(T0930-04-22) A Complete Automation Solution Using an Acoustic Ejection Mass Spectrometry System for Rapid Sample Analysis

Purpose: While using plate readers to measure biochemical assays is the traditional high-throughput approach, it is well-known that fluorescent quenchers, colored compounds, and other mechanisms can interfere with the readout from fluorescent assays. In addition, many library compounds form aggregates onto which enzymes and/or substrates can adhere, resulting in false‑positive inhibition. Data collection using LC-MS can also be slow, taking minutes to hours, and can cause large backlogs of information. Rapid analysis that takes only seconds can alleviate this pain point and prevent sample degradation. Here, an approach is demonstrated that uses a fully automated liquid handler, robotics, and an Acoustic Ejection Mass Spectrometry (AEMS) system to meet the needs of high-throughput compound analysis, including increased demand for sample preparation.

Methods: Sample preparation utilizes the Biomek i7 Automated Workstation from Beckman Coulter, which can be configured with a 96-multichannel pipetting head or Span-8 pod (independent probes) and integrated with devices including a plate shaker, a centrifuge, a robotic transport arm, an incubator, a plate hotel, and a sealer/peeler. Prepared plates were loaded onto the Echo® MS system. The Echo® MS system is controlled by SAMI EX scheduling software, which utilizes the SCIEX OS software control API. An array of commercially available analytes (e.g., triazines, drug molecules, and metabolites) was prepared at 5–500 nM to evaluate the robustness of the system. Data collection and processing were performed using SCIEX OS software.

Results: The purpose of this study was to explore the critical parameters (e.g., carrier solvent composition, chemical additives, and varying matrices) affecting the data quality of AEMS on a high-throughput scale. AEMS utilizes contactless sampling via the Open Port Interface (OPI). Acoustic energy is applied to a liquid surface to generate nanodroplets on the order of 2.5 nL. The 384 or 1536 microtiter well plate moves underneath the fixed OPI position allowing for sampling at a rate of up to 3 samples per second. Nanodroplets are captured in the OPI and subsequently transported via carrier solvent (~ 400 uL/min), into the ionization source for multiple reaction monitoring (MRM) analysis. The second half of this study focused on the increased throughput by automating the Echo® MS system. Preliminary AEMS data, without automation, show extremely fast analysis of compounds, measuring a 384-well plate in just 10 min. Subsequent ejections result in baseline resolution between ejection peaks and no carry-over between measurements. Very high reproducibility is observed across a single plate and when measuring many plates in series, with an average of <10% CV across 146 384-well microtiter plates (~56,000 samples) over the span of 4 working days. The addition of the liquid handler increases workflow and sample throughput by being able to prepare and schedule subsequent plates around the clock. By doing so, the number of samples analyzed drastically increases while still maintaining data quality, reproducibility, and system robustness.

Conclusion: A fully automated system with quality, reproducibility, and system robustness being key indicators of system performance for high-throughput sample analysis