Recent applications and chiral separation development based on stationary phases in open tubular capillary electrochromatography(2019-2022) / 药物分析学报

Capillary electrochromatography(CEC)plays a significant role in chiral separation via the double sepa-ration principle,partition coefficient difference between the two phases,and electroosmotic flow-driven separation.Given the distinct properties of the inner wall stationary phase(SP),the separation ability of each SP differs from one another.Particularly,it provides large room for promising applications of open tubular capillary electrochromatography(OT-CEC).We divided the OT-CEC SPs developed over the past four years into six types:ionic liquids,nanoparticle materials,microporous materials,biomaterials,non-nanopolymers,and others,to mainly introduce their characteristics in chiral drug separation.There also added a few classic SPs that occurred within ten years as supplements to enrich the features of each SP.Additionally,we discuss their applications in metabolomics,food,cosmetics,environment,and biology as analytes in addition to chiral drugs.OT-CEC plays an increasingly significant role in chiral separation and may promote the development of capillary electrophoresis(CE)combined with other instruments in recent years,such as CE with mass spectrometry(CE/MS)and CE with ultraviolet light detector(CE/UV).

Chiral separation methods and the applications in the study of chiral components of traditional Chinese medicines / 药学学报

Chirality is one of the fundamental properties of nature, and most of the important molecules in living organisms contain chiral structures. The efficacy and safety of drugs are often closely related to the chiral structure of compounds, however, there are relatively more studies on synthetic characterization, pharmacology, and toxicology of chiral small molecule chemical drugs, but relatively less studies on chiral compounds contained in natural drugs such as traditional Chinese medicines. Chiral separation, as the basis of chiral research, has a pivotal position in the study of chiral compounds. In this paper, we systematically describe the separation methods of chiral compounds from the classification of chiral splitting methods based on chromatographic and non-chromatographic methods, as well as chromatographic packing materials, chiral additives and chiral derivatization, and review the chiral compounds in natural drugs such as traditional Chinese medicines reported in the past ten years, in order to provide references for the splitting and evaluating the activity of chiral compounds, and the improvement of quality standards of traditional Chinese medicines.

Chiral separation of four acidic drugs by micellar electrokinetic chromatography with ionic liquid-based surfactant / 中国药科大学学报

@#In this paper, micellar electrokinetic chromatography (MEKC) with glucose-β-cyclodextrin (Glu-β-CD) as chiral selector and ionic liquid surfactant N-butyl-N-methyl pyrrolidine lauryl sulfate ([C4MP] [C12SO4]) micelles formed at low pH as a pseudo stationary phase was applied for the chiral separation of four acidic drugs naproxen,warfarin, ketoprofen and ibuprofen.Under the same conditions,significantly improved separation of tested drug enantiomers was achieved with the MEKC system based on [C4MP][C12SO4] compared with the system based on the conventional surfactant sodium dodecyl sulfate (SDS).Several primary parameters affecting enantioseparation such as type and proportion of organic modifier, concentration and pH of the running buffer, concentration of chiral selector,concentration of ionic liquid surfactant and applied voltage were systematically investigated.

Chiral discrimination of amlodipine from pharmaceutical products using capillary electrophoresis

The present work describes the development of a capillary electrophoresis (CE) method for the chiral discrimination of amlodipine (AML) enantiomers using cyclodextrine (CD) derivatives as chiral selectors. A large number of native and derivatized, neutral and ionized CD derivatives were screened to find the optimal chiral selector; and carboximethyl-β-CD (CM-β-CD) was selected for the enantiomeric discrimination. A factorial analysis study was performed by orthogonal experimental design in which several factors were varied at the same time to optimize the separation method. The optimized method (25 mM phosphate buffer, pH = 9.0, 15 mM CM-β-CD, 15 ºC, + 25 kV, 30 mbar/1 second, detection wavelength 230 nm) was successfully applied for the baseline separation of AML enantiomers within 5 minutes. Successful validation and application of the proposed CE method suggest its routine use in enantioselective control of AML in pharmaceutical preparations.

Determination of L-norvaline and L-tryptophan in dietary supplements by nano-LC using an O-2-(methacryloyloxy)-ethylcarbamoyl-10,11-dihydroquinidine-silica hybrid monolithic column / 药物分析学报

An analytical methodology based on an O-[2-(methacryloyloxy)-ethylcarbamoyl]-10,11-dihydroquinidine (MQD)-silica hybrid monolithic column was developed for the enantioseparation of 9-fluorenylmethoxycarbonyl (FMOC) derivatized amino acids by nano-liquid chromatography. The mo-bile phase was optimized including the apparent pH, content of ACN, and concentration of the buffer to obtain a satisfactory enantioresolution performance. 27 FMOC derivatized amino acids including 19 protein and 8 non-protein amino acids were tested, and 19 out of them were enantiomerically discriminated obtaining baseline separation for 11 of them. Analytical characteristics of the method were evaluated for norvaline and tryptophan in terms of linearity, precision, accuracy, limits of detection (LOD) and quantitation (LOQ) showing good performance to be applied to the enantiomeric determination of these amino acids in dietary supplements. LOD and LOQ values were 9.3 and 31μM for norvaline en-antiomers and 7.5 and 25μM for tryptophan enantiomers, respectively. The contents of D-norvaline and D-tryptophan were below their respective LODs in all the analyzed samples. Quantitation of L-tryptophan and L-norvaline showed good agreement with the labeled contents except for one sample which did not show presence of L-norvaline, contrary to the label indication.

Mass spectrometry detection of basic drugs in fast chiral analyses with vancomycin stationary phases / 药物分析学报

Current trends in chiral analysis of pharmaceutical drugs are focused on faster separations and higher separation efficiencies. Core-shell or superficially porous particles (SPP) based chiral stationary phases (CSPs) provide reduced analysis times while maintaining high column efficiencies and sensitivity. In this study, mobile phase conditions suitable for chiral analyses with electrospray ionization LC-MS were systematically investigated using vancomycin as a representative CSP. The performance of a 2.7 μm SPP based vancomycin CSP (SPP-V) 10 cm × 0.21 cm column was compared to that of a corresponding 5 μm fully porous particles based analogue column. The results demonstrated that the SPP-V column provides higher efficiencies, 2–5 time greater sensitivity and shorter analysis time for a set of 22 basic pharma-ceutical drugs. The SPP-V was successfully applied for the analysis of the degradation products of racemic citalopram whose enantiomers could be selectively identified by MS.

Chiral Separation of Manidipine Enantiomers and the Pharmacodynamics of the Hydrochlorides / 中国药学杂志

OBJECTIVE: To investigate the chiral separation of the manidipine enantiomers by the proposed synthetic route, and the blood pressure effect of the hydrochlorides. METHODS: Based on the solubility differences of the salts which are the reaction products of the manidipine enantiomers and the different chiral resolution agents[(+)-Di-p-toluoyl-D-tartaric acid/(-)-Di-p-toluoyl-L-tartaric acid] in the specific solvents, the S and R manidipine enantiomers were obtained respectively, which is called the chemical salting out crystal method. The changes of systolic pressure, diastolic pressure, and mean arterial pressure of spontaneously hypertensive rats(SHR) before and after manidipine hydrochloride medications administration were monitored through implantable physiological signal remote sensing pressure monitoring system (data science international, DSI). RESULTS: The chiral separation process is simple, maneuverability is strong and the ee values and purity of the S and R enantiomorphism isomers are both higher in the synthetic route; in the SHR model, the antihypertensive effect was equivalent between manidipine hydrochloride group and S-manidipine hydrochloride group; R-manidipine hydrochloride did not have the antihypertensive effect. CONCLUSION: The chiral resolution of manidipine enantiomers has certain scientific value, which can improve the efficacy and safety of drug, and which is of great significance to develop manidipine single enantiomeric new drugs.

Chiral Separation of Atorvastatin Calcium and Its Enantiomeric Impurity by Supercritical Fluid Chromatography / 中国药学杂志

OBJECTIVE: To develop a supercritical fluid chromatography method for the separation of atorvastatin calcium and its enantiomer, meanwhile assaying the enantiomer. METHODS: Atorvastatin calcium and its enantiomer were separated on a ACQUITY UPC2 Trefoil CEL2 column(3.0 mm×150 mm, 2.5 μm) maintained at 45 ℃ with the mobile phase containing a mixture of CO2 and methanol with 0.1% TFA(78∶22, V/V) at 1.5 mL·min-1, and the detection wavelength was set at 244 nm. The back pressure was set at 13.8 MPa. RESULTS: The enantiomer and atorvastatin calcium were separated successfully in 5 min with a resolution factor of 4.1. Good linear relationship was established between the peak response and the concentration in the range of 2.5-50 μg·mL-1 for enantiomer(r2=0.999 9, n=6), the quantitative limit(S/N=10) was 2.5 μg·mL-1, and the detection limit(S/N=3) was 1.0 μg·mL-1. The spiked recovery of the enantiomer was 100.40%(n=9). CONCLUSION: The proposed method shows high accuracy, repeatability and stability. It can be employed for the quality control and stability research of the enantiomer of atorvastatin calcium.

Enantiomeric Separation of Three Kinds of Aromatic Amino Acids by C18 Column Tandem Crown Ether Chiral Column / 分析化学

A reversed phase high performance liquid chromatographic ( RP-HPLC ) method was proposed by using C18 column tandem crown ether chiral column for simultaneous separation of three kinds of aromatic amino acid enantiomers. The chromatographic conditions, including mobile phase proportion, pH, column temperature and flow rate, were optimized. The experimental results showed that the three aromatic amino acid enantiomers could be successfully separated under the optimal conditions such as perchloric acid solution-acetonitrile ( 86:14, V/V, pH = 2 ) as mobile phase, column temperature of 20℃ and flow rate of 0. 4 mL/min. The separation under four HPLC column connection modes was further investigated. The results revealed that different kinds of amino acids could be separated on C18 column, but not for amino acid enantiomers; amino acid enantiomers could be separated on crown ether chiral column, but the chromatographic peaks of each amino acid enantiomers overlapped seriously; baseline separation of three amino acid enantiomers could be obtained under the connection of two columns, but the separation under different column connection sequence almost had no difference except the peak shape.

Preparation and application of novel polysulfone chiral membranes for the separation of tryptophan enantiomers / 中国药科大学学报

@#To preparate a novel polysulfone chiral membranes, β-cyclodextrin was functionalized with dodecanoyl chloride, and this modified β-cyclodextrin was then incorporated into polysulfone casting solution to form the dodecanoyl-β-cyclodextrin/polysulfone chiral membrane. Meanwhile, current studies have investigated the effect of adding different amount of dodecanoyl-β-cyclodextrin on the pure water flux, bovine serum albumin(BSA)rejection rate and enantioselectivity of the membranes. The morphology of the dodecanoyl-β-cyclodextrin/polysulfone chiral membrane was characterized by scanning electron microscopy(SEM). With the incorporation of dodecanoyl-β-cyclodextrin, the pore structure of the membrane changed significantly, with more finger-like pore structures appearing in the support layer. So the membrane water flux increased significantly, while the BSA rejection rate decreased. When the addition amount of dodecanoyl-β-cyclodextrin was in the range of 2% to 3. 5%, the enantiomeric excess increased with the addition of dodecanoyl-β-cyclodextrin. A complete separation of racemic tryptophan can be performed using this novel dodecanoyl-β-cyclodextrin/polysulfone chiral membrane-based separation system.

Separation of atropisomers by chiral liquid chromatography and thermodynamic analysis of separation mechanism / 药物分析学报

In the pharmaceutical industry, the analysis of atropisomers is of considerable interest from both scientific and regulatory perspectives. The compound of interest contains two stereogenicaxes due to the hindered rotation around the single bonds connecting the aryl groups, which results in fourpotential configurational isomers (atropisomers). The separation of the four atropisomers was achieved on aderivatized β-cyclodextrin bonded stationary phase. Further investigation showed that low temperatureconditions, including sample preparation (?70 °C), sample storage (?70 °C), and chromatographic separation (6 °C),were critical to preventing interconversion. LC-UV-laser polarimetric analysis identified peaks 1 and 2as a pair of enantiomers and peaks 3 and 4 as another. Thermodynamic analysis of the retention data indicatedthat the separation of the pairs of enantiomers is primarily enthalpy controlled as indicated by the positiveslope of the van't Huff plot. The difference in absolute Δ (Δ H), ranged from 2.20 kJ/mol to 2.42 kJ/mol.

Three pairs of alkaloid enantiomers from the root of Isatis indigotica

Three pairs of enantiomerically pure alkaloids with diverse structure features, named isatindigoticoic acid A and epiisatindigoticoic acid A [(-)-1 and (+)-1], phaitanthrin A and epiphaitanthrin A [(-)-2 and (+)-2], and isatindopyrromizol A and epiisatindopyrromizol A [(-)-3 and (+)-3], respectively, were isolated from an aqueous extract of the roots of Isatis indigotica. Racemic and scalemic mixtures of these enantiomers were separated by HPLC on a chiral semi-preparative column. Their structures including absolute configurations were determined by extensive spectroscopic analysis in conjunction with the calculation of electronic circular dichroism (ECD) spectra. The enantiomer pairs possess parent structures of 2-oxo-1,2,3,4-tetrahydroquinoline-4-carboxylic acid, indolo[2,1-b]quinazolinone, and 3-thioxohexahydro-1H-pyrrolo[1,2-c]imidazol-1-one, respectively. Except for phaitanthrin A [(-)-2] which the configuration was previously undetermined, these compounds are new enantiomeric natural products.

Study on Enantiomer Separation for Atropine by Capillary Electrophoresis / 中国药师

Objective:To investigate the enantiomer separation for atropine by capillary electrophoresis .Methods:Capillary elec-trophoresis was used with an elastic quartz capillary column (60 cm ×75 mm, effective length of 40 cm).The concentration of phos-phate buffer was 30 mmol· L-1 .The high and time of injection was 10 cm and 5 s, respectively.The detection wavelength was 225 nm.The best separation conditions were selected including the type and concentration of chiral resolving agent , pH of the buffer solu-tion, operating voltage and organic solvent.Results:The optimum conditions of separation were as follows:the pH of buffer solution was 7.0, the concentration of S-β-CDP was 10 mg· ml-1 , and the operating voltage was 12 kV.Conclusion: The method is simple and fast, which can be used to se parate the optical isomers of atrpo ine.

Determination of Stereoisomers in Landiolol Hydrochloride by Ultra Performance Convergence Chromatography / 分析化学

A new method for chiral separation and purity inspection of landiolol hydrochloride and its stereoisomers was developed by ultra-performance convergence chromatography ( UPC2 ) . The mobile phase was the mixture of supercritical CO2 and methanol/n-butyl alcohol/acetonitrile (1:1:1, V/V) plus 0. 5%NH3?H2O. The separation was carried out on the Daicel CHIRALPAK? IF column (150 mm × 4. 6 mm, 3 μm) with a flow rate of 2. 8 mL/min at 50℃ using 223 nm as detection wavelength. Under the optimized experimental conditions, for R,R-stereoisomer, R,S-stereoisomer and S,R-stereoisomer, the detection limits (LOD, S/N=3) were 0. 3, 0. 4 and 0. 3 mg/L, the linear ranges were 2-300 mg/L, 5-300 mg/L and 2-300 mg/L, the recoveries of spike samples were 103. 4%±2. 5%, 91. 8%±2. 5% and 101. 7%±1. 5%, and the injection repeatabilities were 0. 06%, 0. 09% and 0. 08% (n=6), respectively. The experimental results demonstrate that the UPC2-based method can be used for the analysis and determination of landiolol hydrochloride and its stereoisomers.

Preparation of bovine serum albumin immobilized chiral monolithic column and its protein bonding quantity / 中国药科大学学报

@#Determination of exact total protein bonding quantity is often a key step in the preparation of protein-immobilized chiral monolith. In this study, we developed and evaluated a bovine serum albumin(BSA)modified monolith based on glycidyl methacrylate(GMA)and ethylene dimethacrylate(EDMA)for chiral separation. The epoxy groups of the polymer were used directly for the covalent bonding of BSA. A Coomassie brilliant blue(CBB)protein assay(Bradford method)was established to determine the protein bonding quantity, and the influence of some key aspects such as ionic strength, pH value and reaction time were studied. The method was validated with respect to linearity, precision, accuracy and robustness. The maximum amount of immobilized BSA was 11. 90 mg/g, obtained using 65 ∶35 cyclohexanol/dodecanol as the porogen. The monolith was successfully applied in the chiral separation of R/S-warfarin and D/L-tryptophan in only 1-20 min. Furthermore, the chromatographic conditions like pH and organic additive of the mobile phase were optimized. The chiral separation performance of this BSA-immobilized monolith is positively correlated to the protein bonding quantity.

Chiral separation of fourteen amino alcohols by nonaqueous capillary electrophoresis / 药学学报

We developed a new method for chiral separation of fourteen amino alcohols by nonaqueous capillary electrophoresis (NACE) with the D-(+)-gluconic acid δ-lactone-boric acid complex as chiral selector. In order to achieve good enantioseparation, the effects of D-(+)-gluconic acid δ-lactone and boric acid concentrations, triethylamine concentration, as well as capillary temperature were systematically investigated. The optimized conditions were identified as follows:an uncoated fused silica capillary of 50 μm ID with a total length (Ltot) of 55 cm and an effective length (Leff) of 45 cm; 200 mmol·L-1 D-(+)-gluconic acid δ-lactone, 80 mmol·L-1 boric acid, and 57.4 mmol·L-1 triethylamine in methanol; positive pressure injection at 2.9 psi for 2 s; capillary temperature, 25 ±0.2℃; applied voltage, +15 kV; detection wavelength, 214 nm. Under the optimized conditions, a good chiral resolution was achieved in most of the tested drugs. This method provides a foundation for the development and application of new chiral selectors of polyhydroxy compound-boric acid complexes in chiral drugs analysis by NACE.

Chiral separation and purity test of levofloxacin-N-oxide / 中国药学杂志

OBJECTIVE: To establish a high performance liquid chromatographic method for chiral separation of levofloxacin-N-oxide enantiomers and purity test of the bulk drug. METHODS: The analysis was carried out on a C18 column with D-phenylalanine-copper sulfate solution-methanol (80:20) as mobile phase. The flow rate was 1.0 mL · min-1, the UV detection wavelength was set at 325 nm, and the column temperature was maintained at 40℃. RESULTS: A baseline separation of levofloxacin-N-oxide enantiomers was achieved and the resolution was 3.2 between the enantiomers under the above chromatographic conditions. CONCLUSION: This method is simple, rapid, and reproducible, and it can be used for the separation and purity test of levofloxacin-N-oxide.

Chiral separation of ezetimibe and R-enantiomer by supercritical fluid chromatography / 中国药学杂志

OBJECTIVE: To develop an isocratic supercritical fluid chromatography method for the separation of ezetimibe and its R-enantiomer. METHODS: Ezetimibe and its R-enantiomer were separated on a Chiralcel OD column (4.6 mm × 250 mm, 10 μm) maitained at 35℃ with the mobile phase containing a mixture of CO2 and methanol with 0.1% TFA-0.1% TEA (90:10, V/V) at 3.0 mL · min-1, and the detection wavelength was set at 235 nm. The back pressure of SF-CO2 was set at 15 MPa. RESULTS: The R-enantiomer and ezetimibe were separated successfully in 15 min with a resolution factor of 1.6. Good linear relationships were established between the peak response and the concentration in the range of 0.010-0.100 mg · mL-1 for each analyte(r =0.999 9 and 0.999 9, respectively, n =7), the quantitation limits (S/N = 10) were 34 and 32 ng, and the detection limits (S/N =3) were 10 and 9 ng, respectively. The spiked recovery of R-enantiomer of ezetimibe was 98.4% (n =9). CONCLUSION: The proposed method shows high accuracy, repeatability and stability. It could be employed for the quality control and stability research of R-enantiomer of ezetimibe and its tablet formulation.

Chiral Separation and Identification of D,L-Histidine Based on Nanochannels Membrane Coupling with Surface Enhanced Raman Scattering Spectroscopy / 分析化学

Gold nanochannels were prepared using Al2 O3 nanotubules membrane as the carrier and modified with chitosan by a classical N-(3-dimethylaminopropyl)-N-ethyl carbodiimide ( EDC)/N-hydroxysuccinimide ( NHS ) coupling reaction. The nanochannels were characterized by field emission scanning electron microscopy ( FESEM) , cyclic voltammetry and AC impedance method. The Au nanochannels modified with chitosan showed a chiral environment and can be used to separate histidine enantiomer. The effects of pore size and solution pH on the separation efficiency of histidine were investigated. To increase the detection sensitivity of D-, L-histidine, Ag nanoparticles were used to enhance the surface enhanced Raman scattering ( SERS) activity. The results showed that the chitosan-modified gold nanochannels can be used to separate chiral histidine based on this unique selective nanochannel membrane. L-Histidine and D-histidine were respectively detected by SERS at wavelengths of 1000 and 1590 cm-1 . The results showed that L-histidine and D-histidine were separated well in the mixture containing 200 μL of histidine, 100 μL of colloidal Ag and 100 μL of 80 mmol/L NaCl ( pH=7 . 59 ) with a separation efficiency of 4 . 91 .

Enantioseparation and Determination of Propranolol in Human Plasma on a New Derivatized β-Cyclodextrin Bonded Phase by HPLC / 分析化学

A 6-azido-β-cyclodextrin was synthesized and derivatized with p-nitrophenyl isocyanate as chiral ligand. Following that the ligand was chemically bonded to mesoporous SBA-15 via a ‘Click Chemistry ’ reaction of the azido group with alkynyl group. A new p-nitrophenylcarbamoylatedβ-cyclodextrin bonded SBA-15 chiral stationary phase ( NPCSP ) for HPLC was obtained. The new stationary phase was first used to enantioseparate propranolol in human plasma under the polar organic solvent mode. The effects of methanol content , additive concentration of glacial acetic acid/triethylamine in mobile phase and the temperature on the enantioseparation were studied. The optimal chromatographic conditions were as follows: mobile phase was acetonitrile/methanol/glacial acetic acid/triethylamine (90:10:1. 25:2. 25, V/V), temperature 288 K, flow rate of 0. 5 mL/min, injection volume of 20 μL, detection wavelength at 290 nm. The resolution was 2. 04 with a short run time (< 15 min) under the above conditions. The composition of propranolol in plasma was quantitatively measured by HPLC-MS selected ion monitoring mode ( [ M +H ]+ m/z 260 . 10 ) with hydrochlorothiazide as internal standard. And linear range was 2. 5-250 μg/L and with a good linear relationship. The detection limit was 1 μg/L according to S/N=3. The experimental results showed that the chiral stationary phase exhibited excellent chiral separation ability to propranolol and the analysis method for propranolol in plasma was sensitive, accurate, simple and fast, which could be used for the determination of propranolol in plasma and pharmacokinetic studies.