Determination of the cephalosporin Ro 13-99041 in plasma,urine, and bile by means of ion-pair reversed phase chromatography

An HPLC method has been developed for the determination of the cephalosporin antibiotic Ro 13-9904 in plasma, urine, and bile of dogs and of human volunteers using the technique of ion-pair chromatography with a LiChrosorb RP-18 column. The three mobile phases employed contained tetrapentyl-, tetraoctyl- and hexadecyltrimethyl-ammonium bromide, respectively, as lipophilic counterions. The chromatographic conditions chosen allowed simple and rapid sample preparation. Plasma was deproteinized with ethanol and the supernatant was directly injected onto the column; urine and bile were diluted with mobile phase and injected without any purification. The detection limit for the cephalosporin was about 0.5 μg/ml for plasma samples and approximately 5 μg/ml for bile and urine.