Abstract
A fully automated on-line solid-phase extraction (SPE) and high-performance liquid chromatography (HPLC) with diode array detection (DAD) method was developed for determination of bavachinin in mouse plasma. Analytical process was performed on two reversed-phase columns (SPE cartridge and analytical column) connected via a Valco 6-port switching valve. Plasma samples (10μL) were injected directly onto a C18 SPE cartridge (MF Ph-1 C18, 10mm×4mm, 5μm) and the biological matrix was washed out for 2min with the loading solvent (5mM NaH2PO4 buffer, pH 3.5) at a flow rate of 1mL/min. By rotation of the switching valve, bavachinin was eluted from the SPE cartridge in the back-flush mode and transferred to the analytical column (Venusil MP C18, 4.6mm×150mm, 5μm) by the chromatographic mobile phase consisted of acetonitrile-5mM NaH2PO4 buffer 65/35 (v/v, pH 3.5) at a flow rate of 1mL/min. The complete cycle of the on-line SPE purification and chromatographic separation of the analyte was 13min with UV detection performed at 236nm. Calibration curve with good linearity (r=0.9997) was obtained in the range of 20–4000ng/mL in mouse plasma. The intra-day and inter-day precisions (RSD) of bavachinin were in the range of 0.20–2.32% and the accuracies were between 98.47% and 102.95%. The lower limit of quantification (LLOQ) of the assay was 20ng/mL. In conclusion, the established automated on-line SPE-HPLC-DAD method demonstrated good performance in terms of linearity, specificity, detection and quantification limits, precision and accuracy, and was successfully utilized to quantify bavachinin in mouse plasma to support the pharmacokinetic (PK) studies. The PK properties of bavachinin were characterized as rapid oral absorption, high clearance, and poor absolute bioavailability.
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