**7.1. Protein precipitation**

Protein precipitation (PP) is simple and straightforward method widely used in bioanalysis of plasma samples. It is accomplished by using organic solvent (typically acetonitrile or methanol) or an acid (typically perchloric or trichloroacetic acid). It is followed by centrifugation to separate proteins from liquid supernatant and additionally, supernatant is sometimes diluted with chromatographically compatible solvent (e.g. mobile phase eluent). Supernatant can be directly injected or pre-concentrated after evaporation and reconstitution. Although only proteins are removed, other endogenous compounds remain which can still cause interferences such as matrix effect in mass spectrometry analyses. PP offers a generic and fast sample preparation technique that can be easily automated. The method has been also extended to quantification of drugs and metabolites from whole blood [65]. The same sample preparation technique in not suitable only for plasma but can be transferred to other biological samples such as urine. Moreover, the absence of proteins in these matrices allows direct injection without sample pretreatment. Nevertheless, it is advisable to dilute and filter or centrifuge the samples to reduce matrix effect and to remove eventually present particles [42]. Many examples for metabolite determination using PP, mainly in serum and urine, can be found in recently published review articles [66-68]. PP is also the most convenient method for less complex biological matrices in pharmacokinetic studies, such as hepatocytes [69] or microsomes [4]. In this case protein precipitation by icecold methanol (triple volume) at the same time terminates the incubation reaction and introduces internal standard to the final sample.

## **7.2. Liquid-liquid extraction**

100 Chromatography – The Most Versatile Method of Chemical Analysis

**7. Sample preparation** 

**7.1. Protein precipitation** 

are then the only media for isolation of those metabolites.

sample injection followed by on-line extraction methods.

introduces internal standard to the final sample.

metabolites cannot be produced by proposed *in vitro* models the *in vivo* biological samples

Adequate sample preparation is a key aspect of quantitative bioanalysis and it is usually the most time consuming part of analyses. Interfering matrix compounds, such as proteins, lipids, salts, other endogenous and background compounds, should be removed in sample pretreatment, not only to avoid column clogging and instrument soiling, but also to improve the sensitivity, selectivity and reliability of analyses. Selection of an appropriate preparation procedure depends upon metabolite characteristics, their expected concentrations, the sample size and matrix, and the availability of analytical techniques for analyte quantification. Insufficiently treated samples may cause interfering peaks when using spectroscopic detection techniques such as UV-absorbance or fluorescence. However, analyses by LC-MS/MS are less prone to sample matrix and therefore usually require less pretentious sample clean up. Commonly and widely applied sample preparation techniques include protein precipitation (PP), liquid-liquid extraction (LLE) and solid-phase extraction (SPE). Manual operations associated with sample treatment may be very labor intensive and time consuming and that could be avoided with automation in 96-well plate format or direct

Protein precipitation (PP) is simple and straightforward method widely used in bioanalysis of plasma samples. It is accomplished by using organic solvent (typically acetonitrile or methanol) or an acid (typically perchloric or trichloroacetic acid). It is followed by centrifugation to separate proteins from liquid supernatant and additionally, supernatant is sometimes diluted with chromatographically compatible solvent (e.g. mobile phase eluent). Supernatant can be directly injected or pre-concentrated after evaporation and reconstitution. Although only proteins are removed, other endogenous compounds remain which can still cause interferences such as matrix effect in mass spectrometry analyses. PP offers a generic and fast sample preparation technique that can be easily automated. The method has been also extended to quantification of drugs and metabolites from whole blood [65]. The same sample preparation technique in not suitable only for plasma but can be transferred to other biological samples such as urine. Moreover, the absence of proteins in these matrices allows direct injection without sample pretreatment. Nevertheless, it is advisable to dilute and filter or centrifuge the samples to reduce matrix effect and to remove eventually present particles [42]. Many examples for metabolite determination using PP, mainly in serum and urine, can be found in recently published review articles [66-68]. PP is also the most convenient method for less complex biological matrices in pharmacokinetic studies, such as hepatocytes [69] or microsomes [4]. In this case protein precipitation by icecold methanol (triple volume) at the same time terminates the incubation reaction and 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 reported [72].

## **7.3. Solid-phase extraction**

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 ability of the sorbent [71].

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 metabolites [66]. Another advantage regarding silica based phase is ease of use, since there is no need to keep those phases moistened to maintain interaction. Mix mode ionexchange and ion-exchange sorbents are even more convenient since strongly retained ionic metabolites allow rigorous washing of cartridge (e.g. 100% methanol) achieving cleaner sample with less matrix interferences [73].

Analytical Methods for Quantification of Drug Metabolites in Biological Samples 103

biological samples based upon their particle sizes and also due the chromatographic interaction. The proteins, that are unable to penetrate into the hydrophobic pores and the hydrophilic outer layer of particles, are first eluted to waste, the smaller molecules penetrate into pores and are additionally retained through the hydrophobic forces [78]. RAM columns may be used either in single column mode, being extraction (SPE) and analytical column at same time, or extraction column in combination with second analytical column. Single column mode approach shows simplicity but is limited due to chromatographic separation power [79]. Monolith phases as extraction sorbents for sample treatment looks promising and has been reviewed recently [77]. Monolith columns may be very convenient as single column mode for high throughput method in LC-

LC-MS/MS has become the predominant bioanalysis method for pharmacokinetic and metabolism studies due to its inherent specificity, sensitivity and speed. A literature survey of analytical methods for metabolite determination in biological samples undoubtedly confirms that fact. However, HPLC coupled with other detector systems or other separation techniques is often used. As an example, analytical methods for determination of antidepressants and their metabolites [67] are shown. HPLC coupled to different detectors (73%), among them the most popular being mass spectrometry (35%) and UV detection (24%), is the most frequently used analytical method. Applications of electrophoretic and gas chromatography methods for analysis of antidepressants and their metabolites in biofluids have seldom been published in literature (13 and 9%, respectively). Since the data were collected in time frame 2000-2010 [67], the frequency of LC-MS/MS methods is believed to be growing and is nowadays significantly higher because mass spectrometers are lately more accessible. In this section the most frequently used separation techniques as well as detectors will be overviewed with emphasis on LC-

Good chromatographic separation is prerequisite for reliable and accurate quantification of metabolites in the biological samples. Baseline resolution must be achieved when liquid chromatography is coupled to non-MS detector. Although extensive chromatographic separation using LC-MS/MS is often not necessary, for certain cases, adequate resolution between drugs and various metabolites is required to avoid mass spectrometric interferences. Different metabolites may share the same MRM transition, such as hydroxylate metabolites [81] or glucuronides [37]. An example is shown in Figure 1. Additionally, unstable metabolites, such as N-oxides or glucuronides may be converted to parent drug by in-source dissociation or thermal degradation [79] or in collision cell (ion channel cross-talk). Interferences with endogenous compounds should also be avoided as

**8. Analytical methods for metabolite quantification** 

MS/MS analysis [80].

MS/MS.

**8.1. Liquid chromatography** 

matrix effect may appear (see 9.1.).

There are now commercially available protein precipitation devices in plate format that allow PP within the plate whilst also removing phospholipids (HybridSPETM and Waters OstroTM). This novel semi automated sample clean up procedure includes combination of PP and SPE. Proteins in sample are firstly precipitated with organic solvent, then transferred to SPE and directly injected into the analytical instrument. Method is simple, fast and almost free from phospholipids [74]. This sample preparation approach has been successfully applied in metabolism studies of various drugs [75].

## **7.4. On-line SPE**

The on-line SPE offers speed, high sensitivity by the pre-concentrating factor, and low extraction cost per sample, but typically require the use of program switch valves and column re-configurations [71]. Biological samples can be directly injected into liquid chromatographic system without any sample preparations except for aliquoting samples, adding the internal standard and sometimes sample diluting and/or centrifugation. On-line SPE is considered as another dilute and injection approach like protein precipitation, however, it provides cleaner extract with reduced chance for matrix effect. Commonly used columns for on-line SPE are packed with large particles (typically > 20µm) of stationary material, such as polymeric and silica based, which work based on reversed phase, ionexchange or mixed mode of separation. The combination of large particle size in these narrow bore columns (typically 50x1 mm) and fast flow (typically 3-5 mL/min), called also as turbulent flow chromatography, promotes the rapid removal of proteins with simultaneous retention of the small-molecular analytes of interest. After flushing all the proteins to waste, the direction of the flow is switched; the analytes are back-flushed onto the analytical column for chromatographic separation and detection. Fully integrated homebuilt or commercial systems enable eluting analytes from the extraction column onto analytical column in narrow bands. That allows multiple injections onto analytical column prior to elution into the instrument detector resulting in better sensitivity [76]. Most on-line SPE approaches use column-switching to couple with the analytical column as well as additional HPLC pump. Various instrument setups and column dimensions can be configured for the fast analysis of drugs and their metabolites in biological matrix at the ng/mL levels or lower [71].

Typically, on-line SPE columns can withstand few hundred injections of diluted plasma or urine samples what depends on the injection volume and sample matrix [77]. Beside mentioned SPE sorbents for turbulent flow chromatography, restricted access materials (RAM), monolithic materials and disposable SPE cartridges are available. The working principle of RAM phases is to isolate macromolecules from the target small molecules in biological samples based upon their particle sizes and also due the chromatographic interaction. The proteins, that are unable to penetrate into the hydrophobic pores and the hydrophilic outer layer of particles, are first eluted to waste, the smaller molecules penetrate into pores and are additionally retained through the hydrophobic forces [78]. RAM columns may be used either in single column mode, being extraction (SPE) and analytical column at same time, or extraction column in combination with second analytical column. Single column mode approach shows simplicity but is limited due to chromatographic separation power [79]. Monolith phases as extraction sorbents for sample treatment looks promising and has been reviewed recently [77]. Monolith columns may be very convenient as single column mode for high throughput method in LC-MS/MS analysis [80].

## **8. Analytical methods for metabolite quantification**

LC-MS/MS has become the predominant bioanalysis method for pharmacokinetic and metabolism studies due to its inherent specificity, sensitivity and speed. A literature survey of analytical methods for metabolite determination in biological samples undoubtedly confirms that fact. However, HPLC coupled with other detector systems or other separation techniques is often used. As an example, analytical methods for determination of antidepressants and their metabolites [67] are shown. HPLC coupled to different detectors (73%), among them the most popular being mass spectrometry (35%) and UV detection (24%), is the most frequently used analytical method. Applications of electrophoretic and gas chromatography methods for analysis of antidepressants and their metabolites in biofluids have seldom been published in literature (13 and 9%, respectively). Since the data were collected in time frame 2000-2010 [67], the frequency of LC-MS/MS methods is believed to be growing and is nowadays significantly higher because mass spectrometers are lately more accessible. In this section the most frequently used separation techniques as well as detectors will be overviewed with emphasis on LC-MS/MS.

## **8.1. Liquid chromatography**

102 Chromatography – The Most Versatile Method of Chemical Analysis

cleaner sample with less matrix interferences [73].

applied in metabolism studies of various drugs [75].

**7.4. On-line SPE** 

ng/mL levels or lower [71].

metabolites [66]. Another advantage regarding silica based phase is ease of use, since there is no need to keep those phases moistened to maintain interaction. Mix mode ionexchange and ion-exchange sorbents are even more convenient since strongly retained ionic metabolites allow rigorous washing of cartridge (e.g. 100% methanol) achieving

There are now commercially available protein precipitation devices in plate format that allow PP within the plate whilst also removing phospholipids (HybridSPETM and Waters OstroTM). This novel semi automated sample clean up procedure includes combination of PP and SPE. Proteins in sample are firstly precipitated with organic solvent, then transferred to SPE and directly injected into the analytical instrument. Method is simple, fast and almost free from phospholipids [74]. This sample preparation approach has been successfully

The on-line SPE offers speed, high sensitivity by the pre-concentrating factor, and low extraction cost per sample, but typically require the use of program switch valves and column re-configurations [71]. Biological samples can be directly injected into liquid chromatographic system without any sample preparations except for aliquoting samples, adding the internal standard and sometimes sample diluting and/or centrifugation. On-line SPE is considered as another dilute and injection approach like protein precipitation, however, it provides cleaner extract with reduced chance for matrix effect. Commonly used columns for on-line SPE are packed with large particles (typically > 20µm) of stationary material, such as polymeric and silica based, which work based on reversed phase, ionexchange or mixed mode of separation. The combination of large particle size in these narrow bore columns (typically 50x1 mm) and fast flow (typically 3-5 mL/min), called also as turbulent flow chromatography, promotes the rapid removal of proteins with simultaneous retention of the small-molecular analytes of interest. After flushing all the proteins to waste, the direction of the flow is switched; the analytes are back-flushed onto the analytical column for chromatographic separation and detection. Fully integrated homebuilt or commercial systems enable eluting analytes from the extraction column onto analytical column in narrow bands. That allows multiple injections onto analytical column prior to elution into the instrument detector resulting in better sensitivity [76]. Most on-line SPE approaches use column-switching to couple with the analytical column as well as additional HPLC pump. Various instrument setups and column dimensions can be configured for the fast analysis of drugs and their metabolites in biological matrix at the

Typically, on-line SPE columns can withstand few hundred injections of diluted plasma or urine samples what depends on the injection volume and sample matrix [77]. Beside mentioned SPE sorbents for turbulent flow chromatography, restricted access materials (RAM), monolithic materials and disposable SPE cartridges are available. The working principle of RAM phases is to isolate macromolecules from the target small molecules in Good chromatographic separation is prerequisite for reliable and accurate quantification of metabolites in the biological samples. Baseline resolution must be achieved when liquid chromatography is coupled to non-MS detector. Although extensive chromatographic separation using LC-MS/MS is often not necessary, for certain cases, adequate resolution between drugs and various metabolites is required to avoid mass spectrometric interferences. Different metabolites may share the same MRM transition, such as hydroxylate metabolites [81] or glucuronides [37]. An example is shown in Figure 1. Additionally, unstable metabolites, such as N-oxides or glucuronides may be converted to parent drug by in-source dissociation or thermal degradation [79] or in collision cell (ion channel cross-talk). Interferences with endogenous compounds should also be avoided as matrix effect may appear (see 9.1.).

Analytical Methods for Quantification of Drug Metabolites in Biological Samples 105

Reversed phase chromatography is most widely used technique in analysis of drugs and their metabolites due to its extensive application to most small molecules which are separated by their degree of hydrophobic interaction with the stationary phase. In most cases, metabolic changes lead to an increased polarity of the metabolite (strong shift for glucuronides and other phase II metabolites as also demonstrated in Figure 1) and therefore decreased retention on this stationary phase in relation to the parent drug [34]. The use of gradient elution is usually required to perform analysis of parent drug and polar metabolites. Common HPLC methods typically use a combination of water and either methanol or acetonitrile containing nonvolatile buffers, such as phosphate buffer and other inorganic additives as mobile phase. However, these nonvolatile additives cannot be recommended for LC-MS/MS because of possible MS contamination and also strong ion suppression effect. Volatile additives are used instead, such as formic or acetic acid (0.1% or lower) or ammonium acetate/formiate (2-10 mM) as salts. In order to maintain consistent chromatographic conditions, the pH of the mobile phase should be two units above or below pKa. C18 column is most commonly used. In some cases for polar metabolites shortchain bonded phases, such as C8, phenyl or cyano are more appropriate. Another effective way to resolve the retention issue is to add ion-paring reagent into mobile phase. The formed neutral ion pars increase retention and also improve peak shape. Among different ion-paring reagents trifluoroacetic acid and other perfluorated acids for basic analytes and for instance nucleoside phosphates for acidic analytes are appropriate for LC-MS/MS analyses [79]. These additives, especially trifluoroacetic acid, should be used at low

HILIC using low aqueous/high organic mobile phase is emerging as a valuable supplement to the reversed phase chromatography for the retention of polar analytes [82]. An appropriate amount of water (usually 5-15%) in the mobile phase is suggested for maintaining a stagnant enriched water layer on the surface of the polar stationary phase where the analytes partite. HILIC separates compounds by eluting with strong organic mobile phase against a hydrophilic stationary phase where elution is driven by increasing the water content in the mobile phase [83]. Although some column companies are marketing columns specific for HILIC, most columns used with normal phases, such as pure silica or cyano columns, can operate in HILIC conditions. The highly volatile organic mobile phases, such as methanol and acetonitrile provide low column backpressure and also increased ionization efficiency for MS detection. It has been reported that the ionization responses for basic and acidic polar compounds were enhanced by 5-8 fold in the positive ionization mode and up to 20-fold in the negative ionization mode by the HILIC LC-MS/MS methods as compared to the reversed phase LC-MS/MS method [84]. Low back-pressure allows higher flow rates and may be used for shortening run times, up to several times [85]. Another advantage of HILIC is the possibility to inject higher volumes of organic solvent

*8.1.1. Reversed phase chromatography* 

concentrations because they cause ion suppression.

*8.1.2. Hydrophilic interaction chromatography (HILIC)* 

**Figure 1.** LC-MS/MS chromatogram of urine sample from a patient receiving raloxifene. MRM transitions represent (A) raloxifene diglucuronide, (B) two raloxifene monoglucuronides (C) parent raloxifene, (D) haloperidol as internal standard. For analysis conditions refer to [37].

#### *8.1.1. Reversed phase chromatography*

104 Chromatography – The Most Versatile Method of Chemical Analysis

**Figure 1.** LC-MS/MS chromatogram of urine sample from a patient receiving raloxifene. MRM transitions represent (A) raloxifene diglucuronide, (B) two raloxifene monoglucuronides (C) parent

raloxifene, (D) haloperidol as internal standard. For analysis conditions refer to [37].

Reversed phase chromatography is most widely used technique in analysis of drugs and their metabolites due to its extensive application to most small molecules which are separated by their degree of hydrophobic interaction with the stationary phase. In most cases, metabolic changes lead to an increased polarity of the metabolite (strong shift for glucuronides and other phase II metabolites as also demonstrated in Figure 1) and therefore decreased retention on this stationary phase in relation to the parent drug [34]. The use of gradient elution is usually required to perform analysis of parent drug and polar metabolites. Common HPLC methods typically use a combination of water and either methanol or acetonitrile containing nonvolatile buffers, such as phosphate buffer and other inorganic additives as mobile phase. However, these nonvolatile additives cannot be recommended for LC-MS/MS because of possible MS contamination and also strong ion suppression effect. Volatile additives are used instead, such as formic or acetic acid (0.1% or lower) or ammonium acetate/formiate (2-10 mM) as salts. In order to maintain consistent chromatographic conditions, the pH of the mobile phase should be two units above or below pKa. C18 column is most commonly used. In some cases for polar metabolites shortchain bonded phases, such as C8, phenyl or cyano are more appropriate. Another effective way to resolve the retention issue is to add ion-paring reagent into mobile phase. The formed neutral ion pars increase retention and also improve peak shape. Among different ion-paring reagents trifluoroacetic acid and other perfluorated acids for basic analytes and for instance nucleoside phosphates for acidic analytes are appropriate for LC-MS/MS analyses [79]. These additives, especially trifluoroacetic acid, should be used at low concentrations because they cause ion suppression.

#### *8.1.2. Hydrophilic interaction chromatography (HILIC)*

HILIC using low aqueous/high organic mobile phase is emerging as a valuable supplement to the reversed phase chromatography for the retention of polar analytes [82]. An appropriate amount of water (usually 5-15%) in the mobile phase is suggested for maintaining a stagnant enriched water layer on the surface of the polar stationary phase where the analytes partite. HILIC separates compounds by eluting with strong organic mobile phase against a hydrophilic stationary phase where elution is driven by increasing the water content in the mobile phase [83]. Although some column companies are marketing columns specific for HILIC, most columns used with normal phases, such as pure silica or cyano columns, can operate in HILIC conditions. The highly volatile organic mobile phases, such as methanol and acetonitrile provide low column backpressure and also increased ionization efficiency for MS detection. It has been reported that the ionization responses for basic and acidic polar compounds were enhanced by 5-8 fold in the positive ionization mode and up to 20-fold in the negative ionization mode by the HILIC LC-MS/MS methods as compared to the reversed phase LC-MS/MS method [84]. Low back-pressure allows higher flow rates and may be used for shortening run times, up to several times [85]. Another advantage of HILIC is the possibility to inject higher volumes of organic solvent

onto the column without impairing peak shapes. Therefore, evaporation and reconstitution step of organic extracts after extraction procedure could be omitted making improvement in sample preparation automation and throughput [86].

Analytical Methods for Quantification of Drug Metabolites in Biological Samples 107

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

studies [4].

*8.2.2. Core-shell column* 

*8.2.3. Monolithic chromatography* 

seconds [71].

phase consumption.

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

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].

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

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
