High-Pressure Liquid Chromatography for Quantitation of Antimicrobial Agents

A high-pressure liquid chromatographic method is described for the analysis of a wide range of cephalosporin congeners, using only three reagents for extraction and drug analysis. Plasma was treated with cold methanol-0.1 M sodium acetate to precipitate protein. Cephalosporins were resolved on a C-18 reverse-phase column, utilizing a mobile phase of various percentages of 0.01 M sodium acetate and acetonitrile-methanol. Compounds analyzed included cephalexin, cefamandole, cephalothin, cefotaxime, cefazolin, cephaloridine, cefoxitin, cefaclor, cephapirin, and cefoperazone. Each antibiotic demonstrated excellent linearity throughout the therapeutic range. The method of standard additions revealed recoveries of 93 to 101%, with detection limits ranging from 0.2 to 1.0 ,ug/ml for these drugs. Retention times ranged from 4 to 6 min. This method offers a rapid and simple means by which this group of cephalosporins may be reliably quantitated.The application of high-pressure liquid chromatography (HPLC) to the analysis of antibiotics introduces a powerful tool for therapeutic drug monitoring as well as clinical research (1). The advantages of short turnaround time, method reliability, method sensitivity, and drug specificity justify the use of HPLC for various groups of antibiotics (8,15).Original reports of cephalosporin quantitation by HPLC typically describe the analysis of a single cephalosporin in biological fluid (2,7,9,10,13,14). Each of these methods presents a unique preparatory and chromatographic protocol for a single cephalosporin or its metabolite or both. The analysis of a wide range of different cephalosporins would be complicated by extensive method changeover of extraction procedure, mobile phase, stationary phase, and UV detection wavelength (6, 15). Research on a large class of compounds such as the cephalosporins would be expedited by the availability of a readily adjustable method of analysis that used a limited number of reagents.With this goal in mind, we describe the development of a reverse-phase HPLC method for the analysis of cephalothin, cephaloridine, cefaclor, cefamandole, cefazolin, cephalexin, cefoperazone, cephapirin, cefoxitin, and cefotaxime in human plasma. MATERIALS AND METHODSCephalosporin standards were prepared fresh in distilled deionized water or 0.45 M phosphate buffer, pH 6.8 (according to the manufacturers' reconstitution instructions), on the day of analysis. Internal standards were prepared fresh daily in absolute methanol at a concentration approximating the mean of the therapeutic range (Tables 1 and 2 All experiments were performed on antibiotic-spiked human plasma from the hospital blood bank. Plasma (0.3 ml) was combined with an equal volume of ice-cold 70% absolute methanol with internal standard-30% 0.1 M sodium acetate, pH 5.2. This mixture was vortexed for 30 s and incubated at -20°C for 10 min. The specimens were centrifuged at 1,500 x g for 10 min, and 10 ,ul of the resulting supernatant was used for analysis.Chromatography was performed at room temperature sin...

“…The advantages of short turnaround time, method reliability, method sensitivity, and drug specificity justify the use of HPLC for various groups of antibiotics (8,15).…”

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confidence: 99%

High-pressure liquid chromatographic method for analysis of cephalosporins

Signs¹,

File²,

Tan³

1984

“…It is a member of the monobactams, naturally occurring 3-lactams produced by bacteria (7). This compound is currently undergoing clinical development, and human pharmacology data have been reported in detail (4,5).The assay of antibiotics in body fluids has traditionally been performed by microbiological assay, but recently high-pressure liquid chromatography (HPLC) has been extensively employed as an alternative procedure (8,11 3.9 mm; length, 3.0 cm). The guard column was packed with Bondapak C18 on Corasil.…”

mentioning

confidence: 99%

“…The assay of antibiotics in body fluids has traditionally been performed by microbiological assay, but recently high-pressure liquid chromatography (HPLC) has been extensively employed as an alternative procedure (8,11 Aztreonam. Aztreonam, prepared in our laboratories, was used as either the dipolar ion form shown in Fig.…”

mentioning

confidence: 99%

High-pressure liquid chromatographic analysis of aztreonam in sera and urine

Pilkiewicz¹,

Remsburg²,

Fisher³

et al. 1983

Aztreonam (SQ 26,776) is a new synthetic monocyclic P-lactam antibiotic which is specifically active against aerobic gram-negative bacteria. High-pressure liquid chromatographic (HPLC) systems were developed for the quantitative analysis of aztreonam in human, monkey, rat, mouse, and rabbit sera and urine. The HPLC conditions employed for these analyses were a ,uBondapak C18 column, a mobile phase made up of 0.005 M tetrabutylammonium hydrogen sulfate at pH 3.0 and acetonitrile or methanol, UV detection at 293 nm, and a flow rate of 2.0 ml/min. For human sera and urine, the mobile phase was 80%o 0.005 M tetrabutylammonium hydrogen sulfate-0.005M (NH4) 2SO4 and 20% acetonitrile (vol/vol). For the range of sera and urine, HPLC analyses were shown to have excellent detector linearity of aztreonam over a concentration range of 1.0 mg/ml to 0.5 ,ug/ml. Correlation coefficients for plots of aztreonam peak area versus its concentration were -0.990. The detection limit of aztreonam was 1.0 jig/ml in sera and 5.0 ,ug/ml in urine. HPLC and microbiological assays of aztreonam in human sera and urine were in good agreement.Aztreonam (SQ 26,776) (Fig. 1) is a totally synthetic monocyclic ,-lactam antibiotic specifically active against aerobic gram-negative bacteria (6). It is a member of the monobactams, naturally occurring 3-lactams produced by bacteria (7). This compound is currently undergoing clinical development, and human pharmacology data have been reported in detail (4,5).The assay of antibiotics in body fluids has traditionally been performed by microbiological assay, but recently high-pressure liquid chromatography (HPLC) has been extensively employed as an alternative procedure (8,11 3.9 mm; length, 3.0 cm). The guard column was packed with Bondapak C18 on Corasil. For studies with both chromatographic systems, the following conditions were employed: a UV detector setting of 293 nm, a solvent flow rate of 2.0 ml/min, and a chart speed of 0.5 cm/min. Quantitation of aztreonam was performed by an external standard method of calculation, and peak area measurements were used.Aztreonam. Aztreonam, prepared in our laboratories, was used as either the dipolar ion form shown in Fig. 1 or the disodium or dipotassium salt formed from the free diacid.

“…Quantitation of antimicrobial agents in serum by high-pressure liquid chromatography (LC) is developing as a rapid, reliable assay method for many compounds (6). An essential step in any LC method for antibiotics in serum or other body fluids is the removal of protein from the sample.…”

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confidence: 99%

“…An essential step in any LC method for antibiotics in serum or other body fluids is the removal of protein from the sample. Deproteinization is usually accomplished by precipitation with acids or organic solvents (6). Ultrafiltration and adsorption to silica gel or to cation-exchange columns (1) have also been used.…”

mentioning

confidence: 99%

Anion-exchange extraction of cephapirin, cefotaxime, and cefoxitin from serum for liquid chromatography

Fasching¹,

Peterson²

1982

An anion-exchange column technique for extraction of antibiotics from serum proteins has been developed for use in the assay of cephapirin, cefotaxime, and cefoxitin by high-pressure liquid chromatography. Anion-exchange extraction of cephapirin from serum samples by this technique was compared with protein precipitation methods, using 6% trichloroacetic acid or absolute ethanol. Column extraction gave improved quantitative drug recovery and reduced background serum interferences in the resultant chromatograms when evaluated against protein precipitation. Comparisons of this method with microbiological assay gave statistically equivalent results. Twelve patient samples were assayed for cephapirin, and no interferences were encountered from the 22 systemic agents these subjects were receiving. The anion-exchange technique for antibiotic extraction provides a rapid, precise, and quantitative antibiotic assay when used with liquid chromatography.Quantitation of antimicrobial agents in serum by high-pressure liquid chromatography (LC) is developing as a rapid, reliable assay method for many compounds (6). An essential step in any LC method for antibiotics in serum or other body fluids is the removal of protein from the sample. Deproteinization is usually accomplished by precipitation with acids or organic solvents (6). Ultrafiltration and adsorption to silica gel or to cation-exchange columns (1) have also been used.Extraction procedures should be rapid and simple and need to provide quantitative drug recovery. Anhalt (1) has found excellent results with carboxymethyl Sephadex cation-exchange columns for protein removal in the assay of aminoglycosides. The purposes of this report were (i) to describe the use of anion-exchange columns with DEAE-A-25 Sephadex for extraction of cephapirin, cefotaxime, and cefoxitin from human serum; (ii) to compare the results obtained by this column extraction method and LC with results obtained by bioassay; and (iii) to compare LC results with an internal and an external standard.MATERIALS AND METHODS Antibiotic extraction technique. Anion-exchange columns were prepared by using 6.0-ml syringe barrels (Monoject, St. Louis, Mo.) with a 12.7-mm filter paper disk (Schleicher & Schuell, Inc., Keene, N.H.) placed to retain Sephadex. DEAE-A-25 Sephadex (Pharmacia Fine Chemicals, Inc., Piscataway, N.J.) in pH 7.2 phosphate-buffered saline (8.0 g NaCl, 1.15 g Na2HPO4, 0.2 g KCl, and 0.2 g KH2PO4 dissolved in 1 liter of distilled water) was used to ifil the syringe barrels, which were subsequently packed to a bed volume of 3.0 ml, and this was washed with phosphate-buffered saline. Another 12.7-mm filter paper disk was added to the top of the column to prevent the bed from being disturbed when samples or reagents were added. After column preparation, 0.5 ml of a serum sample was placed on the column, followed by 0.5 ml of phosphate-buffered saline. Protein and other serum interferences were removed by addition of 4.0 ml of phosphate-buffered saline pH 7.2. The antibiotics retained on the Seph...