(T1530-10-54) High-Speed Compound Quality Assessment Using Acoustic Ejection Mass Spectrometry

Purpose: Efficient compound management involves collecting, storing, and processing thousands of compounds vital to the drug discovery process. The compound integrity, specifically the identity and purity, is essential for developing new therapeutics. The integrity of these compounds is typically confirmed via nuclear magnetic resonance (NMR) spectroscopy or LC-MS in combination with UV absorbance and evaporative light scattering detection (ELSD). Although these techniques provide an accurate way to determine the integrity of a compound, the high cost of NMR systems and the low throughput of LC-MS assays due to long run times make them both inefficient. Here, we show a fast, efficient method to analyze compounds in a library using a high-resolution acoustic ejection mass spectrometer system (AEMS-TOF).

Methods: Compounds of interest were monitored using a AEMS-TOF system. Analytes and their respective internal standards were prepared in DMSO and diluted to a working concentration in DMSO. Sample preparation utilized the Biomek i7 Automated Workstation from Beckman Coulter® Life Sciences, which is configured with a 96-multichannel pipetting head and/or a Span-8 pod and integrated with devices including a plate shaker, robotic transport arm, and a plate hotel. The prepared samples were centrifuged, and 50 µL aliquots were transferred to an Echo® MS Qualified 384-well microplate. The screened compounds were delivered into the mass spectrometer using 0.1% formic acid in methanol as the carrier solvent at a flow rate of 400 µL/min with an ejection volume of 20 nL. Data was collected using a TOF MS method and processed using SCIEX OS software.

Results: The AEMS-TOF system includes an Open Port Interface (OPI) and Acoustic Droplet Ejection (ADE) module coupled with a ZenoTOF 7600 mass spectrometer, controlled with acquisition software.  The OPI-ADE module, in conjunction with the ZenoTOF 7600 system, can analyze compounds at the rate of up to 1 sample per second, making it an ideal system for high-throughput screening. The low volume and no contact sampling eliminates carryover and reduces matrix suppression, which are common issues to overcome using conventional LC-MS/MS. Carrier solvent, flow rate, and ejection volume optimization were performed to identify the appropriate system parameters. Data acquisition and quantitation were done using SCIEX OS software. The DMSO calibration mode on the acoustic ejection sampler allows the analysis of samples in pure DMSO. The AEMS-TOF system supports the screening of compounds in a 384- and 1536-well format, thus reducing the assay volumes to a few microliters and ensuring a larger batch of samples can be analyzed in a single run. Mass accuracy and isotope ratio were used to identify compounds of interest. At the same time, subtraction of the background signal from that of the analyte provided a sample-specific signal that was used for purity assessment. Measurement of a single analyte across a full 384-well plate showed < 5% CV, indicating high precision in these measurements.

Conclusion: Acoustic ejection mass spectrometry provides a rapid and unambiguous technique for screening of compound libraries in early drug discovery settings.

Analysis of 45 compounds dissolved in DMSO using the Echo® MS+ system with the ZenoTOF 7600 system. (A) The
total ion chromatogram (TIC) of the TOF MS data acquired in positive polarity. (B) The barcodes present at the start and end of a run are highlighted in red. The XIC of an analyte at m/z 280.1696 was detected at 0.874 minutes.