High-performance Thin-layer Chromatographic Method Research Articles

Development of HPTLC method for simultaneous determination of quercetin and kaempferol in leaf extract of Hibiscus mutabilis

The aim of this study was to develop and validate a densitometric High-Performance Thin-Layer Chromatography (HPTLC) method for the simultaneous quantification of quercetin (Q) and kaempferol (K) in Hibiscus mutabilis leaf extracts. The analyses were performed on silica gel 60 F254 plates using a mobile phase composed of toluene, formic acid, and ethyl acetate (6:0.4:4, v/v/v). Detection was carried out at a wavelength of 272 nm using a deuterium and tungsten light source. The method exhibited excellent linearity over the concentration range of 100–600 ng/spot for quercetin and 500–3000 ng/spot for kaempferol, with determination coefficients (r2) of 0.9989 and 0.9973, respectively. The method showed no interferences from the plant matrix. The relative standard deviation (RSD) values for intra- and inter-day precision were less than 2% for both flavonoids. Recovery rates ranged from 97.69% to 99.20% for quercetin and from 89.91% to 95.87% for kaempferol. The limits of detection (LOD) were 190.23 ng/spot for quercetin and 187.23 ng/spot for kaempferol, while the limits of quantification (LOQ) were 570.10 ng/spot for quercetin and 566.12 ng/spot for kaempferol. This validated HPTLC method is reliable, precise, and accurate, making it suitable for the quality control of Hibiscus mutabilis leaf extracts. The study’s findings can be broadly applied to the quality control of herbal products, pharmacological research, and the development of nutraceuticals. The method’s ability to provide rapid and accurate quantification makes it an invaluable tool for researchers across various disciplines.

Comprehensive phytochemical profiling of Phyllanthus emblica L. flowers on UHPLC/MS quadrupole time of flight, HPTLC, HPLC, and NMR analytical platforms reveals functional metabolites with potent anti-inflammatory effects in human (THP-1) macrophages.

Phyllanthus emblica L., renowned for its pharmacological benefits found in its fruits and leaves, has received considerable attention. However, there is a notable lack of research on its flowers, specifically on metabolite profiling and pharmacological activity. The present study aims to delineate the phytochemical constituents of hydromethanolic extract of P.emblica flowers by ultra-high-performance liquid chromatography coupled with quadrupole time of flight mass spectrometry (UHPLC-QToF-MS), high-performance thin layer chromatography (HPTLC), high-performance liquid chromatography (HPLC), infrared and nuclear magnetic resonance spectroscopic methods and subsequent evaluation of its anti-inflammatory potential. The identification and characterization of phytochemicals in P.emblica flowers was performed by UHPLC/MS-QToF in both positive and negative ionization modes. Additionally, marker compounds present in flower extract were analyzed using HPTLC, HPLC, FT-IR, and NMR methods. The anti-inflammatory potential was evaluated in lipopolysaccharide-stimulated THP-1 macrophages by evaluating inflammatory biomarkers. UHPLC/MS-QToF analysis facilitated the identification of 51 compounds from P.emblica flowers including gallic acid derivatives, flavonoid glycosides, and tannins based on their fragmentation patterns and previous literature reports. Notably, the study also identified spermidine compounds for the first time in this species. Optimization of HPTLC and HPLC methods marked the presence of corilagin as major compound followed by FT-IR and NMR spectral methods. Moreover, treatment with hydromethanolic extract of P.emblica flowers resulted in decreased levels of proinflammatory cytokines, TNF-α, IL-1β, and IL-6, alongside modulation of nuclear factor-κB activity in lipopolysaccharide-induced THP-1 macrophages. Chromatographic techniques in conjunction with spectral methods found robust prevalence in the identification of signature phytometabolites present in P. emblica flowers, which sets the basis for its anti-inflammatory potentials. The studies established a foundation for further exploration of potential applications of P. emblica flowers across various domains.

High Performance Thin Layer Chromatography (HPTLC) Analysis of Anti-Asthmatic Combination Therapy in Pharmaceutical Formulation: Assessment of the Method's Greenness and Blueness.

A cost-effective, selective, sensitive, and operational TLC-densitometric approach has been adapted for the concurrent assay of Hydroxyzine Hydrochloride (HYX), Ephedrine Hydrochloride (EPH), and Theophylline (THP) in their pure powder and pharmaceutical forms. In the innovative TLC-densitometric approach, HYX, EPH, and THP were efficaciously separated and quantified on a 60F254 silica gel stationary phase with chloroform-ammonium acetate buffer (9.5:0.5, v/v) adjusted to pH 6.5 using ammonia solution as a mobile liquid system and UV detection at 220 nm. The novel TLC method validation has been performed in line with the international conference for harmonization (ICH) standards and has been effectively used for the estimation of the researched medicines in their pharmaceutical formulations without intervention from excipients. Additionally, parameters affecting the chromatographic analysis have been investigated. The new TLC approach's functionality and greenness were appraised using three modern and automated tools, namely the Blue Applicability Grade Index (BAGI), the Analytical Greenness metric (AGREE), and the Green Analytical Procedure Index (GAPI) tools. In short, the greenness characteristics were not achieved as a result of using mandatory, non-ecofriendly solvents such as ammonia and chloroform. On the contrary, the applicability and usefulness of the novel TLC approach were attained via concurrent estimation for the three drugs using simple and straightforward procedures. Moreover, the novel TLC method outperforms previously published HPLC ones in terms of the short run time per sample and moderate pH value for the liquid system. According to the conclusions of comparisons with previously recorded TLC methods, our novel HPTLC method has the highest AGREE score, so it is the greenest HPTLC strategy. Moreover, its functionality and applicability are very appropriate because of the simultaneous assessment of three drugs in one TLC run. Furthermore, no tedious and complicated extraction and evaporation processes are prerequisites.

Extraction, Isolation and Characterization of Bioactive Compounds from Euphorbia neriifolia (L.) Leaf and Evaluation of their Antioxidant Activity

Background: Amidst the identification of numerous secondary chemicals from Euphorbia neriifolia, there is a desperate need for the development of primary metabolite separation techniques. Objectives: In order to do that, bioactive chemicals from Euphorbia neriifolia (L.) leaf were extracted, isolated, and characterized. Subsequently, their antioxidant activity was evaluated. Methods: In this study, the determination of linoleic acid (LA) in petroleum ether extract (PEE) of Euphorbia neriifolia leaf (ENL) was carried out the first time by using thin-layer chromatography (TLC) and high-performance thin-layer chromatography (HPTLC) methods. Results: The chromatographic analysis of the PEE of ENL shows better spots and well-separated peak of LA with 0.49 retention factor (Rf) value and 22.54 ng LA content. The linearity of the calibration curve ranges from 5-25 ng/spot with a high correlation coefficient. The proposed method was characterized by better accuracy close to 99.5%, well robustness, and good precision range from 0.183% (intra-day) to 0.242% (inter-day). The percentage (%) RSD, which determined the stability of standard LA, did not exceed 2% after time period of 12, 24, 36, 48, and 72 h. The GC-MS analysis revealed the presence of different types of low or high-molecular-weight phytocompounds of varying quantities from the fractions of ENL. The FT-IR spectrum of ICs showed various peaks that confirmed the presence of C=C bending, C-H stretching, O-H stretching, CH2 stretching, and a carboxyl group. The 1H-NMR spectrum of the ICs from ENL confirmed the presence of octadecanoic in IC1, L-(+)-ascorbic acid dihexadecanoate in IC2, hexadecanoic acid in IC3, linoleic acid in IC4, and oleic acid in IC5, respectively. IC-4 showed greater antioxidant activity in comparison to other compounds with an IC50 value of 3.9 ± 0.01 μg mL-1. Conclusion: Thus, the present study identified five different phytocompounds that may be utilized as an effective option for the cure of different diseases.

Method development and validation for quantification of Yohimbine in root of Rauwolfia serpentina by HPTLC

Plants are a priceless and vital resource that give us medicine, food, fibre, and all other necessities. The evergreen shrub, Rauwolfia serpentina (L.) Benth. ex Kurz., also known as Indian snakeroot, sarpagandha is a member of the Apocynaceae family. Rauwolfia serpentina has fewer negative effects and an abundance of phytochemicals in the roots and leaves of the plant, it shows a wide range of therapeutic activity. With the goal of developing a densitometric high-performance thin-layer chromatography (HPTLC) method, in order to quantify Yohimbine hydrochloride in Rauwolfia roots, it will help to insure the collection and harvesting time. The five distinct brands of market Rauwolfia root samples are compared with in-house samples prepared in the laboratory. The silica gel 60 F254 high-performance thin-layer chromatography plates were used with toluene, Toluene : Ethyl acetate : Diethylamine (7:1.5:1.5 v/v/v) as a mobile phase. The validation parameters for the developed method were carried out as per the International Conference on Harmonisation (ICH) guideline Q2 (R1). The calibration curve was found to be linear over a range of 1-7 μg/spot for yohimbine. The method involves densitometric detection at 280 nm. The relative standard deviations (RSD) for precision were found to be lower than 2% for both analytes. Percent recoveries for accuracy were performed and found to be 98.5-99.98% for yohimbine, The LOD and LOQ for Yohimbine were 0.08 μg/ml and 0.24 μg/ml, respectively. Validation parameters were carried out as per International Conference on Harmonisation (ICH) guideline Q2 (R1). The Rf value of 0.64 have been observed for Yohimbine. This HPTLC densitometric method is highly suitable for the analysis of Yohimbine present in the dosage of marketed herbal products of sarpagandha root without any interference.

Synchronized Assessment of Lobeglitazone Sulfate and Metformin Hydrochloride in Tablet by Robust, High-performance Thin-layer Chromatographic Method

Background: A combination of fixed-doses containing 0.5 mg lobeglitazone sulfate and 500 mg metformin hydrochloride has demonstrated efficacy in enhancing glycemic control in diabetes. Aims: The projected work aimed to establish and validate a high-performance thin-layer chromatographic methodology for the quantification of both drugs in tablet formulations. Objectives: The task involves creating and validating a method in accordance with ICH guidelines to quantify two particular drugs in tablet formulations accurately. Methods: The high-performance thin-layer chromatographic analysis utilized aluminum plates layered with silica gel 60F254, and the solvent system consisted of acetonitrile, 1 M ammonium acetate (methanol), toluene, and triethyl amine (1.5:2.5:4:0.2 v/v/v/v), followed by densitometric scanning at 237 nm. Results: The methodology exhibited linearity in the range of 100-1500 ng/band for lobeglitazone sulfate and 1000-15000 ng/band for metformin hydrochloride, with correlation coefficients of 0.9991 and 0.9992, correspondingly. Exceptional sensitivity was observed, with detection limits of 8.17 ng/band for lobeglitazone sulfate and 271.34 ng/band for metformin hydrochloride, along with quantification limits of 24.75 ng/band for lobeglitazone sulfate and 822.24 ng/band for metformin hydrochloride. The method demonstrated precision (% relative standard deviation of peak area <2) and accuracy (recovery between 96 and 103%). Conclusion: The suggested methodology is fit for the concurrent quantification of both drugs in tablet formulations, making it applicable for routine quality control assessments in laboratories.

Development and Validation of HPTLC for the Determination of Gefitinib HCl in Tablet Dosage Form

A simple, high-performance thin-layer chromatography (HPTLC) method of gefitinib HCl in pharmaceutical formulation was developed. Separation was done on TLC aluminum plates precoated by silica gel 60GF254 (20 × 20 cm) with 200 μm layer depth. With UV detection set to 254nm, chromatography was conceded out by the mobile phase of dichloromethane and methanol (9:1, v/v). Linearity was found to range of 50 to 300 ng/spot and, the correlation coefficient was found to be 0.99 and the Rf value was found to be 0.46. The system’s suitability was considered to ascertain the performance of ascertaining quality performance of the developed chromatographic method. The method’s accuracy, precision, and specificity were all verified against the International Council of Harmonization (ICH) standards. Application of the approach to the determination of gefitinib in tablet form was successful.

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

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

Development and Validation of High-Performance Thin Layer Chromatographic Method for Estimation of Acyclovir

Development and Validation of High-Performance Thin Layer Chromatographic Method for Estimation of Acyclovir

Enhancing simultaneous determination of some angiotensin II receptor antagonists and amlodipine in plasma using HPTLC with fluorescence densitometry: Independent fluorescence detection of the co-administrative drugs in the mixture across various pH conditions

A novel and highly sensitive high-performance thin-layer chromatographic (HPTLC) method was developed and validated to quantify a combination of five pharmaceutical mixtures spiked to human plasma. The compounds comprised Amlodipine (AML) along with five angiotensin II receptor antagonist drugs (AIIRAs), namely Olmesartan (OLM), Telmisartan (TLM), Candesartan (CAN), Losartan (LOS), and Irbesartan (IRB). HPTLC was performed on silica gel 60 F254 plates using a mobile phase of Toluene: ethyl acetate: methanol: acetone: acetic acid (6:1.5:1:0.5:1, v/v/v/v/v). In a pioneering move, a reflectance/fluorescence detection mode was employed to identify two concurrently administered drugs at different pH levels for the first time. This method utilized the same chromatographic system, incorporating a specific measurement for AML at a neutral medium to achieve its maximum fluorescence at a 360 nm excitation wavelength, and measuring emission using a 540 nm optical filter. The process involved obtaining a very low fluorescence response from AIIRA. Subsequently, to enhance AIIRA’s fluorescence, the plate was sprayed with perchloric acid to transition to a strong acidic medium, ultimately attaining the maximum fluorescence of AIIRA using various excitation wavelengths and a 400 nm emission filter. Through this strategic process, we could optimize the fluorescence signals of both drugs, thereby elevating the sensitivity of detection for this drug combination. AML demonstrated a linear range of 18–300 ng/band, while AIIRAs drugs exhibited a linear range of 6–150 ng/band. The method satisfied the International Conference on Harmonization (ICH) criteria for recovery, precision, repeatability, and robustness, showcasing exceptional sensitivity. The approach was successfully applied to quantify AML and AIIRAs drugs in both bulk drug and plasma samples, achieving high recovery percentages and minimal standard deviations.

Quantification of isoniazid in tablets for tuberculosis treatment by thin layer chromatography with smartphone image capture and ImageJ analysis

Isoniazid (INH) oral dosage tablets are an antibacterial medication commonly used to treat tuberculosis (TB). While this medication can be highly effective in managing and curing TB, manufacturing of poor-quality INH products can lead to the proliferation of drug resistant TB strains – particularly in low- and middle-income countries (LMICs). It is therefore imperative that reliable methods of quality screening of INH tablets are available for areas that have limited access to traditional testing resources. In this study, a thin layer chromatography (TLC) procedure for INH identification is validated in conjunction with smartphone image capture and open-source image analysis software to provide an alternative quantitative quality screening method for INH tablets, primarily intended for applications in LMICs. The method assessed two individual tablet formulations: 100 mg and 300 mg INH, each from different manufacturers. The modified TLC procedure used for the study was based on a validated high-performance thin layer chromatography method for the determination of rifampicin, INH, and pyrazinamide in a fixed dosage tablet. Quantitation was performed by visualizing and photographing TLC plates using a UV lamp, 3D-printed light box, and Apple iPhone SE 2nd Generation (2020) and Google Pixel 4a (5G) smartphones, and images were then analyzed using ImageJ Fiji software. The processed images’ pixel data were used to create a calibration curve from the INH standard spots to determine the concentration of an INH sample spot. Linearity, range, accuracy, repeatability and intermediate precision, specificity, and robustness were evaluated. The method was linear in the range of 0.150–0.450 mg mL−1, with coefficients of determination ≥0.98. The overall accuracy of the method was 99.2 % with an RSD of 2.4 % for image sets captured using the Apple iPhone, and 99.7 % with an RSD of 3.3 % for image sets captured using the Google Pixel. The pooled standard deviations for repeatability and intermediate precision were 2.27 % and 3.25 %, respectively, for the iPhone and 2.62 % and 3.74 %, respectively, for the Pixel. The method demonstrated sufficient specificity and robustness. The results of this method validation suggest that this procedure may provide a feasible alternative to traditional testing as a low-cost means of quality screening in LMICs.

Comparing the Greenness and Validation Metrics of Traditional and Eco-Friendly Stability-Indicating HPTLC Methods for Ertugliflozin Determination.

The literature does not provide any "high-performance thin-layer chromatographic (HPTLC)" techniques for the determination of a novel antidiabetic medicine, ertugliflozin (ERZ). Additionally, there are not many environmentally friendly analytical methods for ERZ measurement in the literature. A rapid, sensitive, and eco-friendly reversed-phase-HPTLC (RP-HPTLC) method was designed and validated in an attempt to analyze ERZ in marketed pharmaceutical tablets more precisely, accurately, and sustainably over the traditional normal-phase HPTLC (NP-HPTLC) method. The stationary phases used in the NP- and RP-HPTLC procedures were silica gel 60 NP-18F254S and 60 RP-18F254S plates, respectively. For NP-HPTLC, a chloroform/methanol (85:15 v/v) mobile phase was used. However, ethanol-water (80:20 v/v) was the preferred method for RP-HPTLC. Four distinct methodologies, including the National Environmental Method Index (NEMI), Analytical Eco-Scale (AES), ChlorTox, and Analytical GREEnness (AGREE) approaches, were used to evaluate the greenness of both procedures. For both approaches, ERZ detection was carried out at 199 nm. Using the NP- and RP-HPTLC techniques, the ERZ measurement was linear in the 50-600 and 25-1200 ng/band ranges. The RP-HPTLC method was found to be more robust, accurate, precise, linear, sensitive, and eco-friendly compared to the NP-HPTLC approach. The results of four greenness tools demonstrated that the RP strategy was greener than the NP strategy and all other reported HPLC techniques. The fact that both techniques can assess ERZ when its degradation products are present implies that they both have characteristics that point to stability-indicating features. 87.41 and 99.28%, respectively, were the assay results for ERZ in commercial tablets when utilizing the NP and RP procedures. Based on several validation and greenness metrics, it was determined that the RP-HPTLC approach was better than the NP-HPTLC method. As a result, it is possible to determine ERZ in pharmaceutical products using the RP-HPTLC approach.

Paris polyphylla Sm. characterized extract infused ointment accelerates diabetic wound healing in In-vivo model

Ethnopharmacological relevanceThe dried rhizome of Paris polyphylla Sm. is extensively used by traditional healers in India, China, and Vietnam to treat skin inflammation, cut wounds, uterine and traumatic bleeding, and cancer. Aim of the studyThe traditional use of P. polyphylla rhizomes for treating wounds and bleeding has been reported previously. However, the potential of P. polyphylla in the treatment of diabetic wounds has not yet been explored. Our present study focused on the investigation of the wound-healing activity of P. polyphylla infused ointment in streptozotocin (STZ)-induced diabetic rats to validate the traditional claim. Materials and methodsHydroalcoholic extract of the dried rhizomes of P. polyphylla were quantified by validated and optimized HPTLC (High-performance thin layer chromatography) method for Paris saponin VII, Dioscin and Polyphyllin V. The extract was used to prepare P. polyphylla ointments (5 and 10%). P. polyphylla ointment was subjected to physiochemical analysis and skin irritation test. Thirty STZ-induced diabetic adult male Wistar albino rats were divided into five groups (n = 6) and a circular excision wound was created. P. polyphylla ointment, ointment base (OB), and standard (STD) (Povidone Iodine 10%) were administered topically. The wound area of all groups were recorded every six days and compared with that of control. The epithelization period of each group was recorded. On day 18, the histopathological study of skin tissues of all groups was performed using hematoxylin and eosin (H&E) and Mallory's trichrome (MT). ResultsMarker analysis and quantification of phytomolecules in hydroalcoholic extract ofP. Polyphylla were found to be of paris saponin VII (3.28 ± 0.08% w/w), dioscin (1.94 ± 0.12% w/w), and polyphyllin V (1.87 ± 0.84% w/w). A physiochemical study of P. polyphylla ointment showed that the prepared ointment was within an acceptable range and was not irritable to the skin. Daily topical administration of 10% P. polyphylla ointment (PP10) for 18 days completely healed the STZ-induced diabetic wounds. On day 18, the 5% P. polyphylla ointment (PP5) showed 99.1 ± 2.9% wound closure, while that of the standard and control was 78.4 ± 7.3% and 18.5 ± 5.9%, respectively. The epithelialization period of PP10 was 18 days, whereas that of the control was 28 days. Histopathological analysis of the progression of PP10 and PP5 wounds showed a decrease in inflammatory cells, regenerated epithelial layer, keratosis layer, hair follicles, fibroblasts, and collagen. Upon collagen intensity quantification of MT stained sections, an increase in collagen density of PP10 and PP5 treated groups was observed, showing accelerated wound healing potential of P. polyphylla extract in diabetic wounds compared to the standard ointment. ConclusionThis study suggested the potential of P. polyphylla rhizomes derived formulation to treat diabetic wounds, although the plant is traditionally used to treat normal wounds. The results indicate the validation of traditional claim, which has been explored commercially in industrial aspect.

Development and validation of a high-performance thin-layer chromatography method for detection of sibutramine in dietary supplements.

In the period between 1997 and 2010, sibutramine-containing drugs were widely prescribed for obesity and over-weight management. Due to safety concerns, in 2010 all medicines containing sibutramine were urgently withdrawn from the USA and European pharmaceutical market. Although sibutramine is no longer available in pharmaceutical products, there have been numerous reports of mislabeled weight-loss dietary supplements containing sibutramine.

Stability indicating high performance thin layer chromatography method development and validation for quantitative determination of tetracycline hydrochloride in tetracycline hydrochloride active pharmaceutical ingredient (API) and its dosage forms

Simple, quick, cost-effective, and environmentally friendly analytical methods for quality assurance and control roles for different medicines, including Tetrcyclines, are most significantly needed. Also, different thin layer chromatography (TLC)-based methods for tetracycline identification exist, but high performance thin layer chromatography methods based on modern state- of- the art equipment are still nonexistent. Thus, in this study, analytical method development and verification were done by high performance thin layer chromatography (HPTLC) (using an automated equipment model) using glass plates coated with silica gel 60 F254 after treating with 10% Na2EDTA. Validation was carried out according to International Council for Harmonization (ICH) guidelines. A mobile phase formed from ethyl acetate, acetonitrile, methanol, and 1% aqueous ammonia in the composition of 4.4:19.6:10:6 volume to volume ratio (V/V) was used. Rf value, percentage recoveries, linearity ranges, limit of detection (LOD), and limit of quantitation (LOQ) for the developed HPTLC method were 0.28, 100.83–106.25%, 160–560 ng/band (r2 values of 0.9999), 31.9 ng/band, and 96.7 ng/band, respectively. The results of the sample assays conducted using the new method and the United States Pharmacopoeia (USP) high performance liquid chromatography (HPLC) method were 91.59% to 108.3% and 90.83% to 102.85%, respectively. The F test for the aforementioned methods was 2.01, which is smaller than the tabulated F value of 5.05 with 5 degrees of freedom at a 95% confidence range, proving that the newly developed HPTLC and HPLC pharmacopoeial methods can be used interchangeably.The newly developed HPTLC method is easy, economical, specific, accurate, and roboust, thus it can be employed in a range of settings that require quality control and assurance activities of tetracycline hydrochloride (TC-HCl) in bulk and ointment dosage forms.

Simultaneous determination of lesinurad and febuxostat in commercial fixed-dose combinations using a greener normal-phase HPTLC method

Abstract So far, no documented method for simultaneously analyzing lesinurad (LND) and febuxostat (FBX) has been reported for either traditional high-performance thin-layer chromatography (HPTLC) or a green HPTLC technique. In order to determine LND and FBX simultaneously in commercially available fixed-dose combo tablets, this study devised a normal-phase HPTLC method that is fast, sensitive, and green. The green eluents for the simultaneous analysis of LND and FBX were a mixture of ethyl acetate:ethanol:water at 70:20:10 (v/v/v) ratio. The new approach’s greenness was predicted utilizing four distinct greenness tools: the National Environmental Method Index, Analytical Eco-Scale, ChlorTox, and Analytical GREENness approaches, and the results revealed a significantly greener profile. The current method operated on a linear scale between 30 and 1,000 ng·band−1. It was confirmed that the current approach is sensitive, accurate, precise, robust, and green. The LND and FBX contents of commercially available tablet products A and B were found to be within the range of 100 ± 2%, indicating that the existing methodology for simultaneously determining LND and FBX in pharmaceutical combination products is applicable. The results of the current methodology indicated that LND and FBX could be consistently measured in pharmaceutical combination products simultaneously using the current approach.

Development and Validation of High-Performance Thin Layer Chromatographic Method for Simultaneous Estimation of Curcumin and Artemisinin

Curcumin and Artemisinin are proposed to be formulated as combination therapy to expand the antimalarial effect in resistant and liable isolates. The present research work was to develop and validate a simple, accurate, precise, high-performance thin layer chromatographic method for simultaneous determination of Curcumin and Artemisinin. The chromatographic separation was carried out on a precoated silica gel aluminum plate, 20 × 20 cm, 100μm thickness, with a mixture of Hexane, Toluene and Ethyl acetate (6:1:3) as mobile phase UV detection was performed at 496nm. The Rf were 0.23 and 0.76 for Curcumin and Artemisinin, respectively. Calibration plots were linear over the concentration range 200-1000ng/band for both Curcumin and Artemisinin. The method was validated in accordance with ICH Q2(R1) guidelines for linearity, sensitivity accuracy, precision, and robustness. The percent recoveries in terms of accuracy for the synthetic mixture were found to be 99.8% to 100.0% and 99.94% to 100.4%, and the pooled percent relative standard deviation for repeatability and intermediate precision studies was found to be 0.015-0.042% and 0.008-0.070% for Curcumin and Artemisinin, respectively. A factorial design was used to optimize the chromatographic conditions for the robustness study. In conclusion, a novel, simple, accurate and reproducible HPTLC method was developed and could be applied for quality control testing of Curcumin and Artemisinin in synthetic mixture.

Robust high-performance thin-layer chromatography (HPTLC) method for stability assessment and simultaneous quantification of epigallocatechin-3-gallate and rosmarinic acid in lipid-based nanoparticles and biological matrices.

Skin cancer poses a significant health risk globally, necessitating effective and safe therapeutic interventions. Epigallocatechin-3-gallate (EGCG) from green tea and rosmarinic acid (RA) from herbs like rosemary offer promising anticancer properties. Combining these compounds may enhance their effectiveness, prompting the need for a reliable analytical method to quantify them. Herein, we present the development and validation of a high-performance thin-layer chromatography (HPTLC) method for concurrent quantification of EGCG and RA in lipid-based nanoparticles and biological samples. The method underwent optimisation through design of experiments (DoE), resulting in the establishment of robust chromatographic conditions. The separation process utilised aluminium HPTLC plates coated with silica gel 60 F254 as the stationary phase, with the mobile phase comprising ethyl acetate, toluene, formic acid, and methanol in a ratio of 4:4:1:1 v/v. The retention factor (Rf) values obtained were 0.38 for EGCG and 0.61 for RA. The method demonstrated linearity over a range of 100-500 ng/band for both compounds with excellent correlation coefficients. Limits of detection and quantification were determined, indicating high sensitivity. Precision evaluations revealed relative standard deviation below 2%, ensuring method reproducibility. Recovery assays in lipid-based nanoparticles, plasma, and urine samples demonstrated excellent recoveries (96.2%-102.1%). Forced degradation studies revealed minimal degradation under various stress conditions, with photolytic degradation showing the least impact. The developed HPTLC method offers a rapid, sensitive, and reliable approach for quantifying EGCG and RA, laying the groundwork for their further investigation as anticancer agents alone and in combination therapies.

Quantitative assessment of paclitaxel in Taxus wallichiana zucc. bark using HPTLC densitometry: Insights for germplasm selection and toxicological studies

Taxus wallichiana Zucc. [Synonym: Taxus baccata subsp. Wallichiana (Zucc.) Pilg.] or Himalayan Yew belonging to the evergreen gymnosperm family Taxaceae, grows in temperate regions and has been used in traditional medicines to treat cancer, bronchial disorders, colds, cough, etc. The plant is known to contain paclitaxel (PTX), a key bioactive component with anticancer properties. High-performance thin layer chromatography (HPTLC) was used in the present study to estimate paclitaxel from the bark of T. wallichiana obtained from 15 different geographical locations in the eastern and western Himalayas of India. HPTLC method was determined to be a reliable and efficient technique for the quantification of paclitaxel, offering rapid outcomes.The bark materials of T. wallichiana chemotype obtained from Sikkim (Samanden) contained the highest concentration of paclitaxel, followed by West Bengal (Sandakphu), Arunachal Pradesh (Dibang Valley), and respectively. The present study also examined the cytotoxicity and genotoxicity of the aqueous bark extract of the TW1 chemotype at various doses on the meristematic cells derived from the roots of Allium cepa L.. The bark extracts demonstrate considerable cytotoxic and mitotic properties. This study could be useful to the pharmaceutical industry for selecting elite (high paclitaxel-producing) chemotypes of T. wallichiana. The toxicological evaluation of the T. wallichiana chemotypes (using Allium cepa test) could also help to determine the genotoxic properties of the bark extract.

High performance thin layer chromatography fingerprint on cyano-bonded stationary phase of selected Stachys and Betonica species and their antioxidant activity with chemometric calculations

The methanolic extracts of selected Stachys and Betonica species were analyzed by high performance thin layer chromatography (HPTLC) method using cyano bonded stationary phase and mobile phase consisted of methanol and water 45:55 (v/v) with Natural product reagent derivatization. The HPTLC method was validated (linearity, precision, accuracy, robustness, LOD, LOQ) and the validation results confirm the usefulness of the method used. The obtained images of fingerprint chromatograms presented as numerical data (using the TLC analyzer program) were next processed using Excel and SpecAlign softwares. The phytochemical similarity between analyzed plant extracts was evaluated using the similarity index (Pearson correlation coefficient) and the results were presented as hierarchical cluster analysis dendrograms and as graph for principal component analysis (PCA). The total phenolic content (TPC) by Folin-Ciocalteu method and the antioxidant activity with Ferric Ion Reducing Antioxidant Potential (FRAP) technique were determined for studied extracts using gallic acid as standards in both methods.