We present a method for finely adjustable electroosmotic flow (EOF) velocity in cathodic direction for the optimization of separations in capillary electrophoresis. To this end, we use surface modification of the separation fused silica capillary by the covalently attached copolymer of acrylamide (AM) and 2-acrylamido-2-methyl-1-propanesulfonate (AMPS), that is, poly(AM-co-AMPS) or PAMAMPS. Coatings were formed by the in-capillary polymerization of a mixture of the neutral AM and anionic AMPS monomers premixed in various ratios in order to control the charge density of the copolymer. EOF mobility varies in the 0 to ∼40 × 10-9 m2 V-1 s-1 interval for PAMAMPS coatings ranging from 0 to 60 mol.% of charged AMPS monomer. For EOF in PAMAMPS-treated capillaries, we observed (i) a negligible dependence on pH in the 2-10 interval, (ii) a minor variance among background electrolytes (BGEs) in function of their components and (iii) its standard decrease with increasing ionic strength of the BGE. Interest in variable cathodic EOF was demonstrated by the amelioration of separation of two kinds of isomeric anionic analytes, that is, monosaccharides phosphates and helquat enantiomers, in counter-EOF mode.
Sensitive electrophoretic determination of 3-hydroxybutyrate (3HB) as an indicator of human ketogenesis is performed in fused silica capillary covalently coated by an anionic copolymer of poly(acrylamide-co-sodium-2-acrylamido-2-methylpropanesulphonate) (PAMAMPS). Baseline separation of 3HB from other components of human serum is achieved in a 20 μm capillary with an effective length of 17 cm covered by 4% PAMAMPS, which generates a cathodic EOF with a mobility of 8.30 ± 0.00 · 10-9 m2/V.s in 80 mM MES/His as background electrolyte. 3HB migrates in counter-current electrophoretic mode against EOF, that effectively improving electrophoretic resolution. Sample pre-treatment is based on adding of 45 μL acetonitrile to 15 μL serum and, after shaking, a 28 mm long zone of supernatant is injected into the capillary, and sharpened after turning on a separation voltage of 20 kV using the technique of large volume sample stacking, where the EOF forces the residual acetonitrile from the capillary. When combined with universal contactless conductivity detection, the achieved LOD and LOQ are 0.43 μM and 1.44 μM, respectively, that are sufficiently low for monitoring the physiological 3HB level. The performed clinical study subsequently showed that serum 3HB increases from a concentration of 71 μM, corresponding to normal food, to level of 1924 μM after 60 h of fasting and returns to the normal physiological concentration 48 h after commencing consumption of high-saccharide food.
- MeSH
- acetonitrily MeSH
- akrylové pryskyřice MeSH
- alkylsulfonany MeSH
- elektroforéza kapilární * metody MeSH
- kyselina 3-hydroxymáselná MeSH
- lidé MeSH
- omezení příjmu potravy * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Electrophoretic stacking is developed for sensitive determination of three zwitterionic antiepileptics, namely vigabatrin, pregabalin and gabapentin, in human serum. CE separation is performed in a 25 μm fused silica capillary covalently coated with the copolymer of acrylamide with 5% content of permanently charged 3-acrylamidopropyl trimethylammonium chloride (PAMAPTAC). In background electrolyte of 500 mM acetic acid, the 5% PAMAPTAC generates an anodic electro-osmotic flow with a magnitude of (-18.6 ± 0.5) · 10-9 m2V-1s-1, which acts against the direction of the electrophoretic migration of the analytes. A sample of the antiepileptic prepared in a 25% v/v infusion solution and 75% v/v acetonitrile is injected into the capillary in a large volume attaining a zone length of up to 270 mm. After turning on the separation voltage, the antiepileptics are isotachophoretically focussed behind the zone of Na+ ions with a sensitivity enhancement factor of 78. For the clinical determination of antiepileptics, the human serum is diluted with acetonitrile in a ratio of 1:3 v/v and a zone with a length of 90 mm is injected into the capillary. The method is linear in the 0.025-2.5 μg/mL concentration range; the attained limit of quantification is in the range 18.3-22.8 nmol/L; the within-day precision for the migration time is 0.8-1.2% and for the peak area 1.5-2.4%.
- MeSH
- antikonvulziva * MeSH
- chloridy * MeSH
- elektroforéza kapilární MeSH
- lidé MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Electroosmotic flow (EOF) plays a pivotal role in optimization of capillary electrophoresis (CE) separations of (bio)molecules and (bio)particles. EOF velocity is directly related to analysis time, peak resolution and separation efficiency. Here, we report a concept of charged polymer coatings of the inner fused silica capillary wall, which allows anodic EOF with mobility ranging from 0 to ∼(30-40) × 10-9 m2V-1s-1. The capillary wall is modified by covalently bound cationic copolymer poly(acrylamide-co-(3-acrylamidopropyl)trimethylammonium chloride) (PAMAPTAC) containing variable ratio of the charged monomer in the 0-60 mol. % interval. The EOF mobility showed minor variability with composition of background electrolyte (BGE) and pH in the 2-10 interval. The coatings were evaluated by CE-UV and nanospray CE-MS in the counter-EOF arrangement for a series of basic drug molecules in acetic acid based acidic BGE. Tunable EOF velocity was demonstrated as a useful tool for optimization of peak resolution, separation efficiency and migration time of analytes. Electrostatic repulsion of positively charged capillary surface was shown as beneficial for suppression of analyte adsorption, notably for hydrophobic cationic analytes.
- MeSH
- adsorpce MeSH
- elektroforéza kapilární * MeSH
- elektroosmóza * MeSH
- kationty MeSH
- polymery MeSH
- Publikační typ
- časopisecké články MeSH
Asymmetric and symmetric dimethylarginines are toxic non-coded amino acids. They are formed by post-translational modifications and play multifunctional roles in some human diseases. Their determination in human blood plasma is performed using capillary electrophoresis with contactless conductivity detection. The separations are performed in a capillary covered with covalently bonded PAMAPTAC polymer, which generates anionic electroosmotic flow and the separation takes place in the counter-current regime. The background electrolyte is a 750 mM aqueous solution of acetic acid with pH 2.45. The plasma samples for analysis are treated by the addition of acetonitrile and injected into the capillary in a large volume, reaching 94.5% of the total volume of the capillary, and subsequently subjected to electrophoretic stacking. The attained LODs are 16 nm for ADMA and 22 nM for SDMA. The electrophoretic resolution of both isomers has a value of 5.3. The developed method is sufficiently sensitive for the determination of plasmatic levels of ADMA and SDMA. The determination does not require derivatization and the individual steps in the electrophoretic stacking are fully automated. The determined plasmatic levels for healthy individuals vary in the range 0.36-0.62 µM for ADMA and 0.32-0.70 µM for SDMA.
In this study, the apparent binding constants and limiting mobilities of the multiply charged complexes of the Δ- and Λ-enantiomers of Ru(II)- and Fe(II)-polypyridyl associates ([Ru(2,2'-bipyridine)3 ]2+ , [Ru(1,10-phenanthroline)3 ]2+ , and [Fe(1,10-phenanthroline)3 ]2+ ) with single-isomer 2,3-diacetylated-6-sulfated-cyclodextrins (CDs) (12Ac-6S-α-CD, 14Ac-7S-β-CD, and 16Ac-8S-γ-CD) were determined by ACE using uncorrected and ionic strength corrected actual mobilities of the species involved. Two limiting models were tested for the ionic strength correction of the actual mobilities based on an empirical relation for the ionic strength correction of multivalent ionic species. In model 1, the nominal values of the charge numbers (zS,nom ) and analytical concentrations (cS,nom ) of the above CD selectors in the BGEs were applied for calculation of the BGE ionic strength, as usual. In model 2, the CD selectors were considered as singly charged species (zS = -1) with |zS,nom |-times higher concentrations in the BGE than their analytical concentrations (cS = |zS,nom | × cS,nom ) in the calculation of the BGE ionic strength. In all three cases-with uncorrected actual mobilities as well as with actual mobilities corrected according to the two limiting models-the measured effective mobilities of the above enantiomers fit well the theoretical curves of their mobility dependences on the CD selectors concentrations in the BGE, with high average coefficients of determination (R2 = 0.9890-0.9995). Nevertheless, the best physico-chemically meaningful values of the apparent binding constants and the limiting mobilities of the enantiomer-CDs complexes with low RSDs were obtained using the actual mobilities of the species involved corrected according to model 2.
Capillary electrophoretic separation of ketamine, norketamine, hydroxynorketamine, and dehydronorketamine was performed in the counter-current regime under the influence of oppositely-directed electroosmotic flow. For this purpose, the fused silica capillaries were covalently coated with the poly(acrylamide-co-3-acrylamidopropyl trimethylammonium chloride) copolymer (PAMAPTAC). The content of the cationic monomer APTAC in the polymerization mixture varied in the range 0-6 mol. % and the generated electroosmotic flow increased continuously in the 0-20 · 10-9 m2V-1s-1 interval. Importantly, it resulted in improved electrophoretic resolution of ketamine/norketamine, which increased from 0.8 for neutral PAM coating (i.e. 0% PAMAPTAC) to 3.0 for 6% PAMAPTAC. The determination of ketamine and its derivates in rat serum was performed in a 4% PAMAPTAC capillary with an inner diameter of 25 μm. The separation was performed in a 500 mM aqueous solution of acetic acid (pH 2.3). The clinical sample was deproteinized by the addition of acetonitrile to the serum and a large volume of the treated sample was injected directly into the capillary. The achieved limit of detection ranged from 2.2 ng/mL for dehydronorketamine to 4.1 ng/mL for hydroxynorketamine; the intra-day repeatability was 1.0-1.5% for the migration time and 2.8-3.3% for the peak area. The developed methodology was employed for time monitoring of ketamines in rat serum after intra venous administration of low doses of anaesthetic at a level of 2 μg per g of body weight.
- Publikační typ
- časopisecké články MeSH
Affinity capillary electrophoresis (ACE) is typically used for the determination of stability constant, Kst, of weak to moderately strong complexes. Sensitive detection such as mass spectrometry (MS) is required for extension of ACE methodology for estimation of Kst of stronger complexes. Consequently, an efficient interface for hyphenation of CE with MS detection is necessary. For evaluation of interfaces for electrospray ionization mass spectrometric (ESI/MS) detection in ACE conditions, potassium-crown ether complexation was used as model system. The effective mobilities of the crown ether ligands and the Kst of their potassium complexes were measured/determined by ACE-ESI/MS using two lab-made interfaces: (i) a sheathless porous tip CE-ESI/MS interface and (ii) a nano-sheath liquid flow CE-ESI/MS interface, and, in turn, compared with those obtained by ACE with UV spectrophotometric detection. Apparent stability constant of potassium-crown ether complexes in 60/40 (v/v) methanol/water mixed solvent, pH* 5.5, was about 1300 L/mol for dibenzo-18-crown-6, 1600 L/mol for benzo-18-crown-6 and 5200 L/mol for 18-crown-6 ligands, respectively. It was observed that electrophoretic mobilities from CE-MS experiments differ from reference values determined by UV detection by ∼7% depending on the CE-MS interface used. Good agreement of CE-MS and CE-UV data was achieved for nano-sheath liquid flow interface, in which the spray potential and the CE separation potential can be effectively decoupled. As for sheathless porous tip interface, a correction procedure involving a mobility marker has been proposed. It provides typically only ca. 1% difference of effective mobilities and Kst values obtained from CE-MS data as compared to those received by the reference ACE-UV method.
A sensitive capillary electrophoretic method with on-line sample preconcentration by large volume sample stacking has been developed for determination of the anti-microbial agent pentamidine. The separation is performed in a fused silica capillary coated with covalently bound hydroxypropyl cellulose, with an internal diameter of 50 μm and length of 31.5 cm; the background electrolyte was 100 mM acetic acid/Tris at pH 4.7. The stacking is tested using a model sample of 1 μM pentamidine dissolved in 25% infusion solution and 75% acidified acetonitrile. Stacking permits the injection of a sample zone with a length of 95% of the total capillary length to achieve an enhancing factor of 77 compared to low injection into 1.8% of the total capillary length, with simultaneous high separation efficiency of approximately 1 350 000 plates/m. Stacking is based on simultaneous application of a separation field and a hydrodynamic pressure to force the acetonitrile zone out of the capillary. This approach allows the determination of pentamidine in rat blood plasma using only 12.5 μL of plasma treated by the addition of acetonitrile in a ratio of 1:3 v/v. The attained LOD is 0.03 μM and the intra-day repeatability is 0.1% for the migration time and 1.0% for the peak area at the injection 28.3% of capillary length. The performed pharmacokinetic study with ten-second scanning of the blood reveals rapid dynamics of pentamidine in the arterial bloodstream, while the changes are much slower in the venous system.
- MeSH
- antiinfekční látky krev MeSH
- elektroforéza kapilární metody MeSH
- krysa rodu rattus MeSH
- limita detekce MeSH
- lineární modely MeSH
- pentamidin krev MeSH
- potkani Wistar MeSH
- reprodukovatelnost výsledků MeSH
- tlak MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
CE using randomly highly sulfated α-, β-, and γ-CDs (S-α-CD, S-β-CD, S-γ-CD), sulfobutylether-β-CD (SBE-β-CD), single isomer (6-O-sulfo) α-, β-, and γ-CDs, and their derivatives as stereoselectors was applied to chiral analysis of polypyridyl complexes of [Ru(bpy)3 ](2+) , [Ru(phen)3 ](2+) , and [Fe(phen)3 ](2+) (bpy = 2,2'-bipyridine; phen = 1,10 phenanthroline). The best separations of Δ- and Λ-enantiomers of the these complexes with high resolution (up to R1,2 = 7.0) and short analysis times (10-20 min) were achieved in the BGE composed of 22 mM NaOH/35 mM H3 PO4 , pH 2.4, containing 1.5-6.0 mM S-α-CD or S-β-CD, or SBE-β-CD as chiral selectors. The developed method was applied to the assessment of enantiomeric purity of several samples of [Ru(bpy)3 ](2+) catalyst. CE experiments were performed in a homemade analyzer equipped with bare or hydroxypropylcellulose-coated fused-silica capillaries (total/effective length 40/29 cm, id/od 50/375 μm) and an UV absorption detector operating at 206 nm. In addition to chiral analysis, apparent binding constants of the complexes of [Ru(bpy)3 ](2+) , [Ru(phen)3 ](2+) , and [Fe(phen)3 ](2+) enantiomers with five sulfated CDs (S-α-CD, S-β-CD, S-γ-CD, SBE-β-CD, and 16Me-8S-γ-CD) were determined from the dependence of their effective electrophoretic mobilities on the concentration of the CDs in the BGE by nonlinear regression analysis. Calculated apparent binding constants of these complexes were found to be in the (1.10-4.66) × 10(3) L/mol range. Moreover, it was shown that at selected concentrations of some S-CDs and suppressed or very low electroosmotic flow, the exceptional enantioseparations with infinite resolution could be achieved.
