Optimization of generic conditions for electromembrane extraction of basic substances of moderate or low polarity.

Generic electromembrane extraction (EME) methods were developed and optimized for basic analytes of moderate or low polarity, employing prototype conductive vial EME equipment. Two generic methods, B1 and B2, were devised for mono- and dibasic compounds with distinct polarity windows: 2.0 < log P < 6.0 for B1 and 1.0 < log P < 4.5 for B2. In B1, 10 µL of 2-nitrophenyl octyl ether served as the liquid membrane, while B2 utilized 10 µL of 2-undecanone. Both methods involved the acidification of 125 µL of human plasma samples with 125 µL of sample diluent (0.5 M HCOOH for B1 and 1.0 M HCOOH for B2). The acceptor phase consisted of 250 µL of 100 mM HCOOH. Extraction was conducted for 30 min with agitation at 800 rpm, employing an extraction potential of 100 V for B1 and 50 V for B2. A set of 90 pharmaceutical compounds was employed as model analytes. Both B1 and B2 demonstrated high recoveries (40%-100%) for the majority of model analytes within their respective polarity windows. Intra-day precision was within 2.2% and 9.7% relative standard deviation. Both extraction systems exhibited stability in terms of current, matrix effect values were between 90% and 109%.

Generic Liquid Membranes for Electromembrane Extraction of Bases with Low or Moderate Hydrophilicity.

For the first time, this paper introduces the idea of generic extraction conditions in electromembrane extraction (EME), where the selection of the liquid membrane is based on the charge (z) and hydrophobicity (log P) of the analyte. A broad range of organic solvents were tested as liquid membranes, and 90 basic pharmaceuticals were used as model analytes (-4.2 < log P < 8.1). 2-Nitrophenyl octyl ether (NPOE) was confirmed as a highly efficient liquid membrane for mono- and dibases (+1.0 ≤ z ≤ +2.0) of low polarity in the log P range of 2.2-6.4. This log P range was set as the extraction window (operational range) of NPOE. NPOE provided very high operational stability. At 50 V, the current was at a 1 µA level, and gas formation and drifting pH due to electrolysis were insignificant. 2-Undecanone was discovered as a new and robust alternative. This solvent extracted monobasic analytes (z = +1) in the log P range of 1.0-5.8 and was efficient even for bases of moderate polarity. The current was at the 1-3 µA level when 2-undecanone was operated at 50 V. Tri(pentyl) phosphate emerged as another new alternative for bases in the log P range of 0.5 to 5.5, providing greater selectivity differences. This solvent provided a higher current (30-50 µA), but the EME system stability was not compromised. 2-Undecanone and tri(pentyl) phosphate extracted protonated bases mainly by hydrogen bond interactions. NPOE, on the other hand, extracted based on a combination of hydrogen bond and π-type interactions and was consequently less selective.

Electromembrane extraction in microfluidic formats.

Electromembrane extraction is a microextraction technique where charged analytes are extracted across a supported liquid membrane and selectively isolated from the sample based on an electrical field. Since the introduction in 2006, there has been continuously increasing interest in electromembrane extraction, and currently close to 50 new articles are published per year. Electromembrane extraction can be performed in different technical configurations, based on standard laboratory glass vials or 96-well plate systems, and applications are typically related to pharmaceutical, environmental, and food and beverages analysis. In addition to this, conceptual research has developed electromembrane extraction into different milli- and microfluidic formats. These are much more early-stage activities, but applications among others related to organ-on-chip systems and smartphone detection indicate unique perspectives. To stimulate more research in this direction, the current article reviews the scientific literature on electromembrane extraction in milli- and microfluidic formats. About 20 original research articles have been published on this subject so far, and these are discussed critically in the following. Based on this and the authors own experiences with the topic, we discuss perspectives, challenges, and future research.

Electromembrane Extraction and Mass Spectrometry for Liver Organoid Drug Metabolism Studies.

Liver organoids are emerging tools for precision drug development and toxicity screening. We demonstrate that electromembrane extraction (EME) based on electrophoresis across an oil membrane is suited for segregating selected organoid-derived drug metabolites prior to mass spectrometry (MS)-based measurements. EME allowed drugs and drug metabolites to be separated from cell medium components (albumin, etc.) that could interfere with subsequent measurements. Multiwell EME (parallel-EME) holding 100 µL solutions allowed for simple and repeatable monitoring of heroin phase I metabolism kinetics. Organoid parallel-EME extracts were compatible with ultrahigh-performance liquid chromatography (UHPLC) used to separate the analytes prior to detection. Taken together, liver organoids are well-matched with EME followed by MS-based measurements.

Selectivity and efficiency of electromembrane extraction of polar bases with different liquid membranes-Link to analyte properties.

In the present fundamental study, selectivity and efficiency of electromembrane extraction of 50 polar basic substances (-6.7 < log P < +1.0) was systematically studied for ten different supported liquid membranes. For each model substance, 23 molecular descriptors were collected and these were investigated as potential parameters for understanding of extraction efficiency and selectivity by means of partial least squares regression. Overall, a highly aromatic deep eutectic solvent composed of coumarin and thymol with addition of 2% ionic carrier (di(2-ethylhexyl) phosphate) provided the highest extraction efficiency with an average extraction yield of 69% from pure water samples, 55% from plasma, and 62% from urine. With this solvent system, ionic, cation-π, and π-π interactions between the supported liquid membrane and analytes were dominant. Supported liquid membranes without aromaticity, however, operated primarily based on hydrogen-bonding interactions. This is the first time the relationship between analyte properties, solvent composition, and extraction yield has systematically been studied for polar bases in electromembrane extraction. This new knowledge represents a first step toward enabling future development and optimization of electromembrane extraction systems for polar bases based on rational design, rather than trial-and-error approaches.

Parallel electromembrane extraction of peptides with monoterpene and medium-length fatty acid deep eutectic solvents.

BACKGROUND: Electromembrane extraction (EME) involves the process of mass transfer of charged analytes from an aqueous sample through an organic liquid membrane into an aqueous acceptor medium under the influence of an electrical field. Successful solvation of the analyte within the liquid membrane is of paramount importance and involves molecular interactions with the liquid membrane. In this comprehensive investigation, parallel EME was examined using a training set of 13 model peptides employing deep eutectic solvents as the liquid membrane. These deep eutectic solvents were formulated by mixing specific monoterpenes (thymol, menthol, camphor) with medium-chain fatty acids (1-octanoic acid and 1-decanoic acid). RESULTS: From an array of different liquid membrane compositions explored, it was revealed that the combination of camphor and 1-decanoic acid (in a 1:1 w/w ratio) with 2% di (2-ethylhexyl) phosphate (DEHP) delivered the most efficient extraction system. The solvation of the model peptides within this liquid membrane predominantly relied on ionic interactions between protonated basic functionalities and DEHP, along with hydrogen bond interactions between the deprotonated acid functionalities (hydrogen bond acceptor) and 1-decanoic acid (hydrogen bond donor). Selectivity was modulated by the pH of the sample and acceptor solutions, with a direct correlation to the polarity and net charge of the model peptides. The ionization of 1-decanoic acid in the interfacial region between the sample and liquid membrane emerged as an important factor influencing the selectivity. SIGNIFICANCE AND NOVELTY: Although parallel EME of peptides has been reported previously, the current liquid membrane provides an extraction system with sufficient stability for the first time. Selective extraction of peptides through EME holds substantial promise within the realm of next-generation environmentally-friendly sample preparation methodologies. The findings presented in this paper contribute significantly to our fundamental understanding of these processes, and may serve as an important reference for the development of future methods in this field.

Extraction performance of electromembrane extraction and liquid-phase microextraction in prototype equipment.

In this comparative study, the performance of liquid-phase microextraction and electromembrane extraction in prototype equipment was evaluated for extraction of ninety basic substances from plasma. Using a commercial EME device based on conductive vials enabled a standardized and comprehensive comparison between the two methods. Extractions were performed from a pH-adjusted donor solution, across an organic liquid membrane immobilized in a porous polypropylene membrane, and into an acidic acceptor solution. In LPME, dodecyl acetate was used as the extraction solvent, while 2-nitrophenyl octyl ether was used for EME with an electric field applied across the system. To assess the extraction performance, extraction recovery plots and extraction time curves were constructed and analyzed. These plots provided insights into the efficiency and effectiveness of LPME and EME, allowing users to make better decisions about the most suitable method for a specific bioanalytical application. Both LPME and EME were effective for substances with 2.0 < log P < 4.0, with EME showing faster extraction kinetics. Small (200 µL) and large vials (600 µL) were compared, showing that smaller vials improved kinetics markedly in both techniques. Carrier-mediated extraction showed improved performance for analytes with log P < 2 in EME, however, with some limitations due to system instability. This is, to our knowledge, the first time LPME was performed in the commercial vial-based equipment. An evaluation of vial-based LPME investigating linearity, precision, accuracy, and matrix effects showed promising results. These findings contribute to a general understanding of the performance differences in vial-based LPME and EME.

Conductive vial electromembrane extraction - Principles and practical operation.

Electromembrane extraction (EME) is a microextraction technique where charged analytes are extracted from an aqueous sample solution, through a liquid membrane, and into an aqueous acceptor, under the influence of an external electric field. The liquid membrane is a few microliters of organic solvent immobilized in a polymeric support membrane. EME is a green technique and provides high selectivity. The selectivity is controlled by the direction and magnitude of the electric field, the chemical composition of the liquid membrane and the pH. Recently, commercial prototype equipment for EME was launched based on the use of conductive vials, and interest in EME is expected to increase. The current article is a tutorial and discusses the principle and practical work with EME. The practical information is related to the commercial prototype equipment but is valid also for other technical configurations of EME. The tutorial is intended to give readers a fundamental understanding of EME, which is required for method development and operation, and for avoiding common pitfalls.

Electromembrane extraction of peptides based on hydrogen bond interactions.

BACKGROUND: Electromembrane extraction (EME) of peptides reported in the scientific literature involve transfer of net positively charged peptides from an aqueous sample, through a liquid membrane, and into an aqueous acceptor solution, under the influence of an electrical field. The liquid membrane comprises an organic solvent, containing an ionic carrier. The purpose of the ionic carrier is to facilitate peptide solvation in the organic solvent based on ionic interactions. Unfortunately, ionic carriers increase the conductivity of the liquid membrane; the current in the system increases, the electrolysis in sample and acceptor is accelerated, and the extraction system tend to be unstable and suffers from drifting pH. RESULTS: In the present work, a broad selection of organic solvents were tested as pure liquid membrane for EME of peptides, without ionic carrier. Several phosphates provided high mass transfer, and tri(pentyl) phosphate was selected since this solvent also provided high operational stability. Among 16 different peptides used as model analytes, tri(pentyl) phosphate extracted those with net charge +1 and with no more than two polar side chains. Tri(pentyl) phosphate served as a very strong hydrogen bond acceptor, while the protonated peptides were hydrogen bond donors. By such, hydrogen bonding served as the primary interactions responsible for mass transfer. Tri(pentyl) phosphate as liquid membrane, could exhaustively extract leu-enkephalin, met-enkephalin, and endomorphin from human blood plasma and detected by LC-MS/MS. Calibration curves were linear (r2 > 0.99) within a concentration range from 1 to 500 ng/mL, and a relative standard deviation within 12% was observed for precision studies. SIGNIFICANCE: The current experiments are important because they indicate that small peptides of low polarity may be extracted selectively in EME based on hydrogen bond interactions, in systems not suffering from electrolysis.

Electromembrane extraction of polar substances - Status and perspectives.

In this article, the scientific literature on electromembrane extraction (EME) of polar substances (log P < 2) is reviewed. EME is an extraction technique based on electrokinetic migration of analyte ions from an aqueous sample, across an organic supported liquid membrane (SLM), and into an aqueous acceptor solution. Because extraction is based on voltage-assisted partitioning, EME is fundamentally suitable for extraction of polar and ionizable substances that are challenging in many other extraction techniques. The article provides an exhaustive overview of papers on EME of polar substances. From this, different strategies to improve the mass transfer of polar substances are reviewed and critically discussed. These strategies include different SLM chemistries, modification of supporting membranes, sorbent additives, aqueous solution chemistry, and voltage/current related strategies. Finally, the future applicability of EME for polar substances is discussed. We expect EME in the coming years to be developed towards both very selective targeted analysis, as well as untargeted analysis of polar substances in biomedical applications such as metabolomics and peptidomics.

Electromembrane extraction of streptomycin from biological fluids.

In this fundamental study, streptomycin was extracted successfully from urine and plasma using electromembrane extraction (EME). Streptomycin is an aminoglycoside with log P -7.6 and was selected as an extremely polar model analyte. EME is a microextraction technique, where charged analytes are extracted under the influence of an electrical field, from sample, through a supported liquid membrane (SLM), and into an acceptor solution. The SLM comprised 2-nitrophenyl pentyl ether (NPPE) mixed with bis(2-ethylhexyl) phosphate (DEHP). DEHP served as ionic carrier and facilitated transfer of streptomycin across the SLM. For EME from urine and protein precipitated plasma, the optimal DEHP content in the SLM was 45-50% w/w. From untreated plasma, the content of DEHP was increased to 75% w/w in order to suppress interference from plasma proteins. Most endogenous substances with UV absorbance were not extracted into the acceptor. Proteins and phospholipids were also discriminated, with <0.6% of proteins and <0.02% of phospholipids found in the acceptor after EME. Thus, despite the fact that the SLM was permeable to more polar molecules, the EME still provided very efficient sample cleanup. Extraction process efficiencies of 98% and 61% were achieved from urine and plasma, respectively, with linear calibration (R2 > 0.9929), absence of significant matrix effects (94-112%), accuracy of 94-125%, and RSD ≤ 15% except at LLOQ. The average current during extractions was 67 µA or less. The findings of this paper demonstrated that EME is feasible for extraction of basic analytes of extreme polarity.

Electromembrane extraction using deep eutectic solvents as the liquid membrane.

In this work, we investigated for the first time hydrophobic deep eutectic solvents (DES) as supported liquid membrane (SLM) for electromembrane extraction (EME). Camphor, coumarin, DL-menthol, and thymol were used as non-ionic DES components. Different DESs compositions were tested, to study systematically the importance of hydrogen bonding and dispersion/aromatic interactions during mass transfer across the SLM. Unexpectedly, mixtures of coumarin and thymol were highly efficient SLMs, and provided exhaustive or near-exhaustive extraction of non-polar bases, non-polar acids, and polar bases. SLMs with such performance for both bases and acids, in a large polarity window, are not found in current literature. The SLMs were highly aromatic, very strong hydrogen bonding donors, and moderately strong hydrogen bonding acceptors. Aromatic (π type) interactions were apparently very important for transfer of bases, while hydrogen bonding were dominant for acids. EME of six polar basic drugs from plasma, with a coumarin and thymol mixture as SLM, and combined with UHPLC-MS/MS analysis, was evaluated to test the potential for analytical applications. Plasma was diluted 1:1 with phosphate buffer pH 2.0. Calibration curves were linear in the therapeutic ranges (0.970 < R2 < 0.999), recoveries ranged between 47 and 93%, and repeatability was within 1.6-10.7% RSD. The clean-up efficiency was excellent and no matrix effects from plasma were seen. Presence of trace levels of coumarin in the acceptor phase was however found to cause some ion enhancement. Based on the current work, we foresee more research on the use of DES in EME.

Electromembrane extraction of peptides using deep eutectic solvents as liquid membrane.

For the first time, we report electromembrane extraction (EME) of peptides using deep eutectic solvent (DES) as supported liquid membrane (SLM). DES were mixtures of coumarin, camphor, DL-menthol and thymol. Sixteen model peptides were extracted from 100 µL 50 mM phosphate buffer solution (pH 3.0), through the SLM, and into 100 µL acceptor solution consisting of 50 mM phosphoric acid (pH 1.8). EME was performed in 96-well format with 30 V to facilitate extraction of positively charged peptides. The model peptides comprised three to 13 amino acids, and differed significantly in terms of acid/base functionalities and polarity. We found pure DES to be inefficient for EME of peptides. However, with addition of a small amount of the ionic carrier di(2-ethylhexyl) phosphate (DEHP) to the DES, the extraction efficiency increased due to ionic interactions. With the most efficient SLM; coumarin and thymol mixed in molar ratio (1:2) with 2.0% (v/v) DEHP, average recovery after 15 min was 55%; five peptides were extracted with recovery > 80%, nine peptides with recoveries in the range 40-80%, and two peptides were not extracted (recovery < 5%). When extraction time was extended to 45 min, average extraction recovery increased to 83%. Extraction recoveries with DES were higher than previously reported in the literature for the same model peptides.