Gas chromatography-mass spectrometry assay of the 18O enrichment of water as trimethyl phosphate

We have developed an assay for determining the O-18 enrichment of water in biological fluids. Urine, plasma, or whole blood is reacted with phosphorous pentachloride to yield phosphoric acid. Derivatization of phosphoric acid with diazomethane generates trimethyl phosphate. The enrichment of trimethyl phosphate is nearly four times that of water and is assayed using gas chromatography-mass spectrometry (electron impact ionization). Yang et al. (1998, Anal. Biochem. 258, 315-321) assayed the H-2 enrichment of body water after exchange with acetone, by gas chromatography-mass spectrometry. The combination of our O-18 method and the H-2 method of Yang et al. allows one to measure energy expenditure via "doubly labeled" water ((HO)-H-2-O-2 + (H2O)-O-18), using small samples of body fluids. These techniques were used to measure energy expenditure in mice, in which the O-18 enrichment of body water can be monitored down to 0.025%. (C) 2002 Elsevier Science (USA).