High-temperature liquid chromatography coupled on-line to a continuous-flow biochemical screening assay with electrospray ionization mass spectrometric detection.
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 degrees 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 IC50 values were obtained. Exposing the inhibitors to high temperatures did not lead to thermal decomposition. The separation of inhibitors and the subsequent biochemical assay was performed either isothermally at various temperatures or by applying various temperature gradients as well as at various flow rates. The results obtained clearly show the compatibility of HTLC with an enzymatic screening assay.
de Boer
Arjen R
AR
Faculty of Sciences, Department of Chemistry and Pharmaceutical Sciences, Section of Analytical Chemistry & Applied Spectroscopy, Vrije Universiteit Amsterdam, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands. a.r.de_boer@lumc.nl
Alcaide-Hidalgo
Juan M
JM
Krabbe
Johannes G
JG
Kolkman
Jeffrey
J
van Emde Boas
Caspar N
CN
Niessen
Wilfried M A
WM
Lingeman
Henk
H
Irth
Hubertus
H
D002401:Cathepsin B / Q000378:metabolism; D002853:Chromatography, Liquid / Q000379:methods*; D004151:Dipeptides / Q000032:analysis; D004355:Drug Stability; D006358:Hot Temperature; D007930:Leucine / Q000031:analogs & derivatives / Q000032:analysis; D007976:Leupeptins / Q000032:analysis; D009862:Online Systems; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D013662:Tea / Q000737:chemistry
D004151:Dipeptides; D007976:Leupeptins; D013662:Tea; C068250:N-(3-propylcarbamoyloxirane-2-carbonyl)-isoleucyl-proline; D002401:Cathepsin B; D007930:Leucine; C032854:leupeptin; C024974:E 64