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Q: Where do you see the benefits of high-resolution mass spectrometry (HRMS) in discovery and development?
A: The advent of HRMS greatly simplifies the analytical workflow to support discovery and development by providing high confidence data with generic analytical workflow. This means user do not need to ‘tune’ the conditions for each analyte, and a generic acquisition method can be applied across a broad range of sample sets. At the MS level, data can be interrogated in one of two ways: using targeted data mining or using an untargeted approach. With accurate mass spectrometry, one can either infer a chemical formula based on the detected signal (an untargeted analysis) or simply mine the data for the presence of a specific compound (a hypothesis-driven, targeted analysis). HRMS can easily distinguish between species that only a few milli-mass units apart which increases the selective detection of compounds and eliminates chemical noise. Additional confirmation can be extracted from the isotope pattern for confident data interpretation, and at the MS/MS level, structural information can be extracted with higher confidence. HRMS has made automated data mining and interpretation far simpler to implement.
Q: Why use LC-HRMS instead of LC-MS/MS for quantification?
A: Over the two decades, considerable advancement have been made in liquid chromatography. The manufacturing of smaller particle size (<3u), and the associated development of LC systems (pumps and autosampler) that can accommodate higher pressure (UPLC), has facilitated separation of isoforms (same chemical formula, different functional group position) even under rapid gradient elution. Because of these advancement, Because of these advancement, LC-HRMS can selectively detect and quantify at the MS level many compounds. However, much if that selectivity comes from the LC separation and the prior knowledge of retention time of the target isoform is required. Distinguishing isoforms is possible at the MSMS level since unique fragment can be generated and identified. Using HRMS at the MS/MS level, can eliminate ambiguities in terms of detection even under co-eluting conditions. For these reasons, acquisition strategies that use MS/MS collection are crucial and still valuable for quantitation.
Q: What are the current limitations of the HRMS technique?
A: The greatest challenge of HRMS remains providing definitive structural information, especially for some positional isoforms. The advent of alternate fragmentation techniques—such as laser-based ultraviolet photodissociation (UVPD) and EAD (electron activated dissociation)—opens the door to this possibility. These new fragmentation techniques complement the conventional collision-induced dissociation (CID) approaches by generating more selective fragmentation, which leads to improved structural interpretation. Other techniques—such as ion mobility spectrometry (IMS) and differential mobility spectrometry (DMS)—assist on the separation side in cases where LC struggles under generic gradient conditions or when it is completely removed from the workflow.
Q: What improvements are needed to increase adoption of the HRMS technique in early discovery and development?
A: At the early discovery stage, HRMS is already well established and implemented. While the list of benefits is quite long, the simplified generic workflow and the improved automated data reduction in particular have had a significant impact on the speed at which relevant information can be obtained with high confidence. At the development stage, there are probably two main barriers to wider adoption of HRMS: sensitivity and cost.
Sensitivity remains a dominant driver in bioanalysis when it comes to quantification. In that area, triple quadrupole systems have a head start of a few decades in terms of development, and they have been optimized to detect fragment ions with high sensitivity over a wide concentration range. While HRMS is typically linear over a shorter range, the technology does offer the ability to match that of a triple quadrupole system by capturing isotope information or complete MS/MS spectra. The development of automated data mining narrows the gap in this area between triple quadrupole systems and HRMS.
As for the cost of converting to HRMS, while it may not be a significant barrier at the discovery stage, there are considerations to bear in mind at the development stage. The cost of changing an entire infrastructure in terms of operation—including training, standard operating procedures (SOPs) and conversion of already-operating methods—during late-stage development, which frequently leads to outsourcing of the analysis, should not be discounted. Converting an entire fleet of instruments to new technologies, even when adjacent, can take some time when factoring in the entire data “supply chain,” including the laboratory where the analysis is outsourced. However, even though converting to HRMS may take time, it is occurring, and its adoption will continue to grow as the sensitivity gap gets narrower with established techniques.