Mass Spectrometry Imaging of Diclofenac and Its Metabolites in Tissues Using Nanospray Desorption Electrospray Ionization.
Glucuronidation is a common phase II metabolic process for drugs and xenobiotics which increases their solubility for excretion. Acyl glucuronides (glucuronides of carboxylic acids) present concerns of toxicity as they have been implicated in gastrointestinal toxicity and hepatic failure. Despite the substantial success in the bulk analysis of these species, little is known about their localization in tissues. Herein, we used nanospray desorption electrospray ionization mass spectrometry imaging (nano-DESI-MSI) to examine the localization of diclofenac, a widely used nonsteroidal anti-inflammatory drug, and its metabolites in mouse kidney and liver tissues. Nano-DESI allows for label-free imaging with high spatial resolution and sensitivity without special sample pretreatment. Using nano-DESI-MSI, ion images for diclofenac and its major metabolites were produced. MSI data acquired over a broad m/z range showed fairly low signals of the drug and its metabolites. At least an order of magnitude improvement in the signals was obtained using selected ion monitoring (SIM), with m/z windows centered around the low-abundance ions of interest. Using nano-DESI MSI in SIM mode, we observed that diclofenac acyl glucuronide is localized to the inner medulla and hydroxydiclofenac to the cortex of the kidney. The distributions observed for both metabolites closely match the previously reported localization of enzymes that process diclofenac into its respective metabolites. The localization of diclofenac acyl glucuronide to medulla likely indicates that the toxic metabolite is being excreted from the tissue. In contrast, a uniform distribution of diclofenac, hydroxydiclofenac and the diclofenac acyl glucuronide metabolite was observed in the liver tissue. Semiquantitative analysis found the metabolite to diclofenac ratios calculated from nano-DESI in agreement to those calculated from liquid chromatography tandem mass spectrometry (LC-MS/MS) experiments. Collectively, our results demonstrate nano-DESI-MSI can be successfully used to image diclofenac and its primary metabolites in dosed liver and kidney tissues from mice and derive semi-quantitative data from localized tissue regions.