Measurement of amoxicillin in plasma and gastric samples using high-performance liquid chromatography with fluorimetric detection

Abstract

A rapid, selective and sensitive HPLC assay has been developed for the routine analysis of amoxicillin in rat plasma, gastric juice aspirate and gastric tissue which is applicable to low concentrations of amoxicillin (<1 μg mL −1) or small sample volumes. Amoxicillin was converted, via an internal rearrangement, to form a fluorescent product which was subsequently recovered using liquid–liquid extraction. A Kromasil ODS 3 μm (150×3.2 mm I.D.) column was maintained at 40 °C and used with a mobile phase consisting of methanol–water (55:45, v/v). Fluorimetric detection was at an λ ex of 365 nm and an λ em of 445 nm. The limits of quantitation for amoxicillin were 0.1 μg mL −1 for gastric juice aspirate (500 μL), 0.5 μg mL −1 for plasma (50 μL) and 0.075 μg g −1 for gastric tissue (250 mg). The method was linear up to at least 15 μg mL −1 in gastric juice aspirate, up to 200 μg mL −1 in plasma and up to 100 μg g −1 in gastric tissue, with inter- and intra-day RSDs being less than 19%. The assay has been applied to the measurement of amoxicillin in rat plasma, gastric juice aspirate and gastric tissue for pharmacokinetic studies in individual rats.