High-performance Thin-layer Chromatographic Method 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.

Method Development and Validation of HPTLC Method for Simultaneous Estimation of Atropine, Piperine and Vasicine in <i>Ayurvedic</i> Formulation

Background: Dhumayoga, an Ayurvedic formulation is used for the management of respiratory disorders, including dyspnea and asthma. Aim: To develop and validate a High-Performance Thin-Layer Chromatography (HPTLC) method for the simultaneous estimation of atropine, vaccine and piperine in Dhumayoga Ayurvedic formulation. Methods: The three biomarkers were resolved using precoated silica gel 60F254 as stationary phase, dioxane: toluene: methanol: ethyl acetate: Aqueous ammonia (4:7:3:5:1v/v/v/v/v) as mobile phase and detected and quantified at 254nm. Results: The Rf values of atropine, vasicine, and piperine were found to be 0.406 ± 0.02, 0.56 ± 0.02, 0.87 ± 0.02, respectively. The calibration plot was linear in the range of 500-2500ng per zone with a correlation coefficient (r2) of atropine, vasicine and piperine was 0.994, 0.9986 and 0.9935, respectively. Conclusion: The developed method was applied for the quantitation of biomarkers in Dhumayoga. The method was validated in terms of linearity, precision, specificity, accuracy and robustness as per International Council for Harmonisation (ICH) guidelines.

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

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.

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

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.

Development of Amla Nutri-bar: Proximate analysis, gallic acid quantification and its validation through HPTLC, and spectroscopy

BACKGROUND: “Ayush Ahara” confluences advanced food technology with Ayurveda to develop dietary products rooted in traditional medicine. Here, Amla (Indian gooseberry), a medicinal fruit well-known for its health benefits, was used to develop a novel dietary product—Amla Nutri-bar (ANB). Furthermore, this research aims to substantiate its therapeutic potential through quality control analysis. METHODS: Three batches of ANB were prepared at the pharmaceutical laboratory, All India Institute of Ayurveda, New Delhi, India. These were evaluated for sensory attributes using a 9-point hedonic scale and underwent physicochemical, phytochemical, total phenolic content, chromatographic, and spectroscopy assays. RESULTS: The sensory evaluation indicated positive feedback, with an overall acceptability score of 8, followed by taste and flavor rated at 7 Physiochemical constants included pH: 2.2, moisture: 11.48%w/w, total ash: 0.17%w/w, water-soluble extractive value: 88.4%w/w, and alcohol-soluble extractive value: 56.65%w/w. Phytochemical screening revealed that the product is rich in flavonoids, tannins, and alkaloids. Nutritional composition per 100 g had energy: 373.76 kcal, proteins: 1.61%, carbohydrates: 88.59% by wt, dietary fiber: 2.72 g, total fat: 1.44%, vitamin C: 300.11 mg, vitamin A: 71.23 pg, iron: 5.63 mg, calcium: 126.69 mg, and potassium: 103.57 mg. Total phenolic content was 4.95 mg gallic acid equivalents/g, with gallic acid quantified at 270.4 μg/100 mg employing a validated high-performance thin layer chromatography method. Fourier transform infrared spectroscopy analysis showed the presence of alcohol, phenol, and alkanes. All the tested pesticide residues were found below detection limits. CONCLUSION: ANB can be a food product that is, not delicious only but can also contribute significantly to overall health and well-being.

Development and Validation of a High-Performance Thin-Layer Chromatography Method for the Qualitative Analysis and Quantification of Hispidulin in Hydroethanol Extract of S. barbata D. Don

Background: Hispidulin, a flavonoid renowned for its diverse pharmacological activities, is abundant in S. barbata D. Don. Given its prevalence, it is essential to establish a dependable analytical method for quantifying hispidulin in this plant. Methods: This study introduces a sensitive and reliable high-performance thin-layer chromatographic (HPTLC) method for both qualitative and quantitative analysis of hispidulin in S. barbata D. Don. Pre-coated TLC aluminum plates were utilized, and after testing various mobile phase combinations, the most effective separation was achieved with Toluene-ethyl acetate-formic acid (5:4:1, v/v/v). The method underwent extensive validation for linearity, specification, instrument precision, precision, limits of LOD and LOQ, accuracy, and robustness. Results: The developed method demonstrated excellent linearity over a specified concentration range, with a determination coefficient (r2) of 0.988. A well-defined spot at Rf 0.7, corresponding to hispidulin, was observed. The concentration of hispidulin in the hydroethanolic extract of S. barbata D. Don was quantified as 13.02 μg/mg of extract. Conclusion: In conclusion, the HPTLC method presented in this study offers a simple, accurate, and robust tool for quantitatively analyzing hispidulin in the hydroethanol extract of S. barbata D. Don. This method is suitable for routine quality control and standardization of herbal formulations containing S. barbata D. Don, thereby contributing to the advancement of research in natural product- based pharmaceuticals.

Green stability-indicating RP-HPTLC technique for determining croconazole hydrochloride

Abstract The objective of the proposed investigation is the development and validation of a green stability-indicating reverse-phase high-performance thin-layer chromatographic method to determine croconazole hydrochloride (CCZ). The developing system used was an 80:20 v/v mixture of acetone and water. The measurement of CCZ was done at 198 nm. With the use of the Analytical Eco-Scale (AES), ChlorTox, and Analytical GREEnness (AGREE) tools, greenness was evaluated. The linearity was demonstrated by the present method in the 25–1,200 ng/band range. The present approach was additionally reliable, accurate, sensitive, precise, and green. An exceptional greenness profile was demonstrated by the AES, total ChlorTox, and AGREE scales, which were determined to be 89, 1.08 g, and 0.82, respectively. The greenness metrics of the present method were much better than the reported high-performance liquid chromatography approach. Under acid and oxidative degradation circumstances, CCZ was shown to be unstable, while under alkaline and thermal-stress settings, it was sufficiently stable. Furthermore, the stability-indicating component determined by analytical method identified CCZ in the presence of its degradation products. Commercial CCZ cream was found to contain 0.98% w/w of CCZ. The investigation’s results suggested that CCZ in commercially available creams might be regularly examined with the help of the recommended green technology.

Development and validation of High-Performance Thin Layer Chromatographic methods for simultaneous determination of antibacterial pharmaceutical formulations containing Clindamycin phosphate.

The main objective of this research is to establish and authenticate the High-Performance Thin-Layer Chromatographic techniques for the simultaneous assessment of Clindamycin phosphate in combination with Tretinoin (formulation-1) and Clindamycin phosphate with Adapalene (formulation-2), both of which are antimicrobial combinations. In the developed HPTLC methods, for combination-1, the optimized mobile phase was a mixture of n-Hexane: Ethyl acetate: ACN: Methanol (5:2:2:1 by volume) with observed Rf values of 0.413 and 0.787 for Clindamycin phosphate and Tretinoin, respectively. Similarly, for combination-2, the mobile phase comprised n-Hexane: Diethyl ether: Dichloromethane: Acetic acid (4:4:2:0.1 by volume) with recorded Rf values of 0.407 and 0.559 for Clindamycin phosphate and Adapalene, respectively. The developed methods are successfully validated as per the regulatory guidelines. This approach proves to be significantly time-saving and costeffective, rendering it suitable for routine analyses of the formulations containing selected antibacterial active pharmaceutical ingredients and commercial formulations.

HPTLC Densitometric Analysis for Quantification of Sennocide-B and Rhein in Leaf Extracts of Senna alexandrina Mill. and Their Commercial Formulations.

The leaf extract of Senna alexandrina Mill., a plant from the Fabaceae family, contains bioactive anthraquinones sennoside-B and rhein, traditionally used in Indian medicine for various health issues. However, standardizing the quality of S. alexandrina and its commercial products is challenging. To address this, we established two high-performance thin-layer chromatography (HPTLC) methods for accurate analysis of these biomarkers in S. alexandrina leaf extract and its commercial formulations. For sennoside-B, a mobile phase combination of solvent for example, butanol, water, and glacial acetic acid (6:3.5:0.5, v/v/v) was used, with detection at 254 nm, producing spots at an Rf value of 0.37 ± 0.006. The method showed linearity between 100 to 2000 ng/spot (R² = 0.9983 ± 0.0017), with a limit of detection (LoD) of 22.84 ± 0.554 ng/band and a limit of quantitation (LoQ) of 69.22 ± 0.859 ng/band. For rhein, a mobile phase combination of solvent toluene, ethyl acetate, and glacial acetic acid in the ratio (7:2.5:0.5, v/v/v) was utilized and was also detected at 254 nm. The obtained spots had Rf value of 0.67 ± 0.004. The linearity was confirmed within the same range (R² = 0.9985 ± 0.0005), with an LoD of 15.49 ± 0.645 ng/spot and an LoQ of 46.94 ± 1.172 ng/spot. Herein, the methods have followed the guidelines of ICH (Q2) R1. These developed HPTLC methods are selective, simple, sensitive, accurate, and economical, making them suitable for everyday assessment of these bioactive markers in S. alexandrina and its commercial products. This study supports the quality control of S. alexandrina, ensuring product consistency, efficacy, and safety.

Stability-indicating high-performance thin-layer chromatography method development and validation for dapagliflozin propanediol monohydrate, vildagliptin, and metformin hydrochloride in active pharmaceutical ingredient and its tablet dosage form

Stability-indicating high-performance thin-layer chromatography method development and validation for dapagliflozin propanediol monohydrate, vildagliptin, and metformin hydrochloride in active pharmaceutical ingredient and its tablet dosage form

An eco-friendly smartphone based HPTLC method versus conventional densitometric one for determination of Naltrexone and Bupropion

The rapid uprising technologies of smartphone applications and software introduced a new era for analytical detection techniques. It has transformed bench-top laboratory methods into simpler ones depending on cost-effective, portable, and widely accessible devices. In this work, two high performance thin layer chromatographic (HPTLC) methods were developed based on smartphone’s camera detection and either ImageJ desktop software or Color-Picker smartphone’s application as alternative techniques to conventional densitometric detection. A mixture of Naltrexone hydrochloride (NAL) and Bupropion hydrochloride (BUP) was chromatographed on HPTLC- plates using ethyl acetate, methanol, acetone, and glacial acetic acid (3:6:1:0.5, by volume) as a developing system. The developed plates were scanned at 203 nm for the densitometric analysis, then visualized by modified Dragendorff’s reagent and shot by a smartphone’s camera. The captured images were uploaded to either ImageJ software or Color-Picker application to detect the separated spots. The results derived from the three detection methods were compared over the concentration range of 0.4–24 & 0.6–18 µg/band for the densitometric method, 0.4–24 & 2–24 µg/band for ImageJ built method and 0.8–20 & 5–20 µg/band for Color Picker built method for NAL and BUP, respectively. The methods were found to be appropriate for assaying both active drug substances in pure forms and combined in marketed pharmaceutical formulations. The excellent sustainability of densitometric and ImageJ-based methods enabled also the assessment of their dosage form content uniformity. The greenness and sustainability of the methods were assessed by three metric tools, namely Green Analytical Procedure Index (GAPI), Analytical GREEnness Metric Approach (AGREE), and White Analytical Chemistry (WAC). The assessments results confirmed the sustainability and superiority of the proposed methods in terms of sample treatment, waste mount, energy consumption, cost, and number of analyzed samples per an hour.

Development and validation of a method for the simultaneous estimation of terpinene-4-ol and 1,8-cineole in Melaleuca alternifolia (Maiden & Betche) Cheel and Rosmarinus officinalis L. oil using HPTLC coupled with a QbD approach

This paper comprehends a Quality by design (QbD) approach for development of a simple and precise HPTLC (High performance thin layer chromatography) method for simultaneous estimation of the phytoconstituents terpinen-4-ol and 1,8-cineole found in Melaleuca alternifolia (tea tree oil) and Rosmarinus officinalis (rosemary oil) respectively. The method was optimized by Design expert software using response surface methodology. The chromatographic condition optimized by using software was, mobile phase as hexane: ethyl acetate: glacial acetic acid in the ratio of 8.5:1.5:0.47 % v/v, stationary phase as TLC plates precoated with silica gel 60 F254 at ambient temperature conditions and saturation time 20 min at 366 nm. Optimized conditions were verified and validated as per ICH Q2 (R1) (International council for harmonization of technical requirements of pharmaceuticals for human use) guidelines. The method developed was validated for the parameters such as specificity, linearity, DL, QL, accuracy and precision. This method was then applied for simultaneous estimation of terpinen-4-ol and 1,8-cineole in Melaleuca alternifolia (tea Tree oil) and Rosmarinus officinalis (rosemary oil) respectively.

Development and Validation of a High-Performance Thin-Layer Chromatography Method for Simultaneous Qualitative and Quantitative Analysis of Scutellarin and Scutellarein in S. barbata D. Don

Background: S. barbata D. Don, a perennial herb from the Lamiaceae family, is renowned for its medicinal properties, with scutellarin and scutellarein being key bioactive constituents. Aim: In this study, we present the development and validation of a High-Performance Thin- Layer Chromatography (HPTLC) method for the simultaneous quantitative and qualitative analysis of scutellarin and scutellarein in the hydroethanol extract of S. barbata D. Don. Methods: The chromatographic conditions were optimized using different solvent systems, and validation was performed as per ICH guidelines. Results: The mobile phase comprising Ethyl acetate: methanol: formic acid: water (20: 2.7: 0.5: 2) and scanning wavelength set at 254 nm was optimized for study, resulting in distinct and well-separated spots for scutellarin and scutellarein with Rf values of 0.31 and 0.96, respectively. The method was rigorously validated to ensure reliability and reproducibility. Calibration curves exhibited excellent linearity for both scutellarin (r² > 0.98) and scutellarein (r² > 0.99) over a concentration range of 10-30 μg/ml. The developed method underwent thorough validation following ICH guidelines. Validation parameters included accuracy, limit of quantification, limit of detection, linearity, precision, and recovery. The determined concentration of scutellarin and scutellarein in the hydroethanolic extract of S. barbata D. Don was found to be 27.00 μg/mg and 20.16 μg/mg of extract, respectively. Conclusion: This proposed method demonstrates its utility for both qualitative and quantitative analyses of scutellarin and scutellarein in the hydroethanolic extract of S. barbata D. Don, offering a reliable and validated approach for quality control and standardization in the herbal product industry.

Method Development and Validation of HPTLC Method for Simultaneous Estimation of Piperine, Bergapten, Plumbagin and Lupeol in <i>Chitrakadi Vati</i>

Background: Chitrakadi Vati is an Ayurvedic formulation containing several phytoconstituents. A reliable method for simultaneous quantification of its key components is essential for quality control and standardisation. Aim: To develop and validate a High-Performance Thin-Layer Chromatography (HPTLC) method for the simultaneous determination of four key phytoconstituents in Chitrakadi Vati: Piperine, Bergapten, Plumbagin and Lupeol. Methods: An HPTLC method was developed using silica gel 60F254 plates with a mobile phase of Petroleum ether: Ethyl acetate: Toluene: Formic acid (7:2:1:0.1 v/v/v/v). The method was validated for linearity, accuracy, precision, specificity, robustness, Limit of Detection (LOD) and Limit of Quantitation (LOQ). Robustness was further evaluated using a 2³ factorial design, examining the effects of mobile phase volume, saturation time, and solvent front on retention factors. Results: The method demonstrated good linearity (R² = 0.9972-0.9989), precision (%RSD <2%), accuracy (97.68-102.80 % recovery) and specificity. LOD and LOQ ranged from 100.8-325 ng/band and 305.5-985 ng/band, respectively. The factorial design revealed that mobile phase volume and solvent front significantly impacted retention factors, while saturation time had minimal effect. Conclusion: The developed and validated HPTLC method proved effective for the simultaneous estimation of Piperine, Bergapten, Plumbagin and Lupeol in Chitrakadi Vati. The comprehensive validation, including the Design of Experiments for robustness evaluation, ensures the method’s reliability for quality control applications.

Development and validation of stability indicating HPTLC method for estimation of Daclatasvir Dihydrochloride in pharmaceutical dosage form

A high-performance thin-layer chromatographic method was developed and validated for estimation of Daclatasvir Dihydrochloride in pharmaceutical dosage form. The proposed method was applied successfully to the pharmaceutical analysis of the recently approved dosage form of Daclatasvir Dihydrochloride which is available in market as a brand name of ‘NALDAC 60’ tablets. The drugs were satisfactorily show peak with RF 0.38 for Daclatasvir Dihydrochloride. Method was validated according to the ICH guidelines. The calibration plot was linear between 50-300 ng per band for Daclatasvir Dihydrochloride. The LOD and LOQ for Daclatasvir Dihydrochloride were found to be 0.171 μg per band and 0.521 μg per band, respectively. Accuracy and precision of the proposed method was evaluated by recovery studies (% recovery for Daclatasvir Dihydrochloride was 99.88%) and intra-day and inter-day precision studies (standard deviation value for precision studies was found to be 0.453). In stability testing, Daclatasvir Dihydrochloride was found susceptible to acid hydrolysis and alkaline degradation. Because the method could effectively separate the drugs from their degradation products, it can be used as a stability indicating method. The proposed validated stability indicating assay for the sensitive determination of the mentioned drugs is suitable for Quality control laboratories as a simple fast economic method. Degradation product of Daclatasvir Dihydrochloride in alkaline condition was carried out.

Anatomic and Phytochemical Investigation of Herbal Tea Bags Sold as Lemon Balm (Melissa officinalis L.) in the Market

Melissa officinalis L., an ethnobotanically valuable plant, has been used for the treatment of several diseases since ancient times. However, different plants with the same name are sold instead of lemon balm in markets that sell herbal products in Turkey. For this purpose, 15 different brands of tea bags in crushed form, sold as lemon balm (Melissa officinalis L.) in markets, were analyzed. The total phenolic, flavonoid and antioxidant capacities of the hydroalcoholic extracts of these tea samples along with the stomatal structure were investigated. Additionally, chemical compositions and rosmarinic acid contents were determined by the high-performance thin layer chromatography method. Among the samples examined, S1, S2, S9 and S12 tea samples were found to meet the eligibility criteria. The leaves of these samples had diacytic stomata and the rosmarinic acid ratio in their phytochemical composition was over 2%. Furthermore, caffeic acid was detected in these samples.

Rapid mitragynine quantification and fingerprinting of products from Mitragyna speciosa Korth. leaf (Kratom) using high-performance thin-layer chromatography.

Kratom (leaves from Mitragyna speciosa Korth.; Rubiaceae) is a herbal medicine known for its analgesic properties and psychoactive effects. Kratom in Thailand is currently legal; however, it is prohibited in some countries and considered a narcotic plant. Our aim was to establish a reliable, simple, and rapid method for quantifying mitragynine in Kratom leaves and related products through a combination of high-performance thin-layer chromatography (HPTLC) and densitometry. A densitometric HPTLC method was developed and validated in terms of specificity, linearity, limit of detection (LOD), limit of quantification (LOQ), accuracy, precision, and robustness. The fingerprints of kratom leaves, Mitragyna spp., and related products were constructed. For HPTLC, samples were applied to silica gel 60 F254 plates, and the mobile phase comprised n-hexane, ethyl acetate, and triethylamine (1:1:0.15, v/v/v). Densitometric detection was carried out under ultraviolet light at a wavelength of 226 nm. The validated method exhibited a range of 14.31-143.10 μg/mL, yielding a correlation coefficient of 0.9993. Spiked recovery rates were within a range of 98.3%-100.9%, and the LOD and LOQ were 3.80 and 11.53 μg/mL, respectively. Kratom samples were analyzed with the developed method, and the correlation coefficient was 0.9641, compared to the high-performance liquid chromatography-diode-array detection (HPLC-DAD) method. The HPTLC fingerprints displayed a distinctive pattern, facilitating discrimination among different plant parts and Mitragyna spp. The established method offers the advantages of simplicity, ease of use, and speed of analysis, serving as a practical alternative for mitragynine quantification in kratom leaf and its related products.

A validated HPTLC method for quantification of a new combination containing Lidocaine HCl and Diltiazem HCl

Regulators approve newer medications and medication combinations for the treatment of various disorders. For the treatment of anal fissures, a novel combination of currently available medications, such as lidocaine HCl and diltiazem HCl, has been approved. The current work represents high-performance thin layer chromatographic method to estimate the amounts of lidocaine hydrochloride and diltiazem hydrochloride in a combined gel dosage form called Crema-L gel (2% W/W of lidocaine and 2% W/W of diltiazem hydrochloride). This method has been validated. The mobile phase in this method consisted of a mixture of toluene, methanol, ethyl acetate (7:2: 1% v/v/v) and two drops of ammonia. The stationary phase was pre-coated aluminium sheet with silica gel G60 F254 (10 × 10 cm, 0.2 mm layer thickness). The drugs’ resolved peaks were found at the Rf values of 0.59 for Lidocaine HCl and 0.48 for Diltiazem HCl. The overlay UV spectra of the two medications were used to determine the analytical wavelength and at 220 nm, both drugs showed appreciable absorbance. The concentration range of 400–1200 ng/band was determined to be the linear approach for both lidocaine HCl and diltiazem HCl, as indicated by their respective correlation coefficients (r2) of 0.9987 and 0.9980. The technique for simultaneously estimating lidocaine hydrochloride and diltiazem hydrochloride in bulk and pharmaceutical dosage form was shown to be robust, accurate, precise, and repeatable.Graphical