Column liquid chromatographic determination of bupivacaine in human serum using solid-phase extraction

A convenient and economical procedure for the extraction of bupivacaine and its major metabolites, desbutylbupivacaine and 4'-hydroxybupivacaine, from serum is described. A 0.25-ml aliquot of 4 micrograms/ml solution of N-pentyl-2,6-pipecoloxylidide used as the internal standard and an equal volume of the serum sample are applied to a 1-ml BondElut C18 silica extraction column which has been conditioned by subsequent washing once with 1 M HCl, twice with methanol and once with water. After passing the sample at a slow rate, the column is washed twice with water and once with acetonitrile. The desired compounds are then eluted with a 0.25-ml aliquot of a mixture of 35% perchloric acid-methanol (1:40, v/v). A 5-microliters aliquot of the eluate is injected onto a 150 x 4.6 mm I.D. column packed with 5-microns C8 silica particles, which is eluted at ambient temperature with a mobile phase of acetonitrile-10 mM KH2PO4 (25:80, v/v) at a flow-rate of 1.5 ml/min. The peaks are detected at 205 nm. The extraction recovery of all the compounds is in the range of 90%. The chromatogram is clean and the desired peaks are well separated from each other and from the extraneous peaks.