Development and validation of an LC-MS/MS method for the determination of DPHB in rat plasma and its application in...

The aim of the present study was to develop a rapid, specific and sensitive LC-MS/MS method for the determination of DPHB [7-(4″-hydroxy-3″-methoxyphenyl)-1-phenyl-4-hepten-3-one] in rat plasma using yakuchinone A as an internal standard (IS). n-Hexane was used for the extraction of DPHB from rat plasma. Chromatographic separation of DPHB was achieved using a Kinetex XB-C₁₈ column (2.10 × 50 mm, 2.6 μm) at 40°C. The mobile phase consisted of water (containing 0.1‰ formic acid, A) and acetonitrile (containing 0.1‰ formic acid, B) under a gradient elution at a flow rate of 0.3 mL min^-1 . Positive electrospray ionization and multiple reaction monitoring mode were used for detection. The selected precursor ion to product ion pairs, m/z 311.3 → 137.0 for DPHB and m/z 313.1 → 137.0 for yakuchinone A, were monitored. Good linearity was observed over the concentration range from 2 to 2000 ng mL^-1 (r = 0.9969). The recovery efficiency of DPHB from rat plasma was 54.8-69.7%, while the matrix effect ranged from 99.7 to 113%. Intra- and inter-day precision and accuracy values were within ±15% at three different quality control concentration levels. This validated method was successfully applied to pharmacokinetic studies in rats after a single p.o. or i.v. dose of DPHB solution. The route of administration significantly influenced systemic exposure to DPHB, and low bioavailability of DPHB was observed. The method developed here will be further improved and used in future pharmacokinetic studies.