Highly routinely reproducible alignment of 1H NMR spectral peaks of metabolites in huge sets of urines

A method to obtain high reproducibility of H-1 NMR chemical shift of peaks of biofluid metabolites, by simple acidification with HCl is evaluated. Biofluid H-1 NMR analysis is indeed spoiled by a strong chemical shift dependence of metabolite peaks on parameters such as ionic strength, concentration of some earth alkali cations and, mostly, on pH of samples. The resulting chemical shift variations, as large as 0.1 ppm, generate misalignments of homogeneous peaks, artifacts and misinterpretations. Reproducible alignment is essential in H-1 NMR based metabonomics, where peak misalignments prevent even very wide bins (i.e., 0.04 ppm, as elsewhere proposed) from being used to integrate spectral data for multivariate statistical analysis. Here is demonstrated that routine acidification with HCl to 1.2 <= pH <= 2.0 ensures highly reproducible peak alignment of urine H-1 NMR spectra. In this respect, simple inspection of citrate peaks in the urine can be used to measure pH, as it will be extensively discussed, in that at such low pH they show no dependency on other urine components as reported at higher pH. Under these conditions, in as many as 493 urine samples, in which concentrations of Ca2+, Mg2+, K+, Na+, Cl-, phosphate, and creatinine and ionic strength measured by means of well standardized conventional procedures, showed very wide ranges, peaks align within a SD always lower than 0.002 ppm, thus allowing the use of integration bins at least five times narrower than 0.04 ppm. (C) 2010 Elsevier B.V. All rights reserved.