The nicotine dosage in tobacco leaves extracted with
water was determined by HPLC. Standard nicotine was
purchased from Sigma (lot no. 093K4121). Analysis was
done on a C18 reverse-phase column (cartridge: 5 m,
250 x 4.6 mm). The separation was done at 35 °C. The
isocratic mobile phase used was a mixture of aqueous phase
(phosphate buffer, pH 6.8, and methanol at a ratio of 35:65,
v/v). The solution was sonicated before use and the flow
rate was 1.2 mL/min. The HPLC method used was based on
work by Tambwekar et al. (2003). A stock solution of nicotine
(1 mg/mL) was prepared by dissolving 100 mg in
100 mL of phosphate buffer (pH 6.8).