Use of tert-butylbenzoylated tartardiamide chiral stationary phase for the enantiomeric resolution of acidic compounds by nano-liquid chromatography

Several racemic acidic compounds of pharmaceutical and environmental interest have been separated into their enantiomers by nano-liquid chromatography (nano-LC) employing a tert-butylbenzoylated tartardiamide chiral stationary phase (CHI-TBB). CHI-TBB was packed into a fused silica capillary of 100 microm id and retained by two frits made with a heated wire; detection was on-column at a window (about 0.5 cm) prepared by removing the polyimide layer. The normal phase mode was selected for eluting the studied acidic compounds and therefore n-hexane/2-propanol/acetic acid (89/10/1, v/v/v) was used as mobile phase. Working at a flow rate of 220 nL/min a good resolution was obtained for mecoprop, dichlorprop, diclofop, fenoxaprop (herbicides) and for DF 1738Y, DF 1770Y, DF 2008Y (drugs under evaluation). In order to optimize the chiral resolution we modified the polarity of the mobile phase by adding several polar additives such as ethyl acetate, dichloromethane, tert-butyl methyl ether. Better results were obtained for some herbicides on working with 2-propanol/CH2Cl2/n-hexane/acetic acid (8/4/87/1, v/v/v/v). The influence of the capillary temperature on chiral resolution was studied for two herbicides with different chemical structures, namely mecoprop and haloxyfop in the temperature range between 10 and 40 degrees C and with n-hexane/2-propanol/1% acetic acid (89/10/1, v/v/v) as the mobile phase. Linear correlation of ln k vs 1/T and In alpha vs 1/ T was observed; deltaH degrees values were negative, demonstrating that retention of analytes was an exothermic process. A decrease in resolution was observed with rising temperature, showing that enantioresolution was mainly influenced by selectivity factors.     

Chiral separation of newly synthesized arylpropionic acids by capillary electrophoresis using cyclodextrins or a glycopeptide antibiotic as chiral selectors

Summary The chiral separation of two newly synthesized arylpropionic acids of pharmaceutical interest, namely 2-[(5′-benzoil-2′-hydroxy)phenyl]-propionic acid (DF-1738y) and 2-[(4′-benzoiloxy-2′-hydroxy)phenyl]-propionic acid (DF-1770y), was performed by Capillary Zone Electrophoresis (CZE) using either cyclodextrins or antibiotics as chiral selectors in coated capillary. In order to optimize the separation, the effect on the migration time and resolution of type and concentration of the chiral selector, the buffer pH and the capillary temperature were studied. Several cyclodextrins, namely the charged 6^A-monomethylamino-β-cyclodextrin (MeNH-β-CD) and the neutral methyl-β-cyclodextrins (M-β-CD) and heptakis-2,3,6-tri-O-methyl-β-cyclodextrin (TM-β-CD), were tested for the enantiomeric separation of aryl propionic acids (APAs) compounds. Of these TM-β-CD provided the highest enantiomeric resolution at pH 5, however only DF-1738y optical isomers were baseline resolved. Using background electrolytes (BGEs) at higher pHs (pH=6–7) supported with the above listed CDs, an enantioresolution increase was recognized only for compound DF-1738y. In contrast DF-1770y exhibited the highest resolution at the lowest pH value studied (pH 4). The macrocyclic antibiotic vancomycin was therefore added to the BGE and tested as chiral selector using the partial filling counter current mode in order to obtain a sensitive analysis, high resolution and reduced antibiotic adsorption on the capillary wall. 5 mM vancomycin dissolved in the BGE at pH 5 and 25°C provided relatively high enantiomeric resolution (R _DF-1738y=3.4,R _DF-1770y=2.22) of both compounds.     

Cationic vesicles as chiral selector for enantioseparations of nonsteroidal antiinflammatory drugs by micellar electrokinetic chromatography

A vesicle-forming chiral cationic surfactant (1R,2S)-(-)-N-dodecyl-N-methyl-ephedrinium bromide was evaluated as a pseudo-stationary phase in micellar electrokinetic chromatography (MEKC) for enantioseparation of eight non-steroidal anti-inflammatory drugs e.g., carprofen, flurbiprofen, fenoprofen, ibuprofen, indoprofen, ketoprofen, naproxen and suprofen by capillary electrophoresis. The effects of varying experimental conditions such as pH and concentration of surfactant in the running buffer on the enantiomer separation of the drugs are reported. A mixture of five of the above drugs was separated and each enantiomeric pair was also separated simultaneously in a single run by use of the surfactant. The strong electrostatic interactions between the analytes and the vesicles seemed to have a major role in the enantiomeric separation of the profens.     

Enantiomeric separation of some demethylated analogues of clofibric acid by capillary zone electrophoresis and nano-liquid chromatography

The enantiomeric separation of some demethylated analogues of clofibric acid, namely 2-(6-chloro-benzothiazol-2-ylsulfanyl)-, 2-(6-methoxy-benzothiazol-2-ylsulfanyl)-, 2-(quinolin-2-yloxy)-, 2-(6-chloro-quinolin-2-yloxy)-, 2-(7-chloro-quinolin-4-yloxy)-propionic acid (compounds A-E, respectively), has been studied by CZE and nano-LC using for the first technique two beta-CD derivatives and vancomycin added to the BGE and vancomycin-modified silica particles for the second one, with the aim to find the optimum experimental conditions for the baseline resolution. The type and the concentration of the chiral selector added to the BGE, the buffer pH, the type of organic modifier and its concentration, the capillary temperature and the applied voltage played a very important role in the enantioresolution of the analysed compounds. The use of 6-monodeoxy-6-monoamino-beta-CD allowed to achieve baseline resolution of four of five clofibric acid derivatives in less than 10 min while heptakis-(2,3,6-tri-O-methyl)-beta-CD partially resolved the same compounds in their enantiomers. Employing vancomycin as the chiral selector in CZE, the counter-current partial filling method was chosen achieving baseline resolution of four analytes. All the studied compounds were enantioresolved employing a capillary column packed with vancomycin stationary phase by nano-LC, and the resolution was strongly influenced by the concentration of the organic modifier and by the pH of the mobile phase. The best results were achieved at pH 4.5 in presence of 60% of methanol (MeOH). However, longer analysis times were observed in the experiments carried out by nano-LC.     

Enantioseparations on amylose tris(5-chloro-2-methylphenylcarbamate) in nano-liquid chromatography and capillary electrochromatography

Amylose tris(5-chloro-2-methylphenylcarbamate) was coated onto native and aminopropylsilanized silica in order to prepare chiral stationary phases (CSP) for enantioseparations using nano-liquid chromatography (nano-LC) and capillary electrochromatography (CEC). The effect of the nature of silica, the particle size and pore diameter, the chiral selector loading onto silica, the mobile phase composition and pH, as well as separation variables such as a linear flow rate of the mobile phase, applied voltage in CEC, etc. on the separation of enantiomers was studied. It was found that CSPs based on amylose tris(5-chloro-2-methylphenylcarbamate) can be used for preparation of stable capillary columns for enantioseparations by nano-LC and CEC in combination with polar organic and aqueous–organic mobile phases. Higher peak efficiency was observed in CEC than in nano-LC.     

Enantiomeric separation of chlorophenoxy acid herbicides by nano liquid chromatography-UV detection on a vancomycin-based chiral stationary phase

Enantiomeric separation of mecoprop, dichlorprop, and fenoprop herbicides in their acid form, commonly used to control the growth of broad-leaved weeds, was carried out by nano-liquid chromatography (nano-LC) at a flow rate of 60 nL/min, using a packed capillary column with vancomycin-modified silica particles of 5 microm. The length of chiral stationary phase was 21 cm, while the total and effective lengths were 43 and 33cm, respectively. Inner diameter was 0.075 mm. Separated peaks were detected at 195 nm. Several mixtures of methanol, water, and 500 mM ammonium acetate buffer at different pH's were tested as mobile phase, and experimental parameters such as resolution (Rs), capacity factor (k), efficiency (N/m), and enantioselectivity factor (alpha) were measured under all the test conditions. Baseline enantiomeric separation was obtained for the three studied herbicides with alpha in the range 1.6-1.9, using as the mobile phase aqueous solutions containing 85% methanol, 5% of 500mM ammonium acetate pH4.5 buffer, and 10% water. Experimental results show that the vancomycin stationary phase presents a great enantiorecognition capability towards chlorophenoxy acid herbicides on using nano-LC.     

卵类粘蛋白柱手性拆分酮基布洛芬

 在卵类粘蛋白柱上 ,考察了酮洛芬对映体在 3种流动相体系下的保留行为 ,建立了同时分离酮洛芬及其甲酯两对对映体的较佳色谱条件。方法可用于酶法立体选择性水解或酯化过程中生成的 4种化合物的同时测定     

Optical isomer separation of flavanones and flavanone glycosides by nano-liquid chromatography using a phenyl-carbamate-propyl-β-cyclodextrin chiral stationary phase

In this paper a phenyl-carbamate-propyl-β-cyclodextrin stationary phase was employed for the enantioseparation of several flavonoids, including flavanones and methoxyflavanones by using nano-liquid chromatography (nano-LC). The same stationary phase was also used for the diastereoisomeric separation of two flavanone glycosides. The compounds: flavanone, 2′-hydroxyflavanone, 4′-hydroxyflavanone, 6-hydroxyflavanone, 7-hydroxyflavanone, 4′-methoxyflavanone, 6-methoxyflavanone, 7-methoxyflavanone, hesperetin, hesperidin, naringenin and naringin were studied using reversed, polar organic and normal elution modes. The effect of the nature and composition of the mobile phase (organic modifier type, buffer and water content in the reversed phase mode) on the enantioresolution (R_s), retention factor (k) and enantioselectivity (α) were investigated. Baseline resolution of all studied flavonoids, with the exception of 2′-hydroxyflavanone and naringin, was achieved in reversed phase mode using a mixture of MeOH/H₂O at different ratios as mobile phase. Good results, in terms of peak efficiency and short analysis time, were obtained adding 1% triethylammonium acetate pH 4.5 buffer to MeOH/H₂O mixture. The separation of the studied compounds was also performed in polar organic mode. By using 100% of MeOH as mobile phase, the resolution was achieved for the studied analytes, except for 7-hydroxyflavanone, 2′-hydroxyflavanone, naringenin, hesperidin and naringin. Normal mode was tested employing a mixture of EtOH/hexane/TFA as mobile phase achieving the enantiomeric and diastereomeric separation of only hesperetin and hesperidin, respectively. The use of nano-LC technique for the resolution of flavanones optical isomers allowed to achieve good resolutions in shorter analysis time compared to the results reported in literature with conventional HPLC.     

人血清白蛋白柱上药物的手性拆分

考察了４种酸性药物和１种中性药物对映体在人血清白蛋白手性固定相上的保留行为。这５种药物与人血清白蛋白结合的亲和力高，难于实现快速分离，作者提出在流动相中加入短链脂肪酸－正己酸，可快速手性拆分非诺洛芬、萘普生和布洛芬。酮基布洛芬对映体分离选择性随乙腈浓度升高而增大，流动相中加入适量异丙醇可使对映体选择性大大增加（α～１．２３），华法令同样可取得很好分离。     

Enantiomeric analysis of drugs in water samples by using liquid–liquid microextraction and nano-liquid chromatography

The nano-LC technique is increasingly used for both fast studies on enantiomeric analysis and test beds of novel stationary phases due to the small volumes involved and the short conditioning and analysis times. In this study, the enantioseparation of 10 drugs from different families was carried out by nano-LC, utilizing silica with immobilized amylose tris(3-chloro-5-methylphenylcarbamate) column. The effect on chiral separation caused by the addition of different salts to the mobile phase was evaluated. To simultaneously separate as many enantiomers as possible, the effect of buffer concentration in the mobile phase was studied, and, to increase the sensitivity, a liquid–liquid microextraction based on the use of isoamyl acetate as sustainable extraction solvent was applied to pre-concentrate four chiral drugs from tap and environmental waters, achieving satisfactory recoveries (>70%).     

几种手性化合物在直链淀粉-三(环己基氨基甲酸酯)手性固定相上高压液相中的拆分

正相条件下, 在自制的涂敷型直链淀粉-三(环己基氨基甲酸酯)手性固定相上, 直接拆分了九种不同类型的手性化合物, 考察了流动相中极性添加剂对手性拆分的影响, 并与直链淀粉-三(3,5-二甲基苯基氨基甲酸酯)进行了比较. 研究发现, 此种手性固定相不仅具有较高的手性识别能力, 而且对某些类型手性化合物的选择性与后者明显不同; 探讨了此种固定相可能的拆分机理.     

Enantiomeric separation of 2‐arylpropionic acid nonsteroidal anti‐inflammatory drugs by HPLC with hydroxypropyl‐β‐cyclodextrin as chiral mobile phase additive

The enantio‐separations of eight 2‐arylpropionic acid nonsteroidal anti‐inflammatory drugs (2‐APA NSAIDs) were established using reversed‐phase high‐performance liquid chromatography with hydroxypropyl‐β‐cyclodextrin (HP‐β‐CD) as chiral mobile phase additive for studying the stereoselective skin permeation of suprofen, ketoprofen, naproxen, indoprofen, fenoprofen, furbiprofen, ibuprofen and carprofen. The effects of the mobile phase composition, concentration of HP‐β‐CD and column temperature on retention and enantioselective separation were investigated. With 2‐APA NSAIDs as acidic analytes, the retention times and resolutions of the enantiomers were strongly related to the pH of the mobile phase. In addition, both the concentration of HP‐β‐CD and temperature had a great effect on retention time, but only a slight or almost no effect on resolutions of the analytes. Enantioseparations were achieved on a Shimpack CLC‐ODS (150 × 4.6 mm i.d., 5 μm) column. The mobile phase was a mixture of methanol and phosphate buffer (pH 4.0–5.5, 20 mM) containing 25 mM HP‐β‐CD. This method was flexible, simple and economically advantageous over the use of chiral stationary phase, and was successfully applied to the enantioselective determination of the racemic 2‐APA NSAIDs in an enantioselective skin permeation study. Copyright © 2009 John Wiley & Sons, Ltd.     

Cyclodextrins as a chiral mobile phase additive in nano-liquid chromatography: comparison of reversed-phase silica monolithic and particulate capillary columns

Enantioseparations of racemic nonsteroidal anti-inflammatory drugs (naproxen, ibuprofen, ketoprofen, flurbiprofen, suprofen, indoprofen, cicloprofen, and carprofen) were performed by nano-liquid chromatography, employing achiral capillary columns and heptakis(2,3,6-tri-O-methyl)-β-cyclodextrin (TM-β-CD) or hydroxylpropyl-β-cyclodextrin (HP-β-CD) as a chiral mobile phase additive (CMPA). Working under the same experimental conditions (in terms of mobile phase and linear velocity), the performance of a RP-C18 monolithic column was compared with that of a RP-C18 packed column of the same dimensions (100 μm i.d. × 10 cm). Utilizing a mobile phase composed of 30% ACN (v/v) buffered with 50 mM sodium acetate at pH 3, and containing 30 mM TM-β-CD, the monolithic column provided faster analysis but lower resolution than the packed column. This behavior was ascribed to the high permeability of the monolithic column, as well as to its minor selectivity. HP-β-CD was chosen as an alternative to TM-β-CD. Employing the monolithic column, the effects of different parameters such as HP-β-CD concentration, mobile phase composition, and pH on the retention factor and the chiral resolution of the analytes were studied. For the most of the analytes, enantioresolution (which ranged from R _s = 1.80 for naproxen to R _s = 0.86 for flurbiprofen) was obtained with a mobile phase consisting of sodium acetate buffer (25 mM, pH 3), 10% MeOH, and 15 mM HP-β-CD. When the same experimental conditions were used with the packed column, no compound eluted within 1 h. Upon increasing the percentage of organic modifier to favor analyte elution, only suprofen eluted within 30 min, with an R _s value of 1.14 (20% MeOH). Replacing MeOH with ACN resulted in a loss of enantioresolution, except for naproxen (R _s = 0.89).     

Capillary electrochromatographic separation of non-steroidal anti-inflammatory drugs with a histidine bonded phase

An open tubular wall-coated capillary column containing histidine functional groups was prepared and employed for the capillary electrochromatographic separation of non-steroidal anti-inflammatory drugs. The anion exchange along with the hydrogen bonding and hydrophobic properties of the surface coating allowed the separation of analytes with very similar ionic mobility. Selectivity and resolution were studied by changing the pH over the range from 3.5 to 5.0 and the concentration of the buffer from 10 to 25 mM, as well as variation of the organic modifier, such as methanol, ethanol and 1-propanol over the range 7.5 to 20%. The optimum experimental conditions for the separation of a drug mixture, which consisted of indoprofen, ketoprofen, suprofen, naproxen, flurbiprofen, fenoprofen and ibuprofen were using a mixture of acetate buffer (20 mM, pH 5.0)-ethanol (1:5, v/v) as background electrolyte and an applied voltage of -20 kV with UV detection at 220 nm. The separation of these drugs could be achieved with an average plate number of 1.0 x 10(5) m(-1).     

酸性外消旋联苯类药物在纤维素-三(3,5-二甲基苯基氨基甲酸酯)固定相上的直接拆分

合成了纤维素 三 ( 3,5 二甲基苯基氨基甲酸酯 ) (CDMPC)衍生物 ,并将其涂敷于自制的球形硅胶上 ,制备出用于高效液相色谱手性拆分的固定相。在正相条件下 ,用该固定相对 5种酸性外消旋联苯类药物进行了直接拆分 ,并考察了流动相组成和样品结构对保留和拆分的影响 ,讨论了固定相对样品的作用模式。结果表明 ,在正己烷 /异丙醇流动相体系中加入少量三氟乙酸 ,可对外消旋羧酸类化合物进行有效的手性拆分。     

A Validated Chiral HPLC Method for the Determination of Enantiomeric Purity of R-β-amino-β-(4-methoxyphenyl) Propionic Acid

A normal phase chiral LC method for chiral purity evaluation of β-amino-β-(4-methoxyphenyl) propionic acid was developed on donor–acceptor (pirkle) column. The chiral stationary phase used was a 250 × 4.6 mm (R, R) Whelk-01 with 5 μm particle size, which was accompanied with a 1 cm long guard column. The “hybrid” pi-electron donor–acceptor based stationary phase (R, R) Whelk-01 was found to be enantiomeric selective for (R) and (S) enantiomers of β-amino-β-(4-methoxyphenyl) propionic acid with a resolution >2.5. The concentration of 2-propanol and TFA in the mobile phase plays an important role on the chrmatographic efficiency and resolution between the enantiomers. The limit of detection and limit of quantification of (S) enantiomer was 0.3 and 1.0 μg mL^-1 for 20 μL injection volume. The percentage RSD of the peak area of six replicate injections of (S) enantiomer at LOQ concentration was 4.5. The percentage recovery of (S) enantiomer from (R) enantiomer samples ranged from 92 to 100. The test solution was observed to be stable up to 24 h after the preparation. The developed method was also checked by different analysts and on different lots of columns, reagents and it was proved to be rugged. The developed normal phase chiral LC method can be used for the determination of the enantiomeric purity of R-β-amino-β-(4-methoxyphenyl) propionic acid.     

高效液相色谱法直接测定几种非甾体类解热镇痛药物中的对映体含量

用WHELK-O1手性色谱柱，在正相条件下测定了几种非甾体类解热镇痛药物萘普生、布洛芬、酮基布洛芬和苯氧布洛芬等中对映体的含量。结果表明：这种手性固定相色谱柱能够以正己烷和异丙醇为流动相，简便、快速、准确地测定非甾类药物中对映体的含量。     

Use of vancomycin silica stationary phase in packed capillary electrochromatography I. Enantiomer separation of basic compounds

Chiral separation of basic compounds was achieved by using 75 or 100 microm ID fused-silica capillaries packed with a vanoomycin-modified diol silica stationary phase. The capillary was firstly packed for about 12 cm with a slurry mixture composed of diolsilica (3:1) then with the vancomycin modified diol-silica (3:1) (23 cm), and finally with diol-silica (3:1) for about 2 cm. Frits were prepared by a heating wire at the two ends of the capillary; the detector window was prepared at 8.5 cm from the end of the capillary where vancomycin was not present. The influence of the mobile phase composition (pH and concentration, organic modifier type and concentration) on the velocity of the electroosmotic flow, chiral resolution and enantioselectivity was studied. Good enantiomeric resolution was achieved for atenolol, oxprenolol, propranolol, and venlafaxine using a mobile phase composition of 100 mM ammonium acetate solution (pH 6)/water/acetonitrile (5:5:90 v/v/v) while for terbutaline a mixture of 5:15:80 v/v/v provided the best separations. The use of methanol instead of acetonitrile caused a general increase of enantiomer resolution of the studied compounds together with a reduction of efficiency and detector response. However, the combination of acetonitrile and methanol in the mobile phase (as, e.g., 10% methanol and 80% acetonitrile) allowed to improve the enantiomer resolution with satisfactory detector response.     

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.     

Preparation and evaluation of a vancomycin-immobilized silica monolith as chiral stationary phase for CEC

Enantiomeric separations in CEC with the macrocyclic antibiotic vancomycin immobilized silica monolith as a chiral stationary phase are presented. The monolithic silica capillary columns were prepared by a sol-gel process in fused-silica capillaries with an inner diameter of 50 mum and subsequently in situ immobilization of vancomycin as a chiral selector by reductive amination. Enantioselectivity was obtained for eight pairs of enantiomers in nonaqueous polar organic or aqueous mobile phases and most of them were baseline-separated with high column efficiencies. It was observed that the organic modifier ratio (MeOH/ACN) in the polar organic mobile phase played a significant role in controlling the resolution and efficiency of the enantiomers. In enantiomeric separation of propranolol, repeatability for column efficiency and resolution in the nonaqueous mobile phase was given in terms of RSD values at 1.1 and 2.3% (n = 5) for run-to-run injections and 7.2 and 9.6% (n = 5) for column-to-column testing while repeatability for the separation of thalidomide in the aqueous mobile phase was given in terms of RSD values at 1.5, 2.8% and 6.1, 10.5%, respectively.