Single Extraction Procedure Research Articles

Microbial metabolomics: welcome to the real world!

In the post-genomics era there has been a growing emphasis on understanding the metabolic consequences of engineering cells to produce desired chemicals. Proteomics measurements have improved our understanding of gene expression, and metabolomics promises to take us a step further. A consequence of the metabolome being further down the line from gene function is that the interpretation of metabolomics data reflects more closely the activities of a cell at the functional level and potentially leads to the revelation of metabolite function. This latter point is of great importance especially when we consider that a given metabolite may come from more than one metabolic pathway and may impact the cell in multiple ways. Further, by accurately quantifying the metabolome, we can begin to understand the stoichiometric significance of metabolite production. Since metabolomics was first coined, it was received with a great deal of fanfare. Like the earlier omics techniques, it was thought that comprehensive metabolomics data sets would be a staple of functional genomics analyses. That was until it was realized that the structural diversity that exists within the metabolome would make it virtually impossible, with current analytical technologies, to characterize the metabolomes of microorganisms fully. To make matters worse, the diverse chemical and physical properties within the metabolome, together with the structural differences in the cell walls of microorganisms, meant that it would be extremely difficult to establish a single sample extraction procedure that was suitable for all metabolites. Added to the fact that the recovery of metabolites would largely depend on the extraction protocol used, meant that the achievement of global metabolite analysis was not a trivial matter. Questions that should have been asked at the time were, how much of the metabolome was necessary to characterize metabolism and what aspects of metabolomics data were truly meaningful? While the goal of complete holistic metabolomics data sets is still what we aspire to achieve, at present we have softened our stance on metabolomics by focusing on local metabolite profiling. In addition to this, we have concentrated our efforts on looking for specific differences in metabolite levels in response to genetic modification, stress responses and environmental change. Even though this is a far cry from global metabolite profiling, these approaches combined with the latest analytical technologies are being used to successfully unravel gene function and to identify biomarkers and unknown metabolites. The most promising techniques that are currently employed in microbial metabolomics research are nuclear magnetic resonance (NMR), array-based mass spectrometry (MS), gas chromatography–MS (GC–MS), liquid chromatography–MS (LC–MS), and capillary electrophoresis–MS (CE–MS). Though NMR can provide high throughput fingerprinting and structural elucidation, cost, sensitivity and quantification are issues that need to be addressed if it is to compete with the MS-based technologies. While arraybased techniques, such as nanostructure-initiator MS (NIMS), are ideal for screening crude microbial extracts, developments in nano surface technology are essential if E. E. K. Baidoo J. D. Keasling (&) Physical Biosciences Division, Joint Bioenergy Institute, Lawrence Berkeley National Laboratory, Berkeley, CA, USA e-mail: jdkeasling@lbl.gov

Sequential Extraction for Evaluating the Behaviour of Selected Chemical Elements in Light weight Aggregates Manufactured From Mining and Industrial Wastes

The chemical extraction of twenty-nine elements in a washing aggregate sludge, a clay-richsediment, a sewage sludge, the mixtures of these materials and the artificial lightweight aggregates (LWAs)manufactured with them was determined by applying the optimized European Community Bureau of Reference sequential extraction procedure in order to establish the effect of the thermal treatment on the extraction of the selected elements. In accordance with the UNE-EN-1744-3 standard (2003), which states a single extraction procedure, preparation of eluates by aggregate leaching was also carried out. All the elements are mainly associated with the residual fraction in the LWAs so their availability is relatively low. The element that presented the highest concentration in the eluates after completion of the single extraction procedure was Fe. All the studied elements are in concentrations below the limit values for inert wastes so, from the standpoint of chemical leaching, the studied LWAs may be used in lightweight concrete manufacturing.

Physico-Chemical Parameters Determining the Variability of Actually and Potentially Available Fractions of Heavy Metals in Fluvial Sediments of the Middle Odra River

Abstract The occurrence of heavy metals (Cd, Cr, Cu, Ni, Pb, Zn) has been determined in the fluvial sediment samples collected along three transects in the Middle Odra River (western Poland) with a width of 360 m. The total concentrations of the metals were obtained after HNO3 microwave digestion and the available fractions of heavy metals were determined by single extraction procedures using two extractants: 0.01M CaCl2 and 0.05M EDTA. The measurement of physico-chemical parameters was also performed. The determination of total and available fractions of heavy metals, except potential available fractions of Cr, revealed high concentrations of studied elements detected in the sediment samples characterized by high content of coarse and very coarse-grained sand fraction and high content of organic matter. It was found that the concentrations of total and available fractions of metals could increase along with the content of organic matter, Eh values and concentrations of H+. Apart from the above, those concentrations become the lowest, the higher the content of medium grain size fractions is. Furthermore, the amounts of CaCl2 and EDTA extractable metals increase in the sediments samples characterized by the lowest total and available concentrations of heavy metals.

Evaluation of methods using celite to concentrate norovirus, adenovirus and enterovirus from wastewater

Enteroviruses, noroviruses and adenoviruses are among the most common viruses infecting humans worldwide. These viruses are shed in the feces of infected individuals and can accumulate in wastewater, making wastewater a source of a potentially diverse group of enteric viruses. In this study, two procedures were evaluated to concentrate noroviruses, adenoviruses and enteroviruses from primary effluent of wastewater. In the first procedure, indigenous enteroviruses, noroviruses and adenoviruses were concentrated using celite (diatomaceous earth) followed by centrifugation through a 30K MWCO filter and nucleic acid extraction. The second procedure used celite concentration followed by nucleic acid extraction only. Virus quantities were measured using qPCR. A second set of primary effluent samples were seeded with Coxsackievirus A7, Coxsackievirus B1, poliovirus 1 or enterovirus 70 before concentration and processed through both procedures for recovery evaluation of enterovirus species representatives. The pairing of the single step extraction procedure with the celite concentration process resulted in 47–98% recovery of examined viruses, while the celite concentration process plus additional centrifugal concentration before nucleic acid extraction showed reduced recovery (14–47%). The celite concentration process followed by a large volume nucleic acid extraction technique proved to be an effective procedure for recovering these important human pathogens from wastewater.

Effect of Inorganic and Organic Copper Fertilizers on Copper Nutrition in Spinacia oleracea and on Labile Copper in Soil

To ensure an optimal concentration of Cu in food crops, the effectiveness of eight liquid Cu fertilizers was studied in a spinach ( Spinacia oleracea L.) crop grown on Cu-deficient soil under greenhouse conditions. Plant dry matter yields, Cu concentrations in spinach plants (total and morpholino acid (MES)- and ethylenediaminedisuccinic acid (EDDS)-extractable), and Cu uptakes were studied. The behavior of Cu in soil was evaluated by both single and sequential extraction procedures. The highest quantities of Cu in labile forms in the soil, total uptakes, and Cu concentrations in the plants were associated with the application of the two sources that contained Cu chelated by EDTA and/or DTPA. The fertilizers containing these Cu chelates represent a promising approach to achieve high levels of agronomic biofortification. The stronger correlations obtained between low-molecular-weight organic acid-extractable Cu in soil and the Cu concentrations and Cu uptakes by the plants show the suitability of this soil extraction method for predicting Cu available to spinach plants.

From soil to leaves – Aluminum fractionation by single step extraction procedures in polluted and protected areas

The paper presents the fractionation of aluminum in the samples of soil and plants of different species using a selective single-step extraction method. The study was conducted in the area located near a chemical plant, which for many years served as a post-crystallization leachate disposal site storing chemical waste (sector I), and in the area around the site: in Wielkopolski National Park, Rogalin Landscape Park and toward the infiltration ponds at the “Dębina” groundwater well-field for the city of Poznań (Poland) (sector II). The results of aluminum fractionation in samples of soil, leaves and plants showed heavy pollution with aluminum, especially in the water soluble aluminum fraction – Alsw (maximum concentration of aluminum in soil extract was 234.8 ± 4.8 mg kg−1, in the leaves of Betula pendula it was 107.4 ± 1.8 mg kg−1 and in the plants of Artemisia vulgaris (root) and Medicago sativa (leaves) it amounted to 464.7 ± 10.7 mg kg-1and 146.8 ± 1.2 mg kg−1 respectively). In addition, the paper presents the problem of organic aluminum fractionation in biological samples and it shows the relationship between aluminum concentration in soil and the analysed woody and herbaceous species.

A sensitive GC/MS method using cold fiber SPME to determine polycyclic aromatic hydrocarbons in spring water

Polycyclic aromatic hydrocarbons (PAH) are carcinogenic to humans and can be found in various environments. This paper describes a cold fiber (CF) solid-phase microextraction (SPME) sampling method with gas chromatography mass spectrometry (GC/MS) to identify 16 PAH in spring water. The method yields good results for some validation parameters. The LOD and LOQ ranged from 7.10 to 57.96ngL−1 and from 23.60 to 193.19ngL−1, respectively. The intra-assay precision shows a coefficient of variation (CV) ranging from 4.38 to 17.65%, and recovery tests varied from 76±13% to 99±8%. The proposed method was attractive, as it extracted higher amounts of PAH in a single extraction procedure in comparison with conventional approaches. The method was applied to the analyses of PAH in twenty-four spring water samples collected in Divinópolis City, Minas Gerais, Brazil. Average PAH concentrations were in the range of 62.80±10.40ngL−1 for Benzo[a]anthracene and 146.58±98.98ngL−1 for Benzo[a]pyrene.

Estimates of viral abundance in soils are strongly influenced by extraction and enumeration methods

Viruses are highly abundant in temperate soils, ranging from 107 to 109 g−1, and outnumbering soil bacteria from 5- to over 1,000-fold. In order to determine the potential impacts of viruses on soil microbial communities, it is important to establish reliable methods for comparing changes in viral abundances within and across soil samples. The goals of this study were to optimize extraction-enumeration methods to accurately determine viral abundances in a range of soil types, to evaluate the feasibility of simultaneously enumerating bacterial cells and virus particles using a single extraction procedure, and to assess the utility of flow cytometry (FCM) for enumerating virus particles in soil extracts. Comparisons of extraction approaches indicated that sonication or blender extraction of soils with potassium citrate buffer yielded the highest viral abundances for most soil types. Combined viral and bacterial extractions underestimate abundances compared to separately-optimized extractions for each. Flow cytometric counts were anywhere between 350- and 1,400-fold higher than epifluorescence microscopy (EFM)-based counts for the same soil. Trends in viral abundance across soil types were different from those via EFM, and different relationships between viral abundance and soil properties were observed depending on the enumeration method. Thus, FCM is not currently recommended for enumeration of viruses in soil extracts. Based on EFM results, soil moisture and organic matter content were the most important factors determining viral abundance in soils.

Estimation of Lead Releasing Characteristics in Contaminated Agricultural Soils by Chelating Agents

Lead (Pb) is a highly toxic element because of its accumulative and persistent character in the environment. Especially, the risks of their potential entry into the food chain and pollution of heavy metal in an agricultural system will threaten to human health. Songsan wastewater irrigation region, a big rice production area of Anshan, Liaoning province, was polluted by containments of heavy metals for more than 80 years. The total Pb concentration in polluted soil sample was higher than in natural soil (value of environmental standard ≤35 mg•kg-1). In the study, the two chelating agents (EDTA and DTPA) were used to release Pb from the polluted soil. On the basis of single extraction procedures of Pb with two extracting agents, we found that EDTA had a significantly higher chelating capacity than DTPA (P<0.05). In addition, among our researches on releasing kinetics of lead with two reagents, the results indicated that the equilibrium action time was about 16 h.

Simultaneous quantitation of azole antifungals, antibiotics, imatinib, and raltegravir in human plasma by two-dimensional high-performance liquid chromatography–tandem mass spectrometry

High-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a standard analytical technique for therapeutic drug monitoring (TDM). A rapid LC-MS/MS method was developed for simultaneous quantitation of 3 antifungals and one active metabolite (posaconazole, voriconazole, itraconazole, and hydroxy-itraconazole), 5 antibiotics (daptomycin, ciprofloxacin, oxacillin, levofloxacin, and rifampicin), an antineoplastic agent (imatinib), and an antiretroviral (raltegravir) in human plasma. Protein precipitation of 10 μL of plasma with acetonitrile was used as a single-extraction procedure. After 2-dimensional LC, all drugs were quantified by electrospray ionization-triple quadrupole mass spectrometry by selected reaction monitoring detection in the positive mode. The method was validated per FDA recommendations including the study of extraction recovery (from 79.3% to 105.9%) and matrix effect via ion suppression/enhancement phenomenon. This method is precise (intra- and inter-assay coefficients of variation of 1.95-12.77%, 2.56-8.16% and 2.12-11.38% for low, medium and high levels of internal quality controls respectively) and accurate (intra- and inter-assay biases of 0.19-12.67%, 0.04 to -12.17% and 0.22-12.98% respectively). This method is an efficient tool for routine TDM and optimization of laboratory resource utilization.

Validation of a Single-Extraction Procedure for Sequential Analysis of Vitamin E, Cholesterol, Fatty Acids, and Total Fat in Seafood

Food lipid major components are usually analyzed by individual methodologies using diverse extractive procedures for each class. A simple and fast extractive procedure was devised for the sequential analysis of vitamin E, cholesterol, fatty acids, and total fat estimation in seafood, reducing analyses time and organic solvent consumption. Several liquid/liquid-based extractive methodologies using chlorinated and non-chlorinated organic solvents were tested. The extract obtained is used for vitamin E quantification (normal-phase HPLC with fluorescence detection), total cholesterol (normal-phase HPLC with UV detection), fatty acid profile, and total fat estimation (GC-FID), all accomplished in <40 min. The final methodology presents an adequate linearity range and sensitivity for tocopherol and cholesterol, with intra- and inter-day precisions (RSD) from 3 to 11 % for all the components. The developed methodology was applied to diverse seafood samples with positive outcomes, making it a very attractive technique for routine analyses in standard equipped laboratories in the food quality control field.

Systematic Evaluation of Extraction Methods for Multiplatform-Based Metabotyping: Application to the Fasciola hepatica Metabolome

Combining data from multiple analytical platforms is essential for comprehensive study of the molecular phenotype (metabotype) of a given biological sample. The metabolite profiles generated are intrinsically dependent on the analytical platforms, each requiring optimization of instrumental parameters, separation conditions, and sample extraction to deliver maximal biological information. An in-depth evaluation of extraction protocols for characterizing the metabolome of the hepatobiliary fluke Fasciola hepatica, using ultra performance liquid chromatography and capillary electrophoresis coupled with mass spectroscopy is presented. The spectrometric methods were characterized by performance, and metrics of merit were established, including precision, mass accuracy, selectivity, sensitivity, and platform stability. Although a core group of molecules was common to all methods, each platform contributed a unique set, whereby 142 metabolites out of 14,724 features were identified. A mixture design revealed that the chloroform:methanol:water proportion of 15:59:26 was globally the best composition for metabolite extraction across UPLC-MS and CE-MS platforms accommodating different columns and ionization modes. Despite the general assumption of the necessity of platform-adapted protocols for achieving effective metabotype characterization, we show that an appropriately designed single extraction procedure is able to fit the requirements of all technologies. This may constitute a paradigm shift in developing efficient protocols for high-throughput metabolite profiling with more-general analytical applicability.

A single extraction and HPLC procedure for simultaneous analysis of phytosterols, tocopherols and lutein in soybeans

A saponification/extraction procedure and high performance liquid chromatography (HPLC) analysis method were developed and validated for simultaneous analysis of phytosterols, tocopherols and lutein (a carotenoid) in soybeans. Separation was achieved on a phenyl column with a ternary, isocratic solvent system of acetonitrile, methanol and water (48:22.5:29.5, v/v/v). Evaporative light scattering detection (ELSD) was used to quantify β-sitosterol, stigmasterol, campesterol, and α-, δ- and γ-tocopherols, while lutein was quantified with visible light absorption at 450nm. Peak identification was verified by retention times and spikes with external standards. Standard curves were constructed (R2>0.99) to allow for sample quantification. Recovery of the saponification and extraction was demonstrated via analysis of spiked samples. Also, the accuracy of results of four soybeans using the described saponification and HPLC analytical method was validated against existing methods. This method offers a more efficient alternative to individual methods for quantifying lutein, tocopherols and sterols in soybeans.

Electro membrane extraction of biological anions with ion chromatographic analysis

A simple and sensitive single step electro membrane extraction (EME) procedure was demonstrated for biological organic anions with determination by ion chromatography (IC). Nitrite, adipate, oxalate, iodide, fumarate, thiocyanate and perchlorate were extracted from aqueous donor solutions, across a supported liquid membrane (SLM) consisting of methanol impregnated in the walls of a porous polypropylene membrane bag and into an alkaline aqueous acceptor solution in the lumen of the propylene envelope by the application of potential of 12V applied across the SLM. The acceptor solution was analyzed by IC. Parameters affecting the extraction performance such as type of SLM, extraction time, pH of the donor and acceptor solution, and extraction voltage were studied. The most favorable EME conditions were methanol as the SLM, extraction time of 5min, pH of acceptor and sample solutions of 12 and 4, respectively, and a voltage of 12V. Portable 12V batteries were used in the study. Under these optimized conditions, all anions had enrichment factors ranging from 3.6 to 36.2 with relative standard deviations (n=3) of between 6.6 and 17.5%. Good linearity ranging from 0.1 to 10μgmL−1 with coefficients of correlation (r) of between 0.9981 and 0.9996 were obtained. The limits of detection of the EME-IC method were from 0.01 to 0.14μgmL−1. The developed methodology was applied to amniotic fluid samples to evaluate the feasibility of the method for real applications.

Single Step Extractions of Metals in Coal Fly Ash

There is an increasing use of coal and subsequently large amounts of fly ash is generated in the thermal power plants in India and elsewhere in the world. Hence it becomes increasingly important to be able to assess the environmental risks involved in the management and disposal of fly ash materials. The fly ashes contain toxic metals that can be released into the environment through the coal combustion processes and through the leaching during disposal/ wet storage/ reuse of fly ash. The evaluation of the optimum time for leaching of the toxic metals and metalloids present in the three fly ashes was determined using the single step extraction procedure. The optimum time of 4 hours agitation was determined for the water soluble step as well as that for ion exchangeable step while the optimum time was 24 hours agitation in the acid soluble and reducible steps.

Quantification of retinoid concentrations in human serum and brain tumor tissues

Retinoic acid signaling is essential for central nervous system (CNS) differentiation and appears to be impaired in tumors. Thus far, there are no established methods to quantify relevant retinoids (all-trans-retinoic acid, 9-cis-retinoic acid, 13-cis retinoic acid, and retinol) in human brain tumors. We developed a single step extraction and quantification procedure for polar and apolar retinoids in normal tissue, lipid-rich brain tumor tissues, and serum. This quantification procedure is based on high performance liquid chromatography (HPLC) with diode-array detection (DAD) using all-trans-acitretin as an internal standard and extraction by liquid–liquid partition with ethyl acetate and borate buffer at pH 9. Recovery with this extraction procedure was higher than earlier (two-step) liquid–liquid extraction procedures based on hexane, NaOH, and HCl. The overall quantification procedure was validated according to Food and Drug Administration (FDA) guidelines and fulfilled all criteria of accuracy, precision, selectivity, recovery, and stability. The overall method accuracy varied between −5.6% and +5.4% for serum and −3.8% and +6.2% for tissues, and overall precision ranged from 3.1% to 6.9% for serum and 2.1% to 8.3% for tissues (%CV batch-to-batch). The lower limit of quantification for all compounds in tumor tissue (and serum) was 3.9ngg−1 (ngmL−1). Using this assay, photodegradation of the retinoids was evaluated and endogenous polar and apolar retinoids were quantified in sera and brain tumor tissues of patients and compared with serum and tonsil tissue concentrations of controls. It may thus serve as a suitable method for the characterization of retinoid uptake and metabolism in the respective compartments.

Simultaneous determination of polycyclic aromatic hydrocarbons and benzene, toluene, ethylbenzene and xylene in water samples using a new sampling strategy combining different extraction modes and temperatures in a single extraction solid-phase microextraction-gas chromatography–mass spectrometry procedure

This study proposes a new optimization approach for the simultaneous determination of polycyclic aromatic hydrocarbons (PAHs) and benzene, toluene, ethylbenzene and xylene isomers (BTEX) from water samples using the solid-phase microextraction technique followed by gas chromatography–mass spectrometry (GC–MS) separation and detection. The objective of the study was to achieve compromise extraction conditions, suitable for all semi-volatile and volatile compounds, under which the amount extracted is maximized for all analytes. This was achieved by careful optimization of the fiber coating, salting-out effect, extraction time and temperature and extraction mode (headspace or direct immersion). With the optimized fiber coating – PDMS/DVB 65μm – the other selected factors were optimized using a response surface methodology through central composite designs. As expected, the optimized results for each class of analytes varied significantly, probably due to the differences in their volatility and the equilibrium constants for the analyte/fiber coating. In order to overcome this issue, a new optimization approach was proposed based on a combination of extraction modes and extraction temperatures in a single extraction procedure. The final optimized procedure was: 48min of extraction in direct immersion mode with the sample maintained at 80°C followed by a further 32min of headspace extraction with the sample temperature kept at 10°C. The proposed procedure was compared with conventional methods based on the use of a single extraction mode and temperature (80min of headspace extraction at 60°C or 80min of direct immersion extraction at 50°C). The newly proposed method was shown to be more attractive as it extracted higher amounts of both semi-volatile and volatile compounds in a single extraction procedure compared to the conventional approaches. The optimized method was validated and excellent results were obtained.

Comparison of two sequential extraction procedures for the fractionation of zinc in agricultural calcareous soils

Twenty soil samples with a wide range of chemical and physical properties were collected from typical uncontaminated soils of south-west Iran, characterised by their high calcium carbonate contents (mean value of 321 g kg−1) and zinc deficiency (mean value of 0.2 mg kg−1 for DTPA-extractable Zn). The extractability and solid-phase fractionation of Zn in the soils was determined by the single extraction procedure (DTPA extractability) and two different sequential extraction procedures: a seven-step Singh method (procedure A) and the modification of three-step BCR (Community Bureau of Reference) method (procedure B). The DTPA extractable zinc levels were low (on average 3.34%). Zinc was mainly contained in the residual fraction (91.43% for procedure A and 75.25% for procedure B). In comparison with procedure B, few relationships were established between the zinc fractions isolated by procedure A and the soil properties. The Singh scheme provided better information than the BCR scheme about Zn distribution in the soils studied.

Heavy metal chemical fractionation and immobilization in lightweight aggregates produced from mining and industrial waste

The fractionation of five heavy metals in a washing aggregate sludge, a sewage sludge, a clay-rich sediment, the mixtures of these materials and the lightweight aggregates manufactured with them has been determined by applying the optimized European Community Bureau of Reference sequential extraction procedure in order to evaluate the effects of the heating process on the extraction of these elements. Additionally, preparation of eluates by aggregate leaching has been performed in accordance with the UNE-EN-1744-3 standard. The availability of all the studied heavy metals has been reduced by the thermal treatment, since most of the heavy metals have become part of the undigested material in the lightweight aggregates. Nickel has been the heavy metal that has presented the highest concentration in the eluates obtained after completion of the single extraction procedure in the lightweight aggregates. The studied lightweight aggregates may be used in lightweight concrete manufacturing from the standpoint of heavy metal leaching.

Assessment of potentially reactive pools of aluminum in Andisols using a five-step sequential extraction procedure

A five-step sequential extraction procedure was developed for the partitioning of soil aluminum (Al) into five fractions: exchangeable, weakly organic bound, strongly organic bound, inorganic non-crystalline and crystalline. The results obtained by the sequential extraction method for Al were compared with those estimated from single extractions using identical operating conditions applied in each individual sequential fraction. The Al content in the extracts was measured by inductively coupled plasma optical emission spectrometry. The results indicated that the first four steps [potassium chloride (KCl), copper chloride (CuCl2), sodium pyrophosphate (Na4P2O7), acid ammonium oxalate ((NH4)2C2O4)] in the sequential procedure could be as effective as single extraction methods at estimating exchangeable Al, weakly organic bound Al, strongly organic bound Al, and non-crystalline Al. However, the crystalline Al content by sequential procedure was not in agreement with single extraction procedures. Further, the sequential extractions resulted in more consistent estimates of the aluminum/silicon (Al/Si) molar ratio and allophane and crystalline Al contents than single extractions method. Results of X-ray diffraction on the soil samples confirm the presence of appreciable amounts of at least three types of crystalline minerals, including gibbsite, vermiculite or halloysite. Our result implies that the sequential method can be more reliable in estimating the various Al forms in Andisols.