RESUMEN
The continuous interest in discovering new bioactive molecules derived from natural products (NP) has stimulated the development of improved screening assays to help overcome challenges in NP-based drug discovery. Here, we describe a unique platform for the online screening of acetylcholinesterase inhibitors without the need for pre-treating the sample. In the current study, we have demonstrated the ability to combine reversed-phase separation with a capillary immobilized enzyme reactor (cIMER) in two-dimensional liquid chromatography system coupled with mass spectrometry detection. We systematically investigated the effects of method parameters that are of practical significance and are known to affect the enzyme assay and interfere in the analysis such as: bioreactor dimensions, loop sizes, amount of immobilized enzyme, second dimension flow rates, reaction time, substrate concentration, presence of organic modifier, limit of detection and signal suppression. The performance of this new platform was evaluated using a mixture containing three known AChE inhibitors (tacrine, galanthamine and donepezil) and an ethanolic extract obtained from the dry bulbs of Hippeastrum calyptratum (Amaryllidaceae) was investigated to provide a proof of concept of the applicability of the platform for the analysis of complex mixtures such as those derived from NPs.
RESUMEN
In recent years there has been increasing interest in the use of HILIC separations in two-dimensional liquid chromatography (2D-LC), mainly because the selectivity of HILIC separations complement that of reversed-phase separations for a variety of molecules. Historically, the re-equilibration of HILIC phases following gradient elution has been perceived as too slow to be useful in the second dimension of 2D-LC separations in particular. Recent studies of re-equilibration of HILIC phases by McCalley and coworkers using a limited set of conditions showed that highly repeatable gradient separations could be obtained with re-equilibration times as short as 4.3 min [1,2]. In this study we aimed to study re-equilibration of HILIC phases under a broader set of conditions, and at much shorter re-equilibration times, in the interest of determining whether or not HILIC separations can be generally considered as a viable option for use in the second dimension of 2D-LC separations. To this end we studied the effects of mobile phase pH, buffer concentration, and preparation method, flow rate, analyte and stationary phase chemistry, column length, and re-equilibration time on retention of a variety of small molecule probe solutes following gradient elution. In general, we have found that excellent separation repeatability can be obtained with quite short (âª10â¯min) re-equilibration times, even when progress toward full equilibration of the column is quite slow (â«10â¯min). In other words, even if the stationary phase is not fully equilibrated, as long as it is partially equilibrated in a highly precise manner, highly repeatable retention times can be obtained. Higher flow rate has a positive effect on both the rate of progress toward full equilibration and the repeatability of separation. No significant, consistent effects of eluent pH or buffer concentration on repeatability of gradient separation were observed for the stationary phases studied. Excellent gradient separation repeatability was obtained with shorter columns (30â¯mm length) with re-equilibration times as short as 3â¯s. A proof-of-concept 2D-LC separation of several small molecule probes using HILIC separations in both dimensions was performed to illustrate that re-equilibration of these columns can be fast enough for HILIC columns to be considered as a viable option for the second dimension of comprehensive 2D-LC separations.