Routine Analytical Chemistry Sub-Group. Technical Report

Transcription

1 Routine Analytical Chemistry Sub-Group Technical Report 2014 Collaborative Study Comparing CRM35 for the Determination of Total Alkaloids (as Nicotine) in Tobacco by Continuous Flow Analysis to a New Method with Safer Chemistry March 2017 Sub-Group Co-ordinator: Linda A. Crumpler, Cerulean, USA Study Co-ordinator: Wei Zhang, China National Tobacco Quality Supervision and Test Center, China Linda A. Crumpler, Cerulean, USA Authors: Wei Zhang, China National Tobacco Quality Supervision and Test Center, China Yanjun Ma, Guorong Du, R&D, Beijing Cigarette Factory, China Linda. A. Crumpler, Cerulean, USA

2 Table of Contents 1. Introduction Organisation Participants Samples Data Sets Raw data Statistical Analysis Numerical outlier technique: Cochran & Grubbs test Outlier results Repeatability and reproducibility estimation Comparison of 2014 Study to Previous Studies Method Validation Conclusions and Recommendations Conclusions Recommendations Appendix A Experimental Protocol Appendix B Chinese KSCN/DCIC Method of Collaborative Test for Total Alkaloids in Tobacco Appendix C Raw Data by Laboratory Appendix D Raw Mean and Standard Deviation Value Appendix E Data Representation Appendix F IUPAC 1994 Harmonized Statistical Procedure

3 1. Introduction To produce a safer method to test total alkaloids in tobacco (as nicotine) the CORESTA Routine Analytical Chemistry Sub-Group (RAC) undertook a collaborative study in 2014 of two methods for the determination of total alkaloids in tobacco by segmented continuous-flow analysis. The two methods are CORESTA Recommended Method N 35 (CRM35) and a new method (NCM) proposed by China National Tobacco Quality Supervision and Test Center (CTQTC). In CRM35 cyanogen chloride is generated in situ by the reaction of potassium cyanide (KCN), a highly toxic compound and chloramine T. NCM proposed by CTQTC eliminates the use of the potassium cyanide by employing potassium thiocyanate (KSCN) with sodium dichloroisocyanurate dihydrate (DCIC). Each method was tested using water extracted tobacco and 5 % acetic acid (HAC) extracted tobacco. As documented in CRM35 it is recommended that 5 % HAC extracts should be used if total alkaloids (as nicotine) and reducing substances or reducing carbohydrate analyses are to be carried out simultaneously. Calibration standards were prepared with the same extraction solutions. Further objectives of this work are: to provide a new CRM reflecting the safer chemistry with flow diagrams for both macro and micro flow instruments to provide a basis for work proposed within ISO TC126 regarding the technical review of the corresponding ISO Standard Organisation 2.1 Participants In total, 19 laboratories participated in the 2014 study using the study protocol and the KSCN/DCIC method attached in Appendices A and B, respectively. The list of laboratories in alphabetical order and the continuous-flow equipment used are shown below in Table 1. Table 1: Participating laboratories and equipment Laboratory Name British American Tobacco, Germany China National Tobacco Quality Supervision and Test Center, China Essentra Scientific Services Global Laboratory Services, Inc. Guizhou tobacco quality supervision and inspection station Landewyck Tobacco s.a. R&D Shandong Industrial Co., Ltd. R&D, Beijing Cigarette Factory R&D, Shenzhen Cigarette Factory R&D, China tobacco Hubei Industrial Co., Ltd. R&D, China tobacco Jiangxi Industrial Co., Ltd. R&D, China tobacco Yunnan Industrial Co., Ltd. R&D, Guangxi International Tobacco Co., Ltd Equipment SEAL Analytical, AA3 SEAL Analytical, AA3 Technicon, AA II Unity Scientific, Discrete Analyzer SEAL Analytical, AA3 SEAL Analytical SEAL Analytical, AA3 SEAL Analytical, AA3 Skalar, San plus System SEAL Analytical, AA3 SEAL Analytical, AA3 SEAL Analytical, AA3 SEAL Analytical, AA3 RAC CTR 2014 Safer Chemistry Collaborative Study March /45

4 Laboratory Name R&D, Hua Huan International Tobacco Co., Ltd R&D, Inner Mongolia Kunming cigarettes Co., Ltd ITG Reemtsma SEAL Analytical Sichuan Tobacco Quality Supervision and Test Station Yunnan Academy of Agricultural Sciences Equipment SEAL Analytical, AA3 SEAL Analytical, AA3 Skalar, San plus System SEAL Analytical, AA3 SEAL Analytical, AA3 SEAL Analytical, AA3 2.2 Samples Samples A-F are ground and distributed to participating laboratories by CTQTC. Samples G- H are also ground and prepared by participating laboratories themselves. Sample descriptions are given in Table 2. Table 2: Sample description Sample Name A B C D E F G H Sample Description Flue-cured tobacco Burley tobacco Oriental tobacco Dark sun-cured tobacco Fire-cured cigarette Blended cigarette CM7 3R4F 2.3 Data sets Six replicates for each method and extraction technique were requested. However, due to some restrictions on the use of KCN, problems with sample transport or technical difficulties some labs failed to provide complete data packages as shown in Table 3. Table 3: Number of results by laboratory Sample Water extracts 5% HAC extracts CRM35 NCM CRM35 NCM A B C D E F G H RAC CTR 2014 Safer Chemistry Collaborative Study March /45

5 3. Raw data The original data of the 19 laboratories can be found in Appendix C. The raw mean and standard deviation values of CRM35 and NCM can be found in Appendix D. In Appendix E, the raw data for CRM35 and NCM with water and 5 % HAC extracts shown in plots, in which the mean values of the 95 % confidence interval are highlighted in colors. The 99 % confidence limits of difference between the average of all laboratories and the average of one laboratory are also presented. 4. Statistical analysis 4.1 Numerical outlier technique: Cochran & Grubbs test The statistical evaluation of data for this collaborative study followed the methods provided by ISO For outlier testing, the Grubbs and Cochran methods were used. The protocol Harmonized statistical procedure defined by IUPAC (International Union of Pure and Applied Chemistry) has been applied as well. It consists of sequential applications of the Cochran and Grubbs tests until no further outliers are detected or until a drop of more than 22.2 % (for this study =2/9) in the original number of laboratories would occur (see flowchart in Appendix F). First apply the Cochran outlier test, and if an outlying laboratory is identified, then a single value Grubbs test is performed on the individual values of this outlying laboratory (Individual Grubbs). If no individual value is identified as outlier, the outlying laboratory is removed. When an individual value is identified as an outlier, only this value is removed. Afterwards single-value Grubbs test is applied and outlying laboratories are removed. If no laboratory is identified as outlier, the pair-value test is applied (two values at the same end). Remove any laboratory flagged by these tests, but stop removal if more than 22.2 % (2 of 9 laboratories) would be removed or until a drop of more than 22.2 % (ca. 2/9) in the original number of laboratories would occur 2. The details of this method can be found in Appendix F. 1 ISO :1994 : Accuracy (trueness and precision) of measurement methods and results Part 2: Basic method for the determination of repeatability and reproducibility of a standard measurement method Collaborative Study of CORESTA Monitors #7 (CM7) for the Determination of Test Piece Weight, TPM, Water, Nicotine, NFDPM, Carbon Monoxide and Puff Count Obtained Under Mainstream ISO and Intense Smoking Regimes, Routine Analytical Chemistry Sub-Group Technical Report, March 19, RAC CTR 2014 Safer Chemistry Collaborative Study March /45

6 4.1.1 Outlier results Table 4: Results of outlier determination Samples Outlier Method CRM35 in water NCM in water CRM35 in HAC NCM in HAC A B C D E F G H Cochran 6 5, , 14, 5 Grubbs 16, Cochran 15, 16 15, 16 15, 17 15, 10, 14 Grubbs Cochran 16, 8 16, 13, 11 16, 9, 4 14, 13, 1 Grubbs Cochran 2 5, 4 6 5, 14, 13 Grubbs Cochran 12, 4, 16 16, 5, , 14, 13 Grubbs Cochran 8, 9, 11 5, 13, 9, , 13, 8 Grubbs Cochran 4, 8 18, 5, 13 17, 3 5, 14, 13 Grubbs Cochran 12 4, , 5, 4 Grubbs The data for each sample and each individual laboratory is shown in Appendix D, Tables D-1 to D-8. Table 4 summarizes the analyses from laboratories that were considered outliers and with higher standard deviations according to the Cochran-Grubbs statistical analysis. RAC CTR 2014 Safer Chemistry Collaborative Study March /45

7 4.2 Repeatability and reproducibility estimation Repeatability (r) and Reproducibility (R) limits are calculated after removal of outliers. Nevertheless, the stragglers are retained for these calculations. r and R values shown in Tables 5-8, are calculated for both the water and 5 % HAC extraction methods. Table 5: CRM35 with water extraction Tobacco Type Mean (%) r rcv (%)* R RCV (%) * Fire-cured Burley Oriental Dark sun-cured Fire-cured cigarette Blended cigarette CM R4F *rcv is r/mean 100% and RCV is R/mean 100% Table 6: NCM with water extraction Tobacco Type Mean (%) r rcv (%) * R RCV (%) * Fire-cured Burley Oriental Dark sun-cured Fire-cured cigarette Blended cigarette CM R4F RAC CTR 2014 Safer Chemistry Collaborative Study March /45

8 Table 7: CRM35 with 5 % HAC extraction Tobacco Type Mean (%) r rcv (%) * R RCV (%) * Fire-cured Burley Oriental Dark sun-cured Fire-cured cigarette Blended cigarette CM R4F Table 8: NCM with 5 % HAC extraction Tobacco Type Mean (%) r rcv (%) * R RCV (%) * Fire-cured Burley Oriental Dark sun-cured Fire-cured cigarette Blended cigarette CM R4F Comparison of 2014 study to previous studies In order to further evaluate the data acquired in this study, data were also compared to earlier collaborative studies performed by the RAC on CRM35. Table 9 lists the r and R values of CRM35 from the 1993 international collaborative study involving 12 laboratories and 3 samples. Table 10 lists the r and R values of CRM35 from the collaborative study involving 17 laboratories and 6 samples. Comparing Table 5, 6, 9 and 10, it can be seen that, for the same tobacco type, the r and R values of CRM35 from the three collaborative studies are close to each other Collaborative Studies for Nicotine, Sugars and Nitrate in Tobacco, May 2008 RAC CTR 2014 Safer Chemistry Collaborative Study March /45

9 Table 9: r and R of total alkaloids in 1993 collaborative study Extraction Method Tobacco Type Mean (%) r rcv * R RCV * Oriental Water 5 % acetic acid Flue-Cured Burley Oriental Flue-Cured Burley Table 10: r and R of total alkaloids in 2006 collaborative study Tobacco Type Mean* (%) r rcv * R RCV * Flue-Cured A Flue-Cured B Flue-Cured C Burley sample D Burley sample E Burley sample F *This CRM recommends that equivalent results are obtained when either water or 5 % HAC are used as the extraction solvents and therefore the results from this study were not segregated in the subsequent data analysis. There was also a study performed in 2013 by a large group of laboratories in China comparing CRM35 and the NCM. Table 11 lists the r and R values from this collaborative study. Again it can be observed that the r and R values in this study are comparable to the current study for the same tobacco type. Table 11: r and R of total alkaloids in 2013 Chinese collaborative study Method Samples Mean (%) r rcv * R RCV * Fire-cured Burley CRM35 KSCN/DCIC Oriental Cigarette A Cigarette B Fire cured Burley Oriental Cigarette A Cigarette B It can be concluded from the analysis above that the data from three previous studies and the current study are consistent and can be used in comparative analysis. RAC CTR 2014 Safer Chemistry Collaborative Study March /45

10 Total Alkaloids (%) Figure 1 shows the r and R values of total alkaloids determined by CRM35 and NCM extraction with water and 5 % HAC extracts, respectively, in 2014 international collaborative study. As shown, r is always marked in yellow, while the R values from the four different measuring methods are displayed in four different colours. Mean values of the four different measuring methods are also presented. It can be observed from above tables and Figure 1 that, for both extracts, the r values of CRM35 are, however, larger than the observed for NCM. The values of the two methods are very similar. The R values for CRM35 extracted in water for samples A, F, and G are lower than those observed for the NCM water extracts. While for samples B, C, D, E and H, the results are reversed. The R values of CRM35 extracted in HAC are lower than those of NCM extracted in HAC for samples A, B, D, E, F, and G, while for C and H, the R values of CRM35 are higher than for 5 % HAC. The difference in R values of both methods is statistically not significant and considered acceptable R of CRM35 in water R of NCM in water R of CRM35 in HAC R of NCM in HAC r Mean values A B C D E F G H Test Samples ID Figure 1: The r and R of total alkaloids determined by CRM35 and NCM extraction with water and 5 % HAC, respectively, in 2014 international collaborative study. RAC CTR 2014 Safer Chemistry Collaborative Study March /45

11 Total Alkaloids (%) Figure 2 shows the r and R of total alkaloids determined by CRM35 and NCM in the 2013 Chinese collaborative study. It can be observed that, the R value of CRM35 is lower than observed for NCM for sample B and E, while for sample A, C and D, the R values of CRM35 are higher than the ones of NCM. The r values of the two methods are similar. The small difference observed in R values for both methods, is acceptable R of CRM R of NCM r Mean values A B C D E Test Samples ID Figure 2: The r and R of total alkaloids determined by CRM35 and NCM in 2013 Chinese collaborative study. 5. Method Validation Validations of CRM35 and NCM, (extraction in water) in 2014 collaborative study are shown in Figure 3. The location of the lab mean is close to y = x line, illustrating that the total alkaloids of CRM35 method are very similar to those from NCM extraction by water method. The correlation coefficient of the two methods is , and the root mean square error (RMSE) is %. RMSE can be calculated by: RMSE N i 1 y NCM y CRM35 where N is the number of data points, y NCM and y CRM representation to the total alkaloids determined by NCM and CRM35, respectively in this work. The ratio performance deviation (RPD) is also used in this work, it can be calculated by: RPD N SD y RMSE CRM35 2 RAC CTR 2014 Safer Chemistry Collaborative Study March /45

12 Total alkaloids (%) by NCM in water If RPD exceeds 3, the consistency of CRM35 and NCM is considered as satisfactory according to Williams and Sobering (1993) 4. Using water extraction, the RPD is , showing good consistency between the two methods. The linear regression equation of the two methods was also calculated, y NCM =0.9962y CRM , where y NCM and y CRM were NCM and CRM extraction in water, respectively. 6 5 y = x Lab mean Total alkaloids (%) by CRM35 in water Figure 3: The relationship of total alkaloids between method CRM35 and NCM by water extraction for the 2014 international collaborative study. 4 P.C. Williams and D.C. Sobering, Comparison of commercial near infrared transmittance and reflectance instruments for analysis of whole grains and seeds, Journal of Near Infrared Spectroscopy, Volume 1 Issue 1, Pages (1993) RAC CTR 2014 Safer Chemistry Collaborative Study March /45

13 Total alkaloids (%) by NCM in 5 % HAC The relationship of CRM and NCM extraction in 5 % HAC in 2014 collaborative study is visualized in Figure 4. The correlation coefficient of two methods is , and the root mean square error (RMSE) is %. The RPD is , showing good consistency of CRM35 and NCM in extraction with 5 % HAC. The linear regression equation of the two method was also calculated, y NCM =0.9951y CRM , where y NCM and y CRM were NCM and CRM35 extraction in 5 % HAC, respectively y = x Lab mean Total alkaloids (%) by CRM35 in 5 % HAC Figure 4: The relationship of total alkaloids between method CRM35 and NCM by 5 % HAC extraction for the 2014 international collaborative study. RAC CTR 2014 Safer Chemistry Collaborative Study March /45

14 Total alkaloids (%) by NCM Figure 5 shows the relationship of CRM35 and NCM for Chinese collaborative study in The correlation coefficient of the two methods is , and the root mean square error (RMSE) is %. The RPD is , showing good consistency of CRM35 and NCM. The linear regression equation of the two method was also calculated, y NCM =0.9788y CRM , where y NCM and y CRM35 were NCM and CRM35 in 2013 Chinese collaborative study, respectively. 6 5 y = x Lab mean Total alkaloids (%) by CRM35 Figure 5: The relationship of total alkaloids between CRM35 and NCM for the 2013 Chinese collaborative study. In order to further validate the agreement of CRM35 to NCM, Bland Altman plots were used. Figure 6 shows the result of CRM35 and NCM in extraction with water. The logarithmic transformation of the dataset using a technique described by Bland and Altman 5 (1986) was applied because the difference of the two methods increased as total alkaloid values increased. The green line is the mean value of the difference of two methods, and the red dash lines represent the 95 % confidence interval. It can be seen that the means for all the eight samples are located within the 95 % confidence interval demonstrating that results obtained for CRM35 and NCM using water extraction are consistent. The mean results of CRM35 and NCM using 5 % acetic acid extraction are shown in Figure 7. Consistent with previous collaborative studies on CRM35 using both water and 5 % HAC extraction solutions, the NCM method gives equivalent results. 5 Statistical methods for assessing agreement between two methods of clinical measurement, JM Bland, DG Altman, The Lancet, 1986, I: RAC CTR 2014 Safer Chemistry Collaborative Study March /45

15 Difference between two measures Difference between two measures Bland Altman plot E H 0 C D B G A F Mean of two measures Figure 6: Bland Altman plot of CRM35 and NCM in extraction with water Bland Altman plot E B A H F G D C Mean of two measures Figure 7: Bland Altman plot of CRM35 and NCM extraction in 5 % HAC RAC CTR 2014 Safer Chemistry Collaborative Study March /45

16 Difference between two measures Difference between two measures 0.07 Bland Altman plot D B G EH 0.02 C F 0.01 A Mean of two measures Figure 8: Bland Altman plot of CRM35 in water and CRM35 in 5% HAC Bland Altman plot B A F G D EH C Mean of two measures Figure 9: Bland Altman plot of NCM in water and NCM in 5 % HAC RAC CTR 2014 Safer Chemistry Collaborative Study March /45

17 6. Conclusions and recommendations 6.1 Conclusions For samples A to H, the contents of total alkaloids determined by CRM35 and NCM with water extract are similar. The correlation coefficient of the two methods is , the root mean square error (RMSE) is % and the RPD is , showing good agreement between CRM35 and NCM in extraction with water. For sample A to H, the contents of total alkaloids determined by CRM35 and NCM with HAC extract are similar. The correlation coefficient of the two methods is , the root mean square error (RMSE) is %, and the RPD is , showing good agreement between CRM35 and NCM in extraction with HAC. It is recommended that 5% HAC extracts should be used if total alkaloids (as nicotine) and reducing substances or reducing carbohydrates are to be carried out simultaneously. NCM method showed comparable r and R values to those published in CRM35 for both water or HAC extraction in this 2014 collaborative study. For NCM, the results showed good consisitent with water and HAC extractions. The results of CRM35 in 2014 international collaborative study showed comparable r and R data to those obtained by the CORESTA RAC reproducibility and repeatability 1993 and 2006 international collaborative studies. 6.2 Recommendations Firstly, the RAC recommends that CRM35 be made obsolete and replaced with a new CRM reflecting the safer chemistry with new flow diagrams for both macro and micro flow instruments. Secondly, once the new CRM is published that it be submitted to ISO TC126. RAC CTR 2014 Safer Chemistry Collaborative Study March /45

18 Appendix A Experimental Protocol Protocol of collaborative test for total alkaloids in tobacco 1. Objective To assess the repeatability and reproducibility of laboratories applying the same analytical method. Besides, to assess the distinction between the Chinese KSCN/DCIC method and the CRM35.The moisture content shall be determined, in order to be able to provide results on a dry weight basis as well as an as-received basis. The proposed method was developed and validated for tobacco, conventional tobacco products. 2. Test coordinator Zhang Wei Senior Engineer China National Tobacco Quality Supervision & Test Centre No. 2 Fengyang Road, Zhengzhou High and New Technology Industries Development Zone, Henan, China, zhangw_jh@163.com Tel: Fax: Test methods Each laboratory shall apply both the provided Chinese KSCN/DCIC and the CRM35 methods to test the total alkaloids in the samples with water and 5% acetic acid extractions. The determination of moisture should be performed according to each participants own method. If possible, a CORESTA or ISO recommended method should be used. 4. Test samples Test samples consist of tobacco products listed in Table 1. The CM7 and 3R4F cigarettes (samples G and H) should be taken from the participants own stock. Tobacco samples A through F have been provided by Zhang Wei, China National Tobacco Quality Supervision & Test Centre. Table 1. Test products for the collaborative study Code A B C D E F Test product Fire cured tobacco Burley tobacco Oriental tobacco Dark sun-cured tobacco Fire-cured cigarette Blended cigarettes G CM 7 H 3R4F RAC CTR 2014 Safer Chemistry Collaborative Study March /45

19 5. Schedule For each sample, 6 replicates should be measured within a single test day. The standard substance should be dissolved by the same solution as the extraction one. The study starts in August The test results should be sent to the test coordinator no later than August 25st, Reporting of test results The test results should be reported using the Excel file CORESTA Total Alkaloids Reporting Sheets. The reporting units are specified in CORESTA Total Alkaloids Reporting Sheets as well as in the Excel template. Please provide the results on an as received and a dry weight basis, after correction for the moisture content. In the General Information worksheet, please provide a brief description of the analytical instruments and parameters you use, as well as any minor modifications to the method. Also, please provide information about the source of your standard materials on the spreadsheet provided. Results should be sent electronically: To: zhangw_jh@163.com Cc: linda.crumpler@cerulean.com RAC CTR 2014 Safer Chemistry Collaborative Study March /45

20 Appendix B Chinese KSCN/DCIC Method of Collaborative Test for Total Alkaloids in Tobacco Unless otherwise specified, all chemicals should be of Analytical grade or equivalent. LIST OF RAW MATERIALS(for KSCN/DCIC Chemistry) Safety classifications: Brij-35*, 30 % solution Sodium phosphate dibasic, Na 2 HPO 4 12H 2 O Sodium phosphate monobasic, NaH 2 PO 4 2H 2 O Sodium citrate, C 6 H 5 Na 3 O 7 2H 2 O Sulphanilic acid, NH 2 C 6 H 4 SO 3 H Potassium thiocyanate, KSCN, harmful Sodium dichloroisocyanurate dihydrate, C3Cl2N3NaO3, harmful Sodium carbonate, Na2CO3, irritant Ferrous sulfate, FeSO4.7H2O, harmful Citric acid, C6H8O7.H2O Nicotine (>98%), highly toxic REAGENTS MAKE UP DI Water refers to high quality reagent water. POTASSIUM THIOCYANATE SOLUTION Dissolve 2.88 g of potassium thiocyanate in DI water. Dilute to 250 ml with DI water and mix well. DCIC WORKING SOLUTION DCIC2.20 g DI water to 250 ml Dissolve 2.20 g of DCIC and dilute to 250 ml with DI water. NEUTRALISATION SOLUTION A Citric acid 1 g Ferrous sulfate 10 g DI water to 1000 ml Dissolve 1 g of citric acid and 10 g of ferrous sulfate in about 500 ml of DI water. Dilute to 1000 ml with DI water and mix well. RAC CTR 2014 Safer Chemistry Collaborative Study March /45

21 NEUTRALISATION SOLUTION B Sodium carbonate 10 g DI Water to 1000 ml Dissolve 10 g of sodium carbonate in about 500 ml of DI water. Dilute to 1000 ml with DI water and mix well. BUFFER SOLUTION A Dissolve 71.6 g of sodium phosphate dibasic and g of sodium citrate in about 500 ml of DI water. Dilute to 1000 ml with DI water, add 1 ml of Brij, 30% solution and mix well. BUFFER SOLUTION B Dissolve 71.6 g of sodium phosphate dibasic, 6.2 g of sodium phosphate monobasic,11.76 g of sodium citrate and7.0 g of sulphanilic acid in about 800 ml of DI water. Dilute to 1000 ml with DI water, add 1 ml of DI water and mix well. Figure 1: Proposed flowchart for macro flow systems RAC CTR 2014 Safer Chemistry Collaborative Study March /45

22 Figure 2: Proposed flowchart for micro flow systems RAC CTR 2014 Safer Chemistry Collaborative Study March /45

23 Appendix C Raw Data by Laboratory Lab 1 Samples CRM35 in water NCM in water CRM35 HAC NCM in HAC A B C D E F G H Lab 2 Samples CRM35 in water NCM in water CRM35 HAC NCM in HAC A B C D E F G H Lab 3 Samples CRM35 in water NCM in water CRM35 HAC NCM in HAC A B C D E F G H RAC CTR 2014 Safer Chemistry Collaborative Study March /45

24 Lab 4 CRM35 in water NCM in water CRM35 HAC NCM in HAC Samples A B C D E F G H Lab 5 CRM35 in water NCM in water CRM35 HAC NCM in HAC Samples A B C D E F G H Lab 6 CRM35 in water NCM in water CRM35 HAC NCM in HAC Samples A B C D E F G H RAC CTR 2014 Safer Chemistry Collaborative Study March /45

25 Lab 7 CRM35 in water NCM in water CRM35 HAC NCM in HAC Samples A B C D E F G H Lab 8 Samples CRM35 in water NCM in water CRM35 HAC NCM in HAC A B C D E F G H Lab 9 Samples CRM35 in water NCM in water CRM35 HAC NCM in HAC A B C D E F G H RAC CTR 2014 Safer Chemistry Collaborative Study March /45

26 Lab 10 Samples CRM35 in water NCM in water CRM35 HAC NCM in HAC A B C D E F G H Lab 11 Samples CRM35 in water NCM in water CRM35 HAC NCM in HAC A B C D E F G H Lab 12 Samples CRM35 in water NCM in water CRM35 HAC NCM in HAC A B C D E F G H RAC CTR 2014 Safer Chemistry Collaborative Study March /45

27 Lab 13 Samples CRM35 in water NCM in water CRM35 HAC NCM in HAC A B C D E F G H Lab 14 CRM35 in water NCM in water CRM35 HAC NCM in HAC Samples A B C D E F G H Lab 15 CRM35 in water NCM in water CRM35 HAC NCM in HAC Samples A B C D E F G H RAC CTR 2014 Safer Chemistry Collaborative Study March /45

28 Lab 16 CRM35 in water NCM in water CRM35 HAC NCM in HAC Samples A B C D E F G H Lab 17 CRM35 in water NCM in water CRM35 HAC NCM in HAC Samples A B C D E F G H Lab 18 Samples CRM35 in water NCM in water CRM35 HAC NCM in HAC A B C D E F G H RAC CTR 2014 Safer Chemistry Collaborative Study March /45

29 Lab 19 Samples CRM35 in water NCM in water CRM35 HAC NCM in HAC A B C D E F G H RAC CTR 2014 Safer Chemistry Collaborative Study March /45

30 Appendix D Raw Mean and Standard Deviation Value Table D-1: Sample A - raw mean and standard deviation value of total alkaloids (as nicotine determined by CRM35 and NCM (KSCN/DCIC) in water and 5 % HAC, respectively Lab ID CRM35 in water NCM in water CRM35 in HAC NCM in HAC Mean SD Mean SD Mean SD Mean SD RAC CTR 2014 Safer Chemistry Collaborative Study March /45

31 Table D-2: Sample B - raw mean and standard deviation value of total alkaloids (as nicotine) in sample B determined by CRM35 and NCM (KSCN/DCIC) in water and 5% HAC, respectively Lab ID CRM35 in water NCM in water CRM35 in HAC NCM in HAC Mean SD Mean SD Mean SD Mean SD RAC CTR 2014 Safer Chemistry Collaborative Study March /45

32 Table D-3: Sample C - raw mean and standard deviation value of total alkaloids (as nicotine) in determined by CRM35 and NCM (KSCN/DCIC) in water and 5% HAC, respectively Lab ID CRM35 in water NCM in water CRM35 in HAC NCM in HAC Mean SD Mean SD Mean SD Mean SD RAC CTR 2014 Safer Chemistry Collaborative Study March /45

33 Table D-4: Sample D - raw mean and standard deviation value of total alkaloids (as nicotine) determined by CRM35 and NCM (KSCN/DCIC) in water and 5% HAC, respectively Lab ID CRM35 in water NCM in water CRM35 in HAC NCM in HAC Mean SD Mean SD Mean SD Mean SD RAC CTR 2014 Safer Chemistry Collaborative Study March /45

34 Table D-5: Sample E - raw mean and standard deviation value of total alkaloids (as nicotine) in sample E determined by CRM35 and NCM (KSCN/DCIC) in water and 5% HAC, respectively Lab ID CRM35 in water NCM in water CRM35 in HAC NCM in HAC Mean SD Mean SD Mean SD Mean SD RAC CTR 2014 Safer Chemistry Collaborative Study March /45

35 Table D-6: Sample F - raw mean and standard deviation value of total alkaloids (as nicotine) in sample F determined by CRM35 and NCM (KSCN/DCIC) in water and 5% HAC, respectively Lab ID CRM35 in water NCM in water CRM35 in HAC NCM in HAC Mean SD Mean SD Mean SD Mean SD RAC CTR 2014 Safer Chemistry Collaborative Study March /45

36 Table D-7: Sample G - raw mean and standard deviation value of total alkaloids (as nicotine) in sample G determined by CRM35 and NCM (KSCN/DCIC) in water and 5% HAC, respectively Lab ID CRM35 in water NCM in water CRM35 in HAC NCM in HAC Mean SD Mean SD Mean SD Mean SD RAC CTR 2014 Safer Chemistry Collaborative Study March /45

37 Table D-8: Sample H - raw mean and standard deviation value of total alkaloids (as nicotine) determined by CRM35 and NCM (KSCN/DCIC) in water and 5 % HAC, respectively Lab ID CRM35 in water NCM in water CRM35 in HAC NCM in HAC Mean SD Mean SD Mean SD Mean SD RAC CTR 2014 Safer Chemistry Collaborative Study March /45

38 Total Alkaloids (%) Total Alkaloids (%) Appendix E Data Representation Oriental tobacco CRM 35 in w ater NCM in w ater Lab value Lab mean Lab ID Dark sun-cured tobacco CRM 35 in w ater NCM in w ater Lab value Lab mean Lab ID RAC CTR 2014 Safer Chemistry Collaborative Study March /45

39 Total Alkaloids (%) Total Alkaloids (%) Fire-cured cigarette CRM 35 in w ater NCM in w ater Lab value Lab mean Lab ID Blended cigarette CRM 35 in w ater NCM in w ater Lab value Lab mean Lab ID RAC CTR 2014 Safer Chemistry Collaborative Study March /45

40 Total Alkaloids (%) Total Alkaloids (%) CM CRM 35 in w ater NCM in w ater Lab value Lab mean Lab ID 3R4F 2.4 CRM 35 in w ater NCM in w ater Lab value Lab mean Lab ID RAC CTR 2014 Safer Chemistry Collaborative Study March /45

41 Total Alkaloids (%) Total Alkaloids (%) Flue-cured tobacco CRM 35 in HAC NCM in HAC Lab value Lab mean Lab ID Burley tobacco 5.5 CRM35 in HAC NCM in HAC Lab value Lab mean Lab ID RAC CTR 2014 Safer Chemistry Collaborative Study March /45

42 Total Alkaloids (%) Total Alkaloids (%) Oriental tobacco 1.3 CRM 35 in HAC NCM in HAC Lab value Lab mean Lab ID Dark sun-cured tobacco 4.2 CRM 35 in HAC NCM in HAC Lab value Lab mean Lab ID RAC CTR 2014 Safer Chemistry Collaborative Study March /45

43 Total Alkaloids (%) Total Alkaloids (%) Fire-cured cigarette CRM 35 in HAC NCM in HAC Lab value Lab mean Lab ID Blended cigarette CRM 35 in HAC NCM in HAC Lab value Lab mean Lab ID RAC CTR 2014 Safer Chemistry Collaborative Study March /45

44 Total Alkaloids (%) Total Alkaloids (%) CM CRM 35 in HAC NCM in HAC Lab value Lab mean Lab ID 3R4F 2.4 CRM 35 in HAC NCM in HAC Lab value Lab mean Lab ID RAC CTR 2014 Safer Chemistry Collaborative Study March /45

45 Appendix F IUPAC 1994 Harmonized Statistical Procedure IUPAC 1994 HARMONIZED STATISTICAL PROCEDURE SCREEN OUT NON VALID DATA START OF LOOP CALCULATE PRECISION MEASURES (C) COCHRAN OUTLYING LAB? YES (Gi) SINGLE GRUBBS OUTLIER ON INDIVIDUAL VALUES OF COCHRAN OUTLYING LAB? YES DROP INDIVIDUAL VALUE NO NO DROP LAB UNLESS OVERALL FRACTION OF LABS DROPPED WOULD EXCEED 2/9 (G) SINGLE GRUBBS OUTLIER? YES DROP LAB UNLESS OVERALL FRACTION OF LABS DROPPED WOULD EXCEED 2/9 NO (G2) PAIRED GRUBBS OUTLIER? NO YES DROP LAB UNLESS OVERALL FRACTION OF LABS DROPPED WOULD EXCEED 2/9 YES ANY LABS DROPPED IN THIS LOOP NO END REPORT ORIGINAL & LAST COMPUTED PRECISION MEASURES RAC CTR 2014 Safer Chemistry Collaborative Study March /45