High-performance Thin-layer Chromatography Plates Research Articles

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.

Mixture Design-driven Statistical Optimization and Method Validation in HPTLC: Targeting Rutin, Quercetin, and Gallic Acid

Rutin (RT), Quercetin (QT), and Gallic Acid (GA) are recognized for their potent antioxidant and anticancer properties, prevalent across numerous plant species. The precise quantification of RT, QT, and GA is pivotal for evaluating the therapeutic potential of plant-based substances. In response, a new, straightforward, cost-effective, and reliable method using High-Performance Thin-Layer Chromatography (HPTLC) has been developed and validated for the quantification of these compounds. The mobile phase optimization employed a mixture design approach, achieving chromatographic separation with a mobile phase mixture of toluene, ethyl acetate, menthol, and formic acid at specific ratios (3.56:3.70:0.94:1.80 v/v/v/v/v/v/v/v). Silica gel 60 F254 HPTLC plates were utilized for the analysis. The retention factors (Rf) observed for RT, GA, and QT were 0.21, 0.58, and 0.74, respectively. This method demonstrated a robust linear relationship for concentrations ranging from 400 to 2000 ng per band, with correlation coefficients (R2) of 0.9921 for RT, 0.9936 for QT, and 0.9912 for GA. The Limits of Detection (LOD) and quantification (LOQ) were established at 100.84, 102.38, and 84.54 ng per band for LOD, and 305.58, 310.25, and 256.18 ng per band for LOQ, respectively, for RT, QT, and GA. This validated HPTLC method developed through a Design of Experiment (DoE) approach was successfully employed for the quantification of GA, QT, and RT from the fruits of Adansonia digitata and leaves of Grewia asiatica ethanolic extracts.

Assessment of adulteration of sage (Salvia sp.) with olive leaves using high‐performance thin‐layer chromatography, image analysis, and multivariate linear modeling

AbstractAccording to the study carried out at the University of Bristol, 60% of oregano spices present on the European Union (EU) market are adulterated with olive, myrtle, cistus, and hazelnut leaves. According to the same authors, the sage products are adulterated by similar bulking agents. The aim of this study was to assess possibilities for detection of sage adulteration by olive leaves using high‐performance thin‐layer chromatography (HPTLC) coupled with digital image analysis and multivariate linear regression/classification (partial least squares and partial least squares discriminant analysis). Twenty‐four samples (4 pure sage leaves, 4 pure olive leaves, and 16 mixtures of olive and sage leaves with content of added olive leaves varying in 5%, 10%, 20%, and 50%) have been prepared, extracted, and analyzed under normal‐phase conditions. Several derivatization methods were tested, and derivatized HPTLC plates were inspected under visible or ultraviolet light. Digital images of chromatograms were recorded. In order to minimize effects of intraplate and interplate peak shifts, background changes, and baseline drifts, correlation‐optimized warping, standard normal variate, and mean centering were applied to acquired signals. Partial least squares and partial least squares discriminant analysis models with moderate complexity (two to four latent variables) based on chromatographic signals obtained after derivatization by FeCl3, anisaldehyde–sulfuric acid, and 2,2‐diphenyl‐1‐picrylhydrazyl demonstrated good statistical performances with R2 ranging 0.894–0.998 and relative prediction error of 4–12%. Misclassification error <4% was obtained in the case of 2,2‐diphenyl‐1‐picrylhydrazyl and anisaldehyde–sulfuric acid derivatization. Therefore, HPTLC combined with multivariate image analysis, signal processing, and linear modeling proved to be promising, cost‐effective chromatographic tool for assessment of sage adulteration by olive leaves.

Online coupling of miniaturized HPLC and high performance thin layer chromatography by a fractionation unit for effect directed analysis

The rising discharge of anthropogenic chemicals into aquatic environments poses a significant threat, necessitating effective monitoring strategies. This study introduces an innovative approach to effect-directed analysis (EDA) by coupling liquid chromatography (LC) with high-performance thin-layer chromatography (HPTLC), utilizing a modified MALDI spotter. The objective is to optimize fractionation parameters for sample application and assess the method's viability in identifying acetylcholinesterase (AChE) inhibitors, specifically malathion, parathion, and chlorfenvinphos.The optimization process involves controlling sample volume, spot shape, and spot distances on HPTLC plates. Successful application is demonstrated by a miniaturized LC system coupled to the HPTLC plate via spotter, allowing effective separation and identification of AChE inhibitors. The study further explores the method's application to water samples from a river with predominantly agricultural drainage area.Spiked samples reveal distinct active spots, identified through extraction and subsequent high-resolution mass spectrometry (HRMS) measurements. However, results indicate the absence of AChE inhibitors in non-spiked water samples, affirming the efficacy of the EU ban on most organophosphate pesticides. The usefulness of HPTLC in separation of coeluting substances from HPLC is emphasized, demonstrating its suitability for the effect-directed analysis of complex samples.This work shows the integration of HPTLC with liquid chromatography for EDA, offering a powerful tool for identifying and monitoring AChE inhibitors in water samples. The approach addresses limitations in current monitoring strategies and provides insights into the presence and impact of chemicals in aquatic ecosystems. The study contributes to ongoing efforts to enhance water quality monitoring, aligning with the principles of the EU Water Framework Directive.

A greener RP-HPTLC-densitometry method for the quantification of apremilast in nanoformulations and commercial tablets: Greenness assessment by analytical eco-scale, ChlorTox, and AGREE methods

The greener “high-performance thin-layer chromatography (HPTLC)” methods for apremilast (APM) analysis in pharmaceutical products and biological fluids are not currently available in the literature. Accordingly, this study involves the development and validation of a rapid, simple, economical, and greener reversed-phase HPTLC methodology for the estimation of APM in prepared nanoparticles (NPs), nanoemulsion, and marketed tablets. The estimation of APM was conducted using “RP-18 silica gel 60 F254S HPTLC plates” as the stationary phase. The combination of ethanol/water (65:35, v/v) was used as the greener mobile phase for APM analysis. The greenness of the method was predicted using three different approaches, namely Analytical Eco-Score (AES), ChlorTox, and the Analytical GREENness (AGREE) approaches. The λmax = 238 nm was used for the APM detection. By contrasting its single band at Rf = 0.61 ± 0.01 with those of pure APM, the HPTLC peaks for APM in an prepared NP formulation, nanoemulsion, and marketed tablets were identified. In the concentration range of 100–700 ng/band, the proposed analytical methodology was linear. The values of AES, ChlorTox, and AGREE were determined to be 93, 0.66 g, and 0.89, respectively, demonstrated an outstanding greener profile for the existing method. The amount of APM in the tablet, NP formulation, and nanoemulsion was found to be 98.40, 101.60, and 99.37 %, respectively. According to the findings of validation tests and pharmaceutical analysis, the suggested analytical technique could be successfully applied for the routine examination of APM in marketed tablets and laboratory generated nanoformulations.

Quantitative Determination and Source Variation of Rutin in Fagopyrum Tataricum

Buckwheat (Fagopyrum sp.) is a traditional crop of Uttarakhand (India) known for its high Rutin content, a bioflavonoid with significant medicinal properties. This study explores the quantitative measurement of Rutin in various buckwheat varieties grown in different regions of Uttarakhand using Thin Layer Chromatography (TLC) and High-Performance Thin Layer Chromatography (HPTLC). Twelve seed sources of Fagopyrum tataricum from the Garhwal and Kumaon regions, along with Fagopyrum esculentum seeds for comparison, were collected. Preliminary phytochemical screening of the extracts revealed the presence of flavonoids, alkaloids, tannins, and carbohydrates, with methanolic extracts showing higher concentrations. TLC analysis was employed to determine the most effective solvent system for Rutin separation, leading to the identification of three samples with substantial Rutin content. HPTLC was then used for quantitative Rutin assessment, with a calibration curve established using standard Rutin solutions. The three selected samples, along with the standard, were applied to HPTLC plates, and the Rutin content was measured based on peak areas and peak heights. The sample collected from Joshimath, Uttarakhand, known as "JOG," exhibited the highest Rutin content. This study provides valuable insights into the phytochemical composition of buckwheat varieties in Uttarakhand, which can inform the development of high-quality functional foods. Rutin, a potent antioxidant with various health benefits, underscores the significance of buckwheat as a naturally occurring functional food source. The results contribute to understanding the Rutin-rich potential of buckwheat grown in Uttarakhand and its applications in functional foods and nutraceuticals. Further research in this field can promote the cultivation and utilization of buckwheat as a source of this valuable bioactive compound with numerous health benefits.

A new universal detection system for high-performance thin-layer chromatography plates using chemiluminescence

High-performance thin-layer chromatography (HPTLC) is a cost-effective method for the separation of small molecules, such as active pharmaceutically ingredients. The method can be used without sophisticated equipment. For example, the detection and quantification of colored compounds on an HPTLC plate can be performed with a CCD camera. For noncolored compounds, a new dipping solution for the generation of chemiluminescence on an HPTLC plates is presented. In combination with a charge-coupled diode (CCD) camera, this can be used as a universal detection system. The efficiency of this new staining reagent is demonstrated by the detection of trimethoprim and sulfamethoxazole from a fixed-dose combination in a ratio of 1:5.

Aloe-emodin Quantification using HPTLC and RP-UHPLC in Extracts and Commercial Herbal Formulations: Evaluation of Antimicrobial and Antioxidant Effects.

High-performance thin-layer chromatography (HPTLC) was developed and validated for the determination of aloe-emodin in accordance with ICH guidelines. In addition, a novel RP-UHPLC method was developed, and both methods were used to analyse the herbal extract and herbal formulation. Separation was carried out on a silica gel 60 F254 HPTLC plate using the mobile phase Toluene: Methanol (9:1). The linearity was good across the 800-4000 ng/spot range. Validation results are within acceptable limits. The percent RSD for accuracy was 0.58-1.77, and precision was 1.10-1.97 and 1.45-1.94 for intraday and interday, respectively. The percentage of aloe-emodin found in the herbal extract and aloe vera capsule was 99.83 ± 1.19 and 99.53 ± 1.29, respectively, using this method. Quantification of aloe-emodin in herbal extract and herbal formulation were done using a novel UHPLC method with chromatographic conditions of orthophosphoric acid Methanol (0.1 percent OPA): Water (65:35, v/v) and pH 3, a flow rate of 1.2 ml/min, and elute detection at 254 nm. At 6.32 minutes, a sharp and symmetric peak was observed. The method developed was validated in accordance with ICH guidelines. The percent RSD numerical value of accuracy was 0.304-0.576, and the inter-day and intraday precision were 0.32-3.08 and 0.51-2.78, respectively. Herbal extract and aloe vera capsule were analysed using the new UHPLC method. Aloe-emodin percentages were reported as 100.3 ± 0.89 and 99.53 ± 1.29, respectively. The antimicrobial and anti-oxidant activities of an aloe-vera herbal formulation were studied, and the results were positive.

Identification of variety-specific metabolites of basil by high performance thin layer chromatography-assisted metabolic profiling techniques

The technological advances of analytical instrumentation and techniques has laid the ground for the rapid expansion of metabolomics or in a wider sense, untargeted analysis applied to life sciences themes. However, the objective of identifying all existing metabolites within organisms remains a daunting challenge. All analytical techniques exhibit varying degrees of sensitivity and versatility for the detection of metabolites and none of the existing analytical platforms can be expected to be ideal for exhaustive chemical profiling. Planar liquid chromatography, and in particular, high performance thin layer chromatography (HPTLC), has been used for chemical profiling of natural products in conjunction with metabolomics. HPTLC has specific advantages which include its ability to generate reliable chemical fingerprinting data and facilitate preparative work for metabolite isolation during later stages of metabolomics analysis. In this study, we investigated the chemical profiles of four commercially available basil cultivars, namely Dolly, Emily, Keira, and Rosie. We used HPTLC as the primary analytical tool for the separation of basil cultivars based on detected metabolites, and then compared the results with those obtained with other analytical platforms. We identified the characteristic marker compounds of each basil cultivar from the HPTLC plates and validated their potential using LC-MS and GC-MS analyses as a metabolomics tool. Firstly, we compared the HPTLC data of the four cultivars, obtained with two systems that used silica gel 60 and two mobile phases composed of toluene-EtOAc (8:2, v/v) and EtOAc-formic acid-acetic acid-water (100:11:11:27, v/v), with 1H NMR data to evaluate their separation power. Despite providing lower resolution and detecting fewer compounds, the HPTLC separation power was comparable, and in some cases even better than that of 1H NMR. Additionally, we investigated the potential of HPTLC as a tool for chemical fingerprinting and demonstrated its suitability for preparative purposes that are essential for identifying metabolites in mixture analysis. Metabolites were easily isolated from sample mixtures, and identified with the assistance of GC-MS, LC-MS, and TLC-densitometry.. Several marker compounds were thus identified, including 2,4 di-tertbutylphenol, palmitic acid, hexadecanamide, 9-octadecenamide, squalene, hentriacontane, methyl 3-(3,5-ditert‑butyl‑4-hydroxyphenyl)propanoic acid, sagerinic acid, and cyanidin-3-O-sophoroside.

Bioactivity profiles of six baobab fruit pulp powders via planar chromatography hyphenated with effect-directed analysis

Baobab (Adansonia digitata) fruit pulp has a high nutrient content and has been traditionally used for medicinal purposes (e.g., as an anti-inflammatory and antioxidant agent) that may help protect against chronic diseases. Six different baobab fruit pulp powders were investigated using three different extractants and analyzed by high-performance thin-layer chromatography (HPTLC) hyphenated with antibacterial bioassays and enzyme inhibition assays. The developed non-target effect-directed screening was performed after extraction with pentyl acetate – ethanol 1:1 (V/V) on the HPTLC plate silica gel 60 using toluene – ethyl acetate – methanol 6:3:1 (V/V/V) as mobile phase system and derivatization via the anisaldehyde sulfuric acid reagent for detection. The physico-chemical profiles of the six baobab fruit pulp powder extracts were comparable, although the intensity of some zones was moderately different. The following effect-directed profiling via tyrosinase, α-glucosidase, and acetylcholinesterase inhibition assays as well as antibacterial Aliivibrio fischeri and Bacillus subtilis bioassays revealed one prominent multipotent bioactive compound zone in common, more or less active in all five studied (bio)assays. Via the recording of high-resolution mass spectra, this compound zone was tentatively assigned to coeluting saturated (palmitic acid 16:0 and stearic acid 18:0), monounsaturated (oleic acid 18:1), and polyunsaturated (linoleic acid 18:2 and linolenic acid 18:3) fatty acids. This finding was confirmed by other studies, which already proved individual activities of fatty acids. The first (bio)activity profiling of baobab fruit pulp powders via HPTLC-effect-directed analysis revealed that the baobab fruit could be considered as a functional food, however, further research is needed to study the impact on health and the influences on the bioactivity arising from different climates, years and soils or regions.

Development and validation of stability indicating thin‐layer chromatography method for simultaneous estimation of Benidipine hydrochloride and Telmisartan

AbstractFor the simultaneous estimation of Benidipine hydrochloride (BEN) and Telmisartan (TEL) in combined pharmaceutical dosage form, a stability‐indicating high‐performance thin‐layer chromatography (HPTLC) method has been developed and validated. It involves the use of HPTLC plates pre‐coated with silica gel 60F254 and a mobile phase comprising of Methanol: Acetonitrile (1:9 v/v). The measurements of both drugs were performed at 237 nm. The drugs were subjected to acid‐alkali hydrolysis, photolytic, thermal, and oxidation degradation. The linearity of BEN was found to be between 200–1000 ng/band (R2 = 0.9917), while for TEL, linearity was found to be between 2000 and 10000 ng/band (R2 = 0.9979). The limit of detection and limit of quantitation were found to be 3.28 and 9.94 ng/spot for BEN and 158.79 and 481.17 ng/spot for TEL. The % relative standard deviation of the precision study was determined to be less than 2%. Various system suitability parameters were determined. The Rf values of BEN and TEL were found to be 0.890 ± 0.0105 and 0.173 ± 0.0151, respectively. The resolution between BEN and TEL peak were found to be 4.48 and capacity factors were found to be 0.21 and 0.74 for BEN and TEL, respectively.

Validation of a high-performance thin-layer chromatography method for the quantitative determination of trehalulose

A novel, simple and reliable high-performance thin-layer chromatography (HPTLC) method was developed and validated for the quantification of trehalulose in stingless bee honey. The chromatographic separation was performed using silica gel 60 F254 HPTLC plates and 1-butanol‒2-propanol‒aqueous boric acid solution (5 mg/mL) (30:50:10, V/V) as the mobile phase. The retardation factor (RF) for trehalulose was found to be 0.045. The method showed linearity over the concentration range of 100–800 ng per band with a coefficient of correlation (R) of 0.9996. The limit of detection and limit of quantification for trehalulose were found to be 20.04 ng per band and 60.72 ng per band, respectively and the mean per cent recovery of trehalulose was 101.8%. The method has been validated for its specificity, linearity, sensitivity, precision, accuracy, repeatability and robustness following the International Council for Harmonisation Q2 (R1), and it has been successfully applied in the determination of trehalulose in stingless bee honey.

Sustainable and smart HPTLC determination of silodosin and solifenacin using a constructed two illumination source chamber with a smartphone camera as a detector: Comparative study with conventional densitometric scanner

The tremendous revolution in the built-in digital camera of smartphones has opened new horizons for its applicability as a portable, easily accessible, and user-friendly detector in different analytical techniques. In this article, we proposed and developed a new and inexpensive detector for simultaneous analysis of silodosin and solifenacin. The innovation makes use of a smartphone camera coupled with high-performance thin layer chromatographic (HPTLC) approach. Firstly, chromatographic separation was attained using HPTLC plates with an ecofriendly mobile phase composed of ethyl acetate, ethanol & 25% w/w ammonia (3.0: 7.0: 0.3, by volume). The developed plates were then visualized using Dragendorff's reagent and then photographed via smartphone's rear-facing camera fixed on a fabricated two-illumination source chamber. The intensities of drug spots were quantified using an open-source image analysis software, ImageJ, over concentration ranges of 2.0–30.0 μg/band. Moreover, the study was extended to compare the obtained results with a benchtop densitometric method at 215.0 nm where the densitometric one gave linear response ranging from 0.1 to 7.0 μg/band for silodosin and 0.1–6.0 μg/band for solifenacin. The fast, simple, reliable and green merits of the proposed HPTLC/smartphone method promted it as an excellent platform for assaying marketed combined tablets and assuring their content uniformity. Moreover, the high sensitivity of the densitometric method was exploited, for the first time, for determining the residual content of the cited drugs on manufacturing equipment surfaces during cleaning validation. Finally, the environmental impact of the methods was appraised via three state-of-the-art greenness metrics.

QUANTITATIVE ANALYSIS OF CYTISINE IN THERMOPSIS ALTERNIFLORAE USING HIGH-PERFORMANCE THIN-LAYER CHROMATOGRAPHY

Objective: An optimized high-performance thin-layer chromatography (HPTLC) method has been established for the quantification of cytisine in Thermopsis alterniflorae Regel and Schmalh.
 Methods: Alcoholic extract of the aerial parts were prepared using Soxhlet extraction method. Separation was achieved on silica gel 60 F254 HPTLC plates using toluene-ethyl acetate-diethyl amine (7:2:1, v/v) as the mobile phase. The quantitation of cytisine was carried out using densitometric scanning at 545 nm after derivatization using Dragendorff’s reagent.
 Results: The linear regression analysis data for the calibration plot showed a good linear relationship (r2 = 0.9849) in the concentration range 10 to 15 µg/spot. The method was validated for precision, repeatability, accuracy, specificity, limit of detection and limit of quantification. The average recovery was 99.0899% indicating good accuracy. The percentage yield of cytisine obtained was 0.5075+0.0135 % w/w.
 Conclusion: The proposed HPTLC method was found to be simple, sensitive, accurate, reproducible, and robust.

Application of the green analytical procedure index to the simultaneous analysis of co-formulated tinidazole and ciprofloxacin in pure form, tablet dosage form, and human plasma using an environmentally friendly micellar high-performance thin-layer chromatographic technology

In individuals with chronic refractory pouchitis and other gastrointestinal disorders, the combination of ciprofloxacin (CIP) and tinidazole (TIN) is beneficial and safe. As a result, a green micellar high-performance thin-layer chromatographic (HPTLC) approach for the immediate analysis of TIN and CIP in pure, spiked human plasma, and co-formulated tablet dosage form has been developed. It is rapid, extremely easy, sensitive, cost-effective, and environmentally friendly. The stationary phase was Merck aluminum HPTLC plates covered with silica gel 60 F254, while the mobile phase was acetone–ethanol–2% watery sodium dodecyl sulfate (3:4:2, V/V). For quantification of both medications, the densitometric scanner was set at 310 nm. For TIN and CIP, this chromatographic separation yielded symmetric, compact peaks with RF values of (0.22 ± 0.009) and (0.42 ± 0.007), respectively. At 310 nm, the separated spots were densitometrically scanned. For TIN and CIP, the detection thresholds were 6.7 ng/band and 25.03 ng/band, respectively. For TIN and CIP, the quantification limits were 20.3 ng/band and 75.25 ng/band, respectively. The approach was validated according to International Council for Harmonisation (ICH) principles and then used to determine the researched medicines in their various pharmaceutical dosage forms, and human plasma yielding an exceptional percent of recovery. In terms of precision and accuracy, the results were in great accordance with the published approach. This method is suitable for the sequential analysis of the two drugs in pure form, tablet dosage forms, and spiked human plasma due to its simplicity, speed, greenness, robustness, and low cost.

Profiling of lipids in Thermus thermophilus HB8 grown under various conditions.

The membrane lipids of Thermus species have unique structures. Only four polar lipid species have so far been identified in Thermus thermophilus HB8; namely, are two phosphoglycolipids and two glycolipids, both of which have three branched fatty acid chains. Other lipid molecules may be present; however, they have not been identified so far. To clarify the whole lipid profile of T. thermophilus HB8, we cultured this organism under four different growth (temperature and/or nutrition) conditions and analyzed the compositions of polar lipids and fatty acids by high-performance thin-layer chromatography (HPTLC) and gas chromatograph-mass spectrometry (GCｰMS), respectively. Thirty-one lipid spots were detected on HPTLC plates and profiled in terms of the presence or absence of phosphate, amino, and sugar groups. Then, we allocated ID numbers to all the spots. Comparative analyses of these polar lipids showed that the diversity of lipid molecules increased under high temperature and minimal medium conditions. In particular, aminolipid species increased under high temperature conditions. As for the fatty acid comparison by GC-MS, iso-branched even-numbered carbon atoms, which are unusual in this organism, significantly increased under the minimal medium condition, suggesting that kinds of branched amino acids at the fatty acid terminus varies under different nutrition conditions. In this study, several unidentified lipids were detected, and elucidation of the lipid structures will provide important information on the environmental adaptation of bacteria.

HPTLC Fingerprinting Profile of Vacha (Acorus calamus Linn.)

Introduction: Vacha (Acorus calamus Linn.) commonly called Sweet flag is a significant herb with well-recognized therapeutic potential. Choorna (powder) of its rhizome is the commonly used dosage form according to Ayurveda. High-performance thin-layer chromatography (HPTLC) fingerprinting of rhizome powder of Vacha was carried out to estimate the active constituents in the dosage form choorna. ß-asarone, which is the major phytoconstituent of rhizome, was used as standard.Materials and Methods: HPTLC plate 5 × 10 cm, pre-coated with silica gel 60 F254 TLC plates, was used as stationary phase. Toluene: ethyl acetate: acetic acid (90:10:2) was used as mobile phase. Visualization was done under ultraviolet light 254 nn and 366 nm.Results and Discussion: HPTLC chromatogram showed the presence of 3 comparable peaks at 254 nm and 2 comparable peaks at 366 nm, in methanolic extract of rhizome powder and standard.Conclusion: This approach creates profile analysis of the plant rhizome and can be helpful for quality control and further researches

Detection and Semi-quantification of Lipids on High-Performance Thin-Layer Chromatography Plate using Ceric Ammonium Molybdate Staining.

It is desirable to quickly check the composition of lipids in small size samples, but achieving this is challenging using the existing staining methods. Herein, we developed a highly sensitive and semi-quantitative method for analysis of lipid samples with ceric ammonium molybdate (CAM) staining. The CAM detection method was systematically evaluated with a wide range of lipid classes including phospholipids, sphingolipids, glycerolipids, fatty acids (FA) and sterols, demonstrating high sensitivity, stability, and overall efficiency. Additionally, CAM staining provides a clean yellow background in high performance thin-layer chromatography (HPTLC) which facilitates quantification of lipids using image processing software. Lipids can be stained with CAM reagent regardless of their head group types, position of the carbon-carbon double bonds, geometric isomerism and the variation in the length of FA chain, but staining is mostly affected by the degree of unsaturation of the FA backbone. The mechanism of the CAM staining of lipids was proposed on principles of the reduction-oxidation reaction, in which Mo(VI) oxidizes the unsaturated lipids into carbonyl compounds on the HPTLC plate upon heating, while itself being reduced to Mo(IV). This method was applied for the separation, identification, and quantification of lipid extracts from porcine brain.

Development and Validation of HPTLC Method for the Estimation of Escitalopram oxalate and Flupentixol Dihydrochloride in Pharmaceutical Formulation

A simple, precise, accurate High-performance thin-layer chromatography (HPTLC) method has been developed and validated for the estimation of escitalopram oxalate (ESC) and flupentixol dihydrochloride (FLU) in pharmaceutical formulation. Separation was achieved on an aluminum HPTLC plate coated with a 0.2 mm layer of silica gel 60 F254 with chloroform: methanol 4:6 (v/v) as mobile phase. The densitometry analysis was carried out in absorbance mode at 254 nm. The Rf values of escitalopram and flupentixol were 0.24 and 0.44 respectively. The linearity of the method was achieved in the range 3-7 µg/spot for ESC with correlation coefficient value 0.998 and 0.3-0.7µg/spot for FLU with correlation coefficient value 0.999. The developed method was validated according to ICH guidelines and the results were statistically validated. The limit of detection and quantification for escitalopram oxalate and flupentixol dihydrochloride was found to be 500 ng/spot and 50 ng/spot, 1000 ng/spot, and 100 ng/spot respectively.

HPTLC determination of ergosterol in wheat and structure elucidation by NMR: Toward confirming method selectivity

Ergosterol (ERG) is considered as an excellent fungal biomarker, therefore it was utilized for the determination of the degree of fungi infection in wheat grains. A novel, fast, cost-effective and selective high performance thin layer chromatography (HPTLC) with reflectance/absorbance densitometry method was developed and validated for ERG determination. Separation was attained on pre-coated silica gel HPTLC plate with ethyl acetate: petroleum ether (6: 4 v/v) as a mobile phase. This system gave compact bands at retardation factor (Rf) of 0.45 ± 0.002 and quantification was performed at 282 nm over concentration range of 40–600 ng/band. The lower detectable limit was 10.79 ng per band which is three times more sensitive than the previously reported TLC method. The developed method could be utilized efficiently for the detection of ERG in pure powders and in fungi infected wheat samples. Resolution of ERG from other compounds in wheat grain extract allowed utilizing preparative TLC for separating and purifying ERG in a sufficient quantity for structure elucidation by nuclear magnetic resonance spectroscopy (NMR) for the first time. H1 and C13 NMR spectra indicated the purity of ERG extracted from the scrapped silica of preparative TLC plate at the Rf of the standard ERG verifying the method selectivity.