Paul Baker received his PhD in biochemistry from Wake Forest University School of Medicine. Baker did his post-doctoral work at the University of Alabama at Birmingham where he helped lead the discovery of a novel class of anti-inflammatory lipid mediators—nitrated lipids. He continued to work on nitrated lipids as an assistant professor at the University of Pittsburgh School of Medicine until June of 2011 when he joined SCIEX. At SCIEX, he helped pioneer the use of differential ion mobility spectrometry and electron impact dissociation for the analysis of lipids. Baker is now the senior staff scientist liaison for lipidomics and metabolomics at SCIEX. 

Q: What are the current challenges for large, quantitative screening analysis by mass spectrometry?

A: There are several challenges in large-panel analyses. One is the speed of the mass spectrometer (MS). As the lists of target molecules grow, the instrument may not be fast enough to analyze all compounds simultaneously. You can mitigate this by scheduling the analysis to focus only on molecules as they elute from the column, but balancing the number of compounds with the instrument's capabilities remains a key issue. Another challenge is the fear of missing out—people worry about missing unknown compounds, which drives interest in untargeted analysis. However, with a well-curated list covering the biochemistry of interest, most questions can be answered. Lastly, quantitation is difficult with large panels because not every compound has an internal standard, which raises concerns about how quantitative the assay truly is. Back in 2015, SCIEX addressed this with the Lipidyzer™ platform (now discontinued, but the workflow lives on using the SCIEX 6500+ with DMS), which provided what we call "accurate quantitation" by using a large spectrum of internal standards related to the target molecules. This allowed for quantitation within a margin of 10–20 percent accuracy.

Q: Can you describe multiple reaction monitoring (MRM)? 

A: MRM is a scan mode on triple quadrupole mass spectrometers. With MRM scans, you monitor a specific precursor ion in Q1, fragment it in Q2 (the collision cell), and detect a specific fragment ion in Q3, ideally unique to the compound of interest. This approach, combined with good chromatography, gives confidence in identifying and quantifying the target molecule. MRM is fast, enabling multiple points across a chromatographic peak, which improves the precision of the assay. It is widely used in many areas of research, from bioanalysis to drug testing in urine to metabolomics, where it’s used to measure compounds involved in pathways like central carbon metabolism. MRM is the most sensitive and specific method for quantitative measurement today.

Q: What are the advantages of fast scanning MRM in the analysis of large panels of analytes?

A: Fast scanning MRM is particularly advantageous when measuring larger panels of analytes. It allows for the analysis of more compounds in a shorter time, which is critical when dealing with large panels. This allows the user to still get enough data points across a chromatographic peak, which is vital for good peak integration and quantitation. As a result, you still get excellent precision, while larger compound lists can be handled. This approach also helps reduce the risk of missing important analytes in targeted assays. Fast MRM maintains the quantitative advantage over untargeted or discovery-based methods, where identification and quantitation are more challenging.

Q: What is the difference between targeted and untargeted MS analyses? Is one more advantageous over the other?

A: Untargeted analysis seeks to discover unknowns by scanning for everything in the sample, while targeted analysis focuses on specific known compounds. Untargeted MS is useful for discovery but lacks the quantitative precision and specificity needed in many biological studies. It typically captures only a few MS/MS spectra per molecule, which is insufficient for reliable quantitation. Targeted MS, especially MRM, provides very accurate quantitation of known molecules, which is crucial in comparing disease versus healthy states or understanding concentrations in metabolic studies. However, the downside of targeted analysis is the risk of missing unexpected compounds. A newer approach, called high-resolution MRM (MRM-HR or PRM), combines elements of both, allowing for quantitation while also enabling discovery by searching the data for unanticipated molecules.

Q: What does the future of targeted quantitative analysis look like?

A: The future of targeted analysis involves faster instruments capable of handling more targets in less time. For instance, the SCIEX 7500+ system can run up to 800 MRMs per second, enabling larger datasets with shorter run times. Beyond speed, advances like electron-activated dissociation (EAD) offer more structurally diagnostic fragmentation patterns compared to traditional collision-induced dissociation (CID). EAD generates many more fragments, each carrying structural information, which can distinguish isomers that are otherwise indistinguishable by traditional CID fragmentation. This could reduce reliance on time-consuming chromatography. Additionally, techniques like ZT Scan DIA (which is the latest iteration of the SCIEX SWATH DIA journey), enables sensitive quantitation of all detectable molecules in a sample, and is emerging as a powerful tool for combining targeted and untargeted workflows, offering both discovery and quantitation in a single run.

Q: Can you elaborate on the recent key advancements, particularly in terms of robustness and the benefits of fast MRM scanning?

A: In addition to speed advancements, a notable feature of the SCIEX 7500+ system is its enhanced robustness due to a redesigned front end. This improvement is achieved through what we’re calling Mass Guard technology. Previously, all ions within the beam would go straight to the analytical quadrupoles, but now they pass through a region with ion-filtering technology, which creates a high m/z cut-off above the m/z of the target precursor ion, removing the unwanted and potentially contaminating species, and narrowing the m/z range of ions transmitted past Q₀. This helps prevent contaminating ions from entering the instrument, as studies have shown that high molecular weight ions are the primary cause of contamination, reducing performance by coating the quadrupole rods. 

To test this, a robustness study was conducted comparing the SCIEX 7500 system and SCIEX 7500+ systems. When analyzing human plasma—a notoriously "dirty" matrix—on the 7500 system, the signal degraded by 50 percent after ~5,000 injections due to contamination. In contrast, the 7500+ system maintained its performance after 10,000 injections. This level of durability is essential for large-scale studies involving complex matrices like urine, plasma, or tissue, as it minimizes downtime for cleaning and ensures continuous productivity.

Additionally, fast MRM scanning offers improved resolution for closely eluting isomers. For instance, two lysophospholipids, differing only in the position of their acyl chains, elute close together in LC but are distinguishable thanks to fast scanning. Slower systems would not be able to separate them, but with more data points across the peak, the 7500+ system's auto-integration can clearly resolve them, which is crucial for accurate bioanalysis. This improved resolution, along with the ability to handle larger analyte panels, makes the fast MRM scanning of the SCIEX 7500+ system a powerful tool for complex biological studies.