This　study　establishes　a　novel　calibration　method　for　pre-equilibrium　hollow-fiber　liquid-phase　microextraction　(PE-HF-LPME),　where　the　time　constant　of　the　extraction　of　the　analyte　from　sample　matrix　to　the　extraction　phase　(organic　solvent)　is　obtained　from　a　simple　concentration　curve.　Comparing　to　the　traditional　kinetic　calibration　method,　where　the　time　constant　was　obtained　from　the　extraction　time　profile,　the　new　calibration　approach　shows　improved　accuracy　and　precision.　More　importantly,　deuterated　standards　are　not　required　in　the　new　method,　thus　significantly　improving　its　cost-effectiveness　and　extending　its　applicability　to　a　wide　range　of　analytes　lack　of　deuterated　analogs　serving　as　internal　standards.　In　addition,　mass　spectrometry　is　not　necessary　for　the　quantification　of　analytes　with　the　new　calibration　method,　which　may　further　extend　the　applicability　of　PE-HF-LPME　to　some　laboratories　without　mass　spectrometers.　This　study　has　been　substantiated　with　both　theoretical　and　experimental　evidences.　Further,　the　feasibility　of　the　method　for　real　biological　samples　was　demonstrated　by　measuring　the　free　concentration　of　flunitrazepam　in　urine　and　plasma　samples　and　its　drug-protein　binding　ratio　in　plasma.　The　results　showed　that　the　method　had　a　short　analysis　time　and　was　easily　implemented　with　high　accuracy　and　good　reproducibility.　(C)　2015　Elsevier　B.V.　All　rights　reserved.