Simultaneous detection of N-acetyl-L-cysteine and physiological low molecular mass thiols in plasma by capillary electrophoresis

N-acetyl-l-cysteine (NAC) is a therapeutic drug widely used as mucolytic agent in the treatment of respiratory diseases. Recently it has been proposed that NAC administration may modify the plasma levels of low molecular weight thiols (LMW) like cysteine, homocysteine and glutathione, though it has been still debated if their plasma concentration increases or decreases during the therapy. Therefore research calls for methods able to analyze simultaneously NAC and the other plasma LMW thiols in order to evaluate if NAC is able to modify plasma thiols concentration and in particular to reduce homocysteine levels in hyperhomocysteinemia. In this paper we present a new capillary electrophoresis method that allows a baseline separation of plasma NAC from the physiological thiols. The proposed method has been utilized to measure the drug and the physiological LMW thiols in NAC administered chronic obstructive broncho-pneumopathy (COPB) disease patients.     

High-speed microchip electrophoresis method for the separation of (R,S)-naproxen

In this research, a capillary electrophoretic method for the fast enantiomeric resolution of (R,S)-naproxen was investigated. Method development involved variation of applied potential, buffer concentration, buffer pH, and cyclodextrin concentration. The optimum electrophoretic separation conditions were 110 mM sodium acetate run buffer (pH 6.0), 30 mM methyl-beta-cyclodextrin, 20% (v/v) acetonitrile, 25 degrees C. The total length of capillary was 48 cm, (50 microm I.D.) with ultra violet (UV) detection at 232 nm. Using these conditions, the number of theoretical plates was close to one million (896,000/m). The possibility of achieving a fast chiral separation of (R,S)-naproxen on a microchip of 2.5 cm in length was investigated. Complete enantiomeric resolution of naproxen was achieved in less than 1 min, on this microchip platform, with linear imaging UV detection. This system had the advantage of real-time separation monitoring, so that enantiomeric resolution could be visually observed, and high-speed chiral analysis was realized. The microchip electrophoresis (MCE) separation was compared with the capillary electrophoresis (CE) separation with regards to speed, efficiency, separation platform, and precision. This work highlights the potential of CE and MCE in future chiral separations.     

An assay for angiotensin-converting enzyme using capillary zone electrophoresis

A sensitive and rapid method was developed for angiotensin-converting enzyme (ACE) activity determination by capillary zone electrophoresis. Hippuryl-l-histidyl-l-leucine, a synthetic tripeptide, was used as the ACE-specific substrate. Capillary zone electrophoresis was employed to separate the products of the enzymatic reaction and the ACE activity was determined by quantification of hippuric acid, a result of the enzymatic reaction on the tripeptide. The capillary electrophoresis was performed in a 27 cm x 75 micrometer i.d. fused-silica capillary using 200 mM boric acid-borate buffer (pH 9.0) as a run buffer with an applied voltage of 8.1 kV at a capillary temperature of 23 degrees C. The electrophoresis was monitored at 228 nm. Each electrophoretic run requires only a nanoliter of the enzymatic reactant solution, at only 6 min, rendering a powerful tool for the ACE assay.     

Detection of a mixed-backbone oligonucleotide (GEM 231) in liver and tumor tissues by capillary electrophoresis

A simple, rapid and sensitive method has been developed and validated for the analysis of a mixed-backbone oligonucleotide (GEM 231) in tumor tissues. The analysis was performed using a capillary electrophoresis (CE) system with UV detection. An extended light path (bubble cell) capillary column of 64.5 cm (effective length 56 cm)×50 μm I.D. is used as the separation column. The optimized chromatographic conditions were background electrolyte: sodium borate buffer (60 mM, pH 9.1), electrokinetic injection: 10 s, applied voltage: 30 kV, detection at λ=210 nm. A linear relationship was observed between the peak area and the amount of GEM 231 in the range of 1.0–1000 μg/ml. The lower detection limit of the drug was 100 pg with an average recovery of about 75±5%. The inter-day and intra-day relative standard deviations were <10%. Assay validation studies revealed that CE method is reproducible and specific for the determination of GEM 231 in tissue homogenates with a run time of less than 5 min.     

High-throughput capillary electrophoresis method for plasma cysteinylglycine measurement: evidences for a clinical application

Summary. Increased levels in plasma homocysteine and cysteine, and more recently, decreased levels in cysteinylglycine have been indicated as a risk factor for vascular diseases. Most assays focused their attention only on homocysteine determination and when also other thiols were measured, analytical times drastically increased. By modifying our previous method for thiols detection, we set up a rapid capillary electrophoresis method for the selective quantification of plasma cysteinylglycine, cutting the analysis time of about 50%. Samples were treated with tri-n-butylphosphine as reducing agent, proteins were precipitated with trichloroacetic acid and released thiols were successively derivatized by the selective thiol laser-induced fluorescence-labeling agent 5-iodoacetamidofluorescein and separated by capillary electrophoresis. A baseline separation between peaks was obtained in about 2 min using 3 mmol/L sodium phosphate/2.5 mmol/L boric acid as electrolyte solution with 75 mmol/L N-methyl-D-glucamine at pH 11.25 in a 47 cm long capillary with a cartridge temperature of 45 °C. The method application was checked by measuring plasma Cys-Gly levels in a group of patients affected by retinal vein occlusion (RVO), an important cause of visual loss in the elderly. The low levels of Cys-Gly found in the RVO patients suggest that these small thiols may have importance in the disease development. Authors’ addresses: Dr. Angelo Zinellu, Dr. Ciriaco Carru, Department Biomedical Sciences, Chair of Clinical Biochemistry, University of Sassari, Viale San Pietro 43/B, 07100 Sassari, Italy     

Capillary electrophoresis-based nanoscale assays for monitoring ecto-5'-nucleotidase activity and inhibition in preparations of recombinant enzyme and melanoma cell membranes

Powerful capillary electrophoresis (CE) methods were developed for monitoring the reaction of ecto-5'-nucleotidase (ecto-5'-NT, CD73), a (patho)biochemically important enzyme that hydrolyzes nucleoside-5'-monophosphates to the corresponding nucleosides. The enzymatic reaction was performed either before injection into the capillary (method A) or directly within the capillary (method B). In method A, separation of substrates and products was achieved within 8 min using an eCAP fused-silica capillary (20 cm effective length, 75 microM i.d., UV detection at 260 nm), 40 mM sodium borate buffer (pH 9.1), normal polarity, and a constant voltage of 15 kV. In method B, the sandwich technique was applied; substrate dissolved in reaction buffer (10mM Hepes [pH 7.4], 2mM MgCl2, and 1mM CaCl2) was hydrodynamically injected into a fused-silica capillary (30 cm, 75 microM i.d.), followed by enzyme (recombinant rat ecto-5'-NT) and subsequent injection of substrate solution. The reaction was initiated by the application of 1 kV voltage for 1 min. The voltage was turned off for 1 min and again turned on at a constant voltage of 15 kV to elute products (nucleosides) within 4 min using borate buffer (40 mM, pH 9.1). Thus, assays could be performed within 6 min, including enzymatic reaction, separation, and quantification of the formed nucleoside. The CE methods were used for measuring enzyme kinetics and for assaying inhibitors and substrates. In addition, the online assay was successfully applied to melanoma cell membrane preparations natively expressing the human ecto-5'-NT.     

Determination of reduced and oxidized homocysteine and related thiols in plasma by thiol-specific pre-column derivatization and capillary electrophoresis with laser-induced fluorescence detection

A new sensitive and rapid capillary electrophoresis (CE) assay for measuring reduced and oxidized thiols in human plasma has been developed. To prevent oxidation of the thiols, whole blood was immediately centrifuged after collection and the plasma proteins were precipitated with perchloric acid. The reduced thiols in the supernatant were derivatized quantitatively at 25°C, pH 7.5 with a fluorescent reagent, fluorescein-5-maleimide (FM). The total plasma concentration of thiols, including the fraction coupled to proteins, was assayed after an initial reduction of the disulfide linkage in plasma with dithiothreitol. The separation of FM-thiols was performed in an acetonitrile/10 mM sodium phosphate–50 mM SDS buffer [25:75 (v/v); pH 7.0] using a fused-silica capillary (57 cm×75 μm I.D.) at 45°C. A 3-mW argon-ion laser (λ_ex 488 nm/λ_em 520 nm) was employed for FM-thiol detection. With the electric field of 530 V/cm, the time needed for the separation of FM-homocysteine, FM-glutathione and FM-N-acetylcysteine was less than 8 min. The lower limit of detection was 3 μM for the total thiols and 10 nM for the reduced thiols. The method was applied to the determination of homocysteine levels in plasma from patients with end-stage renal disease.     

Large-volume sample stacking of selected drugs of forensic significance by capillary electrophoresis

Large-volume sample stacking capillary electrophoresis (LVSS-CE) and conventional capillary electrophoresis (CE) are compared for the separation of drugs of significance to forensic and clinical analyses. LVSS-CE for cations requires the use of an electroosmotic flow (EOF) modifier in conjunction with polarity switching to effect on-column concentration of an analyte and its subsequent migration in the capillary. The run buffer consists of 0.05 mol dm⁻³ disodium tetraborate adjusted to pH 2.2 with orthophosphoric acid, and the EOF modifier is 0.002 mol dm⁻³ cetyltrimethylammonium bromide. CE investigations used an identical run buffer minus the EOF modifier. LVSS-CE and CE investigations used injection times of 30 s and 3 s, respectively. Both modes of capillary electrophoresis are compared in terms of their limits of detection, efficiency, resolution and reproducibility. LVSS-CE is also applied to the analysis of a spiked urine sample.     

Enhanced separation and detection of serum bilirubin species by capillary electrophoresis using a mixed anionic surfactant—protein buffer system with laser-induced fluorescence detection

The four major bilirubin species in serum are separated by capillary electrophoresis and detected using laser-induced fluorescence detection. The optimum buffer system consists of 40 mM sodium dodecyl sulfate (SDS)—0.012 mM bovine serum albumin (BSA). The use of the SDS—BSA mixture in the mobile phase allows for the separation of four major bilirubin species at physiological pH with untreated capillaries. The results show that the use of BSA as a run buffer modifier in SDS solution improves separation efficiency and increases sample solubility via pH changes of the run buffer. The limits of detection for the bilirubin species using laser-induced fluorescence are between 30 and 150 nM, depending on the bilirubin species; not only is this approximately two orders of magnitude lower than with visible-light absorption methods, it allows the bilirubin species in normal sera to be quantitatively measured without sample pretreatment.     

Pre-analytical factors affecting ascorbic and uric acid quantification in human plasma

We have recently described a new capillary electrophoresis assay to measure serum ascorbic and uric acids in which a baseline separation of peaks was obtained in less than 4 min by using a 60.2 cm x 75 microm uncoated capillary with a 100 mmol/L sodium borate running buffer pH 8. Since during sample preparation AA is rapidly oxidized, we employed our new capillary electrophoresis method to analyze the pre-analytical factors affecting its stability. In particular we evaluated how the standard mix preparation, the blood collection (plasma EDTA or serum) and the plasma protein precipitation influence the results of analysis. Our data suggest that standard ascorbate must be dissolved in a solution containing cysteine and EDTA in order to avoid oxidation and that EDTA blood collection is better than serum for AA measurement. Moreover, the type and the quantity of the precipitating compound are critical parameters to obtain a complete recovery of analytes. We performed AA and UA analysis in 32 healthy volunteers with the optimized experimental conditions by using our capillary electrophoresis method and a reference CE assay. Obtained data were compared to Bland-Altman test to verify the accuracy of our CZE method.     

Optimization of a free separation of 30 free amino acids and peptides by capillary zone electrophoresis with indirect absorbance detection: a potential for quantification in physiological fluids

This report describes a rapid, single-run procedure, based on the optimization of capillary electrophoresis (CE) and indirect absorbance detection capabilities, which was developed for the separation and quantification of 30 underivatized physiological amino acids and peptides, usually present in biological fluids. p-Aminosalicylic acid buffered with sodium carbonate at pH 10.2+/-0.1 was used as the running electrolyte. Electrophoresis, carried out in a capillary (87 cm x 75 microm) at 15 kV potential (normal polarity), separated the examined compounds within 30 min. Limits of detection ranged from 1.93 to 20.08 micromol/l (median 6.71 micromol/l). The method was linear within the 50-200 micromol/l concentration range (r ranged from 0.684 to 0.989, median r=0.934). Within run migration times precision was good (median C.V.=0.7%). Less favorable within run peak area precision (median C.V.=6.6%) was obtained. The analytical procedure presented was successfully tested for separation and quantification of amino acids in physiological fluids, such as plasma or supernatant of macrophage cultures. Sample preparations require only a protein precipitation and dilution step.     

Determination of the anticancer drug prospidin in human tissue by high-performance capillary electrophoresis using derivatization

A high-performance capillary electrophoresis (HPCE) method is described for the quantitative determination of the anticancer drug prospidin in human tissue after its derivatization with diethyldithiocarbamic acid sodium salt (DDTC). It was found that absorption of prospidin and its derivatives on the capillary wall due to the strong positive charge in the drug molecules could be eliminated by increasing the methanol content in the run buffer up to 50% and increasing the pH value up to 11.2. While studying the conditions of the interaction between prospidin and DDTC, a molar excess of the latter of 1:9 and 1.5 h of reaction time were found to be enough for complete derivatization. Sample preparation included homogenization of freshly cut papilloma species and deproteinization by methanol addition. Detection was by ultraviolet (UV) absorption at 254 nm. Due to its speed and high performance in separation. Detection was by ultraviolet (UV) absorption at 254 mm. Due to its speed and high practice.     

Determination of prednisolone and the most important associated compounds in ocular and cutaneous pharmaceutical preparations by micellar electrokinetic capillary chromatography

A micellar electrokinetic capillary chromatographic method to separate prednisolone, prednisolone acetate, naphazoline, Zn-bacitracin, sulfacetamide and phenylefrine is described. The separation was carried out by using a fused-silica capillary (57 cmx75 micrometer I.D.) at 25 degrees C and 30 kV, using a 5 mM phosphate-5 mM borate buffer adjusted to pH 8.2, 50 mM sodium dodecyl sulfate (SDS) and 10% methanol-water (v/v) as background electrolyte. Under these conditions, the run time was 8 min and the limits of quantification were about 1.0 mg/l for every component. The method was applied to pharmaceutical preparations and the results provided recoveries close to 100% and the method gave good results when compared with a reference multivariate calibration spectrophotometric method.     

Capillary electrophoresis of methotrexate polyglutamates and its application in evaluation of γ-glutamyl hydrolase activity

A rapid and sensitive procedure for the separation of methotrexate (MTX) polyglutamates² using capillary electrophoresis (CE) is described as it applies to the in vitro assay of the enzyme γ-glutamyl hydrolase (GGH, EC 3.4.22.12). Distinct separation of MTX and polyglutamylated forms (up to glu₄) is achieved within 10 min using a 75 μm I.D. capillary (50 cm, +25 kV), and enables quantitation of both reactant and enzyme products. As activity can be reliably determined using less than 5×10⁵ eukaryotic cells, this new technique can be used to measure GGH in patient tumor samples and investigate the relationship between GGH levels and clinical MTX resistance.     

Determination of camptothecins in DMSO extracts of Nothapodytes foetida by direct injection capillary electrophoresis

A rapid capillary electrophoresis procedure was developed for determining the anti-cancer components, camptothecins, in Nothapodytes foetida. The hydrophobic compound was extracted from plant tissue (ca. 1 mL of DMSO for 100 mg of dried plant tissue) with a water-miscible organic solvent, DMSO, at elevated temperature (60 degrees C). The extract was directly injected into the separation capillary (untreated fused silica, 34 cm in length, 75 microm i.d.) and analysed in MEKC mode (369 nm). Within 5 min of migration, camptothecins were successfully separated and quantified by adding organic modifiers to the running buffer (20% DMSO, 90 mm SDS in 10 mm borate buffer, pH 8.60). The linear dynamic range for camptothecin was from 5 to 400 microg/mL. This method was proven to be very suitable for monitoring the amount of camptothecins during the cultivation of the medicinal plant.     

Evaluation of the use of cyclodextrins in chiral separation of basic drug substances by capillary electrophoresis

The influence of the choice of type and/or concentration of cyclodextrin, other additives, the temperature surrounding the capillary, and buffer pH on the separation of some chiral basic drug substances in capillary zone electrophoresis has been evaluated. It was found that pH of the buffer and type and concentration of cyclodextrin had a major influence on the separation. © 1995 Wiley-Liss, Inc.     

Enantiomeric separation of chiral ruthenium(II) complexes using capillary electrophoresis

Capillary zone electrophoresis (CZE) and micellar capillary electrophoresis (MCE) were applied for the enantiomeric separation of nine mononuclear tris(diimine)ruthenium(II) complexes as well as the separation of all stereoisomers of a dinuclear tris(diimine)ruthenium(II) complex. Nine cyclodextrin (CD) based chiral selectors were examined as run buffer additives to evaluate their effectiveness in the enantiomeric separation of tris(diimine)ruthenium(II) complexes. Seven showed enantioselectivity. Sulfated gamma-cyclodextrin (SGC), with four baseline and three partial separations, was found to be the most useful chiral selector. In CZE mode, the derivatized gamma-CDs were more effective than beta-CDs while sulfated CDs work better than carboxymethyl CDs. In MCE mode, hydroxypropyl beta-CD separated the greatest number of tris(diimine) ruthenium(II) complexes. The effects of chiral selector concentration, run buffer pH and concentration, the concentration ratio between chiral selector and other factors were investigated.     

Investigation of the in vitro biotransformation and simultaneous enantioselective separation of thalidomide and its neutral metabolites by capillary electrophoresis

Reversed-phase liquid chromatography is a long established method for the analysis of drug metabolism. The current investigation demonstrates that micellar electrokinetic capillary chromatography can be an attractive alternative. Two methods were developed using sodium dodecyl sulfate and hexadecyltrimethylammonium bromide for the determination of possible hydroxylated metabolites of the former sedative drug thalidomide (Contergan) in order to study the in vitro metabolism of the drug by incubation with rat liver microsomes. The biotransformation was found to be stereoselective: S-(−)-thalidomide mainly formed 5-hydroxythalidomide, whereas R-(+)-thalidomide was preferentially transformed to two metabolites tentatively assigned to be diastereomers of 5′-hydroxythalidomide.Furthermore, the simultaneous enantioseparation of thalidomide and two of its possible hydroxylated metabolites was achieved using capillary electrophoresis with negatively charged carboxymethyl-β-cyclodextrin. The dependencies of the selectivity of the enantioseparation on the concentration of the chiral additive and the pH of the run buffer were investigated.     

Improved capillary electrophoresis conditions for the separation of kinase substrates by the laser micropipet system

Phosphorylated and nonphosphorylated forms of peptide substrates for protein kinase C (PKC) and calcium-calmodulin activated kinase II (CamKII) were separated by capillary zone electrophoresis. Electrophoresis of the peptide substrates and products in biologic buffer solutions in uncoated capillaries yielded asymmetric analyte peaks with substantial peak tailing. Some of the peptides also exhibited broad peaks with unstable migration times. To improve the electrophoretic separation of the peptides, several strategies were implemented: extensive washing of the capillary with a base, adding betaine to the electrophoretic buffer, and coating the capillaries with polydimethylacrylamide (PDMA). Prolonged rinsing of the capillaries with a base substantially improved the migration time reproducibility and decreased peak tailing. Addition of betaine to the electrophoretic buffer enhanced both the migration time stability as well as the theoretical plate numbers of the peaks. Finally PDMA-coated capillaries brought about significant improvements in the resolving power of the separations. These modifications all utilized an electrophoretic buffer that was compatible with a living biologic cell. Consequently they should be adaptable for the new capillary electrophoresis-based methods to measure kinase activation in single cells.     

Determination of lactate dehydrogenase isoenzymes in single rat glioma cells by capillary electrophoresis with electrochemical detection

A method for determination of lactate dehydrogenase (LDH) isoenzymes in single rat glioma cells (C6) was developed. In this method, a whole cell was electrokinetically injected into the front end of the separation capillary. After that, the cell was lysed by ultrasonication and the isoenzymes in the cell were pre-separated at 20 kV for 5 min and then incubated for 2 min with the enzyme substrates nicotinamide adenine dinucleotide (NAD(+)) and lactate in the capillary electrophoresis running buffer. The electroactive product NADH generated by the isoenzymes through on-capillary enzyme-catalyzed reaction was detected at the outlet of capillary by using the end-capillary amperometric detection with a constant potential mode at a carbon fiber bundle microdisk electrode. Since the amplification of signal via the enzyme reaction, the concentration of nicotinamide adenine dinucleotide (NADH) is much higher than that of LDH. The external standardization was used to quantify isoenzymes in individual cells. Three LDH isoenzymes in single rat glioma cells (C6) were determined and quantified.