Determination of a novel phosphodiesterase-4 inhibitor chlorbipram in mouse plasma and brain by UFLC–MS/MS: Application in pharmacokinetic studies after intravenous administration

• A method coupled with UFLC–MS/MS was first validated for determination of chlorbipram in mouse plasma and brain.
• The method is accurate, precise and meets validation requirements by guideline.
• The method could also be used for preclinical pharmacokinetic study in mice.
• The novel drug could easily cross the blood–brain barrier.

In this study, we evaluated a simple and sensitive method for determination of a novel phosphodiesterase-4 (PDE4) inhibitor, chlorbipram, in mouse plasma and brain using ultra-fast liquid chromatography-tandem mass spectrometry (UFLC–MS/MS). Separation was achieved using an Acquity UPLC BEH C18 column (50 mm × 2.1 mm, particle size 1.7 μm) with a gradient mobile phase consisting of water and methanol at a flow rate of 0.25 ml/min. Detection was performed in the multiple reaction monitoring (MRM) mode using electrospray ionization (ESI) in the positive ion mode. The liquid–liquid extraction method with ethyl acetate was used for both pretreatment of plasma and brain homogenates. The calibration curves of chlorbipram showed good linearity over the concentration range of 0.5–200 ng/ml (R2 > 0.994) for mouse plasma and over the range of 0.25–100 ng/ml (R2 > 0.994) for mouse brain homogenate. The extraction recovery was in the range of 78.3–84.8% for chlorbipram and the internal standard (IS) ZXI14 in two different biological matrices. The intra- and inter-day precision values were less than 13.0% and the accuracy ranged from 97.8% to 106.0% for quality control samples. No noteworthy matrix effects and instability were observed for chlorbipram. This validated method was successfully applied to a pharmacokinetic study of chlorbipram in mice after intravenous administration. The results show that this novel drug crosses the blood-brain barrier and provides the basis for further studies on chlorbipram.