High-Pressure Liquid Chromatographic Assay of Netilmicin in Plasma

Abstract

A high-pressure liquid chromatographic method for the quantitative determination of netilmicin in plasma was developed. The procedures involve acetonitrile protein precipitation, methylene chloride extraction, and dansylation to form the fluorescent dansyl derivative of netilmicin, which is extracted into ethyl acetate and chromatographed on a reverse-phase column with aqueous acetonitrile as the mobile phase. A good linear relationship between peak height measurements and netilmicin concentrations was found. This method is sensitive and reproducible; a netilmicin concentration as low as 0.5 Ag/ml can be measured with only 0.1 ml of plasma sample. The results of assays ofplasma or serum samples by this high-pressure liquid chromatographic method correlate well with those obtained by microbiological assays.Netilmicin is a new semisynthetic derivative of sisomicin. Its antimicrobial spectrum and activity are similar to those of other structurally related aminoglycosidic antibiotics such as gentamicin, sisomicin, and tobramycin (5,6,9). Although compared with gentamicin, netilmicin demonstrated lower nephrotoxicity in animals (3, 4) and less variation in peak levels after intramuscular administration in humans (7), it has potential for plasma level variations and oto-and nephrotoxicity. We wish to report a high-pressure liquid chromatographic method for determining plasma or body fluid levels of netilmicin. Such an assay could be useful in dosage regimen adjustments, in maintaining therapeutic levels in plasma, in averting untoward reactions, and in obtaining useful pharmacokinetic data. Similar benefits have been derived from the assays of gentamicin in plasma (2, 8). MATERIALS AND METHODSNetilmicin was obtained from Schering Corp., Bloomfield, N.J. 5-Dimethylamino-1-naphthalene sulfonyl chloride (dansyl chloride) was purchased from Sigma Chemical Co., St. Louis, Mo. Glassdistilled acetonitrile, methylene chloride, and ethyl acetate were from Burdick and Jackson Laboratory, Muskegon, Mich. Sodium carbonate, sodium bicarbonate, and phosphoric acid were reagent grade from Fisher Scientific Co., Fair Lawn, N.J.Sample preparation. Portions (0.1 ml) of plasma samples were diluted with 0.9 ml of phosphate buffer (0.1 M, alkalinized with sodium hydroxide to pH 11), placed in culture tubes (13 by 100 mm) containing 2.5 ml of acetonitrile, blended in a Vortex mixer, and centrifuged. The supernatant solutions were poured into new tubes, blended in a Vortex mixer with 2 ml of methylene chloride, and centrifuged at 2,000 rpm for about 1 min. Portions (0.5 ml) of the separated aqueous (upper) layer were transferred to new, screw-cap culture tubes containing 4 mg of dansyl chloride in 0.3 ml of acetonitrile.These tubes were closed and incubated in a water bath at 75°C for 5 min. The reaction mixtures were cooled in ice-water, and 0.5 ml of ethyl acetate and 6 ml of carbonate buffer (containing 0.5 M sodium carbonate and bicarbonate, pH 9.5) were added. After Vortex mixing and centrifugation, 1-to 5-,l portions of the organic (u...

High-pressure liquid chromatography analysis and single-dose disposition of tobramycin in human volunteers

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“…limits the applicability of these methods for routine clinical use (1, 2). Others have employed derivatization of aminoglycosides in serum and quantitation by measuring the peak height or peak area of the desired derivative (6,7,10 …”

“…For limits the applicability of these methods for routine clinical use (1,2). Others have employed derivatization of aminoglycosides in serum and quantitation by measuring the peak height or peak area of the desired derivative (6,7,10 ) and a guard column packed with CO: Pell ODS (Whatman, Inc., Clifton, N.J.) were also used. The flow rate was 1.6 ml/min at ambient temperature.…”

High-pressure liquid chromatographic method for determination of Sch 29482 in human serum

“…RESULTS AND DISCUSSION Recently, HPLC methods have been used widely to determine the plasma and serum concentrations of antibiotics such as netilmicin (6) Figure 1 shows the typical liquid chromatogram of Sch 29482 and the internal standard (m-propionamidophenol) extracted from control serum and from the serum spiked with Sch 29482. The retention times for Sch 29482 and the internal standard were 8 and 10 min, respectively.…”

Amikacin Assay in Serum by High-Performance Liquid Chromatography

“…J. Clin. Pathol., in press), netilmicin (10), and a variety of other antimicrobial agents (T. T. Yoshikawa, S. K. Maitra, M. C. Schotz, and L. B. Guze, in G. L. Hawk, ed., Biological/Biomedical Applications of Liquid Chromatography, in press) has been reported. In this report, we describe the application of HPLC for the separation and detection of amikacin.…”