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Abstract
On-line coupled immunoaffinity chromatography and reversed-phase high performance liquid chromatography (on-line IAC-HPLC) with detection by radioimmunoassay (RIA) was developed for 4-[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthyl)-carbamoyl] benzoic acid (Am-80) in human plasma. A 0.05-mL sample was directly loaded onto an immunoaffinity pre-column packed with immobilized polyclonal antibodies against Am-80. The immunoaffinity extract was then automatically introduced to reversed-phase HPLC column for separation by column switching. The immunoreactivity in the HPLC-eluted fraction was measured by competitive RIA.
The within-assay precision and accuracy of the method were 4.0–10.6% and −6.0–+8.3%, respectively, and between-assay precision and accuracy were 6.8–11.9% and −16.0–+9.0%, respectively. The limit of quantitation was 0.5 ng/mL in human plasma. The proposed method yields highly purified analyte in complex matrices, detects it with high sensitivity, not only by radioimmunoassay, but also by other methods, such as mass-spectrometry, and can be used for routine assay of many samples without manual sample extraction and purification.