**6. Approaches for metabolite quantification**

The demand for analyses of low-level drugs in complex biological samples has increased significantly in last years. New pharmaceuticals have typically high potency, so small doses are given and therefore the detection limits of these drugs and their metabolites are of great importance. Selective and sensitive analytical methods for the quantitative evaluation of these analytes are critical for the successful conduction of pharmacological studies. Metabolite quantification is always required when the metabolite is toxic or pharmacologically active or when the concentration of metabolite reaches or exceeds the parent drug concentration in plasma. Different approaches for metabolite determination in biological samples have been used which can be generally divided to direct quantification, indirect quantification through parent drug after metabolite hydrolysis or quantification supported by using response factors between drug and their metabolites. The key role in the selection of the particular approach is driven by the availability of suitable authentic standards. Hence some examples of metabolites production will be also shown here.

Analytical Methods for Quantification of Drug Metabolites in Biological Samples 97

depends on the analyte even at small structural changes. It has been reported that the response in ESI-MS differed by factor 25 for two oxidative isomeric metabolites with same chemical formula [43] or that no signal in contrary to parent drug has been observed for metabolite in positive ionization ESI. Detection of metabolite was in this case possible only in negative ionization mode [26]. However, with the commonly and easily used UV detection, the metabolites have often the same chromophore as the parent drug (but not always [40], hence giving similar response. But the main limitation of this technique in pharmacokinetic studies lies in not sufficient sensitivity and also in lower selectivity as some compounds does not have UV absorption at a wavelength to distinct it from the background. In contrast to UV, fluorescence and electrochemical detection can be very selective and sensitive. For electrochemical detection the response may also be very dependent on structure, especially for phase I metabolites which usually possess changed oxido-reductive properties compared to parent drug [34]. Beside that both detector systems

Direct quantification can also be performed without suitable standards. For that purpose detectors need to give an equimolar response for all compounds of interest. Additionally, such detectors should be highly sensitive with wide dynamic range, robust and easy to use, compatible with reverse-phase gradient elution and not prone to matrix interferences, namely give a response independent of compound [44]. Although there are sophisticated detectors available, few are used routinely for metabolite quantification. Beside radioactivity detector (RAD) which also require suitable standards (radiolabeled compounds) other compound response independent detectors has been recently discussed elsewhere [25, 44- 46]. Such approach has become even more important for metabolite evaluation in the light of recently introduced FDA guidelines on metabolites in safety testing, which recommends that all metabolites greater than 10 percent of parent drug should be examined [1]. Some further examples of metabolite quantification using accelerator MS [47], inductively coupled plasma MS [43], chemiluminescene nitrogen detector [48], quantitative NMR [49] and

Prerequisite to make this approach successful is the chromatographic separation of drug and all metabolites. Quantification is based on using LC-MS/MS in combination with detector that gives an equimolar response independent of the compound, usually with RAD. Response ratio of the metabolite to parent drug on RAD is then correlated to response ratio on LC-MS/MS. Low amounts of metabolites and parent drug in samples are measured by sufficiently sensitive LC-MS/MS, where the analysis of higher amounts allows detection on RAD and due to response factor enables calculating of metabolite concentration. The best way to perform analyses is to combine RAD with MS after liquid chromatography with splitting flow in order to obtain peaks of the metabolites and parent at the same retention times on both detectors [51]. RAD is convenient for such analyses because of the large dynamic range but its use is limited by the availability of radiolabeled standards. However,

are very specific what makes them of limited applicability.

evaporative light-scattering detector [50] are given.

**6.2. Quantification using response factor** 

## **6.1. Direct quantification**

Direct quantification is the most appropriate approach for metabolite determination in biological matrices but two major points need to be considered. Firstly, in general metabolites are much more hydrophilic than parent drug, especially glucuronides [34]. That fact has represented a hindrance for direct metabolite determination because chromatographic separation between these polar analytes and interfering matrix components could not be achieved in many cases. However, this problem has been overcome by advent of powerful liquid chromatography-tandem mass spectrometry instruments which allow direct quantification of these metabolites [42]. LC-MS/MS nowadays play predominant role in bioassays for pharmacokinetic and metabolism studies due to its inherent specificity, sensitivity and speed. Secondly, appropriate authentic standards are needed for reliable and accurate quantification in biological samples. Proper validation of analytical methods includes preparation of calibration and control samples in given biological matrices using suitable reference standards. Authentic metabolite standards are often not commercially available, particularly in the case of new drugs or drugs of abuse. Moreover, available metabolites may be very expensive and therefore not accessible for every research group, especially not in academic sphere. Furthermore, stable isotope labeled standards of metabolites, which are most convenient internal standards for LC-MS/MS analyses, are even less available and/or more expensive than unlabeled metabolites.

In such situation question may arise why not quantitate metabolites concentration based on parent drug calibration curve as this standard are freely accessible. Modified structure of metabolites may change the response to quite diverse extent among various liquid chromatography detection systems. Mass spectrometry using atmospheric pressure ionization sources is very prone to this issue as the intensity of the MS signal strongly depends on the analyte even at small structural changes. It has been reported that the response in ESI-MS differed by factor 25 for two oxidative isomeric metabolites with same chemical formula [43] or that no signal in contrary to parent drug has been observed for metabolite in positive ionization ESI. Detection of metabolite was in this case possible only in negative ionization mode [26]. However, with the commonly and easily used UV detection, the metabolites have often the same chromophore as the parent drug (but not always [40], hence giving similar response. But the main limitation of this technique in pharmacokinetic studies lies in not sufficient sensitivity and also in lower selectivity as some compounds does not have UV absorption at a wavelength to distinct it from the background. In contrast to UV, fluorescence and electrochemical detection can be very selective and sensitive. For electrochemical detection the response may also be very dependent on structure, especially for phase I metabolites which usually possess changed oxido-reductive properties compared to parent drug [34]. Beside that both detector systems are very specific what makes them of limited applicability.

Direct quantification can also be performed without suitable standards. For that purpose detectors need to give an equimolar response for all compounds of interest. Additionally, such detectors should be highly sensitive with wide dynamic range, robust and easy to use, compatible with reverse-phase gradient elution and not prone to matrix interferences, namely give a response independent of compound [44]. Although there are sophisticated detectors available, few are used routinely for metabolite quantification. Beside radioactivity detector (RAD) which also require suitable standards (radiolabeled compounds) other compound response independent detectors has been recently discussed elsewhere [25, 44- 46]. Such approach has become even more important for metabolite evaluation in the light of recently introduced FDA guidelines on metabolites in safety testing, which recommends that all metabolites greater than 10 percent of parent drug should be examined [1]. Some further examples of metabolite quantification using accelerator MS [47], inductively coupled plasma MS [43], chemiluminescene nitrogen detector [48], quantitative NMR [49] and evaporative light-scattering detector [50] are given.

### **6.2. Quantification using response factor**

96 Chromatography – The Most Versatile Method of Chemical Analysis

shown here.

**6.1. Direct quantification** 

than unlabeled metabolites.

**6. Approaches for metabolite quantification** 

The demand for analyses of low-level drugs in complex biological samples has increased significantly in last years. New pharmaceuticals have typically high potency, so small doses are given and therefore the detection limits of these drugs and their metabolites are of great importance. Selective and sensitive analytical methods for the quantitative evaluation of these analytes are critical for the successful conduction of pharmacological studies. Metabolite quantification is always required when the metabolite is toxic or pharmacologically active or when the concentration of metabolite reaches or exceeds the parent drug concentration in plasma. Different approaches for metabolite determination in biological samples have been used which can be generally divided to direct quantification, indirect quantification through parent drug after metabolite hydrolysis or quantification supported by using response factors between drug and their metabolites. The key role in the selection of the particular approach is driven by the availability of suitable authentic standards. Hence some examples of metabolites production will be also

Direct quantification is the most appropriate approach for metabolite determination in biological matrices but two major points need to be considered. Firstly, in general metabolites are much more hydrophilic than parent drug, especially glucuronides [34]. That fact has represented a hindrance for direct metabolite determination because chromatographic separation between these polar analytes and interfering matrix components could not be achieved in many cases. However, this problem has been overcome by advent of powerful liquid chromatography-tandem mass spectrometry instruments which allow direct quantification of these metabolites [42]. LC-MS/MS nowadays play predominant role in bioassays for pharmacokinetic and metabolism studies due to its inherent specificity, sensitivity and speed. Secondly, appropriate authentic standards are needed for reliable and accurate quantification in biological samples. Proper validation of analytical methods includes preparation of calibration and control samples in given biological matrices using suitable reference standards. Authentic metabolite standards are often not commercially available, particularly in the case of new drugs or drugs of abuse. Moreover, available metabolites may be very expensive and therefore not accessible for every research group, especially not in academic sphere. Furthermore, stable isotope labeled standards of metabolites, which are most convenient internal standards for LC-MS/MS analyses, are even less available and/or more expensive

In such situation question may arise why not quantitate metabolites concentration based on parent drug calibration curve as this standard are freely accessible. Modified structure of metabolites may change the response to quite diverse extent among various liquid chromatography detection systems. Mass spectrometry using atmospheric pressure ionization sources is very prone to this issue as the intensity of the MS signal strongly Prerequisite to make this approach successful is the chromatographic separation of drug and all metabolites. Quantification is based on using LC-MS/MS in combination with detector that gives an equimolar response independent of the compound, usually with RAD. Response ratio of the metabolite to parent drug on RAD is then correlated to response ratio on LC-MS/MS. Low amounts of metabolites and parent drug in samples are measured by sufficiently sensitive LC-MS/MS, where the analysis of higher amounts allows detection on RAD and due to response factor enables calculating of metabolite concentration. The best way to perform analyses is to combine RAD with MS after liquid chromatography with splitting flow in order to obtain peaks of the metabolites and parent at the same retention times on both detectors [51]. RAD is convenient for such analyses because of the large dynamic range but its use is limited by the availability of radiolabeled standards. However,

the most straightforward detection technique generally found with LC-MS/MS is UV detection. Metabolites can be (semi)quantified using UV response ratio in cases when the parent drug chromophore offers sufficient selectivity, is not altered by metabolism and the metabolites are well separated from other drug related entities and endogenous compounds [46].

Analytical Methods for Quantification of Drug Metabolites in Biological Samples 99

conditions but quartenary ammonium glucuronides under basic conditions [25]. Additionally, enzymatic hydrolysis of acyl glucuronides may be hindered due to acyl migration what leads to ß-glucuronidase resistant derivates [59]. Nevertheless, if the ßglucuronidase treatment is successful for the metabolite of interest, this procedure should be

Another aspect for quantification using this approach has been shown recently [60]. Different benzodiazepines were determined via their metabolites by using acid hydrolysis of urine samples. The parent drug and all metabolites, conjugated as well as non-conjugated (I phase metabolites), were converted to corresponding benzophenone under studied conditions. Such approach reduces the specificity but at same time the overall sensitivity of

Alternative approach for direct quantification is to obtain authentic metabolite standards. The chemical synthesis is mainly suitable for achieving phase I metabolites, like Odemetylation, N-demetylation, N-oxidation, carbonyl reduction and other. However, synthesis of the phase II metabolites can be cumbersome and stereochemically demanding and hence go beyond possibilities of most laboratories [34]. Versatile alternative to chemical synthesis is enzyme-assisted *in vitro* production of these metabolites using liver homogenates, liver microscale cultures, cell culture lines or microbial systems where each of these methods has its specific drawbacks [61, 62]. Raloxifene, which is metabolized to two distinct monoglucuronides and one diglucuronide, is an illustrative example for in-house production of authentic standards. Glucuronide yield by chemical synthesis was very low and not sufficient enough to characterize those metabolites. On contrary, the biosynthesis with recombinant human UGT enzymes turned out to be successful in converting parent drug to both monoglucuronides [63]. In last attempt the bioproduction of all three metabolites could be accomplished by using the microorganism *Streptomyces sp* [37]*.* For more detail about raloxifene *in vitro* metabolism refer to [19]. Availability of both metabolite standards – unlabeled and stable isotope-labeled internal standards is even more important for reliable quantification using LC-MS/MS. Stable isotope labeled metabolites can be obtained by microsomal incubation of labeled drug, of course if it is available and not too expensive [4]. The alternative approach is to use a labeled UDP-glucuronic acid as cofactor

Moreover, metabolites can be isolated from urine after oral administration and after purification and characterization they can be used as standards. Bisphenol A glucuronide and its deuterated glucuronide were isolated from rat urine [64]. Recently published work dealing with microsomal bioproduction of the same metabolites [4] revealed some drawbacks of the isolation approach. Beside ethical considerations, the yield of both standards was much lower from animal samples (microgram scale) than microsomal incubates (milligram scale). Additionally, urine as matrix requires also more extensive purification procedure in order to obtain highly pure standards. However, in cases where

the method increases, which makes such method suitable for drug abuse monitoring.

the method of choice.

**6.4. Metabolite production** 

in bioproduction of metabolites [62].

This approach may be also reasonable to quantify metabolites in case of limited amounts of authentic standards. After determination of the response factors, metabolites could be then quantified based on calibration curve of parent drug [19]. A constant response factor is absolutely essential and therefore in such cases response factors should be periodically verified. Using the same instrument and without major instrument breakdowns, the response factor seems to be very stable over long periods [52].

## **6.3. Indirect quantification**

Refer to evaluation of glucuronides and other phase II metabolites. These metabolites are determined by cleavage of conjugates to yield the parent drug, which is subsequently detected. This indirect approach has several limitations, including the risk of incomplete hydrolysis, moderate repeatability and time consuming sample preparation [42]. Another drawback is non-selectivity of this procedure toward study of particular metabolite of interest when distinct drug metabolite conjugates are present in sample, like in case of morphine which is transformed to two isomeric metabolites. Morphine-3-glucuronide is an inactive metabolite but morphine-6-glucuronide possesses even greater pharmacological activity than the parent drug [53]. In such cases this approach is not suitable in pharmacokinetic studies as the overall drug concentration including more metabolites is determined in examined biological fluid. However, in the field of toxicology, doping control or drugs of abuse this information may be even more valuable [54, 55]. Nevertheless, direct quantification of metabolites and their indirect quantification via parent drug after metabolite hydrolysis may give comparable results like in case of buprenorphine metabolites [56].

Cleavage of conjugates can be performed by fast chemical hydrolysis or by gentle but time consuming enzyme hydrolysis. Deconjugation by ß-glucuronidase is the predominantly used approach. Different types of enzymes are commercially available but the most frequently used are ß-glucuronidases from *E. coli* or *Helix pomatia*, sometimes combined with arylsulfatase. ß-glucuronidase from *Helix pomatia* provides the advantage of the cleavage of glucuronide and sulfatate conjugates at same time what is important in the field of toxicology [6]. However, the glucuronidase activity is not as high as at *E. coli.* In order to achieve a successful enzyme hydrolysis it is crucial to pay attention on several factors, such as temperature, pH, enzyme origin, enzyme concentration and incubation time [57]. However, cleavage with ß-glucuronidase is not always preferential as for acyl glucuronides (ester conjugates) where alkaline hydrolysis is more suitable [55]. Acid hydrolysis may also be sometimes the better possibility for other glucuronide types [58]. N-glucuronides (primary, secondary and N-hydroxylated amines) are hydrolyzed under mild acidic conditions but quartenary ammonium glucuronides under basic conditions [25]. Additionally, enzymatic hydrolysis of acyl glucuronides may be hindered due to acyl migration what leads to ß-glucuronidase resistant derivates [59]. Nevertheless, if the ßglucuronidase treatment is successful for the metabolite of interest, this procedure should be the method of choice.

Another aspect for quantification using this approach has been shown recently [60]. Different benzodiazepines were determined via their metabolites by using acid hydrolysis of urine samples. The parent drug and all metabolites, conjugated as well as non-conjugated (I phase metabolites), were converted to corresponding benzophenone under studied conditions. Such approach reduces the specificity but at same time the overall sensitivity of the method increases, which makes such method suitable for drug abuse monitoring.

## **6.4. Metabolite production**

98 Chromatography – The Most Versatile Method of Chemical Analysis

response factor seems to be very stable over long periods [52].

**6.3. Indirect quantification** 

metabolites [56].

[46].

the most straightforward detection technique generally found with LC-MS/MS is UV detection. Metabolites can be (semi)quantified using UV response ratio in cases when the parent drug chromophore offers sufficient selectivity, is not altered by metabolism and the metabolites are well separated from other drug related entities and endogenous compounds

This approach may be also reasonable to quantify metabolites in case of limited amounts of authentic standards. After determination of the response factors, metabolites could be then quantified based on calibration curve of parent drug [19]. A constant response factor is absolutely essential and therefore in such cases response factors should be periodically verified. Using the same instrument and without major instrument breakdowns, the

Refer to evaluation of glucuronides and other phase II metabolites. These metabolites are determined by cleavage of conjugates to yield the parent drug, which is subsequently detected. This indirect approach has several limitations, including the risk of incomplete hydrolysis, moderate repeatability and time consuming sample preparation [42]. Another drawback is non-selectivity of this procedure toward study of particular metabolite of interest when distinct drug metabolite conjugates are present in sample, like in case of morphine which is transformed to two isomeric metabolites. Morphine-3-glucuronide is an inactive metabolite but morphine-6-glucuronide possesses even greater pharmacological activity than the parent drug [53]. In such cases this approach is not suitable in pharmacokinetic studies as the overall drug concentration including more metabolites is determined in examined biological fluid. However, in the field of toxicology, doping control or drugs of abuse this information may be even more valuable [54, 55]. Nevertheless, direct quantification of metabolites and their indirect quantification via parent drug after metabolite hydrolysis may give comparable results like in case of buprenorphine

Cleavage of conjugates can be performed by fast chemical hydrolysis or by gentle but time consuming enzyme hydrolysis. Deconjugation by ß-glucuronidase is the predominantly used approach. Different types of enzymes are commercially available but the most frequently used are ß-glucuronidases from *E. coli* or *Helix pomatia*, sometimes combined with arylsulfatase. ß-glucuronidase from *Helix pomatia* provides the advantage of the cleavage of glucuronide and sulfatate conjugates at same time what is important in the field of toxicology [6]. However, the glucuronidase activity is not as high as at *E. coli.* In order to achieve a successful enzyme hydrolysis it is crucial to pay attention on several factors, such as temperature, pH, enzyme origin, enzyme concentration and incubation time [57]. However, cleavage with ß-glucuronidase is not always preferential as for acyl glucuronides (ester conjugates) where alkaline hydrolysis is more suitable [55]. Acid hydrolysis may also be sometimes the better possibility for other glucuronide types [58]. N-glucuronides (primary, secondary and N-hydroxylated amines) are hydrolyzed under mild acidic Alternative approach for direct quantification is to obtain authentic metabolite standards. The chemical synthesis is mainly suitable for achieving phase I metabolites, like Odemetylation, N-demetylation, N-oxidation, carbonyl reduction and other. However, synthesis of the phase II metabolites can be cumbersome and stereochemically demanding and hence go beyond possibilities of most laboratories [34]. Versatile alternative to chemical synthesis is enzyme-assisted *in vitro* production of these metabolites using liver homogenates, liver microscale cultures, cell culture lines or microbial systems where each of these methods has its specific drawbacks [61, 62]. Raloxifene, which is metabolized to two distinct monoglucuronides and one diglucuronide, is an illustrative example for in-house production of authentic standards. Glucuronide yield by chemical synthesis was very low and not sufficient enough to characterize those metabolites. On contrary, the biosynthesis with recombinant human UGT enzymes turned out to be successful in converting parent drug to both monoglucuronides [63]. In last attempt the bioproduction of all three metabolites could be accomplished by using the microorganism *Streptomyces sp* [37]*.* For more detail about raloxifene *in vitro* metabolism refer to [19]. Availability of both metabolite standards – unlabeled and stable isotope-labeled internal standards is even more important for reliable quantification using LC-MS/MS. Stable isotope labeled metabolites can be obtained by microsomal incubation of labeled drug, of course if it is available and not too expensive [4]. The alternative approach is to use a labeled UDP-glucuronic acid as cofactor in bioproduction of metabolites [62].

Moreover, metabolites can be isolated from urine after oral administration and after purification and characterization they can be used as standards. Bisphenol A glucuronide and its deuterated glucuronide were isolated from rat urine [64]. Recently published work dealing with microsomal bioproduction of the same metabolites [4] revealed some drawbacks of the isolation approach. Beside ethical considerations, the yield of both standards was much lower from animal samples (microgram scale) than microsomal incubates (milligram scale). Additionally, urine as matrix requires also more extensive purification procedure in order to obtain highly pure standards. However, in cases where metabolites cannot be produced by proposed *in vitro* models the *in vivo* biological samples are then the only media for isolation of those metabolites.

Analytical Methods for Quantification of Drug Metabolites in Biological Samples 101

To obtain a sensitive analysis for a complex biological media (plasma, urine) liquid-liquid extraction (LLE) or solid phase extraction (SPE) are often required instead of PP. LLE sometimes gives better sample clean up showing less matrix effect in comparison with SPE [70]. Additionally, LLE is in general simpler and may be applicable to almost all laboratories using large variety of available solvents. LLE is also less expensive and flexible as several samples may be prepared in parallels. On the other hand emulsion formation, mutual solubility of analytes in both phases or large volumes of flammable and/or toxic solvents should be considered. In recently published comprehensive overview of methods for measurement of antidepressants and their metabolites in biofluids, many examples of extraction including LLE conditions can be found [67]. Offline methodologies are often very tedious and time consuming, and the risk of sample loss and/or contamination is high. Lack of automation possibilities is therefore another important LLE drawback. However, several research groups have developed different approaches to solve mixing and phase separation problems typically seen in a 96-well plate LLE method [71]. A semi automated LLE procedure using 96-well plates was

SPE has become very popular and is nowadays considered as a basic technique in many laboratories for sample preparation of drugs and their metabolites from biological matrices. SPE offers several advantages over LLE, including higher recoveries, no problems with emulsions, less solvent consumption and a smaller sample volume requirement. Moreover, automation of sample treatment with high speed and feasibility for treatment of numerous samples at one time is possible. However, a drawback often associated with SPE is their high dead volume, which can lead to loss of sample and may cause dilution of applied samples. SPE column lot production variability or column blockage due to sample viscosity or precipitation may also occur. Columns can be supplied as individual units for manual use and also in 96-well plate format for use with robotic sample processors. The column dead volume has been overcome with a novel 96 well SPE plate that was designed to minimize elution volume (< 25µL). The evaporation and reconstitution step that is usually required in SPE is avoided due the concentration

SPE is based on chromatographic separation such as liquid chromatography. Wide variety of cartridge types and solvents make SPE procedure suitable for many polar or nonpolar analytes. The extraction procedure can be a generic protocol or can be optimized if better sample clean up is desired. Beside classical reverse phase (e.g. C8 or C18) also polymer reverse phase (e.g. divinylbenzene, N-vinylbenzene), polymer ionexchange (e.g. weak or strong anion/cation-exchange) or mixed mode ion-exchange sorbents are available. Polymeric reverse phase materials possess both hydrophilic and lipophilic properties and are capable of capturing polar analytes such as drug

**7.2. Liquid-liquid extraction** 

reported [72].

**7.3. Solid-phase extraction** 

ability of the sorbent [71].
