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

Citation
N. Mano et al., Simultaneous detection of cholyl adenylate and coenzyme A thioester utilizing liquid chromatography/electrospray ionization mass spectrometry, ANAL SCI, 17(9), 2001, pp. 1037-1042
Citations number
26
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Spectroscopy /Instrumentation/Analytical Sciences
Journal title
ANALYTICAL SCIENCES
ISSN journal
0910-6340 → ACNP
Volume
17
Issue
9
Year of publication
2001
Pages
1037 - 1042
Database
ISI
SICI code
0910-6340(200109)17:9<1037:SDOCAA>2.0.ZU;2-3
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 fo rmation of amino acid conjugates. Acyl CoA synthetases may be responsible f or this acyl adenylate formation. Recently, we hypothesized the preferentia l formation of cholic acid adenylate, a major bile acid, preceding producti on of the corresponding CoA thioester in incubations with rat liver microso mal fractions. To verify this biosynthetic mechanism, monitoring of the inc ubation mixture of acyl adenylate together with both substrate and acyl CoA thioester is needed. We have developed a detection method for the simultan eous detection of these cholic acid derivatives utilizing liquid chromatogr aphy/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 chelat ing agent, such as EDTA, to the mobile phase and an adjustment of the mobil e phase pH to a weak alkaline effectively removed such chelate formation, p roducing 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 Angstrom particle diameter ODS column and 20 mM ammo nium acetate buffer (pH 9.0)/2-propanol/acetonitrile as the mobile phase ha ve been proved to be preferable. To avoid any degradation of the chemically unstable adenylate produced in the incubation, we employed a direct inject ion of the sample onto a preconcentration column. The obtained results indi cated a high sensitivity of this method.