Dynamic Behavior of Clobazam on High‐Performance Liquid Chromatography Chiral Stationary Phases

Clobazam, a 1,5‐benzodiazepin‐2,4‐dione, is a chiral molecule because its ground state conformation features a nonplanar seven‐membered ring lacking reflection symmetry elements. The two conformational enantiomers of clobazam interconvert at room temperature by a simple ring‐flipping process. Variable temperature HPLC on the Pirkle type (R)‐N‐(3,5‐dinitronenzoyl)phenylglycine and (R,R)‐Whelk‐O1 chiral stationary phases (CSPs) allowed us to separate for the first time the conformational enantiomers of clobazam and to observe peak coalescence‐decoalescence phenomena due to concomitant separation and interconversion processes occurring on the same time scale. Clobazam showed temperature dependent dynamic high‐performance liquid chromatography (HPLC) profiles with interconversion plateaus on the two CSPs indicative of on‐column enantiomer interconversion. (enantiomerization) in the column temperature range between T_col = 10°C and T_col = 30°C, whereas on‐column interconversion was absent at temperature close to or lower than T_col = 5°C. Computer simulation of exchange‐deformed HPLC profiles using a program based on the stochastic model yielded the apparent rate constants for the on‐column enantiomerization and the corresponding free energy activation barriers. At T_col = 20°C the averaged enantiomerization barriers, ΔG^?, for clobazam were found in the range 21.08–21.53 kcal mol^?1 on the two CSPs. The experimental dynamic chromatograms and the corresponding interconversion barriers reported in this article are consistent with the literature data measured by DNMR at higher temperatures and in different solvents. Chirality 28:17–21, 2016. © 2015 Wiley Periodicals, Inc.     

Preparation of a New Chiral Stationary Phase Based on Macrocyclic Amide Chiral Selector for the Liquid Chromatographic Chiral Separations

A new chiral stationary phase (CSP) based on macrocyclic amide receptor was prepared starting from (1R,2R)‐1,2‐diphenylethylenediamine. The new CSP was successfully applied to the resolution of various N‐(substituted benzoyl)‐α‐amino amides with reasonably good separation factors and resolutions (α = 1.75 ~ 2.97 and R_S = 2.89 ~ 6.82 for 16 analytes). The new CSP was also applied to the resolution of 3‐substituted 1,4‐benzodiazepin‐2‐ones and some diuretic chiral drugs including bendroflumethiazide and methylchlothiazide and metolazone. The resolution results for 3‐substituted 1,4‐benzodiazepin‐2‐ones and some diuretic chiral drugs were also reasonably good. Chirality 28:253–258, 2016. © 2016 Wiley Periodicals, Inc.     

Separation of Ofloxacin and Its Six Related Substances Enantiomers by Chiral Ligand‐Exchange Chromatography

A chiral ligand‐exchange high‐performance liquid chromatography method was developed for the enantioseparation of ofloxacin and its six related substances termed impurities A, B, C, D, E, and F. The separation was performed on a conventional C₁₈ column. Different organic modifiers, copper salts, amino acids, the ratio of Cu²⁺ to amino acid, pH of aqueous phase, and column temperature were optimized. The optimal mobile phase conditions were methanol‐water systems consisting of 5 mmol/L copper sulfate and 10 mmol/L L‐isoleucine (L‐Ile). Under such conditions, good enantioseparation of ofloxacin and impurities A, C, E, and F could be observed with resolutions (R_S) of 3.54, 1.97, 3.21, 3.50, and 2.12, respectively. On the relationship between the thermodynamic parameters and structures of analytes, the mechanism of chiral recognition was investigated. It was concluded that ofloxacin and impurities A, C, E, and F were all enthalpically driven enantioseparation and that low column temperature was beneficial to enantioseparation. Furthermore, the structure–separation relationship of these analytes is also discussed. Chirality 27:843–849, 2015. © 2015 Wiley Periodicals, Inc.     

Determination of Tanshinones in Danshen (Salvia miltiorrhiza) by High‐Performance Liquid Chromatography with Fluorescence Detection after pre‐Column Derivatisation

Introduction

Tanshinones are a major class of bioactive ingredients in the traditional herbal medicines, Danshen (Salvia miltiorrhiza). A sensitive and reliable determination method for tanshinones is useful to ensure the quality of Danshen.

Objective

To develop a sensitive and selective analytical method for tanshinones by high‐performance liquid chromatography (HPLC) with fluorescence detection after pre‐column derivatisation.

Methodology

The proposed method depends on derivatisation reaction of tanshinones with 4‐carbomethoxybenzaldehyde and ammonium acetate forming intensely fluorescent imidazole derivative.

Results

The proposed method provided excellent sensitivity with the detection limits of 3.3 nM (66 fmol/injection), 3.2 nM (64 fmol/injection) and 2.0 nM (40 fmol/injection) for cryptotanshinone, tanshinone I and tanshinone IIA, respectively, without the necessity of complicated instrumentations. The developed method is successfully applied to quantify the contents of tanshinones in Danshen.

Conclusion

The developed method is the first analytical method for tanshinones by fluorescence detection. Since the derivatisation reaction is selective for the o‐quinone structure of tanshinone, the developed method will become a suitable mean for the discovering of tanshinone type diterpenoids from herbal samples. Copyright © 2017 John Wiley & Sons, Ltd.     

Separation of Glycopeptides by High Performance Liquid Chromatography

A method is presented for separation of tryptic glycopeptides-containing oligosaccharides of the N-asparagine-linked type. High performance liquid Chromatography (HPLC) of glycopeptides on a C18 reverse-phase system eluted with a gradient of 0%–50% acetonitrile in 0.1 M NaPO₄ pH 2.2 resolves the two major glycosylation sites from the envelope glycoprotein (G) of vesicular stomatitis virus. Glycopeptides containing N-linked oligosaccharides of the complex type coelute with those containing N-linked oligosaccharides of the neutral, high mannose type, indicating that separation is based upon peptide rather than carbohydrate composition. The contribution of the carbohydrate component to glycopeptide elution, as determined by cleavage of the high mannose oligosaccharides with endo-β-Nacetylglucosaminidase H, is that of a significant, but minor, decrease in peptide retention time. Comparison of the tryptic glycopeptide profiles of G isolated from both wild type and mutant strains of VSV illustrates the rapid, reproducible, and quantitative nature of the technique. Through HPLC analysis of appropriately treated glycopeptides, it is possible to explore both the nature and extent of glycosylation at individual sites in glycoproteins in a single step.     

Direct Enantioseparation of Nitrogen‐Heterocyclic Pesticides on Cellulose‐Based Chiral Column by High‐Performance Liquid Chromatography

The enantiomeric separation of eight pesticides including bitertanol ( 1 ), diclobutrazol ( 2 ), fenbuconazole ( 3 ), triticonazole ( 4 ), imazalil ( 5 ), triapenthenol ( 6 ), ancymidol ( 7 ), and carfentrazone‐ethyl ( 8 ) was achieved, using normal‐phase high‐performance liquid chromatography on two cellulosed‐based chiral columns. The effects of isopropanol composition from 2% to 30% in the mobile phase and column temperature from 5 to 40 °C were investigated. Satisfactory resolutions were obtained for bitertanol ( 1 ), triticonazole ( 4 ), imazalil ( 5 ) with the (+)‐enantiomer eluted first and fenbuconazole ( 3 ) with the (—)‐enantiomer eluted first on Lux Cellulose‐2 and Lux Cellulose‐3. (+)‐Enantiomers of diclobutrazol ( 2 ) and triapenthenol ( 6 ) were first eluted on Lux Cellulose‐2. (—)‐Carfentrazone‐ethyl ( 8 ) were eluted first on Lux Cellulose‐2 and Lux Cellulose‐3 with incomplete separation. Reversed elution orders were obtained for ancymidol (7). (+)‐Ancymidol was first eluted on Lux Cellulose‐2 while on Lux Cellulose‐3 (—)‐ancymidol was first eluted. The results of the elution order at different column temperatures suggested that column temperature did not affect the optical signals of the enantiomers. These results will be helpful to prepare and analyze individual enantiomers of chiral pesticides. Chirality 27:32–38, 2015. © 2014 Wiley Periodicals, Inc.     

Superficially Porous Particle Based Hydroxypropyl‐β‐cyclodextrin Stationary Phase for High‐Efficiency Enantiomeric Separations

A superficially porous particle (SPP)‐based hydroxypropyl‐β‐cyclodextrin (HPBCD) chiral stationary phase (CSP) was produced and its chromatographic performance was compared to both 5 µm and 3 µm fully porous particle (FPP)‐based CSPs. The relative surface coverage of the HPBCD chiral selector on each particle was approximately equal, which resulted in equivalent enantiomeric selectivity (α) values on each phase when constant mobile phase conditions were used. Under such conditions, the SPP column resulted in greatly reduced analysis times and three times greater efficiencies compared to the FPP columns. When higher flow rates were used, efficiency gains per analysis times were five times greater for the SPP column compared to the FPP‐based columns. When the mobile phases were altered to give similar analysis times on each column, resolution values were doubled for the SPP column. Finally, the novel SPP based HPBCD column proved to be stable for 500 injections under high flow rate (4.5 mL/min) and high pressure (400 bar) conditions used for an ultrafast (~45 sec) enantiomeric separation. Chirality 27:788–794, 2015. © 2015 Wiley Periodicals, Inc.     

高效液相色谱法测定超滤后血浆中的谷氨酰胺

用高效液相色谱法测定超滤后血浆中的谷氨酰胺, 平均回收率为96.75%, 在130～1300μmol/L范围内呈线性关系(r=0.9906), 健康人正常值男性为(694.97±102.31)μmol/L, 女性为(623.79±99.27)μmol/L, 男女间值有显著差别(P＜0.05)该分析方法简单、迅速, 可用于外科肠道内营养的监测和对肠粘膜结构与功能的研究.     

Stereoselective Determination of a Novel Chiral Insecticide,Sulfoxaflor, in Brown Rice,Cucumber and Apple by Normal‐Phase High‐Performance Liquid Chromatography

An effective high‐performance liquid chromatography method was developed for the stereoselective determination of a new sulfoximines insecticide, sulfoxaflor, in brown rice, cucumber and apple. Target compounds were extracted with acetonitrile and an aliquot cleaned with Cleanert PestiCarb/PSA (primary and secondary amine) cartridge. Five polysaccharide‐based columns were investigated on the separation of sulfoxaflor stereoisomers and the best was achieved on a ChromegaChiral CCA column with n‐hexane/ethanol/methanol (90:2:8, v/v/v) as mobile phase by UV detection at 220 nm at 20ºC. The resolutions of the four stereoisomers were 1.85, 1.54 and 3.08, and the elution order was identified by optical rotation and stereoisomers ratio. The mean recoveries of sulfoxaflor stereoisomers ranged from 77.1% to 99.3%, with relative standard deviations less than 8.9% at three concentration levels in all matrices. The limits of detection for all stereoisomers varied from 0.05 mg/kg to 0.07 mg/kg, while the limit of quantification did not exceed 0.22 mg/kg. The method was then successfully applied to determine the sulfoxaflor stereoisomers in authentic samples, confirming that it is convenient and reliable for stereoselective determination of sulfoxaflor stereoisomers in food. Chirality 26:114–120, 2014. © 2014 Wiley Periodicals, Inc.     

Synthesis and Evaluation of Two Coumarin‐Type Derivatization Reagents for Fluorescence Detection of Chiral Amines and Chiral Carboxylic Acids

The synthesis of two fluorescent coumarin‐type chiral derivatization agents ( 4 and 11 ) is reported. A chiral side chain was introduced at position 7 of the coumarin via Mitsunobu reaction. The two coumarins bear in this side chain either a free amino group or a carboxyl group, making them useful for further transformations. Conjugates of chiral prototype drugs with 4 or 11 were prepared by amide coupling of the analyte's carboxyl group to the reagent's amine group, or vice versa. The separation of seven diastereomeric conjugates through achiral high‐performance liquid chromatography (HPLC) on a common C18 column is demonstrated. Chirality 25:957–964, 2013. © 2013 Wiley Periodicals, Inc.     

High‐Performance Liquid Chromatographic Enantioseparation of Unusual Isoxazoline‐Fused 2‐Aminocyclopentanecarboxylic Acids on (+)‐(18‐Crown‐6)‐2,3,11,12‐Tetracarboxylic Acid‐Based Chiral Stationary Phases

The enantiomers of four unusual isoxazoline‐fused 2‐aminocyclopentanecarboxylic acids were directly separated on chiral stationary phases containing (+)‐(18‐crown‐6)‐2,3,11,12‐tetracarboxylic acid as chiral selector. The nature of the alcoholic modifier (MeOH, EtOH, IPA) exerted a great effect on the retention, whereas the selectivity and resolution did not change substantially. Two types of dependence of retention on alcohol content were detected: k₁ increased continuously with increasing alcohol content or a U‐shaped retention curve was observed. A comparison of the chromatographic data obtained with HCOOH, AcOH, TFA, HClO₄, H₂SO₄, or H₃PO₄ as acidic modifier at a constant concentration demonstrated that in most cases, larger k values were obtained on the application of AcOH or HCOOH, and an increase of the acid content resulted in a decrease of retention. Some mechanistic aspects of the chiral recognition process are discussed with respect to the structures of the analytes and selector. The sequence of elution of the enantiomers was determined in all cases. Chirality 24:817‐824, 2012. © 2012 Wiley Periodicals, Inc.     

Nanocellulose 3, 5‐Dimethylphenylcarbamate Derivative Coated Chiral Stationary Phase: Preparation and Enantioseparation Performance

Nanocrystalline cellulose (NCC) with high surface area and high ordered crystalline structure was prepared from microcrystalline cellulose (MCC) under the hydrolysis of sodium hypochlorite. NCC was further reacted with 3,5‐dimethylphenyl isocyanate to obtain the nanocellulose derivative, and then coated successfully on the surface of silica gel to a prepared NCC‐coated chiral stationary phase (CSP) as a new kind of chiral separation material. Similarly, MCC derivative‐coated CSP was also prepared as contrast. The chiral separation performance of NCC‐based CSP was evaluated and compared with MCC‐based CSP by high‐performance liquid chromatography. Moreover, the effects of the alcohol modifiers, mobile phase additives, and flow rates on chiral separations were investigated in detail. The results showed that 10 chiral compounds were separated on NCC‐based CSP with better peak shape and higher column efficiency than MCC‐based CSP, which confirmed that NCC‐based CSP was a promising packing material for the resolution of chiral compounds.Chirality 28:376–381, 2016. © 2016 Wiley Periodicals, Inc.     

Separation of Tryptophan Enantiomers by Ligand‐Exchange Chromatography With Novel Chiral Ionic Liquids Ligand

Chiral ionic liquids (CILs) with amino acids as cations have been applied as novel chiral ligands coordinated with Cu²⁺ to separate tryptophan enantiomers in ligand exchange chromatography. Four kinds of amino acid ionic liquids, including [L‐Pro][CF₃COO], [L‐Pro][NO₃], [L‐Pro]₂[SO₄], and [L‐Phe][CF₃COO] were successfully synthesized and used for separation of tryptophan enantiomers. To optimize the separation conditions, [L‐Pro][CF₃COO] was selected as the model ligand. Some factors influencing the efficiency of chiral separation, such as copper ion concentration, CILs concentration, methanol ratio (methanol/H₂O, v/v), and pH, were investigated. The obtained optimal separation conditions were as follows: 8.0 mmol/L Cu(OAc)₂, 4.0 mmol/L [L‐Pro][CF₃COO] ,and 20% (v/v) methanol at pH 3.6. Under the optimum conditions, acceptable enantioseparation of tryptophan enantiomers could be observed with a resolution of 1.89. The results demonstrate the good applicability of CILs with amino acids as cations for chiral separation. Furthermore, a comparative study was also conducted for exploring the mechanism of the CILs as new ligands in ligand exchange chromatography. Chirality 26:160–165, 2014. © 2014 Wiley Periodicals, Inc.     

Separation of Nadolol Stereoisomers by Chiral Liquid Chromatography at Analytical and Preparative Scales

The separation of the four nadolol stereoisomers on Chiralpak® AD by chiral liquid chromatography was carried out at both analytical and preparative scales. A screening of possible mobile‐phase compositions was performed using different alcohol–hydrocarbon mixtures. The results obtained confirm the use of 20:80:0.3 ethanol‐hexane‐diethylamine reported by McCarthy (1994) but introduce other possibilities for the complete resolution of the four nadolol stereoisomers at analytical scale, namely, the mixtures 30–40:70–60:0.3 ethanol‐heptane‐diethylamine. Additionally, this work describes how retention and resolution depend on the ethanol content in hexane and heptane mixtures. The separation of nadolol stereoisomers is also carried out at preparative scale and different alcohol–hydrocarbon compositions are proposed, depending on the target component to be obtained. Particularly, this work presents the experimental separation of the more retained nadolol stereoisomer (RSR‐nadolol) by simulated moving bed (SMB) chromatography using an 80:20:0.3 ethanol‐heptane‐diethylamine mobile phase. For a 2 g/l feed concentration, RSR‐nadolol is 100% recovered at the extract outlet stream, 100% pure, and with a system productivity of 0.65 g_{RSR‐nadolol}/(l_bed^.h) and a solvent consumption of 9.6 l_solvent/g_{RSR‐nadolol}. Chirality 25:197–205, 2013. © 2013 Wiley Periodicals, Inc.     

Liquid Chromatographic Resolution of Mexiletine and Its Analogs on Crown Ether‐Based Chiral Stationary Phases

Mexiletine, an effective class IB antiarrhythmic agent, and its analogs were resolved on three different crown ether‐based chiral stationary phases (CSPs), one (CSP 1 ) of which is based on (+)‐(18‐crown‐6)‐2,3,11,12‐tetracarboxylic acid and the other two (CSP 2 and CSP 3 ) are based on (3,3’‐diphenyl‐1,1’‐binaphthyl)‐20‐crown‐6. Mexiletine was resolved with a resolution (R_S) of greater than 1.00 on CSP 1 and CSP 3 containing residual silanol group‐protecting n‐octyl groups on the silica surface, but with a resolution (R_S) of less than 1.00 on CSP 2 . The chromatographic behaviors for the resolution of mexiletine analogs containing a substituted phenyl group at the chiral center on the three CSPs were quite dependent on the phenoxy group of analytes. Namely, mexiletine analogs containing 2,6‐dimethylphenoxy, 3,4‐dimethylphenoxy, 3‐methylphenoxy, 4‐methylphenoxy, and a simple phenoxy group were resolved very well on the three CSPs even though the chiral recognition efficiencies vary with the CSPs. However, mexiletine analogs containing 2‐methylphenoxy group were not resolved at all or only slightly resolved. Among the three CSPs, CSP 3 was found to show the highest chiral recognition efficiencies for the resolution of mexiletine and its analogs, especially in terms of resolution (R_S). Chirality 26:272–278, 2014. © 2014 Wiley Periodicals, Inc.     

Access to Enantiomerically Pure cis‐ and trans‐β‐Phenylproline by High‐Performance Liquid Chromatography Resolution

The preparation of all four stereoisomers of the proline analog that bears a phenyl group attached to the β carbon either cis or trans to the carboxylic acid (cis‐ and trans‐β‐phenylproline, respectively) has been addressed. The methodology developed allows access to multigram quantities of the target amino acids in enantiomerically pure form and suitably protected for use in peptide synthesis. Racemic precursors of cis‐β‐phenylproline and trans‐β‐phenylproline were prepared from easily available starting materials and subjected to high‐performance liquid chromatography enantioseparation. Semipreparative columns (250 × 20 mm) containing chiral stationary phases based on amylose (Chiralpak IA) (Daicel‐Chiral Technologies Europe, Illkirch, France) or cellulose (Chiralpak IC) were used respectively for the resolution of the cis‐ and trans‐β‐phenylproline precursors. Chirality, 24:1082‐1091, 2012. © 2012 Wiley Periodicals, Inc.     

Liquid Chromatographic Resolution of Amino Acid Esters of Acyclovir Including Racemic Valacyclovir on Crown Ether‐Based Chiral Stationary Phases

Valacyclovir, a potential prodrug for the treatment of patients with herpes simplex and herpes zoster, and its analogs were resolved on two chiral stationary phases (CSPs) based on (3,3’‐diphenyl‐1,1’‐binaphthyl)‐20‐crown‐6 covalently bonded to silica gel. In order to find out an appropriate mobile phase condition, various mobile phases consisting of various organic modifiers in water containing various acidic modifiers were applied to the resolution of valacyclovir and its analogs. When 30% acetonitrile in water containing any of 0.05 M, 0.10 M, or 0.15 M perchloric acid was used as a mobile phase, valacyclovir and its analogs were resolved quite well on the two CSPs with the separation factors (α) in the range of 2.49 ~ 6.35 and resolutions (R_S) in the range of 2.95 ~ 12.21. Between the two CSPs, the CSP containing residual silanol protecting n‐octyl groups on the silica surface was found to be better than the CSP containing residual silanol groups. Chirality 27:268–273, 2015. © 2015 Wiley Periodicals, Inc.     

反相高效液相色谱法分析聚乙二醇修饰的水蛭素

目的：采用反相高效液相色谱法（RP-HPLC）对聚乙二醇（PEG）修饰的水蛭素进行分析分离,用以分析修饰产物中不同修饰度水蛭素的组成和比例。方法：色谱柱为Hypersil C18,5μm,4.6mm×150mm;流动相A为H2O＋0.01％的三氟乙酸,流动相B为乙腈＋0.01％的三氟乙酸。40min内由10％-50％流动相B进行梯度洗脱,洗脱流速1mL／min,上样量50μL,检测波长为214nm。结果：在单甲基化PEG-丙酸琥珀酰亚胺和水蛭素摩尔比不同的的反应产物中,PEG1-水蛭素、PEG2-水蛭素均可以达到基线分离,且不同批次的反应产物进行RP-HPLC的重复性良好。结论：RP-HPLC可以有效地对PEG修饰的水蛭素产物进行分析分离,为PEG化水蛭素的长效、缓释剂型的开发提供技术支持。