Rapid and sensitive detection of the inhibitive activities of acetyl- and butyryl-cholinesterases inhibitors by UPLC–ESI-MS/MS

• UPLC–ESI MS/MS method for detection of AChE and BChE inhibition.
• The method is successfully applied to detect inhibitive activities of model drugs.
• The assay is run within 1.2 min with acceptable sensitivity and reproducibility.
• The method is economical for substrate and enzyme consumption.
• The method is suitable for screening AChE and BChE inhibitor from natural product.

Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are legitimate therapeutic targets for Alzheimer's disease. The classical approach for screening potential AChE/BChE inhibitors was developed by Ellman. However, the background color of compounds or plant extracts remained uncertain and frequently interfered with the detection of the secondary reaction, thereby easily yielding false positive or false negative results. Rapid, selective, and sensitive ultra-performance liquid chromatography combined with electrospray ionization tandem mass spectrometry method was developed and used for the detection of AChE and BChE inhibition by directly determining the common product, choline (Ch). Proper separation was achieved for choline and chlormequat (internal standard) within 1.2 min via isocratic elution (0.1% fromic acid:methanol = 98:2) on an HSS T3 column following a simple precipitation of proteins for sample treatment. The relative standard deviations of the intra- and inter-day precisions were below 7.34 and 9.09%, respectively, whereas the mean accuracy for the quality control samples was 100.31 ± 10.93%. The method exhibited the advantages of small total reaction volume (100 μL), short analysis time (1.2 min), high sensitivity (LOQ of 0.036 μM for Ch), and low cost (little consumption enzymes of 0.0035 and 0.008 unit mL−1 for AChE and BChE, and substrates of 5.505 and 7.152 μM for ACh and BCh in individual inhibition, respectively), and without matrix effect (90.00 – 105.03%). The developed method was successfully applied for detecting the AChE and BChE inhibitive activities for model drugs, including galanthamine, tacrine, neostigmine methylsulfate, eserine, as well as β-carboline and quinazoline alkaloids from Peganum harmala.

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