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Gas chromatographic-mass spectrometric determination of isotopic enrichment of 6- 15NH 2 in adenine nucleotides

Authors
  • Lewis, Sydell
  • Yudkoff, Marc
Type
Published Article
Journal
Analytical Biochemistry
Publisher
Elsevier BV
Publication Date
Jan 01, 1985
Volume
145
Issue
2
Pages
354–361
Identifiers
DOI: 10.1016/0003-2697(85)90374-4
Source
Elsevier
Keywords
License
Unknown

Abstract

A gas chromatographic-mass spectrometric method for the determination of isotopic abundance in [6- 15NH 2]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- 15NH 2]adenine-diTBDMS were obtained to identify those ions containing the 6-NH 2 moiety. The base peak ( m z 306) was formed by loss of C 4H 9 (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- 15NH 2 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- 15NH 2]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.

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