DESIGN OF EXPERIMENT UTILIZATION TO DEVELOP AND VALIDATE HIGH PERFORMANCE LIQUID CHROMATOGRAPHY TECHNIQUE FOR ESTIMATION OF PURE DRUG AND MARKETED FORMULATIONS OF ATORVASTATIN IN SPIKED RAT PLASMA SAMPLES

A novel method of estimation and validation of Atorvastatin by Reverse Phase-High Performance Liquid Chromatography coupled with Ultra-violet detection was developed which had high potential in determining drug concentration in rat plasma samples during preclinical studies. Atorvastatin being >99% protein bound, exhibits great challenge in getting extracted from plasma samples. Hence, by treating them with strong protein precipitating agents, that is, initially with 10% w/v perchloric acid and further treating its supernatant with mixture of 2M potassium carbonate and 3M of potassium hydroxide, drug extraction was facilitated. Diclofanac sodium was the selected internal standard. Process of elution was conducted using Phenomenex C₁₈ column and mobile phase comprising of a mixture of methanol: water in 70:30 ratio adjusted to pH 5.5. This precise method was linear between a range of 10 to 1000 ng/ml with limit of detection and quantification as 10ng/ml and 15ng/ml respectively. 2-factor 3-level face centred Central Composite Design was employed using Design Expert Software ver. 8.0.0 to examine effect of independent chromatographic factors like pH of the mobile and methanol: water ratio on the dependent factors like retention time, theoretical plates and tailing factor. The ANOVA studies proved that the model employed for this study was significant. Further, to improve applicability, marketed drug formulation was spiked in rat plasma and developed method was applied for drug detection.