Joint Experiment Technical Study (JETS) Report 17/1 Maleic Hydrazide in Tobacco

Transcription

1 Agrochemical Analysis Sub-Group Joint Experiment Technical Study (JETS) ort 7/ Maleic Hydrazide in Tobacco July 20 Authors: Katiane Oliveira, Souza Cruz, Brazil Alvino Rodrigues, Souza Cruz, Brazil Rodrigo Fontoura, Souza Cruz, Brazil Fernanda Contieri Abad, Souza Cruz, Brazil Statistical Support: Flaviane Brevi, Souza Cruz, Brazil Tiago Camargo Berti, Product Centre AmSSA, Brazil Sub-Group Coordinator: Masahiro Miyoshi, Japan Tobacco Inc., Japan

2 Table of Contents. Introduction Test Material Preparation Homogeneity Distribution Test Procedure Participating Laboratories Limit of Quantitation and Recoveries.... Statistical Evaluation of Results.... Methods overview....2 Spiked sample (sample ) as exclusion criteria.... Tested methods in comparison with ISO method (reference) Analytical Methods Information.... Final Considerations and Conclusions References... APPENDIX : Homogeneity Test... 4 APPENDIX 2: Standard material & Calibration... 7 APPENDIX : Raw Data orted by Participating Laboratories... 2 APPENDIX 4: Comments & Questions... 2 APPENDIX 5: z-scores...

3 . Introduction The ISO 47:0 method (Tobacco and tobacco products - Determination of maleic hydrazide residues) [] was systematically reviewed in 20. Two revision proposals were submitted to the ISO/TC 2 at that time, suggesting adoption of a modified method. Whilst the current version of the ISO method is based on caustic extraction and colorimetric measurement, both submitted proposals are based on an acidic extraction condition. The former proposal was GC-MS detection with an internal standard (IS) (Renaud, 2) [2], and the latter was HPLC without an IS (YC/T ) []. During the annual meeting of the CORESTA Sub-Group Agrochemical Analysis (AA), held in Vienna in 202, an internal trial presented by the Japan Tobacco Group representative, showed different capabilities of the tested methods to extract residues of naturally incurred Maleic Hydrazide (MH) (bound with cell wall fraction, such as lignin; and glucoside conjugates [4]), a fact that still has to be clarified. Similar methods that were submitted to the ISO committee were tested in the mentioned internal trial. Due to the complexity of the distribution of MH throughout the tobacco plant, the AA Sub- Group decided to conduct a Joint Experiment Technical Study (JETS) to assess both the fate of MH and the most appropriate approach for analysis, as an outcome from AA Sub-Group 205 annual meeting, held in Zimbabwe. This study was identified as JETS /2. The JETS /2 was formulated as a mini-proficiency test to evaluate the capacity of different methods - especially those ones submitted to ISO/TC 2 as proposal to replace the current official method - to properly measure MH content in both artificially spiked and naturally incurred tobacco samples [5]. The JETS /2 report was presented at SG AA 20 annual meeting in Thailand. The obtained results showed some indications that YC/T modified method showed a better ability to be compared to the reference results (ISO or ISO modified methods), among the tested methods. However, the limited number of laboratories applying the same method weakened the statistical analysis in achieving conclusive findings. For conclusive findings, an additional study of the YC/T modified method, with an increased number of participating laboratories, was strongly recommended.. The JETS 7/ describes a new mini-proficiency test to evaluate the capacity of the YC/T modified and ISO methods to quantitate MH on tobacco, using both artificially spiked and naturally incurred tobacco sample. AA-0-CTR JETS 7/ Maleic Hydrazide July 20 /7

4 2. Test Material 2. Preparation Five tobacco samples containing naturally-incurred residues of MH (Flue-cured; US, 200 and 202 crops), one tobacco sample artificially spiked with MH (Flue-cured; Japan, 202) and two MH-free tobacco samples (Burley; Japan, 20 and Flue-cured; Japan, 202) were prepared for this JETS, as follows: Blank Samples Test Samples 7/ Blank (Burley; Japan, 20) 7/ Blank 2 (Flue-cured; Japan, 202) 7/ Sample (Flue-cured; Japan, 202), artificially spiked 7/ Sample 4 (Flue-cured; US, 200), naturally incurred 7/ Sample 5 (Flue-cured; US, 202), naturally incurred 7/ Sample (Flue-cured; US, 202), naturally incurred 7/ Sample 7 (Flue-cured; US, 200), naturally incurred 7/ Sample (Flue-cured; US, 202), naturally incurred The selected tobaccos were ground and sieved using a 0. mm mesh screen. The blank samples were provided for recovery testing and for matrix-matched calibration if applied. 2.2 Homogeneity Full report of homogeneity test is available in Appendix. According to the statistical evaluation, samples were considered sufficiently homogeneous for the JETS 7/ purpose. 2. Distribution The eight tobacco samples (approximately 40 g each) were dispatched to 7 participants from April to July Test Procedure. Each test sample was analysed in triplicate (n=) for Maleic Hydrazide (MH), using the laboratory s method(s) of choice described in JETS 7/ participation form (ISO 47:0 method, Part I method, Part II method) AA-0-CTR JETS 7/ Maleic Hydrazide July 20 4/7

5 .2 Two Burley recoveries and two Flue-cured recoveries using the corresponding blank tobaccos supplied were extracted and analysed in parallel with the test samples for most of participants.. Recovery tests were performed by spiking an equivalent level of 0 mg/kg (the current GRL for MH defined in CORESTA Guide Nº [])..4 When matrix-matched calibration standard was used, it was recommended that the calibration curve was prepared from the supplied blank tobacco..5 All sample results were quoted in mg/kg on an as is basis, without recovery or moisture correction.. Results and information on the used method were submitted using the template spreadsheet provided by the JETS coordinators. 4. Participating Laboratories 7 laboratories from different countries confirmed participation in the JETS 7/. Due to clearance issues at customs and other internal reasons, 4 laboratories (2.5 %) did not receive the samples in time, and could not participate. Therefore, laboratories from 0 countries participated in this JETS (Table ). Table : Participants list Laboratory Product Centre Americas Primoris Belgium China National Tobacco Quality Supervision and Test Center Hubei Province Tobacco Quality Supervision and Test Station Sichuan Tobacco Quality Supervision and Testing Station Zhengzhou Tobacco Research Institute of CNTC Eurofins - Dr. Specht Laboratorien Agrolab RDS - Sindos Angler Biochemlab, PT Japan Tobacco Inc. KT&G Research Institute Microbac Laboratories Inc. Tobacco Research Board Country Brazil Belgium China China China China Germany Greece Indonesia Japan Korea USA Zimbabwe Each participating laboratory was coded with a unique lab number once their results were submitted. AA-0-CTR JETS 7/ Maleic Hydrazide July 20 5/7

6 5. Limit of Quantitation and Recoveries Participating laboratories measured and reported two recoveries for the supplied blank samples using chosen method(s). The recovery values, together with limits of quantitation (LOQs) are shown in Tables 2 (Burley tobacco) and (Flue-cured tobacco). Table 2: LOQs and recoveries in Burley tobacco Maleic Hydrazide in Burley Lab number Method used LOQ (mg/kg) Fortification Level (mg/kg) licate (%) licate 2 (%) Mean (%) Part I -* Part II ISO 47: Part I Part II Part II ISO 47: Part I ISO 47: Part I Part II Part I Part II ISO 47: Part I Part II Part I Part II Part I ISO 47: ISO 47: Part II Part I *Not informed by participant AA-0-CTR JETS 7/ Maleic Hydrazide July 20 /7

7 Table : LOQs and recoveries in Flue-cured tobacco Lab number Method used LOQ (mg/kg) Fortification Level (mg/kg) Maleic Hydrazide in Flue-Cured licate (%) licate 2 (%) Mean (%) Part I -* Part II ISO 47: Part I Part II Part II ISO 47: Part I ISO 47: Part I Part II Part I Part II ISO 47: Part I Part II Part I Part II Part I ISO 47: ISO 47: Part II Part I *Not informed by participant Recoveries were measured from 20-0 mg/kg range. The target concentration was 0 mg/kg (the GRL for Maleic Hydrazide) in the protocol. Laboratory 4 reported recovery for the Burley type sample only, and Laboratory reported recoveries of the Flue-cured type sample only, while the other laboratories report the recoveries in both Burley and Flue-cured types. The recoveries between both types were comparable. Laboratory 5 reported recoveries for Flue-cured type sample using YC/T Part I method out of the range of 70 %-20 %, recommended by CORESTA Guide N 5 [7], and the analysis results generated from YC/T Part I method in this laboratory were removed from all statistical evaluations of the JETS 7/. AA-0-CTR JETS 7/ Maleic Hydrazide July 20 7/7

8 . Statistical Evaluation of Results. Methods overview Participating laboratories were grouped according to applied methods (Table 4). laboratories applied the official version of ISO 47:0 as it is. laboratories applied the Modified YC/T Part I method and the same number of laboratories applied the Modified YC/T Part II method. Table 4: Applied methods overview Method Laboratory Number Total Labs ISO 47:0 2, 5,,,, Part I,,, 7,,, 0, Part II,, 4,, 7,,, 2.2 Spiked sample (sample ) as exclusion criteria Trueness of a method is generally assessed by recoveries studies, proficiency testing programmes, and sometimes, when available, reference materials (which often is not the case for agrochemical residue analysis). Proficiency testing programs often use spiked samples; recovery tests of spiked blank samples are recommended, in order to be performed every batch of samples. By taking this background information into account, participant labs performance was evaluated by their ability to properly analyse the spiked sample (sample ) in the JETS 7/, as the criterion to keep the lab results for further statistical evaluation of applied methods, according to this JETS s goal. The assumption is that all applied methods should be able to properly extract and analyse the spiked sample, otherwise the lack of ability to correctly analyse incurred samples with bound MH can be related to performance issues of the lab, and not necessarily due to the method of choice performance. Then, the z-score was used to evaluate the performance of each method in all laboratories to analyse the sample ( according to their average compared to the overall mean. Absolute value of z-score below 2 is considered as satisfactory. AA-0-CTR JETS 7/ Maleic Hydrazide July 20 /7

9 z = x ij x o s o Where: x ij mean of laboratory i applying the method j x o overall mean s o overall deviation Table 5 and Graph show the calculated z-scores for the sample (. Only Laboratory did not obtain a satisfactory evaluation and the analysis results from the laboratory were excluded for further statistical analyses. Table 5: Laboratories Descriptive Results for sample (spiked sample) Lab Number n Maleic Hydrazide 2 Average Std. Dev. (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) z score Part I Part II ISO 47: Part I Part II Part II ISO 47: ISO 47: Part I Part II Part I Part II ISO 47: Part I Part II Part I Part II Part I ISO 47: ISO 47: Part II Part I AA-0-CTR JETS 7/ Maleic Hydrazide July 20 /7

10 Graph : Laboratories z-score for sample (spiked sample) The z-scores of all samples of the JETS 7/ are found in the Appendix. While the z-score evaluate the laboratory performances taking into account the overall mean generated by each sample that were applied, next sections will evaluate the capacity of methods to provide results equivalent to the reference method (ISO 47). The reference method was applied by of participating laboratories, which led to a different statistical approach and conclusions that cannot be comparable to the z-score outcome.. Tested methods in comparison with ISO method (reference) Table shows the comparison of tested methods, by grouping them in the following categories: ISO (reference); I; and II. AA-0-CTR JETS 7/ Maleic Hydrazide July 20 0/7

11 Once ISO method was considered the reference method, Dunnett s test was the statistical method of choice to compare all grouped methods to the reference. Sample Sample Sample 4 Sample 5 Sample Sample 7 Sample Table : Tested methods in comparison to ISO method (reference) Method Maleic Hydrazide (mg/kg) n Average Std. Dev. ISO I 2 5. * 7.5 II *.0 ISO I *.2 II * 5. ISO I * 0.7 II * 2.05 ISO I 2 5. * 2.2 II 24.0 * 2.4 ISO 5..5 I *. II *.54 ISO.7 7. I * 4.72 II * 5.7 *Average statistically different from the ISO method with a 5 % significance level. Both the modified YC/T methods were not comparable to ISO method for the evaluated samples. The standard deviation values for YC/T methods were larger than those of ISO method. One possible reason for these data is the lack of skill in some participants applying the YC/T methods for the first time, since these participants had limited time to validate the methods during this JETS. 7. Analytical Methods Information Submitted information on the methods used for Maleic Hydrazide (MH) analysis are compiled and documented in the following appendices: Appendix 2: Standard material & Calibration AA-0-CTR JETS 7/ Maleic Hydrazide July 20 /7

12 Appendix : Appendix 4: Appendix 5: Raw data reported by participating laboratories Comments & Questions z-scores Regarding participants comments and questions available in Appendix 4, Laboratory informed adjusts performed in YC/T methods; Laboratory 2 informed an alternative method used to evaluate the amount of free MH; Laboratory 5 informed details about sample issues in method YC/T Part I method and solvent evaporation in YC/T part II method; Laboratory and Laboratory 0 informed difficulties to perform ISO method, and Laboratory 0 describe your findings about YC/T method part I, and Laboratory 2 informs details about their LOQ values and curve weighting in YC/T Part II method. Laboratory 2 (for ISO method), Laboratory (for ISO method), Laboratory 2 (for ISO and YC/T Part II methods) and Laboratory (for YC/T Part I method) informed validation details.. Final Considerations and Conclusions Proposals of new methods to replace the current version of the ISO 47:0 have been submitted to ISO/TC 2. Upon these submissions and due to the unclear capacity of methods to extract free and bound Maleic Hydrazide (MH) residues, the CORESTA Sub-group Agrochemicals Analysis (SG AA) has decided to conduct a Joint Experiment Technical Study (JETS) 7/. The JETS 7/ aimed to assess the capacity of different methods to analyse the MH content in both artificially spiked and naturally incurred tobacco samples, with comparable results to the current ISO method. Focus was mainly on performance of submitted methods to ISO/TC 2 - YC/T and was not on the individual performance of participating laboratories. laboratories from 0 countries participated in this JETS. laboratories applied the original version of ISO 47:0. laboratories applied Part II and laboratories applied the Part I. One laboratory that applied Part I (Laboratory ) did not achieve a satisfactory performance in the spiked sample and was removed from all statistical evaluations. No laboratory followed the original version of the method YC/T [], which was submitted to the ISO/TC 2 (YC/T ). Reviewing JETS /2 samples results for ISO and YC/T methods, their behaviours were similar with observed in JETS 7/ samples, with MH reported values for YC/T methods lower than ISO for all samples but the artificially spiked. This might indicate that ISO method is able to extract free and bounded MH in the sample. As a conclusion of this JETS, the YC/T I and YC/T II methods were not able to provide equivalent results to the reference methods (ISO 47) in the evaluated samples. AA-0-CTR JETS 7/ Maleic Hydrazide July 20 2/7

13 . References. ISO 47:0. Tobacco and tobacco products - Determination of maleic hydrazide residues. 2. Renaud, JM; Keller, I; Vuillaume, G. Determination of maleic hydrazide residues in cured tobacco by gas chromatography, Journal of Chromatography, 04 (2) YC/T Tobacco and tobacco products. Determination of multi-pesticide residues. Part 5: Determination of maleic hydrazide residue. High performance liquid chromatography method. 4. CORESTA Agrochemicals Analysis Technical Note, TN #00, Maleic Hydrazide, Plant growth regulator, March CORESTA Agrochemical Analysis Sub-Group, Joint Experiment Technical Study (JETS) ort /2 Maleic Hydrazide in Tobacco, March CORESTA Guide No., Agrochemical Guidance Residue Level, June CORESTA Guide No. 5, Sub-Group on Pesticide Residues, Technical Guideline for Pesticide Residues Analysis on Tobacco and Tobacco Products, February 200. AA-0-CTR JETS 7/ Maleic Hydrazide July 20 /7

14 APPENDIX : Homogeneity Test Objective Tobacco Homogeneity Evaluation for JETS 7/ Maleic Hydrazide To evaluate the homogeneity of the Maleic Hydrazide results (mg/kg) among packages of each tobacco sample to be distributed to laboratories participating in the JETS 7/ Statistical Methodology and Results Homogeneity was evaluated by sampling twice each unit (package) of sample batches. Linear fit, Analysis of Variance was used to evaluate the overall homogeneity. Exploratory Analysis Initially it was performed an exploratory analysis to identify potential atypical values in the results. The values considered outliers by Grubbs test are showed in Table below: Table A.: Values considered outliers by Grubbs test Sample Number Description Reference Code Maleic Hydrazide 4450 V-FL2RMS (202) V-FF2RMS (202) V-FF2RMS (202) Flue-cured spiked 7.4 Homogeneity between Tobacco Portions To evaluate the homogeneity between tobacco portions it was fitted a linear model for each tobacco type as shown in equation (). with, Yi = Maleic Hydrazide result i (mg/kg); Xi = Tobacco sample (reference code) i; β 0 = Linear Coefficient; β = Slope; Y i = β 0 + β X i + ε i () Errors ε i are independently and identically distributed N(0, σ 2 ). AA-0-CTR JETS 7/ Maleic Hydrazide July 20 4/7

15 The following hypothesis about β (slope of linear regression) was tested aiming to verify if there is a trend in portions results, for each sample presented in this study. with, i = {, 2,, 4, 5, } (each tobacco sample) H 0 : β i = 0 H : β i 0 When the slope is considered significant in the fitted model, points will be removed until achieve the homogeneity, and with this to identify the homogeneous slice. These results can be seen on the tables below and the graphs are shown in the end of this Appendix. Table A.2: p-values for β comparisons (First fitted model) p-values for β comparisons (First fitted model) Sample N β p-value V_0FL2RWS_ V_FLXMS_ V_FL2RMS_ < 0.00 V_FLXMS_ < 0.00 V_FF2RMS_ Flue-cured spiked Note: p-value<0.05 indicates that the slope is not zero at the 5 % level of significance Table A.: p-values for β comparisons (Last fitted model) p-values for β comparisons (Last fitted model) Sample N β p-value V_0FL2RWS V_FLXMS_ V_FL2RMS_ V_FLXMS_ V_FF2RMS_ Flue-cured spiked Note: p-value<0.05 indicates that the slope is not zero at the 5 % level of significance After determined the slice of tobacco portion homogeneous it is important to verify the variability behavior, for this it was applied the Score Test for Non-Constant Error Variance. The results of this test can be seen in Table A.4. AA-0-CTR JETS 7/ Maleic Hydrazide July 20 5/7

16 Table A.4: Score Test for Non-Constant Error Variance p-values of Homogeneity Test Sample p-value V_0FL2RWS_ V_FLXMS_ V_FL2RMS_ V_FLXMS_ V_FF2RMS_ Flue-cured spiked 0.5 Note: p-value<0.05 indicates that the slope is not zero at the 5 % level of significance Conclusion Applying this methodology it was possible to identify that the homogeneity was reached for all samples present in this study. Therefore, for V_FLXMS_200 sample it was necessary to exclude half of points to establish the stability, and for V_FL2RMS_202 sample it was excluded points. To obtain a more robust result it is suggested to increase repetitions in all points. There was no increase in terms of variation over the measurements for all samples presented in this study. It was possible to identify a slice of measurements where the results of Maleic Hydrazide are homogeneous for all samples. For V_FLXMS_200 sample it is recommended to use until package 4, for V_FL2RMS_202 sample until package 55 and for other samples all packages can be used. AA-0-CTR JETS 7/ Maleic Hydrazide July 20 /7

17 APPENDIX 2: Standard material & Calibration Table A2.: Standard materials used by each laboratory Lab n Method used Modified YC/T Part I Modified YC/T Part II ISO 47:0 Modified YC/T Part I Modified YC/T Part II Modified YC/T Part II Modified YC/T Part I ISO 47:0 ISO 47:0 Modified YC/T Part I Modified YC/T Part II Modified YC/T Part I Standard Supplier Sigma Aldrich Sigma Aldrich Lot number SZBE5X V SZBE5X V Purity (%) Certi fied. Yes. Yes Solvent used to prepare stock/ working standard ultrapure water (type ) ultrapure water (type ) Internation al used Ehrenstorfer Yes Methanol No Sigma Aldrich Sigma Aldrich Sigma Dr. Ehrenstorfer Dr. Ehrenstorfer Dr. Ehrenstorfer GmbH Dr. Ehrenstorfer GmbH Dr. Ehrenstorfer GmbH Labor Dr. Ehrenstorfer- Schafers SZBD02X V SZBD02X V SZBE5X V. Yes. Yes Yes Yes No No No Yes Ultrapure water Ultrapure water Yes Yes No No. Yes Methanol Yes 0.0N NaOH in H2O 0.0N NaOH in H2O Ultrapure water Ultrapure water Ultrapure water ultrapure water Yes No No Yes Yes Yes AA-0-CTR JETS 7/ Maleic Hydrazide July 20 7/7

18 Lab n Method used Modified YC/T Part II ISO 47:0 Modified YC/T Part II Modified YC/T Part I Modified YC/T Part I Modified YC/T Part II Modified YC/T Part I ISO 47:0 ISO 47:0 Modified YC/T Part II Modified YC/T Part I Standard Supplier Labor Dr. Ehrenstorfer- Schafers Dr. Ehrenstorfer GmbH Dr. Ehrenstorfer GmbH Dr. Ehrenstorfer GmbH Sigma Sigma TCI(Shanghai) Development Co., Ltd Lot number Purity (%) Certi fied Yes No No No SZB2X V SZB2X V PA0Q-0C.0 Yes Chem Service Yes Kanto Chemical Kanto Chemical Solvent used to prepare stock/ working standard ultrapure water deionized water deionized water deionized water Internation al used Yes No Yes Yes. Yes MeOH No. Yes MeOH No ultrapure water 0.N NaOH/DI water Yes No H52.0 No NaOH sol No H52.0 No Ultrapure water Yes Sigma-Aldrich 0252JC.0 Yes Pure water Yes AA-0-CTR JETS 7/ Maleic Hydrazide July 20 /7

19 Lab n Table A2.2: Calibrations applied by each laboratory Method used Type Matrix Part I Part II Matrixmatched Fluecured Matrixmatched Numb er of stand ards r Origin Exclude d Exclude d 2 ISO 47: Part I Part II Part II Part I 5 ISO 47:0 ISO 47: Part I Part II Part I Part II ISO 47: Part II Part I Part I Part II Part I Solvent Solvent Fluecured Matrixmatched Solvent Matrixmatched Fluecured Matrixmatched Solvent Solvent Solvent Solvent Fluecured Matrixmatched Solvent Solvent Fluecured Matrixmatched Matrixmatched Solvent Curve weighti ng None None - 0. Included /X Included /X Burley 0.2 Forced /X Included /X Exclude d Exclude d Exclude d Exclude d Exclude d Exclude d None None /X /X /X /X 0.0 Included None Fluecured & Burley Fluecured & Burley Exclude d Exclude d Exclude d Exclude d Exclude d /X /X /X /X None AA-0-CTR JETS 7/ Maleic Hydrazide July 20 /7

20 Lab n Method used Type Matrix ISO 47:0 2 ISO 47: Part II Part I Solvent Matrixmatched Solvent Matrixmatched Numb er of stand ards r 2 Origin Curve weighti ng Included None Fluecured & Burley - 7 Fluecured Exclude d Exclude d Exclude d None /X None AA-0-CTR JETS 7/ Maleic Hydrazide July 20 20/7

21 APPENDIX : Raw Data orted by Participating Laboratories Table A.: Raw data reported by participating laboratories Results Maleic Hydrazide (mg/kg) Lab Method Sample Part I Part I Part I Part I Part I Part I Part II Part II Part II Part II Part II Part II 2 ISO 47:0 2 ISO 47:0 2 ISO 47:0 2 ISO 47:0 2 ISO 47:0 2 ISO 47: Part I Part I Part I 7/ Sample (artificially 7/ Sample 4 (naturally 7/ Sample 5 (naturally 7/ Sample (naturally 7/ Sample 7 (naturally 7/ Sample (naturally 7/ Sample (artificially 7/ Sample 4 (naturally 7/ Sample 5 (naturally 7/ Sample (naturally 7/ Sample 7 (naturally 7/ Sample (naturally 7/ Sample (artificially 7/ Sample 4 (naturally 7/ Sample 5 (naturally 7/ Sample (naturally 7/ Sample 7 (naturally 7/ Sample (naturally 7/ Sample (artificially 7/ Sample 4 (naturally 7/ Sample 5 (naturally 2 Average SD / Sample (naturally AA-0-CTR JETS 7/ Maleic Hydrazide July 20 2/7

22 Results Maleic Hydrazide (mg/kg) Lab Method Sample Part I Part I Part I Part II Part II Part II Part II Part II Part II Part II Part II Part II Part II Part II Part II Part I Part I Part I Part I Part I Part I 5 ISO 47:0 5 ISO 47: 5 ISO 47:2 7/ Sample 7 (naturally 7/ Sample (naturally 7/ Sample (artificially 7/ Sample 4 (naturally 7/ Sample 5 (naturally 7/ Sample (naturally 7/ Sample 7 (naturally 7/ Sample (naturally 7/ Sample (artificially 7/ Sample 4 (naturally 7/ Sample 5 (naturally 7/ Sample (naturally 7/ Sample 7 (naturally 7/ Sample (naturally 7/ Sample (artificially 7/ Sample 4 (naturally 7/ Sample 5 (naturally 7/ Sample (naturally 7/ Sample 7 (naturally 7/ Sample (naturally 7/ Sample (artificially 7/ Sample 4 (naturally 7/ Sample 5 (naturally 2 Average SD <LO Q <LO Q <LO Q <LO Q <LO Q AA-0-CTR JETS 7/ Maleic Hydrazide July 20 22/7

23 Results Maleic Hydrazide (mg/kg) Lab Method Sample 5 ISO 47: 5 ISO 47:4 5 ISO 47:5 ISO 47:0 ISO 47:0 ISO 47:0 ISO 47:0 ISO 47:0 ISO 47: Part I Part I Part I Part I Part I Part I Part II Part II Part II Part II Part II Part II Part I Part I 7/ Sample (naturally 7/ Sample 7 (naturally 7/ Sample (naturally 7/ Sample (artificially 7/ Sample 4 (naturally 7/ Sample 5 (naturally 7/ Sample (naturally 7/ Sample 7 (naturally 7/ Sample (naturally 7/ Sample (artificially 7/ Sample 4 (naturally 7/ Sample 5 (naturally 7/ Sample (naturally 7/ Sample 7 (naturally 7/ Sample (naturally 7/ Sample (artificially 7/ Sample 4 (naturally 7/ Sample 5 (naturally 7/ Sample (naturally 7/ Sample 7 (naturally 7/ Sample (naturally 7/ Sample (artificially 7/ Sample 4 (naturally <LO Q 2 <LO Q <LO Q Average SD / Sample 5 (naturally AA-0-CTR JETS 7/ Maleic Hydrazide July 20 2/7

24 Results Maleic Hydrazide (mg/kg) Lab Method Sample Part I Part I Part I Part I Part II Part II Part II Part II Part II Part II 7/ Sample (naturally 7/ Sample 7 (naturally 7/ Sample (naturally 7/ Sample (artificially 7/ Sample 4 (naturally 7/ Sample 5 (naturally 7/ Sample (naturally 7/ Sample 7 (naturally 7/ Sample (naturally 2 Average SD Table A.: Raw data reported by participating laboratories (continued) Results Maleic Hydrazide (mg/kg) Lab Method Sample ISO 47:0 ISO 47:0 ISO 47:0 ISO 47:0 ISO 47:0 ISO 47: Part I Part I Part I 7/ Sample (artificially 7/ Sample 4 (naturally 7/ Sample 5 (naturally 7/ Sample (naturally 7/ Sample 7 (naturally 7/ Sample (naturally 7/ Sample (artificially 7/ Sample 4 (naturally 7/ Sample 5 (naturally 2 Average SD / Sample (naturally AA-0-CTR JETS 7/ Maleic Hydrazide July 20 24/7

25 Results Maleic Hydrazide (mg/kg) Lab Method Sample 0 20 Part I Part I Part I Part II Part II Part II Part II Part II Part II Part I Part I Part I Part I Part I Part I Part II Part II Part II Part II Part II Part II Part I 7/ Sample 7 (naturally 7/ Sample (naturally 7/ Sample (artificially 7/ Sample 4 (naturally 7/ Sample 5 (naturally 7/ Sample (naturally 7/ Sample 7 (naturally 7/ Sample (naturally 7/ Sample (artificially 7/ Sample 4 (naturally 7/ Sample 5 (naturally 7/ Sample (naturally 7/ Sample 7 (naturally 7/ Sample (naturally 7/ Sample (artificially 7/ Sample 4 (naturally 7/ Sample 5 (naturally 7/ Sample (naturally 7/ Sample 7 (naturally 7/ Sample (naturally 7/ Sample (artificially 2 Average SD AA-0-CTR JETS 7/ Maleic Hydrazide July 20 25/7

26 Results Maleic Hydrazide (mg/kg) Lab Method Sample Part I Part I Part I Part I Part I ISO 47:0 ISO 47:0 ISO 47:0 ISO 47:0 ISO 47:0 ISO 47:0 2 ISO 47:0 2 ISO 47:0 2 ISO 47:0 2 ISO 47:0 2 ISO 47:0 2 ISO 47: Part II Part II Part II Part II Part II 7/ Sample 4 (naturally 7/ Sample 5 (naturally 7/ Sample (naturally 7/ Sample 7 (naturally 7/ Sample (naturally 7/ Sample (artificially 7/ Sample 4 (naturally 7/ Sample 5 (naturally 7/ Sample (naturally 7/ Sample 7 (naturally 7/ Sample (naturally 7/ Sample (artificially 7/ Sample 4 (naturally 7/ Sample 5 (naturally 7/ Sample (naturally 7/ Sample 7 (naturally 7/ Sample (naturally 7/ Sample (artificially 7/ Sample 4 (naturally 7/ Sample 5 (naturally 7/ Sample (naturally 7/ Sample 7 (naturally 2 Average SD <LO Q <LO Q <LO Q AA-0-CTR JETS 7/ Maleic Hydrazide July 20 2/7

27 Results Maleic Hydrazide (mg/kg) Lab Method Sample Part II Part I Part I Part I Part I Part I Part I 7/ Sample (naturally 7/ Sample (artificially 7/ Sample 4 (naturally 7/ Sample 5 (naturally 7/ Sample (naturally 7/ Sample 7 (naturally 7/ Sample (naturally 2 Average SD AA-0-CTR JETS 7/ Maleic Hydrazide July 20 27/7

28 APPENDIX 4: Comments & Questions Lab number 2 Method used Part I Part II ISO 47:0 Comments It was included an additional step after to get the mixed eluent that consists to transfer the exact volume of mixed eluent to a volumetric flask. The intention of this operation is to reduce the results variability, because the methodology recommends that the sample is eluted in the column and then it is filtered, that is, the final volume is not adjusted. However, the Original YC/T method has a similar procedure to collect the eluent into a 5mL volumetric flask (section 7. "Purification"). Besides the comments in Part I, about the extraction step, it was adapted to use the same apparatus from Part I, but an additional magnetic stirring system was included. It was used a heat reflux with magnetic stirring. The main difference between the modified method Part II and the modified method Part I is the thermodynamic condition. The method Part II has an isovolumetric system, while the Part I has an isobaric system. Maybe, if it were used a kind of erlenmeyer with screw caps under heating, the results could be bigger due the strong extraction condition. Put g of finely ground tobacco into the 250 ml long-neck round flask followed by 50 ml 50 % sodium hydroxide solution and 2- paraffin pellets and some boiling stones. Heat up the open flask at ca. 55 C (not more than C) for 5 minutes. Thereafter, the flask is cooled down to 0 C. The reduction mixture (0.5 g FeCl2 and 5 g Zn) are added into the flask by using a funnel. Immediately after the addition the flask is connected to the distillation bridge. Heated water vapour is drawn thru the solution. At the same time the flask is heated up to 55 C. Never heat above C because otherwise recovery will be lost. The amount How much time did you allow between spiking recovery and the addition of extraction solvent? Methodology validated? Description about the validation steps 0 min No - 0 min No - 0 min Yes hexplicates on spiking levels (2 / 0 / 240 ppm) (ICH Harmonised Tripartite Guideline Validation of Analytical Procedures: Text and Methodology Q2(R)) AA-0-CTR JETS 7/ Maleic Hydrazide July 20 2/7

29 Lab number Method used Part I Part II Comments of water vapour is regulated to obtain 00 ml distillate in ca. 20 minutes. The distillate has to be checked for ph value which must be acidic. The obtained distillate is shaked with 2 g charcoal for minute. Thereafter, it is filtered into a 250 ml round flask (rinsing with some ml water) and is evaporated to ml using a rotary evaporator. The solution is transferred into a 25 ml volumetric flask. ml of freshly prepared.5 % 4- dimethylaminobenzaldehyde solution is added and filled up to 25 ml with water. The obtained solution has to be clear (if not it has to be filtered). The solution is measured with a photometer at 455 nm against ml of freshly prepared.5 % 4- dimethylaminobenzaldehyde filled up to 25 ml with water. Remark: the screening method only covers the free MH. Bound residues of MH are not extracted with the below procedure! Put g of finely ground tobacco into the 50 ml glass jar followed by 0 ml water and 40 ml methanol. Macerate min by using an ultraturrax. Filter thru filter paper into a 50 ml long-neck round flask. Rinse the ultra-turrax rod and the glass jar with 2x0 ml methanol and add to the filter cake. Evaporate the filtered solution down to 2- ml using a rotary evaporator at 70 C. Transfer the remainder into a 0 ml test tube and fill up to an end volume of 5 ml. An aliquot of 2 ml is placed onto a conditioned Chromabond SA Cation-exchange column (500 mg) and sucked thru. The filtrate is collected and column is rinsed with 2 ml water to fill up to 5 ml. The obtained solution is measured with LC-MS/MS negative ESI mode. How much time did you allow between spiking recovery and the addition of extraction solvent? Methodology validated? 0 min Yes Description about the validation steps hexplicates on spiking level (0, ppm) = trigger level to run the first confirmation method - 20 min No min No - AA-0-CTR JETS 7/ Maleic Hydrazide July 20 2/7

30 Lab number Method used Part II ISO 47: Part I Part II ISO 47: Part I Part II Part I Part II ISO 47: Part I Part II Comments How much time did you allow between spiking recovery and the addition of extraction solvent? Methodology validated? Description about the validation steps - 0 min No - The results with recovery correction from the flue cured blank, clean up. Sample.2 /. / 7. /., spike flue cured 2 : no clean up, no results Method part II: After heating, the solvent is completely evaporated. The tests cannot be performed. ISO method is very difficult to handle, when I was asked to apply it in May, I started to use it in my lab for the first time. It takes me many trials before applying it on the JETS samples. In my point of view, there are some comments as follows:. Lots of reagents are consumed, either in types or in amount. 2. The 2.5 mol/l NaOH used is corrosive to the glassy reaction bottle, the bottle even could not be used for the second time considering the high reaction temperatures.. The repeatability of this method is poor, mainly resulting from the complicated pretreatment. 4. The UV-vis detection is simple and fast, but not sensitive enough for low maleic hydrazide residue levels. 0 min No min No min No min No min Yes min Yes min No min No min No - AA-0-CTR JETS 7/ Maleic Hydrazide July 20 0/7

31 Lab number Method used Part I Part II ISO 47: Part I ISO 47:0 ISO 47:0 Comments How much time did you allow between spiking recovery and the addition of extraction solvent? Methodology validated? Description about the validation steps - 5 min No min No - This method is too hard for beginners. The pre-treatment processes are complicated and time-consuming, too many details need to be noticed. The high concentration NaOH solution and the high temperature during distillation and concentration may have security risks. Since we have not got the results, We can't comment on the data. Compared with YC/T , this method is more accurate and sensitive. The use of internal standard makes the extraction more efficient, faster and solvents saving. However, there are still a few problems remain to be resolved. For example, high concentration HCl has not been removed before injection, which may cause damage to the instrument, especially for massive testing. Note: Method adapted from AOAC th Edition. The method that was used was adapted from an AOAC method but is very close to the ISO 47:0 method. Net Absorbance is used for Measurement. Instrumentation and Procedural Standards: Samples solutions are read against a blank at three wavelengths (40 mm, 40 mm and 40 mm). Calculation is based on comparison of the sample Maleic hydrazide net absorbance to a Maleic hydrazide standard curve processed through the method and application of the method dilution factor. 5 min No - 5 min No - Directly after, approximately No LOQ is 0 mg/kg. 0 min Yes Analysts generate a calibration curve to determine the linearity. On a continuing basis, the analyst will spike along the range of the curve. In general, the ICH (International Council for Harmonisation) guidelines are followed for validation. Recovery tests were demonstrated in the range, 70 % -20 % and relative standard deviation 20 % on the two tobacco types, Fluecured and Burley. Fortification level is AA-0-CTR JETS 7/ Maleic Hydrazide July 20 /7

32 Lab number 2 Method used Part II Part I Comments The curve weighting is changed to /x from "none" which was recommended by ordinary YC/T. Preliminary LOQ is 4 mg/kg. How much time did you allow between spiking recovery and the addition of extraction solvent? Methodology validated? 0 min Yes - 0 min Yes Description about the validation steps three points, LOQ, 0xLOQ and GRL. Each data point is n= (n=, 2days). (CORESTA Guide No.5) Recovery tests were demonstrated in the range, 70 % -20 % and relative standard deviation 20 % on the two tobacco types, Fluecured and Burley. Fortification level is three points, LOQ, 0xLOQ and GRL. Each data point is n= (n=, 2days). (CORESTA Guide No.5) g tobacco is weighted and transferred to a 250 ml roundbottom flask,.0 ml of 00 mg/l maleic hydrazide is added to the flask 2. Stay for 0 minutes. 50 ml of 2 % hydrochloric acid and 250 μl of 200 mg/l maleic hydrazide-d2 are added to the flask 4. Continuously heating reflux under 50 C for h. 5. After cooling to room temperature, the mixture is filtered. The mixture is filtered 7. Sample clean-up AA-0-CTR JETS 7/ Maleic Hydrazide July 20 2/7

33 APPENDIX 5: z-scores AA-0-CTR JETS 7/ Maleic Hydrazide July 20 /7

34 AA-0-CTR JETS 7/ Maleic Hydrazide July 20 4/7

35 AA-0-CTR JETS 7/ Maleic Hydrazide July 20 5/7

36 AA-0-CTR JETS 7/ Maleic Hydrazide July 20 /7

37 AA-0-CTR JETS 7/ Maleic Hydrazide July 20 7/7