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? 2023 The AuthorsOligonucleotides are commonly analysed using one dimensional chromatography (1D-LC) to resolve and characterise manufacturing impurities, structural isomers and (in respect to emerging oligonucleotide therapeutic...
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? 2023 The AuthorsOligonucleotides are commonly analysed using one dimensional chromatography (1D-LC) to resolve and characterise manufacturing impurities, structural isomers and (in respect to emerging oligonucleotide therapeutics) drug substance and drug product. Due to low selectivity and co-elution of closely related oligonucleotides using 1D-LC, analyte resolution is challenged. This leads to the requirement for improved analytical methods. Multidimensional chromatography has demonstrated utility in a range of applications as it increases peak capacity using orthogonal separations, however there are limited studies demonstrating the 2D-LC analysis of closely related oligonucleotides. In this study we optimised OGN size and sequence based separations using a variety of 1D-LC methods and coupled these orthogonal modes of chromatography within a 2D-LC workflow. Theoretical 2D-LC workflows were evaluated for optimal orthogonality using the minimum convex hull metric. The most orthogonal workflow identified in this study was ion-pair reversed phase using tributylammonium acetate (IP-RP-TBuAA) coupled with strong anion exchange in conjunction with sodium perchlorate (SAX-NaClO4) at high mobile phase pH. We developed a heart-cut (IP-RP-TBuAA)-(SAX-NaClO4) 2D-LC method for analysis of closely related size and sequence variant OGNs and OGN manufacturing impurities. The 2D-LC method resulted in an increased orthogonality and a reduction in co-elution (or close elution). Application of a UV based reference mapping strategy in conjunction with the 2D-LC method demonstrated a reduction in analytical complexity by reducing the reliance on mass based detection methods.
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Eosin Y is an important tracer in groundwater studies for determining water migration paths and identifying the distribution of environmental pollutants. Spectrofluorimetry, capillary LC/fluorescence, and HPLC-fluorescence are app...
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Eosin Y is an important tracer in groundwater studies for determining water migration paths and identifying the distribution of environmental pollutants. Spectrofluorimetry, capillary LC/fluorescence, and HPLC-fluorescence are approaches to sensitively detect Eosin Y (1, 2). HPLC provides a convenient way to separate eosin from humic substances and interfering tracers. Figure 1 shows the separation of an Eosin standard from impurities obtained with a binary gradient. A gradient is used to reduce peak broadening and improve signal-to-noise ratio. Zero carry-over is assured by wash cycles before and after the injection.
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Propylene carbonate (4-methyl-1,3-dioxolan-2-one) is a five-membered cyclic alkylene carbonate with the chemical formula C4H6O 3. It is most commonly used as a solvent for various synthetic polymers and as an electrochemical solve...
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Propylene carbonate (4-methyl-1,3-dioxolan-2-one) is a five-membered cyclic alkylene carbonate with the chemical formula C4H6O 3. It is most commonly used as a solvent for various synthetic polymers and as an electrochemical solvent. Until this date, few RP-HPLC methods have been developed using propylene carbonate as a mobile phase component. This paper aims to further demonstrate the use of Propylene carbonate in RP-HPLC. Three methods have been developed for the simultaneous estimation of Paracetamol and Lornoxicam in combined dosage forms. All these methods use Propylene carbonate as a mobile phase component but in different proportions. Method I is an isocratic RP-HPLC method, Method II uses a solvent gradient program, whereas Method III uses high temperature for the separation of the two drugs.
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Introduction: Plant-derived salicinoids (conjugates of glucose and salicylate phenolic moieties) are potent modulators of plant-herbivore interactions. We demonstrate the use of micro-high-performance liquid chromatography ( micro...
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Introduction: Plant-derived salicinoids (conjugates of glucose and salicylate phenolic moieties) are potent modulators of plant-herbivore interactions. We demonstrate the use of micro-high-performance liquid chromatography ( micro HPLC) with photodiode-array detection (DAD) for quantification of four salicinoids (salicin, salicortin, hydroxycyclohexen-on-oyl salicortin and tremulacin) in methanolic extracts of Populus. Objective: To develop and implement a solvent-conserving micro HPLC method to quantify salicinoids in methanolic extracts of Populus tissue. Methods: Salicinoids were extracted from Populus tissue into methanol, filtered, and introduced to micro HPLC. Extracted analytes were separated on a Zorbax SB C18-column with a binary gradient of methanol and water (with 2% tetrahydrofuran; 20 micro L/min), and quantified by DAD (274 nm). We confirmed measurement reliability through standard addition, comparison with an accepted method, and assessment of chromatographic peak purity by ultraviolet absorbance spectra. Results: Method detection and quantification limits for the salicinoids as a percentage of dry leaf weight were as follows: salicin (0.1%, 0.2%), salicortin (0.001%, 0.02%), hydroxycyclohexen-on-oyl salicortin (0.02%, 0.06%) and tremulacin (0.0006%, 0.002%). Calibrations by external standardisation were linear over 1.5 orders of magnitude with acceptable accuracy and reproducibility. Conclusion: Micro-HPLC can serve as a solvent-conserving alternative to conventional HPLC for quantification of salicinoids in Populus tissue.
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Algal mass productions are widespread events throughout the world. Most research deals with the environmental impact, ecology and toxicity of these phenomena, but the algae are also promising sources of bioactive natural products,...
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Algal mass productions are widespread events throughout the world. Most research deals with the environmental impact, ecology and toxicity of these phenomena, but the algae are also promising sources of bioactive natural products, and also are potential food additives. In the current study, we aimed to characterize the carotenoid composition of three non-toxic algal isolates with distinct taxonomical position, namely Dunaliella salina, Euglena sanguined and a Nostoc strain. The strains were screened for carotenoid composition with HPLC-UV-APCI-MS with comparison with authentic standards. Carotenoids were purified with open column chromatography, and characterized by LC-UV-MS and NMR. All three species contained a high amount of carotenoids. The composition of the carotenoid pattern was somewhat different compared to literature data. The Dunaliella carotenoid fraction contained lutein (52.1% of total carotenoid), β-carotene (13.1%), violaxanthin (11.5%) and neoxanthin (6.2%) as chief compounds. The chief carotenoids in the Nostoc strain was echinenone (34.9%), while the main components of the Euglena isolate were diatoxanthin (39.0%), lutein (23.7%), an unidentified carotenoid (9.6%) and β-carotene (5.4%). Diatoxanthin is identified by NMR spectroscopy. The carotenoid patterns of the examined strains are somewhat different from the patterns described in the literature. This can be the results of genetic or environmental differences, or combinations thereof. Despite these differences, our study shows the potency of these algae in production of carotenoids, and possibly, usage as food additives.
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Elf Ansammlungen von Cladonia furcata und vierzehn von Cladonia rangiformis, hauptsachlich aus Sachsen-Anhalt (Deutschland), wurden mittels HPLC und HPLC-MS auf ihre Flechtenstoffe analysiert. Die Hauptbestandteile von C. furcata ...
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Elf Ansammlungen von Cladonia furcata und vierzehn von Cladonia rangiformis, hauptsachlich aus Sachsen-Anhalt (Deutschland), wurden mittels HPLC und HPLC-MS auf ihre Flechtenstoffe analysiert. Die Hauptbestandteile von C. furcata sind Fumarprotocetrarsaure, Atranorin, Protocetrarsaure, Rangiformsaure, Bourgeansaure, Norrangiformsaure und 3,4-Dicarboxyhexadecansaure-l-methylester (neu). Cladonia rangiformis ist charakterisiert durch den Gehalt an Atranorin, Rangiformsaure, Norrangiformsaure und 3,4-Dicarboxyhexadecansaure-l-methylester, wahrend Bourgeansaure fehlt.
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An analytical HPLC-MS screening methodology has been developed to improve preparative RP-HPLC-MS purifications in medicinal chemistry laboratories. Although several approaches have been previously described to optimize analytical ...
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An analytical HPLC-MS screening methodology has been developed to improve preparative RP-HPLC-MS purifications in medicinal chemistry laboratories. Although several approaches have been previously described to optimize analytical separations, none of them met our needs for the optimization of preparative conditions. Our screening protocol is based on searching among several orthogonal conditions to find the optimum preparative separation. Five different buffer conditions, from low to high pH, two organic solvents, acetonitrile and methanol, and five stationary phases of different polarities and characteristics were used. The orthogonality of the system was demonstrated using both, a standard mixture and mixtures from synthesis. To carry out the screening one of the analytical " open access" HPLC-MS systems was modified to perform the analytical screening while maintaining the open-access functionality for synthesis reaction monitoring. A software tool for automated sample programming and data reporting was also developed.
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Abstract Photoallergy test of cosmetics and several types of pharmaceutical substances is often necessary for obtaining approval from authorities. However, there are no official test guidelines for photoallergy evaluation. Therefo...
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Abstract Photoallergy test of cosmetics and several types of pharmaceutical substances is often necessary for obtaining approval from authorities. However, there are no official test guidelines for photoallergy evaluation. Therefore, we tried to establish a photoallergy test by utilizing an in chemico alternative sensitization method, amino acid derivative reactivity assay (ADRA). To determine the criteria for judging the photoallergy potential, photo‐ADRA with or without photoirradiation was performed using 60 photoallergenic chemicals, and cysteine and lysine derivatives were detected using high‐performance liquid chromatography either by absorbance or fluorescence measurement. The accuracy of prediction was 81.4% (48 of 59) and 80.0% (48 of 60) using the absorbance and fluorescence methods, respectively. However, as chemicals can breakdown into multiple chemicals during photoirradiation, the absorbance method often cannot perform accurate detection due to co‐elution, whereas the fluorescence method can do this due to lack of co‐elution. Moreover, all eight chemicals that were found to be negative or false‐positive for photoirritation in the 3T3 neutral red uptake phototoxicity test were confirmed as positive for photoallergy using this method. Furthermore, we prepared three types of pseudo‐mixtures where we added one photoallergen along with five nonphotoallergens and performed the photo‐ADRA by the ultraviolet and fluorescence methods. The result of the fluorescence method was almost the same as that obtained with the use of a single photoallergen and hence the outcome was not affected by the mixture. Thus, this study not only showed a method of evaluating the photoallergy potential of a single chemical but also a mixture, making it useful as an in chemico photoallergy alternative test.
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The determination of trace amountsof zirconium and fluoride in various matrixes (tap and bottled water) were investigated by high performance reversed phase liquid chromatography.? High performance liquid chromatography (HPLC) was...
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The determination of trace amountsof zirconium and fluoride in various matrixes (tap and bottled water) were investigated by high performance reversed phase liquid chromatography.? High performance liquid chromatography (HPLC) was used for determinationof? zirconium and fluoride based on their formed complex with 2-(5-bromo-2-pyridylazo)-5-diethylaminophenol (5-Br-PADAP). Solid-phase extraction (SPE) along with high performance liquid chromatographywas used for the analysis. Under optimum conditions, the detection limits for? Zr(IV) ?and flouride? were 0.4 ug/L, and 0.015 ug/L, respectively. Recoveries, 99.74–104.33% for Zr(IV) and 93.33-106.67% for fluoride were obtained. The sensitivity; precision and accuracy of the method are all satisfactory.
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