Precoated TLC Plates Research Articles

Green high‐performance thin layer chromatography‐densitometric approach for tablet formulation containing remogliflozin etabonate and vildagliptin

AbstractA new high‐performance thin‐layer chromatography (HPTLC) method has been developed to analyze Vildagliptin (VLD) and Remogliflozin etabonate (RE) tablets. The procedure uses a mobile phase of methanol, ethyl acetate, toluene, and ammonia (1.5:7:1:0.5, by volume), a stationary phase of silica gel 60 F254 precoated TLC plates, and densitometry to gauge the amount of drug present. The method's linearity, accuracy, precision, and robustness were carefully evaluated. VLD and RE had Rf values of 0.46 and 0.64, respectively, with R2 values of 0.997, indicating good linearity over 10,000–50,000 ng/band for RE and 5000–25,000 ng/band for vildagliptin. Studies on forced deterioration were carried out to assess the probable degradation process. The resolution in the Rf values between the degradant and drug peaks was good. The study revealed that the drug was sensitive to hydrolysis and oxidative stress degradation and was transformed into the active Remogliflozin after the forced degradation tests. The Remogliflozin and RE bands were easily distinguishable. This method can be utilized for analyzing not only the tablet formulation of the drug but also stability samples and standard quality control samples. The Analytical Greenness Calculator methodology aided the greenness evaluation of the method.

Relative estimation of beta-sitosterol in alcoholic extracts of roots and small branches of oroxylum indicum (L.) Kurz by HPTLC for plant part substitution, medicinal uses and conservation

Beta-sitosterol is a common and useful ‘phytosterol’, present in roots and small branches of Oroxylum indicum (L.) Kurz It has mainly cholesterol-lowering, anti-inflammatory, anti-cancer, and dietary properties. O. indicum is a rare medicinal plant in the traditional medicine system. This study comparatively identified and estimated beta-sitosterol in the methanolic extracts of roots and the small branches of O. indicum by the development of a validated HPTLC method. Chromatographic separation was performed on precoated TLC plates by toluene: ethyl acetate: formic acid: water (6:3:1:0.2, v/v/v/v) with standard procedures using HPTLC assembly. Marker was identified at RF 0.60 as prominent bands and quantified at 540 nm with sharp peaks corresponding to beta-sitosterol by calibration method. The method was validated as per ICH guidelines with a good linear relationship i.e. r2 = 0.9973 ± 0.000405 in 100 – 500 ng band−1 with respect to the peak area. The LOD and LOQ were calculated as 35.22 and 106.73 ng/band, respectively. The quantity of marker in extracts of roots and the small branches was found to be 268.7% w/w and 208.5% w/w respectively in 0.5 g of the sample. The method was simple, accurate, and reproducible for rapid screening and quantification of beta-sitosterol in roots and aerial parts of O. indicum. The study may helpful for plant part substitution study, medicinal practises, sustainability, and conservation of such vulnerable plant species. It may useful to establish the quality and efficacy of plant in traditional applications and in routine standardization (quality control) of different commercial preparations of O. indicum.

Analytical Method Development and Validation of Hptlc Method for Simultaneous Estimation of Telmisartan and Azelnidipine in Tablet Dosage Form

Background: A specific, accurate, precise, robust and cost effective HPTLC method was developed and validated for quantitative analysis of Telmisartan and Azelnidipine in fixed dose combination. The separation was carried out on pre-coated TLC plates with Silica gel 60 F254 as stationary phase using Chloroform: Ethyl acetate: Methanol (7.5:2:0.5 v/v/v) as developing system followed by densitometry measurement of bands at 280nm. Result: The separation of Telmisartan and Azelnidipine was found to be at the following RF values 0.3 and 0.63 respectively. Calibration curves were found to be linear over the concentration ranges 400-4000ng/band, 80-800ng/band for Telmisartan and Azelnidipine with correlation coefficient 0.9957 and 0.9966 respectively. Accuracy was found between 98.03% - 102% and 98.72% - 102 % for Telmisartan and Azelnidipine respectively. LOD was found to be 1.04% and 1.6% and LOQ was found to be 3.49% and 5.57% for Telmisartan and Azelnidipine respectively Conclusion: The results showed that the proposed HPTLC method is reliable for routine analysis for simultaneous determination of Telmisartan and Azelnidipine without any interference.

Determination of Diclofenac Sodium in Pharmaceutical Preparations by Computational Image Scanning Densitometry

Diclofenac sodium is a nonsteroidal anti-inflammatory drug which cures by reducing substances in the body that cause pain and inflammation. There is always a risk of heart attack and stroke in case of taking excess of the drug. In present study a simple, fast and cost-effective method is devised for the assay of diclofenac sodium in locally available pharmaceutical preparations. The method is based on the reaction of the drug with 2, 4-DNPH (2, 4-Dinitro phenyl hydrazine). Micro-quantities of the drug gave green coloured spots when mixed with 2,4-DNPH on a pre-coated TLC plate, in the presence of potassium iodate and lithium hydroxide. The spots were scanned by using a flatbed scanner and the images obtained were computationally quantified with the help of custom-made software to measure the optical density. The reaction parameters were optimized, and the results were compared with the standard spectrophotometric method

Differential response for multiple ions: a smart probe to construct optically tunable molecular logic systems.

A rhodamine-based optical probe has been designed through a one-pot synthetic protocol involving phenanthroline as a binding motif. The compound showed a bright pink coloration specifically upon the addition of Cu2+ and Hg2+ ions. However, the appearance of bright red fluorescence was observed only in the presence of Hg2+. Considering both, we can detect and discriminate these two ions even at ppb level concentration. Furthermore, these in situ generated metal complexes were utilized for the selective recognition of CN- and I- ions. Pre-coated TLC plates were developed for rapid on-site detection of these toxic ions even in remote places. Finally, on a single molecular probe based on differential opto-chemical interactions with different ions (Cu2+, Hg2+, CN- and I-), we were able to design numerous trivial (OR, NOR) and non-trivial (INHIBIT, IMPLICATION, COMPLEMENT, TRANSFER, NOT-TRANSFER) logic gates. Most fascinatingly, we can switch the logic response from one type to another by simply tuning only the optical output channel.

DEVELOPMENT AND VALIDATION OF STABILITY INDICATING HPTLC METHOD FOR THE SIMULTANEOUS ESTIMATION OF TINIDAZOLE AND FLUCONAZOLE AND ITS APPLICABILITY IN MARKETED DOSAGE FORM

Objective: The current method is focused on the development and validation of a simple, rapid, precise and robust stability indicating High Performance Thin Layer Chromatography for the simultaneous estimation of anti-infective drugs Tinidazole and Fluconazole in bulk and its pharmaceutical dosage form. The method was tailored to analyse the drugs in their commercial dosage form (tablets) with no interference from ingredients. Methods: Chromatographic separation was performed over precoated TLC plates (60 F254, 20 cm × 10 cm, 250 μm thickness, Merck) via a linear ascending technique using toluene: acetonitrile as the mobile phase in the ratio 6:4 v/v. Detection and quantification was achieved at the isobestic point of the two drugs, which was observed at 263 nm through Spectro-densitometric analysis. Analytical performance of the proposed HPTLC method was validated according to the ICH guidelines with respect to the linearity, accuracy, precision, detection and quantitation limits, robustness and specificity. Results: Tinidazole and Fluconazole were well separated and identified with an Rf value of about 0.46±0.03 and 0.75±0.05, respectively. The calibration curves were linear over a concentration range of 800-1200ng/spot for Tinidazole and 60-90ng/spot for Fluconazole with correlation coefficients (r2) more than 0.998. The above-developed method was validated as per ICH guidelines Q2(R1) and was found to be precise, sensitive, accurate and robust. Conclusion: The validated stability indicating HPTLC method was found to be simple, precise, accurate and sensitive for the concurrent quantification of Tinidazole and Fluconazole in pharmaceutical dosage form and can be released into quality control for regular analysis.

Spectrodensitometric and ultra-performance liquid chromatographic quantification of dapagliflozin and saxagliptin in their dosage form and human plasma


 
 
 
 Purpose: To simultaneously quantify dapagliflozin (DAPA) and saxagliptin (SAX) in a pharmaceutical product and human plasma.
 Methods: Separation and quantification of DAPA and SAX were performed on pre-coated TLC plates in TLC-densitometric method using a solvent system of chloroform, ethyl acetate and methanol at a volume ratio of 8:1:1 as the mobile phase. The developed spots were scanned at 225 and 210 nm in absorbance mode. Moreover, the studied drugs were concurrently determined in human plasma using ultra-performance liquid chromatography (UPLC). The separation process was carried out in WatersTM Acquity C18 BEH column using a solvent system of 0.02 M KH2PO4 buffer, pH 4; MeOH and acetonitrile (2:1:1, v:v:v) isocratically at a flow speed of 0.5 mL/min. The absorbance of each eluent was read at 220 nm.
 Results: Concurrent evaluation of DAPA and SAX was carried without separation using TLC-densitometric method, and it was successful in determination of DAPA and SAX in concentration ranges of 10 – 70 μg/band and 5 – 60 μg/band, respectively. In addition, retardation factor (Rf) values for SAX and DAPA were 0.17 and 0.31, respectively. Furthermore, the studied drugs were concurrently determined in human plasma using UPLC, which was sensitive enough to quantify DAPA and SAX in concentration ranges of 100 – 1000 and 20 – 200 ng/mL, respectively.
 Conclusion: These methods can be utilized for sensitive monitoring of DAPA and SAX in pharmacokinetic and bioequivalence studies.
 
 
 

Forced Degradation Studies of Drospirenone: Isolation and Characterization of Degradation Products

Abstract: Aim and Objectives: To remain safe for further processing or human consumption, study of stressed degradation for the identification of feasible degradants is required. The stability indicating high performance thin layer chromatographic method was developed with Camag HPTLC system. Materials and Methods: Silica C60F254 precoated TLC plates were used as stationary phase for separation of degradation products. The optimized mobile phase system consist of toluene: methanol: diethylamine (7:3:0.1) at 280 nm. Results: From the mass details and IR, NMR interpretation, the plausible structure of alkaline degradation product of drospirenone could be 17α (3-hydroxy propyl)-6β, 7β, 15β, 16β-dimethylene-5β-androstane-3β,5,17β triol and acidic degradation product of drospirenone could be 3-oxo-15α,16α-dihydro-3’H-cyclopropa[15,16]-17α-pregna-4,6- diene-21,17-carbolactone. Also In silico toxicity studies of the degradation products were performed to assess the toxicity profile of the products using Protox online sever. Conclusion: This analytical method can be considered as an alternative practical and inexpensive method for simple, accurate and efficient quantitative detection of drospirenone in the presence of its degraded products. Key words: Drospirenone, Characterization, Forced degradation studies, In silico toxicity study, Degradation products of drospirenone.

Seasonal dynamics of Shatavarin-IV, a potential biomarker of Asparagus racemosus by HPTLC: Possible validation of the ancient Ayurvedic text.

The medicinal property of Asparagus racemosus is primarily attributed to its constituent steroidal saponins, particularly the major component, shatavarin-IV. Thus, it can serve as a biomarker and its level can decide of the utility of the plant cultivar as a drug. Hence, a sensitive, reliable and quantitative High Performance Thin Layer Chromatography (HPTLC) method has been established for quantification of shatavarin-IV in the methanolic extracts of the roots collected in both summer and rainy seasons. The extracts of the powders of dried roots were applied to silica gel 60 F 254 aluminum-supported precoated TLC plates and developed with n-hexane: ethyl acetate: methanol, 80:10:10 (v/v), as the mobile phase. Shatavarin-IV was detected and quantified by densitometry at λ = 336 nm. The accuracy of the method was checked by conducting recovery studies at three different levels of shatavarin-IV. The average recovery was found to be 101% and 107% for summer and rainy seasons respectively. The shatavarin-IV contents, as estimated by the proposed method were 12.5 μg gm -1 and 10.9 μg gm -1 in summer and rainy roots respectively. The entire method was performed six times (n=6) to check the repeatability. The proposed HPTLC method for quantitative monitoring of shatavarin-IV in A. racemosus roots collected in different seasons strictly adhered to the validation issues laid down by the ICH guidelines. The method is reliable reproducible and highly precise and selective.

And validation of HPTLC method for the simultaneous estimation of ascorbic acid and gallic acid in amla juice preparation.

The aim of this study was to asses a simple, selective, precise, and reproducible high performance thin-layer chromatography (HPTLC) method for the simultaneous estimation analysis of ascorbic acid (AA) and gallic acid (GA) in amla juice preparation. The aluminium-based pre-coated TLC plates (Silica gel G 60F 254) were used for the HPTLC fingerprinting analysis. The chromatograms of samples were developed in twin trough glass chamber pre-saturated with mobile phase (toluene: ethyl acetate: methanol: formic acid; 3:3:2:1, v/v/v/v) at room temperature (25 ± 2°C). The densitometric analysis was carried out in absorbance mode at 254 nm. The optimized mobile phase showed compact spots of AA and GA at 0.59 and 0.86 Rf respectively. The linear regression analysis data for the calibration plots of AA and GA showed good linearity (r2= 0.992 and 0.996 respectively) with respect to peak area in the range of 200-1400 ng/spot. The method was validated as per International Conference on Harmonization (ICH) guidelines. The limits of detection and quantification (40 and 140 ng/spot, respectively) were also established. The proposed method has shown the excellent recovery (98.97–99.89%), which supports the suitability of the method for the analysis of AA and GA in the amla juice and other preparations containing these ingredients. Keywords: Amla juice, Ascorbic acid, Gallic acid, HPTLC, ICH guidelines, Validation.

A RAPID VALIDATED UNI-DIMENSIONAL DOUBLE DEVELOPMENT HPTLC-DENSITOMETRY METHOD FOR SIMULTANEOUS ESTIMATION OF METFORMIN HYDROCHLORIDE, GLICLAZIDE AND PIOGLITAZONE HYDROCHLORIDE

Objective: To develop and validate a uni-dimensional double development high-performance thin layer chromatography (UDDD-HPTLC) for estimation of anti-diabetic medicine compromising of metformin (MET) gliclazide (GLZ) and pioglitazone hydrochloride (PIO).
 Methods: The chromatographic separation of these drugs was carried out on precoated TLC plates silica gel 60F254by two mobile phases consisting of Ammonium Sulphate: Methanol: Acetonitrile: Water (4:3:2:1) for MET and PIO and Toluene: Ethyl Acetate: Formic Acid (6:4:0.5) for GLZ respectively for ideal separation and good resolution. The densitometric detection and quantification were carried out at 237 nm for MET and 200 nm for GLZ and PIO. The validation parameters were strictly followed as per the ICH guidelines.
 Results: The linearity range was obtained at 3000-8000ng/spot, 360-960 ng/spot, 90-240 ng/spot for MET, GLZ and PIO with r2value>0.999. The other parameters such as precision, reproducibility, robustness were efficiently obtained within the limits. The proposed method was successfully applied for simultaneous determination of MET, GLZ and PIO in the commercial formulation.
 Conclusion: In simultaneous estimation, the different polarity of drugs makes it more cumbersome to develop and validate any chromatographic method. In the present study, a uni-dimensional double development high-performance thin layer chromatography (UDDD-HPTLC) for estimation of these drugs have been developed and validated to resolve the estimation problem. It is an effortless and speedy method which was developed and validated using ICH guidelines. The developed and validated method using ICH guidelines is effortless and speedy technique.

Identification of Isolated Flavonoid Glycoside From Methanolic Extract of Cucumis dipsaceus Ehrenb. (Fruit)

The present study was objected to investigate isolated flavonoid glycoside from methanolic extract from fruits (Cucumis dipsaceus Ehrenb.) as well as identification. Methanolic extract was already screened for the presence of flavonoid glycoside which was strong antioxidant and hepatoprotective agent. It was first time to isolate these by column chromatography with increasing polarity like Petroleum ether, Chloroform, Ethyl acetate and Methanol solvents of different ratios. Each 20ml elute was collected, analyzed with pre coated Merck F254 TLC plates to compare similar spots and Rf values for same category of contents. The similar elutes were pooled, concentrated dried by vacuum distillation with rotary apparatus. The chloroform ethyl acetate fractions (sticky dark brown mass) were analyzed by pre coated F254 TLC plates and then visualized under visible light, UV 254nm and UV366nm. The developed pre coated TLC plates was further derivatized with 0.5% solution of anisaldehyde - H2SO4 acid and again visualized under visible light and UV366nm.TLC, HPTLC and LCMS methods were used to found and estimate polyphenolic compounds. Spectroscopic methods (IR, 1D and 2D NMR and Mass spectrometry) were used to confirm and elucidate the structure. The concluded structure of flavonoid glycosides was quercetin- 3-rutinoside-7-rhamnoside (M Wt: 756.663g/mol) and plaid with literature data

Stability-Indicating TLC-Densitometric Assay for Methyltestosterone and Quantum Chemical Calculations.

Methyltestosterone is a synthetic testosterone derivative commonly used for the treatment of testosterone deficiency in males and one the anabolic steroids whose use is banned by World Anti-Doping Agency (WADA). This study presents a simple, cost-effective and rapid stability-indicating assay for densitometric quantification of methyltestosterone in pharmaceutical formulation. The developed method employed pre-coated TLC plates with mobile phase hexane:acetone (6.5:3.5 v/v). Limit of detection and limit of quantitation were found to be 2.06 and 6.24 ng/spot, respectively. Stress degradation study of methyltestosterone was conducted by applying various stress conditions such as hydrolysis under acidic, basic and neutral conditions, heating in anhydrous conditions and exposure to light. Methyltestosterone was found to be susceptible to photodegradation, acidic and basic hydrolysis. Degraded products were well resolved with significantly different Rf values. Acid degraded product was identified as 17,17-dimethyl-18-norandrosta-4,13(14)-dien-3-one through spectroscopic methods. The reactivity of methyltestosterone under applied stress conditions was also explained by quantum chemical calculations. The developed method is found to be repeatable, selective and accurate for quantification of methyltestosterone and can be employed for routine analysis.

DEVELOPMENT OF HPTLC METHOD FOR THE ESTIMATION OF PIOGLITAZONE AND ALOGLIPTIN IN SYNTHETIC MIXTURE

A precise and accurate HPTLC method for simultaneous estimation of pioglitazone HCl (PIO) and alogliptin benzoate (ALO) in synthetic mixture has been developed. The stationary phase selected was Pre-coated TLC plates (silica gel G60 – F254 aluminium sheet) and the mobile phase selected was n-butanol : water : acetic acid (6 : 3 :1 V/V/V). The separated spots were densitometrically analyzed in absorbance mode at 270 nm. The Rf value of PIO and ALO were found to be 0.70 and 0.39, respectively. The calibration curve of PIO and ALO were plotted in the range of 400 – 1600 ng/band, which showed the linear correction coefficient (r2) 0.9971 for PIO and 0.998 for ALO, respectively. The stock solutions of both the drugs were subjected to acid and alkali hydrolysis, chemical oxidation, dry heat degradation and photo degradation. The degraded product peaks were well resolved from the pure drug peak with significant difference in their Rf values. Stressed samples were assayed using developed HPTLC method. The method was validated as per ICH guideline Q2 (R1) and applied successfully for determination of both drugs in synthetic mixture.

Development and validation of HPTLC method for simultaneous estimation of curcumin and galangin in polyherbal capsule dosage form

Simple and precise HPTLC methods were developed for the simultaneous estimation of two anti-inflammatory drugs (curcumin and galangin). The method was tailored to analyze both drugs in their commercial dosage form (capsules) with no interference from ingredients. Chromatographic separation was performed over precoated TLC plates (60 F254, 20cm×10cm, 250μm thickness, Merck, Darmstadt, Germany) via a linear ascending technique using n-hexane, ethyl acetate, acetic acid, and methanol as the mobile phase. Detection and quantification was achieved at 404nm through spectro-densitometric analysis. Analytical performance of the proposed HPTLC method was validated according to the ICH guidelines with respect to the linearity, accuracy, precision, detection and quantitation limits, robustness and specificity. The calibration curves were linear with the limits of 80–450 and 200–1200ng/spot for CU and GA, respectively, with correlation coefficients (r2) >0.9998. The limits of detection were 18.31 and 40.50ng/spot for CU and GA, respectively. The validated HPTLC method was successfully applied to the simultaneous determination of CU and GA in the commercial polyherbal formulation.

Development and validation of high-performance thin-layer chromatography method for determination of vicine in herbal extract and formulation

Summary A simple, selective, precise, and reproducible high-performance thin-layer chromatography (HPTLC) method for the analysis of vicine in herbal extracts and formulations containing Momordica charantia was developed. The study was performed on aluminum-based pre-coated TLC plates (silica gel 60F254). The chromatograms of samples were developed in twin trough glass chamber pre-saturated with mobile phase comprised of ethyl acetate-methanol-water-formic acid (7.5:3:1:0.1, v/v/v/v) at room temperature (25 ± 2°C). The densitometric analysis was carried out in absorbance mode at 278 nm. The optimized mobile phase showed a compact spot of vicine (RF = 0.31 ± 0.02) from the other constituents present in the samples. The linear regression analysis data for the calibration plots showed good linearity (r2 = 0.9990) with respect to peak area in the range of 200–1400 ng spot−1. The method was validated as per International Conference on Harmonization (ICH) guidelines. The limits of detection and quantification (50 and 150 ng spot−1, respectively) were also established. The proposed method has shown the excellent recovery (98.01–99.33%), which supports the suitability of the method for the analysis. Statistical analysis of the obtained data showed the selectivity of the proposed method for vicine estimation in the herbal samples.

RP-HPLC and HPTLC Methods for Simultaneous Estimation of Metformin Hydrochloride and Vildagliptin from Bulk and Marketed Formulation: Development and Validation

The reversed-phase high performance liquid chromatography (RP-HPLC) and high performance thin layer chromatography (HPTLC) methods for simultaneous estimation of Metfomin Hydrochloride (MET) and Vildagliptin (VLD) in bulk and their marketed combined dosage form were developed. For RP-HPLC, separation was carried out using HiQsil C18HS (4.6mmo×250mm) analytical column and detection was carried out using variable wavelength detector. The mobile phase was composed of phosphate buffer (pH adjusted to 6 using 3M KOH): methanol: acetonitrile in the ratio of 50:30:20 v/v/v. Flow rate was kept at 0.8ml/min. The drugs- MET and VLD were retained at 3.7 minutes and 4.8 minutes respectively. The HPTLC method was developed using Camag HPTLC system. Silica Gel 60GF254 precoated TLC plates were used as stationary phase. The mobile phase was ammonium acetate in methanol (1% w/v): Toluene; (10:0.5). The detection of spots was carried out densitometrically at 214 nm in absorbance mode. The Rf values for MET and VLD were found to be 0.44 and 0.55 respectively. Performance characteristics of both of these RP-HPLC and HPTLC methods for simultaneous estimation of MET and VLD in bulk and their marketed combined dosage form were statistically validated as per the recommendations of ICH guidelines of analytical method validation. The RP-HPLC method was found to be linear across concentration range of 10-60µg/mL for MET and VLD respectively. For RP-HPLC the LOD values for MET and VLD were 1.09µg/ml and 1.70µg/ml respectively and LOQ values for MET and VLD were 3.32µg/ml and 5.15µg/ml respectively. The HPTLC method was found to be linear with across the range 1000-5000ng/spot and 500-2000ng/spot for MET and VLD respectively. For HPTLC the LOD values for MET and VLD were 17.22ng/spot and 34.60ng/spot respectively and LOQ values for MET and VLD were 52.20ng/spot and 104.85ng/spot respectively. Both of these RP-HPLC and HPTLC methods were found to be simple, specific, linear, accurate, precise and robust, hence any of these methods can be conveniently adopted for routine analysis of the formulations containing MET and VLD, for their simultaneous estimation.

Development of a Sensitive HPTLC Method for Quantification of Nimbolide in Azadirachta indica and Its Dosage Form

An improved and sensitive High Performance Thin-Layer Chromatography (HPTLC) method has been developed for determination of anticancer compound, nimbolide in different parts of Azadirachta indica and its dosage form. A quick and simple ultrasonication technique was used for the preparation of sample solutions. Separation of the components was achieved on precoated TLC plates by using optimized tertiary mobile phase consisting of n-hexane:ethyl acetate:acetic acid (6:4:0.2, v/v/v) with a solvent migration distance of 68 mm. Densitometric quantification was performed after derivatization of the plate with methanol-sulfuric acid reagent in reflection/absorption mode at 515 nm. A linear response of nimbolide was obtained over the range of 200-1400 ng/spot with a correlation coefficient of r(2) = 0.99968, indicating good relationship between concentration and peak area. The method sensitivity was found to be increased by performing the analysis after derivatization with methanol-sulfuric acid reagent. The limit of detection and limit of quantification were found to be 70 and 200 ng/spot, respectively. The obtained recovery range from 96.70 to 98.01% with an average of 97.46% proves excellent accuracy of the method. ICH protocol was followed for validation of the HPTLC method in terms of precision, repeatability and accuracy. The developed method was found to be highly sensitive and the mobile phase efficiently separated nimbolide from other components. The maximum content of nimbolide was found in leaves. Further, the developed HPTLC method can be applied successfully for the marker evaluation of the formulations containing A. indica.

Quantitative Determination of a Novel Pseudoalkaloid Colchatetralene Isolated from the Mycoflora of Gloriosa superba Linn by HPTLC

A High performance thin layer chromatography (HPTLC) method was developed and optimized for determination of colchatetralene, a new bioactive alkaloid isolated from the chloroform extract of an endophytic fungus, isolated from surface sterilized seeds of Gloriosa superba Linn. The development of method involved evaluation and optimization of the various stages of sample preparation, chromatographic separation, detection and quantification. The method employed aluminum backed pre coated TLC plates (silica gel 60F254) as the stationary phase. The mobile phase system consisted of ethyl acetate- methanol-water (10:1.5:1, v/v/v) at room temperature (25 ± 2°C). Densitometric analysis was carried out in the absorbance/reflectance mode at the wavelength of 236nm. The system was found to give compact spots for colchatetralene (Rf value of 0.36 ± 0.03). The linear regression analysis data for the calibration plots showed good linearity (r2 = 0.99601 ± 0.002) in the concentration range 200–1200 ng spot-1. The method was validated according to the International Conference on Harmonization (ICH) guidelines for precision, accuracy, recovery and robustness. The limits of detection and quantification were 20 and 60 ng per spot, respectively.

Development and validation of HPTLC method for quantification of the antidiabetic compound α-amyrin acetate inStreblus asperLour

The bioactive molecule, α-amyrin acetate, was isolated from the nhexane extract Streblus asper stem bark by column chromatography. The structure of the compound was characterized with the help of physical and spectroscopic data. A high-performance thin-layer chromatography (HPTLC) method has been developed and validated for its quantification in S. asper leaf, stem bark, and root. Chromatographic separation of the compound was achieved on high-performance precoated TLC plates by using optimized binary mobile phase consisting of n-hexane-ethyl acetate (9.6:0.4, v/v). HPTLC scanning was performed at 472 nm after derivatization of the plate with methanol-sulphuric acid reagent in reflection/absorption mode. The method was validated according to International Conference on Harmonization (ICH) protocol for specificity, sensitivity, linearity, accuracy, precision, repeatability, and robustness. The developed method is found to be very simple, rapid, precise, sensitive, and accurate for the quantification of α-a...