Stable super(18)O-labeling method for stercobilin and other bilins for metabolomics

Rationale Bilin tetrapyrroles including stercobilin are unique to mammalian waste; they have been used as markers of source water contamination and may have important diagnostic value in human health conditions. Unfortunately, commercial isotopomers for bilins are not available. Thus, there is a need for isotopomer standards of stercobilin and other bilins for quantification in environmental and clinical diagnostic applications. Methods A procedure is described here using H sub(2) super(18)O to label the carboxylic acid groups of bilin tetrapyrroles. Reaction conditions as a function of temperature and reagent volume were found to produce a mixture of isotopomers, as assessed by electrospray ionization and Fourier transform ion cyclotron resonance mass spectrometry (FTICRMS). Stability as a function of storage time and temperature and in conjunction with solid-phase extraction (SPE) was assessed. Results The highest labeling efficiency was achieved at 70 degree C for 8 h, while a stable ratio of the isotopmers could be produced at 60 degree C for 4 h. The stability of the isotopic distribution was maintained under storage (room temperature or frozen) for 20 days. It was also stable throughout SPE. The high mass accuracy and resolving power of FTICRMS enables clear distinction between super(18)O-labeled bilins from other unlabeled bilins present, avoiding a potential interference in quantitation. Conclusions A procedure was developed to label bilins with super(18)O. The final ratio of the super(18)O-labeled bilin isotopomers was reproducible and highly stable for at least 20 days under storage. This ratio was not changed in any statistically significant way even after SPE. Thus a reliable method for producing stable isotopomer ratios for bilins has been achieved.