Combined use of filtered and edited 1H NMR spectroscopy to detect 13C-enriched compounds in complex mixtures

In conventional metabolism and pharmacokinetic studies, radioactive isotopes are used to identify and quantify the breakdown products of xenobiotics. However, the stable isotope 13C provides a cheaper and less hazardous alternative. Metabolites of 13C-enriched xenobiotics can be detected, quantified and identified by 13C-filtered NMR spectroscopy. However, one obstacle to using 13C is its 1.1% natural abundance that produces a background signal in 13C-filtered NMR spectra of crude biological extracts. The signal makes it difficult to distinguish between 13C-enriched xenobiotics resonances from endogenous metabolites unrelated to the xenobiotic. This study proposes that the 13C background signal can be distinguished from resonances of 13C-enriched xenobiotics by the absence of a 12C component in the xenobiotic. This is detected by combined analysis of 13C-filtered and -edited NMR spectra. The theory underlying the approach is described and the method is demonstrated by the detection of sub-microgram amounts of 13C-enriched phenacetin in crude extracts of hepatocyte microsomes. Copyright (c) 2012 John Wiley & Sons, Ltd.