Determination of Amide-type Local Anaesthetics by Direct Injection of Plasma in a Column-switching High-performance Liquid Chromatographic System Using a Pre-column with a Semipermeable Surface

A high-performance liquid chromatographic method using column switching was applied to the direct determination of two local anaesthetics, ropivacaine and bupivacaine, in human plasma. The method is intended to be used in a combined LC-GC system; here only the LC-part is described. After addition of internal standard, the samples were filtered and directly injected into a semipermeable surface (SPS) pre-column where the analytes were strongly retained and separated from many endogenous compounds by a short washing step. The retained analytes were transferred by a buffered methanol phase from the pre-column into a carbonaceous HPLC column and they were detected by UV detection at 254 nm. The SPS pre-column could withstand numerous (> 200) direct injections of plasma samples (10 microliters). The method has a detection limit of 8.2 ng and requires a total assay time of 15 min per plasma sample. Quantitative recoveries were obtained over the range 3.3-114 micrograms/ml with inter-day precisions of 1.6-5.2% (C.V.).