Development and validation of an LC-MS-MS method for determination of methyl kulonate in rat plasma

A sensitive, rapid and specific LC-MS-MS method was established and validated for determination of methyl kulonate, a major bioactive constituent isolated from Meliae Cortex, in rat plasma. Plasma samples were treated by precipitating protein with methanol and were chromatographed using a Capcell Pak C₁₈ column (100 x 4.6 mm, 5 μm) with the mobile phase comprising a mixture of methanol, 10 mM ammonium formate and formic acid (95:5:0.1, v/v/v). Detection and quantification were performed by mass spectrometry in the multiple reaction monitoring mode with positive atmospheric ionization at m/z 467 --> 311 for methyl kulonate, and m/z 469 --> 451 for dubione B (internal standard), respectively. A good linear response was observed over the concentration range 1.00-500 ng/mL with the lower limit of quantification 1.00 ng/mL in rat plasma. The method also afforded satisfactory results base on sensitivity, specificity, precision, accuracy, recovery, freeze-thaw and long-time stability. The validated method was successfully applied to determine the pharmacokinetic properties of methyl kulonate in rats after oral administration at dose of 100 mg/kg. This pharmacokinetic study of methyl kulonate is reported here for the first time.