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

ABSTRACT 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 × 4.6 mm, 5 µm) with the mobile phase comprising a mixture of methanol, 10 m m 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. Copyright © 2011 John Wiley & Sons, Ltd.