Study of RP HPLC Retention Behaviours in Analysis of Carotenoids.

For determination of selected carotenoids, various types of columns for high-performance liquid chromatography (HPLC) with different properties have been used. The characteristics of the laboratory-used packing material containing monomeric alkyl-bonded phases (C18, C30) and phenyl as well as phenyl-hexyl stationary phases were studied. The retention data of the examined compounds were used to determine the hydrophobicity and silanol activity of stationary phases applied in the study. The presence of the polar and carboxyl groups in the structure of the bonded ligand strongly influences the polarity of the stationary phase. Columns were compared according to methylene selectivity using a series of benzene homologues. The measurements were done using a methanol-water mobile phase. Knowledge of the properties of the applied stationary phase provided the possibility to predict the RP HPLC retention behaviours in analysis of carotenoids including lutein, lycopene and ß-carotene. The composition of the mobile phase, the addition of triethylamine and the type of stationary phase had been taken into account in designing the method of carotenoid identification. Also a monolithic column characterised by low hydrodynamic resistance, high porosity and high permeability was applied. The presented results show that the coverage density of the bonded ligands on silica gel packings and length of the linkage strongly influence the carotenoid retention behaviours. In our study, the highest retention parameters for lutein, lycopene and ß-carotene were observed for C30 and C18 stationary phase. This effect corresponds with pore size of column packing greater than 100 Å and carbon content higher than 11 %.

HPLC and TLC enantioseparation of the nitro-positioned aryloxysubstituted aminopropanols.

An enantioseparation study of nitro-substituted aryloxyaminopropanols was performed using HPLC on a teicoplanin chiral stationary phase and TLC impregnated with L-aspartic and L-tartaric acid as chiral selectors. The type of substituent on the nitrogen in the hydrophilic part of molecule is essential for excellent separation by HPLC. L-aspartic acid seems to be a suitable chiral selector for enantioseparation by TLC.

Chemometrical study of the anaesthetical activity of alkoxyphenylcarbamic acid esters.

Several chemometrical techniques were applied to elucidate anaesthetical activity of hydrochlorides of alkoxyphenylcarbamic acid esters. The studied five types of esters contained morpholin-4-ylethyl-, piperidin-1-ylethyl-, piperidin-1-ylpropyl-, azepan-1-ylethyl- and dimethylaminoethyl- groups. The surface anaesthetical activity, designated by A, and the infiltration anaesthetical activity, indicated by B, were correlated to lipophilicity, expressed in different ways--using (a) the logarithm of 1-octanol-water partition coefficient, log P, (b) the logarithm of the HPLC retention factor, log k, (c) the length of the side alkoxy chain represented by the number of carbon atoms, n, (d) molar mass, M. Principal component analysis and cluster analysis were used for close characterization of alkoxyphenylcarbamic acid esters as the potential anaesthetics, and techniques of discrimination analysis were used for predicting the extent of both types of anaesthetic activity. Artificial neural networks were successful in predicting surface anaesthetical activity but prediction of infiltration anaesthetical activity was far less favourable.

Enantioseparation of chiral sulfoxides using teicoplanine chiral stationary phases and kinetic study of decomposition in human plasma.

Teicoplanin chiral stationary phases (CHIROBIOTIC TAG and CHIROBIOTIC T) used in this study are suitable for enantioseparation of chiral sulfoxides in polar-organic phase mode. The method involves determination of chiral sulfoxides in human plasma on teicoplanin chiral stationary phase after the off-line SPE pre-treatment using OASIS HLB cartridges. The limit of determination was in the range of 0.004-0.026 microg/ml for individual racemic mixtures. The S(+) enantiomeric form eluted always as the first, except the 4-(methyl sulfinyl) biphenyl with less retained R(-) enantiomer. It was found that the rate constants of individual chiral sulfoxides depend on the type of halogen substituent. There was no significant difference in rate constants considering the position of Cl-substitution on aromatic ring of sulfoxides. Moreover, the rate constants of R(-) and S(+) forms of enantiomers are significantly different just in the case of 4-fluoro phenyl sulfoxide.

Applications of molecularly imprinted polymers in analytical and pharmaceutical chemistry.

The paper reviews recent developments in the use of molecularly imprinted polymers in several analytical techniques, such as solid-phase extraction, liquid chromatography, capillary electrophoresis, capillary electrochromatography, and as selective sorbents in chemical sensors. Molecular imprinting is achieved by the interaction between complementary groups in a template molecule and functional monomer units. The benefits of imprinted polymers are low cost, storage stability, high mechanical strength, repeated operations without loss of activity, and potential application to a wide range of target molecules.

HPLC separation of enantiomers using chiral stationary phases.

During recent few years, separations of enantiomers have become one of the most important analytical tasks. Modern separation techniques, such as the chromatographic ones, represent a very powerful tool in this respect and nowadays they are commercially and widely used all over the world. The importance of enantioseparation must be emphasized, not just because of drugs, food additives, and pesticides, but also because of other compounds which affect our life. The use of direct chromatographic enantioseparation seems to be a suitable approach not just in the analytical but also the preparative scale. This paper is focused on the separation of enantiomers using the chiral stationary phase as one of the ways of how to perform direct chromatographic separation.

Determination of dissolved sulphides in waste water samples by flow-through stripping chronopotentiometry with a macroporous mercury-film electrode.

Sulphides in water samples were determined by stripping chronopotentiometry in a computer controlled flow system with a flow-through electrochemical cell. The working electrode was a porous glassy carbon electrode coated with Nafion and mercury. The sample was diluted with 0.1 mol L(-1) NaOH and analysed. Sulphides in the sample were collected in the porous electrode as mercury sulphide and then stripped by a current of -500 microA. The limit of detection was found to be 1.6 microg L(-1) and 0.5 microg L(-1) for 1 mL and 5 mL of preconcentrated sample, respectively. The linear range for 1mL sample was found to be 5-400 microg L(-1). The repeatability and reproducibility was found to be 2.6% and 4.8%, respectively. The method was applied to analyses of waste water samples from a tannery.

[Interconversion of stereochemically unstable chiral drugs. Part III: Using beta-cyclodextrin as a chiral stationary phase for HPLC separation of diazepam conformers coupled with an off-line NMR experiment]. / Interkonverzia stereochemicky nestálych chirálnych lieciv. Cast III: Separácia konformérov diazepamu HPLC metodou pouzitim beta-cyklodextrínu ako chirálnej stacionárnej fázy v off-line spojení s NMR experimentom.

Chiral beta-cyclodextrin was used to separate diazepam conformers. Several mobile phases of the composition acetonitrile/acetate buffer 200 mmol/l (pH=3.3, 5.5, 6.5) were employed for this purpose. As follows, the influence of addition of chiral beta-cyclodextrin to the mobile phase on diazepam separation was studied. The interconversion was a concurrence process of separation, resulting from stereolability of the diazepam molecule. The influences of temperature, flow rate, pH, and ionic strength of the mobile phase on interconversion and chromatographic parameters (retention factor and selectivity coefficient) were studied. Complementary off-line NMR measurements were carried out with the goal to confirm the structure of diazepam in the presence of an acid mobile phase.

HPLC determination of morphine, morphine-3-glucuronide and morphine-6-glucuronide in human serum of oncological patients after administration of morphine drugs.

A simultaneous determination of morphine (M) and its two metabolites, morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G), by HPLC in the serum of oncological patients is described. The compounds are extracted from the serum by means of Chromabond C18--EC solid-phase-extraction cartridges, separated on a Symmetry C18 analytical column (150 x 4.9 mm, 5 microm) and detected by a UV detector at 210 nm. The mobile phase consisted of 8% acetonitrile in water, 30 mmol/l phosphate buffer (pH 3) and 1 mmol/l octane sulfonic acid as the ion pairing agent; its flow-rate was 0.8 ml/min. Under these conditions, the detection limits were 10 ng/ml, 60 ng/ml and 90 ng/ml for M, M3G, and M6G, respectively. This paper concerns blood serum concentration levels of M, M3G and M6G in oncological patients, their ratios and their role in pain resistance.

[Interconversion of stereochemically unstable chiral drugs: utilization of chromatographic techniques for the study of enantiomerization. Part II: practical applications]. / Interkonverzia stereochemicky nestálych chirálnych lieciv-- vyuzitie chromatografických techník na stúdium enantiomerizácie cast II: praktické aplikácie.

Enantiomerization is a first-order reaction; it means interconversion of one enantiomer into another (and vice versa). This phenomenon is a typical feature of some configurationally unstable chiral compounds and it complicates separation of racemic mixtures. For the study of enantiomerization, chromatographic separation techniques (GC, SFC, CE, MEKC, HPLC) and NMR can be used. Chromatographic methods suitable for investigation of enantiomerization include dynamic chromatography (if time scales of enantiomerization and separation are the same) and stopped-flow chromatography (time scales of both processes are different). The present paper surveys the research of enantiomerization from 1975, when papers describing enantiomerization in greater detail were published, to the present. Today, mathematical approaches and computer-assisted deconvolution procedures are commonly used for evaluation of enantiomerization--calculation of thermodynamic parameters (deltaGapp, deltaHapp, deltaSapp) and rate constants (klapp, k(-1)app). The aim of many publications was not only determination of the enantiomerization energy barrier, but the influence of the chiral stationary phase on enantiomerization was studied as well. In some cases, enantiomerization was used for preparative purposes--a pure enantiomer was obtained and thus complicated synthesis was excluded.

[Interconversion of stereochemically unstable chiral drugs: utilization of chromatographic techniques for the study of enantiomerization, calculation of thermodynamic parameters. Part I: theoretical aspects]. / Interkonverzia stereochemicky nestálych chirálnych lieciv-- vyuzitie chromatografických techník na stúdium enantiomerizácie, výpocet termodynamických parametrov. Cast I.--teoretické aspekty.

The paper explains the theoretical aspects of the process of enantiomerization and describes its characteristic features (generation of a plateau). In addition, some complications are presented that are produced by enantiomerization either from analytical or pharmacological points of view. It also defines the way of how to calculate energy barriers of enantiomerization according to the methods used for the separation of racemic mixtures (stopped-flow or dynamic chromatography). Mathematic models useful in deconvolution of chromatograms are also described. With the use of dynamic methods it is not possible to quantitatively evaluate the obtained chtromatograms and calculate thermodynamic parameters without computer-assisted deconvolution.

[HPLC separation of racemic basic esters of alkoxyphenylcarbamic acid using two teicoplanin chiral stationary phases]. / HPLC separácia racemátov bazických esterov alkoxyfenylkarbámovej kyseliny pouzitím dvoch teikoplanínových chirálnych stacionárnych fáz.

This paper presents the results obtained with the use of two chiral stationary phases (CSP), based on a glycopeptide antibiotic agent--teicoplanin aglycone (CHIROBIOTIC TAG) and methylated teicoplanin aglycon m-TAG. Twenty-one racemic mixtures of 1-methyl-2-piperidinoethylesters of 2-, 3- and 4-alkoxyphenylcarbamic acid were examined. The investigation included interaction between separated substances and CSP, and the effect of separation of the enantiomers under study on the value of the resolution factor (R(ij)) under identical chromatographic conditions with the use of the method of high-performance liquid chromatography (HPLC). On the basis of obtained results, it is possible to report that CSP-CHIROBIOTIC TAG is more advantageous for these racemates, because it does not contain a saccharide part, with decrease the possibility of non-polar interactions which exert a negative effect on the R(ij) value.

Kinetic study of the degradation of a potential local anesthetic drug in serum using the DNA-based electrochemical biosensor.

DNA-drug association interaction at the DNA modified screen-printed electrode for 1-methyl-2-piperidinoethylester of 2-hexoxyphenylcarbamic acid was found leading to an accumulation of the drug within the DNA layer. A procedure for the determination of drug in blood serum matrix using the protein precipitation and voltammetric measurement of the electroactive drug with the DNA biosensor was obtained and an effort was done to apply it for an assay of the drug enzymatic degradation in human and rabbit sera at 37 degrees C.

HPLC separation of enantiomers of some potential beta-blockers of the aryloxyaminopropanol type using macrocyclic antibiotic chiral stationary phases. Studies of the mechanism of enantioseparation, Part XI.

The macrocyclic antibiotic type chiral stationary phases based on native vancomycin, teicoplanin and teicoplanin aglycone (Chirobiotic V, Chirobiotic T and Chirobiotic TAG) were used for the HPLC separation of enantiomers of potential beta-blockers of the aryloxyaminopropanol type with a morpholino moiety in the hydrophilic part of the molecule. The chromatographic results presented include: retention, separation and resolution factors along with the enantioselective free energy difference corresponding to the separation of the enantiomers. Comparison of the results obtained on the three macrocyclic chiral stationary phases showed that in most cases the teicoplanin aglycone is responsible for enantioseparation of morpholino derivatives. By application of these chiral stationary phase highest resolution factors were achieved with a polar organic mobile phase system.

HPLC enantioseparation of potential beta-blockers of the aryloxyaminopropanol type. Study of the mechanism of enantioseparation, Part VIII.

This paper presents the results of HPLC enantioseparation of derivatives of aryloxyaminopropanols obtained using six chiral stationary phases [macrocyclic antibiotic (vancomycin, teicoplanin, teicoplanin aglycone, and methylated teicoplanin aglycone) and cyclodextrin (beta and gamma)] and a mixture of methanol/acetonitrile/acetic acid/triethylamine (45/55/0.3/0.2) as the mobile phase. No significant difference was observed in the separation of the enantiomers on the vancomycin and teicoplanin chiral stationary phases. Comparing the separation of enantiomers on teicoplanin-based columns the retention factors were increased in the order: native teicoplanin < teicoplanin aglycone < methylated teicoplanin aglycone. The highest values of resolution were obtained on the column containing carbohydrate moieties. The presence of saccharide moieties in the chiral stationary phase plays an important role, together with charge interactions and steric interactions, in the separation of enantiomers of derivatives of aryloxyaminopropanol.

[HPLC determination of diperodon enantiomers in blood serum by using teicoplanin chiral stationary phase]. / HPLC stanovenie enantiomérov diperodonu v krvnom sére s pouzitím teikoplaninovej chiralnej stacionárnej fázy.

The chiral stationary phase on the base of teicoplanin and the polar-organic mobile phase methanol/acetonitrile/acetic acid/triethylamine 45/55/0.3/0.2 was used for the separation of diperodon enantiomers. The developed method was suitable to determine the enantiomers in blood 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.

[Separation of enantiomers of phenylcarbamic acid derivatives by HPLC method]. / Separácia enantiomérov--derivátov kyseliny fenylkarbámovej metódou HPLC.

The chiral stationary phase on the base of beta-cyclodextrin and the mobile phase acetonitrile/0.1 mol/l acetate buffer pH=5.2 (88/12 w/w) were used for the separation of the enantiomers of 1-methoxymethyl, 1-ethoxymethyl, 1-propoxymethyl-2-(1-pyrrolidinyl), (1-piperidino), (1-perhydroazepinyl)ethyl esters of 2-alkoxyphenylcarbamic acid. The influence of the structure of these compounds and the influence of temperature on enantioseparation were studied. The dominant effect on the resolution of enantiomers is exerted by the length of the alkoxysubstituent on the aromatic ring and its position with regard to the stereogenic centre. A decrease in temperature caused an increase in the retention factors of the compounds under study and the resolution values of enantiomers.

Selectivity tuning of serially coupled (S,S) Whelk-O 1 and (R,R) Whelk-O 1 columns in HPLC.

The selectivity tuning of two columns coupled in series is investigated in chiral high-performance liquid chromatography. Two columns with reversal enantioselectivities [(R,R) Whelk-O 1 and (S,S) Whelk-O 1] are coupled in series via a T connector. Selectivity of such a column series is tuned by varying the mobile phase flows in the individual columns. The flow ratio necessary for the required selectivity is calculated on the basis of retention factors measured on the individual columns. The performance of this method for adjusting the required selectivity is studied by the separation of enantiomers of alkoxy substituted esters of phenylcarbamic acid. It is demonstrated that the change of the mobile phase flows in the individual columns enables change in the elution order of enantiomers.

Interaction of some esters of 2-, 3-, 4-alkoxyphenylcarbamic acids with surface-bound DNA at a dsDNA modified electrode. Study of local anaesthetics: Part 164.

An interaction of some derivatives of 2-, 3-, 4-alkoxyphenylcarbamic acids with dsDNA was investigated using the anodic voltammetric signal at a DNA-based electrochemical biosensor. Significant, however, reversible association was found, particularly in weak acidic and neutral solution where the protonated forms of the drug molecules occur which are electrostatically bound to the negatively charged DNA backbone. Total drug liberation from the DNA layer within 1 min. was observed in blank buffer solution without drug molecules.

Kinetic study of derivatives of phenylcarbamic acid enantiomers in rabbit blood serum using an on-line coupled column liquid chromatographic system.

An on-line coupled HPLC system is described for the kinetic study of the enantiomers of 1-methyl-2-piperidinoethylesters of 2-, 3- and 4-alkoxyphenylcarbamic acid in rabbit blood serum. The method involves three steps: (i) off-line pre-concentration and cleanup step, (ii) analytical separation of the racemate on a reversed-phase stationary phase, and (iii) separation of the enantiomers on a teicoplanin aglycone chiral stationary phase (Chirobiotic Tag). The limit of the determination with standard solutions was approximately 5.0 microg/ml. In vitro degradation studies of enantiomers have demonstrated differences in the concentration of the enantiomers after the treatment. The rate constants of R(-)- and S(+)-forms of enantiomers (the same as the position of alkoxychain) were, not significantly different. The number of carbon atoms had an influence on the degradation kinetics.