Increasing donor risk, particularly in liver transplantation, where organs are often marginal, has made dynamic organ preservation techniques and viability assessment essential to safely improve organ quality and increase utilisation. However, existing viability parameters are based on routine clinical assessment in patients with acute liver failure, trauma, or liver resections. These parameters often do not correlate with clinically relevant post-transplant outcomes. This article presents a detailed protocol for the spectrophotometric quantification of Flavin mononucleotide (FMN), a marker of mitochondrial injury. FMN release from mitochondrial complex I was described many decades ago as the initial sign of ischaemia-reperfusion injury, i.e. when oxygen is reintroduced in ischaemic tissues during organ transplantation or machine perfusion. This study describes the detailed FMN quantification in donor plasma and various fluids obtained during machine perfusion, and discusses confounders, challenges, and the role of individual test components. FMN quantification was identified as an immediate organ assessment tool, demonstrating a strong correlation with graft survival and other relevant complications after human liver transplantation. The results highlight FMN quantification as a reliable and standardized method for assessing organ viability, offering significant potential for improving organ selection and better utilisation. This method could provide better a predictive value for transplant outcomes compared to existing parameters currently in use. This research received no external funding but was supported by the Catalyst grant No. CCG0280 at Cleveland Clinic Ohio, U.S. dedicated to A.S.

Ex situ machine perfusion has emerged as a pivotal technique for organ preservation and pre-transplant viability assessment, where the real-time monitoring of mitochondrial biomarkers—flavin mononucleotide (FMN) and nicotinamide adenine dinucleotide (NADH)—could significantly mitigate ischemia-reperfusion injury risks. This study develops a non-invasive optical method combining fluorescence and UV-visible spectrophotometry to quantify FMN and NADH in hypothermic oxygenated perfusion media. Calibration curves revealed linear responses for both biomarkers in absorption and fluorescence (FMN: λ_ex = 445 nm, λ_em = 530–540 nm; NADH: λ_ex = 340 nm, λ_em = 465 nm) at concentrations < 100 μg mL⁻¹. However, NADH exhibited nonlinear fluorescence above 100 μg mL⁻¹, requiring shifted excitation to 365 nm for reliable detection. Spectroscopic analysis further demonstrated how perfusion solution composition alters FMN/NADH fluorescence properties, with consistent reproducibility across media. The method’s robustness was validated through comparative studies in clinically relevant solutions, proposing a strategy for precise biomarker quantification without invasive sampling. These findings establish a foundation for real-time, optical biosensor development to enhance organ perfusion monitoring. By bridging spectroscopic principles with clinical needs, this work advances translational sensor technologies for transplant medicine, offering a template for future device integration.