RP-18 Plates Research Articles

Study of the Lipophilicity and ADMET Parameters of New Anticancer Diquinothiazines with Pharmacophore Substituents.

Lipophilicity is one of the principal parameters that describe the pharmacokinetic behavior of a drug, including its absorption, distribution, metabolism, elimination, and toxicity. In this study, the lipophilicity and other physicochemical, pharmacokinetic, and toxicity properties that affect the bioavailability of newly synthesized dialkylaminoalkyldiquinothiazine hybrids as potential drug candidates are presented. The lipophilicity, as RM0, was determined experimentally by the RP-TLC method using RP18 plates and acetone-TRIS buffer (pH 7.4) as the mobile phase. The chromatographic parameters of lipophilicity were compared to computationally calculated partition coefficients obtained by various types of programs such as iLOGP, XLOGP3, WLOGP, MLOGP, SILCOS-IT, LogP, logP, and milogP. In addition, the selected ADMET parameters were determined in silico using the SwissADME and pkCSM platforms and correlated with the experimental lipophilicity descriptors. The results of the lipophilicity study confirm that the applied algorithms can be useful for the rapid prediction of logP values during the first stage of study of the examined drug candidates. Of all the algorithms used, the biggest similarity to the chromatographic value (RM0) for certain compounds was seen with iLogP. It was found that both the SwissADME and pkCSM web tools are good sources of a wide range of ADMET parameters that describe the pharmacokinetic profiles of the studied compounds and can be fast and low-cost tools in the evaluation of examined drug candidates during the early stages of the development process.

Lipophilicity of tryptophan, its metabolites and derivatives measured by thin-layer chromatography

Fourteen compounds, including tryptophan and its metabolites: kynurenine pathway products (kynurenine, kynurenic acid, 3-hydroxykynurenine), auxin pathway products (indoleacetic acid, indolepropionic acid, indolebutyric acid, indolepyruvic acid), serotonin pathway products (5-hydroxytryptophan, serotonin, melatonin), tryptamine, as well as two synthetic auxin analogs (naphtyl-1-acetamide and naphthalene-1-acetic acid) were chromatographed on RP8 plates using binary mobile phases containing 8 concentrations of methanol as a modifier. The resulting retention data was fitted to linear Soczewiński–Wachtmeister models, obtaining experimental lipophilicity indices, which were then compared with computational lipophilicities calculated with 6 algorithms (ALOGPs, AClogP, ALOGP, MLOGP, XLOGP2 and XLOGP3) using regression and multivariate (principal component analysis) approach. It was confirmed that hydroxylation lowers the lipophilicity, whereas adding one homologous aliphatic CH2, exchange of amino group to keto group (or its loss) increases the lipophilicity. Kynurenic acid has lower lipophilicity than kynurenine under investigated conditions due to its ionization, which is opposite to computational results based on non-ionized molecule. The obtained results can be useful in explaining the biochemical and pharmacological phenomena related to these compounds.

Experimental evaluation of solvent elution strength in reversed-phase thin-layer chromatography on RP2 and RP8 plates

The eluotropic strength on RP2 and RP8 plates was investigated for 7 chromatographic grade solvents miscible with water as a diluent (acetone, acetonitrile, dioxane, ethanol, isopropanol, methanol, and tetrahydrofuran) as well as for water itself. Thirty-five model compounds were used as a test set in the investigation. The use of a modern chemometric mixture design concept allowed separate estimation of each solvent effect from the other results with a relatively small error (about 0.05 of RM value for both adsorbents). Principal component analysis was used as an additional tool and revealed that only one parameter (mean eluotropic strength) is satisfactory to explain the overall variability in the retention datasets. The bootstrapping approach was also used to check the distribution and uncertainty of the obtained values. It can be concluded that all solvents have quite similar elution strength for both adsorbents. The obtained values can be used as a reference during the optimization of thin-layer chro...

A Validated Quantification of Sudan Red Dyes in Spicery using TLC and a 16-bit Flatbed Scanner.

We present a video-densitometric quantification method for Sudan red dyes in spices and spice mixtures, separated by TLC. Application was done band-wise in small dots using a 5μL glass pipette. For separation, the RP-18 plates (20 × 20cm with fluorescent dye; Merck, Germany, 1.05559) were developed in a vertical developing chamber without vapor saturation from the starting point to a distance of 70mm by using acetonitrile, methanol, and aqueous ammonia solution (25%; 8 + 1.8 + 0.2, v/v) as mobile phase. The quantification is based on direct measurements using an inexpensive 16-bit flatbed scanner for color measurements (in red, green, and blue). Evaluation of only the green channel makes the measurements very specific. For linearization, an extended Kubelka-Munk expression for data transformation was used. The range of linearity covers more than two magnitudes and lies between 20 and 500ng. The extraction from a 2g sample with acetonitrile, evaporation, and reconstitution to 200μL with methanol and the band-wise application (7mm) of a 10μL sample allows a statistically defined LOD of less than 500ppb of Sudan red dyes. To perform the analysis, a separation chamber, RP-18 plates, 5μL glass pipettes, and a 16-bit flatbed scanner for 105 € are needed; therefore, the separation method is inexpensive, fast, and reliable.

Can RP-8 plates be used in normal-phase systems? A chemometric approach to estimate the elution strength of popular solvents

During our previous studies, we discovered that reversed-phase thin-layer chromatography (RP-TLC) plates can behave in typical normal-phase manner, which is not widely known nor used in the literature. Therefore, we decided to investigate this behavior more comprehensively on RP-8 plates. We used 35 model compounds, and performed chemometric screening mixture design approach for 20 popular solvents. This gave us the possibility to estimate separate retention effect for each solvent being the measure of average solvent strength. The results were compared to the previous study done on silica gel. It can be concluded that RP-8 plates can be used in typical normal-phase systems and their behavior does not differ substantially from silica gel plates.

Assessment of the chromatographic lipophilicity of eight cephalosporins on different stationary phases

The retention behaviors were investigated for a series of eight cephalosporins in thin-layer chromatography (TLC) using stationary phases of RP-2, RP-8, RP-18, NH2, DIOL, and CN chemically bonded silica gel. Additionally, various binary mobile phases (water/methanol and water/acetone) were used in different volume proportions. The retention behavior of the analyzed molecules was defined by RM0 constant. In addition, reversed phase high performance liquid chromatography (RP-HPLC) was performed in lipophilicity studies by using immobilized artificial membrane (IAM) stationary phase. Obtained chromatographic data (RM0 and logk'IAM) were correlated with the lipophilicity, expressed as values of the log calculated (logPcalc) and experimental (logPexp(shake-flask)) partition coefficient. Principal component analysis (PCA) was applied in order to obtain an overview of similarity or dissimilarity among the analyzed compounds. Hierarchical cluster analysis (HCA) was performed to compare the separation characteristics of the applied stationary phases. This study was undertaken to identify the best chromatographic system and chromatographic data processing method to enable the prediction of logP values. A comprehensive chromatographic investigation into the retention of the analyzed cephalosporins revealed a similar behavior on RP-18, RP-8 and CN stationary phases. The weak correlations obtained between experimental and certain computed lipophilicity indices revealed that RM0 and PC1/RM are relevant lipophilicity parameters and the RP-8, CN and RP-18 plates are appropriate stationary phases for lipophilicity investigation, whereas computational approaches still cannot fully replace experimentation.

TLC Profiles of Selected Cirsium Species with Chemometrics in Construction of Their Fingerprints.

The dried aerial parts of 12 plants of Cirsium species were extracted with the Soxhlet apparatus using dichloromethane and methanol as solvents. Next, the extracts were separated by TLC methods to obtain the fingerprint chromatograms. The analysis was performed on silica gel or RP-18 layers as stationary phases using the following eluents: ethyl acetate/formic acid/acetic acid/water (12/1.5/1.5/4; v/v) for silica gel, and 5% (v/v) aqueous solution of formic acid/methanol (70/30; v/v) for the first development and the same system in the proportion of 50/50 (v/v) for the second development for RP-18. The double development was applied in the case of RP-18 plates. The analysis was performed for all Cirsium methanolic extracts and five selected standards (naringin, apigenin, rutin, caffeic acid and chlorogenic acid). The results were analyzed using chemometrics. The comparison of individual Cirsium species and the identification of unknown species were performed using the similarity indices (Pearson's correlation coefficient, determination coefficient and congruence coefficient), distance indices (Euclidean distance, Manhattan distance and Chebyshev's distance) and Multi-Scale Structural SIMilarity. Based on chemometric analysis, the first extract of the widely grown species is identified as Cirsium arvense and the second one as Cirsium rivulare.

A planar chromatographic method to detect adulteration of vegetable oils in ghee

Reversed-phase thin-layer chromatography (RP-TLC) is a sensitive, reliable, and reproducible technique, which can analyze a number of components simultaneously. This technique was exploited in the present study to identify the adulteration of vegetable oils (groundnut, soybean, and sunflower oil) in ghee, using the fact that β-sitosterol, an unsaponifiable matter, is present in vegetable oils, while absent in pure ghee. For this, RP-TLC of reference standards, unsaponifiable matter of pure ghee (cow and buffalo), pure vegetable oils, and the ghee adulterated with these vegetable oils (≥1%) was carried out on plates with different stationary phases, viz., RP-18, RP-8, and RP-2 plates using solvent system comprising of petroleum ether, acetonitrile, and methanol. The results revealed that adulteration of vegetable oils in ghee can easily be detected at a level of as low as 1 percent on RP-18 and RP-8 plates, while RP-2 plate did not offer any help in detection of adulteration. Thus, with the help of RP-TLC,...

Quantitative Method of Disulfiram Determination by Thin-Layer Chromatographic Image Analysis Technique

A simple and sensitive thin-layer chromatographic (TLC) method coupled with an image analysis technique was developed for the quantitative determination of disulfiram. Separation was performed on RP-18 plates with methanol—water 90:10 (v/v) as mobile phase. Disulfiram was detected using copper(II) ions (producing a yellow complex). Digital images of TLC plate chromatograms were converted into peak chromatograms using TLSee software, and quantitative analysis was conducted. Linearity was obtained in the range of 1.0–10 μg/spot. The limits of detection and quantification were 0.26 μg/spot and 0.86 μg/spot, respectively. Quantitative determination of disulfiram in Anticol was also performed, with a recovery of 100.3%. The method was applied for the determination of disulfiram in a urine-spiked sample.

Revisiting thin-layer chromatography as a lipophilicity determination tool. Part IV — A closer look on extrapolation and averaging

Summary The paper compares linear, quadratic, and cubical regression together with several weighted and robust approaches in the context of lipophilicity determination. The comparison is done on 35 model compounds on data from different modifiers used on RP18, CN, and silica plates. It can be concluded that the use of weighted and moderately robust regression technique increases correlation between extrapolated retention and real lipophilicity, whereas polynomial and very robust techniques give visibly worse results due to their excessive flexibility and higher extrapolation uncertainty. Additionally, we have compared averaging retention from different modifiers by RF, k, and RM values. The results are similar; however, surprisingly, RF averaging performs slightly better to the other approaches.

EXPLORING HIDDEN TRENDS IN CLASSIC AND MICELLAR THIN-LAYER CHROMATOGRAPHIC RETENTION OF MODEL COMPOUNDS BY CHEMOMETRIC METHODS

We present chemometric analysis of the retention of 35 simple model compounds in nonmicellar and micellar TLC systems. Nonmicellar systems were investigated on silica gel, RP18, and CN plates, by use of several modifiers in hexane or water as the mobile phase. Micellar systems were processed on silica and RP8 plates with three surfactants (cetyltrimethylammonium bromide, sodium dodecylsulfate, and Triton-X100) dissolved in water above critical micellar concentration. The resultant dataset was subjected to principal component analysis (PCA) to explore hidden trends in retention. It was concluded that PCA is a valuable tool for factor analysis of retention, as it allows the chromatographer to see and interpret all trends which would be hidden in manual analysis of retention matrix. Retention on each adsorbent can be decomposed to maximally four main independent trends and each compound has be characterized by intensity of these trends, expressed as PCA scores.

Separation of amino acid 2,4-dinitrophenyl-5-l-valine amide diastereomeric derivatives with high-performance planar chromatography and pressurized planar electrochromatography

Summary Separation of 2,4-dinitrophenyl-5-L-valine amide derivatives of some amino acids (leucine, isoleucine, valine, asparagine, cysteine, tryptophane) L and D-enantiomers with high-performance thinlayer chromatography (HPTLC) and pressurized planar electrochromatography (PPEC) is presented. The separation mechanism in the PPEC technique involves two effects (partition and electrophoresis) whereas HPTLC system employs only one (partition). The separation process in both modes was carried out with the HPTLC RP-18 plates. Influence of the aqueous-organic mobile phase composition (acetonitrile concentration, buffer type, its pH) on migration distance of the solute zones is investigated and compared for both techniques. The PPEC system turns out to be more sensitive to buffer type and pH than HPTLC one. Additionally, the electrophoretic effect is responsible for different separation selectivity in PPEC system in comparison to that in HPTLC one. The retention of DL-diastereoisomer derivatives of amino acids is stronger in comparison with its LL-antipodes for both separation techniques. The statistic evaluation of the migration distance in PPEC and TLC systems shows similar RSD.

The efficiency of RP-TLC for lipophilicity assessment. A comparative study on a series of pyrrolyl-acetic acid derivatives, inhibitors of aldose reductase

The lipophilicity of a series of pyrrolyl-acetic acid derivatives, inhibitors of the aldose reductase enzyme, was assessed by reversedphase thin layer chromatography (RP-TLC). The role of the carbon content of the stationary phase in reproducing the octanol-water partitioning was examined using RP-18F254S and RP-8F254S plates. Retention on RP-8 plates was found to be more uniform, while these plates exhibit some advantages for the lipophilicity assessment of acidic compounds, compared to the most commonly used RP-18 plates. However, in both cases, different energetics between octanol-water partitioning and RP-TLC retention seem to be involved. On both plates the influence of ionization of the acid functional group was found to be more pronounced on retention in comparison with previous findings for basic compounds. Both sets of RMw values were compared with HPLC chromatographic indices, reported previously. They were found to correlate better with logkw values, if the latter have been determined in the pr...

A comparative study concerning chromatographic retention and computed partition coefficients of some precursors of peraza crown ethers

Abstract Retention indices for some precursors of peraza crown ethers were determined by reversed phase high-performance thin layer chromatography on RP-18 plates with methanol-water in different volume proportions as mobile phase. The Log P values for the same compounds were calculated using different computer programs: SciQSAR, SciLogP, Chem3D Ultra 8.0, XLOGP (based on atom contributions), Chemaxon and KOWWIN (based on atom/fragment contributions), cLogP (based on fragmental contributions), ALOGPS and IAlogP (based on atom-type electrotopological-state indices and neural network modeling). A comparative study concerning lipophilic parameters (RM0, b and ϕ0) and computed partition coefficients has been developed. Taking into account the correlation coefficients between determined and calculated Log P values, it seems that RM0 and b are less suitable than ϕ0 for estimating lipophilicity of the compounds investigated, and cLogP and ALOGPS provide the best correlations with experimental values.

A versatile method for quantification of aflatoxins and ochratoxin A in dried figs

Thin-layer chromatography is a rapid and reliable working method for quantification of mycotoxins which is suitable for checking EC legislation aflatoxin limits for dried figs without an RP-18 pre-column cleaning step. We describe normal-phase chromatography on silica gel plates with 2.4:0.05:0.1:0.05 ( v/v ) methyl t -butyl ether-water-methanol-cyclohexane as mobile phase and reversed-phase chromatography on RP-18 plates with methanol-4% aqueous ZnSO 4 solution-ethyl methyl ketone 15:15:3 ( v/v ) as mobile phase. Sample pretreatment was by modified QuEChERS (Quick, Easy, Cheap, Effective, Rugged, Safe) extraction with tetrahydrofuran or acetone. NaCl was used as QuEChERS salt. Response was a linear function of amount chromatographed in the ranges 3 to 100 pg per zone for aflatoxins B 2 and G 2 , 10 to 350 pg per zone for the aflatoxins B 1 and G 1 , and 0.25 to 2.5 ng per zone for ochratoxin A. Quantification limits for the aflatoxins were between 13 and 35 pg per zone (equivalent to 1.5 and 2.4 ppb, tak...

Quantitative analysis of gliclazide and glipizide in tablets by a new validated and stability-indicating RPTLC method

A simple, rapid, and stability-indicating RPTLC method has been established and validated for quantitative analysis of two oral antidiabetic agents, the sulfonylureas gliclazide and glipizide, in tablets. Analysis was performed on RP-18 plates with 60% ( v/v ) acetonitrile in pH 2.3 phosphate buffer as mobile phase. Compact spots were obtained for gliclazide ( R F 0.38 ± 0.02, mean ± SD) and glipizide ( R F 0.51 ± 0.03). Detection and quantification were performed by classical densitometry at 215 nm, the wavelength of maximum absorption of gliclazide and glipizide. Calibration plots were constructed in the range 0.8–1.8 μg per 10 μL (per spot) for both drugs and were linear with good correlation coefficients ( r = 0.998 ± 0.001, mean ± SD, for gliclazide and r = 0.993 ± 0.002 for glipizide). The limits of detection and quantification were 50 and 200 ng per spot, respectively, for gliclazide and 60 and 300 ng for glipizide. The method was validated for robustness, precision, accuracy, stability, and specif...

Validation of NP-HPTLC and RP-HPTLC methods with videodensitometric detection for analysis of ziprasidone in pharmaceutical formulations

Two new simple and accurate HPTLC methods for analysis of ziprasidone in capsules have been developed and validated. The first method was normal phase chromatography on silica gel F 254 HPTLC plates with hexane-dioxane-propylamine 1:9:0.4 ( v/v ) as mobile phase. The second method was reversed phase chromatography on RP8 F 254 HPTLC plates with tetrahydrofuran-pH 9.0 phosphate buffer 5:5 ( v/v ) as mobile phase. The silica gel plates were developed to a distance of 9 cm and the RP8 plates to a distance of 4.5 cm. Both analyses were performed in horizontal chambers and the plates were scanned by videodensitometry at 254 nm. The calibration plots were linear in the ranges 0.2–1.2 μg and 0.1–1.1 μg ziprasidone (per spot) for the NP-HPTLC and RP-HPTLC methods, respectively. The precision and accuracy of the methods were fully compared between themselves and also with the control method — classical densitometry at 243 nm. No significant differences were observed. The NP-HPTLC method was also used for study of ...

Direct Analysis of Reversed-Phase High-Performance Thin Layer Chromatography Separated Tryptic Protein Digests Using a Liquid Microjunction Surface Sampling Probe/Electrospray Ionization Mass Spectrometry System

The sampling, ionization and detection of tryptic peptides separated in one-dimension on reversed-phase high-performance thin layer chromatography (HPTLC) plates was performed using liquid microjunction surface sampling probe electrospray ionization mass spectrometry. Tryptic digests of five proteins [cytochrome c, myoglobin, beta-casein, lysozyme and bovine serum albumin (BSA)] were spotted on reversed phase HPTLC RP-8 F254s and HPTLC RP-18 F254s plates. The plates were then developed using 70/30 methanol/water with 0.1M ammonium acetate. A dual purpose extraction/electrospray solution containing 70/30/0.1 water/methanol/formic acid was infused through the sampling probe during analysis of the developed lanes. Both full scan mass spectra and data dependent tandem mass spectra were acquired for each development lane to detect and verify the peptide distributions. Data dependent tandem mass spectra provided both protein identification and sequence coverage information. Highest sequence coverages were achieved for cytochrome c and myoglobin (62.5% and 58.3%, respectively) on reversed phase RP-8 plates. While the tryptic peptides were separated enough for identification, the peptide bands did show some overlap with most peptides located in the lower half of the development lane. Proteins whose peptides were more separated gave higher sequence coverage. Larger proteins such as beta-casein and BSA which were spotted in lower relative amounts gave much lower sequence coverage than the smaller proteins.

Determination of the Lipophilicity of twenty-seven imidazo[2,1-c][1,2,4]triazine derivatives with strong biological activity by reversed-phase TLC. Comparison with results obtained by use of computational algorithms

The lipophilicity, log P , of 27 novel imidazo[2,1- c ][1,2,4]triazine derivatives with strong biological activity has been determined by reversed-phase thin-layer chromatography. The compounds were chromatographed on RP18 plates with methanol-water binary mobile phases containing different proportions of methanol. R M values for pure water were then extrapolated from the linear Soczewinki-Wachtmeister equation. Twelve compounds with known literature log P values acted as a reference calibration data set for computation of experimental log P values. The results obtained were compared, by principal-components analysis, with those obtained by use of nine computational methods.

Lipophilicity Evaluation of the Products Obtained in the Reaction of N3-Substituted Amidrazones with cis-1,2-Cyclohexanedicarboxylic Anhydride

The lipophilicity of the novel 12 products of the reaction of N3-substituted amidrazones with cis-1,2-cyclohexanedicarboxylic anhydride (4 linear, 4 triazole-like and 4 isoindole ones) with potential pharmacologic activity was evaluated by thin layer and liquid chromatography. Using organic-aqueous eluent systems (with methanol or acetonitrile) on RP18 plates and C18 column, a linear relationship between the volume fraction of modifiers and the retention indices was obtained. The retention values, log k or RM were extrapolated to zero organic modifier content to obtain the log kw or RMW values. 12 compounds with known literature lipophilicity were used as a calibration dataset. The results were compared in a multivariate way with in silico methods (ALOGPs, AC_logP, AB/LogP, COSMOFrag, miLogP, ALOGP, MLOGP, KOWWIN, XLOGP3).