Smokeless Tobacco Sub-Group - CORESTA · Tobacco Products by Gas Chromatography (2nd edition) • CRM Nº 61: Determination of 1,2-Propylene Glycol, Glycerol and Sorbitol in Tobacco

Smokeless Tobacco Sub-Group

Technical Report

2012 Collaborative Study on Humectants

February 2015

Author:

Gene Gillman, Ph.D.

Enthalpy Analytical, U.S.A.

Co-Author and Sub-Group Secretary:

Karl Wagner, Ph.D.

Altria Client Services, Inc., U.S.A.

Statistical Analysis:

Alexander Hauleithner

Japan Tobacco, Inc., R&D Ökolab, Austria

STS-CTR 2012 Collaborative Study on Humectants – February 2015 2/39

Table of Contents

1. Summary 3

2. Introduction 3

3. Organisation 4

3.1 Participants 4

3.2 Protocol 4

4. Data – Summary Descriptive Statistics 5

5. Data – Statistical Analysis 5

5.1 Exclusion of Outliers 5

5.2 Calculation of Repeatability and Reproducibility 6

6. Data Interpretation 7

7. Recommendations 10

APPENDIX A: Study Protocol 11

APPENDIX B: Data – Descriptive Statistics 16

APPENDIX C: Raw Data Plots 18

APPENDIX D: Mandel’s h Plots 23

APPENDIX E: Mandel’s k Plots 30

APPENDIX F: Raw Data for Homogeneity Study1 37

APPENDIX G: Homogeneity Testing and Study Data 38


1. Summary

In 2012, the CORESTA Smokeless Tobacco Sub-Group (STS) conducted a collaborative study

for the determination of 1,2-propylene glycol (propylene glycol or PG) and glycerol (Gly) in

smokeless tobacco products. The objective of this study was to evaluate the suitability of the

following CORESTA Recommended Methods (CRMs) for the determination of PG and Gly in

smokeless tobacco products:

• CRM Nº 60: Determination of 1,2-Propylene Glycol and Glycerol in Tobacco and

Tobacco Products by Gas Chromatography (2nd edition)

• CRM Nº 61: Determination of 1,2-Propylene Glycol, Glycerol and Sorbitol in

Tobacco and Tobacco Products by High Performance Liquid Chromatography

(HPLC) (2nd edition)

These CRMs were originally developed with tobacco and did not include smokeless tobacco

products. The current study included two CORESTA Reference Products (CRPs) and three

commercial smokeless tobacco products. Twenty laboratories participated in this study, although

not all laboratories evaluated both methods. Method performance was assessed by calculating

repeatability (r) and reproducibility (R). The r &R results demonstrated that an acceptable level

of variability was observed for both methods. %R values for both methods ranged from

approximately 8% to 23%. However, sample heterogeneity was found to be significant factor in

the interlaboratory reproducibility (R) observed in this collaborative study. The GC-FID method

gave an average %R of 18.7% and 17.8% for PG and Gly, respectively, while the HPLC method

gave an average %R of 13.2% and 23.3% for PG and Gly, respectively. On average, the GC-FID

method yielded slightly lower variability than the HPLC method. As was demonstrated

previously with tobacco, the GC-FID method yielded slightly lower results for smokeless tobacco

products than the HPLC method. On average, PG was 6.4 % lower by GC-FID as compared to

HPLC. A similar trend was also seen for Gly with the values 5% lower by GC-FID as compared

to HPLC.

2. Introduction

In 2010, the CORESTA STS conducted a collaborative study that included the four CRPs and

five commercial smokeless tobacco products. This study was referred to as 2010 Collaborative

and Proficiency Study and included humectants (PG, sorbitol and Gly), nitrates, ammonia,

benzo[a]pyrene, moisture, and a group of trace elements. The purpose of this study was to

calculate the repeatability and reproducibility for each method in order to help prioritize CRM

development within the STS. Participants were directed to use CRM Nº 61 for the determination

of humectants. CRM Nº 61 was originally developed for tobacco and did not include smokeless

tobacco products.

After evaluating the results from the 2010 study, the STS decided to conduct a collaborative study

in 2012 where the participants were requested to use either CRM Nº 60 or CRM Nº 61 for the

determination of PG and Gly in smokeless tobacco products. The intent of this second study was

to calculate repeatability and reproducibility and then update both CRMs to include smokeless

tobacco products.

Data analysis was performed following the statistical model provided by ISO 5725-2 (1994)

(“Basic method for the determination of repeatability and reproducibility of a standard

measurement method”). The raw data were obtained from 20 laboratories. Laboratories were

requested to conduct three replicate determinations; however, not all laboratories provided data

using both CRMs. Analyte yields were reported in units of percent (%). Results were reported on

an as-is basis.


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 as the table below. Not all laboratories provided data for all analytes or both methods.

Laboratory codes that have either a “GC” or “LC” suffix denote GC-FID (CRM Nº 60) or HPLC

(CRM Nº 61), respectively.

Table 1. List of Participating Laboratories

Participants

Altria Client Services, Inc.

Arista Laboratories, Europe

Arista Laboratories, United States

British American Tobacco, Southampton

Essentra Scientific Services

Global Laboratory Services

ITC Limited, ITC R&D Centre

Imperial Tobacco Group, Reemtsma

Imperial Tobacco Group, Seita

Japan Tobacco LTRC

KG ASL-Analytik Service Labor GmbH & Co.

KT&G Research Institute

Labstat International, ULC

Landewyck group

Microbac Laboratories, Inc., Wilson Division

Poeschl Tabak GmbH & Co. KG

RJ Reynolds Tobacco Company

Swedish Match Northern Europe

Swisher International, Inc.

Zhengzhou Tobacco Research Institute of China National Tobacco Corp.

3.2 Protocol

Participants were requested to procure the CRPs from North Carolina State University. The three

commercial products, samples A, B and C, were produced by Swedish Match Northern Europe.

Sample identification and descriptions are shown in Table 2. Prior to distribution, samples A and

B were ground to a smaller particle size than is typical of the product styles. Due to the finer

particle size of sample C, it was distributed without further grinding. Each sample was

thoroughly mixed to ensure homogeneity prior to distributing 100 gram quantities to each

participating laboratory. Homogeneity testing was conducted on these samples by one of the

participating labs as a means to understand the variability in the test samples. The results of the

homogeneity testing are discussed in Section 6, Data Interpretation. Participants were directed to

place the samples in the freezer upon receipt. Samples were thawed at room temperature for at

least 2 hours before use. Samples were stored in the refrigerator after the initial thawing.

Additional details are provided in the study protocol in Appendix A.


Table 2. Sample Identification

Sample Sample Description Contains PG Contains Gly

Sample A American style moist snuff yes no

Sample B American style loose leaf chewing tobacco yes yes

Sample C Swedish style snus - loose tobacco yes yes

CRP 1 Swedish style snus pouch yes no

CRP 4 American style loose leaf chewing tobacco no yes

4. Data – Summary Descriptive Statistics

Tables of the descriptive statistics are provided in Appendix B and the raw data plots are provided

in Appendix C. The data are provided on an as-is basis without any correction for moisture. Not

all samples contained both humectants. Specifically, CRP 4 does not contain added PG and

Sample A and CRP 1 do not contain added Gly; therefore, raw data plots are not provided for

these samples.

5. Data – Statistical Analysis

The statistical analysis was conducted following the recommendations given in ISO 5725-2

(1994). This analysis included outlier detection followed by calculation of repeatability (r) and

reproducibility (R). The statistical analysis is described in greater detail in sections 5.1 and 5.2.

5.1 Exclusion of Outliers

Graphical outlier detection was performed following the recommendations of ISO 5725-2 (1994).

Mandel’s h is used to assess the interlaboratory consistency and Mandel’s k is used to assess the

intralaboratory consistency. Both Mandel’s h and Mandel’s k plots were calculated with 0.95

straggling and 0.99 outlying limits. The Mandel’s h plots and the Mandel’s k plots are given in

Appendices D and E, respectively. Due to scatter of the data, the lower straggling and outlying

limits may be negative (e.g. Mandel’s k plot for Sample A, PG, GC-FID). As described above,

plots are not provided for the samples that do not contain the specific humectant. Outliers

detected with Mandel’s h and Mandel’s k plots were not removed prior to calculation of r & R

because the graphical methods are more likely to detect outliers and lead to excessive data

exclusion, as compared to the numerical methods.

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 Grubbs’ outlier

(single iteration) and Cochran’s tests (multiple iterations). The results for Grubbs’ and Cochran’s

outlier detection methods are presented in Tables 3 and 4, respectively. All numerical outliers

were removed from further analyses while stragglers were retained.


Table 3. Results for Grubbs’ Outlier Test for PG and Gly

GC-FID (CRM Nº 60) HPLC (CRM Nº 61)

Humectant/ Product Outliers Lab # Outliers Lab # P

G

Sample A 7 (straggler) ---

Sample B 20 (straggler) ---

Sample C --- ---

CRP 1 16 (outlier) ---

CRP 4 NA NA

Gly

Sample A NA NA

Sample B --- ---

Sample C --- ---

CRP 1 NA NA

CRP 4 --- ---

The “---“ symbol indicates that no stragglers or outliers were detected.

“NA” indicates that the sample does not contain the humectant.

Table 4: Results for Cochran’s Outlier Test for PG and Gly

GC-FID (CRM Nº 60) HPLC (CRM Nº 61)

Humectant/ Product

1st Iteration

Outliers Lab #

2nd

Iteration Outliers Lab #

3rd

Iteration Outliers Lab #

ǂ

1st Iteration Outliers

Lab # ǂ

PG

Sample A 11 (outlier) 6 (outlier) --- 14 (straggler)

Sample B 20 (straggler) --- --- ---

Sample C 16 (outlier) --- --- 14 (straggler)

CRP 1 14 (outlier) 8 (straggler) --- ---

CRP 4 NA NA NA NA

Gly

Sample A NA NA NA NA

Sample B --- --- --- ---

Sample C 11 (outlier) 1 (outlier) 16 (straggler) 14 (outlier)

CRP 1 NA NA NA NA

CRP 4 --- --- --- ---

1. The “ǂ” symbol indicates that additional iterations of Cochran’s did not identify stragglers or outliers.

2. The “---“ symbol indicates that no stragglers or outliers were detected.

3. “NA” indicates that the sample does not contain the humectant.

5.2 Calculation of Repeatability and Reproducibility

After removal of outlying data based on numerical data consistency methods (Grubbs’ test,

Cochran’s test), the final repeatability and reproducibility (r & R) results were calculated. The r &

R results for GC-FID (CRM Nº 60) are shown in Table 5 while the r & R results for HPLC (CRM

Nº 61) are shown in Table 6.


Table 5: Repeatability (r) and Reproducibility (R) with GC-FID (CRM Nº 60)

Sample

Number of Labs

1

Mean (%)

sr sR r R %R P

G

Sample A 15 0.969 0.0073 0.0446 0.020 0.125 12.9

Sample B 17 0.714 0.0118 0.0643 0.033 0.180 25.2

Sample C 16 3.351 0.0207 0.1643 0.058 0.460 13.7

CRP 1 14 2.754 0.0609 0.2266 0.171 0.634 23.0

CRP 4 NA2 NA

2 -- -- -- -- --

Gly

Sample A NA2 NA

2 -- -- -- -- --

Sample B 17 2.346 0.0456 0.1550 0.128 0.434 18.5

Sample C 14 2.742 0.0452 0.1455 0.126 0.407 14.8

CRP 1 NA2 NA

2 -- -- -- -- --

CRP 4 16 3.822 0.1407 0.2759 0.394 0.772 20.2

1. The number of laboratory data sets remaining after removal of outliers (Grubbs’test, Cochran’s test).

2. The sample did not contain the humectant listed.

Table 6: Repeatability (r) and Reproducibility (R) with HPLC (CRM Nº 61)

Sample Number of Labs

1

Mean (%)

sr sR r R %R

PG

Sample A 7 1.064 0.0227 0.0418 0.0640 0.117 11.0

Sample B 7 0.809 0.0123 0.0465 0.0350 0.130 16.1

Sample C 7 3.489 0.0200 0.1022 0.0560 0.286 8.2

CRP 1 7 2.751 0.0993 0.1696 0.2780 0.475 17.3

CRP 4 NA2 NA

2 -- -- -- -- --

Gly

Sample A NA2 NA

2 -- -- -- -- --

Sample B 7 2.504 0.0182 0.2010 0.0510 0.563 22.5

Sample C 6 2.855 0.0094 0.2343 0.0260 0.656 23.0

CRP 1 NA2 NA

2 -- -- -- -- --

CRP 4 7 4.004 0.0945 0.3500 0.2650 0.980 24.5

1. The number of laboratory data sets remaining after removal of outliers (Grubbs’ test and Cochran’s test).

2. The sample did not contain the humectant listed.

6. Data Interpretation

The values of repeatability (r) and reproducibility (R) are shown for GC-FID (CRM Nº 60) in

Table 5 and for HPLC (CRM Nº 61) in Table 6. These tables also include values for %R for each

sample; which range from approximately 8% to 25%. It was not possible to identify any clear

trends in the r and R values versus concentration, probably due to heterogeneity of the samples

used in this study. On average, the GC-FID method yielded lower variability as compared to the

HPLC method (Table 7). The GC-FID method gave average %R values of 18.7% and 17.8% for

PG and Gly, respectively while the HPLC method gave average %R values of 13.2% and 23.3%

for PG and Gly, respectively.


In addition to lower variability, the GC-FID method (CRM Nº 60) also yielded slightly lower

results as compared to the HPLC method (CRM Nº 61). These results are shown in Table 7. On

average, PG was 6.4 % lower by GC-FID as compared to HPLC. A similar trend was also seen

for Gly with the values 5% lower by GC-FID as compared to HPLC. The difference between the

GC-FID and HPLC methods has been previously demonstrated in the 2007 Routine Analytical

Chemistry (RAC) collaborative study on the determination of PG and Gly in tobacco1.

Table 7: Summary Comparison of CRM Nº 60 and CRM Nº 61 Results

CRM Nº 60 CRM Nº 61 % Difference

between Methods

1 Sample

Mean (%)

%R Mean (%)

%R

PG

Sample A 0.969 12.9 1.064 11.0 9.3

Sample B 0.714 25.2 0.809 16.1 12.5

Sample C 3.351 13.7 3.489 8.2 4.0

CRP 1 2.754 23.0 2.751 17.3 -0.1

Average 18.7 13.2 6.4

Gly

Sample B 2.346 18.5 2.504 22.5 6.5

Sample C 2.742 14.8 2.855 23.0 4.0

CRP 4 3.822 20.2 4.004 24.5 4.7

Average 17.8 23.3 5.1

1. ((CRM Nº 61 − CRM Nº 60) / (Average of CRM Nº 61 and CRM Nº 60)) X 100

The current study data were compared to the 2007 RAC Study1. This comparison is summarized

in Tables 8 and 9 for GC-FID and HPLC, respectively. In the 2007 RAC study, the GC-FID

method (CRM-60) gave an average %R of 13.2% and 17.5% for PG and Gly, respectively while

the HPLC method (CRM-61) gave an average %R of 28.6% and 13.8% for PG and Gly,

respectively. On average, the same level of variability is seen in both the 2007 RAC study and the

current study. It is important to note that efforts were made to prepare homogenous samples for

both the RAC study and the current study in order to reduce sample heterogeneity effects on

overall method variability. CRP 1 and CRP 4 are exceptions as these samples were not further

homogenized before distribution.

1 CORESTA Technical Report on 2007 Joint Experiments to Update Repeatability and Reproducibility Statistics for:

CORESTA Recommended Method 60, Determination of 1,2 Propylene Glycol and Glycerol in Tobacco Products by

Gas Chromatography CORESTA Recommended Method 61, Determination of 1,2 Propylene Glycol and Glycerol in

Tobacco Products by High Performance Liquid


Table 8: Repeatability (r) and Reproducibility (R) from 2007 RAC Study - GC-FID

(CRM Nº 60)1

Sample

Mean (%)

r R %R

PG

Sample 1 0.4040 0.0190 0.0580 14.4

Sample 2 1.1810 0.0290 0.2040 17.3

Sample 3 2.0110 0.0280 0.2100 10.4

Sample 4 3.3030 0.0730 0.3600 10.9

Gly

Sample 1 0.5840 0.0320 0.1780 30.5

Sample 2 1.3650 0.0480 0.1830 13.4

Sample 3 2.1590 0.0940 0.2760 12.8

Sample 4 3.3610 0.1450 0.4440 13.2

Table 9: Repeatability (r) and Reproducibility (R) from 2007 RAC Study - HPLC

(CRM Nº 61)1

Sample

Mean (%)

r R %R

PG

Sample 1 0.4240 0.0500 0.2300 54.2

Sample 2 1.2360 0.0540 0.3900 31.6

Sample 3 2.0720 0.0930 0.4690 22.6

Sample 4 3.4350 0.0750 0.2040 5.9

Gly

Sample 1 0.5580 0.0370 0.1310 23.5

Sample 2 1.4250 0.0590 0.1550 10.9

Sample 3 2.3070 0.0230 0.2450 10.6

Sample 4 3.5550 0.0700 0.3690 10.4

While efforts were taken to prepare homogenous samples in the current study, sample

heterogeneity may have been a significant factor in the variability observed. In order to

understand the effect of sample homogeneity on the reproducibility values seen in this study, a

separate homogeneity study was undertaken. The homogeneity study was conducted by

removing 100 g portions of samples A, B and C from the bulk samples before they were

portioned for distribution. These 100 g samples were sent to one of the participating laboratories

for homogeneity analysis by the HPLC method (CRM-61).

This laboratory removed 10 independent tobacco aliquots from each sample, for analysis. Each of

these aliquots were extracted and the prepared samples were analyzed in duplicate (Appendix F).

The % RSD for the replicate analyses for PG were 2.84% and 4.97% for samples A and B,

respectively, and the % RSD for Gly were 5.88% and 4.60% for samples B and C, respectively.

A comparison of the homogeneity data versus the larger study results are shown graphically in

Appendix G. Sample B was not tested for PG and sample C was not tested for Gly. The

variability found in the samples was almost as great as the variability seen between labs. These

results suggest that sample heterogeneity was a significant factor in the interlaboratory

reproducibility (R) observed in the current collaborative study


7. Recommendations

In 2012, the CORESTA Smokeless Tobacco Sub-Group conducted a collaborative study for the

determination of PG and Gly in smokeless tobacco products using CRM Nº 60 and CRM Nº 61.

The overall level of variability for smokeless tobacco products seen in this study is similar to the

2007 RAC study for ground tobacco samples. The results for the current study showed similar

intra-laboratory and inter-laboratory variability for both tobacco and smokeless tobacco products

which also indicates that this method is appropriate for the analysis of both tobacco and smokeless

tobacco products. No evidence was found for any smokeless tobacco related method

interferences.

This report was discussed at the CORESTA STS meeting held in Quebec, Canada on October 11,

2014. The STS agreed that both CRM Nº 60 and CRM Nº 61 are fit for use for the determination

of PG and Gly in smokeless tobacco products and that the scopes of both CRMs should be

updated to include smokeless tobacco products.


APPENDIX A: Study Protocol

SMOKELESS TOBACCO SUB- GROUP

2012 Collaborative Study for the

Chemical Analysis of Humectants

Humectants Protocol

May, 2012

Author: Amy Walker

Confidentiality Notice: All data in this Collaborative Study should be handled in the strictest of confidence by all

participating laboratories.


1. Introduction

In November 2008, the CORESTA Scientific Commission recommended that a smokeless tobacco Sub-

Group be established. At their 2010 January meeting, an analyte survey was conducted and reviewed. The

reasons given for respondents’ chosen rankings were regulation, process control, research on potential

carcinogenic compounds and to improve current methodologies for consistency. The Sub-Group decided

that metals, B(a)P, humectants, ammonia, nitrates and moisture would be the analytes studied in the spring

of 2010.

Upon review of the data, the STS decided further studies were necessary to determine if homogeneity

testing would explain the large variations in various smokeless products.

2. Objective

The objectives of this small collaborative study are:

To perform homogeneity testing on all samples with goal being to improve “R” values.

(Performed by Eurofins, Sweden)

Evaluate the performance of CRM 60 (GC/FID) and CRM 61 (HPLC) to measure Propylene

Glycol and Glycerol in various smokeless products

Assess the suitability of CRM 60 and CRM 61 to measure Propylene Glycol and Glycerol in

smokeless tobacco products by evaluating the repeatability and reproducibility of both methods

If current CRMS are not acceptable, then altered versions or new base methods will be drafted. The study

will be performed by a number of laboratories (see participant list section 4).

3. Time schedule

NCST – North Carolina State University

AH – Alexander Hauleithner, JTI, Austria,

AW-Amy Walker, Global Laboratory Services

A ctio n Who A ustria

11-Oct 11-Nov 11-Dec 12-Jan 12-Feb 12-M ar 12-Apr 12-M ay 12-Jun 12-Jul 12-Aug 12-Sep

Present draft method and protocol ASW

Confirm participants ASW

Protocol comments ASW

Dispatch samples to homogeneity lab Suppliers

Homogeneity testing Eurofins

Homogeneity report Eurofins

Review any final comments Participants 9-M ay

Dispatch samples to participants TBD

Analysis Participants

Data sent Participants

Organize data and send to AH ASW

Prepare draft report AH

Circulate report to sub group JB

Present results and conclusions ASW


4. Participating Laboratories

The following laboratories have kindly agreed to take part in the study:

Table 1: List of Participants

Participants

Altria Client Services (GC)

Arista laboratories, Europe (GC)

Arista labratories, US (both methods)

British American Tobacco, Southampton (GC)

British American Tobacco, (GC)

Filtrona (GC)

ITC (both methods)

Imperial Tobacco Group, Reemtsma (both methods)

Imperial Tobacco Group, SEITA (both methods)

Japan Tobacco (GC)

JTI Trier (both methods)

JTI-Okolab (GC)

Labstat International (GC)

Landewyck Group sàrl. (both methods)

Microbac (GC)

Philip Morris International R&D (GC)

Poschl (GC)

R.J. Reynolds Tobacco Company (both methods)

Sampoerna (both methods)

Swedish Match Northern Europe (HPLC)

Swedish Match North America(both methods)

Swisher International (both methods)

5. Samples

5.1 Selection

To evaluate the proposed methods by analyzing the CRP products #1 (pouched snus) and #4 (chewing

tobacco), a loose snus sample, a loose leaf sample, and American moist snuff sample which are all products

that contain added Propylene Glycol and/or Glycerol.

5.2 Supply

The samples will be distributed to participating laboratories around first of June, 2012. The CRP products

will need to be ordered from North Carolina State University.


The contact information is listed below:

Karen Andres

NCSU Tobacco Analytical Services Lab

3709 Hillsborough Street Box 8604

Raleigh, NC 27607

919-513-1223 (Tel.)

515-7378 (Fax)

[email protected], www.tobacco.ncsu.edu/strp.html

The other remaining samples will be distributed by Swedish Match Northern Europe. Please send your

contact information to [email protected].

5.3 Receipt and Preparation

The samples shall be signed for immediately upon receipt and be stored in a freezer until the analyses are

performed. The preparation and sample-handling should be carried out according to Coresta Guide No 11

and the attached methods.

6. Analysis

6.1 Analytes

The following analyses should be performed by each participating laboratory.

Table 2: List of Analytes

Analyte Unit

1,2- Propylene Glycol (PG) mg/kg

Glycerol (Gly) mg/kg

6.2 Timing

The homogeneity analyses shall be performed between March 30th and April 30th.

The collaborative study is scheduled to begin the first of July 2012 and results reported two weeks

following.

6.3 Sampling

Each laboratory shall analyze two test portions and inject each extract twice for each sample. It is important

to start the analysis as soon as possible after the thawing, to avoid drying out the samples.

7. Data Reporting

All test results should be reported “as is” (with no correction for moisture content).

Each participating laboratory should perform each extraction in triplicate and inject each extract twice.

As well as the analytical results the following information shall be reported:

Comments from the operators on any deviation from the documented analytical procedure should

be reported in the comments column of the spreadsheet.

Information regarding any irregularities or disturbances during the measurement, reasons for any

missing results.

Results reported to three decimal places.

mailto:[email protected]

http://www.tobacco.ncsu.edu/strp.html


8. Statistical analysis of the data

Statistical analysis of data will be carried out by Alexander Hauleithner, JTI Austria, as per ISO 5725.

9. Presentation of the results from the Collaborative study The draft report of the Collaborative study will be circulated approximately in August, 2012.


APPENDIX B: Data – Descriptive Statistics

Sample A Sample B Sample C CRP 1 CRP 4

1

Method LAB Mean St Dev Mean St Dev Mean St Dev Mean St Dev Mean St Dev

1,2

- P

rop

yle

ne G

lyco

l [

%]

CRM 60 1GC 0.923 0.012 0.746 0.020 3.366 0.020 2.775 0.045 NA --

CRM 60 2GC 0.939 0.006 0.673 0.013 3.299 0.045 2.803 0.007 NA --

CRM 60 3GC 1.047 0.015 0.807 0.014 3.665 0.026 2.990 0.025 NA --

CRM 60 4GC 0.973 0.001 0.773 0.010 3.427 0.012 2.790 0.007 NA --

CRM 60 5GC 0.987 0.003 0.741 0.016 3.431 0.036 3.090 0.019 NA --

CRM 60 6GC 1.025 0.028 0.767 0.007 3.600 0.015 2.860 0.014 NA --

CRM 60 7GC 0.852 0.003 0.642 0.008 2.950 0.013 2.418 0.021 NA --

CRM 60 8GC 0.995 0.010 0.757 0.009 3.148 0.025 2.364 0.133 NA --

CRM 60 9GC 0.952 0.010 0.731 0.004 3.237 0.016 2.599 0.083 NA --

CRM 60 10GC 0.956 0.003 0.722 0.007 3.406 0.013 2.688 0.012 NA --

CRM 60 11GC 0.940 0.035 0.655 0.014 3.335 0.021 NA3

NA NA3 --

CRM 60 14GC 0.988 0.001 0.727 0.003 3.388 0.010 2.678 0.308 NA --

CRM 60 15GC 0.978 0.001 0.729 0.001 3.343 0.006 3.129 0.122 NA --

CRM 60 16GC 1.012 0.008 0.752 0.003 3.823 0.088 3.985 0.010 NA --

CRM 60 17GC 0.965 0.002 0.714 0.001 3.350 0.007 2.718 0.004 NA --

CRM 60 18GC 0.995 0.001 0.672 0.006 3.382 0.009 2.654 0.055 NA --

CRM 60 19GC 2 -- -- -- -- -- -- -- -- NA --

CRM 60 20GC 0.980 0.011 0.536 0.027 3.282 0.012 2.678 0.075 NA --

CRM 61 3LC 1.102 0.020 0.750 0.000 3.372 0.021 2.923 0.013 NA --

CRM 61 6LC 1.092 0.023 0.773 0.022 3.454 0.018 2.639 0.181 NA --

CRM 61 9LC 1.099 0.005 0.804 0.003 3.652 0.002 2.914 0.057 NA --

CRM 61 12LC 1.023 0.005 0.893 0.021 3.434 0.006 2.703 0.055 NA --

CRM 61 13LC 1.007 0.010 0.797 0.005 3.406 0.013 2.758 0.034 NA --

CRM 61 14LC 1.072 0.049 0.825 0.010 3.530 0.043 2.510 0.165 NA --

CRM 61 18LC 1.057 0.013 0.822 0.001 3.579 0.003 2.812 0.039 NA --

1. The sample did not contain PG.

2. The laboratory data were removed due to significant deviations from the CRM.

3. The laboratory did not report this sample.

4. The letters “GC” and “LC” indicate GC-FID (CRM Nº 60) and HPLC (CRM Nº 61), respectively.


Sample A

1 Sample B Sample C CRP 1

1 CRP 4

Method LAB Mean St Dev Mean St Dev Mean St Dev Mean St Dev Mean St Dev

Gly

cero

l [%

]

CRM 60 1GC NA -- 2.373 0.051 2.633 0.143 NA

-- 4.051 0.371

CRM 60 2GC NA -- 2.482 0.050 2.798 0.023 NA -- 3.580 0.246

CRM 60 3GC NA -- 2.398 0.085 2.940 0.065 NA -- 3.847 0.063

CRM 60 4GC NA -- 2.278 0.042 2.785 0.009 NA -- 3.793 0.109

CRM 60 5GC NA -- 2.339 0.043 2.831 0.034 NA -- 3.837 0.012

CRM 60 6GC NA -- 2.697 0.025 3.353 0.035 NA -- 4.224 0.010

CRM 60 7GC NA -- 2.053 0.016 2.417 0.025 NA -- 3.447 0.206

CRM 60 8GC NA -- 2.228 0.062 2.459 0.070 NA -- 3.296 0.158

CRM 60 9GC NA -- 2.337 0.025 2.715 0.019 NA -- 3.894 0.140

CRM 60 10GC NA -- 2.231 0.015 2.749 0.054 NA -- 3.654 0.030

CRM 60 11GC NA -- 2.415 0.064 2.800 0.297 NA3 -- NA

3 --

CRM 60 14GC NA -- 2.371 0.015 2.790 0.007 NA -- 3.724 0.101

CRM 60 15GC NA -- 2.266 0.019 2.739 0.030 NA -- 4.034 0.204

CRM 60 16GC NA -- 2.585 0.017 2.853 0.108 NA -- 4.055 0.046

CRM 60 17GC NA -- 2.279 0.026 2.744 0.023 NA -- 3.975 0.033

CRM 60 18GC NA -- 2.184 0.029 2.805 0.025 NA -- 3.672 0.036

CRM 60 19GC 2

NA -- -- -- -- -- NA -- -- --

CRM 60 20GC NA -- 2.359 0.086 2.757 0.008 NA -- 4.075 0.310

CRM 61 3LC NA -- 2.213 0.033 2.573 0.013 NA -- 3.553 0.003

CRM 61 6LC NA -- 2.505 0.010 2.828 0.012 NA -- 3.891 0.028

CRM 61 9LC NA -- 2.881 0.004 3.289 0.009 NA -- 4.585 0.159

CRM 61 12LC NA -- 2.445 0.028 2.794 0.007 NA -- 4.293 0.030

CRM 61 13LC NA -- 2.419 0.016 2.820 0.011 NA -- 3.994 0.019

CRM 61 14LC NA -- 2.565 0.009 2.888 0.038 NA -- 3.758 0.186

CRM 61 18LC NA -- 2.502 0.003 2.824 0.002 NA -- 3.957 0.022

1. NA: The sample did not contain glycerol.

2. The laboratory data were removed due to significant deviations from the CRM.

3. The laboratory did not report this sample.

4. The letters “GC” and “LC” indicate GC-FID (CRM Nº 60) and HPLC (CRM Nº 61), respectively.


APPENDIX C: Raw Data Plots

STS-CTR Humectants in Smokeless Tobacco Products – February 2015 19/39





APPENDIX D: Mandel’s h Plots

Note: GC and HPLC indicate the instrumental method used

Abbreviations: PG = 1,2-Propylene Glycol and Gly = Glycerol








APPENDIX E: Mandel’s k Plots

Note: GC and HPLC indicate the instrumental method used

Abbreviations: PG = 1,2-Propylene Glycol and Gly = Glycerol








APPENDIX F: Raw Data for Homogeneity Study1

Note: homogeneity testing was only conducted for the samples and analytes listed in the table

below.

Sample A Sample B Sample C Sample C

Replicate Propylene Glycol

(%) Glycerol

(%) Propylene Glycol

(%) Glycerol

(%)

1 1.063 2.473 3.224 2.696

2 1.051 2.059 3.306 2.803

3 1.065 2.241 3.567 3.019

4 1.030 2.259 3.535 2.982

5 0.993 2.605 3.477 2.930

6 1.099 2.276 3.271 2.742

7 1.080 2.368 3.492 2.919

8 1.108 2.194 3.598 2.983

9 1.104 2.177 3.583 2.945

10 1.088 2.348 3.300 2.734

11 1.066 2.310 3.281 2.731

12 1.086 2.432 3.363 2.829

13 1.044 2.449 3.740 3.107

14 1.056 2.248 3.667 3.040

15 1.020 2.443 3.712 3.085

16 1.096 2.269 3.232 2.727

17 1.036 2.468 3.506 2.934

18 1.067 2.478 3.511 2.980

19 1.089 2.363 3.616 2.997

20 1.076 2.561 3.215 2.741

Mean (%) 1.07 2.35 3.46 2.90

Standard Deviation

0.03 0.14 0.17 0.13

% RSD 2.84 5.88 4.97 4.60

1. 10 independent tobacco sample aliquots were removed for analysis. Each of these prepared samples was analyzed in duplicate


APPENDIX G: Homogeneity Testing and Study Data


Documents

Smokeless Tobacco Sub-Group - CORESTA · Tobacco Products by Gas Chromatography (2nd edition) • CRM Nº 61: Determination of 1,2-Propylene Glycol, Glycerol and Sorbitol in Tobacco