Method Development and Validation for the Estimation of Darunavir in Rat Plasma by RP-HPLC
The proposed research work is a simple precise, accurate, selective RP-HPLC method was developed and validated for the quantitative of estimation of darunavir. Sample preparation was performed by liquid-liquid extraction in ethyl acetate. Darunavir was extracted from rat plasma and HPLC analysis was performed using Waters 515 Series pumps combined with a Waters PDA 2998 series photo diode array detector (DAD). The column used was Agilent C18 column (150mm◊4.6mm, particle size 5-micron Agilent, USA). Analysis was isocratic at 1.0 ml/min flow rate with ACN: Tri ethyl amine (0.025M Potassium buffer pH 2.3 (70:30, v/v) as mobile phase. The mobile phase was premixed, filtered through a 0.2 μm nylon membrane filter to remove any particulate matter and degassed by sonication before use. The elution was detected at 210 nm. Each solution was injected in triplicate, and the relative standard deviation (R.S.D.) was measured. The retention times of darunavir 2.24 min. The method was validated over the range of 1.5-3.5 μg/ml. The limit of detection was 0.06μg/ml and the limit of quantification was 0.193μg/ml. Inter-day as well intra-day replicates of darunavir, gave % R.S.D. below 2.07 and 2.001 respectively The absolute recovery of Darunavir was greater than 90% were achieved. This RP-HPLC method was applied for determination of Darunavir in plasma.
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