3.2. Solid phase extraction (SPE)
The recovered concentrations of norethindrone acetate and dydrogesterone were 0.079 and 0.073 mg from human plasma after 30 min. The percentage recoveries of these drugs were calculated and found to be 79% and 73%, respectively. These concentrations were calculated by applying the correction factors due to the blank experiments. The decrease in the concentrations of these drugs (21% of norethindrone acetate and 27% of dydrogesterone) may be due to their binding with plasma proteins. The optimization of SPE includes pH of phosphate buffer, flow rate of plasma sample and eluting solvents, and use of the different eluting solvents. SPE experiment was carried out with pH range of 1.0–8.0 and the maximum recoveries of these drugs were obtained at pH 2.0 for norethindrone acetate and at pH 3.0 for dydrogesterone. The different flow rates of plasma samples (0.10–0.50 mL/min.) were examined to optimize the SPE conditions and poor recoveries were obtained at high flow rate while it was maximum at low flow rate. The same situations were obtained with eluting solvents. The optimized flow rates for both plasma samples and eluting solvents were 0.10 mL/min. Attempts have been made to optimize SPE conditions by using different solvents, such as methanol, ethanol, ethyl acetate and dichloromethane. Besides, the optimization was also carried out by eluting the reported drugs with these solvents with and without different amounts of acetic acid or trifluoroacetic acid. As a result of an extensive experimentation the best eluting solvent was pure methanol for these drugs. It is due to the fact that the polarity of methanol is quite enough to elute these drugs from C18 cartridge under the reported experimental conditions.