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 ) a- Corrected for internal standard recovery QuantificationMethodPercentRecoveryNNNNATNABNNK NG Surrogate Internal Standard (1.60) a 2.24 (1.88) a 0.20 (0.17) a 2.74 (2.30) a NDHAChromatographic Internal Standard (1.59) a 2.00 (1.88) a 0.18 (0.15) a 2.43 (2.29) a ExternalStandard

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

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 CH 2 Cl mL 10 % NaOH using vortex shaker 3. Decant liquid portion of extract onto column containing Na 2 SO 4 and MgSO 4 4. Rinse column with 2 mL CH 2 Cl 2 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 CH 2 Cl 2 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 CH 2 Cl 2, collecting eluent in 250-mL TurboVap™ tube 6. Add 1 mL of 2.0 µg mL -1 NDHA in CH 2 Cl 2 7. Concentrate in TurboVap™ concentrator to  0.5 mL 8. Reconstitute with CH 2 Cl 2 9. GC (DB-1 column) with chemiluminescence detection

Tobacco Specific Nitrosamine Collaborative Study Participating Laboratories ParticipantsMethod 1Method 2 Austria Tabak X Imperial Tobacco LimitedX Japan Tobacco IncorporatedX Labstat International, IncorporatedX Lancaster Laboratories X Lorillard Tobacco CompanyX Philip Morris InternationalXX Philip Morris U.S.AXX R. J. Reynolds Tobacco Company R&DXX Southern Testing & Research Laboratories, IncXX Swedish Match North Europe DivisionX Swedish Match North American DivisionX Swisher International IncorporatedX U. S. Tobacco Mfg. LPXX University of KentuckyXX

Tobacco Specific Nitrosamine Collaborative Study Test Plan Recovery Studies Collaborative Interlaboratory Testing

Tobacco Specific Nitrosamine Collaborative Study Recovery Studies a a - Three tobaccos, three replicates, three nitrosamine levels b - Standards added in buffer, results based on external standard quantification c - Standards added in CH 2 Cl 2, results based on chromatographic standard, NDHA d - Standards added in CH 2 Cl 2, results based on surrogate standard, NG e - Standards added in CH 2 Cl 2, results based on surrogate standard, NDHA LaboratoryAverage(%)Nitrosamine NNN(%)NAT(%)NAB(%)NNK(%) Lab I – Method 2 b Lab I – Method 2 c Lab II – Method 2 d Lab III – Method 1 e Lab IV – Method 1 e

Tobacco Specific Nitrosamine Collaborative Study Samples to be analyzed in replicates of three, on the same day, under repeatability conditions Collaborative Test Design – Analyses

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* TSNALowHigh NNN NNK NAT NAB * 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 95% 99% 95% 99% Stragglers Outliers

Tobacco Specific Nitrosamine Collaborative Study – Method 1 Lab Sdev Mandel k 1% & 5% NNN 95% 99%

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)

Tobacco Specific Nitrosamine Collaborative Study Repeatability Comparisons Coefficient of Variation

Tobacco Specific Nitrosamine Collaborative Study Reproducibility Comparisons Coefficient of Variation

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

Tobacco Specific Nitrosamine Collaborative Study Recovery Lowest Concentration Standard Run as a Sample % Recovery

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)

Tobacco Specific Nitrosamine Collaborative Study Repeatability Comparisons Coefficient of Variation

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.

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