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Munkánk során tenyészedény-kísérlettel teszteltük az elektromos gyökérkapacitás (EC) mérés alkalmazhatóságát szójafajták gyökérnövekedésének és szárazságtűrésének in situ vizsgálata céljából. A kontroll és a szárazságstressznek kitett növények EC-jét rendszeresen mértük, végül biomasszájukat destruktív eljárással meghatároztuk.

Az EC mérésével jól detektálható volt a fajták eltérő gyökérnövekedési dinamikája és biomassza-produkciója. Az EC — fajtától és kortól függő intenzitással — a virágzás kezdetéig folyamatosan emelkedett, majd közel állandóvá vált. A fajták EC-je és gyökértömege szoros korrelációt mutatott a kontroll (R2=0,844) és a szárazságkezelt (R2=0,936) növényeknél egyaránt. A vízhiány 28,8–50,5%-kal csökkentette az egyes fajták EC-jét, ami összhangban állt azok hajtástömegeinek 25,5–49,1%-os, és levélfelületeinek (transzspirációjának) 23,6–51,5%-os csökkenésével, ugyanakkor több fajtánál lényegesen meghaladta a gyökértömegben mutatkozó veszteséget (12,6–47,3%). Ennek oka, hogy a szárazság hatására nőtt a gyökér/hajtás arány (3,9–21,9%-kal), ezért csökkent az egységnyi gyökértömegre eső (fajlagos) vízfelvétel, így a fajlagos EC is. Mindezt alátámasztotta az EC-gyökértömeg regressziós egyenes meredekségének csökkenése (kontroll: 0,437 nF/g gyökér; szárazságkezelt: és 0,317 nF/g gyökér). Megállapítható, hogy az EC a teljes gyökérrendszer felvételi aktivitását jelzi, így — a gyökértömeggel szemben — funkcionális mutatója is a gyökérzet aktuális állapotának.

Eredményeink alapján az EC mérése hatékony módszer a gyökérnövekedési dinamika fajtaspecifikus különbségeinek vizsgálatára, valamint a környezeti hatások okozta biomassza-veszteség becslésére. Az in situ eljárás a hagyományos technikák mellett az agrártudományi kutatások számos területén (pl. fajtaszelekció, stressztűrés-vizsgálatok) haszonnal járhat.

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Ten methanolic extracts of selected Cirsium species were analyzed using two-dimensional thin-layer chromatography (2D-TLC) system with octadecyl reversed-phase (RP-18) chromatographic plate as the stationary phase and two eluents: nonaqueous, consisting of 2-butanone‒toluene‒acetic acid (4.5:5:0.5, v/v) used in the first direction of developing, and aqueous, consisting of methanol—water—formic acid (4:5:1, v/v) used in the second direction. The Naturstoff reagent was used for the derivatization of some phenolic compounds. Five selected standards were analyzed under the same chromatographic conditions, and their retention factor values were used for the confirmation of their presence on selected Cirsium chromatograms. Photographs of ten chromatograms were treated using the ImageJ program. 2D-TLC analysis was also performed to obtain the fingerprint chromatographic profiles of the studied methanolic extracts. The experimental data were objected to principal component analysis (PCA), and the PC2 vs. PC3 graphs were created. Based on the PCA results, the similarity between the selected Cirsium species was confirmed.

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Authors: Boris Ettienne Duffau, Rossana Camila Laurie, Sonia Rojas and René Rocha
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Authors: Sebastián Vecino Mantilla, Álvaro Mancilla Manrique and Paola Gauthier-Maradei

Bio-oil is produced by biomass pyrolysis. It contains hundreds of chemical compounds including alkanes, aromatic hydrocarbons, esters, ethers, ketones, aldehydes, acids, alcohols, and phenols. Phenols are compounds of increasing interest; they can be used as feedstock in many industrial applications such as the production of fuel additives, chemical synthesis, or as food antioxidants. Therefore, the valorization of phenols stemming from bio-oil can be an appropriated alternative to reduce the dependence on petro-based phenols in the chemical industry. The most important phenols in biooil from agricultural wastes are phenol, guaiacol, cresols, syringol, and xylenol. These compounds were separated by silica gel column chromatography technique, using 3 different solvents: a dichloromethane—acetone mixture, ethyl acetate, and methanol. Column elution was followed by thin-layer chromatography (TLC). Phenolic fraction was obtained and not individual phenols. This fraction was analyzed using gas chromatography–flame ionization detector (GC—FID) and gas chromatography—gas chromatography—mass spectrometry (GC—MS) with a DB-1701 column, and it was quantified using the relative response factor. Dichloromethane—acetone mixture was the best eluent to obtain this phenolic fraction, specifically during the first three elution steps.

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Two different botanical sources, Eclipta alba and Wedelia calendulacea are used as “Bhringaraja” in the Ayurvedic s ystem o f medicine. In the present study, an effort has been made to evaluate different sources by using high-performance thin-layer chromatography (HPTLC) as an analytical tool. Wedelolactone, one of the primary constituents of these plants, was taken as the marker compound for the evaluation. An HPTLC method was developed and validated for the evaluation of different sources of “Bhringaraja”. The chromatographic system was developed using silica gel 60 F254 HPTLC plates with the mobile phase toluene–chloroform–ethyl alcohol–formic acid (5:4:1:0.5, v/v). Linearity was found between the concentration ranges of 80 to 280 ng spot−1 (R 2: 0.9994), and limit of detection (LOD) and limit of quantification (LOQ) were found to be 0.36 ng spot−1 and 1.09 ng spot−1. The study has shown the presence of wedelolactone at the concentration of 0.26% w/w and 0.05% w/w in E. alba and W. calendulacea, respectively, whereas it was absent in another closely related species, Wedelia trilobata. At the same time, all the three plants were subjected to evaluation of quality-control parameters as per the World Health Organization (WHO) guidelines. Certain parameters such as foaming index, total alkaloids, and total bitter principles were significantly different in the three plants. Hence, the present HPTLC method development, and the validation and evaluation of quality-control parameters would be helpful in the standardization of individual plants and their formulations.

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Accurate, selective, and sensitive thin-layer chromatography (TLC)—densitometry and reversed-phase high-performance liquid chromatography (RP-HPLC) methods have been developed and validated for the simultaneous determination of vitamin E (VIT E) and vinpocetine (VINP) in the presence of the alkaline-induced degradation product of vinpocetine (DEG). The proposed TLC— densitometric method depends on the separation and quantitation of VIT E, VINP, and VINP alkaline-induced degradation product on TLC silica gel 60 F254 plates, using methanol—chloroform—ethyl acetate—glacial acetic acid—ammonia solution (6:2:2:0.5:0.1, by volume) as the developing system followed by densitometric measurement at 235 nm. The studied components were well resolved from each other with significantly different R f values of 0.81, 0.62, and 0.41 for VIT E, VINP, and DEG, respectively. On the other hand, the developed RP-HPLC method was based on the separation of the studied components using 0.05 M KH2PO4 (adjusted to pH = 3) and methanol in gradient elution mode on C8 column at a flow rate of 1.5 mL min−1 and ultraviolet (UV) detection at 235 nm. The studied components were well resolved from each other with significantly different R t values of 10.90, 2.89, and 1.90 min for VIT E, VINP, and DEG, respectively. The developed methods were validated according to the International Conference on Harmonization (ICH) guidelines demonstrating good accuracy and precision. The results were statistically compared with those obtained by the reported method, and no significant difference was found. The developed methods are the first developed stability-indicating assay methods (SIAMs) for the analysis of the studied binary mixture.

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A high-performance thin-layer chromatography (HPTLC) method for the simultaneous quantitative determination of gallic acid, vanillic acid, protocatechuic acid, and quercetin in methanolic fractions of Limonia acidissima L. fruits was developed for the first time for this species. Methanol was found to be the best for the highest possible recovery of the target analytes. For achieving good separation, a mobile phase of toluene–ethyl acetate–formic acid (5:4:1 v/v) was used. The densitometric determination was carried out at 310 and 254 nm in reflection–absorption mode. The calibration curves were linear in the range of 100–600 ng per spot for gallic acid, vanillic acid, protocatechuic acid, and quercetin. The methanolic fractions of L. acidissima L. fruits showed the presence of gallic acid (0.07%), vanillic acid (0.16%), protocatechuic acid (0.06%), and quercetin (0.14%). The proposed method is simple, precise, specific, and accurate. The statistical analysis of the data obtained proves that the method is reproducible and selective and can be used for the routine analysis of the reported phenolic compounds in crude drug and extracts. The simultaneous quantification of these compounds has not been reported yet in L. acidissima L., which may be utilized for the proper standardization of the drug.

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Authors: Ulka K. Kulkarni, Krishna V. Kulkarni, Rajendra K. Pardeshi and Dhananjay V. Mane
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Authors: Pushpendra Kumar Shukla, Ankita Misra, Manish Kumar, Soundararajan Rajan, Pawan Kumar Agrawal, Ajay Kumar Singh Rawat and Sharad Srivastava

Plant metabolite varies with season and geographic conditions. The present study is aimed at the identification of the potential chemotypes of Coleus forskohlii, available in the natural habitat of Nilgiri hills and adjoining area, in order to provide a basic lead for the industry concerning commercial exploitability, including the location-specific commercial cultivation of the plant. The effect of intra-specific variability in the forskolin content among the populations was estimated using high-performance thin-layer chromatography (HPTLC)—densitometric method. The roots of fourteen naturally occurring populations from the entire hill range were collected, covering the wide topography from foot hills up to the highest peak. The method developed for the quantification of forskolin was validated and found to be linear, specific, and accurate with precision and accuracy. The limit of detection (LOD) and limit of quantification (LOQ) were 1.04 and 3.16 ng spot−1. Precision studies (both inter-day and intra-day) were within the standard limit of relative standard deviation (RSD) (%) less than 3%. The quantification of forskolin within the population revealed that it varied from 0.0046 ± 0.0005 (NBC-36) to 1.156 ± 0.003% (NBC-46). The analysis of variance (ANOVA) suggested that there are significant differences in forskolin content among the populations. A positive correlation (Karl Pearson) was found between the altitude and the forskolin content. The cluster analysis of the population on forskolin content suspected the presence of two chemotypes. The study suggests the presence of chemotaxonomic variation among the populations which can be due to the change in phytogeographical factors.

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The insecticides are broadly divided into organophosphates, organochlorines, carbamates, and pyrethroids. Various chromogenic spray reagents have been reported for the identification of these classes of pesticides and insecticides. Some of them are specific and some are general for a particular class of insecticides. The large volume of insecticides belong still to the organophosphates which can be divided into thio and non-thio insecticides, on the basis of the presence or absence of a sulfur atom. We here present alkaline sodium nitroprusside as a simple chromogenic spray reagent which reacts with only a specific type of organothiophosphorus insecticides and not all organothiophosphorus insecticides.

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An optimized method for the extraction and quantification of artemisinin using high-performance thin-layer chromatography (HPTLC) from dried Artemisia annua L. leaf was established. Seven solvents with different polarities were used for the efficient extraction of artemisinin by hot Soxhlet as well as by conventional soaking method at room temperature. Among these solvents, n-hexane was found to be the superior solvent for both extraction methods. The chromatographic separation was carried out on precoated silica gel plate G60 F254 using n-hexane-ethyl acetate-acetic acid (2:1:0.1) as the mobile phase, and densitometric analysis was carried out in absorbance mode at 540 nm after derivatization with anisaldehyde spraying reagent. The HPTLC method was validated for specificity as well as for recovery. The optimized mobile phase gave well-defined peaks of artemisinin at R f value of 0.43 ± 0.006. The linear regression analysis of the calibration plots using standard artemisinin exhibited good linear relationship with regression coefficient (R 2) of 0.977 within the range of 200–1000 μg spot−1. The method was validated for specificity by overlaying UV spectra and sensitivity with determination of the limit of detection (30 ng spot−1) and limit of quantitation (80 ng spot−1) values. Accuracy was demonstrated by the recovery studies with average percentage recovery of over 80%. Intra-day and inter-day precisions were found to be 0.606 and 0.667 % RSD. The proposed HPTLC method can be applied for robust identification and quantitative determination of artemisinin.

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Authors: Urszula Hubicka and Bernd Spangenberg
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Inula cappa (family Compositae) is used in the Ayurvedic medicinal system for the treatment of bronchitis, diabetes, fever, hypertension, and rheumatism. The proposed high-performance thin-layer chromatography (HPTLC) study offers coherent evaluation of isoalantolactone, germacranolide, β-sitosterol, and lupeol from I. cappa root. Methanolic solutions of isoalantolactone, germacranolide, β-sitosterol, and lupeol were applied on an HPTLC plate and they were scanned at 525 nm. The mobile phase toluene—methanol (9.4:0.6, v/v) was used for all the phytochemicals. After development, all the plates were air-dried at room temperature, derivatized with anisaldehyde–sulfuric acid reagent and heated at 105°C. This study aids the identification of these compounds and provides an easy and simple method for the simultaneous estimation of these markers in the I. cappa roots. The method would serve as an expedient tool in routine analyses to corroborate the drug through good constancy.

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This work represents the validation of a stability-indicating thin-layer chromatographic technique for the simultaneous estimation of metolazone (METO) and spironolactone (SPIRO) from marketed formulation (tablets). Thin-layer chromatography was performed using precoated silica gel plate 60 F254 using ethyl acetate—chloroform—GAA (5:5:0.1 v/v) as the mobile phase for the separation of METO and SPIRO. The stability study forms an integral part of the formulation development process, and its use is also encouraged by various guidelines. Stress study was performed on active pharmaceutical ingredients (APIs) as well as on formulation for establishing a stability-indicating thin-layer chromatographic method for both drugs. The APIs were subjected to change under various environmental conditions such as pH, temperature, oxidation, etc. to determine their effect on the stability of drugs. The developed method was able to resolve drugs and their degradation products formed under the aforementioned conditions. The wavelength selected for quantitation was 238 nm. The method was validated as per the International Conference on Harmonization (ICH) guidelines and found to be linear in the range of 50–300 ng spot−1 for METO and 200–1200 ng spot−1 for SPIRO. The relative standard deviation (% RSD) values of the precision study were <2% which indicated that the developed method was precise; recovery was found to be 99.02–100.58% and 99.26–100.17% for METO and SPIRO, respectively. It could be concluded from the stability study that METO was prone to acidic hydrolysis and photolysis while SPIRO was prone to alkaline degradation.

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Enantioseparation of (RS)-etodolac was achieved by an indirect approach. Extraction of the drug (RS)-etodolac (Etd) was done from commercially available tablets, as its racemic mixture; it was purified, characterized, and was used for enantioseparation studies. Diastereomeric amides of (RS)-Etd were synthesized using a commercial sample of enantiomerically pure (R)-(−)-1-cyclohexylethylamine. Derivatization reactions were carried out under conditions of stirring at room temperature (30°C for 2 h) for (RS)-Etd and under microwave irradiation (MWI). The derivatives (as diastereomeric amides) were separated by thin-layer chromatography (TLC). The successful solvent system for separating the diastereomeric amides of (RS)-Etd was acetonitrile—methanol—dichloromethane—water (6:1:1:0.5, v/v). Spots were located by use of iodine vapor. The diastereomeric amides were recovered by preparative method; these were purified and characterized by recording their m.p., λ max (ultraviolet [UV]), infrared (IR) spectra, specific rotation, and proton nuclear magnetic resonance (1H-NMR) spectra for the verification of the synthesis and for the separation of diastereomeric amides.

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Dispersive liquid–liquid microextraction (DLLME) coupled with gas chromatography equipped with flame photometric detection (GC—FPD) is introduced to extract and determine the fifteen organophosphorus pesticides (OPPs) in hawthorn (Crataegus pinnatifida var. major) juice samples. Some parameters affecting the DLLME efficiency, such as the type and volume of extraction and dispersive solvents, extraction time, and salt concentration, were studied and optimized. The optimized extraction and dispersive solvents are trichloroethane and acetonitrile, respectively. Good linearity was ranged from 0.5 to 100 ng mL−1 with correlation coefficients from 0.9991 to 0.9999. The limits of quantification (LOQs) varied from 0.15 to 0.3 ng mL−1, and the limits of detections (LODs) ranged from 0.05 to 0.1 ng mL−1. The relative standard deviations (RSDs) varied from 1.0% to 2.8% (n = 6) with the relative recoveries in the range of 85.6–119.1%. The method was successfully applied in the determination of OPPs in ten commercial hawthorn juice samples. The chlorpyrifos was detected in one sample.

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Authors: Antimo Gioiello, Valentina Mancino, Paolo Filipponi, Serena Mostarda and Bruno Cerra

The integration of flow systems with statistical design of experiments is emerging as a valuable strategy to develop new synthetic routes towards relevant building blocks, chemical probes, and drug compounds. Optimization by experimental design incorporates statistical algorithms, mathematical models and equations, predicting tools, feedback control, and validation to generate new optimal conditions. Continuous-flow chemistry is ideally suited for this scope, as the integration of in-line analysis is simple; experimental parameters such as temperature, pressure, and flow rate can be easily controlled and fine-regulated; and automation of reaction screening can be accomplished with software assistance. This review article aims to illustrate how the combination of flow synthesizers and design of experiments can be profitable to speed up the development and optimization of more efficient, safer, and reproducible protocols for modern synthetic methods and manufacturing processes.

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An integrated process including continuous-flow syntheses directly coupled to product isolation via continuous crystallization is presented. For the synthesis part, Ce0.495Sn0.495Pd0.01O2—δ was used as heterogeneous catalyst in a custom-made packed-bed reactor (the so-called “Plug and Play” reactor) for continuous Suzuki—Miyaura crosscouplings of various para- and ortho-substituted bromoarenes with phenylboronic acid using environmentally friendly aqueous ethanolic mixtures as reaction solvents. The reactions were stable for up to 30 h without any detectable catalyst deactivation. The desired biaryl products were obtained in gram scale with good to excellent yields and high selectivity. For three methyl-, ketyl-, and nitrile-functionalized biphenyl products, isolation was done using water as antisolvent in an integrated crystallization process as continuous downstream protocol. The desired products could be isolated with high purity and with yields of up to 95% for the overall process.

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Continuous-flow generation of α-diazosulfoxides results in a two- to three-fold increase in yields and decreased reaction times compared to standard batch synthesis methods. These high yielding reactions are enabled by flowing through a bed of polystyrene-supported base (PS-DBU or PS-NMe2) with highly controlled residence times. This engineered solution allows the α-diazosulfoxides to be rapidly synthesized while limiting exposure of the products to basic reaction conditions, which have been found to cause rapid decomposition. In addition to improved yields, this work has the added advantage of ease of processing, increased safety profile, and scale-up potential.

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Prions of the baker’s yeast Saccharomyces cerevisiae allow for the inheritance of complex traits based solely on the acquisition of cytoplasmic protein aggregates and confer distinctive phenotypes to the cells which harbor them, creating heterogeneity within an otherwise clonal cell population. These phenotypes typically arise from a loss-of-function of the prion-forming protein that is unable to perform its normal cellular function( s) while sequestered in prion amyloid aggregates, but the specific biochemical consequences of prion infection are poorly understood. To begin to address this issue, we initiated a direct investigation into the potential control that yeast prions exert over fungal lipid content by utilizing the prions [URE3] and [PSI +], the first two prions discovered in yeast. We utilized silica gel high-performance thin-layer chromatography (HPTLC)—densitometry to conduct pair-wise quantifications of the relative levels of free sterols, free fatty acids, and triacylglycerols [petroleum ether—diethyl ether—acetic acid (80:20:1) mobile phase, phosphomolybdic acid (PMA) detection reagent]; steryl esters and squalene (hexane—petroleum ether—diethyl ether—acetic acid (50:20;5:1), PMA]; and phosphatidylethanolamine, phosphatidylcholine, and phosphatidylinositol (chloroform– diethyl ether—acetic acid (65:25:4.5), cupric sulfate—phosphoric acid) in otherwise clonal prion-infected ([PSI +] or [URE3]) and prion-free ([psi ] or [ure-o]) cells in two growth phases: log-phase and stationary phase. Our analysis revealed multiple statistically significant differences (p < 0.00625) between prion-infected and prion-free cells. Interestingly, prion-induced changes varied dramatically by growth phase, indicating that prions exert differential influences on cell physiology between log and stationary growth. Further experimental replication and extension of the analysis to other prions is expected to resolve additional physiological effects of prion infection. This investigation demonstrates that HPTLC—densitometry is an effective method for studying prion-induced alterations in lipid content in yeast.

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A precise and sensitive reversed phase high-performance thin-layer chromatography (RP-HPLC) method was developed for the determination of nilotinib (NTB) in spiked plasma, urine, and pharmaceutical capsule formulation. The method was based on derivatization NTB with 4-chloro-7-nitrobenzofurazan (NBD-Cl) in the borax buffer (pH 9). The method employs an isocratic elution using acetonitrile and 10 mM orthophosphoric acid (40:60 v/v) as a mobile phase and an C18 column (4.6 mm × 250 mm, 5 μm, Waters Symmetry), with a fluorescence detector (λ ex: 447 nm, λ em: 530 nm). The method validation was performed with respect to linearity, recovery, accuracy, precision, and stability. The linear ranges were 100–600 ng mL−1 in standard solution, plasma, and urine. Correlation coefficients (r 2) were higher than 0.9997 for all of the analytes, indicating good linear relationship. The percentage recovery was 87.89% for plasma, 95.35% for urine, and 96.07% for capsules.

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This paper develops an instrumental analytical approach for detection of fourteen polycyclic aromatic hydrocarbons (PAHs) in edible oil samples using gel permeation chromatography (GPC) and ultra-high performance liquid chromatography (UHPLC) coupled with diode array detector (DAD), and fluorescence detector (FLD). The GPC was used to remove triglycerides from edible oil samples. The extracted samples were then detected using UHPLC—DAD—FLD. In order to obtain good separation and high reproducibility, the UHPLC—DAD—FLD experimental condition was optimized. The PAHs including three groups of isomeric PAHs can be separated completely in 12 min using BEH Shield RP 18 column with a suitable gradient elution program. The mean recoveries were in the range of 73–110% with an acceptable reproducibility (RSD < 10%, n = 3). During real sample analysis, the method can decrease the chance of false positives with both DAD and FLD being used simultaneously. The results indicate that the approach is simple, easy, and acceptably reproducible, thereby showing great potential as a method for detection of fourteen PAHs contained in edible oil samples.

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Authors: Effat Souri, Siavash Mottaghi, Mohammad Zargarpoor, Reza Ahmadkhaniha and Hassan Jalalizadeh

Rivaroxaban is an inhibitor of factor Xa, which is used as an oral anticoagulant for the prevention of thromboembolism. The objective of this study was to develop a stability-indicating high-performance liquid chromatographic method for the quantitative determination of rivaroxaban in pharmaceutical dosage forms. Rivaroxaban was subjected to acidic, basic, oxidative, photolytic, and thermal conditions for forced stress degradation studies. Considerable degradation was observed in all stress degradation tests. Rivaroxaban and its degradation products were separated on a Nova-Pak C8 column utilizing a mixture of acetonitrile and KH2PO4 50 mM (pH 3.0) (40:60, v/v) as the mobile phase, and the chromatogram was recorded at 270 nm using a general ultraviolet (UV) detector.

The developed method was linear over the concentration range of 1–50 μg mL−1 showing acceptable within-day and between-day precision and accuracy values (CV <2% and Error <2%). The dissolution profile of rivaroxaban tablets was also studied in the presence of a surfactant using optimized conditions. The validated method was successfully used for the determination of rivaroxaban in dosage forms and also in dissolution medium indicating the specificity of the assay method.

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Authors: Robert A. Green, Richard C. D. Brown and Derek Pletcher

In recent papers, laboratory microfluidic electrolysis cells with extended channel lengths (0.7–2 m) and narrow interelectrode gap (≤0.5 mm) have been introduced; these cells permit high conversions at a flow rate consistent with the synthesis of products at a rate of multigrams/hour. Such microflow electrolysis cells must be operated with appropriate control parameters if good performance is to be achieved; thus, this paper emphasizes the correct selection of cell current, flow rate, and counter electrode chemistry. It is also shown that, within the limitations, the cells can be used for a number of electrosyntheses in the synthetic laboratory.

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Authors: Caroline Bosch, Pablo López-Lledó, Josep Bonjoch, Ben Bradshaw, Pieter J. Nieuwland, Daniel Blanco-Ania and Floris P. J. T. Rutjes

An Amberlite IR 120 H-promoted one-pot Fischer indolization from a cis-decahydroquinoline using a range of phenylhydrazines led to compounds with the pyrido[2,3-a]carbazole scaffold. The process may be conducted either in batch mode or in a continuous manner in a flow reactor. The stereochemical course of the Fischer indole reaction changed in going from using free phenylhydrazine to the corresponding hydrochloride in batch conditions, whereas, with the short reaction times in continuous flow, no changes due to isomerization processes were observed.

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Authors: Leonardo Degennaro, Claudia Carlucci, Sonia De Angelis and Renzo Luisi

In this review recent examples on the use of flow technology for organometallic-mediated synthesis have been collected. The review focused on synthetic relevant processes and on flow techniques developed for handling reactive intermediates, and conduct synthetic steps difficult to perform using traditional "batch" chemistry.

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Authors: Sergio Rossi, Alessandra Puglisi, Daniela Intrieri and Emma Gallo

The Sandmeyer reaction of anilines to generate aryl azides, followed by the Ru(porphyrin)CO-catalyzed addition to styrenes affording N-aryl aziridines was successfully performed for the first time in mesoreactors, under continuousflow conditions. Mesofluidic technology allowed for a rapid screening of different parameters and a quick identification of the optimized reaction conditions for the two separate steps. The two optimized reactions were then combined in a single continuous process that allowed a safe and efficient synthesis of N-arylaziridines from convenient commercially available starting materials.

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In this Section of the journal, the literature on continuous flow synthesis (primarily organic synthesis and functional materials) from the period of April — June 2016 is presented. All the publications are listed ordered by journal name, with a Review article appearing at the end. In this quarter the number of papers on continuous flow organic synthesis is relatively less as a few special issues are forthcoming. It is interesting to observe that the number of papers on continuous flow oxidation are more than ever before!

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A simple and sensitive liquid chromatography—tandem mass spectrometry method was developed for the quantification of atorvastatin, ortho-hydroxyatorvastatin, para-hydroxyatorvastatin, and atorvastatin lactone in rat plasma. Solid-phase extraction was used for preparation of samples. Rosuvastatin was chosen as an internal standard. Chromatographic separation was achieved on ZORBAX Eclipse C18 Analytical, 4.6 × 100 mm (3.5 μm) column with a gradient mobile phase composed of acetonitrile and 0.1% acetic acid, at a flow rate of 400 μL min−1. The column was kept at constant temperature (25 °C), and autosampler tray temperature was set at 4 °C. The following selected reaction monitoring (SRM) transitions were selected: (m/z, Q1 → Q3, collision energy) atorvastatin (559.47 → 440.03, 22 eV), atorvastatin lactone (541.36 → 448.02, 19 eV), ortho-ohydroxyatorvastatin (575.20 → 440.18, 20 eV), para-hydroxyatorvastatin (575.54 → 440.18, 20 eV), and rosuvastatin (482.25 with selected combination of two fragments 257.77, 31 eV, and 299.81, 35 eV) in positive ion mode. The method was validated over a concentration range of 0.5–20 ng mL−1 for ortho-hydroxyatorvastatin and para-hydroxyatorvastatin and 0.1–20 ng mL−1 for atorvastatin and atorvastatin lactone with excellent linearity (r 2 ≥ 0.99). This method demonstrated acceptable precision and accuracy at four quality control concentration levels. The detection limits were 0.1 and 0.13 ng mL−1 for orth-ohydroxyatorvastatin and para-hydroxyatorvastatin, respectively, and 0.05 ng mL−1 for atorvastatin and atorvastatin lactone. All analytes were found to be stable at examined conditions. Validated method was applied for determination of atorvastatin and its metabolites in plasma of experimental animals.

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Authors: Zine Eddine Hamami, Laurent Vanoye, Pascal Fongarland, Claude de Bellefon and Alain Favre-Reguillon

An efficient and metal-free method for the oxidation of aldehydes to the corresponding carboxylic acids has been developed. In a simple continuous-flow photochemical reactor, the use of camphorquinone (CQ) irradiated with a white light-emitting diode (LED) source enhanced the autoxidation of aldehydes. Under 5 bar of oxygen, visible light, and 0.3 mol% of CQ, the rate of oxidation was increased from 6 times with 2-ethylhexanal to 30 times for n-nonanal. The large interfacial area generated by a segmented flow apparatus associated with radicals formed by photooxidation of CQ ensures metal-free high throughput of carboxylic acids under safe conditions.

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Authors: Eric Mielke, Dominique M. Roberge and Arturo Macchi

Based on previous work studying complex microreactors, it was desired to further improve the mixing efficiency by varying the mixing unit design for fast liquid—liquid reactions. Different flow regimes were studied, including slug flow, parallel flow, and drop flow. The two-phase hydrolysis of 4-nitrophenyl acetate in sodium hydroxide solution was used to evaluate the overall volumetric mass transfer coefficients (K org a) as a function of the average rate of energy dissipation (ε) for each microreactor design and all flow regimes. The liquid—liquid systems investigated used n-butanol or toluene as the organic phase solvent and a 0.5-M NaOH aqueous solution. The use of surfactant was also investigated with the toluene—water system. All microreactor geometry designs were based on contraction—expansion repeating units with asymmetric obstacles to aid the breakup of slugs and desynchronize the recombination of split streams. The investigated designs were chosen to avoid the formation of the parallel flow regime, contrary to curvature-based mixing-unit designs. The microreactor design can then be optimized to reduce the ε required to reach drop flow, since K org a has been found to be constant at equal ε for a given solvent system in this flow regime, regardless of the reactor selection. Additionally, the “3/7th” scaleup rule was applied and confirmed with the LL-Triangle mixer. It was found that, for low interfacial-tension systems (i.e., n-butanol—water), the onset of drop flow occurred at a lower ε for the LL-Triangle mixer when compared with the Sickle or LL-Rhombus mixers.

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Authors: Silvia Garbarino, Javier Guerra, Peter Poechlauer, Bernhard Gutmann and C. Oliver Kappe

The crucial structural motive in viral protease inhibitors such as atazanavir and darunavir is a chiral aminoalcohol structure. The structure is generally introduced during the synthesis of the protease inhibitor via an α-chloroketone intermediate. The α-chloroketone can be synthesized in a multistep sequence from naturally occurring l-phenylalanine. Herein, we report a onepot synthesis of an α-chloroketone starting from N-Boc-l-phenylalanine in a novel type of “tube-in-flask” semi-batch diazomethane generator. Activation of the amino acid to the mixed anhydride was carried out in the flask, while diazomethane was generated from in situ formed N-nitroso-N-methylurea within a gas-permeable tubing contained inside the flask. The diazomethane diffused through the gas-selective membrane into the flask, and reacted with the anhydride to the diazoketone (Arndt—Eistert reaction). The addition of aqueous hydrogen chloride provided the α-chloroketone and destroyed any excess of diazomethane. The desired product was isolated by extraction in excellent purity and yield (90%–96%).

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We converted diverse commercial meta-substituted phenols to the allyl-substituted precursors via nucleophilic substitution using batch technology to allow processing these in microflow by means of the photo-Claisen rearrangement. The latter process is researched on its own, as detailed below, and also prepares the ground for a fully continuous two-step microflow synthesis, as outlined above. It is known that batch processing of electronically deactivated phenols (e.g., bearing a cyano or nitro group) has several orders of magnitude lower reactivity than their parental counterparts [1]. Thus, we here explore if the high quantum yield of microflow, yet at very short residence time, is sufficient to activate the deactivated molecules. In addition, the realization of a true orthogonal two-step flow synthesis can open the door to a large synthetic scope of our approach and possibly overcome limitations due to missing orthogonality of our previously reported thermal approach of combined nucleophilic substitution-Claisen rearrangement in microflow. Consequently, we make for our photo microflow approach an orthogonality check, as previously reported for the thermal approach, and compare both.

To get a broader picture, we have investigated some major parametric sensitivities such as the irradiation intensity, the choice of solvent, the reactant concentration, and, most notably, the influence of the substitution pattern. The irradiation intensity was varied by increasing distance between a lamp and the microflow capillary. In addition, the normal photo-Claisen microflow process (at room temperature) is compared to a high-temperature photo-Claisen microflow process, to check the potential of such novel process window [2]. This is difficult to realize in batch, as the combination of strong ultraviolet (UV) irradiation and high temperature causes a high hazard potential. Yet, under microflow, this can be safely handled.

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The synthetic scope of photo-induced copper-mediated polymerization (photoCMP) in continuous-flow reactors is further explored. A series of monomers, namely, methyl (MA), ethyl (EA), n-butyl (nBA), 2-hydroxyethyl (HEA), and di(ethylene glycol) ethyl ether (DEGA) acrylate are investigated, all showing high livingness (dispersity in the range of 1.1 and linear first order kinetics) in the polymerizations and high conversions within 20-min reaction time. Next to the commonly used solvent (dimethyl sulfoxide [DMSO]), also a water—ethanol mixture was used as greener alternative, without any loss in reaction control. Upscaling the reactor from 2 to 16 mL allows for production of over 200 g of high-definition material (3000 g/mol, 1.1 dispersity) in overnight operation (18 h), demonstrating that the photoprocess can be run under very stable conditions even for extended reaction times. Via coupling of two reactors, direct chain extension of copolymers in a single reaction step is also demonstrated.

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Authors: A. Ochoa, J. Fechine, J.C. Escalona, J. García, S.G. dos Santos and M. Sobral da Silva

Excoecaria lucida Sw. is an evergreen shrub widely distributed in Cuba and throughout the Caribbean region. In spite of its extended traditional use as antiasthmatic and antimicrobial by the local population, scientific reports on the species are almost nonexistent. This paper focuses on the isolation and characterization of compounds present in the crude extract of E. lucida Sw. leaves through the combined use of chromatographic and spectroscopic techniques (medium pressure liquid chromatography, thin-layer chromatography, gas chromatography, nuclear magnetic resonance 1H, and mass spectrometry). A total of 15 nonpolar substances were identified in the four main fractions obtained; some of these substances could be related with the antimicrobial properties attributed to the species.

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Authors: Christiane Schotten, Abdul Hadi Aldmairi, Yerbol Sagatov, Martyn Shepherd and Duncan L. Browne

Herein, we report a continuous-flow process for the preparation of triazenes, whereby diazonium salts are generated and converted into their masked or protected triazene derivatives. Key to realizing the process, which is applicable to a wide range of substrates, is the identification of solvent and reagent parameters that avoid fouling and clogging in the tubing used in these studies. The process has also been applied to prepare the antineoplastic agents mitozolomide and dacarbazine. We also report isolation and differential scanning calorimetry (DSC) analysis of an anthranilic acidderived triazene whose related diazonium salt is a contact explosive. The data highlights improved stability but also suggests that an exothermic process does occur with an onset temperature of 118 °C. Finally, an 18-hour continuous operation of the reaction procedure using high-performance liquid chromatography (HPLC) pumps is reported.

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An accurate and rapid liquid chromatography–electrospray ionizaion– tandem mass spectrometry (LC—ESI—MS/MS) analytical method was developed and validated for the simultaneous determination of antcins A, B, C, H, and K, dehydroeburicoic acid, and 4,7-dimethoxy-5-methyl-1,3-benzodioxole in the extract and capsule of Antrodia cinnamomea (AC) fruiting body. These seven signature compounds were ionized using an electrospray ion source and analyzed by a triple-quadrupole mass analyzer under a multiple reaction monitoring (MRM) mode. The MRM transitions of m/z 453/409 (antcin A), m/z 467/408 (antcin B), m/z 469/425 (antcin C), m/z 485/413 (antcin H), m/z 487/407 (antcin K), m/z 467/337 (dehydroeburicoic acid), and m/z 197/139 (4,7-dimethoxy-5-methyl-1,3-benzodioxole) were used to quantify these seven components, respectively. Their calibration curves presented good linear regressions (R 2 > 0.997) within the tested concentration range. The intra- and inter-day precisions were less than 1.97% and 2.53%, respectively. The overall recovery was in the range of 87.55%–95.41%. This validated high-performance liquid chromatography (HPLC)—MS/MS method offers promising applications for the accurate and rapid quantification of signature compounds in the fruiting body and its commercial products.

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Using a simple flow reactor, the safe use of nitromethane at elevated reaction temperatures was demonstrated in a nitroaldol reaction of different aldehydes. The reaction products were isolated in good yields after a short reaction time.

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Incidence of suicidal attempts presents an explanation for the high prevalence of quetiapine (QTI) in forensic cases. Thus, the interpretation of its concentrations in biological specimens is needed, but in forensic toxicology, potential postmortem changes such as instability of the target drugs should be taken in consideration. High-performance liquid chromatography (HPLC) method has been developed for determination of QTI. This method was based on reversed phase (RP)-HPLC separation of QTI on a C-18 column (150 mm × 4.6 mm, 5 μm) with elution system of acetonitrile—methanol—0.025 M phosphate buffer (pH 2.5), containing 1 mL TEA in each 250 mL, in a ratio of 40:30:30%, v/v, at the flow rate of 1.2 mL min−1 using mirtazapine as internal standard (IS). The proposed method was applied to the determination of QTI in plasma in presence of coadministered drugs. The application of the proposed method was extended for long-term stability study of two different concentration levels of QTI in the whole blood.

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Authors: Lukas Hohmann, Safa Kutup Kurt, Sebastian Soboll and Norbert Kockmann

For complete chemical processes, downstream operation steps are essential, but on a miniaturized scale, they are not so far developed as the microreactors. This contribution presents three different unit operations for phase and component separation. Liquid—liquid extraction is often performed in columns, which were miniaturized for higher separation efficiency and flow rates suitable for processes in flow chemistry. Two-phase mass transfer processes in capillaries benefit from rapid final phase separation, which can be performed in an in-line phase splitter based on different surface wetting behavior. Crystallization is often a final purification step, which is performed in a continuously operated helical tube setup with narrow residence time distribution. For all unit operations, design criteria are shown with typical applications. The methodology of downscaling of known equipment and employing typical microscale phenomena such as good flow control, laminar flow, or dominant surface forces leads to successful equipment design.

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A new and simple method based on high-performance liquid chromatography with ultraviolet detection (HPLC-UV) for the determination of cysteine (Cys) and cysteinylglycine (CysGly) in plasma and urine has been developed. The method involves reduction of disulfide bonds with tris(2-carboxyethyl)phosphine, derivatization of the analytes with 2-chloro-1-methylquinolinium tetrafluoroborate, and separation on Aeris PEPTIDE XB-C18 column (150 mm × 4.6 mm, 3.6 μm, Phenomenex) with UV detection at 355 nm. The calibration lines, obtained with human plasma and urine spiked with Cys- Gly and Cys, were linear in the range of 2.5–50 μmol L−1 and 20–300 μmol L−1, respectively. The intra- and inter-day precision values of the method, expressed as a relative standard deviation, were 0.25–11.1% and 0.71–12.3%, respectively. The analytical recovery varied from 89.7 to 112.3%. The LOQs for total Cys and CysGly were 1.5 pmol and 2.3 pmol in peak, respectively. The method was successfully applied to samples donated by apparently healthy individuals. Concentrations of Cys and CysGly in human plasma from 18 subjects varied from 141.6 to 217.8 μmol L−1 and from 21.1 to 50.9 μmol L−1, respectively. Their concentrations in urine samples (n = 14) ranged from 137.3 to 426.8 μmol L−1 and from 1.6 to 4.9 μmol L−1, respectively.

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A simple and convenient chromatographic method of simultaneous separation, identification, and quantitative determination of thimerosal (TM) (preservative) and aluminum (adjuvant) in vaccines and pharmaceuticals by reversed phase highperformance liquid chromatography (RP-HPLC) with visible (VIS) detection was developed and validated. Due to postcolumn derivatization with dithizone, any interference from matrix was excluded. Similarly, a possibility of on-column decomposition of dithizonates was eliminated. Evaluated detection limits were 0.3 μg TM and 3.0 μg Al, which correspond to the smallest, but possible to recognize, visible peak.

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Authors: Razwan Baber, Luca Mazzei, Nguyen T.K. Thanh and Asterios Gavriilidis

Synthesis of silver nanoparticles (NPs) in an impinging jet reactor (IJR) was investigated due to its unique properties of efficient mixing and lack of channel walls which avoid fouling. Silver NPs were formed at room temperature by reducing silver nitrate with sodium borohydride in the presence of sodium hydroxide. Two types of ligand were used to stabilize the NPs, trisodium citrate, and polyvinyl alcohol (PVA). Weber number, the ratio between inertial forces and surface tension forces, is used to characterize flow in impinging jets. Flow regimes were investigated forWeber numbers in the range of 13–176. A liquid sheet/chain regime was identified at lowerWeber numbers (<90), and an unstable rim structure was identified at higherWeber numbers (>90). Mixing time was found to be in the range 1–7ms, using theVillermaux—Dushman reaction system and interaction by exchange with the mean mixing (IEM) model. Fastest mixing occurred at Weber number ca. 90. Using trisodium citrate as a ligand, NP size decreased from 7.9 ± 5.8 nm to 3.4 ± 1.4 nm when flow rate was increased from 32 mL/min to 72 mL/min using 0.5 mm jets, and from 6.4 ± 3.4 nm to 5.1 ± 4.6 nm when flow rate was increased from 20 mL/min to 32 mL/min using 0.25 mm jets. Using PVA as a ligand, NP size decreased from 5.4 ± 1.6 nm to 4.2 ± 1.1 nm using 0.5 mm jets and stayed relatively constant between 4.3 ± 1 nm and 4.7 ± 1.3 nm using 0.25 mm jets. In general, the size of the NPs decreased when mixing was faster.

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Authors: Laurent Pellegatti, Andreas Hafner and Jörg Sedelmeier

This work describes the manufacturing of ribociclib following the concept of an end-to-end continuous-flow process. The active pharmaceutical ingredient (API) is produced in a two-step telescoped flow process with integrated in-line liquid—liquid extraction and semibatch crystallization.

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Transfer of four thin-layer chromatography (TLC) Global Pharma Health Fund Minilab mobile kit protocols for detecting fake pharmaceutical products to quantitative high-performance TLC (HPTLC)—densitometry methods was carried out using a model process published earlier. The developed and validated methods were for the drugs quinine sulfate, mefloquine, dihydroartemisinin, and piperaquine phosphate. EMD Millipore Premium Purity silica gel 60 F254 glass plates, automated standard and sample solution application with a CAMAG Linomat 4, and automated densitometry with a CAMAG Scanner 3 for detection, identification, and quantification were used. Sample peak identity and purity validation were carried out by spectral comparison checks available in the winCATS software, and accuracy was estimated by the standard addition approach. HPTLC gives better efficiency, selectivity, and resolution than TLC, and the new methods overcome the deficiencies in technology related to manual application and visual zone comparison that do not allow the Minilab TLC procedures to support regulatory compliance actions. These new methods should be fully validated according to International Conference on Harmonization guidelines or by interlaboratory studies if required by their applications. In addition, a previously reported transferred simultaneous HPTLC–densitometry method for lumefantrine and artemether was used to analyze a new combination tablet to demonstrate its applicability.

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In this study, a number of computed or chromatographically measured (RP-18 thin-layer chromatography [TLC]) descriptors are presented. The relationships between these descriptors and the observed (BBobs) and calculated (B2) BBB bioavailability were studied by stepwise multiple regression analysis and discriminant function analysis on a group of 34 compounds of diverse structures. Useful models of the blood—brain distribution given by the equations: BBobs = −1.19 + 2.05 B2 + 3.89 R F − 62.31 R F/PSA + 0.290 log D (R 2 = 0.85, n = 24) and B2 = 4.06 − 1.61 HA − 1.95 HD + 105.49 R F/PSA (R 2 = 0.95, n = 34) were developed and validated. Models for discrimination between CNS+ and CNS− compounds were built on the basis of R F, R F/PSA, HD, and B2 descriptors. The diagnostic power of important parameters was evaluated by cluster analysis. Thirty-four compounds examined throughout this study were successfully assigned to two clusters: CNS+ and CNS−. Analysis of variances for 6 descriptors (HD, HA, R F, R F/PSA, DM, and B2) confirmed the conclusion that the parameters of good differentiating power are B2, HA, HD, R F/PSA ,and R F. The results of the chromatographic analysis proposed in this study (RP-18 TLC) are a source of valuable information on the ability of compounds to cross the BBB. This simple, inexpensive, and very rapid chromatographic technique may be used to assess the BBB permeability of compounds isolated or synthetized on a very small scale. The computed B2 descriptor is a convenient and readily available parameter useful also in the case of theoretical structures.

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A thin-layer chromatographic (TLC) procedure combined with digital image processing analysis was developed for quantitative time-monitoring of the radical- scavenging activity. Reaction of 2,2-diphenyl-1-picrylhydrazyl (DPPH•) with reference antioxidants and selected catecholamine compounds was monitored on RP-18 chromatographic plates using dot-blot technique and different concentrations of standards. Images of the chromatographic plate were recorded after background staining every 5 min for 1 h using a TLC scanner device. ImageDecipher TLC software program was used to convert image data into chromatograms, from which spot area as a function of reaction time was easily monitored for different amounts of antioxidant in all cases. For a comparative evaluation of radical-scavenging activity, all the time-acquired images were documented as a new red, green, and blue (RGB) image after conversion to different color values. Based on the chromatogram digitization results, TLC—time-monitored radical-scavenging profiles of the investigated compounds were obtained. By the developed methodology, different image processing and image analysis procedures were proposed and originally applied for the first time in TLC—time-monitoring of radical-scavenging activity evaluation.

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Ultraviolet—visible (UV—vis) spectrometry, electron paramagnetic resonance spectroscopy, high-performance liquid chromatography, and thin-layer chromatography were used for the evaluation of the antioxidant activity of natural samples. All the methods used free radicals to determine antioxidant capacity and standard antioxidant compounds, usually Trolox or vitamin C, to express the results. In this study, 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) methods for the determination of the antioxidant activity of some wines previously separated by high-performance thin-layer chromatography (HPTLC) are statistically compared. The antioxidant activities were quantified based on the calibration curves obtained after elution under the same chromatographic conditions of different quantities of Trolox. Also, the reaction time was established to be between 10 and 30 min. The results show that the values found for DPPH and ABTS assays are well correlated, the values obtained in the case of ABTS being higher. The ABTS method is more sensitive than DPPH assay and is more useful than DPPH assay in the determination of the antioxidant activity of natural samples. In the case of real samples tested, the antioxidant activities decrease in the order red wines, rose wines, and white wines.

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polarities was investigated by thin-layer chromatography (TLC)—densitometry in the presence and absence of selected metal ions. It was shown that both solvents and ion type have an effect on the degradation process that leads to the generation of some new products with R F values, which are different from R F values for active substance. It was found that acetone and methanol solutions are sensitive to ultraviolet-C (UV-C) radiation as well as acetone—water and methanol—water solutions for cefepime hydrochloride and cefuroxime axetil. The photostability of cefuroxime axetil after UV-C radiation in acetone solution has the strongest effect on photodegradation compared with the photodegradation carried out in methanol. The studied ions enhanced the degradation of cephalosporins in solutions. Fe(III) and Ni(II) ions exerted the strongest effect on photodegradation of cefepime hydrochloride in acetone compared with radiation carried out in the presence of Mn(II), Cu(II), Co(II), Fe(II), and also without these ions. In methanol solutions of cefepime hydrochloride, a stronger effect on photodegradation was observed by addition of Fe(II) ions. Cefuroxime axetil was more stable in the presence of Fe(II), Fe(III), and Ni(II) in all environments than cefepime hydrochloride. Degradation profiles were additionally compared by principal component analysis to explore the main trends in their shape change.

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Authors: Małgorzata Starek, Monika Dąbrowska, Monika Bracha and Włodzimierz Opoka

A study on the effect of oxidizing or reducing agents on the stability testing of piroxicam, tenoxicam, meloxicam, and isoxicam was performed. Detection of the formed oxidation and/or degradation products after reaction with factors such as iodine, potassium manganate( VII), hydrogen peroxide, and ascorbic acid was conducted by thin-layer chromatography (TLC)—densitometry technique. The reacting mixtures were also exposed to increasing temperatures. The chromatographic profiles showed the formation of several new peaks for all oxicams due to the presence of a number of degradation products formed in the presence of analyzed redox agents. The calculated kinetic parameters have confirmed the greatest stability of meloxicam and piroxicam and the smallest stability of isoxicam in the analyzed conditions. In the case of all the analyzed drugs, principal component analysis identified temperature as the main factor responsible for the speed of degradation and the shape of degradation profile.

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Planar chromatography is commonly used for the quality control of herbal medicines due to its many advantages. Its combination with chemometrics was proven to be a fast and reliable tool for the extraction of even more analytical information, such as similarity or dissimilarity between samples, and the identification of marker compounds. To date, depending on image processing procedures, different variables have been obtained as input data, and thus, various preprocessing procedures have been applied. In this study, we converted the chromatogram images of high-performance thin-layer chromatography to form a data matrix, by digitization of the chromatograms. Further, principal component analysis was applied on raw data and investigated after different preprocessing techniques. The proposed preprocessing techniques were successfully applied to improve the differentiation between two types of German propolis. The best multivariate models were observed in the case of warping, standard normal variate, and mean centering/autoscaling.

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The experimental design and the quantitative structure—retention relationship (QSRR) study were applied in order to investigate the retention behavior and to select optimal experimental conditions for the separation of ziprasidone and its five impurities by thin-layer chromatography (TLC). According to a preliminary study, central composite face-centered design was chosen to examine the influence of four factors, i.e., the developing distance, the amount of toluene in the mobile phase, the amount of acetic acid in the mobile phase, and the spot band size, on the retention behavior of the examined compounds. The optimal separation conditions were achieved on the chromatographic plates precoated with silica gel 60 F254 using toluene—methanol—glacial acetic acid (7.5:0.5:0.5, v/v) as the mobile phase in combination with a band width of 6 mm and a developing distance of 110 mm. The retention parameters (hR F) obtained under the selected chromatographic conditions, along with the calculated molecular descriptors, were further used for the QSRR study. Statistically, the best QSRR model (R 2: 0.939, Q 2: 0.916, and RMSEE: 2.98) composed of the three significant variables, i.e., the harmonic oscillator model of aromaticity (HOMA) index, the highest occupied molecular orbital (HOMO), and the lowest unoccupied molecular orbital (LUMO) energy, was developed, using the partial least square methodology. A very good agreement was obtained between the QSRR predicted and the experimentally observed hRf values for an additional ziprasidone impurity (TS1). These results point out to a high prediction potential of the developed QSRR model for the evaluation of the retention behavior of the other ziprasidone impurities.

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Modeling properties of newly synthesized compounds is important for both understanding their activity and predicting their interactions. This paper describes the evaluation of the lipophilicity of the newly synthesized cycloalkylspiro-5-hydantoins by experimental and calculation methods. The chromatographic lipophilicity (R M 0) of the analyzed compounds was determined by reversed-phase liquid chromatographic system consisting of RP-18 stationary phase and methanol—water mobile phase. Correlation coefficients between R M 0 values and the predicted log P were above 0.93, indicating a strong linear relationship between the variables. Multivariate data analysis applied in this study enabled the comparison of the lipophilicity and the structure of the investigated compounds using solely information obtained from thin-layer chromatography. Hierarchical clustering analysis (HCA) reveals a high similarity between R M 0 and miLogP whereas squared log Ps such as ALOGP2 and MLOGP2 were distinct from R M 0. HCA classifies the investigated compounds into three clusters according to lipophilicity. Principal component analysis (PCA) yields two principal components which explained >99.19% total variance. The changes in the lipophilicity of the substituents attached in the molecule are reflected in the value of PCs. An increase in the lipophilicity of spiro substituent results in a decrease of values of both PC1 and PC2. The increase in the lipophilicity of R substituent decreases the value of PC1 and increases the value of PC2. The positive value of PC1 is typical for the compounds with the electron withdrawing groups such as CN, NO 2, or OCH 3.

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Authors: Désirée Humpert, Barbara Milz, Andreas Lotz and Bernd Spangenberg

Phenolic compounds, such as flavonoids and phenolic acids, are very important substances that occur in various medicinal plants. They show different pharmacological activities which might be useful in the therapy of many diseases. Phenolic compounds have achieved an increasing interest over the last years because these compounds are easily oxidized and, thus, act as strong antioxidants. We present the chemiluminescence of different phenolic compounds measured directly on high-performance thin-layer chromatography LiChrospher® plates using the oxalic acid derivative bis(2,4,6-trichlorophenyl) oxalate (TCPO) in conjunction with H2O2. Our results indicate that chemiluminescence intensity increases with an ascending number of phenolic groups in the molecule. The method can be used to detect phenolic compounds in beverages like coffee, tea, and wine.

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Authors: Mirosław A. Hawrył, Anna Hawrył, Ryszard Świeboda, Małgorzata Niemiec and Monika Waksmundzka-Hajnos

Seven different Scutellaria species were analyzed using the extraction procedure (Soxhlet apparatus, dichloromethane, and methanol as solvents) and thin-layer chromatography method. Selected standards of flavonoids and phenolic acids (caffeic acid, chlorogenic acid, ferulic acid, baicalein, wogonin, baicalin, chrysin, quercetin, scutellarin, hesperetin, hesperidin, apigenin, luteolin, rutin, and kaempferol) were separated using silica gel thin-layer chromatography (TLC) plates with the mobile phase consisting of ethyl acetate—toluene—formic acid (5:4.9:0.1, v/v) for dichloromethane and methanolic extracts. Dichloromethane extracts were also developed using cyanopropyl-bonded silica gel with the following mobile phases: propan-2-ol—n-heptane—formic acid (5:4.9:0.1, v/v) and methanol—water—formic acid (6:3.9:0.1, v/v), and after drying, they were sprayed using the anisaldehyde reagent. In the case of methanolic extracts, the same non-aqueous eluent was used and the aqueous eluent consisting of methanol—water—formic acid (4:5.9:0.1, v/v). The presence of selected standards in Scutellaria species was confirmed. The similarities between the obtained fingerprint chromatograms were performed using chemometric methods, the similarity coefficients (Pearson’s correlation coefficient, determination coefficient, and congruence coefficient), distance indices (Euclidean distance, Manhattan distance, and Chebyshev’s distance), and multi-scale structural similarity (MS-SSIM).

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The aim of the work was the chromatographic separation of salicylic acid and their derivatives, i.e., acetylsalicylic acid, salicylanilide, salicylaldehyde, salicylamide, methyl salicylate, phenyl salicylate, 2,5-dihydroxybenzoic acid, salicylhydroxamic acid, 3,5-dinitrosalicylic acid, 3-aminosalicylic acid, 4-aminosalicylic acid, and 5-aminosalicylic acid by use of adsorption thin-layer chromatography (normal-phase thin-layer chromatography [NPTLC]) and partition thin-layer chromatography (reversed-phase thin-layer chromatography/high-performance thin-layer chromatography [RP-TLC/HPTLC]). Three qualitatively and quantitatively different mobile phases were used for the separation of salicylic acid and its derivatives. Cluster analysis (single linkage method, Euclidean distance) allowed the evaluation of the suitability of the chromatographic conditions used to separate the pairs of tested compounds. The cluster analysis data indicate that the composition of the mobile phase is fundamental in the process of separation of the analyzed compounds by use of NP-TLC. The best separation of the studied substances was observed in the case of mobile phase n-hexane—diethyl ether—acetic acid (80%) in different volume ratios. The similarity analysis of the results obtained by use of RP-TLC/HPTLC revealed that the type of chromatographic plates influences significantly the quality of separation of the tested compounds. The best conditions for the separation by RP-TLC were obtained on silica gel RP-18 F254 plates. The present study indicates that the cluster analysis represents a simple-to-use and powerful chemometric tool in the prediction of TLC separation of medically important salicylic acid derivatives under various chromatographic conditions. It can be helpful in the quality control of multicomponent synthetic preparations containing these compounds or in the chemical standardization of plant products consisting of salicylic acid and related compounds.

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