Capillary electrophoresis evidence of the stereoselective ketoreduction of mebendazole and aminomebendazole in echinococcosis patients

An assay for the simultaneous determination of the enantiomers of hydroxymebendazole (OH-MBZ) and hydroxyaminomebendazole (OH-AMBZ) together with aminomebendazole (AMBZ) in human plasma is described for the first time. It is based upon liquid-liquid extraction at alkaline pH from 0.5 mL plasma followed by analysis of the reconstituted extract by CE with reversed polarity in the presence of a 50 mM, pH 4.2 acetate buffer containing 15 mg/mL sulfated beta-CD as chiral selector. For all compounds, detection limits are between 0.01 and 0.04 microg/mL, and intraday and interday precisions evaluated from peak area ratios are <6.9 and <8.5%, respectively. Analysis of 39 samples of echinoccocosis patients undergoing pharmacotherapy with mebendazole (MBZ) revealed that the ketoreduction of MBZ and AMBZ is highly stereoselective. One enantiomer of each metabolite (firstly detected peak in both cases) could only be detected. The CE data revealed that OH-MBZ (mean: 0.715 microg/mL) is the major metabolite followed by AMBZ (mean: 0.165 microg/mL) and OH-AMBZ (mean: 0.055 microg/mL) whereas the MBZ plasma levels (mean: 0.096 microg/mL, levels determined by HPLC) were between those of AMBZ and OH-AMBZ.

Determination of carbohydrate-deficient transferrin in human serum by two capillary zone electrophoresis methods and a direct immunoassay: comparison of patient data

Data obtained with two CZE assays for determining carbohydrate-deficient transferrin (CDT) in human serum under routine conditions, the CAPILLARYS CDT and the high-resolution CEofix (HR-CEofix) CDT methods, are in agreement with patient sera that do not exhibit interferences, high trisialo-transferrin (Tf) levels or genetic variants. HR-CEofix CDT levels are somewhat higher compared to those obtained with the CAPILLARYS method and this bias corresponds to the difference of the upper reference values of the two assays. The lower resolution between disialo-Tf and trisialo-Tf observed in the CAPILLARYS system (mean: 1.24) compared to HR-CEofix (mean: 1.74) is believed to be the key for this difference. For critical sera with high trisialo-Tf levels, genetic variants, or certain interferences in the beta-region, the HR-CEofix approach is demonstrated to perform better than CAPILLARYS. However, the determination of CDT with the HR-CEofix method can also be hampered with interferences. Results with disialo-Tf values larger than 3% in the absence of asialo-Tf should be evaluated with immunosubtraction of Tf and possibly also confirmed with another CZE method or by HPLC. Furthermore, data gathered with the N Latex CDT direct immunonephelometric assay suggest that this assay can be used for screening purposes. To reduce the number of false negative results, CDT data above 2.0% should be confirmed using a separation method.

Determination of ethyl glucuronide in human serum by capillary zone electrophoresis and an immunoassay

Ethyl glucuronide (EtG) is a marker of recent alcohol consumption. For the optimization of the analysis of EtG by CZE with indirect absorbance detection, the use of capillaries with permanent and dynamic wall coatings, the composition of the BGE, and various sample preparation procedures, including dilution with water, ultrafiltration, protein precipitation, and SPE, were investigated. Two validated screening assays for the determination of EtG in human serum, a CZE-based approach and an enzyme immunoassay (EIA), are described. The CZE assay uses a coated capillary, 2,4-dimethylglutaric acid as an internal standard, and a pH 4.65 BGE comprising 9 mM nicotinic acid, epsilon-aminocaproic acid and 10% v/v ACN. Proteins are removed via precipitation with ACN prior to analysis and the LOQ is 0.50 mg/L. The EIA is based upon commercial reagents which are promoted for the determination of urinary EtG. Krebs-Ringer solution containing 5% BSA is used as a calibration matrix. All samples are ultrafiltered prior to analysis of the ultrafiltrate on a Mira Plus analyzer. Assay calibration ranged between 0 and 2 mg/L and the upper reference limit was determined to be 0.05 mg/L. Both assays proved to be suitable for the analysis of samples from different individuals. For EtG levels above 0.50 mg/L, good agreement was observed for the comparison of the results of the two methods.

Dynamic computer simulations of electrophoresis: three decades of active research

Dynamic models for electrophoresis are based upon model equations derived from the transport concepts in solution together with user-inputted conditions. They are able to predict theoretically the movement of ions and are as such the most versatile tool to explore the fundamentals of electrokinetic separations. Since its inception three decades ago, the state of dynamic computer simulation software and its use has progressed significantly and Electrophoresis played a pivotal role in that endeavor as a large proportion of the fundamental and application papers were published in this periodical. Software is available that simulates all basic electrophoretic systems, including moving boundary electrophoresis, zone electrophoresis, ITP, IEF and EKC, and their combinations under almost exactly the same conditions used in the laboratory. This has been employed to show the detailed mechanisms of many of the fundamental phenomena that occur in electrophoretic separations. Dynamic electrophoretic simulations are relevant for separations on any scale and instrumental format, including free-fluid preparative, gel, capillary and chip electrophoresis. This review includes a historical overview, a survey of current simulators, simulation examples and a discussion of the applications and achievements of dynamic simulation.

Genotyping of Francisella tularensis strains by pulsed-field gel electrophoresis, amplified fragment length polymorphism fingerprinting, and 16S rRNA gene sequencing

We evaluated three molecular methods for identification of Francisella strains: pulsed-field gel electrophoresis (PFGE), amplified fragment length polymorphism (AFLP) analysis, and 16S rRNA gene sequencing. The analysis was performed with 54 Francisella tularensis subsp. holarctica, 5 F. tularensis subsp. tularensis, 2 F. tularensis subsp. novicida, and 1 F. philomiragia strains. On the basis of the combination of results obtained by PFGE with the restriction enzymes XhoI and BamHI, PFGE revealed seven pulsotypes, which allowed us to discriminate the strains to the subspecies level and which even allowed us to discriminate among some isolates of F. tularensis subsp. holarctica. The AFLP analysis technique produced some degree of discrimination among F. tularensis subsp. holarctica strains (one primary cluster with three major subclusters and minor variations within subclusters) when EcoRI-C and MseI-A, EcoRI-T and MseI-T, EcoRI-A and MseI-C, and EcoRI-0 and MseI-CA were used as primers. The degree of similarity among the strains was about 94%. The percent similarities of the AFLP profiles of this subspecies compared to those of F. tularensis subsp. tularensis, F. tularensis subsp. novicida, and F. philomiragia were less than 90%, about 72%, and less than 24%, respectively, thus permitting easy differentiation of this subspecies. 16S rRNA gene sequencing revealed 100% similarity for all F. tularensis subsp. holarctica isolates compared in this study. These results suggest that although limited genetic heterogeneity among F. tularensis subsp. holarctica isolates was observed, PFGE and AFLP analysis appear to be promising tools for the diagnosis of infections caused by different subspecies of F. tularensis and suitable techniques for the differentiation of individual strains.

Contactless conductivity detector array for capillary electrophoresis.

A CE system featuring an array of 16 contactless conductivity detectors was constructed. The detectors were arranged along 70 cm length of a capillary with 100 cm total length and allow the monitoring of separation processes. As the detectors cannot be accommodated on a conventional commercial instrument, a purpose built set-up employing a sequential injection manifold had to be employed for automation of the fluid handling. Conductivity measurements can be considered universal for electrophoresis and thus any changes in ionic composition can be monitored. The progress of the separation of Na(+) and K(+) is demonstrated. The potential of the system to the study of processes in CZE is shown in two examples. The first demonstrates the differences in the developments of peaks originating from a sample plug with a purely aqueous background to that of a plug containing the analyte ions in the buffer. The second example visualizes the opposite migration of cations and anions from a sample plug that had been placed in the middle of the capillary.

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.

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 threo-methylphenidate enantiomers in oral fluid by capillary electrophoresis with head-column field-amplified sample injection

Threo-methylphenidate is a chiral psychostimulant drug widely prescribed to treat attention-deficit hyperactivity disorder in children and adolescents. An enantioselective CE-based assay with head-column field-amplified sample stacking for analysis of threo-methylphenidate enantiomers in liquid/liquid extracts of oral fluid is described. Analytes are electrokinetically injected across a short water plug placed at the capillary inlet and become stacked at the interface between plug and buffer. Enantiomeric separation occurs within a few minutes in a pH 3.0 phosphate/triethanolamine buffer containing 20 mg/mL (2-hydroxypropyl)-β-CD as chiral selector. The assay with six point multilevel internal calibration provides a linear response for each enantiomer in the 10-200 ng/mL concentration range, is simple, inexpensive, and reproducible, and has an LOQ of 5 ng/mL. It was applied to oral fluid patient samples that were collected up to 12 h after intake of an immediate release tablet and two different extended release formulations with racemic methylphenidate. Drug profiles could thereby be assessed in a stereoselective way. Almost no levorotary threo-methylphenidate enantiomer was detected after intake of the two extended release formulations, whereas this enantiomer was detected during the first 2.5 h after intake of the immediate release preparation. The noninvasive collection of oral fluid is an attractive alternative to plasma for the monitoring of methylphenidate exposure in the pediatric community.

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.

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.

Development of a method for analysis of ketamine and norketamine enantiomers in equine brain and cerebrospinal fluid by capillary electrophoresis

Ketamine and norketamine are being transported across the blood brain barrier and are also entering from blood into cerebrospinal fluid (CSF). Enantioselective distributions of these compounds in brain and CSF have never been determined. The enantioselective CE based assay previously developed for equine plasma was adapted to the analysis of these compounds in equine brain via use of an acidic pre-extraction of interferences prior to liquid/liquid extraction at alkaline pH. CSF can be treated as plasma. With 100 mg of brain tissue and 0.5 mL of CSF or plasma, assay conditions for up to 30 nmol/g and 6 μM, respectively, of each enantiomer with LOQs of 0.5 nmol/g and 0.1 μM, respectively, were established and the assays were applied to equine samples. CSF and plasma samples analyzed stemmed from anesthetized patient horses and brain, CSF and plasma were obtained from anesthetized horses that were euthanized with an overdose of pentobarbital. Data obtained indicate that ketamine and norketamine enantiomers are penetrating into brain and CSF with those of ketamine being more favorably transported than norketamine, whereas metabolites of norketamine are hindered. More work is required to properly investigate possible stereoselectivities of the ketamine metabolism and transport of metabolites from blood into brain tissue and CSF.

Hematology, serum chemistry, and serum protein electrophoresis ranges for free-ranging roe deer (Capreolus capreolus) in Sweden.

We present the first reference ranges for hematology (n = 35 animals), serum biochemistry (n = 62), and serum protein electrophoresis (n = 32) in physically restrained free-ranging roe deer (Capreolus capreolus). Animals were captured in box traps and physically restrained for blood sampling during the winter in Sweden, 2011-13. No clinically significant sex or age differences were found.

Effects of medetomidine and its active enantiomer dexmedetomidine on N-demethylation of ketamine in canines determined in vitro using enantioselective capillary electrophoresis.

Cytochrome P450 (CYP) enzymes catalyze the metabolism of both, the analgesic and anesthetic drug ketamine and the α2 -adrenergic receptor-agonist medetomidine that is used for sedation and analgesia. As racemic medetomidine or its active enantiomer dexmedetomidine are often coadministered with racemic or S-ketamine in animals and dexmedetomidine together with S- or racemic ketamine in humans, drug-drug interactions are likely to occur and have to be characterized. Enantioselective CE with highly sulfated γ-cyclodextrin as chiral selector was employed for analyzing in vitro (i) the kinetics of the N-demethylation of ketamine mediated by canine CYP3A12 and (ii) interactions occurring with racemic medetomidine and dexmedetomidine during coincubation with ketamine and canine liver microsomes (CLM), canine CYP3A12, human liver microsomes (HLM), and human CYP3A4. For CYP3A12 without an inhibitor, Michaelis-Menten kinetics was determined for the single enantiomers of ketamine and substrate inhibition kinetics for racemic ketamine. Racemic medetomidine and dexmedetomidine showed an inhibition of the N-demethylation reaction in the studied canine enzyme systems. Racemic medetomidine is the stronger inhibitor for CLM, whereas there is no difference for CYP3A12. For CLM and CYP3A12, the inhibition of dexmedetomidine is stronger for the R- compared to the S-enantiomer of ketamine, a stereoselectivity that is not observed for CYP3A4. Induction is observed at a low dexmedetomidine concentration with CYP3A4 but not with CYP3A12, CLM, and HLM. Based on these results, S-ketamine combined with dexmedetomidine should be the best option for canines. The enantioselective CE assay with highly sulfated γ-cyclodextrin as chiral selector is an effective tool for determining kinetic and inhibition parameters of metabolic pathways.