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
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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)
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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]
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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.
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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.
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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.
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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.
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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)
Repeatability Reproducibility
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 %
* The number of laboratory data sets after removal of outliers.
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Table 6: Repeatability (r) and Reproducibility (R) Limits for Nicotine as-is CRM No. 87
Product No. of Labs * Mean (mg/g)
Repeatability Reproducibility
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 %
* The number of laboratory data sets after removal of outliers.
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.
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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
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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.
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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.
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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.
TTPA-193-1-CTR 2019 Collaborative Study for Nicotine – April 2020 15/79
Table 9. Comparison of predicted R% values to study R% for CRM No. 62Hexane
Material R (%) Predicted from
Horowitz equation R (%) from
Study Ratio (R (%) Study/ R
(%) 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
Material R (%) Predicted from
Horowitz equation R (%) from
Study Ratio (R (%) Study/ R
(%) 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
TTPA-193-1-CTR 2019 Collaborative Study for Nicotine – April 2020 16/79
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.
TTPA-193-1-CTR 2019 Collaborative Study for Nicotine – April 2020 17/79
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.
TTPA-193-1-CTR 2019 Collaborative Study for Nicotine – April 2020 18/79
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.
TTPA-193-1-CTR 2019 Collaborative Study for Nicotine – April 2020 19/79
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
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participate.
TTPA-193-1-CTR 2019 Collaborative Study for Nicotine – April 2020 20/79
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
TTPA-193-1-CTR 2019 Collaborative Study for Nicotine – April 2020 21/79
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
University of Kentucky 1 bottle
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
TTPA-193-1-CTR 2019 Collaborative Study for Nicotine – April 2020 22/79
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
University of Kentucky 1 bottle
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
TTPA-193-1-CTR 2019 Collaborative Study for Nicotine – April 2020 23/79
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.
TTPA-193-1-CTR 2019 Collaborative Study for Nicotine – April 2020 24/79
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.
TTPA-193-1-CTR 2019 Collaborative Study for Nicotine – April 2020 25/79
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
3 Cal Level 0 37 CRP4.1-Rep2
4 Cal Level 1 38 CRP4.1-Rep3
5 Cal Level 2 39 Cal Level 0
6 Cal Level 3 40 Cal Level 0
7 Cal Level 4 41 CRP2.1-Rep1
8 Cal Level 5 42 CRP2.1-Rep2
9 Cal Level 0 43 CRP2.1-Rep3
10 Cal Level 0 44 Cal Level 0
11 NIST SRM 3222-Rep1 45 Cal Level 0
12 NIST SRM 3222-Rep2 46 RT6-Rep1
13 NIST SRM 3222-Rep3 47 RT6-Rep2
14 Cal Level 0 48 RT6-Rep3
15 Cal Level 0 49 Cal Level 0
16 VLNCMST-Rep1 50 Cal Level 0
17 VLNCMST-Rep2 51 RT8-Rep1
18 VLNCMST-Rep3 52 RT8-Rep2
19 Cal Level 0 53 RT8-Rep3
20 Cal Level 0 54 Cal Level 0
21 VLNCF1-Rep1 55 Cal Level 0
22 VLNCF1-Rep2 56 CRP3.1-Rep1
23 VLNCF1-Rep3 57 CRP3.1-Rep2
24 Cal Level 0 58 CRP3.1-Rep3
25 Cal Level 0 59 Cal Level 0
26 VLNCF2-Rep1 60 Cal Level 0
27 VLNCF2-Rep2 61 3R4F-Rep1
28 VLNCF2-Rep3 62 3R4F-Rep2
TTPA-193-1-CTR 2019 Collaborative Study for Nicotine – April 2020 26/79
Run Order Sample Name Run Order Sample Name
29 Cal Level 0 63 3R4F-Rep3
30 Cal Level 0 64 Cal Level 0
31 CRP1.1-Rep1 65 Cal Level 0
32 CRP1.1-Rep2 66 RT1-Rep1
33 CRP1.1-Rep3 67 RT1-Rep2
34 Cal Level 0 68 RT1-Rep3
TTPA-193-1-CTR 2019 Collaborative Study for Nicotine – April 2020 27/79
Appendix 2: Modifications required for CRM No 62 when using Hexane 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 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
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.
TTPA-193-1-CTR 2019 Collaborative Study for Nicotine – April 2020 28/79
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.
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
3 Cal Level 0 37 CRP4.1-Rep2
4 Cal Level 1 38 CRP4.1-Rep3
5 Cal Level 2 39 Cal Level 0
6 Cal Level 3 40 Cal Level 0
7 Cal Level 4 41 CRP2.1-Rep1
8 Cal Level 5 42 CRP2.1-Rep2
9 Cal Level 0 43 CRP2.1-Rep3
10 Cal Level 0 44 Cal Level 0
11 NIST SRM 3222-Rep1 45 Cal Level 0
12 NIST SRM 3222-Rep2 46 RT6-Rep1
13 NIST SRM 3222-Rep3 47 RT6-Rep2
14 Cal Level 0 48 RT6-Rep3
15 Cal Level 0 49 Cal Level 0
16 VLNCMST-Rep1 50 Cal Level 0
17 VLNCMST-Rep2 51 RT8-Rep1
18 VLNCMST-Rep3 52 RT8-Rep2
19 Cal Level 0 53 RT8-Rep3
20 Cal Level 0 54 Cal Level 0
21 VLNCF1-Rep1 55 Cal Level 0
22 VLNCF1-Rep2 56 CRP3.1-Rep1
23 VLNCF1-Rep3 57 CRP3.1-Rep2
24 Cal Level 0 58 CRP3.1-Rep3
25 Cal Level 0 59 Cal Level 0
26 VLNCF2-Rep1 60 Cal Level 0
27 VLNCF2-Rep2 61 3R4F-Rep1
28 VLNCF2-Rep3 62 3R4F-Rep2
TTPA-193-1-CTR 2019 Collaborative Study for Nicotine – April 2020 29/79
Run Order Sample Name Run Order Sample Name
29 Cal Level 0 63 3R4F-Rep3
30 Cal Level 0 64 Cal Level 0
31 CRP1.1-Rep1 65 Cal Level 0
32 CRP1.1-Rep2 66 RT1-Rep1
33 CRP1.1-Rep3 67 RT1-Rep2
34 Cal Level 0 68 RT1-Rep3
TTPA-193-1-CTR 2019 Collaborative Study for Nicotine – April 2020 30/79
Appendix 3: Modifications required for CRM No 87
7. 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 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
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.
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.
TTPA-193-1-CTR 2019 Collaborative Study for Nicotine – April 2020 31/79
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.
Follow the run order specified below:
Note: this run order differs from the previous.
Run Order Sample Name Run Order Sample Name
1 Cal Level 0 35 Cal Level 0
2 Cal Level 0 36 CRP2.1-Rep1
3 Cal Level 0 37 CRP2.1-Rep2
4 Cal Level 1 38 CRP2.1-Rep3
5 Cal Level 2 39 Cal Level 0
6 Cal Level 3 40 Cal Level 0
7 Cal Level 4 41 RT6-Rep1
8 Cal Level 5 42 RT6-Rep2
9 Cal Level 0 43 RT6-Rep3
10 Cal Level 0 44 Cal Level 0
11 NIST SRM 3222-Rep1 45 Cal Level 0
12 NIST SRM 3222-Rep2 46 RT8-Rep1
13 NIST SRM 3222-Rep3 47 RT8-Rep2
14 Cal Level 0 48 RT8-Rep3
15 Cal Level 0 49 Cal Level 0
16 VLNCMST-Rep1 50 Cal Level 0
17 VLNCMST-Rep2 51 CRP3.1-Rep1
18 VLNCMST-Rep3 52 CRP3.1-Rep2
19 Cal Level 0 53 CRP3.1-Rep3
20 Cal Level 0 54 Cal Level 0
21 VLNCF1-Rep1 55 Cal Level 0
22 VLNCF1-Rep2 56 3R4F-Rep1
23 VLNCF1-Rep3 57 3R4F-Rep2
24 Cal Level 0 58 3R4F-Rep3
25 Cal Level 0 59 Cal Level 0
26 VLNCF2-Rep1 60 Cal Level 0
27 VLNCF2-Rep2 61 RT1-Rep1
TTPA-193-1-CTR 2019 Collaborative Study for Nicotine – April 2020 32/79
Run Order Sample Name Run Order Sample Name
28 VLNCF2-Rep3 62 RT1-Rep2
29 Cal Level 0 63 RT1-Rep3
30 Cal Level 0 64 Cal Level 0
31 CRP4.1-Rep1 65 Cal Level 0
32 CRP4.1-Rep2 66 CRP1.1-Rep1
33 CRP4.1-Rep3 67 CRP1.1-Rep2
34 Cal Level 0 68 CRP1.1-Rep3
TTPA-193-1-CTR 2019 Collaborative Study for Nicotine – April 2020 33/79
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
TTPA-193-1-CTR 2019 Collaborative Study for Nicotine – April 2020 34/79
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 -- -- --
TTPA-193-1-CTR 2019 Collaborative Study for Nicotine – April 2020 35/79
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
TTPA-193-1-CTR 2019 Collaborative Study for Nicotine – April 2020 36/79
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
TTPA-193-1-CTR 2019 Collaborative Study for Nicotine – April 2020 37/79
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
TTPA-193-1-CTR 2019 Collaborative Study for Nicotine – April 2020 38/79
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
TTPA-193-1-CTR 2019 Collaborative Study for Nicotine – April 2020 39/79
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
TTPA-193-1-CTR 2019 Collaborative Study for Nicotine – April 2020 40/79
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 --
TTPA-193-1-CTR 2019 Collaborative Study for Nicotine – April 2020 41/79
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
TTPA-193-1-CTR 2019 Collaborative Study for Nicotine – April 2020 42/79
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 --
TTPA-193-1-CTR 2019 Collaborative Study for Nicotine – April 2020 43/79
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
TTPA-193-1-CTR 2019 Collaborative Study for Nicotine – April 2020 44/79
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
TTPA-193-1-CTR 2019 Collaborative Study for Nicotine – April 2020 45/79
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
TTPA-193-1-CTR 2019 Collaborative Study for Nicotine – April 2020 46/79
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
TTPA-193-1-CTR 2019 Collaborative Study for Nicotine – April 2020 47/79
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
TTPA-193-1-CTR 2019 Collaborative Study for Nicotine – April 2020 48/79
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
TTPA-193-1-CTR 2019 Collaborative Study for Nicotine – April 2020 49/79
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
TTPA-193-1-CTR 2019 Collaborative Study for Nicotine – April 2020 50/79
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 --
TTPA-193-1-CTR 2019 Collaborative Study for Nicotine – April 2020 51/79
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
TTPA-193-1-CTR 2019 Collaborative Study for Nicotine – April 2020 52/79
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
TTPA-193-1-CTR 2019 Collaborative Study for Nicotine – April 2020 53/79
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
TTPA-193-1-CTR 2019 Collaborative Study for Nicotine – April 2020 54/79
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
TTPA-193-1-CTR 2019 Collaborative Study for Nicotine – April 2020 55/79
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 --
TTPA-193-1-CTR 2019 Collaborative Study for Nicotine – April 2020 56/79
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
TTPA-193-1-CTR 2019 Collaborative Study for Nicotine – April 2020 57/79
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 -- --
TTPA-193-1-CTR 2019 Collaborative Study for Nicotine – April 2020 58/79
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 --
TTPA-193-1-CTR 2019 Collaborative Study for Nicotine – April 2020 59/79
APPENDIX C: Raw Data Plots
• MTBE
■ Hexane
▲GC/MS
• MTBE
■ Hexane
▲GC/MS
TTPA-193-1-CTR 2019 Collaborative Study for Nicotine – April 2020 60/79
• MTBE
■ Hexane
▲GC/MS
• MTBE
■ Hexane
▲GC/MS
TTPA-193-1-CTR 2019 Collaborative Study for Nicotine – April 2020 61/79
• MTBE
■ Hexane
▲GC/MS
• MTBE
■Hexane
▲GC/MS
TTPA-193-1-CTR 2019 Collaborative Study for Nicotine – April 2020 62/79
• MTBE
■ Hexane
▲GC/MS
• MTBE
■ Hexane
▲GC/MS
TTPA-193-1-CTR 2019 Collaborative Study for Nicotine – April 2020 63/79
• MTBE
■ Hexane
▲GC/MS
• MTBE
■ Hexane
▲GC/MS
TTPA-193-1-CTR 2019 Collaborative Study for Nicotine – April 2020 64/79
• MTBE
■ Hexane
▲GC/MS
• MTBE
■ Hexane
▲GC/MS
TTPA-193-1-CTR 2019 Collaborative Study for Nicotine – April 2020 65/79
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)
Calibration Level Nicotine (mg/ml) % Relative Concentration Residual
(%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)
Calibration Level Nicotine (mg/ml) % Relative Concentration Residual
(%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 %
TTPA-193-1-CTR 2019 Collaborative Study for Nicotine – April 2020 66/79
CRM No. 62 MTBE (Lab Code 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 %
CRM No. 87 (Lab Code 2)
Calibration Level Nicotine (mg/ml) % Relative Concentration Residual
(%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 %
CRM No. 62 MTBE (Lab Code 3)
Calibration Level Nicotine (mg/ml) % Relative Concentration Residual
(%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 %
TTPA-193-1-CTR 2019 Collaborative Study for Nicotine – April 2020 67/79
CRM No. 87 (Lab Code 3)
Calibration Level Nicotine (mg/ml) % Relative Concentration Residual
(%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 %
CRM No. 87 (Lab Code 4)
Calibration Level Nicotine (mg/ml) % Relative Concentration Residual
(%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%
CRM No. 62 Hexane (Lab Code 5)
Calibration Level Nicotine (mg/ml) % Relative Concentration Residual
(%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 %
TTPA-193-1-CTR 2019 Collaborative Study for Nicotine – April 2020 68/79
CRM No. 87 (Lab Code 5)
Calibration Level Nicotine (mg/ml) % Relative Concentration Residual
(%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 %
CRM No. 62 MTBE (Lab Code 6)
Calibration Level Nicotine (mg/ml) % Relative Concentration Residual
(%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 %
CRM No. 87 (Lab Code 6)
Calibration Level Nicotine (mg/ml) % Relative Concentration Residual
(%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%
TTPA-193-1-CTR 2019 Collaborative Study for Nicotine – April 2020 69/79
CRM No. 62 Hexane (Lab Code 7)
Calibration Level Nicotine (mg/ml) % Relative Concentration Residual
(%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%
CRM No. 62 MTBE (Lab Code 8)
Calibration Level Nicotine (mg/ml) % Relative Concentration Residual
(%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 %
CRM No. 87 (Lab Code 8)
Calibration Level Nicotine (mg/ml) % Relative Concentration Residual
(%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 %
TTPA-193-1-CTR 2019 Collaborative Study for Nicotine – April 2020 70/79
CRM No. 62 MTBE (Lab Code 9)
Calibration Level Nicotine (mg/ml) % Relative Concentration Residual
(%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 %
CRM No. 87 (Lab Code 9)
Calibration Level Nicotine (mg/ml) % Relative Concentration Residual
(%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 %
CRM No. 62 MTBE (Lab Code 10)
Calibration Level Nicotine (mg/ml) % Relative Concentration Residual
(%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 %
TTPA-193-1-CTR 2019 Collaborative Study for Nicotine – April 2020 71/79
CRM No. 62 Hexane (Lab Code 10)
Calibration Level Nicotine (mg/ml) % Relative Concentration Residual
(%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 %
CRM No. 87 (Lab Code 10)
Calibration Level Nicotine (mg/ml) % Relative Concentration Residual
(%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 %
CRM No. 62 MTBE (Lab Code 11)
Calibration Level Nicotine (mg/ml) % Relative Concentration Residual
(%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
TTPA-193-1-CTR 2019 Collaborative Study for Nicotine – April 2020 72/79
CRM No. 87 (Lab Code 11)
Calibration Level Nicotine (mg/ml) % Relative Concentration Residual
(%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 %
CRM No. 62 MTBE (Lab Code 12)
Calibration Level Nicotine (mg/ml) % Relative Concentration Residual
(%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 %
CRM No. 62 MTBE (Lab Code 13)
Calibration Level Nicotine (mg/ml) % Relative Concentration Residual
(%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 %
TTPA-193-1-CTR 2019 Collaborative Study for Nicotine – April 2020 73/79
CRM No. 62 Hexane (Lab Code 13)
Calibration Level Nicotine (mg/ml) % Relative Concentration Residual
(%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 %
CRM No. 87 (Lab Code 13)
Calibration Level Nicotine (mg/ml) % Relative Concentration Residual
(%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 %
CRM No. 62 MTBE (Lab Code 17)
Calibration Level Nicotine (mg/ml) % Relative Concentration Residual
(%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 %
TTPA-193-1-CTR 2019 Collaborative Study for Nicotine – April 2020 74/79
CRM No. 62 Hexane (Lab Code 17)
Calibration Level Nicotine (mg/ml) % Relative Concentration Residual
(%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 %
CRM No. 87 (Lab Code 17)
Calibration Level Nicotine (mg/ml) % Relative Concentration Residual
(%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%
CRM No. 62 MTBE (Lab Code 19)
Calibration Level Nicotine (mg/ml) % Relative Concentration Residual
(%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 %
TTPA-193-1-CTR 2019 Collaborative Study for Nicotine – April 2020 75/79
CRM No. 87 (Lab Code 19)
Calibration Level Nicotine (mg/ml) % Relative Concentration Residual
(%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 %
CRM No. 62 MTBE (Lab Code 20)
Calibration Level Nicotine (mg/ml) % Relative Concentration Residual
(%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 %
CRM No. 62 Hexane (Lab Code 20)
Calibration Level Nicotine (mg/ml) % Relative Concentration Residual
(%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 %
TTPA-193-1-CTR 2019 Collaborative Study for Nicotine – April 2020 76/79
CRM No. 87 (Lab Code 20)
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 %
CRM No. 62 MTBE (Lab Code 21)
Calibration Level Nicotine (mg/ml) % Relative Concentration Residual
(%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 %
CRM No. 87 (Lab Code 21)
Calibration Level Nicotine (mg/ml) % Relative Concentration Residual
(%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 %
TTPA-193-1-CTR 2019 Collaborative Study for Nicotine – April 2020 77/79
CRM No. 62 MTBE (Lab Code 22)
Calibration Level Nicotine (mg/ml) % Relative Concentration Residual
(%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 %
CRM No. 62 Hexane (Lab Code 22)
Calibration Level Nicotine (mg/ml) % Relative Concentration Residual
(%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 %
CRM No. 87 (Lab Code 22)
Calibration Level Nicotine (mg/ml) % Relative Concentration Residual
(%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 %
TTPA-193-1-CTR 2019 Collaborative Study for Nicotine – April 2020 78/79
CRM No. 87 (Lab Code 23)
Calibration Level Nicotine (mg/ml) % Relative Concentration Residual
(%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 %
CRM No. 62 MTBE (Lab Code 24)
Calibration Level Nicotine (mg/ml) % Relative Concentration Residual
(%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 %
CRM No. 62 Hexane (Lab Code 24)
Calibration Level Nicotine (mg/ml) % Relative Concentration Residual
(%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 %
TTPA-193-1-CTR 2019 Collaborative Study for Nicotine – April 2020 79/79
CRM No. 87 (Lab Code 24)
Calibration Level Nicotine (mg/ml) % Relative Concentration Residual
(%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 %
CRM No. 62 Hexane (Lab Code 25)
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 %