Development of the Analysis of Fecal Stanols in the Oyster Crassostrea gigas and Identification of Fecal Contamination in Shellfish Harvesting Areas

The objective of this work was to study the effects of washing and purification steps on qualitative and quantitative analysis of fecal stanols in the oyster Crassostrea gigas using either single or a combination of lipid purification steps on silica gel or aminopropyl bonded silica gel (NH₂) or a washing step. Among the three analytical pathways compared, the two including water extraction or NH₂ purification did not lead to higher recoveries and decreased repeatabilities of extractions compared to the single purification on silica gel. This latter led to similar recoveries (ca. 80 %) and repeatabilities (ca. 10 %) for both spiked standards (coprostanol and sitostanol). This analytical pathway has been applied to oysters collected in a harvesting area in Brittany (France) where fecal contaminations are important and allowed to quantify eight stanols in oysters. The relative proportions of fecal stanols of these oysters were combined with principal component analysis in order to investigate the usefulness of their stanol fingerprints to record a fecal contamination and to distinguish its source between human, porcine and bovine contaminations. Oysters non-fecally contaminated by Escherichia coli did not present specific stanol fingerprints while oysters fecally contaminated had a bovine fingerprint, suggesting a contamination of these samples by bovine sources. As a consequence, the method developed here allows the use of stanol fingerprints of oysters as a microbial source tracking tool that can be applied to shellfish harvesting areas subjected to fecal contaminations in order to identify the different sources of contamination and improve watershed management.     

Automation of the liquid-phase synthesis of biopolymers

The liquid-phase synthesis of biopolymers is an alternative to the solid-phase methodologies that are widely applied in spite of limitations in the use of heterogeneous media. The advantages of the liquid-phase approach are due to the fact that the support is alternatively soluble in the solvents used for the chemical reactions and insoluble in the mixtures used for the purification steps. Thus, the advantages of both ‘solution’ and ‘solid-phase’ methodologies can be properly integrated. At present the liquid-phase procedure, unlike solid-phase methods that have been highly automated, can be carried out only manually due to technical difficulties imposed by the cyclic repetition of dissolution and precipitation steps. The original apparatus proposed here performs, for the first time, all the steps required by the liquid-phase approach in a fully automatic way controlled by a user-friendly dedicated software. The automated liquid-phase methodology has been tested by synthesizing some sample oligonucleotides and the preliminary results are discussed in the present paper. © 1998 SCI.     

Matrix-assisted laser desorption/ionization imaging mass spectrometry

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is a powerful tool that enables the simultaneous detection and identification of biomolecules in analytes. MALDI-imaging mass spectrometry (MALDI-IMS) is a two-dimensional MALDI-mass spectrometric technique used to visualize the spatial distribution of biomolecules without extraction, purification, separation, or labeling of biological samples. MALDI-IMS has revealed the characteristic distribution of several biomolecules, including proteins, peptides, amino acids, lipids, carbohydrates, and nucleotides, in various tissues. The versatility of MALDI-IMS has opened a new frontier in several fields such as medicine, agriculture, biology, pharmacology, and pathology. MALDI-IMS has a great potential for discovery of unknown biomarkers. In this review, we describe the methodology and applications of MALDI-IMS for biological samples.     

高铁酸盐分析方法研究进展

高铁酸盐可以用作超铁电池储能阴极材料,也是新型绿色有机合成氧化剂和水处理药剂.日益广泛的应用需要精准的分析测试方法.综述了高铁酸盐的定性、定量分析方法,包括红外光谱、紫外可见光谱、滴定分析、XRD、穆斯堡尔谱、电化学分析、荧光分析法等,详述了分析方法原理及适用范围.     

High-Throughput Analysis of Plasma Fatty Acid Methyl Esters Employing Robotic Transesterification and Fast Gas Chromatography

Fatty acid analysis is an important research tool, and indices derived from essential fatty acid contents serve as useful biomarkers related to cardiovascular and other chronic disease risk. Both clinical and basic studies of essential fatty acid composition are becoming ever larger in magnitude leading to delays while the rather laborious lipid analyses are performed. A robotic transesterification procedure has been developed for high-throughput analysis of plasma fatty acid methyl esters. In this approach, robots perform most steps including plasma and reagent transfer, transesterification reaction via heating at 80 °C in open tubes with multiple reagent additions, followed by two-phase extraction and transfer of lipid extracts to GC vials. The vials are then placed directly onto a GC autosampler carousel for robotic sample injection. An improved fast GC method is presented in which the peaks of interest are eluted within 6 min. This method is readily scalable to prepare and analyze 200 samples per day (1,000 samples per week) so that large clinical trials can be accommodated. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Microwave-mediated non-catalytic transesterification of algal biomass under supercritical ethanol conditions

A novel integrated approach has been proposed to convert lipid-rich, dry algae (Nannochloropsis salina) into fatty acid ethyl esters (FAEE) under microwave-mediated supercritical ethanol (MW-SCE) conditions with a non-catalytic transesterification approach. This process enables simultaneous extraction of lipids from algal biomass and conversion/transesterification of them into algal biodiesel in a relatively short reaction time, which may reduce energy consumption versus traditional processes due to simplified separation and purification steps. High conversion rates can be possible when the extractive-transesterification of algal biomass is performed near-critical or supercritical conditions. The use of passive heating elements made of silicon carbide (SiC) to aid the microwave-mediated heating process at higher temperatures is also described. Experimental runs were designed to optimize the process parameters to evaluate the effect on the algal biodiesel under controlled power conditions. The algal biomass characterization and algal biodiesel analysis were performed using various analytical instruments such as FTIR, SEM-EDS, TGA and GC–MS. It was demonstrated in this work that this direct conversion technique has the potential to provide an energy-efficient and economical route for algal biodiesel production.     

生物活性天然产物分离技术的研究进展(英文)

Bioactive natural products are a main source of new drugs, functional foods and food additives. The separation of bioactive natural products plays an important role in transformation and use of biomass. The isolation and purification of bioactive_principle from a complex matrix is often inherent bottleneck for the utilization of natural products, so a series of extraction and separation techniques have been developed. This review covers recent advances in the separation of bioactive natural products with an emphasis on their solubility and diffusion coefficients, recent extraction techniques and isolation techniques. This overview of recent technological advances, dis- cussion of pertinent problems and prospect of current methodologies in the separation of bioactive natural products may provide a driving force for development of novel separation techniques.     

NMR Profiling of Exhaled Breath Condensate Defines Different Metabolic Phenotypes of Non-Cystic Fibrosis Bronchiectasis

Nuclear-magnetic-resonance (NMR) profiling of exhaled breath condensate (EBC) provides insights into the pathophysiology of bronchiectasis by identifying specific biomarkers. We evaluated whether NMR-based metabolomics discriminates the EBC-derived metabolic phenotypes (“metabotypes”) of 41 patients with non-cystic fibrosis (nCF) bronchiectasis of various etiology [24 subjects with Primary Ciliary Dyskinesia (PCD); 17 patients with bronchiectasis not associated with PCD (nCF/nPCD)], who were compared to 17 healthy subjects (HS). NMR was used for EBC profiling, and Orthogonal Projections to Latent Structures with partial least-squares discriminant analysis (OPLS-DA) was used as a classifier. The results were validated by using the EBC from 17 PCD patients not included in the primary analysis. Different statistical models were built, which compared nCF/nPCD and HS, PCD and HS, all classes (nCF/nPCD-PCD-HS), and, finally, PCD and nCF/nPCD. In the PCD-nCF/nPCD model, four statistically significant metabolites were able to discriminate between the two groups, with only a minor reduction of the quality parameters. In particular, for nCF/nPCD, acetone/acetoin and methanol increased by 21% and 18%, respectively. In PCD patients, ethanol and lactate increased by 25% and 28%, respectively. They are all related to lung inflammation as methanol is found in the exhaled breath of lung cancer patients, acetone/acetoin produce toxic ROS that damage lung tissue in CF, and lactate is observed in acute inflammation. Interestingly, a high concentration of ethanol hampers cilia beating and can be associated with the genetic defect of PCD. Model validation with 17 PCD samples not included in the primary analysis correctly predicted all samples. Our results indicate that NMR of EBC discriminates nCF/nPCD and PCD bronchiectasis patients from HS, and patients with nCF/nPCD from those with PCD. The metabolites responsible for between-group separation identified specific metabotypes, which characterize bronchiectasis of a different etiology.     

A Fluorometric Enzymatic Assay for Quantification of Steryl Glucosides in Biodiesel

Steryl glucosides (SG) are common contaminants in biodiesel that form precipitates, which form and cause problems due to fouling during transport and storage. Therefore, their quantification is necessary to assess the quality of this fuel. The methods currently available for SG analysis require expensive instrumentation, need a previous concentration step by solid‐phase extraction (SPE) or are of limited use for the quantitative assessment. We developed an enzymatic method for SG quantification in biodiesel samples based on the hydrolysis of the glucoside catalyzed by a broadly specific beta glucosidase and the subsequent determination of the glucose released by the reaction. The method is non‐expensive, sensitive and was adapted to 96‐well format fluorescence plate reader, making it useful for the parallel assay of multiple samples. The enzymatic assay presented here represent a valuable tool for both quality control and the development of improved biodiesel production and purification procedures.     

Generation and characterization of a novel recombinant antibody against 15-ketocholestane isolated by phage-display

The employment of monoclonal antibodies (Mabs) to identify disease-associated biomarkers in clinical samples represents the underlying principle for many diagnostic tests. To date, these have been principally developed for protein targets with few reported applications for lipids due to their hydrophobicity and poor immunogenicity. Oxysterols represent a family of lipids implicated in diverse human diseases where Mab-based detection assays could have a profound effect on their utility as clinical biomarkers. These are usually identified in patients' samples by mass- spectrometry based approaches. Here, we describe an antibody phage-library based screening methodology for generating a recombinant monoclonal antibody (RAb) targeting the oxysterol-15-ketocholestane (15-KA), a lipid implicated in multiple sclerosis and Autoimmune Encephalomyelitis (EAE). The antibody is highly specific for 15-KA and shows little or no binding activity for other closely related oxysterols. We employ RAb2E9 to address the controversy over whether 15-KA is a true biomarker for MS/EAE and show that 15-KA is undetectable in serum taken from mice with EAE using antibody based detection methodologies; a finding confirmed by mass-spectrometry analysis. This study demonstrates the technical feasibility of using phage display to isolate highly specific antibodies against poorly immunogenic, small molecule lipids.     

Praktische und theoretische Extraktionsstufen in der Fest-Flüssig-Extraktion

Practical and Theoretical Extraction Steps in the Solid-Liquid Extraction A good deal of work has been done on determination of the theoretical number of extraction steps for various problems involving solid-liquid extraction. They permit calculation of the required number of steps, either mathematically or graphically, taking into account the proportion of liquid absorbed in the solid. However, in an actual extractor it is not possible to separate distinctly the miscella at different steps. Therefore the number of separation steps actually required is larger than the one theoretically determined. The study reported deals with the distribution of liquid below the layer of percolation in the extractor as a function of time and space. It has been possible to determine the extent of back-mixing of miscella at various steps in extractors which are provided with separating walls within the layer of solids. Furthermore, the effect of height of solid layer and that of the perforated wall below the solid layer is discussed.     

Assessment of Isoprostanes in Human Plasma: Technical Considerations and the Use of Mass Spectrometry

Oxygenated lipid mediators released from non‐enzymatic peroxidation of polyunsaturated fatty acids (PUFA) are known to have functional roles in humans. Notably, among these lipid mediators, isoprostanes molecules are robust biomarkers of oxidative stress but those from n‐3 PUFA are also bioactive molecules. In order to identify and assess the isoprostanes, the use of mass spectrometry (MS) for analysis is preferable and has been used for over two decades. Gas chromatography (GC) is commonly coupled to the MS to separate the derivatized isoprostanes of interest in biological samples. In order to increase the accuracy of the analytical performance, GC–MS/MS was also applied. Lately, MS or MS/MS has been coupled with high‐performance liquid chromatography to assess multiple isoprostane molecules in a single biological sample without derivatization process. However, there are limitations for the use of LC–MS/MS in the measurement of plasma isoprostanes, which will be discussed in this review.     

Purification of Commercial 2,3-Dimethyl Phenol Using Supercritical Fluid Extraction

The commercial value of phenols is often reduced due to the presence of colored impurities. Several conventional techniques have been used for the purification of phenols. However, conventional purification techniques are tedious and make use of hazardous and expensive organic solvents. In this study, we present a new method for purification of an aged-discolored (orange) commercial 2,3-dimethyl phenol (2,3-DMP) reagent (～97%) using supercritical fluid CO₂ (SCF CO₂), as an extraction solvent. A supercritical fluid extraction (SFE)/purification apparatus was constructed and purification of the reagent under different extraction conditions was investigated. Based on the differential solubility of the 2,3-DMP and the impurities in SCF CO₂, the commercial reagent was successfully purified by SFE; the purified 2,3-DMP was a white solid of high purity (>99.5%). The SFE method was also applied to purify a recently purchased batch of 2,3-DMP reagent. We found that the reagent purified by SFE was of a higher quality than a commercially available analytical standard.     

一种新的绿色化学技术：超临界萃取作为样品制备技术在痕量药检分析中的应

综述了超临界萃取作为样品技术在痕量药检分析中的应用。研究了生物基质对有效萃取目标检测物的影响及提高萃取率的途径。并重点评述了减少类脂杂质、选择性的分离目标检测物的非在线吸附剂收集与在线吸附收集技术。从而展示了ＳＦＥ作为绿色化学技术具有萃取率高、使用溶剂少,分离步骤少,环境无害等明显优于传统方法的特点。     

Preparation of fatty acid methyl esters from brook charr tissues: Comparison of a classical and a direct method

Two methods of preparation (classical and direct) of fatty acid methyl esters (FAME) for gas chromatographic determination were compared for samples of flesh, liver and skin of the brook charr (Salvelinus fontinalis). The classical method is based on the saponification and methylation of a lipid extract from the tissue sample. The direct method is based on the assumption that saponification and methylation of the fatty acids occurs directly in the flask containing the tissues without prior lipid extraction and purification steps. The fatty acid profiles obtained by the two methods are comparable for the liver and the skin, but the direct method seems to be more precise for the flesh samples. In addition, the direct method is more rapid for the routine preparation of the FAME than the classical method and requires a much lower quantity of organic solvents, thereby reducing the costs of analysis and disposal of the used solvents.     

提取分析银杏叶中黄酮类化合物的研究进展

概述了目前银杏叶中黄酮类化合物的提取工艺和分析方法。提取工艺有：水蒸气蒸馏法、有机溶剂萃取法和超临界流体萃取法。分析方法有：分光光度法、气相色谱法、ＨＰＬＣ法和超临界流体色谱法。根据分离与提纯技术的新进展提出了发展方向。     

Extraction and purification of oryzanol from rice bran oil and rice bran oil soapstock

Oryzanol is an important value-added co-product of the rice and rice bran-refining processes. The beneficial effects of oryzanol on human health have generated global interest in developing facile methods for its separation from rice bran oil soapstock, a by-product of the chemical refining of rice bran oil. In this article we discuss the isolation of oryzanol and the effect that impurities have on its extraction and purification. Presented are the principles behind the extraction (solid-liquid or liquid-liquid extraction, and other methods) of these unit operations covered in selected patents. Methods other than extraction such as crystallization or precipitation-based or a combination of these unit operations also are reviewed. The problems encountered and the ways to solve them during oryzanol extraction, such as prior processing and compositional variation in soapstock, resistance to mass transfer, moisture content and the presence of surface active components, which cause emulsion formation, are examined. Engineering inputs required for solving problems such as saponification, increasing mass transfer area, and drying methods are emphasized. Based on an analysis of existing processes, those having potential to work in large-scale extraction processes are presented.     

Single-Tear Proteomics: A Feasible Approach to Precision Medicine

Lacrimal fluid is an attractive source of noninvasive biomarkers, the main limitation being the small sample amounts typically collected. Advanced analytical methods to allow for proteomics profiling from a few microliters are needed to develop innovative biomarkers, with attractive perspectives of applications to precision medicine. This work describes an effective, analytical pipeline for single-tear analysis by ultrahigh-resolution, shotgun proteomics from 23 healthy human volunteers, leading to high-confidence identification of a total of 890 proteins. Highly reproducible quantification was achieved by either peak intensity, peak area, or spectral counting. Hierarchical clustering revealed a stratification of females vs. males that did not emerge from previous studies on pooled samples. Two subjects were monitored weekly over 3 weeks. The samples clustered by withdrawal time of day (morning vs. afternoon) but not by follow-up week, with elevated levels of components of the immune system in the morning samples. This study demonstrates feasibility of single-tear quantitative proteomics, envisaging contributions of this unconventional body fluid to individualized approaches in biomedicine.     

Affinity Purification Coupled to Stable Isotope Dilution LC-MS/MS Analysis to Discover IgG4 Glycosylation Profiles for Autoimmune Pancreatitis

Type 1 autoimmune pancreatitis (AIP) is categorized as an IgG4-related disease (IgG4-RD), where a high concentration of plasma IgG4 is one of the common biomarkers among patients. IgG Fc-glycosylation has been reported to be potential biosignatures for diseases. However, human IgG3 and IgG4 Fc-glycopeptides from populations in Asia were found to be isobaric ions when using LC-MS/MS as an analytical tool. In this study, an analytical workflow that coupled affinity purification and stable isotope dilution LC-MS/MS was developed to dissect IgG4 glycosylation profiles for autoimmune pancreatitis. Comparing the IgG4 and glycosylation profiles among healthy controls, patients with pancreatic ductal adenocarcinoma (PDAC), and AIP, the IgG4 glycosylations from the AIP group were found to have more digalactosylation (compared to PDAC) and less monogalactosylation (compared to HC). In addition, higher fucosylation and sialylation profiles were also discovered for the AIP group. The workflow is efficient and selective for IgG4 glycopeptides, and can be used for clinical biosignature discovery.     

Extraction and purification of Polyhydroxyalkanoates (PHAs): application of Thermoseparating aqueous two-phase extraction

Being biodegradable, non-toxic and renewable as well as having similar or better properties than commercial plastics, polyhydroxyalkanoates (PHAs) can be a potential game changer in the polymer industry. Although viewed as a sustainable alternative to petrochemicals due to its biodegradability, PHAs are plagued with low commercial value due to their high production and recovery costs. Having the benefits of providing a mild environment for bioseparation, being environment-friendly and scalable, together with it its distinctive thermoseparating properties and ease of recyclability, thermoseparating-based aqueous two-phase extraction (ATPE) has provided the eco-friendly and economical solution to the PHA dilemma. ATPE-influencing factors such as types of thermoseparating polymer, concentration of phase-forming components, pH, and effect of centrifugation were investigated. Under the condition of 14 wt/wt% of EOPO 3900 concentration, 14 wt/wt% of ammonium sulfate concentration and pH 6 without the needs for extra centrifugation steps, a recovery yield and a purification factor of up to 72.2% and 1.61 fold can be achieved with the copolymers which can be recycled and reused twice. Thermoseparating ATPE has thus been proven to be a powerful primary purification tool for PHAs.