Development and Validation of RP-HPLC Method for Determination of Propylthiouracil in Hepatic Cells

Background: Propylthiouracil (PTU)-induced liver injury has been concerned in recent years and the mechanisms are largely unknown. Therefore, establishment of an analytical method for PTU detection in hepatic cells would contribute to the investigation of PTU hepatic injury. Objectives: In this study, a simple and rapid high performance liquid chromatography (HPLC) method for the intracellular determination of PTU concentration in rat liver cells (BRL-3A) was first developed and then validated. Methods and Results: The cell samples were pretreated with methanol to precipitate protein and analyzed by HPLC. The mobile phase was methanol-water (40:60) at a rate of 1.0 mL/min. Luna C18 column was adopted with detection wavelength at 272 nm. Specificity of the assay showed that endogenous components and solvents had no interference to PTU measurement. The linearity of method was demonstrated in the range of 0.0833-0.833 μg/mL with the correlation coefficient of 0.9968. The LOD and LOQ were 0.0580 μg/mL and 0.0833 μg/mL, respectively. The average extraction recovery of PTU from cell lysates was 91.9%. Accuracy of the method was found between 98.7% and 103.0%. The precision of intraday and interday (RSD) was less than 5.6%. Conclusion: This method is simple, accurate, and specific, which is suitable for PTU examination in hepatic cells.