**4.1 ADME studies**

*Mass Spectrometry - Future Perceptions and Applications*

trap and TOF-TOF mass spectrometers [54–56].

10-fold higher transfer efficiency in the MS/MS mode.

**3.5 Orbitrap mass analyzer**

fragmentation (**Figure 9**).

technology is so successful for both qualitative and quantitative analysis, TOF analyzers are used as a hybrid platform with quadrupole analyzers. In these hybrid systems, TOF analyzer replaces Q3 of a triple quadrupole system. These hybrid systems are termed as QTOF mass spectrometers. QTOF systems offer high mass resolution (~40,000) and sensitivity. Accurate mass measurements are especially useful in metabolite identification studies and peptide analysis [53]. Various other hydrid TOF platforms have been reported including, linear ion trap, quadrupole ion

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

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

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

**48**

**Figure 9.**

*Schematic representation of operation of orbitrap mass analyzer.*

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 drug disposition [15, 68, 69]. All of these assays have high sensitivity requirements and demand quantification of analytes within a few nanomolar range. For example, in case of permeability assessment, low permeable compounds such as atenolol permeate poorly from apical to basolateral side complicates the quantification of the apparent permeability values, if analyzed with low sensitive detectors. Likewise, fraction unbound values for highly protein compounds such as warfarin were such low that it demands highly sensitive detectors to accurately quantify such low levels. Needless to mention that all of the assays performed to optimize ADME properties of NCE's require highly sensitive detection systems. Hence, with its superior detection sensitivity, mass spectrometer has become an indispensable tool to understand the in vitro ADME properties of NCE's.

As discussed in the previous sections, even though there exist many ionization techniques, atmospheric pressure ionization (API) was more successful in drug metabolism studies, given its rapid, specific and sensitive methodologies for the identification of drugs and its metabolites. Mass spectrometer instruments types used in ADME studies vary from those that provide nominal mass information and accurate mass information. Nominal mass instruments such as triple quadrupoles are useful for quantitative applications, whereas accurate mass instruments including QTOF and Orbitrap are used for both quantitative and qualitative applications.

Of the atmospheric pressure ionization techniques available, ESI and APCI are the most commonly used. While APCI can accommodate high flow rates and produce high sensitivity, nevertheless analytes are subjected to higher temperatures in the evaporation process and hence as an ionization technique is not suitable for thermolabile (esp. glucuronides, N-oxides and sulfates) compounds. However, ESI is comparatively a soft ionization technique and could efficiently ionize these fragile compounds without degradation. As a whole, mass spectrometer exhibits both qualitative and quantitative applications in drug metabolism studies. However, in case of other ADME battery of assays as described above, mass spectrometer is used majorly for quantitative applications.

#### **4.2 Metabolite identification**

Metabolite identification (Met-ID) provides a variety of inputs in drug discovery and development which includes in vitro metabolite profiling in early stages of lead identification/optimization, followed by in vitro/in vivo correlation in late stage lead identification, characterization of putative metabolites, cross-species comparison to identify the right tox preclinical species, understanding drug–drug interactions, and identifying pharmacologically active or toxic metabolites and the mechanisms by which they are formed [70, 71].

Met-ID is quite challenging when it comes to a) identification of vast number of diverse metabolites, b) metabolites that are of low abundance, and c) high throughput analytical requirements to screen majority of early leads in preclinical drug discovery [72]. Even though there exist various platforms such as triple quadrupole, linear ion trap, and Qtrap to quantitatively/qualitatively identify metabolites, they turned obsolete due to nominal mass information they generate and are no longer valuable [73].

The term mass resolution is used to describe the mass resolving power according to the degree to which two analytes with close m/z values can be separated and identified. A practical and convenient way of evaluating the mass resolution of an instrument is the use of the full width at half maximum (FWHM) definition in which the m/z/Δm/z ratio is calculated, where m/z is the mass-to-charge value of an ion peak and Δm/z is the full width at half the maximal height of the peak. Nominal mass instruments generate resolution in low thousands (1000–4000),

**51**

**Figure 10.**

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

which cannot separate isobaric ions with similar nominal m/z value. However, high resolution mass spectrometers provide resolution in the range of higher thousands (10000–100,000), that successfully identify and separate isobaric ions (**Figure 10**). For a given nominal mass, there exists many possible molecular structures for an assigned biotransformation pathway. Hence, for an accurate molecular structure the metabolite has to be scaled up in larger quantities and measured using NMR spectroscopy. However, scaling up metabolites to "mg" quantities need tremendous efforts, resources and is not an economical approach. Later, with the discovery of high-resolution mass spectrometers such as TOF and Orbitrap, that provides accurate mass to the fourth decimal, enabled accurate prediction of molecular structures

In principle, as long as data can be measured accurately, high-resolution data is sufficient to demonstrate the presence or absence of defined species. Apart from high resolution masses, modern mass spectrometers also generate data with higher accuracy. The term mass accuracy is used to define how close the mass measured by the mass spectrometer is to the theoretical exact mass of an ion. Mass accuracy

*Increased resolution separating two closely arranged analytes with similar nominal m/z values.*

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

of metabolites [74–79].

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

which cannot separate isobaric ions with similar nominal m/z value. However, high resolution mass spectrometers provide resolution in the range of higher thousands (10000–100,000), that successfully identify and separate isobaric ions (**Figure 10**).

For a given nominal mass, there exists many possible molecular structures for an assigned biotransformation pathway. Hence, for an accurate molecular structure the metabolite has to be scaled up in larger quantities and measured using NMR spectroscopy. However, scaling up metabolites to "mg" quantities need tremendous efforts, resources and is not an economical approach. Later, with the discovery of high-resolution mass spectrometers such as TOF and Orbitrap, that provides accurate mass to the fourth decimal, enabled accurate prediction of molecular structures of metabolites [74–79].

In principle, as long as data can be measured accurately, high-resolution data is sufficient to demonstrate the presence or absence of defined species. Apart from high resolution masses, modern mass spectrometers also generate data with higher accuracy. The term mass accuracy is used to define how close the mass measured by the mass spectrometer is to the theoretical exact mass of an ion. Mass accuracy

**Figure 10.**

*Increased resolution separating two closely arranged analytes with similar nominal m/z values.*

*Mass Spectrometry - Future Perceptions and Applications*

the in vitro ADME properties of NCE's.

majorly for quantitative applications.

mechanisms by which they are formed [70, 71].

**4.2 Metabolite identification**

drug disposition [15, 68, 69]. All of these assays have high sensitivity requirements and demand quantification of analytes within a few nanomolar range. For example, in case of permeability assessment, low permeable compounds such as atenolol permeate poorly from apical to basolateral side complicates the quantification of the apparent permeability values, if analyzed with low sensitive detectors. Likewise, fraction unbound values for highly protein compounds such as warfarin were such low that it demands highly sensitive detectors to accurately quantify such low levels. Needless to mention that all of the assays performed to optimize ADME properties of NCE's require highly sensitive detection systems. Hence, with its superior detection sensitivity, mass spectrometer has become an indispensable tool to understand

As discussed in the previous sections, even though there exist many ionization techniques, atmospheric pressure ionization (API) was more successful in drug metabolism studies, given its rapid, specific and sensitive methodologies for the identification of drugs and its metabolites. Mass spectrometer instruments types used in ADME studies vary from those that provide nominal mass information and accurate mass information. Nominal mass instruments such as triple quadrupoles are useful for quantitative applications, whereas accurate mass instruments including QTOF and Orbitrap are used for both quantitative and qualitative applications. Of the atmospheric pressure ionization techniques available, ESI and APCI are the most commonly used. While APCI can accommodate high flow rates and produce high sensitivity, nevertheless analytes are subjected to higher temperatures in the evaporation process and hence as an ionization technique is not suitable for thermolabile (esp. glucuronides, N-oxides and sulfates) compounds. However, ESI is comparatively a soft ionization technique and could efficiently ionize these fragile compounds without degradation. As a whole, mass spectrometer exhibits both qualitative and quantitative applications in drug metabolism studies. However, in case of other ADME battery of assays as described above, mass spectrometer is used

Metabolite identification (Met-ID) provides a variety of inputs in drug discovery

Met-ID is quite challenging when it comes to a) identification of vast number of diverse metabolites, b) metabolites that are of low abundance, and c) high throughput analytical requirements to screen majority of early leads in preclinical drug discovery [72]. Even though there exist various platforms such as triple quadrupole, linear ion trap, and Qtrap to quantitatively/qualitatively identify metabolites, they turned obsolete due to nominal mass information they generate and are no longer

The term mass resolution is used to describe the mass resolving power according to the degree to which two analytes with close m/z values can be separated and identified. A practical and convenient way of evaluating the mass resolution of an instrument is the use of the full width at half maximum (FWHM) definition in which the m/z/Δm/z ratio is calculated, where m/z is the mass-to-charge value of an ion peak and Δm/z is the full width at half the maximal height of the peak. Nominal mass instruments generate resolution in low thousands (1000–4000),

and development which includes in vitro metabolite profiling in early stages of lead identification/optimization, followed by in vitro/in vivo correlation in late stage lead identification, characterization of putative metabolites, cross-species comparison to identify the right tox preclinical species, understanding drug–drug interactions, and identifying pharmacologically active or toxic metabolites and the

**50**

valuable [73].

is typically expressed as a relative mass error using the ratio of the difference between the experimental and theoretical m/z values over the theoretical m/z value of an ion.

Metabolite identification studies are typically performed in full scan mode (or) data dependent scan mode. In full scan mode, accurate mass information of parent ions is studied to understand the biotransformation pathways. However, to propose soft spots for metabolites, data dependent scans consisting of combination of full scan and product ion scans are performed. Metabolites in general can be considered as off springs to the parent molecular ions that carry similar fragments as that of parent ion (or) neutral loss fragments. In case of similar fragments to that of parent ion, these fragments can be considered unmodified and are similar to that of parent. Whereas, if the fragment ions are accompanied by a mass change for a given biotransformation pathway, then metabolite soft spot can be proposed with precision to that fragment ion (for example, in case of hydroxylation, neutral loss fragment in metabolite carries an additional mass of 16 amu) [80]. Additionally, apart from fragmentation scans accompanied with full scan, few other specific dependent scans consisting of neutral loss scan, and precursor ion scans are used to study various biotransformation pathways including glucuronidation, glutathione conjugation, sulfation (for example, glucuronidation is accompanied by a specific neutral loss of 176 da and glutathione conjugation by a neutral loss of 129 da in positive mode) [81, 82]. These specific scan functions are helpful in eliminating the background noise and identify the metabolites that exist even at lower abundance.

One major challenge in metabolite identification using LC–MS technology, is quantifying the relative abundance of metabolites. As mass spec quantitation is accompanied by many source/gas and compound dependent parameters that aid in the efficient ionization and detection, minor modifications in the metabolite structures alter the sensitivity by few orders of magnitude. Hence, quantitative results using mass spectrometer for the metabolites for which synthetic standards are not available, is not feasible. Additionally, it is difficult to synthesize each and every metabolite and determine their concentrations accurately. Alternatively, few researchers used LC-UV hyphenated with mass spectrometer to measure the relative abundance of metabolites. However, as majority of metabolites exist in low abundance, it becomes difficult to measure their relative abundance by UV spectroscopy. Also, UV is prone to differences in analytical sensitivities with minor modifications in structure. Hence, future mass spectrometers need to be designed to address these key concerns and facilitate evaluation of both quantitative and qualitative aspects of metabolites.

#### **4.3 Pharmacokinetic analysis**

Screening paradigm in drug discovery includes evaluation of compounds for their ADME properties by various in vitro assays. The datasets obtained from these assays help in rationalizing the synthetic chemistry efforts and make progress towards a pool of lead compounds that exhibit promising in vitro ADME properties. However, in vitro screening consists of unique assay platforms that can only answer a specific question of interest and can never cover all aspects of complex biological systems. Hence, it warrants the screening of selected lead compounds in preclinical species such as rats, mice, dog, pig and monkey, before progressing to clinic [83, 84]. Role of LC–MS/MS in performing bioanalysis of pharmacokinetic samples was well reported in the literature [85–88].

Design of pharmacokinetic studies varies from single route administration with few sampling points followed by multiple route administration with detailed time course evaluation. Samples of various natures ranging from blood, plasma,

**53**

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

serum are collected to analyze the systemic concentration levels. Additionally, to understand the tissue distribution of compounds, various tissues including liver, intestine, brain, spinal cord, heart, lungs, kidneys, skin and adipose tissue are also analyzed. Initially, lead compounds are dosed intravenously to understand the disposition parameters such as volume of distribution, clearance, half-life and mean residence time. Compounds that possess decent pharmacokinetic parameters in intravenous route are further evaluated in alternate (enteral and parenteral) routes to assess bioavailability and exposure parameters (Cmax, Tmax, AUC0-last). To determine plasma concentrations as low as "ng" levels, sensitive analytical detectors are needed. HPLC-UV detector systems are proven robust for analyzing concentrations at higher "μg" level. However, as UV detection is not specific, it suffers from high background noise when biological samples are analyzed. High background noise in turn causes quantitation issues at the lower portion of calibration curve. On the other hand, LC–MS/MS detection system is considered to be highly specific and selective, as background noise can be eliminated by analyzing selected analytes of interest with desired m/z ratios. Also, mass spectrometric detection is considered highly sensitive than UV spectroscopic detection. Altogether, these advanced features, enabled LC–MS/MS to overcome the limitations of UV detection and is more frequently used in drug discovery and development for the bioanalysis of

Of the various mass spectrometers available in the market, triple quadrupole LC–MS/MS systems have demonstrated tremendous success when it comes to quantitative applications. Monitoring specific reaction transitions that consist of parent and fragment ion, with associated source/gas and compound dependent parameters makes the mass spectrometer highly specific, selective and sensitive. Pharmacokinetic study samples are in general monitored for plasma concentrations over the time profile and hence triple quadrupole systems have achieved greater success. However, exploratory studies performed to understand metabolic pattern and biotransformation mechanisms of NCE's again need the assistance of linear ion traps and high-resolution mass spectrometer platforms (Orbitrap and TOF). As pharmacokinetic study samples are of biological origin and complex in nature, extracts obtained after sample preparation complicate the bioanalysis on LC–MS/ MS. This phenomenon in broad terms is termed as Matrix effects. Causes of matrix effects and strategies to mitigate these effects are discussed in detail in the succeed-

Typically, pharmacokinetic studies are performed by administering a single test item in preclinical species. However, screening of single test item is labor intensive, not economical and demand higher turn-around times to generate pharmacokinetic data. Hence, researchers have come up with an alternate strategy, where a pool of compounds are administered in single dose, a technique well-known as cassette dosing or N-in-one dosing. The foundation for designing cassette dosing strategy comes from the ability of LC–MS/MS to analyze multiple test items without any chromatographic separation [89–92]. However, disadvantages with cassette dosing include altered pharmacokinetics due to drug–drug interaction potential, non-feasibility of pooling compounds with close molecular weights and compounds with differing physicochemical properties posing formulatability issues. Compounds with differing physicochemical properties comes with a challenge of formulating the selected pool of compounds in a single formulation vehicle. While issues with physicochemical properties and close molecular weights can be taken care of, drug–drug interaction appears to be of a major concern. One approach to minimize DDI's is by administering the compounds at minimal doses, collectively not exceeding the dose of single test item administration [93]. Also, dosing volumes can be kept as low as possible. Additionally, along with pool of unknown compounds, a quality control compound

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

pharmacokinetic samples.

ing section.

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

serum are collected to analyze the systemic concentration levels. Additionally, to understand the tissue distribution of compounds, various tissues including liver, intestine, brain, spinal cord, heart, lungs, kidneys, skin and adipose tissue are also analyzed. Initially, lead compounds are dosed intravenously to understand the disposition parameters such as volume of distribution, clearance, half-life and mean residence time. Compounds that possess decent pharmacokinetic parameters in intravenous route are further evaluated in alternate (enteral and parenteral) routes to assess bioavailability and exposure parameters (Cmax, Tmax, AUC0-last). To determine plasma concentrations as low as "ng" levels, sensitive analytical detectors are needed. HPLC-UV detector systems are proven robust for analyzing concentrations at higher "μg" level. However, as UV detection is not specific, it suffers from high background noise when biological samples are analyzed. High background noise in turn causes quantitation issues at the lower portion of calibration curve. On the other hand, LC–MS/MS detection system is considered to be highly specific and selective, as background noise can be eliminated by analyzing selected analytes of interest with desired m/z ratios. Also, mass spectrometric detection is considered highly sensitive than UV spectroscopic detection. Altogether, these advanced features, enabled LC–MS/MS to overcome the limitations of UV detection and is more frequently used in drug discovery and development for the bioanalysis of pharmacokinetic samples.

Of the various mass spectrometers available in the market, triple quadrupole LC–MS/MS systems have demonstrated tremendous success when it comes to quantitative applications. Monitoring specific reaction transitions that consist of parent and fragment ion, with associated source/gas and compound dependent parameters makes the mass spectrometer highly specific, selective and sensitive. Pharmacokinetic study samples are in general monitored for plasma concentrations over the time profile and hence triple quadrupole systems have achieved greater success. However, exploratory studies performed to understand metabolic pattern and biotransformation mechanisms of NCE's again need the assistance of linear ion traps and high-resolution mass spectrometer platforms (Orbitrap and TOF). As pharmacokinetic study samples are of biological origin and complex in nature, extracts obtained after sample preparation complicate the bioanalysis on LC–MS/ MS. This phenomenon in broad terms is termed as Matrix effects. Causes of matrix effects and strategies to mitigate these effects are discussed in detail in the succeeding section.

Typically, pharmacokinetic studies are performed by administering a single test item in preclinical species. However, screening of single test item is labor intensive, not economical and demand higher turn-around times to generate pharmacokinetic data. Hence, researchers have come up with an alternate strategy, where a pool of compounds are administered in single dose, a technique well-known as cassette dosing or N-in-one dosing. The foundation for designing cassette dosing strategy comes from the ability of LC–MS/MS to analyze multiple test items without any chromatographic separation [89–92]. However, disadvantages with cassette dosing include altered pharmacokinetics due to drug–drug interaction potential, non-feasibility of pooling compounds with close molecular weights and compounds with differing physicochemical properties posing formulatability issues. Compounds with differing physicochemical properties comes with a challenge of formulating the selected pool of compounds in a single formulation vehicle. While issues with physicochemical properties and close molecular weights can be taken care of, drug–drug interaction appears to be of a major concern. One approach to minimize DDI's is by administering the compounds at minimal doses, collectively not exceeding the dose of single test item administration [93]. Also, dosing volumes can be kept as low as possible. Additionally, along with pool of unknown compounds, a quality control compound

*Mass Spectrometry - Future Perceptions and Applications*

value of an ion.

is typically expressed as a relative mass error using the ratio of the difference between the experimental and theoretical m/z values over the theoretical m/z

Metabolite identification studies are typically performed in full scan mode (or) data dependent scan mode. In full scan mode, accurate mass information of parent ions is studied to understand the biotransformation pathways. However, to propose soft spots for metabolites, data dependent scans consisting of combination of full scan and product ion scans are performed. Metabolites in general can be considered as off springs to the parent molecular ions that carry similar fragments as that of parent ion (or) neutral loss fragments. In case of similar fragments to that of parent ion, these fragments can be considered unmodified and are similar to that of parent. Whereas, if the fragment ions are accompanied by a mass change for a given biotransformation pathway, then metabolite soft spot can be proposed with precision to that fragment ion (for example, in case of hydroxylation, neutral loss fragment in metabolite carries an additional mass of 16 amu) [80]. Additionally, apart from fragmentation scans accompanied with full scan, few other specific dependent scans consisting of neutral loss scan, and precursor ion scans are used to study various biotransformation pathways including glucuronidation, glutathione conjugation, sulfation (for example, glucuronidation is accompanied by a specific neutral loss of 176 da and glutathione conjugation by a neutral loss of 129 da in positive mode) [81, 82]. These specific scan functions are helpful in eliminating the background noise and identify the metabolites that exist even at lower abundance. One major challenge in metabolite identification using LC–MS technology, is quantifying the relative abundance of metabolites. As mass spec quantitation is accompanied by many source/gas and compound dependent parameters that aid in the efficient ionization and detection, minor modifications in the metabolite structures alter the sensitivity by few orders of magnitude. Hence, quantitative results using mass spectrometer for the metabolites for which synthetic standards are not available, is not feasible. Additionally, it is difficult to synthesize each and every metabolite and determine their concentrations accurately. Alternatively, few researchers used LC-UV hyphenated with mass spectrometer to measure the relative abundance of metabolites. However, as majority of metabolites exist in low abundance, it becomes difficult to measure their relative abundance by UV spectroscopy. Also, UV is prone to differences in analytical sensitivities with minor modifications in structure. Hence, future mass spectrometers need to be designed to address these key concerns and facilitate evaluation of both quantitative and

Screening paradigm in drug discovery includes evaluation of compounds for their ADME properties by various in vitro assays. The datasets obtained from these assays help in rationalizing the synthetic chemistry efforts and make progress towards a pool of lead compounds that exhibit promising in vitro ADME properties. However, in vitro screening consists of unique assay platforms that can only answer a specific question of interest and can never cover all aspects of complex biological systems. Hence, it warrants the screening of selected lead compounds in preclinical species such as rats, mice, dog, pig and monkey, before progressing to clinic [83, 84]. Role of LC–MS/MS in performing bioanalysis of pharmacokinetic

Design of pharmacokinetic studies varies from single route administration with few sampling points followed by multiple route administration with detailed time course evaluation. Samples of various natures ranging from blood, plasma,

**52**

qualitative aspects of metabolites.

samples was well reported in the literature [85–88].

**4.3 Pharmacokinetic analysis**

with known pharmacokinetic parameters can be administered. Pharmacokinetic study results of unknown compounds can be considered acceptable, as long as quality control compounds fall within the set acceptance criteria. Compounds with less than 5 da difference in molecular weight are difficult to pool, when triple quadrupole systems are used for analyzing the pharmacokinetic samples. However, this challenge can be overcome by considering high resolution mass spectrometric analysis. Typical pharmacokinetic parameters studied from intravenous administration include half-life (t1/2), clearance (Cl), volume of distribution (Vz), mean residence time (MRT), area under the curve (AUC0-last; AUC0-inf), whereas parameters such as Cmax, Tmax and area under the curve are studies in other routes of administration. When compounds are dosed in multiple routes, along with intravenous route of administration, absolute bioavailability values are calculated.

Current fast LC–MS/MS instruments enable analysis of mixture of analytes with minimal separation, shorter run times and also feasible for hyphenation with ultra-fast liquid chromatography systems. Additionally, latest LC–MS/MS systems are capable of analyzing thousands of samples every week due to higher loading capacity of samples in autosampler, shorter run times and introduction of 96/384 well plate formats.

Test samples are analyzed against a calibration curve and a set of quality control samples. Calibration curve consists of 8–10 known standards and quality controls span the calibration curve at a minimum of 3 levels. Typical accuracy limits for qualifying the calibration and quality control samples is set as ±20%. Typical turnaround times for execution of pharmacokinetic studies right from dosing initiation to generation of pharmacokinetic parameters spans 1–2 weeks. Importance of high throughput bioanalysis and its role in drug discovery is discussed in detail in the section below.

### **4.4 High throughput bioanalysis**

With the combinatorial chemistry efforts leading to synthesis of hundreds of compounds in early phases of drug discovery, there is a constant need for analytical platforms that can quickly churn out data and help in accelerating the discovery process. LC–MS/MS with proven track record as a reliable analytical platform had undergone evolutionary changes to support high throughput demands of drug discovery [60, 70, 87, 94–96]. These strategies include advancements in chromatographic columns, where lower dimensions and microbore HPLC columns cut short run time to one minute per sample. Quick sample run times help in analyzing higher number of samples with in the given stipulated time [97]. Faster gradient methods with LC pumps that can handle higher pressure also enable analysis with shorter run times [98]. With the invention of monolithic HPLC columns that can be operated at high flow rates in the order of 5–6 mL/min, analytical run times were significantly reduced [99–101]. One disadvantage of these columns is that the high flow rates translate in to higher usage of mobile phase, making it an expensive alternative.

Another approach for increasing sample throughput is by the use of parallel HPLC columns, where the effluent from two HPLC systems could be combined and assayed by using the MRM/SRM capabilities of the MS/MS system [102, 103]. One more approach in enhancing the throughput is through staggered analysis approach. Here, multiple HPLC columns are used, but the injection time is staggered such that the "analytical window" can be selected sequentially in order to maximize the use of the MS/MS system and increase sample throughput [104].

**55**

**Figure 11.**

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

analyzed on triple quadrupole systems, care must be taken to have molecular weight differences by at least 5 da. Typically, 4–5 analytes can be pooled and analyzed. Only challenge with pooling/cassette strategy (even with high resolution mass spectrometers) is its non-suitability for isomeric compounds. With advancements in instrumentation technology, modern LC autosamplers are designed to accommodate higher sample load. There are autosamplers that can accommodate as high as twelve 96 well plates. Higher loading capacity of autosamplers enable unattended

When it comes to sample preparation, robotic platforms can be used to screen larger pool of compounds across various in vitro assays. However, this strategy can also be used for processing of in vivo samples, provided if the sample cohort is higher. In general, automated robotic sample preparation platforms are quite often used for screening of compounds in in vitro assays. These robotic platforms help in decreasing the manpower involved and time taken for performing the assays. However, main disadvantage with robotic platforms is the need for preparation of larger volumes of reagents and the cost factor involved. Hence, it is not recommended to use these platforms unless there exists a larger library of compounds.

With the sample nature being biological in origin, supernatants obtained after sample preparation consists of many endogenous components that compete with the analyte of interest and result in either suppression/enhancement of ionization. This process of alteration of the ionization of analytes is termed as matrix effects. The "matrix" refers to all components in the sample other than analyte(s) of interest. Matrix effects are defined as "interference from matrix components that are

*Pictorial representation of LC–MS/MS system with higher loading capacity of autosampler.*

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

analysis of large number of samples (**Figure 11**).

**4.5 Matrix effects**

On the other hand, throughput can also be increased by pooling of samples, provided the sensitivity is not seriously compromised. When these samples are *Mass Spectrometry as a Workhorse for Preclinical Drug Discovery: Special Emphasis on Drug… DOI: http://dx.doi.org/10.5772/intechopen.88385*

analyzed on triple quadrupole systems, care must be taken to have molecular weight differences by at least 5 da. Typically, 4–5 analytes can be pooled and analyzed. Only challenge with pooling/cassette strategy (even with high resolution mass spectrometers) is its non-suitability for isomeric compounds. With advancements in instrumentation technology, modern LC autosamplers are designed to accommodate higher sample load. There are autosamplers that can accommodate as high as twelve 96 well plates. Higher loading capacity of autosamplers enable unattended analysis of large number of samples (**Figure 11**).

When it comes to sample preparation, robotic platforms can be used to screen larger pool of compounds across various in vitro assays. However, this strategy can also be used for processing of in vivo samples, provided if the sample cohort is higher. In general, automated robotic sample preparation platforms are quite often used for screening of compounds in in vitro assays. These robotic platforms help in decreasing the manpower involved and time taken for performing the assays. However, main disadvantage with robotic platforms is the need for preparation of larger volumes of reagents and the cost factor involved. Hence, it is not recommended to use these platforms unless there exists a larger library of compounds.

### **4.5 Matrix effects**

*Mass Spectrometry - Future Perceptions and Applications*

administration, absolute bioavailability values are calculated.

of the MS/MS system and increase sample throughput [104].

On the other hand, throughput can also be increased by pooling of samples, provided the sensitivity is not seriously compromised. When these samples are

well plate formats.

section below.

**4.4 High throughput bioanalysis**

with known pharmacokinetic parameters can be administered. Pharmacokinetic study results of unknown compounds can be considered acceptable, as long as quality control compounds fall within the set acceptance criteria. Compounds with less than 5 da difference in molecular weight are difficult to pool, when triple quadrupole systems are used for analyzing the pharmacokinetic samples. However, this challenge can be overcome by considering high resolution mass spectrometric analysis. Typical pharmacokinetic parameters studied from intravenous administration include half-life (t1/2), clearance (Cl), volume of distribution (Vz), mean residence time (MRT), area under the curve (AUC0-last; AUC0-inf), whereas parameters such as Cmax, Tmax and area under the curve are studies in other routes of administration. When compounds are dosed in multiple routes, along with intravenous route of

Current fast LC–MS/MS instruments enable analysis of mixture of analytes with minimal separation, shorter run times and also feasible for hyphenation with ultra-fast liquid chromatography systems. Additionally, latest LC–MS/MS systems are capable of analyzing thousands of samples every week due to higher loading capacity of samples in autosampler, shorter run times and introduction of 96/384

Test samples are analyzed against a calibration curve and a set of quality control samples. Calibration curve consists of 8–10 known standards and quality controls span the calibration curve at a minimum of 3 levels. Typical accuracy limits for qualifying the calibration and quality control samples is set as ±20%. Typical turnaround times for execution of pharmacokinetic studies right from dosing initiation to generation of pharmacokinetic parameters spans 1–2 weeks. Importance of high throughput bioanalysis and its role in drug discovery is discussed in detail in the

With the combinatorial chemistry efforts leading to synthesis of hundreds of compounds in early phases of drug discovery, there is a constant need for analytical platforms that can quickly churn out data and help in accelerating the discovery process. LC–MS/MS with proven track record as a reliable analytical platform had undergone evolutionary changes to support high throughput demands of drug discovery [60, 70, 87, 94–96]. These strategies include advancements in chromatographic columns, where lower dimensions and microbore HPLC columns cut short run time to one minute per sample. Quick sample run times help in analyzing higher number of samples with in the given stipulated time [97]. Faster gradient methods with LC pumps that can handle higher pressure also enable analysis with shorter run times [98]. With the invention of monolithic HPLC columns that can be operated at high flow rates in the order of 5–6 mL/min, analytical run times were significantly reduced [99–101]. One disadvantage of these columns is that the high flow rates translate in to higher usage of mobile phase, making it an expensive alternative. Another approach for increasing sample throughput is by the use of parallel HPLC columns, where the effluent from two HPLC systems could be combined and assayed by using the MRM/SRM capabilities of the MS/MS system [102, 103]. One more approach in enhancing the throughput is through staggered analysis approach. Here, multiple HPLC columns are used, but the injection time is staggered such that the "analytical window" can be selected sequentially in order to maximize the use

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With the sample nature being biological in origin, supernatants obtained after sample preparation consists of many endogenous components that compete with the analyte of interest and result in either suppression/enhancement of ionization. This process of alteration of the ionization of analytes is termed as matrix effects. The "matrix" refers to all components in the sample other than analyte(s) of interest. Matrix effects are defined as "interference from matrix components that are

**Figure 11.** *Pictorial representation of LC–MS/MS system with higher loading capacity of autosampler.*

unrelated to the analyte" [105, 106]. The process of ion suppression/enhancement is in general referred as matrix effect and is main subject of various published reviews [107–112].Matrix effects result in significant deviation in precision and accuracy of results which in turn debate the reliability of pharmacokinetic parameters of NCE's generated. Matrix effect alters the sensitivity, reproducibility and challenges the reliability of analytical techniques. Although matrix effects occur as a result of various exogenous and endogenous components, one major area of concern is formulation excipients (an exogenous component) used in the preparation of formulations. Dosing vehicles are generally used at high concentrations to solubilize test articles of highly lipophilic nature [113–115]. This in turn can be instrumental in causing matrix effects, thereby questioning the reliability of preclinical PK parameters. This phenomenon has been reported by us in the past for various excipients such as PEG 400 [110, 116, 117], Cremophor EL [111, 118] and Solutol HS15 [112, 119].

Several mechanisms have been proposed to explain matrix effects, but the exact process remains uncertain [120, 121]. Various mechanisms by which matrix components cause ion suppression are as follows:


Reduction of matrix effects can be achieved through various strategies including decreasing the level of matrix components, improving chromatographic separation of interfering materials from the analyte, various sample preparation strategies, lower injection volumes, and even by simple dilution of samples to reduce the overall concentrations of both analyte and co-extracted materials [126, 127]. Switching ionization sources will also help in mitigating the matrix effects [112, 116, 118, 119]. Matrix effects occurring in the early time point samples can be monitored, using another aliquot of the early time point samples analyzed at a higher dilution [128].
