Study of the Distribution of Acetaminophen and Its Metabolites in Rats, from the Whole-Body to Isolated Organ Levels, by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging after On-Tissue Chemical Derivatization

Abstract

During drug development, detailed investigations of the pharmacokinetic profile of the drug are required to characterize its absorption, distribution, metabolism, and excretion properties. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) is an established technique for studies of the distribution of drugs and their metabolites. It has advantages over autoradiography, which is conventionally used for distribution studies: it does not require the radiolabeling of drugs and can distinguish between the drug and its metabolites directly in the tissue. However, its lack of sensitivity in certain cases remains challenging. Novel procedures, such as on-tissue chemical derivatization (OTCD), could be developed to increase sensitivity. We used OTCD to enhance the sensitivity of MALDI-MSI for one of the most widely used drugs, acetaminophen, and to study its distribution in tissues. Without derivatization, this drug and some of its metabolites are undetectable by MALDI-MSI in the tissues of treated rats. We used 2-fluoro-1-methylpyridinium p-toluene sulfonate as a derivatization reagent, to increase the ionization yield of acetaminophen and some of its metabolites. The OTCD protocol made it possible to study the distribution of acetaminophen and its metabolites in whole-body sections at a spatial resolution of 400 μm and in complex anatomical structures, such as the testis and epididymis, at a spatial resolution <50 μm. The OTCD is also shown to be compatible with the quantification of acetaminophen by MALDI-MSI in whole-body tissues. This protocol could be applied to other molecules bearing phenol groups and presenting a low ionization efficiency.

Development of an Automatic Ultrasonic Matrix Sprayer for Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging

“…Accompanied with improvements in methods and techniques, MALDI MSI has been more and more widely used in the field of biomedical research such as disease diagnosis, 2,3 biomarker discovery, 4 and drug development. 5 Matrix selecting and matrix deposition are crucial steps for preparation of samples before MALDI MSI. Various matrixes, such as α-cyano-4-hydroxycinnamic acid (CCA) 6,7 and 2,5dihydroxybenzoic acid (DHB) 8,9 for analysis in positive mode; 9-aminoacridine (9-AA), 10,11 1,5-naphthalenediamine dihydrochloride (1,5-DANHCl), 12 N-(1-naphthyl)ethylenediamine dihydrochloride (NEDC), 13,14 and nitrogen-and sulfur-codoped carbon dots (N,S-co-doped CDs) 15 for analysis in negative mode; and 1,5-naphthalenediamine (1,5-DAN) 16,17 and 3-aminophthalhydrazide (Luminol) 18 for dual-polarity detection, have been used for MALDI MSI in recent years.…”

“…Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI), introduced by Caprioli et al in 1997, is a label-free method which allows in situ localizing of a wide range of biomolecules simultaneously with high sensitivity and molecular specificity from a tissue section in a single run. Accompanied with improvements in methods and techniques, MALDI MSI has been more and more widely used in the field of biomedical research such as disease diagnosis, , biomarker discovery, and drug development …”

Desorption Electrospray Ionization Mass Spectrometry Imaging Allows Spatial Localization of Changes in Acetaminophen Metabolism in the Liver after Intervention with 4-Methylpyrazole

“…Following this early innovative study, sensitivity was improved for oxidative metabolites when using MALDI-MSI on the whole-body section of rats. However, this was at the expenditure of spatial resolution since that was only at 400 μm pixel size. , While this may suggest that the rat may be ideal for MSI analysis of drug metabolism after an APAP overdose, much higher APAP doses are needed to induce toxic effects, and this would have limited clinical relevance since it has been shown that the pathophysiology of APAP hepatotoxicity in the rat is distinct from that of humans and thus the rat is not a clinically relevant model for studying APAP hepatotoxicity . To circumvent these issues with MALDI-MSI for analysis of APAP metabolism, we now show that the label and matrix-free DESI-MSI technique allows us to spatially map changes in APAP metabolism in the liver of the clinically relevant C57BL/6J murine model , after an APAP overdose.…”

“…36,37 Furthermore, the application of the MALDI matrices may also disturb the spatial localization of small molecules. 37,38 Several studies have applied MALDI-MSI to study the spatial distribution of APAP metabolism in mouse liver sections 39 as well as liver 40 or whole-body sections 41 from rats. However, no liver zonation was evident for APAP and its nonoxidative metabolites using the technique, which was unable to detect oxidative metabolites except APAP-GSH.…”

“…However, no liver zonation was evident for APAP and its nonoxidative metabolites using the technique, which was unable to detect oxidative metabolites except APAP-GSH . Though the sensitivity of detection was improved for oxidative metabolites with on-tissue chemical derivatization, this was at the expense of a lower resolution (400 μm) , required to resolve the spatial distribution of analytes within the region of interest in the mouse liver . Thus, it is of utmost importance to have a label-free method for the direct tissue analysis of APAP and its metabolites to evaluate their spatial distribution.…”