The quantitative characterization of free radical sources and traps by electromigration applications

Nowadays, very diverse human activities generate urgent demands for fast, sensitive reliable innovative tools capable of detecting major industrial, military, and other dangerous products. An important part of these compounds are free radicals. Capillary electrophoresis (CE), especially in its miniaturized format (lab-on-a-chip), and other electromigration methods offer special possibilities to resolve this problem. These measurements have a great opportuness because of very wide chemical and biological role of free radicals. Several compounds, e.g. monomers and some biologically important groups (as are nitrones) oppose oxidative challenges by virtue of their trap very rapidly oxygen- or carbon-centered radicals and generating other radical species which are stable and biochemically less harmful than the original ones. In many cases, conventionally, the relative trap capacity is measured against tert.-butylhydroperoxide (TBH). In this lecture are presented numerous important free radical species (active oxygen–, nitrogen- and carbon-centered ones, as HO, NO etc) and their adequate in vitro and in vivo applied bioanalytical methods, including liquid chromatography with electrochemical detection and mass spectrometry, gas chromatography with mass spectrometry, capillary electrophoresis, electron spin resonance and chemiluminescence analysis. A simple and highly sensitive method is the capillary zone electrophoresis with amperometric detection (CZE-AD); It was introduced to determine indirectly OH by analysing its reaction products with salicylic and dihydroxybenzoic acids. Hydroxylated radical products of these acids are often used as a relative measurement in free radical research. Accurate determination of pK(a) values is important for proper characterization of newly synthesized molecules. CZE method was used for determination of their values. Are initiated new research fields as Fenton-, electro-Fenton and photoelectro-Fenton chemistry and foreseen their perspectives.

Nitric oxide is an important cell signaling molecule in physiology and pathophysiology. An indirect method for monitoring nitric oxide (NO) by determining nitrate and nitrite by microchip capillary electrophoresis (CE) with electrochemical (EC) detection has been developed. The amount of nitrite formed in this reaction (analyzed by capillary electrophoresis) was compared with the amount of oxygen consumed (measured by polarography). Were observed a linear relationship between the amount of consumed oxygen and the amount of nitrite formed in the measured range. These results demonstrate that polarographic measurements of the amount of oxygen consumed in the reaction with NO could be used to estimate the concentration of dissolved NO in authentic media. Polarography is an adequate method also to quantitative kinetic study of the free radical activity and of the trapping capacity of different compounds. This method is based on measure of the catalytic polarografic current of Fe(III) in the presence of free radical sources (TBH, hydrogen-peroxydes), and their traps. Personal contribution of the authors in this field is discussed.     

1H-NMR study on the tautomerism of the imidazole ring of histidine residues. I. Microscopic pK values and molar ratios of tautomers in histidine-containing peptides

The 1H-NMR titration curves of chemical shifts versus pH were observed for imidazole, N1-methylimidazole, L-histidine, N1-methyl-L-histidine, N3-methyl-L-histidine, and other related compounds. With these results, the macroscopic pK values of these compounds were determined by a computer curve-fitting for a simple dissociation sequence. From the pK values of imidazole and N1-methylimidazole, the perturbation for the pK of the imidazole ring due to the substitution of a proton with a methyl group was estimated as -0.21 pH unit. The microscopic pK values of the individual tautomers of the imidazole ring were estimated with the pK values of N1-methyl-L-histidine, N3-methyl-L-histidine, and perturbation due to methyl substitution. The estimated pK values were 6.73 for the N1-H tautomer and 6.12 for the N3-H tautomer. These values were in good agreement with those obtained using carboxymethyl derivatives instead of methyl derivatives. Furthermore, the macroscopic pK value (6.02) calculated using the estimated microscopic pK values agreed with that (6.03) observed for the imidazole ring of L-histidine. Thus the method in this work was indicated to be self-consistent. The microscopic pK values of tautomers were also obtained for N alpha-acetyl-L-histidine and N alpha-acetyl-L-histidine methylamide. The molar ratios of tautomers were calculated on the basis of the microscopic pK values of tautomers. The intrinsic (or unperturbed) pK value of imidazole ring and perturbations due to the CO2- and NH3+ were obtained for each of the N1-H and N3-H tautomers.     

Theoretical prediction of relative and absolute pKa values of aminopyridines

This work presents a study aimed at the theoretical prediction of pK(a) values of aminopyridines, as a factor responsible for the activity of these compounds as blockers of the voltage-dependent K(+) channels. To cover a large range of pK(a) values, a total of seven substituted pyridines is considered as a calibration set: pyridine, 2-aminopyridine, 3-aminopyridine, 4-aminopyridine, 2-chloropyridine, 3-chloropyridine, and 4-methylpirydine. Using ab initio G1, G2 and G3 extrapolation methods, and the CPCM variant of the Polarizable Continuum Model for solvation, we calculate gas phase and solvation free energies. pK(a) values are obtained from these data using a thermodynamic cycle for describing protonation in aqueous and gas phases. The results show that the relatively inexpensive G1 level of theory is the most accurate at predicting pK(a) values in aminopyridines. The highest standard deviation with respect to the experimental data is 0.69 pK(a) units for absolute values calculations. The difference increases slightly to 0.74 pK(a) units when the pK(a) is computed relative to the pyridine molecule. Considering only compounds at least as basic as pyridine (the values of interest for bioactive aminopyridines) the error falls to 0.10 and 0.12 pK(a) units for the absolute and relative computations, respectively. The technique can be used to predict the effect of electronegative substituents in the pK(a) of 4-AP, the most active aminopyridine considered in this work. Thus, 2-chloro and 3-chloro-4-aminopyridine are taken into account. The results show a decrease of the pK(a), suggesting that these compounds are less active than 4-AP at blocking the K(+) channel.     

Charge-charge interactions are key determinants of the pK values of ionizable groups in ribonuclease Sa (pI=3.5) and a basic variant (pI=10.2)

The pK values of the titratable groups in ribonuclease Sa (RNase Sa) (pI=3.5), and a charge-reversed variant with five carboxyl to lysine substitutions, 5K RNase Sa (pI=10.2), have been determined by NMR at 20 degrees C in 0.1M NaCl. In RNase Sa, 18 pK values and in 5K, 11 pK values were measured. The carboxyl group of Asp33, which is buried and forms three intramolecular hydrogen bonds in RNase Sa, has the lowest pK (2.4), whereas Asp79, which is also buried but does not form hydrogen bonds, has the most elevated pK (7.4). These results highlight the importance of desolvation and charge-dipole interactions in perturbing pK values of buried groups. Alkaline titration revealed that the terminal amine of RNase Sa and all eight tyrosine residues have significantly increased pK values relative to model compounds.A primary objective in this study was to investigate the influence of charge-charge interactions on the pK values by comparing results from RNase Sa with those from the 5K variant. The solution structures of the two proteins are very similar as revealed by NMR and other spectroscopic data, with only small changes at the N terminus and in the alpha-helix. Consequently, the ionizable groups will have similar environments in the two variants and desolvation and charge-dipole interactions will have comparable effects on the pK values of both. Their pK differences, therefore, are expected to be chiefly due to the different charge-charge interactions. As anticipated from its higher net charge, all measured pK values in 5K RNase are lowered relative to wild-type RNase Sa, with the largest decrease being 2.2 pH units for Glu14. The pK differences (pK(Sa)-pK(5K)) calculated using a simple model based on Coulomb's Law and a dielectric constant of 45 agree well with the experimental values. This demonstrates that the pK differences between wild-type and 5K RNase Sa are mainly due to changes in the electrostatic interactions between the ionizable groups. pK values calculated using Coulomb's Law also showed a good correlation (R=0.83) with experimental values. The more complex model based on a finite-difference solution to the Poisson-Boltzmann equation, which considers desolvation and charge-dipole interactions in addition to charge-charge interactions, was also used to calculate pK values. Surprisingly, these values are more poorly correlated (R=0.65) with the values from experiment. Taken together, the results are evidence that charge-charge interactions are the chief perturbant of the pK values of ionizable groups on the protein surface, which is where the majority of the ionizable groups are positioned in proteins.     

Salt effects on ionization equilibria of histidines in myoglobin

The salt dependence of histidine pK(a) values in sperm whale and horse myoglobin and in histidine-containing peptides was measured by (1)H-NMR spectroscopy. Structure-based pK(a) calculations were performed with continuum methods to test their ability to capture the effects of solution conditions on pK(a) values. The measured pK(a) of most histidines, whether in the protein or in model compounds, increased by 0.3 pH units or more between 0.02 M and 1.5 M NaCl. In myoglobin two histidines (His(48) and His(36)) exhibited a shallower dependence than the average, and one (His(113)) showed a steeper dependence. The (1)H-NMR data suggested that the salt dependence of histidine pK(a) values in the protein was determined primarily by the preferential stabilization of the charged form of histidine with increasing salt concentrations rather than by screening of electrostatic interactions. The magnitude and salt dependence of interactions between ionizable groups were exaggerated in pK(a) calculations with the finite-difference Poisson-Boltzmann method applied to a static structure, even when the protein interior was treated with arbitrarily high dielectric constants. Improvements in continuum methods for calculating salt effects on pK(a) values will require explicit consideration of the salt dependence of model compound pK(a) values used for reference in the calculations.     

Rapid Calculation of Protein pK(a) Values Using Rosetta

We developed a Rosetta-based Monte Carlo method to calculate the pK(a) values of protein residues that commonly exhibit variable protonation states (Asp, Glu, Lys, His, and Tyr). We tested the technique by calculating pK(a) values for 264 residues from 34 proteins. The standard Rosetta score function, which is independent of any environmental conditions, failed to capture pK(a) shifts. After incorporating a Coulomb electrostatic potential and optimizing the solvation reference energies for pK(a) calculations, we employed a method that allowed side-chain flexibility and achieved a root mean-square deviation (RMSD) of 0.83 from experimental values (0.68 after discounting 11 predictions with an error over 2 pH units). Additional degrees of side-chain conformational freedom for the proximal residues facilitated the capture of charge-charge interactions in a few cases, resulting in an overall RMSD of 0.85 pH units. The addition of backbone flexibility increased the overall RMSD to 0.93 pH units but improved relative pK(a) predictions for proximal catalytic residues. The method also captures large pK(a) shifts of lysine and some glutamate point mutations in staphylococcal nuclease. Thus, a simple and fast method based on the Rosetta score function and limited conformational sampling produces pK(a) values that will be useful when rapid estimation is essential, such as in docking, design, and folding.     

Estimating the pKa values of basic and acidic side chains in ion channels using electrophysiological recordings: a robust approach to an elusive problem

As a step toward gaining a better understanding of the physicochemical bases of pK(a)-value shifts in ion channels, we have previously proposed a method for estimating the proton affinities of systematically engineered ionizable side chains from the kinetic analysis of single-channel current recordings. We reported that the open-channel current flowing through mutants of the (cation-selective) muscle nicotinic acetylcholine receptor (AChR) engineered to bear single basic residues in the transmembrane portion of the pore domain fluctuates between two levels of conductance. Our observations were consistent with the idea that these fluctuations track directly the alternate protonation-deprotonation of basic side chains: protonation of the introduced basic group would attenuate the single-channel conductance, whereas its deprotonation would restore the wild-type-like level. Thus, analysis of the kinetics of these transitions was interpreted to yield the pK(a) values of the substituted side chains. However, other mechanisms can be postulated that would also be consistent with some of our findings but according to which the kinetic analysis of the fluctuations would not yield true pK(a)s. Such mechanisms include the pH-dependent interconversion between two conformations of the channel that, while both ion permeable, would support different cation-conduction rates. In this article, we present experimental evidence for the notion that the fluctuations of the open-channel current observed for the muscle AChR result from the electrostatic interaction between fixed charges and the passing cations rather than from a change in conformation. Hence, we conclude that bona fide pK(a) values can be obtained from single-channel recordings.     

pKa predictions with a coupled finite difference Poisson-Boltzmann and Debye-Hückel method

Modeling charge interactions is important for understanding many aspects of biological structure and function, and continuum methods such as Finite Difference Poisson-Boltzmann (FDPB) are commonly employed. Calculations of pH-dependence have identified separate populations; surface groups that can be modeled with a simple Debye-Hückel (DH) model, and buried groups, with stronger resultant interactions that are dependent on detailed conformation. This observation led to the development of a combined FDPB and DH method for pK(a) prediction (termed FD/DH). This study reports application of this method to ionizable groups, including engineered buried charges, in staphylococcal nuclease. The data had been made available to interested research groups before publication of mutant structures and/or pK(a) values. Overall, FD/DH calculations perform as intended with low ΔpK(a) values for surface groups (RMSD between predicted and experimental pK(a) values of 0.74), and much larger ΔpK(a) values for the engineered internal groups, with RMSD = 1.64, where mutant structures were known and RMSD = 1.80, where they were modeled. The weaker resultant interactions of the surface groups are determined mostly by charge-charge interactions. For the buried groups, R(2) for correlation between predicted and measured ΔpK(a) values is 0.74, despite the high RMSDs. Charge-charge interactions are much less important, with the R(2) value for buried group ΔpK(a) values increasing to 0.80 when the term describing charge desolvation alone is used. Engineered charge burial delivers a relatively uniform, nonspecific effect, in terms of pK(a) . How the protein environment adapts in atomic detail to deliver this resultant effect is still an open question.     

Determination of free amino acids and related compounds in amniotic fluid by capillary electrophoresis with contactless conductivity detection

A capillary electrophoretic (CE) method with contactless conductivity detection (CCD) has been developed for the determination of free amino acids (AAs) in the amniotic fluid. Apart from 20 proteinogenic AAs, 12 other biogenic compounds have been identified including ethanolamine, choline, beta-alanine, 2-aminobutyric acid, 4-aminobutyric acid, creatinine, ornithine, carnitine, citrulline, 4-hydroxyproline, 1-methylhistidine and 3-methylhistidine. The running electrolyte consisted of 1.7 M acetic acid and 0.1% hydroxyethyl-cellulose (pH 2.15). An addition of acetonitrile to the sample improved the separation of AAs significantly and permitted an increase in the amount of the sample injected. As a result, the sensitivity of the determination increased and the limit of detection (LOD) decreased by a factor of ca. 4, as compared with our previous study. The LOD values were between 1.5 microM (arginine) and 6.7 microM (aspartic acid). The CE/CCD method has then been applied to clinical analyses of the amniotic fluid collected from 20 pregnant women aged over 35 years and 24 pregnant women with whom abnormal foetus development was suspected. The latter group of women was found to exhibit systematically enhanced amniotic levels of most of the AAs studied.     

Direct pharmaceutical analysis of bisphosphonates by capillary electrophoresis

Bisphosphonate compounds have been studied as a class of potential drugs for the treatment of various bone diseases. However, the analyses of these compounds are problematic because most of them do not contain strong chromophores. Based on the unique structures of these compounds, we have employed a capillary electrophoresis (CE) technique for the characterization of these compounds in pharmaceutical dosage formulations. In this study, two CE methods were developed for the determination of a bisphosphonate compound, 2-thioethane-1,1-bisphosphonic acid. The first method involved the use of an uncoated column, a phosphate buffer, and hydrostatic injection with direct UV absorbance detection. The method showed excellent resolution and precision with a reasonable detection limit of 30 μg/ml. Sensitivity was further improved using a glycerol-coated column, together with a phosphate buffer of higher concentration and electrokinetic injection under sample stacking conditions. This modified method revealed a significant improvement in sensitivity with a detection limit of about 50 ng/ml. Both methods demonstrated high simplicity and excellent reproducibility and were successfully applied to the quantitative analyses of pharmaceutical dosing solutions.     

Analysis of electrostatic interactions in the denatured state ensemble of the N-terminal domain of L9 under native conditions

The pH dependence of protein stability is defined by the difference in the number of protons bound to the folded state and to the denatured state ensemble (DSE) as a function of pH. In many cases, the protonation behavior can be described as the sum of a set of independently titrating residues; in this case, the pH dependence of stability reflects differences in folded and DSE pK(a)'s. pH dependent stability studies have shown that there are energetically important interactions involving charged residues in the DSE of the N-terminal domain of L9 (NTL9), which affect significantly the stability of the protein. The DSE of wild type NTL9 cannot be directly characterized under native conditions because of its high stability. A destabilized double mutant of NTL9, V3AI4A, significantly populates the folded state and the DSE in the absence of denaturant. The two states are in slow exchange on the nuclear magnetic resonance time scale, and diffusion measurements indicate that the DSE is compact. The DSE pK(a)'s of all of the acidic residues were directly determined. The DSE pK(a) of Asp8 and Asp23 are depressed relative to model compounds values. Use of the mutant DSE pK(a)'s together with known native state pK(a)'s leads to a significantly improved agreement between the measured pH dependent stability and that predicted by the Tanford-Wyman linkage relationship. An analysis of the literature suggests that DSE interactions involving charged residues are relatively common and should be considered in discussions of protein stability.     

Development of comparative methods using gas chromatography-mass spectrometry and capillary electrophoresis for determination of endocrine disrupting chemicals in bio-solids

Two analytical separation techniques are being investigated for their potential in determining a wide range of endocrine disrupting chemicals (EDCs) in the environment. Capillary electrophoresis (CE) in the micellar mode in conjunction with a cyclodextrin (CD) modifier is shown to have potential for determination of alkylphenol breakdown products. Gas chromatography with mass spectrometric (GC-MS) detection is being utilised for validation of the CE method development and in addition as a separation technique to optimise preconcentration using solid-phase extraction. GC has demonstrated potential for the separation of 26 priority chemicals suspected as being endocrine disrupting compounds. The challenge of the method development process lies in the fact that these compounds are of differing polarities, size and charge and therefore are difficult to separate in a single run. Capillary electrophoresis in the CD-MEKC (micellar electrokinetic chromatography) mode is showing potential in this regard. Limits of determination are in the low mg/l range for CE and GC, however, using preconcentration it is possible to improve detection sensitivity with >80% recovery for some analytes and up to 100% recovery for most target species.     

Determination of the basicity of Mannich ketones by capillary electrophoresis

Capillary electrophoretic (CE) method to characterise Mannich ketones (MKs) containing morpholine moiety was developed. Basicity (pKa,exp) of the MKs was characterised by measured data derived from the electrophoretic mobility values obtained in the CE separation. The MKs were found to be weaker bases than the parent morpholine molecule itself and the experimentally determined basicity values proved to be dependent on the chemical structure of the MKs. Since the basicity of the MKs has an influence on their reactivity and biological activity it seems to be useful to determine experimentally the pKa,exp values of the newly synthesised compounds to support rational drug design or screening of the molecule libraries.     

The determinants of carboxyl pKa values in turkey ovomucoid third domain

A computational methodology for protein pK(a) predictions, based on ab initio quantum mechanical treatment of part of the protein and linear Poisson-Boltzmann equation treatment of the bulk solvent, is presented. The method is used to predict and interpret the pK(a) values of the five carboxyl residues (Asp7, Glu10, Glu19, Asp27, and Glu43) in the serine protease inhibitor turkey ovomucoid third domain. All the predicted pK(a) values are within 0.5 pH units of experiment, with a root-mean-square deviation of 0.31 pH units. We show that the decreased pK(a) values observed for some of the residues are primarily due to hydrogen bonds to the carboxyl oxygens. Hydrogen bonds involving amide protons are shown to be particularly important, and the effect of hydrogen bonding is shown to be nonadditive. Hydrophobic effects are also shown to be important in raising the pK(a). Interactions with charged residues are shown to have relatively little effect on the carboxyl pK(a) values in this protein, in general agreement with experiment.     

Synthesis and acid ionization constants of cyclic cystine peptides H-Cys-(Gly)n-Cys-OH (n = 0-4)

Cyclic peptide disulfides of the general formula H-Cys-(Gly)n-Cys-OH (n = 0-4) were synthesized from the corresponding peptide derivatives [Boc-Cys(Trt)(Gly)-n-Cys(Trt)-OBut] by oxidation with iodine in methanol and by subsequent removal of the terminal groups with trifluoroacetic acid. Acid ionization constants of the obtained peptides were determined by potentiometric titration in aqueous KCl (0.1 mol/L) medium. All compounds have two dissociable hydrogens, corresponding to carboxyl (pK1 = 2.35-2.84) and to terminal amino group (pK2 = 5.61-6.93); pK1 values show first an upward and then a downward trend with the increase in ring size; the opposite is true for pK2 values. These trends could be tentatively attributed to the intramolecular salt bridge (-COO- ----NH+3-) formation.     

Characterization of electrostatic binding sites of extracellular polymers by linear programming analysis of titration data

Electrostatic binding sites of extracellular polymeric substances (EPS) were characterized from titration data using linear programming analysis. Test results for three synthetic solutions of given solutes comprising amino, carboxyl, and phenolic groups indicated that this method was able to identify the electrostatic binding sites. For the six sites with pK(a) between 3 and 10, the estimated pK(a) deviated 0.11 +/- 0.09 from the theoretical values, and the estimated concentrations deviated 3.0% +/- 0.9% from the actual concentrations. Two EPS samples were then extracted from a hydrogen-producing sludge (HPS) and a sulfate-reducing biofilm (SRB). Analysis of charge excess data in titration from pH 3 to 11 indicated that the EPS of HPS comprised of five electrostatic binding sites with pK(a) ranging from 3 to 11. The pK(a) values of these binding sites and the possible corresponding functional groups were pK(a) 4.8 (carboxyl), pK(a) 6.0 (carboxyl/phosphoric), pK(a) 7.0 (phosphoric), pK(a) 9.8 (amine/phenolic), and pK(a) 11.0 (hydroxyl). EPS of the SRB comprised five of similar binding sites (with corresponding pK(a) values of 4.4, 6.0, 7.4, 9.4, and 11.0), plus one extra site at pK(a) 8.2, which was likely corresponding to the sulfhydryl group. The total electrostatic binding site concentration of EPS extracted from HPS were 10.88 mmol/g-EPS, of which the highest concentration was from the site of pK(a) 11.0. The corresponding values for the EPS extracted from SRB were 16.44 mmol/g-EPS and pK(a) 4.4. The total concentrations of electrostatic binding sites found in this study were 20- to 30-fold of those reported for bacterial cell surface, implying that EPS might be more crucial in biosorption of metals than bacterial cell surface in wastewater treatment and in bioremediation.     

A fast and accurate computational approach to protein ionization

We report a very fast and accurate physics-based method to calculate pH-dependent electrostatic effects in protein molecules and to predict the pK values of individual sites of titration. In addition, a CHARMm-based algorithm is included to construct and refine the spatial coordinates of all hydrogen atoms at a given pH. The present method combines electrostatic energy calculations based on the Generalized Born approximation with an iterative mobile clustering approach to calculate the equilibria of proton binding to multiple titration sites in protein molecules. The use of the GBIM (Generalized Born with Implicit Membrane) CHARMm module makes it possible to model not only water-soluble proteins but membrane proteins as well. The method includes a novel algorithm for preliminary refinement of hydrogen coordinates. Another difference from existing approaches is that, instead of monopeptides, a set of relaxed pentapeptide structures are used as model compounds. Tests on a set of 24 proteins demonstrate the high accuracy of the method. On average, the RMSD between predicted and experimental pK values is close to 0.5 pK units on this data set, and the accuracy is achieved at very low computational cost. The pH-dependent assignment of hydrogen atoms also shows very good agreement with protonation states and hydrogen-bond network observed in neutron-diffraction structures. The method is implemented as a computational protocol in Accelrys Discovery Studio and provides a fast and easy way to study the effect of pH on many important mechanisms such as enzyme catalysis, ligand binding, protein-protein interactions, and protein stability.     

Optimization of a free separation of 30 free amino acids and peptides by capillary zone electrophoresis with indirect absorbance detection: a potential for quantification in physiological fluids

This report describes a rapid, single-run procedure, based on the optimization of capillary electrophoresis (CE) and indirect absorbance detection capabilities, which was developed for the separation and quantification of 30 underivatized physiological amino acids and peptides, usually present in biological fluids. p-Aminosalicylic acid buffered with sodium carbonate at pH 10.2+/-0.1 was used as the running electrolyte. Electrophoresis, carried out in a capillary (87 cm x 75 microm) at 15 kV potential (normal polarity), separated the examined compounds within 30 min. Limits of detection ranged from 1.93 to 20.08 micromol/l (median 6.71 micromol/l). The method was linear within the 50-200 micromol/l concentration range (r ranged from 0.684 to 0.989, median r=0.934). Within run migration times precision was good (median C.V.=0.7%). Less favorable within run peak area precision (median C.V.=6.6%) was obtained. The analytical procedure presented was successfully tested for separation and quantification of amino acids in physiological fluids, such as plasma or supernatant of macrophage cultures. Sample preparations require only a protein precipitation and dilution step.     

Electrophoresis–chemiluminescence detection of phenols catalyzed by hemin

Based on the catalytic activity of hemin, an efficient biocatalyst, an indirect capillary electrophoresis–chemiluminescence (CE‐CL) detection method for phenols using a hemin–luminol–hydrogen peroxide system was developed. Through a series of static injection experiments, hemin was found to perform best in a neutral solution rather than an acidic or alkaline medium. Although halide ions such as Br^? and F^? could further enhance the CL signal catalyzed by hemin, it is difficult to apply these conditions to this CE‐CL detection system because of the self‐polymerization of hemin, as it hinders the CE process. The addition of concentrated ammonium hydroxide to an aqueous/dimethyl sulfoxide solution of hemin–luminol afforded a stable CE‐CL baseline. The indirect CE‐CL detection of five phenols using this method gave the following limits of detections: 4.8 × 10^?8 mol/L (o‐sec‐butylphenol), 4.9 × 10^?8 mol/L (o‐cresol), 5.4 × 10^?8 mol/L (m‐cresol), 5.3 × 10^?8 mol/L (2,4‐dichlorophenol) and 7.1 × 10^?8 mol/L (phenol). Copyright © 2013 John Wiley & Sons, Ltd.     

Determination of salicylate,gentisic acid and salicyluric acid in human urine by capillary electrophoresis with laser-induced fluorescence detection

Acetylsalicylic acid (Aspirin) is rapidly metabolized to salicylic acid (salicylate) and other compounds, including gentisic acid and salicyluric acid. Monitoring of salicylate and its metabolites is of toxicological, pharmacological and biomedical interest. Three capillary electrophoresis (CE) methods featuring alkaline aqueous buffers, laser-induced fluorescence (LIF) detection and no solute extraction or derivatization have been explored. A competitive binding, electrokinetic capillary-based immunoassay is developed that recognizes the presence of salicylate and gentisic acid in urine. Differentiation of the two compounds, however, is problematic. With appropriate ultraviolet excitation, many salicylate-related compounds are fluorescent so that CE with direct urine injection and LIF detection permits the determination of salicylate, gentisic acid and salicyluric acid. Using a HeCd laser with 325 nm produces interference-free monitoring of all three compounds. Using 257 nm excitation from a frequency doubled Ar ion laser, native fluorescence of an endogenous urinary compound that co-migrates with gentisic acid is observed. With wavelength-resolved fluorescence detection, however, the two substances are distinguished. Furthermore, this technique, with comparison to literature data, permits the putative assignment of several peaks to other salicylate metabolites, namely glucuronide conjugates of salicylate and salicyluric acid. All three CE-LIF techniques have been applied to toxicological patient urines and urines collected after ingestion of 500 mg acetylsalicylic acid. CE results compare favorably with those obtained by a commercial fluorescence polarization immunoassay and by a conventional photometric assay.