Simultaneous detection of cholyl adenylate and coenzyme A thioester utilizing liquid chromatography/electrospray ionization mass spectrometry

Nariyasu Mano, Masashi Uchida, Hiroko Okuyama, Ikuko Sasaki, Shigeo Ikegawa, Junichi Goto

Research output: Contribution to journalArticlepeer-review

15 Citations (Scopus)

Abstract

It has been proposed that acyl adenylate is first formed during activation of the carboxy group into the acyl CoA thioester, an intermediate in the formation of amino acid conjugates. Acyl CoA synthetases may be responsible for this acyl adenylate formation. Recently, we hypothesized the preferential formation of cholic acid adenylate, a major bile acid, preceding production of the corresponding CoA thioester in incubations with rat liver microsomal fractions. To verify this biosynthetic mechanism, monitoring of the incubation mixture of acyl adenylate together with both substrate and acyl CoA thioester is needed. We have developed a detection method for the simultaneous detection of these cholic acid derivatives utilizing liquid chromatography/electrospray ionization mass spectrometry. The CoA thioester of cholic acid forms a chelation complex with the divalent cations remaining on the silica gel packed into the analytical column. Both the addition of a chelating agent, such as EDTA, to the mobile phase and an adjustment of the mobile phase pH to a weak alkaline effectively removed such chelate formation, producing a sharp CoA thioester peak. For a simultaneous mass spectrometric analysis of cholic acid, the corresponding adenylate and CoA thioester, the combined use of a 300 Å particle diameter ODS column and 20 mM ammonium acetate buffer (pH 9.0)/2-propanol/acetonitrile as the mobile phase have been proved to be preferable. To avoid any degradation of the chemically unstable adenylate produced in the incubation, we employed a direct injection of the sample onto a preconcentration column. The obtained results indicated a high sensitivity of this method.

Original languageEnglish
Pages (from-to)1037-1042
Number of pages6
Journalanalytical sciences
Volume17
Issue number9
DOIs
Publication statusPublished - 2001

ASJC Scopus subject areas

  • Analytical Chemistry

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