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1.
Nat Commun ; 12(1): 2363, 2021 04 22.
Artículo en Inglés | MEDLINE | ID: mdl-33888690

RESUMEN

Cell-free gene expression (CFE) systems from crude cellular extracts have attracted much attention for biomanufacturing and synthetic biology. However, activating membrane-dependent functionality of cell-derived vesicles in bacterial CFE systems has been limited. Here, we address this limitation by characterizing native membrane vesicles in Escherichia coli-based CFE extracts and describing methods to enrich vesicles with heterologous, membrane-bound machinery. As a model, we focus on bacterial glycoengineering. We first use multiple, orthogonal techniques to characterize vesicles and show how extract processing methods can be used to increase concentrations of membrane vesicles in CFE systems. Then, we show that extracts enriched in vesicle number also display enhanced concentrations of heterologous membrane protein cargo. Finally, we apply our methods to enrich membrane-bound oligosaccharyltransferases and lipid-linked oligosaccharides for improving cell-free N-linked and O-linked glycoprotein synthesis. We anticipate that these methods will facilitate on-demand glycoprotein production and enable new CFE systems with membrane-associated activities.


Asunto(s)
Micropartículas Derivadas de Células/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/citología , Glicoproteínas/biosíntesis , Hexosiltransferasas/metabolismo , Proteínas de la Membrana/metabolismo , Biosíntesis de Proteínas , Membrana Celular/genética , Membrana Celular/metabolismo , Micropartículas Derivadas de Células/genética , Cromatografía Líquida de Alta Presión/métodos , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/aislamiento & purificación , Glicoproteínas/aislamiento & purificación , Hexosiltransferasas/genética , Hexosiltransferasas/aislamiento & purificación , Espectrometría de Masas/métodos , Proteínas de la Membrana/genética , Proteínas de la Membrana/aislamiento & purificación , Oligosacáridos/metabolismo , Ingeniería de Proteínas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
2.
J Chromatogr A ; 1644: 462121, 2021 May 10.
Artículo en Inglés | MEDLINE | ID: mdl-33845425

RESUMEN

In this study, we present results obtained on the enantioseparation of some cationic compounds of pharmaceutical relevance, namely tetrahydro-ß-carboline and 1,2,3,4-tetrahydroisoquinoline analogs. In high-performance liquid chromatography, chiral stationary phases (CSPs) based on strong cation exchanger were employed using mixtures of methanol and acetonitrile or tetrahydrofuran as mobile phase systems with organic salt additives. Through the variation of the applied chromatographic conditions, the focus has been placed on the study of retention and enantioselectivity characteristics as well as elution order. Retention behavior of the studied analytes could be described by the stoichiometric displacement model related to the counter-ion effect of ammonium salts as mobile phase additives. For the thermodynamic characterization parameters, such as changes in standard enthalpy Δ(ΔH°), entropy Δ(ΔS°), and free energy Δ(ΔG°), were calculated on the basis of van't Hoff plots derived from the ln α vs. 1/T curves. In all cases, enthalpy-driven enantioseparations were observed with a slight, but consistent dependence of the calculated thermodynamic parameters on the eluent composition. Elution sequences of the studied compounds were determined in all cases. They were found to be opposite on the enantiomeric stationary phases and they were not affected by either the temperature or the eluent composition.


Asunto(s)
Carbolinas/química , Cromatografía Líquida de Alta Presión/métodos , Resinas de Intercambio Iónico/química , Tetrahidroisoquinolinas/química , Acetonitrilos , Cationes , Metanol , Estereoisomerismo , Relación Estructura-Actividad , Temperatura
3.
Molecules ; 26(6)2021 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-33804019

RESUMEN

Therapeutic success in endodontic treatment depends on successful infection control. Alexidine dihydrochloride (ALX) was recently proposed as a potential alternative to 2% chlorhexidine (CHX) as it possesses similar antimicrobial properties, expresses substantivity and does not produce p-chloroaniline (PCA) when mixed with sodium hypochlorite (NaOCl). However, the products released in this reaction have not been described to date. The aim of this study was to identify detected chemical compounds formed in the reaction of ALX and NaOCl with the ultra-high-performance liquid chromatography-mass spectrophotometry (UHPLC-MS) method and assess whether precipitates and PCA are formed in this reaction. Solutions of ALX were mixed with the equivalent volume of 2% and 5.25% (w/v) NaOCl solutions. As control, 2% (w/v) CHX was mixed with 2% and 5.25% (w/v) NaOCl. Samples were subjected to the UHPLC-MS analysis. The mixture of ALX and NaOCl resulted in a yellowish precipitate formation, the amount of which depended on NaOCl concentration. Interaction of ALX and NaOCl resulted in the production of aliphatic amines. No PCA was formed when NaOCl was mixed with ALX. However, for the first time, we identified the possible products of the interaction. The interaction between NaOCl and ALX results in the formation of aliphatic amines; therefore, these compounds should not be mixed during endodontic treatment.


Asunto(s)
Biguanidas/efectos adversos , Biguanidas/farmacología , Hipoclorito de Sodio/efectos adversos , Hipoclorito de Sodio/farmacología , Aminas/farmacología , Antibacterianos/efectos adversos , Antibacterianos/farmacología , Clorhexidina/farmacología , Cromatografía Líquida de Alta Presión/métodos , Endodoncia/métodos , Humanos
4.
Molecules ; 26(6)2021 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-33804083

RESUMEN

Alkaloids of the Lycopodiaceae family are of great interest to researchers due to their numerous properties and wide applications in medicine. They play a very important role mainly due to their potent antioxidant, antidepressant effects and a reversible ability to inhibit acetylcholinesterase (AChE) enzyme activity. This property is of immense importance due to the growing problem of an increasing number of patients with neurodegenerative diseases in developed countries and a lack of effective and efficient treatment for them. Numerous studies have shown that Lycopodiaceae alkaloids are a rich source of AChE inhibitors. In the obtaining of new therapeutic phytochemicals from plant material, the extraction process and its efficiency is crucial. Therefore, the aim of this work was to optimize the conditions of modern PLE to obtain bioactive alkaloids from two Lycopodium species: L. clavatum L. and L. annotinum L. Five different solvents of different polarity were used for prepared plant extracts in order to compare the alkaloid content in and thereby effectiveness of the entire extraction. PLE parameters were used based on multiple studies conducted that gave the highest alkaloids recovery. Crude extracts were purified using solid-phase extraction (SPE) on Oasis HLB cartridge and examined by HPLC/ESI-QTOF-MS of the highly abundant alkaloids. To the best of our knowledge, this is the first time such high recoveries have been obtained for known Lycopodiaceae alkaloids. The best extraction results of alkaloid-lycopodine were detected in the dichloromethane extract from L. clavatum, where the yield exceeded 45%. The high recovery of annotinine above 40% presented in L. annotinum was noticed in dichloromethane and ethyl acetate extracts. Moreover, chromatograms were obtained with all isolated alkaloids and the best separation and quality of the bands in methanolic extracts. Interestingly, no alkaloid amounts were detected in cyclohexane extracts belonging to the non-polar solvent. These results could be helpful for understanding and optimizing the best conditions for isolating potent AChE inhibitors.


Asunto(s)
Alcaloides/química , Lycopodiaceae/química , Lycopodium/química , Extractos Vegetales/química , Acetilcolinesterasa/química , Antioxidantes/química , Inhibidores de la Colinesterasa/química , Cromatografía Líquida de Alta Presión/métodos , Espectrometría de Masas/métodos , Extracción en Fase Sólida/métodos
5.
Molecules ; 26(6)2021 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-33804205

RESUMEN

In this study, we determined the phytochemical profile of the Spanish "triguero" asparagus landrace "verde-morado" (Asparagus officinalis L.), a wild traditional landrace, and the improved "triguero" HT-801, together with two commercial green asparagus varieties. For comparison, we used reverse-phase high-performance liquid chromatography coupled with diode array electrospray time-of-flight mass spectrometry (RP-HPLC-DAD-ESI-TOF/MS) followed by a permutation test applied using a resampling methodology valid under a relaxed set of assumptions, such as i.i.d. errors (not necessarily normal) that are exchangeable under the null hypothesis. As a result, we postulate that "triguero" varieties (the improved HT-801 followed by its parent "verde-morado") have a significantly different phytochemical profile from that of the other two commercial hybrid green varieties. In particular, we found compounds specific to the "triguero" varieties, such as feruloylhexosylhexose isomers, or isorhamnetin-3-O-glucoside, which was found only in the "triguero" variety HT-801. Although studies relating the phytochemical content of "triguero" asparagus varieties to its health-promoting effects are required, this characteristic phytochemical profile can be used for differentiating and revalorizating these asparagus cultivars.


Asunto(s)
Asparagus (Planta)/química , Fitoquímicos/química , Extractos Vegetales/química , Cromatografía Líquida de Alta Presión/métodos , Flavonoides/química , Flavonoles/química , Saponinas/química , Espectrometría de Masa por Ionización de Electrospray/métodos
6.
J Chromatogr A ; 1645: 462120, 2021 May 24.
Artículo en Inglés | MEDLINE | ID: mdl-33839575

RESUMEN

The quantitative structure-retention relationship (QSRR) models are not only employed in retention behaviour prediction, but also in an in-depth understanding of complex chromatographic systems. The goal of the present research is to enable the comprehensive understanding of retention underlying the separation in ß-cyclodextrin (CD) modified reversed-phase high performance liquid chromatography (RP-HPLC) systems, through the development of mixed QSRR models. Moreover, the amount of ß-CD adsorbed on the stationary phase surface (ß-CDA) is added as the model's input in order to evaluate its contribution to both model performances and retention. Nuclear magnetic resonance (NMR) experiments were conducted to confirm the predicted inclusion complex structures and support the application of in silico tools. The most significant descriptors revealed that retention is governed by the steric factors 7.5 Å distant from the geometrical centre of a molecule, 3D arrangement of atoms determining the molecular size and shape, lipophilicity indicated by topological distances, as well as the unbound system's energy, related to the inclusion complex formation. In addition, a notable effect of the pH of the aqueous phase on the retention of ionizable analytes was shown. In the case of pH of the aqueous phase and ß-CDA the change in retention behaviour of the studied analytes was observed only at the highest ß-CDA value (5.17 µM/m2), but it was not related to the ionization state of analytes. When the analytes did not change the ionization form across the investigated studied pH range, and the acetonitrile content in the mobile phase was 25% (v/v), the retention factor had low values regardless of the ß-CDA; under these circumstances the retention is probably acetonitrile driven.


Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Modelos Químicos , beta-Ciclodextrinas/química , Acetonitrilos/química , Cromatografía de Fase Inversa/métodos , Concentración de Iones de Hidrógeno , Espectroscopía de Resonancia Magnética
7.
Int J Mol Sci ; 22(5)2021 Mar 06.
Artículo en Inglés | MEDLINE | ID: mdl-33800919

RESUMEN

Trypsin inhibitors (TI), a common anti-nutritional factor in soybean, prevent animals' protein digestibility reducing animal growth performance. No commercial soybean cultivars with low or null concentration of TI are available. The availability of a high throughput genotyping assay will be beneficial to incorporate the low TI trait into elite breeding lines. The aim of this study is to develop and validate a breeder friendly Kompetitive Allele Specific PCR (KASP) assay linked to low Kunitz trypsin inhibitor (KTI) in soybean seeds. A total of 200 F3:5 lines derived from PI 547656 (low KTI) X Glenn (normal KTI) were genotyped using the BARCSoySNP6K_v2 Beadchip. F3:4 and F3:5 lines were grown in Blacksburg and Orange, Virginia in three years, respectively, and were measured for KTI content using a quantitative HPLC method. We identified three SNP markers tightly linked to the major QTL associated to low KTI in the mapping population. Based on these SNPs, we developed and validated the KASP assays in a set of 93 diverse germplasm accessions. The marker Gm08_44814503 has 86% selection efficiency for the accessions with low KTI and could be used in marker assisted breeding to facilitate the incorporation of low KTI content in soybean seeds.


Asunto(s)
Genes de Plantas , Fitomejoramiento , Reacción en Cadena de la Polimerasa/métodos , Polimorfismo de Nucleótido Simple , Sitios de Carácter Cuantitativo , Semillas/enzimología , Soja/genética , Inhibidor de la Tripsina de Soja de Kunitz/genética , Alelos , Cromatografía Líquida de Alta Presión/métodos , ADN de Plantas/análisis , ADN de Plantas/genética , Ligamiento Genético , Fenotipo , Hojas de la Planta/química , Soja/enzimología , Inhibidor de la Tripsina de Soja de Kunitz/análisis
8.
J Chromatogr A ; 1645: 462074, 2021 May 24.
Artículo en Inglés | MEDLINE | ID: mdl-33848656

RESUMEN

We prepared two-dimensional (2D) bimetallic metal-organic frameworks (Ni-ZIF-8) nanosheets by a simple solvent-free method at room temperature. The morphology and composition of Ni-ZIF-8 can be controlled through adding different amounts of Ni. And then, the 2D magnetic mesoporous nanosheets (Ni/ZnO@C) were synthesized by directly pyrolyzing Ni-ZIF-8 under argon atmosphere and explored as magnetic solid phase extraction (MSPE) adsorbents for the determination of nitroimidazole antibiotics (NIABs). Magnetic Ni nanoparticles embedded in carbon nanosheets uniformly resulted in high magnetization saturation of Ni/ZnO@C for easy separation. The Ni/ZnO@C can form hydrogen bond and π-π interaction with three NIABs resulting from their rich N-H containing imidazole, π-electron. Due to the high specific surface area and high mass transfer rate of 2D Ni/ZnO@C, the materials showed satisfactory adsorption capacity and rapid adsorption kinetics for NIABs. A rapid and effective method of Ni/ZnO@C-MSPE combined with high-performance liquid chromatography was proposed for the determination of NIABs. Several main parameters affecting MSPE were investigated. Under the optimal conditions, wide linear was achieved ranging from 0.1 to 500 µg⋅L-1 with a low detection limit of 0.025-0.05 µg⋅L-1. The established method has been successfully applied to analyze NIABs from environmental water samples with satisfactory recovery from 74.33 to 105.71%.


Asunto(s)
Antibacterianos , Nanopartículas del Metal/química , Estructuras Metalorgánicas/química , Nitroimidazoles , Extracción en Fase Sólida/métodos , Antibacterianos/análisis , Antibacterianos/aislamiento & purificación , Carbono/química , Cromatografía Líquida de Alta Presión/métodos , Límite de Detección , Modelos Lineales , Imanes/química , Nanoestructuras/química , Níquel/química , Nitroimidazoles/análisis , Nitroimidazoles/aislamiento & purificación
9.
J Chromatogr A ; 1645: 462097, 2021 May 24.
Artículo en Inglés | MEDLINE | ID: mdl-33848664

RESUMEN

Hemp has been an agricultural commodity for millennia, and it has been undergoing a resurgence in interest and production due to its high content of cannabinoids, protein, fiber and other ingredients. For legal possession and use throughout the USA, hemp and hemp products must have delta-9-tetrahydrocannabinol (THC) concentration < 0.3%. As with most crops, pesticides may be applied when farming hemp, which need to be monitored in food, feed, and medicinal products. The aim of this work was to evaluate and validate the recently developed "quick, easy, cheap, effective, rugged, safe, efficient, and robust" (QuEChERSER) sample preparation mega-method to determine pesticide residues in hemp plants, flowers, powders, oils, and pellets. High-throughput analysis of final extracts for 106 targeted pesticides and metabolites from North American monitoring lists entailed: 1) ultrahigh-performance liquid chromatography - tandem mass spectrometry (UHPLC-MS/MS) with column back-flushing, and 2) instrument-top sample preparation + low-pressure gas chromatography (ITSP+LPGC-MS/MS). In QuEChERSER, 2 g sample is extracted with 10 mL 4/1 (v/v) acetonitrile/water by mechanical shaking for 10 min, followed by 3 min centrifugation. For LC, 0.2 mL of extract is taken and solvent exchanged into initial mobile phase followed by 5 min ultra-centrifugation prior to the 10 min analysis. For GC-amenable pesticides, the remaining initial extract is partitioned with 4/1 (w/w) anh. MgSO4/NaCl, and 1 mL is taken for automated ITSP cleanup in parallel with 10 min LPGC analysis. In the former case, the UHPLC column is back-flushed with 1/1 (v/v) methanol/acetonitrile for 3 min between each injection to keep the system clean and avoid ghost peaks. Multi-level, multi-day validation results achieved 70-120% recoveries with RSDs < 20% for more than 80% of the analytes in hemp protein powder, oil, pellets, and fresh plant (dried hemp plant and flower were too complex). Limits of quantification (LOQs) were < 10 ng/g were achieved for nearly all pesticides, yielding 2.8% false negatives among >13,000 analyte results in the spiked samples. The QuEChERSER method was demonstrated to meet the challenge for several complex hemp matrices.


Asunto(s)
Cannabis/química , Residuos de Plaguicidas/análisis , Preparaciones de Plantas/química , Cromatografía Líquida de Alta Presión/métodos , Límite de Detección , Reproducibilidad de los Resultados , Espectrometría de Masas en Tándem/métodos
10.
Molecules ; 26(6)2021 Mar 10.
Artículo en Inglés | MEDLINE | ID: mdl-33802020

RESUMEN

Glycyrrhizae Radix et Rhizoma (GRR) is one of the commonly used traditional Chinese medicines in clinical practice, which has been applied to treat digestive system diseases for hundreds of years. GRR is preferred for anti-gastric ulcer, however, the main active compounds are still unknown. In this study, GRR was used as precursor to synthesize carbon dots (CDs) by a environment-friendly one-step pyrolysis process. GRR-CDs were characterized by using transmission electron microscopy, high-resolution TEM, fourier transform infrared, ultraviolet-visible and fluorescence spectroscopy, X-ray photoelectron spectroscopy, X-ray diffraction and high-performance liquid chromatography. In addition, cellular toxicity of GRR-CDs was studied by using CCK-8 in RAW264.7 cells, and the anti-gastric ulcer activity was evaluated and confirmed using mice model of acute alcoholic gastric ulcer. The experiment confirmed that GRR-CDs were the spherical structure with a large number of active groups on the surface and their particle size ranged from 2 to 10 nm. GRR-CDs had no toxicity to RAW264.7 cells at concentration of 19.5 to 5000 µg/mL and could reduce the oxidative damage of gastric mucosa and tissues caused by alcohol, as demonstrated by restoring expression of malondialdehyde, superoxide dismutase and nitric oxide in serum and tissue of mice. The results indicated the explicit anti-ulcer activity of GRR-CDs, which provided a new insights for the research on effective material basis of GRR.


Asunto(s)
Medicamentos Herbarios Chinos/química , Medicamentos Herbarios Chinos/farmacología , Puntos Cuánticos/química , Animales , Carbono/química , China , Cromatografía Líquida de Alta Presión/métodos , Glycyrrhiza/química , Masculino , Medicina China Tradicional/métodos , Ratones , Espectroscopía de Fotoelectrones/métodos , Pirólisis , Células RAW 264.7 , Espectrometría de Fluorescencia/métodos
11.
Molecules ; 26(6)2021 Mar 10.
Artículo en Inglés | MEDLINE | ID: mdl-33802127

RESUMEN

The aim of this work was to characterize the antioxidant properties of some of the peptides present in bromelain mung bean meal protein hydrolysate (MMPH). The MMPH was subjected to two rounds of bioassay-guided reversed-phase HPLC separation followed by peptide identification in the most potent fractions using tandem mass spectrometry. Twelve antioxidant peptides, namely, HC, CGN, LAN, CTN, LAF, CSGD, MMGW, QFAAD, ERF, EYW, FLQL, and QFAW were identified and assayed for antioxidant properties. CTN, HC, CGN, and CSGD were the most potent (p < 0.05) DPPH radical scavengers with EC50 values of 0.30, 0.29, 0.28, and 0.30 mg/mL, respectively, which are lower than the 0.03 mg/mL obtained for reduced glutathione (GSH). CTN, HC, CGN, and CSGD exhibited the most potent (p < 0.05) scavenging activities against hydroxyl and superoxide radicals with EC50 values that are similar to those of GSH. The cysteine-containing peptides also had stronger ferric reducing antioxidant power and metal chelation activity than peptides devoid of cysteine. In contrast, MMGW, ERF, and EYW had poor radical scavenging and metal chelation activities. We conclude that the availability of the sulfhydryl group may have enhanced antioxidant potency while the presence of bulky groups such phenylalanine and tryptophan had an opposite effect.


Asunto(s)
Péptidos/química , Vigna/enzimología , Vigna/metabolismo , Antioxidantes/química , Antioxidantes/aislamiento & purificación , Quelantes , Cromatografía Líquida de Alta Presión/métodos , Depuradores de Radicales Libres/química , Glutatión/metabolismo , Radical Hidroxilo , Peroxidación de Lípido , Hidrolisados de Proteína/química , Proteínas/química , Superóxidos/química
12.
Molecules ; 26(6)2021 Mar 10.
Artículo en Inglés | MEDLINE | ID: mdl-33802139

RESUMEN

It is usually a tedious task to profile the chemical composition of a given herbal medicine (HM) using high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) due to the time-consuming sample preparation and laborious post-acquisition data processing procedures. Even worse, some labile compounds may face degradation risks when exposed to organic solvents for a relatively long period. As one of the most popular HMs, the promising therapeutic benefits of Epimedii Herba (Chinese name: Yinyanghuo) are well defined; however, the chemical profile, and in particular those flavonoids that have been claimed to be responsible for the efficacy, remains largely unknown. Attempts are devoted here to achieve direct LC-MS measurement and efficient post-acquisition data processing, and chemome comparison among three original sources of Epimedii Herba, such as Epimedium sagittatum (Esa), E. pubescens (Epu), and E. koreanum (Eko) was employed to illustrate the strategy utility. A home-made online liquid extraction (OLE) module was introduced at the front of the analytical column to comprehensively transfer the compounds from raw materials onto the LC-MS instrument. A mass defect filtering approach was programmed to efficiently mine the massive LC-MS dataset after which a miniature database was built involving all chemical information of flavonoids from the genus Epimedium to draw a pentagonal frame to rapidly capture potential quasi-molecular ions (mainly [M-H]-). A total of 99 flavonoids (66 in Esa, 84 in Eko, and 66 in Epu) were captured, and structurally annotated by summarizing the mass fragmentation pathways from the mass spectrometric data of authentic compounds and an in-house data library as well. Noteworthily, neutral loss of 144 Da was firstly assigned to the neutral cleavage of rhamnosyl residues. Significant species-differences didn't occur among their chemical patterns. The current study proposed a robust strategy enabling rapid chemical profiling of, but not limited to, HMs.


Asunto(s)
Epimedium/química , Flavonoides/química , China , Cromatografía Líquida de Alta Presión/métodos , Epimedium/metabolismo , Flavonoides/metabolismo , Medicina China Tradicional/métodos , Plantas Medicinales/química , Espectrometría de Masas en Tándem/métodos
13.
Molecules ; 26(6)2021 Mar 16.
Artículo en Inglés | MEDLINE | ID: mdl-33809511

RESUMEN

Triterpenoids have regained much attention as promising multi-targeting bioactive agents of natural origin in the treatment of numerous disorders. Due to the high potential for phytopharmaceutical development, accurate qualitative and quantitative analysis of triterpenoids for screening and quality control is required. Vaccinium vitis-idaea L. (lingonberry) raw materials have aroused interest as a rich source of triterpenoids. However, currently, no validated, rapid, and easy-to-perform quantification method is available for the routine control of these compounds in lingonberries. This research aimed at developing and validating HPLC-PDA methods for the determination and screening of triterpenoids in extracts of lingonberry leaves, fruits, and flowers. The developed methods were deemed satisfactory by validation, which revealed acceptable analytical specificity, linearity (r2 > 0.9999), precision (RSD < 2%), trueness (94.70-105.81%), and sensitivity (LOD: 0.08-0.65 µg/mL). The real sample analysis demonstrated established methods applicability for quantification of 13 triterpenoids in lingonberries and emphasized differences between raw materials. Lingonberry fruits were distinguished by the richness of ursolic acid; lingonberry flowers by similar profile to fruits, but low content of neutral triterpenoids; whereas lingonberry leaves by the particularly high level of α-amyrin. Thus, the proposed methods proved to be reliable and applicable for quantification and routine analysis of triterpenoids in lingonberry samples.


Asunto(s)
Triterpenos/química , Vaccinium vitis-Idaea/química , Cromatografía Líquida de Alta Presión/métodos , Flores/química , Frutas/química , Triterpenos Pentacíclicos/química , Extractos Vegetales/química , Hojas de la Planta/química , Control de Calidad , Vaccinium myrtillus/química
14.
Molecules ; 26(6)2021 Mar 16.
Artículo en Inglés | MEDLINE | ID: mdl-33809536

RESUMEN

Pyrrolizidine alkaloids (PAs) are a class of natural toxins with hepatotoxicity, genotoxicity and carcinogenicity. They are endogenous and adulterated toxic components widely found in food and herbal products. In this study, a sensitive and efficient ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was used to detect the PAs in 386 kinds of Chinese herbal medicines recorded in the Chinese Pharmacopoeia (2020). The estimated daily intake (EDI) of 0.007 µg/kg body weight (bw)/day was adopted as the safety baseline. The margin of exposure (MOE) approach was applied to evaluate the chronic exposure risk for the genotoxic and carcinogenic potential of PAs. Results showed that PAs was detected in 271 out of 386 samples with a content of 0.1-25,567.4 µg/kg, and there were 20 samples with EDI values above the baseline, 0.007 µg/kg bw/day. Beyond that, the MOE values for 10 out of 271 positive samples were below 10,000. Considering the actual situation, Haber's rule was used to assume two weeks exposure every year during lifetime, and still the MOE values for four out of 271 positive samples were under 10,000, indicating these products may have potential health risk. The developed method was successfully applied to detect the PAs-containing Chinese herbal medicines. This study provides convincing data that can support risk management actions in China and a meaningful reference for the rational and safe use of Chinese herbal medicines.


Asunto(s)
Medicamentos Herbarios Chinos/química , Alcaloides de Pirrolicidina/química , Carcinógenos/química , China , Cromatografía Líquida de Alta Presión/métodos , Medicina de Hierbas/métodos , Humanos , Medición de Riesgo , Espectrometría de Masas en Tándem/métodos
15.
Molecules ; 26(6)2021 Mar 16.
Artículo en Inglés | MEDLINE | ID: mdl-33809747

RESUMEN

The authors developed a 1H qNMR test procedure for identification and quantification of impurity A present in gabapentin active pharmaceutical ingredient (API) and gabapentin products. The validation studies helped to determine the limit of quantitation and assess linearity, accuracy, repeatability, intermediate precision, specificity, and robustness of the procedure. Spike-and-recovery assays were used to calculate standard deviations, coefficients of variation, confidence intervals, bias, Fisher's F test, and Student's t-test for assay results. The obtained statistical values satisfy the acceptance criteria for the validation parameters. The authors compared the results of impurity A quantification in gabapentin APIs and capsules by using the 1H qNMR and HPLC test methods.


Asunto(s)
Contaminación de Medicamentos/prevención & control , Gabapentina/química , Cápsulas/química , Cromatografía Líquida de Alta Presión/métodos , Límite de Detección , Espectroscopía de Resonancia Magnética/métodos , Estándares de Referencia , Reproducibilidad de los Resultados
16.
Arch Insect Biochem Physiol ; 106(3): e21771, 2021 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-33644898

RESUMEN

Antimicrobial proteins (AMPs) are small, cationic proteins that exhibit activity against bacteria, viruses, parasites, fungi as well as boost host-specific innate immune responses. Insects produce these AMPs in the fat body and hemocytes, and release them into the hemolymph upon microbial infection. Hemolymph was collected from the bacterially immunized fifth instar larvae of tasar silkworm, Antheraea mylitta, and an AMP was purified by organic solvent extraction followed by size exclusion and reverse-phase high-pressure liquid chromatography. The purity of AMP was confirmed by thin-layer chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The molecular mass was determined by matrix-assisted laser desorption ionization-time of flight mass spectrometry as 14 kDa, and hence designated as AmAMP14. Peptide mass fingerprinting of trypsin-digested AmAMP14 followed by de novo sequencing of one peptide fragment by tandem mass spectrometry analysis revealed the amino acid sequences as CTSPKQCLPPCK. No homology was found in the database search and indicates it as a novel AMP. The minimum inhibitory concentration of the purified AmAMP14 was determined against Escherichia coli, Staphylococcus aureus, and Candida albicans as 30, 60, and 30 µg/ml, respectively. Electron microscopic examination of the AmAMP14-treated cells revealed membrane damage and release of cytoplasmic contents. All these results suggest the production of a novel 14 kDa AMP in the hemolymph of A. mylitta to provide defense against microbial infection.


Asunto(s)
Péptidos Catiónicos Antimicrobianos , Hemolinfa/metabolismo , Proteínas de Insectos/aislamiento & purificación , Mariposas Nocturnas/metabolismo , Animales , Antibacterianos/química , Antibacterianos/aislamiento & purificación , Antibacterianos/farmacología , Péptidos Catiónicos Antimicrobianos/química , Péptidos Catiónicos Antimicrobianos/aislamiento & purificación , Péptidos Catiónicos Antimicrobianos/farmacología , Candida albicans/efectos de los fármacos , Cromatografía en Gel/métodos , Cromatografía Líquida de Alta Presión/métodos , Escherichia coli/efectos de los fármacos , Proteínas de Insectos/química , Proteínas de Insectos/metabolismo , Proteínas de Insectos/farmacología , Larva/metabolismo , Extracción Líquido-Líquido/métodos , Pruebas de Sensibilidad Microbiana , Staphylococcus aureus/efectos de los fármacos
17.
J Chromatogr A ; 1643: 462079, 2021 Apr 26.
Artículo en Inglés | MEDLINE | ID: mdl-33780878

RESUMEN

In the present study, a comprehensive and sensitive method for simultaneous determination of 21 PIs (nine benzophenones, eight amine co-initiators, and four thioxanthones) in human plasma using high-performance liquid chromatography coupled with tandem mass spectrometry was developed and validated. Two different pre-treatment approaches (liquid-liquid extraction (LLE) and LLE coupled with solid-phase extraction (SPE)) and eight extraction solvents were studied to optimize sample treatment to obtain good recoveries and reduce any matrix effects. The procedure of LLE+SPE was selected as final sample treatment procedure because it obtained higher recoveries as well as lower matrix effects than that performed by LLE alone. The recoveries of 21 target analytes at three spiked concentrations (0.05, 0.5, and 5 ng/mL) ranged from 81% to 109%. The intra- and inter-day relative standard deviations were between 2.5% and 13%. Accuracy and precision data indicated that the detection method was accurate and precise for most of the PIs. The linearities of the labeled dilution calibration curves at 10 concentration levels (iLOQ to 100 ng/mL or iLOQ to 200 ng/mL) were good with correlation coefficients ranged from 0.995 to 0.999. The method quantification limits were in the range of 1.7-16 pg/mL. The analytical method was applied to the analysis of PIs in 14 human plasma samples collected from pregnant women in Guangdong Province, China. Fifteen PIs were detected with total concentrations ranging from 318 to 2772 pg/mL. The ubiquitous contamination of human plasma with PIs suggests that there is widespread exposure to these compounds. Consequently, there should be increased awareness of these pollutants in the environment.


Asunto(s)
Benzofenonas/sangre , Cromatografía Líquida de Alta Presión/métodos , Xantonas/sangre , Adulto , Benzofenonas/aislamiento & purificación , Benzofenonas/normas , Cromatografía Líquida de Alta Presión/normas , Contaminantes Ambientales/sangre , Femenino , Voluntarios Sanos , Humanos , Límite de Detección , Extracción Líquido-Líquido , Embarazo , Control de Calidad , Extracción en Fase Sólida , Espectrometría de Masas en Tándem/métodos , Espectrometría de Masas en Tándem/normas , Tioxantenos/sangre , Tioxantenos/aislamiento & purificación , Tioxantenos/normas , Xantonas/aislamiento & purificación , Xantonas/normas
18.
J Chromatogr A ; 1643: 462081, 2021 Apr 26.
Artículo en Inglés | MEDLINE | ID: mdl-33780883

RESUMEN

In the present work, with the focus on an environmentally-friendly approach, some gels were prepared by synthesizing amine-modified lignin, extracted from sugarcane bagasse, and further esterification and subsequent freeze-drying. These lignin-based gels were implemented as extractive phases in an online micro-solid phase extraction (µSPE) setup in conjunction with high performance liquid chromatography (HPLC) with UV detector. The developed method was used for analytical determination of valsartan and losartan in urine samples. To study the effect of the functionalization process, the efficiency of the unmodified lignin and the functionalized lignin were compared both in the absence and the presence of graphene oxide (GO), presumably as a suitable doping agent. Surprisingly, higher extraction efficiency for the functionalized lignin, compared to both unmodified lignin and GO was observed. The amination process for the prepared gel was analyzed and proved by CHNS elemental analysis and Fourier transform infrared (FT-IR) spectroscopy. The morphology of sorbet was investigated via scanning electron microscope (SEM) imaging and a nanoscale cauliflower feature was observed. The method was optimized and subsequently applied to the analysis of the urine samples. Limits of detection (LOD) of 8 and 6 µg L - 1, limits of quantification (LOQ) of 27 and 20 µg L - 1 and linear dynamic range (LDR) of 27-2000 and 20-2000 µg L - 1 with intraday relative standard deviations (RSD%) of 4 and 3% were obtained for valsartan and losartan, respectively. The whole online µSPE-HPLC setup was conveniently used for the analysis of a patient urine sample and a quantity of 352 µg L - 1 of losartan was found. Acceptable relative recoveries (109-108 and 95-94% for valsartan and losartan) revealed the analytical potential of the method for the determination of drugs in complex urine samples.


Asunto(s)
Lignina/química , Losartán/orina , Microextracción en Fase Sólida/métodos , Valsartán/orina , Aminas/química , Celulosa/metabolismo , Cromatografía Líquida de Alta Presión/métodos , Grafito/química , Humanos , Límite de Detección , Losartán/aislamiento & purificación , Reproducibilidad de los Resultados , Espectrofotometría Ultravioleta , Valsartán/aislamiento & purificación
19.
J Chromatogr A ; 1643: 462088, 2021 Apr 26.
Artículo en Inglés | MEDLINE | ID: mdl-33784502

RESUMEN

Fentanyl analogues used in therapy and a range of highly potent non-pharmaceutical fentanyl derivatives are subject to international control, as the latter are increasingly being synthesized illicitly and sold as 'synthetic heroin', or mixed with heroin. A significant number of hospitalizations and deaths have been reported in the EU and USA following the use of illicitly synthesized fentanyl derivatives. It has been unequivocally demonstrated that the enantiomers of fentanyl derivatives exhibit different pharmaco-toxicological profiles, which makes crucial to avail of suitable analytical methods enabling investigations at a "stereochemical level". Chromatographic methods useful to discriminate the enantioseparation of fentanyls and their derivatives are still missing in the literature. This is the first study in which the enantioseparation of four fentanyl derivatives, that is, (±)-trans-3-methyl norfentanyl, (±)-cis-3-methyl norfentanyl, ß-hydroxyfentanyl, and ß-hydroxythiofentanyl, has been obtained under polar-ionic conditions. Indeed, the use of ACN-based mobile phases with minor amounts of either 2-propanol or ethanol (plus diethylamine and formic acid as ionic additives) allowed obtaining enantioseparation and enantioresolution factors up to 1.83 and 7.02, respectively. For the study, the two chiral stationary phases cellulose tris(3-chloro-4-methylphenylcarbamate) and cellulose tris(4-chloro-3-methylphenylcarbamate) were used, displaying a remarkably different performance towards the enantioseparation of (±)-cis-3-methyl norfentanyl. Chiral LC analyses with a high-resolution mass spectrometry detector were also carried out in order to confirm the obtained data and demonstrate the suitability and compatibility of the optimized mobile phases with mass spectrometric systems.


Asunto(s)
Celulosa/química , Cromatografía Líquida de Alta Presión/métodos , Fentanilo/análisis , Fentanilo/análogos & derivados , Fentanilo/aislamiento & purificación , Espectrometría de Masas , Estereoisomerismo
20.
J Chromatogr A ; 1643: 462069, 2021 Apr 26.
Artículo en Inglés | MEDLINE | ID: mdl-33784503

RESUMEN

The present work reports on the preparation of polythiol-functionalized silica particles by thermally and photo-initiated radical addition reactions using poly(3-mercaptopropyl)methylsiloxane (PMPMS) as sulfhydryl group-rich surface modification reagent. Prior to surface modification with PMPMS, the silica was vinylized with vinyl trimethoxysilane. Finally, the usefulness of the thiolated silica particles was demonstrated by their further modification for various HPLC applications such as argentation chromatography and chiral separations. Aiming at a sulfhydryl group-rich, thin PMPMS layer on the surface of the silica several factors such as quantity of PMPMS, radical starter and reaction time were investigated by a design of experiment (DoE) approach. In thermally induced polymerization reactions 2,2'-azobis(isobutyronitrile) (AIBN) was used as radical starter, in photo-induced reactions 2,2-dimethoxy-2-phenylacetophenone (DMPA) was used instead. The incorporation of PMPMS was evaluated by elemental analysis and reactive and accessible sulfhydryl groups were determined by performing a thiol-disulfide exchange reaction with 2,2'-dipyridyl disulfide (DPDS). Consequently, thiol-functionalized silica particles (200 Å, 5 µm) with 1.81 ± 0.07 µmol sulfhydryl groups per m2 were prepared and further functionalized for silver ion chromatography and chiral separation chromatography clearly proving its utility as platform for further silica functionalization. The fabricated stationary phase for silver ion chromatography showed promising separation abilities for fatty acid methyl esters (FAME) according to the amount of double bonds within the fatty acid residue and cis- and trans-stilbene as model molecule for cis-trans isomerism. After the successful incorporation of O-tert-butylcarbamoyl quinine (tBuCQN) as chiral selector via thiol-ene click chemistry onto the PMPMS layer, the obtained chiral stationary phases (CSP) showed good separation of derivatized amino acids in polar organic elution mode comparable with a column based on commercially available CHIRALPAK QN-AX silica particles (120 Å, 5 µm).


Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Cromatografía por Intercambio Iónico/métodos , Dióxido de Silicio/química , Siliconas/química , Concentración de Iones de Hidrógeno , Plata/química , Estereoisomerismo , Compuestos de Sulfhidrilo/química , Propiedades de Superficie
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