Cooperation Centre for Scientific Research Relative to Tobacco E-Vapour Sub-Group CORESTA Recommended Method No. 84 DETERMINATION OF GLYCERIN, PROPYLENE GLYCOL, WATER, AND NICOTINE IN THE AEROSOL OF E-CIGARETTES BY GAS CHROMATOGRAPHIC ANALYSIS June 2017
CORESTA RECOMMENDED METHOD Nº 84 Title: DETERMINATION OF GLYCERIN, PROPYLENE GLYCOL, WATER, AND NICOTINE IN THE AEROSOL OF E-CIGARETTES BY GAS CHROMATOGRAPHIC ANALYSIS Status: Valid Note: Document history: Date of review Information March 2017 Version 1 June 2017 Version 2 - Re-edition of formula p. 9 CRM No. 84 June 2017 2/11
CORESTA RECOMMENDED METHOD N 84 DETERMINATION OF GLYCERIN, PROPYLENE GLYCOL, WATER, AND NICOTINE IN THE AEROSOL OF E-CIGARETTES BY GAS CHROMATOGRAPHIC ANALYSIS (March 2017) 0. INTRODUCTION In 2015, the CORESTA E-Cigarette Task Force (changed to E-Vapour Sub-Group in 2016) conducted a collaborative study for the determination of glycerin, propylene glycol, nicotine, and water using gas chromatography. Nineteen laboratories participated in the study. The method specified in this study was shown to be appropriate for the analytes of interest for aerosol from e-vapour products. The repeatability and reproducibility values of this method have been assessed according to ISO 5725-2:1994 recommendations and are included. 1. FIELD OF APPLICATION This method is applicable to analysis of glycerin, propylene glycol, nicotine, and water in trapped e-cigarette aerosol. 2. NORMATIVE REFERENCES CORESTA Recommended Method N 81 2015: Routine analytical machine for e-cigarette aerosol generation and collection definitions and standard conditions. 3. PRINCIPLE Aerosol is generated and collected from electronic cigarettes by a standard procedure. The collected matter is dissolved in a solvent and the glycerin, propylene glycol (PG), water, and nicotine content of this solution are determined by gas chromatography. Aerosol collected mass may be determined gravimetrically. Results are expressed as the weight of analyte collected per device, per weight of collected aerosol, per puff, or per puff set as warranted. 4. APPARATUS 4.1. A standard aerosol collection system and trapping devices (i.e. filter pads) complying with CORESTA Recommended Method N 81. 4.2. Typical laboratory glassware such as volumetric flasks and extraction vessels. CRM No. 84 June 2017 3/11
4.3. Mechanical shaker. 4.4. Analytical balance, suitable for measuring to the nearest 0,1 mg. 4.5. A gas chromatograph (GC) equipped with a flame ionization detector (FID) and data collection for glycerin, propylene glycol, or nicotine analysis. 4.6. A gas chromatograph (GC) equipped with a thermal conductivity detector (TCD) and data collection for water analysis. 4.7. Gas chromatography column(s), capillary or packed, capable of distinct separation of the peaks for the solvent, internal standard, analytes of interest from each other and other components of the sample extract. Typically a DB-ALC1 (30 m 0,32 mm 1,8 µm) may be used. 4.8. Gloves (powder-free) and any other personal protective equipment deemed appropriate by the testing laboratory. 5. REAGENTS 5.1. Propan-2-ol (analytical grade, maximum water content 1,0 mg/ml). 5.2. Internal standard(s) for water: 1,4-butanediol, or ethanol, or other appropriate internal standards which have been demonstrated not to co-elute with other peaks and have been found to be consistent for peak area (minimum purity 99 %). A typical concentration for internal standard is 0,2 mg/ml. 5.3. Internal standard(s) for other analytes: n-heptadecane, or quinaldine, n-octadecane, or other appropriate internal standards which have been demonstrated not to co-elute with other peaks and have been found to be consistent for peak area (minimum purity 99 %). A typical concentration for internal standard is 1 mg/ml. 5.4. Gases: hydrogen, nitrogen, and helium and compressed air as necessary for operation of the GC. 5.5. Glycerin (1,2,3-propanetriol, CAS 56-81-5); propylene glycol (1,2-propanediol, CAS 57-55-6); (-)-nicotine (CAS 54-11-5); (minimum purity 99 %) for preparation of standard solutions. Protect from moisture, air, and light as indicated by vendor. 5.6. Water: Type I, or equivalent, distilled or deionized water, minimum resistivity of 18,2 MΩ-cm at 25 C, and free from contamination by analytes of interest. For testing that includes the analysis of water, the bulk solvent (0) container should be fitted with a water trap and all solutions should be kept sealed. Glassware and septa for vials should be stored in a desiccator until use. CRM No. 84 June 2017 4/11
6. CALIBRATION STANDARDS Dissolve glycerin, propylene glycol, water, and nicotine in the solvent to produce a series of at least five calibration solutions including blanks, as warranted, whose concentrations cover the range expected to be found in the samples. A typical range of calibration standards for each analyte is listed in the table below: Table 1 - Calibration standards Analyte Glycerin 0,05 2,0 Propylene glycol 0,05 2,0 Typical range (mg/ml) Water 0 and 0,5 20 Nicotine 0,05 2,0 Note that a blank standard is needed for water and may be warranted for some or all of the other analytes depending on the anticipated concentration range of the samples. Store calibration standards at (4 ± 2) C and protect from light. 7. PROCEDURES 7.1. Gas chromatography (Glycerin, PG, nicotine) Set up and operate the gas chromatograph and equipment according to the manufacturer s instructions. Ensure that peaks of interest are well resolved. Operating conditions, including column type, should be optimized for analyte separation and sensitivity. Typical operating conditions are as follows: Table 2 - Example of operating conditions for gas chromatography (Glycerin, PG, nicotine) Column Carrier Gas Injection Temperature 250 C DB-ALC1 (30 m 0,32 mm 1,8 µm) Helium, 3 ml/min Injection mode Split, 25:1 Injection volume 1 µl Oven temperature Detector Detector Temperature 275 C 90 C (1 min), 15 C/min to 120 C, 40 C/min to 280 C (2 min) Flame Ionization 7.2. Gas chromatography (water) Set up and operate the gas chromatograph and equipment according to the manufacturer s instructions. CRM No. 84 June 2017 5/11
Ensure that peaks of interest are well resolved. Operating conditions, including column type, should be optimized for analyte separation and sensitivity. Typical operating conditions are as follows: Table 3 - Example of operating conditions for gas chromatography (water) Column Carrier Gas DB-ALC1 (30 m 0,32 mm 1,8 µm) Helium, 1.5 ml/min Injection Temperature 225 C Injection mode Split, 10:1 Injection volume 2 µl Oven temperature Detector 90 C (1 min), 15 C/min to 120 C, 40 C/min to 280 C (2 min) Thermal Conductivity Detector Temperature 250 C 7.3. Instrument Calibration of the gas chromatograph(s) Inject aliquots of the standard solutions into the gas chromatograph. Record the peak areas or heights of the analytes and the internal standard. Calculate the ratio of the analyte peak to the internal standard peak from the peak area (or height) data for each of the calibration solutions. Determine the slope (m) and intercept (b) based on linear regression (y = mx +b) for each of the analytes. If the coefficient of determination, R 2, is less than 0,995, calibration should be repeated. The signal (peak area or height) obtained for all test portions must fall within the working range of the calibration curve. If test samples are outside of the working calibration range, the range may be extended if the suitability of the calibration is verified and maintained. Alternatively, samples may be diluted with the solvent (5.4) as appropriate. 7.4. Filter pad handling Filter pads should be stored in the target atmosphere of the test conditions for a minimum of 24 hours prior to determination of pre-testing weights. For all operations, the operator shall prevent contamination from the fingers by wearing gloves of a suitable material (4.8). Between operations, a filter pad holder cap, if available, may be installed to prevent water loss or uptake. Filter pads should be processed as quickly after collection as is feasible to prevent uptake or loss of water. 7.5. Aerosol collection and sample preparation Using CORESTA Recommended Method N 81 set up the aerosol collection system and collect the aerosol onto glass fiber filter pads. Collection parameters such as number of puffs or number of puff sets are dependent on study design. In any case, it should be ensured that the capacity of the filter pads for aerosol is not exceeded. In addition, after aerosol collection, remove holder assemblies from the collection system and process for determination of aerosol mass as necessary (7.6). CRM No. 84 June 2017 6/11
For each filter pad, open the holder and remove filter pad with forceps. Fold the pad twice with the aerosol side toward the inside of the folds, being careful to handle only the edge of the filter pad. Wipe the inside of the filter holder front with two separate quarters of an unused conditioned filter disc and add these to the flask to collect any residual aerosol. Alternatively, wipe the holder with the folded unused side of the filter pad. Transfer to a sample vessel for extraction. Extract the filter pads using a fixed volume of the solvent (5.4) of 20 ml for 44 mm pads or 50 ml for 92 mm pads and 30 min of agitation on a mechanical shaker. The volume of solvent may be adjusted to give analyte concentrations in the range of the calibration standards. The maximum storage time of the extracts is 48 hours under ambient conditions. The storage time may be extended if the stability of the extracts is verified. The extracts must be protected from light. 7.6. Simultaneous determination of Aerosol Collected Mass Optionally, aerosol collected mass (ACM) may be determined gravimetrically. Filter pads in their holder (with or without end caps) and without e-cigarette holders are weighed before and after aerosol collection prior to extraction for analyte analysis. Weights should be determined to the nearest 0,1 mg. Aerosol mass is calculated as: m ACM (mg) = m1 m 0 Where: m = mass 1 = after aerosol collection 0 = before aerosol collection Express the test results in mg/puff, mg/puff set, mg/total puffs or mg/per device as indicated by study design. 7.7. Simultaneous determination of e-cigarette mass loss Optionally, the mass loss of the device may be determined gravimetrically. The test article should be weighed prior to installation on the collection system. Subsequent frequency of mass determination should be as indicated by study design. For example, the test article may be reweighed between each pad set or may be reweighed after completion of the testing session. It is recommended that the mass before collection value be verified if there is any significant time delay between sequential collections (i.e. collection sets that span multiple work days). m ec (mg) = m 1 m 0 Where: m = mass ec = e-cigarette, device 1 = after aerosol collection 0 = before aerosol collection CRM No. 84 June 2017 7/11
7.8. Blank Test for Water Due to the absorption of water by smoke traps and solvent, it is necessary to determine a value for a test blank. At least two (2) test blanks must be included with each collection run. Prepare test blanks by installing a filter pad in its holder as if preparing for aerosol collection. Place the test blanks in close proximity to the collection system during aerosol collection and extract and analyse them with the samples from the associated collection. 7.9. Simultaneous versus sequential analysis for water content The chromatographic methods described herein, or equivalent, may be used in conjunction with each other using two chromatographs or using a single chromatograph equipped with dual columns and dual detectors. A simultaneous automated analysis of water and of glycerin, PG, and nicotine may be achieved by using a GC system equipped with dual detectors in series or with a GC system with dual autosamplers, inlets, columns, and detectors operated in parallel. Example workflow scenarios are shown in the diagram below. When determining analytes from the same sample sequentially, the water determination should be performed first to prevent absorption of water by the sample affecting the final result. Workflow 1 (Simultaneous) Workflow 2 (Sequential) Workflow 3 (Parallel) Optional Determinations: Aerosol Mass, E-Cig Mass Loss Optional Determinations: Aerosol Mass, E-Cig Mass Loss Optional Determinations: Aerosol Mass, E-Cig Mass Loss Extraction of filter pads Extraction of filter pads Extraction of filter pads Single GC (Dual Column) GC-FID, GC-TCD GC-TCD Analysis for Water GC-TCD Analysis for Water GC-FID Analysis for glycerin/pg/nicotine GC-FID Analysis for glycerin/pg/nicotine CRM No. 84 June 2017 8/11
7.10. Determination of analyte content for the samples Inject one or more aliquots of the sample extracts into the gas chromatograph(s) using the conditions described in sections 7.1 and or 7.2. Record the peak areas or heights of analytes and the internal standard(s). Calculate the mean value of the ratio of the peak area or height of analyte to that of the internal standard for the replicate injections. The amount of analyte is determined in mg/ml using an internal standard calibration method. Ensure that the values lie within the range of the standards prepared in section 6. For water analysis, subtract the average value of the test blank(s) associated with the collection from the value for each sample pad: Where: w= water s= sample b = test blank, average water (mg/ml) = w s w b For each analyte, express the test results in mg/mg ACM, mg/puff, mg/puff set, mg/total puffs or mg/per device as indicated by study design. Example calculations are given below: Where: N(mg mg ACM) = C V m ACM N = Nicotine C = the concentration obtained from the calibration curve (mg/ml) m ACM = mass of aerosol collected mass (mg) Vol = the volume of extraction solution added to the sample (20 ml) Where: N(mg puff) = C P V C = the concentration obtained from the calibration curve (mg/ml) P = number of puffs collected V = the volume of extraction solution added to the sample (20 ml) CRM No. 84 June 2017 9/11
8. REPEATABILITY AND REPRODUCIBILITY Results listed in the tables below are based on the study 2015 Aerosol Collaborative Study: Major Constituents Proficiency Study conducted jointly between the E-Vapour Sub-Group (EVAP) and the Routine Analytical Chemistry Sub-Group (RAC) using four commercialvapour products. The average r&r values from these results are listed directly below. The individual results are listed on the following page. Note that due to the higher variability of e- cigarettes relative to cigarettes for yield of these analytes, proportioning to ACM may be a preferred manner of reporting in many cases. Therefore, r&r values are displayed as is and as proportioned to ACM. Table 4 - r&r values for Analytes mg ( as is ) Average of all samples ACM Nicotine PG Glycerin Water r (%) 33.85% 36.97% 36.03% 33.53% 31.35% R (%) 47.75% 46.27% 49.93% 46.40% 84.63% Table 5 - r&r values for Analytes proportioned to ACM Average of all samples Nicotine/ACM PG/ACM Glycerin/ACM Water/ACM r (%) 5.97% 5.55% 6.80% 23.98% R (%) 20.47% 9.90% 14.03% 65.33% Table 6 - r&r values for Analytes mg/total puffs collected as is Individual results for each sample Sample Code ACM Nicotine PG Glycerin Water Sample A Sample B Sample C Sample D r (%) 28.60% -- 31.00% 28.90% 28.40% R (%) 46.60% -- 51.20% 39.50% 78.80% r (%) 33.50% 36.00% 36.10% 32.60% 29.20% R (%) 56.70% 49.40% 57.10% 52.00% 103.40% r (%) 39.40% 40.50% 41.50% 39.10% 35.70% R (%) 47.20% 50.00% 47.40% 47.70% 71.20% r (%) 33.90% 34.40% 35.50% -- 32.10% R (%) 40.50% 39.40% 44.00% -- 85.10% Repeatability and reproducibility calculations were based on a test result being the average of four test items. Calculations were carried out using the robust algorithms in ISO 5725-5; -- = Not calculated. CRM No. 84 June 2017 10/11
Table 7 - r&r values for Analytes proportioned to ACM Individual results for each sample Sample Code ACM Nicotine/ACM PG/ACM Glycerin/ACM Water/ACM Sample A Sample B Sample C Sample D r (%) 28.60% -- 5.30% 6.10% 22.00% R (%) 46.60% -- 9.80% 17.80% 64.70% r (%) 33.50% 5.20% 5.10% 6.10% 29.00% R (%) 56.70% 22.20% 9.60% 13.80% 81.50% r (%) 39.40% 6.50% 6.50% 8.20% 23.90% R (%) 47.20% 17.10% 10.20% 10.50% 62.10% r (%) 33.90% 6.20% 5.30% -- 21.00% R (%) 40.50% 22.10% 10.00% -- 53.00% Repeatability and reproducibility calculations were based on a test result being the average of four test items. Calculations were carried out using the robust algorithms in ISO 5725-5; -- = Not calculated. Similarly, to get a better assessment of the performance of the analytical methods independent of the variability of the devices, repeatability and reproducibility limits were calculated on Sample F, the liquid control sample. Those results are given in the following table. Table 8 - r&r values for Sample F, the liquid control sample Sample Code Nicotine PG Glycerin Water Sample F r (%) 4.37% 4.45% 6.07% 13.6% R (%) 14.8% 10.9% 20.7% 30.5% 9. TEST REPORT The test report shall give the analyte content of the sample as a mass fraction based on the collection parameters of the testing (i.e. per puff, per ACM, per total puffs, per device, etc.). The test report shall also mention all pertinent operating conditions not specified in this CORESTA Recommended Method as well as any circumstances that may have affected the result. CRM No. 84 June 2017 11/11