High-temperature liquid chromatography coupled on-line to a continuous-flow biochemical screening assay with electrospray ionization mass spectrometric detection

A.R. de Boer, J.M. Alcaide-Hidalgo, J.G. Krabbe, J.J. Kolkman, C.N.V. Boas, W.M.A. Niessen, H. Lingeman, H. Irth

Research output: Contribution to JournalArticleAcademicpeer-review

Abstract

The potential of high-temperature liquid chromatography (HTLC) was investigated in an on-line combination with a screening system for bioactive compounds against the enzyme cathepsin B. Samples were separated by HTLC and subsequently analyzed by an on-line continuous-flow enzymatic assay. Detection was performed by electrospray ionization mass spectrometry, revealing both the bioactivity and the molecular mass of the bioactive compounds. Compared to conventional reversed-phase liquid chromatography, the amount of methanol necessary for separation could be decreased to only 10%, which improved the compatibility of LC with a biochemical assay. Sufficient preheating of the mobile phase prior to the separation and postcolumn cooling to prevent deactivation of the enzyme, even at column temperatures as high as 208°C, was achieved as indicated by the reliable peak shapes obtained. The sensitivity was comparable with previously described systems operating at ambient temperatures as similar IC
Original languageEnglish
Pages (from-to)7894-7900
JournalAnalytical Chemistry
Volume77
Issue number24
DOIs
Publication statusPublished - 2005

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