NGHIÊN CỨU ĐỊNH LƯỢNG SILYBIN TRONG HUYẾT TƯƠNG CHÓ BẰNG PHƯƠNG PHÁP SẮC KÝ LỎNG HIỆU NĂNG CAO

Abstract

Objectives: To develop and validate a high-performance liquid chromatography (HPLC) method for quantifying silybin (Si) in canine plasma. Methods: Chromatographic separation was carried out on a Luna C18 column using a gradient mobile phase of methanol:phosphoric acid:water (phase A, 20:0.5:80; phase B, 80:0.5:20). A 100µL injection volume was used. Plasma samples were prepared by precipitating proteins with methanol or perchloric acid. Catechin served as the internal standard. The method was validated according to FDA guidelines, assessing selectivity, specificity, system suitability, linearity, accuracy, precision, limits of detection (LOD) and quantification (LOQ), and sample stability. Results: Optimized sample preparation and chromatographic conditions were established. The method was selective and specific, with linear calibration ranges of 0.118 - 11.758 µg/mL for Si-A and 0.128 - 12.754 µg/mL for Si-B (R² = 0.9997). Relative standard deviations were not more than 3.99% and recoveries were between 85% and 115%. For Si-A, LOD and LOQ were 4.69 ng/mL and 14.20 ng/mL; for Si-B, they were 2.79 ng/mL and 8.46 ng/mL. Silybin concentrations remained stable after 24 hours (recoveries 98.30 - 114.43%) and after three freeze-thaw cycles (recoveries 85.34 - 111.65%). Conclusion: A HPLC method for the quantification of silybin in canine plasma was successfully developed and validated.