Intelligent Recommendation Systems Powered by Consensus Neural Networks: The Ultimate Solution for Finding Suitable Chiral Chromatographic Systems?

The selection of suitable combinations of chiral stationary phases (CSPs) and mobile phases (MPs) for the enantioresolution of chiral compounds is a complex issue that often requires considerable experimental effort and can lead to significant waste. Linking the structure of a chiral compound to a CSP/MP system suitable for its enantioseparation can be an effective solution to this problem. In this study, we evaluate algorithmic tools for this purpose. Our proposed consensus model, which uses multiple optimized artificial neural networks (ANNs), shows potential as an intelligent recommendation system (IRS) for ranking chromatographic systems suitable for the enantioresolution of chiral compounds with different molecular structures. To evaluate the IRS potential in a proof-of-concept stage, 56 structural descriptors for 56 structurally unrelated chiral compounds across 14 different families are considered. Chromatographic systems under study comprise 7 cellulose and amylose derivative CSPs and acetonitrile or methanol aqueous MPs (14 chromatographic systems in all). The ANNs are optimized using a fit-for-purpose version of the chaotic neural network algorithm with competitive learning (CCLNNA), a novel approach not previously applied in the chemical domain. CCLNNA is adapted to define the inner ANN complexity and perform feature selection of the structural descriptors. A customized target function evaluates the correctness of recommending the appropriate CSP/MP system. The ANN-consensus model exhibits no advisory failures and requires only an experimental attempt to verify the IRS recommendation for complete enantioresolution. This outstanding performance highlights its potential to effectively resolve this problem.

Potential of sodium dodecyl sulfate micellar solutions as eluents in magnetic dispersive micro-solid phase extraction with polydopamine-coated magnetite nanoparticles. Application to antidepressant drugs.

In this paper, the potential of micellar solutions of the anionic surfactant sodium dodecyl sulfate (SDS) as eluents in dispersive micro-solid phase extraction (D-µSPE) using polydopamine-coated magnetite nanoparticles (Fe3O4@PDA NPs) for the extraction and preconcentration of seven basic drugs (bupropion, citalopram, fluoxetine, mianserin, nomifensine, trimipramine, and viloxazine) is explored for the first time (to the best to our knowledge) and compared with conventional hydro-organic eluents. The impact of the sample solution pH, Fe3O4@PDA NPs and PDA coating amounts and extraction time on the extraction efficiency (EE), as well as the composition of the eluent on the overall efficiency (OE) are studied. Under the selected experimental conditions (50 mg of Fe3O4@PDA NPs, 100 µL of 1 M NH3, 5 min of extraction time and 0.15 M SDS at pH 2.6 as eluent), EE and OE values were higher than 90% for all compounds and for the most hydrophobic compounds (trimipramine, fluoxetine and mianserin), respectively. The results shown in this paper demonstrate the suitability of Fe3O4@PDA NPs as a sorbent for the extraction of antidepressants as well as the advantages of using SDS micellar solutions over classic hydro-organic eluents containing methanol, acetonitrile or tetrahydrofuran. Finally, the stability and reusability of the Fe3O4@PDA NPs is proven.

Comparative study on retention behaviour and enantioresolution of basic and neutral structurally unrelated compounds with cellulose-based chiral stationary phases in reversed phase liquid chromatography-mass spectrometry conditions.

A comparative study on the retention behaviour and enantioresolution of 54 structurally unrelated neutral and basic compounds using five commercial cellulose-based chiral stationary phases (CSPs) and hydro-organic mobile phases compatible with MS detection is performed. Four phenylcarbamate-type cellulose CSPs (cellulose tris(3,5-dimethylphenylcarbamate), Cell1; cellulose tris(3-chloro-4-methylphenylcarbamate), Cell2; cellulose tris(4-chloro-3-methylphenylcarbamate), Cell4 and cellulose tris(3,5- dichlorophenylcarbamate), Cell5) and one benzoate-type cellulose CSP (cellulose tris(4-methylbenzoate), Cell3) are assayed. Mobile phases consist of binary mixtures of methanol (30-90% MeOH) or acetonitrile (10-98% ACN) with 5 mM ammonium bicarbonate (pH = 8.0). The existence of reversed phase (RPLC) and hydrophilic interaction liquid chromatography (HILIC) retention behaviour domains is explored. In MeOH/H2O mobile phases, for all compounds and CSPs, the typical RPLC retention behaviour is observed. When using ACN/H2O mobile phases, for all compounds in all CSPs (even in the non-chlorinated CSPs) a U-shaped retention behaviour depending on the ACN/H2O content is observed which indicates the coexistence of the RPLC- (< 80% ACN) and HILIC- (∼80-98% ACN) domains. The magnitude of retention changes in both domains is related to the hydrophobicity of the compound as well as to the nature of the CSP. The study of the effect of the nature and concentration of the organic solvent, as well as the nature of the CSP on the enantioresolution reveals that: (i) the use of MeOH/H2O or ACN/H2O greatly affects the enantioselectivity and enantioresolution degree of the chromatographic systems, being, in general, better the results obtained with ACN/H2O mobile phases. (ii) The ACN-RPLC-domain provides much better enantioresolution than HILIC-domain. (iii) Cell2, especially with ACN/H2O mobile phases, is the CSP that allows baseline enantioresolution for a higher number of compounds. (iv) Phenylcarbamate-type CSPs do not offer clear complementary enantioselectivity to that of Cell2. (v) Cell3 is the only CSP that provides marked complementary enantioselectivity to that of Cell2, almost orthogonal in MeOH/H2O mobile phases.

Artificial neural networks to model the enantioresolution of structurally unrelated neutral and basic compounds with cellulose tris(3,5-dimethylphenylcarbamate) chiral stationary phase and aqueous-acetonitrile mobile phases.

Artificial neural networks (ANN; feed-forward mode) are used to quantitatively estimate the enantioresolution (Rs) in cellulose tris(3,5-dimethylphenylcarbamate) of chiral molecules from their structural information. To the best of our knowledge, for the first time, a dataset of structurally unrelated compounds is modelled using ANN, attempting to approach a model of general applicability. After setting a strategy compatible with the data complexity and their relatively limited size (56 molecules), by prefixing initial ANN inner weights and the validation and cross-validation subsets, the ANN optimisation based on a novel quality indicator calculated from 9 ANN outputs allows selecting a proper (predictive) ANN architecture (a single hidden layer of 7 neurons) and performing a forward-stepwise feature selection process (8 variables are selected). Such relatively simple ANN offers reasonable good general performance in predicting Rs (e.g. validation plot statistics: mean squared error = 0.047 and R = 0.98 and 0.92, for all or just the validation molecules, respectively). Finally, a study of the relative importance of the selected variables, combining the estimation from two approaches, suggests that the surface tension (positive overall contribution to Rs) and the -NHR groups (negative overall contribution to Rs) are found to be the main variables explaining the enantioresolution in the current conditions.

Comparative modelling study on enantioresolution of structurally unrelated compounds with amylose-based chiral stationary phases in reversed phase liquid chromatography-mass spectrometry conditions.

Polysaccharide-based chiral stationary phases (CSPs) are the most used chiral selectors in HPLC. These CSPs can be used in normal, polar organic and aqueous-organic mobile phases. However, normal and polar organic mobile phases are not adequate for chiral separation of polar compounds, for the analysis of aqueous samples and for MS detection. In these situations, reversed phase conditions, without the usual non-volatile additives incompatible with MS detection, are preferable. Moreover, in most of the reported chiral chromatographic methods, retention is too large for routine work. In this paper, the chiral separation of 53 structurally unrelated compounds is studied using three commercial amylose-based CSPs -coated amylose tris(3,5-dimethylphenylcarbamate) (Am1), coated amylose tris(5-chloro-2-methylphenylcarbamate) (Am2), and immobilised amylose tris(3-chloro-5-methylphenylcarbamate) (Am3)-. Chiral separations are carried out using acetonitrile/ammonium bicarbonate (pH = 8.0) mixtures, reversed mobile phases compatible with MS detection. To provide realistic conditions for routine analysis, maximum retention factors are set to 15. Retention and enantioresolution behaviour of compounds in those CSPs are compared. On the other hand, to compare and describe the resolution ability of these CSPs, 58 structural variables of the compounds are tested to model for the first time a categorical enantioresolution (CRs) for Am1 and Am3 CSPs. Discriminant partial least squares, for one response categorical variable (DPLS1) is used for feature selection, modelling. The final DPLS1 models showed good descriptive ability.

Reversed phase liquid chromatography for the enantioseparation of local anaesthetics in polysaccharide-based stationary phases. Application to biodegradability studies.

A comprehensive study on the chiral separation of bupivacaine, mepivacaine, prilocaine and propanocaine with eight commercial polysaccharide-based chiral stationary phases (CSPs) in reversed phase conditions compatible with MS detection is performed. Methanol and acetonitrile are used as organic modifiers. Retention and resolution values obtained for each compound in the different CSPs and mobile phases are compared. The polysaccharide-based CSPs tested present different enantioselectivity towards the analytes. From the results, the experimental conditions for determining the enantiomers of bupivacaine, mepivacaine, prilocaine and propanocaine in saline aqueous samples using MS detection are used, for the first time, to perform an enantioselective biodegradability study.

Improved accuracy of environmentally relevant parameter estimates derived from biodegradation assays.

Biodegradation assays involve both biodegradation and analytical processes which can be affected by systematic errors, among others. These errors can affect all the environmentally relevant parameters related to biodegradability, enantioselectivity (in the case of chiral compounds), kinetic parameters and persistence of chemicals. However, such impacts have never been well-characterized. In this work, calculations and models used for a long time are studied by simulating systematic errors at the 5% level, which affect independently the analytical calibration step and the biodegradation process. The impact of these errors is also compared with those obtained from an alternative approach: recently proposed equations and a novel model (a Monod modified version) developed in this work. All simulations are compatible with an environmentally relevant pollutant concentration. The results suggest a high degree of minimization (or even cancelation) of the systematic error impact using the alternative approach respect to the conventional one. These findings can be interpreted either in view of achiral or chiral pollutants. The present work can have a positive impact in the area of risk assessment of new pollutants and hazardous materials.

Anticipating the impact of pitfalls in kinetic biodegradation parameter estimation from substrate depletion curves of organic pollutants.

Accurate and reliable estimation of kinetic parameters of pollutant biodegradation processes is essential for environmental and health risk assessment. Common biodegradation models proposed in the literature, such as the nonlinear Monod equation and its simplified versions (e.g. Michaelis-Menten-like and first-order equations), are problematic in terms of accuracy of kinetic parameters due to the parameter correlation. However, a comparison between these models in terms of accuracy and reliability, related to data imprecision, has not been performed in the literature. This task is necessary, mainly because the model selection cannot be straightforward, as shown in this work. To facilitate the comparison, novel statistics summarising the accuracy and reliability of estimations are introduced. The main objective is to establish relationships between these statistics (trough new diagnostic indicators) to limit the probability of pitfalls or to avoid the negative impact of an improper model selection. Such anticipation is an imperative need in the biodegradation modelling framework and, to the best of our knowledge, it has never been performed. In order to account for accuracy, simulated data in realistic conditions are used to highlight the magnitude of pitfalls related to the model selection for estimation of the main kinetic parameters (Ks, µm and/or Vm). Four scenarios related to model selection are compared for the first time and, diagnostic indicators able to anticipate relevant aspects related to accuracy and reliability are introduced. Moreover, first evidences of the impact of measurement errors in other intrinsic Monod parameters (the initial biomass concentration and the microbial yield coefficient, Y), as well as the impact of the simultaneous µm, Ks and Y estimation, on the accuracy and reliability of the estimations are reported. Despite the pitfalls shown, specific applicability of even unreliable models is highlighted, and suggestions for environmental and health risk modellers are provided accordingly.

Modelling the enantioresolution capability of cellulose tris(3,5-dichlorophenylcarbamate) stationary phase in reversed phase conditions for neutral and basic chiral compounds.

To the best of our knowledge, the prediction of the enantioresolution ability of polysaccharides-based stationary phases in liquid chromatography for structurally unrelated compounds has not been previously reported. In this study, structural information of neutral and basic compounds is used to model their enantioresolution levels obtained from an immobilised cellulose tris(3,5-dichlorophenylcarbamate) stationary phase in reversed phase conditions. Thirty-four structurally unrelated chiral drugs and pesticides, from seven families, are studied. Categorical enantioresolution levels (RsC, 0 = no baseline enantioresolution and 1 = baseline enantioresolution) are established from the experimental enantioresolution values obtained at a fixed experimental conditions. From 58 initial structural variables, three topological parameters (two of them connected to the chiral carbon), and six molecular descriptors (one of them also related with the chiral carbon), are selected after a discriminant partial least squares refinement process. The molar total charge of the molecule at the working pH is the most important variable. The relationships between RsC and the most important structural variables and the drug/pesticide family are evaluated. An explicit model is proposed to anticipate the RsC levels, which provides 100% of correct anticipations. A criterion is introduced to alert about the compounds that should not be anticipated.

Direct chromatographic study of the enantioselective biodegradation of ibuprofen and ketoprofen by an activated sludge.

The quantification of the enantiomeric fraction (EF) during the biodegradation process is essential for environmental risk assessment. In this paper the enantioselective biodegradation of ibuprofen, IBU, and ketoprofen, KET, two of the drugs most consumed, was evaluated. Biodegradation experiments were performed in batch mode using a minimal salts medium inoculated with an activated sludge (collected from a Valencian Waste Water Treatment Plant) and supplemented with the racemate of each compound. The inoculum activity was verified using fluoxetine as reference compound. The experimental conditions used (analyte concentration and volume of inoculum) were chosen according to OECD guidelines. In parallel, the optical density at 600 nm was measured to control the biomass growth and to connect it with enantioselectivity. Two RPLC methods for chiral separations of IBU and KET using polysaccharides-based stationary phases were developed. Novel calculations and adapted models, using directly the chromatographic peak areas as dependent variable, were proposed to estimate significant parameters related to the biodegradation process: biodegradation (BD) and EF values at given time, half-life times of (R)- and (S)-enantiomers, number of days to reach a complete BD and the minimum EF expected. The modelled BD and EF curves fitted adequately the data (R2 > 0.94). The use of these new equations provided similar results to those obtained using concentration data. However, the use of chromatographic peak areas data, eliminates the uncertainty associated to the use of the calibration curves. The results obtained in this paper indicate that an enantiorecognition towards IBU enantiomers by the microorganisms present in the activated sludge used in this study occurred, being the biodegradation of (R)-IBU higher than that of (S)-IBU. For KET, non-enantioselective biodegradation was observed.

Trimeprazine is enantioselectively degraded by an activated sludge in ready biodegradability test conditions.

A great number of available pharmaceuticals are chiral compounds. Although they are usually manufactured as racemic mixtures, they can be enantioselectively biodegraded as a result of microbial processes. In this paper, a biodegradability assay in similar conditions to those recommended in OECD tests of enantiomers of trimeprazine (a phenothiazine employed as a racemate) is carried out. Experiments were performed in batch mode using a minimal salts medium inoculated with an activated sludge (collected from a Valencian Waste Water Treatment Plant, WWTP) and supplemented with the racemate. The concentration of the enantiomers of trimeprazine were monitored by means of a chiral HPLC method using a cellulose-based chiral stationary phase and 0.5 M NaClO4/acetonitrile (60:40, v/v) mobile phases. Experiments were performed at three concentration levels of the racemate. In parallel, the optical density at 600 nm (OD600) was measured to control the biomass growth and to connect it with enantioselectivity. The calculated enantiomeric fractions (EF) offer the first evidence of enantioselective biodegradation of trimeprazine. A simplified Monod equation was used as a curve fitting approach for concentration (S), biodegradation (BD), and for the first time, EF experimental data in order to expand the usefulness of the results. Precision studies on S (repeatability conditions) and, for the first time, EF (intermediate precision conditions) were also performed.

Enantioresolution in electrokinetic chromatography-complete filling technique using sulfated gamma-cyclodextrin. Software-free topological anticipation.

Few papers have tried to predict the resolution ability of chiral selectors in capillary electrophoresis for the separation of the enantiomers of chiral compounds. In a previous work, we have used molecular information available on-line to establish enantioresolution levels of basic compounds using highly sulfated ß-CD (HS-ß-CD) as chiral selector in electrokinetic chromatography-complete filling technique (EKC-CFT). The present study is a continuation of this previous work, introducing some novelties. In this work, the ability of sulfated γ-cyclodextrin (S-γ-CD) as chiral selector in EKC-CFT is modelled for the first time. Thirty-three structurally unrelated cationic and neutral compounds (drugs and pesticides) are studied. Categorical enantioresolution levels (RsC, 0 or 1) are assigned from experimental enantioresolution values obtained at different S-γ-CD concentrations. Novel topological parameters connected to the chiral carbon (C*-parameters) are introduced. Four C*-parameters and a topological parameter of the whole molecule (aromatic atom count) are the most important variables according to a discriminant partial least squares-variable selection process. It suggests the preponderance of the topology adjacent to the chiral carbon to anticipate the RsC levels. A software-free anticipation protocol for new molecules is proposed. Over the current set of molecules evaluated, 100% of correct anticipations (resolved and non-resolved compounds) are obtained, while anticipation of some compounds remains undetermined. A criterion is introduced to alert on compounds which should not be anticipated.

Evaluation of the enantioselective binding of imazalil to human serum albumin by capillary electrophoresis.

In this work, a methodology for the evaluation of enantioselective binding of imazalil (IMA) enantiomers to human serum albumin (HSA) that does not require the separation of free and bound to HSA fractions is developed. This methodology comprises the incubation of IMA-HSA designed mixtures for 30 min directly in the capillary electrophoresis system and the subsequent direct injection and chiral separation of IMA employing highly sulfated ß-cyclodextrin as chiral selector and the complete filling technique. Two mathematical approaches were used to estimate apparent affinity constants (K1), protein binding and enantioselectivity (ES) for both enantiomers of IMA. Moderate enantioselective binding of IMA enantiomers to HSA (ES = 2.0) was shown by the 1:1 stoichiometry and log K1 values of 3.4 ± 0.4 and 3.1 ± 0.3 for the first and second eluted enantiomers, respectively.

Characterizing the interaction between enantiomers of eight psychoactive drugs and highly sulfated-ß-cyclodextrin by counter-current capillary electrophoresis.

The estimation of apparent binding constants and limit mobilities of the complexes of the enantiomers that characterize the interaction of enantiomers with chiral selectors, in this case highly sulfated ß-cyclodextrin, was approached using a simple and economic electrophoretic modality, the complete filling technique (CFT) in counter-current mode. The enantiomers of eight psychoactive drugs, four antihistamines (dimethindene, promethazine, orphenadrine and terfenadine) and four antidepressants (bupropion, fluoxetine, nomifensine and viloxazine) were separated for the first time for this cyclodextrin (CD). Estimations of thermodynamic and electrophoretic enantioselectivies were also performed. Results indicate that, in general, thermodynamic enantioselectivity is the main component explaining the high resolution found, but also one case suggests that electrophoretic enantioselectivity itself is enough to obtain a satisfactory resolution. CFT results advantageous compared with conventional capillary electrophoresis (CE) and partial filling technique (PFT) for the study of the interaction between drugs and chiral selectors. It combines the use of a simple fitting model (as in CE), when the enantiomers do not exit the chiral selector plug during the separation (i.e. mobility of electroosmotic flow larger than mobility of CD), and drastic reduction of the consumption (and cost; ~99.7%) of the CD reagent (as in PFT) compared with the conventional CE.

Fast evaluation of enantioselective drug metabolism by electrophoretically mediated microanalysis: application to fluoxetine metabolism by CYP2D6.

In this work, a capillary electrophoretic methodology for the enantioselective in vitro evaluation of drugs metabolism is applied to the evaluation of fluoxetine (FLX) metabolism by cytochrome 2D6 (CYP2D6). This methodology comprises the in-capillary enzymatic reaction and the chiral separation of FLX and its major metabolite, norfluoxetine enantiomers employing highly sulfated ß-CD and the partial filling technique. The methodology employed in this work is a fast way to obtain a first approach of the enantioselective in vitro metabolism of racemic drugs, with the additional advantage of an extremely low consumption of enzymes, CDs and all the reagents involved in the process. Michaelis-Menten kinetic parameters (Km and Vmax ) for the metabolism of FLX enantiomers by CYP2D6 have been estimated by nonlinear fitting of experimental data to the Michaelis-Menten equation. Km values have been found to be 30 ± 3 µM for S-FLX and 39 ± 5 µM for R-FLX. Vmax estimations were 28.6 ± 1.2 and 34 ± 2 pmol·min(-1) ·(pmol CYP)(-1) for S- and R-FLX, respectively. Similar results were obtained using a single enantiomer (R-FLX), indicating that the use of the racemate is a good option for obtaining enantioselective estimations. The results obtained show a slight enantioselectivity in favor of R-FLX.

Determination of fluoxetine enantiomers in pharmaceutical formulations by electrokinetic chromatography-counter current technique.

In this work, an electrokinetic chromatography-counter current procedure for the separation of fluoxetine enantiomers using highly sulfated ß-cyclodextrin was optimized and applied to the determination of the enantiomers in three pharmaceutical formulations according to the matrix features. Quality criteria were applied to facilitate its transferability to testing laboratories. Fluoxetine was used therapeutically as the racemate, although a stereospecificity associated with its interactions with the neuronal serotonin-uptake carrier was demonstrated. In this context, the development of enantioselective methods for the chiral analysis of pharmaceuticals allowing stereoisomer ratio estimations has increasing interest in pharmaceutical industry. The proposed method allows the quantification of both enantiomers in less than 2 min with high resolution (R(s) = 2.4).

Electrokinetic chromatographic estimation of the enantioselective binding of nomifensine to human serum albumin and total plasma proteins.

This report is the first evidence of enantioselective binding of nomifensine to human serum albumin (HSA) and plasma proteins. The overall process with HSA included: (i) consistent experimental design along two independent sessions; (ii) incubation of nomifensine-HSA designed mixtures; (iii) ultrafiltration for separating the unbound enantiomers fraction; (iv) electrokinetic chromatography (EKC) using heptakis-2,3,6-tri-O-methyl-ß-cyclodextrin as chiral selector to provide experimental data for enantiomers (first, E1, and second, E2, eluted ones); and (v) a recent direct equation allowing univariate tests and robust statistics to provide consistent parameters and uncertainty. A significant enantioselectivity to HSA (2.7 ± 0.1) was encountered, related to a 1:1 stoichiometry and log affinity constants of 3.24 ± 0.10 and 3.67 ± 0.08 for E1 and E2, respectively. The protein binding (PB) estimated at physiological concentration levels was 40 ± 5 and 63 ± 4% for E1 and E2, respectively. The use of synthetic human sera allowed in vitro estimation of the total plasma PB for the racemate (61 ± 5%; coincident with in vivo values), and its enantiomers (58 ± 7 and 64 ± 4% for E1 and E2, respectively). Comparison allowed the relative importance of HSA respect to other plasma proteins for binding nomifensine to be established.

Connecting simulated, bioanalytical, and molecular docking data on the stereoselective binding of (±)-catechin to human serum albumin.

The stereoselective binding of the frequently ingested nutraceutical (±)-catechin, with demonstrated differential biological activity between enantiomers, to human serum albumin (HSA), with the largest complexation and enantioselectivity potential among the plasmatic proteins, is studied by combining simulations to optimize the experimental design, robust in vitro electrokinetic chromatographic data, and molecular docking-chiral recognition estimates. Methodological and mathematical drawbacks in previous reports on (±)-catechin-HSA are detected and eliminated. Recent and novel direct equations extracted from the classical interaction model allows advantageous univariate mathematical data treatment, providing the first evidence of quantitative (±)-catechin-HSA enantioselectivity. Also, the binding site in HSA of the enantiomers is approached, and both the experimental enantioselectivity and the main binding site information are contrasted with a molecular docking approach.

Evaluation of enantioselective binding of fluoxetine to human serum albumin by ultrafiltration and CE--experimental design and quality considerations.

Several pharmacokinetic processes are affected by enantioselectivity (ES). At the level of distribution, protein binding (PB) is one of the most important. The enantioselective binding of fluoxetine (FLX) to HSA has been evaluated in this work by ultrafiltration of FLX­HSA mixtures and chiral analysis of unbound fractions by EKC-CD. PB, affinity constants (K) and ES were obtained for both enantiomers of FLX. In order to improve the consistency of the estimations, the evaluation of affinity constants of each enantiomer was performed using two designs, one keeping constant the total concentration of protein and varying the total concentration of the enantiomers, and the other in the opposite way, in both cases via an unusual short-concentration interval strategy to assure model validity. Different mathematical approaches were compared and characterised and some of them, judged as the most consistent under the experimental conditions used, were selected to provide final estimates. Quality considerations include criteria for three critical aspects: (i) detecting/eliminating outliers, (ii) checking the number of binding sites in the protein and (iii) evaluating the robustness of each approach. The differences on estimates from the selected approaches were used as an uncertainty source to delimit the reported values. The ES of HSA for FLX enantiomers was approximate. Estimates include the assumptions of independent and competitive models. In the last case, a SIMPLEX function was designed capable of simultaneously optimizing the non-linear binding models for both enantiomers, thus improving the consistence of results.

Screening of acetylcholinesterase inhibitors by CE after enzymatic reaction at capillary inlet.

In this study the development of a procedure based on capillary electrophoresis after enzymatic reaction at capillary inlet methodology for the screening and in vitro evaluation of the biological activity of acetylcholinesterase (AChE) inhibitors is presented. The progress of the enzymatic reaction of the hydrolysis of acetylthiocholine at pH 8 in the presence of AChE and the inhibitor studied is determined by measuring at 230 nm the peak area of the reaction product thiocholine (TCh). In the method employed the capillary was first filled with 30 mM borate-phosphate buffer (pH 8.0) and subsequently, plugs of: (i) water, (ii) AChE solution, (iii) substrate solution with or without inhibitor, (iv) AChE solution, and (v) water, were hydrodynamically injected into the capillary, and were allowed to stand (and react) during a waiting period of 2 min. The applicability of the proposed methodology to estimate different kinetic parameters of interest such as inhibition constants K(i), identification of inhibitory action mechanism and IC(50), is evaluated using compounds with known activity, tacrine edrophonium, and neostigmine. The results obtained are compared with bibliographic values and confirm the effectiveness of the methodology proposed. Finally a method for AChE Inhibitor screening is proposed.