High-performance liquid chromatography-mass spectrometry-based acetylcholinesterase assay for the screening of inhibitors in natural extracts.
The present paper describes a High-performance liquid chromatography-mass spectrometry (LC-MS) methodology for the screening of acetylcholinesterase (AChE) inhibitors in natural extracts. AChE activity of sample components is monitored by a post-column biochemical assay that is based on the separate, sequential mixing of AChE and acetylcholine, respectively, with the HPLC eluate. AChE inhibitors are detected by measuring a decrease of product formation using electrospray MS. Ammonium bicarbonate was used as buffer in order to achieve optimum compatibility between biochemical assay and MS detection conditions. The assay is robust and stable for over 13 h and compares favourably with other AChE assays in terms of stability and sensitivity. IC(50) values of 9-aminoacridine, galanthamine, gallamine, (-)-huperzine A and thioflavin T were determined to be 0.12, 0.38, 6.4, 0.46 and 3.2 microM, respectively. The assay was used to effectively identify an AChE inhibitor present in a crude extract of Narcissus c.v. "Bridal Crown".
de Jong
Camiel F
CF
Section Analytical Chemistry and Applied spectroscopy, Department of Chemistry and Pharmaceutical Sciences, Faculty of Sciences, Vrije Universiteit Amsterdam, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands.
Derks
Rico J E
RJ
Bruyneel
Ben
B
Niessen
Wilfried
W
Irth
Hubertus
H
D000110:Acetylcholinesterase / Q000032:analysis*; D002800:Cholinesterase Inhibitors / Q000302:isolation & purification*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004353:Drug Evaluation, Preclinical; D013058:Mass Spectrometry / Q000379:methods*; D031427:Narcissus / Q000737:chemistry; D010936:Plant Extracts / Q000737:chemistry*; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence
D002800:Cholinesterase Inhibitors; D010936:Plant Extracts; D000110:Acetylcholinesterase