beta-Sitosterol and stigmasterol are the most common phytosterols in traditional Chinese medicine. They have been proved to have many important bioactivities. To the best of our knowledge, this is the first report that beta-sitosterol, stigmasterol and ergosterol coexisting in A. roxburghii herbs can be simultaneously extracted by a supercritical fluid extraction (SFE) procedure; then a simple high-performance liquid chromatography/atmospheric pressure chemical ionization ion trap mass spectrometry (HPLC/APCI/MS) method was developed for simultaneous identification and determination of these three compounds. The ion trap MS/MS detector was equipped with an atmospheric pressure chemical ionization source operating in the positive ion mode, APCI(+). The linear responses were obtained in the concentration range of 0.50-150 microg/mL (r = 0.9999) for ergosterol, 5-400 microg/mL (r = 0.9999) for stigmasterol, and 10-2000 microg/mL (r = 0.9998) for beta-sitosterol. An orthogonal L(9) (3(3)) test design was employed for optimization of the SFE process. Under the optimized conditions, i.e. pressure of 25 mPa, temperature of 45 degrees C and ethanol as modifier, the concentrations of sterols in the extract were found to be 2.89% (g/g) for beta-sitosterol, 3.56% (g/g) for stigmasterol and 2.96% (g/g) for ergosterin. The SFE method was also compared with a previously developed Soxhlet extraction. The SFE method produced higher yields of sterols than that of the Soxhlet extraction. The proposed method has been successfully used for identification and quantitation of beta-sitosterol, stigmasterol and ergosterin in a real A. roxburghii sample.

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