We developed a high-performance liquid chromatographic assay to measure ceftriaxone in serum, urine, and cerebrospinal fluid. Ion pairing was used because ceftriaxone is a relatively polar compound which is poorly retained on C18 columns in standard reverse-phase high-performance liquid chromatography and which produces trailing peaks in the absence of ion-pairing agents. The mobile phase was a combination of acetonitrile and water (46:54), adjusted to pH 9.0 with 10 mM K2HPO4, which contained 10 mM hexadecyltrimethylammonium bromide as the ion-pairing agent. Moxalactam (200 ,ug/ml) was used as the internal standard. A silica-packed precolumn (3 cm long) was used to prevent rapid deterioration of the analytical column (30 by 0.4 cm) by the alkaline pH of the mobile phase, and it significantly extended the life of the analytical column. The assay was linear with ceftriaxone concentrations of 1 to 250 ,ug/ml (r = 0.999) and correlated well with an agar diffusion bioassay (r = 0.990). Reproducibility was good, with intrarun coefficients of variation from 2.3 to 6.4% and interrun coefficients of variation from 3.2 to 21.4%. The absolute recoveries of ceftriaxone and moxalactam were 91 to 97 and 96 to 98%, respectively. No interferences were observed with more than 40 commonly prescribed drugs, including 10 cephalosporins (cefotaxime, cefoperazone, ceftazidime, ceftizoxime, cefoxitin, cefamandole, cephalothin, cefazolin, cephapirin, and cephalexin), br with sera from patients with renal or hepatic disease.Ceftriaxone is a new parenteral cephalosporin which exhibits potent activity against a variety of gram-negative and gram-positive bacteria (4), excellent penetration into extravascular spaces (7,12), and an increased resistance to degradation by ,B-lactamases (4). In contrast to other thirdgeneration cephalosporins, ceftriaxone possesses a greatly extended elimination half-life, on the order of 6 to 8 h (7). This prolonged half-life has positive implications for cost-effective antimicrobial therapy, allowing less frequent dosing and thus decreasing personnel and supply costs associated with parenteral drug administration both in the hospital (13) and in outpatient settings (9).Although bioassays represent the traditional approach to measuring antimicrobial concentrations in biological fluids, they vary widely in their turnaround time, specificity, and precision (8). In recent years, high-performance liquid chromatographic (HPLC) assays for ceftriaxone have been described which offer faster, more specific, and more reproducible results than bioassays for both pharmacokinetic studies and routine clinical monitoring (1,3,14). In this report, we describe an ion pair reverse-phase HPLC method for measuring ceftriaxone in serum, urine, and cerebrospinal fluid. In contrast to earlier HPLC assays (1, 3), this method incorporates an internal standard and requires a standard C18 reverse-phase column, which is used more frequently in clinical laboratories than are NH2 normal-phase columns (1). Since ceftriaxone is a highly pol...