Direct injection of large volumes of plasma in a column-switching system for the analysis of local anaesthetics. II. Determination of bupivacaine in human plasma with an alkyldiol silica precolumn.

A column-switching high-performance liquid chromatographic system was applied for the determination of bupivacaine in plasma. A 500-microliter plasma sample was directly introduced onto a C18-alkyl-diol silica (ADS) precolumn separating analytes from proteins and polar endogenous compounds. The fraction containing bupivacaine and ropivacaine (internal standard) was back-flushed and transferred to a conventional reversed-phase column (Kromasil C18) for final separation. A single ADS precolumn could withstand more than 50 ml of plasma injections without changing analytical performance. Quantitative studies showed a broad range of linearity (0.033-3.31 micrograms/ml) and high recovery (95-99.9%) with coefficients of variation less than 3.1%. The advantages of the ADS material are its high capability of sample clean-up, due to rapid elution of plasma proteins and endogenous compounds to waste, and its ability to elicit a stable baseline. As a result, UV detection could be performed at 210 nm and clean chromatograms with baseline separation for desired peaks were obtained within 15 min. The detection limit of this system was 10 ng/ml defined by a signal-to-noise ratio of 3:1. The concentration of bupivacaine in patients determined by this method agreed well with the values obtained from an alternative method, making the technique applicable for pharmacokinetic studies in humans.