OBJECTIVE A novel, rapid and highly sensitive liquid chromatography-tandem mass spectrometry method (LCMS/MS) was developed and validated for the determination and pharmacokinetic investigation of Hydrocortiaone 17-Butyrate in rabbit plasma for the first time.METHODS Hydrocortisone 17-Butyrate and the internal standard (IS), Clobetasol Propionate, were extracted from plasma samples using solid phase extraction with Oasis HLB cartridges.Chromatographic separation was performed on a Ultimat XB-C18 column (150 mm × 4.6 mm i.d., 5 m particle size) using acetonitrile-water(30 mmol· L-1 ammonium acetate buffer, 0.1% formic acid) (70∶30, V/V) as the mobile phase at a flow rate of 0.8 ml·min-1.Detection of Hydrocortisone 17-Butyrate and IS was achieved by tandem mass spectrometry with an electrospray ionization (ESI) interface in positive ion mode.Traditional multiple reaction monitoring (MRM) using the transition of m/z 433.3→m/z 327.3 and m/z 467.2→m/z 373.2 was performed to quantify Hydrocortisone 17-Butyrate and the internal standard, respectively.Eight healthy New Zea land white rabbits (4 males and 4 females) were applied 5g of cream containing 0.1% H-17-B to hairless skin area in the right back for 12 h.Blood samples were collected before and at 1.0, 2.0, 3.0, 4.0, 6.0, 8.0, 10.0, 12.0, 24.0 and 36.0h after drug administration.RESULTS AND CONCLUSION The calibration curves were linear over the range of 0.1-10 ng·ml-1 with the lower limit of quantitation validated at 0.1 ng· ml-1.A typical regression equation was y =0.00826x-0.00229 with acorrelation coefficient (r) of 0.9997, where y represents the peak area ratio of H-17-B to that of IS and x represents the plasma concentration of H-17-B.The intra-and inter-day precisions were within 13.10%, while the accuracy was within ±7.20% of nominal values.At three QC concentration levels 0.25, 1.0 and 5.0 ng· ml-1, the percent extraction recoveries of H-17-B obtained from plasma were (59.6 ± 4.4) % , (63.1 ± 7.6) % and (61.7 ± 5.1)%, respectively.The mean recovery for the internal standard at the concentration employed was (64.5 ± 4.5)%.The matrix effects of H-17-B, evaluated at three concentrations (0.25, 1.0 and 5.0 ng· ml-1), were (92.5 ± 5.1) %, (96.0 ± 10.6) % and (90.1 ± 3.9) % (n =5), respectively.The matrix effect of IS was 90.6 ± 2.9%.The stability results showed that H-17-B spiked into human plasma was stable for at least 12 h at room temperature, for at least 24 h in final extract at 6℃ under autosampler storage condition, for 60 d at-20℃, and during three freeze-thaw cycles, when stored at around-20℃ and thawed to room temperature.The validated LC-MS/MS method was successfully applied to pharmacokinetics study of a new kind of cream formulation cotaining hydrocortisone 17-butyrate 0.1% in rabbit.