Hanne Røberg-Larsen

• Kømurcu, Kristina Sæterdal; Zawadzka, Malgorzata Elzbieta; Meszka, Igor; Aizenshtadt, Aleksandra; Hrušková, Helena & Aakervik, Lydia Emilie [Vis alle 10 forfattere av denne artikkelen] (2026). A Validated Mass Spectrometry Platform for Oxysterol Analysis of Single Human Gastruloids and Liver Organoids. Analytical Chemistry. ISSN 0003-2700. 98(9), s. 6792–6804. doi: 10.1021/acs.analchem.5c07140. Fulltekst i vitenarkiv
• Noreng, Line; Øiestad, Åse Marit Leere; Hansen, Frederik Andre; Røberg-Larsen, Hanne; Wilson, Steven Ray Haakon & Øiestad, Elisabeth Leere (2026). Toward Sustainable Clinical Analysis: Benchmarking Plastic Use in LC–MS Sample Preparation – Exemplified by Ketamine Analogues in Whole Blood. Analytical Chemistry. ISSN 0003-2700. 98(16), s. 11899–11909. doi: 10.1021/acs.analchem.5c08225. Fulltekst i vitenarkiv
• Uhlig, Silvio; So̷rnes, Kristian; Mineeva, Evangeliia; Rise, Frode; Grutle, Lene & Morisbak, Else [Vis alle 9 forfattere av denne artikkelen] (2025). Nucleophilic Addition of Thiols to Methacrylates for Biomedical Applications Revisited. Chemical Research in Toxicology. ISSN 0893-228X. 38(9), s. 1535–1547. doi: 10.1021/acs.chemrestox.5c00179. Fulltekst i vitenarkiv Vis sammendrag

  Composites are popular materials for, among others, restorative dentistry because of their favorable mechanical and esthetic properties and direct-filling applications. The raw materials for such composites usually consist of filler particles embedded in a matrix of dimethacrylate monomers that are polymerized in situ. Because the raw materials cannot polymerize completely, residual monomers leach out over time. The conjugation of methacrylates with sulfur compounds has been recognized as an important reaction as well as a detoxification pathway; thus, leached monomers are expected to undergo chemical reactions with various biomolecules that contain thiol functionalities. To understand the reaction of dental methacrylate monomers with thiols, we studied the reaction of 2-hydroxyethyl methacrylate (HEMA), triethylene glycol dimethacrylate, urethane dimethacrylate, and bisphenol A diglycidyl methacrylate with the model thiol 2-mercaptoethanol using liquid chromatography coupled to low- and high-resolution mass spectrometry (LC–MS and LC–HRMS). The results indicate that thiols react readily with the conjugated double bond, and with methacrylate half-lives of 7–21 h under pseudo-first-order reaction conditions and at neutral pH. Dimethacrylates first formed a monoaddition product, while thiol addition to the second acrylate moiety was observed on a longer time scale. The reaction of HEMA with l-cysteine and l-glutathione was studied in more detail using HRMS and NMR spectroscopy. The reaction rates were substantially higher than for the reaction with mercaptoethanol, and NMR analysis revealed the presence of two isomeric reaction products. Structural characterization also included the identification and assignment of sulfoxides of HEMA-cysteine and HEMA-glutathione. Using the characterized HEMA–thiols as reference standards for LC–HRMS, we demonstrated the presence of HEMA-glutathione, HEMA-cysteine, their sulfoxides, and a putative HEMA-cysteinylglycine in a human osteoblast-like cell line following exposure to HEMA.

• Kogler, Stian; Skottvoll, Frøydis Sved; Hruskova, Helena; Rise, Frode; Aizenshtadt, Aleksandra & Krauss, Stefan Johannes Karl [Vis alle 9 forfattere av denne artikkelen] (2025). Electromembrane Extraction Provides Unprecedented Selectivity for Drugs in Cell Culture Media Used in Organoid and Organ-on-Chip Systems. Analytical Chemistry. ISSN 0003-2700. 97(9), s. 4923–4931. doi: 10.1021/acs.analchem.4c04994. Fulltekst i vitenarkiv Vis sammendrag

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

• Kogler, Stian; Pedersen, Gustav Mathingsdal; Martínez-Ramírez, Felipe; Aizenshtadt, Aleksandra; Busek, Mathias & Krauss, Stefan Johannes Karl [Vis alle 8 forfattere av denne artikkelen] (2024). An FDA-Validated, Self-Cleaning Liquid Chromatography–Mass Spectrometry System for Determining Small-Molecule Drugs and Metabolites in Organoid/Organ-on-Chip Medium. Analytical Chemistry. ISSN 0003-2700. 96(29), s. 12129–12138. doi: 10.1021/acs.analchem.4c02246. Fulltekst i vitenarkiv Vis sammendrag

  As organoids and organ-on-chip (OoC) systems move toward preclinical and clinical applications, there is an increased need for method validation. Using a liquid chromatography–mass spectrometry (LC–MS)-based approach, we developed a method for measuring small-molecule drugs and metabolites in the cell medium directly sampled from liver organoids/OoC systems. The LC–MS setup was coupled to an automatic filtration and filter flush system with online solid-phase extraction (SPE), allowing for robust and automated sample cleanup/analysis. For the matrix, rich in, e.g., protein, salts, and amino acids, no preinjection sample preparation steps (protein precipitation, SPE, etc.) were necessary. The approach was demonstrated with tolbutamide and its liver metabolite, 4-hydroxytolbutamide (4HT). The method was validated for analysis of cell media of human stem cell-derived liver organoids cultured in static conditions and on a microfluidic platform according to Food and Drug Administration (FDA) guidelines with regards to selectivity, matrix effects, accuracy, precision, etc. The system allows for hundreds of injections without replacing chromatography hardware. In summary, drug/metabolite analysis of organoids/OoCs can be performed robustly with minimal sample preparation.

• Hrušková, Helena; Olsen, Christine; Řemínek, Roman; Wang, Chencheng; Aizenshtadt, Aleksandra & Krauss, Stefan Johannes Karl [Vis alle 10 forfattere av denne artikkelen] (2024). Preparative agarose gel electrophoresis for reducing matrix interferences of organoid cell medium prior to LC-MS analysis of insulin. Journal of Chromatography A. ISSN 0021-9673. 1717. doi: 10.1016/j.chroma.2024.464669. Fulltekst i vitenarkiv Vis sammendrag

  Organoids are 3D cell cultures with microanatomies mimicking aspects of real organs, useful for e.g. animal-free studies of development, disease, and drug discovery. The cell medium of organoid models of Langerhans islets, regulating blood glucose levels by insulin secretion, can be analyzed by liquid chromatography-mass spectrometry (LC-MS). However, organoid medium complexity is a major challenge, as matrix interferences can reduce sensitivity and selectivity, even with optimized LC-MS conditions. By applying preparative agarose gel electrophoresis-electrodialysis (PGE-ED), we were able to decrease the cell medium background signal, allowing for reduced interferences affecting LC-MS analysis of human insulin.

• Aizenshtadt, Aleksandra; Wang, Chencheng; Abadpour, Shadab; Menezes, Pedro Duarte; Wilhelmsen, Ingrid & Dalmao-Fernandez, Andrea [Vis alle 15 forfattere av denne artikkelen] (2024). Pump-Less, Recirculating Organ-on-Chip (rOoC) Platform to Model the Metabolic Crosstalk between Islets and Liver. Advanced Healthcare Materials. ISSN 2192-2640. 13(13). doi: 10.1002/adhm.202303785. Fulltekst i vitenarkiv Vis sammendrag

  Type 2 diabetes mellitus (T2DM), obesity, and metabolic dysfunction-associated steatotic liver disease (MASLD) are epidemiologically correlated disorders with a worldwide growing prevalence. While the mechanisms leading to the onset and development of these conditions are not fully understood, predictive tissue representations for studying the coordinated interactions between central organs that regulate energy metabolism, particularly the liver and pancreatic islets, are needed. Here, a dual pump-less recirculating organ-on-chip platform that combines human pluripotent stem cell (sc)-derived sc-liver and sc-islet organoids is presented. The platform reproduces key aspects of the metabolic cross-talk between both organs, including glucose levels and selected hormones, and supports the viability and functionality of both sc-islet and sc-liver organoids while preserving a reduced release of pro-inflammatory cytokines. In a model of metabolic disruption in response to treatment with high lipids and fructose, sc-liver organoids exhibit hallmarks of steatosis and insulin resistance, while sc-islets produce pro-inflammatory cytokines on-chip. Finally, the platform reproduces known effects of anti-diabetic drugs on-chip. Taken together, the platform provides a basis for functional studies of obesity, T2DM, and MASLD on-chip, as well as for testing potential therapeutic interventions.

• Uhlig, Silvio; Cai, Kun; Liskiewicz, Krystyna Anna; Pain, Maria Ronessen; Grutle, Lene & Røberg-Larsen, Hanne [Vis alle 7 forfattere av denne artikkelen] (2024). Profiling of cyclic di-adenyl and -guanyl nucleotides and their precursors and degradation products in bacteria using LC–MS/MS. Separation Science Plus (SSC plus). doi: 10.1002/sscp.202400039. Fulltekst i vitenarkiv
• Kømurcu, Kristina Sæterdal; Wilson, Steven Ray Haakon & Røberg-Larsen, Hanne (2024). LC-MS Approaches for Oxysterols in Various Biosamples. Advances in Experimental Medicine and Biology. ISSN 0065-2598. 1440, s. 57–71. doi: 10.1007/978-3-031-43883-7_4. Fulltekst i vitenarkiv Vis sammendrag

  Oxysterols are involved in a plethora of biological processes, including a wide variety of diseases. Therefore, monitoring oxysterols is important for obtaining a deeper understanding of their biological roles and utilizing them as, for example, biomarkers. However, oxysterols can be challenging compounds to study, as they can be very similar in chemical structure but still have distinct biological roles. In addition, oxysterols may be difficult to detect, even with advanced analytical instrumentation. We here focus on the use of liquid chromatography–mass spectrometry (LC–MS) for the analysis of oxysterols, with an additional focus on the steps needed to prepare oxysterols for LC–MS. Steps can include chemical modification of the oxysterols for improving LC–MS sensitivity and adding chemicals that can reveal if the oxysterol levels have been perturbed during preparation. We then round off with descriptions and applications of various sample preparations for different biological matrices, from blood to cells, and biosamples with emerging attention, for example, exosomes and organoids. Taken together, oxysterol analysis is highly compatible with a wide variety of biosamples, allowing for a deeper understanding of these challenging analytes.

• Johnsen, Mads T. S. Gisle; Olsen, Christine; Røberg-Larsen, Hanne & Wilson, Steven Ray Haakon (2024). Liquid chromatography mass spectrometry-based approaches for determination of pancreatic hormones. Journal of chromatography open. ISSN 2772-3917. 6. doi: 10.1016/j.jcoa.2024.100143. Fulltekst i vitenarkiv
• Kømurcu, Kristina Sæterdal; Wilhelmsen, Ingrid; Thorne, James L.; Krauss, Stefan Johannes Karl; Wilson, Steven Ray Haakon & Aizenshtadt, Aleksandra [Vis alle 7 forfattere av denne artikkelen] (2023). Mass spectrometry reveals that oxysterols are secreted from non-alcoholic fatty liver disease induced organoids. Journal of Steroid Biochemistry and Molecular Biology. ISSN 0960-0760. 232. doi: 10.1016/j.jsbmb.2023.106355. Fulltekst i vitenarkiv Vis sammendrag

  Oxysterols are potential biomarkers for liver metabolism that are altered under disease conditions such as non-alcoholic fatty liver disease (NAFLD). We here apply sterolomics to organoids used for disease modeling of NAFLD. Using liquid chromatography-mass spectrometry with on-line sample clean-up and enrichment, we establish that liver organoids produce and secrete oxysterols. We find elevated levels of 26-hydroxycholesterol, an LXR agonist and the first oxysterol in the acidic bile acid synthesis, in medium from steatotic liver organoids compared to untreated organoids. Other upregulated sterols in medium from steatotic liver organoids are dihydroxycholesterols, such as 7α,26–dihydroxycholesterol, and 7α,25-dihydroxycholesterol. Through 26-hydroxycholesterol exposure to human stem cell-derived hepatic stellate cells, we observe a trend of expressional downregulation of the pro-inflammatory cytokine CCL2, suggesting a protective role of 26-hydroxycholesterol during early-phased NAFLD disease development. Our findings support the possibility of oxysterols serving as NAFLD indicators, demonstrating the usefulness of combining organoids and mass spectrometry for disease modeling and biomarker studies.

• Kogler, Stian; Kømurcu, Kristina Sæterdal; Olsen, Christine Sandsnes; Shoji, Jun-ya; Skottvoll, Frøydis Sved & Krauss, Stefan Johannes Karl [Vis alle 8 forfattere av denne artikkelen] (2023). Organoids, organ-on-a-chip, separation science and mass spectrometry: An update. TrAC. Trends in analytical chemistry. ISSN 0165-9936. 161. doi: 10.1016/j.trac.2023.116996. Fulltekst i vitenarkiv Vis sammendrag

  Organoids are 3D models of organs, grown in the laboratory from stem cells. An organ model grown/placed in microfluidic devices is commonly termed an “organ-on-a-chip”. Organoids and organ-on-a-chip devices are becoming important tools for studying physiology, disease modeling, drug discovery, personalized medicine, toxicology, and organ development/embryogenesis. We review how mass spectrometry is used for studying organoids and organ-on-a-chip-derived material. We first focus on proteomics, metabolomics/lipidomics, and hormones, typically discussing liquid chromatography-mass spectrometry (LC-MS) approaches. We then review work on mass spectrometry imaging (MSI) of organoids and discuss organ-on-a-chip coupled with mass spectrometry. The review focuses on research developments from the past four years. Mass spectrometric analysis of organoids and organ-on-a-chip has allowed novel insights on development and disease of e.g. brain, liver, and tumors, demonstrating potential for replacing or complementing animal models and other traditional model systems. Additional applications are emerging, e.g. related to sports doping and environmental toxicology.

• 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.

• 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
• Websdale, Alex; Kiew, Yi; Chalmers, Philip; Chen, Xinyu; Cioccoloni, Giorgia & Hughes, Thomas A. [Vis alle 14 forfattere av denne artikkelen] (2021). Pharmacologic and genetic inhibition of cholesterol esterification enzymes reduces tumour burden: A systematic review and meta-analysis of preclinical models. Biochemical Pharmacology. ISSN 0006-2952. doi: 10.1016/j.bcp.2021.114731. Fulltekst i vitenarkiv
• Hutchinson, Samantha A.; Websdale, Alex; Cioccoloni, Giorgia; Røberg-Larsen, Hanne; Lianto, Priscilia & Kim, Baek [Vis alle 18 forfattere av denne artikkelen] (2021). Liver x receptor alpha drives chemoresistance in response to side-chain hydroxycholesterols in triple negative breast cancer. Oncogene. ISSN 0950-9232. 40(16), s. 2872–2883. doi: 10.1038/s41388-021-01720-w. Fulltekst i vitenarkiv Vis sammendrag

  Abstract Triple negative breast cancer (TNBC) is challenging to treat successfully because targeted therapies do not exist. Instead, systemic therapy is typically restricted to cytotoxic chemotherapy, which fails more often in patients with elevated circulating cholesterol. Liver x receptors are ligand-dependent transcription factors that are homeostatic regulators of cholesterol, and are linked to regulation of broad-affinity xenobiotic transporter activity in non-tumor tissues. We show that LXR ligands confer chemotherapy resistance in TNBC cell lines and xenografts, and that LXRalpha is necessary and sufficient to mediate this resistance. Furthermore, in TNBC patients who had cancer recurrences, LXRalpha and ligands were independent markers of poor prognosis and correlated with P-glycoprotein expression. However, in patients who survived their disease, LXRalpha signaling and P-glycoprotein were decoupled. These data reveal a novel chemotherapy resistance mechanism in this poor prognosis subtype of breast cancer. We conclude that systemic chemotherapy failure in some TNBC patients is caused by co-opting the LXRalpha:P-glycoprotein axis, a pathway highly targetable by therapies that are already used for prevention and treatment of other diseases.

• 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
• 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.

• Hutchinson, Samantha A; Lianto, Priscilia; Røberg-Larsen, Hanne; Battaglia, Sebastiano; Hughes, Thomas A & Thorne, James L (2019). ER-Negative Breast Cancer Is Highly Responsive to Cholesterol Metabolite Signalling. Nutrients. 11(11), s. 1–18. doi: 10.3390/nu11112618. Fulltekst i vitenarkiv Vis sammendrag

  Interventions that alter cholesterol have differential impacts on hormone receptor positive- and negative-breast cancer risk and prognosis. This implies differential regulation or response to cholesterol within different breast cancer subtypes. We evaluated differences in side-chain hydroxycholesterol and liver X nuclear receptor signalling between Oestrogen Receptor (ER)-positive and ER-negative breast cancers and cell lines. Cell line models of ER-positive and ER-negative disease were treated with Liver X Receptor (LXR) ligands and transcriptional activity assessed using luciferase reporters, qPCR and MTT. Publicly available datasets were mined to identify differences between ER-negative and ER-positive tumours and siRNA was used to suppress candidate regulators. Compared to ER-positive breast cancer, ER-negative breast cancer cells were highly responsive to LXR agonists. In primary disease and cell lines LXRA expression was strongly correlated with its target genes in ER-negative but not ER-positive disease. Expression of LXR’s corepressors (NCOR1, NCOR2 and LCOR) was significantly higher in ER-positive disease relative to ER-negative, and their knock-down equalized sensitivity to ligand between subtypes in reporter, gene expression and viability assays. Our data support further evaluation of dietary and pharmacological targeting of cholesterol metabolism as an adjunct to existing therapies for ER-negative and ER-positive breast cancer patients.

• Becares, Natalia; Cage, Matthew C; Voisin, Maud; Shrestha, Elina; Martin-Gutierrez, Lucia & Liang, Ning [Vis alle 20 forfattere av denne artikkelen] (2019). Impaired LXRα Phosphorylation Attenuates Progression of Fatty Liver Disease. Cell reports. 26(4), s. 984–995.e6. doi: 10.1016/j.celrep.2018.12.094. Fulltekst i vitenarkiv Vis sammendrag

  Non-alcoholic fatty liver disease (NAFLD) is a very common indication for liver transplantation. How fat-rich diets promote progression from fatty liver to more damaging inflammatory and fibrotic stages is poorly understood. Here, we show that disrupting phosphorylation at Ser196 (S196A) in the liver X receptor alpha (LXRα, NR1H3) retards NAFLD progression in mice on a high-fat-high-cholesterol diet. Mechanistically, this is explained by key histone acetylation (H3K27) and transcriptional changes in pro-fibrotic and pro-inflammatory genes. Furthermore, S196A-LXRα expression reveals the regulation of novel diet-specific LXRα-responsive genes, including the induction of Ces1f, implicated in the breakdown of hepatic lipids. This involves induced H3K27 acetylation and altered LXR and TBLR1 cofactor occupancy at the Ces1f gene in S196A fatty livers. Overall, impaired Ser196-LXRα phosphorylation acts as a novel nutritional molecular sensor that profoundly alters the hepatic H3K27 acetylome and transcriptome during NAFLD progression placing LXRα phosphorylation as an alternative anti-inflammatory or anti-fibrotic therapeutic target.

• 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.

• Dias, Irundika H.K.; Wilson, Steven Ray Haakon & Røberg-Larsen, Hanne (2018). Chromatography of oxysterols. Biochimie. ISSN 0300-9084. doi: 10.1016/j.biochi.2018.05.004. 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.

• 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.

• Åstrand, Ove Alexander Høgmoen; Viktorsson, Elvar Örn; Kristensen, Aleksander Lim; Ekeberg, Dag; Røberg-Larsen, Hanne & Wilson, Steven Ray Haakon [Vis alle 12 forfattere av denne artikkelen] (2017). Synthesis, in vitro and in vivo biological evaluation of new oxysterols as modulators of the liver X receptors. Journal of Steroid Biochemistry and Molecular Biology. ISSN 0960-0760. 165, s. 323–330. doi: 10.1016/j.jsbmb.2016.07.010. Fulltekst i vitenarkiv Vis sammendrag

  Liver X Receptor (LXR) modulators have shown potential as drugs since they target genes affecting metabolism and fatty acid synthesis. LXR antagonists are of particular interest since they are able to reduce the synthesis of complex fatty acids and glucose uptake. Based on molecular modeling, five new cholesterol mimics were synthesized, where four contained a hydroxyl group in the 22-S-position. The new compounds were screened in vitro against several genes affecting lipid metabolism. The compound that performed best in vitro was a dimethylamide derivative of 22(S)-hydroxycholesterol and it was chosen for in vivo testing. However, the blood plasma analysis from the in vivo tests revealed a concentration lower than needed to give any response, indicating either rapid metabolism or low bioavailability.

• 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
• Simigdala, Nikiana; Gao, Qiong; Pancholi, Sunil; Røberg-Larsen, Hanne; Zvelebil, Marketa & Ribas, Ricardo [Vis alle 11 forfattere av denne artikkelen] (2016). Cholesterol biosynthesis pathway as a novel mechanism of resistance to estrogen deprivation in estrogen receptor-positive breast cancer. Breast Cancer Research. ISSN 1465-5411. 18:58. doi: 10.1186/s13058-016-0713-5. Fulltekst i vitenarkiv
• Brandtzaeg, Ole Kristian; Johnsen, Elin Follaug; Røberg-Larsen, Hanne; Seip, Knut Fredrik; MacLean, Evan L. & Gesquiere, Laurence R. [Vis alle 9 forfattere av denne artikkelen] (2016). Proteomics tools reveal startlingly high amounts of oxytocin in plasma and serum. Scientific Reports. 6:31693. doi: 10.1038/srep31693. Fulltekst i vitenarkiv Vis sammendrag

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

• Johnsen, Elin Follaug; Brandtzaeg, Ole Kristian; Vehus, Tore; Røberg-Larsen, Hanne; Bogoeva, Vanya & Ademi, Ornela [Vis alle 9 forfattere av denne artikkelen] (2016). A critical evaluation of Amicon Ultra centrifugal filters for separating proteins, drugs and nanoparticles in biosamples. Journal of Pharmaceutical and Biomedical Analysis. ISSN 0731-7085. 120, s. 106–111. doi: 10.1016/j.jpba.2015.12.010. Fulltekst i vitenarkiv
• Vehus, Tore; Røberg-Larsen, Hanne; Waaler, Jo; Aslaksen, Sigrid; Krauss, Stefan & Wilson, Steven Ray Haakon [Vis alle 7 forfattere av denne artikkelen] (2016). Versatile, sensitive liquid chromatography mass spectrometry–Implementation of 10 μm OT columns suitable for small molecules, peptides and proteins. Scientific Reports. 6. doi: 10.1038/srep37507. Fulltekst i vitenarkiv
• Røberg-Larsen, Hanne; Vesterdal, Caroline; Wilson, Steven Ray Haakon & Lundanes, Elsa (2015). Underivatized oxysterols and nanoLC-ESI-MS: A mismatch. Steroids. ISSN 0039-128X. 99, s. 125–130. doi: 10.1016/j.steroids.2015.01.023. Fulltekst i vitenarkiv
• Røberg-Larsen, Hanne; Lund, Kaja; Vehus, Tore; Solberg, Nina; Vesterdal, Caroline & Misaghian, Dorna [Vis alle 10 forfattere av denne artikkelen] (2014). Highly automated nano-LC/MS-based approach for thousand cell-scale quantification of side chain-hydroxylated oxysterols. Journal of Lipid Research. ISSN 0022-2275. 55(7), s. 1531–1536. doi: 10.1194/jlr.D048801. Fulltekst i vitenarkiv Vis sammendrag

  Iso-octyl chain-hydroxylated oxysterols were determined in attomoles per 10,000 cells concentrations in 10,000–80,000 cultured pancreatic adenocarcinoma cells, using a sensitive, highly automated nano-LC-ESI-MS-based method. Identified oxysterols included 24S hydroxycholesterol (24S-OHC), 25 hydroxycholesterol (25-OHC), and 27 hydroxycholesterol (27-OHC), while 20S hydroxycholesterol and 22S hydroxycholesterol were not detected. Lower mass limit of quantification was 23 fg (65 amol) for 25-OHC and 27-OHC (100 times lower than our previous method) and 54 fg (135 amol) for 24S-OHC, after derivatization into Girard T hydrazones and online sample cleanup using simplified and robust automatic filtration and filter back flushing solid phase extraction LC/MS/MS. The instrument configuration was easily installed using a commercial nano-LC/MS system. Recoveries in spiked sample were 96, 97, and 77% for 24S-OHC, 25-OHC, and 27-OHC, with within- and between-day repeatabilities of 1–21% and 2–20% relative SD, respectively. The study demonstrates the potential of nano-LC in lipidomics/sterolomics. This research was originally published in the Journal of Lipid Research. Roberg-Larsen H, Lund K, Vehus T, Solberg N, Vesterdal C, Misaghian D, Olsen PA, Krauss S, Wilson SR, Lundanes E.. Highly automated nano-LC/MS-based approach for thousand cell-scale quantification of side chain-hydroxylated oxysterols. J. Lipid Res. 2014 Jul;55(7):1531-6. © the American Society for Biochemistry and Molecular Biology.

Se alle arbeider i NVA

• Websdale, Alex; Al-Hilali, Yusur; Kim, Baek; Williams, Bethany J; Wastall, Laura & Røberg-Larsen, Hanne [Vis alle 9 forfattere av denne artikkelen] (2025). OXYSTEROLS AND PHYTOSTEROLS IN HUMAN PATHOPHYSIOLOGY: Adipocytes in tumour microenvironment promote chemoresistance in triple-negative breast cancer through oxysterols. Redox Experimental Medicine. 2025(1). doi: 10.1530/rem-25-0006. Fulltekst i vitenarkiv Vis sammendrag

  Graphical abstract Adipocytes-derived oxysterols protect TNBC cells from chemotherapy activating LXRα-Pgp axis. Figure generated with Biorender. Abstract Objective Triple-negative breast cancer (TNBC) patients with excess adipose tissue experience poorer disease-free survival than those with a healthy body mass index. Adipocytes store and release cholesterol, which can be hydroxylated to form oxysterols. These cholesterol derivatives activate the liver X receptor (LXR) pathway. This study tested the hypothesis that adipocytes contribute to an imbalanced tumour microenvironment by exposing cancer cells to elevated oxysterols, mimicking chemotherapy exposure conditions and priming for chemoresistance. Methods Tumour tissue microarray from 148 TNBC patients was assessed using immunohistochemistry for CH25H , CYP46A1 , CYP27A1 , P-glycoprotein ( Pgp/ ABCB1 ) expression and survival outcomes were assessed. Gene expression was compared between tumours from patients (GSE78958) and mouse models (GSE151866) with high versus low adiposity. In vitro , cell lines from lineages found in the tumour microenvironment were evaluated for oxysterol content, secretion, expression of relevant enzymes, and ability to induce ABCB1 expression and drug resistance in TNBC cells. Results In patients, stromal expression of oxysterol-synthesising enzymes correlated with ABCP1 expression in cancer epithelial cells and was associated with shorter disease-free survival. Adipocytes-conditioned media contained significantly higher oxysterol levels than that conditioned by other cell types and induced ABCB1 expression and drug resistance in MDA.MB.468 cells. Obese mice had elevated levels of Abcb1 in tumours compared to lean counterparts. Conclusion Adipocytes secrete oxysterols that promote drug resistance in vitro and correlate with oxysterol: Pgp axis and survival in vivo . Significance statement This study reveals a mechanism by which adipose tissue contributes to drug resistance in ER-negative breast cancers, identifying the oxysterol–Pgp axis as a potential therapeutic target.

• Dundas, Siv Hjorth; Uggerud, Hilde Thelle; Kallenborn, Roland; Enger, Øyvind; Hammer, Stine Eriksen & Røberg-Larsen, Hanne (2025). Svarbrev fra NKS-FAK på nye karakterkrav for Analytisk kjemi kurs ved NMBU. 2, s. 18–20. Fulltekst i vitenarkiv
• Cioccoloni, Giorgia; Dias, Irundika; King, Jenna; Lobaccaro, Jean-Marc; Røberg-Larsen, Hanne & Thorne, James L. (2024). European Network for Oxysterol Research (ENOR). Biochemist. ISSN 0954-982X. 46(6), s. 53–53. doi: 10.1042/BIO_2024_907. Fulltekst i vitenarkiv
• 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
• Røberg-Larsen, Hanne; Kømurcu, Kristina Sæterdal; Kogler, Stian; Olsen, Christine; Skottvoll, Frøydis Sved & Aizenshtadt, Aleksandra [Vis alle 8 forfattere av denne artikkelen] (2023). Organoids, Organ-on-a-chip and Mass Spectrometry. Fulltekst i vitenarkiv
• Midtøy, Lise; Kømurcu, Kristina Sæterdal; Kogler, Stian; wilhelmsen, ingrid; Røberg-Larsen, Hanne & Wilson, Steven Ray Haakon (2023). Sensitive targeted proteomic analysis of cholesterol metabolism enzymes in hepatic stellate cells - going towards single cell analysis. Fulltekst i vitenarkiv
• kvalvik, Eva; Kogler, Stian; Kømurcu, Kristina Sæterdal; Wilson, Steven Ray Haakon & Røberg-Larsen, Hanne (2023). Screening for biomarkers for non-alcoholic fatty liver disease using organ-in-a-column technology. Fulltekst i vitenarkiv
• Haavaag, Jon Erlend; Kogler, Stian; Zawadzka, Malgorzata Elzbieta; Krauss, Stefan Johannes Karl; Wilson, Steven Ray Haakon & Røberg-Larsen, Hanne (2023). Building a high-resolution nano DESI-MS for organoid imaging: Analysis using a built and optimized imaging system. Fulltekst i vitenarkiv
• Pedersen, Gustav Mathingsdal; Kogler, Stian & Røberg-Larsen, Hanne (2023). On-line Sample Preparation for "Organ-in-a-Column". Fulltekst i vitenarkiv
• Tuner, Marius Bull; Kogler, Stian; Hansen, Frederik Andre; Pedersen-Bjergaard, Stig; Røberg-Larsen, Hanne & Wilson, Steven Ray Haakon (2023). Exploring electromembrane extraction and LC-MS for studying liver organoid metabolomics. Fulltekst i vitenarkiv
• Wilson, Steven Ray Haakon; Røberg-Larsen, Hanne; Krauss, Stefan Johannes Karl & Aizenshtadt, Aleksandra (2022). Organ-in-a-column - An automated on-line incubation and analysis system for drug metabolism studies. 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
• Kømurcu, Kristina Sæterdal; Aizenshtadt, Aleksandra; Thorne, James L; Krauss, Stefan Johannes Karl; Wilson, Steven Ray Haakon & Røberg-Larsen, Hanne (2022). Oxysterols are secreted from Non-alcoholic Fatty Liver disease induced organoids. Fulltekst i vitenarkiv
• Kømurcu, Kristina Sæterdal; Aizenshtadt, Aleksandra; Thorne, James L; Wilson, Steven Ray Haakon & Røberg-Larsen, Hanne (2022). Measuring oxysterols using LC-MS for the understanding of non alcoholic fatty liver disease (NAFLD). Fulltekst i vitenarkiv
• Kømurcu, Kristina Sæterdal; Aizenshtadt, Aleksandra; Thorne, James L; Krauss, Stefan Johannes Karl; Wilson, Steven Ray Haakon & Røberg-Larsen, Hanne (2022). Measuring oxysterols secreted from liver organoids by LC-MS for the understanding of Non-alcoholic fatty liver disease. Fulltekst i vitenarkiv
• midtøy, Lise; Kømurcu, Kristina Sæterdal; Kogler, Stian; wilhelmsen, Ingrid; Røberg-Larsen, Hanne & Wilson, Steven Ray Haakon (2022). Sensitive targeted proteomic analysis of cholesterol metabolism enzymes in hepatic stellate cells - going towards single cell analysis. Fulltekst i vitenarkiv
• Kømurcu, Kristina Sæterdal; Aizenshtadt, Aleksandra; Thorne, James L.; Krauss, Stefan Johannes Karl; Wilson, Steven Ray Haakon & Røberg-Larsen, Hanne (2022). Oxysterols are secreted from Non-Alcoholic Fatty Liver Disease (NAFLD) induced organoids. Fulltekst i vitenarkiv
• Tuner, Marius Bull; Kogler, Stian; Hansen, Frederik Andre; Pedersen-Bjergaard, Stig; Røberg-Larsen, Hanne & Wilson, Steven Ray Haakon (2022). Exploring electromembrane extraction for sampling the organoid metabolome. Fulltekst i vitenarkiv
• Røberg-Larsen, Hanne (2022). On-line prøveopparbeidelse – Hva er det og hvorfor trenger vi det? Fulltekst i vitenarkiv
• Kømurcu, Kristina Sæterdal; Aizenshtadt, Aleksandra; Thorne, James L; Krauss, Stefan Johannes Karl; Wilson, Steven Ray Haakon & Røberg-Larsen, Hanne (2022). Measuring oxysterols secreted from liver organoids by LC-MS for the understanding of Non-alcoholic fatty liver disease. 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
• Nordli, Henriette; Aizenshtadt, Aleksandra; Krauss, Stefan Johannes Karl; Wilson, Steven Ray Haakon & Røberg-Larsen, Hanne (2021). Oxysterols secreted from a Non-Alcoholic Fatty Liver Disease (NAFLD) induced liver organoids. Fulltekst i vitenarkiv
• Websdale, Alex; Hutchinson, S.A.; Lianto, P.; Røberg-Larsen, Hanne; Wastall, L.M. & Williams, B. [Vis alle 10 forfattere av denne artikkelen] (2020). Cholesterol side-chain hydroxylation is associated with expression of P-glycoprotein and disease-free survival in triple negative breast cancer patients. Proceedings of the Nutrition Society. ISSN 0029-6651. s. 1–1. doi: 10.1017/S0029665120007429. 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
• Røberg-Larsen, Hanne & Wilson, Steven Ray Haakon (2020). Low-flow Rate Separations of Lipids. I Griffiths, William & Wang, Yuqing (Red.), Lipidomics: Current and Emerging Techniques. Royal Society of Chemistry (RSC) Publishing. ISSN 9781788011600. s. 49–73. doi: 10.1039/9781788013109-00049. Fulltekst i vitenarkiv
• 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
• 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
• Røberg-Larsen, Hanne (2018). Prøvefiltrering - offline eller online? 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
• 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
• 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
• Røberg-Larsen, Hanne; Horne, Hildegunn; Holme, Ane Moe; Holm, Maia Blomhoff; Roland, Marie Cecilie & Michelsen, Trond [Vis alle 8 forfattere av denne artikkelen] (2017). Human in vivo studies of placenta physiology: oxysterols and metabolomic profiling. Fulltekst i vitenarkiv
• Wilson, Steven Ray Haakon; Vehus, Tore; Røberg-Larsen, Hanne & Lundanes, Elsa (2016). “Sub-chip” liquid chromatography-mass spectrometry for measuring oxysterols and related proteins. Fulltekst i vitenarkiv
• Brandtzæg, Ole Kristian Merkesvik; Johnsen, Elin Follaug; Røberg-Larsen, Hanne; Leknes, Siri; Lundanes, Elsa & Wilson, Steven Ray Haakon (2016). Robust nano liquid chromatography mass spectrometry system for targeted determination of oxytocin in human serum/plasma. Fulltekst i vitenarkiv
• Sæterdal, Kristina E.; Vehus, Tore; Røberg-Larsen, Hanne; Krauss, Stefan; Lundanes, Elsa & Wilson, Steven Ray Haakon (2016). CYP27A1 – its relation to oxysterols and cancer, and analytical challenges. Fulltekst i vitenarkiv
• Røberg-Larsen, Hanne; Lund, Kaja; Skottvoll, Frøydis Sved; Gregus, Michael; Lundanes, Elsa & Wilson, Steven Ray Haakon (2016). 27-hydroxycholesterol in cancer exosomes – potential new separation method? Fulltekst i vitenarkiv
• Wilson, Steven Ray Haakon; Lundanes, Elsa; Vehus, Tore & Røberg-Larsen, Hanne (2016). On-line sample preparation and liquid chromatography using "sub-chip" columns for ultra-sensitive proteomics. Fulltekst i vitenarkiv
• Vehus, Tore; Lundanes, Elsa; Røberg-Larsen, Hanne; Brandtzaeg, Ole Kristian & Wilson, Steven Ray Haakon (2016). Open tubular liquid chromatography-mass spectrometry: a powerful and versatile tool for bioanalysis. Fulltekst i vitenarkiv
• Abele, Silvija; Røberg-Larsen, Hanne; Dzabijeva, Diana; Demir, Deniz; Amundsen, Sunniva F. & Wilson, Steven Ray Haakon [Vis alle 8 forfattere av denne artikkelen] (2016). Simultaneous determination of pharmaceutical product residues in water by HPLC-MS/MS using online SPE. Fulltekst i vitenarkiv
• Røberg-Larsen, Hanne; Lund, Kaja; Sæterdal, Kristina Erikstad; Solheim, Stian; Vehus, Tore & Solberg, Nina [Vis alle 9 forfattere av denne artikkelen] (2016). Capillary LC mass spectrometric detection of 27-hydroxycholesterol in breast cancer exosomes. Fulltekst i vitenarkiv
• Røberg-Larsen, Hanne (2015). Highly automatic, sensitive nanoLC based determination of oxysterols in cancer cells. Fulltekst i vitenarkiv
• Wilson, Steven Ray Haakon; Lundanes, Elsa; Vehus, Tore; Røberg-Larsen, Hanne; Johnsen, Elin Follaug & Brandtzaeg, Ole Kristian (2015). Ultra-trace analysis of signal pathways: proteomics, lipidomics and neurotransmitters. Fulltekst i vitenarkiv
• Røberg-Larsen, Hanne; Lund, Kaja; skottvoll, Frøydis S; Krauss, Stefan; Lundanes, Elsa & Wilson, Steven Ray Haakon (2015). Oxysterols in exosomes. Fulltekst i vitenarkiv
• Røberg-Larsen, Hanne; Krauss, Stefan; Greibrokk, Tyge; Lundanes, Elsa & Wilson, Steven Ray Haakon (2014). Investigating the Role of Endogenous Small Molecules on the Hedgehog Signal Pathway: Very Sensitive, Highly Automated Nano-LC Based Determination of Oxysterols in Cancer Cells. Fulltekst i vitenarkiv
• Ruci, Gushi & Røberg-Larsen, Hanne (2025). Optimization of the Sample Preparation for the Chromatographic Separation and Mass Spectrometric Quantification of Oxysterols. Universitetet i Oslo. Fulltekst i vitenarkiv
• Midtøy, Lise & Røberg-Larsen, Hanne (2023). Evaluation of sample preparation methods for protein analysis of liver organoids. Universitetet i Oslo. Fulltekst i vitenarkiv Vis sammendrag

  For mass spectrometry (MS)-based proteomics, different sample preparation methods were evaluated for limited cell samples (10 000-200 000 cells), and the optimized method was used to analyze liver organoids (20-30 organoids, equivalent to approximately 50 000 cells). The sample preparation methods evaluated were urea-based in-solution-digestion (ISD-Urea), a detergent-free sample preparation method named sample preparation by easy extraction and digestion (SPEED), and a method with bead-based sample clean-up called single-pot, solid-phase enhanced sample preparation (SP3). The samples were analyzed with liquid chromatography (LC)-MS using a nanoLC column or a micropillar array column coupled to a trapped ion mobility time of flight (timsTOF)-MS. The samples prepared by SPEED had the overall highest number of protein identifications among all the sample sizes, with a range of 1684-3503 proteins identified for 10 000-100 000 cells. A quantification method for estimation of protein yield was found difficult, but because of the superior performance regarding number of protein identifications, SPEED was chosen as method for analyzing liver organoids of different metabolic states using label-free-quantification. The results showed significant differences between the organoid samples, with many of the hypothesized protein profiles found. The number of protein identifications was in the range of 2140-3203 proteins for the samples consisting of 20-30 organoids each. The method was successfully implemented as a preliminary step for disease studies, e.g. non-alcoholic fatty liver disease (NAFLD).

• Haavaag, Jon Erlend & Røberg-Larsen, Hanne (2023). Towards a high-resolution nano desorption electrospray ionization source for mass spectrometry imaging of organoids. Universitetet i Oslo. Fulltekst i vitenarkiv
• Pedersen, Gustav Mathingsdal; Røberg-Larsen, Hanne & Kogler, Stian (2023). Development of On-line Sample Clean-up System for Organ-in-a-Column. Universitetet i Oslo. Fulltekst i vitenarkiv
• Kvalvik, Eva; Røberg-Larsen, Hanne & Wilson, Steven Ray Haakon (2023). Method Development for Detection of Underivatized Oxysterols in Cell Medium. Universitetet i Oslo. Fulltekst i vitenarkiv Vis sammendrag

  Non-alcoholic fatty liver disease (NAFLD) is a disease caused by the accumulation of fat in the liver, mainly due to obesity and lifestyle. The disease is a worldwide health issue, with high prevalence and a lack of non-invasive biomarkers. To understand the disease, a model system is needed. The use of organoids, stem cell-derived three-dimensional cultures, is suggested as a model reflecting human physiology better than animal models. NAFLD can be induced in liver organoids. This enables the use of “Organ-in-a-Column” technology for studying the disease. Previous research has suggested that oxysterols are potential biomarkers of NAFLD. To make detection easier, oxysterols are usually derivatized in advance of analysis. “Organ-in-a-column” is an on-line system, in which organoids are coupled with Liquid Chromatography – Mass Spectrometry. The on-line approach makes sample preparation in the form of derivatization difficult. Therefore, this study has focused on method development for detecting underivatized oxysterols, with the aim of using the method in an “organ-in-a-column”-setup. Without a derivatization step in the sample preparation, the detection of oxysterols was challenging due to low sensitivity. Attempts of coeluting the groups of hydroxycholesterols and dihydroxycholesterols to establish a steatotic and control fingerprint from organoids with enhanced detection limit were partly successful for the analyte group of dihydroxycholesterols, but not the group of hydroxycholesterols. Sample clean-up was performed on-line using an automated filtration and filter flush solid phase extraction. This ensured robust analysis by the removal of particles before analysis. The optimized experimental parameters included a 5 𝜇L injection volume, a gradient elution utilizing isopropanol (IPA) as the organic modifier (20-65 %), 0.1 % formic acid for pH control in the mobile phase, and the use of a SuperPhenyl hexyl (2.1 mm x 5 cm) column at a temperature of 40 °C. The method allowed for detection of underivatized oxysterols in concentrations down to 0.050 𝜇g/mL in 10:90 IPA:Cell medium. The detection limit was too high to detect oxysterols secreted from liver organoids and the method needs further development before the organ-in-a-column approach can be used for disease monitoring. Also, adjustments to reduce the carry-over would be important to achieve a method providing reliable results.

• 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.

• 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
• 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
• 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
• 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
• 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.

• Sæterdal, Kristina Erikstad; Vehus, Tore; Røberg-Larsen, Hanne; Krauss, Stefan; Lundanes, Elsa & Wilson, Steven Ray Haakon (2016). Determination of CYP27a1 in biological samples using nano liquid chromatography mass spectrometry. Universitetet i Oslo. Fulltekst i vitenarkiv Vis sammendrag

  The cytochrome P450 enzyme, CYP27a1, converts cholesterol into 27-hydroxycholesterol, and both the metabolite and the enzyme are associated with breast cancer, promoting proliferation and metastasis. CYP27A1 is enriched in cancer tissues, and the amount of CYP27a1 correlates to tumor grade. Hence, CYP27a1 is a potential biomarker, and a method for determination and quantification is needed. Antibody assays shows limited performance, and therefore the goal of this work was to develop a nano liquid chromatography mass spectrometry (LC-MS) method for determination of CYP27a1. A sample preparation workflow for CYP27a1 in cell samples was investigated. CYP27a1 was identified by nano LC-MS in MDA-MB-231 breast cancer cells by fractionating cell lysates using gel electrophoresis. Enrichment of CYP27a1 in cell samples using immunoprecipitation, an antibody based method for protein extraction, did not provide adequate identification. However the former method works when looking at other proteins, and this confirms that there are limitations in using antibody based methods for determination and quantification of CYP27a1.

• Røberg-Larsen, Hanne; Lundanes, Elsa; Wilson, Steven Ray Haakon & Krauss, Stefan (2016). High sensitivity measurements of side-chain oxysterols using liquid chromatography mass spectrometry. Universitetet i Oslo. Fulltekst i vitenarkiv
• Vesterdal, Caroline; Røberg-Larsen, Hanne; Wilson, Steven Ray Haakon; Greibrokk, Tyge & Lundanes, Elsa (2015). Comparing methods for sensitive determination of Hedgehog active oxysterols. Universitetet i Oslo. Fulltekst i vitenarkiv Vis sammendrag

  Oxysterols in biological samples of limited size are present in low concentrations thus sensitive methods are advantageous. Derivatization of oxysterols prior to liquid chromatography (LC) mass spectrometry (MS) analysis is a common, however a laborious approach. The possibility to reduce the extent of sample preparation by formation of adducts between oxysterols and mobile phase additives (e.g. ammonium acetate) were investigated. Stable adduct ion signals were not obtained with the use of the MS instruments available. However, loss of water ions ([Oxysterol+H-H2O]+ and [Oxysterol+H-2H2O]+, m/z 385.35 and m/z 367.34) were observed when diluting oxysterol standards with ammonium formate (2.5 mM) and formic acid (0.25%) in methanol (MeOH). Under these conditions no easy recognizable fragmentation pattern was observed in tandem MS mode due to clustering of ions in the low mass area when fragmentation energy was applied. Another challenge with the analysis of native oxysterols with a nanoLC system was adsorption to surfaces, especially to fused silica capillaries. Silanization of the fused silica capillaries reduced the issue, but nowhere near satisfactory. It was considered unfeasible to determine native oxysterols with high sensitivity using nanoLC due to the large carry-over issues. Derivatization is therefore recommended when analyzing oxysterols with nanoLC. An on-line automatic filtration filter back-flush solid phase extraction liquid chromatography tandem mass spectrometry (AFFL-SPE-LC-MS/MS) method for determination of Girard T derivatized 24S-hydroxycholesterol, 25-hydroxycholesterol, 27-hydroxycholesterol, and 22S-hydroxycholesterol was modified for analysis of exosome samples. Best separation of oxysterol isomers was obtained with an ACE 3 C18 (0.1 mm ID × 150 mm, 3μm, 100 Å) column with a column temperature of 15°C and by using a mobile phase gradient from 0.1/25/75 (v/v/v %) FA/H2O/MeOH to 0.1/10/90 (v/v/v %) FA/H2O/MeOH in 25 minutes. The method was used for analyses of exosome samples obtained with the use of different isolation techniques to find a suitable procedure for exosome isolation.

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