Liquid chromatography-tandem mass spectrometry assay for the simultaneous determination of three major flavonoids and their glucuronidated metabolites in rats after oral administration of Artemisia annua L. extract at a therapeutic ultra-low dose.

The traditional antimalarial herb Artemisia annua L., from which artemisinin is isolated, is widely used in endemic regions. It has been suggested that artemisinin activity can be enhanced by flavonoids in A. annua; however, how fast and how long the flavonoids are present in the body remains unknown. In the present study, a rapid and sensitive liquid chromatography with tandem mass spectrometry method was developed and validated for the simultaneous determination of three major flavonoids components, i.e. chrysosplenol D, chrysoplenetin, and artemetin and their glucuronidated metabolites in rats after oral administrations of A. annua extracts at a therapeutic ultra-low dose. The concentration of the intact form was determined directly, and the concentration of the glucuronidated form was assayed in the form of flavonoids aglycones, after treatment with β-glucuronidase/sulfatase. The method was linear in the range of 0.5-300.0 ng/mL for chrysoplenetin and artemetin, and 2-600 ng/mL for chrysosplenol D. All the validation data conformed to the acceptance requirements. The study revealed a significantly higher exposure of the flavonoid constituents in conjugated forms in rats, with only trace intact from. Multiple oral doses of A. annua extracts led to a decreased plasma concentration levels for three flavonoids.