Determination of Sorbic Acid in Whole Tobacco

Health Canada
T-313 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. Tobacco Product Preparation
  10. Smoking Machine Preparation
  11. Sample Generation
  12. Sample Analysis
  13. Quality Control
  14. References
  15. Appendices

1 Scope of Applications

  1. This method is used to determine the amount of sorbic acid added to tobacco as an anti-microbial agent, by reversed phase high performance liquid chromatography (HPLC) and ultra-violet (UV) detection. The method is designed to be used as a routine analysis without the need for derivitization. This is applicable to processed cigarettes, finecut tobacco, chewing tobacco, snuff, cigar samples and pipe tobacco.
  2. This method does not distinguish between the amount of sorbic acid added and the amount of naturally occurring sorbic acid (if any) found in whole tobacco.

2 Normative References

  1. American Society for Testing and Materials (ASTM) D1193-77 - Standard Specification for Reagent Water, Version 1977.
  2. Health Canada Test Method T-115 - Determination of Tar, Water, Nicotine and Carbon Monoxide in Mainstream Tobacco Smoke, 1999-12-31.

3 Method Summary

  1. Whole tobacco is extracted with 50 mL of hot water by sonication.
  2. The tobacco slurry is centrifuged and decanted into a volumetric flask.
  3. The supernatant liquid is cooled and made up to volume, syringe filtered into a 1.5 mL amber autosampler vial and subjected to reversed phase HPLC.
  4. UV Detection monitors the mobile phase and the sorbic acid found in whole tobacco is quantified by comparison to external standard calibrations.

    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. 2000 mL volumetric flask.
  2. 100 mL amber volumetric flasks.
  3. 10 mL amber volumetric flasks.
  4. 50 mL glass graduated measuring cylinder.
  5. 200 × 25 mm screw-top culture tubes.
  6. White polypropylene caps without liners.
  7. Micropipettes for the preparation of analytical standards.
  8. Bottle-top dispensor 10 - 50 mL.
  9. 200 µL micropipette, 25 µL micropipette.
  10. Syringe Filter.
  11. Disposable syringes.
  12. Autosampler Vials.
  13. Screw Cap.
  14. Septa, 8 mm Teflon-faced, 60 ml.
  15. Protective eye glasses.
  16. Rubber Bulbs.
  17. Parafilm.
  18. Centrifuge.
  19. Batch Processor (Robot Coupe RS1-2V or equivalent).
  20. Vortex Mixer.
  21. Analytical balance, capable of reading to four decimal places.
  22. pH Meter.
  23. Sonicator.
  24. PC controlled High Pressure Liquid Chromatography System consisting of:
    1. Solvent Delivery System - ternary gradient pump.
    2. Autosampler with 20 µL sampling loop.
    3. Varian UV Detector or equivalent.
    4. Work Station.
    5. RP 18 e Column.
    6. Disposable Guard Column.

5 Reagents and Supplies

  1. Potassium Sorbate > 99 % purity .
  2. Potassium Dihydrogen Orthophosphate > 99 % purity.
  3. Phosphoric Acid 85 % purity.
  4. Type I water as specified in ASTM D1193.
  5. Methanol, Distilled-in-glass (DIG).
  6. Disposable Glass Pasteur Pipettes.
  7. Disposable gloves.

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. Not applicable to this method.

8 Preparation of Standards

  1. Primary Stock (1°) Sorbate Standards
    1. Weigh 100 mg of the potassium sorbate into a 100 mL volumetric flask.
    2. Dilute to volume with Type I water.
  2. Sorbate Calibration Standards
    1. Take appropriate volumes (0.050 to 1.0 mL) of the sorbate primary stock solution and dilute to 10 mL with Type I water to give calibration standards with approximate sorbate concentrations in the ranges 5-100 ppm.

      The following table displays the details for preparation of sorbic acid calibration standards, including blank 0 and standards 1-5. This includes the initial volume of the primary sorbic acid, final volume of the sample, and the final concentration of the sorbate.

      Sorbic Acid Calibration Standards
      Standard
      ID      		 Vol (mL) 1o
       Sorbic Acid       		Final Volume in 
      mL      		 Sorbate
      [µg/mL] (ppm)
      Blk 0 0 10 0.0
      Std 1 0.050 10 5.0
      Std 2 0.100 10 10.0
      Std 3 0.250 10 25.0
      Std 4 0.5 10 50.0
      Std 5 1.0 10 100.0

      Note 1: The concentration of Sorbate will vary depending on the exact concentration of primary stock prepared.
    2. Transfer to 1.5 mL amber autosampler vials. Rinse vial first and then fill to minimize headspace.
    3. Place vials in a vial file and store at 4 °C protected from light until analyzed.
    4. Prepare new sorbate calibration standards every five working days.

9 Tobacco Product Preparation

  1. Preparation of Test Sample
    1. Tobacco product is to be conditioned as specified in T-115.
    2. The whole tobacco required for analysis must be removed from its original package and inspected for extraneous material.

10 Smoking Machine Preparation

  1. Not applicable to this method.

11 Sample Generation

  1. Finely chop (to a 20 mesh particle size) several grams of tobacco using a batch processor.
  2. Accurately weigh 1g of finely chopped tobacco into a 200 × 25 mm screw cap glass culture tube.
  3. Add 30 mL of hot (50 °C) Type I water to the sample and sonicate for 30 minutes at 50 °C.
  4. Centrifuge at about 1200 rpm for 10 minutes and decant the water into a 100 mL volumetric flask.
  5. Repeat step 11.3 to 11.5 with a second gram of tobacco and decant into the same volumetric flask, pooling the extract from 2 g of tobacco.
  6. Allow sample to cool, then make up to the mark with Type I water.
  7. Mix well, then filter an aliquot into an amber autosampler vial using a disposable syringe filter attached to a disposable syringe. Cap and store at 4 °C.

12 Sample Analysis

  1. Reversed phase HPLC Analysis
    1. Chromatographic Conditions (Reversed Phase Analysis)
      1. Column Temperature: 30 °C.
      2. Mobile Phase: Reagents.
        Solvent A: Methanol, filter and degas. (UHP Helium sparged).
        Solvent B: Prepare 2 L of 1 % IPA adjusted to pH 2.3 with phosphoric acid; filter and degas. (UHP Helium sparged).
      3. Sample Wash: Solvent A.
      4. Mobile Phase: Gradient - Isocratic.

        Flowrate
        Time (minutes)
        1.0 mL/minute
        Composition
        % A % B % C
        0.0 35 65 0
        20.0 35 65 0
        Method End Action: 35 65 0
        (Equilibrate 5 minutes).  
    2. Sample vials are loaded onto the autosampler such that every 10th vial is a standard solution and in such quantities that the total time (Sections 10-11) does not exceed 24 hours at room temperature.
    3. Twenty µL of each sample vial is injected onto the HPLC column and analyzed. Elution pattern should be similar to Figure 1.
  2. Calculations
    1. Construct a Calibration Curve:
      1. Twenty µL of each calibration standard is injected onto the HPLC and analyzed. Do in duplicate. Elution pattern should be similar to Figure 2.
      2. A calibration curve is prepared by plotting the concentrations of the standards versus their respective peak areas.
      3. Determine the response factor from the calibration curve.
    2. Sample Quantification
      1. The amount of sorbic acid in the samples is quantified by the external standard method.
      2. The identification of peaks is by comparison of retention times with standards, and the spiking of samples.
    3. Determination of Sorbic acid Deliveries in µg/cigarette

      Sorbic acid [µg/g] = [Peak Area-Int / Resp. Factor] × [mL of Solution / Wt of Tobacco]

    4. By entering the correct multiplier (overall volume the original sample is diluted to in mL) and divisor (the original sample weight in g) the concentration of Sorbic acid is automatically calculated in µg/g.
    5. To convert this concentration to a percentage (%), the µg/g result must be divided by 10000.
    6. All results are expressed on an "as conditioned" basis. These may be expressed on a dry matter basis using the appropriate moisture result.

13 Quality Control

  1. Typical Chromatogram
    1. See Figure 1.
  2. Recoveries and Levels of Contamination
    1. Each analytical run of whole tobacco should also include:
      1. A Laboratory Reagent Blank (LRB) to evaluate the extent of any interference due to glassware and analyzer effects.
      2. A Laboratory Fortified Blank (LFB) to evaluate the extent of potential analyte loss.
      3. A Laboratory Fortified Matrix (LFM) to assess matrix interference. This is accomplished by spiking a true sample with a known concentration and determining a percent recovery.
  3. Method Detection Limit (MDL) and Limit of Quantitation
    1. Method Detection Limit (MDL)
      The method detection limit is determined by analyzing the lowest level standard at least 10 times as an unknown over several days. The MDL is then calculated as three times the standard deviation of these determinations.
    2. Limit of Quantitation (LOQ)
      The limit of quantification is determined by analyzing the lowest level standard at least 10 times as an unknown over several days. The LOQ is then calculated as 10 times the standard deviation of these determinations.
  4. Stability of Reagents and Supplies
    1. All primary stock Sorbic acid standards are prepared fresh weekly.
    2. All work standards, and extraction solvents are prepared fresh weekly.
    3. All samples are to be analyzed within 24 hours.

14 References

  1. AOAC Official Method 974.10, Fatty Acids (Volatile) in Eggs, Gas Chromatographic Method, AOAC Official Methods of Analysis, 1995, Ch. 34, p. 10-12.
  2. Rosa, Nestor; and C.W. H. Caughill; Effect of Aqueous Ethanol, Sample Particle Size, and Temperature on Extractability of Soluble Acids from Tobacco, Tobacco Science 1, Jan 1984.

15 Appendices

Figure 1: Typical HPLC Chromatogram of Tobacco Sample Analyzed for Sorbate

The following figure displays a typical chromatogram result of tobacco sample analyzed by high performance liquid -chromatography for sorbate. The intensity of absorbance in mili-absorbance units were plotted against time (in minutes).

Figure 1: Typical HPLC Chromatogram of Tobacco Sample Analyzed for Sorbate

Figure 2: Typical HPLC Chromatogram of Sorbate Calibration Standard

The following figure displays a typical chromatogram result of sorbate calibration standard analyzed by high performance liquid chromatogram. The intensity of the absorbance in mili-absorbance units were plotted against time (in minutes).

Figure 2: Typical HPLC Chromatogram of Sorbate Calibration Standard

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