**3. Mass analyzers**

#### **3.1 Quadrupole analyzer**

Quadrupole mass analyzer consists of four hyperbolic or circular rods positioned in parallel and are located diagonally at identical distances from each other. The rods are diagonally connected. Positive direct current (DC; U) is applied to one pair of rods and negative potential is applied to the other pair of rods. Apart from direct current, alternating radiofrequency (RF; *Vcos ωt*) potential is also applied to these rods. The ion trajectory is affected in x and y directions by the total electric field composed by a quadrupolar alternating field and a constant field. Because there is only a two-dimensional quadrupole field the ions accelerated after ionization, maintain their velocity along the z axis.

The motion of ions in the quadrupole can be best described by Mathieu Equations [43]. The ions supposedly travel in a stable trajectory and only those ions that travel in stable trajectory reaches detector. Mass spectrum is obtained by ramping RF and DC voltages in a constant ratio. When DC voltage is set to zero and RF voltage is maintained, all ions pass through quadrupole. It is the DC voltage that helps in filtering out the ions of interest and generate mass spectrum (**Figure 5**). Hence, the quadrupoles that apply only RF voltages just act as ion guides or collision cell. Mass resolution for typical quadrupole analyzers falls in the range 0.6–0.8 da units, which is defined to be a unit resolution. However, current generation high resolution mass spectrometers offer to determine masses within 5–10 ppm error.

#### **3.2 Triple quadrupole mass analyzer**

Triple quadrupole mass analyzer consists of two RF/DC mass analyzers and two RF only mass analyzers. Q0and Q2 (collision cell) were considered to be RF only quadrupoles, whereas Q1 and Q3 falls under RF/DC mass analyzers [44]. Hence, Q0 and Q2 acts as ion guides and Q1 and Q3 acts as mass filters. Q0 acts as an ion guide by focusing all the ions obtained from ionization source to Q1. Q1 even though a RF/ DC mass analyzer, can also be operated in RF only quadrupole depending on the type of analysis. When it comes to qualitative analysis, Q1 acts as a RF only quadrupole, whereas in case of quantitative analysis it acts as RF/DC quadrupole. Similarly, Q3 also operates in both modes based on the analytical requirements. Q2 in addition being a RF only quadrupole, acts as a collision cell to fragment the ions and generate compound specific information, which enables the mass spec to be a more specific and selective detection system (**Figure 6**). Process of generation of fragment ions in the collision cell is termed as collision induced dissociation (CID), which happens with the assistance of neutral argon or nitrogen gas [45, 46].

**45**

**Figure 5.**

**Figure 6.**

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

Based on the modes in which the mass analyzers are operated and the analytical

While full scan modes are useful in understanding the total pool of masses present in the sample analyzed, product ion scan helps in obtaining structural information of a precursor ion. Precursor ion scan is suited to find structural homologs of a selected fragment ion. In multiple reaction monitoring mode (MRM), a selected parent ion (Q1 mass) is fragmented within the collision cell and selected fragment ion analyzed by the detector. Together this series of events forms a reaction where

The quadrupole ion trap and the related quadrupole mass filter were invented by Paul and Steinwedel [47]. A quadrupole ion trap (QIT or 3D-IT) mass spectrometer operates with a three-dimensional quadrupole field. The QIT is formed by three electrodes: a ring electrode with a donut shape placed symmetrically between two end cap electrodes. QIT is a RF only quadrupole that acts a storage device and ions are focused to center of trap by collision with helium gas. Motion of ions in trap is regulated by axial and radial frequencies. The quadrupole ion trap can store only a limited number of ions before space charging occurs. To circumvent this effect, most instruments have an automatic gain control procedure (AGC). This procedure exactly determines the adequate fill time of the trap to maximize sensitivity and minimize resolution losses due to space charge. Ion motion can be modified either by exciting the radial or the axial frequencies by applying a small oscillating potential at the end cap electrodes during the RF ramp. Linear ion trap enables higher sensitivity than triple quadrupole mass spec analyzers in full scan mode, given the

multiple ions are monitored, hence the term multiple reaction monitoring.

capability of ion accumulation before traveling to the detector (**Figure 7**).

requirements, they can be briefly classified as below:

*Schematic diagram of triple quadrupole mass analyzer.*

*Schematic diagram of operation of quadrupole mass analyzer.*

**3.3 Linear ion trap mass analyzer**

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

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

#### **Figure 5.**

*Mass Spectrometry - Future Perceptions and Applications*

**3. Mass analyzers**

**3.1 Quadrupole analyzer**

maintain their velocity along the z axis.

**3.2 Triple quadrupole mass analyzer**

with the assistance of neutral argon or nitrogen gas [45, 46].

soft ionization technique that causes minimal fragmentation of the analyte ions and is also suitable for analysis of large molecules ranging from peptides to proteins, lipids and polymers [35–38]. It is also amenable to high throughput and target sample plates can be readily stored for future use. One major advantage of MALDI-MS is chromatographic separation of analytes is not required. However, due to lack of separation, matrix interferences impact the analytical results [39, 40]. Additionally, MALDI is not suitable for low molecular weight compounds. Also, MALDI needs TOF as an analyzer to cover high mass range in a linear mode, whereas ESI can be coupled with any mass analyzer. Recently, MALDI has been coupled to triple quadrupole and successfully used for the analysis of small molecules [41, 42].

Quadrupole mass analyzer consists of four hyperbolic or circular rods positioned

in parallel and are located diagonally at identical distances from each other. The rods are diagonally connected. Positive direct current (DC; U) is applied to one pair of rods and negative potential is applied to the other pair of rods. Apart from direct current, alternating radiofrequency (RF; *Vcos ωt*) potential is also applied to these rods. The ion trajectory is affected in x and y directions by the total electric field composed by a quadrupolar alternating field and a constant field. Because there is only a two-dimensional quadrupole field the ions accelerated after ionization,

The motion of ions in the quadrupole can be best described by Mathieu Equations [43]. The ions supposedly travel in a stable trajectory and only those ions that travel in stable trajectory reaches detector. Mass spectrum is obtained by ramping RF and DC voltages in a constant ratio. When DC voltage is set to zero and RF voltage is maintained, all ions pass through quadrupole. It is the DC voltage that helps in filtering out the ions of interest and generate mass spectrum (**Figure 5**). Hence, the quadrupoles that apply only RF voltages just act as ion guides or collision cell. Mass resolution for typical quadrupole analyzers falls in the range 0.6–0.8 da units, which is defined to be a unit resolution. However, current generation high resolution mass spectrometers offer to determine masses within 5–10 ppm error.

Triple quadrupole mass analyzer consists of two RF/DC mass analyzers and two RF only mass analyzers. Q0and Q2 (collision cell) were considered to be RF only quadrupoles, whereas Q1 and Q3 falls under RF/DC mass analyzers [44]. Hence, Q0 and Q2 acts as ion guides and Q1 and Q3 acts as mass filters. Q0 acts as an ion guide by focusing all the ions obtained from ionization source to Q1. Q1 even though a RF/ DC mass analyzer, can also be operated in RF only quadrupole depending on the type of analysis. When it comes to qualitative analysis, Q1 acts as a RF only quadrupole, whereas in case of quantitative analysis it acts as RF/DC quadrupole. Similarly, Q3 also operates in both modes based on the analytical requirements. Q2 in addition being a RF only quadrupole, acts as a collision cell to fragment the ions and generate compound specific information, which enables the mass spec to be a more specific and selective detection system (**Figure 6**). Process of generation of fragment ions in the collision cell is termed as collision induced dissociation (CID), which happens

**44**

*Schematic diagram of operation of quadrupole mass analyzer.*

**Figure 6.**

*Schematic diagram of triple quadrupole mass analyzer.*

Based on the modes in which the mass analyzers are operated and the analytical requirements, they can be briefly classified as below:

While full scan modes are useful in understanding the total pool of masses present in the sample analyzed, product ion scan helps in obtaining structural information of a precursor ion. Precursor ion scan is suited to find structural homologs of a selected fragment ion. In multiple reaction monitoring mode (MRM), a selected parent ion (Q1 mass) is fragmented within the collision cell and selected fragment ion analyzed by the detector. Together this series of events forms a reaction where multiple ions are monitored, hence the term multiple reaction monitoring.

#### **3.3 Linear ion trap mass analyzer**

The quadrupole ion trap and the related quadrupole mass filter were invented by Paul and Steinwedel [47]. A quadrupole ion trap (QIT or 3D-IT) mass spectrometer operates with a three-dimensional quadrupole field. The QIT is formed by three electrodes: a ring electrode with a donut shape placed symmetrically between two end cap electrodes. QIT is a RF only quadrupole that acts a storage device and ions are focused to center of trap by collision with helium gas. Motion of ions in trap is regulated by axial and radial frequencies. The quadrupole ion trap can store only a limited number of ions before space charging occurs. To circumvent this effect, most instruments have an automatic gain control procedure (AGC). This procedure exactly determines the adequate fill time of the trap to maximize sensitivity and minimize resolution losses due to space charge. Ion motion can be modified either by exciting the radial or the axial frequencies by applying a small oscillating potential at the end cap electrodes during the RF ramp. Linear ion trap enables higher sensitivity than triple quadrupole mass spec analyzers in full scan mode, given the capability of ion accumulation before traveling to the detector (**Figure 7**).

#### *Mass Spectrometry - Future Perceptions and Applications*


#### **Table 1.**

*Modes of triple quadrupole operation and analytical requirements.*

#### **Figure 7.** *Schematic diagram of linear ion trap mass analyzer.*

There are more than a few important features which impact the time necessary to attain a mass spectrum (duty cycle): (i) injection time (0.5–500.0 ms), (ii) scan speed (5000–20,000 m/z units/s), (iii) separation of the parent ion and fragmentation in tandem MS or MSn . Contrarily to the triple quadrupole, MS/MS is not performed in space but in time. Fragmentation happens with the assistance of helium as collision gas. Also, duty cycles for fragmentation (MS/MS) are much shorter in linear ion trap when compared to triple quadrupole mass analyzer. One major challenge in linear ion trap is to trap precursor ion and fragment in the same space. Often, due to this disadvantage, fragmentation spectra generated in linear ion trap differs from that of triple quadrupole CID. Also, number of MRM transitions that can be monitored in linear ion trap are quite less [4–8] when compared to QqQ mass analyzers (~100 MRM transitions can be monitored).

Due to the high sensitivity in MS<sup>n</sup> mode, ion traps are particularly attractive for qualitative analysis in drug metabolism, metabolomics and proteomics studies. Similar sensitivities to QqQ mass analyzer can be achieved for quantitative analysis on linear ion trap, but at the price of precision and accuracy.

While linear ion traps mainly function on radial ejection, next generation mass analyzers called quadrupole linear ion trap use axial ejection. This led to discovery of hybrid triple quadrupole mass analyzers, where Q3 performs the function of both quadrupole and linear ion trap [48, 49]. Unlike linear ion trap that fragments precursor in time, these hybrid analyzers perform fragmentation in space.

**47**

**Figure 8.**

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

The major advantage of this analyzer is that qualitative and quantitative analysis

Discovered in 1940's, time of flight mass analyzers achieved popularity after 1990's. Time of flight operates on principle of "time that ions need to cross in a field free tube of about 1 m length" [50, 51]. The motion of an ion is characterized by its

through the tube is directly proportional to their m/z value. The velocity of the ions formed is generally low and they are accelerated by strong electric fields (2000– 2030,000 V) in the direction of the detector. Low mass ions reach the detector more rapidly than high mass ions. Due to the short flight time (50–100 msec) and the good transmission, a spectrum can be generated within 100 ms over an almost unlimited mass range. Mass resolution of time of flight mass analyzer depends on the length of flight tube and reduced kinetic energy spread of the ions. Length of flight tube is directly proportional to mass resolution. Kinetic energy spread can be reduced by increasing time delay between ion formation and acceleration, also known as delayed pulse extraction. Also, positioning of electrostatic mirror in the

Briefly, the ions with high energy penetrate deeper into the ion mirror region than those with the same m/z at a lower energy. Because of the different trajectories, all ions of the same m/z reach the detector at the same time. With the reflectron the flight path is increased without changing the physical size of the instrument. Commercial TOF instruments are available to operate in either linear mode or reflectron mode. Even though ESI can be coupled with TOF, but the combination of MALDI and TOF is most popular as both operate on the principle of pulsed technique. Coupling of ESI with TOF needs orthogonal acceleration to drive continuous

Time of flight instruments are designed to use for qualitative analysis with MALDI or atmospheric pressure ionization. MALDI hyphenated with time of flight analyzer enables the identification of large molecules such as proteins, peptides, lipids and polymers. MS/MS information can also be obtained by CID in drift tube with the assistance of nitrogen or argon as collision gas. However, as quadrupole

drift region of ions increases the mass resolution (**Figure 8**).

*Schematics representation of a quadrupole time of flight mass spectrometer.*

(m = mass, v = speed). Therefore, the time ions fly

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

can be performed in the same LC–MS run.

**3.4 Time of flight mass analyzer**

kinetic energy Ec = 0.5 m x v2

beam of ions [52].

The major advantage of this analyzer is that qualitative and quantitative analysis can be performed in the same LC–MS run.
