Determination of benzo[a]pyrene in whole tobacco: T-307

1.1

Applicable to the quantification of Benzo[a]pyrene (B[a]P) content in whole tobacco by high performance liquid chromatography (HPLC) via fluorescence detection.

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 973.30 Polycyclic Aromatic Hydrocarbons and Benzo[a]pyrene in Food, 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

This method is a modification of AOAC Official Method 973.30, Polycyclic Aromatic Hydrocarbons and Benzo[a]pyrene in Food - Spectrophotometric Method. Sample size and subsequent reagent volumes are adjusted to meet the analytical requirements of the sample as well as the availability of the sample and equipment.

4.2

Polycyclic aromatic hydrocarbons are extracted from a sample of tobacco after saponification with an alcoholic KOH solution and partitioning into iso-octane. The solvent is then completely evaporated and the sample is reconstituted in iso-octane. This reconstituted solution is passed through a florisil cartridge and washed with iso-octane. The B[a]P is eluted using benzene, the solvent is evaporated to dryness, and the sample is reconstituted in acetonitrile. The sample is subjected to reversed phase liquid chromatography (HPLC) and quantified via fluorescence detection.

4.3

Some matrices may be too complex to achieve accurate quantification using this solid phase extraction clean-up procedure. These types of samples may require an HPLC column clean-up using normal phase Chromatography on a silica column where the B[a]P fraction may be collected. This fraction may then be concentrated and subjected to reversed phase HPLC.

5.1

Solid phase extraction (SPE) preparation unit or equivalent.

5.2

Condenser, Friedrich-type.

5.3

Argon cylinder with manifold for purging flasks during saponification or equivalent.

5.4

Glass pipettes, 2-50 mL.

5.5

Micro-pipette, 1000 µL or equivalent.

5.6

Glass funnel.

5.7

Glass wool.

5.8

Culture tubes, 16 × 125 mm (20 mL).

5.9

Round bottom flasks, 250 mL or equivalent.

5.10

Separatory funnels, 1000 mL or equivalent.

5.11

Zymark TurboVap Concentrator or equivalent.

5.12

Buchi Rotovap or equivalent.

5.13

Analytical balance measuring to at least 4 decimal places.

5.14

Pasteur pipettes.

5.15

Syringe filters, 0.45 μm, nylon or equivalent.

5.16

Florisil cartridges, 1 g × 6 mL capacity or equivalent.

5.17

Autosampler vials with Teflon-lined caps.

5.18

High Performance Liquid Chromatograph gradient system with fluorescence detector:

5.18.1

Merck, 250 × 4 mm, RP-18e, 5 μm packing, HPLC column or equivalent.

5.18.2

Lichrocart 4-4 Lichrosphere 100 RP-18 endcapped, 5 μm guard column or equivalent.

6.1

All reagents shall be at least analytical reagent grade.

6.2

Acetonitrile - [75-05-8].

6.3

Argon - [7440-37-1] UHP or equivalent.

6.4

Benzene - [71-43-2].

6.5

Benzo[a]pyrene (B[a]P) - [50-32-8].

6.6

Ethanol - [64-17-5].

6.7

Iso-octane - [540-84-1].

6.8

Isopropanol (IPA) - [67-63-0].

6.9

Potassium Hyroxide - [1310-58-3] 50 % (w/v).

6.10

Sodium Chloride - [7647-14-5] saturated solution.

6.11

Sodium Sulphate, anhydrous - [7757-82-6].

6.12

Tetrahydrofuran (THF) - [109-99-9].

6.13

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

Preparation of Working Standards and Spike Solutions

8.1.1

Primary B[a]P Stock: Dissolve 10 mg B[a]P to 50 mL acetonitrile.

8.1.2

Secondary Stock: Pipette 100 µL of Primary Stock to 50 mL acetonitrile.

8.1.3

Working Standards

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

Extraction - Base Saponification

11.1.1

Accurately weigh 2 g of prepared tobacco sample into a 250 mL round bottom flask.

11.1.2

Add 60 mL of ethanol.

11.1.3

Add 4.5 mL of KOH solution.

11.1.4

Purge the round bottom flask with argon.

11.1.5

Set up the refluxing apparatus so that the apparatus is continually purged with argon at a slow rate.

11.1.6

Reflux for 2 hours at a rapid rate.

11.1.7

Allow to cool to room temperature while continuing to purge with argon.

11.1.8

Stopper the flask and store in the dark until ready for partitioning.

11.2

Extraction - Partitioning

11.2.1

Transfer the content of the round bottom flask into a 1000 mL separatory funnel (# I) through a glass funnel containing a plug of glass wool to filter the solids.

11.2.2

Wash the flask with 2 × 20 mL portions of Type I water and transfer into the separatory funnel (# I).

11.2.3

Wash the flask with 2 × 15 mL portions of reagent alcohol and transfer into the separatory funnel (# I).

11.2.4

Wash the flask with a 25 mL portion of iso-octane and transfer into the separatory funnel (# I).

11.2.5

Shake the separatory funnel for 3 minutes or until there is no observed pressure build-up and allow the layers to separate.

11.2.6

Carefully drain the lower layer into a second separatory funnel (# II).

11.2.7

Repeat the above extraction by adding 20 mL of iso-octane to the round bottom flask and transfer into the separatory funnel (# II).

11.2.8

Shake the separatory funnel for 3 minutes or until there is no observed pressure build-up and allow the layers to separate (# II).

11.2.9

Carefully drain the lower layer into a third separatory funnel (# III).

11.2.10

Repeat the above extraction by adding 20 mL of iso-octane to the round bottom flask and transfer into the separatory funnel (# III).

11.2.11

Shake the separatory funnel (# III) for 3 minutes or until there is no observed pressure build-up and allow the layers to separate.

11.2.12

Carefully drain the lower layer to waste and discard.

11.2.13

Wash each iso-octane layer (separatory funnels I, II and III) 3 times with a solution containing 50 mL of warm Type I water and 500 μL saturated NaCl.

11.2.14

Discard the aqueous layer (lower layer) after each washing.

11.2.15

After washing is complete, drain each of the iso-octane layers through a funnel containing anhydrous sodium sulphate into a 250 mL round bottom flask (drain in the order I, II, then III) combining the iso-octane fractions in the same flask.

11.2.16

Rinse the third separatory funnel (# III) with 25 mL of fresh iso-octane and shake.

11.2.17

Transfer the iso-octane into the second separatory funnel (# II) and shake.

11.2.18

Transfer the iso-octane into the first separatory funnel (# I), and shake.

11.2.19

Transfer the iso-octane of the first separatory funnel (# I), com-bining with the previous iso-octane fractions in the same flask.

11.2.20

Repeat this rinsing process (11.2.16 to 11.2.19) with 25 mL of fresh iso-octane starting in the third separatory funnel (# III) once again.

11.2.21

Wash the anhydrous sodium sulphate with a 2 × 5 mL portion of iso-octane.

11.2.22

Evaporate the iso-octane solution in the 250 mL round bottom flask on the rotary evaporator to dryness at approximately 55 °C.

11.2.23

Reconstitute the sample with 2 mL of iso-octane.

11.3

Sample Clean-up

11.3.1

Condition the florisil cartridge by adding approximately 1 g of anhydrous sodium sulphate to the cartridge and then by washing the column with 2 × 5 mL additions of iso-octane (allow to drip via gravity at the rate of approximately one drop per second).

11.3.2

Add the entire reconstituted sample (in 2 mL iso-octane) to the cartridge allowing the eluant to gravimetrically pass through the cartridge to waste.

11.3.3

Wash the flask with 3 × 5 mL additions of iso-octane transferring each fraction to the cartridge, allowing the eluant to gravimetrically pass through the cartridge to waste.

11.3.4

Place 20 mL disposable glass culture tubes beneath each of the cartridges.

11.3.5

Gravimetrically elute the B[a]P from the cartridges with 3 × 5 mL additions of benzene.

11.3.6

Add 1 mL of THF to each tube.

11.3.7

Place the tubes containing the 16 mL of collected eluant into the Zymark TurboVap.

11.3.8

Evaporate the samples to complete dryness.

11.3.9

Pipette 1000 μL of acetonitrile into each of the dried tubes to dissolve the analyte and any residue that may be present.

11.3.10

Vortex the sample at high speed for approximately 15 seconds.

11.3.11

Using a glass transfer pipette, wash down the sides of the tube five times with the sample and transfer to a 2 mL volumetric flask.

11.3.12

Wash the tube with an additional 500 μL acetonitrile as per 11.3.11 and transfer to the 2 mL volumetric flask.

11.3.13

Make to volume with acetonitrile.

11.3.14

Transfer the sample to a 2 mL autosampler vial with a screw cap and Teflon-faced septum.

11.3.15

The samples are ready for HPLC analysis and may be stored at 4 °C until they are analyzed.

12.1

Reversed Phase High Performance Liquid Chromatography Analysis

12.1.1

Jasco Fluorescence Detector Conditions

Excitation Wavelength:

365 nm

Emission Wavelength:

425 nm

Gain:

× 1000

Attenuation:

32

12.1.2

Autosampler : Injection Volume

12.1.2.1

Analyze using a 50 μL injection.

12.1.3

Mobile Phase / Gradient Conditions (Tertiary Gradient System)

Solvent A:

55:45 Acetonitrile/1 % IPA in Type I water (degassed and filtered using 0.45 μm nylon filter)

Solvent B:

Methanol

Solvent C:

Acetonitrile

Flow:

1.5 mL/minute

Gradient:

Example

Equilibration Time: 8.00 minutes

13.1

Determination of Response Factor

13.1.1

Perform an initial calibration by running prepared standards until the response and retention times are constant.

13.1.2

Prepare a calibration curve by plotting the concentration of B[a]P in the standard versus the peak height response from the fluorescence detector.

13.1.3

The Response Factor (RF) is the slope of the line as determined by linear regression (height counts/unit concentration).

13.2

Determination of B[a]P Content [ng/g]

14.1

Typical Control Parameters

14.1.1

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.

14.1.2

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.

14.1.3

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.

14.1.4

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.

14.1.5

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.

14.2

Recoveries and Levels of Contamination

14.2.1

Typical recoveries of Laboratory Fortified Blanks (LFB) and Laboratory Fortified Matrix (LFM) samples range from 75-95 % when a spiked solution (or sample) is carried out through the entire extraction process.

14.2.2

Recoveries lower than 65 % indicate either an insufficient elution of B[a]P from the solid phase extraction cartridges or a change in response factor (RF) of the fluorescence detector. A change in RF must first be investigated before re-processing of samples is initiated.

14.2.3

Typical LRBs range between 0-0.3 ng/g. Contamination of this type is usually associated with an inadequate cleaning of glassware.

14.3

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

14.3.1

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.

14.3.1.1

A typical value for LOD determined in this manner is 0.04 ng/g.

14.3.2

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.

14.3.2.1

A typical value for LOQ determined in this manner is 0.14 ng/g.

14.4

Stability of Reagents and Samples

14.4.1

Store analytical stocks and standards at minus 20 °C.

14.4.2

Stock standards and stock spike solutions remain stable for up to 6 months. Although there is no loss of analyte, evaporation (loss) of solvent may be an issue.

14.4.3

Prepare working standards fresh every 2 months.

14.4.4

Samples are stable at 4 °C for 3 weeks after extraction.

15.1

Tomkins, B. A. et al. 1985. Liquid chromatographic determination of benzo[a]pyrene in total particulate matter of cigarette smoke. J. Assoc. Off. Anal. Chem. 68, 5: 935-940.

15.2

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