Continuous flow isotope ratio mass spectrometry for the measurement of nanomole amounts of (13)CO(2) by a reverse isotope dilution method.

Abstract

A simple method for the determination of nanomole amounts of (13)CO(2) generated from an in vitro reaction is reported. The incubation medium contains a known amount of unlabeled sodium bicarbonate and the gaseous (13)CO(2) enriches the atmosphere upon which a measurement of the isotopic enrichment ((13)CO(2)/(12)CO(2)) is made corresponding to a reverse isotope dilution. The quantification of the (13)CO(2) was performed by gas chromatography/isotope ratio mass spectrometry. This assay was validated in terms of linearity, accuracy and precision using three different substrates which produce (13)CO(2) either by enzymatic reaction [(13)C]urea, sodium [(13)C]formate) or by chemical reaction (sodium [(13)C]bicarbonate). Four calibration curves were tested for each (13)C-labeled substrate, allowing the quantification of (13)CO(2) from 25 pmol to 150 nmol. The dynamics of the assay were obtained as a function of the quantity of unlabeled sodium bicarbonate added to each sample.