Determination of Usnic Acid in Rat Plasma by High Performance Liquid Chromatography

The goal of this study was to develop and validate a high performance liquid chromatography method for determining the usnic acid (UA) concentration in rat plasma. Chromatographic runs were performed using reverse-phase C18 column at 280 nm. The mobile phase, consisting of a mixture of phosphate buffer solution (pH 7.4, 0.02 M) and methanol (60:40, v/v), was delivered at a flow rate of 1.0 ml/min. Proteins of plasma samples were precipitated adding 160 ml of ice-cold methanol/phosphate buffer solution (pH 5.8, 0.2 M, 4:1 v/v). After vortex-mixing for 5 min, the mixtures were centrifuged at 14,000 rpm for 15 min at 20oC ±1oC. Well-defined peaks of UA and no endogenous interfering peaks were observed at the retention time of 5.38 min. The calibration curves were linear over the range from 0.3 to 9.6 mg/mL, with correlation coefficients greater than 0.99. At the low concentration 0.3 mg/mL the precision and accuracy observed were within FDA specified range (>80%). This concentration was thus defined as lower limit of quantification (LLOQ) of the method. The within-day precision ranged from 1.5 to 4.6 % whereas the between-day precision ranged from 1.3 to 3.9 %. The method was thus accurate and precise according to FDA acceptance criteria for analyte measurement in a biological matrix, within ± 15 % at all concentration levels except at the LLOQ. In order to demonstrate the method applicability, male Wistar rats received UA as a single dose (15 mg.kg-1, i.p.). UA plasma concentration of 4.7 ± 0.7 mg.mL-1 was obtained about 1.3 h. The developed method allowed quantifying the UA content up to 36 h, corroborating that it is sensitive to characterize the plasma concentration profile after systemic administration. In conclusion, the developed method is reproducible, accurate and gives adequate validation results without requiring internal standard. Furthermore, this method was successfully applied to the analysis of plasma samples from rats that support a pre-clinical usnic acid pharmacokinetic study. Financial support: CNPq/MCT, FACEPE