Determination of Humectants in Whole Tobacco

Health Canada
T-304 December 31, 1999

Table of Contents

  1. Scope of Applications
  2. Normative References
  3. Method Summary
  4. Apparatus and Equipment
  5. Reagents and Supplies
  6. Preparation of Glassware
  7. Preparation of Solutions
  8. Preparation of Standards
  9. Sampling
  10. Sample Analysis
  11. Quality Control
  12. References
  13. Appendix

1 Scope of Applications

  1. Applicable to the determination of the amount of glycerol, propylene glycol, and triethylene glycol which may be added to tobacco as humectants. The expected range of the individual humectants is to be 0.5 - 4.0 % on an "as received" basis.
  2. This method is not designed to measure trace quantities of contamination from external sources and does not distinguish between the amount of humectant added and any potential naturally occurring humectants (if any).

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.
  2. Health Canada Test Method T-402 - Preparation of Cigarettes, Cigarette Tobacco, Cigars, Kreteks, Bidis, Packaged Leaf, Pipe Tobacco and Smokeless Tobaccos for Testing, 1999-12-31.

3 Method Summary

  1. This method is a gas chromatographic (GC) method using a megabore fused silica column and a flame ionization detector (FID). Four grams of tobacco from a freshly opened source (package or tin of fine cut tobacco) is extracted with 50 mL of a methanol based extraction solvent on a mechanical shaker for 60 minutes. The sample is placed in the dark and allowed to sit for an additional hour until the supernatant is clear. A portion of the supernatant is transferred to a 2 mL autosampler vial and the humectant content evaluated by gas chromatography (GC).
  2. The humectants are analyzed on a megabore fused silica column, which has a polyethylene glycol (PEG) stationary phase. Quantitation is achieved using an internal standard calibration by comparing the FID response of the analytes in the samples against a four-point calibration of the corresponding humectant in the standards.
  3. In order to accurately compare the humectant concentration of one brand to another, a moisture analysis is done and the humectant concentration on a "dry matter" basis is determined.

    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.

4 Apparatus and Equipment

  1. 125 mL Erlenmeyer flasks with stoppers or equivalent.
  2. 10 mL, 100 mL, 2000 mL volumetric flasks.
  3. 20 mL pipette for the preparation of extraction solvent.
  4. Volumetric pipettes for the preparation of standard solutions (various sizes).
  5. Brinkmann Dispensette, 10 - 50 mL or equivalent.
  6. Gas Chromatograph with flame ionization detector (FID).
  7. Autosampler.
  8. 2 mL autosampler vials/caps/Teflon faced septa.
  9. DB-Wax fused silica column 15 m × 0.53 mm × 1 µm (or equivalent).
  10. Mechanical wrist action shaker.
  11. Disposable transfer pipettes.
  12. Analytical balance, capable of measuring to four decimal places.

5 Reagents and Supplies

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

  1. Methanol - Distilled in Glass.
  2. Glycerol.
  3. Triethylene Glycol.
  4. Propylene Glycol.
  5. 1,3-Butanediol (used as an internal standard - ISTD).

6 Preparation of Glassware

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

7 Preparation of Solutions

  1. Extraction Solution (concentration approximately 2 mg/mL 1,3-butanediol)
    1. Accurately weigh 20 g (+/- 0.05 g) of 1,3-butanediol into a 100 mL volumetric flask and make to volume with methanol.
    2. Label as 1,3-butanediol primary stock.
    3. Pipette 20 mL of primary (1o) stock to a 2 L volumetric flask and make up to volume with methanol. Mix well.

8 Preparation of Standards

  1. Glycerol Primary (1°) Standard (approximately 100 mg/mL)
    1. Accurately weigh 10 g (+/- 0.05 g) of glycerol into a 100 mL volumetric flask and make up to volume with extraction solution.
  2. Propylene Glycol Primary (1°) Standard (approximately 50 mg/mL)
    1. Accurately weigh 5 g (+/- 0.05 g) of propylene glycol into a 100 mL volumetric flask and make up to volume with extraction solution.
  3. Triethylene Glycol Primary (1°) Standard (approximately 50 mg/mL)
    1. With an analytical balance, capable of measuring to four decimal places, accurately weigh 5 g (+/- 0.05 g) of triethylene glycol into a 100 mL volumetric flask and make up to volume with extraction solution.
  4. Mixed Secondary (2°) Standard ( Std 4 of working standards)
    1. Pipette a 4 mL aliquot of each of the prepared primary stocks into a 100 mL volumetric flask.
    2. Make up to volume with extraction solution.
  5. Working Standards
    1. All standards are made in volumetric flasks using the dilutions described.

      The following table contains information regarding four working standards. For each sample, the following properties are included: volume of the mixed standards, fixed volume, glycerol concentration, propylene glycol concentration, and triethylene glycol concentration.

      Standard Volume
      of Mixed
      Std (mL)      		 Final
      Volume
      (mL)      		 Glycerol
      [mg/mL]      		 Propylene
      Glycol
      [mg/mL]      		 Triethylene
      Glycol
      [mg/mL]
      Std 1 2 10 0.8 0.4 0.4
      Std 2 4 10 1.6 0.8 0.8
      Std 3 7 10 2.8 1.4 1.4
      Std 4 (2° stock)     4.0 2.0 2.0

    Note 1: Volumetric flasks are made up to volume with extraction solution (which contains the internal standard - ISTD).

    Note 2: Analytical concentrations may vary depending on the concentration of primary stocks and must be calculated in order to prepare an accurate calibration.

    Note 3: If desired, an ultra-low standard may be prepared using 500 µL of the mixed secondary stock to 10 mL with extraction solvent. Although the sensitivity is not a problem, extrapolating beyond these concentrations may lead to misidentified peaks.

9 Sampling

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

10 Sample Analysis

  1. Extraction of Sample
    1. Accurately weigh 4 g of tobacco into a 125 mL Erlenmeyer flask.
    2. Add 50 mL of the extraction solution to the sample.
    3. Stopper the flasks and place on a wrist action shaker for 60 minutes.
    4. Remove samples from shaker, swirling the flask to get all of the tobacco into the solvent.
    5. Allow samples to sit for 30 minutes until the supernatant is clear.
    6. Transfer the supernatant to an autosampler vial and analyze on the GC.
  2. Gas Chromatograph Conditions
    Injector: Split with a split flow of approximately 50 mL/min at 120 °C.
    Column: DB-WAX, 15 m × 0.53 mm × 1.0 µm.
    Detector: Flame Ionization (FID), Channel A, 1 Volt Full Scale, Range:12,
    Atten: 8, Autozero: On.
    Carrier: He at 10.0psi, linear velocity approximately 150 cm/second.
    Relays: Initial relay state: all on.
    Note 1: Detector gas flows set as per manufacturer's specifications for H2 and X-Dry air.
    Note 2: No make-up gas is required because of the high flow rate of carrier gas.
    Temperature Program
    Injector: 220 °C.
    Detector: 260 °C.
    Start Temperature: 120 °C hold for two minutes.
    Rate: 15 °C/minute to 180 °C hold for four minutes.
    Total Run Time: 10.00 minutes,
    Autosampler Conditions: Injection volume: 1.0 µL
    1. Load autosampler vials into rack starting with the highest standard to the lowest before any samples are to be run in order to create a new calibration curve.
      Note: The first standard should be injected a minimum of three times initially to re-condition the column.
    2. Load samples into the rack placing a standard in every 10th position to verify the calibration and the quality of the chromatography.
    3. The DB-WAX column has a polyethylene glycol stationary phase ideal for the separation of glycols. Tailing effects, however, may be very severe in the case of glycerol if the incorrect chromatographic conditions are used. Tailing effects may also be caused by solvent effect and/or reactivity in the injector.
      1. Methanol is generally a poor solvent choice for injecting onto the GC because it causes an enormous amount of tailing. This effect is minimized by having a large split ratio, a very high linear velocity, a thick stationary phase, and deactivated glass injection liners.
      2. Reactivity in the injector is minimized by using deactivated glass inserts. It is necessary that the injection liner be changed between each set of samples (roughly 40 true samples) since the injected solution is quite dirty and creates active sites on the liner after repeated injections of sample.
  3. Calculations
    1. All results are expressed on an "as received" basis. These may be expressed on a dry matter basis using the appropriate moisture result.
      Analytical Result:
      Analyte [mg/g] = (AreaAnalyte Sample / AreaISTD Sample)X RF (mg/mL) × (Multiplier (mL) / Divisor (g)).
      where RF is determined from the calibration curve.
      Conversion of analyte [mg/g] to Percent (as is):
      Analyte (%) "as is" = Result (in mg/g)/10

      Conversion of analyte % (as is) to a Dry Matter Basis
      Analyte (%)dry matter = Analyte (%)"as is"/(1-(% Moisture/100))

      where the % Moisture is determined from the same sample as received for humectant analysis.

11 Quality Control

  1. Typical Chromatogram - see Appendix 1.
  2. Recoveries and Levels of Contamination
    1. This involves the use of a laboratory reagent blank (LRB) and a laboratory fortified blank (LFB) to evaluate the extent of potential analyte loss and reagent interference. Reagents are carefully selected to obtain an LRB as close to zero as possible and LFB as close to 100 % as possible.
    2. Each set of analyses should contain one LRB per batch of up to 20 samples.
    3. A Laboratory Fortified Matrix (LFM) is necessary when evaluating different matrices to determine the recovery of the analyte and potential chromatographic interferences from the sample that may effect quantitation.
    4. A duplicate sample may be analyzed to determine the reproducibility of the procedure.
  3. Method Detection Limit (MDL)/Limit of Quantitation (LOQ)
    1. This involves the use of either a test material with a low level of the analyte or the lowest standard. The standard deviation is determined and the MDL is determined to be three time this standard deviation. The LOQ is determined to be 10 times this standard deviation.
    2. This method was designed to determine relatively high levels of humectants in tobacco matrices. As such, the MDL and LOQ have not been determined. Should such determinations prove necessary, it is essential that they be determined for each set of analyses due to the chromatographic variability of the analytes.
  4. Stability of Reagents and Samples
    1. Stock solutions are stable for at least one month if stored at 4 °C.
    2. Calibration solutions should be made fresh for each new project and if continuing check standards fail.
    3. Extraction Solution can be used indefinitely since fresh calibration standards are made with each new set of samples.

12 References

  1. AOAC Official Method 971.02, Glycerol, Propylene Glycol, and Triethylene Glycol in Cased Cigarette Cut Filler and Ground Tobacco, Gas Chromatographic Method, AOAC Official Methods of Analysis (1995), Ch. 3, p. 30.
  2. AOAC Official Method 968.03, Menthol in Cigarette Filler, Gas Chromatographic Method, AOAC Official Methods of Analysis (1995), Ch. 3, p. 35.

13 Appendix

Appendix 1: Chromatographic Examples

The following figure includes the example chromatogram of a true tobacco sample layered over a standard at 5% offset.

Chromatographic Examples

Figure 1. Example Chromatogram of a true tobacco sample layered over on a standard at a 5 % offset.

Retention Times:

Propylene Glycol: 0.971 minutes.
1,3 - Butanediol (ISTD): 2.153 minutes.
Glycerol: 6.119 minutes.
Triethylene Glycol: 6.220 minutes.

Note: Nicotine in sample chromatogram elutes approximately 3.20 minutes.

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