New and rapid fully automated method for determination of tazobactam and piperacillin in fatty tissue and serum by column-switching liquid chromatography

A sensitive and rapid HPLC assay for determining tazobactam and piperacillin in fatty tissue and serum is described. While the common methods need liquid–liquid extraction before the injection in a automated column switching HPLC, the new method works by direct injection of the filtered tissue extract or diluted serum in a automated column switching HPLC without any other pre-treatment. This was performed by the use of a NH2-precolumn and enrichment/transfer at different pH-level. During the analyses, the NH2-precolumn was automatically regenerated with acetonitrile–water. The chromatogram peaks for piperacillin and tazobactam were identified by the retention time and quantified by peak area. The calibration curve was linear between 1 and 16 μg/ml. The quantification limit of tazobactam was about 1 μg/ml in fatty tissue extracts and in diluted serum (calculated for pure serum 2 μg/ml), respectively. For piperacillin it was less. The described procedure allows sample clean-up and determination of the antibiotic within 35 min. The chromatograms with this easy sample treatment had the same quantity of matrix peaks and in contrast to liquid–liquid extraction no loss of piperacillin. Because of the automatically rinsing of the NH2-precolumn during the chromatographic separation, more than 50 different biological samples could be measured with one NH2-precolumn without loss of performance.