Acetonitrile stacking
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Perampanel is a novel antiepileptic drug used in paediatric patients. Existing methods that determine serum perampanel are of limited practicability. We developed a novel capillary electrophoresis (CE) method using a new version of acetonitrile stacking for on-line sample pre-concentration, and fluorescence detection (FD). CE separations were performed in a fused-silica capillary where the electroosmotic flow was reduced by coating the inner surface using a INST coating solution. The optimised background electrolyte composition was 50 mM chloroacetic acid with addition of 0.5% m/v polyvinylalcohol (pH 2.15) and separation was driven by application of positive voltage + 30 kV. Serum samples (25 μL) treated by the addition of acetonitrile in a ratio of 1:3 v/v were each injected into the capillary at a large volume that corresponded to the length (129 mm) of the sample zone (hydrodynamic pressure impulse 6000 mbars). Acetonitrile stacking is based on the forcing the sample zone out of the capillary with simultaneous application of the separation voltage. Under such conditions, the enhancing factor achieves the value 57 for peak area compared to the small sample injection length (3.2 mm, hydrodynamic pressure impulse 150 mbar.s). A fluorescence detector with a broad excitation filter (240-400 nm) and an emission filter (495 nm) was used for visualisation of the native fluorescence of perampanel. The calibration dependence of the method was linear (in the range of 10-1000 ng mL-1), with adequate accuracy (99.8-103.3 %) and precision (13.1%). LOD and LOQ for perampanel were 2.9 ng mL-1 and 9.5 ng mL-1, respectively. Clinical applicability was validated using serum samples from patients treated with perampanel and the results corresponded with reference LC-MS/MS values. Our method offers a promising alternative for determining serum perampanel with several advantages. In particular, the low quantity of serum (25 μL) required means that testing can be performed on samples obtained for monitoring other antiepileptic medications, and thus reduces the test-burden on paediatric patients.
- MeSH
- acetonitrily chemie MeSH
- antikonvulziva MeSH
- elektroforéza kapilární metody MeSH
- fluorescence MeSH
- kalibrace MeSH
- lidé MeSH
- limita detekce MeSH
- monitorování léčiv metody MeSH
- pyridony krev MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- validační studie MeSH
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
An electrophoretic stacking method has been developed for monitoring the therapeutic level of the antibiotic ceftazidime in blood plasma and microdialysates taken from peripheral soft tissues of the lower limbs of patients with diabetic foot syndrome. The biological samples are treated by addition of acetonitrile in an amount of 75% v/v and injected into a capillary in a large volume; after turning on the separation voltage, the residual acetonitrile is forced out of the capillary by the application of hydrodynamic pressure. The clinical samples were separated in an optimised background electrolyte composed of 50 mM chloroacetic acid +20% v/v methanol +0.5% v/v INST coating solution. The attained LOD for ceftazidime equalled 0.42 μg mL-1(0.8 μM) and the migration time equalled 3.75 min when using a 25 μm capillary with minimum length of 31.5 cm. The separation was controlled by a maximum voltage of +30 kV and the movement of the analyte was accelerated by a pressure of 50 mbar. The RSD values for intra-day repeatability of the migration time and peak area are 0.14% and 3.8%, respectively; the inter-day values equalled 0.25% for the migration time and 7.3% for peak area, respectively. Pharmacological studies revealed that ceftazidime passes from the blood circulation to the peripheral tissues of the lower limbs with an efficiency of 20%. The introduction of CE control of ceftazidime level in diabetic foot represents a very important improvement in achieving the targeted therapeutic effect.
- MeSH
- antibakteriální látky analýza krev MeSH
- ceftazidim analýza krev MeSH
- diabetická noha krev MeSH
- elektroforéza kapilární metody MeSH
- lidé MeSH
- limita detekce MeSH
- mikrodialýza 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
Two CE methods with contactless conductivity detection have been developed for determining the oral antidiabetic drug metformin in human urine and blood. The determination of metformin is performed on a separation capillary with an effective length of 14 cm, using a maximum voltage of 30 kV and with a small injection of 50-fold diluted urine into the capillary. Under these conditions, the migration time of metformin is 35s and the LOD is 0.3 μM. Large-volume sample stacking was used to determine low metformin levels in serum. The injection of a sample of serum deproteinized with acetonitrile was 10 times greater compared to the injected amount of urine. This enabled reduction of the LOD to 0.03 μM and the metformin migration time equalled 86 s. The undesirable solvent from sample zone was forced out of the capillary to ensure rapidity and good repeatability of the determination. The RSD values for the migration time are 0.1% for urine and 0.7% for serum; RSD for the peak areas equalled 1.4% for urine and 2.6% for serum. The developed CE technique was tested on performance of routine analyses of metformin in the urine and serum of patients suffering from type II diabetes mellitus.
A new variant of large-volume sample stacking injection (LVSS) was used in the capillary electrophoresis with capacitively coupled contactless conductivity detection (CE/C(4)D) determination of the neurotransmitters γ-aminobutyric acid (GABA), glycine (Gly) and glutamate (Glu) in microdialysates of periaqueductal gray matter (PAG). The separation capillary was filled to 98% from the injection side with a sample of microdialysate in acetonitrile. Simultaneously with turning on the separation voltage, the sample zone was forced out by the background electrolyte by increasing the pressure in the terminal capillary outlet vessel. As a consequence of the stacking effect, the analyte was concentrated from the large sample volume into a narrow zone at the sample/background electrolyte boundary close to the injection end of the capillary. Under these conditions, LOD values of 9, 10 and 15nM were determined in the model samples for GABA, Gly and Glu, respectively; RSD equalled 0.5% for the migration times and 1.0-1.9% for the peak areas, respectively. In analysis of microdialysates of PAG, LOD values of 29, 29 and 37nM were determined for GABA, Gly and Glu, respectively; RSD equalled 0.5-0.7% for the migration times and 2.6-8.2% for the peak areas, respectively. The determined basal levels of the neurotransmitters in PAG microdialysates are 0.08, 4.7 and 0.8μM for GABA, Gly and Glu, respectively. Carrageenan-induced hyperalgesia increases the Gly and Glu levels and reduces GABA in PAG microdialysate. Peroral administration of paracetamol in hyperalgesia effectively reduces the Gly value and has no effect on Glu and GABA.
- MeSH
- elektroforéza kapilární přístrojové vybavení metody MeSH
- GABA analýza MeSH
- glycin analýza MeSH
- krysa rodu rattus MeSH
- kyselina glutamová analýza MeSH
- mikrodialýza MeSH
- neurotransmiterové látky analýza MeSH
- substantia grisea centralis chemie MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
- práce podpořená grantem MeSH
A simple, sensitive, specific, and cost-effective analytical methodology was developed for the analysis of human plasma samples spiked with imatinib by CZE with on-line UV detection in the context of Therapeutic Drug Monitoring. Several analytical conditions such as the ionic strength (I) and the pH of the BGE composed of citric acid and ε-amino caproic acid were studied in regards of the presence of sodium chloride (NaCl) in plasma samples (1% m/v). Computer simulations (Simul software) were used to confirm the experimental results and to understand imatinib electrophoretic behavior in the presence of NaCl. Furthermore, the advantages of adding ACN to the sample containing NaCl to combine efficient protein precipitation and on-line CZE stacking of imatinib were demonstrated. LOD and LOQ values of 48 and 191 ng/mL were obtained from plasma sample supernatant after protein precipitation with ACN, which is much lower than mean imatinib plasma level observed for patients treated by imatinib mesylate (about 1000 ng/mL). Good linearity was obtained in the concentration range 191-5000 ng/mL (R2 > 0.997). RSD of less than 1.68% and 2.60% (n = 6) for migration times and corrected peak areas, respectively, were observed at the LOQ.
- MeSH
- acetonitrily chemie MeSH
- chlorid sodný chemie MeSH
- elektroforéza kapilární metody MeSH
- imatinib mesylát krev MeSH
- lidé MeSH
- limita detekce MeSH
- lineární modely MeSH
- reprodukovatelnost výsledků MeSH
- software MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem 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.
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
Separation of major environmental pollutants as perfluorooctanoic acid (PFOA) and perfluorooctanesulfonic acid (PFOS) by capillary electrophoresis is reported for the first time. It is not possible to resolve the solutes in an aqueous media. However, the use of methanol and acetonitrile as the background electrolyte (BGE) solvents allowed their rapid separation in an uncoated capillary. A major effort was put into BGE optimization in respect to both separation efficiency and detection for further on-line preconcentration. 5 mmol.L⁻¹ naphthalene-1-sulfonic acid and 10 mmol.L⁻¹ triethylamine dissolved in ACN/MeOH (50:50 v/v) provided best separation and detection conditions. Next, the large-volume sample stacking and the field-amplified sample injection were applied and compared. Large-volume sample stacking improved limits of detection (LODs) with regard to the standard injection by 69 times for PFOA and 143 times for PFOS with LODs of 280 and 230 nmol.L⁻¹, respectively. Field-amplified sample injection improved LODs 624 times for PFOAand 806 times for PFOS with LODs 31 and 40 nmol.L⁻¹, respectively. Both preconcentration methods showed repeatabilities of migration times less than 1.2% RSD intraday and 6.6% RSD interday. The method was applied on PFOA and PFOS analysis in a sample of river water treated with solid-phase extraction, which further improved LOD toward 5.6 × 10⁻¹⁰ mol.L⁻¹ for PFOS and 6.4 × 10⁻¹⁰ mol.L⁻¹ for PFOA and allows the method to be used for river water contamination screening or decomposition studies.
- MeSH
- acetonitrily MeSH
- chemické látky znečišťující vodu izolace a purifikace MeSH
- elektroforéza kapilární metody MeSH
- extrakce na pevné fázi MeSH
- fluorokarbony izolace a purifikace MeSH
- kapryláty izolace a purifikace MeSH
- kyseliny alkansulfonové izolace a purifikace MeSH
- limita detekce MeSH
- methanol MeSH
- řeky chemie MeSH
- rozpouštědla MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
- práce podpořená grantem MeSH
