[Separation of phenylalanine and methionine enantiomers by HPLC method: a comparison of stationary phase types]. / Separácia enantiomérov fenylalanínu a metionínu metódou HPLC: porovnanie typov stacionárnych fáz.

The paper deals with enantioselective separation of amino acids by the high performance liquid chromatography method. Separations of enantiomeric forms were tested on the chiral stationary phases based on ß-cyclodextrine, isopropyl carbamate cyclofructan 6, and the macrocyclic antibiotic teicoplanin. The best enantioseparation was obtained on the teicoplanin-based chiral stationary phase in the reversed-phase mode. UV spectrophotometric detection at 210 nm was used for detection of amino acids. The method was validated with respect to linearity, precision, limit of detection, limit of quantitation, and recovery. Limits of quantitation for phenylalanine and methionine enantiomers were 0.3 and 0.2 µg.ml-1, respectively. The HPLC method with teicoplanin-based chiral stationary phase was applied for analysis of dietary supplements.Key words: separation of enantiomers HPLC phenylalanine methionine.

[Determination of flavonoids in natural materials with HPLC]. / Stanovenie flavonoidov v prírodnom materiáli metódou HPLC.

The paper reviews recent developments in the determination of flavonoids with HPLC. Flavonoids are a group of polyphenolic compounds commonly found in plants. Sample preparation is a very important step in the analytical procedure. The basic concept of a sample preparation method is to convert a real matrix into a sample in a format that is suitable for analysis by separation or analytical technique. In the present paper, attention was focused on the applications of molecularly imprinted polymers, especially on their utilization as selective sorbents in solid-phase extraction in the determination of flavonoids.

Two-dimensional high performance liquid chromatography for determination of homocysteine, methionine and cysteine enantiomers in human serum.

A two-dimensional HPLC system with electrochemical detection was used for determination of homocysteine, methionine and cysteine enantiomers in biological samples. The amino acid separations were not possible only by using a chiral column. The compounds were separated from each other on an achiral column (Purospher RP-18 endcapped 250-4mm, 5µm) and their enantiomers were separated on Chirobiotic TAG (250-4.6mm, 5µm) column in an on-line system. The mobile phase composition and a choice of electrode potentials for detection were investigated. The l-enantiomers always eluted before the d-enantiomers. The proposed method was applied to the analysis of human serum of healthy volunteers and patients with multiple sclerosis. The limit of detection (LOD) and quantitation (LOQ) were defined as the concentration that produced a signal-to-noise ratio (S/N) of 3 and 10. The method LOD values were found to be between 0.05 and 0.50µgmL(-1). The range of LOQ values were between 0.17 and 1.67µgmL(-1), respectively.

Influence of oak wood polyphenols on cysteine, homocysteine and glutathione total levels and PON1 activities in human adult volunteers - a pilot study.

Oxidative stress reflects an imbalance between antioxidants and pro-oxidants. Many diseases like atherosclerosis or heart failure are involved in oxidative stress. Increased oxidative stress is one of the potential contributing factors to aging. The aim of this study was to monitor the total thiol levels as markers of oxidative stress in 20 healthy volunteers after polyphenols intake (extract from the French oak wood Quercus robur - Robuvit® (300 mg/day)). Polyphenols are known as biomodulators with antioxidant activities. Homocysteine, cysteine and glutathione total levels were determined by using HPLC with electrochemical detection. The activity of the antioxidant enzyme paraoxonase-1 toward two substrates was determined by spectrophotometry. The level of thiol compounds and paraoxonase-1 activities were controlled after run-in (week 0), intervention (week 4) and washout (week 6) period. After the intervention period the results showed that Robuvit® had no significant influence on glutathione level (p = 0.382) and paraoxonase activities towards both, arylester and lactone substrates. On the other hand, homocysteine and cysteine levels decreased significantly (p = 0.029; p < 0.001, respectively). The negative correlation between paraoxonase lactonase activity and homocysteine level was noticed. This confirms that paraoxonase might play an important role in homocysteine-thiolactone metabolism.

Using of molecularly imprinted polymers for determination of gallic and protocatechuic acids in red wines by high performance liquid chromatography.

The sorption capacities of gallic- and protocatechuic acid-molecularly imprinted polymers (GA-MIP and PCA-MIP, respectively) and non-imprinted polymer (NIP) have been determined on the piston columns by the frontal analyses (FAs). Mobile phases consisted of MeOH, MeOH/H2O (1:1), 12.5% EtOH or ACN. Solutes concentrations used in FAs were 1µg/mL and 50µg/mL. All sorption capacities were depended on analyte and solvent used. Results obtained from the FAs have shown that both imprinted polymers almost always were preferentially recognized PCA molecule. Only in MeOH, the GA-MIP had ability to recognize its template molecule positively. Surprisingly, in some cases, also the NIP exhibited higher sorption capacities than the MIPs for their templates, e.g. in ACN for GA or in MeOH for PCA. This behaviour indicates that in some solvents, the low affinity sites of the blank polymer can act as strong interacting sites. In the next, prepared MIPs were successfully used as the SPE-sorbents for the extraction and purification of chosen phenolic acids from red wine samples. The recoveries both of MIPs were the highest for PCA, what was in agreement with the experiments carried out in 12.5% EtOH during FAs. Prepared MIP-beads allowed the purification of chosen red wine samples with satisfactory selectivities and high recoveries. The linearity of the method was in the range from 10µg/mL to 70µg/mL and 0.1µg/mL to 4.5µg/mL for GA and PCA, respectively, with the determination coefficients ranging from 0.996-0.999. The LODs (S/N=3) ranged from 0.1µg/mL to 0.4µg/mL. The RSDs for the recoveries varied from 4.0% to 8.1%. The PAs-MIPs and corresponding NIP were also characterized by attenuated total reflectance analysis Fourier transform infrared spectroscopy (ATR-FTIR) and scanning electron analysis (SEM).

HPLC determination of saccharides after pre-column derivatization in honey samples.

The work is focused on the quality evaluation of Slovak honey samples on the basis of minor saccharide contents. The samples included unifloral and multifloral types of honeys. The saccharide contents were determined by HPLC with fluorescence detection. Derivatization of sugars with dansylhydrazine was used to increase detection sensitivity. The C18 type stationary phase was used for the separation of analytes and the mixture of acetonitrile and acetic acid was used as the mobile phase. The determined carbohydrates were saccharose, maltose, turanose, and also the main sugars glucose and fructose. Limits of detection were in the range from 15 to 100 µg.ml-1 and suggest that low quantities of all studied compounds can be detected. Differences in saccharide contents between Slovak samples and samples from different geographical regions (France, Croatia) for maltose, turanose and also for saccharose were obtained. Using multivariate statistical treatment, Slovak honeys can be distinguished from other tested honeys using the saccharide profiles.

Study of physicochemical interaction of aryloxyaminopropanol derivatives with teicoplanin and vancomycin phases in view of quantitative structure-property relationship studies.

The aim of this work was to study the physicochemical interactions between chiral stationary phases and chiral molecules and to elucidate which of the specific interactions are more or less important. The HPLC separation of 58 aryloxyaminopropanols was performed on two chiral stationary phases containing the macrocyclic antibiotics teicoplanin or vancomycin and using a methanol/acetonitrile/acetic acid/triethylamine mobile phase (volume ratios 45/55/0.3/0.2). The resolution of enantiomers (Rij) as the target variable was predicted for the mentioned kind of compounds by means of thoroughly selected descriptors provided by the applied Dragon software. The created QSPR models can be considered as a way to explore and discover new relationships or interactions between the quantitative structure and resolution of enantiomers. For calculation and validation of the QSPR models, different modelling methodologies were applied based on MLR (multiple linear regression) and ANN (artificial neural network) techniques. Both methods exhibit an ability for successful prediction of the enantioresolution characteristics of the studied molecules. The results seem to demonstrate that it is possible to predict resolution values of enantiomeric separations of related compounds on given chromatographic systems.

Application of the van't Hoff dependences in the characterization of molecularly imprinted polymers for some phenolic acids.

Thermodynamic analysis was used to quantify the contribution of entropic and enthalpic terms of the binding processes of selected phenolic acids (PAs), quercetin and diperodon on series of molecularly imprinted polymers (MIPs). All polymers were prepared using acrylamide as functional monomer and acetonitrile as a porogen. The following PAs were used as templates - gallic (GA), gentisic (GeA), syringic (SyrA), protocatechuic (PCA), 4-hydroxybenzoic (pHBA) and vanillic (VA). The assessment was based on quantification by HPLC measurement of the analytes tested at temperature range from 20°C to 60°C in two mobile phases - methanol and porogen. There were determined van't Hoff curves - dependences between logarithms of the retention factors (lnk) and the inverse value of the temperature (1/T). All plots fall along straight lines, what suggests that there were no changes in the sorption mechanisms over the studied temperature range. Determined thermodynamic characteristics helped to specify the nature of molecular recognition on the PAs-MIPs. We found that preferred eluent for analytes sorption on the PAs-MIPs and the NIP was porogen. When methanol as the mobile phase was used there was not documented sorption of the investigated compounds on the NIP. Calculated imprinting factors (IFs) in porogen were highest in the dominant advantage of template molecules used, what confirmed a good molecular imprinting effect. The IF values for PAs studied were as follows: GA=21.98±2.62, PCA=6.07±0.13, pHBA=3.58±0.25, SyrA=2.80±0.17, GeA=2.37±0.34 and VA=2.07±0.10. The results of thermodynamic studies demonstrated that enthalpic term was the dominating driving force for the predominant part of investigated analytes. The exceptions were: SyrA on the NIP and on the GA-MIP, diperodon on the PCA-MIP in acetonitrile and quercetin on the GA-MIP in methanol where a favourable driving force was to be found an entropic term. The PAs-MIPs and NIP were also characterized by attenuated total reflectance analysis Fourier transform infrared spectroscopy (ATR-FTIR) and scanning electron microscopy (SEM).

Thermodynamic study of molecularly imprinted polymer used as the stationary phase in high performance liquid chromatography.

Molecularly imprinted polymer (MIP) and non-imprinted polymer (NIP) on the base of methacrylic acid prepared by a bulk polymerization were used as stationary phases for the HPLC analysis. The thermodynamic processes were carried out to investigate the temperature effects during sorption processes of potential local anaesthetics - morpholinoethyl esters of alkoxy-substituted phenylcarbamic acid (MEsP), local anaesthetic - diperodon, flavonoid - quercetin in methanol, acetonitrile and toluene (porogen) as mobile phases. Mobile phases and corresponding solvents were selected according to the solubility of each analyte. The template was chosen from the set of homologous of MEsP - 2-(morpholin-4-yl)ethyl (2-methoxyphenyl)carbamate. Values of retention factors were measured over the temperature range of 20-60°C. There were determined van't Hoff curves - dependences between logarithms of the retention factors (lnk) and the inverse value of the temperature (1/T). Observed graphs were linear directly indicating that there were no changes of interaction mechanisms in the studied range of temperature. Selectivities (evaluated by the separation factors, α) and sorption selectivities (evaluated by the imprinting factors, IFs) of the MIP and the NIP toward template, related and not-related structures with the template were evaluated chromatographically. The highest separation factors and the imprinting factors (IF=4.73 ± 0.35 for the template) were observed in methanol, not in porogen. Only in the case of quercetin the highest IF was observed in ACN (1.88 ± 0.13). Contrary to expectations, the driving force for the affinity of the target molecules for both of polymers was enthalpic term (with an average of 54%, 82% and 84% contribution of enthalpic term for MeOH, ACN and toluene, respectively on the MIP and 53%, 57% and 65% for MeOH, ACN and toluene, respectively on the NIP). The MIP and NIP were also characterized by attenuated total reflectance analysis Fourier transform infrared spectroscopy (ATR-FTIR) and thermogravimetric analysis (TGA).

QSRR models for potential local anaesthetic drugs using high performance liquid chromatography.

Quantitative structure-retention relationships (QSRR) were proposed for Separon SGX C18 and Separon SGX Phenyl columns using physico-chemical molecular descriptors for the compounds, which are potential local anaesthetic drugs. Chemometrical methods were used for the QSRR studies of the HPLC retention factor k of 59 esters of alkoxyphenylcarbamic acid, which exhibit surface and/or infiltration anaesthetic activity. Four separation systems were used: phenyl column and acetonitrile/water mobile phase, phenyl column and methanol/water mobile phase, C18 column and acetonitrile/water mobile phase, and C18 column and methanol/water mobile phase. The values of logP and logS and ¹³C and ¹H NMR chemical shifts were simulated and utilized in calculating the corresponding QSRR models and predicting the retention factors by artificial neural networks (ANN). In addition, principal component analysis and cluster analysis were used for a closer characterization of alkoxyphenylcarbamic acid esters. The proposed ANN models, based on optimally selected species descriptors, showed a high degree of correlation between k predicted and k measured. The intercepts and the slopes of the obtained dependences were close to the theoretically expected values of 0 and 1, respectively.

Study of selectivity of molecularly imprinted polymers prepared under different conditions.

Molecularly imprinted polymers were prepared and tested in different ways. 1-Methyl-2-piperidinoethylester of 4-decyloxyphenylcarbamic acid was used as the template for imprints formation. Acrylamide, 4-vinylpyridine, and methacrylic acid as monomers and methanol and acetonitrile as a porogene were used. Non-imprinted polymers were prepared for each imprinted polymer by the same procedure. Polymers were employed as sorbents for solid-phase extraction. In this work the influence of polymerization mixture composition on polymer properties, such as capacity and selectivity, was investigated. The influence of alkoxy-chain length and the position on benzene ring on the selectivity of polymers was also investigated.

Determination of the 2,3-pentadienedioic acid enantiomer interconversion energy barrier 1. Classical kinetic approach.

A classical kinetic method was used to determine the energy barrier for the inter-conversion of 2,3-pentadienedioic acid enantiomers. Each individual enantiomer was isolated by collecting the appropriate peaks from the HPLC enantiomeric separation, of racemic 2,3-pentadienedioic acid. The isolated enantiomers were racemized at 22 degrees C using various interconversion times. The ratio of enantiomers in each reaction solution was determined by HPLC at 22 degrees C. The corresponding peak areas of the enantiomers and the interconversion times obtained from the HPLC chromatograms were used to calculate both the interconversion rate constants describing (+)--> (-) and (-) --> (+) interconversions as well as the energy barriers. It was confirmed that the interconversion of 2,3-pentadienedioic acid enantiomers is a firstorder kinetic reaction. Both semiempirical and ab initio methods were used to explore the mechanism of the interconversion of 2,3-pentadienedioic acid enantiomers, and to calculate the interconversion energy barrier. Comparison of the interconversion energy barriers found by the ab initio method (deltaG# = 110.7 kJ/mol) and by classical kinetics in the mobile phase solution at 22 degrees C (delta Gapp = 93.9+/-0.2 kJ/mol) shows a difference which may be attributed to the different conditions assumed in the theoretical calculation (i.e., a gaseous state) and the actual experimental conditions (i. e., liquid solution) and a possible catalytic effect of the solution composition.

Mobile-phase pH influence on the retention of some benzoic acid derivatives in reversed-phase chromatography.

Five end-capped octadecyl RP stationary phases, among which one was a polar embedded stationary phase, were tested for the analysis of benzoic acid derivatives using two mobile phases with or without addition of formic acid (water pH was measured by a common approach; pH of water with addition of formic acid was 3.0 and without formic acid 5.8). The influence of mobile-phase pH on the retention of benzoic acid derivatives was under study. Consequently, Purospher-STAR and Alltima columns provided symmetrical peaks for benzoic acid derivatives at pH 3.0 and also at pH 5.8. Reprosil and Symmetry stationary phases showed poor peak shapes at higher pH of the mobile phase. Differences between the tested columns may be caused by surface heterogeneity. Another reason may be the presence of some atoms creating additional adsorption sites on the surface of Reprosil and Symmetry stationary phases. This can lead to enhanced silanol activity resulting in peak tailing. The addition of formic acid into the mobile phase improved peak shapes. The polar embedded C18 stationary-phase Synergi-Fusion-RP appeared as not a suitable column for the analysis of benzoic acid derivatives. Synergi-Fusion-RP provided asymmetrical peaks even if formic acid was added into the mobile phase.

Determination of the interconversion energy barrier of 2,3-pentadienedioic acid enantiomers by HPLC. 2. On-column interconversion.

In this paper, an HPLC method is used to determine the enantiomerization barrier of 2,3-pentadienedioic acid enantiomers. The racemate of 2,3-pentadienedioic acid was separated by HPLC on a chiral CHIROBIOTIC T column with a 90:10 (100:0.5:0.5 MeOH/HOAc/TEA)/H2O mobile phase. Peak areas of enantiomers prior to (A(+)0, A(-)0) and after the separation (A(+), A(-)), were used for calculation of the rate constants and the enantiomerization barrier, as determined by computer-assisted peak deconvolution of the peak clusters on the chromatograms. The kinetic equation for irreversible reactions was used to determine the apparent enantiomerization rate constants and the interconversion energy barrier. The dependence of the apparent enantiomerization barrier (deltaG1(app), deltaG-1(app)) on temperature was used to determine the apparent activation enthalpy (deltaH1(app), deltaH(-1)app) and entropy (deltaS1(app), deltaS-1(app)) for the interconversion of 2,3-pentadienedioic acid enantiomers, where the coefficients 1 and -1 designate the interconversions (+) --> (-) and (-) --> (+), respectively.

Selective extraction of derivates of p-hydroxy-benzoic acid from plant material by using a molecularly imprinted polymer.

Selective SPE of derivates of p-hydroxybenzoic acid (pHBA) from plant extract of Melissa officinalis is presented using a molecularly imprinted polymer (MIP) made with protocatechuic acid (PA) as template molecule. MIP was prepared with acrylamide as functional monomer, ethylene glycol dimethacrylate as crosslinking monomer and ACN as porogen. MIP was evaluated towards six phenolic acids: PA, gallic acid, pHBA, vanillic acid (VA), gentisic acid (GeA) and syringic acid (SyrA), and then steps of molecularly imprinted SPE (MISPE) procedure were optimized. The best specific binding capacity of MIP was obtained for PA in ACN (34.7 microg/g of MIP). Other tested acids were also bound on MIP if they were dissolved in this solvent. ACN was chosen as solvent for sample application. M. officinalis was extracted into methanol/water (4:1, v/v), the extract was then evaporated to dryness and dissolved in ACN before application on MIP. Water and ACN were used as washing solvents and elution of benzoic acids was performed by means of a mixture methanol/acetic acid (9:1, v/v). pHBA, GA, PA and VA were extracted with recoveries of 56.3-82.1% using this MISPE method. GeA was not determined in plant extract.

Study of the mechanism of enantioseparation part VI: thermodynamic study of HPLC separation of some enantiomers of phenylcarbamic acid derivatives on a (S,S) Whelk-O 1 column.

The enantiomers of thirteen 2-, 3-, and 4-alkoxyphenylcarbamic acid 2-methoxy-1-[(4-methylpiperazino)methyl]ethyl ester were separated on a (S,S) Whelk-O 1 CSP column isothermally in the range of 0-50 degrees C at 10 degrees C increments, using methanol/ water (90/10, v/v) containing 17.5 mmol L(-1) acetic acid and 14.36 mmol L(-1) triethylamine as a mobile phase. The dependence of the natural logarithms of retention and selectivity factors (In k, In alpha, respectively) on the inverse of temperature, 1/T, was used to determine thermodynamic data of enantiomers of alkoxysubstituted phenylcarbamic acid 2-methoxy-1-[(4-methylpiperazino) methyl]ethyl esters. Enthalpyentropy compensation plots showed that all of the compounds in this study separate via the same enthalpy-driven chiral recognition mechanism.