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

INTRODUCTION: 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 Due to safety concerns, in 2010 all medicines containing sibutramine were urgently withdrawn from the USA and European pharmaceutical market.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.

Optimization of ultrasonic pretreatment and analysis of chlorogenic acid in potato leaves.

Chlorogenic acid (CA) is an effective ingredient that can strengthen immunity during following the COVID-19 era. The current cost of CA is high owing to its complex purification process and low yield (approximately 2%). In this study, a one-step path orthogonal experiment was designed based on the results from Gauss calculation, which consisted of acidity, coordination, and hydrolysis in molecules. The optimized extraction conditions were 60 â„ƒ, 60 min, 1:20 liquid ratio, and 40% ethanol in a nitrogen atmosphere controlled using a device of our own design, which led to CA yields of up to 6.35% from potato leaves. The purified CA was analyzed using high-performance liquid chromatography, thin-layer chromatography, ultraviolet-visible spectroscopy, and molecular fluorescence. This accurate and reproducible method can not only be used to obtain high yields of CA but can also be used for the quality control of active plant products and their isomers.

Magnetic field assisted centrifugal acceleration thin-layer chromatography: An approach to investigate the magnetic field effects on separation parameters including retention factor difference, selectivity factor, and resolution.

The effect of internal and external magnetic fields on the separation of antifungal drugs by centrifugal acceleration thin-layer chromatography was reported for the first time. External and internal magnetic fields were applied using neodymium magnets and CoFe2O4@SiO2 ferromagnetic nanoparticles. Separation of ketoconazole and clotrimazole was performed using a mobile phase consisting of n-hexane, ethyl acetate, ethanol, and ammonia (2.0:2.0:0.5:0.2, v/v). The influence of the magnetic field on the entire chromatographic system led to changes in the properties of the stationary and mobile phases and the analytes affecting the retention factor, shape, and width of the separated rings. The extent of this impact depended on the structure of the analyte and the type and intensity of the magnetic field. In the presence of the external magnetic field, there were more significant changes in the chromatographic parameters of the drugs, especially the width of the separated rings, and ketoconazole was more affected than clotrimazole. The changes are conceivably due to the effect of the magnetic field on the analyte distribution between the stationary and mobile phases, which is also caused by the possibility of the magnetic field affecting the viscosity, surface tension, and surface free energy between the stationary and mobile phases.

Quantitative detection of microcystin-LR in Bellamya aeruginosa by thin-layer chromatography coupled with surface-enhanced Raman spectroscopy based on in-situ ZIF-67/Ag NPs/Au NWs composite substrate.

As a hypertoxic natural toxin, the risk of Microcystin-leucine-arginine (MC-LR) residues in Bellamya aeruginosa deserves more attention. Herein, employing the conventional thin-layer chromatography (TLC) technology and a novel surface-enhanced Raman scattering (SERS) substrate, a TLC-SERS chip was fabricated for the purification and quantitative detection of MC-LR in complex samples. The substrate exhibited excellent SERS performance with an enhancement factor of 6.6 × 107, a low detection limit of 2.27 × 10-9 mM for MC-LR, excellent uniformity and reproducibility, as well as a wide linear range. With the application of TLC, the MC-LR was efficiently purified and the concentration was increased to >3 times. Ultimately, recovery rates fluctuated between 93.28% and 101.66% were obtained from the TLC-SERS chip. On balance, the TLC-SERS chip has a robust capacity for achieving rapid and stable quantitative detection of MC-LR, which promises to improve the efficiency of food safety monitoring.

An eco-friendly and cost-effective HPTLC method for quantification of COVID-19 antiviral drug and co-administered medications in spiked human plasma.

The coronavirus-2 has led to a global pandemic of COVID-19 with an outbreak of severe acute respiratory syndrome leading to worldwide quarantine measures and a rise in death rates. The objective of this study is to propose a green, sensitive, and selective densitometric method to simultaneously quantify remdesivir (REM) in the presence of the co-administered drug linezolid (LNZ) and rivaroxaban (RIV) in spiked human plasma. TLC silica gel aluminum plates 60 F254 were used as the stationary phase, and the mobile phase was composed of dichloromethane (DCM): acetone (8.5:1.5, v/v) with densitometric detection at 254 nm. Well-resolved peaks have been observed with retardation factors (Rf) of 0.23, 0.53, and 0.72 for REM, LNZ, and RIV, respectively. A validation study was conducted according to ICH Q2 (R1) Guidelines. The method was rectilinear over the concentration ranges of 0.2-5.5 µg/band, 0.2-4.5 µg/band and 0.1-3.0 µg/band for REM, LNZ and RIV, respectively. The sensitivities of REM, LIN, and RIV were outstanding, with quantitation limits of 128.8, 50.5, and 55.8 ng/band, respectively. The approach has shown outstanding recoveries ranging from 98.3 to 101.2% when applied to pharmaceutical formulations and spiked human plasma. The method's greenness was assessed using Analytical Eco-scale, GAPI, and AGREE metrics.

[Establishment and Optimization of Quality Standards for the Traditional Tibetan Medicine Preparation of Liuwei Nengxiao Pills]. / è—è¯å ­å‘³èƒ½æ¶ˆä¸¸è´¨é‡æ ‡å‡†çš„å»ºç«‹ä¸Žä¼˜åŒ–.

Objective: To establish quality standards for Liuwei Nengxiao pills, to optimize the quality control method, and to provide references for the quality control of Liuwei Nengxiao pills. Methods: Chebula, dried ginger, and Tibetan liqueur root in Liuwei Nengxiao pills of different batch numbers were analyzed by thin layer chromatography (TLC). Then, the content of chrysophanol in the preparation was determined by high performance liquid chromatography (HPLC). Furthermore, a series of methodological validation, including the investigation of the linear relationship, precision, stability, and reproducibility and sample recovery test, were performed to verify the reliability of the results. Results: The TLC identification method was easy to perform and demonstrated high specificity, clear spots, and good separation effect. In addition, the negative controls showed no interference. The HPLC method showed high accuracy. The results of methodological validation showed that the peak area of chrysophanol had a good linear relationship (r2=1.0) in the range of 0.06-0.80 µg, presenting good precision (with the relative standard deviation being lower than 2.0%), good stability and reproducibility (with the relative standard deviation being lower than 1.0%), and an average recovery rate of 100.8%. Conclusion: TLC and HPLC are easy to perform, showing high accuracy and reproducibility. The quality standards established are scientific, reasonable, stable, and feasible, providing references for the quality control of Liuwei Nengxiao pills.

Identification and quantification of pigments in plant leaves using thin layer chromatography-Raman spectroscopy (TLC-Raman).

Carotenoids are yellow, orange, and red pigments commonly found in plants. In leaves, these molecules are essential for photosynthesis, but they also play a major role in plant growth and development. Efficiently monitoring concentrations of specific carotenoids in plant tissues could help to explain plant responses to environmental stressors, infection and disease, fertilization, and other conditions. Previously, Raman methods have been used to demonstrate a correlation between plant fitness and the carotenoid content of leaves. Due to solvatochromatic effects and structural similarities within the carotenoid family, current Raman spectroscopy techniques struggle to assign signals to specific carotenoids with certainty, complicating the determination of amounts of individual carotenoids present in a sample. In this work, we use thin layer chromatography-Raman spectroscopy, or TLC-Raman, to identify and quantify carotenoids extracted from tomato leaves. These quick and accurate methods could be applied to study the relationship between pigment content and a number of factors affecting plant health.

Evaluation of Lipids for the Study of Photosynthetic Membranes.

The biological role of lipids goes far beyond the formation of a structural membrane bilayer platform for membrane proteins and controlling fluxes across the membranes. For example, in photosynthetic thylakoid membranes, lipids occupy well-defined binding niches within protein complexes and determine the structural organization of membrane proteins and their function by controlling generic physicochemical membrane properties. In this chapter, two-dimensional thin-layer chromatography (2D TLC) and gas chromatography (GC) techniques are presented for quantitative analysis of lipid classes and fatty acids in thylakoid membranes. In addition, lipid extraction methods from isolated thylakoid membranes and leaves are described together with a procedure for the derivatization of fatty acids to fatty acid methyl esters (FAME) that is required for GC analysis.

Chiral Thianthrenes.

The absolute configuration and stability of two thianthrene chiral sulfoxides has been determined by means of X-ray single-crystal structure determinations. The analyses and configurations allow verification that the diastereomeric sulfoxides are stable in solution and are not interconverting, which has been suggested in some studies of sulfoxides. The two thianthrene sulfoxides have slightly different Rf values, which allowed their separation using flash chromatography on silica. The spots run back-to-back, which posed a challenge for their separation. The pure, separated compounds in solution remain as separate, single spots on a Thin Layer Chromatography (TLC) plate.

High-Performance Thin-Layer Chromatography Coupled with Single Quadrupole: Application the Identification and Differentiation of Rehmanniae Radix and Its Different Processing Products from Raw Materials to Commercial Products.

The authentication of ingredients in formulas is crucial yet challenging, particularly for constituents with comparable compositions but vastly divergent efficacy. Rehmanniae Radix and its derivatives are extensively utilized in food supplements, which contain analogous compositions but very distinct effects. Rehmanniae Radix, also a difficult-to-detect herbal ingredient, was chosen as a case to explore a novel HPTLC-QDa MS technique for the identification of herbal ingredients in commercial products. Through systematic condition optimization, including thin layer and mass spectrometry, a stable and reproducible HPTLC-QDa MS method was established, which can simultaneously detect oligosaccharides and iridoids. Rehmannia Radix and its processed products were then analyzed to screen five markers that could distinguish between raw and prepared Rehmannia Radix. An HPTLC-QDa-SIM method was further established for formula detection by using the five markers and validated using homemade prescriptions and negative controls. Finally, this method was applied to detect raw and prepared Rehmannia Radix in 12 commercial functional products and supplements.

Screening of α-amylase/trypsin inhibitor activity in wheat, spelt and einkorn by high-performance thin-layer chromatography.

α-Amylase/trypsin inhibitor proteins (ATI) are discussed as possible triggers for non-celiac gluten sensitivity. The potential of high-performance thin-layer chromatography (HPTLC) was studied for the first time to analyse the inhibitory properties of ATIs from flour of wheat, spelt, and einkorn. Inhibition by each flour of the digestive enzymes trypsin or α-amylase was determined by the reduction of released metabolisation products in comparison to non-digested flour, and positive (acarbose) and negative (water) controls. Firstly, amylolysis was carried out in miniaturized form on the HPTLC surface (HPTLC-nanoGIT) after in-vial pre-incubation of the amylase with the inhibitors from flour. α-Amylase inhibition was evident via the reduction of released saccharides, as analysed by normal phase HPTLC. A strong influence of the flour matrix on the assay results (individual saccharides) was evident, caused by an increased amylolysis of further polysaccharides present, making HPTLC analysis more reliable than currently used spectrophotometric sum value assays. The detection and visualization of such matrix influence helps to understand the problems associated with spectrophotometric assays. Only maltotriose was identified as a reliable marker of the amylolysis. The highest α-amylase inhibition and thus the lowest saccharide response was detected for maltotriose in refined spelt, whereas the lowest α-amylase inhibition and thus the highest saccharide response was detected for maltotriose in refined wheat. A comparison of refined and whole grain flours showed no clear trend in the responses. Secondly, trypsin inhibition and proteolysis were performed in-vial, and any inhibition was evident via the reduction of released peptides, analysed by reversed-phase HPTLC. Based on the product pattern of the proteolysis, einkorn and whole wheat showed the highest trypsin inhibition, whereas refined wheat and refined spelt showed the lowest inhibition. Advantageously, HPTLC analysis provided important information on changes in individual saccharides or peptides, which was more reliable and sustainable than spectrophotometric in-vial assays (only sum value) or liquid column chromatography analysis (targeting only the ATI proteins).

Increasing Analytical Quality by Designing a Thin-Layer Chromatography Scanner Method for the Determination of the Radiochemical Purity of Radiopharmaceutical Sodium Iodide 131I Oral Solution.

The goal of this study was to apply the principles of analytical quality by design (AQbD) to the analytical method for determining the radiochemical purity (PQR) of the radiopharmaceutical sodium iodide 131I oral solution, utilizing thin-layer chromatography (TLC) with a radio-TLC scanner, which also enables the evaluation of product quality. For AQbD, the analytical target profile (ATP), critical quality attributes (CQA), risk management, and the method operable design region (MODR) were defined through response surface methodology to optimize the method using MINITAB® 19 software. This study encompassed the establishment of a control strategy and the validation of the method, including the assessment of selectivity, linearity, precision, robustness, detection limit, quantification limit, range, and the stability of the sample solution. Under the experimental conditions, the method parameters of the TLC scanner were experimentally demonstrated and optimized with an injection volume of 3 µL, a radioactive concentration of 10 mCi/mL, and a carrier volume of 40 µL. Statistical analysis confirmed the method's selectivity for the 131I iodide band Rf of 0.8, a radiochemical impurity IO3- Rf of 0.6, a linearity from 6.0 to 22.0 mCi/mL, and an intermediate precision with a global relative standard deviation (RSD) of 0.624%. The method also exhibited robustness, with a global RSD of 0.101%, a detection limit of 0.09 mCi/mL, and a quantification limit of 0.53 Ci/mL, meeting the prescribed range and displaying stability over time (at 0, 2, and 20 h) with a global RSD of 0.362%, resulting in consistent outcomes. The development of a method based on AQbD facilitated the creation of a design space and an operational space, with comprehensive knowledge of the method's characteristics and limitations. Additionally, throughout all operations, compliance with the acceptance criteria was verified. The method's validity was confirmed under the established conditions, making it suitable for use in the manufacturing process of sodium iodide 131I and application in nuclear medicine services.

Colorimetric analysis platform based on thin layer chromatography for monitoring gluten cross-contamination in food industry.

Monitoring of the accidental presence of gluten (Glu), resulting from cross-contamination, is imperative in different industries, in particular food industry. The objective of this study was the development of an analytical platform utilizing thin-layer chromatography (TLC) with colorimetric read-out for making binary (yes/no) decisions on surfaces and/or point of these industries. The composition of the extractive phase was optimized with commercial products used in cleaning processing lines. Subsequently, an exploration of TLC separation and detection was undertaken. CN-modified nanosilica plates and 30:70 acetonitrile:water were used to achieve a selective signal for Glu residues. The study of the detection performance showed that both spectroscopic measurement and image analysis were resulted in satisfactory results for quantitate analysis (RSD = 5 %, LOD = 0.12 mg). The practical application of the proposed methodology on surfaces of the food processing lines. This work demonstrated the operational feasibility in detecting gluten cross-contaminations within the food processing industry.

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 (250 mm × 4.6 mm, 5 µm) with a mobile phase consisting of 0.05 M disodium hydrogen phosphate and methanol in a 40:60% v/v ratio. The flow rate is maintained at 1.0 mL/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: 1.5 v/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 286 nm over the concentration range of 0.1-0.6 g/mL for PNT and 0.4-2.4 g/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 298 nm over the concentration range of 400-2400 ng/spot for ASP and 100-600 ng/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.

Chromatographic Separation and Quantitation of Sphingolipids from the Central Nervous System or Any Other Biological Tissue.

Chromatographic separation and purification of an individual lipid to homogeneity have long been introduced. Using this concept, a more precise method has been developed to identify and characterize the sphingolipid composition(s) using a small amount (30 mg) of biological sample. Sphingolipids (lipids containing sphingosine or dihydrosphingosine) are well-known regulators of the central nervous system development and play a critical role in neurodegenerative diseases. Introducing a silicic acid column chromatography, sphingolipid components have been separated to individual fractions such as ceramide, glucosyl/galactosylceramide, other neutral and acidic glycosphingolipids, including (dihydro)sphingosine and psychosine; as well as phospholipids from which individual components are quantified employing a single or combination of other advanced chromatography procedures such as thin-layer chromatography, gas chromatography-mass spectrometry, and high-performance liquid chromatography-mass spectrometry.

Chromatographic authentication of botanical origin: Herbaceous pollen profiling with HPLC, HPTLC and GC-MS analysis.

This study describes a robust chromatographic authentication methodology for herbaceous pollen, employing gas chromatography-mass spectrometry (GC-MS), high-performance liquid chromatography (HPLC) and high-performance thin liquid chromatography (HPTLC) protocols. The comprehensive profiling of organic compounds not only distinguishes between different botanical sources but also establishes a reliable framework for quality control and assessment of herbaceous pollen authenticity. Traces of quercetin were detectable using HPTLC in Chaenomeles japonica, and the composition of the mobile phase led to distinct phenolic acid tracks in the extracts of free phenolic compounds. In Lonicera nummulariifolia, prominent chlorogenic acid signal and traces of 3,4-dihydroxybenzoic acid were identified, along with the presence of vanillic, trans-ferulic, p-coumaric and p-hydroxybenzoic and sinapic as phenolic acid standards. The HPLC chromatogram identified six peaks representing bioactive phenolic compounds such as gallic acid measuring 5.89 ± 0.56 mg g-1, hydroxybenzoic acid 2.39 ± 0.78 mg g-1 and caffeic acid 2.83 ± 0.11 mg g-1. The combined use of GC-MS, HPTLC and HPLC techniques provides a powerful and reliable means of authenticating the botanical origin of herbaceous pollen, offering valuable insights for quality control and ensuring the accuracy of botanical source identification.

Development of a high-performance thin-layer chromatography-based method for targeted glycerolipidome profiling of microalgae.

The conditionally essential very-long-chain polyunsaturated fatty acids (VLC-PUFAs), such as eicosapentaenoic acid (EPA, C20:5 n-3), play a vital role in human nutrition. Their biological activity is thereby greatly influenced by the distinct glycerolipid molecule that they are esterified to. Here, microalgae differ from the conventional source, fish oil, both in quantity and distribution of VLC-PUFAs among the glycerolipidome. Therefore, the aim of this study was to develop a fast and reliable one-dimensional high-performance thin-layer chromatography (HPTLC)-based method that allows the separation and quantification of the main microalgal glycerolipid classes (e.g., monogalactosyldiacylglycerol (MGDG), sulfoquinovosyl diacylglycerol (SQDG), phosphatidylglycerol (PG)), as well as the subsequent analysis of their respective fatty acid distribution via gas chromatography (GC) coupled to mass spectrometry (MS). Following optimization, method validation was carried out for 13 different lipid classes, based on the International Conference on Harmonization (ICH) guidelines. In HPTLC, linearity was effective between 100 and 2100 ng, with a limit of quantification between 62.99 and 90.09 ng depending on the glycerolipid class, with strong correlation coefficients (R2 > 0.995). The recovery varied between 93.17 and 108.12%, while the inter-day precision measurements showed coefficients of variation of less than 8.85%, close to the limit of detection. Applying this method to crude lipid extracts of four EPA producing microalgae of commercial interest, the content of different glycerolipid classes was assessed together with the respective FA distribution subsequent to band elution. The results showed that the described precise and accurate HPTLC method offers the possibility to be used routinely to follow variations in the glycerolipid class levels throughout strain screening, cultivation, or bioprocessing. Thus, additional quantitative analytical information on the complex lipidome of microalgae can be obtained, especially for n-3 and n-6 enriched lipid fractions.

Densitometric method for assessment of six specialized metabolites in four Sida sp. and its congener Abutilon indicum: Targeted metabolomics, greenness assessment, and chemometrics analysis.

Sida is one of the most diverse genera, with about 200 species distributed in tropical and subtropical regions of the world. Among 18 species distributed in India, Sida acuta, Sida cordifolia, Sida rhombifolia, and Sida cordata are used in traditional medicines along with its possible adulterant Abutilon indicum for several therapeutic uses. The non-availability of marker-based validated methods for the identification and classification of these species leads to adulteration. Indoloquinoline and quinazoline are the major bioactive alkaloids distributed in Sida spp. First time, a simple, economical and high throughput method was developed and validated for the simultaneous determination of 20-hydroxyecdysone (1), vasicine (2), vasicinone (3), cryptolepine (4), quindolinone (5), and cryptolepinone (6) using HPTLC-UV densitometry. The method was validated to meet globally accepted ICH guidelines. The method was sensitive with LOD and LOQ ranging from 0.38-0.63 and 1.57-2.12 µg/band. The samples were spiked at 3 different concentrations, the recovery values were 93.49-98.88%. In addition, the greenness index of the HPTLC method was estimated using four different greenness assessment techniques. Targeted HPTLC analysis indicated the distribution of specialized metabolites in Sida spp. and A. indicum. However, the occurrence of cryptolepine in A. indicum was not reported in the literature, so this was further confirmed by liquid chromatographic studies of the samples from different locations. The chromatographic data was statistically evaluated by principal component analysis (PCA) and hierarchical clustering (HCA). HPTLC-based targeted metabolite quantitation explains the adulteration/substitution in Sida raw material and derived herbal preparations.

Towards non-target proactive food safety: identification of active compounds in convenience tomato products by ten-dimensional hyphenation with integrated simulated gastrointestinal digestion.

Current strategies for non-target food screening focus mainly on known hazardous chemicals (adulterants, residues, contaminants, packaging migrants, etc.) instead of bioactive constituents in general and exclude the biological effect detection. To widen the perspective, a more proactive non-target effect-directed strategy is introduced to complement food safety in order to detect not only known but also unknown bioactive compounds. The developed 10-dimensional hyphenation included on-surface digestion (1D), planar chromatographic separation (2D), visualization using white light (3D), UV light (4D), fluorescence light (5D), effect-directed assay analysis (6D), heart-cut zone elution to an orthogonal reversed phase column chromatography including online desalting (7D) with subsequent diode array detection (8D), high-resolution mass spectrometry (9D), and fragmentation (10D). Metabolism, i.e., intestinal digestion of each sample, was simulated and integrated on the same adsorbent surface to study any changes in the compound profiles. As proof of principle, nine convenience tomato products and a freshly prepared tomato soup were screened via five different planar assays in a non-targeted mode. Non-digested and digested samples were compared side by side. In their effect-directed profiles, 14 bioactive compounds from classes of lipids, plant hormones, spices, and pesticides were identified. In particular, bioactive compounds coming from the lipid class were increased by gastrointestinal digestion, while spices and pesticides remained unaffected. With regard to food safety, the determination of the two dinitrophenol herbicides dinoterb and dinoseb in highly processed tomato products should be given special attention. The hyphenation covered a broad analyte spectrum and showed robust and reliable results.