Rapid Sample Extraction Research Articles

On-line coupling of two-phase microelectroextraction to capillary electrophoresis – Mass spectrometry for metabolomics analyses

We have integrated two-phase electroextraction (EE), capillary electrophoresis (CE) and mass spectrometry (MS) to combine rapid sample extraction, high performance separation and sensitive detection of metabolites. EE took place from a sample vial containing organic donor phase into a ~100 nL droplet of background electrolyte, which was hanging at the inlet of the capillary. In order to enable the EE process while the outlet of the capillary was connected to the MS several modifications were made. A modification was made to the sheath-liquid CE-MS interface, based on the use of a corrosion-resistant titanium ESI sprayer and a 6-port valve to switch between the CE-MS sheath liquid and the EE make-up liquid. Furthermore, a counter-pressure was applied, in order to prevent EOF from retracting the droplet during EE.Then, using five model metabolites (namely, leucine, isoleucine, adenosine, phenylalanine and guanosine) and crystal violet (CV) the extraction time and voltage were optimized and found to be 2000 s and 1 kV, respectively. Using these optimized conditions, the effect of various sodium chloride concentrations was examined to assess the influence of varying salt concentrations in biological samples. A set of 9 amino acids was used to validate the method. The detection limits ranged between 5 and 100 nM. LODs were improved 50–250 times in comparison with conventional CE-MS. Finally, to demonstrate the potential of the EE-CE-MS platform for bioanalysis of volume-limited samples, a urine sample of 300 nL was analyzed. This resulted in detection of 122 putative metabolites. The results indicate that EE-CE-MS could become a powerful tool for metabolomics analyses of volume-limited samples.

High-throughput experimentation for microstructural design in additively manufactured 316L stainless steel

In the present study, a combination of high-throughput (HT) and low-throughput (LT) techniques was used to rapidly determine the processing window and generate processing maps for Selective Laser Melting (SLM) of 316L stainless steel. The HT method includes the fabrication of hundreds of hex nut-shaped specimens, each processed with a unique combination of laser power, scanning speed, and hatch spacing. An easily removable scaffolding permitted rapid sample extraction from the base plate, thus saving machining cost and time. Hardness and immersion density measurements were used for HT characterization to identify a processing window for maximum strength and density. Within the defined processing window, a low-throughput (LT) microstructural interrogation of specimens were performed. The microstructural analysis included quantification at various length scales (i.e., grains size and morphology, texture, primary dendrite arm spacing, and melt pool geometry analysis). Microstructure-based processing maps as a function of volumetric energy density were generated. The combination of HT and LT methods produced a predictive relationship between hardness and primary dendrite arm spacing using a Hall-Petch relationship. A model is proposed to explain the dependence of microstructure on the melt pool geometry. The HT method can be applied for the microstructural design of SLM-fabricated components in other alloys.

High Throughput Metabolomics in Clinical Studies: Review and New Applications to Remote Ischemic Preconditioning.

Metabolomic analysis has made substantial contributions to the understanding of diverse pathological processes and has the potential to improve diagnosis and identify novel therapeutic targets. As early success in perinatal medicine, nutrition, chronic diseases, cancer and trauma demonstrates, metabolomics is approaching feasibility in terms of guiding improvement in population-level diagnosis and treatment. A key barrier to implementing metabolomics as a routine diagnostic tool is rapid sample extraction and data analysis along with the establishment of normal values for novel metabolic markers. This review covers key advancements in clinical metabolomics and applies a high throughput metabolomics method as a proof of principle to identify novel metabolites associated with remote ischemic preconditioning.

A Validation and Estimation of Total Eicosapentaenoic and Docosahexaenoic acids Using LC-MS/MS with Rapid Hydrolysis Enzymatic Method for Hydrolysis of Omega Lipids in Human Plasma and its Application in the Pharmacokinetic Study

Background:In this study, we have developed a novel, rapid enzymatic hydrolysis method for conversion of omega lipids (omega fatty acid triglycerides, phospholipids, omega conjugates) in to free fatty acids at room temperature using lipase and esterase enzymes. </P><P> Objective: To develop simple enzymatic hydrolysis and rapid sample extraction method for quantification of free (un-esterified) and conjugated (esterified) eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) to provide the total EPA and DHA lipids present in human plasma. Quantification of total EPA/DHA was performed using liquid chromatography and tandem mass spectrometer instrument.Methods:The plasma sample is digested with lipase and esterase enzymes and extracted by using combined precipitation and liquid-liquid techniques. The LC-MS/MS method was optimized using EPA-D5 and DHA-D5 as labeled internal standards for EPA/DHA respectively. The analytical method is validated, utilized for simultaneous quantification of total EPA and DHA lipids in plasma collected from healthy human volunteers clinical study.Results:The reproducibility of the established enzymatic hydrolysis method was demonstrated by incurred sample reanalysis and the results for total EPA and DHA lipid were 93.33% and 96.67% respectively. The pharmacokinetic and statistical analysis was performed using baseline corrected concentration of total EPA and DHA lipids.Conclusion:The enzymatic hydrolysis method for conversion of omega fatty acid triglycerides, phospholipids, omega conjugates in to free fatty acid was reported first time for the quantitative application. The shorter time for sample workup procedure, simple enzymatic hydrolysis at room temperature and 3 minutes chromatography run time are well suitable for bioavailability/ bioequivalence studies.

Determination of nitenpyram and 6-chloronicotinic acid in environmental samples by ion chromatography coupled with online photochemically induced fluorescence detector.

A simple, cost-effective, sensitive, and quick method for the determination of nitenpyram and its metabolite 6-chloronicotinic acid in environmental samples was developed by coupling an ion chromatograph with a fluorescence detector and a post-column photochemical reactor. This developed analytical method involved a rapid sample extraction by modified and miniaturized quick, easy, cheap, effective, rugged, and safe method followed by isocratic ion chromatographic separation of nitenpyram and 6-chloronicotinic acid into an IonPac™ AS11-HC column protected by IonPac™ AG11A guard column by running 30mM NaOH + 10% acetonitrile mobile phase. A homemade post-column photochemical reactor was also integrated with the ion chromatographic system for online transformation of both analytes into their respective highly fluorescent photoproduct in basic media without using an extra pump. The developed method was validated by following SANTE/11945/2015 guidelines on analytical quality control and validation procedures. The method showed a good linear response (r>0.999), improved limit of detection (0.101-0.132μg/L), minimum or no matrix effect, excellent recoveries (90.2-100.10%) and relative standard deviations were found to be ≤6.50%.

5-sulfosalicylic Acid Dihydrate-Based Pretreatment for the Modification of Enzyme-Linked Immunoassay of Fluoroquinolones in Fishery Products

A simple, rapid sample extraction method for the determination of FQs was developed. Fishery samples were extracted with 2% of 5-sulfosalicylic acid dihydrate and the extracts were analyzed directly without any further purification or clean-up procedures. The FQs were determined with standards of 2% of 5-sulfosalicylic acid dihydrate in the concentration range of 0.1-25.6 μg L−1, and the limit of detection (LOD) was 0.1 μg L−1. The matrix interference originated from fishery samples was eliminated by 2% of 5-sulfosalicylic acid dihydrate and did not interact with horseradish peroxidase (HRP) labeled IgG in western blotting. No significant matrix interference was observed as samples extracted with 2% of 5-sulfosalicylic acid dihydrate. Recoveries of FQs in fishery muscle were between 72.37–94.35% in the concentrations range of 10–50 μg kg−1.This extraction procedure was much rapider and simpler to conventional ELISA extraction procedure and could be used as a time-saving and cost-effective method for FQs monitoring in fishery samples.

Development of a novel genetically modified bioluminescent-bacteria-based assay for detection of fluoroquinolones in animal-derived foods.

Fluoroquinolones (FQNs) are broad-spectrum antibacterial agents widely used in animal husbandry and aquaculture. The residues and antimicrobial resistance of such antibiotics are a major public health concern. To realize multianalyte detection of FQN residues, a genetically modified bacterium, Escherichia coli pK12 harboring plasmid pRecAlux3, was constructed in this study to develop a bioluminescent-bacteria-based assay for the detection of FQNs in animal-derived foods. This assay was based on the principle of induction of an SOS response by FQNs via inducing the recA-promoter-fused luciferase reporter gene existing on the plasmid pRecAlux3. E. coli pK12 was able to recognize 11 FQNs: difloxacin, enrofloxacin, ciprofloxacin, sarafloxacin, norfloxacin, danofloxacin, ofloxacin, pefloxacin, lomefloxacin, marbofloxacin, and orbifloxacin. This method could be applied to 11 edible tissues, including milk, fish muscle, and the muscles, livers, and kidneys of cattle, chickens, and pigs, with a very simple and rapid sample extraction procedure using only phosphate-buffered saline. The limits of detection of the FQNs were between 12.5 and 100μgkg(-1), all of which were lower than the maximum residue limits. Most of the recoveries of the FQNs were in the range from 60 to 120%, and the interassay coefficients of variation were less than 30%. This method, confirmed by high-performance liquid chromatography, is reliable and can be used as both a screening test and a semiquantitative assay, when the identity of a single type of FQN is known.

A Novel Chip-based Electroelution System for Rapid and Efficient Recovery of Intact Proteins from Polyacylamide Gels

A Novel Chip-based Electroelution System for Rapid and Efficient Recovery of Intact Proteins from Polyacylamide Gels

Quantitative analysis by HPLC‐MS2 of the pyrrolizidine alkaloid adonifoline in Senecio scandens

A quantitative method using HPLC-MS(2) has been developed for the determination of adonifoline, one of the retronecine-type hepatotoxic pyrrolizidine alkaloids in Senecio scandens Buch.-Ham. ex D. Don., a traditional Chinese herb. Using an orthogonal design test, a simple and rapid sample extraction method was developed. HPLC analysis was conducted using a C(18) column as stationary phase and a mixture of acetonitrile and aqueous formic acid as mobile phase. Good linearity for adonifoline was found in the concentration range 0.12-4.18 microg/mL, and the HPLC-MS/MS method was shown to be appropriate, in terms of sensitivity, precision and reproducibility. The quantities of adonifoline in extracts of 18 plant samples from different collection sources and from different parts (flowers, leaves, thick stems, slim stems and roots) of S. scandens were determined using the newly developed HPLC/MS(2) analysis.

Determination of Thiamethoxam residues in honeybees by high performance liquid chromatography with an electrochemical detector and post-column photochemical reactor

A method for the determination of Thiamethoxam in bee samples was set up by means of high performance liquid chromatography with an electrochemical detector and post-column photochemical reactor (HPLC–hν–ED). Analytical method was based on a rapid sample extraction procedure with acetone, followed by chromatographic separation into a C18-RP column isocratically operated by 60 mM phosphate buffer/acetonitrile (75/25) mobile phase at pH 2.7. A photochemical reactor was used as a tool to verify and eventually quantify the presence of Thiamethoxam in the samples by distinguishing it from interference contribution. Detection was performed with a potential of 880 mV after a photoactivation with a 254 nm light. The least detectable dose was 0.002 mg kg −1. Recovery rates ranged between 59.88 and 71.62%.

Highly Sensitive Enzyme-Linked Immunosorbent Assay for Chlorpyrifos. Application to Olive Oil Analysis

Highly sensitive enzyme-linked immunoassays for chlorpyrifos, one of the most applied insecticides, are presented. Several haptens were synthesized for immunoreagent production and ELISA development. The best immunoassays obtained are based on an indirect coated-plate immunoassay format. Two assays were optimized; one shows a limit of detection of 0.3 ng L(-1), an I50 of 271 ng L(-1), and a dynamic range between 4 and 16 474 ng L(-1). The other one has a limit of detection of 0.07 ng L(-1), an I50 of 7 ng L(-1), and a dynamic range between 0.4 and 302 ng L(-1). The assays were used to quantify chlorpyrifos in olive oil using a simple and rapid sample extraction procedure. The good recoveries achieved in both assays (109% mean value) and the agreement with values given by the GC reference method (110% mean value) indicate their potential for either screening or laboratory quantification.

Improved detection of serum estradiol after sample extraction procedure.

The direct measurement of serum estradiol offers advantages over the more traditional sample extraction and immunoassay methods with respect to technical ease of the assay and turnaround time. However, direct assays have not been demonstrated sufficiently sensitive for some important clinical applications, including the measurement of circulating estradiol in women during the early follicular phase of their menstrual cycle, in postmenopausal women, and in men. We report here a rapid sample extraction method developed to improve the detection limit of automated estradiol assays and thus enhance the potential usefulness of these rapid and nonisotopic systems for measuring low concentrations of serum estradiol. Serum estradiol concentrations have long been used in conjunction with ultrasonography and other clinical indices to monitor ovarian stimulation by exogenous gonadotropin therapy during in vitro fertilization (IVF) procedures (1)(2)(3). With the recent use of gonadotropin-releasing hormone agonists, serum estradiol measurement has also been used to assess the adequacy of ovarian suppression before exogenous gonadotropin stimulation. In addition, it has become increasingly important to measure the low initial concentrations of serum estradiol (<150 ng/L) associated with the earlier stages of ovarian follicular stimulation (4)(5). Clinically important measurements of low estradiol concentrations are also required in assessing ovarian activity in perimenopausal women or women experiencing premature ovarian failure and in clinical investigations of the hypothalamic-pituitary-gonadal axis in men (6)(7)(8). Methods for the direct measurement of serum estradiol are replacing the more traditional RIA methods that use preassay sample extraction followed by RIA with antecedent column chromatography. Direct methods are advantageous because they are rapid and less technically demanding. However, direct assays are often inaccurate, particularly for the measurement of relatively low concentrations of serum estradiol (9)(10)(11). The presence of sex-hormone-binding globulin in serum and …

Improved extraction methods for avoiding the interference of copper in the LC determination of ascorbic acid in multivitamin-mineral tablets.

A rapid sample extraction procedure for the determination of ascorbic acid (AA) by high performance liquid chromatography (HPLC) in multivitamin-mineral formulations containing interfering copper has been developed. The method takes special precautions to prevent degradation of AA in contact with high concentrations of interfering elements such as copper. Sample preparation involved addition of pyrogallol, citric acid solution and short time extraction (5 min) under an atmosphere of nitrogen to prevent oxidation of AA. Another sample cleanup based on extraction of the multivitamin-mineral tablet with the extraction solution as described above, but with addition of strong cation exchange sorbent, was also developed. The copper and other minerals in the formula were retained on an ion exchange sorbent and thus represses the speed of oxidation of AA. Extracts of multivitamin-mineral tablets were analyzed by ion-pair reversed phase HPLC and UV-diode array detection. Ten multivitamin formulations containing copper were analyzed by the two proposed methods. The analytical results of AA obtained using the simple sample extraction method and the method involving addition of cation exchange were 89-115% and 95-112%, respectively, of declared concentrations. The parameters for the validation of the methods are given.

A kinetic microassay for glutathione in cells plated on 96-well microtiter plates.

We describe a modified enzymatic, kinetic, glutathione microassay based on the original macroassay described by Tietze and modified by Anderson. It is coupled with the Triton X-100 lysis and an acid extraction that can be performed in 96-well microtiter plates. The microassay can be read in a microplate reader equipped with a standard 405 nm filter. Intracellular glutathione levels are not significantly different when comparing the proposed Triton X-100 lysis and acid extraction method from those found with the cellular homogenization and acid extraction method typically employed. By combining a rapid sample extraction method, which can be done on the microtiter plate, with an assay based on the technology of a microplate reader, we have devised a rapid, reproducible, inexpensive and easy to use GSH microassay that can process several hundred samples daily and will be useful in studies where many replicate samples are required. We have successfully used this method to monitor glutathione status in a human cell line exposed to organochlorine pesticides and cells exposed to naturally occurring mixtures of metals extracted from aquatic sediments.