Knut Fredrik Seip

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.

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 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.

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.

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.

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.

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.

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.

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.

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.