Stability-indicating High-performance Thin-layer Chromatographic Method Research Articles

A novel stability indicating HPTLC method for comparative quantitative estimation of quercetin in herbal preparations

This research introduces a novel stability-indicating HPTLC (High-Performance Thin-Layer Chromatography) method for the comparative quantitative estimation of Quercetin in herbal preparations. Through extensive stability studies, the method is validated, providing a robust framework for evaluating Quercetin's stability in diverse herbal formulations. The developed method ensures accurate and reliable quantification of Quercetin, facilitating comparative analysis across various herbal preparations. Additionally, results reveal that Quercetin degraded under most tested conditions, such as acidic, alkaline, oxidative and neutral conditions underscoring the necessity for intervention by experts to eliminate such challenges and ensure product efficacy and quality in the herbal industry.

DEVELOPMENT AND VALIDATION OF STABILITY-INDICATING HIGH-PERFORMANCE THIN-LAYER CHROMATOGRAPHIC METHOD FOR BUDESONIDE

Objective: A stability-indicating high-performance thin-layer chromatographic method has been developed for budesonide. Methods: Chromatographic separation was achieved on aluminum plates pre-coated with silica gel 60 F254 as the stationary phase using ethyl acetate: toluene (7:3) v/v as the mobile phase. The densitometric evaluation was carried out at 246 nm. The developed method of stability-indicating was validated as per the ICH Q2 (R1) guidelines. Stress degradation studies such as hydrolysis under different pH conditions, photolytic degradation, thermal degradation, and oxidative degradation were performed as per ICH Q1A (R2) and Q1B guidelines. Results: The Rf value of budesonide was found to be 0.48±0.03. The response in terms of peak area was linear over the concentration range of 500–2500 ng/band, with the regression coefficient value greater than 0.99. The LOD and LOQ were 28.04 ng/band and 84.96 ng/band, respectively. Conclusion: This method can conveniently be used for quantitative analysis of budesonide on a routine basis.

Stability-Indicating HPTLC Method for Determination of Remogliflozin Etabonate and Vildagliptin in Tablets.

A sensitive, selective, precise and stability-indicating high-performance thin-layer chromatographic (HPTLC) method is developed for the simultaneous estimation of remogliflozin etabonate and vildagliptin in the presence of their degradation products. The separation was performed in thin layer chromatography plate precoated with silica gel G60F254. The mobile phase consists of methanol:ethyl acetate:toluene:ammonia (1.5:4:4.5:0.1, v/v/v/v). Detection and quantification are performed with densitometer at 212nm. The Rf values of remogliflozin etabonate and vildagliptin are 0.53 and 0.42, respectively. Degradation studies for the two drugs were carried out in acidic, alkaline, neutral, oxidative, photolytic and thermal stress conditions and analyzed. The suitability of this method for the quantitative determination of the compounds is proved by validation in accordance with the requirements laid down by International Conference on Harmonization (ICH). Linearity was found over the concentration range of 200-1000ng/band with correlation coefficient of 0.9936 for remogliflozin etabonate and 100-500ng/band with correlation coefficient of 0.9912 for vildagliptin. The method was successively applied to tablets containing two drugs and found to have no chromatographic interferences from the tablet excipients. The degradant formed was not interfering with estimation of two drugs makes the method more selective for the purpose intended.

Development and Validation of a Stability-Indicating Greener HPTLC Method for the Estimation of Flufenamic Acid

The literature on ecofriendly/greener high-performance thin-layer chromatographic (HPTLC) methods for quantifying flufenamic acid (FFA) is scant. In order to develop and validate a stability-indicating greener HPTLC densitometry assay for FFA determination in marketed products, this research was conducted. The ecofriendly eluent system was composed of ethanol–water (70:30 v/v). FFA was measured at 290 nm of wavelength. The greenness scale of suggested analytical assay was derived using “Analytical GREENness (AGREE)” methodology. The suggested stability-indicating HPTLC assay was linear for FFA determination in 25–1400 ng/band range with a determination coefficient of 0.9974. The suggested analytical assay for FFA analysis was simple, rapid, accurate, precise, robust, selective, stability-indicating, and greener. The AGREE scale for the developed stability-indicating HPTLC assay was derived to be 0.77 utilizing AGREE methodology, indicating an outstanding greenness characteristic of the suggested densitometry technique. The ecofriendly HPTLC technique was able to detect FFA degradation product under forced degradation studies, indicating its stability-indication characteristics and selectivity. The amount of FFA in marketed tablets brand A and B was determined to be 101.28 and 99.17%, respectively, indicating the suitability of the suggested analytical technique in the assay of FFA in marketed products. These results indicated that FFA in marketed products may be routinely measured using the stability-indicating greener HPTLC technique.

Development of a new validated stability-indicating high-performance thin-layer chromatography method for determination of dosulepin HCl in bulk and marketed formulation with characterization of its degradants by liquid chromatography–Mass spectrometry

Aim: Dosulepin hydrochloride is a tricyclic antidepressant. It acts by inhibition of the reuptake of biogenic amine and increasing available neurotransmitter levels at the synaptic cleft. The objective of this work is to develop a new validated stability-indicating high-performance thin-layer chromatography (HPTLC) method for the determination of dosulepin hydrochloric acid (HCl) in bulk and marketed formulation and characterization of the degradation products by liquid chromatography–mass spectrometry (LC–MS). Materials and Methods: The HPTLC method development of dosulepin HCl was performed on aluminum plates precoated with silica gel 60 F254 using dichloromethane: toulene: methanol: glacial acetic acid (GAA) (4:4:2:0.2 v/v) with densitometric detection at 220 nm. The method validation was done using linearity, precision, accuracy, and robustness parameter. Results: Linearity range for dosulepin HCl was found in between 150 and 900 ng/band correlation coefficient was 0.996. The % relative standard deviation for accuracy and precision was found <2%. The developed and validated HPTLC method is simple, accurate, precise, and specific. Conclusion: This study concludes that the dosulepin HCl undergoes degradation to different extent under different stress conditions. From the peak purity profile studies, it was confirmed that the peak of the degradation product was not interfering with the peak of drugs. Major acidic and oxidation degradation products were isolated and characterized by LC–MS and probable degradation pathway for dosulepin HCl was determined.

Simultaneous ultra-sensitive analysis of tamsulosin hydrochloride and tolterodine tartrate binary mixture in their dosage form via high-performance thin-layer chromatography with fluorimetric detection

A simple, sensitive, selective, and stability-indicating high-performance thin-layer chromatography method with fluorescence detection was developed for the analysis of a mixture of tamsulosin hydrochloride and tolterodine tartrate, used in the management of benign prostatic hyperplasia in their laboratory-prepared binary mixture and dosage form. The separation was employed on thin-layer chromatography silica gel 60 aluminum sheets. As developing system, ethyl acetate–n-hexane–diethylamine (9:3:1, V/V) was used. Tamsulosin and tolterodine had retention factors (RF) of 0.40 ± 0.03 and 0.85 ± 0.03, respectively. A 225 nm excitation wavelength was used for the fluorescence detection. Linearity was in the range of 10.0–200.0 and 100.0–900.0 ng/band for tamsulosin and tolterodine, respectively. The method was validated successfully according to the International Council for Harmonisation guidelines. Our suggested method for determining tamsulosin and tolterodine in their dosage forms and in the presence of their degradation products could be conveniently applied in quality-control laboratories.

A rapid, sensitive, and greener stability-indicating normal-phase HPTLC method with univariate calibration for the estimation of chlorhexidine acetate in its commercial formulations

Till date, there is no report on high-performance thin-layer chromatography (HPTLC) analysis of chlorhexidine (CHD) or its salts like CHD acetate (CHD-A), CHD gluconate, and CHD hydrochloride. Therefore, a rapid, sensitive, and greener normal-phase HPTLC method has been reported for the analysis of CHD-A in its four different commercial dosage forms. The quaternary combination of ethyl acetate: ethanol: water: formic acid (75:10:10:5, v v v v−1) was optimized as the green solvent system/mobile phase for CHD-A analysis. The CHD-A detection was carried out at 264 nm. The greener normal-phase HPTLC assay was linear in the range of 10–2000 ng band−1. In addition, the proposed method was accurate, precise, sensitive, and selective for CHD-A analysis. The greener normal-phase HPTLC method was able to detect CHD-A in the presence of its degradation products, suggesting the stability-indicating property of this method. The content of CHD-A in commercial products A, B, C, and D was detected as 0.18, 2.02, 1.46, and 0.19% w w−1, respectively. The greenness scale for the greener normal-phase HPTLC assay was estimated as 0.88 utilizing “analytical GREEnness (AGREE)” calculator, suggested the greener nature of the normal-phase HPTLC assay. Overall, these data suggested that the greener normal-phase HPTLC method can be successfully used for the determination of CHD-A in its commercial products.

Study of the degradation behavior of dapagliflozin propanediol monohydrate and metformin hydrochloride by a stability-indicating high-performance thin-layer chromatographic method

Study of the degradation behavior of dapagliflozin propanediol monohydrate and metformin hydrochloride by a stability-indicating high-performance thin-layer chromatographic method

Stability-indicating high-performance thin-layer chromatography method for the simultaneous estimation of emtricitabine and tenofovir alafenamide fumarate

Stability-indicating high-performance thin-layer chromatography method for the simultaneous estimation of emtricitabine and tenofovir alafenamide fumarate

Stability indicating thin-layer chromatographic method for estimation of antidiabetic drug Remogliflozin etabonate

BackgroundA sensitive, precise, and stability-indicating high-performance thin-layer chromatographic (HPTLC) method has been developed for the analysis of Remogliflozin etabonate in tablet formulation. HPTLC plates precoated with silica gel 60 F254 were used as the stationary phase; methanol: ethyl acetate: toluene: NH3 (2:4:4:0.1, v/v/v) was used as mobile phase, and densitometry was used for the quantitative estimation of the drug. The proposed method was validated with respect to linearity, accuracy, precision, and robustness and applied for the estimation of drug in tablet dosage form.ResultsThe Rf value of Remogliflozin etabonate was observed to be 0.61. The densitometric estimation was performed in reflectance mode at 229 nm. The method was found to be linear in the range of 500–8000 ng/band for Remogliflozin etabonate. The possible degradation pathway was estimated by performing forced degradation studies. The degradant peaks were well resolved from the drug peak with acceptable resolution in their Rf value.ConclusionAn accurate and precise high-performance thin-layer chromatographic method has been developed for the quantification of Remogliflozin etabonate in tablets. Forced degradation studies were performed, and drug was found to be highly susceptible to acid, base hydrolysis, and oxidative stress degradation and gets converted into active drug Remogliflozin. Both Remogliflozin etabonate and Remogliflozin bands were well resolved. The method was applied for the analysis of drug in tablet formulation, and it can be used for routine quality control analysis, as well as for the analysis of stability samples.

DEVELOPMENT AND VALIDATION OF STABILITY-INDICATING SIMULTANEOUS ESTIMATION OF METFORMIN AND ALOGLIPTIN IN TABLETS BY HIGH-PERFORMANCE THIN LAYER CHROMATOGRAPHY

Objective: A simple and stability-indicating high-performance thin-layer chromatographic method was developed and validated for the simultaneous estimation of metformin and alogliptin in tablets.&#x0D; Methods: The method was developed in TLC aluminum plates pre-coated with silica gel 60F254 as the stationary phase and the solvent system consists of methanol: chloroform: 0.5% ammonium sulphate [4:4:2, v/v/v]. The system was found to conferred a compact spot for metformin [Rf value of 0.44±0.02] and alogliptin [Rf value of 0.66 ± 0.22]. Densitometric analysis of metformin and alogliptin was carried out at the wavelength of 254 nm. Forced degradation studies were conducted to know the stability of the drug samples under various stress conditions like acid, base, peroxide, photolytic degradation according to the ICH guidelines.&#x0D; Results: The developed method was found to be suitable for the excellent separation of the drug samples. Calibration curves were linear in the range of 40-200 ng/spot with a correlation coefficient of 0.996 for metformin and calibration curves were linear in the range of 1-5 ng/spot with a correlation co-efficient of 0.997 for alogliptin, respectively. Stability study shows that the chromatograms of samples degraded with acid, base, hydrogen peroxide, dry heat, and photolytic showed well-separated spots of pure metformin and alogliptin as well as some additional peaks at different Rf values. The method was successively applied to pharmaceutical formulation. No chromatographic interference from the tablet excipients was found.&#x0D; Conclusion: The newly developed method can be applied for the identification and quantitative determination of metformin and alogliptin in the combined dosage form.

A Rapid and reliable thin-layer chromatographic method for the simultaneous estimation of celecoxib and diacerein in their binary mixture using nanosilica gel plate

A novel, rapid, and sensitive stability-indicating high-performance thin-layer chromatography method was developed and validated for the simultaneous determination of celecoxib (CEL) and diacerein (DIA) in bulk and a capsule binary mixture. The complete separation was achieved by the proposed method on nanosilica gel F254 plates using n-hexane‒ethyl acetate‒tetrahydrofuran‒glacial acetic acid (7:3:1:0.8, V/V) as the mobile phase. The chromatographic bands were visualized using short-wave ultraviolet light, and the amount of CEL and DIA was determined by scanning densitometry at 254 nm using the peak area. The RF values were 0.59 and 0.37 for CEL and DIA, respectively. From regression plots, it was found that good linearity was achieved in the concentration ranges of 20.00–320.00 ng/band and 5.00–80.00 ng/band with mean percentage recoveries 99.86 ± 0.70 and 99.64 ± 0.96 for CEL and DIA, respectively. The proposed method was validated according to the International Conference on Harmonisation guidelines. The statistical analysis of the results revealed high accuracy and precision, the suggested procedures could be used as a stability-indicating assay for the simultaneous determination of CEL and DIA in bulk drug and their capsule binary mixture in the presence of their degradation products.

Validated stability-indicating HPTLC method for the estimation of adapalene in drugs and the LC–MS identification of its degradation products

A highly sensitive, simple, accurate, and precise high-performance thin-layer chromatographic (HPTLC) method was developed and validated for the quantitative determination of adapalene in the presence of its degradation products. The method employed precoated silica gel G 60 F254 on aluminum sheets using an appropriate solvent system which gives a compact spot of adapalene at RF value 0.58. The densitometric measurement of adapalene bands was carried out at 317 nm in fluorescence mode. The method was validated over a range of 10–100 ng/band. The linear regression data for the calibration plot showed a good relationship with high correlation coefficients. The performance of the method was validated for precision, accuracy, and robustness. The limits of detection and quantification were 1.52 and 4.61 ng/band, respectively. Adapalene was subjected to the ICH-prescribed acidic, alkaline, oxidative, photolytic, and thermal stress conditions, and it undergoes degradation with well-resolved degradation products. These degradation products were further analyzed by mass spectrometry to elucidate the structure of degradation products and proposed the prediction of the degradation pathway.

Validation and eco-scale assessment of stability-indicating HPTLC method for quantitative analysis of carbamazepine and its degradation product, iminostilbene, in pure forms, pharmaceutical preparations, and spiked human plasma

Paying attention to issue of environmental protection is an unavoidable duty which encourages analytical society to balance between establishment of new analytical method and preservation of the environment from the harmful chemicals. By adopting this concept, an excellent green high-performance thin-layer chromatography (HPTLC) method was developed for assessment of carbamazepine, the antiepileptic drug, and iminostilbene, the main degradation product and its official impurity. Separation of the structurally related components was completed on HPTLC sheets using a mixture of petroleum ether: acetone (7:3, v/v) as a developing system with UV detection at 230 nm. To assess the greenness profile of the developed method compared with the official high-performance liquid chromatography (HPLC) method, analytical semi-quantitative eco-scale assessment protocol was applied. The suggested method was confirmed to be greener than the official method with regard to solvent consumption and waste emission. The suggested method was successfully applied to determine carbamazepine in the marketed dosage forms with no intrusion of other co-formulated ingredients. Successful application of the developed method was also proved via simultaneous analysis of carbamazepine and iminostilbene in raw materials and human plasma samples in ranges of 0.1–1.4 μg/band and 0.1–1.2 μg/band for carbamazepine and iminostilbene, respectively. As the maximum plasma concentration of the studied medication was within its calibration range, the proposed HPTLC method can be employed for further pharmacokinetic studies. For comprehensive validation of the presented method, ICH figures of merit were evaluated.

A Validated Stability-Indicating HPTLC Assay for Determination of 10-Hydroxy-2-Decenoic Acid Content in Royal Jelly Products Using Robust Regression Methods.

A new, simple, stability-indicating high-performance thin-layer chromatography method was developed for the quantification of 10-hydroxy-2-decenoic acid (10-HDA) in some royal jelly products marketed in Egypt. The used solvent system was chloroform:acetic acid (10:1, v/v) and the bands were measured densitometrically at 210nm. First- and second-derivative treatments of the data were performed. The present study shows a comparison between three statistical regression methods for handling data: parametric, nonparametric and weighted regression (WR) methods. The developed methods were validated as per International Conference on Harmonization guidelines. To validate the stability-indicating power of the developed analytical method, the royal jelly standard was subjected to forced degradation studies including the effect of hydrolysis, oxidation, photolysis and dry heat. It was found that derivative treatment of the chromatographic response data gives improved quantitation and sensitivity of the chromatographic signals. Weighted regression of the response data is found to be advantageous over the use of both parametric and nonparametric regression models. This was shown by a great enhancement in the accuracy and precision in the analysis of 10-HDA in royal jelly products. The % recovery in case of WR was 99.92±0.16, while % recovery in case of nonparametric and parametric regressions were 99.56±0.25 and 98.63±0.65, respectively.

Novel Stability-Indicating High-Performance Thin Layer Chromatography (HPTLC) Method Development and Validation for Estimation of Daclatasvir Dihydrochloride in Pharmaceutical Dosage Form

Daclatasvir dihydrochloride (DTDH) is a direct-acting antiviral agent used against the hepatitis C Virus. Different analytical methods were reported for the determination of DTDH in pharmaceutical dosage form, including spectrophotometry, high-performance liquid chromatography (HPLC), and stability- indicating HPLC method but to date, no HPTLC method has been reported. A novel, simple, sensitive, selective, precise, and accurate stability-indicating high-performance thin-layer chromatographic (HPTLC) method has been developed for the quantification of Daclatasvir dihydrochloride (DTDH) in pharmaceutical formulation. Chromatography separation was carried out on pre-coated silica gel G60 F254 HPTLC plates using chloroform: methanol (9:1, v/v) as the mobile phase. Scanning and densitometric analysis were done at 316 nm. The band of DTDH were obtain at RF= 0.48±0.02. The calibration curve was plotted in the concentration range of 400– 900 ng/band and the method was found linear with r = 0.995. The LOD and LOQ were found to be 95 ng/band and 313.5 ng/band respectively. The percentage recovery was found to be 97.31-101.81 %. The developed was able to separate peaks of all the degradation products formed in ICH-prescribed stress conditions with a significant difference in their RF values. The developed method was validated according to ICH guidelines and results were found to be within acceptance criteria. The developed stability-indicating method successfully evaluated the DTDH in pharmaceutical tablet dosage form and assay results showed good recovery when statistically compared with the reversed-phase high-performance liquid chromatography (RP-HPLC) method.

Design of Experiment Approach for the Development and Validation of a Stability-Indicating High-Performance Thin-Layer Chromatography Method for the Estimation of Metformin Hydrochloride and Ursodeoxycholic Acid in Pharmaceutical Dosage Form

In the present study, a design of experiment (DoE) approach was used to optimize chromatographic conditions for the development of a high-performance thin-layer chromatography (HPTLC) method for the simultaneous estimation of metformin hydrochloride (MET) and ursodeoxycholic acid (URSO) in pharmaceutical dosage form. The critical factors were identified using a Taguchi design, and after identification of critical factors, optimization was done using Box–Behnken design (BBD). BBD was used to optimize the compositional parameters and to evaluate the main effect, interaction effects, and quadratic effects of the mobile phase compositions, development distance, and saturation time on the retardation factor (RF) of both drugs. HPTLC separation was performed on aluminum plates pre-coated with silica gel 60 F254 as the stationary phase, using toluene-ethanol-acetone-formic acid (4.5:2:2.5:0.85, V/V) as the mobile phase at a wavelength of 234 nm and 700 nm for MET and URSO, respectively. A sharp and well-resolved peak was obtained for MET and URSO at RF values of 0.19 and 0.80 min, respectively. The calibration curve was in the range of 5000–40000 ng per spot and 1500–12000 ng per spot for MET and URSO, with r2 = 0.984 and r2 = 0.980, respectively. The method was validated for linearity, accuracy, precision, limit of detection, limit of quantification, and specificity. To provide a better visualization of the statistically significant factors derived from the statistical analysis, the perturbation plot and response surface plot for the effect of independent variables on the RF of MET and URSO were evaluated. Stability study was performed under different stress conditions such as acid and alkali hydrolysis, oxidation, and temperature. The developed method was able to resolve drugs and their degradation products formed under the afore-mentioned conditions.

Stability-Indicating High-Performance Thin-Layer Chromatographic Method for the Simultaneous Determination of Quercetin and Resveratrol in the Lipid-Based Nanoformulation

Summary A simple, sensitive, specific, rapid, and accurate high-performance thin-layer chromatographic (HPTLC) method has been developed and validated for the simultaneous estimation of quercetin (...

STABILITY-INDICATING HIGH-PERFORMANCE THIN-LAYER CHROMATOGRAPHY METHOD FOR SIMULTANEOUS ESTIMATION OF FORMOTEROL FUMARATE DIHYDRATE AND FLUTICASONE PROPIONATE IN BULK DRUG AND PHARMACEUTICAL DOSAGE FORM

Objective: The objective of the present work was to develop validated stability-indicating high-performance thin-layer chromatographic method for simultaneous estimation of formoterol fumarate dihydrate (FFD) and fluticasone propionate (FP) in bulk drug and pharmaceutical dosage form.&#x0D; Methods: Pre-coated silica gel aluminum plates 60 F-254 were used as stationary phase. The mixture of toluene:ethyl acetate:formic acid (98%) (6:4:0.1; v/v/v) was used as a mobile phase. The densitometric quantification was carried out at 233 nm. The method was validated according to the ICH guidelines. The specificity and stability indicating the capability of the method were proven though degradation studies. Both drugs were subjected to acid (0.1N HCl) and base (0.1N NaOH) hydrolysis, oxidation (3% v/v H2O2), photolytic, and neutral degradation conditions.&#x0D; Results: The selected mobile phase resolved peaks of FFD and FP with Rf values 0.27±0.10 and 0.64±0.10, respectively. Determination coefficients of calibration curves were found to be 0.998 and 0.999 in the range of 1–3.5 μg/spot and 10–60 μg/spot for FFD and FP with an accuracy of 99.09% for FFD and 99.20% for FP. The degradation products of FFD and FP were resolved from the pure drug with significant differences in their retention factor values.&#x0D; Conclusion: The developed method is simple, accurate and can be successfully applied for quantification of FFD and FP in bulk drug and pharmaceutical dosage form, contributing to improve the quality control and assure the therapeutic efficacy.

Stability indicating high-performance thin layer chromatography method for estimation of ascorbic acid in Hibiscus sabdariffa L. aqueous extract

Background: High performance thin layer chromatography (HPTLC) has become a routine analysis technique due to its advantages of low operating cost, high sample throughput and need minimum sample clean up. Aim: To develop and validate an accurate, specific, repeatable and stability-indicating HPTLC method for the determination of ascorbic acid in presence of their degradation products for assessment of stability of the ascorbic acid in Hibiscus sabdariffa L. preparations. Methods: Chromatographic sample separations employed thin layer chromatography plates (20 cm x10 cm) pre-coated with silica gel 60 F254 as a stationary phase. Solvent system consisted of acetone: toluene: water: glacial acetic acid (26:4:4:1 v/v) while scanning wavelength of 269 nm was used. Ascorbic acid was subjected to stress degradation conditions i.e. oxidation, dry heat treatment, photo degradation and hydrolysis under different PH. Method was validated according to international council for harmonisation guidelines. Results: Peaks of products formed were well resolved with different Rf values. The linear regression data for the calibration plots showed good linear relationship with r2 = 0.9912 in the concentration range 2-4 µg/ml. The mean value slope and intercept were -531.72 and 8040.22 respectively. Conclusion: A rapid, simple, accurate and specific HPTLC method for determination of ascorbic acid in Hibiscus sabdariffa L. preparations was developed and validated. In addition, this method separated all the degradants from stress conditions showing that it is a stability indicating method. The developed method could now be used in quality control of H. sabdariffa herb products.