Paweł Mateusz Nowak

Application of capillary electrophoresis in determination of acid dissociation constant values.

The chemical groups undergoing protonation or deprotonation in solution are described by the acid dissociation constant value, the key parameter for physicochemical characterization of biologically- and pharmacologically-important compounds. Capillary electrophoresis (CE) proved to be suitable technique for its determination: it enables automated and accurate measurements even for minute amount of sample, does not require the information about concentration, and handle both the impure and complex samples. In this review, a number of contributions reporting on the application of CE in pKa prediction has been summarized and critically discussed. The reader will find herein the brief introduction of theory, summary of all works published in the last decade, considerations on the most important innovations and achievements, and the discussion of pKa-related issues as e.g. the role of pKa-shifts in the chiral separation mechanism or the elucidation of migration order reversals observed during CE-mediated separations.

An overview of on-line systems using drug metabolizing enzymes integrated into capillary electrophoresis

Enzymatic assays using CE are now among the most noteworthy applications of this analytical technique in pharmacology‐related investigations. Studies of metabolic pathways of new chemical entities mediated by drug metabolizing enzymes are attracting particular attention. Conventional CE‐based enzymatic in vitro assays are generally restricted to the separation of reagents after incubation performed off‐line. EMMA represents an alternative and fully prospective approach, allowing injection, reaction, separation, and detection to be conducted in a single capillary. Such an on‐line system—in contrast to the standard approach—enables automation, miniaturization, and a significant reduction in reagent volumes, resulting in a very robust and cost‐efficient method. Hence, EMMA could be a method of choice for the screening of new drugs, enzymatic inhibitors, and putative drug–drug interactions. This review provides a summary of reports covering the area of EMMA‐based and related methods implemented into in vitro studies of drug metabolizing enzymes. A general description of the EMMA framework, enzyme families, and a concise discussion of the prognosis for the development of this methodology are given as well.

Analytical aspects of achiral and cyclodextrin-mediated capillary electrophoresis of warfarin and its two main derivatives assisted by theoretical modeling

Several distinct analytical issues have been addressed by performing capillary electrophoresis-based separations of the warfarin, 7-hydroxywarfarin and 10-hydroxywarfarin in an achiral and cyclodextrin-containing media. The measurements were conducted across a range of pH in order to find optimum conditions for achiral and chiral separations. The values of acid dissociation constant (pKa) have been determined and compared. Subsequently, after performing a series of mobility shift assays at different pH and cyclodextrin concentration, the pKa values ascribed to diastereomeric complexes with methyl-β-cyclodextrin have been estimated. The significant pKa shifts upon complexation have been noticed for warfarin - up to 1.5 pH units, and only subtle for 10-hydroxywarfarin. A new approach that allows the estimation of association percentage based on the electrophoretic mobility curves has been also demonstrated. The complex mechanism of chiral separation has been found to be responsible for the observed migration profile, relying on a combined equilibrium between complexation/partition and protonation/deprotonation phenomena. The occurrence of the pKa-related migration order reversal has been demonstrated in achiral medium between warfarin and 7-hydroxywarfarin, and in chiral medium between enantiomers, causing a drop in enantioselectivity at specific pH. In parallel, the density functional theory-based calculations have been performed in order to obtain the structures of warfarin and its derivatives as well as to rationalize the shifts in pKa values.

Determination of acid dissociation constant of 20 coumarin derivatives by capillary electrophoresis using the amine capillary and two different methodologies.

In this work capillary electrophoresis has been used to determine acid dissociation constant of 20 structurally diverse coumarin derivatives. For a majority of compounds pKa value has been determined for the first time. The obtained values vary between 4.16-9.10pH unit, pointing to the interesting structure-acidity relationships. The amine permanently coated capillary has been applied for that purpose, because it has turned out to be more effective in pKa determination than the bare silica and other coated capillaries, ensuring good precision and shorter migration times. A traditional methodology relying on measurements in a broad pH range and fitting of a sigmoidal function has been compared to an alternative simplified approach, reported for the first time, where only two electrophoretic mobility values suffice for pKa estimation. The first value corresponds to the partially ionized form and it is measured experimentally, while the second one to the totally ionized form - it is measured experimentally (two-values method) or estimated directly from molecular mass (one-value method). We show that despite a limited measurements number, the alternative approach may be consistent with the traditional methodology, yielding the relatively low pKa deviation. Its reliability has also been confirmed by the analytical predictions, comprising resolution, migration order, migration times and peaks overlapping. Therefore, combination of the amine capillary with the simplified calculation method is an attractive tool for fast and reliable pKa estimation.

Determination of acid dissociation constants of warfarin and hydroxywarfarins by capillary electrophoresis.

In this work the acid dissociation constants--pKa of warfarin and its all important oxidative metabolites have been determined by capillary electrophoresis-based methods. It has resulted in a complete description of two acid-base dissociation equilibria, yet not investigated experimentally for phase I metabolites of warfarin. The capillary electrophoresis (CE) method based on the relation between effective electrophoretic mobilities and pH has proven to be a suitable tool for pKa determination, while the spectrophotometric (CE-DAD) and the internal standard methods (IS-CE), have appeared to be promising alternative approaches. The CE-DAD approach based on the change in absorbance spectra between the acidic and basic forms is a combination between capillary electrophoresis and spectrophotometric titration, and yields very consistent values of pKa1 with CE. The IS-CE, in turn, enables an estimation of pKa1 and pKa2 from only two analytical runs, however, less accurate than CE and CE-DAD. The Debye-Hückel model has been confirmed experimentally as a good predictor of pKa values at various ionic strengths. Therefore, it has been used in determination of thermodynamic pKa1 and pKa2, referring to the zero ionic strength. The results are important from the analytical, pharmacological, and theoretical points of view.

Fast separation of warfarin and 7‐hydroxywarfarin enantiomers by cyclodextrin‐assisted capillary electrophoresis

The enantioseparation of warfarin and its main metabolite has been achieved using several cyclodextrin types and buffers at different pH, including conditions that have not been attempted so far. Methyl-β-cyclodextrin, highly sulfated-β-cyclodextrin and highly sulfated-γ-cyclodextrin were the most efficient chiral selectors. The pH range, within which particular cyclodextrins support chiral separation, has been approximately determined for the first time. By shortening the effective capillary length to 10 cm, the time of analysis has been vastly reduced <2 min. Hence, baseline separations of warfarin and 7-hydroxywarfarin enantiomers have been achieved in times unreported for those species until now. The established conditions are promising for the further development of new highly selective and fast methods involving warfarin, its derivatives, as well as the same cyclodextrin types.

Capillary electrophoresis as a tool for a cost-effective assessment of the activity of plant membrane enzyme chlorophyllase.

The potential of the CE‐based enzymatic assay has been demonstrated in case of a typical plant membrane enzyme – chlorophyllase. An efficient, automated and rapid semi‐quantitative method has been developed, which allowed us to assess the activity of the enzyme via two strategies. Firstly, a reaction conducted in a vial placed directly on the sample tray was combined with the concomitant separation and detection of reagents. The method was used to monitor the reaction progress. Secondly, an online approach was applied using an electrophoretically mediated mixing. The reaction was performed in‑capillary, resulting in an extreme reduction of the reagent amounts required for a single run. Both methods were effective in the assessment of the activity of a membrane enzyme, a member of protein class known to pose experimental difficulties.

Modulation of pKa by cyclodextrins; subtle structural changes induce spectacularly different behaviors

Tuning of acidity-related properties by host–guest complexation is one of the most promising concepts in current supramolecular chemistry. However, still little is known about structural effects which determine direction and magnitude of supramolecular pKa shifts. Here we present the first systematic comparison of cyclodextrin-induced pKa shifts with a focus on minor structural differences between guests – phenolic drug warfarin and its six isomeric derivatives, and hosts – various structurally similar cyclodextrins. Warfarin and five hydroxywarfarins exert upward pKa shifts upon complexation with β-cyclodextrin and its neutral derivatives. However, the magnitude of these shifts depends on the cyclodextrin substituent, and even, on the average substitution degree. The strongest shifts are observed for methyl-β-cyclodextrin, they are among the greatest cyclodextrin-induced pKa shifts noted so far. By contrast, 10-hydroxywarfarin exhibits only minor shifts whose direction is, surprisingly, dependent on temperature. Furthermore, the temperature variations of pKa show that endothermic dissociation is observed for 2-hydroxypropyl-β-cyclodextrin, similarly as in the host-free state, while it becomes exothermic for methyl-β-cyclodextrin. In other words, acid dissociation of two structurally similar host–guest complexes is characterized by dramatically different enthalpic contributions. Finally, some enantioselective effects are also observed. We infer that intramolecular hydrogen bonds and enantioselective interactions with portal cyclodextrin groups are likely crucial for these phenomena. Our work may open up new horizons in understanding of structural effects in the supramolecular pKa tuning.

Seven approaches to elimination of the inherent systematic errors in determination of electrophoretic mobility by capillary electrophoresis

Electrophoretic mobility is a basic parameter that describes the electromigration of an ionized particle, which is used in many fields of analytical and physicochemical science. Its determination by capillary electrophoresis (CE), using a routine method, is intrinsically affected by the generation of Joule heating, entailing a drop in viscosity and possible alteration of the degree of ionization, and also by other commonly overlooked effects: axial electric field distortion and voltage ramping. The objective of this work was to provide the first theoretical overview and experimental comparison of all accessible methods that could be used to prevent these sources of inaccuracy. We have discussed seven independent approaches: (i) extrapolation of mobility to the zero power, (ii) initial buffer resistance-based correction, (iii) rational cooling adjustment, (iv) elimination of the nonthermostated capillary part, (v) inter/extrapolation to the nominal temperature, (vi) internal standard-based correction, and (vii) simple recalculation based on the temperature rise. Two methodologies (v and vi) have been proposed for the first time. Furthermore, we have shown how some approaches can be further developed, obtaining several novel and more sophisticated methods, which are also included in the comparison. Our investigation will help researchers to choose the optimal approach. We have also demonstrated for the first time how to measure the independent impact of four different effects. The outcomes reveal the compensatory character of some phenomena and explain the highly diverse and unpredictable magnitude of the total errors. The use of a correction method seems crucial for ensuring the high reliability of CE-based analyses.

Separation of iron-free and iron-saturated forms of transferrin and lactoferrin via capillary electrophoresis performed in fused-silica and neutral capillaries.

A capillary electrophoresis-based method for the cost-effective and high efficient separation of iron-free and iron-saturated forms of two members of transferrin family: transferrin and lactoferrin has been developed. The proposed qualitative method relying on the SDS application allowed us to separate iron-free and iron-saturated forms of these proteins, as well as human serum albumin, used as an internal standard. Owing to the distinct migration times under established conditions, the combination of transferrin and lactoferrin assays within a single analytical procedure was feasible. The performance of the method using a fused-silica capillary has been compared with the results obtained using the same method but performed with the use of a neutral capillary of the same dimensions. Neutral capillary has been used as an alternative, since the comparable resolution has been achieved with a concomitant reduction of the electroosmotic flow. Despite of this fact, the migration of analytes occurred with similar velocity but in opposite order, due to the reverse polarity application. A quantitative method employing fused-silica capillary for iron saturation study has been also developed, to evaluate the iron saturation in commercial preparations of lactoferrin.