Quantitation of homocysteine in human plasma by capillary electrophoresis and laser-induced fluorescence detection

Homocysteine (Hcy) represents a branching point between the transsulfuration and transmethylation pathway of methionine. A large increase of plasma concentration of Hcy is observed in patients with inherited hyperhomocysteinemia. A moderated increase (above 10 microM) is also observed in various pathological conditions, such as arterial occlusion, hypertension, hyperlipidemia and chronic renal failure. While amino acids were largely studied using capillary electrophoresis with UV or laser-induced fluorescence detection (LIF), thiol-amino acids were not. In this work we present a new approach for testing homocysteine in human plasma using CE-LIF and fluorescein isothiocyanate. The low fluorescence yield of the fluorescein thiocarbamyl (FTC) thiol-amino acids limits, probably, the sensitivity of the detection to 8 x 10(-10) M (instead of 10(-12) M for FTC-arginine).     

Microdialysis-immunoaffinity capillary electrophoresis studies on neuropeptide-induced lymphocyte secretion

A micro-sampling procedure has been developed for studying lymphocyte secretion of biologically important peptides in low cell density cultures. The technique is based on microdialysis recovery of the analytes of interest coupled with immunoaffinity capillary electrophoresis separation of the microdialysis samples and laser-induced fluorescence detection. Although the technique is able to recover secreted materials only at the 5-10 cell level, the detection system has a limit of detection (LOD) in the attomole (10(-18) M) range. This degree of sensitivity indicates that the system has the potential to measure secreted products at the single cell level. An added advantage of this system over other sampling techniques is that the microdialysis probe allows continuous sampling over time.     

General protease assay method coupling solid-phase substrate extraction and capillary electrophoresis

Capillary electrophoresis with laser-induced fluorescence detection was used to develop a universal, highly specific protease assay. In this method, a peptide, biotinylated at the N-terminus, is labeled with fluorescein at a lysine residue near the C-terminus. Impurities are removed from the fluorescence labeling mixture by solid-phase extraction of the substrate on immobilized streptavidin, followed by extensive washing. The purified fluorescent substrate is dissociated from the streptavidin and incubated with the protease. The peptide sequence between the biotin and fluorescent label contains the cleavage sequence of the protease of interest. After cleavage, the fluorescent product does not contain a biotin group. A second solid-phase extraction is used to remove unreacted substrate to dramatically lower the background signal. The product is detected by capillary electrophoresis, which provides powerful discrimination against products generated by nonspecific proteases. With chymotrypsin as a test protease, product was detected with as little as 10 pg/mL (4.6 x 10(-13) M) chymotrypsin, or 5 amol of enzyme in the 10-microL sample volume.     

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

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

Simple sheath flow reactor for post-column fluorescence derivatization in capillary electrophoresis

A system for post-column fluorescence derivatization in capillary electrophoresis is described. The post-column reactor uses a sheath flow detection cell where the reagents, o-phthaldialdehyde and beta-mercaptoethanol, are added to the sheath buffer and mix by diffusion with the analytes effusing from the separation capillary. Reaction progress is monitored and optimized by imaging a large portion of the sheath flow cuvette using an extended UV source and a CCD camera. Significantly, this design provides the ability to switch between the analysis of pre- and post-column derivatized amino acids and peptides easily and without sacrificing system performance. The lack of turbulent flow in this system minimizes post-separation band broadening. The limit of detection for glycine is 9.4 x 10(-8) M (110 amol) with a separation efficiency of 190,000 theoretical plates, without stacking. The performance of the system for a series of amino acids was evaluated using post-column and pre-capillary derivatization.     

Two-dimensional separations: capillary electrophoresis coupled to channel gel electrophoresis

Two-dimensional separations provide extremely high peak capacities. Coupling capillary zone electrophoresis with ultrathin channel gel electrophoresis offers a convenient and efficient way to perform such two-dimensional microseparations. By means of in situ polymerization, high-concentration (up to 50%T) polyacrylamide gels are prepared in 75 mm long, 25 mm wide, and 40 microns thick rectangular channels. By moving the outlet end of the capillary electrophoresis capillary across the entrance of the channel, both separations are completely preserved. Mixtures of peptides labeled by fluorescein isothiocyanate (FITC) are well resolved in less than 15 min, with theoretical plate numbers in the range of 20,000-50,000 for each independent separation. Significant enhancement in separation efficiency and peak capacity over one-dimensional separations are demonstrated by this combination. The two-dimensional separations of a model mixture of peptides, a tryptic digest of trypsinogen, and < 0.05% of an individual B2 neuron from the marine mollusk Aplysia californica are presented.     

Double coupling Edman chemistry for high-sensitivity automated protein sequencing

Capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) is a promising new method for the analysis of protein sequencing products. It gives 10 zmol (1 zmol = 10(-21) mol) limits of detection (3 sigma) for fluorescein thiohydantoin (FTH) amino acids. We have developed a separation for the (FTH)-amino acid products generated from 18 of the 20 coded amino acids. The extremely low volume requirement associated with CE-LIF makes it incompatible with commercial sequencers. For this reason, we have also been developing a miniaturized sequencer that can be more easily coupled to our detection system. Both the CE-LIF system and the miniaturized sequencer are described.     

Isolation and expression of a mouse CB1 cannabinoid receptor gene. Comparison of binding properties with those of native CB1 receptors in mouse brain and N18TG2 neuroblastoma cells

The chiral separation of enantiomeric forms of derivatized amino acids have been achieved based on a metalchelate chiral capillary electrophoretic method and a cyclodextrin mediated host-guest interaction approach in micellar electrokinetic chromatography (MEKC) mode with laser-induced fluorescence detection. This approach has been applied to the determination of enantiomeric forms of amino acids derived from novel depsipeptide antitumor antibiotics, BMY-45012 and its analogs. Amino acids were analyzed by complete hydrolysis and the hydrolysate was derivatized with either dansyl chloride for UV absorbance detection or fluorescein isothiocyanate for laser based fluorescence detection. The presence of several amino acids, serine and beta-hydroxyl-N-methy-valine in the proposed structure have been confirmed as D-serine and L-beta-hydroxyl-N-methy-valine enantiomeric forms by both chiral capillary electrophoresis (chiral CE) and MEKC approaches. A non-chiral amino acid, sarcosine, was also confirmed. These methodologies provide a quick and sensitive approach for the determination of amino acids racemization of pharmaceutical natural products and have proven to be useful for structural elucidation refinement.     

Recent developments in capillary electrophoresis of carbohydrate species

Pesticides are important and diverse environmental and agricultural species. Their determination in pesticide formulations, in feed and food, and in complex environmental matrices (e.g., water, soil, sludge, sediments, etc.) often requires separation methods of high efficiency, unique selectivity and high sensitivity. As shown in this comprehensive review, capillary electrophoresis meets these requirements and has proved to be a suitable microseparation technique for the analysis of a wide variety of chiral and achiral pesticides. It is also shown that by combining selective precolumn derivatization schemes, sensitive detection methods (e.g., laser induced fluorescence detection) and trace enrichment techniques, capillary electrophoresis (CE) is capable of determining pesticides at trace levels as those usually encountered in environmental samples.     

Electrophysiological effects of ketamine on Ca(2+)-activated K+ channel in single rabbit portal vein cell

The effects of ketamine on Ca(2+)-activated K+ channel currents were studied in dispersed single smooth muscle cells from rabbit portal vein using inside-out patch clamp technique. In a near physiological K+ and Ca2+ gradient, three populations of outward rectangular single currents were recorded in isolated cell membrane of rabbit portal vein at +60 mV membrane potential. These currents were judged as Ca(2+)-activated K+ channel currents since application of EGTA or Apamin in the internal solution inhibited these currents. Application of 10(-5)M or 10(-4)M ketamine inhibited the number of occurrences of channel opening and decreased open times, but did not reduce the amplitudes. When the 10(-3)M ketamine was applied, the Ca(2+)-activated K+ channel currents were abolished. We suggest that the depression of Ca(2+)-activated K+ channel currents may explain the continuous contraction observed in rabbit portal vein at a clinical concentration of ketamine from a point of electrophysiological K+ current study.     

Molecular dynamics of the anti-fluorescein 4-4-20 antigen-binding fragment. 2. Time-resolved fluorescence spectroscopy

Time-resolved fluorescence experiments were performed to investigate the dynamic aspects of the antigen-binding fragment (Fab) of a high-affinity monoclonal antibody (4-4-20) which binds the fluorescent hapten fluorescein. Both the unliganded Fab and a complex of the Fab with a nonfluorescent analog of fluorescein (fluoresceinamine, FLM) were examined. A fluorescence polarization probe [5-[[2-[(iodoacetyl)amino]ethyl]amino]naphthalene-1-sulfonic acid, AEDANS] was covalently attached to the C-terminus of the Fab. Experiments were performed at three different temperatures (10, 25, and 35 degrees C), and phase-modulation data sets were collected for five different molar ratios of FLM to Fab at each temperature. Global analyses were then used to extract values for fluorescence lifetime and rotational correlation time from these data. In the lifetime analysis the best fit was obtained when the emission of AEDANS was described by a Lorentzian distribution of lifetimes (tau = 15.6 ns, distribution width = 3.4 ns, both at 25 degrees C), which suggested that the probe experienced a heterogeneous environment. Anisotropy analyses suggested that two different rotational components were present. The first was attributed to the global motion of the Fab and exhibited a rotational correlation time (theta 1) of ca. 33 ns at 25 degrees C. This component was relatively unaffected by antigen binding. The second rotational component was attributed to the local or segmental motion within the Fab and exhibited a rotational correlation time (theta 2) of 1.1 ns at 25 degrees C. This value increased by more than 50% upon antigen binding, a result which was consistent with molecular dynamics simulations of the same Fab--fluorescein system [Lim & Herron (1995) Biochemistry 34, 6962-6974]. Furthermore, statistical analysis showed that this increase was significant at the 95% confidence level.     

Capillary zone electrophoresis with laser-induced fluorescence detection for the determination of nanomolar concentrations of noradrenaline and dopamine: application to brain microdialysate analysis

Determination of catecholamines by capillary zone electrophoresis with laser-induced fluorescence detection was performed on low-concentration samples, which were derivatized with naphthalene-2,3-dicarboxaldehyde to give highly fluorescent compounds. When the borate concentration in the derivatization medium was decreased from 130 to 13 mM, sensitivity for noradrenaline (NA) and dopamine (DA) was greatly enhanced while resolution between these two compounds decreased. A 50 mM borate concentration in derivatization medium was chosen since it provided maximal resolution between NA and DA, together with a high separation efficiency (3.1 million theoretical plates per meter for DA). The injection of 2.4 nL of a NA and DA solution derivatized at 10(-9) M produced peaks with signal-to-noise ratio of 8:1 and 3:1, respectively, corresponding to 1.8 amol of each catecholamine. The calibration curves were linear when NA and DA solutions were derivatized at concentrations ranging from 10(-6) to 10(-9) M. This method was used to determine NA in brain extracellular fluid: a peak corresponding to a basal level of 5 x 10(-9) M endogeneous NA was observed in microdialysates from the medial frontal cortex of the rat, and its nature was confirmed by both electrophoretic and pharmacological validations.     

Ultrasensitive detection of closely related angiotensin I peptides using capillary electrophoresis with near-infrared laser-induced fluorescence detection

A novel near-infrared (NIR) fluorescent dye (NN382, LICOR, Inc.) was evaluated as an ultrasensitive peptide-labeling reagent for use with capillary electrophoresis (CE). Six angiotensin I (Ang-I) variants were selected as model peptides for the derivatization and separation studies. The closely related decapeptides were labeled with the NIR dye, separated using CE, and detected by NIR laser-induced fluorescence. Derivatization of the peptides was achieved under aqueous conditions using 2.5-500 pmol of Ang-I in a 50-microL sample (5 x 10(-8)-1 x 10(-5)M), and between 1.3 and 254 amol of the labeled peptides were injected on column. The fluorescence response was linear over a 200-fold range (correlation r > or = 0.9986). The limit of detection (SNR = 3, signal/RMS noise) ranged from 100 to 300 zmol, for the six Ang-I variants. Four of six peptides were resolved from each other and excess dye using capillary zone electrophoresis with a simple 50 mM phosphate run buffer, pH 7.2. Two pairs of coeluting peptides were successfully resolved using micellar electrokinetic chromatography with a nonionic surfactant, Triton X-100. The NIR amine-labeling reagent NN382 is a viable alternative to using visible fluorophores for CE methods requiring high sensitivity.     

Fluorescence detection of proteins and amino acids in capillary electrophoresis using a post-column sheath flow reactor

A simple laser-induced fluorescence detection method for proteins and amino acids in capillary electrophoresis is reported. A sheath flow cell is utilized as a post-column reactor for fluorescence derivatization of proteins and amino acids by addition of o-phthaldialdehyde-2-mercaptoethanol to the sheath fluid. With the use of a 50 microns I.D. capillary, the limits of detection for carbonic anhydrase are 0.73 nM or 1.8 amol which represents a five- and two-fold improvement, respectively, over the best results previously reported for post-column detection. In addition, separation efficiencies up to 8.07 x 10(5) are achieved and the detector response is linear over three-orders of magnitude. These results demonstrate that mixing is adequate and the reaction kinetics are rapid enough to provide sensitive detection with this approach. Also, because this post-column derivatization scheme requires no instrumental changes to a typical sheath flow cell detector, the system can be used for detection of pre-column labeled analytes and for native fluorescence detection.     

FIA titrations of ephedrine in pharmaceutical formulations with a PVC tetraphenylborate tubular electrode

A flow injection system for the titration of ephedrine in pharmaceutical products with potentiometric detection was developed. For this purpose a tetraphenylborate tubular electrode was constructed. The electrode was prepared without inner reference solution and with a PVC membrane based on tetrapentylammonium tetraphenylborate as ion exchanger and 2-nitrophenylphenyl ether as mediator solvent. Its operational characteristics were evaluated in a low dispersion manifold and compared with more conventionally shaped electrodes using the same sensor. In the pH range 2.5-11.5, the electrodes showed linear response between 3.8 x 10(-6) and 0.1 M with a slope of -56.4 mV/log[BPh4]. Ephedrine determinations in pharmaceutical products were carried out in a single channel manifold with a mixing chamber incorporated and using the tubular electrode as detector. Recovery rates of 98.6 +/- 2.5% were obtained in the analysis of tables, nasal drops and syrups with a sampling rate of about 60 h-1.     

A perfluorosulfonated ionomer end-column electrical decoupler for capillary electrophoresis/electrochemical detection

An end-column electrical decoupler contructed with perfluorosulfonated ionomer (Nafion) is described. This decoupler was fabricated at the cathodic end of the separation capillary by casting liquid Nafion ion exchange powder over a copper-plated tungsten wire. The internal diameter of the flow channel was controlled by adjusting the thickness of the copper plating. This design overcomes problems of conventional end-column detection such as low sensitivity due to low collection efficiency of analytes at the detection electrode, difficulty in precise placement of the detection electrode, and the need to use small-bore capillaries (<-25 micron). The loss of cationic analytes observed with long-cast Nafion on-column decouplers was significantly reduced. The high current shunting capability of the long Nafion decoupler was maintained in this configuration. Elimination of the detection capillary required for on-column electrical decouplers provided higher separation efficiency by maintaining plug-type flow throughout the system. Four model catecholamines were well separated within 5 min with separation efficiency of up to 230 000 plates and migration time reproducibilities of <0.6% RSD. With the optimized experimental conditions, detection limits of 3 nM for the catecholamines were achieved in a Ringer's solution matrix (to model a microdialysis sample).     

Modulation of retinal endothelial barrier in an in vitro model of the retinal microvasculature

The prediabetic/diabetic condition functionally alters the microvascular bed of the eye and the breakdown in the transvascular barrier may be produced by changes in the retinal endothelial barrier. To better understand how retinal microvessel barrier is maintained and is altered in vivo this study applies and extends our previously described in vitro permeability technique to study retinal endothelial monolayers. The model of the retinal microvasculature consists of retinal capillary endothelial cells cultured on porous microcarrier beads and perfused in chromatographic 'cell-columns'. This model design relies on indicator-dilution techniques to measure the permeability of the retinal endothelial monolayer and detects small changes in retinal endothelial permeability produced by treatments. Bovine retinal capillary endothelial cells (RCE) were obtained using an endothelial selective media. RCE were seeded at 3 x 10(4) cells cm-2 of fibronectin-coated gelatin microcarriers. After 7 days of microcarrier culture, microcarriers were poured to form columns 0.66 cm in diameter and 1.6 cm in length. The cell-column elution patterns of coinjected optically absorbing tracers (blue dextran 2 x 10(6) Da; cyanocobalamin 1355 Da; sodium fluorescein 376 Da) were analysed to estimate the permeability of the RCE monolayers covering the microcarriers. Scanning electron microscopic examination showed complete monolayer formation on the surface of the microcarriers. We found that baseline monolayer permeability averaged 7.57 +/- 0.57 x 10(-5) cm sec-1 for cyanocobalamin and 9.29 +/- 0.78 x 10(-5) cm sec-1 for sodium fluorescein (mean +/- S.E.M., n = 39). Permeability did not increase over 2 hr of cell-column perfusion. Permeability was decreased by 1 micron isoproterenol (n = 3) and increased by 1 microgram ml-1 cytochalasin D (n = 5). This is one of the first reports of in vitro permeability values for the transport barrier formed by retinal microvascular endothelial cells. Furthermore, the endothelial component of the retinal barrier is dynamic, and is enhanced by isoproterenol and diminished by cytochalasin D.     

Quantitative assay for epinephrine in dental anesthetic solutions by capillary electrophoresis

A simple and robust method for the separation and quantification of epinephrine in dental anesthetic solutions was developed. The method allows the direct injection of high salt solutions without sample pre-treatment. Large sample plugs (5.7% of the total capillary length) are used for epinephrine determination by selective analyte focusing in capillary electrophoresis. The concentration detection limit for epinephrine is about 5.0 x 10(-7) M (90 ng ml-1) with a commercial UV detector. The separation protocol was validated in terms of its precision, linearity, accuracy and specificity.     

Quantitative analysis of synthetic dyes in lipstick by micellar electrokinetic capillary chromatography

The separation of synthetic dyes, used as color additives in cosmetics, by micellar electrokinetic capillary chromatography (MEKC) is described in this study. The separation of seven dyes, namely eosine, erythrosine, cyanosine, rhodamine B, orange II, chromotrope FB and tartrazine has been achieved in about 3 min in an untreated fused silica capillary containing as background electrolyte a 25 mM tetraborate/phosphate buffer, pH 8.0, and 30 mM sodium dodecyl sulfate. The electrophoretic method exhibits precision and relatively high sensitivity. A detection limit (LOD, signal/noise = 3) in the range of 5-7.5 X 10(-7) M of standard compounds was recorded. Intra-day repeatability of all the studied dye determinations (8 runs) gave the following results (limit values), % standard deviation: 0.24-1.54% for migration time, 0.99-1.24% for corrected peak areas, 0.99-1.24% for corrected peak area ratio (analyte/internal standard) and 1.56-2.74% for peak areas. The optimized method was successfully applied to the analysis of a lipstick sample where eosine and cyanosine were present.     

Determination of amino acids in biological fluids by capillary gas chromatography with nitrogen-phosphorus selective detection

A selective and sensitive method for the determination of protein and non-protein amino acids in biological fluids by capillary gas chromatography (GC) has been developed. The amino acids in the samples were directly converted into their N(O,S)-isobutoxycarbonyl methyl ester derivatives and measured by GC with nitrogen-phosphorus selective detection (NPD) using a DB-17ht capillary column. Using this method, the derivatives of the 21 protein amino acids and the 25 non-protein amino acids provided excellent NPD responses and were quantitatively and reproducibly resolved within 28 min. The lower detection limits of these amino acids, at a signal-to-noise ratio of 3, were ca. 6-150 pg injected. The calibration curves for each amino acid in the range of 0.02-2 micrograms were linear and sufficiently reproducible for quantitative analysis. This method was successfully applied to small urine and serum samples without prior clean-up; there was no evidence of interference from coexisting substances. Overall recoveries of amino acids added to urine and serum samples were 83-112%. The intra-assay and inter-assay R.S.D. of amino acids in these samples were 0.3-8.9% (n = 3) and 1.9-15.8% (n = 3), respectively.