Nicotine concentration was calculated using peak area ratio of internal standard and sample peak. No interfering peaks from tobacco extract were observed.

The retention time for nicotine and metronidazole were 6.42 and 2.95 minutes, respectively. The mobile phase was then filtered and degassed before use, using a vacuum filter system equipped with 0.45 mm filter membrane. Concentrated orthophosphoric acid was used to adjust the pH of the mobile phase to 7.0. The mobile phase was consisted of 12% acetonitrile in 0.01 M phosphate buffer at a flow rate of 1 ml/min. The HPLC column was a reverse phase C18 column (4 µm, 150 ´ 4.6 mm i.d., Nova pack, Waters) operated at ambient temperature (25 ± 1 ☌) in an air conditioned room. The HPLC system consisted of a pump (Model 600E, waters), a variable wavelength detector (Model 484, waters), a U6K injector and a recorder (Model 745B, waters).

There are several analytical methods available for measuring nicotine in cigarette.- In the present study one of the published HPLC methods according to the laboratory condition was chosen and applied for measuring nicotine in cigarettes after some modifications. The test was not blinded to the brands and all the analysis were done in triplicate. However, the same extraction procedure described for cigarette was used to extract nicotine from pipe tobaccos and a solution was prepared for injection into the high performance liquid chromatography (HPLC). The dried tobacco was weighed again and the amounts of moisture in each brand of tobacco were estimated. In orders to measure the nicotine quantity of three different popular pipe tobacco, five grams of each tobacco were placed in a glass plate and left at 70☌ in an oven for 60 minutes to dry. From this solution, 20 µl were injected into the HPLC (n = 3). To 1 ml of the supernatant, 2 ml of metronidazole solution (0.2 mg/ml, using H2O as solvent, as internal standard) was added and the total volume was made up to total of 10 ml, using 0.01 M phosphate buffer (pH 7.0) solution. The mixture ultrasonically vibrated for 1 hour and then centrifuged for 10 minutes. The crashed tobacco of each cigarette was quantitatively suspended in 100 ml of mixture of methanol: 0.1 N NaOH (1:1) solutions. The tobacco of each cigarette was crashed carefully in a blender for one minute. Before any extraction, papers and filters of the cigarettes were removed and the amount of tobacco in each cigarette was weighed. Four pack of each brand of cigarette was chosen randomly and one cigarette from each pack was taken to test.

Cigarette tar and nicotine chart