Aim and Objective: Wedelolactone and demethylwedelolactone are the two major
coumarin constituents of Herba Ecliptae. The objective of this work was to develop and validate a
sensitive, rapid, and robust UPLC-MS/MS method for the simultaneous quantification of
wedelolactone and demethylwedelolactone in rat plasma.
Materials and Methods: Wedelolactone and demethylwedelolactone were extracted from rat
plasma by protein precipitation with acetonitrile. Electrospray ionization in negative mode and
selected reaction monitoring (SRM) were used for wedelolactone and demethylwedelolactone at
the transitions m/z 312.8→298.0 and m/z 299.1→270.6, respectively. Chromatographic separation
was conducted on a Venusil C18 column (50 mm × 2.1 mm, 5 μm) with isocratic elution of
acetonitrile-0.1% formic acid in water (55:45, v/v) at a flow rate of 0.3 mL/min. A linear range was
observed over the concentration range of 0.25–100 ng/mL for wedelolactone and
Results: They reached their maximum plasma concentrations (Cmax, 74.9±13.4 ng/mL for
wedelolactone and 41.3±9.57 ng/mL for demethylwedelolactone) at the peak time (Tmax) of 0.633 h
and 0.800 h, respectively. The AUC0-t value of wedelolactone (260.8±141.8 ng h/mL) was higher
than that of demethylwedelolactone (127.4±52.7 ng h/mL) by approximately 2-fold, whereas the
terminal elimination half-life (t1/2) of wedelolactone (2.20±0.59 h) showed the approximately same
as that of demethylwedelolactone (2.08±0.69 h).
Conclusion: Based on full validation according to US FDA guidelines, this UPLC-MS/MS method
was successfully applied to a pharmacokinetic study in rats.