**9.1. Matrix effect**

110 Chromatography – The Most Versatile Method of Chemical Analysis

low concentrations are expected in such studies [4].

drugs and their metabolites in biological samples [95-97].

**8.5. Other detection techniques** 

mass spectrometry.

in the positive ionization mode in mobile phase containing acetonitrile with formic acid and/or ammonium acetate. But replacement of acetonitrile with methanol in mobile phase gave approximately 25-fold higher response. On the other hand, for the same analyte, mobile phases containing acetonitrile or methanol gave about the same response in negative ionization [79]. Another interesting example is analysis of bisphenol A and its metabolite in biological samples. In order to gain the highest possible sensitivity for bisphenol A and bisphenol A glucuronide, LC-MS/MS conditions were optimized. ESI ionization source operating in negative ionization mode was selected for further optimization of mobile phase. It was found that substitution of acetonitrile/water with methanol/water as mobile phase increased response of parent by approximately two-fold but at the same time decreased response of its metabolite by approximately three-fold. Acetonitrile was selected as organic modifier because metabolite quantification is the main concern of metabolism studies. Additionally, sufficiently high sensitivity is needed for metabolite determination as

UV, fluorescence or electrochemical detectors are usually coupled with liquid chromatograph for determination of drugs and their metabolites. Total analysis time of these methods is often long because baseline chromatographic separation is required for quantification purposes. In terms of reproducibility and robustness, UV and fluorescence detection have an advantage over mass spectrometry. However, methods are less sensitive and specific what requires extensive and time-consuming sample preparation compared to

Before the advent of mass spectrometry, UV was the primary detection technique used in pharmacokinetics for quantification of drugs and their metabolites in biological matrices. Although robust, reliable, simple and easy to use, UV detection provides relatively poor sensitivity, especially when the compound of interest has no significant chromophore [44]. However, HPLC coupled with UV detection is still widely applicable for determination of

In contrast to UV, fluorescence or electrochemical detection can be a very selective and sensitive detection technique. These detectors can extend the sensitivity by 1-3 orders of magnitude if the analyte exhibits, or can be readily derivatized to exhibit, fluorescence or electroactivity [7]. Some drugs such as morphine have good fluorophores which allows its detection without derivatization. For direct determination of morphine and its two glucuronides assays based on liquid chromatography with different detector systems (UV, fluorescence, electrochemical, MS) has been reported. Limits of quantifications for both metabolites were comparable for MS and fluorescence detection but were as expected higher for UV detection [98]. Oxidation vulnerability and native fluorescence properties of most biogenic amines may explain the long history of their quantification by these conventional HPLC detection methods. However, LC-MS/MS methods are rapidly emerging due to its Matrix effect (ME) is a term that describes any changes in the MS response of analyte that can lead to either a reduced response (ion suppression) or an increased response (ion enhancement) of the LC-MS system. ME is caused by molecules originating from the sample matrix or mobile phase that co-elute with the analyte of interest and therefore interfere with the ionization process in the MS ion source. Several approaches have been proposed to evaluate ME. Among them the post column infusion technique is widely used. Use of this qualitative evaluation technique allows the determination of the matrix effect of endogenous components in blank matrix. During analysis of blank matrix, analyte response is monitored to provide information where in the chromatographic run interferences between the analyte and matrix compounds occur. ME is illustrated as response deviation in the otherwise flat response time trace of the continuously post-column infused analyte [104]. This approach is very useful during method development because it provides information on the retention times where ME has to be expected, which can later be avoided for analyte of interest by optimizing chromatographic conditions.

For quantitative estimation of ME another well recognised approach is more suitable. Matuszewski et al. reported practical approach for the assessment of the absolute and relative ME as a part of validation of bioanalytical LC-MS/MS methods [94]. The difference in response between neat solution sample and post-extraction spiked sample is called the absolute ME, while difference between various lots of post-spiked samples is called the relative ME. As such will an absolute ME primary affect the accuracy and relative ME will affect the precision of the method. The determination of a relative ME is much more important than the determination of absolute ME in the evaluation and validation of bioanalytical method in biofluids [94]. The relative ME caused by interindividual variability in the sample matrix is assessed based on at least 5 lots of different matrices. Relative ME can be expressed as a coefficient of variation at particular concentration level or calculated based on slope lines. For the method to be considered reliable and free from the relative ME, the calculated coefficient of variation of determined slopes in different sources of matrices should not exceed 3-4% [105].

Analytical Methods for Quantification of Drug Metabolites in Biological Samples 113

However, the most efficient way to eliminate the influence of ME on the accuracy and precision of a quantitative analytical method is the use of stable isotope labeled analogs as

The selection of a suitable internal standard (IS) is one of the key parameters for establishing a successful LC-MS/MS method. Usually, stable isotope, such as 2H (D, deuterium), 13C, 15N or 17O, labeled standards are most appropriate for compensation of possible loss of analytes during sample clean up and variations in instrument performance (typically caused by matrix effect) since their physicochemical characteristics are practically identical to that of unlabeled analyte. In general, a stable isotope labeled IS is considered to be ideal, since it shows almost same behavior to the analyte of interest in sample preparation, chromatography as well as in ionization process [102]. However, issues like isotopic purity of IS, cross-contamination and cross-talk between MS ion channels, as well as IS stability and isotopic integrity of the label in biological fluid and during sample preparation should

Mass difference between analyte and IS should be at least 3 Da to avoid signal contribution of the natural isotopes to the signal of IS. Although deuterated IS are most frequently used, several disadvantages need to be considered in some cases, such as different extraction recoveries and retention times between such IS and analyte or exchange of deuterium atoms by hydrogen atoms [108]. For example, differences in retention times for deuterium labeled (d16) and unlabeled bisphenol A compounds is shown in Figure 2, where both, labeled metabolite and parent compound eluted slightly before their unlabeled analogs. Interestingly to mention, deuterium labeled bisphenol A (d16) has all 16 hydrogen atoms substituted by deuterium atoms but is actually d14 labeled (mass shift 14 Da in MRM transition, Figure 2) because two deuterium atoms from the functional group (–OD) are easily exchangeable by hydrogen atoms. The observable chromatographic retention time shifts for deuterated analogs depend on the number of deuterated atoms in structure. Deuterated analogs have no retention time shifts up to approximately six deuterium atoms [34] but when more deuterium atoms are included in structure, such as ten, the retention time shift may be significant (up to 1.2 min) [108]. Therefore, other stable isotope labeled IS, such as 13C, are considered as more appropriate

The concentration of IS used for sample preparation is also important and should be approximately at the middle of calibration curve. Optimization of IS concentration is critical to avoid ion suppression by co-eluting analyte leading to standard curve nonlinearity [70]. Even if stable labeled isotope IS is used, ME should still be investigated. Namely if ion suppression significantly reduces the signal of both, analyte and IS, the signal to noise ratio may decrease to a point where accuracy and precision may be

internal standards [105].

be carefully addressed [94].

[108].

negatively affected [102].

**9.2. Selection of internal standard** 

ME is known to be both component and matrix dependent. It was demonstrated that matrix induced ion suppression is especially important for early eluting compounds, such as polar metabolites. Typically, ME more strongly influences lower than higher analyte concentrations [71]. The main source of the commonly observed ME of plasma samples is believed to be endogenous phospholipids and proteins. The lysophospholipids which normally elute earlier in reversed phase chromatography are more likely to cause matrix effects compared to the later eluting phospholipids in spite of the larger concentrations of the latter in plasma [106]. Phospholipids cause ion suppression in both, positive and negative ESI modes and must be removed or resolved chromatographically.

To remove or reduce ME, modification to the sample extraction methodology (SPE or LLE instead of PP) and improved chromatographic separation must be performed. The majority of ME occur in the solvent front of a chromatographic run and if the analytes can be retained to some degree, matrix effects can be minimized. Suitable sample preparation and chromatographic conditions are linked together and form the basis of developing a successful and robust quantitative LC-MS/MS method [102, 103]. Another consideration when dealing with ME is selection of ionization interface. APCI is generally considered to be less prone to ion suppression compared with ESI [94, 105]. However, assay sensitivity and thermal stability of the analyte should be evaluated for eventual APCI application. Reducing the flow rate [20 µl/min or below) directed to ESI source by post column splitting may also reduce or completely remove the ion suppression [107]. Additionally to other approaches UHPLC technology in combination with polymeric mixed-mode SPE and appropriate mobile phase pH may provide significant advantages for reducing ME [73]. Mobile phase additives such as triethylamine and trifluoroacetic acid can also lead to ion suppression. The use of other reagents such as formic or acetic acid, trifluoroacetic acid in conjunction with 10 mM ammonium acetate or addition of 1% propionic acid to the mobile phase may overcome the ME of trifluoroacetic acid. Triethylamine may be replaced with other ion paring reagent such as hexylamine [70].

However, the most efficient way to eliminate the influence of ME on the accuracy and precision of a quantitative analytical method is the use of stable isotope labeled analogs as internal standards [105].

## **9.2. Selection of internal standard**

112 Chromatography – The Most Versatile Method of Chemical Analysis

should not exceed 3-4% [105].

chromatographically.

such as hexylamine [70].

For quantitative estimation of ME another well recognised approach is more suitable. Matuszewski et al. reported practical approach for the assessment of the absolute and relative ME as a part of validation of bioanalytical LC-MS/MS methods [94]. The difference in response between neat solution sample and post-extraction spiked sample is called the absolute ME, while difference between various lots of post-spiked samples is called the relative ME. As such will an absolute ME primary affect the accuracy and relative ME will affect the precision of the method. The determination of a relative ME is much more important than the determination of absolute ME in the evaluation and validation of bioanalytical method in biofluids [94]. The relative ME caused by interindividual variability in the sample matrix is assessed based on at least 5 lots of different matrices. Relative ME can be expressed as a coefficient of variation at particular concentration level or calculated based on slope lines. For the method to be considered reliable and free from the relative ME, the calculated coefficient of variation of determined slopes in different sources of matrices

ME is known to be both component and matrix dependent. It was demonstrated that matrix induced ion suppression is especially important for early eluting compounds, such as polar metabolites. Typically, ME more strongly influences lower than higher analyte concentrations [71]. The main source of the commonly observed ME of plasma samples is believed to be endogenous phospholipids and proteins. The lysophospholipids which normally elute earlier in reversed phase chromatography are more likely to cause matrix effects compared to the later eluting phospholipids in spite of the larger concentrations of the latter in plasma [106]. Phospholipids cause ion suppression in both, positive and negative ESI modes and must be removed or resolved

To remove or reduce ME, modification to the sample extraction methodology (SPE or LLE instead of PP) and improved chromatographic separation must be performed. The majority of ME occur in the solvent front of a chromatographic run and if the analytes can be retained to some degree, matrix effects can be minimized. Suitable sample preparation and chromatographic conditions are linked together and form the basis of developing a successful and robust quantitative LC-MS/MS method [102, 103]. Another consideration when dealing with ME is selection of ionization interface. APCI is generally considered to be less prone to ion suppression compared with ESI [94, 105]. However, assay sensitivity and thermal stability of the analyte should be evaluated for eventual APCI application. Reducing the flow rate [20 µl/min or below) directed to ESI source by post column splitting may also reduce or completely remove the ion suppression [107]. Additionally to other approaches UHPLC technology in combination with polymeric mixed-mode SPE and appropriate mobile phase pH may provide significant advantages for reducing ME [73]. Mobile phase additives such as triethylamine and trifluoroacetic acid can also lead to ion suppression. The use of other reagents such as formic or acetic acid, trifluoroacetic acid in conjunction with 10 mM ammonium acetate or addition of 1% propionic acid to the mobile phase may overcome the ME of trifluoroacetic acid. Triethylamine may be replaced with other ion paring reagent The selection of a suitable internal standard (IS) is one of the key parameters for establishing a successful LC-MS/MS method. Usually, stable isotope, such as 2H (D, deuterium), 13C, 15N or 17O, labeled standards are most appropriate for compensation of possible loss of analytes during sample clean up and variations in instrument performance (typically caused by matrix effect) since their physicochemical characteristics are practically identical to that of unlabeled analyte. In general, a stable isotope labeled IS is considered to be ideal, since it shows almost same behavior to the analyte of interest in sample preparation, chromatography as well as in ionization process [102]. However, issues like isotopic purity of IS, cross-contamination and cross-talk between MS ion channels, as well as IS stability and isotopic integrity of the label in biological fluid and during sample preparation should be carefully addressed [94].

Mass difference between analyte and IS should be at least 3 Da to avoid signal contribution of the natural isotopes to the signal of IS. Although deuterated IS are most frequently used, several disadvantages need to be considered in some cases, such as different extraction recoveries and retention times between such IS and analyte or exchange of deuterium atoms by hydrogen atoms [108]. For example, differences in retention times for deuterium labeled (d16) and unlabeled bisphenol A compounds is shown in Figure 2, where both, labeled metabolite and parent compound eluted slightly before their unlabeled analogs. Interestingly to mention, deuterium labeled bisphenol A (d16) has all 16 hydrogen atoms substituted by deuterium atoms but is actually d14 labeled (mass shift 14 Da in MRM transition, Figure 2) because two deuterium atoms from the functional group (–OD) are easily exchangeable by hydrogen atoms. The observable chromatographic retention time shifts for deuterated analogs depend on the number of deuterated atoms in structure. Deuterated analogs have no retention time shifts up to approximately six deuterium atoms [34] but when more deuterium atoms are included in structure, such as ten, the retention time shift may be significant (up to 1.2 min) [108]. Therefore, other stable isotope labeled IS, such as 13C, are considered as more appropriate [108].

The concentration of IS used for sample preparation is also important and should be approximately at the middle of calibration curve. Optimization of IS concentration is critical to avoid ion suppression by co-eluting analyte leading to standard curve nonlinearity [70]. Even if stable labeled isotope IS is used, ME should still be investigated. Namely if ion suppression significantly reduces the signal of both, analyte and IS, the signal to noise ratio may decrease to a point where accuracy and precision may be negatively affected [102].

Problems may occur when more than one compound is determined in the same analytical method. A number of labeled IS identical to number of analyzed compounds would in this case be required [102]. This is not always practically feasible, especially not for stable isotope labeled drug metabolites as their availability is very limited. In such cases, to assure the suitability of the quantification method for determination of drugs and their metabolites, ME evaluation should be carefully addressed [37]. On the other hand, example for simultaneous determination of parent compound and its metabolite using both labeled IS [4] is shown in Figure 2. It is preferred that the labeled IS product ion used for the MRM transition retains the stable isotope moiety as for m/z 403 → 227 (bisphenol A-glucuronide) versus 417 → 241 (deuterated bisphenol A-glucuronide) in Figure 2.

Analytical Methods for Quantification of Drug Metabolites in Biological Samples 115

during the validation of the method. Carry-over is usually expressed as percentage of detected analyte in blank sample regarding to its limit of quantification [4] and may

**Figure 2.** LC-MS/MS chromatogram in MRM mode for metabolite (BPAG) and parent bisphenol A (BPA) of a typical microsomal incubation. (A) mass transition for BPAGd16 (internal standard for BPAG), (B) mass transition for BPAG, (C) mass transition for BPAd16 (internal standard for BPA), (D) mass

transition for BPA. For analysis conditions refer to [4].

significantly lower the sensitivity of the method [109].

However, labeled ISs are not always available or can be very expensive. As an alternative, structural analogues can be used, with consideration of the structural similarities between the IS and the analyte. To be suitable, the ionization of the analogue must be compared with analyte and should preferably co-elute with the analyte (Figure 1). The selected analog IS should not correspond to any metabolic product of analyte, such as hydroxylated or Ndealkylated metabolites [108]. Nevertheless, in many cases analog ISs or structurally unrelated ISs are not able to compensate the ME [94, 105]. In such situation, other more rigorous approaches to reduce or eliminate ME should be applied (see 9.1.).

## **9.3. LC-MS pitfalls**

Metabolite may produce a molecular ion that is identical to parent ion through in source conversion. Typical metabolites for such conversion are glucuronides, sulfates or N-oxides [79]. N-oxide conversion may also serve for identification purposes as this transformation takes place in APCI source and represent another potential way to differentiate N-oxide from hydroxylated metabolites (both with exact mass) since the later usually do not undergo thermal deoxygenation [23]. Therefore, an inadequate chromatographic separation between parent drug and such metabolites will result in over estimation of parent drug concentration in the presence of these isomeric compounds. The same situation may happen also by ion channel cross-talk in MRM mode, which means that fragment issued from the other scanned transition is still present in collision cell. Metabolites, such as diverse glucuronides, that give the same product ion as parent drug, have to be therefore chromatographically separated. However, for co-eluting compounds, such as analytes and their IS, the absence of cross-talk has to be demonstrated [37].

Carry-over, which is the appearance of an analyte signal in a blank injection subsequently to analysis of high concentration samples, is also a common problem in LC-MS/MS methods. This problem occurs due to retention of analytes by adsorption on active surfaces of the autosamples, solvent lines, extraction columns (e.g. online SPE) or the analytical column. Most carry-over problems can be minimized by an appropriate choice of injector wash solutions and methods, by proper choice of mobile phase and tubings, and by choice of suitable stationary phase and proper variation in the solvent strength [70]. Carry-over becomes prominent after injection of analyte at high concentration and should be assessed during the validation of the method. Carry-over is usually expressed as percentage of detected analyte in blank sample regarding to its limit of quantification [4] and may significantly lower the sensitivity of the method [109].

114 Chromatography – The Most Versatile Method of Chemical Analysis

versus 417 → 241 (deuterated bisphenol A-glucuronide) in Figure 2.

rigorous approaches to reduce or eliminate ME should be applied (see 9.1.).

**9.3. LC-MS pitfalls** 

has to be demonstrated [37].

Problems may occur when more than one compound is determined in the same analytical method. A number of labeled IS identical to number of analyzed compounds would in this case be required [102]. This is not always practically feasible, especially not for stable isotope labeled drug metabolites as their availability is very limited. In such cases, to assure the suitability of the quantification method for determination of drugs and their metabolites, ME evaluation should be carefully addressed [37]. On the other hand, example for simultaneous determination of parent compound and its metabolite using both labeled IS [4] is shown in Figure 2. It is preferred that the labeled IS product ion used for the MRM transition retains the stable isotope moiety as for m/z 403 → 227 (bisphenol A-glucuronide)

However, labeled ISs are not always available or can be very expensive. As an alternative, structural analogues can be used, with consideration of the structural similarities between the IS and the analyte. To be suitable, the ionization of the analogue must be compared with analyte and should preferably co-elute with the analyte (Figure 1). The selected analog IS should not correspond to any metabolic product of analyte, such as hydroxylated or Ndealkylated metabolites [108]. Nevertheless, in many cases analog ISs or structurally unrelated ISs are not able to compensate the ME [94, 105]. In such situation, other more

Metabolite may produce a molecular ion that is identical to parent ion through in source conversion. Typical metabolites for such conversion are glucuronides, sulfates or N-oxides [79]. N-oxide conversion may also serve for identification purposes as this transformation takes place in APCI source and represent another potential way to differentiate N-oxide from hydroxylated metabolites (both with exact mass) since the later usually do not undergo thermal deoxygenation [23]. Therefore, an inadequate chromatographic separation between parent drug and such metabolites will result in over estimation of parent drug concentration in the presence of these isomeric compounds. The same situation may happen also by ion channel cross-talk in MRM mode, which means that fragment issued from the other scanned transition is still present in collision cell. Metabolites, such as diverse glucuronides, that give the same product ion as parent drug, have to be therefore chromatographically separated. However, for co-eluting compounds, such as analytes and their IS, the absence of cross-talk

Carry-over, which is the appearance of an analyte signal in a blank injection subsequently to analysis of high concentration samples, is also a common problem in LC-MS/MS methods. This problem occurs due to retention of analytes by adsorption on active surfaces of the autosamples, solvent lines, extraction columns (e.g. online SPE) or the analytical column. Most carry-over problems can be minimized by an appropriate choice of injector wash solutions and methods, by proper choice of mobile phase and tubings, and by choice of suitable stationary phase and proper variation in the solvent strength [70]. Carry-over becomes prominent after injection of analyte at high concentration and should be assessed

**Figure 2.** LC-MS/MS chromatogram in MRM mode for metabolite (BPAG) and parent bisphenol A (BPA) of a typical microsomal incubation. (A) mass transition for BPAGd16 (internal standard for BPAG), (B) mass transition for BPAG, (C) mass transition for BPAd16 (internal standard for BPA), (D) mass transition for BPA. For analysis conditions refer to [4].

## **9.4. Metabolite stability**

For drugs and metabolites that are unstable, with one converting to other, conditions used during sample preparation and analysis must be optimized in order to minimize such conversion and to achieve accurate quantification of the drug and metabolite. Common factors that affect drug and drug metabolite stability in biological matrices include temperature, light, pH, oxidation and enzymatic degradation [110]. Acyl glucuronides (Oester conjugates) are probably the most commonly encountered problematic metabolites in bioanalysis. Acyl glucuronides are unstable and hydrolyze to release aglycone under neutral and alkaline conditions. Different acyl glucuronides have been shown to have different rates of hydrolysis. However, mildly acidic conditions (pH 3-5) should be the most desirable pH for minimizing the reaction in biological samples [59, 111]. Acyl glucuronides (1-O-acyl glucuronides) are also susceptible to internal migration under both, physiological and alkaline conditions. The rate of migration resulting in 2-, 3- and 4- O-acyl glucuronides increases with increasing pH and temperature. Such isomeric glucuronides are not susceptible to hydrolysis by ß-glucuronidases and may compromise the quantification of metabolites and total parent drug when indirect quantification approach via conjugate cleavage is applied [111]. Similar to O-glucuronides, Nglucuronides can be converted back to parent drug under acidic/basic/neutral pH conditions or at elevated sample processing temperature, but this instability is largely compound dependent. For example olanzapine glucuronide can be readily cleaved under acidic conditions, clozapine and cyclizine glucuronides are unstable at range pH 1-3 and doxepin glucuronide at pH 11 [112]. Lactone is another commonly unstable metabolite function group which may be converted to its open ring hydroxy acid drug. Lactone metabolites, such as atorvastatin metabolite, require optimization of the sample pH (typically 3-5) in order to minimize the hydrolysis of lactone metabolite back to parent drug [113].

Analytical Methods for Quantification of Drug Metabolites in Biological Samples 117

successful monitoring of the drug metabolism, suitable bioanalytical methods have to be developed and validated. Liquid chromatography coupled with mass spectrometry has become the most powerful analytical tool for identification and quantification of drug

The known identity of metabolites is the prerequisite for a suitable metabolic assessment of drugs. Appropriate LC-MS instrumentation is clearly critical to both, detection and structural elucidation. Tandem mass spectrometry instruments are beside their key role for metabolite quantification also well suited for qualitative purposes as they enable different scan possibilities (constant neutral loss, precursor ion, product ion) for structural characterization of metabolites. However, the drive to more versatile and powerful instruments which can perform intelligent data dependent experiments and accurate mass measurements has led to newer high resolution mass analyzers, such as Q-TOF instruments,

Direct quantification of metabolites in biological samples is the most appropriate approach, but also others approaches such as indirect quantification through parent drug after metabolite hydrolysis or quantification supported by using response factors may be used which primary depends on the availability of suitable authentic standards. Analytical methods for metabolite quantification are based on liquid chromatography or other separation techniques coupled with various detector systems where LC-MS/MS plays predominant role in bioassays for pharmacokinetic and metabolism studies due to its

In order to support metabolism experiments in a timely manner, the use of high throughput methods for the analysis of drugs and their metabolites in biological samples has become an essential part, especially in the most time consuming sample preparation. Trend is going toward semi automated off-line sample treatment in 96-well plate format or on-line SPE after direct injection of samples. Additionally, other high throughput approaches can be introduced. Ultra-high performance liquid chromatography with small particles and monolithic chromatography offer improvements in speed, resolution and sensitivity compared to conventional chromatographic techniques. Hydrophilic interaction chromatography (HILIC) or other specialized columns suitable for polar metabolites are emerging as a valuable supplement to classical reversed phase

The main advantage of LC-MS/MS allows development of high throughput methods with little or no sample preparation and minimal chromatographic retention. However, matrix effect may have a significant impact on such analyses. Matrix effect issue is frequently underestimated and should be adequately addressed. Not without reason, matrix effect have been called the Achilles heel of quantitative LC-ESI-MS/MS [103]. The use of stable isotope labeled analog as internal standard is the most efficient way to reduce matrix effect. But normally, additional approaches to reduce or eliminate matrix effect are

which now dominate the metabolite identification field.

inherent specificity, sensitivity and speed.

chromatography.

needed.

metabolites.

N-oxides are also unstable in solutions and biological samples during sample preparation, especially under strong acidic or basic conditions. Light exposure may further accelerate the decomposition of these metabolites. Other compounds are also susceptible to photodegradation such as, catechols, nisoldipine, rifampin and their metabolites and should be protected from light during sample preparation and analysis [112].

## **10. Conclusion**

Current drug discovery efforts have been focused on identification of drug metabolism and pharmacokinetic issues at the earliest possible stage in order to reduce the attrition rate of drug candidates during the developmental phase. Metabolic fate of drugs can be responsible for problems associated with their bioavailability, interindividual variability, drug-drug interactions, pharmacologic activity or the toxicity. Different *in vitro* methods, from subcelullar to organ range, and *in vivo* studies are applied for the clarification of drug metabolism. Among them microsomes and hepatocytes are the most frequently utilized *in vitro* models in drug metabolic profiling and drug interaction studies. For the successful monitoring of the drug metabolism, suitable bioanalytical methods have to be developed and validated. Liquid chromatography coupled with mass spectrometry has become the most powerful analytical tool for identification and quantification of drug metabolites.

116 Chromatography – The Most Versatile Method of Chemical Analysis

For drugs and metabolites that are unstable, with one converting to other, conditions used during sample preparation and analysis must be optimized in order to minimize such conversion and to achieve accurate quantification of the drug and metabolite. Common factors that affect drug and drug metabolite stability in biological matrices include temperature, light, pH, oxidation and enzymatic degradation [110]. Acyl glucuronides (Oester conjugates) are probably the most commonly encountered problematic metabolites in bioanalysis. Acyl glucuronides are unstable and hydrolyze to release aglycone under neutral and alkaline conditions. Different acyl glucuronides have been shown to have different rates of hydrolysis. However, mildly acidic conditions (pH 3-5) should be the most desirable pH for minimizing the reaction in biological samples [59, 111]. Acyl glucuronides (1-O-acyl glucuronides) are also susceptible to internal migration under both, physiological and alkaline conditions. The rate of migration resulting in 2-, 3- and 4- O-acyl glucuronides increases with increasing pH and temperature. Such isomeric glucuronides are not susceptible to hydrolysis by ß-glucuronidases and may compromise the quantification of metabolites and total parent drug when indirect quantification approach via conjugate cleavage is applied [111]. Similar to O-glucuronides, Nglucuronides can be converted back to parent drug under acidic/basic/neutral pH conditions or at elevated sample processing temperature, but this instability is largely compound dependent. For example olanzapine glucuronide can be readily cleaved under acidic conditions, clozapine and cyclizine glucuronides are unstable at range pH 1-3 and doxepin glucuronide at pH 11 [112]. Lactone is another commonly unstable metabolite function group which may be converted to its open ring hydroxy acid drug. Lactone metabolites, such as atorvastatin metabolite, require optimization of the sample pH (typically 3-5) in order to minimize the hydrolysis of lactone metabolite back to parent

N-oxides are also unstable in solutions and biological samples during sample preparation, especially under strong acidic or basic conditions. Light exposure may further accelerate the decomposition of these metabolites. Other compounds are also susceptible to photodegradation such as, catechols, nisoldipine, rifampin and their metabolites and should

Current drug discovery efforts have been focused on identification of drug metabolism and pharmacokinetic issues at the earliest possible stage in order to reduce the attrition rate of drug candidates during the developmental phase. Metabolic fate of drugs can be responsible for problems associated with their bioavailability, interindividual variability, drug-drug interactions, pharmacologic activity or the toxicity. Different *in vitro* methods, from subcelullar to organ range, and *in vivo* studies are applied for the clarification of drug metabolism. Among them microsomes and hepatocytes are the most frequently utilized *in vitro* models in drug metabolic profiling and drug interaction studies. For the

be protected from light during sample preparation and analysis [112].

**9.4. Metabolite stability** 

drug [113].

**10. Conclusion** 

The known identity of metabolites is the prerequisite for a suitable metabolic assessment of drugs. Appropriate LC-MS instrumentation is clearly critical to both, detection and structural elucidation. Tandem mass spectrometry instruments are beside their key role for metabolite quantification also well suited for qualitative purposes as they enable different scan possibilities (constant neutral loss, precursor ion, product ion) for structural characterization of metabolites. However, the drive to more versatile and powerful instruments which can perform intelligent data dependent experiments and accurate mass measurements has led to newer high resolution mass analyzers, such as Q-TOF instruments, which now dominate the metabolite identification field.

Direct quantification of metabolites in biological samples is the most appropriate approach, but also others approaches such as indirect quantification through parent drug after metabolite hydrolysis or quantification supported by using response factors may be used which primary depends on the availability of suitable authentic standards. Analytical methods for metabolite quantification are based on liquid chromatography or other separation techniques coupled with various detector systems where LC-MS/MS plays predominant role in bioassays for pharmacokinetic and metabolism studies due to its inherent specificity, sensitivity and speed.

In order to support metabolism experiments in a timely manner, the use of high throughput methods for the analysis of drugs and their metabolites in biological samples has become an essential part, especially in the most time consuming sample preparation. Trend is going toward semi automated off-line sample treatment in 96-well plate format or on-line SPE after direct injection of samples. Additionally, other high throughput approaches can be introduced. Ultra-high performance liquid chromatography with small particles and monolithic chromatography offer improvements in speed, resolution and sensitivity compared to conventional chromatographic techniques. Hydrophilic interaction chromatography (HILIC) or other specialized columns suitable for polar metabolites are emerging as a valuable supplement to classical reversed phase chromatography.

The main advantage of LC-MS/MS allows development of high throughput methods with little or no sample preparation and minimal chromatographic retention. However, matrix effect may have a significant impact on such analyses. Matrix effect issue is frequently underestimated and should be adequately addressed. Not without reason, matrix effect have been called the Achilles heel of quantitative LC-ESI-MS/MS [103]. The use of stable isotope labeled analog as internal standard is the most efficient way to reduce matrix effect. But normally, additional approaches to reduce or eliminate matrix effect are needed.
