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.
Highlights
The process of drug development is known to be timeconsuming and bear financial uncertainties.[1,2] It is estimated that from 5000 to 10 000 new molecular entities, only one new drug will enter the market.[3]
A conventional ultrahigh-performance liquid chromatography (UHPLC)-mass spectrometry (MS) method used for clinical routine analyses was applied to explore the heroin-metabolizing properties of the parallel-electromembrane extraction (EME) extracted liver organoids
To evaluate the potential of MS for the analysis of liver organoids, heroin was chosen as a model substance due to its familiar phase I metabolism to 6MAM and morphine in the liver
Summary
The process of drug development is known to be timeconsuming and bear financial uncertainties.[1,2] It is estimated that from 5000 to 10 000 new molecular entities, only one new drug will enter the market.[3]. Two stock solutions of the internal standards heroin-d9, 6-MAM-d6, and morphine-d3 were prepared in 5 mM ammonium formate buffer pH 3.1 with analyte concentration of 1.5 μM each and 3 μM each, respectively, and stored at 4 °C. 50 μL of the heroin-exposed liver organoid samples (containing 0.11 M FA) was added to 40 μL of water and 10 μL of the 1.5 μM or 3 μM internal standard solution. Determination of heroin, 6-MAM, and morphine was performed using UHPLC-MS based on a previously described method.[41] The sample extracts were diluted ×10 with 5 mM ammonium formate pH 3.1 and analyzed using an Acquity UHPLC pump coupled to a Xevo. The extracted organoid samples (AG27 iPSC derived) were further diluted ×103 in 5 mM of ammonium formate pH 3.1 buffer, and the injection volume was set to 2 μL.
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