21


Figure 2. Reproducibility of injection across a 384-well plate. Injections of 100 nM dextromethorphan were collected at 1 Hz per sample across the full plate. Peak area reproducibility was 1.98%CV across the full dataset.


Pushing the Throughput Barrier


Researchers at Boehringer Ingelheim successfully applied an AEMS setup for ESI-MS using an ADE-OPI-MS setup for high-throughput drug metabolism and pharmacokinetics analysis using dextromethorphan and d3-dextrorphan (each 100 nM) as simulants [2]. The interface of an ATS-G4P acoustic droplet emission system with a QTRAP® 6500+ mass spectrometer with a modified OptiFlow® Turbo V Ion Source resulted in creation of the ADE-OPI-MS setup. Various modifications and additional equipment resulted in a system that pushes the speed limit of ESI-MS to 3 Hz (or 3 samples per second) for sampling and detection with baseline separation of each sample.


At a sampling rate of 3 Hz, the contactless sample injection method took significantly less time (7.5-fold faster) than the fastest ESI-based systems, and was even faster than the maximum high-throughput screening throughput of MALDI at 2.5 Hz [2]. Furthermore, using lower sampling rates of 1 or 3 Hz, unprocessed samples containing high concentrations of plasma, cell lysate and other matrices (enzyme assay buffer and crude dog plasma) were analysed without observance of ion suppression, suggesting that physiological conditions of samples can be maintained prior to injection [2].


Acceleration of Drug Discovery


With no LC requirements, AEMS eliminates the need to develop separation methods,


troubleshoot LC problems, and wait for LC columns to wash and equilibrate and analytes to elute. Faster turnaround times are possible for the same number of samples and compounds, or more compounds, larger sample sets and bigger cohorts can be analysed in the same time frame as conventional LC-MS. As a result, dead ends and false positives can be found earlier, allowing teams to make critical decisions faster and dramatically accelerate project turnaround times from months to weeks, days or even hours or minutes.


Although there have been significant advances in high-throughput technologies for rapidly screening reaction conditions, collecting analytical data has been an issue. With current MS systems, it can take hours or days to analyse high-density experiments. Combining an ultra-throughput (UT) reader platform with the direct sampling associated with ADE-OPI-MS has been shown by Pfizer scientists to overcome this limitation and support parallel medicinal chemistry and reaction screening efforts [7].


In this study, three sets of experiments were conducted that mimic standard high- throughput experimentation (HTE) and parallel medicinal chemistry (PMC) screening processes:


1. Reaction optimisation for a single transformation (palladium-catalysed C−N coupling of 3-bromopyridine with 4-phenylpiperidine) with variable catalyst and base conditions.


2. Parallel synthesis of a library of drug-like small-molecules involving reaction of common template structures with different monomeric reagents (amidation of two secondary amine templates, each with 96 carboxylic acids for a total of 192 reactions).


3. Similar library synthesis but involving the reaction of a larger template substrate with small-molecule reagents (amide formation between a commercially available DNA fragment possessing an amine reaction point and a set of 96 acids).


The results for each ADE-OPI-MS experiment were compared to those obtained using mainstream high-throughput MS platforms. In all three cases, much less sample (a few nanolitres vs. dozens of nanolitres or microliters) was required, allowing more material to be saved for further analysis. The sampling speed was also an order of magnitude faster, such as ~7.5 minutes for ADE-OPI-MS vs. 5 hours for UHPLC-MS in experiment two.


Importantly, the use of ESI-MS provided accurate quantitative data similar to that obtained using LC-MS and better than that observed with MALDI, without the need for an internal standard, even for reactions involving larger, complex species such as synthetically modified oligonucleotides. In addition, the coverage was greater than that possible with MADLI. Furthermore, use of a high-resolution TOF MS instrument enabled unambiguous identification of both smaller and larger substrates.


While all of the analyses were performed offline after reaction quenching, the researchers suggest that due to the ability to directly sample without any need for sample cleanup, online reaction monitoring should be possible with ADE-OPI-MS, offering the potential to evaluate reaction kinetics in real time.


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