Technical Report. E-Liquid Preliminary Proficiency Study

Transcription

1 E-Cigarette Task Force Technical Report E-Liquid Preliminary Proficiency Study March 2015 Author: Rana Tayyarah Lorillard Tobacco Company Task Force Coordinator: Chuck Garner, Ph.D. R. J. Reynolds Tobacco Company Task Force Secretary: Robert D. Stevens, Ph.D. Lorillard Tobacco Company

2 Table of Contents 1. Summary 3 2. Introduction 3 3. Organisation Participants Protocol Data - Raw 6 4. Data - Statistical Analysis Exclusion of Outliers Calculation of Repeatability and Reproducibility 7 5. Data Interpretation Methods comparison Results for Control Sample Sample Homogeneity Results for Products Nicotine Results for Products Glycerin Results for Products Propylene Glycol Results for Products Water Recommendations Acknowledgements 16 APPENDIX A: Study Protocol and Data Template 17 APPENDIX B: Laboratory Method Summaries 25 APPENDIX C: Raw Data Means 52 APPENDIX D: Nicotine - Raw Data Mean Plots 58 APPENDIX E: Glycerin - Raw Data Mean Plots 61 APPENDIX F: Propylene Glycol - Raw Data Mean Plots 64 APPENDIX G: Water - Raw Data Mean Plots 67 APPENDIX H: Nicotine - Mandel s h and Mandel s k Graphs 69 APPENDIX I: Glycerin - Mandel s h and Mandel s k Graphs 74 APPENDIX J: Propylene Glycol - Mandel s h and Mandel s k Graphs 79 APPENDIX K: Water - Mandel s h and Mandel s k Graphs 84

3 1. Summary In 2014, the CORESTA E-Cigarette Task Force conducted a study that included testing of commercial electronic cigarette e-liquid products. This study was referred to as Electronic Cigarette e-liquid Preliminary Proficiency Study. The laboratory phase of this study was conducted from late 2013 to early 2014 and results were presented to the Task Force in May Nine laboratories participated in the study. Each laboratory tested the same set of 10 commercial e-liquids and 1 study control. The goal of this work was to compare results from multiple laboratories applying their own internal analytical methods for the determination of nicotine, propylene glycol (PG), glycerin and water in e-liquid samples. The study results demonstrate that even without a common method, results were broadly consistent, of low variability, and highly accurate among the participating laboratories. Percent error for nicotine was <5% for all labs. Error for glycerin and PG was generally less than 10% for most labs. For water analysis, Karl Fisher results showed higher accuracy (4% error) compared to GC-TCD results (13% error). There was slight, but non-random trending with data from some labs. Results indicate that establishment of a CORESTA recommended method may be warranted. 2. Introduction One of the short term objectives of the e-cigarette Task Force is to gather and share preliminary data on analysis relevant to e-cigarettes worldwide with a view to making recommendations for product testing. As yet, there are no standardized methods for the characterization of e-liquids. Thus, the Task Force commissioned a study to evaluate the performance of existing methods currently in use by the participating laboratories. Commercial e-liquid products were tested. The analytes and nominal value ranges are listed below: Nicotine: 0 1.6% Glycerin: % PG: 0-100% Water: <~10% A study control was included, as well. The control was a mixture of glycerin (45%), propylene glycol (45%), water (9%), and nicotine (1%) and was prepared by one laboratory using analytical grade chemicals. ECIG CTR E-Liquid Preliminary Proficiency Study March /86

4 3. Organisation 3.1 Participants A list of the participating laboratories is provided in Table 1. The laboratories are listed in alphabetical order. The numerical laboratory codes (1-10) used in this report do not correspond to the same order of the laboratories listed below. Note that one laboratory tested two sets of e-liquid products and the results were coded as 2a and 2b. There is no laboratory 4. All commercial e-liquid products were supplied by Red Kiwi and NicVape. Table 1: List of Participating Laboratories Altria Client Services Arista Laboratories Enthalpy Analytical, Inc. Laboratory Global Laboratory Services, Inc. KT&G R&D Headquarter Labstat International ULC Lorillard Tobacco Co Microbac Laboratories, Inc; Wilson Division RJ Reynolds Tobacco Co. ECIG CTR E-Liquid Preliminary Proficiency Study March /86

5 3.2 Protocol e-liquid products were collected from the manufacturers, labelled with sample numbers, and distributed by Enthalpy. The control samples were made at Lorillard using analytical grade chemicals purchased from Sigma-Aldrich and a 5-place analytical balance. Individual control samples were prepared by weighing the analytes directly into tared sample vials. The control samples were distributed from Enthalpy with the product samples. The commercial samples were distributed in clear 10mL screw-cap containers. Two bottles of each sample were provided in the event a repeat analysis would be required. A single 20mL container of the control sample, Sample 11, was provided. Analytes included in the study were nicotine, glycerin, propylene glycol, and water. The laboratories were instructed to store the e-liquid samples at room temperature for at least 2 hours before sampling. Each laboratory conducted three replicate determinations for each sample using their methods of choice. Sample codes used in the study and the nominal content of the analytes of interested, as provided by the supplier, are listed in Table 2. See Appendix A for the study protocol. See Table 3 for an overview of the study methods. See Appendix B for a summary of each laboratory s methods. Table 2: Sample Identification and Nominal Analyte Content Sample Code Nicotine mg/ml / %w/w* Glycerin PG Water Sample 1 18 mg/ml / 1.57% 40% 50% 5 10% Sample 2 18 / % 50% 5 10% Sample 3 0 / 0 40% 50% 5 10% Sample 4 9 / % 50% 5 10% Sample 5 9 / % 50% 5 10% Sample 6 6 / Sample 7 6 / Sample 8 6 / Sample 9 6 / % 0% -- Sample 10 6 / % 100% -- Sample 11 (Control) -- / % (w/w) 45% (w/w) 9% (w/w) *The nominal values for nicotine for Samples 1 10 were reported in mg/ml. They were converted to % w/w based on Lab 6 s density measurements. -- = Information not provided ECIG CTR E-Liquid Preliminary Proficiency Study March /86

6 Table 3: Analytical Methods Overview General Information across Methods Sample Prep Weigh mg * Dilute with alcohol ( ml) Mix/Shake (10s 3 hours) Weight and volume used varied by analyte Analysis GC-TCD or Karl Fischer for water Packed column if TCD (most) GC-FID for other analytes Capillary column DB-Wax columns for most 4 12 calibration standards (GC) Linear (IS) calibration r 2 >0.99 *Lab 6 measured by volume ( µl) and diluted with 1mL solvent 3.3 Data - Raw The summary descriptive statistics data are listed in Appendix C where each mean represents three replicates. Raw data plots are given in Appendices D-G. Data graphs in Appendices D- G display individual values (gray dots) with mean lines (green lines). To the right of the graph are results from one way analysis of variance with paired comparisons. 4. Data - Statistical Analysis 4.1 Exclusion of Outliers Graphical outlier detection was performed following the recommendations of ISO (1994). Mandel s h is used to assess the inter-laboratory consistency and Mandel s k is used to assess the intra-laboratory consistency. The Mandel s h plots and the Mandel s k plots (0.99 limits) are given in Appendices H-K. Numerical outlier detection was conducted following the recommendations of ISO (1994). The decision on straggling (0.95) and outlying (0.99) data were made using single outlier and Cochran s tests. The results for the Grubbs test to detect outlying means and for the Cochran s test to detect outlying variances are displayed in Table 4. The relatively low variability among, and between, data sets is a likely source of some level of non-practical or false determination of outliers. Note that none of the data were excluded from overall mean calculations. The information is merely provided for the convenience of the participating laboratories. ECIG CTR E-Liquid Preliminary Proficiency Study March /86

7 Table 4. Results for Outlying Means and Variances Nicotine Glycerin Product Cochran's Grubbs' Product Cochran's Grubbs' Sample 1 -- Lab 10 Sample 1 -- Lab 6 Sample 2 -- Lab 2a, 5, 10 Sample 2 -- Lab 1, Lab 7 Sample 3 NA NA Sample 3 Lab 2b Lab 2a Sample Sample Sample 5 -- Lab 5, 10 Sample 5 -- Lab 2b Sample 6 Lab 1 -- Sample Sample Sample Sample 8 Lab 5 Lab 10 Sample Sample 9 -- Lab 5, 10 Sample Sample 10 Lab 2a -- Sample 10 NA NA PG Water Product Cochran's Grubbs' Product Cochran's Grubbs' Sample 1 -- Lab 2a Sample Sample 2 -- Lab 1, 2a Sample 2 -- Lab 7 Sample 3 -- Lab 1 Sample 3 -- Lab 7 Sample Sample 4 Lab 5 Lab 7 Sample 5 -- Lab 8 Sample Sample 6 Lab 5 -- Sample 6 NA NA Sample Sample 7 NA NA Sample 8 -- Lab 2b Sample 8 NA NA Sample 9 NA NA Sample 9 NA NA Sample 10 Lab 5 -- Sample 10 NA NA alpha = 0.01 NA not applicable; the test was not performed -- - no outliers were noted 4.2 Calculation of Repeatability and Reproducibility Each laboratory tested in-common samples using non-standardized methodology. repeatability (r) and reproducibility (R) results were not calculated for this data set. Thus, 5. Data Interpretation 5.1 Methods comparison Although laboratories were not provided standardized methods for use with the study, most aspects of the methodologies used were very similar. All laboratories used an alcohol solvent for dilution, for example. Differences included shake time, which ranged from hand inversion to 180 min of mechanical shaking. Most laboratories used GC-TCD for water analysis, while 3 labs used Karl Fischer. ECIG CTR E-Liquid Preliminary Proficiency Study March /86

8 5.2 Results for Control Sample Given that methodology are not standardized and that there are no reference or monitor e- liquid products available for inclusion in the study, a study control was made and treated in the same manner as the samples. The control sample set was made by using analytical grade chemicals purchased and left unopened prior to preparing the samples. For the highest level of accuracy for the true value of the control, individual samples were made and coded rather than making a larger volume sample and taking aliquots. In this manner, the analytes were weighed directly into a tared vial using a five-place analytical balance. Samples were sealed and stored in the dark prior to shipment for distribution. The target values (%w/w) were: 45%, 45%, 9%, and 1%, for glycerin, propylene glycol, water, and nicotine, respectively. The percent error from the actual value for each laboratories control sample was calculated and was determined to be relatively low for all labs and all analytes. For example, the percent error for nicotine ranged from 0.2% to -4.5%. Precision was high for each laboratory with percent relative standard deviations at <5%. The error was highest for water (<18%error) and the data indicate that the use of Karl Fischer may allow for improved accuracy. The average error for GC-TCD determinations was 13% error while the average for Karl Fischer was 4% error. The mean results for water determinations for the control are displayed in Figure 1 in a bar graph. The bars are colorcoded based on method (Karl Fischer or GC-TCD) to highlight the greater accuracy (i.e. purple bars are closer to blue bars) and greater a precision (i.e. error lines are smaller) for the Karl Fischer determinations. The percent error calculations are shown in Table 5 below. The average error for each analyte is shown as is and as an absolute value (distance from zero without regard for whether the value errs high or low). Mean values are displayed in Appendix C. ECIG CTR E-Liquid Preliminary Proficiency Study March /86

9 Content (w/w %) Table 5. Percent Error for the Control Sample Nicotine Glycerin PG Water Lab 1 2.9% -2.7% 0.2% -15% Lab 2a 0.3% -1.6% -0.7% 4.5%* Lab 2b -2.1% -3.0% -2.4% 3.4%* Lab 3-1.3% 2.3% 0.7% 2.1%* Lab 5-0.5% 5.2% -11% 7.9% Lab 6-0.6% -11.9% -10% -16% Lab 7-0.2% -1.6% -2.4% -10% Lab 8-1.4% -4.6% -5.9% -8.6% Lab 9 1.9% 7.4% 0.9% 18% Lab % -13% -7.5% 2.4%* Average -0.6% -2% -4% -1% Absolute Avg 2% 5% 4% 9% Absolute Max 5% 13% 11% 18% Calculated as %Error = (Measured - Actual) / Actual * 100 *Karl Fischer was used for water analysis; all other labs used GC-TCD 12.00% 11.00% 10.00% 9.00% 8.00% 7.00% 6.00% 5.00% Lab 2a Lab 2b Lab 3 Lab 10 Lab 1 Lab 5 Lab 6 Lab 7 Lab 8 Lab 9 Actual Value Reported - GC/TCD Reported - Karl Fischer Figure 1. Comparison of Actual and Measured Content Values for Water in the Study Control Samples (Sample 11) ECIG CTR E-Liquid Preliminary Proficiency Study March /86

10 5.3 Sample Homogeneity Enthalpy distributed two sample bottles for each product. Laboratories were instructed to test one bottle and retain on bottle as a spare in case repeat analysis was needed. One laboratory, Lab 2, tested both bottles. This was done pre-emptively in case the variability between the labs results was high. If the values between two bottles tested by the same lab were high then the variability would trace back to the samples rather than the labs. The results between Sets 2a and 2b were very consistent; the average percent difference between the matched pairs of samples was 1.6% difference. As is discussed below, there was a high level of consistency of results between all the laboratories, as well. This indicates that the product samples tested were homogenous. 5.4 Results for Products Nicotine The differences between the nominal value and each lab s values were minimal (<10% overall). As with the control samples, these results indicate a consistency among the methods and laboratories. The nominal value compared to the overall average of all the labs data are shown below in Table 6. All results are tabulated in Appendix C and displayed graphically in Appendix D for each sample. Summary graphs are shown below in Figures 2 and 3. Table 6. Overall Nicotine Results compared to the Nominal Values Sample Nominal Value (w/w) Avg of all Labs (w/w)* Sample 1 1.6% 1.48 ± 0.07 Sample 2 1.6% 1.57 ± 0.07 Sample 3 0.0% NR Sample % 0.73 ± 0.03 Sample % 0.72 ± 0.03 Sample % 0.55 ± 0.02 Sample % 0.53 ± 0.01 Sample % 0.54 ± 0.03 Sample % 0.46 ± 0.01 Sample % 0.58 ± 0.02 Sample 11 (Control ) 1.0% 1.03 ± 0.03 *Displayed as mean ± 1 standard deviation NR=Not reportable on average (less than quantification limits) ECIG CTR E-Liquid Preliminary Proficiency Study March /86

11 Content (w/w %) Content (w/w %) 2.0% 1.8% 1.6% 1.4% 1.2% 1.0% 0.8% 0.6% 0.4% 0.2% 0.0% Lab 1 Lab 2a Lab 2b Lab 3 Lab 5 Lab 6 Lab 7 Lab 8 Lab 9 Lab 10 Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Figure 2. Nicotine Content Results for Samples % 0.6% 0.5% 0.4% 0.3% 0.2% Sample 6 Sample 7 Sample 8 Sample 9 Sample % 0.0% Lab 1 Lab 2a Lab 2b Lab 3 Lab 5 Lab 6 Lab 7 Lab 8 Lab 9 Lab 10 Figure 3. Nicotine Content Results for Samples 6-10 ECIG CTR E-Liquid Preliminary Proficiency Study March /86

12 Content (w/w %) 5.5 Results for Products Glycerin A higher level of difference (<20% difference) was noted between laboratories for glycerin. Overall results compared to nominal values are shown in Table 7. Results are displayed in Appendix C and Appendix E. The trending about the nominal values for Samples 1-5 is consistent with the relative results for the control samples. For example, Lab 10 glycerin results were approximately 10% lower than the actual value for the control and are approximately 10% lower than nominal for Samples 1-5. Lab 9 glycerin results for the control were slightly higher than the actual value and the results for Samples 1-5 are about 10% higher than nominal. These results are displayed below in Figure 4. Table 7. Overall Glycerin Results compared to the Nominal Values Sample Nominal Value (w/w) Avg of all Labs (w/w)* Sample 1 40% 42 ± 3 Sample 2 40% 42 ± 3 Sample 3 40% 41 ± 3 Sample 4 40% 40 ± 3 Sample 5 40% 41 ± 3 Sample ± 3 Sample ± 3 Sample ± 4 Sample 9 100% 98 ± 4 Sample 10 0% NR Sample 11 (Control) 45% 44 ± 3 *Displayed as mean ± 1 standard deviation NR=Not reportable on average (less than quantification limits) = information not provided and/or calculated 50.0% 45.0% 40.0% 35.0% 30.0% 25.0% 20.0% 15.0% 10.0% Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 5.0% 0.0% Lab 1 Lab 2a Lab 2b Lab 3 Lab 5 Lab 6 Lab 7 Lab 8 Lab 9 Lab 10 Figure 4. Glycerin Content Results for Samples 1-5. The line at 40% marks the nominal value for the samples. ECIG CTR E-Liquid Preliminary Proficiency Study March /86

13 For Samples 6-8, a nominal value was not provided but all laboratories reported approximately 40% (w/w). For example, the average of all labs was (38 ± 3)% for Sample 6. Sample 9 was approximately 100% and Sample 10 was a no glycerin product. Laboratory results were consistent with each other for Samples 6-10; see Figure 5 below. Figure 5. Glycerin Content Results for Samples Results for Products Propylene Glycol Most samples tested contained 40-60% propylene glycol (PG). There was one 0% and one ~100% sample. All laboratories results are similar for all samples; the overall standard deviation is approximately 2 in most cases as is shown in Table 8 below. All results are shown in Appendix C and Appendix F. Table 8: Overall Propylene Glycol Results compared to the Nominal Values Sample Nominal Value (w/w) Avg of all Labs (w/w)* Sample 1 50% 45 ± 2 Sample 2 50% 45 ± 2 Sample 3 50% 46 ± 2 Sample 4 50% 46 ± 2 Sample 5 50% 46 ± 2 Sample ± 2 Sample ± 2 Sample ± 2 Sample 9 0% NR Sample % 97 ± 4 Sample 11 (Control) *Displayed as mean ± 1 standard deviation NR=Not reportable on average (less than quantification limits) --= Information not provided and/or calculated 45% 43 ± 2 ECIG CTR E-Liquid Preliminary Proficiency Study March /86

14 Content (w/w %) Content (w/w %) Results grouped by the laboratory are displayed in Figures 6 and 7 below. For samples 1-5, all results are below the nominal value provided. As with glycerin, Lab 6 results appear to trend slightly (5-9%) below the overall mean for the analyte. Though, only slightly lower, the trend is consistent with Lab 6 s error for the control sample (-10%). 50.0% 48.0% 46.0% 44.0% 42.0% 40.0% Sample 1 Sample 2 Sample 3 Sample 4 Sample % 36.0% Lab 1 Lab 2a Lab 2b Lab 3 Lab 5 Lab 6 Lab 7 Lab 8 Lab 9 Lab 10 Figure 6. Propylene Glycol Content Results for Samples 1-5. The line at 50% marks the nominal value for the samples % 100.0% 80.0% Sample 6 Sample 7 Sample 8 Sample 9 Sample % 40.0% 20.0% 0.0% Lab 1 Lab 2a Lab 2b Lab 3 Lab 5 Lab 6 Lab 7 Lab 8 Lab 9 Lab 10 Figure 7. Propylene Glycol Content Results for Samples ECIG CTR E-Liquid Preliminary Proficiency Study March /86

15 Content (w/w %) 5.7 Results for Products Water Water results were the least consistent between the laboratories. Whereas, the other analytes had an overall relative standard deviation for pooled data of 3-10 %RSD, the overall %RSD for water was 8-14 %RSD. As with the control sample, some bias in results is apparent based on method choice. Karl Fischer results are more consistent. The level of variability seen in the GC-TCD results may not be sufficient to warrant a change in practice for a given laboratory. Results for samples 1-5 are displayed, grouped by lab and method type, in Figure 8 below. Results are shown in Appendix C and Appendix G. Table 9: Overall Water Results compared to the Nominal Values Sample Nominal Value (w/w) Avg of all Labs (w/w)* Sample % 9.0 ± 0.8 Sample % 8.5 ± 0.9 Sample % 10.2 ± 1.0 Sample % 9.4 ± 1.0 Sample % 9.9 ± 1.4 Sample 11 (Control) 9% 9.0 ± 1.1 *Displayed as mean ± 1 standard deviation NR=Not reportable on average (less than quantification limits) --= Information not provided and/or calculated 14.0% 12.0% 10.0% 8.0% 6.0% 4.0% Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 2.0% 0.0% Lab 2aLab 2b Lab 3 Lab 10 Lab 1 Lab 5 Lab 6 Lab 7 Lab 8 Lab 9 Figure 8. Water Content Results for Samples 1-5. ECIG CTR E-Liquid Preliminary Proficiency Study March /86

16 6. Recommendations The laboratory results were broadly comparable for all analytes. For example, for the control sample, the percent error for nicotine was less than 5% error and averaged 2% for all labs. All laboratories methods appear to be adequate across the concentration range tested for each analyte though there were some instances of non-random trending. For example, Labs 6 and 10 were slightly (<10% from the overall average), but consistently, lower than the other laboratories for glycerin and PG. Also, the laboratories using Karl Fisher for water determination had a slightly higher accuracy for water (4% error) than the laboratories using GC-TCD (13% error). Across the labs, sample preparation techniques were similar with variation in details such as mixing time. For example, all labs used an alcohol solvent. But mixing ranged from 10s mixing to 3 hours of shaking. Pursuit of a formal CORESTA recommended method may be warranted. Additionally, it is recommended that reporting units of per volume (mg/ml) and per weight (mg/mg) be used. This would be achievable by inclusion of weighing a certain volume as part of the sample preparation procedure. 7. Acknowledgements The study team, Drs. Rob Stevens, Narendra Meruva, and Gene Gillman; the participating laboratories; and the product suppliers, Red Kiwi and NicVape, are greatly appreciated. ECIG CTR E-Liquid Preliminary Proficiency Study March /86

17 APPENDIX A: Study Protocol and Data Template CORESTA E-CIGARETTE ANALYTICAL TESTING SUB TEAM (Team Leader Dr. Rob Stevens, Lorillard Tobacco) Project Title: 2013 e-liquid Analysis Protocol Date: Nov 11, 2013 Written by: Naren Meruva, Ph.D. (Altria Client Servies) Phone: Confidentiality Notice: All data generated in the course of this analysis should be handled in the strictest of confidence by all participating laboratories and other CSTS members. ECIG CTR E-Liquid Preliminary Proficiency Study March /86

18 Protocol for a Preliminary Proficiency Study on Determination of Nicotine, Propylene Glycol, Glycerin and Water in e-liquids 1. Objective The key objective of this preliminary proficiency study is to assess the repeatability and reproducibility of participating laboratories applying their own internal analytical methods for determination of nicotine, propylene glycol (PG), glycerin and water in e-liquid samples. Data for these analytes from the participating laboratories will be collected for statistical evaluation purposes. Additionally, data on weight of the aliquot of e-liquid and the volume of extraction solvent used will be collected for individual measurements. 2. Analytes Following analytes are to be measured in e-liquid samples: a. Nicotine b. Propylene Glycol (PG) c. Glycerin d. Water Reporting units mg/mg (as-is) or mg/ml (as-is). Additionally, report the weight (in mg) or volume (in ml) of the aliquots taken from each sample for testing. If aliquots are measured by volume, sample density is to be reported. 3. Test Methods Each laboratory shall apply their own internal analytical methods for the determination of major components (nicotine, PG, glycerin and water) in e-liquid samples. Laboratories are asked to record sample preparation steps and must provide information on analytical method and data collected in the templates distributed with the study protocol. 4. Test Samples Test samples consist of eleven (11) different e-liquids with varying percentage of nicotine (< 2.5%), propylene glycol, glycerin, water and flavor content. The test samples are coded 1 through 11. Two bottles of each sample 1-10 (10 ml in each bottle) and one bottle of Sample 11 (~20mL) will be distributed by Dr. Gene Gillman (Enthalpy Analytical, Inc.) during the first week of December ECIG CTR E-Liquid Preliminary Proficiency Study March /86

19 5. Sample Handling The e-liquid samples need to be stored at room temperature for at least 2 hours before use. Any additional sample storage information needs to be provided in the templates provided with the study protocol. 6. Experimental plan Three (n=3) replicate analysis for each sample are required. If possible, it is requested that all samples be analyzed on a single day. If multiple analysis days are needed it is requested that all three replicates of each sample be analyzed on a single day. 7. Data Reporting The attached templates for e-liquid analysis should be used for data submission. Please provide data in the requested format without creating new cells or rows in the spreadsheet. Results should be reported back to Naren Meruva (narendra.k.meruva@altria.com) and Rana Tayyarah (rtayyarah@lortobco.com) on or before Feb 14 th, 2014 in order to allow sufficient time to evaluate the data. 8. Statistical Data Analysis The data from participating laboratories will be analyzed statistically according to ISO-5725 (organized by Dr. Rob Stevens, Lorillard Tobacco Co.). 9. Timeline Nov 25 th - Dec 15 th Dec 15 th - Feb 14 th Feb 14 th - Feb 21 st Feb 21 st - Mar 28 th April-2014 May-2014 Sample distribution to the participating laboratories Complete testing and reporting data for e-liquid samples Review data from the participating laboratories Statistical analysis of the data Summarize study findings for presentation Study output at the next E-cigarette Task Force Meeting 10. Project Coordinator Naren Meruva narendra.k.meruva@altria.com Phone: ECIG CTR E-Liquid Preliminary Proficiency Study March /86

20 Data Sheet for 2013 Preliminary e-liquid Proficiency Study Laboratory name Company Representative (Title, Name) Address Tel Fax address Note: Please do not modify formatted data sheet for the convenience of data analyses. Note: Laboratory names will not be used for reporting. All labs will be coded. Each lab will know their own code. Note: In this spreadsheet, cells that are shaded blue require input. Cells that are not shaded are calculations or information and should not be edited. Note: Please return this completed spreadsheet to and by February 14, ECIG CTR E-Liquid Preliminary Proficiency Study March /86

21 0 Method Synopsis: please type an overall summary of your method in this box and provide specific details below Method Details (example)* Nicotine Water Glycerin PG Sample Preparation sample storage prior to preparation room temp, in dark Target weight or volume used dilution volume and solvent mixing method Solvent Extraction Additional Filtration 100 (+/- 10)mg 20mL MeOH with IS 10min wrist-action shaker 20 ml MeOH (subambient) wrist-action shaker, 30min PTFE: 0.45um Additional sample processing prior to analysis Additional details of relevance related to sample preparation Analytical Equipment Description the same samples were used for all analytes but were aliquots were taken from original container after hand Agilent GC/MS, EI in SIM mode using detuerated IS; D-5 pyridine (84), pyridine(79) & D-7 quinoline(136) & quinoline(129); Detection Systems + Conditions Additional Details related to analysis Reference Quantitation Information Limit of Quantitation Limit of Detection Number of standards Concentration of each Standard Internal Standard (IS) Calibration Type r 2 Additional Details related to quantitation Column: DBWax 30mX0.25mmX0.25U m, 50oC for 6min, 4oC/min to 160oc, 30oC to 250oC for 5.5min: 10:1 split, flow 15mL/min, total flow 20ml/min, 1ul injection extracts were stored 4C prior to analysis -- Anethole linear, with IS *Note: the example listed is not an e-liquid method. It is just meant to show the requested level of detail for you to list for your e- liquid method. ECIG CTR E-Liquid Preliminary Proficiency Study March /86

22 0 Please paste in a picture of an example chromatogram for a calibration standard and for one or more samples for chromatographic methods Please paste pictures relevant to any unusual observations noted during testing. Please include text explanation as necessary. ECIG CTR E-Liquid Preliminary Proficiency Study March /86

23 0 Please use this sheet if you use volume (rather than weight) to measure samples. Also include density calculations. Density Replicate Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 Sample 7 Sample 8 Sample 9 Sample 10 Sample Nicotine Replicate Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 Sample 7 Sample 8 Sample 9 Sample 10 Sample 11 Actual Vol (ml) Nicotine (mg/ml) Actual Vol (ml) Nicotine (mg/ml) Actual Vol (ml) Nicotine (mg/ml) Actual Vol (ml) Nicotine (mg/ml) Actual Vol (ml) Nicotine (mg/ml) Actual Vol (ml) Nicotine (mg/ml) Actual Vol (ml) Nicotine (mg/ml) Actual Vol (ml) Nicotine (mg/ml) Actual Vol (ml) Nicotine (mg/ml) Actual Vol (ml) Nicotine (mg/ml) Actual Vol (ml) Nicotine (mg/ml) Water Replicate Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 Sample 7 Sample 8 Sample 9 Sample 10 Sample 11 Actual Vol (ml) Water (mg/ml) Actual Vol (ml) Water (mg/ml) Actual Vol (ml) Water (mg/ml) Actual Vol (ml) Water (mg/ml) Actual Vol (ml) Water (mg/ml) Actual Vol (ml) Water (mg/ml) Actual Vol (ml) Water (mg/ml) Actual Vol (ml) Water (mg/ml) Actual Vol (ml) Water (mg/ml) Actual Vol (ml) Water (mg/ml) Actual Vol (ml) Water (mg/ml) Glycerin Replicate Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 Sample 7 Sample 8 Sample 9 Sample 10 Sample 11 Actual Vol (ml) Glycerin (mg/ml) Actual Vol (ml) Glycerin (mg/ml) Actual Vol (ml) Glycerin (mg/ml) Actual Vol (ml) Glycerin (mg/ml) Actual Vol (ml) Glycerin (mg/ml) Actual Vol (ml) Glycerin (mg/ml) Actual Vol (ml) Glycerin (mg/ml) Actual Vol (ml) Glycerin (mg/ml) Actual Vol (ml) Glycerin (mg/ml) Actual Vol (ml) Glycerin (mg/ml) Actual Vol (ml) Glycerin (mg/ml) PG Replicate Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 Sample 7 Sample 8 Sample 9 Sample 10 Sample 11 Actual Vol (ml) PG (mg/ml) Actual Vol (ml) PG (mg/ml) Actual Vol (ml) PG (mg/ml) Actual Vol (ml) PG (mg/ml) Actual Vol (ml) PG (mg/ml) Actual Vol (ml) PG (mg/ml) Actual Vol (ml) PG (mg/ml) Actual Vol (ml) PG (mg/ml) Actual Vol (ml) PG (mg/ml) Actual Vol (ml) PG (mg/ml) Actual Vol (ml) PG (mg/ml) ECIG CTR E-Liquid Preliminary Proficiency Study March /86

24 0 Please use this sheet if you use weight (rather than volume) for samples. Density calculations are not necessary. Nicotine Replicate Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 Sample 7 Sample 8 Sample 9 Sample 10 Sample 11 Actual Wt (mg) Nicotine (mg/mg) Actual Wt (mg) Nicotine (mg/mg) Actual Wt (mg) Nicotine (mg/mg) Actual Wt (mg) Nicotine (mg/mg) Actual Wt (mg) Nicotine (mg/mg) Actual Wt (mg) Nicotine (mg/mg) Actual Wt (mg) Nicotine (mg/mg) Actual Wt (mg) Nicotine (mg/mg) Actual Wt (mg) Nicotine (mg/mg) Actual Wt (mg) Nicotine (mg/mg) Actual Wt (mg) Nicotine (mg/mg) Water Replicate Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 Sample 7 Sample 8 Sample 9 Sample 10 Sample 11 Actual Wt (mg) Water (mg/mg) Actual Wt (mg) Water (mg/mg) Actual Wt (mg) Water (mg/mg) Actual Wt (mg) Water (mg/mg) Actual Wt (mg) Water (mg/mg) Actual Wt (mg) Water (mg/mg) Actual Wt (mg) Water (mg/mg) Actual Wt (mg) Water (mg/mg) Actual Wt (mg) Water (mg/mg) Actual Wt (mg) Water (mg/mg) Actual Wt (mg) Water (mg/mg) Glycerin Replicate Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 Sample 7 Sample 8 Sample 9 Sample 10 Sample 11 Actual Wt (mg) Glycerin (mg/mg) Actual Wt (mg) Glycerin (mg/mg) Actual Wt (mg) Glycerin (mg/mg) Actual Wt (mg) Glycerin (mg/mg) Actual Wt (mg) Glycerin (mg/mg) Actual Wt (mg) Glycerin (mg/mg) Actual Wt (mg) Glycerin (mg/mg) Actual Wt (mg) Glycerin (mg/mg) Actual Wt (mg) Glycerin (mg/mg) Actual Wt (mg) Glycerin (mg/mg) Actual Wt (mg) Glycerin (mg/mg) PG Replicate Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 Sample 7 Sample 8 Sample 9 Sample 10 Sample 11 Actual Wt (mg) PG (mg/mg) Actual Wt (mg) PG (mg/mg) Actual Wt (mg) PG (mg/mg) Actual Wt (mg) PG (mg/mg) Actual Wt (mg) PG (mg/mg) Actual Wt (mg) PG (mg/mg) Actual Wt (mg) PG (mg/mg) Actual Wt (mg) PG (mg/mg) Actual Wt (mg) PG (mg/mg) Actual Wt (mg) PG (mg/mg) Actual Wt (mg) PG (mg/mg) ECIG CTR E-Liquid Preliminary Proficiency Study March /86

25 APPENDIX B: Laboratory Method Summaries Laboratory 1 Method Summary Method Details Nicotine Water Glycerin PG Sample Preparation sample storage prior to preparation Samples were stored at room temperature for 2 hours before use Samples were stored at room temperature for 2 hours before use Samples were stored at room temperature for 2 hours before use Samples were stored at room temperature for 2 hours before use Target weight or volume used 200 (+/- 25)mg 500 (+/- 100)mg 200 (+/- 20)mg 200 (+/- 20)mg dilution volume and solvent 20 ml MeOH with IS 25 ml 2-Propanol with IS 100 ml MeOH with IS 100 ml MeOH with IS mixing method Horizontal shaking for 1 hr at 200 rpm Overnight standing Horizontal shaking for 2 hr at 200 rpm Horizontal shaking for 2 hr at 200 rpm Solvent Extraction Additional Filtration PTFE: 0.45 um PTFE: 0.45 um PTFE: 0.45 um PTFE: 0.45 um Additional sample processing prior to analysis Additional details of relevance related to sample preparation aliquots were taken from original container after hand shaking aliquots were taken from original container after hand shaking aliquots were taken from original container after hand shaking aliquots were taken from original container after hand shaking ECIG CTR E-Liquid Preliminary Proficiency Study March /86

26 Laboratory 1 Method Summary Method Details Nicotine Water Glycerin PG Analytical Equipment Description Agilent GC-FID, Nicotine & Decyl alcohol(istd) Agilent GC-TCD, Water & Ethyl alcohol(istd) Agilent GC-FID Glycerin & Decyl alcohol(istd) Agilent GC-FID PG & Decyl alcohol(istd) Detection Systems + Conditions Column: DB-Wax 30mX0.32mmX0.25um, 100 for 3min, 5 /min to 170, 240 for 10 min: 50:1 split, flow 15 ml/min, total flow 20 ml/min, 1 ul injection detector temperature 275 Column: Porapak Q, 2 m packed 170 for 10 min, 240 for 10 min: Inlet temperature 250, total flow 23 ml/min, 1 ul injection detector temperature 250 Column: DB-Wax 30mX0.32mmX0.25um, equilibration 1 min, 110 for 1 min, 10 /min to 150 for 3 min, 30 /min to 220 for 5 min, post run 240 for 11 min: 50:1 split, flow 1 ml/min, total flow 52.6 ml/min, 2 ul injection, detector temperature 275 Column: DB-Wax 30mX0.32mmX0.25um, equilibration 1 min, 110 for 1 min, 10 /min to 150 for 3 min, 30 /min to 220 for 5 min, post run 240 for 11 min: 50:1 split, flow 1 ml/min, total flow 52.6 ml/min, 2 ul injection, detector temperature 275 Additional Details related to analysis extracts were stored 4 prior to analysis Reference ECIG CTR E-Liquid Preliminary Proficiency Study March /86

27 Laboratory 1 Method Summary Method Details Nicotine Water Glycerin PG Quantitation Information Limit of Quantitation mg/mg mg/mg mg/mg mg/mg Limit of Detection mg/mg mg/mg mg/mg mg/mg Number of standards Concentration of each Standard S1( mg/ml), S2( mg/ml), S3( mg/ml), S4( mg/ml), S5( mg/ml), S1(0.05 mg/ml), S2(0.15 mg/ml), S3(0.30 mg/ml), S4(0.50 mg/ml), S5(0.75 mg/ml), S6(1.00 mg/ml), S7(2.00 mg/ml), S8(3.00 mg/ml) S1( mg/ml), S2( mg/ml), S3( mg/ml), S4( mg/ml), S5( mg/ml), S6( mg/ml), S7( mg/ml), S8( mg/ml) S1( mg/ml), S2( mg/ml), S3( mg/ml), S4( mg/ml), S5( mg/ml), S6( mg/ml), S7( mg/ml), S8( mg/ml) Internal Standard (IS) Decyl alcohol Ethyl alcohol Decyl alcohol Decyl alcohol Calibration Type linear, with IS linear, with IS linear, with IS linear, with IS r Additional Details related to quantitation ECIG CTR E-Liquid Preliminary Proficiency Study March /86

28 Laboratory 2 Method Summary Method Details Nicotine Water Glycerin PG Sample Preparation sample storage prior to preparation Samples were stored at room temperature for 2 hours before use Samples were stored at room temp at least 2 hrs before use Samples were stored at room temperature for 2 hours before use Samples were stored at room temperature for 2 hours before use Target weight or volume used mg 200 to 3500mg dependent on level of water found mg mg dilution volume and solvent 20 ml MeOH with IS directly injection 100 ml MeOH with IS 100 ml MeOH with IS mixing method Wrist-action shaker, 10min Magnetic Stirrer Wrist-action shaker, 10min Wrist-action shaker, 10min Solvent Extraction Additional Filtration Additional sample processing prior to analysis Additional details of relevance related to sample preparation ECIG CTR E-Liquid Preliminary Proficiency Study March /86

29 Laboratory 2 Method Summary Analytical Equipment Method Details Nicotine Water Glycerin PG Description GC-FID Karl Fischer GC-FID GC-FID Detection Systems + Conditions GC: DB-WAX, 15m x 0.25mm x 0.25µm, Column Flow Mode: Ramped Pressure (Agilent)/Pressure Control (Chromeleon). Inlet: Injection Volume = 1.0 µl, Liner: Double taper with glass wool, Temperature: 300 o C, Mode: Split, Split Ratio: 40.0, Pressure Eqil Time: 0.50 min, Gas Saver Flow: 20.0 ml/min, Gas Saver Time: 19.0 min. Detector: Detection: FID, Temperature: 300 o C, Makeup Gas: Helium 50.0 ml/min, Air Flow: 450 ml/min, Hydrogen Flow: 40 ml/min. Oven Parameters: 0.0min = 0.8psi, 60 o C; 0.5min = 0.8psi, 60 o C, 4.5min = 0.8psi, 200 o C, 4.9min = 1.2psi, 240 o C, 6.8min = 1.2psi, 240 o C. Not Applicable Concurrent to Nicotine, same parameters Concurrent to Nicotine, same parameters Additional Details related to analysis GC-MS is used if analyte is trace GC-MS is used if analyte is trace GC-MS is used if analyte is trace Reference ECIG CTR E-Liquid Preliminary Proficiency Study March /86

30 Laboratory 2 Method Summary Method Details Nicotine Water Glycerin PG Quantitation Information Limit of Quantitation 0.05mg/g Limit of Detection 0.005mg/g Number of standards Concentration of each Standard mg/mL 3 replicates mg/mL mg/ml Internal Standard (IS) Anethole 1000mg Anethole Anethole Calibration Type Linear with IS None Linear with IS Linear with IS r 2 >0.995 Titration >0.995 >0.995 Additional Details related to quantitation Not Applicable ECIG CTR E-Liquid Preliminary Proficiency Study March /86

31 Laboratory 3 Method Summary Method Details Nicotine Water Glycerin PG Sample Preparation sample storage prior to preparation Samples were stored at room temp at least 2 hrs before use Samples were stored at room temp at least 2 hrs before use Samples were stored at room temp at least 2 hrs before use Samples were stored at room temp at least 2 hrs before use Target weight or volume used 200 mg 100 to 400mg dependent on level of water found 200 mg 200 mg dilution volume and solvent 20 ml MeOH with IS directly weighed into resevoir containing pretitrated Solvent KN 20 ml MeOH with IS 20 ml MeOH with IS mixing method Mixing the same as extraction Magnetic Stirrer Mixing the same as extraction Mixing the same as extraction Solvent 20 ml MeOH containing IS Not Applicable 20 ml MeOH containing IS 20 ml MeOH containing IS Extraction 45 min with platform shaker Not Applicable 45 min with platform shaker 45 min with platform shaker Additional Filtration None None None None Additional sample processing prior to analysis the same samples used for nicotine, PPG and glycerin None the same samples used for nicotine, PPG and glycerin the same samples used for nicotine, PPG and glycerin Additional details of relevance related to sample preparation Not Applicable Karl Fischer reagent Combi Titrant 5 Keto Not Applicable Not Applicable ECIG CTR E-Liquid Preliminary Proficiency Study March /86

32 Laboratory 3 Method Summary Method Details Nicotine Water Glycerin PG Analytical Equipment Description HP6890 Mettler Toledo DL31 Varian 3800 Varian 3800 Detection Systems + Conditions Column - 2mx3.2 mm OD; 16% Apiezon L, 2%KOH, 2% Carbowax on Chromosorb W Column flow at 20 ml/min; Injector at 230 C; Detector C at 230 C, Oven T at 190 C; Carrier gas: He at 100 psi Not Applicable Column - DB Wax 15mx0.53mmx1um Injector: split with a split flow of ~50 ml/min at 120 C; Temp program: 120 C hold for 2 min, 15 C/min to 180 C, hold for 4 min Column - DB Wax 15mx0.53mmx1um Injector: split with a split flow of ~50 ml/min at 120 C; Temp program: 120 C hold for 2 min, 15 C/min to 180 C, hold for 4 min Additional Details related to analysis Extracts injected in GC immediately Not Applicable Extracts injected in GC immediately Extracts injected in GC immediately Reference ECIG CTR E-Liquid Preliminary Proficiency Study March /86

33 Laboratory 3 Method Summary Method Details Nicotine Water Glycerin PG Quantitation Information Limit of Quantitation mg/mg Not Applicable mg/mg mg/mg Limit of Detection mg/mg Not Applicable mg/mg mg/mg Number of standards 8 3 replicates 4 4 Concentration of each Standard , , , , , , , mg/ml 25µL weighed to 4 decimal places (in g) 0.12, 1.2, 3.0, 14 mg/ml 0.02, 0.2, 0.5, 12 mg/ml Internal Standard (IS) Anethole None Anethole Anethole Calibration Type linear, with IS Titration linear, with IS linear, with IS r Not Applicable Additional Details related to quantitation No additional details No additional details No additional details No additional details ECIG CTR E-Liquid Preliminary Proficiency Study March /86

34 Laboratory 5 Method Summary Method Details Nicotine Water Glycerin PG Sample Preparation sample storage prior to preparation Refrigerated, for 2 h prior to sample preparation Refrigerated, for 2 h prior to sample preparation Refrigerated, for 2 h prior to sample preparation Refrigerated, for 2 h prior to sample preparation Target weight or volume used 250 (+/-30) 250 (+/-30) 250 (+/-30) 250 (+/-30) dilution volume and solvent 40 ml 2-propanol w/ IS 40 ml 2-propanol w/ IS 40 ml 2-propanol w/ IS 40 ml 2-propanol w/ IS mixing method 30 min on platform shaker 30 min on platform shaker 30 min on platform shaker 30 min on platform shaker Solvent 40 ml 2-propanol (ambient) 40 ml 2-propanol (ambient) 40 ml 2-propanol (ambient) 40 ml 2-propanol (ambient) Extraction 30 min on platform shaker 30 min on platform shaker 30 min on platform shaker 30 min on platform shaker Additional Filtration n/a n/a n/a n/a Additional sample processing prior to analysis n/a n/a Dilution with2-propanol w/ IS Dilution with2-propanol w/ IS Additional details of relevance related to sample preparation n/a n/a n/a n/a ECIG CTR E-Liquid Preliminary Proficiency Study March /86

35 Laboratory 5 Method Summary Method Details Nicotine Water Glycerin PG Analytical Equipment Description Perkin Elmer GC/FID Perkin Elmer GC/TCD Perkin Elmer GC/FID Perkin Elmer GC/FID Detection Systems + Conditions WCOT CP-WAX 52CB 25 m x 0.53 mm, guard: 2 m x 0.53 mm, 80 C for 1.75 min, 25 C/min to 140 C, 50 C/min to 190 C for 1.75 min, 30 C/min to 220 C for 1.10 min: 5:1 split, flow 11 ml/min, total flow 31 ml/min, 3 µl injection Porplot Q 10m x 0.53 mm, 125 C for 1.75 min, 30 C/min to 140 C for 1 min, 50 C/min to 190 C, hold 1.75 min: 5:1 split, flow 10 ml/min, total flow 30 ml/min, 3 µl injection WCOT CP-WAX 52CB 25 m x 0.53 mm, guard: 2 m x 0.53 mm, 80 C for 1.75 min, 25 C/min to 140 C, 50 C/min to 190 C for 1.75 min, 30 C/min to 220 C for 1.10 min: 5:1 split, flow 11 ml/min, total flow 31 ml/min, 3 µl injection WCOT CP-WAX 52CB 25 m x 0.53 mm, guard: 2 m x 0.53 mm, 80 C for 1.75 min, 25 C/min to 140 C, 50 C/min to 190 C for 1.75 min, 30 C/min to 220 C for 1.10 min: 5:1 split, flow 11 ml/min, total flow 31 ml/min, 3 µl injection Additional Details related to analysis extracts were stored 4C prior to analysis extracts were stored 4C prior to analysis extracts were stored 4C prior to analysis Reference ECIG CTR E-Liquid Preliminary Proficiency Study March /86

36 Laboratory 5 Method Summary Method Details Nicotine Water Glycerin PG Quantitation Information Limit of Quantitation Limit of Detection Number of standards Concentration of each Standard 5 µg/ml, 10 µg/ml, 25 µg/ml, 50 µg/ml, 100 µg/ml, 250 µg/ml, and 500 µg/ml 0 mg, 1 mg, 2 mg, 4 mg, 10 mg, 20 mg, and 25 mg 10 µg/ml, 20 µg/ml, 50 µg/ml, 100 µg/ml, 200 µg/ml, 500 µg/ml, and 1000 µg/ml 10 µg/ml, 20 µg/ml, 50 µg/ml, 100 µg/ml, 200 µg/ml, and 500 µg/ml Internal Standard (IS) n-heptadecane Ethanol n-heptadecane n-heptadecane Calibration Type linear, with IS linear, with IS linear, with IS linear, with IS r Additional Details related to quantitation ECIG CTR E-Liquid Preliminary Proficiency Study March /86

37 Laboratory 6 Method Summary Method Details Nicotine Water Glycerin PG Sample Preparation sample storage prior to preparation room temperature, dark room temperature, dark room temperature, dark room temperature, dark Target weight or volume used 100µL 100µL 25µL, 50µL, or 100µL 25µL, 50µL, or 100µL dilution volume and solvent to 1mL with isopropanol containing IS to 1mL or 5mL with isopropanol containing IS to 1mL or 5mL with isopropanol containing IS to 1mL or 5mL with isopropanol containing IS mixing method manual mixing/vortex mixer manual mixing/vortex mixer manual mixing/vortex mixer manual mixing/vortex mixer Solvent isopropanol with IS isopropanol with IS isopropanol with IS isopropanol with IS Extraction manual mixing/vortex mixer as needed manual mixing/vortex mixer as needed manual mixing/vortex mixer as needed manual mixing/vortex mixer as needed Additional Filtration none none none none Additional sample processing prior to analysis none none none none Additional details of relevance related to sample preparation none none none none ECIG CTR E-Liquid Preliminary Proficiency Study March /86

38 Laboratory 6 Method Summary Method Details Nicotine Water Glycerin PG Analytical Equipment Description Agilent GC/FID Agilent GC/TCD Agilent GC/FID Agilent GC/FID Detection Systems + Conditions Column: Restek Stabilwax 30mX0.32mmX1.0Um, 1ul injection Column: Supelco Porapak R 6ft. X 1/8 inch ID Column: Restek Stabilwax 30mX0.32mmX1.0Um, 1ul injection Column: Restek Stabilwax 30mX0.32mmX1.0Um, 1ul injection Additional Details related to analysis extracts stored at room temperature extracts stored at room temperature extracts stored at room temperature extracts stored at room temperature Reference ECIG CTR E-Liquid Preliminary Proficiency Study March /86

39 Laboratory 6 Method Summary Method Details Nicotine Water Glycerin PG Quantitation Information Limit of Quantitation Limit of Detection Number of standards Concentration of each Standard , 0.103, 0.257, 0.514, 0.771, 1.03, 1.23 mg/ml 0.495, 1.24, 2.48, 3.71, 4.95, 5.94 mg/ml 0.331, 0.662, 1.66, 3.31, 4.97, 6.62, 7.95, 9.93, 13.2, 33.1 mg/ml 0.255, 0.510, 1.27, 2.55, 3.82, 5.10, 6.11, 7.64, 10.2, 25.5 mg/ml Internal Standard (IS) Heptadecane Ethanol Heptadecane Heptadecane Calibration Type linear, with IS linear, with IS linear, with IS linear, with IS r Additional Details related to quantitation ECIG CTR E-Liquid Preliminary Proficiency Study March /86

40 Laboratory 7 Method Summary Method Details Nicotine Water Glycerin PG Sample Preparation sample storage prior to preparation room temp room temp room temp room temp Target weight or volume used 1 gram ( to the nearest 0.1 mg) 1 gram(to the nearest 0.1 mg) 1 gram(to the nearest 0.1 mg) 1 gram(to the nearest 0.1 mg) dilution volume and solvent 50 ml Isopropanol with IS 50 ml Isopropanol with IS 50 ml Isopropanol with IS 50 ml Isopropanol with IS mixing method 180 min table top shaker 180 min table top shaker 180 min table top shaker 180 min table top shaker Solvent Isopropyl-ethyl- Octadecane Isopropyl-ethyl- Octadecane Isopropyl-ethyl- Octadecane Isopropyl-ethyl- Octadecane Extraction 180 min table top shaker 180 min table top shaker 180 min table top shaker 180 min table top shaker Additional Filtration none none none none Additional sample processing prior to analysis none none none none Additional details of relevance related to sample preparation none none none none ECIG CTR E-Liquid Preliminary Proficiency Study March /86

41 Laboratory 7 Method Summary Method Details Nicotine Water Glycerin PG Analytical Equipment Description Agilent GC 6890 with FID Agilent GC 6890 with TCD Agilent GC 6890 with FID Agilent GC 6890 with FID Detection Systems + Conditions Column: J&W Scientific Co. 5.0m x0.2 mm I.D., 0.4um film thickness, DBWAX ETR. Detector: Temp 300 C, H2 flow 40(ml/min), Detector makeup gas Helium 20(ml/min), Detector Air flow 400ml/min Column: Supelco Inc. 1/8" I.D. stainless steel 80/100 Porapak Q 6ft. 1/8 in x 2.1 mm. Detector: Temp 240 C. Detector makeup gas Helium 30ml/min Column: J&W Scientific Co. 5.0m x0.2 mm I.D., 0.4um film thickness, DBWAX ETR. Detector: Temp 300 C, H2 flow 40(ml/min), Detector makeup gas Helium 20(ml/min), Detector Air flow 400ml/min Column: J&W Scientific Co. 5.0m x0.2 mm I.D., 0.4um film thickness, DBWAX ETR. Detector: Temp 300 C, H2 flow 40(ml/min), Detector makeup gas Helium 20(ml/min), Detector Air flow 400ml/min Additional Details related to analysis NA NA NA NA Reference NA NA NA NA ECIG CTR E-Liquid Preliminary Proficiency Study March /86