Capillary electrophoretic method for the determination of inorganic and organic anions in real samples. Strategies for improving repeatability and reproducibility

Selectivity and robustness of the pyromellitic acid (PMA) based background electrolyte was improved in order to increase its applicability for routine analysis of inorganic and organic anions in real samples. An electrolyte composed of 6.75 mM PMA, 0.5 mM hexamethonium hydroxide as electroosmotic flow (EOF) modifier, Ca(2+) 0.05 mM as complexation agent and pH adjusted to 7.6 with TEA 1M allows for the separation of 22 inorganic and organic anions in less than 17 min. Good RSDs for within-day migration time reproducibility (0.03-0.9%) and day-to-day analyses (0.04-1.4%) were obtained by the use of two internal standards, allowing for an accurate compound identification. The detection limits ranged from 0.1 to 0.4 mgL(-1) (S/N=3) for hydrodynamic injection (1250 mbars). The applicability of the proposed method was demonstrated by the analysis of inorganic and organic anions in diverse real samples. The recoveries obtained ranged from 93 to 106%.     

Hydrogen exchange studies on Alzheimer's amyloid-beta peptides by mass spectrometry using matrix-assisted laser desorption/ionization and electrospray ionization

The conformation and aggregation behavior of synthetic Alzheimer's amyloid peptides (Abeta) has been investigated using hydrogen-deuterium exchange measured by electrospray ionization mass spectrometry and matrix-assisted laser desorption/ionization mass spectrometry. Mass spectrometric fragmentation of deuterated Abeta peptides was carried out by collision-induced dissociation, inlet fragmentation, and post-source decay. In contrast to the C-terminally truncated peptides Abeta(1-40) and Abeta(1-36) showing full hydrogen-deuterium exchange, Abeta(1-42) and the pyroglutamyl peptide Pyr(3)-Abeta(3-42) produced more complex signal patterns resulting from the formation of beta-sheet-structured oligomers having 18-20 strongly protected protons. Using mass spectrometric fragmentation the results show that the reduced isotope exchange of Abeta(1-42) can be attributed to the central part of the chain comprising residues 8-23. This confirms involvement of the hydrophobic binding domain LVFFA in the course of Abeta aggregation and demonstrates that hydrogen-deuterium exchange in combination with mass spectrometry is well suited for structural analysis of monomeric and reversibly associated amyloid peptides using picomole quantities of material.     

Design of peptides that form amyloid-like fibrils capturing amyloid beta1-42 peptides

Amyloid beta-peptide (Abeta) plays a critical role in Alzheimer's disease (AD). The monomeric state of Abeta can self-assemble into oligomers, protofibrils, and amyloid fibrils. Since the fibrils and soluble oligomers are believed to be responsible for AD, the construction of molecules capable of capturing these species could prove valuable as a means of detecting these potentially toxic species and of providing information pertinent for designing drugs effective against AD. To this aim, we have designed short peptides with various hydrophobicities based on the sequence of Abeta14-23, which is a critical region for amyloid fibril formation. The binding of the designed peptides to Abeta and the amplification of the formation of peptide amyloid-like fibrils coassembled with Abeta are elucidated. A fluorescence assay utilizing thioflavin T, known to bind specifically to amyloid fibrils, revealed that two designed peptides (LF and VF, with the leucine and valine residues, respectively, in the hydrophobic core region) could form amyloid-like fibrils effectively by using mature Abeta1-42 fibrils as nuclei. Peptide LF also coassembled with soluble Abeta oligomers into peptide fibrils. Various analyses, including immunostaining with gold nanoparticles, enzyme-linked immunosorbent assays, and size-exclusion chromatography, confirmed that the LF and VF peptides formed amyloid-like fibrils by capturing and incorporating Abeta1-42 aggregates into their peptide fibrils. In this system, small amounts of mature Abeta1-42 fibrils or soluble oligomers could be transformed into peptide fibrils and detected by amplifying the amyloid-like fibrils with the designed peptides.     

Why Is the C-terminus of Abeta(1-42) more unfolded than that of Abeta(1-40)? Clues from hydrophobic interaction

Abeta(1-40) and Abeta(1-42) are the main forms of amyloid beta (Abeta) peptides in the brain of Alzheimer's patients; however, the latter possesses much stronger aggregation and deposition propensity than the former, which is partially attributed to the more unfolded C-terminus of Abeta(1-42) than that of Abeta(1-40). To explore the physical basis underlying the different dynamic behaviors of both Abeta peptides, parallel molecular dynamics (MD) simulations on Abeta(1-40) and Abeta(1-42) were performed to investigate their thermal unfolding processes. It is revealed that the addition of residues 41 and 42 in Abeta(1-42) disrupts the C-terminal hydrophobic core, which triggers the unraveling of the C-terminal helix of Abeta(1-42). This conclusion is supported by the MD simulation on the I41A mutant of Abeta(1-42), in which the C-terminal helix possesses relatively higher conformational stability than that of wild type Abeta(1-42) owing to the change in hydrophobic interaction patterns.     

Electrophoretic separation of amyloid beta peptides in plasma

In this prospective study, for the first time we have separated and quantified amyloid beta (Abeta) peptides in the plasma of patients with Alzheimer's disease (AD, n = 8) and age- and environment-matched healthy controls (n = 9) with urea-based Abeta-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)/immunoblot. In addition to the Abeta peptides 1-37/38/39/40/42, which we recently identified as regular constituents of human cerebrospinal fluid (CSF), we have observed a novel electrophoretic band migrating slightly cathodically to Abeta1-42. Since a standard peptide with the amino acid sequence Abeta2-40 migrates in the same position, we hypothesize that this plasma-specific band may correspond to Abeta2-40. The concentration of Abeta peptides in the plasma has been approximately 100-fold lower compared to the CSF. Interestingly, the concentration of the two shortest peptides and the longest one of these considered here (i.e., Abeta1-37/38/42) have increased significantly when the samples have been frozen at -80 degrees C before immunoprecipitation, while the 'middle-length' peptides (i.e., Abeta1-39/40) have not been affected by this procedure. We have not observed significant differences of the Abeta peptides concentrations between AD and control subjects. Our method can be used to investigate the significance of plasma Abeta peptides in neurodegenerative disorders, and to monitor the efficiency of drugs with beta/gamma-secretase inhibitory potency.     

Quantitative analysis of amyloid beta peptides in cerebrospinal fluid of Alzheimer's disease patients by immunoaffinity purification and stable isotope dilution liquid chromatography/negative electrospray ionization tandem mass spectrometry

The 40 and 42 amino-acid residue forms of amyloid beta (Abeta(1-40) and Abeta(1-42)) in cerebrospinal fluid (CSF) have been proposed as potential biomarkers of Alzheimer's disease (AD). Quantitative analyses of Abeta peptides in CSF have relied almost exclusively on the use of immunoassay-based assays such as the enzyme-linked immunosorbent assay (ELISA) procedure. However, due to the ability of the Abeta peptides to readily self-aggregate or bind to other proteins and glassware, such analyses are extremely challenging. Analyses are further complicated by the potential of the peptides to undergo post-translational modifications and the possibilities for cross-reaction in the ELISA assays with endogenous components of the CSF. An approach based on liquid chromatography/tandem mass spectrometry (LC/MS/MS) has now been developed which overcomes these methodological issues. The key steps in implementing this new approach involved immunoaffinity purification coupled with the use of [15N]-labeled Abeta peptides as internal standards, a basic LC mobile phase, negative ion electrospray ionization, and a basic solvent for dissolving the peptides and washing the injection needle to prevent carryover of analytes during multiple injections on the LC/MS system. The validated method had limits of quantitation of 44 fmol/mL (200 pg/mL) for Abeta(1-42) and 92 fmol/mL (400 pg/mL) for Abeta(1-40). An excellent correlation was found between the LC/MS/MS assay and an ELISA assay for Abeta(1-42) in human CSF (r2 = 0.915), although less correlation was observed for Abeta(1-40) (r2 = 0.644). Mean CSF Abeta(1-42) concentrations for samples collected 2 weeks apart from a limited number of AD patients provided additional confidence in the reproducibility of the LC/MS/MS assay. Concentrations for duplicate samples from AD patients were slightly higher than most previously reported values (mean 1.06 +/- 0.25 ng/mL; n = 7). Abeta(1-40) concentrations in duplicate samples obtained from AD patients were also reproducible but were found to be slightly lower than most previously reported values (mean 6.36 +/- 3.07 ng/mL; n = 7). Consistent with literature reports, mean Abeta(1-42) concentrations were found to be lower in AD patients compared with the normal subjects (mean 1.49 +/- 0.59 ng/mL; n = 7), whereas there was no difference in Abeta(1-40) concentrations between AD patients and normal subjects (mean 5.88 +/- 3.03 ng/mL; n = 7). The accuracy and precision of the LC/MS assay mean that it will be a useful complement to existing ELISA assays for monitoring therapeutic interventions designed to modulate CSF Abeta(1-42) concentrations in individual AD patients. Moreover, the introduction of stable isotope labeled internal standards offers the potential to achieve a more rigorous account of the influence of methodological effects related to sample collection and processing.     

"Click peptide" based on the "o-acyl isopeptide method": control of A beta1-42 production from a photo-triggered A beta1-42 analogue

A clear understanding of the dynamic events of amyloid beta peptide (Abeta) 1-42, such as the folding, self-assembly, and aggregation processes, would be of great significance in Alzheimer's disease (AD) research. However, elucidation of these Abeta1-42 dynamic events is a difficult issue due to uncontrolled polymerization, which also poses a significant obstacle for establishing an experimental system that clarifies the pathological function of Abeta1-42. On the basis of the O-acyl isopeptide method, we herein developed a novel photo-triggered "click peptide" of Abeta1-42, for example, 26-N-Nvoc-26-AIAbeta42, in which the photocleavable 6-nitroveratryloxycarbonyl (Nvoc) group was introduced at the alpha-amino group of Ser26 in 26-O-acyl isoAbeta1-42 (26-AIAbeta42). From the results, (1) the click peptide did not exhibit the self-assembling nature under physiological conditions due to one single modified ester; (2) photoirradiation of the click peptide and subsequent O-N intramolecular acyl migration afforded the intact Abeta1-42 with a quick and one-way conversion reaction (so-called "click"), while the click peptide was stable under nonphotolytic or storage conditions. In addition, it is advantageous that no additional fibril inhibitory auxiliaries were released during conversion to Abeta1-42. This method provides a novel system useful for investigating the dynamic biological functions of Abeta1-42 in AD by inducible activation of Abeta1-42 self-assembly.     

Separation of lidocaine and its metabolites by capillary electrophoresis using volatile aqueous and nonaqueous electrolyte systems

The separation of the basic drug lidocaine and six of its metabolites has been investigated both by using volatile aqueous electrolyte system, at low pH and by employing non-aqueous electrolyte systems. In aqueous systems, the best separation of the compounds under the investigated conditions was achieved by using the electrolyte 60 mM trifluoroacetic acid (TFA)/triethylamine (TEA) at pH 2.5 containing 15% methanol. With this electrolyte, all seven compounds were well separated with high efficiency and migration time repeatability. The separations with bare fused-silica capillaries and polyacrylamide-coated capillaries were compared with higher separation efficiency with the latter. On the other hand, near baseline separation of all the seven compounds was also obtained by employing the non-aqueous electrolyte, 40 mM ammonium acetate in methanol and TFA (99:1, v/v), with comparable migration time repeatability but lower separation efficiency relative to the aqueous system.     

离子液体中卤素离子杂质的离子色谱-直接电导检测法分析

研究了离子色谱-直接电导检测法分离测定离子液体中的卤素离子(F~-、Cl~-、Br~-)杂质.采用Shim-pack IC-A3阴离子交换色谱柱,考察了淋洗液种类及浓度、流速和色谱柱温度对分离测定的影响.最佳色谱条件为:以1.25 mmol/L邻苯二甲酸氢钾为淋洗液,流速1.5 mL/min,色谱柱温45 ℃.在此条件下可以基线分离卤素离子,且NO~-_3、BF~-_4、SO~(2-)_4不干扰测定.该法测定卤素离子的检出限(S/N=3)为0.02 ～0.11 mg/L,峰面积的相对标准偏差(n=5)不大于0.7%,F~-、Cl~- 和Br~- 的标准曲线的线性范围分别为0.1 ～50、0.1 ～50、0.5 ～100 mg/L.将方法用于烷基咪唑四氟硼酸盐离子液体中卤素离子杂质的测定,加标回收率为98% ～102%.     

Simultaneous determination of inorganic and organic anions,alkali, alkaline earth and transition metal cations by capillary electrophoresis with contactless conductometric detection

Simultaneous separation of up to 22 inorganic and organic anions, alkali, alkaline earth and transition metal cations was achieved in less than 3 min in the capillary electrophoresis system with contactless conductometric detector. The sample was injected from both capillary ends (dual opposite end injection) and anionic and cationic species were detected in the center of the separation capillary. The parameters of the separation electrolyte, such as pH, concentration of the electrolyte, concentration of complexing agents and concentration of 18-crown-6 were studied. Best results were achieved with electrolytes consisting of 8 mM L-histidine, 2.8 mM 2-hydroxyisobutyric acid, 0.32 mM 18-crown-6 at pH 4.25 or 9 mM L-histidine, 4.6 mM lactic acid, 0.38 mM 18-crown-6 at pH 4.25. Other electrolytes containing complexing agents such as malic or tartaric acid at various concentrations could also be used. The detection limits achieved for most cations and anions were 7.5 - 62 micro gL(-1) except for Ba2+ (90 micro gL(-1)), Cd 2+, Cr 3+ and F- (125 micro gL(-1)), and fumarate (250 micro gL(-1)). The repeatability of migration times and peak areas was better than 0.4% and 5.9%, respectively. The developed method was applied for analysis of real samples, such as tap, rain, drainage and surface water samples, plant exudates, plant extracts and ore leachates.     

Combined isolation and purification procedures prior to the high-performance liquid chromatographic-ion-trap tandem mass spectrometric determination of estrogens and their conjugates in river sediments

A fast and sensitive analytical procedure has been developed for the simultaneous separation and determination of alpha-estradiol, beta-estradiol, estriol, estrone and ethynylestradiol and their sulfate, glucuronide and acetate conjugates in river sediments. The procedure includes a microwave-assisted extraction (MAE) with aqueous methanol (25:75, v/v) at 100 degrees C in 10 min, a clean-up on Oasis WAX cartridge and a high-performance liquid chromatography-ion-trap tandem mass spectrometry (HPLC-IT-MS/MS) with electrospray ionization. The recovery for each compounds ranged from 83 to 107% and the repeatability represented as RSDs ranged from 4.9 to 9.6%. The limits of detection (LODs) were down to 1 ng g(-1). The analytical performance of the method was evaluated using determination of free and conjugated estrogens in river sediments.     

The amyloid-beta (Abeta) peptide pattern in cerebrospinal fluid in Alzheimer's disease: evidence of a novel carboxyterminally elongated Abeta peptide

The patterns of amyloid beta (Abeta) peptides in human cerebrospinal fluid (CSF) and brain homogenates were studied by surface-enhanced laser desorption/ionization (SELDI) time-of-flight (TOF) mass spectrometry, and the results were compared with those obtained by Abeta-SDS-PAGE/immunoblot. Apart from the peptides known in the literature to occur in the CSF, we postulate the existence of a novel, previously not described peptide, either Abeta1-45 or Abeta2-46. This peptide was observed exclusively in a pool of samples originating from patients with AD, i.e. CSF and postmortem brain homogenates, but not in either the pooled CSF samples nor the pooled brain homogenates of the non-demented controls. Similarly to our previous results, Abeta1-42 was decreased in the CSF in AD. Expectedly, brain homogenates of the control subjects did not show the presence of Abeta peptides. Compared with Abeta-SDS-PAGE/immunoblot, SELDI-TOF enabled more precise analysis of Abeta peptides in the human material. We conclude that SELDI-TOF offers a promising tool for dementia expression pattern profiling using a minute amount of a biological sample.     

Application of dimethyl-beta-cyclodextrin as a chiral selector in capillary electrophoresis for enantiomer separation of ephedrine and related compounds in some drugs

Dimethyl-beta-cyclodextrin (DM-beta-CD) modified capillary electrophoresis has been developed for chiral separation of ephedrine and related compounds, such as (+/-)-norephedrine, (+/-)-N-methylephedrine, (+/-)-ephedrine and (+)-pseudoephedrine. The influence of some crucial parameters such as buffer concentration, pH value, DM-beta-CD concentration, applied voltage and separation temperature on the separation was investigated. Under the optimum conditions, i.e. 40 mM DM-beta-CD in 75 mM Tris (pH 2.5) as the running electrolyte, separation voltage +25 kV and temperature 25 degrees C, a satisfactory separation of the enantiomers was accomplished. The detection limits (S/N = 3) ranged from 65 to 161 ng/mL and the linear range was 0.15 to 101.0 microg/mL for pressure injection. The present method was successfully applied for the analysis of a series of drugs such as anti-tussive, the drug for rheum, the drug for rhinitis and a Chinese traditional herbal medicine, Ephedrae herba (Ma-Huang in Chinese). The recoveries of ephedrine and related compounds in real samples ranged from 97.6 to 103.5%. This method is useful in the simple and rapid analysis of ephedrine derivatives in marketed products.     

The design and synthesis of alanine-based indolicidin derivatives with identical physicochemical properties and their separation using capillary electrophoresis

Four novel alanine-based indolicidin peptide derivatives were designed containing one WPW motif and two alanine residues, resulting in peptides of similar sequence. The separation of these peptides with identical physicochemical properties including molar mass, charge, and secondary structure as characterized by circular dichroism spectroscopy is very difficult; and the separation of peptides with differing physicochemical properties has only previously been reported. Capillary electrophoresis parameters such as separation buffer concentration, separation buffer pH, capillary length, and separation voltage were investigated to optimize the analysis. Using optimized conditions of a background electrolyte containing 5 mM formic acid of pH 2.0, total capillary length of 51 cm and a voltage of 10 kV enabled a baseline separation of the four peptides. The relative standard deviation of the peak areas and migration times for method repeatability (n = 3) were found to be lower than 8% and 3%, respectively. In addition, reasoning for the separation of these peptides is proposed based on the acidity of the formic acid buffer and the hydrophobic grouping of the tryptophan residues in the peptide primary sequence.     

Ex situ atomic force microscopy analysis of beta-amyloid self-assembly and deposition on a synthetic template

The beta-amyloid (Abeta) deposition, which is the conversion of soluble Abeta peptides to insoluble plaques on a surface, is an essential pathological process in Alzheimer's disease (AD). The identification and characterization of possible environmental factors that may influence amyloid deposition in vivo are important to unveil the underlying etiology of AD. According to the amyloid cascade hypothesis, diffuse plaques are initial and visual deposits in the early event of AD, leading to amyloid plaques. To study amyloid deposition and growth in vitro, we prepared a synthetic template by immobilizing Abeta seeds on an N-hydroxysuccinimide ester-activated solid surface. According to our analysis with an ex situ atomic force microscope, the formation of amyloid plaque-like aggregates was mediated by the interaction between Abeta in a solution and on a synthetic template, suggesting that Abeta oligomers function well as seeds for amyloid deposition. It was observed that insoluble amyloid aggregates formed on the template surface serve as a sink of soluble Abeta in a solution as well as mediate the formation of intermediates in the pathway of amyloid fibrillization in a solution. Relative seeding efficiencies of fresh monomers, oligomers, and fully grown fibrils were analyzed by measuring the deposited plaque volume and its height distribution through atomic force microscopy. The result revealed that oligomeric forms of Abeta act more efficiently as seeds than monomers or fibrils do. Fluorescence spectroscopy with thioflavin T confirmed that amyloid aggregate formation proceeds in a concentration-dependent manner. Analysis with Fourier transform infrared spectroscopy indicated a progressive transition of soluble Abeta42 monomer to amyloid fibrils having antiparallel beta-sheet structure on the template. Furthermore, studies on the interaction between Abeta40 and 42, two major variants of Abeta derived from the amyloid precursor protein, showed that amyloid aggregate formation on the surface was accelerated further by the homogeneous association of soluble Abeta42 onto Abeta42 seeds than by other combinations. A slightly acidic condition was found to be unfavorable for amyloid formation. This study gives insight into understanding the effects of environmental factors on amyloid formation via the use of a synthetic template system.     

Cerebrospinal fluid amyloid beta peptide patterns in Alzheimer's disease patients and nondemented controls depend on sample pretreatment: indication of carrier-mediated epitope masking of amyloid beta peptides

A quantitative urea-based amyloid beta (Abeta)-sodium dodecyl sulfate-polyacrylamide gel electrophoresis with Western immunoblot (Abeta-SDS-PAGE/immunoblot) reveals highly conserved and disease-specific Abeta peptide patterns (Abeta 1-37, 1-38, 1-39, 1-40, 1-42) in Alzheimer's disease (AD) patients and nondemented controls. For further standardization of this method, we analyzed cerebrospinal fluid (CSF) of eight probable AD patients and seven nondemented controls using different preanalytical procedures for Abeta-SDS-PAGE/immunoblot and Abeta1-42-enzyme linked immunosorbent assay (ELISA). Both diagnostic groups were discriminated significantly by absolute levels of Abeta1-42 and ratios of Abeta1-42/40, 1-42/38, 1-42/39. Preanalytical freezing of CSF led to a highly significant loss of all Abeta peptide species. This effect was most pronounced for Abeta1-42 and completely prevented by SDS-heat denaturation prior to freezing. Prolonged storage of SDS-heat denatured CSF led to a selective loss of Abeta1-42 and impaired the discrimination of diagnostic groups as measured by Abeta-SDS-PAGE/immunoblot. Neither freezing nor storage significantly affected absolute Abeta1-42 levels as determined by Abeta1-42-ELISA, but both impaired the discrimination of diagnostic groups. Hence, we suggest immediate analysis of samples for Abeta1-42-ELISA, analysis after a short freezing interval for Abeta-SDS-PAGE/immunoblot, and avoidance of prolonged storage intervals. Remarkably, Abeta-SDS-PAGE/immunoblot measured threefold higher levels of Abeta1-42 in CSF than Abeta1-42-ELISA. In summary, our results indicate carrier-mediated epitope masking of Abeta1-42.     

On-column complexation capillary electrophoretic separation of Fe2+ and Fe3+ using 2,6-pyridinedicarboxylic acid coupled with large-volume sample stacking

On-column complexation of Fe2+ and Fe3+ with 2,6-pyridinedicarboxylic acid (2,6-PDCA) formed anionic complexes, which were then separated by capillary zone electrophoresis with direct UV detection at 214 nm. To achieve reasonable separation selectivity and on-column complexation, the conditions such as pH, the concentration of 2,6-PCDA and the EOF modifiers in the electrolyte were examined. The electrolyte contained 5.0 mM 2,6-PDCA, 0.25 mM tetradecyltrimethlammonium bromide (TTAB) and 5% (v/v) acetonitrile at pH 4.0 was optimised for on-column complexation and the separation of Fe[PCDA]2(2-) and Fe[PCDA]2(-). To enhance the detection sensitivity, large-volume sample stacking (LVSS) was used for the on-line preconcentration of Fe[PCDA]2(2-) and Fe[PCDA]2(-). Under the optimised conditions, satisfactory working ranges (0.5-50 microM), lower detection limits (less than 0.1 microM) and good repeatability of the peak areas (R.S.D.: 5.2-7.8%, n = 5) was achieved using LVSS (300 s). With LVSS, the detection sensitivity was enhanced more than 50-fold compared to conventional hydrodynamic injection. The proposed method was used successfully for the determination of Fe2+ and Fe3+ in water samples.     

Efficient and reproducible analysis of peptides by capillary electrophoresis using noncovalently bilayer-coated capillaries

The usefulness of a noncovalent capillary coating consisting of two layers of oppositely charged polymers for the separation of peptides with capillary electrophoresis (CE) was studied. Capillaries were coated simply by subsequently flushing with solutions of 1% m/v Polybrene and 1% v/v poly(vinylsulfonate) (PVS) forming a bilayer, which showed to produce a strong and highly reproducible electroosmotic flow (EOF) at low pH. Using this coating in combination with a background electrolyte (BGE) containing sodium phosphate (pH 2.5) and 0.01% v/v PVS, initially broadened and overlapping peaks were obtained for some test peptides. By omitting the PVS from the BGE, the peak width and shape of the peptides improved resulting in baseline separation. A systematic study of the influence of the BGE composition showed that considerable further enhancement of the separation efficiency was achieved by increasing the ionic strength of the BGE. Using a BGE of 200 mM tris(hydroxymethyl)aminomethane (Tris)-phosphate (pH 2.5) plate numbers for the peptides were in the 300 000-600 000 range and the relative standard deviation of the peptide migration times was less then 0.3% (n = 5). The use of Tris-phosphate instead of sodium phosphate allowed the current to stay within acceptable limits when 30 kV was used as separation voltage. Overall, the bilayer coating showed a remarkable EOF repeatability, as well as long-term stability. Compared to bare fused-silica capillaries the intraday and interday repeatability of migration times was very favorable and coated capillaries could be used for over a month performing analyses with low and high ionic strength BGEs without any performance deterioration. The usefulness of the bilayer-coated capillaries for the analysis of positively charged peptides was demonstrated by the fast and efficient separation of various closely related enkephalins and the baseline separation of an isomeric peptide/peptoid couple exhibiting efficiencies of over 550 000 plates.     

Determination of peptide hormones of brain and intestine by CE with ESI-MS detection

A new method for the determination of the peptide hormones of brain and intestine based on CE coupling with a DAD and ESI-MS was established. Several electrophoretic and ESI-MS parameters were investigated in detail, such as electrolyte nature and concentration, organic solvent and sheath liquid compositions, nebulization gas pressure and the ESI capillary voltage. Optimized conditions were achieved with 25 mM formic acid-ammonium formate (pH 2.9) as the optimal electrolyte, 2 mM formic acid in 80% methanol in water as the sheath liquid, and 20 kV applied voltage. Under the optimized conditions, four protonated peptides were separated by CE and selectively detected by a quadrupole mass spectrometer with a sheath flow ESI interface. LODs for the four peptides (neurotensin hexapeptide, neurotensin, cholecystokinin tetrapeptide, and pentagastrin) were in the range of 0.10-0.60 micromol/L at an S/N of 3. The RSDs (n = 8) of the method were 0.70-1.5% for migration times and 1.6-6.1% for peak areas. This method is simple, rapid, and selective compared with RIA and ELISA techniques, and has been applied to the analysis of rat hypothalamus tissue.     

On-line high-performance liquid chromatography/mass spectrometric investigation of amyloid-beta peptide variants found in Alzheimer's disease

Abeta peptides are the major components of amyloid deposits in Alzheimer's disease. The presence of N-terminally truncated Abeta variants in amyloid may be a critical factor in Alzheimer's disease pathogenesis. These Abeta variants are less soluble and more amyloidogenic than full-length Abeta, making their separation, purification and identification difficult. High-performance liquid chromatography (HPLC) at elevated temperatures, coupled to electrospray ionization (ES) mass spectrometry (MS), enables rapid separation and identification of N-terminally truncated Abeta variants. This methodology provides a potential tool for exploring the importance of these Abeta variants in both the pathogenesis and diagnosis of Alzheimer's disease.