A Collaborative Investigation of

Two Tobacco TSNA Methods

June B. Reece, Charles H. Risner and Walter T. Morgan
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
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
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

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
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)

Quantification Percent NNN NAT NAB NNK

Method Recovery
a a a a
NG Surrogate 84.2 1.91 (1.60) 2.24 (1.88) 0.20 (0.17) 2.74 (2.30)
Internal Standard
a a a a
NDHA 94.3 1.69 (1.59) 2.00 (1.88) 0.18 (0.15) 2.43 (2.29)
Chromatographic
Internal Standard

External -------- 1.57 1.88 0.16 2.28

Standard

a- Corrected for internal standard recovery

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

 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

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
Tobacco Specific Nitrosamine Collaborative Study
Participating Laboratories
Participants Method 1 Method 2

Austria Tabak X
Imperial Tobacco Limited X
Japan Tobacco Incorporated X
Labstat International, Incorporated X
Lancaster Laboratories X
Lorillard Tobacco Company X
Philip Morris International X X
Philip Morris U.S.A X X
R. J. Reynolds Tobacco Company R&D X X
Southern Testing & Research Laboratories, Inc X X
Swedish Match North Europe Division X
Swedish Match North American Division X
Swisher International Incorporated X
U. S. Tobacco Mfg. LP X X
University of Kentucky X X
Tobacco Specific Nitrosamine Collaborative Study

Test Plan

• Recovery Studies
• Collaborative Interlaboratory Testing
Tobacco Specific Nitrosamine Collaborative Study

Recovery Studiesa
Nitrosamine
Laboratory Average NNN NAT NAB NNK
(%)
(%) (%) (%) (%)
b
Lab I – Method 2 91.6 92.2 88.5 94.7 91
c
Lab I – Method 2 97.0 93.4 92.1 106.8 95.8
d
Lab II – Method 2 99.6 100 93.3 109.2 96
e
Lab III – Method 1 105.3 98.4 87.4 100.6
e 92.1
Lab IV – Method 1 88.7 91.1 83.6 81.9
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
 Tobacco Specific Nitrosamine Collaborative Study

Collaborative Test Design – Analyses

Samples to be analyzed in replicates of three,

on the same day, under repeatability conditions
 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
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

Tobacco Specific Nitrosamine Collaborative Study

Analysis

ISO 5725 – Accuracy (trueness and precision)

of measurement methods and results
 Tobacco Specific Nitrosamine Collaborative Study – Method 1
Lab Means
Mandel h
1% & 5% NNN
3
99% Stragglers
2 95%

0 Lab I Lab II Lab III Lab IV Lab V Lab VI Lab VII Lab VIII Lab IX Lab X Lab XI Lab XII

-1
95%
-2 99%

Outliers
-3
 Tobacco Specific Nitrosamine Collaborative Study – Method 1
Lab Sdev
Mandel k
1% & 5% NNN
4

2 99%
95%

0 Lab I Lab II Lab III Lab IV Lab V Lab VI Lab VII Lab VIII Lab IX Lab X Lab XI Lab XII
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
Tobacco Specific Nitrosamine Collaborative Study
Means Comparison Average Log Mean (ng g-1 dry x 10)

2.35
2.15
1.95
1.75
1.55
1.35
1.15
0.95
0.75
NNN NNK NAT NAB
Method 1 Method 2 Surrogate
Method 2 GC Method 2 External
Tobacco Specific Nitrosamine Collaborative Study
Repeatability Comparisons Coefficient of Variation

14
12
10
8
6
4
2
0
NNN NNK NAT NAB
Method 1 Method 2 Surrogate
Method 2 GC Method 2 External
Tobacco Specific Nitrosamine Collaborative Study
Reproducibility Comparisons Coefficient of Variation

50

40

30

20

10

0
NNN NNK NAT NAB
Method 1 Method 2 Surrogate
Method 2 GC Method 2 External
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)

 Tobacco Specific Nitrosamine Collaborative Study
Standard Deviation of Lowest Concentration Standard Run as a Sample
Standard Deviation µg mL-1

0.035
0.03
0.025
0.02
0.015
0.01
0.005
0
NNN NAT NAB NNK
Method 1 Method 2 Surrogate
Method 2 GC Method 2 External
 Tobacco Specific Nitrosamine Collaborative Study
Recovery
Lowest Concentration Standard Run as a Sample
100
90
80
70
% Recovery

60
50
40
30
20
10
0
NNN NAT NAB NNK
Method 1 Method 2 Surrogate
Method 2 GC Method 2 External
 Tobacco Specific Nitrosamine Collaborative Study

Further Analysis

Method 1 Variations

Robotic Sample Preparation

N-nitrosoguvacoline as the Surrogate Internal Standard

Tobacco Specific Nitrosamine Collaborative Study
Means Comparisons Average Log Means (ng g-1 dry x 10)

2.35
2.15
1.95
1.75
1.55
1.35
1.15
0.95
0.75
NNN NNK NAT NAB

Method 1 Method 1 Robotic Method 1 NG

Tobacco Specific Nitrosamine Collaborative Study
Repeatability Comparisons Coefficient of Variation

25

20

15

10

0
NNN NNK NAT NAB

Method 1 Method 1 Robotic Method 1 NG

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.
 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.
 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 R&D

Mark Hathaway, Kim Baughman and Ken Boyer,

Southern Testing & Research Laboratories
 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