E-Cigarette Task Force - CORESTA€¦ · In 2014, the CORESTA E-Cigarette Task Force conducted a study that included testing of commercial electronic cigarette e-liquid products

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

ECIG CTR E-Liquid Preliminary Proficiency Study – March 2015 3/86

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.


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.


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


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.


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.


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.


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


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)


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


0.0%

5.0%

10.0%

15.0%

20.0%

25.0%

30.0%

35.0%

40.0%

45.0%

50.0%


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 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



= 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.


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 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



--= Information not provided and/or calculated


36.0%

38.0%

40.0%

42.0%

44.0%

46.0%

48.0%

50.0%


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%


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.


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 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



--= Information not provided and/or calculated

Figure 8. Water Content Results for Samples 1-5.


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.


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.

mailto:[email protected]


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.


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




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.


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


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.


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


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


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




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







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 ℃


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 ℃


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




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)



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




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



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







Description GC-FID Karl Fischer GC-FID GC-FID


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





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


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




Sample Preparation

sample storage prior to preparation 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


Not Applicable Karl Fischer reagent Combi Titrant 5 Keto

Not Applicable Not Applicable





Description HP6890 Mettler Toledo DL31 Varian 3800 Varian 3800


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





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




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



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

Solvent 40 mL 2-propanol

(ambient) 40 mL 2-propanol



(ambient)

Extraction 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


n/a n/a n/a n/a





Description Perkin Elmer GC/FID Perkin Elmer GC/TCD Perkin Elmer GC/FID Perkin Elmer GC/FID


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



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





Limit of Quantitation

Limit of Detection



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


r2 1.00 1.00 1.00 1.00





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



mixing method 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



Additional Filtration none none none none

Additional sample processing prior to analysis none none none none


none none none none





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



Reference





Limit of Quantitation 0.0514 0.495 0.331 0.255

Limit of Detection 0.00514 0.0495 0.0331 0.0255


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


r2 0.999179 0.999354 0.999452 0.999613





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)



dilution volume and solvent 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



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


none none none none





Description Agilent GC 6890 with FID

Agilent GC 6890 with TCD

Agilent GC 6890 with FID

Agilent GC 6890 with FID


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



Additional Details related to analysis NA NA NA NA

Reference NA NA NA NA





Limit of Quantitation 0.0238 mg/ml 0.103 mg/ml 0.0720 mg/ml 0.0720 mg/ml

Limit of Detection



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




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



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)



Extraction 10 s mixing time on a vortexer

10 s mixing time on a vortexer



Additional Filtration N/A N/A N/A N/A


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




Care must be taken to ensure all glassware used for standard and sample preparation is free of residual water.








Description

Agilent GC equipped with dual hot split/splitless injection ports and dual detectors (FID and TCD).





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.



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.




Reference N/A N/A N/A N/A





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


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




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



N/A N/A N/A N/A


N/A N/A N/A N/A





Description Agilent 7890 GC-FID Agilent 6890 GC-TCD Agilent 7890 GC-FID Agilent 7890 GC-FID


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



Additional Details related to analysis N/A N/A N/A N/A

Reference N/A N/A N/A N/A





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


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




Sample Preparation

sample storage prior to preparation 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 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



N/A aliquots were taken from original container after hand shaking






Description Agilent GC/FID KF Titrator: Aquastar

V5000 Agilent GC/FID Agilent GC/FID


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



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





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



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




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)


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%




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



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



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%



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




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



Water Determinations (% w/w)

Control (Sample 11)


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%




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



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%



*Laboratory used Karl Fischer for water determinations; other labs used GC-TCD


APPENDIX D: Nicotine - Raw Data Mean Plots

Figure 1. Sample 1 Nicotine












APPENDIX E: Glycerin - Raw Data Mean Plots

Figure 1. Sample 1 Glycerin












APPENDIX F: Propylene Glycol - Raw Data Mean Plots

Figure 1. Sample 1 Propylene Glycol












APPENDIX G: Water - Raw Data Mean Plots

Figure 1. Sample 1 Water







APPENDIX H: Nicotine - Mandel’s h and Mandel’s k Graphs















APPENDIX I: Glycerin - Mandel’s h and Mandel’s k Graphs















APPENDIX J: Propylene Glycol - Mandel’s h and Mandel’s k Graphs















APPENDIX K: Water - Mandel’s h and Mandel’s k Graphs








Documents

E-Cigarette Task Force - CORESTA€¦ · In 2014, the CORESTA E-Cigarette Task Force conducted a study that included testing of commercial electronic cigarette e-liquid products