Three phases hollow fiber LPME combined with HPLC-UV for extraction, preconcentration and determination of valerenic acid in Valeriana officinalis

In the present work, the applicability of hollow fiber-based liquid phase microextraction (HF-LPME) was evaluated for the extraction and preconcentration of valerenic acid prior to its determination by reversed-phase HPLC/UV. The target drug was extracted from 5.0 mL of aqueous solution with pH 3.5 into an organic extracting solvent (dihexyl ether) impregnated in the pores of a hollow fiber and finally back extracted into 10 μ L of aqueous solution with pH 9.5 located inside the lumen of the hollow fiber. In order to obtain high extraction efficiency, the parameters affecting the HF-LPME, including pH of the donor and acceptor phases, type of organic phase, ionic strength, the volume ratio of donor to acceptor phase, stirring rate and extraction time were studied and optimized. Under the optimized conditions, enrichment factor up to 446 was achieved and the relative standard deviation (RSD) of the method was 4.36% (n = 9). The linear range was 7.5-850 μg L?1 with correlation coefficient (r2=0.999), detection limits was 2.5 μg L?1 and the LOQ was 7.5 μg L?1. The proposed method was evaluated by extraction and determination of valerenic acid in some Iranian wild species of Valerianaceae.     

Determination of aconitine,hypaconitine and mesaconitine in urine using hollow fiber liquid-phase microextraction combined with high-performance liquid chromatography

Hollow fiber liquid-phase microextraction (HF-LPME) coupled with high-performance liquid chromatography was used to simultaneously determine three Aconitum alkaloids, including aconitine (AC), hypaconitine (HA) and mesaconitine (MA) in human urine sample. Analytes were extracted from 5 mL urine sample containing 1.0 mmol/L NaOH into 1-octanol membrane phase impregnated in the pores of hollow fiber wall, and then back extracted into acidified aqueous solution in the lumen of the hollow fiber. After extraction, 10 μL of the acceptor phase was analyzed directly by HPLC. In this method, some important extraction parameters, such as organic solvent, extraction time, stirring rate, pH of donor phase and acceptor phase, temperature, and the volume of acceptor phase were optimized. This method provided 98- to 288-fold enrichment factors within 60 min of extraction and good repeatability with RSDs of 0.99–7.22%. The calibration curves were linear over the ranges of 16.0–128.0 μg/L for AC, 11.0–88.0 μg/L for HA and 8.1–64.8 μg/L for MA in human urine sample, with correlation coefficients of 0.9949, 0.9969 and 0.9904, respectively. Limits of detection were from 0.7 to 1.5 μg/L, and recoveries from spiked urine sample varied from 84.4% to 106.2% for AC, 77.3% to 85.6% for HA and 90.1% to 100.8% for MA.     

Electrically Assisted Liquid‐Phase Microextraction Combined With Capillary Electrophoresis for Quantification of Propranolol Enantiomers in Human Body Fluids

In this study, electromembrane extraction (EME) combined with cyclodextrin (CD)‐modified capillary electrophoresis (CE) was applied for the extraction, separation, and quantification of propranolol (PRO) enantiomers from biological samples. The PRO enantiomers were extracted from aqueous donor solutions, through a supported liquid membrane (SLM) consisting of 2‐nitrophenyl octyl ether (NPOE) impregnated on the wall of the hollow fiber, and into a 20‐μL acidic aqueous acceptor solution into the lumen of hollow fiber. Important parameters affecting EME efficiency such as extraction voltage, extraction time, pH of the donor and acceptor solutions were optimized using a Box‐Behnken design (BBD). Then, under these optimized conditions, the acceptor solution was analyzed using an optimized CD‐modified CE. Several types of CD were evaluated and best results were obtained using a fused‐silica capillary with ammonium acetate (80 mM, pH 2.5) containing 8 mM hydroxypropyl‐β‐CD as a chiral selector, applied voltage of 18 kV, and temperature of 20°C. The relative recoveries were obtained in the range of 78–95%. Finally, the performance of the present method was evaluated for the extraction and determination of PRO enantiomers in real biological samples. Chirality 26:260–267, 2014. © 2014 Wiley Periodicals, Inc.     

Analysis of amantadine in biological fluids using hollow fiber-based liquid-liquid-liquid microextraction followed by corona discharge ion mobility spectrometry

A method based on liquid-liquid-liquid microextraction combined with corona discharge ion mobility spectrometry was developed for the analysis of amantadine in human urine and plasma samples. Amantadine was extracted from alkaline aqueous sample as donor phase through a thin phase of organic solvent (n-dodecane) filling the pores of the hollow fiber wall and then back extracted into the organic acceptor phase (methanol) located in the lumen of the hollow fiber. All variables affecting the extraction of analyte including acceptor organic solvent type, concentration of NaOH in donor phase, ionic strength of the sample and extraction time were studied. The linear range was 20-1000 and 5-250 ng/mL for plasma and urine, respectively (r(2)≥0.990). The limits of detection were calculated to be 7.2 and 1.6 ng/mL for plasma and urine, respectively. The relative standard deviation was lower than 8.2% for both urine and plasma samples. The enrichment factors were between 45 and 54. The method was successfully applied for the analysis of amantadine in urine and plasma samples.     

Electromembrane extraction of trace amounts of naltrexone and nalmefene from untreated biological fluids

Nalmefene and naltrexone are used to block the effects of narcotics and alcohol. In the present work, for the first time a microextraction technique was presented to reduce matrix interferences and improve detection limits of the drugs in urine and plasma samples. Electromembrane extraction (EME) followed by high performance liquid chromatography (HPLC) coupled with ultraviolet (UV) detection was optimized and validated for quantification of nalmefene and naltrexone from biological fluids. The membrane consists 85% of 2-nitrophenyl octyl ether (NPOE) and 15% di-(2-ethylhexyl) phosphate (DEHP) immobilized in the pores of a hollow fiber. A 100 V electrical field was applied to make the analytes migrate from sample solution with pH 2.0, through the supported liquid membrane (SLM) into an acidic acceptor solution with pH 1.0 which was located inside the lumen of hollow fiber. Extraction recoveries in the range of 54% and 75% were obtained in different biological matrices which resulted in preconcentration factors in the range of 109-149 and satisfactory repeatability (2.0相似文献   

Urine sample preparation of tricyclic antidepressants by means of a supported liquid membrane technique for high-performance liquid chromatographic analysis

Supported liquid membrane (SLM) technique for sample work-up and enrichment was used for determination of tricyclic antidepressant drugs in urine by high-performance liquid chromatography (HPLC) with UV detection. The studied antidepressant drugs were amitriptyline, opipramol, noxiptyline and additionally diethazine was used as possible internal standard. Alkaline phosphoric buffer with urine sample, as the donor solution, was passed over the liquid membrane into which investigated substances were extracted. On the other side of the membrane, analyzed compounds were trapped due to creating non-extractable form in acidic acceptor solution. Enriched and cleaned up drugs were then injected into a HPLC system with ultraviolet detection to analyze of their concentration in acceptor solution. Optimum extraction efficiency was determined by changing acceptor and donor solutions pH, application of different flow rates of donor solution and by using different solvents in the membrane. Also, donor solution volume, extraction time and concentration of analytes were varied to check the linearity of extraction process. The highest extraction efficiency: 43% for opipramol, 56% for noxiptyline, 43% for amitriptyline and 42% for diethazine (R.S.D. values were <6% and n=3) was achieved when 0.05 M phosphate buffer pH 4.0 and 9.5 were used as donor and acceptor solutions, respectively, n-undecane with 5% tri-n-octylphosphine oxide (TOPO) was used as liquid membrane. Limit of quantification (LOQ) for tricyclic antidepressants after enrichment of 100ml of urine sample was about 1 ng/ml.     

Equilibrium sampling through membrane based on a single hollow fibre for determination of drug-protein binding and free drug concentration in plasma

The determination of drug-protein binding and free drug concentration in plasma applying the equilibrium sampling through membrane (ESTM) technique has been studied using supported liquid membrane extraction in a single hollow fibre without any membrane carrier. In the extraction setup, the donor phase (plasma or buffer) was placed in the vial, into which was immersed the hollow fibre with the acceptor phase situated in the lumen. This proposed technique was applied to study the drug-protein binding of five local anaesthetics and two antidepressants as model substances, and the influence of the total drug concentration on the drug-protein binding was investigated. The brief theoretical background for determination of the drug-protein binding under equilibrium conditions is described. The developed method shows a new, improved and simple procedure for determination of free drug concentration in plasma and extent of drug-protein binding.     

Three phase liquid phase microextraction of phenylacetic acid and phenylpropionic acid from biological fluids

Three phase liquid phase microextraction (three phase LPME) technique coupled with HPLC-UV has been applied as a sensitive and efficient sample preparation method to determine phenylacetic acid (PAA) as a biomarker of depressive disorders and phenylpropionic acid (PPA) in biological fluids. The compounds were extracted from 3.0 ml aqueous solution with the adjustment of pH at a fixed value in the range of 2.0-3.5 (donor solution) into an organic phase (1-hexanol) layered on the surface of the donor solution and finally back-extracted into 4.0 microl of the acceptor microdrop (pH 11.1) located at the end of the microsyringe needle. After a prescribed back-extraction time, the acceptor microdrop was withdrawn into the microsyringe and then directly injected into the HPLC system. In order to achieve maximum extraction efficiency, different parameters affecting the extraction conditions were optimized. At the optimum conditions (donor solution: 2.3M Na(2)SO(4), pH 2.0-3.5; organic membrane: 95 microl of 1-hexanol; acceptor solution: 4.0 microl of 0.1M NH(3)/NH(4)(+) with pH 11.1; donor solution temperature: 45-50 degrees C; extraction time: 20 min and back-extraction time: 12 min), up to 110-fold enrichment factor was obtained. The calibration curve for these analytes was linear in the range of 1-5000 microg/l with r(2)>0.998. The intraday and interday RSD% were below 6.5% and the limits of detection (LODs) for both analytes were 0.2 microg/l (based on S/N=3). The proposed technique is a low cost, simple and sensitive method with highly clean-up effect. Finally, this technique was successfully utilized for the detection of target analytes in human urine, serum and plasma.     

Evaluation of progesterone content in saliva using magnetic particle-based immuno supported liquid membrane assay (m-ISLMA)

Progesterone in saliva was monitored using a new method called magnetic particle-based immuno supported liquid membrane assay (m-ISLMA) in a sequential injection (SI) setup, allowing automatic sample cleanup, analyte enrichment, and detection in a single analysis unit. Progesterone (Ag) diffuses from a continuous flowing sample - the donor - into a supported organic liquid membrane (SLM), based on analyte partitioning (solubility) between the aqueous donor and the organic phase. The Ag is re-extracted from the SLM into a second stagnant aqueous acceptor, containing antibodies (Ab) immobilized on magnetic beads, held at the bottom of the acceptor by a magnet. Due to the formation of strong Ag-Ab-bead complexes and a large excess of Ab-beads, the Ag is accumulated and selectively enriched in the acceptor. The extracted progesterone was quantified by injecting into the acceptor a horseradish peroxidase (HRP) labeled analyte tracer, the substrate (luminol, H(2)O(2), and p-iodophenol), and finally detection of the generated chemiluminescence by a photomultiplier tube. After optimization of experimental parameters (e.g., sample flow rate, extraction time, type of organic solvent and antibody-bead concentration in the acceptor), a detection limit of 8.50+/-0.17 fgL(-1) and a dynamic range between 35 fgL(-1) and 10 pgL(-1) was reached. The progesterone level of saliva for three subjects (women in different period of ovarian cycle) was investigated, and the corresponding progesterone concentrations detected with m-ISLMA coincided well with the expected values.     

A one-vial method for routine extraction and quantification of free fatty acids in blood and tissue by HPLC

A one-vial procedure has been developed to quantify free fatty acids in human blood serum and rat heart tissue. To allow routine analysis the system has been constructed to allow simultaneous processing of nine samples (Praechromat). The free fatty acids are extracted with Freon 11 and then derivatized to coumarin esters prior to HPLC. The Freon 11 extraction of the free fatty acids is rapid and complete. Neither hydrolytic degradation of natural fatty acid esters nor oxidative damage of unsaturated fatty acids was observed. Fifteen free fatty acids (FFA) were routinely quantified by isocratic elution with high reproducibility (SD less than 4%) and good recovery (0.1 mM FFA: 98-100%, 0.02 mM: 91-105%). The free fatty acids could be determined in the range from 20 pmol to 20 nmol.     

Supported liquid membrane extraction of peptides

The application of supported liquid membrane (SLM) extraction for the enrichment of short peptides is presented. The extraction efficiency is dependent on the pH of donor phase and salt concentration in acceptor phase. Moreover, the extraction efficiency is also influenced by the peptide amino-acid sequence and hydrophobicity.     

Determination of diprivan in urine by a supported liquid membrane technique and liquid chromatography-electrochemical detection

A supported liquid membrane technique was used for the extraction and enrichment of propofol in a spiked sample of urine. An acidic solution of propofol and thymol as an internal standard was passed over the membrane and after enrichment the acceptor solution was analyzed by LC with an electrochemical detector. The acceptor and donor pH, flow-rate, and volume of donor and different membrane solvents were varied to optimize the extraction efficiency. The detection limit for 100 ml of a spiked urine sample was 10 ppt of propofol.     

On-line extraction of volatile fatty acids in acidogenic chemostat culture using a supported liquid membrane

An on-line extraction of volatile fatty acids (acetic and butyric acids) from acidogenic fermentation in chemostat cultures using a supported liquid membrane (SLM) was investigated in order to overcome end-product inhibition. By using SLM, the high-cell-density retaining dilution rate of the chemostat could be increased, thus enhancing the microbial acidogenesis. To further understand this phenomenon, the growth and extraction kinetics were studied. The effect of substrate concentration was found to obey the Haldane equation. Regarding the inhibition by volatile fatty acids, it turned out that undissociated butyric acid rather than acetic acid severely inhibited the growth, corresponding to non-competitive kinetics. The extraction rates of the acids were proportional to their undissociated concentration as well as to the SLM area/broth volume, and butyric acid extraction was easier than that of acetic acid.     

The origin of 1H NMR-visible triacylglycerol in human neutrophils. Highfatty acid environments result in preferential sequestration of palmitic acid into plasma membrane triacylglycerol.

Human neutrophils incubated for 1 h in vitro with 10% commercial pooled, human serum containing high levels of free fatty acids (1141 microM) displayed a distinct lipid signal, typical of triacylglycerol, in the 1H NMR spectrum. Concurrently their plasma membrane triacylglycerol mass increased 4.6-fold with a selective rise in the content of palmitic and linoleic acids. Although qualitatively similar, these effects were much greater than those observed after incubating neutrophils with 50 microg.mL-1 of lipopolysaccharide in the presence of 10% AB serum with normal free fatty acid content (345 microM, LPS/S). Incubation of neutrophils with an artificial mixture of free fatty acids at concentrations found in commercial serum, or with the fatty acid fraction isolated from commercial serum increased the 1H NMR-detectable triacylglycerol. The signal intensity of the 1H NMR-detectable triacylglycerol depended on the triacylglycerol composition, and correlated with increased membrane triacylglycerol mass. Cellular uptake of 3H-labelled palmitic or oleic acids increased in the presence of commercial serum but not with LPS/S, with little contribution in either case to the triacylglycerol pool that increased in mass. Pulse-chase experiments demonstrated that with LPS/S and commercial serum, radiolabelled palmitic acid was preferentially incorporated into triacylglycerol located in the plasma membrane. This process could occur at the plasma membrane, as cytoplasts efficiently convert exogenous fatty acids into triacylglycerol. We propose that LPS/S and serum containing high levels of free fatty acid, important in conditions of sepsis and inflammation, may facilitate the sequestration of palmitic acid into triacylglycerol by different pathways. This triacylglycerol originates from exogenous and endogenous free fatty acids, is 1H NMR-visible, and may have a role in regulating apoptosis.     

Omega-3 fatty acids modulate ATPases involved in duodenal Ca absorption

Dietary supplementation with fish oil that contains omega-3 polyunsaturated fatty acids has been shown to enhance bone density as well as duodenal calcium uptake in rats. The latter process is supported by membrane ATPases. The present in vitro study was undertaken to test the effect of omega-3 fatty acids on ATPase activity in isolated basolateral membranes from rat duodenal enterocytes. Ca-ATPase in calmodulin-stripped membranes was activated in a biphasic manner by docosahexanoic acid (DHA) (10-30 microg/ml) but not by eicosapentanoic acid (EPA). This effect was blocked partially by 0.5 microM calphostin (a protein kinase C blocker). DHA inhibited Na,K-ATPase (-49% of basal activity, [DHA]=30 microg/ml, P <0.01). This effect could be reversed partially by 50 microM genistein, a tyrosine kinase blocker. EPA also inhibited Na,K-ATPase: (-47% of basal activity, [EPA]=30 microg/ml, P <0.01), this effect was partially reversed by 100 microM indomethacin, a cyclo-oxygenase blocker. Omega-3 fatty acids are thus involved in multiple signalling effects that effect ATPases in BLM.     

Membrane properties modulate the activity of a phosphatidylinositol transfer protein from the yeast, Saccharomyces cerevisiae

A phospholipid transfer protein from yeast (Daum, G. and Paltauf, F. (1984) Biochim. Biophys. Acta 794, 385-391) was 2800-fold enriched by an improved procedure. The specificity of this transfer protein and the influence of membrane properties of acceptor vesicles (lipid composition, charge, fluidity) on the transfer activity were determined in vitro using pyrene-labeled phospholipids. The yeast transfer protein forms a complex with phosphatidylinositol or phosphatidylcholine, respectively, and transfers these two phospholipids between biological and/or artificial membranes. The transfer rate for phosphatidylinositol is 19-fold higher than for phosphatidylcholine as determined with 1:8 mixtures of phosphatidylinositol and phosphatidylcholine in donor and acceptor membrane vesicles. If acceptor membranes consist only of non-transferable phospholipids, e.g., phosphatidylethanolamine, a moderate but significant net transfer of phosphatidylcholine occurs. Phosphatidylcholine transfer is inhibited to a variable extent by negatively charged phospholipids and by fatty acids. Differences in the accessibility of the charged groups of lipids to the transfer protein might account for the different inhibitory effects, which occur in the order phosphatidylserine which is greater than phosphatidylglycerol which is greater than phosphatidylinositol which is greater than cardiolipin which is greater than phosphatidic acid which is greater than fatty acids. Although mitochondrial membranes contain high amounts of negatively charged phospholipids, they serve effectively as acceptor membranes, whereas transfer to vesicles prepared from total mitochondrial lipids is essentially zero. Ergosterol reduces the transfer rate, probably by decreasing membrane fluidity. This notion is supported by data obtained with dipalmitoyl phosphatidylcholine as acceptor vesicle component; in this case the transfer rate is significantly reduced below the phase transition temperature of the phospholipid.     

红树植物红海榄脂肪酸的GC-MS分析

采用索氏提取法对红树植物红海榄(Rhizophora stylosa)叶中的脂肪酸进行提取,甲酯化后利用气相色谱-质谱联用仪对脂肪酸成分进行分离和鉴定,鉴定出了12种脂肪酸,占脂肪酸总量的84.95%,其中主要包括十六酸(棕榈酸)(40.49%),9-十八碳烯酸(油酸)(17.24%),9,12-十八碳二烯酸(亚油酸)(11.24%)等。     

Effects of cholesterol on viability and (n - 6) fatty acid metabolism in cultured human monocyte-like cells (U937).

Effects of supplementation of growth-promoting cholesterol on metabolism of the cytotoxic (n - 6) polyunsaturated fatty acids in cultured human monocyte-like cells (U937) have been examined. U937 cells were incubated in 5% delipidated fetal bovine serum containing 0 or 38.7 microM cholesterol. The rate of uptake and the distribution of metabolites of (n - 6) fatty acids (such as 18:2(n - 6), 18:3(n - 6), and 20:3(n - 6), and 20:4(n - 6)) were examined by adding radiolabelled fatty acid at a level of 1 microgram/mL (3.3 microM for 20-carbon fatty acids and 3.6 microM for 18-carbon-fatty acids). For assessing the cytotoxicity, (n - 6) fatty acids were added to medium at a concentration of 5 micrograms/mL (16.4 microM for 20-carbon fatty acids and 17.9 microM for 18-carbon fatty acids). Cholesterol supplementation suppressed the uptake of all (n - 6) fatty acids and reduced the cytotoxic effects of 18:2(n - 6), 20:3(n - 6), and 20:4(n - 6), but not 18:3(n - 6). In addition, cholesterol supplementation increased peroxide production and metabolism of (n - 6) fatty acids in U937 cells. Thus, the differential suppressive effect of cholesterol on the cytotoxicity of different fatty acids could not be attributed to an inhibitory effect on fatty acid delta 6- and delta 5-desaturation, or to an antioxidant effect on peroxide formation.     

莱菔子脂肪酸成分的GC-MS分析

采用索氏提取法提取莱菔(Raphanussativus)种子中的脂肪酸成分,进行甲酯化处理后用气相色谱-质谱联用技术分离和鉴定脂肪酸成分的组成和含量。结果表明,从莱菔子中共分离鉴定出12种脂肪酸成分,占总量的99.32%,主要包括芥酸(32.47%)、油酸(29.07%)、亚油酸(9.45%)、亚麻酸(8.41%)、棕榈酸(4.31%)和硬脂酸(2.08%)等脂肪酸,其中饱和脂肪酸占9.25%,不饱和脂肪酸高达90.07%。     

Development of a supported emulsion liquid membrane system for propionic acid separation in a microgravity environment

Perstractive fermentation is a good way to increase the productivity of bioreactors. UsingPropionibacteria as the model system, the feasibility of using supported emulsion liquid membrane (SELM) for perstractive fermentation is assessed in this study. Five industrial solvents were considered as the solvent for preparing the SELM. The more polar a solvent is, the higher the partition coefficient. However, toxicity of a solvent also increases with its polarity. CO-1055 (industrial decanol/octanol blend) has the highest partition coefficient toward propionic acid among the solvents that has no molecular toxicity towardPropionibacteria. A preliminary extraction study was conducted using tetradecane as solvent in a hydrophobic hollow fiber contactor. The result confirmed that SELM eliminates the equilibrium limitation of conventional liquid-liquid extraction, and allows the use of a non-toxic solvent with low partition coefficient.