Determination of sodium propionate in whole tobacco: T-312

1.1

This method is to be used to determine the amount of sodium propionate added to whole tobacco as a mold preventative or fungicide by Gas Chromatography with Flame Ionization Detection. The method is designed for use in routine analysis without the need for derivatization.

1.2

This method does not distinguish between the amount of sodium propionate added and the amount of naturally occurring propionic acid (if any) found in tobacco and tobacco products. This method does not distinguish between sodium propionate and propionic acid that may result from some forms of anaerobic fermentation.

Note: This method is not intended for trace analysis, but for the determination of sodium propionate that is used in an additive range up to 0.4%.

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 INTERNATIONAL, AOAC Official Method 971.11 Fatty Acids (Volatile) in Eggs, Gas Chromatographic 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

Tobacco is extracted in a solution of H₂SO₄ in methanol under extremely gentle conditions.

4.2

Calcium chloride (CaCl₂) is used to saturate the ionic species of the solution. The propionate ion's equilibrium with the free acid is quantitatively forced toward the free acid as a consequence of its small K_a value(1.34 X 10^-5 M).

4.3

Iso-butyric acid (K_a = 1.44 X 10^-5 M) is used as an internal standard. (It also has the effect of minimizing the potential pH differences between samples and standards that may affect the ion:free acid ratio).

4.4

The free acid is then analyzed by gas chromatography (GC) using a splitless injection of the methanolic solution onto a Stabilwax-DA fused silica capillary column (or equivalent) with a flame ionization detector (FID).

4.5

Quantification is achieved by internal standard calibration procedures where the relative response of the samples is compared with a calibration of the corresponding acid of the same type of solution.

5.1

Erlenmeyer flasks, 125 mL, Polymethylpentene (PMP) with screw tops or equivalent.

5.2

Volumetric flasks, 100 and 2000 mL.

5.3

Graduated cylinder, 100 mL.

5.4

Screw-top culture tubes with caps, 16 × 25 mm (without liners).

5.5

Finnpipette, 0-5 mL or equivalent.

5.6

Micropipettes, 25 and 200 µL or equivalent.

5.7

Bottletop dispenser, 10-50 mL or equivalent.

5.8

Gas chromatograph with splitless capillary injector and flame ionization detector (FID) or equivalent.

5.9

Autosampler vials with screw caps and Teflon-lined septa.

5.10

Stabilwax-DA, 30 m × 0.32 mm × 1.0 µm; Crossbond Carbowax - PEG for acidic compounds; or Nukol, 30 m × 0.32 mm × 0.25 µm or equivalent.

5.11

Centrifuge.

5.12

Vortex.

5.13

Wrist-action shaker.

6.1

All reagents shall be at least analytical reagent grade.

6.2

Calcium Chloride - [7774-34-7].

6.3

Iso-butyric Acid - [79-31-2].

6.4

Methanol - [67-56-1].

6.5

Sodium Propionate - [137-40-6].

6.6

Sulphuric Acid - [7664-93-9] 95–97%.

6.7

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

6.8

Parafilm^® or equivalent.

7.1

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

8.1

Tobacco Extraction Solution (Aqueous Component)

8.1.1

Add 35 g of CaCl₂ to a 2000 mL volumetric flask.

8.1.2

Add approximately 1 L of Type I water and mix until all of the CaCl₂ is dissolved.

8.1.3

Rinse the neck of the flask to wash down high concentration points of CaCl₂. If a white precipitate forms, re-make the solution taking greater care in washing down the neck of the flask with Type I water.

8.1.4

Dilute 0.54 mL of concentrated H₂SO₄ with approximately 100 mL of Type I water in a glass beaker. Add diluted H₂SO₄ solution to a 2000 mL volumetric flask containing CaCl₂ solution.

8.1.5

Make to volume with Type I water.

8.2

0.01N H₂SO₄

8.2.1

Add 0.27 mL of concentrated H₂SO₄ to approximately 700 mL Type I water in a 1 L volumetric flask.

8.2.2

Make to volume with Type I water.

9.1

Sodium Propionate Primary Standard (Final concentration approximately 20 mg/mL)

9.1.1

Accurately weigh 2 g of sodium propionate into a 100 mL volumetric flask.

9.1.2

Make to volume with 0.01N H₂SO₄.

9.2

Iso-Butyric Acid Standard - ISTD (Final concentration approximately 100 mg/mL)

9.2.1

Accurately weigh 2.5 g of iso-butyric acid into a 25 mL volumetric flask.

9.2.2

Make to volume with aqueous 0.01N H₂SO₄.

9.3

Working standards

9.3.1

Make all standards to 25 mL in volumetric flasks with methanol.

9.3.2

Attempt to maintain a 10% aqueous fraction for each of the standards by adding the appropriate amount of aqueous extraction component (to the nearest 0.05 mL). This is required to maintain a constant aqueous:organic ratio between standards and samples.

10.1

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

10.2

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.

10.3

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.

10.4

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

10.5

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".

11.1

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

12.1

Extraction of Whole Tobacco

12.1.1

Accurately weigh 5 g of test sample into a PMP Erlenmeyer flask.

12.1.2

Add 10 mL of the aqueous extraction component to the sample.

12.1.3

Add 100 µL of iso-butyric acid stock as an internal standard.

12.1.4

Moisten the tobacco by rotating the flask until all the solution is absorbed by the tobacco or the tobacco appears to be saturated.

12.1.5

Add 40 mL of methanol to each of the samples.

12.1.6

Close the flask (seal with Parafilm^® if necessary) and shake the flask for 30 minutes.

12.1.7

Let the samples sit for one hour minimum after shaking.

12.1.8

Shake each sample for an additional 30 seconds after letting the samples sit.

12.1.9

Decant the liquid into a screw-top culture tube and cap so that none of the solution is allowed to evaporate.

12.2

Sample Clean-up

12.2.1

Allow the precipitate to settle in the culture tube by letting it sit for 2 hours. (Alternatively, centrifuge at low speed to settle the precipitate).

12.2.2

Quantitatively transfer 2 mL of the supernatant to a second 16 × 25 mm screw top culture tube.

12.2.3

Quantitatively add 2 mL of methanol to each of the aliquots and cap each tube.

12.2.4

Vortex each of the samples for 10 seconds at high speed.

12.2.5

Centrifuge the diluted sample at medium speed to precipitate any salts that may have formed in the dilution step.

12.2.6

Transfer the supernatant to an autosampler vial to analyze on the GC.

13.1

Gas Chromatograph Configuration

Injector:

Splitless with purge delay of 0.10 minute

Deactivated splitless insert

Detector:

Flame Ionization (FID)

Carrier:

He at 12.0 psi, flow = 2.3 mL/minute at 70 °C

Split (purge flow):

20 mL/minute

Column:

Stabilwax – DA

13.2

Gas Chromatograph Operating Conditions

Injector:

225 °C

Detector:

230 °C

Column Oven Temperature Profile:

Start Temp.:

70 °C, hold for 2 minutes

Rate:

7.5 °C/minute to 155 °C, hold for 2.67 minutes

Rate:

7.5 °C/minute to 205 °C, hold for 4.0 minutes

Tot. Run Time:

25.00 minutes

13.3

Autosampler Conditions

Injection volume: 1.0 µL

14.1

Perform all calculations using an internal standard calibration response factor from the area counts of the standard solutions.

14.2

Use these calibration response factors to calculate the µg/mL concentration of each analyte in the sample, which in turn, with the appropriate multiplier (total volume in mL) and divisor (original sample weight in grams), are used to calculate the concentration of sodium propionate in the sample.

14.3

To convert concentration to a percent (%), divide the [µg/g] result by 10 000.

14.4

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

14.5

Representative Calculations

14.5.1

Analytical Result

where RF is defined from the calibration curve (or as the concentration per unit area).

14.5.2

Conversion to Percent (as is)

14.5.3

Conversion to Dry Matter Basis

where the % moisture is determined from the same sample as received for sodium propionate analysis.

15.1

For a typical chromatogram, see appendix 1.

15.2

Typical Control Parameters

15.2.1

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.

15.2.2

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.

15.2.3

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.

15.2.4

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.

15.2.5

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.

15.3

Recoveries and Levels of Contamination

15.3.1

A typical LRB value is 3.2 ± 2.7 μg/g sodium propionate.

15.3.2

A typical LFB recovery falls in the range 90-110 % recovery.

15.3.3

A typical LFM recovery falls in the range 95-120 % recovery.

15.4

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

15.4.1

The LOD can be determined as 3/10 of the LOQ.

15.4.1.1

A typical value for LOD determined in this manner is 96 µg/g.

15.4.2

The LOQ can be determined from the lowest standard used in preparation of the calibration curve (excluding the blank).

15.4.2.1

A typical value for LOQ determined in this manner is 320 µg/g.

15.5

Stability of Reagents and Supplies

15.5.1

Prepare all primary stock sodium propionate standards fresh weekly.

15.5.2

Prepare all work standards and extraction solvents fresh weekly.

15.5.3

Analyze all samples within one week of extraction.

16.1

CRC Press Inc. CRC Handbook of Chemistry and Physics. 58^th Edition. D-150–151. 1977-1978.

16.2

Fessenden, R. J. and Fessenden, J. S. 1979. Organic Chemistry, 2^nd Edition. Willard Grant Press. 588, 627, 628.

16.3

Merck & Co. Inc. The Merck Index: An Encyclopedia of Chemicals and Drugs, 8^th Edition. 1968.

16.4

RJR Macdonald R & D. Determination of Sorbic Acid in Tobacco. 56-60.

16.5

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