Development and validation of a liquid chromatography-tandem mass spectrometry method for quantification of Lupeol in plasma and its application to pharmacokinetic study in rats.


Bharati Vidyapeeth College of Pharmacy, Kolhapur, Near Chitranagri, Kolhapur 416013, Maharashtra, India. Electronic address: [Email]


Lupeol, a phytosterol and triterpene, possesses numerous medicinal properties against cancer, inflammation, arthritis, diabetes, heart diseases, etc. A novel, sensitive, specific and reproducible method for quantification of Lupeol in rat plasma using liquid chromatography combined with atmospheric pressure chemical ionization (APCI) tandem mass spectrometry (LC-MS/MS) was developed and validated as per regulatory guidelines. Sample preparation was simple and fast which consisted of one-step protein precipitation using acetonitrile. Testosterone was used as an internal standard. HyPurity Advance column was used to develop the chromatography method using 0.1% formic acid in water and acetonitrile as mobile phases by gradient elution. APCI positive ion mode was used for mass spectrometric detection. Multiple reaction monitoring (MRM) transitions of m/z 409.5 [M + H - H2O]+→137.3 for Lupeol and m/z 289.1 [M + H]+→97.1 for Testosterone were used for quantification. The method was validated over a linear concentration range of 5-5000 ng/mL with a correlation coefficient (r2) of ≥ 0.99. This method showed acceptable accuracy (89.52-97.10%), precision (%CV ≤ 10.75%) and recovery with a negligible matrix effect. Lupeol was found to be stable in the stock solution, autosampler condition and also in plasma for four freeze-thaw cycles, 6 h at ambient temperature and 30 days at -20°C. This method was successfully applied to measurement of Lupeol in plasma samples from pharmacokinetic study in rats and can be easily extended to human pharmacokinetic studies.

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