Coupling continuous ultrasound-assisted extraction with ultrasonic probe, solid-phase extraction and high-performance liquid chromatography for the determination of sodium Danshensu and four tanshinones in Salvia miltiorrhiza bunge

A dynamic continuous ultrasound-assisted extraction with high intensity ultrasonic probe (CUAE-HIUP) combined with solid-phase extraction (SPE) for preconcentration and clean-up of the extract prior to high-performance liquid chromatography (HPLC) determination of the main biological active ingredients, sodium Danshensu and four tanshinones (dihydrotanshione I, tanshinone I, cryptotanshinone and tanshinone IIA) from root of Salvia miltiorrhiza bunge has been developed. An experimental design (Plackett–Burman design, 26 × 3/16) was used to optimize the CUAE-HIUP procedure and the SPE step. Detection limits of 1.2–4.2 × 10−6 mg mL−1 were achieved with the preconcentration efficiency of more than 10-folds by the SPE procedure. The repeatability and within-laboratory reproducibility of the whole process, expressed as relative standard deviations (RSDs), were 1.5–3.6%, 1.6–3.1%, respectively. As compared with the conventional extraction techniques such as room temperature extraction (24 h), Soxhlet (4 h) and even microwave-assisted extraction (2 min), CUAE-HIUP achieved the highest extraction yield (98.9%) and the least amount (10 mL) of solvent compared with the other techniques (16 mL) in only 3 min. The method was successfully applied to the determination of the five biological active ingredients in root of S. miltiorrhiza bunge and Danshen-containing pharmaceutical formulations.