Mobile Phase Toluene Research Articles

INFLUENCE OF AUXIN ON BIOMASS PRODUCTION AND WITHANOLIDE ACCUMULATION IN ADVENTITIOUS ROOT CULTURE OF INDIAN RENNET: WITHANIA COAGULANS

Objective: Withanolides are the biologically active, principle compound present in Withania coagulans, which is having a high medicinal value and possesses potent therapeutic activity. The present study was attempted with an objective to investigate a biomass growth and withanolide production in in vitro root tissues of W. coagulans.Methods: High-performance thin layer chromatography (HPTLC) often serves as a method for quantification of major withanolides. In the present study, methanolic withanolide extract of in vitro cultured W. coagulans root tissue were carried out using HPTLC. The HPTLC analysis was performed using precoated silica gel aluminum plate (20 cm × 20 cm) 60F254 (E.MERCK, Germany) with mobile phase toluene: ethyl acetate: formic acid (5:5:1).Results: The optimization of different combination and concentration of plant growth regulators (indole-3-butyric acid [IBA] and indole-acetic acid) was used to stimulate the biomass growth and withanolide production. The maximum biomass growth (7.48±0.25 g/dL) was observed in medium with 4.93 μM−1 IBA. The higher amount of withanolide A (204.98±0.87 μg/L DW) and withaferin A (227.15±0.57 μg/L DW) accumulation was recorded in culture grown on half Murashiga-Skoog media supplemented with 4.93 μM−1 and 2.46 μM−1 IBA.Conclusion: We concluded that in vitro-cultured system could provide unique opportunities for large-scale production of pharmaceutically important compound than field grown plants.

Influence of Demographic Location and Solvent Extraction on Pharmacognostical Assessment and Identification of Conessine Content in Different Parts of Holarrhena antidysentrica Through HPTLC Analysis

Abstract: Objective: The present study is aimed at comparative pharmacognostical studies in terms of macroscopic and quantitative microscopy on different solvent (chloroform, methanol and water) extracted leaves, stem and root parts of HA, procured from the Bangalore soil zone, Karnataka, India. Methods: Initially the soil parameters are checked for the presence of various metals and other physicochemical properties. Microscopy and macroscopic analysis were performed to under the arrangement of anatomical structures of cells and tissues. Thereafter HPTLC study was performed to determine the presence of conessine in various parts of Kurchi. Results and discussion: The results revealed the soil is sandy loam with the pH of 7.40, organic carbon content 0.30%, electrical conductivity (EC) was 13.14 mS cm-1 and the soil redox potential was 17.80 mV. Macroscopical and microscopical evaluation of leaf, stem and root gave special identification characters. Phytochemical investigation reveals the presence of alkaloids, carbohydrate, protein, glycoside, saponin, phytosterols and diterpenes. Thereafter, presence of conessine was identified by HPTLC at 192 nm using mobile phase Toluene, Ethylacetate and Dietylamine (6:3:1) and percentage of conessine resulted higher of 0.51 in methanol bark extract followed by 0.48 % in the methanol root extract. This may be due to the soil nature of Bangalore zone and the effect of solvent where the active constituents are soluble maximized to get more yield. Conclusion: Pharmacognostical parameters and conessine content in different parts of Holarrhena antidysentrica through HPTLC was revealed that was dependent on various factors. Key words: Extracts, Holarrhena antidysentrica, HPTLC, preliminary evaluation, solvents

Estimation of Guggulsterone E and Z in the Guggul-based Commercial Formulations Using High-performance Thin-layer Chromatography.

Background:Guggulsterone (GS) is a plant steroid and bioactive compound present in gum Guggul of Commiphora wightii. An Indian herbal medicine system “Ayurveda” has a long history of use of gum Guggul and plant extract of C. wightii as medicine for the treatment of various illnesses. Complex nature, low availability, and inconsistency of phytoconstituents make its analysis of difficult tasks.Aims:In this work, six different Guggul-based herbal formulations were examined for estimation of GS and their isomers (E and Z) through high-performance thin-layer chromatography technique.Materials and Methods:For that various concentrations of standard E-GS and Z-GS (50 ng–250 ng/spot) with samples (20 μg/spot) were applied on silica gel coated aluminum plate and developed with the mobile phase of toluene: ethyl acetate: formic acid: methanol (6:2:1:0.5). The scanning was performed at 254 nm wavelength and the absorbance (scan) spectrum of E-GS and Z-GS peak was generated at 200 nm–400 nm wavelength range.Results and Conclusions:Rf value and scan spectrum pattern of the samples reveal that they contain either one form of GS (E-GS, Z-GS) or both. The quantity of E-GS and Z-GS within the samples was ranged from 0.230 ± 0.0040–0.926 ± 0.0168% to 0.537 ± 0.0026–0.723 ± 0.0177%, respectively.

Validation and Development of HPTLC Method for Simultaneous Estimation of Apigenin and Luteolin in Selected Marketed Ayurvedic Formulations of ‘Dashmula’ and in Ethyl Acetate Extract of Premna integrifolia L.

Dashmula are specific ayurvedic combination of ten roots used for various disorders of liver, kidney, uterus. Standardisation of herbals are necessity for efficacy and quality parameter as per WHO guidelines. Therefore, through this original research attempt was made to standardise one of ‘Dashmula’, P. integrifolia and selected batches of marketed formulation using Apigenin and Luteolin as active biological marker for simultaneous quantification and fingerprinting through developed and validated HPTLC techniques. Developed mobile phase Toluene: Ethyl acetate: Formic acid (6:4:0.15) gave Rf (Retention factor) 0.39 and 0.29 for Standard Apigenin and Luteolin respectively at 347 nm iso absorptive wavelength. The ethyl acetate extract of P. integrifolia (PI-ET), ‘Dashmularishtha’: Manufactuer 1; 3 coded batches as DF1, DF2, DF3, Manufacturer 2; 3 coded batches -as BF4, BF5, BF6, ‘Dashmulkadha’: Manufacturer 3; 3 coded batches as KF, KF8, KF9 were found to contain : 12.8% w/w, 0.294 mg/ml%, 0.429 mg/ ml%, 0.314 mg/ml%, 0.077 mg/ml%, 0.071 mg/ml%, 0.145 mg/ml%, 0.176 mg/ml%, 0.242 mg/ml%, 0.098 mg/ml% of Apigenin and 4.7% w/w, 0.542 mg/ml%, 0.365 mg/ml%, 0.569 mg/ml%, 0.343 mg/ml%, 0.311 mg/ml%, 0.607 mg/ ml%, 0.812 mg/ml%, 0.828 mg/ml%, 0.439 mg/ml% of Luteolin respectively. In stem powder of P. integrifolia 19.84 mg/gm% Apigenin and 7.433 mg/gm% Luteolin was calculated. The estimation shows variance in manufacturers and even within batches, but will be quality control parameter. The method was validated for specificity, linearity, accuracy, precision, and robustness. It was found to be linear in range of 40-120 ng/band with regression coefficient 0.9983, 0.9997 for Apigenin and luteolin. Percentage recovery study carried out for extract PI-ET and DF1 with spike of Apigenin and Luteolin at 80, 100 and 120% level, carried out for inter and intraday precision, subjected for one way ANOVA and found F value is below tabulated F(2,6) value 5.14, therefore there is no significance variance of obtained values.

Premnine HCl Estimation in Selected Formulations of ‘Dashmul’ and in Chloroform Extract of Premna integrifolia L. by a Selective, Validated and Developed HPTLC Fingerprint Method

HPTLC technique developed as validated method for estimation of Premnine HCl in P. integrifolia chloroform extract (PI-AK) and in selected marketed formulations ‘Dashmul arishta’,‘Dashmul kadha’ in 3 × 3 batches as per ICH guidelines. Premnine HCl (Pr-s) was isolated as per literature from P. integrifolia and focused first time as standard bioactive marker for quantification. Developed mobile phase Toluene: Acetone: Diethylamine (7:2:1) for Pr-S gave Rf 0.59 at λ max 283 nm in densitometric scan, focused for specificity, fingerprint and estimation study in PI-AK and selected formulation successively. Linearity assessed in range of 8 to 16 μg /band with regression coefficient of 0.9983, LOD 0.742 μg/Band, LOQ 2.225, also robust for Pr-S. Accuracy for % recovery performed on extract as well on formulation, further subjected for precision study with application one way ANOVA for finding F value, found within limit therefore no significance of variance. A rapid and selective HPTLC method shows good linearity, recovery and high precision, useful method for analysis of Pr-S and as quality control parameter for raw material as well formulation as per foremost need of WHO, FDA and Pharmacopoeia.

High-performance thin-layer chromatographic analysis for the simultaneous quantification of lupeol and ursolic acid in the methanolic fraction of four different species of Bauhinia

A high-performance thin-layer chromatography (HPTLC) method for the simultaneous quantitative determination of lupeol and ursolic acid in the methanolic fraction of four different species of Bauhinia leaves was developed for the first time. For achieving good separation, a mobile phase of toluene—ethyl acetate—formic acid (8:2:0.1, v/v) was used. The densitometric determination was carried out at 550 nm and 520 nm in reflection—absorption mode for lupeol and ursolic acid, respectively, which were linear in the range of 100–600 ng per band. During the analysis, lupeol (0.15%) and ursolic acid (0.11%) were found to be the highest in the leaves of B. acuminata. The proposed method is simple, precise, specific, and accurate. The statistical analysis of the obtained data proves that the method is reproducible and selective and can be used for the routine analysis of the reported terpenoids in crude drug and extracts. The simultaneous quantification of these compounds has not yet been reported in the leaves of ...

A validated HPTLC method for the quantification of friedelin in Putranjiva roxburghii Wall extracts and in polyherbal formulations

In present study HPTLC method was developed and validated for the determination of friedelin in Putranjiva roxburghii Wall (family: Euphorbiaceae) leaf, bark extract and in polyherbal formulations. Analysis of samples were performed on TLC aluminium precoated plate (60 F254) by using mobile phase toluene: chloroform (9:1v/v). Plate was derivatized with vanillin sulphuric acid and scanned at 580nm. Developed method found to give compact spot for friedelin at Rf value 0.43±0.01. The method was validated using International Council for Harmonization (ICH) guidelines including linearity, precision, accuracy, and robustness. Friedelin was found to be present in leaf extract of Putranjiva roxburghii Wall (0.003%w/w), in bark (0.04%w/w), formulation 1 (0.002%w/w) and formulation 2 (0.035%w/w). A good linearity relationship was found to be (100–500ngspot−1) with correlation coefficient (r2) value of 0.9892 for friedelin. Limit of detection and limit of quantitation was found to be 32.15, 97.44ng/band respectively for friedelin. The developed method was found to be accurate and precise with 0.78%, 0.9% (%RSD) for interday and intraday precision. Accuracy of the method was performed by recovery studies at three different concentration levels and the average percentage recovery was found to be 98.55% for friedelin. The proposed method for the quantitation of friedelin was found to be simple, specific, accurate and robust in Putranjiva roxburghii Wall and polyherbal formulations.

Chemical fingerprinting and quantification of biomarker wedelolactone in different sources of “Bhringaraja” by high-performance thin-layer chromatography and method validation

Two different botanical sources, Eclipta alba and Wedelia calendulacea are used as “Bhringaraja” in the Ayurvedic s ystem o f medicine. In the present study, an effort has been made to evaluate different sources by using high-performance thin-layer chromatography (HPTLC) as an analytical tool. Wedelolactone, one of the primary constituents of these plants, was taken as the marker compound for the evaluation. An HPTLC method was developed and validated for the evaluation of different sources of “Bhringaraja”. The chromatographic system was developed using silica gel 60 F254 HPTLC plates with the mobile phase toluene–chloroform–ethyl alcohol–formic acid (5:4:1:0.5, v/v). Linearity was found between the concentration ranges of 80 to 280 ng spot−1 (R2: 0.9994), and limit of detection (LOD) and limit of quantification (LOQ) were found to be 0.36 ng spot−1 and 1.09 ng spot−1. The study has shown the presence of wedelolactone at the concentration of 0.26% w/w and 0.05% w/w in E. alba and W. calendulacea, respect...

Quantitative analysis of phytochemicals fromInula capparoots

Inula cappa (family Compositae) is used in the Ayurvedic medicinal system for the treatment of bronchitis, diabetes, fever, hypertension, and rheumatism. The proposed high-performance thin-layer chromatography (HPTLC) study offers coherent evaluation of isoalantolactone, germacranolide, β-sitosterol, and lupeol from I. cappa root. Methanolic solutions of isoalantolactone, germacranolide, β-sitosterol, and lupeol were applied on an HPTLC plate and they were scanned at 525 nm. The mobile phase toluene—methanol (9.4:0.6, v/v) was used for all the phytochemicals. After development, all the plates were air-dried at room temperature, derivatized with anisaldehyde–sulfuric acid reagent and heated at 105°C. This study aids the identification of these compounds and provides an easy and simple method for the simultaneous estimation of these markers in the I. cappa roots. The method would serve as an expedient tool in routine analyses to corroborate the drug through good constancy.

Authentication of Piper betle L. folium and quantification of their antifungal-activity

The TLC profiles of intra- and inter-day precision for Piper betle L. (PBL) folium methanol extract was studied for their peak marker recognition and identification. The Numerical chromatographic parameters (NCPs) of the peak markers, the hierarchical clustering analysis (HCA) and the principal component analysis (PCA) were applied to authenticate the PBL. folium extract from other Piper species folium extract and to ensure the antifungal activity quality of the PBL essential oil. The spotted extract was developed with the mobile phase of toluene: ethyl acetate; 93:7, (v/v). The eluted plate was viewed with the TLC-Visualizer, scanned under absorption and fluorescent mode detection, and on each sample the in-situ UV spectra were recorded between 190 to 400 nm.The NCPs profiles of intra- and inter-day precision results offered multi-dimensional chromatogram fingerprints for better marker peak pattern recognition and identification. Using the r-value fingerprints data series generated with this method allowed more precise discrimination the PBL. from other Piper species compared to the marker peak area fingerprint method. The cosine pair comparison was a simple method for authentication of two different fingerprints. The ward linkage clustering and the pair cross-correlation comparison were better chemometric methods to determine the consistency peak area ratio between fingerprints. The first component PCA-loading values of peak marker area fingerprints were correlated linearly to both the bio-marker concentration as well as the antifungal activity. This relationship could be used to control the quality and pharmacological potency. This simple method was developed for the authentication and quantification of herbal medicine.

DEVELOPMENT AND VALIDATION OF SIMPLE, RAPID AND SENSITIVE UV, HPLC AND HPTLC METHODS FOR THE ESTIMATION OF PIRFENIDONE IN TABLET DOSAGE FORM

UV, HPLC and HPTLC methods were developed and validated for the quantitative determination of Pirfenidone, a novel antifibrotic agent used in idiopathic pulmonary fibrosis. Chromatography was carried out by isocratic technique on reversed phase Eclipse XDB-C 18 column (150 x 4.6 mm, 5μm) with mobile phase consisting of phosphate buff: acetonitrile (pH 3.5) 72:28 v/v at flow rate 1mL/min. TLC was carried out by stationary phase precoated Silica Gel 60 F 254 TLC Plate using mobile phase Toluene: Methanol, 8:2 v/v. The UV spectrophotometric determination was performed at 311nm using solvent methanol. The proposed methods were validated according to ICH Q2-(R1) guidelines. The linearity range for Pirfenidone was 5-70 μg/mL for HPLC, 800-1600 ng/spot for HPTLC and 10-60 μg/mL for UV method. These methods were accurate and precise with recoveries in the range of 98.2-102.32 and relative standard deviation < 2%. The developed methods were successfully applied for determination of Pirfenidone in tablets.

A validated inherent stability indicating HPTLC method for estimation of cyclobenzaprine hydrochloride in tablets and use of MS–QTOF in characterization of its alkaline stress degradation product

A simple, selective and sensitive stability indicating high-performance thin-layer chromatographic method was developed and validated for estimation of cyclobenzaprine hydrochloride (CBP) in bulk drug and commercial tablets according to ICH guidelines. A precoated silica gel 60F254 HPTLC plates were used for chromatographic separation using mobile phase toluene: ethyl acetate: methanol: glacial acetic acid in the ratio 4:2:3.5:0.5 v/v/v/v. A TLC scanner was set at detection wavelength 290nm for densitometric analysis of analyte on absorbance mode. The degradants were resolved satisfactorily in a mixture of stressed sample having Rf values 0.48±0.05, 0.52±0.05, 0.86±0.05, 0.66±0.05 and 0.27±0.05. The calibration curve of drug was found linear in the concentration range 200–1000ng/band. The (r2>0.999) correlation coefficient value indicated good correlation between the analyte concentrations and peak areas. The repeatability and intermediate precision study revealed the % RSD value less than 2.0 and was found to be satisfactory. The accuracy of developed method was ascertained by performing the recovery study using standard addition method and expressed by percent recovery (100.32%) which was found satisfactory. CBP was subjected to various stress conditions as per International Conference on Harmonization (ICH) guidelines. The drug showed significant degradation during hydrolytic and oxidative stress condition. CBP remained stable in thermal and photolytic stress testing. The validated chromatographic method was further utilized to isolate the alkaline degradation product using preparative HPTLC technique and extensive FT-IR, ESI-MS/TOF studies were performed to ascertain the structure of degradant.

Analysis of User’s Hair Cannabinoid of Narcotic Type of Marijuana (&amp;lt;i&amp;gt;Cannabis Sativa L.&amp;lt;/i&amp;gt;) Using GCMS Technic

This paper describes the analysis of cannabinoids in the hair of the user of the narcotic type of marijuana <i>(Cannabis sativa L)</i>, using the technique Gas Chromatography Mass Spectroscopy (GCMS) and the validation of the method used. This research was conducted through experiments. Samples were taken from users of narcotic type of marijuana, found in Al-Kamal Sibolangit Rehabilitation Centre, North Sumatra, Indonesia. Marijuana plants were taken from the evidence in Forensic Laboratory of Medan Branch Police. Extraction is done through maceration by sonication at 42 KHz and using methanol – 2 propanol (1:1), kloroform, and petroleum benzene each of 2 ml for 5 minutes. Testing is done with fast blue salt B test and show there is a precipitate Violet. Optimal results obtained at a temperature of 50°C extraction. Identification is done with Thin Layer Chromatography (TLC) in the mobile phase of toluene and n-hexane (1:1) under alkaline conditions of pH 9,5, followed by GC MS techniques for sixteen (16) minutes. The results are contained compounds of cannabidiol, cannabinol, and Δ⁹THC (Tetrahydrocannabinoid) with concentrations ranging from 0.25 up to 2.82 ng/mg in the hair of the user. Validation of GC MS method for the compounds of cannabidiol, cannabinol, and Δ⁹THC produces an accuracy value with <i>%recovery</i> is 103,83, 102,67, and 101,17 respectively. Precision test produces a value of Relatif Standard Deviation (RSD) = 1,3058%, 0,8997%, dan 0,8997% respectively. Linearity test generate r value each of the regression is 0,922, 0,955, and 0,921. Limits of Detection (LOD) is obtained 0,000168 ng/mg, 0,000117 ng/mg, and 0,000164 respectively. Limits of Quantification (LOQ) is 0,00056 ng/mg, 0,00039 ng/mg, and 0,00054 ng/mg respectively. This indicates that the modification process of extraction and GC MS techniques used could produce cannabinoid compounds (cannabidiol, cannabinol, and Δ⁹THC) in hair samples quickly and accurately.

HPTLC Method Development and Validation of Cilnidipine and Metoprolol Succinate in Combined Dosage Form

Introduction and Objectives: The present work involves development and validation of HPTLC method for simultaneous estimation of Cilnidipine and Metoprolol Succinate in their combined tablet dosage form. Method: In HPTLC method, Silica Gel G60 F254 TLC plate as the stationary phase and a mobile phase of Toluene: Chloroform: Methanol: Glacial acetic acid (45: 25: 25: 5 v/v/v/v) was used to resolve CIL and METO. CIL and METO were quantified at 231 nm. The proposed method weas validated according to International Conference on Harmonization. Result and Discussion: Two well-separated and sharp peak for CIL and METO were obtained at Rf values of 0.70 ± 0.01 and 0.34 ± 0.005 respectively. The linearity range obtained for HPTLC method were 100-500 ng/spot and 500-2500 ng/spot for CIL and METO respectively. Conclusion: Method validation was found to be accurate, specific and precise.The developed method was successfully applied for estimation of CIL and METO in combined tablet formulation. Key words: Cilnidipine, Metoprolol succinate, HPTLC, Tablets, Simultaneous estimation, Method validation.

Comparative profiling of biomarker psoralen in antioxidant active extracts of different species of genus &lt;i&gt;Ficus&lt;/i&gt; by validated HPTLC method

Background: A simple but sensitive HPTLC method was developed for the comparative evaluation of psoralen in antioxidant active extracts of leaves of five different species of genus Ficus (Ficus carica, Ficus nitida, Ficus ingens, Ficus palmata and Ficus vasta).Materials and Methods: HPTLC studies were carried out using CAMAG HPTLC system on Glass-backed silica gel 60F254 HPTLC pre-coated plates using selected mobile phase toluene: methanol (9:1). The antioxidant activity was carried out, using DPPH free radical method.Results: Among all the five species of genus Ficus, F. palmata and F. carica exhibited comparatively good antioxidant activity in DPPH assay. The developed HPTLC method was found to give a compact spot for psoralen (Rf = 0.55±0.001) at 305 nm. The regression equation and r2 for psoralen was found to be Y= 4.516X+35.894 and 0.998. The quantification result revealed the presence of psoralen in only two species, F. carica (0.24%, w/w) and F. palmata (1.88%, w/w) which supported their supremacy for anti-oxidant potential over other species. The statistical analysis proved that the developed method was reproducible and selective.Conclusion: 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 psoralen in plasma and other biological fluids.Key words: Ficus species, psoralen, antioxidant, HPTLC, Validation.

A High-Performance Thin Layer Chromatography (HPTLC) Method for Simultaneous Determination of Diphenhydramine Hydrochloride and Naproxen Sodium in Tablets.

A rapid and simple high-performance thin layer chromatography (HPTLC) method with densitometry at 230 nm was developed and validated for simultaneous determination of diphenhydramine hydrochloride (DPH) and naproxen sodium (NPS) from pharmaceutical preparation. The separation was carried out on aluminum plates precoated with silica gel 60 F254 using mobile phase toluene:methanol:glacial acetic acid (7.5:1:0.2, v/v/v). The linearity range lies between 200 and 1200 ng/band for DPH and 1760 and 10,560 ng/band for NPS with correlation coefficients of 0.994 and 0.995, respectively. The Rf value for DPH is 0.20 ± 0.05 and for NPS is 0.61 ± 0.06. % Recoveries of DPH and NPS was in the range of 99.70%–99.95% and 99.63%–99.95%, respectively. Limit of detection value for DPH was 13.21 ng/band and for NPS was 8.03 ng/band. Limit of quantitation value for DPH was 40.06 ng/band and for NPS was 24.34 ng/band. The developed method was validated as per ICH guidelines. In stability testing, DPH was found unstable to acid and alkaline hydrolysis, and DPH and NPS were found unstable to oxidation, whereas both the drugs were stable to neutral and photodegradation. The proposed method was successfully applied for the routine quantitative analysis of dosage form containing DPH and NPS.

COMPARATIVE PROFILING OF BIOMARKER PSORALEN IN ANTIOXIDANT ACTIVE EXTRACTS OF DIFFERENT SPECIES OF GENUS FICUS BY VALIDATED HPTLC METHOD

Background: A simple but sensitive HPTLC method was developed for the comparative evaluation of psoralen in antioxidant active extracts of leaves of five different species of genus Ficus (Ficus carica, Ficus nitida, Ficus ingens, Ficus palmata and Ficus vasta). &#x0D; Materials and Methods: HPTLC studies were carried out using CAMAG HPTLC system on Glass-backed silica gel 60F254 HPTLC pre-coated plates using selected mobile phase toluene: methanol (9:1). The antioxidant activity was carried out, using DPPH free radical method. &#x0D; Results: Among all the five species of genus Ficus, F. palmata and F. carica exhibited comparatively good antioxidant activity in DPPH assay. The developed HPTLC method was found to give a compact spot for psoralen (Rf = 0.55±0.001) at 305 nm. The regression equation and r2 for psoralen was found to be Y= 4.516X+35.894 and 0.998. The quantification result revealed the presence of psoralen in only two species, F. carica (0.24%, w/w) and F. palmata (1.88%, w/w) which supported their supremacy for anti-oxidant potential over other species. The statistical analysis proved that the developed method was reproducible and selective. &#x0D; Conclusion: 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 psoralen in plasma and other biological fluids.

HPTLC METHOD DEVELOPMENT AND VALIDATION FOR SIMULTANEOUS ESTIMATION OF CHLORTHALIDONE AND IRBESARTAN IN COMBINED TABLET DOSAGE FORM

A simple, accurate and rapid high performance thin layer chromatography (HPTLC)-densitometry method was developed for separation and determination of chlorthalidone (CHL) and irbesartan (IBS) in pharmaceutical dosage forms. The compounds were separated on silica gel 60 GF254, HPTLC plates using mobile phase of toluene: ethyl acetate: acetonitrile: methanol: ammonia solution (25%) [5:2:2:1:0.2 v/v/v/v] as compact spots at Rf of 0.57 for chlorthalidone and Rf of 0.36 for irbesartan. Densitometric detection was performed at 254 nm. The method was validated in terms of linearity, precision, accuracy, limit of detection (LOD), and limit of quantification (LOQ). The calibration curves were linear in the range of 12.5-75 ng/spot for CHL and 150-900ng/spot for IBS. For CHL recovery varied in range of 99.26-101.25% and for IBS recovery varied in range of 99.76-100.40%. The LOD was found 1.33 and 11.34 ng/spot for CHL and IBS respectively. The LOQ was found 4.03 and 14.37 ng /spot for CHL and IBS respectively. It was observed that the proposed HPTLC method could be used for efficient analysis of the CHL and IBS in combined tablet dosage forms.

Simultaneous estimation of glycosidic isoflavones in fermented and unfermented soybeans by TLC-densitometric method.

A simple, accurate and rapid high-performance thin-layer chromatographic (HPTLC) method for the simultaneous quantification of three glycosidic isoflavones (daidzin, genistin and glycitin) in soybean (Glycine max L.) has been established and validated. Chromatography was performed on aluminum foil-backed silica gel 60 F254 HPTLC plates and found compact spots for daidzin, genistin and glycitin (Rf value of 0.39, 0.51 and 0.32, respectively) with mobile phase toluene : ethyl acetate : formic acid : acetic acid in the ratio of 1 : 8 : 1 : 0.5, v/v/v/v. Ultraviolet detection was performed densitometrically at the maximum absorbance wavelength, 260 nm. The method was validated for precision, recovery, robustness, specificity, limit of detection (LOD) and limit of quantification (LOQ), in accordance with the ICH guidelines. The LOD (2.9, 19.3 and 3.5 µg mL(-1)), LOQ (9.03, 58.6 and 10.7 µg mL(-1)), recovery (95.9-106.66, 86.97-106.56 and 98.54-105.65%) and precision (≤2.12, ≤0.722 and ≤0.066) were satisfactory for glycosidic form of isoflavones daidzin, genistin and glycitin, respectively. Soybean variety Kh-09 bragg was found to have relatively higher amount of glycosidic isoflavones, namely daidzin, genistin and glycitin 278, 597.5 and 109.4 µg g(-1), respectively, and after fermentation the glycosidic isoflavones concentration in soybean fermented with Bacillus subtilis strain were decreased significantly after 24 h of incubation; conversely, aglycone isoflavones were increased significantly. The method for quantification of isoflavones in unfermented and fermented soybeans, with good resolution has been developed.

HPTLC ANALYSIS OF THE ROOTS OF TAVERNIERA CUNEIFOLIA (ROTH) ALI

Taverniera cuneifolia (Roth) Ali is commonly called as jethimadh (Licorice). High - Performance Thin - Layer Chromatographic analysis wa s carried out for the regional variation of different regions of India, seasonal variation and species variation. The root powder was extracted with Methanol and further analysis was performed. Quantitat ion of β - sitosterol and lupeol was carried out for T. cuneifolia roots collected from different regions of India like Kutch , Rajkot, Jamnagar (Guj a rat), Jodhpur (Rajasthan), Kurnool (Andhra Pradesh) , India . The concentrations of β - sitosterol were found to be 1.98 % in Jamnagar, India, 2.64 % in Rajkot , India, 4.45 % in Andhra Pradesh , India and 1.90 % in Jodhpur , India . The concentrations of lupeol were found to be 2.44 % in Jamnagar, India, 2.21 % in Rajkot , India, 2.29 % in Kutch , India and 2.01 % in Jodh pur , India . Seasonal variation of β - sitosterol was found to be highest in monsoon (7.08 %), winter (3.89 %) and summer (2.64 %). Lupeol was found only in summer (1.42 %). Species variation of Taverniera roots showed only presence of β - sitosterol where Taverniera cuneifolia (10.04 %) and Ta verniera abyssinica (6.89 %). Quantitation was carried out on HPTLC silica gel 60 F254 pre - coated plates with the mobile phase Toluene: Methanol: 8: 1 (v/v). A TLC scanner set at 366 nm in fluorescence / reflectance mode was used for quantitation. Keywor ds : Licorice, Taverniera cuneifolia , HPTLC, Regional, Seasonal .