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CAPILLARY ELECTROPHORESIS | Antibiotics
CAPILLARY ELECTROPHORESIS Antibiotics C. L. Flurer, U.S. Food and Drug Administration, Cincinnati, OH, USA & 2007 Elsevier Ltd. All rights reserved.
Introduction An antibiotic is a substance that has the capacity to inhibit the growth of, and to destroy, bacteria and other microorganisms. This group of pharmaceuticals is one of the most widely prescribed worldwide, and is used in human and veterinary medicine to treat and prevent diseases. New antibiotics are being developed to improve efficacy and to combat the resistant bacteria that are encountered. Capillary electrophoresis (CE) and its related techniques offer effective alternatives to analytical methodologies currently in use for the detection, quantitation, and characterization of these important medications.
Classes of Antibiotics Aminoglycosides
An aminoglycoside antibiotic is comprised of amino sugars that are joined through glycoside linkages. Primary buffer systems for these antibiotics incorporate borate or phosphate–borate combinations as the background electrolyte (BGE). Micellar electrokinetic chromatography (MEKC) has been utilized with sodium pentanesulfonate as the anionic surfactant. One mixed micellar system incorporates sodium dodecyl sulfate (SDS) and Brij 35, and another combines the bile salt sodium deoxycholate and b-cyclodextrin (b-CD). A fundamental difficulty in the detection of aminoglycosides is their lack of a chromophore. One approach to their detection involves the formation of negatively charged, UV-absorbing complexes between borate and the diol moieties of the amino sugars. A number of aminoglycosides and their related substances have been separated and detected this way. Another successful approach is indirect detection, using imidazole in the BGE, and a cationic surfactant for the reversal of the electroosmotic flow (EOF).
Precapillary derivatization has been used successfully with members of this class of antibiotics, when o-phthalaldehyde (OPA) is combined with mercaptoacetic acid, mercaptopropionic acid, or thioglycolic acid. Electrophoretically mediated microanalysis (EMMA) is used for the in-capillary derivatization of aminoglycosides. This involves the introduction of the analyte solution as a ‘sandwich’ between two zones of reagent buffer that contain the derivatizing reagents. A brief application of voltage causes these zones to merge. After an incubation period during which derivatization occurs, the BGE is introduced and component separation proceeds (Figure 1). Another reagent that reacts with primary amines, 1-methoxycarbonylindolizine-3,5-dicarbaldehyde (IDA), produces derivatives that absorb strongly at 409 nm. This reagent has been utilized in an online derivatization mode, with both UV and fluorescence detection. Another approach has incorporated an immunoassay that establishes a competition between free gentamicin and fluorescein-labeled gentamicin for the antibody. Electrochemical detection has been used as an alternative to analyte derivatization, typically with sodium hydroxide as the BGE. Copper or coppermodified carbon fiber microdisk electrodes aligned in wall-jet configurations, or on-capillary electrodes composed of copper or copper-modified gold have been used, generating limits of detection (LODs) in the 1–2 mM range. Another recent approach has been the use of potential gradient detection for the determination of gentamicin components. This detection method measures the mobility differences between sample molecules and the separation buffer’s co-ions, or the potential gradient along the axis of the capillary. Amphenicols
Studies of chloramphenicol, thiamphenicol, and florfenicol have utilized phosphate and borate buffer systems. Because these analytes are neutral, MEKC and capillary electrochromatography (CEC) methods have also been developed. SDS is used as a buffer additive in MEKC and in CEC, where it is added to acetonitrile/water mobile phases, which are combined with XTerra RP18 or Hypersil ODS stationary phases. Detection schemes have incorporated amperometry, with a carbon fiber microdisk array in an end-column configuration, and a carbon disk electrode in a wall-jet configuration. Laser-induced fluorescence (LIF) detection has been used in a
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Figure 1 Electropherogram of a commercial sample of 0.7 mg mL 1 kanamycin sulfate, using CZE-EMMA. Peaks: a, b, degradands; c, d, unknown; 1, reagent; 2, 2-deoxystreptamine; 3, kanamycin D; 4, 6-O-(3-amino-3-deoxy-a-D-glucopyranosyl) deoxystreptamine; 5, 4-O-(6-amino-6-deoxy-a-D-glucopyranosyl) deoxystreptamine þ paromamine; 6, kanamycin A; 7, 1-N-(1-hydroxymethyl2-hydroxyethyl) kanamycin B; 8, kanamycin C; 9, kanamycin B. Separation conditions: capillary, 50 mm 40 cm (31.5 cm effective length); electric field, 588 V cm 1; detection, 335 nm; buffer, 16% (v/v) methanol in 30 mM sodium tetraborate, pH 10.0. (Reproduced with permission from Kaale E, Van Schepdael A, Roots E and Hoogmartens J (2003) Determination of kanamycin by electrophoretically mediated microanalysis with in-capillary derivatization and UV detection. Electrophoresis 24: 1119.)
competitive immunoassay, in which a sample containing chloramphenicol is mixed with rabbit antichloramphenicol antiserum in the presence of a fluorescein–chloramphenicol conjugate. Anthracyclines
These compounds are very powerful antitumor antibiotics. Many of the applications developed have incorporated LIF detection for the monitoring of doxorubicin, daunorubicin, and idarubicin, and their metabolites. Most of these systems utilize combinations of an organic modifier and phosphate or borate, with spermine added to reduce interactions between the analytes and the capillary wall. The argon ion laser line at 488 nm is the most widely used, and can yield limits of quantitation (LOQs) of 0.5 ng mL 1 in serum samples, depending on the sample preparation protocol. CEC is combined with LIF detection for the analysis of doxorubicin labeled with a dicarbocyanine moiety. Electrochemical detection also provides the sensitivity and selectivity needed for clinical applications, such as the determination of mitomycin in serum and daunorubicin in urine. The sensitivity of UV detection can be improved through the use of a sweeping preconcentration method. In this mode, the pH of the BGE is such that the anthracyclines are positively charged. The sample is introduced hydrodynamically until approximately
80% of the capillary is filled. The BGE, which contains SDS, is placed in the inlet vial. In the reversed polarity mode, the positively charged anthracyclines migrate toward the negatively charged micelles. When the zones meet, the anthracyclines are solubilized by the micelles and are drawn toward the detector. LODs on the order of 1 nM can be achieved under these conditions.
b-Lactams
For this class of antibiotics that includes penicillins, cephalosporins, and carbapenems, the most widely used means of component separation is MEKC. Using SDS buffer systems with acetate, phosphate, and/ or borate as the BGE, numerous protocols have been developed for the screening of members within the class, or for the separation of impurities from the primary component. Mixed micellar systems of SDS and Brij 35 or pentanesulfonic acid provide improved resolution through the alteration of the pseudostationary phase. Free-solution CE buffers using phosphate, citrate, borate, and 2-(N-morpholino) ethanesulfonic acid have found applications as screening methods, but these systems are primarily used for dosage form analysis. The free-solution systems are also useful for separating members of the different subgroups from each other, and from other pharmaceuticals that are prescribed in tandem.
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Microemulsion electrokinetic chromatography (MEEKC) has been utilized in the analysis of several compounds within this class of antibiotics. Microemulsions are solutions containing dispersed nanometer-sized droplets of an immiscible liquid. These droplets consist of an immiscible oil suspended in water. The surface tension between the two liquid phases is reduced by adding a surfactant to coat the oil droplets. The surface tension is lowered further by adding a short-chain alcohol, called a cosurfactant, which stabilizes the microemulsion. Microemulsions have been prepared from mixtures of 1-butanol, heptane, sodium glycodeoxycholate, Tween 20, and phosphate buffer. Another study uses a mixture of octane, 1-butanol, SDS, and borate buffer. A system composed of ethyl acetate, 1-butanol, Brij 35, 2-butanol, and 10 mM borate buffer can be used for the separation of eight penicillins (Figure 2). To improve UV sensitivity, a sample stacking technique called the reversed electrode polaritystacking mode (REPSM) is used. After the sample is introduced into the capillary hydrodynamically, a reversed polarity is applied. This preconcentrates the analytes at the interface between the sample zone and the BGE, while the sample matrix is expelled from the capillary by the EOF. A conventional polarity is then applied, and the analysis proceeds. Glycopeptides
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The compounds within this class can be difficult to analyze, because many of them occur as mixtures. For example, bacitracin is a complex mixture of bacitracins A, B, C, D, E, F, G, H, and I, many of which have isomers or amino acid substitutions. A mixed micellar system comprised of the zwitterionic
surfactant 3-(N,N-dimethylhexadecylammonium) propanesulfonate and the nonionic Brij 35 can resolve the numerous components found in bacitracin. Polymyxin B is a cyclic hexapeptide with a tripeptide side chain that differs in the attached fatty acyl residue, giving rise to the components B1, B2, B3, and B4. The presence of methyl-b-CD provides the selectivity to separate the major components as well as minor peaks surrounding polymyxin B1. A similar experimental system is utilized for the analysis of colistin sulfate and its components. Other MEKC separation schemes have used either SDS or cetyltrimethylammonium chloride for the analysis of amphotericin B, zinc bacitracin, vancomycin, avoparcin, ristocetin, and polymyxin B. Vancomycin has also been analyzed in phosphate buffer in 3 min by using a capillary having an effective length of 8.5 cm. Macrolides
Macrolides are antibiotics that are produced by Streptomyces bacteria found in soil, and are comprised of a sugar, an amino sugar, and a 14- or 16member aglycone ring. Although free-solution systems have been utilized, many of the macrolides possess low water solubility; thus, most applications require the use of solubilizing agents in the separation medium. Varying percentages of acetonitrile, methanol, and ethanol have been added to phosphate systems to improve analyte solubility and selectivity. Unlike the alkyl-chain surfactants, bile salts are able to retain their micellar structures in the presence of higher levels of organic solvents. Sodium cholatebased systems, with the addition of acetonitrile to enhance analyte solubility, have been utilized in screening methods. The separations of spiramycins I,
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Figure 2 Electropherogram of a standard mixture of penicillins in a concentration of 500 mg L 1. Peaks: 1, amoxicillin; 2, ampicillin; 3, penicillin G; 4, oxacillin; 5, penicillin V; 6, cloxacillin; 7, nafcillin; 8, dicloxacillin. REPSM injection conditions: 50 mbar for 270 s, 5 kV until 95% of original current reached. Separation conditions: capillary, 75 mm 45 cm (36.5 cm effective length); voltage, 10 kV; detection, 210 nm; buffer, 0.5% ethyl acetate, 1.2% 1-butanol, 2% Brij 35, 10% 2-butanol, and 86.3% 10 mM borate buffer, pH 10.0. (Reproduced with permission from Puig P, Borrull F, Aguilar C and Calull M (2006) Sample stacking for the analysis of penicillins by microemulsion electrokinetic capillary chromatography. Journal of Chromatography B 831: 196).
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II, III, and IV, neospiramycins I, II, and III, and tylosin, erythromycin and their related substances have been achieved in mixed micellar systems containing sodium cholate and the cationic surfactant cetyltrimethylammonium bromide. Methyl-b-CD has also been used in macrolide systems. CEC systems have presented alternatives for the analysis of macrolide antibiotics. Monodisperse poly(glycidyl methacrylate-divinylbenzene) microspheres are derivatized with quaternary ammonium/octadecyl groups to generate a positively charged stationary phase (Figure 3). Another system utilizes capillaries packed with 3.5 mm XTerra RP18 particles, and an SDS/acetonitrile/ammonia mobile phase. Quinolones
Quinolones have emerged as one of the most important classes of synthetic antibiotics, with applications in human and veterinary medicine. As such, numerous screening methods have been developed for pharmaceutical preparations, feed mixes, and animal tissues. Free-solution systems provide adequate separation through the use of borate, boric acid, phosphate, and N-(2-hydroxyethyl)piperazine-N0 -(2ethanesulfonic acid) (HEPES) buffers at different pH values, often in the presence of acetonitrile, methanol, or tetrahydrofuran. An ammonium carbonate buffer is compatible with electrospray ionization (ESI)-MS/MS detection, with sufficient sensitivity to be useful for screening methods. Mi-
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Figure 3 Isocratic CEC of seven macrolides. Peaks: 1, spiramycin; 2, erythromycin; 3, oleandomycin; 4, josamycin; 5, azithromycin; 6, clarithromycin; 7, roxithromycin. Separation conditions: cationic column, 50 mm 30 cm (20 cm effective length); voltage, 15 kV; detection, 206 nm; mobile phase, 45% (v/v) acetonitrile and 10% (v/v) ethanol in 30 mM phosphate buffer, pH 8.0. (Reproduced with permission from Zhang S, Huang X, Yao N and Horva´th Cs (2002) Preparation of monodisperse porous polymethacrylate microspheres and their application in the capillary electrochromatography of macrolide antibiotics. Journal of Chromatography A 948: 193).
cellar systems using SDS, sodium cholate or sodium pentanesulfonate, and a mixed micellar system of sodium cholate and sodium heptanesulfonate have been used for the separation of quinolones. A nonaqueous capillary electrophoresis (NACE) method has also been demonstrated, using a methanol/acetonitrile solution containing hexadimethrine bromide for EOF reversal. Owing to the greater antibacterial activity of S( )-ofloxacin compared with R-( þ )-ofloxacin, an area of interest has been the separation of these enantiomers. The use of cyclodextrins has received considerable attention, primarily methyl-b-CD, sulfobutyl-b-CD, or sulfated b-CD. Another running buffer utilizes the combination of g-CD, zinc sulfate, and D-phenylalanine in ammonium acetate. The lack of optical resolution when one of the buffer components is missing suggests the formation of a diastereomeric ternary complex among the quinolone, Zn2 þ , and phenylalanine, which then forms an inclusion complex with g-CD. Other chiral buffer systems that have been investigated include aminosaccharide derivatives, bovine and human serum albumin, vancomycin, and ( þ )-18-crown6-tetracarboxylic acid. Sulfonamides
The members of this class of antibiotics are widely used in veterinary practices, with both therapeutic and preventative goals. Screening protocols are essential for the monitoring of tissue residues. In free-solution CE systems, the pH of the BGE can be manipulated to alter the separation of the sulfonamides, due to their wide range of pKa values. Citrate buffers, used at a low pH, separate sulfonamides as positively charged species. As the pH of the buffer system is increased, the sulfonamides transform to negatively charged species. Separations have been demonstrated in citrate-, phosphate- and phosphate/ borate-based systems at pH values between 6 and 7. Organic modifiers, SDS, and b-CD have been used as buffer additives to further manipulate separations. Another approach to the separation of sulfonamides, termed argentation electrophoresis, utilizes the complexation that occurs between Ag þ and pelectrons, and between Ag þ and the lone electron pairs of N-, O- and S-containing heterocyclic molecules. In a buffer solution at pH 5, seven of the nine sulfonamides examined comigrate as neutral species. Upon the addition of Ag þ and SDS, complexation occurs to varying degrees, and analyte separation is achieved. Condensation nucleation light scattering detection has been demonstrated as a sensitive and universal
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detection method for CEC. Using sulfanilic acid and sulfanilamide as examples, LODs on the order of 0.4 mg mL 1 are obtained when a diffusion screen is used to modify the size distribution of the spray particles. Without the screen, the LODs improve to approximately 60 ng mL 1. Separations have also been developed for the electrochemical detection of sulfonamides. Carbon electrodes in a wall-jet configuration yield LODs in the range of 0.1–1 mM. In another system, square-wave voltammetry produces responses that are linear between 50 nM and 10 mM for the sulfonamides studied. Tetracyclines
Due to the amphoteric nature of these compounds, analyses can occur under either acidic or alkaline conditions. To prevent metal complexation with the tetracyclines, ethylenediamenetetraacetic acid (EDTA) is added to the BGE, which may be composed of sodium carbonate, sodium phosphate, or sodium borate. Low amounts of an organic modifier such as methanol can also be added to aid in component resolution. Surfactants such as Triton X-100, mixtures of Tween 20 and Tween 80, Triton X-100 and Brij 35, and SDS and Brij 35, also enhance separation. NACE has also been useful in the separation of tetracycline antibiotics. Buffers containing mixtures of methanol/acetonitrile/dimethylformamide, and methanol/acetonitrile have been used. In another NACE system, the complexation between tetracyclines and metal ions is used to create strongly fluorescent metal chelates. Acetate salts of Zn2 þ , Mg2 þ , and Ca2 þ , in the presence of dimethylformamide, dimethyl sulfoxide, or N-methylformamide, provide highly intense fluorescent signals from oxytetracycline–metal complexes. A novel format for CEC involves the etching of the fused silica surface with ammonium hydrogen difluoride, followed by silylation/hydrosilylation and attachment of a C18 moiety. Using this configuration as the stationary phase, tetracyclines can be separated using a phosphate–Tris buffer at pH 2.14.
tubercular agents, rifampicin and pyrazinamide, are separated by MEKC with SDS in a borate buffer. DUP105, an antibacterial in the oxazolidinone class, is a neutral species. A CEC system utilizing C18 particles and an acetonitrile/Tris–HCl mobile phase can separate DUP105, its sulfone and a precursor. MEKC is useful for the analysis of the oxazolidinones linezolid and eperezolid, three congeners, and their impurities. A high-throughput MEKC system, using a 27 cm capillary, separates 2-methyl-4isothiazoli-3-one and 5-chloro-2-methyl-4-isothiazoli-3-one in less than 2 min. When the BGE conditions are adjusted to 0.1 M SDS in 0.05 M borate, pH 9.5, the separation takes place in less than 1 min. Kasugamycin and blasticidin S are antibiotic fungicides that are used to treat rice plant disease. Both are analyzed successfully using a capillary coated with a hydrophilic polymer and an acidic phosphate buffer as the BGE. Validamycin A, another fungicide, has no chromophore, so the addition of aminopyrine in the BGE is required for indirect detection.
Applications It is not possible to list the many ways in which CE has been applied to the practical analysis of antibiotics. Many of the methods described in the preceding sections have been applied to the various dosage forms in which antibiotics occur, and compare favorably with current compendial methods. CE-based protocols have also proven effective in the clinical monitoring of patients undergoing antibiotic therapies, and in the determination of residue levels in food and environmental samples. In many instances, the selectivities of the capillary electrophoretic screening protocols are more informative, because many compendial methods rely on nonspecific microbiological means of identification and assay. The following are meant to serve as illustrations of the various matrices and applications. Finished Dosage Forms
Others
Two electrochemical systems have been used for the detection of the antibacterial isoniazid, hydrazine, and methylhydrazine. One system utilizes a Pd particle-modified carbon fiber microdisk array electrode, and the second uses a Pt electrode modified with 4pyridyl hydroquinone. Alternatively, UV detection of isoniazid utilizes the interaction of salicylaldehyde-5sulfonate with primary amines, under weakly alkaline conditions in the presence of ethanol, to form stable Schiff bases. Isoniazid and two other anti-
Numerous examples exist, describing the assay of antibiotic dosage forms such as tablets, capsules, injectable and intravenous solutions, suspensions, nasal and ocular preparations, and medicated feeds. In many of these cases, the antibiotic is the only active ingredient, and its analysis can be accomplished readily with the methods described previously, without interference from excipient components. These methods also permit the monitoring of impurities and degradants. Products that contain more than one active ingredient, examples of which are found in
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Table 1 Examples of finished dosage pharmaceutical preparations containing multiple active ingredients Dosage form
Ingredients
Tablets
Sulfadiazine, sulfamethazine, and trimethoprim Sulfisoxazole and phenazopyridine Isoniazid, pyrazinamide, and rifampicin Isoniazid and pyrazinamide Sulfacetamide sulfate, prednisolone acetate, and phenylephrine Dexamethasone, trimethoprim, and/ or polymyxin B Prednisolone acetate and sulfacetamide Prednisolone, zinc bacitracin, and phenylephrine Tetracycline and streptomycin Dihydrostreptomycin and penicillin G procaine
Eye drops
Ocular ointment Nasal ointment Wettable powder Intravenous and injectable preparations
Ticarcillin and clavulinic acid Amoxicillin and clavulinic acid Ampicillin and sulbactam Piperacillin and tazobactam
Table 1, have also been analyzed. To improve sample throughput for the analysis of tablets containing sulfamethoxazole and trimethoprim, flow-injection analysis has been coupled to microfluidic CE. In one case, a modified falling drop interface is used for sample introduction. In another, a continuous online preconcentration method is established using a dynamic pH junction to improve detection sensitivity. Chloramphenicol readily hydrolyzes to 2-amino-1(4-nitrophenyl)-propane-1,3-diol. Using amperometric detection, the stability of over-the-counter eye drops containing chloramphenicol has been monitored. Two of the products contained only 85–90% of the declared level of chloramphenicol, 6–9 months prior to the product expiration date; another product contained only 74% of the active on its date of expiration. Another method is used to assess the stability of ceftazidime injectable preparations, indicating that refrigeration for 7 days is acceptable, but room temperature storage causes degradation within 18 h. Food Matrices
The monitoring of antibiotic residue levels in milk has led to the development of a variety of approaches to ensure adequate sensitivity and selectivity. A competitive immunofluorescence assay has been used, whereby chloramphenicol is extracted from milk using ethyl acetate. The sample is mixed with rabbit
antichloramphenicol antiserum in the presence of a fluorescein–chloramphenicol conjugate, and the quantity of unbound conjugate is determined using LIF detection. Following solid-phase extraction (SPE), tetracyclines can be separated using MEKC with LODs on the order of 1–5 ng mL 1. Another approach to the analysis of tetracyclines requires pretreatment of the milk with 10% trichloroacetic acid, followed by SPE and analysis by NACE. Sulfonamides can be derivatized with fluorescamine, separated by MEKC and quantitated using fluorescence detection, with LODs on the order of 1.6– 7.7 nM. Six sulfonamides in milk have been analyzed using CE with nano-electrospray quasi-MS/MS/MS. Using an electrolyte of 0.1 M formic acid, the sulfonamides can be detected at the 20 ppb level by CE/MS/MS in a precursor ion scan mode at m/z ¼ 156. Multiple reaction monitoring of the transitions from MH þ -156 þ yields detection limits at the high part per trillion level for all six sulfonamides. A second mass spectral method utilizes a continuous-flow apparatus to introduce online treated samples into the CE–MS system. A free-solution CE buffer has been used to determine residual streptomycin in egg yolks, with an LOD of 0.12 mg g 1 at 200 nm. Oxolinic acid can be monitored in fish feeds and in sea bass specimens, which are prepared in a pH 7 phosphate buffer, followed by an SPE procedure. This scheme permits UV detection with an LOD of 0.08 mg mL 1. A similar approach is used for the quantitation of oxytetracycline in raw and cooked channel catfish. The quantity of oxytetracycline residue present in the tissue decreases after cooking compared with the raw tissue, and different cooking methods (frying, baking, and smoking) degrade the oxytetracycline residue to different extents. A series of quinolones has been determined in chicken tissue, following sample pretreatment and an SPE procedure. Phosphate and diethylmalonic buffers, typically pH 8, are effective for the separation of analytes from matrix components. Using UV detection, the method is sensitive enough to detect residues at levels below the current maximum residue limits (MRLs) set by the European Union (Figure 4). In another approach, NACE has been used to determine quinolones in pig kidney samples. Oxytetracycline can be analyzed in pig tissue samples using a sodium carbonate buffer, with EDTA added. UV responses are linear from 200 to 2000 mg kg 1 in both kidney and liver, and from 100 to 2000 mg kg 1 in muscle, with LODs below the current MRLs for these tissues. A phosphate-based buffer system is used for the detection of sulfonamides in chicken, beef, and pork samples, following SPE sample
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Minutes Figure 4 Electropherogram of chicken muscle tissue spiked with 100 mg kg 1 for difloxacin, sarafloxacin, and the internal standard, marbofloxacin. Peaks: 1, sarafloxacin; 2, marbofloxacin; 3, difloxacin. Separation conditions: capillary, 75 mm 57 cm (50 cm effective length); voltage, 20 kV; detection, 275 nm; buffer, 35 mM diethylmalonic buffer, pH 8.22. (Reproduced with permission from Barro´n D, Jime´nez-Lozano E, Bailac S and Barbosa J (2002) Determination of difloxacin and sarafloxacin in chicken muscle using solid-phase extraction and capillary electrophoresis. Journal of Chromatography B 767: 313).
preparation. This method is also adequate for monitoring these matrices for sulfonamides at the current MRLs. Clinical Samples
Amoxicillin, florfenicol, and quinolones have been determined in pig plasma, using borate buffers. In the amoxicillin work, the use of field-amplified sample injection lowers the LOD to 280 mg L 1. Detection levels are also improved through the use of capillary isotachophoresis as an on-capillary stacking technique, as is demonstrated in the analysis of quinolones. NACE is used for tetracyclines following sample treatment of pig plasma by SPE. For the detection of aminoglycosides in human serum and plasma, pre- and in-capillary derivatization reactions have been utilized. Amikacin, arbekacin, dibekacin, and kanamycin have been derivatized with IDA, followed by fluorescence detection. Gentamicin, tobramycin, and kanamycin have been derivatized with OPA and either mercaptoacetic acid or mercaptopropionic acid. The ability to monitor peak and trough plasma concentrations of tobramycin or kanamycin in tuberculosis patients is demonstrated. The need for sample pretreatment depends on the matrix, the expected level of the analyte, and the separation system itself. Microdialysate and cerebrospinal fluid samples typically require only dilution and filtration, as is demonstrated by the analysis of
cefazolin and cefpirome in microdialysate samples. Cefepime and ceftazidime have been detected in cerebrospinal fluid to monitor the efficacy of treatments for bacterial meningitis (Figure 5). Using a citrate buffer at pH 6, cephalosporins can be determined in plasma without sample pretreatment. The analysis of quinolones in a HEPES buffer system can proceed after treatment with trichloroacetic acid. The determination of cefotaxime and its metabolite, deacetylcefotaxime, requires protein removal with acetonitrile prior to analysis in a borate buffer. However, using MEKC systems, cefotaxime, cefepime, ceftazidime, and vancomycin can be analyzed with direct injection of serum. MEKC is also useful for the analysis of sulfamethoxazole, cefuroxime, and cefpirome in serum. Nalidixic acid and its metabolites can be analyzed by MEKC after sample acidification and extraction with chloroform. Following protein precipitation with acetonitrile, linezolid can be detected at the 0.5 mg L 1 level. Thiamphenicol has also been determined in serum using an MEKC method. Sample introduction using a sweeping preconcentration approach has been applied to the determination of doxorubicin in plasma. Following sample cleanup, the solution is injected electrokinetically for 5 min at 30 kV. The sample vial is replaced with a vial containing 150 mM SDS dissolved in the BGE. Using UV detection, the levels of doxorubicin can be monitored at 100 min (397 nM) and at 300 min (60 nM) after the administration of a 30 mg m 2 dose of doxorubicin.
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Figure 5 Electropherograms of CSF from a meningitis patient receiving intravenous 2 g of cefepime. (a) Before dosing; (b) after dosing at 8 h. Peaks: 1, cefazolin (internal standard); 2, cefepime. Separation conditions: capillary, 75 mm 31.2 cm (21 cm effective length); voltage, 8 kV; detection, 254 nm; buffer, 200 mM SDS, 20 mM Tris, pH 9.0. (Reproduced with permission from Tseng S-H, Yang Y-H, Chen Y-R and Chen S-H (2004) Determination of cefepime in plasma and cerebrospinal fluid by micellar electrokinetic chromatography with direct sample injection. Electrophoresis 25: 1641).
Another on-capillary preconcentration technique utilizes the ionic nature of the analyte. Blood containing rapamycin is treated by protein precipitation and SPE cleanup. Under alkaline conditions, the lactone bond of the macrolide is cleaved, creating an open-ring, anionic species. Electrokinetic injection under reversed polarity causes the negatively-charged rapamycin to migrate into the capillary, where it meets the acidic BGE. Rapamycin becomes protonated, creating a neutral species that does not migrate further. Focusing is achieved during injection, leading to an LOD of 0.2 mg L 1 (Figure 6). Fosfomycin levels in plasma, microdialysate, and pus samples are determined under reversed EOF conditions, using a cationic surfactant as BGE, and benzoic acid for indirect detection. With LODs ranging from 0.6 to 2 mg mL 1, the detection of fosfomycin in pus samples indicates that the antibiotic is reaching the sites of the abscesses. Electrochemical detection schemes have been used for the determination of mitomycin C and chloramphenicol. Owing to the selectivity of the electrode configurations used, no interfering peaks are observed. Greater sensitivity can also be achieved through the use of LIF detection. This approach is used for
the determination of ciprofloxacin and its metabolite desethyleneciprofloxacin, and moxifloxacin in plasma and microdialysate, with LOQs of the order of 2.5 mg L 1 in plasma. NACE combined with metal complexation and LIF has been utilized in the separation and detection of tetracyclines. Using freesolution CE, idarubicin and its metabolite idarubicinol have been determined, following extraction in chloroform. The LOQ achieved is 0.5 ng mL 1, which permits the monitoring of pediatric oncology patients, and requires only 100 mL of plasma. A similar system has been developed for doxorubicin and doxorubicinol, and idarubicin and doxorubicin have been determined in the serum of patients receiving treatment for ovarian cancer. Additional studies monitor doxorubicin and its metabolites in cell extracts and in single cells, and demonstrate differences in intracellular metabolism between liposomal and free doxorubicin in the treatment of human leukemia cells. Peak plasma concentrations of daunorubicin and daunorubicinol are monitored in children being treated with either free daunorubicin or DaunoXomes, a liposomal formulation of daunorubicin. The total peak plasma levels were
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Figure 6 Electropherograms of a blood sample spiked with 10 mg L 1 rapamycin including the online spectrum (a) of the ring-open rapamycin and (b) a blank blood sample. Separation conditions: capillary, 75 mm 45 cm (36.5 cm effective length); voltage, 15 kV; detection, 278 nm; buffer, 80 mM SDS, 30 mM H3PO4 in methanol/water (20:80, v/v); electrokinetic injection for 300 s at 7.5 kV. (Reproduced with permission from Buchberger W, Ferdig M, Sommer R and Vo TDT (2005) A novel technique for on-capillary preconcentration of anionic compounds applied to the trace analysis of rapamycin in human blood by capillary electrophoresis. Electrophoresis 26: 161).
approximately two orders of magnitude higher after dosing with DaunoXomes than after administration of the free drug. Another method monitors daunorubicin levels in plasma and biopsy material from patients who have Kaposi sarcomas, demonstrating that the daunorubicin is reaching the tumor. In many instances, the analysis of urine can proceed without sample preparation beyond dilution and filtration. The combination of pasiniazide, capreomycin, and ofloxacin is used in the treatment of pulmonary tuberculosis. These analytes can be monitored in urine in a borax buffer containing 10%
ethanol. Cephalosporins can be determined using a citrate buffer. Quinolones can be separated in HEPES or phosphate–borate buffer systems, and separated from pharmaceuticals that may be coadministered. Nalidixic acid and two of its metabolites can be extracted from urine with chloroform, and analyzed using MEKC, with LODs of 0.3–0.7 ng mL 1. Carbon fiber microelectrodes, used in the end-column configuration, are able to detect sulfadiazine and sulfamethoxazole in urine at the 0.1 mM level, and midecamycin at the 0.5 mM level. Daunorubicin can be quantitated using a carbon disc electrode in
10
III / CAPILLARY ELECTROPHORESIS / Antibiotics
the wall-jet configuration at a similar level. Electrochemiluminescence detection, in the presence of Tris(2,20 -bipyridine) ruthenium (II) (Ru(bpy)23 þ ) has been applied to the analysis of lincomycin and ofloxacin in urine, at LODs of 9.0 mM and 500 nM, respectively. LIF has been used for the sensitive detection of ofloxacin and its metabolites. Sulfonamides in urine have been analyzed by coupling an SPE apparatus to the CE instrument via a switching valve. This sample pretreatment and injection protocol yields LODs of 10 ng mL 1. Another means of removing matrix interferences is the use of a channel-coupled microchip electrophoresis device. In the first channel, Na þ and K þ ions in urine are separated from gemifloxacin, and are sent to waste. In the second separation channel, the gemifloxacin enantiomers are separated using only 50 mM of the chiral selector ( þ )-18-crown-6-tetracarboxylic acid. Total analysis time is less than 4 min. Environmental Samples
The elimination of antibiotics in waste products, and their subsequent presence in the environment, is suspected of being responsible for the evolution of antibiotic-resistant bacterial strains. CE and MEKC separation schemes for quinolones have been developed using LIF detection. Photodegradation studies have been conducted on enrofloxacin in water alone, and in the presence of either 20 mg L 1 fulvic acids or 20 mg L 1 humic acids, resulting in differences in the identities and levels of products present. Affinity CE demonstrates that strong binding of some degradation products to the humic acids protects them from degradation. ESI-MS detection has been combined with CE for the determination of penicillin V in river water. A standard solution of penicillin V is analyzed using a 20 mM ammonium acetate buffer at pH 5.1, with LODs of 50 mg L 1 in the positive ionization mode, and 134 mg L 1 in the negative ionization mode. An MEKC approach has also been developed for the monitoring of penicillins in farm water samples. A method for tetracyclines uses a flow manifold coupled online to the CE system. Following preconcentration on an SPE minicolumn, the analytes are introduced into the CE autosampler vial. Using this protocol, UV detection yields sensitivities on the order of 2–20 mg L 1.
Conclusions Capillary electrophoresis and its related techniques have proven to be effective in the analysis of antibiotics. Screening protocols, assay and impurity methods, and clinical and environmental monitoring procedures are viable alternatives to many of the compendial methods that are currently in use. Further developments in sample treatment strategies, either as automated off-line procedures coupled to CE, or as in-capillary approaches, will reduce total analysis time and will enhance detection capabilities. Microfluidic systems offer rapid throughput with low sample volume requirements. The interfacing of CE-related systems to a mass spectrometer offers the unequivocal identification of residues.
See also: II/Capillary Electrophoresis: Microemulsion Electrokinetic Chromatography; Preconcentration Techniques. III/Antibiotics: Liquid Chromatography.
Further Reading Anderson AB, Gergen J and Arriaga EA (2002) Detection of doxorubicin and metabolites in cell extracts and in single cells by capillary electrophoresis with laserinduced fluorescence detection. Journal of Chromatography B 769: 97. Eder AR and Arriaga EA (2005) Micellar electrokinetic capillary chromatography reveals differences in intracellular metabolism between liposomal and free doxorubicin treatment of human leukemia cells. Journal of Chromatography B 829: 115. Herna´ndez M, Borrull F and Calull M (2003) Analysis of antibiotics in biological samples by capillary electrophoresis. Trends in Analytical Chemistry 22: 416. Jime´nez-Lozano E, Roy D, Barro´n D and Barbosa J (2004) Effective sorbents for solid-phase extraction in the analysis of quinolones in animal tissues by capillary electrophoresis. Electrophoresis 25: 65. McCourt J, Bordin G and Rodrı´guez AR (2003) Development of a capillary zone electrophoresis-electrospray ionization tandem mass spectrometry method for the analysis of fluoroquinolone antibiotics. Journal of Chromatography A 990: 259. Santos B, Lista A, Simonet BM, Rı´os A and Valca´rcel M (2005) Screening and analytical confirmation of sulfonamide residues in milk by capillary electrophoresismass spectrometry. Electrophoresis 26: 1567.
Introduction An antibiotic is a substance that has the capacity to inhibit the growth of, and to destroy, bacteria and other microorganisms. This group of pharmaceuticals is one of the most widely prescribed worldwide, and is used in human and veterinary medicine to treat and prevent diseases. New antibiotics are being developed to improve efficacy and to combat the resistant bacteria that are encountered. Capillary electrophoresis (CE) and its related techniques offer effective alternatives to analytical methodologies currently in use for the detection, quantitation, and characterization of these important medications.
Classes of Antibiotics Aminoglycosides
An aminoglycoside antibiotic is comprised of amino sugars that are joined through glycoside linkages. Primary buffer systems for these antibiotics incorporate borate or phosphate–borate combinations as the background electrolyte (BGE). Micellar electrokinetic chromatography (MEKC) has been utilized with sodium pentanesulfonate as the anionic surfactant. One mixed micellar system incorporates sodium dodecyl sulfate (SDS) and Brij 35, and another combines the bile salt sodium deoxycholate and b-cyclodextrin (b-CD). A fundamental difficulty in the detection of aminoglycosides is their lack of a chromophore. One approach to their detection involves the formation of negatively charged, UV-absorbing complexes between borate and the diol moieties of the amino sugars. A number of aminoglycosides and their related substances have been separated and detected this way. Another successful approach is indirect detection, using imidazole in the BGE, and a cationic surfactant for the reversal of the electroosmotic flow (EOF).
Precapillary derivatization has been used successfully with members of this class of antibiotics, when o-phthalaldehyde (OPA) is combined with mercaptoacetic acid, mercaptopropionic acid, or thioglycolic acid. Electrophoretically mediated microanalysis (EMMA) is used for the in-capillary derivatization of aminoglycosides. This involves the introduction of the analyte solution as a ‘sandwich’ between two zones of reagent buffer that contain the derivatizing reagents. A brief application of voltage causes these zones to merge. After an incubation period during which derivatization occurs, the BGE is introduced and component separation proceeds (Figure 1). Another reagent that reacts with primary amines, 1-methoxycarbonylindolizine-3,5-dicarbaldehyde (IDA), produces derivatives that absorb strongly at 409 nm. This reagent has been utilized in an online derivatization mode, with both UV and fluorescence detection. Another approach has incorporated an immunoassay that establishes a competition between free gentamicin and fluorescein-labeled gentamicin for the antibody. Electrochemical detection has been used as an alternative to analyte derivatization, typically with sodium hydroxide as the BGE. Copper or coppermodified carbon fiber microdisk electrodes aligned in wall-jet configurations, or on-capillary electrodes composed of copper or copper-modified gold have been used, generating limits of detection (LODs) in the 1–2 mM range. Another recent approach has been the use of potential gradient detection for the determination of gentamicin components. This detection method measures the mobility differences between sample molecules and the separation buffer’s co-ions, or the potential gradient along the axis of the capillary. Amphenicols
Studies of chloramphenicol, thiamphenicol, and florfenicol have utilized phosphate and borate buffer systems. Because these analytes are neutral, MEKC and capillary electrochromatography (CEC) methods have also been developed. SDS is used as a buffer additive in MEKC and in CEC, where it is added to acetonitrile/water mobile phases, which are combined with XTerra RP18 or Hypersil ODS stationary phases. Detection schemes have incorporated amperometry, with a carbon fiber microdisk array in an end-column configuration, and a carbon disk electrode in a wall-jet configuration. Laser-induced fluorescence (LIF) detection has been used in a
2 III / CAPILLARY ELECTROPHORESIS / Antibiotics 20
Absorbance (mAU)
15
6
10 a
1 b d 3 c 2
5
9
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Figure 1 Electropherogram of a commercial sample of 0.7 mg mL 1 kanamycin sulfate, using CZE-EMMA. Peaks: a, b, degradands; c, d, unknown; 1, reagent; 2, 2-deoxystreptamine; 3, kanamycin D; 4, 6-O-(3-amino-3-deoxy-a-D-glucopyranosyl) deoxystreptamine; 5, 4-O-(6-amino-6-deoxy-a-D-glucopyranosyl) deoxystreptamine þ paromamine; 6, kanamycin A; 7, 1-N-(1-hydroxymethyl2-hydroxyethyl) kanamycin B; 8, kanamycin C; 9, kanamycin B. Separation conditions: capillary, 50 mm 40 cm (31.5 cm effective length); electric field, 588 V cm 1; detection, 335 nm; buffer, 16% (v/v) methanol in 30 mM sodium tetraborate, pH 10.0. (Reproduced with permission from Kaale E, Van Schepdael A, Roots E and Hoogmartens J (2003) Determination of kanamycin by electrophoretically mediated microanalysis with in-capillary derivatization and UV detection. Electrophoresis 24: 1119.)
competitive immunoassay, in which a sample containing chloramphenicol is mixed with rabbit antichloramphenicol antiserum in the presence of a fluorescein–chloramphenicol conjugate. Anthracyclines
These compounds are very powerful antitumor antibiotics. Many of the applications developed have incorporated LIF detection for the monitoring of doxorubicin, daunorubicin, and idarubicin, and their metabolites. Most of these systems utilize combinations of an organic modifier and phosphate or borate, with spermine added to reduce interactions between the analytes and the capillary wall. The argon ion laser line at 488 nm is the most widely used, and can yield limits of quantitation (LOQs) of 0.5 ng mL 1 in serum samples, depending on the sample preparation protocol. CEC is combined with LIF detection for the analysis of doxorubicin labeled with a dicarbocyanine moiety. Electrochemical detection also provides the sensitivity and selectivity needed for clinical applications, such as the determination of mitomycin in serum and daunorubicin in urine. The sensitivity of UV detection can be improved through the use of a sweeping preconcentration method. In this mode, the pH of the BGE is such that the anthracyclines are positively charged. The sample is introduced hydrodynamically until approximately
80% of the capillary is filled. The BGE, which contains SDS, is placed in the inlet vial. In the reversed polarity mode, the positively charged anthracyclines migrate toward the negatively charged micelles. When the zones meet, the anthracyclines are solubilized by the micelles and are drawn toward the detector. LODs on the order of 1 nM can be achieved under these conditions.
b-Lactams
For this class of antibiotics that includes penicillins, cephalosporins, and carbapenems, the most widely used means of component separation is MEKC. Using SDS buffer systems with acetate, phosphate, and/ or borate as the BGE, numerous protocols have been developed for the screening of members within the class, or for the separation of impurities from the primary component. Mixed micellar systems of SDS and Brij 35 or pentanesulfonic acid provide improved resolution through the alteration of the pseudostationary phase. Free-solution CE buffers using phosphate, citrate, borate, and 2-(N-morpholino) ethanesulfonic acid have found applications as screening methods, but these systems are primarily used for dosage form analysis. The free-solution systems are also useful for separating members of the different subgroups from each other, and from other pharmaceuticals that are prescribed in tandem.
III / CAPILLARY ELECTROPHORESIS / Antibiotics 3
Microemulsion electrokinetic chromatography (MEEKC) has been utilized in the analysis of several compounds within this class of antibiotics. Microemulsions are solutions containing dispersed nanometer-sized droplets of an immiscible liquid. These droplets consist of an immiscible oil suspended in water. The surface tension between the two liquid phases is reduced by adding a surfactant to coat the oil droplets. The surface tension is lowered further by adding a short-chain alcohol, called a cosurfactant, which stabilizes the microemulsion. Microemulsions have been prepared from mixtures of 1-butanol, heptane, sodium glycodeoxycholate, Tween 20, and phosphate buffer. Another study uses a mixture of octane, 1-butanol, SDS, and borate buffer. A system composed of ethyl acetate, 1-butanol, Brij 35, 2-butanol, and 10 mM borate buffer can be used for the separation of eight penicillins (Figure 2). To improve UV sensitivity, a sample stacking technique called the reversed electrode polaritystacking mode (REPSM) is used. After the sample is introduced into the capillary hydrodynamically, a reversed polarity is applied. This preconcentrates the analytes at the interface between the sample zone and the BGE, while the sample matrix is expelled from the capillary by the EOF. A conventional polarity is then applied, and the analysis proceeds. Glycopeptides
Absorbance (mAU)
The compounds within this class can be difficult to analyze, because many of them occur as mixtures. For example, bacitracin is a complex mixture of bacitracins A, B, C, D, E, F, G, H, and I, many of which have isomers or amino acid substitutions. A mixed micellar system comprised of the zwitterionic
surfactant 3-(N,N-dimethylhexadecylammonium) propanesulfonate and the nonionic Brij 35 can resolve the numerous components found in bacitracin. Polymyxin B is a cyclic hexapeptide with a tripeptide side chain that differs in the attached fatty acyl residue, giving rise to the components B1, B2, B3, and B4. The presence of methyl-b-CD provides the selectivity to separate the major components as well as minor peaks surrounding polymyxin B1. A similar experimental system is utilized for the analysis of colistin sulfate and its components. Other MEKC separation schemes have used either SDS or cetyltrimethylammonium chloride for the analysis of amphotericin B, zinc bacitracin, vancomycin, avoparcin, ristocetin, and polymyxin B. Vancomycin has also been analyzed in phosphate buffer in 3 min by using a capillary having an effective length of 8.5 cm. Macrolides
Macrolides are antibiotics that are produced by Streptomyces bacteria found in soil, and are comprised of a sugar, an amino sugar, and a 14- or 16member aglycone ring. Although free-solution systems have been utilized, many of the macrolides possess low water solubility; thus, most applications require the use of solubilizing agents in the separation medium. Varying percentages of acetonitrile, methanol, and ethanol have been added to phosphate systems to improve analyte solubility and selectivity. Unlike the alkyl-chain surfactants, bile salts are able to retain their micellar structures in the presence of higher levels of organic solvents. Sodium cholatebased systems, with the addition of acetonitrile to enhance analyte solubility, have been utilized in screening methods. The separations of spiramycins I,
6 4 7
7.5 5 2.5 0 −2.5 −5 −7.5 −10 −12.5
5 2 1
8 3
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8 Time (min)
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Figure 2 Electropherogram of a standard mixture of penicillins in a concentration of 500 mg L 1. Peaks: 1, amoxicillin; 2, ampicillin; 3, penicillin G; 4, oxacillin; 5, penicillin V; 6, cloxacillin; 7, nafcillin; 8, dicloxacillin. REPSM injection conditions: 50 mbar for 270 s, 5 kV until 95% of original current reached. Separation conditions: capillary, 75 mm 45 cm (36.5 cm effective length); voltage, 10 kV; detection, 210 nm; buffer, 0.5% ethyl acetate, 1.2% 1-butanol, 2% Brij 35, 10% 2-butanol, and 86.3% 10 mM borate buffer, pH 10.0. (Reproduced with permission from Puig P, Borrull F, Aguilar C and Calull M (2006) Sample stacking for the analysis of penicillins by microemulsion electrokinetic capillary chromatography. Journal of Chromatography B 831: 196).
4 III / CAPILLARY ELECTROPHORESIS / Antibiotics
II, III, and IV, neospiramycins I, II, and III, and tylosin, erythromycin and their related substances have been achieved in mixed micellar systems containing sodium cholate and the cationic surfactant cetyltrimethylammonium bromide. Methyl-b-CD has also been used in macrolide systems. CEC systems have presented alternatives for the analysis of macrolide antibiotics. Monodisperse poly(glycidyl methacrylate-divinylbenzene) microspheres are derivatized with quaternary ammonium/octadecyl groups to generate a positively charged stationary phase (Figure 3). Another system utilizes capillaries packed with 3.5 mm XTerra RP18 particles, and an SDS/acetonitrile/ammonia mobile phase. Quinolones
Quinolones have emerged as one of the most important classes of synthetic antibiotics, with applications in human and veterinary medicine. As such, numerous screening methods have been developed for pharmaceutical preparations, feed mixes, and animal tissues. Free-solution systems provide adequate separation through the use of borate, boric acid, phosphate, and N-(2-hydroxyethyl)piperazine-N0 -(2ethanesulfonic acid) (HEPES) buffers at different pH values, often in the presence of acetonitrile, methanol, or tetrahydrofuran. An ammonium carbonate buffer is compatible with electrospray ionization (ESI)-MS/MS detection, with sufficient sensitivity to be useful for screening methods. Mi-
Absorbance at 206 nm (mAU)
6 2 4 DMSO
4 6 3 5 7
2 1 0
0
5 Minutes
10
Figure 3 Isocratic CEC of seven macrolides. Peaks: 1, spiramycin; 2, erythromycin; 3, oleandomycin; 4, josamycin; 5, azithromycin; 6, clarithromycin; 7, roxithromycin. Separation conditions: cationic column, 50 mm 30 cm (20 cm effective length); voltage, 15 kV; detection, 206 nm; mobile phase, 45% (v/v) acetonitrile and 10% (v/v) ethanol in 30 mM phosphate buffer, pH 8.0. (Reproduced with permission from Zhang S, Huang X, Yao N and Horva´th Cs (2002) Preparation of monodisperse porous polymethacrylate microspheres and their application in the capillary electrochromatography of macrolide antibiotics. Journal of Chromatography A 948: 193).
cellar systems using SDS, sodium cholate or sodium pentanesulfonate, and a mixed micellar system of sodium cholate and sodium heptanesulfonate have been used for the separation of quinolones. A nonaqueous capillary electrophoresis (NACE) method has also been demonstrated, using a methanol/acetonitrile solution containing hexadimethrine bromide for EOF reversal. Owing to the greater antibacterial activity of S( )-ofloxacin compared with R-( þ )-ofloxacin, an area of interest has been the separation of these enantiomers. The use of cyclodextrins has received considerable attention, primarily methyl-b-CD, sulfobutyl-b-CD, or sulfated b-CD. Another running buffer utilizes the combination of g-CD, zinc sulfate, and D-phenylalanine in ammonium acetate. The lack of optical resolution when one of the buffer components is missing suggests the formation of a diastereomeric ternary complex among the quinolone, Zn2 þ , and phenylalanine, which then forms an inclusion complex with g-CD. Other chiral buffer systems that have been investigated include aminosaccharide derivatives, bovine and human serum albumin, vancomycin, and ( þ )-18-crown6-tetracarboxylic acid. Sulfonamides
The members of this class of antibiotics are widely used in veterinary practices, with both therapeutic and preventative goals. Screening protocols are essential for the monitoring of tissue residues. In free-solution CE systems, the pH of the BGE can be manipulated to alter the separation of the sulfonamides, due to their wide range of pKa values. Citrate buffers, used at a low pH, separate sulfonamides as positively charged species. As the pH of the buffer system is increased, the sulfonamides transform to negatively charged species. Separations have been demonstrated in citrate-, phosphate- and phosphate/ borate-based systems at pH values between 6 and 7. Organic modifiers, SDS, and b-CD have been used as buffer additives to further manipulate separations. Another approach to the separation of sulfonamides, termed argentation electrophoresis, utilizes the complexation that occurs between Ag þ and pelectrons, and between Ag þ and the lone electron pairs of N-, O- and S-containing heterocyclic molecules. In a buffer solution at pH 5, seven of the nine sulfonamides examined comigrate as neutral species. Upon the addition of Ag þ and SDS, complexation occurs to varying degrees, and analyte separation is achieved. Condensation nucleation light scattering detection has been demonstrated as a sensitive and universal
III / CAPILLARY ELECTROPHORESIS / Antibiotics 5
detection method for CEC. Using sulfanilic acid and sulfanilamide as examples, LODs on the order of 0.4 mg mL 1 are obtained when a diffusion screen is used to modify the size distribution of the spray particles. Without the screen, the LODs improve to approximately 60 ng mL 1. Separations have also been developed for the electrochemical detection of sulfonamides. Carbon electrodes in a wall-jet configuration yield LODs in the range of 0.1–1 mM. In another system, square-wave voltammetry produces responses that are linear between 50 nM and 10 mM for the sulfonamides studied. Tetracyclines
Due to the amphoteric nature of these compounds, analyses can occur under either acidic or alkaline conditions. To prevent metal complexation with the tetracyclines, ethylenediamenetetraacetic acid (EDTA) is added to the BGE, which may be composed of sodium carbonate, sodium phosphate, or sodium borate. Low amounts of an organic modifier such as methanol can also be added to aid in component resolution. Surfactants such as Triton X-100, mixtures of Tween 20 and Tween 80, Triton X-100 and Brij 35, and SDS and Brij 35, also enhance separation. NACE has also been useful in the separation of tetracycline antibiotics. Buffers containing mixtures of methanol/acetonitrile/dimethylformamide, and methanol/acetonitrile have been used. In another NACE system, the complexation between tetracyclines and metal ions is used to create strongly fluorescent metal chelates. Acetate salts of Zn2 þ , Mg2 þ , and Ca2 þ , in the presence of dimethylformamide, dimethyl sulfoxide, or N-methylformamide, provide highly intense fluorescent signals from oxytetracycline–metal complexes. A novel format for CEC involves the etching of the fused silica surface with ammonium hydrogen difluoride, followed by silylation/hydrosilylation and attachment of a C18 moiety. Using this configuration as the stationary phase, tetracyclines can be separated using a phosphate–Tris buffer at pH 2.14.
tubercular agents, rifampicin and pyrazinamide, are separated by MEKC with SDS in a borate buffer. DUP105, an antibacterial in the oxazolidinone class, is a neutral species. A CEC system utilizing C18 particles and an acetonitrile/Tris–HCl mobile phase can separate DUP105, its sulfone and a precursor. MEKC is useful for the analysis of the oxazolidinones linezolid and eperezolid, three congeners, and their impurities. A high-throughput MEKC system, using a 27 cm capillary, separates 2-methyl-4isothiazoli-3-one and 5-chloro-2-methyl-4-isothiazoli-3-one in less than 2 min. When the BGE conditions are adjusted to 0.1 M SDS in 0.05 M borate, pH 9.5, the separation takes place in less than 1 min. Kasugamycin and blasticidin S are antibiotic fungicides that are used to treat rice plant disease. Both are analyzed successfully using a capillary coated with a hydrophilic polymer and an acidic phosphate buffer as the BGE. Validamycin A, another fungicide, has no chromophore, so the addition of aminopyrine in the BGE is required for indirect detection.
Applications It is not possible to list the many ways in which CE has been applied to the practical analysis of antibiotics. Many of the methods described in the preceding sections have been applied to the various dosage forms in which antibiotics occur, and compare favorably with current compendial methods. CE-based protocols have also proven effective in the clinical monitoring of patients undergoing antibiotic therapies, and in the determination of residue levels in food and environmental samples. In many instances, the selectivities of the capillary electrophoretic screening protocols are more informative, because many compendial methods rely on nonspecific microbiological means of identification and assay. The following are meant to serve as illustrations of the various matrices and applications. Finished Dosage Forms
Others
Two electrochemical systems have been used for the detection of the antibacterial isoniazid, hydrazine, and methylhydrazine. One system utilizes a Pd particle-modified carbon fiber microdisk array electrode, and the second uses a Pt electrode modified with 4pyridyl hydroquinone. Alternatively, UV detection of isoniazid utilizes the interaction of salicylaldehyde-5sulfonate with primary amines, under weakly alkaline conditions in the presence of ethanol, to form stable Schiff bases. Isoniazid and two other anti-
Numerous examples exist, describing the assay of antibiotic dosage forms such as tablets, capsules, injectable and intravenous solutions, suspensions, nasal and ocular preparations, and medicated feeds. In many of these cases, the antibiotic is the only active ingredient, and its analysis can be accomplished readily with the methods described previously, without interference from excipient components. These methods also permit the monitoring of impurities and degradants. Products that contain more than one active ingredient, examples of which are found in
6 III / CAPILLARY ELECTROPHORESIS / Antibiotics
Table 1 Examples of finished dosage pharmaceutical preparations containing multiple active ingredients Dosage form
Ingredients
Tablets
Sulfadiazine, sulfamethazine, and trimethoprim Sulfisoxazole and phenazopyridine Isoniazid, pyrazinamide, and rifampicin Isoniazid and pyrazinamide Sulfacetamide sulfate, prednisolone acetate, and phenylephrine Dexamethasone, trimethoprim, and/ or polymyxin B Prednisolone acetate and sulfacetamide Prednisolone, zinc bacitracin, and phenylephrine Tetracycline and streptomycin Dihydrostreptomycin and penicillin G procaine
Eye drops
Ocular ointment Nasal ointment Wettable powder Intravenous and injectable preparations
Ticarcillin and clavulinic acid Amoxicillin and clavulinic acid Ampicillin and sulbactam Piperacillin and tazobactam
Table 1, have also been analyzed. To improve sample throughput for the analysis of tablets containing sulfamethoxazole and trimethoprim, flow-injection analysis has been coupled to microfluidic CE. In one case, a modified falling drop interface is used for sample introduction. In another, a continuous online preconcentration method is established using a dynamic pH junction to improve detection sensitivity. Chloramphenicol readily hydrolyzes to 2-amino-1(4-nitrophenyl)-propane-1,3-diol. Using amperometric detection, the stability of over-the-counter eye drops containing chloramphenicol has been monitored. Two of the products contained only 85–90% of the declared level of chloramphenicol, 6–9 months prior to the product expiration date; another product contained only 74% of the active on its date of expiration. Another method is used to assess the stability of ceftazidime injectable preparations, indicating that refrigeration for 7 days is acceptable, but room temperature storage causes degradation within 18 h. Food Matrices
The monitoring of antibiotic residue levels in milk has led to the development of a variety of approaches to ensure adequate sensitivity and selectivity. A competitive immunofluorescence assay has been used, whereby chloramphenicol is extracted from milk using ethyl acetate. The sample is mixed with rabbit
antichloramphenicol antiserum in the presence of a fluorescein–chloramphenicol conjugate, and the quantity of unbound conjugate is determined using LIF detection. Following solid-phase extraction (SPE), tetracyclines can be separated using MEKC with LODs on the order of 1–5 ng mL 1. Another approach to the analysis of tetracyclines requires pretreatment of the milk with 10% trichloroacetic acid, followed by SPE and analysis by NACE. Sulfonamides can be derivatized with fluorescamine, separated by MEKC and quantitated using fluorescence detection, with LODs on the order of 1.6– 7.7 nM. Six sulfonamides in milk have been analyzed using CE with nano-electrospray quasi-MS/MS/MS. Using an electrolyte of 0.1 M formic acid, the sulfonamides can be detected at the 20 ppb level by CE/MS/MS in a precursor ion scan mode at m/z ¼ 156. Multiple reaction monitoring of the transitions from MH þ -156 þ yields detection limits at the high part per trillion level for all six sulfonamides. A second mass spectral method utilizes a continuous-flow apparatus to introduce online treated samples into the CE–MS system. A free-solution CE buffer has been used to determine residual streptomycin in egg yolks, with an LOD of 0.12 mg g 1 at 200 nm. Oxolinic acid can be monitored in fish feeds and in sea bass specimens, which are prepared in a pH 7 phosphate buffer, followed by an SPE procedure. This scheme permits UV detection with an LOD of 0.08 mg mL 1. A similar approach is used for the quantitation of oxytetracycline in raw and cooked channel catfish. The quantity of oxytetracycline residue present in the tissue decreases after cooking compared with the raw tissue, and different cooking methods (frying, baking, and smoking) degrade the oxytetracycline residue to different extents. A series of quinolones has been determined in chicken tissue, following sample pretreatment and an SPE procedure. Phosphate and diethylmalonic buffers, typically pH 8, are effective for the separation of analytes from matrix components. Using UV detection, the method is sensitive enough to detect residues at levels below the current maximum residue limits (MRLs) set by the European Union (Figure 4). In another approach, NACE has been used to determine quinolones in pig kidney samples. Oxytetracycline can be analyzed in pig tissue samples using a sodium carbonate buffer, with EDTA added. UV responses are linear from 200 to 2000 mg kg 1 in both kidney and liver, and from 100 to 2000 mg kg 1 in muscle, with LODs below the current MRLs for these tissues. A phosphate-based buffer system is used for the detection of sulfonamides in chicken, beef, and pork samples, following SPE sample
III / CAPILLARY ELECTROPHORESIS / Antibiotics 7
0.0026 0.0024 0.0022 0.0020 2
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Minutes Figure 4 Electropherogram of chicken muscle tissue spiked with 100 mg kg 1 for difloxacin, sarafloxacin, and the internal standard, marbofloxacin. Peaks: 1, sarafloxacin; 2, marbofloxacin; 3, difloxacin. Separation conditions: capillary, 75 mm 57 cm (50 cm effective length); voltage, 20 kV; detection, 275 nm; buffer, 35 mM diethylmalonic buffer, pH 8.22. (Reproduced with permission from Barro´n D, Jime´nez-Lozano E, Bailac S and Barbosa J (2002) Determination of difloxacin and sarafloxacin in chicken muscle using solid-phase extraction and capillary electrophoresis. Journal of Chromatography B 767: 313).
preparation. This method is also adequate for monitoring these matrices for sulfonamides at the current MRLs. Clinical Samples
Amoxicillin, florfenicol, and quinolones have been determined in pig plasma, using borate buffers. In the amoxicillin work, the use of field-amplified sample injection lowers the LOD to 280 mg L 1. Detection levels are also improved through the use of capillary isotachophoresis as an on-capillary stacking technique, as is demonstrated in the analysis of quinolones. NACE is used for tetracyclines following sample treatment of pig plasma by SPE. For the detection of aminoglycosides in human serum and plasma, pre- and in-capillary derivatization reactions have been utilized. Amikacin, arbekacin, dibekacin, and kanamycin have been derivatized with IDA, followed by fluorescence detection. Gentamicin, tobramycin, and kanamycin have been derivatized with OPA and either mercaptoacetic acid or mercaptopropionic acid. The ability to monitor peak and trough plasma concentrations of tobramycin or kanamycin in tuberculosis patients is demonstrated. The need for sample pretreatment depends on the matrix, the expected level of the analyte, and the separation system itself. Microdialysate and cerebrospinal fluid samples typically require only dilution and filtration, as is demonstrated by the analysis of
cefazolin and cefpirome in microdialysate samples. Cefepime and ceftazidime have been detected in cerebrospinal fluid to monitor the efficacy of treatments for bacterial meningitis (Figure 5). Using a citrate buffer at pH 6, cephalosporins can be determined in plasma without sample pretreatment. The analysis of quinolones in a HEPES buffer system can proceed after treatment with trichloroacetic acid. The determination of cefotaxime and its metabolite, deacetylcefotaxime, requires protein removal with acetonitrile prior to analysis in a borate buffer. However, using MEKC systems, cefotaxime, cefepime, ceftazidime, and vancomycin can be analyzed with direct injection of serum. MEKC is also useful for the analysis of sulfamethoxazole, cefuroxime, and cefpirome in serum. Nalidixic acid and its metabolites can be analyzed by MEKC after sample acidification and extraction with chloroform. Following protein precipitation with acetonitrile, linezolid can be detected at the 0.5 mg L 1 level. Thiamphenicol has also been determined in serum using an MEKC method. Sample introduction using a sweeping preconcentration approach has been applied to the determination of doxorubicin in plasma. Following sample cleanup, the solution is injected electrokinetically for 5 min at 30 kV. The sample vial is replaced with a vial containing 150 mM SDS dissolved in the BGE. Using UV detection, the levels of doxorubicin can be monitored at 100 min (397 nM) and at 300 min (60 nM) after the administration of a 30 mg m 2 dose of doxorubicin.
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Figure 5 Electropherograms of CSF from a meningitis patient receiving intravenous 2 g of cefepime. (a) Before dosing; (b) after dosing at 8 h. Peaks: 1, cefazolin (internal standard); 2, cefepime. Separation conditions: capillary, 75 mm 31.2 cm (21 cm effective length); voltage, 8 kV; detection, 254 nm; buffer, 200 mM SDS, 20 mM Tris, pH 9.0. (Reproduced with permission from Tseng S-H, Yang Y-H, Chen Y-R and Chen S-H (2004) Determination of cefepime in plasma and cerebrospinal fluid by micellar electrokinetic chromatography with direct sample injection. Electrophoresis 25: 1641).
Another on-capillary preconcentration technique utilizes the ionic nature of the analyte. Blood containing rapamycin is treated by protein precipitation and SPE cleanup. Under alkaline conditions, the lactone bond of the macrolide is cleaved, creating an open-ring, anionic species. Electrokinetic injection under reversed polarity causes the negatively-charged rapamycin to migrate into the capillary, where it meets the acidic BGE. Rapamycin becomes protonated, creating a neutral species that does not migrate further. Focusing is achieved during injection, leading to an LOD of 0.2 mg L 1 (Figure 6). Fosfomycin levels in plasma, microdialysate, and pus samples are determined under reversed EOF conditions, using a cationic surfactant as BGE, and benzoic acid for indirect detection. With LODs ranging from 0.6 to 2 mg mL 1, the detection of fosfomycin in pus samples indicates that the antibiotic is reaching the sites of the abscesses. Electrochemical detection schemes have been used for the determination of mitomycin C and chloramphenicol. Owing to the selectivity of the electrode configurations used, no interfering peaks are observed. Greater sensitivity can also be achieved through the use of LIF detection. This approach is used for
the determination of ciprofloxacin and its metabolite desethyleneciprofloxacin, and moxifloxacin in plasma and microdialysate, with LOQs of the order of 2.5 mg L 1 in plasma. NACE combined with metal complexation and LIF has been utilized in the separation and detection of tetracyclines. Using freesolution CE, idarubicin and its metabolite idarubicinol have been determined, following extraction in chloroform. The LOQ achieved is 0.5 ng mL 1, which permits the monitoring of pediatric oncology patients, and requires only 100 mL of plasma. A similar system has been developed for doxorubicin and doxorubicinol, and idarubicin and doxorubicin have been determined in the serum of patients receiving treatment for ovarian cancer. Additional studies monitor doxorubicin and its metabolites in cell extracts and in single cells, and demonstrate differences in intracellular metabolism between liposomal and free doxorubicin in the treatment of human leukemia cells. Peak plasma concentrations of daunorubicin and daunorubicinol are monitored in children being treated with either free daunorubicin or DaunoXomes, a liposomal formulation of daunorubicin. The total peak plasma levels were
III / CAPILLARY ELECTROPHORESIS / Antibiotics 9
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Figure 6 Electropherograms of a blood sample spiked with 10 mg L 1 rapamycin including the online spectrum (a) of the ring-open rapamycin and (b) a blank blood sample. Separation conditions: capillary, 75 mm 45 cm (36.5 cm effective length); voltage, 15 kV; detection, 278 nm; buffer, 80 mM SDS, 30 mM H3PO4 in methanol/water (20:80, v/v); electrokinetic injection for 300 s at 7.5 kV. (Reproduced with permission from Buchberger W, Ferdig M, Sommer R and Vo TDT (2005) A novel technique for on-capillary preconcentration of anionic compounds applied to the trace analysis of rapamycin in human blood by capillary electrophoresis. Electrophoresis 26: 161).
approximately two orders of magnitude higher after dosing with DaunoXomes than after administration of the free drug. Another method monitors daunorubicin levels in plasma and biopsy material from patients who have Kaposi sarcomas, demonstrating that the daunorubicin is reaching the tumor. In many instances, the analysis of urine can proceed without sample preparation beyond dilution and filtration. The combination of pasiniazide, capreomycin, and ofloxacin is used in the treatment of pulmonary tuberculosis. These analytes can be monitored in urine in a borax buffer containing 10%
ethanol. Cephalosporins can be determined using a citrate buffer. Quinolones can be separated in HEPES or phosphate–borate buffer systems, and separated from pharmaceuticals that may be coadministered. Nalidixic acid and two of its metabolites can be extracted from urine with chloroform, and analyzed using MEKC, with LODs of 0.3–0.7 ng mL 1. Carbon fiber microelectrodes, used in the end-column configuration, are able to detect sulfadiazine and sulfamethoxazole in urine at the 0.1 mM level, and midecamycin at the 0.5 mM level. Daunorubicin can be quantitated using a carbon disc electrode in
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the wall-jet configuration at a similar level. Electrochemiluminescence detection, in the presence of Tris(2,20 -bipyridine) ruthenium (II) (Ru(bpy)23 þ ) has been applied to the analysis of lincomycin and ofloxacin in urine, at LODs of 9.0 mM and 500 nM, respectively. LIF has been used for the sensitive detection of ofloxacin and its metabolites. Sulfonamides in urine have been analyzed by coupling an SPE apparatus to the CE instrument via a switching valve. This sample pretreatment and injection protocol yields LODs of 10 ng mL 1. Another means of removing matrix interferences is the use of a channel-coupled microchip electrophoresis device. In the first channel, Na þ and K þ ions in urine are separated from gemifloxacin, and are sent to waste. In the second separation channel, the gemifloxacin enantiomers are separated using only 50 mM of the chiral selector ( þ )-18-crown-6-tetracarboxylic acid. Total analysis time is less than 4 min. Environmental Samples
The elimination of antibiotics in waste products, and their subsequent presence in the environment, is suspected of being responsible for the evolution of antibiotic-resistant bacterial strains. CE and MEKC separation schemes for quinolones have been developed using LIF detection. Photodegradation studies have been conducted on enrofloxacin in water alone, and in the presence of either 20 mg L 1 fulvic acids or 20 mg L 1 humic acids, resulting in differences in the identities and levels of products present. Affinity CE demonstrates that strong binding of some degradation products to the humic acids protects them from degradation. ESI-MS detection has been combined with CE for the determination of penicillin V in river water. A standard solution of penicillin V is analyzed using a 20 mM ammonium acetate buffer at pH 5.1, with LODs of 50 mg L 1 in the positive ionization mode, and 134 mg L 1 in the negative ionization mode. An MEKC approach has also been developed for the monitoring of penicillins in farm water samples. A method for tetracyclines uses a flow manifold coupled online to the CE system. Following preconcentration on an SPE minicolumn, the analytes are introduced into the CE autosampler vial. Using this protocol, UV detection yields sensitivities on the order of 2–20 mg L 1.
Conclusions Capillary electrophoresis and its related techniques have proven to be effective in the analysis of antibiotics. Screening protocols, assay and impurity methods, and clinical and environmental monitoring procedures are viable alternatives to many of the compendial methods that are currently in use. Further developments in sample treatment strategies, either as automated off-line procedures coupled to CE, or as in-capillary approaches, will reduce total analysis time and will enhance detection capabilities. Microfluidic systems offer rapid throughput with low sample volume requirements. The interfacing of CE-related systems to a mass spectrometer offers the unequivocal identification of residues.
See also: II/Capillary Electrophoresis: Microemulsion Electrokinetic Chromatography; Preconcentration Techniques. III/Antibiotics: Liquid Chromatography.
Further Reading Anderson AB, Gergen J and Arriaga EA (2002) Detection of doxorubicin and metabolites in cell extracts and in single cells by capillary electrophoresis with laserinduced fluorescence detection. Journal of Chromatography B 769: 97. Eder AR and Arriaga EA (2005) Micellar electrokinetic capillary chromatography reveals differences in intracellular metabolism between liposomal and free doxorubicin treatment of human leukemia cells. Journal of Chromatography B 829: 115. Herna´ndez M, Borrull F and Calull M (2003) Analysis of antibiotics in biological samples by capillary electrophoresis. Trends in Analytical Chemistry 22: 416. Jime´nez-Lozano E, Roy D, Barro´n D and Barbosa J (2004) Effective sorbents for solid-phase extraction in the analysis of quinolones in animal tissues by capillary electrophoresis. Electrophoresis 25: 65. McCourt J, Bordin G and Rodrı´guez AR (2003) Development of a capillary zone electrophoresis-electrospray ionization tandem mass spectrometry method for the analysis of fluoroquinolone antibiotics. Journal of Chromatography A 990: 259. Santos B, Lista A, Simonet BM, Rı´os A and Valca´rcel M (2005) Screening and analytical confirmation of sulfonamide residues in milk by capillary electrophoresismass spectrometry. Electrophoresis 26: 1567.