The resolution of peaks was performed with the mobile phase consisting of a mixture of 0.272 g of KH2PO4, 0.184 g of sodium n-heptane sulfonate, 820 mL of water, and 180 mL of methanol (HPLC grade). The pH of the mobile phase pH = 3.2 and the flow rate was 1.0 mL/min, and the wavelength was fixed at 256 nm for nicotine and 262 nm for cotinine as per the modified method [26]. Nicotine and cotinine at the concentration of 20 μM/mL were used as standards.