Semi-automated set-up for exhaustive micro-electromembrane extractions of basic drugs from biological fluids.

Manual handling of microliter volumes of samples and reagents is usually prone to errors and may have direct consequence on the overall performance of microextraction process. Direct connection of a syringe pump and a disposable microextraction unit using flexible polymeric tubing was employed for semi-automated liquid handling in micro-electromembrane extraction (µ-EME). A three-phase µ-EME system was formed by consecutive withdrawal of microliter volumes of donor solution, free liquid membrane (FLM) and acceptor solution into the unit. Excellent repeatability and accuracy of the withdrawal sequence was achieved for solution volumes typically used in µ-EME (1-5 µL) as well as excellent correlation between the initially withdrawn and the finally collected solution volumes. µ-EMEs were initiated by application of d.c. electric potential to the terminal aqueous solutions and specific µ-EME parameters were optimized in order to ensure complete transfer of model analytes from donor to acceptor solution. Exhaustive µ-EMEs of three basic drugs, nortriptyline, papaverine and haloperidol, were achieved from 1.3 µL of acidified donor solution (10 mM HCl) across 2.5 µL of FLM (1-ethyl-2-nitrobenzene) into 1.3 µL of acidified acceptor solution (25 mM HCl) in 10 min at 150 V. The three drugs were also exhaustively extracted from salt- and protein-containing standard solutions, human urine and human plasma with extraction recoveries ranging from 79 to 102%. Resulting acceptor solutions were analysed by capillary electrophoresis with ultraviolet detection (CE-UV) and the µ-EME-CE-UV method was characterized by good linearity (coefficients of determination ≥ 0.992), high repeatability (RSD values ≤ 6.5%) and limits of detection ≤ 0.15 mg/L.

Micro-electromembrane extraction using multiple free liquid membranes and acceptor solutions - Towards selective extractions of analytes based on their acid-base strength.

This work investigated selective micro-electromembrane extractions (µ-EMEs) of the colored indicators metanil yellow and congo red (visual proof-of-principle) and the small drug substances nortriptyline, papaverine, mianserin, and citalopram (model analytes) based on their acid-base strength. With two free liquid membranes (FLMs), the target analytes were extracted from aqueous donor solution, across FLM 1 (1-pentanol, 1-ethyl-2-nitrobenzene (ENB) or 4-nitrocumene (4-NC)), into aqueous acceptor solution 1, further across FLM 2 (1-pentanol, ENB or 4-NC), and finally into aqueous acceptor solution 2. All phases had volumes between 1.0 and 1.5 µL and extractions were promoted by 200-300 V d.c. applied across the five-phase µ-EME system formed in a perfluoroalkoxy capillary tubing. The anode was located in acceptor solution 2 and the cathode was located in donor solution for µ-EMEs of acidic analytes, and locations of the electrodes were vice versa for µ-EMEs of basic analytes. After µ-EME, donor solution and acceptor solution 1 and 2 were analyzed by capillary electrophoresis or liquid chromatography-mass spectrometry. The model analytes migrated efficiently in the proposed µ-EME system, their migration behavior was controlled by pH in aqueous solutions and their selective fractionation into acceptor solution 1 and 2 was demonstrated based on their acid-base strength. Under optimal conditions, acceptor solution 2 contained 60% nortriptyline (pKa = 10.5) and less than 1% papaverine (pKa = 6.0) and acceptor solution 1 contained 17% nortriptyline and 27% papaverine after 15 min of µ-EME. The five-phase µ-EME system was also compatible with human plasma samples. Work is in progress to further increase the fractionation capability, and to implement the concept into microfluidic platforms.

Proteomics tools reveal startlingly high amounts of oxytocin in plasma and serum.

The neuropeptide oxytocin (OT) is associated with a plethora of social behaviors, and is a key topic at the intersection of psychology and biology. However, tools for measuring OT are still not fully developed. We describe a robust nano liquid chromatography-mass spectrometry (nanoLC-MS) platform for measuring the total amount of OT in human plasma/serum. OT binds strongly to plasma proteins, but a reduction/alkylation (R/A) procedure breaks this bond, enabling ample detection of total OT. The method (R/A + robust nanoLC-MS) was used to determine total OT plasma/serum levels to startlingly high concentrations (high pg/mL-ng/mL). Similar results were obtained when combining R/A and ELISA. Compared to measuring free OT, measuring total OT can have advantages in e.g. biomarker studies.

Electromembrane extraction of polar basic drugs from plasma with pure bis(2-ethylhexyl) phosphite as supported liquid membrane.

Electromembrane extraction (EME) of polar basic drugs from human plasma was investigated for the first time using pure bis(2-ethylhexyl) phosphite (DEHPi) as the supported liquid membrane (SLM). The polar basic drugs metaraminol, benzamidine, sotalol, phenylpropanolamine, ephedrine, and trimethoprim were selected as model analytes, and were extracted from 300 µL of human plasma, through 10 µL of DEHPi as SLM, and into 100 µL of 10 mM formic acid as acceptor solution. The extraction potential across the SLM was 100 V, and extractions were performed for 20 min. After EME, the acceptor solutions were analyzed by high-performance liquid chromatography-ultraviolet detection (HPLC-UV). In contrast to other SLMs reported for polar basic drugs in the literature, the SLM of DEHPi was highly stable in contact with plasma, and the system-current across the SLM was easily kept below 50 µA. Thus, electrolysis in the sample and acceptor solution was kept at an acceptable level with no detrimental consequences. For the polar model analytes, representing a log P range from -0.40 to 1.32, recoveries in the range 25-91% were obtained from human plasma. Strong hydrogen bonding and dipole interactions were probably responsible for efficient transfer of the model analytes into the SLM, and this is the first report on efficient EME of highly polar analytes without using any ionic carrier in the SLM.

Mass transfer in electromembrane extraction--The link between theory and experiments.

Electromembrane extraction was introduced in 2006 as a totally new sample preparation concept for the extraction of charged analytes present in aqueous samples. Electromembrane extraction is based on electrokinetic migration of the analytes through a supported liquid membrane and into a µL-volume of acceptor solution under the influence of an external electrical field. To date, electromembrane extraction has mostly been used for the extraction of drug substances, amino acids, and peptides from biological fluids, and for organic micropollutants from environmental samples. Electromembrane extraction has typically been combined with chromatography, mass spectrometry, and electrophoresis for analyte separation and detection. At the moment, close to 125 research papers have been published with focus on electromembrane extraction. Electromembrane extraction is a hybrid technique between electrophoresis and liquid-liquid extraction, and the fundamental principles for mass transfer have only partly been investigated. Thus, although there is great interest in electromembrane extraction, the fundamental principle for mass transfer has to be described in more detail for the scientific acceptance of the concept. This review summarizes recent efforts to describe the fundamentals of mass transfer in electromembrane extraction, and aim to give an up-to-date understanding of the processes involved.

Exhaustive and stable electromembrane extraction of acidic drugs from human plasma.

The first part of the current work systematically described the screening of different types of organic solvents as the supported liquid membrane (SLM) for electromembrane extraction (EME) of acidic drugs, including different alcohols, ketones, and ethers. Seven acidic drugs with a wide logP range (1.01-4.39) were selected as model substances. For the first time, the EME recovery of acidic drugs and system-current across the SLM with each organic solvent as SLM were investigated and correlated to relevant solvent properties such as viscosity and Kamlet and Taft solvatochromic parameters. Solvents with high hydrogen bonding acidity (α) and dipolarity-polarizability (π*) were found to be successful SLMs, and 1-heptanol was the most efficient candidate, which provided EME recovery in the range of 94-110%. Both hydrogen bonding interactions, dipole-dipole interactions, and hydrophobic interactions were involved in stabilizing the deprotonated acidic analytes (with high hydrogen bonding basicity and high dipole moment) during mass transfer across the SLM. The efficiency of the extraction normally decreased with increasing hydrocarbon chain length of the SLM, which was mainly due to increasing viscosity and decreasing α and π* values. The system-current during EME was found to be dependent on the type and the volume of the SLM. In contact with human plasma, an SLM of pure 1-heptanol was unstable, and to improve stability, 1-heptanol was mixed with 2-nitrophenyl octyl ether (NPOE). With this SLM, exhaustive EME was performed from diluted human plasma, and the recoveries of five out of seven analytes were over 91% after 10min EME. This approach was evaluated using HPLC-UV, and the evaluation data were found to be satisfactory.

Electromembrane extraction as a rapid and selective miniaturized sample preparation technique for biological fluids.

This special report discusses the sample preparation method electromembrane extraction, which was introduced in 2006 as a rapid and selective miniaturized extraction method. The extraction principle is based on isolation of charged analytes extracted from an aqueous sample, across a thin film of organic solvent, and into an aqueous receiver solution. The extraction is promoted by application of an electrical field, causing electrokinetic migration of the charged analytes. The method has shown to perform excellent clean-up and selectivity from complicated aqueous matrices like biological fluids. Technical aspects of electromembrane extraction, important extraction parameters as well as a handful of examples of applications from different biological samples and bioanalytical areas are discussed in the paper.

Combination of Electromembrane Extraction and Liquid-Phase Microextraction in a Single Step: Simultaneous Group Separation of Acidic and Basic Drugs.

Electromembrane extraction (EME) and liquid-phase microextraction (LPME) were combined in a single step for the first time to realize simultaneous and clear group separation of basic and acidic drugs. Using 2-nitrophenyl octyl ether as the supported liquid membrane (SLM) for EME and dihexyl ether as the SLM for LPME, basic and acidic drugs were extracted and separated simultaneously from a low pH sample by EME and LPME, respectively. After 15 min of extraction, basic drugs (citalopram and sertraline) were exhaustively extracted, whereas the recoveries for acidic drugs (ketoprofen and ibuprofen) were in the range of 76%-86%. Longer extraction time provided higher recoveries for the acidic drugs, but this somewhat deteriorated the group separation. Matrices effects from the coexisting acidic drugs/basic drugs were tested, and we observed that simultaneous EME/LPME was not affected by coexisting drugs at high concentration. This approach was further investigated from human plasma. Extraction recoveries were strongly dependent on dilution of plasma with buffer and on extraction time. Finally, this simultaneous EME/LPME approach was evaluated in combination with liquid chromatography (LC)-MS. The linearity ranges for the basic and acidic drugs were 10-600 ng/mL and 1-60 µg/mL, respectively, with R(2) > 0.997 for all analytes. The repeatability at three different levels for all analytes was less than 15%. The limits of quantification (LOQ, S/N = 10) were found to be 4.0-6.3 ng/mL and 0.6-0.9 µg/mL for basic and acidic drugs, respectively. Simultaneous EME/LPME enabled efficient group separation of basic and acidic analytes under optimum experimental conditions for both EME and LPME.

The potential of electromembrane extraction for bioanalytical applications.

Modern requirements in the field of bioanalysis often involve miniaturized, high-throughput sample preparation techniques that consume low amounts of both sample and potentially hazardous organic solvents. Electromembrane extraction is one technique that meets several of these requirements. In this principle analytes are selectively extracted from a biological matrix, through a supported liquid membrane and into an aqueous acceptor solution. The whole extraction process is facilitated by an electric field across the supported liquid membrane, which greatly reduces the extraction time. This review will give a thorough overview of recent advances in bioanalytical applications involving electromembrane extraction, and discuss both possibilities and challenges of the technique in a bioanalytical setting.

Electromembrane extraction for pharmaceutical and biomedical analysis - Quo vadis.

Electromembrane extraction (EME) was presented as a new microextraction concept in 2006, and since the introduction, substantial research has been conducted to develop this concept in different areas of analytical chemistry. To date, more than 100 research papers have been published on EME. The present paper discusses recent development of EME. The paper focuses on the principles of EME, and discusses how to optimize operational parameters. In addition, pharmaceutical and biomedical applications of EME are reviewed, with emphasis on basic drugs, acidic drugs, amino acids, and peptides. Finally, pros and cons of EME are discussed and future directions for EME are identified. Compared with other reviews focused on EME, the authors have especially highlighted their personal views about the most promising directions for the future, and identified the areas where more fundamental work is required.

Salt effects in electromembrane extraction.

Electromembrane extraction (EME) was performed on samples containing substantial amounts of NaCl to investigate how the presence of salts affected the recovery, repeatability, and membrane current in the extraction system. A group of 17 non-polar basic drugs with various physical chemical properties were used as model analytes. When EME was performed in a hollow fiber setup with a supported liquid membrane (SLM) comprised of 2-nitrophenyl octyl ether (NPOE), a substantial reduction in recovery was seen for eight of the substances when 2.5% (w/v) NaCl was present. No correlation between this loss and the physical chemical properties of these substances was seen. The recovery loss was hypothesized to be caused by ion pairing in the SLM, and a mathematical model for the extraction recovery in the presence of salts was made according to the experimental observations. Some variations to the EME system reduced this recovery loss, such as changing the SLM solvent from NPOE to 6-undecanone, or by using a different EME setup with more favorable volume ratios. This was in line with the ion pairing hypothesis and the mathematical model. This thorough investigation of how salts affect EME improves the theoretical understanding of the extraction process, and can contribute to the future development and optimization of the technique.

Stability and efficiency of supported liquid membranes in electromembrane extraction--a link to solvent properties.

The current work presents a large systematic screening of 61 possible organic solvents used as supported liquid membranes (SLM) in electromembrane extraction (EME). For each organic solvent, recovery, current across the SLM, and stability considerations have been investigated and correlated to relevant solvent properties through partial least square regression analysis. The five unpolar basic drugs pethidine, haloperidol, methadone, nortriptyline, and loperamide were used as model analytes. Efficient EME solvents were found to have a low water solubility (<0.5 g L(-1)) and belonged to cluster 2 of a Kamlet-and-Taft-based solvent classification system (high dipole moments and proton acceptor properties). These parameters were especially found in nitroaromatic compounds and ketones. Small molecules with low log P value and high water solubility were unsuitable, as they tended to give unstable extractions, caused by a high current across the SLM. This was often combined with substantial solvent-related interferences and the generation of an electroosmotic flow across the SLM, with resulting acceptor solution expansion. Large molecules with a high log P value were classified as inefficient. For these solvents, no current was measured across the SLM and no analytes were extracted. This is the first time systematic knowledge on the SLM in EME has been gathered and investigated, and the presented results could be highly beneficial for future development and optimization of EME.

Electromembrane extraction from aqueous samples containing polar organic solvents.

Electromembrane extraction (EME) was performed from aqueous samples and from aqueous samples containing methanol, ethanol, dimethyl sulfoxide, and acetonitrile. The basic drugs pethidine, haloperidol, nortriptyline, methadone and loperamide were used as model analytes. Reversed phase (C18) HPLC with UV (235 nm) and MS detection was used for analysis of the samples. With no organic solvent in the sample, maximum recoveries were obtained after 5-10 min. The maximum recoveries ranged between 83 and 95%. With 50% (v/v) methanol, ethanol, or dimethyl sulfoxide in the sample, recoveries were comparable to those from an aqueous sample, but the time required reaching maximum recovery increased to 15-25 min. With 2-nitrophenyl octyl ether (NPOE) as the supported liquid membrane (SLM), a stable EME system was obtained for 50% (v/v) methanol, 50% (v/v) ethanol, or 75% (v/v) dimethyl sulfoxide in the sample solution. On the other hand, the EME system was unstable with acetonitrile in the sample, as this solvent partly dissolved the SLM. In addition, acetonitrile migrated through the SLM and caused a volume expansion of the acceptor solution. Other SLMs were also tested (ethyl nitrobenzene, isopropyl nitrobenzene, and dodecyl nitrobenzene), but were inferior to NPOE. As a practical example, EME on dried blood spot extracts (80% methanol) were tested, and proved highly successful. These observations showed that EME can be an effective way of preparing aqueous samples containing substantial amounts of an organic solvent.

Electromembrane extraction: distribution or electrophoresis?

This paper presents for the first time a phenomenological theoretical model for the time dependent distribution of analytes during electromembrane extraction (EME). The model was verified experimentally for a range of model drugs and peptides. Analytes were extracted from an acidified aqueous sample solution, through an organic supported liquid membrane (SLM), and into an acidified aqueous acceptor solution. Mass transfer was governed by an applied electric field across the SLM. A rapid depletion was seen in the sample during extractions, with a steady increase in the amount accumulated in the acceptor solution. This was in good accordance with the theoretical model. A deviation from the modeled behavior was seen for some of the peptides where trapping of analyte in the SLM was high. The results demonstrated for the first time that EME behaved like a distribution system, with voltage dependent distribution coefficients. In addition, electrokinetic migration was observed across the SLM, which added an electrophoretic component to the mass transfer. This improved theoretical understanding will be highly beneficial for future optimization and development of applications using EME.

Electromembrane extraction of peptides--fundamental studies on the supported liquid membrane.

A large screening of different components in the supported liquid membrane (SLM) in electromembrane extraction (EME) was performed to test the extraction efficiency on eight model peptides. Electromembrane extraction from a 500 µL acidified aqueous sample containing the model peptides in the concentration 10 µg/mL was used. Extraction time was 5 min with an electric potential of 10 V and 900 rpm agitation of the sample vial. The samples were extracted through a hollow fiber-based SLM with different compositions of organic solvents and carriers. A small volume of acidified acceptor solution (25 µL) was after extraction analyzed directly, or with some dilution, on CE or HPLC. This article has identified mono- or di-substituted phosphate groups as the prominent group of carrier molecules needed to obtain acceptable recoveries. For the organic solvents, primary alcohols and ketones have shown promise regarding recovery and reproducibility, with some differences in selectivity. A new composition of the SLM, namely 2-octanone and tridecyl phosphate (90:10 w/w) has proved to give higher extraction recoveries and lower standard deviation than SLMs previously reported in the literature.

The role of bradykinin and the effect of the bradykinin receptor antagonist icatibant in porcine sepsis.

Bradykinin (BK) is regarded as an important mediator of edema, shock, and inflammation during sepsis. In this study, we evaluated the contribution of BK in porcine sepsis by blocking BK and by measuring the stable BK metabolite, BK1-5, using anesthetized pigs. The effect of BK alone, the efficacy of icatibant to block this effect, and the recovery of BK measured as plasma BK1-5 were first investigated. Purified BK injected intravenously induced an abrupt fall in blood pressure, which was completely prevented by pretreatment with icatibant. BK1-5 was detected in plasma corresponding to the doses given. The effect of icatibant was then investigated in an established model of porcine gram-negative sepsis. Neisseria meningitidis was infused intravenously without any pretreatment (n = 8) or pretreated with icatibant (n = 8). Negative controls received saline only. Icatibant-treated pigs developed the same degree of severe sepsis as did the controls. Both groups had massive capillary leakage, leukopenia, and excessive cytokine release. The plasma level of BK1-5 was low or nondetectable in all pigs. The latter observation was confirmed in supplementary studies with pigs undergoing Escherichia coli or polymicrobial sepsis induced by cecal ligation and puncture. In conclusion, icatibant completely blocked the hemodynamic effects of BK but had no beneficial effects on N. meningitidis-induced edema, shock, and inflammation. This and the fact that plasma BK1-5 in all the septic pigs was virtually nondetectable question the role of BK as an important mediator of porcine sepsis. Thus, the data challenge the current view of the role of BK also in human sepsis.