Self-Cleaning LC–MS in Organoid/Organ-on-Chip Systems for Small Molecule Analysis

Steven Ray Wilson and Hanne Røberg-Larsen, both from the University of Oslo in Norway, discuss the practical application and advantages of their FDA-validated research to analyse small molecule drugs and their metabolites in organoid/organ-on-chip systems that serve as alternatives to animal models.

What was the rationale behind your recently published paper entitled: “An FDA-Validated, Self-Cleaning Liquid Chromatography–Mass Spectrometry (LC–MS) System for Determining Small-Molecule Drugs and Metabolites in an Organoid/Organ-on-Chip Medium (1)?”

Much of our work is dedicated to the study of organoids and organ-on-chip systems, which are laboratory-grown organ models. These models can serve as alternatives to animal models in drug development and disease studies. The number of groups working with these systems is rising in academia, start-ups, contract research organisations, and Big Pharma. There are also several regulatory pushes to reduce animal experiments. As the field is gradually maturing, there is a growing need for analytical chemistry and validated methods to obtain quality measurements. So, we set out to demonstrate the use of LC–MS-based measurements, focusing on obtaining a robust validation for a method featuring minimal sample preparation.

Can you elaborate on why the "self-cleaning" aspect is important?

The system we has features an on-line solid phase extraction. This saves time and often allows the handling of small samples. But, if the sample is complex, this might clog up the on-line system, making it a system that you swear at rather than swear by. But the automated filtration and filter-backflush (AFFL) feature of our system works so that anything that can potentially clog the system, is flushed out in another direction then the one it came from. This is a fairly simple trick with simple plumbing, but is a key to the robustness in our system.

Who did you collaborate with?

We have a long-standing collaboration with top-level biologists and microfluidics experts (Stefan Krauss, Aleksandra Aizenschtadt, and Mathias Busek) in a center of excellence, called the Hybrid Technology Hub. It is important for us to have close ties to biologists, and we often have our chromatography students engage in a bit of cell biology if it aligns with their research interests. This way, the chemists and the biologists get better at understanding each other's scientific dialects, so to speak.

What were the main analytical challenges you encountered and how did you overcome them?

The key challenges of organoids and organ-on-chip systems is that the samples can often be fairly small while being complex. The “blood” of these organ models is the cell culture medium. This matrix is stuffed with proteins, salts, amino acids, vitamins–you name it. We need to remove all of these components before doing our measurements of drugs and metabolites using LC–MS. If not, the columns can get clogged and the mass spectrometer can lose sensitivity.

What were your main findings?

We applied a sample preparation approach called AFFL, which was coupled on-line with solid phase extraction and LC. After extensive optimization, we established conditions that allowed for the complex medium to be injected directly, with the only addition step being the addition of an internal standard. We were pleased that the system could handle approximately 1,000 “messy” samples during the study without needing a single column replacement. If you take care in considering which columns, pore sizes, additives, you can use, it seems that you can actually reduce the number of pre-injection sample preparation steps.

It is great to see also in recent literature that scientists are taking a close look at problems and assumptions we have lived with for decades, for example, column breakthrough and the role of pore sizes and mobile phase additives for retention. Such fundamental studies of chromatography are so important, and we hope that funding agencies will (still) be able to see this.

Our system passed U.S. Food and Drug Administration (FDA) guidelines for method validation, tested with one drug and one metabolite. Obviously, this can´t be extrapolated to all analytes, but we hope that the study also will be a motivator for other scientists in organoids and organ-on-chip systems to prioritize this.

What are you working on next using chromatography?

We are continuing to develop the drug analysis platform and will now begin a downscaling to capillary LC format to reduce solvent consumption and maybe instrument size. We are also focusing on the measurements of pancreatic hormones in islet organoids and sterols in liver organoids. Our aim is to monitor the effects of “forever chemicals” on the body without the use of animals. Also, we are developing chip systems for online drug metabolism monitoring and are also doing more mass spectrometry imaging. But we are quite convinced that separation science is a key to our work, to ensure great selectivity, sensitivity and confidence in identifications. And on that note, we are also exploring simpler analysis methods using detectors such as ultraviolet. Obviously, that is not particularly applicable for “omics” work, but for targeted approaches with great separations a lot can be done.

Anything else you would like to add about this project?

We would like to thank our co-authors, especially Stian Kogler, who first-authored the work. He is quite an amazing guy! Felipe Martínez-Ramírez also made important contributions during a guest stay in our lab. Also, Gustav Mathingsdal Pedersen did a lot of key initial studies during his master's study. There are a lot of excellent young chemists out there!

Reference

(1) Wilson et al. An FDA-Validated, Self-Cleaning Liquid Chromatography–Mass Spectrometry (LC–MS) System for Determining Small-Molecule Drugs and Metabolites in an Organoid/Organ-on-Chip Medium. Anal. Chem. 2024, 96 (29), 12129–12138. DOI: 10.1021/acs.analchem.4c02246

About the Interviewees

Steven Ray Wilson is the research leader at the Department of Chemistry, University of Oslo. Dr. Wilson has during his career explored a number of hyphenated systems, including 2D LC and LC–NMR. Direct correspondence to: s.r.h.wilson@kjemi.uio.no

Hanne Røberg-Larsen is the leader of the bioanalytical chemistry group at the Department of Chemistry, University of Oslo. A key expertise of Dr. Røberg-Larsen is in developing and applying automated sample preparation-LC–MS systems, often with a focus on the role of sterols in disease development.