Compound-SpecificCarbon and Nitrogen Isotopic Analysesof Underivatized Pyrimidine and Purine Nucleobases

We report on a newly developed method for compound-specific isotopic analysis (CSIA) of carbon (δ¹³C) and nitrogen (δ¹⁵N) in underivatized pyrimidine (cytosine, uracil, and thymine) and purine (adenine, guanine, hypoxanthine, and xanthine) nucleobases. The nucleobases are isolated by multistep purification using high-performance liquid chromatography (HPLC). HPLC separation and its wet chemical pretreatments were optimized to isolate these nucleobases without the need for chemical derivatization. Subsequently, the carbon and nitrogen isotopic compositions of these underivatized nucleobases were analyzed by nanoscale elemental analysis/isotope-ratio mass spectrometry (nano-EA/IRMS). The δ¹³C and δ¹⁵N measurements of isolated standard nucleobases indicated minimal isotopic fractionation during the two-step HPLC purification. This HPLC × nano-EA/IMS methodology was utilized to determine the isotopic composition of nucleobases in spinach (δ¹³C: −27.0 to −19.8‰, δ¹⁵N: +2.8 to +6.7‰) and coastal sediment (δ¹³C: −14.9 to −9.2 ‰, δ¹⁵N: +4.5 to +9.0‰) samples. The CSIA of nucleobases can provide insight into how living organisms modulate their biochemical pathways in response to environmental and metabolic variations.