A Validated UPLC–MS/MS Assay for Isosakuranetin Determination in Rat Plasma and Its Application to Pharmacokinetics

Isosakuranetin is a natural flavonoid containing a 4′‐O‐methyl group on the molecular structure and has a broad range of pharmacological activities. In this study, a sensitive and robust UPLC‐ESI‐MS/MS method was developed and fully validated for quantitative determination of isosakuranetin in rat plasma, enabling a comprehensive evaluation of the pharmacokinetics and oral bioavailability of isosakuranetin. Isoscoparin was the internal standard (IS). The plasma samples were prepared by protein precipitation with acetonitrile. Chromatographic separation was conducted with a BEH C18 column and a gradient mobile phase consisting of acetonitrile and 0.1% formic acid aqueous solution. The detection was accomplished in negative electrospray ionization (ESI) by multiple reaction monitoring (MRM) mode. The assay had excellent linearity (r > 0.995) in a linear concentration range of 0.5–800 ng/mL. Precision (intra‐ day and inter‐day) was less than 13%, and extraction recovery was above 89%. Accuracy was between 91% and 113%, and the matrix effects were 93%–104%. Overall, this method was rapid, specific, reproducible, and sufficiently sensitive for in vivo pharmacokinetic studies in rats. After oral administration, the absolute bioavailability of isosakuranetin was calculated to be 64.6%.