Novel cationic coating agent for protein separation by capillary electrophoresis(†).

A novel positively charged surfactant N-dodecyl-N,N-dimethyl-(1,2-propandiol) ammonium chloride was used for the dynamic coating of the inner wall of a silica capillary. This paper covers the evaluation of dynamic coating and study of the influence of the analysis conditions for the magnitude and direction of electroosmotic flow as well as for the effective and selective separation of chosen proteins (ribonuclease A, cytochrome c, lysozyme, and myoglobin). The concentration of 0.1 mM of N-dodecyl-N,N-dimethyl-(1,2-propandiol) ammonium chloride enabled the reversal of the electro-osmotic flow, however, to separate basic as well as neutral proteins the higher concentration of the studied surfactant was necessary. The final conditions for the separation of studied proteins were set at 100 mM sodium acetate pH 5.5 with 10.0 mM of the studied surfactant. The results were also compared with those of two commercially available cationic surfactants, cetyltrimethylammonium bromide and dodecyltrimethylammonium bromide. Additionally, the developed method for protein separation was applied for the determination of lysozyme in a cheese sample. The limits of detection and quantification of lysozyme were 0.9 and 3.0 mg/L, respectively. The mean concentration of lysozyme found in the cheese sample was 167.3 ± 10.3 mg/kg.

Determination and identification of synthetic cannabinoids and their metabolites in different matrices by modern analytical techniques - a review.

Synthetic cannabinoids have gained popularity due to their easy accessibility and psychoactive effects. Furthermore, they cannot be detected in urine by routine drug monitoring. The wide range of active ingredients in analyzed matrices hinders the development of a standard analytical method for their determination. Moreover, their possible side effects are not well known which increases the danger. This review is focused on the sample preparation and the determination of synthetic cannabinoids in different matrices (serum, urine, herbal blends, oral fluid, hair) published since 2004. The review includes separation and identification techniques, such as thin layer chromatography, gas and liquid chromatography and capillary electrophoresis, mostly coupled with mass spectrometry. The review also includes results by spectral methods like infrared spectroscopy, nuclear magnetic resonance or direct-injection mass spectrometry.

Enantiomeric separation of tapentadol by capillary electrophoresis--study of chiral selectivity manipulation by various types of cyclodextrins.

The chiral recognition of the centrally acting analgesic agent tapentadol and its isomers with various cyclodextrins (CDs) was studied by capillary electrophoresis, focusing on the migration order of four stereoisomers. In the case of non-charged hydroxypropylated CDs (2-hydroxypropyl-ß-CD, 2-hydroxypropyl-γ-CD) the beta derivative was able to discriminate the S,R- and R,S-isomers in acidic background electrolyte, whereas the gamma allowed the separation of S,S- and R,R-tapentadol, respectively. Dual CD system containing both hosts was used to separate all of four isomers. Negatively charged sulfated-α-CD at 1.0% (w/v) concentration in 100mM sodium borate buffer (pH 9.5) was capable of separating the isomers with favorable enantiomer migration order and the optimized method was able to determine 0.15% of chiral impurities of tapentadol in the presence of the last migrating clinically important R,R-isomer.

Determination of mushroom toxins ibotenic acid, muscimol and muscarine by capillary electrophoresis coupled with electrospray tandem mass spectrometry.

The CE-ESI-MS/MS method for the identification, separation and determination of mushroom toxins, namely ibotenic acid, muscimol and muscarine, was developed. It proved to be sensitive and thus useful for the real sample analysis with omitting the labor and time consuming pretreatment step. The CE-ESI-MS/MS method was applied on the spiked human urine. The analytical characteristics of the proposed method, such as limits of detection, linearity and repeatability of the peak area and the migration time, were evaluated. The RSD of the migration time and peak area were from 0.93% to 1.60% and from 2.96% to 3.42%, respectively. The obtained LOD values were at the nanomolar concentration level, therefore the developed method is sufficient for the determination and quantification of studied toxins in human urine after mushroom intoxication.

Enantiomeric separation of R,S-tolterodine and R,S-methoxytolterodine with negatively charged cyclodextrins by capillary electrophoresis.

The methods for separation of R,S-tolterodine and R,S-methoxytolterodine enantiomers using sulfated α-, ß-CD and phosphated-γ-CD by CE in acidic BGE based on Tris/phosphate pH 2.5 buffer were developed. Sulfated α- and ß-CD allow anodic detection while phosphated-γ-CD allows only cathodic detection of the separated enantiomers. The influence of chiral selector (CS)'s concentration as well as the influence of composition and concentration of BGE on resolutions were studied. Reversal migration order of tolterodine and methoxytolterodine enantiomers was observed, when sulfated-α- and sulfated-ß-CD were used. The developed methods with all three studied CSs, were validated and compared. All proposed methods enable determination of 0.2% of S-tolterodine as an optical impurity in pills, however the method with phosphated-γ-CD provided lower detection limit, better repeatability of peak areas and migration times, and also lower consumption of CS. Developed method employing phosphated-γ-CD that was applied for the determination of optical purity of R-tolterodine in commercial pills.

Covalent binding of cisplatin impairs the function of Na(+)/K(+)-ATPase by binding to its cytoplasmic part.

This study was aimed at verifying the hypothesis that acute kidney failure accompanying cisplatin administration in the cancer therapy could be due to cisplatin interaction with the cytoplasmic part of Na(+)/K(+)-ATPase. Our results demonstrated that cisplatin-binding caused inhibition of Na(+)/K(+)-ATPase, in contrast to other platinated chemotherapeutics such as carboplatin and oxaliplatin, which are known to be much less nephrotoxic. To acquire more detailed structural information, we performed a series of experiments with the isolated large cytoplasmic segment connecting transmembrane helices 4 and 5 (C45 loop) of Na(+)/K(+)-ATPase. Electrochemistry showed that cisplatin is bound to the cysteine residues of the C45 loop, mass spectrometry revealed a modification of the C45 peptide fragment GSHMASLEAVETLGSTSTICSDK, which contains the conserved phosphorylated residue Asp369. Hence, we hypothesize that binding of cisplatin to Cys367 can cause sterical obstruction during the phosphorylation or dephosphorylation step of the Na(+)/K(+)-ATPase catalytic cycle.

Determination of rosiglitazone and metformin in human serum by CE-ESI-MS.

A new method for the determination of anti-diabetic drugs metformin and rosiglitazone based on the use of capillary electrophoresis with electrospray mass spectrometry was developed. The proposed method allowed their separation within 11 min by using 50 mM formic acid at +20 kV. Positive electrospray ionization and selected ion monitoring [M+H](+) of metformin (m/z=130) and rosiglitazone (m/z=358) were performed. Several important experimental parameters influencing electrospray ionization of metformin and rosiglitazone were studied. The final composition of sheath liquid was water/methanol/formic acid (50:49.5:0.5, v/v/v), at a flow rate of 2 µL/min. The developed method was applied for the determination of metformin and rosiglitazone simultaneously in human serum after protein precipitation with acetonitrile. The limits of detection of developed method were 4.42 and 2.14 ng/mL for rosiglitazone and for metformin, respectively, which is sufficient for therapeutic serum concentration levels monitoring for both studied drugs.

Study of electromigration effects on a pH boundary during the on-line electrokinetic preconcentration by capillary electrophoresis.

A contribution to the description of electrokinetic effects on the pH boundary formed by sodium borate pH 9.5 and sodium phosphate pH 2.5 electrolytes for on-line preconcentration of weak acids is presented in this article. Simulations of electrokinetic injections together with experimental studies using contactless conductivity detection verified that the preconcentration is induced mainly by dissociation changes of analytes on the pH boundary and transient ITP state. Moreover, a study of the addition of organic solvent to the injection electrolyte was performed with impressive results. Subnanomolar LODs of hydroxybenzoic acids were achieved with 80% of methanol in the injection electrolyte which represents more than 70 000-fold preconcentration in comparison with classical CZE method.

Analysis of buserelin in urine by online combination of capillary zone electrophoresis with electrospray mass spectrometry.

A fast and precise analysis of the synthetic peptide buserelin in urine using CZE-ESI-MS method has been demonstrated. Formic acid at 50 mmol/L concentration served as background electrolyte in CZE stage and it is compatible with MS detection in positive ionization mode. Two injection modes were tested, i.e. pressure (50 mbar for 5 s) and electrokinetic injection (5 kV for 5 s), of which electrokinetic injection provided better calibration parameters. Buserelin LODs were 0.47 microg/mL in water and 0.63 microg/mL in ten times diluted urine samples using pressure injection, while they were 0.32 microg/mL in water and 0.34 microg/mL in ten times diluted urine samples using electrokinetic injection. Repeatability of buserelin migration times was below 6% (pressure injection mode) and 1% (electrokinetic injection mode). Repeatability of buserelin peak area in SIM mode (m/z=620.5+/-0.5) was less than 12% (pressure injection mode) and 5.8% (electrokinetic injection mode). In this work, no interferences were observed during the analyses of spiked urine samples.

On-line combination of CE and microscopy: an insight into the migration of microorganisms.

Identification of microbial contamination by CE has interested many researchers mainly because of the high speed of CE analysis. However, the CE separation of such big structures brings a lot of questions mainly about the behavior of microorganisms and about the mechanism of separation. In this work, we constructed a simple apparatus where a microscope was used as one detector and a UV detector was used as the second one and we made the comparison of three typical setups for CE of microorganisms. Saccharomyces cerevisiae was chosen as a model microorganism and was analyzed in bare fused-silica capillaries, covalently modified capillaries and dynamically modified capillaries by poly(ethylene oxide) or CTAB. Results showed that the use of CE instrument directly connected with a microscope could be advantageous for the study of separation mechanisms and/or migration behavior.

Determination of antihyperglycemic drugs in nanomolar concentration levels by micellar electrokinetic chromatography with non-ionic surfactant.

A method for the separation of six selected antihyperglycemic (antidiabetic) drugs (tolbutamide, gliclazide, glimepiride, glibenclamide, repaglinide, and glipizide) was developed with use of micellar electrokinetic chromatography. Two non-ionic poly(ethylene glycol)-based surfactants Genapol X-080 and Triton X-114 (reduced) were studied as neutral pseudostationary phases. High alkaline pH 10.0 was used to obtain negative charges of separated antidiabetic drugs and non-ionic surfactants were employed for selectivity alteration. Both non-ionic surfactants provided good selectivity at concentration 0.2% (v/v) in sodium borate buffer and the separation of six drugs was obtained within 5min. An on-line preconcentration method based on reversed electrode polarity switching was employed for the determination of antihyperglycemic drugs in blood serum after acetonitrile protein precipitation. The limits of detection ranged from 20.8nmolL(-1) for tolbutamide to 6.5nmolL(-1) for glibenclamide, respectively.

Assessment of CE for the identification of microorganisms.

The assessment of capillary electrophoresis for separation and identification of microorganisms is described in this article. The work brings a comparison of the application of uncoated capillaries modified with poly(ethylene oxide) and coated capillaries for the separation of model microorganisms Saccharomyces cerevisiae and Escherichia coli with Tris-borate-EDTA and Tris-citrate-fructose as electrolytes. The best separation was achieved in the coated capillary using 1 mM Tris-citrate-fructose buffer, pH 6.9. A simple identification based on the migration time and UV spectrum was found unsatisfactory and thus a method based on a post-separation cultivation and sequential analysis was developed. Off-line combination with mass spectrometric analysis with the use of desorption electrospray was shown to be an interesting alternative in a study of microorganisms. Mass spectra allowed distinguishing among analyzed cells.

Determination of some phenolic acids in Majorana hortensis by capillary electrophoresis with online electrokinetic preconcentration.

An online accumulation/mobilization preconcentration technique based on a dynamic pH junction technique and electrokinetic injection was employed for analysis of phenolic acids (sinapic, ferulic, coumarinic, caffeic, syringic, vanillic, and 4-hydroxybenzoic acid) in extracts from Majorana hortensis leaves. Samples were extracted by pressurized solvent extraction with acetone at 150 degrees C and 15 MPa. The capillary electrophoretic method employed 50 mmol.L (-1) sodium borate, pH 9.5, as the sample electrolyte, 50 mmol.L (-1) sodium phosphate, pH 2.5, as the background electrolyte, and 50 mmol.L (-1) sodium phosphate, pH 2.5, with 60 mmol.L (-1) sodium dodecyl sulfate as the mobilization electrolyte. The method allowed 720-fold to 5560-fold preconcentration of the phenolic acids during 30 min of electrokinetic accumulation with detection limits from 0.38 to 4.22 ng.mL (-1).

DNA separation methodology based on charge neutralization in a polycationic gel matrix.

A novel method for separation of DNA fragments is here reported, based on migrating the polyanionic DNA fragments in a polycationic polyacrylamide gel, made by incorporating positively charged monomers (the Immobilines used for creating immobilized pH gradients) into the neutral polyacrylamide backbone. Separations can be operated under two working conditions: either against a gradient of positive charges, to allow the various DNA fragments to reach a steady-state position along the migration path and condense (focus) in an environment inducing charge neutralization, or in a plateau gel (i.e., in a gel containing a constant level of positive charges from anode to cathode). In this last case, separation is still obtained due to differential charge modulation of the various DNA fragments. In the 100-1000-bp length, it is shown that separation can be obtained even for fragments differing in length by <0.5%, as shown in the splitting of a 656- and 659-bp doublet, that could not be resolved by conventional polyacrylamide gels. In the 10-100-bp range, it is shown that the present method can resolve single nucleotide polymorphisms, i.e. fragments of identical number of nucleotides but differing by one base substitution. In this last case, separations are obtained only in gradient gels containing a much steeper gradient of charges (0-20 mM Immobiline pK 10.3 and pK 12, as opposed to gradients of only 2-4 mM positive charges for larger size fragments). This novel methodology represents a marked improvement over existing techniques and appears to hold promises for applications in diverse fields, such as molecular biology, forensic medicine, and genetic screening.

Capillary electrophoresis as a verification tool for immunochemical drug screening.

BACKGROUND: The aim of this work was to develop a simple capillary electrophoretic method as the verification and confirmation tool in the screening analysis for amphetamines, opiates, benzodiazepines and cocaine and their metabolites for toxicological applications. METHODS: 50 mM phosphate Tris pH 2.0 with 30% (v/v) of methanol was used as a background electrolyte that enabled fast separation of drugs and their metabolites in saliva and urine. Verification of the data from the electrophoretic method was done by High Performance Thin Layer Chromatography (HPTLC) and the immunochemical screening test QuikScreen. RESULTS: The experimental conditions of the Capillary Electrophoresis (CE) were partially optimized (mainly the influence of concentration and types of additives, e.g. cyclodextrines, organic solvents) and validated; the method was used for analysing samples from drug abusers. CONCLUSIONS: The non-instrumental, immunoassay tests could only confirm qualitative addictions and are mainly employed when the emergency detection of drugs is needed. For quantitative analysis and verification of obtained results the confirmation step is strongly recommended. The simple screening capillary zone electrophoresis method allows recognition of the most abused drugs. The agreement of the results from CE, HPTLC and QuikScreen test was more than 95%.

Combination of large volume sample stacking and dynamic pH junction for on-line preconcentration of weak electrolytes by capillary electrophoresis in comparison with isotachophoretic techniques.

An on-line preconcentration capillary electrophoresis (CE) technique, which combines a large volume sample stacking with a dynamic pH junction technique, is introduced in this paper. This dynamic pH junction with co-electroosmotic migration is formed between sodium borate pH 9.5 and sodium phosphate pH 2.5 with 150 mM sodium dodecylsulfate (SDS). A full capillary based injection allows determination of weak acidic compounds at ppb concentration levels (achieved LOD for benzoic acid was 11 nmol L(-1)). The proposed preconcentration method was compared with ITP/ITP (LOD 120 nmol L(-1)), ITP/CZE (LOD 740 nmol L(-1)) and a simple CZE method (LOD 23,330 nmol L(-1)). The analytical potential of this method was assessed with juice test samples.