**5. Qualitative evaluation of metabolites**

The known identity of metabolites is the prerequisite for a suitable metabolic assessment of drugs. Liquid chromatography coupled with mass spectrometry has become the most powerful analytical tool for screening and identification of drug metabolites in biological matrices. A short overview of analytical strategies for identification of metabolites will be provided. More information regarding metabolite identification can be found in following review articles [7, 23-27]. The selection of suitable LC-MS instrumentation is needed for qualitative evaluation of metabolites. Moreover, this issue is also important for quantitative evaluation of metabolites as discussed in section 8. Additionally, some examples for metabolite identification using LC-MS/MS will be provided in this section.

## **5.1. LC-MS instrumentation**

## *5.1.1. Ionization techniques*

86 Chromatography – The Most Versatile Method of Chemical Analysis

**4.8. Liver slices** 

16, 18, 20].

**4.9. Isolated perfused liver** 

**4.10. Animal and human** *in vivo* **studies** 

culture: medium formulation, extracellular matrix, initial cell suspension and density, drug concentrations. Hepatocytes could also be cultured in a sandwich configuration where hepatocytes are placed between two layers of gelled extracellular matrix. This type of

Liver slices and hepatocytes are the most physiologically relevant *in vitro* techniques used for quantitative and qualitative measurement of hepatic phase I and phase II metabolism of drugs due to full complement of enzymes and cofactors. High-precision tissue slicers (e.g. Krumideck slicer, Brenden-Vitron slicer) are used for the production of liver slices of uniform thickness (less than 250 µm). The advantage of liver slices over hepatocytes lies in the intact structure of liver tissue containing hepatic and non-hepatic cells, normal spatial arrangement and possibility of morphological studies. The described *in vitro* model allows higher throughput compared to isolated perfused liver. Another advantage is the nonrequirement for digestive enzymes and consequently the preservation of intact tissue structure. Moreover, no addition of cofactors is needed for enzyme activity. However, some disadvantages of this model are known: decrease of CYP activity in short time due to impaired diffusion of nutrients and oxygen in the liver slice, damaged cells on the outer sides of the slice, inadequate tissue penetration of the test medium, short viability period (5 days), lack of optimal cryopreservation procedures and a need for expensive equipment [14-

Isolated perfused liver gives an excellent representation of the *in vivo* situation but it is not used frequently due to practical inconveniences. Normally animal liver tissue on a small scale is used, but never human liver tissue. The additional advantages of this *in vitro* model are also three-dimensional architecture, presence of hepatic and non-hepatic cell types, possibility to collect bile. The important disadvantages of this model are: poor reproducibility, functional integrity limited to 3 hours, difficult handling, poor perfusion of cells by nutrients and oxygen, low throughput and no availability of human liver. This

The identity of metabolites present in any matrix of animal or human provides essential information about the biotransformation pathways involved in the clearance of a drug. When the metabolite profiling of a parent drug is similar qualitatively and quantitatively between animal and human, we can assume that potential clinical risks of parent drug and metabolite have been adequately investigated during nonclinical studies. When a difference arises between *in vitro* and *in vivo* findings, the *in vivo* results should always take precedence over *in vitro* studies [21]. The FDA guidance encourages the identification of differences in

model is useful only in case when bile secretion is the subject of research [14-16].

culture retains liver hepatocyte specific functions for a longer period [18, 20].

A LC-MS ion source has the double role of eliminating the solvent from the LC eluent and producing gas-phase ions from the analyte. The application of atmospheric pressure ionization (API) methods has provided a breakthrough for the LC-MS systems and has brought it to the forefront of analytical techniques. Some ion sources such as API operate at atmospheric pressure where others like electron impact (EI) or chemical ionization (CI) operate in vacuum. While soft API interfaces, in particular electrospray, produce molecular ions with minimal fragmentation, high energy sources like EI mostly generate fragment ions. API techniques are most widely used for metabolite detection, identification and quantification [7, 28] due to the ability to operate at atmospheric pressure, good compatibility with reversed phase chromatography and generation of intact molecule ions at very high sensitivity. All three API techniques: electrospay ionization (ESI), atmospheric pressure chemical ionization (APCI) and atmospheric pressure photoionization (APPI) are complementary.

• **Electrospay ionization** is by far the preferred method for metabolite identification and quantification. It is the most universal technique for introducing the molecules into the gas phase and it is most gentle and therefore likely to yield an intact molecular ions. ESI is ideally suited for polar, ionic and thermally labile compounds such as drug metabolites; in particular glucuronides and others phase II metabolites. This technique requires ionization of analytes within solution prior to introduction into ion source and thus works best for fairy basic or acidic compounds. Depending on the voltage polarity, nebulised droplets trapping the ionized analyte will be positively or negatively charged. The reduction in size caused by solvent evaporation accounts for the increase in charge density in the droplet leading to its explosion when repulsive forces between charges exceed the cohesive forces of the droplet. This process occurs repeatedly until gas phase ions are produced [29]. Ions in solution are emitted into gas phase without application of heat making ESI suitable for analysis of thermo labile compounds. Many parameters, such as analyte and solution characteristics: pKa, analyte concentration, other electrolytes in solution, dielectric constant of the solvent, affect the ion formation process [7]. The effects of several mobile phase additives on the ionization efficiency have been reviewed [30] and will be discussed later (section 8). Depending on the chemical structure of an analyte, multiple-charged molecular ions can be formed, which is optimal for the analysis of biological macromolecules (e.g. proteins). Despite the numerous benefits of ESI, it suffers from a shortcoming in that it is susceptible to ion suppression effects from high concentrations of buffer, salts and other endogenous compounds in matrix solutions [23].

Analytical Methods for Quantification of Drug Metabolites in Biological Samples 89

• **Atmospheric pressure photoionization** is relatively new ionization method. This technique can be used for ionization of analytes that are not easily ionizable by ESI and APCI. APPI has similar application range as APCI but slightly extended toward nonpolar compounds [32]. The APPI ion source is very similar to APCI source, except the APCI corona discharge needle is replaced by photoionzation lamp. Depending on the analyte proton affinity relative to the composition of the mobile phase, either a radical molecular ion (typically for nonpolar compounds) or a protonated molecular ion (typically for polar compound) is obtained. APPI has a potencial in the analysis of drug metabolites but more research is needed to fully understand the important parameters

The function of mass analyzer is the separation of ions formed in ionization source according to their different mass-to-charge (m/z) ratios. The quality of mass separation is characterized by the degree to which close m/z values can be separated in the mass analyzer. Mass analyzers are classified regarding resolution into low and high resolution instruments. The later ones are associated with another important parameter, mass accuracy, which allows determination of elemental formula of particular analyte. The selection of suitable analyzer is driven by the purpose of the analysis and the instrument

• **Triple quadrupole instruments (QQQ)** are the most common mass spectrometers in analytical laboratories, having most often been acquired for their evident strengths in high sensitivity quantitative analysis of known analytes. These instruments have been often applied also for metabolite identification due to wide availability and excellent tandem mass (MS/MS) properties. In QQQ, the first quadrupole filters ions of interest, the second quadrupole also called collision cell fragments these ions and further the fragment ions are filtered by third quadrupole before reaching the mass detector. Such QQQ configuration allows performing different scans such as full scan, product ion scan, precursor ion scan (PI), constant neutral loss scan (CNL), single ion monitoring (SIM) and selected reaction monitoring (SRM) or multiple reaction monitoring (MRM). PI and CNL are particularly useful in metabolite identification since both scanning modes do not require previous knowledge about the molecular weight of metabolites. High sensitivity for quantitative purposes is retained only when working in MRM mode, however, the detection sensitivity decreases dramatically when wide mass range is analyzed in a scanning mode. This is one of the major disadvantages of using QQQ

• **Ion trap instruments (IT)** are like QQQ relatively inexpensive and compatible with wide range of ionization interfaces. These analyzers utilize ion trap chamber where ions are trapped and then selectively ejected from the chamber. Additionally, the resonance excitation applied in the trap provides efficient dissociation of the precursor ions to product ions. IT provides more sensitivity for structural elucidation than QQQ due to

performance but also depends on the instrument availability and cost effectiveness.

and factors that affect the ionization efficiency [33].

for the screening of drug metabolites.

*5.1.2. Mass analyzers* 

• **Atmospheric pressure chemical ionization** is more suited for less polar compounds. Certain classes of compound such as heavily halogenated analoges and highly aromatic compounds will run readily on APCI while giving no or a weak response on ESI [7]. APCI like ESI produces ions based on the API strategy, but thought a completely different process. Here, the liquid eluent is sprayed into heated chamber [450-550°C) where the high temperature of a nebulizer gas flow causes the immediate evaporation of the solvent and the analyte. In addition to volatility at the applied temperature, thermal stability of the analyte is also a prerequisite for the successful application of APCI (e.g. glucuronides may break down and appear in the form of protonated aglycone [31]. Ionization of analytes takes place in gas phase where due to high flux of electrons from corona discharge needle, solvent molecules initially react with electrons and form ions that produce protonated solvent ions through secondary reactions. These protonated solvent ions then transfer a proton to form protonated analytes. For efficient ionization, the employed mobile phase should be volatile and also amenable to gas phase acid-base reactions. APCI technique is less prone to ion suppression and provides a wider dynamic detection range than ESI due to ionization that occurs mainly in gas phase. Also, typically higher flow rate is used with APCI [1-2 mL/min) then that in conventional ESI (0.1- 0.5 mL/min) [23].

• **Atmospheric pressure photoionization** is relatively new ionization method. This technique can be used for ionization of analytes that are not easily ionizable by ESI and APCI. APPI has similar application range as APCI but slightly extended toward nonpolar compounds [32]. The APPI ion source is very similar to APCI source, except the APCI corona discharge needle is replaced by photoionzation lamp. Depending on the analyte proton affinity relative to the composition of the mobile phase, either a radical molecular ion (typically for nonpolar compounds) or a protonated molecular ion (typically for polar compound) is obtained. APPI has a potencial in the analysis of drug metabolites but more research is needed to fully understand the important parameters and factors that affect the ionization efficiency [33].

## *5.1.2. Mass analyzers*

88 Chromatography – The Most Versatile Method of Chemical Analysis

solutions [23].

0.5 mL/min) [23].

• **Electrospay ionization** is by far the preferred method for metabolite identification and quantification. It is the most universal technique for introducing the molecules into the gas phase and it is most gentle and therefore likely to yield an intact molecular ions. ESI is ideally suited for polar, ionic and thermally labile compounds such as drug metabolites; in particular glucuronides and others phase II metabolites. This technique requires ionization of analytes within solution prior to introduction into ion source and thus works best for fairy basic or acidic compounds. Depending on the voltage polarity, nebulised droplets trapping the ionized analyte will be positively or negatively charged. The reduction in size caused by solvent evaporation accounts for the increase in charge density in the droplet leading to its explosion when repulsive forces between charges exceed the cohesive forces of the droplet. This process occurs repeatedly until gas phase ions are produced [29]. Ions in solution are emitted into gas phase without application of heat making ESI suitable for analysis of thermo labile compounds. Many parameters, such as analyte and solution characteristics: pKa, analyte concentration, other electrolytes in solution, dielectric constant of the solvent, affect the ion formation process [7]. The effects of several mobile phase additives on the ionization efficiency have been reviewed [30] and will be discussed later (section 8). Depending on the chemical structure of an analyte, multiple-charged molecular ions can be formed, which is optimal for the analysis of biological macromolecules (e.g. proteins). Despite the numerous benefits of ESI, it suffers from a shortcoming in that it is susceptible to ion suppression effects from high concentrations of buffer, salts and other endogenous compounds in matrix

• **Atmospheric pressure chemical ionization** is more suited for less polar compounds. Certain classes of compound such as heavily halogenated analoges and highly aromatic compounds will run readily on APCI while giving no or a weak response on ESI [7]. APCI like ESI produces ions based on the API strategy, but thought a completely different process. Here, the liquid eluent is sprayed into heated chamber [450-550°C) where the high temperature of a nebulizer gas flow causes the immediate evaporation of the solvent and the analyte. In addition to volatility at the applied temperature, thermal stability of the analyte is also a prerequisite for the successful application of APCI (e.g. glucuronides may break down and appear in the form of protonated aglycone [31]. Ionization of analytes takes place in gas phase where due to high flux of electrons from corona discharge needle, solvent molecules initially react with electrons and form ions that produce protonated solvent ions through secondary reactions. These protonated solvent ions then transfer a proton to form protonated analytes. For efficient ionization, the employed mobile phase should be volatile and also amenable to gas phase acid-base reactions. APCI technique is less prone to ion suppression and provides a wider dynamic detection range than ESI due to ionization that occurs mainly in gas phase. Also, typically higher flow rate is used with APCI [1-2 mL/min) then that in conventional ESI (0.1The function of mass analyzer is the separation of ions formed in ionization source according to their different mass-to-charge (m/z) ratios. The quality of mass separation is characterized by the degree to which close m/z values can be separated in the mass analyzer. Mass analyzers are classified regarding resolution into low and high resolution instruments. The later ones are associated with another important parameter, mass accuracy, which allows determination of elemental formula of particular analyte. The selection of suitable analyzer is driven by the purpose of the analysis and the instrument performance but also depends on the instrument availability and cost effectiveness.


its better sensitivity in full scan mode and efficient dissociation of the precursor ions which allows multiple stages mass spectrometry (MSn). Recently, to address classical ion traps (called also 3D IT) shortcomings of insufficient ion storage efficiency, capacity and deterioration of the mass spectrum and dynamic response range, linear IT has been developed [25]. The detection sensitivity in linear IT is at least two orders of magnitude higher than that in 3D IT. Because of these advantages, linear IT will probably in near future totally replace old 3D IT [23].

Analytical Methods for Quantification of Drug Metabolites in Biological Samples 91

MS methodology is the most suitable approach for metabolite identification as commonly low concentrations of drug metabolites are present in complex biological matrices. Appropriate LC-MS instrumentation is clearly critical to both, detection and structural elucidation, although alternative non-MS approaches may also be important in cases when MS data alone are not sufficient. Tandem mass spectrometry instruments are beside their key role for metabolite quantification also well suited for qualitative purposes. Tandem mass spectrometry experiments, which allow different scan mode possibilities, are by far most informational techniques for structural characterization of metabolites [23]. But these experiments require a set of injections to perform full scan and other scan analyses to identify metabolites of interest. The drive to more versatile and powerful instruments which can perform intelligent data dependent experiments has led to newer mass analyzers, such as high resolution Q-TOF instruments, which now dominate the metabolite identification

The non-selective nature of full mass scan acquisition enables detection of practically all ionizable metabolites and giving most complete information in terms of metabolite molecular mass. However, two major disadvantages arise by this approach. Firstly, detection sensitivity using QQQ decreases dramatically when wide mass range is scanned. This obstacle can be overcome by using IT analyzers as its full scan is much more sensitive or even better by using TOF instruments which additionally enable accurate mass determination [23]. In case when only QQQ is available, a practical approach may be applied to improve sensitivity; the whole mass range should be divided to narrow scanning ranges by performing multiple analyses of the same sample. Secondly, other non-metabolite matrix compound may interfere with obtained MS data. A common procedure for metabolite detection involves analysis of test and control samples what then allows subtraction of control sample data. This approach is less successful when complex biological samples, such as plasma and urine, are examined. Expected metabolites in studied samples may be predicted based on biotransformation pathways of parent drugs what enables focused search of these compounds. The most common changes in mass caused by

PI and CNL are more specific approaches for identification of unknown metabolites. This scan mode is only possible for tandem mass spectrometers and therefore suffers at sensitivity like other QQQ scanning acquisitions. In PI scan mode, the second quadrupole mass filter is set to pass only the selected product ions, while the first quadrupole mass filter scans a range of m/z values. In CNL scan mode, both quadrupoles are scanning m/z values while the m/z difference between the quadrupoles is kept constant. Several phase II metabolites at fragmentation lose a distinct neutral group that can be used for specific

**5.2. Strategies for metabolite identification** 

field.

*5.2.1. Full scan* 

biotransformation are shown in Table 1.

*5.2.2. Precursor ion and constant neutral loss scan* 


## **5.2. Strategies for metabolite identification**

MS methodology is the most suitable approach for metabolite identification as commonly low concentrations of drug metabolites are present in complex biological matrices. Appropriate LC-MS instrumentation is clearly critical to both, detection and structural elucidation, although alternative non-MS approaches may also be important in cases when MS data alone are not sufficient. Tandem mass spectrometry instruments are beside their key role for metabolite quantification also well suited for qualitative purposes. Tandem mass spectrometry experiments, which allow different scan mode possibilities, are by far most informational techniques for structural characterization of metabolites [23]. But these experiments require a set of injections to perform full scan and other scan analyses to identify metabolites of interest. The drive to more versatile and powerful instruments which can perform intelligent data dependent experiments has led to newer mass analyzers, such as high resolution Q-TOF instruments, which now dominate the metabolite identification field.

### *5.2.1. Full scan*

90 Chromatography – The Most Versatile Method of Chemical Analysis

future totally replace old 3D IT [23].

PI, CNL and very high sensitive MRM data acquisition.

acquisition speed makes them ideal for fast chromatography [24].

chromatography because it suffers from a slow data acquisition [24].

the means of most laboratories involved in drug metabolism studies [7].

positive/negative switching in one run [24].

its better sensitivity in full scan mode and efficient dissociation of the precursor ions which allows multiple stages mass spectrometry (MSn). Recently, to address classical ion traps (called also 3D IT) shortcomings of insufficient ion storage efficiency, capacity and deterioration of the mass spectrum and dynamic response range, linear IT has been developed [25]. The detection sensitivity in linear IT is at least two orders of magnitude higher than that in 3D IT. Because of these advantages, linear IT will probably in near

• **Triple quadrupole-linear ion traps (QTrap)** combine sensitive QQQ technology with high capacity of linear IT incorporating high trapping efficiencies. In this instrument, the last quadrupole of QQQ is replaced with a linear ion trap, which operates as a mass resolving quadrupole or a linear ion trap. This provides clearly increased metabolite screening capabilities compared to traditional IT or QQQ. QTrap enables high sensitivity, wide range mass scanning and MSn together with QQQ capabilities, such as

• **Time of flight (TOF)** analyzers are the most suitable high resolution mass spectrometers for fast and cost-efficient metabolite identification. TOF are relatively simple and capable of recording all formed ions on a microsecond time scale offering high sensitivity detection. Ions are accelerated from the ion interface to a fixed kinetic energy and then pass through a field-free tube to the detector. The time needed for ion to reach the detector is proportional to its m/z ratio. TOF strength lies in its very high detection sensitivity when acquiring wide range data, enabling the simultaneous detection of data for all metabolites of interest in one run. High mass resolution and mass accuracy (< 3-5ppm) enable reliable and accurate identification of metabolites by determination of elemental formula of a metabolite. Additionally, the very high

• **Triple quadrupole-time of flight (Q-TOF)** instruments combine first mass filter and collision cell of QQQ with TOF as the second mass analyzer. These instruments can operate as true tandem MS while providing accurate mass of the product ions. Most modern Q-TOFs have good linear response and are therefore also suitable for quantitative purposes. However, TOF instruments have not the ability to perform

• **Orbitrap** is another high resolution analyzer which is a hybrid composed of a linear IT and Fourier transform mass spectrometer. It is an effective alternative to the TOF instruments used for metabolite profiling. Orbitrap is capable of high sensitivity screening over wide mass range, MSn and tandem mass spectrometry with accurate mass data for both parent and fragment ion. However, it is not suitable for fast

• **Fourier transform-ion cyclotron resonance (FT-ICR)** is the third high resolution mass analyzer. The high sensitivity, accurate mass measurements, high mass resolution and MS/MS capabilities of FT-ICR make it attractive for structural determination of ions. However, the combined requirement of ultra-high vacuum system, superconducting magnets as well as sophisticated data system place the cost of these instruments beyond The non-selective nature of full mass scan acquisition enables detection of practically all ionizable metabolites and giving most complete information in terms of metabolite molecular mass. However, two major disadvantages arise by this approach. Firstly, detection sensitivity using QQQ decreases dramatically when wide mass range is scanned. This obstacle can be overcome by using IT analyzers as its full scan is much more sensitive or even better by using TOF instruments which additionally enable accurate mass determination [23]. In case when only QQQ is available, a practical approach may be applied to improve sensitivity; the whole mass range should be divided to narrow scanning ranges by performing multiple analyses of the same sample. Secondly, other non-metabolite matrix compound may interfere with obtained MS data. A common procedure for metabolite detection involves analysis of test and control samples what then allows subtraction of control sample data. This approach is less successful when complex biological samples, such as plasma and urine, are examined. Expected metabolites in studied samples may be predicted based on biotransformation pathways of parent drugs what enables focused search of these compounds. The most common changes in mass caused by biotransformation are shown in Table 1.

### *5.2.2. Precursor ion and constant neutral loss scan*

PI and CNL are more specific approaches for identification of unknown metabolites. This scan mode is only possible for tandem mass spectrometers and therefore suffers at sensitivity like other QQQ scanning acquisitions. In PI scan mode, the second quadrupole mass filter is set to pass only the selected product ions, while the first quadrupole mass filter scans a range of m/z values. In CNL scan mode, both quadrupoles are scanning m/z values while the m/z difference between the quadrupoles is kept constant. Several phase II metabolites at fragmentation lose a distinct neutral group that can be used for specific

identification of these conjugates. Glucuronides, sulfates and glutathione conjugates are often detected by CNL of m/z 176, 80 and 129, respectively. Typical PI for some drug conjugates in negative ionization mode like aliphatic sulfates, sulfonates and phosphates are m/z 97, 81 and 79, respectively [28]. Although PI and CNL provide high selectivity for identification of metabolites, the methods are based on predicted fragmentation behavior of metabolites what depends to some extent also on abilities of the analyst. Therefore, metabolites with unexpected fragmentation can be missed. Nevertheless, in combination with full scan data, PI and CNL is a powerful tool for metabolite identification.

Analytical Methods for Quantification of Drug Metabolites in Biological Samples 93

Although the use of PI and CNL data acquisition improves the selectivity of metabolite detection when comparing with full scan acquisition, all three approaches have reduced sensitivity. For this reason, specific MRM screening may serve as alternative approach for metabolite detection. MRM is the most appropriate acquisition method for quantification of analytes. In this mode, the first quadrupole is set to pass only the selected precursor ion that is fragmented in collision cell and usually the most abundant fragment (product ion) is then filtered in a second quadrupole. Monitoring of specific transition for each analyte yields a superior signal-to-noise ratio with significantly higher selectivity. Utilizing metabolism prediction and knowledge of the tandem mass fragmentation of the parent drug, the approach gives a significant increase in sensitivity and enables a wide range of potential MRM transitions to be targeted. Although the possibility to overlook metabolites remains the targeting MRM is a powerful alternative for metabolite detection when sensitivity is an issue. Single ion monitoring is is another option to overcome low sensitivity of QQQ screening techniques. SIM is less specific and sensitive acquisition compared to MRM but may provide advantages when the potential metabolite fragmentation pattern cannot be predicted correctly. In this case a multiple SIM transitions of the predicted metabolites are performed, which are set accordingly to the expected nominal mass changes regarding to

The most widespread analyzer providing high mass accuracy (TOF, Orbitrap, FT-ICR) used in metabolite identification is TOF instrument. The specificity in the detection of metabolites with high resolution is significantly higher than that with unit resolution QQQ or IT instruments where the ion chromatograms can be recorded using a 0.1 mass unit window. The high selectivity provides also better sensitivity for the detection of metabolites. It was reported that detection limits for several drugs were 5-25 times better with accurate mass TOF, than with nominal mass TOF (same unit level than at QQQ) [28]. Accurate mass measurements enable to determine the elemental formula of metabolites. Moreover, exact mass shift enables the establishment of the change in molecular formula of the parent drug. For example, metabolites formed by hydroxylation and dehydrogenation (at same time) are, in this way separated from those formed via methylation, in spite that both reaction increase the molecular weight by 14 (Table 1) [24]. The benefit of reliable accurate mass measurements for structural elucidation of unknown metabolites is therefore extremely high. However, metabolites with the same exact mass cannot be distinguished by analyzers. In this case other approaches are needed. Ion mobility time-of-flight mass spectrometry (IM-MS), which separates ions on the basis of their m/z ratios as well as their interactions with a buffer gas, is very convenient. The main advantage of IM-MS is the potential for separation of metabolite isomers without chromatographic separation which makes it a powerful

*5.2.4. Multiple reaction monitoring* 

parent drug (Table 1).

*5.2.5. High resolution mass spectrometry* 

analytical tool for investigation of complex samples [35].


**Table 1.** The nominal mass changes in biotransformation of drugs by common metabolic reactions [28, 34]

## *5.2.3. Product ion scan*

Product ion scan is used for structural characterization of the detected metabolites. In product ion mode, a precursor ion (metabolite) is selected in first quadrupole, fragmented in collision cell and the product ions are then scanned in second quadrupole. Structural information is obtained by interpretation of the fragmentation patterns for both metabolite and parent drug. Complete structural characterization of metabolites may be hindered by the absence of useful product ions in tandem mass spectrometry. To obtain more specific structural data, the use of multistage (MSn) scan by using ion trap instruments can be provided. The selected product ion can be selectively isolated and further fragmented, narrowing the potential sites of modification and providing a more complete assessment of the metabolite structure.

## *5.2.4. Multiple reaction monitoring*

92 Chromatography – The Most Versatile Method of Chemical Analysis

identification of these conjugates. Glucuronides, sulfates and glutathione conjugates are often detected by CNL of m/z 176, 80 and 129, respectively. Typical PI for some drug conjugates in negative ionization mode like aliphatic sulfates, sulfonates and phosphates are m/z 97, 81 and 79, respectively [28]. Although PI and CNL provide high selectivity for identification of metabolites, the methods are based on predicted fragmentation behavior of metabolites what depends to some extent also on abilities of the analyst. Therefore, metabolites with unexpected fragmentation can be missed. Nevertheless, in combination

Biotransformation Change in molecular formula Change in mass (Da)

Dehydration - H2O -18 Demethylation - CH2 -14 Dehydrogenation - H2 -2 Hydrogenation + H2 +2 Methylation + CH2 +14 Hydroxylation + O +16 Epoxidation + O +16 S/N-oxidation + O +16 Hydration + H2O +18 Dihydroxylation + O2 +32 Acetylation +C2H2O +42 Sulfation +SO3 +80 Glucuronidation +C6H8O6 +176

Glutathione conjugation +C10H15O6N3S +305

modification and providing a more complete assessment of the metabolite structure.

*5.2.3. Product ion scan* 

**Table 1.** The nominal mass changes in biotransformation of drugs by common metabolic reactions [28, 34]

Product ion scan is used for structural characterization of the detected metabolites. In product ion mode, a precursor ion (metabolite) is selected in first quadrupole, fragmented in collision cell and the product ions are then scanned in second quadrupole. Structural information is obtained by interpretation of the fragmentation patterns for both metabolite and parent drug. Complete structural characterization of metabolites may be hindered by the absence of useful product ions in tandem mass spectrometry. To obtain more specific structural data, the use of multistage (MSn) scan by using ion trap instruments can be provided. The selected product ion can be selectively isolated and further fragmented, narrowing the potential sites of

with full scan data, PI and CNL is a powerful tool for metabolite identification.

Although the use of PI and CNL data acquisition improves the selectivity of metabolite detection when comparing with full scan acquisition, all three approaches have reduced sensitivity. For this reason, specific MRM screening may serve as alternative approach for metabolite detection. MRM is the most appropriate acquisition method for quantification of analytes. In this mode, the first quadrupole is set to pass only the selected precursor ion that is fragmented in collision cell and usually the most abundant fragment (product ion) is then filtered in a second quadrupole. Monitoring of specific transition for each analyte yields a superior signal-to-noise ratio with significantly higher selectivity. Utilizing metabolism prediction and knowledge of the tandem mass fragmentation of the parent drug, the approach gives a significant increase in sensitivity and enables a wide range of potential MRM transitions to be targeted. Although the possibility to overlook metabolites remains the targeting MRM is a powerful alternative for metabolite detection when sensitivity is an issue. Single ion monitoring is is another option to overcome low sensitivity of QQQ screening techniques. SIM is less specific and sensitive acquisition compared to MRM but may provide advantages when the potential metabolite fragmentation pattern cannot be predicted correctly. In this case a multiple SIM transitions of the predicted metabolites are performed, which are set accordingly to the expected nominal mass changes regarding to parent drug (Table 1).

#### *5.2.5. High resolution mass spectrometry*

The most widespread analyzer providing high mass accuracy (TOF, Orbitrap, FT-ICR) used in metabolite identification is TOF instrument. The specificity in the detection of metabolites with high resolution is significantly higher than that with unit resolution QQQ or IT instruments where the ion chromatograms can be recorded using a 0.1 mass unit window. The high selectivity provides also better sensitivity for the detection of metabolites. It was reported that detection limits for several drugs were 5-25 times better with accurate mass TOF, than with nominal mass TOF (same unit level than at QQQ) [28]. Accurate mass measurements enable to determine the elemental formula of metabolites. Moreover, exact mass shift enables the establishment of the change in molecular formula of the parent drug. For example, metabolites formed by hydroxylation and dehydrogenation (at same time) are, in this way separated from those formed via methylation, in spite that both reaction increase the molecular weight by 14 (Table 1) [24]. The benefit of reliable accurate mass measurements for structural elucidation of unknown metabolites is therefore extremely high. However, metabolites with the same exact mass cannot be distinguished by analyzers. In this case other approaches are needed. Ion mobility time-of-flight mass spectrometry (IM-MS), which separates ions on the basis of their m/z ratios as well as their interactions with a buffer gas, is very convenient. The main advantage of IM-MS is the potential for separation of metabolite isomers without chromatographic separation which makes it a powerful analytical tool for investigation of complex samples [35].

Q-TOFs are the key high resolution instruments for drug metabolism research. Q-TOF instruments provide sufficient mass resolution (up to 40,000) and accurate mass measurements (below 1 ppm). In addition, they can operate at relatively high scanning rates, which are considered as the main drawback of most of the Orbitrap based instruments. On the other hand, Orbitrap analyzers provide a resolving power of up to 100,000 with mass accuracy below 1 ppm. FT-ICR analyzers provide ultrahigh mass resolving power greater than 200,000 but high purchasing and maintenance cost are beyond financial capabilities of most routine laboratories [27].

Analytical Methods for Quantification of Drug Metabolites in Biological Samples 95

Another important point had to be considered, good chromatography was needed in order to separate both monoglucuronides since they cannot be distinguished based on MS. Representative LC-MS/MS chromatogram, using MRM acquisitions for quantitative purposes

Identification of bisphenol A glucuronide and deuterated bisphenol A glucuronide in microsomal incubations [4] is another example. Twin peaks of metabolites with known mass difference [14] (Da in this case) are helpful for studying fragmentation paths. Product ion scan in ESI negative ionization mode for bisphenol A glucuronide (m/z 403) showed fragments m/z 227 (bisphenol A), 212 (bisphenol A fragment - loss of CH3) and 113. The molecular ion of deuterated bisphenol A glucuronide fragments from m/z 417 to m/z 241 (deuterated bisphenol A), 223 (fragment - additional loss of CD3) and 113. Fragment m/z 113, which is present in both cases represent a glucuronic acid fragment in negative

In case of reactive metabolite studies there are typical approaches to identify glutathione conjugates: increased mass shift 305 Da according to the parent, constant neutral loss of pyroglutamic acid (m/z 129) in the positive ionization mode and/or precursor ion of m/z 272 in the negative ionization mode [34, 38]. Recently, an *in vitro* bioactivation study using these identification approaches has confirmed that bazedoxifene does not show the formation of glutathione conjugates compared to raloxifene what offers an improved safety profile of this

The glucuronide metabolites may be also simply verified by using ß-glucuronidase which provides the conversion of the glucuronide to its aglycone (see next section). If the conversion is complete, this approach is valid for determination of the metabolite stock solution concentrations when small amounts of glucuronide standards are obtained or

However, for more demanding application QQQ is usually not satisfactory. Identification of phase I and phase II metabolites of two antineoplastic agents was demonstrated by use of Q-TOF [40]. In this study, 32 metabolites for dimefluron and 28 metabolites for benfluron were detected in the rat urine within 25 min chromatographic run. The identification of individual biotransformation was performed using high mass accuracy measurements for both full scan and tandem mass spectra by extracted ion chromatograms for expected masses of metabolites together with the information about characteristic neutral loss. Another study compared QQQ, linear IT (QTrap), TOF and Orbitrap instruments for identification of microsomal metabolites of verapamil and amitriptyline [41]. Only TOF found all 28 amitriptyline and 69 verapamil metabolites; both expected and unexpected. The TOF offered sensitivity and high mass resolution and also lowest overall time consumption together with the Orbitrap. Orbitrap also showed good mass resolution but was less sensitive, resulting in some metabolites not being observed. Approaches with QQQ and Q-Trap provided the highest amount of fragment ion data for structural elucidation, but being unable to produce very high important accurate mass data, they suffered from many false

negatives and especially with the QQQ from very high overall time consumption.

of raloxifene and its three metabolites in urine sample is shown in Figure 1.

ionization with subsequent loss of H2O and CO2.

available [37].

third generation drug relative to other available SERMs [39].

## *5.2.6. Other approaches*

Other approaches can be applied to provide specific structural information in cases when MS data are not sufficient to determine metabolite structure. Hydrogen/deuterium exchange LC-MS allows studying mechanisms of MS fragment ion formation and metabolic pathways of drugs, as well as differentiate the structures of isomeric metabolites [7]. Metabolites can be isolated and purified from the incubations, followed by structural analysis by NMR. Alternatively, LC-NMR analysis can be performed on biological samples with minimal sample processing but certain limitations occur with this technique, such as lower sensitivity compared with LC-MS and the requirements of relatively expensive deuterated buffers in mobile phase. More recently, LC-NMR has been coupled with MS which enables simultaneous metabolite structure elucidation [25]. Tentatively identified structure of metabolites may also be synthesized and LC-MS data for these compounds are compared with data from the actual metabolites.

## **5.3. Examples of metabolite identification**

Tandem mass spectrometry is well suited for identification of phase II metabolites [36]. As example for this approach, the elucidation of three raloxifene glucuronides in urine as well as their identity confirmation after bioproduction by using QQQ is provided [37]. Chromatograms of each bioproduced glucuronide standard obtained in ESI positive full scan mode gave only one chromatographic peak where MS spectra of each peak showed strong molecular ions at m/z 650, 650 and 826 for two raloxifene monoglucuronides and diglucuronide, respectively. Nominal mass shift of 176, 176 and 2 x 176 Da compared to parent drug (m/z 474) is characteristic for the structure of monoglucuronide and diglucuronide metabolites (Table 1). Product ion scan showed the same mass spectra for both predicted monoglucuronides: fragmentation of the parent ion m/z 650 to 474 and 112. Product ion spectra confirmed also diglucuronide structure by two subsequent m/z 176 neutral losses from the parent molecular ion (m/z 826), giving fragments of monoglucuronide (m/z 650) and of parent raloxifene (m/z 474) as well as additional m/z 112 fragment of raloxifene (N-ethyl-piperidine). Additionally, constant neutral loss scan (m/z 176) and precursor ion scans (m/z 112 and 474) in urine sample have been performed. The analysis in all three cases gave three distinct peaks in chromatograms at retention times for the diglucuronide, and both monoglucuronides (data not shown, but same retention times as in Figure 1) confirming again the structure of metabolites. Another important point had to be considered, good chromatography was needed in order to separate both monoglucuronides since they cannot be distinguished based on MS. Representative LC-MS/MS chromatogram, using MRM acquisitions for quantitative purposes of raloxifene and its three metabolites in urine sample is shown in Figure 1.

94 Chromatography – The Most Versatile Method of Chemical Analysis

financial capabilities of most routine laboratories [27].

*5.2.6. Other approaches* 

with data from the actual metabolites.

**5.3. Examples of metabolite identification** 

Q-TOFs are the key high resolution instruments for drug metabolism research. Q-TOF instruments provide sufficient mass resolution (up to 40,000) and accurate mass measurements (below 1 ppm). In addition, they can operate at relatively high scanning rates, which are considered as the main drawback of most of the Orbitrap based instruments. On the other hand, Orbitrap analyzers provide a resolving power of up to 100,000 with mass accuracy below 1 ppm. FT-ICR analyzers provide ultrahigh mass resolving power greater than 200,000 but high purchasing and maintenance cost are beyond

Other approaches can be applied to provide specific structural information in cases when MS data are not sufficient to determine metabolite structure. Hydrogen/deuterium exchange LC-MS allows studying mechanisms of MS fragment ion formation and metabolic pathways of drugs, as well as differentiate the structures of isomeric metabolites [7]. Metabolites can be isolated and purified from the incubations, followed by structural analysis by NMR. Alternatively, LC-NMR analysis can be performed on biological samples with minimal sample processing but certain limitations occur with this technique, such as lower sensitivity compared with LC-MS and the requirements of relatively expensive deuterated buffers in mobile phase. More recently, LC-NMR has been coupled with MS which enables simultaneous metabolite structure elucidation [25]. Tentatively identified structure of metabolites may also be synthesized and LC-MS data for these compounds are compared

Tandem mass spectrometry is well suited for identification of phase II metabolites [36]. As example for this approach, the elucidation of three raloxifene glucuronides in urine as well as their identity confirmation after bioproduction by using QQQ is provided [37]. Chromatograms of each bioproduced glucuronide standard obtained in ESI positive full scan mode gave only one chromatographic peak where MS spectra of each peak showed strong molecular ions at m/z 650, 650 and 826 for two raloxifene monoglucuronides and diglucuronide, respectively. Nominal mass shift of 176, 176 and 2 x 176 Da compared to parent drug (m/z 474) is characteristic for the structure of monoglucuronide and diglucuronide metabolites (Table 1). Product ion scan showed the same mass spectra for both predicted monoglucuronides: fragmentation of the parent ion m/z 650 to 474 and 112. Product ion spectra confirmed also diglucuronide structure by two subsequent m/z 176 neutral losses from the parent molecular ion (m/z 826), giving fragments of monoglucuronide (m/z 650) and of parent raloxifene (m/z 474) as well as additional m/z 112 fragment of raloxifene (N-ethyl-piperidine). Additionally, constant neutral loss scan (m/z 176) and precursor ion scans (m/z 112 and 474) in urine sample have been performed. The analysis in all three cases gave three distinct peaks in chromatograms at retention times for the diglucuronide, and both monoglucuronides (data not shown, but same retention times as in Figure 1) confirming again the structure of metabolites. Identification of bisphenol A glucuronide and deuterated bisphenol A glucuronide in microsomal incubations [4] is another example. Twin peaks of metabolites with known mass difference [14] (Da in this case) are helpful for studying fragmentation paths. Product ion scan in ESI negative ionization mode for bisphenol A glucuronide (m/z 403) showed fragments m/z 227 (bisphenol A), 212 (bisphenol A fragment - loss of CH3) and 113. The molecular ion of deuterated bisphenol A glucuronide fragments from m/z 417 to m/z 241 (deuterated bisphenol A), 223 (fragment - additional loss of CD3) and 113. Fragment m/z 113, which is present in both cases represent a glucuronic acid fragment in negative ionization with subsequent loss of H2O and CO2.

In case of reactive metabolite studies there are typical approaches to identify glutathione conjugates: increased mass shift 305 Da according to the parent, constant neutral loss of pyroglutamic acid (m/z 129) in the positive ionization mode and/or precursor ion of m/z 272 in the negative ionization mode [34, 38]. Recently, an *in vitro* bioactivation study using these identification approaches has confirmed that bazedoxifene does not show the formation of glutathione conjugates compared to raloxifene what offers an improved safety profile of this third generation drug relative to other available SERMs [39].

The glucuronide metabolites may be also simply verified by using ß-glucuronidase which provides the conversion of the glucuronide to its aglycone (see next section). If the conversion is complete, this approach is valid for determination of the metabolite stock solution concentrations when small amounts of glucuronide standards are obtained or available [37].

However, for more demanding application QQQ is usually not satisfactory. Identification of phase I and phase II metabolites of two antineoplastic agents was demonstrated by use of Q-TOF [40]. In this study, 32 metabolites for dimefluron and 28 metabolites for benfluron were detected in the rat urine within 25 min chromatographic run. The identification of individual biotransformation was performed using high mass accuracy measurements for both full scan and tandem mass spectra by extracted ion chromatograms for expected masses of metabolites together with the information about characteristic neutral loss. Another study compared QQQ, linear IT (QTrap), TOF and Orbitrap instruments for identification of microsomal metabolites of verapamil and amitriptyline [41]. Only TOF found all 28 amitriptyline and 69 verapamil metabolites; both expected and unexpected. The TOF offered sensitivity and high mass resolution and also lowest overall time consumption together with the Orbitrap. Orbitrap also showed good mass resolution but was less sensitive, resulting in some metabolites not being observed. Approaches with QQQ and Q-Trap provided the highest amount of fragment ion data for structural elucidation, but being unable to produce very high important accurate mass data, they suffered from many false negatives and especially with the QQQ from very high overall time consumption.
