Development and validation of UFLC–MS/MS method for determination of bosentan in rat plasma

• A sensitive UFLC–MS/MS method for assay of bosentan in rat plasma was developed.
• This method was fully validated according to FDA guidance.
• It was successfully applied to the real rat plasma samples.

A rapid, simple and sensitive UFLC–MS/MS method was developed and validated for the determination of bosentan in rat plasma using etodolac as an internal standard (IS) after liquid–liquid extraction with diethyl ether–chloroform (4:1, v/v). Bosentan and IS were detected using electrospray ionization in positive ion multiple reaction monitoring mode by monitoring the transitions m/z 551.90 → 201.90 and 288.20 → 172.00, respectively. Chromatographic separation was performed on the inertsil ODS-4 column with a gradient mobile phase, which consisted of 0.1% acetic acid with 5 mM ammonium acetate in water for solvent A and 5 mM ammonium acetate in acetonitrile–methanol (50:50, v/v) for solvent B at a flow rate of 0.3 mL/min. The method was sensitive with 0.5 ng/mL as the lower limit of quantitation (LLOQ) and the standard calibration curve for bosentan was linear (r > 0.997) over the studied concentration range (0.5–2000 ng/mL). The proposed method was fully validated by determining specificity, linearity, LLOQ, precision and accuracy, recovery, matrix effect and stability. The validated method was successfully applied to plasma samples obtained from rats.

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