Validated HPTLC Method Research Articles

Validated HPTLC Method for Simultaneous Estimation of Lafutidine and Domperidone in a Tablet Dosage Form

A reliable simple, precise, accurate and rapid high performance thin layer chromatographic method has been developed and validated for estimation of lafutidine (LF) and domperidone (DP) in combined tablet dosage forms. The analysis was performed on 10×20 cm aluminium-precoated plates coated with 0.2 mm layers of silica gel 60 F 254 (E-Merck, Germany) with ethyl acetate:methanol:toluene:acetone:glacial acetic acid (1.0:1.5:4.0:2.0:0.2 v/v/v/v/v) as mobile phase. Camag TLC Scanner III was used for the UV densitometric scanning at 285 nm. This system was found to give a compact spot of LF and DP at retention factor (R f ) value of 0.32±0.03 and 0.65±0.02 respectively. Linear regression analysis data for the calibration curve showed good relationship with respect to peak area in the concentration 100-500ng per spot for both LF and DP with (r 2 = 0.9993) and (r 2 = 0.9985) for LF and DP respectively. Limit of detection (15.51 ng/spot for LF and 23.04 ng/spot for DP), limit of quantication (47.03 ng/spot for LF and 69.83 ng/spot for DP) were found. The proposed HPTLC method has potential applications in determination of lafutidine and domperidone in tablet formulations.

Phytochemical investigation and simultaneous estimation of bioactive lupeol and stigmasterol in Abutilon indicum by validated HPTLC method

Md. Sarfaraj Hussain*, Sheeba Fareed, Mohammad Ali, Md. Sarfaraz Alam, Md. Akhlakquer Rahman, A. K. Srivastava Faculty of Pharmacy, Integral University, Dasauli, Kursi Road, Lucknow-226026, Uttar Pradesh, India Department of Pharmacognosy & Phytochemistry, Faculty of Pharmacy, Hamdard University, Hamdard Nagar, New Delhi-110062, India Department of Biotechnology, Integral University, Dasauli, Kursi Road, Lucknow-226026, Uttar Pradesh, India College of Pharmacy, Jazan University, Jazan, K.S.A. Journal of Coastal Life Medicine 2014; 2(5): 394-401

Optimization of Ultrasonic-Assisted Extraction of Gymnemic Acid from Gymnema sylvestre Leaves Using Response Surface Methodology

Gymnemic acid (GA) the main phytoconstituent of Gymnema sylvestre is known to possess diverse pharmacological activities, that prompted the development of ultrasonic-assisted extraction (UAE) for its better utilisation. The main aim of the present study was to maximize the extraction of GA from the leaves of G. sylvestre and its estimation as gymnemagenin by HPTLC. Response surface methodology (RSM) was used for the optimization of UAE conditions. The studied factor included: solvent composition, extraction temperature and time. RSM was based on a three-level, three-variable Box-Behnken design (BBD). The optimal conditions for UAE predicted by BBD were: extraction with methanol-water (81:19 v/v), at 49°C, for 50 min. Confirmation trials under slightly adjusted conditions yielded 121.81±1.86 mg/g, which was in a good agreement with the predicted value of 123.49 mg/g of GA (on dry weight basis of leaves). The results of UAE were also compared with Soxhlet extraction method and former was found 3 folds more efficient than latter in extracting GA. A facile, safe and quick GA extraction strategy using ultrasonication was developed and the extraction efficiency of the process was established by a simple, validated HPTLC method.

Exploring validated HPTLC method of Rivaroxaban in tablet dosage form

Exploring validated HPTLC method of Rivaroxaban in tablet dosage form

Comparison of Conventional and Novel Extraction Techniques for the Extraction of Scopoletin from Convolvulus Pluricaulis

Conventional extraction techniques like soxhlet extraction and reflux extraction and novel extraction techniques like Supercritical Fluid extraction (SFE), Ultrasonic Assisted Extraction (UAE) and microwave assisted extraction method (MAE) were used for the extraction of Convolvulus Pluricaulis. The scopoletin content in the extracts obtained using different extraction techniques was estimated using pre validated HPTLC method. The conventional and novel extraction techniques were compared on the basis of various parameters like extraction time, organic solvent consumption and extraction yield. The novel extraction techniques were found to be better than conventional methods as they require less time, consume less solvent and give higher yields of scopoletin. From amongst the novel extraction techniques used Microwave assisted extraction was found to give highest yield of scopoletin.

Screening and determination of sibutramine in adulterated herbal slimming supplements by HPTLC-UV densitometry

The adulteration of herbal supplements is of growing importance, especially when they contain undeclared compounds like sibutramine that are unsafe drugs. Sibutramine was withdrawn from US and European markets in 2010. In this study, an HPTLC-UV densitometric method was developed for the quantification of sibutramine in herbal diet foods. Sample extracts were directly applied onto HPTLC silica gel plates and separated with a mobile phase made of a toluene–methanol mixture. Sibutramine was quantified at 225 nm and its unequivocal identification was confirmed by MS using a TLC-MS interface. During two surveys, 52 weight loss supplements obtained via the Internet were screened. Half of those were adulterated with sibutramine at amounts reaching up to 35 mg per capsule. The results of this validated HPTLC method were compared with those obtained by HPLC-UV and HPLC-MS/MS. The results were not significantly different with the three methods.

Quantification of Physiologically Available Glycyrrhizin in Anti-Stress Herbal Formulations by Validated HPTLC Method

Quantification of Physiologically Available Glycyrrhizin in Anti-Stress Herbal Formulations by Validated HPTLC Method

Estimation of ursolic acid from Urtica dioica L. using validated HPTLC method

Quantitative analysis of valsartan in tablets formulations by High Performance Thin-Layer ChromatographyDella Grace Thomas Parambi, Molly Mathew, V.Ganesan

Development and Validation of HPTLC Method for Simultaneous Estimation of Metolazone and Spironolactone in Bulk Drug and Pharmaceutical Dosage Form

A simple, precise and accurate HPTLC method has been developed for the simultaneous analysis of Metolazone and Spironolactone in bulk drugs and tablet formulation. Chromatographic separation was carried out on Merck HPTLC aluminium plates of Silica gel 60 F254, using n-propanol : triethylamine (7:3 v/v) as the mobile phase. HPTLC separation of the two drugs followed by densitometric measurement was performed in the absorbance mode at 240 nm. The drugs were resolved with R f values of 0.60 and 0.69 for Metolazone and Spironolactone respectively. The linear regression analysis data for the calibration plots showed good linear relationship with r value 0.99886 and 0.99862 for Metolazone and Spironolactone, respectively in the concentration range of 300 -700 ng/spot for Metolazone and 300-700 ng/spot for Spironolactone. The method was validated following the ICH guidelines in terms of accuracy, precision, specificity and robustness. The limit of detection and quantitation were 200 and 600 ng/spot respectively for Metolazone and 200 and 600 ng/spot for Spironolactone. The proposed developed and validated HPTLC method can be utilised for identification and quantitative analysis of Metolazone and Spironolactone in bulk drugs and pharmaceutical dosage form.

Validated Hptlc Method for Simulteneous Determination of Ceftriaxone Sodium and Sulbactam Sodium in Combined Dosage Form

Validated Hptlc Method for Simulteneous Determination of Ceftriaxone Sodium and Sulbactam Sodium in Combined Dosage Form

Novel validated HPTLC method for estimation of Withaferin A in polyherbal formulations

A novel, accurate, rapid and cost effective quantification method was developed for Withaferin A in herbal combined dosage forms using a destructive reagent. The separation was carried out using mobile phase of toluene : ethyl acetate : formic acid (5 : 5 : 3 v/v/v) at a detection wavelength of 440 nm. The method was validated as per the ICH guideline. The calibration range was found to be 90–630 ng per band. The method was found to be accurate in triplicate results at different standard addition levels. The LOD and LOQ were 2.59 and 7.87 ng respectively. The percentage relative standard deviation was less than 2 for the system suitability testing. The efficiency of the method for separation and quantification was confirmed by applying the same method, without modification, to 11 formulations.

Validated Hptlc Method for Simulteneous Determination of Cefoperazone Sodium and Sulbactam Sodium in Combined Dosage Form

Validated Hptlc Method for Simulteneous Determination of Cefoperazone Sodium and Sulbactam Sodium in Combined Dosage Form

Validated Hptlc Method for Simulteneous Determination of Citicoline sodium and Piracetam in Combined Dosage Form

A simple, accurate and precise HPTLC method for simultaneous estimation of Citicoline sodium and Piracetam in a bulk and in combined dosages form is described in this paper. After optimization a mobile phase of Methanol: Water (16:4 v/v) was used during validation of an analytical method. Aluminum plate coated with the silica Gel 60 F254 was used as stationary phase. Densitometric evaluation of the separated bands was performed at wavelength 212 nm. The Rf values of Citicoline sodium and Piracetam were 0.30 and 0.68 respectively. The validated method was linear over the concentration range of 400 ng to 3200 ng /spot and 1200 ng to 3600 ng/spot of Citicoline sodium and Piracetam respectively. Precision of the method was evaluated by inerday and intraday RSD. The results were Citicoline sodium: Inter day RSD of peak response 0.62 % and Intraday RSD f peak response 1.05 % and for Piracetam : Interday RSD of peak response 1.44 % and Intraday RSD of peak response 1.06 %. Accuracy was determined in terms of percentage recovery at three concentration levels. The results are for Citicoline sodium: 97.66 %, 99.70 % and 97.91 % and for Piracetam: 98.52 %, 97.61 and 96.64 % respectively. Specificity was determined by spectral analysis of Citicoline sodium and Piracetam and overlaying the standard spectra and sample spectra respectively .There was no any interference of mobile phase and diluents at the Rf values of Citicoline sodium and Piracetam. Validation was done in accordance with the ICH Guidelines.

Validated Hptlc Method for Simulteneous Determination of Ceftriaxone Sodium and Sulbactam Sodium in Combined Dosage Form

A simple, accurate and precise HPTLC method for simultaneous estimation of Cefoperazone sodium and Sulbactam sodium as bulk and dry powder for injection in combined dosages form is described in this paper. A mobile phase of Chloroform: Ethyl alcohol: Diethyl amine: Water (14: 16:8:1.2 v/v) was used after optimization at different concentrations for the development of densitogram. Aluminum plate coated with the silica Gel 60 F254 was used as stationary phase. Densitometric evaluation of the separated bands was performed at 274 nm. The Rf values of Cefoperazone sodium and Sulbactam sodium were 0.41 ± 0.01 and 0.56 ± 0.01respectively. The validated method was linear over the concentration range of 200 ng to 900 ng /spot and 400 ng to 1800 ng/spot of Cefoperazone sodium and Sulbactam sodium respectively. Precision of the method was evaluated by inerday and intraday RSD. The results were Cefoperazone sodium: Inter day RSD of peak response 1.25 % and Intraday RSD 1.73 % and for Sulbactam sodium Interday RSD of peak response 1.54 % and Intraday RSD 0.98 %. Accuracy was determined in terms of percentage recovery at three concentration levels for Cefoperazone sodium RSD 99.20 %, 99.50 % and 100.32 % and for sulbactam sodium RSD 101.25 %, 100.40 % and 100.60 % respectively. Specificity was determined by spectral analysis of cefoperazone sodium and sulbactam sodium and overlaying the standard spectra and sample spectra respectively .There was no any interference of mobile phase and diluents at the RF values of Cefoperazone sodium and sulbactam sodium. Validation was done in accordance with the ICH Guidelines.

Determination and Quantification of Ferulic acid in Ferula asafoetida by a Validated HPTLC Method

Abstract: The present study deals with the evaluation of mucilage isolated from fenugreek (Trigonella foenum-graecum L.) seeds (commonly named methi) as an innovative and cheap suspending agent. Zinc oxide suspensions (20 % w/v) were prepared using fenugreek seed mucilage as a suspending agent and it was evaluated to find its stability using the parameters like, sedimentation profile, degree of flocculation and redispersibility. The effect of the tested mucilage on suspension was compared with various commonly used suspending agents, like gum tragacanth, gum acacia, and bentonite at concentrations of 0.5, 1.0 and 2.0 % w/v. The results obtained indicated that the fenugreek seed mucilage could be used as a good suspending agent and was found to be superior than gum tragacanth, gum acacia, and bentonite in performance. Keywords: suspension, suspending agent, mucilage, fenugreek seed

Development of validated HPTLC method for quantification of stigmasterol from leaf and stem of Bryophyllum pinnatum

This study presents the report of TLC densitometric method, which has been developed and validated for quantification of stigmasterol from petroleum ether extract of leaves and stems of Bryophyllum pinnatum. The separation was performed on TLC aluminum plates precoated with silica gel 60 F254. Good separation was achieved in mobile phase using Chloroform:Ethanol (9.8:0.2v/v). Determination and quantitation were performed by densitometric scanning at 490nm in reflection/absorbance mode. This method gave compact spots at Rf 0.45 corresponding to stigmasterol. The method was validated using ICH guidelines in terms of precision, repeatability and accuracy. Linearity range for stigmasterol was 50–300ng/spot with correlation coefficient R2±SD=0.990±5.05% in the concentration range 50–300ng/spot. The LOD and LOQ were found to be 15 and 45ng, respectively. The contents in crude extract obtained from leaf and stem were found to be 0.1545±0.005% w/w and 0.1383±0.002% w/w respectively. HPTLC method was found to be simple, precise, accurate and convenient method for rapid screening of active constituents present in petroleum ether extracts of plant and can be used for analysis and routine quality control of herbal material and formulations containing B. pinnatum.

High-performance thin-layer chromatographic analysis of antioxidants present in different parts of Saraca asoca (Roxb.) de Wilde

ObjectiveGallic acid, ellagic acid and quercetin are the important antioxidant compounds of Saraca asoca (Roxb.) de Wilde (Caesalpiniaceae). This report describes the determination of free radical scavenging activity in different plant parts (of S. asoca) by DPPH assay with correlation, and quantification of gallic acid, ellagic acid and quercetin in different plant parts by a simple, sensitive and validated HPTLC method. MethodologyMethanolic extracts of bark, leaf and flowers of S. asoca were investigated for their antioxidant potential with the aid of DPPH assay followed by HPTLC analysis for quantitative evaluation of antioxidants (gallic acid, ellagic acid, and quercetin) in different plant parts. ResultsHPTLC analysis exhibited high amount of gallic acid and quercetin in flower (0.320% and 0.11% w/w respectively) and high amount of ellagic acid in bark (0.4805% w/w) which corresponds to low IC50 values of 6.83 ± 0.07 μg/ml and 6.6 ± 0.10 μg/ml respectively, indicating the remarkable antioxidant activity of these two plant parts whereas moderate amount of gallic acid (0.164 %w/w) and very low amount of quercetin (0.0445% w/w) and ellagic acid (0.04% w/w) correlate high IC50 value of 28.6 ± 0.62 μg/ml representing its poor antioxidant potential. ConclusionConsumption of bark and flower of S. asoca could be beneficial by virtue of its high antioxidant activity. The flower and bark may be considered as a source of gallic acid and ellagic acid respectively. The presence of moderate amount of gallic acid in leaf can be an alternative source.

Gastroprotective effect of standardized extract of Amukkara choornam on experimental gastric ulcer in rats

Amukkara choornam ethanolic extract (ACE) was investigated for phytochemical screening, content of total phenolics and flavonoids, in vitro radical scavenging activity (RSA), quantification of various antiulcer marker compounds (i.e., eugenol, piperine, trans-caryophyllene, and withaferine A) by a validated HPTLC method, and evaluated for its in vivo gastroprotective ability against ethanol (EtOH)-induced and pylorus ligation (PL)-induced ulcer models in rats. Phytochemical screening revealed the presence of flavonoids, saponins, phenols, bitter principles, and steroids. Total phenolic and flavonoid content was found to be 61.12 ± 0.72 mg GAE/g of ACE and 24.06 ± 1.07 mg RE/g of ACE, respectively; this was found to be very high in plant extracts showing very good antioxidant and antiulcerogenic effect. RSA of ACE appeared significantly (p < 0.05) lower than that of ascorbic acid (AA), but higher than that of ranitidine (RAN). In vivo the pretreatment of rats with RAN (100 mg/kg) and 50, 100, and 200 mg/kg doses of ACE significantly reduced the ulcer index in a dose-dependant manner in both the models by blocking lipid peroxidation and by significant increases in superoxide dismutase and catalase activity. But rats treated with AA (200 mg/kg) did not have any effect on the ulcer induced by EtOH or PL as it has very good in vitro and in vivo antioxidant activity. HPTLC analysis showed the presence of 0.198 ± 0.01 μg/g, 0.754 ± 0.06 mg/g, 3.50 ± 0.04, and 0.854 ± 0.04 μg/g of eugenol, piperine, trans-caryophyllene, and withaferine A per gram of Amukkara choornam (AC). So the antiulcerogenic activity of ACE might be due to a possible synergistic antioxidant, supported by the holistic approach of polyherbal formulations, i.e., systematism, multi-target and multi-channel owing to their complex chemical constituents and antihistaminic-like effects.

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.

Evaluation of reversible contraceptive potential of Cordia dichotoma leaves extract

Considering the safety-risk ratio of steroidal contraceptives, the present work was carried out to evaluate ethno-contraceptive use of Cordia dichotoma G. Forst., Boraginaceae, leaves (LCD). Preliminary pharmacological screening was performed on post-coital female albino rats. The leaves extract (LD50 5.50g/ kg bw) showed 100% anti-implantation activity (n=10) at 800mg/kg dose level. (2-hydroxypropyl)-β-cyclodextrin (BCD) was used as bioavailability enhancer to form LCD-BCD complex, characterized by DLS, SEM and XRD analyses. The LCD-BCD complex (1:1, w/w) exhibited 100% pregnancy interception (n=20) at the dose level of 250mg/kg and also showed strong estrogenic potential with a luteal phase defect. Qualitative and quantitative phytochemical analyses were carried out. The LCD extract was standardized by a validated HPTLC method and two contraceptive phytoconstituents, apigenin and luteolin were isolated. A detailed pharmacological analyses followed by chronic toxicity study were performed to predict the reversible nature of the developed phytopharmaceutical. The histological and biochemical estimations detected the reversible contraceptive potential after withdrawal. The observations suggested that the developed phyto-pharmaceutical has potential antifertility activity with safety aspects.