A Collaborative Investigation of

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A Collaborative Investigation of Two Tobacco
TSNA Methods June B. Reece, Charles H. Risner
and Walter T. Morgan
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Tobacco Specific Nitrosamine Collaborative Study
Background
Method 1 - QUANTIFICATION OF TOBACCO SPECIFIC
NITROSAMINES IN TOBACCO USING
ALKALINE METHYLENE CHLORIDE
EXTRACTION
Method 2 - QUANTIFICATION OF TOBACCO SPECIFIC
NITROSAMINES IN TOBACCO USING
BUFFER EXTRACTION
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Tobacco Specific Nitrosamine Collaborative Study
Background

• Method 1 - Quantification of Tobacco Specific
  Nitrosamines in Tobacco Using Alkaline Methylene
  Chloride Extraction
• Best Day-to-Day Reproducibility (Robotic
  Preparation)
• Lowest Within-day Standard Deviation (Robotic
  Preparation)
• Minimal Solvent
• Economical Extraction
• High Sample Throughput
• Three Laboratories Performing Similar
  Methodologies

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Tobacco Specific Nitrosamine Collaborative Study
Background

• Method 2 - Quantification of Tobacco Specific
  Nitrosamines in Tobacco Using Buffer Extraction
• Basic Method Utilized by 6 Laboratories in the
  2000 Study

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Tobacco Specific Nitrosamine Collaborative Study
Objectives

• Demonstrate method(s) that will be applicable to
  a variety of tobacco types and tobacco products
• Validate the accuracy and precision of TSNA
  method(s)
• Establish a procedure as a reference method

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Tobacco Specific Nitrosamine Collaborative Study
Recovery Studies
Calculation of TSNAs from a Flue-cured Tobacco
Extract Using NG (Surrogate Internal Standard),
NDHA (Chromatographic Internal Standard) and
External Standard Quantification (µg g-1)
a- Corrected for internal standard recovery
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Tobacco Specific Nitrosamine Collaborative Study
Methods
Method 1 - QUANTIFICATION OF TOBACCO SPECIFIC
NITROSAMINES IN TOBACCO USING
ALKALINE METHYLENE CHLORIDE
EXTRACTION
Method 2 - QUANTIFICATION OF TOBACCO SPECIFIC
NITROSAMINES IN TOBACCO USING
BUFFER EXTRACTION Surrogate NG Surrogate
Internal Standard GC NDHA Gas
Chromatographic Standard External External
Standard Calibration
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Tobacco Specific Nitrosamine Collaborative Study
 Method 1 1. Weigh 1.5 g sample in test
tube   2. Extract in 10 mL 0.4 µg mL-1 NDHA in
CH2Cl2 0.5 mL 10 NaOH using vortex
shaker   3. Decant liquid portion of extract onto
column containing Na2SO4 and MgSO4   4. Rinse
column with 2 mL CH2Cl2   5. Evaporate eluent
with air   6. Reconstitute in chloroform   7. GC
(DB-5 column) with chemiluminescence detection
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Tobacco Specific Nitrosamine Collaborative Study
Method 2   1.  Weigh 1 g sample in 125-mL
flask   2. Add 1 mL 2.0 µg mL-1 NG in CH2Cl2   3.
Extract in 50 mL citrate-phosphate-ascorbic acid
buffer   4. Pour entire extract onto head of a
Chem Elut tube   5. Rinse Chem Elut tube with
two 150 mL portions of CH2Cl2, collecting eluent
in 250-mL TurboVap tube   6. Add 1 mL of 2.0 µg
mL-1 NDHA in CH2Cl2   7. Concentrate in
TurboVap concentrator to ? 0.5 mL   8.
Reconstitute with CH2Cl2   9. GC (DB-1 column)
with chemiluminescence detection
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Tobacco Specific Nitrosamine Collaborative Study
Participating Laboratories
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Tobacco Specific Nitrosamine Collaborative Study
Test Plan

• Recovery Studies
• Collaborative Interlaboratory Testing

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Tobacco Specific Nitrosamine Collaborative Study
Recovery Studiesa
a -Three tobaccos, three replicates, three
nitrosamine levels b -Standards added in buffer,
results based on external standard
quantification c -Standards added in CH2Cl2,
results based on chromatographic standard, NDHA d
-Standards added in CH2Cl2, results based on
surrogate standard, NG e -Standards added in
CH2Cl2, results based on surrogate standard, NDHA
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Tobacco Specific Nitrosamine Collaborative Study
Collaborative Test Design Analyses
Samples to be analyzed in replicates of three,
on the same day, under repeatability conditions
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Tobacco Specific Nitrosamine Collaborative Study
Collaborative Test Design Tobacco Types

• Burley
• Flue-cured
• Low TSNA Flue-cured
• Turkish
• 2S3 Kentucky Reference Moist Snuff
• 1R4F Reference Cigarette Tobacco
• Burley Stems
• Composite Commercial Cigarette Blend

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Tobacco Specific Nitrosamine Collaborative Study
Test Design TSNA Range
TSNA Low High NNN 0.24 9.06 NNK
0.10 1.56 NAT 0.12 4.06 NAB 0.03
0.21
Analysis via Method 1 in units of µg g-1 dry
weight
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Tobacco Specific Nitrosamine Collaborative Study
Analysis
ISO 5725 Accuracy (trueness and precision)
of measurement methods and results
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Tobacco Specific Nitrosamine Collaborative Study
Method 1
Lab Means Mandel h 1 5
NNN
Stragglers
99
95
95
99
Outliers
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Tobacco Specific Nitrosamine Collaborative Study
Method 1
Lab Sdev Mandel k 1 5
NNN
99
95
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Tobacco Specific Nitrosamine Collaborative Study
Statistical Method Comparisons

• Analysis of Variance Ryan-Einot-Gabriel-Welsch
  Multiple Range Test
• Analysis of the Log of Sample Means
• Analysis of the Coefficients of Variation for
  Repeatability and Reproducibility

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Tobacco Specific Nitrosamine Collaborative Study
Means Comparison Average Log Mean (ng g-1
dry x 10)
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Tobacco Specific Nitrosamine Collaborative Study
Repeatability Comparisons Coefficient of
Variation
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Tobacco Specific Nitrosamine Collaborative Study
Reproducibility Comparisons Coefficient of
Variation
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Tobacco Specific Nitrosamine Collaborative Study
Further Analysis
Limits of Detection and Quantitation Limit
of Detection (LOD) 3 x SDev (Lowest Standard)
Limit of Quantitation (LOQ) 10 x SDev
(Lowest Standard)
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Tobacco Specific Nitrosamine Collaborative Study
Standard Deviation of Lowest Concentration
Standard Run as a Sample
Standard Deviation µg mL-1
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Tobacco Specific Nitrosamine Collaborative Study
Recovery Lowest Concentration
Standard Run as a Sample
Recovery
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Tobacco Specific Nitrosamine Collaborative Study
Further Analysis
Method 1 Variations Robotic Sample
Preparation N-nitrosoguvacoline as the
Surrogate Internal Standard
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Tobacco Specific Nitrosamine Collaborative Study
Means Comparisons Average Log Means (ng g-1
dry x 10)
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Tobacco Specific Nitrosamine Collaborative Study
Repeatability Comparisons Coefficient of
Variation
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Tobacco Specific Nitrosamine Collaborative Study
Conclusions

• Both Method 1 and Method 2 were demonstrated to
  be applicable to a variety of tobacco types and
  tobacco products.
• Accuracy of the TSNA methods were validated. In
  the study, Method 2 showed a higher average
  recovery, 99.6 vs 92.1 for Method 1.
• ISO Standard 5725 guidelines were applied to
  establish the repeatability and reproducibility
  of each method. Repeatability was not different
  between the two methods. The COV for Method 2
  reproducibility was 6 lower for NNN and 12
  lower for NNK.

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Acknowledgements Hubert Klus, Helmut Begutter
and Anton Pachinger, Austria Tabak Christine
A. Rouse and C. Roy Taylor, Brown Williamson
Tobacco Corporation Jacques Dumont, Imperial
Tobacco Limited Takeshi Sakaki, Hideyuki
Tomita, Hideki Takahashi and Hitoshi Saito, Japan
Tobacco Inc. Bill Rickert, Mehran Sharifi and
Peter Joza, Labstat International, Inc.
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Acknowledgements
Richard Entz and Richard Shober, Lancaster
Laboratories Jim Morgan and Cynthia Williard,
Lorillard Tobacco Company Jean-Marc Renaud and
Roxanne Boudoux, Philip Morris
International Chorng Huang, Philip Morris
USA Mike Borgerding, Christy Fishel, and Angela
Synder, R. J. Reynolds Tobacco Company
RD Mark Hathaway, Kim Baughman and Ken Boyer,
Southern Testing Research Laboratories
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Acknowledgements
Lennart Johansson and Susanne Back, Swedish
Match North Europe Division Steve Terrell,
Swedish Match North American Division Thomas
Losty, John Townend, Paul Moser and Ahad Majeed,
Swisher International, Inc. Cliff B. Bennett,
Carl Midgett, Rusty Owens and Kathleen Johnson,
U. S. Tobacco Harold Burton and Naewanna Dye,
University of Kentucky