Elsa Lundanes

• Olsen, Christine; Wang, Chencheng; Aizenshtadt, Aleksandra; Abadpour, Shadab; Lundanes, Elsa & Skottvoll, Frøydis Sved [Vis alle 11 forfattere av denne artikkelen] (2023). Simultaneous LC–MS determination of glucose regulatory peptides secreted by stem cell–derived islet organoids. Electrophoresis. ISSN 0173-0835. 44(21-22), s. 1682–1697. doi: 10.1002/elps.202300095. Fulltekst i vitenarkiv Vis sammendrag

  Abstract For studying stem cell–derived islet organoids (SC‐islets) in an organ‐on‐chip (OoC) platform, we have developed a reversed‐phase liquid chromatography–tandem mass spectrometry (RPLC–MS/MS) method allowing for simultaneous determination of insulin, somatostatin‐14, and glucagon, with improved matrix robustness compared to earlier methodology. Combining phenyl/hexyl‐C18 separations using 2.1 mm inner diameter LC columns and triple quadrupole mass spectrometry, identification and quantification were secured with negligible variance in retention time and quantifier/qualifier ratios, negligible levels of carryover (<2%), and sufficient precision (±10% RSD) and accuracy (±15% relative error) with and without use of an internal standard. The obtained lower limits of quantification were 0.2 µg/L for human insulin, 0.1 µg/L for somatostatin‐14, and 0.05 µg/L for glucagon. The here‐developed RPLC–MS/MS method showed that the SC‐islets have an insulin response dependent on glucose concentration, and the SC‐islets produce and release somatostatin‐14 and glucagon. The RPLC–MS/MS method for these peptide hormones was compatible with an unfiltered offline sample collection from SC‐islets cultivated on a pumpless, recirculating OoC (rOoC) platform. The SC‐islets background secretion of insulin was not significantly different on the rOoC device compared to a standard cell culture well‐plate. Taken together, RPLC–MS/MS method is well suited for multi‐hormone measurements of SC‐islets on an OoC platform.

• Olsen, Christine; Wiborg, Elisa; Lundanes, Elsa; Abadpour, Shadab; Scholz, Hanne & Wilson, Steven Ray Haakon (2022). On-line reduction of insulin disulfide bonds with photoinduced radical reactions, upstream to nano liquid chromatography-mass spectrometry. Separation Science Plus (SSC plus). 5(6), s. 220–227. doi: 10.1002/sscp.202200022. Fulltekst i vitenarkiv
• Olsen, Christine; Wang, Chencheng; Abadpour, Shadab; Lundanes, Elsa; Hansen, Audun Skau & Skottvoll, Frøydis Sved [Vis alle 8 forfattere av denne artikkelen] (2022). Determination of insulin secretion from stem cell-derived islet organoids with liquid chromatography-tandem mass spectrometry. Journal of chromatography. B. ISSN 1570-0232. 1215. doi: 10.1016/j.jchromb.2022.123577. Fulltekst i vitenarkiv Vis sammendrag

  Organoids are laboratory-grown 3D organ models, mimicking human organs for e.g. drug development and personalized therapy. Islet organoids (typically 100–200 µm), which can be grown from the patient́s own cells, are emerging as prototypes for transplantation-based therapy of diabetes. Selective methods for quantifying insulin production from islet organoids are needed, but sensitivity and carry-over have been major bottlenecks in previous efforts. We have developed a reverse phase liquid chromatography-tandem mass spectrometry (RPLC-MS/MS) method for studying the insulin secretion of islet organoids. In contrast to our previous attempts using nano-scale LC columns, conventional 2.1 mm inner diameter LC column (combined with triple quadrupole mass spectrometry) was well suited for sensitive and selective measurements of insulin secreted from islet organoids with low microliter-scale samples. Insulin is highly prone to carry-over, so standard tubings and injector parts were replaced with shielded fused silica connectors. As samples were expected to be very limited, an extended Box-Behnken experimental design for the MS settings was conducted to maximize performance. The finale method has excellent sensitivity, accuracy and precision (limit of detection: ≤0.2 pg/µL, relative error: ≤±10%, relative standard deviation: <10%), and was well suited for measuring 20 µL amounts of Krebs buffer containing insulin secreted from islet organoids.

• Berg, Henriette Engen; Halldórsso, Skarphéðinn; Bakketeig, Eris Aas; Thiede, Bernd; Sandberg, Cecilie & Lundanes, Elsa [Vis alle 8 forfattere av denne artikkelen] (2022). Micro‐pillar array columns (µPAC): An efficient tool for comparing tissue and cultured cells of glioblastoma. Journal of chromatography open. ISSN 2772-3917. 2. doi: 10.1016/j.jcoa.2022.100047. Fulltekst i vitenarkiv
• Kogler, Stian; Aizenshtadt, Aleksandra; Harrison, Sean; Skottvoll, Frøydis Sved; Berg, Henriette Engen & Abadpour, Shadab [Vis alle 14 forfattere av denne artikkelen] (2022). "Organ-in-a-Column" Coupled On-line with Liquid Chromatography-Mass Spectrometry. Analytical Chemistry. ISSN 0003-2700. 94(50), s. 17677–17684. doi: 10.1021/acs.analchem.2c04530. Fulltekst i vitenarkiv Vis sammendrag

  Organoids, i.e., laboratory-grown organ models developed from stem cells, are emerging tools for studying organ physiology, disease modeling, and drug development. On-line analysis of organoids with mass spectrometry would provide analytical versatility and automation. To achieve these features with robust hardware, we have loaded liquid chromatography column housings with induced pluripotent stem cell (iPSC) derived liver organoids and coupled the “organ-in-a-column” units on-line with liquid chromatography-mass spectrometry (LC-MS). Liver organoids were coloaded with glass beads to achieve an even distribution of organoids throughout the column while preventing clogging. The liver organoids were interrogated “on column” with heroin, followed by on-line monitoring of the drug’s phase 1 metabolism. Enzymatic metabolism of heroin produced in the “organ-in-a-column” units was detected and monitored using a triple quadrupole MS instrument, serving as a proof-of-concept for on-line coupling of liver organoids and mass spectrometry. Taken together, the technology allows direct integration of liver organoids with LC-MS, allowing selective and automated tracking of drug metabolism over time.

• Skottvoll, Frøydis Sved; Aizenshtadt, Aleksandra; Hansen, Frederik Andre; Martinez, Mikel Amirola; Andersen, Jannike Mørch & Bogen, Inger Lise [Vis alle 11 forfattere av denne artikkelen] (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. Fulltekst i vitenarkiv Vis sammendrag

  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.

• Zawadzka, Malgorzata Elzbieta; Racz, Beatrix; Ambrosini, Dario; Görbitz, Carl Henrik; Morth, Jens Preben & Wilkins, Alistair L. [Vis alle 12 forfattere av denne artikkelen] (2021). Searching for a UV-filter in the eyes of high-flying birds. Scientific Reports. 11(1). doi: 10.1038/s41598-020-79533-2. Fulltekst i vitenarkiv Vis sammendrag

  Abstract The eye lens is a unique organ as no cells can be replaced throughout life. This makes it decisive that the lens is protected against damaging UV-radiation. An ultraviolet (UV)-absorbing compound of unknown identity is present in the aqueous humor of geese (wild and domestic) and other birds flying at high altitudes. A goose aqueous humor extract, that was believed to contain the UV protective compound which was designated as “compound X”, was fractionated and examined using a variety of spectroscopic techniques including LC–MS and high field one- and two dimensional-NMR methods. A series of compounds were identified but none of them appeared to be the UV protective “compound X”. It may be that the level of the UV protective compound in goose aqueous humor is much less than the compounds identified in our investigation, or it may have been degraded by the isolation and chromatographic purification protocols used in our investigations.

• Skottvoll, Frøydis Sved; Hansen, Frederik; Harrison, Sean; Boger, Ida Caroline Sneis; Mrsa, Ago & Restan, Magnus Saed [Vis alle 14 forfattere av denne artikkelen] (2021). Electromembrane extraction and mass spectrometry for liver organoid drug metabolism studies. Analytical Chemistry. ISSN 0003-2700. 93, s. 3576–3585. doi: 10.1021/acs.analchem.0c05082. Fulltekst i vitenarkiv Vis sammendrag

  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.

• Røberg-Larsen, Hanne; Lundanes, Elsa; Nyman, Tuula Anneli; Berven, Frode Steingrimsen & Wilson, Steven Ray Haakon (2021). Liquid chromatography, a key tool for the advancement of single-cell omics analysis. Analytica Chimica Acta. ISSN 0003-2670. 1178:338551, s. 1–9. doi: 10.1016/j.aca.2021.338551. Fulltekst i vitenarkiv
• Røberg-Larsen, Hanne; Wilson, Steven Ray Haakon & Lundanes, Elsa (2021). Recent advances in on-line upfront devices for sensitive bioanalytical nano LC methods. TrAC. Trends in analytical chemistry. ISSN 0165-9936. 136, s. 1–10. doi: 10.1016/j.trac.2021.116190. Fulltekst i vitenarkiv
• Ambaw, Yohannes Abere; Dahl, Sandra Rinne; Chen, Yan; Greibrokk, Tyge; Lundanes, Elsa & Lazraq, Issam [Vis alle 11 forfattere av denne artikkelen] (2021). Tailored polymer-based selective extraction of lipid mediators from biological samples. Metabolites. 11(8), s. 1–14. doi: 10.3390/metabo11080539. Fulltekst i vitenarkiv Vis sammendrag

  Lipid mediators, small molecules involved in regulating inflammation and its resolution, are a class of lipids of wide interest as their levels in blood and tissues may be used to monitor health and disease states or the effect of new treatments. These molecules are present at low levels in biological samples, and an enrichment step is often needed for their detection. We describe a rapid and selective method that uses new low-cost molecularly imprinted (MIP) and non-imprinted (NIP) polymeric sorbents for the extraction of lipid mediators from plasma and tissue samples. The extraction process was carried out in solid-phase extraction (SPE) cartridges, manually packed with the sorbents. After extraction, lipid mediators were quantified by liquid chromatography–tandem mass spectrometry (LC–MSMS). Various parameters affecting the extraction efficiency were evaluated to achieve optimal recovery and to reduce non-specific interactions. Preliminary tests showed that MIPs, designed using the prostaglandin biosynthetic precursor arachidonic acid, could effectively enrich prostaglandins and structurally related molecules. However, for other lipid mediators, MIP and NIP displayed comparable recoveries. Under optimized conditions, the recoveries of synthetic standards ranged from 62% to 100%. This new extraction method was applied to the determination of the lipid mediators concentration in human plasma and mouse tissues and compared to other methods based on commercially available cartridges. In general, the methods showed comparable performances. In terms of structural specificity, our newly synthesized materials accomplished better retention of prostaglandins (PGs), hydroxydocosahexaenoic acid (HDoHE), HEPE, hydroxyeicosatetraenoic acids (HETE), hydroxyeicosatrienoic acid (HETrE), and polyunsaturated fatty acid (PUFA) compounds, while the commercially available Strata-X showed a higher recovery for dihydroxyeicosatetraenoic acid (diHETrEs). In summary, our results suggest that this new material can be successfully implemented for the extraction of lipid mediators from biological samples.

• Tsjokajev, Ahmad; Røberg-Larsen, Hanne; Wilson, Steven Ray Haakon; Dyve, Lingelem Anne Berit; Skotland, Tore & Sandvig, Kirsten [Vis alle 7 forfattere av denne artikkelen] (2020). Mass spectrometry-based measurements of cyclic adenosine monophosphate in cells, simplified using reversed phase liquid chromatography with a polar characterized stationary phase. Journal of chromatography. B. ISSN 1570-0232. 1160, s. 1–6. doi: 10.1016/j.jchromb.2020.122384. Fulltekst i vitenarkiv Vis sammendrag

  3′, 5′ – Cyclic adenosine monophosphate (cAMP) is a ubiquitous second messenger that is involved in many cellular functions and biological processes. In several cell types, cholera toxin will increase the level of cAMP, which mediates toxic effects on cells. In this context, we have developed a fast and simple method based on extraction with 5% trichloroacetic acid (TCA) and quantitation with liquid chromatography-mass tandem spectrometry (LC-MS/MS) for measuring cAMP in cells. A main feature of the LC-MS method was employing a reversed phase C18 column (2.1 mm × 50 mm, 1.6 µm particles) compatible with a 100% aqueous mobile phase, providing retention of the highly polar analyte. Isocratic separations allowed for fast subsequent injections. Negative mode electrospray ionization detection was performed with a triple quadrupole (QqQ)MS. cAMP was extracted from cell samples (~106 cells per well) and spiked with a labelled internal standard, using 200 µL of 5% TCA. The extraction solvent was fully compatible for direct injection onto the reversed phase column. After 10 min incubation, the supernatant was removed, and 10 µL of the supernatant was directly analysed by LC-MS. The method was characterized by the simplicity of the extraction, and the speed (3 min retention time of cAMP), sensitivity (250 pg/mL detection limit), and selectivity (separation from interferences e.g. isomeric compounds) of the LC-MS method, and could be used for quantitation of cAMP in the range 1–500 ng/mL cell extract.

• Kogler, Stian; Harrison, Sean; Aizenshtadt, Aleksandra; Skottvoll, Frøydis Sved; Abadpour, Shadab & Krauss, Stefan [Vis alle 10 forfattere av denne artikkelen] (2020). “Organoid-in-a-column” coupled on-line with liquid chromatography-mass spectrometry. BioRxiv. ISSN 0362-4331. doi: 10.1101/2020.09.08.282756. Fulltekst i vitenarkiv
• Hvinden, Ingvild Comfort; Berg, Henriette Engen; Sachse, Daniel; Skaga, Erlend; Skottvoll, Frøydis Sved & Lundanes, Elsa [Vis alle 10 forfattere av denne artikkelen] (2019). Nuclear Magnetic Resonance Spectroscopy to Identify Metabolite Biomarkers of Nonresponsiveness to Targeted Therapy in Glioblastoma Tumor Stem Cells. Journal of Proteome Research. ISSN 1535-3893. 18(5), s. 2012–2020. doi: 10.1021/acs.jproteome.8b00801. Fulltekst i vitenarkiv
• Wilson, Steven Ray Haakon; Olsen, Christine & Lundanes, Elsa (2019). Nano liquid chromatography columns. The Analyst. ISSN 0003-2654. 144(24), s. 7090–7104. doi: 10.1039/c9an01473j. Fulltekst i vitenarkiv Vis sammendrag

  Nano liquid chromatography (nanoLC), with columns having an inner diameter (ID) of ≤100 μm, can provide enhanced sensitivity and enable analysis of limited samples. NanoLC has become an established tool in omics research, and is gaining ground in other applications as well. There are several variants and formats of nanoLC columns, including packed columns, monoliths, open tubular columns, and the pillar array format. Most applications are done with packed columns, while e.g. the monolith and open tubular columns are still less established as routine tools. The pillar array format is a new variant with excellent resolution and low backpressure, and has recently been commercialized and used for bio-applications. In this minireview, we summarize and discuss recent research on nanoLC column development and uses, focusing on literature between 2016 and medio 2019.

• Olsen, Christine; Skottvoll, Frøydis Sved; Brandtzaeg, Ole Kristian; Schnaars, Christian; Rongved, Pål & Lundanes, Elsa [Vis alle 7 forfattere av denne artikkelen] (2019). Investigating Monoliths (Vinyl Azlactone-co-Ethylene Dimethacrylate) as a Support for Enzymes and Drugs, for Proteomics and Drug-Target Studies. Frontiers in Chemistry. 7. doi: 10.3389/fchem.2019.00835. Fulltekst i vitenarkiv
• Solheim, Stian; Hutchinson, Samantha A; Lundanes, Elsa; Wilson, Steven Ray Haakon; Thorne, James L & Røberg-Larsen, Hanne (2019). Fast liquid chromatography-mass spectrometry reveals side chain oxysterol heterogeneity in breast cancer tumour samples. Journal of Steroid Biochemistry and Molecular Biology. ISSN 0960-0760. 192. doi: 10.1016/j.jsbmb.2019.02.004. Fulltekst i vitenarkiv Vis sammendrag

  Oxysterols can contribute to proliferation of breast cancer through activation of the Estrogen Receptors, and to metastasis through activation of the Liver X Receptors. Endogenous levels of both esterified and free sidechain-hydroxylated oxysterols were examined in breast cancer tumours from Estrogen Receptor positive and negative breast tumours, using a novel fast liquid chromatography tandem mass spectrometry method. Multiple aliquots of five milligram samples of 22 tumours were analysed for oxysterol content to assess intra- and inter-tumour variation. Derivatization was performed with Girard T reagent (with and without alkaline hydrolysis) and sample clean-up was performed using a robust automatic on-line column switching system (“AFFL”). Oxysterols were separated isocratically on a 2.1 mm inner diameter column packed with ACE SuperPhenylHexyl core shell particles using a mobile phase consisting of 0.1% formic acid in H2O/methanol/acetonitrile (57/10/33, v/v/v) followed by a wash out step (0.1% formic acid in methanol/acetonitrile, 50/50, v/v). The total analysis time, including sample clean-up and column reconditioning, was 8 min (80% time reduction compared to other on-line systems). Analysis revealed large intra-tumour variations of sidechain oxysterols, resulting in no significant differences in endogenous oxysterols levels between Estrogen Receptor positive and Estrogen Receptor negative breast cancers. However, a correlation between esterified and free 27-hydroxycholesterol was observed. The same correlation was not observed for 24S-hydroxycholesterol or 25-hydroxycholesterol. The oxysterol heterogeneity of tumour tissue is a critical factor when assessing the role of these lipids in cancer.

• Poothong, Somrutai; Papadopoulou, Eleni Zoumpoulia; Lundanes, Elsa; Sanchez, Juan Antonio Padilla; Thomsen, Cathrine & Haug, Line Småstuen (2019). Dried blood spots for reliable biomonitoring of poly- and perfluoroalkylsubstances (PFASs). Science of the Total Environment. ISSN 0048-9697. 655, s. 1420–1426. doi: 10.1016/j.scitotenv.2018.11.214. Fulltekst i vitenarkiv
• Skottvoll, Frøydis Sved; Berg, Henriette Engen; Bjørseth, Kamilla; Lund, Kaja; Roos, Norbert & Bekhradnia, Sara [Vis alle 13 forfattere av denne artikkelen] (2018). Ultracentrifugation versus kit exosome isolation: nanoLC-MS and other tools reveal similar performance biomarkers, but also contaminations. Future science OA. 5(1). doi: 10.4155/fsoa-2018-0088. Fulltekst i vitenarkiv Vis sammendrag

  Aim: For isolation of exosomes, differential ultracentrifugation and an isolation kit from a major vendor were compared. Materials & methods: ‘Case study’ exosomes isolated from patient-derived cells from glioblastoma multiforme and a breast cancer cell line were analyzed. Results: Transmission electron microscopy, dynamic light scattering, western blotting, and so forth, revealed comparable performance. Potential protein biomarkers for both diseases were also identified in the isolates using nanoLC–MS. Western blotting and nanoLC–MS also revealed negative exosome markers regarding both isolation approaches. Conclusion: The two isolation methods had an overall similar performance, but we hesitate to use the term ‘exosome isolation’ as impurities may be present with both isolation methods. NanoLC–MS can detect disease biomarkers in exosomes and is useful for critical assessment of exosome enrichment procedures.

• Levernæs, Maren Christin Stillesby; Brandtzaeg, Ole Kristian; Amundsen, Sunniva Furre; Reubsaet, Leon; Lundanes, Elsa & Halvorsen, Trine Grønhaug [Vis alle 7 forfattere av denne artikkelen] (2018). Selective Fishing for Peptides with Antibody Immobilized Acrylate Monoliths, Coupled Online with NanoLC-MS. Analytical Chemistry. ISSN 0003-2700. 90, s. 13860–13860. doi: 10.1021/acs.analchem.8b00935. Fulltekst i vitenarkiv
• Reinholds, I; Pugajeva, Iveta; Zacs, Dz; Lundanes, Elsa; Rusko, Janis & Perkons, Ingus [Vis alle 7 forfattere av denne artikkelen] (2017). Determination of acidic non-steroidal anti-inflammatory drugs in aquatic samples by liquid chromatography-triple quadrupole mass spectrometry combined with carbon nanotubes-based solid-phase extraction. Environmental Monitoring and Assessment. ISSN 0167-6369. 189(11). doi: 10.1007/s10661-017-6304-9. Fulltekst i vitenarkiv
• Røberg-Larsen, Hanne; Abele, Silvija; Demir, Deniz; Dzabijeva, Diana; Amundsen, Sunniva Furre & Wilson, Steven Ray Haakon [Vis alle 8 forfattere av denne artikkelen] (2017). Rugged Large Volume Injection for Sensitive Capillary LC-MS Environmental Monitoring. Frontiers in Chemistry. AUG(2017). doi: 10.3389/fchem.2017.00062. Fulltekst i vitenarkiv Vis sammendrag

  A rugged and high throughput capillary column (cLC) LC-MS switching platform using large volume injection and on-line automatic filtration and filter back-flush (AFFL) solid phase extraction (SPE) for analysis of environmental water samples with minimal sample preparation is presented. Although narrow columns and on-line sample preparation are used in the platform, high ruggedness is achieved e.g., injection of 100 non-filtrated water samples did not result in a pressure rise/clogging of the SPE/capillary columns (inner diameter 300 μm). In addition, satisfactory retention time stability and chromatographic resolution were also features of the system. The potential of the platform for environmental water samples was demonstrated with various pharmaceutical products, which had detection limits (LOD) in the 0.05–12.5 ng/L range. Between-day and within-day repeatability of selected analytes were <20% RSD.

• Silva, Meire Ribeiro da; Brandtzaeg, Ole Kristian; Vehus, Tore; Lancas, Fernando Mauro; Wilson, Steven Ray Haakon & Lundanes, Elsa (2017). An automated and self-cleaning nano liquid chromatography mass spectrometry platform featuring an open tubular multi-hole crystal fiber solid phase extraction column and an open tubular separation column. Journal of Chromatography A. ISSN 0021-9673. 1518, s. 104–110. doi: 10.1016/j.chroma.2017.08.071. Fulltekst i vitenarkiv
• Skjærvø, Øystein; Brandtzaeg, Ole Kristian; Lausund, Kristian Blindheim; Pabst, Oliver; Martinsen, Ørjan Grøttem & Lundanes, Elsa [Vis alle 7 forfattere av denne artikkelen] (2017). Exploring bioimpendance instrumentation for the characterization of open tubular liquid chromatography columns. Journal of Chromatography A. ISSN 0021-9673. 1534, s. 195–200. doi: 10.1016/j.chroma.2017.12.060. Fulltekst i vitenarkiv
• Pugajeva, Iveta; Rusko, Janis; Perkons, Ingus; Lundanes, Elsa & Bartkevics, Vadims (2017). Determination of pharmaceutical residues in wastewater using high performance liquid chromatography coupled to quadrupole-Orbitrap mass spectrometry. Journal of Pharmaceutical and Biomedical Analysis. ISSN 0731-7085. 133, s. 64–74. doi: 10.1016/j.jpba.2016.11.008. Fulltekst i vitenarkiv
• Poothong, Somrutai; Lundanes, Elsa; Thomsen, Cathrine & Haug, Line Småstuen (2017). High throughput online solid phase extraction-ultra high performance liquid chromatography-tandem mass spectrometry method for polyfluoroalkyl phosphate esters, perfluoroalkyl phosphonates, and other perfluoroalkyl substances in human serum, plasma, and whole blood. Analytica Chimica Acta. ISSN 0003-2670. 957, s. 10–19. doi: 10.1016/j.aca.2016.12.043. Fulltekst i vitenarkiv Vis sammendrag

  A rapid, sensitive and reliable method was developed for the determination of a broad range of poly- and perfluoroalkyl substances (PFASs) in various blood matrices (serum, plasma, and whole blood), and uses only 50 μL of sample material. The method consists of a rapid protein precipitation by methanol followed by high throughput online solid phase extraction (SPE), ultra-high performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS), and negative electrospray ionization detection. The method was developed for simultaneous determination of twenty-five PFASs, including polyfluoroalkyl phosphate esters (PAPs; 6:2, 8:2, 6:2/6:2, and 8:2/8:2), perfluoroalkyl phosphonates (PFPAs; C6, C8, and C10), perfluoroalkyl sulfonates (PFSAs; C4, C6, C7, C8, and C10), perfluoroalkyl carboxylates (PFCAs; C5C14), and perfluoroalkyl sulfonamides (FOSAs; C8, N-methyl, and N-ethyl). High linearity of matrix-matched calibration standards (correlation coefficients, R = 0.99–0.999) were obtained in the range of 0.006–45 ng mL−1 blood. Excellent sensitivity was achieved with method detection limits (MDLs) between 0.0018 and 0.09 ng mL−1, depending on the compound and matrix. The method was validated for serum, plasma, and whole blood (n = 5 + 5) at six levels in the range 0.0180–30 ng mL−1. The accuracy (n = 5) was on average 102± 12%. The intermediate precision (n = 10) ranged from 2 to 40% with an average between-batch of analyses difference of 10± 10%. Two human serum samples from a former interlaboratory comparison were analyzed and the differences between the applied method and the consensus values were below ≤22% (n = 5). The method was also successfully applied to samples of human plasma and whole blood with coefficients of variation in the range 0.8–15.2% (n = 5).

• Røberg-Larsen, Hanne; Lund, Kaja; Sæterdal, Kristina Erikstad; Solheim, Stian; Vehus, Tore & Solberg, Nina [Vis alle 9 forfattere av denne artikkelen] (2017). Mass spectrometric detection of 27-hydroxycholesterol in breast cancer exosomes. Journal of Steroid Biochemistry and Molecular Biology. ISSN 0960-0760. 169, s. 22–28. doi: 10.1016/j.jsbmb.2016.02.006. Fulltekst i vitenarkiv
• Gregus, Michael; Røberg-Larsen, Hanne; Lundanes, Elsa; Foret, Frantisek; Kubán, Petr & Wilson, Steven Ray Haakon (2017). Non-aqueous capillary electrophoretic separation of cholesterol and 25-hydroxycholesterol after derivatization with Girard P reagent. Chemistry and Physics of Lipids. ISSN 0009-3084. 207, s. 87–91. doi: 10.1016/j.chemphyslip.2017.05.014. Fulltekst i vitenarkiv
• Gundersen, Cathrine Brecke; Zhu, Liang; Lindahl, Sofia; Wang, Shiyu; Wilson, Steven Ray Haakon & Lundanes, Elsa (2017). LC-MS/MS Method for Simultaneous Determination of Monoethanol- and Dimethylnitramine in Aqueous Soil Extracts. Chromatographia. ISSN 0009-5893. 80(9), s. 1475–1481. doi: 10.1007/s10337-017-3355-6. Fulltekst i vitenarkiv

Se alle arbeider i NVA

• Kogler, Stian; Aizenshtadt, Aleksandra; Harrison, Sean Philip; Sullivan, Gareth John; Lundanes, Elsa & Bogen, Inger Lise [Vis alle 9 forfattere av denne artikkelen] (2023). Organ-in-a-column – automated on-line incubation, sampling, and LC-MS analysis for measurement of drug metabolites. Fulltekst i vitenarkiv
• Skottvoll, Frøydis Sved; Aizenshtadt, Aleksandra; Harrison, Sean; Krauss, Stefan Johannes Karl; Sullivan, Gareth & Bogen, Inger Lise [Vis alle 9 forfattere av denne artikkelen] (2022). Organ-in-a-column - On-line incubation and LC-MS-analysis for drug metabolism studies. Fulltekst i vitenarkiv
• Nordli, Henriette; Røberg-Larsen, Hanne; Lundanes, Elsa & Wilson, Steven Ray Haakon (2021). Determination of sterols in liver organoids using liquid chromatography-mass spectrometry (LC-MS). Fulltekst i vitenarkiv
• Moe, Harald; Lundanes, Elsa; Wilson, Steven Ray Haakon; Lund, Reidar & Røberg-Larsen, Hanne (2021). Releasing anitmicrobial peptides through liposomal nanocarriers to fight multidrug-resistant bacteria. Fulltekst i vitenarkiv
• Kogler, Stian; Wilson, Steven Ray Haakon; Røberg-Larsen, Hanne; Krauss, Stefan Johannes Karl; Lundanes, Elsa & Skottvoll, Frøydis Sved (2021). Organ-in-a-column. Fulltekst i vitenarkiv
• Wiborg, Elisa; Hermansen, Astrid; Olsen, Christine; Wilson, Steven Ray Haakon & Lundanes, Elsa (2021). Silica-based narrow inner diameter open tubular liquid chromatography columns for use in proteomics. Fulltekst i vitenarkiv Vis sammendrag

  In the world of proteomics, which is the study of proteins and peptides, the samples are usually small and/or with low analyte concentration. To analyse this type of samples it is beneficial to use liquid chromatography (LC) columns with narrow inner diameter (ID) coupled with an electrospray mass spectrometer (ESI-MS). The use of narrow columns in an LC-ESI-MS system makes it possible to achieve high sensitivity [1, 2], due to less radial dilution of the analytes [3]. Such narrow columns can be porous layer open tubular (PLOT) columns, either organic polymer based [2] or silica-based [1, 4, 5]. A silica-based PLOT can be made by sol-gel synthesis [5]. In the present study, both 10 µm and 5 µm ID silica-based PLOT columns have been prepared according to Hara et. al. [4, 5] In this preparation method, a solution of tetramethyl orthosilicate (TMOS), polyethylenglycol (PEG), urea and 0.01 M acetic acid is prepared and filled into pre-treated silica capillaries, with 10 µm or 5 µm ID, either by a pressure bomb system or with a pump. The polymerization into a porous layer structure (silica skeleton) takes place in a water bath at 25 ℃, and a following heat treatment at 80 ℃ makes the mesopores in the silica skeleton. After the porous layer has been formed, functionalization with C18 is preformed to obtain a reversed phase PLOT column. The columns made are used in a nano LC ESI-MS system for ultrasensitive analysis.

• Olsen, Christine; Lundanes, Elsa & Wilson, Steven Ray Haakon (2021). Disulfide bond reduction upfront liquid chromatography mass spectrometry for protein analysis. Fulltekst i vitenarkiv Vis sammendrag

  Microfluidic devices containing human cells or organoids (3D multicellular tissue derived in vitro from human pluripotent stem cells) constitute an exponentially growing technology for online analysis of cell responses and human organ functionality [1, 2]. Human pancreas-on-a-chip (PoC) is a relatively new technology, intended for the study of the endocrine tissue, the islets of Langerhans, which are responsible for regulation of the blood glucose levels [3]. These pancreatic islets consist of five major cell types, where the β-cells are responsible for the production of insulin [4]. For patients with type 1 diabetes, where the β-cells are destructed and unable to produce insulin, a common treatment is β-cell replacement therapy. Such therapy includes donor transplantation of either the entire pancreas or isolated islets. Proteins, which are important compounds regarding quality of donor tissue or cells and the state of the isolated and purified islets may be studied in real-time analysis [3]. Normally, liquid chromatography with electrospray ionization mass spectrometry (LC-ESI-MS) is employed to measure the proteins in complex cell samples. Prior to analysis, the proteins must be digested to peptides, as peptides are far more easily detected and identified with MS. Immobilized enzyme reactors are becoming more common for inclusion of online protein digestion, however, a remaining bottleneck to achieving complete online analysis is that time-consuming reduction and alkylation of the disulfide bonds need to be executed prior to analysis. Therefore, a platform consisting of a photochemical system upfront an LC-MS system has been set up for achieving online reduction of the disulfide bonds [5]. Insulin has been used as a model protein as it consists of two relatively short chains, A and B, connected by two disulfide bonds. Additionally, there is an internal disulfide bond in chain A. The protein was dissolved in a solution of water and isopropanol (1+1, v/v) added 1% of acetone as the photoinitiator. As insulin was irradiated by UV-light at 254 nm, the MS revealed that intact insulin (present as m/z 1452 and m/z 1162) was broken into two chains (A chain with m/z 1191 and B chain with m/z 858), due to the reduction of the disulfide bonds. The set-up may be used for fast measurements of small proteins with post-translational modifications without protein digestion.

• Bakketeig, Eris Aas; Olsen, Christine; Berg, Henriette Engen; Nyman, Tuula Anneli; Thiede, Bernd & Lundanes, Elsa [Vis alle 8 forfattere av denne artikkelen] (2021). Optimization of concatenation of fractions in comprehensive two dimensional liquid chromatography bottom-up proteomics. Fulltekst i vitenarkiv Vis sammendrag

  Two dimensional liquid chromatography (2D-LC) has long been a commonly used technique in bottom-up proteomics, and is as relevant today as ever [1, 2]. To maximize the number of protein identifications, analytical chemists have strived to obtain 2D-LC systems with a high degree of orthogonality, which is needed to reach the required high peak capacities. A system utilizing reversed-phase chromatography (RP) in both column dimensions with a large difference in pH has proven to yield a higher peak capacity for peptides, even with a lower degree of orthogonality compared to other combinations [3]. The present study is based on Reubsaet, et al. [4]. The study is a comprehensive offline RPRP 2D-LC system with high pH (>9) in the first dimension and low pH (3) in the second dimension. The method’s purpose is to maximise protein identifications from one trypsinated protein sample. To achieve maximum identifications, Reubsaet incorporated the concept of concatenation of fractions. In practice, concatenation is applied through collecting the column output from the first dimension in eight numbered vials, where the 1st, 9th, 17th, … fractions are collected in the first vial, the 2nd, 10th, 18th, … are collected in the second, etc. [4]. In the present study, the fractionation scheme is examined by changing the total number of vials in which samples are concatenated, the number of fractions in each vial, and the collection time for each fraction. As the method demands high precision and is very timeconsuming, automatization is practically essential. Automatization is achieved with the spider fractionation scheme, proposed and shown by Kulak, et al.[5]. Here, a 10-port injector connects the first dimension column to each sample vial, and switches between them automatically, reducing the strain on the operator drastically, while removing human errors. The optimization of the concatenation will lead to higher numbers of identified proteins in proteomics.

• Flatval, Jonas; Berg, Henriette Engen; Lillerud, Karl Petter; Olsbye, Unni; Nielsen, Claus Jørgen & Wilson, Steven Ray Haakon [Vis alle 7 forfattere av denne artikkelen] (2020). Retention of nitramines on UiO-66 ZrMOF materials and silica-based reversed phase materials. Fulltekst i vitenarkiv
• Røberg-Larsen, Hanne; Websdale, Alex; Solheim, Stian Kjønnås; Sande, Maria Therese; Hutchinson, Samantha A & Lundanes, Elsa [Vis alle 8 forfattere av denne artikkelen] (2020). Fast LC-MS for determination of oxysterols in breast cancer tumours. Fulltekst i vitenarkiv
• Skottvoll, Frøydis Sved; Harrison, Sean; Hansen, Frederik; Pedersen-Bjergaard, Stig; Sullivan, Gareth & Bogen, Inger Lise [Vis alle 8 forfattere av denne artikkelen] (2020). Liver mini-organ analyses using liquid chromatography mass spectrometry. Fulltekst i vitenarkiv Vis sammendrag

  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.

• Olsen, Christine; Scholz, Hanne; Wilson, Steven Ray Haakon & Lundanes, Elsa (2020). Towards online reduction and protein digestion of secreted proteins from pancreatic islets of Langerhans related to diabetes mellitus type I. Fulltekst i vitenarkiv Vis sammendrag

  What is diabetes mellitus type 1, and how can liquid chromatography assist in the search for a cure? The human pancreas is involved in control of metabolism and energy consumption by both an exocrine and an endocrine function. In the exocrine tissue, a multitude of digestive enzymes are produced and secreted into the small intestine to assist in breaking down proteins, fats and carbohydrates. The endocrine tissue, islets of Langerhans (5% of the total pancreatic mass) are responsible for regulation of the blood glucose levels. The vital metabolic hormone insulin is synthesized in one of five major cell types in the islets, called β-cells [1]. However, there are two types of chronic diseases related to insulin deficiency; an autoimmune disease causes selectively destruction of the β-cells in the islets or the body cannot effectively use the produced insulin, called diabetes mellitus type 1 and type 2, respectively. Diabetes mellitus type 1 or type 2 have been diagnosed in 250 000 Norwegians (approx. 4.7% of the Norwegian population), where 28 000 have been diagnosed with type 1 diabetes mellitus (0.5%) [2]. The most common treatment is exogenous insulin therapy. When insulin was discovered in 1920s the mortality of diabetes type 1 was reduced from a guaranteed fatal diagnosis to a manageable condition with a normal lifespan within the 1950s. However, secondary complications due to diabetic nephropathy, retinopathy and neuropathy led to a renewed interest in β-cell replacement therapy. Progress in β-cell replacement with either pancreas or islet transplantation has led to impressive 1- and 5-year insulin independence rates of approximately 85% and 50 %, respectively [3]. To assist in further progress in β-cell replacement therapy, liquid chromatography with mass spectrometry detection (LC-MS) of the secreted proteins from the pancreas and the islets will aid in the understanding of the treatment of diabetes mellitus type 1. In this work, we seek to enable fully online determination of proteins (in a bottom-up approach) by combining reduction and protein digestion upstream LC-MS (Figure 1). Online protein digestion has been successfully executed with immobilized enzyme reactors (IMERs) in different formats [4-6], but most of the samples have been reduced and alkylated prior to online digestion. We hope to enable real-time determination of proteins with disulfide bonds (such as insulin) by incorporation of a photochemical system for rapid reduction of disulfide bonds [7].

• Hermansen, Astrid; Skottvoll, Frøydis Sved; Olsen, Christine; Desmet, Gert; Wilson, Steven Ray Haakon & Lundanes, Elsa (2020). Preparation of porous layer open tubular columns for sensitive proteomics. Fulltekst i vitenarkiv Vis sammendrag

  The newly introduces 3D liver cellular cluster model, known as liver organoids, is a promising tool to probe disease and human biology. The composition and function of liver organoids is not yet fully understood, calling for sensitive proteomic analysis due to limited sample size and availability. The use of narrow inner diameter (ID) liquid chromatography (LC) separation columns is beneficial for increased sensitivity. A relevant column format to explore is the porous layer open tubular (PLOT), which with narrow ID and low backpressure permit the use of longer columns for higher separation power. The goal of this work is to prepare silica-based C18 PLOT columns with narrow ID (10 and 5 µm) suitable for peptide and intact protein separation and find the optimal conditions for proteomics of liver organoids and their media with mass spectrometry detection. Initial analysis of HeLa cell digest using a PLOT column (C18, 10 µm ID x 56 cm) made by Hara et al., has been performed. HeLa cell digest has also been analyzed using a commercially available packed column (C18, 75 µm x 15 cm) for comparison. Two approaches for the preparation of silica-based C18 PLOT columns with narrow ID were explored, based on methods established by Hara et al. and Vehus et al., respectively. We experienced challenges with regards to clogging of the fused silica capillaries during the formation of the porous silica layer when using the method established by Vehus et al., thus only the method of Hara et al. was further explored. Scanning electron micrographs indicate that a porous layer was present with both preparation methods.

• Mrša, Ago; Skottvoll, Frøydis Sved; Harrison, Sean; Lundanes, Elsa; Sullivan, Gareth & Wilson, Steven Ray Haakon (2020). Miniaturized liquid chromatography systems for small molecule analysis of organoids. Fulltekst i vitenarkiv Vis sammendrag

  Liver organoids are three-dimensional tissue models (typically in the millimetre scale or below) derived from human-induced pluripotent stem cells [1]. They are intended to e.g. represent the physiological functions of a patient’s liver. The liver is the main metabolizing organ in the human body [2]; thus, an important application of liver organoids is to map drug metabolism in vitro, as liver organoids can be more patient-specific compared to traditional biomaterials. The aim of the study is to develop miniaturized liquid chromatography (LC) systems capable of analyzing very small samples, e.g secretion of liver organoids. Emphasis is placed on the determination of small molecules, i.e metabolites. Due to the small size of organoids, there are significant challenges in performing sensitive analysis of the organoid itself and it's minute secretion. Hence a nanoLC system has been explored, capable of sensitive analysis of model substance heroin and its well-known metabolites morphine and 6-Monoacetylmorphine (6-MAM). Using a 50 µm x 5 cm C18 pre-column and a 50 µm x 12 cm C18 analytical column, coupled up with a triple quadrupole mass spectrometer (TQMS) with electrospray ionization, a limit of detection of 0.7 fg was achieved for heroin. Further method development was done using commercially packed C18 columns typically used in proteomics. Separation and detection of heroin, morphine and 6-MaM were achieved in less than 8 minutes and with detection limits in the sub fg area. The method was successfully transferred to liver organoid samples that had been incubated with 1.5 µM heroin for 1 hour, cleaned using electromembrane extraction and further diluted 1:1000. Deuterated internal standards were implemented into the method for future work with organoid samples and the next step is to map the metabolism of liver organoids incubated within various time frames. Taken together, nanoLC-MS proves to be a viable tool for analysis of small molecules and the achievd results are promising for further work with organoid samples.

• Schüller, Maria; Skottvoll, Frøydis Sved; Lundanes, Elsa & Wilson, Steven Ray Haakon (2020). Bottom-up proteomics of biomarkers to investigate drug-induced hepatotoxicity in human liver organoids as part of preclinical development. Fulltekst i vitenarkiv Vis sammendrag

  Drug-induced liver injury (DILI) is an ongoing issue in the development of drugs. It is a major cause of drug withdrawal during development, as well as being the leading cause for post-authorization withdrawal. DILI is commonly discovered in later stages of clinical trials and poses a risk to trial subjects involved in the study. European Medicines Agency (EMEA) provide guidelines for non-clinical assessment of DILI, however regulatory authorities do not require this assessment for the approval of the Investigational New Drug (IND) application for testing in human subjects. This is possibly due to lack of research and validity of current DILI practices which, at worst, could hinder promising therapeutics to enter the market. In recent years, the use of 3D liver cellular clusters, commonly known as liver organoids, has alongside other advances, emerged as a promising tool to supplement the nonclinical assessment of drug candidates. Liver organoids closely resemble human liver physiology and emulate basic liver function. In combination with well-established clinical protein biomarkers, they might revolutionize the drug industry and strengthen patient safety. Organoids and organoid medium are complex in nature and limited in size and availability. Implementing nanoLC for analytical method development for liver organoid analysis will achieve improved sensitivity and therefore capture the detection of minor fluctuations in protein biomarkers upon drug exposure. The goal of this work is to develop a targeted proteomics method for the absolute quantification of alanine aminotransferase (ALT) in organoid medium based on unique peptide MRM transitions with nanoLC-tandem MS. Pilot experiments using human serum albumin (HSA) have been conducted to create a basic outline of experiments which are reasonable to conduct when approaching the main goal. HSA is a readily available and relatively affordable pure protein source, well suited for preliminary testing. The optimal linear gradient for eluting HSA peptides within 48 min was 1% - 15% mobile phase component B (0.1% formic acid and 10% water in acetonitrile). A positive correlation between SSRCalc values (hydrophobicity factor) and elution time has been established and can be implemented in developing the optimal gradient for peptides of ALT. The most abundant peptides have been optimized with respect to collision energy (CE) using a scheduled MRM approach. Regression analysis was performed to establish a relationship between CE and precursor m/z, which can be implemented in ALT analysis. HSA was spiked into HeLa cell lysate to investigate matrix suppression effects, as well as to investigate the effects of gradient length on ion suppression. Planned experiments will revolve around selecting signature peptides for ALT, quantify ALT with suitable quantification methods and measure ALT levels in organoid medium upon exposure to known hepatotoxins.

• Boger, Ida Caroline Sneis; Nerem, Elisabeth; Skottvoll, Frøydis Sved; Harrison, Sean; Sullivan, Gareth & Lundanes, Elsa [Vis alle 8 forfattere av denne artikkelen] (2020). Determination of liver organoid-induced drug metabolites using liquid chromatography-mass spectrometry (LC-MS)”. Fulltekst i vitenarkiv Vis sammendrag

  Liver organoids are three-dimensional tissue models typically derived from adult and human induced pluripotent stem cells. They are intended to e.g. represent the physiological functions of a patient´s liver [1]. The liver is the main metabolizing organ in the human body [2]; thus, an important application of liver organoids is to map drug metabolism in vitro as liver organoids can be more patient-specific compared to traditional biomaterials, e.g. human liver microsomes (HLM). The aim of this study is to explore liver organoid drug metabolism in vitro using liquid chromatography-mass spectrometry (LC-MS). To establish a standardized conventional approach for metabolism studies for later comparison with organoids, heroin metabolism studies in HLMs and S9 fraction were carried out. Quantification of model substance heroin and its well-known metabolites, 6-Monoacetylmorphine (6-MAM) and morphine, was done using UHPLC-MS/MS. The heroin metabolism method was miniaturized to make it transferable to the small organoid samples. This was done by decreasing the microsome amount as much as possible while still observing heroin metabolism (HLM amount decreased from 0.2 mg to 0.01 mg; Heroin concentration was decreased from 10 µM to 0.1 µM). This miniaturized experiment was also transferred to S9 fraction Preliminary experiments with organoids showed that the organoids metabolize heroin to 6-MAM and morphine, as well as phase II biotransformation metabolites morphine-3-glucuronide and morphine-6-glucuronide. These results show that the studied organoids have metabolizing properties. Taken together, LC-MS can be a valuable tool for studying metabolism properties of organoids.

• Andersen, Kari Anne; Flatval, Jonas; Mrša, Ago; Berg, Henriette Engen; Chavan, Sachin Maruti & Olsbye, Unni [Vis alle 8 forfattere av denne artikkelen] (2020). Metal organic framework material UiO-66 as a stationary phase in liquid chromatography. Fulltekst i vitenarkiv Vis sammendrag

  Metal organic framework materials (MOFs) are interesting candidates for applications in multiple disciplines, where chromatography is one of the fields where possible applications might exist. The porous structure and large surface area are key features that make MOFs potential candidates for future stationary phases. In the present study, the MOF UiO-66 has been investigated as a stationary phase in a nano-liquid chromatography (LC) system. UiO-66 is composed of zirconium oxide ion nodes and benzene dicarboxylate (BDC) linkers which form a face-centred cubic structure with octahedral pores (opening 6 Å, cavity 11 Å) and tetrahedral pores (opening 6 Å, cavity 7.5 Å) in a 1+2 ratio. Figure adapted from Cliffe et al., Nature communications volume 5 (2014)1. Columns with a 100 µm inner diameter have been packed in-house and tested in a simple LC-UV system for several test substances. The UiO-66 particles pack rapidly and yield columns with low back pressure. We observe reversed phase selectivity, but our experimental data also suggest other interaction mechanisms, phosphate group adsorption to zirconium oxide nodes among them.

• Berg, Henriette Engen; Halldórsson, Skarphéðinn; Thiede, Bernd; Vik-Mo, Einar O.; Lundanes, Elsa & Wilson, Steven Ray Haakon (2020). Micro pillar array columns for comprehensive proteomics. Fulltekst i vitenarkiv Vis sammendrag

  Pillar array nano liquid chromatography columns can potentially replace packed nano liquid chromatography (nLC) columns for analyses of complex samples due to high chromatographic performance and low backpressure. In our study, pillar array columns are used for the detection of potential biomarkers in Glioblastoma Multiforme (GBM), which is the most aggressive form of brain cancer in adults. Pillar array columns consist of several micropillars etched into silicon wafers using lithographic micromachining. The interest of these columns in nLC format has increased as an option to packed nLC columns as the chromatographic efficiency could outperform nLC columns packed with sub-2 µm particles while maintaining the sensitivity [1]. In contrast to packed columns, where eddy dispersion is the main source of band broadening due to irregular flow paths between the particles, the perfectly ordered pillars in pillar array columns eliminate the eddy dispersion to a minimum. The low backpressure of pillar array columns compared to that of packed columns also permits the use of exceptionally long columns, increasing the chromatographic performance further. Using pillar array columns, plate numbers over 1 million and reduced plate heights (h) even <1 µm have been achieved [2-4]. High chromatographic performance is especially important in comprehensive proteomics. Pre-fractionation (using e.g. gel electrophoresis) is often added in proteomic protocols to increase the throughput [5]. However, performing pre-fractionation can be time-consuming in addition to the highly increased instrument time required. Recently, a micro-Chip pillar array nLC column (µPACTM) compatible with standard nLC instrumentation has been commercialized (Figure 1) [6]. Our question was; could the use of µPAC nLC columns reduce the need for pre-fractionation in comprehensive proteomics of GBM biopsies?

• Berg, Henriette Engen; Hvinden, Ingvild Comfort; Sachse, Daniel; Skaga, Erlend; Lundanes, Elsa & Sandberg, Cecilie [Vis alle 9 forfattere av denne artikkelen] (2020). Observation of brain cancer cells’ molecular responses to chemotherapy. Fulltekst i vitenarkiv
• Hermansen, Astrid; Schüller, Maria; Skottvoll, Frøydis Sved; Hara, T; Desmet, Gert & Lundanes, Elsa [Vis alle 7 forfattere av denne artikkelen] (2019). Protein Analysis of Liver Oganoids with Minaturized LC-MS: Nano and PLOT Format. Fulltekst i vitenarkiv Vis sammendrag

  In recent years, 3D liver cellular clusters, commonly known as liver organoids, have emerged as promising tools to replace 2D cell cultures and animal testing in the development of drugs and the understanding of diseases. The composition and function of liver organoids is yet not fully understood, calling for proteomic analysis. Organoids and the medium in which they secrete into are complex in nature and limited in size and availability; therefore, the use of narrow inner diameter separation columns is beneficial to achieve better sensitivity. Conventional packed columns can have limitations regarding sensitivity and separation power. Consequently, the employment of other column formats can be explored. One such format is the porous layer open tubular (PLOT) column for liquid chromatography (LC), which have low backpressures and smaller inner diameters, permitting longer columns for expanding separation power. The goal of this work is to find the optimal conditions for bottom-up proteomics of liver organoids and their media, comparing reversed-phase LC with commercially packed columns and PLOT columns with 1 / 2 mass spectrometry (MS) detection. Initial analysis of liver organoids with commercially packed columns and conventional run times revealed liver traits as phase 1 and phase 2 metabolism enzymes (aldehyde dehydrogenase mitochondrial, cytochrome P450 1B1 and glutathione S-transferase Mu 3, UDP-glucuronosyltransferase 1, catechol Omethyltransferase), proteins related to nitrogen and glutamate- (glutamate dehydrogenase) and amino acid metabolism (aspartate aminotransferase). Regarding 75 µm inner diameter/15 cm long packed columns, there was a significant difference (α = 0.05) in protein ID numbers when comparing 2 h with 4 and 8 h linear gradients from 2.7-15.3% ACN. The 4 h gradient did not significantly differ from an 8 h gradient, implying that the protein ID numbers capacity was reached at 4 h. We have now proceeded to explore the possibilities of PLOT LC-MS for organoid proteomics, using a 10 µm inner diameter/56 cm long column.

• Mrša, Ago; Boger, Ida Caroline Sneis; Nerem, Elisabeth; Skottvoll, Frøydis Sved; Harrison, Sean & Lundanes, Elsa [Vis alle 9 forfattere av denne artikkelen] (2019). Studying organoid drug metabolism using UHPLC/nanoLC-MS. Fulltekst i vitenarkiv Vis sammendrag

  Liver organoids are three-dimensional tissue models typically derived from adult and human induced pluripotent stem cells. They are intended to e.g. represent the physiological functions of a patient´s liver [1]. The liver is the main metabolizing organ in the human body [2]; thus, an important application of liver organoids is to map drug metabolism in vitro as liver organoids can be more patient-specific compared to traditional biomaterials, e.g. human liver microsomes (HLM). The aim of this study is to explore liver organoid drug metabolism in vitro using liquid chromatography-mass spectrometry (LC-MS). To establish a standardized conventional approach for metabolism studies for later comparison with organoids, heroin metabolism studies in HLMs were carried out. Quantification of model substance heroin and its well-known metabolites, 6-Monoacetylmorphine (6-MAM) and morphine, was done using UHPLC-MS/MS. The heroin metabolism method was miniaturized to make it transferable to the small organoid samples. This was done by decreasing the microsome amount as much as possible while still observing heroin metabolism (HLM amount decreased from 0.2 mg to 0.01 mg; Heroin concentration was decreased from 10 µM to 0.1 µM). Preliminary experiments with organoids showed that the organoids metabolize heroin to 6-MAM and morphine, as well as phase II biotransformation metabolites morphine-3-glucuronide and morphine-6-glucuronide. These results show that the studied organoids have metabolizing properties. We are also exploring sensitive nano-LC-MS (traditionally used for proteomics) for studying organoid drug metabolism. Using a 50 µm (I.D) x 5 cm C18 pre-column and a 50 µm (I.D) x 12 cm C18 analytical column, coupled up with a triple quadrupole MS (QMS) using electrospray ionization (ESI), a LoD of 0.7 fg heroin was achieved. The LoD of the nano-LC-MS method gives a good foundation for future work with detection of trace liver-organoid induced drug metabolites. Taken together, LC-MS can be a valuable tool for studying metabolism properties of organoids. [1] Eisenstein, M., Organoids: the body builders, Nature Methods, (2018) 19. [2] Brandon, E. F., Raap, C. D., Meijerman, I., Beijnen, J. H., Schellens, J. H., An update on in vitro test methods in human hepatic drug biotransformation research: pros and cons, Toxicology and applied pharmacology, (2003) 233-246

• Wilson, Steven Ray Haakon; Berg, Henriette Engen; Røberg-Larsen, Hanne & Lundanes, Elsa (2019). Hyphenations of one-dimensional capillary liquid chromatography with mass spectrometry: state-of-the-art applications. I Tranchida, Peter & Mondello, Luigi (Red.), Hyphenations of Capillary Chromatography with Mass Spectrometry. Elsevier. ISSN 9780128096383. s. 319–358. Fulltekst i vitenarkiv
• Hassine, Esma Ben; Berg, Henriette Engen; Brandtzaeg, Ole Kristian; Vehus, Tore; Wilson, Steven Ray Haakon & Lundanes, Elsa (2018). Capillary poly(styrene-co-octadecene-co-divinylbenzene) monolithic columns. Fulltekst i vitenarkiv
• Brandtzaeg, Ole Kristian; Levernæs, Maren Christin Stillesby; Halvorsen, Trine Grønhaug; Reubsaet, Leon; Røen, Bent Tore & Vehus, Tore [Vis alle 10 forfattere av denne artikkelen] (2018). Online sample processing systems for targeted nano liquid chromatography mass spectrometry bioanalysis. Fulltekst i vitenarkiv
• Røberg-Larsen, Hanne; Sande, Maria Therese; Hutchinson, Samantha A; Solheim, Stian Kjønnås; Lundanes, Elsa & Thorne, James L [Vis alle 7 forfattere av denne artikkelen] (2018). Rapid determination of oxysterols in breast cancer tumors. Fulltekst i vitenarkiv
• Skjærvø, Øystein; Pilarova, Veronika; Khalikova, Maria; Reubsaet, Leon; Halvorsen, Trine Grønhaug & Gjelstad, Astrid [Vis alle 10 forfattere av denne artikkelen] (2018). Those Who Can, Teach: Pharma Stars. The Analytical Scientist. ISSN 2051-4077. 1118, s. 40–45. Fulltekst i vitenarkiv
• Skjærvø, Øystein; Pilarova, Veronika; Khalikova, Maria; Reubsaet, Leon; Halvorsen, Trine Grønhaug & Gjelstad, Astrid [Vis alle 10 forfattere av denne artikkelen] (2018). Those Who Can, Teach: Pharma Stars. The Analytical Scientist. ISSN 2051-4077. 1118, s. 40–45. Fulltekst i vitenarkiv
• Berg, Henriette Sjånes; Bjørseth, Kamilla; Sæterdal, Kristina Erikstad; Smetop, Tone; Skottvoll, Frøydis Sved & Vehus, Tore [Vis alle 8 forfattere av denne artikkelen] (2018). Evaluation of isolation methods of glioma exosomes for biomarker discovery. Fulltekst i vitenarkiv Vis sammendrag

  Gliomas are the most common form of brain cancer, where the sub-group glioblastoma multiforme (GBM) is the most aggressive and the most common in adults [1]. For diagnostic, prognostic, and treatment monitoring, fast and reliable analytical methods for detection of glioblastoma tumors are essential. Non-invasive monitoring of circulating biomarkers in blood (liquid biopsy) is a desirable possibility [2]. Nano LC-MS can provide the high sensitivity, selectivity and low false positive rate needed for the proteomic analysis of exosomes, which are extracellular vesicles released from the tumor and into the bloodstream. We have optimized the packing of 50 µm capillaries (2.6 µm C18 core-shell particles) with peak capacities comparable to similar commercial nano-LC columns (75 µm ID) for peptide separations of minute samples (e.g. exosomes). These columns have previously been successfully used for the targeted detection of CYP27a (a potential breast cancer biomarker) in the MDA-MB-231 cell line. In the present study, these in-house packed columns have been applied for proteins in exosomes isolated from GBM cell culture medium. The aim of this study was to compare isolation methods of GMB exosomes for biomarker discovery and study proteins related to GBM in exosomes. Ultracentrifugation (“golden standard” in exosome isolation) was compared to a Total Exosome Isolation Reagent from Invitrogen (Thermo Fisher Scientific) regarding sample volume, total protein amount, purity, and the presence of exosome markers and GBM biomarker candidates. References 1. Molina, J.R., Y. Hayashi, C. Stephens, and M.M. Georgescu, Invasive Glioblastoma Cells Acquire Stemness and Increased Akt Activation. Neoplasia. 12 (2010) 453-U37. 2. Best, M.G., N. Sol, S. Zijl, J.C. Reijneveld, P. Wesseling, and T. Wurdinger, Liquid biopsies in patients with diffuse glioma. Acta Neuropathologica. 129 (2015) 849-865.

• Gundersen, Cathrine Brecke; Vogt, Rolf David; Lundanes, Elsa; Andersen, Tom & Breedveld, Gijs D. (2018). Environmental Fate and Determination of Nitramines Formed from Amine-based CO2 Capture. Fulltekst i vitenarkiv
• Sätra, Jenny Marie Skytte Af; Nielsen, Claus Jørgen; Bavel, Albert Van; Lundanes, Elsa & Wilson, Steven Ray Haakon (2018). Developing procedures for trace analysis of small amines in water, using LC-MS and GC-MS. Fulltekst i vitenarkiv
• Hvinden, Ingvild Comfort; Rise, Frode; Berg, Henriette Engen; Lundanes, Elsa & Wilson, Steven Ray Haakon (2018). Approaches to untargeted metabolomics of glioblastoma: NMR and MS. Fulltekst i vitenarkiv
• Olsen, Christine; Brandtzaeg, Ole Kristian; Lundanes, Elsa & Wilson, Steven Ray Haakon (2018). On-line trapping of proteins with monolith-bound drugs, as a tool in drug target discovery. Fulltekst i vitenarkiv
• Brandtzaeg, Ole Kristian; Røen, Bent Tore; Lundanes, Elsa & Wilson, Steven Ray Haakon (2018). Qualitative detection of ricin using a multichannel enzyme reactor coupled online with nano liquid chromatography with mass spectrometry detection. Fulltekst i vitenarkiv
• Røberg-Larsen, Hanne; Hutchinson, Sam Alex; Solheim, Stian; Wilson, Steven Ray Haakon; Lundanes, Elsa & Thorne, James L (2018). Oxysterols in breast cancer. Fulltekst i vitenarkiv
• Racz, Beatrix; Zawadzka, Malgorzata Elzbieta; Sätra, Jenny Marie Skytte Af; Ringvold, Amund; Rise, Frode & Lundanes, Elsa [Vis alle 7 forfattere av denne artikkelen] (2018). Identification of “compound X” in goose aqueous humour. Fulltekst i vitenarkiv
• Kular, Ramneet Kaur; Wilson, Steven Ray Haakon; Røberg-Larsen, Hanne & Lundanes, Elsa (2018). Towards ultrafast liquid chromatography of 3’-phosphoadenosine-5’-phosphosulfate using a short hydrophilic interaction chromatography column. Fulltekst i vitenarkiv
• Berg, Henriette Engen; Skottvoll, Frøydis Sved; Bjørseth, Kamilla; Sæterdal, Kristina Erikstad; Brandtzaeg, Ole Kristian & Vehus, Tore [Vis alle 11 forfattere av denne artikkelen] (2018). Development of nanoLC column technology for proteomic studies. Fulltekst i vitenarkiv Vis sammendrag

  Background NanoLC has emerged as a major technique in proteomics due to improved sensitivity, which enables analysis of small sample volumes and low concentrations. In our group (Bioanalytical chemistry, BACH) we focus on the development of column technology, including porous layer open tubular (PLOT) columns and self-packing of columns for proteomic applications. Methods The PLOT columns were used for both on-line digestion (enzyme reactors) and separations (10 µm ID, PS-DVB with ODS). Enzyme reactors (20 µm ID and multi-PLOT columns (~8 µm ID)) were immobilized with trypsin and Lys-C for protein digestion. We also compared self-packed columns of 50 µm ID (2.6 µm C18 core-shell particles) to a commercial column (75 µm ID). The columns were used for detection of CYP27a1 in MDA-MB-231 (breast cancer) cells, and biomarker candidates in MDA-MB-231 and glioblastoma multiforme (brain cancer) exosomes. For exosome isolation (cell culture medium), ultracentrifugation (“golden standard”) was compared to a Total Exosome Isolation Reagent from Thermo Fisher regarding protein yield and purity. Results Our PLOT columns were successfully employed for proteins, metabolites and peptides with attogram detection limits and peak capacities around 250 [1]. The enzyme reactors provided sufficient digestion in ~5 minutes compared to overnight digestions (common in off-line protocols) [2]. Furthermore, a quantitative method of the neurotoxin ricin was developed using the multi-PLOT to reduce manual sample handling [3] . The self-packed columns obtained peak capacities comparable to the commercial nanoLC column for peptide separations [4]. The self-packed columns were used for the targeted detection of CYP27A1 (potential breast cancer biomarker) in MDA-MB-231 cells. In the recent exosome study, exosomes were present and biomarker candidates were identified. However, the characterization techniques are in our hands not satisfactory. Conclusions We have successfully developed sensitive nanoLC column formats for proteomic applications.

• Solheim, Stian; Røberg-Larsen, Hanne; Lundanes, Elsa & Wilson, Steven Ray Haakon (2017). Optimizing selectivity and sensitivity for oxysterol measurements using liquid chromatography-mass spectrometry (LC-MS). Fulltekst i vitenarkiv
• Skottvoll, Frøydis Sved; Lund, Kaja; Røberg-Larsen, Hanne; Berg, Henriette Sjånes; Thiede, Bernd & Lundanes, Elsa [Vis alle 7 forfattere av denne artikkelen] (2017). Diagnostic potential of exosomes: The purification issue. Fulltekst i vitenarkiv
• Demir, Deniz; Abele, Silvija; Røberg-Larsen, Hanne; Wilson, Steven Ray Haakon & Lundanes, Elsa (2017). Development of an ultrasensitive method for multiresidue screening of pharmaceutical products in environmental water samples. Fulltekst i vitenarkiv
• Reinholds, Ingars; Pugajeva, Iveta; Perkons, Ingus; Lundanes, Elsa; Rusko, Janis & Ķizāne, Gunta K. [Vis alle 11 forfattere av denne artikkelen] (2017). Erratum to: Decomposition of multi-class pharmaceutical residues in wastewater by exposure to ionising radiation (International Journal of Environmental Science and Technology, (2017), 10.1007/s13762-017-1290-6). International Journal of Environmental Science and Technology. ISSN 1735-1472. 14(8), s. 1821–1821. doi: 10.1007/s13762-017-1325-z. Fulltekst i vitenarkiv
• Røberg-Larsen, Hanne; Skottvoll, Frøydis Sved; Solheim, Stian; Bjørseth, Kamilla; Thorne, James L & Lundanes, Elsa [Vis alle 7 forfattere av denne artikkelen] (2017). Oxysterols in cancer. Fulltekst i vitenarkiv
• Berg, Henriette Engen; Wilson, Steven Ray Haakon; Lundanes, Elsa; Thiede, Bernd & Vik-Mo, Einar Osland (2022). Exploring miniaturized chromatography formats and sample preparation methods for shotgun proteomics in limited biological samples. Department of Chemistry, Faculty of Mathematics and Natural Sciences, University of Oslo. Fulltekst i vitenarkiv Vis sammendrag

  A significant challenge in medical diagnostics is the development of simple but efficient tools for the detection/quantification of several biomarkers simultaneously using non-invasive sampling techniques. In this regard, the analysis of proteins (proteomics) is essential for understanding cellular processes and biomarker discovery. However, proteins vary greatly in terms of concentration levels and chemical properties in biological materials. Further, low sample sizes of modern biological models (e.g., patient-derived cell cultures, exosomes, and organoids) remain a big analytical challenge. The present work has focused on the brain cancer glioblastoma, which is in great need of increased knowledge and non-invasive sampling techniques. In addition, human organoids, which could act as a future in vitro model for disease modeling and personalized medicine, have been investigated. We have used high-resolution mass spectrometry for protein identification, exploring a selection of miniaturized liquid chromatography formats (for separation) and sample preparation techniques. By implementing these techniques, we have been able to study exosomes, 2D/3D cell cultures, and organoids, identifying over 6300 proteins in a single run using less than 5 µg of protein. The work has provided important insight into the possibilities and challenges of several novel models. It represents a development toward deeper proteomic profiling focusing on maintaining a high protein yield and time efficiency.

• Bakketeig, Eris Aas; Wilson, Steven Ray Haakon; Lundanes, Elsa; Berg, Henriette Engen; Olsen, Christine & Reubsaet, Leon [Vis alle 8 forfattere av denne artikkelen] (2021). The setup and optimization of a complete global proteomics two-dimensional liquid chromatography system, using Spider Fractionation. Universitetet i Oslo. Fulltekst i vitenarkiv
• Wiborg, Elisa & Lundanes, Elsa (2021). Preparation and characterization of silica-based porous layer open tubular columns. Universitetet i Oslo. Fulltekst i vitenarkiv
• Erlandsson, Tonje Monica; Wilson, Steven Ray Haakon; Skottvold, Frøydis Sved & Lundanes, Elsa (2021). Investigation of CYP activity in liver organoids by establishing a liquid chromatography-mass spectrometry method for measuring drug metabolism. Universitetet i Oslo. Fulltekst i vitenarkiv
• Nordli, Henriette; Wilson, Steven Ray Haakon; Lundanes, Elsa & Røberg-Larsen, Hanne (2021). Towards method miniaturization for determination of sterols in liver organoids: an investigation of nano liquid chromatography-mass spectrometry (nLC-MS). Universitetet i Oslo. Fulltekst i vitenarkiv Vis sammendrag

  Obesity and inactivity can lead to a cluster of conditions called metabolic syndrome, which again can lead to non-alcoholic fatty liver disease (NAFLD). NAFLD consists of several stages, where the liver accumulates fats, potentially leading to liver fibrosis and cirrhosis. Cirrhosis is considered a terminal state and requires liver transplantation to save the life of the patient. The only diagnostic tool for NAFLD today is liver biopsy. Hence, less invasive diagnostic tools are currently lacking, like biomarkers, which can be advantageous for early diagnosis. Liver organoids are a promising research model used for disease modeling and recapitulate the human NAFLD in vivo environment. Oxysterols are endogenous cholesterol metabolites seen in some of the NAFLD pathways and are proposed as potential biomarker candidates. However, oxysterols are low abundant in biological samples and need a highly sensitive LC-MS method for quantification. Miniaturizing the inner diameter (ID) of the LC column is one way to enhance the method sensitivity. Additionally, a trap column can further enhance method sensitivity by allowing large volume injections, and an automatic filtration and filter backflushing system (AFFL) enhances the robustness of the platform. Few studies have investigated the use of miniaturized LC columns in oxysterol determination. The aim of this study was to develop a highly sensitive nLC-MS method with core-shell particles, combined with an on-line sample clean-up in the form of a trap column and an AFFL-system, towards quantification of the oxysterols 22R-, 24S-, 25-, and 27-OHC in healthy and NAFLD-induced liver organoids. An automated nLC-MS method combined with an on-line sample clean-up was successfully assembled. 2.5 m core-shell Super Phenyl-Hexyl (SPH) and 5 m phenyl-hexyl (PH) particles were chosen and successfully packed in fused silica capillaries (75 m ID) as the analytical- and trap column, respectively. Significant band broadening was a problem, and a Butterfly Portfolio column heater was included in the platform to enhance column efficiency. It was discovered that the 5 m PH particles in the trap column were partly responsible for the band broadening observed due to incompatibility with the 2.5 m SPH particles. Hence, the 2.5 m SPH particles were found to be suitable in the trap column instead. Relatively efficient nLC columns were packed; however, they proved to be challenging to reproduce and lacked robustness due to short lifetimes, making the oxysterol isomer separation challenging. Core-shell particles continue to prove to be challenging to pack in narrow bore capillaries. Therefore, customizing the packing procedure for these particles should be performed further by, e.g., adding a frit on both ends of the column and optimizing the slurry concentration.

• Moe, Harald Røsand; Wilson, Steven Ray Haakon; Lundanes, Elsa; Røberg-Larsen, Hanne & Lund, Reidar (2021). Establishing a liquid chromatography-mass spectrometry method for the quantification of colistin A, a last line of defense against antibiotic resistance. Universitetet i Oslo. Fulltekst i vitenarkiv Vis sammendrag

  Antibiotic resistance has become an increasingly pressing issue in recent times. With a dwindling discovery rate of new antibacterials, gram-negative bacteria threaten to return humanity to the pre-antibiotic era where mere paper cuts could result in fatal outcomes. The antimicrobial peptide colistin is our present-day last line of defense against gram-negative infections. Nevertheless, despite the recent surge in use and research on colistin, research on its biological effects and chemical behavior remains somewhat scarce. Colistin was initially abandoned due to numerous reports of neuro- and nephrotoxicity. Slow leakage from colistin-loaded liposomes may reduce this toxicity significantly. In this work, an LC-MS method for separating and quantifying colistin and polymyxin B1 was developed to measure colistin leakage from loaded liposomes over time. Isocratic elution provided adequate separation of polymyxin E1 and the internal standard, polymyxin B1, and measurements were done using a triple-quadrupole MS. Widely unreported and uninvestigated adsorption and degradation mechanisms of colistin were observed and addressed by adding a 100 mM ammonium formate buffer at pH 3 to solutions of colistin. The LC-MS method provides a reliable and accurate approach for measuring colistin at lower concentrations (&lt; 10 µg/mL) in ammonium formate buffered solutions, and the future challenges and potential approaches for further research upon colistin are discussed. The LC-MS method described serves as a solid platform for further research on colistin, colistin toxicity negation, and more may be built upon.

• Flatval, Jonas; Lundanes, Elsa; Wilson, Steven Ray Haakon; Olsbye, Unni; Nielsen, Claus Jørgen & Berg, Henriette Engen (2020). Retention of nitramines on silica-based reversed phase materials and UiO-66 MOF materials. Universitetet i Oslo. Fulltekst i vitenarkiv Vis sammendrag

  Capturing and storing of CO2 can reduce the emissions of CO2 in the atmosphere. However, such facilities may emit amines that can be transformed into the potentially carcinogenic nitramines dimethylnitramine (DMA-NO2), monoethanolnitramine (MEA-NO2) and monomethylnitramine (MMA-NO2) which may be released into the environment. Presently, methods for trace determination of these compounds are lacking. The goal of the study was to find a material that can give sufficient retention to provide an enrichment of the nitramines before liquid chromatography electrospray ionization-mass spectrometry determination. The retention of the nitramines was examined on various C18 materials and the metal-organic frameworks UiO-66 and UiO-66 1,4-benzenedicarboxylate-NH2 (UiO-66-NH2), which were packed in 100 µm inner diameter columns. Of the tested materials, UiO-66-NH2 showed the highest retention factors for the nitramines. UiO-66-NH2 with 0.1% formic acid adjusted to pH 7 as mobile phase gave the best compromise of retention factors for DMA-NO2 and MEA-NO2, as the intended liquid chromatography-mass spectrometry method was evaluated for DMA-NO2 and MEA-NO2 only. MMA-NO2 had the highest retention factor on UiO-66-NH2 with 0.05% dibutylamine in the mobile phase. In conclusion, the retention factors achieved were in general higher on the UiO-66 materials than on the C18 materials. However, the retention obtained is probably not large enough to give limit of quantification down to 4 ng/L, albeit, not tested, due to extraordinary circumstances.

• Schüller, Maria; Wilson, Steven Ray Haakon; Lundanes, Elsa & Skottvoll, Frøydis Sved (2020). A targeted proteomics approach for the investigation of drug-induced liver injury in hepatic organoids using nano liquid chromatography mass spectrometry. Universitetet i Oslo. Fulltekst i vitenarkiv Vis sammendrag

  Late-stage drug attrition and post-market withdrawals attributed to drug-induced liver injury (DILI) has been a prevailing concern for the pharmaceutical industry. The current gaps in preclinical safety assessment impede the termination of DILI drug candidates to earlier stages of drug development, with the consequence of possibly exposing patients to hepatotoxic agents. Recent advances in liver-emulating models, with the development of hepatic organoids, seem promising in filling these gaps. The purpose of this study was to look into opportunities for the advancement of the preclinical safety assessment of the hepatic system. This was approached by combining the liver-emulating power of hepatic organoids with the well-studied protein biomarker alanine aminotransferase isoform 1 (ALT1) to investigate their compatibility with nano liquid chromatography mass spectrometry (nanoLC-MS). A targeted proteomics approach for the absolute quantification of ALT1 was developed by a thorough assessment of signature peptide candidates and corresponding multiple reaction monitoring (MRM) transitions. NanoLC-MS platform optimization was performed to maximize detection sensitivity and reliability. From empirical nanoLC-MS platform optimization, the greatest improvement was achieved from collision energy optimization, with an increase of mean peptide peak area of 22%. ALT1 peptide assessment showed that the peptide LLVAGEGHTR with the MRM fragments y8+ and y7+ were best suited to infer the presence of ALT1 in absolute quantification. The finalized method was applied for the investigation of drug-induced liver injury in hepatic organoids, where acetaminophen was used as the model drug. This work has shown that ALT1 quantification with a nanoLC-MS platform is feasible and has great potential to support preclinical DILI detection. Further studies must be conducted to give reliable statements on the applicability of the method for drug-induced ALT1 release in hepatic organoids.

• Hermansen, Astrid; Skottvoll, Frøydis Sved; Wilson, Steven Ray Haakon & Lundanes, Elsa (2020). Development and implementation of open tubular columns for high-sensitivity liquid chromatography-mass spectrometry proteomics of liver organoids. Universitetet i Oslo. Fulltekst i vitenarkiv
• Andersen, Kari Anne; Lundanes, Elsa; Berg, Henriette Engen; Wilson, Steven Ray Haakon; Chavan, Sachin Maruti & Lillerud, Karl Petter [Vis alle 7 forfattere av denne artikkelen] (2020). UiO-66 metal-organic framework materials as stationary phases in liquid chromatography. Universitetet i Oslo. Fulltekst i vitenarkiv
• Boger, Ida Caroline Sneis; Wilson, Steven Ray Haakon; Skottvoll, Frøydis Sved; Lundanes, Elsa & Bogen, Inger Lise (2020). Determination of liver organoid- and spheroid induced drug metabolites using liquid chromatography-mass spectrometry. Universitetet i Oslo. Fulltekst i vitenarkiv Vis sammendrag

  The liver is the main metabolizing organ in the human body, and several in vitro models have been developed to resemble hepatic drug metabolism. However, commonly used in vitro models such as human liver microsomes (HLMs), S9 fraction, and hepatocytes lack the complexity of the corresponding in vivo tissue, which limits the in vivo resemblance. Liver organoids are three-dimensional tissue models typically derived from induced pluripotent stem cells (iPSCs) and are intended to recapitulate physiological functions of the human liver. Liver spheroids are similar 3D culture systems but lack the multicellularity that characterizes the organoids. Few studies have investigated drug metabolism in liver organoids- and spheroids. The aim of this study was, therefore, to explore liver organoid- and spheroid drug metabolism using liquid chromatography-mass spectrometry (LC-MS) as the liver organoids- and spheroids can be more representative to the in vivo situation. Additionally, an LC-MS method was to be established at the Department of Chemistry for drug metabolite detection. Drug incubated samples were analyzed using a validated LC-MS method at the Department of Forensic Sciences or by the LC-MS method established at the Department of Chemistry. Heroin was chosen as the model drug, and heroin metabolism studies were initially carried out in HLMs to establish a standardized conventional approach for later drug metabolism studies in organoids and spheroids. The concentrations of heroin and its phase I metabolites, 6-monoacetylmorphine (6-MAM) and morphine were measured over time (20 minutes). The heroin metabolism method was downscaled by decreasing the HLM concentration (from 2 mg/mL to 0.1 mg/mL) and the heroin concentration (from 1 μM to 0.1 μM). The downscaled method was applied to the S9 fraction and later executed with the addition of exogenous cofactors to look for the phase II metabolites morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G). With the addition of cofactors, M3G could be quantified in the S9 fraction. Preliminary heroin metabolism studies in iPSC derived liver organoids showed that the organoids metabolized heroin (1 μM) to 6-MAM and morphine. The phase II metabolites M3G and M6G were also identified but below the limit of quantification (LOQ). Both the phase I- and phase II metabolites were detected above LOQ in liver spheroid samples after incubation in 10 M heroin. To sum up, the detection of heroin drug metabolites using LC-MS showed that the liver organoids- and spheroids had heroin metabolizing properties. Additionally, LC-MS proved to be a valuable tool for detection of liver organoid induced drug metabolites.

• Hunnes, Linn Maria; Lundanes, Elsa; Lindberg, Diana & Afseth, Nils Kristian (2020). Classical biotechnological and analytical techniques in combination with FTIR spectroscopy: A study of protein hydrolysates from poultry residual raw materials. Universitetet i Oslo. Fulltekst i vitenarkiv
• Sande, Maria Therese; Wilson, Steven Ray Haakon; Lundanes, Elsa & Røberg-Larsen, Hanne (2019). Enzymatic hydrolysis with cholesterol esterase in the presence of surfactant improves efficiency for sample preparation of side-chain oxysterols in biosamples. Universitetet i Oslo. Fulltekst i vitenarkiv
• Tveit, Svein; Lundanes, Elsa; Fægri, Karoline & Skaugrud, Brit (2019). Egnethet av en minigasskromatograf i kjemi programfag i norsk videregående skole. Universitetet i Oslo. Fulltekst i vitenarkiv Vis sammendrag

  Praktisk gjennomføring av kromatografi i videregående skole blir vanligvis gjort ved at elevene får gjennomføre papir- eller tynnsjiktkromatografi. Med økt tilgang på billige gasskromatografer til undervisningsformål bør muligheten for å bruke disse i videregående skole undersøkes. Målet med denne studien var å undersøke om en kommersielt tilgjengelig minigasskromatograf fra Vernier, var egnet til bruk i undervisning i kjemi programfag i norsk videregående skole, og er så vidt forfatteren vet, den første studien som ser på bruken av dette instrumentet i videregående skole. En kvalitativ og en kvantitativ analyseoppgave, hver med en tidsramme på 90 minutter, ble videreutviklet og prøvd ut av elever og lærere i videregående skole. Studien viste at de fleste elevene klarte å bruke instrumentet til å gjennomføre en kvalitativ analyseoppgave og at en del av elevene klarte å gjennomføre en kvantitativ analyseoppgave. Elevene vektla læring av praktiske og eksperimentelle ferdigheter når de oppga hva de lærte av å jobbe med instrumentet, og over halvparten av elevenes svar var knyttet til motivasjon når de oppga hva de syntes var bra med å jobbe med instrumentet. Lærerne som ble spurt, mente i hovedsak at instrumentet og den kvalitative analyseoppgaven var godt egnet til bruk i kjemi programfag, men flere hadde forbehold knyttet til pris. I studien kom det også frem at det ikke påløp for store utgifter til drift av instrumentet. Mye tyder på at instrumentet og den kvalitative analyseoppgaven er egnet til bruk i kjemi programfag, men at den kvantitative oppgaven er for tidkrevende og ikke like sentral i forhold til læreplanen. Pris på instrumentet trekkes frem som en utfordring. Videre arbeid bør søke å finne flere og mindre tidkrevende laboratorieoppgaver der minigasskromatografen kan brukes i kjemi programfag, og undersøke skoleledere og læreres villighet til å investere i et slikt instrument.

• Olsen, Christine; Wilson, Steven Ray Haakon; Skottvoll, Frøydis Sved; Brandtzaeg, Ole Kristian; Rongved, Pål & Lundanes, Elsa (2019). Synthesis and immobilization of linked Wnt-signaling pathway inhibitor on organic monoliths as a proof-of-concept of a capti remedium ad monolitus reactor for online drug deconvolution. Universitetet i Oslo. Fulltekst i vitenarkiv Vis sammendrag

  A challenge in drug discovery is the identification of the drug target, called drug deconvolution. Additionally, off-target effects are considered as one of the reasons many developed drugs fail in the clinical trials. The goal of this work was to develop a solid support, displaying low secondary interactions, for immobilization of drugs (named by author as a CRAM reactor) suitable for incorporation online liquid chromatography mass spectrometry set-ups. The hypothesis was that selective purification on the online reactor would allow identification of low abundant drug targets as a consequence of reduced handling time, contamination and loss of the sample. As a proof-of-concept, an ethylene dimethacrylate-co-vinyl azlactone (EDMA-co-VDM) monolith, prepared in 180 µm inner diameter (ID) or 250 µm ID polyimide-coated fused silica capillaries, would be immobilized with Wnt-signaling pathway inhibitor 161. The 161-immobilized CRAM reactor would then attempt to selectively trap and release a low abundant protein target, tankyrase 2 (TNKS2). The EDMA-co-VDM monolith was successfully prepared in 250 µm ID capillaries. The Wnt-inhibitor 161 was rejected based on MS characterization and LDW639, a structural analogue of Wnt-inhibitor XAV939, was successfully synthesized by the author. To improve availability of LDW639 after immobilization, a linker was attached to LDW639 during synthesis. The linked LDW639 showed 50% inhibition of the Wnt-signaling pathway at a concentration of 11 µM after 24 hours incubation in cells. The EDMA-co-VDM monolith showed secondary interactions towards proteins, but the issues were resolved by quenching the reactive VDM monomer with either monoethanolamine (MEA) or an excess of linked LDW639. Immobilization of the linked LDW639 was found to be successful based on measured UV-Vis absorbance of solutions containing LDW639 was reduced by flushing a monolith, but not by monoliths already flushed with MEA (MEA monolith). The linked LDW639-immobilized CRAM reactors and the MEA monolith were not able to trap and release TNKS1/2 from human embryonic kidney 293 cells after cell lysis with a non-denaturing buffer. Showing that the identification of the drug target from complex matrices remained a challenge, even with tailored materials.

• Brandtzaeg, Ole Kristian; Wilson, Steven Ray Haakon; Lundanes, Elsa; Reubsaet, Leon; Krauss, Stefan & Martinsen, Ørjan Grøttem (2018). Online sample Preparation Platforms for Targeted Nano Liquid Chromatography Mass Spectrometry Bioanalysis. Universitetet i Oslo. Fulltekst i vitenarkiv
• Vehus, Tore; Wilson, Steven Ray Haakon; Lundanes, Elsa; Krauss, Stefan; Halvorsen, Trine Grønhaug & Morth, Jens Preben (2018). Nano Liquid Chromatography-Tandem Mass Spectrometry Platforms for Determination of Low Abundant Wnt/Beta-Catenin Proteins in Cancer Research. Universitetet i Oslo. Fulltekst i vitenarkiv
• Kular, Ramneet Kaur; Wilson, Steven Ray Haakon; Røberg-Larsen, Hanne & Lundanes, Elsa (2018). Towards ultrafast liquid chromatography of 3’-phosphoadenosine-5’-phosphosulfate using zwitterionic hydrophilic interaction liquid chromatography-mass spectrometry. Universitetet i Oslo. Fulltekst i vitenarkiv
• Hassine, Esma Ben; Lundanes, Elsa; Wilson, Steven Ray Haakon; Vehus, Tore; Brandtzaeg, Ole Kristian & Berg, Henriette Engen (2018). Capillary poly(styrene-co-octadecene-co-divinylbenzene) monolithic trap columns for bioanalytical analysis. Universitetet i Oslo. Fulltekst i vitenarkiv Vis sammendrag

  The increasing demand for faster and more efficient separations for biological samples have driven organic polymer-based monolithic columns back into the spotlight. The nature of the monolithic structure allows the use of higher flow rates without increasing the system backpressure. They are also easier to contain inside the column body, opposed to particle packed columns, which are held in place by frits. These are some of the advantages that monolithic columns offer compared to particle packed columns. Poly(styrene-co-octadecene-co-divinylbenzene) (PS-OD-DVB) monolithic columns have been prepared and characterized using the neuropeptide oxytocin, “the love hormone” as test compound. The columns, 50 µm inner diameter x 100 mm in length, were to be used as trap columns in a miniaturized liquid chromatography-mass spectrometry (LC-MS) column switching system for bottom-up proteomics analysis. The monolithic PS-OD-DVB columns were prepared in situ (inside the fused silica capillary), using a polymerization mixture consisting of an initiator (lauroyl peroxide), a crosslinker (divinylbenzene), monomers (styrene and 1-octadecene), a good porogen (N,N-dimethylformamide (DMF)) and a bad porogen (1-decanol). Polymerization time, polymerization temperature, initiator concentration, and monomer to porogen ratios were investigated in order to obtain a monolithic structure with a high surface area and good permeability. The best PS-OD-DVB monolithic column was prepared using a mixture of 1.9% lauroyl peroxide (weight percentage of monomer amount), 14.2% divinylbenzene, 15.2% monomers, 11.9% DMF and 57.5% 1-decanol. The column was polymerized for one hour at 73°C and gave a plate height of 62 µm, a backpressure of 23 bar at a flow rate of 500 nL/min with a mobile phase consisting of 20% B (acetonitrile (ACN), water, formic acid (FA), 80/20/0.1, v/v/v) and 80% A (water with 0.1% (v/v) FA) and a retention time repeatability of 2.6% (relative standard deviation). The repeatability of the columns made by various individuals was satisfactory. However, when preparing twenty-one columns by the same individual, the repeatability revealed to be questionable. Hence, further improvements in the polymerization procedure do seem necessary and might be able to enhance the repeatability of the columns.

• Solheim, Stian Kjønnås; Lundanes, Elsa; Wilson, Steven Ray Haakon & Røberg-Larsen, Hanne (2018). Exploring selectivity and sensitivity for oxysterol measurements using liquid chromatography-mass spectrometry (LC-MS). Universitetet i Oslo. Fulltekst i vitenarkiv
• Poothong, Somrutai; Haug, Line Småstuen; Thomsen, Cathrine & Lundanes, Elsa (2018). Poly- and perfluoroalkyl substances (PFASs): from external exposure to human blood. Universitetet i Oslo. Fulltekst i vitenarkiv Vis sammendrag

  This thesis contributed to the field of human exposure to environmental pollutants called poly- and perfluoroalkyl substances (PFASs). Daily intakes of PFASs via ingestion, inhalation, and dermal absorption were estimated and compared to the internal doses. Overall, diet contributed most to the daily intake of PFASs, followed by house dust, indoor air, and dermal absorption, but some variations were observed on an individual basis. Significant associations between concentrations of PFASs measured in serum, and estimated intakes based on direct and indirect (precursor) exposure were observed. Measured serum concentrations and modelled serum concentrations based on external exposure estimates were in the same order of magnitude. The estimated daily intakes of PFASs in this study were lower than the health-based guidance values. Also, in this study, analytical methods have been developed and validated for the determinations of a broad range of PFASs in serum, plasma, whole blood, dried blood spot, and hand wipes. This study underlines the importance of performing studies considering multiple exposure pathways on an individual basis.

• Gundersen, Cathrine Brecke; Vogt, Rolf David; Lundanes, Elsa; Breedvelt, Gijs & Andersen, Tom (2018). Environmental fate of nitramines formed during amine-based post-combustion CO2 capture. Universitetet i Oslo. Fulltekst i vitenarkiv Vis sammendrag

  There is now international political agreement to reduce greenhouse gas emissions to limit global warming by 2 °C1. Technology for amine-based CO2 capture offers the opportunity of reducing emission from already existing large-scale point sources. A thorough risk assessment of the technology is lacking. Ongoing investigations point to the formation of potentially carcinogenic and stable nitramines as the greatest risk. This study contributes to the risk assessment by investigating the soil sorption of nitramines and the response from environmental bacteria to the nitramines. Soil sorption is the most important process for retaining chemicals in the terrestrial environment. Bacteria are of interest since they can potentially degrade nitramines, but not if the nitramines pose toxic effects on the bacteria. Focus was on MEA (monoethanol)-, MMA (monomethyl)-, and DMA (dimethyl)-nitramines. To enable these studies, a method for nitramine determination in relevant sample matrices had to be optimized. Soil sorption was studied using a batch experimental setup and quantified indirectly by measuring loss of nitramine from the aqueous phase after equilibrium had been established with the soil. Potential toxic effect on bacteria was assessed by studying the response parameters of aerobic respiration, carbon utilization, and growth. During these experiments the biodegradability of the nitramines was also tested. Both environmental bacterial communities and pure cultures of isolated strains were included. Liquid chromatography–tandem mass spectrometry (LC-MS/MS) was used for the determination of nitramines. The final optimized version of the method, presented in Paper 2, is the first thorough validation for the simultaneous determination of multiple nitramines available in peer-reviewed literature. The method consists of reversed phase separation (C18) and electrospray ionisation with polarity switching mode, and can be used to detect low µg L-1 level nitramines in various aqueous matrices. Paper 3 provides evidence for a weak binding of MEA- and DMA-nitramines to soils rich in organic matter. Thus, accumulation can occur in organic rich soil layers, potentially hindering the nitramines from reaching e.g. the ground water. Different types of environmental bacteria were found to exert an ambiguous response towards the nitramines. For example, toxic effect was observed towards MEA-nitramine in a bacterial lake community (Paper 4). Contrary to this, no toxic response, but approximately 10% biodegradation of all three nitramines occurred in a pure culture of Pseudomonas fluorescens (Paper 5). The research presented in Papers 1-5 has been conducted using nitramine exposure levels at least three orders of magnitude higher than the levels that can be expected in precipitation in the vicinity of amine-based CO2 capture facilities. Future studies should aim at improving the sensitivity of the LC-MS/MS method. This would allow for detection of nitramines at environmentally relevant conditions, both from amine-based CO2 capture and from other alternative sources of nitramine formation.

• Amundsen, Sunniva Furre; Wilson, Steven Ray Haakon; Berg, Henriette Engen; Røberg-Larsen, Hanne & Lundanes, Elsa (2018). Online entrapment of epitope-containing peptides from integrins using immobilized antibodies. Universitetet i Oslo. Fulltekst i vitenarkiv
• Racz, Beatrix; Wilson, Steven Ray Haakon; Rise, Frode & Lundanes, Elsa (2018). Identification of a major UV absorbing compound in goose aqueous humour. Universitetet i Oslo. Fulltekst i vitenarkiv Vis sammendrag

  About 5% of solar radiation comes from the ultraviolet (UV) region. It carries high energy and can do serious damage to living organisms. The eye of animals and humans is a critically exposed area and contains UV absorbing compounds like proteins, tryptophan, tyrosine, ascorbic acid and uric acid in the aqueous humour (AH) for protection. In addition, an unknown component (compound X) which causes red-shift in the UV absorbance spectrum at 254 nm has been observed in heavily exposed species like goose, which migrates at 10 000 m altitude. X is a major absorber. UV measurements confirmed the presence of a compound in geese which has higher absorption at 254 nm compared to other species. Compound X also has fluorescence activity that resembled indole-functionality. Aqueous humour samples from different species (goose, chicken, turkey) were measured by nuclear magnetic resonance spectroscopy (NMR). The proton NMR spectra did not show any specific high abundant compound in the goose eye at the aromatic region compared to the other species, thus NMR could not reveal what made the difference between in UV absorbance. Mass spectrometry with atmospheric pressure photoionization (APPI) has earlier showed that the molar mass of the compound X could be 149 g/mole, which suggests that compound X might be the indole: 5,6-dihydroxyindole (DHI). DHI has a central role in the biosynthesis of melanin which takes place in melanocyte cells. DHI tends to easily be polymerized and one of the results is a notable change in its solubility. It was not possible to obtain a suitable external standard which did not undergo auto-polymerization, mirroring the observations with the isolated compound.To further test the hypothesis of DHI being compound X, reversed phase (RP) high performance liquid chromatography (HPLC) and hydrophilic interaction liquid chromatography (HILIC) were performed. Ascorbic acid co-eluted with the most abundant peak (compound X) in RP-HPLC. In HILIC the most abundant peak was not ascorbic acid. Hence, compound X is not ascorbic acid, and subsequent experiments showed that it was neither the UV absorbing compounds tryptophan, tyrosine or uric acid. After using several analytical tools, the hypothesis that compound X is 5,6-dihydroxyindole was strengthened. However an external standard is still necessary to provide accurate information.

• Hvinden, Ingvild Comfort; Wilson, Steven Ray Haakon; Rise, Frode; Berg, Henriette Engen & Lundanes, Elsa (2018). Nuclear magnetic resonance spectroscopy based metabolomics discovers biomarkers of glioblastoma drug response. Universitetet i Oslo. Fulltekst i vitenarkiv Vis sammendrag

  Glioblastoma is the most common and aggressive form of brain cancer. Even with comprehensive treatment regimes, patients face an expected survival of only 15 months. Currently, methods for assessing treatment response are lacking; it is difficult to accurately determine the efficacy of a treatment. The goal of the present study was to contribute to treatment assessment by scouting for metabolic biomarkers occurring in response to exposure to chemotherapeutic agents temozolomide (TMZ) and sepantronium bromide (YM155). Untargeted metabolomics of lysate from cultured glioblastoma cells was carried out with liquid state proton nuclear magnetic resonance (NMR) spectroscopy at resonance frequency 800 MHz. Spectral data were analyzed with two different multivariate statistical methods: principal component analysis (PCA) and partial least squares (PLS) regression. For YM155, two biomarker candidates were found: citric and lactic acid. Citric acid appeared to increase most in samples from cell lines less sensitive to YM155. Lactic acid decreased in all cell lines and was considered a more general biomarker of treatment exposure. TMZ-treated samples were not distinguishable from control samples, most likely due to too short exposure time (24 hours). Analyses with nano hydrophilic interaction liquid chromatography coupled with mass spectrometry (MS) corroborated the findings by NMR spectroscopy and statistical analyses. Both citric acid and lactic acid are biomarker candidates, but a more detailed understanding of their fluctuations in glioblastoma during treatment is needed. Nevertheless, they represent genuine candidates and should be considered for further in vivo magnetic resonance spectroscopy (MRS) studies. In the future, the biomarkers could be monitored with MRS, allowing a more unambiguous and personalized assessment of response to treatment in individual patients.

• Bjørseth, Kamilla; Wilson, Steven Ray Haakon; Brandtzaeg, Ole Kristian & Lundanes, Elsa (2017). Evaluation of exosome isolation techniques for hydrophilic interaction liquid chromatography-mass spectrometry based metabolomics. Universitetet i Oslo. Fulltekst i vitenarkiv
• Skottvoll, Frøydis Sved; Berg, Henriette Engen; Lund, Kaja; Røberg-Larsen, Hanne; Wilson, Steven Ray Haakon & Lundanes, Elsa (2017). Critical evaluation of isolation and characterization techniques of breast cancer exosomes. Universitetet i Oslo. Fulltekst i vitenarkiv Vis sammendrag

  Exosomes represent a distinct class of extracellular vesicles of endocytic origin secreted by multiple cell types, including tumour cells. An increased release of exosomes, which appears to be a rich source of biomarkers, has been reported from tumour cells. However, current strategies concerning the isolation (and characterization) of exosomes from fluids differ significantly and no consensus method is established. The goal of this work was to evaluate two different exosome isolation methods with two different breast cancer cell lines in culture: differential ultracentrifugation and commercial isolation kit. Evaluation was done using a bicinchoninic acid assay (protein concentration), transmission electron microscopy (morphology), dynamic light scattering (hydrodynamic size), western blotting (targeted protein exosome markers) and nano liquid chromatography tandem mass spectrometry (comprehensive protein identification). The characterization techniques confirmed the isolation of exosomes with both isolation kit and ultracentrifugation. However, the isolated samples did contain contaminations, and there was a clear difference in the protein amount, particle size and populations identified with the two isolation methods. In addition, the majority of the characterization techniques provided poor repeatability, reproducibility and/or demanded extensive optimization. The evaluation showed that the exosome isolation procedures used in this thesis appear to be far from mature. Additionally, the majority of the characterization techniques used in this study provided challenges.

• Demir, Deniz; Lundanes, Elsa; Wilson, Steven Ray Haakon; Abele, Silvija & Røberg-Larsen, Hanne (2017). Automatic capillary liquid chromatography tandem mass spectrometry method for pharmaceutical products in environmental water samples. Universitetet i Oslo. Fulltekst i vitenarkiv Vis sammendrag

  Abstract The use of active pharmaceutical ingredients (APIs) is increasing continuously and thereby constitutes a potential source of surface water contamination. Environmental concentrations of APIs are usually at trace levels, generally in low ng/L concentrations, making them challenging to detect. However, these low concentrations can be sufficient to induce toxic effects on the aquatic environment. This project focuses on selected APIs, which are probably the most common in the aquatic environment. Several of the compounds selected are on The Norwegian Institute of Public Health’s (NIPH’s) list over the most prescribed APIs in Norway throughout 2014. A rapid, sensitive and selective method was developed and validated for screening of seven APIs (acetaminophen, atenolol, fluoxetine, sulfapyridine, sulfamethoxazole, trimethoprim and xylazine) in surface water. The method include an on-line automatic filter and filter back flush (AFFL) solid phase extraction (SPE) in combination with capillary liquid chromatography (capLC) coupled to a triple quadrupole mass analyser (MS). By combining large volume injection (100 μL) and the AFFL-SPE-system, a rugged and high throughput switching system was obtained. The analytes were loaded under non-eluting condition and further trapped on a reversed phase (RP) Kromasil (C18, 5 mm x 1 mm ID, 5 μm) SPE-column with a flow rate of 150 μL/min. A 10 minutes gradient was applied using an ACE C18 column (150 mm x 0.3 mm ID, 3 μm) as analytical column with a flow rate of 4 μL/min. The mobile phases (MP) for loading and MP A consisted of type 1 water with 0.1 % formic acid (FA) and the organic MP (MP B) consisted of 100 % methanol with 0.1 % FA (pH 2.7). The total analysis time was 15 minutes including 6 minutes reconditioning. This study demonstrates that the AFFL-SPE-capLC system combined with a triple quadrupole MS enables the detection of selected classes of APIs in surface water in a concentration range of 10-100 ng/L. The calculated concentration limit of detection (cLOD) for the selected APIs was in the 2– 18 ng/L range and calculated concentration limit of quantification (cLOQ) in the 5 – 54 ng/L range. Acceptable linearity (R2 = 0.9777-0.9998) and generally high apparent recovery values (from 69-288 %) with generally relative standard deviation (RSD %) lower than 20 % were found.

• Johnsen, Elin Follaug; Lundanes, Elsa; Wilson, Steven Ray Haakon & Greibrokk, Tyge (2017). High performance analytical tools for small molecules in biosamples. Universitetet i Oslo. Fulltekst i vitenarkiv

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