A new method for the quantitative determination of bosentan monohydrate in male rabbit plasma was developed by Reverse Phase HPLC as per standard regulatory guidelines. Precipitation method was used for extracting the drug from the drug spiked plasma using mobile phase. The analyte extracted was injected into ODS column C18 (250 x 4.6 mm, 5µm), the temperature was maintained at 250C and monitoring of effluent was performed at 273 nm. The mobile phase consists acetonitrile and phosphate buffer (pH 4.6) in 80:20 ratio. The method developed shown high specificity for bosentan monohydrate. The calibration curve for bosentan monohydrate was linear from 0.1-5 µg/0.5mL in plasma (R2=0.9999). The lower limit of detection is 0.1µg/0.5mL. The developed method was verified for precision in intra-day and inter-day and was found within the limits. The accuracy of the developed Reverse Phase HPLC method was found to be 99.8±1.25-98.7±1.29% and is found to be reproducible. The method developed was proved to be adequate, sensitive and reproducible for the determination of bosentan monohydrate in rabbit plasma.
Key words: Bosentan monohydrate, Reverse Phase HPLC, ICH guidelines, rabbit plasma, standard regulatory guidelines.
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