**2. Atmospheric pressure ionization**

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 pharmacokinetics are very limited.

**41**

**Figure 1.**

*Mechanism of electrospray ionization process.*

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

*Mass Spectrometry as a Workhorse for Preclinical Drug Discovery: Special Emphasis on Drug…*

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

*DOI: http://dx.doi.org/10.5772/intechopen.88385*

**2.1 Electrospray ionization (ESI)**

*Mass Spectrometry as a Workhorse for Preclinical Drug Discovery: Special Emphasis on Drug… DOI: http://dx.doi.org/10.5772/intechopen.88385*
