E-Vapour Sub-Group
Technical Report
2019 Collaborative Study for the Determination of Formaldehyde and Acetaldehyde in E-Vapour Product Aerosol
November 2020
Study Project Leader and Author: Gene Gillman, Ph.D., Enthalpy Analytical LLC, U.S.A. Co-Author and Statistical Analysis: Michael Morton, Ph.D., Altria Client Services LLC, U.S.A. Table of Contents
1. Summary ...... 3 2. Introduction ...... 3 3. Organization ...... 4 3.1 Participants ...... 4 3.2 Protocol ...... 4 4. Study Data ...... 6 5. Statistical Analysis ...... 6 5.1 Exclusion of Outliers ...... 6 5.2 Calculation of Repeatability and Reproducibility ...... 7 6. Data Interpretation ...... 8 7. Recommendations ...... 9
APPENDIX A: Study Protocol ...... 10 APPENDIX B: Analysis Method ...... 16 APPENDIX C: Raw Data ...... 23 APPENDIX D: Raw Data Plots ...... 31
1. Summary
At the October 2018 CORESTA E-Vapour Sub-Group (EVAP) meeting held in Kunming, China, the Sub-Group initiated a collaborative study for the determination of carbonyl compounds in e-vapour product aerosol generated under CORESTA Recommended Method N° 81 puffing conditions. The target compounds for this study included formaldehyde and acetaldehyde, which were determined following a proposed CORESTA Recommended Method (CRM) for the determination of carbonyl compounds in e-vapour product aerosol. The results of the study demonstrate that the proposed CRM is suitable for the determination of formaldehyde and acetaldehyde in e-vapour product aerosol generated under CRM N° 81 puffing conditions. The EVAP Sub-Group recommends that a New Work Item Proposal (NWIP) be submitted to CORESTA for the creation of a Recommended Method for the determination of formaldehyde and acetaldehyde in e-vapour product aerosol generated under CRM N° 81 puffing conditions.
2. Introduction
A 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 in June 2016. At the fall 2016 EVAP Sub-Group meeting in Berlin, three analytical methods were presented for consideration to be the basis of a collaborative study. 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 e-liquid portion of the project was completed and published on the CORESTA website in May 2018. The Sub-Group decided that the results from the e-liquid study did not support the creation of a CORESTA Recommended Method due to the poor r & R values, which were attributed to issues with the instability of these analytes in the e-liquid matrix and the length of time to complete the study (two months). Due to the stability issues of carbonyl compounds in the e-liquid matrix, the Sub-Group decided to move forward with a collaborative study on determination of carbonyls in e-vapour product aerosol. The analyte list was agreed upon at the meeting and included only the following carbonyl compounds: • Formaldehyde • Acetaldehyde During the October 2018 meeting the timeline and study overview for the carbonyl compounds in aerosol study was approved by the Sub-Group. The participating laboratories agreed to determine the amounts of the analytes of interest in the aerosol generated from the combination of an Aspire Nautilus and a variable power battery provided by Evolv LLC. Samples would be collected from aerosol generated from three e-liquids. In addition to the native e-liquids, the Sub-Group also decided to fortify each e-liquid sample with the target compounds. Fortifying the e-liquid with the target compounds was done to ensure that a measurable amount of each compound would be present in the resulting aerosol.
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The collaborative study was executed between January 2019 and December 2019 with 11 participating laboratories. Preliminary results of the collaborative study were presented at the EVAP Sub-Group meeting in April 2020. 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-vapour product aerosol.
3. Organization
3.1 Participants 16 laboratories agreed to participate in the study with 11 laboratories reporting results. 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. Table 1: List of Participating Laboratories
Participating Laboratories Altria Client Services ASL Analytic Service Laboratory GmbH British American Tobacco Enthalpy Analytical LLC, Durham NC Enthalpy Analytical LLC, Richmond VA Eos Scientific Essentra KT&G Japan Tobacco, Inc. JT International GmbH JTI-Ökolab (Austria Tabak) Labstat International Inc Philip Morris R&D R.J. Reynolds Tobacco Company Reemtsma Imperial Brands Zhengzhou Tobacco Research Institute of CNTC
3.2 Protocol The collaborative study protocol is provided in Appendix A and specific details from the protocol are described below. 3.2.1 Study Samples Study e-liquids were provided by Alternative Ingredients, Inc. Laboratories were requested to store the samples at ambient conditions. The samples are identified in Table 2.
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Table 2: Sample Identification
Target Target Target PG Target Gly Sample ID Flavour Profile Nicotine Added H O (%w/w) (%w/w) 2 (%w/w) (%w/w) CS_Carb01 unflavored 1.5 47 47 0 CS_Carb02 tobacco 1.5 47 47 0 CS_Carb03 tobacco menthol 1.5 47 47 0
3.2.2 Analysis Participants were requested to conduct three independent replicate analyses for each sample, under CRM 81 puffing conditions. A replicate was defined as one port. Participants were asked to analyse samples according to the method given in Appendix B. Participants were requested to prepare fortified samples of the three e-liquids, each at three levels. Participating laboratories were requested to use the fortification solution from AccuStandard (Prod# S-78185; Lot# 218051164), which contains 50 µg/mL of the formaldehyde and acetaldehyde in propylene glycol. Laboratories could also use the fortification solution(s) of their choice as long as the fortification solution(s) provided fortified samples with acceptable accuracy for the analytes of interest. The samples were stored at ambient temperature prior to fortification. Four levels of each sample are to be tested as shown in Table 3, according to the amounts detailed below when using the AccuStandard fortification solution. Table 3: Sample Fortification Levels
Sample Fortification Total E- Concentration of Approximate Amount Solution LIQUID Analyte in E-LIQUID PG:VG Ratio (mg) (mg) (mg) (µg /g) 2000 0 2000 0 50:50 1400 600 2000 15 65:35 1000 1000 2000 25 75:25 600 1400 2000 35 85:15
3.2.3 Deviations Participating laboratories were requested to document any deviations from the protocol and the applicable CORESTA methods and submit any deviations with their results. Two participating laboratories reported results that were more than ten times greater than the other participating laboratories. In both cases, the study results support that either the devices were defective, mishandled or contained insufficient e-liquid, leading to the production of elevated levels of carbonyl compounds. Both data sets were removed from the study before statistical analysis. Three laboratories reported minor deviations: • Lab 7: Three (3) impingers were used to collect aerosol samples. Data was not reported for sample unflavoured replicate 3, at the fortification level 35 µg/g. Pre-column was not used during analysis. • Lab 9: E-Liquid C (Menthol/Tobacco) was not tested due to an insufficient number of tanks. • Lab 10: Reported using an alternate column, Thermo Acclaim RSCL Carbonyls column (2.2 µm 2.1 x 150 mm)
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4. Study Data
The raw data set, without removal of outliers, is provided in Appendix C. Each analysis includes three replicates.
5. Statistical Analysis
The statistical analysis was conducted in basic conformance with ISO 5725-2:1994 and ISO/TR 22971:2005. 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, without removal of outliers, are shown in Appendix D.
5.1 Exclusion of Outliers An adaptation of Levene’s Test[1] was used for eliminating laboratories with overly large repeatability standard deviations and Grubbs’ Test was used to eliminate laboratories with outlying mean values. The results are shown in Table 4. ISO 5725(2) 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. However, the critical values associated with Mandel’s h and k statistics do not 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. Table 4: Outliers
Levene’s Grubbs’ Sample Analyte* Added Outliers Lab Outliers Lab Unflavored Acetaldehyde 25 7 – Unflavored Acetaldehyde BS 25 7 – Menthol/Tobacco Acetaldehyde 25 – 7 Menthol/Tobacco Acetaldehyde BS 25 – 7 All Formaldehyde 0 – 9 All Formaldehyde 15 – 9 All Formaldehyde 25 – 9 All Formaldehyde 35 – 9 Unflavored Formaldehyde BS 15 – 9 Unflavored Formaldehyde BS 35 – 9 The (–) symbol indicates there was not an outlier of that type. * BS in the analyte name means blank subtracted and represents the yield at the indicated fortification level minus the yield with no fortification.
[1] The approach is discussed in detail by Michael Morton in “Within-Laboratory Variance Outlier Detection: An Alternative to Cochran’s Test” in Beiträge zur Tabakforschung International, Vol 27 No. 7, pp 135-144.
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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 Tables 5 and 6 for acetaldehyde and formaldehyde, respectively. Tables 7 and 8 show the values for the same two variables after blank subtraction, that is, after subtracting the analyte yield with no fortification. The r & R results reflect both laboratory variability and product consistency. Table 5: Repeatability (r) and Reproducibility (R) Limits for Acetaldehyde (μg/g) under CRM 81 Conditions
Added Product N Labs* Average r r % R R % (μg/g) Menthol/Tobacco 0 8 2.33 1.32 56.7 % 4.80 206 % Tobacco 0 9 2.43 1.36 55.9 % 5.06 208 % Unflavored 0 9 4.03 2.42 60.1 % 6.98 173 % Menthol/Tobacco 15 8 9.06 3.86 42.6 % 10.50 116 % Tobacco 15 9 11.45 3.51 30.6 % 15.34 134 % Unflavored 15 9 13.23 4.15 31.4 % 17.34 131 % Menthol/Tobacco 25 7 15.79 5.31 33.6 % 18.00 114 % Tobacco 25 9 19.24 9.65 50.1 % 21.19 110 % Unflavored 25 8 17.97 5.45 30.3 % 19.32 108 % Menthol/Tobacco 35 8 23.20 7.86 33.9 % 28.72 124 % Tobacco 35 9 24.96 12.97 52.0 % 33.36 134 % Unflavored 35 9 26.59 13.28 49.9 % 35.64 134 % * The number of laboratory data sets after removal of outliers.
Table 6: Repeatability (r) and Reproducibility (R) Limits for Formaldehyde (μg/g) under CRM 81 Conditions
Added Product N Labs* Average r r % R R % (μg/g) Menthol/Tobacco 0 7 8.46 3.16 37.4 % 12.51 148 % Tobacco 0 8 8.29 4.33 52.2 % 11.48 139 % Unflavored 0 8 11.57 3.95 34.1 % 14.41 124 % Menthol/Tobacco 15 7 17.26 3.27 19.0 % 17.02 99 % Tobacco 15 8 16.72 3.52 21.1 % 13.67 82 % Unflavored 15 8 20.93 2.68 12.8 % 12.70 61 % Menthol/Tobacco 25 7 27.98 12.41 44.3 % 18.56 66 % Tobacco 25 8 25.78 6.19 24.0 % 13.28 52 % Unflavored 25 8 28.55 5.90 20.7 % 17.31 61 % Menthol/Tobacco 35 7 32.64 10.21 31.3 % 17.20 53 % Tobacco 35 8 30.40 7.39 24.3 % 19.10 63 % Unflavored 35 8 33.63 6.98 20.7 % 13.25 39 % * The number of laboratory data sets after removal of outliers.
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Table 7: Repeatability (r) and Reproducibility (R) Limits for blank subtracted Acetaldehyde (μg/g) under CRM 81 Conditions
Added Product N Labs* Average r r % R R % (μg/g) Menthol/Tobacco 15 8 6.73 3.86 57.3 % 7.28 108 % Tobacco 15 9 9.01 3.51 38.9 % 12.13 135 % Unflavored 15 9 9.20 4.15 45.1 % 12.45 135 % Menthol/Tobacco 25 7 13.17 5.31 40.3 % 14.90 113 % Tobacco 25 9 16.81 9.65 57.4 % 19.33 115 % Unflavored 25 8 13.58 5.45 40.1 % 16.48 121 % Menthol/Tobacco 35 8 20.87 7.86 37.7 % 29.77 143 % Tobacco 35 9 22.53 12.97 57.6 % 34.48 153 % Unflavored 35 9 22.57 13.28 58.8 % 36.85 163 % * The number of laboratory data sets after removal of outliers.
Table 8: Repeatability (r) and Reproducibility (R) Limits for blank subtracted Formaldehyde (μg/g) under CRM 81 Conditions
Added Product N Labs* Average r r % R R % (μg/g) Menthol/Tobacco 15 8 7.44 22.71 305.2 % 22.76 306 % Tobacco 15 9 7.89 9.77 123.7 % 11.10 141 % Unflavored 15 8 9.35 2.68 28.6 % 3.99 43 % Menthol/Tobacco 25 8 18.45 11.70 63.4 % 20.27 110 % Tobacco 25 9 16.79 28.15 167.6 % 28.15 168 % Unflavored 25 9 12.15 5.82 47.9 % 45.91 378 % Menthol/Tobacco 35 8 23.53 9.81 41.7 % 14.12 60 % Tobacco 35 9 20.67 9.56 46.3 % 21.95 106 % Unflavored 35 8 22.05 6.98 31.6 % 16.65 76 % * The number of laboratory data sets after removal of outliers.
6. Data Interpretation
For each of the products tested, the amount of formaldehyde and acetaldehyde was corrected for the total aerosol mass produced and reported on a per gram of aerosol basis. This allowed the data to be compared regardless of the total aerosol mass produced or the number of puffs per replicate. The results for the native e-liquid samples demonstrate that the test devices are producing low levels of formaldehyde and acetaldehyde (Table 5 and Table 6) with all native e-liquids yielding average values of approximately 2-4 µg/g and 8-11 µg/g of acetaldehyde and formaldehyde respectively. Given the low native levels of acetaldehyde and formaldehyde produced, the observed average reproducibility for e-liquids, which ranged from 196 % and 137 %, respectively, was not unexpected. E-vapour devices are well known to be highly
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variable for the production of acetaldehyde and formaldehyde[1,2]. When the e-liquids were fortified with formaldehyde and acetaldehyde, the observed reproducibility of the study results was greatly improved. For formaldehyde, reproducibility was improved as the fortification level increased. At the 35 µg/gram fortification level, the reproducibility of formaldehyde in the aerosol was found to be 53 %, 63 % and 39 % for the menthol/tobacco, tobacco and unflavoured e-liquids respectively. For acetaldehyde, reproducibility was also improved as the fortification level increased with the greatest improvement seen at the 25 µg/g fortification level. At the 25 µg/g fortification level, the reproducibility of acetaldehyde in the aerosol was found to be 114 %, 110 % and 108 % for the menthol/tobacco, tobacco and unflavoured e-liquids respectively. These results are comparable to the reproducibility observed for formaldehyde in mainstream cigarette smoke, under the intense smoking regime[3], which ranged from 47 % to 98 %. The fortified study results were blank corrected for the amount measured in the native samples. This was performed in an attempt to correct for differences in potential in the devices provided to each laboratory. Results for the fortified samples were corrected, within each laboratory, by subtracting the average native amount of formaldehyde and acetaldehyde at each fortification level. The blank subtracted results did not show improved reproducibility over the non-blank subtracted data and generally lead to poorer r and R values versus the non-blank corrected results. These results indicate that blank values are not predictive of the measured values of the fortified samples. The fortification data does show that formaldehyde and acetaldehyde added to the e-liquids is transferred to the resulting aerosol with a transfer efficiency of approximately 60 %.
7. Recommendations
The study results were reviewed during the April 2020 EVAP Sub-Group meeting. During that meeting it was decided that this collaborative study supported the publication of a CORESTA Recommended Method for the analysis of formaldehyde and acetaldehyde in e-vapour aerosol.
[1] Belushkin M, Tafin Djoko D, Esposito M, et al. Selected Harmful and Potentially Harmful Constituents Levels in Commercial e-Cigarettes. Chem Res Toxicol. 2020;33(2):657-668. [2] Gillman IG, Pennington ASC, Humphries KE, Oldham MJ. Determining the impact of flavored e-liquids on aldehyde production during Vaping. Regul Toxicol Pharmacol. 2020;112:104588. [3] CORESTA. Recommended Method No. 74: Determination of Selected Carbonyls in Mainstream Cigarette Smoke by HPLC. 2014; http://www.coresta.org/Recommended_Methods/CRM_74.pdf. Accessed May 2018.
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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 2, 2019
Written by: Matt S. Melvin (Altria Client Services, Study Coordinator) and I. Gene Gillman (Enthalpy Analytical, SG Secretary)
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1. Introduction
This protocol describes the initial study to identify a method for the analysis of carbonyl compounds in e-vapour product aerosols. The analytes of interest include the following: • Formaldehyde • Acetaldehyde
2. Objective
The participating laboratories are to determine the amounts of the analytes of interest in the aerosol generated from the combination of an Aspire Nautilus with 1.8 Ω tank coil and an battery provided by Evolve. Aspire CF G-Power or CF Passthrough battery (See Appendix A for pictures) have also been shown to be effective for this study if availability is an issue. The participating laboratories will acquire the samples for this study from Alternative Ingredients (see below for information) and fortify these samples in house according to the details provided below. The participating laboratories are requested to use the provided method in this collaborative study.
3. Time schedule and Data Reporting
Date Activity January 3, 2019 Distribute draft protocol and method Laboratories state their intention to participate and order January 14, 2019 supplies January 15, 2019 Distribute final protocol, method, and data reporting sheet January 15 to February 17, 2019 Participants order and receive the samples February 18 to March 14, 2019 Laboratories conduct the study Laboratories submit results by this date Statistical evaluation and preparation of results Discuss results at 2019 Fall EVAP meeting
This time schedule will allow for tabulation of the results, completion of the statistical analysis, and presentation at the 2019 Fall EVAP meeting which is scheduled for October 2019 in Hamburg, Germany.
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4. Participating Laboratories
Following receipt of this protocol, the participating laboratories in this study will confirm and notify Matt Melvin and Gene Gillman of their active participation.
Participating Laboratories Altria ASL Analytic Service Laboratory GmbH British American Tobacco Enthalpy Analytical LLC, Durham NC Enthalpy Analytical LLC, Richmond Va Eos Scientific Essentra KT&G Japan Tobacco, Inc. JT International GmbH JTI-Ökolab (Austria Tabak) Labstat International Inc Philip Morris R&D R.J. Reynolds Tobacco Company Zhengzhou Tobacco Research Institute of CNTC
5. Samples
5.1 Selection Each participating laboratory should request the following e-liquid products. The samples will be fortified at three different levels with formaldehyde and acetaldehyde in-house by the participating laboratories. Unfortified samples will also be analyzed.
2019 CORESTA Carbonyl Aerosol CS Products
Flavor Target Nicotine Target PG Target Gly Target Added Sample ID Profile (%w/w) (%w/w) (%w/w) H2O (%w/w) CS_Carb01 unflavored 1.5 47 47 0 CS_Carb02 tobacco 1.5 47 47 0 tobacco CS_Carb03 1.5 47 47 0 menthol
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5.2 Supplies and Shipping
E-Liquids: Shipments of the 2019 Carbonyl CS products may be obtained through Eduardo Berea from Alternative Ingredients Inc. 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 Eduardo Berea.
Please use the following table:
Company Name Address
City State & ZIP Country Attention Phone Fax E-mail 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. Aspire Nautilus Tank System: Nils Rose from Borgwaldt has secured the Aspire Nautilus I device. These devices can be ordered directly from Borgwaldt (Lea Jansen) with the following product numbers: Part#: 80436010 – Smoke machine adaptor - €20,00 (if required) Part#: 80436020 – Nautilus Test Kit – Two each - €50,00 plus freight (recommend purchasing two sets for a total of four devices) Batteries: John Bellinger from Evolve has agreed to supply regulated batteries for the study. Please send him your request at the above email address with the same shipping information provided to Eduardo.
5.3 Receipt Upon receipt, the samples are to be stored at ambient temperature.
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5.4 Within Laboratory Sample Preparation
FORTIFICATION SOLUTION Participating laboratories are encouraged to use the fortification solution from AccuStandard (Prod# S-78185; Lot# 218051164) which contains 50 µg/mL of the analytes in propylene glycol. Laboratories may also use the fortification solution(s) of their choice as long as the fortification solution(s) provide fortified samples with acceptable accuracy for the analytes of interest.
SAMPLE FORTIFICATION The samples are to be stored at ambient temperature prior to fortification. Four levels of each sample are to be tested, one level per day, according to the amounts detailed below when using the Accustandard fortification solution. Larger amounts of fortified sample may be prepared as long as the appropriate scaling is applied. If a fortification solution other than the Accustandard solution is used then the laboratories will have to develop the appropriate fortification scheme to reach the indicated levels. The fortified and unfortified samples should be analyzed for analyte content prior to aerosol generation and the results reported. Samples should be mixed thoroughly prior to adding the two millilitres of liquid to the Aspire tank device.
Concentration of Sample Amount Fortification Total E-LIQUID Approximate Analyte in E- (mg) Solution (mg) (mg) PG:VG Ratio LIQUID (µg /g)
2000 0 2000 0 50:50 1400 600 2000 15 65:35 1000 1000 2000 25 75:25 600 1400 2000 35 85:15
AEROSOL GENERATION AND COLLECTION The air flow on the tank system should be set to the smallest opening with an orifice of 0.9 mm. There is a detailed diagram in the manual that comes with the device. The collections are to be performed at a 12 W power setting. Aerosol collection shall be performed using the CORESTA recommended puff regime of 55 mL puff volume, 3 sec puff duration, and 30 sec puff period under a square wave puff profile. It is recommended that all samples are collected between 45 to 90 degrees from vertical. Puff collections of 30 to 35 puffs per collection shall be collected which should generate 300 mg of aerosol. The number of puffs per block to achieve the 300 mg of aerosol per collection should be verified by the laboratories and the number of puffs adjusted to meet the 300 mg of collected aerosol target. The same coil must be used for each liquid type (unflavoured, tobacco etc). Coils must be cleaned between each spiking level. It is recommended that coils are removed from the tank, at the end of each n=3 collection, and rinsed with methanol and allowed to dry overnight. The coils should be allowed to equilibrate in the liquid for at least 30 minutes before sample collection. Participants are to verify that the unfortified samples are producing low levels of analyte ( ≤ 10 µg/ g of aerosol). Any coil that produces greater values should be discarded.
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6. Analysis
Samples will be collected in triplicate by collecting 3 puff blocks from the same device. Replicate one will be puffs 1 through 30, replicate two will be puffs 31 through 60, and replicate three is puffs 61 through 90. This collection shall be done for each sample type and each fortification level. The samples will be analyzed by the provided method. Samples where the values of analyte are greater than ten times the expected value are to be considered outliers and the analysis repeated.
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 Gene Gillman. 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 ( 8. Statistical Analysis A statistical analysis in general conformance with ISO 5725-2:1994 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 2019 EVAP Sub-Group meeting. EVAP-127-2-CTR Determination of Formaldehyde and Acetaldehyde – November 2020 15/37 APPENDIX B: Analysis Method 2019 EVAP Sub-Group Carbonyl Collaborative Study Carbonyl Method of Analysis for Liquids and E-Vapour Product Aerosol: Phase II - Aerosols EVAP-127-2-CTR Determination of Formaldehyde and Acetaldehyde – November 2020 16/37 1.0 Scope and Application: The purpose of this document is to describe the procedures used for quantitation of formaldehyde and acetaldehyde in electronic cigarette (e-cigarette) aerosols utilizing HPLC-UV. E-cigarette devices used in this method may be manual (push-button) or automatic. The device e-liquids used to generate aerosol samples may be unflavored or flavored and include extracted tobacco derived flavors. This method may be applied to e-liquid samples as well. 2.0 Summary of Method: E-cigarettes are smoked using a smoking machine. The aerosol is passed through a sampling train consisting of a Cambridge filter pad and a single fritted-tipped impinger containing a derivatization solution. The derivatization solution generates a colored adduct that can be visualized on an HPLC with a UV-Vis Diode Array Detector or Variable Wavelength Detector. Samples are collected using a standard puffing regimen. 3.0 Definitions: 3.1 Reagent Blank – an aliquot of the reagent (acetonitrile) used to prepare the standards and/or dilutions. The reagent blank is analyzed to ensure that no contamination is introduced by the acetonitrile. 3.2 Aerosol Blank – an aliquot of quenched impinger solution associated with a puff block collected from an empty port during aerosol sample collection. The Aerosol Blank is carried through all of the same sample preparation and analysis steps as the samples. 4.0 Safety: 4.1 All laboratory personnel handling chemicals used in this procedure should familiarize themselves with the appropriate MSDSs for health and safety information. Personnel should wear appropriate personal protective equipment when handling hazardous substances. This equipment should include safety glasses, gloves, and/or laboratory coat. 4.2 There are a number of hazardous chemicals used in this method, including the following: 4.2.1 Acetonitrile is toxic and flammable. 4.2.2 Several of the analytes are toxic and irritants. 4.2.3 Tetrahydrofuran is flammable and should be handled with nitrile gloves. 4.2.4 Dry DNPH is explosive, may cause liver damage, and can be absorbed transdermally. 4.2.5 Isopropanol is an irritant and flammable. 4.2.6 Pyridine has a strong odor and is an irritant. 5.0 Equipment and Supplies: Note that equivalent supplies and equipment may be used provided that the equivalence is demonstrated. 5.1 Balance, 4 to 5-place, 0.01 to 0.1 mg precision. 5.2 Class-A glassware: graduated cylinders, volumetric pipettes, and volumetric flasks. 5.3 Adjustable pipettes. EVAP-127-2-CTR Determination of Formaldehyde and Acetaldehyde – November 2020 17/37 5.4 Amber vials with caps, 8mL. 5.5 Glass autosampler vials with crimp or screw tops. 5.6 Bottle-top dispenser capable of dispensing 20 mL (±2 %). 5.7 Impinger: Bubbler Bottle, 24/40 outer joint, 215 mm, O.L. Hgt x 30 mm O.D. 5.8 Stem: Bubbler insert with fritted tip, 24/40 inner o-ring and 12/5 socket/ball o-ring connections. 5.9 HPLC columns: 5.9.1 Guard Column: Waters XTerra MS C18 20 mm x 3.9 mm x 5µm or equivalent. 5.9.2 Zorbax SB-C18, 4.6mm x 15cm, 1.8µm, 600bar or equivalent. 5.10 Instrument: HPLC system with UV detection capable of performing the method below. 6.0 Reagents and Standards: Note that equivalent reagents and reference materials may be used provided that the equivalence is demonstrated. 6.1 Deionized Water (Resistance ≥ 10 MΩ•cm). 6.2 Acetonitrile (ACN). 6.3 Phosphoric acid (H3PO4, 85 %, or 10 % (v/v) Aqueous Solution). 6.4 DNPH-HCL, DNPH may also be re-crystallized in-house to improve purity, if necessary. 6.5 Pyridine. 6.6 Aldehydes/Ketones DNPH Standard, Restek 15µg/mL in acetonitrile (P/N 31808). 6.7 Solution Preparation: Prepare appropriately proportioned amounts of the solutions listed below. All solutions must be equilibrated to room temperature prior to use. Use graduated cylinders and calibrated pipettes to combine components. 6.7.1 10 % H3PO4: Prepare by bringing 117.6 mL of 85 % H3PO4 to 1 L with DIUF. Vendor-prepared 10 % H3PO4 may be used instead. 6.7.2 Prepare trapping solution by dissolving 1.0 g DNPH-HCL in 500 mL of ACN, combine with 40 mL of 10 % H3PO4 and bring to 1 L with DIUF. Store the solution in an amber glass bottle at room temperature. Discard the solution when the Aerosol Blank contains an analyte concentration greater than that of the LOQ. The use of DNPH free base containing water may also be used with the appropriate mass scaling to provide the same final concentration of DNPH solution 6.8 Reference Standard Preparation: 6.8.1 Using the stock solution(s) in section(s) 6.6, prepare at least 5 working calibration standards using volumetric glassware at nominal concentrations that will bracket expected sample concentrations. 6.8.2 Store standards in amber glass containers at 4 °C (±2 °C). EVAP-127-2-CTR Determination of Formaldehyde and Acetaldehyde – November 2020 18/37 7.0 Sample Preservation, Storage, and Handling: 7.1 Analyze the e-cigarette aerosol solutions within 2 days, if stored at room temperature; or analyze within 7 days when stored at 4 °C (±2 °C). 8.0 Calibration: 8.1 Initial Calibration: 8.1.1 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 0.995. The curve type is linear, the origin is ignored, and the weight is linear (amt). The initial calibration standards are acceptable if they are within 15 % of their assigned values for the LOQ (low standard) and within 10 % for all other standards. 8.1.2 If the calibration does not meet criteria in section 8.1.1, check the instrument for problems; analyze fresh standard aliquots and/or fresh standards prepared from stock solutions. 9.0 Procedure: 9.1 Sample Preparation: 9.1.1 Label any and all tubes and vials needed for sample preparation. Labels will be generated by a computer or hand-written on labels that are resistant to smearing by solvent etc. 9.1.2 Using the bottle top dispenser, add 35 mL of trapping solution to a separate impinger with a fritted stem insert for each sample to be collected. 9.1.3 Assemble trapping system of the smoke machine as follows: preweighed Cambridge filter pad/holder → impinger → backup filter pad → smoking machine. Aerosol collection is carried out as specified in the study protocol. 9.1.4 After aerosol collection is complete, weight the Cambridge filter pad/holder for determination of collected aerosol mass. 9.1.5 Transfer the Cambridge filter pad to a 60 mL vial. 9.1.6 Mix the contents of the impinger. Using a clean disposable pipette, rinse the inside of the impinger stem five (5) times being sure to wet the inside. 9.1.7 Transfer the impinger solution to the 60 mL flask containing the Cambridge filter pad. 9.1.8 Extract the filter pad by mechanical shaking for 20 min. 9.1.9 Transfer 5 mL of impinger solution into an 8 mL glass vial containing 0.25 mL of pyridine. This solution may become cloudy and can be filtered into an auto-sampler vial using a 0.45µm PTFE syringe filter and appropriate disposable syringe. 9.1.10 Transfer an aliquot to an auto-sampler vial and close the vial with crimp- cap. The samples are now ready for analysis. 9.1.11 Samples may be diluted in neutralized trapping solution if necessary. Document the dilution factor and dilution preparation. 9.1.12 Place impingers and stems in hot soapy water after use. EVAP-127-2-CTR Determination of Formaldehyde and Acetaldehyde – November 2020 19/37 9.2 Instrument conditions using the Zorbax SB-C18 column. 9.2.1 Mobile Phase A – 100 % DIUF H2O 9.2.2 Mobile Phase B – 100 % Acetonitrile 9.2.3 Flow Rate: 1.0 mL/min 9.2.4 Run Time: 12 min with a 2 min post time 9.2.5 Pump Timetable: Time (min) % Mobile Phase A % Mobile Phase B 0.00 35 65 1.49 35 65 1.50 45 55 3.00 45 55 3.01 35 65 3.56 35 65 7.50 25 75 8.00 0 100 9.50 0 100 9.55 35 65 12.00 35 65 9.2.6 Injection Volume: 5µL 9.2.7 Column Compartment Temperature: 32 ºC 9.2.8 DAD: Signal A: 360 nm, 16 nm slit, reference 510 nm, 100 nm slit UV and Vis lamp is required Pre-run and post-run balancing Margin for negative absorbance: 100 mAU 9.2.9 VWD: Signal wavelength: 360 nm Signal peakwidth: > 0.1 min Prerun balancing: Yes Margin for negative absorbance: 100 mAU Signal Polarity: Positive Enable analysis when lamp is off: No 9.3 Sample Analysis: 9.3.1 Sample concentrations shall be within the instrument’s calibration range; if sample concentrations exceed the range, dilute the impinger sample using neutralized trapping solution. EVAP-127-2-CTR Determination of Formaldehyde and Acetaldehyde – November 2020 20/37 10.0 Data Analysis and Calculations: 10.1 Acetaldehyde may elute as two peaks because its corresponding hydrazone exists in two isomers. The peak areas should be integrated consistently in both the standards and samples. This may be accomplished by drawing a single baseline tangent across both peaks. 10.2 Samples are neutralized with pyridine after collection. This “dilution” must be accounted for in sample calculations. When method volumes are followed, multiply the calculated carbonyl concentration by total sample volume to determine yield in µg/mL as follows: (S - B) * total sample volume = yield (µg/mL) Where S is the sample concentration and B is the blank concentration. 11.0 References: 11.1 Determination of Selected Carbonyls in Mainstream Tobacco Smoke, Health Canada, Tobacco Control Programme 11.2 UK Smoke Constituents Study: Part 2 Method: Determination of Eight Carbonyl Yields in Cigarette Smoke by High Performance Liquid Chromatography 11.3 ISO 3308:2000 Routine analytical cigarette-smoking machine – definitions and standard conditions 11.4 Determination of Carbonyl Compounds by Reversed-Phase-High-Performance Liquid Chromatography – Dionex Application Note 97 ISO 3308:1991 Sections 4.2 – 4.4 11.5 Intorp M, Purkis S, Whittaker M, Wright W. Determination of “Hoffmann Analytes” in Cigarette Mainstream Smoke. The CORESTA 2006 Joint Experiment. Contributions to Tobacco Research 2009;23-4:161–202, https://content.sciendo.com/view/journals/cttr/23/4/cttr.23.issue-4.xml 12.0 Tables, Diagrams, and Flow Charts: 12.1 Appendix A – Example Chromatogram – Agilent Zorbax SB-C18 column EVAP-127-2-CTR Determination of Formaldehyde and Acetaldehyde – November 2020 21/37 Analysis Method - Appendix A Example Standard Chromatogram – Agilent Zorbax SB-C18 Example Sample Chromatogram – Agilent Zorbax SB-C18 EVAP-127-2-CTR Determination of Formaldehyde and Acetaldehyde – November 2020 22/37 APPENDIX C: Raw Data No Spiking Aerosol Aerosol Product Lab Formaldehyde Acetaldehyde Puffs Mass Mass Spiking (μg/g) (μg/g) (mg) (mg/puff) Menthol/Tobacco 1 50 444.9 8.90 0 6.05 1.91 Menthol/Tobacco 1 50 456.5 9.13 0 6.11 1.83 Menthol/Tobacco 1 50 424.9 8.50 0 6.54 2.55 Menthol/Tobacco 2 45 382.2 8.49 0 8.30 1.57 Menthol/Tobacco 2 45 428.4 9.52 0 8.17 1.39 Menthol/Tobacco 2 50 424.0 8.48 0 8.11 1.63 Menthol/Tobacco 3 25 348.5 13.94 0 5.98 1.90 Menthol/Tobacco 3 28 294.6 10.52 0 6.34 1.95 Menthol/Tobacco 3 25 293.7 11.75 0 6.45 2.06 Menthol/Tobacco 4 25 305.2 12.21 0 6.26 1.32 Menthol/Tobacco 4 25 274.3 10.97 0 8.71 1.47 Menthol/Tobacco 4 25 309.5 12.38 0 9.86 2.49 Menthol/Tobacco 6 37 345.9 9.35 0 9.35 1.97 Menthol/Tobacco 6 37 349.3 9.44 0 9.47 2.42 Menthol/Tobacco 6 35 339.2 9.69 0 9.48 2.33 Menthol/Tobacco 7 50 348.7 6.97 0 5.85 0.61 Menthol/Tobacco 7 50 358.6 7.17 0 3.20 0.00 Menthol/Tobacco 7 50 271.8 5.44 0 1.44 0.38 Menthol/Tobacco 8 30 305.9 10.20 0 18.02 2.88 Menthol/Tobacco 8 30 322.3 10.74 0 16.62 2.49 Menthol/Tobacco 8 30 322.4 10.75 0 17.44 2.54 Menthol/Tobacco 9 32 227.9 7.12 0 78.95 6.61 Menthol/Tobacco 9 32 223.4 6.98 0 82.68 6.75 Menthol/Tobacco 9 31 223.1 7.20 0 68.00 4.95 Tobacco 1 50 396.4 7.93 0 6.50 1.76 Tobacco 1 50 395.0 7.90 0 6.09 2.25 Tobacco 1 50 416.1 8.32 0 5.80 2.04 Tobacco 2 45 425.8 9.46 0 8.01 1.56 Tobacco 2 45 431.9 9.60 0 8.04 1.30 Tobacco 2 45 410.8 9.13 0 7.75 1.55 Tobacco 3 30 312.4 10.41 0 5.09 2.22 Tobacco 3 30 282.3 9.41 0 11.40 2.38 Tobacco 3 25 304.0 12.16 0 9.37 2.19 Tobacco 4 25 272.6 10.90 0 7.55 2.02 Tobacco 4 25 301.2 12.05 0 6.71 1.22 Tobacco 4 25 314.6 12.58 0 8.41 0.70 Tobacco 5 75 865.9 11.55 0 7.82 2.73 Tobacco 5 30 346.2 11.54 0 8.53 3.09 Tobacco 5 35 368.7 10.53 0 8.92 4.68 EVAP-127-2-CTR Determination of Formaldehyde and Acetaldehyde – November 2020 23/37 No Spiking Aerosol Aerosol Product Lab Formaldehyde Acetaldehyde Puffs Mass Mass Spiking (μg/g) (μg/g) (mg) (mg/puff) Tobacco 6 37 332.9 9.00 0 7.84 1.93 Tobacco 6 37 375.9 10.16 0 6.75 1.91 Tobacco 6 35 355.8 10.17 0 6.40 1.91 Tobacco 7 50 342.6 6.85 0 6.12 0.00 Tobacco 7 50 412.7 8.25 0 2.90 0.25 Tobacco 7 50 353.7 7.07 0 1.73 1.12 Tobacco 8 30 327.0 10.90 0 18.54 2.69 Tobacco 8 30 337.0 11.23 0 16.83 2.23 Tobacco 8 30 342.3 11.41 0 15.88 2.21 Tobacco 9 32 336.2 10.51 0 116.31 6.94 Tobacco 9 32 345.5 10.80 0 108.64 6.35 Tobacco 9 32 317.5 9.92 0 118.63 6.45 Unflavored 1 50 417.5 8.35 0 8.57 3.92 Unflavored 1 50 406.6 8.13 0 9.77 4.35 Unflavored 1 50 418.8 8.38 0 10.10 4.26 Unflavored 2 45 373.5 8.30 0 14.40 3.34 Unflavored 2 45 355.1 7.89 0 13.66 2.63 Unflavored 2 50 419.9 8.40 0 14.34 3.10 Unflavored 3 28 344.8 12.31 0 7.73 2.52 Unflavored 3 25 314.2 12.57 0 7.37 2.46 Unflavored 3 25 310.5 12.42 0 7.66 2.40 Unflavored 4 25 294.7 11.79 0 15.71 4.24 Unflavored 4 25 249.8 9.99 0 19.71 6.77 Unflavored 4 25 297.0 11.88 0 15.34 4.58 Unflavored 5 75 748.7 9.98 0 13.06 4.15 Unflavored 5 30 265.9 8.86 0 13.33 5.13 Unflavored 5 35 323.6 9.25 0 13.16 4.43 Unflavored 6 37 357.1 9.65 0 10.50 4.12 Unflavored 6 37 406.1 10.98 0 9.50 3.57 Unflavored 6 35 388.5 11.10 0 9.32 3.55 Unflavored 7 50 356.4 7.13 0 5.68 3.31 Unflavored 7 50 398.1 7.96 0 2.04 0.00 Unflavored 7 50 372.6 7.45 0 1.97 0.00 Unflavored 8 30 322.4 10.75 0 20.58 2.43 Unflavored 8 30 329.1 10.97 0 17.72 2.70 Unflavored 8 30 334.8 11.16 0 16.57 2.39 Unflavored 9 40 332.0 8.30 0 128.95 10.20 Unflavored 9 40 333.6 8.34 0 135.40 9.57 Unflavored 9 40 320.0 8.00 0 127.77 8.64 EVAP-127-2-CTR Determination of Formaldehyde and Acetaldehyde – November 2020 24/37 Spiking=15 (μg/g) Aerosol Aerosol Product Lab Formaldehyde Acetaldehyde Puffs Mass Mass Spiking (μg/g) (μg/g) (mg) (mg/puff) Menthol/Tobacco 1 50 457.9 9.16 15 17.85 11.05 Menthol/Tobacco 1 50 457.0 9.14 15 15.23 9.11 Menthol/Tobacco 1 50 471.6 9.43 15 16.60 10.10 Menthol/Tobacco 2 45 445.3 9.90 15 19.60 8.57 Menthol/Tobacco 2 45 424.6 9.44 15 19.63 9.28 Menthol/Tobacco 2 45 565.4 12.56 15 17.57 7.24 Menthol/Tobacco 3 25 304.7 12.19 15.15 16.70 11.56 Menthol/Tobacco 3 25 318.2 12.73 15.15 16.88 10.54 Menthol/Tobacco 3 25 330.2 13.21 15.15 17.89 11.71 Menthol/Tobacco 4 25 287.6 11.51 15 18.27 7.28 Menthol/Tobacco 4 25 289.5 11.58 15 19.17 9.14 Menthol/Tobacco 4 25 305.3 12.21 15 18.90 9.03 Menthol/Tobacco 6 35 314.4 8.98 15 13.79 6.55 Menthol/Tobacco 6 35 329.8 9.42 15 14.32 6.57 Menthol/Tobacco 6 35 329.3 9.41 15 15.85 7.09 Menthol/Tobacco 7 50 471.6 15.00 9.43 8.68 4.18 Menthol/Tobacco 7 50 427.7 15.00 8.55 7.38 3.88 Menthol/Tobacco 7 50 325.0 15.00 6.5 5.73 5.91 Menthol/Tobacco 8 30 401.1 13.37 15 27.33 7.27 Menthol/Tobacco 8 30 420.5 14.02 15 25.93 6.17 Menthol/Tobacco 8 30 416.8 13.89 15 29.09 6.37 Menthol/Tobacco 9 40 345.3 8.63 15 61.38 12.71 Menthol/Tobacco 9 39 408.2 10.47 15 61.42 17.33 Menthol/Tobacco 9 35 328.6 9.39 15 100.77 18.78 Tobacco 1 50 454.4 9.09 15 17.58 17.49 Tobacco 1 50 450.0 9.00 15 16.69 15.82 Tobacco 1 50 484.8 9.70 15 17.56 16.01 Tobacco 2 45 423.0 9.40 15 18.30 15.27 Tobacco 2 45 478.3 10.63 15 19.33 13.93 Tobacco 2 45 463.2 10.29 15 18.89 13.53 Tobacco 3 25 412.3 16.49 15.02 12.01 14.00 Tobacco 3 25 284.6 11.38 15.47 16.70 12.00 Tobacco 3 25 320.5 12.82 15.47 16.88 9.11 Tobacco 4 25 283.1 11.32 15 18.43 8.05 Tobacco 4 25 305.5 12.22 15 19.13 8.18 Tobacco 4 25 308.4 12.34 15 19.07 9.30 Tobacco 5 35 352.5 10.07 15 15.83 12.06 Tobacco 5 35 322.7 9.22 15 15.59 10.99 Tobacco 5 35 338.4 9.67 15 15.88 11.42 Tobacco 6 35 336.3 9.61 15 12.08 6.45 Tobacco 6 35 383.7 10.96 15 13.98 7.33 EVAP-127-2-CTR Determination of Formaldehyde and Acetaldehyde – November 2020 25/37 Spiking=15 (μg/g) Aerosol Aerosol Product Lab Formaldehyde Acetaldehyde Puffs Mass Mass Spiking (μg/g) (μg/g) (mg) (mg/puff) Tobacco 6 35 378.7 10.82 15 14.51 7.86 Tobacco 7 50 434.5 8.69 15 10.72 5.82 Tobacco 7 50 422.6 8.45 15 7.68 4.13 Tobacco 7 50 408.1 8.16 15 8.32 3.25 Tobacco 8 30 434.3 14.48 15 25.82 7.53 Tobacco 8 30 438.0 14.60 15 25.04 7.24 Tobacco 8 30 435.1 14.50 15 25.37 7.12 Tobacco 9 32 338.6 10.58 15 113.52 19.62 Tobacco 9 32 287.9 9.00 15 129.40 22.49 Tobacco 9 32 295.8 9.24 15 111.39 23.04 Unflavored 1 50 420.3 8.41 15 20.52 20.39 Unflavored 1 50 429.3 8.59 15 20.34 19.67 Unflavored 1 50 434.5 8.69 15 21.32 19.60 Unflavored 2 45 417.1 9.27 15 24.18 10.77 Unflavored 2 45 345.1 7.67 15 24.26 10.65 Unflavored 2 45 416.3 9.25 15 23.79 9.88 Unflavored 3 25 332.1 13.28 15.07 16.96 11.99 Unflavored 3 25 331.3 13.25 15.07 18.35 11.99 Unflavored 3 25 375.6 15.02 15.07 18.13 11.38 Unflavored 4 25 289.6 11.58 15 24.49 9.52 Unflavored 4 25 286.5 11.46 15 25.53 11.29 Unflavored 4 25 289.3 11.57 15 25.92 10.04 Unflavored 5 35 333.4 9.53 15 22.93 18.82 Unflavored 5 35 354.6 10.13 15 21.30 15.03 Unflavored 5 35 328.0 9.37 15 21.39 14.79 Unflavored 6 35 344.4 9.84 15 19.58 10.72 Unflavored 6 35 383.8 10.97 15 17.24 10.15 Unflavored 6 35 366.8 10.48 15 16.13 7.71 Unflavored 7 50 410.2 8.20 15 14.62 6.66 Unflavored 7 50 402.7 8.05 15 11.89 5.94 Unflavored 7 50 365.9 7.32 15 13.59 7.50 Unflavored 8 30 428.5 14.28 15 26.47 10.80 Unflavored 8 30 430.8 14.36 15 26.83 7.97 Unflavored 8 30 436.2 14.54 15 26.47 7.78 Unflavored 9 40 337.6 8.44 15 119.00 28.34 Unflavored 9 40 323.0 8.08 15 119.12 24.76 Unflavored 9 40 325.6 8.14 15 129.36 23.07 EVAP-127-2-CTR Determination of Formaldehyde and Acetaldehyde – November 2020 26/37 Spiking=25 (μg/g) Aerosol Aerosol Product Lab Formaldehyde Acetaldehyde Puffs Mass Mass Spiking (μg/g) (μg/g) (mg) (mg/puff) Menthol/Tobacco 1 50 457.9 9.16 25 17.90 11.10 Menthol/Tobacco 1 50 475.5 9.51 25 21.14 16.60 Menthol/Tobacco 1 50 431.2 8.62 25 21.48 14.59 Menthol/Tobacco 2 45 407.0 9.04 25 25.80 14.26 Menthol/Tobacco 2 45 445.0 9.89 25 28.48 14.73 Menthol/Tobacco 2 45 461.0 10.24 25 28.37 14.05 Menthol/Tobacco 3 25 350.5 14.02 24.59 25.38 17.77 Menthol/Tobacco 3 25 345.3 13.81 24.59 26.23 18.51 Menthol/Tobacco 3 25 336.4 13.46 24.59 25.08 19.08 Menthol/Tobacco 4 25 327.8 13.11 25 29.49 16.26 Menthol/Tobacco 4 25 338.1 13.52 25 25.87 14.13 Menthol/Tobacco 4 25 310.4 12.42 25 27.82 15.98 Menthol/Tobacco 6 35 334.4 9.55 25 22.20 7.75 Menthol/Tobacco 6 35 344.7 9.85 25 22.76 8.05 Menthol/Tobacco 6 35 331.6 9.47 25 28.32 10.36 Menthol/Tobacco 7 50 194.3 3.89 25 47.95 134.83 Menthol/Tobacco 7 50 439.8 8.80 25 31.61 58.79 Menthol/Tobacco 7 50 401.5 8.03 25 27.92 52.04 Menthol/Tobacco 8 30 359.2 11.97 25 32.77 12.15 Menthol/Tobacco 8 30 316.0 10.53 25 35.85 11.22 Menthol/Tobacco 8 30 308.4 10.28 25 35.18 10.70 Menthol/Tobacco 9 34 332.7 9.79 25 86.23 32.27 Menthol/Tobacco 9 34 327.3 9.63 25 87.11 25.89 Menthol/Tobacco 9 34 340.2 10.01 25 89.14 26.18 Tobacco 1 50 422.3 8.45 25 23.29 27.21 Tobacco 1 50 498.0 9.96 25 24.20 26.35 Tobacco 1 50 471.9 9.44 25 23.16 24.91 Tobacco 2 45 364.4 8.10 25 26.69 24.42 Tobacco 2 45 470.0 10.44 25 26.48 23.36 Tobacco 2 45 445.3 9.90 25 28.29 25.16 Tobacco 3 25 334.3 13.37 24.67 28.81 19.70 Tobacco 3 25 326.1 13.04 24.67 27.20 18.64 Tobacco 3 25 295.4 11.82 24.67 28.03 23.52 Tobacco 4 25 298.4 11.94 25 27.83 13.79 Tobacco 4 25 306.8 12.27 25 28.03 15.21 Tobacco 4 25 293.8 11.75 25 28.02 15.13 Tobacco 5 35 300.6 8.59 25 20.05 16.51 Tobacco 5 35 262.8 7.51 25 28.32 23.86 Tobacco 5 35 356.0 10.17 25 27.38 22.03 Tobacco 6 35 347.0 9.91 25 14.35 6.94 Tobacco 6 35 393.5 11.24 25 15.83 8.41 EVAP-127-2-CTR Determination of Formaldehyde and Acetaldehyde – November 2020 27/37 Spiking=25 (μg/g) Aerosol Aerosol Product Lab Formaldehyde Acetaldehyde Puffs Mass Mass Spiking (μg/g) (μg/g) (mg) (mg/puff) Tobacco 6 35 406.3 11.61 25 18.45 9.95 Tobacco 7 50 495.3 9.91 25 27.70 20.77 Tobacco 7 50 445.0 8.90 25 24.10 13.89 Tobacco 7 50 402.7 8.05 25 30.78 18.23 Tobacco 8 30 381.9 12.73 25 30.02 12.64 Tobacco 8 30 330.6 11.02 25 29.91 9.97 Tobacco 8 30 318.6 10.62 25 31.84 10.07 Tobacco 9 32 375.2 11.73 25 92.53 38.98 Tobacco 9 32 252.7 7.90 25 135.77 24.62 Tobacco 9 32 253.8 7.93 25 148.93 25.17 Unflavored 1 50 365.3 7.31 25 22.19 26.64 Unflavored 1 50 473.8 9.48 25 24.43 26.91 Unflavored 1 50 473.3 9.47 25 27.76 31.26 Unflavored 2 45 397.7 8.84 25 25.53 12.56 Unflavored 2 45 557.4 12.39 25 26.02 13.96 Unflavored 2 45 453.3 10.07 25 31.85 16.15 Unflavored 3 25 358.1 14.32 24.86 26.53 19.40 Unflavored 3 25 353.3 14.13 24.86 25.77 18.87 Unflavored 3 25 402.6 16.10 24.86 24.95 18.20 Unflavored 4 25 272.7 10.91 25 30.05 13.47 Unflavored 4 25 297.8 11.91 25 32.46 16.04 Unflavored 4 25 292.1 11.68 25 32.97 15.60 Unflavored 5 35 309.0 8.83 25 25.23 19.42 Unflavored 5 35 365.4 10.44 25 24.33 15.46 Unflavored 5 35 357.1 10.20 25 26.71 19.78 Unflavored 6 35 374.4 10.70 25 18.36 8.79 Unflavored 6 35 358.3 10.24 25 21.08 11.13 Unflavored 6 35 392.8 11.22 25 21.57 11.37 Unflavored 7 50 383.7 7.67 25 40.17 50.80 Unflavored 7 50 414.6 8.29 25 39.34 72.76 Unflavored 7 50 380.7 7.61 25 38.24 58.44 Unflavored 8 30 407.0 13.57 25 30.20 12.18 Unflavored 8 30 338.8 11.29 25 34.40 10.94 Unflavored 8 30 264.2 8.81 25 35.01 11.26 Unflavored 9 37 354.3 9.58 25 106.23 30.58 Unflavored 9 40 364.1 9.10 25 103.87 26.96 Unflavored 9 40 369.5 9.24 25 102.61 24.34 EVAP-127-2-CTR Determination of Formaldehyde and Acetaldehyde – November 2020 28/37 Spiking=35 (μg/g) Aerosol Aerosol Product Lab Formaldehyde Acetaldehyde Puffs Mass Mass Spiking (μg/g) (μg/g) (mg) (mg/puff) Menthol/Tobacco 1 50 467.7 9.35 35 27.91 19.48 Menthol/Tobacco 1 50 614.8 12.30 35 24.05 18.38 Menthol/Tobacco 1 50 581.2 11.62 35 26.05 18.96 Menthol/Tobacco 2 45 430.3 9.56 35 44.73 27.04 Menthol/Tobacco 2 45 564.7 12.55 35 34.91 21.13 Menthol/Tobacco 2 45 542.0 12.04 35 38.59 20.12 Menthol/Tobacco 3 25 348.8 13.95 34.79 31.79 25.44 Menthol/Tobacco 3 25 351.6 14.06 34.79 31.94 26.32 Menthol/Tobacco 3 25 345.9 13.84 34.79 32.73 26.56 Menthol/Tobacco 4 25 361.4 14.45 35 33.97 16.27 Menthol/Tobacco 4 25 359.9 14.40 35 34.20 18.48 Menthol/Tobacco 4 25 341.2 13.65 35 32.85 18.09 Menthol/Tobacco 6 35 412.3 11.78 35 30.08 13.95 Menthol/Tobacco 6 35 383.1 10.95 35 28.59 14.96 Menthol/Tobacco 6 34 383.1 11.27 35 28.59 14.87 Menthol/Tobacco 7 50 491.4 9.83 35 27.27 44.59 Menthol/Tobacco 7 50 561.8 11.24 35 22.50 37.04 Menthol/Tobacco 7 50 408.4 8.17 35 35.33 49.57 Menthol/Tobacco 8 30 300.5 10.02 35 34.81 11.8 Menthol/Tobacco 8 30 302.4 10.08 35 40.63 11.36 Menthol/Tobacco 8 30 283.8 9.46 35 43.89 14.92 Menthol/Tobacco 9 33 312.7 9.48 35 97.61 30.81 Menthol/Tobacco 9 34 344.7 10.14 35 93.16 29.44 Menthol/Tobacco 9 30 328.4 10.95 35 95.89 27.31 Tobacco 1 50 365.9 7.32 35 32.51 40.04 Tobacco 1 50 471.6 9.43 35 29.07 34.3 Tobacco 1 50 500.9 10.02 35 28.69 31.93 Tobacco 2 45 425.8 9.46 35 35.56 32.01 Tobacco 2 45 653.1 14.51 35 29.29 28.36 Tobacco 2 45 505.7 11.24 35 33.71 32.6 Tobacco 3 25 330.5 13.22 35.14 32.71 26.04 Tobacco 3 25 321.7 12.87 35.14 32.65 27.52 Tobacco 3 25 318.8 12.75 35.14 32.51 26.45 Tobacco 4 25 357.9 14.31 35 35.12 17.25 Tobacco 4 25 369.0 14.76 35 35.46 20.12 Tobacco 4 25 349.4 13.98 35 35.34 20.19 Tobacco 5 35 403.7 11.53 35 19.14 13.87 Tobacco 5 35 389.0 11.11 35 21.12 14.01 Tobacco 5 35 320.4 9.15 35 17.27 3.06 Tobacco 6 35 421.4 12.04 35 20.58 12.73 Tobacco 6 35 403.8 11.54 35 23.71 15.2 EVAP-127-2-CTR Determination of Formaldehyde and Acetaldehyde – November 2020 29/37 Spiking=35 (μg/g) Aerosol Aerosol Product Lab Formaldehyde Acetaldehyde Puffs Mass Mass Spiking (μg/g) (μg/g) (mg) (mg/puff) Tobacco 6 34 411.9 12.11 35 22.22 14.01 Tobacco 7 50 499.7 9.99 35 30.94 47.52 Tobacco 7 50 497.4 9.95 35 28.53 31.29 Tobacco 7 50 451.9 9.04 35 39.69 51.97 Tobacco 8 30 297.6 9.92 35 38.44 15.38 Tobacco 8 30 343.4 11.45 35 37.42 12.66 Tobacco 8 30 334.8 11.16 35 38.05 13.4 Tobacco 9 30 288.2 9.61 35 127.16 32.39 Tobacco 9 32 346.5 10.83 35 115.57 31.69 Tobacco 9 30 292.2 9.74 35 128.21 27.95 Unflavored 1 50 455.4 9.11 35 27.76 35.52 Unflavored 1 50 514.5 10.29 35 28.39 33.53 Unflavored 1 50 470.1 9.40 35 35.57 42.52 Unflavored 2 45 475.3 10.56 35 35.22 17.93 Unflavored 2 45 527.8 11.73 35 32.38 17.41 Unflavored 2 45 638.0 14.18 35 31.74 15.4 Unflavored 3 25 359.6 14.38 35.14 34.82 29.97 Unflavored 3 25 394.8 15.79 35.14 31.84 25.97 Unflavored 3 25 292.6 11.70 35.14 32.62 25.32 Unflavored 4 25 331.1 13.24 35 37.07 15.32 Unflavored 4 25 343.0 13.72 35 35.46 15.53 Unflavored 4 25 329.3 13.17 35 37.05 16.41 Unflavored 5 35 426.2 12.18 35 32.10 32.15 Unflavored 5 35 389.3 11.12 35 32.56 30.07 Unflavored 5 35 458.8 13.11 35 30.32 22.74 Unflavored 6 35 393.1 11.23 35 26.50 15.85 Unflavored 6 35 434.1 12.40 35 25.57 15.7 Unflavored 6 − − − 35 − − Unflavored 7 50 437.8 8.76 35 36.76 47.43 Unflavored 7 50 470.1 9.40 35 33.97 43.3 Unflavored 7 50 404.7 8.09 35 41.46 60.71 Unflavored 8 30 327.5 10.92 35 38.68 14.23 Unflavored 8 30 342.5 11.42 35 40.08 15.41 Unflavored 8 30 346.2 11.54 35 43.10 16.58 Unflavored 9 38 300.1 7.90 35 128.06 40.62 Unflavored 9 38 301.8 7.94 35 118.37 29.51 Unflavored 9 40 348.4 8.71 35 107.23 27.15 EVAP-127-2-CTR Determination of Formaldehyde and Acetaldehyde – November 2020 30/37 APPENDIX D: Raw Data Plots EVAP-127-2-CTR Determination of Formaldehyde and Acetaldehyde – November 2020 31/37 EVAP-127-2-CTR Determination of Formaldehyde and Acetaldehyde – November 2020 32/37 EVAP-127-2-CTR Determination of Formaldehyde and Acetaldehyde – November 2020 33/37 EVAP-127-2-CTR Determination of Formaldehyde and Acetaldehyde – November 2020 34/37 EVAP-127-2-CTR Determination of Formaldehyde and Acetaldehyde – November 2020 35/37 EVAP-127-2-CTR Determination of Formaldehyde and Acetaldehyde – November 2020 36/37 EVAP-127-2-CTR Determination of Formaldehyde and Acetaldehyde – November 2020 37/37