Determination of 20(R)-25-Methoxyl-dammarane-3,12,20-triol and Its Active Metabolite in Beagle Dog Plasma by LC–MS–MS and Its Application to Pharmacokinetics Studies

A rapid and sensitive liquid chromatography-tandem mass spectrometric method (LC–MS–MS) has been developed for simultaneous determination of 20(R)-25-methoxyl-dammarane-3,12,20-triol (25-OCH3-PPD) and its active metabolite, 20(R)-dammarane-3,12,20,25-tetrol (25-OH-PPD) in beagle dog’s plasma. Diazepam was employed as an internal standard. Sample preparation involved a liquid–liquid extraction with diethyl ether and dichloromethane (3:2, v/v). Chromatographic separation was achieved on a Phenomenex C18 material (50 × 2.0 mm i.d., 4 μm) using methanol–water-formic acid (124:26:0.3, v/v/v) as the mobile phase at a flow rate of 0.25 mL min−1. The present method exhibited good linearity over the concentration range of 2–500 ng mL−1 for 25-OCH3-PPD and 2–60 ng mL−1 for 25-OH-PPD. The lower limit of quantification was 2 and 2 ng mL−1 for 25-OCH3-PPD and 25-OH-PPD, respectively. The intra- and inter-day precision values for 25-OCH3-PPD and 25-OH-PPD met the requirements of the FDA guidance, and the accuracy (RE) ranged from −4.1 to 5.6% calculated from QC samples. No endogenous compound was found to interfere with the analysis. 25-OCH3-PPD and 25-OH-PPD were stable during all storage, pretreatment and analytical periods. The validated method was successfully used to study the pharmacokinetics of 25-OCH3-PPD in beagle dogs after oral administration at a dose of 12 mg kg−1.