**3.5 Orbitrap mass analyzer**

Orbitrap mass analyzer operates on principle of Fourier transform, where orbital trapping of ions around an electrode system is achieved with the assistance of electrical field [57]. The orbitrap is formed by a central spindle-like electrode surrounded by an electrode with a barrel-like shape to create an electrostatic potential. The m/z is a reciprocal proportionate to the frequency of the ions oscillating along the z-axis. Detection is performed by measuring the current image of the axial motion of the ions around the inner electrode. The mass spectrum is obtained after Fourier transformation of the image current. The orbitrap provides a mass resolving power exceeding 100,000 and a mass accuracy ~3 ppm. To be operational as a mass spectrometer the orbitrap requires external ion accumulation, cooling and fragmentation (**Figure 9**).

The first commercial instrument to utilize this capability, LTQ Orbitrap Classic, was introduced by Thermo Fisher Scientific in 2005, which later underwent many innovations with the addition of a collision cell after the C-trap in LTQ Orbitrap XL, addition of electron transfer dissociation (ETD) capabilities, followed by MALDI source operating at reduced pressure with high-end LTQ Orbitrap XL MALDI instrument, and finally a stacked ring rf ion guide (so called S-lens) brought about 10-fold higher transfer efficiency in the MS/MS mode.

Typically, the highest resolving powers available in TOF devices are several times lower than the resolution in Orbitrap, although recent multipass TOF devices

**49**

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

are capable of ultrahigh resolution (R ≥ 100,000 at m/z 400) [58, 59]. While TOF accompanies similar resolution in both MS and MS/MS modes, orbitrap suffers from low resolution in MS/MS mode. Orbitrap technology will endure to progress towards increased resolving power, acquisition speed, sensitivity and mass accuracy. These developments will indisputably open the arena for new applications as the Orbitrap instruments are getting more prevalent and exploring into new areas

Drug discovery research was solely driven by chemists and pharmacologist in early 1990's, when very little is known about drug absorption, distribution, metabolism and elimination (ADME). However, it did not take much time before researchers realized the importance of optimizing ADME properties of NCE's for successfully driving drug discovery programs [60]. In this section, we highlighted importance of ADME in drug discovery and its relation to mass spectrometry.

Drug metabolism also known as xenobiotic biotransformation is the process by which lipophilic compounds gets eliminated from the body after getting converted to hydrophilic species that are easily filtered through kidney. While metabolism is desired in few cases where metabolites are the active species producing efficacy, there are metabolites that are toxic in nature. In such cases, where toxic by-products are produced, metabolism is not desired. Metabolism as a discipline drawn its first attention after the publication of RT Williams on Detoxification mechanisms [61]. Drug metabolism over the years with the aid of mass spectrometry technology has evolved in understanding the metabolic pathways of NCE's and also to identify the metabolites (both desired and undesired) [62–65]. Mass spectrometry was initially hyphenated with gas chromatography to understand the metabolic behavior of NCE's. Gas chromatography worked well for analyzing volatile compounds and its metabolites, however it did not suit for nonvolatile and thermolabile compounds. With the advent of liquid chromatography that can handle and separate components without subjecting to evaporation, it became prevalent as an analytical tool for understanding drug metabolism in drug discovery and development [66, 67]. As a part of understanding the metabolic properties, NCE's will be initially screened for metabolic stability in across species (human/rat/dog/mouse/monkey) and in various matrices including microsomes/S9 fractions/cytosol/hepatocytes, plasma, tissue homogenates, and buffer. If metabolism is not desired then compounds will be screened for their stability in relevant matrices and compounds with moderate to high stability (defined by half-life and intrinsic clearance) are further optimized for additional ADME properties. Various Phase 1 metabolic reactions including oxidation, demethylation, hydroxylation and phase 2 metabolic reactions covering glucuronidation, sulfation, methylation, amino acids conjugation and glutathione conjugation can be quantitatively and qualitatively studied using LC–MS/ MS. Additionally, for compounds that are unstable, understanding the soft spots responsible for instability helps medicinal chemists to make relevant structural modifications in order to stabilize the unstable compounds. Understanding the soft spots precisely, needs the assistance of high-resolution mass spectrometry instruments such as TOF and Orbitrap. With the accurate mass information obtained from these mass spec's, identifying a metabolite structure will be spot on.

Similarly, other in vitro parameters such as permeability, protein binding, solubility, lipophilicity, CYP inhibition and CYP induction also play a key role in

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

**4. Applications in drug discovery**

of research.

**4.1 ADME studies**

**Figure 9.** *Schematic representation of operation of orbitrap mass analyzer.*

are capable of ultrahigh resolution (R ≥ 100,000 at m/z 400) [58, 59]. While TOF accompanies similar resolution in both MS and MS/MS modes, orbitrap suffers from low resolution in MS/MS mode. Orbitrap technology will endure to progress towards increased resolving power, acquisition speed, sensitivity and mass accuracy. These developments will indisputably open the arena for new applications as the Orbitrap instruments are getting more prevalent and exploring into new areas of research.
