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E-Cigarette Task Force
Technical Report
E-Liquid Preliminary Proficiency Study
March 2015
Author:
Rana Tayyarah
Lorillard Tobacco Company
Task Force Coordinator:
Chuck Garner, Ph.D.
R. J. Reynolds Tobacco Company
Task Force Secretary:
Robert D. Stevens, Ph.D.
Lorillard Tobacco Company
Table of Contents
1. Summary 3
2. Introduction 3
3. Organisation 4
3.1 Participants 4
3.2 Protocol 5
3.3 Data - Raw 6
4. Data - Statistical Analysis 6
4.1 Exclusion of Outliers 6
4.2 Calculation of Repeatability and Reproducibility 7
5. Data Interpretation 7
5.1 Methods comparison 7
5.2 Results for Control Sample 8
5.3 Sample Homogeneity 10
5.4 Results for Products – Nicotine 10
5.5 Results for Products – Glycerin 12
5.6 Results for Products – Propylene Glycol 13
5.7 Results for Products – Water 15
6. Recommendations 16
7. Acknowledgements 16
APPENDIX A: Study Protocol and Data Template 17
APPENDIX B: Laboratory Method Summaries 25
APPENDIX C: Raw Data Means 52
APPENDIX D: Nicotine - Raw Data Mean Plots 58
APPENDIX E: Glycerin - Raw Data Mean Plots 61
APPENDIX F: Propylene Glycol - Raw Data Mean Plots 64
APPENDIX G: Water - Raw Data Mean Plots 67
APPENDIX H: Nicotine - Mandel’s h and Mandel’s k Graphs 69
APPENDIX I: Glycerin - Mandel’s h and Mandel’s k Graphs 74
APPENDIX J: Propylene Glycol - Mandel’s h and Mandel’s k Graphs 79
APPENDIX K: Water - Mandel’s h and Mandel’s k Graphs 84
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1. Summary
In 2014, the CORESTA E-Cigarette Task Force conducted a study that included testing of
commercial electronic cigarette e-liquid products. This study was referred to as Electronic
Cigarette e-Liquid Preliminary Proficiency Study. The laboratory phase of this study was
conducted from late 2013 to early 2014 and results were presented to the Task Force in May
2014. Nine laboratories participated in the study. Each laboratory tested the same set of 10
commercial e-liquids and 1 study control. The goal of this work was to compare results from
multiple laboratories applying their own internal analytical methods for the determination of
nicotine, propylene glycol (PG), glycerin and water in e-liquid samples. The study results
demonstrate that even without a common method, results were broadly consistent, of low
variability, and highly accurate among the participating laboratories. Percent error for
nicotine was <5% for all labs. Error for glycerin and PG was generally less than 10% for
most labs. For water analysis, Karl Fisher results showed higher accuracy (4% error)
compared to GC-TCD results (13% error). There was slight, but non-random trending with
data from some labs. Results indicate that establishment of a CORESTA recommended
method may be warranted.
2. Introduction
One of the short term objectives of the e-Cigarette Task Force is “…to gather and share
preliminary data on analysis relevant to e-cigarettes worldwide with a view to making
recommendations for product testing”. As yet, there are no standardized methods for the
characterization of e-liquids. Thus, the Task Force commissioned a study to evaluate the
performance of existing methods currently in use by the participating laboratories.
Commercial e-liquid products were tested. The analytes and nominal value ranges are listed
below:
Nicotine: 0 – 1.6%
Glycerin: 0 - 100 %
PG: 0 - 100%
Water: <~10%
A study control was included, as well. The control was a mixture of glycerin (45%),
propylene glycol (45%), water (9%), and nicotine (1%) and was prepared by one laboratory
using analytical grade chemicals.
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3. Organisation
3.1 Participants
A list of the participating laboratories is provided in Table 1. The laboratories are listed in
alphabetical order. The numerical laboratory codes (1-10) used in this report do not
correspond to the same order of the laboratories listed below. Note that one laboratory tested
two sets of e-liquid products and the results were coded as 2a and 2b. There is no laboratory
4. All commercial e-liquid products were supplied by Red Kiwi and NicVape.
Table 1: List of Participating Laboratories
Laboratory
Altria Client Services
Arista Laboratories
Enthalpy Analytical, Inc.
Global Laboratory Services, Inc.
KT&G R&D Headquarter
Labstat International ULC
Lorillard Tobacco Co
Microbac Laboratories, Inc; Wilson Division
RJ Reynolds Tobacco Co.
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3.2 Protocol
e-Liquid products were collected from the manufacturers, labelled with sample numbers, and
distributed by Enthalpy. The control samples were made at Lorillard using analytical grade
chemicals purchased from Sigma-Aldrich and a 5-place analytical balance. Individual control
samples were prepared by weighing the analytes directly into tared sample vials. The control
samples were distributed from Enthalpy with the product samples. The commercial samples
were distributed in clear 10mL screw-cap containers. Two bottles of each sample were
provided in the event a repeat analysis would be required. A single 20mL container of the
control sample, Sample 11, was provided. Analytes included in the study were nicotine,
glycerin, propylene glycol, and water. The laboratories were instructed to store the e-liquid
samples at room temperature for at least 2 hours before sampling. Each laboratory conducted
three replicate determinations for each sample using their methods of choice. Sample codes
used in the study and the nominal content of the analytes of interested, as provided by the
supplier, are listed in Table 2. See Appendix A for the study protocol. See Table 3 for an
overview of the study methods. See Appendix B for a summary of each laboratory’s
methods.
Table 2: Sample Identification and Nominal Analyte Content
Sample Code Nicotine
mg/mL / %w/w* Glycerin PG Water
Sample 1 18 mg/mL / 1.57% 40% 50% 5 – 10%
Sample 2 18 / 1.57 40% 50% 5 – 10%
Sample 3 0 / 0 40% 50% 5 – 10%
Sample 4 9 / 0.79 40% 50% 5 – 10%
Sample 5 9 / 0.79 40% 50% 5 – 10%
Sample 6 6 / 0.54 -- -- --
Sample 7 6 / 0.53 -- -- --
Sample 8 6 / 0.53 -- -- --
Sample 9 6 / 0.48 100% 0% --
Sample 10 6 / 0.58 0% 100% --
Sample 11 (Control)
-- / 1.00 45% (w/w) 45% (w/w) 9% (w/w)
*The nominal values for nicotine for Samples 1 – 10 were reported in mg/mL.
They were converted to % w/w based on Lab 6’s density measurements.
-- = Information not provided
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Table 3: Analytical Methods Overview
General Information across Methods
Sample Prep
• Weigh 100 – 1000 mg * • Dilute with alcohol (10 – 100 mL) • Mix/Shake (10s – 3 hours) • Weight and volume used varied by analyte
Analysis
• GC-TCD or Karl Fischer for water • Packed column if TCD (most)
• GC-FID for other analytes • Capillary column • DB-Wax columns for most
• 4 – 12 calibration standards (GC) • Linear (IS) calibration
• r2
>0.99
*Lab 6 measured by volume (25 – 100 µL) and diluted with 1mL solvent
3.3 Data - Raw
The summary descriptive statistics data are listed in Appendix C where each mean represents
three replicates. Raw data plots are given in Appendices D-G. Data graphs in Appendices D-
G display individual values (gray dots) with mean lines (green lines). To the right of the
graph are results from one way analysis of variance with paired comparisons.
4. Data - Statistical Analysis
4.1 Exclusion of Outliers
Graphical outlier detection was performed following the recommendations of ISO 5725-2
(1994). Mandel’s h is used to assess the inter-laboratory consistency and Mandel’s k is used
to assess the intra-laboratory consistency. The Mandel’s h plots and the Mandel’s k plots
(0.99 limits) are given in Appendices H-K.
Numerical outlier detection was conducted following the recommendations of ISO 5725-2
(1994). The decision on straggling (0.95) and outlying (0.99) data were made using single
outlier and Cochran’s tests. The results for the Grubbs’ test to detect outlying means and for
the Cochran’s test to detect outlying variances are displayed in Table 4.
The relatively low variability among, and between, data sets is a likely source of some level
of non-practical or false determination of outliers. Note that none of the data were excluded
from overall mean calculations. The information is merely provided for the convenience of
the participating laboratories.
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Table 4. Results for Outlying Means and Variances
Nicotine
Glycerin
Product Cochran's Grubbs' Product Cochran's Grubbs'
Sample 1 -- Lab 10 Sample 1 -- Lab 6
Sample 2 -- Lab 2a, 5, 10 Sample 2 -- Lab 1, Lab 7
Sample 3 NA NA Sample 3 Lab 2b Lab 2a
Sample 4 -- -- Sample 4 -- --
Sample 5 -- Lab 5, 10 Sample 5 -- Lab 2b
Sample 6 Lab 1 -- Sample 6 -- --
Sample 7 -- -- Sample 7 -- --
Sample 8 Lab 5 Lab 10 Sample 8 -- --
Sample 9 -- Lab 5, 10 Sample 9 -- --
Sample 10 Lab 2a -- Sample 10 NA NA
PG
Water
Product Cochran's Grubbs' Product Cochran's Grubbs'
Sample 1 -- Lab 2a Sample 1 -- --
Sample 2 -- Lab 1, 2a Sample 2 -- Lab 7
Sample 3 -- Lab 1 Sample 3 -- Lab 7
Sample 4 -- -- Sample 4 Lab 5 Lab 7
Sample 5 -- Lab 8 Sample 5 -- --
Sample 6 Lab 5 -- Sample 6 NA NA
Sample 7 -- -- Sample 7 NA NA
Sample 8 -- Lab 2b Sample 8 NA NA
Sample 9 NA NA Sample 9 NA NA
Sample 10 Lab 5 -- Sample 10 NA NA
alpha = 0.01
NA – not applicable; the test was not performed
-- - no outliers were noted
4.2 Calculation of Repeatability and Reproducibility
Each laboratory tested in-common samples using non-standardized methodology. Thus,
repeatability (r) and reproducibility (R) results were not calculated for this data set.
5. Data Interpretation
5.1 Methods comparison
Although laboratories were not provided standardized methods for use with the study, most
aspects of the methodologies used were very similar. All laboratories used an alcohol solvent
for dilution, for example. Differences included shake time, which ranged from hand inversion
to 180 min of mechanical shaking. Most laboratories used GC-TCD for water analysis, while
3 labs used Karl Fischer.
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5.2 Results for Control Sample
Given that methodology are not standardized and that there are no reference or monitor e-
liquid products available for inclusion in the study, a study control was made and treated in
the same manner as the samples. The control sample set was made by using analytical grade
chemicals purchased and left unopened prior to preparing the samples. For the highest level
of accuracy for the ‘true value’ of the control, individual samples were made and coded rather
than making a larger volume sample and taking aliquots. In this manner, the analytes were
weighed directly into a tared vial using a five-place analytical balance. Samples were sealed
and stored in the dark prior to shipment for distribution. The target values (%w/w) were:
45%, 45%, 9%, and 1%, for glycerin, propylene glycol, water, and nicotine, respectively.
The percent error from the actual value for each laboratories control sample was calculated
and was determined to be relatively low for all labs and all analytes. For example, the percent
error for nicotine ranged from 0.2% to -4.5%. Precision was high for each laboratory with
percent relative standard deviations at <5%.
The error was highest for water (<18%error) and the data indicate that the use of Karl Fischer
may allow for improved accuracy. The average error for GC-TCD determinations was 13%
error while the average for Karl Fischer was 4% error. The mean results for water
determinations for the control are displayed in Figure 1 in a bar graph. The bars are color-
coded based on method (Karl Fischer or GC-TCD) to highlight the greater accuracy (i.e.
purple bars are closer to blue bars) and greater a precision (i.e. error lines are smaller) for the
Karl Fischer determinations.
The percent error calculations are shown in Table 5 below. The average error for each analyte
is shown as is and as an absolute value (distance from zero without regard for whether the
value errs high or low). Mean values are displayed in Appendix C.
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Table 5. Percent Error for the Control Sample
Nicotine Glycerin PG Water
Lab 1 2.9% -2.7% 0.2% -15%
Lab 2a 0.3% -1.6% -0.7% 4.5%*
Lab 2b -2.1% -3.0% -2.4% 3.4%*
Lab 3 -1.3% 2.3% 0.7% 2.1%*
Lab 5 -0.5% 5.2% -11% 7.9%
Lab 6 -0.6% -11.9% -10% -16%
Lab 7 -0.2% -1.6% -2.4% -10%
Lab 8 -1.4% -4.6% -5.9% -8.6%
Lab 9 1.9% 7.4% 0.9% 18%
Lab 10 -4.5% -13% -7.5% 2.4%*
Average -0.6% -2% -4% -1%
Absolute Avg 2% 5% 4% 9%
Absolute Max 5% 13% 11% 18%
Calculated as %Error = (Measured - Actual) / Actual * 100
*Karl Fischer was used for water analysis; all other labs used GC-TCD
Figure 1. Comparison of Actual and Measured Content Values for Water in the Study
Control Samples (Sample 11)
5.00%
6.00%
7.00%
8.00%
9.00%
10.00%
11.00%
12.00%
Lab 2a Lab 2b Lab 3 Lab 10 Lab 1 Lab 5 Lab 6 Lab 7 Lab 8 Lab 9
Co
nte
nt
(w/w
%)
Actual Value Reported - GC/TCD Reported - Karl Fischer
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5.3 Sample Homogeneity
Enthalpy distributed two sample bottles for each product. Laboratories were instructed to test
one bottle and retain on bottle as a spare in case repeat analysis was needed. One laboratory,
Lab 2, tested both bottles. This was done pre-emptively in case the variability between the
labs’ results was high. If the values between two bottles tested by the same lab were high
then the variability would trace back to the samples rather than the labs. The results between
Sets 2a and 2b were very consistent; the average percent difference between the matched pairs
of samples was 1.6% difference. As is discussed below, there was a high level of consistency
of results between all the laboratories, as well. This indicates that the product samples tested
were homogenous.
5.4 Results for Products – Nicotine
The differences between the nominal value and each lab’s values were minimal (<10%
overall). As with the control samples, these results indicate a consistency among the methods
and laboratories. The nominal value compared to the overall average of all the labs’ data are
shown below in Table 6. All results are tabulated in Appendix C and displayed graphically in
Appendix D for each sample. Summary graphs are shown below in Figures 2 and 3.
Table 6. Overall Nicotine Results compared to the Nominal Values
Sample Nominal Value (w/w) Avg of all Labs (w/w)*
Sample 1 1.6% 1.48 ± 0.07
Sample 2 1.6% 1.57 ± 0.07
Sample 3 0.0% NR
Sample 4 0.79% 0.73 ± 0.03
Sample 5 0.79% 0.72 ± 0.03
Sample 6 0.54% 0.55 ± 0.02
Sample 7 0.53% 0.53 ± 0.01
Sample 8 0.53% 0.54 ± 0.03
Sample 9 0.48% 0.46 ± 0.01
Sample 10 0.58% 0.58 ± 0.02
Sample 11 (Control ) 1.0% 1.03 ± 0.03
*Displayed as mean ± 1 standard deviation
NR=Not reportable on average (less than quantification limits)
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Figure 2. Nicotine Content Results for Samples 1-5
Figure 3. Nicotine Content Results for Samples 6-10
0.0%
0.2%
0.4%
0.6%
0.8%
1.0%
1.2%
1.4%
1.6%
1.8%
2.0%
Lab 1 Lab 2aLab 2b Lab 3 Lab 5 Lab 6 Lab 7 Lab 8 Lab 9 Lab 10
Co
nte
nt
(w/w
%)
Sample 1
Sample 2
Sample 3
Sample 4
Sample 5
0.0%
0.1%
0.2%
0.3%
0.4%
0.5%
0.6%
0.7%
Lab 1 Lab 2a Lab 2b Lab 3 Lab 5 Lab 6 Lab 7 Lab 8 Lab 9 Lab 10
Co
nte
nt
(w/w
%)
Sample 6
Sample 7
Sample 8
Sample 9
Sample 10
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0.0%
5.0%
10.0%
15.0%
20.0%
25.0%
30.0%
35.0%
40.0%
45.0%
50.0%
Lab 1 Lab 2a Lab 2b Lab 3 Lab 5 Lab 6 Lab 7 Lab 8 Lab 9 Lab 10
Co
nte
nt
(w/w
%)
Sample 1
Sample 2
Sample 3
Sample 4
Sample 5
5.5 Results for Products – Glycerin
A higher level of difference (<20% difference) was noted between laboratories for glycerin.
Overall results compared to nominal values are shown in Table 7. Results are displayed in
Appendix C and Appendix E. The trending about the nominal values for Samples 1-5 is
consistent with the relative results for the control samples. For example, Lab 10 glycerin
results were approximately 10% lower than the actual value for the control and are
approximately 10% lower than nominal for Samples 1-5. Lab 9 glycerin results for the
control were slightly higher than the actual value and the results for Samples 1-5 are about
10% higher than nominal. These results are displayed below in Figure 4.
Table 7. Overall Glycerin Results compared to the Nominal Values
Sample Nominal Value (w/w) Avg of all Labs (w/w)*
Sample 1 40% 42 ± 3
Sample 2 40% 42 ± 3
Sample 3 40% 41 ± 3
Sample 4 40% 40 ± 3
Sample 5 40% 41 ± 3
Sample 6 -- 38 ± 3
Sample 7 -- 38 ± 3
Sample 8 -- 41 ± 4
Sample 9 100% 98 ± 4
Sample 10 0% NR
Sample 11 (Control) 45% 44 ± 3
*Displayed as mean ± 1 standard deviation
NR=Not reportable on average (less than quantification limits)
= information not provided and/or calculated
Figure 4. Glycerin Content Results for Samples 1-5. The line at 40% marks the nominal
value for the samples.
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For Samples 6-8, a nominal value was not provided but all laboratories reported
approximately 40% (w/w). For example, the average of all labs was (38 ± 3)% for Sample 6.
Sample 9 was approximately 100% and Sample 10 was a no glycerin product. Laboratory
results were consistent with each other for Samples 6-10; see Figure 5 below.
Figure 5. Glycerin Content Results for Samples 6-10
5.6 Results for Products – Propylene Glycol
Most samples tested contained 40-60% propylene glycol (PG). There was one 0% and one
~100% sample. All laboratories results are similar for all samples; the overall standard
deviation is approximately 2 in most cases as is shown in Table 8 below. All results are
shown in Appendix C and Appendix F.
Table 8: Overall Propylene Glycol Results compared to the Nominal Values
Sample Nominal Value (w/w) Avg of all Labs (w/w)*
Sample 1 50% 45 ± 2
Sample 2 50% 45 ± 2
Sample 3 50% 46 ± 2
Sample 4 50% 46 ± 2
Sample 5 50% 46 ± 2
Sample 6 -- 57 ± 2
Sample 7 -- 56 ± 2
Sample 8 -- 44 ± 2
Sample 9 0% NR
Sample 10 100% 97 ± 4
Sample 11 (Control)
45% 43 ± 2
*Displayed as mean ± 1 standard deviation
NR=Not reportable on average (less than quantification limits)
--= Information not provided and/or calculated
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36.0%
38.0%
40.0%
42.0%
44.0%
46.0%
48.0%
50.0%
Lab 1 Lab 2a Lab 2b Lab 3 Lab 5 Lab 6 Lab 7 Lab 8 Lab 9 Lab 10
Co
nte
nt
(w/w
%)
Sample 1
Sample 2
Sample 3
Sample 4
Sample 5
0.0%
20.0%
40.0%
60.0%
80.0%
100.0%
120.0%
Lab 1 Lab 2a Lab 2b Lab 3 Lab 5 Lab 6 Lab 7 Lab 8 Lab 9 Lab 10
Co
nte
nt
(w/w
%)
Sample 6
Sample 7
Sample 8
Sample 9
Sample 10
Results grouped by the laboratory are displayed in Figures 6 and 7 below. For samples 1-5,
all results are below the nominal value provided. As with glycerin, Lab 6 results appear to
trend slightly (5-9%) below the overall mean for the analyte. Though, only slightly lower, the
trend is consistent with Lab 6’s error for the control sample (-10%).
Figure 6. Propylene Glycol Content Results for Samples 1-5.
The line at 50% marks the nominal value for the samples.
Figure 7. Propylene Glycol Content Results for Samples 6-10.
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0.0%
2.0%
4.0%
6.0%
8.0%
10.0%
12.0%
14.0%
Lab 2aLab 2b Lab 3 Lab 10 Lab 1 Lab 5 Lab 6 Lab 7 Lab 8 Lab 9
Co
nte
nt
(w/w
%)
Sample 1
Sample 2
Sample 3
Sample 4
Sample 5
5.7 Results for Products – Water
Water results were the least consistent between the laboratories. Whereas, the other analytes
had an overall relative standard deviation for pooled data of 3-10 %RSD, the overall %RSD
for water was 8-14 %RSD. As with the control sample, some bias in results is apparent based
on method choice. Karl Fischer results are more consistent. The level of variability seen in
the GC-TCD results may not be sufficient to warrant a change in practice for a given
laboratory. Results for samples 1-5 are displayed, grouped by lab and method type, in Figure
8 below. Results are shown in Appendix C and Appendix G.
Table 9: Overall Water Results compared to the Nominal Values
Sample Nominal Value (w/w) Avg of all Labs (w/w)*
Sample 1 5 – 10% 9.0 ± 0.8
Sample 2 5 – 10% 8.5 ± 0.9
Sample 3 5 – 10% 10.2 ± 1.0
Sample 4 5 – 10% 9.4 ± 1.0
Sample 5 5 – 10% 9.9 ± 1.4
Sample 11 (Control)
9% 9.0 ± 1.1
*Displayed as mean ± 1 standard deviation
NR=Not reportable on average (less than quantification limits)
--= Information not provided and/or calculated
Figure 8. Water Content Results for Samples 1-5.
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6. Recommendations
The laboratory results were broadly comparable for all analytes. For example, for the control
sample, the percent error for nicotine was less than 5% error and averaged 2% for all labs.
All laboratories’ methods appear to be adequate across the concentration range tested for each
analyte though there were some instances of non-random trending. For example, Labs 6 and
10 were slightly (<10% from the overall average), but consistently, lower than the other
laboratories for glycerin and PG. Also, the laboratories using Karl Fisher for water
determination had a slightly higher accuracy for water (4% error) than the laboratories using
GC-TCD (13% error). Across the labs, sample preparation techniques were similar with
variation in details such as mixing time. For example, all labs used an alcohol solvent. But
mixing ranged from 10s mixing to 3 hours of shaking.
Pursuit of a formal CORESTA recommended method may be warranted. Additionally, it is
recommended that reporting units of per volume (mg/mL) and per weight (mg/mg) be used.
This would be achievable by inclusion of weighing a certain volume as part of the sample
preparation procedure.
7. Acknowledgements
The study team, Drs. Rob Stevens, Narendra Meruva, and Gene Gillman; the participating
laboratories; and the product suppliers, Red Kiwi and NicVape, are greatly appreciated.
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APPENDIX A: Study Protocol and Data Template
CORESTA E-CIGARETTE ANALYTICAL TESTING SUB TEAM
(Team Leader – Dr. Rob Stevens, Lorillard Tobacco)
Project Title: 2013 e-Liquid Analysis Protocol
Date: Nov 11, 2013
Written by: Naren Meruva, Ph.D. (Altria Client Servies)
Email: [email protected]
Phone: 1-804-335-2455
Confidentiality Notice: All data generated in the course of this analysis should be handled in
the strictest of confidence by all participating laboratories and other CSTS members.
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Protocol for a Preliminary Proficiency Study on Determination of Nicotine,
Propylene Glycol, Glycerin and Water in e-Liquids
1. Objective
The key objective of this preliminary proficiency study is to assess the repeatability and
reproducibility of participating laboratories applying their own internal analytical methods for
determination of nicotine, propylene glycol (PG), glycerin and water in e-liquid samples.
Data for these analytes from the participating laboratories will be collected for statistical
evaluation purposes. Additionally, data on weight of the aliquot of e-liquid and the volume of
extraction solvent used will be collected for individual measurements.
2. Analytes
Following analytes are to be measured in e-liquid samples:
a. Nicotine
b. Propylene Glycol (PG)
c. Glycerin
d. Water
Reporting units – mg/mg (as-is) or mg/mL (as-is). Additionally, report the weight (in mg) or
volume (in mL) of the aliquots taken from each sample for testing. If aliquots are measured
by volume, sample density is to be reported.
3. Test Methods
Each laboratory shall apply their own internal analytical methods for the determination of
major components (nicotine, PG, glycerin and water) in e-liquid samples.
Laboratories are asked to record sample preparation steps and must provide information on
analytical method and data collected in the templates distributed with the study protocol.
4. Test Samples
Test samples consist of eleven (11) different e-liquids with varying percentage of nicotine (<
2.5%), propylene glycol, glycerin, water and flavor content.
The test samples are coded 1 through 11. Two bottles of each sample 1-10 (10 mL in each
bottle) and one bottle of Sample 11 (~20mL) will be distributed by Dr. Gene Gillman
(Enthalpy Analytical, Inc.) during the first week of December 2013.
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5. Sample Handling
The e-liquid samples need to be stored at room temperature for at least 2 hours before use.
Any additional sample storage information needs to be provided in the templates provided
with the study protocol.
6. Experimental plan
Three (n=3) replicate analysis for each sample are required. If possible, it is requested that all
samples be analyzed on a single day. If multiple analysis days are needed it is requested that
all three replicates of each sample be analyzed on a single day.
7. Data Reporting
The attached templates for e-liquid analysis should be used for data submission. Please
provide data in the requested format without creating new cells or rows in the spreadsheet.
Results should be reported back to Naren Meruva ([email protected]) and Rana
Tayyarah ([email protected]) on or before Feb 14th
, 2014 in order to allow sufficient
time to evaluate the data.
8. Statistical Data Analysis
The data from participating laboratories will be analyzed statistically according to ISO-5725
(organized by Dr. Rob Stevens, Lorillard Tobacco Co.).
9. Timeline
Nov 25th - Dec 15
th Sample distribution to the participating laboratories
Dec 15th - Feb 14
th Complete testing and reporting data for e-liquid samples
Feb 14th - Feb 21
st Review data from the participating laboratories
Feb 21st - Mar 28
th Statistical analysis of the data
April-2014 Summarize study findings for presentation
May-2014 Study output at the next E-cigarette Task Force Meeting
10. Project Coordinator
Naren Meruva
Email: [email protected]
Phone: 1-804-335-2455
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Laboratory name
Company Representative (Title, Name)
Address
Tel
Fax
E-mail address
Data Sheet for 2013 Preliminary e-Liquid Proficiency Study
Note: Please do not modify formatted data sheet for the convenience of data analyses.
Note: Laboratory names will not be used for reporting. All labs will be coded. Each lab will know their
own code.
Note: In this spreadsheet, cells that are shaded blue require input. Cells that are not shaded are
calculations or information and should not be edited.
Note: Please return this completed spreadsheet to [email protected] and
[email protected] by February 14, 2014.
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Method Details (example)* Nicotine Water Glycerin PGSample Preparationsample storage prior to
preparation room temp, in dark
Target weight or volume
used 100 (+/- 10)mg
dilution volume and solvent20mL MeOH with IS
mixing method 10min wrist-action
shaker
Solvent 20 mL MeOH (sub-
ambient)
Extraction wrist-action shaker,
30min
Additional FiltrationPTFE: 0.45um
Additional sample
processing prior to analysis
the same samples
were used for all
analytes but were Additional details of
relevance related to sample
preparation
aliquots were taken
from original
container after hand
Analytical EquipmentDescription
Agilent GC/MS, EI in
SIM mode using
detuerated IS; D-5
pyridine (84),
pyridine(79) & D-7
quinoline(136) &
quinoline(129);
Detection Systems +
Conditions Column: DBWax
30mX0.25mmX0.25U
m, 50oC for 6min,
4oC/min to 160oc,
30oC to 250oC for
5.5min: 10:1 split,
flow 15mL/min, total
flow 20ml/min, 1ul
injection
Additional Details related to
analysisextracts were stored
4C prior to analysis
Reference--
Quantitation
InformationLimit of Quantitation
Limit of Detection
Number of standards
Concentration of each
Standard
Internal Standard (IS) Anethole
Calibration Type linear, with IS
r2
Additional Details related to
quantitation
0
Method Synopsis:
*Note: the example listed is not an e-liquid method. It is just meant to show the requested level of detail for you to list for your e-
liquid method.
please type an overall summary of your method in this box and provide specific details below
ECIG CTR E-Liquid Preliminary Proficiency Study – March 2015 22/86
Please paste in a picture of an example chromatogram for a calibration
standard and for one or more samples for chromatographic methods
0
Please paste pictures relevant to any unusual observations noted
during testing. Please include text explanation as necessary.
ECIG CTR E-Liquid Preliminary Proficiency Study – March 2015 23/86
Density Replicate Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 Sample 7 Sample 8 Sample 9 Sample 10 Sample 11
1
2
3
Nicotine Replicate
Actual Vol (mL) Nicotine (mg/mL) Actual Vol (mL) Nicotine (mg/mL) Actual Vol (mL) Nicotine (mg/mL) Actual Vol (mL) Nicotine (mg/mL) Actual Vol (mL) Nicotine (mg/mL) Actual Vol (mL) Nicotine (mg/mL) Actual Vol (mL) Nicotine (mg/mL) Actual Vol (mL) Nicotine (mg/mL) Actual Vol (mL) Nicotine (mg/mL) Actual Vol (mL) Nicotine (mg/mL) Actual Vol (mL) Nicotine (mg/mL)
1
2
3
Water Replicate
Actual Vol (mL) Water (mg/mL) Actual Vol (mL) Water (mg/mL) Actual Vol (mL) Water (mg/mL) Actual Vol (mL) Water (mg/mL) Actual Vol (mL) Water (mg/mL) Actual Vol (mL) Water (mg/mL) Actual Vol (mL) Water (mg/mL) Actual Vol (mL) Water (mg/mL) Actual Vol (mL) Water (mg/mL) Actual Vol (mL) Water (mg/mL) Actual Vol (mL) Water (mg/mL)
1
2
3
Glycerin Replicate
Actual Vol (mL) Glycerin (mg/mL) Actual Vol (mL) Glycerin (mg/mL) Actual Vol (mL) Glycerin (mg/mL) Actual Vol (mL) Glycerin (mg/mL) Actual Vol (mL) Glycerin (mg/mL) Actual Vol (mL) Glycerin (mg/mL) Actual Vol (mL) Glycerin (mg/mL) Actual Vol (mL) Glycerin (mg/mL) Actual Vol (mL) Glycerin (mg/mL) Actual Vol (mL) Glycerin (mg/mL) Actual Vol (mL) Glycerin (mg/mL)
1
2
3
PG Replicate
Actual Vol (mL) PG (mg/mL) Actual Vol (mL) PG (mg/mL) Actual Vol (mL) PG (mg/mL) Actual Vol (mL) PG (mg/mL) Actual Vol (mL) PG (mg/mL) Actual Vol (mL) PG (mg/mL) Actual Vol (mL) PG (mg/mL) Actual Vol (mL) PG (mg/mL) Actual Vol (mL) PG (mg/mL) Actual Vol (mL) PG (mg/mL) Actual Vol (mL) PG (mg/mL)
1
2
3
Sample 8
0
Please use this sheet if you use volume (rather than weight) to measure samples. Also
include density calculations.
Sample 1 Sample 2 Sample 3 Sample 9 Sample 10 Sample 11
Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 Sample 7 Sample 8 Sample 9
Sample 4 Sample 5 Sample 6 Sample 7
Sample 7
Sample 10 Sample 11
Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 Sample 7
Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 Sample 8 Sample 9 Sample 10 Sample 11
Sample 9 Sample 10 Sample 11Sample 8
ECIG CTR E-Liquid Preliminary Proficiency Study – March 2015 24/86
Nicotine Replicate
Actual Wt (mg) Nicotine (mg/mg) Actual Wt (mg) Nicotine (mg/mg) Actual Wt (mg) Nicotine (mg/mg) Actual Wt (mg) Nicotine (mg/mg) Actual Wt (mg) Nicotine (mg/mg) Actual Wt (mg) Nicotine (mg/mg) Actual Wt (mg) Nicotine (mg/mg) Actual Wt (mg) Nicotine (mg/mg) Actual Wt (mg) Nicotine (mg/mg) Actual Wt (mg) Nicotine (mg/mg) Actual Wt (mg) Nicotine (mg/mg)
1
2
3
Water Replicate
Actual Wt (mg) Water (mg/mg) Actual Wt (mg) Water (mg/mg) Actual Wt (mg) Water (mg/mg) Actual Wt (mg) Water (mg/mg) Actual Wt (mg) Water (mg/mg) Actual Wt (mg) Water (mg/mg) Actual Wt (mg) Water (mg/mg) Actual Wt (mg) Water (mg/mg) Actual Wt (mg) Water (mg/mg) Actual Wt (mg) Water (mg/mg) Actual Wt (mg) Water (mg/mg)
1
2
3
Glycerin Replicate
Actual Wt (mg) Glycerin (mg/mg) Actual Wt (mg) Glycerin (mg/mg) Actual Wt (mg) Glycerin (mg/mg) Actual Wt (mg) Glycerin (mg/mg) Actual Wt (mg) Glycerin (mg/mg) Actual Wt (mg) Glycerin (mg/mg) Actual Wt (mg) Glycerin (mg/mg) Actual Wt (mg) Glycerin (mg/mg) Actual Wt (mg) Glycerin (mg/mg) Actual Wt (mg) Glycerin (mg/mg) Actual Wt (mg) Glycerin (mg/mg)
1
2
3
PG Replicate
Actual Wt (mg) PG (mg/mg) Actual Wt (mg) PG (mg/mg) Actual Wt (mg) PG (mg/mg) Actual Wt (mg) PG (mg/mg) Actual Wt (mg) PG (mg/mg) Actual Wt (mg) PG (mg/mg) Actual Wt (mg) PG (mg/mg) Actual Wt (mg) PG (mg/mg) Actual Wt (mg) PG (mg/mg) Actual Wt (mg) PG (mg/mg) Actual Wt (mg) PG (mg/mg)
1
2
3
Sample 8
0
Please use this sheet if you use weight (rather than volume) for samples. Density
calculations are not necessary.
Sample 1 Sample 2 Sample 3 Sample 9 Sample 10 Sample 11
Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 Sample 7 Sample 8 Sample 9
Sample 4 Sample 5 Sample 6 Sample 7
Sample 7
Sample 10 Sample 11
Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 Sample 7
Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 Sample 8 Sample 9 Sample 10 Sample 11
Sample 9 Sample 10 Sample 11Sample 8
ECIG CTR E-Liquid Preliminary Proficiency Study – March 2015 25/86
APPENDIX B: Laboratory Method Summaries
Laboratory 1 Method Summary
Method Details Nicotine Water Glycerin PG
Sample Preparation
sample storage prior to preparation
Samples were stored at room temperature for 2 hours before use
Samples were stored at room temperature for 2 hours before use
Samples were stored at room temperature for 2 hours before use
Samples were stored at room temperature for 2 hours before use
Target weight or volume used
200 (+/- 25)mg 500 (+/- 100)mg 200 (+/- 20)mg 200 (+/- 20)mg
dilution volume and solvent 20 mL MeOH with IS 25 mL 2-Propanol with IS 100 mL MeOH with IS 100 mL MeOH with IS
mixing method Horizontal shaking for 1 hr
at 200 rpm Overnight standing
Horizontal shaking for 2 hr at 200 rpm
Horizontal shaking for 2 hr at 200 rpm
Solvent
Extraction
Additional Filtration PTFE: 0.45 um PTFE: 0.45 um PTFE: 0.45 um PTFE: 0.45 um
Additional sample processing prior to analysis
Additional details of relevance related to sample preparation
aliquots were taken from original container after hand shaking
aliquots were taken from original container after hand shaking
aliquots were taken from original container after hand shaking
aliquots were taken from original container after hand shaking
ECIG CTR E-Liquid Preliminary Proficiency Study – March 2015 26/86
Laboratory 1 Method Summary
Method Details Nicotine Water Glycerin PG
Analytical Equipment
Description
Agilent GC-FID, Nicotine & Decyl alcohol(ISTD)
Agilent GC-TCD, Water & Ethyl alcohol(ISTD)
Agilent GC-FID Glycerin & Decyl alcohol(ISTD)
Agilent GC-FID PG & Decyl alcohol(ISTD)
Detection Systems + Conditions
Column: DB-Wax 30mX0.32mmX0.25um,
100 ℃ for 3min, 5 ℃/min to
170 ℃, 240 ℃ for 10 min: 50:1 split, flow 15 mL/min, total flow 20 mL/min, 1 uL injection detector temperature 275
℃
Column: Porapak Q, 2 m packed
170 ℃ for 10 min, 240 ℃ for 10 min: Inlet temperature 250
℃, total flow 23 mL/min, 1 uL injection detector temperature
250 ℃
Column: DB-Wax 30mX0.32mmX0.25um,
equilibration 1 min, 110 ℃
for 1 min, 10 ℃/min to 150 ℃ for 3 min, 30 ℃/min to
220 ℃ for 5 min, post run
240 ℃ for 11 min: 50:1 split, flow 1 mL/min, total flow 52.6 mL/min, 2 uL injection, detector
temperature 275 ℃
Column: DB-Wax 30mX0.32mmX0.25um,
equilibration 1 min, 110 ℃
for 1 min, 10 ℃/min to 150 ℃ for 3 min, 30 ℃/min to
220 ℃ for 5 min, post run
240 ℃ for 11 min: 50:1 split, flow 1 mL/min, total flow 52.6 mL/min, 2 uL injection, detector
temperature 275 ℃
Additional Details related to analysis extracts were stored 4 ℃ prior to analysis
Reference
ECIG CTR E-Liquid Preliminary Proficiency Study – March 2015 27/86
Laboratory 1 Method Summary
Method Details Nicotine Water Glycerin PG
Quantitation Information
Limit of Quantitation 0.002 mg/mg 0.0005 mg/mg 0.002 mg/mg 0.002 mg/mg
Limit of Detection 0.0004 mg/mg 0.00013 mg/mg 0.0005 mg/mg 0.0005 mg/mg
Number of standards 5 8 8 8
Concentration of each Standard
S1(0.02133 mg/mL), S2(0.06399 mg/mL), S3(0.10665 mg/mL), S4(0.14931 mg/mL), S5(0.21330 mg/mL),
S1(0.05 mg/mL), S2(0.15 mg/mL), S3(0.30 mg/mL), S4(0.50 mg/mL), S5(0.75 mg/mL), S6(1.00 mg/mL), S7(2.00 mg/mL), S8(3.00 mg/mL)
S1(0.10015 mg/mL), S2(0.20029 mg/mL), S3(0.40058 mg/mL), S4(0.80116 mg/mL), S5(1.20174 mg/mL), S6(1.60232 mg/mL), S7(2.00290 mg/mL), S8(4.00580 mg/mL)
S1(0.10015 mg/mL), S2(0.20029 mg/mL), S3(0.40058 mg/mL), S4(0.80116 mg/mL), S5(1.20174 mg/mL), S6(1.60232 mg/mL), S7(2.00290 mg/mL), S8(4.00580 mg/mL)
Internal Standard (IS) Decyl alcohol Ethyl alcohol Decyl alcohol Decyl alcohol
Calibration Type linear, with IS linear, with IS linear, with IS linear, with IS
r2 0.9999 0.9981 0.9998 0.9999
Additional Details related to quantitation
ECIG CTR E-Liquid Preliminary Proficiency Study – March 2015 28/86
Laboratory 2 Method Summary
Method Details Nicotine Water Glycerin PG
Sample Preparation
sample storage prior to preparation Samples were stored at room temperature for 2 hours before use
Samples were stored at room temp at least 2 hrs before use
Samples were stored at room temperature for 2 hours before use
Samples were stored at room temperature for 2 hours before use
Target weight or volume used 20-120mg 200 to 3500mg
dependent on level of water found
20-120mg 20-120mg
dilution volume and solvent 20 mL MeOH with IS directly injection 100 mL MeOH with IS 100 mL MeOH with IS
mixing method Wrist-action shaker,
10min Magnetic Stirrer
Wrist-action shaker, 10min
Wrist-action shaker, 10min
Solvent
Extraction
Additional Filtration
Additional sample processing prior to analysis
Additional details of relevance related to sample preparation
ECIG CTR E-Liquid Preliminary Proficiency Study – March 2015 29/86
Laboratory 2 Method Summary
Method Details Nicotine Water Glycerin PG
Analytical Equipment
Description GC-FID Karl Fischer GC-FID GC-FID
Detection Systems + Conditions
GC: DB-WAX, 15m x 0.25mm x 0.25µm, Column Flow Mode: Ramped Pressure (Agilent)/Pressure Control (Chromeleon).
Inlet: Injection Volume = 1.0 µL, Liner: Double taper with glass wool, Temperature:
300oC, Mode: Split, Split Ratio: 40.0,
Pressure Eqil Time: 0.50 min, Gas Saver Flow: 20.0
ml/min, Gas Saver Time: 19.0 min.
Detector: Detection: FID, Temperature: 300
oC, Makeup Gas: Helium 50.0
ml/min, Air
Flow: 450 ml/min, Hydrogen Flow: 40 ml/min.
Oven Parameters: 0.0min = 0.8psi, 60
oC; 0.5min = 0.8psi, 60
oC,
4.5min = 0.8psi, 200oC,
4.9min = 1.2psi, 240oC, 6.8min = 1.2psi,
240oC.
Not Applicable Concurrent to Nicotine, same parameters
Concurrent to Nicotine, same parameters
Additional Details related to analysis GC-MS is used if analyte is trace GC-MS is used if analyte is trace
GC-MS is used if analyte is trace
Reference
ECIG CTR E-Liquid Preliminary Proficiency Study – March 2015 30/86
Laboratory 2 Method Summary
Method Details Nicotine Water Glycerin PG
Quantitation Information
Limit of Quantitation 0.05mg/g 0.90 0.015 0.016
Limit of Detection 0.005mg/g 0.40 0.005 0.0016
Number of standards 6 5 6
Concentration of each Standard
0.00026-1.637mg/mL 3 replicates 0.0251-2.514mg/mL 0.00065-4.046 mg/mL
Internal Standard (IS) Anethole 1000mg Anethole Anethole
Calibration Type Linear with IS None Linear with IS Linear with IS
r2 >0.995 Titration >0.995 >0.995
Additional Details related to quantitation Not Applicable
ECIG CTR E-Liquid Preliminary Proficiency Study – March 2015 31/86
Laboratory 3 Method Summary
Method Details Nicotine Water Glycerin PG
Sample Preparation
sample storage prior to preparation Samples were stored at room temp at least 2 hrs before use
Samples were stored at room temp at least 2 hrs before use
Samples were stored at room temp at least 2 hrs before use
Samples were stored at room temp at least 2 hrs before use
Target weight or volume used 200 mg 100 to 400mg
dependent on level of water found
200 mg 200 mg
dilution volume and solvent 20 mL MeOH with IS directly weighed into
resevoir containing pre-titrated Solvent KN
20 mL MeOH with IS 20 mL MeOH with IS
mixing method Mixing the same as
extraction Magnetic Stirrer
Mixing the same as extraction
Mixing the same as extraction
Solvent 20 mL MeOH containing
IS Not Applicable
20 mL MeOH containing IS
20 mL MeOH containing IS
Extraction 45 min with platform
shaker Not Applicable
45 min with platform shaker
45 min with platform shaker
Additional Filtration None None None None
Additional sample processing prior to analysis the same samples used for nicotine, PPG and glycerin
None the same samples used for nicotine, PPG and glycerin
the same samples used for nicotine, PPG and glycerin
Additional details of relevance related to sample preparation
Not Applicable Karl Fischer reagent Combi Titrant 5 Keto
Not Applicable Not Applicable
ECIG CTR E-Liquid Preliminary Proficiency Study – March 2015 32/86
Laboratory 3 Method Summary
Method Details Nicotine Water Glycerin PG
Analytical Equipment
Description HP6890 Mettler Toledo DL31 Varian 3800 Varian 3800
Detection Systems + Conditions
Column - 2mx3.2 mm OD; 16% Apiezon L, 2%KOH, 2% Carbowax on Chromosorb W Column flow at 20 mL/min; Injector at 230 C; Detector C at 230 C, Oven T at 190 C; Carrier gas: He at 100 psi
Not Applicable
Column - DB Wax 15mx0.53mmx1um Injector: split with a split flow of ~50 mL/min at 120 C; Temp program: 120 C hold for 2 min, 15 C/min to 180 C, hold for 4 min
Column - DB Wax 15mx0.53mmx1um Injector: split with a split flow of ~50 mL/min at 120 C; Temp program: 120 C hold for 2 min, 15 C/min to 180 C, hold for 4 min
Additional Details related to analysis Extracts injected in GC
immediately Not Applicable
Extracts injected in GC immediately
Extracts injected in GC immediately
Reference
ECIG CTR E-Liquid Preliminary Proficiency Study – March 2015 33/86
Laboratory 3 Method Summary
Method Details Nicotine Water Glycerin PG
Quantitation Information
Limit of Quantitation 0.000112 mg/mg Not Applicable 0.025 mg/mg 0.0125 mg/mg
Limit of Detection 0.00003367 mg/mg Not Applicable 0.0075 mg/mg 0.00375 mg/mg
Number of standards 8 3 replicates 4 4
Concentration of each Standard 0.0226, 0.0389, 0.0648, 0.1297, 0.2431, 0.3241, 0.6483, 0.81038 mg/mL
25µL weighed to 4 decimal places (in g)
0.12, 1.2, 3.0, 14 mg/mL
0.02, 0.2, 0.5, 12 mg/mL
Internal Standard (IS) Anethole None Anethole Anethole
Calibration Type linear, with IS Titration linear, with IS linear, with IS
r2 1.00 Not Applicable 1.00 1.00
Additional Details related to quantitation No additional details No additional details No additional details No additional details
ECIG CTR E-Liquid Preliminary Proficiency Study – March 2015 34/86
Laboratory 5 Method Summary
Method Details Nicotine Water Glycerin PG
Sample Preparation
sample storage prior to preparation Refrigerated, then @RT for 2 h prior to sample preparation
Refrigerated, then @RT for 2 h prior to sample preparation
Refrigerated, then @RT for 2 h prior to sample preparation
Refrigerated, then @RT for 2 h prior to sample preparation
Target weight or volume used 250 (+/-30) 250 (+/-30) 250 (+/-30) 250 (+/-30)
dilution volume and solvent 40 mL 2-propanol w/ IS 40 mL 2-propanol w/ IS 40 mL 2-propanol w/ IS 40 mL 2-propanol w/ IS
mixing method 30 min on platform
shaker 30 min on platform
shaker 30 min on platform
shaker 30 min on platform
shaker
Solvent 40 mL 2-propanol
(ambient) 40 mL 2-propanol
(ambient) 40 mL 2-propanol
(ambient) 40 mL 2-propanol
(ambient)
Extraction 30 min on platform
shaker 30 min on platform
shaker 30 min on platform
shaker 30 min on platform
shaker
Additional Filtration n/a n/a n/a n/a
Additional sample processing prior to analysis n/a n/a Dilution with2-Propanol
w/ IS Dilution with2-Propanol
w/ IS
Additional details of relevance related to sample preparation
n/a n/a n/a n/a
ECIG CTR E-Liquid Preliminary Proficiency Study – March 2015 35/86
Laboratory 5 Method Summary
Method Details Nicotine Water Glycerin PG
Analytical Equipment
Description Perkin Elmer GC/FID Perkin Elmer GC/TCD Perkin Elmer GC/FID Perkin Elmer GC/FID
Detection Systems + Conditions
WCOT CP-WAX 52CB 25 m x 0.53 mm, guard: 2 m x 0.53 mm, 80°C for 1.75 min, 25°C/min to 140°C, 50°C/min to 190°C for 1.75 min, 30°C/min to 220°C for 1.10 min: 5:1 split, flow 11 mL/min, total flow 31 mL/min, 3 µL injection
Porplot Q 10m x 0.53 mm, 125°C for 1.75 min, 30°C/min to 140°C for 1 min, 50°C/min to 190°C, hold 1.75 min: 5:1 split, flow 10 mL/min, total flow 30 mL/min, 3 µL injection
WCOT CP-WAX 52CB 25 m x 0.53 mm, guard: 2 m x 0.53 mm, 80°C for 1.75 min, 25°C/min to 140°C, 50°C/min to 190°C for 1.75 min, 30°C/min to 220°C for 1.10 min: 5:1 split, flow 11 mL/min, total flow 31 mL/min, 3 µL injection
WCOT CP-WAX 52CB 25 m x 0.53 mm, guard: 2 m x 0.53 mm, 80°C for 1.75 min, 25°C/min to 140°C, 50°C/min to 190°C for 1.75 min, 30°C/min to 220°C for 1.10 min: 5:1 split, flow 11 mL/min, total flow 31 mL/min, 3 µL injection
Additional Details related to analysis extracts were stored 4C prior to analysis
extracts were stored 4C prior to analysis
extracts were stored 4C prior to analysis
Reference
ECIG CTR E-Liquid Preliminary Proficiency Study – March 2015 36/86
Laboratory 5 Method Summary
Method Details Nicotine Water Glycerin PG
Quantitation Information
Limit of Quantitation
Limit of Detection
Number of standards 7 7 7 6
Concentration of each Standard
5 µg/mL, 10 µg/mL, 25 µg/mL, 50 µg/mL, 100 µg/mL, 250 µg/mL, and 500 µg/mL
0 mg, 1 mg, 2 mg, 4 mg, 10 mg, 20 mg, and 25 mg
10 µg/mL, 20 µg/mL, 50 µg/mL, 100 µg/mL, 200 µg/mL, 500 µg/mL, and 1000 µg/mL
10 µg/mL, 20 µg/mL, 50 µg/mL, 100 µg/mL, 200 µg/mL, and 500 µg/mL
Internal Standard (IS) n-Heptadecane Ethanol n-Heptadecane n-Heptadecane
Calibration Type linear, with IS linear, with IS linear, with IS linear, with IS
r2 1.00 1.00 1.00 1.00
Additional Details related to quantitation
ECIG CTR E-Liquid Preliminary Proficiency Study – March 2015 37/86
Laboratory 6 Method Summary
Method Details Nicotine Water Glycerin PG
Sample Preparation
sample storage prior to preparation room temperature, dark room temperature, dark room temperature, dark room temperature, dark
Target weight or volume used 100µL 100µL 25µL, 50µL, or 100µL 25µL, 50µL, or 100µL
dilution volume and solvent to 1mL with isopropanol containing IS
to 1mL or 5mL with isopropanol containing IS
to 1mL or 5mL with isopropanol containing IS
to 1mL or 5mL with isopropanol containing IS
mixing method manual mixing/vortex mixer
manual mixing/vortex mixer
manual mixing/vortex mixer
manual mixing/vortex mixer
Solvent isopropanol with IS isopropanol with IS isopropanol with IS isopropanol with IS
Extraction manual mixing/vortex mixer as needed
manual mixing/vortex mixer as needed
manual mixing/vortex mixer as needed
manual mixing/vortex mixer as needed
Additional Filtration none none none none
Additional sample processing prior to analysis none none none none
Additional details of relevance related to sample preparation
none none none none
ECIG CTR E-Liquid Preliminary Proficiency Study – March 2015 38/86
Laboratory 6 Method Summary
Method Details Nicotine Water Glycerin PG
Analytical Equipment
Description Agilent GC/FID Agilent GC/TCD Agilent GC/FID Agilent GC/FID
Detection Systems + Conditions Column: Restek Stabilwax 30mX0.32mmX1.0Um, 1ul injection
Column: Supelco Porapak R 6ft. X 1/8 inch ID
Column: Restek Stabilwax 30mX0.32mmX1.0Um, 1ul injection
Column: Restek Stabilwax 30mX0.32mmX1.0Um, 1ul injection
Additional Details related to analysis extracts stored at room temperature
extracts stored at room temperature
extracts stored at room temperature
extracts stored at room temperature
Reference
ECIG CTR E-Liquid Preliminary Proficiency Study – March 2015 39/86
Laboratory 6 Method Summary
Method Details Nicotine Water Glycerin PG
Quantitation Information
Limit of Quantitation 0.0514 0.495 0.331 0.255
Limit of Detection 0.00514 0.0495 0.0331 0.0255
Number of standards 7 6 10 10
Concentration of each Standard 0.0514, 0.103, 0.257, 0.514, 0.771, 1.03, 1.23 mg/mL
0.495, 1.24, 2.48, 3.71, 4.95, 5.94 mg/mL
0.331, 0.662, 1.66, 3.31, 4.97, 6.62, 7.95, 9.93, 13.2, 33.1 mg/mL
0.255, 0.510, 1.27, 2.55, 3.82, 5.10, 6.11, 7.64, 10.2, 25.5 mg/mL
Internal Standard (IS) Heptadecane Ethanol Heptadecane Heptadecane
Calibration Type linear, with IS linear, with IS linear, with IS linear, with IS
r2 0.999179 0.999354 0.999452 0.999613
Additional Details related to quantitation
ECIG CTR E-Liquid Preliminary Proficiency Study – March 2015 40/86
Laboratory 7 Method Summary
Method Details Nicotine Water Glycerin PG
Sample Preparation
sample storage prior to preparation room temp room temp room temp room temp
Target weight or volume used 1 gram ( to the nearest 0.1 mg)
1 gram(to the nearest 0.1 mg)
1 gram(to the nearest 0.1 mg)
1 gram(to the nearest 0.1 mg)
dilution volume and solvent 50 ml Isopropanol with
IS 50 ml Isopropanol with
IS 50 ml Isopropanol with
IS 50 ml Isopropanol with
IS
mixing method 180 min table top shaker 180 min table top shaker 180 min table top shaker 180 min table top shaker
Solvent Isopropyl-ethyl-Octadecane
Isopropyl-ethyl-Octadecane
Isopropyl-ethyl-Octadecane
Isopropyl-ethyl-Octadecane
Extraction 180 min table top shaker 180 min table top shaker 180 min table top shaker 180 min table top shaker
Additional Filtration none none none none
Additional sample processing prior to analysis none none none none
Additional details of relevance related to sample preparation
none none none none
ECIG CTR E-Liquid Preliminary Proficiency Study – March 2015 41/86
Laboratory 7 Method Summary
Method Details Nicotine Water Glycerin PG
Analytical Equipment
Description Agilent GC 6890 with FID
Agilent GC 6890 with TCD
Agilent GC 6890 with FID
Agilent GC 6890 with FID
Detection Systems + Conditions
Column: J&W Scientific Co. 5.0m x0.2 mm I.D., 0.4um film thickness, DBWAX ETR . Detector: Temp 300°C, H2 flow 40(ml/min), Detector makeup gas Helium 20(ml/min), Detector Air flow 400ml/min
Column: Supelco Inc. 1/8" I.D. stainless steel 80/100 Porapak Q 6ft. 1/8 in x 2.1 mm. Detector: Temp 240°C. Detector makeup gas Helium 30ml/min
Column: J&W Scientific Co. 5.0m x0.2 mm I.D., 0.4um film thickness, DBWAX ETR . Detector: Temp 300°C, H2 flow 40(ml/min), Detector makeup gas Helium 20(ml/min), Detector Air flow 400ml/min
Column: J&W Scientific Co. 5.0m x0.2 mm I.D., 0.4um film thickness, DBWAX ETR . Detector: Temp 300°C, H2 flow 40(ml/min), Detector makeup gas Helium 20(ml/min), Detector Air flow 400ml/min
Additional Details related to analysis NA NA NA NA
Reference NA NA NA NA
ECIG CTR E-Liquid Preliminary Proficiency Study – March 2015 42/86
Laboratory 7 Method Summary
Method Details Nicotine Water Glycerin PG
Quantitation Information
Limit of Quantitation 0.0238 mg/ml 0.103 mg/ml 0.0720 mg/ml 0.0720 mg/ml
Limit of Detection
Number of standards 6 8 6 6
Concentration of each Standard
Level 1: 0.0238 mg/ml Level 2: 0.0990 mg/ml Level3: 0.2970 mg/ml Level4: 1.188 mg/ml Level 5: 1.980 mg/ml Level 6: 5.000 mg/ml
Level 1: Blank Level 2:0.200 mg/ml Level 3:1.000 mg/ml Level 4: 2.000mg/ml Level 5: 3.000mg/ml Level 6 5.000mg/ml Level 7: 8.000mg/ml Level 8:10.000mg/ml
Level 1: 0.0720 mg/ml Level 2: 0.3000 mg/ml Level 3: 0.9000 mg/ml Level 4: 3.600 mg/ml Level 5: 6.000 mg/ml Level 6: 15.000 mg/ml
Level 1: 0.0720 mg/ml Level 2: 0.3000 mg/ml Level 3: 0.9000 mg/ml Level 4: 3.600 mg/ml Level 5: 6.000 mg/ml Level 6: 15.000 mg/ml
Internal Standard (IS) Octadecane 200 Proof Ethyl Alcohol Octadecane Octadecane
Calibration Type linear with IS linear with IS linear with IS linear with IS
r2 0.999986 0.999219 0.999629 0.999887
Additional Details related to quantitation none none none none
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Laboratory 8 Method Summary
Method Details Nicotine Water Glycerin PG
Sample Preparation
sample storage prior to preparation Room temperature Room temperature Room temperature Room temperature
Target weight or volume used 500±100 mg 500±100 mg 500±100 mg 500±100 mg
dilution volume and solvent 25 mL of dry n-propanol extraction solution
25 mL of dry n-propanol extraction solution
25 mL of dry n-propanol extraction solution
25 mL of dry n-propanol extraction solution
mixing method 10 s mixing on a vortexer 10 s mixing on a vortexer 10 s mixing on a vortexer 10 s mixing on a vortexer
Solvent Dry n-propanol with IS (Quinoline and 1,4-Butanediol)
Dry n-propanol with IS (Quinoline and 1,4-Butanediol)
Dry n-propanol with IS (Quinoline and 1,4-Butanediol)
Dry n-propanol with IS (Quinoline and 1,4-Butanediol)
Extraction 10 s mixing time on a vortexer
10 s mixing time on a vortexer
10 s mixing time on a vortexer
10 s mixing time on a vortexer
Additional Filtration N/A N/A N/A N/A
Additional sample processing prior to analysis
Same amount of sample used for all analytes, however, the sample extracts were diluted 1:10 with extraction solution for PG and Glycerin
Same amount of sample used for analytes, however, the sample extracts were diluted 1:10 with extraction solution for PG and Glycerin
Same amount of sample used for analytes, however, the sample extracts were diluted 1:10 with extraction solution for PG and Glycerin
Same amount of sample used for analytes, however, the sample extracts were diluted 1:10 with extraction solution for PG and Glycerin
Additional details of relevance related to sample preparation
Care must be taken to ensure all glassware used for standard and sample preparation is free of residual water.
Care must be taken to ensure all glassware used for standard and sample preparation is free of residual water.
Care must be taken to ensure all glassware used for standard and sample preparation is free of residual water.
Care must be taken to ensure all glassware used for standard and sample preparation is free of residual water.
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Laboratory 8 Method Summary
Method Details Nicotine Water Glycerin PG
Analytical Equipment
Description
Agilent GC equipped with dual hot split/splitless injection ports and dual detectors (FID and TCD).
Agilent GC equipped with dual hot split/splitless injection ports and dual detectors (FID and TCD).
Agilent GC equipped with dual hot split/splitless injection ports and dual detectors (FID and TCD).
Agilent GC equipped with dual hot split/splitless injection ports and dual detectors (FID and TCD).
Detection Systems + Conditions
Columns: Two DB-ALC1 capillary columns (front and back), 30m x 0.32 mm ID, 1.8 µm film thickness. Oven Programming: 90°C for 1min, 15°C/min to 120°c, 40°C to 280°C for 2min, Bake time 2 min, total run time 9 min, Injection port temperatures of 250°C, GC-FID - 1µL injection at 25:1 split and carrier gas flow of 3mL/min.
Columns: Two DB-ALC1 capillary columns (front and back), 30m x 0.32 mm ID, 1.8 µm film thickness. Oven Programming: 90°C for 1min, 15°C/min to 120°c, 40°C to 280°C for 2min, Bake time 2 min, total run time 9 min, Injection port temperatures of 225°C, GC-TCD - 2µL injection at 10:1 split and carrier gas flow of 1.5mL/min.
Columns: Two DB-ALC1 capillary columns (front and back), 30m x 0.32 mm ID, 1.8 µm film thickness. Oven Programming: 90°C for 1min, 15°C/min to 120°c, 40°C to 280°C for 2min, Bake time 2 min, total run time 9 min, Injection port temperatures of 250°C, GC-FID - 1µL injection at 25:1 split and carrier gas flow of 3mL/min.
Columns: Two DB-ALC1 capillary columns (front and back), 30m x 0.32 mm ID, 1.8 µm film thickness. Oven Programming: 90°C for 1min, 15°C/min to 120°c, 40°C to 280°C for 2min, Bake time 2 min, total run time 9 min, Injection port temperatures of 250°C, GC-FID - 1µL injection at 25:1 split and carrier gas flow of 3mL/min.
Additional Details related to analysis
n-Propanol extraction solvent is stored dried with molecular sieves (5 g per liter of extraction solution) to remove any trace levels of water contamination.
n-Propanol extraction solvent is stored dried with molecular sieves (5 g per liter of extraction solution) to remove any trace levels of water contamination.
n-Propanol extraction solvent is stored dried with molecular sieves (5 g per liter of extraction solution) to remove any trace levels of water contamination.
n-Propanol extraction solvent is stored dried with molecular sieves (5 g per liter of extraction solution) to remove any trace levels of water contamination.
Reference N/A N/A N/A N/A
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Laboratory 8 Method Summary
Method Details Nicotine Water Glycerin PG
Quantitation Information
Limit of Quantitation 51.1 µg/mL 516 µg/mL 50.7 µg/mL 49.3 µg/mL
Limit of Detection 16.9 µg/mL 170 µg/mL 16.7 µg/mL 16.3 µg/mL
Number of standards 6 6 6 6
Concentration of each Standard 50, 100, 250, 500, 1000,
2000 500, 1000, 2500, 5000,
10000, 20000 50, 100, 250, 500, 1000,
2000 50, 100, 250, 500, 1000,
2000
Internal Standard (IS) Quinoline (FID) 1,4-Butanediol (TCD) 1,4-Butanediol (FID) 1,4-Butanediol (FID)
Calibration Type Linear, with IS Quadratic, with IS Quadratic, with IS Linear, with IS
r2 >0.990 >0.990 >0.990 >0.990
Additional Details related to quantitation N/A N/A N/A N/A
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Laboratory 9 Method Summary
Method Details Nicotine Water Glycerin PG
Sample Preparation
sample storage prior to preparation room temp, in dark room temp, in dark room temp, in dark room temp, in dark
Target weight or volume used 150 mg 150 mg 150 mg 150 mg
dilution volume and solvent 10 mL IPA w/ ISTD 10 mL EtOAc w/ ISTD 10 mL IPA w/ ISTD 10 mL IPA w/ ISTD
mixing method 10 min mechanical shaker 10 min mechanical shaker 10 min mechanical shaker 10 min mechanical shaker
Solvent N/A N/A N/A N/A
Extraction N/A N/A N/A N/A
Additional Filtration N/A N/A N/A N/A
Additional sample processing prior to analysis
N/A N/A N/A N/A
Additional details of relevance related to sample preparation
N/A N/A N/A N/A
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Laboratory 9 Method Summary
Method Details Nicotine Water Glycerin PG
Analytical Equipment
Description Agilent 7890 GC-FID Agilent 6890 GC-TCD Agilent 7890 GC-FID Agilent 7890 GC-FID
Detection Systems + Conditions
Column: RTXWax (30mx0.32mmx1µm); Split 20:1; 120 °C to 250 °C (11 °C/min ramp); 1 minute hold; 1 µL injection volume
Column: DBWax (30mx0.53mmx1µm); 50 °C to 240 °C (20 °C/min ramp); 1 minute hold; 0.5 µL injection volume
Column: RTXWax (30mx0.32mmx1µm); Split 20:1; 120 °C to 250 °C (11 °C/min ramp); 1 minute hold; 1 µL injection volume
Column: RTXWax (30mx0.32mmx1µm); Split 20:1; 120 °C to 250 °C (11 °C/min ramp); 1 minute hold; 1 µL injection volume
Additional Details related to analysis N/A N/A N/A N/A
Reference N/A N/A N/A N/A
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Laboratory 9 Method Summary
Method Details Nicotine Water Glycerin PG
Quantitation Information
Limit of Quantitation 1 µg/mL 120 µg/mL 5 µg/mL 2 µg/mL
Limit of Detection N/A N/A N/A N/A
Number of standards 12 8 12 12
Concentration of each Standard 1-1000 µg/mL 120 - 2000 µg/mL 5-15000 µg/mL 2 - 15000 µg/mL
Internal Standard (IS) 1,3-butanediol 1,3-butanediol 1,3-butanediol 1,3-butanediol
Calibration Type Linear; 1/x2 Linear; 1/x Linear; 1/x
2 Linear; 1/x
2
r2 ≥0.99950 ≥0.99950 ≥0.99950 ≥0.99950
Additional Details related to quantitation N/A N/A N/A N/A
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Laboratory 10 Method Summary
Method Details Nicotine Water Glycerin PG
Sample Preparation
sample storage prior to preparation Room Temp, Ambient Lighting
Room Temp, Ambient Lighting
Room Temp, Ambient Lighting
Room Temp, Ambient Lighting
Target weight or volume used 0.5 ± 0.05 g 0.1-2 g 0.5 ± 0.05 g 0.5 ± 0.05 g
dilution volume and solvent 10 mL MeOH with IS, further diluted 100 fold in MeOH with IS
N/A 10 mL MeOH with IS, further diluted 100 fold in MeOH with IS
10 mL MeOH with IS, further diluted 100 fold in MeOH with IS
mixing method Mixed by inversion N/A Mixed by inversion Mixed by inversion
Solvent MeOH with IS MeOH MeOH with IS MeOH with IS
Extraction N/A N/A N/A N/A
Additional Filtration N/A N/A N/A N/A
Additional sample processing prior to analysis the same samples were used for all analytes
N/A the same samples were used for all analytes
the same samples were used for all analytes
Additional details of relevance related to sample preparation
aliquots were taken from original container after hand shaking
N/A aliquots were taken from original container after hand shaking
aliquots were taken from original container after hand shaking
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Laboratory 10 Method Summary
Method Details Nicotine Water Glycerin PG
Analytical Equipment
Description Agilent GC/FID KF Titrator: Aquastar
V5000 Agilent GC/FID Agilent GC/FID
Detection Systems + Conditions
ColumnL Restek Rtx-35 (30m x 0.25 mm, 1.0 µm df); 4mm Split liner with glass wool, Injector Temp: 250 °C; Detector Temp: 275 °C; Split ≈ 50:1; Oven temp: 110 °C, hold 1 min, increase to 150 at 10 °C, hold 0.5 min, increase to 240 °C at 60 °C, hold 3 min.
N/A
ColumnL Restek Rtx-35 (30m x 0.25 mm, 1.0 µm df); 4mm Split liner with glass wool, Injector Temp: 250 °C; Detector Temp: 275 °C; Split ≈ 50:1; Oven temp: 110 °C, hold 1 min, increase to 150 at 10 °C, hold 0.5 min, increase to 240 °C at 60 °C, hold 3 min.
ColumnL Restek Rtx-35 (30m x 0.25 mm, 1.0 µm df); 4mm Split liner with glass wool, Injector Temp: 250 °C; Detector Temp: 275 °C; Split ≈ 50:1; Oven temp: 110 °C, hold 1 min, increase to 150 at 10 °C, hold 0.5 min, increase to 240 °C at 60 °C, hold 3 min.
Additional Details related to analysis Solutions stored at 2-8
°C N/A
Solutions stored at 2-8 °C
Solutions stored at 2-8 °C
Reference
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Laboratory 10 Method Summary
Method Details Nicotine Water Glycerin PG
Quantitation Information
Limit of Quantitation 2 mg
Limit of Detection N/A
Number of standards 8 N/A 8 8
Concentration of each Standard 100, 200, 401, 601, 802, 1002, 1203, 1403 µg/mL
N/A 99, 199, 398, 597, 796, 995, 1194, 1393 µg/mL
100, 200, 400, 599, 799, 999, 1199, 1399 µg/mL
Internal Standard (IS) 1, 4 Butanediol N/A 1, 4 Butanediol 1, 4 Butanediol
Calibration Type linear, with IS N/A linear, with IS linear, with IS
r2 1.00 N/A 0.99 1.00
Additional Details related to quantitation
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APPENDIX C: Raw Data Means
Raw data are presented as means ± 1 standard deviation
Nicotine Determinations (% w/w)
Control (Sample 11)
Reported value Actual value %Error
Lab Average St dev
1 1.048 0.005 1.018 2.9%
2a 1.037 0.006 1.033 0.3%
3 1.008 0.001 1.022 -1.3%
2b 0.980 0.036 1.001 -2.1%
5 1.033 0.012 1.038 -0.5%
6 1.037 0.003 1.044 -0.6%
7 1.054 0.002 1.057 -0.2%
8 1.044 0.002 1.058 -1.4%
9 1.060 0.003 1.040 1.9%
10 0.980 0.010 1.027 -4.5%
Sample 1 Sample 2 Sample 3 Sample 4 Sample 5
Lab Average Stdev Average Stdev Average Stdev Average Stdev Average Stdev
Nominal 1.572 -- 1.579 -- 0 -- 0.790 -- 0.793 --
Overall Avg
1.481 -- 1.568 -- NR -- 0.730 -- 0.725 --
1 1.566 0.007 1.595 0.001 NR -- 0.772 0.008 0.759 0.005
2a 1.480 0.010 1.520 0.017 NR -- 0.726 0.006 0.741 0.024
3 1.491 0.004 1.535 0.004 NR -- 0.729 0.002 0.710 0+.012
2b 1.510 0.010 1.563 0.015 NR -- 0.743 0.006 0.740 0.009
5 1.527 0.015 1.583 0.040 NR -- 0.747 0.006 0.733 0.006
6 1.309 0.008 1.756 0.054 NR -- 0.654 0.005 0.663 0.015
7 1.466 0.011 1.508 0.005 NR -- 0.732 0.010 0.723 0.003
8 1.478 0.004 1.520 0.002 NR -- 0.727 0.006 0.720 0.000
9 1.511 0.003 1.562 0.010 NR -- 0.741 0.009 0.743 0.004
10 1.477 0.006 1.533 0.006 NR -- 0.730 0.000 0.717 0.006
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Sample 6 Sample 7 Sample 8 Sample 9 Sample 10
Lab Average Stdev Average Stdev Average Stdev Average Stdev Average Stdev
Nominal 0.536 -- 0.534 -- 0.532 -- 0.480 -- 0.577 --
Overall Avg
0.547 -- 0.534 -- 0.544 -- 0.456 -- 0.578 --
1 0.589 0.024 0.562 0.010 0.557 0.005 0.467 0.002 0.579 0.005
2a 0.551 0.009 0.537 0.005 0.518 0.012 0.475 0.011 0.565 0.024
3 0.521 0.001 0.533 0.001 0.520 0.005 0.451 0.001 0.555 0.003
2b 0.555 0.006 0.538 0.006 0.526 0.001 0.462 0.007 0.574 0.002
5 0.557 0.015 0.533 0.006 0.610 0.044 0.467 0.006 0.570 0.000
6 0.517 0.006 0.513 0.004 NR1 -- 0.442 0.006 0.610 0.011
7 0.544 0.004 0.537 0.002 0.564 0.014 0.461 0.002 0.571 0.006
8 0.540 0.001 0.522 0.000 0.528 0.005 0.458 0.001 0.568 0.000
9 0.550 0.001 0.538 0.001 0.556 0.010 0.457 0.003 0.591 0.004
10 0.547 0.006 0.530 0.000 0.533 0.006 0.423 0.006 0.593 0.006
n=3, NR= Not reportable; <method limits, NR1 = Not reported due to interfering peak
‘-- = Value not provided or not calculated
Glycerin Determinations (% w/w)
Control (Sample 11)
Reported value Actual value %Error
Lab Average Stdev
1 43.7 0.9 44.9 -2.7%
2a 44.1 1.4 44.8 -1.6%
3 45.9 0.1 44.9 2.3%
2b 43.6 2.4 45.0 -3.0%
5 46.2 0.8 43.9 5.2%
6 39.5 0.4 44.8 -11.9%
7 44.2 0.2 44.9 -1.6%
8 42.8 0.6 44.8 -4.6%
9 48.1 0.1 44.8 7.4%
10 39.3 0.3 45.0 -12.7%
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Sample 1 Sample 2 Sample 3 Sample 4 Sample 5
Lab Average Stdev Average Stdev Average Stdev Average Stdev Average Stdev
Nominal 40.0 -- 40.0 -- 40.0 -- 40.0 -- 40.0 --
Overall Avg
41.6 -- 41.6 -- 40.7 -- 40.3 -- 40.7 --
1 42.1 1.1 41.3 0.6 40.8 0.8 40.3 1.0 40.8 1.0
2a 42.0 0.8 41.6 1.3 41.8 0.8 39.3 1.1 42.4 2.2
3 43.2 0.2 43.2 1.0 42.0 0.7 42.2 0.3 41.3 1.3
2b 43.6 0.7 44.6 1.2 42.2 2.4 41.7 0.6 41.6 1.0
5 46.2 1.7 45.4 0.9 44.1 1.0 46.1 0.2 44.7 1.0
6 37.1 0.5 36.7 0.1 36.1 0.2 35.7 0.1 36.7 0.3
7 41.3 0.2 41.7 0.1 40.7 0.3 40.3 0.4 40.7 0.2
8 40.1 0.0 40.0 0.3 39.2 0.3 39.0 0.4 39.3 0.1
9 45.1 0.2 45.5 0.2 44.7 0.2 43.5 0.8 44.4 0.4
10 35.5 0.1 35.9 0.1 35.3 0.2 35.2 0.4 35.6 0.5
Sample 6 Sample 7 Sample 8 Sample 9 Sample 10
Lab Average Stdev Average Stdev Average Stdev Average Stdev Average Stdev
Nominal -- -- -- -- -- -- 100.0 -- 0 --
Overall Avg
38.4 -- 38.4 -- 40.5 -- 98.1 -- NR --
1 38.3 0.8 38.6 0.7 39.2 2.2 96.5 1.3 NR --
2a 39.0 1.7 39.5 0.8 39.0 2.1 92.5 3.8 NR --
3 39.2 0.4 39.5 0.3 40.1 1.9 100.2 1.2 NR --
2b 39.2 1.9 39.0 0.9 39.8 0.3 99.5 3.3 NR --
5 43.1 2.1 41.7 0.3 47.1 1.9 102.9 0.8 NR --
6 34.3 0.3 34.5 0.1 37.5 0.7 98.3 1.5 NR --
7 38.1 0.4 38.3 0.2 40.3 0.7 97.4 0.5 NR --
8 37.0 0.2 37.6 0.3 38.2 1.2 92.2 0.3 NR --
9 42.4 0.1 42.0 0.2 48.6 0.9 105.7 0.8 NR --
10 33.0 0.2 33.7 0.5 35.6 1.1 95.6 0.3 NR --
n=3, NR= Not reportable; <method limits
‘-- = Value not provided or not calculated
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Propylene Glycol Determinations (% w/w)
Control (Sample
11) Reported value Actual value %Error
Lab Average Stdev
1 45.1 0.3 45.0 0.2%
2a 44.9 0.3 45.2 -0.7%
3 45.5 0.1 45.2 0.7%
2b 43.9 0.8 45.0 -2.4%
5 40.1 1.5 45.2 -11.4%
6 40.5 0.5 45.1 -10.1%
7 43.9 0.1 45.0 -2.4%
8 42.3 0.7 44.9 -5.9%
9 45.4 0.2 45.0 0.9%
10 41.6 0.5 45.0 -7.5%
Sample 1 Sample 2 Sample 3 Sample 4 Sample 5
Lab Average Stdev Average Stdev Average Stdev Average Stdev Average Stdev
Nominal 50.0 -- 50.0 -- 50.0 -- 50.0 -- 50.0 --
Overall Avg
44.9 -- 45.0 -- 45.9 -- 46.5 -- 46.0 --
1 47.0 0.6 46.6 0.9 47.1 0.9 47.7 0.7 46.9 0.9
2a 46.3 0.4 46.2 0.5 47.4 0.3 47.2 0.4 47.6 1.1
3 46.9 0.0 47.1 0.1 47.7 0.5 48.6 0.1 47.5 0.5
2b 46.8 0.4 47.2 0.4 47.7 1.1 48.0 0.2 47.7 0.7
5 43.1 1.1 43.7 1.1 45.2 0.5 47.5 0.3 46.2 0.4
6 41.1 0.7 40.9 0.1 41.8 0.3 42.1 0.1 42.3 0.4
7 45.1 0.3 45.4 0.2 46.2 0.3 46.6 0.7 46.2 0.2
8 43.5 0.2 43.3 0.3 44.2 0.2 44.8 0.4 44.2 0.1
9 46.5 0.1 47.1 0.3 47.9 0.1 47.8 0.6 47.9 0.2
10 42.8 0.4 43.0 0.3 44.1 0.1 44.6 0.3 43.8 0.8
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Sample 6 Sample 7 Sample 8 Sample 9 Sample 10
Lab Average Stdev Average Stdev Average Stdev Average Stdev Average Stdev
Nominal -- -- -- -- -- -- 0 -- 100.0 --
Overall Avg
56.6 -- 56.3 -- 44.5 -- NR -- 96.5 --
1 58.4 1.2 57.0 0.4 46.1 2.0 NR -- 95.8 1.1
2a 57.9 1.1 58.0 0.3 45.2 1.5 NR -- 98.3 2.6
3 57.6 0.1 57.9 0.2 45.4 1.9 NR -- 98.1 0.1
2b 58.6 0.3 57.8 0.9 45.4 0.7 NR -- 99.7 0.3
5 57.5 2.5 55.5 0.8 40.2 1.8 NR -- 102.0 4.4
6 51.6 0.5 51.7 0.3 43.5 0.8 NR -- 88.5 1.3
7 56.5 0.4 56.5 0.2 46.0 0.8 NR -- 95.3 1.1
8 54.4 0.3 55.0 0.4 43.3 1.2 NR -- 93.0 0.3
9 59.2 0.1 58.7 0.1 45.6 1.1 NR -- 100.0 0.5
10 54.9 0.7 54.8 0.4 43.9 0.3 NR -- 94.8 2.1
n=3, NR= Not reportable; <method limits
‘-- = Value not provided or not calculated
Water Determinations (% w/w)
Control (Sample 11)
Reported value Actual value %Error
Lab Average Stdev
1 7.7 0.2 9.0 -15.2%
2a* 9.4 0.1 9.0 4.5%
3* 9.1 0.0 8.9 2.1%
2b* 9.2 0.2 8.9 3.4%
5 10.6 0.5 9.8 7.9%
6 7.6 0.3 9.0 -15.6%
7 8.2 0.2 9.1 -10.0%
8 8.4 0.2 9.2 -8.6%
9 10.8 0.6 9.1 17.9%
10* 9.3 0.1 9.0 2.4%
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Sample 1 Sample 2 Sample 3 Sample 4 Sample 5
Lab Average Stdev Average Stdev Average Stdev Average Stdev Average Stdev
Nominal 5-10 -- 5-10 -- 5-10 -- 5-10 -- 5-10 --
Overall Avg
9 -- 8.5 -- 10.2 -- 9.4 -- 9.9 --
1 8.2 0.2 7.6 0.0 8.8 0.4 8.1 0.1 8.2 0.3
2a* 9.4 0.1 9.1 0.2 10.5 0.1 9.9 0.1 10.0 0.1
3* 8.7 0.4 8.8 0.1 10.2 0.1 9.5 0.1 9.7 0.1
2b* 9.4 0.1 9.0 0.1 10.7 0.1 9.9 0.2 10.0 0.1
5 10.0 0.3 9.9 0.8 11.8 0.1 11.1 0.5 10.3 0.6
6 8.9 0.6 7.8 0.1 9.9 0.5 9.0 0.2 13.2 0.6
7 8.1 0.2 7.1 0.1 8.5 0.1 7.9 0.2 8.3 0.3
8 8.3 0.1 7.7 0.0 9.6 0.4 8.6 0.2 8.8 0.1
9 10.3 0.3 9.4 0.4 11.2 0.2 10.1 0.3 10.6 0.2
10* 9.2 0.2 8.8 0.7 10.5 0.1 9.7 0.1 10.1 0.1
Sample 6 Sample 7 Sample 8 Sample 9 Sample 10
Lab Average Stdev Average Stdev Average Stdev Average Stdev Average Stdev
Nominal -- -- -- -- -- -- -- -- -- --
Overall Avg
60.0% -- 90.0% -- 80.0% -- 70.0% -- 50.0% --
1 0.4% 0.1% 0.4% 0.1% 0.6% 0.1% 0.2% 0.0% 0.2% 0.0%
2a* NR -- 0.7% 0.0% 0.6% 0.0% 0.4% 0.0% 0.0% --
3* 0.2% 0.0% 0.6% 0.0% 0.6% 0.0% 0.4% 0.0% 0.1% 0.0%
2b* NR -- 0.6% 0.0% 0.6% 0.0% 0.3% 0.0% 0.0% --
5 1.4% 0.3% 1.7% 0.2% 1.6% 0.2% 1.9% 0.1% 1.7% 0.5%
6 0.6% 0.0% 1.2% 0.1% 0.5% 0.1% 0.7% 0.0% 0.0% 0.0%
7 0.9% 0.2% 1.2% 0.2% 1.4% 0.2% 1.3% 0.2% 1.0% 0.2%
8 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0%
9 1.1% 0.1% 1.6% 0.1% 1.3% 0.1% 1.2% 0.2% 0.8% 0.1%
10* 0.3% 0.1% 0.7% 0.0% 0.7% 0.1% 0.7% 0.1% 0.2% 0.0%
n=3, NR= Not reportable; <method limits
‘-- = Value not provided or not calculated
*Laboratory used Karl Fischer for water determinations; other labs used GC-TCD
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APPENDIX D: Nicotine - Raw Data Mean Plots
Figure 1. Sample 1 Nicotine
Figure 2. Sample 2 Nicotine
Figure 3. Sample 4 Nicotine
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Figure 4. Sample 5 Nicotine
Figure 5. Sample 6 Nicotine
Figure 6. Sample 7 Nicotine
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Figure 7. Sample 8 Nicotine
Figure 8. Sample 9 Nicotine
Figure 9. Sample 10 Nicotine
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APPENDIX E: Glycerin - Raw Data Mean Plots
Figure 1. Sample 1 Glycerin
Figure 2. Sample 2 Glycerin
Figure 3. Sample 3 Glycerin
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Figure 4. Sample 4 Glycerin
Figure 5. Sample 5 Glycerin
Figure 6. Sample 6 Glycerin
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Figure 7. Sample 7 Glycerin
Figure 8. Sample 8 Glycerin
Figure 9. Sample 9 Glycerin
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APPENDIX F: Propylene Glycol - Raw Data Mean Plots
Figure 1. Sample 1 Propylene Glycol
Figure 2. Sample 2 Propylene Glycol
Figure 3. Sample 3 Propylene Glycol
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Figure 4. Sample 4 Propylene Glycol
Figure 5. Sample 5 Propylene Glycol
Figure 6. Sample 6 Propylene Glycol
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Figure 7. Sample 7 Propylene Glycol
Figure 8. Sample 8 Propylene Glycol
Figure 9. Sample 9 Propylene Glycol
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APPENDIX G: Water - Raw Data Mean Plots
Figure 1. Sample 1 Water
Figure 2. Sample 2 Water
Figure 3. Sample 3 Water
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Figure 4. Sample 4 Water
Figure 5. Sample 5 Water
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APPENDIX H: Nicotine - Mandel’s h and Mandel’s k Graphs
Figure 1. Sample 1 Nicotine
Figure 2. Sample 2 Nicotine
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Figure 3. Sample 4 Nicotine
Figure 4. Sample 5 Nicotine
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Figure 5. Sample 6 Nicotine
Figure 6. Sample 7 Nicotine
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Figure 7. Sample 8 Nicotine
Figure 8. Sample 9 Nicotine
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Figure 9. Sample 10 Nicotine
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APPENDIX I: Glycerin - Mandel’s h and Mandel’s k Graphs
Figure 1. Sample 1 Glycerin
Figure 2. Sample 2 Glycerin
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Figure 3. Sample 3 Glycerin
Figure 4. Sample 4 Glycerin
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Figure 5. Sample 5 Glycerin
Figure 6. Sample 6 Glycerin
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Figure 7. Sample 7 Glycerin
Figure 8. Sample 8 Glycerin
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Figure 9. Sample 9 Glycerin
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APPENDIX J: Propylene Glycol - Mandel’s h and Mandel’s k Graphs
Figure 1. Sample 1 Propylene Glycol
Figure 2. Sample 2 Propylene Glycol
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Figure 3. Sample 3 Propylene Glycol
Figure 4. Sample 4 Propylene Glycol
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Figure 5. Sample 5 Propylene Glycol
Figure 6. Sample 6 Propylene Glycol
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Figure 7. Sample 7 Propylene Glycol
Figure 8. Sample 8 Propylene Glycol
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Figure 9. Sample 10 Propylene Glycol
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APPENDIX K: Water - Mandel’s h and Mandel’s k Graphs
Figure 1. Sample 1 Water
Figure 2. Sample 2 Water
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Figure 3. Sample 3 Water
Figure 4. Sample 4 Water
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Figure 5. Sample 5 Water