mg/kg) Peak area E E E E E E E E E+04 Linear regression: y = x (zero value included, 1/x weighting) r = Where x = concentration (ng/ml) y = peak area r = correlation coefficient 20

24 Table 3 Typical standard calibration for NK-1375 by LC-MS/MS (quantitation transition monitored) Standard Equivalent concentration concentration (ng/ml) in matrix (mg/kg) Peak area E E E E E E E E E+05 Linear regression: y = x (zero value included, 1/x weighting) r = Where x = concentration (ng/ml) y = peak area r = correlation coefficient Table 4 Typical standard calibration for NK-1375 by LC-MS/MS (confirmation transition monitored) Standard Equivalent concentration concentration (ng/ml) in matrix (mg/kg) Peak area E E E E E E E E E E+04 Linear regression: y = x (zero value included, 1/x weighting) r = Where x = concentration (ng/ml) y = peak area r = correlation coefficient 21

25 Table 5 Validation data IKI-3106 in grapes (quantitation transition monitored) Fortification Replicate Concentration Recovery Mean CV level detected (% ) (% ) (% ) (mg/kg) (mg/kg) Control A ND - Control B ND A B C D E A B C D E Overall Mean (%) 95 Overall CV (%) 4.5 Sample used: 09/00/9376 ND Not Detected (less than the limit of detection of mg/kg) 22

26 Table 6 Validation data IKI-3106 in grapes (confirmation transition monitored) Fortification Replicate Concentration Recovery Mean CV level detected (% ) (% ) (% ) (mg/kg) (mg/kg) Control A ND - Control B ND A B C D E A B C D E Overall Mean (%) 91 Overall CV (%) 7.5 Sample used: 09/00/9376 ND Not Detected (less than the limit of detection of mg/kg) 23

27 Table 7 Validation data NK-1375 in grapes (quantitation transition monitored) Fortification Replicate Concentration Recovery Mean CV level detected (% ) (% ) (% ) (mg/kg) (mg/kg) Control A ND - Control B ND A B C D E A B C D E Overall Mean (%) 95 Overall CV (%) 6.4 Sample used: 09/00/9376 ND Not Detected (less than the limit of detection of mg/kg) 24

28 Table 8 Validation data NK-1375 in grapes (confirmation transition monitored) Fortification Replicate Concentration Recovery Mean CV level detected (% ) (% ) (% ) (mg/kg) (mg/kg) Control A ND - Control B ND A B C D E A B C D E Overall Mean (%) 94 Overall CV (%) 7.6 Sample used: 09/00/9376 ND Not Detected (less than the limit of detection of mg/kg) 25

29 Table 9 Validation data IKI-3106 in wine (quantitation transition monitored) Fortification Replicate Concentration Recovery Mean CV level detected (% ) (% ) (% ) (mg/kg) (mg/kg) Control A ND - Control B ND A B C D E A B C D E Overall Mean (%) 93 Overall CV (%) 7.4 Sample used: 08/00/0296 ND Not Detected (less than the limit of detection of mg/kg) 26

30 Table 10 Validation data IKI-3106 in wine (confirmation transition monitored) Fortification Replicate Concentration Recovery Mean CV level detected (% ) (% ) (% ) (mg/kg) (mg/kg) Control A ND - Control B ND A B C D E A B C D E Overall Mean (%) 93 Overall CV (%) 9.2 Sample used: 08/00/0296 ND Not Detected (less than the limit of detection of mg/kg) 27

31 Table 11 Validation data NK-1375 in wine (quantitation transition monitored) Fortification Replicate Concentration Recovery Mean CV level detected (% ) (% ) (% ) (mg/kg) (mg/kg) Control A ND - Control B ND A B C D E A B C D E Overall Mean (%) 94 Overall CV (%) 9.0 Sample used: 08/00/0296 ND Not Detected (less than the limit of detection of mg/kg) 28

32 Table 12 Validation data NK-1375 in wine (confirmation transition monitored) Fortification Replicate Concentration Recovery Mean CV level detected (% ) (% ) (% ) (mg/kg) (mg/kg) Control A ND - Control B ND A B C D E A B C D E Overall Mean (%) 98 Overall CV (%) 10.0 Sample used: 08/00/0296 ND Not Detected (less than the limit of detection of mg/kg) 29

33 Table 13 Validation data IKI-3106 in peaches (quantitation transition monitored) Fortification Replicate Concentration Recovery Mean CV level detected (% ) (% ) (% ) (mg/kg) (mg/kg) Control A ND - Control B ND A B C D E A B C D E Overall Mean (%) 100 Overall CV (%) 6.2 Sample used: 12/00/2692 ND Not Detected (less than the limit of detection of mg/kg) 30

34 Table 14 Validation data IKI-3106 in peaches (confirmation transition monitored) Fortification Replicate Concentration Recovery Mean CV level detected (% ) (% ) (% ) (mg/kg) (mg/kg) Control A ND - Control B ND A B C D E A B C D E Overall Mean (%) 94 Overall CV (%) 8.8 Sample used: 12/00/2692 ND Not Detected (less than the limit of detection of mg/kg) 31

35 Table 15 Validation data NK-1375 in peaches (quantitation transition monitored) Fortification Replicate Concentration Recovery Mean CV level detected (% ) (% ) (% ) (mg/kg) (mg/kg) Control A ND - Control B ND A B C D E A B C D E Overall Mean (%) 97 Overall CV (%) 8.3 Sample used: 12/00/2692 ND Not Detected (less than the limit of detection of mg/kg) 32

36 Table 16 Validation data NK-1375 in peaches (confirmation transition monitored) Fortification Replicate Concentration Recovery Mean CV level detected (% ) (% ) (% ) (mg/kg) (mg/kg) Control A ND - Control B ND B C D E A B C D E Overall Mean (%) 93 Overall CV (%) 11.7 Sample used: 12/00/2692 ND Not Detected (less than the limit of detection of mg/kg) 33

37 Table 17 Validation data IKI-3106 in oilseed rape seeds (quantitation transition monitored) Fortification Replicate Concentration Recovery Mean CV level detected (% ) (% ) (% ) (mg/kg) (mg/kg) Control A ND - Control B ND A B C D E A B C D E Overall Mean (%) 95 Overall CV (%) 7.6 Sample used: 12/00/2980 ND Not Detected (less than the limit of detection of mg/kg) 34

38 Table 18 Validation data IKI-3106 in oilseed rape seeds (confirmation transition monitored) Fortification Replicate Concentration Recovery Mean CV level detected (% ) (% ) (% ) (mg/kg) (mg/kg) Control A ND - Control B ND A B C D E A B C D E Overall Mean (%) 95 Overall CV (%) 5.8 Sample used: 12/00/2980 ND Not Detected (less than the limit of detection of mg/kg) 35

39 Table 19 Validation data NK-1375 in oilseed rape seeds (quantitation transition monitored) Fortification Replicate Concentration Recovery Mean CV level detected (% ) (% ) (% ) (mg/kg) (mg/kg) Control A ND - Control B ND A B C D E A B C D E Overall Mean (%) 94 Overall CV (%) 7.7 Sample used: 12/00/2980 ND Not Detected (less than the limit of detection of mg/kg) 36

40 Table 20 Validation data NK-1375 in oilseed rape seeds (confirmation transition monitored) Fortification Replicate Concentration Recovery Mean CV level detected (% ) (% ) (% ) (mg/kg) (mg/kg) Control A ND - Control B ND A B C D E A B C D E Overall Mean (%) 100 Overall CV (%) 6.7 Sample used: 12/00/2980 ND Not Detected (less than the limit of detection of mg/kg) 37

41 Table 21 Validation data IKI-3106 in dry beans (quantitation transition monitored) Fortification Replicate Concentration Recovery Mean CV level detected (% ) (% ) (% ) (mg/kg) (mg/kg) Control A ND - Control B ND A B C D E A B C D E Overall Mean (%) 91 Overall CV (%) 4.7 Sample used: 11/00/2988 ND Not Detected (less than the limit of detection of mg/kg) 38

42 Table 22 Validation data IKI-3106 in dry beans (confirmation transition monitored) Fortification Replicate Concentration Recovery Mean CV level detected (% ) (% ) (% ) (mg/kg) (mg/kg) Control A ND - Control B ND A B C D E A B C D E Overall Mean (%) 95 Overall CV (%) 6.2 Sample used: 11/00/2988 ND Not Detected (less than the limit of detection of mg/kg) 39

43 Table 23 Validation data NK-1375 in dry beans (quantitation transition monitored) Fortification Replicate Concentration Recovery Mean CV level detected (% ) (% ) (% ) (mg/kg) (mg/kg) Control A ND - Control B ND A B C D E A B C D E Overall Mean (%) 96 Overall CV (%) 7.0 Sample used: 11/00/2988 ND Not Detected (less than the limit of detection of mg/kg) 40

44 Table 24 Validation data NK-1375 in dry beans (confirmation transition monitored) Fortification Replicate Concentration Recovery Mean CV level detected (% ) (% ) (% ) (mg/kg) (mg/kg) Control A ND - Control B ND A B C D E A B C D E Overall Mean (%) 95 Overall CV (%) 7.6 Sample used: 11/00/2988 ND Not Detected (less than the limit of detection of mg/kg) 41

45 Table 25 Final extract stability data Sample ID Analyte Matrix Analyte detected (ng/ml) Day 0 Day 7 Mean stability Grape Procedural recovery (%) N/A 97 Mean stability Wine Procedural recovery (%) N/A 98 Mean stability IKI-3106 Peach Procedural recovery (%) N/A 100 Mean stability Oilseed rape Procedural recovery (%) seed N/A 105 Mean stability Dry bean Procedural recovery (%) N/A 97 Mean stability Grape Procedural recovery (%) N/A 98 Mean stability Wine Procedural recovery (%) N/A 100 Mean stability NK-1375 Peach Procedural recovery (%) N/A 100 Mean stability Oilseed rape Procedural recovery (%) seed N/A 106 Mean stability Dry bean Procedural recovery (%) N/A 99 N/A not applicable Stability data is the mean of two replicate samples Sample ID (grapes) : 09/00/9376 Sample ID (wine) : 08/00/0296 Sample ID (peach) : 12/00/2692 Sample ID (oilseed rape seeds) : 12/00/2980 Sample ID (dry beans) : 11/00/2988 Note: all samples were fortified with 25 ng of each analyte in 5 ml control final extract ( 5 ng analyte/ml final extract). Stability samples analysed following initial fortification and after the storage period. Procedural recovery samples were fortified just prior to second analysis (following storage of stability samples). 42

46 Figure 1 Typical standard calibration for IKI-3106 by LC-MS/MS (quantitation transition monitored) _B6a.rdb (IKI / 284): "Linear" Regression ("1 / x" weighting): y = x (r = ) 8.5e4 8.0e4 7.5e4 7.0e4 6.5e4 6.0e4 5.5e4 Area, counts 5.0e4 4.5e4 4.0e4 3.5e4 3.0e4 2.5e4 2.0e4 1.5e4 1.0e Concentration, ng/ml 43

47 Figure 2 Typical standard calibration for IKI-3106 by LC-MS/MS (confirmation transition monitored) _B6a.rdb (IKI / 177): "Linear" Regression ("1 / x" weighting): y = x (r = ) 5.2e4 5.0e4 4.5e4 4.0e4 3.5e4 Area, counts 3.0e4 2.5e4 2.0e4 1.5e4 1.0e Concentration, ng/ml 44

48 Figure 3 Typical standard calibration for NK-1375 by LC-MS/MS (quantitation transition monitored) _B6a.rdb (NK / 498): "Linear" Regression ("1 / x" weighting): y = x (r = ) 1.6e5 1.5e5 1.4e5 1.3e5 1.2e5 1.1e5 1.0e5 Area, counts 9.0e4 8.0e4 7.0e4 6.0e4 5.0e4 4.0e4 3.0e4 2.0e4 1.0e Concentration, ng/ml 45

49 Figure 4 Typical standard calibration for NK-1375 by LC-MS/MS (confirmation transition monitored) _B6a.rdb (NK / 266): "Linear" Regression ("1 / x" weighting): y = x (r = ) 7.5e4 7.0e4 6.5e4 6.0e4 5.5e4 5.0e4 4.5e4 Area, counts 4.0e4 3.5e4 3.0e4 2.5e4 2.0e4 1.5e4 1.0e Concentration, ng/ml 46

50 Figure 5 Mass spectrum daughter ion scan of m/z 602 (IKI-3106) +MS2 (602.00) CE (70): to min from Sample 1 (Cal 1 ng/ml) of _SCAN202.wiff (Turbo Spray... Max. 4.2e5 cps. 4.2e5 4.0e5 3.8e5 3.6e5 3.4e5 3.2e5 3.0e5 2.8e5 2.6e5 2.4e5 2.2e5 2.0e5 1.8e5 1.6e5 1.4e5 1.2e5 1.0e5 8.0e4 6.0e4 4.0e4 2.0e m/z, amu 47

51 Figure 6 Mass spectrum daughter ion scan of m/z 566 (NK-1375) +MS2 (566.00) CE (30): to min from Sample 1 (Cal 1 ng/ml) of _SCAN102.wiff (Turbo Spray... Max. 3.0e5 cps e5 2.6e5 2.4e e5 2.0e5 1.8e e5 1.4e5 1.2e5 1.0e e e4 4.0e4 2.0e m/z, amu 48

52 Figure 7 Typical chromatogram of a 10 ng/ml calibration standard (quantitation transition monitored) Sample Name: "Cal 10 ng/ml" Sample ID: "" File: "_B6a18.wiff" Peak Name: "IKI / 284" Mass(es): "602.0/284.0 amu" Sample Type: Standard Concentration: 10.0 ng/ml Calculated Conc: 9.73 ng/ml Acq. Date: 12/06/2012 Acq. Time: 18:01:18 Modified: No Proc. Algorithm: IntelliQuan - IQA Min. Peak Height: cps Min. Peak Width: 5.00 sec Smoothing Width: 5 points RT Window: 10.0 sec Expected RT: min Use Relative RT: No 2.9e4 2.8e4 2.7e4 2.6e4 2.5e4 2.4e Int. Type: Valley Retention Time: min Area: e+004 counts Height: 2.91e+004 cps Start Time: min End Time: min 2.3e4 2.2e4 2.1e4 2.0e4 1.9e4 1.8e4 1.7e4 1.6e4 1.5e4 1.4e4 1.3e4 1.2e4 1.1e4 1.0e IKI-3106 Sample Name: "Cal 10 ng/ml" Sample ID: "" File: "_B6a18.wiff" Peak Name: "NK / 498" Mass(es): "566.0/498.0 amu" Sample Type: Standard Concentration: 10.0 ng/ml Calculated Conc: 9.98 ng/ml Acq. Date: 12/06/2012 Acq. Time: 18:01:18 Modified: No Proc. Algorithm: IntelliQuan - IQA Min. Peak Height: cps Min. Peak Width: 5.00 sec Smoothing Width: 5 points RT Window: 10.0 sec Expected RT: min Use Relative RT: No 5.0e4 4.8e4 4.6e4 4.4e4 4.2e Int. Type: Valley Retention Time: min Area: e+005 counts Height: 5.09e+004 cps Start Time: min End Time: 1.14 min 4.0e4 3.8e4 3.6e4 3.4e4 3.2e4 3.0e4 2.8e4 2.6e4 2.4e4 2.2e4 2.0e4 1.8e4 1.6e4 1.4e4 1.2e4 1.0e NK

53 Figure 8 Typical chromatogram of a 10 ng/ml calibration standard (confirmation transition monitored) Sample Name: "Cal 10 ng/ml" Sample ID: "" File: "_B6a18.wiff" Peak Name: "IKI / 177" Mass(es): "602.0/177.0 amu" Sample Type: Standard Concentration: 10.0 ng/ml Calculated Conc: 9.72 ng/ml Acq. Date: 12/06/2012 Acq. Time: 18:01:18 1.7e4 1.6e Modified: No Proc. Algorithm: IntelliQuan - IQA Min. Peak Height: cps Min. Peak Width: 5.00 sec Smoothing Width: 5 points RT Window: 10.0 sec Expected RT: min Use Relative RT: No Int. Type: Valley Retention Time: min Area: e+004 counts Height: 1.68e+004 cps Start Time: min End Time: min 1.6e4 1.5e4 1.5e4 1.4e4 1.4e4 1.3e4 1.3e4 1.2e4 1.2e4 1.1e4 1.1e4 1.0e IKI-3106 Sample Name: "Cal 10 ng/ml" Sample ID: "" File: "_B6a18.wiff" Peak Name: "NK / 266" Mass(es): "566.0/266.0 amu" Sample Type: Standard Concentration: 10.0 ng/ml Calculated Conc: 9.96 ng/ml Acq. Date: 12/06/2012 Acq. Time: 18:01:18 Modified: Yes Proc. Algorithm: IntelliQuan - IQA Min. Peak Height: cps Min. Peak Width: 5.00 sec Smoothing Width: 7 points RT Window: 10.0 sec Expected RT: min Use Relative RT: No 2.3e4 2.2e4 2.1e4 2.0e4 1.9e Int. Type: Manual Retention Time: min Area: e+004 counts Height: 2.34e+004 cps Start Time: min End Time: 1.11 min 1.8e4 1.7e4 1.6e4 1.5e4 1.4e4 1.3e4 1.2e4 1.1e4 1.0e NK

54 Figure 9 Typical chromatogram of a 1 ng/ml calibration standard (quantitation transition monitored) Sample Name: "Cal 1 ng/ml" Sample ID: "" File: "_B6a11.wiff" Peak Name: "IKI / 284" Mass(es): "602.0/284.0 amu" Sample Type: Standard Concentration: 1.00 ng/ml Calculated Conc: 0.99 ng/ml Acq. Date: 12/06/2012 Acq. Time: 17:31: Modified: No Proc. Algorithm: IntelliQuan - IQA Min. Peak Height: cps Min. Peak Width: 5.00 sec Smoothing Width: 5 points RT Window: 10.0 sec Expected RT: min Use Relative RT: No Int. Type: Valley Retention Time: min Area: e+003 counts Height: 3.02e+003 cps Start Time: min End Time: min IKI-3106 Sample Name: "Cal 1 ng/ml" Sample ID: "" File: "_B6a11.wiff" Peak Name: "NK / 498" Mass(es): "566.0/498.0 amu" Sample Type: Standard Concentration: 1.00 ng/ml Calculated Conc: 1.06 ng/ml Acq. Date: 12/06/2012 Acq. Time: 17:31: Modified: No Proc. Algorithm: IntelliQuan - IQA Min. Peak Height: cps Min. Peak Width: 5.00 sec Smoothing Width: 5 points RT Window: 10.0 sec Expected RT: min Use Relative RT: No Int. Type: Valley Retention Time: min Area: e+004 counts Height: 5.19e+003 cps Start Time: min End Time: 1.05 min NK

55 Figure 10 Typical chromatogram of a 1 ng/ml calibration standard (confirmation transition monitored) Sample Name: "Cal 1 ng/ml" Sample ID: "" File: "_B6a11.wiff" Peak Name: "IKI / 177" Mass(es): "602.0/177.0 amu" Sample Type: Standard Concentration: 1.00 ng/ml Calculated Conc: 1.04 ng/ml 1800 Acq. Date: 12/06/2012 Acq. Time: 17:31: Modified: No Proc. Algorithm: IntelliQuan - IQA Min. Peak Height: cps Min. Peak Width: 5.00 sec Smoothing Width: 5 points RT Window: 10.0 sec Expected RT: min Use Relative RT: No Int. Type: Valley Retention Time: min Area: e+003 counts Height: 1.83e+003 cps Start Time: min End Time: min IKI-3106 Sample Name: "Cal 1 ng/ml" Sample ID: "" File: "_B6a11.wiff" Peak Name: "NK / 266" Mass(es): "566.0/266.0 amu" Sample Type: Standard Concentration: 1.00 ng/ml Calculated Conc: 1.01 ng/ml Acq. Date: 12/06/2012 Acq. Time: 17:31: Modified: No Proc. Algorithm: IntelliQuan - IQA Min. Peak Height: cps Min. Peak Width: 5.00 sec Smoothing Width: 7 points RT Window: 10.0 sec Expected RT: min Use Relative RT: No Int. Type: Valley Retention Time: min Area: e+003 counts Height: 2.35e+003 cps Start Time: min End Time: 1.09 min NK

56 Figure 11 Typical chromatogram of a ng/ml calibration standard (quantitation transition monitored) Sample Name: "Cal ng/ml" Sample ID: "" File: "_B6a27.wiff" Peak Name: "IKI / 284" Mass(es): "602.0/284.0 amu" Sample Type: Standard Concentration: ng/ml Calculated Conc: 0.05 ng/ml Acq. Date: 12/06/2012 Acq. Time: 18:39: Modified: Yes Proc. Algorithm: IntelliQuan - IQA Min. Peak Height: cps Min. Peak Width: 5.00 sec Smoothing Width: 5 points RT Window: 10.0 sec Expected RT: min Use Relative RT: No Int. Type: Manual Retention Time: min Area: e+002 counts Height: 1.48e+002 cps Start Time: min End Time: min IKI-3106 Sample Name: "Cal ng/ml" Sample ID: "" File: "_B6a27.wiff" Peak Name: "NK / 498" Mass(es): "566.0/498.0 amu" Sample Type: Standard Concentration: ng/ml Calculated Conc: 0.03 ng/ml Acq. Date: 12/06/2012 Acq. Time: 18:39:19 Modified: Yes Proc. Algorithm: IntelliQuan - IQA Min. Peak Height: cps Min. Peak Width: 5.00 sec Smoothing Width: 5 points RT Window: 10.0 sec Expected RT: min Use Relative RT: No Int. Type: Manual Retention Time: min Area: e+002 counts Height: 2.18e+002 cps Start Time: min End Time: min NK