Determination of illicit and/or abused drugs and compounds of forensic interest in biosamples by capillary electrophoretic/electrokinetic methods

The application of capillary electrophoresis (CE) methods in forensic toxicology for the determination of illicit and/or misused drugs in biological samples is reviewed in the present paper. Sample pretreatments and direct injection modes used in CE for analysis of drugs in biological fluids are briefly described. Besides, applications of separation methods based on capillary zone electrophoresis or micellar electrokinetic chromatography with UV absorbance detection to (i) analysis of drugs of abuse, (ii) analysis of other drugs and toxicants of potential forensic interest and (iii) for metabolism studies are reviewed. Also, alternative CE methods are briefly discussed, including capillary isotachophoresis and separation on mixed polymer networks. High sensitivity detection methods used for forensic drug analysis in biological samples are then presented, particularly those based on laser induced fluorescence. A glimpse of the first examples of application of CE-mass spectrometry in forensic toxicology is finally given.     

Analysis of compounds containing carboxyl groups in biological fluids by capillary electrophoresis

Capillary electrophoresis (CE) is one of the suitable separation techniques used to analyze drugs or metabolites in complicated sample matrices such as plasma, serum and urine. It sometimes requires only a simple process of sample pretreatment, deproteinization, dilution or extraction for biological fluids, otherwise no pretreatment is necessary. Various metabolic disorders concerning the compounds which possess carboxyl groups such as organic acids have been monitored by CE. Drug metabolism in the body can be monitored by the same technique. Recent publications suggest the feasibility of an automated system for diagnosis based on CE technique.     

Activity-dependent changes in voltage-dependent calcium currents and transmitter release

The use of capillary electrophoresis (CE) for clinically relevant assays is attractive since it often presents many advantages over contemporary methods. The small-diameter tubing that holds the separation medium has led to the development of multicapillary instruments, and simultaneous sample analysis. Furthermore, CE is compatible with a wide range of detectors, including UV-Vis, fluorescence, laser-induced fluorescence, electrochemistry, mass spectrometry, radiometric, and more recently nuclear magnetic resonance, and laser-induced circular dichroism systems. Selection of an appropriate detector can yield highly specific analyte detection with good mass sensitivity. Another attractive feature of CE is the low consumption of sample and reagents. However, it is paradoxical that this advantage also leads to severe limitation, namely poor concentration sensitivity. Often high analyte concentrations are required in order to have injection of sufficient material for detection. In this regard, a series of devices that are broadly termed 'analyte concentrators' have been developed for analyte preconcentration on-line with the CE capillary. These devices have been used primarily for non-specific analyte preconcentration using packing material of the C18 type. Alternatively, the use of very specific antibody-containing cartridges and enzyme-immobilized microreactors have been demonstrated. In the current report, we review the likely impact of the technology of capillary electrophoresis and the role of the CE analyte concentrator-microreactor on the analysis of biomolecules, present on complex matrices, in a clinical laboratory. Specific examples of the direct analysis of physiologically-derived fluids and microdialysates are presented, and a personal view of the future of CE in the clinical environment is given.     

Nonaqueous capillary electrophoretic separation and thermo-optical absorbance detection of five tricyclic antidepressants and metabolism of amitriptyline by Cunninghamella elegans

We developed a technique based on nonaqueous capillary electrophoresis and laser-based thermo-optical absorbance detection to assay five antidepressants with similar structures and mass-to-charge ratios. A mixture of methanol and acetonitrile with ammonium acetate was essential to achieve baseline resolution of these compounds. We investigated the effects of ammonium acetate concentration, temperature, applied voltage, and capillary length on separation efficiency. The nonaqueous capillary electrophoresis and laser-based thermo-optical absorbance detection technique was used to study the metabolism of amitriptyline by Cunninghamella elegans. Sample preparation procedures were simplified for fast screening of the parent drug and its metabolites. Reproducible electropherograms were obtained from replicate cultures of C. elegans growing in the presence of amitriptyline.     

Capillary electrophoresis procedures for serum protein analysis: comparison with established techniques

Methods using automated capillary electrophoresis (CE) instrumentation are available for serum protein electrophoresis with monoclonal band quantitation, isoelectric focusing and sodium dodecyl sulphate-polyacrylamide gel electrophoresis separations. The advantages of CE over previous gel methods relate to the time and labour saved by the automated instrumentation. High pI monoclonal bands and cryoglobulin specimens can be successfully analysed by CE. However, if the CE application uses a standard company supplied kit, then the cost savings are often negated by the high cost of the kit. Improvements such as the inclusion of both a UV-Vis as well as a fluorescence detector as standard within the one commercial instrument, the production of coated IEF capillaries with a useful life of at least 100 samples, and the introduction of a capillary array into all commercial instrumentation would ensure greater use of CE within both the clinical and other protein laboratories.     

Investigation of the stereoselective metabolism of praziquantel after incubation with rat liver microsomes by capillary electrophoresis and liquid chromatography-mass spectrometry

Two different separation methods for the antischistosomal drug praziquantel and its metabolites by capillary electrophoresis are described. Achiral separation was obtained by micellar electrokinetic capillary chromatography using sodium dodecyl sulfate as micelle-forming surfactant. On the other hand, the negatively charged sulfobutylether-beta-cyclodextrin as a chiral selector enabled the separation of the drug and its metabolites as well as their enantioseparation. Based on this separation, the enantioselectivity of the metabolism of praziquantel was studied by incubation of the drug with rat liver microsomes. Whereas trans- and cis-4-hydroxypraziquantel were mainly formed from the R-(-)-enantiomer, another, different monohydroxylated metabolite was only formed from the S-(+)-enantiomer. Information about the structure of these metabolites was obtained, using LC-MS.     

Systematic development of on-line membrane preconcentration-capillary electrophoresis-mass spectrometry for the analysis of peptide mixtures

A unique method for analyte preconcentration on-line with CE and CE-mass spectrometry (CE-MS) that can concentrate large sample volumes (> 100 microL) is described. A cartridge containing a suitably impregnated membrane is installed at the inlet of the CE capillary, and this approach is termed membrane preconcentration-CE-MS (mPC-CE-MS). The authors describe the mPC-CE-MS analysis of dilute peptide mixtures (approximately 1 fmol/nL) that are concentrated onto the membrane and subsequently subjected to on-line sample cleanup prior to elution and ultimately CE-MS. The systematic development of mPC-CE-MS is demonstrated, and the necessity of minimizing the adsorptive phase in order to ensure efficient peptide analyte recovery and minimization of organic elution solvent is described. The authors compare and contrast mPC-CE and mPC-CE-MS with PC-CE and solid-phase preconcentration-CE-MS (PC-CE-MS), and describe several limitations of the latter approach, which includes analyte zone broadening and compromised analyte resolution. Finally, the authors show that the use of mPC-CE-MS in conjunction with transient isotachophoresis after the peptides have been eluted from the adsorptive membrane affords optimal performance, and theoretical plate values of up to approximately 2.6 x 10(6) are observed for the analysis of a nine-component peptide mixture using this approach. It is conservatively estimated that, in conjunction with an MS array detector, a practical concentration limit of detection (CLOD) for peptides is approximately 500 fg-1 pg/mL using mPC-CE-MS.     

Tubular-wire dual electrode for detection of thiols and disulfides by capillary electrophoresis/electrochemistry

A new dual-electrode detector for capillary electrophoresis is described. The detector consists of an integrated gold tubular electrode as the generator electrode and a gold wire electrode for detection. The detector configuration, including electrode size and position, has been optimized in terms of detection sensitivity and separation efficiency. After amalgamation of the dual electrode with mercury, the capillary electrophoresis/electrochemistry system was employed for simultaneous detection of thiols and disulfides. The response of cystine was found to be linear from 1 microM to 1 mM with a LOD of 0.5 microM (S/N = 3) and sensitivity of 60 pA/microM. The detection limits represent 200-fold improvement over previously reported dual-electrode designs for the detection of disulfides. The use of this detector for identification of thiol- and disulfide-containing peptides was demonstrated with a tryptic digest of ribonuclease A.     

Capillary electrophoretic purity method for the novel metal chelator TMT-NCS

A capillary electrophoretic method (CE) was developed for determining the purity of the novel metal chelator TMT-NCS. The separation of TMT-NCS from its degradation products, synthetic intermediates and by-products was accomplished using free solution CE in an aqueous-organic solvent system. This compound exhibits a complex impurity profile with the potential for over 30 degradants/impurities. The CE separation was optimized with respect to buffer type and concentration, pH, organic solvent and competitive chelator additive, allowing the resolution of all impurities in under 20 min. The specificity was established by examining stressed samples and evaluating peak purity using a diode-array detector. The sensitivity for low level impurities was optimized using sample stacking. Preliminary validation data were accumulated to support the utility of this method for estimating the purity of this drug intermediate.     

End-column amperometric detection in capillary electrophoresis: influence of separation-related parameters on the observed half-wave potential for dopamine and catechol

Capillary electrophoresis (CE) was coupled to a micro-electrode-based end-column amperometric detector. The influences of separation voltage, CE buffer concentration, and capillary-to-electrode distance on the observed hydrodynamic voltammetry of dopamine and catechol were studied using a separation capillary with an i.d. of 25 microns. It was found that an increased CE voltage, increased buffer concentration, or decreased capillary-to-electrode distance resulted in a positive shift of the observed half-wave potentials for both dopamine and catechol. At a constant separation current of 1.6 microA, the observed half-wave potential was found to increase with applied separation voltage. Furthermore, when experiments were carried out with a platinum quasi-reference electrode instead of a Ag/AgCl reference electrode, similar shifts in half-wave potential were observed. These results indicate that the observed shifts are an effect of the separation voltage rather than the separation current or a change in the reference potential. The characteristics of end-column detection with and without a fracture decoupler were compared. It was found that the effects of separation voltage, CE buffer concentration, and capillary-to-electrode distance were minimized by the use of a decoupling device. The observed half-wave potentials for dopamine and catechol were more positive when a CE capillary without a decoupler was employed compared to when a decoupler was used. Additionally, using the fracture decoupler, the observed half-wave potentials for both dopamine and catechol were approximately the same as when no CE voltage was applied (i.e., when the hydrodynamic voltammograms were recorded under flow injection conditions).     

Hemodynamic cerebral ischemia. An appeal for systematic data gathering prior to a new EC/IC trial

Capillary electrophoresis (CE) equipped with a diode-array detector have been used to determine diagnostic metabolites occurring in urine of patients with various diseases. The urine samples were injected directly onto the CE instrument without any pretreatment. Identification of abnormal metabolites was based on relative migration times and characteristic diode-array spectra. The CE-method readily diagnosed alkaptonuria, neuroblastoma and liver failure due to galactosemia. The simple and automated CE method appears to be suitable for implementation as a screening test in analytical systems aimed at diagnosis of metabolic disorders.     

Affinity capillary electrophoresis in biomolecular recognition

Affinity capillary electrophoresis is a new method for studies of biomolecular recognition. Applications reported in the literature include chiral separation of racemic biomolecules, measurement of binding constants, estimation of kinetic on- and off-rate constants, determination of binding stoichiometries (a useful tool in examining electrostatic interactions), estimation of effective charges and molecular weights of proteins, characterization of enzymatic activities and library screening for tight-binding drug candidates in solution. This technique demands only small amounts of sample (nanolitre injection volumes, picograms of proteins), involves no radiolabelled materials or chemically immobilized ligands, and does not require changes in spectroscopic characteristics upon binding. This paper reviews the most recent applications of affinity capillary electrophoresis and its use in the analysis of biomolecules.     

Precolumn affinity capillary electrophoresis for the identification of clinically relevant proteins in human serum: application to human cardiac troponin I

An approach has been developed to the on-line extraction and identification of clinical disease-state marker proteins in human serum. Fabrication of capillaries with integral packed beds for the online determination of human cardiac troponin I (cTnI), a diagnostic marker for myocardial infarction, at clinically relevant levels (2 nmol/L) in serum is demonstrated. The technique, termed precolumn affinity capillary electrophoresis (PA-CE), utilizes a short (approximately 5 mm) packed bed of porous silica containing covalently immobilized monoclonal anti-cTnI antibodies directly integrated within a separation capillary for the selective retention of cTnI from a complex matrix. Following a rinsing step to eliminate nonspecifically bound serum proteins and other impurities from the column, desorption of the antigen into the separation region of the PA-CE capillary for subsequent measurement of femto-molar amounts of cTnI by CE is effected by the injection of an appropriate elution buffer. Advantages of this approach over previously reported affinity preconcentration techniques, related applications for PA-CE technology, and its potential for use in the development of a certified reference material for cTnI in serum are discussed.     

Behavior of peptides in capillary electrophoresis: effect of peptide charge, mass and structure

In the last decade the large potential of capillary electrophoresis as a technique for separation and characterization of peptides has been demonstrated extensively. In this field, a large number of chemical structures has to be taken into consideration, for which very often no data or even standards are available. As a result, there has been a strong desire to relate electrophoretic behavior to molecular properties and structure of the compounds. The activities in that direction, in the area of capillary zone electrophoresis, are critically reviewed. Special attention is paid to peptide charge, mass, hydrophobicity and structure, and their influence on the selectivity of the separation. Also, some complexation phenomena are discussed.     

Analysis of peptides, proteins, protein digests, and whole human blood by capillary electrophoresis/electrospray ionization-mass spectrometry using an in-capillary electrode sheathless interface

An in-capillary electrode sheathless interface was applied to the capillary electrophoresis/electrospray ionization-mass spectrometry (CE/ESI-MS) analysis of mixtures of small peptides, proteins, and tryptic digests of proteins. The effects of different experimental parameters on the performance of this CE/ESI-MS interface were studied. The distance of the in-capillary electrode from the CE outlet and the length of the electrode inside the capillary had no significant effects on the CE separation and ESI behavior under the experimental conditions used. However, significant enhancement of the sensitivity resulted from the use of narrower CE capillaries. Using a quadrupole mass spectrometer, an aminopropylsilane-coated capillary, and a wide scan mass-to-charge ratio range of 500-1400, detection limits of approximately 4, 1, and 0.6 fmol for cytochrome c and myoglobin were achieved for 75-, 50-, and 30-micron inner diameter capillaries, respectively. Approximately one order of magnitude lower detection limits were achieved under the multiple-ion monitoring mode. The application of the in-capillary electrode sheathless interface to real-world samples was demonstrated by CE/ESI-MS analysis of a human blood sample.     

Angioplasty and primary stenting of the subclavian, innominate, and common carotid arteries in 83 patients

A new macrocyclic of the bis(benzylisoquinoline) alkaloid family, d-(+)-tubocurarine chloride (DTC), has been evaluated as a chiral selector for the separation of optical isomers of organic carboxylates using capillary electrophoresis (CE). The pertinent physicochemical properties, such as absorption spectrum, isoionic point, and solution conformation, of DTC were determined. The effects of varying such experimental parameters as DTC concentration, pH, and methanol content in the running buffer were assessed. CE separation of the enantiomers of 18 different compounds was achieved using DTC as the chiral selector under optimized background electrolytic conditions.     

Post-column derivatization for fluorescence and chemiluminescence detection in capillary electrophoresis

Instrumental developments and applications of post-column derivatization for fluorescence and chemiluminescence detection in capillary electrophoresis (CE) are reviewed. Various systems to merge the reagent solution with the separation medium have been developed, including coaxial capillary reactors, gap reactors and free solution or end-column systems. For all reactor types the geometry of the system, as well as the method to propel the reaction mixture (by pressure or by voltage) appeared to be critical to preserve the separation efficiency. Plate numbers of over 100,000 could be realised with different reactors. The strict requirements on the rate of post-column derivatization reactions to be applied in CE limit the number of different reagents that have been used. For fluorescence detection, with laser or lamps as the excitation source, so far mainly o-phthalaldehyde and its naphthalene analogue have been used as reagent. Derivatization systems that are based on complexation reactions also showed good promise for application in CE. Detection limits could be obtained that were comparable to those obtained after pre-column derivatization. Various reagents for chemiluminescence detection (e.g. the luminol and peroxyoxalate systems) have been studied. The often complicated chemistry involved made application of these reagents in CE even more difficult. Results obtained so far, in terms of sensitivity, have not been up to expectation, with detection limits usually in the order of micromol l(-1).     

On-line nonaqueous capillary electrophoresis and electrospray mass spectrometry of tricyclic antidepressants and metabolic profiling of amitriptyline by Cunninghamella elegans

An on-line nonaqueous capillary electrophoresis-electrospray mass spectrometry (ESI-MS) technique was developed using a commercial ion spray interface. The nonaqueous capillary electrophoresis ESI-MS system was used to profile tricyclic antidepressants of similar structures and mass-to-charge ratios. We found that pure methanol can be used as a sheath liquid to obtain stable ion spray from nonaqueous capillary electrophoresis. The flow rate of the coaxial nebulizing gas affected baseline signals, separation efficiency, and migration times. Other nonaqueous capillary electrophoresis operating conditions and electrospray parameters were optimized for enhanced baseline separation and high sensitivity detection. The effect of sample stacking on separation and detection was evaluated. The calculated detection limits were approximately 3 pg injected onto the capillary. ESI mass spectra of tricyclic antidepressants from a single quadrupole MS were obtained and elucidated. The information was used to propose fragmentation pathways of the tricyclic antidepressants. The method was also used to analyze the metabolites of amitriptyline produced by the fungus Cunninghamella elegans. Sixteen metabolites were detected and most of them were tentatively identified as demethylated and/or hydroxylated, and/or N-oxidized products.     

Chiral surfactants in micellar electrokinetic capillary chromatography

Most biologically active molecules contain one or more chiral centres, giving rise to stereoisomeric forms which can behave differently in a chiral environment. Thus, only one of the enantiomers may show the desired physiological activity, whereas the other enantiomers may either be considerably less active or even show undesireable side effects. Establishing that a chiral drug consists of one single enantiomer is nowadays essential before it can be given to patients. Analytical tools that can discriminate between enantiomers play a very important role in determining the stereoisomeric composition of chiral molecules. Until recently chromatographic techniques were the most popular for enantiomeric separations. The increased use of capillary electrophoresis (CE) has provided complementary methodology for chiral discrimination. One mode of CE that has been used for this purpose is micellar electrokinetic capillary chromatography (MECC), where natural or synthetic chiral surfactants are added to the separation buffer.