Single Extraction Protocol Research Articles

Conventional and nonconventional extraction techniques for optimal extraction processes of rosmarinic acid from six Lamiaceae plants as determined by HPLC-DAD measurement

The goal of this study was to improve the extraction efficiency of rosmarinic acid (RA) from Lamiaceae herbs (lemon balm, peppermint, oregano, rosemary, sage, and thyme) using various extraction techniques (maceration with stirring, MACS; heat reflux, HRE; and microwave-assisted extraction, MAE) and extraction conditions (solvent acidity, solvent type, extraction time and temperature). The RA content was measured by high-performance liquid chromatography with diode-array detection (HPLC-DAD) under test conditions. Our results showed that extraction with acidified aqueous ethanol (EtOH-H2O-HCl, 70:29:1, v/v/v) was the best choice for the recovery of RA compared to other solvent systems. Further study suggested the following optimal extraction times for the different techniques: 120 min at 25 °C with MACS, 15 min at boiling point with HRE, and 5 min at 50 °C and 80 °C with MAE. Based on our results, we demonstrated that by careful adjustment of the extraction conditions, it is possible to set up a single extraction protocol to extract RA from different plants.

Drugs of abuse extraction from whole blood using supported liquid extraction (SLE) and Extrahera automation prior to UPLC-MS/MS analysis

Whole blood continues to be a valuable tool in forensic toxicology for the immediate and near-term detection of illicit drugs and in cases where no other sample is available. Screening drugs of abuse can be complicated due to the wide variation of functional groups associated with different analyte classes. Most extraction techniques cannot extract all analytes using a single procedure without using non-optimal extraction protocols resulting in compromised extract cleanliness. Supported liquid extraction allows for the simultaneous analysis of cross functional analytes in a single extraction protocol without forfeiting extract cleanliness. The objective was to develop an automatable, common extraction procedure for multiple drug panels from whole blood matrix using supported liquid extraction (SLE) prior to UPLC-MS/MS analysis. The drug suites included amphetamines, benzodiazepines, cocaine, opiates, ketamine, buprenorphine, Z-drugs and THC-COOH. Blank whole blood was spiked with drugs from various panels at 13 ng/mL for the analytes and appropriate deuterated internal standards. Whole blood extraction was performed using ISOLUTE SLE+ 1 mL capacity columns evaluating pH control and optimum extraction solvent combinations. The resultant extracts were evaporated to dryness and reconstituted in mobile phase for subsequent UPLC-MS/MS analysis. Samples were analyzed using a Waters ACQUITY UPLC coupled to a Quattro Premier XE triple quadrupole mass spectrometer (Waters). Positive ions were acquired using electrospray ionisation in the MRM mode. Initial investigations focused on sample treatment. Extraction conditions were evaluated using spiked whole blood pre-treated 1:1 (v/v) with various ammonium hydroxide (aq) concentrations from 0.1 % up to 2 %. 0.1 % NH 4 OH (aq) was selected as the pre-treatment as many of the drugs are basic in nature, however an environment at this pH value also allowed concomitant extraction of THC-COOH. Loading 1 mL of pre-treated whole blood led to the extraction of matrix components in the form of red blood cells. Evaluation of various loading volumes from 500 to 900 μL resulted in selection of 750 μL (1:1, v:v) as the optimal loading volume for extract cleanliness. Dichoromethane, dichloromethane/isopropanol acid and methyl tert-butyl ether combinations with various modifiers were evaluated for extraction efficiency. Benzoylecgonine could only be extracted efficiently when using dichloromethane as the extraction solvent. The optimum extraction protocol provided recoveries greater than 70 % for nearly all drugs spiked into whole blood. Benzoylecgonine and buprenorphine recovered 47 % and 52 % respectively. RSD values for all drugs were under 10 %. Lower recoveries for benzoylecgonine and buprenorphine did not prevent quantitation at sub-ng/mL concentrations. Evaporative effects were eliminated using 0.05 M HCl in methanol prior to evaporation. Calibration curves constructed from 1–500 ng/mL demonstrated excellent linearity and r 2 > 0.99 for all analytes. LLOQs were determined to be sub-ng/mL for each analyte when extracting 375 μL of whole blood, with the exception of THC-COOH (5 ng/mL). MRM analysis for common phospholipid molecules demonstrated the optimum method yields extremely clean extracts. This method was transferred to the Extrahera sample automation platform for repeat investigations into recovery, reproducibility and linearity and in nearly all analytes, the automation approach improves upon the manual approach for acquired data while also saving operator time. This poster describes the suitability of ISOLUTE SLE+ for the rapid and reliable extraction of multiple analyte panels from whole blood in a single, fully-automatable assay on the Extrahera platform, prior to UPLC-MS/MS analysis.

An efficient modified method for plant leaf lipid extraction results in improved recovery of phosphatidic acid

BackgroundLipidomics plays an important role in understanding plant adaptation to different stresses and improving our knowledge of the genes underlying lipid metabolism. Lipidomics involves lipid extraction, sample preparation, mass spectrometry analysis, and data interpretation. One of the practical challenges for large-scale lipidomics studies on plant leaves is the requirement of an efficient and rapid extraction method.ResultsA single-extraction method with a polar solvent mixture gives results comparable to a widely used, multi-extraction method when tested on both Arabidopsis thaliana and Sorghum bicolor leaf tissue. This single-extraction method uses a mixture of 30 parts chloroform, 25 parts isopropanol, 41.5 parts methanol, and 3.5 parts water (v/v/v/v) and a 24-h extraction time. Neither inclusion of ammonium acetate nor inclusion of acetic acid increased extraction efficiency.ConclusionsThe extract produced by this method can be used for analysis by mass spectrometry without a solvent evaporation step. The amount of lipid extracted, including phosphatidic acid, is comparable to widely used, more labor-intensive methods. The single-extraction protocol is less laborious, reducing the potential for human error.

Systematic Assessment of Seven Solvent and Solid-Phase Extraction Methods for Metabolomics Analysis of Human Plasma by LC-MS.

The comparison of extraction methods for global metabolomics is usually executed in biofluids only and focuses on metabolite coverage and method repeatability. This limits our detailed understanding of extraction parameters such as recovery and matrix effects and prevents side-by-side comparison of different sample preparation strategies. To address this gap in knowledge, seven solvent-based and solid-phase extraction methods were systematically evaluated using standard analytes spiked into both buffer and human plasma. We compared recovery, coverage, repeatability, matrix effects, selectivity and orthogonality of all methods tested for non-lipid metabolome in combination with reversed-phased and mixed-mode liquid chromatography mass spectrometry analysis (LC-MS). Our results confirmed wide selectivity and excellent precision of solvent precipitations, but revealed their high susceptibility to matrix effects. The use of all seven methods showed high overlap and redundancy which resulted in metabolite coverage increases of 34–80% depending on LC-MS method employed as compared to the best single extraction protocol (methanol/ethanol precipitation) despite 7x increase in MS analysis time and sample consumption. The most orthogonal methods to methanol-based precipitation were ion-exchange solid-phase extraction and liquid-liquid extraction using methyl-tertbutyl ether. Our results help facilitate rational design and selection of sample preparation methods and internal standards for global metabolomics.

XANES speciation of mercury in three mining districts – Almadén, Asturias (Spain), Idria (Slovenia)

The mobility, bioavailability and toxicity of mercury in the environment strongly depend on the chemical species in which it is present in soil, sediments, water or air. In mining districts, differences in mobility and bioavailability of mercury mainly arise from the different type of mineralization and ore processing. In this work, synchrotron-based X-ray absorption near-edge spectroscopy (XANES) has been taken advantage of to study the speciation of mercury in geological samples from three of the largest European mercury mining districts: Almadén (Spain), Idria (Slovenia) and Asturias (Spain). XANES has been complemented with a single extraction protocol for the determination of Hg mobility. Ore, calcines, dump material, soil, sediment and suspended particles from the three sites have been considered in the study. In the three sites, rather insoluble sulfide compounds (cinnabar and metacinnabar) were found to predominate. Minor amounts of more soluble mercury compounds (chlorides and sulfates) were also identified in some samples. Single extraction procedures have put forward a strong dependence of the mobility with the concentration of chlorides and sulfates. Differences in efficiency of roasting furnaces from the three sites have been found.

Broad-Range Bacterial and Fungal DNA Amplification on Vitreous Humor from Suspected Endophthalmitis Patients

Prompt and accurate diagnosis of infectious endophthalmitis is crucial for rapid and effective treatment. By identifying whether the causative pathogen is bacterial or fungal, a rational approach for the use of antibacterials or corticosteroids, respectively, can be followed. To assess the clinical utility of broad-range bacterial and fungal DNA amplification in the detection of endophthalmitis (postoperative, posttraumatic, and endogenous). In a prospective study, vitreous humor samples from 70 patients with the clinical diagnosis of presumed endophthalmitis, and from 30 patients undergoing surgery for non-infectious causes, were subjected to routine microbiologic and molecular investigation. DNA extracted from a 50 microL sample was amplified by primers targeting the conserved 16S and 18S ribosomal RNA gene sequences of bacteria and fungi, respectively. Reagents for bacterial DNA amplification were decontaminated of endogenous DNA using 8-methoxypsoralen and long wave UV treatment. A total of 35 specimens were positive for bacteria or fungi by culture. Of these, Gram-positive organisms were isolated in 19 specimens, Gram-negative organisms in 13 specimens and fungi in 3 specimens. Pseudomonas species, coagulase-negative Staphylococcus, and Streptococcus species were the main etiological agents isolated. Bacterial DNA amplification resulted in 49 positive specimens, compared with 32 positive specimens by culture; and fungal DNA amplification resulted in 11 positive specimens, compared with 3 positive specimens by culture. All control specimens were negative for both culture and DNA amplification. DNA extracted using a single-extraction protocol from 50 microL of vitreous humor and amplified with broad-range bacterial and fungal primers will enable the rapid differentiation (within 14 hours) between bacterial and fungal endophthalmitis and allow tailoring of therapy to individual patients.

Complexation efficiency of differently fixed 8-hydroxyquinoline and salicylic acid ligand groups for labile aluminium species determination in soils—comparison of two methods

Two methods utilizing the complexation of labile Al species by 8-hydroxyquinoline (HQN) and salicylic acid (SA) ligand groups were developed for aluminium operationally defined fractionation in acid soils. First, the solid phase extraction (SPE) procedure by a short-term ion-exchange batch reaction with chelating resins Iontosorb Oxin and Iontosorb Salicyl containing both ligand groups was used previously. Second, the 8-hydroxyquinoline, salicylic acid and ammonium salicylate agents with different concentrations by a single extraction protocol were applied in this paper. The flame atomic absorption spectrometry (FAAS) and optical emission spectrometry with inductively coupled plasma were used for aluminium quantification. The comparison of results from both methods show the possibility to supersede the first laborious method for the second simpler one in Al environmental risk assessment. The use of 1% 8-hydroxyquinoline in 2% acetic acid and 0.2% salicylic acid by a single extraction protocol without a need of sample filtration can supersede the SPE procedure in the Al pollution soil monitoring. Finally, the new scheme usable in a laboratory and moreover, directly in a field was proposed for Al fractionation in solid and liquid environmental samples. The labile Al species in soils and sediments are separated after their single leaching by 8-hydroxyquinoline or salicylic acid without a need of sample filtration. The labile Al species in soil solutions and natural waters are separated after their ultrafiltration followed by the SPE procedure with Iontosorb Oxin or Iontosorb Salicyl.

Discriminating between intra‐ and extracellular metals using chemical extractions

Several washing agents (EDTA, CDTA, NTA, 8 HQS, Ti‐EDTA‐citrate, Ca2+, and H+) were tested for their ability to extract adsorbed metal from biological surfaces to distinguish between internalized and total cellular metal (Pb, Ni, Cd, Zn, and Cu) for a green alga, Chlorella kesslerii. Although a single extraction protocol cannot be provided that would be acceptable for all biological species and metals, several guidelines are proposed that should facilitate the interpretation of metal bioaccumulation data. It was shown that for the studied metals, a thermodynamic explanation was generally sufficient to explain the metal extraction results. At pH 6.0, a solution of 5 × 10−3 M ethylenediaminetetraacetic acid (EDTA) was an acceptable washing agent in most cases, whereas Ca or proton exchange solutions did not appear to quantitatively remove adsorbed metals. Conditions of ligand excess could be attained with relatively small concentrations of added ligand, effectively minimizing potential damage to the organism, including modifications in membrane permeability. Potential deleterious effects of the wash agents were verified. For example, a Ti‐EDTA‐citrate or an EDTA wash at high concentrations resulted in perturbations of algal membrane permeability. In spite of potential difficulties interpreting extraction data, the usefulness of the extraction protocols was clearly demonstrated for several organisms.

Single extraction protocol for the analysis of 8-hydroxy-2′-deoxyguanosine (oxo 8dG) and the associated activity of 8-oxoguanine DNA glycosylase

Determination of the promutagenic base 8-hydroxy-2′-deoxyguanosine (oxo 8dG) has been the hallmark of studies aimed to determine oxidative damage to DNA. Different techniques including HPLC, GC–mass spectrometry, DNA sensitive sites and histology have been used to quantify oxo 8dG levels in samples from different sources. The most accepted and well-established methods are based on HPLC and the ability of oxo 8dG to be oxidized with an electrochemical detector. Considerable concerns have been raised in the ability of different labs to utilize a process of DNA extraction that reduces the levels of artifactual oxo 8dG formed during sample workup. Here, we present a fully detailed protocol that has been extensively used in our Lab to extract and analyze DNA and has little or no impact in the basal levels of oxo 8dG. Additionally, this protocol allows for the determination of the activity of mOgg1, the enzyme responsible for the initial step in the repair of the accumulated oxo 8dG, in the same sample in which oxo 8dG is detected.