Performance Thin Layer Chromatographic Method Research Articles

High performance thin layer chromatography fingerprint on cyano-bonded stationary phase of selected Stachys and Betonica species and their antioxidant activity with chemometric calculations

The methanolic extracts of selected Stachys and Betonica species were analyzed by high performance thin layer chromatography (HPTLC) method using cyano bonded stationary phase and mobile phase consisted of methanol and water 45:55 (v/v) with Natural product reagent derivatization. The HPTLC method was validated (linearity, precision, accuracy, robustness, LOD, LOQ) and the validation results confirm the usefulness of the method used. The obtained images of fingerprint chromatograms presented as numerical data (using the TLC analyzer program) were next processed using Excel and SpecAlign softwares. The phytochemical similarity between analyzed plant extracts was evaluated using the similarity index (Pearson correlation coefficient) and the results were presented as hierarchical cluster analysis dendrograms and as graph for principal component analysis (PCA). The total phenolic content (TPC) by Folin-Ciocalteu method and the antioxidant activity with Ferric Ion Reducing Antioxidant Potential (FRAP) technique were determined for studied extracts using gallic acid as standards in both methods.

Development and Validation of HPTLC and First Derivative Spectrophotometric Methods for Determination of Levetiracetam in Bulk and Tablet Dosage Form

A simple, sensitive high performance thin layer chromatographic (HPTLC) method and first order derivative spectrophotometric methods were developed for estimation of US FDA drug levetiracetam in bulk and its tablet dosage form. Chromatographic estimation was carried out on silica gel 60 F245 TLC plate as stationary phase and toluene: methanol: Ethyl acetate (4:4:2v/v/v) as mobile phase. Densitometric analysis was carried out at wavelength 218nm. The Rf values for Levetiracetam was found to be 0.47±0.03. Linearity was observed over range of 400-3200ng/spot for Levetiracetam (R2 0.998). The %recovery of Levetiracetam was found to be 99.25-100.7%. The LOD and LOQ were found to be 0.0917ng/ml and 0.278ng/ml respectively indicating sensitivity of the method. The first order derivative spectra were obtained at n=4, Δλ=3nm and determinations was made at 215 nm using methanol as solvent. Linearity was observed in the range of 80μg/ml - 280μg/ml with good regression values. Percent mean recovery of Levetiracetam was obtained as 101.5%. Both proposed methods were simple, fast, accurate, precise and reproducible and hence can be applied for routine quality control analysis of levetiracetam in bulk and its tablet dosage form.

Novel HPTLC method for simultaneous estimation from ternary mixture of chlorpheniramine maleate, dextromethorphan hydrobromide and phenylephrine hydrochloride in syrup formulations

A synergic antihistamine, cough suppressant, and decongestant combination of chlorpheniramine, dextromethorphan, and phenylephrine is used to treat acute respiratory infections caused by seasonal viruses. The effective qualitative and quantitative methods require the simultaneous measurement of a ternary combination in the pharmaceutical syrup dosage form. Therefore, a new, simple, fast and robust high performance thin layer chromatographic (HPTLC) method has been developed and validated for chlorpheniramine maleate (CPM), dextromethorphan hydrobromide (DEXO) and phenylephrine hydrochloride (PE). The chromatographic separation was carried out on precoated aluminium plates with silica gel 60 F254 as the stationary phase. Mobile phase used was chloroform: methanol: ammonia (2.5:7.5:0.3, v/v/v) for proper separation. The detection was carried out at 270nm wavelength in absorbance mode. Developed method was validated as per International Council for Harmonization (ICH) Q2 (R1) guideline. The linearity range is 400 to 1400ng/band for CPM, 3000 to 11500ng/band for DEXO and 1000 to 3500ng/band for PE with correlation coefficient ≥0.995. The consistent lower values of relative standard deviation (RSD, %) for precision and robustness study indicate the method reliability. The percent recovery ranged from 97.82 to 102.03% indicates the good accuracy of the method. The proposed method was complying for the analytical method validation parameters suggested by the ICH Q2 (R1) guideline. The method was found to be simple, rapid and reliable for the simultaneous estimation of CPM, DEXO and PE from its pharmaceutical syrup dosage form. The method was successfully applied to quantify these analytes from the several pharmaceutical syrup dosage form.

Isolation, In-silico Studies, and Biological Evaluation of Higenamine from Annona squamosa L. against Breast Cancer

The efficacy of Annona squamosa L. in cancer treatment has been documented, inspiring the authors to investigate the plant further for potential novel anticancer compounds. Therefore, the current study aimed to isolate and characterize Higenamine from leaves extract of A. squamosa L. and studied for the in-vitro cancer cell line and molecular docking. The high performance liquid chromatography (HPLC) and thin layer chromatography (TLC) method development of A. squamosa L. leaves extract furnished Higenamine of which characterization was established by HPLC, infrared radiation (IR), liquid chromatography-mass spectrometry (LC-MS) and its acute toxicity study and cytotoxic properties using MCF-7 cell lines by MTT assay were assessed. Molecular docking was done using Auto Dock Vina software to validate the anticancer activity. The extraction batches by ultrasonication and maceration methods showed good results using 80% methanol. Acute toxicity studies, after a single dose (2000 mg/kg) administration of methanolic extract don’t show clinical symptoms of toxicity and mortalities. From the extract, Higenamine was eluted and the dried fraction showed 66% of enrichment of Higenamine. The developed HPLC and TLC methods showed Higenamine (HGN) peak at 6.21 minutes band at Rf 0.61, respectively. The isolated compound was identified as Higenamine with FTIR spectroscopic analysis which revealed various characteristic band values with functional groups. HPLC-MS showed m/z at 272.1 (M+H+) corresponding to the molecular formula C16H17NO3. The IC50 value for Higenamine was 42.39 μg/mL were observed in MCF-7 breast cancer which was better than 5-Fluorouracil having 39.22 μg/mL. In-silico studies explored that higenamine showed good binding affinity to breast cancer PDBIDs and forms a stable complex. Furthermore, absorption, distribution, metabolism, and excretion (ADME) properties of isolated compound was calculated using the Swiss ADME online tool in which higenamine was found to have a good pharmacokinetic profile as compared with 5-fluorouracil and doxorubicin. Higenamine has been isolated and identified as a potential anticancer agent. Further research can explore its effectiveness through pharmaceutical formulation and in vitro cell line studies.

Analytical Method Development, Validation, and Applications of the HPTLC Densitometric Approach for Simultaneous Quantification of Azelnidipine and Chlorthalidone

For the simultaneous quantification of Azelnidipine and Chlorthalidone in synthetic combination, a high performance thin layer chromatographic method was devised that is quick, accurate, selective, and exact. The mobile phase used in the chromatographic analysis was in the ratio of 5: 4.7: 0.3: 0.1 v/v/v/v, and the stationary phase utilised was silica gel 60 F254 as the precoated stationary phase on aluminium plates. A 10 x 20 cm TLC chamber with a 15-minute saturation period was utilised. Azelnidipine and chlorthalidone were found to have retardation factors (RF) of 0.43 0.03 and 0.30 0.02, respectively. At 242 nm, densitometric analysis was performed. Following the ICH Q2 (R1) standard, a validation study was conducted. The calibration plots' regression data revealed a strong linear association with R2 = 0.999 for the concentration ranges of azelnidipine and chlorthalidone, 400–1200 ng band-1 and 600–1800 ng band-1, respectively. The method's precision, accuracy, and robustness were all validated. For azelnidipine and chlorthalidone, the minimum detectable levels were determined to be 26.71 ng band-1 and 38.39 ng band-1, respectively, and the limits of quantitation were found to be 80.94 ng band-1 and 116.033 ng band-1. Azelnidipine and chlorthalidone can be estimated simultaneously for routine analysis, in drug formulations, and in biological matrices, in short, using the proven analytical approach.

Quality by design optimization and validation of a HPTLC-MS method for simultaneous estimation of paracetamol and prochlorperazine from bulk and formulation

Study reports that the, stability indicating high-performance thin layer chromatographic technique (HPTLC) was developed as well as validated for estimating Paracetamol and Prochlorperazine in bulk and dosage form. These drugs are largely used in migraine and first line treatment of schizophrenia. Selected drug combination employed for Quality by design based Box Behnken design by high performance thin layer chromatography method utilizing aluminium plates; pre-coated using silica gel 60F254 as stationary phase by using acetone: toluene: methanol (4:4:2 v/v/v) as a mobile phase. Densitogram shows Rf values for Paracetamol at 0.74 and for Prochlorperazine at 0.32 evaluated by densitometry measurement bands at 248 nm. Paracetamol and Prochlorperazine marketed formulation of 700 mg by using various excipients prepared at lab scale and it was evaluated for various types of tablet tests, and it showed the accurate result with respect to standard guidelines. The approach has been validated in accordance with International Conference on Harmonization (ICH) requirements. The correlation coefficient was found to be 0.992 and 0.998 at a concentration range of 100–300 μg/band and 1000–3000 ng/band for Paracetamol and Prochlorperazine, respectively. The method had an accuracy of 100.51 % and 100.53 % for Paracetamol and Prochlorperazine, respectively. The degradation of Paracetamol and Prochlorperazine was carried out in an acid, alkaline, oxidative, thermal and photolytic environment. The stress degraded product was successfully separated using the HPTLC and using high resonance mass spectroscopy technique (HRMS-MS), the degradation product was discovered.

Quality control of marketed products of Andrographis paniculata by HPLC analysis

Andrographis paniculata is one of the most important medicinal plants having several pharmaceutical properties. Various pharmaceutical products and raw powders of this herb are sold in the market. The high demand for A. paniculata poses the problem of adulteration in their products. Therefore, it becomes necessary to check the quality in terms of their major bioactive compounds. In the present study, several marketed products were analyzed for their quality assessment i.e. for the presence of desired bioactive constituent, andrographolide. Ten tested products of A. paniculata showed the presence of andrographolide. A validated High Performance Thin Layer Chromatography (HPLC) method has been used for the quantitative estimation of andrographolide. andrographolide was detected at a retention time (Rt) of 17.8±0.05 and was quantified using a linear regression equation. The amount of andrographolide in the marketed products was in the range of 0.01 to 7.08 %. The chromatographic fingerprints were developed in all marketed products in the study. Thus, the present study dealing with the detection and quantification of andrographolide through HPLC analysis would have enough significance in the quality control of marketed products of andrographis paniculata.

HPTLC METHOD DEVELOPMENT AND VALIDATION FOR THE QUANTIFICATION OF ALECTINIB HYDROCHLORIDE IN BULK POWDER AND CAPSULE PREPARATIONS

The high performance thin layer chromatography method described here enables simple, accurate and precise estimation of alectinib hydrochloride content in capsule formulation. To achieve adequate separation, G60 - F254 pre-coated silica gel sheets of aluminium were used as the stationary phase. A mobile phase comprising a solvent mixture of methanol and water in a ratio of 80:20 (V/V) was utilized with a chamber saturation duration of 30 minutes. The retardation factor value was found to be 0.47 at the detection wavelength 341 nm. The method’s linearity was confirmed across a concentration range from 600 - 2100 ng band-1, exhibiting a high correlation coefficient of 0.9992. The accuracy range was observed between 98.50 % – 100.45 %. The lowest detection limits of drug were observed to be 17.49 ng band-1 and lowest quantitation limits of drug were found to be 53.01 ng band-1. This developed methodology was effectively employed for the quantification of alectinib hydrochloride in capsule preparations

Green HPTLC - Densitometric approach for quantitation of Ruxolitinib in bulk and marketed formulation

Background: This paper describes the development of HPTLC method for the quantitative determination of Ruxolitinib in bulk and tablet dosage form using a systemic approach. A. simple, precise, accurate and specific high performance thin layer chromatographic method has been developed and validated for the Ruxolitinib in bulk and marketed formulation. Methods: The solvent system was 8.0:2.0:0.05 v/v/v of chloroform, methanol, and formic acid. This system was found to give compact spots for Ruxolitinib (Rf value of (0.71±0.02) Densitometric analysis of Ruxolitinib was tested in the absorbance mode at 236nm. Results: The developed method produced linear results with R2 = 0.998 for a range of 100 - 600ng/band. The accuracy of the method was determined at 80, 100, and 120% level. The % recoveries were found to be 100.01%, 99.13%, and 99.82% which are within the limit of 99% to 101%. The LOD and LOQ were found to be 8.19 ng/band and 25.06ng/band indicating the sensitivity of the method. Using the developed method, it was found that intraday and interday RSD values were less than 2%. The method was also found to be robust as indicated by the % RSD values less than 2%. Conclusion: The present method was validated according to the ICH guidelines and it is applied successfully for the determination of Ruxolitinib in tablets.

High Performance Thin Layer Chromatographic Method for The Simultaneous Quantification of Glimepiride and 3-Acetyl-11-Keto-Beta-Boswellic acid along with Robustness study using Fractional Factorial Design

A simple, specific, and quantitative high-performance thin-layer chromatography method has been developed for the simultaneous determination of Glimepiride and 3-acetyl-11-keto-beta-boswellic acidin the formulation. Chromatographic development was performed using n-hexane: Ethyl acetate: Toluene: Glacial acetic acid (4:2:4:0.2, % v/v/v/v) as optimized mobile phase on TLC aluminum plate precoated withsilica gel 60 F254 and further densitometric measurement at 242nm.The drugs were satisfactorily resolved with Rfvalues of 0.38±0.02, 0.65±0.02, for Glimepiride and 3-acetyl-11-keto-beta-boswellic acid, respectively. The linear ranges were found to be 100-600 and 4000-24000ng/band for Glimepiride and 3-acetyl-11-keto-beta-boswellic acid. The accuracy and precision measured were less than 2% relative standard deviation for both drugs. The sensitivity of the method in terms of limit of detection and limit of quantitation were found to be 11.22 and 34ng/band for Glimepiride and 439.92 and 1333.102ng/band for 3-acetyl-11-keto-beta-boswellic acid respectively. 24-1 fractional factorial design was applied for robustness study to evaluate the small difference in independent variables. Among four factors, the significant factor found was the volume of n-hexane that resulted in a change in the Rf of drugs. The proposed method was found to be accurate, precise, reproducible, robust, and specific.

Comparative HPTLC study for simultaneous determination of ivabradine and metoprolol using UV and fluorescence detectors

New, simple, accurate, sensitive and validated high performance thin layer chromatographic (HPTLC) method coupled with UV absorbance mode and fluorescence (FL) detectors which were used for simultaneous determination of ivabradine (IVA) and metoprolol (MET) in their bulk and pharmaceutical dosage form using TLC silica 60 F254 plates and non-fluorescent TLC silica gel 60 plates. The developing system was chloroform: methanol: formic acid: ammonia (8.5:1.5:0.2:0.1, v/v). Desnitometric analysis in UV absorbance mode was set at λ = 275 nm. While, fluorescence mode was performed with excitation at 260 nm for quantitative simultaneous determination of IVA and MET using optical filter K320. The retention factors values were 0.45 ± 0.05 and 0.89 ± 0.01 of IVA and MET, respectively. UV absorbance mode, linearity was 50.0–600.0 ng/band for IVA and 50.0–900.0 ng/band for MET. For fluorescence mode, the linearity ranges were 18.0–400.0 ng/band and 50.0–550.0 ng/band for IVA and MET; respectively. ICH guidelines were followed in respect to linearity and range, accuracy, precision and selectivity, limit of detection (LOD), limit of quantitation (LOQ) and robustness. The analytical eco-scale, green analytical procedure index (GAPI) and analytical greenness metric tools were used to assess the suggested method. The quantitative proposed method results showed there was no statistically significant difference at 95% confidence when compared to the reported method of high performance liquid chromatography (HPLC).

A high performance thin layer chromatography (HPTLC) method for the quality assessment of citronella oil: application in commercial sample analysis

Citronella oil, extracted from Cymbopogon species (winterianus and nardus) is a commercially valuable essential oil used in personal-care products and insect repellents. Routine analysis in gas chromatography is incapable of detecting high-boiling adulterants therein. In this study, an HPTLC technique was developed for the absolute quantification of citronellal (characteristic chemical marker) and triglyceride (main constituent of vegetable oil adulterant) in citronella oil for its quality assessment. It was validated in terms of specificity, linearity, sensitivity, accuracy and precision. Further, the developed method was employed to quantify citronellal and triglyceride in twenty commercial samples. The results showed a wide variation in citronellal content (trace to 30.65% w/w) and could differentiate its two chemotypes. Also, it revealed the possibility of vegetable oil adulteration through the detection and quantification of triglyceride in selected samples. It can be a simple and rapid technique for the quality control of citronella oil.

Modulation of IL-6 in ameliorating arthritic conditions in rats by Rosa alba L flower extract and fraction

Introduction: rheumatoid arthritis characterized by joint inflammation and pain,affecting millions of peoples around the world. Traditional system of medicine hadproven to be preventive and treating physical and mental illness. Objective: theobjective of the study is to assess anti-arthritic potential of the plant Rosa alba L byconsidering CFA, formaldehyde and turpentine oil induced arthritic model. Materialand Method: ethanolic extract and its ethyl acetate fraction were considered for thestudy and quercetin was quantitatively estimated by high performance thin layer chromatographic(HPTLC) method. Moreover, hematological and biochemical studies(IL-6) were performed in blood and serum of wistar albino rat. Then histopathologicalstudies had also been performed on rat hind paw joint. Result: phytochemicalscreening estimated the presence of carbohydrates, phenolic compounds, flavonoids,phytosterols, amino acids and fixed oil in ethanolic extract and ethyl acetate fraction ofthe plant. Amount of quercetin in the flowers of the plant was found to be 0.26% w/wby quantitative HPTLC method. The level of proinflammatory cytokines, interleukin(IL-6) was considerably decreased (p<0.001) in the fraction treated group of rats at400 mg/kg b.w by considering complete freund’s adjuvant (CFA) induced arthritic model. Conclusion: Ameliorated hematological,hematological, biochemical and histopathologicalparameters confirmed the antiarthritic potential of the plant.

Corrigendum to: High performance thin layer chromatography (HPTLC) method development and validation for the simultaneous determination of paracetamol, caffeine, chlorpheniramine and phenylepherine in tablet formulation

"Corrigendum to: High performance thin layer chromatography (HPTLC) method development and validation for the simultaneous determination of paracetamol, caffeine, chlorpheniramine and phenylepherine in tablet formulation" published on 11 Aug 2022 by Akadémiai Kiadó.

High performance thin layer chromatography (HPTLC) method development and validation for the simultaneous determination of paracetamol, caffeine, chlorpheniramine and phenylepherine in tablet formulation

AbstractA rapid, selective, and precise high performance thin layer chromatographic method was developed and validated for the simultaneous analysis of paracetamol, caffeine, phenylephrine and chlorpheniramine in tablets. The chromatographic analysis was carried out on glass plates pre-coated with silica gel 60 F254 as a stationary phase. The optimized mobile phase was methanol : n-butanol : toluene : acetic acid (8:6:4:0.2 v/v). TLC chamber of 10 × 20 cm was used with saturation time of 15 min. The retardation factor (RF) for chlorpheniramine, phenylephrine, caffeine and paracetamol was found to be 0.15 ± 0.02, 0.29 ± 0.02, 0.50 ± 0.02, 0.68 ± 0.02 respectively. Detection was carried out at 212 nm. Validation study was done following ICH Q2 (R1) guideline. The regression data for the calibration plots showed good linear relationship with R2 = 0.997 over the concentration range of 300–1,500 ng band−1 for caffeine, R2 = 0.996 over the concentration range of 100–500 ng band−1 for phenylephrine, R2 = 0.996 over the concentration range of 200–600 ng band−1 for chlorpheniramine, R2 = 0.998 over the concentration range of 400–2,400 ng band−1 for paracetamol. The method was validated for precision, accuracy and recovery. Minimum detectable amounts were found to be 304.9 ng band−1, 87.88 ng band−1, 117.18 ng band−1 and 143.06 ng band−1 for caffeine, phenylephrine, chlorpheniramine, and paracetamol respectively while the limit of quantification was found to be 923.95 ng band−1, 266.32 ng band−1, 355.11 ng band−1, and 433.53 ng band−1 in the same order. The method was successfully applied to analyze two marketed tablets in a selective and reproducible manner.

DEVELOPMENT AND VALIDATION OF HIGH-PERFORMANCE THIN LAYER CHROMATOGRAPHY METHOD FOR THE ESTIMATION OF POSACONAZOLE IN FORMULATION

Objective: The study aims to developed and validate novel high performance thin layer chromatographic method for estimation of posaconazole in tablet dosage form and validate as per the international conference on harmonization (ICH) guidelines. Methods: The method was developed on silica gel 60 F254 HPTLC plates and the mobile phase was used as toluene: chloroform: ethanol in ratio (5:4:1 V/V/V). Densitometric analysis of Posaconazole was performed at the wavelength 265 nm. Results: The RF of Posaconazole was found to be 0.331. The calibration curve was found to be linear in the concentration range of 100–500 ng/band. The correlation coefficient is 0.9991 for Posaconazole. The precision result was found to be satisfactory, which indicate that the method is precise. The recovery value lies in the range of 98–102% indicating the accuracy of the method. Conclusion: A new, simpleaccurate, fast, economic, precise, and robust high performance thin layer chromatographic method was developed for the estimation of Posaconazole. The method is selective, specific it was applied in pharmaceutical analysis of Posaconazole tablet dosage form.

Quality control of marketed herbal products of Asparagus racemosus Willd. through high performance thin layer chromatography (HPTLC) analysis

Asparagus racemosus Willd. is a valuable medicinal plant which is used all over the world. There are several marketed products of A. racemosus. The high demand for this herb has increased the risk of adulteration in its commercial products. The adulterated herbal products might pose serious ill effects on health. Therefore, it is necessary to check the quality of marketed products in terms of the presence of their major bioactive compounds. The present study aimed to carry out the qualitative and quantitative analysis of Shatavarin IV in marketed products of A. racemosus through a validated high performance thin layer chromatography (HPTLC) method. Ten marketed products were analysed and all of them had shown the presence of Shatavarin IV which was quantified. The identification and quantification were done by taking a standard Shatavarin IV as reference. The Shatavarin IV was detected at Rf 0.4±0.05 and showed maximum absorption at 425 nm. The Shatavarin IV was quantified using a 6-point calibration curve having a standard deviation of 3.89 % with an R2 value of 0.9968. The amount of Shatavarin IV varied between 1.47±0.25 to 2.69±0.51 mg/g on a dry weight basis which is a normal range in the raw plant materials. Thus, the present findings would be a simple, reliable and cost-effective method for the quality determination of herbal products of A. racemosus. The developed HPTLC chromatograms would serve as a reference for the quality assessment of commercial products of A. racemosus in future.

An eco-friendly separation-based framework for quantitative determination and purity testing of an antihypertensive ternary pharmaceutical formulation

Designing new, verified methodologies with a focus on sustainability, analytical efficiency, simplicity, and the environment has become a major priority for pharmaceutical quality control units. In this way, sustainable and selective separation-based methodologies were designed and validated for the concurrent estimation of amiloride hydrochloride (AML), hydrochlorothiazide (HCT) and timolol maleate (TIM) in their fixed dose formulation (Moducren® Tablets) along with hydrochlorothiazide potential impurities, salamide (DSA) and chlorothiazide (CT). The first method is a high performance thin layer chromatographic method (HPTLC-densitometry). The first developed method employed silica gel HPTLC F254 plates as stationary phase using a chromatographic developing system composed of ethyl acetate–ethanol–water–ammonia (8.5:1:0.5:0.3, by volume). The separated drug bands were densito-metrically measured at 220.0 nm for AML, HCT, DSA and CT and at 295.0 nm for TIM. The linearity was assessed over a wide concentration range, 0.5–10 µg/band, 1.0–16.0 µg/band and 1.0–14 µg/band for AML, HCT and TIM, in order and 0.05–1.0 µg/band for each of DSA and CT. The second method is capillary zone electrophoresis (CZE). The electrophoretic separation was achieved using background electrolyte (BGE), borate buffer 40.0 mM with pH 9.0 ± 0.2, at applied voltage of + 15 kV with on-column diode array detection at 200.0 nm. The method linearity was reached over the concentration range of 20.0–160.0 µg/mL, 10.0–200.0 µg/mL, 10.0–120.0 µg/mL for AML, HCT and TIM, respectively and 10.0–100.0 µg/mL for DSA. The suggested methods were optimized to achieve best performance and validated agreeing with the ICH guidelines. Assessment of methods’ sustainability and greenness was performed using different greenness assessment tools.Graphical

Analytical Quality by Design (AQBD) Assisted Development and Validation of HPTLC Method for Estimation of Rottlerin in Topical Patch Formulation

Pharmacognosy Research,2023,15,2,267-276.DOI:10.5530/pres.15.2.029Published:February 2023Type:Original Article Authors:Kaumudee Bodas, Vaibhav M Shinde, Deshmukh Vishal, and Deshmukh Sheetal Author(s) affiliations:Kaumudee Bodas1, Vaibhav M Shinde1,*, Deshmukh Vishal2, Deshmukh Sheetal2 1Centre for Advanced Research in Pharmaceutical Sciences, Poona College of Pharmacy, Pune, Maharashtra, INDIA. 2Bharati Vidyapeeth (Deemed to be University), Yashwantrao Mohite Institute of Management, Karad, Maharashtra, INDIA. Abstract:Background: Rottlerin, a natural polyphenolic ketone is a major constituent of Mallotus philippensis (Lam.) Mull. Arg. plant. Although, less explored it has diverse array of therapeutic applications. Objectives: The current work aimed to develop novel, simple, speedy, accurate, potent High Performance Thin Layer Chromatography Method (HPTLC) to separate, and quantify rottlerin from the herbal topical patch In-House formulation. Materials and Methods: This was the foremost attempt to develop and validate HPTLC method using Analytical Quality by design (AQbD) approach. Box Behnken design (BBD) coupled with response surface methodology approach was followed for the systematic optimization. The end results of toluene content, chamber conditioning time, and solvent front on Rf value of rottlerin was studied. The optimised method was validated as per ICH guidelines. Results: For HPTLC, stationary phase used was aluminium plates 60F254 coated with silica gel as. AQbD optimised conditions were mobile phase proportion namely toluene: ethyl acetate: methanol: ammonia (5:4:2:0.2), development distance 80 mm, band width 6mm, and chamber saturation time 10 min. The method generated well resolved and compact bands at Rf of 0.41±0.05. The developed method exhibited high accuracy, precision, robustness, and recovery. The quantity of Rottlerin in developed herbal patch was found to be 134.8 ng/band. Conclusion: In a nutshell, the present study undeniably vouches for a QbD based method development approach for Rottlerin and can be extrapolated to estimate it in other herbal extracts, or marketed formulations Keywords:Analytical Quality by design (AQbD), HPTLC, Mallotus philippensis (Lam.) Mull. Arg., Rottlerin, Topical patch.View:PDF (1.15 MB)

Simultaneous densitometric determination of β-sitosterol and lupeol through validated HPTLC method in different plant parts of Uraria picta (Jacq.) Desv. ex DC. – A dashmool species

Abstractβ-sitosterol (BS) and lupeol (LU) exhibit a number of biological activities and are the important bioactive marker compounds in pharmaceutical science. In the present study, a simple, precise, accurate and validated high performance thin layer chromatographic (HPTLC) method was developed for simultaneous quantification of these two compounds in leaves, stem and roots of Uraria picta, a critically endangered medicinal plant and one of the important constituents of ten plants ayurvedic formulation called Dashmoola. Standards and test samples were applied on TLC aluminum plate precoated with 0.2 mm layer of silica gel 60F254. The plate was run in a twin glass chamber comprising toluene: methanol: chloroform (8:1:1, v/v/v) as a mobile phase. The plates were immersed in anisaldehyde-sulfuric reagent and then heated at 105 °C for 5 min in CAMAG TLC plate heater for appearance of bands. Densitometric scanning was performed at λmax = 525 nm using tungsten light source in CAMAG TLC Scanner4 armed with WinCATS software. RF values of BS and LU standards and those of test samples were found to be 0.53 ± 0.01 and 0.63 ± 0.01 respectively. The method was further validated by following the International Conference of Harmonization (ICH) guidelines. For BS and LU, the linear regression data for the calibration plots revealed a satisfactory linear association with r2 = 0.995 and 0.998, respectively. Linear range for both BS and LU was 200–600 ng/band. Accuracy of the method was evaluated by performing recovery study at three different levels by standard addition methods with an average recovery of 99.86% and 101.07%. The results revealed that the leaf samples of U. picta contained highest concentration of BS (0.150 ± 0.02%) while its root samples confined the highest concentration of LU (0.149 ± 0.01%). The developed method can be applied for routine and quality control analysis in different herbal formulations containing U. picta species.