One-step Extraction Method Research Articles

A simplified method for the measurement of nonmetabolized 2-[18F]F-A-85380 in blood plasma using solid-phase extraction

Quantification of alpha(4)beta(2)* nicotinic acetylcholine receptors using 2-[(18)F]fluoro-3-(2(S)-azetidinylmethoxy)pyridine (2-[(18)F]FA) and positron emission tomography (PET) imaging requires measurement of nonmetabolized radioligand in blood plasma, which was previously accomplished using high-performance liquid chromatography (HPLC). Here, we introduce a one-step solid-phase extraction (SPE) method for measuring the concentration of nonmetabolized 2-[(18)F]FA. This method allows many samples to be processed in a short period of time. SPE effectively separated 2-[(18)F]FA from radioactive metabolites typically observed in blood plasma after administration of radioligand in humans. Measurements of the 2-[(18)F]FA parent fraction in healthy human volunteers obtained using the SPE method were nearly identical to those obtained using HPLC (1.3+/-5% average underestimation of SPE), and reproducibility was good within and between runs (2% and 6% coefficient of variation, respectively). SPE recovery of 2-[(18)F]FA from blood plasma was not appreciably diminished (3+/-0.6%) by a larger volume of blood plasma loaded onto the cartridge, suggesting the possibility of increasing the plasma sample volume at later times in a PET study to improve measurement sensitivity. 2-[(18)F]FA was stable in blood stored on ice over 8 h and in saline at low concentrations (<2 MBq/ml) but not at high concentrations (ca. 130 MBq/ml). Using SPE, the elimination half-life and full body distribution volume of 2-[(18)F]FA in healthy human volunteers were estimated as 4.2+/-0.8 h and 220+/-70 L, respectively. These results suggest that SPE is the method of choice for the determination of the plasma 2-[(18)F]FA concentration when measurement of individual metabolites is not required.

Determination of low levels of perchlorate in lettuce and spinach using ion chromatography-electrospray ionization mass spectrometry (IC-ESI-MS).

A sample preparation method was developed to quantify environmentally relevant (low micrograms per liter) concentrations of perchlorate (ClO4(-)) in leafy vegetables using IC-ESI-MS. Lettuce and spinach were macerated, centrifuged, and filtered, and the aqueous extracts were rendered water-clear using a one-step solid-phase extraction method. Total time for extraction and sample preparation was 6 h. Ion suppression was demonstrated and was likely due to unknown organics still present in the extract solution after cleanup. However, this interference was readily eliminated using a Cl(18)O4(-) internal standard at 1 microg/L in all standards and samples. Hydroponically grown perchlorate-free butterhead lettuce was spiked to either 10.3 or 37.7 microg/kg of fresh weight (FW), and recoveries were between 91 and 98% and between 93 and 101%, respectively. Five types of lettuce and spinach from a local grocery store were then analyzed; they contained from 0.6 to 6.4 microg/kg of FW. Spike recoveries using the store-bought samples ranged from 89 to 100%. The method detection limit for perchlorate in plant extracts is 40 ng/L, and the corresponding minimum reporting limit is 200 ng/L or 0.8 microg/kg of FW.

Designing a purification process for chromium-, copper- and arsenic-contaminated wood

The disposal of chromated copper arsenate (CCA)-treated wood is becoming a serious problem in many countries due to increasing levels of contamination by the hazardous elements, chromium, copper and arsenic. The present experiment was conducted as a preliminary step toward one-step solvent extraction of CCA-treated wood. Because chromium, copper and arsenic have different chemical characteristics, it is best to consider them separately prior to designing a one-step extraction process. As a basis, various two-step extraction processes were first designed and tested experimentally to determine feasibility. Among these combinations, the treatment combining oxalic acid as the 1st step and a sodium oxalate solution under acidic conditions (pH 3.2) as the 2nd step was found to be an effective way of extracting CCA elements from treated wood. Extraction efficiency reached 100% for arsenic and chromium and 95.8% for copper after a 3-h sodium oxalate treatment, following a 1-h pre-extraction process with oxalic acid. On the other hand, the same combination under alkaline conditions (pH 11.2) during the 2nd step was ineffective for copper removal, indicating that pH plays an important role in complexation with sodium oxalate solution. The present results suggest that the extraction of CCA elements using a combination of oxalic acid and acidic sodium oxalate solution is a promising basis for application to a one-step extraction method.

High-efficiency protein extraction from polyacrylamide gels for molecular mass measurement by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry

A simple and fast method of protein extraction from Coomassie Brilliant Blue (CBB)-stained polyacrylamide gels suited for molecular mass measurement of proteins by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) is reported. Proteins in CBB-stained gel pieces were extracted by a 10-min soaking in 0.1 M NaOH at 25 degrees C. The recovery of this one-step extraction method was 34-73% for proteins <67 kDa. CBB adduction to proteins during mass spectrometric analysis was avoided by a destaining step before the alkaline extraction. The molecular mass values of the extracted proteins coincided with those of purified proteins within +/-0.01-0.10% deviation for all the proteins <36 kDa. Because of the high extraction recovery, mass measurement was possible for the proteins extracted from CBB-stained gels with loaded protein quantities as little as 34 ng for cytochrome c, alpha-lactalbumin, myoglobin, beta-lactoglobulin, trypsinogen, and carbonic anhydrase (12.4-29.0 kDa), 340 ng for glyceraldehyde-3-phosphate dehydrogenase (35.6 kDa) and albumin (66.3 kDa). This method provides a highly efficient approach to utilize CBB-stained one- or two-dimensional gels for whole protein analysis using MALDI-TOF-MS.

Comparison of a rhizosphere-based method with other one-step extraction methods for assessing the bioavailability of soil metals to wheat

There is no method recognized as a universal approach for evaluation of bioavailability of heavy metals in soil. Based on the simulation of the rhizosphere soil conditions and integration of the combined effects of root–soil interactions as a whole, a rhizosphere-based method has been proposed. Wet fresh rhizosphere soil was extracted by low-molecular-weight organic acids (LMWOAs) to fractionate metal fractions of soil pools, which were then correlated with the metal contents of wheat roots and shoots. The rhizosphere-based method was compared with other one-step extraction methods using DTPA, EDTA, CaCl 2, and NaNO 3 as extractants and the first step of the Community Bureau of Reference (BCR) method. Simple correlation and stepwise multiple regression analysis were used for the comparison. Simple correlation indicated that the extractable Cu, Zn, Cr, and Cd of soils by the rhizosphere-based method were significantly correlated with the metal contents of wheat roots. For DTPA, BCR1 and EDTA methods there was a relatively poor correlation between the extractable Cu, Zn and Cd of soil and metal contents of wheat roots. Stepwise multiple regression analysis revealed that the equation of the rhizosphere-based method was the simplest one, and no soil properties variables needed to be added. In contrast, the equations of other one-step extraction methods were more complicated, and soil properties variables needed to be entered. The most distinct feature of the rhizosphere-based method was that the recommended method was suitable for acidic, neutral and near alkaline soils. However, the DTPA and EDTA extraction methods were suitable for calcareous soils only-or-only for acidic soils. The CaCl 2, and NaNO 3 extraction methods were only suitable for exchangeable metals. In short, the rhizosphere-based method was the most robust approach for evaluation of bioavailability of heavy metals in soils to wheat.

Le jardin botanique: une image de la nature

Nowadays, the safety of cosmetics is a widespread concern. Amines are common cosmetic additives. Some of them such as amino acids are beneficial. Another kind of amines, however, ε-aminocaproic acid (EACA) is prohibited to add into cosmetics for its adverse reactions. In this study, a simple, rapid, sensitive and eco-friendly one-step ultrasonic-assisted extraction and derivatization (UAE-D) method was developed for determination of EACA and amino acids in cosmetics by coupling with high-performance liquid chromatography (HPLC). By using this sample preparation method, extraction and derivatization of EACA and amino acids were finished in one step in ultrasound field. During this procedure, 4-fluoro-7-nitrobenzofurazan (NBD-F)was applied as derivatization reagent. The extraction conditions including the amount of NBD-F, extraction and derivatization temperature, the ultrasonic vibration time and pH value of the aqueous phase were evaluated. Meanwhile, the extraction mechanism was investigated. Under optimized conditions, the method detection limits were 0.086–0.15 μg/L, and method quantitation limits were 0.29–0.47 μg/L with RSDs less than 3.7% (n = 3). The recoveries of EACA and amino acids obtained from cosmetic samples were in range from 76.9% to 122.3%. Amino acids were found in all selected samples and quantified in range from 1.9 ± 0.9 to 677.2 ± 17.9 μg/kg. And EACA was found and quantified with the contents of 1284.3 ± 22.1 μg/kg in a toner sample. This UAE-D-HPLC method shortened and simplified the sample pretreatment as well as enhanced the sensitivity of analytical method. In our record, only 10 min was needed for the total sample preparation process. And the method detection limits were two orders of magnitude less than literature reports. Furthermore, we reduced the consumption of solvent and minimized the usage of organic solvents, which made our method moving towards green analytical chemistry. In brief, our UAE-D-HPLC method is a simple, rapid, sensitive and eco-friendly analytical method for the determination of EACA and amino acids in cosmetics.

Distribution of Fatty Acid Ethyl Esters in Long Evans Rats Following Acute Ethanol Ingestion

Summary: Fatty acid ethyl esters (FAEEs) are members of a class of nonoxidative metabolites of ethanol. These lipophilic moieties have been detected in various animal and human tissues in vivo and in vitro. A one-step liquid-liquid extraction method with subsequent analysis by GC/MS is described for the identification and quantitation of palmitic acid ethyl ester and stearic acid ethyl ester in blood, liver, and brain from Long Evans rats administered ethanol by feeding needle. Animals received one of four doses of ethanol (1, 2, 4, and 5 g/kg) as a 25% w/v gavage solution. Control animals received tap water. One hour after gavage, a blood specimen (3-5 mL) was collected by cardiac puncture and the animals were euthanized in accordance with guidelines promulagated by the Institutional Animal Care and Utilization committee. Necropsies included the complete resection of liver and removal of whole brain. Blood ethanol concentrations (BECs) were determined by GC headspace with n-propanol as internal standard. Heptadecanoic acid ethyl ester was added as internal standard to 1.0 mL aliquots of blood or 2 g liver or whole brain homogenates and extracted in toluene-hexane (1:1) at pH 5. Extraction mixtures were throroughly mixed and centrifuged. Solvent layers were removed and evaporated to dryness under nitrogen. Reconstituted residues were injected into a GC/MS equipped with an HP INNOWax GC column. Selected ions in the SIM mode included 88, 101, and 157. BEC data and FAEE concentrations for blood, liver, and brain are reported. These results suggest that a one-step extraction method coupled with GC/MS analysis could be used in identifying and quantitating nonoxidative fatty acid ethyl ester metabolites of ethanol in vivo.

Quantitation of N,N-dimethyltryptamine and harmala alkaloids in human plasma after oral dosing with ayahuasca.

Harmine, harmaline, tetrahydroharmine (THH), and N,N-dimethyltryptamine (DMT) were quantitated in plasma from 15 healthy male volunteers after the ingestion of ayahuasca, a beverage that has been used for religious purposes in Brazil since pre-Columbian times. A growing awareness of the interest in this ancient shamanistic practice in modern urban cultures and the widespread popular dissemination of the inebriant effects and type and sources of the plant admixtures used to prepare the beverage have provided additional impetus for this study. The three harmala alkaloids were quantitated from protein-precipitated plasma by high-performance liquid chromatography using fluorescence detection. Recovery from blank human plasma was quantitative, and the limit of quantitation (LOQ) was below 2 ng/mL of plasma for each of the harmala alkaloids. Standard concentrations ranged from 10 to 250 ng/mL for harmine and THH and from 1.0 to 25.0 ng/mL for harmaline, respectively. Linearity was observed for harmine, harmaline, and THH within these respective ranges. The highest concentrations of harmala alkaloids in human plasma were found to be 222.3 ng/mL for harmine, 134.5 ng/mL for THH, and 9.4 ng/mL for harmaline. DMT was quantitated by gas chromatography using nitrogen-phosphorus detection after liquid-liquid extraction with diphenhydramine as an internal standard. DMT recovery was quantitative, and the limit of detection and LOQ were 0.5 and 5 ng/mL, respectively. Linearity for DMT was observed from 5 to 1000 ng/mL. The one-step extraction method for DMT and the protein precipitation method for the three harmala alkaloids afford rapid, sensitive, and quantitative analyses of these alkaloids with minimal analyte loss. The analytical methods also may be applicable to other matrices, including whole blood and urine samples and homogenized tissue specimens. These are the first reported observations of DMT and harmala alkaloids in plasma after ritual ingestion of ayahuasca.

One-step isolation of α1-Acid glycoprotein

α 1-Acid glycoprotein could be isolated by a one-step extraction method from human sera and plasma. Protein recovered in the water phase after extraction with phenol at 70°C for 20 min was verified as human α 1-acid glycoprotein when it was compared with the reference standard human α 1-acid glycoprotein by Ouchterlony double immunodiffusion, sodium dodecylsulfatepolyacrylamide gel electrophoresis, Western blot analysis, and periodic acid-Schiff stain. The present isolation procedure is simple and fast, and can extract about 81% of the total α 1-acid glycoprotein in the sera and plasma, as determined by radial immunodiffusion.

A monoclonal antibody-based enzyme-linked immunosorbent assay of aflatoxin B1 in peanut products

An improved enzyme-linked immunosorbent assay (ELISA) combined with monoclonal antibody (MAb) and one-step extraction method was established for the estimation of aflatoxin B1 (AFB1) in a peanut product. AFB1 was converted to AFB1-oxime, and then conjugated with bovine serum albumin (BSA). Spleen cells from mice immunized with AFB1-BSA conjugates were fused with myeloma cells. After double selection with AFB1-ovalbumin (OVA) and carbodiimide-modified OVA, five stable hybridoma cells secreting anti-AFB1 MAbs (AF1, AF 2, AF 3, AF 4, and AF5) were cloned. Using these anti-AFB1 MAbs, we developed the indirect competitive ELISA (cELISA) with alkaline phosphatase (ALP) - labeled sheep anti - mouse IgG as marker and the direct cELISA with AFBi-oxime horseradish peroxidase (POD) as marker.The minimum detectable limits of the indirect cELISA with AF 1, 2, 3, 4, and 5 were 5, 5, 5, 5, and 50 pg of standard AFB1 per assay, respectively, and those of the direct cELISA with AF 1, 3, 4, and 5 were 2.5, 5, 25, and 100 pg of standard AFB1 per assay, respectively. The cross reactivity of each toxins with these MAbs in the indirect cELISA was as follows: (a) AF 1 and AF 2 were reactive with AFB2 as well as AFB1, weakly with AFG2 > AFG1 > aflatoxicol II (COL II) > aflatoxicol I (COL I) and less weakly with other aflatoxins; (b) AF 3 and AF 4 were reactive with COL II as well as AFB1, weakly with COLI > AFQ1 and less weakly with others; (c) AF 5 was AFQ1 as well as AFB1 weakly with COL II > AFG2 > COL I and less weakly with others.The 60% aqueous methanol extracts of oil-roasted blanched peanuts ("butter peanut"), naturally contaminated with AFB1, were assayed by the direct cELISA without further purification. The direct cELISA with the most sensitive MAb AF 1 was allowed to determine 1 ng of AFB1 per g samples.