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Slide 1 - Welcome to the ITC Con


									 Product Testing and Monitoring
          Richard J. O‟Connor, Ph.D.
      Roswell Park Cancer Institute, Buffalo, NY

           Clifford H. Watson, Ph.D.
Centers for Disease Control and Prevention, Atlanta, GA
        What will we cover?
1) The importance of product surveillance
  and monitoring to the Framework
  Convention on Tobacco Control
2) Equipment and methods for
  characterizing and assessing product
3) How to become involved in TobLabNet
  and/or the International Tobacco Product
Tobacco Product Regulation under

   FCTC provides a basic framework for
    regulating the manufacture, distribution,
    and emissions of tobacco products

   Regulations should serve public health
                     Article 9
   Regulation of the contents of tobacco products
   “The Conference of the Parties … shall propose
    guidelines for testing and measuring the
    contents and emissions of tobacco products, and
    for the regulation of these contents and
   “Each Party shall, where approved by competent
    national authorities, adopt and implement
    effective legislative, executive and administrative
    or other measures for such testing and
    measuring, and for such regulation.”
                      Article 10
   Regulation of tobacco product disclosures
   “Each Party shall, in accordance with its national law,
    adopt and implement effective legislative, executive,
    administrative or other measures requiring
    manufacturers and importers of tobacco products to
    disclose to governmental authorities information about
    the contents and emissions of tobacco products.”
   “Each Party shall further adopt and implement effective
    measures for public disclosure of information about the
    toxic constituents of the tobacco products and the
    emissions that they may produce.”
                  Article 11
   Packaging and labeling of tobacco
   Bans „light,‟ „mild,‟ and other misleading
   Requires minimum standards for warning
    labels (size, placement, etc.)
   Requires information about contents and
    emissions to be disclosed on the pack
    Current product regulations…
   Vary widely from country to country, but most
       ISO smoke chemistry testing
       Disclosure of yields on packs
       Some type of health warning on packs
   Governments and public health officials
    generally have little control or knowledge of
       additives
       emissions under more realistic smoking conditions
       Physical properties and design features of products
       Contents of smokeless tobacco products
Smoke Chemistry Testing
 Research – Chemical, in vitro,
            animal, human

       Understanding the product
       Assessing design features
       Hypothesis generating

 Testing -- Product characterization

       Large sample size
       Repetitive testing
       Aid regulation/enforcement
  Basic Cigarette Smoke Nomenclature

Sidestream Smoke                   Vapor Phase

                         Mainstream Smoke

                                 Particulate Phase
A. Necessary equipment, initial costs, continuing costs
1. Smoking machines

   i. Linear (1, 10, 20 ports common)
         Useful for analyzing multiple
         brands simultaneously.
         Cost range $ 12,000 (single port)
         to $ 150,000 USD (20-port).

   ii. Rotary (1 port)
         Useful for generating continuous
         smoke for analysis/exposure studies on
         single brand. Cost ~$100,000

   Required supplies and testing equipment:
   Glass filter pads ($500/yr), extra filter pad
   holders (~$200 each),
   extra gas collection bags, CO standards

   Air flow velocity anemometer (~$5000)
   Puff volume analyzer (~$2000)
2.   Environmental chamber
     Conditioning cigarettes 24 hrs prior to smoking at constant temperature
     and humidity (ISO: 22C, 60% RH).
     Small chamber for conditioning tobacco products only ($4,000 to
     Room size chamber for smoking machine and tobacco products
     ($40,000 to $60,000).
     Thermometer, hydrometer, barometer (~$100 to $1000).

3.   Analytical instrumentation (and solvents, vials, glassware, and
     i.       Precision balance (~$2000)
     ii.      Gas chromatography with thermal conductivity detector and
              flame ionization detector for water and nicotine analysis
B. Testing Regimes using linear smoking machine:

Routine analysis of tar, nicotine, and carbon monoxide (TNCO)

                                                                      High tar

                                  Total particulate matter (TPM):       Low tar
                                  “tar”, nicotine, and water.
                                  Smoking Regimens
                                  FTC/ISO (35 ml/60s)
                                  Massachusetts (45ml/30s 50% FV)      Unused
                                  Health Canada (55ml/30s 0% FV)

      Suggested: FTC/ISO for historical comparison and an
      “intense” regimen for upper exposure limit estimate
     FTC/ISO smoking regime:          Intense smoking regimen:
     35 ml puff volume,               55 ml puff volume, 2 s puff,
     2 s puff, 60 s interval.         30 s interval, 0% filter ventilation
    iii.   GC with thermal energy analyzer (TEA) for analysis of tobacco specific
           nitrosoamines (TSNAs) (~$95,000)

    iv.    Mass spectrometer(s)

           GC/MS – Volatile organic chemicals (VOCs), semi-volatiles, flavors,
           polyaromatic hydrocarbons (PAHs) ,free-base nicotine
           (~$80,000 to $140,000).

           LC/MS/MS – Phenols, high molecular weight PAHs, TSNAs
           (~$320,000 to $400,000)

           ICP/ MS – Heavy metals (~$250,000)

   v.      Service contracts (Estimate 10% of equipment costs for yearly service).

4. Facilities and personnel

   Laboratory space (preparation lab with workbench, fume hood, chemical
   storage cabinets, refrigerator/freezer for sample storage, sink, ultrasonic bath,
   platform shaker)

   Personnel (1-3 covers basics, more as activities expand)
C. The basics: Tar, nicotine, and carbon monoxide (TNCO)

    General Steps: Condition cigarettes, weight filter pads, smoke (20 sets of 5) under FTC, fewer
    with intense regimen, determine total particulate matter (TPM), extract nicotine and water,
    analyze with GC, TPM – nicotine – water = “tar”

                                     Particulate phase collected CFP
                                                                               Vapor phase gas sampling bag

                Cigarette smoke

                            nicotine, “tar”,
                            TSNAs, PAHs, Phenols,                                Carbon monoxide, NOx,
                            Heavy metals                                         volatile organic compounds

Useful References: The FTC cigarette test method for determining tar, nicotine, and carbon monoxide yields of U.S.
cigarettes: report of the NCI Expert Committee (Smoking and tobacco control monograph no. 7). Bethesda, Maryland: US
Department of Health and Human Services, National Cancer Institute, 1996 (NIH Publication No. 96-4028).
Determination of “Tar”, Nicotine and Carbon Monoxide in Mainstream Tobacco Smoke, Health Canada, Official Method, International Organization for
Standardization (ISO) Methods: ISO 8243:1991 (Sampling), ISO 3308:1991 and ISO 3402:1991 (Cigarette Analysis,
definitions and standard conditions), ISO 4387:1991(Tar), ISO 8454:1995 (CO), ISO 10315:1991 (Nicotine),
ISO 10362:1991 (Water)
D. Advanced: TSNAs, PAHs, Phenols, VOCs, free-nicotine,
   and heavy metals

                                    N                     N              N             N

                        N           CH3            N      H        N     H       N     H

 Main                   Nicotine                  Nornicotine     Anabasine      Anatabine
                        O       N       CH3
              N   O
        O                           N
              N                               O
                  CH3                                     N              N             N

    N                       N                       N     N   O    N     N   O   N     N   O

        NNK                 NNA                         NNN            NAB           NAT

Analysis in smokeless tobacco products, cigarette, cigar, pipe tobacco filler, and
smoke using:

 GC with thermal energy analyzer (GC/TEA) - lower cost

 Liquid chromatography tandem mass spectrometer detection (LC/MS) - higher cost.
                Typical TSNA Sample Preparation

                          Mainstream                IS addition

                                       Filter pad                 DCM extraction

0.1N HCl            1N NaOH

                  Aqueous phase

                   Organic phase
                                                            Chemical analysis:
                            Solid phase extraction          GC/TEA or LC/MS
     Liq-Liq extraction
Analysis of volatile organic compounds (VOCs) in smoke

VOCs (Typically vapor phase constituents passing through the particulate matter
filer. Samples are typically collected in clean gas sampling bags, impingers, or cold

VOCs can also be measured in the filler. One application is to provide a “chemical
fingerprint” for contraband or counterfeit detection.

ISO and Health Canada methods are available.
                                     GC/MS VOC profile of Marlboro cigarettes
                     Marlboro (Customs)

                     Marlboro (US for export)   Propylene Glycol
Relative Abundance


                     Marlboro (US for export)                          1-Acetoxy-2-propanol

                     Marlboro (US for export)

                       8.0             9.0           10.0              11.0             12.0
                                                Retention time (min)
Polyaromatic hydrocarbons (PAHs) and Phenols

PAHs and Phenols typically have low concentration in the tobacco filler and
originate as combustion products.

Both are example of semi-volatiles and are mainly present as constituents
in the particulate matter collected on the filter pad.

                          Typical PAH analysis method

           Collect                        SPE                           GC/MS
            TPM                        Clean-up                         Analysis

                     Typical Phenols analysis method

Collect                  Derivatization                Headspace                   GC/MS
  TPM                                                                              Analysis

ISO and Health Canada methods are available for benzo[a]pyrene and common phenolic compounds.
      Polyaromatic Hydrocarbon GC/MS Profiles
                                                             1.     Naphthalene                  11.    Benzo[b]fluoranthene
                               4                             2.     Acenaphthylene               12.    Benzo[k]fluoranthene
                                                             3.     Acenaphthene
                                                                                                 13.    Benzo[e]pyrene
                                                             4.     Fluorene
                                                             5.     Phenanthrene                 14.    Benzo[a]pyrene
                                                             6.     Anthracene                   15.    Indeno[1,2,3-c,d]-
PAH stock                            5                       7.     Fluoranthene                        pyrene
            1                                                8.     Pyrene
                                                                                                 16.    Dibenz[a,h]anthracene
                                                 7           9.     Benz[a]anthracene
                                           6                 10.    Chrysene                     17.    Benzo[g,h,i]perylene
                                                                                12 13                       16 17
                                                             9     10      11            14


                6.00       8.00    10.00       12.00       14.00        16.00    18.00        20.00    22.00   24.00
Heavy Metals

Present smokeless tobacco products and tobacco filler of cigarettes,
cigars, and pipe tobacco.

In mainstream cigarette smoke most metals tend towards partitioning in the
particulate matter.

Typical glass filter have high metal background. Use of other filter materials
or smoke collection techniques such as quartz fiber or impingers advised.

For inductively coupled plasma mass spectrometry (ICP-MS) tobacco samples
are dissolved in strong acid or prepared using microwave digestion.

Metal content in filler and smoke usually reflect metal content in soil where
tobacco was grown.

Available analytical methodologies include:

Atomic Absorption Spectroscopy (AAS)
Inductively Coupled Plasma – Atomic Emission Spectroscopy (ICP/AES), and
Inductively Coupled Plasma – Mass Spectrometry (ICP/MS)
Nicotine exists in 1 of 3 forms, di-protonated, mono-protonated, or
non-protonated. Protonated and free-base forms are typically present in
tobacco or smoke. Free-base nicotine absorbs more rapidly.

                                                    +                         +
                      N                             N                         N
                                                       H                            H
                      CH3                           CH3            +          CH3
          N                             N                          N
      Free-base or                      Protonated                Di-Protonated
      Unprotonated                      Nicotine                  Nicotine
      Nicotine (volatile)               (less volatile)           (less volatile)

              Total Nicotine as measured with
              standard TNCO method

Developments of ISO (and others) standard methods are underway.
                      Free-base nicotine

Fraction of Species

                       70                                   N
                                                                  H                         N
                                                  N         CH3
                       50                                                          N        CH3
                            0   1    2    3   4       5     6         7   8    9       10   11

                                                          Free-base nicotine
                        “Effective pH” = pKa2 + log
                                                          Protonated nicotine
Design Features and Physical Properties

    a. Effects of design features on human smoking behavior

    b. Ventilation, pressure drop, paper porosity

    c. Tobacco analysis – weight, density, moisture,
       nicotine content, blend, pH

    d. Filter analysis – weight, density, fiber fallout

    e. Lower-cost options
a. Effects of design features on human smoking behavior

     Typical Low Delivery Cigarette

                                During a puff, air entering vent
                                holes dilutes smoke stream

                                                          Tobacco filler
                                                          (~0.6 grams)
                Filter ventilation

                        Air dilution through filter vent holes significantly
                        reduces mainstream smoke deliveries.
                        People don’t smoke like machines

               Location of
             Filter vent holes

                                                               Finger or lip blockage increases
Air dilution through filter
                                                               mainstream smoke deliveries(1).
vent holes reduces machine
smoked mainstream deliveries.
                                                               Air dilution may result in larger or more
                                                               frequent puffs(2) increasing delivery(3).

 1.   Kozlowski LT, Rickert WS, Pope MA, Robinson JC, Frecker RC (1982) British Journal of Addiction 77, 159-165.
 2.   Kozlowski LT, O’Connor RJ (2002), Tobacco Control, 11, i40-i50.
 3.   Kozlowski LT, Sweeney CT, Pillitteri, JL (1996) Experimental and Clinical Psychoparmacology, 4, 404-408.
                                       Effect of vent blockage on machine smoked deliveries
                                           of an ultralight cigarette (80% filter ventilation)

                          1.6                                                           25

                                Nicotine                                                     Tar
nicotine (mg/cigarette)

                                                                   tar (mg/cigarette)
                            1                                                           15




                            0                                                            0
                                   0            50           100                              0             50             100
                                    % filter vent blockage                                        % filter vent blockage

                                Nicotine and tar levels increase ~10x as filter vent holes are blocked.
b. Ventilation, Paper porosity, pressure drop

Ventilation: % air dilution of mainstream smoke from filter vent holes and
air permeability of paper wrapper (porosity)

                  Air enters the tobacco rod through the porous paper
  Vent holes

    Total pressure drop: Limited range of consumer acceptability
                         Determines draw resistance

    Filter pressure drop: Proportional to filter efficiency
              Ventilation apparatus
(Filter ventilation, Rod ventilation, Pressure drop)

                                   Adjustable seal


                                   Adjustable seal
                                   Adjustable seal

              Draw Resistance/pressure drop
                  Pressure drop often reported as PDO or PDC
            Air                        Air

                                                    Pressure drop:
                                                    Pressure difference at the
Air                     Air   Air            Air    cigarette’s two ends
                                                    with 17.5ml/sec air flow.

                                                    Pressure differential ~ airflow
                                                    A lower air flow (i.e., highly
      Air         Air                               ventilated filter), reduces
                                                    pressure drop.
 Pressure drop                      Pressure drop
 open (PDO)                         closed (PDC)
How to measure ventilation and
pressure drop…
   Several lab
   Cost US$15,000-
   Digital instruments
    easy to use
c. Tobacco Analysis – weight, density, moisture, nicotine
                      content, blend, pH
                         Weight: Gravimetric analysis
                                 Precision balance + 0.1 mg

                         Density: Weight / Tobacco rod volume
                                   Lighter delivery cigarettes often contain
                                  “expanded” filler having less density

                         Moisture: Measured via IR or gravimetrically after drying
                                    Influences consumer acceptability and deliveries

                         Nicotine content: Influenced by blend, grade, stalk position
                                            Determined from solvent extraction/GC

                         pH: Aqueous pH measurement of supernatant from
                         tobacco suspension
Typical US blend:

Bright   Burley   Stems   Oriental Recon
(30-40%) (20-30%) (2-10%) (10-15%)  (Bandcast or Paper 5-15%)
d. Filter Analysis – weight, density, fiber fallout

                                      Filter materials include cellulose
                                      acetate (most common), paper, and

                                      Filters may also contain activated
                                      charcoal (cavity or Dalmatian style) or
                                      other components.

                                      Loose filter fibers could be inhaled and
                                      deposit in lungs.

                                      Fallout analysis useful to access
                                      filter’s mechanical integrity.
e. Lower cost options

 1. Collaborate with others
         a. Independent tobacco laboratories
         b. Universities
         c. Government laboratories

 2. Use independent contract laboratory

 3. Develop expertise with least expensive methods
         a. Physical properties (length, diameter, weight)
         b. Microscopy (confirm filter ventilation, blend analysis)
         c. Electrode methods (pH, ammonia, nitrite)
         d. Ion chromatography (ammonia, perchlorate, selected metals)
         e. HPLC/UV and GC/FID instrumentation
Ways to get involved in product monitoring and research

a. International Tobacco Products Repository

• Currently houses cigarette samples from 10 countries
   • US, UK, Canada, Australia, Czech Republic, Greece,
   China, Malaysia, Thailand, Mexico
Ways to get involved in product monitoring and research

• We are looking to expand by 10-12 countries this year
  • If you are interested in knowing more about the
    cigarettes available in your country, we can assess
    design features and arrange for ISO smoke chemistry
    testing at an independent lab

• Grants of up to $3500 available to facilitate purchases
  and testing

• See Dr. O’Connor or Craig Steger for application
International Tobacco Products Repository

• What we do for you
• Provide packaging materials and pre-labeled specimen
  bags to make things easy.
• Provide suggestions to help identify leading brands in
  your country.
• Provide unique information about brands in your country
  and comparisons to other countries

b. What you do for us
• Identify leading brands in your country
• Purchase 18 packs of each brand variety in 3 distinct
   locations within each country
• Provide additional samples for comparison among
• Add to the overall value of the repository

    TobLabNet Goals

Establish global tobacco testing and research capacity to test tobacco products
for regulatory compliance.

Research and develop harmonized standards for contents and emissions

Share tobacco research and testing standards and results.

Inform risk assessment activities related to use of tobacco products.

Develop harmonized reporting of such results so that data can be transformed
into meaningful trend information that can be compared across countries and
over time.
TobLabNet Activities
Provide remedial support for labs
Develop a method compendium
Train laboratory staff in new analytical methodologies
Develop common materials for standardization, PT and QC
Identify best laboratory practices
Test new methods using multiple laboratories
Carry out projects for global and regional products and
Perform collaborative research
Provide access to world-wide expertise
Provide means for electronic communications between labs
TobLabNet Contacts

Gemma Vestel
Tobacco Free Initiative
World Health Organization
20 Ave. Appia
1211 Geneva, Switzerland
+4122 791 4705
C. CDC training in smoke chemistry analysis (web-based

      Current methods are being evaluated.

      Appropriate methods will hopefully be available on-line (2007).

      Focus on method for product testing.

      New method will continue to be added as needed.

      For more information about CDC’s tobacco program see:

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