*8.1.3. Chromatographic approaches for polar metabolites*

Metabolites, in particular glucuronides, have typically higher polarity than their parent drugs (Figures 1 and 2). This is the reason that classical reversed phase chromatography (e.g. C18) is sometimes not sufficient enough to maintain appropriate chromatographic retention of these analytes. In such cases already mentioned approaches like short-chain bonded reversed phases and ion-paring reagents (8.1.1.) or HILIC (8.1.2.) may be used. Additionally, mixed-mode columns with an embedded ion-paring group in the reversed phase stationary phase provide the capability for both ion-exchange and hydrophobic interactions in the mobile phase to retain ionizable polar analytes. The mixed-mode column allows retaining hydrophobic analytes by the reversed phase mechanism and hydrophilic analytes by the ion exchange mechanism at higher organic content in the mobile phase [87]. Normal phase chromatography may also be used for retention of polar analytes but due to limited amount of water allowed in the mobile phase, normal phase chromatography interfaced with MS requires complex pretreatment steps for biological samples and therefore has much fewer applications than reversed phase LC-MS/MS [88].

The use of special packing material known as porous graphic carbon (PGC) is another alternative to achieve retention and separation of polar analytes. PGC chromatography commonly employs water, acetonitrile and methanol as the mobile phase but provides markedly greater retention and selectivity for polar analytes than reversed phase columns. For analyte elution PGC normally requires larger organic content in the mobile phase than reversed phase chromatography what consequently results in favorable sensitivity with MS detection [79, 83, 89].

Derivatization of polar analytes results in the reduction of polarity and is therefore another possibility to enhance the chromatographic retention. But this approach is disadvantaged as it is not going toward high throughput, especially in case when the primary purpose of the derivatization is not the detection or stability improvement of the analyte.

## **8.2. Strategies for high-throughput improvement in liquid chromatography**

Current trend in pharmaceutical analysis is the reduction of the analysis time and the increase in sample throughput without sacrificing the separation selectivity. Highthroughput bioanalytical assays are typically based on LC-MS/MS but may also be successfully extended to classical HPLC analyses. Approaches to achieve faster analyses include sample preparation (on-line automation or offline semi automation, section 7) and fast liquid chromatography. The later may be in general improved by three approaches: smaller particle size, shorter columns and higher mobile phase flow rates.

## *8.2.1. Ultra-high performance liquid chromatography (UHPLC)*

Reducing the particle diameter from 5.0 µm to 1.7 µm will, in principle, result in a 3-fold increase of efficiency, 1.7-fold increase in resolution, a 1.7-fold in sensitivity, and 3-fold increase in speed [79]. For fast analyses using sub-2µm particle column dimensions are typically 50x2 mm. An additional benefit of UHPLC is the low consumption of mobile phase, where it saves at least 80% compared to HPLC [90]. The high back-pressure resulting in decreased particle size need appropriately designed chromatographic system that would withstand such high pressure (instruments nowadays up to 1200 bars) and also provide at least possible extra column effects. To prevent clogging, manufacturers of UHPLC recommend filtration of both samples and solvents through 0.2 µm filter. Advantages as enhanced separation efficiency, short analysis time and high detection sensitivity make UHPLC coupled with MS/MS an even more powerful analytical support in pharmacokinetic studies [4].

## *8.2.2. Core-shell column*

106 Chromatography – The Most Versatile Method of Chemical Analysis

sample preparation automation and throughput [86].

MS/MS [88].

detection [79, 83, 89].

*8.1.3. Chromatographic approaches for polar metabolites* 

onto the column without impairing peak shapes. Therefore, evaporation and reconstitution step of organic extracts after extraction procedure could be omitted making improvement in

Metabolites, in particular glucuronides, have typically higher polarity than their parent drugs (Figures 1 and 2). This is the reason that classical reversed phase chromatography (e.g. C18) is sometimes not sufficient enough to maintain appropriate chromatographic retention of these analytes. In such cases already mentioned approaches like short-chain bonded reversed phases and ion-paring reagents (8.1.1.) or HILIC (8.1.2.) may be used. Additionally, mixed-mode columns with an embedded ion-paring group in the reversed phase stationary phase provide the capability for both ion-exchange and hydrophobic interactions in the mobile phase to retain ionizable polar analytes. The mixed-mode column allows retaining hydrophobic analytes by the reversed phase mechanism and hydrophilic analytes by the ion exchange mechanism at higher organic content in the mobile phase [87]. Normal phase chromatography may also be used for retention of polar analytes but due to limited amount of water allowed in the mobile phase, normal phase chromatography interfaced with MS requires complex pretreatment steps for biological samples and therefore has much fewer applications than reversed phase LC-

The use of special packing material known as porous graphic carbon (PGC) is another alternative to achieve retention and separation of polar analytes. PGC chromatography commonly employs water, acetonitrile and methanol as the mobile phase but provides markedly greater retention and selectivity for polar analytes than reversed phase columns. For analyte elution PGC normally requires larger organic content in the mobile phase than reversed phase chromatography what consequently results in favorable sensitivity with MS

Derivatization of polar analytes results in the reduction of polarity and is therefore another possibility to enhance the chromatographic retention. But this approach is disadvantaged as it is not going toward high throughput, especially in case when the primary purpose of the

Current trend in pharmaceutical analysis is the reduction of the analysis time and the increase in sample throughput without sacrificing the separation selectivity. Highthroughput bioanalytical assays are typically based on LC-MS/MS but may also be successfully extended to classical HPLC analyses. Approaches to achieve faster analyses include sample preparation (on-line automation or offline semi automation, section 7) and fast liquid chromatography. The later may be in general improved by three approaches:

**8.2. Strategies for high-throughput improvement in liquid chromatography** 

derivatization is not the detection or stability improvement of the analyte.

smaller particle size, shorter columns and higher mobile phase flow rates.

An emerging alternative to porous particles are porous layer beads, known as core-shell or fused-core particles. The high separation efficiency of core-shell particles is a result of a faster analyte mass transfer from the mobile phase to outer porous layer of the particle. The improved dynamics of analyte movement through these columns result in higher effective peak capacities and separation efficiencies comparable to those fully porous sub-2µm but with advantage of lower back-pressure [91]. This technology is comparable to UHPLC in terms of chromatographic performance but demands neither expensive UHPLC instrumentation nor new laboratory protocols [88]. Commonly available columns, such as Ascentis, Poroshell and Kinetex, use different stationary phases and particle sizes (e.g. Kinetex 1.7 and 2.6 µm) and are widely used with classical HPLC instruments, also in our laboratories. Core-shell columns in combination with UHPLC-MS/MS exhibit excellent performance, as demonstrated in quantification of raloxifene and its three glucuronides [37].

### *8.2.3. Monolithic chromatography*

The use of single rod monolith column is an alternative approach to the chromatographic columns packed with fine particles. The high permeability allows the use of higher flow rates and therefore shorter chromatographic runs, as demonstrated for the separation of bupropion metabolites in 23 seconds or for methylphenidate and its metabolite in 15 seconds [71].

High flow rates may require flow splitting before entering MS. An attractive approach using monolith separation is to combine it with high flow on-line extraction, which allows fast extraction and separation of samples [77]. Current limitations in the application of these columns are the small pH range [2-8], poor temperature resistance, limited column dimensions and stationary phases (C8 and C18) as well as higher costs due to higher mobile phase consumption.

## **8.3. Other separation techniques**

Gas chromatography with mass spectrometry (GC-MS) is most useful for the analysis of trace amounts of organically extractable, non-polar, volatile compounds and highly volatile compounds that may undergo headspace analysis. The GC-MS analysis of polar compounds, such as metabolites, from biological matrices requires analyte extraction into a volatile organic solvent either directly or after chemical derivatization, which typically enhances the volatility of previously non-volatile organic compounds [25]. Most analytes need extensive time-consuming sample preparation including derivatization to become stable, volatile and amenable to the ionization technique. This drawback in throughput necessitated the direction of GC-MS to LC-MS. LC-MS has an advantage over GC-MS method in drug metabolism studies, particularly for low dosed and large drugs, and of course for the analysis of phase II metabolites. However, GC-MS may also have advantages, especially in clinical and forensic toxicology or doping control [54]. GC-MS has been frequently applied for quantification of glucuronides in biological samples but only after treatment with ß-glucuronidase in order to obtain parent drug before analysis [92]. The GC-MS technique is receiving wider acceptance in various classes of antidepressant agents, representing 6% of overall analytical methods for determination of antidepressants and their metabolites [67].

Analytical Methods for Quantification of Drug Metabolites in Biological Samples 109

transitions cannot be established. Alternatively, a precursor → precursor scan for reducing

Also other analyzers, such as ion traps and TOF, have been widely and increasly used for metabolite quantification. Especially hybrid instruments which combine a QQQ (Q1 and colission cell) and ion trap or TOF (Qtrap, Q-TOF). These instruments can operate as true tandem mass spectrometry and are usually applied for this purposes. Q-TOF can also operate as TOF and thus provide accurate mass measurments. Additonaly, high resolution of TOF instruments allows the resolution of chromatographic peak from background interferences achieveing better sensitivity. However, it does apear that QQQ using MRM

The selection of an appropriate ionization technique depends on the analyte characteristics, such as the structure, polarity or molecular weight. In LC-MS/MS analysis three atmospheric pressure ionization techniques cover the whole range of compound polarities and molecular weight: ESI, APCI and APPI. Moreover, the polarity mode can be chosen according to the acidic, neutral or basic properties of the analytes. If the right choice of ionization technique and the polarity mode is not so obvious, all available possibilities should be considered in order to obtain the best response for tested analytes. The softest ionization technique, ESI, is the method of choice for polar and ionic compounds. The advantage of soft ionization is in providing reliable information about molecular weight of the phase II metabolites in comparison to other ionization techniques [42]. For parent drug and phase I metabolites with a lower polarity, APCI and APPI may provide better ionization efficiency and sensitivity [34]. APPI has a similar application range as APCI, but slightly extended toward nonpolar compounds [32]. ESI is generally more susceptible to matrix ionization suppression than APCI [94]. In case of neutral steroids or other poorly ionizable analytes, derivatization can be employed in order to increase detection sensitivity, but additionally the chromatographic retention enhancement of such derivatizated analytes may therefore provide less matrix effect. On the other hand, the disadvantage of derivatization lies in an additional time consuming step for sample preparation [30]. The adjustment of the mobile phase for improving analyte response is much easier compared to derivatization. The effect of mobile phase on ESI efficiency is not well understood and hence the behavior of an analyte in different mobile phase conditions cannot be routinely predicted. Various factors can affect the ionization of analytes in ESI, such as pH, mobile phase additives, flow rate, solvent composition and concentration of electrolytes. It is recommended to evaluate the use of additives (e.g. formic acid, ammonium acetate) and organic modifiers in mobile phase to maximize the ionization efficiency of the analyte, which is highly dependent on its chemical structure. Acidic conditions often promote positive mode ionization of basic compounds and conditions, which are slightly below neutral, neutral or basic, promote negative

A dramatic difference in the ESI response can be found even when acetonitrile is replaced by methanol in mobile phase. It was reported that an analyte gave only weak ESI response

remain about three to five times more sensitive than TOF [93].

noise can be employed [70].

ionization of acidic compounds.

Capillary electrophoresis (CE) is another separation method for quantification of metabolites. This method offers very high resolution capability, high efficiency and short time of analysis. Moreover, CE in many instances can have distinct advantages over HPLC in terms of simplicity, rapid method development, solvent saving and minimal sample requirement [10-30 nL injected) making this technique very interesting for rapid and practical analyses in the biomedical field. However, the main disadvantage is low sensitivity. For this reason, application of CE for analysis of antidepressants and their metabolites is not so widely reported [67]. Applicability of CE using UV-absorbance or mass spectrometry detection was reported for determination of tamoxifene and its phase I metabolites [68].

## **8.4. Mass spectrometry**

Currently, the QQQ using single or multiple reaction monitoring is most often used for quantitative analysis of small molecules in the pharmaceutical industry. QQQ or single stage MS, operating in SIM, is not anymore recommended for reliable bioanalytical quantification, because it suffers from insufficient selectivity in comparison with MRM. SIM can provide the selected ion at certain m/z value, but the matrix or impurity interferences may occur at the same m/z value. Beside lower selectivity, SIM shows also much lower sensitivity in comparison to MRM due to much higher background noise. However, in some specific cases of good chromatographic resolution and the absence of matrix interferences, SIM may be considered as an alternative quantification method. Occasionally, due to the nature of dissociation pathways, resulting in low molecular weight product ions, radial ejection preceding dissociation and/or charge stripping, reliable precursor → product ion transitions cannot be established. Alternatively, a precursor → precursor scan for reducing noise can be employed [70].

108 Chromatography – The Most Versatile Method of Chemical Analysis

Gas chromatography with mass spectrometry (GC-MS) is most useful for the analysis of trace amounts of organically extractable, non-polar, volatile compounds and highly volatile compounds that may undergo headspace analysis. The GC-MS analysis of polar compounds, such as metabolites, from biological matrices requires analyte extraction into a volatile organic solvent either directly or after chemical derivatization, which typically enhances the volatility of previously non-volatile organic compounds [25]. Most analytes need extensive time-consuming sample preparation including derivatization to become stable, volatile and amenable to the ionization technique. This drawback in throughput necessitated the direction of GC-MS to LC-MS. LC-MS has an advantage over GC-MS method in drug metabolism studies, particularly for low dosed and large drugs, and of course for the analysis of phase II metabolites. However, GC-MS may also have advantages, especially in clinical and forensic toxicology or doping control [54]. GC-MS has been frequently applied for quantification of glucuronides in biological samples but only after treatment with ß-glucuronidase in order to obtain parent drug before analysis [92]. The GC-MS technique is receiving wider acceptance in various classes of antidepressant agents, representing 6% of overall analytical methods for determination of antidepressants and their

Capillary electrophoresis (CE) is another separation method for quantification of metabolites. This method offers very high resolution capability, high efficiency and short time of analysis. Moreover, CE in many instances can have distinct advantages over HPLC in terms of simplicity, rapid method development, solvent saving and minimal sample requirement [10-30 nL injected) making this technique very interesting for rapid and practical analyses in the biomedical field. However, the main disadvantage is low sensitivity. For this reason, application of CE for analysis of antidepressants and their metabolites is not so widely reported [67]. Applicability of CE using UV-absorbance or mass spectrometry detection was reported for determination of tamoxifene and its phase I

Currently, the QQQ using single or multiple reaction monitoring is most often used for quantitative analysis of small molecules in the pharmaceutical industry. QQQ or single stage MS, operating in SIM, is not anymore recommended for reliable bioanalytical quantification, because it suffers from insufficient selectivity in comparison with MRM. SIM can provide the selected ion at certain m/z value, but the matrix or impurity interferences may occur at the same m/z value. Beside lower selectivity, SIM shows also much lower sensitivity in comparison to MRM due to much higher background noise. However, in some specific cases of good chromatographic resolution and the absence of matrix interferences, SIM may be considered as an alternative quantification method. Occasionally, due to the nature of dissociation pathways, resulting in low molecular weight product ions, radial ejection preceding dissociation and/or charge stripping, reliable precursor → product ion

**8.3. Other separation techniques** 

metabolites [67].

metabolites [68].

**8.4. Mass spectrometry** 

Also other analyzers, such as ion traps and TOF, have been widely and increasly used for metabolite quantification. Especially hybrid instruments which combine a QQQ (Q1 and colission cell) and ion trap or TOF (Qtrap, Q-TOF). These instruments can operate as true tandem mass spectrometry and are usually applied for this purposes. Q-TOF can also operate as TOF and thus provide accurate mass measurments. Additonaly, high resolution of TOF instruments allows the resolution of chromatographic peak from background interferences achieveing better sensitivity. However, it does apear that QQQ using MRM remain about three to five times more sensitive than TOF [93].

The selection of an appropriate ionization technique depends on the analyte characteristics, such as the structure, polarity or molecular weight. In LC-MS/MS analysis three atmospheric pressure ionization techniques cover the whole range of compound polarities and molecular weight: ESI, APCI and APPI. Moreover, the polarity mode can be chosen according to the acidic, neutral or basic properties of the analytes. If the right choice of ionization technique and the polarity mode is not so obvious, all available possibilities should be considered in order to obtain the best response for tested analytes. The softest ionization technique, ESI, is the method of choice for polar and ionic compounds. The advantage of soft ionization is in providing reliable information about molecular weight of the phase II metabolites in comparison to other ionization techniques [42]. For parent drug and phase I metabolites with a lower polarity, APCI and APPI may provide better ionization efficiency and sensitivity [34]. APPI has a similar application range as APCI, but slightly extended toward nonpolar compounds [32]. ESI is generally more susceptible to matrix ionization suppression than APCI [94]. In case of neutral steroids or other poorly ionizable analytes, derivatization can be employed in order to increase detection sensitivity, but additionally the chromatographic retention enhancement of such derivatizated analytes may therefore provide less matrix effect. On the other hand, the disadvantage of derivatization lies in an additional time consuming step for sample preparation [30]. The adjustment of the mobile phase for improving analyte response is much easier compared to derivatization. The effect of mobile phase on ESI efficiency is not well understood and hence the behavior of an analyte in different mobile phase conditions cannot be routinely predicted. Various factors can affect the ionization of analytes in ESI, such as pH, mobile phase additives, flow rate, solvent composition and concentration of electrolytes. It is recommended to evaluate the use of additives (e.g. formic acid, ammonium acetate) and organic modifiers in mobile phase to maximize the ionization efficiency of the analyte, which is highly dependent on its chemical structure. Acidic conditions often promote positive mode ionization of basic compounds and conditions, which are slightly below neutral, neutral or basic, promote negative ionization of acidic compounds.

A dramatic difference in the ESI response can be found even when acetonitrile is replaced by methanol in mobile phase. It was reported that an analyte gave only weak ESI response

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 low concentrations are expected in such studies [4].

Analytical Methods for Quantification of Drug Metabolites in Biological Samples 111

specificity, sensitivity and high throughput [99]. Electrochemical detection is also very suitable for determination of antioxidants, such as ascorbic acid or glutathione, in biological

When analytes do not exhibit fluorescence, electroactivity or have poor UV detection, derivatization can be performed to enhance their detection. In addition, chromatographic retention is enhanced by derivatization what is a very convenient in analysis of polar drug metabolites. Derivatization is an additional step in sample preparation where consideration regarding the stability of derivatizated analyte to solvolysis and thermal degradation need to be addressed. Nevertheless, fluorescence detection is still widely

LC-MS/MS is currently considered as the method of choice for quantitative analysis of drugs and their metabolites. The advantages of using this technique in MRM mode due to high sensitivity, selectivity and speed allow developing high throughput methods with little or no sample preparation and minimal chromatographic retention. However, matrix effect may have a significant impact on such LC-MS/MS analyses [94, 102, 103]. Therefore, the evaluation of matrix effect as well as strategies for its elimination or minimization needs to be adequately addressed. Another important parameter for analytical quality is the selection of an appropriate internal standard for the compensation of possible loss of analytes during sample clean up and variations in instrument performance. Other LC-MS/MS issues, such as ion channel cross-talk and carry-over should also not be overlooked. Moreover, metabolite instability may have an influence on the analytical performance and will be additionally

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

samples [100].

used [67, 68, 101].

addressed here.

**9.1. Matrix effect** 

**9. Aspects of analytical quality** 

optimizing chromatographic conditions.

## **8.5. Other detection techniques**

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 mass spectrometry.

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

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 specificity, sensitivity and high throughput [99]. Electrochemical detection is also very suitable for determination of antioxidants, such as ascorbic acid or glutathione, in biological samples [100].

When analytes do not exhibit fluorescence, electroactivity or have poor UV detection, derivatization can be performed to enhance their detection. In addition, chromatographic retention is enhanced by derivatization what is a very convenient in analysis of polar drug metabolites. Derivatization is an additional step in sample preparation where consideration regarding the stability of derivatizated analyte to solvolysis and thermal degradation need to be addressed. Nevertheless, fluorescence detection is still widely used [67, 68, 101].
