An on-tissue chemical derivatization method for MALDI-MSI analysis of anthraquinones in mouse tissues

Acquisition of tissue spatial distribution information is essential for identifying sites of action, assessing pharmacological effects and elucidating mechanisms, thereby providing evidence for drug development. Anthraquinones, including rhein, emodin, aloe-emodin, chrysophanol and physcion, are the main pharmacologically active constituents of rhubarb. However, as exogenous compounds, most anthraquinones are difficult to detect by conventional matrix-assisted laser desorption/ionization time-of-flight mass spectrometry imaging (MALDI-TOF-MSI) because of low tissue abundance, poor ionization efficiency and strong ion suppression from endogenous matrices. To overcome these limitations, we established a highly sensitive on-tissue chemical derivatization (OTCD) workflow for MALDI-TOF-MSI analysis of anthraquinones in mouse tissues. By optimizing matrix selection and OTCD labeling conditions, we first visualized the spatial distributions of rhein and chrysophanol in mouse small intestine and kidney tissues. The derivatization reagent 2-fluoro-1-methylpyridinium p-toluenesulfonate (FMPTS) selectively labels phenolic hydroxyl groups, enabling rapid and efficient in situ derivatization of anthraquinones on tissue sections. In on-tissue spiking experiments (1 µL per spot), the FMPTS-OTCD method achieved a practical lowest tested concentration producing a signal of 100 pg per spot, with diagnostic ions clearly distinguishable above the local tissue background. In contrast, the underivatized workflow required a higher amount (≥10 ng per spot) for detectable signals, demonstrating that FMPTS-OTCD effectively overcomes the low-response bottleneck of the traditional MALDI-TOF-MSI method for anthraquinones. The resulting derivatization increased the signal to noise ratio by 20-1154 times and enabled reliable in situ visualization of rhein and chrysophanol in mouse tissues.