Capillary electrophoresis for the determination of drugs in biological fluids

C H A P T E R

4 Capillary electrophoresis for the determination of drugs in biological fluids Wolfgang Thormann* Clinical Pharmacology Laboratory, Institute for Infectious Diseases, University of Bern, Bern, Switzerland *Corresponding author.

Abstract Capillary electrophoresis (CE) is an effective and economic tool for analysis of licit and illicit drugs and their metabolites in biological samples. This chapter provides a brief overview of the principles of CE, the features of CE instrumentation and the key aspects of CE-based drug assays that were developed for therapeutic drug monitoring, clinical and forensic toxicology and the assessment of drug metabolism and pharmacokinetics.

4.1 Introduction Capillary electrophoresis (CE) encompasses a family of techniques carried out under the influence of a DC electric field in capillaries of small radii or microchannels. Capillaries are either filled with a buffer or comprise a chromatographic support together with an appropriate buffer. The electric power applied along the column induces two electrokinetic phenomena, electrophoresis and electroosmosis. Electrophoresis comprises the transport of charged particles or ions relative to the fluid, and electroosmosis represents the movement of the entire liquid within the capillary. Analytes separate when their transport velocities differ. CE exploits numerous separation principles and can thus be applied to the separation and analysis of a broad spectrum of compounds ranging from small molecules and ions to large molecules and particles. The specific CE techniques used for drug monitoring comprise (1) capillary zone electrophoresis (CZE) which refers to separations in aqueous media, (2) nonaqueous CE (NACE) which refers to zone electrophoretic separations in liquid media other than water, (3) capillary isotachophoresis (CITP) which comprises analyses in a discontinuous

Methods of Therapeutic Drug Monitoring including Pharmacogenetics https://doi.org/10.1016/B978-0-444-64066-6.00004-6

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buffer system, (4) capillary electrochromatography (CEC) which encompasses electrophoretic separations in a capillary that contains a chromatographic stationary phase, and (5) a range of electrokinetic capillary chromatography (EKC) techniques, including micellar electrokinetic capillary chromatography (MEKC). EKC techniques operate with two distinct phases (e.g., an aqueous and a micellar phase) and permit separation of neutral and charged molecules. For neutral molecules, separation is based upon differential partitioning between the two phases that are transported at different velocities through the capillary [1e6]. Although CITP analyses were performed in plastic capillaries and microchannels in the 1970s and 1980s [1], the determination of drugs in biological fluids by CE began to spread after commercial instrumentation with fused-silica capillaries became available some 30 years ago [7e26]. The purpose of this chapter is to present the principles, current status, and future outlook of CE for the determination of drugs and metabolites in biological fluids. Instead of a detailed list of references, topical review papers or textbooks which provide the relevant literature are typically cited only. Selected examples in all major fields of drug analysis are mentioned, including those associated with therapeutic drug monitoring (TDM), toxicology, drug metabolism, and pharmacokinetics.

4.2 Instrumentation and methods CE instrumentation comprises a sampler with sample and buffer vials, the capillary, and an on- or off-column detector for analyte detection and identification (Fig. 4.1A). The central part is typically a fused-silica capillary of 25e75 mm I.D. and 5e100 cm length which is mounted between two vials filled with buffer which house the driving electrodes. After filling the capillary with buffer and applying a small amount of sample at one column end, a high-voltage DC electric field is applied along the column which induces the electrokinetic transport due to the combined action of electrophoresis and electroosmosis. Electroosmosis is dependent on the surface charge of the inner capillary wall, as well as the ionic strength and composition (type of buffer components and solvent) of the background electrolyte (BGE). No electroosmosis is observed in an uncharged (neutral) capillary. Electroosmotic flow (EOF) is characterized by a flat flow profile (also referred to as a plug profile) and not a parabolic distribution that is associated with pressure-driven hydrodynamic flow within a capillary tube. Thus, there is hardly any analyte dispersion due to EOF. Sample zone broadening in CE is mainly caused by longitudinal diffusion, electrophoretic dispersion, thermal effects, and analyte sorption to the capillary walls. EOF and sample-wall interactions can be favorably influenced or minimized via the buffer selection (including its pH), permanent modification of the inner walls of capillaries, or a dynamic coating of the wall with agents added to the buffer. Application of a modest hydrodynamic flow to reduce or elongate the presence of the analytes within the capillary can be used without causing appreciable zone dispersion [27]. For CE separations, small amounts of samples (typically nL volumes or pmol analyte quantities) are introduced by electrokinetic (application of power for a specified time interval) or hydrodynamic techniques (application of pressure, vacuum, or gravity for a short time interval) while the capillary end is dipped into the sample vial. Upon application of power (about 5e30 kV, 1e150 mA, 0.1e6 W/m), samples are transported through the capillary and detected on-column via direct or indirect absorbance, direct or indirect fluorescence, or conductivity, as well as endcolumn via mass spectrometry (MS). Fully automated instruments with optical and/or MS

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FIGURE 4.1 (A) Schematic representation of an open tubular capillary electrophoresis (CE) setup with a fusedsilica capillary and on-column solute detection toward the cathodic capillary end. The electroosmotic flow (EOF) is toward the cathode. In the capillary zone electrophoresis (CZE) mode, this configuration permits the detection of cations, neutrals (not separated), and anions whose electrophoretic mobilities are smaller than the electroosmotic mobility. Letters C, N, and A refer to cation, neutral, and anion. (B) CZE electropherogram of a model mixture of cations and anions simultaneously detected by absorbance at 220 nm. The analysis was executed at room temperature in a homebuilt instrument using a 75 mm ID plain fused-silica capillary of 70 cm total (50 cm effective) length, a pH 7.79 buffer composed of 100 mM N-2-aminoethanesulfonic acid (ACES) and 90 mM NaOH, and a constant voltage of 15 kV (current: 80 mA; power level 1.71 W/m). The sample comprised tryptamine (Tra), procaine (Proc), L-tyrosyl-L-a-lysine (Tyr-Lys), tryptophan (Trp), L-tryptophyl-L-glutamic acid (Trp-Glu), salicylurate and salicylate. EO denotes the electroosmotic void peak. From W. Thormann, Theoretical principles of capillary electromigration methods, in: C.F. Poole (Ed.), Handbook of Separation Science, Elsevier, Amsterdam, 2018, pp. 21e44.

detection are currently available from a number of manufacturers, including Sciex (Framingham, MA, USA; formerly instruments of Beckman Coulter) and Agilent Technologies (Waldbronn, Germany). For the monitoring of drugs in body fluids, hair, and tissues, free-fluid CE methods, including CZE, NACE, and MEKC, are mainly employed. CZE and NACE are conducted with a continuous buffer where the samples are the only discontinuities present. Under the influence of an electric field, sample zones migrate without exhibiting any steady-state behavior and thus their shape and position continuously change with time. In these techniques, analyte separation is based upon differences in electrophoretic mobilities. Such analyses can be performed in

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capillaries without any buffer flow or in presence of EOF and/or deliberately applied hydrodynamic flow. Configurations with flow permit the simultaneous determination of cations and anions in the same run (Fig. 4.1B). Separations primarily depend on the buffer and solvent properties which determine both the charge and the solubility of the analytes. Neutral compounds can only be separated if charged complexation agents are added to the buffer and there is a difference in complexation. In MEKC, a combination of electrophoresis and chromatography, two distinct phases are used, an aqueous and a pseudostationary phase. In its most common form, the micellar phase is formed by employing buffers containing charged surfactants (e.g., sodium dodecyl sulfate [SDS]) which are added above their critical micellar concentration. Micelles are dynamic structures which are in equilibrium with the monomer. An MEKC analysis is typically performed in a capillary which exhibits electroosmosis under current flow. Thus, both the movement of the entire liquid and migration of the charged micelles are occurring concurrently. The two phases migrate at different velocities which permit chromatographic separations. Nonionic solutes partition between the two phases and elute with zone velocities between those of the two phases. For that case, separation is solely of chromatographic nature and elution order is based on the degree of partitioning (i.e., in the order of increasing hydrophobicity). For ionic solutes, separation is based on chromatography and charge effects, including electrophoresis. MEKC separations are dependent on micelle formation, buffer composition, and pH, as well as the presence of modifiers, such as small amounts of organic solvents. MEKC drug analysis can also be performed in coated capillaries that do not exhibit an EOF when reversed voltage is applied. In this approach, analytes are detected in the order of decreasing hydrophobicity [28]. This is illustrated with the example depicted in Fig. 4.2. The presented data were obtained with the extract of a quality control serum containing 14 different drugs that was analyzed after liquid/liquid extraction at alkaline pH using a neutral capillary and multiwavelength absorption detection. Peak assignment was accomplished via spectral analysis of the detected peaks. In that approach, normalized spectra of the peaks are compared to those obtained with standards (Fig. 4.2D). The data revealed that this assay can be employed for the simultaneous MEKC analysis of four hydrophobic antiepileptic drugs, carbamazepine (CBZ), carbamazepine-10,11-epoxide (CBZE), phenytoin, and lamotrigine within about 6 min [28]. Other methods employed include CITP, which is performed in a discontinuous buffer system and results in forming a steady-state migrating pattern of consecutive zones between the leader and the terminator [1], and CEC. CEC is a hybrid between CZE and micro-HPLC (high performance liquid chromatography). It comprises capillary columns that are partially packed with a porous solid support (small particles or continuous bed), sorptive interactions between the solutes and the support, and electroosmosis for transport of sample and buffer through the capillary column [29,30]. CEC provides unique selectivities, higher peak efficiencies than in HPLC, and high peak capacities. CITP and CEC are rarely used for the determination of drugs in biological samples.

4.3 Sample preparation Sample preparation for the determination of drugs in a biological specimen is either absent or includes simple liquid handling procedures (e.g., centrifugation, dilution, filtering), the

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FIGURE 4.2 Micellar electrokinetic capillary chromatography (MEKC) electropherograms in absence of electroosmotic flow (EOF) for analysis of hydrophobic drugs. (A) One-dimensional (210 nm) and (B,C) three-dimensional (195e320 nm; 5 nm interval) MEKC electropherograms of an extract of a quality control serum analyzed in a neutral eCAP capillary (50 mm ID, 44.5 cm to the detector) at pH 7.6 with a phosphate/tetraborate buffer containing 60 mM SDS and 4% 2-propanol. Hydrodynamic sample injection was effected at 6 psi x s and the applied voltage was
20 kV. Key (concentration in mM): 4, lamotrigine (12.1); 5, phenytoin (31.7); 6, carbamazepine-10,11-epoxide (CBZE, 9.9); 7, carbamazepine (CBZ, 54.9). M refers to the micelle peak and IS to the internal standard p-bromoacetanilide. Other experimental conditions are described in Ref. [28]. Panel D depicts spectral identity proofs for CBZ, CBZE, phenytoin, lamotrigine, and the IS.

release of the analyte from the biological matrix (e.g., hydrolysis, sonication), the removal of matrix components (e.g., precipitation, ultrafiltration, and extraction), as well as the enhancement of sensitivity by analyte concentration and/or derivatization. The possibility of directly injecting a tiny amount of a body fluid onto the capillary is an appealing feature of electrokinetic capillary techniques. The sample plugs applied are in the 1e20 nL range. Thus, this technology allows drug determinations to be performed in nanoliter samples of body fluids. Proteinaceous fluids, such as serum and plasma, cannot be analyzed by CZE and NACE with direct sample application as the proteins cause deleterious effects, including adsorption to capillary walls, precipitation, and capillary clogging. MEKC with

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dodecyl sulfate, however, was shown to allow direct injection. The proteins are solubilized by dodecyl sulfate and eluted (as a very broad zone) after uric acid. Drugs which elute outside the solubilized proteins can be recognized by UV absorbance or fluorescence detection. Fully validated serum assays for different drugs, including phenobarbital, ethosuximide, flucytosine, antipyrine, felbamate, theophylline, and cefepime, have been published [9,10,13,14,31]. Similarly, drugs can be analyzed in ultrafiltered serum, a method which leads to the determination of the free (unbound) fraction of a drug, saliva, urine, and other body fluids. For UV absorption, direct injection of a body fluid requires that drug concentrations be at or higher than the mg/mL (mM) level. Lower concentrations can be monitored by lamp-based fluorescence or laser-induced fluorescence (LIF) detection for cases with highly fluorescing tags and the availability of optimized excitation wavelengths. For drugs that fluoresce without a tag, the concentration range accessible with fluorescence detection can be comparable to that observed with absorbance detection [32]. Sample dilution can be used for specimens that contain really high amounts of drugs and metabolites. This is often possible for urine analysis. The sample matrix can be simplified by protein precipitation (e.g., with acetonitrile, methanol, or trichloroacetic acid) and injection of the supernatant after centrifugation. This approach is quick, simple, and inexpensive and is applicable to CZE, NACE, and MEKC. It provides, however, diluted samples and is thus only applicable to solutes that are present at high concentrations and/or to configurations with highly sensitive detection. In the case of protein precipitation using acetonitrile, an inherent electrokinetic solute concentration (stacking) effect provides detection limits that are about the same as with direct injection of the body fluid [14]. This is illustrated with a brief description of the CZE-based assay for lamotrigine in serum [33] that was introduced in our laboratory with proper quality assurance for routine use some 20 years ago [34]. After deproteination, the acidified supernatant is analyzed in a buffer composed of 130 mM sodium acetate that was adjusted with acetic acid to pH 4.8. Sample preparation comprises (1) mixing of 50 mL of serum or plasma with 100 mL of acetonitrile containing 30 mg/mL tyramine as internal standard, (2) centrifugation, and (3) mixing of 100 mL of the clear supernatant with 100 mL of 0.9 M acetic acid followed by hydrodynamic injection of an aliquot of this mixture. The assay is based upon five-point internal calibration in the range of 1e10 mg/mL (3.9e39 mM) using corrected peak areas (peak areas divided by detection time) for data evaluation [34]. Using the same approach as for lamotrigine but in combination with LIF detection, Möller et al. described a routine method for the determination of moxifloxacin in human body fluids in the concentration range between 2.5 and 5000 ng/mL [35] and Hempel et al. reported TDM of doxorubicin in pediatric oncology where 10e100 mL plasma samples are to be measured [36]. Alternatively, Theurillat et al. developed an MEKC assay for cefepime in serum and plasma [37,38]. It is based on protein precipitation using SDS at pH 4.5, optional removal of hydrophobic compounds with dichloromethane, and analysis in a pH 9.1 borate/phosphate buffer comprising 75 mM SDS. Sample preparation comprises (1) mixing of 100 mL of serum or plasma with 20 mL of internal standard solution (400 mg/mL ceftazidime in 10-fold diluted pH 4.5 acetate buffer), 50 mL of sample preparation reagent (pH 4.5 acetate buffer with 75 mM SDS), and 50 mL of water, (2) optional addition of 250 mL of dichloromethane to the mixture, and (3) centrifugation followed by hydrodynamic injection of an aliquot of the clear supernatant. The assay is based upon six-point internal calibration in the range of 1e60 mg/mL of cefepime using corrected peak areas (peak areas divided by detection time) for data evaluation.

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The limit for quantitation is 1 mg/mL (smallest calibrator) and the detection limit is 0.5 mg/mL. Intraday and interday drug level repeatabilities are 3.5% and 6.0%, respectively. The MEKC assay is used for TDM in sera from severely ill patients, including those of patients that were simultaneously treated with cefepime and co-trimoxazole. For the latter sera, the use of dichloromethane for removal of sulfamethoxazole and other hydrophobic compounds is recommended. Cefepime levels monitored by MEKC were found to compare well with those obtained by LCeMS [37]. Liquideliquid and solid phase (solideliquid) extraction schemes are greatly simplifying the sample matrix, and, when commenced with large amounts of body fluids, analytes can simultaneously be concentrated by one to two orders of magnitude. Due to the relatively high sample concentrations required for electrokinetic capillary analysis, the latter effect is very important. In most of the applications reported, standard approaches were employed, including the use of disposable cartridges for solid phase extraction [13e15,23,25]. Solid phase extraction was extensively applied to the analysis of multiple classes of drugs in urine and is discussed here briefly. A two-step solid phase extraction scheme from a copolymeric sorbent followed by analyzing aliquots of the two concentrated extracts by MEKC (for most drugs) or CZE (for methadone and its primary metabolite 2-ethylidene-1,5-dimethyl3,3-diphenylpyrrolidine [EDDP]) at alkaline pH and on-column polychrome solute detection was developed [39,40]. The spectral information obtained in that approach is employed for identification of substances (see Fig. 4.2). Methylene chloride was applied as first elutant and contained barbiturates, some benzodiazepines, 11-nor-delta9-tetrahydrocannabinol9-carboxylic acid (THC-COOH), and methaqualone and some of its metabolites. The second elutant, methylene chloride/isopropyl alcohol (80:20) with 3%e5% NH3, contained opiates, cocaine and benzoylecgonine, selected benzodiazepines or their metabolites, methadone, EDDP, diphenhydramine, some methaqualone metabolites, amphetamines, and lysergic acid diethylamide (LSD). Commencing with 5 mL urine, this comprehensive screen was demonstrated to provide a sensitivity of about 50 ng/mL for each compound. This is equal or better than those of most commercial immunoassays typically used for rapid urine screening. This procedure is useful for most drugs of abuse. It is, however, not sensitive enough for LSD which requires a cutoff value of 5 ng/mL. Low ppb concentrations of LSD have been shown to be accessible using electroinjection from a different extract and CE with LIF [41].

4.4 Analyte detection, identification, and quantification Analyte detection is effected on-column by UV absorbance at a single or multiple wavelengths (including fast forward scanning multiwavelength monitoring or diode array detection), fluorescence (lamp-based fluorescence or LIF), conductivity (contactless detector), or off-column by hyphenation with MS. Because of the short optical path length within the detection cell, the lowest detectable concentration in CE (without preconcentration of solutes) with UV absorption detection is in the 1e10 mM range. Using a bubble, a Z-shaped or a multireflection detection cell to increase the path length, about a 10-fold improvement, is obtained. However, any path extension along the capillary axis is accompanied by a loss in column efficiency. Conductivity detection provides detection sensitivities that are comparable with those of UV absorption detection whereas using MS, fluorescence and LIF sensitivity can be 10- to

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1000-fold better. These latter detection modes provide increased selectivity which is useful for identification of analytes. Furthermore, nonabsorbing and nonfluorescing analytes can be detected with indirect absorbance or indirect fluorescence detection, conductivity, and MS. The two indirect detection methods are based on the use of an absorbing or fluorescing BGE compound of like charge. Upon current flow, its concentration deviates from the value in the BGE at the location of an analyte which provides the basis for the localization of the analyte. Analytes can be concentrated via extraction (e.g., up to 50-fold), electrokinetic injection (e.g., up to 100-fold), or a combination of both approaches. Lower sample concentrations can typically be detected when electrokinetic instead of hydrodynamic sample injection is employed [42,43]. Electrokinetic injection from a sample of very low conductivity prepared by extraction has been shown to easily provide a 1000-fold sensitivity enhancement [44e47]. With UV absorption detection, this approach leads to a ppb (ng/mL) solute detection capability from a rather small initial sample volume [45,47]. Alternatively, on-column chromatographic drug concentration methods have been proposed and repeatedly highlighted [48,49]. However, no validated application of such an approach has been reported to date. Analyte identification is based upon detection time and detection response, including absorption spectrum [39], fluorescence spectrum [50], or m/z values in MS. Intraday and interday detection time repeatability is typically <1%. Hyphenations of CE with MS (CEeMS, commercially available [17,23,46,51]) and NMR (CEeNMR, no commercial instrumentation available [52]) are approaches for providing structural information which are important for the identification of drugs and their metabolites. The detection response obtained by conductivity detection does not provide any structural information but can also be employed for drug monitoring, particularly in the case of compounds that cannot be detected by optical means [53]. The same is true for non- or poorly absorbing drugs using CE with indirect detection. Alternatively, such drugs may be chemically derivatized and then detected by UV absorbance and/or fluorimetry [54]. Quantification is typically performed by multilevel internal calibration using peak areas (or peak areas divided by detection time) and by running the samples only once. For analysis by direct injection of the body fluid, no internal standard has to be included and multilevel external calibration can be employed. Intraday and interday imprecisions are on the 1%e5% and 2%e8% levels, respectively. Moreover, high-quality data obtained by CE have also been manifested via analysis of external quality control samples [9,10,14,33e36].

4.5 Analyte separability and assay development In addition to sample preparation and detection, CE assays can be tailored via proper selection of the composition of the BGE, particularly its pH. Other parameters, like solvent (aqueous, binary, or nonaqueous media), buffer concentration, buffer additives, and temperature are also important. Two compounds separate while migrating under the influence of the applied electric field when their mobilities are different. For weak electrolytes, the effective mobility includes the degree of dissociation for a particular ionic species. For example, the effective mobility of a monovalent weak acid or base is equal to the product of the ionic mobility of the fully charged ionic species and the degree of dissociation which reflects the molar fraction of this species. Thus, the separation of acids, bases, and ampholytes can be tuned

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via variation of buffer pH. Acid/base dissociation and association reactions are very fast compared to the time scale of electromigration separation processes such that species of a constituent (e.g., the four species of phosphoric acid or the three species of tryptophan) migrate together and do not separate under the influence of the applied electric field. The involvement of complexation equilibria between analytes and one or several buffer components can be employed to separate similar compounds as long as the complexation reactions are fast. For example, addition of a chiral selector such as a b-cyclodextrin to the BGE in CZE and MEKC provides a simple, inexpensive, and effective approach for the separation of drug enantiomers and other stereoisomers. CE is particularly well suited to monitor the optical isomers of a drug together with its metabolites in one run and this for many drugs of interest in TDM, toxicology, doping control, and drug metabolism studies [19e21,26,55]. The example presented in Fig. 4.3 refers to the CZE separation of the stereoisomers of the weak base ketamine and its metabolites norketamine, 5,6-dehydronorketamine and 6-hydroxynorketamine [47]. In absence of the chiral selector, ketamine, norketamine, and 5,6-dehydronorketamine could not be completely separated in the chosen BGE. The internal standard was detected first and 6-hydroxynorketamine last (Fig. 4.3A). With 0.66% highly sulfated g-cyclodextrin in the BGE, all compounds are being strongly retarded by complexation and all stereoisomers can be separated (Fig. 4.3B). In these experiments, analytes were electrokinetically injected across a 50 mM buffer plug without a chiral selector. This plug is required to avoid a contact of the cationic analytes and the negatively charged chiral selector during electrokinetic injection [47]. Furthermore, the combination of immunochemistry and CE has led to the emergence of competitive binding drug immunoassays using fluorescently labeled drugs as tracers. Most of these procedures have in common that small amounts of body fluids (20e50 mL), antibody solution, and tracer are incubated prior to application of a tiny aliquot of the mixture onto the capillary and separation of the unbound fluorescent tracer and antibodyetracer complex by CZE or MEKC with on-column LIF detection. Single- and multianalyte immunoassays have been described [15,23,56,57]. Assay development includes optimization of sample preparation and composition of the BGE for all analytes of interest, finding of an appropriate internal standard, tests for separability of the analytes from endogenous compounds and metabolites that are not targeted, detection sensitivity of the analytes of interest, and reproducibility of the anticipated result.

4.6 Applications The measurement of drugs, their metabolites, and other exogenous compounds in body fluids and tissues is essential in modern drug therapy, diagnostics, clinical and forensic toxicology, assessment of drug abuse, and research. CE-based assays are being used in all these fields. A number of validated CE-based TDM assays have been described in the literature (for early overviews see Refs. [9,13,14,17,18]). CE assays for single parameters that can accurately and specifically be monitored with automated immunoassays are typically not attractive for routine purposes. However, assays for multiple drugs (e.g., anticonvulsants) or drugs and their pharmacologic active metabolites (e.g., CBZ and CBZE, see Fig. 4.2) are well suited.

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ABSORBANCE (AU)

(A) 0.009 IST

0.006 1,2

0.000

(B) ABSORBANCE (AU)

5,8 3,4 6,7

0.003

4.0

4.5

5.0

5.5

6.0

0.009 IST

0.006 12

0.003

345

6

7

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0.000 5

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9 11 TIME (min)

13

15

FIGURE 4.3 CZE electropherograms of drug standards (about 91 ng/mL each) dissolved in 91 mM phosphoric acid solution and applied via a 15 s electrokinetic injection at 6 kV across a 50 mM phosphate buffer plug (pH 3.0) of 2.68 cm length. A 50 mm ID plain fused-silica capillary of 45 cm total length (effective length 35 cm) was used and detection occurred at 200 nm. The BGE comprised (A) 100 mM phosphate buffer (pH 3.0) and (B) the same buffer to which 0.66% of highly sulfated g-CD was added. The applied voltage and cartridge temperature were 20 kV and 25 C, respectively. The currents were (A) 55 mA and (B) 64 mA. Key: (1) (2R,6R)-hydroxynorketamine, (2) (2S,6S)-hydroxynorketamine, (3) R-norketamine, (4) S-norketamine, (5) S-5,6-dehydronorketamine, (6) R-ketamine, (7) S-ketamine, and (8) R-5,6-dehydronorketamine. IST refers to the internal standard (D-(þ)-norephedrine). The experimental conditions are the same as described in Ref. [47].

Furthermore, if commercial immunoassays are not available or if drugs have to be monitored in small-scale biological samples (i.e., microliter [36,45], submicroliter [58], and even nanoliter [assays with direct injection of body fluids] amounts of body fluids), CE is an attractive alternative to HPLC and LCeMS. This has particularly been shown for TDM of flucytosine in plasma (MEKC assay with direct sample injection [59]), the antiepileptic drug lamotrigine (CZE assay with protein precipitation [17,33,34]), the antibiotic drug cefepime (MEKC assay with protein precipitation [37,38]), the anthracycline cytostatic drugs doxorubicin [36] and daunorubicin [60] (CZE assays with protein precipitation or extraction), the immunosuppressant drug mycophenolic acid (MEKC [61] and CZE [62] assays with protein precipitation), and the anticancer drug imatinib (CZE assays with extraction [63,64]), to name but a few. CE has been applied to comprehensive screening for drugs in body fluids, extracts of organs and hair. Achiral and chiral MEKC and CZE assays with no or minimal sample preparation or with solute extraction, hydrolysis, and stacking have been described and applied to clinical and forensic samples. Assays described include those for amphetamine and analogs in urine and hair, benzodiazepines in urine, barbiturates in urine, and serum, anticonvulsants in

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serum, THC-COOH in urine, opioids in urine, serum, and hair, methaqualone in hair, urine, blood, and gastric content, LSD in blood and urine, methadone in serum, urine, and hair, atropine, strychnine, and tetracaine in blood and gastric content, acetylsalicylic acid and metabolites in plasma and urine, paracetamol and metabolites in urine and serum, and nonopioid analgesics in urine, to name but a few [15e18,22,23,39,40,65e68]. A scheme for analysis of multiple classes of drugs of abuse in urine comprising sequential elution from a copolymeric sorbent followed by analyzing concentrated extracts of the fractions by MEKC (for most drugs) or CZE using on-column polychrome solute detection was designed and successfully applied to a patient and quality control samples [39,40]. The spectral information of a peak is employed for identification of substances (see Fig. 4.2). Alternatively, Hudson et al. developed a comprehensive screen for over 650 drugs of forensic interest in whole blood [68]. In that approach, basic drugs were extracted by liquid/liquid extraction and acidic drugs by solid phase extraction and the extracts were injected by electrokinetic and hydrodynamic principles, respectively. Two CZE buffers were employed. Basic drugs were analyzed at low pH (100 mM phosphate at pH 2.38) whereas acidic drugs were monitored at alkaline pH (100 mM borate at pH 8.5). This methodology was successfully applied to positive whole blood case extracts and quality control. Other noteworthy developments include chiral CE for enantiomer screening of misused, abused, and banned substances, multianalyte CE-based immunoassays for urinary drugs of abuse and analysis and confirmation of urinary drugs and metabolites by CZEeMS. These approaches have successfully been applied to external quality control and patient urines [16,23,65]. CE-based assays are used to characterize and explore the metabolism and pharmacokinetics of many drugs [19e21,26,69e71]. The electrokinetic capillary methodology applied has either provided an attractive alternative to currently available chromatographic methods or has solved an analytical problem. Examples to the latter aspect include (1) the monitoring of drug enantiomers in body fluids [19e21,26], (2) the determination of drugs in small sample environments [36,45,58,72], and (3) the characterization of the pharmacokinetics of macromolecular drug-targeting preparations [73] or antisense drugs [74]. Furthermore, the simplicity of performing chiral analyses by CE has lead to a fair amount of validated enantioselective assays [20,26]. It provided an efficient technology to explore the stereoselectivity of the ketamine metabolism of different species both in vivo [75e78] and in vitro [78e81]. Stereoselective drug metabolism and pharmacokinetics studies not only provide fundamental contributions to drug discovery, development, and regulatory guidelines, they also contribute to the optimization of pharmacotherapy. The chiral assay for verapamil was employed for the assessment of its bioavailability and is claimed to have been validated for enantioselective drug monitoring with more than 100 quality control samples and to have been employed for over 1000 human plasma samples [82].

4.7 Concluding remarks With the advent of fused-silica capillary instrumentation, drug analysis by CE (mainly based upon CZE and MEKC) became popular because of the high resolution achieved, the applicability to hydrophilic and hydrophobic substances, and the possibility to analyze the targeted analytes in small biosamples. CE is a versatile low-cost analytical methodology.

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The possibility of direct sample injection or minimal sample preparation is an appealing feature for analysis of drugs and metabolites in body fluids. On the other hand, the concentration sensitivity of CE is somewhat lower than that of other separation techniques (including HPLC), this often calling for effective on-line or off-line preconcentration of analytes that allow the analysis of drugs and metabolites on the ppb level [43e48]. Buffer consumption and the amount of hazardous chemicals (e.g., organic solvents) are low which make CE assays attractive from economical, health, and environmental points of view. CE methods are robust and reproducible. Imprecisions for single determinations are on the 4%e12% levels, numbers that compare well with those of other methodologies. With a single capillary instrument, sample throughputs range from about 3 to 10 per h. Having instrumentation with multiple capillaries, sample throughputs can be increased to an extent that they become comparable to those obtained in clinical autoanalyzers. CE is complementary to other analytical methods, including HPLC, GC, and high-throughput, automated immuno- and photometric assays. For analysis of drugs and metabolites in biosamples, CE provides meaningful data of clinical and forensic interest and can be applied to drug monitoring in the routine arena [17,18]. CE is currently successfully used for TDM of lamotrigine (since 20 years [34]) and cefepime (since 5 years [37,38]) in Bern. Other routine applications are not known to the author or were discontinued some years ago. As mentioned in this chapter, CE is a useful tool for screening of drugs in body fluids and tissues and to monitor therapeutic drugs in samples of metabolic studies. The latter aspect is used in research laboratories for analysis of drugs and metabolites in biofluids, tissues, and microsomal preparations. Major interest is thereby focused on the elucidation of stereoselective aspects of drug metabolism [19e21,26,71,75e81]. CE permits chiral separations and analyses to be performed very efficiently. Separation conditions can be changed rapidly via a simple buffer change, an approach that does not require lengthy column conditioning procedures. CE enables the simultaneous enantioselective determination of a drug together with its metabolites in real world samples. For such enantioselective analyses, CE offers clear advantages over HPLC.

Acknowledgments The author would like to acknowledge the valuable contributions made by research colleagues, students, postdocs and laboratory technicians during the past 30 years, as well as the support obtained by Bio-Rad Laboratories, Mundipharma Pharmaceuticals, the Liver Foundation in Bern, and the Swiss National Science Foundation.

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