Fast and precise quantitative analysis of metabolic mixtures by 2D 1H INADEQUATE NMR

Non-linear effects in quantitative 2D NMR of polysaccharides: Pitfalls and how to avoid them

“…Even though the determination coefficient of the regression curve is excellent for all experiments and all signals, only 1D NMR leads to acceptable quantification results (error lower than 7%), while the deviation to the target concentration obtained by 2D NMR is always higher than 25%. These results clearly indicate that the standard addition approach is not suitable to quantify PRP by 2D NMR, contrary to what we recently described for small molecules [8,17]. Several hypotheses have been made in order to understand why the concentration was underestimated by 2D NMR.…”

“…1 [7,37]. Note that the INADEQUATE experiment is generally used to establish 13 C-13 C connectivities, but that its 1 H-1 H version is also a powerful analytical tool to simplify the homonuclear 2D spectra of complex mixtures [8,10], an approach similar to the Maximum Quantum strategy described by Caldarelli and co-workers [38]. The three experiments cited above have been characterized by excellent analytical performances (repeatability and linearity) on small molecules, and detailed information about the corresponding experiments can be found in the references mentioned above.…”

“…It has been shown that when standard additions or calibration curves are used, it is possible to work under partial saturation conditions, provided that T 1 s do not vary over the studied concentration range [8,17]. In order to ensure that it was not the case here, we measured the T 1 s of the different molecular sites on the initial solution and after the last standard addition.…”

“…From the quantitative point of view, 2D NMR has the high advantage of offering a much better discrimination of resonances than 1D NMR, as the peaks are spread along an additional orthogonal dimension. The use of 2D NMR for quantitative analysis has been particularly expanding during the last decade [5][6][7][8][9][10][11], a late development which is explained by two main drawbacks. The first one is the long experiment duration inherent to the time incrementation procedure associated with the acquisition of the indirect dimension.…”

“…It also makes it difficult to study samples whose composition evolves in the course of time under the effect of kinetic or dynamic processes. Fortunately, a number of strategies have been proposed to reduce the duration of quantitative 2D NMR experiments, from the careful optimization of acquisition and processing parameters [7,8,13,14] or the use of covariance spectroscopy [15,16] to the use of different sampling strategies such as ultrafast 2D NMR [12,17].…”