Calcium-dependent modulation of the agonist affinity of the mammalian olfactory cyclic nucleotide-gated channel by calmodulin and a novel endogenous factor

Capillary electrophoresis has been successfully employed to determine the level of drugs in a variety of pharmaceutical preparations. A large number of reports have shown agreement between CE results and HPLC data or with label claim. Currently the use of CE for main component assays constitutes 26% of the routine usage of CE within drug companies and is the most frequent application. The choice between adopting CE or HPLC for a particular application is very dependent upon the relative merits of each technique to the individual assay. Often, CE can have advantages in terms of reduced sample pretreatment, consumable costs, and analysis time. The ability to separate a wide range of solutes using a single set of operating conditions is a strong advantage of CE. This paper extensively reviews the literature reports of the use of CE for main peak assay and indicates the validation performance data achieved. The specific requirements relating to optimized accuracy and precision in CE assay are discussed in some detail. The use of an appropriate internal standard to improve performance for precision, accuracy, and linearity, and to reduce the impact of sample matrix effects, is experimentally shown by results from the analysis of levothyroxine samples. The applications are subdivided into those samples analyzed by free solution capillary electrophoresis (FSCE) at low or high pH or those separated by micellar electrokinetic capillary electrophoresis (MECC).     

Capillary electrophoresis and microdialysis: current technology and applications

Microdialysis sampling has become an important method for the continuous monitoring from an in vivo environment. This technique has been used to monitor many endogenous molecules, such as neurotransmitters, as well as exogenous species such as drug substances. Microdialysis samples have traditionally been analyzed by liquid chromatographic (LC) methods to gain resolution and quantification of the molecules of interest. However, LC separations have a relatively large injection volume requirement which, as a consequence, increases microdialysis sampling times. Capillary electrophoresis (CE), with its very small sample volume requirements and high resolving power, has therefore gained popularity as an alternative to LC. Reviewed here are many of the technologies currently available for CE and examples of how this technique has been effectively applied to the analysis of microdialysis samples.     

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.     

Capillary electrophoresis combined with matrix-assisted laser desorption/ionization mass spectrometry; continuous sample deposition on a matrix-precoated membrane target

Capillary electrophoresis (CE) and matrix-assisted laser desorption/ionization mass spectrometry (MALDI/MS) were combined in an off-line arrangement to provide separation and mass analysis of peptide and protein mixtures in the attomole range. A membrane target, precoated with MALDI matrix, was used for the continuous deposition of effluent exiting from a CE device. A sample track was produced by linear movement of the target during the electrophoretic separation and this track was subsequently analyzed by MALDI/MS. The technique is effective for peptides and proteins, having limits of detection (signal-to-noise >3) of about 50 amol for neurotensin (1673 Da) and 250 amol for cytochrome c (12361 Da) and apomyoglobin (16951 Da). The electrophoretic separation achieved from the membrane target, as measured by theoretical plate numbers from the mass spectrometric data, can be as high as 80-90% of that achieved by on-line UV detection under optimal conditions, although band broadening occurs and with some loss of separation efficiency. Non-volatile buffers such as 10-50 mM phosphate can also be used in the electrophoresis process and directly deposited on the membrane. The use of post-source decay techniques is shown for peptides in the CE sample track in order to obtain sequence verification. The effectiveness of this method of integration of CE and MALDI/MS is demonstrated with both peptide and protein mixtures and with the analysis of a tryptic digest of a protein.     

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.     

Capillary electrophoresis interfaced to inductively coupled plasma mass spectrometry for element selective detection in arsenic speciation

A method is presented to separate and detect six arsenic species by capillary electrophoresis (CE) interfaced to inductively coupled plasma mass spectrometry (ICP-MS). CE was used as a highly resolving separation system, whereas ICP-MS served as an element selective detector providing low detection limits. The special mode of operation included sample stacking and a differentiation of separation and detection. This provided separation and detection of six As species, uncharged and anionic, to be monitored within a single run. Detection limits were calculated according to IUPAC recommendation at 15 microg As/L for As (III), dimethyl arsinic acid (DA), monomethyl arsonic acid (MA) and As (V), or 65 microg As/L for arsenobetaine (AsB) and arsenocholine (AsC). Investigations were focused on possibly occurring interferences, e.g., ArCl+ interference at the monoisotope 75As. Finally, real samples from biomedical field (urine) and environmental field (sewage sludge) were analyzed.     

Separation and quantitation of monoclonal antibodies in cell growth medium using capillary zone electrophoresis

IgG1 is separated from its impurities in cell growth medium under simple CZE conditions without specific sample pretreatment. Linearity, limit of quantitation, limit of detection, precision and accuracy for the method are demonstrated. The quantitation for IgG1 in the cell growth medium is obtained by generating a calibration curve and by using standard additions. This CE method can offer a good alternative to conventional HPLC methods. Attempts are also made to separate the heterogeneous species in monoclonal antibodies using both CZE and MECC.     

Development of a capillary electrophoretic method for the separation of the macrolide antibiotics, erythromycin, josamycin and oleandomycin

Capillary electrophoresis (CE) provides high separation efficiency and thus is suitable for the analysis of complex mixtures of structurally similar compounds. The versatile nature of CE can be realised by controlling the chemistry of the inner capillary wall, by modifying the electrolyte composition and by altering the physicochemical properties of the analyte. A CE method has been developed for the separation of three macrolide antibiotics, erythromycin, oleandomycin and josamycin. A systematic approach was used to maximise analyte differential electrophoretic mobility by manipulating electrolyte pH, molarity and composition. In addition, some instrumental parameters such as capillary length and diameter and applied voltage were varied. The effect of the sample solvent and on-capillary concentrating techniques such as field amplified sample injection were investigated. Also, the influence of the injection of a water plug on the quantity of sample injected was demonstrated. The macrolides were completely resolved in less than 30 min in a 100 cm x 75 microm I.D. fused-silica uncoated capillary with a Z-shaped flow cell of path-length 3 mm. The analysis was performed in a 75 mM phosphate buffer (pH 7.5) with 50% (v/v) methanol and an applied voltage of 25 kV was selected to effect the separation.     

Monocular enucleation prevents retinal ganglion-cell loss following neonatal visual cortex damage in cats

Studies were conducted to assess the utility of free solution capillary electrophoresis (CE) for monitoring the effects of selected excipients on the thermal denaturation of a model protein (Ribonuclease A, RNase A) at low pH. Thermal denaturation/unfolding experiments were conducted via temperature-controlled CE using a run buffer of 20 mM citric acid in the pH range of 2.3-3.1, with a marker peptide incorporated to correct for temperature-induced changes in endoosmotic flow. The effects of selected excipients on the thermal unfolding of RNase A were then evaluated by adding either sorbitol, sucrose, polyethylene glycol 400 (PEG 400) or 2-methyl-2,4-pentanediol (MPD) to the electrophoretic run buffer (pH 2.3). Confirmatory denaturation experiments were conducted under the same solution conditions using circular dichroism (CD) spectropolarimetry. Using temperature-controlled CE, an increase in solution pH from 2.3 to 2.7 and 3.1 resulted in an increase in transition temperatures of RNase A by approximately 8 and 13 degrees C, respectively. Similar shifts in transition temperatures were observed when thermal denaturation transitions were monitored by far-UV CD. Sorbitol (0.55-1.1 M) and sucrose (0.55 M) each shifted the denaturation transition temperatures of RNase A to higher values, whereas PEG 400 and MPD had minimal effect on the unfolding transition midpoint at the concentrations evaluated (0.55 M for each). The observed changes in the transition temperatures for RNase A as a function of pH and selected excipients were similar when measured by either CE or far-UV CD. These results support the utility of CE for monitoring the effects of neutral excipients on the thermal denaturation of a model protein under selected conditions. The widespread utility of the technique may be limited by the narrow temperature range of most commercial CE instruments and the need to use extreme pH conditions to monitor the complete denaturation transition.     

Chemiluminescence detection in capillary electrophoresis

Capillary electrophoresis (CE) and related techniques yield highly efficient separations while requiring only minute amounts of sample. Thus, these techniques are amenable to analyses of complex samples in diverse matrices and in situations where sample is extremely limited. The constraints of on-column detection generally result in poor detection limits and have reduced the overall application of CE. One logical approach to increased sensitivity in CE detection has been the development of chemiluminescence (CL)-based detectors. The current state of post-column detector development, CL applications, and limitations of the technique are reported herein.     

Strategies for capillary electrophoresis: method development and validation for pharmaceutical and biological applications

This review is in support of the development of selective, reproducible and validated capillary electrophoretis (CE) methods. Focusing on pharmaceutical and biological applications, the successful use of CE is demonstrated by more than 800 references, mainly from 1994 until 1998. Approximately 80 recent reviews have been catalogued. These articles sum up the existing strategies for method development in CE, especially in the search for generally accepted concepts, but also looking for new, promising reagents and ideas. General strategies for method development were derived not only with regard to selectivity and efficiency, but also with regard to precision, short analysis time, limit of detection, sample pretreatment requirements and validation. Standard buffer recipes, surfactants used in micellar electrokinetic capillary chromatography (MEKC), chiral selectors, useful buffer additives, polymeric separation media, electroosmotic flow (EOF) modifiers, dynamic and permanent coatings, actions to deal with complex matrices and aspects of validation are collected in 20 tables. Detailed schemes for the development of MEKC methods and chiral separations, for optimizing separation efficiency, means of troubleshooting, and other important information for key decisions during method development are given in 19 diagrams. Method development for peptide and protein separations, possibilities to influence the EOF and how to stabilize it, as well as indirect detection are considered in special sections.     

On-column sample preconcentration using sample matrix switching and field amplification for increased sensitivity of capillary electrophoretic analysis of physiological samples

An on-line sample concentration method using sample matrix switching and field amplification peak stacking has been developed. A microbore LC guard column is used to slightly retain the analytes in order to switch from a high ionic strength sample matrix (the physiological fluid) to a low ionic strength matrix (the LC mobile phase). The eluted LC peak is then trapped in a CE system and preconcentrated by field amplification peak stacking. The concentrated sample peak is then analyzed by CE. Compared to normal hydrodynamic injection, the sensitivity was increased by more than 500-fold without loss in resolution. A limit of detection of less than 10 nM for a physiological sample was achieved using UV adsorption detection. This method can be used for negatively or positively charged analytes.     

Capillary electrophoresis: a powerful microanalytical technique for biologically active molecules

Capillary electrophoresis (CE) is a versatile microanalytical technique that has gained much attention, particularly from those working with biologically active molecules. Its appealing characteristics include unprecedented sensitivity and the ability for automating the rapid electrophoretic separation of a number of low-volume samples in a reproducible manner, with relatively short analysis times. The picomole-femtomole (10(-12)-10(-15) mol) sensitivity of UV-CE has been enhanced tremendously by the interfacing of detection systems such as laser-induced fluorescence, which has extended the sensitivity into the attomole-zeptomole (10(-18)-10(-21) mol) range. Fluorescence detection has shown great potential for the CE analysis of a wide range of biomolecules including peptides, proteins and DNA. CE research and development has taken on directions focused primarily on improving detection, understanding and exploiting the basic chemistry of CE and devising new applications.     

On-line dialysis solid-phase extraction coupled to capillary electrophoresis

A fully automated dialysis solid-phase extraction (SPE) sample preparation procedure is coupled on-line to capillary electrophoresis (CE) for the first time. The system is used to determine sulfonamides in serum and urine. The dialysis unit serves to remove proteins and particulate matter. Reconcentration of the analytes is performed with a small SPE column while (in)organic salts and other interferences are removed simultaneously. Finally, the analytes are desorbed and injected, via a homemade interface, into the CE system. Limits of detection (LOD) of 0.05-0.1 and 0.05-0.3 microg/mL are obtained in urine and serum, respectively. The within-day and between-day precisions are in the range of 2-6% and 3-8%, respectively, for a concentration of five times the LOD. The dialysis SPE-CE system was used over a period of six months for the analysis of over 500 serum and urine samples without problems such as clogging of the CE capillary or SPE column.     

Detection of gamma-aminobutyric acid in a living rat brain using in vivo microdialysis-capillary electrophoresis/mass spectrometry

Coupling of in vivo microdialysis with capillary electrophoresis/mass spectrometry (MD-CE/MS) was demonstrated. A microdialysis probe was inserted in the striatum of a rat brain and gamma-aminobutyric acid (GABA) in the brain was detected by CE/MS without any chemical treatment of the sample. MD-CE/MS is expected to be a useful technique for analyzing brain substances, because of the relatively small damage it causes to a living structure and the high selectivity of the analytical technique.     

Hemodynamic cerebral ischemia. An appeal for systematic data gathering prior to a new EC/IC trial

Capillary electrophoresis (CE) equipped with a diode-array detector have been used to determine diagnostic metabolites occurring in urine of patients with various diseases. The urine samples were injected directly onto the CE instrument without any pretreatment. Identification of abnormal metabolites was based on relative migration times and characteristic diode-array spectra. The CE-method readily diagnosed alkaptonuria, neuroblastoma and liver failure due to galactosemia. The simple and automated CE method appears to be suitable for implementation as a screening test in analytical systems aimed at diagnosis of metabolic disorders.     

Cue exposure in moderation drinking: A comparison with cognitive–behavior therapy.

To date, the published controlled trials on exposure to alcohol cues have had an abstinence treatment goal. A modification of cue exposure (CE) for moderation drinking, which incorporated priming doses of alcohol, could train participants to stop drinking after 2 to 3 drinks. This study examined the effects of modified CE within sessions, combined with directed homework practice. Nondependent problem drinkers who requested a moderation drinking goal were randomly allocated to modified CE or standard cognitive-behavior therapy (CBT) for alcohol abuse. Both interventions were delivered in 6 90-min group sessions. Eighty-one percent of eligible participants completed treatment and follow-up assessment. Over 6 months, CE produced significantly greater reductions than CBT in participants' reports of drinking frequency and consumption on each occasion. No pretreatment variables significantly predicted outcome. The modified CE procedure appears viable for nondependent drinkers who want to adopt a moderate drinking goal. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Preoperative diagnosis of a patent foramen ovale: rational use of transthoracic and transesophageal contrast echocardiography

BACKGROUND: The detection or ruling out of a patent foramen ovale (PFO) can be determined noninvasively by contrast echocardiography (CE). The transesophageal technique is superior to the transthoracic technique regarding sensitivity, whereas the specificity of both methods is equally high. This prospective study shows the rational use of transesophageal CE for the detection of a PFO, in patients without cardiovascular disorders. METHODS: 165 patients (92 female, 73 male, age 48 +/- 18 years) with planned neuro-surgery in a sitting position, underwent CE to rule out a PFO. If the CE was positive, an alternative position was selected in order to avoid a paradoxical air embolism. RESULTS: Initially, a transthoracic CE was performed in all patients resulting in 21 patients (13%) being positive and 39 patients (24%) being negative by sufficient image quality. A transesophageal CE was performed in 96 of the remaining 105 patients (63%). Here, further 25 patients showed a positive CE in the sense of a PFO. The combined use of transthoracic and transesophageal CE revealed a PFO in 46 of 165 patients (28%). CONCLUSION: The use of both, transthoracic and transesophageal CE is an efficient approach to the preoperative detection of a PFO in the sense of quality and economics. Depending upon the image quality, the use of a transesophageal examination could be avoided in one third of the cases.     

分子印迹技术在分析化学中的应用进展

分子印迹技术具有专一性和高选择性的特点,其技术关键是功能单体和模板分子的筛选、聚合及模板分子的洗脱。分子印迹聚合物的制备方法主要有:包埋法、亲和印迹法、冷冻干燥法、表面印迹法等。在分析化学中分子印迹技术广泛用于色谱分析、环境痕量分析、膜分离、手性物质拆分,也可用于分离/富集金属离子、样品前处理、传感器、中药有效成分筛选等。从分子水平上认识分子印迹和识别的机理,探索新的合成方法,实现分子印迹技术从有机相向水相过渡、从非极性溶剂环境转向极性溶剂环境,不断扩展新的应用领域是该技术今后的研究方向。