Determination of a new orally active cephalosporin in human plasma and urine by high-performance liquid chromatography using automated column switching.

A sensitive method for the determination of a new cephalosporin in human serum and urine is described. The sensitivity of the procedure is derived from a high-performance liquid chromatographic separation which utilizes the different selectivities of two columns. Partial separation of the agent from deproteinized serum or diluted urine is achieved by an anion-exchange column. To concentrate the large volume of the eluent fraction containing the compound from the anion-exchange column, a reversed-phase short column is placed between the anion-exchange column and a reversed-phase analytical column. The separation is completed by switching the eluent fraction containing the compound from the second column to the analytical column. The compound is detected by ultraviolet absorption at 295 nm. Quantitation is possible down to 0.05 microgram/ml using 300 microliter of serum and down to 0.5 microgram/ml using 50 microliter of urine. The coefficients of variation of the method are 6.8% and 0.6% in serum when spiked at the 0.05 microgram/ml and 1.0 microgram/ml level, respectively. One assay can be completed in 16 min. Serum levels and urinary excretion data obtained with this method are given for three healthy volunteers who had received a 100-mg oral dose of the compound.