Teicoplanin-based Chiral Stationary Phase Research Articles

Retention mechanisms of dipeptides on superficially porous particle vancomycin- and teicoplanin-based chiral stationary phases

Chromatographic behavior of new chiral stationary phases (CSPs) Chiral-T and Chiral-V with teicoplanin and vancomycin antibiotics grafted onto superficially porous silica particles was studied in relation to dipeptide (DP) stereoisomers. The unbuffered water–methanol solutions were used as mobile phases (MPs). The effects of physical properties and molecular structure of analytes and selectors on retention and separation of DP stereoisomers are discussed herein. Chiral-T was evinced to exhibit high enantioselectivity, with highest α values attaining 16.5, 18.8 and 20.4 for Gly-Leu, dd/ll-Phe-Leu and ld/dl-Ala-Ala. At this point, Chiral-V did not exhibit enantioselectivity towards DP stereoisomers. The effect of MP composition on retention and enantioseparation of DPs was investigated. Lipophilicity of DPs was found to be an essential factor in the dependence of their retention vs. methanol concentration in МPs. Lipophobic DPs were eluted more quickly by water-rich solvents, with lipophilic DPs exhibiting an asymmetric U-shaped, or a descending dependence of retention factor vs. the methanol percentage on Chiral-T or Chiral-V, respectively. A theoretical model taking into account interaction of both solvents of a binary MP with both an analyte and adsorption sites was successfully applied so as to approximate and interpret the dependences of DP retention (monotonic and U-shaped) vs. a modifier content in MP. Water molecules were evinced to predominantly participate in competitive adsorption with DP molecules. The model predicted better solvation of lipophilic DPs by methanol and better solvation of lipophobic DPs by water. An attempt was made to verify the possibility of modeling by molecular docking the processes occurring during interaction between DP stereoisomers and CSPs, including consideration of the influence of competitive binding of eluent molecules in selector cavity.

Enantiomeric Separation and Molecular Modelling of Bioactive 4-Aryl-3,4-dihydropyrimidin-2(1H)-one Ester Derivatives on Teicoplanin-Based Chiral Stationary Phase

The enantiomeric separation of 15 racemic 4-aryl-3,4-dihydropyrimidin-2(1H)-one (DHP) alkoxycarbonyl esters, some of which proved to be highly active as A2B adenosine receptor antagonists, was carried out by HPLC on ChirobioticTM TAG, a chiral stationary phase (CSP) bearing teicoplanin aglycone (TAG) as the chiral selector. The racemic compounds were separated under polar organic (PO) conditions. Preliminarily, the same selectands were investigated on three different Pirkle-type CSPs in normal-phase (NP) conditions. A baseline separation was successfully obtained on TAG-based CSPs for the majority of compounds, some of which achieved high enantioselectivity ratios (α > 2) in contrast with the smaller α values (1–1.5) and the lack of baseline resolution observed with the Pirkle-type CSPs. In particular, the racemic tetrazole-fused DHP ester derivatives, namely compounds 8 and 9, were separated on TAG-based HPLC columns with noteworthy α values (8.8 and 6.0, respectively), demonstrating the potential of the method for preparative purposes. A competition experiment, carried out with a racemic analyte (6) by adding N-acetyl-d-alanine (NADA) to the mobile phase, suggested that H-bonding interactions involved in the recognition of the natural dipeptide ligand d-Ala-d-Ala into the TAG cleft should be critical for enantioselective recognition of 4-aryl DHPs by TAG. The X-ray crystal structure of TAG was elucidated at a 0.77 Å resolution, whereas the calculation of molecular descriptors of size, polar, and H-bond interactions, were complemented with molecular docking and molecular dynamics calculations, shedding light on repulsive (steric effects) and attractive (H-bond—polar and apolar) interactions between 4-aryl DHP selectands and TAG chiral selectors.

Surface Design of Enantiomeric HPLC Separation on Vancomycin and Teicoplanin-Based Stationary Phases, a Tool for Chiral Recognition of Model β-Blockers.

A quality-by-design approach was adopted for enantioseparation of atenolol on Vancomycin and Teicoplanin-based chiral stationary phases using reversed phase (RP) mode and polar ionic mode (PIM), respectively to account for major forces involved in enantiorecognition of β-blockers on macrocyclics. A fractional factorial screening design for the two modes; followed by a central composite optimization design and regression analysis were able to point out critical factors and chromatographic responses and robust surface of the design. Within the studied range of flow the optimal was 0.3 mL/min for Chirobiotic T and 1 mL/min for Chirobiotic V. In PIM, a composition of 100% methanol was mandatory to compromise between best separation and least retention with equal amounts of the acid and base modifiers for enantiomers of atenolol, as model drug in addition to metoprolol and pindolol as structurally related compounds for possible extrapolation of results on members of the same class. However, in RP mode, only triethylamine acetate was needed as buffer for atenolol enantiomers. Chiral recognition of atenolol in both elution modes, further confirmed via extrapolation of the models on the two other β-blockers showed that ionic interactions rather than any other forces governed chiral recognition on the two macrocyclic stationary phases in both modes.

Direct analysis of chiral active pharmaceutical ingredients and their counterions by ultra high performance liquid chromatography with macrocyclic glycopeptide-based chiral stationary phases

In this work the simultaneous separation of chiral active pharmaceutical ingredients (API) in salt form from their counterions has been performed by using different high-efficiency macrocyclic glycopeptide-based chiral stationary phases (CSPs). Not only a new zwitterionic vancomycin-based CSP has been prepared (similarly to what was done for teicoplanin) but macrocyclic selectors have also been bonded to sub-2 μm fully porous silica particles through traditional ureidic linkage to obtain versions of CSPs suitable for ultra-high performance applications. The direct separation of chiral APIs and counterions is particularly attracting since it simplifies the workflow traditionally used with reduction of analysis time and costs. The wide selection of macrocyclic antibiotics CSPs now available has allowed to manage different cases that can happen in the simultaneous separation of APIs and their counterions (either cations or anions). Indeed, while inorganic cations are retained on traditional vancomycin- and teicoplanin-based CSPs, inorganic anions are almost unretained (due to Donnan’s effect). On the other hand, cations and anions can be both retained on the zwitterionic versions of these CSPs. Afterwards, zwitterionic CSPs allowed the separation of other compounds including N-derivative amino-acids, profens, polyols, sugar anomers, oligosaccharides and inorganic anions/cations opening new perspectives in the use of this family of CSPs.

Comparison of HPLC Separation of Phenylalanine Enantiomers on Different Types of Chiral Stationary Phases

HPLC separation of phenylalanine by using chiral stationary phases (CSPs) with chiral selectors β-cyclodextrin, isopropylcarbamate cyclofructan 6, teicoplanin, and ristocetin were studied. The effects of mobile phase composition and column temperature from 0 to 30 °C on the retention and resolution of enantiomers were investigated. β-cyclodextrin and isopropylcarbamate cyclofructan 6 CSPs showed chiral recognition in normal phase separation mode. The sufficient enantioseparations (resolution values 1.59 and 2.75) were reached using teicoplanin and ristocetin-based stationary phases at 23 °C in reversed-phase mode with mobile phases consist of acetonitrile and water at ratios 75/25 and 60/40 (v/v), respectively. Chiral HPLC system coupled with circular dichroism and polarimetry detector could be used to determine enantiomeric elution order. The optimized and validated HPLC-UV method with teicoplanin-based chiral stationary phase was applied for determination of phenylalanine in real samples. Both of the enantiomeric forms were detected in samples of dietary supplements and L-phenylalanine in samples of energy drinks. Obtained recoveries were higher than 82% with an RSD less than 10%. The HPLC method showed good linearity (correlation coefficients > 0.998) in concentration range 0.1–500 μg mL−1. The limit of detection was 0.1 μg mL−1 for both enantiomers.

Ultra-fast high-efficiency enantioseparations by means of a teicoplanin-based chiral stationary phase made on sub-2 μm totally porous silica particles of narrow size distribution

A new ultra-high performance teicoplanin-based stationary phase was prepared starting from sub-2μm totally porous silica particles of narrow size distribution. Columns of different lengths were packed at high pressure and a deep and systematic evaluation of kinetic performance, in terms of van Deemter analysis, was performed under different elution conditions (HILIC, POM, RP and NP) by using both achiral and chiral probes. For the achiral probes, the efficiency of the columns at the minimum of the van Deemter curves were very high leading to some 278000, 270000, 262000 and 232000plates/m in hydrophilic interaction liquid chromatography (HILIC), polar organic mode (POM), normal phase (NP) and reversed phase (RP) respectively. The lowest plate height, Hmin=3.59μm (h(/)=1.89), was obtained under HILIC conditions at a flow rate of 1.4mL/min. Efficiency as high as 200000–250000plates/m (at the optimum flow rate) was obtained in the separation of the enantiomers of chiral probes under HILIC/POM conditions. N-protected amino acids, α-aryloxy acids, herbicides, anti-inflammatory agents were baseline separated on short (2-cm) and ultra-short (1-cm) columns, with analysis time in the order of 1min. The enantiomers of N-BOC-d,l-methionine were successfully baseline separated in only 11s in HILIC mode. Several examples of fast and efficient resolutions in sub/supercritical fluid chromatography were also obtained for a range of chiral carboxylic acids.

Enantioselective separation of unusual amino acids by high performance liquid chromatography

Unusual amino acids, i.e. amino acids not encoded by DNA, play fundamental roles in many scientific fields. Since the single enantiomeric form can cause different and often serious response of organisms, chiral separations of unusual amino acids are irreplaceable tools in their study and their employment. Two types of chiral stationary phases, two teicoplanin-based and four polysaccharide-based columns, were used. Separation conditions of reversed phase mode, polar organic mode and hydrophilic interaction chromatography were evaluated and compared. All columns used exhibited significantly different enantioselectivities. Teicoplanin-based chiral stationary phases, especially Chirobiotic T column, were able to separate almost all enantiomers tested, with the exception of Z-D,L-4-F-phenylalanine ethyl ester. No partial enantioseparation of this analyte was obtained on teicoplanin-based chiral stationary phases, while baseline enantioresolution was achieved on polysaccharide-based columns. Change of elution order of L- and D-enantiomers was proved regarding the chiral stationary phase used.

A molecular model of the enantioselective liquid chromatographic separation of (R,S)-ifosfamide and its N-dechloroethylated metabolites on a teicoplanin aglycon chiral stationary phase

The enantioselective separations of the chiral oxazaphosphorines (R,S)-ifosfamide (IF), (R,S)-2-N-dechloroethyl-IF (2-DCE-IF) and (R,S)-3-N-dechloroethyl-IF (3-DCE-IF) were achieved on teicoplanin-based chiral stationary phase using isopropanol:methanol (60:40, v/v) as the mobile phase. Computational models of the teicoplanin and teicoplanin aglycon (TAG) chiral selectors were constructed and used in docking experiments to examine the chiral recognition mechanism associated with the observed resolutions. Initial data showed no significant differences between the simulated selector–selectand complexes using teicoplanin and TAG, and the full study was conducted using TAG. The data from the study indicate that hydrophobic interactions arise between the chlorine atom present in the cholorethyl moieties of the oxazaphosphorine molecules and hydrophobic pockets within the TAG basket and that these interactions anchored and positioned the selectands within the selector–selectand complexes. The complexes were stabilized through the formation of a network of hydrogen bond and cation–π interactions, in which the latter involved the phosphorous atom of the phosphoramide moiety and aromatic components of the TAG aglycon basket. The chirality of the oxazaphosphorine molecule determined the number and strength of the stabilizing interactions which resulted in significant differences in the relative mean binding energies between the complexes formed by the (R) and (S) enantiomers of the selectands. These differences were consistent with the observed chromatographic enantioselectivity and suggest a multi-step chrial recognition mechanism involving the tethering of the selectand to the selector followed by conformational adjustments and stabilization of the selectand–selector complex.

Binding of Dipeptides and Amino Acids to Teicoplanin Chiral Stationary Phase: Apparent Homogeneity of Some Heterogeneous Systems

The binding between Teicoplanin glycopeptide antibiotic and some dipeptides and amino acids has been studied by nonlinear liquid chromatography. A Teicoplanin-based chiral stationary phase, specifically designed to achieve maximum selectivity and loading by reducing non-specific interactions, has been prepared and packed into a microbore column. The adsorption isotherms of the enantiomers of proline, alanine, and alanine-alanine (Ala-Ala) have been measured through frontal analysis. The experimental binding data have been interpreted in the context of the ordinary homogeneous Michaelis-Menten model and by considering an heterogeneous model that accounts for a broad adsorption energy distribution (AED). AED has been achieved by the analysis of adsorption isotherms. Besides confirming the importance of the terminal D-Ala-D-Ala moiety in the molecular recognition between the dipeptide and the macrocyclic antibiotic Teicoplanin (it was found that Teicoplanin behaves as a molecular filter toward the enantiomers of Ala-Ala), this study shows that a heterogeneous adsorption model is needed for the correct interpretation of binding data.

Comparison of enantioselective HPLC separation of structurally diverse compounds on chiral stationary phases with different teicoplanin coverage and distinct linkage chemistry

Enantioseparation performance of two teicoplanin-based chiral stationary phases, Chirobiotic T and Chirobiotic T2, with different teicoplanin coverage and distinct linkage chemistry was compared. Three structurally diverse groups of analytes, amino alcohols (beta-blockers), chlorophenoxypropionic acids, and branched-chain amino acids, in various mobile phase compositions/separation modes were examined. The amino alcohols showed better enantioresolution on Chirobiotic T2 in reverse-phase, hydrophilic interaction chromatography, and polar-ionic mode separation systems. The best results with these analytes were obtained in the polar-ionic mobile phase. In contrast, the chlorophenoxypropionic acids and the branched-chain amino acids yielded an improved enantioresolution on the chiral stationary phase with lower amount of teicoplanin in the hydrophilic interaction chromatography system. The comparison of separation of the chlorophenoxypropionic acids enantiomers in the reverse-phase and hydrophilic interaction chromatography environments showed completely opposite results. While better enantioresolution of chlorophenoxypropionic acids was achieved on Chirobiotic T2 in mobile phases with low methanol content, high methanol concentration was needed to reach baseline enantioseparation on Chirobiotic T.

Comparative study of three teicoplanin-based chiral stationary phases using the linear free energy relationship model

Teicoplanin (T) is a macrocyclic glycopeptide that is highly effective as a chiral selector for enantiomeric separations. In this study, we used three teicoplanin-based chiral stationary phases (CSPs) – native teicoplanin, teicoplanin aglycon (TAG) and recently synthesized methylated teicoplanin aglycon (MTAG). In order to examine the importance of various interaction types in the chiral recognition mechanism the three related CSPs were evaluated and compared using a linear free energy relationship (LFER). The capacity factors of 19 widely different solutes, with known solvation parameters, were determined on each of the columns under the same mobile phase conditions used for the chiral separations. The regression coefficients obtained revealed the magnitude of the contribution of individual interaction types to the retention on the compared columns under those specific experimental conditions. Statistically derived standardized regression coefficients were used to evaluate the contribution of individual molecular interactions within one stationary phase. It has been concluded that intermolecular interactions of the hydrophobic type significantly contribute to retention on all the CSPs studied here. Other retention increasing factors are n- and π-electron interactions and dipole–dipole or dipole-induced dipole ones, while hydrogen donating or accepting interactions are more predominant with the mobile phase than with the stationary phases. However, these types of interactions are not equally significant for all the CSPs studied.

Chiral separation of beta-adrenergic antagonists, profen non-steroidal anti-inflammatory drugs and chlorophenoxypropionic acid herbicides using teicoplanin as the chiral selector in capillary liquid chromatography

Three groups of structurally diverse chiral compounds were used to study the interaction mechanism responsible for stereoselective recognition with teicoplanin as chiral selector in capillary liquid chromatography. Teicoplanin-based chiral stationary phase (CSP) was used. The effect of the variation of mobile phase composition on retention and enantioselective separation was studied. The mobile phase composition suitable for enantioresolution of the various chiral compounds differed according to the interaction forces needed for chiral recognition. Mobile phases with high buffer portion (70–90 vol.%) were preferred for separation of enantiomers of profen non-steroidal anti-inflammatory drugs and chlorophenoxypropionic acid herbicides that require hydrophobic interactions, inclusion and π–π interactions for stereoselective recognition with teicoplanin. Higher concentration triethylamine in the buffer (0.5–1.0%) increased resolution of these acids. On the other hand, H-bonding and electrostatic interactions are important in stereoselective interaction mechanism of β-adrenergic antagonists with teicoplanin. These interaction types predominate in the reversed phase separation mode with high organic modifier content (95% methanol) and in polar organic mobile phases. For this reason β-adrenergic antagonists were best enantioresolved in the polar organic mode. The mobile phase composed of methanol/acetic acid/triethylamine, 100/0.01/0.01 (v/v/v), provided enantioresolution values of all the studied β-adrenergic antagonists in the range 1.1–1.9. Addition of teicoplanin to the mobile phase, which was suitable for enantioseparation of certain compounds on the CSP, was also investigated. This system was used to dispose of nonstereoselective interactions of analytes with silica gel support that often participate in the interaction with CSPs. Very low concentration of teicoplanin in the mobile phase (0.1 mM) resulted in enantioselective separation of 2,2- and 2,4-chlorophenoxypropionic acids.

Comparative study of the instrumental couplings of high performance liquid chromatography with microwave-assisted digestion hydride generation atomic fluorescence spectrometry and inductively coupled plasma mass spectrometry for chiral speciation of selenomethionine in breast and formula milk

A comparison of chiral separation and analysis of selenomethionine in breast and formula milk, using high performance liquid chromatography (HPLC) on a glycopeptide teicoplanin-based chiral stationary phase (Chirobiotic T), coupled to atomic fluorescence spectrometry (AFS) and inductively coupled plasma (ICP) MS detectors has been performed. The coupling HPLC-microwave-assisted digestion hydride generation requires on-line post-column analytes treatment, and a severe sample clean-up for fat and proteins elimination using centrifugation and ultrafiltration. Underivatized dl-selenomethionine enantiomers were completely resolved in 10 min using unbuffered water mobile phase at 1 ml min −1 flow. Good selectivity and sensitivities (detection limits 3.1 and 3.5 ng ml −1 as Se for l- and d-selenomethionine, respectively) were obtained, and method robustness and simplicity, together to the low cost of AFS detector, makes it suitable for infant milk routine analysis. HPLC–ICP-MS coupling exhibits very low detection limits (0.9 ng ml −1, as Se) for each dl-selenomethionine enantiomers, but the method suffers from matrix influence, that produces a poor S/N ratio and low reliability. The methods were applied to breast and formula milk samples with recoveries of 80% of the total selenium presence, which is attributable to the existence of other unknown species. l-Selenomethionine was the only isomer present in breast milk, but a 30% of d-selenomethionine was also detected in formula milk.

Potential of fermentation profiling via rapid measurement of amino acid metabolism by liquid chromatography–tandem mass spectrometry

Monitoring amino acid metabolism during fermentation has significant potential from the standpoint of strain selection, optimizing growth and production in host strains, and profiling microbial metabolism and growth state. A method has been developed based on rapid quantification of underivatized amino acids using liquid chromatography–electrospray tandem mass spectrometry (LC–MS–MS) to monitor the metabolism of 20 amino acids during microbial fermentation. The use of a teicoplanin-based chiral stationary phase coupled with electrospray tandem mass spectrometry allows complete amino acid analyses in less than 4 min. Quantification is accomplished using five isotopically labeled amino acids as internal standards. Because comprehensive chromatographic separation and derivatization are not required, analysis time is significantly less than traditional reversed- or normal-phase LC-based amino acid assays. Intra-sample precisions for amino acid measurements in fermentation supernatants using this method average 4.9% (R.S.D.). Inter-day (inter-fermentation) precisions for individual amino acid measurements range from 4.2 to 129% (R.S.D.). Calibration curves are linear over the range 0–300 μg/ml, and detection limits are estimated at 50–450 ng/ml. Data visualization techniques for constructing semi-quantitative fermentation profiles of nitrogen source utilization have also been developed and implemented, and demonstrate that amino acid profiles generally correlate with observed growth profiles. Further, cellular growth events, such as lag-time and cell lysis can be detected using this methodology. Correlation coefficients for the time profiles of each amino acid measured illustrate that while several amino acids are differentially metabolized in similar fermentations, a select group of amino acids display strong correlations in these samples, indicating a sub-population of analytes that may be most useful for fermentation profiling.

Study of mechanisms of chiral discrimination of amino acids and their derivatives on a teicoplanin-based chiral stationary phase

The behavior of a series of amino acids and some of their methyl ester hydrochloride, N-acetyl and N- tert-butyloxycarbonyl derivatives has been investigated on a teicoplanin-based chiral stationary phase by changing the chromatographic conditions, namely, the type and amount of mobile phase organic modifier and the ionic strength of the solutions. By using species with significantly different characteristics and chemical reactivity, some general conclusions regarding the chiral recognition process on this kind of stationary phase have been formulated. The importance of the carboxylic moiety for the formation of the complex between enantiomers and the aglycone basket of teicoplanin has been demonstrated via chromatography. Additionally, the increased possibility to make an hydrogen bond between the amidic hydrogen of the acetylated compounds and an amidic group on the stationary phase has been proposed to be pivotal for the stability of the complex aglycone d-enantiomer. Phenomena leading to the exclusion from the chiral stationary phase of one or both enantiomers have been rationalized by considering the ionic interactions between stationary phase, molecules to be separated and the surrounding medium and/or steric hindrance effects. The understanding of some of the observed phenomena may be important for optimizing the performance of the separation on aglycone-based media.

Simultaneous analysis of underivatized chiral amino acids by liquid chromatography–ionspray tandem mass spectrometry using a teicoplanin chiral stationary phase

Simultaneous chiral separations of underivatized amino acids have been performed using a teicoplanin-based chiral stationary phase and ionspray tandem mass spectrometry for their ionisation and detection. Different amino acid enantiomer pairs were separated simultaneously, including those of positional isomeric amino acids (e.g., l, d-Leu/Ile, or l, d-Val/Iva). Due to the specificity of tandem mass spectrometry, co-eluting enantiomers of different amino acids could also be determined. Fifteen chiral underivatized proteinogenic and non-proteinogenic amino acids were analysed simultaneously under isocratic conditions (acetonitrile–water, 75:25) in less than 25 min. For maximum sensitivity, post-column addition of 500 m M aqueous HCOOH was necessary. Detection limits varied from 2.5 to 50 μg l −1 depending on the amino acid. The signal vs. concentration relationship was linear for all d- and l-amino acids (0.9995≤ r 2≤1) for three orders of magnitude.

MODIFICATION OF THE CHIRAL BONDING PROPERTIES OF TEICOPLANIN CHIRAL STATIONARY PHASE BY ORGANIC ADDITIVES. HPLC SEPARATION OF ENANTIOMERS OF ALKOXYSUBSTITUTED ESTERS OF PHENYLCARBAMIC ACID

The behaviour of teicoplanin-based chiral stationary phase (CHIROBIOTIC T) towards changes in organic and ionic modifiers in mobile phase was investigated in order to deduce suitable conditions for the liquid chromatographic enantioseparations of a series of alkoxysubstituted esters of phenylcarbamic acid. Methanol and acetonitrile were the non-ionic modifiers tested in the mobile phase, while different aliphatic carboxylic acids (formic acid, acetic acid, propionic acid, hexanoic acid) and bases (triethylamine, trimethylamine, diethylamine) were used as ionic modifiers. The influence of the nature and concentration of the modifiers on retention, selectivity, and resolution of enantiomers was investigated. Under these conditions, enantiomeric separations could be obtained for 3- and 4-alkoxysubstituted derivatives. The elution order of enantiomers was also determined.

Enantioseparation of selected N- tert.-butyloxycarbonyl amino acids in high-performance liquid chromatography and capillary electrophoresis with a teicoplanin chiral selector

Enantioseparation of N- tert.-butyloxycarbonyl amino acids ( N- t-Boc-Aas) with teicoplanin chiral selector was performed in two different separation systems: A teicoplanin-based chiral stationary phase (CSP-TE) was used in reversed-phase HPLC, and the same chiral selector (CS) was added into a background electrolyte (BGE) in HPCE. The enantioselective interaction with the same CSP/CS can be influenced by several factors, such as mobile phase/background electrolyte composition: the buffer concentration, pH, the CS concentration, the presence of organic modifiers. In addition, the charge of the chiral selector related to the charge of the analyte and to EOF are important variables in CE. The effect of these parameters on enantioselectivity and enantioseparation of selected N- t-Boc-Aas was studied. The presence of a sufficient concentration (1% solution) of a triethylamine acetate buffer in the mobile phase was shown to be essential for enantioseparation of these blocked amino acids in HPLC. A certain concentration of teicoplanin aggregates (along with teicoplanin molecules) in the BGE is required to obtain enantioseparation of N- t-Boc-Aas in HPCE.

Chiral speciation and determination of selenomethionine enantiomers in selenized yeast by HPLC-ICP-MS using a teicoplanin-based chiral stationary phase

Chiral separation of underivatized selenoaminoacids enantiomers by high performance liquid chromatography (HPLC) on a glycopeptide antibiotic teicoplanin-based chiral stationary phase (Chirobiotic T) was investigated. The effect of methanol content, pH and ionic strength of the mobile phase on retention and resolution of the enantiomers was examined to optimize the chromatographic conditions. Underivatized DL-selenomethionine and DL-selenoethionine enantiomers were completely resolved within 8 min using a 2% (v/v) methanol–water (unbuffered) mobile phase delivered at 1 mL min−1. Very good selectivity and excellent detection limits of 1.9 µg L−1 (0.8 µg L−1 as Se) for each DL-selenomethionine enantiomer were obtaining by on-line coupling the chiral HPLC separation with selenium-specific detection by inductively coupled plasma quadrupole mass spectrometry (ICP-MS). The HPLC-ICP-MS method was successfully applied to the chiral speciation and determination of D- and L- selenomethionine enantiomers in real samples of selenized yeast.

Comparison of enantioselective separation of N- tert.-butyloxycarbonyl amino acids and their non-blocked analogues on teicoplanin-based chiral stationary phase

A teicoplanin-based chiral stationary phase (CSP) was tested for enantioseparation of underivatized amino acids and their N- tert.-butyloxycarbonyl ( t-Boc) derivatives, important precursors in peptide synthesis. Mobile phase composition was optimized for organic modifier and triethylamine acetate buffer (TEAA) contents, and retention and enantioresolution of t-Boc-amino acids and their non-blocked analogues were compared. The importance of the amino group of amino acids in the interaction mechanism was evaluated. Native amino acids have better possibility for interaction with teicoplanin; they are more retained on the CSP and better enantioresolved than the blocked amino acids. Presence of triethylamine in the aqueous portion of the mobile phase was shown to be important for separation of enantiomers of t-Boc-amino acids; while native amino acids were almost not affected by the addition of TEAA. Results obtained for blocked amino acid on teicoplanin CSP with mobile phase composed of 1% triethylamine acetate, pH 4.1 and organic modifier were compared to those got on hydroxypropyl-β-cyclodextrin CSP. Good enantioseparation in a reasonable analysis time was obtained on the teicoplanin-based chiral stationary phase.