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A rapid, inexpensive, and stereoselective densitometric—thin-layer chromatographic (TLC) method using l-(+)-tartaric acid and l-histidine—Cu complex as chiral mobile phase additive for the enantioseparation of atracurium besylate and atropine sulfate, respectively, and quantitative determination of their chiral switching (eutomer) isomers, cisatracurium besylate and hyoscyamine sulfate, were used in this study. The effect on resolution of different chiral selector concentrations, temperatures, and pH values was investigated. The spots were detected with ultraviolet (UV) lamp followed by densitometric measurements at 280 nm and 215 nm for cisatracurium besylate and hyoscyamine sulfate, respectively. The mobile phases enabling successful resolution were acetonitrile—methanol—dichloromethane—glacial acetic acid—H2O containing 70 mg l-(+)-tartaric acid (7:1:0.5:0.7:1, by volume), pH 5 for atracurium besylate, and methanol—H2O containing 40 mmol l-histidine and 20 mmol copper(II) acetate (8.8:1.2, by volume), pH 6.5 for atropine sulfate. Thermodynamic parameters, enthalpy ΔH and entropy ΔS were investigated to study the effect of temperature on the enantioseparation using the Van’t Hoff plot.

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Authors: Mohamed Abdel Tawab Korany, Marwa Said Moneeb, Dina Ahmed Selim, Aya Mohamed Asaad and Nadia Abdel Aziz El-Sebakhy
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The healthy properties of functional food are associated with the presence of a wide range of phenolic antioxidants. Thus, the aim of this study was to develop a simple, rapid, and reproducible high-performance thin-layer chromatographic (HPTLC) method combined with a direct 2,2′-diphenyl-1-picrylhydrazyl (DPPH) assay, to compare antioxidant activity in terms of free radical scavenging activity in functional food. We wanted to quantify caffeic acid, chlorogenic, and gallic acid, three natural phenolic antioxidants commonly found in functional food, and determine their contribution to the total free radical scavenging activity. Antioxidant activities of organic cacao, green coffee, chia seeds, goji berries, spirulina, and chlorella were evaluated and compared. The most potent free radical scavenger was found to be chlorogenic acid. Free radical scavenging activity in the functional foods tested was mostly affected by the presence of chlorogenic and caffeic acid, while gallic acid, although present in higher amounts, was a less potent free radical scavenger.

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Impregnation of a stationary phase by organic and inorganic agents in high-performance thin-layer chromatography (HPTLC) may result in higher separation selectivity and resolution. This work is devoted to the application of novel core–shell polymers consisting of hyperbranched (poly(ethyleneimine)) core surrounded by oligosaccharide shell (PEI-OS) as a component of silica stationary phase in HPTLC for the separation of water-soluble vitamins, amino acids, and chiral β-blocker enantiomers. The influence of polymer structure (degree of substitution of PEI terminal amino groups with oligosaccharides and molecular weight of dendritic core [5 or 25 kDa]) as well as the content of PEI-OS in the stationary phase and a method of modification of the stationary phase on the efficiency of vitamin and amino acid determination and on the enantioselectivity factors of β-blockers separation were investigated. It was established that such polymers can be used as modifying agents of HPTLC systems for on-line preconcentration of vitamins (B2) and amino acids (lysine, tryptophan, and glutamic acids). These polymers can also be recommended as chiral selectors for the effective TLC separation of β-blockers.

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A sensitive, specific, and accurate stability-indicating high-performance thin-layer chromatographic method for the analysis of ambroxol hydrochloride and doxofylline, for both bulk drug and formulation, was developed and validated according to the International Conference on Harmonization (ICH) guidelines. The densitometric analysis of both drugs was carried out in the absorbance mode at 276 nm using diethyl ether‒n-butanol‒ammonia (9:0.9:0.1, v/v) as the solvent system. This system was found to give compact spots for ambroxol hydrochloride at R F of 0.74 ± 0.01 and doxofylline at 0.41 ± 0.01. Moreover, both drugs were subjected to acid and alkali hydrolysis, oxidation, and photodegradation. Also, the degraded products were well resolved from the pure drug with different R F values. Linearity was found in the range of 20–100 and 100–500 ng band−1 for ambroxol hydrochloride and doxofylline. The limit of detection (LOD) and limit of quantitation (LOQ) for ambroxol hydrochloride and doxofylline were 1.17 and 3.57 ng band−1 and 30.68 and 92.97 ng band−1, respectively. “Bartlett’s test” and “Lack of fit” applied on peak area for linearity additionally proved validity of the linearity of the developed method. Good accuracy and precision were obtained as revealed from percent relative standard deviation (% RSD) less than 2. Moreover, robustness testing was performed applying fractional factorial design, 24–1. All the four factors: volume of diethyl ether, volume of n-butanol, solvent front, and chamber saturation time, evaluated in the robustness testing, were found to have an insignificant effect on the retention factor. Similarly, no interference was observed from common excipients in tablet formulation as well as degradation product, indicating specificity of the method. As the method could effectively separate both drugs from their degradation products, it can be used as a stability-indicating method.

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Authors: Joanna Nowakowska, Krzesimir Ciura, Wiktoria Struck-Lewicka, Piotr Pikul, Michał Jan Markuszewski and Piotr Kawczak

The chromatographic properties of eight steroids: trans-andros-terone, methyltestosterone, testosterone, progesterone, cortisone, hydrocortisone, estrone, and β-estradiol were studied by planar chromatography by use of aluminum oxide plates as well as of four different binary mobile phases (acetonitrile-water, acetonitrile-DMSO, acetone-petroleum ether, and acetone-water). Correlations between chromatographic data and physicochemical parameters were presented as results of established quantitative structure-retention relationships (QSRRs). Principal component analysis (PCA) and multiple linear regression (MLR) showed the possible relationships between the retentions and the physicochemical parameters of the studied compounds. The evaluation of thin-layer chromatographic (TLC) separation power, based on ΔhR F, was also discussed. Finally, two chromatographic systems for the effective separation of glucocorticoids and other analyzed steroid hormones were recommended.

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The works reported in the literature (1994–2015) on the use of surfactants as separation modifiers of organic and inorganic compounds by thin-layer chromatography (TLC) is discussed. A number of adsorbents such as silica gel (plain and modified with surfactants and other compounds), stannous silicate, aminoplast, soil, urea-formaldehyde polymer with cellulose binder, and bismuth silicate have been used as layer materials. Surfactants used for the modification of the mobile and stationary phases in TLC have opened new opportunities for realizing novel separations of analytical importance. The salient features of TLC systems used in the analysis of organic and inorganic mixtures of substances have been provided in the tabular form.

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This paper reviews the use of thin-layer chromatography (TLC) for the analysis of neutral lipids and phospholipids in medically and economically important snails (gastropod molluscs). It updates and fills in unintentionally neglected papers from earlier reviews on this topic published in 1990 and 2006 in this journal, and extends coverage to lipophilic pigments for the first time. The review discusses all steps of the TLC procedure, including sample preparation, sample and standard application, layers and mobile phases, detection of zones, and quantification. Significant findings on the effects of temperature, larval parasitism, diet, and environmental changes on the lipid and lipophilic pigment content in the snails as determined by TLC as well as the results of other miscellaneous studies and advantages and future prospects are presented.

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A validated online over-pressured layer chromatography (OPLC) method was developed for the rapid, cost-effective, and efficient separation of gallic acid. A reversed-phase (RP) ultraviolet (UV)–online OPLC analytical method was developed and validated as per the International Conference on Harmonization (ICH) guidelines. Methanolic extract of Annona muricata (fruit) was prepared by cold maceration. Separation of marker was achieved on RP-OPLC plates (5 × 20 cm) with isocratic solvent system of 0.1% acetic acid, water and methanol (30:70) at a flow rate of 0.15 mL min−1; detection was carried out at λmax 270 nm. The base peak of standard gallic acid was at 5.441 min with good linearity (r 2 > 0.999), precision, and accuracy. The limit of detection (LOD) (521.84 mg mL−1) and limit of quantification (LOQ) (1581.336 mg mL−1) values reflect that the method is sensitive with high peak purity; the recoveries of analyte were 99 to 103%. The achievement of the method was the early retention time of gallic acid which in turn increased the efficiency of the quantification of the targeted marker in a short duration of time for even larger number of samples in plant extract as well as biological fluids for pharmacokinetic studies. The application of the method can be extended in regulatory guidelines for the quality control of herbal drugs/products and formulations. The method is rapid and economical in terms of solvent consumption and, hence, can be preferred over other high-performance thin-layer chromatographic or liquid chromatographic methods.

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Authors: Hiroshi Yamaguchi, Takeshi Honda and Masaya Miyazaki

The microreaction technology which is an interdisciplinary science and engineering, have attracted attention in many fields in the past years. Several microreactors have been developed. Enzyme is one of the catalysts, which is useful in substance production in an environmentally friendly way, and has high potential for the preparation of chiral compounds. These features are suitable for pharmaceutical process. However, not so many enzymatic processes were commercialized, because of problems in stability of enzyme molecule, cost, and efficiency of the reactions. Thus, there have been demands for innovation in process engineering particularly for enzymatic reactions, and microreaction devices can be a strong tool for the development of enzyme processes. In this minireview, we summarize fundamental immobilization techniques to develop enzyme microreactor. Some important applications of this technology toward the chemical processing are also included.

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Possibilities have been studied of using a solvation model to predict the retention behavior of solutes in liquid chromatography mobile phases of water-acetonitrile and water-methanol with the organic solvent content varying from 1 to 100 vol%. Twenty-one test solutes, both aliphatic and aromatic compounds, have been selected on the basis of two-level factorial designs. Using the multiple linear regression analysis, regression coefficients, which are characteristics of the stationary and mobile-phase system, were calculated for different mobile phases in the solvation model. Regression coefficients have been used for the prediction of the retention behavior. Unbiased results have been obtained by using two sets, one training and the other the testing set. The predicted retention has been compared with the experimental data. The methanol-water system provided good results at low and medium methanol concentrations; the retention prediction was unsatisfactory for mobile phases containing more than 90% of methanol. The acetonitrile-water system yielded similar results, but the retention prediction ceased to be at acetonitrile concentrations greater than 80%. The retention has primarily been determined by cohesive and acid-base interactions. The dependences of the regression coefficients on the mobile-phase composition were similar for the acetonitrile-water and methanol-water systems.

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Providing new fluorogenic substrates with designed enzyme-labile moieties for microfluidic live cell analysis is an innovative complementary approach to conventional cultivation based methods of bacterial diagnostics. The advance of their integrated application in microfluidic devices is presented in comparison to established approaches. A comprehensive insight on recent implementation is given and highlighted with a commercially available example.

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This study investigated the survival and catalytic potential of a single species Pseudomonas taiwanensis VLB120ΔC biofilm for the conversion of styrene to (S)-styrene oxide in a multiphasic capillary microreactor containing the highly toxic substrate styrene as a pure phase. The catalytic biofilm was cultivated under high fluidic stress in a continuous three-phase segmented flow system comprising aqueous medium, air, and styrene. This concept required an adaptation period of 7 days, during which P. taiwanensis VLB120?C developed a biofilm exhibiting a remarkable cellular integrity with nearly 70% intact cells. In a three-phase segmented flow biofilm microreactor, an average specific styrene epoxidation rate of 10 g/Ltube/day was achieved continuously for a period of 20 days without any clogging problems. Overall, this note highlights the robustness of biofilms as a promising biocatalyst format for the conversion/synthesis of toxic organic chemicals and the applicability of multiphasic capillary microreactors for biofilm based catalysis.

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Microchannel-based hemodialysis has a potential to improve survival rates and quality of life for end-stage renal disease patients compared to conventional hemodialysis technology. Characterization of hydrodynamic behavior in microchannel geometries is necessary for improving flow uniformity, a critical challenge in realizing a commercial device. A test loop was developed for measuring the impulse response of a tracer dye injected into a dialyzer test article for the purpose of developing residence time distributions (RTD) to characterize lamina design. RTD variance tended to lower for designs that are more dominated, volume-wise, by the microchannel array versus the headers. RTD results also emphasize how defect issues can significantly impact a microchannel device via discrepancies between conceptual and operational devices. A multisegmented CFD model, developed for pairing with the impulse response test loop and dialyzer, showed good agreement between visual observation of the tracer in simulations and experiments, and the shape and peak of the output profiles.

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A rapid and sensitive method for the identification and quantification of phillyrin (POG) in Forsythia suspense is described. The phillyrin standard solution was directly infused into the ion trap mass spectrometers (IT-MS) for collecting the MSn spectra. The electrospray ionization (ESI) mass spectral fragmentation pathway of phillyrin was proposed, and the ESI-MSn fragmentation behavior of phillyrin was deduced in detail. The major product ion at m/z 355 belongs to furofuran, which was formed by loss the glucopyranoside (180 Da), and the characteristic fragment ions m/z 473, 395, 337, 309, and 249 were observed. The loss of 18 Da could arise from two different fragmentation pathways, and the observed ion was composed of a mixture of two different structural ions. Quantification of phillyrin was assigned in positive-ion mode at a product ion at m/z 557 → 355 by liquid chromatography-mass spectrometry (LC-MS). The LC-MS method was validated for linearity, sensitivity, accuracy, and precision and then used to determine the content of the phillyrin. Lastly, the LC-MS method was successfully applied to determine phillyrin in real sample F. suspense and three of its medicinal preparations in the positive mode at the first time.

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The utilization of continuous-flow biochemical reactors, including biocatalysis, biotransformation, and biochemical interaction based flow-analytical systems, and enzyme reactors are recently the focus of attention to produce fine biochemicals and also show great potential in bioanalytical applications. Continuous-flow biochemical processes implemented in microstructured reactors enable short development time to production scale utilizing enzymatic processes to efficiently fulfill the current needs of the fine biochemical and pharmaceutical industry. Immobilization of the enzymes is preferable because it usually enhances their stability, and in some instances, immobilized enzymes can even be reused multiple times. In this review on the continuous-flow biochemical reactors, first the enzyme immobilization strategies will be briefly discussed followed by summarizing the recent developments in the field of immobilized enzyme microflow reactors for biocatalysis, bioconversion and bioanalytical purposes.

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The performance of the Corning AFR™ Low Flow (LF) fluidic module for Candida antarctica lipase B-catalyzed isoamyl acetate synthesis in an n-heptane–buffer two-liquid phase system was evaluated. Obtained flow regime of dispersed n-heptane droplets in a continuous buffer phase, which enables in situ extraction of the produced isoamyl acetate to the n-heptane phase, provides a very large interfacial area for the esterification catalyzed by an amphiphilic lipase B, which positions itself on the n-heptane–buffer interface. Productivities obtained were the highest reported so far for this reaction and indicate that Corning Advanced-Flow Reactor™ (AFR™) modules are also very efficient for carrying out biotransformations in two-phase systems. Additionally, for the separation of the n-heptane from the aqueous phase, a membrane separator consisting of a hydrophobic PTFE membrane was integrated, which enabled the reuse of biocatalyst in several consecutive biotransformations.

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The different separation patterns of petroleum ether extract of leaves of Heiba (Ficus pomifera Wall.) were studied with open column chromatography, highperformance liquid chromatography (HPLC), and liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS). A new method of locating and estimating newly discovered compounds by LC-ESI-MS was developed. Compound 1, a triterpenoid of mass 398, was identified by spectral analysis. It was located in the chromatogram by comparison of mass spectrum of the isolated compound to mass spectra of in mass spectral data of LC-ESI-MS. The concentration of the triterpenoid was found to be 8.64 ppm in 2000 ppm of the extract, assuming that all the components are eluted.

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Biodegradable and bioresorbable macromolecular conjugates of paclitaxel were prepared. The release of drug from conjugates has been carried out in vitro at 37 °C in phosphate buffered saline solution (PBS, pH 7.4), with 0.1% (w/v) Cremophor® EL. Periodically, all the solution in the samples was removed and replaced with fresh buffer until limit of detection (LOD) of paclitaxel was released. The quantity of released drug was analyzed by means of high-performance liquid chromatography (HPLC) method. Chromatographic separations were conducted using the NUCLEODUR C18 Gravity column with a guard column at 30 °C. Mobile phase consisted of a mixture of acetonitrile, methanol, and deionized water (60:2:38). The flow rate was 1.0 mL min−1, and paclitaxel was detected at 229 nm, retention time of 3.5 min. The applied analytical method was validated according to International Conference on Harmonization (ICH) procedures or recommendations. The chromatographic separation was excellent. The linearity in the range 0.1–4.5 μg mL−1 was found to be very good (R 2 = 0.9999). LOD and limit of quantification (LOQ) were calculated to be 0.023 μg mL−1 and 0.068 μg mL−1, respectively. The release profiles were evaluated and compared. The process of paclitaxel release from Paclcon-2 conjugate seems to be the most interesting. Paclitaxel is released the longest and the most evenly.

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Micro-thin-layer chromatography in two dimensional (2D-mTLC) mode in normal and reversed phase systems by use of diol bonded stationary phase was applied to make fingerprints of 11 species of Mentha genus and two finished pharmaceutical products.

Nonaqueous eluents (propan-2-ol or ethyl acetate dissolved in n-heptane) were used in normal phase systems. Mixtures of acetonitrile with water were used in reversed phase chromatographic systems.

Optimization of one dimensional systems was performed by determining of R F vs. composition of mobile phases dependencies for standards occurring in various species of Mentha. Most selective eluents were chosen to optimize two-dimensional systems by creating R F in normal-phase (NP) systems vs. R F in reversed-phase (RP) systems correlations.

2D-mTLC on diol polar bonded stationary phase were optimized to separate phenolic compounds and make fingerprints of examined plant materials and this method was never applied earlier in the chromatographic analysis.

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Biotransformation of l-phenylalanine (l-1a) and five unnatural substrates (rac-1bf) by phenylalanine ammonia-lyase (PAL) was investigated in a novel microfluidic device (Magne-Chip) that comprises microliter volume reaction cells filled with PAL-coated magnetic nanoparticles (MNPs). Experiments proved the excellent reproducibility of enzymecatalyzed biotransformation in the chip and the excellent reusability of the enzyme layer during 14 h continuous measurement (>98% over 7 repetitive measurements with l-1a). The platform also enabled fully automatic multiparameter measurements with a single biocatalyst loading of about 1 mg PAL-MNP. Computational fluid dynamics (CFD) calculations were used to study the flow field in the chambers and the effect of unintended bubble formation. Optimal flow rate for l-1a reaction and specific activities for rac-1bf under these conditions were determined.

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Authors: Søren Heintz, Aleksandar Mitic, Rolf H. Ringborg, Ulrich Krühne, John M. Woodley and Krist V. Gernaey

A microfluidic toolbox for accelerated development of biocatalytic processes has great potential. This is especially the case for the development of advanced biocatalytic process concepts, where reactors and product separation methods are closely linked together to intensify the process performance, e.g., by the use of in-situ product removal (ISPR). This review provides a general overview of currently available tools in a microfluidic toolbox and how this toolbox can be applied to the development of advanced biocatalytic process concepts. Emphasis is placed on describing the possibilities and advantages of the microfluidic toolbox that are difficult to achieve with conventional batch-processbased technologies. Application of this microfluidic toolbox will potentially make it possible to intensify biocatalytic reactions and thereby facilitate the development towards novel and advanced biocatalytic processes, which in many cases have proven too difficult in conventional batch equipment.

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Authors: Anita Šalić, Katarina Pindrić, Gordana Hojnik Podrepšek, Nikolina Novosel, Maja Leitgeb and Bruno Zelić

In this study, magnetic nanoparticles (MNPs) of maghemite (γ-Fe2O3) were synthesized and characterized. The method of multifactor experimental design and evolutionary operation (EVOP) was used to optimize immobilization of the alcohol dehydrogenase (ADH) enzyme on MNPs. Optimal operating conditions for the immobilization process were determined (γADH = 0.08 mg/mL, 2% glutaraldehyde for surface activation, t = 28 h), and in such conditions, a specific activity of S.A. = 118 ± 6 U/mg and immobilization efficiency of η = 84.97 ± 3.67% were achieved. Compared to the native enzyme, ADH immobilized on MNPs retained 66.45 ± 3.66% of the initial activity. ADH immobilized on MNPs at optimal conditions was used as a biocatalyst for model reaction—NADH oxidation. NADH oxidation was performed in two different magnetic microreactor configurations: (1) microreactor equipped with permanent square magnets and (2) microreactor equipped with an electromagnet and an oscillating magnetic field that enables magnetic particles movement in the microreactor. In the system with the oscillating magnetic field, equal conversion (X = 100%) was achieved in 2-fold shorter residence time.

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High-performance liquid chromatography-mass spectrometry (HPLC-MS) method coupled with radical reaction for screening active ingredients from perennial fujimoto bean whole herb was established. The active ingredients, present in perennial fujimoto bean whole herb, possess scavenging effects towards 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical, superoxide, peroxy radical, and hydroxyl radical. The radical scavenging abilities of these active ingredients were evaluated based on the relative peak areas in the HPLC chromatogram. The results indicate that potent antioxidants are present in the anhydrous methanol extract of perennial fujimoto bean whole herb. Based on HPLC-MS analysis, it was found that the scavenging ability can be mostly attributed to the presence of three compounds: cyanidin-3-o-β-d-glucopyranoside, troxerutin, and rutin. The structures were identified based on the MS and nuclear magnetic resonance (NMR) data. Free radical scavenging activity decreased in the following order: troxerutin > rutin > cyanidin-3-o-β-d-glucopyranoside.

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A new, sensitive, and selective high-performance liquid chromatography-tandem mass spectrometric method (HPLC-MS/MS) has been developed for the quantification of six flavonoids (sophoricoside, genistin, genistein, rutin, quercetin, and kaempferol) in rat bile and urine. The sample pretreatment was simple by liquid-liquid extraction. Sulfamethalazole was used as internal standard (IS). During method development, the effect of extraction volume, mobile phase composition, column temperature, and injection volume were varied to optimize sensitivity and achieve a run time as short as possible. Chromatographic separation was accomplished on a C18 column with a simple linear gradient elution within 9 min. Full validation of the assay was in accordance with the requirement of the validation of the method in vivo and implemented including specificity, linearity, accuracy, precision, recovery, and matrix effect. This is the first report on determination of the major flavones in rat bile and urine after oral administration of Fructus Sophorae extract. The method has been used successfully in excretion studies of six major flavonoids in rat bile and urine.

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Citri Grandis Exocarpium (CGE) is a traditional Chinese medicine with a variety of biological activities. For efficient quality control of CGE, a simple, rapid, and accurate high-performance liquid chromatographic (HPLC) method was developed for simultaneous determination of four main compounds (naringin, rhoifolin, meranzin hydrate, and isoimperatorin) in this herb. These four compounds were separated on a C18 column by gradient elution with methanol and water. The flow rate was 1.0 mL·min−1, and the detection wavelength was 324 nm. The recoveries of the method ranged from 96.32% to 103.71%, and good linear relationships (r 2 > 0.9998) over relative wide concentration ranges were obtained. Then this validated method was successfully applied to the analysis of nine batches of CGE samples.

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A simple stability-indicating high-performance liquid chromatography-diode array detection (HPLC-DAD) method has been developed for the simultaneous determination of triamterene (TRI) and xipamide (XIP) in presence of the degradation products generated in studies of forced decomposition. Drugs were subjected to stress by hydrolysis (acidic, alkaline, and neutral), oxidation, photolysis (254 and 365 nm), and dry and wet heat treatments. Degradation occurs under acidic and alkaline conditions (TRI only), oxidative stress (TRI and XIP), and by photolysis (XIP only), but both drugs were stable under other stress conditions investigated. Separation of the two drugs from all the degradant peaks was achieved within 11 min using C8 column (250 × 4.6 mm, 5 μm) and mobile phase consisting of acetonitrile and 0.05 M phosphate buffer adjusted to pH 4 delivered at a flow rate of 1 mL min−1 using gradient elution system. The drugs were quantified at 220 nm using photodiode array detector, based on peak area. Peak homogeneity of the two drugs was checked using diode array detector, and the purity angle was within the purity threshold limit in all of the stressed samples. The calibration graphs for each drug were rectilinear in the range of 0.2–50 and 0.1–20 μg mL−1 for TRI and XIP, respectively. The method was validated in compliance with International Conference on Harmonization (ICH) guidelines; in terms of linearity, accuracy, precision, robustness, limit of detection, and limit of quantitation. The proposed method was successfully applied for the determination of the investigated drugs in their tablet without interference from excipients with acceptable accuracy and precision; the label claim percentages were 100.23 ± 0.70% and 100.75 ± 1.11% for TRI and XIP, respectively.

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Many phytochemical investigations have been focused recently on the antioxidant activity of herbal extracts which can be used in phytotherapy. The previous study revealed antioxidative properties of Mutellina purpurea extract, but the constituents responsible for this action were not described yet. The aim of this study was activityguided separation and identification of antioxidant compounds from M. purpurea herb. Thin-layer chromatography-2,2-diphenyl-1-picrylhydrazyl (TLC-DPPH) assay was used to detect compounds of interest; liquid chromatography-diode array detection-mass spectrometry (LC-DAD-MS) analysis allowed to identify antioxidants. The active fractions analyzed with LC-DAD-MS contained as a main compound chlorogenic acid accompanied with p-coumaric acid, ferulic acid, three dicaffeoylquinic acids, and caffeoylferuloylquinic acid. The fast TLC-DPPH assay with LC-DAD-MS identification enabled the accurate identification of antioxidants in M. purpurea herb, which was done for the first time.

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Authors: Brae V. Petersen, Paul S. Powell, Jeffery D. St. Jeor, Tanner H. Anderson, Zachary T. Greenlee, Jared B. Breakall, Kishor Prasain and David C. Collins

Thin-layer chromatography and electrophoresis, with their long histories of simple and effective characterization of chemical mixtures, have motivated an effort to combine these techniques. Simultaneous chromatography and electrophoresis (SCE) utilizes an electric field orthogonal to capillary action or pressure flow to achieve a single-step two-dimensional separation. In this work, plate conditioning and pressurized simultaneous chromatography and electrophoresis (pSCE) are introduced. These improvements reduce separation times and concurrently increase or maintain separation quality as described by visual comparisons, nearest neighbor distance descriptors, and relative spot capacities.

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The separation of 1-fluoro-2,4-dinitrophenyl-5-l-valine amide (FVDA) diastereomeric derivatives of aspartic acid, cysteine, and histidine by means of high-performance thin-layer chromatography (HPTLC) as well as pressurized planar electrochromatography (PPEC) techniques in systems with HPTLC RP-18W plates and the various acetonitrile—buffer mobile phases is presented. The influence of the mobile phase components, i.e., acetonitrile concentration and buffer kind on migration distance of the solute zones, was investigated. The effect of mono (formic) and various dicarboxylic acids (oxalic, malonic, maleic, malic, succinic, tartaric, and pimelic) as the mobile phase buffer components on the solute retention was studied. It is observed that an increase of acetonitrile content of the mobile phase affects the solute zone migration and retention in PPEC and HPTLC. What is more, the separation selectivity in the latter and former techniques differs. The PPEC mode presents a higher efficiency in comparison with HPTLC. The solute separation with electromigrational system is more fragile on the kind of acid used as mobile phase buffer component than with chromatographic method. Nevertheless, the influence of the kind of mobile phase buffer component on solute selectivity and retention in both techniques was determined. The electrokinetic (zeta) potential of the stationary—mobile phase interface was measured and compared with the solute retention data of both techniques.

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Authors: Virginia Coman, Ştefan Kreibik, Mihaela Vlassa, Florina Copaciu, Ioana Perhaiţa and Miuţa Filip

In our previous papers, we defined layer (planar) dielectrochromatography and we presented its fundamentals (theoretical aspects) such as the displacement of dielectric liquids under nonuniform external alternating electric fields (dielectroosmotic flow [DEOF] effect), the displacement of solute particles or polarized granules (dielectrophoresis [DEP] effect) in electric fields generated by armatures, the theoretical evaluation of the electric intensity generated in the stratified dielectrics, etc. Ready-to-use plates of alumina, silica gel, and cellulose were used for experiments. The obtained results have encouraged us to create our own TLC plates based on alumina enriched with compounds of high dielectric constants like barium sulfate, barium titanate, and titanium dioxide. In this paper, we present the preparation of seven plates containing increasing amounts (1, 2, 3, 4, 6, 8, 10) g of barium titanate in 35 g alumina, the methodology used in their characterization, as well as the obtained results and the perspectives of using this ingredient.

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Authors: Heidrun Gruber-Woelfler, Marie-G. Braunbruck, Rene Laskowski, Peter W. Feenstra, Johannes G. Khinast and Hans-Jörg Bart

The synthesis and characterization of novel stationary phases for analytical applications as well as for planar electrochromatography are presented. The stationary phases are C8-functionalized silica-based monoliths, which are supported by silica particles in order to avoid shrinkage and to increase mechanical stability. After optimizing the separation conditions in capillaries for capillary electrochromatography (CEC), the novel materials were implemented in a planar test cell and tested for the electrochromatochraphic separation of polyaromatic hydrocarbons (PAHs). Although the change of geometries implied a scale-up of the filling procedure, high separation performances with theoretical plate numbers of up to 30,000 m−1 and throughputs of 50 mL h−1 could be achieved.

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In the presented paper, the influence of the static electric field on retention, and in consequence separation, of tropane alkaloids present in Datura innoxia Mill. extract was demonstrated. Thin-layer electrochromatography (ETLC) on initially dry layers was used as the investigation method. The results obtained by ETLC were compared with the data obtained in thin-layer chromatography (TLC) analogous systems. Pseudo-reversed-phase systems (stationary phase — silica gel, mobile phase buffer at different pH and methanol mixture with different content of the methanol) were used. The presence of the electric field influences the retention parameters of the investigated compounds. The influence depends on the pH of the buffered mobile phase, composition of the mobile phase, and turn of the electric field lines in relation to the mobile phase migration direction. In the mobile phase, in which some of the alkaloids are in the ionic form, the significant differences between retention in cathode side and anode side were observed due to electrophoretic migration of the solutes, so direction of the electric field lines can be one of the systems’ optimization factor. The experiment proves that full separation of the investigated mixture ingredients is possible in ETLC only.

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Thin-layer chromatography (TLC) and pressurized planar electrochromatography (PPEC) of amino acids in normal-phase system is presented. The results have been obtained for various normal-phase high-performance thin-layer chromatography (HPTLC) and PPEC systems with the mobile phase that comprised acetonitrile in the concentration ranges 40–90% and 20–90%, respectively, and HPTLC silica gel 60 F254s plates from Merck. The data obtained show differences in separation selectivity between HPTLC and PPEC systems. The respective separation selectivities have been obtained for HPTLC and PPEC systems with the mobile phase buffer pH in the range 2.6–9.0. The retention of amino acids in HPTLC systems has demonstrated minor dependence on buffer pH, while, in analogous PPEC systems, migration distances of the solutes have shown considerable changes. The differences of separation selectivity in HPTLC and PPEC systems are interpreted in terms of solute partition in the former and solute partition and electrophoresis in the latter.

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A comparison of the chromatographic behavior of benzoic acids on normal (silica gel), reversed (RP-18), and polyamide-11 plates using thin-layer chromatography (TLC) with controlled gas phase inside the chamber has been performed. This variant of TLC is based on the use of a gas phase moving over the TLC plate for regulation of the stationary and mobile phases as well as the acid—base properties of analytes during the separation process. The feasibility of such an approach is illustrated by the separation of benzoic acid derivatives using carbon dioxide, ammonia, and acetic acid vapor. It was shown that a gradual change of mobile phase acidity makes it possible to enhance separation efficiency and selectivity, this effect being dependent on the type of the stationary phase. The most considerable change in the retention of benzoic acid derivatives was observed for normal-phase plates with silica gel or silica sol, or starch binders used as the stationary phase. An alteration of surface acidity for polyamide and RP-18 plates is not so pronounced as for silica gel ones so that a smaller change in chromatographic parameters of benzoic acid derivatives was observed.

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We present a videodensitometric quantification method for methadone in syrup, separated by thin-layer chromatography (TLC). The quantification is based on a derivation reaction with Dragendorff reagent. Measurements were carried out using a 16-bit flatbed scanner. The range of linearity covers two magnitudes of power using the Kubelka-Munk expression for data transformation. The separation method is inexpensive, fast, and reliable.

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To evaluate the quality of Fructus Arctii, an accurate and reliable method of high-performance liquid chromatography/diode array detection—electrospray ionization—mass spectrometry (HPLC/DAD—ESI—MS) was developed. Nine compounds, including chlorogenic acid, caffeic acid, trans-p-hydroxycinnamic acid, arctiin, arctignan A, ethyl caffeate, matairesinol, arctigenin, and lappaol B, were determined simultaneously in 19 batches of Fructus Arctii samples collected from different localities. Nineteen common peaks were identified or tentatively assigned by comparing their mass spectrometric data with reference compounds, self-established compound library, and published literatures. Also, the 19 common peaks were selected as characteristic peaks to assess the similarity of chromatographic fingerprinting of these samples. Moreover, hierarchical clustering analysis (HCA) and principal components analysis (PCA) were successfully applied to demonstrate the variability of samples. The results indicated the content of nine compounds that varied greatly among the samples, and 19 samples collected from different localities could be discriminated. Furthermore, chlorogenic acid, arctiin, and arctigenin were found to be chemical markers for evaluating the quality of Fructus Arctii.

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Authors: M. Mori, T. Masuno, D. Kozaki, N. Nakatani, K. Tanaka and H. Itabashi

Ion chromatography of inorganic cations using a phosphate-coated zirconia stationary phase (PZ) was first attempted. The retentions of cations to PZ increased by elevating the column temperature and the reproducibility of the separation could improve at the higher temperature. The PZ functioned as a cation-exchanger from changes in the retention factor of cations as a function of eluent pH. Furthermore, the Gibbs free energies of cations were estimated from enthalpy and entropy using the retention factors of cations as a function of the column temperature. The reaction was based on the endothermic reaction.

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There is no doubt that high-performance thin-layer chromatography (HPTLC) can be applied as a quantitative method if the technique is properly used. Densitometry is a commonly used detection mode for quantitation in HPTLC. The influence of instrumental settings on signal intensity, peak resolution, and peak positioning was rarely described in literature. Especially, quantitation of adjacent substance zones was critical when improper combinations of these settings merge. Future trends regarding ultrathin-layer chromatography and hyphenation to scanning or imaging mass spectrometry required the consideration of these delicate points. The influence of different instrumental settings on the obtained signal intensities was demonstrated for four separated parabens (each 150 ng band−1). The maximum mean signal deviations of all four compounds were 6.9% by the optical system, 16.8% by the scan slit dimension, 7.5% by the scan speed, and 1.5% by the data resolution. The influence of these settings on the quantitation of three parabens in two skin protection creams was investigated. Depending on the selected settings, deviations of the calculated substance amount of up to 5.6% were yielded, whereby determination coefficients of the polynomial calibration curves (60–300 ng band−1) varied between 0.9985 and 0.9999. The setting of integration markers between two adjacent peaks was demonstrated to be deficient if low spatial data resolution is applied; however, this challenging task will rise in interest due to the trend towards miniaturization.

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Authors: O. Gazdag, L. Ködöböcz, T. Szili-Kovács and A. Murányi
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The use of matrix solid-phase dispersion (MSPD) was tested to extract some coumarins (umbelliferone, xanthotoxin, isopimpinellin, bergapten, and pimpinellin) from Pimpinella roots. The method was compared with liquid—solid extraction methods (LSE) such as accelerated solvent extraction (ASE), ultrasound-assisted extraction (USAE) and Soxhlet extraction followed by solid-phase extraction (SPE). Several methanol concentrations and temperature conditions were examined during the liquid—solid extraction techniques. The analysis was performed by high-performance liquid chromatography with diode array detector (HPLC—DAD). MSPD extracted furanocoumarins with satisfactory recoveries ranging from 91.65% to 97.55% and relative standard deviations lower than 5,0415%. The results presented in the paper reveal that MSPD is an efficient, fast, simple and easy-to-perform method suitable for the isolation of furanocoumarins from herbs.

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The aim of this study was to compare the antioxidant activity of crude extracts of differing polarities of the fruits of Chaenomeles speciosa (Sweet) Nakai. The antioxidant compositions of the extracts were analyzed qualitatively and quantitatively, respectively. The 75% ethanol extract of the dried fruits was fractionated by sequential extraction using petroleum ether, ethyl acetate, and n-butyl alcohol. The antioxidant effectiveness of the components of differing polarities was examined by 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging method and compared with two reference substances: ascorbic acid and butylated hydroxytoluene (BHT). The total phenolic content of the extracts was analyzed using the Folin–Ciocalteu method and expressed as gallic acid equivalents. The active compounds were analyzed by thin-layer chromatography (TLC)–bioautography and ultra-performance liquid chromatography (UPLC). The order of antioxidant capacities of various solvent extracts from the fruit of C. speciosa was found to be ethyl acetate ≥ n-butyl alcohol > petroleum ether. The ethyl acetate extract was more active than the reference substances ascorbic acid and BHT. The radical-scavenging capacity of the extracts decreased as the total phenolic content decreased. TLC–bioautography revealed that the ethyl acetate extract contained many antioxidant spots that can remove DPPH radical, and protocatechuic acid and chlorogenic acid were the major antioxidant components. UPLC analysis confirmed that protocatechuic acid and chlorogenic acid were mainly distributed in the ethyl acetate fraction. The study demonstrated that the ethyl acetate extract had excellent antioxidant capacity. The total phenolic, protocatechuic acid, and chlorogenic acid contents of this extract were higher than those of the other two solvent extracts. These results showed that the ethyl acetate extract from the fruit of C. speciosa could be considered as a potential source of natural antioxidant agent.

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This work presents a comparative study on the development and validation of two analytical techniques applied for the simultaneous determination of hydrocortisone acetate (HCA), fusidic acid (FSA), methyl paraben (MPB), and propyl paraben (PPB) formulated as a topical cream. The first technique was thin-layer chromatography (TLC)–densitometric method, which was developed by separating the four components on silica gel 60 F254 using methylene chloride–methanol–benzene in the ratio of 10:2:5, v/v, as the developing system, followed by densitometric measurement of the bands at 240 nm. The second technique was the chemometric method using two models: principle component regression model (PCR) and partial least squares (PLS). The suggested techniques were validated in compliance with the International Conference on Harmonization (ICH) guidelines and were successfully applied for the determination of the quaternary mixtures as prepared synthetically in laboratory and in the commercial topical pharmaceutical formulation.

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A high-performance thin-layer chromatographic (HPTLC) method for the analysis of the protostemonine from Stemona sessilifolia (Miq.) Miq. has been developed and validated. According to the HPTLC fingerprint, 12 common peaks were selected to evaluate the similarities among 10 batches of samples with R F values ranging from 0.02 to 0.95. Ten different batches of samples were stable and reliable in quality, all the similarities of which are more than 0.98. The results indicated that this method was sensitive and precise for the quality evaluation of S. sessilifolia samples.

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Herein, an optimized microfluidic device for manufacturing encapsulating water-in-oil-in-water (w/o/w) double emulsions is reported. The adjustability of the microfluidic device allows on-demand formation of oil shells with different thicknesses during the w/o/w double emulsion formation while maintaining the same core size. This was achieved by manipulation of the separation distance between the cylindrical tubes constituting the flow-focusing part of the device, the middle flow rate of the middle phase, and the outer flow rate of the continuous phase, all at the same time. By incorporating lipids in the oil shell, the w/o/w double emulsions serve as templates for the formation of monodisperse encapsulating liposomes. Thus, liposomes with different shell properties can be generated after evaporation of the oil that can be collected either separately or pooled together in a single sample batch using only one experimental step. The w/o/w double emulsions are highly monodisperse, generated with a throughput of more than 10 Hz, having water core diameters ranging from 130 to 290 μm and different oil shell thicknesses varying from 5 to 13 μm. Moreover, double emulsions with diameters down to 10 μm are reported; however, at this size, the dispersity is less controllable. The microfluidic device is composed of commercially available parts with only minor modifications required, thus, facilitating the manufacturing of encapsulating w/o/w double emulsions.

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Authors: I. Kádár, P. Ragályi, A. Murányi, L. Radimszky and A. Gajdó
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Authors: Sofie Seghers, Frederik E.A. Van Waes, Ana Cukalovic, Jean-Christophe M. Monbaliu, Jeroen De Visscher, Joris W. Thybaut, Thomas S.A. Heugebaert and Christian V. Stevens

Despite extensive research into peptide synthesis, coupling of amino acids with weakly nucleophilic heterocyclic amines remains a challenge. The need for microwave technology to promote this coupling interferes with the scalability of the process. By applying the microwave-to-flow paradigm, a library of (α-aminoacyl)amino-substituted heterocycles was continuously produced at near quantitative conversions and the reaction was scaled up successfully. Various N-Cbz-protected amino acids were activated using BtH/SOCl2 under continuous-flow conditions with excellent yields. Their coupling with heterocyclic amines was accomplished in MeCN—NMP on a preparative scale. However, performing both steps in-line resulted in an inconvenient work-up. Therefore, a two-step approach was taken, isolating the intermediate Bt-activated amino acid via simple filtration. This allows for a solvent switch to DMSO for the coupling reaction which led to excellent conversions for a broad range of substrates.

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A rapid high-performance liquid chromatography (HPLC) method for chiral purity determination of tenofovir disoproxil fumarate in raw material and pharmaceutical formulations was developed. The (S)-enantiomer appears to be as an impurity and pharmacologically inactive. The effects of various stationary phases, mobile phase composition, and column temperature on enantiomeric separation of tenofovir disoproxil were investigated and optimized. Chromatography resolution of tenofovir disoproxil enantiomers was performed on NUCLEOCEL ALPHA-RP S column (250 × 4.6 mm i.d., 5 μm). The elution was achieved by using 95:5% (υ/υ) methanol—acetonitrile, containing 0.1% triethylamine at a flow rate of 0.8 mL min−1. The ultraviolet (UV) detector was set at 260 nm. Calibration curves were linear in the range of 1–100 μg mL−1 and 0.2–20 μg mL−1 for (R)-tenofovir disoproxil and (S)-enantiomer, respectively. Limits of detection and quantitation for (S)-enantiomer were 0.06 and 0.2 μg mL−1. The run time of analysis was less than 7.0 min. The proposed method was used successfully for separation and quantification of tenofovir disoproxil enantiomers in raw material and pharmaceutical formulations.

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