A liquid chromatography/electrospray ionization tandem mass spectrometric method for quantification of asiatic acid from plasma: application to pharmacokinetic study in rats.

Asiatic acid (AA), a pentacyclic triterpene from Centella asiatica (L.) Urban, has shown numerous therapeutic activities. However, none of the published works to date has used high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) for determination of AA from biological fluids. Therefore, the present paper describes a sensitive HPLC/electrospray ionization (ESI)-MS/MS method for quantification of AA in rat plasma. Ammonium adduct formation of AA was essential in the development of a sensitive method with the rat plasma samples being pre-treated by a simple solid-phase extraction method. The separation was achieved on a Cosmosil C(18) column using a gradient mobile phase flow. Detection was performed using an Applied Biosystems API Q-Trap 2000 mass spectrometer equipped with an ESI source operated in positive mode with colchicine used as internal standard. An eight-point calibration curve over the concentration range of 1.02-407.88 ng/mL for AA from rat plasma provided an optimum linear detector response (with r(2) >0.9983). The mean percentage recovery (n = 3) for the low, middle and high quality control samples was 91.23 ± 1.88%, 90.36 ± 0.55% and 89.71 ± 0.21%, respectively. The intra-day and inter-day precision and accuracy of the quality control samples were within ≤5% and ±7% correspondingly. The developed method was validated as per US FDA guidelines and applicability demonstrated by successful measurement of AA from plasma following oral administration of C. asiatica extracts to Wistar rats. The results suggest that the method could be applied to therapeutic monitoring of AA and pharmacokinetic studies in human volunteers.