Category: Automation and High-Throughput Technologies
Label-free Liquid Chromatography/Mass Spectrometry (LC/MS) based screening technology is routinely used in pharmaceutical industries for hit discovery and various Absorption, Distribution, Metabolism, and Excretion (ADME) profiling applications. Although the current analysis speed of fewer than 30 seconds per sample is quite promising, it still cannot match the throughput provided by plate-reader based High Throughput Screening (HTS) platforms. Acoustic droplet ejection (ADE) is a droplet transfer technology capable of high speed, reproducibility and absolute accuracy. In this work, we successfully couple ADE and the standard Electrospray Ionization (ESI) ion source of a mass spectrometer with the open-port probe (OPP) sampling interface. Screening speeds as fast as 0.4 seconds-per-sample are demonstrated with high sensitivity, high reproducibility, good quantitation capability, no matrix ion suppression effect, and broad compound coverage from small molecules to peptides and antibodies.
In this study, an Echo® acoustic liquid handler has been modified to drive a custom stage that moves a 384-well microplate to position wells over a piezoelectric transducer for acoustic sampling. The OPP sampling interface is positioned above the plate so that 2.5-nL sized droplets ejected by ADE are quantitatively captured at the sampling end of the OPP. This interface uses a vertically aligned, co-axial tube arrangement enabling solvent delivery down to the sampling end of the device through the tubing annulus with solvent aspiration up the center tube and into the standard ESI ion source.
We analyzed various parameters relevant to the performance of the integrated ADE-OPP-ESI-MS system. The “classic” ESI ion source for MS analysis yields a perfectly Gaussian-shaped signal peak with baseline width of 0.8-1.5 seconds. High sensitivity (attomole loading for small molecules, or sub-femtomole for intact antibody) and high reproducibility (Coefficient of Variation CV less than 8%) was demonstrated for this platform, showing good linearity over at least three orders of magnitude even without internal standard. The 2.5-nL sized droplet was diluted within the sampling end of the OPP by several hundred fold, thus no matrix effect was observed even with incubation buffers containing detergents. The continuous flow of the carrier solvent maintains ionization stability and also actively cleans the entire flow system, thus eliminating carry over. In addition, the “classic” ESI ion source used in this setup enables detection of a broad range of analytes including small drug molecules, peptides (MW>1500 Da) and large proteins (up to 150 kDa). HTS applications were successfully demonstrated with a robust Z’>0.7. The screening throughput can be further improved by multiplexing. 0.4 seconds-per-well sampling rates were demonstrated by the judicious arrangement of samples in consecutive wells, and this is limited only by the speed of mechanical stage movement.
Chang Liu– Research Scientist, Sciex, Concord, ON, Canada