Espectrometria de Massa e Técnicas de Purificação
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Re-Validation of New Develop Highly Sensitive, Simple LCMS/MS Method for the Estimation of Rohitukine and its Application in ADME/Pre-Clinical Pharmacokinetics

Amarinder Singh, Priya Wazir, Pankaj Chibber, Nitika Kapoor, Amit Kumar, Utpal Nandi, Sumit G Gandhi, Surjeet Singh, Manoj Kumar Tikoo, Ram Vishwakarma and Gurdarshan Singh

The purpose of the research was to develop a simple, rapid, accurate, reproducible and sensitive liquid chromatography–tandem mass spectrometry (LC-MS/MS) method for determination of Rohitukine, a chromone alkaloid in plasma. The chromatographic separation was achieved with high resolution RP18e Chromolith column (100 × 4.6 mm, 2 μm) employing a isocratic composition of organic solvent acetonitrile with 0.1% (v/v) formic acid (80:20, %v/v) at a flow rate of 0.5 mL/min. Triple quadrupole mass spectrometry with positive electrospray ionization (ESI) technique operating in multiple reaction monitoring used to estimate MS/MS ion transitions like 306.05>245.10 and 306.05>231.05 for Rohitukine and 330.30>97.0 for IS. Simple single step protein precipitate method was used for sample preparation. The method was validated for specificity, linearity, accuracy, precision, recovery, matrix effect and stability as per FDA guidelines. Linearity of the analyte was acquired throughout the concentration range from 0.1 ng/mL to 1000 ng/mL in mice plasma. Pharmacokinetic study was performed on female BALB/c mice through oral (20 mg/kg) and intravenous (2 mg/kg) route where the oral bioavailability of Rohitukine obtained was 84%. The bioanalytical method was successfully used for determination of plasma protein binding study, permeability and microsomal stability in mouse, rat and human liver microsomes.