Frederik Andre Hansen

• Noreng, Line; Øiestad, Åse Marit Leere; Hansen, Frederik Andre; Røberg-Larsen, Hanne; Wilson, Steven Ray Haakon & Øiestad, Elisabeth Leere (2026). Toward Sustainable Clinical Analysis: Benchmarking Plastic Use in LC–MS Sample Preparation – Exemplified by Ketamine Analogues in Whole Blood. Analytical Chemistry. ISSN 0003-2700. 98(16), p. 11899–11909. doi: 10.1021/acs.analchem.5c08225. Full text in Research Archive
• He, Junling; Peng, Shaoyun; Xie, Shangjun; Tang, Yong; Lin, Chiliang & Pedersen-Bjergaard, Stig [Show all 10 contributors for this article] (2026). Development of electromembrane extraction using deep eutectic solvents for 22 representative veterinary drug residues in honey, milk, and eggs. Analytical and Bioanalytical Chemistry. ISSN 1618-2642. doi: 10.1007/s00216-026-06497-z. Full text in Research Archive
• Hay, Anne Oldeide & Hansen, Frederik Andre (2025). Electromembrane extraction of tetracyclines in foods and human plasma prior to LC-MS/MS analysis: Impact of sample matrix on extraction performance. Advances in Sample Preparation. 17. doi: 10.1016/j.sampre.2025.100227. Full text in Research Archive
• Samkumpim, Thidarat; Dowlatshah, Samira; Alahmad, Waleed; Varanusupakul, Pakorn; Hruskova, Helena & Hansen, Frederik Andre [Show all 7 contributors for this article] (2025). 96-Well Agarose-Gel Electromembrane Extraction. Analytical Chemistry. ISSN 0003-2700. 98(1), p. 1090–1097. doi: 10.1021/acs.analchem.5c06783. Full text in Research Archive Show summary

  For the first time, gel electromembrane extraction was demonstrated in a 96-well system. Gel membranes of 3% w/v agarose and with a thickness of 3.5 mm were immobilized in hydrophilic polyvinylidene fluoride (PVDF) filters in a 96-well filter plate. The filters provided mechanical support for the gel membranes, and were important for the stability and robustness of the system. A selection of 90 basic pharmaceuticals in the polarity range −4.2 < log P < 8.1 was used as model analytes (compounds). The compounds were extracted from 200 μL sample (water or human plasma) adjusted to pH 4.0 with dilute formic acid, through the gel membrane, and into 200 μL of 100 mM formic acid as acceptor. The extraction potential was 25 V, and the extraction time was 20 min for careful operation to limit Joule heating and electroendosmosis. The majority of the compounds in the polarity range −4.0 < log P < 3.0 were extracted with high recovery (40–100%). Compounds with log P > 3.0 were discriminated due to interactions with the gel membrane. Proteins and phospholipids were not extracted, and the system therefore provided efficient cleanup from human plasma samples. The 96-well agarose-gel electromembrane extraction (EME) system showed great potential. Selectivity was controlled by interactions with the aqueous gel membrane. This is fundamentally very different from traditional EME with oil membranes, where selectivity is controlled by electro-assisted partition in and out of the oil membrane. 96-Well electromembrane extraction with gel membranes of agarose is favorable in terms of greenness and efficiency for polar analytes, as compared with systems based on oil membranes.

• He, Junling; Tang, Yong; Lin, Chiliang; Peng, Shaoyun; Pedersen-Bjergaard, Stig & Hansen, Frederik Andre [Show all 7 contributors for this article] (2025). Electromembrane extraction of multi-class prohibited/restricted veterinary drugs from food samples – Exploring liquid membrane composition. Food Chemistry. ISSN 0308-8146. 470. doi: 10.1016/j.foodchem.2024.142680. Full text in Research Archive
• Dong, Xue-Man; Hansen, Frederik Andre; Wu, Pu; Wu, Yelu; He, Sile & Tang, Weiyang (2025). Sample preparation of polar metabolites in biological samples: Methodologies and technological insights. TrAC. Trends in analytical chemistry. ISSN 0165-9936. 189. doi: 10.1016/j.trac.2025.118244. Full text in Research Archive
• Kogler, Stian; Skottvoll, Frøydis Sved; Hruskova, Helena; Rise, Frode; Aizenshtadt, Aleksandra & Krauss, Stefan Johannes Karl [Show all 9 contributors for this article] (2025). Electromembrane Extraction Provides Unprecedented Selectivity for Drugs in Cell Culture Media Used in Organoid and Organ-on-Chip Systems. Analytical Chemistry. ISSN 0003-2700. 97(9), p. 4923–4931. doi: 10.1021/acs.analchem.4c04994. Full text in Research Archive Show summary

  The use of organoids and organ-on-chip technologies as nonanimal methodologies in drug discovery and personalized medicine is rapidly expanding. However, the complexity and small volumes of organoid culture samples present significant analytical challenges, e.g., in drug analysis using liquid chromatography–mass spectrometry (LC–MS). Essentially an electrophoresis across an oil membrane, electromembrane extraction (EME) offers a promising approach for measuring drugs, as it is, for example, compatible with small samples such as organoid and organ-on-chip formats. Given the potential of the technology, there is a need to assess the extraction purity of EME extracts to ensure EME’s compatibility with high-throughput, downstream analysis. This study evaluates the effectiveness of EME for sample cleanup in various common cell culture media used for organoids and organs-on-chips. The media were spiked with 90 small-molecule drugs. Using gel electrophoresis (sodium dodecyl sulfate polyacrylamide gel electrophoresis), high-resolution nuclear magnetic resonance spectroscopy, and LC–MS, we demonstrate that EME provides exhaustive removal of unwanted medium components (proteins, polar molecules, and apolar/neutral molecules) while selectively extracting the spiked small-molecule drugs. The approach was demonstrated with human stem-cell-derived liver organoids, allowing simple detection and monitoring of telltale cytochrome P450 metabolism. Taken together, our observations highlight an unprecedented ability of EME to provide sample cleanup for drug analysis in matrixes compatible with organoids and organ-on-chip technology.

• Dowlatshah, Samira; Hay, Anne Oldeide; Noreng, Line & Hansen, Frederik Andre (2025). A practical tutorial for optimizing electromembrane extraction methods by response surface methodology. Advances in Sample Preparation. 14. doi: 10.1016/j.sampre.2025.100170. Full text in Research Archive Show summary

  Microextraction techniques, such as electromembrane extraction (EME), have greatly advanced analytical chemistry by providing efficient, cost-effective, and eco-friendly sample preparation methods. EME is especially promising for ionizable analytes, with its success relying on optimizing multiple experimental variables. Traditionally, optimization has employed a one-variable-at-a-time (OVAT) approach, which is time-consuming and may overlooks variable interactions. This tutorial introduces response surface methodology (RSM) as a superior alternative for optimizing EME methods. RSM allows for the simultaneous variation of multiple factors, offering a comprehensive understanding of their effects and interactions with fewer experiments. This tutorial covers the fundamental principles of EME, essential experimental parameters, and the application of RSM for method optimization. It includes guidance on software selection, design choice, factor settings, response definition, and the optimization of multiple responses. Practical steps for conducting experiments, analyzing data, and interpreting models are detailed, with real-life examples illustrating the process. The tutorial aims to make RSM accessible to both novice and moderately experienced users, promoting its broader application in analytical chemistry. By simplifying the complex optimization process, this tutorial supports the development of robust, efficient, and green EME methods.

• Zhou, Chen; Dowlatshah, Samira; Hansen, Frederik Andre & Pedersen-Bjergaard, Stig (2024). Generic conditions for electromembrane extraction of polar bases. Talanta: The International Journal of Pure and Applied Analytical Chemistry. ISSN 0039-9140. 267. doi: 10.1016/j.talanta.2023.125215. Full text in Research Archive Show summary

  The current paper reports the first generic conditions for electromembrane extraction (EME) of polar bases within −2.0 < log P < 1.0 from human plasma. Generic conditions are important for inter-lab transferability and reproducibility, and were accordingly developed in commercially available EME equipment. In previous work, generic methodology was proposed for the extraction of bases of moderate and low polarity in the range 1.0 < log P < 6.0. The current paper extends this range of hydrophobicity downward. For polar bases, the composition of the liquid membrane is critical, in order to obtain high extraction recoveries, and to keep the extraction current as low as possible. The final liquid membrane was based on the deep eutectic solvent formed upon mixing 6-methyl coumarin and thymol in a 1:2 w/w ratio. The deep eutectic solvent was mixed with 2-undecanone in a 1:1 volumetric ratio. The latter decreased the extraction current, and improved the stability of the EME system. Finally, di(2-ethylhexyl) phosphate (DEHP) was added as ionic carrier to the liquid membrane to improve the mass transfer of polar bases. With this liquid membrane, the following generic conditions were established for mono- and dibases in the range −2.0 < log P < 4.5; plasma samples were diluted with an equal volume of 900 mM HCOOH, the acceptor was 450 mM HCOOH, voltage was 40 V, and the extraction time was 30 min. With this development, generic EME methods are now available for mono- and dibases in the polarity range −2.0 < log P < 6.0.

• Fabris, André L.; Hansen, Frederik Andre; Yonamine, Mauricio; Pedersen-Bjergaard, Stig & Øiestad, Elisabeth Leere (2024). An efficient and green method for the analysis of synthetic cathinones in whole blood using 96-well electromembrane extraction and LC-MS/MS. Sustainable Chemistry and Pharmacy. 38, p. 1–12. doi: 10.1016/j.scp.2024.101494. Full text in Research Archive Show summary

  Electromembrane extraction (EME) is a miniaturized technique in which charged molecules are extracted from biological samples, through a porous membrane, into an acceptor solution. This process occurs in the presence of an electric field that enables increased analyte recovery in short extraction times. Studies are being carried out to establish recommended extraction conditions for different groups of compounds based on their physicochemical properties. In this work, pre-established generic liquid membranes for the extraction of basic compounds were tested using synthetic cathinones within a wide log P range as basic model analytes. First, recommended organic solvents for the chosen analytes were evaluated in terms of recovery. The best candidates were included in an optimization study with other relevant EME conditions (voltage, extraction time, and agitation rate). All analyses were performed using a UPLC-MS/MS with the multiple reaction monitoring mode. 2-Undecanone was chosen as the liquid membrane providing the best extraction efficiency in combination with 40 V, 35 min, and 725 rpm. Validation showed r2 > 0.99 in the 1–500 ng/mL range, accuracy of ±12%, imprecision of ≤19%, 1 ng/mL as limit of quantitation, 0.1–0.5 ng/mL as limit of detection, matrix effect 97–118%, and recovery 63–110%. Among the generic liquid membranes studied in this work, 2-Undecanone was the best choice for the target basic analytes. High recoveries and low matrix effects were achieved, thus demonstrating the applicability of recommended generic liquid membranes in the 96-well plate setup and with whole blood samples. Our findings contribute to further establishing EME as an actual sample preparation technique for routine analyses that could be further explored in a commercial format.

• Song, Chenchen; Zhou, Chen; Hansen, Frederik Andre; Hay, Anne Oldeide & Pedersen-Bjergaard, Stig (2024). Optimization of generic conditions for electromembrane extraction of basic substances of moderate or low polarity. Journal of Separation Science. ISSN 1615-9306. 47(3). doi: 10.1002/jssc.202300801. Full text in Research Archive Show summary

  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%.

• Rye, Torstein Kige; Lee, Chien-Yun; Zellner, Andreas; Moen, Sara Haglund; Dowlatshah, Samira & Halvorsen, Trine Grønhaug [Show all 8 contributors for this article] (2024). Electromembrane extraction of peptides based on charge, hydrophobicity, and size – A large-scale fundamental study of the extraction window. Journal of Separation Science. ISSN 1615-9306. 47(15). doi: 10.1002/jssc.202400292. Full text in Research Archive Show summary

  This study investigated the capability of electromembrane extraction (EME) as a general technique for peptides, by extracting complex pools of peptides comprising in total of 5953 different substances, varying in size from seven to 16 amino acids. Electromembrane extraction was conducted from a sample adjusted to pH 3.0 and utilized a liquid membrane consisting of 2-nitrophenyl octyl ether and carvacrol (1:1 w/w), containing 2% (w/w) di(2-ethylhexyl) phosphate. The acceptor phase was 50 mM phosphoric acid (pH 1.8), the extraction time was 45 min, and 10 V was used. High extraction efficiency, defined as a higher peptide signal in the acceptor than the sample after extraction, was achieved for 3706 different peptides. Extraction efficiencies were predominantly influenced by the hydrophobicity of the peptides and their net charge in the sample. Hydrophobic peptides were extracted with a net charge of +1, while hydrophilic peptides were extracted when the net charge was +2 or higher. A computational model based on machine learning was developed to predict the extractability of peptides based on peptide descriptors, including the grand average of hydropathy index and net charge at pH 3.0 (sample pH). This research shows that EME has general applicability for peptides and represents the first steps toward in silico prediction of extraction efficiency.

• Dowlatshah, Samira; Rye, Torstein Kige; Hansen, Frederik Andre; Halvorsen, Trine Grønhaug & Pedersen-Bjergaard, Stig (2024). Parallel electromembrane extraction of peptides with monoterpene and medium-length fatty acid deep eutectic solvents. Analytica Chimica Acta. ISSN 0003-2670. 1297. doi: 10.1016/j.aca.2024.342360. Full text in Research Archive Show summary

  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.

• Rye, Torstein Kige; Dowlatshah, Samira; Hansen, Frederik Andre; Halvorsen, Trine Grønhaug & Pedersen-Bjergaard, Stig (2024). Electromembrane extraction of peptides – Looking retrospectively into published studies. Advances in Sample Preparation. 9. doi: 10.1016/j.sampre.2024.100106. Full text in Research Archive Show summary

  Electromembrane extraction (EME) has been developed into an attractive microextraction concept for extraction of pharmaceuticals and related substances from blood and urine. In EME, charged substances are extracted across a liquid membrane (organic) under the influence of an electrical field. Recently, commercial equipment for EME was launched, and this is expected to increase the interest for the technique. EME has also been explored for extraction of peptides, but this is more complicated due to the challenges related to the solvation of peptides in the organic liquid membrane. In this paper, we review the literature on EME of peptides, and we retrospectively look closer into published studies based on our current understanding of the concept. By such, we can explain early observations in more detail, and we can improve our fundamental understanding.

• Rye, Torstein Kige; Hansen, Frederik Andre; Halvorsen, Trine Grønhaug & Pedersen-Bjergaard, Stig (2024). Electromembrane extraction of human chorionic gonadotropin – A case study on mass transfer of intact protein versus signature peptide. Advances in Sample Preparation. 11. doi: 10.1016/j.sampre.2024.100126. Full text in Research Archive Show summary

  For the first time, we present targeted protein detection by tryptic digestion of human chorionic gonadotropin (hCG) followed by electromembrane extraction (EME). Operational parameters were optimized, and urine and serum samples spiked with hCG underwent tryptic digestion followed by EME of the βT5 signature peptide. The liquid membrane comprised nitrophenyl octyl ether (NPOE), carvacrol, and di(2-ethyl hexyl) phosphate (DEHP) at ratios of 49:49:2 (w/w/w). Extractions were performed in a conductive vial format for 45 min at 5 V. Even from highly complex digested samples of serum and urine, the signature peptide βT5 was extracted by EME and detected by LC-MS/MS. While attempts to extract intact hCG protein were unsuccessful, the extraction of the signature peptide was efficient. The extraction recovery from undigested and digested urine was 71 % (RSD = 17 %) and 116 % (RSD = 17 %), respectively. For serum, the extraction recoveries were 11 % (RSD = 23 %) for undigested samples and 110 % (RSD = 14 %) for digested samples. This study demonstrates both the potential and challenges of EME for protein analysis. Experiments regarding EME of intact proteins provided new insights into protein phase distribution. This fundamental case study underscores the potential of EME as a sample preparation technique for the targeted determination of protein biomarkers and drugs.

• Hay, Anne Oldeide & Hansen, Frederik Andre (2024). Exploring electromembrane extraction coupled to fast LC-MS/MS as a high-throughput platform for determination of 12 polar endogenous metabolites in human plasma. Journal of Chromatography A. ISSN 0021-9673. 1737. doi: 10.1016/j.chroma.2024.465451. Full text in Research Archive Show summary

  High efficiency in the analytical workflow, including fast sample preparation and LC-MS/MS analysis, is an advantage when analyzing a high number of samples. It can however be a challenge when determining polar analytes in complex, biological samples, and one must expect to make a compromise between a simple sample preparation followed by a long chromatographic separation, or vice versa, to limit matrix effects. In this proof-of-concept work, a one-step 96-well (parallel extraction) electromembrane extraction (EME) method was coupled to flow injection-MS/MS of 0.7 min per sample, allowing a very high-throughput analysis of 12 polar, endogenous metabolites from unprecipitated plasma of limited dilution. The throughput of the EME method matched the subsequent analysis. Recoveries ranged from 6 to 93 %, and repeatability and linearity were 2–15 % and R2 ≥ 0.9949, respectively, for all but two compounds. Matrix effects were approximately 50 % after EME and varied <11 % between 6 plasma donors, which represented a major improvement relative to a simple protein precipitation where signals were entirely suppressed. The work demonstrates a potential for EME coupled to flow injection-MS/MS to serve as a high-throughput platform for bioanalysis, not just of polar analytes, but also hydrophobic drugs both basic and acidic.

• Song, Chenchen; Dowlatshah, Samira; Gaznawi, Somayeh; Hay, Anne Oldeide; Hasvold, Grete & Hansen, Frederik Andre (2024). New robust and efficient liquid membranes for conductive vial electromembrane extraction of acids with low to moderate hydrophilicity in human plasma. Analytical and Bioanalytical Chemistry. ISSN 1618-2642. doi: 10.1007/s00216-024-05503-6. Full text in Research Archive Show summary

  The current paper reports two new, robust, and efficient conditions for electromembrane extraction of acidic substances from human plasma. Two systems were developed based on eutectic solvents: A1 (“A” for acid) comprised dodecyl methyl sulfoxide and thymol in 1:2 ratio (w/w) as liquid membrane, while A2 used [6-methylcoumarin:thymol (1:2)]:2-nitrophenyl octyl ether in 2:1 ratio (w/w). The performance of A1 and A2 was characterized by extraction of 31 acidic model analytes (pharmaceutical drugs and nutrients) spiked into 100 µL human plasma diluted 1:1 (v/v) with phosphate buffer pH 7.4. The acceptor solution was 50 mM NH4HCO3 buffer pH 10.0, and extraction was performed at an agitation rate of 750 RPM. Voltage and extraction time were 30 V for 30 min and 10 V for 20 min for A1 and A2, respectively. Under optimal conditions, A1 extracted analytes with 1.8 ≤ log P ≤ 6.0 with an average recovery (R) of 85.1%, while A2 extracted in a range of 0.5 ≤ log P ≤ 6.0 with an average recovery of 79.9%. Meanwhile, extraction current was low at 9 and 26 µA, respectively, which is indicative of good system robustness. Using UHPLC-MS/MS analysis of the acceptor solution, repeatability of the A1 and A2 methods was determined to be 2.8–7.7% and 3.3–9.4% for R > 40%, matrix effects were 82–117% and 84–112%, respectively, and linear calibration curves were obtained. The performance and compatibility with human plasma represent a major improvement over previous state-of-the-art liquid membranes for acidic analytes, namely 1-octanol.

• Schüller, Maria; Hansen, Frederik Andre & Pedersen-Bjergaard, Stig (2023). Extraction performance of electromembrane extraction and liquid-phase microextraction in prototype equipment. Journal of Chromatography A. ISSN 0021-9673. 1710. doi: 10.1016/j.chroma.2023.464440. Full text in Research Archive
• Schüller, Maria; Hansen, Frederik Andre; Skaalvik, Tonje Gottenberg & Pedersen-Bjergaard, Stig (2023). Conductive vial electromembrane extraction – Principles and practical operation. Analytical Science Advances (ASA). 4(7-8), p. 236–243. doi: 10.1002/ansa.202200065. Full text in Research Archive Show summary

  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.

• Dowlatshah, Samira; Hansen, Frederik Andre; Zhou, Chen; Ramos-Payán, María; Halvorsen, Trine Grønhaug & Pedersen-Bjergaard, Stig (2023). Electromembrane extraction of peptides based on hydrogen bond interactions. Analytica Chimica Acta. ISSN 0003-2670. 1275. doi: 10.1016/j.aca.2023.341610. Full text in Research Archive Show summary

  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.

• Zhou, Chen; Dowlatshah, Samira; Hay, Anne Oldeide; Schüller, Maria; Pedersen-Bjergaard, Stig & Hansen, Frederik Andre (2023). Generic Liquid Membranes for Electromembrane Extraction of Bases with Low or Moderate Hydrophilicity. Analytical Chemistry. ISSN 0003-2700. 95(23), p. 8982–8989. doi: 10.1021/acs.analchem.3c01052. Full text in Research Archive Show summary

  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.

• Hay, Anne Oldeide; Hansen, Frederik Andre; Psillakis, Elefteria & Pedersen-Bjergaard, Stig (2022). Liquid-phase microextraction in bioanalysis – How green can it be? Green Analytical Chemistry. 3. doi: 10.1016/j.greeac.2022.100028. Full text in Research Archive
• Hansen, Frederik Andre & Pedersen-Bjergaard, Stig (2022). Electromembrane extraction – looking closer into the liquid membrane. Advances in Sample Preparation. 2. doi: 10.1016/j.sampre.2022.100020. Full text in Research Archive
• Hansen, Frederik Andre; Petersen, Nickolaj Jacob; Kutter, Jörg P. & Pedersen-Bjergaard, Stig (2022). Electromembrane extraction in microfluidic formats. Journal of Separation Science. ISSN 1615-9306. 45(1), p. 246–257. doi: 10.1002/jssc.202100603. Full text in Research Archive Show summary

  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.

• Hay, Anne Oldeide; Trones, Roger; Herfindal, Lars; Skrede, Silje & Hansen, Frederik Andre (2022). Determination of methotrexate and its metabolites in human plasma by electromembrane extraction in conductive vials followed by LC-MS/MS. Advances in Sample Preparation. 2. doi: 10.1016/j.sampre.2022.100011. Full text in Research Archive
• Primdahl, Karoline Gangestad; Hansen, Frederik Andre; Solum, Eirik Johansson; Nolsøe, Jens Mortansson Jelstrup & Aursnes, Marius (2022). Introduction to Preparative Chromatography: Description of a Setup with Continuous Detection. Journal of Chemical Education. ISSN 0021-9584. 99(6), p. 2372–2377. doi: 10.1021/acs.jchemed.1c00917. Full text in Research Archive
• Skottvoll, Frøydis Sved; Aizenshtadt, Aleksandra; Hansen, Frederik Andre; Martinez, Mikel Amirola; Andersen, Jannike Mørch & Bogen, Inger Lise [Show all 11 contributors for this article] (2022). Direct Electromembrane Extraction-Based Mass Spectrometry: A Tool for Studying Drug Metabolism Properties of Liver Organoids. Analysis & Sensing. 2(2). doi: 10.1002/anse.202100051. Full text in Research Archive Show summary

  This work introduces a strategy for organoid analysis - direct Electromembrane Extraction based Mass Spectrometry (dEME-MS) – for coupling liver organoids with mass spectrometry (MS). dEME-MS comprises electrophoresis of selected small molecules from a culture chamber across an oil membrane, and to a MS compatible solution. This enables clean micro-extraction of drugs and their metabolites as produced in the liver organoids to capillary liquid chromatography-mass spectrometry. Applying dEME-MS, proof-of-concept of directly measuring methadone metabolism is demonstrated on adult liver organoids. With 50 liver organoids and 1 μM methadone, methadone metabolism was monitored from 0 to 24 hours (11 time points). All analytes had <0.4 % variance in retention times with >100 measurements. dEME-MS is capable of automated and selective monitoring of drug metabolism in liver organoids, and could serve as a valuable tool for automated drug discovery efforts.

• Skottvoll, Frøydis Sved; Hansen, Frederik; Harrison, Sean; Boger, Ida Caroline Sneis; Mrsa, Ago & Restan, Magnus Saed [Show all 14 contributors for this article] (2021). Electromembrane extraction and mass spectrometry for liver organoid drug metabolism studies. Analytical Chemistry. ISSN 0003-2700. 93, p. 3576–3585. doi: 10.1021/acs.analchem.0c05082. Full text in Research Archive Show summary

  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.

• Eie, Linda Vårdal; Rye, Torstein Kige; Hansen, Frederik; Halvorsen, Trine Grønhaug & Pedersen-Bjergaard, Stig (2021). Electromembrane extraction of peptides and amino acids – status and perspectives. Bioanalysis. ISSN 1757-6180. 13(4), p. 277–289. doi: 10.4155/bio-2020-0285. Full text in Research Archive
• Eie, Linda Vårdal; Pedersen-Bjergaard, Stig & Hansen, Frederik Andre (2021). Electromembrane extraction of polar substances – Status and perspectives. Journal of Pharmaceutical and Biomedical Analysis. ISSN 0731-7085. 207. doi: 10.1016/j.jpba.2021.114407. Full text in Research Archive Show summary

  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.

• Rye, Torstein Kige; Martinovic, Gordana; Eie, Linda Vårdal; Hansen, Frederik Andre; Halvorsen, Trine Grønhaug & Pedersen-Bjergaard, Stig (2021). Electromembrane extraction of peptides using deep eutectic solvents as liquid membrane. Analytica Chimica Acta. ISSN 0003-2670. 1175. doi: 10.1016/j.aca.2021.338717. Full text in Research Archive Show summary

  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.

• Hansen, Frederik Andre; Tirandaz, Shima & Pedersen-Bjergaard, Stig (2021). Selectivity and efficiency of electromembrane extraction of polar bases with different liquid membranes—Link to analyte properties. Journal of Separation Science. ISSN 1615-9306. 44(13), p. 2631–2641. doi: 10.1002/jssc.202100167. Full text in Research Archive
• Hansen, Frederik & Pedersen-Bjergaard, Stig (2021). Electromembrane extraction of streptomycin from biological fluids. Journal of Chromatography A. ISSN 0021-9673. 1639. doi: 10.1016/j.chroma.2021.461915. Full text in Research Archive Show summary

  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.

• Hansen, Frederik; Øiestad, Elisabeth Leere & Pedersen-Bjergaard, Stig (2020). Bioanalysis of pharmaceuticals using liquid-phase microextraction combined with liquid chromatography–mass spectrometry. Journal of Pharmaceutical and Biomedical Analysis. ISSN 0731-7085. 189, p. 1–12. doi: 10.1016/j.jpba.2020.113446. Full text in Research Archive
• Hansen, Frederik; Santigosa-Murillo, Elia; Payan, Maria Ramos; Munoz, Maria; Øiestad, Elisabeth Leere & Pedersen-Bjergaard, Stig (2020). Electromembrane extraction using deep eutectic solvents as the liquid membrane. Analytica Chimica Acta. ISSN 0003-2670. 1143, p. 109–116. doi: 10.1016/j.aca.2020.11.044. Full text in Research Archive Show summary

  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.

• Drobnjak, Milena; Hansen, Frederik; Øiestad, Elisabeth Leere; Løvli, Trond; Trones, Roger & Martinsen, Ørjan Grøttem [Show all 7 contributors for this article] (2020). Electromembrane extraction with vials of conducting polymer. LC GC North America. ISSN 1527-5949. 38(8), p. 435–439. Full text in Research Archive
• Hansen, Frederik; Jensen, Henrik & Pedersen-Bjergaard, Stig (2020). Electromembrane extraction using sacrificial electrodes. Analytical Chemistry. ISSN 0003-2700. 92(7), p. 5595–5603. doi: 10.1021/acs.analchem.0c00626. Full text in Research Archive
• Hansen, Frederik; Kuban, Pavel; Øiestad, Elisabeth Leere & Pedersen-Bjergaard, Stig (2020). Electromembrane extraction of highly polar bases from biological samples – Deeper insight into bis(2-ethylhexyl) phosphate as ionic carrier. Analytica Chimica Acta. ISSN 0003-2670. 1115, p. 23–32. doi: 10.1016/j.aca.2020.04.027. Full text in Research Archive
• Hansen, Frederik; Jaghl, Mohamad Fadi; Øiestad, Elisabeth Leere; Jensen, Henrik; Pedersen-Bjergaard, Stig & Huang, Chuixiu (2020). Impact of ion balance in electromembrane extraction. Analytica Chimica Acta. ISSN 0003-2670. 1124, p. 129–136. doi: 10.1016/j.aca.2020.05.039. Full text in Research Archive
• Hansen, Frederik & Pedersen-Bjergaard, Stig (2019). Emerging extraction strategies in analytical chemistry. Analytical Chemistry. ISSN 0003-2700. 92(1), p. 2–15. doi: 10.1021/acs.analchem.9b04677. Full text in Research Archive

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• Tuner, Marius Bull; Kogler, Stian; Hansen, Frederik Andre; Pedersen-Bjergaard, Stig; Røberg-Larsen, Hanne & Wilson, Steven Ray Haakon (2023). Exploring electromembrane extraction and LC-MS for studying liver organoid metabolomics. Full text in Research Archive
• Tuner, Marius Bull; Kogler, Stian; Hansen, Frederik Andre; Pedersen-Bjergaard, Stig; Røberg-Larsen, Hanne & Wilson, Steven Ray Haakon (2022). Exploring electromembrane extraction for sampling the organoid metabolome. Full text in Research Archive
• Skottvoll, Frøydis Sved; Harrison, Sean; Hansen, Frederik; Pedersen-Bjergaard, Stig; Sullivan, Gareth & Bogen, Inger Lise [Show all 8 contributors for this article] (2020). Liver mini-organ analyses using liquid chromatography mass spectrometry. Full text in Research Archive Show summary

  Organoids (self-organized multi-cellular tissue derived from e.g. patient-derived stem cells) are emerging in vitro 3D representations of human organ functionality, predicted to have a game-changing impact on drug discovery, disease modeling and personalized medicine [1]. Integrating the organoids in a microfluidic chip (i.e. organoids-on-a-chip) would allow for controllable perfusion of nutrients and manipulation of the organoids by the addition of e.g. drugs [2]. Coupling the organoid-on-a-chip online to liquid chromatography mass spectrometry (LC-MS) would enable real-time measurements of the biochemical processes that occur on-chip. However, limited sample sizes, complex matrices and the presence of low abundant analytes also create analytical challenges [3]. Moreover, the organoids are still at the developmental stage, thus fundamental questions on the functionality of the developed organoids remain to be answered. Liver organoid functionality was assessed by protein characterization of adult liver tissue and liver organoids using nanoLC-MS/MS. The proteins identified from the liver organoids covered 70-84% (1003 proteins) of the liver protein expression from adult liver tissue derived from five patients. The proteins that were shared between the adult liver tissue and liver organoids could also relate to central liver pathways (e.g. amino acid biosynthesis and glycolysis). The sample preparation technique electromembrane extraction (i.e. electrophoretic separation across an oil membrane, EME) has shown to have on-chip analytical potential [4] and was thus used in the study of heroin drug metabolism in liver organoids. Multi-well EME (100 µL solutions) allowed for simple and repeatable monitoring of heroin metabolism kinetics without interference from cell medium components (e.g. albumin). The EME extracts were also compatible with different analytical approaches (LC-MS, nanoLC-MS, capillary electrophoresis-UV). Hence, the hyphenation of EME with nanoLC-MS would be a natural next step. References 1. Method of the Year 2017: Organoids. Nature Methods. Vol. 15 (2018) 1-1. 2. Park, S.E., A. Georgescu, and D. Huh, Organoids-on-a-chip. Science. Vol. 364 (2019) 960. 3. Lin, A., F. Sved Skottvoll, S. Rayner, S. Pedersen-Bjergaard, G. Sullivan, S. Krauss, S. Ray Wilson, and S. Harrison, 3D cell culture models and organ-on-a-chip: Meet separation science and mass spectrometry. ELECTROPHORESIS. Vol. 0 (2019). 4. Hansen, F.A., D. Sticker, J.P. Kutter, N.J. Petersen, and S. Pedersen-Bjergaard, Nanoliter-Scale Electromembrane Extraction and Enrichment in a Microfluidic Chip. Analytical Chemistry. Vol. 90 (2018) 9322-9329.

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