Lucia Veizerová

Monastrol analogs: A synthesis of pyrazolopyridine, benzopyranopyrazolopyridine, and oxygen-bridged azolopyrimidine derivatives and their biological screening

A synthesis of novel pyrazolopyridine, benzopyranopyrazolopyridine, and oxygen-bridged pyrazolo-, tetrazolo-, benzimidazo-, and thiazolopyrimidines via Hantzsch- and Biginelli-like condensations has been developed. The ability of these compounds to inhibit Eg5 activity has been examined. The results indicate that synthetic manipulations in the monastrol thiourea moiety are inefficient.

Multidrug analysis of pharmaceutical and urine matrices by on-line coupled capillary electrophoresis and triple quadrupole mass spectrometry

The present work illustrates potentialities of CE hyphenated with MS/MS for the simultaneous determination and identification of a mixture of simultaneously acting drugs in pharmaceutical and biological matrices. Here, the hyphenation was provided by ESI interface, while the MS/MS technique was based on the triple quadrupole configuration. Three drugs, namely pheniramine, phenylephrine, and paracetamol were determined and identified with high reliability due to their characterization in three different dimensions, i.e. electrophoresis and MS/MS, that prevented practically any interference. Appropriately selected transitions of the analytes (parent ion-quantifier product ion-qualifier product ion) provided their selective determination at maximum S/N. The proposed CE-MS/MS method was validated (LOD/LOQ, linearity, precision, recovery, accuracy) and applied for (i) the multidrug composition pharmaceuticals, namely Theraflu®, and (ii) human urine taken after per-oral administration of the same pharmaceutical preparation. The method was applied also for the investigation of potential weak associates of the drugs and monitoring of predicted (bio)degradation products of the drugs. Successful validation and application of the proposed method suggest its routine use in highly effective and reliable advanced drug control and biomedical research.

Determination of quinine in beverages by online coupling capillary isotachophoresis to capillary zone electrophoresis with UV spectrophotometric detection

The present study illustrates the possibilities of capillary isotachophoresis (CITP) online coupled with capillary zone electrophoresis (CZE) and hyphenated with fiber-based spectrophotometric diode array detection (DAD) for the direct, highly reliable, and ultrasensitive determination of quinine (QUI) in real multicomponent ionic matrices (beverages). Here, the CITP provided an effective online sample pretreatment (preseparation and preconcentration) prior to the CZE separation. Due to the CITP sample preconcentration, a simple UV-visible absorbance spectrophotometric detection was sufficient for obtaining very low concentration limits of detection (~2.3 ng/mL). Enhanced separation selectivity due to the combination of different separation mechanisms (CITP vs. CZE) enabled to obtain a pure analyte zone, suitable for its detection and quantitation in the directly injected real samples. The spectrophotometric DAD, unlike single wavelength UV detection, enabled to characterize the purity (i.e. spectral homogeneity) of the analyte zone and preliminary data indicate structurally related compounds via characteristic spectra recorded in the interval of 200-600 nm. The proposed CITP-CZE-DAD method was characterized by favorable performance parameters (sensitivity, linearity, precision, recovery, accuracy, robustness, and selectivity) and successfully applied to the control of QUI and potential QUI impurities in commercial beverages. This method is proposed as a routine automatized method for the highly reliable quality food control.

On-line column coupled isotachophoresis-capillary zone electrophoresis hyphenated with tandem mass spectrometry in drug analysis: Varenicline and its metabolite in human urine

A new highly advanced analytical approach, based on two-dimensional column coupled CE (ITP-CZE) hyphenated with tandem mass spectrometry (MS/MS, here triple quadrupole, QqQ) was developed, evaluated and applied in biomedical field in the present work. Capillary isotachophoresis (ITP) coupled on-line with capillary zone electrophoresis (CZE) used in hydrodynamically closed separation system was favorable for increasing the sample load capacity, increasing the analyte concentration, and removing the deteriorative highly conductive major matrix constituents. These factors considerably reduced the concentration limits of detection (cLOD) and external sample preparation (comparing to single column CZE), and, by that, provided favorable conditions for the mass spectrometry (enhanced signal to noise ratio, reproducibility of measurements, working life of MS). Here, the CZE-ESI combination provided more effective interfacing than ITP-ESI resulting in both a higher obtainable intensity of MS detection signal of the analyte as well as reproducibility of measurements of the analytes peak area. The optimized ITP-CZE-ESI-QqQ method was successfully evaluated as for its performance parameters (LOD, LOQ, linearity, precision, recovery/accuracy) and applied for the direct identification and ultratrace (pgmL(-1)) determination of varenicline and, in addition, identification of its targeted metabolite, 2-hydroxy-varenicline, in unpretreated/diluted human urine. This application example demonstrated the real analytical potential of this new analytical approach and, at the same time, served as currently the most effective routine clinical method for varenicline.

Lycopus europaeus: phenolic fingerprint, antioxidant activity and antimicrobial effect on clinical Staphylococcus aureus strains

Lycopus europaeus L. leaves water extract (LEL) was subjected to phytochemical analysis, and evaluated for its antibacterial and antioxidant effects. Antibacterial activity testing was performed on Staphylococcus aureus clinical strains from catheter-related and skin infections by broth microdilution test. LEL showed bactericidal activity at concentrations from 2500 to 5000 μg/mL against all, including methicillin resistant and polyresistant nosocomial, strains. Antioxidant activity was examined using DPPH and ABTS (11.3 and 9.8 μg/mL, respectively) and by ferric reducing ability of the plasma method (891 μmol AAE/g dry extract). Phytochemical analysis of LEL was performed by LC-DAD-MS/MS. Ten phenolic compounds were identified; two minor compounds (glucopyranosyl rosmarinic acid and sagerinig acid) have not been described in Lycopus yet. The major compounds, considered to be responsible for biological activities detected in the study, were determined as rosmarinic acid (76 mg/g) and luteolin-7-O-glucuronide (23 mg/g). L. europaeus arises from our study as a promising source of antibacterial agent for topical usage.

On-line coupled capillary isotachophoresis-capillary zone electrophoresis in hydrodynamically closed separation system hyphenated with laser induced fluorescence detection

An analytical method, based on a column coupling capillary ITP and CZE in a hydrodynamically closed separation mode hyphenated with the detection in the modular arrangement, was developed in this work. Analytical possibilities of this approach are demonstrated on the direct and ultrasensitive quantitative determination of quinine (QUI) in diluted real multicomponent ionic matrices (beverages, urine). The detection cell interface, with the rectangular arrangement of the optical channels inside, connected the separation capillary with the LIF detector via optical fibers in the on‐column detection arrangement. ITP enabled the direct large volume (30 μL) injections of the diluted real matrices with an on‐line sample pretreatment (preseparation, preconcentration) so that no external sample preparation (except for the dilution) was necessary for the separation of the analyte in the multicomponent ionic matrices. Due to the ITP sample preconcentration and intrinsic sensitivity of the LIF detection, very low concentration LOD (as low as 77 pg/mL), were reached at the same time. This was ca. two orders lower than the corresponding LOD achieved by the same 2D separation system with UV absorbance detection. Compared to the single column CE‐LIF methods applied for this model analyte and matrix, this method was found to be superior in terms of concentration LOD, with acceptable selectivity and benefits of the on‐line sample preparation. A food control and bioanalytical application clearly illustrates great practical possibilities and routine use of the proposed modular ITP–CZE–LIF technique.

2D Capillary Electrophoresis Hyphenated with Spectral Detection for the Determination of Quinine in Human Urine

The possibilities of a column coupling two-dimensional capillary electrophoresis (2D CE) combined with fiber-based diode array detection (DAD) for the direct, highly reliable and ultrasensitive quantitative determination of quinine in real multicomponent ionic matrices (human urine) are demonstrated in this work. The capillary isotachophoresis (CITP) stage provided an on-line sample pretreatment (elimination of interfering matrix constituents, preseparation and preconcentration of the analyte) before the capillary zone electrophoresis (CZE) separation. Due to the large volume (30 µL) sample injection and CITP sample preconcentration, a simple absorbance photometric detection was sufficient for obtaining very low concentration limits of detection (∼8.6 ng/mL). The combination of the different separation mechanisms (CITP and CZE) resulted in enhanced separation selectivity. This enabled us to obtain a pure analyte zone in the directly injected real samples suitable for qualitative and quantitative evaluation. The spectral DAD allowed (i) characterization of the purity (i.e., spectral homogeneity) of the analyte zone; and (ii) preliminary indication of structurally related compounds (i.e., potential biodegradation products of quinine), via characteristic spectra recorded in intervals of 200-800 nm. The CITP-CZE-DAD method was characterized by favorable performance parameters that are suitable for its routine biomedical use. One of the primary benefits of the CITP-CZE-DAD method is the possibility of performing direct injections of real biological samples while avoiding external sample preparation procedures and, therefore, enhancing the reliability and applicability of analyses and the potential for method automatization and miniaturization.

HPLC-QTOF-MS Method for Identification and Determination of Bleomycin A2 and B2 Fractions

Bleomycin is a very important drug in oncology used as first-line treatment for many cancers. The development of high-performance liquid chromatography—tandem mass spectrometry method for the separation, identification, and determination of two major structurally related analogs of this antibiotic drug, namely bleomycin A2 and B2, is presented in this work. The proposed method is based on a hydrophilic interaction chromatography (HILIC) stationary phase that enables one to avoid an ion-pairing reagent in the separation system (formerly used in mobile phase for bleomycin) in order to properly separate these highly polar bleomycin analogs. The ion-pairing reagent-free HILIC separation system is suitable for coupling the chromatographic and mass spectrometry stages (for the first time for bleomycin). Some performance parameters, namely, limit of detection (ng/mL-pg/mL), limit of quantification, linearity, precision, and recovery/accuracy, were evaluated showing high reliability, selectivity, and sensitivity of the method. It was successfully applied for the quality drug control determining bleomycin A2 and B2 fractions in commercial pharmaceuticals (Bleomedac infusions). In addition, the high-performance liquid chromatography–quadrupole-time of flight mass spectrometry (HPLC-QTOF-MS) method was able to determine an accurate molecular weight of bleomycin A2 and B2 fractions, confirming an identity/quality of the (commercial) drug. The possibilities of the HPLC-QTOF-MS method to identify bleomycin A2 and B2 in model plasma samples were also demonstrated.

(5R*,11R*)-5-Methyl-1,2-dihydro-5,11-methano-5H,11H-1,3-thia­zolo[2,3-d][1,3,5]benzoxadiazo­cine

The title compound, C(13)H(14)N(2)OS, crystallizes as a racemate in a non-chiral space group. It represents a conformationally restricted analogue of so-called Biginelli compounds known to exhibit multiple pharmacological activities and was selected for a single-crystal X-ray analysis in order to probe the chemical and spatial requirements of some kinds of activity. It was found that the state of hybridization of the formally aminic nitro-gen of the heterocycle is between sp(2) and sp(3) with the lone-pair electrons partially delocalized through conjugation with the sulfur atom rather than the double bond of the pyrimidine nucleus. As a result, the thia-zolo ring adopts a flat-envelope conformation and the puckering of the central pyrimidine ring is close to a half-chair. The critical phenyl ring is fixed in a pseudo-axial and perpendicular [dihedral angle 84.6 (1)°] orientation with respect to the pyrimidine ring via an oxygen bridge.

Ethyl 6-ethoxy-carbonyl-methyl-4-(2-hydroxy-phen-yl)-2-oxo-1,2,3,4-tetra-hydro-pyrimidine-5-carboxyl-ate.

The title compound, C17H20N2O6, belongs to the monastrol-type of anticancer agents and was selected for crystal structure determination in order to confirm its molecular structure and explore some aspects of its structure–activity relationships. The central tetrahydropyrimidine ring has a flat-envelope conformation. The 4-hydroxyphenyl group occupies a pseudo-axial position and is inclined at an angle of 87.7 (2)° to the mean plane of the heterocyclic ring. Of the two ethyl ester groups, one (in the 5-position) is in a coplanar and the other (in the 6-position) is in a perpendicular orientation with respect to the heterocyclic plane. There is a three-dimensional hydrogen-bonding network in which all hydrogen-bond donors and acceptors are involved.