High-performance Thin-layer Chromatographic Method Research Articles

Estimation of Active Components in Gokshura Tablet and Pushyanug Churna Formulation using High-performance Thin Layer Chromatography Method

Gokshura tablet is an ayurvedic formulation with gokhru (Tribulus terrestris) as best fixing recommended for building vitality levels. It enhances life, sexual want and drive. Pushyanug churna is an ayurvedic polyherbal formulation, hence this seems essential to explain the material institutionalization, various bioactive markers, blends exhibit in the polyherbal ayurveda compositions such as pushyanug churna. Point of the exhibit effort has been to create what’s more, approve a high-performance thin layer chromatography (HPTLC) strategy for assurance of diosgenin present in gokshura tablet. Mangiferin and chlorogenic acid are present in pushyanug churna. Diosgenin, a biomarker chemical found in gokshura tablets, and mangiferin, a biomarker compound found in pushyanug churna, were standardized using recently developed easy and accurate HPTLC procedures. Pre-coated silica gel 60-F254 was employed at the stationary phase and a mixture of toluene, ethyl acetate, and formic acid (in the proportions 5:4:1) was employed for the mobile phase in the development methodology for diosgenin. In the mobile phase, mangiferin, ethyl acetate and methanol were added in a ratio of 40:60 v/v were utilized. In the chlorogenic acid mobile phase, ethyl acetate:formic acid:acetic acid:water (10:1.1:1.1:2.6 v/v). It was determined that the Rf value of the marker chemical was 0.77 (diosgenin) in gokhsura tablet and 0.23 mangiferin, 0.75 chlorogenic acid in pushyanug churna. For bioactive marker chemicals found in in-house and commercially available formulations, the developed HPTLC approach has shown to be straightforward, sensitive, specific, and dependable.

Rapid Smartphone-based Image Reading for Assessing Quality of Active Pharmaceutical Ingredients of Commonly Prescribed Drugs-Lusaka Zambia

Third world countries suffer from supply of substandard therapeutic drugs and due to lack of robust quality testing facilities, many of these drugs find themselves in the dispensaries and various outlets for over the counter purchase. In order to ensure quality, simple yet informative tests are available and one of them is high performance thin layer chromatography. High Performance Thin Layer Chromatography (HPTLC) coupled with image acquisition by TLC software application on a smart phone and analysis by freeware Digitally enhanced Thin Layer Chromatography (DeTLC) respectively was performed using the minilab protocols for assessing quality of drugs dispensed at the University of Zambia clinic as well as drugs randomly bought from different pharmacies around Lusaka, Zambia. The quality of drugs was assessed by comparing retardation factors with standards and approximate percent active ingredient amounts were compared using the maximal K intensities derived by the DeTLC software for image analysis. Normalizing the spots to the 100% spot, mean values with respective standard deviations were : atenelol (85.6±0.5), ciprofloxacin (95.6±1.1), ketoconazole (78.2±1.1), mebendazole (95.5±1.1), nevirapine (96.7±1.1), penicillin v (100.5±1.1), quinine (92.9±1.1), rifampicin (99.3±0.7) and isoniazid (72.0±4.1). All the drugs tested were within the acceptable range of concentrations of the active pharmaceutical ingredient ( 0 to 5.3% Rf error). Thus, the simple robust HPTLC method coupled with simple to use digital imaging software provides a quick means to check on quality of the drugs dispensed.

Development of a validated high-performance thin-layer chromatography method for the standardization of an Ayurvedic formulation using berberine and ursolic acid

Development of a validated high-performance thin-layer chromatography method for the standardization of an Ayurvedic formulation using berberine and ursolic acid

Development and validation of a novel high-performance thin-layer chromatography method for the quantitative estimation of neohesperidin from Citrus aurantium peel extract

Development and validation of a novel high-performance thin-layer chromatography method for the quantitative estimation of neohesperidin from Citrus aurantium peel extract

Estimation of betulinic acid from wild fruit extracts of Ziziphus mauritiana and Ziziphus nummularia from different regions of North India by a validated high-performance thin-layer chromatography method

Estimation of betulinic acid from wild fruit extracts of Ziziphus mauritiana and Ziziphus nummularia from different regions of North India by a validated high-performance thin-layer chromatography method

Development and validation of a high-performance thin-layer chromatography method for the estimation of bromfenac in ophthalmic solution

Development and validation of a high-performance thin-layer chromatography method for the estimation of bromfenac in ophthalmic solution

Development and validation of a high-performance thin-layer chromatography method for the simultaneous quantification of rosmarinic acid, quercetin, glycyrrhizin and betulinic acid in polyherbal immunostimulant formulation

Development and validation of a high-performance thin-layer chromatography method for the simultaneous quantification of rosmarinic acid, quercetin, glycyrrhizin and betulinic acid in polyherbal immunostimulant formulation

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.

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 SIMPLE AND VALIDATED HP-TLC METHOD FOR SIMULTANEOUS ANALYSIS OF ETHNO-MEDICINE GALLIC ACID AND EUGENOL

A new method has been developed and validated for the simultaneous estimation of gallic acid and eugenol for the poly-herbal formulation. The HP-TLC (High Performance Thin Layer Chromatography) method was developed using pre-coated silica gel 60 F254 on aluminum plate and the solvent system used was iso propyl alcohol : hexane: ethyl acetate: glacial acetic acid (50:30:30:1 V/V) and scanned at 254 nm wavelength. The Rf value for gallic acid was 0.608±0.041 and 0.752±0.035 for eugenol. The range of linearity detected for gallic acid and eugenol was 2-10 ng µL-1. The method was validated in accordance with ICH (International Council for Harmonization) guidelines for linearity, range, LOD, LOQ, precision, specificity and robustness. The developed method was found to be easy, simple, selective, specific and robust.

ICH and US-FDA validated HPTLC methods with greenness assessments for the assay of mixtures prescribed in stroke prophylaxis: application to pharmaceutical preparations and human plasma

The importance of the binary mixtures of the novel oral anticoagulants (NOACs): apixaban (APX), edoxaban tosylate (EDX) and rivaroxaban (RIV) with the lipid-lowering statin, rosuvastatin calcium (ROS) is highly emerging to save lives of cardiovascular patients as these combinations are used in prophylaxis from stroke. A high-performance thin-layer chromatography (HPTLC) method was developed for the quantitative assay of these life-saving mixtures in tablets and human plasma. Two mobile phases were developed for the assay in bulk and tablets; the first one: toluene‒ethyl acetate‒methanol‒25% ammonia (3.5:4.5:2:0.2, V/V) (method I) used for the three mixtures, and the second one: methanol‒25% ammonia (9.95:0.05, V/V) (method II) used for EDX/ROS mixture only. For analysis in human plasma, APX was used as internal standard in RIV/ROS and EDX/ROS mixtures using methods I and II, respectively, while RIV was used as internal standard in APX/ROS mixture using method I; the methods were validated according to the Food and Drug Administration (FDA) regulation for analysis in biological fluids. The method selectivity was demonstrated by its ability to simultaneously analyze the drugs in the presence of dosage form excipients and in the presence of plasma interferences (analysis in biological fluid) at single wavelength (291 nm) by use of the internal standard.

Screening of elite germplasms for industrially valuable medicinal crop Stevia rebaudiana for stevioside and rebaudioside A production: An HPTLC-linked chemotaxonomic assessment

Stevia rebaudiana Bertoni (Asteraceae) is a South American perennial herb native to the Paraguay region. It is high-value commercial crop in the food industry because of an array of bioactive diterpenoid glycosides, the most important of which are the steviol glycosides, stevioside (Stev) and rebaudioside A (Reb A). Standardized extract from Stevia leaves, which is high in steviol glycosides, can be used as a high-potency sweetener. The current study proposes a high-performance thin layer chromatography (HPTLC) approach for estimating stevioside and rebaudioside that is rapid, verified, and repeatable. A collection of 32 chemotypes of Stevia rebaudiana were collected from 32 districts across nine Indian states. Stevioside and rebaudioside A content ranged between 1.43 - 5.88 % and 0.77 - 1.95 % respectively across 32 chemotypes of S. rebaudiana . Among all the studied samples, SrP1 chemotype was found to possess the highest quantity of stevioside (5.88 %) and rebaudioside A (1.95 %), followed by SrH11 (5.28 % Stev; 1.89 % Reb A) chemotypes obtained from Jalandhar, Punjab and Palampur, Himachal Pradesh respectively. Chemotype SrWb32 (1.43 % Stev; 0.77 % Reb A) collected from Bardhaman, West Bengal exhibited least amount of stevioside and rebaudioside A. The current fast, reproducible, and validated HPTLC method presents an efficacious toolkit to explore the quantitative variation in the stevioside and rebaudioside A contents in natural S. rebaudiana chemotypes. This methodology may be employed to screen high stevioside and rebaudioside A yielding elite genotypes for future propagation as well as commercialization of these highly potent sweeteners to deal with the ever-expanding demand for alternative yet natural sweeteners in food industries. • Thirty two populations or chemotypes of Stevia rebaudiana were analysed to detect steviol glycosides. • Chemotypes of the plant revealed altitudinal variation in steviosides and rebaudisides A content. • Elite chemotypes can further be used for propagation and commercial utilization.

Development and validation of a high-performance thin-layer chromatographic method for the simultaneous estimation of berberine, gallic acid, mangiferin, and quercetin in Amritamehari churnam

Development and validation of a high-performance thin-layer chromatographic method for the simultaneous estimation of berberine, gallic acid, mangiferin, and quercetin in Amritamehari churnam

High-performance thin-layer chromatography method development and validation for quantification of naringin in different extracts of Citrus sinensis L. and its antioxidant activity

High-performance thin-layer chromatography method development and validation for quantification of naringin in different extracts of Citrus sinensis L. and its antioxidant activity

Cytochrome P450 3A4-mediated pharmacokinetic interaction study between tadalafil and canagliflozin using high-performance thin-layer chromatography.

Diabetes is a known risk factor for sexual dysfunction in men; diabetic men have an increased risk of erectile dysfunction compared to non-diabetic men. Canagliflozin is one of the common antidiabetic drugs that is readily used in the treatment of type-2 diabetes. Concomitantly phosphodiesterase 5 inhibitors, such as tadalafil, can be given to the patient to alleviate erectile dysfunction. Canagliflozin is reported to be one of the cytochrome P450 3A4 enzyme inhibitors, that might seriously influence blood concentration levels of tadalafil but there is no study till now, discussing this interaction. Therefore, a fast, simple, and sensitive high-performance thin-layer chromatographic method was developed, validated, and applied for the simultaneous determination of tadalafil and canagliflozin in spiked and real human plasma. The limit of detection for tadalafil was 0.14ng/band and for canagliflozin was 0.16ng/band. The limit of quantitation value for tadalafil was 0.43ng/band and for canagliflozin was 0.47ng/band. Tadalafil and canagliflozin were determined simultaneously in real human plasma using the described procedure and the method was applied for in vivo pharmacokinetic drug interaction study between the studied drugs, which proved significant interaction between them when administered simultaneously.

Comparison of Validation Parameters for the Determination of Vitamin D3 in Commercial Pharmaceutical Products Using Traditional and Greener HPTLC Methods

Several analytical methods are documented for the estimation of vitamin D3 (VD3) in pharmaceuticals, food supplements, nutritional supplements, and biological samples. However, greener analytical methods for VD3 analysis are scarce in the literature. As a consequence, attempts were made to design and validate a greener “high-performance thin-layer chromatography (HPTLC)” method for VD3 estimation in commercial pharmaceutical products, as compared to the traditional HPTLC method. The greenness indices of both approaches were predicted by utilizing the “Analytical GREENness (AGREE)” method. Both traditional and greener analytical methods were linear for VD3 estimation in the 50–600 ng band−1 and 25–1200 ng band−1 ranges, respectively. The greener HPTLC strategy outperformed the traditional HPTLC strategy for VD3 estimation in terms of sensitivity, accuracy, precision, and robustness. For VD3 estimation in commercial tablets A–D, the greener analytical strategy was better in terms of VD3 assay over the traditional analytical strategy. The AGREE index of the traditional and greener analytical strategies was estimated to be 0.47 and 0.87, respectively. The AGREE analytical outcomes suggested that the greener analytical strategy had a superior greener profile to the traditional analytical strategy. The greener HPTLC strategy was regarded as superior to the traditional HPTLC methodology based on a variety of validation factors and pharmaceutical assays.

Phytochemical analysis of Clerodendron infortunatum Linn. leaves in various seasons by different solvent and extraction techniques

Clerodendron infortunatum Linn is widely used for the treatment of malaria and vermifuge, scorpion and snake bites, tumors, inflammation, bronchitis and in wound healing. The present study was carried out to estimate the variation in the amounts of Gallic acid, Rutin and Quercetin in Clerodendron infortunatum Linn.leaves extracts collected in different seasons using high-performance thin layer chromatography (HPTLC).In this study, leaves of C. infortunatum were subjected to methanol extractions using Soxhlet method and cold maceration. The leaves were collected in different seasons in the months of December (Sample A -Shishir rutu-Winter season) and July (Sample B -Varsha Rutu -Rainy season). The methanolic extracts were subjected to HPTLC study & using CAmag HPTLC system, employing a mixture of toluene:ethyl acetate:formic acid:methanol(3:6:1.6:0.4 ) as the mobile phase with densitometric scanning at 254nm. Quantity of Quercetin is found in both samples, extracted by Soxhlet method. Its quantity is higher in the winter season. Quantity of Gallic acidis found in both samples. Its quantity is the same in Sample B by Soxhlet extraction method & maceration method respectively. Quantity of Rutin is found in both the samples extracted by Soxhlet & maceration methods. It is higher in the sample-A. The present HPTLC method is used for the detection and quantization of Gallic acid, Quercetin and Rutin of C. infortunatum leaves. It needs to study the season wise and region wise variation of phytochemicals in useful parts of all other plants by different extraction methods.

An HP-TLC densitometric method and fingerprinting for estimating capecitabine and thymoquinone simultaneously and its application in nanoscience using Box–Behnken design

World Health Organization estimates that 10% of medications are of inferior quality, thus dangerous to human health. One way to avoid such a problem is to create an adequate, economical, and competitive analytical system. As a result, the study’s goal is to create a High-Performance Thin Layer Chromatography (HP-TLC) method for determining capecitabine (CAP) and thymoquinone (TQ) simultaneously. To the best of our knowledge, no such method for determining CAP and TQ simultaneously exists today. The method was created by implementing an analytical quality-by-design approach based on the Box–Behnken design (BBD) to optimize the chromatographic conditions and a combination of factors such as toluene volume (A), solvent front (B), and chamber saturation time (C), all of which were likely to affect the R f of CAP and TQ, respectively, later validated using TLC-silica coated plate 60 F254. The validated parameters were within an acceptable range, according to ICH guidelines. BBD design revealed that the volume of toluene and solvent front had a greater effect on all of the responses studied and thus needs to be controlled. The developed method of the analysis was found to be facile, dependable, expeditious, cost-effective, and could be used to quantify CAP and TQ in Nanoformulation.

Phytoconstituents of Nigella Sativa and Quantitative Densitometric Analysis of its Bioactive Compound Thymoquinone

Herbal plants are a reservoir of potential phytochemical compounds and the richest bioresource of drugs for traditional systems of medicine, nutraceuticals, food supplements, modern medicines, pharmaceutical intermediates, folk medicines, and chemical entities for synthetic drugs. In the present study we find out phytoconstituents of Nigella sativa and quantitative densitometric analysis of its bioactive compound thymoquinone in the different solvent extracts. It was found that Nigella sativa seeds were extracted with ethanol, methanol, and benzene as solvents. Phytochemical analysis showed the presence of potent bioactive constituents such as alkaloids, phenols, tannins, trepenoid, saponins, and steroids in methanol extract. Benzene extracts have only alkaloids and steroids. While ethanol extract showed the presence of alkaloids, phenols, tannins, proteins, amino acids, flavonoids, terpenoids, saponins, and steroids. The high-performance thin layer chromatographic method (HPTLC) was employed to quantify and densitometrically analyze thymoquinone in methanol, ethanol, and benzene extract of Nigella sativa. The analysis was performed on an aluminum plate with a mobile phase of n-hexane: ethyl acetate: methanol (7:2:1 v/v/v) and a densitometric measurement using a TLC scanner (CAMAG) at 254 nm. The ethanol extract of N. Sativa exhibited single sharp peak of thymoquinone with 0.85 Rf value, the highest area of the band 8137.6, and a total recovery of was 98.08% which is nearly equal to the standard thymoquinone with Rf value (0.85), the highest area of the band 8789.4 and total recovery was obtained 100%. The present research indicated that purified thymoquinone from N. sativa is a potential source for therapeutic application. Keywords: Phytoconstituents, Herbal extract, Essential oil, Quantification, Chromatography.

Genome size and gas chromatography-mass spectrometry (GC-MS) analysis of field-grown and in vitro regenerated Pluchea lanceolata plants.

Pluchea lanceolata is a threatened pharmacologically important plant from the family Asteraceae. It is a source of immunologically active compounds; large-scale propagation may offer compounds with medicinal benefits. Traditional propagation method is ineffective as the seeds are not viable; and root sprout propagation is a slow process and produces less numbers of plants. Plant tissue culture technique is an alternative, efficient method for increasing mass propagation and it also facilitate genetic improvement. The present study investigated a three-way regeneration system in P. lanceolata using indirect shoot regeneration (ISR), direct shoot regeneration (DSR), and somatic embryo mediated regeneration (SER). Aseptic leaf and nodal explants were inoculated on Murashige and Skoog (MS) medium amended with plant growth regulators (PGRs), 2,4-dichlorophenoxy acetic acid (2,4-D), 1-naphthalene acetic acid (NAA), and 6-benzyl amino purine (BAP) either singly or in combinations. Compact, yellowish green callus was obtained from leaf explants in 1.0 mg/l BAP (89.10%) added medium; ISR percentage was high, i.e., 69.33% in 2.0 mg/l BAP + 0.5 mg/l NAA enriched MS with 4.02 mean number of shoots per callus mass. Highest DSR frequency (67.15%) with an average of 5.62 shoot numbers per explant was noted in 0.5 mg/l BAP added MS medium. Somatic embryos were produced in 1.0 mg/l NAA fortified medium with 4.1 mean numbers of somatic embryos per culture. On BAP (1.0 mg/l) + 0.5 mg/l gibberellic acid (GA3) amended medium, improved somatic embryo germination frequency (68.14%) was noted showing 12.18 mean numbers of shoots per culture. Histological and scanning electron microscopic (SEM) observation revealed different stages of embryos, confirming somatic embryogenesis in P. lanceolata. Best rooting frequency (83.95%) of in vitro raised shootlets was obtained in 1.0 mg/l IBA supplemented half MS medium with a maximum of 7.83 roots per shoot. The regenerated plantlets were transferred to the field with 87% survival rate. The 2C genome size of ISR, DSR, and SER plants was measured and noted to be 2.24, 2.25, and 2.22 pg respectively, which are similar to field-grown mother plant (2C = 2.26 pg). Oxidative and physiological events suggested upregulation of enzymatic activities in tissue culture regenerated plants compared to mother plants, so were photosynthetic pigments. Implementation of gas chromatography-mass spectrometry (GC–MS) technique on in vivo and in vitro raised plants revealed the presence of diverse phyto-chemicals. The yields of alpha amyrin and lupeol (medicinally important triterpenoids) were quantified using high-performance thin-layer chromatography (HPTLC) method and enhanced level of alpha amyrin (2.129 µg g−1 dry wt) and lupeol (1.232 µg g−1 dry wt) was noted in in vitro grown leaf tissues, suggesting in vitro conditions act as a potential trigger for augmenting secondary metabolite synthesis. The present protocol represents a reliable mass propagation technique in producing true-to-type plants of P. lanceolata, conserving 2C DNA and ploidy successfully without affecting genetic homogeneity.Supplementary InformationThe online version contains supplementary material available at 10.1007/s13353-022-00727-7.