1、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 - QUA
2、NTIFICATION 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 ExtractionBest Day-to-Day Reproducibility (Robotic Prepa
3、ration) 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 i
4、n 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(
5、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 (g g-1),a- Cor
6、rected for internal standard recovery,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 EXTRACTIONSurro
7、gate NG Surrogate Internal StandardGC NDHA Gas Chromatographic StandardExternal External Standard Calibration,Methods,Tobacco Specific Nitrosamine Collaborative Study,Method 11. Weigh 1.5 g sample in test tube2. Extract in 10 mL 0.4 g mL-1 NDHA in CH2Cl2 + 0.5 mL 10 % NaOH using vortex shaker3. Deca
8、nt liquid portion of extract onto column containing Na2SO4 and MgSO44. Rinse column with 2 mL CH2Cl25. Evaporate eluent with air 6. Reconstitute in chloroform7. GC (DB-5 column) with chemiluminescence detection,Tobacco Specific Nitrosamine Collaborative Study,Method 21. Weigh 1 g sample in 125-mL fl
9、ask2. Add 1 mL 2.0 g mL-1 NG in CH2Cl23. Extract in 50 mL citrate-phosphate-ascorbic acid buffer 4. Pour entire extract onto head of a Chem Elut tube5. Rinse Chem Elut tube with two 150 mL portions of CH2Cl2, collecting eluent in 250-mL TurboVap tube6. Add 1 mL of 2.0 g mL-1 NDHA in CH2Cl2 7. Concen
10、trate in TurboVap concentrator to 0.5 mL8. Reconstitute with CH2Cl2 9. GC (DB-1 column) with chemiluminescence detection,Tobacco Specific Nitrosamine Collaborative Study,Participating Laboratories,Tobacco Specific Nitrosamine Collaborative Study,Test Plan,Recovery StudiesCollaborative Interlaborator
11、y Testing,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 quantificationc -Standards added in CH2Cl2, results based on chromatographic standard, NDHA d -S
12、tandards 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,Samples to be analyzed in replicates of three, on the same day, under repeatability conditions,Collaborative Test
13、 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 C
14、ollaborative Study,Test Design TSNA Range*,TSNA Low HighNNN 0.24 9.06 NNK 0.10 1.56NAT 0.12 4.06 NAB 0.03 0.21,* Analysis via Method 1 in units of g g-1 dry weight,Tobacco Specific Nitrosamine Collaborative Study,Analysis,ISO 5725 Accuracy (trueness and precision) of measurement methods and results,
15、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 SdevMandel k1% & 5%,NNN,95%,99%,Tobacco Specific Nitrosamine Collaborative Study,Statistical Method Comparisons,A
16、nalysis of Variance: Ryan-Einot-Gabriel-Welsch Multiple Range TestAnalysis of the Log of Sample MeansAnalysis 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 N
17、itrosamine 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 QuantitationLimit of Detec
18、tion (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 g mL-1,Tobacco Specific Nitrosamine Collaborative Study,RecoveryLow
19、est Concentration Standard Run as a Sample,% Recovery,Tobacco Specific Nitrosamine Collaborative Study,Further Analysis,Method 1 VariationsRobotic Sample PreparationN-nitrosoguvacoline as the Surrogate Internal Standard,Tobacco Specific Nitrosamine Collaborative Study,Means Comparisons Average Log M
20、eans (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. Ac
21、curacy 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 C
22、OV for Method 2 reproducibility was 6% lower for NNN and 12% lower for NNK.,AcknowledgementsHubert Klus, Helmut Begutter and Anton Pachinger, Austria TabakChristine A. Rouse and C. Roy Taylor, Brown & Williamson Tobacco CorporationJacques Dumont, Imperial Tobacco LimitedTakeshi Sakaki, Hideyuki Tomi
23、ta, Hideki Takahashi and Hitoshi Saito, Japan Tobacco Inc.Bill Rickert, Mehran Sharifi and Peter Joza, Labstat International, Inc.,Richard Entz and Richard Shober, Lancaster LaboratoriesJim Morgan and Cynthia Williard, Lorillard Tobacco CompanyJean-Marc Renaud and Roxanne Boudoux, Philip Morris Inte
24、rnationalChorng Huang, Philip Morris USAMike Borgerding, Christy Fishel, and Angela Synder, R. J. Reynolds Tobacco Company R&DMark Hathaway, Kim Baughman and Ken Boyer, Southern Testing & Research Laboratories,Acknowledgements,Lennart Johansson and Susanne Back, Swedish Match North Europe DivisionSteve Terrell, Swedish Match North American DivisionThomas Losty, John Townend, Paul Moser and Ahad Majeed, Swisher International, Inc.Cliff B. Bennett, Carl Midgett, Rusty Owens and Kathleen Johnson, U. S. TobaccoHarold Burton and Naewanna Dye, University of Kentucky,Acknowledgements,
