Reversed-phase Thin-layer Chromatography Research Articles

Determination of Trans-Anethole in Essential Oil, Methanolic Extract and Commercial Formulations of Foeniculum vulgare Mill Using a Green RP-HPTLC-Densitometry Method

Due to the lack of ecofriendly/green reversed-phase high-performance thin-layer chromatography (RP-HPTLC) methods for trans-anethole (TAL) and its simplicity over routine analytical techniques, there was a necessity to establish a suitable HPTLC methodology for the quantitative analysis of TAL. Therefore, the first objective of this research was to develop an accurate, rapid and green RP-HPTLC densitometry methodology for the quantitative analysis of TAL in essential oil, traditional and ultrasound-assisted extracts of Foeniculum vulgare Mill and commercial formulations. The second objective was to compare the traditional method of extraction of TAL with its ultrasound-assisted method of extraction. The chromatogram of TAL from essential oil and traditional and ultrasound-assisted extracts of fennel and commercial formulations was verified by recoding its single spectra at Rf = 0.31 ± 0.01 in comparison to standard TAL. The proposed analytical methodology has been found to be superior in terms of linearity, accuracy and precision compared to most of the reported analytical methods for TAL analysis. The amount of TAL in the essential oil of fennel was recorded as 8.82 mg per g of oil. The content of TAL in traditional extracts of fennel, formulation 1 (dietary supplement 1) and formulation 2 (dietary supplement 2), was recorded as 6.44, 4.88 and 4.48 mg per g, respectively. The amount of TAL in ultrasound-assisted extracts of fennel, formulation 1 and formulation 2, was recorded as 8.34, 6.46 and 5.81 mg per g, respectively. The ultrasound method of extraction of TAL was found to be better than the traditional method of extraction. The results of validation studies and phytochemical analysis showed that the proposed methodology could be efficiently utilized for the quantification of TAL in the wide range of products having TAL as a component.

Separation and analysis of heavy metal ions by thin-layer chromatography (TLC)—a mini-review (2000–2019)

This review article describes the work reported on the separation and analysis of heavy metal ions by different modes of thin-layer chromatography (TLC) such as soil, micellar, and reversed-phase TLC during 2000–2019. The history of different modes of TLC has been briefly mentioned. The literature survey reveals that the number of papers reported during 2000–2019 on the separation of heavy metal ions by different modes of TLC follows the trend: micellar TLC > soil TLC > reversed-phase TLC.

Structure–retardation factor relationship of natural amino acids in two different mobile phases of RP-TLC

Separation of amino acids (AAs) and their simple and inexpensive determination/identification is an interesting topic in biological and protein science, different food industries, and drug factories. Also, the presentation of the chromatographic behavior of compounds in a predictive model can be effective to estimate the structural/chemical properties of analyte and mobile phases. In this work for the first time, retardation factor (RF) of 42 AAs in reversed-phase thin layer chromatography (RP-TLC) was modeled. Acetonitrile-sodium azide solution and 1,2 dioxane-sodium azide solution were two mobile phases which have been studied in this work. Results showed that the values of RF are correlated with the structural properties of AAs and these properties had some similarities and differences in two noted mobile phases. For the TLC data in two mobile phases, five parametric linear models were suggested (R2train = 0.93 and 0.97; R2test = 0.93 and 0.99). The models were also evaluated with different statistical approaches. It was shown that increasing the sum of geometrical distances between N and O in AAs causes decreasing their RF in RP-TLC using both mobile phases. Other structural effects of AAs on their separation in the desired RP-TLC system were also discussed.

Heterocycles 51: Liphophilicity investigation of some thiazole chalcones and aurones by experimental and theoretical methods

Reversed-phase thin-layer chromatography and reversed-phase high-performance liquid chromatography were used for lipophilicity determination of a library of 30 thiazole chalcones and aurones previously synthetized in our laboratory. The experimental lipophilicity data have been compared with theoretical lipophilicity parameters estimated by various computational methods. Good correlations between the experimental and calculated lipophilicity parameters have been found for both investigated classes of compounds. Correlations between the lipophilicity of the thiazole chalcones and aurones and their antiproliferative activity were discussed. The methodologies and data gathered in this study will contribute to the lipophilicity studies of chalcones and aurones derivatives, two important classes of compounds in medicinal chemistry.

Production and Purification of N‐Hydroxy Succinyl Cadaverine (HSC) By Synthetic and Biosynthetic Methods

Optimum conditions for the production and purification of N‐Hydroxy Succinyl Cadaverine, HSC, were investigated. HSC is not commercially available and is essential for research with an enzyme known as DesD. This enzyme will allow HSC to be trimerized and macrocyclized into a siderophore. The siderophore is capable of absorbing iron in the bloodstream that antibiotic‐resistant bacteria use as a life‐sustaining nutrient. Small‐scale organic synthesis was involved in the process of producing the precursor to HSC, known as SC. SC, succinyl cadaverine, is non‐hydroxylated HSC. A deprotection of Boc‐protected succinyl cadaverine was performed. Regular phase thin‐layer chromatography (TLC) was used to check reaction progress while reverse phase TLC was used to determine solvent conditions for purification of SC. Previous attempts to create SC yielded orange‐red puddles of oil. Reaction conditions were changed to theoretically yield white crystals, which indicates purity. After decreasing the equivalencies of TFA, changing the solvent of the reaction, and increasing the run time, white crystals were achieved. 21.5 milligrams of SC were produced in total. The appearance of the crystals increased confidence that the product was fairly pure. Regardless, purification was necessary. The solvent condition for purification of SC was determined to be 100% dichloromethane. This solvent condition yielded a Rf of ~0.5 on a reverse‐phase TLC plate. With this year being the fifth year of research on this project, the organic synthesis of HSC has proven to be very difficult and time‐consuming. New approaches have been considered to make the mechanism more efficient. A biochemical pathway of synthesis will now be investigated. High pressure liquid chromatography will be used to explore the separation conditions of materials between steps. The trimerized and macrocyclized version of HSC, known as DFO‐E, is $400/mg if purchased commercially, and HSC is not available at all. If DesD, a potential new antibiotic drug target, is to be fully investigated, efficient, and cost‐effective, means of creating HSC will be required.Support or Funding InformationThis research was supported in part by the National Science Foundation (NSF‐RUI grant 1716986 to K.M.H.), and by the John Stauffer Charitable Trust.

Lipophilicity Determination of Antifungal Isoxazolo[3,4-b]pyridin-3(1H)-ones and Their N1-Substituted Derivatives with Chromatographic and Computational Methods.

The lipophilicity of a molecule is a well-recognized as a crucial physicochemical factor that conditions the biological activity of a drug candidate. This study was aimed to evaluate the lipophilicity of isoxazolo[3,4-b]pyridine-3(1H)-ones and their N1-substituted derivatives, which demonstrated pronounced antifungal activities. Several methods, including reversed-phase thin layer chromatography (RP-TLC), reversed phase high-performance liquid chromatography (RP-HPLC), and micellar electrokinetic chromatography (MEKC), were employed. Furthermore, the calculated logP values were estimated using various freely and commercially available software packages and online platforms, as well as density functional theory computations (DFT). Similarities and dissimilarities between the determined lipophilicity indices were assessed using several chemometric approaches. Principal component analysis (PCA) indicated that other features beside lipophilicity affect antifungal activities of the investigated derivatives. Quantitative-structure-retention-relationship (QSRR) analysis by means of genetic algorithm—partial least squares (GA-PLS)—was implemented to rationalize the link between the physicochemical descriptors and lipophilicity. Among the studied compounds, structure 16 should be considered as the best starting structure for further studies, since it demonstrated the lowest lipophilic character within the series while retaining biological activity. Sum of ranking differences (SRD) analysis indicated that the chromatographic approach, regardless of the technique employed, should be considered as the best approach for lipophilicity assessment of isoxazolones.

Separation of 9-Fluorenylmethyloxycarbonyl Amino Acid Derivatives in Micellar Systems of High-Performance Thin-Layer Chromatography and Pressurized Planar Electrochromatography

The problems with separation of amino acid mixtures in reversed-phase mode are the result of their hydrophilic nature. The derivatisation of the amino group of mentioned above solutes leads to their solution. For this purpose, 9-fluorenylmethoxycarbonyl chloroformate (f-moc-Cl) as the derivatisation reagent is often used. In our study, the separation of some f-moc- amino acid derivatives (alanine, phenylalanine, leucine, methionine, proline and tryptophan) with the use of micellar systems of reversed-phase high-performance thin-layer chromatography (HPTLC) and pressurized planar electrochromatography (PPEC) is investigated. The effect of surfactant concentration, its type (anionic, cationic and non-ionic) and mobile phase buffer pH on the discussed above solute migration distances are presented. Our work reveals that the increase of sodium dodecylsulphate concentration in the mobile phase has a different effect on solute retention in HPTLC and PPEC. Moreover, it also affects the order of solutes in both techniques. In PPEC, in contrast to the HPTLC technique, the mobile phase pH affects solute retention. The type of surfactant in the mobile phase also impacts solute retention and migration distances. A mobile phase containing SDS improves system efficiency in both techniques. Herein, such an effect is presented for the first time.

Lipophilicity Determination of Quaternary (Fluoro)Quinolones by Chromatographic and Theoretical Approaches.

Lipophilicity is a vital physicochemical parameter of a molecule, which affects several biological processes such as absorption, tissue distribution, and pharmacokinetic properties. In this study, evaluation of lipophilicities of a series of novel fluoroquinolone-Safirinium dye hybrids using chromatographic and computational methods is presented. Fluoroquinolone-Safirinium dye hybrids have been synthesized as new dual-acting hydrophilic antibacterial agents. Reversed phase thin-layer chromatography and micellar electrokinetic chromatography experiments were carried out. Furthermore, logP values of the target structures were predicted by means of different software platforms and algorithms. In order to assess similarities and dissimilarities of the obtained lipophilicity indexes, cluster analysis and sum of ranking differences were performed. The significant differences of calculated logP values (α = 0.05, p < 0.001) indicated that an experimental approach is necessary for lipophilicity prediction of this class of antibiotics. Chromatographic data indicated that the newly synthesized hybrid (fluoro)quinolone-based quaternary ammonium derivatives show less lipophilic character than the parent (fluoro)quinolones. Additionally, the chromatographically obtained lipophilicity indexes were evaluated for possible application in quantitative retention–activity relationships. The established lipophilicity models have the potential to predict antimicrobial activities of a series of quaternary (fluoro)quinolones against Bacillus subtilis, Escherichia coli, and Proteus vulgaris.

Influence of flavonoids' lipophilicity on platelet aggregation.

Flavonoids are natural polyphenolic compounds present in a wide spectrum of plants that have a beneficial effect on human health. In the context of cardiovascular diseases related to plaque and thrombus formation, flavonoids exhibit an anti-aggregatory effect. Previously, it has been reported that all tested flavonoids exhibit an antiaggregatory effect on platelet aggregation when measured by impedance aggregometry on whole blood, in the test of aggregation induced by adenosine diphosphate (ADP). As not all flavonoids have the same targets within signaling pathways, an assumption of a common non-specific mechanism related to lipophilicity is to be considered. To test this hypothesis, reverse-phase thin layer chromatography was used to assess the lipophilicity of flavonoids; impedance aggregometry was used for testing of platelet aggregation and flow cytometry to monitor the influence of flavonoids on platelet activation. Lipophilicity analysis showed a highly negative correlation of logP and MINaAC for groups of flavones and flavanones. As determined by flow cytometry, the exposition of receptors necessary for the promotion of platelet activation and primary clot formation was diminished, i.e., lowered expression of the activated form of integrin αIIbβ3 was observed in the presence of flavanone. Platelet membrane stabilization by flavonoids as a mechanism of antiaggregatory effect has been supported by impedance aggregometry experiments when specific inhibitors of platelet aggregation signaling pathways (U73122, indomethacin, verapamil) were used in the presence of a weak (ADP) and a strong (TRAP-6) agonist of aggregation. While individual flavonoids can have specific targets within aggregation signaling pathways, all flavonoids share a common non-specific mechanism of platelet aggregation inhibition related to their lipophilicity and membrane stabilization that, to some extent, contributes to their antiaggregatory effect.

Synthesis, antimicrobial activity, and determination of the lipophilicity of ((cyclohex-3-enylmethylene)hydrazinyl)thiazole derivatives

Synthesis and investigation of antimicrobial activity of fifteen novel thiazoles containing cyclohexene moiety are presented. Among the derivatives, compounds 3a–3d, 3f, 3n, and 3o showed very strong activity against the reference Candida spp. strains with MIC = 0.015–3.91 µg/ml. The activity of these compounds is similar and even higher than the activity of nystatin used as positive control. Compounds 3d, 3f, 3n, 3o showed the highest activity with very strong effect towards most of yeasts isolated from clinical materials with MIC = 0.015–7.81 µg/ml. The cytotoxicity studies for the most active compounds showed that Candida spp. growth was inhibited at noncytotoxic concentrations for the mammalian L929 fibroblast. In addition, a good correlation was obtained between lipophilicity of compounds determined using reversed phase thin-layer chromatography and their antifungal activity.

Factor Optimization Study to Develop and Validate a Reversed-Phase Thin-Layer Chromatography Method for the Determination of Trimetazidine Dihydrochloride and Its Reported Impurities in Pharmaceuticals

A validated reversed-phase thin-layer chromatography (RP-TLC)—densitometry method was developed and optimized for the determination of trimetazidine dihydrochloride (TMZ) and its potential impurities listed in the British Pharmacopoeia, namely, piperazinecarboxaldehyde (Y-145), trimethoxybenzyl alcohol (Y-235), and trimethoxybenzaldehyde (Y-234). Chromatographic separation was performed on aluminum plates pre-coated with silica gel 60 RP-18F using a mixture of acetonitrile—methanol—0.1% aqueous ortho-phosphoric acid pH 6.2 (4.5:4.5:1, V/V) as the developing system. Different factors affecting resolution were studied and optimized. Successful resolution was observed with significant difference in the RF values of 0.21 ± 0.02, 0.35 ± 0.02, 0.5 ± 0.02, and 0.85± 0.02 for TMZ and its impurities, respectively. Densitometric measurement was done at 215 nm over the range of 0.05–10, 0.05–1.1, 0.05–1.2, and 0.04–1.0 µg per spot with lower limits of detection (LOD) and quantification (LOQ) of 20 and 50 ng per spot for TMZ, Y-145, and Y-235, respectively, and 15 and 40 ng per spot for Y-234 impurity. Good accuracy was obtained with mean percentage recoveries of 99.55 ± 1.06, 100.50 ± 1.16, 100.07 ± 1.24, and 99.41 ± 1.11 for TMZ and its impurities, respectively. The developed method was used to investigate the impurity profile of TMZ in drug substance and different products and it was validated as per the International Conference on Harmonisation (ICH) guidelines.

Application of reversed-phase thin layer chromatography and QSRR modelling for prediction of protein binding of selected β-blockers

Chromatographic properties of sixteen β-blockers were studied using planar chromatography. The aim of presented study was to investigate influence of different organic solvents (acetonitrile, methanol, dioxin and tetrahydrofurane) on β-blockers’ retention on C18 bonded silica gel stationary phase. Group of sixteen, diverse in terms of structure, beta blockers was used as a model set. The main goal of this study was to compare chromatographically estimated lipophilicity parameters with values obtained with the use of computational methods. Furthermore, in order to understand molecular mechanisms of retention better, quantitative structure-retention relationships (QSRR) analysis was performed. The next step was focused on application of chromatographically obtained lipophilicity parameters for prediction of protein binding (PB), based on quantitative retention–activity relationships (QRAR) approach. The obtained results showed that reversed-phase thin layer chromatography (RP-TLC), especially with tetrafydrofurane used as an organic modifier of mobile phase, is a useful tool for lipophilicity estimation, as well as for prediction of β-blockers’ biological properties. The QRAR model included C0 parameters for tetrahydrofuran-water as a mobile phase, as well as maximal projection area, and can be easily applied for prediction of systematically synthesized β-blockers structures’ PB.

Lipophilicity estimation of anti-proliferative and anti-inflammatory 6-substituted 9-fluoroquino[3,2-b]benzo[1,4]thiazines

The lipophilicity parameters (logPcalcd, RM0 and logPTLC) of 17 new anti-proliferative and anti-inflammatory tetracyclic 6-substituted 9-fluoroquinobenzothiazines were determined theoretically using computational methods and experimentally by reversed-phase thin-layer chromatography. The experimental parameter RM0 was determined on the RP-18 silica plates with acetone-aqueous TRIS buffer as mobile phase. The obtained parameter RM0 was further transformed into parameter logPTLC by use of the calibration curve. The theoretical logPcalcd values differed depending on the substituents and nature of calculating programs. Fluoroquinobenzothiazines turned out to be rather middling or highly lipophilic compounds. The parameter RM0 and specific hydrophobic surface area b were meaningly intercorrelated showing congeneric two classes of quinobenzothiazines. The calculated logPcalcd and experimental logPTLC values were compared and intercorrelated to show different prediction power of the computational programs. The lipophilicity was correlated with molecular descriptors (molar mass, molar volume, and molar refractivity), biomolecular descriptors (HIA, PB, MDCK, Caco-2, and BBB) and in vitro tested biological activities (tumor necrosis factor alpha inhibition and antiproliferative activity).

Evaluation of [18F]pitavastatin as a positron emission tomography tracer for in vivo organic transporter polypeptide function

IntroductionTo understand the pathways involved in drug clearance from the body, quantitative evaluations of the hepatobiliary transport of drugs are important. The organic anion transporting polypeptide (OATP) family transporter, particularly OATP1B1 and 1B3, are considered to play an important role in hepatic uptake of organic anion compounds. Pitavastatin is a substrate of OATP, and it includes a fluorine group. Therefore, it represents an acceptable positron-emission tomography (PET) tracer using fluorine-18 to image in vivo hepatic transporter functions. Method[18F]Pitavastatin was synthesized using the method we previously reported. To evaluate the potential of [18F]pitavastatin in PET imaging of in vivo OATP functions, we investigated the hepatic uptake with/without rifampicin as an OATP inhibitor after administration in normal SD rats. [18F]Pitavastatin metabolite was evaluated using reverse-phase thin-layer chromatography (TLC) autoradiography. We subsequently analyzed the PET image results and demonstrated that [18F]pitavastatin selectively accumulated in the liver post-administration.Result and discussionIn metabolite analysis using reverse-phase TLC, we found that the radioactivity detected in the plasma, liver (>90% intact), and bile mostly originated from the parent pitavastatin of the PET study (~40 min). [18F]pitavastatin's hepatic uptake decreased (approximately 76%) with rifampicin co-administration in PET analysis. Because [18F]pitavastatin has lower clearance in rats than other previously reported OATP1B PET s, it holds the potential of an imaging tracer that has a higher sensitivity in monitoring hepatic OATP1B function's changes. ConclusionCompared with the previously reported OATP imaging tracers, [18F]pitavastatin is more suitable for the sensitive detection of functional changes in OATP transporters. We believe that [18F]pitavastatin enables quantitative analysis of the hepatobiliary transport system for organic anion compounds.

Investigation of lipophilicity and pharmacokinetic properties of 2-(methoxy)phenylpiperazine dopamine D2 ligands

Reversed-phase thin-layer chromatography and micellar thin-layer chromatography were used in order to investigate retention behaviour and to determine lipophilicity of series of 2-(methoxy)phenylpiperazine dopamine D2 ligands with different size, shape and rigidity. The retention mechanism was discussed. The lipophilicity parameters obtained in conventional reversed-phase systems expressed as RM0 and C0, as well as RM values determined in microemulsion reversed-phase systems were correlated with in silico determined lipophilicity values. In silico pharmacokinetic properties of 2-(methoxy)phenylpiperazine dopamine D2 ligands revealed the importance of experimentally determined lipophilicity values besides the molecular weight, on the blood–brain barrier permeability process. Also, the experimentally determined lipophilicity was found as a very important factor in plasma protein binding process of 2-(methoxy)phenylpiperazine dopamine D2 ligands. Besides, the Lipinski's rule of five indicates that examined ligands satisfy the criterion of drug-like molecules. The principal component analysis was performed on the experimentally determined and calculated lipophilicity values as well on the molecular descriptors which describe the pharmacokinetic properties in order to provide basic insights into similarities among the studied ligands.

Identification of Apigenin and Luteolin in Artemisia annua L. for the Quality Control

Objective: To identify active compounds and establish the chemical fingerprint of Artemisia annua L. for the quality control. Methods: Thin-layer chromatography (TLC) conditions were developed to screen for 2 common flavonoids (apigenin and luteolin). Three mobile phases were used to isolate these flavonoids in 80% ethanolic extract of A. annua .Hexane : ethyl acetate : acetic acid (31:14:5, v/v) and toluene : 1,4-dioxane : acetic acid (90:25:4, v/v) were used in normal phase TLC (NP-TLC), and 5.5% formic acid in water : methanol (50:50, v/v) were used in reverse phase TLC (RP-TLC). Chromatograms were visualized under visible light after spraying with Fast Blue B Salt. Apigenin and luteolin bands were checked by comparing their Rf values and UV-Vis absorption spectra with reference markers. Results: Apigenin and luteolin were simultaneously detected with good specificity in RP-TLC condition, while only apigenin was detected in NP-TLC condition. Apigenin band intensity was higher than luteolin band intensity in both conditions. Conclusion: This knowledge can be applied to the development of quality control assessments to ensure product efficacy and consistency.

Lipophilicity as a Central Component of Drug-Like Properties of Chalchones and Flavonoid Derivatives.

Lipophilcity is an important physico-chemical parameter that influences membrane transport and binding ability to action. Migration distance following complete elution of compounds was used to calculate different lipophilicity-related parameters. The aim of this study is to show that lipophilicity is a central component of thiazole chalcones and flavonoid derivatives regarding their drug-like properties. Experimental and computational methods were used. This study considers 44 previously synthesized compounds (thiazole chalcones, flavanones, flavones, 3-hydroxyflavones, and their acetylated derivatives). The concerned compounds have shown antitumoral hallmarks and antibacterial activity in vitro. The experimental method used to determine compounds’ lipophilicity was the reverse-phase thin layer chromatography (RP-TLC). Lipophilicity related parameters—isocratic retention factor (RM), relative lipophily (RM0), slope (b), chromatographic hydrophobic index (φ0), scores of principal components (PC1/RM)—were determined based on reverse-phase chromatography results.

Experimental evaluation of solvent elution strength in reversed-phase TLC on medium polarity phases

Seven chromatographic grade solvents miscible with water (acetone, acetonitrile, dioxane, ethanol, isopropanol, methanol, tetrahydrofuran), as well as water itself were investigated in the context of their eluotropic strength in reversed-phase chromatography on four medium-polar adsorbents (cellulose, CN, NH2 and DIOL). The test set for this research consisted of 35 model compounds. A modern chemometric mixture design concept allowed to estimate each solvent effect separately with a relatively small error (about 0.05 of RM value for all adsorbents). The solvent effects were analyzed chemometrically in multivariate manner and a final model was fitted to achieve the solvent strength. The estimation error was computed both from regression and bootstrap approach.

Chromatographic descriptors in QSAR study of barbiturates

Barbiturate derivatives were evaluated for their parameters of biological activity by applying linear regression and two multivariate methods (Cluster analysis and Principal component analysis). The lipophilicity of the studied barbiturates was determined on the modified carriers C18 in mixtures of water and four organic modifiers separately (methanol, n-propanol, acetone and tetrahydrofuran) by performing reversed phase thin layer chromatography and by applying relevant software packages. Chromatographic and computational lipophilicity of the examined barbiturates was correlated with the selected pharmacokinetic and toxicological predictors and good relationships were obtained. More concrete results were obtained by multivariate methods which showed that the polarity of the substituent has the greatest influence, and its electronic effects to a lesser extent on the tested parameters of the barbiturate derivatives. Results obtained by multivariate methods also suggest that the chromatographic retention constant, RM0, shows a greater resemblance to the parameters of lipophilicity. The chromatographic parameter m, exhibits better agreement with the toxicity parameters.

Evaluation of Hydrophobicity for Fatty Acids Using Reversed-phase Thin Layer Chromatography.

Hydrophobicity of ingredients is important for designing food, pharmaceuticals, and cosmetics. Here, we evaluated the hydrophobicity of fatty acids and showed the effect of the alkyl length and the unsaturation degree using reversed-phase thin layer chromatography (RP-TLC). A linear relation was obtained between the methanol concentration in a mobile phase and the Rm value. The linear regression analysis was achieved and the hydrophobicity value Rmw was obtained using the robust regression (MM-estimator). The hydrophobicity of fatty acids depends on the structure of alkyl chain as follows: a longer alkyl chain in fatty acid increased the hydrophobicity. Additionally, the hydrophobicity increased as the number of unsaturated parts increased. Fatty acid with branched structures were less hydrophobic than that with straight chained structures.