Determination of isoorientin levels in rat plasma by ultra-high performance liquid chromatography coupled with diode array detector and its application to a pharmacokinetic study

Abstract

Background: Isoorientin is a C-glycosylflavone and a pharmacologically active ingredient found in various medicinal plants. Objective: The aim of this study is to develop a specific and reproducible ultra-high-performance liquid chromatography-diode array detector (UHPLC-DAD) based method to quantify isoorientin in rat plasma and to apply the devised method to a pharmacokinetic study in rats. Materials and Methods: Simple protein precipitation with methanol was utilized to extract isoorientin and rutin (the internal standard) from rat plasma. Analytes were separated on an UHPLC Phenomenex Luna Omega Polar C18 column (100 mm x 2.1 mm, 1.6 mm) by gradient elution using a mobile phase containing 1% aqueous acetic acid and 100% acetonitrile at the flow rate of 0.25 mL/min. Results: The developed UHPLC-DAD method showed good linearity (R-2 = 0.9993) over the concentration range 20u5000 ng/mL with a lower limit of quantification of 20 ng/mL. Intra- and inter-day precisions were <10.7% and accuracy were within the range of 89.4%u101.3%. The recovery of isoorientin from plasma was in acceptable range (89.4%u95.5%). The devised method was successfully applied to a pharmacokinetic study after a single oral administration of isoorientin to rats. Conclusion: This is the first report of a simple, rapid, and cost-effective UHPLC-DAD-based method for quantifying isoorientin in rat plasma and its application to a pharmacokinetic study. Abbreviations used: UHPLC: Ultra-high-performance liquid chromatography; DAD: Diode array detector; LC-MS/MS: Liquid chromatography-tandem mass spectrometry; LLOQ: Lower limit of quantification; IS: Internal standard; QC: Quality control; RSD: Relative standard deviation; Cmax: The maximum plasma concentration; Tmax: The time point to reach the maximum concentration; AUClast: The area under the curve from time zero to the last measurable point, AUCinf: The area under the curve from time zero to infinity; ke: Terminal elimination rate constant; t1/2: Terminal half-life; CL/F: Oral clearance; Vd/F: Apparent volume of distribution after oral administration; SD: Standard deviation; SPE: Solid-phase extraction.