HR-MS – when does it make a difference?

In addition to knowledge and knowhow regarding the early phase ADME studies, also the analytical instrumentation applied plays a big role in their success. The importance of using liquid chromatography coupled to high resolution mass spectrometry (LC/HR-MS) in drug discovery and preclinical phase ADME studies has been emphasized for few years, but when does it really make a difference? To elucidate the topic further, we interviewed an expert in the field, Dr Ari Tolonen.

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What is the main difference between LC/MS/MS and LC/HR-MS?

While with the triple quadrupole mass spectrometers the MS/MS-detection at unit mass resolution is based on monitoring compound-specific MS/MS fragmentation reactions only; the HR-MS acquires data over wide mass range (e.g. m/z 100 – 1500). Thus HR-MS collects all the time the data of all compounds ionisable under LC/MS-conditions employed. Instead of the selective fragmentation patterns, the detection selectivity in LC/HR-MS is based on ion chromatograms generated post data acquisition with very narrow m/z window (e.g. 5 ppm), which in turn is enabled by high mass resolving power (i.e. very narrow mass spectral peaks). Also and on the contrary to triple quadrupole MS instruments, HR-MS instruments can collect a full scan data (over wide m/z-range) with high detection sensitivity.

For what kind of studies HR-MS brings the biggest value?

It is for in vitro and in vivo DMPK studies, clearly. Even though in the first place one would be analysing in vitro clearance or in vivo pharmacokinetics of the test compound itself, the very same LC/MS data acquired can later on be also used for metabolite identification, without a need for any new sample preparation or analyses. Thus, the time and cost savings may be high in the met id phase. The same principle applies all other screening type studies as well, as HR-MS enables checking of various other things from the very same data, e.g. inhibitor levels or presence of inhibitory metabolites from the data acquired for CYP inhibition assay with liver microsomes or hepatocytes.

In which applications HR-MS brings the most cost/time savings?

Like said above, it is the identification of metabolites or degradation products from stability studies; as the LC/MS data for this is directly available already after the stability assay. Yet, the use of LC with HR-MS saves time also when analysing samples from various screening assays with high number of compounds per batch, because due to the full scan data acquisition there is no need to optimize compound-specific MS/MS detection reactions. But it is also to note that nowadays there is quite a number of different high res-mass specs from various vendors, and they may differ in their optimum applications, as their sensitivity, dynamic range, data collection rate and ability to collect data dependent fragment ion data does differ.

Data interpretation – is it different between LC/HR-MS and LC/MS/MS?

No, not for the analysis of targeted compounds. But like said earlier, the HR-MS is much more information rich and can be used to check a variety of other things than only the targeted compounds; in the way that would bring more light to interpretation of the results from the assay for which the samples are collected.

When you would recommend traditional triple quadrupole mass spectrometry?

In the case of targeted bioanalysis that requires maximum detection sensitivity, i.e the lowest possible lower limits of detection and quantification. If comparing the same generation high end triple quadrupole mass specs, they tend generally to be a bit more sensitive than corresponding high res mass specs, although the difference is necessarily not high. Triple quads also produce much lower amount of data (as megabytes), so those are data storage-wise more convenient when there is no the need to later mine something else from the same data, e.g. plasma protein binding or routine CYP inhibition assays.

Written by Minna Komu & Ari Tolonen

PS. You may also have a look on very recent poster “High resolution mass spectrometry (HR-MS) with N-in-one CYP IC50 shift TDI assay”  to see how HR-MS is used in CYP inhibition assay.