Molecular Clefts Derived from 9,9′-spirobi[9H-fluorene] for enantioselective complexation of pyranosides and dicarboxylic acids

The molecular clefts (R)- and (S)- 3 , incorporating 9,9′-spirobi[9H-fluorene] as a spacer and two N-(5,7-dimethyl-1,8-naphthyridin-2-yl)carboxamide (CONH(naphthy)) units as H-bonding sites were prepared via the bis(succinimid-N-yl esters) of (R)-and (S)-9,9′-spirobi[9H-fluorene]-2,2′-dicarboxylic acid ( 5 ). Derivative 6 , with one CONH(naphthy) unit and one succinimid-N-yl ester residue allowed easy access to spirobifluorene clefts with two different H-bonding sites, as exemplified by the synthesis of 4 . Binding studies with (R)- and (S)- 3 and optically active dicarboxylic acids in CDCl₃ exhibited differences in free energy of the formed diastereoisomeric complexes (Δ(ΔGº)) between 0.5 and 1.6 kcal mol^?1 (T 300 K). Similar enantioselectivities were observed with the spirobifluorene clefts (R)- and (S)- 1 , bearing two N-(6-methylpyridin-2-yl)carboxamide (CONH(py)) H-bonding sites. The thermodynamic quantities ΔHº and ΔSº for the recognition processes with (R)- and (S)- 1 were determined by variable-temperature ¹H-NMR titrations and compared to those with (R)- and (S)- 2 , which have two CONH(py) moieties attached to the 6,6′-positions of a conformationally more flexible 1,1′-binaphthyl cleft. All association processes showed high enthalpic driving forces which are partially compensated by unfavorable changes in entropy. Pyranosides bind to the optically active clefts 1 and 3 in CDCl₃ with ?ΔGº = 3.0–4.3 kcal mol^?1. Diastereoisomeric selectivities up to 1.2 kcal mol^?1 and enantioselectivities up to 0.4 kcal mol^?1 were observed. Cleft 4 and N-(5,7-dimethyl-1,8-naphthyridin-2-yl)acetamide ( 25 ) complexed pyranosides 22–24 as effectively as 3 indicating that only one CONH(naphthy) site in 3 associates strongly with the sugar derivatives. Based on the X-ray crystal structure of 3 , a computer model for the complex between (S)- 3 and pyranoside 22 was constructed. Molecular-dynamics (MD) simulations showed that differential geometrical constraints are at the origin of the high enantioselectivity in the complexation of dicarboxylic acid (S)- 7 by (R)- and (S)- 1 and (R)- and (S)- 3 .     

Enantioseparation of selected chiral sulfoxides in high‐performance liquid chromatography with polysaccharide‐based chiral selectors in polar organic mobile phases with emphasis on enantiomer elution order

The separation of the enantiomers of 17 chiral sulfoxides was studied on polysaccharide‐based chiral columns in polar organic mobile phases. Enantiomer elution order (EEO) was the primary objective in this study. Two of the six chiral columns, especially those based on amylose tris(3,5‐dimethylphenylcarbamate) and cellulose tris(4‐chloro‐3‐methylphenylcarbamate) (Lux Cellulose‐4) proved to be most successful in the separation of the enantiomers of the studied sulfoxides. Interesting examples of EEO reversal were observed depending on the chiral selector or the composition of the mobile phase. For instance, the R‐(+) enantiomer of lansoprazole eluted before the S‐(?) enantiomer on Lux Cellulose‐1 in both methanol or ethanol as the mobile phase, while the elution order was opposite in the same eluents on amylose tris(3,5‐dimethylphenylcarbamate) with the S‐(?) enantiomer eluting before the R‐(+) enantiomer. The R‐(+) enantiomer of omeprazole eluted first on Lux Amylose‐2 in methanol but it was second when acetonitrile was used as the mobile phase with the same chiral selector. Several other examples of reversal in EEO were observed in this study. An interesting example of the separation of four stereoisomers of phenaminophos sulfoxide containing chiral sulfur and phosphor atoms is also reported here.     

HPLC separation of the enantiomers of racemic benzergoline derivatives on a chiral binaphthalene covalently bonded to silica gel

A novel chiral stationary phase (CSP) derived from the atropisomeric enantiomer S-3,3′-dicarboxy-2,2′-dihydroxy-1,1′-binaphthyl (S-DDBN) has been synthesized and its use for the separation of enantiomers demonstrated. The chiral selector is covalently bonded to amino-functionalized silica gel, thus enabling the use of alcohols as mobile phases. Good chiral discrimination was obtained for the pharmacologically interesting class of benzergoline derivatives which act as selective dopamine D₁ receptor agonists. This paper reports the successful separtion of fifteen structurally related benzergoline racemates with separation factors up to 3.5. The influence of small differences in molecular structure on chiral discrimination was examined.     

Unusually high enantioselectivity in high-performance liquid chromatography using cellulose tris(4-methylbenzoate) as a chiral stationary phase

The cellulose tris(4-methylbenzoate) chiral stationary phase (CSP) (commercially known as Chiralcel OJ-H) exhibited an extremely high enantioselectivity when used in the HPLC resolution of N-thiocarbamoyl-3-(4′-prenyloxy)-phenyl-5-phenyl-4,5-dihydro-(1H) pyrazole (Compound 1), in both normal-phase and polar organic conditions. Using n-hexane–ethanol (80:20, v/v) as a mobile phase, an enantioseparation factor value of 138.5 was found. In order to modulate the elution time of the longer retained enantiomer, a simple HPLC procedure was developed. The optimized analytical protocol was based on the stopped flow technique and did not involve any change in mobile phase composition. The stronger interaction energy of the (S) enantiomer compared to that of the (R) enantiomer was mainly attributed to the formation of a hydrogen bonding between the amino group of the thiocarbamoyl moiety and the carbonyl oxygen of the CSP.     

Comparison of Coated and Immobilized Chiral Stationary Phases Based on Amylose tris-[(S)-α-Methylbenzylcarbamate] for the HPLC Enantiomer Separation of α-Lipoic Acid and Its Reduced Form

The couple of chiral sulfur compounds α-lipoic acid (ALA)/α-dihydrolipoic acid (DHALA) has attracted considerable attention in recent years owing to its remarkable anti-inflammatory and antioxidant properties. It is well known that the chirality of the C6 plays a key role in determining the biological activity of ALA. The natural occurring (R)-ALA enantiomer is an essential cofactor for key oxidative metabolism enzyme complexes and, after oral administration of the racemic mixture, it shows higher plasma concentration than (S)-ALA. Differently, the in vivo enantioselective action difference between the enantiomers of DHALA has not yet been studied. This lacking is perhaps due to the unavailability of analytical methods capable of determining the enantiomeric composition of biological samples during pharmacokinetic and pharmacodynamic events. In the present work, the direct and baseline enantioresolution of both chiral acids by HPLC on two amylose-derived chiral stationary phases is presented. The proposed chiral enantioselective protocol, therefore, does not require pre- or on-column derivatization. The performance of the coated Chiralpak AS-H CSP and the new immobilized Chiralpak IH-3 CSP, which have the same chiral selector amylose tris-[(S)-α-methylbenzylcarbamate], were compared using conventional normal-phase mobile phases containing ethanol or 2-propanol as alcoholic solvents and a fixed percentage of trifluoroacetic acid. Nonconventional eluents containing dichloromethane, ethyl acetate, and 2-methyltetrahydrofuran as organic cosolvents were applied in the separation of the enantiomers of two carboxylic acids on the immobilized Chiralpak IH-3 CSP. The effect of the column temperature was carefully evaluated in order to improve enantioselectivity. Adequate amounts of enantiomers were isolated by an analytical-size Chiralpak IH-3 column and submitted to chiroptical measurements. The absolute configuration assignment of the isolated enantiomers was determined by a multidisciplinary procedure based on the comparison of the experimental and calculated chiroptical properties.     

脲衍生物型手性固定相拆分噻利洛尔对映体

用脲衍生物型手性固定相直接拆分了药物对映体噻利洛尔。优化的正相色谱流动相的组成为正己烷／１,２－二氯乙烷／乙醇（７７∶２１∶２,Ｖ／Ｖ／Ｖ）。实验表明,流动相中乙醇含量的改变对分离度产生了较大的影响;不同极性调节剂的使用表现出了不同的拆分效果。最后讨论了异构体的出峰次序,认为异构体与固定相的手性中心之一的氢键作用力是造成异构体分离的主要因素。     

Supercritical fluid extraction: a novel method for the resolution of tetramisole

A new resolution method, based on the selective distribution of enantiomers between a chiral solid and an achiral supercritical fluid phase, is reported. The chiral solid phase is formed from the optically active dicarboxylic acid derivative, (2R,3R)-O,O′-dibenzoyltartaric acid, and the racemic base (tetramisole). A new method is also described for the enrichment of enantiomeric mixtures which have an enantiomeric ratio other than 1:1. This is based on the partial salt formation of the enantiomeric mixture with an achiral substance, which is then followed by supercritical fluid extraction of the free enantiomer. The extract has an enantiomeric composition which is different from the starting mixture. The method is applied to an enantiomeric mixture of tetramisole with hydrochloric acid.     

HPLC of fluoroquinolone antibacterials using chiral stationary phase based on enantiomeric (3,3′‐diphenyl‐1,1′‐binaphthyl)‐20‐crown‐6

A residual silanol group‐protecting chiral stationary phase (CSP) based on optically active (3,3′‐diphenyl‐1,1′‐binaphthyl)‐20‐crown‐6 was successfully applied to the resolution of fluoroquinolone compounds including gemifloxacin mesylate. The chiral recognition ability of the residual silanol group‐protecting CSP was generally greater than that of the residual silanol group‐containing CSP. From these results, it was concluded that the simple protection of the residual silanol groups of the latter CSP with lipophilic n‐octyl groups can improve its chiral recognition ability for the resolution of racemic fluoroquinolone compounds. The chromatographic resolution behaviors were investigated as a function of the content and type of organic and acidic modifiers and the ammonium acetate concentration in aqueous mobile phase and the column temperature. Especially, the addition of ammonium acetate to the mobile phase was found to be a quite effective means of reducing the enantiomer retentions without sacrificing the chiral recognition efficiency of the CSP.     

新型含苯并噻唑α-氨基膦酸酯类化合物在淀粉类手性固定相上的液相色谱分离

本文运用涂敷型(Chiralpak AD-H)和键合型(Chiralpak IA)两种淀粉类手性固定相高效液相色谱法,进行了新型含苯并噻唑α-氨基膦酸酯类化合物的手性分离。从色谱分离的保留因子(k)、分离系数(α)和分离度(Rs)三个方面考察了两种类型色谱柱的分离性能,上述化合物在Chiralpak IA柱上能够得到较好的基线分离。同时,讨论了温度、流动相极性和目标分析物的结构等因素对Chiralpak IA柱分离性能的影响。由于键合型固定相较稳定的性能,使某些非常规的溶剂(如THF)成功地应用于手性α-氨基膦酸酯类化合物的分离。     

Enantioseparation of selected chiral sulfoxides using polysaccharide-type chiral stationary phases and polar organic, polar aqueous-organic and normal-phase eluents

HPLC enantioseparation of selected chiral sulfoxides was studied using cellulose and amylose phenylcarbamate derivatives as chiral stationary phases (CSPs). The contributions of various functional groups of a chiral analyte as well as the polysaccharide derivatives in the analyte retention and chiral recognition were evaluated. A very high enantioseparation factor exceeding 110 was observed in the enantioseparation of 2-(benzylsulfinyl)benzamide (BSBA) on cellulose tris(3,5-dichlorophenylcarbamate) (CDCPC) CSP by using 2-propanol as a mobile phase. The enantiomer elution order was opposite on cellulose and amylose phenylcarbamates. For the polysaccharide-type CSPs, pure alcohols such as methanol, ethanol and 2-propanol represent a valuable alternative to more common alcohol-hydrocarbon and reversed-phase eluents.     

Exclusive Observation of the (132R)-Enantiomer of Chlorophyll-c from a Diatom Chaetoseros calcitrans

Chiral high-performance liquid-chromatography (HPLC) for quantitative analysis of optically active chlorophyll(Chl)-c molecules, which are seen in many marine photosynthetic organisms, was developed. Chls-c have a single asymmetric carbon at the 13²-position, so their stereoisomers are (13²R)- and (13²S)-enantiomers. After the separation of each enantiomer, the stereochemistry was unambiguously characterized using its circular dichroism spectrum in comparison with that of the structure-related compound, protochlorophyllide-a. Moreover, Chls-c were carefully extracted from the cells of a diatom Chaetoseros calcitrans without racemization and were subjected to the chiral HPLC. The results clearly demonstrated that naturally occurring Chl-c molecules are enantiomerically pure (13²R)-forms, which are generally found in photosynthetically active chlorophyllous pigments.     

Separation and quantitation of oxprenolol in urine and pharmaceutical formulations by HPLC using a Chiralpak IC and UV detection

Abstract  

A stereoselective HPLC method has been developed for the simultaneous determination of oxprenolol enantiomers in urine and pharmaceutical products. Enantiomeric resolution of oxprenolol was achieved on cellulose tris(3,5-dichlorophenylcarbamate) immobilized onto a 5 μm spherical porous silica chiral stationary phase (CSP) known as Chiralpak IC with UV detection at 273 nm. The mobile phase consisted of n-hexane:isopropanol:triethylamine 70:30:0.1 (v/v/v) at a flow rate of 1.0 cm³/min. The method was validated for its linearity, accuracy, precision, and robustness. The calibration curves were linear over the range of 0.5–75 μg/cm³, with a detection limit of 0.1 μg/cm³ for each enantiomer. An average recovery of 99.0% and a mean relative standard deviation of 2.6% at 40.0 μg/cm³ for S-(−)- and R-(+)-enantiomers were obtained. The overall recoveries of oxprenolol enantiomers from pharmaceutical formulations were in the range 97.5–99.0%, with RSDs ranging from 0.6 to 0.8%. The mean extraction efficiency of oxprenolol from urine was in the range of 86.0–93.0% at 0.5–5 μg/cm³ for each enantiomer. The assay method proved to be suitable as a chiral quality control for oxprenolol formulations using HPLC and for therapeutic drug monitoring.     

Optimization of the HPLC enantioseparation of 3,3′‐dibromo‐5,5′‐disubstituted‐4,4′‐bipyridines using immobilized polysaccharide‐based chiral stationary phases

The HPLC enantioseparation of nine atropisomeric 3,3′,5,5′‐tetrasubstituted‐4,4′‐bipyridines was performed in normal and polar organic (PO) phase modes using two immobilized polysaccharide‐based chiral columns, namely, Chiralpak IA and Chiralpak IC. The separation of all racemic analytes, the effect of the chiral selector, and mobile phase (MP) composition on enantioseparation and the enantiomer elution order (EEO) were studied. The beneficial effect of nonstandard solvents, such as tetrahydrofuran (THF), dichloromethane (DCM), and methyl t‐butyl ether on enantioseparation was investigated. All selected 4,4′‐bipyridines were successfully enantioseparated on Chiralpak IA under normal or PO MPs with separation factors from 1.14 to 1.70 and resolutions from 1.3 to 6.5. Two bipyridines were enantioseparated at the multimilligram level on Chiralpak IA. Differently, Chiralpak IC was less versatile toward the considered class of compounds and only five bipyridines out of nine could be efficiently separated. In particular, on these columns, the ternary mixture n‐heptane/THF/DCM (90:5:5) as MP had a positive effect on enantioseparation. An interesting phenomenon of reversal of the EEO depending on the composition of the MP for the 3,3′‐dibromo‐5,5′‐bis‐(E)‐phenylethenyl‐4,4′‐bipyridine along with an exceptional enantioseparation for the 3,3′‐dibromo‐5,5′‐bis‐ferrocenylethynyl‐4,4′‐bipyridine (α = 8.33, R_s = 30.6) were observed on Chiralpak IC.     

Chiral Separation of (R,R)-Tadalafil and Its Enantiomer in Bulk Drug Samples and Pharmaceutical Dosage Forms by Chiral RP-LC

A new simple isocratic chiral RP-LC method has been developed for the separation and quantification of the enantiomer of (R,R)-tadalafil in bulk drugs and dosage forms with an elution time of about 20 min. Chromatographic separation of (R,R)-tadalafil and its enantiomer was achieved on a bonded macro cyclic glycopeptide stationary phase. The method resolves the (R,R)-tadalafil and its enantiomer with a resolution (R _s) greater than 2.4 in the developed chiral RP-LC. The mobile phase used for the separation and quantification of (R,R)-tadalafil and its enantiomer involves a simple mixture of reverse phase solvents and the cost of analysis was drastically decreased. The test concentration is 0.4 mg mL⁻¹ in the mobile phase. This method is capable of detecting the enantiomer of (R,R)-tadalafil up to 0.0048 μg wrt test concentration 400 μg mL⁻¹ for a 20 μL injection volume. The drug was subjected to stress conditions of hydrolysis, oxidation, photolysis and thermal degradation. There was no interference of degradants with (R,R)-tadalafil and its enantiomer in the developed method. The developed chiral RP-LC method was validated with respect to linearity, accuracy, precision and robustness. The percentage recovery for the enantiomer of (R,R)-tadalafil in bulk drug samples and in dosage forms ranged from 97.0 to 102.5%. The test solution was found to be stable in the mobile phase for 48 h after preparation.     

基于(R)-1,1’-联萘硫脲的手性荧光受体: 合成与手性识别

Two chiral fluorescent receptors 1 and 2 based on (R)-1,1‘-binaphthylene-2,2‘-bisthiourea were synthesized, and their chiral recognition properties for enantiomeric mandelate anions were studied by fluorescence spectra and molecular modeling. Addition of the L- and D-mandelate anions caused considerable fluorescent increases in the fluorescent intensity of the host solution. The L-enantiomer can enhance the fluorescence intensity of 1 much more than the D-enantiomer can do, and 1 shows a better enantioselective recognition ability than 2.     

几种手性醇在纤维素键合的SBA-15基手性固定相上的直接拆分

在强酸性条件下, 以三嵌段聚醚P123为模板, 合成了孔径大且粒径均匀的SBA-15介孔二氧化硅微球. 将含有少量三乙氧硅丙基氨基甲酸酯残基的纤维素-三(3,5-二甲基苯基氨基甲酸酯)通过分子间缩聚作用固载到氨丙基化的SBA-15微球上, 制得手性固定相; 采用常规和非常规的流动相模式, 对一些芳香醇的消旋体进行了手性拆分. 实验结果表明, 所制备的SBA-15微球不仅分散性良好, 具有规则的二维六方孔道结构, 而且消除了微孔; 所制备的键合手性固定相不仅固载手性选择剂的量大, 而且经六甲基二硅胺烷封端后可有效改善拖尾现象, 对实验选用的手性醇具有较高的拆分能力; 与大孔硅胶为基质的同类纤维素键合手性固定相相比, 该固定相对同种手性消旋体的分离因子明显提高.     

Analogs of Cinchona Alkaloids Incorporating a 9,9′-Spirobifluorene Moiety

The Cinchona alkaloid analogs (+)- and (?)- 5 with a quinuclidine-2-methanol residue attached to C(2) of a 9,9′-spirobifluorene moiety were prepared as a racemic mixture by reacting lithiated 2-bromo-9,9′-spirobifluorene 7 with (2-ethoxycarbonyl)quinuclidine (±)- 6 to give ketone (±)- 8 , followed by diastereoselective reduction with diisobutylaluminum hydride (DIBAL-H). The absolute configuration at C(9) and C(8), i.e., at the methanol bridge and the adjacent quinuclidine C-atom, in the two enantiomers of 5 is identical to the configuration at the corresponding centers in (?)-quinine ( 1 ) and (+)-quinidine ( 2 ), respectively. For the optical resolution of (±)- 5 , a chiral stationary phase for HPLC was prepared by covalently bonding quinine via a thiol spacer to a silica-gel surface. The enantiomer separation was accomplished at an α value of 1.61 with (±)- 5 being eluted last, in agreement with ¹H-NMR studies in CDCl₃ which showed that (+)- 5 underwent a more stable host-guest association with quinine than (?)- 5 . ¹H{¹H} Nuclear Overhauser effect (NOE) difference spectroscopical analysis of the host-guest associations with quinine in CDCl₃, combined with computer-model examinations, allowed the assignment of the absolute configurations as (+)-(8R,9S)- 5 and (?)-(8S,9R)- 5 . A detailed conformational analysis displayed excellent agreement between the results of computational methods (Monte Carlo multiple minimum simulations, analyses of the total energy as a function of the flexible dihedral angles in the molecule) and ¹H{¹H}-NOE difference spectroscopical data. It was found that (?)- 5 and (+)- 5 differ significantly in their conformational preference from their natural counterparts quinine ( 1 ) and quinidine ( 2 ). Whereas the natural alkaloids prefer the ‘open’ conformation, with the quinuclidine N-atom pointing away from the quinoline ring, analog (±)- 5 adopts preferentially (by ca. 4 kcal mol^?1) a ‘closed’ conformation, in which the quinuclidine N-atom points into the cleft of the 9,9′-spirobifluorene moiety. Since the basic quinuclidine N-atom in the ‘closed’ conformation is sterically shielded from forming strong H-bonds, the new Cinchona alkaloid analogs form less stable host-guest associations via H-bonding than quinine or quinidine.     

键合型聚丙烯酰胺衍生物手性固定相的制备与手性识别性能

高效液相色谱(HPLC)被广泛认为是分离制备光学纯单一对映体的最有效方法。在高效液相色谱手性拆分中,手性固定相(CSP)的性能直接影响到色谱柱的手性分离能力。在众多手性固定相中,键合型手性固定相具有溶剂耐受性好,分离模式灵活等优点,已经发展成为一类重要的手性固定相。本文通过两步化学反应合成了新型的光学活性丙烯酰胺衍生物--(S)-1-丙烯酰-2-(N-苯基甲酰胺基)吡咯烷((S)-APACP),采用核磁共振氢谱表征了(S)-APACP的化学结构;通过3步化学反应制备了键合型聚丙烯酰胺衍生物手性固定相,采用热重分析法表征了聚合物的键合量,采用HPLC评价了键合型手性固定相的识别能力,分析了影响其手性识别能力的因素。研究结果表明,APACP聚合物成功地键合到硅胶表面制备了具有良好溶剂耐受性的键合型手性固定相,其聚合物键合量为10.2%~11.8%,该键合型手性固定相对若干种对映体显示了较好的手性识别能力。     

LC with a Teicoplanin Aglycone Chiral Sorbent for the Separation of the Enantiomers of Non-Steroidal Anti-Inflammatory Drugs: An Evaluation of Chiral Capillary Columns

A capillary column with a teicoplanin aglycone (TAG) stationary phase (CSP) was used for enantioselective separation of selected profen non-steroidal anti-inflammatory drugs in capillary liquid chromatography (cLC). The effect of variations in the mobile phase composition on the retention and enantioselective separation was examined. The best resolution was attained in the mobile phase composed of 40/60 (v/v) methanol/1.0% triethylamine acetate buffer, pH 4.0 or 4.5. Under the optimized separation conditions, five of the set of eight analytes were enantioresolved with resolution values better than 0.9. Only fenoprofen was not enantioseparated in any system tested. The optimized separation conditions were used for evaluation of three chiral capillary columns (all prepared in the same way in our laboratory) in terms of the repeatability and reproducibility of the results. The run-to-run repeatability was expressed in terms of the relative standard deviation (RSD) values, obtained from ten independent measurements, for the following parameters: the retention factor for the first eluted enantiomer (k ₁), the selectivity (α), the enantioresolution (R), the theoretical plate count per meter for the first eluted enantiomer (N ₁) and the elution curve asymmetry for the first eluted enantiomer (As ₁). None of the RSD values exceeded 8%. The column-to-column reproducibility of these parameters ranged between 1 and 9%. The results obtained with TAG based CSPs in cLC (a laboratory packed capillary column) were compared with those obtained by classical high-performance liquid chromatography (HPLC) with a commercially available column. The cLC procedure provided a better enantioresolution and the elution curves had a better symmetry.

     

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.