Characterization of interactions between bile salts and drugs by micellar electrokinetic capillary chromatography. Part I

PURPOSE: The general properties of micellar electrokinetic capillary chromatography (MECC) were utilized to characterize the strength of interactions between bile salts and biological active substances. METHODS: For that purpose various bile salts were used as micellar pseudostationary phase in the background electrolyte. Furthermore, a physicochemical model was applied and the effective partition coefficients between micellar and water phase were calculated in order to evaluate the strength of interactions between bile acids and the drugs. RESULTS: It was found that the interactions between the selected drugs and bile salts depend both on the lipohilicity of the drugs and on the charge of the components. Only hydrophobic, cationic drugs such as quinine and propranolol are able to interact with these surface active agents. CONCLUSIONS: MECC is a valuable method to characterize interactions such occurring between drugs and bile salts.     

Interaction between bile salts and beta-adrenoceptor antagonists

Chronic suppurative otitis media (CSOM) in profoundly deaf patients is a contraindication for cochlear implantation. Eight (6%) of the 126 patients referred to cochlear implantation at this center between 1986 and 1992 became deafened as a result of bilateral CSOM but were otherwise suitable candidates. This study details the methods used in four patients to prepare the septic ear for a sterile device. Two patients had wet radical cavities with residual cholesteatoma, and two had discharging safe perforations resistant to surgical repair. Obliteration of the middle ear cleft with blind pit closure of the ear canal was attempted in all four patients, and cochlear implants were installed at a second operation 3 to 6 months later. The hearing results were as good as in implanted patients without CSOM, and the only complication has been the finding of a cholesteatoma pearl at the second operation in one patient. Fat obliteration of the mastoid and middle ear with blind pit closure of the ear canal can be adapted to make most chronic ears fit for implantation, if the patient is prepared to undergo two operations.     

Analysis of traditional Chinese anticancer drugs by capillary electrophoresis

The recognition and removal of human apoptotic peripheral lymphocytes in selected populations of periportal and perivenous endothelial cells was studied in in situ and in vitro experiments. Apoptotic peripheral blood lymphocytes once injected into the liver circulation were retained by the sinusoids showing a large heterogeneity of distribution: apoptotic cells are found in the periportal tract double the amount found in the perivenous region. Apoptotic PBL adhesion was lowered to a sixth of the control after preinjection with a sugar mixture (Mannose, N-acetylgalactosamine, N-acetylglucosamine, D-galactose), as suggested by the expression of modified surface glycoconjugates on the plasma membrane of apoptotic cells. A bimodal profile of the distribution of the hepatic sinusoidal cell population, regarding the number of galactose and mannose receptors and the porosity index, was found. Two endothelial cell subsets were present: low porosity cells (average index 14 +/- 6%; periportal tract) with a high number of carbohydrate binding sites, and high porosity cells (average index 26 +/- 7%; perivenous tract), with a low number of carbohydrate binding sites.     

Effect on the partition equilibrium of various drugs by the formation of mixed bile salt/phosphatidylcholine/fatty acid micelles. A characterization by micellar affinity capillary electrophoresis. Part IV

Mixed micelles, which mimic the bile containing fatty acids in the gastrointestinal tract, were used as a pseudostationary phase in capillary electrophoresis. The mixed micellar system studied contained the dihydroxy bile salts sodium glycodeoxycholate or sodium taurodeoxycholate or the trihydroxy bile salt sodium taurocholate, in association with different sodium salts of fatty acids including lauric, myristic, palmitic, oleic, stearic and linoleic acid and lecithin or dipalmitoylphosphatidylcholine as phospholipid. The determination of the changing mobilities of ionic analytes in the presence of mixed micelles reflected interactions between the used drugs and the mixed micelles. These were determined as dependence on the fatty acid concentration in the bile salt/fatty acid micelles and the mixed bile salt/phosphatidylcholine/fatty acid micelles. The capacity factor kappa MMC, for the partition between mixed micellar and aqueous phase was calculated. The partition equilibrium of basic and acidic drugs depends considerably on shape and charge of the mixed micelles (dependent on the fatty acid concentration) as well as on the acid-base properties of the drug. The mobility of the micelle aggregates was determined as an important reference value to the calculations of kappa MMC. This paper also describes the use of laser-induced fluorescence detection and electrospray mass spectrometry and tandem mass spectrometry for the characterization of the mixed micelle composition.     

Use of capillary electrophoresis in the study of ligand-DNA interactions

The presence and genetic content of integrons were investigated for 37 epidemiologically unrelated multiple-drug-resistant strains of Salmonella enterica serotype Typhimurium from humans. All isolates were resistant to ampicillin, chloramphenicol, kanamycin, streptomycin, sulfonamides, and trimethoprim, as well as to tetracycline and/or nalidixic acid; 20% of them were also resistant to gentamicin and amikacin. Three different class 1 integrons (In-t1, In-t2, and In-t3) were identified by Southern blot hybridization, PCR, and DNA sequencing, and these integrons were found to carry the aadB, catB3, oxa1, aadA1a, aacA4, and aacC1 gene cassettes. Integrons In-t1 (aadB and catB3) and In-t2 (oxa1 and aadA1a) were both located on a conjugative IncFI plasmid of 140 kb. In-t3 (aacA4, aacC1, and aadAIa) was located on an IncL/M plasmid of 100 kb which was present, in association with the IncFI plasmid, in gentamicin- and amikacin-resistant isolates. Despite the extensive similarity at the level of the antibiotic resistance phenotype, integrons were not found on the prototypic IncFI plasmids carried by epidemic Salmonella strains isolated during the late 1970s. The recent appearance and the coexistence of multiple integrons on two conjugative plasmids in the same Salmonella isolate are examples of how mobile gene cassettes may contribute to the acquisition and dissemination of antibiotic resistance.     

Quantitative analysis of non-steroidal anti-inflammatory drugs by capillary zone electrophoresis

The potential utility of capillary zone electrophoresis (CZE) for the separation and quantitative determination of some non-steroidal anti-inflammatory drugs (NSAIDs) was investigated. The influence of different parameters on migration times, peak symmetry, efficiency and resolution was studied; these parameters included the nature and concentration of the anionic and cationic components of the separation buffer. A buffer consisting of 75 mM glycine adjusted to pH 9.1 with triethanolamine was found to provide a very efficient and stable electrophoretic system for the CZE analysis of NSAIDs, giving RSD values of about 0.1 and 0.5% for the within-day reproducibility of migration times and peak areas, respectively at a concentration of 25 micrograms ml-1 (n = 5). Response was linear from 2-100 micrograms ml-1 for both sulindac and tiaprofenic acid, for which the LOQ values were 2.8 and 1.9 micrograms ml-1, respectively, using UV detection at 280 nm. Accuracy for each drug was 102-103%.     

Choice of capillary electrophoresis systems for the impurity profiling of drugs

In order to develop a strategy for the impurity profiling of drugs, the possibilities of some capillary electrophoresis systems were investigated. A mixture containing a drug and some of its possible impurities has been used as a model problem. The test compounds were investigated by capillary zone electrophoresis (CZE) and by micellar electrokinetic chromatography (MEKC). The pH of the CZE buffer was varied, but the two stereoisomers could not be separated. Moreover, CZE is not suitable for neutral compounds. In MEKC, two different types of surfactants, sodium dodecyl sulphate (SDS) and cetyltrimethylammonium bromide (CTAB), have been used and the effect of type and concentration modifier on the separation and the elution window was studied. In the SDS system, both the resolution and the elution window could be increased considerably by the addition of modifier. The use of two MEKC systems of different selectivity seems to be a combination with high potential for the impurity profiling of drugs.     

Application of capillary zone electrophoresis in cephalosporin analysis

Cephalosporins have structures and antibiotic activity similar to those of penicillins which represent a class of compounds with closely related structures. Most of the cephalosporins contain aromatic groups and show distinctive UV spectra. Separating the different types of cephalosporins is a challenging task for HPLC. but the resolving power of capillary zone electrophoresis (CZE) makes this separation fast and simple. The present study reports the application of CZE for cephalosporin analysis and the separation of cephalosporins from plasma. Both field strength and temperature were shown to influence the plate number. The influence of injection time on the peak height was studied. Furthermore, the influence of pH value on the separation of cephalosporins by CZE was investigated. The low sample amount required and the relatively short analysis time are the main advantages of this method.     

Use of affinity capillary electrophoresis for the study of protein and drug interactions

Protein-drug interactions were studied using affinity capillary electrophoresis (ACE). The initial study was performed using a model system, fibronectin-heparin interaction. Two distinct binding constants, 21 and 641 nM, were derived from the Scatchard plots. The results are consistent with reported data obtained using other analytical techniques. The ACE binding assay was applied for studying molecular interactions between kedarcidin chromophore and apoprotein. Conditions with an organic solvent as buffer component were examined to establish a suitable binding assay. It appears that the electrophoretic behavior of the protein shows little distortion in the presence of either dimethyl sulfoxide (up to 10%) or acetonitrile (ACN) (up to 30%). The binding assay was initially conducted in aqueous buffer phase. The saturation concentration of chomophore was found to be around 15 microM. A linear Scatchard plot was derived from binding data with a correlation coefficient of 0.94. The binding constant was determined as Kd = 5.6 microM. The effects of organic solvent content ranging from 0 to 30% ACN on the constant were examined. The binding constants were determined as Kd = 11, 12.5 and 16.2 microM for 5, 10 and 30% ACN, respectively. It appeared that the binding affinity between kedarcidin chromophore and apoprotein is reduced as the organic solvent content in the aqueous phase is increased.     

Use of capillary electrophoresis methods to characterize the pharmacokinetics of antisense drugs

As antisense drugs become mature for clinical trials, analytical techniques to analyze antisense DNA in biological media for characterization of their pharmacokinetics will be in demand. Due to the superior resolving power of capillary gel electrophoresis (CGE), CGE will likely be a preferred method in quantifying intact oligonucleotides as well as the putative metabolic products. Nonetheless, biological mediums can influence the stability of the gel column, making a CGE assay time-consuming. In one approach, high-performance liquid chromatography (HPLC) was used to quantify the total amount of antisense compounds to increase the sample throughput and CGE was used to determine the relative percentage of the intact and metabolic species on specific samples. Alternatively, extensive sample pretreatment procedures were performed and the samples were quantified and characterized directly by CGE alone with the use of an internal standard. Both methods have been used to characterize the pharmacokinetics of antisense compounds. This review focuses on the instrumental and technical aspects of analyzing antisense DNA in biological mediums using CGE either as a single or a combined method towards better understanding of the pharmacokinetics of antisense DNA. Moreover, the newer analytical technologies of capillary electrophoresis (CE), which hold great potential to be used for pharmacokinetic applications, such as the replenishable sieving matrix combined with an innovative coupling approach and microchip CE, will also be explored.     

Enantioseparation of anaesthetic drugs by capillary zone electrophoresis using cyclodextrin-containing background electrolytes

The enantiomers of five racemic anaesthetic drugs were resolved with cyclodextrins using capillary zone electrophoresis. Parameters which affected the chiral resolution, such as type and concentration of cyclodextrin, temperature, and addition of organic modifier were investigated. The results show that the enantiomeric discrimination of the solutes is influenced by the structural shape of the solute molecules, separation temperature, and type of cyclodextrin. It was found that alpha-cyclodextrin was the best enantioselector for resolution of prilocaine and ketamine, while the enantiomers of mepivacaine, ropivacaine, and bupivacaine were resolved with beta-cyclodextrin and/or modified beta-cyclodextrins, i.e., methyl- and 2-hydroxypropyl-beta-cyclodextrin, as chiral selectors. The length of the alkyl chain on the amino group of the drug molecule had a strong effect on the enantioresolution of mepivacaine, ropivacaine, and bupivacaine. Baseline separation of racemic ketamine was achieved with alpha- and methyl-beta-cyclodextrin at 15 degrees C. Addition of 5 M urea to the running buffer containing beta-cyclodextrin at high concentrations resulted in the enantioseparation of prilocaine, mepivacaine, and ketamine. Enantioresolution was improved upon the addition of 10% methanol to the buffer containing urea and beta-cyclodextrin. Generally, the complex formed between the S-enantiomers and modified beta-cyclodextrins was stronger than the corresponding R-forms. An exception was prilocaine where the R-form gave a more stable complex both with alpha- and beta-cyclodextrin.     

Synergism of capillary isotachophoresis and capillary zone electrophoresis

Commercially pure titanium (CPT) substrate was subjected to porcelain firing and bond strengths under three-point bending mode (span length: 15 mm; crosshead speed: 0.5 mm/min) were evaluated. Experimental variables included surface treatments of CPT and porcelain firing schedules. Variables for the surface treatments were (1) sandblasting, (2) mono- and triple-layered nitridation, and (3) mono-layered chrome-doped nitridation. Variables for the porcelain firing schedule included (4) bonding agent application, (5) bonding agent plus gold bonding agent application, and (6) Procera porcelain application. All together eleven sample groups were prepared with different combination of aforementioned experimental variables. Statistically all of them exhibited no significant differences. Hence, we employed two further criteria; (I) the minimum bond strength should exceed the maximum porcelain strength per se, and (II) the CPT substrate should not be heated close to the beta-transus temperature. After applying these criteria, it was concluded that mono-layered nitridation and mono-layered application of chrome-doped nitridation on both (with and without) sandblasted and non-sandblasted surfaces were the most promising conditions for a successful Titanium-Porcelain System.     

Application of capillary zone electrophoresis for analysis of thyreostatics

Capillary zone electrophoresis was optimized for the separation of thiouracil, methylthiouracil and propylthiouracil. Methylthiouracil could be determined in various types of urine (human, bovine, horse), either without any pretreatment or in ethyl acetate extracts, within 15 min. For identification, the simultaneous detection at three UV wavelengths (216, 245 and 278 nm) was advantageously used while for quantification the wavelength of the absorbance maximum at 278 nm was preferred. Under optimized conditions a linear response of the detector in the concentration range 0.1-100 ppm was obtained. On analysis of untreated urine, a detection limit of 0.5 ppm was found; for urine extracts the detection limit was 0.1 ppm. Univocal peak identification, based on absorption at three wavelength, was only possible above 2 ppm. Relative standard deviation for standard solutions of methylthiouracil, diluted in the background electrolyte, was 1%; for methylthiouracil in extracts dissolved in the background electrolyte it was 4.5%, and for methylthiouracil in untreated urine, 12.7%.     

Application of spherical and other polymers in capillary zone electrophoresis: separation of antiviral drugs and deoxyribonucleoside phosphates by different principles

Soluble polymers of linear chains with limited branching and spherical polymers (limit dextrins and sucrose, such as Dextran and Ficoll (Pharmacia Chemicals), yielding lower viscosities, are examined here for the separation of different nucleotides and several anti-AIDS drugs by capillary zone electrophoresis (CZE). The linear polymer forms a network but spherical polymers appear to create a second pseudo-phase. In general, they tend to enhance the solute mobility and reduce peak width; thus, they improve the column efficiency. We observe that the beads of a spherical polymer produce a pseudo-phase even in a very low polymer concentration. The proposed method involving a spherical polymer yields the best separation for twelve deoxyribonucleoside mono-, di- and triphosphates in ca. 10 min. Common anti-AIDS drugs (ddA, ddC, ddI, d4T, AZT) and an AZT metabolite (AZT-glucuronate) are resolved by using conventional micellar electrokinetic capillary chromatography (MEKC). These results not only offer fast and highly sensitive detection techniques for the pharmacokinetics of nucleotides, drugs, and their metabolites, but they also demonstrate an application of the proposed second pseudo-phase involving spherical polymer beads in CZE separations.     

Application of capillary electrophoresis and related techniques to drug metabolism studies

The use of capillary electrophoresis (CE) for the separation of small organic molecules such as pharmaceutical agents and drug/xenobiotic metabolites has become increasingly popular. This has arisen, at least in part, from the complimentary mode of separation afforded by CE when compared to the more mature technique of HPLC. Other qualities of CE include relative ease of method of development, rapid analysis, and low solvent consumption. The recent introduction of a variety of detector systems (including UV diode array, laser-induced fluorescence, conductivity) and the demonstrated coupling of CE to MS have also aided acceptance of this technology. In the present report, we review the role of CE coupled to various detector systems including a mass spectrometer for the characterization of both in vitro and in vivo derived drug metabolite mixtures. Attributes of CE for this application are demonstrated by discussion of metabolism studies of the neuroleptic agent haloperidol. Various aspects of the development and use of CE and CE-MS for the characterization of haloperidol metabolites, including criteria for selection of parameters such as pH, ionic strength, extent of organic modification, and the use of nonaqueous capillary zone electrophoresis are discussed. We also consider potential limitations of CE and CE-MS for drug metabolism research and describe the introduction of membrane preconcentration-CE (mPC-CE) and mPC-CE-MS as a solution that overcomes the rather poor concentration limits of detection of CE methods without compromising the resolution of analytes or separation efficiency of this technique.     

High resolution multichannel fluorescence detection for capillary electrophoresis. Application to multicomponent analysis

A wavelength-resolved fluorescence detector for laser-induced fluorescence detection in capillary electrophoresis (CE) is described that uses a charge injection device (CID) array detector Post-column fluorescence detection occurs using a sheath flow cell. The limit of detection for fluorescein is 4.8 x 10(-11) M (29,000 molecules), the spectral resolution is 0.56 nm/pixel, and the spectrograph/CID monitors a 250 nm spectrum throughout the 250-875 nm range. Custom array readout, data manipulation and data processing methods are described to convert wavelength/spatial CID images into electropherograms. The application of the system to characterizing bilirubins in human serum is described, demonstrating the ability to match electrophoretic peaks to standards using spectral information.     

Migration behavior and separation of sulfonamides in capillary zone electrophoresis. I. Influence of buffer pH and electrolyte modifier

UDPG-pyrophosphorylase (EC 2.7.7.9) from Saccharomyces cerevisiae was studied and the presence of isoforms investigated. Its activity was monitored during growth of cultures in rich media containing glucose, galactose, sucrose, maltose or glycerol as carbon sources. The results suggest that UDPG-pyrophosphorylase is subject to both catabolite repression and catabolite inactivation. The inactivation process seems to be complex: in order to produce maximum inactivation, glucose and ammonium sulfate must be added together. Addition of glucose or ammonium sulfate separately produced little effect upon enzyme activity. Adsorption to and elution from a DEAE-Sephacel column of a crude protein extract prepared from yeast cells collected in stationary phase from a glucose medium showed three activity peaks, which we denominated isoform I, II, and III. Isoform I is constitutive, it was the only form present during exponential growth on glucose medium, and did not suffer any alteration after glucose exhaustion, heat shock or by growing cells on maltose. On the other hand, isoforms II and III were shown to be repressed by glucose, and induced by heat shock. Furthermore, isoform II of UDPG-pyrophosphorylase was present together with isoform I when yeast cells were grown on maltose. The presence of a MAL4C allele rendered isoform II constitutive. Interestingly, a gal3 mutant strain had low UDPG-pyrophosphorylase activity and isoforms I and II were not expressed. These results are discussed in relation to trehalose metabolism.     

Separation of tetracyclines by high-performance capillary electrophoresis and capillary electrochromatography

Separations of various tetracycline mixtures by high-performance capillary electrophoresis (HPCE) and a new form of electrochromatography (CEC) are compared. The new CEC method involves etching the inner wall of the capillary surface with an appropriate reagent (ammonium dihydrogen fluoride) in order to produce a significant increase in surface area. The etched surface is then modified by a silation/hydrosilation reaction sequence to first produce a hydride intermediate which is then further reacted to attach a C18 moiety. The bare and hydride capillaries are tested under HPCE conditions while the C18 capillary functions in the CEC mode. The effects of pH and the presence of an organic modifier (methanol) are also studied. Detection limits ( < 10 micrograms/ml) are comparable to previous HPLC and HPCE results. Resolutions for mixtures which simulate real analytical problems are equal to or better than those reported for separations on polymeric and diol columns by HPLC and in earlier studies by HPCE and MECC.     

Analysis of disease-causing genes and DNA-based drugs by capillary electrophoresis. Towards DNA diagnosis and gene therapy for human diseases

Rapid progress in the Human Genome Project has stimulated investigations for gene therapy and DNA diagnosis of human diseases through mutation or polymorphism analysis of disease-causing genes and has resulted in a new class of drugs, i.e., DNA-based drugs, including human gene, disease-causing gene, antisense DNA, DNA vaccine, triplex-forming oligonucleotide, protein-binding oligonucleotides, and ribozyme. The recent development of capillary electrophoresis technologies has facilitated the application of capillary electrophoresis to the analysis of DNA-based drugs and the detection of mutations and polymorphism on human genes towards DNA diagnosis and gene therapy for human diseases. In this article the present state of studies on the analysis of DNA-based drugs and disease-causing genes by capillary electrophoresis is reviewed. The paper gives an overview of recent progress in the Human Genome Project and the fundamental aspects of polymerase chain reaction-based technologies for the detection of mutations and polymorphism on human genes and capillary electrophoresis techniques. Attention is mainly pad to the application of capillary electrophoresis to polymerase chain reaction analysis, restriction fragment length polymorphism, single strand conformational polymorphism, variable number of tandem repeat, microsatellite analysis, hybridization technique, and monitoring of DNA-based drugs. Possible future trends are also discussed.