PMID: 11334353May 4, 2001Paper

Determination of inhibitors' potency (IC50) by a direct high-performance liquid chromatographic method on an immobilised acetylcholinesterase column

Journal of Chromatography. B, Biomedical Sciences and Applications
Vincenza AndrisanoG Felix

Abstract

An immobilised acetylcholinesterase (AChE) stationary phase was prepared by using an in situ AChE immobilisation procedure. A stainless steel column packed with epoxide silica was connected to the HPLC system and the enzyme solution at pH 5.8 was recycled through the column at a flow-rate of 0.5 ml/min for 24 h. The activity of the immobilised AChE was determined by injecting the substrate acetylthiocholine, using as mobile phase 0.1 M phosphate buffer (pH 7.4) containing Ellman's reagent [5,5'-dithio-bis(2-nitrobenzoic acid)] and measuring the area of the obtained peak with UV detection at 412 nm. The effect of AChE inhibitors tacrine, edrophonium and donepezil were evaluated by the simultaneous injection of each inhibitor with the substrate. The resulting decrease in the AChE activity, as expressed by the decrease of the peak area detected at 412 nm, was related to the concentration and potency of the solutes. The obtained IC50 values were compared with those derived by the conventional spectrophotometric method. This immobilized enzyme reactor, included in a chromatographic system, can be used for the rapid screening for new inhibitors allowing for the on-line determination of a compound's inhibitory potency. The advantages ...Continue Reading

Citations

Mar 12, 2010·Journal of the American Society for Mass Spectrometry·Jian PeiRobert T Kennedy
Nov 25, 2003·Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences·C BertucciV Andrisano
Mar 31, 2005·Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences·Anna Maria Girelli, Enrico Mattei
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