Technical Report Collaborative Study on Carbonyl Containing Compounds in Electronic Cigarette Liquids

Transcription

1 E-Vapour Sub-Group Technical Report 2017 Collaborative Study on Carbonyl Containing Compounds in Electronic Cigarette Liquids May 2018 Author and Study Project Leader: Matt S. Melvin, Ph.D., Altria Client Services LLC, U.S.A. Co-Author: Gene Gillman, Ph.D., Enthalpy Analytical, U.S.A. Statistical Analysis: Michael Morton, Ph.D., Altria Client Services LLC, U.S.A.

2 Table of Contents 1. Summary Introduction Organization Participants Protocol Study Samples Within Laboratory Sample Preparation Sample Analysis and Data Reporting Deviations Data Summary Descriptive Statistics Data Statistical Analysis Exclusion of Outliers Calculation of Repeatability and Reproducibility Data Interpretation Stability of compounds in sample matrix Recommendations APPENDIX A: Study Protocol APPENDIX B: Analytical Method APPENDIX C: Raw Data Plots... 35

3 1. Summary A New Work Item Proposal (NWIP) was submitted to the Scientific Commission (SC) by the E-Vapour Sub-Group (EVAP) for the development of an analytical method suitable for the analysis of carbonyl containing compounds in electronic cigarette liquids (e-liquids) and aerosols. The NWIP was approved by the SC at the June 2016 meeting. At the fall 2016 EVAP meeting in Berlin, three analytical methods were presented for consideration to be the basis of a collaborative study. Consensus was not reached on which method to proceed with and a poll was conducted in December A method proposed based on high-performance liquid chromatography with ultra-violet detection (HPLC-UV) and 2,4-dinitrophenylhydrazine (DNPH) derivatization was selected by the group. Also at the Berlin meeting, it was decided to perform the project in two parts separating liquid analysis from aerosol analysis. The analyte list was agreed upon at the meeting and includes the following carbonyl compounds: Formal Butyral Acetal MEK Acetone Diacetyl (2,3 butanedione) Acrolein 2,3-Pentanedione (Acetyl Propionyl) Crotonal Acetoin Propional The collaborative study was executed between February 2017 and April 2017 with ten participating laboratories. Preliminary results of the collaborative study were presented at the EVAP meeting in May The purpose of this study was to evaluate repeatability and reproducibility (r&r) values of the methodology and draft a new CORESTA Recommended Method (CRM) for the determination of carbonyls in e-liquids. The Sub-Group decided that the results do not support the creation of a CRM at this time due to the poor r&r values which are attributed to issues with sample integrity. This Technical Report details the results and findings of the collaborative study for the analysis of carbonyls in e-liquids. 2. Introduction In the Spring of 2017, the CORESTA EVAP Sub-Group conducted a collaborative study for the determination of carbonyl containing compounds in e-liquids. Since no reference or monitor e-liquid samples are available, a member of the Sub-Group agreed to provide four samples fortified with the analytes of interest for the collaborative study. These samples included four flavour categories; unflavoured, tobacco, tobacco plus menthol, and sweet. The purpose of this study was to evaluate the repeatability and reproducibility for the stated HPLC- UV method. The carbonyl content of the samples was determined by derivatizing approximately 100 mg of sample in 1 ml of the DNPH solution described in the method. After sufficient time had passed for complete derivatization, the samples were treated with 50 µl of pyridine and analyzed by HPLC-UV. Data analysis was in basic conformance with the recommendations of ISO :1994 and ISO/TR 22971:2005. The raw data were obtained from 10 laboratories. Analyte levels were reported in units µg/g of e-liquid on an as-is basis. EVAP CTR Collaborative Study on Carbonyls in E-liquids May /36

4 3. Organization 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 used in this report do not correspond to the same order as shown in the table below. Lab code H was withdrawn because the study protocol method was not followed. Lab H analyzed the samples by LC-MS instead of LC-UV. 3.2 Protocol Altria Client Services Table 1: List of Participating Laboratories Enthalpy Analytical Inc Durham Enthalpy Analytical Inc. Richmond Essentra Scientific Japan INC. JTI Ökolab Labstat International ULC R.J. Reynolds Participants Shanghai New Products Research Institute of CNTC Zhengzhou Research Institute of CNTC Study Samples All fortified samples were provided and shipped to the participating laboratories. Samples were to be stored at 4 C prior to starting the study. The study was to be conducted from February 2017 to April Laboratories were requested to submit data by April 15, Most of the data, including re-checks, were received by mid-april 2017, with one sample received later. The samples are identified in Table 2. Sample ID Flavor Profile Table 2: Sample Identification Target Nicotine (%w/w) Target PG (%w/w) Target Gly (%w/w) Target Added H2O (%w/w) CS_Carb CS_Carb CS_Carb03 Menthol CS_Carb04 Fruit/Sweet EVAP CTR Collaborative Study on Carbonyls in E-liquids May /36

5 3.2.2 Within Laboratory Sample Preparation The laboratories were directed to remove the samples from cold storage prior to testing and to allow the samples to equilibrate to ambient temperature. The samples were prepared by treating 100 mg with 1 ml of the DNPH solution and treating with 50 µl of pyridine prior to analysis by HPLC-UV Sample Analysis and Data Reporting Laboratories were requested to conduct three replicate analyses for each sample. The replicates should be determined from independent sample preparations. Data were reported in units of µg/g, on an as-is basis Deviations Labs A and I did not report acetoin and formal due to co-elution of peaks. 4. Data Summary Descriptive Statistics The full data set is listed in Table 3. The results are presented on an as-is basis, without correction for moisture. Each analysis includes three replicates. Raw data plots that include all replicates, without removal of outliers, are given in Appendix C. Outliers are discussed in 5.1. Data eventually dropped as outliers are included in Table 3, but were eliminated prior to r&r calculation. EVAP CTR Collaborative Study on Carbonyls in E-liquids May /36

6 Table 3: Full Data Set (results are presented on an as-is basis) The carbonyl values are provided on an as-is µg/g basis without correction for moisture. Lab Product Replicate Units Formal Acetal Acetone Acrolein Propional A 1 µg/g NR 2.441* 9.47 <LOQ 9.58 A 2 µg/g NR 2.971* 8.38 <LOQ 9.17 A 3 µg/g NR 3.337* <LOQ 9.62 A 1 µg/g NR 3.040* <LOQ A 2 µg/g NR <LOQ <LOQ A 3 µg/g NR <LOQ 8.09 <LOQ A A A Tobacc o Tobacc o Tobacc o 1 µg/g NR 3.623* <LOQ µg/g NR 3.381* <LOQ µg/g NR <LOQ <LOQ A Sweet 1 µg/g NR 4.610* <LOQ A Sweet 2 µg/g NR 4.487* <LOQ A Sweet 3 µg/g NR 5.557* <LOQ B 1 µg/g B 2 µg/g B 3 µg/g B 1 µg/g B 2 µg/g B 3 µg/g B B B Menthol / 1 µg/g µg/g µg/g B Sweet 1 µg/g B Sweet 2 µg/g B Sweet 3 µg/g C 1 µg/g <LOQ C 2 µg/g <LOQ C 3 µg/g <LOQ C 1 µg/g C 2 µg/g C 3 µg/g C 1 µg/g EVAP CTR Collaborative Study on Carbonyls in E-liquids May /36

7 Lab Product Replicate Units Formal Acetal Acetone Acrolein Propional C C 2 µg/g µg/g C Sweet 1 µg/g <LOQ C Sweet 2 µg/g <LOQ C Sweet 3 µg/g <LOQ D 1 µg/g <LOQ D 2 µg/g <LOQ D 3 µg/g <LOQ D 1 µg/g <LOQ D 2 µg/g <LOQ D 3 µg/g <LOQ D D D 1 µg/g <LOQ µg/g <LOQ µg/g <LOQ D Sweet 1 µg/g <LOQ D Sweet 2 µg/g <LOQ D Sweet 3 µg/g <LOQ E 1 µg/g <LOQ E 2 µg/g <LOQ E 3 µg/g <LOQ E 1 µg/g E 2 µg/g E 3 µg/g E E E 1 µg/g µg/g µg/g E Sweet 1 µg/g <LOQ E Sweet 2 µg/g <LOQ E Sweet 3 µg/g <LOQ F 1 µg/g <LOD 9.65 F 2 µg/g <LOD 9.80 F 3 µg/g <LOD 9.61 F 1 µg/g EVAP CTR Collaborative Study on Carbonyls in E-liquids May /36

8 Lab Product Replicate Units Formal Acetal Acetone Acrolein Propional F 2 µg/g <LOQ F 3 µg/g <LOQ F F F 1 µg/g <LOD µg/g <LOD µg/g <LOD 9.15 F Sweet 1 µg/g NR <LOD F Sweet 2 µg/g NR <LOD F Sweet 3 µg/g NR <LOD G 1 µg/g <LOQ G 2 µg/g <LOQ G 3 µg/g <LOQ G 1 µg/g <LOQ G 2 µg/g <LOQ G 3 µg/g <LOQ G G G 1 µg/g <LOQ 2 µg/g <LOQ 3 µg/g <LOQ G Sweet 1 µg/g <LOQ G Sweet 2 µg/g <LOQ G Sweet 3 µg/g <LOQ I 1 µg/g NR I 2 µg/g NR I 3 µg/g NR I 1 µg/g NR I 2 µg/g NR I 3 µg/g NR I I I 1 µg/g NR µg/g NR µg/g NR I Sweet 1 µg/g NR NR I Sweet 2 µg/g NR NR I Sweet 3 µg/g NR NR EVAP CTR Collaborative Study on Carbonyls in E-liquids May /36

9 Lab Product Replicate Units Formal Acetal Acetone Acrolein Propional J 1 µg/g < LOD J 2 µg/g < LOD J 3 µg/g < LOD J 1 µg/g J 2 µg/g J 3 µg/g J J J 1 µg/g µg/g µg/g J Sweet 1 µg/g <LOD J Sweet 2 µg/g <LOD J Sweet 3 µg/g <LOD K 1 µg/g <LOQ K 2 µg/g <LOQ K 3 µg/g <LOQ 16.3 K 1 µg/g <LOQ 9.13 K 2 µg/g <LOQ 9.32 K 3 µg/g <LOQ 8.96 K K K 1 µg/g <LOQ µg/g <LOQ µg/g <LOQ 11 K Sweet 1 µg/g <LOQ K Sweet 2 µg/g <LOQ 13.9 K Sweet 3 µg/g <LOQ 13.5 EVAP CTR Collaborative Study on Carbonyls in E-liquids May /36

10 Lab Product Repli cate Units Crotonal Methylethyl ketone Butyral Diacetyl 2,3- Pentane dione Acetoin A A A 1 µg/g NR 2 µg/g NR 3 µg/g NR A 1 µg/g * NR A 2 µg/g * NR A 3 µg/g * NR A A A 1 µg/g NR 2 µg/g NR 3 µg/g NR A Sweet 1 µg/g NR A Sweet 2 µg/g NR A Sweet 3 µg/g NR B B B 1 µg/g <LOQ <LOQ <LOQ 2 µg/g <LOQ <LOQ <LOQ 3 µg/g <LOQ <LOQ <LOQ B 1 µg/g <LOQ <LOQ <LOQ B 2 µg/g <LOQ <LOQ <LOQ B 3 µg/g <LOQ <LOQ <LOQ B B B 1 µg/g <LOQ <LOQ <LOQ 2 µg/g <LOQ <LOQ <LOQ 3 µg/g <LOQ <LOQ <LOQ B Sweet 1 µg/g <LOQ <LOQ B Sweet 2 µg/g <LOQ <LOQ B Sweet 3 µg/g <LOQ <LOQ C C C 1 µg/g <LOD <LOD 2 µg/g <LOD <LOD 3 µg/g <LOD <LOD EVAP CTR Collaborative Study on Carbonyls in E-liquids May /36

11 Lab Product Repli cate Units Crotonal Methylethyl ketone Butyral Diacetyl 2,3- Pentane dione Acetoin C 1 µg/g <LOD <LOD C 2 µg/g <LOD <LOD C 3 µg/g <LOD <LOD C C C 1 µg/g <LOD <LOD 2 µg/g <LOD <LOD 3 µg/g <LOD < LOD C Sweet 1 µg/g <LOD <LOD C Sweet 2 µg/g <LOD <LOD C Sweet 3 µg/g <LOD <LOD D D D 1 µg/g * <LOQ <LOQ 2 µg/g * <LOQ <LOQ 3 µg/g * <LOQ <LOQ D 1 µg/g * <LOQ <LOQ D 2 µg/g * <LOQ <LOQ D 3 µg/g * <LOQ <LOQ D D D 1 µg/g * <LOQ <LOQ 2 µg/g * <LOQ <LOQ 3 µg/g * <LOQ <LOQ D Sweet 1 µg/g * <LOQ <LOQ D Sweet 2 µg/g * <LOQ <LOQ D Sweet 3 µg/g * <LOQ <LOQ E E E 1 µg/g 0.672* <LOQ <LOQ 2 µg/g 0.475* <LOQ <LOQ 3 µg/g 0.563* <LOQ <LOQ E 1 µg/g 0.347* <LOQ 0.646* E 2 µg/g 0.378* <LOQ 0.687* E 3 µg/g 0.460* <LOQ 0.612* E 1 µg/g 0.488* <LOQ 0.384* EVAP CTR Collaborative Study on Carbonyls in E-liquids May /36

12 Lab Product Repli cate Units Crotonal Methylethyl ketone Butyral Diacetyl 2,3- Pentane dione Acetoin E E 2 µg/g 0.518* <LOQ 0.342* 3 µg/g 0.526* <LOQ 0.398* E Sweet 1 µg/g 0.434* <LOQ <LOQ E Sweet 2 µg/g 0.231* <LOQ <LOQ E Sweet 3 µg/g 0.456* <LOQ <LOQ F F F 1 µg/g <LOQ <LOD <LOD 2 µg/g <LOQ <LOD <LOD 3 µg/g <LOQ <LOD <LOD F 1 µg/g <LOQ <LOD <LOQ F 2 µg/g <LOQ <LOD <LOQ F 3 µg/g <LOQ <LOD <LOQ F F F 1 µg/g <LOD <LOD <LOQ 2 µg/g <LOD <LOD <LOQ 3 µg/g <LOD <LOD <LOQ F Sweet 1 µg/g <LOD <LOD <LOD F Sweet 2 µg/g <LOD <LOD <LOD F Sweet 3 µg/g <LOD <LOD <LOD G G G 1 µg/g <LOQ <LOQ 2 µg/g <LOQ <LOQ 3 µg/g <LOQ <LOQ G 1 µg/g <LOQ <LOQ <LOQ G 2 µg/g <LOQ <LOQ <LOQ G 3 µg/g <LOQ <LOQ <LOQ G G G 1 µg/g <LOQ <LOQ 2 µg/g <LOQ <LOQ 3 µg/g <LOQ <LOQ G Sweet 1 µg/g <LOQ G Sweet 2 µg/g <LOQ EVAP CTR Collaborative Study on Carbonyls in E-liquids May /36

13 Lab Product Repli cate Units Crotonal Methylethyl ketone Butyral Diacetyl 2,3- Pentane dione Acetoin G Sweet 3 µg/g <LOQ I I I 1 µg/g < NR 2 µg/g < NR 3 µg/g < NR I 1 µg/g < NR I 2 µg/g < NR I 3 µg/g < NR I I I 1 µg/g < NR 2 µg/g < LOQ NR 3 µg/g < LOQ NR I Sweet 1 µg/g < LOQ NR I Sweet 2 µg/g < LOQ NR I Sweet 3 µg/g < LOQ NR J J J 1 µg/g < LOD < LOD 2 µg/g 0.298* < LOD 0.626* 3 µg/g 0.251* < LOD 0.490* J 1 µg/g 0.220* < LOD < LOD J 2 µg/g 0.268* < LOD < LOD J 3 µg/g 0.242* < LOD 0.472* J J J 1 µg/g 0.150* < LOD < LOD 2 µg/g 0.228* < LOD 0.376* 3 µg/g 0.310* < LOD < LOD J Sweet 1 µg/g 0.328* < LOD < LOD J Sweet 2 µg/g < LOD < LOD J Sweet 3 µg/g 0.172* < LOD < LOD K K 1 µg/g <LOQ <LOQ <LOQ 2 µg/g <LOQ <LOQ <LOQ EVAP CTR Collaborative Study on Carbonyls in E-liquids May /36

14 Lab K Product Unflavor- ed Repli cate Units Crotonal Methylethyl ketone Butyral Diacetyl 2,3- Pentane dione Acetoin 3 µg/g <LOQ <LOQ <LOQ K 1 µg/g <LOQ <LOQ 0.84 K 2 µg/g <LOQ <LOQ 0.9 K 3 µg/g <LOQ <LOQ 0.96 K K K 1 µg/g <LOQ <LOQ <LOQ 2 µg/g <LOQ <LOQ <LOQ 3 µg/g <LOQ <LOQ <LOQ K Sweet 1 µg/g <LOQ <LOQ <LOQ K Sweet 2 µg/g <LOQ <LOQ <LOQ K Sweet 3 µg/g <LOQ <LOQ <LOQ NR indicates that the laboratory did not provide data for that sample. Values between the limit of quantification and the limit of detection were reported as estimates and indicated as such by an *. 5. Data Statistical Analysis A statistical analysis was conducted in basic conformance with ISO :1994 and ISO/TR 22971:2005. A summary of the results from outlier detection and the calculated results for repeatability (r) and reproducibility (R) are given below in sections 5.1 and 5.2, respectively. Raw data plots that include all replicates shown in Table 3, prior to removal of outliers, are given in Appendix C. 5.1 Exclusion of Outliers Procedures outlined in ISO :1994 and ISO/TR 22971:2005 were generally used for the exclusion of outliers. An adaptation of Levene s Test was used for eliminating laboratories with overly large repeatability standard deviations and Grubbs Test was used to eliminate laboratories with outlying mean values. ISO also recommends the use of Mandel s h and k plots. Mandel s h statistic is the same as the statistic used in Grubbs Test. Similarly Mandel s k statistic, associated with within lab standard deviation, is statistically equivalent to the c-value calculated in Cochran s Test ( k = n c ). However, the critical values associated with Mandel s h and k statistics do not labs make allowance for multiple testing and can therefore, give a false impression of statistical significance. Thus, Mandel s h and k statistics do not add fundamentally new information and as typically employed may lead to incorrect conclusions. For those reasons, we do not include Mandel s h and k plots. The intent of ISO :1994 is to eliminate outliers that exceed a 1 % critical value. This was accomplished by an adaptation of Levene s Test. Levene s Test is preferable to Cochran s Test, which is recommended in ISO :1994, because of Cochran s Test s extreme sensitivity to deviations from normality. Grubbs Test and an adaptation of Levene s Test were applied at the standard nominal 1 % significance level to determine outliers and the results are EVAP CTR Collaborative Study on Carbonyls in E-liquids May /36

15 shown in Table 4. Levene s Test is mentioned in ISO/TR 22971:2005 as an alternative to Cochran s Test. However, Levene s Test does not directly apply without adaptation. For more details, see the footnote below. 1 Preliminary graphs of the data showed that Laboratory A was an outlier for acetal and diacetyl for all samples and Laboratory G was an outlier for acrolein and crotonal. Both of those laboratories were eliminated for those analytes prior to initial examination of the data for outliers. Outliers identified by the outlier tests are shown in Table 4. Table 4: Outliers Sample Analyte Levene s Outliers Lab Grubbs Outliers Lab Formal K Acetal K Formal D The ( ) symbol indicates an outlier was not detected. 5.2 Calculation of Repeatability and Reproducibility After removal of outlying data based on numerical data consistency methods discussed above (Grubbs Test and Levene s Test), the final repeatability and reproducibility (r & R) results were calculated and are shown in Table 5. In general it should be understood that the r & R results reflect both laboratory variability and product consistency. That is particularly true in this instance in light of the stability findings shown in section 6.1 below. Note that the mean values for each compound are well below the targeted 25 µg/ml (roughly 21.9 µg/g). 1 Levene s Test is commonly used to determine if each of several subpopulations have the same variance. Since it was designed to test for overall differences, not to determine if the largest variance is significantly greater than the others, some adaptation is necessary to use the approach to eliminate laboratories whose within lab variation is too large. Levene s Test was adapted to this purpose by Morton, who presented the approach utilized in this report at the 2014 CORESTA Congress (Quebec, Canada, presentation ST28, October 14, 2014). Specifically, the approach taken here is a two-step process with a lab being eliminated as an outlier if both steps are statistically significant. First, Levene s Test was run at a nominal -level of Second a comparison of the largest variance to the remaining variances is carried out at a one-sided nominal level of =0.01/number of labs. Dividing by the number of labs is to account for multiple testing, since it is not known a priori which lab will have the largest variance. Simulation studies were carried out by Morton and presented at the 2014 CORESTA Congress and these results demonstrated that this process has an overall -level near 0.01 and is robust to deviations from normality. EVAP CTR Collaborative Study on Carbonyls in E-liquids May /36

16 Table 5: Repeatability (r) and Reproducibility (R) Limits for Carbonyls on an as-is basis (µg/g) Sample Analyte Mean (µg/g) No. Labs r (%) R (%) Formal % 18.2 % Formal % 19.8 % Formal % 21.4 % Sweet Formal % 24.3 % Acetal % 31.0 % Acetal % 44.4 % Acetal % 28.8 % Sweet Acetal % 43.1 % Acetone % 99.8 % Acetone % 91.6 % Acetone % 56.3 % Sweet Acetone % 67.5 % Acrolein % 242 % Propional % 58.9 % Propional % 52.3 % Propional % 50.1 % Sweet Propional % 40.9 % Crotonal % 206 % Crotonal % 244 % Crotonal % 229 % Sweet Crotonal % 247 % Methylethylketone % 50.6 % Methylethylketone % 69.1 % Methylethylketone % 56.1 % Sweet Methylethylketone % 30.2 % Butyral % 60.4 % Butyral % 44.4 % Butyral % 44.5 % Sweet Butyral % 33.0 % Diacetyl % 62.7 % Diacetyl % 58.3 % Diacetyl % 48.2 % Sweet Diacetyl % 62.3 % EVAP CTR Collaborative Study on Carbonyls in E-liquids May /36

17 6. Data Interpretation The values for repeatability (r) and reproducibility (R) are shown in Table 5. The values for r&r demonstrate similar repeatability and reproducibility for both flavoured and unflavoured liquid samples. This table also includes values for % R for each sample, which range from approximately 18 % to over 200 %. These results indicate a similar level of variability for the determination of carbonyl compounds in both flavoured and unflavoured e-liquid samples. Acetal, formal and butyral gave R values in between 18 % to 49 % while diacetyl, MEK and acetone gave R values between 32 % to 73 % and acrolein and crotonal where either not detected in the samples or gave R values greater than 200%. Acetoin and 2,3-Pentanedione were not added to any of the study samples and were excluded from the data analysis. 6.1 Stability of compounds in sample matrix One possible explanation for the study results was lack of compound stability in the sample matrix. This would explain the low repeatability seen in each laboratory and also the high reproducibility seen across the study. In order to understand the stability of the compounds in matrix, one of the participating laboratories agreed to perform a study assessing the stability of the target compounds in the sample matrix. In this study, the target compounds were added to each flavour sample at 25 g/ml which is approximately equivalent to 21.9 g/mg. The samples were prepared at the time of spiking and all compounds were found to be within 10 % of the target concentrations. These samples were used as the time zero time point. These samples were held at ambient conditions and analysed at discrete time points over the next 46 days. The concentration of each compound was normalized to the time zero-time point. Data for each flavour sample are presented in tables 6a to 6d. Time points with a change greater than 20 % are shown in bold. Table 6a: Percent remaining over 46 days for the target compounds in the unflavoured sample Compound D0 D2 D4 D6 D14 D21 D31 D46 Acetal Acetoin Acetone Acrolein ND ND ND ND ND ND Methylethylketone Butyral Crotonal Diacetyl Formal ,3-Pentanedione Propional EVAP CTR Collaborative Study on Carbonyls in E-liquids May /36

18 Table 6b: Percent remaining over 46 days for the target compounds in the unflavoured sample Compound D0 D2 D4 D6 D14 D21 D31 D46 Acetal Acetoin Acetone Acrolein ND ND ND ND ND ND ND Methylethylketone Butyral Crotonal Diacetyl Formal ,3-Pentanedione Propional Table 6c: Percent remaining over 46 days for the target compounds in the unflavoured sample Compound D0 D2 D4 D6 D14 D21 D31 D46 Acetal Acetoin Acetone Acrolein ND ND ND ND ND ND ND Methylethylketone Butyral Crotonal Diacetyl Formal ,3-Pentanedione Propional EVAP CTR Collaborative Study on Carbonyls in E-liquids May /36

19 Table 6d: Percent remaining over 46 days for the target compounds in the unflavoured sample Compound D0 D2 D4 D6 D14 D21 D31 D46 Acetal Acetoin Acetone Acrolein ND ND ND ND ND ND ND Methylethylketone Butyral Crotonal Diacetyl Formal ,3-Pentanedione Propional The data presented in Tables 6a-6d show that several of the target compounds are highly unstable in the e-liquid matrix. These include acrolein and crotonal which were found to decrease by greater than 60 % within two days of sample preparation. After 46 days of storage, only acetoin and acetone were within 20 % of the initial spiked concentration. The study samples were prepared and shipped to laboratories located around the world and since the majority of the compounds were not stable in the sample matrices, it is probable that the repeatability and reproducibility values seen in this study do not accurately represent the repeatability and reproducibility of the analytical method. 7. Recommendations The EVAP Sub-Group decided at the October meeting in Kitzbühel to stop phase one of the NWIP with the technical report and not write a CRM due to the r&r values generated in the study. EVAP CTR Collaborative Study on Carbonyls in E-liquids May /36

20 APPENDIX A: Study Protocol CORESTA E-VAPOUR SUB-GROUP Project Number: 127 Project Title: Carbonyl Method for Liquids and E-Vapour Product Aerosol Type of Document: Protocol Revision Date: January 10, 2017 Written by: Matt S. Melvin (Altria Client Services, Study Coordinator) and I. Gene Gilman (Enthalpy Analytical, SG Secretary) EVAP CTR Collaborative Study on Carbonyls in E-liquids May /36

21 1. Introduction This protocol describes the initial study to identify a method for the analysis of carbonyl compounds in liquids and e-vapour product aerosols. The analytes of interest include the carbonyl compounds contained in CRM N 74 as well as the diacetyl-type flavouring compounds listed below. 2. Objective Formal Methylethylketone Acetal Butyral Acetone Diacetyl Acrolein 2,3-pentanedione Propional Acetoin Crotonal The participating laboratories are to determine the amounts of the analytes of interest in liquid samples intended for use in e-vapour products. The participating laboratories are requested to use the provided method in this initial collaborative study. Data submitted by other methods is encouraged but will be excluded from the study as they do not support the study objective. 3. Time schedule and Data Reporting Date December 31, 2016 January 13, 2017 January 20, 2017 January 27, 2017 End January to February 17, 2017 February March 31, 2017 April 1, 2017 April 2017 May 2017 Activity Participants select initial method. Distribute draft protocol, method, and data reporting sheet Laboratories state their intention to participate and order supplies Distribute final protocol, method, and data reporting sheet Participants order and receive the samples Laboratories conduct the study Laboratories submit results by this date Statistical evaluation and preparation of results Discuss results at 2017 Spring EVAP meeting This time schedule will allow for tabulation of the results, completion of the statistical analysis, and presentation at the 2017 Spring EVAP meeting which is scheduled for May, 2017 in Charlottesville, VA, USA. 4. Participating Laboratories Following receipt of this protocol, the participating laboratories in this study will confirm or notify Matt Melvin (Matt.S.Melvin(@)altria.com) and Gene Gillman (Gene.Gillman(@)enthalpy.com) of their active participation. Laboratories are encouraged, but not required to submit data for all analytes. EVAP CTR Collaborative Study on Carbonyls in E-liquids May /36

22 Essentra Scientific Participating Laboratories ASL Analytic Service Laboratory GmbH R.J. Reynolds Zhengzhou Research Institute of CNTC JTI Ökolab JAPAN TOBACCO INC. Labstat International ULC JTI UK 5. Samples 5.1 Selection Global Laboratory Services Altria Client Services Enthalpy Analytical Inc RDU Enthalpy Analytical Inc. RVA Lauterbach & Associates Shanghai New Products Research Institute of CNTC Laboratoire National de Métrologie et D'essais Each participating laboratory should request the following e-liquid products and quantities stated. The samples will be fortified with all or some of the analytes of interest in a concentration range between µg/ml by the supplier CORESTA Carbonyl CS Products Sample ID Flavor Profile Target Nicotine (%w/w) Target PG (%w/w) Target Gly (%w/w) Target Added H2O (%w/w) CS_Carb01 unflavored CS_Carb02 tobacco CS_Carb03 tobacco menthol CS_Carb04 Fruit/sweet E-Liquid Supply and Shipping Shipments of the 2017 Carbonyl Collaborative Study products may be obtained through Eduardo Berea from Alternative Ingredients Inc. Product versions have also been formulated that do not contain nicotine. Please request these if shipping nicotine containing products is a problem in your location. Please fortify the products with the indicated amount of nicotine prior to analysis. Each participating laboratory will send its shipping address, person to whom delivery should be made, shipping account (FedEx International, DHS,UPS) arrangements, and any special delivery information to the following address: Eberea(@)ALTERNATIVEINGREDIENTS.COM Eduardo Berea will inform the laboratories of the actual shipping date and tracking information so that the receiving laboratories can prepare for receipt of the samples. Samples will be shipped at ambient conditions. Laboratories should not submit data if they question the integrity of the samples they received. EVAP CTR Collaborative Study on Carbonyls in E-liquids May /36

23 5.3 Receipt Upon receipt, the samples must immediately be stored at approximately 4 C. 5.4 Within Laboratory Sample Preparation Samples should be removed from the refrigerator a minimum of 2 hours prior to opening for analysis. The samples should not be opened during the time the samples are equilibrating to ambient temperature. 6. Analysis Each participating laboratory should conduct three independent replicates following the procedure outlined in the Carbonyl collaborative study provided method. Data collected with other methods or with methods that have significant deviations from the provided method will not be included in the study. 7. Data Reporting Enter the final results and any comments into the provided data reporting spreadsheet. The final reporting spreadsheet and any questions should be forwarded to the study coordinator. (Matt.S.Melvin(@)altria.com): Data shall be reported to four significant figures. If data are below a quantitation limit, report the estimated analytical result for the specific analyte and make a note beside the cell that the result is below the quantitation limit. If data are below a detection limit, report the result for the specific analyte as below the quantitation limit (<LOD). All results shall be reported as is (with no correction for moisture content). Report any relevant deviations. 8. Statistical Analysis A statistical analysis in general conformance with ISO :1994 and ISO/TR 22971:2005 will be conducted. 9. Tabulation and Presentation of the Data The data will be coded by laboratory number rather than laboratory identity. The code will be provided to the respective participating laboratory along with the tabulated data. Results will be presented at the spring EVAP meeting. EVAP CTR Collaborative Study on Carbonyls in E-liquids May /36

24 APPENDIX B: Analytical Method 2017 EVAP SG Carbonyl Collaborative Study Carbonyl Method of Analysis for Liquids and E-Vapour Product Aerosol: Phase I - Liquids TABLE OF CONTENTS 1. PURPOSE SCOPE METHOD SUMMARY CHEMICALS STANDARDS ANCILLARY CHEMICALS EQUIPMENT REAGENT PREPARATION % H 3PO 4 SOLUTION DNPH WORKING SOLUTION MOBILE PHASE SOLUTION STANDARDS AND QUALITY CONTROLS PREPARATION CALIBRATION STOCKS AND STANDARDS QC STOCKS AND STANDARDS REAGENT BLANK PREPARATIONS STANDARD STABILITY PROCEDURE SAMPLE PREPARATION SAMPLE STABILITY SAMPLE RUN ORDER INSTRUMENT PARAMETERS DATA INTERPRETATION AND STATISTICAL ANALYSIS CALIBRATION AND QUALITY CONTROL STANDARD ACCEPTANCE CRITERIA DATA REDUCTION OF RAW DATA FROM HPLC-UV EQUATIONS REPORTING OF ASSAY RESULTS DEFINITIONS REFERENCES EVAP CTR Collaborative Study on Carbonyls in E-liquids May /36

25 1. PURPOSE This document describes the method for determination carbonyls in e-liquid solutions. Note that the term carbonyl in this analytical method refers to the following elements: acetoin, diacetyl, 2,3-pentanedione, formal, acetal, acetone, acrolein, propional, crotonal, 2-butanone (methyl ethyl ketone), and butyral. 2. SCOPE This method is applicable to the quantitation of selected carbonyls in e-liquids and solutions of collected aerosol from e-cigarettes. The validation included linearity, LOQ analysis, instrument precision and accuracy (for samples and standards), method precision and accuracy, as well as sample and standard stability. 3. METHOD SUMMARY This method describes the multi-element determination of select carbonyls by HPLC-UV equipped with a diode array detector or a variable wavelength detector. Carbonyls are quantitated from a derivatization of approximately 100 µl of e-liquid. Derivatization of e-liquid is accomplished at room temperature using 2,4- dinitrophenylhydrazine (DNPH). The carbonyls are measured in units of mass-to-volume (µg/ml). The measured concentration, the amount of sample derivatized, and the sample solution volume(s) are used to calculate the total analyte mass on a per gram (µg/g) or per ml (µg/ml) of sample basis. 4. CHEMICALS 4.1 Standards Equivalent reagents and standards may be used provided that the equivalence is demonstrated. Calibration Standards: Custom Standard Carbonyl-DNPH mix Restek (P/N ) ,3-Butanedione (Diacetyl) Sigma-Aldrich (P/N B85307) ,3-Pentanedione Sigma Aldrich (P/N ) Acetoin (3-hydroxy-2-butanone) Sigma-Aldrich (P/N U) Formal (37 %) Sigma Aldrich (P/N ) 4.2 Ancillary Chemicals Deionized water (Type-1 H2O) Millipore (Resistance 18 MΩ cm) EVAP CTR Collaborative Study on Carbonyls in E-liquids May /36

26 4.2.2 DNPH Derivatization reagent DNPH (50 % in water) TCI America (P/N D0845) (may be re-crystallized in house to improve purity if necessary) or DNPH Hydrochloride (DNPH-HCl) TCI America (P/N D0846) Acetonitrile (ACN) (HPLC grade) [CAS #: ] Tetrahydrofuran (HPLC grade) [CAS #: ] Isopropanol (distilled in glass) [CAS #: ] Pryridine (99 %, ACS reagent grade) [CAS #: ] Phosphoric acid H3PO4 Sigma-Aldrich (P/N38081) (85 %, ACS reagent grade) or (10 % v/v aqueous solution) Ricca (P/N 58501) 5. EQUIPMENT HPLC system consisting of the following, or equivalent: o Quaternary pump o Column oven o UV detector (PDA or UV/Vis) Balance 4 to 5-place, 0.01 to 0.1 mg precision Class A glassware, graduated cylinders, volumetric pipettes, and volumetric flasks Adjustable pipettes Note: Positive displacement pipettes are highly recommended for handling e-liquid samples. Amber vials with caps, 8 ml Glass autosampler vials with screw tops Whatman Mini-UniPrep G2 filter vials with single or 8 vial press Bottle-top dispenser capable of dispensing 50 ml (±2 %) Perkin Elmer Brownlee Choice C18 5µm 150 x 4.6 mm (P/N N ) 6. REAGENT PREPARATION % H3PO4 Solution Bring ml of 85 % H3PO4 to 1 L with DIUF H2O. Note: Solution expiration: 6 months 6.2 DNPH Working Solution Preparation from 50 % DNPH in water Dissolve 18.0 g DNPH (50 %) in approximately 300 ml of DIUF H2O. Combine solution with 40 ml of 10 % H3PO4 then bring to 500 ml with DIUF H2O. Finally bring the entire solution up to 1 L with ACN. Store at room temperature in an amber glass bottle. Note: Solution expiration: Maximum of 6 months Note: Discard the solution if the reagent blank exceeds acceptance criteria. EVAP CTR Collaborative Study on Carbonyls in E-liquids May /36

27 6.2.2 Preparation from DNPH Hydrochloride (DNPH-HCl) Dissolve 4 g DNPH-HCl in approximately 300 ml of DIUF H2O. Combine solution with 40 ml of 10 % H3PO4 then bring to 500 ml with DIUF H2O. Finally bring the entire solution up to 1 L with ACN. Store at room temperature in an amber glass bottle. Note: Solution expiration: Maximum of 6 months Note: Discard the solution if the reagent blank exceeds acceptance criteria. 6.3 Mobile Phase Solution Mobile Phase A: Prepare a solution containing water:acetonitrile:tetrahydrofuran:isopropanol at the following ratio 63:27:9:1 to the volume necessary for the number of samples being analyzed. Store at room temperature. Note: Solution expiration: 6 months. Mobile Phase B: Prepare a solution containing water:acetonitrile:tetrahydrofuran:isopropanol at the following ratio 40:58:1:1 to the volume necessary for the number of samples being analyzed. Store at room temperature. Note: Solution expiration: 6 months. Mobile Phase C: Prepare a volume of 100 % acetonitrile necessary for the number of samples being analyzed. Store at room temperature. Note: Solution expiration: 6 months. 7. STANDARDS AND QUALITY CONTROLS REPARATION 7.1 Calibration Stocks and Standards Primary Carbonyl Standard: This standard is a custom ISO Guide 34 certified mix containing 100 µg/ml of Formal-DNPH and Crotonal-DNPH, 200 µg/ml of Acrolein- DNPH, Propional-DNPH, Methyl ethyl ketone-dnph, and n- Butyral-DNPH, 1000 µg/ml of Acetone-DNPH, and 2000 µg/ml of Acetal-DNPH. Note: Concentrations are listed on the free al or ketone basis Primary Diketone Standard (Underivatized): Weight out 100 µl of diacetyl into a 10 ml volumetric flask. Dilute to volume with acetonitrile. In a separate 10 ml volumetric flask, weight out 100 µl of 2,3- pentanedione and dilute to volume with acetonitrile. Weigh 0.05 g of acetoin into another separate 10 ml volumetric flask, and dilute to volume with acetonitrile Primary Diketone Standard (Derivatized): Aliquot 1.0 ml of each diacetyl and 2,3-pentanedione underivatized stock into separate 25 ml volumetric flasks. Dilute to volume with the DNPH solution described in section 7.2. Allow the solutions to derivatize for 20 ±1 minute before adding 1.25 ml of pyridine. Be sure to account for the extra volume of pyridine EVAP CTR Collaborative Study on Carbonyls in E-liquids May /36

28 when calculating the concentration. For acetoin, add 2.0 ml of acetoin underivatized stock to a 25 ml volumetric flask and dilute to volume with the same DNPH solution. Allow standard to derivatize for 20 ±1 minute before adding 1.25 ml of pyridine. Note: Each laboratory must verify the time required to form the diketones DNPH derivatives by analysis of the primary stock. Reaction times have been reported to vary from 5 to 20 minutes depending on temperature and source of DNPH. The same reaction time must be used for both standards and samples Secondary Calibration Standard (Standard 8): Aliquot 2.0 ml (2 ampules) of the primary carbonyl standard mix into a 20 ml volumetric flask. Aliquot 4.0 ml of the primary diketone derivatized standard to the same 20 ml volumetric flask. Dilute to volume with acetonitrile Working Calibration Standards: Using the Secondary Calibration Standard, prepare seven working calibration standards according to the table below: Standard Volume of 2 (ml) Final Volume (ml) Standard Dilution to the final volume should be done with acetonitrile. This will result in approximate concentrations described in the tables below: Volume of 2 Final Volume Formal Acetal Acetone Acrolein (ml) (ml) [µg/ml] [µg/ml] [µg/ml] [µg/ml] Secondary Standard EVAP CTR Collaborative Study on Carbonyls in E-liquids May /36

29 Standard Volume of 2 Final Volume Propionaldehyd e Crotonal Butyral 2-Butanone (ml) (ml) [µg/ml] [µg/ml] [µg/ml] [µg/ml] Secondary Standard Standard Volume of 2 Final Volume Acetoin Diacetyl 2,3-Pentanedione (ml) (ml) [µg/ml] [µg/ml] [µg/ml] Secondary Standard Note: Standard 8 is the Secondary Calibration Standard. 7.2 QC Stocks and Standards Primary Carbonyl Standard: This standard is a custom ISO Guide 34 certified mix containing 100 µg/ml of Formal-DNPH and Crotonal-DNPH, 200 µg/ml of Acrolein- DNPH, Propional-DNPH, Methyl ethyl ketone-dnph, and n- Butyral-DNPH, 1000 µg/ml of Acetone-DNPH, and 2000 µg/ml of Acetal-DNPH. If possible a different lot should be used than the calibration stock Primary Diketone Standard (Underivatized): Weight out 100 µl of diacetyl into a 10 ml volumetric flask. Dilute to volume with acetonitrile. In a separate 10 ml volumetric flask, weight out 100 µl of 2,3- pentanedione and dilute to volume with acetonitrile. Weigh 0.05 g of acetoin into another separate 10 ml volumetric flask, and dilute to volume with acetonitrile Primary Diketone Standard (Derivatized): Aliquot 0.5 ml of each diacetyl and 2,3-pentanedione underivatized stock into separate 25 ml volumetric flasks. Dilute to volume with the DNPH solution described in section 7.2. Allow the solutions to derivatize for 20 ±1 minute before adding 1.25 ml of pyridine. Be sure to account for the extra volume of pyridine when calculating the concentration. For acetoin, add 1.0 ml of acetoin underivatized stock to a 25 ml volumetric flask and dilute to volume with the EVAP CTR Collaborative Study on Carbonyls in E-liquids May /36

30 same DNPH solution. Allow standard to derivatize for 20 ±1 minute before adding 1.25 ml of pyridine. Note: Each laboratory must verify the time required to form the diketones DNPH derivatives by analysis of the primary stock. Reaction times have been reported to vary from 5 to 20 minutes depending on temperature and source of DNPH. The same reaction time must be used for both standards and samples Secondary Calibration Standard: Aliquot 1.0 ml (1 ampules) of the primary carbonyl standard mix into a 10 ml volumetric flask. Aliquot 1.0 ml of the primary diketone standard (derivatized) to the same 10 ml volumetric flask. Dilute to volume with acetonitrile Working QC Standard: Using the Secondary Calibration Standard, prepare one quality control standard according to the table below: Standard Volume of 2 (ml) Final Volume (ml) QC Dilution to the final volume should be done with acetonitrile. This will result in an approximate concentration of: Standard Formal Acetal Acetone Acrolein [µg/ml] [µg/ml] [µg/ml] [µg/ml] Standard Propional Crotonal Butyral 2-Butanone [µg/ml] [µg/ml] [µg/ml] [µg/ml] Standard Acetoin Diacetyl 2,3- Pentanedione [µg/ml] [µg/ml] [µg/ml] 7.3 Reagent Blank Preparations Reagent Blank The reagent blank shall be prepped with every batch of 18 samples. Analytes detected in the blank at concentrations greater than the lower standard shall be subtracted from the calculated concentrations of the samples. Analyze a reagent blank with each analytical batch. EVAP CTR Collaborative Study on Carbonyls in E-liquids May /36

31 7.4 Standard Stability The stability of all certified stocks is dictated by vendor specifications. All primary and secondary stocks are stable for up to 6 months. Working QCs and calibration standards are stable for up to 6 months, but may expire earlier based on the expiration dates of the stocks from which they were prepared. All standards and associated QCs should be stored at -20 C and upon usage, thawed to room temperature and vortexed thoroughly to mix. 8. PROCEDURE 8.1 Sample Preparation For e-liquid samples: Pipette 100µL of sample into an autosampler vial Record the mass of each sample Add 1 ml of DNPH derivatizing solution Cap and vortex well, then allow sample to derivatize at room temperature for 20 ±1 min. Note: Reaction time must be the same as used for preparation of analytical standards Quench the reaction with 50 µl pyridine, filter samples into an autosampler vial using a 0.45 M PTFE syringe filter or Whatman Mini- UniPrep G2 filter vials Analyze filtered samples by HPLC-UV. 8.2 Sample Stability Samples, when sealed and stored at room temperature, are stable for 2 days from the date of derivatization. Samples, when stored at 4 C (±2 C) are stable for 7 days from the date of derivatization. Note: If samples are stored at 4 C (±2 C) they must the allowed to warm to room temperature and vortexed before analysis. 8.3 Sample Run Order Calibration Standards Analyze the initial calibration consisting of at least 5 consecutive standard concentrations per analyte. The regression is linear and should have a coefficient of determination (r2) of at least The curve type is linear, the origin is ignored, and the weighting is 1/X. The result of each calibration point shall be within 10 % of its nominal value Initial QC (Calibration Verification) The working QC standard in section shall be analyzed after an acceptable calibration curve is established. The results of this analysis shall be within 10 % of its nominal values. EVAP CTR Collaborative Study on Carbonyls in E-liquids May /36