Capillary electrophoresis on-line hyphenated with mass spectrometry for analysis of insulin-like growth factor-1 in pharmaceutical preparations.

Insulin-like growth factor-1 (IGF-1) is a 70-amino acid single-chain polypeptide, which has found application in diagnostics as a biomarker of growth hormone disorders and as a therapy for growth failure in children and adolescents. Due to its strong anabolic effects, it is often abused by athletes for doping purposes. Here, we developed an on-line hyphenated method based on capillary zone electrophoresis (CZE) and triple quadrupole mass spectrometry (MS) detection with electrospray ionization (CZE-electrospray ionization source-MS [CZE-ESI-MS]) for the determination of IGF-1 in pharmaceutical matrices. We achieved a highly efficient, accurate, repeatable, sensitive, and selective analysis of IGF-1 with favorable migration times (<15 min). Optimized and validated CZE-ESI-MS method was successfully applied for the determination of IGF-1 in injectable solutions (Increlex®), and its presence was also confirmed in nutritional preparations (tablets and liquid colostrum). This is the first validated CZE-ESI-MS method for the determination of IGF-1 in pharmaceutical matrices revealing the potential of capillary electrophoresis for its use in drug quality control laboratories with benefits, such as high separation efficiency, high-speed analysis, low sample consumption, as well as environmental and cost aspects.

Capillary electrophoresis-mass spectrometry for intact protein analysis: Pharmaceutical and biomedical applications (2018-March 2023).

Capillary electrophoresis is recognized as a valued separation technique for its high separation efficiency, low sample consumption, good economic and ecological aspects, reproducibility, and complementarity to traditional liquid chromatography techniques. Capillary electrophoresis experiments are generally performed utilizing optical detection, such as ultraviolet or fluorescence detectors. However, in order to provide structural information, capillary electrophoresis hyphenated to highly sensitive and selective mass spectrometry has been developed to overcome the limitations of optical detections. Capillary electrophoresis-mass spectrometry is increasingly popular in protein analysis, including biopharmaceutical and biomedical research. It is frequently applied for the determination of physicochemical and biochemical parameters of proteins, offers excellent performance for in-depth characterizations of biopharmaceuticals at various levels of analysis, and has been also already proven as a promising tool in biomarker discovery. In this review, we focus on the possibilities and limitations of capillary electrophoresis-mass spectrometry for protein analysis at their intact level. Various capillary electrophoresis modes and capillary electrophoresis-mass spectrometry interfaces, as well as approaches to prevent protein adsorption and to enhance sample loading capacity, are discussed and the recent (2018-March 2023) developments and applications in the field of biopharmaceutical and biomedical analysis are summarized.

Simultaneous determination of caffeine and its metabolites in rat plasma by UHPLC-MS/MS.

Caffeine is a widely consumed psychostimulant with several mechanisms of action and various positive and negative effects on organisms. Caffeine undergoes extensive hepatic metabolism to form main metabolites such as theobromine, theophylline, paraxanthine, and 1,3,7-trimethyluric acid. However, interspecies diversities have been observed in caffeine metabolism. In the present study, we developed a sensitive and straightforward ultra-high-performance liquid chromatography-tandem mass spectrometry method to quantify caffeine and its primary metabolites, namely theobromine, theophylline, paraxanthine, and 1,3,7-trimethyluric acid in rat plasma. After extraction of analytes using micro solid-phase extraction plate, analytes were separated by elution gradient on the Acquity UPLC HSS T3 (50 × 2.1 mm, 1.8 µm) column over 4 min. The detection was done on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring modes using a positive electrospray ionization interface. The method was successfully validated according to the European Medicine Agency guideline over a concentration range of 5-1500 ng/ml for caffeine, 5-1200 ng/mL for theobromine, and 2.5-1200 ng/mL for theophylline, paraxanthine, and 1,3,7-trimethyluric acid. The developed method was applied to analyze samples from animal experiments focusing on the metabolism and effects of caffeine and caffeine-containing beverages.

Sample preparation and fractionation techniques for intact proteins for mass spectrometric analysis.

The analysis of proteins in biological samples is highly desirable, given their connection to myriad biological functions and disease states, as well as the growing interest in the development of protein-based pharmaceuticals. The introduction and maturation of "soft" ionization methods, such as electrospray ionization and matrix-assisted laser desorption/ionization, have made mass spectrometry an indispensable tool for the analysis of proteins. Despite the availability of powerful instrumentation, sample preparation and fractionation remain among the most challenging aspects of protein analysis. This review summarizes these challenges and provides an overview of the state-of-the-art in sample preparation and fractionation of proteins for mass spectrometric analysis, with an emphasis on those used for top-down proteomic approaches. Biological fluids, particularly important for clinical and pharmaceutical applications and their characteristics are also discussed. While immunoaffinity-based methods are addressed, more attention is given to non-immunoaffinity-based methods, such as precipitation, coacervation, size exclusion, dialysis, solid-phase extraction, and electrophoresis. These techniques are presented in the context of a significant number of studies where they have been developed and utilized.

Effect of difluoroacetic acid and biological matrices on the development of a liquid chromatography-triple quadrupole mass spectrometry method for determination of intact growth factor proteins.

In biological systems, variable protein expression is a crucial marker for numerous diseases, including cancer. The vast majority of liquid chromatography-triple quadrupole mass spectrometry-based quantitative protein assays use bottom-up methodologies, where proteins are subjected to proteolytic cleavage prior to analysis. Here, the effect of difluoroacetic acid and biological matrices on the developement of a multiple reaction monitoring based top-down reversed-phase liquid chromatography-triple quadrupole mass spectrometry method for analysis of cancer-related intact proteins was evaluated. Seven growth factors (5.5-26.5 kDa; isoelectric points: 4.6-9.9) were analyzed on a wide-pore C4 column. The optimized method was performed at 30°C, using a 0.2 mL/min flow rate, a 10 %B/min gradient slope, and 0.05% v/v difluoroacetic acid as a mobile phase modifier. The increase of mass spectrometry sensitivity due to the difluoroacetic acid (estimated limits of detection in biological matrices 1-500 ng/mL) significantly varied for proteins with lower and higher charge state distributions. Matrix effects, as well as the specificity of the method were assessed for variable biological samples and pretreatment methods. This work demonstrates method development to improve the ability to target intact proteins directly by more affordable triple quadrupole mass spectrometry instrumentation, which could be beneficial in many application fields.

Profiling of Amino Acids in Urine Samples of Patients Suffering from Inflammatory Bowel Disease by Capillary Electrophoresis-Mass Spectrometry.

Urine represents a convenient biofluid for metabolomic studies due to its noninvasive collection and richness in metabolites. Here, amino acids are valuable biomarkers for their ability to reflect imbalances of different biochemical pathways. An impact of amino acids on pathology, prognosis and therapy of various diseases, including inflammatory bowel disease (IBD), is therefore the subject of current clinical research. This work is aimed to develop a capillary electrophoresis-tandem mass spectrometry (CE-MS/MS) method for the quantification of the 20 proteinogenic amino acids in human urine samples obtained from patients suffering from IBD and treated with thiopurines. The optimized CE-MS/MS method, with minimum sample preparation (just "dilute and shoot"), exhibited excellent linearity for all the analytes (coefficients of determination were higher than 0.99), with inter-day and intra-day precision yielding relative standard deviations in the range of 0.91-15.12% and with accuracy yielding relative errors in the range of 85.47-112.46%. Total analysis time, an important parameter for the sample throughput demanded in routine practice, was shorter in ca. 17% when compared to established CE-MS methods. Favorable performance of the proposed CE-MS/MS method was also confirmed by the comparison with corresponding ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) method. Consistent data for the investigated amino acid metabolome were obtained using both methods. For the first time, the amino acid profiling by CE-MS approach was applied on the clinical IBD samples. Here, significant differences observed in the concentration levels of some amino acids between IBD patients undergoing thiopurine treatment and healthy volunteers could result from the simultaneous action of the disease and the corresponding therapy. These findings indicate that amino acids analysis could be a valuable tool for the study of mechanism of the IBD treatment by thiopurines.

Comparison of hydrodynamically closed two-dimensional capillary electrophoresis coupled with ultraviolet detection and hydrodynamically open capillary electrophoresis hyphenated with mass spectrometry in the bioanalysis of varenicline.

Two capillary electrophoresis methods for monitoring renally excreted varenicline, a highly effective drug prescribed for smoking cessation, in human urine were developed and compared. A method combining capillary electrophoresis with mass spectrometry was proposed for the fast analysis of varenicline (analysis time up to 7 min). Here, mass spectrometry was a prerequisite for achieving high sensitivity and selectivity of the analysis suitable for the quantification of a 15 ng/mL level of varenicline in un-pretreated urine matrices. An alternative approach, two-dimensional (column-coupled) capillary electrophoresis with enhanced sample load capacity and ultraviolet detection, was proposed as a low-cost alternative to capillary electrophoresis with mass spectrometry. The isotachophoresis on-line sample treatment included simple elimination of the major matrix constituents and stacking of the sample in a large volume so that threefold lower quantitation limits could be easily achieved in comparison to the capillary electrophoresis with mass spectrometry. On the other hand, longer analysis time (ca. 4.5-fold) and more complex electrolyte system in the coupled zone electrophoresis step (including two additives enhancing separation selectivity, i.e. isopropanol and cyclodextrin) were prerequisites for the complete separation of varenicline from the sample matrix. Anyway, both the developed methods were validated according to the Food and Drug Administration guidelines showing favorable performance parameters, suitable for their routine biomedical use.

Two-Dimensional Capillary Electrophoresis with On-Line Sample Preparation and Cyclodextrin Separation Environment for Direct Determination of Serotonin in Human Urine.

An advanced two-dimensional capillary electrophoresis method, based on on-line combination of capillary isotachophoresis and capillary zone electrophoresis with cyclodextrin additive in background electrolyte, was developed for effective determination of serotonin in human urine. Hydrodynamically closed separation system and large bore capillaries (300-800 µm) were chosen for the possibility to enhance the sample load capacity, and, by that, to decrease limit of detection. Isotachophoresis served for the sample preseparation, defined elimination of sample matrix constituents (sample clean up), and preconcentration of the analyte. Cyclodextrin separation environment enhanced separation selectivity of capillary zone electrophoresis. In this way, serotonin could be successfully separated from the rest of the sample matrix constituents migrating in capillary zone electrophoresis step so that human urine could be directly (i.e., without any external sample preparation) injected into the analyzer. The proposed method was successfully validated, showing favorable parameters of sensitivity (limit of detection for serotonin was 2.32 ng·mL-1), linearity (regression coefficient higher than 0.99), precision (repeatability of the migration time and peak area were in the range of 0.02-1.17% and 5.25-7.88%, respectively), and recovery (ranging in the interval of 90.0-93.6%). The developed method was applied for the assay of the human urine samples obtained from healthy volunteers. The determined concentrations of serotonin in such samples were in the range of 12.4-491.2 ng·mL-1 that was in good agreement with literature data. This advanced method represents a highly effective, reliable, and low-cost alternative for the routine determination of serotonin as a biomarker in human urine.

Capillary Electrophoresis Hyphenated with Mass Spectrometry for Determination of Inflammatory Bowel Disease Drugs in Clinical Urine Samples.

Azathioprine is the main thiopurine drug used in the treatment of immune-based inflammations of gastrointestinal tract. For the purpose of therapy control and optimization, effective and reliable analytical methods for a rapid drug monitoring in biological fluids are essential. Here, we developed a separation method based on the capillary electrophoresis (CE) hyphenated with tandem mass spectrometry (MS/MS) for the simultaneous determination of azathioprine and its selected metabolites (6-thioguanine, 6-mercaptopurine, and 6-methylmercaptopurine) as well as other co-medicated drugs (mesalazine, prednisone, and allopurinol). The optimized CE-MS/MS conditions provided a very efficient and stable system for the separation and sensitive detection of these drugs in human urine matrices. The developed method was successfully applied for the assay of the targeted drugs and their selected metabolites in urine samples collected from patients suffering from inflammatory bowel disease (IBD) and receiving azathioprine therapy. The developed CE-MS/MS method, due to its reliability, short analysis time, production of complex clinical profiles, and favorable performance parameters, evaluated according to FDA guidelines for bioanalytical method validation, is proposed for routine clinical laboratories to optimize thiopurine therapy, estimate enzymatic activity, and control patient compliance with medication and co-medication.

Enantioselective column coupled electrophoresis employing large bore capillaries hyphenated with tandem mass spectrometry for ultra-trace determination of chiral compounds in complex real samples.

A new multidimensional analytical approach for the ultra-trace determination of target chiral compounds in unpretreated complex real samples was developed in this work. The proposed analytical system provided high orthogonality due to on-line combination of three different methods (separation mechanisms), i.e. (1) isotachophoresis (ITP), (2) chiral capillary zone electrophoresis (chiral CZE), and (3) triple quadrupole mass spectrometry (QqQ MS). The ITP step, performed in a large bore capillary (800 µm), was utilized for the effective sample pretreatment (preconcentration and matrix clean-up) in a large injection volume (1-10 µL) enabling to obtain as low as ca. 80 pg/mL limits of detection for the target enantiomers in urine matrices. In the chiral CZE step, the different chiral selectors (neutral, ionizable, and permanently charged cyclodextrins) and buffer systems were tested in terms of enantioselectivity and influence on the MS detection response. The performance parameters of the optimized ITP - chiral CZE-QqQ MS method were evaluated according to the FDA guidance for bioanalytical method validation. Successful validation and application (enantioselective monitoring of renally eliminated pheniramine and its metabolite in human urine) highlighted great potential of this chiral approach in advanced enantioselective biomedical applications.

Transient isotachophoresis-Capillary zone electrophoresis-Mass spectrometry method with off-line microscale solid phase extraction pretreatment for quantitation of intact low molecular mass proteins in various biological fluids.

Quantification of proteins is still predominantly done by the traditional bottom-up approach. Targeting of intact proteins in complex biological matrices is connected with multiple challenges during the sample pretreatment, separation, and detection step of the analytical workflow. In this work, we focused on the development of an on-line hyphenated capillary zone electrophoresis-mass spectrometry method employing off-line microscale solid-phase extraction based on hydrophilic lipophilic balance (HLB) sorbent as a sample pretreatment step for the analysis of low molecular mass intact proteins (<20 kDa) spiked in various biological fluids (human serum, plasma, urine, and saliva). A detailed optimization process involved the selection of a suitable capillary surface, background electrolyte (BGE), and comparison of two in-capillary preconcentration methods, namely transient isotachophoresis (tITP) and dynamic pH junction (DPJ), to enhance the sensitivity of the method. Optimum separation of the analytes was achieved using uncoated bare fused silica capillary employing 500 mM formic acid (pH 1.96) + 5 % (v/v) acetonitrile as BGE. tITP was utilized as an optimum preconcentration technique, achieving a 19- to 127-fold increase in the signal intensity when using 200 mM ammonium formate (adjusted to pH 4.00) as the leading electrolyte and BGE as the terminating electrolyte. Off-line microscale solid-phase extraction with various eluate treatment procedures was evaluated to ensure the compatibility of the sample pretreatment method with the selected in-capillary preconcentration, separation, and detection process. Achieved extraction recoveries of spiked proteins were in the range of 76-100 % for urine, 12-54 % for serum, 21-106 % for plasma, and 25-98 % for saliva when the eluate was evaporated and reconstituted in the solution of the leading electrolyte to achieve the tITP process. The optimum method was validated across different biological matrices, offering good linearity, accuracy, and precision, and making it suitable for proteomic studies (e.g., therapeutic drug monitoring, biomarker research) in different biological samples.

On-line coupled capillary isotachophoresis-capillary zone electrophoresis in hydrodynamically closed separation system hyphenated with laser induced fluorescence detection.

An analytical method, based on a column coupling capillary ITP and CZE in a hydrodynamically closed separation mode hyphenated with the detection in the modular arrangement, was developed in this work. Analytical possibilities of this approach are demonstrated on the direct and ultrasensitive quantitative determination of quinine (QUI) in diluted real multicomponent ionic matrices (beverages, urine). The detection cell interface, with the rectangular arrangement of the optical channels inside, connected the separation capillary with the LIF detector via optical fibers in the on-column detection arrangement. ITP enabled the direct large volume (30 µL) injections of the diluted real matrices with an on-line sample pretreatment (preseparation, preconcentration) so that no external sample preparation (except for the dilution) was necessary for the separation of the analyte in the multicomponent ionic matrices. Due to the ITP sample preconcentration and intrinsic sensitivity of the LIF detection, very low concentration LOD (as low as 77 pg/mL), were reached at the same time. This was ca. two orders lower than the corresponding LOD achieved by the same 2D separation system with UV absorbance detection. Compared to the single column CE-LIF methods applied for this model analyte and matrix, this method was found to be superior in terms of concentration LOD, with acceptable selectivity and benefits of the on-line sample preparation. A food control and bioanalytical application clearly illustrates great practical possibilities and routine use of the proposed modular ITP-CZE-LIF technique.

Multidrug analysis of pharmaceutical and urine matrices by on-line coupled capillary electrophoresis and triple quadrupole mass spectrometry.

The present work illustrates potentialities of CE hyphenated with MS/MS for the simultaneous determination and identification of a mixture of simultaneously acting drugs in pharmaceutical and biological matrices. Here, the hyphenation was provided by ESI interface, while the MS/MS technique was based on the triple quadrupole configuration. Three drugs, namely pheniramine, phenylephrine, and paracetamol were determined and identified with high reliability due to their characterization in three different dimensions, i.e. electrophoresis and MS/MS, that prevented practically any interference. Appropriately selected transitions of the analytes (parent ion-quantifier product ion-qualifier product ion) provided their selective determination at maximum S/N. The proposed CE-MS/MS method was validated (LOD/LOQ, linearity, precision, recovery, accuracy) and applied for (i) the multidrug composition pharmaceuticals, namely Theraflu®, and (ii) human urine taken after per-oral administration of the same pharmaceutical preparation. The method was applied also for the investigation of potential weak associates of the drugs and monitoring of predicted (bio)degradation products of the drugs. Successful validation and application of the proposed method suggest its routine use in highly effective and reliable advanced drug control and biomedical research.

[Determination of varenicline in the preparation Champix® with the use of two-dimensional capillary electrophoresis in connection with UV detection]. / Stanovenie vareniklínu v lieku Champix® dvojdimenzionálnou kapilárnou elektroforézou v spojení s UV detekciou.

This paper deals with an analytical method based on two dimensional capillary electrophoresis (CITP-CZE coupling) with simple UV-detection for the determination of a highly effective drug - varenicline. The method was elaborated with the possibility of its future connection with an advanced detection ending - mass spectrometry. The electrolytes for the CITP (LE = 10 mM NH4Ac + 5 mM HAc + 0,05% m-HEC; TE = 10 mM HAc) and CZE (BGE = 20 mM HAc) separation of varenicline were selected considering such requirements. The UV detector was set at the constant wavelength of 237 nm. The presented CITP-CZE-UV combination enabled rapid and effective evaluation of varenicline in the dosage forms with LOD 5.92 ng/ml.

Solid phase extraction as sample pretreatment method for top-down quantitative analysis of low molecular weight proteins from biological samples using liquid chromatography - triple quadrupole mass spectrometry.

Targeting and quantifying intact proteins from biological samples is still a very challenging research area. Several crucial steps exist in the analytical workflow, including development of a reliable sample preparation method. Here, we developed and applied for the first time a non-immunoaffinity sample preparation method based on a generally widely available micro-elution solid phase extraction (µSPE) strategy for the extraction of multiple lower molecular weight intact proteins (<30 kDa) from various biological matrices. Omission of a time-consuming drying and reconstitution step after extraction resulted in a more simple and rapid sample preparation procedure. A model set of eleven intact proteins (molecular weights: 5.5-29 kDa; isoelectric points: 4.5-11.3) were analyzed in multiple biological fluids using reversed-phase liquid chromatography with a triple quadrupole mass spectrometer operated in multiple reaction monitoring mode. Various sample pre-treatment reagents, sorbent types, and washing and elution solvents were experimentally tested and optimized to obtain the µSPE clean-up condition for a broad mixture of intact proteins having variable physicochemical properties. 1% trifluoroacetic acid and 0.2% Triton 100-X were selected as suitable sample pre-treatment reagents for releasing protein-protein interactions in human serum/plasma and human urine, respectively. Hydrophilic lipophilic balanced µSPE sorbent was selected as a high performing stationary phase. Addition of 1% trifluoroacetic acid to all washing and elution solutions showed the most beneficial effect for the extraction recovery of the proteins. Under the optimized conditions, reproducible extraction recoveries >65% for all targeted proteins (up to 30 kDa) in human urine and >50% for most of the proteins in serum/plasma were achieved. The selected conditions were applied also for the analysis of clinical serum and urine samples to demonstrate the feasibility of the developed method to target intact proteins directly by more affordable µSPE sample preparation and triple quadrupole mass spectrometry, which could be beneficial in many application fields.

Determination of quinine in beverages by online coupling capillary isotachophoresis to capillary zone electrophoresis with UV spectrophotometric detection.

The present study illustrates the possibilities of capillary isotachophoresis (CITP) online coupled with capillary zone electrophoresis (CZE) and hyphenated with fiber-based spectrophotometric diode array detection (DAD) for the direct, highly reliable, and ultrasensitive determination of quinine (QUI) in real multicomponent ionic matrices (beverages). Here, the CITP provided an effective online sample pretreatment (preseparation and preconcentration) prior to the CZE separation. Due to the CITP sample preconcentration, a simple UV-visible absorbance spectrophotometric detection was sufficient for obtaining very low concentration limits of detection (~2.3 ng/mL). Enhanced separation selectivity due to the combination of different separation mechanisms (CITP vs. CZE) enabled to obtain a pure analyte zone, suitable for its detection and quantitation in the directly injected real samples. The spectrophotometric DAD, unlike single wavelength UV detection, enabled to characterize the purity (i.e. spectral homogeneity) of the analyte zone and preliminary data indicate structurally related compounds via characteristic spectra recorded in the interval of 200-600 nm. The proposed CITP-CZE-DAD method was characterized by favorable performance parameters (sensitivity, linearity, precision, recovery, accuracy, robustness, and selectivity) and successfully applied to the control of QUI and potential QUI impurities in commercial beverages. This method is proposed as a routine automatized method for the highly reliable quality food control.

Simultaneous determination of twelve biogenic amines in human urine as potential biomarkers of inflammatory bowel diseases by capillary electrophoresis - tandem mass spectrometry.

Biogenic amines (BA) are a broad group of biologically active substances, the presence of which in the human body can provide important diagnostic information for many various pathologies, including chronic inflammation. In this work, a capillary electrophoresis (CE) hyphenated with tandem mass spectrometry (MS/MS) method was developed for the simultaneous determination of twelve BA (histamine, serotonin, dopamine, norepinephrine, epinephrine, putrescine, cadaverine, spermine, spermidine, tyramine, tryptamine, phenylethylamine) in human urine as potential biomarkers of inflammatory bowel diseases (IBD). The electrophoretic separations were carried out in an uncoated fused silica capillary (I.D. 50 µm) using 50 mM formic acid (pH 2.0) as a background electrolyte. A reliable identification of the analytes was based on the combination of time resolution in CE and mass resolution in triple quadrupole MS/MS. The total analysis time of the proposed CEMS/MS method was less than 10 min with the limits of detection in the range of 4.47-144 ng/mL. The intra- and inter-day accuracy ranged in the intervals 89.75-109.4% and 89.99-110.2%, respectively, with the RSD values for the intra- and inter-day precision lower than 14 and 13 %, respectively. The recovery values for the samples spiked at three concentration levels ranged from 81.73-105.6% with a precision not exceeding 9.9 %. The favorable performance parameters of the CEMS/MS method highlighted its usefulness for routine clinical applications. In this work, the CEMS/MS method was applied, for the first time, to the analytical profiling of the BA in clinical human samples. The obtained results showed a statistically significant decrease of serotonin and norepinephrine, and an increase of histamine and spermidine, in the studied group of IBD patients when compared with the control group. These findings could be utilized in studying and clarifying the mechanisms of IBD or relevant therapy.

Advanced CE for chiral analysis of drugs, metabolites, and biomarkers in biological samples.

An analysis of recent trends indicates that CE can show real advantages over chromatographic methods in ultratrace enantioselective determination of biologically active compounds in complex biological matrices. It is due to high separation efficiency and many applicable in-capillary electromigration effects in CE (countercurrent migration, stacking effects) enhancing significantly (enantio)separability and enabling effective sample preparation (preconcentration, purification, analyte derivatization). Other possible on-line combinations of CE, such as column coupled CE-CE techniques and implementation of nonelectrophoretic techniques (extraction, membrane filtration, flow injection) into CE, offer additional approaches for highly effective sample preparation and separation. CE matured to a highly flexible and compatible technique enabling its hyphenation with powerful detection systems allowing extremely sensitive detection (e.g. LIF) and/or structural characterization of analytes (e.g. MS). Within the last decade, more as well as less conventional analytical on-line approaches have been effectively utilized in this field and their practical potentialities are demonstrated on many new application examples in this article. Here, three basic areas of (enantioselective) drug bioanalysis are highlighted and supported by a brief theoretical description of each individual approach in a compact review structure (to create integrated view on the topic), including (i) progressive enantioseparation approaches and new enantioselective agents, (ii) in-capillary sample preparation (preconcentration, purification, derivatization), and (iii) detection possibilities related to enhanced sensitivity and structural characterization.