Liquid chromatographic assay of antibiotics

C I l n l"c a l M"lCrO b lology

Newsletter Copyright © 1981 by G. K. Hall & Co.

December I, 1981

ISSN 0196-4399

Liquid Chromatographic Assay o f Antibiotics John P. Anhalt Department of Laboratory Medicine Mayo Clinic and Mayo Foundation Rochester, Minnesota 55901 Liquid chromatography has become established during the past decade as a powerful analytic technique for separation, identification, and quantitation of drugs and drug nSetabolites in biologic fluids. Implementation of this technique for antibiotic assays in clinical laboratories is just beginning and will probably grow as the need to provide more specific and rapid assays is established. The published methods for antibiotic assays include virtually every antibiotic that is used clinically, and for many drugs, several methodologies have been proposed (2, 5). I hope to give an overview that will help those who are just beginning to choose from among the many published methods a methodology to establish in their own laboratory.

Theory Liquid chromatography is basically a method for separating complex mixtures. Separation occurs as result of differences in the interactions of the molecules of each component in the sample with a mobile phase and a stationary phase. The mobile phase is usually a simple mixture of methanol or acetonitrile with water or an aqueous buffer. The stationary phase is typically a C, or C~, hydrocarbon chemically bonded to a microparticulate silica support. The support particles average 5-10/~m in diameter and are densely packed into a stainless steel column with an internal bore of

2-5 mm and a length of 10-25 cm. The combination of a mobile phase that is more polar than the relatively nonpolar stationary phase is termed "reversed-phase" chromatography. The mobile phase is forced through the column at a flow rate of 1-3 ml per minute, which requires pressures in excess of 1000 lb/inL A solution to be analyzed (20-100 tal) is introduced into the mobile phase from an injector valve at one end of the column. The mobile phase carries the sample components through the column and into a detector. The effluent from the detector is usually directed to waste, but a device for collecting the separated components can be used. Components of a mixture that are retarded by the column to different extents will be separated and detected at different times. The retention time of a particular compound is characteristic, highly reproducible, and forms the basis for chromatographic identification of compounds in a mixture. Horvath and coworkers (7) have described retention behavior on reversed-phase columns as being due to "solvophobic" interactions (i.e., repulsion by the mobile phase forcing sample molecules to be retained on the stationary phase). Alternative theories that relate retention to other mechanisms have been proposed (4). Empirically, the more nonpolar a compound, the longer it will be retained by a reversed-phase column. Conversely, when the mobile phase is made less polar, as by addition of methanol or acetonitrile, the retention of a compound will be reduced. By manipulation of the mobile phase composition, retention times can

often be adjusted so that the compounds of interest are completely separated from other substances in the sample and elute in a relatively short time (e.g., 4-15 min). Once established, the composition of the mobile phase for a particular analysis rarely needs to be changed significantly. (On occasion, a little more or less of methanol or acetonitrile may be needed to adjust retention times after replacing a column.) For this reason, I see little need in routine clinical applications for systems that, at greatly increased cost, provide for continuously varying the composition of the mobile phase. In our laboratory, we use a simple valve to select which of six mobile phases enters the pump.

Quantitation Quantitation is achieved by analysis of the separated moieties of a mixture as they emerge from the chromatography column. This analysis can take many forms, including bioassay and immunoassay; however, in clinical laboratories, quantitafion is usually based on the response of a spectrophotometer. By

In This Issue Liquid Chromatographic Assay of Antibiotics . . . . . . . . . . . . . . . . . A review o f H P L C and its applications

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Vibrio Cholera . . . . . . . . . . . . . . . . The significance o f an endemic focus within the United States

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Letters . . . . . . . . . . . . . . . . . . . . . . .

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Laboratory Management Survey

Table 1 Common Methods for Sample Preparation

Method

Advantages

Disadvantages

Protein precipitation

Simple Excellent analytic recovery

Removes few interferences Analyte diluted

Solvent extraction

Complements analytic separation Relatively simple

Variable analytic recovery Requires internal standard Requires solvent evaporation

Selective adsorption and elution

Often best complements analytic separation Good analytic recovery

Requires most care Analyte often diluted May require solvent evaporation

appropriate selection of wavelength, sensitivity for the compounds of interest can be optimized while minimizing response to potential interferences. The height or area of the chronlatographic peak can be related directly to the concentration of drug in the sample if the dilution and response factors are known. Because these factors may vary, the most accurate results are usually obtained by adding an internal standard to the specimen before analysis (12). The internal standard should be chemically similar to the analyte so that changes affecting dilution or response factors will affect each compound to the same degree. By knowing only the amount of standard added and the relative reponse of the standard and analyte from calibration standards, the analyte can be quantitated.

Sample Preparation Accurate results are obtained only when the analyte and internal standard are separated completely from potential interferences. Adjustment of chromatographic conditions and use of more selective detectors, such as fluorescence detectors, can decrease interferences. These approaches affect the entire chromatographic system and may decrease utility for other drugs. For example, use of an ion-pairing reagent in the mobile phase to avoid an interference with one assay may render the system unsuitable for analysis of another drug without

first changing the column. Alternatively, various methods of sample preparation can be used to remove interferences. Because these procedures are done separately from the chromatography, they do not affect the utility of the whole system. Sample preparation for many drugs consists simply of precipitating protein or filtering the specimen (Table 1). These methods primarily prevent the analytic column from becoming plugged by particulate matter or by proteins that would precipitate when mixed with the mobile phase. Potential interferences are not removed. One failure that we have found with some published methods for antibiotics is that these methods were developed for pharmacokinetic studies in essentially healthy volunteers under well controlled conditions. Simple procedures that work well in this situation may have inadequate specificity for specimens from acutely ill patients who receive a variety of other drugs of which the laboratory is most likely not informed. Our laboratory, doing antimicrobial assays, for example, is rarely informed when a patient is receiving diuretics or analgesics-drugs that can have similar chromatographic properties to cephalosporins, penicillins, and chloramphenicol. Solvent extraction usually involves dilution of the sample with a buffer and addition of an internal standard, which is often contained in I

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the dilution buffer to minimize pipetting. An organic solvent such as ethyl acetate, diethyl ether, or methylene chloride is then mixed with the diluted sample and the phases are allowed to separate. The organic phase, which now contains the drug of interest, is either evaporated to a residue that is dissolved in a suitable solvent or "back-extracted" under different conditions into an aqueous solution. For example, a weak acid can often be extracted into ethyl acetate from an acidic solution (pH ~< pK a of the analyte -1.5) and then backextracted into an alkaline buffer. In either case, the solvent eventually used for the sample must be compatible with the mobile phase and detector. Conditions for solvent extraction can be manipulated to remove many interferences from a sample. Because retention time in reversed-phase chromatography is largely controlled by nonpolar differences between compounds, sample preparation based on acid-base reactions complements rather than simply extends the analytic separa tion. The disadvantages of solvent extraction are that more time is required, an internal standard is essential, and some of the solvents pose health risks unless handled with appropriate care. Selective adsorption and elution from small chromatography columns prior to analytic chromatography is a third method of sample preparation that can be used _ _ ,

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when solvent extraction cannot be. For example, the aminoglycosides are too polar to be extracted into an organic solvent but can be extracted from serum by adsorption to an ion-exchange resin (I) or to silica gel (10) followed by elution. The use of extraction involving a different mode of separation than the analytic column (i.e., ionexchange or adsorption versus reversed-phase) may be the best way to remove a difficult interference. However, the technique is not as widely applied and suffers from some of the same disadvantages as solvent extraction. Both methods of extraction can be automated, as illustrated by the Technicon FASTLC for solvent extraction and the DuPont Prep I for column extraction.

Specific Applications Aminoglycosides Among the antibiotics for which assays are most often performed, the aminoglycosides present the greatest analytic challenge for liquid chromatography. They are polar, water-soluble compounds that do not absorb ultraviolet light appreciably at a wavelength greater than 200 nm. All of the assays that have been developed for them depend on some form of derivatization prior to detection (2). Our laboratory has used a chromatographic assay for several years, but with an increasing work load and the availability of rapid and automated homogeneous immunoassays, liquid chromatography is no longer the method of choice. The principal advantages that chromatography may have are in the study of new aminoglycosides and in the investigation of therapeutic accidents (e.g., parenteral administration of neomycin) for which immunoassays may not be available or may fail to give necessary qualitative information.

Chlorarnphenicol Chloramphenicol is the single, most important example of an antibiotic that can be assayed by liquid chromatography. Chloramphenicol

is administered intravenously as a prodrug, chloramphenicol sodium succinate, and following administration, appreciable quantities of the prodrug and metabolites can be detected in serum (6, 8). The need to assay chloramphenicol for accurate dosage adjustment and the complexity of its pharmacology have apparently been a stimulus to the development of liquid chromatographic assays. At least thirteen assays have been reported (2). Six of these assays use protein precipitation; the remainder use some form of solvent extraction for sample preparation. We found that several drugs (e.g., phenacetin, phenobarbital, and oxacillin) could interfere with chloramphenicol when using an aqueous acetonitrile mobile phase and therefore prefer a method using solvent extraction. We use either a modification of the method of Thies and Fischer (13) in which extraction is at pH 10.4 or a modification of the method of Koup et al. (9) using extraction at pH 4.6. In either case, extraction is with ethyl acetate, which is evaporated; the residue is dissolved in mobile phase for analysis. The available internal standards used in published methods were evaluated, and none fit our criteria of stability, lack of volatility, similar ultraviolet spectrum, and elution after chloramphenicol. (Elution after chloramphenicol avoids potential interference from chloramphenicol succinate, metabolites, and some diuretics.) We prepared and used the pivaloyl analogue of chloramphenicol for an internal standard. The two extraction conditions differ principally in whether chloramphenicol succinate will be extracted. The alkaline extraction does not extract the succinate ester, and in a very few cases we have avoided chromatographic interferences in the alkaline extracts that were evident in the acidic extracts. The magnitude of the interference was small, however.

Vancomycin Vancomycin can be assayed easily following extraction with a small

column containing a weak cationexchange resin (CM-Sephadex) and elution with a borate buffer (14). Ristocetin is used as the internal standard, and detection is with ultraviolet absorption at 210 nm.

Flucytosine As for chloramphenicol and vancomycin, liquid chromatography is probably the method of choice for flucytosine. Several methods have been published. We have used the method of Miners et al. (11), which uses protein precipitation for sample preparation and 5-methylcytosine as internal standard, but our experience is limited to less than 10 assays.

fl-Lactanl Antibiotics As a class, the fl-lactams represent the largest and most frequently used group of antibiotics. It is not necessary to routinely monitor these relatively safe drugs; however, unusual circumstances do arise where measurement is necessary. Unfortunately, these are often complex cases, with the patients receiving several antibiotics, which preclude the obvious choice of bioassay as a methodology. Liquid chromatographic procedures have been published for almost all of these drugs (5). With few exceptions, these assay procedures have used protein precipitation as sample preparation and have been used for pharmacokinetic studies under controlled conditions. They have not been well documented for absence of interferences and use with acutely ill patients. A recent procedure by Brisson and Fourtillan (3) uses an extraction method for parenteral cephalosporins. This method may be prone to fewer interferences, but again, clinical evaluation has been limited. Because of the expected low work load and the large number of different drugs to be assayed, the /3-1actam antibiotics would appear to be ideal candidates for development of liquid chromatographic assays. Summary The advantages of liquid chromatography over alternative

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biologic, immunologic, and chemical methods for antibiotic assays are high specificity and the capability for analysis of several drugs with only minor modification, if any, in methodology. The principal disadvantages are the lengthy procedures sometimes needed for sample preparation and the fact that samples are processed sequentially through the time-consuming chromatography step. Immunoassay is the method of choice for aminoglycosides because of the large volume of tests. For small numbers of tests and when metaboIites may interfere with other methodologies, liquid chromatography is the method of choice. Further development is needed in /3-1actam assays to provide methods applicable in a clinical laboratory. References 1. Anhalt, J. P., and S. D. Brown.

1978. High-performance liquid chromatographic assay of aminoglycoside antibiotics in serum. Clin. Chem. 24:1940-1947. 2. Anhait, J. P., and T. P. Moyer. 1980. The role of gas-liquid

chromatography and liquid chromatography in therapeutic drug monitoring. Lab. Med. 11:797-806. 3. Brisson, A. M., and J. B. Fourtillan. 1981. Determination of

cephalosporins in biological material by reversed-phase liquid column chromatography. J. Chromatogr. 223:393-399. 4. Colin, H., and G. Guiochon. 1977. Introduction to reversed-phase highperformance liquid chromatography. J. Chromatogr. 141:291-312. 5. Gerson, B., and J. P. Anhalt. 1980.

High-pressure liquid chromatography and therapeutic drug monitoring, pp. 81-162. American Society of Clinical Pathologists, Chicago. 6. Glazko, A. J. 1967. Identification of chloramphenicol metabolites and some factors affecting metabolic disposition, pp. 655-665. In Antimicrobial Agents and Chemotherapy--1966. American Society for Microbiology, Washington, D.C. 7. Horvath, C., and W. Melander.

1977. Liquid chromatography with hydrocarbonaceous bonded phases; theory and practice of reversed phase chromatography. J. Chromatogr. Sci. 15:393-404. 8. Kauffman, R. E., et ai. 1981. Phar-

macokinetics of chloramphenicol and chloramphenicol succinate in infants and children. J. Pediatr. 98:315-320. 9. Koup, J. R., et al. 1978. Highperformance liquid chromatographic assay of chloramphenicol in serum. Antimicrob. Agents Chemother. 14:439-443. 10. Maitra, S. K., et al. 1977. Serum gentamicin assay by highperformance liquid chromatography. Clin. Chem. 23:2275-2278. 11. Miners, J. O., T. Foenander, and D. J. Birkett. 1980. Liquid-

chromatographic determination of 5-fluorocytosine. Clin. Chem. 26:117-119. 12. Snyder, L. R., and S. van der Wal.

1981. Precision of assays based on liquid chromatography with prior solvent extraction of the sample. Anal. Chem. 53:877-884. 13. Thies, R. L., and L. J. Fischer.

1978. High-performance liquid chromatographic assay for chloramphenicol in biological fluids. Clin. Chem. 24:778-781. 14. Uhl, J. R., and J. P. Anhalt. 1979. High-performance liquid chromatographic assay of vancomycin in serum. Therapeutic Drug Monitoring 1:75-83.

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Editorial The Significance of Vibrio cholerae in the United Stales

Myron M. Levine, M.D., D.T.P.H. James B. Kaper, Ph.D. Centerfor Vaccine Development University of Maryland School of Medicine Balthnore, Maryland 21201 Cholera is a diarrheal infection that can occur in explosive epidemics and lead to rapid dehydration and death. Until recently, most American microbiologists and clinicians regarded it as an exotic tropical infection of little practical significance. However, there is now an endemic focus of cholera along the Gulf of Mexico of the United States, which requires that we gain familiarity with the disease and its causative agent. Vibrio cholerae, the etiologic

agent of cholera, induces diarrhea by the effects of an enterotoxin on mucosa of the small intestine. Clinical infection follows ingestion of contaminated food and water, and humans are the only known natural hosts. Much confusion exists about V. cholerae as a taxonomic entity because the species covers a diverse array of organisms that includes nonpathogenic free-living water vibrios as well as strains responsible for epidemic diarrhea. Strains associated with large-scale epidemics and pandemics are uniformly enterotoxigenic and manifest a common group O1 antigen, which makes them serologically distinct from biochemically similar (but nonpathogenic) V. cholerae of environmental sources and from certain pathogenic strains that cause sporadic cases of diarrhea and extraintestinal infection. These

latter diarrhea-causing vibrios possess O serogroups other than O1 and in the past were referred to by the misnomers "non-cholera vibrios" or "non-agglutinable (NAG) vibrios." Within the O1 serogroup are two biotypes (classic and El ToO and two common serotypes (Inaba and Ogawa). The current (seventh) pandemic of cholera, caused by the El Tor biotype, began in 1961 in Indonesia and by 1971 had spread throughout Asia, Africa, and Oceania. El Tor strains isolated early in the pandemic (1961 to 1962) were highly hemolytic but, beginning in 1963, nonhemolytic strains appeared, and after 1966 El Tor strains were exclusively nonhemolytic. In 1973 a case of cholera occurred along the Gulf coast, in Port LaVaca, Texas, in an American who