technical report 2015 collaborative study for …...e-vapour sub-group technical report 2015...

E-Vapour Sub-Group

Technical Report

2015 Collaborative Study for Determination of Glycerin,

Propylene Glycol, Water and

Nicotine in Collected Aerosol of E-Cigarettes

March 2017

Sub-Group Coordinator:

Chuck Garner, Ph.D., RAI Services Co.

Sub-Group Secretary:

Robert D. Stevens, Ph.D., ITG Brands, LLC

Author:

Rana Tayyarah, ITG Brands, LLC

Statistician:

Mike Morton, Ph.D., Altria Client Services

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

5. Data Interpretation .............................................................................................................. 9

5.1 Aerosol Collection ................................................................................................... 9

5.2 Measured Analytes ................................................................................................ 10

5.2.1 Results for Control Sample (Sample F) ..................................................... 10

5.2.2 Results for the Blank Sample (Sample E) ................................................. 12

5.2.3 Verification of E-Cigarette Content .......................................................... 12

5.3 Analyte Results – Normalisation to ACM Yields ................................................. 12

5.4 Analyte Results for Products ................................................................................. 13

5.5 General Viability of the Analytical Method .......................................................... 16

6. Recommendations ............................................................................................................ 17

7. Acknowledgements .......................................................................................................... 17

APPENDIX A: Study Protocol And Data Template ............................................................... 18

APPENDIX B: Analytical Method ......................................................................................... 26

APPENDIX C: Raw Data Means ............................................................................................ 33

EVAP-086-1-CTR 2015 Coll. Study glycerin, propylene glycol, water and nicotine – March 2017 3/52

1. Summary

In 2015, the CORESTA E-Cigarette Task Force (changed to E-Vapour Sub-Group in 2016)

conducted a study that included testing of commercial electronic cigarette products. This

study was referred to as “2015 Aerosol Collaborative Study: Major Constituents”.

Preliminary results were presented to the Sub-Group in October 2015. Eighteen laboratories

participated in the study. Each laboratory tested the same set of 4 commercial e-cigarette

products and 1 study control using the same collection and analytical methods. The goal of

this work was to compare results from multiple laboratories applying CRM N° 81 for aerosol

collection and provide a draft analytical method for the determination of nicotine, propylene

glycol (PG), glycerin, and water in collected aerosol. The study results demonstrate that

aerosol delivery for the study samples was relatively variable and that the analyte deliveries

for a given product tend to be proportional to aerosol collected mass (ACM) deliveries.

Evaluation of analyte results normalized to ACM delivery yielded acceptable r&R values.

Results from the study control supported the accuracy of the method. Based on these results,

the draft analytical CORESTA recommended method will be presented for approval by the

Scientific Commission.

2. Introduction

One of the short term objectives of the E-Cigarette Task Force was “… to gather and share

preliminary data on analysis relevant to e-cigarettes worldwide with a view to making

recommendations for product testing”. From previous Task Force (TF) studies, it was

determined that participating laboratories’ methods were quite similar and yielded similar

results. For example, most laboratories conducted aerosol collection under conditions and

with supplies and equipment established for cigarette smoking. Laboratories used various

alcohols for extraction and GC-FID and GC-TCD for analysis. Thus, the TF established

CRM N° 81 and developed a draft analytical CRM based on the methodology employed in

previous TF studies. At the June 2015 TF meeting, key specifications for the draft method

were defined and the TF commissioned a study to evaluate the performance of the collection

and analytical recommended methods. Commercial e-cigarette products matched for e-liquid

lot number but with varied content of major constituents were used to conduct the study. The

analytes and nominal value ranges are listed below:

Nicotine: 0 – 5.4 %

Glycerin: PG 70:30 or 100:0

Water: Content not specified

A study control was included, as well. The control was a mixture of glycerin (43 %),

propylene glycol (43 %), water (12 %), and nicotine (2 %) 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 used in this report do not correspond to

the same order of the laboratories listed below. Note that any breaks in sequence are due to a

laboratory for which a lab code was provided prior to the laboratory declining participation.

All commercial e-cigarette products were supplied by White Cloud Electronic Cigarettes.

Table 1: List of Participating Laboratories

Laboratory

Altria Client Services

Borgwaldt KC GmbH

Enthalpy Analytical

Essentra

Global Laboratory Services Inc.

Godfrey Phillips India Ltd

ITG Brands, LLC

Japan Tobacco Inc.

Japan Tobacco International

JTI-ÖKOLAB Gesellschaft

Laboratoire National de Métrologie et d'Essais

Labstat International ULC

PMI Research & Development

Reemtsma Cigarettenfabriken GmbH

RJ Reynolds Tobacco Company

SEITA Imperial Tobacco

Shanghai Tobacco Group. CNTC

Zhengzhou Tobacco Research Institute

Laboratoire National de Métrologie et d'Essais


3.2 Protocol

E-cigarette products were collected and distributed directly from the manufacturer. The

control samples (Sample F) were made at ITG Brands 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 and were

subsequently distributed directly to the participating laboratories. Analytes included in the

study were nicotine, glycerin, propylene glycol, and water. The laboratories were instructed

to store the samples unopened in the laboratory conditions to be used for testing prior to

testing. Each laboratory conducted eight replicate determinations in a specified run order for

each sample using the collection and analytical methods provided. Sample codes used in the

study and the nominal content of the analytes of interest, 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

analytical method and any significant deviations noted. See Appendix B for the analytical

method provided to the laboratories.

Table 2: Sample Identification and Nominal Analyte Content

Sample Code Nicotine

%w/w

Glycerin

%w/w

PG

%w/w

Glycerin:PG

Ratio

Water

%w/w

Sample A 0 % -- -- 70:30 --

Sample B 2.4 % -- -- 70:30 --

Sample C 5.4 % -- -- 70:30 --

Sample D 2.4 % -- -- 100:0 --

Sample E 0 % 0 % 0 % 0 % 0 %

Sample F 2.0 % 43 % 43 % 50:50 12 %

-- = Information not provided


Table 3: Analytical Method Overview

General Information across Methods Of Note

Aerosol Collection

Aerosol collection by CORESTA CRM N° 81 In addition,

allowable Temp target 20 – 25 °C

allowable RH target 40-70 % RH

Samples stored sealed prior to collection

Collection across three pad sets in the

same day

80 puffs total in 3 segments (10 then 20

then 50 puffs)

Run order by replicate not by sample

A range of traditional

cigarette smoking machines

and e-cigarette vaping

machines were used

Temp / RH at 22 °C 60 % for

most labs, Lab 14 used a 75

% RH target

Sample Prep Pad extraction with Propan-2-ol, 20 mL and shaking

A range of alcohols were used

Solvent volume was varried 10-20 mL

Analysis

GC-TCD for water

GC-FID for other analytes

DB-ALC1 30 m x 0,32 mm x 1,8 µm

A range of capilary columns

Some labs used packed columns for water

3.3 Data - Raw

The summary descriptive statistics data are listed in Appendix C.

4. Data - Statistical Analysis

Data were statistically analysed in conformance with ISO 5725(5) and ISO 13528. ISO

5725(5) was used instead of 5725(2) because it uses an approach less strongly affected by

outliers and does not attempt to delete outliers prior to analysis.

In order to improve r&R values, a test result was defined to be the average of 4 pads of the

same type (i.e. 4 pad 1s) similar to the treatment of tar, nicotine, and CO under ISO smoking.

The difference from the standard treatment of tar, nicotine, and CO values is that rather than

having replicate sets of 4 pads, the standard deviations were mathematically adjusted using an

approach similar to that found in ISO 5725(6) section 4.2.1.

Each analyte was examined as is and as a proportion or percentage of ACM (analyte/ACM).

r&R values for normalized yield and on an ‘as is’ basis are shown below.


Table 4: r&R values for Analytes proportioned to ACM

Sample Code ACMmg Nicotine/

ACM PG/ACM

Glycerin/ ACM

Water/ ACM

Sample A

Mean 148 -- 55.60% 25.50% 16.40%

Indiv 30.2 -- 2.11% 1.11% 2.58%

sr 15.1 -- 1.06% 0.56% 1.29%

sR 24.6 -- 1.94% 1.62% 3.80%

r (%) 28.60% -- 5.30% 6.10% 22.00%

R (%) 46.60% -- 9.80% 17.80% 64.70%

Sample B

Mean 127 2.03% 54.70% 25.90% 16.50%

Indiv 30.3 0.08% 1.98% 1.13% 3.42%

sr 15.2 0.04% 0.99% 0.56% 1.71%

sR 25.7 0.16% 1.87% 1.27% 4.80%

r (%) 33.50% 5.20% 5.10% 6.10% 29.00%

R (%) 56.70% 22.20% 9.60% 13.80% 81.50%

Sample C

Mean 109 4.40% 52.60% 24.00% 17.10%

Indiv 30.8 0.20% 2.43% 1.40% 2.92%

sr 15.4 0.10% 1.22% 0.70% 1.46%

sR 18.4 0.27% 1.91% 0.90% 3.80%

r (%) 39.40% 6.50% 6.50% 8.20% 23.90%

R (%) 47.20% 17.10% 10.20% 10.50% 62.10%

Sample D

Mean 142 2.25% 79.50% -- 15.00%

Indiv 34.4 0.10% 2.99% -- 2.26%

sr 17.2 0.05% 1.49% -- 1.13%

sR 20.5 0.18% 2.84% -- 2.84%

r (%) 33.90% 6.20% 5.30% -- 21.00%

R (%) 40.50% 22.10% 10.00% -- 53.00%

Sample F

Mean -- 2.25% 79.50% -- 15.00%

Indiv -- 0.10% 2.99% -- 2.26%

sr -- 0.05% 1.49% -- 1.13%

sR -- 0.18% 2.84% -- 2.84%

r (%) -- 6.20% 5.30% -- 21.00%

R (%) -- 22.10% 10.00% -- 53.00%

Repeatability and reproducibility calculations based on a test result being the average of four test items. Indiv is

the standard deviation (sd) among individual values (i.e., not the average of 4); Calculations carried out using the

robust algorithms in ISO 5725-5; -- = Not calculated, note that percentage is used as a unit for mean yields of

analytes normalized to ACM and as the percentage of the mean for r and R values


Table 5: r&R values calculated on an ‘as is’ basis (mg)

Sample Code ACM Nicotine PG Glycerin Water

Sample A

Mean 148 -- 82.1 37.5 23

Indiv 30.2 -- 18.2 7.74 4.66

Sr 15.1 -- 9.1 3.87 2.33

sR 24.6 -- 15 5.29 6.47

r (%) 28.60% -- 31.00% 28.90% 28.40%

R (%) 46.60% -- 51.20% 39.50% 78.80%

Sample B

Mean 127 2.59 69.9 32.6 19

Indiv 30.3 0.667 18 7.59 3.69

Sr 15.2 0.334 9 3.8 1.98

sR 25.7 0.457 14.3 6.06 7.03

r (%) 33.50% 36.00% 36.10% 32.60% 29.20%

R (%) 56.70% 49.40% 57.10% 52.00% 103.40%

Sample C

Mean 109 4.86 58.2 26.1 17.6

Indiv 30.8 1.408 17.3 7.29 4.5

Sr 15.4 0.704 8.6 3.65 2.25

sR 18.4 0.869 9.9 4.46 4.48

r (%) 39.40% 40.50% 41.50% 39.10% 35.70%

R (%) 47.20% 50.00% 47.40% 47.70% 71.20%

Sample D

Mean 142 3.2 113.2 -- 20.7

Indiv 34.4 0.786 28.7 -- 4.74

Sr 17.2 0.393 14.4 -- 2.37

sR 20.5 0.45 17.8 -- 6.29

r (%) 33.90% 34.40% 35.50% -- 32.10%

R (%) 40.50% 39.40% 44.00% -- 85.10%

Sample F

Mean -- 2.08 43.10 43.73 11.96

sr -- 0.03 0.69 0.95 0.58

sR -- 0.11 1.67 3.23 1.30

r (%) -- 4.37% 4.45% 6.07% 13.60%

R (%) -- 14.80% 10.90% 20.70% 30.50%

Repeatability and reproducibility calculations based on a test result being the average of four test items. Indiv is

the sd among individual values (i.e., not the average of 4); Calculations carried out using the robust algorithms

in ISO 5725-5; -- = Not calculated


5. Data Interpretation

5.1 Aerosol Collection

Aerosol collection can serve as an indicator of overall performance for the test session and the

devices tested. Aerosol collection was conducted under the puffing parameters specified in

CRM N° 81 and is determined by simple weight difference of the trapping media before and

after collection. Simultaneous determination of device mass loss is complementary in that the

device loss and the trapping media uptake should be approximately the same. Significant

differences in these values can indicate experimental error.

Aerosol from the devices was collected in segmented collections. Collection 1 was the first

10 puffs collected onto a weighed filter pad. Collection 2 was the next 20 puffs onto a

separate weighed filter pad. Collection 3 was the next 50 puffs onto a third weighed filter

pad. Varied puff number per collection was established to ensure a range of analyte deliveries

for improved evaluation of the analytical method. Collection 1 was kept to a low number of

puffs to allow for efficient removal and replacement of non-performing product from the test

set, if warranted. Mean values are displayed in Appendix C. Select information is discussed

below to highlight trends noted in the results.

Collection of aerosol under the parameters specified in CRM N° 81 and using the specified

puff numbers yielded sufficient ACM for analysis.

Typically Device Weight loss and ACM yields were comparable. This indicates that there

was no significant loss of aerosol due to collection and that there were minimal experimental

errors that would be associated with differences in these values. See the Table below for a

summary of information for Product Sample A. See Appendix C for detailed information for

each product.


Table 6: Sample A Summary of Device Loss versus ACM Yields Comparison

Lab Device Mass Loss ACM Yields Mass Loss: ACM Yields

mg mg

Lab 1 135 146 0.92

Lab 2 141 150 0.93

Lab 3* 99 108 0.92

Lab 5 121 127 0.95

Lab 6 67 69 0.97

Lab 7 124 130 0.95

Lab 8 131 139 0.94

Lab 9 149 157 0.95

Lab 10 134 146 0.92

Lab 11* 167 173 0.96

Lab 13 148 155 0.95

Lab 14 157 167 0.94

Lab 15 141 152 0.93

Lab 16 110 149 0.81

Lab 17 138 145 0.95

Lab 18 168 179 0.94

Lab 19 167 174 0.96

Lab 20 153 152 1.00

*purposeful break in sequence; some laboratories were unable to report results after lab numbers were assigned

Variability of collection was approximately 30%. Based on previous experience with a range

of products and collection regimes, this level of variability is not unusual for e-cigarette

devices. On the other hand, in evaluation of method performance and calculation of analyte

r&R values, measures, such as analyte normalization to ACM, should be employed to help

distinguish analytical variability from variability of the devices.

5.2 Measured Analytes

Evaluation of the draft analytical CRM begins with placing the analyte results in context.

Contextual considerations are: the results from the control sample, results for the blank

samples, isolating the analyte results from the ACM results, and the impact of water on the

results of the other analytes.

5.2.1 Results for Control Sample (Sample F)

Given the relative variability of e-cigarette aerosol collection particularly for segmented puff

set collection, a study control was made and analyzed in a similar manner to the samples to

aid evaluation of the analytical method independent of aerosol collection. 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: 43 %, 43 %, 12 %, and 2 %, for glycerin,

propylene glycol, water, and nicotine, respectively.

The error from the actual value for each laboratory’s control sample was calculated and was

determined to be relatively low for all labs and all analytes. For example, the actual values

for nicotine content range from 1.9 % to 2.3 %.

The actual error calculations are shown in Table 7 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.

Simply based on the results for the control sample, the results show a high degree of accuracy

for the method for all labs. The errors are typically less than one unit for each of the analytes.

Percent error for all analytes was typically less than 10%.

Table 7. Error for the Control Sample

Nicotine Glycerin PG Water

Target 2% 43% 43% 12%

Average of all Labs 2.0% 43.9% 43.5% 11.9%

Lab 1 -0.1 0 0 -1

Lab 2 0.2 4 5 0

Lab 3 0.2 4 0 1

Lab 5 0.2 1 1 0

Lab 6 0.0 6 2 0

Lab 7 0.1 2 0 0

Lab 8 0.0 0 0 0

Lab 9 0.1 5 6 2

Lab 10 -0.1 1 1 0

Lab 11 -0.1 -1 1 -2

Lab 13 0.1 -1 0 0

Lab 14 -0.2 -1 0 -2

Lab 15 0.1 3 1 -1

Lab 16 0.1 -2 -2 2

Lab 17 0.0 -2 -2 -1

Lab 18 0.0 -1 -1 -1

Lab 19 -0.1 -4 -2 1

Lab 20 0.0 -2 0 0

Average Error 0.0 1 0 0

Average Absolute Error

0.1 2 1 1

Max Absolute Error 0.2 6 6 2

Calculated as %Error = (Measured - Actual) / Actual * 100, presented without regard for statistical significance


5.2.2 Results for the Blank Sample (Sample E)

Blank samples were included in the study to aid understanding of any potential anomalies.

Blanks were puffed through empty ports in the same manner as the samples followed by

extraction and analysis of the collection pads in the same manner as the samples. The blank

samples were found to be free of analytes except water. Water content was typically

approximately 2 mg/80 puffs. This is in contrast to results for Samples A-D which were

approximately 20 mg water per 80 puffs of collected aerosol. Thus no correction, for results

from the blank samples was necessary for evaluation of the study results.

5.2.3 Verification of E-Cigarette Content

Limited testing to verify the composition of the devices’ e-liquid was conducted. One

laboratory analyzed unused devices that were extras from testing. This was primarily done to

estimate the water content of the devices since a nominal value for water was not provided.

Results are displayed below. Analytes’ measured and nominal content are in agreement. The

water content was significant at approximately 7-8 %.

Table 8: Nominal versus Measured Analyte Content


%w/w

PG

%w/w

Glycerin

%w/w

Glycerin:PG

Ratio

Water

%w/w

Sample A Nominal 0% -- -- 70:30 --

Measured <LOQ 67.2 ± 0.4 24.8 ± 0.3 73:27 8.1 ± 0.4

Sample B Nominal 2.40% -- -- 70:30 --

Measured 2.3 ± 0.1 67.6 ± 1.4 24.5 ± 0.4 73:27 6.7 ± 1.8

Sample C Nominal 5.40% -- -- 70:30 --

Measured 4.9 ± 0.2 63.3 ± 2.2 22.6 ± 0.8 74:26 7.4 ± 0.1

Sample D Nominal 2.40% -- -- 100:0 --

Measured 2.2 ± 0.0 89.1 ± 1.3 <LOQ 100:0 8.3 ± 1.3

n=5; -- = Information not provided, LOQ = Limit of Quantitation

5.3 Analyte Results – Normalisation to ACM Yields

The analytes under study trend with ACM delivery. In other words, when a device delivers

higher or lower yields, or with high or low variability, these analytes deliver in higher or

lower amounts with more or less variability. See the aerosol yield results displayed in Figure

1 below as an example: Data points that are relatively high for ACM are also relatively high

for the other analytes. If one were to compare the PG results, for example, one would have

difficulty evaluating the method and or laboratories’ performance for PG because the yield

from one device to another is quite different.


Figure 1: Yield results comparison demonstrating trending with ACM (mg)

For the objectives of the study, trends analysis with analytes adjusted for differences that are

strictly related to ACM collection is included by proportioning the analyte yield to the ACM

yield.

5.4 Analyte Results for Products

A summary of results from all laboratories is shown in the table below. Propylene glycol is

the major component of the products’ e-liquid. Proportions of propylene glycol to glycerin

content are consistent with nominal values and with the e-liquid content estimates. Nicotine

results are consistent with expected values. Average total yields are presented graphically

below. Detailed results are displayed in Appendix C.

Water is more varied between the laboratories than the other analytes and appears to be higher

in aerosol samples than e-liquid measurements. A review of laboratory method information

revealed that all laboratories targeted approximately 22 °C and most laboratories targeted 60

% RH. Lab 14 room RH was 74 %. The water data appears to trend by laboratory though no

correlation based on laboratory conditions, such as room humidity or choice of solvents, could

be ascertained.


Table 9: Nominal versus Measured Analyte Content


%w/w

PG

%w/w

Glycerin

%w/w

PG:Glycerin

Ratio

Water

%w/w

Sample A

Nominal 0% -- -- 70:30 --

e-liquid est* <LOQ 67 25 73:27 8

Measured <LOQ 56 26 68:32 16

Sample B

Nominal 2.40% -- -- 70:30 --

e-liquid est* 2.3 67 25 73:27 7

Measured 2.0 55 26 68:32 16

Sample C

Nominal 5.40% -- -- 70:30 --

e-liquid est* 4.9 63 23 74:26 7

Measured 4.4 53 24 69:31 16

Sample D

Nominal 2.40% -- -- 100:0 --

e-liquid est* 2.2 89 <LOQ 100:0 8

Measured 2.2 80 <LOQ 100:0 14

*See Table 8 above, Aerosol measurements are presented as %ACM, -- = Information not provided, LOQ =

Limit of Quantitation

Figure 2: Propylene Glycol - Total Yield Results Comparison among Laboratories


Figure 3: Glycerin - Total Yield Results Comparison among Laboratories

Figure 4: Nicotine - Total Yield Results Comparison among Laboratories


Figure 5: Water - Total Yield Results Comparison among Laboratories

5.5 General Viability of the Analytical Method

The proposed analytical method uses equipment (GC) and tools available in a typical

analytical laboratory. Sample preparation is relatively straight-forward for the proposed

method. It is basically a ‘shake and shoot’ method using low-hazard solvents. The

chromatography provides analyte peaks that should yield no or low level subjectivity in

integration. Example chromatograms are displayed below.

The linear dynamic range of the method was sufficient to quantify the analytes across the

added range even for the low (10 puff) samples. Laboratories reported R2 values of >0.99 for

calibration standards of at least 5 levels.

Given the relatively low %error for the range of laboratories in the study for the study control

(Sample F), this method appears to be fit for purpose.

Figure 6: Example Chromatogram (PG, Glycerin, and Nicotine)


Figure 7: Example Chromatogram (Water)

6. Recommendations

Results of this study were presented to the E-Cigarette Sub-Group at the October 2015

meeting. The group was in agreement that the analytical method was suitable for the

determination of nicotine, propylene glycol, glycerin, and water. Thus, it is recommended

that the draft analytical CRM be published for use. Additionally, evaluation of CRM N° 81

under a range of control RH conditions with a variety of product types (i.e. e-liquid

compositions) may be warranted to better understand the impact of ambient water on results.

7. Acknowledgements

The E-Vapour Sub-Group appreciates the support of the RAC Sub-Group and the

participating laboratories, and White Cloud Electronic Cigarettes for providing the products.


APPENDIX A: Study Protocol and Data Template

CORESTA E-CIGARETTE TASK FORCE

Project Title: 2015 Aerosol Collaborative Study: Major Constituents

Type of Document: Protocol

Date: June 10, 2015

Written by:

Rana Tayyarah (ITG Brands)

Bus. +1 336 335-6613

e-mail: [email protected]

mailto:[email protected]


1. Objective

The objective of this study is to provide an initial assessment of methodology in CORESTA

Recommended Method N°81 and the attached draft CORESTA Recommended Method for

the determination of select analytes in e-cigarette aerosol. The E-Cigarette Task Force and the

Routine Analytical Sub-Group are working in collaboration to achieve the study objective and

finalize the draft analytical CRM.

2. Time Schedule

Date Activity

July 31, 2015 Product volunteers to ship samples to the laboratories by this date

September 18, 2015 Laboratories to submit results prior to this date

October TF Meeting Preliminary reporting of results at Task Force meeting

December 15, 2015 Statistical analysis and final report submitted to Scientific Commission

3. Methods

3.1 Test Products

Sample Description

Sample A Commercial cigalike1 type e-cigarette, rechargeable

Sample B Commercial cigalike type e-cigarette, rechargeable

Sample C Commercial cigalike type e-cigarette, rechargeable

Sample D Commercial cigalike type e-cigarette, rechargeable

Sample E Study Blank

Sample F Study Control - Laboratory-prepared e-liquid sample

Samples A – D will be supplied by White Cloud Electronic Cigarettes and will be

shipped from Florida in the US. A minimum of 132 units per product for the study for

each volunteer laboratory. All units for a given product shall be from the same

manufacturing lot as is feasible. If it is necessary to ship multiple lots for a given

sample, lot codes will be cross referenced with the laboratory codes by the product

volunteer.

1 Where cigalike refers to an electronic cigarette as defined in CRM N° 81 that is similar in shape and size to a

conventional cigarette product 2 This number may be rounded up as necessary for shipping convenience.


The product volunteers shall ship units directly to each laboratory. Packages will be

clearly marked “For Laboratory Use Only” and according to any special instructions

required for a given testing laboratory.

Instructions and sufficient batteries, chargers and charging stations for testing will be

provided. Batteries and chargers will be tested prior to dispatch.

Each laboratory shall verify the lot and condition of the products and notify the product

supplier of any issues. A sample receipt template will be provided to document sample

conditions, lot number, etc. This template shall be sent to [email protected]

upon receipt of the samples.

Sufficient product for limited retesting will be provided. Any retains shall be held in

original packaging under laboratory conditions until completion of the study. Each

laboratory shall dispose of retained samples in accordance with their own internal

practices for e-cigarette disposal.

3.2 Study Blank

The study blank, Sample E, is a sample with no devices in the collection port. The study

blank is to be treated in the same manner as all other samples in the study as described

below. The collection run order design shall be established to have at least one “port” of

Sample E with each run. The study blank is to be reported as a sample. The blank’s

values are not to be subtracted from other samples’ values.

3.3 Study Control

At this time there is no standardized reference product for e-cigarette aerosol analysis.

A laboratory prepared e-liquid sample, Sample F, will be provided to all participating

laboratories. The target composition of Sample F will be 43 %, 43 %, 12 %, and 2 %

glycerin, PG, water, and nicotine, respectively. Analytical grade chemicals will be used

for preparation of the samples. The e-liquid sample will not be subjected to the aerosol

collection prescribed in the protocol. For each replicate, 100 mg of e-liquid will be

added to an extraction vessel and diluted with 20 mL of extraction solvent. The

resulting solution shall be treated in the same manner as the aerosol collection samples.

Eight (8) replicates of the study reference shall be prepared and tested. These study

control will be shipped directly to the testing volunteers from North Carolina in the

US.

3.4 Testing Atmosphere

The laboratory in which the aerosol collection will be conducted shall be maintained at

a target of 20-25 °C and 40-70 % RH. The temperature and RH shall be maintained in

accordance with CRM N° 81 clause 5.8. Target temperature and RH values shall be

reported in the data reporting template.

3.5 Sample Handling

All samples shall be stored under the laboratory conditions that will be used for testing

and remain in their original packaging prior to testing. If packaging for cartomizers

must be breached to allow for charging of batteries, cartomizers shall be transferred to

sealed, non-hygroscopic, containers and analysed within 24 hours.

Rechargeable devices shall be fully charged within 24 hours prior to testing according

to manufacturer instructions before use. If battery supplies are sufficient, a single

battery charge will be used for each new e-cigarette device. Otherwise, if batteries must

be recharged, follow manufacturer instructions and spread repeat uses across the full set



of batteries available3. All testing for a given device should occur within the same

working day. In cases where this is not feasible and there is a break in collection across

working days, batteries should be recharged prior to continued collection. Note that this

practice would be considered a deviation to protocol and should be clearly documented

if implemented.

Between puff groups, it is best if the position of the devices is maintained. For example,

if the collection position is horizontal, the device should be kept horizontal during pad

changes, if possible.

Glass fiber filter pads to be used for aerosol collection shall be stored under conditions

equivalent to the testing atmosphere for a minimum of 24 hours before use.

3.6 Replicates and Run Order

For purposes of this study, a replicate consists of the measurement of a single e-

cigarette unit.

The analysis of eight (8) units shall be attempted for each sample. If product failures or

experimental errors are such that there are fewer than 5 reportable results, repeat

analysis is requested if there is sufficient product retain.

Sample run order shall be organized by replicate number rather than sample number. In

other words, all first replicates for all samples shall be collected and analysed together,

all second replicates for all samples shall be collected and analysed together, etc.

If the capacity of the collection apparatus is sufficient for testing more than one

replicate set, more than one replicate may be tested together.

If fewer collection ports than the number of samples are available, replicates shall be

collected sequentially. In these cases, the replicate run order shall be established to

allow for at least one replicate of the blank sample E to be included with each run.

See Table 1 below for replicate organization examples that support grouping by

replicate rather than by sample. These designs are an example not a specification for the

study.

There are no timing requirements for spacing of collection runs. On the other hand, all

80 puffs (three puff sets) for a given device are to be collected in the same working day.

3 For example rather than using Battery 1 four times and Battery 2 one time, use Battery 1 two times and Battery

2 two times.

EVAP-086-1-CTR-2015 Coll. Study glycerin, propylene glycol, water and nicotine – March 2017 22/52

Table 1 – Organization of Aerosol Collection Grouped by Replicate – Example Run Order Designs

20 Port Collection System 8-10 Port Collection System 1-4 Port Collection System

Run # 1 2 Run # 1 2 3 4

Run # 1 2 3

port # port # port #

1 A1 B5

1 A1 B3 C5 D7

1 A1 D1 C2 etc

2 B1 C5

2 B1 C3 D5 E7

2 B1 A2 D2

3 C1 D5

3 C1 D3 E5 A8

3 C1 B2 A3

4 D1 E5

4 D1 E3 A6 B8

4 E1 E2 E3

5 E1 A6

5 E1 A4 B6 C8

6 A2 B6

6 A2 B4 C6 A7

7 B2 C6

7 B2 C4 A5 B7

8 C2 D6

8 C2 A3 B5 C7

9 D2 E6

9

10 E2 A7

10

11 A3 B7

12 B3 C7

13 C3 D7

14 D3 E7

15 E3 A8

16 A4 B8

17 B4 C8

18 C4 D8

19 D4 E8

20 E4 A5

The example assumes 4 products (A-D) and 1 blank E, 8 replicates (1-8) for each product, and at least 1 replicate of E with each run,

Run = full collection experiment for a given set of devices

EVAP-086-1-CTR 2015 Coll. Study glycerin, propylene glycol, water and nicotine – March 2017 23/52 23/52

3.7 Data Resolution and Puff Count

Aerosol from each device shall be collected in three puff groups. All puffs from a puff

group shall be collected onto a single glass fiber filter pad (44mm) and subjected to

analysis.

Puff groups are:

Puff Group Puffs to collect4

1 1-10

2 11-30

3 31-80

If a device reaches end of life prior to completion of all three puff groups, aerosol

collection shall be terminated and the actual puff count recorded for the last (partial)

puff group. End of life may be based on indications noted in CRM N° 81 or, for

purposes of this protocol, when the mass of aerosol collected for a complete group falls

below 0.25 mg/puff do not collect subsequent puff groups. Note that determination of

end of life based on ACM yield drop off may result in an overestimate of puff count and

should be noted in the comments for the collection.

The aerosol traps (filter pad in holder) shall be weighed before and after each puff

group. The e-cigarette device shall be weighed before puff group 1 and after the last

puff group. All puff groups for a given device shall be collected on the same working

day.

3.8 Aerosol Collection

E-Cigarette aerosol collection shall be in accordance with CRM N° 81.

For cylindrical products 4.5-9 mm diameter and collection systems with cigarette

holders described in ISO3308, neoprene washers shall not be used in this study.

As applicable to the collection system, machine warm-up and system test procedures

must be as prescribed in ISO 4387 clause 7.6.3.

The weight of aerosol collected matter (ACM) for each sample will be determined

gravimetrically by measuring the trapping system before and after each collection event.

The total amount of aerosol collected will be determined by difference in mass (pad

mass after collection – pad mass before collection). Record ACM values as mg/pad in

the data template, along with puff count per pad.

Filter pad holders shall be cleaned and dried or replaced between collection sets.

4 A range of puffs per puff group are specified to allow for a range of analyte levels among and between sample

sets


3.9 E-cigarette Mass Loss

The amount of material lost from each e-cigarette will be determined by measuring the

e-cigarette mass just prior to the start of aerosol collection and after the final collection

event (puff group 3).

E Cigarette mass loss calculation:

e-Cigarette mass loss (mg) = massafter pad 3 aerosol collection – massbefore pad 1 aerosol

collection

Record mass loss as mg in the data template.

3.10 Extraction and Analysis

Follow the procedures outlined in the draft analytical CRM that is provided with this

protocol. In cases where strict adherence to the protocol is not possible, please record

deviations in the comments area of the data reporting template.

3.11 Analytes

Report results for puff count (per pad) and e-cigarette mass loss, ACM, propylene

glycol, glycerin, nicotine, and water (mg/pad) using the data template provided.

3.12 Replicates and Puff Count Determination

Eight (8) replicates for each e-cigarette are recommended. If a defective product is

encountered, that product shall be removed from the test pool. Provided that at least five

(5) replicates are successfully collected, the laboratory shall report data for that

particular product. If fewer than five replicates are successfully collected, repeat testing

if possible.

4. Data Submission

The attached template shall be used for data submission. Please supply data in the

requested format without creating new cells or rows in the spreadsheet.

The sample log spreadsheet shall be sent [email protected] upon receipt of

samples.

Results shall be reported back to [email protected] on or before September

18, 2015.

5. Data Analysis

The data will be analysed statistically in accordance with appropriate ISO guidelines by

the study statistical coordinator.


mailto:rana.tayyarah@itgbrands


6. Reference Documents

Collection parameters

Coresta Recommended Method Nº 81

Routine analytical machine for e-cigarette aerosol generation and collection – definitions and standard conditions

(file attached to protocol)

ISO 3308 – Routine analytical cigarette-smoking machine – Definitions and standard conditions

ISO 4387:2000 Cigarettes — Determination of total and nicotine-free dry particulate matter using a routine analytical smoking machine

Analytical Testing

Draft Analytical CRM

Determination of glycerin, propylene glycol, water, and nicotine in the aerosol of e-cigarettes by gas chromatographic analysis


Sample Log Template Excel spreadsheet provided


Data reporting Template Excel spreadsheet provided



APPENDIX B: Analytical Method

CORESTA RECOMMENDED METHOD (draft)

DETERMINATION OF GLYCERIN, PROPYLENE GLYCOL, WATER,

AND NICOTINE IN THE AEROSOL OF E-CIGARETTES BY GAS

CHROMATOGRAPHIC ANALYSIS

(month year)

1. FIELD OF APPLICATION

This method is applicable to analysis of glycerin, propylene glycol, nicotine, and

water in trapped e-cigarette aerosol.

2. NORMATIVE REFERENCES

CORESTA Recommended Method N° 81 2015:

Routine analytical machine for e-cigarette aerosol generation and collection – definitions

and standard conditions.

3. PRINCIPLE

Aerosol is generated and collected from electronic cigarettes by a standard procedure. The

collected matter is dissolved in a solvent and the glycerin, propylene glycol (PG), water, and

nicotine content of this solution are determined by gas chromatography. Aerosol collected

mass may be determined gravimetrically. Results are expressed as the weight of analyte

collected per device, per weight of collected aerosol, per puff, or per puff set as warranted.

4. APPARATUS

4.1 A standard aerosol collection system and trapping devices (i.e. filter pads) complying

with CORESTA Recommended Method N° 81.

4.2 Typical laboratory glassware such as volumetric flasks and extraction vessels

4.3 Mechanical shaker

4.4 Analytical balance, suitable for measuring to the nearest 0,1 mg.

4.5 A gas chromatograph (GC) equipped with a flame ionization detector (FID) and data

collection for glycerine, propylene glycol, and/or nicotine analysis.

4.6 A gas chromatograph (GC) equipped with a thermal conductivity detector (TCD and

data collection for water analysis.

4.7 Gas chromatography column(s), capillary or packed, capable of distinct separation of

the peaks for the solvent, internal standard, analytes of interest from each other and

other components of the sample extract, typically a DB-ALC1 (30 m x 0,32 mm x 1,8


µm) may be used. Gloves (powder-free) and any other personal protective equipment

deemed appropriate by the testing laboratory

4.8 Gloves (powder-free) and any other personal protective equipment deemed appropriate

by the testing laboratory

5. REAGENTS

5.1 Propan-2-ol (analytical grade, maximum water content 1,0 mg/mL).

5.2 Internal standard(s) for water: 1, 4 butanediol, or ethanol, or other appropriate internal

standard which have been demonstrated not to co-elute with other peaks and have been

found to be consistent for peak area (minimum purity 99%). A typical concentration for

internal standard is 0.2 mg/mL.

5.3 Internal standard(s) for other analytes: n-heptadecane, or quinaldine, n-octadecane, or

other appropriate internal standard which have been demonstrated not to co-elute with

other peaks and have been found to be consistent for peak area (minimum purity 99 %).

A typical concentration for internal standard is 1 mg/mL.

5.4 Prepared solvent for sample and calibration standard preparation: propan-2-ol (0)

containing an appropriate concentration of internal standard(s) (0 and/or 0).

5.6 Gases: hydrogen, nitrogen, and/or helium and compressed air as necessary for operation

of the GC.

5.7 Glycerin (1,2,3-propanetriol, CAS 56-81-5); propylene glycol (1,2-propanediol, CAS

57-55-6); (-)-nicotine (CAS 54-11-5); (minimum purity 99 %) for preparation of

standard solutions. Protect from moisture, air, and light as indicated by vendor.

5.8 Water: Type I, or equivalent, distilled or dionized water, minimum resistivity of 18,2

MΩ-cm @25 °C, and free from contamination by analytes of interest.

5.9 Solvent and solutions stored below 20 °C shall be allowed to equilibrate to (22 ± 2) °C

before use.

5.10 For testing that includes the analysis of water, the bulk solvent (0) container should be

fitted with a water trap and all solutions should be kept sealed. Glassware and septa for

vials should be stored in a desiccator until use.


6. CALIBRATION STANDARDS

Dissolve glycerin, propylene glycol, water, and nicotine in the solvent, described in section

0, to produce a series of at least five calibration solutions including blanks, as warranted,

whose concentrations cover the range expected to be found in the samples. A typical range

of calibration standards for each analyte is listed in the table below:

Analyte Typical range (mg/mL)

Glycerin 0,05 – 2,0

Propylene Glycol 0,05 – 2,0

Water 0 and 0,5 – 20

Nicotine 0,05 – 2,0

Note that a blank standard is needed for water and may be warranted for some or all of the

other analytes depending on the anticipated concentration range of the samples.

Store calibration standards at 0 °C to +4 °C and protect from light.

7. PROCEDURES

7.1 Gas chromatography (Glycerin, PG, nicotine)

Set up and operate the gas chromatograph and equipment according to the

manufacturer’s instructions.

Ensure that peaks of interest are well resolved.

Operating conditions, including column type, should be optimized for analyte

separation and sensitivity. Typical operating conditions are as follows:

Column DB-ALC1 (30 m x 0,32 mm x 1,8 µm)

Carrier Gas: Helium, 3 mL/min

Injection Temperature: 250 °C

Injection mode: Split, 25:1

Injection volume: 1 µL

Oven temperature: 90 °C (1min), 15 °C/min to 120 °C, 40 °C/min to 280 °C (2 min)

Detector: Flame Ionization

Detector Temperature: 275 °C


7.2 Gas chromatography (water)

Set up and operate the gas chromatograph and equipment according to the

manufacturer’s instructions.

Ensure that peaks of interest are well resolved.

Operating conditions, including column type, should be optimized for analyte

separation and sensitivity. Typical operating conditions are as follows:

Column DB-ALC1 (30 m x 0,32 mm x 1,8 µm)

Carrier Gas: Helium, 1.5 mL/min

Injection Temperature: 225 °C

Injection mode: Split, 10:1

Injection volume: 2 µL

Oven temperature: 90 °C (1min), 15 °C/min to 120 °C, 40 °C/min to 280 °C (2 min)

Detector: Thermal Conductivity

Detector Temperature: 250 °C

7.3 Instrument Calibration of the gas chromatograph(s)

Inject aliquots of the standard solutions into the gas chromatograph. Record the peak

areas or heights of the analytes and the internal standard.

Calculate the ratio of the analyte peak to the internal standard peak from the peak area

(or height) data for each of the calibration solutions. Determine the slope (m) and

intercept (b) based on linear regression (y = mx +b) for each of the analytes.

If the correlation coefficient R2 is less than 0,99, calibration should be repeated. If an

individual calibration point differs by 10 % or more from the expected value (estimated

by linear regression), it should be omitted. The signal (peak area or height) obtained for

all test portions must fall within the working range of the calibration curve. If test

portions are outside of the working calibration range, the range may be extended is the

suitability of the calibration is maintained. Alternatively, samples may be diluted with

the solvent (5.4) as appropriate.

A mid-level standard may be treated as a check standard and injected after every 20th

run. A maximum of 5% deviation from target is acceptable for water and a maximum

of 3% deviation from target is acceptable for all other analytes.

7.4 Filter pad handling

Filter pads should be stored in the target atmosphere of the test conditions for a

minimum of 24 hours prior to determination of pre-testing weights.

For all operations, the operator shall prevent contamination from the fingers by wearing

gloves of a suitable material (4.8). Between operations, filter pad holder cap, if

available, may be installed to prevent water loss or uptake. Filter pads should be

processed as quickly after collection as is feasible to prevent uptake or loss of water.


7.5 Aerosol collection and sample preparation

Using CORESTA Recommended Methods N° 81 set up the aerosol collection system

and collect the aerosol onto glass fiber filter pads. Collection parameters such as

number of puffs or number of puff sets are dependent on study design. In any case, it

should be ensured that the capacity of the filter pads for aerosol are not exceeded. In

addition, after aerosol collection, remove holder assemblies from the collection system

and process for determination of aerosol mass as necessary (7.6).

For each filter pad, open the holder and remove filter pad with forceps. Fold the pad

twice with the aerosol side toward the inside of the folds, being careful to handle only

the edge of the filter pad. Wipe the inside of the filter holder front with two separate

quarters of an unused conditioned filter disc and add these to the flask the filter pad to

collect any residual aerosol. Alternatively, wipe the holder with the folded unused side

of the filter pad. Transfer to a sample vessel for extraction.

Extract the filter pads using a fixed volume of the solvent (5.40) of 20mL for 44mm

pads or 50mL for 92 mm pads and 30 min of agitation on a mechanical shaker. The

volume of solvent may be adjusted to give analyte concentrations in the range of the

calibration standards. The maximum storage time of the extracts is 48 hours under

ambient conditions. The extracts must be protected from light.

7.6 Simultaneous determination of Aerosol Collected Mass

Optionally, aerosol collected mass (ACM) is determined gravimetrically. Filter pads in

their holder (with or without end caps) and without e-cigarette holders are weighed

before and after aerosol collection prior to extraction for analyte analysis. Weights

should be determined to the nearest 0,1mg.

Aerosol mass is calculated as:

ACM (mg) = massafter aerosol collection – massbefore aerosol collection

Express the test results in mg/puff, mg/puff set, mg/total puffs and/or mg/per device as

indicated by study design.

7.7 Simultaneous determination of e-Cigarette mass loss

Optionally, the mass loss of the device may be determined gravimetrically. The test

article should be weighed prior to installation on the collection system. Subsequent

frequency of mass determination should be as indicated by study design. For example,

the test article may be reweighed between each pad set or may be reweighed after

completion of the testing session. It is recommended that the mass before collection

value be verified if there is any significant time delay between sequential collections

(i.e. collection sets that span multiple work days).

e-Cigarette mass loss (mg) = massafter aerosol collection – massbefore aerosol collection

7.8 Blank Test for Water

Due to the absorption of water by smoke traps and solvent, it is necessary to determine

a value for a test blank. At least two (2) test blanks must be included with each

collection run. Prepare test blanks by installing a filter pad in its holder as if preparing

for aerosol collection. Place the test blanks in close proximity to the collection system

during aerosol collection and extract and analyse them with the samples from the

associated collection.


7.9 Simultaneous versus sequential analysis for water content

The chromatographic methods described herein, or equivalent, may be used in

conjunction with each other using two chromatographs or using a single chromatograph

equipped with dual columns and dual detectors. A simultaneous automated analysis of

water and of glycerin, PG, and nicotine may be achieved by using a splitting system or

an autosampler with two injection positions. Example workflow scenarios are shown in

the diagram below.

When determining analytes from the same sample sequentially, the water determination

is performed first to prevent absorption of water by the sample affecting the final result.

Optional Determinations:

Aerosol Mass, E-Cig Mass Loss

Single GC (Dual Column)

GC-FID, GC-TCD



GC-TCD Analysis for Water

GC-FID Analysis for glycerin/PG/nicotine



GC-TCD Analysis for Water

GC-FID Analysis for glycerin/PG/nicotine

Extraction of filter pads



Workflow 1(Simultaneous)

Workflow 3(Parallel)

Workflow 2(Sequential)

7.10 Determination of analyte content for the samples

Inject replicate aliquots of the sample extracts into the gas chromatograph(s) using the

conditions described in sections 7.1 and or 7.2. Record the peak areas or heights of

analytes and the internal standard(s).

Calculate the mean value of the ratio of the peak area or height of analyte to that of the

internal standard for the replicate injections.

Using the calibration produced, determine the concentration of each analyte in the

extracts as mg/mL. Ensure that the values lie within the range of the standards prepared

in section 6.

For water analysis, subtract the average value of the test blank(s) associated with the

collection from the value for each sample pad:

[water, mg/mL]sample – [water, mg/mL]test blank average

For each analyte, express the test results in mg/mg ACM, mg/puff, mg/puff set,

mg/total puffs and/or mg/per device as indicated by study design.


8. REPEATABILITY AND REPRODUCIBILITY

Information is pending completion of a collaborative study to be launched July 2015.

9. TEST REPORT

The test report shall give the analyte content of the sample as a mass fraction based on the

collection parameters of the testing (i.e. per puff, per pad, per total puffs, per device, etc). The

test report shall also mention all pertinent operating conditions not specified in this

CORESTA Recommended Method as well as any circumstances that may have affected the

result.


APPENDIX C: Raw Data Means

Raw data are presented as means ± 1 standard deviation

Nicotine Determinations (% w/w)

Control

(Sample F) Reported value Actual value Error

Lab Average St dev

Lab 1 2.06 0.08 2.12 -0.06

Lab 2 2.29 0.19 2.12 0.18

Lab 3 2.24 0.03 2.02 0.22

Lab 5 2.27 0.08 2.03 0.25

Lab 6 2.08 0.01 2.05 0.02

Lab 7 2.13 0.35 2.03 0.10

Lab 8 2.00 0.03 2.02 -0.02

Lab 9 2.12 0.16 2.02 0.10

Lab 10 2.00 0.06 2.08 -0.08

Lab 11 2.00 0.02 2.06 -0.06

Lab 13 2.13 0.06 2.02 0.10

Lab 14 2.00 0.02 2.17 -0.17

Lab 15 2.15 0.09 2.03 0.11

Lab 16 2.10 0.00 2.00 0.09

Lab 17 2.00 0.00 1.98 0.02

Lab 18 2.00 0.02 2.04 -0.03

Lab 19 2.00 0.01 2.06 -0.06

Lab 20 1.95 0.01 2.00 -0.05

n=8, typically, Error = Reported value – Actual value


Glycerin Determinations (% w/w)

Control


Lab Average St dev

Lab 1 43.4 1.8 43.0 0.4

Lab 2 47.4 3.8 43.0 4.4

Lab 3 47.1 1.1 43.4 3.7

Lab 5 43.4 0.9 42.5 0.9

Lab 6 49.5 0.4 43.0 6.5

Lab 7 44.5 1.8 42.8 1.7

Lab 8 43.0 2.3 42.9 0.1

Lab 9 48.2 3.7 42.8 5.4

Lab 10 44.0 1.1 43.0 1.0

Lab 11 43.0 0.8 43.5 -0.5

Lab 13 41.6 1.0 42.8 -1.2

Lab 14 42.0 0.5 43.0 -1.0

Lab 15 46.5 1.6 43.0 3.5

Lab 16 41.1 0.6 42.9 -1.8

Lab 17 41.0 0.1 43.5 -2.5

Lab 18 41.9 0.5 42.8 -0.9

Lab 19 39.0 0.2 42.8 -3.8

Lab 20 41.6 0.4 43.4 -1.8



Propylene Glycol Determinations (% w/w)

Control


Lab Average St dev

Lab 1 42.7 1.6 42.9 -0.2

Lab 2 47.8 2.9 42.9 4.9

Lab 3 42.4 0.4 42.7 -0.3

Lab 5 44.7 0.9 43.7 1.1

Lab 6 44.4 0.3 42.9 1.5

Lab 7 43.0 0.5 43.2 -0.2

Lab 8 43.0 0.7 43.0 0.0

Lab 9 49.0 3.7 43.0 6.1

Lab 10 44.0 1.1 42.9 1.1

Lab 11 43.0 0.7 42.5 0.5

Lab 13 42.7 1.1 43.1 -0.5

Lab 14 43.0 0.3 42.8 0.2

Lab 15 44.0 1.5 43.0 1.0

Lab 16 40.9 0.5 43.2 -2.3

Lab 17 41.0 0.1 42.6 -1.6

Lab 18 42.5 0.3 43.1 -0.6

Lab 19 41.0 0.1 43.1 -2.1

Lab 20 42.5 0.7 42.6 -0.1



Water Determinations (% w/w)

Control


Lab Average St dev

Lab 1 10.7 0.9 12.0 -1.3

Lab 2 12.2 1.2 12.0 0.3

Lab 3 13.3 0.4 11.9 1.4

Lab 5 12.2 0.6 11.8 0.4

Lab 6 12.4 0.1 12.0 0.4

Lab 7 11.8 0.5 12.0 -0.2

Lab 8 12.0 0.9 12.0 0.0

Lab 9 14.0 1.4 12.2 1.8

Lab 10 12.0 0.5 11.9 0.1

Lab 11 10.0 1.1 11.9 -1.9

Lab 13 12.5 0.3 12.0 0.5

Lab 14 10.0 0.6 12.0 -2.0

Lab 15 11.4 0.3 12.0 -0.6

Lab 16 13.4 0.3 11.9 1.5

Lab 17 11.0 0.7 11.9 -0.9

Lab 18 11.3 0.2 12.0 -0.7

Lab 19 13.0 0.7 12.0 1.0

Lab 20 11.9 0.5 12.0 -0.1



Sample A - Analyte Determinations (mg/80 puffs)

Lab Yields

(80 Puffs) Wt Loss ACM PG Glycerin Nicotine Water

Mass Loss: ACM

Analyte Sum: ACM

mg mg mg mg mg mg

1 average 135.1 146.1 81.8 37.5 0.01 15.54 0.92 0.92

stdev 26.2 28.4 16.7 8.4 0.01 4.63

%RSD 19% 19% 20% 22% 80% 30%

2 average 140.5 150.4 87.1 40.0 0.05 27.47 0.93 1.03

stdev 31.5 32.3 20.4 8.6 0.01 4.91

%RSD 22% 21% 23% 21% 14% 18%

3 average 99.4 108.4 59.1 26.6 NR 24.06 0.92 1.01

stdev 29.8 30.6 18.7 8.8 NR 3.73

%RSD 30% 28% 32% 33% NR 15%

5 average 120.9 127.4 74.4 33.2 NR 21.52 0.95 1.01

stdev 28.2 30.0 19.0 7.6 NR 3.58

%RSD 23% 24% 25% 23% NR 17%

6 average 66.6 68.6 38.2 19.1 NR 11.31 0.97 1.00

stdev 41.6 43.3 25.4 12.6 NR 6.39

%RSD 62% 63% 67% 66% NR 57%

7 average 124.0 130.0 71.9 33.9 NR 20.98 0.95 0.98

stdev 36.0 37.8 20.9 8.9 NR 5.37

%RSD 29% 29% 29% 26% NR 26%

8 average 131.0 139.0 78.0 37.8 NR 23.37 0.94 1.00

stdev 14.2 15.2 9.6 4.1 NR 2.84

%RSD 11% 11% 12% 11% NR 12%

9 average 148.8 156.9 91.2 41.7 NR 27.17 0.95 1.02

stdev 28.8 27.4 17.4 6.4 NR 3.22

%RSD 19% 17% 19% 15% NR 12%

n=5-8 NR= Not reportable; <method limits

‘-- = Value not provided or not calculated


Sample A - Analyte Determinations (mg/80 puffs) (continued)

Lab Yields


Mass Loss: ACM

Analyte Sum: ACM

mg mg mg mg mg mg

10 average 134.5 145.8 80.2 38.3 NR 22.55 0.92 0.97

stdev 43.4 46.0 28.2 12.0 NR 5.99

%RSD 32% 32% 35% 31% NR 27%

11 average 166.5 172.9 96.1 40.0 0.07 30.15 0.96 0.96

stdev 28.5 28.6 16.7 6.3 0.00 6.48

%RSD 17% 17% 17% 16% 1% 22%

13 average 147.6 155.3 83.3 39.7 NR 25.41 0.95 0.96

stdev 23.2 23.9 14.5 6.4 NR 3.71

%RSD 16% 15% 17% 16% NR 15%

14 average 157.5 166.9 99.3 44.0 NR 9.45 0.94 0.92

stdev 15.2 14.5 10.7 4.9 NR 3.46

%RSD 10% 9% 11% 11% NR 37%

15 average 141.1 152.1 85.4 40.6 0.11 23.37 0.93 0.98

stdev 22.8 24.3 15.2 6.4 0.01 3.76

%RSD 16% 16% 18% 16% 9% 16%

16 average 109.5 148.6 80.3 38.0 NR 31.56 0.81 1.01

stdev 13.3 25.9 10.6 4.3 NR 3.47

%RSD 12% 17% 13% 11% NR 11%

17 average 137.6 144.7 80.1 37.3 NR 18.67 0.95 0.94

stdev 16.9 18.0 11.1 4.0 NR 3.27

%RSD 12% 12% 14% 11% NR 18%

18 average 168.4 178.7 101.6 45.0 NR 23.43 0.94 0.95

stdev 48.3 50.1 31.2 13.5 NR 6.62

%RSD 29% 28% 31% 30% NR 28%

19 average 167.3 173.6 97.3 39.9 NR 26.6 0.96 0.94

stdev 31.0 28.5 17.5 6.4 NR 2.96

%RSD 19% 16% 18% 16% NR 11%

20 average 152.6 152.3 78.1 35.0 NR 29.29 1.00 0.93

stdev 25.1 26.0 17.7 7.6 NR 5.22

%RSD 16% 17% 23% 22% NR 18%


Sample B - Analyte Determinations (mg/80 puffs)

Lab Yields


Mass Loss: ACM

Analyte Sum: ACM

mg mg mg mg mg mg

1 average 117.8 128.7 70.3 32.3 2.43 10.67 0.92 0.90

stdev 42.8 45.1 27.6 11.2 0.96 2.74

%RSD 36% 35% 39% 35% 39% 26%

2 average 127.7 137.5 77.8 36.2 2.81 23.08 0.93 1.02

stdev 30.1 30.8 19.0 7.7 0.66 3.65

%RSD 24% 22% 24% 21% 23% 16%

3 average 87.2 95.6 50.6 23.3 2.18 21.72 0.91 1.02

stdev 28.4 30.0 17.3 7.0 0.66 3.56

%RSD 33% 31% 34% 30% 30% 16%

5 average 94.9 102.4 59.0 27.4 2.35 17.16 0.93 1.03

stdev 33.7 36.2 22.2 8.9 0.89 5.02

%RSD 35% 35% 38% 32% 38% 29%

6 average 40.5 44.0 23.1 12.2 0.80 7.94 0.92 1.00

stdev 13.0 14.8 7.9 4.4 0.27 2.30

%RSD 32% 34% 34% 36% 33% 29%

7 average 98.0 105.1 58.7 28.4 2.51 17.01 0.93 1.01

stdev 28.3 29.8 17.9 8.5 0.69 4.80

%RSD 29% 28% 30% 30% 27% 28%

8 average 99.5 108.6 61.1 30.1 2.23 16.20 0.92 1.01

stdev 22.0 23.6 15.0 6.0 0.53 3.22

%RSD 22% 22% 25% 20% 24% 20%

9 average 173.3 171.1 101.7 45.7 3.42 26.71 1.01 1.04

stdev 69.0 49.5 30.2 13.0 1.02 4.79

%RSD 40% 29% 30% 29% 30% 18%




Sample B - Analyte Determinations (mg/80 puffs) (continued)

Lab Yields


Mass Loss: ACM

Analyte Sum: ACM

mg mg mg mg mg mg

10 average 136.5 151.3 83.4 38.6 3.08 21.23 0.90 0.97

stdev 22.9 24.3 15.6 5.6 0.53 3.15

%RSD 17% 16% 19% 15% 17% 15%

11 average 132.1 144.5 79.6 33.9 2.88 39.16 0.91 1.08

stdev 12.4 12.1 8.9 2.9 0.28 20.39

%RSD 9% 8% 11% 8% 10% 52%

13 average 145.1 156.1 83.5 39.8 3.17 22.62 0.93 0.96

stdev 35.4 38.5 22.1 9.3 0.85 3.76

%RSD 24% 25% 26% 23% 27% 17%

14 average 126.6 136.6 80.0 35.8 2.69 6.21 0.93 0.91

stdev 24.8 26.2 16.0 6.7 0.61 3.99

%RSD 20% 19% 20% 19% 23% 64%

15 average 125.0 138.8 76.0 36.9 2.08 16.40 0.90 0.96

stdev 21.6 23.1 13.4 6.5 1.42 6.60

%RSD 17% 17% 18% 18% 68% 40%

16 average 156.8 139.8 73.9 35.6 2.60 29.16 1.12 1.01

stdev 38.2 19.0 10.4 5.1 0.38 10.43

%RSD 24% 14% 14% 14% 15% 36%

17 average 139.7 147.2 81.6 38.6 3.09 17.59 0.95 0.96

stdev 31.5 32.5 19.7 8.7 0.70 4.03

%RSD 23% 22% 24% 22% 23% 23%

18 average 122.7 130.7 70.6 32.9 2.50 20.79 0.94 0.97

stdev 25.7 26.5 15.5 6.2 0.52 3.82

%RSD 21% 20% 22% 19% 21% 18%

19 average 130.4 135.3 74.1 32.8 2.66 20.73 0.96 0.96

stdev 40.8 46.8 27.1 10.7 0.97 1.87

%RSD 31% 35% 37% 32% 37% 9%

20 average 111.5 117.0 62.5 30.2 2.29 19.62 0.95 0.98

stdev 30.9 33.7 17.5 8.0 0.69 4.08

%RSD 28% 29% 28% 26% 30% 21%




Sample C - Analyte Determinations (mg/80 puffs)

Lab Yields


Mass Loss: ACM

Analyte Sum: ACM

mg mg mg mg mg mg

1 average 106.1 114.8 60.0 27.1 4.81 12.94 0.92 0.91

stdev 30.1 32.4 18.6 7.7 1.54 6.79

%RSD 28% 28% 31% 29% 32% 52%

2 average 108.4 115.9 63.1 28.9 5.19 22.05 0.94 1.03

stdev 35.2 36.3 21.9 9.2 1.77 5.36

%RSD 32% 31% 35% 32% 34% 24%

3 average 67.6 74.0 37.2 16.4 3.38 18.43 0.91 1.02

stdev 20.7 23.1 11.8 6.1 1.01 2.84

%RSD 31% 31% 32% 37% 30% 15%

5 average 109.2 113.7 63.2 27.8 5.70 19.65 0.96 1.02

stdev 38.0 40.5 25.7 9.5 2.25 7.72

%RSD 35% 36% 41% 34% 39% 39%

6 average 37.5 36.1 18.6 8.7 1.45 6.70 1.04 0.98

stdev 13.8 15.4 8.6 4.0 0.70 2.82

%RSD 37% 43% 46% 46% 49% 42%

7 average 88.1 91.5 50.2 22.2 4.57 15.76 0.96 1.01

stdev 51.0 52.2 29.6 13.4 2.58 7.96

%RSD 58% 57% 59% 60% 56% 50%

8 average 114.6 123.3 66.7 30.4 5.67 19.31 0.93 0.99

stdev 39.6 41.3 24.9 9.8 2.08 6.61

%RSD 35% 33% 37% 32% 37% 34%

9 average 117.9 125.4 69.3 33.1 5.21 22.14 0.94 1.03

stdev 33.0 35.7 20.6 6.3 1.54 7.22

%RSD 28% 28% 30% 19% 30% 33%




Sample C - Analyte Determinations (mg/80 puffs) (continued)

Lab Yields


Mass Loss: ACM

Analyte Sum: ACM

mg mg mg mg mg mg

10 average 104.6 115.0 60.0 28.0 4.96 18.51 0.91 0.97

stdev 35.1 37.6 20.6 9.0 1.73 4.80

%RSD 34% 33% 34% 32% 35% 26%

11 average 89.0 94.2 49.4 21.9 4.12 19.67 0.94 1.01

stdev 28.8 31.8 17.7 5.3 1.42 7.56

%RSD 32% 34% 36% 24% 35% 38%

13 average 120.8 127.2 64.8 30.4 5.59 20.17 0.95 0.95

stdev 19.1 20.3 11.2 5.1 0.99 2.96

%RSD 16% 16% 17% 17% 18% 15%

14 average 110.6 117.4 65.7 28.4 5.01 4.55 0.94 0.88

stdev 15.1 14.8 8.8 4.5 0.80 2.12

%RSD 14% 13% 13% 16% 16% 47%

15 average 115.2 125.4 69.0 31.8 5.72 18.67 0.92 1.00

stdev 34.9 36.3 22.3 9.8 1.92 4.50

%RSD 30% 29% 32% 31% 34% 24%

16 average 167.3 122.3 63.2 28.7 5.66 24.28 1.37 1.00

stdev 42.9 30.4 11.4 5.6 1.12 6.25

%RSD 26% 25% 18% 20% 20% 26%

17 average 99.0 103.7 54.4 25.7 4.54 13.20 0.95 0.94

stdev 25.6 27.5 15.2 6.5 1.25 2.02

%RSD 26% 27% 28% 25% 28% 15%

18 average 105.6 111.6 57.5 25.9 4.63 17.59 0.95 0.95

stdev 29.3 30.2 17.1 6.8 1.43 3.56

%RSD 28% 27% 30% 26% 31% 20%

19 average 114.7 121.0 63.5 25.6 5.2 19.7 0.95 0.94

stdev 16.0 17.4 9.8 5.3 0.8 2.3

%RSD 14% 14% 16% 21% 16% 12%

20 average 100.7 104.9 56.8 25.9 4.54 18.41 0.96 1.01

stdev 22.5 25.2 17.4 7.5 1.15 3.23

%RSD 22% 24% 31% 29% 25% 18%




Sample D - Analyte Determinations (mg/80 puffs)

Lab Yields


Mass Loss: ACM

Analyte Sum: ACM

mg mg mg mg mg mg

1 average 144.9 155.5 125.1 0.2 3.28 12.93 0.93 0.91

stdev 45.8 47.2 41.2 0.1 1.09 6.19

%RSD 32% 30% 33% 40% 33% 48%

2 average 155.6 163.7 137.6 0.0 3.52 27.37 0.95 1.03

stdev 41.4 42.7 42.5 0.0 0.98 5.88

%RSD 27% 26% 31% 36% 28% 21%

3 average 126.9 135.1 107.2 NR 3.21 24.38 0.94 1.00

stdev 28.4 30.7 24.4 NR 0.55 4.73

%RSD 22% 23% 23% NR 17% 19%

5 average 146.6 152.0 125.1 NR 3.74 23.11 0.96 1.00

stdev 29.3 30.4 27.6 NR 0.76 4.36

%RSD 20% 20% 22% NR 20% 19%

6 average 97.6 103.6 82.9 NR 2.10 13.97 0.94 0.96

stdev 13.3 14.3 14.0 NR 0.43 2.08

%RSD 14% 14% 17% NR 21% 15%

7 average 126.2 131.7 106.8 NR 3.38 19.85 0.96 0.99

stdev 28.5 29.6 25.6 NR 0.82 3.66

%RSD 23% 22% 24% NR 24% 18%

8 average 130.3 138.7 115.1 1.6 3.17 19.82 0.94 1.00

stdev 25.0 24.6 22.3 0.9 0.59 5.35

%RSD 19% 18% 19% 58% 19% 27%

9 average 132.1 138.1 114.3 NR 2.90 22.11 0.96 1.01

stdev 30.9 33.2 27.5 NR 0.71 6.09

%RSD 23% 24% 24% NR 25% 28%




Sample D - Analyte Determinations (mg/80 puffs) (continued)

Lab Yields


Mass Loss: ACM

Analyte Sum: ACM

mg mg mg mg mg mg

10 average 128.8 140.4 111.6 NR 3.13 20.32 0.92 0.96

stdev 45.4 47.6 39.7 NR 1.08 5.67

%RSD 35% 34% 36% NR 34% 28%

11 average 139.2 146.7 110.9 NR 3.18 30.65 0.95 0.99

stdev 20.9 21.4 25.9 NR 0.48 12.69

%RSD 15% 15% 23% NR 15% 41%

13 average 152.3 160.7 122.9 NR 3.48 22.62 0.95 0.93

stdev 25.1 26.3 21.5 NR 0.57 1.96

%RSD 16% 16% 17% NR 16% 9%

14 average 126.7 134.0 110.8 1.0 2.89 5.71 0.95 0.89

stdev 44.8 46.6 41.7 1.1 1.05 1.82

%RSD 35% 35% 38% 111% 37% 32%

15 average 149.8 161.8 130.8 NR 3.59 21.21 0.93 0.96

stdev 31.0 32.5 26.9 NR 0.68 3.22

%RSD 21% 20% 21% NR 19% 15%

16 average 162.4 174.2 136.5 4.9 4.02 28.81 0.93 0.98

stdev 30.8 27.9 16.7 0.7 0.44 4.11

%RSD 19% 16% 12% 14% 11% 14%

17 average 128.0 132.8 103.9 NR 2.92 15.81 0.96 0.92

stdev 29.7 30.1 24.5 NR 0.63 4.60

%RSD 23% 23% 24% NR 21% 29%

18 average 135.1 142.5 113.1 NR 3.05 20.58 0.95 0.96

stdev 33.0 34.7 29.8 NR 0.75 4.07

%RSD 24% 24% 26% NR 24% 20%

19 average 140.5 147.8 116.6 NR 3.1 21.6 0.95 0.96

stdev 49.1 51.5 42.2 NR 1.1 6.5

%RSD 35% 35% 36% NR 35% 30%

20 average 115.5 119.7 89.8 1.4 2.93 20.13 0.96 0.94

stdev 31.0 34.7 21.7 NR 0.77 3.61

%RSD 27% 29% 24% NR 26% 18%




Sample A - Analyte Determinations (mg)

Lab

ACM

PG

Glycerin

Nicotine

Water

Puffs average stdev average stdev average stdev average stdev average stdev

1 0-10 19.22 3.24 10.56 1.73 4.45 1.05 0.00 0.00 2.19 0.63

11-30 36.04 4.80 19.51 3.14 9.41 1.41 0.00 0.01 5.12 1.03

31-80 90.86 20.84 51.72 12.24 23.68 6.08 0.00 0.00 8.22 4.46

2 0-10 18.13 5.23 10.54 3.20 4.49 1.60 0.02 0.00 4.01 0.89

11-30 37.70 7.84 20.72 4.60 10.56 1.98 0.02 0.00 7.30 1.20

31-80 94.54 20.91 55.81 13.51 24.98 5.47 0.02 0.00 16.15 3.16

3 0-10 15.21 3.69 8.29 2.18 3.00 1.23 NR -- 5.41 0.77

11-30 27.19 8.04 14.22 4.57 6.81 2.34 NR -- 6.78 1.09

31-80 66.03 19.10 36.63 12.24 16.74 5.42 NR -- 11.88 2.33

5 0-10 16.56 4.30 9.20 3.10 3.96 1.35 NR -- 3.32 0.46

11-30 31.36 7.11 17.20 4.38 8.20 1.86 NR -- 5.68 0.84

31-80 79.43 19.28 48.05 12.03 21.07 4.69 NR -- 12.93 2.11

6 0-10 10.60 7.77 6.09 4.41 2.45 2.04 NR -- 1.94 1.62

11-30 17.93 12.04 9.89 6.79 5.08 3.55 NR -- 2.97 1.75

31-80 40.10 23.71 22.20 14.33 11.61 7.15 NR -- 6.41 3.16

7 0-10 16.86 4.23 9.89 2.49 4.00 1.14 NR -- 3.48 1.09

11-30 32.70 9.94 15.99 5.37 8.19 2.37 NR -- 5.55 1.77

31-80 80.46 23.77 46.03 14.82 21.71 6.06 NR -- 11.95 2.88

8 0-10 18.41 3.09 9.98 1.62 4.56 1.22 NR -- 3.11 1.21

11-30 33.83 3.81 18.74 2.42 9.71 1.45 NR -- 6.14 1.46

31-80 86.75 9.86 49.28 6.35 23.57 2.20 NR -- 14.12 1.07

9 0-10 20.77 4.70 12.07 2.85 5.43 1.07 NR -- 4.29 0.77

11-30 38.43 8.36 22.00 5.05 10.75 2.25 NR -- 7.09 0.87

31-80 97.69 14.90 57.15 9.88 25.53 3.25 NR -- 15.79 2.06

10 0-10 18.36 5.47 10.02 3.03 4.40 1.74 NR -- 3.18 0.81

11-30 35.24 11.99 18.62 6.86 9.67 3.32 NR -- 5.73 1.68

31-80 92.20 28.69 51.58 18.40 24.21 6.98 NR -- 13.64 3.56




Sample A - Analyte Determinations (mg) (continued)

Lab

ACM

PG

Glycerin

Nicotine

Water


11 0-10 21.26 3.47 11.92 1.85 4.15 0.84 0.00 0.00 3.18 0.58

11-30 40.93 7.67 22.27 4.53 10.84 1.99 0.00 0.00 6.47 1.06

31-80 110.74 18.14 61.88 10.55 25.03 3.78 0.04 0.03 20.49 6.10

13 0-10 19.08 2.56 9.86 1.56 4.40 0.73 NR -- 3.69 0.92

11-30 36.78 5.65 18.79 3.31 9.65 1.39 NR -- 6.09 1.07

31-80 99.46 15.97 54.61 9.92 25.62 4.37 NR -- 15.63 2.40

14 0-10 21.38 1.29 12.39 0.90 5.29 0.41 NR -- 0.90 --

11-30 41.21 4.17 23.61 2.89 11.36 1.32 NR -- 2.43 2.41

31-80 104.30 10.21 63.27 7.43 27.38 3.40 NR -- 8.73 1.90

15* 0-10 19.16 3.06 -- -- -- -- -- -- -- --

11-30 37.98 7.27 -- -- -- -- -- -- -- --

31-80 94.93 14.24 85.40 15.16 40.63 6.41 0.11 0.01 23.37 3.76

16 0-10 19.94 3.31 11.20 1.86 4.86 0.92 NR -- 6.31 1.17

11-30 37.07 5.66 19.07 2.75 10.12 1.57 NR -- 8.27 0.47

31-80 91.61 18.02 50.01 10.11 22.99 3.91 NR -- 16.98 3.16

17 0-10 19.93 3.00 11.22 1.56 4.88 0.76 NR -- 2.54 0.62

11-30 36.01 5.48 19.42 3.31 9.71 1.28 NR -- 4.43 0.98

31-80 88.77 11.20 49.48 7.02 22.73 2.54 NR -- 11.70 1.82

18 0-10 23.56 3.24 10.56 1.73 4.45 1.05 NR -- 2.19 0.63

11-30 44.84 4.80 19.51 3.14 9.41 1.41 NR -- 5.12 1.03

31-80 110.25 20.84 51.72 12.24 23.68 6.08 NR -- 8.22 4.46

19 0-10 21.24 3.35 11.94 1.96 4.94 0.76 NR -- 4.00 0.43

11-30 43.79 6.87 23.37 4.09 10.19 1.75 NR -- 6.89 0.78

31-80 108.56 18.93 61.99 11.68 24.74 4.33 NR -- 15.71 2.03

20 0-10 21.23 4.89 11.34 2.60 4.24 0.91 NR -- 4.37 1.08

11-30 38.63 6.19 20.20 3.48 9.25 1.73 NR -- 7.63 1.24

31-80 92.45 15.66 46.55 12.72 21.52 5.50 NR -- 17.29 3.07



*Data presented in the 31-80 row for Lab 15 is for 80 puffs


Sample B - Analyte Determinations (mg)

Lab

ACM

PG

Glycerin

Nicotine

Water

Puffs average stdev average stdev avera ge stdev avera ge stdev average stdev

1 0-10 15.84 4.56 8.53 2.46 3.74 1.23 0.29 0.08 1.93 0.75

11-30 31.34 11.17 16.36 6.34 8.12 2.96 0.56 0.22 3.99 0.77

31-80 81.52 29.57 45.43 18.95 20.43 7.13 1.58 0.66 4.75 3.43

2 0-10 17.05 3.71 9.63 2.15 4.51 0.89 0.36 0.08 3.50 0.63

11-30 33.34 6.83 18.19 4.08 9.30 1.73 0.67 0.15 6.09 0.83

31-80 87.11 21.04 50.02 13.13 22.35 5.31 1.78 0.44 13.49 2.55

3 0-10 11.65 4.31 6.35 2.18 2.38 1.05 0.34 0.10 4.77 0.77

11-30 23.98 7.36 12.38 4.31 5.92 1.95 0.57 0.17 6.00 0.94

31-80 59.99 18.72 31.91 10.94 15.02 4.25 1.28 0.41 10.96 2.52

5 0-10 13.86 4.20 8.23 2.27 3.63 1.10 0.33 0.09 2.84 0.66

11-30 26.46 9.30 14.33 5.55 7.12 2.39 0.57 0.22 4.55 1.52

31-80 62.11 23.24 36.47 14.82 16.66 5.52 1.45 0.59 9.78 2.94

6 0-10 5.91 1.51 3.19 0.76 1.21 0.42 0.11 0.02 1.08 0.38

11-30 10.37 3.90 5.49 1.88 2.96 1.13 0.19 0.07 2.01 0.69

31-80 27.76 9.66 14.46 5.50 8.02 2.87 0.50 0.19 4.84 1.46

7 0-10 13.18 3.48 7.81 1.81 2.93 1.14 0.37 0.11 2.78 1.02

11-30 26.44 7.64 14.35 4.62 7.41 2.11 0.66 0.18 4.61 1.68

31-80 65.49 18.93 36.58 11.55 18.08 5.44 1.48 0.44 9.63 2.29

8 0-10 12.17 3.10 6.84 1.59 3.05 0.72 0.26 0.06 2.38 0.78

11-30 25.56 6.45 13.81 3.84 7.59 1.68 0.51 0.14 4.24 1.29

31-80 70.82 15.05 40.42 9.79 19.42 4.06 1.46 0.34 9.58 1.93

9 0-10 21.40 4.74 12.80 2.87 5.90 1.27 0.45 0.10 4.10 1.07

11-30 40.36 15.17 24.37 8.36 11.64 3.76 0.84 0.29 6.86 1.60

31-80 109.38 29.72 64.52 19.10 28.16 8.07 2.13 0.64 15.74 2.40

10 0-10 18.82 2.98 10.07 1.65 4.65 0.88 0.39 0.06 3.15 0.76

11-30 37.95 5.90 20.28 3.54 10.26 1.48 0.76 0.13 5.68 0.74

31-80 94.58 16.17 53.09 10.86 23.66 3.56 1.93 0.36 12.41 2.10




Sample B - Analyte Determinations (mg) (continued)

Lab

ACM

PG

Glycerin

Nicotine

Water

Puffs average stdev average stdev average stdev avera ge stdev average stdev

11 0-10 17.64 2.30 9.54 1.45 3.54 0.49 0.37 0.05 2.66 0.40

11-30 33.30 3.86 17.90 2.63 8.73 1.22 0.67 0.08 5.10 0.50

31-80 93.51 10.20 52.13 7.04 21.66 2.13 1.84 0.23 31.41 19.91

13 0-10 19.60 5.17 10.15 2.72 4.89 1.32 0.39 0.11 4.04 1.47

11-30 37.86 9.78 19.71 5.66 10.05 2.56 0.75 0.22 5.37 1.07

31-80 98.65 24.01 53.64 14.29 24.89 5.77 2.03 0.54 13.21 2.07

14 0-10 18.17 3.24 10.43 1.90 4.59 0.94 0.34 0.09 0.35 0.31

11-30 33.93 7.59 19.63 4.01 9.40 1.75 0.66 0.16 1.33 1.54

31-80 84.49 16.47 49.92 10.87 21.82 4.26 1.70 0.39 5.46 2.80

15* 0-10 17.49 3.00 -- -- -- -- -- -- -- --

11-30 34.57 6.01 -- -- -- -- -- -- -- --

31-80 86.79 14.56 75.97 13.38 36.88 6.52 2.08 1.42 16.40 6.60

16 0-10 17.50 3.05 9.28 1.68 4.49 0.91 -- -- 11.14 1.28

11-30 34.80 4.53 18.03 2.06 9.45 1.20 0.71 0.11 8.44 2.32

31-80 87.54 12.38 46.64 7.58 21.70 3.27 1.88 0.28 15.15 2.69

17 0-10 18.22 2.77 10.28 1.39 4.88 0.86 0.45 0.05 2.28 0.56

11-30 36.80 6.90 19.66 4.24 10.02 1.76 0.77 0.15 4.16 1.27

31-80 92.20 23.31 51.61 14.19 23.70 6.11 1.87 0.51 11.15 2.36

18 0-10 16.01 4.56 8.53 2.46 3.74 1.23 0.29 0.08 1.93 0.75

11-30 31.01 11.17 16.36 6.34 8.12 2.96 0.56 0.22 3.99 0.77

31-80 83.65 29.57 45.43 18.95 20.43 7.13 1.58 0.66 4.75 3.43

19 0-10 17.67 6.72 9.67 3.46 4.33 1.76 0.36 0.13 3.35 0.90

11-30 36.06 13.60 19.07 7.87 9.23 3.17 0.69 0.29 5.87 1.54

31-80 86.10 25.31 47.69 15.29 20.45 5.48 1.70 0.54 12.25 2.62

20 0-10 16.08 4.48 8.45 2.26 3.91 1.08 0.29 0.08 3.22 0.77

11-30 29.41 8.14 15.38 4.44 7.92 2.11 0.54 0.17 5.05 1.17

31-80 71.49 21.68 38.67 11.14 18.35 5.01 1.46 0.44 11.35 2.30





Sample C - Analyte Determinations (mg)

Lab

ACM

PG

Glycerin

Nicotine

Water


1 0-10 16.50 4.14 8.77 2.29 3.37 0.98 0.69 0.19 1.74 0.67

11-30 29.66 8.21 15.00 4.64 7.09 1.95 1.19 0.38 4.12 1.46

31-80 68.60 20.99 36.22 12.19 16.66 4.96 2.93 1.01 7.08 5.16

2 0-10 15.03 5.84 8.43 3.52 3.36 1.94 0.70 0.30 3.57 1.28

11-30 30.91 11.51 16.44 6.84 8.08 2.97 1.38 0.57 6.17 1.76

31-80 69.91 22.71 38.22 13.84 17.41 5.43 3.11 1.09 12.31 3.10

3 0-10 11.54 3.33 5.92 1.37 1.92 0.95 0.58 0.13 4.50 0.48

11-30 19.39 6.35 9.41 3.17 4.29 1.72 0.90 0.28 5.24 1.05

31-80 43.05 13.75 21.87 7.43 10.17 3.59 1.91 0.62 8.68 1.76

5 0-10 15.95 5.22 8.93 2.69 3.75 1.18 0.80 0.26 3.12 1.07

11-30 27.96 10.90 14.89 6.53 6.91 2.50 1.35 0.59 5.16 2.62

31-80 69.74 24.81 39.34 16.71 17.10 5.93 3.55 1.43 11.37 4.07

6 0-10 6.64 2.20 3.39 1.22 1.13 0.46 0.26 0.10 1.37 0.73

11-30 9.86 4.52 5.22 2.56 2.47 1.24 0.41 0.20 1.80 0.80

31-80 19.60 8.82 10.00 4.90 5.10 2.30 0.78 0.41 3.53 1.62

7 0-10 12.84 5.36 7.63 2.98 2.58 1.47 0.75 0.30 2.75 1.39

11-30 24.00 13.25 12.63 7.23 5.93 3.34 1.20 0.63 4.23 2.13

31-80 54.70 33.91 29.94 19.57 13.70 8.66 2.62 1.66 8.79 4.55

8 0-10 16.52 4.84 8.68 2.65 3.60 1.17 0.76 0.23 2.77 1.16

11-30 30.08 12.04 15.92 6.81 7.87 2.98 1.36 0.58 4.83 2.12

31-80 76.71 25.48 42.07 15.81 18.90 6.05 3.55 1.32 11.72 3.62

9 0-10 18.28 4.10 10.10 2.30 4.47 1.04 0.76 0.18 3.85 1.70

11-30 31.32 8.03 17.24 4.85 8.01 1.87 1.31 0.37 5.72 1.93

31-80 75.80 23.91 42.00 13.75 20.59 4.43 3.14 1.02 12.57 3.72

10 0-10 15.85 5.28 8.07 2.64 3.57 1.40 0.69 0.23 2.66 0.96

11-30 29.02 10.40 14.81 5.65 7.33 2.53 1.23 0.47 5.30 1.34

31-80 70.10 22.08 37.10 12.47 17.06 5.09 3.04 1.04 10.55 2.64




Sample C - Analyte Determinations (mg) (continued)

Lab

ACM

PG

Glycerin

Nicotine

Water


11 0-10 12.30 3.18 6.43 1.67 2.15 0.51 0.56 0.13 1.90 0.61

11-30 21.90 6.05 11.02 3.02 5.00 1.42 0.95 0.26 3.77 1.05

31-80 60.04 24.86 31.98 13.72 14.74 3.75 2.61 1.09 14.00 7.05

13 0-10 17.81 2.52 9.03 1.40 3.86 0.74 0.78 0.12 3.53 0.67

11-30 32.05 5.02 15.73 2.88 7.87 1.27 1.37 0.25 5.07 0.95

31-80 77.34 13.20 40.04 7.15 18.64 3.27 3.44 0.63 11.58 1.71

14 0-10 17.52 2.27 10.82 3.27 3.42 1.40 0.71 0.14 -- --

11-30 30.81 3.27 16.50 1.99 7.84 0.94 1.29 0.16 -- --

31-80 69.05 10.36 38.34 6.86 17.12 2.55 3.01 0.54 4.55 2.12

15* 0-10 17.76 3.60 -- -- -- -- -- -- -- --

11-30 32.91 8.42 -- -- -- -- -- -- -- --

31-80 74.76 25.07 68.98 22.26 31.76 9.84 5.72 1.92 18.67 4.50

16 0-10 18.34 4.25 9.79 1.99 3.90 1.36 0.86 0.19 9.03 1.32

11-30 31.24 8.41 16.16 4.25 7.59 2.07 1.42 0.40 7.17 1.76

31-80 72.77 19.42 37.25 10.11 17.21 4.28 3.37 0.95 12.59 2.79

17 0-10 14.44 2.77 8.04 1.38 3.47 0.70 0.71 0.11 1.95 0.55

11-30 25.86 6.32 13.11 3.51 6.70 1.49 1.11 0.28 2.95 0.44

31-80 63.36 18.85 33.21 10.68 15.49 4.42 2.72 0.88 8.30 1.64

18 0-10 15.83 4.14 8.77 2.29 3.37 0.98 0.69 0.19 1.74 0.67

11-30 27.95 8.21 15.00 4.64 7.09 1.95 1.19 0.38 4.12 1.46

31-80 67.84 20.99 36.22 12.19 16.66 4.96 2.93 1.01 7.08 5.16

19 0-10 16.05 3.47 8.78 1.92 3.51 0.90 0.74 0.17 3.17 0.59

11-30 31.37 5.58 15.84 2.63 6.91 1.07 1.32 0.22 5.28 0.71

31-80 73.62 9.35 38.87 5.88 15.23 3.59 3.19 0.49 11.27 1.47

20 0-10 16.05 3.86 8.01 2.01 3.41 0.79 0.65 0.16 3.29 0.99

11-30 26.91 5.52 13.75 3.31 6.64 1.43 1.11 0.27 4.80 0.95

31-80 61.91 17.23 35.02 13.24 15.85 6.03 2.78 0.76 10.32 2.02





Sample D - Analyte Determinations (mg)

Lab

ACM

PG

Glycerin

Nicotine

Water


1 0-10 22.08 4.07 17.30 3.66 0.08 0.03 0.47 0.10 2.44 0.88

11-30 39.10 12.02 31.13 10.15 0.04 0.06 0.82 0.27 4.42 1.78

31-80 94.30 32.19 76.70 28.25 0.06 0.03 1.99 0.74 6.06 5.44

2 0-10 25.15 4.40 20.37 3.80 NR -- 0.57 0.10 4.78 0.93

11-30 41.25 12.02 34.37 11.54 NR -- 0.92 0.27 7.63 1.63

31-80 97.26 26.67 82.86 27.66 0.02 0.01 2.03 0.62 14.96 3.52

3 0-10 21.45 5.42 15.84 3.47 NR -- 0.56 0.06 5.28 1.67

11-30 35.45 7.59 27.97 7.24 NR -- 0.88 0.17 6.85 1.41

31-80 78.15 18.89 63.42 15.11 NR -- 1.77 0.36 12.25 1.89

5 0-10 24.19 4.87 19.25 3.82 NR -- 0.60 0.14 4.05 0.52

11-30 41.41 6.16 33.04 5.79 NR -- 1.01 0.16 6.53 1.47

31-80 86.39 22.32 72.81 20.42 NR -- 2.13 0.55 12.53 3.00

6 0-10 15.69 2.69 12.16 2.29 NR -- 0.32 0.08 2.55 0.67

11-30 25.83 3.21 20.91 3.17 NR -- 0.54 0.10 3.70 0.58

31-80 62.13 8.97 49.88 8.86 NR -- 1.24 0.26 7.72 1.03

7 0-10 22.20 2.61 18.13 2.38 NR -- 0.63 0.12 4.06 0.87

11-30 35.10 6.83 28.11 5.94 NR -- 0.93 0.20 5.43 1.03

31-80 74.35 20.45 60.60 17.67 NR -- 1.82 0.52 10.36 2.53

8 0-10 24.87 4.44 20.24 3.57 NR -- 0.57 0.10 3.57 1.34

11-30 37.42 7.25 30.63 6.42 NR -- 0.86 0.17 5.08 1.65

31-80 76.44 17.06 64.21 15.20 1.58 0.92 1.75 0.41 11.17 2.76

9 0-10 23.05 3.65 18.50 2.85 NR -- 0.49 0.09 3.98 1.15

11-30 35.94 7.69 30.06 6.35 NR -- 0.78 0.17 5.85 1.71

31-80 79.15 22.40 65.75 18.84 NR -- 1.63 0.47 12.28 3.41

10 0-10 26.59 4.83 20.84 3.89 NR -- 0.61 0.11 4.09 1.43

11-30 38.65 10.60 30.49 8.76 NR -- 0.87 0.23 5.83 1.28

31-80 75.21 32.73 60.23 27.51 NR -- 1.65 0.75 10.40 3.28




Sample D - Analyte Determinations (mg) (continued)

Lab

ACM

PG

Glycerin

Nicotine

Water


11 0-10 24.21 2.85 19.52 2.52 NR -- 0.55 0.07 3.33 0.33

11-30 39.05 8.46 31.37 7.29 NR -- 0.86 0.19 5.55 1.30

31-80 83.48 14.90 60.05 20.62 NR -- 1.77 0.35 21.78 12.17

13 0-10 26.19 3.90 19.65 2.90 NR -- 0.57 0.09 4.01 0.76

11-30 43.29 7.84 33.11 6.18 NR -- 0.96 0.18 6.32 0.98

31-80 91.26 16.67 70.18 13.88 NR -- 1.95 0.36 12.29 1.27

14 0-10 23.14 5.33 17.99 5.81 0.57 1.02 0.51 0.13 -- --

11-30 36.85 12.61 30.65 10.94 0.21 0.09 0.79 0.28 0.09 0.01

31-80 74.00 29.02 62.17 26.43 0.22 0.09 1.59 0.67 5.68 1.83

15* 0-10 27.24 4.34 -- -- -- -- -- -- -- --

11-30 45.63 7.66 -- -- -- -- -- -- -- --

31-80 88.94 23.19 130.78 26.94 NR -- 3.59 0.68 21.21 3.22

16 0-10 28.10 3.88 21.51 3.17 4.95 0.67 0.66 0.10 6.14 0.46

11-30 46.41 7.02 36.19 5.86 NR -- 1.09 0.17 6.14 0.46

31-80 99.69 18.24 78.81 15.04 NR -- 2.27 0.42 16.53 4.23

17 0-10 23.89 4.52 19.08 3.70 NR -- 0.58 0.10 2.81 0.61

11-30 38.39 7.83 30.45 6.48 NR -- 0.88 0.16 4.32 1.20

31-80 70.50 20.60 54.31 17.31 NR -- 1.46 0.45 8.69 3.13

18 0-10 25.01 4.07 17.30 3.66 0.08 0.03 0.47 0.10 2.44 0.88

11-30 39.63 12.02 31.13 10.15 0.04 0.06 0.82 0.27 4.42 1.78

31-80 77.83 32.19 76.70 28.25 0.06 0.03 1.99 0.74 6.06 5.44

19 0-10 28.03 6.53 21.68 4.84 NR -- 0.61 0.14 4.46 0.82

11-30 45.03 15.74 34.80 13.14 NR -- 0.96 0.36 6.42 1.93

31-80 74.70 30.50 60.07 25.31 NR -- 1.57 0.63 10.71 3.87

20 0-10 21.93 3.97 16.46 3.31 -- -- 0.45 0.09 4.03 0.85

11-30 33.70 9.22 25.72 6.64 -- -- 0.73 0.21 5.51 1.22

31-80 64.09 22.71 47.58 13.24 -- -- 1.75 0.68 10.59 2.43


