Determination of sorbic acid in whole tobacco: T-313

1.1

This method is used to determine the amount of sorbic acid added to whole tobacco as an anti-microbial agent, by reversed phase high performance liquid chromatography (HPLC) and ultraviolet (UV) detection. The method is designed to be used as a routine analysis without the need for derivatization.

2.1

Health Canada Official Method T-115. Determination of Tar, Water, Nicotine and Carbon Monoxide in Mainstream Tobacco Smoke, 2016.

2.2

Health Canada Official Method T-402. Preparation of Sample for Testing of Cigarettes, Tobacco Sticks, Cigarette Tobacco, Cigars, Little Cigars, Kreteks, Bidis, Leaf, Pipe and Smokeless Tobacco, 2016.

2.3

International Organization for Standardization, ISO 8243 Cigarettes - Sampling. 2013.

2.4

International Organization for Standardization, ISO 15592-1 Fine-Cut tobacco and smoking articles made from it - Methods of sampling, conditioning and analysis - Part 1: Sampling. 2001.

2.5

AOAC INTERNATIOANAL, AOAC Official Method 974.10 Sorbic Acid in Dairy Products; Spectrophotometric Method, Official Methods of Analysis of AOAC INTERNATIONAL, 20^th Ed., 2016.

3.1

Refer to T-301 for definitions of terms used in this document.

4.1

Whole tobacco is extracted with 25 mL of hot water by sonication.

4.2

The extraction solution is cooled, syringe filtered into an amber autosampler vial and subjected to reversed phase HPLC.

4.3

Ultraviolet detection monitors the mobile phase and the sorbic acid found in whole tobacco is quantified by comparison with external standard calibrations.

5.1

Volumetric flask, 2000 mL.

5.2

Amber volumetric flasks, 10 and 100 mL.

5.3

Glass graduated measuring cylinder, 50 mL.

5.4

Screw-top culture tubes, 200 × 25 mm.

5.5

White polypropylene caps without liners.

5.6

Micropipettes, 25 and 200 μL or equivalent.

5.7

Disposable glass Pasteur pipettes.

5.8

Bottletop dispenser, 10-50 mL or equivalent.

5.9

Syringe filter.

5.10

Disposable syringes.

5.11

Autosampler vials, caps and Teflon-lined septa.

5.12

Rubber bulbs.

5.13

Parafilm^®.

5.14

Batch processor, Robot Coupe RS1-2V or equivalent.

5.15

Vortex mixer.

5.16

Analytical balance measuring to at least 4 decimal places.

5.17

pH meter.

5.18

Sonicator.

5.19

High Pressure Liquid Chromatography System with UV Detector.

5.19.1

RP 18e column or equivalent.

5.19.2

Disposable guard column.

6.1

All reagents shall be at least analytical reagent grade.

6.2

Methanol - [67-56-1] Distilled-In-Glass.

6.3

Phosphoric Acid - [7664-38-2] 85 % purity.

6.4

Potassium Dihydrogen Orthophosphate - [7778-77-0] > 99 % purity.

6.5

Potassium Sorbate - [590-00-1] 99 % purity.

6.6

Water, Type I (as outlined in ASTM D1193, Table 1: Processes for Reagent Water Production, Note A).

7.1

Clean and dry glassware in a manner to ensure that contamination from residues on glassware does not occur.

8.1

Primary Stock Sorbate Standards

8.1.1

Weigh 134 mg of potassium sorbate into a 100 mL volumetric flask.

8.1.2

Convert the potassium sorbate to sorbic acid by multiplying the weight of potassium sorbate by the molecular weight of sorbic acid (112.14) and dividing by the molecular weight of potassium sorbate (150.22).

8.1.3

Dilute to volume with Type I water.

8.2

Sorbic Acid Calibration Standards

8.2.1

Take appropriate volumes (0.01-1.0 mL) of the potassium sorbate primary stock solution and dilute to 10 mL with Type I water to give calibration standards with approximate sorbic acid concentrations in the ranges 0.2-100 ppm.

Sorbic Acid Calibration Standards

8.2.2

Transfer to amber autosampler vials. Rinse the vials first, then fill to minimize headspace.

8.2.3

Store at 4 °C, protected from light, until analyzed.

8.2.4

Prepare new sorbic acid calibration standards every 5 days.

9.1

The sampling of cigarettes for the purpose of testing shall be in accord with ISO 8243.

9.2

The sampling of kreteks, little cigars, bidis, tobacco sticks for the purpose of testing shall be in accord with ISO 8243, but modified such that the term "cigarette" is substituted with "kreteks", "little cigars", "bidis" or "tobacco sticks", whereby the term "carton" is equivalent to 200 units.

9.3

The sampling of cigars for the purpose of testing shall be in accord ISO 8243, but modified such that the term "cigarette" is substituted with "cigar", whereby 200 units of cigarette is equivalent to 200 grams of cigar.

9.4

The sampling of cigarette tobacco for the purpose of testing shall be in accord with ISO 15592-1.

9.5

The sampling of leaf tobacco, pipe tobacco or smokeless tobacco shall be in accord with ISO 15592-1 but modified such that the term "fine-cut" is substituted with "leaf tobacco", "pipe tobacco" or "smokeless tobacco".

10.1

The preparation of tobacco products for the purpose of testing shall be as specified in T-402.

11.1

Weigh 0.5 g of finely chopped tobacco into a glass culture tube.

11.2

Add 25 mL of hot (50 °C) Type I water to the sample and sonicate for 30 minutes at 50 °C.

11.3

Allow the sample to cool.

11.4

Mix well, then filter an aliquot into an amber autosampler vial using a 45 μm disposable syringe filter attached to a disposable syringe. Cap and store at 4 °C, protected from light, until analyzed.

12.1

Chromatographic Conditions (Reversed Phase HPLC Analysis)

• Column Temperature: 30 °C
• 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)
• Sample Wash: Solvent A
• Mode: Isocratic
• Flow Rate: 1.0 mL/minute
• UV Wavelength: 259 nm

Equilibration Time: 5 minutes

12.2

Load the sample vials onto the autosampler.

12.3

Inject 20 μL of each sample vial onto the HPLC column. The elution pattern should be similar to appendix 1, figure 1.

13.1

Constructing a Calibration Curve

13.1.1

Inject 20 μL of each calibration standard onto the HPLC. The elution pattern should be similar to appendix 1, figure 2.

13.1.2

Prepare a calibration curve by plotting the concentrations of the standards versus their respective peak areas.

13.1.3

Determine the response factor from the calibration curve.

13.2

Sample Quantification

13.2.1

The amount of sorbic acid in the samples is quantified by the external standard method.

13.2.2

The identification of peaks is by comparison of retention times with standards, and the spiking of samples.

13.3

Determination of Sorbic Acid Deliveries in μ g/g

13.3.1

Calculate the concentration of sorbic acid automatically by entering the correct multiplier (overall volume the original sample is diluted to in mL) and divisor (the original sample weight in g) to find the μg/g.

13.3.2

To convert this concentration to a percent, divide the μg/g result by 10 000.

13.3.3

All results are expressed on an 'as received' basis. These may be expressed on a 'dry matter' basis using the appropriate moisture result.

14.1

For typical chromatograms, see appendix 1, figure 1 and figure 2.

14.2

Typical Control Parameters

14.2.1

Laboratory Reagent Blank (LRB)

To detect potential contamination during the sample preparation and analysis processes, include a laboratory reagent blank (LRB). The LRB consists of all reagents and materials used in performing the analysis on test samples and is analyzed as a test sample.

14.2.2

Laboratory Fortified Blank (LFB)

To detect potential loss of analyte during the sample preparation and analysis processes, include a laboratory fortified blank (LFB). The LFB consists of all reagents and materials used in performing the analysis on test samples plus fortification with a known concentration of at least one of the analytes of interest. The level of fortification should reflect the range of typical results for that sample. The LFB is then analyzed as a test sample.

14.2.3

Laboratory Fortified Matrix (LFM)

To detect potential matrix interferences, include a laboratory fortified matrix (LFM). During the sample preparation and/or analysis processes, divide a test sample and fortify an aliquot with at least one of the analytes of interest in known concentration. The level of fortification should reflect the range of typical results for that sample. The LFM is then analyzed as a test sample.

14.2.4

Laboratory Control Sample

To assess the overall performance of an analysis, a control sample is analyzed. The results of the control sample should be compared, using appropriate statistical techniques, to 'expected values' generated by the laboratory or, if none exist, to values found in literature. This provides information to the laboratory, on test accuracy and precision.

14.2.5

Standard as Sample

To assess the stability of the analytical system, a standard is analyzed as a sample. The results of this standard should be compared, using appropriate statistical techniques, to expected concentrations.

14.3

Recoveries and Levels of Contamination

14.3.1

Typical LRB values average 0.1 ± 0.5 μg/g sorbic acid.

14.3.2

Typical LFB and LFM recoveries fall in the range 85–105 % recovery.

14.4

Limit of Detection (LOD) and Limit of Quantification (LOQ)

14.4.1

The LOD can be determined as 3 times the standard deviation of results obtained by analyzing the lowest standard level a minimum of 10 times over several days.

14.4.1.1

A typical value for LOD is 75 μg/g as analyzed.

14.4.2

The LOQ can be determined as 10 times the standard deviation of results obtained by analyzing the lowest standard level a minimum of 10 times over several days.

14.4.2.1

A typical value for LOQ is 250 μg/g as analyzed.

14.5

Stability of Reagents and Supplies

14.5.1

Prepare all primary stock standards fresh weekly from potassium sorbate.

14.5.2

Prepare all work standards and extraction solvents fresh weekly.

14.5.3

Analyze all samples within 48 hours, keep stored in the dark and at refrigerator temperature to reduce the potential loss of sorbic acid through microbial growth.

15.1

Rosa, N. and H. Caughill, C. W. 1984. Effect of aqueous ethanol, sample particle size, and temperature on extractability of soluble acids from tobacco. Tobacco Science. 1: 24-25.

15.2

ASTM International, ASTM Standard D1193-06(2011). Standard Specifications for Reagent Water.