Pipette tip solid-phase extraction and high-performance liquid chromatography for the determination of flavonoids from Epimedii herba in rat serum and application of the technique to pharmacokinetic studies

• Home-made pipette tip extraction was developed for the flavonoids analysis in serum.
• The tip extraction had faster extraction time, less sample and less solvent.
• The method was used to study the pharmacokinetics of the flavonoids in rats.

Epimedii herba is a traditional Chinese medicine for the treatment of osteoporosis. Epimedin A, B and C and icariin are the primary effective ingredients of this medicine. In this study, a simple and low-cost method based on pipette tip solid-phase extraction, high-performance liquid chromatography separation, and diode array detection has been developed for the simultaneous analysis of four flavonoids (epimedin A, B and C and icariin) from Epimedii herba in rat serum samples. In this novel extraction configuration, the sorbents were placed between a filter (hollow fiber) and the pipette tip. Pipette tip solid-phase extraction has several advantages compared to conventional extraction methods: faster extraction time (6.0 min); lower sample volume (100 μL); lower solvent volume (100 μL); and less solvent waste. Under the optimum extraction conditions, the method showed good linearity (0.05–10.0 μg mL−1), acceptable intra- and inter precision (RSD < 6%), low limits of quantification (0.027–0.045 μg mL−1) and satisfactory relative recoveries (98.63–103.18%). This method was successfully applied to investigate the pharmacokinetics of the major flavonoids in Epimedii herba extract after oral administration to rats (10 g kg−1 body weight). The primary pharmacokinetic parameters for rats were determined as follows: Cmax, 0.45–4.11 μg mL−1; Tmax, 0.21–0.26 h; t1/2α, 0.06–0.12 h; t1/2β, 2.02–3.48 h; AUC0–∞: 0.50–2.58 μg h mL−1; CL, 19.53–44.72 L kg−1 h−1; and MRT0–∞, 2.25–3.77 h. The developed method has the potential to promulgate the pharmacokinetics and provide more information for clinical applications.