**Contribution of Mass Spectrometry to the Study of Antimalarial Agents**

Ana Raquel Sitoe, Francisca Lopes, Rui Moreira, Ana Coelho and Maria Rosário Bronze

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/56225

### **1. Introduction**

Mass spectrometry (MS) has become a powerful analytical tool for qualitative and quantita‐ tive applications, providing information about the structure and purity of compounds, and also about the chemical composition of complex samples.

The most recent applications of mass spectrometry are oriented towards biochemical appli‐ cations such as proteome, metabolome and drug discovery. During the last decade, mass spectrometry has progressed rapidly and an evolution has been observed in the type of ap‐ plications, software and equipments. Atmospheric pressure ionization sources are now used, an analyser based on a new concept (the orbitrap) was recently developed, existing ones were modified, and new hybrid instruments were developed using combinations of different analysers, depending on applications. One of the major trends was the transition to high resolution/accurate mass analysis, made routine by new MS instruments. The use of separation techniques as gas chromatography (GC), liquid chromatography (LC) and capil‐ lary electrophoresis (CE) coupled with mass spectrometry and tandem mass spectrometry, expanded the interest in this methodology.

In this chapter are presented general aspects related with characteristics of mass spectrome‐ try equipments. The contribution of this technique to new discoveries concerning one of the major infectious diseases in man, malaria, is also discussed.

### **2. Basics of mass spectrometry**

Mass spectrometry is a technique used to analyse from small inorganic molecules to biologi‐ cal macromolecules and relies on the formation of gas-phase ions (positively or negatively

charged) that are isolated based on their mass-to–charge ratio (m/z). In order to achieve this state, the sample must be volatilized and this may become a problem to biological samples, as biomolecules have usually high molecular mass and high polarity, factors that limit their volatility.

All mass spectrometers share common components as an ionization source, a mass analyser and a detector (Fig. 1). As there are available equipments with different specifications, even from the same supplier, it is necessary to choose carefully the most adequate equipment for each type of application.

**Figure 1.** Basic components in a mass spectrometer. ESI, electrospray ionization, APCI, atmospheric-pressure chemical ionization; MALDI, Matrix-Assisted Laser Desorption ionization; DESI: Desorption Electrospray Ionization ; DART: Direct Analysis in Real Time; FT-ICR: Fourier transform ion cyclotron resonance (adapted from Glish & Vachet, 2003)

In MALDI, ions are produced by pulsed-laser irradiation (e.g. nitrogen lasers) of the sample co-crystallized with an organic matrix (e.g. gentisic, sinapic or ferulic acid) and operating in the vacuum or more recently, at atmospheric pressure. MALDI ionization uses a low amount of sample but low molecular mass molecules (below 500 Da) are difficult to analyse, due to

**Table 1.** Characteristics of the most used ionization sources. FAB: Fast Atomic Bombardment; MALDI: Matrix Assisted Laser Desorption Ionization; ESI: Electrospray Ionization; DESI: Desorption Electrospray Ionization ; DART: Direct

**method Analytes Sample introduction Mass range Type of ionization**

with a matrix

compounds Direct injection, LC Large range

compounds Direct injection, LC <5000 Da Soft

Analysis of a surface Large range

compounds Analysis of a surface Less broad than DESI Simple mass spectra

500-500 000 Da Very soft. Generates mainly

Contribution of Mass Spectrometry to the Study of Antimalarial Agents

single charged ions

http://dx.doi.org/10.5772/56225

63

Softer than MALDI. Generates multiple charged ions

Generates single or multiple charged molecular ions from small or large analytes

(molecular ion)

The development of other soft ionization techniques has become crucial to the analysis of biomolecules dissolved in a mixture of water and a volatile organic solvent (e.g. methanol, acetonitrile). Techniques as ESI and MALDI make MS methodologies versatile as both techniques accomplish the conversion into gas-phase ions of non-volatile and thermally labile large molecules, allowing the study of biological compounds. Both techniques produce protonated peptide and protein ions, deprotonated deoxyribonucleic acids (DNA) and ribonucleic acids (RNA). Some reviews have been published on the covalent and nonconvalent interactions between drug molecules with DNA and RNA, protein and enzyme targets for drug action and toxicity (Feng, 2004). When using ESI, proteins are ionized as they have several sites of protonation or deprotonation, and this multiple charging enables mass spectrometers with limited *m/z* ranges to analyse higher molecular mass molecules. However, ion suppression may occur when solutions contain high concentrations of salt, or when the target analytes are present in low concentration in matrices with high content of other analytes. Strategies based on the type of analyte, ionization reaction, ionization efficiency, analyte solution composition and pH, have been described for producing positive or negative ion modes when operating with an ESI source (Feng, 2004). APCI, is less susceptible to matrix interferences from salts, and is used for monitoring weakly polar compounds. However, labile

strong interferences of the organic matrix ions.

**Ionization**

DESI

DART

Analysis in Real Time

FAB Organometallic

ESI Organic and inorganic

Small non-polar and large polar molecules (peptides and proteins)

Low molecular mass

MALDI Biomolecules Sample co-crystallized

In order to achieve this state, the sample must be volatilized and this may become a problem to biological samples, as biomolecules have usually high molecular mass and high polarity, factors that limit their volatility.

Different ionization techniques may be used in mass spectrometry equipments, depending on the need of molecule disruption for the induction of ion formation. These techniques may perform strong and soft ionization processes. Soft ionization methods, like fast atomic bombardment (FAB), liquid secondary ion mass spectrometry (LSIMS), matrix-assisted laser desorption ionization (MALDI) and electrospray ionization (ESI) allow the detection of molecular ions and are more suitable for the analysis of biomolecules and non-purified analytes. The ions are generated by protonation, deprotonation or formation of adducts. In table 1 are summarized some of the main characteristics of the most used ionization methods.

The use of FAB is useful to assign the molecular ion peaks. The sample is dissolved in a suitable liquid matrix, with low vapour pressure (e.g. diethanolamine, triethanolamine, glycerol, thioglycerol or 3-nitrobenzyl alcohol), inserted into the mass spectrometer and bombarded with high energy argon or xenon atoms, providing efficient means to analyze polar, ionic, thermally labile, energetically labile, and high molecular mass compounds (El-Aneed et al., 2009).


charged) that are isolated based on their mass-to–charge ratio (m/z). In order to achieve this state, the sample must be volatilized and this may become a problem to biological samples, as biomolecules have usually high molecular mass and high polarity, factors that limit their

All mass spectrometers share common components as an ionization source, a mass analyser and a detector (Fig. 1). As there are available equipments with different specifications, even from the same supplier, it is necessary to choose carefully the most adequate equipment for

**Figure 1.** Basic components in a mass spectrometer. ESI, electrospray ionization, APCI, atmospheric-pressure chemical ionization; MALDI, Matrix-Assisted Laser Desorption ionization; DESI: Desorption Electrospray Ionization ; DART: Direct Analysis in Real Time; FT-ICR: Fourier transform ion cyclotron resonance (adapted from Glish & Vachet, 2003)

In order to achieve this state, the sample must be volatilized and this may become a problem to biological samples, as biomolecules have usually high molecular mass and high polarity,

Different ionization techniques may be used in mass spectrometry equipments, depending on the need of molecule disruption for the induction of ion formation. These techniques may perform strong and soft ionization processes. Soft ionization methods, like fast atomic bombardment (FAB), liquid secondary ion mass spectrometry (LSIMS), matrix-assisted laser desorption ionization (MALDI) and electrospray ionization (ESI) allow the detection of molecular ions and are more suitable for the analysis of biomolecules and non-purified analytes. The ions are generated by protonation, deprotonation or formation of adducts. In table 1 are summarized some of the main characteristics of the most used ionization methods.

The use of FAB is useful to assign the molecular ion peaks. The sample is dissolved in a suitable liquid matrix, with low vapour pressure (e.g. diethanolamine, triethanolamine, glycerol, thioglycerol or 3-nitrobenzyl alcohol), inserted into the mass spectrometer and bombarded with high energy argon or xenon atoms, providing efficient means to analyze polar, ionic, thermally labile, energetically labile, and high molecular mass compounds (El-Aneed et al.,

volatility.

each type of application.

62 Tandem Mass Spectrometry - Molecular Characterization

factors that limit their volatility.

2009).

**Table 1.** Characteristics of the most used ionization sources. FAB: Fast Atomic Bombardment; MALDI: Matrix Assisted Laser Desorption Ionization; ESI: Electrospray Ionization; DESI: Desorption Electrospray Ionization ; DART: Direct Analysis in Real Time

In MALDI, ions are produced by pulsed-laser irradiation (e.g. nitrogen lasers) of the sample co-crystallized with an organic matrix (e.g. gentisic, sinapic or ferulic acid) and operating in the vacuum or more recently, at atmospheric pressure. MALDI ionization uses a low amount of sample but low molecular mass molecules (below 500 Da) are difficult to analyse, due to strong interferences of the organic matrix ions.

The development of other soft ionization techniques has become crucial to the analysis of biomolecules dissolved in a mixture of water and a volatile organic solvent (e.g. methanol, acetonitrile). Techniques as ESI and MALDI make MS methodologies versatile as both techniques accomplish the conversion into gas-phase ions of non-volatile and thermally labile large molecules, allowing the study of biological compounds. Both techniques produce protonated peptide and protein ions, deprotonated deoxyribonucleic acids (DNA) and ribonucleic acids (RNA). Some reviews have been published on the covalent and nonconvalent interactions between drug molecules with DNA and RNA, protein and enzyme targets for drug action and toxicity (Feng, 2004). When using ESI, proteins are ionized as they have several sites of protonation or deprotonation, and this multiple charging enables mass spectrometers with limited *m/z* ranges to analyse higher molecular mass molecules. However, ion suppression may occur when solutions contain high concentrations of salt, or when the target analytes are present in low concentration in matrices with high content of other analytes. Strategies based on the type of analyte, ionization reaction, ionization efficiency, analyte solution composition and pH, have been described for producing positive or negative ion modes when operating with an ESI source (Feng, 2004). APCI, is less susceptible to matrix interferences from salts, and is used for monitoring weakly polar compounds. However, labile compounds can be thermally decomposed, and due to its high sensitivity, the solvents used with this technique must have higher purity.

DART and DESI are well established open-air ionization techniques, as no sample preparation is required, making these techniques suitable for screening a large number of samples (Fernández et al., 2006). The DART ion source produces a heated stream of protonated reactant ions and the analytes in the sample are ionized, producing protonated molecules [M+H]+ or deprotonated molecules [M-H] in the open air of the laboratory environment, making possible the analysis of organic compounds directly, in real time, without time-consuming analytical protocols and destruction of the sample. The method may detect concentrations of analytes as low as femtomole (Arnaud, 2007). Due to these characteristics, DART has become an ionization method useful for rapid screening of pharmaceutical products. In DESI analysis, a high-speed charged liquid spray is directed to the sample (Takats et al., 2005). The DESI spray dissolves the material from the sample and the charged droplets are sampled downstream by a mass spectrometer. Desolvation and evaporation from these droplets creates ions that will generate a mass spectrum of the sample components.

Following the ionization process, the selected ions are extracted, accelerated, and analyzed. A mass analyser is characterized by its mass range limit, analysis speed, transmission, mass accuracy and resolution, expressed as full width at half maximum (FWHM). The most used analysers and their characteristics are summarized in Table 2.

Quadrupoles are widely used mass analysers, where ions are separated according to the stability of their trajectories in the oscillating electric fields applied between the four parallel rods. The QTOF, a hybrid quadrupole time of flight mass spectrometer, is a high-resolution mass spectrometer with MS/MS capability, and has been often used in drug studies (Nyunt et al., 2005). FT-ICR is also a high resolution and high mass accuracy analyser that enables the study of the binding of ligands (drugs) to RNA targets (Hofstadler et al., 1999; Masselon et al., 2000). The Orbitrap mass analyzer employs electrostatic trapping and it bears similarities to FT-ICR as both belong to the same Fourier Transform MS (FTMS) family of instruments. Orbitrap mass spectrometry is expected to provide maximum resolving powers of 100,000– 200,000. A modified Orbitrap instrument has shown that this technology is capable of a resolution of 1,000,000 for m/z < 300–400, which makes it compatible to be used with chroma‐ tographic separation techniques (Denisov et al., 2012).

Liquid chromatography coupled to mass spectrometry (Triple Quadrupole TQ, QTOF, Linear ionTrap and Linear QTRAP analyzers) is today a well established methodology used due to its high sensitivity, speed, selectivity, versatility and ease of automation. Recently the advan‐ tages of using mass spectrometry in comprehensive liquid chromatography (LC X LC system) have been discussed and different applications have been described for pharmaceutical

Malaria is caused by *Plasmodium* parasites, which are transmitted through the bite of an infected female *Anopheles* mosquito, and remains one of the major infectious disease in man. Five species from the genus *Plasmodium* namely: *P. falciparum*, *P. vivax P. ovale*, *P. malariae*, and *P. knowlesi* cause infection in humans. Of these, *P. falciparum* and *P. vivax* account for more than 95% of malaria cases in the world, with *P. falciparum*, being responsible for most of the deaths caused by malaria every year. The species of human malaria differ in the periodicity of their

compounds (Donato et al., 2012).

**Quadrupole Ion Trap**

*m/z* (resonance frequency)

MSn Fragments Low-energy collision

**Table 2.** Comparison of mass analysers (adapted from Hoffmann & Stroobant, 2002)

*m/z* (trajectory stability)

MS/MS Fragments Precursors Neutral loss Low-energy collision

Principle of separation

Mass limit

Resolution FWHM (m/z 1000)

Accuracy

Pressure

Tandem mass spectrometry

**Time-offlight**

Velocity (flight time)


**Time-of-flight reflectron**

Velocity

(flight time) Momentum

2 000 4 000 5 000 20 000 100 000 500 000 100 000

Symbol Q IT TOF TOF B FT-ICR FT-OT

(Th) 4 000 6 000 >1 000 000 4 000 20 000 30 000 50 000

(ppm) <sup>100</sup> <sup>100</sup> <sup>200</sup> <sup>10</sup> <10 <5 <5 Ion sampling continuous pulsed pulsed pulsed continuous pulsed pulsed

(Torr) 10-5 10-3 10-6 10-6 10-6 10-10 10-10

MS/MS Fragments Low-energy collision

**Magnetic sector**

Contribution of Mass Spectrometry to the Study of Antimalarial Agents

MS/MS Fragments Precursors Neutral loss High-energy collision

**Fourrier Transform ion cyclotron resonance**

http://dx.doi.org/10.5772/56225

m/z (resonance frequency)

MSn Fragments Low-energy collision

**Fourrier Transform Orbitrap**

65

m/z (resonance frequency)


**3. An overview of malaria**

Tandem mass spectrometry (MSn) development was crucial for the structural analysis of compounds. In tandem experiments, a molecular ion is selectively isolated and fragmented in a controlled environment. With this type of analysers, it is possible to perform different types of experiments (e.g. parent scan, daughter scan, neutral loss) and data obtained, will allow to identify or quantify the analytes, even in complex matrices (e.g. natural product extracts and biological fluids). Multiple Reaction Monitoring (MRM), has become an important tool, used for quantification purposes, allowing an increment of methods specificity and sensitivity.

Finally the signal obtained in the detector will produce a mass spectrum, the x-coordinate represents m/z values and the y-axis indicates total ion counts.


**Table 2.** Comparison of mass analysers (adapted from Hoffmann & Stroobant, 2002)

Liquid chromatography coupled to mass spectrometry (Triple Quadrupole TQ, QTOF, Linear ionTrap and Linear QTRAP analyzers) is today a well established methodology used due to its high sensitivity, speed, selectivity, versatility and ease of automation. Recently the advan‐ tages of using mass spectrometry in comprehensive liquid chromatography (LC X LC system) have been discussed and different applications have been described for pharmaceutical compounds (Donato et al., 2012).

### **3. An overview of malaria**

compounds can be thermally decomposed, and due to its high sensitivity, the solvents used

DART and DESI are well established open-air ionization techniques, as no sample preparation is required, making these techniques suitable for screening a large number of samples (Fernández et al., 2006). The DART ion source produces a heated stream of protonated reactant ions and the analytes in the sample are ionized, producing protonated molecules [M+H]+ or

the analysis of organic compounds directly, in real time, without time-consuming analytical protocols and destruction of the sample. The method may detect concentrations of analytes as low as femtomole (Arnaud, 2007). Due to these characteristics, DART has become an ionization method useful for rapid screening of pharmaceutical products. In DESI analysis, a high-speed charged liquid spray is directed to the sample (Takats et al., 2005). The DESI spray dissolves the material from the sample and the charged droplets are sampled downstream by a mass spectrometer. Desolvation and evaporation from these droplets creates ions that will generate

Following the ionization process, the selected ions are extracted, accelerated, and analyzed. A mass analyser is characterized by its mass range limit, analysis speed, transmission, mass accuracy and resolution, expressed as full width at half maximum (FWHM). The most used

Quadrupoles are widely used mass analysers, where ions are separated according to the stability of their trajectories in the oscillating electric fields applied between the four parallel rods. The QTOF, a hybrid quadrupole time of flight mass spectrometer, is a high-resolution mass spectrometer with MS/MS capability, and has been often used in drug studies (Nyunt et al., 2005). FT-ICR is also a high resolution and high mass accuracy analyser that enables the study of the binding of ligands (drugs) to RNA targets (Hofstadler et al., 1999; Masselon et al., 2000). The Orbitrap mass analyzer employs electrostatic trapping and it bears similarities to FT-ICR as both belong to the same Fourier Transform MS (FTMS) family of instruments. Orbitrap mass spectrometry is expected to provide maximum resolving powers of 100,000– 200,000. A modified Orbitrap instrument has shown that this technology is capable of a resolution of 1,000,000 for m/z < 300–400, which makes it compatible to be used with chroma‐

Tandem mass spectrometry (MSn) development was crucial for the structural analysis of compounds. In tandem experiments, a molecular ion is selectively isolated and fragmented in a controlled environment. With this type of analysers, it is possible to perform different types of experiments (e.g. parent scan, daughter scan, neutral loss) and data obtained, will allow to identify or quantify the analytes, even in complex matrices (e.g. natural product extracts and biological fluids). Multiple Reaction Monitoring (MRM), has become an important tool, used for quantification purposes, allowing an increment of methods specificity and sensitivity.

Finally the signal obtained in the detector will produce a mass spectrum, the x-coordinate

in the open air of the laboratory environment, making possible

with this technique must have higher purity.

64 Tandem Mass Spectrometry - Molecular Characterization

a mass spectrum of the sample components.

analysers and their characteristics are summarized in Table 2.

tographic separation techniques (Denisov et al., 2012).

represents m/z values and the y-axis indicates total ion counts.

deprotonated molecules [M-H]-

Malaria is caused by *Plasmodium* parasites, which are transmitted through the bite of an infected female *Anopheles* mosquito, and remains one of the major infectious disease in man.

Five species from the genus *Plasmodium* namely: *P. falciparum*, *P. vivax P. ovale*, *P. malariae*, and *P. knowlesi* cause infection in humans. Of these, *P. falciparum* and *P. vivax* account for more than 95% of malaria cases in the world, with *P. falciparum*, being responsible for most of the deaths caused by malaria every year. The species of human malaria differ in the periodicity of their life cycle, as well as in the outcomes of the disease. Generally clinical manifestations can include fever, chills, prostration and anemia. Severe disease can include delirium, metabolic acidosis, cerebral malaria and multi-organ system failure, coma and death may ensue. (Kantele & Jokiranta, 2011)

**3.2. Agents with antimalarial activity**

nate malaria. (Hobbs, C. & Duffy, P., 2011)

resistant *Plasmodium falciparum* malaria.

identical efficacy. (Capela et al., 2011)

2012):

to insecticides;

targets the parasite;

The public health problem of malaria has been addressed by different approaches (Biot et al.,

Contribution of Mass Spectrometry to the Study of Antimalarial Agents

http://dx.doi.org/10.5772/56225

67

**•** use of insecticides to control the mosquito vector, but mosquitoes are developing resistance

**•** vaccines, but in spite of all the efforts there is yet not available a vaccine that effectively

**•** chemotherapy to control malaria has relied mainly on a restricted number of chemically

Increasing resistance of *P. falciparum* to the commonly used drugs is recognized as one of the major problems in eradication of the disease. The severe malaria situation under‐ scores the continuing need of research for new classes of antimalarial agents with new mechanisms of action or re-utilization of the existing drugs with new types of therapies. The existing drug armamentarium is insufficient to answer the call for malaria eradica‐ tion. The first line of treatment for malaria currently relies on a single class, the artemisi‐ nins. To overcome this problem scientists are exploring many approaches, targeting different stages of the parasite life cycle, to find agents that will prevent, cure or elimi‐

Many antimalarial agents contain a 4-aminoquinoline, 8-aminoquinoline or quinolone methanol scaffolds (Rosenthal, 2001). Chloroquine and amodiaquine are 4-aminoquino‐ lines used to treat and prevent malaria, while primaquine is the single 8-aminoquinoline clinically approved to treat relapsing malaria caused by *P. vivax*. Mefloquine (Fig. 3) is a quinoline methanol antimalarial structurally similar to quinine, the first pure substance used to treat malaria and extracted from the bark of the cinchona tree. Other relevant classes of antimalarial agents include the antifolates (e.g. pyrimethamine and proguanyl), phenanthrene methanols (e.g. halofantrine), and naphthoquinones (e.g. atovaquone). More recently, artemisinin (Fig. 3), a sesquiterpene lactone isolated from the *Artemisia an‐ nua* chinese herb, and its analogues were a major breakthrough in malaria chemotherapy because they produce a very rapid therapeutic response, particularly against multidrug-

With exception of primaquine, (Vale et al., 2009) most available antimalarials are active against the blood stage of the disease. However to achieve the eradication goal, new compounds with new modes of action are needed to block parasite transmission and eliminate the asymptomatic and latent hepatic forms. (Rodrigues et al., 2012) A strategy used to address this major goal is to combine two chemotypes - each one targeting a spe‐ cific stage of the parasite's life cycle in a single chemical entity, to develop effective hy‐ brid antimalarials capable of killing both the blood and liver-stage parasites with

related drugs belonging to either the quinoline or the antifolate groups.

The World Health Organization (WHO) estimated 225 million cases of malaria and about 800,000 deaths worldwide in 2010 (WHO, 2010). Due to the rapid evolution and spread of multi-resistant parasites to the current antimalarial drugs, both, chemotherapy and prophy‐ laxis are at risk of impairment. Malaria is most prevalent in developing countries of tropical areas such as sub-Saharan Africa, East Asia and South America (Rodrigues et al., 2010; Eisenstein, 2012).

### **3.1. Life cycle of the malaria parasite**

The malaria parasite exhibits a complex life cycle (Fig. 2) involving an insect vector (mosquito) and a vertebrate host (human). It includes an asexual cycle in humans, encompassing an asymptomatic liver-stage, a symptomatic blood-stage and a sexual cycle in a mosquito.

The liver or hepatic stage (A) is initiated when sporozoites injected through the bite of a mosquito travel to the liver and infect hepatocytes, where a clinically silent asexual multipli‐ cation takes place, generating thousands of merozoites. The release of merozoites into the bloodstream (B) marks the beginning of the erythrocytic stage of infection (C), during which parasites infect red blood cells, undergo repeated asexual replication cycles, and give rise to clinical illness. Some merozoites differentiate into gametocytes (D) that can be taken up by the mosquito during a posterior blood meal. Within the mosquito, gametocytes undergo a sexual development to form sporozoites that migrate to the salivary glands and can infect another host trough another bite (E).

**Figure 2.** *Plasmodium* life cycle

### **3.2. Agents with antimalarial activity**

life cycle, as well as in the outcomes of the disease. Generally clinical manifestations can include fever, chills, prostration and anemia. Severe disease can include delirium, metabolic acidosis, cerebral malaria and multi-organ system failure, coma and death may ensue. (Kantele &

The World Health Organization (WHO) estimated 225 million cases of malaria and about 800,000 deaths worldwide in 2010 (WHO, 2010). Due to the rapid evolution and spread of multi-resistant parasites to the current antimalarial drugs, both, chemotherapy and prophy‐ laxis are at risk of impairment. Malaria is most prevalent in developing countries of tropical areas such as sub-Saharan Africa, East Asia and South America (Rodrigues et al., 2010;

The malaria parasite exhibits a complex life cycle (Fig. 2) involving an insect vector (mosquito) and a vertebrate host (human). It includes an asexual cycle in humans, encompassing an asymptomatic liver-stage, a symptomatic blood-stage and a sexual cycle in a mosquito.

The liver or hepatic stage (A) is initiated when sporozoites injected through the bite of a mosquito travel to the liver and infect hepatocytes, where a clinically silent asexual multipli‐ cation takes place, generating thousands of merozoites. The release of merozoites into the bloodstream (B) marks the beginning of the erythrocytic stage of infection (C), during which parasites infect red blood cells, undergo repeated asexual replication cycles, and give rise to clinical illness. Some merozoites differentiate into gametocytes (D) that can be taken up by the mosquito during a posterior blood meal. Within the mosquito, gametocytes undergo a sexual development to form sporozoites that migrate to the salivary glands and can infect another

**C**

**A**

**B**

Jokiranta, 2011)

Eisenstein, 2012).

**3.1. Life cycle of the malaria parasite**

66 Tandem Mass Spectrometry - Molecular Characterization

host trough another bite (E).

**Figure 2.** *Plasmodium* life cycle

**D**

**E**

The public health problem of malaria has been addressed by different approaches (Biot et al., 2012):


Increasing resistance of *P. falciparum* to the commonly used drugs is recognized as one of the major problems in eradication of the disease. The severe malaria situation under‐ scores the continuing need of research for new classes of antimalarial agents with new mechanisms of action or re-utilization of the existing drugs with new types of therapies. The existing drug armamentarium is insufficient to answer the call for malaria eradica‐ tion. The first line of treatment for malaria currently relies on a single class, the artemisi‐ nins. To overcome this problem scientists are exploring many approaches, targeting different stages of the parasite life cycle, to find agents that will prevent, cure or elimi‐ nate malaria. (Hobbs, C. & Duffy, P., 2011)

Many antimalarial agents contain a 4-aminoquinoline, 8-aminoquinoline or quinolone methanol scaffolds (Rosenthal, 2001). Chloroquine and amodiaquine are 4-aminoquino‐ lines used to treat and prevent malaria, while primaquine is the single 8-aminoquinoline clinically approved to treat relapsing malaria caused by *P. vivax*. Mefloquine (Fig. 3) is a quinoline methanol antimalarial structurally similar to quinine, the first pure substance used to treat malaria and extracted from the bark of the cinchona tree. Other relevant classes of antimalarial agents include the antifolates (e.g. pyrimethamine and proguanyl), phenanthrene methanols (e.g. halofantrine), and naphthoquinones (e.g. atovaquone). More recently, artemisinin (Fig. 3), a sesquiterpene lactone isolated from the *Artemisia an‐ nua* chinese herb, and its analogues were a major breakthrough in malaria chemotherapy because they produce a very rapid therapeutic response, particularly against multidrugresistant *Plasmodium falciparum* malaria.

With exception of primaquine, (Vale et al., 2009) most available antimalarials are active against the blood stage of the disease. However to achieve the eradication goal, new compounds with new modes of action are needed to block parasite transmission and eliminate the asymptomatic and latent hepatic forms. (Rodrigues et al., 2012) A strategy used to address this major goal is to combine two chemotypes - each one targeting a spe‐ cific stage of the parasite's life cycle in a single chemical entity, to develop effective hy‐ brid antimalarials capable of killing both the blood and liver-stage parasites with identical efficacy. (Capela et al., 2011)

### **4. Study of antimalarials agents by mass spectrometry**

Structural and stability information is fundamental for any drug study, including antimalar‐ ials. In fig. 3 are presented currently available antimalarial drugs. Due to the rapid emergence and spread of resistant parasites to well-established antimalarial drugs, there is an urgent need for novel drugs. Studies performed on new antimalarial compounds using mass spectrometry are scarce but are useful for the elucidation of structures, also for prediction of compound stability and properties, isomer characterization, and detection of counterfeit products. Furthermore, studies using this technique coupled to chromatographic methods have been conducted for the evaluation of pharmacokinetics, metabolite identification, and detection of impurities.

### **4.1. Structural elucidation**

One of the main applications of mass spectrometry is the structural elucidation of molecules. Based on the molecular ion peaks and their fragmentation patterns, the structure of com‐ pounds can then be proposed.

Among the different equipments that can be used for these purposes, FAB ion sources are frequently described. Applications can be found in the study of the oxidation products of primaquine, 5,5-di-[6-methoxy-8-(4-amino-1-butyl amino)] quinoline (PI), 6-methoxy-5,8-di- [4-amino-1-methyl butyl amino] quinoline (PII) and 5,5-di-[7-hydroxy-6-methoxy-8(4 amino-1-methyl butylamino)] quinoline (PIII) (Fig. 4) (Sinha & Dua 2004). The mass spectrum of PI with molecular formula C30H40N6O2 presents the molecular ion at *m/z* 517 confirming the molecular mass of the compound, and a fragment at *m/z* 500 is detected due to the presence of a terminal amino group at position 4´.

An LC-MS/MS method was developed for the analysis of bulaquine (BQ) 3-[1-[4-[(6-me‐ thoxy-8-quinolinylamino] pentylamino] ethylidene]dihydro-2 (3H)-furanone (Fig. 5) and its metabolite primaquine in monkey plasma. Protonated species at *m/z* 370 and 260 were detected for bulaquine and primaquine, respectively. MS/MS conditions were optimized generating product ions through fragmentation of the molecular ions. Based on the fragmentation spectra obtained from [M+H]+ under the established analytical conditions, a fragmentation pattern was presented for these compounds. This type of study is important to establish analytical conditions for the quantification of drugs and their metabolites in biological fluids. (Nitin et al., 2003)

An ESI-Ion trap mass spectrometer was used to perform MSn analyses, in the study of imidazolidin-4-one peptidomimetic derivatives of primaquine. (Vale et al., 2008a)

analytical method which is reliable, reproducible, sensitive, selective, and if possible, compat‐

**Atovaquone Artemisinin**

**Cl**

Drug efficacy requires adequate drug concentration at the site of action. Monitoring drugs and their metabolites in biological samples (*in vivo* studies), is fundamental in order to control the intake of the antimalarials by the infected populations. Mass spectrometry has been success‐ fully used for this purposes, coupled with liquid chromatography, and analytical methods

ible with high-throughput pharmacokinetic approaches.

**N**

**HN**

**N**

**CF3**

**Cl NH**

**O**

**O**

**Figure 3.** Currently available antimalarial drugs

**CF3 <sup>N</sup>**

**HN**

**OH**

**Proguanyl**

**HO**

**Cl**

**HN**

**N**

**N**

**Chloroquine Amodiaquine Primaquine**

**Mefloquine Quinine Pyrimethamine**

**N**

**H**

**Cl**

**O**

**NH**

**NH**

**HN**

**HO H**

**HN**

**OH N**

**N**

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**N N**

**NH2**

**NH2**

69

**HN**

**H2N**

**O**

Contribution of Mass Spectrometry to the Study of Antimalarial Agents

**Cl**

**HO**

**F3C**

**O O**

**Cl**

**Halofantrine**

**O**

**H H**

**H <sup>O</sup> <sup>O</sup>**

**Cl**

**N**

### **4.2. Pharmacokinetic studies**

Pharmacokinetic (PK) studies provide a mathematical basis to assess the time course of drug in the body. It enables to quantify absorption, distribution, metabolism and excretion of the drug and their metabolites. The primary requirement to undertake a PK study, is to have an Contribution of Mass Spectrometry to the Study of Antimalarial Agents http://dx.doi.org/10.5772/56225 69

**Figure 3.** Currently available antimalarial drugs

**4. Study of antimalarials agents by mass spectrometry**

68 Tandem Mass Spectrometry - Molecular Characterization

impurities.

**4.1. Structural elucidation**

pounds can then be proposed.

of a terminal amino group at position 4´.

obtained from [M+H]+

**4.2. Pharmacokinetic studies**

al., 2003)

Structural and stability information is fundamental for any drug study, including antimalar‐ ials. In fig. 3 are presented currently available antimalarial drugs. Due to the rapid emergence and spread of resistant parasites to well-established antimalarial drugs, there is an urgent need for novel drugs. Studies performed on new antimalarial compounds using mass spectrometry are scarce but are useful for the elucidation of structures, also for prediction of compound stability and properties, isomer characterization, and detection of counterfeit products. Furthermore, studies using this technique coupled to chromatographic methods have been conducted for the evaluation of pharmacokinetics, metabolite identification, and detection of

One of the main applications of mass spectrometry is the structural elucidation of molecules. Based on the molecular ion peaks and their fragmentation patterns, the structure of com‐

Among the different equipments that can be used for these purposes, FAB ion sources are frequently described. Applications can be found in the study of the oxidation products of primaquine, 5,5-di-[6-methoxy-8-(4-amino-1-butyl amino)] quinoline (PI), 6-methoxy-5,8-di- [4-amino-1-methyl butyl amino] quinoline (PII) and 5,5-di-[7-hydroxy-6-methoxy-8(4 amino-1-methyl butylamino)] quinoline (PIII) (Fig. 4) (Sinha & Dua 2004). The mass spectrum of PI with molecular formula C30H40N6O2 presents the molecular ion at *m/z* 517 confirming the molecular mass of the compound, and a fragment at *m/z* 500 is detected due to the presence

An LC-MS/MS method was developed for the analysis of bulaquine (BQ) 3-[1-[4-[(6-me‐ thoxy-8-quinolinylamino] pentylamino] ethylidene]dihydro-2 (3H)-furanone (Fig. 5) and its metabolite primaquine in monkey plasma. Protonated species at *m/z* 370 and 260 were detected for bulaquine and primaquine, respectively. MS/MS conditions were optimized generating product ions through fragmentation of the molecular ions. Based on the fragmentation spectra

was presented for these compounds. This type of study is important to establish analytical conditions for the quantification of drugs and their metabolites in biological fluids. (Nitin et

An ESI-Ion trap mass spectrometer was used to perform MSn analyses, in the study of

Pharmacokinetic (PK) studies provide a mathematical basis to assess the time course of drug in the body. It enables to quantify absorption, distribution, metabolism and excretion of the drug and their metabolites. The primary requirement to undertake a PK study, is to have an

imidazolidin-4-one peptidomimetic derivatives of primaquine. (Vale et al., 2008a)

under the established analytical conditions, a fragmentation pattern

analytical method which is reliable, reproducible, sensitive, selective, and if possible, compat‐ ible with high-throughput pharmacokinetic approaches.

Drug efficacy requires adequate drug concentration at the site of action. Monitoring drugs and their metabolites in biological samples (*in vivo* studies), is fundamental in order to control the intake of the antimalarials by the infected populations. Mass spectrometry has been success‐ fully used for this purposes, coupled with liquid chromatography, and analytical methods **No. Delete** 

**Page No.** 

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**11** 

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**Lin e**

**PROOF CORRECTIONS FORM** 

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**Lin e**

Fig. 4. The oxidative antimalarial primaquine analogous (PI, PII and PIII)

**Replace with** 

healthy volunteers during a clinical efficacy trial, using a LC-MS/MS equipment.(Doyle et al.,

**N**

**CH3**

**N**

**CH3**

**PIII**

**NH2**

For NPC1161 (Fig. 7), an 8-aminoquinoline analog (*8-[(4-amino-1-methylbutyl)amino]-5-[3,4 dichloro-phenoxyl]-4-methyl-quinoline)* and their metabolites, a LC-MS method using an electro‐

Fig. 7. NPC1161, an 8-aminoquinoline

Using mass spectrometry other antimalarial molecules as α-/β-diastereomers of arteether (AE), sulphadoxine (SDX) and pyrimethamine (PYR) (Sabarinath et al., 2006) and three novel trioxane antimalarials (Fig. 8) (Singh et al., 2008) were determined in rat plasma. A *N*-alkyla‐ midine compound, M64, and its corresponding bioprecursors were measured in human and rat plasma (Margout et al., 2011) (Fig. 9). Artemisinin class compounds and its active *in-vivo*

> **O OO**

**<sup>A</sup> <sup>B</sup> <sup>C</sup>**

**O OO**

**OCH3 . HO2C**

**CO2H**

Contribution of Mass Spectrometry to the Study of Antimalarial Agents

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71

**NH2**

**N**

**HN**

**HN**

**HN**

**O**

**H3CO**

**H3CO**

**HO**

**H3CO**

**HO**

spray ionization source and a TOF analyzer, was implemented.

analog.

**O OO**

**Figure 8.** Chemical structures of three novel trioxane antimalarials

metabolites were analysed in monkey plasma (Singh et al., 2009).

**Figure 7.** NPC1161, an 8-aminoquinoline analog

**F3C**

2002)

Fig. 4. The oxidative antimalarial primaquine analogous (PI, PII and PIII)

**Replace with** 

**N**

**CH3**

**PII**

**Delete** 

**HN**

**HN**

**H3CO**

**PROOF CORRECTIONS FORM** 

**No. Delete** 

**N**

**CH3**

**HN**

**Figure 6.** Tafenoquine

**NH2**

**NH2**

**NH2 CH3 NH2**

Fig. 4. The oxidative antimalarial primaquine analogous (PI, PII and PIII) **Figure 4.** The oxidative antimalarial primaquine analogous (PI, PII and PIII)

**Page** 

**Lin**

**Figure 5.** Bulaquine

have been optimized and validated for quantification of different drugs and metabolites in biological fluids. Fig. 7. NPC1161, an 8-aminoquinoline **11** 

A LC-MS/MS method was used to study 14 antimalarial drugs, which are the components of the current first-line combination treatments for malaria (artemether, artesunate, dihydroar‐ temisinin, amodiaquine, N-desethyl-amodiaquine, lumefantrine, desbutyl-lumefantrine, piperaquine, pyronaridine, mefloquine, chloroquine, quinine, pyrimethamine and sulfadox‐ ine). The best conditions for mass spectrometry analysis were optimized (Hodel et al., 2009) and the method developed was implemented, and used to analyse samples from an *in vivo* study with 125 Southeast Asian patients from two regions of Cambodia: one region with a high level of antimalarial drug resistance and another region with moderate levels of drug resistance (Hodel et al., 2010). The 14 antimalarial drugs were measured in plasma samples from the patients, and results showed that for half of them, an antimalarial drug was detected namely mefloquine, piperaquine, chloroquine or quinine. However all patients reported either not having taken any antimalarial before or not knowing to have taken. These results are important, as they show that it is urgent to ensure appropriate use of antimalarials among the populations. Fig. 7. NPC1161, an 8-aminoquinoline analog. analog. **Delete**  Fig. 7. NPC1161, an 8-aminoquinoline analog.

Tafenoquine (8-[(4-amino-1-methylbutyl)amino]-2,6-dimethoxy-4-methyl-5-(3-trifluorometh‐ yl-phenoxy) quinoline succinate) (Fig. 6) was measured in human plasma from patients and healthy volunteers during a clinical efficacy trial, using a LC-MS/MS equipment.(Doyle et al., 2002) **H3CO**

**CH3**

**HN**

**H3CO**

**HO**

**HO**

**N**

**Figure 6.** Tafenoquine

**NH2**

**NH2**

**NH2 CH3 NH2**

Fig. 4. The oxidative antimalarial primaquine analogous (PI, PII and PIII)

**Replace with** 

**N**

**CH3**

**PII**

**HN**

**HN**

**H3CO**

have been optimized and validated for quantification of different drugs and metabolites in

Fig. 4. The oxidative antimalarial primaquine analogous (PI, PII and PIII)

**Replace with** 

**N**

**CH3**

**PII**

**NH2**

**NH**

**O O**

**NH2 NH2 CH3 NH2**

**H3CO**

**H3CO**

**HN**

**HN**

**No. Delete Replace with** 

**Lin e**

**NH**

**N**

**H3CO**

**N**

**N**

**CH3**

**NH2**

Fig. 4. The oxidative antimalarial primaquine analogous (PI, PII and PIII)

**NH2**

**PROOF CORRECTIONS FORM** 

**No. Delete** 

**CH3**

**PIII**

Fig. 7. NPC1161, an 8-aminoquinoline

Fig. 7. NPC1161, an 8-aminoquinoline analog.

analog.

**HN**

**PI**

**CH3**

**HN**

**N**

**N**

**CH3**

**H3CO**

**HO**

**H3CO**

**HN**

**HN**

**HO**

**PROOF CORRECTIONS FORM** 

10

**Page No.** 

**Lin e**

**No. Delete** 

**Page No.** 

10

**Page No.** 

**11** 

**Lin e**

**Lin e**

> A LC-MS/MS method was used to study 14 antimalarial drugs, which are the components of the current first-line combination treatments for malaria (artemether, artesunate, dihydroar‐ temisinin, amodiaquine, N-desethyl-amodiaquine, lumefantrine, desbutyl-lumefantrine, piperaquine, pyronaridine, mefloquine, chloroquine, quinine, pyrimethamine and sulfadox‐ ine). The best conditions for mass spectrometry analysis were optimized (Hodel et al., 2009) and the method developed was implemented, and used to analyse samples from an *in vivo* study with 125 Southeast Asian patients from two regions of Cambodia: one region with a high level of antimalarial drug resistance and another region with moderate levels of drug resistance (Hodel et al., 2010). The 14 antimalarial drugs were measured in plasma samples from the patients, and results showed that for half of them, an antimalarial drug was detected namely mefloquine, piperaquine, chloroquine or quinine. However all patients reported either not having taken any antimalarial before or not knowing to have taken. These results are important, as they show that it is urgent to ensure appropriate use of antimalarials among the

**Delete** 

Tafenoquine (8-[(4-amino-1-methylbutyl)amino]-2,6-dimethoxy-4-methyl-5-(3-trifluorometh‐ yl-phenoxy) quinoline succinate) (Fig. 6) was measured in human plasma from patients and

biological fluids.

**Figure 5.** Bulaquine

Fig. 7. NPC1161, an 8-aminoquinoline analog.

**N**

**N**

**CH3**

70 Tandem Mass Spectrometry - Molecular Characterization

**NH2**

Fig. 4. The oxidative antimalarial primaquine analogous (PI, PII and PIII)

**O**

**11** 

**Page No.** 

**Figure 4.** The oxidative antimalarial primaquine analogous (PI, PII and PIII)

**CH3**

**PI**

**HN**

**HN**

**H3CO**

**H3CO**

populations.

For NPC1161 (Fig. 7), an 8-aminoquinoline analog (*8-[(4-amino-1-methylbutyl)amino]-5-[3,4 dichloro-phenoxyl]-4-methyl-quinoline)* and their metabolites, a LC-MS method using an electro‐ spray ionization source and a TOF analyzer, was implemented. **No. Delete Replace with** 

**Figure 7.** NPC1161, an 8-aminoquinoline analog

Fig. 7. NPC1161, an 8-aminoquinoline analog. **Delete**  Using mass spectrometry other antimalarial molecules as α-/β-diastereomers of arteether (AE), sulphadoxine (SDX) and pyrimethamine (PYR) (Sabarinath et al., 2006) and three novel trioxane antimalarials (Fig. 8) (Singh et al., 2008) were determined in rat plasma. A *N*-alkyla‐ midine compound, M64, and its corresponding bioprecursors were measured in human and rat plasma (Margout et al., 2011) (Fig. 9). Artemisinin class compounds and its active *in-vivo* metabolites were analysed in monkey plasma (Singh et al., 2009).

**Figure 8.** Chemical structures of three novel trioxane antimalarials

M64, X=H; M64AH, X=OH; M6 4-S-Me, X=OSO2CH3

**Figure 9.** Chemical structures of compound, M64, and its corresponding bioprecursors

A method was also developed and validated according to FDA guidelines for simultaneous determination of two mono-thiazolium compounds in plasma, whole blood and red blood cells from human and rat (Taudon et al., 2008). More recently a rapid (3 min analysis) and sensitive UPLC-MS/MS method using a triple quadrupole tandem mass spectrometer in positive ESI mode, has been implemented for the analysis of ARB-89 (7α-hydroxy artemisinin carbamate) (Fig. 10) in rat serum, for pharmacokinetics studies, (Pabbisetty et al., 2012).

of the five metabolites of piperaquine in urine samples. Two of the metabolites (a carboxylic and a mono N-oxidated piperaquine metabolite) were considered as the most relevant as they were detected in the serum/plasma samples collected up to 93 days, and also in urine 123 days after administration of the drug. Other two monohydroxylated metabolites and a di-N-

**QM12-QM7 QM17-QM20**

A major impurity (8-(4-amino-4-methylbutylamino)-6 methoxyquinoline) associated with primaquine drug samples (Fig.12), obtained from European Pharmacopoeia and other commercial sources, was detected by gas chromatography-electron impact-mass spectrometry

Mass spectrometry can also be conducted in order to contribute to study the properties of compounds, through knowledge of their stability and fragmentation mechanisms, under the gas-phase conditions of a mass spectrometer. This type of studies can have, in the future,

oxidized metabolite were also detected in urine samples.

**O O O O**

> **O O O O**

> > **O O**

**Figure 11.** Proposed metabolic pathways for QHS *in vitro* and *in vivo* (Liu et al., 2011)

**O**

**O**

**O**

**CH3**

**CH3**

**CH3**

**CH3**

**H3C**

**H3C**

**H3C**

**O**

**CH3**

**OH OH**

**O O O O**

**H3C**

**QM13 QM26**

**H3C**

**QM14 QM1 QM8-QM12 QM21-QM25**

**H3C**

**OH O OGlu**

**O**

**O O O O**

> **O O**

> > **O**

**O**

**CH3**

**CH3**

**CH3**

**OH**

**O O O O**

**H3C**

**O**

http://dx.doi.org/10.5772/56225

73

**CH3**

**OGlu**

**CH3**

**CH3**

**CH3**

**OGlu OH**

Contribution of Mass Spectrometry to the Study of Antimalarial Agents

**CH3**

**CH3**

important implications in drug analysis and development.

**4.3. Detection of impurities**

**O O**

**O**

**H3C**

**O**

**CH3**

**CH3**

(GC–EI-MS) (Dongre et al., 2005).

**4.4. Chemical stability studies**

**Figure 10.** ARB-89 (7α-hydroxy artemisinin carbamate)

Mass spectrometry has proven to be particularly useful in identifying complex metabolites as those arising from phase I (e.g. those involving cytochrome P450 monooxygenases) and phase II (e.g. conjugation with glucuronic acid, sulfonates, glutathione or amino acids) reactions. In a study published by Liu et al., 2011, metabolites of artemisinin, also known as Qing-hao-su (QHS), and its active derivative dihydroartemisinin (DHA) were identified in *in vitro* and *in vivo* biological samples using a LTQ-Orbitrap mass spectrometer in tandem with H/D ex‐ change. The authors were able to show that artemisinin drugs mainly undergo hydroxylation and loss of oxygen in the phase I metabolic pathway and can form glucuronides in the phase II processes, as shown in Fig. 11. Based on MS data it was proposed a metabolic pathway for these metabolites.

Piperaquine was synthesized for the first time about 50 years ago, but seems to be a suitable partner drug in artemisinin-based combination treatments. In a paper published by Tarning et al., 2006, the main metabolites of piperaquine were characterized in a 16-h human urine, after a single p.o. administration of a fixed combination of dihydroartemisinin-piperaquine, with a fatty meal. A LC method in tandem with a QTRAP system was used to analyse piperaquine and its metabolites and a FT-ICR/MS equipment was used to obtain mass spectra Contribution of Mass Spectrometry to the Study of Antimalarial Agents http://dx.doi.org/10.5772/56225 73

**Figure 11.** Proposed metabolic pathways for QHS *in vitro* and *in vivo* (Liu et al., 2011)

of the five metabolites of piperaquine in urine samples. Two of the metabolites (a carboxylic and a mono N-oxidated piperaquine metabolite) were considered as the most relevant as they were detected in the serum/plasma samples collected up to 93 days, and also in urine 123 days after administration of the drug. Other two monohydroxylated metabolites and a di-Noxidized metabolite were also detected in urine samples.

### **4.3. Detection of impurities**

A method was also developed and validated according to FDA guidelines for simultaneous determination of two mono-thiazolium compounds in plasma, whole blood and red blood cells from human and rat (Taudon et al., 2008). More recently a rapid (3 min analysis) and sensitive UPLC-MS/MS method using a triple quadrupole tandem mass spectrometer in positive ESI mode, has been implemented for the analysis of ARB-89 (7α-hydroxy artemisinin carbamate) (Fig. 10) in rat serum, for pharmacokinetics studies, (Pabbisetty et al., 2012).

**N H**

M64, X=H; M64AH, X=OH; M6 4-S-Me, X=OSO2CH3

**12**

**N N X X**

**CH3**

**N**

Mass spectrometry has proven to be particularly useful in identifying complex metabolites as those arising from phase I (e.g. those involving cytochrome P450 monooxygenases) and phase II (e.g. conjugation with glucuronic acid, sulfonates, glutathione or amino acids) reactions. In a study published by Liu et al., 2011, metabolites of artemisinin, also known as Qing-hao-su (QHS), and its active derivative dihydroartemisinin (DHA) were identified in *in vitro* and *in vivo* biological samples using a LTQ-Orbitrap mass spectrometer in tandem with H/D ex‐ change. The authors were able to show that artemisinin drugs mainly undergo hydroxylation and loss of oxygen in the phase I metabolic pathway and can form glucuronides in the phase II processes, as shown in Fig. 11. Based on MS data it was proposed a metabolic pathway for

Piperaquine was synthesized for the first time about 50 years ago, but seems to be a suitable partner drug in artemisinin-based combination treatments. In a paper published by Tarning et al., 2006, the main metabolites of piperaquine were characterized in a 16-h human urine, after a single p.o. administration of a fixed combination of dihydroartemisinin-piperaquine, with a fatty meal. A LC method in tandem with a QTRAP system was used to analyse piperaquine and its metabolites and a FT-ICR/MS equipment was used to obtain mass spectra

**N**

**O**

**O**

**H**

**O O O**

**O**

**O H**

**O O H**

**Figure 10.** ARB-89 (7α-hydroxy artemisinin carbamate)

these metabolites.

**O**

**O**

72 Tandem Mass Spectrometry - Molecular Characterization

**O**

**O**

**H3C N**

**Figure 9.** Chemical structures of compound, M64, and its corresponding bioprecursors

**H**

**O**

**H N**

**H**

A major impurity (8-(4-amino-4-methylbutylamino)-6 methoxyquinoline) associated with primaquine drug samples (Fig.12), obtained from European Pharmacopoeia and other commercial sources, was detected by gas chromatography-electron impact-mass spectrometry (GC–EI-MS) (Dongre et al., 2005).

### **4.4. Chemical stability studies**

Mass spectrometry can also be conducted in order to contribute to study the properties of compounds, through knowledge of their stability and fragmentation mechanisms, under the gas-phase conditions of a mass spectrometer. This type of studies can have, in the future, important implications in drug analysis and development.

**14** 

**Page No.** 

**Lin e**

**Replace with** 

Fig. 12. Fragmentation pattern of primaquine and the corresponding impurity **Figure 12.** Fragmentation pattern of primaquine and the corresponding impurity

may become a problem to biological samples, as biomolecules have usually **Figure 13.** PQ imidazolidin-4-ones, PQAAPro and PQProAA mimetic derivatives of primaquine

**<sup>1</sup>**28 (*m/z*). (m/z). In order to achieve this state, the

sample must be volatilized and this

high molecular mass and high polarity,

**4.5. Detection of counterfeit drugs**

The quality of commercially available drugs varies among countries. The WHO/International Medical Products Anti-Counterfeiting Taskforce estimates that in some less developed countries, the counterfeit drugs are up to 50% of the total drugs supplied to the popula‐ tions(Hall et al., 2006). Due to the lack of regulations and poor quality control practices, the amount of the active ingredient may be incorrect, as a result of chemical degradation that occurs due to poor storage conditions, especially in warm and humid tropical environments. In some cases, expired drugs are repackaged with new expiration dates and put in the market. Also some drugs can be contaminated or replaced by other substances and people consume

Contribution of Mass Spectrometry to the Study of Antimalarial Agents

http://dx.doi.org/10.5772/56225

75

The report published by WHO in 2011, about the quality of antimalarial drugs in 6 countries of sub-Saharan Africa (Cameroon, Ethiopia, Ghana, Kenya, Nigeria and the United Republic of Tanzania) resumes the results obtained from the analysis of 935 samples and gives an idea

As an incorrect intake can result in a low bioavailability of the drug in the individual, leading to drug-resistance strains, provoking a therapeutic failure, reliable methods of analysis must be available to determine the quality of antimalarials commercialized, and LC coupled with mass spectrometry can be used. However this technique is expensive, requires training, technological support, and sample preparation. Actually, DART (Fernández et al., 2006) and DESI (Haiss et al., 2007) methodologies are often used, as they produce results rapidly, because they do not require sample preparation. Results obtained from a DESI MS method were used to validate Fourier-transform infrared imaging for characterization of counterfeit antimalarial pharmaceutical in tablets (Ricci et al., 2007). A DESI MS method was also applied for the quantitative screening of counterfeit antimalarial tablets containing artesunate (Nyadong et al., 2008), and more recently DESI and DART methods were used to validate results from the application of FT-Raman spectroscopy for *in situ* screening for potentially counterfeit artesu‐

The rapid diagnosis of malaria infection can also be performed by mass spectrometry. Hemozoin, the malaria pigment, can be detected by laser desorption mass spectrometry (LDMS) in human blood. (Scholl et al., 2004) Detection of malaria in 45 asymptomatic pregnant Zambian women was performed by this technique. Detection of *Plasmodium falciparum* malaria during pregnancy is complicated by sequestration of parasites in the placenta reducing peripheral blood microscopic detection. LDMS was able to detect <10 parasites/uL cultured in human blood and provided a more rapid mean of screening for infection than the technique

The information obtained from the use of mass spectrometry in the study of antimalarials agents is important, in order to understand all the mechanisms of the illness, malaria, and the

used currently for this purpose, light microscopy (Nyunt et al., 2005).

sawdust, paint and other toxic or inert substances (Kaur et al., 2009).

about the quality and counterfeits that more often occur.

nate antimalarial tablets (Ricci et al., 2008).

**4.6. Rapid diagnosis of malaria**

**5. Conclusions**

Studies on primaquine derived imidazolidin-4-ones using an ESI-ion trap mass spectrometer have allowed to find a correlation between the stability of the ions in the nozzle-skimmer region during the CID (Collision Induced Dissociation) analyses and reactivity in both isotonic buffer and human plasma (Vale et al., 2008). The same authors (Vale et al., 2008a) studied imidazolidin-4-one peptidomimetic derivatives of primaquine, PQAAPro and PQProA (Fig. 13) and they also found the parallelism between compound reactivity to hydrolysis and stability during CID analysis. Using CID and MS/MS experiments to study the peptidomimetic imidazolidin-4-one derivatives of primaquine, it was possible to conclude that CID spectra reflected the reactivity of compounds under physiological conditions, and the relative abundances of MS/MS generated fragments were correlated with the Charton steric parame‐ ters associated to amino acid side chain in the molecule (Vale et al., 2009). factors that limit their volatility. **2** 2 specifications even Specifications, even **2** 6 Figure 1. Example of basic Figure 1. Basic **2** 7 ionization;; ionization; **2** 8 Real Time (adapted Real Time; FT-ICR: Fourier transform

From the results obtained, ESI-MS proved to be a reliable tool for stability prediction of compounds towards hydrolysis at physiological pH and temperature although the mecha‐ nisms in water and in the gas-phase are not comparable. This type of studies were, for the first time, approached by these authors.

### **4.5. Detection of counterfeit drugs**

The quality of commercially available drugs varies among countries. The WHO/International Medical Products Anti-Counterfeiting Taskforce estimates that in some less developed countries, the counterfeit drugs are up to 50% of the total drugs supplied to the popula‐ tions(Hall et al., 2006). Due to the lack of regulations and poor quality control practices, the amount of the active ingredient may be incorrect, as a result of chemical degradation that occurs due to poor storage conditions, especially in warm and humid tropical environments. In some cases, expired drugs are repackaged with new expiration dates and put in the market. Also some drugs can be contaminated or replaced by other substances and people consume sawdust, paint and other toxic or inert substances (Kaur et al., 2009).

The report published by WHO in 2011, about the quality of antimalarial drugs in 6 countries of sub-Saharan Africa (Cameroon, Ethiopia, Ghana, Kenya, Nigeria and the United Republic of Tanzania) resumes the results obtained from the analysis of 935 samples and gives an idea about the quality and counterfeits that more often occur.

As an incorrect intake can result in a low bioavailability of the drug in the individual, leading to drug-resistance strains, provoking a therapeutic failure, reliable methods of analysis must be available to determine the quality of antimalarials commercialized, and LC coupled with mass spectrometry can be used. However this technique is expensive, requires training, technological support, and sample preparation. Actually, DART (Fernández et al., 2006) and DESI (Haiss et al., 2007) methodologies are often used, as they produce results rapidly, because they do not require sample preparation. Results obtained from a DESI MS method were used to validate Fourier-transform infrared imaging for characterization of counterfeit antimalarial pharmaceutical in tablets (Ricci et al., 2007). A DESI MS method was also applied for the quantitative screening of counterfeit antimalarial tablets containing artesunate (Nyadong et al., 2008), and more recently DESI and DART methods were used to validate results from the application of FT-Raman spectroscopy for *in situ* screening for potentially counterfeit artesu‐ nate antimalarial tablets (Ricci et al., 2008).

### **4.6. Rapid diagnosis of malaria**

Studies on primaquine derived imidazolidin-4-ones using an ESI-ion trap mass spectrometer have allowed to find a correlation between the stability of the ions in the nozzle-skimmer region during the CID (Collision Induced Dissociation) analyses and reactivity in both isotonic buffer and human plasma (Vale et al., 2008). The same authors (Vale et al., 2008a) studied imidazolidin-4-one peptidomimetic derivatives of primaquine, PQAAPro and PQProA (Fig. 13) and they also found the parallelism between compound reactivity to hydrolysis and stability during CID analysis. Using CID and MS/MS experiments to study the peptidomimetic imidazolidin-4-one derivatives of primaquine, it was possible to conclude that CID spectra reflected the reactivity of compounds under physiological conditions, and the relative abundances of MS/MS generated fragments were correlated with the Charton steric parame‐

Fig. 12. Fragmentation pattern of primaquine and the corresponding impurity

**Replace with** 

Fig. 12. Fragmentation pattern of primaquine and the corresponding impurity

**N**

**PQ imidazolidin-4-ones**

**N**

**R3 R2**

**NH**

**R1**

**O**

analysers, depending on applications.

and tandem mass spectrometry,

**NH**

sample must be volatilized and this may become a problem to biological samples, as biomolecules have usually high molecular mass and high polarity, factors that limit their volatility.

**PQProAA**

**N N**

**R3 R2**

**O**

**O**

**NH2 R4 R1**

expanded

**OCH3**

**No. Delete Replace with** 

**OCH3**

**1** 17 using combinations of analysers. using combinations of different

**1** 22 In this chapter will be discussed In this chapter are presented

**1** 23 equipments and the contribution Equipments. The contribution

**Figure 13.** PQ imidazolidin-4-ones, PQAAPro and PQProAA mimetic derivatives of primaquine

**2** 2 specifications even Specifications, even

**2** 6 Figure 1. Example of basic Figure 1. Basic

**2** 7 ionization;; ionization;

**1** 24 in man, malaria. In man, malaria, is also discussed.

**<sup>1</sup>**28 (*m/z*). (m/z). In order to achieve this state, the

**N N**

**2** 8 Real Time (adapted Real Time; FT-ICR: Fourier transform

**1** 20 (CE) in tandem with mass spectrometry expanded (CE) coupled with mass spectrometry

**NH**

**1** 10 analytes compounds

**<sup>N</sup> NH R2 R3**

**R1**

**Figure 12.** Fragmentation pattern of primaquine and the corresponding impurity

From the results obtained, ESI-MS proved to be a reliable tool for stability prediction of compounds towards hydrolysis at physiological pH and temperature although the mecha‐ nisms in water and in the gas-phase are not comparable. This type of studies were, for the first

ters associated to amino acid side chain in the molecule (Vale et al., 2009).

time, approached by these authors.

**14** 

**Page No.** 

**Lin e**

**OCH3**

**NH**

**NH**

**PQAAPro**

74 Tandem Mass Spectrometry - Molecular Characterization

**O O**

**R4**

The rapid diagnosis of malaria infection can also be performed by mass spectrometry. Hemozoin, the malaria pigment, can be detected by laser desorption mass spectrometry (LDMS) in human blood. (Scholl et al., 2004) Detection of malaria in 45 asymptomatic pregnant Zambian women was performed by this technique. Detection of *Plasmodium falciparum* malaria during pregnancy is complicated by sequestration of parasites in the placenta reducing peripheral blood microscopic detection. LDMS was able to detect <10 parasites/uL cultured in human blood and provided a more rapid mean of screening for infection than the technique used currently for this purpose, light microscopy (Nyunt et al., 2005).

### **5. Conclusions**

The information obtained from the use of mass spectrometry in the study of antimalarials agents is important, in order to understand all the mechanisms of the illness, malaria, and the way the different drugs interact in the human organism. Due to its characteristics (sensitivity, speed, possibility to be automated, possibility to combine with separation techniques) and diversity of equipments available, mass spectrometry can be used in the structural identifica‐ tion of new molecules, in the study of many phases of drug discovery, for assessment of compound stability, pharmacokinetic studies monitoring the concentrations of antimalarials and metabolites in biological matrices, the studies of cell permeability and plasma protein binding, and finally, in the quality control of commercial drugs.

[6] Dongre, V. G, Karmuse, P. P, Nimbalkar, M. M, Singh, D, & Kumar, A. (2005). Appli‐ cation of GC-EI-MS for the identification and investigation of positional isomer in primaquine, an antimalarial drug. Journal of Pharmaceutical and Biomedical Analy‐

Contribution of Mass Spectrometry to the Study of Antimalarial Agents

http://dx.doi.org/10.5772/56225

77

[7] Doyle, E, Fowles, S. E, Summerfield, S, & White, T. J. (2002). Rapid determination of tafenoquine in small volume human plasma samples by high-performance liquid chromatography-tandem mass spectrometry. Jounal of Chromatography B Analyti‐

[8] Eisenstein, M. (2012). Drug development holding Holding out for reinforcements.

[9] El-Aneed, A, Cohen, A, & Banoub, J. (2009). Mass Spectrometry, Review of the Ba‐ sics: Electrospray, MALDI, and Commonly Used Mass Analyzers. Applied Spectro‐

[10] Feng, W. Y. (2004). Mass spectrometry in drug discovery: a current review. Current

[11] Fernández, F. M, Cody, R. B, Green, M. D, Hampton, C. Y, Mcgready, R, Sengaloun‐ deth, S, White, N. J, & Newton, P. N. (2006). Characterization of Solid Counterfeit Drug Samples by Desorption Electrospray Ionization and Direct-analysis-in-real-time

Coupled to Time-of-flight Mass Spectrometry. ChemMedChem , 1(7), 702-705.

first century. Nature Reviews Drug Discovery , 2(2), 140-150.

something borrowed. F1000 Biology Reports, 3(24), 1-9.

[12] Glish, G. L, & Vachet, R. W. (2003). The basics of mass spectrometry in the twenty-

[13] Haiss, W, Thanh, N. T, Aveyard, J, & Fernig, D. G. (2007). Determination of size and concentration of gold nanoparticles from UV-vis spectra. Analytical Chemistry ,

[14] Hall, K. A, & Newton, P. N. Green, M.D; De Veij, M; Vandenabeele, P.; Pizzanelli, D.; Mayxay, M.; Dondorp, A. & Fernandez, F.M. ((2006). Characterization of counterfeit artesunate antimalarial tablets from southeast Asia. The American Journal Tropical

[15] Hobbs, C, & Duffy, P. (2011). Drugs for malaria: something old, something new,

[16] Hodel, E. M, Genton, B, Zanolari, B, Mercier, T, Duong, S, Beck, H. P, Olliaro, P, De‐ costerd, L. A, & Ariey, F. (2010). Residual antimalarial concentrations before treat‐ ment in patients with malaria from Cambodia: indication of drug pressure. Journal

[17] Hodel, E. M, Zanolari, B, Mercier, T, Biollaz, J, Keiser, J, Olliaro, P, Genton, B, & De‐ costerd, L. A. LC-tandem mass spectrometry method for the simultaneous determi‐ nation of 14 antimalarial drugs and their metabolites in human plasma. Journal of

cal Technologies Biomedical Life Sciences , 769(1), 127-132.

sis 39(1-2), 111-116.

Nature 484(7395): SS18., 16.

scopy Reviews , 44(3), 210-230.

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The use of mass spectrometry to predict stability of compounds in physiological conditions may become an important tool.

### **Author details**

Ana Raquel Sitoe1 , Francisca Lopes1 , Rui Moreira1 , Ana Coelho2 and Maria Rosário Bronze1,2

1 Research Institute for Medicines and Pharmaceutical Sciences (iMed.UL), Faculty of Phar‐ macy, University of Lisbon, Portugal

2 Instituto de Tecnologia Química e Biológica, Oeiras, Portugal

### **References**


[6] Dongre, V. G, Karmuse, P. P, Nimbalkar, M. M, Singh, D, & Kumar, A. (2005). Appli‐ cation of GC-EI-MS for the identification and investigation of positional isomer in primaquine, an antimalarial drug. Journal of Pharmaceutical and Biomedical Analy‐ sis 39(1-2), 111-116.

way the different drugs interact in the human organism. Due to its characteristics (sensitivity, speed, possibility to be automated, possibility to combine with separation techniques) and diversity of equipments available, mass spectrometry can be used in the structural identifica‐ tion of new molecules, in the study of many phases of drug discovery, for assessment of compound stability, pharmacokinetic studies monitoring the concentrations of antimalarials and metabolites in biological matrices, the studies of cell permeability and plasma protein

The use of mass spectrometry to predict stability of compounds in physiological conditions

, Rui Moreira1

1 Research Institute for Medicines and Pharmaceutical Sciences (iMed.UL), Faculty of Phar‐

[1] Arnaud, C. H. (2007). Taking mass spec into the open: Open-air ionization methods minimize sample pre and widen range odf mass spectrometry applications. Chemi‐

[2] Biot, C, Castro, W, & Navarro, M. (2012). The therapeutic potential of metal-based antimalarial agents: Implications for the mechanism of action. Dalton Transactions ,

[3] Capela, R, Cabal, G. G, Rosenthal, P. J, Gut, J, Mota, M. M, Moreira, R, Lopes, F, & Prudêncio, M. (2011). Design and Evaluation of Primaquine-Artemisinin Hybrids as a Multistage Antimalarial Strategy. Antimicrobial Agents and Chemotherapy ,

[4] Denisov, E. Damoc; Lange, O. & Makarov, A. ((2012). Orbitrap mass spectrometry with resolving powers above 1,000,000. International Journal of Mass Spectrometry

[5] Donato, P. Cacciola; Tranchida, F.P.; Dugo, P. & Mondello, L. ((2012). Mass spec‐ trometry detection in comprehensive liquid chromatography: basic concepts, instru‐ mental aspects, applications and trends. Mass Spectrometry Reviews , 31(5), 523-559.

, Ana Coelho2

and Maria Rosário Bronze1,2

binding, and finally, in the quality control of commercial drugs.

, Francisca Lopes1

cal & Engineering News , 85(41), 13-18.

2 Instituto de Tecnologia Química e Biológica, Oeiras, Portugal

may become an important tool.

76 Tandem Mass Spectrometry - Molecular Characterization

macy, University of Lisbon, Portugal

41(21), 6335-6349.

55(10), 4698-4706.

325-327(0), 80-85.

**Author details**

Ana Raquel Sitoe1

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**Chapter 4**

**Mass Spectrometry Strategies for Structural Analysis of**

**Mass spectrometry strategies for structural analysis of** 

Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Paraná,

Carbohydrates are compounds rich in hydroxyl groups, being a monosaccharide a building block for complex carbohydrates. Beside the large amounts of hydroxyl groups, two chemical functions define the organic class, and even the simplest carbohydrate must contain either an

monosaccharide a building block for complex carbohydrates. Beside the large amounts of hydroxyl groups, two chemical functions define the organic class, and even the simplest carbohydrate must contain either an aldehyde (polyhydroxyaldehyde) or

Carbohydrates are compounds rich in hydroxyl groups, being a

Figure 1 – Functional groups defining carbohydrates (A) aldose (B) ketose

Carbohydrates can be naturally found as free monomers named

© 2013 Sassaki and Souza; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 Sassaki and Souza; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Laires et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons

monosaccharides (e.g. glucose, fructose), linked itself forming oligosaccharides such as the disaccharides sucrose and lactose, or larger structures containing hundreds of monosaccharides, the polysaccharides. Carbohydrates are the most abundant biomolecules worldwide, since they are found as structural matrix of plants (cellulose) and invertebrates (chitin). Other biological roles of carbohydrates are storage and transport of energy (e.g. starch, glycogen and sucrose). Despite many roles of carbohydrates are not well understood, it is well accepted that carbohydrates contain the codes for cell-cell recognition. To understand the importance of carbohydrates as

Carbohydrates can be naturally found as free monomers named monosaccharides (e.g. glucose, fructose), linked itself forming oligosaccharides such as the disaccharides sucrose and lactose, or larger structures containing hundreds of monosaccharides, the polysaccharides. Carbohydrates are the most abundant biomolecules worldwide, since they are found as structural matrix of plants (cellulose) and invertebrates (chitin). Other biological roles of

aldehyde (polyhydroxyaldehyde) or ketone (polyhydroxyketone) functions (Fig. 1).

**A B**

**Carbohydrates and Glycoconjugates**

Guilherme L. Sassaki and Lauro Mera de Souza

**carbohydrates and glycoconjugates** 

Guilherme L. Sassaki<sup>1</sup> and Lauro Mera de Souza<sup>1</sup>

Additional information is available at the end of the chapter

ketone (polyhydroxyketone) functions (Fig. 1).

**Figure 1.** Functional groups defining carbohydrates (A) aldose (B) ketose

http://dx.doi.org/10.5772/55221

Curitiba-PR, 19046, Brazil;

**1. Introduction**

1

