Silica Gel 60F254 Research Articles

Use of TLC-Densitometric Method for Determination of Valproic Acid in Capsules.

Determination of valproic acid in the drug was carried out on the aluminum silica gel 60F254 plates and using acetone–water–chloroform–ethanol–ammonia at a volume ratio of 30:1:8:5:11 as the mobile phase, respectively. Two methods of detection of valproic acid were used. The first was a 2% aqueous CuSO4×5H2O solution, and the second was a 2′,7′-dichlorofluorescein-aluminum chloride-iron (III) chloride system. The applied TLC-densitometric method is selective, linear, accurate, precise, and robust, regardless of the visualizing reagent used for the determination of valproic acid in Convulex capsules. It has low limits of detection (LOD) and limits of quantification (LOQ), which are equal to 5.8 μg/spot and 17.4 μg/spot using a 2% aqueous CuSO4×5H2O solution as visualizing agent and also 0.32 μg/spot and 0.97 μg/spot using a 2′,7′-dichlorofluorescein-aluminum chloride-iron (III) chloride system as visualizing reagent, respectively. The described analytical method can additionally be used to study the identity of valproic acid in a pharmaceutical preparation. The linearity range was found to be 20.00–80.00 μg/spot and 1.00–2.00 μg/spot for valproic acid detected on chromatographic plates using a 2% aqueous CuSO4×5H2O solution and the 2′,7′-dichlorofluorescein-aluminum chloride-iron (III) chloride system, respectively. A coefficient of variation that was less than 3% confirms the satisfactory accuracy and precision of the proposed method. The results of the assay of valproic acid equal 96.2% and 97.0% in relation to the label claim that valproic acid fulfill pharmacopoeial requirements. The developed TLC-densitometric method can be suitable for the routine analysis of valproic acid in pharmaceutical formulations. The proposed TLC-densitometry may be an alternative method to the modern high-performance liquid chromatography and square wave voltammetry in the control of above-mentioned substances, and it can be applied when other analytical techniques is not affordable in the laboratory.

Chromatographic Fingerprinting of an Ayurvedic Formulation by High-Performance Thin Layer Chromatography Method

In the present study, an attempt has been made to develop a high-performance thin layer chromatography fingerprinting of gallic acid, piperine and curcumin from an Ayurvedic formulation, Dekofcyn tablet. Chromatographic separation was achieved on the plate pre-coated with silica gel 60F254 as the stationary phase. The solvent system consisted of toluene:ethyl acetate:formic acid:glacial acetic acid in the ratio (5:3.5:1:1 v/v/v/v) respectively. The retardation factor value was calculated at 0.35 (±0.02), 0.66 (±0.02) and 0.89 (±0.02) for gallic acid, piperine and curcumin respectively. Densitometric analysis was carried out in the isoabsorptive mode at 295 nm. The regression analysis was carried out in the range of 100-700 ng/spot for gallic acid and piperine, 400-1600 ng/spot for curcumin and the regression coefficient for all three markers was found to be 0.99 respectively. Validation of the method was performed according to international conference on hominization guidelines Q2 (R1). This method can also be used to evaluate other formulations containing these markers as one of the constituents, thus conforming to the need for ensuring the quality of an Ayurvedic formulation.

Development and Validation of a Novel High Performance Thin Layer Chromatography Method for Simultaneous Estimation of Mangiferin and Berberine in an Ayurvedic Medicine

Setebid tablet is the natural antihyperglycemic agent with powerful antioxidant action. A highperformance thin layer chromatography method was developed for simultaneous estimation of mangiferin and berberine from setebid tablets. Camag Linomat 5 sample applicator was used for application of sample. Chromatographic separation of drugs was performed over thin layered chromatography plates pre-coated with silica gel 60F254 using toluene:acetone:formic acid (3.5:5.5:1 v/v/v) as the mobile phase via a linear ascending technique in twin trough chamber. Detection and quantification were carried out at an iso-absorptive wavelength of 261 nm using thin layered chromatography scanner III. This method showed good resolution for both drugs with retention factor 0.39±0.03 and 0.80±0.03 for mangiferin and berberine, respectively. The calibration curves were linear in the range of 400-800 and 100-500 ng/spot for mangiferin and berberine and correlation coefficients (r2) were 0.9995 and 0.9991, respectively. The method was validated according to the international conference on harmonisation Q2 R1 guidelines for accuracy, precision, limit of detection, linearity, limit of quantification, robustness and specificity. The limits of detection were 26.18 and 18.50 ng/spot for mangiferin and berberine, respectively. In conclusion, the developed method was rapid, simple, reliable and specific for the identification and quantification of mangiferin and berberine.

HPTLC Quantification of Withaniasomnifera (L.) Dunal

At present study a simple, selective, easy, robust, sensitive and precise method is developed to investigate the outcomes of HPTLC for the simultaneous determination of 3 key withanolides, namely withaferin A, withanolide A and withanolide B in Ashwagandha; Withaniasomnifera plant part samples. The method is analyzed simultaneously on Camag aluminum per-coated silica gel 60-F254 HPTLC plates, using toluene: ethylacetate: formic acid: ethanol (6:3:0.1:0.6 v/v/v/v) as mobile phase. The Quantitative evaluation of withanolides was performed by densitometry in the reflection-absorption mode at 530 nm. The method was validated for recovery, precision, accuracy, robustness, limit of detection, limit of quantitation, and specificity conferring to International Conference on Harmonization guidelines. Specificity of quantification was confirmed using retention factor (Rf) values, UV Visual spectral correlation, and electrospray ionization mass spectra of marker compounds in sample tracks. This combination of techniques has been applied, for the first time, for the estimation of withaferin-A, withanolide A and withanolide B in fruit, leaf, stem and root parts samples of Withaniasomnifera. Present method may be beneficial in a quality control setting as it would allow rapid qualitative analysis of herbal material while sustaining accurate quantification of extract composition and can be used for routine analysis as well as for quality control of raw materials and herbal formulations.

Stability Indicating HPTLC Method for Simultaneous Determination of Amlodipine Besylate and Lisinopril in Combined Dose Tablet Formulation

A combined dose tablet formulation containing Amlodipine besylate and Lisinopril is used for the treatment of essential hypertension. The present study reports development and validation of stability indicating high performance thin layer chromatographic method for simultaneous estimation of these drugs in combined dose tablet formulation. The two drugs were satisfactorily resolved on aluminum plates precoated with silica gel 60F254 using n-butanol : methanol: ammonia (4:4:1 v/v/v) as mobile phase. The Rf value for lisinopril and amlodipine besylate were 0.27±0.02 and 0.62±0.02, respectively. Densitometric evaluation of the separated bands was performed at 215nm. The calibration curves for lisinopril and amlodipine besylate were found to be linear in the concentration range of 1000-6000ng/band. The method was validated as per ICH guidelines for accuracy, precision, robustness, specificity, limit of detection and limit of quantitation. Statistical analysis proves that the method is suitable for simultaneous analysis of Lisinopril and Amlodipine besylate in pharmaceutical formulation without any interference from the excipients/degradant. The developed method offers several advantages such as sensitive, rapid, cost effective and less time consuming as compared to the reported methods. As the method could effectively separate the drugs from its degradation products, it can be employed as a stability indicating method.

A Validated HPTLC Densitometric Method for The Quantitative Determination of Ubidecarenone in Bulk and in Capsule Formulation

A new, simple, precise, accurate and rapid high performance thin layer chromatographic method has been developed and validated for the estimation of ubidecarenone in bulk and in capsule formulation. The chromatographic separation was performed on aluminium TLC plates precoated with silica gel 60F254 as a stationary phase and methanol:water (7:3) as a mobile phase. Detection was performed densitometrically in the absorbance mode at 280nm for the evaluation of chromatograms. The system has given well sharp peak of ubidecarenone (Rf=0.51±0.02). The linearity of the method was established in the range of 1-6 ng/µL with correlation coefficient (r2) of 0.9995. The method was validated for precision, accuracy, robustness, ruggedness, LOD, and LOQ as per ICH guidelines. The limit of detection was found to be 0.0392 ng/µL, whereas the limit of quantitation was found to be 0.1189 ng/µL. The percentage label claim for ubidecarenone in the capsule formulation was found to be 99.96±0.4703. The accuracy of the method was confirmed by recovery studies. The percentage recovery was found to be in the range of 100.10-101.45% for ubidecarenone. The % RSD value was found to be less than 2. The low %RSD value indicates that there is no interference due to excipients used in the formulation. Hence, the developed method was found to be simple, precise, accurate, and rapid for the analysis of ubidecarenone in bulk and pharmaceutical formulation and it can be effectively applied for the quality control analysis of ubidecarenone in bulk and pharmaceutical formulation.

Densitometric Quantification and Optimization of Polyphenols in Phyllanthus maderaspatensis by HPTLC

Quantifying and optimizing the polyphenol content of Phyllanthus maderaspatensis was accomplished using a single-solvent HPTLC system. Analyzing hydroalcoholic extracts for kaempferol, rutin, ellagic acid, quercetin, catechin, and gallic acid, we simultaneously quantified and optimized their concentration. In the experiment, the methanol to water ratio (%), temperature (°C), and time of extraction (min) were all optimized using a Box-Behnken statistical design. Kaempferol, rutin, ellagic acid, quercetin, catechin, and gallic acid were among the dependent variables analyzed. In the HPTLC separation, silica gel 60F254 plates were used, and toluene, ethyl acetate, and formic acid (5:4:1) made up the mobile phase. For kaempferol, rutin, ellagic acid, quercetin, catechin, and gallic acid, densitometric measurements were carried out using the absorbance mode at 254 nm. Hydroalcoholic extract of P. maderaspatensis contains rutin (0.344), catechin (2.62), gallic acid (0.93), ellagic acid (0.172), quercetin (0.0108) and kaempferol (0.06). Further, it may be affected by more than one factor at a time, resulting in a varying degree of reaction. A negative correlation was found between X1 (extraction time (min)) and X2 (temperature), as well as X1 and X3 (solvent ratios). Taking these characteristics into consideration, the method outlined here is a validated HPTLC method for measuring kaempferol, rutin, ellagic acid, quercetin, catechin, and gallic acid.

Application of Central Composite Design for screening and Optimization of HPTLC method for simultaneous quantitation of Aprepitant, Dexamethasone and Ondansetron in their synthetic mixtures

New HPTLC method was developed and optimized for estimation of Ondansetron (OND), Dexamethasone (DEX) and Aprepitant (APT) in laboratory prepared ternary mixtures by using Central composite design (CCD). The independent variables used for the optimization were the acetone content in mobile phase (%mL), distance of developing solvent (cm) and saturation time (min). HPTLC Separation was performed on Precoated silica gel F254 aluminum plate (10X10 cm, 100μm thickness) with a mobile phase consisting of chloroform: methanol: acetone: ethyl acetate: ammonia (9:4:2:5:0.2 % v/v/v/v). Quantification of OND, APT and DEX were achieved based on a Densitometric analysis over the concentration range of 200-1200 ng/band, 500-1000 ng/band and 1000-2000 ng/band, respectively, at 254nm. The method was yielded dense and well-resolved bands at Rf values of 0.54± 0.02, 0.79±0.02 and 0.23±0.01 for OND, APT and DEX, respectively. The linear regression analysis for the calibration plots produced r2= 0.9997, r2= 0.9998 and r2=0.9997 for OND, APT and DEX, respectively. The method was validated according to the ICH guidelines. The robustness test was determined that the selected factors have an insignificant effect on the responses. The results indicated that the method is suitable for the routine quality control testing of OND, APT and DEX in their bulk form.

Rapid and sensitive High-Performance Thin-Layer Chromatographic (HPTLC) method for identification and quantification of luteolin by densitometry in Kasamarda (Cassia occidentalis L.)

Kasamarda (C. occidentalis L.) is a traditional herb recently recognized as a potential nutraceutical in bone health. The current botanical nutraceutical regulations require consistent standardization for biological applications. The present study reported the standardization of bioactive flavonoid luteolin from Cassia occidentalis L. using validated high-performance thin-layer chromatographic (HPTLC) densitometric (DS) method. The mobile phase composition of toluene, ethyl acetate, and formic acid was optimized to separate and identify luteolin using silica gel 60F254 aluminum plates. The densitometric (DS) scanning was performed at 353 nm. This HPTLC-DS method was further validated as per ICH guidelines. The linearity was 200–700 ng/band with a correlation coefficient value of 0.994. The LOD and LOQ were found to be 54.06 ng/band and 163.84 ng/band, respectively. The recovery (88.38% and 100.72%) and precision (RSD,<5%) indicated method performance is robust and accurate for the routine analysis. Further, this bioactive flavonoid presence was confirmed and quantified by UV-spectrumin the sample matrix using this validated HPTLC-DS method. This HPTLC-DS method was robust, precise and accurate for quality control of active constituents present in C. occidentalis L.

Quantification of β-Adrenergic Receptor Agonist Drug Mirabegron in Presence of Degradants by High Performance Thin Layer Chromatography

A sensitive, selective, accurate, precise and robust thin layer chromatographic method has been developed and validated for the estimation of β-Adrenergic Receptor agonist drug Mirabegron in bulk and pharmaceutical formulation. Aluminium supported plates pre-coated with silica gel 60F-254 was used as stationary phase while methanol: acetonitrile: triethylamine (4: 6: 0.1, v/v/v) mixture saturated in twin trough chamber was used as mobile phase. The Rf values of Mirabegron was observed to be 0.58 ± 0.0089. The detection was carried out in absorbance mode at 247 nm. Linear regression analysis data showed linear relationship for Mirabegron over a concentration range of 100-1200 ng/band. Forced degradation study showed drug was susceptible to acid and alkali hydrolysis, chemical oxidation and photo degradation. The degraded product peaks of Mirabegron were well resolved from the parent peak as data obtained from their significant Rf values. The method was validated and data from forced degradation study showed that the method can be used for quantifying Mirabegron in marketed formulation.

Total Flavonoids Content of Polar Extracts of Cayratia trifolia Leaves

Cayratia trifolia leaves contain flavonoids compound that can dissolve well in polar solvents. This study aimed to evaluate the total flavonoids content of polar extracts which extracted sequentially from C. trifolia leaves. The dried C. trifolia leaves were macerated continually using n-hexane, ethyl acetate, and ethanolic solvent. The total flavonoids content was determined using spectrophotometric methods with a AlCl3 reagent. TLC chromatogram of the extracts were evaluate using chloroform: aceton: formic acid (10:2:1) as a mobile phase and silica gel F254 as a stationary phase. The citroborate and ammonia vapour were used as detection spray reagents. The highest total flavonoids content (27.95±0.62 mgQE/g) were found in 70% ethanol extract of C. trifolia leaves compared to ethyl acetate extract (17.98 ± 0.89 mgQE/g). The ethanolic extract is found six compound spots, while the ethyl acetate extract found four compound spots which were thought to be a flavonoid group. Thus, it can be concluded that the different polar extracting solvents are able to attract flavonoid compounds with different levels. The flavonoids in C. trifolia extract need to be separated to determine the types.

Development and validation of HPTLC method for simultaneous estimation of bioactive components in combined extracts of three hepatoprotective plants

Picroside-I, andrographolide and silybin exhibit manifold biological activities, present as bioactive components in Picrorhiza kurroa (roots), Andrographis paniculata (aerial parts) and Silybum marianum (seeds), respectively. A validated HPTLC method was developed for the simultaneous determination of picroside-I, andrographolide and silybin in the combined hydroalcoholic extract of the plants as per ICH guidelines. On silica gel F254 HPTLC plates, the combined hydroalcoholic extract of plants and standard solutions were applied. The plates were run in twin chamber consisting n-butanol: glacial acetic acid: water (6:1:3, v/v/v) as a mobile phase and documented at 254 and 366 nm using Camag TLC scanner III with WinCATS software. The analysis revealed that a good linear relationship was established between response (peak area) and amount in the range of 60–600 ng/spot. The LOD (ng/spot) and LOQ (ng/spot) for estimation of picroside-I, andrographolide and silybin were found to be 15.089, 21.969, 25.702 and 45.725, 66.573, 77.885, respectively. Recovery experiments showed 97.75–103.74%, 99.75–101.12% and 98.96–101.69% for picroside-I, andrographolide and silybin, respectively. The HPTLC method showed good linearity, recovery and high precision of all the three bioactive components, which can be applied for the routine analysis of polyherbal formulations containing these plants as their ingredients.

Phytochemical Screening and High-performance Thin-layer Chromatography Quantification of Vitex trifolia Leaves Hydro-alcoholic Extract: Potential Anti-inflammatory Properties

Aims: The present study aims to evaluate the phytochemical composition of Vitex trifolia (V. trifolia) leaves hydro-alcoholic extract and to report for the first time, its phenolic content using a validated high-performance thin-layer chromatography (HPTLC) method.&#x0D; Study Design: Qualitative phytochemical analysis and HTLC densitometric quantitative analysis.&#x0D; Place and Duration of Study: The study was carried out at the Faculty of Pharmacy, Universiti Teknologi MARA (UiTM), Malaysia, from March 2020 to December 2020.&#x0D; Methodology: The preliminary phytochemical screening was carried out qualitatively. The HPTLC analysis was performed on glass-backed 60 F254 silica gel plates using a two steps gradient elution method of the mobile phase. In the first step, methanol was used to develop the plates until 40 mm of developing distance, while in the second step, plates were developed with n-hexane:ethyl acetate:acetic acid (20:9:1, v/v/v) until 80 mm of developing distance. Detection and quantification were performed by densitometric analysis at 254 nm. The method was validated as per the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) guideline in terms of linearity, precision, accuracy, the limit of detection (LOD), and the limit of quantification (LOQ).&#x0D; Results: The preliminary phytochemical screening of V. trifolia leaves hydro-alcoholic extract showed the presence of alkaloids, flavonoids, phenols, phytosterols, and terpenoids. The developed HPTLC method was proved to be linear, precise, and accurate. The LOD and LOQ of the method were determined to be 2.01 µg/band and 6.08 µg/band, respectively. The total phenolic content of the extract was calculated from the standard gallic acid calibration plot and found to be 136.94 ± 4.02 mg gallic acid equivalent (GAE)/g of dried extract.&#x0D; Conclusion: This preliminary study revealed that V. trifolia has a considerable amount of phenolic compounds, which can potentially contribute to its anti-inflammatory, antioxidant, and anticancer activities. Further pharmacological investigations are being carried out to support the folkloric claims.

A converged pharmaceutical analysis supported with hydrotropy & DoE with dual HPTLC and stress studies for estimation of tolvaptan

The article introduces an inventive, eco-friendly, simple, consistent and coalescing TLC-Densitometry approach to pharmaceutical estimation of Tolvaptan in bulk and tablet matrix. Towards as successful sample preparation and further analysis in both normal and reverse- phases, hydrotropic solubilization was chosen. The determination was performed by employing densitometric estimation using ultraviolet exposure at 271 nm. The separation was achieved on (10 × 10 cm) aluminum backed silica gel 60-F254 and RP-18 F254S as stationary phases separately. Optimized mobile phases were carbon tetrachloride: methanol (3.5: 1.5 v / v); acetonitrile: water (3: 2 v / v) for normal and reverse phase modes respectively. Dual quantitation was conducted over a concentration range of 300–1800 ng/band. The compact and well-resolved bands for the Tolvaptan standard and samples were obtained in both methods at a retention factor (Rf) value of 0.39 ± 0.2 and 0.42 ± 0.2 respectively. In order to examine the stability-indicating characteristics of the process, the compound was subjected to acidic and alkaline, oxidative, film, thermal (dry heat) and neutral stress conditions. Extensive degradation has been observed in conditions with acidic, alkaline and oxidative stress. Box-Behnken design was applied as a DoE approach to evaluate method robustness and to ensure system efficiency. The methods developed were validated according to the International Conference on Harmonization (ICH) for accuracy, robustness and ruggedness.

Phyto-pharmacognostical evaluation and HPTLC finger printing profile of Gulbanafsha (Viola odorata L.) flower

Viola odorata Linn. (Family Violaceae) is popularly known as Sweet Violet or English Violet and Gulbanafsha in Hindi. V. odorata is used as remedy for coughs, sore throat, hoarseness and tonsillitis. It is valued as an expectorant, antioxidant, diaphoretic, antibacterial, antipyretic and diuretic. Pharmacological studies have revealed the role of V. odorata in Unani drugs for treatment of common cold, asthma and cough associated diseases. The present study focuses on the phyto-pharmacognostical evaluation and HPTLC finger printing profile of Gulbanafsha (Viola odorata L.) flower - white variety. Physicochemical parameters were determined and phytochemical screening done by standard procedures. HPTLC finger-printing profile of the methanolic extracts of dried flowers, were performed on precoated TLC silica gel 60F254 plateswith salicylic acid as marker. Phytochemical screening revealed presence of flavonoids, glycosides, phenolic compounds/tannins.

ANTIBACTERIAL ACTIVITY AND TLC-BIOAUTOGRAPHY ANALYSIS OF THE ACTIVE FRACTIONS OF Muntingia calabura L. LEAVES AGAINST Staphylococcus aureus

This study aims to determine the antibacterial activity and TLC-bioautography profile of the active fractions of Muntingia calabura L. leaves against Staphylococcus aureus. Muntingia calabura L. leaves were macerated with ethanol 96% then fractionated with n-hexane, ethyl acetate, and methanol solvent, respectively. The antibacterial activity was tested by the Kirby-Bauer method to determine the most active fraction and the lowest concentration that inhibited the growth of Staphylococcus aureus. TLC-bioautography was tested using chloroform: ethyl acetate (2:8) as the mobile phase and silica gel F254 as the stationary phase. Antibacterial activity test of n-hexane and ethyl acetate fractions at a concentration of 10% w/v showed activities with inhibition zone diameter of 0.33±0.288 and 9.66±5.77 mm, respectively. At the same time, the methanol fraction showed no activity. The lowest concentration of ethyl acetate fraction which still showed the inhibition zone was 0.312% w/v. The TLC-bioautography profile showed active spots with an Rf value of 0.82 and had an inhibitory zone diameter of 4.013±0.864 mm. It can be concluded that the ethyl acetate fraction was the most active fraction that inhibited the growth of Staphylococcus aureus and had one active spot on the bioautography test.

Ecofriendly Validated Chromatographic Methods for Quantitation of Cyclizine and Its Toxic Impurities in Its Parenteral Formulation

Cyclizine (CYZ) abuse is commonly reported, either through oral or intravenous routes, for its euphoric or hallucinatory effects. The concomitant misuse of CYZ among addicted teenagers leads to life-threatening neuromuscular disorders. Consequently, two green and validated chromatographic methods were developed for the determination of CYZ, an antiemetic drug, in its parenteral formulation in the presence of 1-methyl piperazine and diphenylmethanol (benzhydrol) as the pharmacopeial stated impurities of CYZ. The first method was TLC-densitometry that relied on using a mixture consisting of ethyl acetate:isopropanol:ammonia (9.5:1:0.5, by volume) as a developing system, TLC 60 F254 silica gel plates as a stationary phase and detection of the scanned bands was performed at 210.0 nm. On the other hand, the second method was UPLC which based on the separation of CYZ and its impurities using a mobile phase consisting of ethanol and acidic water at pH 5 adjusted by phosphoric acid in the ratio of (60:40, %v/v) at flow rate 0.2 mL min−1 and UV detection were carried out at 210.0 nm. The greenness profile of the established methods was evaluated and calculated for the first time for CYZ and its toxic impurities through different assessment tools like analytical eco-scale, analytical method volume intensity and greenness profile methods. Validation of the developed methods was carried out in accordance with the guidelines of the International Conference on Harmonization. The suggested methods were accurate, reliable, time and cost-saving. Therefore, they could be used in quantification of CYZ abuse and its toxic impurities in parenteral formulation for routine quality control. The results achieved by applying the suggested methods were statistically analyzed and compared with those given by reported one and no significant differences were obtained regarding both precision and accuracy.

DEVELOPMENT AND VALIDATION OF STABILITY-INDICATING SIMULTANEOUS ESTIMATION OF METFORMIN AND ALOGLIPTIN IN TABLETS BY HIGH-PERFORMANCE THIN LAYER CHROMATOGRAPHY

Objective: A simple and stability-indicating high-performance thin-layer chromatographic method was developed and validated for the simultaneous estimation of metformin and alogliptin in tablets.&#x0D; Methods: The method was developed in TLC aluminum plates pre-coated with silica gel 60F254 as the stationary phase and the solvent system consists of methanol: chloroform: 0.5% ammonium sulphate [4:4:2, v/v/v]. The system was found to conferred a compact spot for metformin [Rf value of 0.44±0.02] and alogliptin [Rf value of 0.66 ± 0.22]. Densitometric analysis of metformin and alogliptin was carried out at the wavelength of 254 nm. Forced degradation studies were conducted to know the stability of the drug samples under various stress conditions like acid, base, peroxide, photolytic degradation according to the ICH guidelines.&#x0D; Results: The developed method was found to be suitable for the excellent separation of the drug samples. Calibration curves were linear in the range of 40-200 ng/spot with a correlation coefficient of 0.996 for metformin and calibration curves were linear in the range of 1-5 ng/spot with a correlation co-efficient of 0.997 for alogliptin, respectively. Stability study shows that the chromatograms of samples degraded with acid, base, hydrogen peroxide, dry heat, and photolytic showed well-separated spots of pure metformin and alogliptin as well as some additional peaks at different Rf values. The method was successively applied to pharmaceutical formulation. No chromatographic interference from the tablet excipients was found.&#x0D; Conclusion: The newly developed method can be applied for the identification and quantitative determination of metformin and alogliptin in the combined dosage form.

Development and validation of HPTLC method for identification and quantification of sterols from leaves of Erythroxylum monogynum Roxb. and in vitro evaluation of anti-oxidant and anti-glycation activities

The petroleum ether extract from leaves of Erythroxylum monogynum when subjected to phytochemical analysis and chromatographic purification, resulted in the isolation of two new sterols namely 4-methyl ergosta-7, 23‑dien‑3β-ol and 4-methyl ergosta-7, 24 (28)‑dien‑3β-ol. Their structure was elucidated by NMR and Mass Spectrometry. The two sterols were separated on a silica gel 60F254 HPTLC plates impregnated with AgNO3 using dichloromethane: methanol (9:1 v/v) as a mobile phase. Upon densitometric scanning at 254 nm in a reflectance mode, the two sterols separated as sharp and compact bands with Rf of 0.47±0.02 and 0.52±0.01 respectively for 4-methyl ergosta-7, 23‑dien‑3β-ol and 4-methyl ergosta-7, 24 (28)‑dien‑3β-ol. Linear regression and calibration plots constructed (n = 6) showed excellent linearity at 100–500 ng per spot with a correlation coefficient, r = 0.9979±0.0004 and 0.9974±0.0007 for 4-methyl ergosta-7,23‑dien‑3β-ol and 4-methyl ergosta-7,24 (28)‑dien‑3β-ol, respectively. The Limits of detection and quantification were found to be 21.07 ng and 57.23 ng/band for 4-methyl ergosta-7,23‑dien‑3β-ol and 25.18 ng and 63.86 ng/band for 4-methyl ergosta-7,24 (28)‑dien‑3β-ol, respectively. Both the sterols demonstrated good anti-oxidant and anti-glycation activities where, the IC50 values obtained were comparable with the positive control compounds, ascorbic acid and aminoguanidine. The 4-methyl ergosta-7,23‑dien‑3β-ol displayed 100% anti-oxidant activity at 100 µg/ml for both DPPH and H2O2 scavenging assays. In addition, both the sterols presented more than 90% inhibition of Advanced Glycation End products at 100 µg/ml concentration. The results obtained are remarkable indicating that these plant extracts could serve as a natural alternative to the conventional oral hypoglycemic molecules.

The Effect of Methanolic Extract and Ethyl Acetate Fraction of Kepel Fruit (Stelechocarpus burahol) to α-Glutathione S-Transferase Enzyme Concentration of Rat Liver and Blood Induced by CCl4

Degenerative diseases are mostly caused by high free radical concentrations in the body. Kepel fruit(Stelechocarpus burahol) is known to contain flavonoid compounds, a class of compounds that has free radicalscavenging activity in the body and could affect the activity of antioxidant enzymes. There have been reportsthat methanolic extract and ethyl acetate fractions of Kepel fruit have the lowest IC50 values, compared toextracts or fractions which are made using other organic solvents, on in vitro assays so that flavonoids aresuspected as responsible compounds to the free radical scavenging activity. This study aimed to identify thepresence of flavonoid content in methanolic extract and the fraction of ethyl acetate of Kepel fruit and also itseffect on rat alpha Glutathione S-Transferase (α-GST) enzyme concentration for the function to prevent hepaticcell damage due to carbon tetrachloride exposure. The identification of flavonoids content used a series ofcolor chemical reaction tests and thin layer chromatography that used silica gel 60F254 as the solid phase andmixture of ethyl acetate, formic acid, glacial acetic acid, and water (100: 11 : 11: 20 v/v) as the mobile phase.This study used 30 Sprague-Dawley female rats (age 2-3 months) that were grouped into 5 groups as solventcontrol (CMC-Na) group, negative control (CCl4) group, positive control (vitamin C) group, methanolic extractgroup, and ethyl acetate fraction group. All groups were induced with carbon tetrachloride (CCl4) compoundsexcept for the CMC-Na solvent control group. Blood sampling at 0 h, 24 h, and 72 h, while liver sampling at 24h and 72 h, with 3 samples each group. The analysis of rat α-GST enzyme concentrations used theEnzyme-linked Immunosorbent Assay (ELISA) method. Based on the phytochemical test, the methanolicextract and the ethyl acetate fraction of Kepel fruit have been identified contains flavonoids compound. Basedon the in vivo study, the positive control group's α-GST enzyme concentration was decreased, while thenegative control group's α-GST enzyme concentration was increased. Administration of methanolic extractand ethyl acetate fraction also decreased both liver and blood rat α-GST enzyme concentrations, althoughwithout significant correlation, and still could prevent the hepatic cell damage due to carbon tetrachlorideexposure.