**Abstract**

Mass spectrometry imaging (MSI) is a powerful analytical method for the simultaneous analysis of hundreds of compounds within a biological sample. Despite the broad applicability of this technique, there is a critical need for advancements in methods for small molecule detection. Some molecular classes of small molecules are more difficult than others to ionize, e.g., neurotransmitters (NTs). The chemical structure of NTs (i.e., primary, secondary, and tertiary amines) affects ionization and has been a noted difficulty in the literature. In order to achieve detection of NTs using MSI, strategies must focus on either changing the chemistry of target molecules to aid in detection or focus on new methods of ionization. Additionally, even with new strategies, the issues of delocalization, chemical background noise, and ability to achieve high throughput (HTP) must be considered. This chapter will explore previous and up-and-coming techniques for maximizing the detection of NTs.

**Keywords:** mass spectrometry imaging, neurotransmitters, gold nanoparticles, derivatization, sample preparation

### **1. Introduction**

Mass Spectrometry Imaging (MSI) is an incredibly powerful label-free technique that can determine qualitative and quantitative information of hundreds of compounds in a tissue section in one experiment [1, 2]. Small molecule detection, especially of neurotransmitters (NTs), currently relies heavily on histochemical, immunohistochemical, and ligand-based assays. Antibody-based methods suffer from limitations in cost and availability of antibodies, lack of specificity for target molecules of interest, and low throughput [3, 4]. The development of MSI has overcome many of these challenges and will be discussed throughout. The basic methodology of MSI is to section tissue using a cryostat to approximately 10-20 μm thickness; tissues may or may not be embedded in a cryomatrix such as Shandon™ M-1 (ThermoFisher Scientific). Next, matrix must be deposited on the tissue section, which is most often done by spray-coating the tissue using a pneumatic sprayer. Variations on typical organic matrices, such as inorganic nanoparticles (NPs), have been explored by numerous researchers and will be commented on here. Mass spectral data is collected at discrete locations on the sample surface via a raster pattern, which can then be assembled into a heat-map image of molecule location. **Figure 1** depicts the typical MSI scheme [5]. A number of overall reviews of MSI have appeared in recent years [6] that address broad topics like ionization of small molecules [7], clinical applications [2], and high-resolution analyses [5].

#### **Figure 1.**

*General scheme of the mass spectrometry imaging process. (a) The tissue section is covered with matrix and irradiated by a pulsed laser beam. (b) Mass spectrum acquired from one spatial location on the tissue section. (c) MS images of different m/z peaks compiled from all spatial locations. Reprinted with permission from Ref. [5]. Copyright 2013 Springer.*

The broader scientific community is not yet fully utilizing MSI as there are still challenges to be overcome [7, 8], including: (i) low ionization efficiency for small molecules, (ii) chemical noise interferents/overlapping signals of small molecules with traditional matrices [9], (iii) reproducibility issues across laboratories which limits universal procedures for MSI in pre-/clinical research, (iv) limits to lateral spatial resolution inherent to the matrix crystallization process which affects the ability to clearly define tissue margins, and (v) delocalization of analyte molecules during sample preparation. This chapter will focus on the analysis of small molecules, specifically neurotransmitters (NTs), due to the complex biological processes that occur in the brain and have broad implications in disease states and overall health. This chapter is broken down in two main categories, as strategies to improve ionization must either focus on (i) the chemical nature of the analyte and changing its properties to better facilitate ionization, or (ii) on utilizing a different mechanism of ionization to favor small molecules of interest.
