Sensitive assay for measuring amoxicillin in human plasma and middle ear fluid using solid-phase extraction and reversed-phase high-performance liquid chromatography.

We developed a sensitive assay to measure amoxicillin in human plasma and middle ear fluid (MEF) using solid-phase extraction and reversed-phase HPLC. Amoxicillin and cefadroxil, the internal standard, were extracted from 50-200 microliters of sample with Bond Elut C18 cartridges. The extract was analyzed on a 15 cm x 2 mm, 5 micron Keystone MOS Hypersil-1 (C8) column with UV detection at 210 nm. The mobile phase was 6% acetonitrile in 5 mM phosphate buffer (pH = 6.5) and 5 mM tetrabutylammonium. The average absolute recovery of amoxicillin and cefadroxil were 91.2 +/- 16.6% and 91.0 +/- 6.8%, respectively. The limit of quantitation was 0.125 microgram/ml with 200 microliters sample size. The linear range was from 0.125 to 35.0 micrograms/ml with correlation coefficients greater than 0.999. These analytic conditions produced a highly sensitive amoxicillin assay in human body fluids without derivatization.