An automated 1D 1H and 2D 1H-13C HSQC NMR platform for high-field (900 MHz) metabolite profiling of cerebrospinal fluid

NMR spectra from cerebrospinal fluid (CSF) samples (n=25) were acquired using a high-field spectrometer at 900 MHz. The digital platform BioMetIQ was developed for 1D 1 H and 2D 1 H- 13 C HSQC experiments to characterize the metabolome in biofluids. To identify and quantify metabolites, the platform integrates an adaptive pseudo-Voigt synthetic peak model and an algorithm for matching metabolite peaks to spectral databases. Up to 54 metabolites were identified, including 48 by 1 H analysis, establishing the first fully annotated 1 H NMR spectrum of CSF at 900 MHz. Six metabolites were explicitly identified using 2D 1 H- 13 C HSQC spectra, resolving assignment ambiguities in dense 1 H spectral regions. Among the identified metabolites, 35 were present in all samples, demonstrating the method's robustness. In addition, 2-hydroxyisobutyric acid, not previously reported in CSF, was consistently detected in all samples. The 48 metabolites quantified by 1 H NMR exhibited concentrations ranging from 0.07 ± 0.03 μM (trimethylamine) to 2810 ± 990 μM (lactic acid). Across clinically heterogeneous cohorts, inter-individual coefficients of variation were lower than previous reports, consistent with higher precision for low metabolite concentrations in the 1-50 μM range. By offering a documented and reproducible approach to spectral analysis, BioMetIQ, combined with high-field NMR spectroscopy, establishes a new milestone for the characterization of the biofluid metabolome.