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　P-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　mu　g/mL　(r　=　0.9999)　for　ergosterol,　5-400　mu　g/mL　(r　=　0.9999)　for　stigmasterol,　and　10-2000　mu　g/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.　Copyright　(c)　2007　John　Wiley　&　Sons,　Ltd.