HRMS in Drug Development: An Interview with Nicholas Ingram of IQVIA Laboratories

High-resolution mass spectrometry (HRMS) is a technique that provides the highest precision in measuring molecules’ mass-to-charge (m/z) ratio. HRMS can discriminate compounds with the same nominal mass by precisely measuring their specific mass defects. This technique is valuable for identifying compounds and analyzing complex samples in research settings.

LCGC International recently sat down with Nicholas Ingram, the method development and validation group leader for regulated work at IQVIA Laboratories’ bioanalytical lab in Indianapolis, Indiana. Ingram spoke about research he presented earlier this year at the AAPS Summer Scientific Forum on utilizing HRMS to identify and overcome interferences, monitor ex-vivo reactions, and to push detection limits. He also discussed his research using HRMS as a tool for targeted stabilization and improved LLOD.

Can you define HRMS for our readers?

HRMS is high-resolution mass spectrometry. We use HRMS and associated techniques for analysis of drugs, metabolites and biomarkers in biological samples. Working in a regulated and validated analytical testing environment, we apply HRMS primarily for targeted analyte quantification in support of pharmacokinetic (PK) and pharmacodynamic (PD) studies. Separately, HRMS is also used for a broad scope of qualitative investigations in metabolic identification (Met ID) activities.

HRMS has proven an adaptable quantitative tool following our initial use of the technology for metabolism Met ID. For such applications, we use a hybrid Quadrupole-Orbitrap that delivers high resolution mass analysis along with the option to leverage the selectivity of MS/MS. It has advantages over traditional triple quadrupole mass spectrometry for the right applications. It is complimentary to traditional triple quadrupole mass spectrometry but in the right hands, and applied wisely, it has found a permanent place in our regulated bioanalytical tool-box. The benefits to our clients and our operations when faced with unique challenges, and often with the more complex compounds, has heavily influenced our investments and service offerings as a contract bioanalytical laboratory.

Can you talk about some of those advantages a little bit more?

The high-resolution aspect is really the differentiator over triple quadrupole LC-MS. Mass resolution of 1 ppm or better provides selectivity unmatched by any triple quadrupole instrument. Such selectivity/specificity can deliver increased sensitivity over traditional approaches. Such capabilities also extend from small molecule chemical entity compounds to application to biological analytes including peptides, proteins and oligonucleotides. As such, HRMS has proven application to bioanalytical programs that may have previously challenged ligand-binding assay (LBA) approaches. The signal to noise benefits of HRMS are inherently related to the selectivity afforded by high-resolution mass separation. Also related to the resolution benefits is the ability to apply to higher mass/charge ratios where triple quadrupole instrument resolution cannot differentiate isotope mass separation. This is key to the biologic analyte applications. Fundamentally this is all molecule dependent and it’s critical that you use the right tool for the job.

Is HRMS often hyphenated with chromatography, in your experience?

For our applications, it is almost always used in combination with on-line chromatography. Future potential of chromatography-free bioanalytical applications is interesting to consider. However, the selectivity dimension afforded by chromatography in combination with mass separation of HRMS delivers the practical solution for the quantitative measurement of analytes in complex biological samples. You could obviously direct infuse and get some crude information but in terms of quantitation, we always couple with liquid chromatography (LC). Metabolite interferences are a classic example where chromatography helps address the bioanalytical challenge. You must balance what your expectations are with what kind of data you can glean from the unknowns that might present in raw data. For example, identifying esters in the breast milk method that I discussed in the presentation. We didn't know what that chromatography was going to look like. In the example that I gave, the chromatographic separation is not complete, but we can tell it's related to the target analyte.

In the first study (3), you specifically investigated drug stability. Can you tell us a little bit more about the project, what type of HRMS was used?

What we observed and suspected was the drug undergoing esterification in sample preparation, so we had to look for ways we could reduce or prevent this conventionally. We employed a set of stability screening techniques to perform experimental combinations of acidifications, solvents and stabilizers. It can be a very long, drawn out process, but our analyst suggested, “What if we use the Q-Exactive (QE), a quadrupole-orbitrap instrument, and see if we can use more targeted approaches to identify exactly what the molecule is changing to?” Following this, we were able target exactly what stabilizing technique needs to be implemented in a more efficient manner. For example, adding isopropyl alcohol (IPA) to the samples made a significant difference. We were using the QE to acquire stability data before and after the parent ion loss.

Parallel reaction monitoring (PRM) is a technique analogous to MS/MS on a triple quadrupole instrument. For identification purposes, we started with a full scan, to identify parent only, and from there, you can use PRM and a generic fragmentation method to identify products. Even then, determining what the fragmentation pattern is going to be and what the abundance is going to be, is all very different than to a triple quadrupole. For example, on a triple quadrupole, you might infuse your analyte, and then throughout that infusion process, you can adjust parameters and try to tease out different fragmentations and different abundances. With a QE, if you're running PRM, you set a collision energy and run an injection, and then you can tease out every single fragment that was created from the raw data. The QE gives you a clear snapshot of relative abundances across the board from a single injection. When you have that information, you can simply quantify the exact product you want or sum multiple products. In full scan mode, you can do an “everything-all-the-time” approach, where you know what your target mass is going to be, but you can scan in a very wide range. You won’t use all of it, but you can go back and data mine which is particularly useful in troubleshooting. This is an approach you cannot practically do on a triple quadrupole due to scanning speed in full scan and the lack of selectivity under unit-mass resolution constraints. For the triple quadrupole you must be more targeted with a precursor-product combination.

Can you give me a little overview of that approach? What your findings were, and any other important details?

This one was incredibly complicated. It's another proprietary compound. It has a quinone and a hydroquinone metabolite associated with it. The trick with these is that there's all kinds of interconversion and stability problems happening. In the body, they will form acyl-glucuronides, which can be notoriously unstable on the bench. Even during analysis, acyl-glucuronides can fragment in the ion source of the mass spectrometer, and you won't be able to differentiate between the acyl-glucuronide and the parent, unless you resolve them chromatographically. The quinone and hydroquinone would interconvert through an auto-redox reaction, and with the acyl-glucuronides present, it was difficult to understand stability of each component. Essentially, we ended up with too many variables in this assay, where we can stabilize acyl-glucuronides, but that acidification process drives the auto redox reaction out of equilibrium, and eventually, we're converting our metabolite of interest back into the parent, but we're keeping the acyl-glucuronides intact. The acyl-glucuronides are not our interest, we're just trying to prevent them from contributing to our targets. It's all this complex interplay - how do you stabilize all of them without disrupting equilibrium on the bench? Essentially, what we want from the study is a freeze frame, so that as soon as that sample leaves the patient body, everything stops. We want the quinone to stop converting to the hydroquinone. We want the acyl-glucuronides to remain stable. We want everything to just hold still, but nothing will on the bench without modification. We were able to stabilize the acyl-glucuronides or the hydroquinone redox reaction, but we couldn't do both.

We had a conversation with a sponsor and had to ask, “What is more important? Are you really worried about differentiating the quinone from the hydroquinone, or are you more concerned about just stabilizing the acyl-glucuronides, so you don't have to worry about contribution there?” Ultimately, what we decided was that we just couldn’t find a good approach to prevent the auto redox fully. We were able to control it one direction or the other. We were able to control all the components’ stability, but we were unable to control the oxidation portion of the auto redox reaction. We could stop everything else. So essentially, what was decided with our sponsor was that it was more important to stabilize the acyl-glucuronides and prevent any other metabolite contribution to those targets. We concluded that we should go ahead and drive that redox reaction to form all quinone. Basically, taking the metabolite and reforming it into the quinone and quantifying that as what we call a total assay. So instead of giving a quinone result and a hydroquinone result, we were able to deliver a total (quinone plus hydroquinone) result. To the client, it was more important to maintain the acyl glute stability so that there's no contribution to the parent molecule. That was more important than having all of these teased out. In the presentation, I structure out exactly what kind of approach needed to weed out the variables, and how you can use HRMS to this effect and address the analyte stability in sample preparation. That's all thanks to the HRMS. To build the assay flexibility needed into a triple quad method, would be very difficult. This way, we have one injection, one data acquisition, and again, we can go back in and data mine for all kinds of information that we don’t know. For example, it would let us look for mystery offenders if we saw a 20% drop in one species, but we didn't see a 20% increase anywhere else, we could look to the raw data and try to find exactly what biotransformation was likely happening. This was all a great example of the power of HRMS in the hands of the modern bioanalyst.

References

(1) High Resolution Mass Spectrometry. ScienceDirect 2016. https://www.sciencedirect.com/topics/chemistry/high-resolution-mass-spectrometry-hrms (accessed 2024-10-30)

(2) Q² Solutions is now IQVIA Laboratories. IQVIA Laboratories 2024. https://labs.iqvia.com/ (accessed 2024-9-20)

(3) Ingram, N. HRMS as a Tool for Targeted Stabilization and Improved LLOD. AAPS Summer Scientific Forum program 2024.

(4) Ingram, N. Utilizing HRMS to Identify and Overcome Interferences, Monitor Ex-Vivo Reactions, and to Push Detection Limits. In AAPS Summer Scientific Forum, Kansas City, Missouri, USA, July 22–25, 2024.