Quantitative nuclear magnetic resonance (NMR) for isotopic
measurements,
known as irm-NMR (isotope ratio measured by NMR), is well suited for
the quantitation of 13C-isotopomers in position-specific
isotope analysis and thus for measuring the carbon isotope composition
(δ13C, mUr) in C-atom positions. Irm-NMR has already
been used with glucose after derivatization to study sugar metabolism
in plants. However, up to now, irm-NMR has exploited a “single-pulse”
sequence and requires a relatively large amount of material and long
experimental time, precluding many applications with biological tissues
or extracts. To reduce the required amount of sample, we investigated
the use of 2D-NMR analysis. We adapted and optimized the NMR sequence
so as to be able to analyze a small amount (10 mg) of a glucose derivative
(diacetonide glucofuranose, DAGF) with a precision better than 1 mUr
at each C-atom position. We also set up a method to correct raw data
and express 13C abundance on the usual δ13C scale (δ-scale). In fact, due to the distortion associated
with polarization transfer and spin manipulation during 2D-NMR analyses,
raw 13C abundance is found to be on an unusual scale. This
was compensated for by a correction factor obtained via comparative
analysis of a reference material (commercial DAGF) using both previous
(single-pulse) and new (2D) sequences. Glucose from different biological
origins (CO2 assimilation metabolisms of plants, namely,
C3, C4, and CAM) was analyzed with the two sequences
and compared. Validation criteria such as selectivity, limit of quantification,
precision, trueness, and robustness are discussed, including in the
framework of green analytical chemistry.