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 microg/ml. The quantification limit of tazobactam was about 1 microg/ml in fatty tissue extracts and in diluted serum (calculated for pure serum 2 microg/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.