Comparing ¹³C methyl and deuterated methyl isotopic labeling for the quantification of methyl cellulose patterns...

The methyl substitution along and among the polymer chains of methyl cellulose (MC) is commonly analyzed by ESI-MS after perdeuteromethylation of the free-OH groups and partial hydrolysis to cello-oligosaccharides (COS). This method requires a correct quantification of the molar ratios of the constituents belonging to a particular degree of polymerization (DP). However, isotopic effects are most pronounced for H/D since their mass difference is 100%. Therefore, we investigated whether more precise and accurate results could be obtained for the methyl distribution of MC by MS of ¹³CH₃ instead of CD₃-etherified O-Me-COS. Internal isotope labeling with ¹³CH₃ makes the COS of each DP chemically and physically much more similar, reducing mass fractionation effects, but at the same time requires more complex isotopic correction for evaluation. Results from syringe pump infusion ESI-TOF-MS with ¹³CH₃ and CD₃ as isotope label were equal. However, in the case of LC-MS with a gradient system, ¹³CH₃ was superior to CD₃. In the case of CD₃, the occurrence of a partial separation of the isotopologs of a particular DP resulted in slight distortion of the methyl distribution since the signal response is significantly dependent on the solvent composition. Isocratic LC levels this problem, but one particular eluent-composition is not sufficient for a series of oligosaccharides with increasing DP due to peak broadening. In summary, ¹³CH₃ is more robust to determine the methyl distribution of MCs. Both syringe pump and gradient-LC-MS measurements are possible, and the more complex isotope correction is not a disadvantage.