Three different dutasteride extraction methods were compared and a method based on liquid-liquid extraction (LLE) using methyl tert-butyl ether and methylene chloride was proved to be more effective than others for the extraction of dutasteride and finasteride, the internal standard (IS), from rat plasma. Additionally, a method composed of the LLE extraction, liquid chromatography, and multiple reaction monitoring (MRM) to target dutasteride and IS was validated by assessing specificity, linearity (r 2 = 0.9993, 5 -400 ng/mL), sensitivity (the limit of detection: 4.03 ng/mL; the limit of quantitation: 12.10 ng/mL), accuracy (intra-day: 89.4 -105.9%; inter-day: 84.9 -100.9%), precision (intra-day: 0.8 -6.9%; inter-day: 2.9 -15.9%), and recovery (84.7 -107.8%). Since the validated method was successfully applied to a pharmacokinetic study of dutasteride, it can be useful for the pharmacokinetic evaluation of newly developed dutasteride formulations.

Tacrolimus (TAC) is an immunosuppressant widely used in organ transplantation, but its extremely low aqueous solubility causes poor intestinal absorption. There have been efforts to develop an alternative TAC formulation with an improved dissolution rate and oral bioavailability (BA), and the development of a rapid and sensitive analytical method for its in vivo pharmacokinetic study is an essential prerequisite. Thus, here, we develop a novel method to determine TAC in rat whole blood based on liquid chromatography and tandem mass spectrometry, and liquid-liquid extraction (LLE) with mild temperature ultrasonication. For rapid and efficient separation of TAC from other hydrophobic compounds, a C8 column was chosen with isocratic mobile phase elution. With the help of the high specificity and the high sensitivity of multiple reaction monitoring in positive ion mode, the present method showed good performance including specificity, linearity (r(2) ≥ 0.996 within 1-200 ng/mL), sensitivity (the lower limit of quantitation at 1 ng/mL), intra- and inter-day accuracy (88.7-104.5 %) and precision (≤10.3 %), and recovery (94.7-102.6 %). Also, the stability of TAC and ascomycin, the internal standard, in rat whole blood was confirmed before and after the sample preparation. The validated method was satisfactorily applied to a pharmacokinetic study to determine TAC in rat whole blood following oral administration of the marketed product (Prograf(®), Astellas Pharma). In the present study, LLE with mild temperature ultrasonication was successfully expanded to the determination of a drug from whole blood or plasma for the first time. Therefore, the present method can contribute to the rapid in vivo evaluation of novel TAC formulations, and will be able to contribute to the development of TAC formulations with a higher dissolution rate and a higher BA.