Determination of paeonol O-demethylation activities in vitro by ultra-performance liquid chromatography with MS detection (UPLC-MS)

O-demethylation of paeonol has been implicated as an important mechanism of paeonol elimination. To characterize paeonol metabolism by O-demethylation, a rapid and specific ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS) method was developed for the qualitative and quantitative determination of paeonol O-demethylation activity in human liver microsomes (HLMs). HLMs incubation system with paeonol resulted in the formation of a single product peak. Following the purification, this peak was confirmed as O-demethylated metabolite of paeonol (2,4-Dihydroxyacetophenone, DHA) by MS and NMR. The method was validated for the determination of DHA with respect to specificity, linearity, detection limit, recovery, stability, precision and accuracy. The chromatographic separation was achieved on a UPLC BEH C18 column (50 mm × 2.1 mm i.d., 1.7 μm), with phase of acetonitrile-water (ratio 30:70). Selective ion reaction (SIR) monitor was specific for paeonol, DHA and I.S.. The method was linear over the concentration range 0.5-100 μM for DHA in spiked HLMs. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. DHA were stable at 4°C for at least 72 h in spiked liver microsomes samples. The method was successfully used to determine the kinetics of DHA activities toward paeonol in liver microsomes from different speices