**2.1 Electrospray ionization (ESI)**

*Mass Spectrometry - Future Perceptions and Applications*

drug discovery and development.

emphasis on drug metabolism and pharmacokinetic assays.

**2. Atmospheric pressure ionization**

higher flow rates of liquid sample. Additionally, ionization techniques such as chemical and electron impact ionization did not suit for thermolabile and high molecular weight compounds [2, 3]. Advancement in ionization techniques such as fast atom bombardment that suited for analysis of large molecules, thermospray and particle beam ionization which were efficient for small molecules enabled the fruitful hyphenation of liquid chromatography with mass spectrometry [4–7]. Thermospray in general forms ammonium adducts, while particle beam generated electron impact spectra. Within a few years thermospray was succeeded by atmospheric pressure ionization techniques such electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), atmospheric pressure photoionization (APPI) and atmospheric pressure matrix assisted laser desorption ionization (AP-MALDI). Mass spectrometer operates on the principle of ionization of analytes followed by their separation based on their mass-to-charge ratio [8]. Various mass spectrometer analyzers ranging from linear trap, ion trap, triple quadrupole, time of flight and orbitrap have specific applications for sample analysis in preclinical

Medicinal chemistry efforts in drug discovery is majorly focused on understanding the right combination of new chemical entity (NCE) properties that helps in cherry-picking the compounds with promising properties to progress from discovery to development phase. In the process of lead optimization, NCE's are subjected to a series of drug metabolism and pharmacokinetic assays to assess the druggable properties and mitigate late stage failures. Almost 40% of failures in development phase were due to poor pharmacokinetic properties of NCE's [9]. However, this percentage had gradually decreased to 10% as major pharmaceutical companies incorporated drug metabolism and pharmacokinetic screening (DMPK) in lead optimization phase of drug discovery. In this view, compounds must pass through a series of screens that scrutinize the problematic compounds until a small number have been selected for more rigorous testing in the development phase. Hence, lead optimization typically is an iterative process that uses the DMPK data to optimize the druggable properties of NCE's. Regardless of the screening panel, qualitative and quantitative analytical results to understand absorption, distribution, metabolism and excretion (ADME) properties were generated using liquid chromatography–tandem mass spectrometry (LC–MS/MS) [10–17]. In this chapter, we have briefly discussed on various atmospheric pressure ionization techniques/mass analyzers, and applications of mass spectrometer in drug discovery with special

Ionization in atmospheric ionization sources occur at atmospheric pressure and ions then gets transferred into the vacuum. As the liquid completely converts in to gas phase in the ionization source, those ionization techniques that use atmospheric pressure ionization are more convenient to hyphenate with liquid chromatography. These ionization techniques include electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), and atmospheric pressure photoionization (APPI) and are most widely used. Additionally, MALDI that also uses atmospheric pressure ionization is getting popular with its unique feature in performing mass spectrometry imaging (MSI) and analysis of large molecules [18]. Also, other ionization techniques such as desorption electrospray (DESI) or direct analysis in real time (DART) are becoming popular for the analysis of surface or solid samples [19, 20]. However, their applications in the field of drug metabolism and pharmaco-

**40**

kinetics are very limited.

In ESI, analytes initially get charged with the assistance of electrical energy and charged ions transfer from solution to gaseous phase, before subjecting to mass spectrometric analysis. Ionic species in solution can be analyzed as such, whereas neutral compounds can be converted to ionic species and studied by ESI-MS. Electrospray ionization occurs in four stages: 1) charging of analytes in the capillary tube 2) formation of fine spray of charged droplets 3) solvent evaporation 4) columbic explosion/Rayleigh scattering of ions from the droplet (**Figure 1**). The liquid effluent moves from liquid chromatography to the mass spectrometer through a fused silica capillary maintained at voltage of 2.5–6.0 KV. In negative mode, to avoid discharge the range is lower (3–4 KV) than positive mode. ESI is a condensed phase ionization process and the ions have to be already present in solution. To generate ions, the pH has to be adjusted in such a way that ionizable groups are either protonated or deprotonated. In some cases, neutral molecules can be analyzed by the formation of adducts with ions such as ammonium, sodium, potassium, acetate or silver. Charged droplets undergo nebulization in the presence of nebulizer gas. After nebulization, charged droplets further reduce in size with the assistance of heat and breakdown in to minute droplets. Finally, as the droplets grow smaller and smaller, ions get released in to gaseous phase by a mechanism called rayleigh scattering/columbic explosion. The emitted ions are sampled by a sampling skimmer cone and are then accelerated into the mass analyzer for subsequent measurement of molecular mass and ion intensity [21–24]. An important characteristic of ESI-MS is it works as a concentration-dependent detector, which means MS response is directly proportional to concentration of analyte. Hence, irrespective of flow rate of mobile phase post column to the ionization source, response remains the same as long as the source-gas conditions are optimal for the flow rate. ESI technique is suitable for the analysis of polar to moderately polar molecules.

### **2.2 Atmospheric pressure chemical ionization (APCI)**

In atmospheric pressure chemical ionization, unlike ESI, sample evaporation occurs first, followed by ionization in gas phase through corona discharge needle (**Figure 2**) [25]. The ionization principle is mostly similar to chemical ionization; however, it occurs at atmospheric pressure. APCI-MS can also be called as masssensitive detector, as the higher flow that goes in to ionization source, the higher will

**Figure 1.** *Mechanism of electrospray ionization process.*

be the peak response. One important requirement in APCI for optimal sensitivity is the sample has to be completely evaporated, before subjecting to ionization. First an aerosol of mobile phase is formed with the assistance of nebulizer gas. This aerosol is further subjected to heating at 200–550°C in a ceramic tube enabling complete evaporation. Even though higher temperatures are employed, the actual temperature felt by analyte molecules is way lesser due to a phenomenon called evaporative cooling effect/evaporation enthalpy. Next, analyte molecules in gas phases were bombarded with electrons formed from corona discharge needle [26, 27]. In positive mode primary ions such as N2+ are formed by electron impact. These ions further react with water in several steps by charge transfer to form H3O+ . Ionization of the analytes occurs then by proton transfer from H3O+ . In negative mode ions are formed either by: (i) resonance capture (AB to AB− ), (ii) dissociative capture (AB to B− ) or (iii) ion–molecule reaction (BH to B− ). One disadvantage with APCI when compared to ESI is, APCI is not suitable for thermolabile compounds as typical temperatures experienced by the analyte molecules are ~150°C. However, in case of ESI, molecules encounter temperatures ~40°C in the process of evaporation.
