Rapid Screening of Acetylcholinesterase Inhibitors by Effect-Directed Analysis Using LC × LC Fractionation, a High Throughput in Vitro Assay, and Parallel Identification by Time of Flight Mass Spectrometry

Abstract

Effect-directed analysis (EDA) is a useful tool to identify bioactive compounds in complex samples. However, identification in EDA is usually challenging, mainly due to limited separation power of the liquid chromatography based fractionation. In this study, comprehensive two-dimensional liquid chromatography (LC × LC) based microfractionation combined with parallel high resolution time of flight (HR-ToF) mass spectrometric detection and a high throughput acetylcholinesterase (AChE) assay was developed. The LC × LC fractionation method was validated using analytical standards and a C18 and pentafluorophenyl (PFP) stationary phase combination was selected for the two-dimensional separation and fractionation in four 96-well plates. The method was successfully applied to identify AChE inhibitors in a wastewater treatment plant (WWTP) effluent. Good orthogonality (>0.9) separation was achieved and three AChE inhibitors (tiapride, amisulpride, and lamotrigine), used as antipsychotic medicines, were identified and confirmed by two-dimensional retention alignment as well as their AChE inhibition activity.

Automated high-throughput in vitro screening of the acetylcholine esterase inhibiting potential of environmental samples, mixtures and single compounds

“…Another way to develop routine EDA could be to implement a powerful and unique chemical analysis step directly on-line with a very fine fractionation step. This is what Ouyang et al (2016) developed with a two dimensional LC fractionation step coupled with an on-line ToF-MS. Although very efficient to enhance EDA throughput it seems difficult to implement additional chemical analysis using another technique in order to ensure a good success rate when analysing different samples.…”

“…Furthermore, this format can be directly linked with chemical fractionation with the help of a special fraction collector (e.g. Paper IV; Ouyang et al, 2016). Finally, the cost of a bioassay can be lowered when the assay is simplified, does not require keeping animals in the laboratory, and most of the time when it is downscaled as well (as it requires less solvent use and more samples can be run in a day).…”

“…In addition, HPLC and UHPLC systems can be used together in the same EDA study to perform a very fine fractionation step. For example, Ouyang et al (2016) identified AChE inhibitors in a WWTP effluent using a two dimensional fractionation step with a HPLC system as first dimension and a UHPLC system as second dimension. This fractionation protocol generated 384 fractions in one step, making any additional fractionation unnecessary.…”

Integrating chemical analysis and bioanalysis to evaluate the contribution of wastewater effluent on the micropollutant burden in small streams

“…The effect of DOC has been previously assessed for cell based assays, with negligible effect found in agonist mode (Neale and Escher, 2014), though DOC can potentially interfere with assays run in antagonist mode (Neale et al, 2015b). Additional sample pre-treatment steps, such as fractionation, may help to reduce experimental artifacts associated with DOC (Ouyang et al, 2016).…”

Recent trends in water analysis triggering future monitoring of organic micropollutants

“…This approach is very time-consuming, and therefore an interesting alternative is a microfractionation system that combines LC or even LC×LC separations with a simultaneous collection of fractions in microtiter plates for effect analysis that allows for linking of effect and chemical identity [42–44]. The applicability of such a system has already been demonstrated for high-throughput screening of several end points relevant in aquatic systems.…”