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Clifford Watson, Ph.D.Centers for Disease Control and PreventionNational Centers for Environmental HealthDivision of Laboratory Sciences

June 8-9, 2010Gaithersburg, Maryland

Analytical methodologies for measuring harmful/potentially harmful chemicals in tobacco products/tobacco smoke

The findings and conclusions in this presentation have not been formally disseminated by the Centers for Disease Control and Prevention and should not be construed to represent any agency determination or policy.

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Review general sources for tobacco products/tobacco smoke analytical methods

Terms, abbreviations, general overview of common analytical procedures

Review some commonly used methods for specific chemicals in tobacco/smoke

Multiple methods: Assessing equivalency, criteria to review relative strengths and weaknesses

Summary

Objectives

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International Organization for Standardization (ISO)

Cooperation Centre for Scientific Research Relative to Tobacco (CORESTA)

Health Canada

Commercial Labs (Labstat, Arista, Filtrona Scientific Services)

Centers for Disease Control and Prevention (CDC)

Tobacco Industry Phillip Morris: Counts et al. (Reg. Tox and Pharm 39 (2004) 111-134)

British American Tobacco

Benchmark Study for Massachusetts

Sources for established tobacco products/tobacco smoke methods

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Terms and abbreviations

Method A standardized analytical laboratory procedure used to determine the amount or concentration of a specific chemical or group of chemicals

Analyte A specific chemical (e.g., benzene) or mixture (e.g., tar) that is determined in an analytical laboratory procedure

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Example methods for specific chemicals in tobacco products/tobacco smoke

Not an exhaustive listing

Often multiple methods are available

Not an endorsement

Inclusion strictly illustrative

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General analytical procedure outline (common abbreviations)

Generation (to be discussed at next meeting)

Separation Gas chromatography (GC)High performance liquid chromatography (HPLC)Ion chromatography (IC)

Detection Mass spectrometry (MS)Tandem mass spectrometry (MS/MS)Thermal energy analyzer (TEA)Thermal conductivity (TC)Flame ionization (FID)Conductivity (CD)Infrared Spectrometry (IR)Ultraviolet/Visible Spectroscopy (UV)Inductively Coupled Plasma (ICP)Atomic Absorption Spectroscopy (AAS)

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Analyte GC (FID) GC/MS GC (TC) Gravimetric NDIRd

Nicotine 1,2 3Water 1,4TPMa

1,5

Tarb

COc 1,6

Example methods for specific analytes

a) Total particulate matter; b) Tar is defined as TPM less the nicotine and water content; c) Carbon monoxide;

d) Non-dispersive infrared detection

1) Health Canada T-115 2) ISO 10315:1991; 3) ISO 10362-1:1991; 4) Stanfill 2009; 5) ISO 4387:1991; 6) ISO 8454:1995

Analyte GC/MSAcrylonitrile 1-4Benzene 1-41,3-Butadiene 1-4Isoprene 1-4Styrene 1-4Toluene 1-4

1) Health Canada T-116; 2) Arista Labs; 3) Dong et al. 2000; 4) Polzin et al. 2007

Example methods, cont.

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Analyte HPLC-UV GC/MSAcetaldehyde 1 2Acetone 1 2Acrolein 1 2Butyraldehyde 1Crotonaldehyde 1Formaldehyde 1Methyl ethyl ketone 1 2Propionaldehyde 1 3

1) Health Canada T-104; 2) Polzin et al. 2007

Example methods, cont.

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Analyte HPLC/Fluorescence GC/MS

Catechol 1 2

Hydroquinone 1 2

Phenol 1 2

Resorcinol 1 2

Cresols 1 2

1) Health Canada T-114; 2) Vaughan et al. 2008

Example methods, cont.

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1) Hoffmann et al. 1979; 2) Truker et al. 1991; 3) Wu W et al. 2003; 4) Wagner, et al. 2005;5) Wu J, et al. 2008.

Example methods, cont.

Analyte HPLC/TEA GC/TEA HPLC/MS/MSNAB 1 2 3-5NAT 1 2 3-5NNK 1 2 3-5NNN 1 2 3-5

NNK: 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone; NNN: N’-nitrosonornicotine NAB: N’-nitrosoanabasine; NAT: N’-nitrosoanatabine

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Example methods, cont.

1) Health Canada T-209; 2) Rhoades et al. 1987; 3) Health Canada T-206; 4) Pappas et al. 2006

Analyte AAS or ICP-AES ICP-MSArsenic 1,2Cadimum 1 4Chromium 1,2Lead 1,2 4Mercury 3Nickel 1,2Selenium 1

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Analyte GC/MS1-aminonaphthalene 12-aminonaphthalene 13-aminobiphenyl 14-aminobiphenyl 1pyridine 2quinoline 2

Example methods, cont.

1) Health Canada, T-102; 2) Health Canada, T-112

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Analyte GC/MSanabasine 1,2anatabine 1,2nornicotine 1,2myosmine 1,2

1) Health Canada, T-301, 1999; 2) Wu (2003)

Example methods, cont.

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Example methods, cont.

1) Health Canada T-101; 2) Health Canada T-110; 3) Health Canada T-313; 4) Wilson et al. 2008

Analyte HPLC/UV IC GC/MS Chemiluminescenceammonia 1nitrogen oxides 2sorbic acid 3chlorinated dioxins/furans 4

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AnalyteGC (Flame ionization) HPLC

Colorimetric analyzer GC/MS

glycerol 1propylene glycol 1triethylene glycol 1benzo[a]pyrene 2 3

hydrogen cyanide 4

eugenol 5 6nitrate 7sodium propionate 8triacetin 9menthol 10

Example methods, cont.

1) Health Canada T-304; 2) Health Canada T-103; 3) Ding et al. (2005); 4) Health CanadaT-1075) Health Canada T-105; 6) ) Stanfill and Ashley (2000); 7) Health Canada T-308;8) Health Canada T-312; 9) Health Canada T-311; 10) Celebucki et al (2005)

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Multiple methods

Often different methods can provide comparable or equivalent results

Standard criteria to establish equivalency exist:

Representative sample set for comparison

Analytical determination

Statistical comparison of results

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Some considerations for selecting specific analytical methods

Applicability

Selectivity/Specificity

Universal or chemical specific detection

Analytical merit

Quantification range, Linearity, Recovery, Robustness, Accuracy, Precision, Reproducibility,…

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Summary

Analytical methods exist to quantify chemical levels for analytes on example list

In many cases multiple analytical methods are available for measuring specific analytes

Numerous factors guide decisions for using a specific method when multiple methods are available

Means exist to compare analytical data from differentmethods to ensure comparability

Clarifying Questions?

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ISO www.iso.org

CORESTA www.coresta.org

Health Canada www.hc-sc.gc.ca

British American Tobacco www.bat-science.com

Labstat www.labstat.com/

Arista Labs www.aristalabs.com

Useful Web links for quick referenceon tobacco/smoke method compilations

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References

Arista Labs, http://www.aristalabs.com/customtesting_mainstream.htm

Celebucki CC, et al. (2005) “Characterization of measured menthol in 48 U.S. cigarette sub-brands,” Nicotine & Tobacco Research 7(4) 523–531.

Ding YS, et al. (2005), “Determination of 14 Polycyclic Aromatic Hydrocarbons in Mainstream Smoke from Domestic Cigarettes,” Env. Sci. Tech., 39(2), 471-478.

Dong JZ, et al. (2000), “A simple GC-MS technique for the analysis of vapor phase mainstream cigarette smoke. J. Microcolumn Separations 12, 142-148.

Health Canada (1999) Determination of Ammonia in Mainstream Tobacco Smoke, Method T-101

Health Canada (1999) determination of Ni, Pb, Cd, Cr, As and Se in mainstream tobacco smoke, Method T-109

Health Canada (1999) Determination of 1- and 2- Aminonaphthalene and 3- and 4- Aminobiphenyl in Mainstream Tobacco Smoke, Method T-102

Health Canada (1999) Determination of Benzo[A]pyrene in Mainstream Tobacco Smoke, Official Method T-103

Health Canada (1999) Determination of Selected Carbonyls in Mainstream Tobacco Smoke, Method T-104

Health Canada (1999) Determination of Eugenol in Mainstream Tobacco Smoke, T-105

Health Canada (1999) Determination of Hydrogen Cyanide in Mainstream Tobacco Smoke, Method T-107

Health Canada (1999) Determination of Oxides of Nitrogen in Mainstream Tobacco Smoke, Method T-110

Health Canada (1999) Determination of Pyridine, Quinoline and Styrene in Mainstream Tobacco Smoke, Method T-112

Health Canada (1999) Determination of Phenolic Compounds in Mainstream Tobacco Smoke, Method T-114

Health Canada (1999) Determination of 1,3-Butadiene, Isoprene, Acrylonitrile, Benzene, and Toluene in Mainstream Tobacco Smoke, Method T-116

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References, cont.

Health Canada (1999) Determination of Tar, Water, Nicotine and Carbon Monoxide in Mainstream Smoke, Method T-119

Health Canada (1999) Determination of mercury in sidestream tobacco smoke, Method T-206

Health Canada (1999) Determination of oxides of nitrogen in sidestream tobacco smoke, Method T-208

Health Canada (1999) Determination of Alkaloids in Whole Tobacco, Method T-301

Health Canada (1999) Determination of Humectants in Whole Tobacco, Method T-304

Health Canada (1999) Determination of Nitrate from Whole Tobacco, Method T-308

Health Canada (1999) Determination of Sodium Propionate in Whole Tobacco, Method T-312

Health Canada (1999) Determination of Triacetin in Whole Tobacco, Method T-311

Health Canada (1999) Determination of Sodium Propionate in Whole Tobacco, Method T-312

Health Canada (1999) Determination of Sorbic Acid in Whole Tobacco, Method T-313

Hoffmann et al. (1979), Assessment of Tobacco-specific N-Nitrosamines in Tobacco Products” Cancer Research, 39, 2505-2509

ISO 4387:1991, Determination of the total particulate matter present in the smoke.

ISO 10362-1:1991, Water content of the total particulate.

ISO 10315:1991, Nicotine content of the total particulate.

ISO 8454:1995, Measurement of carbon monoxide present in the vapour phase of smoke

Pappas RS, et al. (2006) Cadmium, lead, and thallium in mainstream tobacco smoke particulate. Food and Chemical Toxicology 44(5): 714-723.

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References, cont.

Polzin et al. (2007), Analysis of volatile organic compounds in mainstream cigarette smoke. Env. Sci. Tech. 41, 1297-1303.

Rhoades CB, et al.(1997) Mainstream smoke collection by electrostatic precipitation for acid dissolution in a microwave digestion system prior to trace metal determination. Journal of AOAC International 80(6):1320-1331.

Stanfill SB, et al. (2000), “Quantification of flavor-related alkenylbenzenes in tobacco smoke particulate by selected ion monitoring gas chromatography mass spectrometry,” J. Arig. Food Chem., 48(4). 1298-1306.

Truker et al. (1991), “N-Nitroso compounds in cigarette tobacco and their occurrence in mainstream tobacco smoke,” Carcinogenesis, 12(2), 257-261.

Vaughan C, et al. (2008). Automated determination of seven phenolic compounds in mainstream tobacco smoke. Nicotine Tob Res. 10(7):1261-8.

Wagner, et al. (2005), “Development of a Quantitative Method for the Analysis of Tobacco-Specific Nitrosamines in Mainstream Cigarette Smoke Using Isotope Dilution Liquid Chromatography/Electrospray Ionization Tandem Mass Spectrometry,” Anal. Chem., 77, 1001-1006.

Wilson CL, et al. (2008), “Assessment of dioxin and dioxin-like compounds in mainstream smoke from selected US brands and reference cigarettes,” Food and Chem. Tox. 46, 1721-1733

Wu J, et al. (2008), “Quantitative Method for the Analysis of Tobacco Specific Nitrosamines in Cigarette Tobacco and Mainstream Cigarette Smoke by use of Isotope Dilution Liquid Chromatography Tandem Mass Spectrometry,” Anal. Chem, 80, 1341-1345

Wu W, et al.(2003) “Simultaneous Determination of Five Tobacco Specific Nitrosamines in Mainstream Cigarette Smoke by Isotope Dilution Liquid chromatography/Electrospray Ionization Tandem Mass Spectrometry,” Anal. Chem., 75(18), 4827 4832.