Enantioselective determination of oxprenolol and its metabolites in human urine by cyclodextrin-modified capillary zone electrophoresis

A stereospecific capillary electrophoresis assay for oxprenolol enantiomers and their basic metabolites in human urine has been developed using hydroxypropyl-beta-CD as a chiral selector in the mobile phase. The bioassay method has been validated and the detection limit from spiked urine samples is 0.2 micrograms/ml. The calibration curves are linear from 0.4 to 16 micrograms/ml. Extraction recovery ranged from 84.7 to 96.4% for all the compounds studied. The influence of various parameters on the chiral separation of oxprenolol and its basic metabolites have been investigated. Urinary excretion profiles of oxprenolol enantiomers and those of two metabolites have also been studied, following a single oral dose of racemic oxprenolol.     

Use of cyclodextrins in capillary electrophoresis: resolution of tramadol enantiomers

Capillary zone electrophoresis was successfully applied to the enantiomeric resolution of racemic tramadol. Both uncoated and polyacrylamide-coated capillaries were tested for method optimization using either negatively charged or native cyclodextrins (CD) added to the background electrolyte (BGE). The resolution was strongly influenced by the CD type and concentration as well as by the pH and the concentration of the BGE. Among the CDs tested, carboxymethylated-beta-cyclodextrin allowed the baseline separation of tramadol enantiomers. After the method was optimized, it was validated in a coated capillary for enantiomeric analysis of tramadol enantiomers in pharmaceutical formulation, including specificity and elution order, linearity, accuracy and precision, determination of limit of detection (LOD) and quantification (LOQ), enantiomeric purity linearity, freedom from interference, and stability of sample solutions. Precision at the target concentration was less than 2%, with an accuracy higher than 99%. Furthermore, the method was able to detect 0.3% and to quantify 1% of the minor enantiomer in the presence of the major one at the target value.     

High-performance capillary electrophoresis of hyaluronic acid: determination of its amount and molecular mass

Individual GABAergic interneurons in hippocampus can powerfully inhibit more than a thousand excitatory pyramidal neurons. Therefore, control of interneuron excitability provides control over hippocampal networks. We have identified a novel mechanism in hippocampus that weakens excitatory synapses onto GABAergic interneurons. Following stimulation that elicits long-term potentiation at neighboring synapses onto excitatory cells, excitatory synapses onto inhibitory interneurons undergo a long-term synaptic depression (interneuron LTD; iLTD). Unlike most other forms of hippocampal synaptic plasticity, iLTD is not synapse specific: stimulation of an afferent pathway triggers depression not only of activated synapses but also of inactive excitatory synapses onto the same interneuron. These results suggest that high frequency afferent activity increases hippocampal excitability through a dual mechanism, simultaneously potentiating synapses onto excitatory neurons and depressing synapses onto inhibitory neurons.     

Separation of enantiomers of drugs by capillary electrophoresis. Part 4: hydroxypropyl-gamma-cyclodextrin as chiral solvating agent

In an extended chiral drug screening program, enantioseparation of 86 racemic drugs was tested with hydroxypropyl-gamma-cyclodextrin as chiral solvating agent (CSA). A total of 30 drugs out of 86 could be resolved in this straightforward approach. The number of experiments performed under identical conditions allows a statistical treatment of the data. The enantioseparation of the analytes is correlated with their interaction strength with the CSA. Hence, the concentration of the CSA is a crucial parameter for optimization of the enantioseparation, as shown by a subset of 23 examples.     

Analysis of trifluoroacetic acid in lyophilized natural products by capillary electrophoresis

A rapid and simple method for the capillary electrophoretic determination of residual trifluoroacetic acid in lyophilized natural products is described. The technique utilizes 2,6-naphthalenedicarboxylic acid as a separation buffer additive providing indirect ultraviolet absorption detection. Using this method, acceptable precision, accuracy, selectivity, range and linearity were achieved.     

Determination of kynurenic acid by capillary electrophoresis with laser-induced fluorescence detection

We have investigated the influence of three structurally different but functionally related compounds [1, 10 ortho-phenanthroline (phenanthroline), Rifampicin and aurin tricarboxylic acid (ATA)] on the rate and the extent of proliferation of progesterone-responsive T47D human breast cancer cells. These compounds have previously been used in this laboratory and have been shown to modulate properties of nucleic acid binding proteins. Because p53 and the progesterone receptor (PR) are both DNA binding proteins that appear to regulate proliferation of breast cells, alterations in T47D cell p53 and PR levels were examined to determine their relevance in cell proliferation. T47D cells were grown in the absence of phenol red and in the presence of 5% fetal calf serum with or without charcoal stripping in the presence of the inhibitors. The rate of proliferation of cells grown in Rifampicin containing medium exhibited nearly 70% inhibition. Phenanthroline, a known metal chelator, was an effective inhibitor of proliferation at 3 mM reducing the cell number by more than 75%. ATA (0.24-2.4 micrograms/ml) inhibited the growth of the cells by nearly 50%. Analysis of the mechanism of action of these compounds revealed that treatment with these compounds caused specific changes in the molecular composition of T47D cell PR. Whereas ATA caused increased stability of PR isoforms, Rifampicin induced a upshift in the mobility of PR in SDS gels-a phenomenon associated with hyperphosphorylation of steroid receptors (SRs). Phenanthroline treatment (> 2 mM) caused a complete down-regulation of PR and the tumor suppressor protein, p53. The downregulation of p53 paralleled the changes in the molecular composition of PR. We propose that the inhibition of T47D cell proliferation by phenanthroline, Rifampicin and ATA results from a number of cellular changes that include regulation of p53 and PR.     

Detection of nucleic amino acid enantiomers by HPLC with preliminary modification by o-phthalic aldehyde

The use of reversed-phase HPLC with UV detection at 254 nm for the separation of stereoisomers of nucleoamino acids, namely, pyrimidyl and purinyl derivatives of alanine, after premodification with o-phthalic aldehyde and N-acetyl-L-cysteine was studied. The use of 0.01 M phosphate buffer (pH 7.0) containing 5% acetonitrile as a mobile phase resulted in satisfactory separation. The range of the detectable amounts of nucleoamino acids was 0.08-1.0 nmol. This method was used for monitoring the formation of stereoisomers in the hydrolysis of N-phenylacetylcytosinylalanine.     

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 antibiotics by capillary electrophoresis

The broad category of antibiotics encompasses some of the most widely prescribed pharmaceuticals in the world. As is the case with any pharmaceutical, an antibiotic must be characterized in terms of its potency or activity, and the presence and quantity of impurities. Additionally, any residue or metabolite that may be present as a result of its use must be monitored. Many capillary electrophoretic techniques have been utilized in the analysis of antibiotics, addressing the various aspects of their quantitation, profiling, and monitoring. Some of the more recent applications are summarized in this review article.     

Analysis of nucleotides by capillary electrophoresis

Capillary electrophoresis is a useful tool for the analysis of nucleotides. Methods have been optimized for both CZE and MECC modes. A variety of CZE buffers, such as borate, carbonate and phosphate were used successfully. The pH of the buffer changes the charge on the nucleotides. Therefore, the selectivity of the analytes can be controlled by the acidity of the buffer solution. CE separations of nucleotides have been performed at all pH levels, in both CZE and MECC modes. SDS was the most commonly used modifier in MECC separations, but other additives have been added to optimize selectivity. In addition, nucleotides have been quantified in different matrices, including tissue and cell extracts and several DNA and RNA sources. This paper summarizes the methods used for the optimization of nucleotides by CE and includes the most recent techniques to improve selectivity, reproducibility and sensitivity. A summary of CE methods is used in analyses of nucleotides in biological matrices is included.     

Determination of glycyrrhizin and glycyrrhetinic acid in traditional Chinese medicinal preparations by capillary electrophoresis

A simple, rapid, accurate and reproducible capillary electrophoretic method was developed for the assay of glycyrrhizin and glycyrrhetinic acid in traditional Chinese medicinal preparations. The buffer solution used in this method was acetonitrile and 0.02 M sodium dihydrogen-phosphate solution adjusted to pH 7.5 with 0.05 M sodium hydroxide. The linear calibration range was 0.04-2.00 mg/ml (r = 0.9988) for glycyrrhizin and 0.007-0.35 mg/ml (r = 0.9985) for glycyrrhetinic acid and recoveries were 98.1-101.3% for glycyrrhizin and 98.5-101.4% for glycyrrhetinic acid. The relative standard deviations were 1.02% (n = 6) for glycyrrhizin and 0.91% (n = 6) for glycyrrhetinic acid. The content of these two acids in Glycyrrhizae Radix and Glycyrrhizae Radix-containing Chinese medicinal preparations was successfully determined within 10 min.     

Resolution of enantiomers of amino acids by HPLC

Application of HPLC as a prime tool in the area of enantiomeric resolution has opened doors of success and varied interest. Use of chiral reagents either indirectly (as derivatization reagent) or directly (added to stationary or mobile phase) has led to achieve resolution of a wide range of compounds. Amino acids, being important molecules with simple structure and easy availability, have been extensively studied. A bibliographic survey on HPLC resolution of amino acids and derivatives along with a brief discussion on general methods of enantiomeric separation has been presented.     

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.     

Separation of the enantiomers of ibuprofen and its major phase I metabolites in urine using capillary electrophoresis

This report describes a simple, rapid, automated microassay for measuring in vitro changes of oxidative burst of phagocytes following challenge with metals for orthopedic devices. The production of reactive oxygen species (ROS) by polymorphonuclear leukocytes (PMNs) was measured using 2',7'-dichlorofluorescin-diacetate (DCFH-DA) as fluorescent probe. DCFH-DA enters the cells and is oxidized by ROS to fluorescent DCF. The DCF generated was directly proportional to ROS produced intracellularly: The fluorescence intensity was read and converted to an index of ROS production by cells. In our experimental system, granulocytes (PMNs) were isolated from normal human blood and seeded in microplates. To verify if metals could influence ROS production, chromium, cobalt, nickel, molybdenum, titanium, aluminum, and vanadium prepared as aqueous extracts in phosphate-buffered saline were tested onto PMNs using phorbolmyristate acetate (PMA) as positive control. Molybdenum, aluminum, and vanadium increased ROS generation by PMNs, while signals not different from unstimulated PMNs were recorded for chromium, cobalt, nickel, and titanium. The DCFH-DA microplate-based assay provides an in vitro tool for the detection of oxygen-reactive species generated by PMNs as a response to metals.     

Detection of 2'-deoxyoxanosine by capillary electrophoresis

We have investigated capillary electrophoresis separation of oxanine (Oxa), a base moiety of 2'-deoxyoxanosine (Figure 1), from various bases. Oxa was not well separated from the others by micelle electrokinetic chromatography which is a general method for separating of the bases. On the other hand, capillary zone electrophoresis showed much improved separation when used pH 12 where the six-membered ring of Oxa is opened.     

Characterization of DNA standards by capillary electrophoresis

We have used capillary electrophoresis to evaluate commercial DNA size standards and have found that it can provide an efficient assessment of size. However, the accuracy of the determination is adversely affected by anomalous migration times due to specific interactions of the DNA with the gel matrix as well as conformational differences in the DNA due to sequence heterogeneity. These anomalous migration times are strongly dependent on the choice of gel matrix. For example, the anomalous migration times that are observed in a 1 kilobase standard DNA ladder can be minimized using nongel hydroxyethylcellulose. In addition, the peak resolution can be increased and the anomalous migration can be reduced by the addition of the intercalating dye, ethidium bromide. However, in the case of the D1S80 allelic ladder, some of the DNA fragments possess nucleotide sequences which do not interact equivalently with the dye and produce irregular migration times. These measurements yield preliminary information useful in evaluating DNA size standards which may be used for a wide range of DNA diagnostic applications.     

Enantioseparation of amino acids and dipeptides using vancomycin as chiral selector in capillary electrophoresis

Vancomycin was applied as chiral selector for the enantiomeric separation of derivatized amino acids and dipeptides. The influence of vancomycin concentration, pH and presence of 2-propanol in the buffer were examined in order to find optimal separation conditions. Optimization was by factorial design. Further, chiral separation of derivatives prepared with three different reagents was compared. These reagents were 9-fluorenylmethyl chloroformate (FMOC), 2-(9-anthryl)ethyl chloroformate (AEOC) and dansyl chloride (dansyl). Optimum resolution was at high vancomycin concentrations, while optimum efficiency was at low vancomycin concentrations. As a consequence of the very high enantioselectivity of vancomycin, the vancomycin concentration below the amount necessary for maximal resolution can be used. Separation efficiency was relatively low, and this could be attributed to adsorption of the selector at the capillary wall. Three factors led to decreased adsorption: application of a pH above the zero mobility pH value, low vancomycin concentrations and the presence of 2-propanol. For amino acids, the resolutions of the different derivatives were: dansyl > AEOC > FMOC, while for dipeptides, the highest selectivity was with AEOC.     

Separation of chiral compounds by capillary electrophoresis

This review presents the different chiral selectors used in capillary electrophoresis (CE) for the separation of enantiomers. The use of charged cyclodextrins, crown ethers, polysaccharides, proteins, natural and synthetic micelles, macrocyclic antibiotics and ergot alkaloids is discussed in detail. Neutral native and derivatized cyclodextrins are not treated because several review articles have already been published on this topic. Recent developments like the application of two chiral selectors in the same background electrolyte are highlighted.     

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.     

Drug analysis by capillary electrophoresis and laser-induced fluorescence

This review briefly presents the different laser-induced fluorescence detectors, outlines the different dyes used to derivatize molecules which are used with capillary electrophoresis/laser-induced fluorescence (CE-LIF), and provides an overview and current status of CE-LIF in drug analysis.