NMR spectroscopy for mixture analysis – both a blessing and a curse: dissecting cell growth media as an example

Nuclear magnetic resonance (NMR) spectroscopy is a powerful non-targeted analytical tool for compositional profiling of complex mixtures, such as cell culture media, offering high-throughput capabilities that support rapid assessment across large sample cohorts. Its sensitivity to proton-containing metabolites enables the detection of both expected and unforeseen components. However, its application can evoke a ‘Pandora’s box’ effect, revealing unanticipated complexities that challenge conventional interpretative frameworks. Here, NMR analysis identified unexpected media ‘ingredients’ such as substantial ethanol concentrations (up to >10 mM) across all samples, likely resulting from rigorous efforts to avoid bacterial contamination. This highlights the critical vulnerability of small-volume systems, such as multi-well plates, to ethanol contamination by routine disinfection that can disproportionately affect medium composition, potentially compromising cellular viability. Furthermore, NMR spectra revealed time-dependent degradation of L-glutamine into pyroglutamic acid, providing a tool to assess media freshness and experimental reproducibility. Surprisingly, significant lactate levels were already detected in unspent media, ultimately traced to foetal bovine serum, a commonly used but ethically debated supplement. Alongside these unexpected outcomes, NMR was successfully utilised to analyse pyruvate and citric acid to understand culture metabolic state and to validate core metabolic assumptions. Our findings also underscore the necessity of critically evaluating multivariate data through direct spectral inspection; overlapping or shifting peaks may otherwise be misinterpreted. In conclusion, even though NMR spectroscopy is an intrinsically insensitive method and signal overlap of similar molecules in mixtures can hamper unambiguous assignment, it still remains one of the most powerful tools in mixture analysis – even if it may unveil complicating variables constantly raising new questions. Its comprehensive, quantitative nature far outweighs its challenges, offering invaluable insights into media integrity and cellular environments.