Search

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 β-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.

A membrane protein VanZTei which is encoded by the gene vanZ from the vanA glycopeptide resistance gene cluster is a part of the large family of VanZ proteins. VanZTei confers resistance to teicoplanin in Enterococcus faecalis without the presence of other proteins encoded by the cluster. The aim of my work was to compare the ability of two orthologous proteins VanZTei and VanZg (from the genome of Enterococcus faecium) to confer resistance to glycopeptides in Staphylococcus aureus RN4220 and Enterococcus faecium. We have shown that VanZg increases resistance to teicoplanin (Tei) 8 to 16 times the and also to dalbavancin (Dalb) 8 times. VanZTei also confers resistance to Tei and Dalb, but the increase is only twofold. Conversely VanZTei confers resistance to newly synthetized glycopeptides more effectively than VanZg (fourfold increase of resistance confered by VanZTei and two to fourfold increase of resistance confered by VanZg). It suggests that both proteins have different specificity to antibiotics. In despite the mutants of S. aureus RN4220 VanZTei pRMC2 with increased resistance to teicoplanin (MICTei> 8 µg/ml) in which the resistance is dependent on vanZTei expression were selected. These resistant mutants do not carry mutation in a gene vanZTei or in its ribosomal binding site. Neither of the...

The aim of this work was to develop and to optimize HPLC method for enantioseparation of newly synthesized derivatives of amino acids. The set of these analytes contained four N- blocked derivatives of D,L-Phenylalanine, three N-unblocked derivatives of D,L- Phenylalanine, , one methylated derivative of D,L-Tyrosine and D,L-Tyrosine. Two separation modes i.e. reversed phase and polar-organic modes and two columns i.e. Chirobiotic® T and Chirobiotic® T2 were used. Chiral stationary phases of these columns were composed of macrocyclic antibiotic teicoplanin coated on silica gel support. As mobile phases in revesed phase mode, methanol and acetate buffer were used with Chirobiotic® T column. The most suitable concentration were 20 mM and the most suitable pH value were 4.00. Under the above mentioned conditions eight analytes of ten were separated and the optimal conditions were found. Polar-organic mode and Chirobiotic® T column were suitable only for enantioseparation of three N-blocked derivatives of D,L- Phenylalanine. Mobile phases were composed of methanol with small additions of triethylamine and acetic acid. Chirobiotic® T2 column in reversed phase mode was not suitable for tested set of analytes. Only partial separation of D,L-Tyrosine derivative and one N-unblocked D,L-Phenylalanine...

In this work, an enantioselective HPLC method with UV and fluorimetric detection was developed and subsequently optimized for chiral separation of four aminoacids (D/L-alanine, D/L-valine, D/L-leucine, D/L-isoleucine) in native and particularly in derivatized form with an emphasis on enantioseparation of D-analogues. Retention and enantioseparation behavior of studied analytes was investigated on three chiral stationary phases based on teicoplanin (Chirobiotic T, Chirobiotic T2) and teicoplanin aglycone (Chirobiotic TAG). At the Chirobiotic T column, enantioseparations of underivatized aminoacids were performed with UV detection at 205 nm in the mobile phases methanol/water with different volume ratios. Baseline separation of L- and D-forms was achieved, however, the sensitivity of detection was very low. In order to increase detection sensitivity, derivatization of aminoacids was performed using 9-fluorenylmethyl chloroformate (FMOC-Cl) and the derivatization procedure was monitored on Chirobiotic T column with fluorimetric detection (λEx = 254 nm, λEm = 314 nm) in a buffered mobile phase methanol/0.5% TEAA buffer, pH 6.0 40/60 (v/v). In terms of derivatization, volume ratio D/L-aminoacid/derivatization agent 1/1 with ten times higher concentration of derivatization agent was found to be the most suitable....

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.