Tobacco and Tobacco Products Analytes Sub-Group€¦ · determination of nicotine in 13 sample types including cigarette filler, leaf, smokeless tobacco products (STPs), cigar, and

Tobacco and Tobacco Products Analytes Sub-Group

Technical Report

2019 Collaborative Study for the

Determination of Nicotine in

Tobacco and Tobacco Products

April 2020

Study Project Leaders:

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

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

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.

Table of Contents

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

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

2.1 Objective ................................................................................................................. 4

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

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

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

3.2.1 Study Samples ............................................................................................. 5

3.2.2 Within Laboratory Sample Preparation ....................................................... 6

3.2.3 Sample Analysis and Data Reporting .......................................................... 7

3.2.4 Deviations .................................................................................................... 7

4. Data – Summary Descriptive Statistics ............................................................................ 8

5. Data – Statistical Analysis ................................................................................................ 8

5.1 Exclusion of Outliers ............................................................................................... 8

5.2 Calculation of Repeatability and Reproducibility ................................................... 9

6. Data Interpretation .......................................................................................................... 10

7. Recommendations ........................................................................................................... 16

APPENDIX A: Study Protocol ................................................................................................ 18

APPENDIX B: Raw Data ......................................................................................................... 33

APPENDIX C: Raw Data Plots ................................................................................................. 59

APPENDIX D: Standards, %RCR ............................................................................................ 65

TTPA-193-1-CTR 2019 Collaborative Study for Nicotine – April 2020 3/79

1. Summary

At the October 2018 CORESTA Tobacco and Tobacco Products Analytes Sub-Group (TTPA)

meeting held in Kunming, the Sub-Group initiated a collaborative study for the determination

of nicotine in traditional and very low nicotine (VLN) tobacco and tobacco products. The intent

of this study was to lower the calibration range for two CORESTA Recommended Methods,

CRM No. 62 (GC-FID with MTBE and Hexane extractions) and CRM No. 87 (GC-MS), for

the analysis of VLN tobaccos and tobacco products. This project is important to ensure these

two CRMs are relevant for emerging regulations to reduce nicotine for combusted tobacco

products[1]. In early 2018, the United States Food and Drug Administration (FDA) issued the

advance notice of proposed rulemaking (ANPRM) to obtain information for consideration in

developing a product standard to set the maximum nicotine level for cigarette filler[2]. The

collaborative study described herein involved 25 laboratories; 16 labs provided data for

CRM No. 62 MTBE, 11 labs provided data for CRM No. 62 Hexane, and 18 labs provided data

for CRM No. 87. The results of the study demonstrate that, with modifications, CRM No. 62

and CRM No. 87 are suitable for the analysis of traditional and VLN tobacco and tobacco

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

values. The TTPA Sub-Group recommends that the calibration ranges of the two CRMs be

lowered to include VLN tobacco and tobacco products.

2. Introduction

In 1999 to 2000, the Nicotine Subcommittee of the Tobacco Science Research Conference

Analytical Methods Committee (TSRC-AMC), the CORESTA RAC Sub-Group, and

Deutsches Institut für Normung (DIN) coordinated an international collaborative study

involving 37 laboratories to assess the repeatability and reproducibility of five methods for the

determination of nicotine in 13 sample types including cigarette filler, leaf, smokeless tobacco

products (STPs), cigar, and pipe tobacco[3]. The intent of the study was to standardize a

reference method for nicotine analysis in tobacco and tobacco products. Between 9 and 18

laboratories provided data using GC-FID based on n-Hexane extraction using capillary column

or packed-column, GC-FID based on methanol/ammonia extraction using capillary column,

continuous flow analysis (CFA), and GC-FID based on MTBE extraction using capillary

column that was a modified version of the CDC method published in the Federal Register[4].

The three capillary GC methods provided consistent results for mean nicotine and variance

characteristics. The results for GC-FID MTBE and n-Hexane extraction methods of this

collaborative study were the basis for CRM No. 62 (Determination of Nicotine in Tobacco and

Tobacco Products by Gas Chromatographic Analysis).

[1] World Health Organization (2015) Advisory Note: Global Nicotine Reduction Strategy - ISBN 978 92 4 150932 9,

WHO Study Group on Tobacco Product Regulation (TobReg),

(https://www.who.int/tobacco/publications/prod_regulation/nicotine-reduction/en/). [2] Tobacco Product Standard for Nicotine Level of Combusted Cigarettes, 83 Federal Register (March 16, 2018). [3] Franke, J.E., Bennett, C.B, Davis, R.E., Thomsen, H.V., Johnston, K.S., and Shanmugan, S.M.: Determination of

Nicotine in Tobacco: Collaborative Study; Beiträge zur. Tabakforschung International 19 (2001), 251-265. [4] Annual Submission of the Quantity of Nicotine Contained in Smokeless Tobacco Products Manufactured, Imported,

or Packaged in the United States Requirement, 64 Federal Register (March 23, 1999)

https://www.who.int/tobacco/publications/prod_regulation/nicotine-reduction/en/


In late 2015 to early 2016, the CORESTA RAC and Smokeless Tobacco Sub-Group (STS)

(now named TTPA) conducted a collaborative study involving 19 laboratories to evaluate the

repeatability and reproducibility of a GC-MS method based on methanol extraction for the

determination of nicotine in tobacco and tobacco products[5]. The intent of the study was to

develop a Recommended Method that did not require standard addition, as is the case with

CRM No. 62 (MTBE). This study found no evidence of interferences with nicotine or the

internal standard with flavour compounds present in a variety of tobacco products. The results

of this study were the basis for CRM No. 87 (Determination of Nicotine in Tobacco and

Tobacco Products by GC-MS).

In early 2018, FDA issued an ANPRM for a Tobacco Product Standard for Nicotine Level of

Combusted Cigarettes and sought public comment for consideration in developing a potential

product standard to lower nicotine in cigarettes. This ANPRM included a maximum nicotine

level of 0,3 to 0,5 mg/g in cigarette filler. This ANPRM sought comment for a potential

analytical method to determine nicotine at the maximum nicotine level in tobacco suggested in

the ANPRM. Although several CORESTA Recommended Methods and instrumentation

platforms were listed for consideration, none of the methods’ scopes cited in the ANPRM

include VLN tobacco or tobacco products at the 0,3 to 0,5 mg/g (VLN) concentration.

2.1 Objective

In late 2018 to early 2019, the CORESTA TTPA Sub-Group conducted a collaborative study

to support scope expansion of CRM No. 62 and CRM No. 87 to include the determination of

nicotine in VLN tobacco and tobacco products and provide an assessment of inter-laboratory

capability. In addition to products with very low nicotine content, conventional products were

included to verify that changes in the method required for VLN samples did not negatively

affect the ability to analyze samples with conventional nicotine contents. The laboratories were

to provide analytical results for nicotine in a variety of test samples on an as-is basis. This work

was conducted using the applicable CRMs with lower calibration ranges referenced in the

protocol, which is provided in Appendix A. The protocol for this study was distributed in

December 2018 and the study was conducted in January 2019 through June 2019. 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

3. Organization

3.1 Participants

Twenty-five laboratories participated in the study; 18 provided data based on CRM No. 87, 16

based on CRM No. 62 MTBE and 11 based on n-Hexane. 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 shown in the table

below.

[5] CORESTA Routine Analytical Chemistry and Smokeless Tobacco Sub-Groups Technical Report: 2016 Collaborative

Study on Nicotine in Tobacco Products, February 2017 [RAC-STS-056-1-CTR]


Table 1: List of Participating Laboratories

Participants

Altria Client Services LLC, United States

American Snuff Company, United States

British American Tobacco, Brazil

British American Tobacco, Germany

Enthalpy Analytical Durham, United States

Enthalpy Analytical Richmond, United States

Essentra, United Kingdom

Global Laboratory Services, Inc., United States

Imperial Tobacco Laboratory, The Netherlands

ITC Life Sciences & Technology Centre, India

ITG Brands LLC, United States

Japan Tobacco Inc., Japan

KT&G, South Korea

Labstat International ULC, Canada

Liggett Group, United States

RJ Reynolds Tobacco Company, United States

Schweitzer-Mauduit International, France

Swedish Match, Northern Europe, Sweden

Swedish Match, Owensboro, United States

Swisher International, United States

University of Kentucky, United States

Philip Morris International, Brazil

Philip Morris International Sampoerna, Indonesia

C.I.T. Montepaz S.A., Uruguay

Shanghai New Tobacco Product Research Institute Co. Ltd., China

3.2 Protocol

The Protocol is provided in Appendix A and specific details from the protocol are described

below.

3.2.1 Study Samples

Laboratories were responsible for procuring NIST 3222 from the National Institute of Standards

and Technology, VLNCMST from the American Snuff Co., VLNCF1 and VLNCF2 from Altria

Client Services, 2016 CRPs from North Carolina State University (NCSU), and RT6, RT8,

3R4F, RT1 from the University of Kentucky. Laboratories were requested to store the samples

at approximately 4 °C upon receipt if the analyses would be conducted within one week or to

store the samples at approximately -20 °C if the analyses would be delayed. The study was to

be conducted from December 2018 through March 2019. Laboratories were requested to

submit data by March 1, 2019. The final data, including re-checks, were received by June 2019.

The samples are identified in Table 2.


Table 2: Sample Identification

Sample Name Description

NIST SRM 3222

VLNC Cigarette Tobacco Filler

VLNCMST VLNC American-style loose moist snuff – experimental prototype produced in

limited quantity

VLNCF1 VLNC American blended cigarette filler - experimental prototype produced in

limited quantities

VLNCF2 VLNC American blended cigarette filler - experimental prototype produced in

limited quantities

CRP1.1 Swedish-style Snus pouch

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

CRP2.1 American-style loose moist snuff

RT6 Cigar filler, flavored, ground

RT8 Cigar filler, unflavored, ground

CRP3.1 American-style dry snuff powder

3R4F 3R4F Reference Cigarette

RT1 1R6F Filler, American blended cigarette filler, ground

3.2.2 Within Laboratory Sample Preparation

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

unopened samples in a refrigerator at approximately 4 °C for a minimum of 24 hours to ensure

water was fully equilibrated. Next, they were directed to remove the unopened samples from

the refrigerator to equilibrate to ambient conditions for a minimum of 1 hour prior to opening

for analysis. Once samples were opened, the samples could be stored in a tightly sealed

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

which differ from those specified in the CRMs, are described below:

• NIST SRM 3222: The filler shall be ground before analysis. Grind the contents of one

jar that contains 10 g of cigarette tobacco filler and then remove the three replicates from

the ground material. Laboratories should use their standard grinding procedure but keep

grinding to the minimum necessary to produce a homogenous sample.

• 3R4F Cigarette: The filler shall be ground before analysis. Grind the filler from 40

cigarettes (2 packs) after removal of the paper and filter materials. Laboratories should

use their standard grinding procedure but keep grinding to the minimum necessary to

produce a homogenous sample.

• CRP1.1: The snus pouches shall be cut into 2 halves and added directly to the extraction

vessel (include both the tobacco and paper). Although CRM No. 87 calls for 0,25 g of

sample, an entire ~1 g CRP1.1 snus pouch shall be analyzed.

• The VLNCMST, VLNCF1, VLNCF2, CRP2.1, CRP3.1, CRP4.1, RT6, RT8, and RT1

did not require sample grinding and were to be analysed as received.


3.2.3 Sample Analysis and Data Reporting

The participating laboratories were requested to conduct three (3) replicate analyses from

independent tobacco extractions for each sample. Data were reported in units of mg/g, on an

as-is basis using the current versions of the CRMs with the method modifications stated in the

protocol and summarized below.

• CRM No. 62, Determination of Nicotine in Tobacco and Tobacco Products using Gas

Chromatographic Analysis, February 2015

o The calibration range for MTBE and n-Hexane extractions were lowered from 5,0

and 4,8 mg/g to 0,025 and 0,024 mg/g, respectively. These modifications lowered

the calibration range by a factor of 200. Participants were instructed to prepare a

series of at least five nicotine standard solutions and use a linear regression model

with 1/x weighting, x being concentration. The protocol stated a criterion for the

linear regression equation: R2 > 0,990 and nicotine standard level 1 < 15 % relative

error and for the remaining nicotine standard levels <10 % relative error. These

criteria were consistent with those presented in CRM No. 62.

o Additional modifications included changing CRM No. 62 gas chromatography

injection mode from split to split-less to increase sensitivity.

• CRM No. 87, Determination of Nicotine in Tobacco and Tobacco Products using GC-MS,

April 2018

o The calibration range was lowered, by a factor of 10, from 0,64 mg/g to 0,064 mg/g.

3.2.4 Deviations

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 would be excluded from the study.

Deviations reported by the laboratories are identified below. Five labs reported deviations.

However, only Lab 16 provided results identified as a major deviation, specifically for not

following the method principle for CRM No. 62 n-Hexane.

− Lab 9: Minor deviation was reported for CRM No. 87 where a quadratic regression was

used instead of a linear regression as specified in the CRM. The data were included.

− Lab 11: Minor deviation was reported for CRM No. 87 where a quadratic regression

was used instead of a linear regression as specified in the CRM. The data were

included.

− Lab 3: Minor deviation was reported for CRM No. 87 where a GC/MS/MS (triple quad)

was used instead of the GC/MS specified in the CRM. The data were included.

− Lab 8: Minor deviation was reported for CRM No. 62 MTBE where a hyphenated

GC-FID/MS was used instead of the GC-FID specified in the CRM. The data from the

FID were included.

− Lab 16: Major deviation was reported for CRM No. 62 n-Hexane where the laboratory

did not add base to the sample and used methanol as extraction solvent. The data were

not included.


4. Data – Summary Descriptive Statistics

The raw data set, without removal of outliers, is provided in Appendix B. The results are

presented on an as-is basis, without correction for moisture. 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, provided in Table 3.

5. Data – Statistical Analysis

The statistical analysis was conducted in basic conformance with ISO 5725-2:1994 and ISO/TR

22971:2005. 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 C.

5.1 Exclusion of Outliers

An adaptation of Levene’s Test[6] was used for eliminating laboratories with overly large

repeatability standard deviations and Grubbs’ Test was used to eliminate laboratories with

outlying mean values. In general, Levene’s Test tends to identify fewer outliers than Cochran’s

Test and in some instances may result in a larger estimated r value than if Cochran’s Test was

used. There is not a consistent effect on the estimated R value.

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.

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. Thus,

Mandel’s h and k statistics do not add fundamentally new information and as typically employed

may lead to incorrect conclusions. For those reasons, we do not include Mandel’s h and k plots.

The initial examination for outliers indicated that Lab 8 tended to give outlying results for

MTBE. For that reason, Lab 8 MTBE results were dropped from the analysis and the data were

again examined for outliers with Lab 8 MTBE results excluded. For NIST SRM 3222, Lab 20

rep 1 using GC/MS was a single-point outlier and that point was eliminated.

Table 3: Outliers

Sample Levene’s Outliers Lab Grubbs’ Outliers Lab

NIST 3222 – Lab 11, MTBE

VLNCF1 – Lab 11, MTBE

CRP 2.1 Lab 17, MTBE –

CRP 3.1 Lab 11, MTBE –

RT8 Lab 11, MTBE –

Additional Outliers: Lab 8 dropped for all MTBE results. Lab 20 for GC/MS, rep 1 was dropped as a single point outlier.

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

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

Alternative to Cochran’s Test” in Beiträge zur Tabakforschung International, Vol 27 No. 7, pp135-144.


5.2 Calculation of Repeatability and Reproducibility

After removal of outlying data based on numerical data consistency methods discussed above

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

were calculated and are shown in Table 4. The r & R results reflect both laboratory variability

and product consistency.

Table 4: Repeatability (r) and Reproducibility (R) Limits for Nicotine as-is CRM No. 62 MTBE

Product No. of Labs * Mean

(mg/g)

Repeatability Reproducibility

r r (%) R R (%)

NIST SRM 3222 14 0,152 0,029 18,8 % 0,057 37,7 %

VLNCF1 13 0,391 0,034 8,8 % 0,083 21,1 %

VLNCMST 14 0,617 0,041 6,7 % 0,149 24,1 %

VLNCF2 14 0,926 0,047 5,1 % 0,225 24,3 %

CRP1.1 16 7,44 0,616 8,3 % 1,719 23,1 %

CRP4.1 16 8,72 0,446 5,1 % 1,361 15,6 %

CRP2.1 15 10,47 0,599 5,7 % 1,608 15,4 %

RT6 14 10,91 0,366 3,4 % 1,125 10,3 %

RT8 13 13,84 0,644 4,7 % 1,573 11,4 %

CRP3.1 15 16,75 0,508 3.00 % 2,162 12,9 %

3R4F 15 17,92 0,666 3,7 % 2,607 14,5 %

RT1 14 18,04 0,489 2,7 % 2,648 14,7 %

* The number of laboratory data sets after removal of outliers.

Table 5: Repeatability (r) and Reproducibility (R) Limits for Nicotine as-is CRM No. 62 Hexane

Product No. of Labs * Mean

(mg/g)


r r (%) R R (%)

NIST SRM 3222 8 0,156 0,03 19,4 % 0,075 47,8 %

VLNCF1 10 0,396 0,036 9.0 % 0,083 21.0 %

VLNCMST 10 0,593 0,043 7,3 % 0,109 18,4 %

VLNCF2 10 0,906 0,061 6,7 % 0,141 15,5 %

CRP1.1 11 6,97 1,240 17,8 % 2,306 33,1 %

CRP4.1 11 8,71 0,212 2,4 % 1,289 14,8 %

CRP2.1 11 10,09 0,361 3,6 % 2,684 26,6 %

RT6 9 10,84 0,371 3,4 % 2,041 18,8 %

RT8 9 13,52 0,222 1,6 % 1,558 11,5 %

CRP3.1 11 16,41 1,632 9,9 % 2,835 17,3 %

3R4F 10 17,37 1,890 10,9 % 2,826 16,3 %

RT1 9 17,70 0,607 3,4 % 1,840 10,4 %



Table 6: Repeatability (r) and Reproducibility (R) Limits for Nicotine as-is CRM No. 87

Product No. of Labs * Mean (mg/g)


r r (%) R R (%)

NIST SRM 3222 15 0,207 0,054 25,9 % 0,114 54,9 %

VLNCF1 17 0,399 0,035 8,7 % 0,112 28.0 %

VLNCMST 17 0,671 0,076 11,3 % 0,171 25,5 %

VLNCF2 17 0,928 0,054 5,8 % 0,241 26.0 %

CRP1.1 18 7,48 0,891 11,9 % 1,488 19,9 %

CRP4.1 18 9,13 0,563 6,2 % 1,912 20,9 %

CRP2.1 18 10,80 0,771 7,1 % 2,402 22,2 %

RT6 16 11,24 1,419 12,6 % 1,981 17,6 %

RT8 16 14,64 0,975 6,7 % 2,324 15,9 %

CRP3.1 18 17,22 1,262 7,3 % 2,927 17.0 %

3R4F 18 18,16 0,88 4,8 % 3,593 19,8 %

RT1 16 18,71 0,722 3,9 % 2,631 14,1 %


6. Data Interpretation

For each of the products tested, the GC/MS (CRM No. 87) method average was greater than

the MTBE (CRM No. 62) and the Hexane (CRM No. 62) methods. Figure 1 shows a graph of

each method divided by the average of the three methods. The reason for normalizing by the

average is to allow all of the samples to be included in a single graph. In each instance, the

GC/MS method is greater than both the MTBE and Hexane methods. The difference is

somewhat exaggerated for the NIST SRM 3222 material because of the extremely low levels.

In Table 7 the average relative differences between the three methods are shown, both including

and excluding NIST SRM 3222, along with the p-values of a paired t-test statistically

comparing them to one another. From Table 7 all three methods are statistically significantly

different[7] from one another with the results trending in the following order: GC/MS > MTBE

> Hexane. The difference is the average percent difference between the two methods and the

p-value is based on a two-sided paired t-test comparing the two methods. Nominally a

p-value<0,05 is considered statistically significant.

As can be seen in Table 7, the estimated difference between the MTBE method and the Hexane

method, though statistically significant, is small – around 2 %. Interestingly in the published

report on which CRM No. 62 was based1, the opposite conclusion was reached. Namely there

was a small but statistically significant difference with the Hexane method giving higher results

than the MTBE method. Both studies estimated small differences and few single studies are

ever definitive. A reasonable conclusion would be that there is little if any difference in mean

between the MTBE and Hexane methods.

[7] If one adjusts for multiple comparisons the picture is less clear-cut. In that case, including NIST SRM 3222, only the

Hexane vs. GC/MS comparison is significant. If NIST SRM 3222 is excluded, all of the comparisons are still statistically

significant.


Table 7: Statistical comparison of the three methods.

Comparison Difference p-value

Including all samples shown in Tables 4-6

MTBE vs. Hexane +1,9 % 0,021

MTBE vs. GC/MS –5,5 % 0,040

Hexane vs. GC/MS –7,4 % 0,004

Excluding NIST SRM 3222

MTBE vs. Hexane +2,3 % 0,005

MTBE vs. GC/MS –3,2 % 0,001

Hexane vs. GC/MS –5,5 % 0,0001

The GC/MS method has previously been seen to result in higher values than the MTBE or

Hexane methods[8], possibly because the CRM No. 87 method principle provides a single-phase

solution where the CRM No. 62 method principles provide a biphasic solution with nicotine

partitioning into the organic phase. An alternate hypothesis is the methanol solvent gives

somewhat more complete extraction than either Hexane or MTBE.

Figure 1. Each Method Compared to the Average of the Three Methods.

[8] CORESTA Routine Analytical Chemistry and Smokeless Tobacco Sub-Groups Technical Report: 2016 Collaborative

Study on Nicotine in Tobacco Products, February 2017 [RAC-STS-056-1-CTR]

• MTBE

■ Hexane

▲GC/MS


Graphs of the R (%) values[9] for the three methods are compared to their respective values in

the current CRM in Figures 2 - 4. Generally, the r and R values for the MTBE and Hexane

methods are somewhat larger than the values published in CRM No. 62 based on a 1999

collaborative study and the GC/MS values appear comparable to the 2016 results. However,

there may be other factors besides the extension of the method calibration range that explain

the apparent differences in precision for the MTBE and Hexane methods.

1. In the 1999 study, several of the samples were ground and homogenized prior to

shipment in the study on which CRM No. 62 was based. That approach has definite

advantages because it reduces the influence of sample variability but is not the common

procedure for collaborative studies conducted within CORESTA. It would increase the

homogeneity of products and would be expected to reduce lab-to-lab variability (hence

reproducibility) and to a lesser extent within lab variability (repeatability). This makes

a direct comparison of the calculated precision values potentially misleading.

2. In the 1999 study, the Hexane methods all used a single nicotine salicylate standard that

was shipped with the samples. This would remove a potential source of variability and

reduce lab-to-lab variation.

3. For the MTBE portion of the 1999 study, a large number of outliers were eliminated

(16,8 % of the data). This is within IUPAC guidelines, but is more than is common and,

in addition to the factors already mentioned, could have contributed to that study having

smaller r & R values than observed in the current study. In the MTBE portion of this

study 8,9 % of the data were eliminated as outliers.

[9] The values in Tables 1 and 2 of CRM No. 62 are on a dry weight basis. The values were converted to as-is using the

average of the 15 g and 50 g moisture (OV) values given in Franke et al. (2001) Table 1.


The r (%) and R (%) values in Tables 4-6 were compared to one another using paired t-tests.

The only statistically significant differences were that the MTBE method had lower r and R

values overall than the GC/MS method.

The calibration range for CRM No. 62 was lowered by a factor of 200 and the calibration range

in CRM No. 87 was lowered by a factor of 10 to bracket the VLN samples. Two additional

modifications to CRM No. 62 were suggested in the protocol: 1) The gas chromatography

injection mode was changed from split to split-less to allow for an increase in detector response

i.e. method sensitivity and 2) the linear regression model was changed from equal weighting to

1/x weighting, x being concentration. CRM No.87 did not require any additional modifications.

To assess the modifications to the calibration range, participants were asked to provide the

nicotine concentrations per calibration standard levels, coefficient of linear regression, %RCR

(i.e. percent error) for each level and signal to noise ratios (S/N) for the lowest standard levels.


The laboratories were requested to use 1/x weighting to reduce the error at the low end of the

calibration range and this was used by most laboratories. Most laboratories reported correlation

coefficients R2 > 0.995 and reported < 20 % RCR from the expected value for calibration level 1

and ≤ 10 % RCR for the remaining calibration levels. These values are provided in Appendix D.

Six participants were not able to demonstrate this level of accuracy for calibration standards 1

and 2, which are below the nicotine content of the NIST 3222 sample. There were two labs

that observed >10 %RCR values for midlevel standards. Considering the fact that most labs

demonstrated acceptable %RCR (i.e. < 20 % RCR from the expected value for calibration level

1 and ≤ 10 % RCR for the remaining calibration levels), there is good reason to believe that all

labs could do the same with more experience. Two labs used quadratic regression and

demonstrated < 10 % RCR for all calibration levels.

A comparison of the Predicted R (%) values derived from the Horowitz equation[10] are

compared to the three methods R% values in Tables 8-10. A ratio [R (%) study / R (%)

predicted from the Horowitz equation] is provided to determine if each method is fit for purpose

to determine nicotine content in the study samples.

Table 8. Comparison of predicted R% values to study R% for CRM No. 62 MTBE

Material R (%) Predicted from

Horowitz equation R (%) from

Study Ratio (R (%) Study/ R

(%) Predicted)

NIST SRM 3222 21,0 % 37,7 % 1,8

VLNCF1 18,2 % 21,1 % 1,2

VLNCMST 17,0 % 24,1 % 1,4

VLNCF2 16,0 % 24,3 % 1,5

CRP1.1 11,7 % 23,1 % 2,0

CRP4.1 11,4 % 15,6 % 1,4

CRP2.1 11,1 % 15,4 % 1,4

RT6 11,1 % 10,3 % 0,9

RT8 10,7 % 11,4 % 1,1

CRP3.1 10,4 % 12,9 % 1,2

3R4F 10,3 % 14,5 % 1,4

RT1 10,2 % 14,7 % 1,4

[10] Horwitz equation translated to provide R% = 5.6*C^(-0.1505). C is sample concentration g/g basis.


Table 9. Comparison of predicted R% values to study R% for CRM No. 62Hexane




(%) Predicted)

NIST SRM 3222 20,9 % 47,8 % 2,3

VLNCF1 18,2 % 21,0 % 1,2

VLNCMST 17,1 % 18,4 % 1,1

VLNCF2 16,1 % 15,5 % 1,0

CRP1.1 11,8 % 33,1 % 2,8

CRP4.1 11,4 % 14,8 % 1,3

CRP2.1 11,2 % 26,6 % 2,4

RT6 11,1 % 18,8 % 1,7

RT8 10,7 % 11,5 % 1,1

CRP3.1 10,4 % 17,3 % 1,7

3R4F 10,3 % 16,3 % 1,6

RT1 10,3 % 10,4 % 1,0

Table 10. Comparison of predicted R% values to study R% for CRM No. 87




(%) Predicted)

NIST SRM 3222 20,1 % 54,9 % 2,7

VLNCF1 18,2 % 28,0 % 1,5

VLNCMST 16,8 % 25,5 % 1,5

VLNCF2 16,0 % 26,0 % 1,6

CRP1.1 11,7 % 19,9 % 1,7

CRP4.1 11,4 % 20,9 % 1,8

CRP2.1 11,1 % 22,2 % 2,0

RT6 11,0 % 17,6 % 1,6

RT8 10,6 % 15,9 % 1,5

CRP3.1 10,3 % 17,0 % 1,6

3R4F 10,2 % 19,8 % 1,9

RT1 10,2 % 14,1 % 1,4


Generally, for each method the ratio of [R (%) / R (%) predicted from Horowitz equation] is

consistent across methods and within method over a broad range of nicotine content in study

samples, varying from 0,4 to 18,9 mg/g. The average ratio for the three methods ranged from

1,4 to 1,8 (CRM No. 87 > Hexane > MTBE) and indicates the methods are fit for the

determination of nicotine in the study sample matrices[11]. The Horowitz equation is at best just

a rough empirical guide and we took as a rule of thumb that a ratio of less than two to be

acceptable. However, there were samples such as NIST3222 for CRM No. 62 Hexane and

CRM No. 87 where the ratios were higher than was predicted. There were other specific

samples such as CRP1.1 for CRM No. 62 MTBE and Hexane that the ratio was higher than its

nicotine content would suggest. A possible explanation could be CRP1.1 nicotine content is

more variable relative to reference tobacco products with similar nicotine content. This is

plausible considering CRP1.1 is a portioned product.

The NIST3222 sample was the lowest nicotine content sample in the study and is the only

sample in the study with a certified value. The NIST3222 is a standard reference material

(SRM) from the National Institute of Standards and Technology (NIST) with an assigned value

of 0,117 mg /g ± 0,018 mg/g expanded uncertainty at approximately 95 %[12]. In this study

every reported value for each of the three methods from each of the laboratories was above the

assigned value and 101 out of the 117 reported values were above the upper confidence limit

for the assigned value (0,135 mg/g).

NIST used data based on five different analytical methodologies to assign the value for

NIST3222[13]. Four of the methodologies used isotopic dilution liquid chromatography with

tandem mass spectrometry (ID-LC-MS/MS) and one data set, from the Tobacco Products

Laboratory for the Centers for Disease Control and Prevention (CDC), used a sample

preparation procedure similar to CRM No. 62. The CDC reported a mean nicotine content

0,137 mg/g that is within the range of values reported by laboratories employing CRM No. 62

in this study. So, the difference between the values reported here in comparison to the NIST

assigned value may be, at least partially, a difference in analytical methodology.

7. Recommendations

In 2019, TTPA and RAC conducted an inter-laboratory study for the determination of nicotine

in traditional tobacco and tobacco products and VLN moist smokeless tobacco and VLN cut

and ground cigarette filler. The CRMs listed below were originally developed for the analysis

of traditional nicotine content tobacco and tobacco products.

− CRM No. 62, Determination of nicotine in tobacco and tobacco products by gas

chromatographic analysis

− CRM No. 87, Determination of nicotine in tobacco products by GC-MS

[11] Boyer, K.W., W. Horwitz, and R. Albert, Interlaboratory variability in trace element analysis, Anal. Chem. 57 (1985)

454-459. [12] Certificate of Analysis Standard Reference Material 3222 Cigarette Tobacco Filler, National Institute of Standards

& Technology, United States of America Department of Commerce [13] Sander L.C., Pritchett J.S, Daniels Y.C., Wood L.J., Lang B.E., Wise S.A., et al., Development of a Cigarette Tobacco

Filler Standard Reference Material, Anal. Chem., 2017 Vol. 89, 10461-10467.


In this study the two CRMs’ calibration ranges were expanded to accommodate VLN tobaccos,

anticipated to go as low as 0,117 mg/g or less in the study. Participants bracketed sample

nicotine concentrations in test portions using nicotine standards to generate a linear regression

model with 1/x weighting for acceptable accuracy at the VLN concentration. The study results

show CRM No. 87 produces, on average, higher results than CRM No. 62 Hexane and MTBE.

This difference was proportionally larger on the NIST 3222 material. It is not known which

method provides nicotine results closest to the true value.

Generally, the three methods produced similar reproducibility statistics and compared

reasonably to previous studies as well to predicted R (%) values derived from the Horowitz

equation. It does not appear that the extension of the calibration range negatively impacts the

analysis of conventional samples. These observations support the TTPA’s recommendation to

update the calibration ranges of CRM No. 62 and CRM No. 87 to include VLN tobacco and

tobacco products.

Acknowledgments

The authors appreciate the support of Dr. Huihua Ji and Ms. Erin Pyrek at the Kentucky

Tobacco Research & Development Center (KTRDC) for distributing tobacco samples to

participants in the study. Additional gratitude to the 25 participants that provided data,

specifically to Dr. John Bunch at American Snuff Co.


APPENDIX A: Study Protocol

CORESTA TOBACCO and TOBACCO PRODUCTS

ANALYTES SUB-GROUP

Project Title: Collaborative study for the Determination of Nicotine in Tobacco

and Tobacco Products Using CRM No. 62 and CRM No. 87

Type of Document: Collaborative Study Protocol

Date: November 29, 2018

Written by: Anthony Brown and Regina Ballentine - Study Coordinators

Confidentiality Notice: All data submitted by participating laboratories will be coded and

kept confidential.


1. Introduction

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

on April 17th, 2018 in Guildford, the group proposed to expand the scopes of CORESTA

Recommended Methods No. 62 and 87 to include very low nicotine content (VLNC) tobaccos

and tobacco products.

2. Objective

The objective of this collaborative study is to expand the scope of the following CORESTA

Recommended Methods (CRM) beyond traditional tobacco and tobacco products to include

VLNC tobacco products. This will necessitate modifications to the CRMs.

• Nicotine: CRM No. 62, Determination of nicotine in tobacco and tobacco products by

gas chromatographic analysis

• Nicotine: CRM No. 87, Determination of nicotine in tobacco products by GC-MS

The final output will be a presentation of the results at the spring 2019 TTPA meeting, one

technical report, including repeatability (r) and reproducibility (R) values and revisions to CRM

62 and 87.

Note: Since the purpose of this study is to expand the scopes of the CRMs and calculate

new r&R values, it is imperative that participants follow the specified CRMs and follow

the CRM modifications detailed in this protocol.

3. Time schedule

Table 1: Study timeline

Date Activity

December 2018 Participants order samples

December-February Laboratories conduct the study

March 1, 2019 Laboratories submit results by this date

April 10, 2019 Discuss results at spring TTPA SG meeting in Richmond, Virginia

4. Participating Laboratories:

The laboratories listed in Table 2 have kindly agreed to take part in the study. Other

laboratories are encouraged to participate and should notify Anthony Brown

aaaaaaaaaaaaaaaaaaaaanat and Karl Wagner kkkkkkkkkkkkkkkkk of their interest to

participate.


Table 2: Participating Laboratories

Participating Laboratories CRM 62

MTBE

CRM 62

Hexane CRM 87

Altria Client Services LLC, United States X X X

American Snuff Company, United States X X

British American Tobacco, Brazil X ?

British American Tobacco, Germany X

British American Tobacco, Sweden X

CNTC Zhengzhou Tobacco Research Institute, China X X

Enthalpy Analytical Durham, United States X X

Enthalpy Analytical Richmond, United States X X

Essentra, United Kingdom X X

Global Laboratory Services, Inc., United States X X

Imperial Tobacco, Germany X X X

ITG Brands LLC, United States X X X

Japan Tobacco Inc., Japan X X

KT&G, South Korea X X

Labstat International ULC, Canada X X X

Liggett Group, United States X X X

RJ Reynolds tobacco Company, United States X X X

Schweitzer-Mauduit International, France X

Swedish Match, Northern Europe, Sweden X X

Swisher International, United States X X

University of Kentucky, United States X X

Inciensa, Costa Rica X

Philip Morris International, Brazil X

Philip Morris International Sampoerna, Indonesia X

C.I.T. Momtepaz S..A., Uruguay X X

China National Tobacco Quality Supervision and Test Center, China

X X X

Shanghai New Tobacco Product Research Institute Co. Ltd., China

X X X

Qingzhou Tobacco Institute of CNTC, China X X


5. Samples

Order the samples from the suppliers listed in Table 3. The approximate nicotine concentration

is provided for each sample type in order to show the range for the various samples. Ordering

information is shown below:

NIST SRM 3222: Ordering Information can be found at https://www-

s.nist.gov/srmors/view_detail.cfm?srm=3222

• 2016 CRPs: The CRPs are distributed by North Carolina State University (NCSU). It

is critical that the CRPs have been stored at the recommended temperature of -20 °C

prior to analysis. Ordering Information can be found at https://strp.wordpress.ncsu.edu/

• RT6, RT8, 3R4F, RT1: Ordering Information can be found at the University of

Kentucky website for reference products. https://ctrp.uky.edu/home

• VLNCF1 and VLNCF2 samples: These samples will also be distributed by the

University of Kentucky. You must indicate in your University of Kentucky order

for RT6, RT8, 3R4F, and RT1 that the order is for the CORESTA nicotine Study

using CRM No. 62 and CRM No. 87 so that the VLNCF1 and VLNCF2 samples

will be included.

• VLNCMST sample: Request sample from John Bunch by providing your shipping

information to John at bbbbbbbbbbbbbbbb.

Note: Upon receipt samples should be stored in the refrigerator at approximately 4 °C if

they will be analyzed within 2 weeks. Samples held longer than 2 weeks prior to analysis

should be stored in the freezer at approximately −20 °C. All remaining samples should

be retained in sealed containers at −20 °C as they may be used for future collaborative

studies for other analytes.

Table 3: Samples

Approximate Nicotine

(µg/g)

Sample Name

Description Provider Quantity

to Request

150 NIST SRM 3222

VLNC Cigarette Tobacco Filler NIST 1pack

(20x10g)

400 VLNCMST VLNC American-style loose moist snuff – experimental prototype produced in limited quantity

American Snuff Co. 2 cans

400 VLNCF1

VLNC American blended cigarette filler - experimental prototype produced in limited quantities

University of Kentucky 1 bottle

1000 VLNCF2

VLNC American blended cigarette filler - experimental prototype produced in limited quantities


8000 CRP1.1 Swedish-style Snus NCSU 2 cans

9000 CRP4.1 American-style chopped loose-leaf chewing tobacco

NCSU 2 cans

11000 CRP2.1 American-style loose moist snuff NCSU 2 cans

11000 RT6 Cigar filler, flavored, ground University of Kentucky 1 bottle

https://www-s.nist.gov/srmors/view_detail.cfm?srm=3222

https://www-s.nist.gov/srmors/view_detail.cfm?srm=3222

https://strp.wordpress.ncsu.edu/

https://ctrp.uky.edu/home


Approximate Nicotine

(µg/g)

Sample Name

Description Provider Quantity

to Request

13000 RT8 Cigar filler, unflavored, ground University of Kentucky 1 bottle

17000 CRP3.1 American-style dry snuff powder NCSU 1 can

18000 3R4F 3R4F Reference Cigarette University of Kentucky 1 Carton

19000 RT1 1R6F Filler, American blended cigarette filler, ground


Note: The RT1 ground filler must be ordered from the University of Kentucky. Do not use filler

that has been removed from 1R6F cigarettes.

6. Analysis

Note: Since the purpose of this study is to expand the scopes of the CRMs and calculate new

r&R values, it is imperative that participants follow the CRMs specified below and follow the

modifications detailed in this protocol appendix 1-3:

6.1 Methods:

The following CRMs will be used for the determination of nicotine:

• CRM No. 62, Determination of nicotine in tobacco and tobacco products by gas

chromatographic analysis

o Using MTBE as a solvent, see appendix 1 for required modifications

o Using Hexane as a solvent, see appendix 2 for required modifications

• CRM No. 87, Determination of nicotine in tobacco products by GC-MS

o See appendix 3 for required modifications

6.2 Replicates: Conduct three (3) independent replicate analyses for each sample. The

replicates should be determined from independent tobacco extractions.

6.3 Sample equilibration: The samples shall be transferred from the freezer to the

refrigerator a minimum of 24 hours before analysis. Transfer the samples from the

refrigerator to room temperature at least 2 hours prior analysis. This procedure will ensure

there is enough time for water to fully re-equilibrate within the product.

Note: Insufficient equilibration time has been identified as a source of variability.

6.4 Sample preparation:

6.4.1 NIST SRM 3222: The filler shall be ground before analysis. Grind the contents

of one jar that contains 10 g of cigarette tobacco filler and then remove the three

replicates from the ground material. Laboratories should use their standard

grinding procedure, but keep grinding to the minimum necessary to produce a

homogenous sample.

6.4.2 3R4F Cigarette: The filler shall be ground before analysis. Grind the filler from

40 cigarettes (2 packs) after removal of the paper and filter materials.

Laboratories should use their standard grinding procedure, but keep grinding to

the minimum necessary to produce a homogenous sample.

6.4.3 CRP1.1: The snus pouches shall be cut into 2 halves and added directly to the

extraction vessel (include both the tobacco and paper). Although CRM 87 calls


for 0.25 g of sample, an entire ~1 g CRP1.1 snus pouch shall be analyzed. The

1 g CRP1.1 snus pouch will be within the calibration range.

6.4.4 No grinding or additional sample preparation shall be used for the other study

samples.

6.5 Sample Analysis:

The sample run order specified in the data reporting sheet must be followed. This is to

limit the chance of carryover due to the extremely wide range of nicotine in the test

samples.

6.6 Data Reporting:

Email the completed data reporting sheet to Anthony Brown

aaaaaaaaaaaaaaaaaaaaaaaa Karl Wagner kkkkkkkkkkkkkkkkkkkkk, and Johan

Lindholm jjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjj

Additionally:

• Data shall be reported in mg/g and be reported to three decimal places.

• If data are below a quantitation limit, report the estimated analytical result and note

that the analytical result is an estimate beside the cell.

• All test results shall be reported as-is (with no correction for moisture content).

• For CRM No. 62 and CRM No. 87: Submit standard curves and chromatograms for

Cal 1 through Cal 3 along with the other information listed in the reporting

spreadsheet of the “2018 Nicotine Data Reporting Worksheet”.

7. Statistical Analysis

A statistical analysis in general conformance with ISO 5725-2:1994 and ISO/TR 22971:2005

will be conducted. Repeatability and reproducibility values will be calculated and results from

the three CRMs will be compared.

8. Presentation of the Results

The results will be a presented for discussion at the spring 2019 TTPA meeting.


Appendix 1: Modifications required for CRM No 62 when using MTBE as

the solvent

6. STANDARDS

Prepare a series of at least five nicotine standard solutions whose concentrations cover the range

(Calibration Levels 1 through 9) expected to be found in the test portion, as in the example

given below. Prepare the calibration level 0, i.e. solvent blank with internal standard, and use

as a quality control sample to gauge possible carry over.

Table 1. Summary of Modified Calibration Standards

Calibration Level

Volume of Nicotine Stock (ml)

Nicotine (mg/ml)a Quinoline (ISTD)

(mg/ml)b

0 0,0 0,000 0,4

1 0,0025 0,0005 0,4

2 0,005 0,001 0,4

3 0,025 0,005 0,4

4 0,25 0,05 0,4

5 0,5 0,1 0,4

6 1,0 0,2 0,4

7 2,0 0,4 0,4

8 3,0 0,6 0,4

9 4,0 0,8 0,4

1. Calibration levels 0-4 are new levels not shown in the published CRM

2. a Based on 10,00 mg/ml Nicotine Stock Solution concentration and volume of Nicotine Stock

diluted to a final volume of 50 ml using extraction solution containing quinoline (0,400 mg/ml).

3. b Based on 0,400 mg/ml extraction solution concentration containing quinoline.

7. PROCEDURES

7.4 Gas Chromatography

Injection Mode: Use a splitless injection; use an appropriate sized splitless liner i.e.

Glass 4,0 mm I.D. deactivated liner with glass wool.

Column recommended: Use a typical column with the following dimensions: 30 m x

0.25 mm ID x 0.25 µm df to improve method sensitivity.

Prime the GC system: To be performed after maintenance of the GC injection port.

Use a sample extract, inject ~10 aliquots at an elevated GC oven temperature. This

shall be performed before calibration of the gas chromatograph.

7.5 Calibration of the gas chromatograph

Use a linear regression model with 1/x weighting: Calculate the ratio of the nicotine

peak to the internal standard peak (Y = Anicotine/AIS) from the peak area (or height) data

for each of the nicotine standards including the calibration level 1 to be used as a solvent

blank. Only plot the graph of the concentrations of added nicotine (X axis) in accordance

with the area ratios (Y axis). Calculate a linear regression equation (Y = a + bx) using 1/x

weighting, x being concentration, from this data. Use both the slope (b) and the intercept

(a) of the linear regression.


If the correlation coefficient R2 is less than 0,990, then the calibration should be repeated.

If the calibration level 1 differs by 15 % or more from the expected value or another

calibration level differs by 10 % or more from the expected value (estimated by linear

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

portions must fall within the working range of the calibration curve.

7.6 Determination of the nicotine content of samples

Use Calibration level 0 to evaluate carry over and system performance: Inject three

replicates of calibration level 0 before and after calibration of the gas chromatograph.

Inject three (3) replicates of calibration level 0 after three (3) independent replicate

analyses for each sample.

Follow the run order specified below:

Run Order Sample Name Run Order Sample Name

1 Cal Level 0 35 Cal Level 0

2 Cal Level 0 36 CRP4.1-Rep1









11 NIST SRM 3222-Rep1 45 Cal Level 0

12 NIST SRM 3222-Rep2 46 RT6-Rep1


14 Cal Level 0 48 RT6-Rep3


16 VLNCMST-Rep1 50 Cal Level 0

17 VLNCMST-Rep2 51 RT8-Rep1




21 VLNCF1-Rep1 55 Cal Level 0

22 VLNCF1-Rep2 56 CRP3.1-Rep1





27 VLNCF2-Rep2 61 3R4F-Rep1




29 Cal Level 0 63 3R4F-Rep3


31 CRP1.1-Rep1 65 Cal Level 0

32 CRP1.1-Rep2 66 RT1-Rep1




Appendix 2: Modifications required for CRM No 62 when using Hexane as

the solvent

6. STANDARDS






Calibration Level

Volume of Nicotine Stock (ml)

Nicotine (mg/ml)a n-heptadecane (ISTD)

(mg/ml)b

0 0,0 0,000 0,5

1 0,005 0,0006 0,5

2 0,010 0,0012 0,5

3 0,050 0,006 0,5

4 0,500 0,060 0,5

5 1,000 0,120 0,5

6 2,000 0,240 0,5

7 3,000 0,360 0,5

8 5,000 0,600 0,5

9 8,000 0,960 0,5

1. Calibration levels 0-4 are new levels not shown in the published CRM.

2. a Based on 2,4 mg/ml nicotine stock solution concentration and volume of nicotine stock

solution diluted to a final volume of 20 ml using extraction solution containing n-heptadecane

(0,500 mg/ml).

3. b Based on 0,500 mg/ml extraction solution concentration containing n-heptadecane.

7. PROCEDURES

7.4 Gas Chromatography

Injection Mode: Use a splitless injection; use an appropriate sized splitless liner i.e.

Glass 4,0 mm I.D. deactivated liner with glass wool.

Column recommended: Use a typical column with the following dimensions: 30 m x

0.25 mm ID x 0.25 µm df to improve method sensitivity


Use a sample extract, inject ~10 aliquots at an elevated GC oven temperature. This shall

be performed before calibration of the gas chromatograph.

7.5 Calibration of the gas chromatograph

Use a linear regression model with 1/x weighting: Calculate the ratio of the nicotine

peak to the internal standard peak (Y = Anicotine/AIS) from the peak area (or height) data

for each of the nicotine standards including the calibration level 1 to be used as a solvent

blank. Only plot the graph of the concentrations of added nicotine (X axis) in accordance

with the area ratios (Y axis). Calculate a linear regression equation (Y = a + bx) using

1/x weighting, x being concentration, from this data. Use both the slope (b) and the

intercept (a) of the linear regression.


If the correlation coefficient R2 is less than 0,990, then the calibration should be

repeated. If the calibration level 1 differs by 15 % or more from the expected value or

another calibration level differs by 10 % or more from the expected value (estimated by

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

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

7.6 Determination of the nicotine content of samples

Use calibration level 0 to evaluate carry over and system performance: Inject three

replicates of calibration level 0 before and after calibration of the gas chromatograph.

Inject three (3) replicates of calibration level 0 after every three (3) independent

replicate analyses for each sample.








































Appendix 3: Modifications required for CRM No 87

7. STANDARDS






Calibration Level

Volume of Secondary

Nicotine Stock (ml)

Volume of WISS (ml)

Nicotine (µg/ml)a Quinoline (µg/ml)b

0 0 0,250 0 40

1 0,010 0,250 0,4 40

2 0,025 0,250 1,0 40

3 0,100 0,250 4,0 40

4 0,200 0,250 8,0 40

5 0,500 0,250 20,0 40

6 1,00 0,250 40,0 40

7 3,00 0,250 120,0 40

8 5,00 0,250 200,0 40

9 10,00 0,250 400,0 40

1. Calibration levels 2-3 are new levels not shown in the published CRM

2. a Based on 1,0 mg/ml nicotine stock solution concentration and volume of nicotine stock

solution diluted to a final volume of 25 ml using methanol

3. b Based on 4,0 mg/ml WISS concentration and volume of WISS diluted to a final volume of

25 ml using methanol

9. SAMPLE ANALYSIS

9.2 System Suitability


Use a sample extract, inject ~10 aliquots at an elevated GC oven temperature. This shall

be performed before calibration of the gas chromatograph.

9.3 Calibration of the GC-MS

Use a linear regression model with 1/x weighting: Create an internal standard

calibration method in the instrument operating software. A calibration curve is

generated by calculating a linear regression of the area ratios of nicotine to quinoline

(y) as a function of the concentration ratios of nicotine to quinoline (x) using 1/x

weighting.

If the correlation coefficient R2 is less than 0,990, then the calibration should be

repeated. If the calibration level 1 differs by 15 % or more from the expected value or

another calibration level differs by 10 % or more from the expected value (estimated by

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

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


9.4 Determination of the concentration of Nicotine

Use calibration level 0 to evaluate carry over and system performance: Inject three

replicates of calibration level 0 before and after calibration of the GC-MS. Inject three

(3) replicates of calibration level 0 after every three (3) independent replicate analyses

for each sample.


Note: this run order differs from the previous.


















17 VLNCMST-Rep2 51 CRP3.1-Rep1

18 VLNCMST-Rep3 52 CRP3.1-Rep2









27 VLNCF2-Rep2 61 RT1-Rep1



28 VLNCF2-Rep3 62 RT1-Rep2




32 CRP4.1-Rep2 66 CRP1.1-Rep1

33 CRP4.1-Rep3 67 CRP1.1-Rep2



APPENDIX B: Raw Data

Lab Code Product

CRM No. 62 MTBE

CRM No. 62 Hexane

CRM No. 87

Nicotine

mg/g

Nicotine

mg/g

Nicotine

mg/g

1 NIST SRM 3222 0,142 0,143 0,171

1 NIST SRM 3222 0,141 0,133 0,173

1 NIST SRM 3222 0,140 0,137 0,169

2 NIST SRM 3222 0,172 -- 0,189

2 NIST SRM 3222 0,153 -- 0,182

2 NIST SRM 3222 0,153 -- 0,168

3 NIST SRM 3222 0,140 -- 0,166

3 NIST SRM 3222 0,135 -- 0,175

3 NIST SRM 3222 0,130 -- 0,183

4 NIST SRM 3222 -- -- 0,202

4 NIST SRM 3222 -- -- 0,193

4 NIST SRM 3222 -- -- 0,210

5 NIST SRM 3222 -- 0,117 0,161

5 NIST SRM 3222 -- 0,126 0,154

5 NIST SRM 3222 -- 0,127 0,152

6 NIST SRM 3222 0,139 -- 0,185

6 NIST SRM 3222 0,135 -- 0,168

6 NIST SRM 3222 0,137 -- 0,177

7 NIST SRM 3222 -- -- --

7 NIST SRM 3222 -- -- --

7 NIST SRM 3222 -- -- --

8 NIST SRM 3222 0,200 -- 0,277

8 NIST SRM 3222 0,234 -- 0,259

8 NIST SRM 3222 0,267 -- 0,245

9 NIST SRM 3222 0,170 -- --

9 NIST SRM 3222 0,168 -- --

9 NIST SRM 3222 0,211 -- --

10 NIST SRM 3222 0,145 0,153 0,203

10 NIST SRM 3222 0,146 0,151 0,206

10 NIST SRM 3222 0,148 0,158 0,204

11 NIST SRM 3222 0,258 -- 0,216

11 NIST SRM 3222 0,260 -- 0,222

11 NIST SRM 3222 0,266 -- 0,214


Lab Code Product

CRM No. 62 MTBE

CRM No. 62 Hexane

CRM No. 87

Nicotine

mg/g

Nicotine

mg/g

Nicotine

mg/g

12 NIST SRM 3222 0,168 -- --

12 NIST SRM 3222 0,164 -- --

12 NIST SRM 3222 0,168 -- --

13 NIST SRM 3222 0,170 0,156 0,209

13 NIST SRM 3222 0,155 0,160 0,210

13 NIST SRM 3222 0,155 0,156 0,211

14 NIST SRM 3222 0,130 -- --

14 NIST SRM 3222 0,120 -- --

14 NIST SRM 3222 0,130 -- --

15 NIST SRM 3222 -- -- --

15 NIST SRM 3222 -- -- --

15 NIST SRM 3222 -- -- --

16 NIST SRM 3222 -- -- --

16 NIST SRM 3222 -- -- --

16 NIST SRM 3222 -- -- --

17 NIST SRM 3222 0,188 0,174 0,336

17 NIST SRM 3222 0,149 0,174 0,256

17 NIST SRM 3222 0,145 0,174 0,272

18 NIST SRM 3222 -- -- --

18 NIST SRM 3222 -- -- --

18 NIST SRM 3222 -- -- --

19 NIST SRM 3222 0,133 -- 0,241

19 NIST SRM 3222 0,125 -- 0,233

19 NIST SRM 3222 0,133 -- 0,200

20 NIST SRM 3222 0,188 0,159 0,448

20 NIST SRM 3222 0,189 0,198 0,272

20 NIST SRM 3222 0,190 0,212 0,247

21 NIST SRM 3222 0,155 -- 0,180

21 NIST SRM 3222 0,157 -- 0,174

21 NIST SRM 3222 0,150 -- 0,174

22 NIST SRM 3222 -- -- --

22 NIST SRM 3222 -- -- --

22 NIST SRM 3222 -- -- --

23 NIST SRM 3222 -- -- --

23 NIST SRM 3222 -- -- --


Lab Code Product

CRM No. 62 MTBE

CRM No. 62 Hexane

CRM No. 87

Nicotine

mg/g

Nicotine

mg/g

Nicotine

mg/g

23 NIST SRM 3222 -- -- --

24 NIST SRM 3222 0,144 0,132 0,262

24 NIST SRM 3222 0,147 0,124 0,194

24 NIST SRM 3222 0,143 0,131 0,170

25 NIST SRM 3222 -- 0,196 --

25 NIST SRM 3222 -- 0,181 --

25 NIST SRM 3222 -- 0,178 --

1 VLNCF1 0,383 0,383 0,415

1 VLNCF1 0,403 0,404 0,432

1 VLNCF1 0,388 0,451 0,408

2 VLNCF1 0,356 -- 0,397

2 VLNCF1 0,350 -- 0,413

2 VLNCF1 0,345 -- 0,423

3 VLNCF1 0,410 -- 0,319

3 VLNCF1 0,405 -- 0,303

3 VLNCF1 0,395 -- 0,306

4 VLNCF1 -- -- 0,370

4 VLNCF1 -- -- 0,384

4 VLNCF1 -- -- 0,383

5 VLNCF1 -- 0,349 0,392

5 VLNCF1 -- 0,363 0,415

5 VLNCF1 -- 0,354 0,422

6 VLNCF1 0,403 -- 0,380

6 VLNCF1 0,404 -- 0,373

6 VLNCF1 0,407 -- 0,381

7 VLNCF1 -- 0,394 --

7 VLNCF1 -- 0,380 --

7 VLNCF1 -- 0,382 --

8 VLNCF1 0,347 -- 0,412

8 VLNCF1 0,349 -- 0,394

8 VLNCF1 0,380 -- 0,404

9 VLNCF1 0,421 -- 0,406

9 VLNCF1 0,421 -- 0,416

9 VLNCF1 0,418 -- 0,421

10 VLNCF1 0,410 0,411 0,405


Lab Code Product

CRM No. 62 MTBE

CRM No. 62 Hexane

CRM No. 87

Nicotine

mg/g

Nicotine

mg/g

Nicotine

mg/g

10 VLNCF1 0,409 0,409 0,403

10 VLNCF1 0,406 0,406 0,397

11 VLNCF1 0,515 -- 0,511

11 VLNCF1 0,531 -- 0,497

11 VLNCF1 0,533 -- 0,476

12 VLNCF1 0,366 -- --

12 VLNCF1 0,368 -- --

12 VLNCF1 0,354 -- --

13 VLNCF1 0,364 0,388 0,402

13 VLNCF1 0,375 0,392 0,421

13 VLNCF1 0,385 0,404 0,416

14 VLNCF1 0,336 -- --

14 VLNCF1 0,350 -- --

14 VLNCF1 0,358 -- --

15 VLNCF1 -- 0,445 0,408

15 VLNCF1 -- 0,449 0,382

15 VLNCF1 -- 0,446 0,397

16 VLNCF1 -- 0,496 --

16 VLNCF1 -- 0,477 --

16 VLNCF1 -- 0,465 --

17 VLNCF1 -- -- --

17 VLNCF1 -- -- --

17 VLNCF1 -- -- --

18 VLNCF1 -- 0,371 --

18 VLNCF1 -- 0,358 --

18 VLNCF1 -- 0,360 --

19 VLNCF1 0,401 -- 0,399

19 VLNCF1 0,414 -- 0,414

19 VLNCF1 0,415 -- 0,425

20 VLNCF1 0,415 0,417 0,381

20 VLNCF1 0,433 0,437 0,397

20 VLNCF1 0,444 0,415 0,385

21 VLNCF1 0,423 -- 0,469

21 VLNCF1 0,412 -- 0,423

21 VLNCF1 0,408 -- 0,415


Lab Code Product

CRM No. 62 MTBE

CRM No. 62 Hexane

CRM No. 87

Nicotine

mg/g

Nicotine

mg/g

Nicotine

mg/g

22 VLNCF1 -- -- --

22 VLNCF1 -- -- --

22 VLNCF1 -- -- --

23 VLNCF1 -- -- 0,344

23 VLNCF1 -- -- 0,348

23 VLNCF1 -- -- 0,357

24 VLNCF1 0,403 0,377 0,371

24 VLNCF1 0,340 0,380 0,382

24 VLNCF1 0,351 0,376 0,371

25 VLNCF1 -- 0,398 --

25 VLNCF1 -- 0,390 --

25 VLNCF1 -- 0,387 --

1 VLNCMST 0,703 0,619 0,708

1 VLNCMST 0,708 0,625 0,738

1 VLNCMST 0,702 0,618 0,721

2 VLNCMST 0,655 -- 0,670

2 VLNCMST 0,651 -- 0,688

2 VLNCMST 0,644 -- 0,601

3 VLNCMST 0,635 -- 0,546

3 VLNCMST 0,625 -- 0,566

3 VLNCMST 0,645 -- 0,555

4 VLNCMST -- -- 0,601

4 VLNCMST -- -- 0,641

4 VLNCMST -- -- 0,613

5 VLNCMST -- 0,567 0,658

5 VLNCMST -- 0,561 0,691

5 VLNCMST -- 0,557 0,720

6 VLNCMST 0,579 -- 0,597

6 VLNCMST 0,584 -- 0,593

6 VLNCMST 0,601 -- 0,603

7 VLNCMST -- 0,557 --

7 VLNCMST -- 0,574 --

7 VLNCMST -- 0,570 --

8 VLNCMST 0,672 -- 0,695

8 VLNCMST 0,730 -- 0,740


Lab Code Product

CRM No. 62 MTBE

CRM No. 62 Hexane

CRM No. 87

Nicotine

mg/g

Nicotine

mg/g

Nicotine

mg/g

8 VLNCMST 0,500 -- 0,721

9 VLNCMST 0,651 -- 0,717

9 VLNCMST 0,647 -- 0,723

9 VLNCMST 0,645 -- 0,732

10 VLNCMST 0,509 0,562 0,526

10 VLNCMST 0,522 0,561 0,616

10 VLNCMST 0,525 0,529 0,629

11 VLNCMST 0,674 -- 0,753

11 VLNCMST 0,606 -- 0,765

11 VLNCMST 0,584 -- 0,788

12 VLNCMST 0,536 -- --

12 VLNCMST 0,538 -- --

12 VLNCMST 0,541 -- --

13 VLNCMST 0,609 0,588 0,647

13 VLNCMST 0,620 0,592 0,694

13 VLNCMST 0,609 0,596 0,714

14 VLNCMST 0,549 -- --

14 VLNCMST 0,550 -- --

14 VLNCMST 0,552 -- --

15 VLNCMST -- 0,574 0,630

15 VLNCMST -- 0,575 0,647

15 VLNCMST -- 0,581 0,672

16 VLNCMST -- 0,621 --

16 VLNCMST -- 0,674 --

16 VLNCMST -- 0,670 --

17 VLNCMST -- -- --

17 VLNCMST -- -- --

17 VLNCMST -- -- --

18 VLNCMST -- 0,591 --

18 VLNCMST -- 0,569 --

18 VLNCMST -- 0,509 --

19 VLNCMST 0,681 -- 0,614

19 VLNCMST 0,663 -- 0,658

19 VLNCMST 0,652 -- 0,687

20 VLNCMST 0,631 0,631 0,706


Lab Code Product

CRM No. 62 MTBE

CRM No. 62 Hexane

CRM No. 87

Nicotine

mg/g

Nicotine

mg/g

Nicotine

mg/g

20 VLNCMST 0,652 0,640 0,748

20 VLNCMST 0,629 0,631 0,757

21 VLNCMST 0,616 -- 0,693

21 VLNCMST 0,611 -- 0,681

21 VLNCMST 0,620 -- 0,688

22 VLNCMST -- -- --

22 VLNCMST -- -- --

22 VLNCMST -- -- --

23 VLNCMST -- -- 0,676

23 VLNCMST -- -- 0,647

23 VLNCMST -- -- 0,688

24 VLNCMST 0,643 0,667 0,671

24 VLNCMST 0,643 0,659 0,704

24 VLNCMST 0,652 0,653 0,700

25 VLNCMST -- 0,604 --

25 VLNCMST -- 0,614 --

25 VLNCMST -- 0,610 --

1 VLNCF2 0,926 0,891 0,922

1 VLNCF2 0,946 0,901 0,961

1 VLNCF2 0,946 0,909 0,954

2 VLNCF2 0,762 -- 0,886

2 VLNCF2 0,751 -- 0,827

2 VLNCF2 0,764 -- 0,834

3 VLNCF2 0,955 -- 0,671

3 VLNCF2 0,930 -- 0,704

3 VLNCF2 0,925 -- 0,702

4 VLNCF2 -- -- 0,841

4 VLNCF2 -- -- 0,858

4 VLNCF2 -- -- 0,881

5 VLNCF2 -- 0,833 1,001

5 VLNCF2 -- 0,854 0,988

5 VLNCF2 -- 0,858 1,016

6 VLNCF2 0,935 -- 0,883

6 VLNCF2 0,935 -- 0,875

6 VLNCF2 0,964 -- 0,889


Lab Code Product

CRM No. 62 MTBE

CRM No. 62 Hexane

CRM No. 87

Nicotine

mg/g

Nicotine

mg/g

Nicotine

mg/g

7 VLNCF2 -- 0,918 --

7 VLNCF2 -- 0,899 --

7 VLNCF2 -- 0,895 --

8 VLNCF2 0,962 -- 0,961

8 VLNCF2 0,773 -- 0,974

8 VLNCF2 0,650 -- 0,972

9 VLNCF2 0,966 -- 0,992

9 VLNCF2 0,982 -- 0,970

9 VLNCF2 0,968 -- 0,997

10 VLNCF2 0,980 0,900 0,862

10 VLNCF2 0,960 0,955 0,954

10 VLNCF2 0,950 0,974 0,907

11 VLNCF2 1,048 -- 1,075

11 VLNCF2 1,090 -- 1,075

11 VLNCF2 1,096 -- 1,078

12 VLNCF2 0,887 -- --

12 VLNCF2 0,888 -- --

12 VLNCF2 0,867 -- --

13 VLNCF2 0,915 0,928 0,911

13 VLNCF2 0,895 0,916 0,955

13 VLNCF2 0,899 0,904 0,944

14 VLNCF2 0,830 -- --

14 VLNCF2 0,838 -- --

14 VLNCF2 0,832 -- --

15 VLNCF2 -- 0,943 0,978

15 VLNCF2 -- 0,937 0,968

15 VLNCF2 -- 0,954 0,963

16 VLNCF2 -- 1,007 --

16 VLNCF2 -- 1,020 --

16 VLNCF2 -- 0,979 --

17 VLNCF2 -- -- --

17 VLNCF2 -- -- --

17 VLNCF2 -- -- --

18 VLNCF2 -- 0,792 --

18 VLNCF2 -- 0,790 --


Lab Code Product

CRM No. 62 MTBE

CRM No. 62 Hexane

CRM No. 87

Nicotine

mg/g

Nicotine

mg/g

Nicotine

mg/g

18 VLNCF2 -- 0,870 --

19 VLNCF2 0,997 -- 0,956

19 VLNCF2 0,986 -- 0,984

19 VLNCF2 0,976 -- 0,978

20 VLNCF2 0,988 0,983 0,973

20 VLNCF2 0,985 0,993 0,982

20 VLNCF2 0,993 0,957 0,974

21 VLNCF2 0,935 -- 0,953

21 VLNCF2 0,941 -- 0,989

21 VLNCF2 0,930 -- 0,991

22 VLNCF2 -- -- --

22 VLNCF2 -- -- --

22 VLNCF2 -- -- --

23 VLNCF2 -- -- 0,882

23 VLNCF2 -- -- 0,900

23 VLNCF2 -- -- 0,878

24 VLNCF2 0,826 0,935 0,890

24 VLNCF2 0,885 0,919 0,875

24 VLNCF2 0,802 0,916 0,874

25 VLNCF2 -- 0,897 --

25 VLNCF2 -- 0,881 --

25 VLNCF2 -- 0,891 --

1 CRP1.1 7,497 6,981 7,470

1 CRP1.1 7,428 6,723 7,362

1 CRP1.1 7,379 6,742 7,436

2 CRP1.1 8,166 -- 8,254

2 CRP1.1 8,147 -- 8,523

2 CRP1.1 8,494 -- 8,833

3 CRP1.1 7,405 -- 7,503

3 CRP1.1 7,350 -- 7,773

3 CRP1.1 7,098 -- 7,723

4 CRP1.1 -- -- 6,676

4 CRP1.1 -- -- 6,759

4 CRP1.1 -- -- 6,732

5 CRP1.1 -- 7,125 7,781


Lab Code Product

CRM No. 62 MTBE

CRM No. 62 Hexane

CRM No. 87

Nicotine

mg/g

Nicotine

mg/g

Nicotine

mg/g

5 CRP1.1 -- 7,035 7,927

5 CRP1.1 -- 6,839 7,505

6 CRP1.1 7,543 -- 5,769

6 CRP1.1 7,374 -- 7,240

6 CRP1.1 7,275 -- 6,534

7 CRP1.1 -- 6,188 --

7 CRP1.1 -- 6,633 --

7 CRP1.1 -- 7,103 --

8 CRP1.1 7,795 -- 6,741

8 CRP1.1 5,311 -- 7,783

8 CRP1.1 7,485 -- 7,766

9 CRP1.1 8,441 -- 7,583

9 CRP1.1 8,738 -- 7,978

9 CRP1.1 8,464 -- 8,158

10 CRP1.1 7,094 6,301 7,149

10 CRP1.1 6,961 4,949 7,197

10 CRP1.1 7,138 4,447 7,132

11 CRP1.1 6,941 -- 7,054

11 CRP1.1 7,835 -- 7,700

11 CRP1.1 7,754 -- 6,855

12 CRP1.1 7,078 -- --

12 CRP1.1 6,691 -- --

12 CRP1.1 7,024 -- --

13 CRP1.1 7,064 7,108 7,937

13 CRP1.1 6,917 6,812 8,046

13 CRP1.1 6,996 5,832 8,134

14 CRP1.1 6,506 -- --

14 CRP1.1 6,340 -- --

14 CRP1.1 6,296 -- --

15 CRP1.1 -- 7,175 7,316

15 CRP1.1 -- 7,173 6,792

15 CRP1.1 -- 7,212 7,398

16 CRP1.1 -- 7,574 --

16 CRP1.1 -- 7,859 --

16 CRP1.1 -- 7,533 --


Lab Code Product

CRM No. 62 MTBE

CRM No. 62 Hexane

CRM No. 87

Nicotine

mg/g

Nicotine

mg/g

Nicotine

mg/g

17 CRP1.1 7,247 7,696 7,340

17 CRP1.1 6,967 6,546 7,709

17 CRP1.1 7,804 7,105 7,801

18 CRP1.1 -- -- --

18 CRP1.1 -- -- --

18 CRP1.1 -- -- --

19 CRP1.1 7,446 -- 7,115

19 CRP1.1 8,118 -- 7,313

19 CRP1.1 7,803 -- 7,244

20 CRP1.1 6,436 7,436 7,385

20 CRP1.1 6,612 7,093 7,809

20 CRP1.1 6,741 6,592 7,714

21 CRP1.1 8,191 -- 7,588

21 CRP1.1 7,986 -- 7,600

21 CRP1.1 8,046 -- 7,388

22 CRP1.1 7,904 8,223 7,776

22 CRP1.1 7,957 8,296 7,395

22 CRP1.1 7,960 8,337 7,881

23 CRP1.1 -- -- --

23 CRP1.1 -- -- --

23 CRP1.1 -- -- --

24 CRP1.1 7,360 7,492 7,835

24 CRP1.1 7,586 7,436 7,687

24 CRP1.1 7,581 7,528 7,060

25 CRP1.1 -- 7,423 --

25 CRP1.1 -- 7,123 --

25 CRP1.1 -- 7,346 --

1 CRP4.1 8,706 8,633 8,882

1 CRP4.1 8,829 8,617 9,097

1 CRP4.1 8,761 8,709 9,116

2 CRP4.1 9,470 -- 10,987

2 CRP4.1 9,567 -- 10,726

2 CRP4.1 9,354 -- 10,535

3 CRP4.1 8,595 -- 7,666


Lab Code Product

CRM No. 62 MTBE

CRM No. 62 Hexane

CRM No. 87

Nicotine

mg/g

Nicotine

mg/g

Nicotine

mg/g

3 CRP4.1 8,555 -- 7,931

3 CRP4.1 8,655 -- 8,119

4 CRP4.1 -- -- 8,669

4 CRP4.1 -- -- 9,005

4 CRP4.1 -- -- 8,906

5 CRP4.1 -- 7,974 9,394

5 CRP4.1 -- 8,101 10,507

5 CRP4.1 -- 8,032 9,659

6 CRP4.1 8,917 -- 8,260

6 CRP4.1 8,847 -- 8,427

6 CRP4.1 8,871 -- 8,332

7 CRP4.1 -- 8,127 --

7 CRP4.1 -- 8,130 --

7 CRP4.1 -- 8,167 --

8 CRP4.1 7,829 -- 8,829

8 CRP4.1 7,237 -- 9,014

8 CRP4.1 7,087 -- 8,955

9 CRP4.1 8,689 -- 8,810

9 CRP4.1 8,679 -- 9,126

9 CRP4.1 8,891 -- 9,065

10 CRP4.1 9,029 9,423 8,674

10 CRP4.1 8,981 9,185 9,078

10 CRP4.1 8,979 9,226 9,032

11 CRP4.1 8,235 -- 10,041

11 CRP4.1 7,654 -- 9,846

11 CRP4.1 7,582 -- 9,778

12 CRP4.1 8,178 -- --

12 CRP4.1 8,185 -- --

12 CRP4.1 7,703 -- --

13 CRP4.1 8,611 8,548 9,489

13 CRP4.1 8,571 8,548 9,346

13 CRP4.1 8,451 8,516 9,565


Lab Code Product

CRM No. 62 MTBE

CRM No. 62 Hexane

CRM No. 87

Nicotine

mg/g

Nicotine

mg/g

Nicotine

mg/g

14 CRP4.1 8,220 -- --

14 CRP4.1 8,230 -- --

14 CRP4.1 8,260 -- --

15 CRP4.1 -- 8,680 9,084

15 CRP4.1 -- 8,546 8,809

15 CRP4.1 -- 8,455 9,151

16 CRP4.1 -- 9,538 --

16 CRP4.1 -- 9,625 --

16 CRP4.1 -- 9,550 --

17 CRP4.1 8,284 8,625 8,399

17 CRP4.1 8,285 8,677 8,417

17 CRP4.1 8,452 8,751 8,424

18 CRP4.1 -- -- --

18 CRP4.1 -- -- --

18 CRP4.1 -- -- --

19 CRP4.1 9,114 -- 8,454

19 CRP4.1 9,134 -- 8,764

19 CRP4.1 9,000 -- 8,835

20 CRP4.1 8,788 9,112 9,014

20 CRP4.1 8,695 9,083 9,243

20 CRP4.1 8,670 9,064 9,179

21 CRP4.1 8,868 -- 9,199

21 CRP4.1 8,631 -- 9,479

21 CRP4.1 8,856 -- 9,370

22 CRP4.1 9,844 9,503 9,842

22 CRP4.1 9,696 9,617 9,944

22 CRP4.1 9,227 9,605 9,806

23 CRP4.1 -- -- --

23 CRP4.1 -- -- --

23 CRP4.1 -- -- --

24 CRP4.1 8,861 8,652 9,004

24 CRP4.1 8,756 8,892 8,933


Lab Code Product

CRM No. 62 MTBE

CRM No. 62 Hexane

CRM No. 87

Nicotine

mg/g

Nicotine

mg/g

Nicotine

mg/g

24 CRP4.1 9,038 8,710 9,002

25 CRP4.1 -- 8,559 --

25 CRP4.1 -- 8,468 --

25 CRP4.1 -- 8,553 --

1 CRP2.1 10,573 10,311 10,471

1 CRP2.1 10,563 10,217 10,761

1 CRP2.1 10,377 10,232 10,736

2 CRP2.1 11,346 -- 11,151

2 CRP2.1 11,276 -- 11,786

2 CRP2.1 11,072 -- 12,260

3 CRP2.1 10,230 -- 9,319

3 CRP2.1 10,165 -- 9,380

3 CRP2.1 9,990 -- 9,315

4 CRP2.1 -- -- 10,182

4 CRP2.1 -- -- 10,386

4 CRP2.1 -- -- 10,466

5 CRP2.1 -- 9,498 11,033

5 CRP2.1 -- 9,614 11,281

5 CRP2.1 -- 9,679 11,125

6 CRP2.1 10,455 -- 9,805

6 CRP2.1 10,454 -- 9,998

6 CRP2.1 10,288 -- 10,016

7 CRP2.1 -- 9,564 --

7 CRP2.1 -- 9,562 --

7 CRP2.1 -- 9,601 --

8 CRP2.1 10,218 -- 11,717

8 CRP2.1 9,916 -- 11,120

8 CRP2.1 9,608 -- 11,385

9 CRP2.1 11,230 -- 11,077

9 CRP2.1 11,335 -- 11,741

9 CRP2.1 11,370 -- 11,636

10 CRP2.1 10,467 10,648 10,949

10 CRP2.1 10,450 10,293 11,036

10 CRP2.1 10,339 10,907 10,424

11 CRP2.1 9,414 -- 11,470


Lab Code Product

CRM No. 62 MTBE

CRM No. 62 Hexane

CRM No. 87

Nicotine

mg/g

Nicotine

mg/g

Nicotine

mg/g

11 CRP2.1 9,734 -- 11,597

11 CRP2.1 9,743 -- 11,656

12 CRP2.1 9,754 -- --

12 CRP2.1 9,742 -- --

12 CRP2.1 9,838 -- --

13 CRP2.1 9,794 10,144 11,228

13 CRP2.1 10,016 10,112 11,447

13 CRP2.1 9,792 10,064 11,491

14 CRP2.1 10,130 -- --

14 CRP2.1 10,220 -- --

14 CRP2.1 10,020 -- --

15 CRP2.1 -- 10,008 11,342

15 CRP2.1 -- 9,971 11,082

15 CRP2.1 -- 9,932 10,893

16 CRP2.1 -- 10,575 --

16 CRP2.1 -- 10,429 --

16 CRP2.1 -- 10,628 --

17 CRP2.1 7,365 7,893 8,627

17 CRP2.1 7,471 7,814 8,486

17 CRP2.1 7,394 7,729 8,670

18 CRP2.1 -- -- --

18 CRP2.1 -- -- --

18 CRP2.1 -- -- --

19 CRP2.1 11,150 -- 10,175

19 CRP2.1 11,351 -- 10,359

19 CRP2.1 11,059 -- 10,587

20 CRP2.1 10,242 10,645 10,648

20 CRP2.1 10,448 10,606 11,152

20 CRP2.1 10,386 10,634 11,158

21 CRP2.1 10,214 -- 10,841

21 CRP2.1 10,395 -- 10,766

21 CRP2.1 10,463 -- 10,878

22 CRP2.1 11,000 11,945 10,757

22 CRP2.1 10,657 11,665 11,610

22 CRP2.1 12,014 11,534 12,006


Lab Code Product

CRM No. 62 MTBE

CRM No. 62 Hexane

CRM No. 87

Nicotine

mg/g

Nicotine

mg/g

Nicotine

mg/g

23 CRP2.1 -- -- --

23 CRP2.1 -- -- --

23 CRP2.1 -- -- --

24 CRP2.1 10,447 10,427 11,153

24 CRP2.1 10,509 10,394 11,378

24 CRP2.1 10,612 10,460 11,087

25 CRP2.1 -- 10,353 --

25 CRP2.1 -- 10,285 --

25 CRP2.1 -- 10,075 --

1 RT6 10,156 10,368 10,971

1 RT6 10,764 10,384 11,192

1 RT6 10,704 10,382 11,204

2 RT6 11,095 -- 11,871

2 RT6 11,240 -- 12,252

2 RT6 11,238 -- 12,578

3 RT6 10,880 -- 9,968

3 RT6 11,070 -- 10,114

3 RT6 10,960 -- 10,371

4 RT6 -- -- 10,811

4 RT6 -- -- 10,931

4 RT6 -- -- 10,697

5 RT6 -- 10,135 11,319

5 RT6 -- 10,208 11,845

5 RT6 -- 10,179 12,097

6 RT6 11,070 -- 10,060

6 RT6 11,091 -- 10,478

6 RT6 11,101 -- 10,584

7 RT6 -- 10,235 --

7 RT6 -- 10,197 --

7 RT6 -- 10,146 --

8 RT6 6,388 -- 11,091

8 RT6 7,070 -- 10,937

8 RT6 7,845 -- 10,881

9 RT6 11,119 -- 11,206

9 RT6 11,128 -- 11,443


Lab Code Product

CRM No. 62 MTBE

CRM No. 62 Hexane

CRM No. 87

Nicotine

mg/g

Nicotine

mg/g

Nicotine

mg/g

9 RT6 11,091 -- 11,329

10 RT6 10,808 12,219 9,336

10 RT6 10,823 12,246 11,649

10 RT6 10,918 12,590 12,632

11 RT6 11,369 -- 11,884

11 RT6 11,445 -- 11,995

11 RT6 11,114 -- 12,143

12 RT6 10,464 -- --

12 RT6 10,471 -- --

12 RT6 10,491 -- --

13 RT6 10,669 10,608 11,510

13 RT6 10,667 10,616 11,975

13 RT6 10,739 10,680 11,896

14 RT6 10,370 -- --

14 RT6 10,370 -- --

14 RT6 10,290 -- --

15 RT6 -- 10,640 11,508

15 RT6 -- 10,680 11,437

15 RT6 -- 10,688 11,420

16 RT6 -- 10,446 --

16 RT6 -- 10,127 --

16 RT6 -- 9,896 --

17 RT6 -- -- --

17 RT6 -- -- --

17 RT6 -- -- --

18 RT6 -- -- --

18 RT6 -- -- --

18 RT6 -- -- --

19 RT6 11,576 -- 10,227

19 RT6 11,630 -- 10,724

19 RT6 11,692 -- 10,732

20 RT6 11,091 11,232 11,584

20 RT6 11,094 11,225 11,750

20 RT6 11,089 11,206 10,501

21 RT6 11,044 -- 11,334


Lab Code Product

CRM No. 62 MTBE

CRM No. 62 Hexane

CRM No. 87

Nicotine

mg/g

Nicotine

mg/g

Nicotine

mg/g

21 RT6 11,097 -- 11,582

21 RT6 11,077 -- 11,577

22 RT6 -- -- --

22 RT6 -- -- --

22 RT6 -- -- --

23 RT6 -- -- --

23 RT6 -- -- --

23 RT6 -- -- --

24 RT6 10,414 10,334 11,709

24 RT6 10,507 10,783 11,082

24 RT6 10,013 10,142 11,017

25 RT6 -- 11,500 --

25 RT6 -- 11,435 --

25 RT6 -- 11,487 --

1 RT8 13,406 12,940 14,025

1 RT8 13,583 13,077 14,253

1 RT8 13,762 13,175 14,365

2 RT8 14,209 -- 15,733

2 RT8 14,448 -- 16,433

2 RT8 15,275 -- 16,569

3 RT8 13,980 -- 14,012

3 RT8 13,910 -- 14,009

3 RT8 13,865 -- 13,941

4 RT8 -- -- 13,968

4 RT8 -- -- 14,028

4 RT8 -- -- 14,030

5 RT8 -- 12,998 14,751

5 RT8 -- 12,988 15,714

5 RT8 -- 12,748 15,462

6 RT8 13,886 -- 12,791

6 RT8 13,667 -- 13,316

6 RT8 13,949 -- 13,569

7 RT8 -- 12,901 --

7 RT8 -- 12,986 --

7 RT8 -- 13,019 --


Lab Code Product

CRM No. 62 MTBE

CRM No. 62 Hexane

CRM No. 87

Nicotine

mg/g

Nicotine

mg/g

Nicotine

mg/g

8 RT8 10,377 -- 13,992

8 RT8 12,553 -- 14,099

8 RT8 9,853 -- 14,122

9 RT8 14,312 -- 14,648

9 RT8 14,160 -- 14,772

9 RT8 14,209 -- 14,709

10 RT8 13,756 14,639 15,194

10 RT8 13,775 14,590 13,872

10 RT8 13,681 14,666 13,383

11 RT8 15,989 -- 15,326

11 RT8 13,562 -- 15,456

11 RT8 14,280 -- 15,576

12 RT8 13,290 -- --

12 RT8 13,347 -- --

12 RT8 13,267 -- --

13 RT8 14,031 13,680 15,199

13 RT8 13,581 13,660 15,583

13 RT8 13,096 13,728 15,786

14 RT8 12,990 -- --

14 RT8 12,970 -- --

14 RT8 13,030 -- --

15 RT8 -- 13,548 15,180

15 RT8 -- 13,601 14,863

15 RT8 -- 13,568 15,009

16 RT8 -- 13,658 --

16 RT8 -- 13,736 --

16 RT8 -- 13,428 --

17 RT8 -- -- --

17 RT8 -- -- --

17 RT8 -- -- --

18 RT8 -- -- --

18 RT8 -- -- --

18 RT8 -- -- --

19 RT8 14,608 -- 13,508

19 RT8 14,968 -- 14,099


Lab Code Product

CRM No. 62 MTBE

CRM No. 62 Hexane

CRM No. 87

Nicotine

mg/g

Nicotine

mg/g

Nicotine

mg/g

19 RT8 14,752 -- 13,658

20 RT8 14,169 14,095 14,763

20 RT8 13,890 13,982 15,206

20 RT8 14,098 13,911 14,985

21 RT8 14,272 -- 14,486

21 RT8 14,037 -- 14,799

21 RT8 13,949 -- 14,960

22 RT8 -- -- --

22 RT8 -- -- --

22 RT8 -- -- --

23 RT8 -- -- --

23 RT8 -- -- --

23 RT8 -- -- --

24 RT8 13,248 13,555 15,121

24 RT8 13,204 13,603 14,570

24 RT8 13,105 13,637 14,738

25 RT8 -- 13,280 --

25 RT8 -- 13,199 --

25 RT8 -- 13,346 --

1 CRP3.1 16,352 16,016 16,935

1 CRP3.1 16,417 15,987 17,111

1 CRP3.1 16,329 15,982 17,117

2 CRP3.1 17,846 -- 18,250

2 CRP3.1 18,457 -- 18,434

2 CRP3.1 18,824 -- 18,547

3 CRP3.1 16,460 -- 16,448

3 CRP3.1 16,520 -- 17,052

3 CRP3.1 16,540 -- 16,667

4 CRP3.1 -- -- 16,567

4 CRP3.1 -- -- 16,263

4 CRP3.1 -- -- 16,151

5 CRP3.1 -- 15,569 17,637

5 CRP3.1 -- 16,049 19,147

5 CRP3.1 -- 15,985 19,437

6 CRP3.1 16,873 -- 15,868


Lab Code Product

CRM No. 62 MTBE

CRM No. 62 Hexane

CRM No. 87

Nicotine

mg/g

Nicotine

mg/g

Nicotine

mg/g

6 CRP3.1 16,814 -- 16,103

6 CRP3.1 16,839 -- 15,832

7 CRP3.1 -- 15,903 --

7 CRP3.1 -- 15,878 --

7 CRP3.1 -- 16,783 --

8 CRP3.1 15,348 -- 16,680

8 CRP3.1 12,956 -- 16,705

8 CRP3.1 12,302 -- 16,596

9 CRP3.1 17,169 -- 17,655

9 CRP3.1 17,215 -- 17,579

9 CRP3.1 17,190 -- 16,776

10 CRP3.1 16,680 17,603 16,541

10 CRP3.1 16,641 17,707 16,939

10 CRP3.1 16,552 17,753 18,312

11 CRP3.1 16,339 -- 18,406

11 CRP3.1 18,425 -- 18,450

11 CRP3.1 17,693 -- 18,548

12 CRP3.1 16,110 -- --

12 CRP3.1 16,136 -- --

12 CRP3.1 15,998 -- --

13 CRP3.1 16,324 16,336 18,225

13 CRP3.1 16,563 16,308 18,499

13 CRP3.1 16,737 16,336 18,564

14 CRP3.1 15,680 -- --

14 CRP3.1 15,750 -- --

14 CRP3.1 15,700 -- --

15 CRP3.1 -- 16,387 17,405

15 CRP3.1 -- 16,174 17,685

15 CRP3.1 -- 16,245 16,914

16 CRP3.1 -- 17,249 --

16 CRP3.1 -- 17,318 --

16 CRP3.1 -- 17,599 --

17 CRP3.1 15,281 14,942 15,194

17 CRP3.1 15,276 15,062 15,317

17 CRP3.1 15,194 15,101 15,418


Lab Code Product

CRM No. 62 MTBE

CRM No. 62 Hexane

CRM No. 87

Nicotine

mg/g

Nicotine

mg/g

Nicotine

mg/g

18 CRP3.1 -- -- --

18 CRP3.1 -- -- --

18 CRP3.1 -- -- --

19 CRP3.1 17,577 -- 16,036

19 CRP3.1 17,514 -- 16,214

19 CRP3.1 17,646 -- 16,233

20 CRP3.1 16,961 13,869 16,820

20 CRP3.1 16,951 17,034 16,843

20 CRP3.1 16,945 17,068 17,291

21 CRP3.1 16,811 -- 17,396

21 CRP3.1 16,977 -- 17,567

21 CRP3.1 16,731 -- 17,367

22 CRP3.1 17,636 18,316 15,915

22 CRP3.1 17,434 18,267 16,827

22 CRP3.1 17,011 18,348 17,984

23 CRP3.1 -- -- --

23 CRP3.1 -- -- --

23 CRP3.1 -- -- --

24 CRP3.1 16,876 16,649 18,428

24 CRP3.1 16,757 16,658 18,677

24 CRP3.1 17,251 16,689 18,213

25 CRP3.1 -- 16,162 --

25 CRP3.1 -- 16,200 --

25 CRP3.1 -- 16,104 --

1 3R4F 16,261 16,238 17,254

1 3R4F 16,923 15,900 17,320

1 3R4F 16,703 16,485 16,673

2 3R4F 19,633 -- 20,299

2 3R4F 18,669 -- 19,448

2 3R4F 19,751 -- 19,818

3 3R4F 17,770 -- 17,649

3 3R4F 17,775 -- 17,906

3 3R4F 17,635 -- 18,025

4 3R4F -- -- 16,779

4 3R4F -- -- 16,741


Lab Code Product

CRM No. 62 MTBE

CRM No. 62 Hexane

CRM No. 87

Nicotine

mg/g

Nicotine

mg/g

Nicotine

mg/g

4 3R4F -- -- 17,007

5 3R4F -- 17,206 19,492

5 3R4F -- 16,746 19,240

5 3R4F -- 16,465 19,746

6 3R4F 17,146 -- 16,061

6 3R4F 17,414 -- 16,270

6 3R4F 17,470 -- 16,688

7 3R4F -- 16,623 --

7 3R4F -- 16,645 --

7 3R4F -- 16,653 --

8 3R4F 15,903 -- 17,305

8 3R4F 16,052 -- 17,704

8 3R4F 14,689 -- 17,495

9 3R4F 18,625 -- 19,079

9 3R4F 18,606 -- 19,222

9 3R4F 18,812 -- 19,212

10 3R4F 18,641 19,275 17,618

10 3R4F 18,393 19,100 17,852

10 3R4F 18,482 18,836 17,857

11 3R4F 18,884 -- 19,131

11 3R4F 18,822 -- 19,703

11 3R4F 19,066 -- 20,047

12 3R4F 17,678 -- --

12 3R4F 17,644 -- --

12 3R4F 17,668 -- --

13 3R4F 17,177 17,872 18,961

13 3R4F 16,583 17,864 19,545

13 3R4F 16,682 17,756 19,529

14 3R4F 16,880 -- --

14 3R4F 16,860 -- --

14 3R4F 16,750 -- --

15 3R4F -- 16,830 17,740

15 3R4F -- 16,753 18,072

15 3R4F -- 16,736 17,606

16 3R4F -- 18,015 --


Lab Code Product

CRM No. 62 MTBE

CRM No. 62 Hexane

CRM No. 87

Nicotine

mg/g

Nicotine

mg/g

Nicotine

mg/g

16 3R4F -- 18,237 --

16 3R4F -- 18,448 --

17 3R4F 17,349 17,520 17,230

17 3R4F 17,225 17,300 15,878

17 3R4F 17,161 17,224 16,372

18 3R4F -- -- --

18 3R4F -- -- --

18 3R4F -- -- --

19 3R4F 18,298 -- 16,889

19 3R4F 18,414 -- 17,128

19 3R4F 18,378 -- 17,180

20 3R4F 19,384 17,218 19,639

20 3R4F 19,329 16,706 19,881

20 3R4F 19,605 20,245 20,183

21 3R4F 17,518 -- 17,943

21 3R4F 17,803 -- 18,559

21 3R4F 17,860 -- 18,694

22 3R4F -- -- --

22 3R4F -- -- --

22 3R4F -- -- --

23 3R4F -- -- 16,776

23 3R4F -- -- 16,826

23 3R4F -- -- 16,775

24 3R4F 17,852 17,607 19,215

24 3R4F 17,171 18,724 19,428

24 3R4F 17,696 17,258 19,748

25 3R4F -- 17,196 --

25 3R4F -- 17,060 --

25 3R4F -- 17,140 --

1 RT1 17,691 17,272 18,260

1 RT1 17,613 17,087 18,416

1 RT1 17,677 17,395 18,207

2 RT1 19,967 -- 19,227

2 RT1 19,715 -- 19,631

2 RT1 20,026 -- 19,847


Lab Code Product

CRM No. 62 MTBE

CRM No. 62 Hexane

CRM No. 87

Nicotine

mg/g

Nicotine

mg/g

Nicotine

mg/g

3 RT1 17,835 -- 17,766

3 RT1 17,870 -- 18,416

3 RT1 17,770 -- 17,835

4 RT1 -- -- 17,692

4 RT1 -- -- 17,955

4 RT1 -- -- 17,858

5 RT1 -- 16,773 19,372

5 RT1 -- 17,184 19,826

5 RT1 -- 17,584 19,841

6 RT1 18,197 -- 17,159

6 RT1 18,327 -- 17,146

6 RT1 18,231 -- 17,305

7 RT1 -- 16,829 --

7 RT1 -- 16,791 --

7 RT1 -- 16,789 --

8 RT1 16,598 -- 17,664

8 RT1 17,187 -- 18,206

8 RT1 16,177 -- 18,099

9 RT1 17,900 -- 19,068

9 RT1 17,950 -- 19,496

9 RT1 18,159 -- 19,049

10 RT1 18,346 19,325 18,987

10 RT1 18,254 18,541 18,916

10 RT1 18,175 18,669 18,901

11 RT1 19,449 -- 19,511

11 RT1 19,439 -- 20,101

11 RT1 19,565 -- 19,959

12 RT1 17,177 -- --

12 RT1 17,166 -- --

12 RT1 17,291 -- --

13 RT1 16,856 18,016 19,554

13 RT1 16,517 18,044 19,972

13 RT1 16,797 18,148 19,953

14 RT1 17,350 -- --

14 RT1 17,320 -- --


Lab Code Product

CRM No. 62 MTBE

CRM No. 62 Hexane

CRM No. 87

Nicotine

mg/g

Nicotine

mg/g

Nicotine

mg/g

14 RT1 17,320 -- --

15 RT1 -- 17,819 19,389

15 RT1 -- 17,728 19,503

15 RT1 -- 17,828 19,190

16 RT1 -- 18,012 --

16 RT1 -- 18,404 --

16 RT1 -- 18,231 --

17 RT1 -- -- --

17 RT1 -- -- --

17 RT1 -- -- --

18 RT1 -- -- --

18 RT1 -- -- --

18 RT1 -- -- --

19 RT1 18,765 -- 16,854

19 RT1 18,976 -- 17,422

19 RT1 18,762 -- 16,912

20 RT1 18,005 18,218 18,186

20 RT1 18,054 18,304 18,340

20 RT1 18,130 18,259 18,349

21 RT1 18,193 -- 18,642

21 RT1 18,068 -- 18,914

21 RT1 18,627 -- 18,875

22 RT1 -- -- --

22 RT1 -- -- --

22 RT1 -- -- --

23 RT1 -- -- --

23 RT1 -- -- --

23 RT1 -- -- --

24 RT1 17,168 17,731 19,608

24 RT1 16,563 17,993 19,819

24 RT1 16,251 17,579 18,818

25 RT1 -- 17,236 --

25 RT1 -- 17,337 --

25 RT1 -- 17,355 --


APPENDIX C: Raw Data Plots

• MTBE

■ Hexane

▲GC/MS

• MTBE

■ Hexane

▲GC/MS


• MTBE

■ Hexane

▲GC/MS

• MTBE

■ Hexane

▲GC/MS


• MTBE

■ Hexane

▲GC/MS

• MTBE

■Hexane

▲GC/MS


• MTBE

■ Hexane

▲GC/MS

• MTBE

■ Hexane

▲GC/MS


• MTBE

■ Hexane

▲GC/MS

• MTBE

■ Hexane

▲GC/MS


• MTBE

■ Hexane

▲GC/MS

• MTBE

■ Hexane

▲GC/MS


APPENDIX D: Standards, %RCR

CRM No. 62 MTBE (Lab Code 1)

Calibration Level Nicotine (mg/ml) % Relative Concentration Residual

(%RCR)

0 0 NA

1 0,0004 5,5 %

2 0,0010 1,0 %

3 0,0030 -1,2 %

4 0,0060 -0,1 %

5 0,0118 -1,9 %

6 0,0401 0,2 %

7 0,1197 -0,2 %

8 0,2804 0,1 %

9 0,4013 0,3 %

10 0,7986 -0,2 %

CRM No. 62 Hexane (Lab Code 1)


(%RCR)

0 0 NA

1 0,0006 -5,0

2 0,0012 -7,0

3 0,0060 4,7

4 0,0603 1,6

5 0,1206 6,0

6 0,2412 0,4

7 0,4824 0,6

8 0,9648 -1,3

CRM No. 87 (Lab Code 1)


(%RCR)

0 0,000 NA

1 0,0004 9,8 %

2 0,001 -0,03 %

3 0,004 -3,4 %

4 0,010 -2,5 %

5 0,020 -2.7 %

6 0,040 -1,9 %

7 0,120 0,7 %

8 0,200 0,7 %

9 0,400 -0,2 %



Calibration Level Nicotine (mg/ml) % Relative Concentration

Residual (%RCR)

0 0 NA

1 0,0004 -17,8 %

2 0,0008 -5,9 %

3 0,0051 6,5 %

4 0,0468 12,5 %

5 0,0957 6,2 %

6 0,1953 1,6 %

7 0,3930 0,2 %

8 0,5896 -2,7 %

9 0,7768 0,2 %



(%RCR)

0 0 NA

1 0,0003 -15,8 %

2 0,0009 5,5 %

3 0,0039 4,0 %

4 0,0080 8,7 %

5 0,0205 1,5 %

6 0,0370 -9,4 %

7 0,1016 -7,4 %

8 0,1848 0,7 %

9 0,3700 2,7 %



(%RCR)

0 0 NA

1 0,0005 -15,2 %

2 0,001 -8,8 %

3 0,005 -4,9 %

4 0,05 35,3 %

5 0,1 0,4 %

6 0,2 -3,5 %

7 0,4 -4,0 %

8 0,6 -2,1 %

9 0,8 2,2 %




(%RCR)

0 0 NA

1 0,0004 55,7 %

2 0,001 1,3 %

3 0,004 -12,6 %

4 0,008 -24,3 %

5 0,02 -17,9 %

6 0,04 -7,8 %

7 0,12 5,8 %

8 0,2 -1,5 %

9 0,4 1,3 %



(%RCR)

0 0 NA

1 0,0004 14,1 %

2 0,0010 1,3 %

3 0,0040 6,1 %

4 0,0080 23,3 %*

5 0,0199 6,3 %

6 0,0398 4,3 %

7 0,1195 5,2 %

8 0,1992 1,2 %

9 0,3985 0,2 %

* Omitted during data analysis due to %RCR > 10%



(%RCR)

0 0 NA

1 0,0006 1,8 %

2 0,0012 -2,1 %

3 0,0599 0,4 %

4 0,1198 -0,0 %

5 0,2397 -0,5 %

6 0,3595 0,6 %

7 0,5991 -0,1 %

8 0,9586 -0,0 %




(%RCR)

0 0 NA

1 0,0004 -13,0 %

2 0,0010 2,8 %

3 0,0040 2,7 %

4 0,0080 6,7 %

5 0,0199 -2,4 %

6 0,0398 2,7 %

7 0,1194 0,6 %

8 0,1990 1,1 %

9 0,3980 -1,0 %



(%RCR)

0 0 NA

1 0,0005 5,9 %

2 0,0047 -6,3 %

3 0,1011 0,5 %

4 0,4020 -0,1 %

5 0,8047 0,0 %



(%RCR)

0 0 NA

1 0,0004 12,2%

2 0,0401 -9,9%

3 0,1203 -5,2%

4 0,2006 -1,2%

5 0,4012 3,1%




(%RCR)

0 0 NA

1 0,0006 4,7 %

2 0,0012 -2,3 %

3 0,0060 -6,3 %

4 0,0604 1,6 %

5 0,1208 0,4 %

6 0,2415 3,1 %

7 0,3623 -0,4 %

8 0,6038 -0,2%

9 0,9661 -0,6%



(%RCR)

0 0 NA

1 0,0005 1,8 %

2 0,001 5,5 %

3 0,005 -2,0 %

4 0,05 -6,5 %

5 0,1 -0,9 %

6 0,2 1,9 %

7 0,4 0,2 %

8 0,6 0,1 %

9 0,8 -0,2 %



(%RCR)

0 0 NA

1 0,0004 -4,4 %

2 0,001 11,7 %

3 0,004 -0,6 %

4 0,008 -6,4 %

5 0,02 1,5 %

6 0,04 -0,2 %

7 0,12 -2,6 %

8 0,2 0,2 %

9 0,4 0,8 %




(%RCR)

0 0 NA

1 0,0010 2,2 %

2 0,0048 1,5 %

3 0,0961 1,7 %

4 0,1922 1,9 %

5 0,5767 -0,9 %



(%RCR)

0 0 NA

1 0,0004 -2,6 %

2 0,001 -1,1 %

3 0,004 -0,1 %

4 0,008 -0,4 %

5 0,020 2,4 %

6 0,040 2,8 %

7 0,120 1,4 %

8 0,200 -1,6 %



(%RCR)

0 0 NA

1 0,0005 0,0 %

2 0,0010 2,8 %

3 0,0050 -3,4 %

4 0,0503 0,2 %

5 0,101 0,6 %

6 0,201 0,0 %

7 0,402 -0,1 %

8 0,603 -0,3 %

9 0,805 0,2 %




(%RCR)

0 0 NA

1 0,0006 12,0 %

2 0,0012 -9,5 %

3 0,0061 -2,0 %

4 0,0608 3,0 %

5 0,1217 -4,4 %

6 0,2433 -1,0 %

7 0,6083 0,9 %

8 0,9732 -0,7 %



(%RCR)

0 0 NA

1 0,0004 12,4 %

2 0,0010 -3,5 %

3 0,0040 -8,5 %

4 0,0202 -7,8 %

5 0,0402 7,8 %

6 0,1213 0,6 %

7 0,2022 -1,0 %



(%RCR)

0 0 NA

1 0,0005 *

2 0,001 *

3 0,005 0.5 %

4 0,0499 *

5 0,0998 -0.4 %

6 0,1996 2.8 %

7 0,3992 -4.4 %

8 0,5988 -1.0 %

9 0,7983 2.1 %

* Not included




(%RCR)

0 0 NA

1 0,0004 -1,1 %

2 0,0010 -4,5 %

3 0,0039 4,1 %

4 0,0079 -5,2 %

5 0,0197 3,3 %

6 0,0394 6,8 %

7 0,1180 -3,7 %

8 0,1970 0,2 %

9 0,3940 0,3 %



(%RCR)

0 0 NA

1 0,0005 5,9 %

2 0,001 2,9 %

3 0,005 -2,9 %

4 0,05 -2,3 %

5 0,1 -2,1 %

6 0,2 0,7 %

7 0,4 0,1 %

8 0,6 -0,1 %

9 0,8 0,6 %



(%RCR)

0 0 NA

1 0,0010 1,9 %

2 0,0050 6,7 %

3 0,0495 -4,5 %

4 0,0990 -2,7 %

5 0,1980 -1,4 %

6 0,3960 -1,6 %

7 0,5940 -0,2 %

8 0,7920 1,8 %




(%RCR)

0 0 NA

1 0,0012 15,8 %

2 0,0059 -10,1 %

3 0,0594 -3,4 %

4 0,1189 -2,1 %

5 0,2378 -0,8 %

6 0,3567 0,0 %

7 0,5945 -0,2 %

8 0,9515 0,8 %



(%RCR)

0 0 NA

1 0,0010 18 %

2 0,0040 6 %

3 0,0080 9 %

4 0,0199 5 %

5 0,0399 2 %

6 0,1196 2 %

7 0,1994 4 %

8 0,3988 1 %



(%RCR)

0 0 NA

1 0,0005 14,7 %

2 0,0010 -9,3 %

3 0,0050 -6,4 %

4 0,0497 -5,2 %

5 0,0995 -7,3 %

6 0,2000 1,0 %

7 0,3994 1,0 %

8 0,5989 0,6 %

9 0,7987 0,1 %




(%RCR)

0 0 NA

1 0,0006 20,5 %

2 0,0012 -7,4 %

3 0,0060 -16,0 %

4 0,0597 -9,4 %

5 0,1201 -3,5 %

6 0,2397 0,4 %

7 0,3597 1,5 %

8 0,6007 1,2 %

9 0,9612 -0,2 %



(%RCR)

0 0 NA

1 0,0004 15,0 %

2 0,0010 0,0 %

3 0,0042 -7,1 %

4 0,0084 -9,5 %

5 0,0209 -9,1 %

6 0,0421 -1,0 %

7 0,1260 1,0 %

8 0,2104 0,7 %

9 0,4182 0,2%



(%RCR)

0 0 NA

1 0,0005 -1,6 %

2 0,0010 8,3 %

3 0,0049 -2,3 %

4 0,0492 -0,7 %

5 0,0984 -0,5 %

6 0,1968 -3,1 %

7 0,3936 -2,6 %

8 0,5904 1,3 %

9 0,7872 1,2 %




(%RCR)

0 0 NA

1 0,0004 9,3 %

2 0,0010 1,5 %

3 0,0041 -3,2 %

4 0,0082 -6,1 %

5 0,0204 -4,9 %

6 0,0409 2,9 %

7 0,1226 1,0 %

8 0,2043 -0,6 %

9 0,4087 0,1 %



(%RCR)

0 0 NA

1 0,0005 72.0 %

2 0,001 19.8 %

3 0,005 9.6 %

4 0,05 -1.6 %

5 0,1 -1.2 %

6 0,2 0.1 %

7 0,4 0.1 %

8 0,6 0.0 %

9 0,8 0.1 %



(%RCR)

0 0 NA

1 0,0006 65,6 %

2 0,0012 18,0 %

3 0,006 4,1 %

4 0,06 -0,8 %

5 0,12 -0,6 %

6 0,24 0,9 %

7 0,36 -1,3 %

8 0,6 0,4 %

9 0,96 0,0 %



Calibration Level Nicotine (mg/m l) % Relative Concentration Residual

(%RCR)

0 0 NA

1 0,0004 53,2 %

2 0,001 4,6 %

3 0,004 7,2 %

4 0,008 8,8 %

5 0,0201 6,6 %

6 0,0402 5,9 %

7 0,1205 1,9 %

8 0,2009 0,8 %

9 0,4017 2,1 %



(%RCR)

0 0 NA

1 0,000502 NA

2 0,001004 14,3 %

3 0,005019 -8,0 %

4 0,036139 -3,1 %

5 0,100386 -3,8 %

6 0,200772 -0,5 %

7 0,301158 0,3 %

8 0,401544 0,2 %

9 0,803088 0,6 %



(%RCR)

0 0 NA

1 0,00039 13,2 %

2 0,00097 -1,3 %

3 0,00390 -5,0 %

4 0,00780 -5,1 %

5 0,03899 -3,2 %

6 0,09748 0,9 %

7 0,19495 0,5 %




(%RCR)

0 0 NA

1

2

3

4 0,0509 3,7 %

5 0,1018 3,7 %

6 0,2036 4,3 %

7 0,4072 5,6 %

8 0,6108 1,3 %

9 0,8144 2,5 %



(%RCR)

0 0 NA

1

2

3

4

5 0,130 0,1 %

6 0,260 0,4 %

7 0,390 0,2 %

8 0,650 0,7 %

9 1,040 0,3 %



(%RCR)

0 0 NA

1 0,0004072 13,4 %

2 0,001018 1,8 %

3 0,008144 3,6 %

4 0,02036 7,3 %

5 0,04072 6,0 %

6 0,1273 1,0 %

7 0,2036 2,6 %




(%RCR)

0 0 NA

1 0,0004001 20,0 %

2 0,0010003 -2,0 %

3 0,0040013 23,0 %

4 0,0080027 2,5 %

5 0,0200067 -0,5 %

6 0,0400133 6,0 %

7 0,1200399 0,2 %



(%RCR)

0 0 NA

1 0,001 3,4 %

2 0,005 1,9 %

3 0,100 1,4 %

4 0,200 1,0 %

5 0,400 0,9 %

6 0,601 0,04 %

7 1,001 0,01 %



(%RCR)

0 0 NA

1 0,001 5,0 %

2 0,005 4,7 %

3 0,099 0,3 %

4 0,198 0,2 %

5 0,397 0,6 %

6 0,595 0,2 %

7 0,992 0,4 %




(%RCR)

0 0 NA

1 0,0004 11,1 %

2 0,0040 -7,6 %

3 0,0198 -3,9 %

4 0,0397 -1,1 %

5 0,1191 2,0 %

6 0,1985 0,6 %


Calibration Level Nicotine (mg/ml) % Relative Concentration

Residual (%RCR)

0 0 NA

1 0,0006 156 %

2 0,0012 77 %

3 0,0061 8,4 %

4 0,0606 0,4 %

5 0,1212 -0,6 %

6 0,2424 -0,3 %

7 0,4849 -0,3 %

8 0,6061 -0,4 %

9 0,9697 -0,2 %

Documents

Tobacco and Tobacco Products Analytes Sub-Group€¦ · determination of nicotine in 13 sample types including cigarette filler, leaf, smokeless tobacco products (STPs), cigar, and