Gas chromatographic-mass spectrometric determination of isotopic enrichment of 6-15NH2 in adenine nucleotides.
A gas chromatographic-mass spectrometric method for the determination of isotopic abundance in [6-15NH2]adenine nucleotides is described. The method involves formation of the di-t-butyldimethylsilyl (TBDMS) derivative of adenine following isolation of the nucleotide fraction with solid-phase ion-exchange chromatography and subsequent acid hydrolysis of nucleotides to free base. Mass spectra for both adenine-diTBDMS and [6-15NH2]adenine-diTBDMS were obtained to identify those ions containing the 6-NH2 moiety. The base peak (m/z 306) was formed by loss of C4H9 (57) and constitutes approximately one-third of the total ion current. Using selected ion monitoring of the m/z 306/m/z 307 ratio, levels of isotopic abundance of 1.0-50.0 mol% excess could be measured reproducibly with the injection of 10-20 pmol of the adenine-diTBDMS derivative obtained from isolated rat hepatocytes. Confirmation that measured isotopic abundance was referable to labeling of the 6-15NH2 group was obtained by oxidation of adenine to hypoxanthine and determination of enrichment in the hypoxanthine-diTBDMS derivative. The method was used to study the formation of [6-15NH2]adenine nucleotides during the incubation of isolated rat hepatocytes with [15N]alanine. A level of approximately 6.0 mol% excess was observed at 60 min incubation.
D000225:Adenine / Q000378:metabolism; D000227:Adenine Nucleotides / Q000032:analysis*; D000641:Ammonia / Q000378:metabolism; D000818:Animals; D008401:Gas Chromatography-Mass Spectrometry; D019271:Hypoxanthine; D007042:Hypoxanthines / Q000032:analysis; D007553:Isotope Labeling; D008099:Liver / Q000378:metabolism; D009587:Nitrogen Isotopes; D051381:Rats
D000227:Adenine Nucleotides; D007042:Hypoxanthines; D009587:Nitrogen Isotopes; D019271:Hypoxanthine; D000641:Ammonia; D000225:Adenine