Bioanalysis of drugs and their metabolites by chiral electromigration techniques (2010-2020).

The further development and application of capillary electromigration techniques for the enantioselective determination of drugs and their metabolites in body fluids, tissues, and in vitro preparations during the 2010 to 2020 time period continued to proof their usefulness and attractiveness in bioanalysis. This review discusses the principles and important aspects of capillary electrophoresis- based chiral drug bioassays, provides a survey of the assays reported during the past 10 years and presents an overview of the key achievements encountered in that time period. For systems with charged chiral selectors, special attention is paid on assays that feature field-amplified sample injection to enable the determination of ppb levels of analytes and optimized online incubation procedures for the rapid assessment of a metabolic pathway. Applications discussed encompass the pharmacokinetics of drug enantiomers in vivo and in vitro, the impact of inhibitors on metabolic steps, the elucidation of the stereoselectivity of drug metabolism in vivo and in vitro, and drug enantiomers in toxicological, forensic, and doping analysis.

Computer simulation of the isotachophoretic migration and separation of norpseudoephedrine stereoisomers with a free or immobilized neutral chiral selector.

A detailed computer simulation study of the isotachophoretic migration and separation of norpseudoephedrine stereoisomers for cases with the neutral selector added to the leader, immobilized to the capillary wall or support, or partially present in the separation column is presented. The electrophoretic transport of the analytes from the sampling compartment into the separation medium with the selector, the formation of a transient mixed zone, the separation dynamics of the stereoisomers with a free or immobilized selector, the dependence of the leader pH, the ionic mobility of norpseudoephedrine, the complexation constant and selector immobilization on steady-state plateau zone properties, and zone changes occurring during the transition from the chiral environment into a selector free leader are thereby visualized in a hitherto unexplored way. For the case with the selector dissolved in the leading electrolyte, simulation data are compared to those observed in experimental setups with coated fused-silica capillaries that feature minimized electroosmosis and zone detection with conductivity and absorbance detectors.

High-resolution capillary zone electrophoresis and mass spectrometry for distinction of undersialylated and hypoglycosylated transferrin glycoforms in body fluids.

High-resolution capillary zone electrophoresis is used to distinguish transferrin glycoforms present in human serum, cerebrospinal fluid, and serum treated with neuraminidase and N-glycosidase F. The obtained data are compared to mass spectrometry data from the literature. The main focus is on the analysis of the various asialo-transferrin, monosialo-transferrin, and disialo-transferrin molecules found in these samples. The features of capillary zone electrophoresis and mass spectrometry are reviewed and highlighted in the context of the analysis of undersialylated and hypoglycosylated transferrin molecules. High-resolution capillary zone electrophoresis represents an effective tool to assess the diversity of transferrin patterns whereas mass spectrometry is the method of choice to elucidate structural identification about the glycoforms. Hypoglycosylated transferrin glycoforms present in sera of alcohol abusers and normal subjects are structurally identical to those in sera of patients with a congenital disorder of glycosylation type I. Asialo-transferrin, monosialo-transferrin and disialo-transferrin observed in sera of patients with a type II congenital disorder of glycosylation or a hemolytic uremic syndrome, in cerebrospinal fluid and after treatment of serum with neuraminidase are undersialylated transferrin glycoforms with two N-glycans of varying structure. Undersialylated disialo-transferrin is also observed in sera with high levels of trisialo-transferrin.

Contemporary chiral simulators for capillary zone electrophoresis.

For separation of enantiomers in presence of a chiral selector, data obtained with the 1D dynamic simulators SIMUL5complex and GENTRANS are compared to data predicted by PeakMaster 6, a recently released generalized model of the linear theory of electromigration. Four electrophoretic systems with stereoisomers of weak bases were investigated. They deal with the estimation of input data for complexation together with the elucidation of the origin of observed system peaks, the interference of analyte and system peak migration, the change of enantiomer migration order as function of the selector concentration and the inversion of analyte migration direction in presence of a multiply negatively charged selector. For all systems, data predicted with PeakMaster 6 are in agreement with those of the dynamic simulators and simulation data compare well with experimental data that were monitored with setups featuring conductivity and/or UV absorbance detection along the capillary. SIMUL5complex and GENTRANS provide the full dynamics of any buffer and sample arrangement and require very long execution time intervals. PeakMaster 6 is restricted to conventional CZE, is based on an approximate solution of the transport equations, provides data for realistic experimental conditions within seconds and represents a practical tool for an experimentalist.

Inverse cationic ITP for separation of methadone enantiomers with sulfated ß-cyclodextrin as chiral selector.

Chiral ITP of the weak base methadone using inverse cationic configurations with H+ as leading component and multiple isomer sulfated ß-CD (S-ß-CD) as leading electrolyte (LE) additive, has been studied utilizing dynamic computer simulation, a calculation model based on steady-state values of the ITP zones, and capillary ITP. By varying the amount of acidic S-ß-CD in the LE composed of 3-morpholino-2-hydroxypropanesulfonic acid and the chiral selector, and employing glycylglycine as terminating electrolyte (TE), inverse cationic ITP provides systems in which either both enantiomers, only the enantiomer with weaker complexation, or none of the two enantiomers form cationic ITP zones. For the configuration studied, the data reveal that only S-methadone migrates isotachophoretically when the S-ß-CD concentration in the LE is between about 0.484 and 1.113 mM. Under these conditions, R-methadone migrates zone electrophoretically in the TE. An S-ß-CD concentration between about 0.070 and 0.484 mM results in both S- and R-methadone forming ITP zones. With >1.113 mM and < about 0.050 mM of S-ß-CD in the LE both enantiomers are migrating within the TE and LE, respectively. Chiral inverse cationic ITP with acidic S-ß-CD in the LE is demonstrated to permit selective ITP trapping and concentration of the less interacting enantiomer of a weak base.

High-resolution capillary zone electrophoresis for transferrin glycoform analysis associated with congenital disorders of glycosylation.

High-resolution capillary zone electrophoresis is used to assess the transferrin profile in serum of patients with eight different congenital disorders of glycosylation that represent type I, type II, and mixed type I/II disorders. Capillary zone electrophoresis data are compared to patterns obtained by gel isoelectric focusing. The high-resolution capillary zone electrophoresis method is shown to represent an effective tool to assess the diversity of transferrin patterns. Hypoglycosylated disialo-, monosialo-, and asialo-transferrin in type I cases can be distinguished from the corresponding underdesialylated transferrin glycoforms present in type II disorders. The latter can be separated from and detected ahead of their corresponding hypoglycosylated forms of type I patients. Both types of glycoforms are detected in sera of mixed type I/II patients. The assay has the potential to be used as screening method for congenital disorders of glycosylation. It can be run with a few microliters of serum when microvials are used.

Monitoring of transferrin isoforms in biological samples by capillary electrophoresis.

Work dealing with the monitoring of transferrin isoforms in human serum and other body fluids by capillary electrophoresis is reviewed. It comprises capillary zone electrophoresis and capillary isoelectric focusing efforts that led to the exploration and use of assays for the determination of carbohydrate-deficient transferrin as a marker for excessive alcohol intake, genetic variants of transferrin, congenital disorders of glycosylation and ß-2-transferrin, which is a marker for cerebrospinal fluid leakage. This paper provides insight into the development, specifications, strengths, weaknesses, and routine use of the currently known capillary electrophoresis based assays suitable to detect transferrin isoforms in body fluids. The achievements reached so far indicate that capillary zone electrophoresis is an attractive technology to monitor the molecular forms of transferrin in biological specimens as the assays do not require an elaborate sample pretreatment and thus can be fully automated for high-throughput analyses on multicapillary instruments. Assays based on capillary isoelectric focusing are less attractive. They require immunoextraction of transferrin from the biological matrix and mobilization after focusing if instrumentation with a whole-column imaging detector is not available. Interactions of the carrier ampholytes with the iron of transferrin may prevent iron saturation and thus provide more complicated isoform patterns.

Computer simulation and enantioselective capillary electrophoresis to characterize isomer mixtures of sulfated ß-cyclodextrins.

The enantiomeric separation of methadone in the presence of multiple isomer mixtures of sulfated ß-cyclodextrin (S-ß-CD) was studied experimentally with CZE and theoretically using computer simulation. Experiments were performed over many years with several lots of S-ß-CD from the same manufacturer with a specified degree of substitution of 7-11. Large differences in the migration patterns were observed between certain lots and it was concluded that the extent of labelling in lots released after a transition time was higher than originally specified. The migration pattern was observed to be associated with (i) the ionic strength increase resulting from using S-ß-CDs with a higher charge state and (ii) differences in buffer composition. Apparent binding constants between methadone and the S-ß-CD and complex mobilities were determined for different lots of S-ß-CD at varying ionic strength using phosphate and 3-morpholino-2-hydroxypropanesulfonic acid buffers. The obtained values were used as input for simulations. For a given ionic strength, agreement between predicted and experimentally observed behavior was obtained for different buffers. R-methadone has a stronger interaction with S-ß-CD than S-methadone. For any given configuration there is a distinct S-ß-CD concentration range which results in the cationic migration of S-methadone while the migration direction of R-methadone is reversed. This configuration was demonstrated to be applicable for micropreparative CZE separations.

Analysis of genetic variants of transferrin in human serum after desialylation by capillary zone electrophoresis and capillary isoelectric focusing.

Capillary electrophoresis analysis of transferrin in human serum is used to assess genetic variants after desialylation with neuraminidase and iron saturation to reduce the complexity of the transferrin pattern and thus facilitate the recognition of transferrin polymorphisms. Asialo-transferrin forms are analyzed by capillary zone electrophoresis using assay conditions as for the monitoring of carbohydrate-deficient transferrin or by capillary isoelectric focusing in a pH 5-8 gradient which requires immunoextraction of transferrin prior to analysis. With the carrier ampholytes used, peaks for iron saturated and iron depleted transferrin are monitored which indicates complexation of iron ions by carrier ampholytes. For BC, CD, and BD genetic variants, the expected peaks for B, C, and D forms of transferrin were detected with both methods. Monitoring of CC patterns revealed three cases, namely those producing double peaks in both methods, a double peak in capillary isoelectric focusing only and a double peak in capillary zone electrophoresis only. For all samples analyzed, data obtained by capillary isoelectric focusing could be confirmed with gel isoelectric focusing. The two capillary electrophoresis methods are shown to represent effective tools to assess unusual transferrin patterns, including genetic variants with dissimilar abundances of the two forms.

Validation of CE modeling with a contactless conductivity array detector.

Dynamic computer simulation data are compared for the first time with CE data obtained with a laboratory made system comprising an array of 8 contactless conductivity detectors (C(4) Ds). The experimental setup featured a 50 µm id linear polyacrylamide (LPA) coated fused-silica capillary of 70 cm length and a purpose built sequential injection analysis manifold for fluid handling of continuous or discontinuous buffer configurations and sample injection. The LPA coated capillary exhibits a low EOF and the manifold allows the placement of the first detector at about 2.7 cm from the sample inlet. Agreement of simulated electropherograms with experimental data was obtained for the migration and separation of cationic and anionic analyte and system zones in CZE configurations in which EOF and other column properties are constant. For configurations with discontinuous buffer systems, including ITP, experimental data obtained with the array detector revealed that the EOF is not constant. Comparison of simulation and experimental data of ITP systems provided the insight that the EOF can be estimated with an ionic strength dependent model similar to that previously used to describe EOF in fused-silica capillaries dynamically double coated with Polybrene and poly(vinylsulfonate). For the LPA coated capillaries, the electroosmotic mobility was determined to be 17-fold smaller compared to the case with the charged double coating. Simulation and array detection provide means for quickly investigating electrophoretic transport and separation properties. Without realistic input parameters, modeling alone is not providing data that match CE results.

Impact of Taylor-Aris diffusivity on analyte and system zone dispersion in CZE assessed by computer simulation and experimental validation.

Application of pressure-driven laminar flow has an impact on zone and boundary dispersion in open tubular CE. The GENTRANS dynamic simulator for electrophoresis was extended with Taylor-Aris diffusivity which accounts for dispersion due to the parabolic flow profile associated with pressure-driven flow. Effective diffusivity of analyte and system zones as functions of the capillary diameter and the amount of flow in comparison to molecular diffusion alone were studied for configurations with concomitant action of imposed hydrodynamic flow and electroosmosis. For selected examples under realistic experimental conditions, simulation data are compared with those monitored experimentally using modular CE setups featuring both capacitively coupled contactless conductivity and UV absorbance detection along a 50 µm id fused-silica capillary of 90 cm total length. The data presented indicate that inclusion of flow profile based Taylor-Aris diffusivity provides realistic simulation data for analyte and system peaks, particularly those monitored in CE with conductivity detection.

Computer simulation of electrophoretic aspects of enantiomer migration and separation in capillary electrochromatography with a neutral selector.

A computer simulation study describing the electrophoretic separation and migration of methadone enantiomers in presence of free and immobilized (2-hydroxypropyl)-ß-CD is presented. The 1:1 interaction of methadone with the neutral CD was simulated by using experimentally determined mobilities and complexation constants for the complexes in a low-pH BGE comprising phosphoric acid and KOH. The use of complex mobilities represents free solution conditions with the chiral selector being a buffer additive, whereas complex mobilities set to zero provide data that mimic migration and separation with the chiral selector being immobilized, that is CEC conditions in absence of unspecific interaction between analytes and the chiral stationary phase. Simulation data reveal that separations are quicker, electrophoretic displacement rates are reduced, and sensitivity is enhanced in CEC with on-column detection in comparison to free solution conditions. Simulation is used to study electrophoretic analyte behavior at the interface between sample and the CEC column with the chiral selector (analyte stacking) and at the rear end when analytes leave the environment with complexation (analyte destacking). The latter aspect is relevant for off-column analyte detection in CEC and is described here for the first time via the dynamics of migrating analyte zones. Simulation provides insight into means to counteract analyte dilution at the column end via use of a BGE with higher conductivity. Furthermore, the impact of EOF on analyte migration, separation, and detection for configurations with the selector zone being displaced or remaining immobilized under buffer flow is simulated. In all cases, the data reveal that detection should occur within or immediately after the selector zone.

Determination of genetic transferrin variants in human serum by high-resolution capillary zone electrophoresis(†).

High-resolution capillary zone electrophoresis in the routine arena with stringent quality assurance is employed for the determination of carbohydrate-deficient transferrin in human serum. The assay comprises mixing of human serum with a Fe(III) -containing solution prior to analysis of the iron-saturated mixture in a dynamically double-coated capillary using a commercial buffer at alkaline pH. In contrast to other assays, it provides sufficient resolution for proper recognition of genetic transferrin variants. Analysis of 7290 patient sera revealed 166 isoform patterns that could be assigned to genetic variants, namely, 109 BC, 53 CD, one BD and three CC variants. Several subtypes of transferrin D can be distinguished as they have large enough differences in pI values. Subtypes of transferrin C and B cannot be resolved. However, analysis of the detection time ratios of tetrasialo isoforms of transferrin BC and transferrin CD variants revealed multimodal frequency histograms, indicating the presence of subtypes of transferrin C, B and D. The data gathered over 11 years demonstrate the robustness of the high-resolution capillary zone electrophoresis assay. This is the first account of a capillary zone electrophoresis based carbohydrate-deficient transferrin assay with a broad overview on transferrin isoform patterns associated with genetic transferrin variants.

Dynamic high-resolution computer simulation of isotachophoretic enantiomer separation and zone stability.

The development of electrophoretic computer models and their use for simulation of electrophoretic processes has increased significantly during the last few years. Recently, GENTRANS and SIMUL5 were extended with algorithms that describe chemical equilibria between solutes and a buffer additive in a fast 1:1 interaction process, an approach that enables simulation of the electrophoretic separation of enantiomers. For acidic cationic systems with sodium and H3 0(+) as leading and terminating components, respectively, acetic acid as counter component, charged weak bases as samples, and a neutral CD as chiral selector, the new codes were used to investigate the dynamics of isotachophoretic adjustment of enantiomers, enantiomer separation, boundaries between enantiomers and between an enantiomer and a buffer constituent of like charge, and zone stability. The impact of leader pH, selector concentration, free mobility of the weak base, mobilities of the formed complexes and complexation constants could thereby be elucidated. For selected examples with methadone enantiomers as analytes and (2-hydroxypropyl)-ß-CD as selector, simulated zone patterns were found to compare well with those monitored experimentally in capillary setups with two conductivity detectors or an absorbance and a conductivity detector. Simulation represents an elegant way to provide insight into the formation of isotachophoretic boundaries and zone stability in presence of complexation equilibria in a hitherto inaccessible way.

Determination of carbohydrate-deficient transferrin in human serum by capillary zone electrophoresis: evaluation of assay performance and quality assurance over a 10-year period in the routine arena.

The performance of high-resolution CZE for determination of carbohydrate-deficient transferrin (CDT) in human serum based on internal and external quality data gathered over a 10-year period is reported. The assay comprises mixing of serum with a Fe(III) ion-containing solution prior to analysis of the iron saturated mixture in a dynamically double-coated capillary using a commercial buffer at alkaline pH. CDT values obtained with a human serum of a healthy individual and commercial quality control sera are shown to vary less than 10%. Values of a control from a specific lot were found to slowly decrease as function of time (less than 10% per year). Furthermore, due to unknown reasons, gradual changes in the monitored pattern around pentasialo-transferrin were detected, which limit the use of commercial control sera of the same lot to less than 2 years. Analysis of external quality control sera revealed correct classification of the samples over the entire 10-year period. Data obtained compare well with those of HPLC and CZE assays of other laboratories. The data gathered over a 10-year period demonstrate the robustness of the high-resolution CZE assay. This is the first account of a CZE-based CDT assay with complete internal and external quality assessment over an extended time period.

Monitoring of alcohol markers by capillary electrophoresis.

Work dealing with the monitoring of alcohol markers by CE performed during the past two decades led to the development of assays for carbohydrate-deficient transferrin (CDT), ethyl sulfate, ethyl glucuronide, and phosphatidylethanol in body fluids and first attempts for the detection of the urinary 5-hydroxytryptophol/5-hydroxyindoleacetic acid ratio and stable hemoglobin acetaldehyde adducts. Most notably are assays for CDT that have been commercialized and are being used in many laboratories under routine conditions. This paper provides insight into the development, specifications, and use of the currently known CE-based assays suitable to detect alcohol markers. The achievements reached so far indicate that CE is an attractive technology for monitoring alcohol markers. This is particularly seen with the CDT assays that do not require an elaborate sample pretreatment and thus could be fully automated for high-throughput analyses on multicapillary instruments.

Transferrin immunoextraction for determination of carbohydrate-deficient transferrin in human serum by capillary zone electrophoresis.

CZE-based assays for carbohydrate-deficient transferrin (CDT) in which serum is mixed with an Fe(III) ion-containing solution prior to analysis are effective approaches for the determination of CDT in patient samples. Sera of patients with progressed diseases, however, are prone to interferences comigrating with transferrin (Tf) that prevent the proper determination of CDT by CZE in these samples. The need of a simple and economic approach to immunoextract Tf from human serum prompted us to investigate the use of a laboratory-made anti-Tf spin column containing polyclonal rabbit anti-human Tf antibodies linked to Sepharose 4 Fast Flow beads. This article reports extraction column manufacturing and column characterization with sera having normal and elevated CDT levels. The developed procedure was applied to a number of relevant hepatology and dialysis patient samples and could thereby be shown to represent an effective method for extraction and concentration of all Tf isoforms. Furthermore, lipemic sera were delipidated using a mixture of diisopropyl ether and butanol prior to immunoextraction. CDT could unambiguously be determined in all pretreated samples.

Dynamic high-resolution computer simulation of electrophoretic enantiomer separations with neutral cyclodextrins as chiral selectors.

GENTRANS, a comprehensive one-dimensional dynamic simulator for electrophoretic separations and transport, was extended for handling electrokinetic chiral separations with a neutral ligand. The code can be employed to study the 1:1 interaction of monovalent weak and strong acids and bases with a single monovalent weak or strong acid or base additive, including a neutral cyclodextrin, under real experimental conditions. It is a tool to investigate the dynamics of chiral separations and to provide insight into the buffer systems used in chiral capillary zone electrophoresis (CZE) and chiral isotachophoresis. Analyte stacking across conductivity and buffer additive gradients, changes of additive concentration, buffer component concentration, pH, and conductivity across migrating sample zones and peaks, and the formation and migration of system peaks can thereby be investigated in a hitherto inaccessible way. For model systems with charged weak bases and neutral modified ß-cyclodextrins at acidic pH, for which complexation constants, ionic mobilities, and mobilities of selector-analyte complexes have been determined by CZE, simulated and experimentally determined electropherograms and isotachopherograms are shown to be in good agreement. Simulation data reveal that CZE separations of cationic enantiomers performed in phosphate buffers at low pH occur behind a fast cationic migrating system peak that has a small impact on the buffer composition under which enantiomeric separation takes place.

Confirmation analysis of ethyl glucuronide and ethyl sulfate in human serum and urine by CZE-ESI-MS(n) after intake of alcoholic beverages.

CZE coupled to sheath liquid-based electrospray ionization (ESI) and multiple-stage ion trap mass spectrometry (MS(n) ) was used for the confirmation analysis of ethyl glucuronide (EtG) and ethyl sulfate (EtS) in human serum and urine collected after intake of alcoholic beverages. Electrophoretic separations were performed in uncoated fused-silica capillaries using a pH 9.5 ammonium acetate background electrolyte and normal polarity. MS detection of EtG and EtS occurred after negative ionization using a spray liquid containing 0.5% v/v ammonia in isopropanol/water (60:40%, v/v). CZE-MS and CZE-MS² results obtained after injection of solid-phase extracts for EtG and EtS and of diluted urine confirmed the presence of EtG and EtS in samples whose levels were previously determined by CZE with indirect UV detection. Detection limits of each compound were estimated to be around 2.0 (injection of diluted urine) and 0.2 µg/mL (extracts).

Stereoselective determination of drugs and metabolites in body fluids, tissues and microsomal preparations by capillary electrophoresis (2000-2010).

During the past two decades, chiral capillary electrophoresis (CE) emerged as a promising, effective and economic approach for the enantioselective determination of drugs and their metabolites in body fluids, tissues and in vitro preparations. This review discusses the principles and important aspects of CE-based chiral bioassays, provides a survey of the assays developed during the past 10 years and presents an overview of the key achievements encountered in that time period. Applications discussed encompass the pharmacokinetics of drug enantiomers in vivo and in vitro, the elucidation of the stereoselectivity of drug metabolism in vivo and in vitro, and bioanalysis of drug enantiomers of toxicological, forensic and doping interest. Chiral CE was extensively employed for research purposes to investigate the stereoselectivity associated with hydroxylation, dealkylation, carboxylation, sulfoxidation, N-oxidation and ketoreduction of drugs and metabolites. Enantioselective CE played a pivotal role in many biomedical studies, thereby providing new insights into the stereoselective metabolism of drugs in different species which might eventually lead to new strategies for optimization of pharmacotherapy in clinical practice.