Support matrix effects in the reversed-phase thin-layer chromatography of some peptides.

The retentions of 28 peptides in reversed-phase thin-layer chromatography (RPTLC) were determined on cellulose and on impregnated cellulose and alumina layers with 1-propanol as the organic component of the mobile phase. Each peptide showed a support matrix effect: their RM values first decreased to a minimum, then increased with increasing 1-propanol concentration. On cellulose layers only the increasing phase was observed. The retention behaviour of peptides was adequately described with a quadratic or linear function, but the slope value of the linear function had a positive value. The results demonstrate that the support matrix effect can be observed on non-silica supports and it may occur in reversed-phase chromatography in the case of polar solutes and supports with free adsorptive centres on their surfaces. Both the intercept and slope values of the function are needed to describe the lipophilicity of peptides, but the correlation is not strong enough for the determination of the lipophilicity of peptides by RPTLC. Principal component analysis showed that the peptides form distinct clusters on the basis of their retention characteristics: peptides containing a basic amino acid, peptides with a ring structure in the amino acid side-chain and peptides containing uncharged amino acids.     

Lipophilicity determination of some new crown ethers by reversed-phase thin-layer chromatography

Summary The lipophilicity of 28 modified crown ether derivatives was determined by reversed-phase thin-layer chromatography (RPTLC) using various organic phases and supports. The lipophilicity values determined in different RPTLC systems showed good correlations, however the quality of the organic phase (methanol, acetone, acetonitrile) and the support characteristics influenced to a small extent the determination.     

阴离子型胶束液相色谱的溶质保留行为

以SDS阴离子表面活性剂作流动相,酸性、中性及两性药物为受试药物,运用三相平衡理论考察影响阴离子型胶束液相色谱（AMLC）溶质保留行为的几个因素。保留由溶质与胶束相及修饰后固定相的综合作用决定。有机调节剂正丙醇的加入改变了溶质从水相到固定相或到胶束相的平衡,保留取决于溶质疏水性和静电性间的平衡。此外对羟基苯甲酸酯类同系物的亲脂性与3种细菌最小抑菌浓度具有显著相关性,提示其抑菌机理主要取决于药物与生物膜的亲和性。     

模拟生物膜色谱用于预测药物的小肠吸收

采用涂敷磷脂的硅胶为模拟生物膜色谱固定相,与传统的凝胶体相比固定相的机械强度及磷脂固载能力均有较大提高,且有较好的稳定性。研究发现氢化可的松等6种药物在模拟生物膜色谱上pH5.4、pH7.0及pH7.4三种缓冲环境下的色谱保留值迭加的结果能够与药物的肠吸收指数押送好的拟合,线性相关系数为0.9365,该结果高于单一pH条件下拟合结果,并将此结果与药物在C18反相色谱上的保留值及辛醇-水系统下的分配系数与药物的小肠吸收的拟合结果进行比较,表明与这两种模型相比模拟生物膜色谱预测药物的小肠吸收有更高的准确度。     

Octanol/water partitioning simulation by reversed-phase high performance liquid chromatography for structurally diverse acidic drugs: Effect of n-octanol as mobile phase additive

The role of n-octanol as mobile phase additive for the lipophilicity assessment of 45 structurally diverse acidic drugs both at neutral (pH 2.5) and ionized form (pH 7.4) was investigated. Extrapolated retention factors logk(w) were determined on a BDS C18 column using methanol as organic modifier and different amounts of n-octanol as mobile phase additive. For more polar compounds, the effect of n-octanol in retention was found to decrease as their lipophilicity increased. In the case of carboxylic acids and oxicams, the differentiation in retention, in presence and absence of n-octanol, could be further attributed to the attenuation of polar interactions, concerning mainly hydrogen bonding. At pH 2.5, the use of n-octanol saturated buffer, without further addition of n-octanol in the mobile phase, led to 1:1 correlation with logP. At physiological pH, 1:1 correlation was obtained between logD(7.4) and logk(w)(oct) indices upon addition of 0.25% n-octanol, in the case of weak acids. For strongly ionized compounds, a good correlation was also established under the same conditions. The corresponding equation, however, possessed a large negative intercept and a slope lower than unity.     

Lipophilicity analysis of newly synthetized quinobenzothiazines by use of TLC

Lipophilicity is a very important property of chemical compound taking into consideration in drugs design. Relationships between biological activity, among others lipophilicity, and chemical structure (QSAR) of the compound are very often used by researches. Especially important is the kind of substituents connected to the basic structural fragment and how it changes the lipophilicity of the compound. The aim of this study was to determine the parameters of lipophilicity of quinobenzothiazine derivatives using reversed phase - thin-layer chromatography (RP-TLC), which would enable one to determine the structure–activity relationship. The objective of our work is a series of 15 newly synthetized quinobenzothiazines. They were analyzed by thin-layer chromatography (TLC) with the use of two different mobile phases consisting of methanol or acetone as organic modifiers. For all compounds investigated, the values of lipophilicity obtained from computational method were also determined. Cluster analysis was carried out too for all data of lipophilicity obtained. Low correlation was found between values of experimental lipophilicity and lipophilicity from computational methods for newly synthetized compounds.     

Retention of structurally diverse drugs in human serum albumin chromatography and its potential to simulate plasma protein binding

The retention behavior of 39 structurally diverse neutral, basic and acidic drugs was investigated on an HSA stationary phase using PBS buffer (pH 7.0) and acetonitrile or 2-propanol as organic modifiers. Extrapolated or directly measured log k_w values as well as isocratic retention factors were correlated with plasma protein binding data taken from the literature. Retention factors determined in the presence of 10% acetonitrile led to high quality 1:1 correlation with apparent log K_HSA values. The derived reference equation was successfully validated using a secondary set of 24 drugs. Further analysis of HSA retention into more fundamental properties revealed the involvement of anionic species in solute-stationary phase interactions, expressed by the negatively charged fraction, besides the partitioning mechanism which was reflected by lipophilicity. Protonation of basic drugs, although less important, may also influence retention, leading to reduced partitioning into the HSA surface as a net effect, while it seems to have no effect on HSA binding. The above results were further confirmed by linear solvation energy relationships (LSER).     

Affinity partitioning for membrane purification exploiting the biotin-NeutrAvidin interaction. Model study of mixed liposomes and membranes

The linear-solvent strength (LSS) model of gradient elution has been applied to estimate parameters of lipophilicity and acidity of a series of drugs and model chemicals. Apparent pKa values and log kw values for individual analytes were determined in 2-3 gradient runs. The first experiment (or first two experiments) uses a wide-range organic modifier gradient with pH chosen for suppressed ionization of the analyte. The result of this experiment allows an estimate of contents of organic modifier of the mobile phase (%B) providing the required retention coefficient, k, for the non-ionized analyte. The following experiment is carried out with the latter %B and a pH-gradient of the aqueous component of the eluent that is sufficient to overlap the possible pKa-value of the analyte. The initial pH of the buffer used to make the mobile phase is selected to insure that the analyte is in non-ionized form. The resulting retention time allows an estimate of PKa in a solvent of the selected %B. At the same time, estimates of log kw can also be obtained. The log kw parameter obtained from gradient HPLC by the approach proposed correlated well with the corresponding value obtained by standard procedure of extrapolation of retention data determined in a series of isocratic measurements. Correlation between log kw and the reference parameter of lipophilicity, log P, was very good for a series of test analytes and satisfactory for a structurally diverse series of drugs. The approach supported with specific detection procedures can be recommended for fast screening of lipophilicity of individual components of complex mixtures like those produced by combinatorial chemistry. The values of pKa obtained in a study were found to correlate with the literature pKa data determined in water for a set of aniline derivatives studied. In case of a series of drugs the correlation was less than moderate if the general procedure of pKa determination was applied.     

Influence of pH and organic modifiers on the dissociation constants of selected drugs using reversed‐phase thin‐layer chromatography: Comparison with other techniques and computational tools

Knowledge of the acid–base dissociation constants of drugs is the key to understanding their biopharmaceutical characteristics. In the present work, the effect of pH and organic modifiers (acetonitrile and methanol) was investigated in the determination of dissociation constants (pK_a) of nine representative drugs (atenolol, betahistine, clarithromycin, deferiprone, diclofenac, ibuprofen, metoprolol, naproxen and propranolol) using reversed‐phase thin‐layer chromatography. Mobile phase consisting of various buffers and methanol–acetonitrile (10, 20, 30, 40, 50 and 60%, v/v) was used to evaluate the retention pattern on reversed‐phase plates. Compared with methanol, acetonitrile gave better results for the experimentally determined pK_a values by extrapolation to zero organic modifier volume fractions. To assess the effectiveness of the developed method the results were correlated using principal component analysis and hierarchical cluster analysis. The calculated values of the aqueous dissociation constant were compared with those reported previously using potentiometry and capillary electrophoresis and also with different computational platforms like ACD/Lab, ChemAxon and Jchem calculator. The results obtained by the RPTLC method were in good agreement with potentiometric methods for pK_a determination.     

Comprehensive evaluation of lipophilicity of biogenic amines and related compounds using different chemically bonded phases and various descriptors

The retention behavior for a series of biogenic amines and related sympathomimetic drugs has been investigated in reversed-phase thin-layer chromatography using RP-2, RP-8, RP-18W, and Diol stationary phase and mixtures of phosphate buffer (pH = 7.10) and methanol in different proportions as mobile phases. Several methodologies like arithmetic mean of experimental retention values, extrapolation to zero methanol concentration procedure and principal component analysis were applied to retention data values (R(M)) in order to determine relevant parameters (mean of R(M) - mR(M), R(M0), and scores corresponding to the first principal component - PC1/R(M) respectively) encoding information on the lipophilic behavior of compounds. High similarities in lipophilicity behavior of investigated amines were highlighted by mR(M) and PC1/R(M) lipophilicity indices for all of the studied stationary phases. The experimental results were compared with some computed lipophilicity parameters expressed as distribution coefficients at working pH (logD), partition coefficients (logP(N), logP(I), and diff(logP(N-I))) concerning both neutral and fully protonated species and difference between both species, and also with various lipophilicity values (logP) generated by different commonly used software. Significant correlations were observed between the experimental lipophilicity indices mR(M) respectively PC1/R(M) and diff(logP(N-I) ) values in all cases.     

胶束溶液体系对毛细管电动色谱酸性化合物分离的影响

以阴离子表面活性剂十二烷基硫酸钠（SDS）、非离子表面活性剂吐温20（ Tween 20）及两者组成的混合胶束体系作为毛细管胶束电动色谱（MECC）的分离介 质，进行4种结构相似的酸性化合物的MECC分离研究，考察了胶束的类型、表面活 性剂的浓度、缓冲溶液的pH值及有机改性剂乙醇对分离的影响。结果表明各因素对 酸性药物的MECC分离有不同的影响规律。SDS胶束体系对溶质的保留值最大， Tween 20体系的保留值最小，二者的分离选择性正好相反，混合胶束体系的分离行 为则介于两者之间；在SDS和Tween 20体系中，表面活性剂浓度增加，溶质的保留 时间均随之递增，混合胶束体系中，总浓度一定，随Tween 20配比的增加，溶质的 保留时间先减少后增加；缓冲溶液的pH值增大，使溶质的分离效果均能变差；有机 改性剂乙醇的加入对容量因子的影响主要与溶质的疏水性有关，并对分离作用机理 进行了探讨。在SDS和Tween 20 MECC体系下，分别进行了实样测定，取得了满意的 结果。     

Relationships between LC retention, octanol-water partition coefficient, and fungistatic properties of 2-(2,4-dihydroxyphenyl)benzothiazoles

The retention behavior of newly synthesized compounds with antimycotic activity from the 2-(2,4-dihydroxyphenyl)benzothiazole group by high-performance liquid chromatography has been investigated. RP-18 stationary phase and methanol-acetate buffer aqueous mobile phases at pH 4 and 7.4 have been used. In the case of the mobile phase at pH 7.4, higher concentrations of water can be applied than at pH 4. The studied compounds showed regular retention behavior, their log k values decreasing linearly with an increasing concentration of methanol in the mobile phase. On the basis of these relationships, the lipophilicity (log kw), specific hydrophobic surface area (S), and isocratic chromatographic hydrophobicity index (psi0) were determined. Similar log kw values and sensitivity to changes in the structure of compounds studied for both mobile phases have been found. Moderate correlations between the chromatographic parameters and the calculated octanol-water log P values were found. Finally, the lipophilicity parameters were compared with the fungistatic properties of compounds expressed by log MIC (minimum inhibitory concentration) values to find quantitative structure activity relationship equations.     

Chromatographic and computational screening of anisotropic lipophilicity and pharmacokinetics of newly synthesized 1-aryl-3-ethyl-3-methylsuccinimides

The present study is focused on a series of newly synthesized 1-aryl-3-ethyl-3-methylsuccinimide derivatives, as potential anticonvulsants. The retention behavior of eleven succinimide derivatives was determined by using reversed phase high performance liquid chromatography (RP-HPLC) and reversed phase high performance thin layer chromatography (RP-HPTLC). The estimated retention behavior was correlated with partition (logP) and distribution coefficients (logD). These high correlations pointed out that the determined retention parameters (logk₀ and R_M⁰) can be considered chromatographic (anisotropic) lipophilicity of the studied succinimide derivatives. The structural properties, which dominantly affect the chromatographic lipophilicity, were determined as well. The significant correlations between the chromatographic lipophilicity and plasma protein binding (PPB), Madin-Darby Canine Kidney (MDCK) cells permeability, volume of distribution (Vd) and absorption constant (Ka) indicate the strong influence of lipophilicity on pharmacokinetics of 1-aryl-3-ethyl-3-methylsuccinimide derivatives. These derivatives have also been tested applying Comprehensive Medicinal Chemistry (CMC) drug-like rules which confirmed their drug-like properties. Besides, their blood-brain penetration (BBB) ability has been estimated applying the set of Clark’s rules and by using Pre-ADMET software. Regarding toxicity, it was predicted that only one compound from the set might have toxic effects by blocking the hERG potassium channel. The present study reveals which molecular features in the structure of novel succinimide derivatives could be crucial for their lipophilicity, and consequently for their pharmacokinetic properties. The results indicate that the newly synthesized series of succinimide derivatives should be further considered in design of novel anticonvulsants.     

High-performance liquid chromatography of some basic drugs on a n-octadecylphosphonic acid modified magnesia-zirconia stationary phase

The high-performance liquid chromatographic behavior of some basic drugs was studied on a n-octadecylphosphonic acid modified magnesia-zirconia (C18PZM) stationary phase. The effect of mobile phase variables such as methanol content, ionic strength, and pH on their chromatographic behavior was investigated. The retention mechanism of basic drugs on the stationary phase was elucidated. The results indicate that both hydrophobic and cation-exchange interactions contribute to solute retention under most chromatographic conditions. The inherent Br?nsted-acid sites and also the adsorbed Lewis base anionic buffer constituents on accessible ZM surface Lewis acid sites play a role in the retention of ionized solutes by cation-exchange interaction. However, especially at high mobile phase pH, the retention of basic drugs depends mainly on hydrophobic interactions between solutes and support. Separations of the basic drugs on the C18PZM phase by a predominantly reversed-phase retention mode were very promising. The mixed-mode retention feature on this phase, as a result of the adsorbed Lewis base anionic buffer constituents acting as sites for cation-exchange, could also be very useful, e.g. for enhancing the chromatographic selectivity of such analytes. The C18PZM seems to be an excellent alternative to silica-based reversed-phase stationary phase for the separation of strongly basic solutes.     

Use of unmodified silica with buffered aqueous mobile phase mixtures for selective chromatography of basic drugs

Summary The characteristic selectivity of unmodified silica using mixed aqueous-methanolic mobile phase systems for the separation of basic drugs was studied. It was found that low buffer concentration-high methanol eluents show increased chromatographic performance over mobile phase systems lower in methanol and higher in ion strength. Regarding an assumed ion-exchanger retention mechanism, the capacity factors of an amino group containing drugs is primarily dependent on the concentration of the cation and the pH-value of the eluent. By increasing the pH-value of the eluent, elution order of primary and secondary to tertiary amines is reversed. By only using eluents with buffering capability, a very high stability and reproducibility of retention of the basic drugs could be observed. Comparing three different brands of unmodified silica materials, with different pore size and specific surface area, only small differences in the selectivity for the tested drugs could be found.     

Relationship between the lipophilicity and specific hydrophobic surface area of some pesticides by RP-HPLC and HPTLC

Summary Using methanol-water mixtures as the mobile phases, the retention behaviors of thirty-seven pesticides were determined in RP-HPLC and RP-HPTLC. Regular retention behavior was observed for all the investigated pesticides: theirR _m and logk values decreasing linearly with increasing concentration of methanol in the mobile phase. The lipophilicity and specific hydrophobic surface area values for each compound were obtained and they have a good linear relationship. Although the chemical structures of these pesticides were different, factor analysis proved that the lipophilicity and specific hydrophobic surface area of these compounds have much in common, and the insecticides, fungicides and herbicides could not be distinguished from each other according to their lipophilicity parameters obtained from chromatography method.     

HPTLC and magnetochromatography of new complexes of carboxylates with transition metals or rare earth elements and their ligands – study of lipophilicity

Nineteen new complexes of carboxylates with transition and rare elements as central ions and their ligands were characterized by chromatographic analyses. The parameter of relative lipophilicity (R _M0) of the tested compounds was determined experimentally by the reversed‐phase high‐performance thin layer chromatography method with mixtures of various organic modifiers (acetonitrile, acetone, dioxane) and water as a mobile phase. The extrapolated R _M0 values were compared with the logP values calculated from the molecular structures of tested solutes. Similarities between the lipophilicity indices were analysed by principal component analysis and linear regression. Thin‐layer chromatography combined with a magnetic field has been proposed as a complementary method for determination of lipophilicity of the investigated compounds. The chromatograms in the field and outside it were developed simultaneously in two identical chromatographic chambers. One of them was placed in the external magnetic field of 0.4 T inductivity. We proved that chelation causes a drastic change in compound lipophilicity, but all complexes did not exhibit enhanced activity as compared with the parent ligand. Also in the magnetic field the retention of some complexes changed, which means that the presence of the field influences the physicochemical properties of the compounds and their interactions with the stationary phase.     

Investigation of polar stationary phases for the separation of sympathomimetic drugs with nano-liquid chromatography in hydrophilic interaction liquid chromatography mode

In this study, the retention and selectivity of a mixture of basic polar drugs were investigated in hydrophilic interaction chromatographic conditions (HILIC) using nano-liquid chromatography (nano-LC). Six sympathomimetic drugs including ephedrine, norephedrine, synephrine, epinephrine, norepinephrine and norphenylephrine were separated by changing experimental parameters such as stationary phase, acetonitrile (ACN) content, buffer pH and concentration, column temperature. Four polar stationary phases (i.e. cyano-, diol-, aminopropyl-silica and Luna HILIC, a cross-linked diol phase) were selected and packed into fused silica capillary columns of 100 μm internal diameter (i.d.). Among the four stationary phases investigated a complete separation of the all studied compounds was achieved with aminopropyl silica and Luna HILIC stationary phases only. Best chromatographic results were obtained employing a mobile phase composed by ACN/water (92/8, v/v) containing 10 mM ammonium formate buffer pH 3. The influence of the capillary temperature on the resolution of the polar basic drugs was investigated in the range between 10 and 50 °C. Linear correlation of ln k vs. 1/T was observed for all the columns; ΔH° values were negative with Luna HILIC and positive with aminopropyl- and diol-silica stationary phases, demonstrating that different mechanisms were involved in the separation.To compare the chromatographic performance of the different columns, Van Deemter curves were also investigated.     

Simultaneous isolation and determination of esculin and rutin in natural materials using SPE and HPLC

Summary Esculin (ESC) and rutin (RUT) have been simultaneously isolated from pharmaceutical natural materials by solid phase extraction (SPE). Determination of both substances was performed by reversed phase high performance liquid chromatography (RPHPLC) with UV detection. Optimization of the separation conditions showed that simultaneous isolation and determination of rutin and esculin from pharmaceutical material was possible. The recovery obtained was not lower than 95±2%.     

Hydrophilic interaction liquid chromatography for the separation of acidic agricultural compounds

Organic acids with very low pK_a require extremely low pH conditions to achieve adequate retention in reversed‐phase liquid chromatography, but an extremely low pH mobile phase can cause instrument reliability problems and limit the choice of columns. Hydrophilic interaction chromatography is a potential alternative to reversed‐phase liquid chromatography for the separation of organic acids using more moderate conditions. However, the hydrophilic interaction chromatography separation mechanism is known to be very complex and involves multiple competing mechanisms. In the present study, a hydrophilic interaction chromatography column packed with bare silica core–shell particles was used as the separation column and six agricultural organic acids were used as model analytes to evaluate the effects of buffer concentration, buffer pH, and temperature on sample loading capacity, selectivity, retention, and repeatability. It was found that using a higher concentration of buffer can lead to a significant improvement in the overall performance and reproducibility of the separation. Investigation of column equilibration time revealed that a very long equilibration time is needed when changing mobile phase conditions in between runs. This limitation needs to be acknowledged in hydrophilic interaction chromatography method development and sufficient equilibration time needs to be allowed in method scouting.