Studies On Aromatic Metabolites By LC-MS And NMR

Aromatic metabolites in the urine are mainly derived from the digestion and absorption of aromatic amino acids and polyphenols in intestine. Tryptophan, phenylalanine and tyrosine are the three aromatic amino acids. Standard curves were made by quantitative studies of29compouds related to the pathway of the three aromatic amino acids by LC-MS. Then, different forms of indole-3-pyruvic acid which is unstable in different solvents and its degradation products were investigated by multi-dimensional NMR techniques. The main contents include:1. We compared the separation of aromatic metabolites on three columns from different companies and studied the effect of temperature on separation. The Agilent C18column has the best performance and the chromatographic peaks displayed evenly. As the temperature increase, the retention of the metabolites decrease slightly and the separation trend of the overlapped peaks became obvious. In addition, we optimized the MS parameters in order to obtain the largest signal response from each compoud. Furthermore, Standard curves of29aromatic metablites were made based on the optimized separation method and MS method.2. We studied the different forms of indole-3-pyruvic acid in different solvens and its degradation products by multi-dimensional NMR techniques. The results showed that the enol-form of indole-3-pyruvic acid was the dominant form in acetontrile whereas the keto-form was the main form in water. Enol indole-3-pyruvic acid in acetontrile reacted with oxygeon partially to produce indole-3-carbaldehyde acid and oxalic acid and the existence of water would accelerate this reaction. Keto indole-3-pyruvic acid in the neutral or alkaline aqueous solution reacted with oxygen to produce indoleacetic acid and carbon dioxide.4-hyddroxyphenylpyruvic acid in aqueous solution has the same reaction with oxygeon to produce4-hydroxyphenylbenzaldehyde and4-hydroxyphenylacetic acid. In addition, there are signals of4-hydroxymandelic acid in2D NMR spectra. All these phenomena indicated that aromatic pyruvic acids are unstable in aqueous solution and they acted as reductive agent to react with oxygen.