Biological Activities and ADMET-Related Properties of Novel Set of Cinnamanilides.

A series of nineteen novel ring-substituted N-arylcinnamanilides was synthesized and characterized. All investigated compounds were tested against Staphylococcus aureus as the reference strain, two clinical isolates of methicillin-resistant S. aureus (MRSA), and Mycobacterium tuberculosis. (2E)-N-[3-Fluoro-4-(trifluoromethyl)phenyl]-3-phenylprop-2-enamide showed even better activity (minimum inhibitory concentration (MIC) 25.9 and 12.9 µM) against MRSA isolates than the commonly used ampicillin (MIC 45.8 µM). The screening of the cell viability was performed using THP1-Blue™ NF-κB cells and, except for (2E)-N-(4-bromo-3-chlorophenyl)-3-phenylprop-2-enamide (IC50 6.5 µM), none of the discussed compounds showed any significant cytotoxic effect up to 20 µM. Moreover, all compounds were tested for their anti-inflammatory potential; several compounds attenuated the lipopolysaccharide-induced NF-κB activation and were more potent than the parental cinnamic acid. The lipophilicity values were specified experimentally as well. In addition, in silico approximation of the lipophilicity values was performed employing a set of free/commercial clogP estimators, corrected afterwards by the corresponding pKa calculated at physiological pH and subsequently cross-compared with the experimental parameters. The similarity-driven property space evaluation of structural analogs was carried out using the principal component analysis, Tanimoto metrics, and Kohonen mapping.

Development of a precolumn derivatization HPLC method with diode-array detection for the determination of amino sugars in peat and soil humic acids.

The work is focused on the development of a high-performance liquid chromatography method with diode-array detection for the separation and quantitation of the three most abundant amino sugars; d-glucosamine, d-galactosamine, and d-mannosamine. The high-performance liquid chromatography separation was carried out by reversed-phase chromatography on Chromolith Performance RP-18e monolithic column after acid hydrolysis (5 M HCl) and precolumn derivatization of samples using diethyl ethoxymethylenemalonate. Gradient elution and a mobile phase composed of ammonium formate buffer solution (10 mmol/L, pH 3.60) and methanol with flow rate of 1.0 mL/min were used. The monitoring wavelength was set at 280 nm. The limits of detection and quantitation for analytes ranged from 0.017 to 0.122 mg/L and from 0.057 to 0.407 mg/L, respectively. The proposed method was successfully applied for the determination of amino sugars in samples of humic acids isolated from different soils and peat.

Characterization of Klason lignin samples isolated from beech and aspen using microbore column size-exclusion chromatography.

The present study is focused on the separation and characterization of lignin samples isolated by Klason method from European beech (Fagus sylvatica) broadleaf hardwood and European aspen (Populus tremula) broadleaf softwood by size-exclusion chromatography. The separation was carried out using dimethylformamide as major component of the mobile phase and a 3 mm id microbore column packed with hydroxyethyl methacrylate gel, calibrated with polystyrene standards. The influence of mobile phase composition and sample solvent composition on the chromatographic behavior and molar mass distributions was investigated.

Fast ultra-high-performance liquid chromatography with diode array and mass spectrometry method for determination of tadalafil drug substance and its impurities.

Tadalafil is used for the treatment of erectile dysfunction. Its related patents expired in 2016, and so related generic drug production is predicted to be increased. This work is focused on developing a fast ultra-high-performance liquid chromatography with diode array detection and/or mass spectrometry detection for the separation and determination of tadalafil and its impurities in pharmaceutical samples. A modern reversed-phase stationary phase with sub-2 µm particle size, Zorbax StableBond Rapid Resolution High Definition with octylsilane chemically bonded phase to totally porous silica particles, was used for the solving this problem. Column temperature was set at 40 ± 0.1°C. A mobile phase consisting of acetonitrile and aqueous solution of 0.1% (v/v) trifluoroacetic acid for diode array detection detection and 0.05% (v/v) formic acid, both running at a flow rate of 0.62 mL/min, were used to achieve the required separation of all components within a 5 min run. The limit of detection was 3.5 µg/L and the limit of quantification was 10.0 µg/L for the method for both UV and MS detectors. Accurate mass spectra of tadalafil's related impurities are shown for advanced confirmation. The method is directly transferable to routine analysis of tadalafil in pharmaceutical and control laboratories.

On-line flow-through extraction-preconcentration-large volume injection-RP LC for trace determination of pyrethroids in Slovak soil.

A rapid micro-analytical multiresidue method was developed for analysis of pyrethroids (kadethrin K, cypermethrin C and permethrin P) in soil micro-sample (200 mg). It uses on-line flow-through extraction of soil micro-samples (packed into a short glass column) with a methanol-aqueous citric acid buffer mixture, successive on-line SPE preconcentration of analytes from the extract and on-line RP-HPLC analysis with UV photometric detection. The separation of pyrethroids is performed on a Purospher RP-18e column with methanol/water as mobile phase. Effects of sorbent placed at the bottom of a short column holding the soil sample and different kinds of on-line SPE columns were tested. Besides, the influence of volume of the effluent on the pyrethroids recovery was also studied. Calibration curves were linear over the range assayed from 0.01 to 0.2 microg/mL with correlation coefficients of linear regression (least-squares method) in the range 0.998-0.999. Recovery studies were carried out at 0.25-1.00 microg/g dry soil fortification level and obtained recoveries were for K 81-84%, C 56-59% and for P 58-63%. Achieved LOD (confidence band) of studied pyrethroids were for large-volume injection (1 mL) 4.5 ng K, 3.7 ng C, 3.6 ng P or 27 ng/g K, 32 ng/g C and 29 ng/g P in dry soil "solid sampling HPLC".

Reversed phase liquid chromatography trace analysis of pesticides in soil by on-column sample pumping large volume injection and UV detection.

The idea of utilization of one hydraulic line of a common commercial HPLC pump for direct on-column sample pumping injection of large sample volumes, 20 mL, was further investigated with the aim to develop multicomponent pesticides trace residues HPLC method in gram soil samples. Target pesticides group involve asulam, atrazine, 2,4-D, PCA, propazine, simazine, 4-chloro-2-methylphenoxyacetic acid, 2-(4-chloro-2-tolyloxy) propionic acid, chlortoluron, metoxuron, epoxiconazole. The results proved the applicability of this approach in experiments with mixtures of analytes at low ng/mL levels. Analysis of 20 mL of soil leachates and extracts of fortified soil samples containing these pesticides at the 10-50 ng/g level (in dry soil) revealed good figures of merit, also in the presence of large excess of humics. LODs achieved by detection at 220 nm evaluated from calibration runs of spiked soil extracts by Hubaux et al. method ranged from 5-12 ng per injected volume. For 20 mL large volume injection it represents 0.25-0.6 ng/mL of diluted soil extract, or 2.5-6 ng/mL of crude extract, or 6-5 ng/g dry soil. Recoveries of pesticides at concentration levels approaching half of maximum allowable concentration of pesticides in soil (100 ng/g) ranged from 85 to 98% with acceptable reproducibility, except asulam and metoxuron.

Reversed-phase liquid chromatographic characterization and analysis of air particulates humic (-like) substances in presence of pollens.

Newly developed method for characterisation and analysis of humic substances (HS) and humic-like substances (HULIS) in air dust particles was tested for potential interferences caused by abundant co-sampled pollens (common dandelion, common wormwood-absinth, apple tree). RP-HPLC using 10-step gradient of dimethylformamide (DMF) in a buffered aqueous mobile phase and a wide-pore (30 nm) octadecylsilica column has been applied to the analysis of HS and HULIS using tandem of spectrophotometric (DAD) and fluorimetric detection (FLD). Achieved results suggest that the devised method is reliable for characterisation, fractionation and analysis of terrestrial HS, air dust HS and air particulate HULIS in liquid extracts at a trace concentration levels. Fluorimetric detection (ex. 470 nm/em. 530 nm; LOD, 3.1 microg/ml) enables sensitive, highly selective and interference free determination of HS and HULIS regardless the presence of pollen constituents, whereas spectrophotometric detection is susceptible to interferences in UV region above 260 nm. However, even in this case, the interfering substances can be revealed by both different pattern and shape of their peaks, as well as by spectral features different from HS or HULIS. Analytical procedure based on air sampling, extraction and above-mentioned HPLC method enables characterisation and analysis of HS and/or HULIS at relative concentration levels down to 0.1% (m/m) in 10 mg mass scale of sampled air dust and particulate material.

Rapid analysis of porphyrins at low ng/l and microg/l levels in human urine by a gradient liquid chromatography method using octadecylsilica monolithic columns.

Rapid gradient RP-HPLC method with fluorimetric detection for trace analysis of diagnostically significant porphyrins in human urine was developed for clinical and diagnostic purposes. Results show that optimized high-pressure gradient elution and monolithic column Chromolith SpeedRod RP18e enabled separation of seven urine porphyrins including baseline separation of I and III positional isomers of uro- and coproporphyrins within 3.2 min. Problems associated with high metal cation complexing ability of the analytes and common stainless steel based instrumentation were substantially reduced by use of 0.1 mol/l ammonium citrate buffer (pH 5.47) and methanol as a mobile phase components. Good reproducibilities of retention times (within +/- 0.36% RSD) and peak areas (from +/- 0.6 to +/- 2.5% RSD) at 5-20 microg/l level of the analytes were achieved. Determined LOQ (10 x S/N) values of diagnostically important porphyrins using fluorimetric detection (ex.405 nm/em.620 nm) were 82 pmol/l (65 ng/l, 1.30 pg/injection) for uroporphyrin I, 44 pmol/l (33 ng/l, 0.66 pg/injection) for uroporphyrin III, 50 pmol/l (40 ng/l, 0.80 pg/injection) for coproporphyrin I and 47 pmol/l (39 ng/l, 0.78 pg/injection) for coproporphyrin III. Attained LOQ concentration level is approximately 20-120 times lower than concentration of porphyrins in a urine of healthy person. Calculated LOD's (3 x S/N) were at a low ng/l levels, what enabled quantification of carry-over effect to be from 2.0% to 0.2% in each of three consecutive blank runs and from 2.5% to 7% in total after injection of mixed standard of porphyrins with 5-20 microg/l concentrations. Recovery of porphyrins at low microg/l concentration levels was from 93% to 97.5%. Devised method increases productivity of clinical laboratory from 2 to 10 times in dependence of duration of currently used method.

Short-column anion-exchange chromatography for soil and peat humic substances profiling by step-wise gradient of high pH aqueous sodium ethylenediaminetetraacetate.

Novel anion-exchange liquid chromatographic method with step gradient of aqueous EDTA(4-) based mobile phase elution has been developed to profile available Slovak soil humic substances and alkaline extracts of various soils. The method utilize short glass column (30mm×3mm) filled in with hydrolytically stable particles (60µm diameter) Separon HEMA-BIO 1000 having (diethylamino)ethyl functional groups. Step gradient was programmed by mixing mobile phase composed of aqueous solution of sodium EDTA (pH 12.0; 5mmolL(-1)) and mobile phase constituted of aqueous solution of sodium EDTA (pH 12.0, 500mmolL(-1)). The FLD of HSs was set to excitation wavelength 480nm and emission wavelength 530nm (λem). Separation mechanism was studied by use of selected aromatic acids related to humic acids with the aid of UV spectrophotometric detection at 280nm. The proposed method benefits from high ionic strength (I=5molL(-1)) of the end mobile phase buffer and provides high recovery of humic acids (98%). Accurate and reproducible profiling of studied humic substances, alkaline extracts of various types of soils enables straightforward characterization and differentiation of HSs in arable and forest soils. Selected model aromatic acids were used for separation mechanism elucidation.

Novel stepwise gradient reversed-phase liquid chromatography separations of humic substances, air particulate humic-like substances and lignins.

RP-HPLC using stepwise gradients of dimethylformamide (DMF) in buffered aqueous mobile phase and a wide-pore (30 nm) octadecylsilica column has been applied to the analysis of soil, peat and air particulate humic and humic-like substances (HSs), as well as lignin, in order to demonstrate the usefulness of the approach for their characterization even at trace concentration level. Tandem combination of spectrophotometric (DAD) and fluorimetric detection was used to get more detailed information on chromatographic behaviour of HSs. The results showed that ten-step gradient can induce distinct features of HSs and lignins. Combination of very good DMF solvating and disaggregating properties for HSs and lignins together with wide pore RP sorbent improves surface interactions of the analytes and suppresses influence of size exclusion effects. Thus it provides reproducibility of characterisation profiles and robustness of the method. Very good reproducibility of retention times (from +/-0.12 to +/-0.36% RSD), of peaks enforced by the step gradient shape supports well defined characterization and/or fractionation of HSs. Evaluated limit of quantitation (S/N = 10) of selected soil humic acid working standard using fluorimetric detection (ex.470 nm/em.530 nm) was 9.7 microg/ml. This corresponds to 0.5 microg of the humic acid per injection. Calculated limit of detection (S/N = 3) was 3.3 microg/ml, what corresponds to 0.15 microg of the humic acid per injection and enabled determination of 0.1% (m/m) humic-like substances in 20 mg of air particulates under defined rules and conditions.

Role of biological matrices during the analysis of chiral drugs by liquid chromatography.

The review article covers advances of chiral drugs analysis by high-performance liquid chromatography (HPLC) methods achieved during last 10 years. Emphasis is given to various aspects of influence of biological matrix in pharmacodynamics, pharmacokinetics, HPLC analysis. Discussed is composition of main biological matrices from the point of view of potential interferences to above-mentioned fields of study. Beside typical analytical approaches to chiral recognition in HPLC, sample pretreatment and/or clean-up by conventional extraction procedures, column switching (CSW) techniques using restricted access materials (RAMs), microdialysis (MCD) is discussed. Measurement of unbound drug concentration and discussion of column maintenance and remedy is an additional source of information and field where knowledge on complex properties and interactions of biological matrix is usefully applicable.

Characterization and analysis of soil humic acids by off-line combination of wide-pore octadecylsilica column reverse phase high performance liquid chromatography with narrow bore column size-exclusion chromatography and fluorescence detection.

Liquid chromatography method arranged around RP-HPLC using step-wise gradients of dimethylformamide (DMF) in pH 3.0 aqueous phosphate buffered mobile phase and a wide-pore (30 nm) 4mm inner diameter (I.D.) octadecylsilica column was applied to the fractionation separation of soil and peat humic substances (HS), respectively. Combination of acceptable DMF solvating properties for HSs together with wide pore RP sorbent improved surface interactions of the analytes and suppressed influence of size-exclusion effects in RP-HPLC. Individual fractions collected from the RP-HPLC were off-line analyzed by size-exclusion chromatographic method (SEC) using 99/1 DMF/aqueous phosphate buffer pH 3.0 with Spheron HEMA 100 stationary phase filled in a 2.2 mm I.D. column. Both methods provided reproducibility of characterization profiles and robustness resulting from excellent reproducibility of HSs fraction retention times (±0.5% RSD) of peaks enforced by the step gradient shape. Obtained results indicate that the methods can be combined in a compatible, compact, automatic, 2D orthogonal separation system for fluorimetric characterization of such complicated natural substances as are examined humic acids and obtain so more information about their character.

Some theoretical and practical aspects in the separation of humic substances by combined liquid chromatography methods.

Permanent need to understand nature, structure and properties of humic substances influences also separation methods that are in a wide scope used for fractionation, characterization and analysis of humic substances (HS). At the first glance techniques based on size-exclusion phenomena are the most useful and utilized for relating elution data to the molecular mass distribution of HS, however, with some limitations and exceptions, respectively, in the structural investigation of HS. The second most abundant separation mechanism is reversed-phase based on weak hydrophobic interactions beneficially combined with the step gradients inducing distinct features in rather featureless analytical signal of HS. Relatively great effort is invested to the developments of immobilized-metal affinity chromatography mimicking chelate-forming properties of HS as ligands in the environment. Surprisingly, relatively less attention is given to the ion-ion interactions based ion-exchange chromatography of HS. Chromatographic separation methods play also an important role in the examination of interactions of HS with pesticides. They allow us to determine binding constants and the other data necessary to predict the mobility of chemical pollutants in the environment. HS is frequently adversely acting in analytical procedures as interfering substance, so more detailed information is desired on manifestation of its numerous properties in analytical procedures. The article topic is covered by the review emphasizing advances in the field done in the period of last 10 years from 2000 till 2010.

Method of fast trace microanalysis of the chiral pesticides epoxiconazole and novaluron in soil samples using off-line flow-through extraction and on-column direct large volume injection in reversed-phase high performance liquid chromatography.

An analytical method combining off-line flow-through extraction of a soil micro-sample (mass around 100 mg, packed into a short HPLC glass column) and direct on-column large-volume injection (LVI up to 1.00 mL) of a methanol-water soil extract onto a conventional C18 RP HPLC column enabled fast (within 3.5 minutes) trace micro-analysis of the relatively new chiral pesticides epoxiconazole (E) and novaluron (N), respectively. Linear calibration curves were evaluated from UV detection (230 nm) data in the range from 0.1 to 5 mg/kg in three most abundant Slovak agricultural soils. LOD (confidence band) at the levels 0.08-0.11 mg/kg and LOQ 0.4-0.6 mg/kg and LOD (S/N = 3) at the levels 0.007-0.018 mg/kg and LOQ (S/N = 10) 0.024-0.060 mg/kg, respectively, of dry soil were achieved. Recovery of pesticides in the overall LVI method including flow-through 130-200 mg soil micro-sample extraction was: for epoxiconazole from 74 to 85% and from 56% to 90% for novaluron with reproducibility within +/- 6% RSD. This fast (30 min) and simple method consists of just three steps which are short column filling with a solid micro-sample; flow-through liquid extraction and direct large-volume injection RP HPLC DAD analysis. The method is prepared for automation and further analysis of enantiomers of both investigated pesticides by achiral-chiral column switching techniques.

Method of analysis of a selected group of pyrethroids in soil samples using off-line flow-through extraction and on-column direct large-volume injection in reversed phase high performance liquid chromatography.

An analytical method using flow-through extraction of a soil sample filled in a short HPLC column with methanol or methanol-water mixtures and large-volume injection in RP-HPLC has been developed for the simultaneous determination of residues of three pyrethroids--kadethrin, cypermethrin, and permethrin--from soil samples. The developed RP-HPLC method enables separation of four diastereoisomers of cypermethrin into three peaks and resolution of two diastereoisomers of permethrin. The UV photometric detection limits of direct on-column large-volume injection of 1.00 mL of extract were 30 ng/mL of kadethrin, 37 ng/mL of total content of cypermethrin, and 65 ng/mL of trans-permethrin, which corresponds to a pyrethroid soil content of around 0.3 mg/kg. Effects of extractant flow rate and optimal extractant volume on the percentage recovery of pyrethroids from Slovak soil samples were studied. Recovery studies were performed at 0.5- 5.0 microg/g fortification level of kadethrin and 1.0-2.5 microg/g fortification level of cypermethrin and permethrin in a soil sample. Recoveries ranged from 83 to 90% for kadethrin, from 87 to 94% for total cypermethrin, and from 85 to 98% for trans-permethrin. This work comprises a basic study aimed at elaboration of an RP-HPLC method of direct analysis of pyrethroids in a soil matrix at low concentration levels achieved by a "solid sample injection" in HPLC--on-line interfacing of analyte extraction, extract clean-up, and analysis.

Characterization of Klason lignins by reversed-phase high-performance liquid chromatography using wide-pore octadecylsilica and stepwise gradients of dimethylformamide in water.

RP-HPLC using stepwise gradients of N,N-dimethylformamide (DMF) in a phosphate-buffered (pH 3.00) aqueous mobile phase and a wide-pore (30 nm) octadecylsilica column has been applied to the analysis of a variety of lignins--organocell lignin, dioxane lignin, Björkman lignin, and Klason lignins from both softwoods and hardwoods, respectively, in order to demonstrate the usefulness of the approach for their characterization. Tandem combination of spectrophotometric diode array detection and fluorimetric detection was used to acquire more detailed information about the chromatographic behavior of lignins. The results show that a ten-step gradient can reveal distinct features of lignins and humic substances. Combination of good solvating properties of DMF for lignins together with a wide-pore RP sorbent improves surface interactions of the analytes and suppresses the influence of size exclusion effects. Thus it provides reproducibility of characterization profiles and robustness of the method. The calculated repeatability of the retention time of selected peaks was +/- 0.46% RSD. The reproducibility of the data within one week (set of seven data) was +/- 1.1% RSD. These data are also representative of the other well-shaped peaks of analyzed substances.

New approach to large-volume injection in reversed-phase high performance liquid chromatography: determination of atrazine and hydroxyatrazine in soil samples.

A well established method of direct injection of larger than conventional sample volumes ranging from 0.1 mL to 10 mL in HPLC is the injection valve method in which a loop of tubing is totally or partially filled with sample. Recent HPLC pumps have a flow-rate setting accuracy of +/- 1-2% over a flow-rate range from 0.1 mL/min to 10 mL/min and the flow stability is 0.2% or less. Quarternary low pressure gradient pumps are widely available and used, but all their hydraulic lines are seldom utilised. The idea of using one line of a common commercial HPLC quaternary low-pressure pump for direct on-column injection (pumping) of large sample volumes ranging from 1 mL to 100 mL was tested. This approach was evaluated during practical work on the development of an RP-HPLC method of determination of residual atrazine and hydroxyatrazine. In lysimetric environmental experiments hydroxyatrazine was formed in situ in a soil column by hydrolysis of atrazine. The results proved the applicability of this approach not only in experiments with model mixtures of analytes at microg/L levels in solutions. Analysis of 20 mL of soil leachates and extracts of soil samples containing atrazine and hydroxyatrazine at the 10 microg/kg level (in dry soil) revealed that good figures-of-merit were preserved, even in the presence of a large excess of humic substances.