Fast,selective, and sensitive analysis of low-abundance peptides in human plasma by electromembrane extraction

A totally new concept based on electrokinetic migration was evaluated for the extraction of three biologically active peptides from human plasma. Angiotensin 2, leu-enkephalin, and endomorphin 1 migrated from a diluted human plasma sample (2 mL, positive electrode), through a supported liquid membrane (SLM) of 1-octanol, di-isobutylketon, and di-(2-ethylhexyl) phosphate (DEHP) (55:35:10, w/w/w), and into an acidified acceptor solution (25 μL 50 mM HCl, negative electrode) by the application of an electrical potential (20 V) across the SLM. After only five min of extraction, the acceptor solution was injected and analyzed directly by liquid chromatography. The three peptides were quantified by tandem mass spectrometry, with acceptable linearity ranging from 100.0 to 1000.0 pg mL⁻¹ (r² in the range 0.9736–0.9988), and repeatability (RSD) ranging between 15% and 24% (n = 5), using plasma spiked with the three peptides in 100 pg mL⁻¹ concentration. The estimated detection limits (S/N ratio of 3:1) for angiotensin 2, leu-enkephalin, and endomorphin 1, were 60, 24, and 24 pg mL⁻¹, respectively. With this novel approach based on electromembrane extraction (EME) coupled to LC–MS/MS, endogenous concentrations of the peptides were detected in non-spiked human plasma samples, with a total analysis time less than 50 min. These experimental findings were highly interesting, and showed the opportunities for EME with regard to future peptide extractions.     

Comparison of extraction methods for the recovery, amplification and species-specific analysis of DNA from bone and bone meals

We report the effect of several parameters on the efficiency of recovery of DNA from animal bones. The effects of preheating the samples (at either 60 degrees C or 100 degrees C) at different intervals (from 1 h to overnight) in different media (water, 0.5 M ethylenediaminetetraacetic acid (EDTA), or 0.5 M EDTA + 0.05% sodium dodecyl sulfate (SDS) were investigated. The effect of slight (5 min) or intense (30 min) pretreatments with ultrasound was also evaluated. Several different treatments with proteinase K (ranging from 200 to 800 microg, and lasting from 1 to 3 h) at 65 degrees C were also considered. Additionally, two different DNA extraction methods (based on silica resins and purification columns, respectively) were evaluated. The recovery of DNA from the samples was 40% higher when the bones were preheated in 0.5 M EDTA at 60 degrees C for 1 h, this being followed by treatment with 800 microg of proteinase K for 3 h. The DNA thus obtained was successfully amplified by polymerase chain reaction (PCR) using a set of primers specific to a 359 bp region of the mitochondrial cytochrome b gene, and the species of origin were identified by visualizing the restriction fragment length polymorphism (RFLP) with the endonucleases PalI and MboI.     

Comparative evaluation of extraction methods for simultaneous mass-spectrometric analysis of complex lipids and primary metabolites from human blood plasma

Metabolomic results on human blood plasma largely depend on the sample preparation protocols employed for protein precipitation and metabolite extraction. Five different extraction methods were examined, which can be grouped into two categories, liquid-liquid extraction and protein precipitation methods, including long-standing protocols such as the Folch extraction and Bligh-Dyer extraction in comparison to modern methods such as the Matyash protocol and two global metabolite extraction methods. Extracts were subjected to analysis of blood plasma lipids and primary metabolites by using chip-based direct infusion nanoelectrospray tandem mass spectrometry and gas chromatography coupled to time-of-flight mass spectrometry, respectively. Optimal extraction schemes were evaluated based on the number of identified metabolites, extraction efficiency, compound diversity, reproducibility, and convenience for high-throughput sample preparations. Results showed that Folch and Matyash methods were equally valid and robust for lipidomic assessments while primary metabolites were better assessed by the protein precipitation methods with organic solvent mixtures. Graphical Abstract

Schematic workflow of five extraction methods and subsequent mass spectrometry analysis using GC-TOF MS and nanoelectrospray direct-infusion ion trap MS/MS?     

Kinetic electro membrane extraction under stagnant conditions—Fast isolation of drugs from untreated human plasma

Amitriptyline, citalopram, fluoxetine, and fluvoxamine were isolated by electro membrane extraction (EME) from 70 μl of untreated plasma (pH 7.4), through a supported liquid membrane (SLM) of 1-ethyl-2-nitrobenzene immobilized in the pores of a porous polypropylene hollow fiber, and into 30 μl of 10 mM HCOOH as acceptor solution inside the lumen of the hollow fiber. The driving force of the extraction was a 9 V potential sustained over the SLM with a common battery, with the positive electrode placed in the plasma sample and the negative electrode placed in the acceptor solution. Extractions were performed under totally stagnant conditions with a very simple device for 1 min (kinetic regime), and subsequently the acceptor solution was analyzed directly by liquid chromatography–mass spectrometry (LC–MS). Recoveries were 12, 13, 22, and 17% for fluoxetine, amitriptyline, citalopram, and fluvoxamine, respectively. Sample clean-up was comparable to reversed-phase solid-phase extraction (SPE), but EME required substantially less time than SPE. The time advantage of EME was further improved by parallel extraction of three samples (for 1 min) with the same 9 V battery. EME from plasma combined with LC–MS provided limits of quantification (S/N = 10) in the range 0.4–2.3 ng/ml, linearity in the range 1–1000 ng/ml with r²-values of 0.998–0.999, and repeatability in the range 3.2–8.9% RSD in the mid-therapeutic window (100 ng/ml).     

Fast ultrasound-assisted extraction of copper,iron, manganese and zinc from human hair samples prior to flow injection flame atomic absorption spectrometric detection

A dynamic ultrasound-assisted extraction procedure utilizing diluted nitric acid was developed for the determination of copper, iron, manganese and zinc in human hair taken from workers in permanent contact with a polluted environment. The extraction unit of the dynamic ultrasound-assisted extraction system contains a minicolumn into which a specified amount of hair (5–50 mg) is placed. Once inserted into the continuous manifold, trace metals were extracted at 3 mL min⁻¹ with 3 mol L⁻¹ nitric acid under the action of ultrasound for 2 min for zinc and 3 min for copper, iron and manganese determination, and using an ultrasonic water-bath temperature of 70 °C for zinc and 80 °C for copper, iron and manganese determination. The system permits the direct analysis of hair and yields concentrations with relative standard deviations of <3% (n = 11). The applicability of the procedure was verified by analysing human hair samples from workers exposed to welding fumes, and its accuracy was assessed through comparison with a conventional sample dissolution procedure and the use of a certified reference material (BCR 397, human hair).     

Electromembrane extraction of basic drugs from untreated human plasma and whole blood under physiological pH conditions

The present work describes the first systematic study of electromembrane extraction (EME) from biological matrices under physiological conditions. Six basic drugs with protein binding in the range of 20–97% were extracted from untreated human plasma and whole blood through a supported liquid membrane (SLM) consisting of 1-ethyl-2-nitrobenzene impregnated in the walls of a hollow fiber, and into an acidified aqueous solution inside the lumen of the fiber. The electrical potential difference over the membrane reduced the protein binding of the drugs and transported the free drug fraction over the membrane. Recoveries in the range 25–65% were obtained with 10-min extraction time and an applied voltage of only 10 V over the SLM. Interday precision better than 20% RSD and linearity in the range 0.5–10 μg/mL were obtained for nortriptyline and methadone. Extraction from untreated whole blood was also demonstrated with recoveries in the range 19–51%.     

Fast high-performance liquid chromatographic analysis of mianserin and its metabolites in human plasma using monolithic silica column and solid phase extraction

A fast high-performance liquid chromatography (HPLC) method was developed and validated for the simultaneous determination of mianserin (MIAN) and its metabolites desmethylmianserin (DMM), 8-hydroxymianserin (HM) and mianserin-N-oxide (MNO) in human plasma. Each compound, together with internal standard (propranolol) was extracted from the plasma matrix using solid phase extraction. Chromatographic resolution of the analytes was performed on a Chromolith Speed Rod monolithic silica column ( mm i.d.) under isocratic conditions using a mobile phase of 74:26 (v/v) 25 mM phosphate buffer (pH 5.3 adjusted with phosphoric acid): acetonitrile. The elution of the analytes were monitored at 292 mm and conducted at ambient temperature. Because of high column efficiency the mobile phase was pumped at a flow rate of 3.5 ml/min. The total run time of the assay was 5 min. The method was validated over the range of 10-200 ng/ml for MIAN, 10-150 ng/ml for DMM, 20-300 ng/ml for HM and 25-500 ng/ml for MNO. The method proved to be precise (within-run precision ranging from 1.6 to 6.9% R.S.D. and between-run precision ranging from 1.3 to 7.2% R.S.D.) and accurate (within-run accuracies ranged from 1.4 to 6.4% and between-run accuracies ranging from 1.5 to 4.5%). The mean absolute recoveries were 95.7, 94.8, 99.6, and 102.6% for MIAN, DMM, HM and MNO, respectively. The limit of quantitation (LOQ) for MIAN and DMM was 10 ng/ml and for HM and MNO were 20 and 25 ng/ml in human plasma, respectively. The limit of detection (LOD) for MIAN, DMM, HM and MNO was 5, 2.5, 10 and 15 ng/ml, respectively. The described method demonstrates the feasibility for employing monolithic columns to effect rapid bioanalytical HPLC analysis for the quantitative determination of MIAN and its major metabolites in human plasma.     

PIXE eleemental analysis of erythrocyte and blood plasma samples from human pregnancies

Elemental concentrations of P, S, Cl, K, Ca, Fe, Ni, Cu, Zn, Br, Rb have been determined in erythrocyte and blood plasma samples from normal and diabetic human pregnancies. Average values, the dependence of the concentrations on the time during gestation period, the correlation coefficients for pairs of elements as well as for the same elements in plasma and erythrocyte samples are given. A marked difference appeared in a number of cases between normal and diabetic pregancies.     

Effect of nucleic acid binding dyes on DNA extraction,amplification, and STR typing

We report on the effects of six dyes used in the detection of DNA on the process of DNA extraction, amplification, and detection of STR loci. While dyes can be used to detect the presence of DNA, their use is restricted if they adversely affect subsequent DNA typing processes. Diamond? Nucleic Acid Dye, GelGreen?, GelRed?, RedSafe?, SYBR^® Green I, and EvaGreen? were evaluated in this study. The percentage of dye removed during the extraction process was determined to be: 70.3% for SYBR^® Green I; 99.6% for RedSafe?; 99.4% for EvaGreen?; 52.7% for Diamond? Dye; 50.6% for GelRed?, and; could not be determined for GelGreen?. It was then assumed that the amount of dye in the fluorescent quantification assay had no effect on the DNA signal. The presence of all six dyes was then reviewed for their effect on DNA extraction. The t‐test showed no significant difference between the dyes and the control. These extracts were then STR profiled and all dyes and control produced full DNA profiles. STR loci in the presence of GelGreen^TM at 1X concentration showed increased amplification products in comparison to the control samples. Full STR profiles were detected in the presence of EvaGreen? (1X), although with reduced amplification products. RedSafe? (1X), Diamond? Dye (1X), and SYBR^® Green I (1X) all exhibited varying degrees of locus drop‐out with GelRed? generating no loci at all. We provide recommendations for the best dye to visualize the presence of DNA profile as a biological stain and its subsequent amplification and detection.     

Matrix solid-phase dispersion extraction of sulfonamides from blood

Matrix solid-phase dispersion extraction was applied to the extraction of sulfadiazine, sulfamerazine, and sulfamethazine from human and animal bloods. The separation and determination of the analytes were carried out by high-performance liquid chromatography. The effects of the types of the dispersion adsorbents and elution solvents were investigated, and the highest recovery was obtained when diatomaceous earth was used as the dispersion adsorbent, while acetone was used as the elution solvent. Under the optimal conditions, the linear range for determining the sulfonamides in blood samples was 0.020-10.0 μg/mL, and the average recoveries of the three sulfonamides were higher than 87.5%.     

Ultrasound-assisted extraction, HPLC analysis, and antioxidant activity of polyphenols from unripe apple

The polyphenols were extracted from the unripe apple assisted by a highly efficient and simple method of the ultrasound. Response surface methodology was used to investigate the effects of processing parameters, including ultrasound power, extraction time, temperature, and ethanol concentration on total polyphenols yield and polyphenols composition was analyzed by HPLC. Antioxidant activity of the polyphenols was evaluated as 2, 2-diphenyl-1-picrylhydracyl scavenging activity and inhibition activity of lipid peroxidation. The results showed that 10-100 times higher total polyphenols yield was obtained from the unripe apple than those from the reported apple pomace. The optimum extraction conditions were ultrasonic power of 519.39 W, extraction time of 30 min, extraction temperature 50°C, ethanol concentration of 50% gave the total polyphenols yield of 13.26 ± 0.56 mg GAE/g. HPLC analysis indicated that (-)-epicatechin, procyanidin B2, chlorogenic acid, and procyanidin B1 were the predominant polyphenols in unripe apple, which contributed to the higher antioxidant activity to 2, 2-diphenyl-1-picrylhydracyl of unripe apple polyphenols than other apple polyphenols. The extracted polyphenols had higher ability to inhibit lipid peroxidation than butylated hydroxy toluene, which demonstrated that the unripe apple polyphenols have the potential to be used as a substitute of some synthetic antioxidants.     

Extraction of local anaesthetics from human blood by direct immersion-solid phase micro extraction (SPME)

Summary Local anaesthetics have been shown to be extractable from human whole blood samples by direct immersion (DI)-solid phase micro extraction (SPME). After deproteinization with perchloric acid, the pH of the clear supernatants of human whole blood samples containing the drugs were adjusted to about 7 with 10 M NaOH in the presence of NaCl; a polydimethylsiloxanecoated SPME fiber was then immersed directly into the sample solution to allow adsorption of the drugs before capillary gas chromatography (GC) with flame ionization detection. The DI-SPME for 1-mL whole blood gave peaks for all the drugs; only a small amount of background noise appeared and this gave no problems in the detection of the drugs. Recoveries of the ten drugs from human whole blood was 0.74–19.7 %. The calibration curves for seven drugs showed linearity in the range of 0.25–12 g mL^–1 whole blood, with detection limits of 54–158 ng mL^–1.     

GC-MS analysis of metabolites from soxhlet extraction,ultrasound-assisted extraction and supercritical fluid extraction of Salacca zalacca flesh and its alpha-glucosidase inhibitory activity

Abstract

Different extraction processes were employed to extract bioactive metabolites from Salacca zalacca flesh by a range of aqueous and organic solvents. The highest extraction yield was obtained by 50% ethanol extract of SE (73.18?±?4.35%), whereas SFE_1 showed the lowest yield (0.42?±?0.08%). All extracts were evaluated for in vitro α-glucosidase inhibitory activity, measured by their IC₅₀ values in comparison to that of quercetin, the positive control (IC₅₀ = 2.7?±?0.7?μg/mL). The lowest α-glucosidase inhibitory activity was indicated by water extract of SE (IC₅₀ = 724.3?±?42.9?μg/mL) and the highest activity was demonstrated by 60% ethanol extract by UAE (IC₅₀ = 16.2?±?2.4?μg/mL). All extracts were analysed by GC-MS and identified metabolites like carbohydrates, fatty acids, organic acids, phenolic acids, sterols and alkane-based compounds etcetera that may possess the potential as α-glucosidase inhibitor and may attribute to the α-glucosidase inhibitory activity.     

Solid phase DNA extraction on PDMS and direct amplification

In this paper we report an innovative use of Poly(DiMethyl)Siloxane (PDMS) to design a microfluidic device that combines, for the first time, in one single reaction chamber, DNA purification from a complex biological sample (blood) without elution and PCR without surface passivation agents. This result is achieved by exploiting the spontaneous chemical structure and nanomorphology of the material after casting. The observed surface organization leads to spontaneous DNA adsorption. This property allows on-chip complete protocols of purification of complex biological samples to be performed directly, starting from cells lysis. Amplification by PCR is performed directly on the adsorbed DNA, avoiding the elution process that is normally required by DNA purification protocols. The use of one single microfluidic volume for both DNA purification and amplification dramatically simplifies the structure of microfluidic devices for DNA preparation. X-Ray Photoelectron Spectroscopy (XPS) was used to analyze the surface chemical composition. Atomic Force Microscopy (AFM) and Field Emission Scanning Electron Microscopy (FESEM) were employed to assess the morphological nanostructure of the PDMS-chips. A confocal fluorescence analysis was utilized to check DNA distribution inside the chip.     

Rapid extraction of codeine and morphine in whole blood for HPLC analysis

Summary A rapid and efficient procedure is described for the extraction and analysis of codeine and morphine in whole blood. Red blood cells were fragmented by sonication and the blood sample extracted by passing through a bonded silica column (Bond Elute^?). The adsorbed drugs were washed and eluted followed by analysis by HPLC. Recoveries were between 95–100% at 5 ng/ml concentrations.     

A device for extraction, manipulation and stretching of DNA from single human chromosomes

We describe the structure and operation of a micro/nanofluidic device in which individual metaphase chromosomes can be isolated and processed without being displaced during exchange of reagents. The change in chromosome morphology as a result of introducing protease into the device was observed by time-lapse imaging; pressure-driven flow was then used to shunt the chromosomal DNA package into a nanoslit. A long linear DNA strand (>1.3 Mbp) was seen to stretch out from the DNA package and along the length of the nanoslit. Delivery of DNA in its native metaphase chromosome package as well as the microfluidic environment prevented DNA from shearing and will be important for preparing ultra-long lengths of DNA for nanofluidic analysis.     

Fast and selective extraction of nicotine from human plasma based on magnetic strong cation exchange resin followed by liquid chromatography-tandem mass spectrometry

In the study, a fast and selective method based on magnetic separation has been developed for the extraction of nicotine from human plasma using magnetic strong cation exchange (MSCX) resins as adsorbent. MSCX resins were prepared using hydrophobic Fe₃O₄ magnetite as magnetically susceptible component, styrene and acrylic acid as polymeric matrix components, and acetyl sulfonate as the sulfonation agent. The extraction procedure was carried out in a single step by stirring the mixture of diluted plasma sample and MSCX resins in the vortex for 5 min. Then, the resins with adsorbed nicotine were separated from the sample matrix by applying an appropriate magnetic field. Main factors affecting the extraction of nicotine such as the amount of MSCX resins, pH value of the extraction solvent, extraction time, and washing and eluting conditions were optimized. The nicotine eluted from the resins was determined by liquid chromatography–tandem mass spectrometry. The calibration curve obtained by analyzing matrix-matched standards shows excellent linear relationship (r ² = 0.9998) in the concentration range of 10–2,500 ng mL⁻¹. The limit of detection and quantification obtained are 2.9 and 9.7 ng mL⁻¹, respectively. The relative standard deviations of intra- and inter-day obtained are in the range of 1.9–6.9% and 2.5–7.8% with the recoveries ranging from 78.7% to 99.1%. The proposed method was successfully applied to determine nicotine in human plasma phlebotomized from ten male smokers. Nicotine was detectable with the contents ranging from 44.4 to 221.9 ng mL⁻¹ in five samples.     

High-pressure liquid chromatographic analysis of diazepam, oxazepam and n-desmethyldiazepam in human blood

We describe a rapid method for precisely measuring concentrations of diazepam, oxazepam and N-desmethyldiazepam in blood by high-pressure liquid chromatography. The drugs, together with an internal standard, prazepam, are extracted from 2 ml of blood and analyzed isocratically on a reversed-phase column with a mobile phase consisting of acetonitrile-0.01 M sodium acetate buffer (35:65 v/v). The eluted drugs are detected by their absorption at 240 nm. The sensitivity of this method is 30 microgram/l for oxazepam and N-desmethyldiazepam, 40 microgram/l for diazapam, for 2-ml blood samples. Relative recovery of added drugs to blood varies from 91 to 110%. The day-to-day precision (coefficient of variation) established by 10 replicate analyses was 2.8 to 9.6%.     

Design,synthesis, and characterization of molecularly imprinted solid phase extraction for alpha Mangostin analysis in blood sample

Low levels of α-mangostin (AM) in biological fluids require adequate sample preparation to be analyzed. Molecularly imprinted polymers can serve as sorbents in solid phase extraction, enabling concentration and extraction of α-mangostin from complex matrices, such as biological fluids. To date, there are no molecular imprinted polymers for the analysis of α-mangostin in biological fluids. In this study, AM molecular imprinted polymer (MIP) was designed using molecular modeling, molecular dynamic simulations, and prepared by bulk polymerization and suspension polymerization methods. The geometry optimization results showed that acrylamide (AAM) monomer forms the most stable complex with AM at the pre-polymerization with the most negative Gibbs free energy (ΔG) of −6.91818 Kcal/mol. Radial distribution function (RDF) in molecular dynamics simulation of AM:AAM:ethylene glycol dimethacrylate (EGDMA) with mol ratio 1:4:20 shows the complex with the best composition through the formation of four stable hydrogen bonds. Based on the experimental results, molecularly imprinted polymers in suspension exhibit better characteristics, selectivity, and adsorption capacity than in bulk. The suspension polymerization method showed a high recovery (85.88% ± 2.5), which was higher than C18 SPE cartridge (24.19% ± 1.47). Hence, it can be concluded that the MIPs from MD simulations were accessible and could be used in practice, such as in the separation and detection of AM in blood serum.