Rapid separation of pharmaceutical enantiomers using electrokinetic chromatography with a novel chiral microemulsion

A novel oil-in-water microemulsion incorporating the chiral surfactant dodecoxycarbonylvaline (DDCV) was used to achieve the rapid enantiomeric separation of pharmaceutical drugs by electrokinetic chromatography (EKC). Incorporation of DDCV into a microemulsion resulted in an elution range more than double that provided the micellar form of the surfactant aggregate. Interestingly, for the same compounds the enantioselectivity provided by the chiral DDCV microemulsions ranged from 1.06-1.30 for the neutral and cationic drugs, which was slightly higher than that provided by chiral DDCV micelles. The use of a low surface tension oil (ethyl acetate) permitted a much lower concentration of chiral surfactant to be employed; this, together with the use of a zwitterionic buffer (ACES) resulted in a very low conductivity microemulsion that allowed a higher separation voltage to be utilized, resulting in rapid enantiomeric separations (< 8 min.). Mobility matching of the buffer cation(s) was used to improve peak shape and efficiencies. In our limited survey of the phase diagram, the optimum composition of the microemulsion buffer was 1.0% (w/v) DDCV (30 mM), 0.5% (v/v) ethyl acetate, 1.2% (v/v) 1-butanol and 50 mM ACES buffer at pH 7.     

Two-chiral component microemulsion EKC - chiral surfactant and chiral oil. Part 2: diethyl tartrate

In this second study on dual-chirality microemulsions containing a chiral surfactant and a chiral oil, a less hydrophobic and lower interfacial tension chiral oil, diethyl tartrate, is employed (Part 1, Foley, J. P. et al.., Electrophoresis, DOI: 10.1002/elps.200600551). Six stereochemical combinations of dodecoxycarbonylvaline (DDCV: R, S, or racemic, 2.00% w/v), racemic 2-hexanol (1.65% v/v), and diethyl tartrate (D, L, or racemic, 0.88% v/v) were examined as pseudostationary phases (PSPs) for the enantioseparation of six chiral pharmaceutical compounds: pseudoephedrine, ephedrine, N-methyl ephedrine, metoprolol, synephrine, and atenolol. Average efficiencies increased with the addition of a chiral oil to R-DDCV PSP formulations. Modest improvements in resolution and enantioselectivity (alpha(enant)) were achieved with two-chiral-component systems over the one-chiral-component microemulsion. Slight enantioselective synergies were confirmed using a thermodynamic model. Results obtained in this study are compared to those obtained in Part 1 as well as those obtained with chiral MEEKC using an achiral, low-interfacial-tension oil (ethyl acetate). Dual-chirality microemulsions with the more hydrophobic oil dibutyl tartrate yielded, relative to diethyl tartrate, higher efficiencies (100,000-134,000 vs. 80,800-94,300), but lower resolution (1.64-1.91 vs. 2.08-2.21) due to lower enantioselectivities (1.060-1.067 vs. 1.078-1.081). Atenolol enantiomers could not be separated with the dibutyl tartrate-based microemulsions but were partially resolved using diethyl tartrate microemulsions. A comparable single-chirality microemulsion based on the achiral oil ethyl acetate yielded, relative to diethyl tartrate, lower efficiency (78 300 vs. 91 600), higher resolution (1.99 vs. 1.83), and similar enantioselectivities.     

Effect of surfactant concentration and buffer selection on chromatographic figures of merit in chiral microemulsion electrokinetic chromatography

The enantiomeric resolution of 15 different pharmaceutical compounds was explored using chiral microemulsion electrokinetic chromatography (MEEKC). The microemulsion employed was comprised of the chiral surfactant dodecoxycarbonylvaline (DDCV), 1-butanol, and ethyl acetate, at an initial composition of 1% w/v:1.2% v/v:0.5% v/v, respectively. The effect of varying the background buffer composition, voltage, and ultimately the surfactant concentration and/or aggregate phase ratio were examined. Changing from a zwitterionic buffer ((2-[2-amino-2-oxoethyl)amino]ethanesulfonic acid, ACES) to the same concentration of phosphate buffer improved the efficiency and decreased overall analysis time, but also resulted in a decrease in chiral resolution. Furthermore, using phosphate buffer while simultaneously increasing the percent DDCV from 1 to 4% increased the efficiencies from a range of 34,000 to 59,000 N/m to a range of 160,000 to 400,000 N/m. While the enantioselectivities did not change significantly, the improvement in efficiencies, elution range, and retention factors provided an increase in both resolution and the number of enantiomers that were separated. Using an optimized microemulsion comprised of phosphate buffer and 4% DDCV, chiral separation was achieved for all 11 pairs of enantiomers, with a resolution ranging from 0.90 to 4.71. Moreover, the average resolution doubled in going from nonoptimized to optimized conditions for five of the eleven compounds. Finally, a comparison was made of the effect of increasing only the surfactant concentration by a factor of 4 versus increasing the overall composition (or phase ratio) by a factor of 4. Ultimately, the microemulsion containing 4% DDCV provided a larger elution range, greater resolution, and more optimal retention than that provided by the 4x phase increase.     

Comparison of dodecoxycarbonylvaline microemulsion, solvent-modified micellar and micellar pseudostationary phases for the chiral analysis of pharmaceutical compounds

A direct comparison of dodecoxycarbonylvaline (DDCV) microemulsion, micellar and butanol-modified micellar aggregate systems was performed employing both 2 and 4% DDCV. With respect to either DDCV concentration, use of the micellar system provided the largest elution range, followed by the butanol-modified micellar system and ultimately the microemulsion. Using 2% DDCV, all three aggregate analyses yielded similar values for enantioselectivity, resolution and retention factors that were slightly better using the micellar phase, but efficiencies were consistently better using either the microemulsion or butanol-modified micellar phases. Largely, the microemulsion and butanol-modified micellar phases behaved fairly similar, although use of the butanol-modified micelle provided resolution and efficiency that were slightly better for all but two of the compounds. While reasonable separations were achieved using 2% DDCV, the results using 4% DDCV for the microemulsion system were far superior. Analyses using analogous micellar and butanol-modified micellar aggregates were unstable, making them unsuitable for use at that surfactant concentration.     

Effect of microemulsion component purity on the chromatographic figures of merit in chiral microemulsion electrokinetic chromatography

Numerous combinations of one-, two-, and three-chiral-component microemulsions have been previously prepared in our group, using N-dodecoxycarbonylvaline (DDCV), 2-hexanol, and ethyl acetate, dibutyl tartrate, or diethyl tartrate. A few results of the various formulations investigated suggested the possible presence of minor impurities in one or more components of the microemulsion. In this study, the purity of the current lots of R- and S-surfactant were measured, as was the subsequent effect of minor impurities on the relevant chromatographic figures of merit (CFOMs) that describe a chiral separation, i.e., efficiency, enantioselectivity, retention, migration window (elution range), and resolution. Two related methods are proposed for correcting enantioselectivities measured in the presence of chiral impurities in the chiral microemulsion.     

Two-chiral-component microemulsion electrokinetic chromatography-chiral surfactant and chiral oil: part 1. dibutyl tartrate

The first simultaneous use of a chiral surfactant and a chiral oil for microemulsion EKC (MEEKC) is reported. Six stereochemical combinations of dodecoxycarbonylvaline (DDCV: R, S, or racemic, 2.00% w/v), racemic 2-hexanol (1.65% v/v), and dibutyl tartrate (D, L, or racemic, 1.23% v/v) were examined as chiral pseudostationary phases (PSPs) for the separation of six pairs of pharmaceutical enantiomers: pseudoephedrine, ephedrine, N-methyl ephedrine, metoprolol, synephrine, and atenolol. Subtle differences were observed for three chromatographic figures of merit (alpha(enant), alpha(meth), k) among the chiral microemulsions; a moderate difference was observed for efficiency (N) and elution range. Dual-chirality microemulsions provided both the largest and smallest enantioselectivities, due to small positive and negative synergies between the chiral microemulsion components. For the ephedrine family of compounds, dual-chiral microemulsions with surfactant and oil in opposite stereochemical configurations provided higher enantioselectivities than the single-chiral component microemulsion (RXX), whereas dual-chiral microemulsions with surfactant and oil in the same stereochemical configurations provided lower enantioselectivities than RXX. Slight to moderate enantioselective synergies were confirmed using a thermodynamic model. Efficiencies observed with microemulsions comprised of racemic dibutyl tartrate or dibutyl-D-tartrate were significantly higher than those obtained with dibutyl-L-tartrate, with an average difference in plate count of about 25 000. Finally, one two-chiral-component microemulsion (RXS) provided significantly better resolution than the remaining one- and two-chiral-component microemulsions for the ephedrine-based compounds, but only slightly better or equivalent resolution for non-ephedrine compounds.     

Temperature effects on chiral microemulsion electrokinetic chromatography employing the chiral surfactant dodecoxycarbonylvaline

Dodecoxycarbonylvaline (DDCV) microemulsions (1% and 4%, w/v) were employed to evaluate the retention mechanism of a series of enantiomers over a temperature range of 15-35 degrees C. From the acquired retention data, van't Hoff plots were constructed and enthalpy and entropy of transfer were calculated from the slope and intercept, respectively. Resolution, enantioselectivity, distribution coefficients and Gibb's free energy were also calculated, as well as between enantiomer differences in enthalpy, entropy and Gibb's free energy. Finally, comparisons were made between the microemulsion thermodynamic data and a corresponding set of micellar data. While the 4% DDCV microemulsion did not provide a linear van't Hoff relationship, the 1% DDCV microemulsion was linear over a temperature range of 15-30 degrees C. For the 1% DDCV microemulsion, the enthalpic contribution to retention was consistently favorable (deltaH < 0), whereas the entropic contribution varied from compound to compound. Finally, while the achiral attraction of the analytes was greater for the micellar phase, the microemulsion seemed to provide a suitable difference in entropy (and Gibb's free energy) between enantiomers to achieve chiral discrimination.     

Influence of microemulsion chirality on chromatographic figures of merit in EKC: results with novel three-chiral-component microemulsions and comparison with one- and two-chiral-component microemulsions

Novel microemulsion formulations containing all chiral components are described for the enantioseparation of six pairs of pharmaceutical enantiomers (atenolol, ephedrine, metoprolol, N-methyl ephedrine, pseudoephedrine, and synephrine). The chiral surfactant dodecoxycarbonylvaline (DDCV, R- and S-), the chiral cosurfactant S-2-hexanol, and the chiral oil diethyl tartrate (R- and S-) were combined to create four different chiral microemulsions, three of which were stable. Results obtained for enantioselectivity, efficiency, and resolution were compared for the triple-chirality systems and the single-chirality system that contained chiral surfactant only. Improvements in enantioselectivity and resolution were achieved by simultaneously incorporating three chiral components into the aggregate. The one-chiral-component microemulsion provided better efficiencies. Enantioselective synergies were identified for the three-chiral-component nanodroplets using a thermodynamic model. Additionally, two types of dual-chirality systems, chiral surfactant/chiral cosurfactant and chiral surfactant/chiral oil, were examined in terms of chromatographic figures of merit, with the former providing much better resolution. The two varieties of two-chiral-component microemulsions gave similar values for enantioselectivity and efficiency. Lastly, the microemulsion formulations were divided into categories based on the number of chiral microemulsion reagents and the average results for each pair of enantiomers were analyzed for trends. In general, enantioselectivity and resolution were enhanced while efficiency was decreased as more chiral components were used to create the pseudostationary phase (PSP).     

Chiral microemulsion electrokinetic chromatography with two chiral components: Improved separations via synergies between a chiral surfactant and a chiral cosurfactant

In this study, the combination of two chiral components in a microemulsion formulation for the separation of enantiomers via microemulsion EKC (MEEKC) was successfully accomplished. Previous publications of chiral microemulsions have utilized only one chiral entity; the surfactant, cosurfactant, or oil was chiral. This is the first study, to date, of the effects of using two chiral species in a single pseudostationary phase (PSP). The chiral surfactant dodecoxycarbonylvaline (DDCV) was used in conjunction with the chiral cosurfactant S-2-hexanol. Ethyl acetate was incorporated as the oil core of the microemulsion and the buffer was 50 mM phosphate at a pH of 7. Additionally, a microemulsion prepared with racemic 2-hexanol was used for comparison to a previous DDCV microemulsion and as a baseline for the newly formulated dual chiral microemulsion. The efficiencies, resolutions, and enantioselectivities for the S-2-hexanol, racemic 2-hexanol, and original 1-butanol DDCV microemulsions are compared. The hexanol-based PSPs provide improved efficiencies and resolutions. To evaluate the combination of each DDCV enantiomer (R and S) with S-2-hexanol, changes in Gibb's free energy were calculated. A synergistic effect was found when two chiral components were combined to form a microemulsion.     

Chiral cyclodextrin-modified microemulsion electrokinetic chromatography

Cyclodextrin (CD)-modified microemulsion electrokinetic chromatography (MEEKC) or CD-MEEKC has not previously been applied to the area of chiral separations. Herein, the results of investigations of various microemulsions with CD additives are presented. Two different microemulsions are explored: an ethyl acetate sodium dodecyl sulfate microemulsion, and a chiral dodecoxycarbonylvaline (DDCV) microemulsion. Each microemulsion is paired separately with a neutral CD (hydroxypropyl-beta-CD) and an anionic CD (sulfated-beta-CD). In addition, the chiral DDCV microemulsion is investigated in both the R- and S- form. By varying simple parameters such as buffer system, applied voltage, surfactant enantiomer, and type of cyclodextrin, dramatic improvements in the chiral separations were noted. Resolution was found to be highly dependent on buffer identity and concentration, and somewhat dependent on whether the CDs used were randomly or highly sulfated. Under optimized conditions, the resolution ranged from 0.8 to 4.8, with plate counts ranging from 4000 to 26 000. Additionally, S- and R-levetiracetam, which had never before been enantioseparated via capillary electrophoresis (CE) methodologies, were separated in less than 8 min, with a resolution of 1.1.     

Investigation of the chiral surfactant N-dodecoxycarbonylvaline in electrokinetic chromatography: improvements in elution range and pH stability via mixed micelles and vesicles, and the hydrophobicity determination of basic pharmaceutical drugs

The chiral surfactant dodecoxycarbonylvaline (DDCV) has proven to be an effective pseudostationary phase for the separation of many enantiomeric pharmaceutical compounds. In this study the elution range and the prediction of octanol-water partitioning for the DDCV micellar system was examined. Through incorporation of DDCV in mixed micelles and unilamellar vesicles, enhancement of the elution range was observed. The mixed micelles contained a second anionic surfactant, sodium dodecyl sulfate (SDS), while the vesicles were composed of DDCV and the cationic surfactant cetyltrimethylammonium bromide (CTAB). Enantioselectivity, as well as other chromatographic and electrophoretic parameters, were compared between the mixed micelles, vesicles, and DDCV micelles. The hydrophobicity of the DDCV system was also evaluated as a predictor of n-octanol-water partition coefficients for 15 beta amino alcohols. The correlation between the logarithm of the retention factor (log k) and log P(ow) for seven hydrophobic beta-blockers and eight beta-agonists were r2 = 0.964 and r2 = 0.814, respectively.     

Validation of an oil-in-water microemulsion liquid chromatography method for analysis of perindopril tert-butylamine and its impurities

This paper describes the development and validation of a microemulsion liquid chromatography (MELC) method for simultaneous determination of perindopril tert-butylamine and its impurities in bulk active substances and the pharmaceutical dosage form of tablets. An appropriate resolution with reasonable retention times was obtained for a microemulsion containing 0.24% (w/v) butyl acetate, 0.30% (w/v) ethyl acetate, 2% (w/v) sodium dodecyl sulfate, 7.75% (w/v) n-butanol, and 20.0 mM potassium dihydrogen phosphate, the pH of which was adjusted to 3.70 with 85% orthophosphoric acid. Separations were performed on a Nucleosil 120-5 butyl modified (C4), 250 x 4 mm, 5 microm particle size silica column at 40 degrees C, with a mobile phase flow rate of 1.25 mL/min. UV detection was performed at 254 nm. The established method was subjected to method validation, and required validation parameters were defined. Robustness testing, an important part of method validation, was performed as well. Since robustness validation can be conducted using different experimental designs, the Plackett-Burman design was applied due to its possibility of testing many factors at the same time. The validated MELC method was found to be suitable for the simultaneous determination of perindopril tert-butylamine and its impurities in pharmaceuticals.     

Chiral microemulsion electrokinetic chromatography: Effect of cosurfactant identity on enantioselectivity, methylene selectivity, resolution, and other chromatographic figures of merit

The effect of cosurfactant identity on microemulsion size, elution range, retention factor, enantioselectivity, methylene selectivity, efficiency, and resolution in chiral microemulsion formulations was examined. The chiral surfactant dodecoxycarbonylvaline was used in conjunction with the cosurfactants 1-butanol, 1-pentanol, 2-pentanol, 1-hexanol, 2-hexanol, cyclopentanol, and cyclohexanol. The millimolar concentration of cosurfactant was held constant regardless of identity. Ethyl acetate was incorporated as the microemulsion oil core and the buffer utilized was 50 mM phosphate at a pH of 7.0. In general, secondary alcohols improved enantioselectivities and primary alcohols had the opposite effect, with the exception of the 1-butanol. The trends observed varied slightly depending on analyte. Of the six chiral analytes tested, cyclopentanol provided the best enantioselectivity for three, 1-butanol for two compounds, and 2-pentanol for one analyte. The lowest enantioselectivities were achieved with 1-pentanol or 1-hexanol for all compounds. Methylene selectivity was found to decrease with reductions in alcohol chain length. Among equal carbon number alcohols, methylene selectivity was lower for secondary alcohols. Efficiency and resolution values varied with different cosurfactants and depended on analyte identity.     

Sample stacking microemulsion electrokinetic capillary chromatography induced by reverse migrating pseudostationary phase for the quantification of phenobarbital and its p-hydroxyphenobarbital metabolite in rat urine

For the first time, a capillary electrophoretic (CE) method with sample stacking induced by a reverse migrating pseudostationary phase (SRMP) technique has been developed and validated for sensitive determination of phenobarbital (PB) and its p-hydroxyphenobarbital (PHPB) metabolite in rat urine samples. Separation and determination were optimized on a fused-silica capillary with a total length of 50 cm (effective length 40 cm) and 75 μm ID. The microemulsion background electrolyte consisted of 0.8% (v/v) ethyl acetate, 6.6% (v/v) butan-2-ol, 1.0% (v/v) acetonitrile, 2.0% (w/v) sodium n-dodecyl sulfate (SDS), and 89.6% (v/v) of 7.5 mM ammonium formate at pH 8. When this preconcentration technique was used, the sample stacking and the separation processes took place successively with changing the voltage with an intermediate polarity switching step. For practical application, a solid-phase extraction (SPE), C(18) sorbent with n-hexane/ethyl acetate (1?:?1%, v/v) as the elution solvent was used for sample purification and concentration. The SPE method gave good extraction yields for all the analytes, with absolute recovery values of 96.9% and 99.1% for PB and PHPB, respectively. The regression equations for PB and PHPB showed excellent linearity over a concentration range of 55-1386 ng mL(-1) for PB and PHPB (r = 0.998). The developed microemulsion electrokinetic capillary chromatography (MEEKC) method for separation of the studied compounds with SRMP as the electrophoretic preconcentration technique allowed detection limits in urine samples at 16.8 ng mL(-1) for PB and PHPB which are 15-fold lower than the reported CE method in the literature. The precision results, expressed by the intra-day and inter-day relative standard deviation (RSD) values range from 3.6 to 7.1% (repeatability) and from 3.2 to 7.2% (intermediate precision) for PB and PHPB, respectively, which were in line with Food and Drug Administration (FDA) criteria.     

Centrifugal partition chromatography directly interfaced with mass spectrometry for the fast screening and fractionation of major xanthones in Garcina mangostana

Xanthones are well known for their interesting phytochemical properties, which make them attractive to the pharmaceutical and medicinal industry. We have therefore developed a method to analyse the major xanthones in Garcina mangostana. The xanthones were extracted by pressurized liquid extraction with ethanol and separated at the semi-preparative scale by centrifugal partition chromatography (CPC) with a biphasic solvent system consisting of heptane/ethyl acetate/methanol/water (2:1:2:1, v/v/v/v). A CPC-electrospray ionisation MS coupling was performed and used to simultaneously separate and identify the compounds. Thanks to a variable flow splitter and an additional stream of ethanol/1 mol L⁻¹ ammonium acetate (95:5, v/v), all the compounds were ionised, detected and monitored whatever the solvents used in mobile phase for the CPC separation. The dual mode or elution–extrusion which are less solvent-consuming and faster than the elution mode were used without loss of ionisation and detection.     

An efficient high-speed counter-current chromatography method for the preparative separation of potential antioxidants from Paeonia lactiflora Pall. combination of in vitro evaluation and molecular docking

Paeonia lactiflora Pall., one of the most famous classical herbal medicine, has been used to treat diseases for over 1200 years. In this research, the functional ingredients were purified by online-switch two-dimensional high-speed counter-current chromatography combined with inner-recycling and continuous injection mode. The antioxidant activity was evaluated by investigating the 2,2′-azobis (2-amidinopropane) dihydrochloride-induced oxidant damage in vitro and confirmed through molecular docking. n-Butanol/ethyl acetate/water (2:3:5, v/v) solvent system was used for the first-dimensional separation and optimized the sample loading. Two pure compounds and a polyphenol-enriched fraction were separated. The polyphenol-enriched fraction was separated with a solvent system n-hexane/ethyl acetate/methanol/water (2:8:4:6, v/v) with continuous injection mode. Five compounds were successfully separated, including gallic acid ( 1 ), methyl gallate ( 2 ), albiflorin ( 3 ), paeoniflorin ( 4 ), and ethyl gallate ( 5 ). Their structures were identified by mass spectrometry and NMR spectroscopy. The results from the antioxidant effect showed that albiflorin had stronger antioxidant activity. Molecular docking results indicated that the affinity energy of the identified compounds ranged from -3.79 to -8.22 kcal/mol and albiflorin showed the lowest affinity energy. Overall, all those findings suggested that the strong antioxidant capacity of albiflorin can be potentially used for the treatment of diseases caused by oxidation.     

超高效液相色谱-串联质谱法同时测定食品中4种常用香精

建立了超高效液相色谱-串联质谱法同时测定食品中香兰素、乙基香兰素、麦芽酚和乙基麦芽酚4种香精的方法。样品用水提取,固相萃取小柱净化,目标化合物采用UPLC^TMBEH C18色谱柱(50 mm×2.1 mm, 1.7 μm)分离,以甲醇和含0.002 mol/L乙酸铵及0.1%(v/v)甲酸的水溶液为流动相进行梯度洗脱,采用电喷雾离子源电离、正离子多反应监测模式质谱检测。4种香精在5~500 μg/L或10~1000 μg/L质量浓度范围内线性良好,相关系数均在0.9995~0.9998之间;回收率为75.8%~116%,相对标准偏差(RSD, n=6)为1.58%~4.01%。该方法灵敏、准确,检测范围广,分析速度快,适合食品中香兰素、乙基香兰素、麦芽酚和乙基麦芽酚4种香精的检测。     

Sample stacking and sweeping in microemulsion electrokinetic chromatography under pH-suppressed electroosmotic flow

Two on-line sample concentration techniques, sample stacking and sweeping under pH-suppressed electroosmotic flow, were evaluated in microemulsion electrokinetic chromatography. The concept of stacking with anion selective electrokinetic injection and a water plug in a reverse-migrating microemulsion (SASIW-RMME) was brought forward in this article. Six flavonoids were concentrated using a microemulsion consisting of 80 mM sodium dodecyl sulfate, 1.2% (v/v) ethyl acetate, 0.6% (v/v) 1-butanol, 10% acetonitrile (v/v) and 50 mM phosphoric acid (pH* 1.8). Significant detector response improvements were achieved. The limits of detection were in the low ng/ml level. Finally, the sample of Fructus aurantii Immaturus was analyzed using sweeping technique.     

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.     

Separation of nine compounds from Salvia plebeia R.Br. using two‐step high‐speed counter‐current chromatography with different elution modes

Nine compounds were successfully separated from Salvia plebeia R.Br. using two‐step high‐speed counter‐current chromatography with three elution modes. Elution–extrusion counter‐current chromatography was applied in the first step, while classical counter‐current chromatography and recycling counter‐current chromatography were used in the second step. Three solvent systems, n‐hexane/ethyl acetate/ethanol/water (4:6.5:3:7, v/v), methyl tert‐butyl ether/ethyl acetate/n‐butanol/methanol/water (6:4:1:2:8, v/v) and n‐hexane/ethyl acetate/methanol/water (5:5.5:5:5, v/v) were screened and optimized for the two‐step separation. The separation yielded nine compounds, including caffeic acid ( 1 ), 6‐hydroxyluteuolin‐7‐glucoside ( 2 ), 5,7,3′,4′‐tetrahydroxy‐6‐methoxyflavanone‐7‐glucoside ( 3 ), nepitrin ( 4 ), rosmarinic acid ( 5 ), homoplantaginin ( 6 ), nepetin ( 7 ), hispidulin ( 8 ), and 5,6,7,4′‐tertrahydroxyflavone ( 9 ). To the best of our knowledge, 5,7,3′,4′‐tetrahydroxy‐6‐methoxyflavanone‐7‐glucoside and 5,6,7,4′‐tertrahydroxyflavone have been separated from Salvia plebeia R.Br. for the first time. The purities and structures of these compounds were identified by high‐performance liquid chromatography, electrospray ionization mass spectrometry, ¹H and ¹³C NMR spectroscopy. This study demonstrates that high‐speed counter‐current chromatography is a useful and flexible tool for the separation of components from a complex sample.