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.

Department of Analytical Chemistry Faculty of Science Charles University Prague CZ 128 43 Czech Republic

Hybrid Technology Hub Centre of Excellence Institute of Basic Medical Sciences Faculty of Medicine University of Oslo Oslo 0372 Norway

Section for Chemical Life Sciences Department of Chemistry University of Oslo Oslo NO 0315 Norway

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Zhao Z.; Chen X.; Dowbaj A. M.; Sljukic A.; Bratlie K.; Lin L.; Fong E. L. S.; Balachander G. M.; Chen Z.; Soragni A.; Huch M.; Zeng Y. A.; Wang Q.; Yu H. Organoids. Nat. Rev. Methods Primers 2022, 2, 94.10.1038/s43586-022-00174-y. PubMed DOI PMC

Leung C. M.; de Haan P.; Ronaldson-Bouchard K.; Kim G.-A.; Ko J.; Rho H. S.; Chen Z.; Habibovic P.; Jeon N. L.; Takayama S.; Shuler M. L.; Vunjak-Novakovic G.; Frey O.; Verpoorte E.; Toh Y.-C. A guide to the organ-on-a-chip. Nat. Rev. Methods Primers 2022, 2, 33.10.1038/s43586-022-00118-6. DOI

Vandana J. J.; Manrique C.; Lacko L. A.; Chen S. Human pluripotent-stem-cell-derived organoids for drug discovery and evaluation. Cell Stem Cell 2023, 30, 571–591. 10.1016/j.stem.2023.04.011. PubMed DOI PMC

Shoji J.; Davis R. P.; Mummery C. L.; Krauss S. Global Meta-Analysis of Organoid and Organ-on-Chip Research. Adv. Healthcare Mater. 2023, 2301067.10.1002/adhm.202301067. PubMed DOI

FDA . No Longer Needs to Require Animal Tests before Human Drug Trials; Science. https://www.science.org/content/article/fda-no-longer-needs-require-animal-tests-human-drug-trials.

Ingber D. E. Human organs-on-chips for disease modelling, drug development and personalized medicine. Nat. Rev. Genet. 2022, 23, 467–491. 10.1038/s41576-022-00466-9. PubMed DOI PMC

Kogler S.; Kømurcu K. S.; Olsen C.; Shoji J.; Skottvoll F. S.; Krauss S.; Wilson S. R.; Røberg-Larsen H. Organoids, organ-on-a-chip, separation science and mass spectrometry: An update. TrAC, Trends Anal. Chem. 2023, 161, 116996.10.1016/j.trac.2023.116996. DOI

Olsen C.; Wang C.; Aizenshtadt A.; Abadpour S.; Lundanes E.; Skottvoll F. S.; Golovin A.; Busek M.; Krauss S.; Scholz H.; Wilson S. R. Simultaneous LC-MS determination of glucose regulatory peptides secreted by stem cell-derived islet organoids. Electrophoresis 2023, 44, 1682–1697. 10.1002/elps.202300095. PubMed DOI

Kogler S.; Aizenshtadt A.; Harrison S.; Skottvoll F. S.; Berg H. E.; Abadpour S.; Scholz H.; Sullivan G.; Thiede B.; Lundanes E.; Bogen I. L.; Krauss S.; Røberg-Larsen H.; Wilson S. R. Organ-in-a-Column” Coupled On-line with Liquid Chromatography-Mass Spectrometry. Anal. Chem. 2022, 94, 17677–17684. 10.1021/acs.analchem.2c04530. PubMed DOI PMC

Hrušková H.; Olsen C.; Řemínek R.; Wang C.; Aizenshtadt A.; Krauss S.; Scholz H.; Røberg-Larsen H.; Foret F.; Wilson S. R. Preparative agarose gel electrophoresis for reducing matrix interferences of organoid cell medium prior to LC-MS analysis of insulin. J. Chromatogr. A 2024, 1717, 464669.10.1016/j.chroma.2024.464669. PubMed DOI

Skottvoll F. S.; Aizenshtadt A.; Hansen F. A.; Martinez M. A.; Andersen J. M.; Bogen I. L.; Kutter J. P.; Pedersen-Bjergaard S.; Lundanes E.; Krauss S.; Wilson S. R. Direct Electromembrane Extraction-Based Mass Spectrometry: A Tool for Studying Drug Metabolism Properties of Liver Organoids. Analysis Sensing 2022, 2, e20210005110.1002/anse.202100051. DOI

Svendsen K. O.; Larsen H. R.; Pedersen S. A.; Brenna I.; Lundanes E.; Wilson S. R. Automatic filtration and filter flush for robust online solid-phase extraction liquid chromatography. J. Sep. Sci. 2011, 34, 3020–3022. 10.1002/jssc.201100553. PubMed DOI

Kømurcu K. S.; Wilhelmsen I.; Thorne J. L.; Krauss S.; Wilson S. R.; Aizenshtadt A.; Røberg-Larsen H. Mass spectrometry reveals that oxysterols are secreted from non-alcoholic fatty liver disease induced organoids. J. Steroid Biochem. Mol. Biol. 2023, 232, 106355.10.1016/j.jsbmb.2023.106355. PubMed DOI

LaLone V.; Aizenshtadt A.; Goertz J.; Skottvoll F. S.; Mota M. B.; You J.; Zhao X.; Berg H. E.; Stokowiec J.; Yu M.; Schwendeman A.; Scholz H.; Wilson S. R.; Krauss S.; Stevens M. M. Quantitative chemometric phenotyping of three-dimensional liver organoids by Raman spectral imaging. Cells Rep. Methods 2023, 3, 100440.10.1016/j.crmeth.2023.100440. PubMed DOI PMC

Busek M.; Aizenshtadt A.; Koch T.; Frank A.; Delon L.; Martinez M. A.; Golovin A.; Dumas C.; Stokowiec J.; Gruenzner S.; Melum E.; Krauss S. Pump-less, recirculating organ-on-a-chip (rOoC) platform. Lab Chip 2023, 23, 591–608. 10.1039/d2lc00919f. PubMed DOI

FDA’s Guidelines for Bioanalytical Method Validation. https://www.fda.gov/media/70858/download.

Abadpour S.; Niemi E. M.; Orrhult L. S.; Hermanns C.; de Vries R.; Nogueira L. P.; Haugen H. J.; Josefsen D.; Krauss S.; Gatenholm P.; et al. Adipose-Derived Stromal Cells Preserve Pancreatic Islet Function in a Transplantable 3D Bioprinted Scaffold. Adv. Healthcare Mater. 2023, 12, 2300640.10.1002/adhm.202300640. PubMed DOI PMC

The Fitness for Purpose of Analytical Methods A Laboratory Guide to Method Validation and Related Topics by EuChem, n.d. EuChem webpage. https://www.eurachem.org/images/stories/Guides/pdf/MV_guide_2nd_ed_EN.pdf.