Tobacco and Tobacco Products Analytes Sub-Group · 2018-08-20 · • Lab 17: Used an oven temperature of 99 °C ± 0,5 °C. The study deviations for Karl Fischer analysis for Lab

Tobacco and Tobacco Products Analytes Sub-Group

Technical Report

2018 Moisture (OV), Water by Karl

Fischer and Gas Chromatography

Interlaboratory Study

August 2018

Author:

Anthony Brown, Altria Client Services LLC, U.S.A.

Co-Author and Statistical Analysis:

Michael Morton, Ph.D., Altria Client Services LLC, U.S.A.

Study Project Leaders:

Karl Wagner, Altria Client Services LLC, U.S.A.

Anthony Brown, Altria Client Services LLC, U.S.A.

Table of Contents

1. Summary ........................................................................................................................... 3

2. Introduction ....................................................................................................................... 3

2.1 Objective ........................................................................................................................... 3

3. Organization ...................................................................................................................... 4

3.1 Participants ........................................................................................................................ 4

3.2 Protocol ............................................................................................................................. 5

3.2.1 Sample Shipment .................................................................................................. 5

3.2.2 Within Laboratory Sample Preparation ................................................................ 5

3.2.3 Sample Analysis and Data Reporting ................................................................... 6

4. Data – Raw ........................................................................................................................ 7

5. Data – Statistical Analysis ................................................................................................ 7

5.1 Exclusion of Outliers ............................................................................................ 7

5.2 Calculation of Repeatability (r) and Reproducibility (R) ..................................... 8

5.3 Z-Scores .............................................................................................................. 11

5.4 Method Comparison ............................................................................................ 14

6. Data Interpretations ......................................................................................................... 16

7. Recommendations ........................................................................................................... 17

APPENDIX A: Full Data Set ................................................................................................... 18

APPENDIX B: Raw Data Plots ............................................................................................... 35

TTPA-171-1-CTR Water by KF and GC and OV Interlaboratory – August 2018 3/37

1. Summary

At the October 2017 CORESTA Tobacco and Tobacco Products Analytes Sub-Group (TTPA)

meeting held in Kitzbühel, Austria, the Sub-Group initiated an interlaboratory study for the

determination of water by Karl Fischer method (KF), water by gas chromatography (GC), and

moisture content (also referred to as oven volatiles or OV) in smokeless tobacco products,

cigarette filler, and cigar filler. The purpose of this study is to support laboratory

accreditations and to make a formal comparison of water and moisture results for a variety of

tobacco products.

2. Introduction

At the CORESTA Tobacco and Tobacco Products Analytes Sub-Group (TTPA) meeting held

in Kitzbühel, Austria (October 2017), it was decided to conduct an interlaboratory study for

the determination of water by Karl Fischer method, water by GC, and moisture content (also

referred to as oven volatiles or OV) by using the applicable CORESTA Recommended

Methods (CRMs) or ISO standards. The ISO standards were developed from the CRMs and

are essentially equivalent to the corresponding CRMs.

• Water by Karl Fischer:

o CRM N° 56, Determination of Water in Tobacco and Tobacco Products by Karl

Fischer Method, May 2011;

o ISO 6488:2004, Tobacco and tobacco products – Determination of water content

– Karl Fischer method

• Water by gas chromatography:

o CRM N° 57, Determination of Water in Tobacco and Tobacco Products by Gas

Chromatographic Analysis, December 2002;


– Gas-chromatographic method

• Moisture content (Oven Volatiles):

o CRM N° 76, Determination of Moisture Content (Oven Volatiles) of Tobacco and

Tobacco Products, July 2017

The collaborative study involved 19 laboratories and will support laboratory accreditations.

Tabulated data are presented along with repeatability (r) and reproducibility (R) values.

2.1 Objective

This study was conducted to support laboratory accreditation and to provide an assessment of

inter-laboratory variability for a variety of tobacco products for the determination of water by

Karl Fischer method, water by GC, and moisture content (oven volatiles). This study

included a variety of different types of smokeless tobacco products, cigarette filler, and

ground cigar filler. Data were collected from the participating laboratories and statistically

evaluated in basic conformance with the recommendations of ISO 5725-2:1994 and ISO/TR

22971:2005. For further reference and to provide additional support for laboratories’

accreditation, z-scores were also calculated in basic conformance with ISO 13528:2015.


3. Organization

3.1 Participants

A list of the participating laboratories is provided in Table 1. Not all laboratories provided

data for all analyses nor did all laboratories analyze all samples. The laboratories are listed in

alphabetical order. Laboratory codes were assigned to each laboratory and do not correspond

to the order shown in the table below.

Table 1: List of Participating Laboratories in the 2018 Interlaboratory Study

Participating Laboratories KF

CRM N° 56/ ISO6488

GC CRM N° 57/ ISO16632

OV CRM N° 76

Altria Client Services LLC, United States X X X

American Snuff Company, United States X

British American Tobacco (Bayreuth), Germany X X

China National Tobacco Quality Supervision and Test Center, China

X

CNTC Shanghai Tobacco Group Co., China X

Compañia Industrial de Tabacos Montepaz S.A., Uruguay

X X

Enthalpy Analytical, United States X X

Global Laboratory Services, United States X X X

ITG Brands, United States X

JTI Ökolab, Austria X

KT&G Research Institute, South Korea X X X

Labstat International ULC, Canada X X X

Liggett Group, LLC, United States X X

R.J. Reynolds Tobacco Company, United States X X X

Swedish Match, North America, United States X X

Swedish Match, Northern Europe, Sweden X X

Swisher International, United States X

University of Kentucky, United States X X

Zhengzhou Tobacco Research Institute of CNTC, China

X


3.2 Protocol

Specific details from the protocol are described below:

3.2.1 Sample Shipment

Laboratories were responsible for procuring 1R6F (RT1), RT6, Cigar Filler #1-11/17 and

Cigar Filler #2-11/171 from University of Kentucky, CRPs from the North Carolina State

University (NCSU) Tobacco Analytical Services Lab, mentholated cigarette from Altria

Client Services LLC, and two loose moist snuff samples from American Snuff Company.

Laboratories were requested to store the samples at approximately −20 °C after receipt.

Laboratories were requested to conduct the study in December through February and report

data by February 16, 2018. The samples are identified in Table 2.

Table 2: Sample Identification

Product Type

CRP1.1 - Swedish-style Snus

CRP2.1 - American-style loose moist snuff

CRP3.1 - American-style dry snuff powder

CRP4.1 - American-style chopped loose-leaf chewing tobacco

1R6F ground filler (Lot/Batch Number RT1) - Unflavoured American Blended cigarette filler, ground

Mentholated cigarette - Flavoured American blended cigarette

RT6 - Flavoured cigar Filler, ground

Cigar Filler #1-11/17 - Flavoured cigar filler, ground

Cigar Filler #2-11/17 - Unflavoured cigar filler, ground

MS-W - American-style loose moist snuff - wintergreen

MS-M - American-style loose moist snuff - mint

3.2.2 Within Laboratory Sample Preparation

The laboratories were directed to remove samples from the −20 °C freezer and place the

unopened samples in a refrigerator for a minimum of 24 hours to ensure water was fully

equilibrated. Samples were to be removed from the refrigerator for a minimum of 1 hour

prior to opening for analysis. Once samples were opened, they were to be stored in a tightly

sealed container at approximately 4 ºC for up to one week. Special handling requirements,

which differ from those specified in the CRMs and/or ISO standards, are described below:

• CRP1.1: The Snus pouches shall be cut into two halves and both the paper and

tobacco shall be added directly into the extraction vessel or OV container.

• Mentholated Cigarette: Immediately prior to analysis, open three packs of cigarettes,

remove the filler and place the filler in a tightly sealed bottle and mix well. This

process should be completed without delay to avoid volatile losses. This composite

sample should be discarded after one week.

• All other samples may be analyzed by removing aliquots directly from the container

after mixing with a spatula.


3.2.3 Sample Analysis and Data Reporting

The participating laboratories were instructed to conduct triplicate replicate analyses

(individual tobacco weighing) for the following:

• Water by Karl Fischer method:

o CRM N° 56, Determination of Water in Tobacco and Tobacco Products by Karl

Fischer Method, May 2011;


– Karl Fischer method

• Water by Gas chromatography:

o CRM N° 57, Determination of Water in Tobacco and Tobacco Products by Gas

Chromatographic Analysis, December 2002;


– Gas-chromatographic method

• Moisture content (Oven Volatiles):

o CRM N° 76, Determination of Moisture Content (Oven Volatiles) of Tobacco and

Tobacco Products, July 2017

Participating laboratories were requested to document any deviations from the protocol and

the CRMs and submit the deviations with their results. As stated in the protocol, data

submitted with significant deviations from the applicable CRM were excluded from the study.

Deviations reported by the laboratories are identified below and those considered significant

deviations are noted. To facilitate comparison among the three methods the results were all

expressed on a percent basis.

The following deviations for the determination of water by Karl Fischer method (CRM

N° 57/ISO 16632) were reported:

• Lab 9: Used a Karl Fischer apparatus using coulometric detection instead of

volumetric detection. This was considered a significant deviation; therefore, the

results were excluded from the r & R portion of the study.

• Lab 17: Extracted 0,6 g sample in 60 ml of dry methanol instead of the typical ratio of

5 g sample to 250 ml of dry methanol specified in the CRM.

• Lab 18: Used sample amounts of 0,1 g weighed in 20 ml vials. The vials were sealed

and heated to 175 °C and the water was purged into Karl Fischer reaction vessel for

analysis. This was considered a significant deviation; therefore, the results were

excluded from the r & R portion of the study.

The following deviations for the determination of water by gas chromatography (CRM

N° 56/ISO 6488) were reported:

• Lab 1: Extracted 1 g sample in 20 ml of dry methanol instead of the typical 5 g sample

to 100 ml of dry methanol.

• Lab 8: Used a higher concentration of internal standard (~6 ml/l) in dry methanol

instead of the recommended internal standard concentration of 2 ml/l.

• Lab 14: Used a higher concentration of internal standard (~20 ml/l) in dry methanol.


• Lab 2: Extracted 1 g sample in 25 ml of dry methanol and used a higher concentration

of internal standard (~5ml/l instead of 2 ml/l) in dry methanol.

• Lab 17: Extracted 0,6 g sample in 60 ml of dry methanol and extraction was

performed using a mechanical shaker for 30 minutes instead of the recommended

3 hours.

The following deviations for the determination of moisture content (OV) (CRM N° 76) were

reported:

• Lab 3: Used an oven temperature of 105 °C ± 5 °C instead of the specified

temperature of 100 °C ± 1 °C. This was considered a significant deviation; therefore,

the results were excluded from the r & R portion of the study.

• Lab 17: Used an oven temperature of 99 °C ± 0,5 °C.

The study deviations for Karl Fischer analysis for Lab 9 and Lab 18 were considered

significant (i.e. likely to impact testing results) and their water data by Karl Fischer method

were excluded from the r&R portion of the study. The study deviation for moisture content

(OV) determination of Lab 3 was considered significant and their OV data were excluded

from the r & R portion of the study. All other deviations were considered minor and

therefore, the data were included. All test results were to be reported to a minimum of two

decimal places. The study results and the comments were sent by e-mail to the study

coordinators.

4. Data – Raw

The full data set for the study is provided in Appendix A. Each analysis includes three

replicates. Not all laboratories provided data for all analyses or all samples. Data sets were

removed from the repeatability (r) and reproducibility (R) (r & R) portion of the study if the

data were identified as outlying data. Those data are included in Appendix A, but were

eliminated prior to the r & R analysis. Raw data plots that include all replicates, without

removal of outliers, are given in Appendix B. The data sets submitted with significant

deviations from the protocol, and removed from the r & R analysis, are included in

Appendices A and B.

5. Data – Statistical Analysis

The statistical analysis for the collaborative study was conducted in basic conformance with

ISO 5725-2:1994 and ISO/TR 22971:2005. The z-scores were calculated using Algorithm A

in basic conformance with ISO 13528:2015. A summary of the results from outlier detection

and the calculated results for repeatability (r) and reproducibility (R) are given below in

sections 5.1 and 5.2, respectively. Raw data plots that include all replicates, without removal

of outliers, are shown in Appendix B.

5.1 Exclusion of Outliers

Procedures outlined in ISO 5725-2:1994 and ISO/TR 22971:2005 were generally used for the

exclusion of outliers. An adaptation of Levene’s Test was used for eliminating laboratories


with overly large repeatability standard deviations1 and Grubbs’ Test was used to eliminate

laboratories with outlying mean values.

ISO 5725(2) also recommends the use of Mandel’s h and k plots. Mandel’s h statistic is the

same as the statistic used in Grubbs’ Test. Similarly Mandel’s k statistic, associated with

within lab standard deviation, is statistically equivalent to the c-value calculated in Cochran’s

Test ( cnk labs= ). However, the critical values associated with Mandel’s h and k statistics

do not make allowance for multiple testing and can therefore, give a false impression of

statistical significance. For those reasons, we did not include Mandel’s h and k plots.

Based on initial examination of graphs of the data and preliminary outlier identification, it

was noted that the Lab 1 GC water values tended to be lower than the other laboratories’

values and would frequently have been dropped by the Grubbs’ Test as outliers. Based on

those findings, the Lab 1 GC water values were dropped prior to the final outlier analysis. It

was also noted that Lab 3 had a single-point outlier for moisture for CRP1.1 which was

dropped prior to formal outlier evaluation. The remaining outliers are shown in Table 3.

Table 3: Outliers*

Product Analyte Levene’s Outlier Lab Grubbs’ Outlier Lab

CRP1.1 Moisture – 18



CRP3.1 GC Water – 18

Cigar Filler #2-11/17 Moisture 13 –

The (-) symbol indicates an outlier was not detected.

* As noted above, Lab 1 moisture results were also dropped and Lab 3’s replicate 3 for moisture was dropped as

a single point outlier for CRP1.1.

5.2 Calculation of Repeatability (r) and Reproducibility (R)

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

and Levene’s Test), the final repeatability and reproducibility (r & R) results were calculated.

The r & R results are shown in Table 4 – 6. The full description of the product types is

provided in Table 2. The r & R results reflect both laboratory variability and product

consistency. Note that the final number of labs that analyzed the samples by the Karl Fischer

method is relatively small and consequently the values (particularly for reproducibility)

should not be over-interpreted.

1 The approach is discussed in detail by Michael Morton in “Within-Laboratory Variance Outlier Detection: An

Alternative to Cochran’s Test” in Beitrage zur Tabakforschung International, Vol 27, No. 7, pp 135-144.


Table 4: Repeatability (r) and Reproducibility (R) Limits for Moisture (OV)

Product Type N° of

Labs*

Mean

(%)

Repeatability Reproducibility

r

r

R

R

(% of mean)

(% of mean)

1R6F ground filler (Lot/Batch Number RT1) - Unflavoured American Blended cigarette filler

16 11,92 0,43 3,58 0,61 5,13

CRP1.1 - Swedish-style Snus 15 54,02 1,55 2,88 2,37 4,39


14 51,44 0,52 1,01 0,84 1,63


15 7,91 0,19 2,39 1,26 15,9


16 24,58 0,75 3,05 2,73 11,1

Cigar Filler #1-11/17- Flavoured cigar filler, ground

15 12,97 0,30 2,31 0,53 4,05

Cigar Filler #2-11/17- Unflavoured cigar filler, ground

15 12,60 0,32 2,52 1,00 7,97

Mentholated Cigarette- Flavoured American blended cigarette

14 11,74 0,25 2,15 2,15 18,3

MS-M, American-style loose moist snuff - Mint

13 52,64 0,34 0,64 1,05 2,00

MS-W, American-style loose moist snuff - Wintergreen

14 52,43 0,79 1,51 1,46 2,79

RT6 - Flavoured Cigar Filler 16 12,54 0,24 1,94 0,82 6,51

*This is the number of laboratory data sets reported after removal of outliers.

Table 5: Repeatability (r) and Reproducibility (R) Limits for Water by Gas Chromatography

Product Type N° of

Labs*

Mean

(%)


r

r

R

R

(% of mean)

(% of mean)


10 10,56 1,09 10,3 2,67 25,3



10 48,01 2,50 5,22 5,59 11,6


9 6,29 0,56 8,83 1,28 20,4


10 21,05 1,67 7,94 4,87 23,1


Product Type N° of

Labs*

Mean

(%)


r

r

R

R

(% of mean)

(% of mean)


10 11,41 0,41 3,60 1,89 16,5


10 11,45 0,52 4,56 2,46 21,5


10 9,93 0,78 7,89 2,56 25,8


10 48,55 2,54 5,23 7,87 16,2


10 47,30 2,27 4,79 8,05 17,0



Table 6: Repeatability (r) and Reproducibility (R) Limits for Water by Karl Fischer Method

Product Type N° of

Labs*

Mean

(%)


R

r

R

R

(% of mean)

(% of mean)


6 10,04 1,17 11,7 2,33 23,2



6 51,25 0,97 1,9 7,69 15,0


6 6,21 0,45 7,3 2,30 37,0


6 21,10 0,92 4,3 1,41 6,7


6 11,66 0,19 1,6 0,94 8,1


6 11,90 0,46 3,8 1,06 8,9


6 9,90 0,42 4,2 2,75 27,8


5 50,66 1,54 3,0 5,98 11,8


5 48,90 0,66 1,4 4,29 8,8




5.3 Z-Scores

Graphs of the z-scores are given in Figures 1 – 3 and the corresponding z-statistics are given

in Tables 7 – 9. As noted above, for this analysis, the assigned mean and standard deviation

for each product were calculated using Algorithm A. As described in ISO 13528 and

ISO 5725-5, this is a robust estimation procedure that mitigates the influence of outliers rather

than explicitly eliminating them. As a general rule, the z-scores are regarded as reasonable if

-2 < z < 2 and generally thought to be unacceptable if |z|>3. Laboratories should consider |z|

between 2 and 3 as a warning signal. Laboratories should particularly be aware of the pattern

of their results. For example a single z-value signal should be looked into, but a pattern of

problematic z-scores in the same direction for the same analyte should be taken much more

seriously still.

Figure 1: Z-Scores for Moisture (Oven Volatiles)


Figure 2: Z-Scores for Water by Gas Chromatography

Figure 3: Z-Scores for Water by Karl Fischer Method


Table 7. Z-Scores for Moisture (Oven Volatiles)

Lab CRP1.1 CRP2.1 CRP3.1 CRP4.1 1R6F/RT1

CF#1

-11/17

CF#2

-11/17

Moist Snuff- Mint

Moist Snuff- WG

Men Cigt

RT6

1 -0,38 -0,37 -0,67 -0,12 1,51 -0,03 0,25 1,07 0,96 0,51 0,62

2 -0,42 -0,32 -0,72 -0,86 -1,42 -1,45 -0,99 0,62 0,31 -0,98 -1,05

3 0,78 1,23 0,34 0,64 1 1,86 0,07 0,32 0,06 1,45 -0,23

4 -1,32 -0,58 -1,11 -0,72 -1,92 -0,59 -1,04 -1,98 -1,16 0,44 -1,52

5 2,68 -0,49 -1,05 -1,14 -0,24 -1,21 -2,71 0 0,02 -0,13 -1,05

6 -0,19 0,29 1,73 0,75 0,88 -0,8 0,42 -0,58 -2,77 0,45 0,01

7 1,04 -2,57 2,12 0 0,85 1,18 0,95 − − -1,43 1,31

8 -0,89 -2,23 -0,12 -1,34 -0,53 0,48 0,88 1,03 1,48 -1,5 -0,12

10 -0,46 -0,37 0,66 0,95 -0,03 -0,1 -0,98 0,97 0,56 0,61 -1,52

11 0,17 -0,24 -0,98 2,02 -0,68 0,33 -0,23 -0,47 -0,65 0,5 -0,65

12 -0,07 0,05 0,35 -0,13 0,62 0,98 0,13 0,78 0,46 0,79 0,49

13 0,57 0,14 -0,08 0,7 -0,08 0,61 0,28 0,41 0,61 0,82 0,74

14 0,2 0,82 -0,18 0,53 -0,25 0,77 0,96 -0,23 0,42 0,51 0,5

15 -0,69 -0,32 -0,57 -0,31 -1,02 -0,76 0,33 -1,15 -1,82 -0,71 -0,02

17 2,96 1,81 -0,59 0,87 -0,46 -0,8 2,03 0,17 0,37 -0,86 0,49

18 -7,69 9,84 6,95 -2,43 0,64 -0,11 -1,03 -2,11 -0,44 -0,46 0,98

19 0,16 0,17 0,22 0,19 0,73 − − − − − 0,96

Mean* 53,88 51,47 7,94 24,64 11,94 12,99 12,61 52,66 52,47 11,87 12,54

StDev* 0,594 0,295 0,461 0,882 0,194 0,204 0,300 0,333 0,407 0,917 0,309

* Mean and standard deviation calculated from the lab means using Algorithm A, a robust estimation procedure. – Not Available

Table 8. Z-Scores for GC Water


CF#1

-11/17

CF#2

-11/17

Moist Snuff-Mint

Moist Snuff-

WG

Men Cigt

RT6

1 -6,05 -1,34 -3,86 -3,08 -2,96 -3,45 -3,21 -0,82 -1,01 -2,16 -4,21

2 0,31 0,26 0,18 0,29 0,10 -0,07 -0,21 0,56 0,46 -0,26 0,22

5 1,25 0,57 -0,40 -0,87 -1,50 -1,46 -2,00 0,38 0,77 -0,60 -1,39

6 -0,27 -0,48 -0,37 -0,64 -0,59 0,47 0,31 0,40 0,70 -0,46 0,51

8 -0,05 -0,32 0,65 -0,32 0,56 -0,03 -0,28 0,72 0,00 -0,25 0,08

11 -0,60 -0,92 0,60 1,13 0,62 0,74 0,80 -1,30 -0,77 1,45 0,71

13 0,23 -0,55 -0,54 -0,33 -0,15 -0,50 -0,43 0,05 -0,85 0,81 -0,53

14 0,27 0,66 -0,13 1,65 0,68 0,65 0,85 0,27 0,75 1,02 0,43

15 1,43 1,16 0,96 0,56 1,22 0,88 1,02 1,42 1,63 0,46 1,51

17 0,06 -0,48 -0,96 0,42 0,07 -0,24 0,62 -0,22 -0,49 -0,89 -0,21

18 -1,13 1,45 5,91 -0,24 0,51 1,05 0,33 -1,47 -1,06 0,22 0,18

Mean* 48,20 47,70 6,29 20,70 10,39 11,28 11,36 48,30 46,94 9,76 11,38

StDev* 2,83 2,31 0,67 2,09 1,17 0,86 0,97 3,07 3,16 1,15 0,88

* Mean and standard deviation calculated from the lab means using Algorithm A, a robust estimation procedure.


Table 9. Z-Scores for Karl Fischer Water


CF#1

-11/17

CF#2

-11/17

Moist Snuff-Mint

Moist Snuff-

WG

Men Cigt

RT6

2 1,27 1,84 0,86 -0,27 0,60 0,42 -0,28 1,95 0,74 -0,35 0,90

4 0,95 0,44 -0,42 -0,83 -2,49 -0,04 -0,60 0,72 1,39 -0,24 -1,39

5 0,44 -0,76 -0,64 -1,53 -0,11 -0,61 -1,44 -0,46 -0,63 0,03 -0,36

9 0,13 -0,25 0,13 1,40 -0,14 0,40 0,42 -0,37 -0,31 0,32 0,14

15 -0,21 -0,25 0,41 0,02 0,17 0,24 0,63 -0,34 -0,19 -0,77 -0,19

16 -1,28 -0,43 -0,97 0,38 0,14 -1,64 -0,33 – – 0,87 -0,08

17 -0,36 -1,14 -0,87 0,28 -0,65 -0,42 0,10 -1,19 -1,27 -1,31 -0,51

18 -0,95 0,90 5,25 0,51 2,55 2,09 1,69 0,15 0,27 1,45 5,29

Mean* 49,53 51,38 6,51 21,28 10,30 11,82 12,07 50,43 48,89 10,29 11,85

StDev* 3,71 2,56 1,09 0,58 0,68 0,45 0,51 1,71 1,43 1,32 0,29

* Mean and standard deviation calculated from the lab means using Algorithm A, a robust estimation procedure. – Not Available

5.4 Method Comparison

The data from both the reproducibility calculations and from the standard deviations

calculated during the generation of the z-scores indicate that the moisture results show less

lab-to-lab variation than either the GC water results or the Karl Fischer water results,

particularly at higher moisture/water levels. The variability differences are illustrated with the

R-limits of the methods graphed in Figure 4. Generally speaking the R-limits were much

larger for GC water and Karl Fischer water than for moisture.


Figure 4. Comparison of R limits for Moisture (OV), Water by GC and by Karl Fischer Method

The moisture (OV) method tended to result in higher values that either of the water-specific

methods. These results are explained by the fact that the measurement of moisture (OV)

includes volatile components (e.g. volatile flavors, humectants, etc.) other than just water.

The Karl Fischer water results were on average higher than the GC water results and the

difference was statistically significant2. The difference was more evident at higher water

levels. A 2009 collaborative study conducted by the Smokeless Tobacco Sub-Group also

found the Karl Fischer method to result in higher values than the GC method for samples with

higher water levels. However, the 2002 collaborative study on which the two CRMs are

based found both methods to give comparable results, even at higher water levels. As

mentioned earlier, the final number of labs that analyzed the samples by the Karl Fischer

method is relatively small and consequently the values (particularly for reproducibility)

should not be over-interpreted.

A graph comparing the results is given in Figure 5. In the graph, the water results are plotted

on the horizontal axis as the average the Karl Fischer and GC water results and the difference

between the two is plotted on the vertical axis. Looking at the results for the 2009 and current

studies, it can be seen that the results are predominantly above zero, particularly at higher

water levels, indicating that the Karl Fischer method is giving higher results. By contrast the

results for the 2002 study are close to the zero line and roughly balanced above and below the

zero line, indicating that the two methods were (on average) giving similar results.

2 The statistical comparison was carried out multiple ways and, though the p-values varied quite a lot, the

approaches all showed that the difference was statistically significant. The simplest approach is to simply

conduct paired t-tests with the averages either in the r&R tables or the z-score tables. A slightly more

complicated method is to carry out a two-way analysis of variance with the individual lab averages after outlier

removal – this shows that the amount of the difference varies from product to product, but did show an overall

statistically significant difference.


Figure 5. Comparison of Karl Fischer and GC Water Results

The variability seen for water by the GC method and water by the Karl Fischer method in this

study was greater than reported in the CRMs based on a collaborative study conducted in

2002. R values calculated from the results shown in the CRM average 9,4 % (of their

respective mean values) for Karl Fischer water and 12,7 % for GC water. From this study the

corresponding R-values expressed as a percent of their respective mean values averaged

15.7 % for the Karl Fischer method and 18,9 % for the GC method. In the 2002 study there

were 17 participating laboratories carrying out GC water and 11 laboratories carried out the

Karl Fischer method. In the 2009 study, eleven laboratories provided results for water using

the Karl Fischer method and seven labs provided results for water using Gas

Chromatography.

6. Data Interpretations

As noted above, the lab-to-lab variation was much less for moisture (oven volatiles) than for

either of the water-specific methods. The variability seen for water in this study was

somewhat greater than reported in the CRMs of the respective methods. As expected, the

values obtained for moisture tended to be larger than the measure of water. Since moisture

would be sensitive to water plus any other volatiles, that difference was anticipated. The Karl

Fischer water method tended overall to give values that were a little larger than the GC water

method, particularly at higher water levels. This finding is consistent with a 2009 study

carried out by the STS Sub-Group, but not consistent with the findings of the 2002 study on

which the two CRMs are based which found the Karl Fischer and GC methods to give (on

average) equivalent results.


7. Recommendations

The CRMs and ISO standards listed below were originally developed for the analysis of

smokeless tobacco, raw tobacco, pipe tobacco, and cigarette products:

• Water: CRM N° 56, Determination of Water in Tobacco and Tobacco Products by

Karl Fischer Method, May 2011; ISO 6488:2004, Tobacco and tobacco products –

Determination of water content – Karl Fischer method

• Water: CRM N° 57, Determination of Water in Tobacco and Tobacco Products by Gas

Chromatographic Analysis, December 2002; ISO 16632:2003, Tobacco and tobacco

products – Determination of water content – Gas-chromatographic method

The results of this study demonstrate that the CRMs are also fit for the analysis of cigar filler.

The TTPA recommends the scope of the CRMs be updated to include the r & R data from this

study. The TTPA also recommends to propose to ISO/TC 126 that the ISO standards be

updated during the next systematic review cycle.


APPENDIX A: Full Data Set

Full Data Set (results are presented on an as-is basis)

Lab Code Product Moisture GC Water Karl Fischer Water

% mg/g mg/g

1 1R6F ground filler/RT1 12,13 69,4 –



2 1R6F ground filler/RT1 11,64 104,0 106,3



3 1R6F ground filler/RT1 12,30 – –



4 1R6F ground filler/RT1 11,50 – 95,3















9 1R6F ground filler/RT1 – – 102,0












% mg/g mg/g
























1 CRP1.1 53,89 300,6 –

1 CRP1.1 53,38 310,0 –

1 CRP1.1 53,71 321,4 –

2 CRP1.1 52,93 491,7 541,4

2 CRP1.1 54,38 491,3 542,1

2 CRP1.1 53,59 489,4 544,3

3 CRP1.1 54,30 – –

3 CRP1.1 54,40 – –

3 CRP1.1 45,60 – –

4 CRP1.1 53,00 – 538,2

4 CRP1.1 53,50 – 527,2

4 CRP1.1 52,80 – 526,7

5 CRP1.1 55,75 518,6 514,8



% mg/g mg/g

5 CRP1.1 55,19 529,5 512,7

5 CRP1.1 55,49 503,9 506,9

6 CRP1.1 53,62 470,7 –

6 CRP1.1 53,78 482,9 –

6 CRP1.1 53,91 469,3 –

7 CRP1.1 54,94 – –

7 CRP1.1 53,15 – –

7 CRP1.1 55,42 – –

8 CRP1.1 53,31 481,1 –

8 CRP1.1 53,65 481,3 –

8 CRP1.1 53,11 478,8 –

9 CRP1.1 – – 499,7

9 CRP1.1 – – 500,6

9 CRP1.1 – – 500,2

10 CRP1.1 53,49 – –

10 CRP1.1 53,79 – –

10 CRP1.1 53,55 – –

11 CRP1.1 53,59 468,5 –

11 CRP1.1 53,98 466,8 –

11 CRP1.1 54,38 460,0 –

12 CRP1.1 54,02 – –

12 CRP1.1 53,76 – –

12 CRP1.1 53,74 – –

13 CRP1.1 54,23 495,8 –

13 CRP1.1 54,21 494,6 –

13 CRP1.1 54,23 475,3 –

14 CRP1.1 54,32 492,8 –

14 CRP1.1 53,90 484,0 –

14 CRP1.1 53,79 492,1 –

15 CRP1.1 53,24 516,5 493,4

15 CRP1.1 53,39 516,8 482,5

15 CRP1.1 53,80 534,0 486,6

16 CRP1.1 – – 448,9

16 CRP1.1 – – 440,9

16 CRP1.1 – – 453,6

17 CRP1.1 57,23 472,2 487,5



% mg/g mg/g

17 CRP1.1 54,92 472,7 477,9

17 CRP1.1 54,78 505,7 480,4

18 CRP1.1 48,89 467,6 473,2

18 CRP1.1 49,45 447,9 455,9

18 CRP1.1 49,61 434,5 451,5

19 CRP1.1 53,96 – –

19 CRP1.1 53,83 – –

19 CRP1.1 54,14 – –

1 CRP2.1 51,44 452,0 –

1 CRP2.1 51,45 438,3 –

1 CRP2.1 51,20 448,1 –

2 CRP2.1 51,80 484,6 560,5

2 CRP2.1 51,31 484,4 560,7

2 CRP2.1 51,02 480,1 561,4

3 CRP2.1 51,40 – –

3 CRP2.1 51,60 – –

3 CRP2.1 52,50 – –

4 CRP2.1 51,30 – 524,8

4 CRP2.1 51,30 – 526,6

4 CRP2.1 51,30 – 523,6

5 CRP2.1 51,23 494,4 494,9

5 CRP2.1 51,38 480,0 499,2

5 CRP2.1 51,37 496,5 488,6

6 CRP2.1 51,47 468,4 –

6 CRP2.1 51,64 478,3 –

6 CRP2.1 51,56 450,9 –

7 CRP2.1 50,64 – –

7 CRP2.1 50,04 – –

7 CRP2.1 51,46 – –

8 CRP2.1 50,56 469,3 –

8 CRP2.1 50,76 468,7 –

8 CRP2.1 51,12 470,8 –

9 CRP2.1 – – 510,3

9 CRP2.1 – – 508,1

9 CRP2.1 – – 503,7

10 CRP2.1 51,33 – –



% mg/g mg/g

10 CRP2.1 51,50 – –

10 CRP2.1 51,26 – –

11 CRP2.1 51,53 455,2 –

11 CRP2.1 51,41 454,9 –

11 CRP2.1 51,26 457,2 –

12 CRP2.1 51,61 – –

12 CRP2.1 51,57 – –

12 CRP2.1 51,28 – –

13 CRP2.1 51,33 468,2 –

13 CRP2.1 51,87 469,0 –

13 CRP2.1 51,34 456,2 –

14 CRP2.1 51,82 490,1 –

14 CRP2.1 51,61 494,2 –

14 CRP2.1 51,71 492,3 –

15 CRP2.1 51,43 510,3 508,4

15 CRP2.1 51,41 502,9 506,5

15 CRP2.1 51,29 498,7 507,6

16 CRP2.1 – – 504,6

16 CRP2.1 – – 496,8

16 CRP2.1 – – 506,7

17 CRP2.1 52,07 468,7 487,9

17 CRP2.1 52,01 456,8 485,2

17 CRP2.1 51,94 472,0 480,7

18 CRP2.1 54,32 532,5 535,9

18 CRP2.1 54,34 498,7 539,5

18 CRP2.1 54,46 500,2 535,0

19 CRP2.1 51,43 – –

19 CRP2.1 51,72 – –

19 CRP2.1 51,42 – –

1 CRP3.1 7,76 37,1 –

1 CRP3.1 7,45 37,7 –

1 CRP3.1 7,70 36,1 –

2 CRP3.1 7,64 63,9 75,9

2 CRP3.1 7,59 63,3 73,7

2 CRP3.1 7,60 65,1 73,7

3 CRP3.1 8,10 – –



% mg/g mg/g

3 CRP3.1 8,20 – –

3 CRP3.1 8,00 – –

4 CRP3.1 7,50 – 59,1

4 CRP3.1 7,30 – 61,1

4 CRP3.1 7,50 – 61,3

5 CRP3.1 7,46 60,6 56,8

5 CRP3.1 7,44 60,9 58,2

5 CRP3.1 7,48 59,1 59,3

6 CRP3.1 8,73 55,5 –

6 CRP3.1 8,72 62,0 –

6 CRP3.1 8,78 63,7 –

7 CRP3.1 8,95 – –

7 CRP3.1 8,88 – –

7 CRP3.1 8,94 – –

8 CRP3.1 7,88 69,5 –

8 CRP3.1 7,81 66,9 –

8 CRP3.1 7,97 65,3 –

9 CRP3.1 – – 66,0

9 CRP3.1 – – 68,6

9 CRP3.1 – – 65,0

10 CRP3.1 8,30 – –

10 CRP3.1 8,23 – –

10 CRP3.1 8,22 – –

11 CRP3.1 7,53 66,0 –

11 CRP3.1 7,54 67,9 –

11 CRP3.1 7,40 66,8 –

12 CRP3.1 8,06 – –

12 CRP3.1 8,15 – –

12 CRP3.1 8,10 – –

13 CRP3.1 7,98 61,0 –

13 CRP3.1 7,87 58,5 –

13 CRP3.1 7,87 58,2 –

14 CRP3.1 7,84 61,9 –

14 CRP3.1 7,88 63,0 –

14 CRP3.1 7,86 61,2 –

15 CRP3.1 7,71 69,1 72,4



% mg/g mg/g

15 CRP3.1 7,71 70,3 68,7

15 CRP3.1 7,63 68,4 67,6

16 CRP3.1 – – 54,8

16 CRP3.1 – – 54,1

16 CRP3.1 – – 54,5

17 CRP3.1 7,71 57,7 56,9

17 CRP3.1 7,63 57,9 56,6

17 CRP3.1 7,68 53,7 53,2

18 CRP3.1 11,14 102,3 127,4

18 CRP3.1 11,23 103,0 120,4

18 CRP3.1 11,08 102,3 119,5

19 CRP3.1 8,04 – –

19 CRP3.1 8,04 – –

19 CRP3.1 8,06 – –

1 CRP4.1 24,33 144,4 –

1 CRP4.1 24,70 139,0 –

1 CRP4.1 24,55 144,5 –

2 CRP4.1 24,05 215,2 210,6

2 CRP4.1 23,76 222,3 212,4

2 CRP4.1 23,81 202,1 210,9

3 CRP4.1 24,20 – –

3 CRP4.1 26,00 – –

3 CRP4.1 25,40 – –

4 CRP4.1 23,90 – 204,6

4 CRP4.1 24,30 – 215,3

4 CRP4.1 23,80 – 204,2

5 CRP4.1 23,34 190,3 205,7

5 CRP4.1 23,66 185,3 202,7

5 CRP4.1 23,88 191,1 203,5

6 CRP4.1 24,95 197,4 –

6 CRP4.1 25,27 188,8 –

6 CRP4.1 25,67 194,9 –

7 CRP4.1 24,33 – –

7 CRP4.1 24,96 – –

7 CRP4.1 24,62 – –

8 CRP4.1 23,79 196,3 –



% mg/g mg/g

8 CRP4.1 23,45 201,8 –

8 CRP4.1 23,11 202,9 –

9 CRP4.1 – – 225,0

9 CRP4.1 – – 216,2

9 CRP4.1 – – 221,8

10 CRP4.1 25,23 – –

10 CRP4.1 25,51 – –

10 CRP4.1 25,68 – –

11 CRP4.1 26,52 235,1 –

11 CRP4.1 26,43 225,4 –

11 CRP4.1 26,30 231,5 –

12 CRP4.1 24,54 – –

12 CRP4.1 24,45 – –

12 CRP4.1 24,56 – –

13 CRP4.1 24,77 202,3 –

13 CRP4.1 25,57 199,3 –

13 CRP4.1 25,42 198,7 –

14 CRP4.1 25,01 241,7 –

14 CRP4.1 25,03 242,7 –

14 CRP4.1 25,28 240,4 –

15 CRP4.1 25,00 208,1 213,8

15 CRP4.1 24,14 231,8 213,5

15 CRP4.1 23,94 216,1 211,6

16 CRP4.1 – – 213,6

16 CRP4.1 – – 214,6

16 CRP4.1 – – 217,0

17 CRP4.1 25,48 215,4 218,8

17 CRP4.1 25,32 216,5 214,2

17 CRP4.1 25,40 215,4 210,5

18 CRP4.1 22,40 197,8 224,6

18 CRP4.1 22,56 208,7 218,2

18 CRP4.1 22,51 199,6 204,7

19 CRP4.1 24,79 – –

19 CRP4.1 24,82 – –

19 CRP4.1 24,81 – –

1 Cigar Filler #1-11/17 12,96 82,4 –



% mg/g mg/g

1 Cigar Filler #1-11/17 13,09 82,4 –

1 Cigar Filler #1-11/17 12,89 84,1 –

2 Cigar Filler #1-11/17 12,66 114,3 119,1

2 Cigar Filler #1-11/17 12,71 111,3 120,4

2 Cigar Filler #1-11/17 12,70 110,9 120,7

3 Cigar Filler #1-11/17 13,50 – –

3 Cigar Filler #1-11/17 13,30 – –

3 Cigar Filler #1-11/17 13,30 – –

4 Cigar Filler #1-11/17 12,80 – 118,3

4 Cigar Filler #1-11/17 12,80 – 117,7

4 Cigar Filler #1-11/17 13,00 – 118,0

5 Cigar Filler #1-11/17 12,84 101,2 115,7

5 Cigar Filler #1-11/17 12,72 98,6 115,6

5 Cigar Filler #1-11/17 12,66 100,7 115,1

6 Cigar Filler #1-11/17 12,95 118,6 –

6 Cigar Filler #1-11/17 12,66 116,6 –

6 Cigar Filler #1-11/17 12,86 115,4 –

7 Cigar Filler #1-11/17 13,29 – –

7 Cigar Filler #1-11/17 13,21 – –

7 Cigar Filler #1-11/17 13,18 – –

8 Cigar Filler #1-11/17 13,12 112,8 –

8 Cigar Filler #1-11/17 12,90 112,7 –

8 Cigar Filler #1-11/17 13,23 112,2 –

9 Cigar Filler #1-11/17 – – 122,1

9 Cigar Filler #1-11/17 – – 119,7

9 Cigar Filler #1-11/17 – – 118,1

10 Cigar Filler #1-11/17 13,03 – –

10 Cigar Filler #1-11/17 13,00 – –

10 Cigar Filler #1-11/17 12,87 – –

11 Cigar Filler #1-11/17 13,04 119,7 –

11 Cigar Filler #1-11/17 13,08 117,3 –

11 Cigar Filler #1-11/17 13,04 120,7 –

12 Cigar Filler #1-11/17 13,03 – –

12 Cigar Filler #1-11/17 13,12 – –

12 Cigar Filler #1-11/17 13,41 – –

13 Cigar Filler #1-11/17 13,27 108,2 –



% mg/g mg/g

13 Cigar Filler #1-11/17 12,95 109,0 –

13 Cigar Filler #1-11/17 13,11 108,2 –

14 Cigar Filler #1-11/17 13,16 119,2 –

14 Cigar Filler #1-11/17 13,15 117,4 –

14 Cigar Filler #1-11/17 13,12 118,6 –

15 Cigar Filler #1-11/17 12,76 118,0 118,9

15 Cigar Filler #1-11/17 12,91 122,7 120,2

15 Cigar Filler #1-11/17 12,82 120,6 118,7

16 Cigar Filler #1-11/17 – – 110,6

16 Cigar Filler #1-11/17 – – 110,1

16 Cigar Filler #1-11/17 – – 111,9

17 Cigar Filler #1-11/17 12,74 111,1 115,7

17 Cigar Filler #1-11/17 12,87 109,9 116,5

17 Cigar Filler #1-11/17 12,86 111,3 116,8

18 Cigar Filler #1-11/17 12,95 123,5 125,3

18 Cigar Filler #1-11/17 12,94 122,2 125,6

18 Cigar Filler #1-11/17 13,00 120,0 131,6

1 Cigar Filler #2-11/17 12,92 80,5 –

1 Cigar Filler #2-11/17 12,52 83,9 –

1 Cigar Filler #2-11/17 12,62 83,0 –

2 Cigar Filler #2-11/17 12,34 111,1 118,0

2 Cigar Filler #2-11/17 12,27 111,9 120,3

2 Cigar Filler #2-11/17 12,33 111,6 119,5

3 Cigar Filler #2-11/17 12,70 – –

3 Cigar Filler #2-11/17 12,60 – –

3 Cigar Filler #2-11/17 12,60 – –

4 Cigar Filler #2-11/17 12,30 – 113,7

4 Cigar Filler #2-11/17 12,30 – 119,8

4 Cigar Filler #2-11/17 12,30 – 119,3

5 Cigar Filler #2-11/17 11,77 96,4 112,1

5 Cigar Filler #2-11/17 11,78 93,0 113,5

5 Cigar Filler #2-11/17 11,84 93,2 114,4

6 Cigar Filler #2-11/17 12,73 118,2 –

6 Cigar Filler #2-11/17 12,79 118,1 –

6 Cigar Filler #2-11/17 12,69 113,3 –

7 Cigar Filler #2-11/17 13,03 – –



% mg/g mg/g

7 Cigar Filler #2-11/17 12,88 – –

7 Cigar Filler #2-11/17 12,78 – –

8 Cigar Filler #2-11/17 13,06 110,8 –

8 Cigar Filler #2-11/17 12,88 111,0 –

8 Cigar Filler #2-11/17 12,69 110,9 –

9 Cigar Filler #2-11/17 – – 121,1

9 Cigar Filler #2-11/17 – – 123,7

9 Cigar Filler #2-11/17 – – 123,7

10 Cigar Filler #2-11/17 12,39 – –

10 Cigar Filler #2-11/17 12,42 – –

10 Cigar Filler #2-11/17 12,14 – –

11 Cigar Filler #2-11/17 12,56 122,6 –

11 Cigar Filler #2-11/17 12,54 119,7 –

11 Cigar Filler #2-11/17 12,53 121,5 –

12 Cigar Filler #2-11/17 12,61 – –

12 Cigar Filler #2-11/17 12,66 – –

12 Cigar Filler #2-11/17 12,68 – –

13 Cigar Filler #2-11/17 12,45 109,2 –

13 Cigar Filler #2-11/17 12,93 108,7 –

13 Cigar Filler #2-11/17 12,71 110,3 –

14 Cigar Filler #2-11/17 12,91 121,8 –

14 Cigar Filler #2-11/17 12,89 121,8 –

14 Cigar Filler #2-11/17 12,90 121,7 –

15 Cigar Filler #2-11/17 12,67 124,8 123,5

15 Cigar Filler #2-11/17 12,76 121,6 123,2

15 Cigar Filler #2-11/17 12,70 124,0 124,9

16 Cigar Filler #2-11/17 – – 120,0

16 Cigar Filler #2-11/17 – – 118,4

16 Cigar Filler #2-11/17 – – 118,7

17 Cigar Filler #2-11/17 13,30 117,9 120,8

17 Cigar Filler #2-11/17 13,24 120,2 121,6

17 Cigar Filler #2-11/17 13,12 120,4 121,2

18 Cigar Filler #2-11/17 12,33 121,0 129,6

18 Cigar Filler #2-11/17 12,31 116,1 129,5

18 Cigar Filler #2-11/17 12,27 113,1 128,8

1 Mentholated Cigarette 12,57 69,2 –



% mg/g mg/g



2 Mentholated Cigarette 10,95 95,8 98,6



3 Mentholated Cigarette 13,30 – –



4 Mentholated Cigarette 12,30 – 99,3















9 Mentholated Cigarette – – 106,9















% mg/g mg/g


















1 Moist Snuff Mint 53,08 458,5 –



2 Moist Snuff Mint 52,75 501,8 528,2

2 Moist Snuff Mint 52,92 497,1 542,7

2 Moist Snuff Mint 52,93 501,4 541,9

3 Moist Snuff Mint 52,60 – –



4 Moist Snuff Mint 51,90 – 514,2



5 Moist Snuff Mint 52,73 485,1 496,7

5 Moist Snuff Mint 52,60 491,2 495,2

5 Moist Snuff Mint 52,65 507,9 497,5







% mg/g mg/g



9 Moist Snuff Mint – – 503,4


















15 Moist Snuff Mint 52,38 528,4 498,3

15 Moist Snuff Mint 52,28 525,9 489,9

15 Moist Snuff Mint 52,17 525,2 507,0

17 Moist Snuff Mint 52,63 496,2 481,1

17 Moist Snuff Mint 52,78 463,6 485,3

17 Moist Snuff Mint 52,74 468,9 485,3

18 Moist Snuff Mint 51,90 453,5 502,9

18 Moist Snuff Mint 52,02 420,3 498,9

18 Moist Snuff Mint 51,95 440,4 518,5

1 Moist Snuff Wintergreen 52,81 433,5 –



2 Moist Snuff Wintergreen 52,58 485,4 502,1



3 Moist Snuff Wintergreen 52,40 – –



% mg/g mg/g



4 Moist Snuff Wintergreen 52,00 – 510,5












9 Moist Snuff Wintergreen – – 497,4
























% mg/g mg/g






1 RT6 - Flavoured Cigar Filler 12,60 77,0 –



2 RT6 - Flavoured Cigar Filler 12,25 115,9 121,1



3 RT6 - Flavoured Cigar Filler 12,60 – –



4 RT6 - Flavoured Cigar Filler 12,10 – 115,9















9 RT6 - Flavoured Cigar Filler – – 120,1









% mg/g mg/g



























The (–) symbol indicates the laboratory did not submit a value for that sample analysis,


APPENDIX B: Raw Data Plots



Documents

Tobacco and Tobacco Products Analytes Sub-Group · 2018-08-20 · • Lab 17: Used an oven temperature of 99 °C ± 0,5 °C. The study deviations for Karl Fischer analysis for Lab