High-performance Thin-layer Chromatographic Method Research Articles

A novel stability indicating HPTLC method for comparative quantitative estimation of quercetin in herbal preparations

This research introduces a novel stability-indicating HPTLC (High-Performance Thin-Layer Chromatography) method for the comparative quantitative estimation of Quercetin in herbal preparations. Through extensive stability studies, the method is validated, providing a robust framework for evaluating Quercetin's stability in diverse herbal formulations. The developed method ensures accurate and reliable quantification of Quercetin, facilitating comparative analysis across various herbal preparations. Additionally, results reveal that Quercetin degraded under most tested conditions, such as acidic, alkaline, oxidative and neutral conditions underscoring the necessity for intervention by experts to eliminate such challenges and ensure product efficacy and quality in the herbal industry.

Paris polyphylla Sm. characterized extract infused ointment accelerates diabetic wound healing in In-vivo model

Ethnopharmacological relevanceThe dried rhizome of Paris polyphylla Sm. is extensively used by traditional healers in India, China, and Vietnam to treat skin inflammation, cut wounds, uterine and traumatic bleeding, and cancer. Aim of the studyThe traditional use of P. polyphylla rhizomes for treating wounds and bleeding has been reported previously. However, the potential of P. polyphylla in the treatment of diabetic wounds has not yet been explored. Our present study focused on the investigation of the wound-healing activity of P. polyphylla infused ointment in streptozotocin (STZ)-induced diabetic rats to validate the traditional claim. Materials and methodsHydroalcoholic extract of the dried rhizomes of P. polyphylla were quantified by validated and optimized HPTLC (High-performance thin layer chromatography) method for Paris saponin VII, Dioscin and Polyphyllin V. The extract was used to prepare P. polyphylla ointments (5 and 10%). P. polyphylla ointment was subjected to physiochemical analysis and skin irritation test. Thirty STZ-induced diabetic adult male Wistar albino rats were divided into five groups (n = 6) and a circular excision wound was created. P. polyphylla ointment, ointment base (OB), and standard (STD) (Povidone Iodine 10%) were administered topically. The wound area of all groups were recorded every six days and compared with that of control. The epithelization period of each group was recorded. On day 18, the histopathological study of skin tissues of all groups was performed using hematoxylin and eosin (H&E) and Mallory's trichrome (MT). ResultsMarker analysis and quantification of phytomolecules in hydroalcoholic extract ofP. Polyphylla were found to be of paris saponin VII (3.28 ± 0.08% w/w), dioscin (1.94 ± 0.12% w/w), and polyphyllin V (1.87 ± 0.84% w/w). A physiochemical study of P. polyphylla ointment showed that the prepared ointment was within an acceptable range and was not irritable to the skin. Daily topical administration of 10% P. polyphylla ointment (PP10) for 18 days completely healed the STZ-induced diabetic wounds. On day 18, the 5% P. polyphylla ointment (PP5) showed 99.1 ± 2.9% wound closure, while that of the standard and control was 78.4 ± 7.3% and 18.5 ± 5.9%, respectively. The epithelialization period of PP10 was 18 days, whereas that of the control was 28 days. Histopathological analysis of the progression of PP10 and PP5 wounds showed a decrease in inflammatory cells, regenerated epithelial layer, keratosis layer, hair follicles, fibroblasts, and collagen. Upon collagen intensity quantification of MT stained sections, an increase in collagen density of PP10 and PP5 treated groups was observed, showing accelerated wound healing potential of P. polyphylla extract in diabetic wounds compared to the standard ointment. ConclusionThis study suggested the potential of P. polyphylla rhizomes derived formulation to treat diabetic wounds, although the plant is traditionally used to treat normal wounds. The results indicate the validation of traditional claim, which has been explored commercially in industrial aspect.

Quantification of isoniazid in tablets for tuberculosis treatment by thin layer chromatography with smartphone image capture and ImageJ analysis

Isoniazid (INH) oral dosage tablets are an antibacterial medication commonly used to treat tuberculosis (TB). While this medication can be highly effective in managing and curing TB, manufacturing of poor-quality INH products can lead to the proliferation of drug resistant TB strains – particularly in low- and middle-income countries (LMICs). It is therefore imperative that reliable methods of quality screening of INH tablets are available for areas that have limited access to traditional testing resources. In this study, a thin layer chromatography (TLC) procedure for INH identification is validated in conjunction with smartphone image capture and open-source image analysis software to provide an alternative quantitative quality screening method for INH tablets, primarily intended for applications in LMICs. The method assessed two individual tablet formulations: 100 mg and 300 mg INH, each from different manufacturers. The modified TLC procedure used for the study was based on a validated high-performance thin layer chromatography method for the determination of rifampicin, INH, and pyrazinamide in a fixed dosage tablet. Quantitation was performed by visualizing and photographing TLC plates using a UV lamp, 3D-printed light box, and Apple iPhone SE 2nd Generation (2020) and Google Pixel 4a (5G) smartphones, and images were then analyzed using ImageJ Fiji software. The processed images’ pixel data were used to create a calibration curve from the INH standard spots to determine the concentration of an INH sample spot. Linearity, range, accuracy, repeatability and intermediate precision, specificity, and robustness were evaluated. The method was linear in the range of 0.150–0.450 mg mL−1, with coefficients of determination ≥0.98. The overall accuracy of the method was 99.2 % with an RSD of 2.4 % for image sets captured using the Apple iPhone, and 99.7 % with an RSD of 3.3 % for image sets captured using the Google Pixel. The pooled standard deviations for repeatability and intermediate precision were 2.27 % and 3.25 %, respectively, for the iPhone and 2.62 % and 3.74 %, respectively, for the Pixel. The method demonstrated sufficient specificity and robustness. The results of this method validation suggest that this procedure may provide a feasible alternative to traditional testing as a low-cost means of quality screening in LMICs.

Enhancing simultaneous determination of some angiotensin II receptor antagonists and amlodipine in plasma using HPTLC with fluorescence densitometry: Independent fluorescence detection of the co-administrative drugs in the mixture across various pH conditions

A novel and highly sensitive high-performance thin-layer chromatographic (HPTLC) method was developed and validated to quantify a combination of five pharmaceutical mixtures spiked to human plasma. The compounds comprised Amlodipine (AML) along with five angiotensin II receptor antagonist drugs (AIIRAs), namely Olmesartan (OLM), Telmisartan (TLM), Candesartan (CAN), Losartan (LOS), and Irbesartan (IRB). HPTLC was performed on silica gel 60 F254 plates using a mobile phase of Toluene: ethyl acetate: methanol: acetone: acetic acid (6:1.5:1:0.5:1, v/v/v/v/v). In a pioneering move, a reflectance/fluorescence detection mode was employed to identify two concurrently administered drugs at different pH levels for the first time. This method utilized the same chromatographic system, incorporating a specific measurement for AML at a neutral medium to achieve its maximum fluorescence at a 360nm excitation wavelength, and measuring emission using a 540 nm optical filter. The process involved obtaining a very low fluorescence response from AIIRA. Subsequently, to enhance AIIRA’s fluorescence, the plate was sprayed with perchloric acid to transition to a strong acidic medium, ultimately attaining the maximum fluorescence of AIIRA using various excitation wavelengths and a 400 nm emission filter. Through this strategic process, we could optimize the fluorescence signals of both drugs, thereby elevating the sensitivity of detection for this drug combination. AML demonstrated a linear range of 18–300 ng/band, while AIIRAs drugs exhibited a linear range of 6–150 ng/band. The method satisfied the International Conference on Harmonization (ICH) criteria for recovery, precision, repeatability, and robustness, showcasing exceptional sensitivity. The approach was successfully applied to quantify AML and AIIRAs drugs in both bulk drug and plasma samples, achieving high recovery percentages and minimal standard deviations.

Development and validation of a high-performance thin-layer chromatography method for detection of sibutramine in dietary supplements.

In the period between 1997 and 2010, sibutramine-containing drugs were widely prescribed for obesity and over-weight management. Due to safety concerns, in 2010 all medicines containing sibutramine were urgently withdrawn from the USA and European pharmaceutical market. Although sibutramine is no longer available in pharmaceutical products, there have been numerous reports of mislabeled weight-loss dietary supplements containing sibutramine.

Quantification of the main constituents of “sitopaladi churna—Ayurvedic multi-herbal preparation” using a validated high-performance thin-layer chromatography method

Quantification of the main constituents of “sitopaladi churna—Ayurvedic multi-herbal preparation” using a validated high-performance thin-layer chromatography method

Stability indicating high performance thin layer chromatography method development and validation for quantitative determination of tetracycline hydrochloride in tetracycline hydrochloride active pharmaceutical ingredient (API) and its dosage forms

Simple, quick, cost-effective, and environmentally friendly analytical methods for quality assurance and control roles for different medicines, including Tetrcyclines, are most significantly needed. Also, different thin layer chromatography (TLC)-based methods for tetracycline identification exist, but high performance thin layer chromatography methods based on modern state- of- the art equipment are still nonexistent. Thus, in this study, analytical method development and verification were done by high performance thin layer chromatography (HPTLC) (using an automated equipment model) using glass plates coated with silica gel 60 F254 after treating with 10% Na2EDTA. Validation was carried out according to International Council for Harmonization (ICH) guidelines. A mobile phase formed from ethyl acetate, acetonitrile, methanol, and 1% aqueous ammonia in the composition of 4.4:19.6:10:6 volume to volume ratio (V/V) was used. Rf value, percentage recoveries, linearity ranges, limit of detection (LOD), and limit of quantitation (LOQ) for the developed HPTLC method were 0.28, 100.83–106.25%, 160–560 ng/band (r2 values of 0.9999), 31.9 ng/band, and 96.7 ng/band, respectively. The results of the sample assays conducted using the new method and the United States Pharmacopoeia (USP) high performance liquid chromatography (HPLC) method were 91.59% to 108.3% and 90.83% to 102.85%, respectively. The F test for the aforementioned methods was 2.01, which is smaller than the tabulated F value of 5.05 with 5 degrees of freedom at a 95% confidence range, proving that the newly developed HPTLC and HPLC pharmacopoeial methods can be used interchangeably.The newly developed HPTLC method is easy, economical, specific, accurate, and roboust, thus it can be employed in a range of settings that require quality control and assurance activities of tetracycline hydrochloride (TC-HCl) in bulk and ointment dosage forms.

Simultaneous determination of lesinurad and febuxostat in commercial fixed-dose combinations using a greener normal-phase HPTLC method

Abstract So far, no documented method for simultaneously analyzing lesinurad (LND) and febuxostat (FBX) has been reported for either traditional high-performance thin-layer chromatography (HPTLC) or a green HPTLC technique. In order to determine LND and FBX simultaneously in commercially available fixed-dose combo tablets, this study devised a normal-phase HPTLC method that is fast, sensitive, and green. The green eluents for the simultaneous analysis of LND and FBX were a mixture of ethyl acetate:ethanol:water at 70:20:10 (v/v/v) ratio. The new approach’s greenness was predicted utilizing four distinct greenness tools: the National Environmental Method Index, Analytical Eco-Scale, ChlorTox, and Analytical GREENness approaches, and the results revealed a significantly greener profile. The current method operated on a linear scale between 30 and 1,000 ng·band−1. It was confirmed that the current approach is sensitive, accurate, precise, robust, and green. The LND and FBX contents of commercially available tablet products A and B were found to be within the range of 100 ± 2%, indicating that the existing methodology for simultaneously determining LND and FBX in pharmaceutical combination products is applicable. The results of the current methodology indicated that LND and FBX could be consistently measured in pharmaceutical combination products simultaneously using the current approach.

Robust high-performance thin-layer chromatography (HPTLC) method for stability assessment and simultaneous quantification of epigallocatechin-3-gallate and rosmarinic acid in lipid-based nanoparticles and biological matrices.

Skin cancer poses a significant health risk globally, necessitating effective and safe therapeutic interventions. Epigallocatechin-3-gallate (EGCG) from green tea and rosmarinic acid (RA) from herbs like rosemary offer promising anticancer properties. Combining these compounds may enhance their effectiveness, prompting the need for a reliable analytical method to quantify them. Herein, we present the development and validation of a high-performance thin-layer chromatography (HPTLC) method for concurrent quantification of EGCG and RA in lipid-based nanoparticles and biological samples. The method underwent optimisation through design of experiments (DoE), resulting in the establishment of robust chromatographic conditions. The separation process utilised aluminium HPTLC plates coated with silica gel 60 F254 as the stationary phase, with the mobile phase comprising ethyl acetate, toluene, formic acid, and methanol in a ratio of 4:4:1:1 v/v. The retention factor (Rf) values obtained were 0.38 for EGCG and 0.61 for RA. The method demonstrated linearity over a range of 100-500 ng/band for both compounds with excellent correlation coefficients. Limits of detection and quantification were determined, indicating high sensitivity. Precision evaluations revealed relative standard deviation below 2%, ensuring method reproducibility. Recovery assays in lipid-based nanoparticles, plasma, and urine samples demonstrated excellent recoveries (96.2%-102.1%). Forced degradation studies revealed minimal degradation under various stress conditions, with photolytic degradation showing the least impact. The developed HPTLC method offers a rapid, sensitive, and reliable approach for quantifying EGCG and RA, laying the groundwork for their further investigation as anticancer agents alone and in combination therapies.

Quantitative assessment of paclitaxel in Taxus wallichiana zucc. bark using HPTLC densitometry: Insights for germplasm selection and toxicological studies

Taxus wallichiana Zucc. [Synonym: Taxus baccata subsp. Wallichiana (Zucc.) Pilg.] or Himalayan Yew belonging to the evergreen gymnosperm family Taxaceae, grows in temperate regions and has been used in traditional medicines to treat cancer, bronchial disorders, colds, cough, etc. The plant is known to contain paclitaxel (PTX), a key bioactive component with anticancer properties. High-performance thin layer chromatography (HPTLC) was used in the present study to estimate paclitaxel from the bark of T. wallichiana obtained from 15 different geographical locations in the eastern and western Himalayas of India. HPTLC method was determined to be a reliable and efficient technique for the quantification of paclitaxel, offering rapid outcomes.The bark materials of T. wallichiana chemotype obtained from Sikkim (Samanden) contained the highest concentration of paclitaxel, followed by West Bengal (Sandakphu), Arunachal Pradesh (Dibang Valley), and respectively. The present study also examined the cytotoxicity and genotoxicity of the aqueous bark extract of the TW1 chemotype at various doses on the meristematic cells derived from the roots of Allium cepa L.. The bark extracts demonstrate considerable cytotoxic and mitotic properties. This study could be useful to the pharmaceutical industry for selecting elite (high paclitaxel-producing) chemotypes of T. wallichiana. The toxicological evaluation of the T. wallichiana chemotypes (using Allium cepa test) could also help to determine the genotoxic properties of the bark extract.

High-performance thin-layer chromatography method development and validation for quantification of rutin in different parts of Capparis zeylanica Linn. plant

High-performance thin-layer chromatography method development and validation for quantification of rutin in different parts of Capparis zeylanica Linn. plant

High performance thin layer chromatography fingerprint on cyano-bonded stationary phase of selected Stachys and Betonica species and their antioxidant activity with chemometric calculations

The methanolic extracts of selected Stachys and Betonica species were analyzed by high performance thin layer chromatography (HPTLC) method using cyano bonded stationary phase and mobile phase consisted of methanol and water 45:55 (v/v) with Natural product reagent derivatization. The HPTLC method was validated (linearity, precision, accuracy, robustness, LOD, LOQ) and the validation results confirm the usefulness of the method used. The obtained images of fingerprint chromatograms presented as numerical data (using the TLC analyzer program) were next processed using Excel and SpecAlign softwares. The phytochemical similarity between analyzed plant extracts was evaluated using the similarity index (Pearson correlation coefficient) and the results were presented as hierarchical cluster analysis dendrograms and as graph for principal component analysis (PCA). The total phenolic content (TPC) by Folin-Ciocalteu method and the antioxidant activity with Ferric Ion Reducing Antioxidant Potential (FRAP) technique were determined for studied extracts using gallic acid as standards in both methods.

Characterization of Secondary Metabolites of Leaf Buds from Some Species and Hybrids of Populus by Gas Chromatography Coupled with Mass Detection and Two-Dimensional High-Performance Thin-Layer Chromatography Methods with Assessment of Their Antioxidant Activity.

Poplars provide medicinal raw plant materials used in pharmacy. Leaf buds are one of the herbal medicinal products collected from poplars, having anti-inflammatory and antiseptic properties, but there are no quality standards for their production and there is a need to determine their botanical sources. Therefore, the chemical compositions of the leaf buds from four species and varieties of poplars, Populus balsamifera, P. × berolinensis, P. × canadensis 'Marilandica', and P. wilsonii were investigated and compared using gas chromatography coupled with mass detection (GC-MS) and two-dimensional high-performance thin-layer chromatography (2D-HPTLC) in order to search for taxa characterized by a high content of biologically active compounds and with a diverse chemical composition that determines their therapeutic effects. The presence of 163 compounds belonging to the groups of flavonoids, phenolic acids derivatives, glycerides, and sesquiterpenes was revealed. Moreover, the conditions for the separation and identification of biologically active compounds occurring in analyzed leaf buds using 2D-HPTLC were optimized and used for metabolomic profiling of the studied poplars, enabling their fast and simple botanical identification. The total phenolic (TPC) and flavonoid (TFC) contents of examined extracts were determined and their antioxidant capacities were estimated by spectrophotometric DPPH, ABTS, and FRAP assays. Based on the analysis of phytochemicals and antioxidant activity, P. × berolinensis buds were selected as the raw plant material for medicinal purposes with the highest content of active compounds and the strongest antioxidant activity.

Mixture Design-driven Statistical Optimization and Method Validation in HPTLC: Targeting Rutin, Quercetin, and Gallic Acid

Rutin (RT), Quercetin (QT), and Gallic Acid (GA) are recognized for their potent antioxidant and anticancer properties, prevalent across numerous plant species. The precise quantification of RT, QT, and GA is pivotal for evaluating the therapeutic potential of plant-based substances. In response, a new, straightforward, cost-effective, and reliable method using High-Performance Thin-Layer Chromatography (HPTLC) has been developed and validated for the quantification of these compounds. The mobile phase optimization employed a mixture design approach, achieving chromatographic separation with a mobile phase mixture of toluene, ethyl acetate, menthol, and formic acid at specific ratios (3.56:3.70:0.94:1.80 v/v/v/v/v/v/v/v). Silica gel 60 F254 HPTLC plates were utilized for the analysis. The retention factors (Rf) observed for RT, GA, and QT were 0.21, 0.58, and 0.74, respectively. This method demonstrated a robust linear relationship for concentrations ranging from 400 to 2000 ng per band, with correlation coefficients (R2) of 0.9921 for RT, 0.9936 for QT, and 0.9912 for GA. The Limits of Detection (LOD) and quantification (LOQ) were established at 100.84, 102.38, and 84.54 ng per band for LOD, and 305.58, 310.25, and 256.18 ng per band for LOQ, respectively, for RT, QT, and GA. This validated HPTLC method developed through a Design of Experiment (DoE) approach was successfully employed for the quantification of GA, QT, and RT from the fruits of Adansonia digitata and leaves of Grewia asiatica ethanolic extracts.

Development and Validation of High-performance Thin layer Chromatography Method for Simultaneous Determination of Gallic Acid and Quercetin from Annona squamosa L. leaf Extract

The objective of this research was to develop a validated high performance thin layer chromatography (HPTLC) procedure for identification, estimation and standardization of two biomarker compound Quercetin and gallic acid from Annona squamosa L. Leaf hydro alcoholic Extract. The chromatographic quantification were conducted on normal HPTLC (20 cm x 10 cm glass backed silica gel 60 F 254) with Toluene: Ethyl acetate: Formic Acid (5.4:3.8:0.8 v/v/v) used as a mobile phase . The detection and evaluation were performed by densitometric scanning at 254nm. The system was found to give a well resolved, compact and intense peaks for quercetin (Rf 0.45±0.05) and gallic acid ( Rf 0.28±0.05) at 360nm and 280nm respectively from other constituents present in the extract of Annona squamosa L .The proposed analytical method for both the biomarker compound was found to be handy, simple, precise, linear (%RSD =0.673808 and 0.896642 for GA and Quercetin respectively), accurate, reliable and sensitive for analysis of both the biomarkers. The LOD/ LOQ (ng) for Gallic acid and Quercetin were recorded as 15/45 and 12/36, respectively in the linearity range of 200-1200ng per spot. The obtained result showed quantities of Gallic acid and Quercetin in leaf hydroalcoholic extract as 0.023% w/w and 0.048 w/w, respectively. The developed HPTLC was found to be suitable for the routine analysis of both of these biomarkers in the hydroalcoholic extract of Annona squamosa L. and can be further employed in the process quality control of herbal formulations containing the mentioned biomarkers.

Development and Validation of HPTLC and First Derivative Spectrophotometric Methods for Determination of Levetiracetam in Bulk and Tablet Dosage Form

A simple, sensitive high performance thin layer chromatographic (HPTLC) method and first order derivative spectrophotometric methods were developed for estimation of US FDA drug levetiracetam in bulk and its tablet dosage form. Chromatographic estimation was carried out on silica gel 60 F245 TLC plate as stationary phase and toluene: methanol: Ethyl acetate (4:4:2v/v/v) as mobile phase. Densitometric analysis was carried out at wavelength 218nm. The Rf values for Levetiracetam was found to be 0.47±0.03. Linearity was observed over range of 400-3200ng/spot for Levetiracetam (R2 0.998). The %recovery of Levetiracetam was found to be 99.25-100.7%. The LOD and LOQ were found to be 0.0917ng/ml and 0.278ng/ml respectively indicating sensitivity of the method. The first order derivative spectra were obtained at n=4, Δλ=3nm and determinations was made at 215 nm using methanol as solvent. Linearity was observed in the range of 80μg/ml - 280μg/ml with good regression values. Percent mean recovery of Levetiracetam was obtained as 101.5%. Both proposed methods were simple, fast, accurate, precise and reproducible and hence can be applied for routine quality control analysis of levetiracetam in bulk and its tablet dosage form.

Development and Validation of Chromatographic Methods for Simultaneous Estimation of Aspirin and Pantoprazole Sodium in Pure and Mixture Form.

This technique is employed for the simultaneous quantification of aspirin (ASP) and pantoprazole sodium in both pure powder form and formulations. The high performance liquid chromatography (HPLC) method uses a C-18 column (250mm × 4.6mm, 5μm) with a mobile phase consisting of 0.05M disodium hydrogen phosphate and methanol in a 40:60% v/v ratio. The flow rate is maintained at 1.0mL/min. On a layer of silica gel 60F254 with an aluminum backing, the high performance thin layer chromatography (HPTLC) separation was carried out with ethyl acetate and methanol (8: 1.5v/v) as the mobile phase. With a mean recovery of 100.54% and 99.55% for ASP and PNT, respectively, quantification was accomplished using the HPLC method with UV detection at 286nm over the concentration range of 0.1-0.6g/mL for PNT and 0.4-2.4g/mL for ASP. With a mean recovery of 99.44% and 99.01% for ASP and PNT, respectively, quantification was achieved using the HPTLC method with UV detection at 298nm over the concentration range of 400-2400ng/spot for ASP and 100-600ng/spot for PNT, respectively. The methods can be used for the simultaneous determination of ASP and PNT in pure powder form and formulations as they are simple, accurate and sensitive.

Green innovation in analytical chemistry: A sustainable densitometric HPTLC approach for the distinctive separation and quantification of structurally related abused drugs - tramadol, tapentadol, and venlafaxine - in seized pharmaceutical dosage forms

An innovative ecofriendly high-performance thin layer chromatographic (HPTLC) method with spectrophotometric detection for simultaneous determination of Tramadol (TMD), Tapentadol (TAP), and Venlafaxine (VEN) in seized dosage forms was presented. Our method was conducted to achieve separation following the optimal conditions: pre-coated silica gel plates using a green mobile phase (heptane: acetone: ammonia, 7:3:0.5 v/v), with absorbance scanning at 272 nm. The validation of the method was done following International Conference on Harmonization (ICH) guidelines, demonstrates linearity, accuracy, precision, selectivity, robustness, and system suitability. Separation was achieved with a detection limit of 0.34, 0.16, and 0.084 (ug/band) for TMD, TAP, and VEN, respectively, the method successfully analyzes seized samples. Trueness is confirmed through a high degree of similarity between HPTLC and gas chromatography results. The study's ecofriendly approach, simplicity, and selectivity position it as a promising method for efficient, on-site monitoring of seized samples.

Quantitative analysis of rutin in flavonoid-rich plant raw material by HPTLC

Introduction. Due to the increasing requirements for the quality of pharmaceutical products, the requirements for standardization of herbal medicines are also growing, which in turn necessitates the development of approaches to quality control of medicinal plant raw materials. The use of such an advanced method as high-performance thin-layer chromatography (HPTLC) will improve the quality of drug standardization, which is associated with the advantages of this method: minimized sample preparation, parallel determination of small volumes, elution time, high sensitivity, efficiency and speed. Objective. Quantitative determination of rutin by high-performance thin-layer chromatography, as well as comparison of the results of quantitative determination of the amount of flavonoids in terms of rutin by the spectrophotometric method. Material and methods. Medicinal plant raw materials – violet herb – were subjected to extraction using a developed method with 70 and 96% ethanol. Quantitative determination of rutin in extracts was carried out by HPTLC and spectrophotometry. Results. The developed method for the quantitative determination of rutin was validated in terms of linearity, specificity and accuracy. The difference in the extractive ability of the solvents used was shown. The percentage of rutin determined using the HPTLC method in an ethanol (96%) extract (3 µl) is 3.0826%, in an ethanol (70%) extract (3 µl) – 1.5911%. The percentage of rutin determined using the HPTLC method in an ethanol (96%) extract (5 µl) is 3.0894%, in an ethanol (70%) extract (5 µl) – 1.6064%. The percentage of total flavonoids in terms of rutin determined using the spectrophotometric method in the ethanol (96%) extract is 3.4289%, in the ethanol (70%) extract – 2.4176%. Conclusion. As a result of the study, a method for the quantitative determination of rutin was developed and validated, which can be used in the standardization of violet herb.

Novel HPTLC method for simultaneous estimation from ternary mixture of chlorpheniramine maleate, dextromethorphan hydrobromide and phenylephrine hydrochloride in syrup formulations

A synergic antihistamine, cough suppressant, and decongestant combination of chlorpheniramine, dextromethorphan, and phenylephrine is used to treat acute respiratory infections caused by seasonal viruses. The effective qualitative and quantitative methods require the simultaneous measurement of a ternary combination in the pharmaceutical syrup dosage form. Therefore, a new, simple, fast and robust high performance thin layer chromatographic (HPTLC) method has been developed and validated for chlorpheniramine maleate (CPM), dextromethorphan hydrobromide (DEXO) and phenylephrine hydrochloride (PE). The chromatographic separation was carried out on precoated aluminium plates with silica gel 60 F254 as the stationary phase. Mobile phase used was chloroform: methanol: ammonia (2.5:7.5:0.3, v/v/v) for proper separation. The detection was carried out at 270nm wavelength in absorbance mode. Developed method was validated as per International Council for Harmonization (ICH) Q2 (R1) guideline. The linearity range is 400 to 1400ng/band for CPM, 3000 to 11500ng/band for DEXO and 1000 to 3500ng/band for PE with correlation coefficient ≥0.995. The consistent lower values of relative standard deviation (RSD, %) for precision and robustness study indicate the method reliability. The percent recovery ranged from 97.82 to 102.03% indicates the good accuracy of the method. The proposed method was complying for the analytical method validation parameters suggested by the ICH Q2 (R1) guideline. The method was found to be simple, rapid and reliable for the simultaneous estimation of CPM, DEXO and PE from its pharmaceutical syrup dosage form. The method was successfully applied to quantify these analytes from the several pharmaceutical syrup dosage form.