Electromembrane Extraction and Mass Spectrometry for Liver Organoid Drug Metabolism Studies

Abstract

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.

Metabolomics-based mass spectrometry methods to analyze the chemical content of 3D organoid models

“…The potential of using liver organoids with LC-MS measurements to perform drug metabolism studies has been shown by Skottvoll et al 38 To study the phase I metabolism of heroin to 6-monoacetylmorphine (6-MAM) and morphine, liver organoids were exposed to the drug for 1, 3, 6, and 24 hours, with the concentrations of heroin and 6-MAM tracked during those timepoints. The authors developed an electromembrane extraction approach to separate drug metabolites from components of cell culturing media, allowing for simple and repeatable measurements.…”

Direct Electromembrane Extraction‐Based Mass Spectrometry: A Tool for Studying Drug Metabolism Properties of Liver Organoids

“…E) Drug studies performed on chip (single-sample dEME) with continuous EME extraction and no manual sample handling prior to LC-MS analysis (this Communication). Figure adapted/expanded from our previous work [12] (further permissions related to the material excerpted should be directed to the American Chemical Society). solution was continuously pumped through the acceptor channel.…”

“…We have recently shown that off-line EME is excellent for simple and parallel extraction of drugs and metabolites from organoids in a 96-well format, reducing sample preparation steps (Figure 1D). [12] However, on-line coupling of EME and MS would allow for a substantially higher degree of automation and throughput. EME has been briefly explored for on-line coupling to MS for microsome-based studies, [13][14][15] encouraging us to hypothesize that EME is a potential tool for the bridging of liver organoids and MS.…”

“…C) Schematic overview of drug studies performed with simple sample handling using centrifugation and manual pipetting prior to analysis [6]. D) Drug studies performed with high throughput sample handling using parallel-EME with reduced steps of sample handling prior to analysis (multi-sample EME) [12]. E) Drug studies performed on chip (single-sample dEME) with continuous EME extraction and no manual sample handling prior to LC-MS analysis (this Communication).…”

“…After successful initial tests, the cell culture chamber would be filled with 50 liver organoids and mixed with methadone, typically in the low μM range, in accordance with our previous work. [6,12] The system was used to test drug metabolism of methadone to its metabolites EDDP and EMDP by liver organoids. Figure 4 shows clean LC-MS chromatograms of the methadone, EDDP and EMDP 24 hours after liver organoid exposure to 1 μM methadone, illustrating activity of the CYP3A4 enzyme.…”