A Collaborative Investigation of Two Tobacco TSNA Methods June B. Reece, Charles H. Risner and Walter T. Morgan

A Collaborative Investigation of Two Tobacco TSNA MethodsJune 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


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


Background

• Method 2 - Quantification of Tobacco Specific Nitrosamines in Tobacco Using Buffer Extraction

•Basic Method Utilized by 6 Laboratories in the 2000 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


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


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

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


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


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


Test Plan

• Recovery Studies

• Collaborative Interlaboratory Testing


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


Samples to be analyzed in replicates of three,

on the same day, under repeatability conditions

Collaborative Test Design – Analyses


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


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


Analysis

ISO 5725 – Accuracy (trueness and precision) of measurement methods and results

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

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%


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

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


Means Comparison Average Log Mean (ng g-1 dry x 10)

0

2

4

6

8

10

12

14

NNN NNK NAT NABMethod 1 Method 2 SurrogateMethod 2 GC Method 2 External


Repeatability Comparisons Coefficient of Variation

0

10

20

30

40

50

NNN NNK NAT NABMethod 1 Method 2 SurrogateMethod 2 GC Method 2 External


Reproducibility Comparisons Coefficient of Variation


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)

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


Standard Deviation of Lowest Concentration Standard Run as a SampleS

tan

dar

d D

evia

tio

n µ

g m

L-1

0102030405060708090

100

NNN NAT NAB NNKMethod 1 Method 2 Surrogate

Method 2 GC Method 2 External


Recovery Lowest Concentration Standard Run as a Sample

% R

eco

very


Further Analysis

Method 1 Variations

Robotic Sample Preparation

N-nitrosoguvacoline as the Surrogate Internal Standard

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


Means Comparisons Average Log Means (ng g-1 dry x 10)

0

5

10

15

20

25

NNN NNK NAT NAB

Method 1 Method 1 Robotic Method 1 NG


Repeatability Comparisons Coefficient of Variation


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.

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

Acknowledgements

Documents

A Collaborative Investigation of Two Tobacco TSNA Methods June B. Reece, Charles H. Risner and Walter T. Morgan