technical report coresta reference products 2019 analysis · 2020. 3. 25. · similar to the 2009...

Tobacco and Tobacco Products Analytes Sub-Group

Technical Report

CORESTA Reference Products

2019 Analysis

March 2020

Author and Study Coordinator:

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. Organisation .................................................................................................................... 4

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

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

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

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

3.2.3 Sample Analysis and Data Reporting ........................................................ 5

4. Data – Raw ...................................................................................................................... 6

5. Data – Statistical Analysis .............................................................................................. 6

5.1 Exclusion of Outliers ............................................................................................. 6

5.2 Calculation of Repeatability (r) and Reproducibility (R) ...................................... 7

5.3 Calculation of Z-Scores ....................................................................................... 10

5.4 CRP Stability Assessment ................................................................................... 12

6. Data Interpretations ....................................................................................................... 13

7. Recommendations ......................................................................................................... 14

APPENDIX A: Raw Data (results presented on an as-is basis and outliers are included) .... 15

APPENDIX B: Raw Data Plots for 2019 Study .................................................................... 20

APPENDIX C: Graphs Comparing 2016 and 2019 Results .................................................. 34

TTPA-220-CTR 2019 Analysis of CRPs – March 2020 3/47

1. Summary

In April 2019, the CORESTA Tobacco and Tobacco Products Analytes Sub-Group (TTPA)

initiated an interlaboratory study designed to assess the stability of the four CORESTA

Reference Products (CRPs) manufactured in 2016, and to provide repeatability (r) and

reproducibility (R) results and z-scores to support laboratory accreditation. Thirteen

laboratories participated in the study. The participating laboratories reported the levels of

nicotine, pH, moisture (oven volatiles), and tobacco specific nitrosamines (TSNAs) in the CRPs

using CORESTA Recommended Methods (CRMs). Tabulated data are presented along with

r and R estimates and z-scores.

The results from this 2019 stability analyses generally compared well to the 2016 stability

analyses. The only statistically significant difference from the 2016 results was an increase in

moisture in CRP3.1, similar to what had been previously observed with CRP3 from the 2009

CRPs[1].

2. Introduction

In November 2008, the Smokeless Tobacco Sub-Group (STS), now named Tobacco and

Tobacco Product Analytes Sub-Group (TTPA), was established by recommendation of the

CORESTA Scientific Commission. In 2009, STS Working Group Three (WG3) cooperated to

design and manufacture four CORESTA Reference Products (CRPs) referred to as CRP1,

CRP2, CRP3, and CRP4. These products were intended as replacements for the Smokeless

Tobacco Research Products: 2S3 (Moist Snuff), 1S2 (Dry Snuff) and 2S1 (Loose-leaf Chewing

Tobacco), which were more than ten years old. The STS recommended monitoring the stability

of the 2009 CRPs on an annual basis, by determining the levels of nicotine, pH, moisture (oven

volatiles) and TSNAs using CRMs or draft CRMs[2]. These studies were conducted through

2015 and demonstrated that the CRPs were stable when held at the recommended storage

conditions of -20 °C.

In 2015 the supply of the 2009 CRPs decreased to the point that the STS decided to

remanufacture the CRPs. Similar to the 2009 production, four CRPs were manufactured in

2016 and included Swedish snus pouch (CRP1.1), American-style loose moist snuff (CRP2.1),

American-style loose dry snuff powder (CRP3.1), and American-style chopped loose-leaf

chewing tobacco (CRP4.1). CRP4.1 was produced in a chopped format in 2016 to improve

within package sample homogeneity as compared to the 2009 loose leaf format. Even though

the 2016 CRPs included the four product styles that were produced in 2009, it was anticipated

the chemistry would differ between the two productions considering different tobaccos were

used and different manufacturers produced three of the reference products. In 2016, an initial

characterization of the 2016 CRPs was reported for levels of nicotine, pH, moisture, TSNAs,

ammonia and B[a]P using the applicable CRMs[3]. This report serves as the second

characterization of the 2016 CRPs by reporting the levels of nicotine, pH, moisture, and TSNAs

using the applicable CRMs.

The 2016 CRPs are stored at -20 °C and distributed by the North Carolina State University

(NCSU) Tobacco Analytical Services Laboratory.

[1] STS Technical Report: 2009 Collaborative Study (Smokeless Tobacco r&R Study), updated January 2016. [2] STS Technical Report: CORESTA Reference Products 2010 Analysis, February 2014. [3] STS Technical Report: CORESTA Reference Products (Smokeless Tobacco) – 2016 Analysis.


2.1 Objective

The 2019 participating laboratories were to provide analytical results for nicotine, pH, moisture

(oven volatiles), the four TSNAs N-nitrosonornicotine (NNN), N-nitrosoanatabine (NAT),

N-nitrosoanabasine (NAB) 4-N-nitrosomethylamino-1-3-pyridyl-1-butanone (NNK) for the

purpose of examining the stability of the CRPs. Results were reported on an as-is basis and not

corrected for moisture. This work was conducted using the applicable CRMs referenced in

Section 3.

This study was conducted to support the characterization of the 2016 CRPs, to provide an

assessment of inter-laboratory variability, and to examine the stability of the CRPs. 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. Additionally, z-

scores were calculated as a measure of each laboratory’s performance as compared to the results

of other laboratories. The results from this year’s evaluation were compared to the results from

2016 using a t-test with p<0,0125 as the criterion for statistical significance. The

0,0125 criterion is based on adjusting the standard 0,05 criterion to account for the four

reference products being compared.

3. Organisation

3.1 Participants

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

for all analyses. The laboratories are listed in alphabetical order. Letter codes were assigned

to each laboratory and do not correspond to the order in the table below.

Table 1: List of Participating Laboratories

Participating Laboratories

Altria Client Services LLC, United States

American Snuff Company, United States

Enthalpy Analytical Richmond, United States

Global Laboratory Services, United States

British American Tobacco (BAT), Germany

Scandinavian Tobacco Group, Eersel, The Netherlands

Japan Tobacco, Inc., Japan

Cia. Industrial de Tabacos Monte Paz S.A., Uruguay

R.J. Reynolds Tobacco Company, United States

Swedish Match Northern Europe, Sweden

Swedish Match, Owensboro, United States

Swisher International, United States

University of Kentucky, United States


3.2 Protocol

Specific details from the protocol are described below.

3.2.1 Sample Shipment

Laboratories were responsible for procuring each of the CRPs from NCSU immediately before

starting the study. 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. Laboratories were requested to conduct

the study in May through August and report data by August 20, 2019. The samples are

identified in Table 2.

Table 2: Sample Identification

Product Type

CRP1.1 - Swedish style snus pouch

CRP2.1 - American-style loose moist snuff

CRP3.1 - American-style loose dry snuff powder

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

3.2.2 Laboratory Sample Preparation

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

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

equilibrated. Samples could then be removed from the refrigerator for a minimum of 2 hours

prior to opening for analysis. Once samples were opened, the samples could be stored in a

tightly sealed container and stored at approximately 4 °C for up to one week. Handling

requirements for the CRPs are described below:

3.2.2.1 The snus pouch (CRP1.1) was to be cut into two halves and placed directly into

the extraction vessel. Both the tobacco and pouch material were included in the

analysis.

3.2.2.2 The moist snuff (CRP2.1), dry snuff product (CRP3.1), and chopped loose-leaf

(CRP4.1) did not require sample grinding and were to be analyzed as received.

3.2.3 Sample Analysis and Data Reporting

The participating laboratories were instructed to conduct triplicate analyses for the following:

nicotine, pH, moisture (oven volatiles), and TSNAs. The laboratories were requested to use the

current versions of the following CRMs:

3.2.3.1 pH: CRM N° 69, Determination of pH in Smokeless Tobacco Products

3.2.3.2 Moisture (oven volatiles): CRM N° 76, Determination of Moisture Content (Oven

Volatiles) of Smokeless Tobacco Products

3.2.3.3 Nicotine:

3.2.3.3.1 CRM N° 62, Determination of Nicotine in Tobacco and Tobacco Products

by Gas Chromatographic Analysis. Either the methyl tert-butyl ether

(MTBE) or n-hexane extraction may be used. The extraction method shall

be specified in the data reporting sheet.

or

3.2.3.3.2 CRM No. 87, Determination of Nicotine in Tobacco Products by GC/MS


3.2.3.4 TSNAs: CRM N° 72, Determination of Tobacco Specific Nitrosamines in

Smokeless Tobacco Products by Liquid Chromatography - Tandem Mass

Spectrometry

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

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

with significant deviations from the applicable CRM were excluded from the r&R portion of

the study. Deviations reported by the laboratories are identified below as well as if the data

were excluded from the study.

• Lab H: Major deviations were reported for nicotine where CRM N° 85-Tobacco,

Determination of the content of total alkaloids as Nicotine-Continuous Flow Analysis

Method was used instead of CRM N° 62 or CRM N° 87. These results were not

included in the study.

All test results were to be reported on an as-is basis with no correction for moisture content.

The results were not to be rounded and ideally reported to at least one more digit than typically

required.

The study results and the comments were to be sent by e-mail to the study coordinators

(Anthony Brown and Karl Wagner).

4. Data – Raw

The full data set for the study, including outliers, is listed in Appendix A. 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. Raw data plots that include all

replicates, without removal of outliers, are given in Appendix B.

5. Data – Statistical Analysis

A statistical analysis was conducted in basic conformance with ISO 5725-2:1994 and

ISO/TR 22971:2005. A summary of the results from outlier detection and the calculated results

for repeatability (r) and reproducibility (R) are given below in sections 5.1 and 5.2, respectively.

Even though ISO 5725-2:1994 does not suggest calculation of z-scores, z-scores are presented

in section 5.3 so that the participating laboratories would have an additional measure of their

performance compared to their peers. Raw data plots that include all replicates, without

removal of outliers, are shown in Appendix B.

5.1 Exclusion of Outliers

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

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

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

laboratories with outlying mean values.

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

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

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

Test. 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


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

The intent of ISO 5725-2:1994 is to eliminate outliers that exceed a 1 % critical value. This

was accomplished by an adaptation of Levene’s Test. Levene’s Test is preferable to Cochran’s

Test, which is recommended in ISO 5725-2:1994, because of Cochran’s Test’s extreme

sensitivity to deviations from normality. Grubbs’ Test and an adaptation of Levene’s Test were

applied at the standard nominal 1 % significance level to determine outliers and the results are

shown in Table 3. Levene’s Test is mentioned in ISO/TR 22971:2005 as an acceptable

alternative to Cochran’s Test. However, Levene’s Test does not directly apply without

adaptation. For more details, see the footnote below.[4]

Table 3: Outliers

Analyte Product Levene’s Outlier Lab Grubbs’ Outlier Lab

pH CRP3.1 K –

NAB CRP4.1 – B

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

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

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

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

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

product consistency.

Table 4: Repeatability (r) and Reproducibility (R) Limits

Parameter Product N° of

Labs* Mean

Repeatability Reproducibility

r % r R % R

Nicotine (mg/g) CRP1.1 11 7,30 0,77 10,6 % 1,29 17,7 %

Nicotine (mg/g) CRP2.1 11 10,16 0,29 2,8 % 1,59 15,6 %

Nicotine (mg/g) CRP3.1 11 16,44 0,44 2,7 % 1,90 11,5 %

Nicotine (mg/g) CRP4.1 11 8,77 0,13 1,5 % 0,89 10,1 %

pH CRP1.1 13 8,19 0,053 NA 0,353 NA

pH CRP2.1 13 7,68 0,029 NA 0,170 NA

pH CRP3.1 12 6,89 0,052 NA 0,203 NA

pH CRP4.1 13 6,06 0,027 NA 0,191 NA

[4] Levene’s Test is commonly used to determine if each of several subpopulations have the same variance. Since

it was designed to test for overall differences, not to determine if the largest variance is significantly greater than

the others, some adaptation is necessary to use the approach to eliminate laboratories whose within lab variation

is too large. Levene’s Test was adapted to this purpose by Morton, who presented the approach utilized in this

report at the 2014 CORESTA Congress (Quebec, Canada, presentation ST28, October 14, 2014). Specifically, the

approach taken here is a two-step process with a lab being eliminated as an outlier if both steps are statistically

significant. First, Levene’s Test was run at a nominal -level of 0,02. Second a comparison of the largest variance

to the remaining variances is carried out at a one-sided nominal level of =0,01/number of labs. Dividing by the

number of labs is to account for multiple testing, since it is not known a priori which lab will have the largest

variance. Simulation studies were carried out by Morton and presented at the 2014 CORESTA Congress and these

results demonstrated that this process has an overall -level near 0,01 and is robust to deviations from normality.


Parameter Product N° of

Labs* Mean

Repeatability Reproducibility

r % r R % R

Moisture (%) CRP1.1 12 53,56 1,45 2,7 % 1,59 3,0 %

Moisture (%) CRP2.1 12 51,22 0,74 1,4 % 1,36 2,7 %

Moisture (%) CRP3.1 12 7,87 0,23 3,0 % 1,58 20,1 %

Moisture (%) CRP4.1 12 24,12 0,75 3,1 % 3,03 12,6 %

NNN (µg/g) CRP1.1 10 0,199 0,035 17,7 % 0,060 30,4 %

NNN (µg/g) CRP2.1 10 3,49 0,218 6,3 % 0,867 24,8 %

NNN (µg/g) CRP3.1 10 5,74 0,269 4,7 % 1,792 31,2 %

NNN (µg/g) CRP4.1 10 3,57 0,303 8,5 % 0,706 19,8 %

NNK (µg/g) CRP1.1 10 0,05 0,015 29,0 % 0,036 71,2 %

NNK (µg/g) CRP2.1 10 2,07 0,108 5,2 % 0,343 16,6 %

NNK (µg/g) CRP3.1 10 2,59 0,205 7,9 % 0,438 16,9 %

NNK (µg/g) CRP4.1 10 0,82 0,101 12,4 % 0,136 16,6 %

NAT (µg/g) CRP1.1 10 0,14 0,028 19,6 % 0,037 25,9 %

NAT (µg/g) CRP2.1 10 4,12 0,279 6,8 % 0,833 20,2 %

NAT (µg/g) CRP3.1 10 4,25 0,264 6,2 % 0,723 17,0 %

NAT (µg/g) CRP4.1 10 1,59 0,173 10,9 % 0,310 19,4 %

NAB (µg/g) CRP1.1 9 0,009 0,0027 30,6 % 0,0053 59,6 %

NAB (µg/g) CRP2.1 10 0,268 0,0274 10,2 % 0,0465 17,4 %

NAB (µg/g) CRP3.1 10 0,309 0,0394 12,8 % 0,0569 18,4 %

NAB (µg/g) CRP4.1 9 0,115 0,0091 7,9 % 0,0130 11,3 %

* This is the number of laboratory data sets reporting numerical values and after removal of outliers.

NA = Since pH is not a proportional scale, it is not appropriate to calculate % r or % R.

The r&R values from this study are compared to the values obtained in the 2016 study in Table 5

and the results from this study are generally comparable to or slightly less variable than the

2016 results.


Table 5. Comparison of Repeatability and Reproducibility

Parameter Product

Current Study1

Previous Study2

Current Study1

Previous Study2

Current Study1

Previous Study2

Mean Mean % r % r % R % R

Nicotine (mg/g) CRP1.1 7.30 7.62 10.6 % 11.0 % 17.7 % 16.8 %

Nicotine (mg/g) CRP2.1 10.16 10.69 2.8 % 5.0 % 15.6 % 24.8 %

Nicotine (mg/g) CRP3.1 16.44 17.22 2.7 % 1.9 % 11.5 % 12.2 %

Nicotine (mg/g) CRP4.1 8.77 8.86 1.5 % 7.9 % 10.1 % 13.1 %

pH CRP1.1 8.19 8.30 NA NA NA NA




Moisture (%) CRP1.1 53.56 53.95 2.7 % 2.1 % 3.0 % 3.4 %

Moisture (%) CRP2.1 51.22 51.41 1.4 % 0.6 % 2.7 % 1.4 %

Moisture (%) CRP3.1 7.87 7.33 3.0 % 2.6 % 20.1 % 10.1 %

Moisture (%) CRP4.1 24.12 24.19 3.1 % 2.5 % 12.6 % 8.9 %

NNN (µg/g) CRP1.1 0.199 0.19 17.7 % 22.1 % 30.4 % 35.8 %

NNN (µg/g) CRP2.1 3.492 3.391 6.3 % 6.3 % 24.8 % 25.8 %

NNN (µg/g) CRP3.1 5.736 5.631 4.7 % 5.7 % 31.2 % 27.3 %

NNN (µg/g) CRP4.1 3.567 3.394 8.5 % 5.8 % 19.8 % 21.8 %

NNK (µg/g) CRP1.1 0.050 0.052 29.0 % 25.2 % 71.2 % 56.6 %

NNK (µg/g) CRP2.1 2.069 2.059 5.2 % 10.2 % 16.6 % 25.5 %

NNK (µg/g) CRP3.1 2.586 2.512 7.9 % 5.5 % 16.9 % 22.6 %

NNK (µg/g) CRP4.1 0.816 0.79 12.4 % 11.4 % 16.6 % 33.1 %

NAT (µg/g) CRP1.1 0.141 0.14 19.6 % 22.9 % 25.9 % 50.7 %

NAT (µg/g) CRP2.1 4.123 4.237 6.8 % 8.8 % 20.2 % 21.0 %

NAT (µg/g) CRP3.1 4.251 4.327 6.2 % 2.7 % 17.0 % 20.2 %

NAT (µg/g) CRP4.1 1.594 1.588 10.9 % 6.8 % 19.4 % 23.8 %

NAB (µg/g) CRP1.1 0.009 0.009 30.6 % 35.9 % 59.6 % 63.2 %

NAB (µg/g) CRP2.1 0.268 0.265 10.2 % 10.2 % 17.4 % 25.4 %

NAB (µg/g) CRP3.1 0.309 0.303 12.8 % 10.8 % 18.4 % 28.0 %

NAB (µg/g) CRP4.1 0.115 0.115 7.9 % 9.4 % 11.3 % 37.9 %

1. The columns labelled “Current Study“ include data from the current 2019 study for the 2016 CRPs.

2. The columns labelled “Previous Study“ include data from the previous 2016 study for the 2016 CRPs[5].

[5] STS Technical Report: CORESTA Reference Products 2016 Analysis, January 2017.


5.3 Calculation of Z-Scores

Although calculation of z-scores is not suggested in ISO 5725-2:1994, z-scores were calculated

so that the participating laboratories could compare their results to those of their peers. As

mentioned above, data sets were removed from the r&R portion of the study due to significant

deviations from the study protocol or if the data were identified as outlying data. The assigned

value and standard deviation for proficiency assessment were calculated after removing

outliers; however, z-scores were calculated for the outliers. Generally speaking, absolute

z-scores less than 2 are acceptable, absolute z-scores between 2 and 3 constitute a warning, and

those above 3 should be investigated.

Final summary tables of z-scores are presented in Tables 6a through 6d for the study.

Table 6a: Z-Scores

Lab Nicotine pH

CRP1.1 CRP2.1 CRP3.1 CRP4.1 CRP1.1 CRP2.1 CRP3.1 CRP4.1

A 0,76 0,17 0,25 0,00 0,93 -0,28 0,24 0,20

B – – – – -1,04 -1,28 0,43 0,15

C -0,08 -0,05 0,52 -0,15 0,40 0,61 1,79 -0,34

D 0,54 0,87 1,28 1,26 -0,83 1,33 -0,47 -1,13

E 1,55 0,34 0,35 -0,40 1,28 -1,50 0,43 -0,39

F 0,14 0,52 -0,04 -0,49 -1,71 -0,09 -0,31 0,87

G 0,65 -2,27 -2,14 -0,97 -0,14 -0,28 0,24 0,50

H – – – – -0,46 0,22 0,80 0,94

I -0,70 -1,22 -0,88 -0,46 1,23 1,78 -0,19 -0,39

J -0,79 0,88 -0,04 1,37 -1,20 -1,39 -2,40 -2,51

K 0,86 0,94 1,09 1,15 -0,16 -0,06 5,32 0,99

L -1,21 -0,61 -1,00 -1,83 0,91 0,16 0,19 1,04

M -1,72 0,42 0,62 0,52 0,79 0,79 -0,74 0,08

The (–) symbol indicates the laboratory did not submit data for that sample analysis.


Table 6b: Z-Scores

Lab Moisture NNN


A 0,59 -0,52 -0,24 -0,16 – – – –

B – – – – 0,66 0,36 0,14 0,12

C -0,52 0,32 -0,02 0,12 -0,26 -0,68 -0,46 -0,40

D -2,11 -1,73 -2,24 -1,48 0,52 0,13 0,23 -0,17

E 0,80 0,86 1,53 2,66 0,70 0,23 0,33 -0,46

F 0,77 -0,65 0,80 -0,28 0,92 0,19 0,11 0,27

G 1,33 0,19 0,35 0,07 1,47 1,76 1,87 1,59

H 0,04 -0,01 -0,65 -0,68 – – – –

I 0,65 2,09 0,59 0,62 – – – –

J -0,01 0,45 -0,19 -0,76 -1,25 -0,10 -0,11 0,86

K -0,02 -1,27 -1,03 -0,03 -1,36 -2,26 -2,25 -2,25

L 0,06 -0,21 0,98 0,27 -1,11 0,28 0,10 0,00

M -1,57 0,48 0,10 -0,35 -0,28 0,09 0,06 0,43


Table 6c: Z-Scores

Lab NNK NAT


A – – – – – – – –

B 2,16 -0,89 0,05 0,59 0,28 -2,08 -2,22 -1,26

C -0,02 -0,29 -0,02 0,48 1,12 0,63 1,25 0,80

D 0,24 0,06 0,21 -0,41 -0,20 -0,36 0,09 -1,43

E 0,30 1,51 0,30 0,63 0,63 0,54 -0,29 0,82

F 0,64 1,23 1,39 0,70 0,28 -0,12 -0,45 -0,59

G -0,37 -1,41 -1,62 -2,51 0,77 0,41 0,81 0,38

H – – – – – – – –

I – – – – – – – –

J -0,54 -0,09 -0,45 -0,22 -2,04 -0,36 -0,26 -0,27

K -1,67 -1,24 -1,44 -0,50 0,80 1,84 1,21 1,82

L 0,00 0,26 0,16 0,75 -0,37 -0,17 0,06 0,17

M -0,74 0,86 1,41 0,48 -1,27 -0,32 -0,20 -0,43



Table 6d: Z-Scores

Lab NAB

CRP1.1 CRP2.1 CRP3.1 CRP4.1

A – – – –

B 0,02 1,86 0,88 -2,62

C 0,02 0,89 1,27 2,07

D 0,12 0,96 0,98 -0,37

E 0,18 -0,47 -1,13 0,77

F 2,35 -0,33 0,49 0,34

G -0,17 0,35 0,60 0,43

H – – – –

I – – – –

J * -1,40 -1,72 -0,76

K -0,56 -1,02 -0,63 -0,88

L -0,68# -0,31 -0,09 -0,62

M -1,28 -0,52 -0,66 -0,98


The (#) symbol indicates one of the three replicate values was reported as below the

lab reporting threshold and was omitted.

The (*) symbol indicates all three replicates were below the lab reporting threshold.

5.4 CRP Stability Assessment

The data were evaluated for stability by comparing the results of the testing in 2016 with the

testing in 2019 and the analysis is summarized in Table 7. Graphs comparing the 2016 and

2019 results are shown in Appendix C.

Table 7: Summary of Stability Analysis

Product Variable 2016 Avg.

Test Results

2019 Avg.

Test Results p-value1

CRP1.1 Nicotine 7,62 7,30 0,062

CRP2.1 Nicotine 10,69 10,16 0,111

CRP3.1 Nicotine 17,23 16,44 0,013

CRP4.1 Nicotine 8,86 8,77 0,521

CRP1.1 Moisture 53,95 53,56 0,046

CRP2.1 Moisture 51,41 51,22 0,132

CRP3.1 Moisture 7,33 7,87 0,002

CRP4.1 Moisture 24,19 24,12 0,845

CRP1.1 pH 0,14 0,14 0,937

CRP2.1 pH 0,27 0,27 0,374

CRP3.1 pH 0,30 0,31 0,546


Product Variable 2016 Avg.

Test Results

2019 Avg.

Test Results p-value1

CRP4.1 pH 1,59 1,59 0,917

CRP1.1 NNN 8,300 8,190 0,084

CRP2.1 NNN 7,740 7,680 0,099

CRP3.1 NNN 6,960 6,890 0,143

CRP4.1 NNN 6,080 6,060 0,560

CRP1.1 NNK 0,190 0,199 0,306

CRP2.1 NNK 3,391 3,492 0,456

CRP3.1 NNK 5,631 5,736 0,679

CRP4.1 NNK 3,394 3,567 0,127

CRP1.1 NAT 0,052 0,050 0,672

CRP2.1 NAT 4,237 4,123 0,878

CRP3.1 NAT 2,512 2,586 0,339

CRP4.1 NAT 0,790 0,816 0,398

CRP1.1 NAB 0,009 0,009 0,814

CRP2.1 NAB 2,059 2,069 0,729

CRP3.1 NAB 4,327 4,251 0,582

CRP4.1 NAB 0,115 0,115 0,960

1. P-values are declared statistically significant if p < 0,0125. 0,0125 is derived by dividing the nominal 0,05

level by 4 to account for testing multiplicity per analyte.

Because of the large number of comparisons being made in Table 7, some consideration should

be made to allow for testing multiplicity. Our practice for stability studies has been to adjust

by analyte. That means a comparison is judged to be statistically significant if the p-value is

less than 0,05 / 4 = 0,0125, as noted in the footnote to Table 7. The divisor of four is based on

four comparisons being made per analyte, one for each CRP. Only moisture for CRP3.1 reaches

statistical significance by that criterion. That is consistent with what was seen in CRP3 from

the 2009 group of CRPs. Nicotine in CRP3.1 was near statistical significance but did not quite

meet the criterion. Along with the other analytes, it will be examined in future studies to

determine if there is further evidence of a trend.

6. Data Interpretations

In terms of stability, only moisture for CRP3.1 showed a statistically significant difference in

comparison to the testing conducted in 2016. That increase was consistent with the pattern seen

for CRP3 from the 2009 CRPs. The CRP3.1 moisture increase from the time of manufacture

is approximately 0,5 %. As mentioned earlier, results are presented on an as-is basis; however,

this negligible shift in moisture for CRP3.1 would not be evident in the other chemical analyses

considering the negligible change in moisture in comparison with lab-to-lab method variability.

A comparison of the r & R results was presented in Table 5. Generally speaking both the

repeatability and reproducibility in this study were comparable or slightly less than the

corresponding values seen in the 2016 study of these CRPs.


7. Recommendations

The results of this study were consistent with those in studies of the 2009 CRPs. Most analytes

were not statistically significantly different from the results in 2016. The exception was

moisture for CRP3.1 which was also observed for CRP3. The TTPA recommends that the

stability of the 2016 CRPs be continued to be monitored on a biennial basis.


APPENDIX A: Raw Data (results are presented on an as-is basis and outliers are included)

CODE Sample Replicate Nicotine

pH

Oven volatiles

NAB NAT NNK NNN

mg/g % µg/g µg/g µg/g µg/g

A CRP1.1 1 7,804 8,330 54,348 – – – –

A CRP1.1 2 7,389 8,300 53,604 – – – –

A CRP1.1 3 7,634 8,300 53,382 – – – –

B CRP1.1 1 – 8,070 – 0,0110 0,141 0,065 0,214

B CRP1.1 2 – 8,060 – 0,0070 0,151 0,080 0,220

B CRP1.1 3 – 8,060 – 0,0090 0,140 0,083 0,200

C CRP1.1 1 7,234 8,230 53,190 0,0089 0,153 0,051 0,194

C CRP1.1 2 7,308 8,240 53,630 0,0089 0,153 0,051 0,196

C CRP1.1 3 7,265 8,260 53,260 0,0092 0,152 0,048 0,192

D CRP1.1 1 7,583 8,070 52,529 0,0089 0,138 0,051 0,206

D CRP1.1 2 7,493 8,110 53,490 0,0098 0,138 0,055 0,216

D CRP1.1 3 7,486 8,090 52,247 0,0088 0,140 0,053 0,205

E CRP1.1 1 8,207 8,340 52,630 0,0094 0,138 0,049 0,199

E CRP1.1 2 7,540 8,330 53,993 0,0105 0,164 0,064 0,216

E CRP1.1 3 8,032 8,390 54,952 0,0079 0,141 0,048 0,221

F CRP1.1 1 7,290 7,980 53,902 0,0135 0,143 0,061 0,225

F CRP1.1 2 7,499 7,970 53,931 0,0127 0,143 0,055 0,209

F CRP1.1 3 7,287 7,990 53,710 0,0128 0,146 0,057 0,215

G CRP1.1 1 7,595 8,160 54,650 0,0090 0,154 0,047 0,230

G CRP1.1 2 7,689 8,180 53,170 0,0090 0,161 0,047 0,242

G CRP1.1 3 7,413 8,190 54,360 0,0080 0,132 0,043 0,208

H CRP1.1 1 – 8,130 53,480 – – – –

H CRP1.1 2 – 8,130 53,750 – – – –

H CRP1.1 3 – 8,150 53,480 – – – –

I CRP1.1 1 7,040 8,340 53,520 – – – –

I CRP1.1 2 6,880 8,350 53,960 – – – –

I CRP1.1 3 7,140 8,350 53,920 – – – –

J CRP1.1 1 7,180 8,070 53,630 – 0,128 0,051 0,182

J CRP1.1 2 6,897 8,000 53,120 – 0,108 0,039 0,154

J CRP1.1 3 6,880 8,060 53,900 * 0,125 0,041 0,189

K CRP1.1 1 7,690 8,150 53,940 0,0080 0,159 0,033 0,172



pH

Oven volatiles

NAB NAT NNK NNN


K CRP1.1 2 7,810 8,190 53,510 0,0090 0,157 0,030 0,193

K CRP1.1 3 7,440 8,180 53,190 0,0070 0,132 0,027 0,154

L CRP1.1 1 6,817 8,320 53,710 0,0082 0,145 0,049 0,180

L CRP1.1 2 6,858 8,290 53,530 0,0074 0,123 0,046 0,163

L CRP1.1 3 6,778 8,310 53,500 * 0,144 0,055 0,190

M CRP1.1 1 7,464 8,284 53,040 0,0075 0,131 0,040 0,197

M CRP1.1 2 6,135 8,295 53,240 0,0063 0,126 0,039 0,195

M CRP1.1 3 6,227 8,299 52,600 0,0065 0,127 0,044 0,189

A CRP2.1 1 10,233 7,650 51,019 – – – –

A CRP2.1 2 10,170 7,680 51,074 – – – –

A CRP2.1 3 10,375 7,670 50,877 – – – –

B CRP2.1 1 – 7,610 – 0,3100 3,506 2,027 3,679

B CRP2.1 2 – 7,600 – 0,2930 3,658 1,978 3,578

B CRP2.1 3 – 7,610 – 0,2810 3,417 1,887 3,547

C CRP2.1 1 10,090 7,730 51,310 0,2752 4,307 1,994 3,269

C CRP2.1 2 10,170 7,720 51,450 0,2834 4,375 2,041 3,265

C CRP2.1 3 10,140 7,710 51,310 0,2832 4,229 2,071 3,321

D CRP2.1 1 10,710 7,770 50,611 0,2799 4,022 2,078 3,548

D CRP2.1 2 10,620 7,760 50,367 0,2850 4,014 2,107 3,575

D CRP2.1 3 10,619 7,760 50,410 0,2799 4,021 2,045 3,472

E CRP2.1 1 10,456 7,600 51,242 0,2460 4,088 2,250 3,570

E CRP2.1 2 10,487 7,590 51,599 0,2680 4,440 2,210 3,596

E CRP2.1 3 10,123 7,590 51,926 0,2680 4,304 2,282 3,522

F CRP2.1 1 10,528 7,683 50,975 0,2497 4,048 2,227 3,482

F CRP2.1 2 10,437 7,661 50,863 0,2885 4,146 2,229 3,590

F CRP2.1 3 10,401 7,690 50,959 0,2501 4,069 2,189 3,575

G CRP2.1 1 9,097 7,660 51,410 0,2710 4,186 1,896 3,912

G CRP2.1 2 8,822 7,670 51,220 0,2750 4,298 1,917 4,113

G CRP2.1 3 8,759 7,670 51,260 0,2720 4,239 1,895 4,055

H CRP2.1 1 – 7,690 51,290 – – – –

H CRP2.1 2 – 7,680 51,160 – – – –

H CRP2.1 3 – 7,720 51,180 – – – –

I CRP2.1 1 9,470 7,790 51,910 – – – –

I CRP2.1 2 9,410 7,790 51,880 – – – –



pH

Oven volatiles

NAB NAT NNK NNN


I CRP2.1 3 9,550 7,790 52,590 – – – –

J CRP2.1 1 10,698 7,600 52,050 0,2509 3,994 2,080 3,421

J CRP2.1 2 10,624 7,590 51,500 0,2441 4,000 2,083 3,449

J CRP2.1 3 10,638 7,610 50,680 0,2466 4,066 2,012 3,521

K CRP2.1 1 10,630 7,670 50,530 0,2540 4,769 1,929 2,790

K CRP2.1 2 10,780 7,680 50,710 0,2480 4,548 1,920 2,897

K CRP2.1 3 10,650 7,690 50,750 0,2560 4,631 1,917 2,738

L CRP2.1 1 9,919 7,700 51,110 0,2660 4,120 2,070 3,400

L CRP2.1 2 9,809 7,690 51,020 0,2580 3,900 2,100 3,640

L CRP2.1 3 9,736 7,690 51,240 0,2650 4,200 2,130 3,690

M CRP2.1 1 10,410 7,736 51,470 0,2584 4,022 2,145 3,504

M CRP2.1 2 10,404 7,730 51,400 0,2608 3,975 2,230 3,471

M CRP2.1 3 10,385 7,726 51,400 0,2608 4,093 2,136 3,583

A CRP3.1 1 16,616 6,910 7,785 – – – –

A CRP3.1 2 16,653 6,910 7,715 – – – –

A CRP3.1 3 16,532 6,910 7,713 – – – –

B CRP3.1 1 – 6,930 – 0,2970 3,665 2,683 5,943

B CRP3.1 2 – 6,920 – 0,3440 3,778 2,643 5,658

B CRP3.1 3 – 6,920 – 0,3300 3,669 2,455 5,866

C CRP3.1 1 16,730 7,050 7,862 0,3347 4,545 2,575 5,390

C CRP3.1 2 16,750 7,020 7,852 0,3308 4,474 2,592 5,525

C CRP3.1 3 16,860 6,990 7,868 0,3248 4,656 2,584 5,416

D CRP3.1 1 17,262 6,860 6,519 0,3262 4,283 2,645 5,985

D CRP3.1 2 17,312 6,840 6,537 0,3278 4,342 2,638 5,772

D CRP3.1 3 17,284 6,880 6,799 0,3217 4,191 2,569 5,887

E CRP3.1 1 16,710 6,930 8,652 0,3100 4,072 2,576 5,980

E CRP3.1 2 16,659 6,890 8,746 0,3040 4,398 2,664 5,800

E CRP3.1 3 16,629 6,950 8,795 0,2560 4,072 2,648 6,062

F CRP3.1 1 16,428 6,881 8,347 0,3206 4,119 2,784 5,790

F CRP3.1 2 16,385 6,873 8,326 0,3208 4,150 2,843 5,832

F CRP3.1 3 16,406 6,859 8,294 0,3097 4,154 2,738 5,789

G CRP3.1 1 14,783 6,910 8,130 0,3170 4,432 2,342 6,931

G CRP3.1 2 14,672 6,910 8,090 0,3230 4,466 2,355 6,921

G CRP3.1 3 15,586 6,910 7,990 0,3170 4,452 2,359 6,921



pH

Oven volatiles

NAB NAT NNK NNN


H CRP3.1 1 – 6,940 7,540 – – – –

H CRP3.1 2 – 6,950 7,500 – – – –

H CRP3.1 3 – 6,960 7,490 – – – –

I CRP3.1 1 15,770 6,870 8,340 – – – –

I CRP3.1 2 15,940 6,850 8,160 – – – –

I CRP3.1 3 15,850 6,920 8,100 – – – –

J CRP3.1 1 16,429 6,720 7,700 0,2646 4,204 2,522 5,617

J CRP3.1 2 16,367 6,740 7,830 0,2839 4,092 2,430 5,724

J CRP3.1 3 16,429 6,710 7,760 0,2917 4,262 2,613 5,658

K CRP3.1 1 17,160 7,270 7,160 0,3080 4,627 2,474 4,220

K CRP3.1 2 17,130 7,250 7,320 0,3030 4,540 2,374 4,456

K CRP3.1 3 17,190 7,290 7,410 0,2840 4,481 2,286 4,242

L CRP3.1 1 15,779 6,910 8,450 0,3160 4,410 2,660 5,880

L CRP3.1 2 15,747 6,910 8,470 0,3060 4,270 2,480 5,830

L CRP3.1 3 15,791 6,900 8,350 0,3000 4,120 2,690 5,680

M CRP3.1 1 16,861 6,845 7,950 0,3004 4,190 2,803 5,727

M CRP3.1 2 16,865 6,842 7,980 0,2999 4,184 2,742 5,854

M CRP3.1 3 16,818 6,836 7,850 0,2933 4,231 2,828 5,734

A CRP4.1 1 8,813 6,080 23,896 – – – –

A CRP4.1 2 8,758 6,080 24,113 – – – –

A CRP4.1 3 8,730 6,070 23,856 – – – –

B CRP4.1 1 – 6,070 – 0,1180 1,479 0,862 3,507

B CRP4.1 2 – 6,080 – 0,1070 1,465 0,849 3,685

B CRP4.1 3 – 6,070 – 0,0900 1,464 0,806 3,594

C CRP4.1 1 8,780 6,060 23,970 0,1212 1,675 0,821 3,461

C CRP4.1 2 8,700 6,030 24,050 0,1228 1,687 0,805 3,461

C CRP4.1 3 8,682 6,030 24,740 0,1249 1,654 0,879 3,498

D CRP4.1 1 9,175 5,980 22,404 0,1120 1,420 0,795 3,460

D CRP4.1 2 9,160 5,990 22,490 0,1132 1,441 0,808 3,574

D CRP4.1 3 9,158 5,990 22,780 0,1157 1,498 0,799 3,548

E CRP4.1 1 8,642 6,030 26,874 0,1220 1,840 0,822 3,484

E CRP4.1 2 8,601 6,030 26,789 0,1140 1,644 0,866 3,534

E CRP4.1 3 8,687 6,050 27,171 0,1180 1,538 0,834 3,356

F CRP4.1 1 8,608 6,119 23,800 0,1157 1,503 0,843 3,559



pH

Oven volatiles

NAB NAT NNK NNN


F CRP4.1 2 8,597 6,126 23,805 0,1209 1,581 0,873 3,755

F CRP4.1 3 8,634 6,120 23,862 0,1124 1,522 0,813 3,579

G CRP4.1 1 8,489 6,100 24,100 0,1140 1,632 0,717 3,942

G CRP4.1 2 8,380 6,090 24,320 0,1160 1,612 0,680 3,826

G CRP4.1 3 8,521 6,100 24,160 0,1200 1,649 0,763 4,062

H CRP4.1 1 – 6,130 23,430 – – – –

H CRP4.1 2 – 6,130 23,380 – – – –

H CRP4.1 3 – 6,120 23,410 – – – –

I CRP4.1 1 8,660 6,040 24,510 – – – –

I CRP4.1 2 8,660 6,030 24,310 – – – –

I CRP4.1 3 8,550 6,040 25,510 – – – –

J CRP4.1 1 9,242 5,900 23,430 0,1163 1,616 0,821 3,712

J CRP4.1 2 9,169 5,880 23,440 0,1084 1,539 0,801 3,603

J CRP4.1 3 9,190 5,900 23,080 0,1117 1,546 0,802 3,998

K CRP4.1 1 9,130 6,130 23,740 0,1100 1,847 0,847 3,033

K CRP4.1 2 9,160 6,140 24,320 0,1120 1,668 0,707 3,118

K CRP4.1 3 9,100 6,120 24,200 0,1130 1,802 0,838 2,960

L CRP4.1 1 8,193 6,120 24,280 0,1080 1,610 0,813 3,440

L CRP4.1 2 8,216 6,130 24,460 0,1170 1,610 0,872 3,700

L CRP4.1 3 8,168 6,150 24,490 0,1130 1,610 0,850 3,560

M CRP4.1 1 8,953 6,075 23,870 0,1123 1,561 0,847 3,672

M CRP4.1 2 8,992 6,060 23,750 0,1086 1,533 0,842 3,586

M CRP4.1 3 8,854 6,071 23,650 0,1129 1,558 0,815 3,748


The * symbol indicates that the value was below their reporting limit and was excluded from the analysis.


APPENDIX B: Raw Data Plots for 2019 Study


APPENDIX C: Graphs Comparing 2016 and 2019 Results

technical report coresta reference products 2019 analysis · 2020. 3. 25. · similar to the 2009...

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