Katarzyna E. Stępnik

The use of biopartitioning micellar chromatography and immobilized artificial membrane column for in silico and in vitro determination of blood–brain barrier penetration of phenols

Biopartitioning Micellar Chromatography (BMC) is a mode of micellar liquid chromatography that uses C18 stationary phases and micellar mobile phases of Brij35 under adequate experimental conditions and can be useful to mimic human drug absorption, blood-brain barrier distribution or partitioning processes in biological systems. BMC system can be useful in constructing good predictive models because the characteristics of the BMC system are similar to biological barriers and extracellular fluids. Immobilized Artificial Membrane (IAM) chromatography uses stationary phase which consists of a monolayer of phosphatidylcholine covalently immobilized on an inert silica support. IAM columns are thought to mimic very closely a membrane bilayer and are used in a HPLC system with a physiological buffer as eluent. In this paper the usefulness of BMC and IAM system for in silico and in vitro determination of blood-brain barrier (BBB) penetration of phenols has been demonstrated. The most important pharmacokinetic parameters of brain have been obtained for the determination of BBB penetration, i.e. BBB permeability - surface area product (PS), usually given as a logPS, brain/plasma equilibration rate (log(PS×fu,brain)) and fraction unbound in plasma (Fu). Moreover, the relationships between retention of eighteen phenols and different parameters of molecular size, lipophilicity and BBB penetration were studied. Extrapolated to pure water values of the logarithms of retention factors (logkw) have been compared with the corresponding octanol-water partition coefficient (logPo-w) values of the solutes. In addition, different physicochemical parameters from Foleys equation for BMC system have been collated with the chromatographic data. The Linear Solvation Energy Relationship (LSER) using Abraham model for the describing of phenols penetration across BBB has been used. Four equations were developed as a multiple linear regression using retention data from IAM and BMC system (QRAR models) and molecular volume parameter (Vm) and Abraham descriptors to correlate the logBB values. Moreover, in order to establish the relationships between different variables, the principal components analysis (PCA) has been done. The results of PCA were obtained using chromatographic data from IAM and BMC systems as well as from the structures of tested phenols. The four parameters: logkwIAM(exp), logkwBMC(exp), analyte-micelle association constant (Kma) and logPo-w have been checked.

in vitro and in silico determination of oral, jejunum and Caco-2 human absorption of fatty acids and polyphenols. Micellar liquid chromatography.

In this investigation chosen saturated, mono- and polyunsaturated fatty acids as well as polyphenols have been analyzed. The main aim of this study was to determine oral, jejunum and Caco-2 human absorption of chosen fatty acids and polyphenols using in vitro and in silico methods. For in vitro determination of human drug absorption, the usefulness of Micellar Liquid Chromatography (MLC) with mobile phases containing different surfactants (including Brij35-Biopartitioning Micellar Chromatography (BMC)) has been confirmed. On the basis of Foleys equation, 1/k vs. CM correlations for the tested compounds have been done. Satisfactory linearity of the relationships was found over the whole eluents composition range studied with R(2) approximately 0.99 in each case. Moreover, the analyte-micelle association constants (Kma) from Foleys equation have been compared for different micellar environments, containing Brij35, SDS and CTAB as a main component of micellar mobile phases. Completely new models describing human oral as well as Caco-2 and jejunum absorption have been constructed and compared with the cited models. These models are based on the Abraham descriptors and lipophilicity parameters as well as steric descriptors. Furthermore, many different correlations between physicochemical parameters and human intestinal absorption have been done, e.g. the correlation between human jejunum permeability estimated in silico and received using LSER parameters was excellent (R(2) nearly 0.99). Chromatographic parameters have been collated with steric, electronic and physicochemical ones using QRAR (Quantitative Retention - Activity Relationships) and QSAR (Quantitative Structure - Activity Relationships) models. Moreover, retention BMC data have been compared with lipophilicity parameter logPo/w (n-octanol-water partition coefficient). The influence of lipophilicity on oral absorption (%) has been checked. The correlation between predicted oral absorption (%) and logPo/w has been done. Obtained R(2) was 0.82. On the basis of chromatographic, lipophilicity, steric and different physicochemical parameters, the principal components analysis (PCA) has been done.

A new application of micellar liquid chromatography in the determination of free ampicillin concentration in the drug–human serum albumin standard solution in comparison with the adsorption method

The determination of free drug concentration is a very important issue in the field of pharmacology because only the unbound drug fraction can achieve a pharmacological effect. Due to the ability to solubilize many different compounds in micellar aggregates, micellar liquid chromatography (MLC) can be used for direct determination of free drug concentration. Proteins are not retained on the stationary phase probably due to the formation of protein - surfactant complexes which are excluded from the pores of stationary phase. The micellar method is simple and fast. It does not require any pre-preparation of the tested samples for analysis. The main aim of this paper is to demonstrate a completely new applicability of the analytical use of MLC concerning the determination of free drug concentration in the standard solution of human serum albumin. The well-known adsorption method using RP-HPLC and the spectrophotometric technique was applied as the reference method. The results show that the free drug concentration value obtained in the MLC system (based on the RP-8 stationary phase and CTAB) is similar to that obtained by the adsorption method: both RP-HPLC (95.83μgmL(-1), 79.86% of free form) and spectrophotometry (95.71μgmL(-1), 79.76%). In the MLC the free drug concentration was 93.98μgmL(-1) (78.3%). This indicates that the obtained results are within the analytical range of % of free ampicillin fraction and the MLC with direct sample injection can be treated like a promising method for the determination of free drug concentration.

A short review of applications of micellar liquid chromatography to study biologically active compounds

The features of micellar systems are outstanding compared with conventional RP-LC ones. Therefore, the unique properties of micellar chromatography (MLC) are widely recognized. In this short review the applicability of MLC as an in vitro method for the determination of biological activity is discussed. For this purpose many specific examples of MLC applications supported by the theoretical backgrounds of the cited biological activity areas as well as the factors affecting them are presented. This study collects and organizes the most important references of bioactivity determination which were created both recently and in the past, using the MLC method. Although there are many papers on the MLC there is no literature review focused particularly on its applicability in the study of biological activity of various compounds. This work can be treated as a significant review of so far published papers which particularly emphasizes the importance of MLC as in vitro method for determination of bioactivity of different compounds. Copyright

Analysis of Some Biogenic Amines by Micellar Liquid Chromatography

Micellar liquid chromatography (MLC) with the use of high performance liquid chromatography (HPLC) was used to determine some physicochemical parameters of six biogenic amines: adrenaline, dopamine, octopamine, histamine, 2-phenylethylamine, and tyramine. In this paper, an influence of surfactant’s concentration and pH of the micellar mobile phase on the retention of the tested substances was examined. To determine the influence of surfactant’s concentration on the retention of the tested amines, buffered solutions (at pH 7.4) of ionic surfactant—sodium dodecyl sulfate SDS (at different concentrations) with acetonitrile as an organic modifier (0.8/0.2 v/v) were used as the micellar mobile phases. To determine the influence of pH of the micellar mobile phase on the retention, mobile phases contained buffered solutions (at different pH values) of sodium dodecyl sulfate SDS (at 0.1 M) with acetonitrile (0.8/0.2 v/v). The inverse of value of retention factor (1/𝑘) versus concentration of micelles (𝐶𝑀) relationships were examined. Other physicochemical parameters of solutes such as an association constant analyte—micelle (𝐾ma)—and partition coefficient of analyte between stationary phase and water (hydrophobicity descriptor) (𝑃swΦ) were determined by the use of Foley’s equation.

A comparative study of the lipophilicity of 1,2,4-triazoles by reversed-phase and micellar TLC and OPLC

Lipophilicities of 21 newly synthesized 1,2,4-triazoles, compounds of potential importance as fungicides, have been determined by micellar and reversed-phase planar chromatography. Different stationary and mobile phases were used in the measurements. Chromatographic lipophilicities (log km and RM0) were correlated with partition coefficients log P (Alog Ps, AClog P, Alog P, Mlog P, KowWin, xlog P2 and xlog P3) calculated from molecular structures. Principal component analysis allowed a more objective comparison of different lipophilicity descriptors determined from various chromatographic systems and calculated theoretically as log P parameters.

Determination of binding properties of ampicillin in drug-human serum albumin standard solution using N-vinylpyrrolidone copolymer combined with the micellar systems

It is well-known that only the unbound (free) drug fraction can achieve a pharmacological effect. Therefore the determination of free drug concentration is a very important issue in the field of pharmacology. In this study poly-1-vinyl-2-pyrrolidone (VP) crosslinked with divinylbenzene (DVB) compared with the micellar liquid chromatography (MLC) with and without pre-made drug adsorption was used for quantitative analysis of free ampicillin concentration in the standard solution of drug-human serum albumin owing to its ability to block protein adsorption. The commonly recognized adsorption method based on drug adsorption on VP-DVB has been compared to the entirely new application of MLC with direct sample injection (DSI) not requiring pre-made adsorption. Micellar aggregates are able to solubilize various compounds therefore micellar environment can be used for direct determination of free drug concentration. The obtained results show that the free drug concentration values obtained in the micellar systems based on cetyltrimethylammonium bromide (CTAB) (93.98μgL-1, 78.3%) as well as on polyoxyethylene (23) lauryl ether (Brij35) (91.15μgL-1, 75.9%) are similar to those obtained after the drug adsorption on VP-DVB using both RP-HPLC (95.85μgmL-1, 79.9%) and spectrophotometry (96.47μgmL-1, 80.4%). However, only %PPB (% plasma protein binding) value calculated on the basis of Brij35 retention factor is similar to the literature data. The obtained results are within the analytical range of % of free drug concentration. Therefore N-vinylpyrrolidone copolymer as well as micellar system based on the non-ionic surfactant can be successfully applied for determination of free drug concentration. Moreover, the new application of MLC with DSI can be recognized as a promising, fast and simple method for quantitative determination of free drug concentration.

Skin–mimetic chromatography for prediction of human percutaneous absorption of biologically active compounds occurring in medicinal plant extracts

The main aim of this study was to predict quantitatively human percutaneous absorption of chosen compounds commonly occurring in plants which can be used as medicinal extracts in the drug and beauty industries. The most important human percutaneous descriptors, i.e. logKp (logarithm of the water/skin partition coefficient) and logJmax (logarithm of the maximum flux of solutes penetrating the skin), of fatty acids and polyphenols were determined using both in vitro and in silico methods. For in vitro determination of human percutaneous absorption, micellar liquid chromatography based on hexadecyltrimethylammonium bromide, sodium dodecyl sulfate and polyoxyethylene (23) lauryl ether (Brij35) was used. Human percutaneous absorption was characterized by entirely new QSAR/QRAR models based on retention, lipophilic, steric and electronic data as well as on the linear free energy relationship parameters. Many different correlations between human skin absorption and different physicochemical parameters were performed, e.g. the in silico estimated logKp value was correlated with the retention parameter logkw (logarithm of the retention factor extrapolated to pure water) from the systems imitating a cutaneous environment (R2  = 0.92). Moreover, the influence of lipophilicity on percutaneous absorption was examined. The obtained correlation was excellent (R2  = 0.95).

Micellar liquid chromatography of plant extracts components

Raw materials quality assessment is a very important factor, enabling products creation up to the international standards. In this investigation, the qualitative composition of bioactive plant extracts has been checked. The tested extracts have been obtained by the use of supercritical carbon dioxide extraction and can be utilized as raw materials in a commercial production of a wide range of many different goods. Plant extracts of raspberry (Rubus idaeus), strawberry (Fragaria ananassa), blackcurrant (Ribes nigrum), aronia (Aronia Medik.), Japanese rose (Rosa rugosa Thunb.) seeds, and palmetto palm (Sabal minor) fruit have been examined. In these extracts, saturated, monounsaturated, and polyunsaturated fatty acids as well as polyphenols have been identified using thin-layer chromatography (TLC) with aqueous—organic mobile phases. Appropriate content of these components tends to be a determinant of human organism operation. In our investigation, the plant extract components, especially saturated and unsaturated fatty acids and polyphenols, have been tested using micellar chromatography. Different micellar environments, containing polioxyethylene (23) lauryl ether (Brij35), sodium dodecyl sulfate (SDS), and cetyltrimethylammonium bromide (CTAB) as a main component of micellar mobile phases, have been utilized. The analyte—micelle association constants (Kma) from Foley’s equation have been compared for each of these environments.