Standard Addition as a Method for Quantitative Mass Spectrometry Imaging.

In mass spectrometry imaging (MSI), analytes are desorbed and ionized directly from a complex and unique chemical microenvironment in each pixel, which makes their quantification challenging. Matrix effects have been addressed by the use of isotopically labeled internal standards (IS), either included in the solvent or sprayed over the tissue section, for pixel-by-pixel relative quantification. However, in addition to requiring preselection, isotopically labeled IS may be costly or unavailable. Here, we introduce a novel approach for quantification in MSI, based on the standard addition method. We report a workflow for both acquiring and processing quantitative data. Furthermore, we compare the detected concentrations obtained by standard addition to the detected concentrations obtained using both IS quantification and external calibration. Finally, we show the applicability of using molecules extracted from tissue as an easily accessible standard mixture for standard addition quantification in MSI. The possibility of using analytical standards and readily available endogenous analytes as a source of calibration standards makes our standard addition-based quantitative approach cost-effective, accessible, and versatile.