A simple, rapid, accurate, and ecofriendly thin-layer chromatography (TLC)–densitometric method was developed and validated for the simultaneous determination of telmisartan (TEL), amlodipine besylate (AML), and indapamide (IND) in their recently approved ternary combination. The method is based on TLC separation of the three drugs followed by densitometric measurements of their bands at 326 nm. The separation was conducted on precoated TLC aluminum sheets of silica gel 60 GF254 using ethyl acetate–hexane–ethanol–ammonia solution (1:1:1:0.4, V/V) as the mobile phase. Calibration curves were linear over the ranges 1–12 μg/band, 0.2–1.8 μg/band, and 0.3–1.1 μg/band for TEL, AML, and IND, respectively. The method was subsequently applied to both lab-prepared mixtures and tablet formulations. Method validation was performed in accordance with the International Council for Harmonisation (ICH) Q2 guidelines, demonstrating excellent linearity, accuracy (100 ± 2
A validated instrumental thin-layer chromatography (TLC) method was developed and applied for the simultaneous quantitation of esomeprazole and naproxen in fixed-dose combination pharmaceutical tablets. The method employed a silica gel plate and a mixture of chloroform–ethyl acetate–methanol–glacial acetic acid (3:7:1:0.2, V/V) as the mobile phase. Samples were applied on plate as 6-mm long bands. The band-to-band distance was 9 mm. The distance between the bands and the bottom of the plate was 8 mm. The applied volume was 7.0 µL/band. The developing distance was 85 mm. After development, the plates were scanned at 302 nm to detect spots and to record chromatograms. The method was proved to be accurate (overall recovery ranges were from 97.7
Standardization of Ayurvedic polyherbal formulations remains a major challenge owing to their complex phytoconstituents composition. Panchasama Churna is a traditional classical polyherbal formulation containing therapeutical important phytoconstituents such as scopoletin from Opericulum turpentum, ellagic acid from Terminalia chebula and other sources, and zingerone from Zingiber officinale. Although various chromatographic methods have been reported for individual phytoconstituents, no validated high-performance thin-layer chromatography (HPTLC) method has been reported for their simultaneous estimation in Panchasama Churna. The present study aimed to develop and validate a simple, rapid, accurate, and cost-effective HPTLC‒densitometric method for the simultaneous quantification of zingerone, ellagic acid, and scopoletin in Panchasama Churna as per the International Council for Harmonisation (ICH) Q2(R2) guidelines. Chromatographic separation was achieved on pre-coated silica gel 60 F254 HPTLC using toluene‒ethyl acetate‒methanol‒formic acid (6:2.7:1.3:0.5, V/V) as the optimized mobile phase. Densitometric scanning was carried out at 254 nm for scopoletin and ellagic acid and at 540 nm for zingerone after derivatization with anisaldehyde–sulphuric acid reagent. The developed method showed well resolved peaks with RF values of 0.35 for ellagic acid, 0.60 for scopoletin, and 0.72 for zingerone. The calibration curve showed excellent linearity within the range of 2000–10,000 ng/band with correlation coefficients (R2) greater than 0.999. Validation parameters including precision, accuracy, robustness, limit of detection and limit of quantification were found within acceptable limits. The validated HPTLC method was found to be simple, precise, reliable and economical for the simultaneous quantification of selected phytoconstituents in Panchasama Churna. This method may be effectively applied for routine quality control and standardization of Ayurvedic herbal formulation.
Standardization plays a crucial role in enhancing the global acceptance of herbal drugs by ensuring their therapeutic effectiveness and safety. The present work aimed to develop a simple and precise high-performance thin-layer chromatography (HPTLC) method for the simultaneous estimation of berberine, gallic acid, ferulic acid, and piperine in Chandraprabha Vati. The separation of phytoconstituents was carried out on silica gel 60 F254 plates via a linear ascending technique using toluene–ethyl acetate–ethanol–formic acid (4.5:3.5:1:1, V/V) as the mobile phase. Densitometric scanning was carried out at 340 nm. The RF values of berberine, gallic acid, ferulic acid, and piperine were found to be 0.2025, 0.4023, 0.5625, and 0.6221, respectively. The proposed HPTLC method was validated as per the International Council for Harmonisation (ICH) guidelines for linearity, accuracy, precision, limit of detection, limit of quantification, robustness, and specificity. The coefficients of determination (R2) of berberine, gallic acid, ferulic acid, and piperine were found to be 0.9910, 0.9964, 0.9950, and 0.9949, respectively. The developed method was successfully applied for the concurrent estimation of berberine, gallic acid, ferulic acid, and piperine in different brands of Chandraprabha Vati.
Currently, green chemistry’s primary criteria are the removal of dangerous compounds and the reduction of their harmful impacts on human health and the environment. Therefore, developing new analytical techniques necessitates taking these factors into consideration. Empagliflozin, linagliptin, and linagliptin impurity 5 ternary mixture were quantified in both pharmaceutical formulations and in their pure forms using two environmentally benign, simple, and reproducible chromatographic methods. The proposed thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) methods enable the concurrent determination of the two active drugs along with a related impurity of linagliptin in a single analysis, an aspect that remains limited in previously reported chromatographic methods. Separation was performed using the proposed TLC method. Silica gel F254 plates were employed as the stationary phase, while a mixture of methanol and acetone (8:2, V/V) was used as the mobile phase for band development. The bands were visualized under an ultraviolet (UV) lamp at 220 nm. The calculated retention factor (RF) values were 0.58 for empagliflozin, 0.25 for linagliptin, and 0.72 for linagliptin impurity 5. Calibration curves were constructed over the concentration ranges of 0.1–4, 0.1–2, and 0.1–1.2 µg/band for empagliflozin, linagliptin, and linagliptin impurity 5, respectively. Additionally, a complementary HPLC method was employed. Separation was achieved using a reversed-phase C18 column (250 mm × 4.6 mm, 5 µm particle size). Isocratic elution was performed using a mobile phase consisting of methanol and water (80:20, V/V). The flow rate was maintained at 1.2 mL/min, and detection was carried out at 220 nm. The mean retention times were 2.67 min for empagliflozin, 4.34 min for linagliptin, and 9.17 min for linagliptin impurity 5. The method exhibited linearity over the concentration ranges of 5–50, 2–30, and 1–15 µg mL−1 for the three components, respectively. Successful application to Empacyrl 10/5® tablets yielded results that were statistically comparable to those obtained from a previously cited procedure.
The potential of pollen from Indian bees as a practical therapeutic agent is still largely unknown. It is crucial to carefully assess, evaluate, and set national standards for the quality of Indian bee pollen because of the variations in chemical composition across different geographic regions. The chemical composition of bee pollen samples varies significantly between countries, and there are no common criteria for assessing the qualities of bee pollen. Therefore, the present study aimed to develop and validate a method for the simultaneous estimation of rutin, rosmarinic acid, kaempferol, and caffeic acid in Indian coconut bee pollen with the help of thin-layer chromatography–densitometric technique. A mixture of toluene–ethyl acetate–methanol–formic acid–water (3:6:1:0.5:0.5, V/V) was used as the mobile phase to accomplish good chromatographic separation using an ultraviolet–visible (UV–Vis) detector at wavelengths of 366 and 328 nm. The retardation factors for rutin, rosmarinic acid, caffeic acid, and kaempferol were set as 0.090, 0.553, 0.610, and 0.683, respectively. A good linear relationship was achieved for rutin, rosmarinic acid, caffeic acid, and kaempferol in the linear regression data for the calibration curves over the concentration ranges of 400–900 (R2 = 0.9979), 1000–4500 (R2 = 0.9995) 500–3000 (R2 = 0.9909), and 1500–4000 ng/band (R2 = 0.9979), respectively. The established method was found to be exact, accurate, and resilient. Following the method’s application to a commercial formulation (Natural Bee Pollen Capsules), the percentage amounts of rosmarinic acid and kaempferol were determined to be 4.13 ± 0.0001 and 0.205 ± 0.001, respectively.
High-performance thin-layer chromatography with multi-detection, comprising chemical and effect-directed detections, has been scaled down. The latest open-source 2LabsToGo-Eco has successfully merged the instrumental capabilities of two laboratories into a miniaturized, all-in-one system. Using it, not only known but also unknown hazardous or beneficial compounds can be discovered in complex samples. The do-it-yourself concept of the open-source 2LabsToGo-Eco affords traceability of the necessary improvements made. Its operation was sped up by integrating the latest operating system, Debian 13, the newest Python web framework, Django 5, and the comparatively faster single-board computer, the Raspberry Pi 5. After successfully resolving the challenges related to the system modifications, the software’s proper functioning was demonstrated and verified through 2LabsToGo-Eco analysis of 13 cannabidiol-containing oils, which showed red zones for cannabinoids detected after derivatization with the Fast Blue B salt reagent. Loading the software tabs and saving the methods was impressively fast.
A simple and reliable high-performance thin-layer chromatography (HPTLC) assay was developed and validated in accordance with the International Council for Harmonisation (ICH) guidelines for the quantification of progesterone in pharmaceutical formulations. The assay was carried out on silica gel 60 F254 HPTLC plates, hexane–ethyl acetate (3:1, V/V) was chosen as mobile phase, and the analysis performed at 255 nm. The method was found to meet ICH requirements for specificity, linearity, sensitivity, precision, accuracy, repeatability, and robustness. Progesterone presented as a sharp band at RF 0.13, and there was good linearity between the 25 and 200 ng/band concentration range (r2 = 0.999). Limit of detection and limit of quantification were calculated to be 7.78 ng and 23.57 ng, respectively; the mean drug recovery was found to be between 100.58
In this study, a straightforward, rapid, and cost-effective preliminary screening method is described for the estimation of melatonin, using xanthydrol as a chromogenic reagent in thin-layer chromatography (TLC). Melatonin is a neurohormone predominantly produced by the pineal gland, which is essential for maintaining circadian rhythm regulation. Even though melatonin is a commonly used sleep aid, the detection of this compound is increasingly important, as it is widely misused in criminal activities. Although melatonin is widely regarded as safe, analytical identification may be required in certain forensic scenarios where its presence in questioned samples needs evaluation. This approach involves the derivatization of melatonin with xanthydrol to generate a stable blue-colored product, facilitating enhanced detection and visualization, employing TLC as an analytical method. The visual detection limit was observed at approximately 0.5 µg/mL. Selected potentially interfering compounds did not exhibit an identical RF–color response under the optimized conditions. The method is intended as a rapid preliminary screening tool and does not replace confirmatory instrumental techniques such as liquid chromatography–tandem mass spectrometry (LC–MS/MS). Limitations regarding selectivity toward other amide-containing compounds are acknowledged.
High-performance thin-layer chromatography (HPTLC) is increasingly recognised as a versatile, low-solvent technique for routine quality control of food and herbal and pharmaceutical products. Recent developments in green analytical chemistry (GAC), blue analytical chemistry (BAC) and white analytical chemistry (WAC) have accelerated the transition of HPTLC from solvent-intensive workflows to sustainability-driven, decision-oriented platforms. The current review describes the basics of HPTLC but in the context of a single GAC–BAC–WAC framework, including the use of solvents that are eco-friendly, miniaturisation and greener methods of sample preparation, including quick, easy, cheap, effective, rugged and safe (QuEChERS), pressurised fluid extraction, dispersive liquid–liquid microextraction, ultrasound-assisted extraction and solid-phase microextraction. Sustainable validation practices, including plate miniaturisation, chamber pre-saturation, automated spotting, binary low-toxicity mobile phases and low-volume derivatisation, are critically discussed with respect to solvent consumption, analytical performance and regulatory robustness. Particular emphasis is placed on contemporary greenness and whiteness assessment tools (Green Analytical Procedure Index [GAPI]; Analytical Greenness Metric [AGREE]; Analytical Eco-Scale; National Environmental Methods Index [NEMI]; red, green, blue (RGB); Greenness Evaluation Metric for Analytical Methods [GEMAM]; and Blue Applicability Grade Index [BAGI]) and their application to HPTLC methods. This review examines software-assisted HPTLC workflows, persistent gaps (limited life-cycle assessments [LCA], trace sensitivity, digital standardisation), and prospects for smart software integration with GAC–BAC–WAC principles.
Niacinamide is widely incorporated into cosmetic formulations owing to its broad spectrum of topical benefits. However, its safety profile is often compromised by the unintentional or deliberate addition of hydroquinone, a potent skin‑lightening agent prohibited in several regions, including the European Union and the Gulf Standardization Organization (GSO). Conventional reversed-phase high-performance liquid chromatography methods frequently fail to achieve sufficient resolution between these two highly polar analytes, increasing the risk of co‑elution and false‑positive detection. This study aims to develop and validate an orthogonal, thin‑layer chromatography (TLC) method for the simultaneous quantification of niacinamide and hydroquinone in complex cosmetic matrices. Chromatographic separation was performed on precoated silica gel 60 F254 plates using a mobile phase of chloroform–ethanol (9:1, V/V). Quantification was achieved by densitometric scanning at 280 nm, complemented by postchromatographic derivatization with anisaldehyde–sulfuric acid (ASA) reagent. Method specificity was confirmed through superimposable reflectance spectra and three-dimensional spectral overlays of standards and commercial cream samples. Distinct separation was obtained with retardation factor (RF) values of 0.22 for niacinamide and 0.27 for hydroquinone. The method exhibited excellent linearity across the tested ranges. Limits of detection (LOD) were determined as 0.45 µg for niacinamide and 3.38 µg for hydroquinone. Recovery values ranged from 98.57 to 102.5
In this study, a thin-layer chromatographic approach was developed for the enantioresolution of three chiral amine drugs, viz., mexiletine, flecainide, and tocainide, using azithromycin (a macrocyclic antibiotic) as a chiral selector. Azithromycin was incorporated into the stationary phase on thin-layer chromatography (TLC) plates to create a stereochemically interactive microenvironment capable of promoting multiple molecular interactions, including hydrogen bonding, π–π stacking, dipole interactions, and steric recognition. The chromatographic conditions were optimized to achieve successful resolution of the racemic analytes. The limit of detection and limit of quantification values were found to be in the range of 0.072–0.108 µg/spot and 0.216–0.321 µg/spot, respectively, for the three analytes. This work demonstrates the successful application of azithromycin-modified TLC plates for the chiral resolution of the studied amine drugs, offering a simple, cost-efficient alternative for enantioseparation of chiral analytes and supporting further application in routine quality control and stereoselective pharmacokinetic studies.
A novel, precise, robust, and stability-indicating instrumental thin-layer chromatography (TLC) method was developed and validated for the quantitative estimation of tegoprazan in its marketed tablet formulation (Ki-CAB, 50 mg). Chromatographic separation was achieved using TLC silica gel 60 F254 glass plates and a mobile phase comprising toluene–ethyl acetate–ethanol–ammonia (70:20:10:0.1, V/V). Detection was performed densitometrically at 224 nm. The optimized method produced a sharp, symmetrical peak for tegoprazan with an RF value of 0.30 ± 0.02. The method was developed in compliance with the International Council for Harmonisation (ICH) Q2(R2) and ICH Q14 guidelines to achieve the desired analytical target profile—ensuring drug quantification with an accuracy of ± 2
Clindamycin (CLIN) is widely used for respiratory and skin infections, particularly in capsule dosage forms. Despite its widespread use, no image-assisted thin-layer chromatography (TLC) method integrating sustainability concepts from Green Analytical Chemistry (GAC), White Analytical Chemistry (WAC), and Click Analytical Chemistry (CAC) has been reported. The objective of this work was to develop and validate an eco-efficient, stability-indicating TLC method for CLIN capsules using digital image analysis, combining sustainability, practical efficiency, and modular analytical design. A silica gel plate, microsyringe, and a mobile phase of purified water–ethanol (3:7, V/V) were used, and spot detection was achieved by derivatization (visualization) with 1.2
In this work, a novel thin-layer chromatography (TLC) method for the simultaneous estimation of two biomarker compounds: ferulic acid (FA) and caffeic acid (CA) in the alcoholic extract of Ipomoea carnea Jacq. is established and validated. The developed method comprised a ternary mobile phase consisting of toluene–ethyl acetate–formic acid (5.5:4.5:0.5, V/V). Densitometric ultraviolet detection was carried out at 320 nm, the absorption maxima of both markers. In compliance with the International Council for Harmonisation (ICH) criteria, the developed method was validated for precision, recovery, robustness, specificity, limit of detection, and limit of quantification. This mobile phase resulted in well resolved bands for CA and FA at RF values of 0.44 and 0.51, respectively. The limit of detection (1.042 and 1.753 ng), limit of quantification (3.160 and 5.310 ng), and recovery (86.05
The increased demand for medicinal plants such as Rheum emodi Wall. Ex Meisn. has intensified exploitation owing to its therapeutic properties. The plant is in high demand, often leading to adulteration or substitution with related species. This study aimed to develop a validated thin-layer chromatography (TLC) method for identification of key variables for precise species authentication and adulteration detection. Samples were initially identified using macroscopic and microscopic analysis, but high microscopic similarity highlights the need for standardization using advanced analytical technique. The ethanolic extracts of the test and standard samples were evaluated using the TLC technique, which was tested for linearity, specificity, precision, and accuracy, in compliance with the International Council for Harmonisation (ICH) standards. The quantitative assessment was carried out using pure emodin as external standard. By comparing the test samples with the reference standard, the chemicals’ identities were verified, which could help in distinguishing the original drug from its adulterants. The mobile phase hexane–ethyl acetate–formic acid (7:2.5:0.5, V/V) helped in the analysis of emodin in R. emodi and its adulterants at 254 nm. The technical data generated clearly showed that the genuine R. emodi sample contained the highest amount of the principal compound emodin, thus guaranteeing the genuineness and quality of R. emodi.
Diabetes mellitus is a widespread chronic disorder commonly associated with poor dietary habits and elevated stress levels. The US Food and Drug Administration (FDA) has recently approved a novel fixed-dose combination therapy containing metformin hydrochloride (MET), sitagliptin phosphate (SITA), and pioglitazone hydrochloride (PIO), providing a new strategy for managing Type 2 diabetes mellitus. This has created a need to establish a precise, sensitive, and validated method for their simultaneous quantification. A literature review revealed that no stability-indicating high-performance thin-layer chromatography (HPTLC) method has been reported for this combination, highlighting the significance of this work. For the separation process, thin-layer chromatography plates coated with silica gel 60 F254 were used as the stationary phase. A mixture of methanol‒toluene‒ammonia‒acetic acid (7:2.5:0.4:0.2, V/V) was used as the mobile phase. Detection was carried out densitometrically at a wavelength of 254 nm. The peaks corresponding to MET, SITA, and PIO were well separated at retention factors (RF) of 0.45, 0.63, and 0.82, respectively. The developed method demonstrated linearity across the concentration ranges of 1500‒4500 ng/band, 300‒900 ng/band, and 45‒135 ng/band with correlation coefficient (r2) of 0.9992, 0.9993, and 0.9993 for MET, SITA, and PIO, respectively. The limits of detection (LOD) were determined to be 420.50, 84.50, and 4.73 ng/band and the limits of quantification (LOQ) were determined to be 893.50, 179.60, and 10.04 ng/band for MET, SITA, and PIO, respectively. The stability study was conducted under acidic, basic, oxidative, neutral, photolytic, and thermal conditions. MET and PIO were found stable in thermal and photolytic conditions, while SITA was stable in neutral condition. The proposed method complies with all International Council for Harmonisation (ICH) guidelines, demonstrating that it is accurate, simple, appropriate, and reliable for determining MET, SITA, and PIO in their combined dosage form.
Phenolics, withanolides, and triterpenoids are prominent bioactive classes found in various adaptogenic botanicals and have potential applications as phytogenic feed additives. In the present study, a robust and reproducible instrumental thin-layer chromatography (TLC) method, coupled with densitometric analysis, was developed and validated for the simultaneous detection and quantification of these phytoconstituents in selected medicinal plant extracts. Method optimization involved selecting suitable mobile phases and derivatization reagents to achieve efficient resolution and reliable detection. Validation was carried out in accordance with the International Council for Harmonisation (ICH) guidelines, assessing parameters including specificity, linearity, precision, accuracy, limit of detection, and limit of quantification. The developed instrumental TLC method was successfully applied to standardize multiple adaptogenic botanical extracts, confirming the presence and concentration range of the targeted compounds. This approach facilitates quality control and ensures consistency in formulations intended for use as phytogenic feed additives. The study underscores the relevance of instrumental TLC as a cost-effective and high-throughput analytical tool for the phytochemical standardization of complex herbal matrices.
Brinzolamide has been quantified in bulk and pharmaceutical dose form using an analytical quality by design (AqbD)-based instrumental thin-layer chromatography (TLC) approach that is sensitive, accurate, precise, and robust. The Box‒Behnken design was applied to the chromatographic conditions in TLC for better efficiency and reliability. The method used TLC aluminum plates precoated with silica gel 60 F254 as the stationary phase and toluene‒acetone‒methanol (6.5:2:1.5, V/V) as the mobile phase. The retardation factor (RF) was recorded as 0.53, indicating the reliability of the chromatographic results. The densitometric analysis was carried out in absorbance mode at a wavelength of 254 nm. The independent variables used in the optimization were mobile phase composition, chamber saturation time, and development distance. The linearity was determined to be between 250 and 1500 ng/band, with a correlation coefficient (R) of 0.9983. The limit of detection (LOD) and limit of quantification (LOQ) were determined to be 50 and 152 ng/band, respectively. The proposed technique was validated in accordance with the International Council for Harmonisation (ICH) guidelines Q2(R1). The proposed approach for forced degradation research was tested on brinzolamide under stress conditions such as acid and base hydrolysis, photolytic, thermal, and oxidative stress degradation to create the stability-indicating method. The statistical study supports the method’s suitability for evaluating brinzolamide in pharmaceutical formulations, with no influence from excipients, as per ICH recommendations.
The fixed-dose combination of nirmatrelvir and ritonavir is a frontline therapy for coronavirus disease 2019 (COVID-19), with ritonavir acting as a pharmacokinetic enhancer for nirmatrelvir. Reliable analytical methods are essential to ensure the stability, safety, and efficacy of this combination. This study reports the development and validation of a novel instrumental thin-layer chromatography (TLC) method for the simultaneous estimation of nirmatrelvir and ritonavir in bulk and combined dosage forms. Chromatographic separation was achieved using silica gel 60 F254 TLC plates with a mobile phase of chloroform–methanol (8:2, V/V), and densitometric detection at 235 nm. The method was validated in accordance with the International Council for Harmonisation (ICH) Q2(R1) guidelines. It demonstrated excellent linearity in the ranges of 300–1800 ng/band for nirmatrelvir and 200–1200 ng/band for ritonavir, with correlation coefficients (R2) above 0.998. Recovery values confirmed high accuracy, while percentage relative standard deviation (
