Strategies for high-precision nitrogen and carbon position -specific isotope analysis.

摘要

High precision measurement of subtle changes in intramolecular isotope distribution of a given compound at natural abundance level reflects its origin conditions and may permit the reconstruction of metabolic fluxes and pools. The conventional intramolecular techniques, mass spectrometry and NMR cannot sensitively measure natural carbon isotope variations with sufficient precision. Recently, high-precision measurements were made easier to achieve for carbon by the advent of a novel online instrumental approach for position-specific isotope analysis (PSIA). However, no high-precision approach has been reported for N-PSIA. This thesis describes in depth the extension of the online PSIA strategy to the measurement of nitrogen and carbon position-specific isotope analysis while developing a method specific to a class of environmentally important compounds, short chain organic acids (SCOA). Further, it introduces an enzymatic alternative to achieve the N-PSIA goal by using commercially available online systems.;The underlying principle of this PSIA strategy is based on online separation of the desired analyte from the sample, its pyrolytic fragmentation, followed by a second stage separation of the fragments and the subsequent measurements of the isotope ratios of specific positions or moieties. Four SCOA were investigated using this strategy for the measurement of their carboxyl carbon isotope ratio. Precision achieved averaged SD(delta13C) < 0.7‰. Histamine was used as a test molecule for N-PSIA. Nitrogen isotope ratio measurements were determined for the fragments detected with high to moderate precision, while carbon isotope ratio measurements of the same fragments were performed with high precision. However, N-PSIA by pyrolysis is a challenge due to the complexity of the instrument setup and the negative effects of even tiny amounts of atmospheric nitrogen.;An alternative enzymatic strategy for N-PSIA is also described and is implemented on commercially available compound-specific isotope analysis systems. This strategy necessitates chemical derivatization for some of the deaminated compounds to enhance volatility for GC analysis. High-precision measurements were achieved for the two non-equivalent N positions of lysine (SD(delta 15N) < 0.75‰). Enzymatic schemes for the five remaining polynitrogenous amino acids are proposed.