Determination of dopamine and methoxycatecholamines in patient urine by liquid chromatography with electrochemical detection and by capillary electrophoresis coupled with spectrophotometry and mass spectrometry

The applicability of capillary electrophoresis (CE) with UV and mass spectrometric (MS) detection for the determination of dopamine and methoxycatecholamines in urine was evaluated in comparison with the liquid chromatography-electrochemical detection (LC-EC) method widely used in catecholamine analysis. The catecholamines in urine were deconjugated with acid or enzyme hydrolysis, purified by cation exchange (CEX) or solid-phase extraction (SPE) with a copolymer of N-divinylpyrrolidone and divinylbenzene and analyzed by LC-EC, CE-UV, and CE-MS. Acid hydrolysis was more effective in the deconjugation than enzymatic hydrolysis with Helix pomatia. However, the recoveries of HMBA, DA and NMN from spiked samples were less than 30% after acid hydrolysis and SPE purification. The CEX purification was more efficient than SPE in removing matrix compounds from the urine samples. The limits of detection were lower in LC-EC analysis than in CE-UV or CE-MS. Many factors in the analytical procedure caused deviations in the concentrations measured for urinary dopamine and methoxycatecholamines. The recovery of HMBA, which was used as the internal standard, was poor after acid hydrolysis and SPE purification. The purification methods were validated in conjunction with the analytical methods and therefore cross analysis was unsuccessful. The LC-EC method was the most sensitive, but CE-UV and CE-MS were sensitive enough for the determination of dopamine and methoxycatecholamines even in healthy patient urine. The EC and MS detections were superior to the UV detection in specificity since, after acid hydrolysis, some matrix compounds were migrating close to I.S., DA and 3MT.     

Analysis of Aβ (1-40) and Aβ (1-42) monomer and fibrils by capillary electrophoresis

A method based on capillary electrophoresis (CE) with UV absorbance detection is presented to characterize synthetic amyloid beta (Aβ) peptide preparations at different aggregation states. Aggregation of Aβ (1-40) and Aβ (1-42) is closely linked to Alzheimer's disease (AD), and studying how Aβ peptides self-assemble to form aggregates is the focus of intense research. Developing methods capable of identifying, characterizing and quantifying a wide range of Aβ species from monomers to fully formed fibrils is critical for AD research and is a major analytical challenge. Monomer and fibril samples of Aβ (1-40) and Aβ (1-42) were prepared and characterized for this study. The monomer-equivalent concentration for each sample was determined by HPLC-UV, and aggregate formation was confirmed and characterized by transmission electron microscopy. The same samples were studied using CE with UV absorbance detection. Analysis by mass spectrometry of collected CE fractions was used to confirm the presence of Aβ for some CE-UV peaks. The CE-UV method reported here clearly indicates that monomeric and aggregated Aβ were electrophoretically separated, and substantial differences in the electrophoretic profiles between samples of Aβ (1-40) and Aβ (1-42) were observed. This CE-UV method can differentiate between Aβ monomer, oligomeric intermediates, and mature fibrils.     

On-column preconcentration of glutathione and glutathione disulfide using pH-mediated base stacking for the analysis of microdialysis samples by capillary electrophoresis

Capillary electrophoresis (CE) has become a useful analytical tool for the analysis of microdialysis samples. However, CE with UV detection (CE-UV) does not provide detection limits sufficient to quantify glutathione (GSH) and glutathione disulfide (GSSG) in biological samples such as liver microdialysates, because of the small optical path length in the capillary. To overcome this limitation, an on-column preconcentration technique, pH-mediated base stacking, was used in this study to improve the sensitivity of CE-UV. This stacking technique allowed large volumes of high ionic strength sample injection without deterioration of the separation efficiency and resolution. A 26-fold increase in sensitivity was achieved for both GSH and GSSG using the pH-mediated base stacking, relative to normal injection without stacking. The limit of detection for GSH and GSSG was found to be 0.75 microM (S/N=6) and 0.25 microM (S/N=6), respectively. The developed method was used to analyze GSH and GSSG in liver microdialysates of anesthetized Sprague Dawley male rats. The basal concentrations of GSH and GSSG in the liver microdialysates of male rats were found to be 4.73+/-2.08 microM (n=7) and 5.52+/-3.66 microM (n=7), respectively.     

Near infrared spectroscopy compared to liquid chromatography coupled to mass spectrometry and capillary electrophoresis as a detection tool for peptide reaction monitoring

Peptide interaction is normally monitored by liquid chromatography (LC), liquid chromatography coupled to mass spectrometry (LC-MS), mass spectrometric (MS) methods such as MALDI-TOF/MS or capillary electrophoresis (CE). These analytical techniques need to apply either high pressure or high voltages, which can cause cleavage of newly formed bondages. Therefore, near infrared reflectance spectroscopy (NIRS) is presented as a rapid alternative to monitor the interaction of glutathione and oxytocin, simulating physiological conditions. Thereby, glutathione can act as a nucleophile with oxytocin forming four new conjugates via a disulphide bondage. Liquid chromatography coupled to UV (LC-UV) and mass spectrometry via an electrospray ionisation interface (LC-ESI-MS) resulted in a 82% and a 78% degradation of oxytocin at pH 3 and a 5% and a 7% degradation at pH 6.5. Capillary electrophoresis employing UV-detection (CE-UV) showed a 44% degradation of oxytocin. LC and CE in addition to the NIRS are found to be authentic tools for quantitative analysis. Nevertheless, NIRS proved to be highly suitable for the detection of newly formed conjugates after separating them on a thin layer chromatography (TLC) plate. The recorded fingerprint in the near infrared region allows for a selective distinct qualitative identification of conjugates without the need for expensive instrumentation such as quadrupole or MALDI-TOF mass spectrometers. The performance of the established NIRS method is compared to LC and CE; its advantages are discussed in detail.     

Kinetics of phosphorothioate oligonucleotide metabolism in biological fluids.

The in vitro stability and metabolism of GEM[91, a 25mer phosphorothioate antisense oligonucleotide complementary to the gag mRNA region of HIV-1, was investigated using capillary electrophoresis (CE). The in vitro degradation of the parent compound at 37 degrees C was followed over the course of 120 h in human plasma. A CE method using laser-induced fluorescence detection was able to detect 5'-end intact metabolites including the parent compound extracted from biological fluids. Because the primary metabolic pathway is believed to be via 3'-exonuclease activity, the results of this study were compared with the stability of the compound in a solution containing 3'-exonuclease. The numerical solution of sequential first-order reactions was used to obtain kinetic parameters. Exonuclease digestion of the parent compound, as measured using an automated CE-UV instrument, yielded striking similarities between the two in vitro systems as well as between in vitro and in vivo systems.     

Tetrabutylammonium phosphate-assisted separation of multiplex polymerase chain reaction products in non-gel sieving capillary electrophoresis

A method based on non-gel sieving capillary electrophoresis (NGS–CE) with ultraviolet (UV) detection has been developed for the separation of multiplex polymerase chain reaction (PCR) products of three pathogenic bacteria in which hydroxypropylmethylcellulose was used as the sieving medium and dynamic capillary coating. In the method, an ion pair reagent, tetrabutylammonium phosphate (TBAP), was first used in NGS–CE to improve the detection sensitivities and resolutions of DNA fragments. The interaction of TBAP and DNA was proved using the UV spectra of DNA with and without TBAP. Field-enhanced sample injection was used as an on-line preconcentration method to improve the detection sensitivity. The separation of DNA fragments ranging from 100 to 1000 bp was accomplished in 30 min. Three pairs of primers and three PCR products of bacteria were successfully separated in 25 min using the developed method. The intraday relative standard deviations (RSDs) for the migration time and peak area for each PCR product were less than 2.4% (n = 5), and the interday RSDs were less than 6.1% (n = 15).     

水柱法场扩大提高CE-UV检测核酸灵敏度

目的：采用水柱法场扩大堆积技术,提高毛细管电泳紫外分析核酸灵敏度。方法：已知浓度的DNA Marker为标准样品,TE缓冲液递度稀释,压力进样前加一段去离子水柱（0.5psi,20s）,观察不同浓度下压力进样紫外检测图谱。结果：在对分离度无明显影响下,将压力进样时间延长至0.5psi,990s,与常规的压力进样（0.5psi,10s）相比,灵敏度提高了94.4倍。与时间延长后的压力进样（0.5psi,90s）相比,灵敏度提高8.2倍,最低检测总浓度为1ng/μl,DNA的检测限降至80ng/ml（S/N=3）,比前人报道的7ng/μl检测限提高了87.5倍。结论：验证了水柱法场扩大堆积注射可以有效提高毛细管电泳紫外检测核酸灵敏度。     

Analysis of gamma radiation-induced damage to plasmid DNA using dynamic size-sieving capillary electrophoresis

Bacterial plasmids and the chromosomal DNA of many organisms adopt naturally the negatively supercoiled conformation. Therefore, the irradiation of such plasmids could be used to model conformational changes of chromosomal DNA associated with externally-induced damage. We have applied dynamic size-sieving capillary electrophoresis (CE) to monitor the damage of three DNA plasmids, over an unprecedented base pair (bp) size range (2870–27 500 bp), upon exposure to γ-radiation (20–400 Gy). Predominantly, CE with UV absorbance detection in the absence of DNA intercalating dyes was employed to preclude undesirable, induced plasmid conformational changes. Plasmid samples and their enzymatic digestion products were analyzed using both CE and slab gel electrophoresis (SGE) in order to verify the conformation of sample components. Relative to SGE, CE analyses revealed more fine structural features of plasmid degradation.     

Capillary electrophoretic analysis of advanced glycation endproducts formed from the reaction of reducing sugars with the amino group of glucosamine

Glucosamine (GlcN) is an amino sugar sold over-the-counter and is widely used as a dietary supplement to relieve symptoms of osteoarthritis. It is not known whether it is the GlcN alone or one of its many possible nonenzymatic glycation products that is responsible for this effect. The current study demonstrates that reducing sugars form advanced glycation endproducts (AGEs) with GlcN and, as a result, decrease GlcN autocondensation by reducing the availability of the GlcN amino group. Capillary electrophoresis (CE) was used to analyze the in vitro Maillard reaction of GlcN with glyceraldehyde (GA), glucose (Glc), and fructose (Fru) as well as their inhibition of GlcN autocondensation under physiological conditions. Formation of AGEs was monitored by UV and fluorescence spectroscopy. Major components were separated by CE using a bare capillary and UV detection at 214 nm. AGE species were separated by HPLC and were complementary to the CE results. The effects of sugar concentration and incubation time on the AGE profile are also reported for each of the GlcN reducing sugar model systems. A simple and rapid CE method was developed to analyze the AGE formation in this initial report of the reaction of reducing sugars with the amino group of GlcN.     

Application of a novel chemiluminescence-based DNA detection method to single-vector and multiplex DNA sequencing

A chemiluminescent DNA detection method is described and its application shown for both single-vector and multiplex DNA sequencing using the standard dideoxy chain-termination process. This recently developed detection method, which utilizes the light emitted by an enzyme-catalyzed dioxetane reaction, is highly sensitive and affords significant advantages in safety and speed over the traditional radioactive labeling method. When adapted to a multiplex strategy, this chemiluminescent detection method constitutes a safe, simple and rapid method for increasing the throughput of DNA sequencing procedures.     

Surface plasmon field-enhanced fluorescence spectroscopy in PCR product analysis by peptide nucleic acid probes

Surface plasmon field-enhanced fluorescence spectroscopy (SPFS) was recently developed for PCR product analysis, which allowed for real-time monitoring of hybridization processes and for the detection of trace amounts of PCR products, with a detection limit of 100 fmol on the peptide nucleic acid (PNA) probe surface, and 500 fmol on the DNA probe surface. By selectively labeling the strands of PCR-amplified DNA, it was shown that the heat denaturation process in combination with the application of low-salt condition substantially reduced the interference from the antisense strands and thus simplified the surface hybridization. Furthermore, SPFS was demonstrated to be capable of quantitatively discriminating the difference induced by single nucleotide substitution, even within one minute of contact time.     

Characterization and inhibition study of MurA enzyme by capillary electrophoresis

A capillary electrophoresis-based enzyme assay for UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) is described. This method, based on UV detection, provides baseline separation of one of the reaction products, enolpyruvyluridine 5'-diphospho-N-acetylglucosamine (EP-UDP-GlcNAc), from substrates phosphoenolpyruvate (PEP) and uridine 5'-diphospho-N-acetylglucosamine (UDP-GlcNAc) within 4 min. The other product, phosphate, is not detectable by UV at 200 nm. Quantitation of individual components, substrates or product, can be accomplished based on the separated peaks. This methodology was used to determine the Michaelis constant, Km, and product formation rate constant, Kcat, for MurA. Additionally, the CE method was used to evaluate the inhibition effects on MurA using one specific compound as an example. By following similar procedures, the apparent Km values in the presence of different inhibitor concentrations were determined. The inhibition constant, Ki, can be determined from these apparent Km values. In addition, this CE method can be used to study the inhibition mechanism. The principle of this approach is generally applicable to other enzyme studies.     

Analytical methods to determine phytoestrogenic compounds

The analytical methods for the determination of phytoestrogenic compounds in edible plants, plant products and biological matrices are reviewed. The detection, qualitative and quantitative methods based on different chromatographic separations of gas chromatography (GC), high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE) coupled with various detections by ultraviolet absorption (UV), electrochemical detection (ED), fluorescence detection, mass spectrometry (MS) and nuclear magnetic resonance spectroscopy (NMR), as well as non-chromatographic immunoassay are each extensively examined and compared. An overview on phytoestrogen chemistry, bioactivities and health effects, plant precursors, metabolism and sample preparation is also presented.     

Analysis of recombinant human growth hormone by capillary electrophoresis with bilayer-coated capillaries using UV and MS detection

The characterization of recombinant human growth hormone (rhGH; somatropin) by capillary electrophoresis (CE) with UV-absorbance and mass spectrometric (MS) detection using capillaries noncovalently coated with polybrene (PB) and poly(vinyl sulfonic acid) (PVS) is demonstrated. Compared with bare fused-silica capillaries, PB-PVS coated capillaries yielded more favorable migration-time reproducibilities and higher separation efficiencies. Optimal separation conditions for the bilayer-coated capillaries comprised a background electrolyte (BGE) of 400 mM Tris phosphate (pH 8.5) yielding migration-time R.S.D.s of less than 1.0% and plate numbers above 300,000 for intact rhGH. The protein was also analyzed using the CE method described in the European Pharmacopoeia (Ph. Eur.) monograph. The pharmacopoeial method gave much longer analysis times (22 min versus 8 min), lower resolution and plate numbers, and consecutive shifts in migration time for rhGH, indicating possible interactions between the protein and the inner capillary wall. Due to stable migration times obtained with the coated capillaries, reliable profiling and quantification of rhGH and its byproducts in time was possible. Analysis of thermally degraded rhGH revealed the formation of two main degradation products. CE-mass spectrometry (MS) of this sample, using a PB-PVS coated capillary and a BGE of 75 mM ammonium formate (pH 8.5), suggests that these products are desamido forms of rhGH. Analyses of expired rhGH preparations with CE-UV and CE-MS indicated the presence of both deamidation and oxidation products.     

基于焦磷酸测序技术的 基因突变检测在精准医疗中的应用研究进展

基因突变检测在肿瘤等疾病的早期诊断、个体化给药指导、疾病治疗进程与耐药监控等方面具有极其重要的意义。随着测序技术的 不断发展,DNA 突变的检测与分析已为病毒感染、血液病和实体瘤等疾病的个体化诊治提供重要参考。焦磷酸测序技术是一种基于生物发 光法测定焦磷酸盐的实时 DNA 测序技术,其用于 DNA 序列分析时不需电泳和荧光标记,定量性能好,结果准确,易于实现自动化,在基 因突变检测分析与肿瘤等疾病诊治中发挥巨大作用。综述基于焦磷酸测序技术的基因突变检测在分子靶向个体化治疗和疾病诊断中的应用 研究进展。     

Colorimetric-detected DNA sequencing

A sensitive, colorimetric method for visualizing the band pattern of DNA sequencing reaction is described. The enzymatic incorporation of radioactive nucleotides commonly used for the band detection is replaced by biotin conjugated to the 5'-terminus of a synthetic oligonucleotide by chemical synthesis. The oligonucleotide so labeled is used as a primer for dideoxy DNA sequencing in a primer extension reaction. The products of the sequencing reactions are analyzed on a denaturing polyacrylamide gel using the direct blotting electrophoresis technique. This technique makes it possible to transfer the band pattern during the electrophoresis onto an immobilizing matrix, on which it is made visible by an enzymatic reaction in less than 3 h. This biotin-based detection method is so sensitive that the sequencing reactions can be performed under the same conditions and concentrations as those for the radioactive detection.     

Detection of DNA by tritiated actinomycin D on ultrathin frozen sections

Ultrathin frozen sections of fresh liver tissue were floated on actinomycin D-³H. Quantitative high resolution radioautography was performed to determine the value of the method for detection of DNA by electron microscopy. A complete series of control experiments involving various treatments of frozen sections with enzymes (pronase, DNase) and 0.1 N HCl were also carried out to determine the specificity of the labeling. The results indicate the value of the method for detection of DNA directly on ultrathin frozen sections. Short treatments with pronase followed by DNase reduce the labeling to zero, whereas removal of chromosomal proteins with HCl increases the amount of radioactivity in the nucleus considerably. The results are discussed in view of the future applications opened by ultracryotomy, since radioautographic detection of various macromolecules and cellular components by labeled compound with specific affinities will now be possible.     

Sensitive and specific single-molecule sequencing of 5-hydroxymethylcytosine

We describe strand-specific, base-resolution detection of 5-hydroxymethylcytosine (5-hmC) in genomic DNA with single-molecule sensitivity, combining a bioorthogonal, selective chemical labeling method of 5-hmC with single-molecule, real-time (SMRT) DNA sequencing. The chemical labeling not only allows affinity enrichment of 5-hmC-containing DNA fragments but also enhances the kinetic signal of 5-hmC during SMRT sequencing. We applied the approach to sequence 5-hmC in a genomic DNA sample with high confidence.     

Screening Human Genes for Small Alterations Performing an Enzymatic Cleavage Mismatched Analysis (ECMA) Protocol

Many human diseases are caused by small alterations in the genes and in the majority of cases sophisticated protocols are required for their detection. In this study we estimated the efficacy of an enzymatic protocol, which using a new mismatch-specific DNA plant endonuclease from celery (CEL family) recognizes and cleaves mismatched alleles between mutant and normal PCR products. The protocol was standardized on a variety of known mutations, in 11 patients with cystic fibrosis (CF), Fabry’s disease (FD), steroid 21-hydroxylase deficiency (21-HD) and Duchenne/Becker muscular dystrophy (DMD/BMD). The method does not require special equipment, labeling or standardization for every PCR product, since conditions of heteroduplex formation and enzyme digestion are universal for all products. The results showed that the method is rapid, effective, safe, reliable, and very simple, as the mutations are visualized on agarose or nusieve/agarose gels. The protocol was furthermore evaluated in three DMD patients with the detection of three alterations which after sequencing, were characterized as disease causative mutations. The proposed assay, which was applied for the first time in a variety of monogenic disorders, indicates that point mutation identification is feasible in any conventional molecular lab even for cases, where other techniques have failed.     

Automated polymerase chain reaction product sample preparation for capillary electrophoresis analysis

The analysis of crude polymerase chain reaction (PCR) products by capillary electrophoresis (CE) is often compromised due to the presence of a high concentration of salt. Salt interferes with the electrokinetic injection and induces localized heating within the column; hence, PCR products must be desalted or cleaned-up prior to CE analysis. A variety of commercial clean-up systems are available that have been traditionally used to prepare PCR products for cloning, sequencing and digestion with restriction enzymes. These systems were tested for their effectiveness in preparing PCR products for CE analysis and were evaluated based on CE resolution, salt removal, DNA recovery, processing time and cost. One particularly effective clean-up system, membrane dialysis, was automated using a robotic workstation.