Determination of rifabutin by high-performance liquid chromatography using on-line concentration and column switching

A simple HPLC method has been developed that allows the sensitive determination of rifabutin (RBT) in human serum using on-line concentration and column switching. After pretreatment of the serum with acetonitrile and centrifugation, the samples were applied to a concentration column (CC) (Zorbax CN). Washing with phosphate buffer-methanol removed most of the contaminating substances. Via a six-port valve the CC was switched to the analytical mode. RBT was separated on a Chromspher RP 8 column (acetonitrile-phosphate buffer pH 7.4/sodium chloride) and determined photometrically at 278 nm. The lower limit of quantification for 200 microliters serum precipitated with 200 microliters acetonitrile and after injection of 2 x 150 microliters was 33 micrograms/l and linearity was observed up to 27 mg/l. Different modes of sample application (single, repeated, and different injection volume portions), as well as washing time, cycle time and different CC materials were investigated.