Extraction of Azole Antifungal Drugs from Milk and Biological Fluids Using a New Hollow Fiber Liquid-Phase Microextraction and Analysis by GC-FID

A simple, rapid and sensitive high-performance liquid chromatography LC (HPLC) method of determining the concentration of the novel betulinic acid derivative DRF-4012 (5??-chloro-2,3-didehydroindolo2??,3??:2,3]betulinic acid) in rat plasma for pharmacokinetic and toxicokinetic purposes has been developed and validated. A simple and fast protein precipitation was performed, and then an extraction using an ethyl acetate:methanol (75:25 v/v) mixture was used to extract DRF-4012 and an internal standard (IS, DRF-4015) from rat plasma. Chromatographic separation was achieved using a Zorbax Eclipse XDB-C8 reversed-phase column with UV detection at 235 nm. The isocratic mobile phase, phosphate buffer (water adjusted to pH 3 with 20% o-phosphoric acid) and acetonitrile (15:85, v/v), was run at a flow rate of 1.2 mL min^?1. The assay was linear (r ² > 0.99) over the concentration range 0.040?C75.0 ??g mL^?1, and presented limits of detection and quantification of 0.020 and 0.040 ??g mL^?1, respectively, in rat plasma. The absolute recovery of both the analyte and the IS was >85% from rat plasma. The intraday accuracy ranged from 99.25 to 102.67% with a precision of 2.62?C4.48%, and the interday accuracy ranged from 98.48 to 104.56% with a precision of 3.87?C5.68%. This developed and validated method was successfully used to determine the DRF-4012 concentration in rat plasma for a pharmacokinetic and toxicokinetic study after the intravenous administration of a 1 mg mL^?1 DRF-4012 nanoparticle formulation at doses of 2?C10 mg kg^?1 in Wistar rats.