An on-line clean-up procedure for large sample volume analysis of serum aminoglycoside antibiotics by reversed-phase high-performance liquid chromatography.

Abstract

A reversed-phase (RP) liquid chromatography procedure using column switching was applied to assay one of the aminoglycoside antibiotics, sisomicin. The drug in serum was derivatized with o-phthalaldehyde (OPA) and β-mercaptopropionic acid (β-MP) in 0.05M borate buffer (pH 9.0), then directly injected onto a RP pre-column (ODS-silica, 50×4 mm i.d.) to remove protein and interfering compounds (confirmed by ultraviolet monitoring at 280nm). The sisomicin derivatives retained on the pre-column were desorbed by back-flushing onto an analytical column (5μm ODS-silica 200×4mm i.d.) with 0.05M potassium dihydrogen phosphate-0.05M disodium hydrogen phosphate buffer (pH 7) and were separated by elution with a mixture of methanol, ethylene glycol and counter ion solution (sodium 1-heptanesulfonate/acetic acid/water, 2.5g/42ml/208ml). This system could be utilized for more than 50 samples with a 1ml injection volume. A good linear response curve was found between the injected sample amounts and the peak heights for the concentration range of 0.5 to 5μg/ml of sisomicin. The limit of detection for sisomicin in serum was 62.5.ng/ml for 0.1ml of sample. The mean recovery from serum was 97.5%.