A sensitive approach for screening acetylcholinesterase inhibition of water samples using ultra-performance liquid chromatography-tandem mass spectrometry

A sensitive assay was developed to evaluate inhibitory effects of aqueous solution on acetylcholinesterase (AChE) activity via measuring hydrolysis rates of acetylcholine (ACh) based on ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Upon having identified precursor ions and product ions of the ACh and its hydrolysis products choline (Ch), the separation chromatogram for these two analytes has been established using a 50 mm reverse-phase BEH Shield RP18 column. The total chromatographic separation time is 7 min; limits of detection (LODs) for ACh and Ch are 0.14 µg L^-1 and 0.12 µg L^-1, respectively. A simple method for inactivation of AChE and optimization of operational parameters were then sequentially performed. It was found that adjusting solution pH to 2.5 not only can terminate the enzymatic reaction but also solve band shifting and broadening caused by aqueous matrices in chromatographic separation during UPLC-MS/MS detection. Under conditions of 0.00075 U mL^-1 AChE, initial concentration of ACh at 100 µg L^-1 and 20 min observation time, IC₅₀ values of the proposed assay for chlorpyrifos-oxon, diazoxon, malaoxon, methidathion oxon, omethoate and paraoxon were 3.5 nM, 16.8 nM, 2.4 nM, 6.8 nM, 270 nM and 36.9 nM, respectively. They are 4.5-51.9 times smaller than those reported in a LC-MS based method, and >120 times lower than those obtained by the traditional Ellman method. The results suggested that, the proposed assay significantly increases the sensitivity of commercial AChE. In addition, inhibition efficiencies of three surface waters, a groundwater and four commercial brands of bottled drinking water samples on AChE activity were firstly measured using this UPLC-MS/MS based method. These water samples were proved to have different inhibitory effects on AChE activity, and the inhibition efficiencies dependent on concentrations of dissolved organic carbon (DOC) but are independent of UV absorbance at 254 nm (UV₂₅₄) values. These results indicate that the proposed method has advantages of high sensitivity over all other conventional methods. It may become a promising AChE inhibition assay for assessing toxicity of aqueous solution containing neurotoxicity contaminants such as organophosphorus pesticides (OPPs) at low levels, or used to evaluate potential inhibition effects of natural waters on AChE activity.