The use of high-performance liquid chromatography (HPLC) to measure ribavirin in serum and other biological fluids has been limited by endogenous interfering substances. We report an HPLC procedure based on the extraction of ribavirin from serum, plasma, or cerebrospinal fluid with a boronate affinity gel, which uses a 3-methylcytidine internal standard. This assay is sensitive (to 0.4 i,M), specific (no interference with 34 commonly prescribed drugs), reproducible (coefficients of variation from 5.4 to 22.4%), and linear (r = 0.999) over the range of clinically relevant concentrations in serum (from 0.5 to 50.0 ,uM). It also correlates well with the ribavirin radioimmunoassay (r = 0.992). This HPLC assay should be useful for measuring ribavirin in serum and other body fluids during clinical trials.Ribavirin (1-,-D-ribofuranosyl-1,2,4-triazole-3-carboxamide; Virazole) is a purine nucleoside analog which resembles guanosine (13). It is active in vitro against a wide variety of DNA and RNA viruses (9,18). Controlled in vivo studies have shown that ribavirin is effective against respiratory syncytial virus (8, 16), influenza A and B virus (7), and Lassa fever virus (11) infections. Ribavirin is being studied currently in clinical trials to further evaluate its activity against human immunodeficiency virus infections (5). Oral ribavirin also has been employed in the treatment of measles (2) and acute hepatitis A (17) infections.Hematological toxicity, consisting of intravascular erythrocyte losses and reticulocytosis, has been associated with the use of oral ribavirin. Headache, insomnia, fatigue, and exertional dyspnea have been reported in patients receiving large oral or parenteral doses (10,11,14). These and other observations suggest that ribavirin toxicity is dose related and reversible. Sites of toxicity include circulating erythrocytes, the bone marrow, and the central nervous system.Methods available currently for quantitating ribavirin in serum or plasma include bioassay (19), radioimmunoassay (RIA) (1), gas chromatography-mass spectroscopy (15), and high-performance liquid chromatography (HPLC) (3, 20). The use of HPLC to measure ribavirin has advantages over bioassay and gas chromatography-mass spectroscopy in terms of speed (turnaround time) and ease of performance. HPLC does not require the use of radioisotopes, as does the RIA. In this report we describe a rapid, sensitive, and specific HPLC assay for ribavirin in serum, plasma, and cerebrospinal fluid. This technique is potentially applicable to both pharmacokinetic studies and routine clinical assays. A stock solution of ribavirin, corrected for drug potency, was prepared at a concentration of 1.0 mM in distilled water and stored at -70°C until needed. Reference samples containing 0.5, 1.0, 10.0, 30.0, and 50.0 ,uM concentrations of ribavirin were prepared by diluting this stock solution with drug-free pooled human sera. These reference samples were used to assess intrarun and interrun precision. A single calibration standard was prepared in pooled ...

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...

We compared the precision and accuracy of five methods used to measure the concentration of vancomycin in serum: bioassay, high-pressure liquid chromatography, fluorescence polarization immunoassay (FPIA [TDX; Abbott Laboratories, North Chicago, Ill.]), radioimmunoassay (RIA), and fluorescence immunoassay. Based on an analysis of seven standards and of 106 patient samples, all five methods were accurate, and four (bioassay, high-pressure liquid chromatography, FPIA, and RIA) were also precise. The FPIA was the most precise and the fluorescence immunoassay was the least precise of the methods tested; intrarun coefficients of variation for these two methods were 0.9 to 3.0% versus 8.9 to 14.5%, and interrun coefficients of variation were 2.8 to 8.1 % versus 12.2 to 16.2%, respectively. The RIA was inconvenient because it required an extra dilution of the specimen being tested and an additional (64 ,ug/ml) vancomycin standard for specimens with 32 to 64 ,ug of vancomycin per ml. Based on its rapid turnaround time and the stability of its standard curve, we believe that the FPIA is the best method currently available to quantitate vancomycin in the clinical laboratory.

We developed a rapid, sensitive, high-pressure liquid chromatographic (HPLC) procedure which incorporates a commercially available internal standard, 1-amino-4-nitronaphthalene, to measure amphotericin B in serum. Recovery was quantitative (.90%), and the standard curve was linear from 0.04 to at least 10.0 ,ug/ml. The reproducibility of the assay was good, with intrarun coefficients of variation from 2.0 to 6.8% and interrun coefficients of variation from 4.9 to 10.0%. Comparison by linear regression analysis of the HPLC assay with an agar well diffusion bioassay gave a correlation coefficient of 0.942, with the HPLC assay exhibiting greater precision and sensitivity. No interference was encountered from over 20 drugs and three amphotericin B analogs. However, serum specimens that contained high concentrations of conjugated bilirubin (>3 mg/dl) produced interfering peaks in both this assay and other previously reported HPLC assays for amphotericin B. We also describe a solid-phase extraction procedure which effectively removes this interference and uses an alternative internal standard (N-acetyl amphotericin B).Amphotericin B is a polyene antifungal agent that is presently the drug of choice for the treatment of most severe systemic fungal infections. Use of the drug, however, is limited by its toxicity, particularly by its impairment of renal function. The pharmacokinetics of amphotericin B are complex, involving a rapid initial decrease in serum levels for approximately 24 h, followed by a long (15-day half-life) terminal elimination phase (1,4,8). Other pharmacokinetic properties, such as elimination pathways, are not completely understood. Renal and biliary excretion have each been estimated to account for up to 20% of a dose of amphotericin B (6, 16). The lack of a clearly defined relationship between serum levels and toxicity or clinical outcome has prevented the development of a rational, systematic approach to amphotericin B therapy.Many of the problems associated with pharmacokinetic studies of amphotericin B arise from difficulty in obtaining rapid, accurate, and reproducible measurements of drug levels in various biological fluids. Most researchers have used bioassay systems that involve either serial tube dilution or plate diffusion with a variety of indicator organisms, including Paecilomyces varioti, Candida tropicalis, and Saccharomyces cerevisiae (2-4, 6-8, 17-19). Bioassays, however, vary widely in their accuracy, precision, sensitivity, and specificity, making the comparison of data from different papers difficult.Several high-pressure liquid chromatographic (HPLC) assays have been reported recently which offer faster and more accurate and reproducible alternatives to bioassays for both pharmacokinetic studies and routine clinical use (9, 10, 13-15, 17, 20). HPLC also offers improved sensitivity and specificity and is easier to standardize than bioassays are. In this report we describe an HPLC method which incorporates an internal standard for the measurement of amphotericin B in serum and a...

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