The reliability and robustness of metabolite assignments in 1H NMR is complicated by numerous factors including variations in temperature, pH, buffer choice, ionic strength, and mixture composition that led to peak overlap and spectral crowding. As sample conditions fluctuate, peak drift and line broadening further complicate peak deconvolution and subsequent chemical assignment. We present a collection of 1D 1H NMR spectra of 54 common metabolites at varied pH (6.0 to 8.0 in 0.5 step increments) and temperature (290 K to 308 K) to quantify chemical shift variability to facilitate automated metabolite assignments. Our results illustrate the fundamental challenges with accurately assigning NMR peaks under varied environmental conditions prevalent in complex mixtures. Phosphorylated metabolites showed a larger variation in chemical shifts due to pH, whereas; amino acids showed a higher variation due to temperature. Mixtures of phosphorous compounds showed a consistently poor reliability in achieving an accurate assignment. Phosphorylated cholines, amino acids, and glycerols yielded a 40 % false negative rate for 7 out of 9 mixture conditions. Amino acids had a false negative rate of 57 % at 298 K and pH 8. Our results demonstrate that the automated assignments of complex biofluid mixtures require an expert to intervene to confirm the accuracy of metabolite assignments. Our analysis also indicates the need for reference databases to include spectra under a variety of conditions that includes mixtures and a range of pH and temperature to improve the accuracy and reproducibility of metabolite assignments.
Keywords: (1)H NMR; Automated metabolite assignment; Biofluids; Temperature; pH.
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