A degradation study of cefepime hydrochloride in solutions under various stress conditions by TLC–densitometry

A rapid, accurate and sensitive thin‐layer chromatography (TLC) method with densitometric detection has been developed and validated for the determination of cefepime in pharmaceuticals. Chromatographic separation was achieved on a silica gel TLC F₂₅₄ plates with a mobile phase consisting of ethanol–2‐propanol–glacial acetic acid 99.5%–water (4:4:1:3, v/v). Densitometric detection was carried out at wavelength of 266 nm in reflectance/absorbance mode. The validation of the method was found to be satisfactory with high accuracy (from 99.24 to 101.37%) and precision (RSD from 0.06 to 0.36%). Additionally, the stability of cefepime in solution was investigated, including the effect of pH, temperature and incubation time. Favorable retention parameters (R_f, R_s, α) were obtained under the developed conditions, which guaranteed good separation of the studied components. The degradation process of cefepime hydrochloride was described by kinetic and thermodynamic parameters (k, t_0.1, t_0.5 and E_a). Moreover, the chemical properties of degradation products were characterized by the R_f values, absorption spectra, HPLC‐MS/MS and TLC‐densitometry analysis. As the method could effectively separate the active substance from its main degradation product (1‐methylpyrrolidine), it can be employed as a method to indicate the stability of this drug. Copyright © 2014 John Wiley & Sons, Ltd.     

High performance thin layer chromatographic determination of erythromycin in pharmaceutical preparations

Summary A high performance thin layer chromatographic method was developed for the determination of erythromycin. The drug was separated on a silica gel 60 plate and developed in methanol by means of an automatic multiple development. The chromatogram was sprayed with 10% sulphuric acid solution and heated at 100°C for 10–15 minutes. The area of the spot was quantified by a TLC scanner at a wavelength of 410 nm. A linear calibration curve was established over the range of 4–6 μg in 10μl of erythromycin. The relative standard deviation for five replicate determinations was found to be 1.45% for 5 μg in 10 μl of erythromycin standard. The average percentage recovery was found to be 99.87. The method has been applied to the determination of erythromycin in various pharmaceutical dosage forms. Common excipients in formulations do not interfere. After optimizing the solvent system, it was found that the use of silica gel 60 F₂₅₄ TLC plate with a DVS composed of ethyl acetate, ethanol and 10% sodium acetate pH 9.5 (9:7:8) led to the differentiation and quantitation of erythromycins A, B and C with an R.S.D. of less than 2.0%. The method is simple, precise and inexpensive. It should be used for routine analysis.     

Determination of platinum, palladium and rhodium in Pt-catalysts by high performance thin layer chromatography using densitometry

The determination of palladium and platinum in alumina-supported bimetallic Pt-Re, Pd and Pt catalysts at the 0.03 and 0.3% (m/m) levels, respectively, and of rhodium and platinum in platinum sieve catalysts at the 0.04% (m/m) level and in the 4.75–19.75% (m/m) range, respectively, is described. The platinum group metals in fresh or spent catalysts are determined chromatographically using densitometry by forming theirN,N-diethyl-N-benzoylthiourea complexes. The precision of the method, %RSD, is 0.4–3.5, 2.2–6.7, and 4.2–6.0 for Pt, Pd and Rh, respectively. The accuracy was tested using alumina-based and active carbon platinum and palladium standards. The results were also compared with those obtained by FAAS. There was at most 8% difference between the results obtained with these methods, except for one active carbon based Pd standard.     

Stability-indicating high performance thin layer chromatography determination of Paroxetine hydrochloride in bulk drug and pharmaceutical formulations

A simple selective precise and stability-indicating high performance thin layer chromatographic method of analysis of Paroxetine hydrochloride both as a bulk drug and in formulations was developed and validated. The method employed TLC (Thin Layer Chromatography) aluminum precoated with silica gel 60F-254 as the stationary phase. The solvent system consisted of butanol:acetic acid:water (8:2:0.5, v/v/v). This system was found to give compact spots for Paroxetine HCl (Rf, retardation factor, value-0.48 ± 0.02). Paroxteine HCl was subjected to acid and alkali hydrolysis, oxidation and photodegradation, where the degraded product was well separated from the pure drug. Densitometric analysis of Paroxetine hydrochloride was carried out in the absorbance mode at 295 nm. The linear regression analysis data for the calibration spots showed good relationship with (regression) r² = 0.9903 in the amount range of 300-1500 ng (nanogram) per spot. The mean value of co-relation co-efficient, slope and intercept were 0.9903 ± 0.001, 5.38 ± 0.058 and 182.5 ± 2.16 respectively. The method was validated for precision, recovery and robustness. The limits of detection and quantitation were 50 and 150 ng, respectively. The drug doesnot undergo degradation with oxidation, but gets affected in acidic and alkaline conditions. The acid and alkali degradation showed extra peaks at 0.4 and 0.08 Rf, respectively. This indicates that the drug is susceptible to acidic and alkaline medium. As the method could effectively separate the drug from its degradation products, it can be employed as a stability-indicating one.     

手性薄层色谱评述

根据作者团队近八年的手性薄层色谱文献的再现实验以及进一步研究,从实用性的角度系统总结了手性薄层色谱的9个特点。并且根据作者团队的再现实验结果,介绍了目前的手性薄层色谱报道情况、已有商品手性薄层色谱板的拆分性质及作者团队的万古霉素薄层色谱板的研究。     

Contour diagrams for the presentation of separation quality in two-dimensional thin layer chromatography

Several methods for evaluating separation quality in two-dimensional thin layer chromatography are discussed. These include using either the distance between spot centers for the pair of solutes that is the most difficult to separate in a mixture, or the value of a suitable, separation function, as criteria of overall separation quality. Contour diagrams may be constructed in which solvent compositions for each of the two-dimensional developments are the independent variables and spot separation or the value of a suitable function is the dependent variable. Such diagrams indicate which are the best and the worst regions of the two-dimensional solvent domain. These diagrams should be of value for comparing different solvent systems for two-dimensional thin layer chromatography.     

High-performance thin layer chromatography method for estimation of andrographolide in herbal extract and polyherbal formulations

A new, simple, sensitive, precise and robust high-performance thin layer chromatographic (HPTLC) method was developed for the estimation of andrographolide in herbal extracts and pharmaceutical dosage forms. Analysis of andrographolide was performed on TLC aluminium plates pre-coated with silica gel 60F-254 as stationary phase. Linear ascending development was carried out in twin trough glass chamber saturated with chloroform:toluene:methanol (66:26:8, v/v/v) at room temperature (25 ± 2 °C). The R_f value of andrographolide was found to be 0.49. Camag TLC scanner III was used for spectrodensitometric scanning and analysis in absorbance mode at 229 nm. The system was found to give compact spots for andrographolide (R_f value of 0.49). The data for calibration plots showed good linear relationship with r² = 0.9986 in the concentration range of 200 ng to 1000 ng with respect to peak area. The present method was validated by precision, recovery, robustness and reproducibility according to ICH guidelines. The limits of detection and quantification were determined and it was found to be 3.5 and 11.7 ng, respectively. Statistical analysis of the data showed that the method is reproducible and selective for estimation of andrographolide.     

Quantitative determination of fluoxetine in human serum by high performance thin layer chromatography

A high performance thin layer chromatographic method was developed and validated for the quantification of fluoxetine in human serum. Fluoxetine was extracted by liquid–liquid extraction method with diethyl ether as extraction solvent. Imipramine was used as internal standard. The chromatographic separation was achieved on precoated silica gel F 254 high performance thin layer chromatographic plates using a mixture of toluene/acetic acid glacial (4:5 v/v) as mobile phase. 4‐Dimethylamino‐azobenzene‐4‐sulphonyl chloride was used as derivatization reagent. Densitometric detection was done at 272 nm. The method was linear between 12.5 and 87.5 ng/spot, corresponding to 0.05 and 0.35 ng/μL of fluoxetine in human serum after extraction process and applying 25 μL to the chromatographic plates. The method correlation coefficient was 0.999. The intra‐assay and inter‐assay precisions, expressed as the RSD, were in the range of 0.70–2.01% (n=3) and 0.81–3.90% (n=9), respectively. The LOD was 0.23 ng, and the LOQ was 0.70 ng. The method proved be accurate, with a recovery between 94.75 and 98.95%, with a RSD not higher than 3.61% and was selective for the active principle tested. This method was successfully applied to quantify fluoxetine in patient serum samples. In conclusion, the method is useful for quantitative determination of fluoxetine in human serum.     

Digitally enhanced thin layer chromatography for simultaneous determination of norfloxacin and tinidazole with the aid of Taguchi orthogonal array and desirability function approach: Greenness assessment by analytical Eco‐Scale

In this study, an eco‐friendly fast simple method was developed for simultaneous determination of norfloxacin and tinidazole based on thin‐layer chromatography and image‐processing analysis. The binary mixture was separated using reversed phase ‐ thin layer chromatography plates and 30% trifluoroacetic acid only as mobile phase. Mobile phase composition was optimized using Taguchi orthogonal array and Derringer's desirability function. The plates were viewed under UV lamp and photographed by iPhone camera followed by image processing with Fiji software using integrated density as the measured response. As decreasing illumination increases the sensitivity of the method, this method was applied on two different ranges for each drug. The first one was 0.6–6.0 and 0.9–9.0 µg/spot for norfloxacin and tinidazole, respectively measured on the original image with normal illumination. The second one was measured after decreasing the illumination of the captured images at 0.06–0.60 and 0.09–0.90 µg/spot for norfloxacin and tinidazole, respectively. The proposed method was successfully applied for the determination of both drugs in tablets dosage form without interference from the commonly encountered excipients. Analytical Eco‐Scale was used to evaluate the greenness profile of the proposed method and it was found to be excellent green analytical method.     

Comparison between micellar liquid chromatography and capillary zone electrophoresis for the determination of hydrophobic basic drugs in pharmaceutical preparations

The determination of highly hydrophobic basic compounds by means of conventional reversed-phase liquid chromatographic methods has several drawbacks. Owing to the characteristics of micellar liquid chromatography (MLC) and capillary electrophoresis (CE), these techniques could be advantageous alternatives to reversed-phase chromatographic methods for the determination of these kinds of compounds. The objective of this study was to develop and compare MLC and CE methods for the determination of antipsychotic basic drugs (amitryptiline, haloperidol, perphenazine and thioridazine) in pharmaceutical preparations. The chromatographic determination of the analytes was performed on a Kromasil C(18) analytical column; the mobile phase was 0.04 m cetyltrimethylammonium bromide (CTAB), at pH 3, containing 5% 1-butanol, at a flow rate of 1 mL/min. The CE separation was performed in a fused-silica capillary with a 50 mm tris-(hydroxymethyl)-aminomethane buffer, pH 7, at an applied voltage of 20 kV, using barbital as internal stardard. The proposed methods are suitable for a reliable quantitation of these compounds in the commercial tablets and drops in terms of accuracy and precision and require a very simple pre-treatment of the samples. By comparing the performance characteristics and experimental details of the MLC and CE methods we conclude that CE seems to be slightly better than MLC in the determination of highly hydrophobic compounds in pharmaceuticals in terms of resolution and economy, taking into account that the limits of detection are not a handicap in pharmaceutical samples.     

Analysis of levofloxacin in pharmaceutical preparations by high performance thin layer chromatography

A new simple, precise, rapid, and selective high‐performance thin layer chromatography (HPTLC) method has been developed for the analysis of levofloxacin in pharmaceutical formulations. The method uses lamotrigine as an internal standard. The stationary phase was silica gel 60F₂₅₄ prewashed with methanol; water‐methanol‐n‐butanol‐ammonia solution 5 + 5 + 5 + 0.4 (v/v) was used as mobile phase. Detection and quantification were performed densitometrically at λ = 298 nm. The linear range of the analysis was 0.8–3.0 μg and the percentage recovery was 99.90%.     

A simple and rapid method for the determination of taxol produced by fungal endophytes from medicinal plants using high performance thin layer chromatography

 Taxol is an important anticancer drug used widely in the clinical field. In this study, some endophytic fungi were isolated from selected medicinal plants, and were screened for their potential in the production of taxol, using a rapid separation technique of high performance thin layer chromatography (HPTLC). Of the 20 screened fungi, only 13 fungal species produced taxol in the artificial culture medium. The results of HPTLC showed that the 13 fungal species had identical ultraviolet (UV) characteristics, positive reactivity with a spray reagent, yielding a blue spot, which turned to dark gray after 24 hours, and had Rf values identical to that of the authentic taxol. The amount of taxol was also quantified by comparing the peak area and the peak height of the fungal samples with those of authentic taxol.     

毒鼠强薄层色谱分析的检出限

建立薄层色谱法快速对毒鼠强进行简易分析的方法,利用薄层色谱进行层析分离,采用特殊显色方法,对毒鼠强进行显色,以能正常观察到斑点的色泽状态,辅助GC-MS方法确定检出极限。薄层色谱法分析毒鼠强检出限可达5~10μg。能用此方法对大多数中毒案件提取的检材进行定性分析。     

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.     

Estimation of the breakthrough volume of selected steroids for C‐18 solid‐phase extraction sorbent using retention data from micro‐thin layer chromatography

SPE method is a very popular technique, and is commonly used for the prepurification, concentration, and isolation of different organic compounds from variable matrices. In this work, the optimization of SPE process was carried out. The breakthrough volume of solid sorbents based on octadecylsilane was determined and three methods were compared: (1) calculation one – the breakthrough volume was calculated using retention factor k determined with micro‐TLC method, frontal analysis – (2) breakthrough volume was determined as volume of whole elution peak, and (3) breakthrough volume was determined as the center of peak gravity. For calculation method, the k values of key estrogens and progestogens were derived from the micro‐TLC experiment reported previously. By combining these three methods, we can point the start of elution, the maximum concentration of analyte in eluate, and the whole eluent volume, which is necessary to achieve an appropriate selectivity and high extraction recovery. Proposed calculation method allows to estimate the beginning of the steroid peak, when the analyte appears in the eluate flowing from the sorbent. Such observation advances the SPE optimization protocol that was described before and was based on the correlation between raw k_SPE and k_micro‐TLC data.     

Liquid phase microextraction‐ion exchange‐high performance thin layer chromatography for the preconcentration,separation, and determination of plasticizers in aqueous samples

Liquid phase microextraction combined with ion‐exchange‐high performance thin layer chromatography has been developed for analysis of four plasticizers in aqueous samples. After their preconcentration by liquid phase microextraction, fast separation on thin layers of inorganic ion‐exchanger stannic silicate has been developed using a mixture of toluene + ethyl acetate (10:1, v/v) as mobile phase. Consequently, densitometric quantitative determination of the plasticizers has been made at λ = 280 nm in reflection–absorption mode by Camag TLC scanner‐3. The effects of type and volume of extraction solvent, stirring rate, extraction time, and ionic strength in the microextraction method have been also evaluated and optimized. The results show that the proposed method provides enhanced accuracy, linear range, LOD, and LOQ, and is very effective for analyzing the target compounds in water samples. Under the optimized conditions, preconcentration factor of 149–279 and extraction efficiency of 31–59% have been obtained. Repeatability (5.67–7.26%) and intermediate precision (6.21–8.17%) were in acceptable range. The relative recovery obtained for each analyte in different water samples was higher than 82.3% at three fortification levels with RSD <7.9%.     

Preliminary results for 2‐D separation with high‐performance thin‐layer chromatography and pressurized planar electrochromatography

Preliminary results of 2‐D separation of test dye mixture using high‐performance thin‐layer chromatography (HPTLC) and pressurized planar electrochromatography (PPEC) are demonstrated. The advantage of 2‐D HPTLC/PPEC separation is based on different separation selectivities obtained in both HPTLC and PPEC systems. HPTLC RP18 W plates of 5×20 cm from Merck were used in the investigations. In the first dimension, a HPTLC process was performed using 5 cm length of the plate and in the second dimension PPEC separation was obtained applying plate of 20 cm length. PPEC process followed prewetting the chromatographic plate with sample zones on it, which were partly separated after first dimensional (HPTLC) separation. In the experiments, the modified version of PPEC device for 20 cm long chromatographic plate and the reservoir for prewetting the adsorbent layer were applied.     

Comparative study on the inclusion behaviour of cyclodextrin derivatives with venoruton and rutin by thin layer chromatography

The interaction of rutin and venoruton (troxerutin), with alpha-, beta- and gamma-cyclodextrin (CD), hydroxypropyl-beta-cyclodextrin (HP-beta-CD) and methyl-beta-cyclodextrin (M-beta-CD) was investigated by reversed-phase thin layer chromatography on polyamide plates. A mobile phase consisted of NH(4)OH; NH(4)Cl buffer solution containing various CD concentrations (pH = 9.7, 20 degrees C) was used as mobile phase. The equilibrium constants (K(f)) and the retention factor (R(f)) were determined and used to study the inclusion process. The in fluence of CDs on the solubility of rutin and venoruton was characterized by R(M) values and the increasing hydrophilicity of drugs. The results show that the inclusion capacity of cyclodextrins follows the order HP-beta-CD > M-beta-CD > beta-CD > gamma-CD, and rutin is more easily included by the studied cyclodextrins than venoruton. In addition, the thermodynamic parameters (Delta H, Delta S) for the formation of complexes were obtained from the van't Hoff equation, displaying the enthalpy-entropy compensation effect.     

Coupling protocol for thin layer chromatography/matrix-assisted laser desorption ionization

Summary This paper reports the investigation and optimization of the thin layer chromatography/matrix-assisted laser desorption/ionization (TLC-MALDI) coupling protocol. The fundamental coupling parameters which influence sensitivity and lateral analyte spreading are extraction solvent selection, extraction time, and pressure. Selection of the solvent is dependent upon its extraction efficiency, which has been correlated with extraction solventR _f value. Maximum extraction efficiency occurs after two minutes for the analyte/solvent system studied. High extraction efficiency solvents cause significant lateral spreading of analyte; up to a three-fold increase in initial analyte spot size. Analyte recovery was found to be limited by the silica gel inter-partice porosity and the solvent extraction efficiency. For maintaining chromatographic resolution and maximizing signal intensity, extraction solvents withR _f values between 0.4 to 0.6 are optimal. The upper analyte recovery limit, using extraction solvents within thisR _f range, is estimated at 22%. Dedicated to Professor Werner Haerdi on the occasion of his 70^th birthday.     

Development of a highly sensitive high‐performance thin‐layer chromatography method for the screening and simultaneous determination of sofosbuvir,daclatasvir, and ledipasvir in their pure forms and their different pharmaceutical formulations

The combination of sofosbuvir and daclatasvir or sofosbuvir and ledipasvir is now widely used as an ideal treatment for hepatitis C virus infection. For this purpose, a simple, sensitive, accurate, economic, and precise high‐performance thin‐layer chromatography was developed and validated for the determination of sofosbuvir, daclatasvir, and ledipasvir in their pure form as well as their different pharmaceutical products. The method used Merck high‐performance thin‐layer chromatography aluminum plates precoated with silica gel 60 F254 as a stationary phase and mobile phase consisting of methylene chloride/methanol/ethyl acetate/ammonia (25%) (6:1:4:1, v/v/v/v). This system was found to give compact symmetric peaks of sofosbuvir, daclatasvir, and ledipasvir with retardation factors of 0.27 ± 0.01, 0.50 ± 0.007, and 0.68 ± 0.008, respectively. The densitometric scanner was set at 275 nm using a deuterium lamp. The calibration curves were linear over the range of 100–3000 ng/spot for sofosbuvir, and daclatasvir, and range of 50–3000 ng/spot for ledipasvir. The detection limits were 22.5, 31.90, and 15.80 for sofosbuvir, daclatasvir, and ledipasvir. The quantitation limits were 67.50, 95.60, and 47.50 for sofosbuvir, daclatasvir, and ledipasvir. The proposed method was validated according to International Conference on Harmonization (ICH) guidelines and the results were acceptable.