Improved Micromethod for Mezlocillin Quantitation in Serum and Urine ...

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Aug 14, 1984 - ureidopenicillins azlocillin, mezlocillin and Bay K4999 in plasma by high performance liquid chromatography. Br. J. Pharmacol. 8:589-592. 5.
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Nov. 1984, p. 775-777

Vol. 26, No. 5

0066-4804/84/110775-03$02.00/0 Copyright C 1984, American Society for Microbiology

Improved Micromethod for Mezlocillin Quantitation in Serum and Urine by High-Pressure Liquid Chromatography DARIO FIORE,* FRANCOIS A. AUGER, GEORGE L. DRUSANO, VIJAY R. DANDU, AND LAWRENCE J. LESKO Clinical Pharmacokinetics Laboratory, School of Pharmacy, Department of Pharmaceutics, University of Maryland at Baltimore, Baltimore, Maryland 21201 Received 11 May 1984/Accepted 14 August 1984 A rapid, sensitive, and specific method of analysis for mezlocillin in serum and urine by high-pressure liquid chromatography is described. A solid-phase extraction column was used to remove interfering substances from samples before chromatography. Quantitation included the use of an internal standard, nafcillin. Mezlocillin was chromatographed with a phosphate buffer-acetonitrile (73:27) mobile phase and a C-18 reverse-phase column and detected at a wavelength of 220 nm. The assay had a sensitivity of 1.6 ,ug/ml and a linearity of up to 600 ,ug/ml and 16 mg/ml in serum and urine, respectively, with only 0.1 ml of sample. The interday and intraday coefficients of variation for replicate analyses of spiked serum and urine specimens were less than 6.5%.

stock solution of 73 mg/ml to contain 0.5, 1.0, 3.0, 6.0, 9.2, or 16.2 mg of mezlocillin per ml. Portions of the serum (0.25 ml) and urine (0.25 ml after 1:10 dilution with buffer) standards were stored at -25°C in polypropylene tubes, where they were stable for at least 3 weeks. A volume of 0.10 ml of serum or urine (after 1:10 dilution with buffer) was added to a 0.50-ml disposable polypropylene tube containing 0.10 ml of internal standard solution and was then vortexed for 10 s. The mixture was then transferred to a solid-phase extraction column (Bondelut; catalog no. 607101, Analytichem International, Harbor City, Calif.) supported in a Vac-elut (Analytichem International) 10-sample vacuum manifold; the tube had been activated by flushing with two 1.0-ml volumes of CH3CN followed by 1.0 ml of buffer. After the sample was loaded, the column was flushed with 1.0 ml of buffer and 0.5 ml of 15% CH3CN in buffer, which were then discarded. The sample was eluted with 0.50 ml of 70% CH3CN in buffer into 1.5-ml disposable polypropylene tubes. A 10-pl portion of the final eluant was analyzed by HPLC assay. Samples were analyzed with an HPLC system consisting of an automatic prograrmmable sample injector (model 710B), a dual-piston reciprocating pump (model 6000A), and a variable-wavelength detector (model 480), all from Waters Associates, Milford, Mass. The HPLC column was a 0.4- by 30-cm ,uBondapak C-18 (Waters Associates) preceded by a 0.4- by 4.5-cm guard column which had been slurry packed with Spherisorb 10-p.m C-18 packing material (Phase Separations, Hauppauge, N.Y.). The mobile phase, CH3CN-0.05 M potassium dihydrogen phosphate, pH 7.0 (27:73, vol/vol), was delivered at 1.1 ml/min, and the eluant was monitored at 220 nm (6). The detector output was recorded by an integrator-plotter (model 3390A; Hewlett Packard Co., Baltimore, Md.) at a chart speed of 0.2 cm/min and at attenuations of 5 for serum and 6 for urine. Retention times were 4.2 min for mezlocillin and 7.5 min for nafcillin. Mezlocillin was quantitated by the peak height ratio (mezlocillin/nafcillin) with a least-squares linear regression analysis of a standard curve plotted daily with the unknown samples. Intraday assay precision was determined by analysis in triplicate of standard curves for mezlocillin in serum and urine. Interday precision was determined by analysis of medium, low, and high mezlocillin concentration controls

Mezlocillin is a relatively new member of the ureidopenicillin class of antibiotics. It possesses a broader spectrum of activity against aerobic and anaerobic bacteria than the older semisynthetic penicillins. The pharmacokinetics of mezlocillin must be understood to design dosage regimens which provide drug concentrations in serum above the MIC for the target organism. In addition, it may be important in some instances to determine the mezlocillin concentration at or near the site of infection, for example, in urine or cerebrospinal fluid (1, 2, 7, 8). High-pressure liquid chromatographic (HPLC) assays are frequently used for pharmacokinetic studies of antibiotics because of their specificity, sensitivity, speed, and range of linearity. A number of HPLC methods for determination of mezlocillin levels have been described previously (3-6). One method requires a time-consuming extraction step and results in poor recovery (