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Development and validation of HPLC-UV based bioanalytical method for the quantification of atorvastatin in rat plasma

A highly selective, sensitive, and fast HPLC based bioanalytical method was developed and validated for the estimation of atorvastatin in rat plasma which can be further employed in pharmacokinetic studies. The developed analytical procedure utilized protein precipitation approach to extract atorvastatin from rat plasma using ice-cold acetonitrile as an extraction solvent and diclofenac as IS. The chromatographic separation of atorvastatin was achieved on a LiChrospher RP C-18 column (250 × 4.6 mm, 5 μm) using isocratic elution of  Methanol: water (containing 0.05 % glacial acetic acid) (70:30, v/v pH 3.0) as mobile phase with 1mL min-1 flow rate and further detected by UV absorbance at 248 nm. Atorvastatin eluted within 3.093 min, whereas IS eluted at 8.953 min. The developed method was validated as per USFDA guidelines. Linearity was observed in concentration between 15.62 ng mL-1 to 2000 ng mL-1 with r2 equals to 0.9999 and LOQ 10.45 ng mL-1. The developed method follows the all acceptance criteria concerning its linearity, accuracy, precision, selectivity, dilution integrity, and recovery. Also, atorvastatin was demonstrated to be stable in the battery of stability studies such as freeze-thaw, bench-top, autosampler, short-term, and long-term stability. Thus the developed bioanalytical assay can be used in pharmacokinetic studies.