Developed High-performance Thin-layer Chromatography Method Research Articles

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.

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.

Validated HPTLC Analysis for Estimation of Quercetin in Seeds of Anethum graveolens

A high-performance thin layer chromatography (HPTLC) method for the quantification of quercetin was developed on methanolic extract of dill (Anethum graveolens) seeds and subsequently validated. A suitable mobile phase was used to establish the HPTLC method, ethyl acetate, toluene, methanol, chloroform, and formic acid (2:3:3:2). A densitometric analysis was done at 366 nm of wavelength. Quercetin has an Rf value of 0.55. In the dilution range of 100 to 800 ng per band, quercetin revealed a linear connection with r2= 0.9938 in the calibration’s linear regression analysis. By conducting replication analysis on 2 separate days and one day, accuracy was verified. The standard addition method was used to conduct recovery studies to validate accuracy. The quercetin recovery rate was 98.60% on average. Five replicates of each of the three standards were used to detect the system suitability parameter. With regard to both the peak area and the Rf value, the %RSD was observed to be under 2%. The mobile phase concentration was altered from Toluene: Ethyl acetate: Chloroform: Methanol (2:3:3:2) to (3:2:2:3) with few drops of formic acid. It was found to have a %RSD of peak area below 10%, the robustness of the method was assessed. The developed HPTLC method was discovered to be easy to use, precise, accurate, suitable, and robust for estimating quercetin from dill seed extract.

Simultaneous quantification of pharmacological markers quercetin, berberine HCl and curcumin using High-performance thin-layer chromatography (HPTLC) from polyherbal formulation

Quercetin, curcumin and berberine HCl have anti-acne, antimicrobial, anti-inflammatory, antioxidant properties. In the present study an attempt has been made to develop a simple, precise, rapid, selective and cost-effective high-performance thin-layer chromatographic (HPTLC) method for simultaneous estimation of quercetin, curcumin and berberine HCl from polyherbal gel prepared for the treatment of Acne. The method was developed using HPTLC silica gel GF254 precoated aluminium plate as a stationary phase. The solvent system consisted of Toluene, Ethyl acetate, Methanol, Formic acid. Densitometric analysis was carried out in the absorbance mode at 254nm. This system was found to give compact spots for quercetin, curcumin and berberine HCl at specific Rf values. The linearity of quercetin, curcumin and berberine HCl were obtained between 200 and 1000 ng/spot with good correlation coefficient values. The % of quercetin, curcumin and berberine HCl from polyherbal gel was found to be 99.42±0.98, 98.67±0.76% and 99.6±1.06% respectively, which was well within the limit. It was concluded that the developed HPTLC method would be an important tool in the quality control of polyherbal formulations.

A developed high-performance thin-layer chromatography method for the determination of baicalin in Oroxylum indicum L. and its antioxidant activity

A simple, accurate, precise, and specific high-performance thin-layer chromatography (HPTLC) method for the quantitative determination and validation of baicalin in different extracts of Oroxylum indicum has been developed for the first time. The mobile phase of acetone‒ethyl acetate‒water‒formic acid (2:10:0.5:0.5, V/V) was used for achieving good separation. Densitometric determination was carried out at 318 nm. The calibration curves were found to be linear in the range between 200 and 1000 ng per spot. During the analysis, the ethanolic extract of O. indicum showed higher content of baicalin than acetone, DMSO, and DMF extracts. Further, the antioxidant potential of different extracts of O. indicum were assessed with the 2,2-diphenyl-1-picrylhydrazyl (DPPH) method. The developed method of HPTLC was validated for specificity, accuracy, precision and linearity. The ethanolic extract has unveiled significant antioxidant activity with a percentage inhibition of 67.34%.Graphic

Simultaneous Determination of Pharmacological Active Phytocompound Gallic Acid and Quercetin in Aerial Parts of Solanun indicum and Solanum xanthocarpum by Validated High Performance Thin‑layer Chromatography (HPTLC) Method

Objective: Quantitative standardization of plant‑based products is challenging albeit essential to maintain their quality. This study aims to develop and validate high‑performance thin‑layer chromatography (HPTLC) method for the simultaneous determination of gallic acid and quercetin from aerial parts of Solanum indicum and Solanum xanthocarpum. Materials and Methods: The stock solution (1-mg/mL) of standard, gallic acid and quercetin in methanol: Water (1:1) was serially diluted and spotted (5 µL) on silica gel 60 F254 thin‑layer chromatography plates. Toluene: Ethyl acetate: Formic acid: Methanol (3.5:4.8:1.0.:0.7, v/v/v/v) was selected as mobile phase for analysis at 280 nm. Results: The developed method was robust and resolved gallic acid and quercetin at Rf0.34 ± 0.03 and 0.55 ± 0.01, respectively. The limit of detection (26 and 47 ng band-1 ) limit of quantification (78 and 141 ng.band-1 ), recovery (99.6–99.8 and 98.5–99.7%), and precision (≤1.98 and 1.97) were satisfactory for gallic acid and quercetin respectively. Linearity range for gallic acid and quercetin were 100–1000 (r 2 = 0.9993) and 150–900 ng band-1 (r 2 = 0.9956) and the contents estimated as 0.63 ± 0.01% and 0.57 ± 0.01% w/w in SI and 0.51 ± 0.01% and 0.69 ± 0.01% w/w for gallic acid and quercetin respectively. Conclusion: The developed HPTLC method was rapid, accurate, precise, reproducible, and specific for the concurrent estimation of gallic acid and quercetin. The method has been successfully applied in the analysis and routine quality control of herbal material and formulations containing Solanum Species.

Development and Validation of Thin Layer Chromatography-Densitometric Method for Quantification of Kaempferol and Chlorogenic Acid in Methanolic Extract of Dragea Volubilis

The present study aims to prepare a methanolic extract of Dragea volubilis and to quantitatively estimate kaempferol and chlorogenic acid using newly developed and validated method of high-performance thin layer chromatography (HPTLC) using TLC densitometry technique. Dried leaves obtained from D. volubilis were defatted with petroleum ether and extracted in a Soxhlet extractor with methanol. The solvent was evaporated using a Rota evaporator. Quantitative analysis of the extract was done using TLC and HPTLC. A mixture of toluene: ethyl acetate: glacial acetic acid (5.5:4:0.5) for kaempferol and ethyl acetate: acetic acid: water (7:1.5:1.5) for chlorogenic acid was used as a mobile phase. The chromatography was performed on a TLC plate precoated with silica gel GF254 and the developed TLC plates were visualized and quantified at 367 nm and 327 nm for kaempferol and chlorogenic acid, respectively. The percentage yield was found to be 0.0397 ± 0.0001 and 0.0755 ± 0.0000 for kaempferol and 0.0755 ± 0.0000 for chlorogenic acid. Rf. values 0.95 and 0.61 were observed for kaempferol and chlorogenic acid, respectively. The method was validated for instrumental precision, repeatability, coefficient of determination (r2), linearity range (ng), LOD (ng), LOQ (ng), and intra-day and inter-day precision for both kaempferol and chlorogenic acid, and all the parameters were found to be within the acceptable range as per the ICH guidelines. The newly developed HPTLC method was found to be prompt, cost-effective, precisely accurate, and reproducible for the qualitative as well as quantitative analysis of kaempferol and chlorogenic acid in methanolic extract of D. volubilis.

Application of Chemometry and Design of Experiments to Green HPTLC Method for Synchronous Estimation of Multiple FDCs of Cilnidipine.

Numerous fixed-dose combinations (FDCs) of cilnidipine (CIL) with chlorthalidone (CLT) and azilsartan medoxomil (AZL) are used for the treatment of hypertension. Numerous reverse-phase high-pressure liquid chromatography (RP-HPLC) and high-performance thin-layer chromatography (HPTLC) methods have been reported in the literature for the estimation of FDCs of CIL. But no HPTLC method has been reported yet for synchronous estimation of multiple FDCs of CIL to save time, organic solvent, and cost of analysis. In the recent scenario of green chemistry, the minimum usage of organic solvent in the development of the chromatographic method is highly desirable for the safety of the environment and analysts. Hence, a robust and green HPTLC method has been developed for the synchronous estimation of multiple FDCs of CIL using the analytical quality by design (AQbD) approach. The chemometric tool was applied for the identification of the critical method variables (CMVs) and critical analytical attributes (CAAs) for the HPTLC method development. The design of experiments (DoE) was applied for the response surface analysis of identified CMVs and CAAs using the Box-Behnken design. The analytical design space and control strategy was framed for the lifecycle management of the HPTLC method. The chromatographic separation was performed using silica gel G60 F254 as the stationary phase and toluene-ethyl acetate-methanol (6.5 + 2 + 1.5, v/v) as the mobile phase. The method was validated as per the International Council for Harmonization Q2 (R1) guideline. The developed method was applied for the synchronous estimation of multiple FDCs of CIL, and results were found in compliance with labeled claims. The developed HPTLC method can be used as a green, economical, and rapid analytical tool for routine analysis and quality control of multiple FDCs of CIL in the pharmaceutical industry. Development of a HPTLC method for synchronous estimation of multiple FDCs of CIL using the AQbD approach based on principles of chemometry and DoE. Application of the developed method for synchronous assay of multiple FDCs of CIL to save time, cost, and solvent for analysis.

A Green High-Performance Thin-Layer Chromatography Method for the Determination of Caffeine in Commercial Energy Drinks and Formulations.

The literature on green analytical approaches for caffeine estimation is limited. As a consequence, this study aimed to establish a reverse-phase high-performance thin-layer chromatography (HPTLC) technique for caffeine estimation in a variety of commercial energy drinks (ED) and pharmaceutical formulations that is rapid, sensitive, and green. The combination of ethanol-water (55:45 v v−1) was used as a mobile phase. The detection of caffeine was carried out at 275 nm. The green reverse-phase HPTLC method was linear in the concentration range of 50–800 ng band−1. Furthermore, the developed method for caffeine estimation was simple, quick, economical, accurate, precise, robust, sensitive, and green. The amount of caffeine in different marketed ED (ED1–ED10) was recorded in the range of 21.02–37.52 mg 100 mL−1 using the developed HPTLC method. However, the amount of caffeine in different commercial formulations (F1–F3) was estimated as 10.63–20.30 mg 100 mL−1 using the same method. The “analytical GREEnness (AGREE)” scale for the developed analytical method was predicted to be 0.80, utilizing 12 distinct components of green analytical chemistry, indicating the HPTLC approach’s excellent greener profile. Overall, the developed method for estimating caffeine in marketed ED and dosage forms was found to be reliable.

A High-Performance Thin-Layer Chromatographic Method for the Simultaneous Determination of Curcumin I, Curcumin II and Curcumin III in Curcuma longa and Herbal Formulation

Curcuma longa (turmeric) has traditionally been used in Ayurvedic, Unani and herbal drugs to cure numerous ailments. Due to the high demand, the quantitative standardization of herbal products is challenging to maintain their quality. We aim to develop a rapid, sensitive and validated high-performance thin-layer chromatography (HPTLC) method for the simultaneous determination and quantification of curcumin I, curcumin II and curcumin III in C. longa and herbal formulation. The three standards were separated using centrifugal preparative thin-layer chromatography (CPTLC) silica gel and identified by different spectroscopic methods. The developed HPTLC method was validated by following ICH guidelines (linearity; limit of detection, LOD; limit of quantitation; accuracy; precision; and robustness). The calibration curves of both the compounds were linear (50–500 ng/spot), with a correlation coefficient (r2) of >999. The developed HPTLC method was effectively applied to the concurrent detection and quantification of curcumins I–III in fresh, dry rhizomes and the herbal formulation of C. longa extracts was obtained by hot and cold extraction methods.

Rapid Screening of Lipase Inhibitors from Ophiopogonis Radix Using High-Performance Thin Layer Chromatography by Two Step Gradient Elution Combined with Bioautographic Method.

In this study, a high-performance thin layer chromatography (HPTLC) method by two step gradient elution with two mobile phases was developed for the simultaneous analysis of seven constituents in Ophiopogonis Radix. The chromatography was performed on silica gel 60 F254 plate with dichloromethane-methanol-ethyl acetate-water (70:25:12:3, v/v/v/v) and dichloromethane-methanol (300:1, v/v) as the mobile phase for two step gradient elution. Then, the HPTLC profiles were observed after derivatization with 10% sulfuric acid in ethanol solution. The obtained HPTLC images were further analyzed by chemometric approaches and the samples could be clustered based on regions and/or growth years, which were two important factors affecting the constituents in Ophiopogonis Radix. Furthermore, five compounds including ophiopogonin D, ophiopojaponin C, ophiopogonin D’, ophiopogonin C’ and methylophiopogonanone B were screened as potential lipase inhibitors from Ophiopogonis Radix by the HPTLC-bioautographic method. The binding modes and interactions between the five compounds and lipase were further explored by molecular docking analysis. The developed HPTLC method could be used for quality control of Ophiopogonis Radix and screening of the potential lipase inhibitors.

Development of HPTLC method for determination of α-terpinyl acetate, and evaluation of antioxidant properties of essential oils in Elettaria cardamomum

Purpose: To develop a simple and reliable protocol for high performance thin layer chromatography (HPTLC) quantification of α-terpinyl acetate in oils extracted from Elettaria cardamomum, and to study relative antioxidant potential of oils obtained from three varieties of fruits of E. cardamomum.
 Methods: Essential oil was extracted separately from the fruits of three varieties of E. cardamomum, viz, Valley-green, Palakuzhi, and ICRI-2, using hydro-distillation method. In the development of an HPTLC method, standard α-terpinyl acetate was subjected to chromatography on aluminium-backed silica gel 60 F254 plates using a mobile phase of n-hexane: ethyl acetate (8:2, v/v), and quantified at 665 nm through densitometric analysis. The antioxidant property of essential oil of each cultivar was determined with respect to 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azino-bis(3- ethylbenzothiazoline-6-sulphonic acid (ABTS) radical scavenging assays, as well as ferric reducing assay.
 Results: The developed HPTLC method showed a good resolution for α-terpinyl acetate, with Rf of 0.64 ± 0.01, and also showed good linearity of the calibration plots (r2 = 0.9982). The HPTLC method was validated as per ICH guidelines, and used for the determination of α-terpinyl acetate in the essential oils. The valley green variety of E. cardamomum had the highest content of α-terpinyl acetate (55.36 ± 1.33 %w/w). The inhibitory capacity (IC50 value) for the oil of valley green variety as determined using DPPH and ABTS methods was 378.2 and 19.87 μg/mL, respectively.
 Conclusion: The proposed HPTLC method is suitable for routine analysis of α-terpinyl acetate in medicinal herbs, and it reveals the role of α-terpinyl acetate in the antioxidant efficacy of cardamom oil.

A Validated, Rapid and Cost-Efficient HPTLC Method for Quantification of Gamma-Linolenic Acid in Borage Oil and Evaluation of Antioxidant Activity.

Borage oil that is extracted from (Borago officinalis Linn.) is a well-known medicinal plant having various medicinal benefits. In this work, an affordable, simple, reliable, rapid and easily accessible high-performance thin-layer chromatography (HPTLC) method was developed for the estimation of gamma-linolenic acid (GLA) in borage oil. HPTLC method employs thin-layer chromatography (TLC) aluminum plates precoated with silica gel (G60F254) as the stationary phase, and the mixture of hexane:toulene:glacial acetic acid (3:7:1, v/v/v) was used as the mobile phase. Densitometric analysis of the TLC plates was carried out at 200nm. The developed method showed well-resolved spots with retention factor (Rf) value of 0.53 ± 0.04 for GLA. Various experimental conditions like saturation time for chamber, solvent phase migration and width of the band were studied intensely for selecting the optimum conditions. The method validation was performed for parameters like linearity, accuracy, specificity and precision. The values of limit of detection and limit of quantification for GLA were found to be 0.221 and 0.737μg/band, respectively. In nutshell, the developed HPTLC method was found to be highly sensitive for the estimation of GLA in the herbal oil samples and formulations.

Phytochemical Screening and High-performance Thin-layer Chromatography Quantification of Vitex trifolia Leaves Hydro-alcoholic Extract: Potential Anti-inflammatory Properties

Aims: The present study aims to evaluate the phytochemical composition of Vitex trifolia (V. trifolia) leaves hydro-alcoholic extract and to report for the first time, its phenolic content using a validated high-performance thin-layer chromatography (HPTLC) method.
 Study Design:  Qualitative phytochemical analysis and HTLC densitometric quantitative analysis.
 Place and Duration of Study: The study was carried out at the Faculty of Pharmacy, Universiti Teknologi MARA (UiTM), Malaysia, from March 2020 to December 2020.
 Methodology:  The preliminary phytochemical screening was carried out qualitatively. The HPTLC analysis was performed on glass-backed 60 F254 silica gel plates using a two steps gradient elution method of the mobile phase. In the first step, methanol was used to develop the plates until 40 mm of developing distance, while in the second step, plates were developed with n-hexane:ethyl acetate:acetic acid (20:9:1, v/v/v) until 80 mm of developing distance. Detection and quantification were performed by densitometric analysis at 254 nm. The method was validated as per the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) guideline in terms of linearity, precision, accuracy, the limit of detection (LOD), and the limit of quantification (LOQ).
 Results: The preliminary phytochemical screening of V. trifolia leaves hydro-alcoholic extract showed the presence of alkaloids, flavonoids, phenols, phytosterols, and terpenoids. The developed HPTLC method was proved to be linear, precise, and accurate. The LOD and LOQ of the method were determined to be 2.01 µg/band and 6.08 µg/band, respectively. The total phenolic content of the extract was calculated from the standard gallic acid calibration plot and found to be 136.94 ± 4.02 mg gallic acid equivalent (GAE)/g of dried extract.
 Conclusion:  This preliminary study revealed that V. trifolia has a considerable amount of phenolic compounds, which can potentially contribute to its anti-inflammatory, antioxidant, and anticancer activities. Further pharmacological investigations are being carried out to support the folkloric claims.

Development of a HPTLC method with iterative calibration approach for quantitative evaluation of kinsenoside content in different types of Anoectochilus roxburghii

Anoectochilus roxburghii (Wall.) Lindl. (Orchidaceae) or more commonly known as Jin Xian Lian is a widely adapted medicinal herb in the Asian continent. However, till date there are limited rapid analytical method such as thin layer chromatography method that acts as standard for quantifying the active ingredients of A. roxburghii. In this research, a simple and rapid method high performance thin layer chromatography (HPTLC)-anisaldehyde-sulfuric acid derivative with iterative calibration method was developed and validated to quantify the non UV sensitive bioactive compound kinsenoside in wild, cultivated and tissue cultured A. roxburghii plants. The optimum mobile phase identified for kinsenoside in A. roxburghii was chloroform/methanol/water (45:30:4,%v/v/v). It was also proved that HPTLC method for determination of kinsenoside showed good accuracy, precision, stability and repeatability, with wide working concentration range of 0.099–6.365 mg/mL. Intra-day and inter-day precision (% RSD) of the developed HPTLC method were 5.08–7.57% and 6.72–8.72%, respectively. The % RSD of iterative calibration method was 3.139%. Through this study, it can be concluded that the proposed HPTLC method with iterative calibration approach was successfully applied for quantitative analysis of kinsenoside in three types of A. roxburghii plants.

Novel HPTLC-densitometric method for concurrent quantification of linalool and thymol in essential oils

Monoterpenes, linalool and thymol have been widely used in not only the treatments of several diseases but also food flavouring, dental, perfumery, and toilet products. Plants of the several Lamiaceae family are good sources of linalool and thymol. A rapid, simple, and precise high-performance thin layer chromatography (HPTLC) method was developed for the concurrent estimation of linalool and thymol, and this method was orchestrated using the essential oils extracted from two Lamiaceae plants leaves i.e. O. basilicum and T. vulgaris. Two standards were separated using pre-coated thin-layer chromatography glass plates. The developed HPTLC method was validated by following ICH guidelines (linearity; limit of detection, LOD; limit of quantitation; specificity; accuracy; precision; and robustness). The calibration curves of both the compounds were linear (100–700 ng/spot), with a correlation coefficient (r2) of >999. The developed HPTLC method was effectively applied to the concurrent detection and quantification of linalool and thymol in commercial essential oils.

HPTLC METHOD FOR ESTIMATION AND QUANTIFICATION OF β-SITOSTEROL FROM IN-VIVO AND IN-VITRO SAMPLES OF MERREMIA AEGYPTIA AND MERREMIA DISSECTA

Objective: A normal-phase high-performance thin-layer chromatography (HPTLC) method has been developed and validated for estimation and quantitation of beta-sitosterol from the methanolic fraction of different plant parts of two medicinally important plants viz. Merremia aegyptia and Merremia dissecta. These plants have been reported to possess antimicrobial, antioxidant, and anti-inflammatory activities.
 Methods: Chromatographic separation of beta-sitosterol from the methanolic extracts of plant parts of M. aegyptia and M. dissecta was performed on TLC aluminum plates pre-coated with silica gel 60F254 using a suitable mobile phase. The densitometric scanning was done after derivatization at ????-580 nm for ????-sitosterol.
 Result: Only M. dissecta leaf sample was reported to contain ????-sitosterol (4.6 ng/μl), whereas other samples such as seed, stem, and callus extracts of M. aegyptia and M. dissecta did not showed its presence.
 Conclusion: The developed HPTLC method is simple, rapid, and precise and can be used for routine analysis and quantification of ????-sitosterol and other useful plant bioactives that are phytopharmaceutically important.

Development of a novel HPTLC fingerprint method for simultaneous estimation of berberine and rutin in medicinal plants and their pharmaceutical preparations followed by its application in antioxidant assay

The present study was designed to develop and validate a high-performance thin-layer chromatography (HPTLC) system for the simultaneous quantitative determination of berberine and rutin in Tinospora cordifolia extract and their pharmaceutical preparations. Chromatographic development was done using a blend of n-hexane, ethyl acetate, glacial acetic acid and methanol (10:1.1:1.1:2.5, v/v) as the mobile phase. Detection was completed densitometrically at 254 nm. The RF estimation of berberine and rutin was observed to be 0.67 ± 0.02 and 0.47 ± 0.02, respectively. The developed HPTLC method was validated according to ICH guidelines; the method was specific, linear and accurate and can be used to determine berberine and rutin in marketed herbal preparations. The Tinospora cordifolia plant extract was further evaluated for antioxidant activity using HPTLC, and berberine was found to be more active than rutin during DPPH (2,2-diphenyl-1-picrylhydrazyl) scavenging activity. The method was found simple, rapid, accurate, specific and robust for the analysis of berberine and rutin in crude drug using the same method.

Central composite design expert-supported development and validation of HPTLC method: Relevance in quantitative evaluation of protopine in Fumaria indica

The current study was executed for method development, validation and to estimate the concentration of protopine in methanolic extract of Fumaria indica by high-performance thin-layer chromatography (HPTLC). Isolation of bioactive compounds was carried out using the mobile phase, toluene:ethyl acetate:diethyl amine (8:2.5:0.5 v/v/v), and detected at wavelength 290 nm. This method was validated for precision, specificity, linearity, limit of detection (LOD), limit of quantification (LOQ), etc. The calibration range was found to be 500–5000 ng/spot for the bioactive compound. Protopine was separated with an Rf value of 0.22 ± 0.03. The method was validated for linearity (r2 ≥ 0.996 ± 0.082), accuracy 98.75–102.12%), and RSD of precision (0.49–2.07) with a calibration curve range of 500.00–5000.00 ng/spot. The LOD and LOQ were found to be 83.92 ng/spot and 254.30 ng/spot., respectively. The Central Composite design expert was applied for the validation of robustness. Three independent variables such as the volume of toluene in solvent system, chamber saturation time and wavelength were investigated. The results indicated that a slight change in these variables had no significant effect on the peak response. This developed HPTLC method is simple, precise, robust, specific, rapid, and cost effective. It could be used for quality control study and quantification of protopine in the plant extract and different herbal formulations containing the plant species.

Analytical Quality by Design (AQbD) Approach Based HPTLC Method for Quantification of Fisetin with Superior Recovery in Formulations

Background: A stability indicating high-performance thin layer chromatography (HPTLC) method was developed for the evaluation of fisetin (FIS) in active pharmaceutical ingredient (API) and marketed capsule formulation in accordance with the ICH guidelines. Methods: The mobile phase combination toluene: ethyl acetate: formic acid: methanol (3: 5.5: 1: 0.5 v/v/v/v) was optimized with the aid of AQbD approach. The absorbance mode at 254 nm was chosen for densitometric analysis as it gives a compact spot of FIS at Rf value of 0.74. Results: The R2 value obtained from the linear regression equation of calibration plots made by taking the spot in the concentration range of 100-1400 ng/spot was found to be 0.9993. The observed LOD and LOQ value was found to be 29.8 ng/spot and 98.5 ng/spot, respectively. The exposure of FIS to various stress conditions revealed the fact that the drug is stable in photochemical and dry heat stress conditions without any degradation. The drug-exposed to acidic, alkaline and oxidative stress was found to be degraded into different degradation products with the highest degradation was found in alkaline stress. All degradation products were observed to be fairly separated from well-resolved parent peak of FIS. Conclusion: The developed HPTLC method have shown well-resolved peaks and also shown good recovery in the compound FIS as well as FIS formulation.