A compound-specific isotope method for measuring the stable nitrogen isotopic composition of tetrapyrroles

The stable nitrogen isotopic signature of natural tetrapyrroles such as chloro- and pheo-pigments and alkylporphyrins is a potentially valuable proxy for the reconstruction of the nitrogen cycle of modern aquatic environments and of the geological past. However, its application has been limited due to analytical difficulties. In this study, we developed a method to determine stable nitrogen isotopic composition of tetrapyrroles by a combination of chemical conversion into monopyrrole units (i.e. maleimides) and their isotope analysis by gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS). Two authentic (pyropheophorbide a and mesoporphyrin IX methylester) and four natural (chlorophyll a, deoxophylloerythroetioporphyrin (DPEP), and two 17-nor-DPEP) tetrapyrroles were converted to maleimides by HCl treatment and chromic acid oxidation. The stable nitrogen isotopic composition of the maleimides can be determined by GC/C/IRMS, with a standard deviation (1σ) of better than ± 0.5‰ and the minimum sample amount of 0.8 nmol N. The isotopic composition of the maleimides is consistent with that of the original tetrapyrroles, suggesting nitrogen isotopic fractionation during the chemical treatment was insignificant. Further, no substantial difference is observed in the isotopic composition among the maleimides derived from a single tetrapyrrole. The developed method is applicable for the nitrogen isotope analysis of natural tetrapyrroles in various biological and geological samples.