Technical Report Determination of Nitrate in …...Smokeless Tobacco Sub-Group Technical Report Determination of Nitrate in Smokeless Tobacco Products by Continuous Flow Analysis 2010

Smokeless Tobacco Sub-Group

Technical Report

Determination of Nitrate in Smokeless Tobacco Products by

Continuous Flow Analysis

2010 Collaborative and Proficiency Studies

January 2015

Author and Sub-Group Secretary:

Karl Wagner, Ph.D. Altria Client Services, Inc., U.S.A.

Sub-Group Coordinator:

John Bunch, Ph.D. American Snuff Company, U.S.A.

Statistical Analysis:

Alexander Hauleithner Japan Tobacco, Inc., R&D Ökolab, Austria

STS-CTR Nitrate in Smokeless Tobacco Products – January 2015 2/24

Table of Contents

1. Summary 3

2. Introduction 3

3. Organisation 3

3.1 Participants 3

3.2 Protocol 4

4. Data – Summary Descriptive Statistics 5

5. Data – Statistical Analysis 5

5.1 Exclusion of Outliers 5

5.2 Calculation of Repeatability and Reproducibility 7

6. Data Interpretation 8

7. Recommendations 9

APPENDIX A ‒ Raw Data Plots 10

APPENDIX B ‒ Mandel’s h Plots 15

APPENDIX C ‒ Mandel’s k Plots 20


1. Summary In 2010, the CORESTA Smokeless Tobacco Sub-Group conducted a collaborative study for the determination of nitrate in smokeless tobacco products. The goal of this work was to calculate repeatability and reproducibility and update the current CORESTA Recommended Method (CRM) to include smokeless tobacco products as the CRM was developed with ground tobacco only. The study results demonstrated that the method was also suitable for the analysis of smokeless tobacco products and CRM N° 36, Determination of Nitrate in

Tobacco and Smokeless Tobacco Products by Reduction to Nitrite and Continuous Flow

Analysis was updated in August 2011 to include smokeless tobacco products.

2. Introduction In 2010, the CORESTA Smokeless Tobacco Sub-Group conducted a collaborative study that included the four CORESTA Reference Products (CRPs) and five commercial smokeless tobacco products. This study was referred to as 2010 Collaborative and Proficiency Studies. The laboratory phase of this study was conducted in the May/June timeframe of 2010. This study included the determination of nitrate and several other analytes (ammonia, humectants, benzo[a]pyrene, select trace metals, and moisture content). Seventeen laboratories participated in the larger study; however, only 11 labs provided data for the nitrate portion of the study. This study specified the use of CRM N° 36 for the determination of nitrate. The purpose of this collaborative study was to determine if CRM N° 36, which was originally developed for tobacco, was also suitable for smokeless tobacco products. The focus of this report is the interlaboratory study that was conducted in support of this goal.

The nitrate content of smokeless tobacco products was determined by aqueous extraction of the samples followed by reduction of nitrate to nitrite with hydrazinium sulphate in the presence of a copper catalyst. This was followed by reaction with sulphanilamide to form the diazo compound which was subsequently reacted with N-1-naphthylethylenediamine dihydrochloride to form a coloured complex. The absorbance of the coloured complex was measured at 520 nm. It should be noted that if nitrite is also present in the tobacco, it will be detected by this method and may thus increase the apparent nitrate concentration. However, the presence of nitrite typically is not an issue because nitrite is typically present at a concentration two orders of magnitude lower that nitrate.

Data analysis of the nitrate content for CRP 1 – CRP 4 and the 5 commercial smokeless tobacco products was performed following the statistical model provided by ISO 5725-2 (1994) (“basic method for the determination of repeatability and reproducibility of a standard measurement method”). Three replicates for nitrate were determined by each participating laboratory. Nitrate was reported in units of percent (%), on a wet weight basis. CRM Nº 36 was followed except that the CRM recommends that samples contain at least 0.2% nitrate and this study included samples with less nitrate. Additionally, CRM Nº 36 specifies a calibration range from 0.05% to 2% nitrate while this study included smokeless tobacco products where nitrate was in the range of 0.05% to 5%.

3. Organisation

3.1 Participants A list of the participating laboratories is provided in Table 1. The laboratories are listed in alphabetical order. The numerical laboratory codes (1-15) used in this report do not correspond to the same order as the list below. Although 17 laboratories initially agreed to


participate in this study, only 11 laboratories provided data for nitrate. Laboratories 4, 8, 10, and 13 did not participate in the nitrate study; however, these laboratories provided data for other analyses; therefore, these codes are included in this report as well, since the same laboratory codes were used for all analytes included in the larger study.

Table 1. List of Participating Laboratories

Laboratory

Arista Laboratories, Inc.

Arnold André Cigars

British American Tobacco

China National Tobacco Company

Eurofins Food & Agro Sweden AB

Global Laboratory Services

Imperial Tobacco

Imperial Tobacco Group, SEITA

ITC Limited

Japan Tobacco International

KT&G

Labstat International

Philip Morris International

Pöschl Tabak GmbH & Co. KG

Swedish Match Northern Europe

Swedish Match North America

Swisher International

3.2 Protocol As discussed above, the protocol describes the work conducted for a larger collaborative study that included several analytical methods; however, this report only discusses the results from the nitrate portion of the study.

Nine smokeless tobacco samples were provided to all participants by two participating organizations. The commercial smokeless tobacco products were reduced in size to <4mm, homogenized and then distributed to each participant. The CRPs were shipped to the participants without sample grinding or homogenization. Approximately 150 g of each sample type was provided. Participants were directed to place the samples in the freezer upon receipt, until the analyses could be conducted. Samples were thawed at room temperature for at least 2 hours before use. Samples were stored in the refrigerator after the initial thawing.

Each laboratory conducted three replicate determinations of nitrate for each of the nine samples listed in Table 2. The laboratories were requested to determine the three replicates under repeatability conditions; i.e. within a short time interval and by the same operator using the same equipment.


Table 2. Sample Identification

Sample ID Product Type

A Snus – loose

B CRP1 - Swedish style snus pouch

C CRP2 - American-style loose moist snuff

D CRP4 - American-style loose-leaf chewing tobacco

E Chewing Tobacco Bits – rolled tobacco pellet1

F Hard Snuff/Pellet

G Nasal Snuff

H Chewing - Gutkha flake

I CRP3 - American-style loose dry snuff powder

1. Referred to as ‘Chewing Tobacco – Twist’ in the study protocol

4. Data – Summary Descriptive Statistics The summary descriptive statistics are listed in Table 3; where each mean represents three replicates. Laboratories 4, 8, 10, and 13 did not provide nitrate data. Raw data plots that include all replicates, without exclusion of outliers, are given in Appendix A. All data are presented on a wet weight basis.

5. Data – Statistical Analysis A statistical analysis was conducted following the recommendations given in ISO 5725-2 (1994). A summary of the results from outlier detection and the calculated results for repeatability (r) and reproducibility (R) are given in sections 5.1 and 5.2, respectively.

5.1 Exclusion of Outliers Graphical outlier detection was performed following the recommendations of ISO 5725-2 (1994). Mandel’s h is used to assess the inter-laboratory consistency and Mandel’s k is used to assess the intra-laboratory consistency. Both Mandel’s h and Mandel’s k plots use the 0.95 straggling and 0.99 outlying limits. The Mandel’s h plots and the Mandel’s k plots are given in Appendices B and C, respectively. Outliers identified with Mandel’s h and Mandel’s k plots were not removed prior to calculation of r & R because the graphical methods are more likely to detect outliers, and lead to excessive data exclusion, as compared to the numerical methods.

Numerical outlier detection was conducted following the recommendations of ISO 5725-2 (1994). The decision on straggling (0.95) and outlying (0.99) data were made using Grubbs’ single outlier and Cochran’s tests. The results for the Grubbs’ test to detect straggling and outlying means are displayed in Table 4. The results for the Cochran’s test to detect straggling and outlying variances are displayed in Table 5.


Table 3. Summary Descriptive Statistics

1. All data are presented on a wet weight basis. 2. “StDev“ indicates standard deviation. 3. Laboratories 4, 8, 10, and 13 did not participate in the nitrate study. 4. “<LOQ“ indicates that nitrate was reported as less than the limit of quantitation or was not detected in the sample.

Sample A Sample B Sample C Sample D Sample E Sample F Sample G Sample H Sample I

Snus-Loose CRP1 CRP2 CRP4 Chewing

Tobacco Bits Hard

Snuff/Pellet Nasal Snuff

Chewing - Gutkha flake

CRP3

Lab Code

Mean1

(%) StDev

2

(%) Mean (%)

StDev (%)

Mean (%)

StDev (%)

Mean (%)

StDev (%)

Mean (%)

StDev (%)

Mean (%)

StDev (%)

Mean (%)

StDev (%)

Mean (%)

StDev (%)

Mean (%)

StDev (%)

1 2.313 0.056 0.698 0.003 2.117 0.001 1.226 0.001 1.730 0.049 0.813 0.001 0.099 0.006 1.605 0.018 2.170 0.001 2 0.517 0.003 0.641 0.004 1.485 0.003 1.180 0.053 0.438 0.004 0.110 0.006 1.109 0.009 <LOQ -- 4.698 0.004 3 0.505 0.005 0.634 0.013 1.477 0.009 1.137 0.026 <LOQ -- <LOQ -- 1.081 0.002 <LOQ -- 4.845 0.025 4 -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- 5 0.533 0.001 0.650 0.004 1.476 0.001 1.160 0.001 0.464 0.000 0.133 0.003 1.131 0.004 0.029 0.000 4.643 0.018 6 0.535 0.002 0.646 0.009 1.433 0.005 1.222 0.016 0.427 0.006 0.097 0.000 1.099 0.002 4.617 0.016 7 0.578 0.003 0.712 0.013 1.649 0.013 1.298 0.046 0.488 0.024 0.149 0.001 1.232 0.004 0.030 0.002 5.249 0.011 8 -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- 9 0.537 0.011 0.669 0.015 1.476 0.016 1.208 0.029 0.480 0.011 0.196 0.003 1.106 0.014 0.115 0.001 4.663 0.061

10 -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- 11 0.569 0.006 0.685 0.013 1.536 0.005 1.227 0.015 0.478 0.002 0.146 0.001 1.190 0.004 0.036 0.001 4.933 0.037 12 0.545 0.002 0.659 0.016 1.511 0.031 1.178 0.033 0.496 0.003 0.163 0.001 1.127 0.006 0.027 0.001 4.506 0.074 13 -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- 14 0.596 0.006 0.672 0.016 1.596 0.012 1.198 0.041 0.451 0.004 0.161 0.010 1.139 0.004 0.076 0.002 4.702 0.113 15 0.503 0.003 0.594 0.006 1.310 0.012 0.848 0.015 0.102 0.003 <LOQ -- 0.888 0.021 <LOQ -- 5.448 0.039


Table 4. Results of Grubbs’ Test for Straggling and Outlying Means

Product Lab #

Snus – Loose 1 (outlier)

CRP1 ---

CRP2 1 (outlier)

CRP4 15 (outlier)

Chewing Tobacco Bits 1 (outlier), 15 (outlier)

Hard Snuff/ Pellet 1 (outlier)

Nasal Snuff 1 (outlier), 15 (straggler)

Chewing- Gutkha Flake 1 (outlier)

CRP3 1 (outlier)

The “-- “ indicates that no outliers were detected.

Table 5. Results of Cochran’s Tests for Straggling and Outlying Variances

Product 1

st Iteration Lab #

2

st Iteration Lab #

3

st Iteration Lab #

Snus – Loose 1 (outlier) 9 (straggler) --

CRP1 -- -- --

CRP2 12 (outlier) -- --

CRP4 -- -- --

Chewing Tobacco Bits 1 (outlier) 7 (outlier) 9 (straggler)

Hard Snuff/ Pellet 14 (outlier) 2 (straggler) --

Nasal Snuff 15 (straggler) -- --

Chewing- Gutkha Flake 1 (outlier) -- --

CRP3 14 (straggler) -- --

The “-- “ indicates that no outliers were detected.

5.2 Calculation of Repeatability and Reproducibility After removal of outlying data based on numerical data consistency methods (Grubbs’ test, Cochran’s test), the final repeatability (r) and reproducibility (R) results were calculated. The r & R results are shown in Table 6. As mentioned above, results are presented on an as-is or wet weight basis.


Table 6. Repeatability (r) and Reproducibility (R) Results for all Test Samples

No of Labs

1

Nitrate (wwb)

2,3

Repeatability Reproducibility

Sample Type N (%) r %r R %R

Snus – loose 10 0.542 0.014 2.58 0.087 16.05 CRP1 - Swedish style snus pouch 11 0.660 0.032 4.85 0.095 14.39 CRP2 - American-style loose moist snuff 9 1.493 0.027 1.81 0.272 18.22 CRP4 - American-style loose-leaf chewing tob. 10 1.204 0.087 7.23 0.144 11.96

Chewing Tobacco Bits – rolled tobacco pellet4 7 0.462 0.015 3.25 0.070 15.15

Hard Snuff/Pellet 7 0.142 0.008 5.63 0.093 65.49 Nasal Snuff 10 1.110 0.025 2.25 0.253 22.79 Chewing - Gutkha flake 6 0.052 0.003 5.77 0.101 194.23 CRP3 - American-style loose dry snuff powder 10 4.831 0.143 2.96 0.851 17.62

1. The number of laboratory data sets included after removal of outliers. 2. Results presented on an as-is or wet weight basis (wwb). 3. Moisture data are published in the Smokeless Tobacco Sub-Group Technical Report, Analysis

of Moisture Content (Oven Volatiles) of Smokeless Tobacco Products 2010 Collaborative and Proficiency Studies, March 2014.

4. Referred to as ‘Chewing Tobacco – Twist’ in the study protocol.

6. Data Interpretation As shown in Table 6, there was not a correlation between nitrate content and within-lab relative variability (%r) which ranged from 1.8% to 7.2%. Even the two samples with very low nitrate content (Chewing - Gutkha flake and Hard Snuff/Pellet) have %r values similar to high nitrate samples, showing good within-lab variability for samples with a wide range of nitrate content. Between-lab variability (R) was poorer for the two samples that contained very low levels of nitrate. This is not unexpected as relative variability often increases as the analyte level approaches the method limit of quantitation. Labs 2, 3, and 15 reported less than the limit of quantitation (<LOQ) for at least one of the low nitrate samples. %R values for the remaining seven samples ranged from approximately 12% to 23% and variability did not correlate with nitrate level. These seven samples covered a range from 0.46% nitrate to 4.8% nitrate.

Since the purpose of this study was to determine if CRM N° 36 was also suitable for the determination of nitrate in smokeless tobacco products, the results from this study were compared to the most recent Routine Analytical Chemistry (RAC) Study, which was conducted in 20061. The summary results for the RAC study are shown in Table 7. In contrast to the current study that included smokeless tobacco products, the RAC study only included ground flue-cured and burley tobaccos and included samples with a narrower range of nitrate content. Specifically, the current study included samples containing 0.052% to 4.83% nitrate on a wet weight basis while the RAC study included samples containing 0.13% to 1.5% nitrate on a wet weight basis.

When considering the samples with similar levels of nitrate, within-lab variability (r) was slightly better for the current study on smokeless tobacco products as compared to the RAC study while between-lab variability (R) was similar for each study. The results demonstrate that CRM N° 36 is also fit for use for the determination of nitrate in smokeless tobacco products.


Table 7. Repeatability (r) and Reproducibility (R) Results from 2006 Routine Analytical Chemistry Study1

No of Labs

1

Nitrate (wwb)

2

Repeatability Reproducibility

Sample N (%) r %r R %R

A (Flue-Cured) 10 0.22 0.03 13.6 0.06 27.3 B (Flue-Cured) 10 0.13 0.02 15.4 0.05 38.5 C (Burley) 11 1.50 0.06 4.0 0.26 17.3 D (Burley) 10 1.39 0.06 4.3 0.10 7.2 E (Flue-Cured) 11 0.65 0.11 16.9 0.12 18.5 A (Flue-Cured) 11 1.46 0.06 4.1 0.16 11.0

1. The number of laboratory data sets included after removal of outliers. 2. Results presented on an as-is or wet weight basis (wwb).

7. Recommendations This study demonstrates that CRM N° 36, which was originally developed for the analysis of ground tobacco, is also fit for the analysis of smokeless tobacco products. The method showed good repeatability and reproducibility for smokeless tobacco samples containing nitrate from 0.462% to 4.831%. Although repeatability was excellent for the samples containing very low levels of nitrate (0.05% – 0.14%), reproducibility was poorer and interlaboratory data for samples in this range should be considered questionable. Laboratories using the CRM for samples containing very low levels of nitrate should first determine if the method is fit for use using their specific operating conditions and testing objectives. As mentioned in the Introduction, CRM N° 36, Determination of Nitrate in Tobacco and

Smokeless Tobacco Products by Reduction to Nitrite and Continuous Flow Analysis has been updated to include smokeless tobacco products and the revised version was published in August 2011.

1 Routine Analytical Chemistry Sub-Group Technical Report, 2006 Collaborative Studies for Nicotine, Sugars and Nitrate in Tobacco, May 2008


APPENDIX A ‒ Raw Data Plots Outliers are included. Laboratories 4, 8, 10, and 13 did not provide nitrate data.






APPENDIX B ‒ Mandel’s h Plots

Stragglers (0.95 limit) and outliers (0.99 limit) are marked with a red symbol. Laboratories 4, 8, 10, and 13 did not provide nitrate data. The mean value presented in each plot was calculated prior to removal of outliers.






APPENDIX C ‒ Mandel’s k Plots Stragglers (0.95 limit) and outliers (0.99 limit) are marked with a red symbol. Laboratories 4, 8, 10, and 13 did not provide nitrate data. The mean value presented in each plot was calculated prior to removal of outliers.

MANDEL's k STD DEV Nitrates (g/100g) SAMPLE A

MANDEL's k STD DEV Nitrates (g/100g) SAMPLE B


MANDEL's k STD DEV Nitrates (g/100g) SAMPLE C

MANDEL's k STD DEV Nitrates (g/100g) SAMPLE D


MANDEL's k STD DEV Nitrates (g/100g) SAMPLE E

MANDEL's k STD DEV Nitrates (g/100g) SAMPLE F


MANDEL's k STD DEV Nitrates (g/100g) SAMPLE G

MANDEL's k STD DEV Nitrates (g/100g) SAMPLE H


MANDEL's k STD DEV Nitrates (g/100g) SAMPLE I

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Technical Report Determination of Nitrate in …...Smokeless Tobacco Sub-Group Technical Report Determination of Nitrate in Smokeless Tobacco Products by Continuous Flow Analysis 2010