Densitometric High-performance Thin-layer Chromatographic Method Research Articles

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.

Development and validation of a high-performance thin-layer chromatography‒densitometric method and mass spectroscopy profiling for the determination of bioactive phytosterol from Manilkara zapota L. P. Royen leaves and correlating its antioxidant and antiinflammatory potential

Development and validation of a high-performance thin-layer chromatography‒densitometric method and mass spectroscopy profiling for the determination of bioactive phytosterol from Manilkara zapota L. P. Royen leaves and correlating its antioxidant and antiinflammatory potential

Isolation, identification, and quantification of stigmasterol in Hygrophila schulli plant by a validated high-performance thin-layer chromatography‒densitometric method

Isolation, identification, and quantification of stigmasterol in Hygrophila schulli plant by a validated high-performance thin-layer chromatography‒densitometric method

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.

Development and validation of a high-performance thin-layer chromatography densitometric method for the simultaneous determination of novel 1-acridinyl-1,2,3-triazole derivatives

High-performance thin-layer chromatographic (HPTLC) method provides a simple, sensitive, and accurate analytical method for the simultaneous determination of certain synthesized 1-acridinyl-1,2,3-triazole derivatives without interference from starting materials and intermediates. Separation was carried out on Merck HPTLC silica gel 60F254 plates, using chloroform‒methanol (9:1, V/V) and hexane‒ethyl acetate (3:2, V/V) as mobile phases. Validation of the method was performed based on the basis of the International Council for Harmonisation (ICH) guidelines in terms of linearity, sensitivity, limit of detection, limit of quantification, precision, selectivity, and specificity. Least-square equations were calculated for the studied compounds in the ranges of 25–500 and 10–500 ng/spot for ultraviolet (UV) and fluorescence measurements, respectively. Correlation coefficients (r) values were found ranging from 0.9913 to 0.9992 for analytes. The method provides selectivity and specificity which ensure that synthesized compounds are in the pure form without the interference of starting materials and intermediates. The detection limits for the studied compounds ranged from 11.02 to 51.09 ng/spot and 3.84 to 31.95 ng/spot and quantification limits were 33.39–154.82 ng/spot and 11.63–73.67 ng/spot for both spectrophotometric and spectrofluorimetric methods, respectively, indicating applicability for good qualitative and quantitative determination of members of this series at the nanogram concentration levels in biological fluids.

HPTLC-Densitometric Method for Simultaneous Estimation of Olmesartan medoxomil and Chlorthalidone in Tablet Dosage Form

The principle motive behind this work is the densitometric high-performance thin-layer chromatography (HPTLC) method has been developed and validated for the simultaneous estimation of Olmesartan medoxomil and Chlorthalidone in three different brands of the tablet dosage form. The method involves silica gel 60 F254 high-performance thin-layer chromatography and densitometric detection at 238 nm using Toluene: Ethyl acetate: Methanol: Glacial acetic acid (5:4.7:0.3:0.1 % v/v/v/v) as a mobile phase. The calibration curve was linear over a range of 100-1200 ng/band for Olmesartan medoxomil and 62.5-750 ng/band for Chlorthalidone. Precision provided relative standard deviations (RSD) lower than 2 % of both analytes. Percent recoveries were performed and found to be 99.53-99.90 % for Olmesartan medoxomil and 98.18-99.49 % for Chlorthalidone. Validation of the method was carried out as per International Conference on Harmonization (ICH) guideline Q2 (R1). The HPTLC densitometric driven method is highly suitable for analysis of Olmesartan medoxomil and Chlorthalidone in dosage form without any interference.

A Validated HPTLC Densitometric Method for Determination of Lupeol, β-Sitosterol and Rotenone in Tephrosia purpurea: A Seasonal Study.

Tephrosia purpurea (L.) Pers., commonly known as "sarpunkha" and "wild indigo", is being used in traditional systems of medicine to treat liver disorders, spleen and kidney. In the present study, a validated High Performance Thin Layer Chromatography (HPTLC) method was established for the estimation of lupeol, β-sitosterol and rotenone in various extracts of T. purpurea with the aim to see the effect of seasons on the quantity of aforesaid phytoconstituents. The plant material was collected in summer (April), rainy (August) and winter (December) during 2013-2014 from Lucknow, India. The method was validated in terms of precision, repeatability, specificity, sensitivity linearity and robustness. The method permits reliable quantification and showed good resolution on silica gel with toluene-ethyl acetate-formic acid (9:1:1v/v/v) as mobile phase, and characteristic bands of β-sitosterol, rotenone and lupeol were observed at Rf 0.38, 0.45 and 0.52, respectively. The content of aforesaid phytoconstituents varies from season to season and extract to extract. Our finding indicated that winter season (December) may not be appropriate for collection of T. purpurea for the preparation of therapeutic formulations because of the high content of rotenone, a known insecticide that is responsible for Parkinson's disease and associated with heart failure, fatty liver and liver necrosis.

Jatropha curcas poisoning in children in western Kenya – a case report

A new HPTLC densitometric method for the assay of griseofulvin tablets was developed and validated according to ICH guidelines and USP specifications. The method was developed using a mobile phase prepared with an environmentally friendly solvent system, diethyl ether: toluene (4:1) on pre-coated TLC silica gel 60F254 glass plates with a saturation time of 25 min. Development time of 10 min was required for a migration distance of 70 mm. The best detection wavelength was 299 nm. The Rf value was 0.22 and there were no interferences from the excipients or solvents. With regard to repeatability and intermediate precision, the RSD values were 1.43 and 1.58, respectively. Linearity testing gave the polynomial R2 values, calculated on three consecutive days, of 0.9910, 0.9801 and 0.9840, respectively. The accuracy values tested at 80 %, 100 % and 120 % concentrations were between 98.11-102.66 %. The developed HPTLC densitometric method for the assay of griseofulvin tablets is simple, selective, accurate, reproducible, high throughput, cost-effective and environmentally friendly.Keywords: High Performance Thin Layer Chromatography (HPTLC), griseofulvin, densitometry, validation

English

Essential oils of the fruits of three cultivars of Amomum subulatum (Family-Zingiberaceae), such as varlangy, seremna and sawney were isolated. Antioxidant potential was determined using 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) scavenging and FeCl3-reducing models and the presence of 1,8-Cineole was quantified using the developed high performance thin layer chromatography (HPTLC) method. In the DPPH scavenging model, the IC50 value of the essential oil of the seremna, varlangy and sawney were found to be 172.3, 216.9 and 274.3 μg/ml, respectively, while in the ABTS scavenging model, the IC50 value of the seremna, varlangy and sawney were found to be 27.96, 31.34 and 32.49 μg/ml respectively. The antioxidant power of the FeCl3 reducing model, the absorbance value of the essential oil of the seremna was found comparatively higher than the essential oils of varlangy and sawney. The content of 1,8-cineole was determined by the developed HPTLC densitometric method, and the value of the percentage (mean ± SD, % w/w) content of the essential oils  of the seremna, varlangy and sawney were found to be 69.59 ± 1.45%, 48.78 ± 3.21 and  47.84 ± 1.76 respectively. The highest antioxidant value of seremna cultivar may be due to the presence of high content of 1, 8-cineole. The HPTLC method, therefore confirms that monoterpene 1,8-cineole is the main antioxidant compound present in the fruits of A. subulatum.   Key words: Amomum subulatum, cultivars, essential oil, 1,8-cineole, high performance thin layer chromatography (HPTLC), antioxidants. &nbsp

High-Performance Thin-Layer Chromatography of Four Sterols in a Medicinally Important Grass Species – Heteropogon contortus (L.) Beauv.

Presently, sterols like stigmasterol, β-sitosterol, campesterol, and ergosterol are identified and quantified from leaves, stem, and inflorescence of Heteropogon contortus. (L.) Beauv (family: Poaceae) from Rajasthan, India, using high-performance thin-layer chromatography (HPTLC). H. contortus is reported with various medicinal properties. In the present study, a HPTLC–densitometric method was developed for the quantification of sterols by using toluene‒methanol‒formic acid (9:1:0.3 v/v) as the mobile phase. Post-derivatization of the plates was done by using freshly prepared anisaldehyde–sulfuric acid, heated at 110–120°C and scanned at 530 nm absorbing wavelength. The maximum contents of stigmasterol, campesterol, and ergosterol were estimated in inflorescence extracts (11.64 ± 017, 19.90 ± 0.18, and 16.64 ± 0.81 mg g−1 of dry wt. w/w, respectively), whereas β-sitosterol was found maximum in leaves, i.e., 56.52 ± 1.25 mg g−1 of dry wt (w/w). This HPTLC method is found to be simple, precise, specific, s...

Simultaneous Quantification of Syringic Acid and Kaempferol in Extracts of Bergenia Species Using Validated High-Performance Thin-Layer Chromatographic-Densitometric Method.

A rapid, sensitive, selective and robust quantitative densitometric high-performance thin-layer chromatographic method was developed and validated for separation and quantification of syringic acid (SYA) and kaempferol (KML) in the hydrolyzed extracts of Bergenia ciliata and Bergenia stracheyi. The separation was performed on silica gel 60F254 high-performance thin-layer chromatography plates using toluene : ethyl acetate : formic acid (5 : 4: 1, v/v/v) as the mobile phase. The quantification of SYA and KML was carried out using a densitometric reflection/absorption mode at 290 nm. A dense spot of SYA and KML appeared on the developed plate at a retention factor value of 0.61 ± 0.02 and 0.70 ± 0.01. A precise and accurate quantification was performed using linear regression analysis by plotting the peak area vs concentration 100-600 ng/band (correlation coefficient: r = 0.997, regression coefficient: R(2) = 0.996) for SYA and 100-600 ng/band (correlation coefficient: r = 0.995, regression coefficient: R(2) = 0.991) for KML. The developed method was validated in terms of accuracy, recovery and inter- and intraday study as per International Conference on Harmonisation guidelines. The limit of detection and limit of quantification of SYA and KML were determined, respectively, as 91.63, 142.26 and 277.67, 431.09 ng. The statistical data analysis showed that the method is reproducible and selective for the estimation of SYA and KML in extracts of B. ciliata and B. stracheyi.

Simultaneous quantification of biomarkers bergenin and menisdaurin in the methanol extract of aerial parts of Flueggea virosa by validated HPTLC densitometric method.

A simple, sensitive high-performance thin-layer chromatography (HPTLC) method was developed for the simultaneous quantification of biomarker bergenin and menisdaurin in the methanol extracts of aerial parts of Flueggea virosa (FVME). Chromatography was performed on glass-backed silica gel 60F254 HPTLC plates using dichloromethane: methanol as mobile phase. Scanning and quantification was done at UV absorption maxima of 260 nm. The system was found to give compact spot for bergenin and menisdaurin at Rf = 0.29 ± 0.01 and 0.16 ± 0.01, respectively. The linearity ranges for bergenin and menisdaurin were found to be the same (100-800 ng/spot) with correlation coefficients (R(2) values) of 0.997 and 0.999, respectively. The limit of detection for bergenin and menisdaurin was found to be 27 and 36.2 ng/band, respectively, while the limit of quantification was found to be 81 and 108 ng/band, respectively. Intra- and interday precisions (n = 6) for bergenin and menisdaurin were found to be 1.41-1.71 and 1.65-1.87%, and 1.68-1.89 and 1.75-1.93%, respectively. The percent recoveries were found to be 98.7-99.4 and 99.5-99.9%, respectively, for bergenin and menisdaurin. The percentage of bergenin and menisdaurin was found to be 15.25 and 4.22% (w/w), respectively, in FVME. The developed method permitted the simultaneous quantification of bergenin and menisdaurin and showed good resolution and separation from other constituents of extract; hence, the method can be used to standardize herbal formulations as well as bulk drugs for bergenin and menisdaurin.

Development of a densitometric high-performance thin-layer chromatographic method for the quantitative analysis of biomarker lupeol in the leaves of different species of genusFicus

Summary A simple and sensitive high-performance thin-layer chromatographic (HPTLC) method was developed for the evaluation of biomarker lupeol in the leaves of five different species of genus Ficus (Ficus carica, Ficus nitida, Ficus ingens, Ficus palmata, and Ficus vest) belonging to family Moraceae. Chromatography was performed on glass-backed silica gel 60 F 254 precoated HPTLC plates with solvents toluene–methanol (9:1, v/v) as the mobile phase. After development, the HPTLC plate was derivatized with p-anisaldehyde reagent, scanned, and quantified at 540 nm. The system was found to give compact spot for lupeol at RF = 0.32 ± 0.01. The precisions and accuracy (n = 6) for lupeol were found to be 1.47–1.64% and 1.63–1.86%, and 99–100% and 99.4–99.7%, respectively, for inter-day and intra-day. Lupeol was found to be present in four species, i.e., F. carica (0.4%, w/w), F. nitida (1.4%, w/w), F. palmata (0.33%, w/w), and F. vest (0.59%, w/w), while it was absent in F. ingens. The statistical analysis proved that the developed method is reproducible and selective. The developed method can be used as an important tool to assure the therapeutic dose of active ingredients in herbal formulations as well as for standardization and quality control of bulk drugs and in-process formulations. This method can also be employed for the further study of degradation kinetics and determination of lupeol in plasma and other biological fluids.

Simultaneous quantitation of piperine and piperlongumine in the fruit ofPiper longumLinn. by validated high-performance thin-layer chromatography-densitometric method

The fruit of Piper longum Linn. (family: Piperaceae), known as pippali in India, is a reputed drug of Ayurveda (the Indian system of medicine). It contains many amides of piperic acid among which piperine and piperlongumine are important from biological point of view. In the present work, we quantified these two marker compounds from the fruits of P. longum by high-performance thin-layer chromatography (HPTLC) with densitometry. The method was found to be precise. Relative standard deviation (RSD) values for intra-day analyses were in the range of 1.05 to1.07% (piperine) and 1.12 to 1.16% (piperlongumine) and, for inter-day analyses, the values were in the range of 1.11 to 1.21% (piperine) and 1.02 to1.18% (piperlongumine). Instrumental RSD values were 0.64 and 0.78% for piperine and piperlongumine, respectively. Accuracy of the method was evaluated by carrying a recovery study at three different levels for the two compounds. Average recoveries were found to be 99.23% for piperine and 99.26% for piperlong...

Stability-indicating densitometric high-performance thin-layer chromatographic method for the quantitative analysis of biomarker naringin in the leaves and stems ofRumex vesicariusL.

A simple, sensitive, and stability-indicating high-performance thinlayer chromatography (HPTLC)-densitometric method was developed for the quantification of biomarker naringin in the methanol extracts of stems and leaves of Rumex vesicarius. Chromatography was performed on glass-backed silica gel 60 F254 high-performance thin-layer chromatography (HPTLC) plates with ethyl acetateglacial acetic acid-MeOH-H2O (30:10:5:1, ½/½) as mobile phase. Scanning and quantification were done at 275 nm. The system was found to give compact spot for naringin at RF = 0.46 ± 0.001. The linear regression analysis data for the calibration plots showed good linear relationship with r2 = 0.998 with respect to area in the concentration range of 100–1000 ng. The regression equation of standard was found to be Y = 3.438X + 38.485. Naringin was subjected to acid and alkali hydrolysis, peroxide oxidation, photodegradation, dry heat, moist heat, and ultraviolet (UV) treatment. The drug undergoes complete degradation under acidic tre...

Simultaneous Quantification of Diarylheptanoids in Alnus nepalensis Using a Validated HPTLC Method

Platyphylloside, oregonin and hirsutanonol 5-O-β-D-glucopyranoside are known bioactive metabolites in Alnus nepalensis. In this article, the aforementioned three markers were isolated and simultaneously quantified by the thin-layer chromatography densitometric method. A sensitive, selective and robust qualitative and quantitative densitometric high-performance thin-layer chromatographic method was developed and validated for the determination of above markers in the leaves of A. nepalensis. The separation was performed on silica gel 60F254 high-performance thin-layer chromatography plates using chloroform:methanol:formic acid (75:25:2, v/v) as mobile phase. The quantitation of diarylheptanoids was carried out using the densitometric reflection/absorption mode at 610 nm after post-chromatographic derivatization with vanillin-sulfuric acid reagent. A precise and accurate quantification can be performed in the linear working concentration range of 333-3330 ng/spot with good correlation (r(2) = 0.999). The method was validated for peak purities, precision, robustness, limit of detection and quantitation, etc., as per ICH guidelines. Specificity of quantitation was confirmed using retention factor and spectra correlation of markers in standard and sample tracks.

Antifilarial diarylheptanoids from Alnus nepalensis leaves growing in high altitude areas of Uttarakhand, India

Lymphatic filariasis continues to be a major health problem in tropical and subtropical countries. A macrofilaricidal agent capable of eliminating adult filarial parasites is urgently needed. Platyphyllenone (A), alusenone (B), hirustenone (C) and hirsutanonol (D) are important biologically active diarylheptanoids present in Alnus nepalensis. In the present study, we report the antifilarial activity in diarylheptanoids isolated from the leaves of A. nepalensis. Out of four compounds (A–D) tested in vitro one has shown promising anti-filarial activity both in vitro and in vivo studies. This is the first ever report on antifilarial efficacy of a compound of the plant and warrants further studies around this scaffold.In addition, a sensitive, selective and robust densitometric high-performance thin-layer chromatographic method was developed and validated for the above four biomarker compounds. The separation was performed on silica gel 60F254 high-performance thin layer chromatography plates using chloroform:methanol (9:1, v/v) as mobile phase. The quantitation of marker compounds was carried out using densitometric reflection/absorption mode at 600nm after post-chromatographic derivatization using vanillin–sulfuric acid reagent. The method was validated for peak purity, precision, robustness, limit of detection (LOD) and quantitation (LOQ) etc., as per the International Conference on Harmonization (ICH) guidelines.

An HPTLC-densitometry method for the quantification of pharmacologically active alkaloids inSceletium tortuosumraw material and products

Mesembrine-type alkaloids are reported to be responsible for the psychoactive properties of Sceletium tortuosum. The aim of the study was to develop a simple, rapid, and precise high-performance thin-layer chromatography densitometric method for the simultaneous determination of mesembrenol, mesembranol, mesembrine, and mesebrenone in S. tortuosum raw materials and commercial products. Silica gel 60 F254 pre-coated glass plates were used with a mobile phase of dichloromethane-MeOH-10% NH3 (90:10:0.1, v/v). Densitometric quantification was performed in absorbance mode at 280 nm, and good separation was obtained using a single mobile phase. Regression analyses of the calibration data showed good linear relationships with regression coefficient (r2) values ranging from 0.994 to 0.999. The method was validated for linearity, accuracy, limit of detection, limit of quantification, and precision (repeatability). The results showed good correlation with data obtained from gas chromatography/mass spectrometry anal...

Simultaneous quantification of five marker compounds of Betula utilis stem bark using a validated high‐performance thin‐layer chromatography method

A sensitive, selective and robust densitometric high-performance thin-layer chromatographic method was developed and validated for five marker compounds, namely betulin, lupeol, oleanolic acid, 3-acetyloleanolic acid and β-sitosterol, known for their various therapeutic activities. The marker compounds have been isolated from the stem bark of Betula utilis, well characterized by the spectral analysis, and their simultaneous quantitative determination carried out by high-performance thin-layer chromatography (HPTLC) method. The resolution of marker compounds was carried out on silica-gel 60 plates, using n-hexane:ethyl acetate (8:2 v/v) as the mobile phase. The HPTLC densitometry was performed at 500-nm wavelength after the post chromatographic derivatization with ceric ammonium sulfate reagent. The optimized method provided good linear relation (r>0.9960) for all the investigated analytes. The method is simple, and reproducible, which may be applied for quantitative analysis of the above-mentioned marker compounds.

Effective separation and analysis of E- and Z-guggulsterones in Commiphora mukul resin, guggulipid and their pharmaceutical product by high performance thin-layer chromatography-densitometric method

A high performance thin-layer chromatographic (HPTLC) method for the simultaneous determination of the hypolipidemic agents, E- and Z-isomers of guggulsterone in Commiphora mukul resin, guggulipid (ethyl acetate extract of resin), and its pharmaceutical formulation, was developed. The developed system was efficient enough to separate both isomers from their conger, 17,20-dihydroguggulsterone ( 3). HPTLC glass plates, pre-coated with silica gel 60F-254, were used as a stationary phase. The mobile phase consisted of toluene:acetone (9.3:0.7, v/v) which gave well resolved spots for E- and Z-guggulsterones ( R f: 0.52 ± 0.01, and 0.67 ± 0.01, respectively) following double development of chromatoplate with the same mobile phase under unsaturated conditions. The analyte stability towards the developed chromatographic procedure was also investigated by two-dimensional (2D) HPTLC analysis. 17,20-Dihydroguggulsterone ( 3) was identified by the electrospray ionization quadrupole time-of-flight tandem mass spectrometry (ESI–QTOF–MS/MS) analysis.