Determination of 1,3-Butadiene, Isoprene, Acrylonitrile, Benzene and Toluene in Mainstream Tobacco Smoke

Health Canada
T-116 December 31, 1999

Table of Contents

  1. Scope of Applications
  2. Normative references
  3. Definitions
  4. Method Summary
  5. Apparatus and Equipment
  6. Reagents and Supplies
  7. Preparation of Glassware
  8. Preparation of Solutions
  9. Preparation of Standards
  10. Sampling
  11. Tobacco Product Preparation
  12. Smoking Machine Preparation
  13. Sample Generation
  14. Sample Analysis
  15. Quality Control
  16. Modifications for Intense Smoking Conditions
  17. References
  18. Appendix

1 Scope of Applications

  1. Applicable to the isolation and quantitation of the 1,3-Butadiene, Isoprene, Acrylonitrile, Benzene, and Toluene ("Volatiles") content of mainstream tobacco smoke by gas chromatograph/mass spectrometer (GC/MS).

2 Normative References

  1. Health Canada Test Method T-115 - Determination of Tar, Water, Nicotine and Carbon Monoxide in Mainstream Tobacco Smoke, 1999-12-31.

3 Definitions

  1. Refer to T-115 for definitions of terms used in this document.

4 Method Summary

  1. Volatiles are collected by passing the mainstream smoke of 10 cigarettes* through a 92 mm glass fibre filter disc (pad) and into cryogenic traps containing methanol. The impinger solutions are spiked with D6-benzene and injected onto a gas chromatograph/mass spectrometer (GC/MS) for quantitation.

    *For other tobacco products, select a number such that breakthrough does not occur.

    Note: The testing and evaluation of certain products against this test method may require the use of materials and or equipment that could potentially be hazardous and this document does not purport to address all the safety aspects associated with its use. Anyone using this test method has the responsibility to consult with the appropriate authorities and to establish health and safety practices in conjunction with any existing applicable regulatory requirements prior to its use.

5 Apparatus and Equipment

  1. Equipment needed to perform smoking as specified in T-115.
  2. Equipment needed to perform conditioning as specified in T-115.
  3. Equipment needed to perform marking for butt length as specified in T-115.
  4. Analytical balance measuring to at least four decimal places.
  5. 70 mL glass impingers with extra-coarse frits.
  6. Tygon tubing with connectors.
  7. Dewar flask.
  8. Thermometer (-100 to 40 °C).
  9. 10, 25, 50 and 100 mL volumetric flasks.
  10. Volumetric pipettes or gas-tight syringes for range 100 to 1000 µL.
  11. Varian Saturn I GC/MS system consisting of an 8100 autosampler, a 3400 GC with a 1077 split/splitless injector and an ion trap detector (or equivalent).
  12. J&W Scientific 60 m × 0.32 mm × 1 µm DB-5MS column (or equivalent) with 1m × 0.25 mm deactivated fused silica transfer line.
  13. Glassware oven.
  14. Vortex Mixer.
  15. Spectrophotometer.

6 Reagents and Supplies

Note: All reagents shall be, at the least, recognized as analytical reagent grade in quality.

  1. Dry ice.
  2. Propan-2-ol (IPA).
  3. Methanol - Distilled-in-Glass, or equivalent.
  4. Reagent Alcohol.
  5. D6 - Benzene - greater than or equal to 99 % pure.
  6. 1,3-Butadiene.
  7. Isoprene.
  8. Acrylonitrile.
  9. Benzene.
  10. Toluene.

7 Preparation of Glassware

  1. Glassware should be cleaned and dried in such a manner to ensure that contamination from glassware does not occur.

8 Preparation of Solutions

  1. Not applicable.

9 Preparation of Standards

  1. Preparation of Standards (except 1,3-butadiene)
    1. Four primary stock solutions are prepared by accurately weighing 100 µL each of isoprene, acrylonitrile, benzene and toluene into four 10 mL volumetric flasks, filling each flask to the mark with methanol and mixing well.
    2. A combined secondary stock solution is prepared by transferring appropriate aliquots of each of the primary stock solutions into a 25 mL volumetric flask, filling it to the mark with methanol and mixing well.
    3. A stock solution of D6-benzene is prepared by transferring the contents of a 1 g ampoule to a 10 mL volumetric flask, filling the flask to the mark with methanol and mixing well.
    4. An internal standard spiking solution is prepared by diluting 4 mL of the stock (9.1.3) to 100 mL with methanol and mixing well. Aliquots of this spiking solution are stored in 25 mL vials with Teflon-lined caps and at minus 20 °C.
    5. Five calibration standard solutions are prepared by adding 100 µL ISTD to each of five 10 mL volumetric flasks. The sides are rinsed with methanol, then appropriate aliquots of the secondary stock solution are added to each flask. The flasks are filled to the mark with methanol and mixed well.
    6. The solutions are transferred to a series of labelled autosampler vials, capped with Teflon-lined septa and stored at minus 20 °C until use.
      Note: Each vial is only used once.
  2. Preparation of 1,3-Butadiene Standards
    1. Attach a piece of Tygon tubing to the valve of a 1,3-butadiene cylinder. Place a Pasteur pipette on the other end and immerse the tip of the pipette into a 100 mL volumetric flask containing methanol up to the base of the neck of the flask. Open the valve and gently bubble the 1,3- butadiene into the methanol for about five minutes. Make the volume to the mark with methanol and mix well.
    2. Pipette 1 mL of the stock solution into a clean 100 mL volumetric flask and make to the mark with methanol and mix well. This is the secondary stock solution.
    3. Determination of secondary stock concentration
      1. Pipette 1 mL of the secondary stock solution into a 100 mL volumetric flask and make to the mark with reagent alcohol and mix well.
      2. Measure the absorbance of the solution against a reagent alcohol blank on the spectrophotometer at 217 nm. Make dilutions as necessary so that the absorbance (A) falls between 0.2 and 0.6.
      3. Calculate the concentration of the secondary stock solution according to the following:
      4. Conc. (µg/mL) = [A / 20893 L/mole] × 54 g/mole × [1000 mg/g /1000 mL/L] × [100 mL / 1mL] × 1000 µg/mg.
    4. Once the concentration of the secondary stock solution is known, make a minimum of four calibration standard solutions in the range appropriate for the expected delivery levels (typically 5 to 50 µg/mL).
    5. Internal standard solution (100 µL) is added to each 10 mL volumetric flask. The appropriate aliquot of secondary stock solution is also added and the volume made up to the mark with methanol.
    6. The solutions are transferred to a series of labelled autosampler vials, capped with Teflon-lined septa and stored at minus 20 °C until use. Note: Each vial is only used once.

10 Sampling

  1. The sampling of tobacco products for the purpose of testing shall be as specified in T-115.

11 Tobacco Product Preparation

  1. Product is to be conditioned as specified in T-115.
  2. Cigarettes, cigarette equivalent, bidis, and cigars are to be marked for butt length as specified in T-115.
  3. Cigarettes to be smoked under intense smoking conditions shall be prepared as specified in T-115.

12 Smoking Machine Preparation

  1. Ambient Conditions
    1. The ambient conditions for smoking shall be as those specified in T-115.
  2. Machine Conditions
    1. The machine conditions shall be as those specified in T-115.

13 Sample Generation

  1. Cigarettes shall be smoked and TPM collected as specified in T-115 with the following modifications as detailed below:
    1. Smoking is conducted on a 20 port rotary machine. See diagram.

      This figure displays a schematic drawing of the apparatus set up of the 20 port rotary smoking machine, which uses two impingers in a cryogenic bath.


      Fig 3: Set-up using two 70ml impingers in a cryogenic bath (-70)oC
    2. Prepare the impingers by adding 10 mL of methanol into each impinger.
    3. Immerse the impingers into a dry-ice/IPA bath (temperature at or below minus 70 °C).
    4. The impinger frits must be completely immersed in the methanol.
    5. Insert a glass fibre filter disc (pad) holder with pad into the syringe of the smoking machine, and then hook up in series the two impingers to the pad holder. Attach the first impinger to the large 92 mm pad holder.

14 Sample Analysis

  1. Spike the solution in each impinger with 100 µL of the ISTD solution, and vortex for approximately 10 seconds.
  2. Decant aliquots of each impinger solution into two labelled autosampler vials (four vials in total). Fill each vial to the base of the neck and cap with a Teflonlined septum. DO NOT OVERFILL VIALS.
  3. Store samples in the freezer (minus 20 °C) for up to 48 hours prior to analysis.
  4. Instrument Analysis: GC/MS Conditions
    Injector temperature 220 °C.
    Column temperature 35 °C for six minutes
    20 °C per minute to 225 °C, hold six minutes.
    Column pressure: 13 psi.
    Transfer line temperature: 200 °C.
    Manifold temperature: 240 °C.
    One µL of the methanol solution is injected onto the GC/MS, which is run in the split mode (Split flow 30 mL/minute). The GC/MS is operated in full-scan mode (50 to 200 amu). The following ion peak areas are used for quantitation:
    1,3-Butadiene 51+52
    Isoprene 67
    Acrylonitrile 52
    Benzene 78
    D6-benzene 84
    Toluene 91.
    Note: The assignment of these masses is based on selection of the best response (i.e. the base peak) and the need to avoid possible contamination from interfering peaks which may contain similar ions. The choice of quantitation ions may be different for different instrument configurations.
  5. Calculations
    1. Calibration Curve
      1. A calibration curve is generated at the beginning of each sample set or "project". Each standard solution is injected once and a calibration file built using the method for internal standard quantitation available with the Saturn quantitation software.
    2. Sample Calculation
      1. The software on the GC/MS is used to generate results for each analyte based on the concentrations of the standard solutions. The results are reported in µg/mL. To calculate the final results, the following calculation is used:
        Analyte (μg/cigarette) = [Conc. of Analyte in Sample (μg/mL) × Volume (mL)]/# of cigarettes

15 Quality Control

  1. Typical Chromatogram
    1. 15.1.1 See Appendix.
  2. Recoveries and Levels of Contamination
    1. To determine trapping efficiency, the contents of the two impingers are treated as two separate samples and the amount of volatiles in each determined. The amount of each analyte is determined in the second impinger and is reported as a % of the amount in the first. Typically, this "carryover" is less than 1 % for all analytes except 1,3-butadiene and Isoprene, which have typical "carryover" values of 3 % or less. In most cases, it is sufficient to only analyze the contents of the first impinger.
    2. Laboratory reagent blanks (LRB) should be analyzed every 20 samples. There is occasionally a small amount of toluene present in methanol and this should be monitored closely. A laboratory fortified blank (LFB) is not necessary for this analysis as there is no sample work-up.
  3. Method Detection Limit (MDL)/Limit of Quantitation (LOQ)
    1. The MDL can be defined as the level that gives a signal to noise ratio of three to one. The LOQ can be defined as the level that gives a signal to noise ratio of 10 to one. Because of chromatographic differences and the effect of solvent on the 1,3-butadiene peak, each analyte has different MDL and LOQ. They are estimated as follows (units are in µg/mL):
        MDL LOQ
      1,3-butadiene 0.3 1
      Isoprene 0.05 0.2
      Acrylonitrile 0.3 1
      Benzene 0.05 0.2
      Toluene 0.05 0.2

    Note: The DB-5 column used in this method would not be the best choice for analyzing acrylonitrile alone. It gives a poor peak shape and this is reflected in the much higher MDL and LOQ for this analyte. A more polar column would be more suitable for acrylonitrile, but for most purposes, the detection limits are acceptable for the matrices covered by this test method.
  4. Stability of Reagents and Samples
    1. Calibration standards are stable for at least one week if kept at minus 20 °C. Once punctured, the isoprene is lost rapidly so each vial is used only once.
    2. 1,3-butadiene standards are stable for approximately one week if kept at minus 20 °C. Once punctured, the vial should be discarded.
    3. Volatile stock solutions should be made fresh at the beginning of every project and can be stored in the freezer for at least two weeks to be used for working standards.
    4. Acrylonitrile, benzene and toluene are significantly less volatile than 1,3- butadiene and isoprene, and stock solutions may be stable for up to a month if kept at minus 20 °C.
    5. The secondary stock solution for 1,3-butadiene can be re-used almost indefinitely as the actual concentration of this solution is determined every time working standards are prepared from it.
    6. Samples are stable at minus 20 °C for up to 48 hours if the septum has not been punctured. It is essential that at least two vials be prepared for each sample as the vial is discarded once punctured.

16 Modifications for Intense Smoking Conditions

  1. Under intense smoking conditions, five cigarettes are smoked into 20 mL of methanol/impinger. Each impinger is spiked with 200 mL of ISTD solution.

17 Reference

  1. Byrd, G.D., K.W. Fowler, R.D. Hicks, M.E. Lovette and M.F. Borgerding. Isotope dilution gas chromatography-mass spectrometry in the determination of benzene, toluene, styrene and acrylonitrile in mainstream cigarette smoke, J. Chromat., 503, 1990, p. 359-368.
  2. Brunnemann, K.D., M.R. Kagan, J.E. Cox, and D. Hoffmann. Analysis of 1,3- butadiene and other selected gas-phase components in cigarette mainstream and sidestream smoke by gas chromatography-mass selective detection, Carcinogenesis, 11, 1990, p. 1863-1868.
  3. Brunnemann, K.D., M.R. Kagan, J.E. Cox, and D. Hoffmann. Determination of benzene, toluene and 1,3-butadiene in cigarette smoke by GC-MSD. Analysis of 1,3-butadiene and other selected gas-phase components in cigarette mainstream and sidestream smoke by gas chromatography-mass selective detection, Exp. Pathol., 11, 1989, p. 108-113.

Appendix

Appendix: Typical Chromatogram

Chromatogramme Plot
Comment : DB5 60x0. 32xi. 0 10 PSI SPLIT 1.0 UL INJ
Scan No: 1199
Retention time: 19:59
Mass Range: 50 - 199
Plotted : 1à 1199
Range : 1 à 1199

This figure displays a typical chromatogram plot of various target analytes peaks. These peaks correspond to the following analytes: 1,3-butadiene, isoprene, acrylonitrile, 2,6-benzene, benzene, toluene and styrene.

Typical Chromatogram

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