**2. Small molecule basics for MSI**

Small molecule NTs are the chemical messengers of the central nervous system. Having a complete picture of NT location and abundance will aid in understanding of many different disease states and developmental processes. NTs are difficult to detect *in situ* via mass spectrometry due to their low physiological abundance (e.g., nM to pM concentration) within a complex biological tissue with many different classes of biomolecules, and overlapping low molecular mass range with most traditional matrices. Prior to the analyses discussed here, NTs were localized based on their protein-receptors or transporters, which does not always give an accurate accounting of present location.

Instrumentation used for MSI can vary widely, but most laser-based work is performed by time-of-flight (TOF) instruments. The other common setup is using desorption electrospray ionization (DESI) as an ion source, which is not the focus here, but is another option gaining in popularity [10, 11]. Other hardware

**101**

molecules, specifically NTs.

*Mass Spectrometry Imaging of Neurotransmitters DOI: http://dx.doi.org/10.5772/intechopen.93835*

adequately explored this topic [6, 7].

optimum crystallization.

configurations can favor small molecules (e.g., ion mobility, triple quadrupole instruments, Fourier transform – ion cyclotron resonance) and so there is no onesize-fits-all set-up for small molecule MSI experiments. In contrast to instrument choice, sample preparation/derivatization and ionization conditions are areas that can be standardized in order to achieve similar results across different platforms. The focus here is not on the many instrument combinations as other reviews have

Sample handling and preparation of tissue sections are integral to maintaining sample integrity; after cryo-sectioning, tissue is typically thaw mounted onto a solid surface. The surface must be conductive in order to apply a potential to the sample and accelerate ions out of the instrument source. Common materials include coated metal targets (expensive, cannot be archived, and not histology compatible) or indium-tin oxide (ITO) coated glass slides. Matrix application to the tissue section is ideally a homogenous coating of small crystals that provide optimum extraction conditions of analyte. After application, the key process is the co-crystallization that must occur between matrix and analyte. Spraying parameters affect the "wetness" of the surface of the tissue and are a balance between molecular diffusion and effective extraction. Crystal size is one of the more critical factors for a successful MSI experiment and multiple studies determined the parameters important for

Commonly used organic acid matrices for MSI are shown in **Table 1** and include sinapinic acid (SA), 2,5-dihydroxybenzoic acid (DHB), and α-cyano-4-hydroxycinnamic acid (CHCA). These matrices work for a broad variety of biomolecules including peptides and lipids, but do not always translate well to small molecule detection. The introduction of N-(1-napthyl)ethylenediamine dihydrochloride (NEDC) and 1,5-diaminonapthalene (DAN) have improved the detection of small molecules, though these matrices have different preparation needs (e.g., sublimation and recrystallization) which increases the time required for sample processing [12, 19]. DAN and NEDC matrices have not been fully explored yet in the literature. Moreover, the propensity for organic matrices to self-ionize and create chemical noise in the low mass range prevents effective analysis of most metabolites. Recently, this has led toward the rational design [35] or selection [36] of matrices that can address this, but the lack of consistency in performance can still be an issue. Matrix applications are notorious for behaving differently across laboratories, and significant research in the past 25 years has been devoted to identi-

fying preparation methods that result in the most consistent data [37–39].

Sublimation procedures, mentioned *vida supra*, require that matrix and sample are placed in a vacuum chamber which is evacuated [32]. The sample is cooled while the matrix is heated, resulting in sublimation of the matrix which will condense on the cool sample surface. Recrystallization of the matrix is often coupled with this technique. There are advantages and disadvantages to all of the aforementioned matrix application techniques, which have been discussed thoroughly in the literature [40–42]. Automated sprayers have become increasingly popular and help with consistency of matrix application, though the size and spacing of the matrix droplets will ultimately affect spatial resolution of the experiment. This topic has been frequently discussed and reviewed in the literature [2, 6], so only a basic introduction is given here.

Despite these challenges, there are a few examples of successful metabolomics imaging experiments, though they have utilized purposefully designed matrices that do not generate interfering signals [35] or have used high-resolution instruments that have the high mass accuracy to distinguish between isobaric signals [43, 44]. These approaches are not an all-encompassing solution, and the next sections explore other strategies to achieve broader success with MSI of small

#### *Mass Spectrometry Imaging of Neurotransmitters DOI: http://dx.doi.org/10.5772/intechopen.93835*

*Mass Spectrometry in Life Sciences and Clinical Laboratory*

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

*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* 

Small molecule NTs are the chemical messengers of the central nervous system. Having a complete picture of NT location and abundance will aid in understanding of many different disease states and developmental processes. NTs are difficult to detect *in situ* via mass spectrometry due to their low physiological abundance (e.g., nM to pM concentration) within a complex biological tissue with many different classes of biomolecules, and overlapping low molecular mass range with most traditional matrices. Prior to the analyses discussed here, NTs were localized based on their protein-receptors or transporters, which does not always give an accurate

Instrumentation used for MSI can vary widely, but most laser-based work is performed by time-of-flight (TOF) instruments. The other common setup is using desorption electrospray ionization (DESI) as an ion source, which is not the focus here, but is another option gaining in popularity [10, 11]. Other hardware

mechanism of ionization to favor small molecules of interest.

**2. Small molecule basics for MSI**

accounting of present location.

**100**

**Figure 1.**

*Ref. [5]. Copyright 2013 Springer.*

configurations can favor small molecules (e.g., ion mobility, triple quadrupole instruments, Fourier transform – ion cyclotron resonance) and so there is no onesize-fits-all set-up for small molecule MSI experiments. In contrast to instrument choice, sample preparation/derivatization and ionization conditions are areas that can be standardized in order to achieve similar results across different platforms. The focus here is not on the many instrument combinations as other reviews have adequately explored this topic [6, 7].

Sample handling and preparation of tissue sections are integral to maintaining sample integrity; after cryo-sectioning, tissue is typically thaw mounted onto a solid surface. The surface must be conductive in order to apply a potential to the sample and accelerate ions out of the instrument source. Common materials include coated metal targets (expensive, cannot be archived, and not histology compatible) or indium-tin oxide (ITO) coated glass slides. Matrix application to the tissue section is ideally a homogenous coating of small crystals that provide optimum extraction conditions of analyte. After application, the key process is the co-crystallization that must occur between matrix and analyte. Spraying parameters affect the "wetness" of the surface of the tissue and are a balance between molecular diffusion and effective extraction. Crystal size is one of the more critical factors for a successful MSI experiment and multiple studies determined the parameters important for optimum crystallization.

Commonly used organic acid matrices for MSI are shown in **Table 1** and include sinapinic acid (SA), 2,5-dihydroxybenzoic acid (DHB), and α-cyano-4-hydroxycinnamic acid (CHCA). These matrices work for a broad variety of biomolecules including peptides and lipids, but do not always translate well to small molecule detection. The introduction of N-(1-napthyl)ethylenediamine dihydrochloride (NEDC) and 1,5-diaminonapthalene (DAN) have improved the detection of small molecules, though these matrices have different preparation needs (e.g., sublimation and recrystallization) which increases the time required for sample processing [12, 19]. DAN and NEDC matrices have not been fully explored yet in the literature. Moreover, the propensity for organic matrices to self-ionize and create chemical noise in the low mass range prevents effective analysis of most metabolites. Recently, this has led toward the rational design [35] or selection [36] of matrices that can address this, but the lack of consistency in performance can still be an issue. Matrix applications are notorious for behaving differently across laboratories, and significant research in the past 25 years has been devoted to identifying preparation methods that result in the most consistent data [37–39].

Sublimation procedures, mentioned *vida supra*, require that matrix and sample are placed in a vacuum chamber which is evacuated [32]. The sample is cooled while the matrix is heated, resulting in sublimation of the matrix which will condense on the cool sample surface. Recrystallization of the matrix is often coupled with this technique. There are advantages and disadvantages to all of the aforementioned matrix application techniques, which have been discussed thoroughly in the literature [40–42]. Automated sprayers have become increasingly popular and help with consistency of matrix application, though the size and spacing of the matrix droplets will ultimately affect spatial resolution of the experiment. This topic has been frequently discussed and reviewed in the literature [2, 6], so only a basic introduction is given here.

Despite these challenges, there are a few examples of successful metabolomics imaging experiments, though they have utilized purposefully designed matrices that do not generate interfering signals [35] or have used high-resolution instruments that have the high mass accuracy to distinguish between isobaric signals [43, 44]. These approaches are not an all-encompassing solution, and the next sections explore other strategies to achieve broader success with MSI of small molecules, specifically NTs.

**Table 1.** *Common MSI organic acid matrices and their applications.*
