**4.3. Cytosolic fraction**

Cytosolic fraction is an *in vitro* model that has not been used very often so far. Like HLM, cytosol is produced by differential centrifugation of liver homogenate. Soluble enzymes of phase II such as NAT, GST, SULT, carboxylesterase, soluble epoxide hydrolase, diamine oxidase, xanthine oxidase and alcohol dehydrogenase are expressed in cytosolic fraction, but only first three are expressed at higher concentration. This *in vitro* model requires cofactors like acetyl coA, dithiothreitol and acetyl coA-regenerating system for NAT, PAPS for SULT, glutathione for GST activity [14-16, 18].

Analytical Methods for Quantification of Drug Metabolites in Biological Samples 85

concentration. On the other hand, the absence or low expression of most important phase I and phase II drug metabolizing enzymes limits the application of this *in vitro* model. Moreover, metabolites are not easily detected and it is difficult to investigate individual

Transgenic cell line is a cell line that recombinantly expresses human phase I and/or phase II enzymes. All important human CYPs and UGTs have been expressed in this way to overcome the limitations of liver cell lines. Cell lines may be transfected at high efficiency using protoplast fusion. The main advantages are the ease of culturing, high expression of CYP and UGT isoenzymes, possibility to study single enzyme reactions and the influence of one isoenzyme or a combination of a number of isoenzymes. This *in vitro* model can also be used in the study of metabolite structures, pharmacological elucidation and to assess drugdrug interactions. The main drawback is that only one or a few of isoenzymes are expressed, therefore the complete *in vivo* situation cannot be reflected. Moreover, transgenic cell lines

Hepatocytes are well-established, well-characterized and frequently used *in vitro* model in drug biotransformation research. This *in vitro* model could be employed for the evaluation of metabolic stability, metabolite profiling and identification, drug efficacy, hepatic proliferation, hepatotoxicity and drug-drug interactions. Phase I and phase II drug metabolism pathways can be studied by the use of primary hepatocytes and cultured hepatocytes. Like with microsomes interindividual variation can be observed with hepatocytes. This can be overcome by using mixture of hepatocytes from different donors. Cryopreservation of hepatocytes offers many advances in the experimentation, namely

Primary hepatocytes are obtained by collagenase perfusion of whole liver or a part of liver. This *in vitro* system has strong resemblance of *in vivo* situation due to heterogeneity of enzyme expression in human liver and preservation of drug metabolizing enzymes at *in vivo* levels. Another advantage of primary hepatocytes is the ease of use and high throughput. The important disadvantage is the drop of hepatocytes viability during incubation period (viable 2-4 hrs). Moreover, lack of liver non-hepatocyte cells which may be necessary for cofactor supply, lack of cell polarity, cell-cell and cell-matrix contacts limits the *in vivo*

After isolation, hepatocytes can be cultured in a monolayer in order to prolong the viability to 4 weeks. This characteristic in combination with the prolonged regulatory pathways allows the use of this *in vitro* model in studies of up-regulation or down-regulation of metabolic enzymes. However, cultured hepatocytes gradually lose viability and liver specific function. Many factors influence the morphology and functions of hepatocytes in

are more expensive than other enzyme-based technologies [14-16, 18].

activity of most phase I and phase II enzymes is retained.

enzymes due to their low expression level [14-16].

**4.6. Transgenic cell lines** 

**4.7. Hepatocytes** 

resemblance [14-18, 20].

The main advantage is the presence of only aforementioned enzymes at higher concentrations than in liver S9 fraction. The biotransformation by NAT, GST or SULT can be studied separately or in combination depending on the cofactors added. The main disadvantage is the absence of UGT and therefore glucuronidation cannot be studied by this model [14-16, 18].

## **4.4. S9 fractions**

S9 fraction contains both microsomal and cytosolic fractions and consequently expresses a wide range of metabolic enzymes – CYP, FMO, carboxylesterases, epoxide hydrolases, UGT, SULT, methyl transferases, acethyltransferases, GST and others. This *in vitro* model could be employed for metabolic, toxicity and mutagenicity studies. Similar to upper mentioned *in vitro* models the addition of cofactors is needed; NADPH or NRS for CYP, UDPGA for UGT, acetyl coA, dithiothreitol and acetyl coA-regenerating system for NAT, PAPS for SULT and glutathione for GST [14-16, 18, 20].

The main advantage over microsomes and cytosolic fraction is a more complete representation of the metabolic profile due to the presence of phase I and phase II enzymes. However, a disadvantage is the overall lower enzyme activity in the S9 fraction compared to microsomes and cytosol, which may leave some metabolites unnoticed [14-16, 18].

## **4.5. Cell lines**

This *in vitro* model is less popular than other described models due to dedifferentiated cellular characteristics and lack of complete expression of all families of metabolic enzymes. The sources of cell lines are primary tumors of liver parenchyma. Currently available cell lines are Hep G2, Hep 3B, SNU-398, SNU-449, SNU-182, SNU-475, BC2, PLC/PRE/5, C3A, SK-Hep-1 and among them Hep G2 cell line is most frequently used for biotransformation studies. The metabolic activity of liver cell lines is generally low compared to freshly isolated human hepatocytes. Metabolic activity of some metabolic enzymes is even not detected. The problem of low activity could be partly overcome by the pretreatment of cell lines by inducers of various metabolic enzymes. But still the induced activity is below the enzymatic activity in freshly isolated human hepatocytes. Liver cell lines require appropriate culture medium, whose composition significantly influences the metabolic activity. The described *in vitro* model is easy to culture and have stable enzyme concentration. On the other hand, the absence or low expression of most important phase I and phase II drug metabolizing enzymes limits the application of this *in vitro* model. Moreover, metabolites are not easily detected and it is difficult to investigate individual enzymes due to their low expression level [14-16].

## **4.6. Transgenic cell lines**

84 Chromatography – The Most Versatile Method of Chemical Analysis

for SULT, glutathione for GST activity [14-16, 18].

Cytosolic fraction is an *in vitro* model that has not been used very often so far. Like HLM, cytosol is produced by differential centrifugation of liver homogenate. Soluble enzymes of phase II such as NAT, GST, SULT, carboxylesterase, soluble epoxide hydrolase, diamine oxidase, xanthine oxidase and alcohol dehydrogenase are expressed in cytosolic fraction, but only first three are expressed at higher concentration. This *in vitro* model requires cofactors like acetyl coA, dithiothreitol and acetyl coA-regenerating system for NAT, PAPS

The main advantage is the presence of only aforementioned enzymes at higher concentrations than in liver S9 fraction. The biotransformation by NAT, GST or SULT can be studied separately or in combination depending on the cofactors added. The main disadvantage is the absence of UGT and therefore glucuronidation cannot be studied by this

S9 fraction contains both microsomal and cytosolic fractions and consequently expresses a wide range of metabolic enzymes – CYP, FMO, carboxylesterases, epoxide hydrolases, UGT, SULT, methyl transferases, acethyltransferases, GST and others. This *in vitro* model could be employed for metabolic, toxicity and mutagenicity studies. Similar to upper mentioned *in vitro* models the addition of cofactors is needed; NADPH or NRS for CYP, UDPGA for UGT, acetyl coA, dithiothreitol and acetyl coA-regenerating system for NAT, PAPS for SULT and

The main advantage over microsomes and cytosolic fraction is a more complete representation of the metabolic profile due to the presence of phase I and phase II enzymes. However, a disadvantage is the overall lower enzyme activity in the S9 fraction compared to

This *in vitro* model is less popular than other described models due to dedifferentiated cellular characteristics and lack of complete expression of all families of metabolic enzymes. The sources of cell lines are primary tumors of liver parenchyma. Currently available cell lines are Hep G2, Hep 3B, SNU-398, SNU-449, SNU-182, SNU-475, BC2, PLC/PRE/5, C3A, SK-Hep-1 and among them Hep G2 cell line is most frequently used for biotransformation studies. The metabolic activity of liver cell lines is generally low compared to freshly isolated human hepatocytes. Metabolic activity of some metabolic enzymes is even not detected. The problem of low activity could be partly overcome by the pretreatment of cell lines by inducers of various metabolic enzymes. But still the induced activity is below the enzymatic activity in freshly isolated human hepatocytes. Liver cell lines require appropriate culture medium, whose composition significantly influences the metabolic activity. The described *in vitro* model is easy to culture and have stable enzyme

microsomes and cytosol, which may leave some metabolites unnoticed [14-16, 18].

**4.3. Cytosolic fraction** 

model [14-16, 18].

**4.4. S9 fractions** 

**4.5. Cell lines** 

glutathione for GST [14-16, 18, 20].

Transgenic cell line is a cell line that recombinantly expresses human phase I and/or phase II enzymes. All important human CYPs and UGTs have been expressed in this way to overcome the limitations of liver cell lines. Cell lines may be transfected at high efficiency using protoplast fusion. The main advantages are the ease of culturing, high expression of CYP and UGT isoenzymes, possibility to study single enzyme reactions and the influence of one isoenzyme or a combination of a number of isoenzymes. This *in vitro* model can also be used in the study of metabolite structures, pharmacological elucidation and to assess drugdrug interactions. The main drawback is that only one or a few of isoenzymes are expressed, therefore the complete *in vivo* situation cannot be reflected. Moreover, transgenic cell lines are more expensive than other enzyme-based technologies [14-16, 18].

## **4.7. Hepatocytes**

Hepatocytes are well-established, well-characterized and frequently used *in vitro* model in drug biotransformation research. This *in vitro* model could be employed for the evaluation of metabolic stability, metabolite profiling and identification, drug efficacy, hepatic proliferation, hepatotoxicity and drug-drug interactions. Phase I and phase II drug metabolism pathways can be studied by the use of primary hepatocytes and cultured hepatocytes. Like with microsomes interindividual variation can be observed with hepatocytes. This can be overcome by using mixture of hepatocytes from different donors. Cryopreservation of hepatocytes offers many advances in the experimentation, namely activity of most phase I and phase II enzymes is retained.

Primary hepatocytes are obtained by collagenase perfusion of whole liver or a part of liver. This *in vitro* system has strong resemblance of *in vivo* situation due to heterogeneity of enzyme expression in human liver and preservation of drug metabolizing enzymes at *in vivo* levels. Another advantage of primary hepatocytes is the ease of use and high throughput. The important disadvantage is the drop of hepatocytes viability during incubation period (viable 2-4 hrs). Moreover, lack of liver non-hepatocyte cells which may be necessary for cofactor supply, lack of cell polarity, cell-cell and cell-matrix contacts limits the *in vivo* resemblance [14-18, 20].

After isolation, hepatocytes can be cultured in a monolayer in order to prolong the viability to 4 weeks. This characteristic in combination with the prolonged regulatory pathways allows the use of this *in vitro* model in studies of up-regulation or down-regulation of metabolic enzymes. However, cultured hepatocytes gradually lose viability and liver specific function. Many factors influence the morphology and functions of hepatocytes in

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

Analytical Methods for Quantification of Drug Metabolites in Biological Samples 87

drug metabolism between animals and humans as early as possible during the drug development process in order to find unique human metabolites and major metabolites [1, 21]. FDA defines that metabolites will need to undergo additional safety evaluation when steady-state systemic exposure to metabolite in humans exceeds 10% of parent drug

The results of aforementioned *in vitro* studies can be correlated to *in vivo* situation and vice versa. This multidisciplinary approach of translational medicine yields an insight into complex mechanisms of drug disposition. The principle of translational medicine is presented on raloxifene, a selective estrogen receptor modulator, which exhibits quite large and unexplained interindividual variability in pharmacokinetics and pharmacodynamics [2, 3, 19, 22]. The gained knowledge about drug pharmacokinetics and pharmacodynamics

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

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

insures a safer and more effective treatment strategy in the clinical setting.

metabolite identification using LC-MS/MS will be provided in this section.

exposure (disproportionate metabolite) [1].

**5. Qualitative evaluation of metabolites** 

**5.1. LC-MS instrumentation** 

*5.1.1. Ionization techniques* 

complementary.

## **4.8. Liver slices**

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- 16, 18, 20].

## **4.9. Isolated perfused liver**

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

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

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 drug metabolism between animals and humans as early as possible during the drug development process in order to find unique human metabolites and major metabolites [1, 21]. FDA defines that metabolites will need to undergo additional safety evaluation when steady-state systemic exposure to metabolite in humans exceeds 10% of parent drug exposure (disproportionate metabolite) [1].

The results of aforementioned *in vitro* studies can be correlated to *in vivo* situation and vice versa. This multidisciplinary approach of translational medicine yields an insight into complex mechanisms of drug disposition. The principle of translational medicine is presented on raloxifene, a selective estrogen receptor modulator, which exhibits quite large and unexplained interindividual variability in pharmacokinetics and pharmacodynamics [2, 3, 19, 22]. The gained knowledge about drug pharmacokinetics and pharmacodynamics insures a safer and more effective treatment strategy in the clinical setting.
