Quantification of Bullatine A and Bullatine B in Rat Plasma Using UPLC-MS/MS and Application to Pharmacokinetics.

A highly specific and sensitive UPLC-MS/MS assay was established and validated for simultaneous determination of bullatine A and bullatolide B in rat plasma. Midazolam was employed as the internal standard (IS). Separation was performed on a Waters UPLC BEH C18 column using a gradient mobile phase consisting of methanol and 0.1% aqueous formic acid. Detection was carried out in the mode of multiple reaction monitoring (MRM), m/z 344.3 → 105.0 (bullatine A), m/z 438.6 → 154.2 (bullatine B), and m/z 326.2 → 291.4 (IS). Plasma proteins were effectively precipitated with acetonitrile prior to analysis. Good linear calibration curves (r2 > 0.995) were obtained in a range of 0.5-600 ng/mL for both analytes, with a limit of quantification of 0.5 ng/mL. Both intra- and inter-day precision (percent RSD) were less than 14% for bullatine A and less than 13% for bullatine B. Accuracy (percentage nominal concentration) was within the range of 91%-109% for all quality control levels. The proposed method exhibited excellent sensitivity and selectivity and was successfully applied to compare the pharmacokinetics following intravenous and oral administration in rats. The absolute oral bioavailability was determined to be 4.3% for bullatine A and 12.8% for bullatine B.