Sample stacking in CZE using dynamic thermal junctions I. Analytes with low dpKa/dT crossing a single thermally induced pH junction in a BGE with high dpH/dT.

The possibility to compress analyte bands at the beginning of CE runs has many advantages. Analytes at low concentration can be analyzed with high signal-to-noise ratios by using the so-called sample stacking methods. Moreover, sample injections with very narrow initial band widths (small initial standard deviations) are sometimes useful, especially if high resolutions among the bands are required in the shortest run time. In the present work, a method of sample stacking is proposed and demonstrated. It is based on BGEs with high thermal sensitive pHs (high dpH/dT) and analytes with low dpK(a)/dT. High thermal sensitivity means that the working pK(a) of the BGE has a high dpK(a)/dT in modulus. For instance, Tris and Ethanolamine have dpH/dT=-0.028/ degrees C and -0.029/ degrees C, respectively, whereas carboxylic acids have low dpK(a)/dT values, i.e. in the -0.002/ degrees C to+0.002/ degrees C range. The action of cooling and heating sections along the capillary during the runs affects also the local viscosity, conductivity, and electric field strength. The effect of these variables on electrophoretic velocity and band compression is theoretically calculated using a simple model. Finally, this stacking method was demonstrated for amino acids derivatized with naphthalene-2,3-dicarboxaldehyde and fluorescamine using a temperature difference of 70 degrees C between two neighbor sections and Tris as separation buffer. In this case, the BGE has a high pH thermal coefficient whereas the carboxylic groups of the analytes have low pK(a) thermal coefficients. The application of these dynamic thermal gradients increased peak height by a factor of two (and decreased the standard deviations of peaks by a factor of two) of aspartic acid and glutamic acid derivatized with naphthalene-2,3-dicarboxaldehyde and serine derivatized with fluorescamine. The effect of thermal compression of bands was not observed when runs were accomplished using phosphate buffer at pH 7 (negative control). Phosphate has a low dpH/dT in this pH range, similar to the dK(a)/dT of analytes. It is shown that mid R:dK(a)/dT-dpH/dTmid R>>0 is one determinant factor to have significant stacking produced by dynamic thermal junctions.

Sample stacking in CZE using dynamic thermal junctions II: analytes with high dpKa/dT crossing a single thermal junction in a BGE with low dpH/dT.

In a previous work [M. Mandaji, et al., this issue] a sample stacking method was theoretically modeled and experimentally demonstrated for analytes with low dpK(a)/dT (analytes carrying carboxylic groups) and BGEs with high dpH/dT (high pH-temperature-coefficients). In that work, buffer pH was modulated with temperature, inducing electrophoretic mobility changes in the analytes. In the present work, the opposite conditions are studied and tested, i.e. analytes with high dpK(a)/dT and BGEs that exhibit low dpH/dT. It is well known that organic bases such as amines, imidazoles, and benzimidazoles exhibit high dpK(a)/dT. Temperature variations induce instantaneous changes on the basicity of these and other basic groups. Therefore, the electrophoretic velocity of some analytes changes abruptly when temperature variations are applied along the capillary. This is true only if BGE pH remains constant or if it changes in the opposite direction of pK(a) of the analyte. The presence of hot and cold sections along the capillary also affects local viscosity, conductivity, and electric field strength. The effect of these variables on electrophoretic velocity and band stacking efficacy was also taken into account in the theoretical model presented. Finally, this stacking method is demonstrated for lysine partially derivatized with naphthalene-2,3-dicarboxaldehyde. In this case, the amino group of the lateral chain was left underivatized and only the alpha amino group was derivatized. Therefore, the basicity of the lateral amino group, and consequently the electrophoretic mobility, was modulated with temperature while the pH of the buffer used remained unchanged.

Cattle tick Boophilus microplus salivary gland contains a thiol-activated metalloendopeptidase displaying kininase activity.

This work reports on the characterization of a metalloendopeptidase kininase present in Boophilus microplus salivary glands. Using the guinea pig ileum assay, salivary gland whole extracts (SGE) were found to have a potent kininase activity. Ion-exchange chromatography separated two kininase activities from SGE. The major enzymatic component, eluted at lower ionic strength, was named BooKase (Boophilus Kininase). Analysis of the hydrolysis products by capillary electrophoresis identified Phe5-Ser6 as the only hydrolyzable peptide bond in bradykinin after BooKase treatment. This is the same specificity as the mammalian thimet oligoendopeptidase (EC 3.4.24.15). Like this enzyme, BooKase is also a metallo-peptidase (requires Mn2+) and is activated by-SH protecting reagents. In addition, BooKase was partially inhibited by cFP-AAF-pAB, a specific inhibitor of thimet oligopeptidase. Contrary to other kininases, BooKase had no activity upon angiontensin I. Our results show that BooKase behaves as a typical peptidase with kinase activity.

Validation of non-aqueous capillary electrophoresis for simultaneous determination of four tricyclic antidepressants in pharmaceutical formulations and plasma samples.

We present the validation of a method using non-aqueous capillary electrophoresis (NACE) for quantitative analysis of four tricyclic antidepressants (TADs) in pharmaceutical formulations and plasma. The method presented high resolution allowing the separation of the TADs in 4.3 min at optimized conditions: 50 mM ammonium acetate, 1 M acetic acid in acetonitrile, capillary with 48 cm in length, 40 cm to the detector, and voltage of 30 kV. Acceptable precision (relative standard deviation R.S.D.14.1% from plasma samples) and linearity were achieved using the internal standard (IS) method. The limits of quantification determined for plasma, after liquid-liquid extraction (LLE), were between 30 and 50 ng ml-1. These values are beyond the plasmatic therapeutic concentration. Our results were found comparable or better than those described in the literature for high performance liquid chromatography (HPLC)-based methods.

In vitro monitoring of GTPase activity and enzyme kinetics studies using capillary electrophoresis.

A capillary electrophoresis (CE)-based method for the in vitro detection and monitoring of nucleotide-triphosphatase activity is described. This robust and reproducible method was used to investigate GTPase activity of a recombinant protein construct containing the catalytic domain of Human SEPT4/Bradeion beta (GST-rDGTPase). This example application demonstrates that the CE technique can replace classical radioactive methods for GTPase activity assays and may be used as a routine analytical tool. Enzyme kinetics of GST-rDGTPase was studied and yielded the following kinetic parameters: v(max) = 1.7 microM min(-1) +/- 0.1, Km = 1.0 mM +/- 0.3, and apKcat = 9 x 10(-3) s(-1). In addition the effect of co-factors such as Mg2+ and Mn2+ on the catalytic activity was investigated. The described analytical method was also shown to be useful to analyze diphosphated and triphosphated forms of other nucleotides.

Performance of an ultraviolet light-emitting diode-induced fluorescence detector in capillary electrophoresis.

The performance of a fluorescence detector in capillary electrophoresis (CE) using a light-emitting diode (LED) as excitation source is reported. An ultraviolet LED pulsed at a repetition rate of 500 Hz, combined with a time-discrimination and averaging acquisition system, was used. Limits of detection of 3 and 18 fmoles (at a signal-to-noise ratio equal to 3) were achieved for fluorescamine-derivatized bradykinin and lysine, respectively. This system exhibited a linear response for a concentration range between 54 and 417 microM for derivatized lysine, and between 1.81 and 23.58 microM for derivatized bradykinin. This detection system showed to be very convenient for routine analytical applications.