Effect-Directed Profiling of Akebia quinata and Clitoria ternatea via High-Performance Thin-Layer Chromatography, Planar Assays and High-Resolution Mass Spectrometry.

Two herbal plants, Akebia quinata D. leaf/fruit and Clitoria ternatea L. flower, well-known in traditional medicine systems, were investigated using a non-target effect-directed profiling. High-performance thin-layer chromatography (HPTLC) was combined with 11 different effect-directed assays, including two multiplex bioassays, for assessing their bioactivity. Individual active zones were heart-cut eluted for separation via an orthogonal high-performance liquid chromatography column to heated electrospray ionization high-resolution mass spectrometry (HPLC-HESI-HRMS) for tentative assignment of molecular formulas according to literature data. The obtained effect-directed profiles provided information on 2,2-diphenyl-1-picrylhydrazyl scavenging, antibacterial (against Bacillus subtilis and Aliivibrio fischeri), enzyme inhibition (tyrosinase, α-amylase, ß-glucuronidase, butyrylcholinesterase, and acetylcholinesterase), endocrine (agonists and antagonists), and genotoxic (SOS-Umu-C) activities. The main bioactive compound zones in A. quinata leaf were tentatively assigned to be syringin, vanilloloside, salidroside, α-hederin, cuneataside E, botulin, and oleanolic acid, while salidroside and quinatic acids were tentatively identified in the fruit. Taraxerol, kaempherol-3-rutinoside, kaempferol-3-glucoside, quercetin-3-rutinoside, and octadecenoic acid were tentatively found in the C. ternatea flower. This straightforward hyphenated technique made it possible to correlate the biological properties of the herbs with possible compounds. The meaningful bioactivity profiles contribute to a better understanding of the effects and to more efficient food control and food safety.

Simultaneous green TLC determination of nirmatrelvir and ritonavir in the pharmaceutical dosage form and spiked human plasma.

Quantitative analysis of pharmaceutical compounds up to Nano gram levels is highly recommended to introduce feasible and sensitive tool for determination of the compounds in the pharmaceutical and biological samples. Nirmatrelvir plus ritonavir was recently approved in the US, the UK and Europe as a new co-packaged dosage form for the treatment of COVID-19. The objective of this work was to develop a more sensitive TLC method based on using ß-cyclodextrin as a chiral selector additive in the mobile phase for simultaneous determination of nirmatrelvir and ritonavir in pure form, pharmaceutical formulation and spiked human plasma. The analysis procedures were developed using TLC aluminum silica gel plates and methanol-water- 2% urea solution of ß-cyclodextrin (40:10:.5, by volume) as a mobile phase with UV detection at 215 nm. The developed method was successfully applied over a linearity range of 10-50 ng/band for both nirmatrelvir and ritonavir. The method was validated for limits of detection and quantitation, accuracy, precision, specificity, system suitability, and robustness. Furthermore, the eco-friendliness of the proposed method was assessed using the analytical eco-scale and the green analytical procedure index. The described method exhibited compliance with green analytical chemistry principles based on common green metric values.

Urinary Glycosaminoglycans: Characterization and Quantification.

The growing body of evidence supports the potential of using urinary glycosaminoglycans (uGAGs) levels as biomarkers to guide diagnosis and as predictive biomarkers of treatment efficacy. Recently, studies have shown that, in addition to MPS, the prognosis and treatment of cancers and viral infections, including COVID-19, are enabled by characterization and/or traits by GAGs. Reliable and accessible detection and assay protocols of urinary GAGs are therefore of great support for laboratory workers and clinicians. Here we describe a semiquantitative and quantitative urinary glycosaminoglycans determination using 1,9-dimethylmethylene blue (DMB) and the characterization of uGAGs using thin layer chromatography (TLC).

A Non-Instrumental Green Analytical Method Based on Surfactant-Assisted Dispersive Liquid-Liquid Microextraction-Thin-Layer Chromatography-Smartphone-Based Digital Image Colorimetry(SA-DLLME-TLC-SDIC) for Determining Favipiravir in Biological Samples.

Favipiravir (FAV) has become a promising antiviral agent for the treatment of COVID-19. Herein, a green, fast, high-sample-throughput, non-instrumental, and affordable analytical method is proposed based on surfactant-assisted dispersive liquid-liquid microextraction (SA-DLLME) combined with thin-layer chromatography-digital image colourimetry (TLC-DIC) for determining favipiravir in biological and pharmaceutical samples. Triton X-100 and dichloromethane (DCM) were used as the disperser and extraction solvents, respectively. The extract obtained after DLLME procedure was spotted on a TLC plate and allowed to develop with a mobile phase of chloroform:methanol (8:2, v/v). The developed plate was photographed using a smartphone under UV irradiation at 254 nm. The quantification of FAV was performed by analysing the digital images' spots with open-source ImageJ software. Multivariate optimisation using Plackett-Burman design (PBD) and central composite design (CCD) was performed for the screening and optimisation of significant factors. Under the optimised conditions, the method was found to be linear, ranging from 5 to 100 µg/spot, with a correlation coefficient (R2) ranging from 0.991 to 0.994. The limit of detection (LOD) and limit of quantification (LOQ) were in the ranges of 1.2-1.5 µg/spot and 3.96-4.29 µg/spot, respectively. The developed approach was successfully applied for the determination of FAV in biological (i.e., human urine and plasma) and pharmaceutical samples. The results obtained using the proposed methodology were compared to those obtained using HPLC-UV analysis and found to be in close agreement with one another. Additionally, the green character of the developed method with previously reported protocols was evaluated using the ComplexGAPI, AGREE, and Eco-Scale greenness assessment tools. The proposed method is green in nature and does not require any sophisticated high-end analytical instruments, and it can therefore be routinely applied for the analysis of FAV in various resource-limited laboratories during the COVID-19 pandemic.

Highly sensitive high-performance thin-layer chromatography method for the simultaneous determination of molnupiravir, favipiravir, and ritonavir in pure forms and pharmaceutical formulations.

Favipiravir, molnupiravir, and ritonavir have been recently approved as the first oral antivirals for treatment of SARS-CoV-2 viral infections. Their combination was reported in several clinical studies, alternatively, to enhance the viral eradication and improve patient's recovery times and rates. Being all orally administered, therefore, the development of new sensitive and validated methodologies for their simultaneous determination is a necessitate. In the proposed research, a sensitive, selective, and simple high-performance thin layer chromatography method was developed and validated for determination of favipiravir, molnupiravir, and ritonavir. Silica gel 60F254 thin layer chromatography plates were used as stationary phase for this separation using mobile phase composed of methylene chloride:ethyl acetate:methanol:25% ammonia (6:3:4:1, v/v/v/v). Densitometric detection was performed at wavelength 289 nm. Peaks of favipiravir, molnupiravir, and ritonavir were resolved at retention factors 0.22, 0.42, and 0.63, respectively. The proposed method was found linear within the specified ranges of 3.75-100.00 µg/mL for molnupiravir and favipiravir, and 2.75-100.00 µg/mL for ritonavir. Limits of detection were found to be 1.12, 1.21, and 0.89 µg/mL for favipiravir, molnupiravir, and ritonavir, respectively. This is the first method to be reported for the simultaneous determination of the cited three antiviral drugs. The method was assessed on novel greenness metrics.

US FDA-validated TLC method with four greenness assessment evaluations for simultaneous determination of prednisolone and esomeprazole in spiked human plasma.

Recently, prednisolone has been used in treating many medical conditions, such as autoimmune diseases and cancer. It is also prescribed to mitigate the respiratory complications caused by COVID-19 infection. It can cause some health complications, such as GIT ulcers, so it should be co-administered with proton-pump inhibitors, such as esomeprazole, to prevent the risk of ulcers. This work aims to develop an ecofriendly and sensitive TLC method for simultaneous determination of esomeprazole and prednisolone in their binary mixtures and spiked human plasma. Separation was performed using a mixture of ethyl acetate, methanol, and ammonia (9.5:0.5:0.1, v/v/v) as an eluting system with UV scanning at 245 nm. Dapoxetine was used as an internal standard to correct the variation during sampling. The resulting Rf values for plasma, esomeprazole, prednisolone, and dapoxetine were 0.03, 0.51, 0.72 and 0.85, respectively. Four greenness assessment tools-national environmental method index, eco-scale assessments, analytical greenness metric approach (AGREE), and green analytical procedure index (GAPI)-were used to evaluate the greenness characteristics of the proposed method to the environment, and the results were acceptable and satisfactory. Validation parameters were checked according to the US FDA guidelines to achieve the international requirements for bioanalytical method validation, and the results were within the accepted ranges.

Phytometabolite profiling of Coronil, a herbal medicine for COVID-19, its identification by mass-spectroscopy and quality validation on liquid chromatographic platforms.

Coronil is a tri-herbal medicine consisting of immunomodulatory herbs, Withania somnifera, Tinospora cordifolia, and Ocimum sanctum. The formulation has been developed specifically as the supporting measure for COVID-19. Current investigation is aimed to identify the phytoconstituents in Coronil utilizing ultra-performance liquid chromatography-mass spectrometry coupled with quadrapole time of flight and to establish its quality standardization using high-performance liquid chromatography and high performance thin layer chromatography. Out of 52 identified compounds, cordifolioside A, magnoflorine, rosmarinic acid, palmatine, withanoside IV, withanoside V, withanone, betulinic acid, and ursolic acid were quantified in 15 different batches of Coronil on validated high-performance liquid chromatography method. Similarly, withanoside IV, withaferin A, magnoflorine, palmatine, rosmarinic acid, and ursolic acid were analyzed on high performance thin layer chromatography. Methods were validated as per the International Council for Harmonization guidelines. These methods were specific, reproducible, accurate, precise, linear (r2 > 0.99), and percent recoveries were within the prescribed limits. The content uniformity of Coronil was ascertained using Fourier transform infrared spectroscopy. Results indicated that, validated methods were fit for their intended use and the analytical quality of Coronil was consistent across the batches. Taken together, these developed methods could drive the analytical quality control of herbal medicines such as Coronil, and other formulations containing similar chemical profiles.

An Evolving Technology That Integrates Classical Methods with Continuous Technological Developments: Thin-Layer Chromatography Bioautography.

Thin-layer chromatography (TLC) bioautography is an evolving technology that integrates the separation and analysis technology of TLC with biological activity detection technology, which has shown a steep rise in popularity over the past few decades. It connects TLC with convenient, economic and intuitive features and bioautography with high levels of sensitivity and specificity. In this study, we discuss the research progress of TLC bioautography and then establish a definite timeline to introduce it. This review summarizes known TLC bioautography types and practical applications for determining antibacterial, antifungal, antitumor and antioxidant compounds and for inhibiting glucosidase, pancreatic lipase, tyrosinase and cholinesterase activity constitutes. Nowadays, especially during the COVID-19 pandemic, it is important to identify original, natural products with anti-COVID potential compounds from Chinese traditional medicine and natural medicinal plants. We also give an account of detection techniques, including in situ and ex situ techniques; even in situ ion sources represent a major reform. Considering the current technical innovations, we propose that the technology will make more progress in TLC plates with higher separation and detection technology with a more portable and extensive scope of application. We believe this technology will be diffusely applied in medicine, biology, agriculture, animal husbandry, garden forestry, environmental management and other fields in the future.

Development and validation of a rapid high-performance thin-layer chromatographic method for quantification of gallic acid, cinnamic acid, piperine, eugenol, and glycyrrhizin in Divya-Swasari-Vati, an ayurvedic medicine for respiratory ailments.

Divya-Swasari-Vati is a calcium containing polyherbal ayurvedic medicine prescribed for the lung-related ailments observed in the current pandemic of Severe Acute Respiratory Syndrome Coronavirus 2 infections. The formulation is a unique quintessential blend of nine herbs cited in Ayurvedic texts for chronic cough and lung infection. Analytical standardization of herbal medicines is the pressing need of the hour to ascertain the quality compliance. This persuaded us to develop a simple, rapid, and selective high-performance thin-layer chromatographic method for Divya-Swasari-Vati quality standardization. The developed method was validated for the quantification of marker components, gallic acid, cinnamic acid, piperine, eugenol and glycyrrhizin, against reference standards in five different batches of Divya-Swasari-Vati. The analytes were identified by visualization at 254 nm, and by matching their retention factor with authentic standards. The developed method was validated as per the guidelines recommended by the International Council for Harmonization for parameters like, linearity, limit of detection, limit of quantification, accuracy, and precision. Therefore, the developed novel high-performance thin-layer chromatographic process could be employed for rapid standardization of Divya-Swasari-Vati and other related herbal formulation, which would aid in quality manufacturing and product development.

Dual-Mode Gradient HPLC and TLC Densitometry Methods for the Simultaneous Determination of Paracetamol and Methionine in the Presence of Paracetamol Impurities.

BACKGROUND: Paracetamol (PC) is one of the most widely used analgesic and antipyretic drugs and has recently been integrated into the supportive therapy of COVID-19. Pharmaceuticals containing methionine (MT) with PC may contribute to avoid hepatotoxicity and eventual PC overdose-dependent death. OBJECTIVE: The current work purposes to develop and validate two chromatographic methods for the simultaneous determination of MT and PC in the presence of two PC impurities (4-nitrophenol [NP] and 4-aminophenol [AP]). METHOD: Two chromatographic methods were established and validated according to the International Conference on Harmonization guidelines. The first one was an RP-HPLC/UV method based on applying a "dual-mode" gradient elution. The separation was realized via varying both the composition of the ternary mobile phase (acetonitrile-methanol-water) and its flow rate. This strategy enabled a relatively rapid analysis with a satisfactory resolution, although the investigated compounds exhibit a significant difference in lipophilicity. The second one relied on TLC-densitometry, where the optimum separation was realized using a quaternary mobile phase system composed of butanol-dioxane-toluene-methanol (8:2.5:3.5:0.3, by volume). Both methods were monitored at 220 nm. RESULTS: The developed methods were proven to be robust, accurate, specific, and appropriate for the routine analysis of PC in its pure form or in pharmaceutical formulations with MT in quality control laboratories. CONCLUSIONS: The corresponding methods are suitable to determine MT and PC in the presence of PC impurities. HIGHLIGHTS: The study achieves the analysis of MT and PC in the presence of PC impurities via the application of HPLC and TLC-densitometry methods.

Simultaneous Determination of Paracetamol, Propyphenazone and Caffeine in Presence of Paracetamol Impurities Using Dual-Mode Gradient HPLC and TLC Densitometry Methods.

Two chromatographic methods were validated for the determination of the widely prescribed analgesic and antipyretic drug combination of paracetamol (PC) (recently integrated into the supportive treatment of COVID-19), propyphenazone (PZ) and caffeine (CF) in the presence of two PC impurities, namely 4-aminophenol and 4-nitrophenol. A "dual-mode" gradient high-performance liquid chromatography method was developed, where the separation was achieved via "dual-mode" gradient by changing both the ternary mobile phase composition (acetonitrile: methanol: water) and the flow rate. This enables a good resolution within a relatively shorter analysis time. The analysis was realized using Zorbax Eclipse XDB column C18, 5 µm (250 × 4.6 mm) and the UV detector was set at 220 nm. The other method is a thin-layer chromatography densitometry method, where the separation was achieved using a mobile phase composed of chloroform: toluene: ethyl acetate: methanol: acetic acid (6: 6: 1: 2: 0.1, by volume). Densitometric detection was performed at 220 nm on silica gel 60 F254 plates. The developed methods were fully validated as per the ICH guidelines and proved to be accurate, robust, specific and suitable for application as purity indicating methods for routine analysis of PC in pure form or in pharmaceuticals with PZ and CF in quality control laboratories.

Identification of Falsified Chloroquine Tablets in Africa at the Time of the COVID-19 Pandemic.

Reports that chloroquine and hydroxychloroquine may be effective against COVID-19 have received worldwide attention, increasing the risk of the introduction of falsified versions of these medicines. Five different types of falsified chloroquine tablets were discovered between March 31, 2020 and April 4, 2020, in Cameroon and the Democratic Republic of Congo by locally conducted thin layer chromatographic analysis. Subsequent investigation by liquid chromatography and mass spectrometry in Germany proved the absence of detectable amounts of chloroquine and the presence of undeclared active pharmaceutical ingredients, that is, paracetamol and metronidazole, in four of the samples. The fifth sample contained chloroquine, but only 22% of the declared amount. Such products represent a serious risk to patients. Their occurrence exemplifies that once medicines or vaccines against COVID-19 may be developed, falsified products will enter the market immediately, especially in low- and middle-income countries (LMICs). Timely preparations for the detection of such products are required, including the establishment of appropriate screening technologies in LMICs.