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Determination of Selected Carbonyls in Mainstream Tobacco Smoke
10 Sampling
10.1 The sampling of tobacco products for the purpose of testing shall be as specified in T-115.
11 Tobacco Product Preparation
11.1 Product shall be conditioned as specified in T-115.
11.2 Cigarettes, cigarette equivalents, bidis, kreteks and cigars shall be marked for butt length as specified in T-115.
11.3 Cigarettes to be smoked under intense smoking conditions shall be prepared as specified in T-115.
12 Smoking Machine Preparation
12.1 Ambient Conditions
12.1.1 The ambient conditions for smoking shall be as those specified in T-115.
12.2 Machine Conditions
12.2.1 The machine conditions shall be as those specified in T-115 (with the following modifications as detailed below:)
12.2.1.1 It is important to ensure that the mainstream tobacco smoke is characteristic of the test sample before proceeding with the analysis. Because the mainstream total particulate matter (TPM) is not filtered, no filter pad is present; puff count information must then be used to characterise the smoke extract samples and monitor the smoking process.
12.2.1.2 Assemble the carbonyl mainstream apparatus on 12 alternating ports of the linear smoking machine without using the filter pads and holders.
12.2.1.3 Connect the special glass cigarette holder (6.5cm in length and 8.0 mm internal diameter) at the back by Nalgene tubing to the 250 mL impinger and trap. See diagram.
12.2.1.4 Check and adjust the puff volume drawn by the smoking machine in all of the 12 ports as per T-115. Volumes are checked at the cigarette end of the port (with fritted impinger and DNPH in line) via the brass restrictor.
12.2.1.5 The same impinger can be used to adjust each port before smoking begins. Discard the DNPH solution after puff volumes have been measured and adjusted.
12.2.1.6 Add 80 mL of fresh 2,4-DNPH solution to each impinger.
13 Sample Generation
13.1 Cigarettes (two cigarettes*/observation) shall be smoked per observation as specified in T-115.
*For other tobacco products, select a number such that breakthrough does not occur.
14 Sample Analysis
14.1 Mainstream Smoke Extract Solution
14.1.1 One run consists of 12 DNPH smoke extract samples. Process 12 samples at a time but not more than two runs or 24 samples per day. Do not smoke more than can be analysed in a 24 hour period.
14.1.2 Rinse the cigarette holder and the Nalgene tubing with the impinger solution by forcing the impinger solution back up the impinger as far as the glass cigarette holder using positive air pressure and then with negative air pressure until air is forced back through the solution.
14.1.3 Repeat this rinsing procedure at least three times for each impinger to dissolve any smoke condensate in the gas transfer lines.
14.1.4 Allow the DNPH smoke extract solution to sit for least five minutes before continuing with sample preparation.
14.1.5 Pipette 6 mL of 1 % Trizma base solution into a 10 mL volumetric flask.
14.1.6 Add 4 mL of syringe-filtered DNPH smoke extract to the volumetric flask.
14.1.7 Mix the volumetric flask well. Transfer a portion of this solution by Pasteur pipette to autosampler vials in duplicate (a and b). (Rinse each vial first with a few drops, fill to minimise head space).
14.1.8 Cap the vials with Teflon faced septa and stored at 4 °C until analysed.
14.1.9 Repeat 14.1.5 to 14.1.8 for each smoke extract sample.
14.2 Preparation of Controls and Blanks
14.2.1 Prepare at least one LRB, LFB, and one LFM per day of activity as follows to demonstrate that interference from the analytical system, glassware, and reagents are not present.
14.3 Laboratory Reagent Blank (LRB)
14.3.1 Pipette 6 mL of the 1 % Trizma base dilution solution into a 10 mL volumetric flask.
14.3.2 Add 4 mL of fresh filtered DNPH solution to the volumetric flask. Cap the flask and mix well.
14.3.3 Transfer to two autosampler vials (a and b), cap and store at 4 °C until ready to analyse.
14.4 Laboratory Fortified Blank (LFB)
14.4.1 Add 1 mL of the Carbonyl Spiking Solution and 79 mL of DNPH solution to the 250 mL impinger. Mix well.
14.4.2 Pipette 6 mL of the 1 % Trizma base dilution solution into a 10 mL volumetric flask.
14.4.3 Add 4 mL of the filtered DNPH/Spiking solution (14.4.1) to the volumetric flask. Cap the flask and mix well.
14.4.4 Transfer to two autosampler vials (a and b), cap and store at 4 °C until ready to analyse.
14.5 Laboratory Fortified Matrix (LFM)
14.5.1 Pipette 5 mL of the 1 % Trizma base dilution solution into a 10 mL volumetric flask.
14.5.2 Add 1 mL of the Carbonyl Spiking Solution to the 10 mL volumetric flask.
14.5.3 Add 4 mL of filtered DNPH/smoke extract solution from a control brand to the 10 mL volumetric flask. Cap the flask and mix well.
14.5.4 Transfer to two autosampler vials (a and b), cap and store at 4 °C until ready to analyse.
14.6 Reversed Phase High Performance Liquid Chromatography
14.6.1 Chromatographic Conditions
14.6.1.1 Column Temperature: 30 °C.
14.6.1.2 Injection volume: 20 mL.
14.6.1.3 UV detection at 365 nm.
14.6.1.4 Mobile Phase: Reagents
Solvent A: Prepare 2 L of 30 % Acetonitrile, 10 % THF, 1 % IPA in Type I water, filter and degas. (UHP Helium sparged).
Solvent B: Prepare 2 L of 65 % Acetonitrile, 1 % THF, 1 % IPA in Type I water, filter and degas. (UHP Helium sparged).
Solvent C: Acetonitrile (UHP Helium sparged).
14.6.1.5 Sample Wash: Solvent A.
14.6.1.6 Mobile Phase: Gradient.
| Flowrate Time (minutes) | Composition 1.5 mL/minute | ||
|---|---|---|---|
| % A | % B | % C | |
| 0.0 | 100 | 0 | 0 |
| 8.0 | 70 | 30 | 0 |
| 20.0 | 47 | 53 | 0 |
| 27.0 | 0 | 100 | 0 |
| 30.0 | 0 | 0 | 100 |
| 32.0 | 0 | 0 | 100 |
| 34.0 | 95 | 5 | 0 |
| Method End Action: | 100 | 0 | 0 |
| (Equilibrate 10 minutes) | |||
14.6.2 Sample vials are loaded onto the autosampler such that every 8th vial is a standard solution and in such quantities that the total analysis time (14.1 - 14.6) does not exceed 24 hours.
14.6.3 Inject 20 mL of vial (a) of each sample onto the HPLC column and analyze as per the chromatographic conditions listed in 14.6.1. Vial b is the backup sample in the event of a problem.
14.6.4 Elution pattern should be similar to Figure 1.
14.7 Calculations
14.7.1 Construct a Calibration Curve:
14.7.1.1 Twenty mL of each calibration standard is injected onto the HPLC column and analysed as per the chromatographic conditions listed in 12.7. Do in duplicate. Elution pattern should be similar to Figure 2.
14.7.2 Determination of Response Factor
14.7.2.1 A calibration curve for each individual carbonyl is prepared by plotting the concentration of the standards versus their respective peak areas.
14.7.2.2 Response factors are calculated for each individual carbonyl compound from the calibration curves.
14.7.3 Sample Quantification
14.7.3.1 The amount of the various carbonyl compounds in smoke samples is quantified by the external standard method. The identification of peaks is by comparison of retention times with standards, and the spiking of smoke samples.
14.7.3.2 Carbonyl concentrations are reported in (mg/mL) by the software.
14.7.3.3 Determination of Mainstream Carbonyl Deliveries in [μg/cigarette]
e.g. Carbonyl [mg/cigarette] =[ Peak Area/ Resp. Factor] X [DF/ No. of Cigarettes]
where DF is the dilution factor. The response factor shall be determined from the calibration curve.
15 Quality Control
15.1 Typical Chromatogram
15.1.1 See Figures1, 2. .
15.2 Recoveries and Levels of Contamination
15.2.1 Each analytical run of test cigarettes should include:
A Laboratory Reagent Blank (LRB) to evaluate the extent of any interference due to glassware, trapping reagents, glass fibre filter discs (pads), and analyzer effects.
A Laboratory Fortified Matrix (LFM) to evaluate the extent of potential analyte loss.
A standard run as a sample to verify the calculation process and validate the calibration.
15.3 Method Detection Limit (MDL) and Limit of Quantitation
15.3.1 Method Detection Limit (MDL)
15.3.1.1 The method detection limit is determined by analysing 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.
15.3.2 Limit of Quantitation (LOQ)
15.3.2.1 The limit of quantification is determined by analysing the lowest level standard at least 10 times as an unknown over several days. The LOQ is then calculated as ten times the standard deviation of these determinations.
15.4 Stability of Reagents and Supplies
15.4.1 All primary Carbonyl standards are prepared as required.
15.4.2 All work standards are to be prepared every 20 days.
15.4.3 All samples are to be analysed as soon as they are prepared and within four hours of the cigarettes being smoked.
16 Modifications for Intense Smoking Conditions
16.1 No modifications for intense smoking are necessary.
17 Reference
17.1 Houlgate, P. R., Dhingra, K. S., Nash, J. S., and Evans, W. H., 1989: Determination of Formaldehyde and Acetaldehyde in Mainstream Cigarette Smoke by high-performance Liquid Chromatography; Analyst 114, p. 355-360.
17.2 Manning, D.L., Maskerinec, M.P., Jenkins, R.A., and Marshall, A.H. "High Performance Liquid Chromatographic Determinations of Selected Gas Phase Carbonyls in Tobacco Smoke" Journal of Assoc of Anal Chem ., 66, p. 8-12.
Appendices
Appendix 1 - Typical Calibration Standards
(b): Carbonyl Working Standards **
Figure 1: Analytical Chromatogram of Volatile Carbonyls in DNPH Extract of Mainstream Tobacco Smoke
Figure 2: Chromatogram of a typical combined Carbonyl Calibration Standard
