title: Thin Layer Chromatography-Autography-High Resolution Mass Spectrometry Analysis: Accelerating the Identification of Acetylcholinesterase Inhibitors.
abstract: BACKGROUND
  The prevailing treatment for Alzheimer's disease is the use of acetylcholinesterase (AChE) inhibitors. Natural extracts are the principal source of AChE's inhibitors. However, their chemical complexity demands for simple, selective and rapid assays.
  
  
  OBJECTIVE
  To develop a strategy for identification of AChE inhibitors present in mixtures employing high resolution mass spectrometry (HRMS) and thin layer chromatography (TLC)-biological staining.
  
  
  METHODS
  The strategy uses an autographic assay based on the α-naphthyl acetate - fast blue B system for the detection of AChE activity. The immobilisation of AChE in agar allowed the extraction of the compounds for analysis by HRMS. Three TLC experiments employing different solvent systems were used in parallel and the mass spectra of the compounds extracted from the inhibition halos, were compared. The analysis was performed under MatLab environment.
  
  
  RESULTS
  The strategy was used to detect the presence of physostigmine in an extract of Brassica rapa L. spiked with the inhibitor. Similarly, caffeine was straightforwardly spotted as responsible for the inhibitory properties of an extract of Ilex paraguariensis Saint-Hilaire. Comparison of the HRMS profiles lead to the facile identification of the [M+H](+) and [M+Na](+) of the compounds responsible for the inhibition.
  
  
  CONCLUSIONS
  The proposed methodology, coupling TLC-AChE autography-HRMS, illustrates the feasibility of assigning molecular formulas of active compounds present in complex mixtures directly from autography. The new AChE agar-immobilised assay presented a more homogenous colour and a better definition than direct spraying methods, reducing the cost of the assay and improving its sensitivity.
authors.affiliation: Instituto de Investigaciones para el Descubrimiento de Fármacos de Rosario (IIDEFAR/CONICET-UNR), Universidad Nacional de Rosario, Ocampo y Esmeralda 2000 Rosario, Argentina.
authors.forename: I Ayelen
authors.initials: IA
authors.lastname: Ramallo
authors.affiliation: Instituto de Investigaciones para el Descubrimiento de Fármacos de Rosario (IIDEFAR/CONICET-UNR), Universidad Nacional de Rosario, Ocampo y Esmeralda 2000 Rosario, Argentina.
authors.forename: Mario O
authors.initials: MO
authors.lastname: Salazar
authors.affiliation: Instituto de Investigaciones para el Descubrimiento de Fármacos de Rosario (IIDEFAR/CONICET-UNR), Universidad Nacional de Rosario, Ocampo y Esmeralda 2000 Rosario, Argentina.
authors.forename: Ricardo L E
authors.initials: RL
authors.lastname: Furlan
mesh_terms: D001345:Autoradiography / Q000191:economics / Q000379:methods*; D001688:Biological Products / Q000737:chemistry*; D029741:Brassica rapa / Q000737:chemistry; D002110:Caffeine / Q000302:isolation & purification; D002800:Cholinesterase Inhibitors / Q000032:analysis*; D002855:Chromatography, Thin Layer / Q000191:economics / Q000379:methods*; D055808:Drug Discovery; D030019:Ilex paraguariensis / Q000737:chemistry*; D013058:Mass Spectrometry / Q000191:economics / Q000379:methods*; D010830:Physostigmine / Q000032:analysis
chemical_list: D001688:Biological Products; D002800:Cholinesterase Inhibitors; D002110:Caffeine; D010830:Physostigmine
keywords: Acetylcholinesterase; HRMS; bioautography; thin layer chromatography