Petr Kubáň

Sensitive determination of glutathione in biological samples by capillary electrophoresis with green (515 nm) laser-induced fluorescence detection.

A new sensitive capillary electrophoretic method with laser-induced fluorescence (LIF) was developed for quantitation of glutathione (GSH) in biological samples. Eosin-5-maleimide was used to label the GSH molecule and the formed conjugate was separated in a 15 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid electrolyte at pH 7.0 in less than 3 min. The conjugate was detected with an in-house built LIF system, utilizing an inexpensive 515 nm diode laser module. Studies were performed to optimize the derivatization (the ratio of reagent to analyte, the reaction time, pH, etc.) and separation conditions. Sensitive detection of GSH at concentrations as low as 0.18 nM was obtained. The method was applied in the analysis of biological fluids (exhaled breath condensate, saliva) and was found to be suitable for determination of GSH in these samples at trace levels below 1 nM. To the best of our knowledge, this is the first report on determination of GSH in exhaled breath condensate by capillary electrophoresis (CE).

Fingerprinting postblast explosive residues by portable capillary electrophoresis with contactless conductivity detection

A portable capillary electrophoretic system with contactless conductivity detection was used for fingerprint analysis of postblast explosive residues from commercial organic and improvised inorganic explosives on various surfaces (sand, concrete, metal witness plates). Simple extraction methods were developed for each of the surfaces for subsequent simultaneous capillary electrophoretic analysis of anions and cations. Dual‐opposite end injection principle was used for fast (<4 min) separation of 10 common anions and cations from postblast residues using an optimized separation electrolyte composed of 20 mM MES, 20 mM l‐histidine, 30 μM CTAB and 2 mM 18‐crown‐6. The concentrations of all ions obtained from the electropherograms were subjected to principal component analysis to classify the tested explosives on all tested surfaces, resulting in distinct cluster formations that could be used to verify (each) type of the explosive.

Portable capillary electrophoresis instrument with contactless conductivity detection for on-site analysis of small volumes of biological fluids

A novel, easy to use and portable capillary electrophoretic instrument for injection of small volumes of biological fluids equipped with contactless conductivity detection was constructed. The instrument is lightweight (<5 kg), all necessary parts including a tablet computer are accommodated in a plastic briefcase with dimensions 20 cm × 33 cm × 17 cm (w × l × h), allows hydrodynamic injection of small sample volumes and can continuously operate for at least 10 hours. The semi-automated hydrodynamic sample injection is accomplished via a specially designed PMMA interface that is able to repeatedly inject sample aliquots from a sample volume as low as 10 μL, with repeatability of peak areas below 5%. The developed interface and the instrument were optimized for the injection of biological fluids. Practical utility was demonstrated on the determination of formate in blood serum samples from acute methanol intoxication patients and on the analysis of ionic profile (nitrosative stress markers, including nitrite and nitrate) in the exhaled breath condensate from one single exhalation.

Monitoring of HPLC profiles of selected polyphenolic compounds in sea buckthorn (Hippophaë rhamnoides L.) plant parts during annual growth cycle and estimation of their antioxidant potential

AbstractPresented work summarizes the data about polyphenolic profiles in various plant parts (leaves, shoots, berries) of sea buckthorn (Hippophaë rhamnoides L.) during the annual growth cycle. A reversed-phase high performance liquid chromatography method (RP-HPLC) coupled with diode-array detection (DAD) was optimized for determination of catechin, epicatechin, gallic acid, p-coumaric acid, caffeic acid, ferulic acid, rutin (quercetin 3-rutinoside) and quercitrin (quercetin 3-rhamnoside). The content of these polyphenolic compounds was monitored in extracts of sea buckthorn plant samples from April to October. The total antioxidant activity was determined using scavenging of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonate) cation radical (ABTS·+) and 1,1-diphenyl-2-picrylhydrazyl radical (DPPH·). The total content of polyphenols was estimated by conventional spectrophotometric method using Folin-Ciocalteu reagent. The monitoring of temporal changes of selected polyphenolic compounds by RP-HPLC showed that catechin, epicatechin and gallic acid were the most abundant analytes in annual green shoots and leaves, and their content varied significantly during the studied period.n

Capillary electrophoresis with contactless conductometric detection for rapid screening of formate in blood serum after methanol intoxication

A new method for rapid, direct determination of formate in blood serum samples by capillary electrophoresis with contactless conductometric detection is presented. A selective separation of formate was achieved in approximately 1 min using an electrolyte system comprising 10 mM L-histidine, 15 mM glutamic acid and 30 μM cetyltrimethylammonium bromide at pH 4.56. The only sample preparation was dilution (1:100) with deionized water. The limit of detection and limit of quantitation was 2.2 μM and 7.3 μM, respectively, which corresponds to 0.22 mM and 0.73 mM in undiluted blood serum. The method provides a simple and rapid diagnostic test in suspected methanol intoxication cases. The method has been successfully tested on determination of formate in blood of a patient admitted to the hospital under acute methanol intoxication. The peak concentration of formate detected in the patient blood serum was 12.4mM, which is 10- to 100-fold higher than the normal values in healthy population. The developed method presents the fastest test currently available to detect formate in blood samples.

Monitoring the ionic content of exhaled breath condensate in various respiratory diseases by capillary electrophoresis with contactless conductivity detection.

The analysis of an ionic profile of exhaled breath condensate (EBC) by capillary electrophoresis with contactless conductivity detection and double opposite end injection, is demonstrated. A miniature sampler made from a 2u2009ml syringe and an aluminium cooling cylinder was used for the fast collection of EBC (under one minute). Analysis of the collected EBC was performed in a 60u2009mM 2-(N-morpholino)ethanesulfonic acid, 60u2009mM L-histidine background electrolyte with 30u2009µM cetyltrimethylammonium bromide and 2u2009mM 18-crown-6 at pH 6, and excellent repeatability of migration times (RSDu2009u2009<1.3% (n = 7)) and peak areas (RSDu2009u2009<u2009u20097% (n = 7)) of 14 ions (inorganic anions, cations and organic acids) was obtained. It is demonstrated that the analysis of EBC samples obtained from patients with various respiratory diseases (chronic obstructive pulmonary disease, asthma, pulmonary fibrosis, sarcoidosis, cystic fibrosis) is possible in less than five minutes and the ionic profile can be compared with the group of healthy individuals. The analysis of the ionic profile of EBC samples provides a set of data in which statistically significant differences among the groups of patients could be observed for several clinically relevant anions (nitrite, nitrate, acetate, lactate). The developed collection system and method provides a highly reproducible and fast way of collecting and analyzing EBC, with future applicability in point-of-care diagnostics.

Capillary electrophoresis in the analysis of biologically important thiols

In this review article, CE methods for analysis of biologically important thiols are overviewed. The article covers the period from the previously published comprehensive review in 2004 until mid‐2016, with emphasis on various detection modes, novel approaches for sample preconcentration, and applications in clinical practice. The most commonly used detection methods, such as conductometry or absorbance detection, although universally applicable and available in most commercial instruments have low sensitivity and have only limited use in thiol analysis. Amperometric and MS detection are more sensitive and have their steady place in thiol analysis, although the mainstay remains CE with LIF detection, reaching nanomolar concentration sensitivities for most of the thiols. Novel probes for CE‐LIF have been developed and tested. The preconcentration approaches using modified gold nanoparticles reaching excellent sensitivities in the picomolar range and various sample stacking methods are also reviewed. Finally, significant clinical applications of the developed methods are discussed with critical insights into the future of CE analysis of thiols.

Inorganic analysis using CE: Advanced methodologies to face old challenges

Inorganic species still remain of secondary importance when CE is used as an analytical separation tool. Despite considerable efforts of great many groups over last 20 years, plenty of competition, mostly from ion chromatography and elemental MS, restrains the routine use of CE in inorganic analysis. If there is yet hope clinging to make CE a viable alternative in the field, success would ultimately be attained through research activities on negating the disadvantages and amplifying the advantages of the method. Being the latest update of a series of reviews covering the time period from 1990 to 2010, this paper will comment on how and to what extent these challenges of inorganic CE analysis have been addressed in two recent years.

Double opposite end injection capillary electrophoresis with contactless conductometric detection for simultaneous determination of chloride, sodium and potassium in cystic fibrosis diagnosis

A novel approach for diagnosis of cystic fibrosis is presented. A simple and fast procedure to obtain sweat sample was developed. It consists of repeatedly wiping the skin of the forearm with deionized water moisturized cotton swab and extraction in 1mL of deionized water. Double opposite end injection capillary electrophoresis with contactless conductometric detection is used for the analysis of the extract. Chloride, sodium and potassium as the three target ions that participate in the ion transfer across the cellular membranes, and are affected by CF, are simultaneously determined in approximately 3min in a background electrolyte containing 20mM 2-(N-morpholino)ethanesulfonic acid, 20mM l-histidine and 2mM 18-crown-6. By using the target ion ratios rather than the concentrations of each individual ion combined with principal component analysis, the diagnosis of CF can be made more accurately and greatly reduce the number of false positive or negative results as is often the case when single ion (chloride) is analyzed.

Single‐breath analysis using a novel simple sampler and capillary electrophoresis with contactless conductometric detection

The analysis of ionic content of exhaled breath condensate (EBC) from one single breath by CE with C4D is demonstrated for the first time. A miniature sampler made from a 2‐mL syringe and an aluminum cooling cylinder for collection of EBC was developed. Various parameters of the sampler that influence its collection efficiency, repeatability, and effect of respiratory patterns were studied in detail. Efficient procedures for the cleanup of the miniature sampler were also developed and resulted in significant improvement of sampling repeatability. Analysis of EBC was performed by CE‐C4D in a 60 mM MES/l‐histidine BGE with 30 μM CTAB and 2 mM 18‐crown‐6 at pH 6 and excellent repeatability of migration times (RSD < 1.3% (n = 7)) and peak areas (RSD < 7% (n = 7)) of 12 inorganic anions, cations, and organic acids was obtained. It has been shown that the breathing pattern has a significant impact on the concentration of the analytes in the collected EBC. As the ventilatory pattern can be easily controlled during single exhalation, the developed collection system and method provides a highly reproducible and fast way of collecting EBC with applicability in point‐of‐care diagnostics.