Multicenter Validation Study of Quantitative Imaging Mass Spectrometry.

The aim of this study was to assess potential sources of variability in quantitative imaging mass spectrometry (IMS) across multiple sites, analysts, and instruments. A sample from rat liver perfused with clozapine was distributed to three sites for analysis by three analysts using a predefined protocol to standardize the sample preparation, acquisition, and data analysis parameters. In addition, two commonly used approaches to IMS quantification, the mimetic tissue model and dilution series, were used to quantify clozapine and its major metabolite norclozapine in isolated perfused rat liver. The quantification was evaluated in terms of precision and accuracy with comparison to liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The results of this study showed that, across three analysts with six replicates each, both quantitative IMS methods achieved relative standard deviations in the low teens and accuracies of around 80% compared to LC-MS/MS quantification of adjacent tissue sections. The utility of a homogeneously coated stable-isotopically labeled standard (SIL) for normalization was appraised in terms of its potential to improve precision and accuracy of quantification as well as qualitatively reduce variability in the sample tissue images. SIL normalization had a larger influence on the dilution series, where the use of the internal standard was necessary to achieve accuracy and precision comparable to the non-normalized mimetic tissue model data. Normalization to the internal standard appeared most effective when the intensity ratio of the analyte to internal standard was approximately one, and thus precludes this method as a universal normalization approach for all ions in the acquisition.