查看更多>>摘要：Therapeutic monoclonal antibodies (mAbs) are inherently heterogeneous and hence generally studied and controlled by an array of orthogonal separation methods. During drug candidate development, fractionation by HPLC is regularly employed to assist peak identification and product understanding. One overlooked challenge is the protein oxidation introduced by the fractionation process. In this study, we report the extent of fractionation-induced protein oxidation, which tends to complicate data interpretation and peak assignments. Higher-energy detectors such as fluorescence detectors and lower fraction concentration were found to exacerbate the oxidation artifacts. Other contributing factors than the detector induced photostress were also found to contribute significantly to protein oxidation. Furthermore, our study showed that collecting fractions into a solution with oxidation scavengers, such as histidine and methionine, was effective in eliminating the oxidation artifacts introduced by detector exposure and fraction processing steps. Through an example, we demonstrate that the modified fractionation workflow improves the accuracy of peak assignments. (c) 2021 Elsevier B.V. All rights reserved.
查看更多>>摘要：A method is proposed for measuring the relative contribution of extracolumn and column effects to the detrimental interactions which occur between metal-sensitive solutes and the complete HPLC system. The method involves the substitution of a length of narrow bore silica tubing for the column and measuring the extracolumn contribution, which is subtracted from the total bandspreading measured with a column in place to yield the column contribution. The investigation focussed on HILIC separations, which have been relatively little studied compared with similar effects in RPLC. Metal-solute interactions can lead to tailing peaks and reduced sensitivity or even irreversible adsorption of particularly challenging solutes such as mono-, di- and triphosphorylated nucleotides, which show strong interactions between their phosphate groups and metals. A deactivated HILIC column, treated by a vapour deposition procedure gave generally good results when using high pH (pH 9.0) mobile phases, which suppress the effects of metals. The addition of metal complexing agents such as citrate at low millimolar concentration gave further improvements in peak shape at high pH, and even micromolar concentrations of citrate or medronic acid showed good results. These lower concentrations are more favourable for LC-MS. Addition of the higher concentration of citrate gave acceptable results for the nucleotides even at low pH (pH 3.0). With the standard UHPLC instrument used, loss of efficiency due to metal solute interactions was 25% or less, with most losses due to interactions with the column, although this result will depend on the condition and design of the instrument, which is easily assessed by the proposed procedure. (C) 2021 Elsevier B.V. All rights reserved.
查看更多>>摘要：High-throughput screening of inhibitors from natural products is an efficient approach to target key enzymes in diabetes progression. In this study, an on-line detection system was established for the first time to rapidly screen inhibitors of alpha-amylase and alpha-glucosidase from Prunus mume . Among 28 identified compounds, 26 and 21 compounds showed strong inhibitory effect against alpha-amylase and alpha-glucosidase, respectively. Their inhibitory effects were validated by in vitro enzyme assay and fluorescence quenching which demonstrated that these inhibitors effectively interfered enzyme active sites. The inhibition kinetics suggested that chemical structures are of great importance for interfering the enzyme structures and their microenvironment polarity. Among evaluated compounds, isorhamnetin-3-O-glucoside (19) showed the strongest binding activities to alpha-amylase and alpha-glucosidase (6.34 x 10(6).nmol(-1) and 6.28 x 10(6).nmol(-1) , respectively) by the on-line detection system. Its IC50 values were 0.16 +/- 0.06 and 0.09 +/- 0.01 mu M against alpha-amylase and alpha-glucosidase, respectively. 19 gave a much higher K-i for alpha-amylase (0.1307 mM) than alpha-glucosidase (0.0063 mM), indicating its selectivity towards alpha-glucosidase. This reported method was rapid and reliable to identify prototype inhibitors against key enzymes in diabetes, and thus might serve as a general platform to screen enzyme inhibitors from natural products. (C) 2021 Elsevier B.V. All rights reserved.
Nakai, Diogo K.Ribeiro, Jose A. A.Martins, Pedro A.Soares, Itania P....
8页
查看更多>>摘要：A fast method based on reversed-phase ultra-high-performance liquid chromatography using evaporative light scattering detection (RP-UHPLC-ELSD) was developed for monitoring the intermediates and end products of biodiesel production. Gradient elution of water, acetonitrile, and a mixture of acetonitrile:2propanol:n-hexane was used. With a minimal and easy sample preparation, fatty acid methyl esters (FAME), fatty acid ethyl esters (FAEE), free fatty acids (FFA), monoacylglycerols (MAG), diacylglycerols (DAG), and triacylglycerols (TAG) were successfully separated. The developed method was used to monitor an ethylic enzymatic transesterification of soybean oil and to characterize the ester content of methyl and ethyl biodiesel. The ester content obtained was compared with the reference method by gas chromatography and flame ionization detector (GC-FID), with similar results for both methyl and ethyl biodiesel. The presented method is a simple and fast alternative, a 17 min run, to monitor the transesterification process, simultaneously quantifying all the analytes produced in the reaction: biodiesel and its intermediates. Limits of detection (LOD, between 29 and 307 ng) and quantification (LOQ, between 48 and 614 ng), linearity (R 2 > 0.99), precision (between 0.30 and 6.58%), and accuracy (between 81.6 and 119.9%) were determined for the twenty-one compounds. (c) 2021 Elsevier B.V. All rights reserved.
查看更多>>摘要：A simple and efficient dispersive solid-phase extraction (D-SPE) method combined with gas chromatography tandem mass spectrometry (GC-MS/MS) was developed to determine organochlorine pesticides (OCP) in honey. Firstly, a type of hybrid nanocomposite (CD-MOF/TiO 2 ) was prepared by grafting a metalorganic framework material synthesized with cyclodextrin as an organic ligand onto titanium dioxide. Then, the CD-MOF/TiO 2 was used as a D-SPE adsorbent to extract the OCP, and the effects of the amount of adsorbent, ultrasonic time, vortex time, pH, and salinity on the extraction were investigated using Plackett-Burman design and Box-Behnken Design. Under the optimized adsorption and desorption conditions, an analysis method that combined D-SPE with GC-MS/MS was established. The linear ranges of 14 OCP are 1-500 mu g kg -1 and the correlation coefficients are between 0.9991 and 1.0 0 0. The limits of detection and quantification vary from 0.01 to 0.04 mu g kg -1 and 0.04 to 0.12 mu g kg -1 , respectively. The intra-day and inter-day precision of this method are suitable (RSDs% less than 11.3%). The established CD-MOF/TiO 2 / D-SPE method was used for the extraction of OCP in honey samples with recovery in the range of 76.4 to 114.3%. The results demonstrate that the CD-MOF/TiO 2 has a good selective enrichment ability for OCP and is suitable for the D-SPE pretreat of honey sample analysis. (c) 2021 Elsevier B.V. All rights reserved.
查看更多>>摘要：A micellar liquid chromatographic method was developed to assist in the modeling of the skin permeability of pharmaceutical and cosmetic compounds. The composition of the mobile phase was determined by means of a two-factor central composite design, after which it was tested on both a particle-based and monolithic column. The latter provided the opportunity to increase the flow rate from 1 to 8 mL/min without reaching too high backpressures. The micellar conditions allowed analyzing a large test set of compounds with diverse characteristics with just one mobile-phase composition. The obtained experimental chromatographic descriptors besides two sets of theoretical molecular descriptors were used to model the skin permeability coefficient log K-p, applying multiple linear regression and partial least squares regression approaches. The micellar method on the monolithic column provided useful models with similar or even slightly better performance parameters than the method on the particle-based column. Furthermore, a much faster analysis can be achieved when applying a flow rate of 8 mL/min, making the micellar monolithic method ideal to estimate skin permeability. (C) 2021 Elsevier B.V. All rights reserved.
查看更多>>摘要：In this study, the first example of a polytetrafluoroethylene (PTFE)-based magnet coated with weak anion exchange (WAX) monolith as novel support for stir bar sorptive extraction (SBSE) is presented. Firstly, the PTFE magnets were properly modified and vinylized in order to immobilize polymer monoliths onto its surface. Then, a glycidyl methacrylate monolith was prepared and modified with ethylenediamine (EDA) to create weak anion exchanger via ring opening reaction of epoxy groups. The prepared covalently immobilized EDA-modified monoliths onto PTFE magnet exhibited good stability and reusability. Application of resulting material as stir bar for SBSE was investigated for a series of acidic compounds that includes acesulfame, saccharin, diclofenac or ibuprofen, among others as target compounds. Firstly, the SBSE conditions were optimized to promote the WAX interactions with the target compounds achieving recoveries from 37 to 75% and enable the selective extraction of these compounds as it provided values of% matrix effect from 17 to -13% when they were determined by SBSE followed by liquid chromatography tandem mass spectrometry. The analytical methodology, was then validated and applied for the determination of the target solutes in environmental water samples, which were found at concentration up to 2500 ng L-1 in river waters. (C) 2021 Elsevier B.V. All rights reserved.
查看更多>>摘要：We evaluate here different analytical strategies for the chromatographic separation and determination of N-acetyl-5-methoxytryptamine (MEL) and its oxidative metabolites N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK), N1-acetyl-5-methoxykynuramine (AMK) and cyclic 3-hydroxymelatonin (c3OHM) in cell culture samples. Two dimensional liquid chromatography (2D-LC) in the multiple heart-cutting mode was compared with regular 1D chromatographic separations of MEL and its oxidative metabolites. Our results showed that the use of trifluoroacetic acid (TFA) as mobile phase modifier was required to obtain a satisfactory resolution and peak shapes particularly for c3OHM. As TFA is not compatible with ESI ionization the application of the MHC mode was mandatory for a proper chromatographic separation. We evaluate also different internal standardization approaches based on the combined use of a surrogate standard (5-methoxytryptophol) and an internal standard (6-methoxytryptamine) for MEL quantification in cell culture samples obtaining unsatisfactory results both by 1D- and 2D-LC-ESI-MS/MS (from 9 +/- 2 to 186 +/- 38%). We demonstrate that only the application of isotope dilution Mass Spectrometry through the use of an in house synthesized C-13 isotopically labelled analogue provided quantitative MEL recoveries both by using 1D- and 2D-LC-ESI-MS/MS (99 +/- 1 and 98 +/- 1. Respectively) in androgen-insensitive human prostate carcinoma PC3 cells. (C) 2021 The Author(s). Published by Elsevier B.V.
查看更多>>摘要：The continuous quest for chromatographic supports offering kinetic performance properties superior to that of the packed bed of spheres has pushed the field to consider alternative formats such as for example monolithic and pillar array columns. This quest seems bound to culminate in the use of 3D printing technology, as this intrinsically offers the possibility to produce supports with a perfect uniformity and with a size and shape that is fully optimized for the chromatographic separation process. However, to be competitive with the current state-of-the-art, structures with sub-micron feature sizes are required. The present contribution therefore investigates the use of the 3D printing technology with the highest possible resolution available today, i.e. , two-photon polymerization (2PP). It is shown that 2PP printing is capable of achieving the required <= 1 mu m printing resolution. Depending on the laser scan speed, the lower limit through-pore size for a tetrahedral skeleton monolith with a theoretical 80% external porosity was found to be at 800 nm, when printing at a scan speed of 50 mm/s with a laser power of 10%. For a scan speed of 10 mm/s, the minimal through-pore size dropped to 500 nm. However, this very high resolution comes at the cost of excessively long printing times. The total printing time for a column volume equivalent to that of a typical nano-LC column (75 mu m i.d. cylindrical tube with length L = 15 cm) has been determined to correspond to 330 and 470 h for the 50 mm/s and the 10 mm/s scan speed respectively. Other issues remaining to be solved are the need to clad the printed skeleton with a suitable mesoporous layer for chromatographic retention and the need to add a top-wall to the printed channels after the removal of the non-polymerized resin. It is therefore concluded that 2PP printing is not ready yet to replace the existing column fabrication methods. (c) 2021 Elsevier B.V. All rights reserved.
查看更多>>摘要：Since conventional biological wastewater treatments are not admittedly effective to convert pharmaceutical active compounds (PhACs) into nontoxic products, natural abiotic mechanisms such as solar photolysis arises as an important degradation process, especially for halogenated molecules. In the present work, photolysis simulation was carried out in-lab for precursors and their respective photo-transformation products (photo-TPs), which were analyzed through reversed-phase ultra-high performance liquid chromatography coupled to high resolution mass spectrometry (RP-UHPLC-HRMS). An in-house library was created in order to provide reference information for target (precursors) and suspect screening (photo-TPs) analysis of freshwater samples from impacted aquatic environments. Strategies in the use of data-dependent acquisition (DDA) and data-independent acquisition (DIA), as well as the data processing software are discussed here for the identification of 6 PhACs and photo-TPs. Because no standards of photo-TPs were available, only the target compounds, i.e. sitagliptin (398 +/- 2 ng L-1), iohexol (209 +/- 5 ng L-1), lamotrigine (103 +/- 10 ng L-1), losartan (43 +/- 10 ng L-1), ofloxacin (28 +/- 7 ng L-1), and sertraline (25 +/- 7 ng L-1) could be quantified through multiple standard additions. (C) 2021 Elsevier B.V. All rights reserved.
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