Mobilization in two-step capillary isoelectric focusing: Concepts assessed by computer simulation.

The mobilization step in a two-step capillary isoelectric focusing protocol is discussed by means of dynamic computer simulation data for systems without and with spacer compounds that establish their zones at the beginning and end of the focusing column. After focusing in an electroosmosis-free environment (first step), mobilization (second step) can be induced electrophoretically, by the application of a hydrodynamic flow, or by a combination of both means. Dynamic simulations provide insight into the complexity of the various modes of electrophoretic mobilization and dispersion associated with hydrodynamic mobilization. The data are discussed together with the relevant literature.

Electrophoretic mobilization in capillary isoelectric focusing by a weak acid or an acidic ampholyte as catholyte assessed by computer simulation.

Capillary isoelectric focusing (CIEF) with cationic electrophoretic mobilization induced via replacing the catholyte with the anolyte or a solution of another acid or amino acid was investigated by computer simulation for a wide range pH gradient bracketed between two amphoteric spacers and short electrode vials with a higher id than the capillary. Dynamic simulations provide insight into the complexity of the mobilizing process in a hitherto inaccessible way. The electrophoretic mobilizing process begins with the penetration of the mobilizing compound through the entire capillary, is followed by a gradual or steplike decrease of pH, and ends in an environment with a non-homogenous solution of the mobilizer. Analytes do not necessarily pass the point of detection in the order of decreasing pI values. Cationic mobilization encompasses an inherent zone dispersing and refocusing process toward the capillary end. This behavior is rather strong with phosphoric acid and citric acid, moderate with aspartic acid, glutamic acid (GLU), formic acid, and acetic acid and less pronounced in the absence of the cathodic spacer. The data reveal that optical detectors should not be placed before 90% of capillary length. Aspartic acid, GLU, formic acid, and acetic acid provide an environment with a continuously decreasing pH that explains their successful use in optimized two-step CIEF protocols.

Enantioselective CE-MS analysis of ketamine metabolites in urine.

The chiral drug ketamine has long-lasting antidepressant effects with a fast onset and is also suitable to treat patients with therapy-resistant depression. The metabolite hydroxynorketamine (HNK) plays an important role in the antidepressant mechanism of action. Hydroxylation at the cyclohexanone ring occurs at positions 4, 5, and 6 and produces a total of 12 stereoisomers. Among those, the four 6HNK stereoisomers have the strongest antidepressant effects. Capillary electrophoresis with highly sulfated γ-cyclodextrin (CD) as a chiral selector in combination with mass spectrometry (MS) was used to develop a method for the enantioselective analysis of HNK stereoisomers with a special focus on the 6HNK stereoisomers. The partial filling approach was applied in order to avoid contamination of the MS with the chiral selector. Concentration of the chiral selector and the length of the separation zone were optimized. With 5% highly sulfated γ-CD in 20 mM ammonium formate with 10% formic acid and a 75% filling the four 6HNK stereoisomers could be separated with a resolution between 0.79 and 3.17. The method was applied to analyze fractionated equine urine collected after a ketamine infusion and to screen the fractions as well as unfractionated urine for the parent drug ketamine and other metabolites, including norketamine and dehydronorketamine.

Dynamic computer simulations of electrophoresis: 2010-2020.

The transport of components in liquid media under the influence of an applied electric field can be described with the continuity equation. It represents a nonlinear conservation law that is based upon the balance laws of continuous transport processes and can be solved in time and space numerically. This procedure is referred to as dynamic computer simulation. Since its inception four decades ago, the state of dynamic computer simulation software and its use has progressed significantly. Dynamic models are the most versatile tools to explore the fundamentals of electrokinetic separations and provide insights into the behavior of buffer systems and sample components of all electrophoretic separation methods, including moving boundary electrophoresis, CZE, CGE, ITP, IEF, EKC, ACE, and CEC. This article is a continuation of previous reviews (Electrophoresis 2009, 30, S16-S26 and Electrophoresis 2010, 31, 726-754) and summarizes the progress and achievements made during the 2010 to 2020 time period in which some of the existing dynamic simulators were extended and new simulation packages were developed. This review presents the basics and extensions of the three most used one-dimensional simulators, provides a survey of new one-dimensional simulators, outlines an overview of multi-dimensional models, and mentions models that were briefly reported in the literature. A comprehensive discussion of simulation applications and achievements of the 2010 to 2020 time period is also included.

The effect of pH adjusted electrolytes on capillary isoelectric focusing assessed by high-resolution dynamic computer simulation.

The effect of the composition of electrolytes on capillary IEF is assessed for systems with carrier ampholytes covering two pH units and with catholytes of decreased pH, anolytes of increased pH, and both electrode solutions with adjusted pH values. For electrolytes composed of formic acid as anolyte and ammonium hydroxide as catholyte, simulation is demonstrated to provide the expected IEF system in which analytes with pI values within the formed pH gradient are focused and become immobile. Addition of formic acid to the catholyte results in the formation of an isotachophoretic zone structure that migrates toward the cathode. With ammonium hydroxide added to the anolyte migration occurs toward the anode. In the two cases, all carrier components and amphoteric analytes migrate isotachophoretically as cations or anions, respectively. The data reveal that millimolar amounts of a counter ion are sufficient to convert an IEF pattern into an ITP system. With increasing amounts of the added counter ion, the overall length of the migrating zone structure shrinks, the range of the pH gradient changes, and the migration rate increases. The studied examples indicate that systems of this type reported in the literature should be classified as ITP and not IEF. When both electrolytes are titrated, a non-uniform background electrolyte composed of formic acid and ammonium hydroxide is established in which analytes migrate according to local pH and conductivity without forming IEF or ITP zone structures. Simulation data are in qualitative agreement with previously published experimental data.

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.

Instabilities of the pH gradient in carrier ampholyte-based isoelectric focusing: Elucidation of the contributing electrokinetic processes by computer simulation.

Electrokinetic processes that lead to pH gradient instabilities in carrier ampholyte-based IEF are reviewed. In addition to electroosmosis, there are four of electrophoretic nature, namely (i) the stabilizing phase with the plateau phenomenon, (ii) the gradual isotachophoretic loss of carrier ampholytes at the two column ends in presence of electrode solutions, (iii) the inequality of the mobilities of positively and negatively charged species of ampholytes, and (iv) the continuous penetration of carbonate from the catholyte into the focusing column. The impact of these factors to cathodic and anodic drifts was analyzed by simulation of carrier ampholyte-based focusing in closed and open columns. Focusing under realistic conditions within a 5 cm long capillary in which three amphoteric low molecular mass dyes were focused in a pH 3-10 gradient formed by 140 carrier ampholytes was investigated. In open columns, electroosmosis displaces the entire gradient toward the cathode or anode whereas the electrophoretic processes act bidirectionally with a transition around pH 4 (drifts for pI > 4 and pI < 4 typically toward the cathode and anode, respectively). The data illustrate that focused zones of carrier ampholytes have an electrophoretic flux and that dynamic simulation can be effectively used to assess the magnitude of each of the electrokinetic destabilizing factors and the resulting drift for a combination of these effects. Predicted drifts of focused marker dyes are compared to those observed experimentally in a setup with coated capillary and whole column optical imaging.

Stereoselective methadone disposition after administration of racemic methadone to anesthetized Shetland ponies assessed by capillary electrophoresis.

The enantioselectivity of the pharmacokinetics of methadone was investigated in anesthetized Shetland ponies after a single intravenous (0.5 mg/kg methadone hydrochloride; n = 6) or constant rate infusion (0.25 mg/kg bolus followed by 0.25 mg/kg/h methadone hydrochloride; n = 3) administration of racemic methadone. Plasma concentrations of l-methadone and d-methadone and their major metabolites, l- and d-2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), respectively, were analyzed by CE with highly sulfated γ-cyclodextrin as chiral selector and electrokinetic analyte injection from liquid/liquid extracts prepared at alkaline pH. In both trials, the d-methadone concentrations were lower than those of l-methadone and the d-EDDP levels were lower than those of L-EDDP. For the case of a single intravenous bolus injection, the plasma concentration versus time profile of methadone enantiomers was analyzed with a two-compartment pharmacokinetic model. l-methadone showed a slower elimination rate constant, a lower body clearance, and a smaller steady-state volume of distribution than d-methadone. d-methadone and d-EDDP were eliminated faster than their respective l-enantiomers. This is the first study that outlines that the disposition of racemic methadone administered to anesthetized equines is enantioselective.

Antinociceptive effects of levomethadone in standing horses sedated with romifidine.

OBJECTIVE: To evaluate the antinociceptive effect of a bolus of intravenous levomethadone administered to horses during romifidine constant rate infusion (CRI). STUDY DESIGN: Prospective, randomized, masked, crossover experimental study. ANIMALS: A group of eight adult Warmblood horses (seven geldings, one mare) aged 6.6 ± 4.4 years, weighing 548 ± 52 kg [mean ± standard deviation (SD)]. METHODS: Levomethadone 0.1 mg kg-1 or an equivalent volume of saline (control) was administered intravenously to standing horses 60 minutes after starting a romifidine CRI. Blood samples to quantify romifidine and levomethadone plasma concentrations by capillary electrophoresis were collected up to 150 minutes after levomethadone administration. The nociceptive withdrawal reflex threshold (NWRT) was determined continuously using an automated threshold tracking device. Sedation and cardiopulmonary variables were assessed at regular intervals. A pharmacokinetic-pharmacodynamic (PK-PD) model was elaborated. Data are presented as mean ± SD or median (interquartile range, 25%-75%) where appropriate. Differences between groups were considered statistically significant for p < 0.05. RESULTS: Horses exhibited higher NWRTs after levomethadone administration than after saline (123 ± 9% versus 101 ± 9% relative to baseline, p < 0.05). The PK-PD model identified a contribution of levomethadone to the NWRT increase. Effect size was variable among individuals. No adverse reactions to levomethadone administration were observed. A slight effect of levomethadone on sedation scores was evident for the 60 minutes following its administration. CONCLUSIONS AND CLINICAL RELEVANCE: A single injection of levomethadone has the potential to increase the NWRT during romifidine CRI in horses and can be administered in combination with α2-adrencoceptor agonists to enhance antinociception in horses. However, individual variation is marked.

Pharmacokinetics and pharmacodynamics of l-methadone in isoflurane-anaesthetized and mechanically ventilated ponies.

OBJECTIVE: To evaluate the pharmacokinetics and selected pharmacodynamic effects of a commercially available l-methadone/fenpipramide combination administered to isoflurane anaesthetized ponies. STUDY DESIGN: Prospective single-group interventional study. ANIMALS: A group of six healthy adult research ponies (four mares, two geldings). METHODS: Ponies were sedated with intravenous (IV) detomidine (0.02 mg kg-1) and butorphanol (0.01 mg kg-1) for an unrelated study. Additional IV detomidine (0.004 mg kg-1) was administered 85 minutes later, followed by induction of anaesthesia using IV diazepam (0.05 mg kg-1) and ketamine (2.2 mg kg-1). Anaesthesia was maintained with isoflurane in oxygen. Baseline readings were taken after 15 minutes of stable isoflurane anaesthesia. l-Methadone (0.25 mg kg-1) with fenpipramide (0.0125 mg kg-1) was then administered IV. Selected cardiorespiratory variables were recorded every 10 minutes and compared to baseline using the Wilcoxon signed-rank test. Adverse events were recorded. Arterial plasma samples for analysis of plasma concentrations and pharmacokinetics of l-methadone were collected throughout anaesthesia at predetermined time points. Data are shown as mean ± standard deviation or median and interquartile range (p < 0.05). RESULTS: Plasma concentrations of l-methadone showed a rapid initial distribution phase followed by a slower elimination phase which is best described with a two-compartment model. The terminal half-life was 44.3 ± 18.0 minutes, volume of distribution 0.43 ± 0.12 L kg-1 and plasma clearance 7.77 ± 1.98 mL minute-1 kg-1. Mean arterial blood pressure increased from 85 (±16) at baseline to 100 (±26) 10 minutes after l-methadone/fenpipramide administration (p = 0.031). Heart rate remained constant. In two ponies fasciculations occurred at different time points after l-methadone administration. CONCLUSIONS AND CLINICAL RELEVANCE: Administration of a l-methadone/fenpipramide combination to isoflurane anaesthetized ponies led to a transient increase in blood pressure without concurrent increases in heart rate. Pharmacokinetics of l-methadone were similar to those reported for conscious horses administered racemic methadone.

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.

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.

Pharmacokinetic-pharmacodynamic modelling of the antinociceptive effect of a romifidine infusion in standing horses.

OBJECTIVE: To evaluate the effect of a romifidine infusion on antinociception and sedation, and to investigate its relationship with plasma concentration. STUDY DESIGN: Prospective, experimental, nonrandomized trial. ANIMALS: A total of 10 healthy adult warmblood horses. METHODS: Romifidine (loading dose: 0.08 mg kg-1, infusion: 0.03 mg kg-1 hour-1) was administered intravenously over 120 minutes. Romifidine plasma concentrations were determined by capillary electrophoresis. Sedation quality and nociceptive thresholds were evaluated at regular time points before, during and after romifidine administration. The nociceptive withdrawal reflex was elicited by electrical stimulation at the thoracic limb using a dedicated threshold tracking algorithm and recorded by electromyography at the deltoid muscle. A pharmacokinetic-pharmacodynamic model was established and correlation between romifidine plasma concentration and main output variables tested. RESULTS: A two compartmental model best described the romifidine pharmacokinetic profile. The nociceptive thresholds increased compared with baseline in all horses from 10 to 146 minutes after romifidine administration (p < 0.001). Peak effect reached 5.7 ± 2.3 times the baseline threshold (mean ± standard deviation). The effect/concentration relationship followed a counter-clockwise hysteresis loop. The mean plasma concentration was weakly correlated to nociceptive thresholds (p < 0.0071, r = 0.392). The sedative effects were significant until 160 minutes but variable, not correlated to plasma concentration (p = 0.067), and weakly correlated to nociceptive thresholds (p < 0.0001, r = 0.33). CONCLUSIONS AND CLINICAL RELEVANCE: Romifidine elicited a marked antinociceptive effect. Romifidine-induced antinociception appeared with a delayed onset and lasted longer than sedation after discontinuing its administration.

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.

Enantioselective capillary electrophoresis for pharmacokinetic analysis of methadone and 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine in equines anesthetized with ketamine and isoflurane.

An enantioselective assay for the determination of methadone and its main metabolite 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine in equine plasma based on capillary electrophoresis with highly sulfated γ-cyclodextrin as chiral selector and electrokinetic analyte injection is described. The assay is based on liquid/liquid extraction of the analytes at alkaline pH from 0.1 mL plasma followed by electrokinetic sample injection of the analytes from the extract across a buffer plug without chiral selector. Separation occurs cationically at normal polarity in a pH 3 phosphate buffer containing 0.16% (w/v) of highly sulfated γ-cyclodextrin. The developed assay is precise (intra- and interday RSD < 4% and < 7%, respectively), is capable to determine enantiomer levels of methadone and 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine in plasma down to 2.5 ng/mL, and was successfully applied to monitor enantiomer drug and metabolite levels in plasma of a pony that was anesthetized with racemic ketamine and isoflurane and received a bolus of racemic methadone and a bolus followed by constant rate infusion of racemic methadone. The data suggest that the assay is well suited for pharmacokinetic purposes.

Computer simulation of the enantioselective separation of weak bases in an online capillary electrophoresis based microanalysis configuration comprising sulfated cyclodextrin as selector.

Computer simulation was utilized to characterize the electrophoretic processes occurring after reactant mixing in an online assay format used for monitoring the enantioselective N-demethylation of ketamine to norketamine in the presence of highly sulfated γ-cyclodextrin (HS-γ-CD). The incubated reaction mixture (at pH 7.4 and without chiral selector) is bracketed by a low pH BGE containing 2% HS-γ-CD as chiral selector, thereby forming a discontinuous buffer system. Upon power application, simulation provides insight into the formation of moving boundaries and new zones together with the prediction of the behavior of ketamine and norketamine enantiomers. The analytes first migrate cationically in a zone electrophoretic manner until they come in contact with HS-γ-CD upon which enantioseparation is initiated. Complexation has a focusing effect and the electrophoretic transport becomes reversed, that is, toward the anode. Simulation revealed that the initial conditions for the chiral separation, including buffer components concentrations, pH, and ionic strength, are different than those in the BGE. As a consequence thereof, the experimentally determined complexation parameters for the BGE were unable to correctly describe the migration behavior of the analytes in this column section. An increase in the input binding constants by a factor of two to four, as a result of the decreased ionic strength, resulted in simulation data that agreed with experimental findings.

Enantioselective capillary electrophoresis provides insight into the phase II metabolism of ketamine and its metabolites in vivo and in vitro.

Glucuronidation catalyzed by uridine-5'-diphospho-glucuronosyl-transferases (UGTs) is the most important reaction in phase II metabolism of drugs and other compounds. O-glucuronidation is more common than N-glucuronidation. The anesthetic, analgesic and antidepressive drug ketamine is metabolized in phase I by cytochrome P450 enzymes to norketamine, hydroxynorketamine (HNK) diastereomers and dehydronorketamine (DHNK). Equine urine samples collected two hours after ketamine injection were treated with ß-glucuronidase and analyzed with three enantioselective capillary electrophoresis assays. Concentrations of HNK diastereomers and norketamine were significantly higher in comparison to untreated urine and an increase of ketamine and DHNK levels was found in selected but not all samples. This suggests that O-glucuronides of HNK and N-glucuronides of the other compounds are formed in equines. N-glucuronidation of norketamine was studied in vitro with liver microsomes of different species and the single human enzyme UGT1A4. With equine liver microsomes (ELM) a stereoselective N-glucuronidation of norketamine was found that compares well to the results obtained with urines collected after ketamine administration. No reaction was observed with canine liver microsomes, human liver microsomes and UGT1A4. Incubation of ketamine and DHNK with ELM did not reveal any glucuronidation. Enantioselective CE is suitable to provide insight into the phase II metabolism of ketamine and its metabolites.

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.

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.

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.