查看更多>>摘要：? 2022 Elsevier B.V.Extra virgin olive oil is a potentially vulnerable foodstuff that can be mixed with other vegetal edible oils including poorer quality olive oils in order to obtain illicit profits. These unauthorized operations may take place at any stage of the production process and radically affect the chemical composition. In this paper, the analysis of different virgin olive oil samples before and after blending with other lower-grade olive oils in different proportions were performed. The direct analysis of the samples by (NP)HPLC-DAD in a wavelength range between 190 and 700 nm allowed the simultaneous analysis of several compound families responsible of the colour including chlorophylls, pheophytins, carotenes and tocopherols, the first three responsible for the olive oil colour. Unsupervised pattern recognition techniques applied on the chromatography-agnostic fingerprints of unblended virgin olive oil samples clearly showed the occurrence of groupings according to the sample hue (green and yellow). Two strategies, based on revealing changes in the spectrum-chromatographic fingerprints, are tested in order to detect the occurrence of such fraudulent blends: two-input class classification methods (SIMCA) and similarity analysis. The SIMCA strategy was effective only for detecting blends carried out on virgin olive oils with a greenish hue (high chlorophyll/pheophytin content). Furthermore, the similarity profile, developed and applied for the first time in this study evidences the blending in all cases irrespective of the original olive oil hue.
查看更多>>摘要：? 2022Two-dimensional liquid chromatography (2DLC) offers great separation power for complex mixtures. The frequently encountered incompatibility of two orthogonal separation systems, however, makes its application complicated. Active-modulation strategies can reduce such incompatibility issues considerably. Stationary-phase-assisted modulation (SPAM) is the most-common of these techniques, but also the least robust due to the major disadvantage that analytes may elute prematurely. The range of liquid chromatography (LC) applications continues to expand towards ever more complex mixtures. Retention modelling is increasingly indispensable to comprehend and develop LC separations. In this research, a tool was designed to assess the feasibility of applying SPAM in 2DLC. Several parameters were investigated to accurately predict isocratic retention of analytes on trap columns under dilution-flow conditions. Model parameters were derived from scanning-gradient experiments performed on analytical columns. The trap-to-trap repeatability was found to be similar to the prediction error. Dead volumes for the trap columns could not be accurately determined through direct experimentation. Instead, they were extrapolated from dead-volume measurements on analytical columns. Several known retention models were evaluated. Better predictions were found using the quadratic model than with the log-linear (“linear-solvent-strength”) model. Steep scanning gradients were found to result in inaccurate predictions. The impact of the dilution flow on the retention of analytes proved less straightforward than anticipated. Under certain conditions dilution with a weaker eluent was found to be counter productive. A tool was developed to quantify the effect of the dilution flow and to predict whether SPAM could be applied in specific situations. For nine different analytes under 36 different sets of conditions and with three different modulation times, the SPAM tool yielded a correct assessment in more than 95% of all cases (less than 5% false positives plus false negatives).
查看更多>>摘要：? 2022 Elsevier B.V.Exosomes can reflect the physiological state of parent cells and are potential disease biomarkers. In this study, we developed an innovative hydrophilic material by post-synthesis of covalent organic frameworks with dual hydrophilic groups of glutathione and cysteine (denoted as COF-S@Au@GC) to detect glycosylated exosomes in human serum. COF-S@Au@GC enriched glycosylated exosomes in human serum due to glutathione and cysteine (GC) hydrophilicity. Our results show that COF-S@Au@GC has a detection limit of 5 amol μL?1, selectivity of 1:2000, size-exclusion effect of 1:10,000, repeatability of 10 cycles, recovery of 98.3 ± 0.5%, and loading capacity of 50 mg g?1 for glycopeptides. In addition, 182 glycopeptides were detected after enrichment with COF-S@Au@GC from renal carcinoma serum, demonstrating the feasibility of enriching glycopeptides from complex biological samples. Furthermore, COF-S@Au@GC successfully captured glycosylated exosomes in the serum of renal cancer patients, with their 161 glycopeptides detected by nano liquid chromatography with tandem mass spectrometry (LC-MS/MS). This study provides a new heuristic strategy for isolating exosomes and contributes to further functional analysis of exosomes.
查看更多>>摘要：? 2022Traditional Western blots are commonly used to separate and assay proteins; however, they have limitations including a long, cumbersome process and large sample requirements. Here, we describe a system for Western blotting where capillary gel electrophoresis is used to separate sodium dodecyl sulfate-protein complexes. The capillary outlet is threaded into a piezoelectric inkjetting head that deposits the separated proteins in a quasi-continuous stream of <100 pL droplets onto a moving membrane. Through separations at 400 V/cm and protein capture on a membrane moving at 2 mm/min, we are able to detect actin with a limit of detection at 8 pM, or an estimated 5 fg injected. Separation and membrane capture of sample containing 10 proteins ranging in molecular weights from 11 – 250 kDa was achieved in 15 min. The system was demonstrated with Western blots for actin, β-tubulin, ERK1/2, and STAT3 in human A431 epidermoid carcinoma cell lysate.
查看更多>>摘要：? 2022 Elsevier B.V.Belamcandae Rhizoma is a widely used traditional Chinese herbal medicine with isoflavones as the main active ingredient. In this paper, an integrated strategy was developed to discover and identify new isoflavones in Belamcandae Rhizoma by an ultra-high-performance liquid chromatography coupled with high resolution multistage mass spectrometry. Different characterization methods were used based on structural features of isoflavone aglycones and glycosides. On one hand, we adopted a data-dependent acquisition mode incorporated into intelligent AcquireX deep scan algorithms to analyze crude extract, and used a mass defect filtering technique to filter out two kinds of isoflavone aglycones from the extract. On the other hand, neutral-loss-triggered MSn was used to analyze isoflavone glycosides, and under this acquisition mode, MSn scan only took place when chemical components exhibited specific neutral losses. Identification of isoflavones was achieved either by comparison with reference compounds or analysis of characteristic product ions based on MS2 or MSn fragmentation patterns. UV absorbance spectra also contributed to the confirmation of isoflavones. As a result, a total of 65 isoflavone aglycones (42 new aglycones) and 142 isoflavone glycosides (122 new glycosides) were discovered, including a number of trace components. Meanwhile, modifications of new sugar moieties, such as xylose, rhamnose and 6-O-(4?hydroxy-3,5-dimethoxybenzoyl)-β-D-glucose, were discovered in Belamcandae Rhizoma for the first time. These results indicated the feasibility of this established strategy for in-depth identification of new isoflavone aglycones and glycosides.
查看更多>>摘要：? 2022Protein A chromatography with a high salt wash usually leads to robust clearance of host cell proteins (HCPs) in most recombinant monoclonal antibodies (mAbs), but a small subset of recalcitrant mAbs show significant HCP copurification. In this study, we carried out systematic studies using 4 different mAbs to explore the HCP copurification mechanism. HCP identification results revealed that the 3 high-HCP mAbs had many common HCPs which do not copurify with the low-HCP mAb, suggesting a similar mechanism is at play. Through wash evaluation, surface patch analysis, chain-swapping, domain evaluation, and structure-guided mutations, several charged residues in each mAb were found which correlated with HCP copurification. Surprisingly, these residues are also critical for self-association propensity. We observed an inverse correlation between diffusion interaction parameter and HCP copurification. Each of the high-HCP mAbs could form dynamic clusters consisting of 3～6 mAb molecules. Therefore, a mAb cluster can exhibit higher net positive charges on the order of 3 to 6, compared with the individual mAb. In Protein A chromatography, high-HCP mAbs had elution tailing which contained high level of HCPs. Addition of Arginine-HCl or point mutations preventing cluster formation effectively reduced HCP copurification and elution tailing. Based on these results, we propose a novel HCP-copurification mechanism that formation of mAb clusters strengthens charge-charge interactions with HCPs and thus compromises HCP removal by Protein A chromatography. Besides arginine, histidine under acidic pH conditions prevented cluster formulation and resulted in effective HCP removal. Finally, structure-guided protein engineering and solution screening by using cluster size as indicator are useful tools for managing mAbs with high-HCP issues.
查看更多>>摘要：? 2022An anti-inflammatory skin condition is treated with fluocinolone acetonide (FLA), a synthetic corticoid. The current study aims to develop a stability-indicating UPLC method for the determination of impurities present in fluocinolone acetonide and its topical oil formulation. The method development was performed by implementing Analytical Quality by Design (AQbD) and green chemistry principles. A detailed risk assessment was conducted based on the cause-and-effect relationship. D-optimal split-plot design was employed to screen the critical method parameters (CMPs). The central composite design (CCD) was employed to optimize the final method conditions. p-values for the model and lack of fit were <0.0001 and >0.05, respectively, which indicates the best fit statistical model for the studied responses (peak resolutions R1 – R5). The critical method attributes (CMAs) and CMPs such as the ratio of ACN: Water in mobile phase-B as 600:400 (v/v), the ratio of mobile phase-A & B in initial gradient program as 60:40, flow rate as 0.3 mL min?1, and column oven temperature as 50 °C were optimized from the CCD. The best possible separation among all components was achieved with a gradient elution using Waters Acquity UPLC HSS C18, 100 mm × 2.1 mm, 1.8 μm analytical column. The optimized gradient program is time (min)/%B: 0.0/40, 1.5/40, 6.0/60, 8.0/70, 9.0/80, 12.0/100, 15.0/100, 15.1/40 & 18.0/40. Optimization of diluent is highly critical for any oil-based formulations. The experimental results show that acetonitrile is the most suitable diluent for the current study. The method validation was executed in compliance with ICH and USP 〈1225〉 guidelines. Mean recovery of the impurities ranged between 95.7 and 105.7%, the correlation coefficient(r) was> 0.999, the RSD values (n = 6) ranged between 0.9 – 3.2% across the range for LOQ – 150% levels. The peaks from the specificity study did not interfere with the known and active analyte peaks. The major degradation products were identified as Imp-C, B, and A, and established their degradation pathways from FLA based on the stress studies. The method greenness was evaluated using GAPI, AGREE and analytical eco scale and found that the method is green.
查看更多>>摘要：? 2022 Elsevier B.V.Prolyl hydroxylase 2 (PHD2) is a key oxygen receptor regulating oxygen homeostasis in human body, and it is one of the important targets for drug research and development of hypoxia related diseases. In PHD2 enzymatic reaction, the structure of substrate (HIF-1α556–574) and product (hydroxylated HIF-1α) peptide only differ from one oxygen atom (MW>2000), which makes it a great challenge to separate them accurately and efficiently. In this work, the direct separation and detection of HIF-1α and hydroxylated HIF-1α has been firstly reported based on micellar electrokinetic chromatography (MEKC). Under optimized conditions, the intraday RSD of peak area and apparent electrophoretic mobility of hydroxylated HIF-1α were 1.87% and 0.81% respectively, and the interday RSD were 2.01% and 1.03% respectively. The LOD and LOQ of the MEKC method were 10 μM and 50 μM respectively, and the recoveries was 98.42–105.38%. Subsequently, the feasibility and accuracy of MEKC method to screen PHD2 inhibitors were confirmed by using roxadustat, and the IC50 (10.36 μM) and inhibitor type (competitive) were consistent with literature. Finally, the method was used to screen the PHD2 inhibitory activity of five traditional Chinese medicines (TCMs). The present work not only overcomes the difficulties of direct quantitative detection of hydroxylated HIF-1α, but also provides technical support for exploring and discovering new drug leads for hypoxia-related diseases from complex matrix such as TCMs.
查看更多>>摘要：? 2022Reversed-phase (RP) HPLC separation of peptides labeled with amine-reacting tags for relative protein quantitation (iTRAQ4, iTRAQ8 - isobaric tag for relative and absolute quantitation, TMT - tandem mass tag) has been investigated using large-scale proteomics derived retention datasets. These tags have similar chemistry but use linkers of different length and hydrophobicity, moving the positively charged functional groups further from peptide backbone. Peptide hydrophobicity (RP HPLC retention), on average, increases in the following order: non-labeled < iTRAQ4 < iTRAQ8 < TMT under both low pH (0.1% formic acid) and pH 10 eluent conditions. At the same time, the interplay between hydrophobicity and length of the labeling group drives the deviations from this order. Thus, longer and less hydrophobic iTRAQ8 moiety results in greater retention increase for peptides carrying amphipathic helical structures at the N-terminus. Development of a peptide retention prediction models for these modifications was achieved by predicting correspondent retention shifts ΔHI (hydrophobicity index,% acetonitrile) between unmodified and labelled peptide pairs.
查看更多>>摘要：? 2022 Elsevier B.V.Veterinary drug residues in food samples of animal origin are currently analyzed by target analysis using high-performance liquid chromatography combined with sophisticated mass spectrometers. Since the results are only partially consistent with the microbiological results and positive findings occur rarely (in the per mil range in Germany), the potential of a simple planar bioassay screening was studied in the field of veterinary drug residue analysis. Using only a simple dilution of the milk for sample preparation, it was challenging to meet the maximum residue limits for antibiotic drug residues, exemplarily shown for the screening of two fluoroquinolones. However, the potential was evident for a simple, rapid, eco-friendly, and non-target screening without expensive instrumentation. Regardless of whether it is an active metabolite, contaminant, degradation product, or veterinary drug residue, the effect indicated on the planar surface due to bioassay detection will most likely also affect the human microbiome when consumed. The non-target screening of the milk samples revealed compounds with substantial antibacterial effects, which were not in the previous focus of interest. These antibacterial compounds will most likely also affect the human microbiome. Is it only the regulated antibiotic residues or generally all antibiotic compounds in a sample that count for consumer protection? The current prevailing understanding of food safety and antimicrobial resistance, based on the results of target (rather than effect) analyses, is being challenged. Non-target planar bioassay screening has been shown to fill a current gap by providing an understanding of inconsistencies and complementing routine target analysis of veterinary drug residues. As a highlight, it provides the full picture of the real levels of active compounds, regardless of the permitted limits of antibiotics.
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