Direct-injection HPLC assay for the determination of a new carbapenem antibiotic in human plasma and urine.

Abstract Reversed-phase high-performance liquid chromatography (RP-HPLC) assays using ultraviolet (UV) absorbance detection have been developed for the determination of a new carbapenem antibiotic I in human plasma and urine. A column-switching technique is employed in the HPLC methods to perform on-line extraction and separation for each sample. Each plasma sample is thawed, centrifuged, stabilized, and then injected onto an in-line reversed-phase extraction column using a methanol (8%)/phosphate buffer, pH 6.5. After 3 min, the analytes are back-flushed off the extraction column with a mixture of acetonitrile (5.5%) and methanol (10%)/phosphate buffer (pH 6.5) for 3 min onto a BDS Hypersil 3 μm C18 (100×4.6 mm i.d.) analytical column. The sample preparation and HPLC conditions for the urine assay are similar to the plasma assay, except that a CN extraction column is used. Both assays are specific with respect to endogenous material and the major metabolite II , and both are linear over the concentration range of 0.25–50, and 2–200 μg/ml, respectively. The assays were successfully applied to a clinical dose-ranging study. One limitation of the on-line extraction method is that the extraction column needs to be replaced regularly every 100–150 plasma samples and every 200–300 urine samples. Subsequently, the urine method was modified to an ion-pair HPLC assay for the simultaneous determination of both the antibiotic I and its metabolite II .