Chemo- and enantio-selective reversed-phase HPLC analysis of rosuvastatin using a cellulose-based chiral stationary phase in gradient elution mode.

A direct reversed-phase high-performance liquid chromatographic (HPLC) method was developed for determining the content of the enantiomeric impurity of the chiral statin rosuvastatin calcium salt (RSV) in commercial tablets. The baseline enantioseparation was achieved using the Lux Cellulose-2 column and a binary linear gradient of acetonitrile and trifluoroacetic acid 0.05% in an aqueous solution. The flow rate of the mobile phases and column temperature were set at 1.0 mL min- 1 and 40 °C, respectively. In comparison with the isocratic HPLC method reported in the European Pharmacopoeia (EP) monograph for RSV, the gradient elution method offered improved chemo-and enantio-selectivity and reduced analysis times. The limits of quantitation and detection of the enantiomeric impurity were found to be 0.15 and 0.05 µg mL-1.

Simultaneous enantio- and diastereo-selective high-performance liquid chromatography separation of paroxetine on an immobilized amylose-based chiral stationary phase under green reversed-phase conditions.

A simple and green high-performance liquid chromatography method for the separation of paroxetine from its enantiomeric and diastereomeric impurities has been developed. The simultaneous chromatographic resolution was carried out on the amylose-based Chiralpak IA-3 chiral stationary phase using the mixture ethanol-water-diethylamine 80:20:0.1 (v/v/v) as a mobile phase. The effects of substitution of ethanol with methanol or acetonitrile and changes in column temperature on selectivity have been carefully investigated. The optimized single-run HPLC protocol allows the baseline separation of the enantiomers of paroxetine without suffering from interference from five other chiral and achiral impurities reported in the monograph of the European Pharmacopoeia.

Direct separation of the enantiomers of ramosetron on a chlorinated cellulose-based chiral stationary phase in hydrophilic interaction liquid chromatography mode.

Ramosetron is an enantiopure active pharmaceutical ingredient marketed in Japan since 1996 and later in a few Southeast Asian countries predominantly as an antiemetic for patients receiving chemotherapy. In this study, a simple and rapid high-performance liquid chromoatography method for the separation of ramosetron and its related enantiomeric impurity by using hydrophilic interaction liquid chromatography mode is presented. Chiral resolution was performed on an analytical column (100 mm × 4.6 mm id) packed with 3 µm particles of cellulose-based Chiralpak IC-3 chiral stationary phase. Using a mobile phase containing acetonitrile-water-diethylamine (100:10:0.1, v/v/v) and setting the column temperature at 35°C, the resolution value was 7.35. At a flow rate of 1 mL/min, the enantioseparation was completed within 5 min. The proposed method was partially validated and it has proven to be sensitive with limit of detection and limit of quantitation of the (S)-enantiomer impurity of 44.5 and 133.6 ng/mL.

Single-run reversed-phase HPLC method for determining sertraline content, enantiomeric purity, and related substances in drug substance and finished product.

A direct enantio-, diastereo-, and chemo-selective high-performance liquid chromatographic method was developed for determining the content, enantiomeric purity, and related substances of the chiral antidepressant drug sertraline HCl in a single chromatographic run. The separation was achieved on a chiral stationary phase based on amylose tris(3-chloro-5-methylphenylcarbamate) under reversed-phase conditions. The method was optimized by evaluating the influence of the temperature and mobile phase composition on the retention and selectivity. The application of the single-run approach allowed to baseline resolve all investigated species in less than 15 min, without using buffers or tandem-coupled columns. The chromatographic method was validated according to the guidelines of the Official Medicines Control Laboratory and applied to control the content of sertraline HCl and related chiral substances in a generic antidepressant formulation.

Temperature and eluent composition effects on enantiomer separation of carvedilol by high-performance liquid chromatography on immobilized amylose-based chiral stationary phases.

Carvedilol is a chiral drug with potent antihypertensive and antianginal activities. Although it is clinically used as a racemic mixture, its enantiomers show different pharmacokinetic and pharmacodynamic profiles. Here, the direct chiral separation of racemic drug by high performance liquid chromatography using two immobilized-type amylose-based chiral stationary phases is presented. Some chromatographic parameters, such as retention and selectivity, were determined under multimodal eluent conditions and different temperatures. A temperature-dependent inversion of the elution order of enantiomers was observed in the operative temperature range of chiral chromatographic support. Finally, an effective direct enantioselective method was successfully applied to the separation of the enantiomers of carvedilol on a semipreparative scale.

Unusual retention behavior of omeprazole and its chiral impurities B and E on the amylose tris (3-chloro-5-methylphenylcarbamate) chiral stationary phase in polar organic mode.

Recent reports have demonstrated that the new commercially available immobilized-type chiral stationary phases (CSPs) containing amylose tris(3-chloro-5-methylphenylcarbamate) (ACMPC) as a selector exhibit not only an exceptionally high enantioselectivity in high-performance liquid chromatography (HPLC) but they are also applicable to a wide range of chiral analytes. Herein, we report the results obtained in the HPLC analysis of omeprazole and its impurities B and E on the ACMPC-based Chiralpak IG-3 CSP (CSP) under polar organic conditions. A systematic evaluation of the retention characteristics of the selected benzimidazole chiral probes was carried out by changing the composition of the mobile phase and the column temperature. It is worth emphasizing that the high affinity of both enantiomers of all analytes recorded in pure methanol mode dramatically decreased incorporating small volumes of either basic or acid additives in the mobile phase. Unspecified sites of the IG-3 CSP presumably involved in strong and non-stereoselective H-bonding contacts with chiral analytes are assumed responsible for the unproductive retention process.

Development of a high-performance liquid chromatography method for the simultaneous determination of chiral impurities and assay of (S)-clopidogrel using a cellulose-based chiral stationary phase in methanol/water mode.

A simple reversed-phase high-performance liquid chromatography method for the chiral separation of the active pharmaceutical ingredient (S)-clopidogrel has been developed on the cellulose-based Chiralcel OJ-RH chiral stationary phase. The S enantiomer was baseline resolved from its R impurity (impurity C) with a mobile phase consisting of methanol/water (100:15) without any interference coming from the other two potential chiral impurities A and B. The enantio- and chemoselective method was partially validated and compared with that reported in the United States Pharmacopoeia for the drug product. The versatility of the Chiralcel OJ-RH allowed separating the enantiomers of the impurity B also under normal phase and setting up an efficient strategy to convert the racemic sample into the enantiomeric S form on a semipreparative scale.

Enantiomers of triclabendazole sulfoxide: Analytical and semipreparative HPLC separation, absolute configuration assignment, and transformation into sodium salt.

Direct HPLC separation of the enantiomers of triclabendazole sulfoxide (TCBZ-SO), which is the main metabolite of the anthelmintic drug triclabendazole, was carried out using the polysaccharide-based Chiralpak AS-H and Chiralpak IF-3 chiral stationary phases (CSPs). The chromatographic behaviour of both CSPs was evaluated and compared using normal-phase and reversed-phase eluents at different column temperatures. The eluent mixture of n-hexane-2-propanol-trifluoroacetic acid 70:30:0.1 (v/v/v) and a column temperature of 40°C were identified as the best operational conditions to carry out semipreparative enantioseparations on a 1-cm I.D. AS-H column. Under these conditions, 12.5mg of racemic sample were resolved in a single chromatographic run within 15min. Comparison of calculated and experimental chiroptical properties provided the absolute configuration assignment at the sulfur atom. The salification of the isolated enantiomers of TCBZ-SO by reaction with sodium hydroxide solution produced water-soluble Na salts which are potentially useful in the development of new anthelmintic enantiomerically pure formulations.

Analytical and semipreparative high performance liquid chromatography enantioseparation of bicalutamide and its chiral impurities on an immobilized polysaccharide-based chiral stationary phase.

Direct HPLC separation of enantiomers of Bicalutamide (BCT), a non-steroidal antiandrogen used for the treatment of prostate cancer, was performed by using the immobilized amylose-based Chiralpak IA chiral stationary phase (CSP). Enantioselective conditions were achieved using standard normal phase mixtures n-hexane-alcohol (ethanol or 2-propanol) and a "non-standard" mobile phase containing ethyl acetate (EA). The chromatographic behaviour of the IA CSP under these elution modes was evaluated and compared at different temperatures. The eluent mixture n-hexane-EA-ethanol 100-30-5 (v/v/v) and the column temperature of 40°C were identified as the best operational conditions to carry out semipreparative enantioseparations on a 1-cm I.D. IA column. Using this protocol, about 960mg of (R)-BCT, which is the enantiomer with the almost entire anti-androgenic activity of BCT, per day could be isolated. The analytical and semipreparative HPLC resolution of chiral impurities of BCT, and their empiric absolute configuration assignment by circular dichroism correlation method are also presented.

Green high-performance liquid chromatography enantioseparation of lansoprazole using a cellulose-based chiral stationary phase under ethanol/water mode.

A simple and environmentally friendly reversed-phase high-performance liquid chromatography method for the separation of the enantiomers of lansoprazole has been developed. The chromatographic resolution was carried out on the cellulose-based Chiralpak IC-3 chiral stationary phase using a green and low-toxicity ethanol-aqueous mode. The effects of water content in the mobile phase and column temperature on the retention of the enantiomers of lansoprazole and its chiral and achiral related substances have been carefully investigated. A mixed-mode hydrophilic interaction liquid chromatography and reversed-phase retention mechanism operating on the IC-3 chiral stationary phase allowed us to achieve simultaneous enantioselective and chemoselective separations in water-rich conditions. The enantiomers of lansoprazole were baseline resolved with a mobile phase consisting of ethanol/water 50:50 without any interference coming from chiral and achiral impurities within 10 min.

Comparison of reversed-phase enantioselective HPLC methods for determining the enantiomeric purity of (S)-omeprazole in the presence of its related substances.

A simple analytical high-performance liquid chromatography (HPLC) method was applied for the enantiomeric excess determination of esomeprazole ((S)-OME), the enantiopure active ingredient contained in drug products, in the presence of its potential organic impurities A-E. The enantioselective separation was accomplished on the immobilized-type Chiralpak ID-3 chiral stationary phase (CSP) under reversed-phase conditions. The results were evaluated and compared with those obtained by the official enantioselective method of European Pharmacopoeia used as the reference for checking the enantiomeric excess of (S)-OME. It has been established that the use of the Chiralpak ID-3 CSP allows the determination of the enantiomeric purity of (S)-OME without any interference coming from its chiral and achiral related substances. The analytical procedure of the drug regulatory agencies based on the AGP CSP suffered instead from poor specificity due to overlap of the peaks pertinent to the achiral impurity A and the chiral impurity (R)-OME (impurity F).

Determination of the Enantiomerization Barrier of the Residual Enantiomers of C3 -Symmetric Tris[3-(1-Methyl-2-Alkyl)Indolyl]Phosphane Oxides: Case Study of a Multitasking HPLC Investigation Based on an Immobilized Polysaccharide Stationary Phase.

The residual enantiomers of three tris-(3-indolyl)-phosphane oxides bearing different alkyl groups (methyl, ethyl or i-propyl) in position 2 of the indole rings constituting the blades were separated on the immobilized type Chiralpak IC column in polar organic and reversed-phase modes. The good enantioselectivity and versatility of the IC CSP allowed easy isolation of the enantiomerically highly enriched samples suitable for configurational stability studies. The enantiomerization barriers of residual phosphane oxides were evaluated both by off-column techniques (CD signal and enantiomeric purity decay kinetics) and by dynamic enantioselective high-performance liquid chromatography (HPLC).

Indolylarylsulfones carrying a heterocyclic tail as very potent and broad spectrum HIV-1 non-nucleoside reverse transcriptase inhibitors.

We synthesized new indolylarylsulfone (IAS) derivatives carrying a heterocyclic tail at the indole-2-carboxamide nitrogen as potential anti-HIV/AIDS agents. Several new IASs yielded EC50 values <1.0 nM against HIV-1 WT and mutant strains in MT-4 cells. The (R)-11 enantiomer proved to be exceptionally potent against the whole viral panel; in the reverse transcriptase (RT) screening assay, it was remarkably superior to NVP and EFV and comparable to ETV. The binding poses were consistent with the one previously described for the IAS non-nucleoside reverse transcriptase inhibitors. Docking studies showed that the methyl group of (R)-11 points toward the cleft created by the K103N mutation, different from the corresponding group of (S)-11. By calculating the solvent-accessible surface, we observed that the exposed area of RT in complex with (S)-11 was larger than the area of the (R)-11 complex. Compounds 6 and 16 and enantiomer (R)-11 represent novel robust lead compounds of the IAS class.

Direct separation of the enantiomers of oxaliplatin on a cellulose-based chiral stationary phase in hydrophilic interaction liquid chromatography mode.

(R,R)-oxaliplatin is an anticancer enantiopure active pharmaceutical ingredient. Little attention has been devoted to the analysis of its enantiomeric composition. The enantioselective HPLC method reported in the current Pharmacopoeias shows clear disadvantages with regard to the low resolution and long elution times. In this work, it has been proven the applicability of a last generation polysaccharide-based chiral stationary phase (CSP), i.e. the Chiralpak IC-3, in the enantioseparation of oxaliplatin. Experimental results demonstrated the benefits arising from the development of enantioselective hydrophilic interaction liquid chromatography (HILIC) based strategies. A baseline separation with resolution factor of 5.8 was achieved using a 100mm×4.6mm I.D. IC-3 column set at the temperature of 40°C and a mobile phase consisting of acetonitrile-water 100:5 mixture. At a flow rate of 1mLmin(-1) the separation was completed within 8min. The optimized method was proven to be sensitive with LOD and LOQ of the enantiomeric impurity of 0.07 and 0.21µgmL(-1), respectively.

Effect of the water content on the retention and enantioselectivity of albendazole and fenbendazole sulfoxides using amylose-based chiral stationary phases in organic-aqueous conditions.

Four commercially available immobilized amylose-derived CSPs (Chiralpak IA-3, Chiralpak ID-3, Chiralpak IE-3 and Chiralpak IF-3) were used in the HPLC analysis of the chiral sulfoxides albendazole (ABZ-SO) and fenbendazole (FBZ-SO) and their in vivo sulfide precursor (ABZ and FBZ) and sulfone metabolite (ABZ-SO2 and FBZ-SO2) under organic-aqueous mode. U-shape retention maps, established by varying the water content in the acetonitrile- and ethanol-water mobile phases, were indicative of two retention mechanisms operating on the same CSP. The dual retention behavior of polysaccharide-based CSPs was exploited to design greener enantioselective and chemoselective separations in a short time frame. The enantiomers of ABZ-SO and FBZ-SO were baseline resolved with water-rich mobile phases (with the main component usually being 50-65% water in acetonitrile) on the IF-3 CSP and ethanol-water 100:5 mixture on the IA-3 and IE-3 CSPs. A simultaneous separation of ABZ (or FBZ), enantiomers of the corresponding sulfoxide and sulfone was achieved on the IA-3 using ethanol-water 100:60 (acetonitrile-water 100:100 for FBZ) as a mobile phase.

Retention behavior of proton pump inhibitors using immobilized polysaccharide-derived chiral stationary phases with organic-aqueous mobile phases.

In the present study, the chromatographic behavior of two immobilized polysaccharide-derived chiral stationary phases (CSPs), the Chiralpak ID-3 and Chiralpak IE-3, under aqueous mobile phases conditions is presented. Four proton pump inhibitors (PPIs) (omeprazole, lansoprazole, pentaprazole and rabeprazole) were selected as test compounds. The effect of the concentration of water in the mobile phase was investigated with respect to its contribution to enantioselectivity and retention. Under acetonitrile-water mobile phase conditions, retention behavior evidenced an interesting pattern. At lower water content, the retention factors decreased with increasing water and at higher water content a reversed trend was observed. These findings support the hypothesis that two retention mechanisms operated successively on the same CSP: the HILIC (with water-poor eluents) and RPLC (with water-rich eluents) modes. The retention factors were minimum in the intermediate region, corresponding to a water concentration of about 20%. Interestingly, the baseline separation of all PPIs investigated was optimized under organic-aqueous mobile phases containing a high water content (from about 50 to 65%). Thus, the dual retention behavior of the PPIs on the Chiralpak ID-3 and Chiralpak IE-3 made it possible to reach greener and harmless enantioselective conditions in a short analysis time.

3-methylcyclohexanone thiosemicarbazone: determination of E/Z isomerization barrier by dynamic high-performance liquid chromatography, configuration assignment and theoretical study of the mechanisms involved by the spontaneous, acid and base catalyzed processes.

Here, we report on the simultaneous direct HPLC diastereo- and enantioseparation of 3-methylcyclohexanone thiosemicarbazone (3-MCET) on a polysaccharide-based chiral stationary phase under normal-phase conditions. The optimized chromatographic system was employed in dynamic HPLC experiments (DHPLC), as well as detection technique in a batch wise approach to determine the rate constants and the corresponding free energy activation barriers of the spontaneous, base- and acid-promoted E/Z diastereomerization of 3-MCET. The stereochemical characterization of four stereoisomers of 3-MCET was fully accomplished by integrating the results obtained by chemical correlation method with those derived by theoretical calculations and experimental investigations of circular dichroism (CD). As a final goal, a deepened analysis of the perturbing effect exercised by the stationary phase on rate constant values measured through DHPLC determinations as a function of the chromatographic separation factor α of the interconverting species was successfully accomplished. This revealed quite small deviations from the equivalent kinetic values obtained by off-column batch wise procedure, and suggested a possible effective correction of rate constants measured by DHPLC approach.

Computer-aided molecular design of asymmetric pyrazole derivatives with exceptional enantioselective recognition toward the Chiralcel OJ-H stationary phase.

A computer-aided design of novel asymmetric pyrazoles with improved enantioselective properties was performed by docking experiments starting from a model of Chiralcel OJ chiral in the stationary phase. Synthesis and HPLC experiments confirmed the theoretical prediction and led to a detailed investigation of the enantioselective recognition process. For the first time, looking at the time spent by each enantiomer in contact with the CSP during long molecular dynamic simulations, the experimental analytical trend has been reproduced.

Application of 3 µm particle-based amylose-derived chiral stationary phases for the enantioseparation of potential histone deacetylase inhibitors.

In this work, we report on the difference in performance of the two 3 µm particle-based Chiralpak IA-3 and Chiralpak AD-3 chiral stationary phases (CSPs) in the direct resolution of four racemic cinnamyl 2-aminoanilides, endowed with histone deacetylase inhibitory activity. The 3 µm CSPs were explored to determine if they could provide an effective resolution of enantiomers in presence of alcoholic eluents such as pure methanol, ethanol and 2-propanol. Temperature variable enantioselective HPLC and subsequent van't Hoff analysis were performed. In most of cases the van't Hoff plots were found to show a non-linear behaviour. The knowledge of the enantiomeric elution order associated with the data coming from enantioselective HPLC permitted to advance some hypothesis about the groups involved in chiral recognition mechanism.

A chromatographic study on the exceptional enantioselectivity of cellulose tris(4-methylbenzoate) towards C5-chiral 4,5-dihydro-(1H)-pyrazole derivatives.

A set of ten C5-chiral 4,5-dihydro-(1H)-pyrazole derivatives was synthesized and analyzed by high-performance liquid chromatography (HPLC) on the polysaccharide-based Chiralcel OJ-H chiral stationary phase (CSP). The enantioseparations were carried out using pure ethanol as eluent. Different structural elements of the investigated compounds were recognized for obtaining a very high enantioselectivity. In order to clarify some aspects of the chiral discrimination process, the thermodynamic parameters associated to the enantiorecognition and the enantiomer elution order were established.