Direct enantiomeric high performance liquid chromatographic separation of aminoglutethimide and its major metabolite on a series of Chiralcel OD and Chiralcel OJ columns and its application to biological fluids

A direct, isocratic, sensitive and precise liquid chromatographic method is presented for the enantiomeric separation of aminoglutethimide (AG) and its acetylated metabolite (AcAG) using cellulose tris-3,5-dimethyl phenyl carbamate (Chiralcel OD) and cellulose tris(4-methylphenyl benzoate) ester (Chiralcel OJ) columns in series. The enantiomeric elution order is determined by separate chromatography of the racemate AG and racemate AcAG and of their separate enantiomers under similar conditions. This method has been used to determine and identify the enantiomers of AG and AcAG in the urine sample collected from a metastatic breast cancer patient after administration of AG for 24 h. Large amounts of (+)-R-AG are excreted unchanged in the urine together with smaller quantities of (+)-R-AcAG, while most of the (-)-S-AG is metabolically converted into (-)-S-AcAG.     

Direct liquid chromatographic resolution of racemic aminoglutethimide and its acetylated metabolite using a chiral alpha 1-acid glycoprotein column

A 10-cm long alpha 1-acid glycoprotein column is used for the enantiomeric resolution of the clinically used racemic aminoglutethimide (+/- AG) and its acetylated metabolite (+/- AAG). A direct liquid chromatographic resolution of racemic aminoglutethimide and its acetylated metabolite is accomplished without any derivatizations. Maximum resolutions of 1.37 and 0.73 are obtained for the enantiomers of aminoglutethimide and its acetylated metabolite, respectively. The effect of the 2-propanol content in mobile phase on retention and enantioselectivity of aminoglutethimide and its acetylated metabolite is demonstrated. The variation of the separation factors (alpha) with pH in enantiomeric separation of aminoglutethimide is also shown.     

Enantiomeric separations of chiral polychlorinated biphenyls on three polysaccharide-type chiral stationary phases by supercritical fluid chromatography

Enantiomeric separations of 18 chiral polychlorinated biphenyls (PCBs) were investigated on three polysaccharide-type chiral stationary phases (CSPs; Sino-Chiral OJ, Chiralpak IB, and Chiralcel OD) by supercritical fluid chromatography (SFC). With these commonly used polysaccharide CSPs, 17 PCBs except PCB 135 (R(S) = 0.81) were well resolved (R(S) > 1.5) under appropriate mobile phases and temperatures. Using Sino-Chiral OJ, 14 PCBs could be baseline-separated, while only one and nine PCBs could be completely separated using Chiralpak IB and Chiralcel OD, respectively. The influence of column temperature was studied for the optimization of resolution, as well as for the type and percentage of organic modifier in the mobile phase. The resolution decreased as the temperature increased in the range of 26-40 °C in which the enantiomeric separations were an enthalpy-driven process. The addition of modifiers in the mobile phase decreased the resolution of the PCB enantiomers, but it clearly shortened their retention time. These separation results indicate that SFC is a promising chromatographic technique for chiral separation and enantiopure standard preparation.     

Comparative study between the polysaccharide-based chiralcel OJ and chiralcel OD CSPSs in chromatographic enantioseparation of imidazole analogues of fluoxetine and miconazole

The enantiomeric separation of a series of imidazole analogues of Fluoxetine and Miconazole endowed with potent antifungal activity was performed using cellulose tris(4-methylbenzoate) (Chiralcel OJ) and cellulose tris(3,5-dimethylphenylcarbamate) (Chiralcel OD) as chiral stationary phases. Binary mixtures of n-hexane and alcohol as well as pure alcohols (ethanol or 2-propanol) were used as eluents. The enantiomer elution order was monitored by chiroptical detectors based on on-line optical rotation and circular dichroism measurements. For some of the compounds studied very high enantioseparation factor values (alpha > 7) on Chiralcel OJ CSP were observed. In order to study the chiroptical characteristics of the two most biologically active compounds, chromatographic resolutions were carried out on a semipreparative scale. Assignment of the absolute configuration was empirically established by comparing the CD spectra of the separated enantiomers with those obtained from the enantiomers of Miconazole.     

High-performance liquid chromatographic separation of imidazolinone herbicide enantiomers and their methyl derivatives on polysaccharide-coated chiral stationary phases

Many chiral pesticides exhibit enantioselectivity in biotransformation and ecotoxicity in the environment. A significant class of chiral pesticides is imidazolinone herbicides, of which enantioselectivity has not been well studied. Development of efficient chiral separation methods is the first step for allowing characterization of enantioselectivity in environmental processes. In this study, we attempted to resolve enantiomers of imidazolinone herbicides using reversed-phase and normal-phase high-performance liquid chromatography with polysaccharide-type chiral columns. Enantiomers of imazethapyr, imazaquin, and imazamox were separated on a Chiralcel OD-R column using 50mM phosphate buffer-acetonitrile as mobile phase. Enantiomers of imazapyr, imazapic, imazethapyr, imazamox and imazaquin were resolved on a Chiralcel OJ column using n-hexane (0.1% trifluoroacetic acid)-alcohol as mobile phase. The enantiomers of five methyl derivatives of imidazolinone herbicides were also resolved on the Chiralcel OJ column. The Deltak' values revealed a structure-enantioselectivity relationship for the separation behaviors of the enantiomers on the OJ column. The described method was successfully applied for chiral analysis of two imidazolinone herbicides (imazapyr and imazaquin) in spiked soil samples.     

Direct enantiomeric separations of tris(2-phenylpyridine) iridium (III) complexes on polysaccharide derivative-based chiral stationary phases

A convenient method is presented for the first time for the direct separation of enantiomers of a tris(2-phenylpyridine) iridium (III) and an analog substituted with long alkoxy chains on polysaccharide derivative-based chiral stationary phases by HPLC. Tris(2-phenylpyridine) iridium (III) was separated on the immobilized amylose 3,5-dimethylphenylcarbamate (Chiralpak IA) using hexane/CHCl3/CH2Cl2 (75:20:5) as an eluent, and the analog could be separated on the coated cellulose 3,5-dimethyl-phenylcarbamate (Chiralcel OD) and cellulose 4-methylbenzoate (Chiralcel OJ) using hexane/2-propanol (96:4) as the eluent. CD spectra of the eluted HPLC fractions were also recorded, and the observed mirror image patterns confirm their enantioseparations.     

Enantiomeric resolution of new aromatase inhibitors by liquid chromatography on cellulose chiral stationary phases

Analytical HPLC methods using derivatized cellulose chiral stationary phases were developed for the direct enantioseparation of substituted [1-(imidazo-1-yl)-1-phenylmethyl)]-benzothiazolinone and benzoxazolinone derivatives with one chiral center. Those analogues of fadrozole constitute new potent nonsteroidal inhibitors of aromatase (P450 arom). The separations were made using normal phase methodology with a mobile phase consisting of n-hexane-alcohol (ethanol, 1-propanol, or 2-propanol) in various proportions, and a silica-based cellulose tris-3,5-dimethylphenylcarbamate (Chiralcel OD-H), or tris-methylbenzoate (Chiralcel OJ). The effects of concentration of various aliphatic alcohols in the mobile phase were studied. A better separation was achieved on cellulose carbamate phase compared with the cellulose ester phase. The effects of structural features of the solutes along with the temperature of the column on the discrimination between the enantiomers were examined. Baseline separation (Rs > 1.5) was easily obtained in many cases.     

Enantiomeric separation of precursors of rho-kinase inhibitors by HPLC on polysaccharide-based chiral stationary phases

Summary The separation of enantiomers of substituted cyclohexanecarboxamides, benzamides and chemical precursors of Rho-kinase inhibitors was achieved using derivatized polysaccharide-based chiral stationary phases. Separations were by normal phase HPLC with a mobile phase ofn-hexane-alcohol (methanol, ethanol or 2-propanol) in various proportions, and a silica-based cellulose tris-3,5-dimethylphenylcarbamate (Chiralcel OD-H), tris-methylbenzoate (Chiralcel OJ), a silica-based amylose tris-(S)-1-phenylethylcarbamate (Chiralpak AS), or tris-3,5-dimethylphenylcarbamate (Chiralpak AD). The effects of cencentration of various aliphatic alcohols in the mobile phase were investigated. The effect of structural features on the discrimination between the enantiomers was examined. The isolation of milligram amounts of enantiomers of two derivatives was performed on an analytical column by multiple repetitive injections under overload conditions.     

Liquid chromatographic enantiomer resolution of N-fluorenylmethoxycarbonyl alpha-amino acids and their ester derivatives on polysaccharide-derived chiral stationary phases

The liquid chromatographic enantiomer separation of N-fluorenylmethoxycarbonyl (FMOC) protected alpha-amino acids and their ethyl ester derivatives was performed on polysaccharide-derived chiral stationary phases, Chiralcel OD, Chiralpak AD, and Chiralpak AS. In general, Chiralcel OD and Chiralpak AD showed good performance for resolution of N-FMOC alpha-amino acids and their ethyl esters, respectively. All investigated N-FMOC alpha-amino acid enantiomers were baseline separated on Chiralcel OD or Chiralpak AD, whereas N-FMOC alpha-amino acid ethyl ester enantiomers were baseline resolved (alpha = 1.15-3.03) on Chiralpak AD, except for two analytes. The L-enantiomers of all examined FMOC alpha-amino acid ethyl ester derivatives are preferentially retained on Chiralpak AD, while the elution orders of the other enantiomer separations are not consistent.     

在多糖衍生物类色谱柱上手性拆分β-氨基醇及β-羟基 硫醚类化合物

在以正己烷-异丙醇为移动相的体系中,用ChiralcelOD,ChiralcelOJ及ChiralpakAD作为手性固定相对13种β-氨基醇及β-羟基硫醚类化合物对映体进行HPLC手性拆分,这些化合物至少能在一支柱上得到基线级分离。考察了它们于不同浓度配比的这类洗脱体系中在柱上的色谱行为。实验表明化合物取代基的性质明显影响它们在手性柱上的拆分。手性固定相与外消旋样品上的极性基团之间的氢键作用和π-π作用可能是进行手性识别的主要原因。方法已用于非手性环氧化合物不对称开环反应产物β-氨基醇及β-羟基硫醚类化合物的光学纯度鉴定。     

Enantiomers of New Synthetic Pyrrolylphenylethanoneamine Mono-Amino Oxidase Inhibitor Compounds: Analytical and Semipreparative HPLC Separations, and Chiroptical Characteristics

The polysaccharide chiral stationary phases (CSPs) Chiralcel OD and Chiralpak AD, and the brush-type (R,R)-Whelk-01 chiral stationary phases have been evaluated to separate new synthetic pyrrolylphenylethanoneamine racemic compounds, potentially monoamine oxidase (MAO) inhibitors, under various mobile phase compositions, using various temperatures. The enantioseparation was evaluated by comparing the (R,R)-Whelk-01 column performance with those of Chiralpak AD and Chiralcel OD. Significant differences were observed in their chiral recognition, as revealed from their retention, selectivity, resolution and elution order. Performances of the Chiralpak AD column were superior to those of the Chiralcel OD and (R,R)-Whelk-01 columns. Some of the racemic compounds were resolved by semipreparative chromatography on Chiralpak AD column in order to study the chiroptical proprieties of the single enantiomers.     

含磷手性化合物在多聚糖类手性固定相上的手性分离

在纤维素 三(3,5 二甲基苯基氨基甲酸酯)(ChiralcelOD)和直链淀粉 三(3,5 二甲基苯基氨基甲酸酯)(ChiralpakAD H)手性固定相上,采用高效液相色谱正相条件,分离了系列含磷手性化合物。考察了流动相中有机改性剂的种类及浓度对手性分离的影响;研究了化合物的结构与保留及对映体选择性的关系;并探讨了手性识别机理。     

Enantiomer separaton of tifluadom and analogues by liquid chromatography with cellulose-based chiral stationary phases

Chromatographia - The resolution of nine recamic mixtures of tifluadom analogues has been evaluated using the chiral stationary phases Chiralcel OD and Chiralcel OJ. The separation was performed on...     

High-performance liquid chromatographic separation of the enantiomers of organophosphorus pesticides on polysaccharide chiral stationary phases.

High-performance liquid chromatographic separation of the individual enantiomers of 12 organophosphorus pesticides (OPs) was obtained on polysaccharide enantioselective HPLC columns using alkane-alcohol mobile phase. The OP pesticides were crotoxyphos, dialifor, fonofos, fenamiphos, fensulfothion, isofenphos, malathion, methamidophos, profenofos, crufomate, prothiophos and trichloronate. The enantiomers of fenamiphos, fensulfothion, profenofos and crufomate were separated on CHIRALPAK AD; the enantiomers of fenamiphos were also separated on CHIRALPAK AS; the enantiomers of methamidophos, crufomate and trichloronate were separated on CHIRALCEL OD; the enantiomers of crotoxyphos, dialifor, fonofos, malathion, prothiophos and trichloronate were separated on CHIRALCEL OJ; and the enantiomers of isofenphos were separated on CHIRALCEL OG. Baseline or partial separation of the enantiomers of six of these OP pesticides was obtained on CHIRALCEL OJ. In continued method development, the separation of the enantiomers of the 12 OPs was investigated more extensively on CHIRALCEL OJ to determine whether the mobile phase composition, flow-rate and column temperature could be optimized to yield at least partial separation of the enantiomers. Chromatographic conditions were found that gave either baseline or near baseline separations of the enantiomers of the 12 OPs on the CHIRALCEL OJ column.     

Chiral Resolution of Enantiomers of Homocamptothecin Derivatives, Antitumor Topoisomerase I Inhibitors, Using High Performance Liquid Chromatography on Polysaccharide-Based Chiral Stationary Phases

Analytical HPLC methods using derivatized cellulose and amylose chiral stationary phases were developed for the separation of the enantiomers of homocamptothecin (hCPT) derivatives which constitute a promising series of potent anticancer agents targeting DNA topoisomerase I. The resolutions were performed using a normal phase methodology with two silica-based celluloses tris-3,5-dimethylphenylcarbamate (Chiralcel OD-H) and tris-methylbenzoate (Chiralcel OJ) or two amyloses tris-3,5-dimethylphenylcarbamate (Chiralpak AD) and tris-(S)-1-phenylethylcarbamate (Chiralpak AS). The mobile phase and the chiral stationary phase were varied to achieve the best resolution. Different types and concentration of aliphatic alcohols in the mobile phase were also tested along with the temperature dependence. An optimal baseline separation (Rs > 1.5) was readily obtained in most cases. The different columns gave complementary results in term of resolution. The limits of detection and quantification were between 0.08–0.40 M and 0.24–1.80 M, respectively and the enantiomeric purity was superior to 99.9%.     

纤维素-三（3,5-二甲苯基氨基甲酸酯）手性固定相拆分氨鲁米特对映体

以纤维素-三（3,5-二甲苯基氨基甲酸酯）为手性固定相（Chiralcel OD-H）在高效液相色谱上拆分了氨鲁米特对映体。通过测定氨鲁米特在正己烷/乙醇和正己烷/异丙醇中的溶解度,优选了对样品溶解度大的流动相体系,并考察了流动相添加剂乙醇胺对拆分效果的影响。在此基础上进一步研究了流动相中乙醇含量、柱温和进样量对分离因子、分离度、不对称因子和理论板数的影响,从而确定了最佳的拆分条件：固定相为Chiralcel OD-H,流动相为正己烷/乙醇/乙醇胺（体积比为30：70：0.1）,柱温25℃。本文所得结果可为工业放大提供基础数据。     

Simultaneous enantioselective determination of triazole fungicide difenoconazole and its main chiral metabolite in vegetables and soil by normal-phase high-performance liquid chromatography

Herein is reported, for the first time, a simple and highly sensitive chiral high-performance liquid chromatography (HPLC) method for the simultaneous quantitative determination of difenoconazole stereoisomers and their hydroxylated metabolite difenoconazole alcohol (CGA-205375) enantiomers in vegetables and soil matrix. The separation of difenoconazole and CGA-205375 including their simultaneous enantioseparation was studied using four different polysaccharide-type chiral stationary phases (CSPs) in combination with n-hexane-polar organic alcohols mobile phase. Chiralcel OJ consisting of 25?% of cellulose tris(4-methylbenzoate) coated on wide-pore polysaccharide silica gel exhibited higher resolving ability compared to cellulose tris(3,5-dimethylphenylcarbamate) (Chiralcel OD) as well as to its similar amylose derivative (Chiralpak AD) CSPs for this particular set of chiral analytes. Baseline separation and simultaneous enantioseparation of difenoconazole and its metabolite CGA-205375 could be achieved under optimized separation conditions. Based on the established HPLC method, enantioselective analysis method for this fungicide and its main chiral metabolite in vegetables and soil matrix were developed and validated. Parameters including the matrix effect, linearity, precision, accuracy, and stability were evaluated. Under the optimal conditions, the mean recoveries from cucumber, tomato, and soil matrix ranged from 81.65 to 94.52?%, with relative standard deviations in the range of 1.05-8.32?% for all stereoisomers. Coefficients of determination R (2)?≥?0.998 were achieved for each enantiomer in the cucumber, tomato and soil matrix calibration curves within the range of 0.5-50?μg mL(-1). The limits of quantification for all enantiomers in three matrices were all below 0.1?μg mL(-1). The methodology was successfully applied for simultaneous enantioselective analysis of difenoconazole stereoisomers and their metabolite in the real samples, indicating its efficacy in investigating the environmental stereochemistry of difenoconazole in food and environmental matrix.     

高效液相色谱法对反式-1,2-二取代环丙烷类化合物的手性拆分

在ＣｈｉｒａｌｃｅｌＯＤ和ＣｈｉｒａｌｃｅｌＯＪ柱上,以各种配比的正己烷／异丙醇为洗脱剂,对１３种反式－１,２－二取代环丙烷类化合物的对映体进行了手性拆分。考察了这些外消旋物在这两种柱上的色谱行为。实验表明带芳环的反式－１,２－二取代环丙烷类化合物在ＯＤ及ＯＪ柱上的拆分能力明显地与芳环上取代基的性质和位置有关。另一方面,一些带有脂族取代基的反式－１,２－二取代环丙烷类化合物也能在这两种柱上得到拆分。     

The solute molecule—Rigid or partially flexible? calculation of benzodiazepineR F values as an example

Summary The liquid-chromatographic separation of the enantiomers of amino acid esters as benzophenone Schiff-base derivatives on polysaccharide-derived chiral stationary phases (CSPs) is described. The performance of Chiralcel OF was superior to that of the other CSPs for resolution of benzophenone imine derivatives of amino acid ethyl and methyl esters. The enantiomers of most of the amino acid esters examined as their benzophenone imine derivatives were resolved to baseline on Chiralcel OF. The L-(−) enantiomers of all the analytes were preferentially retained on Chiralcel OF. The resolution of several imine derivatives of amino acid esters was investigated, as was the effect of eluent composition on the resolution of amino acid ethyl esters as their benzophenone imine derivatives.     

Chromatographic separation and enantiomeric resolution of flurbiprofen and its major metabolites

Summary The chromatographic separation and resolution of the enantiomers of flurbiprofen and its two major metabolites, 4′-hydroxyflurbiprofen and 3′-hydroxy-4′-methoxyflurbiprofen was investigated using four different approaches: reversed-phase HPLC after pre-column derivatization with (R)-1-(naphthen-1-yl)ethylamine; reversed-phase HPLC using hydroxypropyl-β-cyclodextrin as a chiral mobile phase additive; chiral-phase HPLC using either an α₁-acid glycoprotein CSP (Chiral-AGP) or an amylose tris(3,5-dimethylphenylcarbamate) CSP (Chiralpak AD). Of all the approaches, only the direct method using the Chiralpak AD CSP demonstrated separation and enantiomeric resolution of all three analytes within an acceptable run time of 45 minutes. Enantiomeric resolution values of 1.67,3.67 and 3.44 were obtained for flurbiprofen, 4′-hydroxyflurbiprofen and 3′-hydroxy-4′-methoxyflurbiprofen respectively. Semi-preparative isolation of the individual enantiomers of both metabolites, followed by CD analysis, revealed that the elution order on the AD CSP wasR-beforeS-enantiomer for both metabolites and the same as that observed for flurbiprofen. The metabolite elution order was subsequently confirmed on the analysis of urine samples obtained from a healthy volunteer following oral administration of the individual drug enantiomers.