Pharmacokinetic Study: Liquid Chromatographic Assay of Apigenin and Analogues in Rat Plasma after Oral Administration.

Abstract

Introduction: Various methods are available for estimating apigenin, but no methods have been applied to the pharmacokinetic study of its prepared analogues, such as polymorphs, co-crystals, and complexes. This study aims to develop a method for the pharmacokinetic assessment of apigenin and its analogues using HPLC-UV.

Methods: The internal standardization approach was utilized to analyse apigenin and its derivatives using UV detection at 340 nm, with chromatographic separation achieved under isocratic conditions on a Phenomenex C18 column (250 mm × 4.6 mm id, 5 μm). The optimal mobile phase consisted of a mixture of 0.3% formic acid and methanol (30:70, v/v) at a 1.5 mL/min flow rate. The system demonstrated a significant and well-resolved peak for apigenin and the internal standard quercetin, with retention times of 4.2 and 2.9 minutes, respectively. The calibration curve's linear regression analysis revealed a robust linear relationship over the concentration range of 2-10. 0 μg/mL; R² was determined to be 0.9995.

Results: The findings indicated that the limit of quantification (LOQ) and limit of detection (LOD) were 0.0803μg and 0.0265μg, respectively. The pharmacokinetics in rats were evaluated using this approach. Cmax, the plasma concentration of apigenin and its analogs, was reached following an oral dose of 60 mg/kg/rat. The collected data were utilized to calculate all pharmacokinetic parameters.

Discussion: The pharmacokinetic study revealed that apigenin analogues (A2-A7 and C1) significantly improved the oral bioavailability of apigenin (AP), as indicated by elevated Cmax and AUC_0-24 values relative to AP alone. Specifically, analogues A6 and A7 reached peak plasma concentrations more swiftly, implying a quicker onset of action, whereas A3 exhibited a delayed Tmax, reflecting slower absorption.

Conclusion: Following oral administration of a single dose of apigenin, this method was deemed suitable for quantifying the levels of apigenin and its derivatives in rats. This HPLC methodology could be an effective tool for determining apigenin analogues in plasma due to its excellent sensitivity, accuracy, linearity, and specificity.