A New LC/MS/MS Method for the Analysis of Phyllanthin in Rat Plasma and its Application on Comparative Bioavailability of Phyllanthin in Different Formulations after Oral Administration in Rats

Introduction: A simple, short UPLC/MS/MS method for quantitation of phyllanthin in rat plasma in less than 2 minutes have been developed and fully validated. The validated method was used to investigate the pharmacokinetic properties of phyllanthin in PA extract and phospholipid complex of PA extract in rat. Methods: The separation was carried out on Acquity C₁₈ (50 x 2.1 mm; 1.7 μm), with a mobile phase of 10 mM aqueous amonium acetate and acetonitrile (10:90; v/v), at a flow rate of 0.2 mL/min. Felodipin was used as internal standard. Phyllanthin is extracted from a small volume of rat plasma (100 μl) by means of liquid-liquid extraction method with tert butyl methyl ether. Electrospray ionization (ESI) mass spectrometry was applied in positive mode at capillary voltage of 4000 V for both phyllanthin and IS, cone voltage of 24 V for phyllanthin and 20 V for IS, desolvation temperature of 360oC, cone gas flow of 25 L/h, collision energy of 12 V for phyllanthin and 10 V for IS. Multiple reaction monitoring (MRM) was used to monitor the transitions at m/z (Q1/Q3) 436.41/355.36 for phyllanthin and 384.20/352.18 for IS. Results: The linear calibration curve of phyllanthin was obtained over the concentration range of 0.5 – 100 ng/mL. The intra‐ and inter‐day precisions were less than 7.08 % and the accuracies were within ± 7.55%. The Cmax values of phyllanthin from two different preparations in rat plasma after oral administration of 2.0 mg/kg were 11.44 and 31.44 ng/ml, and the AUC values were 18.07 and 41.43 h.ng/ml, respectively. Conclusion: A simple, short UPLC/MS/MS method for quantitation of phyllanthin in rat plasma in less than 2 minutes have been developed and fully validated. The bioavailability of phyllanthin from the phospholipid complex of PA extract in rat plasma was significantly improved compared with that of raw PA extract after oral administration.