Asthekeypointinsexhormoneanalysis,samplepre-treatmenttechnologyhasattractedscientists’attentionallovertheworld,andthedevelopmenttrendofsamplepreparationforwardedtofasterandmoreefficienttechnologies.Takingeconomicandenvironmentalconcernsintoaccount,subcriticalfluidextractionasafasterandmoreefficientmethodhasstoodoutasasamplepre-treatmenttechnology.Thisnewextractiontechnologycanovercometheshortcomingsofsupercriticalfluidandachievehigherextractionefficiencyatrelativelylowpressuresandtemperatures.Inthisexperiment,asimple,sensitiveandefficientmethodhasbeendevelopedforthedeterminationofdiethylstilbestrol（DES）infishtissueusingsubcritical1,1,1,2-tetrafluoroethane（R134a）extractionincombinationwithgaschromatography-massspectrometry（GC-MS）.Afterextraction,freezing-lipidfiltrationwasutilizedtoremovefattyco-extract.FurtherpurificationstepswereperformedwithC18andNH2solidphaseextraction（SPE）.Finally,theanalytewasderivedbyheptafluorobutyricanhydride（HFBA）,followedbyGC-MSanalysis.Responsesurfacemethodology（RSM）wasemployedtooptimizingtheextractioncondition,andtheoptimizedwasasfollows:extractionpressure,4.3MPa;extractiontemperature,26℃;amountofco-solventvolume,4.7mL.Underthiscondition,ataspikedlevelof1,5,10μgkg-1,themeanrecoveryofDESwasmorethan90%withrelativestandarddeviations（RSDs）lessthan10%.Finally,thedevelopedmethodhasbeensuccessfullyusedtoanalyzingtherealsamples.
