Jozef Čižmárik

Examination of the Chemical Composition of Propolis 2. Isolation and Identification of 4-Hydroxy-3-Methoxycinnamic Acid (Ferulic Acid) from Propolis

SummaryA component of propolis has been identified as 4-hydroxy-3-methoxycinnamic acid (ferulic acid). It was found in the course of the systematic investigation of the chemical composition of propolis, using UV, IR and NMR spectrometry and paper and thin-layer chromatography. The isolation and identification of the compound are described, and reference made to its pharmacological activity.

Relationship between physicochemical properties, lipophilicity parameters, and local anesthetic activity of dibasic esters of phenylcarbamic acid

The basic physicochemical properties, lipophilicity parameters of dibasic alkyloxy-substituted phenylcarbamic acids were estimated. For the prepared set of compounds the experimentally obtained solubility, acidity, and lipophilicity parameters were correlated with those computed using various computer programs based on the associative artificial neural network and fragmental methods. The results of pharmacological evaluation were used as entry data for the complex correlations.

Study of the Mechanism of Enantioseparation. Part XII. Comparison Study of Thermodynamic Parameters on Separation of Phenylcarbamic Acid Derivatives by HPLC using Macrocyclic Glycopeptide Chiral Stationary Phases

Abstract Four macrocyclic glycopeptide chiral stationary phases (CSPs) based on native teicoplanin (Chirobiotic T), methylated teicoplanin aglycone (Chirobiotic m‐TAG), teicoplanin aglycone (Chirobiotic TAG), and vancomycin (Chirobiotic V) were compared for the high performance liquid chromatographic (HPLC) separation of enantiomers of 1‐methyl‐2‐piperidinoethylesters of 2‐, 3‐and 4‐ alkoxyphenylcarbamic acid (potential local anaesthetic drugs). The enthalpies (ΔH i ), entropies (ΔS i ), and Gibbs energies (ΔG i ) of transfer were evaluated for the separation of these compounds. The enantiomers were separated isothermally in the range of 0–50°C with 10°C increments, in the polar organic mode. The thermodynamic parameters were calculated in order to gain an understanding of the driving forces for retention in this chromatographic system. From these results, it is evident that the elongation of the alkoxy‐chain has major influence on the values of (ΔS i ) when using the vancomycin CSP and on the values of (ΔH i ) using the teicoplanin‐type CSPs.

Application of umbelliferone molecularly imprinted polymer in analysis of plant samples

The molecularly imprinted polymers (MIPs) were synthesised and the influence of the type of porogen, the nature of sample solvent, and the binding capacity of material were tested by high-performance liquid chromatography (HPLC). Umbelliferone was used as the template for imprint formation. Methacrylic acid was used as the monomer and acetonitrile, ethanol, and chloroform as porogen. Non-imprinted polymers (NIPs) were prepared by the same procedure. The highest value of the specific binding capacity (269 μg of umbelliferone per 100 mg of polymer) was obtained for polymers prepared in chloroform as porogen and methanol/water (φr = 1: 1) as the sample solvent. The group-selective MIP was used as sorbent for the SPE pre-treatment of umbelliferone from plant extracts prior to HPLC analysis. Analysis of the spiked samples showed good recoveries (> 77 %). The limit of detection, limit of determination, and repeatability of the method were also calculated.

In vitro study of enzymatic hydrolysis of diperodon enantiomers in blood serum by two-dimensional LC.

An on-line coupled HPLC system is described for the determination of the enantiomers of diperodon in blood serum. The method involves three steps: (i) off-line preconcentration and clean-up, (ii) separation of the diperodon enantiomers from the matrix components on a reversed-phase stationary phase, and (iii) separation of the racemate from the reversed-phase column on a teicoplanin chiral stationary phase. The method is suitable for simultaneous determination of both enantiomers in serum up to 0.5 microg/ml. The degradation of diperodon enantiomers was studied in serum by an in vitro method and the experimental rate constants were determined. The enantiomeric hydrolysis rates and half-lives for diperodon in serum are different.

Determination of Quinic and Shikimic Acids in Products Derived from Bees and their Preparates by HPLC

Abstract An HPLC method was developed for determination of some organic acids in propolis, honey, and pastilles. The on‐line coupled Separon SGX C18 and Polymer IEX H‐form column with mobile phase composed of sulphuric acid 9 mmol/L and methanol (95:5) at a flow rate 0.8 mL/min and spectrophotometric detection at 215 nm were used for the determination of quinic acid and shikimic acid. Limit of detection of quinic acid was 10 µg/mL and shikimic acid 0.43 µg/mL. Limits of quantitation were 30 µg/mL for quinic acid and 1.26 µg/mL for shikimic acid. Shikimic acid concentrations from 4.2 to 309.0 µg/g and quinic acid concentrations from 0.2 to 6.2 mg/g were determined in all tested bees products. The differences in the acid concentrations were observed for propolis samples from East and West Slovakia.

Permeability profiles of M-alkoxysubstituted pyrrolidinoethylesters of phenylcarbamic acid across caco-2 monolayers and human skin.

AbstractPurpose. The purpose of the present research was to study 10 m-alkoxysubstituted pyrrolidinoethylesters of phenylcarbamic acid—potential local anesthetics. The relationships between the structure of the molecule, its physicochemical parameters (log Doct, log k, RM, solubility) were correlated to the permeability data obtained from permeation experiments in Caco-2 monolayers and excised human skin in vitro. Methods. The extent and mechanism(s) of permeability of the series were studied through a Caco-2 monolayer in the apical-to-basolateral (a-b) and basolateral-to-apical (b-a) directions. The MTT test was performed to determine cellular damage. In vitro transdermal permeability data were obtained from permeation experiments on excised human skin by using side-by-side chambers. Passive diffusion and iontophoretically enhanced permeability were measured. Results. In Caco-2 monolayers, similar results in the shape of the permeability curves were obtained for the two directions. In the b-a direction, the values of Papp were ∼2-6 times greater than in the a-b direction. A plot of drug permeability vs. the number of carbons in the alkoxychain plateaued first, after which the permeability decreased by the increasing lipophilicity of the drug. If the log Doct of the ester was ≥ 3.4 and the MW > 385 Da, no measurable Caco-2 permeability was found. Cell damage was also higher by the more lipophilic compounds. In excised human skin, the relationship between the passive diffusion of the drugs and the number of carbons in the alkoxychain was parabolic (r2 = 0.95). Introducing low-level electrical current (iontophoresis), transdermal permeability of the more hydrophilic phenylcarbamic acid esters increased clearly. Conclusions. Lipophilicity and solubility of a compound have crucial roles in the permeation process. A very high lipophilicity has, however, a negative influence on the permeability, both intestinally and transdermally. Iontophoresis significantly increases the diffusion of small and less lipophilic compounds.

COMPARISON HPLC AND FLUORESCENCE SPECTROMETRY METHODS FOR DETERMINATION OF COUMARIN DERIVATIVES IN PROPOLIS

This paper illustrates the composition of propolis samples obtained from two regions in the Slovak Republic and focuses on the determination of coumarins. The HPLC method with on-line spectrophotometric and fluorescent detection was used for the separation and the determination of coumarins from the subgroup of simple coumarins (esculin, daphnetin, fraxetin, umbelliferone, 4-methylumbelliferone, 4-hydroxycoumarin, scoparone, coumarin, herniarin) and cinnamyl alcohol. The results confirmed that the quantity of umbelliferone, 4-methylumbelliferone, and scoparone in the propolis depends on the area of collection. The HPLC results were compared with those obtained by the spectrofluorimetric method developed for the determination of total coumarins (calculated as scoparone). For the quantification of total coumarins in propolis extracts we found 15% higher values of coumarin content by fluorescence spectrometry than by HPLC, which can be ascribed to the higher matrix effects on fluorescence spectra.

Determination of Some Phenolic Acids in Propolis by an HPLC Method

Abstract The liquid chromatographic method was developed for determination of some organic acid in ethanol and water extracts of propolis from Slovakia. Analysis of chlorogenic acid, p‐hydroxybenzoic acid, and rosmarinic acid was realized with the chromatographic column Symmetry Shield RP18 type and using a gradient of mobile phase methanol‐water (pH=2.5 adjusted with formic acid) and spectrophotometric and fluorescence detection (for p‐hydroxybenzoic acid). Chlorogenic acid in the range from 2.7 to 56.6 µg/g and p‐hydroxybenzoic acid from 3.9 to 150.3 µg/g was determined in tested propolis samples. Rosmarinic acid concentration in all tested propolis samples was below the limit of detection in this method. The significant differences in the acids concentrations were observed for propolis samples from east and west of Slovakia. It is probably the effect of different conditions of the collection of the resin and secrets by bees.

HPLC Separation of Diazepam Conformers Coupled with Off‐Line NMR Experiment

Abstract The HPLC analysis of diazepam conformers was carried out in the temperature range 273–313 K, increment 10 K. The pH of the mobile phase was 3.3; 5.5; 5.5 (Ic=const; 6.5; 6.5 Ic=const.). The various flow rates of 0.2; 0.5; 1.0; mL/min were used. Chiral stationary phase based on β‐cyclodextrin (ChiraDex) was used for separation of diazepam. The influence of temperature, flow rate, pH of mobile phase, and ionic strength on the retention and elution profile on two peaks of diazepam conformers were studied. After that, the chiral β‐cyclodextrin (0.001 mol/L; 0.003 mol/L; 0.005 mol/L) was added into the mobile phase. The increasing amount of β‐cyclodextrin in the mobile phase caused the disappearance of the first peak. The complete chiral separation of diazepam was not possible because of simultaneous interconversion. The plateau between two peaks of diazepam appeared in the whole range studied, any inhibitory effects of chromatographic conditions (pH of mobile phase, temperature, flow rate, ionic strength) on interconversion was not observed, only the change of the area ratio between two peaks appeared. Off‐line standard 1H and COSY NMR experiments were used to study the diazepam structure in the achiral mobile phase. The results of 1H and COSY NMR experiments confirmed that there is no pH dependent open‐ring reaction of 1,4‐benzodiazepine ring that could occur during the residence time of diazepam in the achiral mobile phase.