Stable isotope determination of ester and ether methyl moieties in plant methoxyl groups

Plant methoxyl groups of lignin and pectin have both distinct stable hydrogen isotope (δ²H) and carbon isotope (δ¹³C) values that can be used for studying environmental processes and for investigating the origin and authenticity of biomaterials. Up to now, the reported methods have been applied only to determine isotope values of the bulk plant methoxyl pool. In this work, we have applied several methods to distinguish between stable isotope ratios of methoxyl groups of pectin and the bulk plant methoxyl pool. Our results demonstrate that by applying alkaline hydrolysis to specifically cleave off the ester methyl moiety (pectin-like), we can distinguish δ²H and δ¹³C values of the pectin methoxyl pool from the bulk methoxyl pool. No measureable isotope discrimination was observed either during sample preparation or during analytical measurement. Furthermore, using this method, no major isotope difference in either the hydrogen or carbon isotope signature of the methoxyl groups of plant pectin and bulk matter from plant species such as leaves from trees, apples, carrots and potatoes was noted. We show the methanol released during alkaline hydrolysis of plant material and subsequently treated with hydriodic acid to be an excellent procedure to measure specifically and precisely the δ¹³C and δ²H isotope values of plant pectin-like methoxyl groups. This method is particularly advantageous when plant matter with a low methoxyl content has to be analysed.