Organoids and organ-on-a-chip (OoC) are human-induced pluripotent stem cell-derived miniaturized organs that recapitulate organ specific functions. They open up a plethora of opportunities to change the way we test drugs, research diseases, and treat patients. In this context, liquid chromatography (LC) and mass spectrometry (MS) will have to be used, e.g. for monitoring drug metabolism, toxicity screens and organ function. On-line coupling of these technologies would allow for automatized, high-throughput systems. By downsizing the LC-MS part, low abundant analytes can also be determined. In this study, an on-line sampling and analysis system for OoC-technology has been developed. The system features hepatic organoids in a flow-through tube with continuous medium flow (organ-in-a-column, OiC), a two-loop valve system for sampling, reverse phase liquid chromatography (LC) analysis, and electrospray ionization mass spectrometry (ESI-MS). Phase 1 metabolism of heroin to its metabolite morphine was successfully measured on-line with the described system. Live/dead double staining revealed the potential of long-term cultivation of organoids in the system. In parallel, tin oxide coated fused silica nanoelectrospray ionization (nanoESI) emitters suitable for the most narrow bore chromatographic columns have been developed with HF etching and an atomic layer deposition (ALD) approach. The emitters have been tested against commercially available stainless steel (SS) emitters by injections of heroin and showed similar signal-to-noise ratios. These emitters will find a use in a future miniaturized version of the system described herein. In summary, a proof-of-concept system for on-line measurement of heroin metabolites has successfully been developed for a simple OoC-like device. NanoESI emitters for the most narrow columns have also been developed with a conductive ALD-coating of SnO2.