Szymon Dziomba

Field-amplified sample stacking-sweeping of vitamins B determination in capillary electrophoresis.

A capillary electrophoretic method for determination of five water soluble vitamins B along with baclofen as an internal standard has been developed and assessed in context of precision, accuracy, sensitivity, freedom from interference, linearity, detection and quantification limits. On-line preconcentration technique, namely field-amplified sample stacking (FASS)-sweeping, has been employed in respect to obtain more sensitive analysis. Separation conditions received after optimization procedure were as following background electrolyte (BGE), 10 mM NaH(2)PO(4), 80 mM SDS, (pH 7.25); sample matrix (SM), 10 mM NaH(2)PO(4) (pH 4.60); uncoated fused silica capillary (50 μm i.d. × 67 cm length); UV spectrophotometric detection at 200 nm; injection times: 10s and 30s at 3.45 kPa; applied voltage 22 kV; temperature 22°C. Validation parameters, namely precision, accuracy and linearity, were considered as satisfactory. Under the optimized conditions, it has been also successfully applied for vitamins B determination in bacterial growth medium and commercially available Ilex paraguariensis leaves.

Micelle to solvent stacking of tricyclic psychiatric drugs in capillary electrophoresis.

A capillary electrophoresis (CE) method with UV-detection based on preconcentration with on-line micelle to solvent stacking (MSS) was optimized and validated to the simultaneous determination of tricyclic psychiatric drugs in urine samples. Sample preparation was based on liquid-liquid extraction (LLE) of analyzed drugs from biological samples. The electrophoretic conditions were: separation buffer composed of 40 mM sodium dihydrogen phosphate (pH 3.7) and 60% methanol (v/v), uncoated fused silica capillary (75 μm i.d.× 57 cm length), UV spectrophotometric detection at 254 nm, injection time 90 s at 3.45 kPa, applied voltage 25 kV, and temperature 22 °C. The advantage of proposed MSS strategy is its improved limit of detection of analytes compared to traditional CE technique. This is important for the analysis of drugs presented at very low concentration in biological samples. The developed CE method allows determination of psychotropic drugs at ng/ml level in human urine. This is the first report, presenting the possibility of determination of these drugs in urine samples by MSS-CE.

Field-amplified sample injection coupled with pseudo-isotachophoresis technique for sensitive determination of selected psychiatric drugs in human urine samples after dispersive liquid-liquid microextraction.

A field-amplified sample injection (FASI) technique was elaborated for fast and sensitive determination of selected central nervous system drugs in human urine samples. Factors affecting the sensitivity enhancement, such as background electrolyte (BGE) and the analytical matrix composition were optimized and discussed. Pseudo-isotachophoresis (p-ITP) mechanism contribution in preconcentration mechanism was discussed. All separations were performed in uncoated fused silica capillaries 50 μm × 57 cm at 22 kV. The optimized analytical matrix was composed of 0.25 mM HCOOH in 90% (v/v) methanol, while BGE contained 45 mM TRIS/HCl (pH 2.20). The head-column injection was performed in 0.25 mM HCOOH water solution (3s, 3.45 kPa). Sample was introduced into the capillary by electrokinetic injection (70 s, 5 kV) followed by short BGE plug (3s, 3.45 kPa). Seven psychiatric drugs (olanzapine, prochlorperazine dimaleate, trifluoperazine dihydrochloride, perphenazine, promazine hydrochloride, clomipramine hydrochloride, and chlorprothixene hydrochloride) were separated in about 6 min. The elaborated method was additionally supported with dispersive liquid-liquid microextraction (DLLME) technique which in summary with FASI provided about 8000-13,000-fold sensitivity enhancement in comparison to the capillary zone electrophoresis (CZE) method with standard hydrodynamic injection (5s, 3.45 kPa).

The advances of electromigration techniques applied for alkaloid analysis

Alkaloids are natural products of metabolism found mainly in plants. Their diversified pharmacological activities as medical agents and extensive occurrence in nutritional products demand the development of reliable and sensitive analytical tools. This review presents the developments in the field of electromigration techniques and specifically capillary and microchip electrophoresis. We include the main aspects of interest to researches over the past 12 years. The scope of this review covers detection, on- and off-line preconcentration techniques, chiral separation and developments in the field of microchip electrophoresis. Their applications in alkaloid determination, capillary electrochromatography and inter-molecular interactions are also examined.

Thin layer chromatography in drug discovery process

The review is mainly focused on application of thin layer chromatography (TLC) as simple, rapid and inexpensive method for lipophilicity assessment. Among separation techniques, TLC is still one of the most popular for lipophilicity measurement. The principles and methodology of Quantitative Structure Retention Relationship (QSRR) employed to lipophilicity prediction from retention data are presented. Moreover, applications of TLC retention constants in Quantitative Structure Activity Relationship (QSAR) studies were critically overviewed. The paper concerns also bioautography as a TLC method complementary to QSAR studies. In the article, the advantages and limitations of well established and less common planar chromatography modes applied for drug discovery process were discussed.

Optimization of a Pre-MEKC Separation SPE Procedure for Steroid Molecules in Human Urine Samples

Many steroid hormones can be considered as potential biomarkers and their determination in body fluids can create opportunities for the rapid diagnosis of many diseases and disorders of the human body. Most existing methods for the determination of steroids are usually time- and labor-consuming and quite costly. Therefore, the aim of analytical laboratories is to develop a new, relatively low-cost and rapid implementation methodology for their determination in biological samples. Due to the fact that there is little literature data on concentrations of steroid hormones in urine samples, we have made attempts at the electrophoretic determination of these compounds. For this purpose, an extraction procedure for the optimized separation and simultaneous determination of seven steroid hormones in urine samples has been investigated. The isolation of analytes from biological samples was performed by liquid-liquid extraction (LLE) with dichloromethane and compared to solid phase extraction (SPE) with C18 and hydrophilic-lipophilic balance (HLB) columns. To separate all the analytes a micellar electrokinetic capillary chromatography (MECK) technique was employed. For full separation of all the analytes a running buffer (pH 9.2), composed of 10 mM sodium tetraborate decahydrate (borax), 50 mM sodium dodecyl sulfate (SDS), and 10% methanol was selected. The methodology developed in this work for the determination of steroid hormones meets all the requirements of analytical methods. The applicability of the method has been confirmed for the analysis of urine samples collected from volunteers—both men and women (students, amateur bodybuilders, using and not applying steroid doping). The data obtained during this work can be successfully used for further research on the determination of steroid hormones in urine samples.

Repetitive injection field-amplified sample stacking for cationic compounds determination.

The development of a field-amplified sample stacking technique is presented. Sensitivity enhancement in this technique was obtained by repetitive injections of a sample followed by steps of sample matrix removal through the application of counter-pressure. Under optimized conditions the background electrolyte (BGE) was composed of 80 mM H3PO4 while the sample matrix contained 0.5mM H3PO4 and 30% (v/v) methanol. The elaborated method enabled a 4-fold effective injection of the sample (53 s, 0.5 psi). Each injection was followed by a focusing step during which the application of a voltage (2 kV) and counter-pressure (-1 psi) was performed for 0.65 min. The method was developed for the determination of six psychiatric drugs (opipramol, hydroxyzine, promazine, amitriptyline, fluoxetine, and thioridazine). The elaborated method was applied for analysis of human urine samples after a simple liquid-liquid extraction procedure. The detection limits obtained were in the range of 2.23-6.21 ng/mL.

Sweeping of hydrophobic amines under inhomogeneous electric field and low surfactant concentration in micellar electrokinetic chromatography.

The influence of sample matrix on sample sweeping in MEKC was examined in the presented manuscript. Significant focusing effect was observed for relatively hydrophobic cationic compounds (emetine, strychnine and quinine) using high ionic strength sample matrix (900 mM H3PO4/720 mM Tris) which conductivity was about ninefold higher than utilized BGE. Moreover, the results were obtained using BGE composed of comparatively low surfactant concentration (10 mM SDS) and 40 mM H3PO4/32 mM Tris buffer solution. About 200 to 300‐fold preconcentration of analytes was reached with the presented method. Basing on experimental results and computer simulation using Simul5 software, hypothetical mechanism of observed phenomenon was proposed.

Simultaneous determination of scopolamine, hyoscyamine and anisodamine in in vitro growth media of selected Solanaceae hairy roots by CE method.

An electrophoretic method for fast separation of three tropane alkaloids (hyoscyamine, anisodamine and scopolamine) was presented. The substances were complete resolved in less than one minute due to utilization of relatively short capillary (20.2cm effective length) and high voltage (25kV). Detector probing frequency was found among the parameters that significantly affected the detection sensitivity. The performed experiments showed insufficient available probing frequency of used commercial spectrophotometric detector according to capillary electrophoresis (CE) separation potential. Under the optimized conditions the background electrolyte (BGE) was composed of 20mM Tris, 6mM HCl and 20mM NaCl (pH 8.50). All analyses were carried out in fused silica capillaries of 50μm (inner diameter) and 31.2cm (total capillary length). Samples were injected hydrodynamically (5s; 3.45kPa) without any sample preparation step and separation was performed at 25kV. The elaborated method was applied in plant cultures growth media analysis after incubation with hairy roots of selected Solanaceae species. The performed experiments proved the usefulness of CE in quality control of biotechnological processes.

Gold nanoparticles dispersion stability under dynamic coating conditions in capillary zone electrophoresis

Capillary zone electrophoresis (CZE) of unmodified gold nanoparticles (Au NPs) was investigated in terms of dispersion stability in a presence of buffering counter-ions in background electrolyte (BGE). Capillary length, migration time and electric field strength were identified among factors influencing particles CZE. Moreover, BGE electrolysis was found to significantly affect analyses repeatability. The adsorption of NPs to capillary wall was recognized as the main problem. It was shown that this inconvenience can be overcome by the application of relatively big counter-ions. According to this observation, steric stabilization of NPs suspension by BGE components during CZE was hypothesized. In result, repeatable CZE of bare Au NPs under dynamic coating conditions was shown.