Column-switching technique for the sensitive determination of ertapenem in human cerebrospinal fluid using liquid chromatography and ultraviolet absorbance detection

Abstract

A sensitive reversed-phase high-performance liquid chromatographic (RP-HPLC) assay with on-line extraction was developed for quantifying ertapenem in human cerebrospinal fluid (CSF). This assay is at least five times more sensitive than previously published ertapenem methods with a lower limit of quantitation at 0.025 μg/ml. In this assay, a CSF sample is extracted on-line using a RP extraction column and an aqueous acidic mobile phase (0.1% formic acid) to wash away polar endogenous materials, while ertapenem is retained on the column. Ertapenem is then back-flushed off the extraction column and directed to a RP analytical column using an acidic mobile phase with an organic modifier (acetonitrile/0.1% formic acid, 15:85 (v/v)) and detected using UV absorbance. The acidic mobile phase provided a sharper chromatographic peak and on-line extraction allowed large injection volumes (≥150 μl) of buffered CSF to be injected without compromising column integrity. These assay conditions were necessary to quantify ertapenem at levels expected to be found in human CSF (<0.05 μg/ml). The method was successfully validated and implemented for a clinical study: intraday precision and accuracy of the CSF assay for calibration standards (0.025–10 μg/ml) and quality control samples (0.1, 0.5, and 2.5 μg/ml) were <6.2% coefficient of variation and 96.8–104.0% of nominal concentration, respectively.