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.
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 (
Morinda tinctoria Roxb. (Rubiaceae) is an important herb traditionally used for the treatment of dyspepsia, stomatitis, diarrhea, fevers, and gout. The present work covers a new, accurate, and precise thin-layer chromatography (TLC)–densitometry method for the quality assurance of five therapeutically important biomarkers, namely, dehydromethoxygaertneroside (1), β-sitosterol-3-O-glucoside (2), 14-deoxy-11,12-didehydroandrographolide (3), rotundioic acid (4), and stigmasterol (5) from the leaves of M. tinctoria. All the five phytoconstituents are highly attributed with a variety of pharmacological activities. The precoated aluminum plates with silica gel 60 F254 (10 cm × 10 cm), using toluene–diethyl ether–methanol–chloroform (5:0.5:1.5:3.5, V/V), were used for separation of markers. The identification of compounds (1–5) was achieved on TLC plate by derivatization using vanillin–sulfuric acid reagent and then heating for 3 min at 125 °C. The concentration range was 0.2–1.0 μg/band with excellent correlation (0.9991–0.9997) for all the selected markers. The limit of detection and limit of quantification of the selected compounds varied between 20–33 ng/band and 66–110 ng/band, respectively. The validated method was precise, having recoveries vary from 93.09
Metformin and evogliptin are antidiabetic medications. Metformin works by lowering glucose production in the liver, delaying the absorption of sugar (glucose) from the intestines and increasing the body’s sensitivity to insulin. Evogliptin works by increasing the release of insulin from the pancreas and decreasing the hormones that raise blood sugar levels. This reduces fasting and post-meal sugar levels. An accurate and precise high-performance thin-layer chromatographic (HPTLC) method has been developed for the estimation of metformin and evogliptin. Aluminum HPTLC plates measuring 10 × 10 cm and 20 × 10 cm precoated with silica gel F254 was used for the chromatographic separation as the stationary phase, and a mixture of chloroform–methanol–ethyl acetate–formic acid (2:5.5:2:0.5, V/V) was used as the mobile phase. The method was linear in the concentration range of 1000–6000 ng/band and 10–60 ng/band of metformin and evogliptin, respectively, with a correlation coefficient of 0.99. The
A high-performance thin-layer chromatography (HPTLC) method was developed and validated for the simultaneous quantification of formoterol fumarate (FF) and glycopyrrolate (GP) in pharmaceutical formulations. The method was developed using stationary phase precoated silica gel HPTLC plates 60 F254 with a mobile phase of toluene–methanol–ethyl acetate–glacial acetic acid (8:2:1:1, V/V). Densitometric scanning was performed at 228 nm using a CAMAG TLC Scanner 4. The retardation factors (RF) were 0.43 for FF and 0.33 for GP, with a resolution of 1.5. Linearity was established over concentration ranges of 400–1400 ng/band for FF and 800–2400 ng/band for GP, with correlation coefficients (r2) of 0.998 and 0.997, respectively. The limit of detection and limit of quantification were 94.85 ng/band and 189.65 ng/band for FF, and 287.35 ng/band and 574 ng/band for GP, respectively. Intraday and interday precision showed percentage relative standard deviation (
This study presents the development and validation of a robust, stability-indicating instrumental thin-layer chromatography (TLC) method for the quantification of itraconazole in tablet dosage form using a quality by design (QbD) approach. Central composite design (CCD) was employed to optimize key chromatographic parameters such as saturation time, solvent front, and mobile phase ratio (toluene–ethyl acetate–formic acid, 60:40:0.5, V/V). The method demonstrated excellent linearity over the range of 1000–5000 ng/band with a correlation coefficient (r2) of 0.9998. Precision studies showed percentage relative standard deviation (
Etizolam is an emerging new psychoactive substance (NPS) with sedative effects similar to benzodiazepines, which is increasingly being detected in illicit markets. In Chile, its recent appearance in seized samples underscores the necessity for straightforward, expeditious, and cost-effective analytical instruments for routine forensic screening. The present study proposes a validated high-performance thin-layer chromatography (HPTLC) method that facilitates rapid identification and quantification of etizolam with minimal sample preparation and reduced solvent consumption. The method exhibited adequate selectivity, linearity, precision, and accuracy, thus fulfilling validation requirements for the intended forensic purpose. Its straightforward implementation and low cost make it especially suitable for routine analysis of seized materials in forensic laboratories with limited resources.
Naloxone hydrochloride, a life-saving opioid antagonist, plays a vital role in emergency overdose management. While numerous high-performance liquid chromatography and ultraviolet methods exist for its estimation, a critical gap remains in the use of high-performance thin-layer chromatography (HPTLC) for its quantitative analysis and degradation profiling. This study focuses on the development and validation of a robust, environmentally friendly HPTLC method for the quantification of naloxone hydrochloride, guided by the principles of quality by design (QbD). The method also encompasses the detection and characterization of degradation products under stress conditions recommended by the International Council for Harmonisation. A Box–Behnken design was applied to optimize critical chromatographic variables. Stress degradation studies were performed under all the conditions. Characterization of the degradation products was carried out using high-resolution mass spectrometry and infrared spectroscopy (IR). The method’s environmental sustainability and practical utility were assessed using Analytical GREEnness Metric, Green Analytical Procedure Index, and Blue Applicability Index assessment tools. The method exhibited excellent linearity (250–1500 ng/band, R2 = 0.9986), high precision, and robust recovery (98.93–100.97
