HPTLC Research Articles

Advanced HPTLC Method Development for Silibinin Analysis in Nanoformulated Scaffolds: A Box-Behnken Approach.

Silibinin (silybin), a bioactive component derived from the seeds of milk thistle (Silybum marianum), is recognized for its diverse pharmacological properties, including antioxidant, anti-inflammatory, and hepatoprotective effects. Given its therapeutic significance, accurately quantifying silybin in various formulations is essential. High-performance thin-layer chromatography (HPTLC) is a powerful analytical technique frequently used for this purpose. In this study, an HPTLC method was validated according to the International Council for Harmonization (ICH) guidelines to determine the concentration of silybin. The design of experiments (DoE), specifically the Box-Behnken design, was employed to optimize and understand the influence of critical method variables. The HPTLC method validation was performed using silica gel F254 HPTLC plates. The variables investigated included the composition of the mobile phase (% v/v), saturation time (minutes), and temperature in degree Celsius (°C), with the Box-Behnken design for optimization. The mobile phase consisted of chloroform, acetone, and formic acid in a 7:2:1 (v/v) ratio. Both the formulated scaffold and standard drug were applied to the plates, which were then processed in a twin chamber. After development, the plates were scanned at 288 nm using the Camag TLC Scanner IV with Vision CATS software. The validated HPTLC method demonstrated a strong linear relationship within the silybin concentration range of 2-10 μg/mL. The limit of detection (LOD) and limit of quantification (LOQ) for silybin were determined to be 0.469 and 1.423 μg/mL, respectively. Recovery studies indicated that the method provided accurate quantification, with recovery rates ranging from 97.53% to 99.82%. These results confirm the method's high accuracy, outstanding linearity, and reliability for the quantification of silybin in formulations. The validated HPTLC method proved to be a reliable analytical tool for the quantification of silybin in various formulations, particularly those containing polymers. The method's strong linearity, precision, and accuracy align with the ICH guidelines, making it suitable for routine analysis in quality control laboratories. The use of the Box-Behnken design for method optimization highlights the importance of systematic experimentation in achieving robust analytical outcomes.

Sustainable and efficient monitoring of tryptophan and tyrosine serum levels: a green HPTLC method as a biomarker for type 2 diabetes

In recent years, there has been considerable interest in using amino acids like tryptophan (Trp) and tyrosine (Tyr) as biomarkers for various diseases, including type 2 diabetes mellitus (T2D). In diseases like T2D, the metabolism of Trp and Tyr is altered. The activity of enzymes involved in Trp metabolism increases, leading to a decrease in its serum level. On the other hand, the serum level of Tyr increases due to the suppressed activity of its metabolizing enzymes. These observations suggest that Trp and Tyr metabolism may play a crucial role in the pathophysiology of type 2 diabetes. Our study highlights the potential utility of Trp and Tyr as biomarkers for the early detection, prognosis, and monitoring of this metabolic disorder. Given these observations, we aimed to develop a high-performance thin-layer chromatographic (HPTLC) method that is sensitive, selective, rapid, and environmentally friendly for estimating the concentrations of Trp and Tyr in biological fluids, particularly serum samples. To evaluate the method, we performed analysis using serum samples from controlled and streptozotocin-induced diabetic rats. Our main objective was to develop a method that is sensitive and selective for precisely determining Trp and Tyr serum levels, which could serve as potential biomarkers for T2D. Fluorescence and absorption modes were employed for densitometry scanning. We assessed the precision and high separation efficiency of the chromatographic system by calculating parameters such as separation and resolution factors, number of theoretical plates, and height equivalent to theoretical plates. To evaluate the environmental impact of our proposed method, we employed the AGREE (Analytical GREEnness metric) and GAPI (Green Analytical Procedure Index) greenness assessment tools. The results confirmed that our method is environmentally friendly and exhibits superior eco-friendliness and greenness compared to other reported methods.Graphical

High-Performance Thin Layer Chromatography (HPTLC) Analysis for the Quantification of Constituents in Ficus religiosa

High-Performance Thin Layer Chromatography (HPTLC) Analysis for the Quantification of Constituents in Ficus religiosa

Production of phenolic compounds, flavonoids, and lupeol by Lycium schweinfurthii suspension cultures in shake flasks and a rocking-motion bioreactor

AbstractLycium schweinfurthii, a wild shrub of the Solanaceae family, has received increasing attention in the last decade for its therapeutic potential in traditional medicine due to its diverse array of secondary metabolites, including phenolic substances and terpenoids. The aim of this study was to investigate the accumulation of phenolics, flavonoids, and the terpenoid lupeol in L. schweinfurthii cell suspension shake flask cultures and a single-use 2-dimensional rocking motion bioreactor. Three different media formulations were compared for in vitro cell cultures. Various parameters, such as biomass accumulation, settled cell volume, cell viability (assessed via a 2,3,5-triphenyl tetrazolium chloride assay), and sucrose consumption were determined as indicators of cell activity and growth. Total phenolic and flavonoid contents were estimated spectrophotometrically, lupeol was quantified via High-Performance Thin Layer Chromatography (HPTLC). Although a higher fresh biomass concentration of 464 g L− 1 was obtained in MS medium supplemented with a combination of each, 1 mg L− 1 of 2,4-Dichlorophenoxyacetic acid (2,4-D) and 1-Naphthaleneacetic acid (NAA), the rocking-motion bioreactor cultivation was performed with 2 mg L− 1 NAA due to its superior reproducibility in viability, productivity, and content of bioactive compounds (e.g., phenolics, flavonoids, lupeol). A final fresh biomass concentration of 185 g L− 1 was achieved in a 16 L cultivation scale with a notable increase in the concentration of phenolics (1.4-fold) and flavonoids (1.7-fold). Most importantly, the concentration of lupeol, a pentacyclic triterpenoid known for its anti-inflammatory, antibacterial, and anti-atherogenic properties, exhibited a remarkable 5.5-fold increase in the bioreactor cultivation (585 µg g− 1) compared to shake flask cultivations (106 µg g− 1). The current study demonstrated the profound impact of media composition and non-limited fed-batch conditions in a rocking-motion bioreactor on the accumulation of bioactive compounds. The findings are also relevant to other plant cell cultures.

A high-performance thin-layer chromatography densitometric method for the separation of isomeric ceftriaxone in powder for injection formulation

The aim of this study was to develop and validate a High-Performance Thin Layer Chromatographic (HPTLC) method for simultaneous determination of ceftriaxone and ceftriaxone e-isomer in powder for injection formulation. Ceftriaxone sodium injection is an antibiotic that used globally. It has Z/E geometrical conformation, in which ceftriaxone sodium and 3 ene-isomer have Z- conformation while (E)-isomer has E- conformation and the potential toxicity of ceftriaxone (E)-isomer has been reported. Thus, to safeguard the public health, a simple and easy to use, rapid and reliable method was developed for qualitative and quantitative determination of ceftriaxone sodium and its (E)–isomer. Samples were applied on HPTLC glass plates precoated with silica gel 60F254 by using Linomat semi-auto sampler. Separation was carried out using acetone, triethyl amine, water, chloroform and ethyl acetate as a mobile phase in different ratios. The Rf values of separated compounds were 0.51 ± 0.01 and 0.62 ± 0.01 for ceftriaxone sodium and ceftriaxone (E)-isomer respectively. The method was validated by studying Specificity, Linearity, Accuracy, Precision, Robustness, Limit of Detection (LOD) and Limit of Quantification (LOQ) and Solution stability. The developed method was successfully, sensitive, simple, precise, accurate, robust and applicable for the simultaneous determination of ceftriaxone sodium and ceftriaxone (E)-isomer in powder for injection formulation.

Investigating Flavonoids by HPTLC Analysis Using Aluminium Chloride as Derivatization Reagent.

This is the first study to report on high performance thin layer chromatography (HPTLC) generated spectrophotometric data to systematically capture flavonoid compounds using optimized derivatization with either AlCl3 or NaNO2-AlCl3-NaOH as visualisation reagents. While the traditional AlCl3 colorimetric method using UV-Vis analysis provides valuable insights into the presence of flavonoids and allows derivation of the total flavonoid content (TFC) of a sample, HPTLC fingerprints obtained after spraying with AlCl3 or NaNO2-AlCl3-NaOH enable the visualization of the various flavonoids present in a sample based on their respective absorption shifts, thus complementing the traditional TFC assay. In this study, 40 different flavonoids representing different classes (flavonols, flavanolols, flavan-3-ol, flavones, flavanones, and isoflavonoids) were analysed. Upon derivatization with AlCl3 most of the investigated flavonoids recorded bathochromic shifts, yielding characteristic λmax values between 370 and 420 nm, while spraying with NaNO2-AlCl3-NaOH triggered hyperchromic shifts, and thus an increase in absorbance intensity in flavonoids with particular substitution patterns. A few non-flavonoid components with structural similarities to flavonoids (e.g., rosmarinic acid, gallic acid, aspirin, salicylic acid) served as the negative control in this study to determine whether the derivatization reagents allowed exclusive detection of flavonoids. The method was then applied to the analysis of flavonoid containing supplements as well as red clover honey to demonstrate the method's application in the analysis of natural products.

Preparation and evaluation of soft gelatin capsules loaded with astaxanthin, niacin and garlic oil for removal of plague in atherosclerosis

Atherosclerosis, a chronic inflammatory disease, is the leading cause of cardiovascular diseases. In atherosclerosis, the arterial walls are characterized by plaque deposits on the inside of arteries. This study focuses on preparing a fill formulation to remove plaque in atherosclerosis, then analyse the pharmaceutical ingredients used and later produce soft gelatin capsules. Soft gelatin capsules are hermetically sealed, one-piece capsule which has a suspension or semi-solid as fill. Oxidative stress and inflammation are factors responsible for the development of atherosclerosis. Hence, the active pharmaceutical ingredients (APIs) chosen for fill formulation need to possess effective anti-oxidant and anti-inflammatory properties. A novel combination of APIs: Astaxanthin, Niacin and Garlic oil were chosen for the removal of plaque in atherosclerosis. Each of these APIs is known to possess a specific function in the control of plaque development. The fill formulation prepared was analysed for its recovery using various analytical techniques like UV spectrophotometry, high-performance liquid chromatography (HPLC) and thin-layer chromatography (TLC). This fill formulation was encapsulated within the gelatin shell prepared to produce soft gelatin capsules. The encapsulated soft gelatin capsules were evaluated based on various parameters which include average net weight, disintegration test, loss on drying and dissolution.

Research work of Prof. De-Eknamkul, including the mechanistic synergy of hair growth promotion by the Avicennia marina extract and its active constituent (avicequinone C) in dermal papilla cells isolated from androgenic alopecia patients

Androgenetic alopecia (AGA) is a genetically-predetermined disorder that affects many people around the world, leading to progressive hair loss. This baldness can negatively impact people’s perceptions of themselves and scientists are searching for a cure. There are only two US Food and Drug Administration-approved drugs used to treat AGA and they both have side effects. If a natural anti-AGA product could be developed with the ability to be effective minus negative side effects, this could be a game-changer for people with AGA. Professor Wanchai De-Eknamkul, Department of Pharmacognosy and Pharmaceutical Botany, Chulalongkorn University, Thailand, an expert in plant natural product biotechnology, is working to develop a natural anti-AGA product. He is a professor at the University’s Natural Product Biotechnology Research Unit, where research is underway to: elucidate the biosynthesis of bioactive natural products by searching for enzymes and genes involved in their biosynthetic pathways; discover bioactive natural compounds from medicinal plants by either enzyme-based or cell-based screening followed by studying their mechanism of actions; and develop high-performance thin-layer chromatography (HPTLC) as a tool for quick screening and identification of bioactive compounds. De-Eknamkul have been working to shed light on the genes and enzymes involved in the biosynthesis of the diterpenoid ‘plaunotol’ in Croton stellatopilosus. In another line of investigation, they are searching for natural compounds with anti-AGA and anti-grey hair activities and a further area of focus is the development of a technique for direct detection of compounds with anti-diabetes, anti-gonorrhoea and anti-AGA activities.

Stability-indicating high-performance thin-layer chromatography method development and validation for the simultaneous estimation of azithromycin, fluconazole, and secnidazole in active pharmaceutical ingredient and its tablet dosage form

Stability-indicating high-performance thin-layer chromatography method development and validation for the simultaneous estimation of azithromycin, fluconazole, and secnidazole in active pharmaceutical ingredient and its tablet dosage form

Phytochemistry of Petroselinum crispum (Mill.) Fuss, Murraya koenigii (L.) Spreng. and Cinnamomum tamala (Buch.-Ham.) T. Nees & C. H. Eberm. and in silico studies of the role of their bioactive components against cancer

Phytochemical profiling using reliable equipment and validated methods helps us know the medicinal value of the plants. Since natural compounds have fewer side effects, they can serve as replacements for synthetic drugs that are being used in the treatment of challenging chronic diseases like cancer. The present study focuses on the bioactive phytochemical profiling of Petroselinum crispum (Mill.) Fuss, Murraya koenigii (L.) Spreng., and Cinnamomum tamala (Buch.-Ham.) T. Nees & C. H. Eberm. and in silico studies to check the anticancer potential of their bioactive components. In thin-layer chromatography (TLC) plate analysis, it was found that the methanolic extracts of plants contained the maximum number of components. Gas chromatography–mass spectrometry (GC-MS) analysis of the methanolic extracts of plants showed the presence of 22 bioactive components. High-performance thin-layer chromatography (HPTLC) analysis of the extracts of these plants showed the important chromatographic peak, apigenin. In silico studies showed the binding efficacy of selected bioactive components observed in the analysis of plant extracts. Amongst them, apigenin was found to be most effective at binding to the receptors of targeted cancer cells, viz., hepatocellular carcinoma, lung, and breast cancer. After the analysis of the study, it was arrived at the conclusion that the plants, viz., P. crispum, M. koenigii, and C. tamala, possess various bioactive components, and some of these components have anticancer potential. Therefore, in vivo and in vitro studies should be essentially conducted for the development of cancer-preventive drugs.

Greenness and whiteness appraisal for bioanalysis of quetiapine, levodopa and carbidopa in spiked human plasma by high performance thin layer chromatography.

A sustainable HPTLC-densitometric method was developed for quantitative determination of Quetiapine (QUET), Levodopa (LD) and Carbidopa (CD) in presence of Dopamine (DOP) as an internal standard. This applicable technique was achieved by spiking human plasma and extraction was performed using the protein precipitation approach. The mobile phase used was acetone, dichloromethane, n-butanol, glacial acetic acid and water (3: 2.5: 2: 2: 1.75, by volume). Method validation was done according to US-FDA guidelines and was able to quantify Quetiapine, Levodopa and Carbidopa in the ranges of 100-4000, 200-8000 and 30-1300 ng/mL, respectively. Bioanalytical method validation parameters were assessed for the studied drugs. Finally, the analytical suggested methodology was evaluated using various green and white analytical chemistry metrics and other tools, such as the green solvent selection tool, analytical eco-scale, green analytical procedure index, analytical greenness metric approach and the red-green-blue algorithm tool. The results revealed that the applied analytical method had a minor impact on the environment and is a relatively greener option than other previously reported chromatographic methods.

Analysis of quality parameters and preservative concentrations in Sahacharadi Kwatha: A comparative study of three commercial brands

BackgroundSahacharadi Kwatha is traditionally employed in Ayurvedic therapy for "vata" related conditions such as back pain, herniated disc, palsy, sciatica, and paralysis. Classical Ayurvedic texts recommend the use of freshly prepared Kwatha for optimal patient benefits. However, in response to the commercialization of Ayurveda and the demand for convenient over-the-counter (OTC) formulations, various commercial preservatives have been incorporated by Ayurvedic manufacturers to facilitate OTC preparation and prolong shelf life. ObjectivesThis study aims to comprehensively analyse and compare the quality parameters and preservative content in three prominent brands of Sahacharadi Kwatha available in the Indian market. Materials and methodsOrganoleptic and physicochemical properties, phytochemical content, and microbial load of the samples were analyzed following standardized procedures. Sodium benzoate levels in the samples were determined using both titrimetric and High-Performance Liquid Chromatography (HPLC) methods. High-Performance Thin-Layer Chromatography (HPTLC) profiles were compared to discern differences among the samples. ResultsThe study revealed significant variations in organoleptic and physicochemical properties, HPTLC profiles, and microbial load among the tested samples. Sodium benzoate levels in all samples exceeded the FDA and API-approved limit. Additionally, substantial variations were noted in the phytochemical content of the samples. ConclusionThis investigation underscores noteworthy disparities in quality parameters and preservative content within the tested market variants of Sahacharadi Kwatha. The findings emphasize the existence of unregulated standards in the preparation of Ayurvedic medicines available in the market, highlighting the imperative for standardization and validation of Ayurvedic formulations. Such measures are essential for enhancing consumer acceptability and fostering the overall development and growth of the Ayurveda industry.

Determining Some Chemical and Microbiological Changes in the Ripening Process of Kashar Cheese

In the present study, some microbial, chemical, and physicochemical characteristics occurring during ripening were observed in unpackaged and vacuum-packed Kashar cheese samples. Some microbial and chemical properties of Kashar cheese samples were studied Also, the free fatty acid ratio was determined with the SDS-Page Electrophoresis to determine proteolysis during the ripening period and with the High-Performance Thin Layer Chromatography (HPTLC) for lipolysis. Physical, chemical, and microbiological analyzes were made at 0, 15, 30, 45, 60, and 75 days of ripening during which the changes in the number of microbiologically total aerobic mesophilic bacteria (TAMB), Lactobacillus spp., Lactococcus spp., Pseudomonas spp., yeast-mold, lipolytic, and proteolytic bacteria were determined. In the present study, % lactic acid, pH, dry matter percentage, color parameters (L, a and b values) and water activity (aw) were analyzed during ripening and the changes during storage were defined. Lactobacillus spp., Lactococcus spp., TAMB, and proteolytic bacteria counts and % lactic acid ratios were higher in vacuum-packed Kashar cheeses. It was found that lipolysis and proteolysis were higher in cheese samples stored open during ripening.

Recent applications of high-performance thin-layer chromatography (HPTLC) in forensic drug analysis

Recent applications of high-performance thin-layer chromatography (HPTLC) in forensic drug analysis

Isolation, Development and Validation of Chromatographic Methods for the Estimation of Linoleic Acid from Different Parts of Euphorbia Neriifolia Linn.

Objectives: This is the first report on the development and validation of thin-layer chromatography (TLC) and high-performance thin-layer chromatography (HPTLC)-densitometric methods for the identification of linoleic acid (LA) in petroleum ether extract (PEE) of Euphorbia neriifolia (EN) stem (ST), latex (LX), and bark (BA). Methodology: Chromatographic analyses were performed on silica gel-G and silica gel 60 F254 plates and the antioxidant activities of isolated compounds were investigated by 2,2-diphenyl-1- picrylhydrazyl (DPPH) spectrophotometric assay. Results: The chromatographic analyses revealed better spots and well-separated peaks of LA with retention factor (Rf) values at 0.54 (ST), 0.40 (LX), and 0.64 (BA), respectively. The linearity of the calibration curve ranges from 10-50 ng/spot (ST), 10-100 ng/spot (LX), and 50-200 ng/spot (BA). The proposed method was characterized by better accuracy, better robustness, and good precision, ranging from 0.173 to 0.372% (intra-day) and 0.185 to 0.205% (inter-day). The value of the limit of detection and quantification equal to 1.04 and 3.16 ng/spot in ST, 0.87 and 2.64 ng/spot in LX, and 0.177 and 0.53 ng/spot in BA determined the sensitivity of the method. In the obtained chromatogram, no peak was observed other than the LA which determined the specificity of the method. The % RSD of < 2% after periods of 12, 24, 36, 48, and 72 h determined the stability of standard LA. Conclusion: Thus, the fingerprinting method is valuable in determining the adulterants and in routine quality control of formulations and herbal drugs.

Eco-friendly High Performance Thin Layer Chromatography Technique for Quantification of Kojic Acid in Bulk and Formulations

Eco-friendly High Performance Thin Layer Chromatography Technique for Quantification of Kojic Acid in Bulk and Formulations

Stability Indicating HPTLC Method for Estimation of Cubebin in Crude Drug and Marketed Formulations

Background: Cubebin is a naturally occurring lactone lignan isolated from the plant species Piper cubeba. It exhibits a range of biological activities, including anti-inflammatory, antimicrobial, and anticancer properties. This compound has garnered significant interest for its potential therapeutic applications in traditional and modern medicine. Aim: This study aimed to develop a novel stability-indicating High-Performance Thin Layer Chromatography (HPTLC) technique that is simple yet precise in estimating cubebin in crude drug and marketed formulations. Method: Cubebin was estimated using precoated TLC silica gel 60 F254 plate as a stationary phase and n-hexane: ethyl acetate: formic acid: methanol (6:3.5:0.5:0.3 v/v/v/v) as a mobile phase with the chamber saturation time of 20 mins. Key Findings: Cubebin was detected and quantified at the wavelength of 283nm. The Rf value of cubebin in standard solution, Piper cubeba extract, Dhanwantari Vati, and Khadiradi Vati was found to be 0.494±0.0015, 0.492±0.002, 0.490±0.0020, 0.499±0.001 respectively. The calibration curve exhibited linearity within the range of 3000-13000 ng per band with a correlation coefficient (r2) of Cubebin was 0.9968. The method developed was utilized for the quantitation of cubebin in Piper cubeba and both the marketed formulations. Conclusion: A novel HPTLC method was developed and validated in accordance to ICH guidelines for the analysis of cubebin in crude drug and marketed formulations. The method was found to be simple, precise, accurate, and specific. This method is suitable for quality control and stability testing of cubebin-containing formulations.

A Review on Advancement in Analytical Quality by Design (AQbD)

Analytical Quality by Design (AQbD) represents a transformative methodology in pharmaceutical development, anchored in a systematic, risk-based, and data-driven framework. This approach optimizes analytical methods, fostering heightened product quality, efficient regulatory compliance, and informed decision-making. The industry's increasing acceptance of AQbD principles signifies a paradigm shift towards enhanced efficiency, sustainability, and global harmonization. This review comprehensively explores AQbD principles, regulatory perspectives, and its applications, particularly in analytical method development, including high-performance liquid chromatography (HPLC) and high-performance thin-layer chromatography (HPTLC). Emphasis is placed on the symbiotic relationship between AQbD and analytical method validation (AMV), elucidating their collective role in ensuring reliable and accurate analytical results. Integrating AQbD in method transfer, automation, and control strategies underscores its pivotal role in achieving robust, efficient, and compliant analytical processes. The review delves into lifecycle management and continuous improvement, coupled with AQbD principles, ensuring sustained method reliability throughout the pharmaceutical product lifecycle. AQbD's significant contribution to pharmaceutical lifecycle management, optimization, and change control is explored, emphasizing its systematic, data-driven, and risk-based approach to method development, validation, and ongoing enhancement. This review illuminates AQbD's transformative impact on pharmaceutical analysis, aligning with industry trends toward quality, efficiency, and regulatory compliance.

Determination of acute sertraline intoxication by high-performance thin-layer chromatography

Determination of acute sertraline intoxication by high-performance thin-layer chromatography

High-performance thin-layer chromatography and high-performance liquid chromatography determination of two anthocyanins in medicine mulberry

High-performance thin-layer chromatography and high-performance liquid chromatography determination of two anthocyanins in medicine mulberry