Sample Preparation And Residue Isolation

Sample preparation requirements are dependent on the particular analysis that is to be performed. For example, ba or ia procedures for antibiotics may require little or no sample preparation, whereas sample preparation for tic, lc, and gc procedures can be a major limiting factor of the analyses.

Liquid samples such as milk, blood, urine, saliva, or other body fluids can be assayed directly for antibiotics by ba or ia techniques, or the samples may require only minor cleanup steps (centrifugation, pH adjustment, or a protein precipitation) prior to the assay. In some cases the antibiotic residues that may be present in the sample at low concentrations may require an analyte-concentration step prior to the analysis.

Residue enrichment methods that concentrate the antibiotic residue prior to an analysis may involve solvent extractions, column chromatography, or solid-phase extraction (spe) techniques. The utility of solvent-solvent extractions is limited because of the polar characteristics of many antibiotics such as beta-lactams, aminoglycosides, macrolides, polyether ionophores, and tetracyclines. Ionexchange column chromatography techniques utilized to isolate polar ionizable antibiotics are only marginally effective. Solid-phase extraction appears to hold more promise as a routine residue enrichment approach. The application of this technique proved very effective in cleaning and concentrating penicillin residues in animal tissue prior to HPLC analysis (7). Methods that attempt to circumvent cleanup steps by assaying samples for antibiotics directly are only rarely efficient, except for ba or ia techniques of some liquid samples such as milk.

Supercritical fluid extraction (SFE), a relatively new extraction technique, is also superior to conventional sample preparation methods as no cleaning is required prior to analysis. SFE minimized sample manipulation and eliminated the use of organic solvents when used to extract sulfamethazine in chicken eggs (8). This advantage was also evident when supercritical C02 modified with acetonitrile was used to recover sulfonamide from chicken liver, beef liver, and egg yolk (9).

Sample-preparation steps may not be necessary in some cases for antibiotic determinations but their use can often facilitate more accurate analyses. Therefore, it is incumbent on the analyst to obtain the cleanest sample extract possible. Clean extracts from some liquids and most nonliquid matrices such as muscle or organ tissues are more difficult to obtain and generally require an extraction step and multiple manipulations to isolate the antibiotic residue from the sample with high percentage recoveries. Unfortunately, sample extracts obtained in this manner may contain naturally occurring inhibitors or interferences that could affect the analysis. Thus further cleanup of extracts is usually required prior to performing the analysis. Because of the polar nature of many antibiotics, buffered aqueous solutions are routinely employed for antibiotic extractions and it is difficult, if not impossible, to isolate antibiotic residues from these aqueous extracts by partitioning with organic solvents. Thus drying of the aqueous extract, a time-consuming process, or the use of other preparative steps such as column chromatography or spe techniques may be required before an analytical determination can be made. A direct assay of the aqueous extract may be possible if the concentration of the antibiotic in the extract is sufficiently high or if the analytical method is particularly sensitive. As the complexity of the sample and the number of sample cleanup steps increases, the utility of techniques such as ba, ia, and tic for quick antibiotic screening purposes decreases. In addition, extracts suitable for ba or ia procedures may not be sufficiently clean for more sophisticated tic, lc, or gc determinations, thereby requiring additional and different residue isolation procedures for confirmatory techniques.

Sophisticated techniques based on lc, gc, lc-mass spectrometry (ms), and gc-ms usually require more rigorous sample preparation to isolate the antibiotic free from interferences found in the sample extract. These classic isolation techniques can be laborious and time-consuming. Classic isolation techniques for aminoglycoside (10), fi-lactam (11-13), chloramphenicol (14), ionophore (15), mac-rolide (13,16), sulfonamide (17-20), and tetracycline (21) antibiotics generally involve their extraction from biological matrices with large volumes of extracting solvents, chemical manipulations such as pH adjustments and protein precipitations, centrifugations, back washing, and the evaporation of large volumes of organic solvents. This approach limits the usefulness of many classic isolation techniques for multiresidue determinations by lc and gc.

Analytical capability is, therefore, limited to a large extent by interferences present in the sample extract. While state-of-the-art analytical techniques can detect picogram levels of pure antibiotic standard compounds, this same level of detection may not be achievable for antibiotics obtained from the extraction of biological samples. Specifically, coextracted interferences may hinder antibiotic determinations by tic, lc, or gc because they may have similar detector and chromatographic characteristics or they may exist in such a large quantity that they overwhelm the detection method used. Interferences in ba or ia techniques can contribute to cross-reactions that may lead to false-positive or false-negative determinations. Thus a major limiting factor associated with antibiotic determinations is not the available analytical capability but the sample-preparation steps required to extract the antibiotics. The sample-preparation steps should ideally result in clean biological extracts that contain the antibiotic residue with high percentage recoveries and that have minimal interferences that might limit the choice of analysis.

The need to test for more antibiotic residues in more foods requires rapid, rugged, and multiresidue isolation techniques allowing the analyst to test for multiple drugs in the same sample. Classic residue isolation techniques have not been able to meet this challenge. Matrix solidphase dispersion (mspd) techniques (22-28), recently developed for the isolation of drug residues from animal matrices, have the potential to greatly enhance many antibiotic residue isolation protocols. In mspd the sample (0.5 g) is dispersed onto octadecasilyl polymeric—derived silica beads [C-18 reversed-phase packing material (2 g), 1,000 m2 surface area, theoretical]. The dispersion mechanism, utilizing a mortar and pestle, involves the disruption, unfolding, and rearrangement of matrix constituents by mechanical and hydrophobic forces onto the C-18 beads. Lipid and lipophilic materials associate with the lipophilic C-18, allowing the more hydrophilic components and protein regions to extend outward away from the nonpolar, inner C-18—lipid region. Water and more polar constituents preferentially associate with the hydrophilic ends. A column fashioned from the C-18—sample matrix blend can be eluted sequentially with solvents (8 mL) of different polarities to effectively remove interferences in one solvent and elute the target residue in a different solvent. The process can be envisioned as an exhaustive extraction process whereby a large volume of solvent is passed over a thin layer of sample. Mspd has been utilized for the isolation of beta-lactams from beef tissue (23); sulfonamides from milk (24), infant formula (25), and pork muscle tissue (26); as well as for chloramphenicol (27) and tetracycline (28) isolations from milk. The theoretical aspects describing the disruption, unfolding, and rearrangement of matrix constituents onto the C-18 have been published (22-28).

Advances in multiclass—multiresidue isolation procedures, which are rapid, rugged, generic in nature, and free from interferences and which facilitate the isolation of multiple antibiotic residues from one sample, will greatly enhance analytical determinations of antibiotics.


Bioassays are used routinely to test for violative levels of antibiotics in milk, animal tissues, and feeds. Bioassays involve the inhibition of growth of specific bacterial spores or viable bacteria in the presence of a sample or sample extract that contains antibiotic residues. Bioassays may also utilize the measurement of labeled antibiotic analyte bound to receptors, a ligand assay, on vegetative bacterial cells. Bioassays have been utilized to detect aminoglycosides (29-32), ^-lactams (30,32-34), chloramphenicol (32,35-37), ionophore (15), sulfonamide (29,30), and tetracycline (29,30,32,33,38) antibiotics in food.

For example, the swab test on premises (stop) procedure (30) is used by federal meat inspectors to test for chloramphenicol, tetracycline, aminoglycoside, penicillin G, and sulfonamide antibiotics. The stop method involves taking a sterile cotton swab and macerating the target organ or tissue with the noncotton end. The size of the macerated zone should be slightly larger in diameter and deeper than the cotton end of the swab. The cotton end is then inserted into the disrupted area and allowed to absorb fluids (30 min). The fluid-soaked cotton swab is then placed onto a nutrient agar plate that has been inoculated with a lawn of specific bacterial spores and the plates are then incubated (16-22 h). Standards of known concentration are run in parallel. A zone of microbial growth inhibition on the sample plate is an indication of the presence of an antibiotic and the size of the zone of inhibition can be semicor-related with the size of the zone of inhibition for a given concentration of pure antibiotic standard. The sample is positive if the size of the zone of inhibition is similar to that for a known pure standard. Unfortunately, the stop procedure detects only the presence of inhibitors, not their specific identity. Other antibiotics or naturally occurring inhibitors may contribute to the size of the zone of growth inhibition observed, indicating a positive sample although the amount of the individual antibiotic compounds present may be less than the violative level.

The classic disk assay procedure (34) is similar to the stop procedure for the same antibiotics except that a filter paper disk is placed on the inoculated nutrient agar, the liquid sample or suitable extract is added to the disk and then the plate is incubated for a minimum of 2.5 h or until a zone of inhibition can be observed. The zone of inhibition may be enhanced by dyeing techniques (39,40), which aid in its detection. Semiquantitative determinations by the disk assay can be accomplished, provided incubation times are increased (16-22 h). The disk assay procedure is the official method described in the Pasteurized Milk Ordinance (PMO) (41) for antibiotic testing in milk. The disk assay procedure suffers limitations similar to those of the stop procedure. However, the PMO allows for the use of any method that gives results equivalent to the disk assay method; therefore, many states utilize alternative techniques such as the color reaction test (crt) to test for antibiotics in milk. The Delvo test (39) is an example of a crt technique used to determine antibiotics, specifically beta-lactams, in milk. The test involves placing the milk sample onto agar containing a viable strain of Bacillus, nutrients, and pH indicators. If the color of the agar changes from purple (basic) to yellow (acid) after incubation (1.5 h) then no penicillin is present to prevent the outgrowth of the acid-producing bacteria. Color reaction tests can be rapid and simple to perform.

The microbial receptor assay (mra) (Charm test) technique (38) can be used to detect beta-lactam, macrolide, and aminoglycoside antibiotic classes. The mra method involves the use of C-14 or tritium isotopically labeled ana-lyte to displace nonlabeled analyte from the bacterial receptors located on vegetative cells under a standard set of conditions. An equilibrium condition between unlabeled and isotopically labeled analyte results, allowing quantitative measurement of the amount of antibiotic present by an appropriate radiometric method. However, the identity of the compound is not known and must be determined by other methodology.

The thin layer chromatography—bioautography of tlb bioassay technique (15,32,40) uses traditional thin layer chromatography to separate sample constituents on silica or microcrystalline cellulose chromatography (mcc) plates. Different antibiotics will migrate on the plates according to their chemical characteristics and the developing solvent utilized. The developed plates are covered with a spore-inoculated nutrient agar and then incubated (16-24 h). Zones of growth inhibition observed for different locations on the plate after incubation indicate the presence of antibiotics. Aminoglycoside (32), ^-lactam (32), chloramphenicol (32), ionophore (15,32), macrolide (32,40), and tetracycline (32) antibiotics have been assayed by tlb. The tlb method is more specific and sensitive (36) than either the stop or classical disk assay procedures because it takes advantage of the ability of tic to separate 14 different antibiotics from each other as well as from other impurities that may be present in the sample. Therefore, zones of inhibition can be more closely correlated to different antibiotics. The tic-developing solutions are designed to optimize separations between similar antibiotics. However, the separation of up to 14 different antibiotic residues can only be accomplished by utilizing three separate tic plates. Furthermore, extensive sample-preparation and extraction steps are employed in the tlb procedure that are similar to those employed for more sophisticated analytical determinations. Because of the incubation time required the tlb procedure is not as rapid as the crt or the disk assay methods for screening purposes. However, the tlb procedure has an advantage over the stop or disk assay procedures for screening purposes because it can provide for more precise determinations between different antibiotics. Absolute confirmation of the antibiotics is not possible by tlb and time requirements are in excess of many sophisticated, more definitive, liquid and gas chromatographic techniques.

The stop, disk assay, crt, and tlb procedures are valuable screening tools. The first three techniques are relatively cheap, easy to perform, and require a minimal amount of equipment and technician training. However, they lack specificity and sensitivity and/or they may be subject to interpretive errors in the presence of naturally occurring inhibitors. Furthermore, each of these four methods requires confirmational testing of positives. The mra method using isotopically labeled antibiotics can be expensive and requires special handling and equipment; but it can provide for quantitative determinations. These bioassays, if used judiciously, can minimize the number of samples screened by more costly analytical methods, but they cannot replace such methods for absolute confirmations.


Immunoassay techniques are based on classic antibody-antigen reactions whereby the antibody will bind with its corresponding antigen (antibiotic) and result in visible turbidity if reacted in solution or form visible immunoprecip-itation in a gel at the location where the antigen and antibody meet. Visible end point immunoprecipitation that occurs at low antibiotic concentrations may be difficult to see and may require detection by more sensitive nonvisual means.

Immunoassay techniques such as enzyme immunoassay (eia), radio immunoassay (ria), enzyme-linked immunosorbent assay (elisa), fluorescence polarization immunoassay (fpia), particle-concentration immunoassay (pcia), particle-concentration fluorescence immunoassay (pcfia), quenching fluoroimmunoassay (fia), and latex-agglutination inhibition immunoassay (laia) require the measurement of by-products produced by linked enzyme systems or the measurement of radioactive, fluorescence, metal, or latex labels that have been attached to one of the reactants (42). The displacement between the unlabeled and labeled antigen or antibody allows for true measurement of the immunoprecipitate concentration, which corresponds to the concentration and type of analyte present in the sample extract. Ria determinations of chloramphenicol (43,44) as a residue in eggs, milk, and meat were comparable to values obtained by gc. The eia determination of monensin

(45), a polyether antibiotic in urine, serum, and fecal extracts, may be applicable to food extracts as well. A review of ia techniques such as ria, fia, fpia, and laia for aminoglycoside determinations in body fluids has been published

(46). The recent use of a monoclonal-antibody—based agglutination test (spot test) for beta-lactams in milk (47) and elisa for chloramphenicol determinations in swine tissue (48), milk (49), and sulfamethazine in swine tissue (50) has been adapted to other antibiotics including spectino-mycin (51,52), enrofloxacin, (53) and gentamicin (54).

Immunoassays can be sensitive, class specific, accurate, and provide a means for rapidly screening samples for antibiotics; the future development of monoclonal antibodies will allow for more specificity in immunoassay determinations. The growth in the use of immunoassay-based detection has lead to the development of commercial kits allowing for on-site detection of chemical contaminants (5557). Radioimmunoassay techniques that require radioisotopes may be subject to specific regulations, require expensive counting equipment, and pose disposal problems. Reagent kits can be relatively expensive and have a limited shelf life. In this regard, nonisotopic immunoassays such as elisa, fpia, pcia, or pcfia, and monoclonal-based ias will in all likelihood play an increasingly important role in antibiotic screening immunoassay determinations.

At present, ia techniques for antibiotics approved for use in animal production are limited and have minimal utility for aminoglycoside, /?-lactam, chloramphenicol, and sulfonamide determinations, but they have the potential for use as screens for all the major antibiotic classes. Im-

munoassay techniques have been limited to liquid samples such as milk because of the difficulties associated with the isolation of antibiotic residues from matrices such as muscle or organ tissues. Increased use of ia techniques to screen for antibiotics in tissues is directly dependent on the development of rapid residue isolation techniques that isolate the antibiotic from the tissue and provide for an extract that is free from cross-reacting components and nonspecific binding (NSB) factors. The development of such isolation techniques will further the use of ia techniques for the screening of antibiotics in tissues.

Immunoassay techniques are more specific than microbiological based bioassays and would allow for the rapid screening of large numbers of samples. With the need to screen more samples for more drugs, the ia techniques will become more important in residue control protocols. Immunoassay techniques can greatly reduce the number of samples that are presently screened by more sophisticated analytical techniques and have the potential to replace many bioassay and tic methods, provided rapid and efficient antibiotic extraction procedures can be developed. As a regulatory tool, ia techniques for antibiotic screening coupled to highly specific and accurate techniques, such as lc, gc, lc-ms, or gc-ms for confirmations of positives, mark the future with respect to a rapid and reliable residue control strategy.

Thin Layer Chromatography

Thin layer chromatography techniques have been used in chemical separations for decades. Thin layer chromatography is rapid and inexpensive, can be highly sensitive depending on the compound examined and the visualization technique employed, and is easy to use and versatile. It can be adapted to separations of all classes of antibiotics by utilizing different sorbents and solvent-developing systems. Aminoglycoside (31,32), ^-lactam (32,48,58), chloramphenicol (32), ionophore (15,32), macrolide (16,32,40), sulfonamide (59,60), and tetracycline (38,61,62) antibiotics have been successfully assayed by tic. However, the utility of tic for the detection of picogram levels of antibiotics is limited and thus tic techniques have come to play only a minor role in antibiotic analyses.

The sulfa on site (sos) tic procedure for sulfonamide determinations in swine urine was developed by the USDA— FSIS and is presently in use in 100 of the largest swine-slaughtering facilities in the United States (63). This method was adopted in 1988 as the official method for inplant testing of swine for sulfamethazine (64). Thin layer chromatography techniques can complement other antibiotic assay techniques such as tic-bioautography bioassays (15,32,40). TLC/bioautography qualitative and semiquantitative methods are routinely used in Austria for the analysis of antibiotic residues in poultry, meat, and fish (65). This method is limited to a large degree by the cleanliness of the sample that is to be analyzed. Impurities in the sample can interfere with the chromatography of the antibiotic by altering the migration of the antibiotic on the tic plates compared to pure standards, and these impurities can negate visualization techniques. Thin layer chromatography determinations also require confirmations of suspect residues isolated from the tic plates to determine if the residue is indeed an antibiotic. In this regard bioassay and immunoassay techniques provide for a more precise determination of the presence of antibiotics in a sample extract. Thin layer chromatography will perhaps play a decreasing role in antibiotic residue control strategies where low-level detection is required for some antibiotics, but should not be eliminated from the analyst's tools utilized for the isolation and purification of compounds from animal-derived matrices.

Liquid Chromatography

The convenience and versatility of liquid chromatography has led to its adoption as the analytical method of choice for the determination of many drugs, especially antibiotics. Liquid chromatography, using selective detectors, can give reproducible results that are specific, sensitive, and precise. The polar characteristics of antibiotics make them well suited to lc procedures, in which mobile phase solvent systems and columns can be varied to facilitate specific antibiotic determinations.

Recent publications describing lc methods for the analysis of aminoglycoside (10,31,66), /?-lactam (1113,16,23,67-69), chloramphenicol (14,27,70,71), ionophore (15,16), macrolide (13,16,72), sulfonamide (17,19,2426,73-75), and tetracycline (21,36,61,68,76-80) antibiotics underscore the utility of lc determinations for the analysis of these compounds as residues in foods and other biological matrices.

Food extracts containing residues of sulfonamide (15,18,24-27), yg-lactam (11,16,23,67), chloramphenicol (14,27), tylosine (16) and spiramycin (13) macrolides, and tetracycline (21,36,61,68,78) antibiotics may be analyzed directly by ultraviolet (uv) detection at picogram— nanogram levels because they have characteristic uv ab-sorbances and large extinction coefficients. Photodiode array uv detection of antibiotics can provide the analyst with uv spectra of suspect peaks (81) and thus serve as a pre-confirmational screening tool. However, some beta-lactams and macrolides, which have a maximum absorbance in the low uv (210-240 nm) range or relatively small extinction coefficients, can be more difficult to analyze by lc—uv because of coextracted interferences that absorb readily in this range. Optimizing lc chromatographic conditions to separate a particular antibiotic residue from interferences that may be present in the extract severely limits lc techniques in terms of multiresidue antibiotic determinations.

Aminoglycoside and most macrolide antibiotics that have low or nonexistent uv-absorbing properties may require the formation of derivatives to aid in their detection or the use of alternative detection methods. Benzene sul-fonyl chloride (82) and l-fluoro-2,4-dinitrobenzene (83) have been used to prepare uv derivatives of aminoglycosides for analyses by lc. Detection methods for antibiotics that do not require making derivatives are ideal because reaction conditions for many derivatizations can be difficult to optimize. However, until improved or new detection methods are developed for antibiotics, derivatives will continue to be used to facilitate sensitive and selective detections, especially for aminoglycosides.

The analyses of aminoglycoside (10), /?-lactam (12), mac-rolide (16), and tetracycline (38) antibiotics can also be facilitated by the formation of fluorescent derivatives. The ionophore lasalocid has native fluorescence due to its sal-icyclic acid-type aromatic moiety and, therefore, can be analyzed directly in food extracts at nanogram levels by fluorescence detection (15). Fluorescent derivatives of tetracyclines (38) can be made by simply complexing the tetracycline with different metal ions. The tetracycline— metal ion complexes have unique excitation and emission wavelengths that can be measured to quantitatively determine tetracycline concentrations in the food extract. In the case of aminoglycoside antibiotics, which do not have native fluorescence, fluorescent derivatives must be made to facilitate their detection. The preparation of fluorescent derivatives can require exacting reaction conditions, additional equipment in the form of reactors and delivery pumps, and in many cases the removal of reaction reagents before an analysis can be made. However, this cannot be avoided in the case of aminoglycosides because no other suitable chromatographic method presently exists for their detection at nanogram levels. Fluorescence detection of o-phthalaldehyde (OPA) or fluorescamine derivatives has been shown to give accurate and reliable values for aminoglycoside residues in animal tissue (10,31). The deriva-tization of the aminoglycoside amino group with a fluoro-genic agent can be accomplished precolumn or postcolumn. The added cost of the postcolumn reactor and solvent pump needed to deliver the derivatization solution may be disadvantageous. Alternatively, precolumn derivatization reaction mixtures may require the removal of derivatizing reagents prior to the analyses. Specific applications may require either approach and are dependent on the type of food that is to be extracted and the inherent interferences present.

Fluorescent derivatives (12) of penicilloaldehyde products obtained by enzymatic hydrolysis of beta-lactam rings and reaction with dansyl hydrazine have been reported for eight neutral beta-lactams. This is a novel approach for beta-lactam determinations and might be applicable to other antibiotics that are inherently unstable and difficult to analyze. Techniques that serve to characterize unique enzymatic or degradative products of antibiotics may be a useful tool for many antibiotic determinations.

Liquid chromatography of antibiotics will continue to be the method of choice for most antibiotic determinations. Coupling of photodiode array uv, fluorescence, electrochemical and other yet to be developed detectors in tandem can provide information in terms of retention times, structures, and characteristic uv and fluorescent spectra. Information obtained by this approach may be sufficient to confirm the presence of specific antibiotics if coupled with positive results obtained by immunoassay techniques for the specific antibiotic in question. Liquid chromatography—mass spectrometry is not presently suitable for low-level detection of the major antibiotic classes, but advances in this area, the use of tandem detection methods, and specific monoclonal-based immunoassay screening techniques would contribute significantly to overall antibiotic residue control strategies needed to insure a safe and wholesome food supply.

Gas Chromatography

Gas chromatography coupled to specific detectors can provide valuable information about the retention time, structure, chemical characteristics, and identity of compounds. Gas chromatography has been utilized for antibiotic determinations but is limited to some extent by the molecular weight, high polarity and the relative lack of thermal stability of many antibiotics. Chemical derivatives can serve to impart greater stability and volatility to antibiotics. However, controlling reaction conditions to insure the formation of consistent derivative products with high yields can be difficult to accomplish. These difficulties have limited the usefulness of gc for routine antibiotic determinations.

Presently there are no suitable gc methods for the routine determination of aminoglycoside, beta-lactam, and most ionophore, macrolide, and tetracycline antibiotics. Gas chromatography of trimethylsilyl (TMS) derivatives of aminoglycoside (84), lasalocid ionophore (14), and tetracycline (85) antibiotics have been reported. However, the usefulness of this technique for low-level determinations of these antibiotics in food extracts is limited because of the need to control silylating reaction conditions carefully, the formation of multiple TMS antibiotic derivative products, the abundance of TMS interferences contributed by the sample, and the lack of stability of TMS derivatives.

Gas chromatography methods for chloramphenicol have been reviewed (14). Gas chromatography utilizing electron capture (ECD), flame ionization (FID) and thermionic (TID) detection of TMS and heptafluorobutyl derivatives of chloramphenicol isolated from muscle, liver, kidney, and milk resulted in picogram—nanogram detection giving results comparable to radioimmunoassay determinations (43). Gas chromatography of chloramphenicol residues in food extracts can provide for confirmations of suspect residues and complement lc determinations.

Gas chromatography methods for sulfonamides have also been reviewed (17). Sulfonamide determinations in animal tissues (20,86) by gc have been accomplished by analyzing volatile methylated or acylated derivatives. Methylation of sulfonamides at the AM position followed by acylation at the N-4 position can provide for stable volatile derivatives suitable for gc analysis. However, it can be difficult to control reaction conditions necessary to optimize this two-step derivative reaction. Low yields and the formation of multiple derivative products may result in nonrepresentative and misleading data. Although methods for the quantitative determination of sulfonamides (18,20,75) have been reported, it has been noted (18) that some sulfonamides determined in this manner resulted in low and variable recoveries. Because of this variability, it is necessary to be careful when quantitatively evaluating data obtained by this technique.

Advances in supercritical fluid chromatography (sfc) may eventually lead to applications involving antibiotic determinations. At present, sfc is limited as a routine analytical tool to nonpolar substances (87). Analysis of polar antibiotics by sfc will require advances in sfc hardware and columns. In addition, the solubility characteristics of different antibiotics in different supercritical media will re quire extensive laboratory investigation to define optimal conditions in terms of pressures and polar mobile phase modifiers that may be required:

Thus gas chromatography methods have minimal utility for antibiotic determinations. Gas chromatography will remain a supplemental analytical tool for antibiotic residue determinations until new derivatization schemes, reagents, and refined reaction conditions are developed. The development of such innovations can, however, advance present marginal gc antibiotic techniques to full-fledged quantitative antibiotic confirmatory methods, which will contribute significantly to an integrated antibiotic residue control strategy.

Lc-ms and Gc-ms

Mass detectors coupled to chromatographic techniques such as lc and gc can provide for the unequivocal identification of compounds. Sensitivity limitations associated with lc-mc precluded its use in antibiotic residue analyses when low-level determinations were required. However, in recent years lc-ms has been developed to permit such determinations. Keever, Voysker, and Tyczkowska (88) reported a quantitative procedure using lc-electrospray ms to detect ceftiofur in milk at levels down to 10 ppb. An interlaboratory study to monitor pirlimycin residues in bovine milk using lc-thermospray ms was able to detect as low as 0.4 ppm in milk and 0.5 ppm in liver tissue (89). Gc-ms techniques for antibiotics have been predominantly applied to colatile sulfonamide derivatives (17,20,75,86) but may provide for confirmation of suspect residues, although it may be inadequate as a regulatory tool. However, gc-ms procedures were reported suitable for identifying and quantifying chloramphenicol residues with detection limits of 0.1-1.0 ng/kg (90). This method compared well with results obtained using radioimmunoassay and enzyme immunoassay and was considered to be a good screening test. Nagota and Oka (91) reported a capillary gc-ms procedure capable of measuring antibiotic residues in yellowtail fish including chloramphenicol, florfenicol, and thamphenicol. Recovery of each antibiotic was greater than 65% with a detection limit of 5 ppb. Further refinement of lc-ms and gc-ms techniques, advances in ms sensitivity, more efficient lc-ms interfaces, and future developments may allow ms to become a routine confirmatory tool that will greatly enhance antibiotic residue determinations.

Homemade Pet Food Secrets

Homemade Pet Food Secrets

It is a well known fact that homemade food is always a healthier option for pets when compared to the market packed food. The increasing hazards to the health of the pets have made pet owners stick to containment of commercial pet food. The basic fundamentals of health for human beings are applicable for pets also.

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