routine analytical chemistry sub-group - coresta · routine analytical chemistry sub-group ......

Routine Analytical Chemistry Sub-Group

Technical Report

2014 Collaborative Study

Comparing CRM35 for the

Determination of Total Alkaloids

(as Nicotine) in Tobacco by Continuous Flow Analysis to a

New Method with Safer Chemistry

March 2017

Sub-Group Co-ordinator:

Linda A. Crumpler, Cerulean, USA

Study Co-ordinator:

Wei Zhang, China National Tobacco Quality Supervision and Test Center, China

Linda A. Crumpler, Cerulean, USA

Authors:

Wei Zhang, China National Tobacco Quality Supervision and Test Center, China

Yanjun Ma, Guorong Du, R&D, Beijing Cigarette Factory, China

Linda. A. Crumpler, Cerulean, USA

Table of Contents

1. Introduction ....................................................................................................................... 3

2. Organisation ...................................................................................................................... 3

2.1 Participants .............................................................................................................. 3

2.2 Samples ................................................................................................................... 4

2.3 Data Sets .................................................................................................................. 4

3. Raw data ........................................................................................................................... 5

4. Statistical Analysis ............................................................................................................ 5

4.1 Numerical outlier technique: Cochran & Grubbs test ............................................. 5

4.1.1 Outlier results ................................................................................................. 6

4.2 Repeatability and reproducibility estimation .......................................................... 7

4.3 Comparison of 2014 Study to Previous Studies ...................................................... 8

5. Method Validation .......................................................................................................... 11

6. Conclusions and Recommendations ............................................................................... 17

6.1 Conclusions ........................................................................................................... 17

6.2 Recommendations ................................................................................................. 17

Appendix A – Experimental Protocol ...................................................................................... 18

Appendix B – Chinese KSCN/DCIC Method of Collaborative Test for Total Alkaloids in

Tobacco ............................................................................................................ 20

Appendix C – Raw Data by Laboratory ................................................................................... 23

Appendix D – Raw Mean and Standard Deviation Value ....................................................... 30

Appendix E – Data Representation .......................................................................................... 38

Appendix F – IUPAC 1994 Harmonized Statistical Procedure ............................................... 45

.

RAC-052-1-CTR 2014 Safer Chemistry Collaborative Study – March 2017 3/45

1. Introduction

To produce a safer method to test total alkaloids in tobacco (as nicotine) the CORESTA

Routine Analytical Chemistry Sub-Group (RAC) undertook a collaborative study in 2014 of

two methods for the determination of total alkaloids in tobacco by segmented continuous-flow

analysis. The two methods are CORESTA Recommended Method N° 35 (CRM35) and a new

method (NCM) proposed by China National Tobacco Quality Supervision and Test Center

(CTQTC). In CRM35 cyanogen chloride is generated in situ by the reaction of potassium

cyanide (KCN), a highly toxic compound and chloramine T. NCM proposed by CTQTC

eliminates the use of the potassium cyanide by employing potassium thiocyanate (KSCN)

with sodium dichloroisocyanurate dihydrate (DCIC). Each method was tested using water

extracted tobacco and 5 % acetic acid (HAC) extracted tobacco. As documented in CRM35 it

is recommended that 5 % HAC extracts should be used if total alkaloids (as nicotine) and

reducing substances or reducing carbohydrate analyses are to be carried out simultaneously.

Calibration standards were prepared with the same extraction solutions.

Further objectives of this work are:

to provide a new CRM reflecting the safer chemistry with flow diagrams for both

macro and micro flow instruments

to provide a basis for work proposed within ISO TC126 regarding the technical

review of the corresponding ISO Standard 15152

2. Organisation

2.1 Participants

In total, 19 laboratories participated in the 2014 study using the study protocol and the

KSCN/DCIC method attached in Appendices A and B, respectively. The list of laboratories in

alphabetical order and the continuous-flow equipment used are shown below in Table 1.

Table 1: Participating laboratories and equipment

Laboratory Name Equipment

British American Tobacco, Germany SEAL Analytical, AA3

China National Tobacco Quality Supervision and Test Center, China SEAL Analytical, AA3

Essentra Scientific Services Technicon, AA II

Global Laboratory Services, Inc. Unity Scientific, Discrete Analyzer

Guizhou tobacco quality supervision and inspection station SEAL Analytical, AA3

Landewyck Tobacco s.a. SEAL Analytical

R&D Shandong Industrial Co., Ltd. SEAL Analytical, AA3

R&D, Beijing Cigarette Factory SEAL Analytical, AA3

R&D, Shenzhen Cigarette Factory Skalar, San plus System

R&D, China tobacco Hubei Industrial Co., Ltd. SEAL Analytical, AA3

R&D, China tobacco Jiangxi Industrial Co., Ltd. SEAL Analytical, AA3

R&D, China tobacco Yunnan Industrial Co., Ltd. SEAL Analytical, AA3

R&D, Guangxi International Tobacco Co., Ltd SEAL Analytical, AA3


Laboratory Name Equipment

R&D, Hua Huan International Tobacco Co., Ltd SEAL Analytical, AA3

R&D, Inner Mongolia Kunming cigarettes Co., Ltd SEAL Analytical, AA3

ITG Reemtsma Skalar, San plus System

SEAL Analytical SEAL Analytical, AA3

Sichuan Tobacco Quality Supervision and Test Station SEAL Analytical, AA3

Yunnan Academy of Agricultural Sciences SEAL Analytical, AA3

2.2 Samples

Samples A-F are ground and distributed to participating laboratories by CTQTC. Samples G-

H are also ground and prepared by participating laboratories themselves. Sample descriptions

are given in Table 2.

Table 2: Sample description

Sample Name Sample Description

A Flue-cured tobacco

B Burley tobacco

C Oriental tobacco

D Dark sun-cured tobacco

E Fire-cured cigarette

F Blended cigarette

G CM7

H 3R4F

2.3 Data sets

Six replicates for each method and extraction technique were requested. However, due to

some restrictions on the use of KCN, problems with sample transport or technical difficulties

some labs failed to provide complete data packages as shown in Table 3.

Table 3: Number of results by laboratory

Sample Water extracts 5% HAC extracts

CRM35 NCM CRM35 NCM

A 17 18 17 17

B 17 18 17 17

C 17 18 17 17

D 17 18 17 17

E 17 18 17 17

F 17 18 17 17

G 16 17 16 16

H 16 17 16 16


3. Raw data

The original data of the 19 laboratories can be found in Appendix C. The raw mean and

standard deviation values of CRM35 and NCM can be found in Appendix D. In Appendix E,

the raw data for CRM35 and NCM with water and 5 % HAC extracts shown in plots, in which

the mean values of the 95 % confidence interval are highlighted in colors. The 99 %

confidence limits of difference between the average of all laboratories and the average of one

laboratory are also presented.

4. Statistical analysis

4.1 Numerical outlier technique: Cochran & Grubbs test

The statistical evaluation of data for this collaborative study followed the methods provided

by ISO 5725-21. For outlier testing, the Grubbs and Cochran methods were used.

The protocol “Harmonized statistical procedure” defined by IUPAC (International Union of

Pure and Applied Chemistry) has been applied as well. It consists of sequential applications of

the Cochran and Grubbs tests until no further outliers are detected or until a drop of more than

22.2 % (for this study =2/9) in the original number of laboratories would occur (see flowchart

in Appendix F).

First apply the Cochran outlier test, and if an outlying laboratory is identified, then a single

value Grubbs test is performed on the individual values of this outlying laboratory (Individual

Grubbs). If no individual value is identified as outlier, the outlying laboratory is removed.

When an individual value is identified as an outlier, only this value is removed.

Afterwards single-value Grubbs test is applied and outlying laboratories are removed. If no

laboratory is identified as outlier, the pair-value test is applied (two values at the same end).

Remove any laboratory flagged by these tests, but stop removal if more than 22.2 % (2 of 9

laboratories) would be removed or until a drop of more than 22.2 % (ca. 2/9) in the original

number of laboratories would occur2. The details of this method can be found in Appendix F.

1 ISO 5725-2:1994 : Accuracy (trueness and precision) of measurement methods and results – Part 2: Basic

method for the determination of repeatability and reproducibility of a standard measurement method.

2 2013 Collaborative Study of CORESTA Monitors #7 (CM7) for the Determination of Test Piece Weight, TPM,

Water, Nicotine, NFDPM, Carbon Monoxide and Puff Count Obtained Under Mainstream ISO and Intense

Smoking Regimes, Routine Analytical Chemistry Sub-Group Technical Report , March 19, 2014.


4.1.1 Outlier results

Table 4: Results of outlier determination

Samples Outlier Method

CRM35 in water

NCM in water CRM35 in

HAC NCM in HAC

A

Cochran 6 5, 14 17 13, 14, 5

Grubbs 16, 11 - 16 -

B

Cochran 15, 16 15, 16 15, 17 15, 10, 14

Grubbs - - 16 -

C

Cochran 16, 8 16, 13, 11 16, 9, 4 14, 13, 1

Grubbs - - - -

D

Cochran 2 5, 4 6 5, 14, 13

Grubbs - - 16 -

E

Cochran 12, 4, 16 16, 5, 7 17 5, 14, 13

Grubbs - - 16 -

F

Cochran 8, 9, 11 5, 13, 9, 8 8 14, 13, 8

Grubbs 6 - 16 -

G

Cochran 4, 8 18, 5, 13 17, 3 5, 14, 13

Grubbs - - 16 -

H

Cochran 12 4, 8 4 14, 5, 4

Grubbs - - -

The data for each sample and each individual laboratory is shown in Appendix D, Tables D-1

to D-8. Table 4 summarizes the analyses from laboratories that were considered outliers and

with higher standard deviations according to the Cochran-Grubbs statistical analysis.


4.2 Repeatability and reproducibility estimation

Repeatability (r) and Reproducibility (R) limits are calculated after removal of outliers.

Nevertheless, the stragglers are retained for these calculations.

r and R values shown in Tables 5-8, are calculated for both the water and 5 % HAC extraction

methods.

Table 5: CRM35 with water extraction

Tobacco Type Mean (%) r rCV (%)* R RCV (%)*

Fire-cured 1.76 0.05 2.87 0.12 6.67

Burley 4.77 0.11 2.35 0.61 12.76

Oriental 0.98 0.04 4.07 0.24 24.10

Dark sun-cured 3.71 0.08 2.19 0.48 12.89

Fire-cured cigarette 2.11 0.06 2.71 0.34 15.90

Blended cigarette 2.02 0.03 1.59 0.20 9.65

CM 7 2.2 0.05 2.41 0.28 12.48

3R4F 2.15 0.06 2.90 0.29 13.70

*rCV is r/mean × 100% and RCV is R/mean × 100%

Table 6: NCM with water extraction

Tobacco Type Mean (%) r rCV (%)* R RCV (%)

*

Fire-cured 1.81 0.04 2.37 0.27 14.81

Burley 4.77 0.11 2.23 0.52 10.97

Oriental 0.98 0.03 3.51 0.19 19.21

Dark sun-cured 3.71 0.06 1.73 0.45 12.02



CM 7 2.22 0.05 2.29 0.30 13.43

3R4F 2.12 0.06 2.61 0.24 11.52


Table 7: CRM35 with 5 % HAC extraction


*

Fire-cured 1.74 0.04 2.39 0.13 7.51

Burley 4.53 0.11 2.39 0.41 8.99

Oriental 0.96 0.04 4.04 0.14 14.66

Dark sun-cured 3.51 0.07 2.06 0.26 7.46



CM 7 2.11 0.05 2.33 0.17 8.19

3R4F 2.08 0.06 2.80 0.26 12.55

Table 8: NCM with 5 % HAC extraction


*

Fire-cured 1.76 0.04 2.28 0.19 11.04

Burley 4.53 0.08 1.87 0.56 12.35

Oriental 1.00 0.03 3.38 0.14 13.70

Dark sun-cured 3.60 0.06 1.60 0.44 12.29



CM 7 2.17 0.04 1.95 0.27 12.25

3R4F 2.10 0.05 2.34 0.25 11.82

4.3 Comparison of 2014 study to previous studies

In order to further evaluate the data acquired in this study, data were also compared to earlier

collaborative studies performed by the RAC on CRM35. Table 9 lists the r and R values of

CRM35 from the 1993 international collaborative study involving 12 laboratories and 3

samples. Table 10 lists the r and R values of CRM35 from the 20063 collaborative study

involving 17 laboratories and 6 samples. Comparing Table 5, 6, 9 and 10, it can be seen that,

for the same tobacco type, the r and R values of CRM35 from the three collaborative studies

are close to each other.

3 2006 Collaborative Studies for Nicotine, Sugars and Nitrate in Tobacco, May 2008


Table 9: r and R of total alkaloids in 1993 collaborative study

Extraction Method

Tobacco Type Mean (%) r rCV* R RCV

*

Water

Oriental 1.17 0.05 4.27 0.19 16.24

Flue-Cured 2.90 0.08 2.76 0.41 14.14

Burley 3.97 0.12 3.02 0.55 13.85

5 % acetic acid

Oriental 1.17 0.07 5.98 0.21 17.95

Flue-Cured 2.90 0.11 3.79 0.67 23.10

Burley 3.97 0.13 3.27 0.97 24.43

Table 10: r and R of total alkaloids in 2006 collaborative study

Tobacco Type Mean* (%) r rCV* R RCV

*

Flue-Cured A 3.19 0.17 5.33 0.53 16.61

Flue-Cured B 2.86 0.09 3.15 0.47 16.43

Flue-Cured C 0.70 0.03 4.29 0.16 22.86

Burley sample D 3.30 0.12 3.64 0.67 20.30

Burley sample E 1.51 0.08 5.30 0.32 21.19

Burley sample F 0.69 0.03 4.35 0.12 17.39

*This CRM recommends that equivalent results are obtained when either water or 5 % HAC are used as the

extraction solvents and therefore the results from this study were not segregated in the subsequent data analysis.

There was also a study performed in 2013 by a large group of laboratories in China

comparing CRM35 and the NCM. Table 11 lists the r and R values from this collaborative

study. Again it can be observed that the r and R values in this study are comparable to the

current study for the same tobacco type.

Table 11: r and R of total alkaloids in 2013 Chinese collaborative study

Method Samples Mean (%) r rCV* R RCV

*

CRM35

Fire-cured 1.71 0.03 1.75 0.25 14.62

Burley 4.31 0.07 1.62 0.60 13.92

Oriental 0.94 0.04 4.26 0.17 18.09

Cigarette A 2.04 0.04 1.96 0.37 18.14

Cigarette B 1.98 0.04 2.02 0.24 12.12

KSCN/DCIC

Fire cured 1.73 0.04 2.31 0.22 12.72

Burley 4.35 0.06 1.38 0.64 14.71

Oriental 0.97 0.04 4.12 0.12 12.37

Cigarette A 2.03 0.05 2.46 0.30 14.78

Cigarette B 2.00 0.04 2.00 0.27 13.50

It can be concluded from the analysis above that the data from three previous studies and the

current study are consistent and can be used in comparative analysis.


Figure 1 shows the r and R values of total alkaloids determined by CRM35 and NCM

extraction with water and 5 % HAC extracts, respectively, in 2014 international collaborative

study. As shown, r is always marked in yellow, while the R values from the four different

measuring methods are displayed in four different colours. Mean values of the four different

measuring methods are also presented. It can be observed from above tables and Figure 1 that,

for both extracts, the r values of CRM35 are, however, larger than the observed for NCM. The

values of the two methods are very similar. The R values for CRM35 extracted in water for

samples A, F, and G are lower than those observed for the NCM water extracts. While for

samples B, C, D, E and H, the results are reversed. The R values of CRM35 extracted in HAC

are lower than those of NCM extracted in HAC for samples A, B, D, E, F, and G, while for C

and H, the R values of CRM35 are higher than for 5 % HAC. The difference in R values of

both methods is statistically not significant and considered acceptable.

Figure 1: The r and R of total alkaloids determined by CRM35 and NCM extraction

with water and 5 % HAC, respectively, in 2014 international collaborative study.

A B C D E F G H 0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

5.5

Test Samples ID

R of CRM35 in water

R of NCM in water

R of CRM35 in HAC R of NCM in HAC

r

Mean values

To

tal A

lka

loid

s (

%)


Figure 2 shows the r and R of total alkaloids determined by CRM35 and NCM in the 2013

Chinese collaborative study. It can be observed that, the R value of CRM35 is lower than

observed for NCM for sample B and E, while for sample A, C and D, the R values of CRM35

are higher than the ones of NCM. The r values of the two methods are similar. The small

difference observed in R values for both methods, is acceptable.

Figure 2: The r and R of total alkaloids determined by CRM35 and NCM in 2013

Chinese collaborative study.

5. Method Validation

Validations of CRM35 and NCM, (extraction in water) in 2014 collaborative study are shown

in Figure 3. The location of the lab mean is close to y = x line, illustrating that the total

alkaloids of CRM35 method are very similar to those from NCM extraction by water method.

The correlation coefficient of the two methods is 0.9970, and the root mean square error

(RMSE) is 0.0924 %. RMSE can be calculated by:

NRMSE

N

i

1

2

CRM35NCM yy

where N is the number of data points, yNCM and yCRM representation to the total alkaloids

determined by NCM and CRM35, respectively in this work. The ratio performance deviation

(RPD) is also used in this work, it can be calculated by:

RMSE

RPD CRM35SD y

A B C D E 0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

Test Samples ID

To

tal A

lka

loid

s (

%)

R of CRM

R of NCM r

Mean values


If RPD exceeds 3, the consistency of CRM35 and NCM is considered as satisfactory

according to Williams and Sobering (1993)4. Using water extraction, the RPD is 12.8849,

showing good consistency between the two methods. The linear regression equation of the

two methods was also calculated, yNCM=0.9962yCRM+0.0145 , where yNCM and yCRM were

NCM and CRM extraction in water, respectively.

Figure 3: The relationship of total alkaloids between method CRM35 and NCM by

water extraction for the 2014 international collaborative study.

4 P.C. Williams and D.C. Sobering, Comparison of commercial near infrared transmittance and reflectance

instruments for analysis of whole grains and seeds, Journal of Near Infrared Spectroscopy, Volume 1 Issue 1,

Pages 25–32 (1993)

0 1 2 3 4 5 6 0

1

2

3

4

5

6

Total alkaloids (%) by CRM35 in water

To

tal a

lkalo

ids (

%)

by N

CM

in w

ate

r

y = x

Lab mean


The relationship of CRM and NCM extraction in 5 % HAC in 2014 collaborative study is

visualized in Figure 4. The correlation coefficient of two methods is 0.9964, and the root

mean square error (RMSE) is 0.0916 %. The RPD is 11.3798, showing good consistency of

CRM35 and NCM in extraction with 5 % HAC. The linear regression equation of the two

method was also calculated, yNCM=0.9951yCRM+0.0354, where yNCM and yCRM were NCM and

CRM35 extraction in 5 % HAC, respectively.

Figure 4: The relationship of total alkaloids between method CRM35 and NCM by 5 %

HAC extraction for the 2014 international collaborative study.

0 1 2 3 4 5 0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

5.5

Total alkaloids (%) by CRM35 in 5 % HAC

To

tal a

lkalo

ids (

%)

by N

CM

in

5 %

HA

C

y = x

Lab mean


Figure 5 shows the relationship of CRM35 and NCM for Chinese collaborative study in 2013.

The correlation coefficient of the two methods is 0.9977, and the root mean square error

(RMSE) is 0.0727 %. The RPD is 14.4239, showing good consistency of CRM35 and NCM.

The linear regression equation of the two method was also calculated,

yNCM=0.9788yCRM35+0.0359 , where yNCM and yCRM35 were NCM and CRM35 in 2013

Chinese collaborative study, respectively.

Figure 5: The relationship of total alkaloids between CRM35 and NCM for the 2013

Chinese collaborative study.

In order to further validate the agreement of CRM35 to NCM, Bland Altman plots were used.

Figure 6 shows the result of CRM35 and NCM in extraction with water. The logarithmic

transformation of the dataset using a technique described by Bland and Altman5 (1986) was

applied because the difference of the two methods increased as total alkaloid values increased.

The green line is the mean value of the difference of two methods, and the red dash lines

represent the 95 % confidence interval. It can be seen that the means for all the eight samples

are located within the 95 % confidence interval demonstrating that results obtained for

CRM35 and NCM using water extraction are consistent. The mean results of CRM35 and

NCM using 5 % acetic acid extraction are shown in Figure 7. Consistent with previous

collaborative studies on CRM35 using both water and 5 % HAC extraction solutions, the

NCM method gives equivalent results.

5 Statistical methods for assessing agreement between two methods of clinical measurement, JM Bland, DG

Altman, The Lancet, 1986, I: 307-310

0 1 2 3 4 5 6 0

1

2

3

4

5

6

Total alkaloids (%) by CRM35

To

tal a

lka

loid

s (

%)

by N

CM

y = x

Lab mean


Figure 6: Bland Altman plot of CRM35 and NCM in extraction with water

Figure 7: Bland Altman plot of CRM35 and NCM extraction in 5 % HAC

0 0.5 1 1.5

-0.04

-0.03

-0.02

-0.01

0

0.01

0.02

0.03

A

BC D

E

F

G

H

Bland Altman plot

Mean of two measures

Diffe

ren

ce

be

twe

en

tw

o m

ea

su

res

0 0.5 1 1.5-0.05

-0.04

-0.03

-0.02

-0.01

0

0.01

A

B

C

D

E

F

G

H

Bland Altman plot


Diffe

ren

ce

be

twe

en

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res


Figure 8: Bland Altman plot of CRM35 in water and CRM35 in 5% HAC

Figure 9: Bland Altman plot of NCM in water and NCM in 5 % HAC

0 0.5 1 1.5

0

0.01

0.02

0.03

0.04

0.05

0.06

0.07

A

B

C

D

E

F

G

H

Bland Altman plot


Diffe

ren

ce

be

twe

en

tw

o m

ea

su

res

0 0.5 1 1.5-0.03

-0.02

-0.01

0

0.01

0.02

0.03

0.04

0.05

0.06

A

B

C

D

E

F G

H

Bland Altman plot


Diffe

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ea

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res


6. Conclusions and recommendations

6.1 Conclusions

▪ For samples A to H, the contents of total alkaloids determined by CRM35 and NCM

with water extract are similar. The correlation coefficient of the two methods is 0.9970,

the root mean square error (RMSE) is 0.0924 % and the RPD is 12.8849, showing good

agreement between CRM35 and NCM in extraction with water.

▪ For sample A to H, the contents of total alkaloids determined by CRM35 and NCM with

HAC extract are similar. The correlation coefficient of the two methods is 0.9964, the

root mean square error (RMSE) is 0.0916 %, and the RPD is 11.3798, showing good

agreement between CRM35 and NCM in extraction with HAC. It is recommended that

5% HAC extracts should be used if total alkaloids (as nicotine) and reducing substances

or reducing carbohydrates are to be carried out simultaneously.

▪ NCM method showed comparable r and R values to those published in CRM35 for both

water or HAC extraction in this 2014 collaborative study.

▪ For NCM, the results showed good consisitent with water and HAC extractions.

▪ The results of CRM35 in 2014 international collaborative study showed comparable r

and R data to those obtained by the CORESTA RAC reproducibility and repeatability

1993 and 2006 international collaborative studies.

6.2 Recommendations

Firstly, the RAC recommends that CRM35 be made obsolete and replaced with a new CRM

reflecting the safer chemistry with new flow diagrams for both macro and micro flow

instruments.

Secondly, once the new CRM is published that it be submitted to ISO TC126.


Appendix A – Experimental Protocol

Protocol of collaborative test for total alkaloids in tobacco

1. Objective

To assess the repeatability and reproducibility of laboratories applying the same

analytical method. Besides, to assess the distinction between the Chinese KSCN/DCIC

method and the CRM35.The moisture content shall be determined, in order to be able

to provide results on a dry weight basis as well as an as-received basis.

The proposed method was developed and validated for tobacco, conventional tobacco

products.

2. Test coordinator

Zhang Wei

Senior Engineer

China National Tobacco Quality Supervision ＆ Test Centre

No. 2 Fengyang Road, Zhengzhou High and New Technology Industries Development

Zone, Henan, China, 450001

E-mail: [email protected]

Tel: +86 371 67672617 Fax: +86 371 67672625

3. Test methods

Each laboratory shall apply both the provided Chinese KSCN/DCIC and the CRM35

methods to test the total alkaloids in the samples with water and 5% acetic acid

extractions.

The determination of moisture should be performed according to each participants own

method. If possible, a CORESTA or ISO recommended method should be used.

4. Test samples

Test samples consist of tobacco products listed in Table 1. The CM7 and 3R4F

cigarettes (samples G and H) should be taken from the participants’ own stock. Tobacco

samples A through F have been provided by Zhang Wei, China National Tobacco

Quality Supervision ＆ Test Centre.

Table 1. Test products for the collaborative study

Code Test product

A Fire cured tobacco

B Burley tobacco

C Oriental tobacco

D Dark sun-cured tobacco

E Fire-cured cigarette

F Blended cigarettes

G CM 7

H 3R4F


5. Schedule

For each sample, 6 replicates should be measured within a single test day.

The standard substance should be dissolved by the same solution as the extraction one.

The study starts in August 2014. The test results should be sent to the test coordinator

no later than August 25st, 2014.

6. Reporting of test results

The test results should be reported using the Excel file “CORESTA Total Alkaloids

Reporting Sheets”.

The reporting units are specified in “CORESTA Total Alkaloids Reporting Sheets” as

well as in the Excel template. Please provide the results on an “as received” and a dry

weight basis, after correction for the moisture content.

In the “General Information” worksheet, please provide a brief description of the

analytical instruments and parameters you use, as well as any minor modifications to

the method.

Also, please provide information about the source of your standard materials on the

spreadsheet provided.

Results should be sent electronically:

To: [email protected]

Cc: [email protected]


Appendix B – Chinese KSCN/DCIC Method of Collaborative Test for Total Alkaloids in Tobacco

Unless otherwise specified, all chemicals should be of Analytical grade or equivalent.

LIST OF RAW MATERIALS(for KSCN/DCIC Chemistry) Safety classifications:

Brij-35*, 30 % solution

Sodium phosphate dibasic, Na2HPO4·12H2O

Sodium phosphate monobasic, NaH2PO4·2H2O

Sodium citrate, C6H5Na3O7•2H2O

Sulphanilic acid, NH2C6H4SO3H

Potassium thiocyanate, KSCN, harmful

Sodium dichloroisocyanurate dihydrate, C3Cl2N3NaO3, harmful

Sodium carbonate, Na2CO3, irritant

Ferrous sulfate, FeSO4.7H2O, harmful

Citric acid, C6H8O7.H2O

Nicotine (>98%), highly toxic

REAGENTS MAKE UP

DI Water refers to high quality reagent water.

POTASSIUM THIOCYANATE SOLUTION

Dissolve 2.88 g of potassium thiocyanate in DI water. Dilute to 250 mL with DI water and mix well.

DCIC WORKING SOLUTION

DCIC2.20 g

DI water to 250 mL

Dissolve 2.20 g of DCIC and dilute to 250 mL with DI water.

NEUTRALISATION SOLUTION A

Citric acid 1 g

Ferrous sulfate 10 g

DI water to 1000 mL

Dissolve 1 g of citric acid and 10 g of ferrous sulfate in about 500 mL of DI water. Dilute to 1000 mL with DI water and mix well.


NEUTRALISATION SOLUTION B

Sodium carbonate 10 g

DI Water to 1000 mL

Dissolve 10 g of sodium carbonate in about 500 mL of DI water. Dilute to 1000 mL with DI water and mix well.

BUFFER SOLUTION A

Dissolve 71.6 g of sodium phosphate dibasic and 11.76 g of sodium citrate in about 500 mL of DI water. Dilute to 1000 mL with DI water, add 1

mL of Brij, 30% solution and mix well.

BUFFER SOLUTION B

Dissolve 71.6 g of sodium phosphate dibasic, 6.2 g of sodium phosphate monobasic,11.76 g of sodium citrate and7.0 g of sulphanilic acid in

about 800 mL of DI water. Dilute to 1000 mL with DI water, add 1 mL of DI water and mix well.

Figure 1: Proposed flowchart for macro flow systems


Figure 2: Proposed flowchart for micro flow systems


Appendix C – Raw Data by Laboratory

Lab 1

Samples CRM35 in water NCM in water CRM35 HAC NCM in HAC

1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6

A 1.79 1.77 1.76 1.78 1.77 1.78 1.85 1.83 1.83 1.83 1.84 1.85 1.69 1.70 1.71 1.70 1.71 1.70 1.75 1.75 1.75 1.74 1.76 1.75

B 4.92 4.90 4.92 4.92 4.90 4.88 4.85 4.84 4.80 4.88 4.84 4.77 4.40 4.45 4.46 4.49 4.47 4.47 4.49 4.46 4.50 4.53 4.52 4.52

C 0.89 0.92 0.87 0.92 0.91 0.91 0.98 0.99 0.97 1.01 1.00 1.00 0.88 0.91 0.93 0.89 0.93 0.93 0.91 0.91 0.97 0.91 0.97 0.96

D 3.88 3.84 3.83 3.83 3.84 3.84 3.87 3.87 3.85 3.85 3.85 3.85 3.50 3.53 3.52 3.53 3.51 3.55 3.59 3.64 3.61 3.60 3.60 3.63

E 2.11 2.12 2.12 2.10 2.11 2.12 2.15 2.16 2.15 2.16 2.14 2.15 2.01 2.02 2.02 2.01 2.01 2.02 2.00 2.01 2.01 2.01 2.01 2.01

F 2.14 2.10 2.12 2.11 2.13 2.14 2.17 2.15 2.16 2.18 2.16 2.18 1.98 1.98 1.98 2.00 1.97 1.99 2.03 2.03 2.02 2.04 2.02 2.04

G 2.25 2.25 2.24 2.24 2.25 2.25 2.28 2.27 2.27 2.29 2.28 2.29 2.09 2.11 2.11 2.09 2.09 2.08 2.14 2.15 2.16 2.14 2.14 2.14

H 2.17 2.17 2.21 2.21 2.19 2.22 2.19 2.19 2.23 2.21 2.23 2.23 2.04 2.05 2.08 2.05 2.05 2.05 2.09 2.10 2.13 2.11 2.12 2.11

Lab 2


1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6

A 1.73 1.72 1.70 1.72 1.73 1.68 1.81 1.81 1.79 1.80 1.80 1.76 1.69 1.68 1.66 1.69 1.69 1.69 1.72 1.73 1.71 1.73 1.74 1.72

B 4.67 4.67 4.79 4.71 4.66 4.63 4.82 4.80 4.91 4.82 4.83 4.82 4.36 4.41 4.38 4.43 4.45 4.42 4.46 4.49 4.45 4.47 4.52 4.49

C 0.95 0.96 0.95 0.94 0.96 0.95 1.04 1.06 1.06 1.05 1.07 1.07 0.96 0.93 0.92 0.93 0.93 0.93 1.00 1.00 1.01 1.01 1.01 1.01

D 3.50 3.57 3.56 3.60 3.58 3.67 3.74 3.78 3.75 3.77 3.76 3.86 3.43 3.42 3.44 3.48 3.44 3.44 3.53 3.54 3.51 3.53 3.53 3.52

E 2.02 2.08 2.05 2.09 2.05 2.04 2.13 2.18 2.15 2.18 2.18 2.18 2.00 2.00 2.00 2.00 1.99 1.98 2.02 2.02 2.02 2.02 2.01 2.01

F 1.98 1.97 1.97 1.97 2.05 1.98 2.16 2.11 2.10 2.09 2.10 2.10 1.93 1.93 1.94 1.93 1.94 1.94 1.99 1.98 1.98 1.99 1.98 1.99

G 2.07 2.07 2.07 2.11 2.11 2.07 2.16 2.14 2.14 2.14 2.18 2.16 2.04 2.03 2.04 2.05 2.02 2.02 2.10 2.09 2.09 2.08 2.08 2.06

H 2.10 2.09 2.12 2.11 2.09 2.09 2.17 2.19 2.19 2.17 2.15 2.17 2.01 2.02 2.01 2.00 2.01 2.02 2.06 2.06 2.06 2.06 2.04 2.05

Lab 3


1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6

A 1.71 1.73 1.72 1.74 1.72 1.72 1.73 1.76 1.75 1.76 1.73 1.74 1.72 1.70 1.71 1.73 1.69 1.73 1.72 1.70 1.72 1.72 1.72 1.72

B 4.51 4.45 4.51 4.53 4.52 4.45 4.61 4.58 4.63 4.64 4.58 4.54 4.55 4.56 4.53 4.51 4.48 4.47 4.48 4.47 4.50 4.49 4.52 4.48

C 0.96 0.98 0.95 0.95 0.97 0.96 0.94 0.94 0.93 0.92 0.93 0.91 0.96 0.94 0.95 0.95 0.96 0.98 0.98 0.97 0.97 0.96 0.96 0.97

D 3.50 3.53 3.54 3.51 3.51 3.53 3.54 3.56 3.59 3.30 3.58 3.52 3.49 3.50 3.53 3.51 3.51 3.54 3.54 3.54 3.53 3.57 3.54 3.55

E 2.01 1.98 1.98 1.98 2.02 2.03 1.99 1.98 1.96 1.98 1.99 1.99 2.02 2.05 2.04 2.04 2.04 2.00 1.98 2.00 2.00 1.98 2.00 2.01

F 2.02 2.02 2.01 2.03 2.02 2.01 2.00 2.01 2.02 2.01 1.99 1.98 1.99 1.99 2.03 2.01 2.01 2.03 2.01 1.99 2.01 2.00 1.99 1.98

G 2.12 2.17 2.11 2.17 2.17 2.16 2.14 2.16 2.14 2.16 2.17 2.14 2.15 2.15 2.18 2.21 2.15 2.13 2.12 2.12 2.15 2.15 2.14 2.14

H 2.06 2.06 2.09 2.09 2.03 2.09 2.06 2.03 2.06 2.03 2.04 2.05 2.08 2.08 2.06 2.07 2.06 2.08 2.07 2.09 2.08 2.08 2.10 2.09


Lab 4


1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6

A 1.78 1.76 1.78 1.77 1.76 1.71 1.75 1.74 1.77 1.74 1.71 1.73 1.73 1.73 1.72 1.72 1.70 1.67 1.68 1.70 1.69 1.67 1.65 1.63

B 4.84 4.84 4.86 4.82 4.86 4.77 4.67 4.58 4.66 4.57 4.62 4.57 4.18 4.35 4.27 4.23 4.31 4.22 4.14 4.19 4.21 4.20 4.23 4.20

C 0.98 1.01 0.99 1.02 0.99 1.02 0.95 0.93 0.94 0.92 0.91 0.91 0.95 1.02 1.02 1.02 0.99 1.00 0.97 0.99 0.98 0.98 0.97 0.97

D 3.65 3.63 3.68 3.68 3.66 3.66 3.57 3.45 3.50 3.51 3.53 3.49 3.38 3.37 3.31 3.35 3.35 3.40 3.41 3.41 3.34 3.37 3.38 3.35

E 2.02 2.14 2.08 2.02 1.97 1.97 1.86 1.88 1.86 1.88 1.85 1.83 1.94 1.96 1.94 1.97 1.98 1.93 1.88 1.89 1.91 1.90 1.91 1.88

F 1.95 1.95 1.99 1.97 1.96 1.97 1.91 1.95 1.93 1.94 1.95 1.93 1.97 1.97 1.97 1.97 1.96 2.00 1.98 1.99 1.99 1.98 1.98 2.00

G 2.22 2.31 2.31 2.36 2.34 2.16 2.16 2.20 2.20 2.15 2.14 2.20 2.09 2.10 2.08 2.08 2.09 2.08 2.13 2.12 2.11 2.12 2.14 2.09

H 2.18 2.20 2.17 2.25 2.23 2.19 2.01 2.03 2.10 2.13 2.13 2.07 2.00 2.08 2.02 2.03 1.97 2.07 2.02 2.07 2.05 2.03 1.95 2.06

Lab 5


1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6

A 1.77 1.78 1.75 1.76 1.76 1.73 1.77 1.71 1.73 1.80 1.84 1.77 1.66 1.69 1.68 1.69 1.70 1.69 1.64 1.70 1.69 1.62 1.67 1.66

B 4.96 4.91 4.92 4.90 4.89 4.87 4.62 4.70 4.67 4.65 4.61 4.57 4.56 4.56 4.54 4.53 4.51 4.49 4.26 4.30 4.37 4.39 4.38 4.38

C 0.81 0.85 0.83 0.85 0.84 0.85 0.88 0.97 0.91 0.91 0.90 0.90 0.96 0.97 0.97 0.92 0.93 0.93 0.96 0.98 0.96 0.96 0.98 0.96

D 3.81 3.79 3.84 3.82 3.84 3.86 3.68 3.56 3.58 3.72 3.73 3.63 3.44 3.46 3.37 3.36 3.39 3.39 3.54 3.54 3.57 3.56 3.67 3.66

E 2.14 2.14 2.15 2.17 2.06 2.11 2.02 2.01 2.11 2.04 1.97 2.02 2.02 1.98 1.96 2.02 1.98 1.95 2.04 1.96 1.96 1.99 2.04 1.93

F 2.01 2.01 2.01 2.02 2.01 2.07 2.07 2.09 2.03 2.09 2.01 2.00 1.84 1.80 1.79 1.84 1.82 1.86 2.00 1.96 2.00 1.95 1.98 1.97

G 2.20 2.22 2.25 2.21 2.24 2.23 2.16 2.11 2.15 2.07 2.03 2.13 1.97 1.95 1.99 2.01 2.00 2.01 2.11 2.14 2.06 2.14 2.04 2.17

H 2.17 2.16 2.15 2.16 2.14 2.11 2.04 2.02 2.06 2.13 2.07 2.04 1.93 1.88 1.91 1.96 1.98 1.98 2.05 2.09 2.04 1.98 1.97 2.02

Lab 6


1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6

A 2.20 2.23 2.18 2.16 2.14 2.15 1.89 1.86 1.86 1.84 1.84 1.83 1.83 1.80 1.76 1.75 1.77 1.78 1.76 1.76 1.73 1.71 1.73 1.74

B 5.16 5.12 5.12 5.18 5.14 5.14 4.98 4.99 5.02 5.01 5.01 4.99 4.65 4.56 4.52 4.55 4.60 4.60 4.52 4.48 4.47 4.51 4.48 4.48

C 1.17 1.20 1.20 1.20 1.22 1.20 0.97 0.97 0.98 0.95 0.96 0.95 1.09 1.06 1.05 1.05 1.06 1.07 1.01 0.98 0.98 0.98 1.00 1.01

D 4.08 3.98 3.99 4.05 4.06 4.05 3.80 3.81 3.81 3.82 3.85 3.85 3.60 3.54 3.57 3.55 3.49 3.45 3.58 3.56 3.57 3.58 3.57 3.53

E 2.39 2.42 2.45 2.41 2.42 2.40 2.09 2.09 2.10 2.07 2.09 2.08 2.18 2.11 2.06 2.12 2.12 2.14 2.02 1.99 2.00 2.01 1.99 2.00

F 2.34 2.35 2.37 2.36 2.37 2.36 2.04 2.06 2.06 2.06 2.08 2.06 2.03 2.07 2.06 2.08 2.05 2.02 1.98 1.97 1.97 1.97 1.97 1.97

G 2.38 2.38 2.43 2.41 2.37 2.36 2.18 2.20 2.22 2.18 2.19 2.22 2.17 2.14 2.16 2.15 2.18 2.17 2.10 2.09 2.11 2.14 2.13 2.13

H 2.31 2.31 2.29 2.29 2.34 2.28 2.15 2.15 2.14 2.16 2.16 2.12 2.11 2.10 2.09 2.11 2.16 2.11 2.05 2.04 2.02 2.03 2.03 2.02


Lab 7


1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6

A

B

C

D

E

F

G

H

Lab 8


1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6

A

B

C

D

E

F

G

H

Lab 9


1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6

A

B

C

D

E

F

G

H


Lab 10


1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6

A

B

C

D

E

F

G

H

Lab 11


1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6

A

B

C

D

E

F

G

H

Lab 12


1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6

A

B

C

D

E

F

G

H


Lab 13


1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6

A

B

C

D

E

F

G

H

Lab 14


1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6

A

B

C

D

E

F

G

H

Lab 15


1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6

A

B

C

D

E

F

G

H


Lab 16


1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6

A

B

C

D

E

F

G

H

Lab 17


1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6

A

B

C

D

E

F

G

H

Lab 18


1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6

A

B

C

D

E

F

G

H


Lab 19


1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6

A

B

C

D

E

F

G

H


Appendix D – Raw Mean and Standard Deviation Value

Table D-1: Sample A - raw mean and standard deviation value of total alkaloids (as

nicotine determined by CRM35 and NCM (KSCN/DCIC) in water and 5 % HAC,

respectively

Lab ID CRM35 in water NCM in water CRM35 in HAC NCM in HAC

Mean SD Mean SD Mean SD Mean SD

1 1.77 0.01 1.84 0.01 1.70 0.01 1.75 0.01

2 1.71 0.02 1.80 0.02 1.68 0.01 1.73 0.01

3 1.72 0.01 1.74 0.01 1.71 0.02 1.72 0.01

4 1.76 0.03 1.74 0.02 1.71 0.02 1.67 0.03

5 1.76 0.02 1.77 0.05 1.69 0.01 1.66 0.03

6 2.18 0.04 1.85 0.02 1.78 0.03 1.74 0.02

7 1.77 0.02 1.89 0.01 1.81 0.01 1.94 0.01

8 1.85 0.01 1.92 0.01 1.73 0.01 1.82 0.01

9 1.75 0.03 1.75 0.02 1.78 0.01 1.78 0.01

10 1.82 0.02 1.80 0.03 1.75 0.01 1.74 0.01

11 1.95 0.03 1.97 0.02 1.73 0.03 1.75 0.02

12 1.74 0.02 1.74 0.02 1.78 0.04 1.70 0.02

13 - - 1.64 0.02 - - 1.69 0.04

14 1.72 0.03 1.73 0.03 1.73 0.01 1.74 0.04

15 1.73 0.02 1.71 0.01 1.71 0.02 1.74 0.02

16 1.90 0.02 1.95 0.03 2.19 0.02 1.82 0.02

17 - - - - 1.75 0.03 - -

18 1.77 0.01 1.81 0.01 - - - -

19 1.75 0.01 1.74 0.01 1.82 0.01 1.79 0.00


Table D-2: Sample B - raw mean and standard deviation value of total alkaloids (as

nicotine) in sample B determined by CRM35 and NCM (KSCN/DCIC) in water and 5%

HAC, respectively



1 4.91 0.02 4.83 0.04 4.46 0.03 4.50 0.03

2 4.69 0.06 4.83 0.04 4.41 0.03 4.48 0.02

3 4.49 0.03 4.60 0.04 4.52 0.04 4.49 0.02

4 4.83 0.03 4.61 0.04 4.26 0.06 4.20 0.03

5 4.91 0.03 4.64 0.05 4.53 0.03 4.35 0.05

6 5.14 0.02 5.00 0.01 4.58 0.05 4.49 0.02

7 4.65 0.02 4.88 0.02 4.78 0.02 5.01 0.02

8 4.93 0.05 4.99 0.05 4.54 0.07 4.70 0.02

9 4.55 0.06 4.67 0.05 4.67 0.02 4.55 0.03

10 4.67 0.02 4.81 0.01 4.48 0.07 4.56 0.09

11 5.08 0.02 5.08 0.02 4.53 0.04 4.47 0.02

12 5.00 0.06 4.97 0.03 4.43 0.03 4.37 0.03

13 - - 4.60 0.06 - - 4.49 0.06

14 4.46 0.06 4.49 0.06 4.41 0.15 4.59 0.21

15 4.31 0.11 4.19 0.11 4.26 0.22 4.21 0.22

16 4.98 0.08 4.86 0.18 5.59 0.04 4.76 0.02

17 - - - - 4.80 0.08 - -

18 4.62 0.04 4.75 0.03 - - - -

19 4.69 0.03 4.55 0.01 4.75 0.01 4.61 0.01


Table D-3: Sample C - raw mean and standard deviation value of total alkaloids (as

nicotine) in determined by CRM35 and NCM (KSCN/DCIC) in water and 5% HAC,

respectively



1 0.90 0.02 0.99 0.01 0.91 0.02 0.94 0.03

2 0.95 0.01 1.06 0.01 0.93 0.01 1.01 0.01

3 0.96 0.01 0.93 0.01 0.96 0.01 0.97 0.01

4 1.00 0.02 0.92 0.02 1.00 0.03 0.98 0.01

5 0.84 0.01 0.91 0.03 0.95 0.02 0.97 0.01

6 1.20 0.01 0.96 0.01 1.07 0.01 0.99 0.02

7 0.98 0.01 1.10 0.00 1.00 0.00 1.09 0.00

8 1.00 0.03 1.08 0.01 0.95 0.01 1.08 0.01

9 1.03 0.01 0.95 0.01 0.97 0.04 1.00 0.01

10 0.93 0.01 0.96 0.01 0.93 0.02 0.96 0.02

11 1.05 0.02 1.10 0.02 0.95 0.02 1.01 0.02

12 0.90 0.04 0.88 0.01 1.04 0.01 0.97 0.01

13 - - 0.85 0.03 - - 0.87 0.03

14 0.96 0.02 1.00 0.02 0.94 0.01 0.98 0.04

15 0.96 0.01 0.97 0.02 0.94 0.01 0.98 0.02

16 0.88 0.11 0.91 0.12 1.27 0.06 1.07 0.05

17 - - - - 0.96 0.01 - -

18 1.03 0.02 1.05 0.01 - - - -

19 0.95 0.01 0.99 0.01 0.90 0.00 0.93 0.00


Table D-4: Sample D - raw mean and standard deviation value of total alkaloids (as

nicotine) determined by CRM35 and NCM (KSCN/DCIC) in water and 5% HAC,

respectively



1 3.84 0.02 3.86 0.01 3.52 0.02 3.61 0.02

2 3.58 0.05 3.78 0.04 3.44 0.02 3.53 0.01

3 3.52 0.02 3.52 0.11 3.51 0.02 3.54 0.01

4 3.66 0.02 3.51 0.04 3.36 0.03 3.38 0.03

5 3.83 0.02 3.65 0.07 3.40 0.04 3.59 0.06

6 4.03 0.04 3.82 0.02 3.53 0.05 3.57 0.02

7 3.64 0.01 3.80 0.02 3.73 0.01 3.96 0.01

8 3.81 0.05 3.89 0.03 3.53 0.02 3.80 0.01

9 3.68 0.03 3.69 0.02 3.45 0.03 3.65 0.02

10 3.75 0.02 3.71 0.02 3.59 0.01 3.57 0.01

11 3.98 0.02 3.93 0.01 3.52 0.04 3.51 0.02

12 3.74 0.04 3.78 0.03 3.47 0.03 3.48 0.04

13 - - 3.52 0.02 - - 3.65 0.04

14 3.43 0.05 3.50 0.01 3.47 0.03 3.61 0.05

15 3.49 0.03 3.40 0.02 3.47 0.04 3.51 0.01

16 3.74 0.03 3.81 0.04 4.07 0.04 3.80 0.03

17 - - - - 3.60 0.06 - -

18 3.61 0.01 3.71 0.03 - - - -

19 3.54 0.01 3.55 0.02 3.56 0.02 3.53 0.01


Table D-5: Sample E - raw mean and standard deviation value of total alkaloids (as

nicotine) in sample E determined by CRM35 and NCM (KSCN/DCIC) in water and 5%

HAC, respectively



1 2.11 0.01 2.15 0.01 2.01 0.00 2.01 0.00

2 2.05 0.03 2.17 0.02 1.99 0.01 2.01 0.00

3 2.00 0.02 1.98 0.01 2.03 0.02 2.00 0.01

4 2.03 0.06 1.86 0.02 1.95 0.02 1.90 0.01

5 2.13 0.04 2.03 0.05 1.98 0.03 1.99 0.05

6 2.42 0.02 2.09 0.01 2.12 0.04 2.00 0.01

7 2.03 0.00 1.97 0.40 2.09 0.01 2.24 0.01

8 2.19 0.04 2.24 0.02 2.04 0.01 2.20 0.02

9 2.12 0.01 2.02 0.02 2.02 0.02 2.12 0.02

10 2.04 0.01 2.06 0.00 1.97 0.01 2.02 0.02

11 2.26 0.01 2.22 0.01 2.05 0.02 1.99 0.02

12 2.03 0.08 2.04 0.01 2.09 0.02 2.00 0.01

13 - - 2.08 0.02 - - 2.06 0.04

14 2.01 0.02 1.99 0.02 2.00 0.02 2.03 0.04

15 2.03 0.01 1.94 0.01 2.03 0.01 2.01 0.02

16 2.24 0.05 2.23 0.05 2.44 0.01 2.17 0.01

17 - - - - 2.09 0.04 - -

18 2.00 0.01 2.10 0.01 - - - -

19 2.14 0.01 2.05 0.02 2.17 0.01 2.07 0.00


Table D-6: Sample F - raw mean and standard deviation value of total alkaloids (as

nicotine) in sample F determined by CRM35 and NCM (KSCN/DCIC) in water and 5%

HAC, respectively



1 2.13 0.02 2.17 0.01 1.98 0.01 2.03 0.01

2 1.99 0.03 2.11 0.03 1.94 0.00 1.98 0.00

3 2.02 0.01 2.00 0.02 2.01 0.02 2.00 0.01

4 1.97 0.01 1.93 0.01 1.97 0.02 1.98 0.01

5 2.02 0.03 2.05 0.04 1.83 0.02 1.98 0.02

6 2.36 0.01 2.06 0.02 2.05 0.02 1.97 0.01

7 2.04 0.01 2.16 0.00 2.06 0.00 2.23 0.01

8 2.16 0.04 2.27 0.02 1.97 0.03 2.19 0.03

9 2.08 0.03 2.05 0.03 2.02 0.02 2.11 0.01

10 2.00 0.01 2.03 0.01 1.93 0.01 1.99 0.01

11 2.26 0.02 2.27 0.01 1.99 0.02 1.95 0.02

12 2.04 0.01 2.05 0.01 1.98 0.02 1.96 0.01

13 - - 1.98 0.04 - - 1.80 0.04

14 1.95 0.02 1.97 0.03 1.93 0.02 2.02 0.05

15 1.96 0.01 1.93 0.01 1.94 0.02 1.97 0.02

16 2.19 0.02 2.21 0.01 2.33 0.02 2.13 0.01

17 - - - - 2.01 0.03 - -

18 2.02 0.01 2.08 0.01 - - - -

19 2.02 0.00 2.02 0.00 2.05 0.00 2.02 0.01


Table D-7: Sample G - raw mean and standard deviation value of total alkaloids (as

nicotine) in sample G determined by CRM35 and NCM (KSCN/DCIC) in water and 5%

HAC, respectively



1 2.25 0.01 2.28 0.01 2.09 0.01 2.14 0.01

2 2.08 0.02 2.15 0.02 2.03 0.01 2.08 0.01

3 2.15 0.03 2.15 0.01 2.16 0.03 2.14 0.01

4 2.28 0.08 2.18 0.03 2.09 0.01 2.12 0.02

5 2.23 0.02 2.11 0.05 1.99 0.02 2.11 0.05

6 2.39 0.03 2.20 0.02 2.16 0.02 2.12 0.02

7 2.15 0.01 2.25 0.01 2.20 0.01 2.37 0.01

8 2.27 0.04 2.44 0.01 2.14 0.01 2.34 0.01

9 2.19 0.02 2.12 0.01 2.16 0.03 2.20 0.02

10 2.14 0.01 2.16 0.01 2.06 0.01 2.11 0.01

11 2.38 0.01 2.39 0.02 2.17 0.02 2.14 0.02

12 2.20 0.03 2.24 0.02 2.09 0.02 2.10 0.02

13 - - 2.10 0.04 - - 2.08 0.03

14 2.11 0.02 2.13 0.03 2.12 0.03 2.18 0.04

15 2.12 0.02 2.08 0.02 2.12 0.03 2.13 0.02

16 2.29 0.02 2.28 0.02 2.44 0.02 2.26 0.01

17 - - - - 2.15 0.06 - -

18 2.16 0.02 2.17 0.06 - - - -

19 - - - - - - - -


Table D-8: Sample H - raw mean and standard deviation value of total alkaloids (as

nicotine) determined by CRM35 and NCM (KSCN/DCIC) in water and 5 % HAC,

respectively



1 2.20 0.02 2.21 0.02 2.05 0.01 2.11 0.01

2 2.10 0.01 2.17 0.01 2.01 0.01 2.05 0.01

3 2.07 0.03 2.04 0.01 2.07 0.01 2.08 0.01

4 2.20 0.03 2.08 0.05 2.03 0.04 2.03 0.04

5 2.15 0.02 2.06 0.04 1.94 0.04 2.03 0.05

6 2.30 0.02 2.15 0.02 2.11 0.02 2.03 0.01

7 2.11 0.01 2.23 0.01 2.16 0.02 2.27 0.01

8 2.27 0.04 2.37 0.04 2.09 0.04 2.27 0.04

9 2.05 0.02 2.04 0.01 2.06 0.02 2.10 0.02

10 2.08 0.01 2.09 0.02 2.00 0.01 2.06 0.01

11 2.36 0.03 2.28 0.02 2.09 0.03 2.04 0.03

12 2.10 0.04 2.09 0.01 2.05 0.01 2.03 0.02

13 - - 2.09 0.01 - - 2.01 0.03

14 2.01 0.02 2.01 0.04 2.04 0.01 2.07 0.08

15 2.07 0.01 2.01 0.00 2.06 0.02 2.07 0.01

16 2.20 0.02 2.20 0.01 2.33 0.02 2.17 0.04

17 - - - - 2.16 0.03 - -

18 2.10 0.01 2.14 0.01 - - - -

19 - - - - - - - -


Appendix E – Data Representation

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

0.7

0.8

0.9

1

1.1

1.2

1.3

Lab ID

To

tal A

lka

loid

s (

%)

Oriental tobacco

CRM 35 in w ater

NCM in w ater

Lab value

Lab mean

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

3.3

3.4

3.5

3.6

3.7

3.8

3.9

4

4.1

Lab ID

To

tal A

lka

loid

s (

%)

Dark sun-cured tobacco

CRM 35 in w ater

NCM in w ater

Lab value

Lab mean


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

1.8

1.9

2

2.1

2.2

2.3

2.4

Lab ID

To

tal A

lka

loid

s (

%)

Fire-cured cigarette

CRM 35 in w ater

NCM in w ater

Lab value

Lab mean

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

1.8

1.9

2

2.1

2.2

2.3

2.4

Lab ID

To

tal A

lka

loid

s (

%)

Blended cigarette

CRM 35 in w ater

NCM in w ater

Lab value

Lab mean


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

1.95

2

2.05

2.1

2.15

2.2

2.25

2.3

2.35

2.4

2.45

Lab ID

To

tal A

lka

loid

s (

%)

CM7

CRM 35 in w ater

NCM in w ater

Lab value

Lab mean

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

1.9

2

2.1

2.2

2.3

2.4

Lab ID

To

tal A

lka

loid

s (

%)

3R4F

CRM 35 in w ater

NCM in w ater

Lab value

Lab mean


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

1.5

1.6

1.7

1.8

1.9

2

2.1

2.2

2.3

Lab ID

To

tal A

lka

loid

s (

%)

Flue-cured tobacco

CRM 35 in HAC

NCM in HAC

Lab value

Lab mean

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

4

4.5

5

5.5

Lab ID

Burley tobacco

CRM35 in HAC NCM in HAC Lab value Lab mean

Tota

l A

lkalo

ids (

%)


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

0.8

0.9

1

1.1

1.2

1.3

Lab ID

To

tal A

lka

loid

s (

%)

Oriental tobacco

CRM 35 in HAC

NCM in HAC

Lab value

Lab mean

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

3.2

3.4

3.6

3.8

4

4.2

Lab ID

To

tal A

lka

loid

s (

%)

Dark sun-cured tobacco

CRM 35 in HAC

NCM in HAC

Lab value

Lab mean


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

1.8

1.9

2

2.1

2.2

2.3

2.4

Lab ID

To

tal A

lka

loid

s (

%)

Fire-cured cigarette

CRM 35 in HAC

NCM in HAC

Lab value

Lab mean

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

1.8

1.9

2

2.1

2.2

2.3

2.4

Lab ID

To

tal A

lka

loid

s (

%)

Blended cigarette

CRM 35 in HAC

NCM in HAC

Lab value

Lab mean


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

1.9

2

2.1

2.2

2.3

2.4

Lab ID

To

tal A

lka

loid

s (

%)

CM7

CRM 35 in HAC

NCM in HAC

Lab value

Lab mean

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

1.9

2

2.1

2.2

2.3

2.4

Lab ID

To

tal A

lka

loid

s (

%)

3R4F

CRM 35 in HAC

NCM in HAC

Lab value

Lab mean


Appendix F – IUPAC 1994 Harmonized Statistical Procedure

IUPAC 1994 HARMONIZED STATISTICAL PROCEDURE

SCREEN OUT NON

VALID DATA

START OF LOOP

CALCULATE PRECISION

MEASURES

(C)

COCHRAN

OUTLYING LAB?

(G)

SINGLE

GRUBBS

OUTLIER?

(G2)

PAIRED

GRUBBS

OUTLIER?

END REPORT

ORIGINAL & LAST

COMPUTED

PRECISION

MEASURES

DROP LAB UNLESS OVERALL

FRACTION OF LABS DROPPED

WOULD EXCEED 2/9

DROP INDIVIDUAL VALUE



WOULD EXCEED 2/9

ANY LABS

DROPPED IN

THIS LOOP

YES

YES

YES

YES

NO

NO

NO

NO

(Gi)SINGLE GRUBBS

OUTLIER ON INDIVIDUAL VALUES

OF COCHRAN OUTLYING

LAB?

YES



WOULD EXCEED 2/9

NO

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