Measuring deuterium enrichment of glucose hydrogen atoms by gas chromatography/mass spectrometry.

We developed a simple and accurate method for determining deuterium enrichment of glucose hydrogen atoms by electron impact gas chromatography mass spectrometry (GC/MS). First, we prepared 18 derivatives of glucose and screened over 200 glucose fragments to evaluate the accuracy and precision of mass isotopomer data for each fragment. We identified three glucose derivatives that gave six analytically useful ions: (1) glucose aldonitrile pentapropionate (m/z 173 derived from C4-C5 bond cleavage; m/z 259 from C3-C4 cleavage; m/z 284 from C4-C5 cleavage; and m/z 370 from C5-C6 cleavage); (2) glucose 1,2,5,6-di-isopropylidene propionate (m/z 301, no cleavage of glucose carbon atoms); and (3) glucose methyloxime pentapropionate (m/z 145 from C2-C3 cleavage). Deuterium enrichment at each carbon position of glucose was determined by least-squares regression of mass isotopomer distributions. The validity of the approach was tested using labeled glucose standards and carefully prepared mixtures of standards. Our method determines deuterium enrichment of glucose hydrogen atoms with an accuracy of 0.3 mol %, or better, without the use of any calibration curves or correction factors. The analysis requires only 20 μL of plasma, which makes the method applicable for studying gluconeogenesis using deuterated water in cell culture and animal experiments.