Optimized Extraction Protocol Research Articles

Work fluctuation and its optimal extraction with time dependent harmonic potential from a non-Markovian bath

We study the optimal work extraction protocol for a Brownian particle described by generalized Langevin dynamics from a single reservoir with the aid of position measurement. The particle is confined in a time dependent harmonic potential. In the overdamped situation, we show that memory effects typically reduce the extracted work, and full information-work conversion can only be achieved by manipulating both the stiffness and center of the potential quasistatically. In the non-Markovian underdamped regime, we investigate the exponential tail of work distribution using the path integral formalism, memory effects on the characteristic work production is also revealed.

Optimization of extraction and analytical protocol for mass spectrometry-based metabolomics analysis of hepatotoxicity.

Drug-induced liver injury is a clinically leading side-effect of drugs. In the present study, a liquid chromatography mass spectrometry-based metabolomics protocol was optimized for extraction and analysis of endogenous metabolites from liver tissue during hepatotoxicity. Various extraction solutions, resuspension solutions, extraction folds and dissolution methods for the supernatant were compared using the number of extracted total ions, relative response and relative extraction efficiency of targeted metabolites from liver tissue. The polar and nonpolar endogenous metabolites associated with liver injury were analyzed by hydrophilic interaction chromatography and reversed-phase liquid chromatography with UPLC-QTOFMS. The results indicated that extraction with 10-fold 50% acetonitrile in water and the supernatant diluted (1:1) with 100% acetonitrile rather than resuspension was the optimal extraction protocol. Subsequently, the optimized method was able to examine the change in metabolites in mouse liver tissue resulting from treatment with a toxic natural product, toosendanin. Taken together, the optimized extraction and analytical protocol provides high reliability and reproducibility for polar and nonpolar metabolites in liver tissue and may be suitable for metabolomics analysis of liver injury induced by drugs or chemicals.

Determination of Doxycycline, 4-epidoxycycline, and 6-epidoxycycline in Aquatic Animal Muscle Tissue by an Optimized Extraction Protocol and Ultra-performance Performance Liquid Chromatography with Ultraviolet Detection

A fast ultra-performance liquid chromatography method was developed for the separation and quantification of doxycycline, 4-epidoxycycline, and 6-epidoxycycline in the muscles of channel catfish, shrimp, eel and turtle. The analytes were separated by comprehensive optimization of the chromatographic conditions. The extraction method was performed using a modified QuEChERS methodology. 2 g of muscle sample were mixed with 5 mL Mcllvaine buffer (pH 4), and 4 g of sodium sulfate and 2 g of sodium chloride were added for liquid–liquid partitioning. The mixture was agitated for 30 s and extracted with 10 mL of acidified acetonitrile. The resulting extract was purified using 200 mg of a C18-silica adsorbent. The samples were analyzed using ultra-performance liquid chromatography with ultraviolet detection using a BEH C18 column (2.1 mm ×100 mm, 1.7 μm). The limits of detection were less than 25 μg/kg and the limits of quantitation were less than 50 μg/kg for all analytes. The recoveries of the analytes were from 62.3% to 89.0% with relative standard derivations below 10.0%. The method provided efficient extraction and purification that enabled rugged and low-cost determination of the analytes.

Optimizing the Maximum Recovery of Dihydromyricetin from Chinese Vine Tea, Ampelopsis grossedentata, Using Response Surface Methodology.

This work provides an optimized extraction approach intended to maximize the recovery of dihydromyricetin (DHM) from Chinese vine tea (Ampelopsis grossedentata) leaves. The presented work adopts a Box-Behnken design as a response surface methodology to understand the role and influence of specific extraction parameters including: time, temperature, and solvent composition/ethanol (%) on DHM final yields. Initially, single factor experiments were used to delineate the role of above factors (temperature, time, and solvent composition) before proceeding with three factors-three levels Box-Behnken design with 17 separate runs to assess the effect of multifactorial treatments on DHM recovery rates. The collected data shows that independent variables (solvent composition, time, and temperature) can significantly affect DHM recovery rates with maximum yields resulting from a combined 60 °C, 60% aqueous ethanol, and 180 min treatment. From the empirical point of view, the above optimized extraction protocol can substantially enhance processing and profitability margins with a minimum need of interventions or associated costs.

Resolving Species Level Changes in a Representative Soil Bacterial Community Using Microfluidic Quantitative PCR.

Rapid advances in genome sequencing technologies enable determination of relative bacterial abundances and community composition, yet, changes at the species level remain difficult to detect despite importance for certain ecological inferences. We present a method for extraction and direct quantification of species composition of a predefined multispecies bacterial community using microfluidic-based quantitative real-time PCR (qPCR). We employ a nested PCR approach based on universal 16S rRNA gene pre-amplification followed by detection and quantification of absolute abundance of bacterial species using microfluidic array of parallel singleplex qPCR reactions. Present microfluidic qPCR supports 2,304 simultaneous reactions on a single chip, while automatic distribution of samples and reactants minimizes pipetting errors and technical variations. The utility of the method is illustrated using a synthetic soil bacterial community grown in two contrasting environments – sand microcosms and batch cultures. The protocol entails extraction of total nucleic acid, preparation of genomic DNA, and steps for qPCR assessment of bacterial community composition. This method provides specific and sensitive quantification of bacterial species requiring only 2 ng of community DNA. Optimized extraction protocol and preamplification step allow for rapid, quantitative, and simultaneous detection of candidate species with high throughput. The proposed method offers a simple and accurate alternative to present sequencing methods especially when absolute values of species abundance are required. Quantification of changes at the species level contributes to the mechanistic understanding of the roles of particular species in a bacterial community functioning.

Chromatographic analysis of tryptophan metabolites.

The kynurenine pathway generates multiple tryptophan metabolites called collectively kynurenines and leads to formation of the enzyme cofactor nicotinamide adenine dinucleotide. The first step in this pathway is tryptophan degradation, initiated by the rate‐limiting enzymes indoleamine 2,3‐dioxygenase, or tryptophan 2,3‐dioxygenase, depending on the tissue. The balanced kynurenine metabolism, which has been a subject of multiple studies in last decades, plays an important role in several physiological and pathological conditions such as infections, autoimmunity, neurological disorders, cancer, cataracts, as well as pregnancy. Understanding the regulation of tryptophan depletion provide novel diagnostic and treatment opportunities, however it requires reliable methods for quantification of kynurenines in biological samples with complex composition (body fluids, tissues, or cells). Trace concentrations, interference of sample components, and instability of some tryptophan metabolites need to be addressed using analytical methods. The novel separation approaches and optimized extraction protocols help to overcome difficulties in analyzing kynurenines within the complex tissue material. Recent developments in chromatography coupled with mass spectrometry provide new opportunity for quantification of tryptophan and its degradation products in various biological samples. In this review, we present current accomplishments in the chromatographic methodologies proposed for detection of tryptophan metabolites and provide a guide for choosing the optimal approach.

Standardization of Protocols for Extraction of Aloe vera and Cinnamon Bark Extracts

The present study was envisaged to optimize the extraction protocols viz. time, temperature and solvent concentration for extraction of phyto-extracts from aloe vera and cinnamon bark based on antioxidant efficacy in-vitro. These were optimized using Box-Behnken Design (BBD) and completely randomized design (CRD) of Response Surface Methodology (RSM). A total number of 17 different combinations of time, temperature and ethanol concentration in BBD and 13 different combinations of time, temperature in CRD were considered for optimizing extraction protocols for aloe vera and cinnamon respectively. The in-vitro 1, 1 diphenyl-2picrylhydrazyl (DPPH % inhibition) and 2, 2′-Azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) ABTS+ radical scavengering activity of aqueous extract of cinnamon bark was recorded highest for the combination of 99% concentration at 80°C temperature for 3h amongst all 13 runs. The 90% ethanolic extract of aloe vera and cinnamon bark exhibited higher in-vitro antioxidant ability than aqueous extract at time, temperature combination of 15 min for 65°C and 9h for 60°C respectively.

Optimization Protocol for the Microwave-Assisted Extraction of Antioxidant Components from Pinus elliottii Needles Using Response Surface Methodology

Response surface methodology (RSM) based on a Box–Behnken rotatable design was used to determine the optimum conditions for the microwave-assisted extraction of antioxidant compounds from Pinus elliottii needles. Four process variables were evaluated at three levels (29 experimental conditions): ethanol (50, 70, and 90%), solvent:solute ratio (25:1, 20:1, and 15:1), extraction temperature (60, 70, and 80 °C), and ultrasonic power (100, 150, and 200 W). Using RSM, a quadratic polynomial equation was obtained by multiple regression analysis to predict the optimized extraction protocol. The radical scavenging capacity was determined by O2−, ·OH, and DPPH methods. For the microwave-assisted extraction of antioxidant compounds from Pinus elliottii needles, the optimum process used ethanol at 72%, a solvent:solute ratio of 21:1 mL/g, an extraction temperature of 67 °C, and an ultrasonic power of 200 W. The results indicated good correlation between total polyphenols content and O2−, ·OH, and DPPH radical scavenging activities.

Comparison of capture and storage methods for aqueous macrobial eDNA using an optimized extraction protocol: advantage of enclosed filter

Summary Aqueous environmental DNA (eDNA) is an emerging efficient non‐invasive tool for species inventory studies. To maximize performance of downstream quantitative PCR (qPCR) and next‐generation sequencing (NGS) applications, quality and quantity of the starting material is crucial, calling for optimized capture, storage and extraction techniques of eDNA. Previous comparative studies for eDNA capture/storage have tested precipitation and ‘open’ filters. However, practical ‘enclosed’ filters which reduce unnecessary handling have not been included. Here, we fill this gap by comparing a filter capsule (Sterivex‐GP polyethersulfone, pore size 0·22 μm, hereafter called SX) with commonly used methods. Our experimental set‐up, covering altogether 41 treatments combining capture by precipitation or filtration with different preservation techniques and storage times, sampled one single lake (and a fish‐free control pond). We selected documented capture methods that have successfully targeted a wide range of fauna. The eDNA was extracted using an optimized protocol modified from the DNeasy® Blood & Tissue kit (Qiagen). We measured total eDNA concentrations and Cq‐values (cycles used for DNA quantification by qPCR) to target specific mtDNA cytochrome b (cyt b) sequences in two local keystone fish species. SX yielded higher amounts of total eDNA along with lower Cq‐values than polycarbonate track‐etched filters (PCTE), glass fibre filters (GF) or ethanol precipitation (EP). SX also generated lower Cq‐values than cellulose nitrate filters (CN) for one of the target species. DNA integrity of SX samples did not decrease significantly after 2 weeks of storage in contrast to GF and PCTE. Adding preservative before storage improved SX results. In conclusion, we recommend SX filters (originally designed for filtering micro‐organisms) as an efficient capture method for sampling macrobial eDNA. Ethanol or Longmire's buffer preservation of SX immediately after filtration is recommended. Preserved SX capsules may be stored at room temperature for at least 2 weeks without significant degradation. Reduced handling and less exposure to outside stress compared with other filters may contribute to better eDNA results. SX capsules are easily transported and enable eDNA sampling in remote and harsh field conditions as samples can be filtered/preserved on site.

Identification and efficient extraction method of phlorotannins from the brown seaweed Macrocystis pyrifera using an orthogonal experimental design

The brown seaweed contains a type polyphenol compound characteristic of its species, the phlorotannins, which are produced from the polymerization of phloroglucinol units. They have been extensively studied due to their pharmacological and nutraceutical properties, but there is still a need for an optimized extraction protocol. In this study, the brown seaweed Macrocystis pyrifera was employed to determine the best conditions for extraction of phlorotannins. A set of different variables were evaluated such as the use of pre-treatment, type of solvent, drying temperature, particle size, temperature and extraction time as well as the solid/liquid ratio upon extraction. The optimal conditions for the extraction of phlorotannins were: pre-treatment with hexane, extraction with water, drying temperature 40°C, particle size below 1.4mm, at 55°C for 4h and a solid-to-liquid ratio of 1:15. Under these conditions, the concentration of phlorotannins achieved in the extract was 200.5±5.6mg gallic acid equivalent (GAE)/100g dry seaweed (DS) and total antioxidant activity of the extract of 38.4±2.9mg trolox equivalent (TE)/100g DS. Further, it was possible to identify two phlorotannins through HPLC-ESI-MS analyses: phloroeckol and a tetrameric phloroglucinol. These phlorotannins have been reported in the literature to have an antidiabetic effect and prevention of Alzheimer's disease for phloroeckol, and free radical scavenging ability and antiallergic effect for tetrameric phloroglucinol. Therefore, the extract of phlorotannins has potential as medicinal foods or therapeutics for human health applications.

New Horizons for Ninhydrin: Colorimetric Determination of Gender from Fingerprints

In the past century, forensic investigators have universally accepted fingerprinting as a reliable identification method via pictorial comparison. One of the most traditional detection methods uses ninhydrin, a chemical that reacts with amino acids in the fingerprint content to produce the blue-purple color known as Ruhemann's purple. It has recently been demonstrated that the amino acid content in fingerprints can be used to differentiate between male and female fingerprints. Here, we present a modified approach to the traditional ninhydrin method. This new approach for using ninhydrin is combined with an optimized extraction protocol and the concept of determining gender from fingerprints. In doing so, we are able to focus on the biochemical material rather than exclusively the physical image.

Comparable efficiency of different extraction protocols for wheat and rye prolamins

The identification and quantification of cereal storage proteins is of interest of many researchers. Their structural or functional properties are usually affected by the way how they are extracted. The efficiency of extraction process depends on the cereal source and working conditions. Here, we described various commonly used extraction protocols differing in the extraction conditions (pre-extraction of albumins/globulins, sequential extraction of individual protein fractions or co-extraction of gluten proteins, heating or non-heating, reducing or non-reducing conditions). The total protein content of all fractions extracted from commercially available wheat and rye flours was measured by the Bradford method. Tris-Tricine SDS-PAGE was used to determine the molecular weights of wheat gliadins, rye secalins and high-molecular weight glutelins which are the main triggering factors causing celiac disease. Moreover, we were able to distinguish individual subunits (α/β-, γ-, ω-gliadins and 40k-γ-, 75k-γ-, ω-secalins) of wheat/rye prolamins. Generally, modified extraction protocols against classical Osborne procedure were more effective and yields higher protein content in all protein fractions. Bradford measurement led into underestimation of results in three extraction procedures, while all protein fractions were clearly identified on SDS-PAGE gels. Co-extraction of gluten proteins resulted in appearance of both, low-molecular weight fractions (wheat gliadins and rye secalins) as well as high-molecular weight glutelins which means that is not necessary to extract gluten proteins separately. The two of three extraction protocols showed high technical reproducibility with coefficient of variation less than 20%. Carefully optimized extraction protocol can be advantageous for further analyses of cereal prolamins.

Selection of a protein solubilization method suitable for phytopathogenic bacteria: a proteomics approach.

BackgroundFinding the best extraction method of proteins from lysed cells is the key step for detection and identification in all proteomics applications. These are important to complement the knowledge about the mechanisms of interaction between plants and phytopathogens causing major economic losses. To develop an optimized extraction protocol, strains of Acidovorax citrulli, Pectobacterium carotovorum subsp. carotovorum and Ralstonia solanacearum were used as representative cells in the study of phytopathogenic bacteria. This study aims to compare four different protein extraction methods, including: Trizol, Phenol, Centrifugation and Lysis in order to determine which are more suitable for proteomic studies using as parameters the quantity and quality of extracted proteins observed in two-dimensional gels.ResultsThe bacteria studied showed different results among the tested methods. The Lysis method was more efficient for P. carotovorum subsp. carotovorum and R. solanacearum phytobacteria, as well as simple and fast, while for A. citrulli, the Centrifugation method was the best. This evaluation is based on results obtained in polyacrylamide gels that presented a greater abundance of spots and clearer and more consistent strips as detected by two-dimensional gels.ConclusionsThese results attest to the adequacy of these proteins extraction methods for proteomic studies.

Qualitative and spatial metabolite profiling of lichens by a LC-MS approach combined with optimised extraction.

Lichens are self-sustaining partnerships comprising fungi as shape-forming partners for their enclosed symbiotic algae. They produce a tremendous diversity of metabolites (1050 metabolites described so far). A comparison of metabolic profiles in nine lichen species belonging to three genera (Lichina, Collema and Roccella) by using an optimised extraction protocol, determination of the fragmentation pathway and the in situ localisation for major compounds in Roccella species. Chemical analysis was performed using a complementary study combining a Taguchi experimental design with qualitative analysis by high-performance liquid chromatography coupled with mass spectrometry techniques. Optimal conditions to obtain the best total extraction yield were determined as follows: mortar grinding to a fine powder, two successive extractions, solid:liquid ratio (2:60) and 700 rpm stirring. Qualitative analysis of the metabolite profiling of these nine species extracted with the optimised method was corroborated using MS and MS/MS approaches. Nine main compounds were identified: 1 β-orcinol, 2 orsellinic acid, 3 putative choline sulphate, 4 roccellic acid, 5 montagnetol, 6 lecanoric acid, 7 erythrin, 8 lepraric acid and 9 acetylportentol, and several other compounds were reported. Identification was performed using the m/z ratio, fragmentation pathway and/or after isolation by NMR analysis. The variation of the metabolite profile in differently organised parts of two Roccella species suggests a specific role of major compounds in developmental stages of this symbiotic association. Metabolic profiles represent specific chemical species and depend on the extraction conditions, the kind of the photobiont partner and the in situ localisation of major compounds.

Extraction and quantification of microphytobenthic Chl a within calcareous reef sands

Calcareous reef sands are characterized by high concentrations of photosynthetic pigments that extend well below the sediment surface, as well as by high variability in concentrations between study sites. An important contributor to the observed variability may be differences in extraction protocols, further complicated by variability in calcareous sand characteristics. We tested the effects of freeze‐drying, grinding, sonication, extraction temperature, and extraction time on quantification of Chl a content within calcareous sands. The resulting optimized extraction protocol consists of freeze‐drying, grinding with a mortar and pestle for 30 s, and extracting with 100% acetone at −20°C for at least 20 h, yielding a 39% increase in Chl a content over frozen, unground samples. Using this protocol, we measured and compared ten sedimentary Chl a profiles taken in close proximity to test for relationships between surface and sub‐surface concentrations. Sedimentary Chl a content at a back‐reef location on the south shore of O'ahu varied between 4.33‐14.25 µg g−1 dw, with distinctly higher values occurring in a relatively enriched surface layer (0–1 cm). Surface Chl a concentrations varied between 86‐307 mg m−2, depending on the depth of integration (0.5–2 cm), with 73% of the full‐core (0‐8 cm) Chl a concentration occurring below 2 cm. The concentrations of surface and subsurface layers were significantly correlated between cores, allowing for the use of plug sampling when profile generation is not feasible or necessary to determine the magnitude of the subsurface microphytobenthic biomass and its variability over scales of meters.

Optimised protocols for the metabolic profiling of S. cerevisiae by 1H-NMR and HRMAS spectroscopy

An optimised extraction protocol for the analysis of Saccharomyces cerevisiae aqueous and organic metabolites by nuclear magnetic resonance spectroscopy that allows the identification and quantification of up to 50 different compounds is presented. The method was compared with other metabolic profiling protocols for S. cerevisiae, where generally different analytical techniques are applied for metabolite quantification. In addition, the analysis of intact S. cerevisiae cells by HRMAS was implemented for the first time as a complementary method. The optimised protocols were applied to study the metabolic effect of glucose and galactose on S. cerevisiae growth. Furthermore, the metabolic reaction of S. cerevisiae to osmotic stress has been studied.

Evaluation of extraction protocols for simultaneous polar and non-polar yeast metabolite analysis using multivariate projection methods.

Metabolomic and lipidomic approaches aim to measure metabolites or lipids in the cell. Metabolite extraction is a key step in obtaining useful and reliable data for successful metabolite studies. Significant efforts have been made to identify the optimal extraction protocol for various platforms and biological systems, for both polar and non-polar metabolites. Here we report an approach utilizing chemoinformatics for systematic comparison of protocols to extract both from a single sample of the model yeast organism Saccharomyces cerevisiae. Three chloroform/methanol/water partitioning based extraction protocols found in literature were evaluated for their effectiveness at reproducibly extracting both polar and non-polar metabolites. Fatty acid methyl esters and methoxyamine/trimethylsilyl derivatized aqueous compounds were analyzed by gas chromatography mass spectrometry to evaluate non-polar or polar metabolite analysis. The comparative breadth and amount of recovered metabolites was evaluated using multivariate projection methods. This approach identified an optimal protocol consisting of 64 identified polar metabolites from 105 ion hits and 12 fatty acids recovered, and will potentially attenuate the error and variation associated with combining metabolite profiles from different samples for untargeted analysis with both polar and non-polar analytes. It also confirmed the value of using multivariate projection methods to compare established extraction protocols.

Development of a microwave-assisted extraction for the analysis of phenolic compounds from Rosmarinus officinalis

For potent biological activity natural phenols are considered as compounds with perspective of being isolated in their pure form from natural sources. For analytical purposes, as well as for potential industrial application, it is important to apply extraction procedure that allows efficient and time-consuming extraction of polyphenols. In this study a microwave-assisted extraction procedure has been optimized in respect to the recovery of rosemary polyphenols. The optimization was performed in respect to total polyphenols, flavonoids, total anthocyanins, monomeric and condensed anthocyanins. The efficiency of different solvents was investigated for different classes of polyphenols, as well as the influence of the magnetron power and the extraction time. The efficiency of the extraction using optimized extraction protocol for total phenolics was compared with that achieved by Soxhlet and ultrasound-assisted extraction. The present work proposes an optimal microwave-assisted extraction program for the determination of total polyphenolics, flavonoids, total anthocyanins, monomeric and condensed anthocyanins from rosemary.

Using an In Vitro Model of Lipid Deposition to Assess the Efficiency of Hydrogen Peroxide Solutions to Remove Lipid from Various Contact Lens Materials

Purpose: To test the ability of two commercially available hydrogen peroxide disinfection solutions, one containing a surfactant and one without, to remove lipid from various contact lens materials using in vitro radiochemical experiments.Methods: Etafilcon A, senofilcon A and balafilcon A contact lens materials were incubated in an artificial tear solution (ATS) containing a mixture of lipids, proteins, mucin and either 14C-cholesterol or 14C-phosphatidylcholine for 8 h. Following incubation, the lenses were removed, rinsed, and placed for 16 h in either a surfactant-containing peroxide solution (ClearCare®), a peroxide solution devoid of a surfactant (AOSept®) or stored without solution (control). This process was repeated every day for 1 week. The lenses were extracted with a previously optimized extraction protocol, evaporated, re-suspended, fluor added and counted for their radioactive signals. Masses of lipids deposited were calculated based on standard calibration curves, the disinfection solutions were compared and repeated measures ANOVA and post hoc statistical analysis was completed using Statistica 9.Results: The results of this experiment found that daily disinfection with hydrogen peroxide solutions reduced the amount of cholesterol and phosphatidylcholine deposited on the three contact lens materials examined, however in many cases the reduction in deposition was less than 15% when compared to the control. Disinfection with the solution containing the surfactant (ClearCare), resulted in the least deposited cholesterol and phosphatidylcholine for all materials, however not all of the comparisons were statistically significant.Conclusions: Overall, ClearCare hydrogen peroxide disinfection solution containing Pluronic 17R4 removed the most lipid from lenses when compared to the non-surfactant containing AOSept or the control, for both lipids and all lens materials. However, the differences found were quite small at times and whether these differences are clinically significant are yet to be determined.

Optimization of Extraction Method for GC-MS based Metabolomics for Filamentous Fungi

Metabolite extraction is considered as one of the most important for quality controlling steps in metabolomic research. In the present study, optimal extraction protocol for fungal intracellular metabolites was investigated. Two extraction protocols with different solvent systems, methanol extraction and biphasic extraction under different pH conditions were compared. Number of detected peaks, sample-to-sample variation, and throughput of whole process were taken into account as criteria. Extracted metabolites were analyzed by gas chromatography-mass spectrometry (GC-MS) and principal component analysis (PCA). The extraction protocol using methanol as extraction solvent provided the highest number of detected peaks (~ 300 peaks) with the ease and speed, suggesting that the methanol-extraction method was suitable for intracellular metabolites extraction from P. chrysosporium. However, depending on the nature of metabolites of interest, the biphasic extraction method under different pH conditions is suitable for a targeted analysis.