Development and Validation of an HPLC-MS/MS Method for the Simultaneous Quantification of Vitexin and Isovitexin in...

Vitexin and isovitexin are natural flavone C-glucosides that have numerous benefits for human health. However, their low oral bioavailability and poor gastrointestinal absorption dramatically restrict their potential medicinal uses. To overcome this challenge, chitosan-coated alginate microcapsules were prepared for intragastrical administration to rabbits. An LC-MS/MS method was developed and validated for the simultaneous determination of vitexin and isovitexin in the plasma of treated rabbits, using salicylic acid as the internal standard. Raw rabbit plasma samples were deproteinized using acetonitrile as a precipitation agent. Chromatographic separation was performed on a reversed-phase C18 column (100 mm × 4.6 mm, 3.5 µm), with an isocratic mobile solvent system comprising methanol and 0.1% acetic acid (40:60) as the mobile phase. All the analytes and the internal standard were ionized on a triple quadrupole mass spectrometer and electrospray ionization, operating in negative mode and multiple reaction monitoring. The analytical approach developed underwent validation in terms of system suitability, specificity, selectivity, LLOQ of 2 ng/mL, linearity (2.0-200 ng/mL, R² > 0.99), accuracy (the intra- and inter-day from 94 to 110% with the relative standard deviations no more than 8.7%, precision with the recoveries from 97% to 102%, matrix effect (90-100%), carry-over, dilution integrity (2 times), and stability at room and frozen temperature for up to 1 month, and all the parameters met FDA and EMA requirements for bioanalytical methods. The validated procedure was applied to measure the absorption of vitexin and isovitexin from encapsulated extracts in a pilot pharmacokinetic study on rabbit plasma. Compared to the raw traditional extracts, the microcapsules enhanced the bioavailability of vi-texin and isovitexin regarding C_max and AUC values.