Development and Validation of RP-HPLC Method for Simultaneous Determination of Tramadol hydro chloride, Paracetamol and Dicyclomine hydro chloride by using Design of Experiment Software (DOE)

simple, fast and sensitive RP-HPLC method was developed for determination of tramadol hydrochloride, paracetamol and dicyclomine hydrochloride by using DOE statistical design. The isocratic separations of analytes were achieved using Kromasil C18 (250×4.6 mm, 5µ) column at a flow rate 0.5 ml/min. Mobile phase used was MEOH: water containing 0.1 %trifluoro acetic acid and10 % tetrahydrofuran (90:10 % v/v), pH adjusted to 3.0 with triethylamineto minimize the tailing effects. The chromatographic detection was carried out at 281 nm. The method was validated for specificity, linearity, precision, accuracy and robustness. The ranges of the independent variables used for the optimization were MEOH: 60-90%, pH: 2.5-3.5 and flow rate: 0.5-0.9 ml/min. The influences of these independent variables on the tailing factor (Tf) and resolutions (Rs) were evaluated. Using this strategy, mathematical model was defined and response surface was derived for the separation. The two responses were simultaneously optimized by using DOE.