Separation of the enantiomers of substituted dihydrofurocoumarins by HPLC using macrocyclic glycopeptide chiral stationary phases

Enantiomer separations by HPLC using the macrocyclic glycopeptides teicoplanin (Chirobiotic T), teicoplanin aglycon (Chirobiotic TAG), and ristocetin A (Chirobiotic R) chiral stationary phases (CSP) have been achieved on a unique series of potentially biologically active racemic analogues of dihydrofurocoumarin. The macrocyclic glycopeptides have proven to be very selective for this class of compound. All of the 28 chiral analogues examined afforded baseline separation on at least one of the macrocyclic glycopeptide CSP. The teicoplanin CSP showed the broadest enantioselectivity with 24 of the compounds baseline separated. The TAG and the R CSP produced 23 and 14 baseline separations respectively. All three mobile phase modes, i.e. normal phase (NP), reversed phase (RP), and new polar organic modes (PO), have been evaluated. The NP mode proved to be most effective for the separation of chiral dihydrofurocoumarins on all CSP tested. In the reversed phase (RP) mode, all three CSP separated a similar number of compounds. It was observed that the structural characteristics of the analytes and steric effects are very important factors leading to chiral recognition. Hydrogen bonding was found to play a secondary role in chiral discrimination in the normal phase and polar organic modes. Hydrophobic interactions are important for chiral separation in the reversed-phase mode. Chromatographic retention data does not provide information on the absolute configuration of these chiral dihydrofurocoumarin derivatives. However, when coupled with circular dichroism using the exciton coupling chirality method, the enantiomer elution order and the absolute configuration of some chiral dihydrofurocoumarins were successfully determined.     

Chiral Separation of Duloxetine and Its R-Enantiomer by LC

The direct enantioseparation of duloxetine and its R-enantiomer was achieved by HPLC using hydroxypropyl-β-cyclodextrin (HP-β-CD) as a chiral selector and a vancomycin chiral stationary phase (Chirobiotic V). Operational parameters, such as the concentration of HP-β-CD, buffer pH, organic modifiers, temperature and flow rate, were varied in order to achieve the desired retention time and resolution. These two enantioseparation methods developed gave a baseline resolution of the enantiomers. Finally, the HPLC-CSP method was selected to determine the enantiomeric purity of duloxetine drug substance due to its much shorter analysis time and better resolution. The limit of detection of this method was 0.06 μg mL⁻¹.     

Comparison of the separation efficiencies of chirobiotic T and TAG columns in the separation of unusual amino acids

Two macrocyclic antibiotic type chiral stationary phases (CSPs), based on native teicoplanin and teicoplanin aglycone, Chirobiotic T and Chirobiotic TAG, respectively, were evaluated for the high-performance liquid chromatographic separation of enantiomers of 15 unnatural conformationally constrained alpha-amino acids, Phe and Tyr analogs, and 12 beta-amino acids having cycloalkane or cycloalkene skeletons. The chromatographic results are given as the retention, separation and resolution factors along with the enantioselective free energy difference corresponding to the separation of the enantiomers. It is clearly established that in most cases the aglycone is responsible for the enantioseparation of amino acids. The difference in enantioselective free energy between the aglycone CSP and the teicoplanin CSP was between 0.02 and 0.30 kcal mol(-1) for these particular amino acids. The resolution factors are higher with the aglycone CSP. Although the sugar units generally decrease the resolution of amino acid enantiomers, they can contribute significantly to the resolution of some unusual amino acid analogs. By application of these two CSPs excellent resolutions were achieved for most of the investigated compounds by using reversed phase or polar organic mobile mode systems. The separation conditions were optimized by variation of the mobile phase composition.     

肝糖选择剂-毛细管电泳手性拆分盐酸度洛西汀对映体及分离机制

以肝糖为毛细管电泳手性选择剂,对盐酸度洛西汀对映体的分离进行研究,建立了拆分方法.考察了手性选择剂浓度、运行缓冲液的离子强度、pH值及分离电压对手性分离的影响.在3.0% (m/V)肝糖、50 mmol/L Tris-H3PO4(pH 3.0)的运行缓冲液中,分离电压25 kV时,盐酸度洛西汀两对映体分离度达1.84.对分离机制进行了初步探讨.     

Enantioseparation of vesamicol and novel vesamicol analogs by high-performance liquid chromatography on different chiral stationary phases

High-performance liquid chromatography enantioseparation of vesamicol and six novel azaspirovesamicols (amino alcohols) was accomplished on different chiral stationary phases (CSPs) by using an optical rotation based chiral detector for identification of the resolved enantiomers. The Pirkle-type column Reprosil Chiral-NR was found to be most suitable for chiral resolution in normal phase (NP) mode; all compounds could be enantioseparated successfully. Also the cellulose-based column Reprosil Chiral-OM showed appropriate separation properties by using NP conditions. The amylose-type column Reprosil Chiral-AM-RP was most suitable for enantioseparation in reversed phase (RP) mode; five out of seven compounds were resolved. This CSP showed a considerably higher capability for chiral recognition of vesamicol derivatives in RP mode than the corresponding cellulose-based column Reprosil Chiral-OM-RP. Enantioseparation with the teicoplanin aglycone-based column Reprosil Chiral-AA was successful under polar ionic mobile phase conditions.     

Comparison of Separation Efficiency of Macrocyclic Glycopeptide-Based Chiral Stationary Phases for the LC Enantioseparation of β-Amino Acids

Direct reversed-phase high-performance liquid chromatographic methods were developed for the separation of enantiomers of eighteen unnatural β-amino acids, including several β-3-homo-amino acids. The direct separations of the underivatized analytes were performed on chiral stationary phases containing macrocyclic glycopeptide antibiotics such as teicoplanin (Chirobiotic T and T2), teicoplanin aglycone (Chirobiotic TAG), vancomycin (Chirobiotic V and V2), and ristocetin A (Chirobiotic R) as chiral selectors. The effects of the organic modifier, mobile phase composition and pH on the separations were investigated. A comparison of the separation performances of the macrocyclic glycopeptide stationary phases revealed that the Chirobiotic T2 column exhibited better selectivity than the Chirobiotic T column for the separation of β-3-homo-amino acid enantiomers; vancomycin or ristocetin A exhibited lower selectivity. The elution sequence was determined in some cases: the S enantiomers eluted before the R enantiomers, with the exception of the Chirobiotic R column, where the elution sequence R < S was observed.     

Super/subcritical fluid chromatography chiral separations with macrocyclic glycopeptide stationary phases

The chiral recognition capabilities of three macrocyclic glycopeptide chiral selectors, namely teicoplanin (Chirobiotic T), its aglycone (Chirobiotic TAG) and ristocetin (Chirobiotic R), were evaluated with supercritical and subcritical fluid mobile phases. A set of 111 chiral compounds including heterocycles, analgesics (nonsteroidal antiinflamatory compounds), beta-blockers, sulfoxides, N-protected amino acids and native amino acids was separated on the three chiral stationary phases (CSPs). All separations were done with an outlet pressure regulated at 100 bar, 31 degrees C and at 4 ml/min. Various amounts of methanol ranging from 7 to 67% (v/v) were added to the carbon dioxide along with small amounts (0.1 to 0.5%, v/v) of triethylamine and/or trifluoroacetic acid. The Chirobiotic TAG CSP was the most effective closely followed by the Chirobiotic T column. Both columns were able to separate, partially or fully, 92% of the enantiomers of the compound set. The ristocetin chiral selector could partially or baseline resolve only 60% of the enantiomers tested. All separations were done in less than 15 min and 70% were done in less than 4 min. The speed of the separations is the main advantage of the use of SFC compared to normal-phase HPLC. In addition, SFC is advantageous for preparative separations with easy solute recovery and solvent disposal.     

HPLC enantioseparation of β2‐homoamino acids using crown ether‐based chiral stationary phase

RP high‐performance liquid chromatographic methods were developed for the enantioseparation of eleven unusual β²‐homoamino acids. The underivatized analytes were separated on a chiral stationary phase containing (+)‐(18‐crown‐6)‐2,3,11,12‐tetracarboxylic acid as chiral selector. The effects of organic (alcoholic) and acidic modifiers, the mobile phase composition and temperature on the separation were investigated. The structures of the substituents in the α‐position of the analytes substantially influenced the retention and resolution. The elution sequence was determined in some cases: the S enantiomers eluted before the R enantiomers.     

Comparison of enantioseparation of selected benzodiazepine and phenothiazine derivatives on chiral stationary phases based on β‐cyclodextrin and macrocyclic antibiotics

Enantioselective separation of some phenothiazine and benzodiazepine derivatives was studied on six different chiral stationary phases (CSPs) in HPLC. Selected CSPs, with respect to the structure of the separated compounds, were either based on β‐cyclodextrin chiral selectors – underivatized β‐cyclodextrin and hydroxypropyl ether β‐cyclodextrin, or on macrocyclic antibiotics – vancomycin, teicoplanin, teicoplanin aglycone, and ristocetin A. Measurements were carried out in a reversed‐phase separation mode. The influence of mobile phase composition on retention and enantioseparation was studied. Benzodiazepines could be enantioresolved with almost all the chiral stationary phases used, except for the vancomycin‐bonded CSP. Peak coalescence of oxazepam and lorazepam was observed if separation was carried out at laboratory temperature. Reduced temperature was required in some instances in order to avoid the on‐column racemization. Separation systems composed of teicoplanin‐bonded CSP and buffer‐methanolic or pure methanolic mobile phases were shown to be suitable even for preparative purposes due to high resolution values of the enantiomers. Enantioseparation of phenothiazine derivatives was more difficult to achieve but it was successful, at least partly, also with both types of the CSPs used (except for levomepromazine).     

3，5-二甲基苯基异氰酸酯替考拉宁和苯基异氰酸酯替考拉宁手性固定相的评价与比较

用大环抗生素替考拉宁手性固定相(TE CSP)分别与3,5-二甲基苯基异氰酸酯和苯基异氰酸酯反应得到了两种新型的高效液相色谱手性固定相----3,5-二甲基苯基异氰酸酯替考拉宁手性固定相(DMP-TE CSP)和苯基异氰酸酯替考拉宁手性固定相(Ph-TE CSP)。用十八个手性化合物在反相及极性流动相模式对这两种CSP的对映体分离能力进行了评价和比较。在反相流动相中,十二个化合物（包括八个氨基酸和四个非氨基酸化合物----对羟基苯甘氨酸,拉米夫定,醇酸和去甲羟安定）的对映体在这两种手性固定相上都获得了分离,大部分的溶质在DMP-TE上获得了更强的保留和稍好的手性分离效果。在极性流动相中,六个氨基醇类化合物在DMP-TE上获得了更强的保留,但它们在两种CSP上的选择因子几乎没有区别。对自制的替考拉宁衍生物手性固定相进行评价和比较,将有助于大环糖肽类抗生素手性固定相手性识别机理的研究。     

LC Enantioseparation of β-Lactam and β-Amino Acid Stereoisomers and a Comparison of Macrocyclic Glycopeptide- and β-Cyclodextrin-Based Columns

Direct reversed-phase high-performance liquid chromatographic methods were developed for the separation of the enantiomers of tricyclic β-lactams, cis-3,4-benzo-6-azabicyclo[3.2.0]heptan-7-one, cis-4,5-benzo-7-azabicyclo[4.2.0]-octan-8-one, cis-5,6-benzo-8-azabicyclo[5.2.0]nonan-9-one and new bicyclic β-amino acids, the six- and seven-membered homologues of cis-1-amino-4,5-benzocyclopentane-2-carboxylic acid (benzocispentacin), cis-1-amino-5,6-benzocyclohexane-2-carboxylic acid and cis-1-amino-6,7-benzocycloheptane-2-carboxylic acid. The direct separations of the analytes were performed on chiral stationary phase (CSP) columns containing the macrocyclic glycopeptide antibiotic teicoplanin (Chirobiotic T), teicoplanin aglycone (Chirobiotic TAG), vancomycin (Chirobiotic V), vancomycin aglycone (Chirobiotic VAG), ristocetin A (Chirobiotic R) or a new dimethylphenyl carbamate-derivatized β-cyclodextrin-based Cyclobond DMP. The results achieved with the different methods were compared in systematic chromatographic examinations. The effects of an organic modifier and of the mobile phase composition on the separation and the separation efficiency of different columns were investigated. The difference in enantioselective free energy between the aglycone CSP and the teicoplanin CSP for these β-lactams and β-amino acids ranged between 0.3 and −1.1 kJmol⁻¹. Better enantioseparations were attained in most cases on the aglycone CSP.

     

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 pi-pi 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 beta-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 beta-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 beta-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.     

Enantioseparation of organic acids of pharmaceutical interest using eremomycin as a chiral selector

Strong adsorption of eremomycin on the fused-silica capillary wall was used for separation of enantiomers by CE. The capillary with adsorbed chiral selector was shown to be easily prepared and has reproducible properties. The effect of the chiral selector concentration, pH and composition of the BGE, and applied voltage on enantioseparation of acidic compounds, such as profens and aromatic carboxylic acids, was investigated. Two native α-amino acids, aspartic acid and glutamic acid, were enantioseparated. Fourteen tested compounds (including amino acids) were baseline resolved. Good selectivity of separation (α>1.09) was achieved. The migration order of ibuprofen and ketoprofen enantiomers was determined. The procedures were proposed for the analysis of flurbiprofen and warfarin in pharmaceuticals. Linearity was achieved in the concentration range of 4.0×10(-5)-2.0×10(-3) M for flurbiprofen and 3.2×10(-6)-4.9×10(-6) M for warfarin. The detection limits were found to be about 1×10(-5) M for flurbiprofen and 1×10(-6) M for warfarin.     

Chiral resolution of flurbiprofen and ketoprofen enantiomers by HPLC on a glycopeptide-type column chiral stationary phase

Vancomycin is an amphoteric, glycopeptide, macrocyclic antibiotic. When attached to 5 microspherical silica gel, vancomycin proved to be an effective chromatographic chiral stationary phase that could be used in the reversed-phase mode. In this study, a bonded vancomycin chiral stationary phase (Chirobiotic Vtrade mark) was investigated for the chiral liquid chromatography analysis of ketoprofen and flurbiprofen. The selectivity factor (alpha) and the chiral resolution factor (RS) of Chirobiotic Vtrade mark were evaluated first as a function of the buffer pH and molarity, and second as a function of organic modifier type and composition of the mobile phase. Four organic modifiers (tetrahydrofuran, 2-propanol, 1,4-dioxane and methanol) have been tested for their selectivity. Optimized conditions using 20% of tetrahydrofuran in ammonium nitrate (100 mM, pH 5) were selected for the enantioseparation of flurbiprofen and ketoprofen from their racemic forms. At pH 5, these acidic compounds are almost negatively charged, while the chiral selector possesses a positive charge allowing it to interact electrostatistically with the analytes. Using these chromatographic conditions, the column stability was excellent over several months of experiments.     

High-performance liquid chromatographic enantioseparation of beta-3-homo-amino acid stereoisomers on a (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid-based chiral stationary phase

High-performance liquid chromatographic methods were developed for the separation of the enantiomers of thirteen unusual beta-3-homo-amino acids and three of its ethyl esters on a chiral stationary phase containing (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid as chiral selector. The effects of the mobile phase composition and the acidic modifiers on the separation were investigated. The structures of the substituents in beta-position substantially influenced the retention and enantioseparation. The influence of ionic strength on the enantioseparation was established experimentally. The elution sequence was determined in all cases.     

Direct high-performance liquid chromatographic separation of unusual secondary amino acids and a comparison of the performances of Chirobiotic T and TAG columns

Two macrocyclic glycopeptide antibiotic-type chiral stationary phases (CSPs) based on native teicoplanin and teicoplanin aglycone, Chirobiotic T and TAG, respectively, were evaluated with regard to the high-performance liquid chromatographic separation of the enantiomers of 10 secondary alpha-amino acids (imino acids). The chromatographic results are given as the retention, separation and resolution factors, together with the enantioselective free energy difference corresponding to the separation of the enantiomers. By application of these two CSPs, excellent resolutions were achieved for the investigated compounds by using reversed-phase mobile mode systems. The separation conditions were optimized by variation of the mobile phase composition. The difference in enantioselective free energy between the aglycone CSP and the teicoplanin CSP for these particular amino acids ranged between 0.70 and -1.83 kJ mol(-1). It was established that better enantioseparations of the secondary alpha-amino acids were attained in most cases on the aglycone CSP.     

Chiral stationary phase designed for beta-blockers.

A chiral stationary phase (CSP) derived from an N-3,5-dinitrobenzoyl-alpha-amino phosphonate was prepared for the direct separation of the enantiomers of underivatized beta-blockers. Structure-chromatographic activity relationships for beta-blockers and closely related analogues are reported for this CSP and are found to be consistent with the model used to design this CSP. The effect of temperature on the chromatographic behavior of beta-blocker enantiomers is unusual. A reduction in temperature reduces the retention of the less retained enantiomer and increases the retention of the more retained enantiomer without appreciable band broadening.     

双选择体手性固定相中选择体的构型对分离性能的影响

为研究选择体的构型对双选择体固定相手性识别的影响,以(1S,2S)-(~)-二苯基乙二胺及L-(~)-二苯甲酰酒石酸为手性源,合成了一种新的双选择体固定相,并用不同结构的手性样品测试了其手性分离能力。结果表明,这种固定相与以(1R,2R)-(+)-二苯基乙二胺及L(~)-二苯甲酰酒石酸为手性源制备的双选择体固定相有相当的手性分离能力,但这两种固定相所能分离的化合物不尽相同。对双选择体固定相中两个选择体的构型对固定相手性识别的影响进行了探讨。在手性识别中,以不同手性源制备的两个选择体的立体构型不能同时与一个手性样品的立体构型相匹配,从而导致相应的双选择体固定相手性分离能力的下降。     

Novel Pirkle-type quinine 3,5-dinitrophenylcarbamate chiral stationary phase implementing click chemistry

A new Pirkle-anion exchange hybrid-type chiral stationary phase (CSP-1) has been synthesized by immobilizing 10,11-didehydroquinine 3,5-dinitrophenylcarbamate onto 3-azidopropyl silica gel using click chemistry (1,3-dipolar Huisgen cycloaddition). This chiral selector and CSP contain a strong π-accepting 3,5-dinitrophenyl residue besides the π-basic quinoline group and an ionizable tertiary amino group. In concert with ion pairing it offers π-donor-π-acceptor interactions resulting in an enhancement of the selectivity toward specific π-donating analytes such as aryloxypropionic acids and profens. A representative set of these analytes has been investigated under various chromatographic conditions (polar-organic, reversed- and normal-phase) leading to base-line enantioseparations with selectivity (α) values up to 1.8. Control experiments with related quinine tert-butylcarbamate phase grafted onto the surface either by thioether (Chiralpak QN-AX) or 1,2,3-triazole linker revealed the impact of the additional aromatic moiety in the chiral selector motif.     

High-performance liquid chromatographic enantioseparation of beta-amino acid stereoisomers on a (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid-based chiral stationary phase

Direct reversed-phase high-performance liquid chromatographic methods were developed for the separation of enantiomers of 14 unnatural beta-amino acids, including several beta-3-homo-amino acids on a chiral stationary phase containing (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid bonded to 3-aminopropyl silica gel as chiral selector. The effects of the organic and the acidic modifiers and the mobile phase composition on the separation were investigated. The natures and positions of the substituents on the aromatic ring substantially influenced the retention and enantioseparation. The elution sequence in most cases was determined and the R enantiomers were eluteted before the S enantiomers.