A New Dimensional Perspective on Drug R&D: Mass Spectrometry Imaging Reshapes the Paradigm of Pharmacokinetic Research

Pharmacokinetic (PK) studies are introducing a new analytical dimension. Traditional PK studies involve collecting blood and tissue samples at different time points for mass spectrometry analysis to obtain time-concentration curves for drugs and metabolites; however, they are unable to answer the key question of “how the drug is distributed within an organ”. The advent of mass spectrometry imaging has expanded PK research from the temporal to the spatial dimension—not only can we determine how drug concentrations change over time, but we can also visualise the spatial distribution of the drug within different regions of a tissue. This shift is reshaping the analytical paradigm of drug discovery and development.

Information gaps in traditional PK studies

Pharmacokinetic (PK) studies are one of the core components of new drug development. The basic workflow of traditional PK studies is as follows: collection of blood and organ tissue samples at different time points following drug administration → homogenisation and extraction → quantitative analysis via LC-MS/MS → plotting of time-concentration curves for the drug and its metabolites.

Whilst this workflow answers the question of “how does the concentration of a drug in the body change over time?”, it suffers from a fundamental information gap: tissue homogenisation yields the “average concentration” of the entire organ, completely obscuring the variations in drug distribution across different regions within the organ.

Take the liver as an example: it is composed of hepatic lobules, with significant differences in enzyme expression and metabolic capacity between different regions. Drug concentrations around the central vein and around the portal tract within a hepatic lobule may differ by a factor of several, yet tissue homogenisation can only provide an average value. This lack of spatial information may lead to misjudgements regarding the drug’s tissue distribution and metabolic behaviour.

Spatial information is equally critical for drug toxicity assessment. If a drug becomes abnormally concentrated in a specific region of an organ, even if the overall average concentration remains within safe limits, the localised high concentration may cause tissue damage—this “spatial toxicity” cannot be detected at all in traditional PK studies.

Mass spectrometry imaging bridges the spatial information gap

Mass spectrometry imaging directly fills the information gap in traditional PK studies by scanning tissue sections point by point to obtain the spatial distribution of drugs and metabolites.

At different time points following drug administration, frozen sections are prepared from animal organs, and the mass spectrometry imaging system scans the sections to obtain mass spectrometry data for each pixel. Once signals with the same mass-to-charge ratio are reconstructed in spatial coordinates, they form a spatial distribution map of the drug or metabolite within the organ. Overlaying the spatial distribution maps from different time points provides a complete picture of the drug’s spatiotemporal distribution in vivo.

This analytical approach delivers enhancements in three key areas:

Visualisation of drug spatial distribution—it is immediately apparent in which regions within an organ the drug is concentrated and in which it is scarce. Spatial tracking of metabolites—the spatial distribution of drug metabolites within an organ can be compared with that of the parent drug, revealing the spatial characteristics of metabolic transformation. Spatial toxicity early warning—unintended accumulation of the drug in off-target tissues can be detected at an early stage, providing spatial evidence for toxicological assessment.

The impact of mass spectrometry imaging ion sources on drug analysis

The effectiveness of mass spectrometry imaging in drug PK studies depends on the detection capability of the mass spectrometry imaging ion source for drug molecules.

Drug molecules exhibit a wide range of polarity — from highly polar, water-soluble drugs to low-polarity, fat-soluble drugs, covering the entire polarity spectrum. Many important drug classes (steroidal drugs, fat-soluble vitamins, macrolide antibiotics) are weakly polar or non-polar compounds; traditional DESI mass spectrometry imaging ion sources are unable to effectively ionise and detect these molecules.

This issue is particularly pronounced in pharmacokinetic (PK) studies. If a mass spectrometry imaging ion source exhibits polarity discrimination, the drug molecules themselves or their metabolites may be missed, resulting in incomplete spatial distribution maps or even misleading results.

The introduction of photo-ionisation technology has changed this situation. The MSI DPI electrospray/photochemical ionisation mass spectrometry imaging source from Hefei Xinceyuan Instrument Technology Co., Ltd. employs a dual-ionisation architecture combining DESI with photochemical secondary ionisation, The ionisation energy of VUV photons (Kr lamp, 10.0/10.6 eV) covers the ionisation energy range of the vast majority of organic compounds, without introducing selectivity based on molecular polarity—weakly polar and non-polar drug molecules, such as steroid drugs and fat-soluble vitamins, are afforded the same opportunities for ionisation and detection as polar drugs.

In terms of sensitivity, the signal intensity in the DESI/PI mode of MSI DPI is 1–3 orders of magnitude higher than that of conventional DESI. For low-concentration metabolites commonly encountered in PK studies, this increase in sensitivity means that more trace metabolites can be detected and spatially localised, leading to a more comprehensive elucidation of drug metabolism pathways.

The reduction in ion suppression is equally important for drug quantification. In traditional DESI, ion suppression in complex biological matrices leads to inaccurate drug quantification and requires cumbersome sample purification. MSI DPI’s post-photoionisation utilises the direct interaction between photons and neutral molecules, without relying on charge competition; this significantly reduces ion suppression, allowing complex tissue samples to be analysed directly, thereby improving the accuracy of drug quantification.

Spatial analysis of herbal mixtures

Pharmacokinetic (PK) studies of traditional Chinese medicines and natural product-based drugs face a particular challenge: herbal extracts are multicomponent mixtures containing hundreds of chemical constituents, each with distinct distribution and metabolic behaviour within the body. Traditional PK studies require the development of separate detection methods for each target constituent, which is labour-intensive and prone to overlooking non-target active components.

The non-targeted detection capabilities of mass spectrometry imaging (MSI) provide an efficient solution for the spatial analysis of herbal mixtures. A single MSI experiment can simultaneously capture the spatial distribution of multiple constituents and their metabolites within organs, without the need to predefine detection targets.

The full-polarity detection capability of the MSI DPI mass spectrometry imaging source is particularly important in this context. Active constituents in herbal medicines encompass a wide range of chemical classes, including aglycones (weakly polar), glycosides (polar), terpenes (non-polar) and alkaloids (with significant variations in polarity). Whilst traditional DESI can only cover a portion of these, the DESI/PI mode of MSI DPI is capable of simultaneously detecting these constituents of varying polarities.

In mass spectrometry imaging studies of the traditional Chinese medicinal herb Atractylodes macrocephala, the MSI DPI has demonstrated its ability to spatially detect polyacetylenes and sesquiterpenes (results published in *Molecular Horticulture*, 2025, 5:11); this capability can be extended to pharmacokinetic (PK) studies of the in vivo distribution of Atractylodes macrocephala’s active constituents.

Integration from research to the drug development process

The application of mass spectrometry imaging in drug development is evolving from “validation studies” towards “process integration”.

During the new drug discovery phase, mass spectrometry imaging can assess the tissue permeability of drug candidates—can the drug reach the target tissue? Is the concentration in the target tissue sufficient? Answers to these questions from a spatial perspective aid in the early screening and optimisation of candidate drug molecules.

During the preclinical research stage, mass spectrometry imaging can track changes in the spatial distribution of drugs across different organs over time, identifying unexpected accumulation in non-target tissues and providing spatial evidence for toxicological assessment.

In studies of drug metabolism mechanisms, mass spectrometry imaging can simultaneously capture the spatial distribution of both the parent drug and its metabolites within tissues, revealing the spatial characteristics of metabolic transformation—which areas are metabolically active? In which areas do metabolites accumulate?

Hefei Xinceyuan’s MSI LDPI laser/photochemical ionisation mass spectrometry imaging source provides cell-level drug distribution analysis with a spatial resolution of 2–3 μm. In tumour tissue imaging, MSI LDPI can distinguish differences in metabolite distribution between tumour regions and normal tissue, offering unique value for assessing the penetration and distribution of anti-tumour drugs within the tumour microenvironment.

Industry Drivers

The advancement of mass spectrometry imaging applications in drug discovery is driven by several factors.

From a technical perspective, the post-photoionisation mass spectrometry imaging ion source resolves the issues of polarity discrimination and low sensitivity associated with traditional imaging sources, enabling the spatial detection of drug molecules (particularly weakly polar and non-polar drugs). The launch of the MSI DPI and MSI LDPI products provides drug R&D institutions with domestically manufactured equipment options.

In terms of cost, the MSI DPI mass spectrometry imaging source is compatible with the three major mass spectrometry brands—Agilent, AB SCIEX and Thermo. Drug R&D laboratories already equipped with mass spectrometers can acquire mass spectrometry imaging capabilities simply by retrofitting an imaging source, thereby lowering the barrier to entry. The product comes with a 12-month warranty, and localised technical support offers rapid response times, whilst maintenance costs are lower than those of imported products.

From an academic perspective, a series of findings published by Professor Pan Yang’s research group in journals such as *Analytical Chemistry* have provided academic validation for the application of mass spectrometry imaging in pharmaceutical analysis. At the 4th National Conference on Mass Spectrometry Analysis, several pharmaceutical companies and drug R&D institutions expressed an interest in collaborating on the MSI DPI and MSI LDPI mass spectrometry imaging sources.

Challenges and Outlook

The widespread application of mass spectrometry imaging in pharmaceutical PK research still faces certain challenges. Standardised methods for quantitative analysis have yet to be established, and there are discrepancies in imaging conditions and data processing workflows across different laboratories. There remains a gap between imaging speed and the throughput requirements of pharmaceutical PK research. The balance between improving spatial resolution and detection sensitivity needs to be optimised for different drug types.

Nevertheless, the value of mass spectrometry imaging technology in extending PK research from the temporal to the spatial dimension has already been validated. With the continuous improvement in the detection capabilities of domestically produced mass spectrometry imaging ion sources and the gradual increase in the prevalence of such equipment, mass spectrometry imagers are expected to become standard analytical tools in the drug R&D process, providing spatial analytical support to enhance the efficiency of new drug development.

Hefei Xinceyuan has stated that the company will continue to advance the validation and optimisation of mass spectrometry imaging sources in drug R&D scenarios, whilst collaborating with drug R&D institutions and CROs to drive the integration and implementation of mass spectrometry imaging technology into the drug R&D process.