**5. Quantitation**

MSI has been applied to quantitative analysis of drugs [90, 91], metabolites [92], and biomarkers in tissue [93] using pneumatic sprayers and sublimation techniques described in this chapter. Nearly all of these demonstrations have utilized organic acid matrices such as DHB, CHCA, and trihydroxyacetophenone (THAP), with one research group utilizing TiO2 NPs [94, 95]. Methods of quantitation are still being investigated [96], as many of the consistency issues with MALDI-MSI that have been discussed in this chapter are even more relevant with quantitative MSI (qMSI). **Figure 6** shows a summary of two of the more common methods used for generation of a calibration curve for qMSI, on-tissue spotting and tissue mimetic models which feature spiking of tissue homogenates [96].

On-tissue spotting uses either a standard molecule that is chemically similar to the analyte or a stable isotope of the analyte for making the calibration curve. Ion intensities between the analyte and standard are used to estimate the drug concentration in dosed tissue. Disadvantages include difficulty in maintaining uniform application of standards and differences in ionization for sprayed on standards vs. analyte molecules embedded within tissue. Advantages are that this method is fast and straightforward. The tissue mimetic model uses a surrogate tissue that is homogenized and spiked with the analyte of interest, frozen, sectioned, then prepared with matrix. The advantage of this method is that there is

**Figure 6.**

*Description of qMSI experiments where on-tissue spotting and homogenate spiking are two popular methods. Reprinted with permission from Ref. [94]. Copyright 2019 Elsevier Ltd.*

better representation of the ionization process for analyte embedded within tissue. However, it is more time consuming, labor intensive, and requires more tissue for the calibration curve. Each method has been correlated with LC-MS data, the current primary method used for quantification [97].

Specific examples with clinical relevancy are briefly described here. First, epertinib and lapatinib were quantified in a metastatic brain cancer mouse model using stable isotope labeling, and with liquid chromatography (LC)-MS validation [91]. The topical drugs roflumilast, tofacitinib, ruxolitinib, and LEO 29102 were examined in human skin explants to determine drug penetration and evaluate lipid markers [90]. qMSI data had a much lower quantitation range than LC-MS data of individual skin layers. Rifampicin in mouse liver tissue was quantified via a fragment ion of the intact molecule. The method used an in-house synthesized stable isotope and correlated the results with LC-MS/MS [98]. Lastly, there is one example that specifically focused on comprehensive mapping of NTs in Parkinson's disease lesioned mouse brain and demonstrated quantitation of dopamine using a stable isotope [87]. All of the drug molecules listed in this paragraph are above the general size range that NTs and metabolites fall within, ranging from 400 to 800 Da, but present possible future avenues of research for the NT-focused methods discussed in this chapter.

The tissue mimetic model first gained popularity with examination of lapatinib and nevirapine in mouse liver by Groseclose and Castellino [99]. In addition to demonstrating high spatial resolution, they examined reproducibility and drug distribution within the homogenate. Fewer applications of the tissue mimetic model have been done, especially with small molecules as opposed to lipids [100]. A notable example includes the determination of the spatial distribution of gemcitabine, a chemotherapeutic agent, and its metabolites in mouse model pancreatic tumors using AuNPs and a traditional matrix as comparison [101]. Further experiments also work on the computational side of MSI and determining the best ways to normalize spectra [102, 103].
