Optimization of NMR analysis of biological fluids for quantitative accuracy

Abstract

With the rising interest in the use of nuclear magnetic resonance (NMR) for the study of biological fluids such as urine and serum for metabonomic or diagnostic purposes, new challenges have arisen concerning the efficacy of NMR data acquisition and analysis. In particular the quantification of sample constituents such as metabolites is of great importance. This study compares five one-dimensional proton NMR pulse sequences using synthetic urine samples to determine appropriate acquisition parameters for reasonable sample throughput and accuracy. Each pulse sequence has its own advantages and limitations with respect to solvent suppression, stable baseline, exchangeable protons, and quantization of resonances near the residual water peak. Hardware issues such as low-pass filters, unique to each spectrometer, also impact quantitation accuracy. Metabolite concentrations were determined using integration referenced to an added internal standard, and using the Chenomx NMR Suite software package. Since nuclei in different metabolites and the internal standard all have different longitudinal relaxation rates (T 1 ) we included a mathematical correction factor for quantitation.

Dolphin: a tool for automatic targeted metabolite profiling using 1D and 2D 1H-NMR data

“…On the other hand, and due to the shape constraints of its spectral library, Chenomx is the tool that usually presents higher quantifying errors for these metabolites. Chenomx uses reference deconvolutions to adjust the shim for the spectra in its library for all metabolites, but it is only effective in removing line-shape distortions that affect all signals in the spectrum equally [33]; in addition, there are other factors affecting the shape of he signals [34,35], especially if they are unusually intense and, consequently, the Chenomx library signals rarely fit accurately such peaks present in the real spectrum. This lack of shape flexibility makes it difficult to achieve a precise quantification and increases error.…”

Metabolite profiling of the intraerythrocytic malaria parasite Plasmodium falciparum by 1H NMR spectroscopy

“…The intracellular concentrations for over 40 of these compounds were estimated using Chenomx NMR suite software. This software allowed estimates of the concentrations of compounds in the extracts to be made from the complex 1D 1 H NMR spectra in a more convenient and reliable way than with the traditional approach of integration of specific NMR signals (13,31,41). The NMR techniques used in this study are less sensitive than the alternative mass spectrometry methods, but do offer the advantage that prior chromatography and/or derivatisation procedures are not required.…”

Ubiquinone-binding Site Mutations in the Saccharomyces cerevisiae Succinate Dehydrogenase Generate Superoxide and Lead to the Accumulation of Succinate

“…All of the spectra were acquired on a 600-MHz Inova NMR spectrometer at 25°C using the tn noesy pulse sequence (circa Vnmr 6.1B software, Varian Inc). All of the spectra had an acquisition time of 4 s, a preacquisition delay of 1 s, a mixing time of 0.1 s, a sweep width of 7200 Hz, and 256 transients (42). All of the spectra were analyzed using the Chenomx NMR Suite Professional software version 4.5.…”

“…Cytochrome spectra of standards, and then the absolute concentrations were calculated by comparison with the added reference peak (42). We identified succinate and fumarate in the 1 H NMR spectra of the culture medium after growth of the strains.…”