Rapid High-performance Thin-layer Chromatography Research Articles

A rapid HPTLC fingerprinting technique for identifying the various geo-floral origins of honeys

Honey has several nutritional and therapeutic uses due to the presence of different bioactive compounds.These substances are derived from floral nectar. Therefore, it becomes essential to identify the distinct chemical profiles of honey samples. The aim of this research was to provide an accurate, straightforward, and sensitive HPTLC approach for different types of honey verification. Eight mono floral honeys (mustard, eucalyptus, litchi, orange, tea, Indian plum, black plum and pineapple) were collected from different origin of West Bengal (eastern India) and examined. Standard procedures were followed to check the quality of each honey.The flora was identified by microscopically examining the pollen found in honeys. High-performance thin layer chromatography (HPTLC) was used to analyze lipophilic fractions of each honey. Chromatographic results identified distinct patterns of bands with specific Rf values for each type of mono floral honey. HPTLC is a simple and effective method for routine analysis and verification. It can serve as authentication for different types of honey.

A greener RP-HPTLC-densitometry method for the quantification of apremilast in nanoformulations and commercial tablets: Greenness assessment by analytical eco-scale, ChlorTox, and AGREE methods

The greener “high-performance thin-layer chromatography (HPTLC)” methods for apremilast (APM) analysis in pharmaceutical products and biological fluids are not currently available in the literature. Accordingly, this study involves the development and validation of a rapid, simple, economical, and greener reversed-phase HPTLC methodology for the estimation of APM in prepared nanoparticles (NPs), nanoemulsion, and marketed tablets. The estimation of APM was conducted using “RP-18 silica gel 60 F254S HPTLC plates” as the stationary phase. The combination of ethanol/water (65:35, v/v) was used as the greener mobile phase for APM analysis. The greenness of the method was predicted using three different approaches, namely Analytical Eco-Score (AES), ChlorTox, and the Analytical GREENness (AGREE) approaches. The λmax = 238 nm was used for the APM detection. By contrasting its single band at Rf = 0.61 ± 0.01 with those of pure APM, the HPTLC peaks for APM in an prepared NP formulation, nanoemulsion, and marketed tablets were identified. In the concentration range of 100–700 ng/band, the proposed analytical methodology was linear. The values of AES, ChlorTox, and AGREE were determined to be 93, 0.66 g, and 0.89, respectively, demonstrated an outstanding greener profile for the existing method. The amount of APM in the tablet, NP formulation, and nanoemulsion was found to be 98.40, 101.60, and 99.37 %, respectively. According to the findings of validation tests and pharmaceutical analysis, the suggested analytical technique could be successfully applied for the routine examination of APM in marketed tablets and laboratory generated nanoformulations.

Quantification of Suvorexant in Human Urine Using a Validated HPTLC Bioanalytical Method.

Suvorexant (SUV) is a new sedative/hypnotic medicine that is recommended to treat insomnia. It is an important medicine from a forensic point of view due to its sedative/hypnotic and depressant effects. To the best of our knowledge, high-performance thin-layer chromatography (HPTLC) bioanalytical methods have not been published to measure SUV in human urine and pharmaceutical samples. Accordingly, this study was designed and validated a sensitive and rapid bioanalytical HPTLC method to determine SUV in human urine samples for the very first time. The densitometric measurement of SUV and the internal standard (IS; sildenafil) was performed on glass-coated silica gel normal-phase-60F254S TLC plates using a mixture of chloroform and methanol (97.5:2.5 v/v) as the eluent system. Both the SUV and IS were detected at a wavelength of 254 nm. Both analytes were extracted using the protein precipitation technique utilizing methanol as the solvent. For the IS and SUV, the Rf values were 0.09 and 0.45, respectively. The proposed bioanalytical method for SUV was linear in the 50-1600 ng/band range. The current bioanalytical technique was linear, precise (% RSD = 3.28-4.20), accurate (% recovery = 97.58-103.80), robust (% recovery = 95.31-102.34 and % RSD = 2.81-3.15), rapid, and sensitive (LOD = 3.73 ng/band and LOQ = 11.20 ng/band). These findings suggested that the current bioanalytical method can be regularly used to determine SUV in wide varieties of urine samples.

A Rapid and Sensitive Stability-Indicating Eco-Friendly HPTLC Assay for Fluorescence Detection of Ergotamine.

Eco-friendly liquid chromatographic methods for measuring ergotamine (EGT) are scant in the published database. Accordingly, the goal of the current study was to develop a high-performance thin-layer chromatography (HPTLC) method for fluorescence detection of EGT in commercially available tablets. This approach was based on the application of ethyl alcohol-water (80:20 v/v) as the eco-friendly eluent mixture. The fluorescence detection of EGT was carried out at 322 nm. The greenness score of the present approach was evaluated by "Analytical GREENness (AGREE)" technology. The present approach for measuring EGT in the 25-1000 ng band-1 range was linear. The present assay for fluorescence detection of EGT was validated successfully by ICH guidelines for various parameters. The method was found to be rapid, sensitive, eco-friendly, and stability-indicating. The computed AGREE index for the current strategy was 0.84, displaying outstanding greenness features. The present methodology successfully separated the EGT degradation products under forced-degradation circumstances, exhibiting its stability-indicating qualities and selectivity. An amount of 99.33% of EGT was found in commercial formulations, indicating the validity of the current method for pharmaceutical analysis of EGT in commercial products. The results showed that EGT in commercial products might be regularly measured by the existing method.

Quality control assessment of Aegle marmelos (L.) Correa: A combined approach using high-performance thin-layer chromatography, heavy metal, pesticide and aflatoxin analysis

Aegle marmelos or bael is a medicinally important tree species used in various Indian traditional systems of medicines. In the present study, a sensitive and rapid high-performance thin-layer chromatography method was developed for the simultaneous determination of marmelosin, umbelliferone, and psoralen from the four different aerial parts of A. marmelos. The method uses HPTLC silica gel 60 RP-18 W plates as stationary phase, and acetonitrile: 0.1% orthophosphoric acid in water (1:1 v/v) as solvent system. The developed method is validated for specificity, precision, repeatability, and accuracy in accordance with International Conference on Harmonisation (ICH) guidelines. Further, the estimation of heavy metal suggested that the A. marmelos bark sample was highly contaminated with cadmium (3.8 ± 0.7 µg/g) which was far from the permissible range as described by World Health Organization (WHO). The observed chromium concentration was higher in the bark and the fruit than the normal range recorded in the plants. Arsenic and lead content was within the permissible range, while mercury was not detected in any of the tested samples. Studied samples were further tested for the presence of organochlorine and pyrethroid pesticides, as well as organophosphate and carbamates. Results suggested that gamma-hexachlorocyclohexane (γ-HCH) was more prominent and detected in the bark and the young twig samples. Carbamates were not detected in any of the samples. Aflatoxins were absent from all the samples analyzed through ultra-performance liquid chromatography - fluorescence (UPLC-FLR) analysis. The present study demonstrated that phytochemicals and physicochemical estimation are highly required for maintaining the quality of the raw drugs before the preparation of any formulations.

High performance thin layer chromatography (HPTLC) method development and validation for determination of doxycycline hyclate in capsule and tablet formulations

Abstract According to World Health Organization (WHO) 10% of the medicines in the Low and Middle Income Countries (LMICs) are of poor quality posing a major public health threat. One way to circumvent such problem is the development and deployment of rapid, economical and efficient analytical methods. Hence this research aims to develop a High-Performance Thin Layer Chromatography (HPTLC) method for the determination of doxycycline hyclate. A rapid and simple HPTLC method with densitometry detection at 360 nm to determine doxycycline hyclate in capsules and tablet formulations was developed and validated. HPTLC was performed on glass plates coated with C18 reverse phase silica gel 60 F254 and pretreated with 0.27 M ethylenediaminetetraaceticacid (EDTA) solution. The mobile phase was dichloromethane: methanol: acetonitrile: 1% aqueous ammonia in the ratio of 10:22:53:15 (v/v). The linearity range lies between 200 and 1,000 ng/spot with correlation coefficient of 0.997. The Rf value is 0.5 ± 0.02%. Recoveries were in the range of 94.50–100.5%. Limit of detection and limit of quantitation values for doxycycline hyclate were 40 and 160 ng/spot respectively. The developed method was validated as per ICH guidelines. Thus, it was found to be accurate, precise, specific and robust. In forced degradation study, doxycycline hyclate was found to degrade in acidic and alkaline media, and through oxidative stress. The drug was found to be relatively stable to heat and photo degradation. The method was successfully applied for the routine quantitative analysis of dosage forms containing doxycycline hyclate. The developed method offered comparable results (as confirmed by F-test) with that of the HPLC pharmacopoeial (BP) analysis method.

Response surface methodology and artificial neural network modelling for optimization of solid-liquid extraction and rapid HPTLC analysis of glycyrrhizin in Glycyrrhiza glabra root

Glycyrrhizin, a major bioactive compound of Glycyrrhiza glabra has been used in traditional medicine system to treat several diseases. In the past few decades, its demand has been increased significantly for the active ingredient glycyrrhizin as well as for being a zero calorie sweetener. In the current study, extraction of glycyrrhizin from dry roots of G. glabra was established by reflux method. Quantitative estimation of glycyrrhizin content in the extracted samples was analysed by using high performance thin layer chromatography (HPTLC) system. In this study, response surface methodology (RSM) and artificial neural network (ANN) were used for the first time as a model to optimize the extraction conditions of glycyrrhizin from G. glabra roots in order to compare and establish effective prediction models. The extraction process variables including the time, temperature, solvent composition, particle size, solvent to solute ratio, pH and extraction steps were optimized using a Plackett-Burman design (PBD), Central Composite design (CCD), artificial neural network (ANN) and Multiple Layer Perceptron (MLP) on glycyrrhizin yield. The optimum extraction conditions were 55°C (extraction temperature), 45 min (extraction time), 55% (v/v) (ethanol concentration) and 30 ml/g (solvent to solute ratio) to obtain the maximum glycyrrhizin yield. The contributions of the quadratic model with high determination coefficient (R2=0.9806) were observed and showed good consistency between the experimental value 7.6 mg/g (0.76%) and predicted value 7.51 mg/g (0.751%). Response values were close to the predicted values with prediction accuracy as ANN > RSM, indicating that ANN model has higher prediction accuracy than RSM. The study suggests that RSM and ANN model system can be manipulated for the optimization and production of valuable bioactive compounds.

High-Performance Thin-Layer Chromatography (HPTLC) Method for Identification of Meloxicam and Piroxicam

Background: High-performance thin-layer chromatography (HPTLC) is an advantageous, modern analytical technique based on the principles of thin-layer chromatography (TLC), which provides essential benefits, such as improved sample application, better and faster separation, and less mobile phase usage. The aim of this work was to develop a simple and rapid HPTLC method that could be used for the identification of meloxicam and piroxicam. Methods: HPTLC. The analysis was carried out using silica gel 60 F254 glass TLC plates and as the mobile phase: hexane: ethyl acetate: glacial acetic acid, in a ratio of 65:30:5 v/v/v. For the standard solution preparation, ethanol was used. Front: 60 mm. Detection was performed at 366 nm. Results: The Rf value for meloxicam was 0.81 and the Rf value for piroxicam was 0.57. The proposed method can be used in the detection of the analyzed compounds in very low concentrations. It was established that the detection limit of meloxicam was 0.04 μg per band and that of piroxicam was 0.05 μg per band. It was also established that the quantitation limit of meloxicam was 0.12 μg per band and that of piroxicam was 0.15 μg per band. Conclusions: The proposed method is simple, sensitive, stable, cost effective, and eco-friendly. It could be used in research or for routine quality control purposes.

Simultaneous Determination of Caffeine and Paracetamol in Commercial Formulations Using Greener Normal-Phase and Reversed-Phase HPTLC Methods: A Contrast of Validation Parameters.

There has been no assessment of the greenness of the described analytical techniques for the simultaneous determination (SMD) of caffeine and paracetamol. As a result, in comparison to the greener normal-phase high-performance thin-layer chromatography (HPTLC) technique, this research was conducted to develop a rapid, sensitive, and greener reversed-phase HPTLC approach for the SMD of caffeine and paracetamol in commercial formulations. The greenness of both techniques was calculated using the AGREE method. For the SMD of caffeine and paracetamol, the greener normal-phase and reversed-phase HPTLC methods were linear in the 50–500 ng/band and 25–800 ng/band ranges, respectively. For the SMD of caffeine and paracetamol, the greener reversed-phase HPTLC approach was more sensitive, accurate, precise, and robust than the greener normal-phase HPTLC technique. For the SMD of caffeine paracetamol in commercial PANEXT and SAFEXT tablets, the greener reversed-phase HPTLC technique was superior to the greener normal-phase HPTLC approach. The AGREE scores for the greener normal-phase and reversed-phase HPTLC approaches were estimated as 0.81 and 0.83, respectively, indicated excellent greenness profiles for both analytical approaches. The greener reversed-phase HPTLC approach is judged superior to the greener normal-phase HPTLC approach based on numerous validation parameters and pharmaceutical assays.

A rapid, sensitive, and greener stability-indicating normal-phase HPTLC method with univariate calibration for the estimation of chlorhexidine acetate in its commercial formulations

Till date, there is no report on high-performance thin-layer chromatography (HPTLC) analysis of chlorhexidine (CHD) or its salts like CHD acetate (CHD-A), CHD gluconate, and CHD hydrochloride. Therefore, a rapid, sensitive, and greener normal-phase HPTLC method has been reported for the analysis of CHD-A in its four different commercial dosage forms. The quaternary combination of ethyl acetate: ethanol: water: formic acid (75:10:10:5, v v v v−1) was optimized as the green solvent system/mobile phase for CHD-A analysis. The CHD-A detection was carried out at 264 nm. The greener normal-phase HPTLC assay was linear in the range of 10–2000 ng band−1. In addition, the proposed method was accurate, precise, sensitive, and selective for CHD-A analysis. The greener normal-phase HPTLC method was able to detect CHD-A in the presence of its degradation products, suggesting the stability-indicating property of this method. The content of CHD-A in commercial products A, B, C, and D was detected as 0.18, 2.02, 1.46, and 0.19% w w−1, respectively. The greenness scale for the greener normal-phase HPTLC assay was estimated as 0.88 utilizing “analytical GREEnness (AGREE)” calculator, suggested the greener nature of the normal-phase HPTLC assay. Overall, these data suggested that the greener normal-phase HPTLC method can be successfully used for the determination of CHD-A in its commercial products.

Enrichment of alliin in different in vitro grown tissues of Allium sativum through CdCl2 elicitation as revealed by high performance thin layer chromatography (HPTLC)

Plant cell tissue and organ culture techniques provide an efficient way for the biosynthesis of high-value secondary metabolites in which elicitation has been recognized to be a potent approach in enriching the bioactive compounds. In this study, an effort was made to assess the influence of Cadmium chloride (CdCl2) on alliin yield of different in vitro grown tissues such as callus, somatic embryos, leaves and roots of Allium sativum. The tissues were cultured in MS amended with various levels of CdCl2 [control (0 mM), T1 (0.05 mM), T2 (0.1 mM), T3 (0.15 mM) and T4 (0.2 mM)] and the yield of alliin was quantified by using high performance thin layer chromatography (HPTLC). Biochemical analyses revealed that sample tissues under increasing CdCl2 conditions accumulated higher levels of sugar, protein and proline with maximum content of sugar (19.61 mg g−1 FW), protein (6.01 mg g−1 FW) and proline (12.19 mg g−1 FW) in leaves at T3 after 4 days of elicitor treatment. Changes within the activities of varied antioxidant enzymes like catalase (CAT), superoxide dismutase (SOD) and ascorbate peroxidase (APX) was assayed as the addition of CdCl2 in the medium causes stress. We noted that with an elevated level of CdCl2, antioxidant enzyme activity increased linearly with the highest activity observed in leaves at T4 of 6 days of elicitation (4.97 EU min−1 mg−1 protein CAT, 6.30 EU min−1 mg−1 protein SOD and 1.125 EU min−1 mg−1 protein APX). Increased activities of enzymes are involved in negating the cellular stress triggered due to the overproduction of ROS. The yield of alliin in response to varying levels of CdCl2 was estimated by using a simple, rapid, and sensitive HPTLC method. The best separation was achieved using butanol:propanol:acetic acid:water (3:1:1:1) as mobile phase. For analysis of alliin, the plates were scanned at 550 nm after spraying with ninhydrin and a good resolution was achieved with Rf value of 0.66. In our study, the alliin yield enhanced in all tested cultures with the elicitor level up to T3. In leaves, maximum accumulation of alliin (3.37 μg g−1 dry wt.) was noted on 0.15 mM (T3) CdCl2 added medium. Our data indicated the increase of alliin in CdCl2 added cultures, which suggests that the use of elicitors as a potential strategy for augmenting secondary metabolite synthesis.

Rapid HPTLC determination of patulin in fruit-based baby food in Turkey

Mycotoxin contamination is an ongoing problem for all kinds of food commodity, and it is necessary to investigate the extent of this problem in baby food products. It is seen that factors such as developing technology, urbanization, and intense work tempo change dietary habits in every age group. The aim of this study was to provide a current overview on patulin (PAT) contamination of fruit-based baby food in Turkey. Safety levels of products were evaluated based on the provisional maximum tolerable daily intake (PMTDI) value predefined by the regulatory agencies. A total of 58 baby foods from Turkey market were investigated in this study. A high-performance thin-layer chromatography (HPTLC) method was developed by using acidic QUECHERS (quick-easy-cheap-effective-rugged-safe) extraction process. PAT was detected in 12 of 58 with a concentration between 0.13 and 2.98 mg/L; the mean value and the median value were found 1.71 mg/L and 1.92 mg/L, respectively. Based on PMTDI values recommended as 0.4 μg/kg bm/day, it was found that 11 of the 12 PAT-containing products were above the legal limit. The presence of PAT-containing baby food above the permitted values will be the subject of acute or chronic intoxications in the clinic, forensics in the consumer courts, and financial obligations of the questioned producers during the importation of the product at customs.

Qualitative and quantitative analysis of arctiin and arctigenin in Arctium tomentosum Mill. by high-performance thin-layer chromatography

A sensitive, reliable and rapid high-performance thin-layer chromatography (HPTLC) method for the determination of arctiin and arctigenin in Arctium tomentosum Mill. was established. A. tomentosum Mill. extract was used for chromatographic analysis. The ratio of chloroform and methanol was 48:5 as mobile phase. Temperature is 20–23 °C and humidity is less than 30%. The scanning wavelength is 280 nm. The results showed that arctiin had a good linear relationship in the range of 0.5315–5.8465 μg, r = 0.9982; arctigenin had a good linear relationship in the range of 0.5654–6.2194 μg, r = 0.9951. Precision analysis showed that the RSD < 3.0%. The stability study showed that the sample was stable within 24 h at room temperature, RSD < 2.0%. The average recoveries were 103.07 ± 1.57% and 98.55 ± 2.71%, respectively. The antioxidant activity of Arctium tomentosum Mill. was also identified. The results showed that the antioxidant component identified by thin-layer chromatography–1, 1-diphenyl-2-trinitrophenylhydrazine (TLC-DPPH) was arctigenin not arctiin. The proposed HPTLC is a simple and accurate method for the qualitative and quantitative analysis of arctiin and arctigenin in Arctium tomentosum Mill. from different areas.

Impact of seasonal variation on ‘daidzein’ accumulation in callus and in vivo parts of Pueraria tuberosa (Willd.) DC

Daidzein-natural isoflavones found in Pueraria tuberosa (Willd.) DC. (Fabaceae) which is a potent medicinal plant. It acts as signal carriers and responds to the pathogenic attacks and reduces pain during menopause, osteoporosis and also antidiabetic in nature. In the present work, in vitro callus development from tuber explants during both rainy and summer seasons was conducted. Further, a simple unambiguous and rapid high-performance thin-layer chromatography method was established for quantitative estimation of daidzein in callus and in vivo parts to assess the impact of seasonal change on valuable phytochemical accumulation. Maximum callusing (90%) was obtained on MS medium fortified with a combination of 6N-Benzylamino purine (2 mg/l) and 2,4-dichlorophenoxyacetic acid (2 mg/l) during the summer season from the tuber explant. During the HPTLC method validation, the linearity range obtained was 100–1000 ng/spot with a regression value (r) value of 0.99845. All the parts were found to contain a significant amount of daidzein. The maximum daidzein (2112.567±0.35 ng/g) content was obtained from a young tuber bark followed by callus (171.903±0.33 ng/g) during the summer season compared to rainy season parts and callus. Thus, it can be concluded that in vitro callus is an alternative source of daidzein without destroying the natural plant and the developed HPTLC method could be used for quality control analysis and recommended for daidzein quantification for different herbal formulation and drug preparation.

DEVELOPMENT AND VALIDATION OF A HIGH-PERFORMANCE THIN-LAYER CHROMATOGRAPHY FOR THE DETERMINATION OF TERBUTALINE SULFATE, BROMHEXINE HYDROCHLORIDE, AND ETOPHYLLINE IN PHARMACEUTICAL DOSAGE FORM

Objective: The study aimed to development and validation of simple, precise, and reliable high-performance thin-layer chromatography (HPTLC) for the determination of terbutaline sulfate (TBS), bromhexine hydrochloride (BRH), and etophylline (ETP) in pharmaceutical dosage form.&#x0D; Methods: A simple, precise, rapid, and accurate HPTLC method was developed for the estimation of TBS, BRH, and ETP in pharmaceutical dosage form. Pre-coated silica gel G60 F254 aluminum sheet (10 cm2×10 cm2 and thickness 0.2 mm) was used as stationary phase while mobile phase consisting of benzene: methanol:glacial acetic acid 8:0.5:1.5 v/v/v detection at 275 nm. The present method had validated according to ICH guidelines.&#x0D; Results: Migration distance found 80 mm at 275 nm. The retention factor found to be 0.24, 0.57, and 0.68, respectively. The detector response was linear in the concentration range of 60–210 ng/band, 2400–8400 ng/band, and 96–336 ng/band, respectively. The linear regression equation being Y=32.20x−562.9, Y=11.79x−1711, and Y=1.756x−5636, respectively. The limit of detection for TBS 0.677 μg, for BRH 8.123 μg, and for ETP 57.915 μg and limit of quantification to be 2.053, 24.617, and 175.5 μg, respectively, were found. The developed method validated by ICH guideline, i.e., accuracy, precision, robustness, specificity, and system suitability.&#x0D; Conclusion: In this study, we had developed a simple, fast, and reliable HPTLC method for the determination of TBS, BRH, and ETP in pharmaceutical dosage form.

Modeling and optimization of ultrasound-assisted green extraction and rapid HPTLC analysis of stevioside from Stevia Rebaudiana

Stevioside is the major constituent of the Stevia Rebaudiana plant. The aim of this study is to appraise optimum procedure conditions for extraction of stevioside from Stevia Rebaudiana by Peleg’s mathematical methodology. Stevioside from dried leaves of Stevia Rebaudiana was extracted by means of probe type sonication system using glycerol as a green and generally recognized as safe (GRAS) solvent. High Performance Thin Layer Chromatography (HPTLC) was used as a very efficient, rapid and precise method for stevioside detection and analysis. Results demonstrated that the optimum conditions for ultrasound-assisted extraction (UAE) of stevioside from Stevia Rebaudiana are glycerol as solvent, temperature of 70 °C, plant to solvent proportion of 1:30, plant powder particle size of 0.075 mm, 200 W ultrasound irradiation power and 20 kHz ultrasound irradiation frequency. The comparing experimental results for Ceq of extracted stevioside with calculated values from Peleg’s mathematical model showed good and acceptable agreement. As a consequence, it was concluded that the ultrasound-assisted extraction is an efficient, fast, and economic method for extraction of stevioside from Stevia Rebaudiana in comparing with Soxhlet and maceration methods. Moreover, selected mathematical model displays the system with an acceptable accuracy which can predict the optimal conditions for extraction of stevioside those are close enough to experimental results.Practical Applications This investigation presented a mathematic model and experimental approach for the optimization of stevioside content of Stevia Rebaudiana using the ultrasound-assisted extraction and HPTLC analysis. Compared with the classic methods for optimizing of stevioside using the mathematic approaches or chemical experiments, the Peleg’s method is more precise and convenient. Moreover using ultrasound and HPTLC as extraction and analysis method, respectively, provides safer, rapid, and simple way for their stevioside extraction. Therefore, the method demonstrated in the present investigation could be used to extract the stevioside of Stevia Rebaudiana in food and beverage industries.

Stability indicating high-performance thin layer chromatography method for estimation of ascorbic acid in Hibiscus sabdariffa L. aqueous extract

Background: High performance thin layer chromatography (HPTLC) has become a routine analysis technique due to its advantages of low operating cost, high sample throughput and need minimum sample clean up. Aim: To develop and validate an accurate, specific, repeatable and stability-indicating HPTLC method for the determination of ascorbic acid in presence of their degradation products for assessment of stability of the ascorbic acid in Hibiscus sabdariffa L. preparations. Methods: Chromatographic sample separations employed thin layer chromatography plates (20 cm x10 cm) pre-coated with silica gel 60 F254 as a stationary phase. Solvent system consisted of acetone: toluene: water: glacial acetic acid (26:4:4:1 v/v) while scanning wavelength of 269 nm was used. Ascorbic acid was subjected to stress degradation conditions i.e. oxidation, dry heat treatment, photo degradation and hydrolysis under different PH. Method was validated according to international council for harmonisation guidelines. Results: Peaks of products formed were well resolved with different Rf values. The linear regression data for the calibration plots showed good linear relationship with r2 = 0.9912 in the concentration range 2-4 µg/ml. The mean value slope and intercept were -531.72 and 8040.22 respectively. Conclusion: A rapid, simple, accurate and specific HPTLC method for determination of ascorbic acid in Hibiscus sabdariffa L. preparations was developed and validated. In addition, this method separated all the degradants from stress conditions showing that it is a stability indicating method. The developed method could now be used in quality control of H. sabdariffa herb products.

High-performance thin-layer chromatography marker-based standardization of Piperine, Asiaticoside, and Withanolide-A in the developed polyherbal formulation and in vitro evaluation of acetylcholinesterase inhibition

Background: Preparation of highly standardized polyherbal formulation with its chief active chemical constituents supported by therapeutic efficacy in vitro is a valuable approach in the field of pharmaceutical sciences. Objective: The present work aims to develop the high-performance thin-layer chromatography (HPTLC) marker-based standardization of polyherbal formulation using Piperine, Asiaticoside, and Withanolide-A and in vitro acetylcholinesterase inhibition activity. Materials and Methods: For successful standardization, the HPTLC quantification of Piperine, Asiaticoside, and Withanolide-A was carried out. Suitable solvent systems were optimized to achieve the better resolution of the marker compounds, extracts, and sample formulation. The reproducibility of the methods was also confirmed by repeating the procedure twice. The identity of the bands in the sample formulation was confirmed by comparing the Rf value with those of their respective reference standards. In vitro acetylcholinesterase inhibition assay was done on the different crude extracts as well as tablet formulation. Results: HPTLC quantification of formulation for Piperine content showed 1.97% content w/w, whereas Piper longum extract showed 2.44% w/w. Similarly, Asiaticoside showed 0.71% in formulation and in Centella asiatica extract, it was 1.33% w/w. Contrary to the above, Withanolide-A content was 2.12% w/w in formulation and in Withania somnifera extract, it was only 0.81% w/w. In vitro acetylcholinesterase inhibition assay exhibited significant inhibition with IC50 value of 70 μg for tablet formulation. Conclusion: The presence of Piperine, Asiaticoside, and Withanolide-A in formulation was identified by rapid HPTLC quantification method. The method is very precise, reproducible, and accurate. The developed method can be used to quantify the marker compounds in the formulations available in the market. The formulation also evaluated for its acetylcholinesterase inhibition assay and showed significant activity. Hence, it could be used in the treatment of memory-related disorders such as Alzheimer's disease.

Stability-indicating high-performance thin-layer chromatographic determination of ondansetron in pure form and pharmaceutical formulations

A new, simple, precise, accurate, stability-indicating, and rapid high-performance thin-layer chromatography (HPTLC) method was developed and validated for the determination of ondansetron hydrochloride in pure form and pharmaceutical formulations. The method used Merck HPTLC aluminum plates precoated with silica gel 60 F254 as the stationary phase. The mobile phase consisted of chloroform–methanol–ethyl acetate (7:2:1, v/v). This system was found to give a compact spot of ondansetron (RF value of 0.67 ± 0.011); ondansetron was subjected to acid and alkali hydrolysis, oxidation, and photodegradation. The wavelength of TLC scanner was set at 302 nm for both detection and quantitation. The calibration curves were linear over the range of 25–500 ng spot−1. The limit of detection was 4.9 ng spot−1, and the limit of quantitation was 14.7 ng spot−1. The proposed analytical method was validated according to the International Conference on Harmonization (ICH) guidelines, and the results were acceptable. The propo...

High-performance thin-layer chromatography method for the simultaneous determination of itopride, pantoprazole, and mosapride in their formulations and spiked human plasma

The combination of itopride (ITP), pantoprazole (PAN), and mosapride (MS) is widely used in the treatment of many gastrointestinal tract (GIT) disorders. For that purpose, a new, simple, precise, accurate, and rapid high-performance thin-layer chromatography (HPTLC) method was developed and validated for the simultaneous determination of ITP, PAN, and MS in their pharmaceutical formulations. The method used Merck HPTLC aluminum plates precoated with silica gel 60 F254 as the stationary phase. The mobile phase consisted of methylene chloride–ethyl acetate–methanol– ammonia (25%) (12:2:0.8:0.2, v/v); this system was found to give compact spot of itopride (RF value of 0.22 ± 0.008), pantoprazole (RF value of 0.41 ± 0.006), and mosapride (RF value of 0.62 ± 0.029). The wavelength of thin-layer chromatography (TLC) scanner was set at 289 nm for both detection and quantitation. The calibration curves were linear over the range of 100–1500 ng spot−1 for ITP and MS, and 70–1500 ng spot−1 for PAN. The detection li...