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A Collaborative Investigation of Two Tobacco TSNA MethodsJune 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
Quantification Quantification MethodMethod
Percent Percent RecoveryRecovery
NNNNNN NATNAT NABNAB NNKNNK
NG Surrogate NG Surrogate Internal StandardInternal Standard
84.284.2 1.91 (1.60)1.91 (1.60)aa 2.24 (1.88)2.24 (1.88)a a 0.20 (0.17)0.20 (0.17)aa 2.74 (2.30)2.74 (2.30)aa
NDHA NDHA Chromatographic Chromatographic Internal StandardInternal Standard
94.394.3 1.69 (1.59)1.69 (1.59)a a 2.00 (1.88)2.00 (1.88)aa 0.18 (0.15)0.18 (0.15)aa 2.43 (2.29)2.43 (2.29)aa
External External StandardStandard
---------------- 1.571.57 1.881.88 0.160.16 2.282.28
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Tobacco Specific Nitrosamine Collaborative Study
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
Methods
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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 LaboratoriesParticipants 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
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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
LaboratoryLaboratory AverageAverage(%)(%)
NitrosamineNitrosamine
NNNNNN(%)(%)
NATNAT(%)(%)
NABNAB(%)(%)
NNKNNK(%)(%)
Lab I – Method 2Lab I – Method 2bb 91.691.6 92.292.2 88.588.5 94.794.7 9191
Lab I – Method 2Lab I – Method 2cc 97.097.0 93.493.4 92.192.1 106.8106.8 95.895.8
Lab II – Method 2Lab II – Method 2dd 99.699.6 100100 93.393.3 109.2109.2 9696
Lab III – Method 1Lab III – Method 1ee
92.192.1105.3105.3 98.498.4 87.487.4 100.6100.6
Lab IV – Method 1Lab IV – Method 1ee 88.788.7 91.191.1 83.683.6 81.981.9
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Tobacco Specific Nitrosamine Collaborative Study
Samples to be analyzed in replicates of three,
on the same day, under repeatability conditions
Collaborative Test Design – Analyses
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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 1Lab Means Mandel h1% & 5%
-3
-2
-1
0
1
2
3
Lab I Lab II Lab III Lab IV Lab V Lab VI Lab VII Lab VIII Lab IX Lab X Lab XI Lab XII
NNN
95%
99%
95%
99%
Stragglers
Outliers
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Tobacco Specific Nitrosamine Collaborative Study – Method 1Lab Sdev Mandel k 1% & 5%
0
1
2
3
4
Lab I Lab II Lab III Lab IV Lab V Lab VI Lab VII Lab VIII Lab IX Lab X Lab XI Lab XII
NNN
95%99%
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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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0.750.95
1.151.35
1.551.75
1.952.15
2.35
NNN NNK NAT NAB
Method 1 Method 2 SurrogateMethod 2 GC Method 2 External
Tobacco Specific Nitrosamine Collaborative Study
Means Comparison Average Log Mean (ng g-1 dry x 10)
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0
2
4
6
8
10
12
14
NNN NNK NAT NABMethod 1 Method 2 SurrogateMethod 2 GC Method 2 External
Tobacco Specific Nitrosamine Collaborative Study
Repeatability Comparisons Coefficient of Variation
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0
10
20
30
40
50
NNN NNK NAT NABMethod 1 Method 2 SurrogateMethod 2 GC Method 2 External
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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0
0.005
0.01
0.015
0.02
0.025
0.03
0.035
NNN NAT NAB NNK
Method 1 Method 2 SurrogateMethod 2 GC Method 2 External
Tobacco Specific Nitrosamine Collaborative Study
Standard Deviation of Lowest Concentration Standard Run as a SampleS
tan
dar
d D
evia
tio
n µ
g m
L-1
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0102030405060708090
100
NNN NAT NAB NNKMethod 1 Method 2 Surrogate
Method 2 GC Method 2 External
Tobacco Specific Nitrosamine Collaborative Study
Recovery Lowest Concentration Standard Run as a Sample
% R
eco
very
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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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0.750.95
1.151.35
1.551.75
1.952.15
2.35
NNN NNK NAT NAB
Method 1 Method 1 Robotic Method 1 NG
Tobacco Specific Nitrosamine Collaborative Study
Means Comparisons Average Log Means (ng g-1 dry x 10)
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0
5
10
15
20
25
NNN NNK NAT NAB
Method 1 Method 1 Robotic Method 1 NG
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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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
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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
Acknowledgements