ANYL 191

A number of endogenous metabolites characterized by their mass/retention time pairs were identified as important markers of variance from a metabolomic analysis of urine using advanced statistical methods. Many of these compounds can be identified by simple database searching. However, some of these markers are unknown and their structure cannot be elucidated by mass spectrometry alone. One such compound has an m/z of 297.1450 Da and is a common unidentified constituent of mammalian urine. An LC/MS fraction collection system was used to isolate this important and novel endogenous metabolite from urine samples. Employing an automated approach, metabolites identified by their mass/retention time pairs, are entered into a list and collected into SPE plates without user intervention. The same metabolite from multiple injections was collected into a single well to achieve adequate sample quantity. The use of sub-2µm packing material produced extremely sharp chromatographic peaks which were successfully collected using a sampling frequency of 3 seconds. Thus the isolation of a reasonably pure fraction of the metabolites of interest was achieved in a relatively quick and efficient manner. These purified samples were then analyzed by NMR which, in conjunction with the previously obtained mass spectral data allowed for the elucidation of the structure of the unknown.