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

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

June 8-9, 2010Gaithersburg, Maryland

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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Objectives

Review general sources for tobacco products/tobacco smoke anal tical methods

Objectives

smoke analytical methods

Terms, abbreviations, general overview of common analytical proceduresanalytical procedures

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

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

Summary

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Sources for established tobacco products/tobacco

I t ti l O i ti f St d di ti (ISO)

psmoke methods

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

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Terms and abbreviationsTerms and abbreviations

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

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

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Example methods for specific chemicals in tobaccoExample 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)(common abbreviations)

Generation (to be discussed at next meeting)

Separation Gas chromatography (GC)High performance liquid chromatography (HPLC)g p q g p y ( )Ion chromatography (IC)

Detection Mass spectrometry (MS)Tandem mass spectrometry (MS/MS)Thermal energy analyzer (TEA)Thermal conductivity (TC)Flame ionization (FID)Flame ionization (FID)Conductivity (CD)Infrared Spectrometry (IR)Ultraviolet/Visible Spectroscopy (UV)

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Inductively Coupled Plasma (ICP)Atomic Absorption Spectroscopy (AAS)

Example methods for specific analytesp p y

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

Nicotine 1,2 3Water 1 4Water 1,4

TPMa 1,5Tarb

COc 1 6COc 1,6a) Total particulate matter; b) Tar is defined as TPM less the nicotine and water content; c) Carbon

monoxide; d) Non-dispersive infrared detectiond) Non-dispersive infrared detection

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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

Example methods cont

Analyte GC/MS

Example methods, cont.

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

1) Health Canada T-116; 2) Arista Labs; 3) Dong et al 2000; 4) Polzin et al 20071) Health Canada T-116; 2) Arista Labs; 3) Dong et al. 2000; 4) Polzin et al. 2007

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Example methods, cont.

Analyte HPLC-UV GC/MSAcetaldehyde 1 21 2Acetone 1 2Acrolein 1 2Butyraldehyde 1Butyraldehyde 1Crotonaldehyde 1Formaldehyde 1Methyl ethyl ketone 1 2Propionaldehyde 1 3

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

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Example methods, cont.

Analyte HPLC/Fluorescence GC/MSAnalyte HPLC/Fluorescence GC/MSCatechol 1 2

Hydroquinone 1 2

Phenol 1 2

Resorcinol 1 2

Cresols 1 2

1) H lth C d T 114 2) V h t l 20081) Health Canada T-114; 2) Vaughan et al. 2008

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Example methods contExample methods, cont.

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

NNK 4 ( h l i i ) 1 (3 id l) 1 b NNN N’ i i iNNK: 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone; NNN: N’-nitrosonornicotine NAB: N’-nitrosoanabasine; NAT: N’-nitrosoanatabine

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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 contExample methods, cont.

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

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

Example methods, cont.

A l t GC/MS

p

Analyte GC/MS1-aminonaphthalene 12-aminonaphthalene 1p3-aminobiphenyl 14-aminobiphenyl 1pyridine 2pyridine 2quinoline 2

1) H lth C d T 102 2) H lth C d T 112

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

Example methods contExample methods, cont.

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

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

Example methods contExample methods, cont.

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

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

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) ; ) ; ) ; )

Example methods, cont.

AnalyteGC (Flame ionization) HPLC

Colorimetric analyzer GC/MSy ) y

glycerol 1propylene glycol 1triethylene glycol 1y g ybenzo[a]pyrene 2 3hydrogen cyanide 4eugenol 5 6eugenol 5 6nitrate 7sodium propionate 8triacetin 9menthol 10

1) Health Canada T-304; 2) Health Canada T-103; 3) Ding et al. (2005); 4) Health CanadaT-1075) H lth C d T 105 6) ) St fill d A hl (2000) 7) H lth C d T 308

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5) 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)

Multiple methodsMultiple methods

Often different methods can provide comparable or equivalent results

Standard criteria to establish equivalency exist:

Representative sample set for comparisonRepresentative sample set for comparison

Analytical determination

Statistical comparison of results

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Some considerations for selecting specificSome considerations for selecting specific analytical methods

Applicability

Selectivity/Specificity

Universal or chemical specific detectionp

Analytical merit

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

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SummarySummary

Analytical methods exist to quantify chemical levels f l t l li tfor analytes on example list

In many cases multiple analytical methods are y p yavailable for measuring specific analytes

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

M i t t l ti l d t f diff tMeans exist to compare analytical data from differentmethods to ensure comparability

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Cl if i Q ti ?Clarifying Questions?

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Useful Web links for quick referenceqon tobacco/smoke method compilations

ISO www.iso.org

CORESTA CORESTA www.coresta.org

Health Canada www.hc-sc.gc.ca

British American Tobacco www.bat-science.com

Labstat www.labstat.com/Labstat www.labstat.com/

Arista Labs www.aristalabs.com

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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-brandsCelebucki 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 109Health 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-103Health 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

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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

References, cont.

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

H lth C d (1999) D t i ti f i id t t b k M th d T 206Health 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

H lth C d (1999) D t i ti f S bi A id i Wh l T b M th d T 313Health 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 smokeISO 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.

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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.

References, cont.

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

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Anal. Chem., 75(18), 4827 4832.