High-performance Thin-layer Chromatographic Method Research Articles

A high-performance thin-layer chromatography densitometric method for the separation of isomeric ceftriaxone in powder for injection formulation

The aim of this study was to develop and validate a High-Performance Thin Layer Chromatographic (HPTLC) method for simultaneous determination of ceftriaxone and ceftriaxone e-isomer in powder for injection formulation. Ceftriaxone sodium injection is an antibiotic that used globally. It has Z/E geometrical conformation, in which ceftriaxone sodium and 3 ene-isomer have Z- conformation while (E)-isomer has E- conformation and the potential toxicity of ceftriaxone (E)-isomer has been reported. Thus, to safeguard the public health, a simple and easy to use, rapid and reliable method was developed for qualitative and quantitative determination of ceftriaxone sodium and its (E)–isomer. Samples were applied on HPTLC glass plates precoated with silica gel 60F254 by using Linomat semi-auto sampler. Separation was carried out using acetone, triethyl amine, water, chloroform and ethyl acetate as a mobile phase in different ratios. The Rf values of separated compounds were 0.51 ± 0.01 and 0.62 ± 0.01 for ceftriaxone sodium and ceftriaxone (E)-isomer respectively. The method was validated by studying Specificity, Linearity, Accuracy, Precision, Robustness, Limit of Detection (LOD) and Limit of Quantification (LOQ) and Solution stability. The developed method was successfully, sensitive, simple, precise, accurate, robust and applicable for the simultaneous determination of ceftriaxone sodium and ceftriaxone (E)-isomer in powder for injection formulation.

Stability-indicating high-performance thin-layer chromatography method development and validation for the simultaneous estimation of azithromycin, fluconazole, and secnidazole in active pharmaceutical ingredient and its tablet dosage form

Stability-indicating high-performance thin-layer chromatography method development and validation for the simultaneous estimation of azithromycin, fluconazole, and secnidazole in active pharmaceutical ingredient and its tablet dosage form

Stability Indicating HPTLC Method for Estimation of Cubebin in Crude Drug and Marketed Formulations

Background: Cubebin is a naturally occurring lactone lignan isolated from the plant species Piper cubeba. It exhibits a range of biological activities, including anti-inflammatory, antimicrobial, and anticancer properties. This compound has garnered significant interest for its potential therapeutic applications in traditional and modern medicine. Aim: This study aimed to develop a novel stability-indicating High-Performance Thin Layer Chromatography (HPTLC) technique that is simple yet precise in estimating cubebin in crude drug and marketed formulations. Method: Cubebin was estimated using precoated TLC silica gel 60 F254 plate as a stationary phase and n-hexane: ethyl acetate: formic acid: methanol (6:3.5:0.5:0.3 v/v/v/v) as a mobile phase with the chamber saturation time of 20 mins. Key Findings: Cubebin was detected and quantified at the wavelength of 283nm. The Rf value of cubebin in standard solution, Piper cubeba extract, Dhanwantari Vati, and Khadiradi Vati was found to be 0.494±0.0015, 0.492±0.002, 0.490±0.0020, 0.499±0.001 respectively. The calibration curve exhibited linearity within the range of 3000-13000 ng per band with a correlation coefficient (r2) of Cubebin was 0.9968. The method developed was utilized for the quantitation of cubebin in Piper cubeba and both the marketed formulations. Conclusion: A novel HPTLC method was developed and validated in accordance to ICH guidelines for the analysis of cubebin in crude drug and marketed formulations. The method was found to be simple, precise, accurate, and specific. This method is suitable for quality control and stability testing of cubebin-containing formulations.

Development of Amla Nutri-bar: Proximate analysis, gallic acid quantification and its validation through HPTLC, and spectroscopy

BACKGROUND: “Ayush Ahara” confluences advanced food technology with Ayurveda to develop dietary products rooted in traditional medicine. Here, Amla (Indian gooseberry), a medicinal fruit well-known for its health benefits, was used to develop a novel dietary product—Amla Nutri-bar (ANB). Furthermore, this research aims to substantiate its therapeutic potential through quality control analysis. METHODS: Three batches of ANB were prepared at the pharmaceutical laboratory, All India Institute of Ayurveda, New Delhi, India. These were evaluated for sensory attributes using a 9-point hedonic scale and underwent physicochemical, phytochemical, total phenolic content, chromatographic, and spectroscopy assays. RESULTS: The sensory evaluation indicated positive feedback, with an overall acceptability score of 8, followed by taste and flavor rated at 7 Physiochemical constants included pH: 2.2, moisture: 11.48%w/w, total ash: 0.17%w/w, water-soluble extractive value: 88.4%w/w, and alcohol-soluble extractive value: 56.65%w/w. Phytochemical screening revealed that the product is rich in flavonoids, tannins, and alkaloids. Nutritional composition per 100 g had energy: 373.76 kcal, proteins: 1.61%, carbohydrates: 88.59% by wt, dietary fiber: 2.72 g, total fat: 1.44%, vitamin C: 300.11 mg, vitamin A: 71.23 pg, iron: 5.63 mg, calcium: 126.69 mg, and potassium: 103.57 mg. Total phenolic content was 4.95 mg gallic acid equivalents/g, with gallic acid quantified at 270.4 μg/100 mg employing a validated high-performance thin layer chromatography method. Fourier transform infrared spectroscopy analysis showed the presence of alcohol, phenol, and alkanes. All the tested pesticide residues were found below detection limits. CONCLUSION: ANB can be a food product that is, not delicious only but can also contribute significantly to overall health and well-being.

Green stability-indicating RP-HPTLC technique for determining croconazole hydrochloride

Abstract The objective of the proposed investigation is the development and validation of a green stability-indicating reverse-phase high-performance thin-layer chromatographic method to determine croconazole hydrochloride (CCZ). The developing system used was an 80:20 v/v mixture of acetone and water. The measurement of CCZ was done at 198 nm. With the use of the Analytical Eco-Scale (AES), ChlorTox, and Analytical GREEnness (AGREE) tools, greenness was evaluated. The linearity was demonstrated by the present method in the 25–1,200 ng/band range. The present approach was additionally reliable, accurate, sensitive, precise, and green. An exceptional greenness profile was demonstrated by the AES, total ChlorTox, and AGREE scales, which were determined to be 89, 1.08 g, and 0.82, respectively. The greenness metrics of the present method were much better than the reported high-performance liquid chromatography approach. Under acid and oxidative degradation circumstances, CCZ was shown to be unstable, while under alkaline and thermal-stress settings, it was sufficiently stable. Furthermore, the stability-indicating component determined by analytical method identified CCZ in the presence of its degradation products. Commercial CCZ cream was found to contain 0.98% w/w of CCZ. The investigation’s results suggested that CCZ in commercially available creams might be regularly examined with the help of the recommended green technology.

Development and Validation of a High-Performance Thin-Layer Chromatography Method for the Qualitative Analysis and Quantification of Hispidulin in Hydroethanol Extract of S. barbata D. Don

Background: Hispidulin, a flavonoid renowned for its diverse pharmacological activities, is abundant in S. barbata D. Don. Given its prevalence, it is essential to establish a dependable analytical method for quantifying hispidulin in this plant. Methods: This study introduces a sensitive and reliable high-performance thin-layer chromatographic (HPTLC) method for both qualitative and quantitative analysis of hispidulin in S. barbata D. Don. Pre-coated TLC aluminum plates were utilized, and after testing various mobile phase combinations, the most effective separation was achieved with Toluene-ethyl acetate-formic acid (5:4:1, v/v/v). The method underwent extensive validation for linearity, specification, instrument precision, precision, limits of LOD and LOQ, accuracy, and robustness. Results: The developed method demonstrated excellent linearity over a specified concentration range, with a determination coefficient (r2) of 0.988. A well-defined spot at Rf 0.7, corresponding to hispidulin, was observed. The concentration of hispidulin in the hydroethanolic extract of S. barbata D. Don was quantified as 13.02 μg/mg of extract. Conclusion: In conclusion, the HPTLC method presented in this study offers a simple, accurate, and robust tool for quantitatively analyzing hispidulin in the hydroethanol extract of S. barbata D. Don. This method is suitable for routine quality control and standardization of herbal formulations containing S. barbata D. Don, thereby contributing to the advancement of research in natural product- based pharmaceuticals.

Development and validation of High-Performance Thin Layer Chromatographic methods for simultaneous determination of antibacterial pharmaceutical formulations containing Clindamycin phosphate.

The main objective of this research is to establish and authenticate the High-Performance Thin-Layer Chromatographic techniques for the simultaneous assessment of Clindamycin phosphate in combination with Tretinoin (formulation-1) and Clindamycin phosphate with Adapalene (formulation-2), both of which are antimicrobial combinations. In the developed HPTLC methods, for combination-1, the optimized mobile phase was a mixture of n-Hexane: Ethyl acetate: ACN: Methanol (5:2:2:1 by volume) with observed Rf values of 0.413 and 0.787 for Clindamycin phosphate and Tretinoin, respectively. Similarly, for combination-2, the mobile phase comprised n-Hexane: Diethyl ether: Dichloromethane: Acetic acid (4:4:2:0.1 by volume) with recorded Rf values of 0.407 and 0.559 for Clindamycin phosphate and Adapalene, respectively. The developed methods are successfully validated as per the regulatory guidelines. This approach proves to be significantly time-saving and costeffective, rendering it suitable for routine analyses of the formulations containing selected antibacterial active pharmaceutical ingredients and commercial formulations.

HPTLC Densitometric Analysis for Quantification of Sennocide-B and Rhein in Leaf Extracts of Senna alexandrina Mill. and Their Commercial Formulations.

The leaf extract of Senna alexandrina Mill., a plant from the Fabaceae family, contains bioactive anthraquinones sennoside-B and rhein, traditionally used in Indian medicine for various health issues. However, standardizing the quality of S. alexandrina and its commercial products is challenging. To address this, we established two high-performance thin-layer chromatography (HPTLC) methods for accurate analysis of these biomarkers in S. alexandrina leaf extract and its commercial formulations. For sennoside-B, a mobile phase combination of solvent for example, butanol, water, and glacial acetic acid (6:3.5:0.5, v/v/v) was used, with detection at 254 nm, producing spots at an Rf value of 0.37 ± 0.006. The method showed linearity between 100 to 2000 ng/spot (R² = 0.9983 ± 0.0017), with a limit of detection (LoD) of 22.84 ± 0.554 ng/band and a limit of quantitation (LoQ) of 69.22 ± 0.859 ng/band. For rhein, a mobile phase combination of solvent toluene, ethyl acetate, and glacial acetic acid in the ratio (7:2.5:0.5, v/v/v) was utilized and was also detected at 254 nm. The obtained spots had Rf value of 0.67 ± 0.004. The linearity was confirmed within the same range (R² = 0.9985 ± 0.0005), with an LoD of 15.49 ± 0.645 ng/spot and an LoQ of 46.94 ± 1.172 ng/spot. Herein, the methods have followed the guidelines of ICH (Q2) R1. These developed HPTLC methods are selective, simple, sensitive, accurate, and economical, making them suitable for everyday assessment of these bioactive markers in S. alexandrina and its commercial products. This study supports the quality control of S. alexandrina, ensuring product consistency, efficacy, and safety.

Stability-indicating high-performance thin-layer chromatography method development and validation for dapagliflozin propanediol monohydrate, vildagliptin, and metformin hydrochloride in active pharmaceutical ingredient and its tablet dosage form

Stability-indicating high-performance thin-layer chromatography method development and validation for dapagliflozin propanediol monohydrate, vildagliptin, and metformin hydrochloride in active pharmaceutical ingredient and its tablet dosage form

An eco-friendly smartphone based HPTLC method versus conventional densitometric one for determination of Naltrexone and Bupropion

The rapid uprising technologies of smartphone applications and software introduced a new era for analytical detection techniques. It has transformed bench-top laboratory methods into simpler ones depending on cost-effective, portable, and widely accessible devices. In this work, two high performance thin layer chromatographic (HPTLC) methods were developed based on smartphone’s camera detection and either ImageJ desktop software or Color-Picker smartphone’s application as alternative techniques to conventional densitometric detection. A mixture of Naltrexone hydrochloride (NAL) and Bupropion hydrochloride (BUP) was chromatographed on HPTLC- plates using ethyl acetate, methanol, acetone, and glacial acetic acid (3:6:1:0.5, by volume) as a developing system. The developed plates were scanned at 203 nm for the densitometric analysis, then visualized by modified Dragendorff’s reagent and shot by a smartphone’s camera. The captured images were uploaded to either ImageJ software or Color-Picker application to detect the separated spots. The results derived from the three detection methods were compared over the concentration range of 0.4–24 & 0.6–18 µg/band for the densitometric method, 0.4–24 & 2–24 µg/band for ImageJ built method and 0.8–20 & 5–20 µg/band for Color Picker built method for NAL and BUP, respectively. The methods were found to be appropriate for assaying both active drug substances in pure forms and combined in marketed pharmaceutical formulations. The excellent sustainability of densitometric and ImageJ-based methods enabled also the assessment of their dosage form content uniformity. The greenness and sustainability of the methods were assessed by three metric tools, namely Green Analytical Procedure Index (GAPI), Analytical GREEnness Metric Approach (AGREE), and White Analytical Chemistry (WAC). The assessments results confirmed the sustainability and superiority of the proposed methods in terms of sample treatment, waste mount, energy consumption, cost, and number of analyzed samples per an hour.

Development and validation of a method for the simultaneous estimation of terpinene-4-ol and 1,8-cineole in Melaleuca alternifolia (Maiden & Betche) Cheel and Rosmarinus officinalis L. oil using HPTLC coupled with a QbD approach

This paper comprehends a Quality by design (QbD) approach for development of a simple and precise HPTLC (High performance thin layer chromatography) method for simultaneous estimation of the phytoconstituents terpinen-4-ol and 1,8-cineole found in Melaleuca alternifolia (tea tree oil) and Rosmarinus officinalis (rosemary oil) respectively. The method was optimized by Design expert software using response surface methodology. The chromatographic condition optimized by using software was, mobile phase as hexane: ethyl acetate: glacial acetic acid in the ratio of 8.5:1.5:0.47 % v/v, stationary phase as TLC plates precoated with silica gel 60 F254 at ambient temperature conditions and saturation time 20 min at 366 nm. Optimized conditions were verified and validated as per ICH Q2 (R1) (International council for harmonization of technical requirements of pharmaceuticals for human use) guidelines. The method developed was validated for the parameters such as specificity, linearity, DL, QL, accuracy and precision. This method was then applied for simultaneous estimation of terpinen-4-ol and 1,8-cineole in Melaleuca alternifolia (tea Tree oil) and Rosmarinus officinalis (rosemary oil) respectively.

Development and validation of stability indicating HPTLC method for estimation of Daclatasvir Dihydrochloride in pharmaceutical dosage form

A high-performance thin-layer chromatographic method was developed and validated for estimation of Daclatasvir Dihydrochloride in pharmaceutical dosage form. The proposed method was applied successfully to the pharmaceutical analysis of the recently approved dosage form of Daclatasvir Dihydrochloride which is available in market as a brand name of ‘NALDAC 60’ tablets. The drugs were satisfactorily show peak with RF 0.38 for Daclatasvir Dihydrochloride. Method was validated according to the ICH guidelines. The calibration plot was linear between 50-300 ng per band for Daclatasvir Dihydrochloride. The LOD and LOQ for Daclatasvir Dihydrochloride were found to be 0.171 μg per band and 0.521 μg per band, respectively. Accuracy and precision of the proposed method was evaluated by recovery studies (% recovery for Daclatasvir Dihydrochloride was 99.88%) and intra-day and inter-day precision studies (standard deviation value for precision studies was found to be 0.453). In stability testing, Daclatasvir Dihydrochloride was found susceptible to acid hydrolysis and alkaline degradation. Because the method could effectively separate the drugs from their degradation products, it can be used as a stability indicating method. The proposed validated stability indicating assay for the sensitive determination of the mentioned drugs is suitable for Quality control laboratories as a simple fast economic method. Degradation product of Daclatasvir Dihydrochloride in alkaline condition was carried out.

Anatomic and Phytochemical Investigation of Herbal Tea Bags Sold as Lemon Balm (Melissa officinalis L.) in the Market

Melissa officinalis L., an ethnobotanically valuable plant, has been used for the treatment of several diseases since ancient times. However, different plants with the same name are sold instead of lemon balm in markets that sell herbal products in Turkey. For this purpose, 15 different brands of tea bags in crushed form, sold as lemon balm (Melissa officinalis L.) in markets, were analyzed. The total phenolic, flavonoid and antioxidant capacities of the hydroalcoholic extracts of these tea samples along with the stomatal structure were investigated. Additionally, chemical compositions and rosmarinic acid contents were determined by the high-performance thin layer chromatography method. Among the samples examined, S1, S2, S9 and S12 tea samples were found to meet the eligibility criteria. The leaves of these samples had diacytic stomata and the rosmarinic acid ratio in their phytochemical composition was over 2%. Furthermore, caffeic acid was detected in these samples.

Rapid mitragynine quantification and fingerprinting of products from Mitragyna speciosa Korth. leaf (Kratom) using high-performance thin-layer chromatography.

Kratom (leaves from Mitragyna speciosa Korth.; Rubiaceae) is a herbal medicine known for its analgesic properties and psychoactive effects. Kratom in Thailand is currently legal; however, it is prohibited in some countries and considered a narcotic plant. Our aim was to establish a reliable, simple, and rapid method for quantifying mitragynine in Kratom leaves and related products through a combination of high-performance thin-layer chromatography (HPTLC) and densitometry. A densitometric HPTLC method was developed and validated in terms of specificity, linearity, limit of detection (LOD), limit of quantification (LOQ), accuracy, precision, and robustness. The fingerprints of kratom leaves, Mitragyna spp., and related products were constructed. For HPTLC, samples were applied to silica gel 60 F254 plates, and the mobile phase comprised n-hexane, ethyl acetate, and triethylamine (1:1:0.15, v/v/v). Densitometric detection was carried out under ultraviolet light at a wavelength of 226 nm. The validated method exhibited a range of 14.31-143.10 μg/mL, yielding a correlation coefficient of 0.9993. Spiked recovery rates were within a range of 98.3%-100.9%, and the LOD and LOQ were 3.80 and 11.53 μg/mL, respectively. Kratom samples were analyzed with the developed method, and the correlation coefficient was 0.9641, compared to the high-performance liquid chromatography-diode-array detection (HPLC-DAD) method. The HPTLC fingerprints displayed a distinctive pattern, facilitating discrimination among different plant parts and Mitragyna spp. The established method offers the advantages of simplicity, ease of use, and speed of analysis, serving as a practical alternative for mitragynine quantification in kratom leaf and its related products.

A validated HPTLC method for quantification of a new combination containing Lidocaine HCl and Diltiazem HCl

Regulators approve newer medications and medication combinations for the treatment of various disorders. For the treatment of anal fissures, a novel combination of currently available medications, such as lidocaine HCl and diltiazem HCl, has been approved. The current work represents high-performance thin layer chromatographic method to estimate the amounts of lidocaine hydrochloride and diltiazem hydrochloride in a combined gel dosage form called Crema-L gel (2% W/W of lidocaine and 2% W/W of diltiazem hydrochloride). This method has been validated. The mobile phase in this method consisted of a mixture of toluene, methanol, ethyl acetate (7:2: 1% v/v/v) and two drops of ammonia. The stationary phase was pre-coated aluminium sheet with silica gel G60 F254 (10 × 10 cm, 0.2 mm layer thickness). The drugs’ resolved peaks were found at the Rf values of 0.59 for Lidocaine HCl and 0.48 for Diltiazem HCl. The overlay UV spectra of the two medications were used to determine the analytical wavelength and at 220 nm, both drugs showed appreciable absorbance. The concentration range of 400–1200 ng/band was determined to be the linear approach for both lidocaine HCl and diltiazem HCl, as indicated by their respective correlation coefficients (r2) of 0.9987 and 0.9980. The technique for simultaneously estimating lidocaine hydrochloride and diltiazem hydrochloride in bulk and pharmaceutical dosage form was shown to be robust, accurate, precise, and repeatable.Graphical

Development of HPTLC method for simultaneous determination of quercetin and kaempferol in leaf extract of Hibiscus mutabilis

The aim of this study was to develop and validate a densitometric High-Performance Thin-Layer Chromatography (HPTLC) method for the simultaneous quantification of quercetin (Q) and kaempferol (K) in Hibiscus mutabilis leaf extracts. The analyses were performed on silica gel 60 F254 plates using a mobile phase composed of toluene, formic acid, and ethyl acetate (6:0.4:4, v/v/v). Detection was carried out at a wavelength of 272 nm using a deuterium and tungsten light source. The method exhibited excellent linearity over the concentration range of 100–600 ng/spot for quercetin and 500–3000 ng/spot for kaempferol, with determination coefficients (r2) of 0.9989 and 0.9973, respectively. The method showed no interferences from the plant matrix. The relative standard deviation (RSD) values for intra- and inter-day precision were less than 2% for both flavonoids. Recovery rates ranged from 97.69% to 99.20% for quercetin and from 89.91% to 95.87% for kaempferol. The limits of detection (LOD) were 190.23 ng/spot for quercetin and 187.23 ng/spot for kaempferol, while the limits of quantification (LOQ) were 570.10 ng/spot for quercetin and 566.12 ng/spot for kaempferol. This validated HPTLC method is reliable, precise, and accurate, making it suitable for the quality control of Hibiscus mutabilis leaf extracts. The study’s findings can be broadly applied to the quality control of herbal products, pharmacological research, and the development of nutraceuticals. The method’s ability to provide rapid and accurate quantification makes it an invaluable tool for researchers across various disciplines.

Comprehensive phytochemical profiling of Phyllanthus emblica L. flowers on UHPLC/MS quadrupole time of flight, HPTLC, HPLC, and NMR analytical platforms reveals functional metabolites with potent anti-inflammatory effects in human (THP-1) macrophages.

Phyllanthus emblica L., renowned for its pharmacological benefits found in its fruits and leaves, has received considerable attention. However, there is a notable lack of research on its flowers, specifically on metabolite profiling and pharmacological activity. The present study aims to delineate the phytochemical constituents of hydromethanolic extract of P.emblica flowers by ultra-high-performance liquid chromatography coupled with quadrupole time of flight mass spectrometry (UHPLC-QToF-MS), high-performance thin layer chromatography (HPTLC), high-performance liquid chromatography (HPLC), infrared and nuclear magnetic resonance spectroscopic methods and subsequent evaluation of its anti-inflammatory potential. The identification and characterization of phytochemicals in P.emblica flowers was performed by UHPLC/MS-QToF in both positive and negative ionization modes. Additionally, marker compounds present in flower extract were analyzed using HPTLC, HPLC, FT-IR, and NMR methods. The anti-inflammatory potential was evaluated in lipopolysaccharide-stimulated THP-1 macrophages by evaluating inflammatory biomarkers. UHPLC/MS-QToF analysis facilitated the identification of 51 compounds from P.emblica flowers including gallic acid derivatives, flavonoid glycosides, and tannins based on their fragmentation patterns and previous literature reports. Notably, the study also identified spermidine compounds for the first time in this species. Optimization of HPTLC and HPLC methods marked the presence of corilagin as major compound followed by FT-IR and NMR spectral methods. Moreover, treatment with hydromethanolic extract of P.emblica flowers resulted in decreased levels of proinflammatory cytokines, TNF-α, IL-1β, and IL-6, alongside modulation of nuclear factor-κB activity in lipopolysaccharide-induced THP-1 macrophages. Chromatographic techniques in conjunction with spectral methods found robust prevalence in the identification of signature phytometabolites present in P. emblica flowers, which sets the basis for its anti-inflammatory potentials. The studies established a foundation for further exploration of potential applications of P. emblica flowers across various domains.

Development and Validation of a High-Performance Thin-Layer Chromatography Method for Simultaneous Qualitative and Quantitative Analysis of Scutellarin and Scutellarein in S. barbata D. Don

Development and Validation of a High-Performance Thin-Layer Chromatography Method for Simultaneous Qualitative and Quantitative Analysis of Scutellarin and Scutellarein in S. barbata D. Don

High Performance Thin Layer Chromatography (HPTLC) Analysis of Anti-Asthmatic Combination Therapy in Pharmaceutical Formulation: Assessment of the Method's Greenness and Blueness.

A cost-effective, selective, sensitive, and operational TLC-densitometric approach has been adapted for the concurrent assay of Hydroxyzine Hydrochloride (HYX), Ephedrine Hydrochloride (EPH), and Theophylline (THP) in their pure powder and pharmaceutical forms. In the innovative TLC-densitometric approach, HYX, EPH, and THP were efficaciously separated and quantified on a 60F254 silica gel stationary phase with chloroform-ammonium acetate buffer (9.5:0.5, v/v) adjusted to pH 6.5 using ammonia solution as a mobile liquid system and UV detection at 220 nm. The novel TLC method validation has been performed in line with the international conference for harmonization (ICH) standards and has been effectively used for the estimation of the researched medicines in their pharmaceutical formulations without intervention from excipients. Additionally, parameters affecting the chromatographic analysis have been investigated. The new TLC approach's functionality and greenness were appraised using three modern and automated tools, namely the Blue Applicability Grade Index (BAGI), the Analytical Greenness metric (AGREE), and the Green Analytical Procedure Index (GAPI) tools. In short, the greenness characteristics were not achieved as a result of using mandatory, non-ecofriendly solvents such as ammonia and chloroform. On the contrary, the applicability and usefulness of the novel TLC approach were attained via concurrent estimation for the three drugs using simple and straightforward procedures. Moreover, the novel TLC method outperforms previously published HPLC ones in terms of the short run time per sample and moderate pH value for the liquid system. According to the conclusions of comparisons with previously recorded TLC methods, our novel HPTLC method has the highest AGREE score, so it is the greenest HPTLC strategy. Moreover, its functionality and applicability are very appropriate because of the simultaneous assessment of three drugs in one TLC run. Furthermore, no tedious and complicated extraction and evaporation processes are prerequisites.

Extraction, Isolation and Characterization of Bioactive Compounds from Euphorbia neriifolia (L.) Leaf and Evaluation of their Antioxidant Activity

Background: Amidst the identification of numerous secondary chemicals from Euphorbia neriifolia, there is a desperate need for the development of primary metabolite separation techniques. Objectives: In order to do that, bioactive chemicals from Euphorbia neriifolia (L.) leaf were extracted, isolated, and characterized. Subsequently, their antioxidant activity was evaluated. Methods: In this study, the determination of linoleic acid (LA) in petroleum ether extract (PEE) of Euphorbia neriifolia leaf (ENL) was carried out the first time by using thin-layer chromatography (TLC) and high-performance thin-layer chromatography (HPTLC) methods. Results: The chromatographic analysis of the PEE of ENL shows better spots and well-separated peak of LA with 0.49 retention factor (Rf) value and 22.54 ng LA content. The linearity of the calibration curve ranges from 5-25 ng/spot with a high correlation coefficient. The proposed method was characterized by better accuracy close to 99.5%, well robustness, and good precision range from 0.183% (intra-day) to 0.242% (inter-day). The percentage (%) RSD, which determined the stability of standard LA, did not exceed 2% after time period of 12, 24, 36, 48, and 72 h. The GC-MS analysis revealed the presence of different types of low or high-molecular-weight phytocompounds of varying quantities from the fractions of ENL. The FT-IR spectrum of ICs showed various peaks that confirmed the presence of C=C bending, C-H stretching, O-H stretching, CH2 stretching, and a carboxyl group. The 1H-NMR spectrum of the ICs from ENL confirmed the presence of octadecanoic in IC1, L-(+)-ascorbic acid dihexadecanoate in IC2, hexadecanoic acid in IC3, linoleic acid in IC4, and oleic acid in IC5, respectively. IC-4 showed greater antioxidant activity in comparison to other compounds with an IC50 value of 3.9 ± 0.01 μg mL-1. Conclusion: Thus, the present study identified five different phytocompounds that may be utilized as an effective option for the cure of different diseases.