Chiral separation of the β2‐sympathomimetic fenoterol by HPLC and capillary zone electrophoresis for pharmacokinetic studies

The development of methods for the separation of the enantiomers of fenoterol by chiral HPLC and capillary zone electrophoresis (CZE) is described. For the HPLC separation precolumn fluorescence derivatization with naphthyl isocyanate was applied. The resulting urea derivatives were resolved on a cellulose tris(3,5‐dimethylphenylcarbamate)‐coated silica gel column employing a column switching procedure. Detection was carried out fluorimetrically with a detection limit in the low ng/mL range. The method was adapted to the determination of fenoterol enantiomers in rat heart perfusates using liquid–liquid extraction. As an alternative a CE method was used for the direct separation of fenoterol enantiomers comparing different cyclodextrin derivatives as chiral selectors. Copyright © 2010 John Wiley & Sons, Ltd.     

Separation of enantiomers of deprenyl with various CDs in CE and the effect of enantiomer migration order on enantiomeric impurity determination of selegiline in active ingredients and tablets

Opposite affinity pattern of enantiomers of the antiparkinsonian chiral drug deprenyl (DEP) was observed towards various neutral and charged derivatives of -CD. The effect of the enantiomer migration order on the LOD of enantiomeric impurity of R-DEP (selegiline) was studied for the standard substances and in the tablets from three different suppliers. The influence of injection mode on the LOD of a minor enantiomeric impurity was also studied and the CE method was compared with the pharmacopoeial HPLC method using a commercially available chiral column Chiralcel OD-H. The optimized CE method was more suitable for low-level enantiomeric impurity determination in selegiline compared to the pharmacopoeial HPLC method.     

Detection and Separation of Free Amino Acid Enantiomers by Capillary Electrophoresis with a Chiral Crown Ether and Indirect Photometric Detection

18-Crown-6 tetracarboxylic acid (18C6H₄) has been successfully used as a chiral selector for capillary electrophoretic (CE), high-performance liquid chromatographic (HPLC), and gas chromatographic (GC) separation of the enantiomers of DL-amino compounds. We have previously used X-ray crystallographic analysis and HPLC with an immobilized 18C6H₄ chiral stationary phase to study chiral recognition by 18C6H₄ of several DL amino acids (DL-AA). In this study CE was used for chiral recognition of several DL-AA in electrolyte solution containing 18C6H₄, in which the analyte (D or L amino acid) interacts freely. Among 14 DL-AA investigated, the enantiomers of nine (Glu, Ile, Met, PheG, Phe, Ser, Tyr, Val, and Thr) were successfully recognized in 4-15 mM 18C6H₄. Indirect photometric detection with a cationic dye, chrysoidine, was used to monitor non-chromophoric DL-AA. Among nine successfully recognized DL-AA, the D forms of Ser, Thr and Met migrated faster than the corresponding L forms. The strengths of interactions predicted from the order of migration of each enantiomer in CE were different from those in HPLC analysis. The different enantiomer recognition probably can be ascribed to the difference between CE in which the selector is not immobilized and HPLC in which the selector is immobilized by means of a spacer.     

Enantiomer separation of N-protected amino acids by non-aqueous capillary electrophoresis and high-performance liquid chromatography with tert.-butyl carbamoylated quinine in either the background electrolyte or the stationary phase

A non-aqueous CE method was developed for evaluating the chiral discrimination potential of cinchona alkaloids and different kinds of carbamoylated derivatives of quinine and quinidine type chiral selectors towards acidic analytes, in particular a series of various Bz (benzoyl), DNB (3,5-dinitrobenzoyl) and DNZ (3,5-dinitrobenzyloxycarbonyl) amino acid derivatives. In this study, the enantioselectivity values obtained in non-aqueous CE with tert.-butyl carbamoylated quinine as chiral additive have been compared with the values found for the same series of selectands in HPLC using the same selector immobilized onto silica as chiral stationary phase. Similarly to the background electrolyte used in CE an ethanol-methanol mixture (60:40, v/v) containing 100 mM octanoic acid and 12.5 mM ammonia has been selected as HPLC mobile phase. Under these conditions, a good correlation (r = 0.954) between the enantioselectivities observed with the two techniques has been obtained. Thus the non-aqueous CE method can be applied as a screening tool for the rapid evaluation of the chiral discrimination potential of a large set of newly developed chiral selectors derived from quinine and related alkaloids.     

Enatiomeric analysis of simendan by CE with beta-CD as chiral selector compared with CMPA-HPLC

The chiral separation of simendan enantiomers using capillary electrophoresis was studied with beta-cyclodextrin (beta-CD) as chiral selector. The influences of the concentration and pH of borate buffer solution, beta-CD concentration and methanol content in the background electrolyte were investigated. These factors were compared with those in an HPLC with beta-CD as chiral mobile phase additive (CMPA-HPLC). The quantification properties of the developed CE method were examined. A baseline separation of simendan enantiomers was achieved in the background electrolyte of 20 mmol/L borate buffer (pH 11.0) containing 12 mmol/L beta-CD-methanol (50:50 in volume ratio). The CE method is comparable with CMPA-HPLC in chiral resolution, although the optimal pH in CE (11.0) is much higher than that (6.0) in CMPA-HPLC. This chiral CE method is applicable to the quantitative ananlysis and enantiomeric excess value determination of L-simendan.     

High-performance liquid chromatographic and capillary electrophoretic methods for the quantitation of the (S)-terbutaline in (R)-terbutaline

Summary The resolution of chiral drugs, metabolites and related substances continues to be an important area in pharmaceutical analysis. Two methods for the optical purity testing of (R)-(-)-terbutaline were developed, namely capillary electrophoresis using hydroxypropyl-β-cyclodextrin and high-performance liquid chromatography using a chiral stationary phase. Validation data such as linearity, recovery, detection limit, and precision of the two methods are presented. The detection limit of (S)-terbutaline in (R)-terbutaline was 0.05% by the HPLC method and 0.03% by the CE method. The was generally good agreement between the HPLC and CE results. These methods were found to be applicable as a practical quality control method for the enantiomeric purity determination of(R)-terbutaline.     

Enantiomeric Resolution of Benzoxazolinonic Aminoalcohols, Potential Adrenergic Ligands, by LC and CE

Benzoxazolinone amino alcohols are chiral derivatives that carry one asymmetric center. Both capillary electrophoresis and high-performance liquid chromatography (LC) techniques were used to separate enantiomers with high resolution and thus permitted the determination of enantiomeric excess. Highly sulfated and anionic cyclodextrins were used as chiral selectors under CE conditions. Amylose and cellulose chiral stationary phases were used for LC for the enantioseparation of five racemic compounds. Separations were carried out on a Chiralpak AS column with various polar organic modifiers. The HPLC method appeared more to be more sensitive and was subsequently validated in terms of limits of detection and quantification. LC detection was based on UV and evaporating light scattering.     

苯丙胺类、氯胺酮、卡西酮类毒品手性分离的研究进展

对映异构体在自然界中普遍存在,在药物化学领域尤为突出。虽然手性药物的对映异构体之间具有相同的化学结构,但它们在药理、毒理、药代动力学、代谢等生物活性方面存在明显差异。苯丙胺类、氯胺酮、卡西酮类毒品也是如此,这3类毒品的手性分离研究在常见毒品中具有代表性。目前常用的手性分离色谱方法有气相色谱法(GC)、高效液相色谱法(HPLC)和毛细管电泳法(CE)。苯丙胺类、氯胺酮、卡西酮类毒品使用以上3种方法进行的手性分离研究具有一定共性:GC较多使用N-三氟乙酰-L-脯胺酰氯和(+)R-α-甲氧基α-三氟甲基苯乙酸两种典型的手性衍生化试剂,HPLC主要应用蛋白质类、多聚糖类和大环抗生素类3种手性固定相,CE中环糊精及其衍生物是最常用的手性选择剂。然而这3种手性分离方法存在各自的不足,GC存在手性衍生化引入杂质、反应温度高影响手性分离等问题,HPLC的应用范围比较有限,成本较高,CE没有明确的方法判断哪种物质是合适的手性选择剂。近年来,这3类毒品的手性分离研究在法医毒物学领域的应用有各自的特点,苯丙胺类毒品的手性分离研究多用于推断市场上毒品的原型及合成路线,氯胺酮的手性分离研究涉及多种生物检材,卡西酮类毒品侧重于手性分离方法的广泛适用性。该文主要遴选近10年国内外核心期刊的文献,对苯丙胺类、氯胺酮、卡西酮类毒品的手性异构体特点及色谱法的手性识别机理进行简单介绍,重点对已有研究的共性以及手性分离在法医毒物学中的应用等内容进行综述。基于以上研究,该文提出未来可以从以下3个方面进行深入研究:一是利用计算机技术建立分子模型深入探究手性识别机理;二是研发新型技术,对超临界流体法进行商用研究;三是将手性分离应用于司法实践、医药研发等实际工作领域。     

High-performance liquid chromatographic and capillary electrophoretic enantioseparation of plant growth regulators and related indole compounds using macrocyclic antibiotics as chiral selectors

Enantioseparation of plant growth regulators, such as 3-(3-indolyl)-butyric acid, abscisic acid and structurally related molecules including a variety of substituted tryptophan compounds, has been achieved by HPLC and/or CE. The covalently bonded macrocyclic antibiotics, teicoplanin, ristocetin A and vancomycin, were used as chiral stationary phases (CSPs) in HPLC. Most of the racemates were baseline resolved in the reversed-phase mode (EtOH-H2O) using the teicoplanin CSP. The chiral recognition mechanism is discussed in regard to the structure of the analytes. In CE, the three aforementioned macrocyclic antibiotics were used as chiral additives in a phosphate run buffer. The effect of pH and the concentration of the organic modifiers were considered. The results obtained by HPLC and CE were compared.     

Mechanistic Principles in Chiral Separations Using Liquid Chromatography and Capillary Electrophoresis

Due to the importance of chiral separations of drugs, pharmaceuticals, agrochemicals and xenobiotics by high performance liquid chromatography (HPLC) and capillary electrophoresis (CE), it is important to have the knowledge of the enantiomeric recognition mechanisms so that scientists may design and module the new chiral selectors for rapid, inexpensive and reproducible chiral separations; specially at preparative scale. The mechanisms of the chiral separation by HPLC and CE using polysaccharides, cyclodextrins, macrocyclic glycopeptide antibiotics, Pirkle type, ligand exchangers, crown ethers and other several types of chiral selectors have been discussed. Various complex formation and different types of interactions responsible for chiral resolution have been presented in detail.     

Optimization of a capillary electrophoresis method to determine the chiral purity of a drug

Capillary electrophoresis (CE) using hydroxypropyl-β-cyclodextrin (HP-β-CD) in the separation buffer was investigated to determine the overall chiral purity of a drug containing a single stereogenic center. The effects of primary factors —pH, buffer components, buffer concentration, cyclodextrin concentration and sample amount (concentration and injection volume) — on the resolution of the enantiomers were investigated. Secondary factors such as the HP-β -CD source, lot and degree of substitution that were expected to affect the robustness of the assay were investigated also. The linearity, quantitation limit for the trace enantiomer and the precision of the measurements were determined. This study shows that understanding and optimizing the assay conditions leads to a chiral CE separation that is comparable to that obtained by chiral HPLC. However, chiral CE separations achieved with buffer additives have the advantages of shorter run times, higher numbers of theoretical plates (greater resolution), smaller amounts of chiral additive (less cost) and greater ruggedness (separation virtually independent of column properties unlike HPLC).     

高效液相色谱法测定1,1’-联萘-2,2’-二酚的光学纯度

用高效液相色谱法测定了1,1’-联禁-2,2’-二酚的光学纯度。色谱柱:Si-5柱,流动相:石油醚／乙醇（93／7,V／V）。方法简便、快速、准确,准确度为98.6％～101.6％,相对平均偏差为1.01％。     

Simultaneous separation of the stereoisomers of 1-amino-2-hydroxy and 2-amino-1-hydroxypropane phosphonic acids by stereoselective capillary electrophoresis employing a quinine carbamate type chiral selector

A stereoselective nonaqueous capillary electrophoresis (CE) method utilizing O-(tert-butylcarbamoyl) quinine as chiral ion-pair agent and additive to the non aqueous background electrolyte was evaluated for the simultaneous separation of the enantiomers and diastereomers of 1 -amino-2-hydroxypropane phosphonic acid besides the corresponding beta-aminophosphonic acid analogs, the stereoisomers of 2-amino-1-hydroxypropane phosphonic acid, in a single run. The separations have been carried out using the partial filling technique to avoid strong background signal from the quinine selector. It conveniently allowed the baseline separation of all eight components of interest (alpha- as well as beta-aminophosphonic acids) as N-2,4-dinitrophenyl derivatives in a single run. Moreover, the absolute configurations of all eight peaks were identified. Compared to the quinine carbamate selector, the corresponding 'pseudo-enantiomeric' O-(tert-butylcarbamoyl) quinidine selector exhibited reserved elution order and nearly identical resolutions. The proposed CE method turned out to be advantageous over stereoselective high-performance liquid chromatography (HPLC) with a quinine carbamate type stationary phase, which showed high enantioselectivity, but failed to simultaneously separate all eight components.     

Separation of Tic-hydantoin enantiomers,potent sigma-1 agonists,by high performance liquid chromatography and capillary electrophoresis

Stereospecific separations of seven Tic-hydantoin sigma-1 agonists were performed by both HPLC method using derivatized cellulose and amylose chiral stationary phases and capillary electrophoresis (CE) method using neutral and anionic cyclodextrins added in the background electrolyte (BGE). An optimal baseline separation (R_s > 3.3 with analysis times < 25 min) was readily obtained with all silica-based celluloses and amyloses using a normal-phase methodology. CE was used as an alternative technique to HPLC for the Tic-hydantoin derivatives separation. The enantiomers were fully resolved with highly sulfated β-cyclodextrins at pH 2.5 (R_s > 1.5 with analysis times <11 min). Both methods were validated in terms of linearity, detection and quantification limits. They were used to check the enantiomeric purity of the enantiomers.     

Analytical and semipreparative enantioseparation of 9-hydroxyrisperidone, the main metabolite of risperidone, using high-performance liquid chromatography and capillary electrophoresis. Validation and determination of enantiomeric purity

The HPLC semipreparative enantioseparation of 9-hydroxyrisperidone (9-OHRisp) was studied by optimizing various experimental conditions: the nature of the chiral stationary phase (CSP), mobile phase composition, temperature and analyte loading. This semipreparative enantioseparation was successfully completed using the polysaccharide Chiralcel OJ chiral stationary phase and a n-hexane/ethanol/methanol (50/35/15, v/v/v) ternary mobile phase. To assess the enantiomeric purity of both isolated isomers, three analytical methods using UV detection were developed and validated: one CE method using dual cyclodextrin mode and two HPLC methods using either the Chiralcel OJ CSP in normal-phase mode or the alpha-acid glycoprotein (alpha-AGP) CSP in reversed-phase mode. The three methods make it possible to obtain excellent enantioseparations (R(s) >3) with analysis times lower than 15 min, and the calculated limits of detection allow for the determination of minor enantiomeric impurities (0.1%). Enantiomeric purity obtained for dextrorotatory and levorotatory enantiomers was superior to 99.9% and equal to 98.9%, respectively, which proved the success of the semipreparative enantioseparation. A brief comparison of the performances of the analytical methods completes this work.     

Metabolic study of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone to the enantiomers of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol in vitro in human bronchial epithelial cells using chiral capillary electrophoresis

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) with one chiral center at the carbinol is a major metabolite of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). As tobacco specific N-nitrosamines (TSNAs), NNK and NNAL are the most pulmonary carcinogens in tobacco products and smoke. In this paper, a chiral CE method modified with highly sulfated β-cyclodextrin (S-β-CD) was developed to investigate the stereoselective formation of NNAL from NNK in vitro in normal human bronchial epithelial (NHBE) cells. Combined with solid phase extraction (SPE) of the cell samples, NNK and NNAL enantiomers were baseline separated under the proposed CE conditions, with satisfactory recoveries (72.5-113% for NNK and (±)-NNAL) and low limits of detection (LOD, 2.5-3 μg/mL for NNK and (±)-NNAL). The cytotoxicity of NNK in NHBE cells was investigated through the cell counting kit (CCK) assay and proved to be highly dependent on the NNK's concentration. The metabolic results obtained from CE analysis demonstrated that NNK was preferentially metabolized to (+)-NNAL through carbonyl reduction. Meanwhile, the ratio of [(+)-NNAL]/[(-)-NNAL] was independent of NHBE cells' incubation time with NNK, but could be changed according to the original incubation concentration of NNK. This chiral CE method could be useful for the study on toxicology and metabolic transformations of related TSNAs.     

Chiral separations using the macrocyclic antibiotics: a review

The macrocyclic antibiotics have recently gained popularity as chiral selectors in CE, HPLC and TLC. The macrocyclic antibiotics used for chiral separations include the ansamycins, the glycopeptides, and the polypeptide antibiotic thiostrepton. Although not strictly considered macrocyclic antibiotics, the aminoglycosides are antibiotics that have been used for chiral separations in CE. More chiral analytes have been resolved using the glycopeptides than with the other macrocyclic antibiotics combined. The glycopeptides vancomycin, ristocetin A and teicoplanin have been used extensively as chiral selectors in CE, with ristocetin A appearing to be the most useful chiral selector followed by vancomycin and teicoplanin, respectively. The macrocyclic antibiotics have also been used as chiral bonded phases in HPLC, and HPLC stationary phases based on vancomycin, ristocetin A and teicoplanin have been commercialized. Ristocetin A seems to be the most useful glycopeptide HPLC bonded phase, but its greater expense can be a drawback. The macrocyclic antibiotics have been used with micelles to improve efficiency, provide unique selectivity, and extend the range of separations to neutral solutes. Changing the macrocyclic antibiotic used in CE or HPLC can significantly alter the enantioselectivity of the separations. In fact, the glycopeptide antibiotics are complementary to one another, where if a partial enantioresolution is obtained with one glycopeptide, there is a high probability that a baseline or better separation can be obtained with another.     

Separation of enantiomers by capillary electrochromatography

The current popularity of capillary electrochromatography (CEC) has led to an increasing number of studies on the development and evaluation of enantioselective CEC systems. These studies clearly demonstrate that the most prominent advantage of electrically driven separation methods, the vastly increased column efficiency as compared to pressure-driven chromatography, can also be experimentally achieved for the separations of enantiomers. In analogy to high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE), several approaches have been used. The addition of a chiral selector to the mobile phase is the simplest method. Less erroneous and more elegant approaches are those that use open-tubular, conventional packed, and monolithic columns containing chiral stationary phases that stereoselectively interact with enantiomers. This review evaluates the new techniques and compares them to enantioselective HPLC and CE. Further, it describes the various concepts of enantioselective CEC and focuses on the current ‘state-of-the-art' column technology.     

Separation of 3'-azido-2',3'-dideoxythymidine pronucleotide diastereoisomers in biological samples by CZE with cyclodextrin addition

The possibility of using capillary electrophoresis as an alternative technique to HPLC for the separation of pronucleotide diastereoisomers of AZT was investigated. In the pH range 6.2-7.2 where the analytes are stable, a chiral additive, carboxymethyl-beta-CD, was found appropriate to enable the separation of the uncharged diastereoisomers. An experimental design strategy was used to study the influence of several parameters (CD and phosphate buffer concentration, methanol content of the electrolyte, injected volume, capillary length, electric field and separation temperature) on the separation and find suitable analytical conditions for monitoring the prodrugs in cell extracts. The diastereoisomers of the three tBuSATE phenylphosphotriester derivatives of AZT studied could be fully resolved within short analysis time (less than 10 min). Method validation results showed satisfactory results for linearity, accuracy and repeatability.     

Comparison of capillary electrophoresis and reversed-phase liquid chromatography for determination of the enantiomeric purity of an M3 antagonist

The chiral separation of an M3 antagonist was investigated using capillary electrophoresis (CE) with various sulfated cyclodextrins and by reversed-phase liquid chromatography with derivatized cellulose, derivatized amylose, and two protein stationary phases. Operational parameters for each technique, such as the concentration of the chiral selectors, background electrolyte (or mobile phase) pH and type, organic modifiers, injection mode and temperature were varied in order to achieve a desired elution order and to meet a 0.1% limit of quantitation (LOQ) criteria. Based on the advantages and disadvantages of each technique, a practical CE method using sulfated gamma-cyclodextrin was selected. The method was validated in terms of linearity, LOQ, accuracy, ruggedness and precision.