Determination of Ammonia in Mainstream Tobacco Smoke

Health Canada
T-101 December 31, 1999

Table of Contents

  1. Scope
  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
  17. References
  18. Appendix

1 Scope

  1. Applicable to the isolation and quantitation of ammonia in mainstream tobacco smoke by ion chromatography.

2 Normative References

  1. American Society for Testing and Materials (ASTM) D 1193-77 - Standard Specifications for Reagent Water, Version 1977.
  2. Health Canada Test Method T-115 - Determination of Tar, Water, Nicotine and Carbon Monoxide in Mainstream Smoke, 1999-12-31.

3 Definitions

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

4 Method Summary

  1. 10 cigarettes* are smoked on a rotary smoking machine through a 92 mm glass fiber filter disc (pad) with two impingers containing 15 mL of 0.1N H2SO4 placed in series between the pad and the drawing syringe. The pad, after being weighed for total particulate matter (TPM) is placed into a 125 mL Erlenmeyer flask to which the contents of the impingers are also added.
  2. Smoking may also be done on a linear type smoking machine. In this case 10 cigarettes* are smoked using 44 mm glass fiber filter discs (pads) with single impingers containing 30 mL of 0.1N H2SO4 placed between the pads and the drawing syringes. In this case the pad must be changed after five cigarettes in order to prevent overloading of the pad (breakthrough), as evidenced by brown stains on the back of the pad.

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

    Note: The adequacy of the required impingers must first be evaluated by creation of a smoke train with three impingers in series. All components of the smoke train must be analyzed separately in order to determine trapping efficiencies. If breakthrough to the second and third impinger is >3% of the first impinger, these must be included in the smoke train. Trapping efficiencies may differ slightly dependent on impinger design.
  3. The pad (or pads) is extracted with the diluted impinger solution on a wrist action shaker for 30 minutes. This mixture is then filtered through a 0.45 µm syringe filter into a scintillation vial where the sample may be stored at 4 °C for up to 48 hours.
  4. An aliquot of the filtered sample is then transferred to an autosampler vial and is analyzed by cation exchange chromatography
  5. A 35 µL volume of sample is injected onto a cation exchange analytical column that uses a Carboxylic acid/Phosphonic acid functional group to achieve separation of ammonium and monovalent cations. In order to adequately resolve sodium from the ammonium cation for quantitation, a 0.003N (Normal) methane sulfonic acid solution is used as the mobile phase. After the ammonium ion has eluted, a gradient using concentrations of 0.2N H2SO4 to a 0.05N H2SO4 is used to remove any divalent cations and quaternary amines that may be present in the sample.
  6. Detection of cations is achieved using a suppressed conductivity detector in external water mode. This method of detection reduces background conductivity from the mobile phase, thus increasing the sensitivity of the detector for the analyte.
  7. Quantitation is obtained from a five point external standard calibration using the peak height response of ammonium sulphate. The amount of ammonia per cigarette is determined by calculating the amount of ammonia present in the analytical solution, then multiplying by the appropriate multiplier (impinger volume × dilutions) and divisor (# of cigarettes).

    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 conditioning as specified in T-115.
  2. Equipment needed to perform marking for butt length as specified in T-115.
  3. Equipment needed to perform smoking of tobacco products as specified in T-115.
  4. Analytical balance measuring to at least four decimal places.
  5. Tweezers and gloves for transferring pads.
  6. 125 mL polymethylpentene (PMP) Erlenmeyer Flasks with screw - caps.
  7. Wrist-action shaker.
  8. Syringe filter: glass fibre (25 mm × 0.45 µm).
  9. 70 mL impingers without frits.
  10. 25, 50, and 100 mL volumetric flasks.
  11. Disposable 5 cc syringe .
  12. 7 mL screw top vials with aluminium lined cap.
  13. Autosampler vials, caps and Teflon-lined septa.
  14. High Performance Liquid Chromatograph (HPLC) consisting of:
    1. Refrigerated autosampler with 100 µL partial fill loop.
    2. Tertiary gradient system.
    3. Column heater.
    4. Dionex ED-40 conductivity detector or equivalent.
    5. Dionex CTC-1 cation trap or equivalent.
    6. Dionex CSRS-II conductivity suppresser in external water mode or equivalent.
    7. Data collection system.
    8. Dionex IonPac CS12A cation exchange analytical column (250 mm × 4 mm) or equivalent.
    9. Dionex IonPac CG12A cation exchange guard column (50 mm × 4 mm) or equivalent.

6 Reagents and Supplies

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

  1. Ammonium Sulphate > 99 %.
  2. Sulphuric Acid 96 %.
  3. Methanesulphonic Acid (MSA) 100 %.
  4. Type I water (Meets ASTM D 1193-77 specifications).

7 Preparation of Glassware

  1. Glassware should be cleaned and dried in such a manner to ensure that contamination from glassware does not occur.
  2. Immediately prior to use, all impingers are rinsed two times with 0.1N H2SO4, then three times with Type I water.

8 Preparation of Solutions

  1. Sulphuric Acid, 0.10N - Impinger Solution
    1. Carefully add 5.108 g of H2SO4 (96 % w/w) to 900 mL of Type I water.
    2. Mix and dilute to 1 L with Type I water.
  2. Sulphuric Acid, 0.20N - Solution C (Ion Chromatography)
    1. Carefully add 10.216 g of H2SO4 (96 % w/w) to 900 mL of Type I water.
    2. Mix and dilute to 1 L with Type I water.
  3. MSA 0.003N - Solution A (Ion Chromatography)
    1. Carefully add 0.2883g of Methanesulphonic Acid (MSA) to 900 mL of Type I water.
    2. Mix and dilute to 1 L with Type I water.

9 Preparation of Standards

  1. Primary (1°) Ammonium Stock:
    1. Accurately weigh 0.20 g of ammonium sulphate into a 50 mL volumetric flask.
    2. Dissolve in 0.10N H2SO4.
    3. Mix and dilute to 50 mL with Type I water.
    4. Prepare fresh every 10 working days.
      Note: This corresponds to a 1.0898 mg/mL NH4+ ion stock solution.
  2. The following table summarizes the set of standards concentration ranging from 0 to 6 µg/ml; and the corresponding volume of the primary standard, and the final volume and concentration of the solutions required to achieve each standard.
    Working standards
    Standard # [μg/mL] Volume of 1o (μL) Final Volume Concentration (mL)
    0 0 25 0.000
    1 250 25 10.898
    2 175 25 7.6283
    3 75 25 3.2693
    4 75 50 1.6346
    5 50 100 0.5449
    6 20 100 0.2180

Note: All working standards are made to volume in order to achieve a concentration of 50mN H2SO4. Prepare fresh every five working days.

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 shall be conditioned as specified in T-115.
  2. Cigarettes, cigarette equivalents, bidis, kreteks and cigars shall 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 noting the following:
    2. Specific to Smoking on a Rotary Machine:
      1. Connect two 70 mL impingers, each containing 15 mL of 0.1N H2SO4, between the 92 mm filter and the back-up glass fiber filter cassette on the pneumatics panel using Tygon tubing.
      2. The pneumatic panel for smoking machine, is adjusted for a 35 mL (+/- 0.2 mL) puff volume (with the Filter and impingers in place) and 1.85 seconds sweep-time, using the supplied timer to measure the adjusted sweep-time.
    3. Specific to Smoking on a Linear Machine:
      1. Connect one 70 mL impinger, containing 30 mL of 0.1N H2SO4, between the 44 mm filter holder and the solenoid of the smoking machine.
      2. The smoking machine, is adjusted for a 35 mL (+/- 0.2 mL) puff volume (with the filter and impinger in place).

13 Sample Generation

  1. Cigarettes shall be smoked and TPM collected as specified in T-115.
    1. After smoking is complete, the 92 mm cassette is weighed and recorded, and the TPM yield per cigarette is calculated using the formula found in the Calculations section.

      Note: Smoking on a linear machine requires the appropriate changes previously mentioned in 12.2.3. In addition to these changes, the glass fiber filter pad must be changed after five cigarettes in order to prevent breakthrough. A single clearing puff is taken at the end of smoking each individual cigarette plus an additional two clearing puffs must be taken before removing the cassette holder (at the end of the fifth and 10th cigarette).

14 Sample Analysis

  1. Extraction of Filter Pads
    1. After smoking, remove the 92 mm (or 44 mm - linear) filter and fold into quarters.
    2. Place the pad (or pads - linear) into a 125 mL Erlenmeyer flask.
    3. Add the solution from the impinger(s).
    4. Rinse the impinger(s) with an equal volume of water (i.e. 30 mL impinger volume uses 30 mL of Type I water), and add to the Erlenmeyer flask.
    5. Close the flask, and shake on the wrist action shaker for 30 minutes.
  2. Sample Clean-up
    1. Filter the solution through a syringe filter into a storage vial noting to rinse the vial initially with approximately 1 mL of sample.
    2. Transfer the filtered extract to an autosampler vial.
    3. Samples can be stored at 4 °C for up to 48 hours prior to analysis.
  3. Instrument Analysis - HPLC Analysis
    1. Dionex ED-40 Conditions
      Suppresser Conductivity (SRS): 100 mA
      Scale: 20 uS
      Output: Offset
      Offset: 1 % of Full Scale
    2. Autosampler : Injection Volume
      1. Analyse using a 100 µL sample loop with the parameter for injection volume in the sample list at 35 µL with a 60 µL wash.
    3. Column Temperature: 30 °C.
    4. Mobile Phase / Gradient Conditions (Tertiary Gradient System)
      Solvent A: 0.003N MSA
      Solvent B: Type I water
      Solvent C: 0.2N H2SO4
      Flow: 1.5 mL/minute
      Gradient: Minor adjustments may be required depending on column conditions and resolution of analyte.

      The following table provides information on the mobile phases and gradient run used for the HPLC analysis. There are three mobile phases used: Solvent A 0.003N MSA, Solvent B Type I water, and Solvent C 0.2N H2SO4. Analyte separation is achieved in 25 minutes during varied gradient and equilibration time of 8 minutes.

      Time (minutes) Composition
      % A % B % C
      0.00 100 0 0
      13.00 100 0 0
      13.01 0 80 20
      14.00 0 80 20
      14.01 0 90 10
      19.00 0 90 10
      19.01 0 99 1
      20.00 0 99 1
      25.00 99 1 0
      25.00 Method End Action: Equilibrate

      Equilibration Time: 9.00 minutes.
  4. Calculations
    1. Determination of Response Factor (RF)
      1. An initial calibration is performed by running prepared standards from high concentration to low concentration (injecting the very first standard a minimum of two times until the response and retention time are constant).
      2. A calibration curve is prepared by plotting the concentration of NH4+ ion in the standard vs. the peak height response from the conductivity detector.
      3. The Response factor is the slope of the line as determined by linear regression (Height counts / unit concentration).
    2. Determination of Ammonium Ion
      NH4+ [µg/cigarette] = [peak height × volume extractant (mL) × final volume (mL)] / [RF × # cigarettes smoked × aliquot volume (mL)]
      where the aliquot volume (mL) is the volume quantitatively transferred to the autosampler vial (if necessary).
    3. Determination of Ammonia
      NH3 [µg/cigarette] = NH4+ [µg/cigarette] × (17/18).
      where 17/18 corrects for molecular weight.
    4. Determination of Total Particulate Matter (TPM)
      TPM[mg/cigarette] = ([Filter pad and holder after smoking (g) − Filter pad and holder before smoking (g)] × 1000 mg/g) / (# of cigarettes)

15 Quality Control

  1. Typical Chromatogram
    1. See Appendix 1.
  2. Typical Control Parameters
    1. Laboratory Reagent Blank (LRB): Before processing any samples, the analyst should demonstrate, through the analysis of a reagent water blank, that interferences from the analytical system, glassware, and reagents are not present.
    2. For each analytical batch, a LRB and laboratory fortified blank (LFB) must be analysed. The blank and spiked samples must be carried through all stages of the sample preparation and measurement steps.
    3. Laboratory fortified matrix (LFM): Analyse a LFM to assess matrix interference. Spike a true sample with a known concentration and determine the % Recovery.
  3. Recoveries and Levels of Contamination
    1. Typical recoveries of Laboratory Fortified Blanks (LFB) and Laboratory Fortified Matrix (LFM) samples range from 85 - 110 % when a spiked solution (or sample) is carried out through the entire extraction process.
    2. Typical Laboratory Reagent Blanks (LRB) have a value of 0 µg/cigarette. Contamination of this type is usually associated with contamination of the filter pad during conditioning or an inadequate cleaning of glassware.
  4. Method Detection Limits (MDL) / Limit of Quantitation (LOQ)
    1. The method detection limit (MDL) is determined by analysing the lowest standard level a minimum of 10 times as an unknown over several days. The MDL is calculated as three times the standard deviation of these determinations.
    2. The MDL (on a ng/cigarette basis) can be varied by modifying the number of cigarettes smoked and the volumes used for extraction and clean up in the procedure.
    3. The practical limit of quantitation (LOQ) is determined by analysing the lowest standard level a minimum of 10 times as an unknown over several days. The LOQ is calculated as 10 times the standard deviation of these determinations.
    4. For true samples, the MDL/LOQ is dependent on the resolution and the amount of sodium ion present in the sample, since the tail of a huge sodium peak may mask any ammonium ion present.
  5. Stability of Reagents and Samples
    1. Primary standards should be prepared fresh every 10 working days and stored at 4 °C.
    2. Run standards should be prepared fresh from the stock solution weekly and stored at 4 °C.
    3. Diluted Samples must be run within 48 hours of preparation.

16 Modifications for Intense Smoking

  1. Under intense smoking conditions, five cigarettes are smoked instead of 20.

17 References

  1. Risner, C.H., Conner, J.M. Collection of Ammonia in Indoor Air by Means of a Weak Cation Exchange Cartridge, Environmental Toxicology and Chemistry, Vol. 10, 1991, p. 1417-1423.
  2. Nanni, E.J., Lovette, M.e., Hicks, R.D., Fowler, K.W. and Borgerding, M.F. Separation and Quantitation of Monovalent and Cationic Species in mainstream Cigarette Smoke Aerosols by High-Performance Ion Chromatography, Journal of Chromatographic Science, Vol. 28, August 1990.
  3. IonPac CS12A Analytical Column, Installation Instructions and Troubleshooting Guide, Document No. 031132, Revision 01, Dionex Corporation, 1995.

Appendix

Appendix 1: Typical Chromatogram

This Figure demonstrates a chromatogram of a standard and a reference cigarette. A 5% offset is observed for the Reference cigarette peaks

An overlay of a standard and a Reference cigarette with a 5 % offset.

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