Determination of 15NH3 by gas chromatography-mass spectrometry. Application to the measurement of putrescine oxidation by human plasma.
A convenient and sensitive method for the determination of 15NH3 has been developed. Ammonia was purified from sample solutions by a modified microdiffusion method, derivatized with pentafluorobenzoyl chloride to pentafluorobenzamide (PFBA) and determined by gas chromatography-mass spectrometry using a multiple ion detector. PFBA was eluted from the gas chromatographic column within 2 min and resulted in a simple mass fragmentation pattern. The 15N/14N ratio was accurately determined with picomole amounts of PFBA by measuring the molecular ions of PFBA and [15N]PFBA. The method was applied to the assay of putrescine oxidation by human plasma. 15NH3 was produced by incubating 15N-labelled putrescine with plasma. The 15NH3 production was time dependent and strongly inhibited by the addition of aminoguanidine, an inhibitor of diamine oxidase. Exceedingly high 15NH3 production from [15N]putrescine was observed in the plasma from pregnant women. In contrast, only trace amounts of 15NH3 were formed in the plasma from normal men and non-pregnant women. The method seems to be applicable to various biological systems that produce ammonia as a metabolic product.
D006631:Amine Oxidase (Copper-Containing) / Q000097:blood; D000641:Ammonia / Q000032:analysis*; D008401:Gas Chromatography-Mass Spectrometry; D006801:Humans; D009587:Nitrogen Isotopes; D010084:Oxidation-Reduction; D011700:Putrescine / Q000097:blood*
D009587:Nitrogen Isotopes; D000641:Ammonia; D006631:Amine Oxidase (Copper-Containing); D011700:Putrescine