Computational simulation of ¹H NMR profiles of complex biofluid analyte mixtures at differential operating frequencies: Applications to low‐field benchtop spectra

Abstract

Estimations of accurate and reliable NMR chemical shift values, coupling patterns and constants within a reasonable timeframe remain significantly challenging, and the unavailability of reliable software strategies for the prediction of low-field (e.g., 60 MHz) spectra from those acquired at higher operating frequencies hampers their direct comparison. Hence, this study explored the applications of accessible software options for predicting these parameters in the 1 H NMR profiles of analytes as a function of magnetic field strength; this was performed for individual analytes and also for complex biofluid matrices featured in metabolomics investigations. For this purpose, results from the very first successful experimental acquisition and simulation of the 1 H NMR profiles of intact human salivary supernatant samples on a 60 MHz benchtop spectrometer were evaluated. Using salivary metabolite concentrations determined at 400 MHz, it was demonstrated that simulation of the low-field spectra of five biomolecules with the most prominent 1 H resonances detectable allowed multiple component fits to be applied to experimental spectra. Hence, these salivary 1 H NMR profiles could be successfully predicted throughout the 45-600 MHz operating frequency range. With the exception of propionate resonance multiplets, which revealed more complex coupling patterns at low field and required more astute computational and fitting options, valuable quantitative metabolomics data on salivary acetate, formate, methanol and glycine could be attained from low-field spectrometres. These studies are both timely and pertinent in view of the recent advancement of low-field benchtop NMR facilities for diagnostically significant biomarker tracking in biofluids. Experiments performed with added ammonium chloride to facilitate the release of salivary metabolites from biopolymer binding sites provided evidence that a small but nevertheless significant proportion of propionate, but not lactate, was bound to such sites, an observation of much relevance to biomolecule quantification in salivary metabolomics investigations.

Updates and Original Case Studies Focused on the NMR-Linked Metabolomics Analysis of Human Oral Fluids Part III: Implementations for the Diagnosis of Non-Cancerous Disorders, Both Oral and Systemic

“…Another alternative, very recently developed approach involves the future application of newly developed low-field (60–100 MHz), near-portable benchtop NMR spectrometers, which are cryogen-free, far less expensive, and very simple to operate and manage, even by previously untrained staff [ 1 , 105 ]. Such ‘state-of-the-art’ devices are easily installed and utilisable at patient ‘point-of-contact’ sites, e.g., local general practice and hospital clinics, dental surgeries, and community pharmacies, etc.…”

“…Such ‘state-of-the-art’ devices are easily installed and utilisable at patient ‘point-of-contact’ sites, e.g., local general practice and hospital clinics, dental surgeries, and community pharmacies, etc. However, although 12 or more metabolites are detectable in WMSS samples using this technique, currently limits of quantification dictate that only five of these (acetate, formate, propionate, glycine and methanol) may be determined in this biofluid when applied at an operating frequency of 60 MHz [ 105 , 106 ]. Indeed, for this biofluid, currently LF NMR spectroscopy is limited to metabolites with the most prominent signals, specifically those with clear singlets or simple first-order coupling patterns located within relatively uncrowded spectral regions.…”

Perspectives on Nuclear Magnetic Resonance Spectroscopy in Drug Discovery Research

“…An inspection of recently published articles shows benchtop NMR data contributing to the quantification of oxidation products, 123 mechanistic insights of synthetic reactions (Figure 33), 124 cycloaddition reaction monitoring, 125 and complex biofluid mixture analysis. 126 Throughout this perspective, we see that numerous NMR workflows are underpinned by computational methods to accelerate data analysis. Robust software solutions for processing, predicting, and analyzing NMR data as well as programs capable of generating 3D structures from NMR parameters are continually sought.…”

NMR Metabolomics Methods for Investigating Disease

“…This is because the spectral patterns and the number of observed peaks change as a function of field strength. Recently, a simulation of 1 H NMR spectra of metabolites was made to predict spectra of five dominant salivary metabolites from 45 to 600 MHz . A comparison of the simulated and experimental spectra obtained at 60 and 400 MHz was made.…”

“…Recently, a simulation of 1 H NMR spectra of metabolites was made to predict spectra of five dominant salivary metabolites from 45 to 600 MHz. 46 A comparison of the simulated and experimental spectra obtained at 60 and 400 MHz was made. Prediction of singlet peaks arising from acetate, formate, methanol, and glycine was straightforward.…”