The crashed tobacco of each cigarette was quantitatively suspended in 100 ml of mixture of methanol: 0.1 N NaOH (1:1) solutions. The mixture ultrasonically vibrated for 1 selleck chemical Axitinib hour and then centrifuged for 10 minutes. 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. From this solution, 20 ��l were injected into the HPLC (n = 3). 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��C in an oven for 60 minutes to dry. The dried tobacco was weighed again and the amounts of moisture in each brand of tobacco were estimated.
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 test was not blinded to the brands and all the analysis were done in triplicate. Chromatographic conditions There are several analytical methods available for measuring nicotine in cigarette.18-21 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.22 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). The HPLC column was a reverse phase C18 column (4 ��m, 150 �� 4.
6 mm i.d., Nova pack, Waters) operated at ambient temperature (25 �� 1 ��C) in an air conditioned room. The mobile phase was consisted of 12% acetonitrile in 0.01 M phosphate buffer at a flow rate of 1 ml/min. Concentrated orthophosphoric acid was used to adjust the pH of the mobile phase to 7.0. The mobile phase was then filtered and degassed before use, using a vacuum filter system equipped with 0.45 mm filter membrane. The absorbance was monitored at 261 nm. The retention time for nicotine and metronidazole were 6.42 and 2.95 minutes, respectively. No interfering peaks from tobacco extract were observed. Nicotine concentration was calculated using peak area ratio of internal standard and sample peak.
Nicotine content was expressed as the concentration of nicotine in tobacco and also as the total amount of nicotine in one entire cigarette. Standard Solutions Stock solution (0.2 mg/ml) of nicotine and metronidazole (internal standard) were prepared by dissolving accurately weighed quantities of pure compounds separately in distilled water. The stock Carfilzomib solution remained stable for more than a month when stored at -20��C. Working standard solutions of nicotine (different concentrations of 10, 15, 20, 23 and 30 ��g/ml) were prepared by dilution of the stock solution with distilled water.
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