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

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-C-18 column (2.10 x 50 mm, 2.6 mu m) at 40 degrees C. The mobile phase consisted of water (containing 0.1 parts per thousand formic acid, A) and acetonitrile (containing 0.1 parts per thousand 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 bio-availability of DPHB was observed. The method developed here will be further improved and used in future pharmacokinetic studies.