Nano liquid chromatography (nanoLC), with columns having an inner diameter (ID) of ≤100 μm, can provide enhanced sensitivity and enable analysis of limited samples.

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Prior to mass spectrometry, on-line sample preparation can be beneficial to reduce manual steps, increase speed, and enable analysis of limited sample amounts. For example, bottom-up proteomics sample preparation and analysis can be accelerated by digesting proteins to peptides in an on-line enzyme reactor. We here focus on low-backpressure 100 μm inner diameter (ID) × 160 mm, 180 μm ID × 110 mm or 250 μm ID × 140 mm vinyl azlactone-co-ethylene dimethacrylate [poly(VDM-co-EDMA)] monoliths as supports for immobilizing of additional molecules (i.e., proteases or drugs), as the monolith was expected to have few unspecific interactions. For on-line protein digestion, monolith supports immobilized with trypsin enzyme were found to be suited, featuring the expected characteristics of the material, i.e., low backpressure and low carry-over. Serving as a functionalized sample loop, the monolith units were very simple to connect on-line with liquid chromatography. However, for on-line target deconvolution, the monolithic support immobilized with a Wnt pathway inhibitor was associated with numerous secondary interactions when exploring the possibility of selectively trapping target proteins by drug-target interactions. Our initial observations suggest that (poly(VDM-co-EDMA)) monoliths are promising for e.g., on-line bottom-up proteomics, but not a “fit-for-all” material. We also discuss issues related to the repeatability of monolith-preparations.

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For studying stem cell–derived islet organoids (SC‐islets) in an organ‐on‐chip (OoC) platform, we have developed a reversed‐phase liquid chromatography–tandem mass spectrometry (RPLC–MS/MS) method allowing for simultaneous determination of insulin, somatostatin‐14, and glucagon, with improved matrix robustness compared to earlier methodology. Combining phenyl/hexyl‐C18 separations using 2.1 mm inner diameter LC columns and triple quadrupole mass spectrometry, identification and quantification were secured with negligible variance in retention time and quantifier/qualifier ratios, negligible levels of carryover (<2%), and sufficient precision (±10% RSD) and accuracy (±15% relative error) with and without use of an internal standard. The obtained lower limits of quantification were 0.2 µg/L for human insulin, 0.1 µg/L for somatostatin‐14, and 0.05 µg/L for glucagon. The here‐developed RPLC–MS/MS method showed that the SC‐islets have an insulin response dependent on glucose concentration, and the SC‐islets produce and release somatostatin‐14 and glucagon. The RPLC–MS/MS method for these peptide hormones was compatible with an unfiltered offline sample collection from SC‐islets cultivated on a pumpless, recirculating OoC (rOoC) platform. The SC‐islets background secretion of insulin was not significantly different on the rOoC device compared to a standard cell culture well‐plate. Taken together, RPLC–MS/MS method is well suited for multi‐hormone measurements of SC‐islets on an OoC platform.