New approaches to chiral discrimination in coupling between molecules

Results in the coupling of chiral molecules are reviewed from elementary points of view and some new results are given. We show that interactions between chiral molecules can be treated by using molecular quantum electrodynamics in electric and magnetic dipole approximation in ways different from standard diagrammatic perturbation theory. The interactions are the dispersive coupling of ground-state chiral molecules and excitation transfer, with emphasis on chiral discrimination. For ground-state molecules the coupling is dealt with first by calculating the coupling, at all separation distances, of electric and magnetic dipoles induced in the two molecules by fluctuations in the vacuum radiation field. The second method is the response by one chiral molecule to the field generated by the other. Excitation transfer is treated as the response by the accepting ground-state molecule to the dipole field of the donor. A novel variant in finding the rate of excitation transfer is by using Poynting's theorem. Received: 17 June 1998 / Accepted: 6 October 1998 / Published online: 16 March 1999     

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.     

Chiral Separation of Duloxetine and Its R-Enantiomer by LC

The direct enantioseparation of duloxetine and its R-enantiomer was achieved by HPLC using hydroxypropyl-β-cyclodextrin (HP-β-CD) as a chiral selector and a vancomycin chiral stationary phase (Chirobiotic V). Operational parameters, such as the concentration of HP-β-CD, buffer pH, organic modifiers, temperature and flow rate, were varied in order to achieve the desired retention time and resolution. These two enantioseparation methods developed gave a baseline resolution of the enantiomers. Finally, the HPLC-CSP method was selected to determine the enantiomeric purity of duloxetine drug substance due to its much shorter analysis time and better resolution. The limit of detection of this method was 0.06 μg mL⁻¹.     

Optimization of the Synthesis of 2,6–Dinitro-4-trifluoromethylphenyl Ether Substituted Cyclodextrin Bonded Chiral Stationary Phases

The comparisons of five different chiral stationary phases (CSPs) based on 2,6-dinitro-4-trifluoromethylphenyl (DNP-TFM) ether substituted β-cyclodextrin are presented. The five CSPs differ from each other in the linkage/spacer chemistry, or on the position of the substituents on β-cyclodextrin, or in the sequence of the synthetic procedure. The results show that there are two optimum combinations: (1) DNP-TFM randomly substituted on the β-cyclodextrin as the chiral selector along with a carbamate linkage chain bonding it to the silica support; and (2) β-cyclodextrin derivatized by DNP-TFM substituents only on the C-2 and C-3 positions of the cyclodextrin with an ether linkage chain anchoring it to the silica gel. These two combinations show complementary separations for some enantiomers. The spacer chain effect is much more pronounced for the CSP based on the β-cyclodextrin derivatives with DNP-TFM substituents only on C-2 and C-3 positions than its randomly substituted counterpart. The sequence of derivatizing the cyclodextrin and attaching it to silica gel also affects its selectivity and efficiency. The β-cyclodextrin should be derivatized before it is linked to the silica gel.     

A Phenyl α-Nitronyl Nitroxide with a Forced Chiral Conformation

Summary.  A new phenyl α-nitronyl nitroxide bearing a hydroxyl group at position 2 of the aromatic ring and a chiral methyl lactate substituent at position 5 has been synthesized with the aim of combining the magnetic properties of this kind of radical with the optical properties endowed by the chiral group. The optically active compound forms intramolecular hydrogen bonds between the OH group and one of the NO groups and shows a large torsion angle between the two rings when compared with similar radicals with no substituent in this position. Therefore, the optical properties are distinct. The optical and magnetic properties of the new radical in both solution and solid state are presented. Received June 23, 2000. Accepted (revised) September 18, 2000     

Thermal characterization of antimicrobial drug ornidazole and its compatibility in a solid pharmaceutical product

The ornidazole drug substance presents melt at approximately 90 °C (∆T = 85–98 °C), which is critical for its use on pharmaceutical manufacturing process. This work aimed the thermal characterization of ornidazole raw-material synthesized by three different manufacturers from India, China, and Italy, using the thermoanalytical techniques of DTA, DSC, and TG, besides the verification of its stability and compatibility as a solid pharmaceutical product by the analysis of its binary mixtures (BM) with excipients and a tablet formulation. The characterization includes the thermal decomposition kinetic investigation by Ozawa model using Arrhenius equation and drug purity determination by Van’t Hoff equation. The DSC purity determination and precision were compared with results from UV–Vis spectrophotometric and liquid chromatography, showing an adequate correlation before being recommended as a general method for purity assay. The drug raw-materials presented similar quality and zero-order kinetic behavior, besides showing differences on thermal stability. The drug presented compatibility with the tested excipients since the BM studied presented melting at the same temperature range as the drug and a decomposition temperature similar to the drug for two of the BM, and at a higher temperature for the others three of the BM evaluated, which presented excipients with higher molecular structure, capable of spatial coating on the small drug molecule promoting a physical interaction pharmaceutical acceptable. The tablet was processed by wet granulation and compressed under normal conditions of pressure and temperature, maintaining the physical properties of solid drug approving the manufacturing process used. In this study, the thermal analysis was used with success as an alternative method to characterize, quantify, and perform a preformulation study.     

Comprehensive two-dimensional gas chromatography applied to illicit drug analysis

Multidimensional gas chromatography (MDGC), and especially its latest incarnation—comprehensive two-dimensional gas chromatography (GC × GC)—have proved advantageous over and above classic one-dimensional gas chromatography (1D GC) in many areas of analysis by offering improved peak capacity, often enhanced sensitivity and, especially in the case of GC × GC, the unique feature of ‘structured’ chromatograms. This article reviews recent advances in MDGC and GC × GC in drug analysis with special focus on ecstasy, heroin and cocaine profiling. Although 1D GC is still the method of choice for drug profiling in most laboratories because of its simplicity and instrument availability, GC × GC is a tempting proposition for this purpose because of its ability to generate a higher net information content. Effluent refocusing due to the modulation (compression) process, combined with the separation on two ‘orthogonal’ columns, results in more components being well resolved and therefore being analytically and statistically useful to the profile. The spread of the components in the two-dimensional plots is strongly dependent on the extent of retention ‘orthogonality’ (i.e. the extent to which the two phases possess different or independent retention mechanisms towards sample constituents) between the two columns. The benefits of ‘information-driven’ drug profiling, where more points of reference are usually required for sample differentiation, are discussed. In addition, several limitations in application of MDGC in drug profiling, including data acquisition rate, column temperature limit, column phase orthogonality and chiral separation, are considered and discussed. Although the review focuses on the articles published in the last decade, a brief chronological preview of the profiling methods used throughout the last three decades is given.     

Chiral separation of raltitrexed by cyclodextrin-modified micellar electrokinetic chromatography

A rapid and effective method was developed for the chiral separation of raltitrexed (RD) enantiomers by carboxymethyl-beta-cyclodextrin (CM-β-CD)-modified micellar electrokinetic chromatography (MEKC). Optimization of conditions including the type and concentration of the chiral selector, concentration of sodium dodecyl sulfate (SDS), pH and concentration of the background electrolyte (BGE), capillary temperature, and applied voltage was investigated. The enantiomers of raltitrexed could be separated with satisfactory resolution and linear response by using 75 mM Tris-phosphate at pH 8.0 containing 30 mM SDS and 8 mM CM-β-CD as buffer system. Furthermore, the usefulness of this method was demonstrated in a purity test of a real synthetic drug sample. Figure Chiral separation of raltitrexed by CM-β-CD MEKC was optimized and applied to test the purity of a synthetic drug sample     

Enantiomer separation by CEC

Three chiral compounds were successfully separated in a short time with two enantiomer separation models on packed-capillary electrochromatography (CEC). (i) 75 μm I.D. capillaries were packed with 5 μm β-cyclodextrin (βCD) chiral stationary phase (CSP). Effects of voltage, pH and concentration of organic modifier on electroosmotic flow (EOF) and chiral separations were investigated systematically. Enantiomers of a neutral compound (benzoin) and a neutral drug (mephenytoin) were separated within a short time with high efficiency. Efficiency of 32 000 theoretical plates per meter and resolution (R8) of 1.42 were achieved for enantiomers of benzoin using a βCD packed column with 6.2crn packed length. Efficiency of 45 000 theoretical plates per meter andR8 of 3.40 were obtained for enantiomers of mephenytoin. Especially, the enantiomer separation of mephenytion was performed in just 3.4 min with R₈ of 2.60. (ii) 75 μm I.D. capillary was packed with octadecylsilica particles (ODs). Chiral separation of a basic drug, propranolol, was studied with chiral agent, via addition of the dimethyl-β-cyclodextrin (DM β-CD) directly into the mobile phase on this column. Baseline separation and efficiency of 81 000 theoretical plates per meter were achieved for propranolol. Project supported by the Natural Science Foundation of Liaoning Province, China, the National Natural Science Foundation of China (Grant No.29875030), and the Excellent Young Scientist Award from the National Natural Science Foundation of China. (Grant No.29725512).     

Separation of lansoprazole enantiomers in human serum by HPLC

Summary A new and simple HPLC method is described for the separation and quantitative determination of the (+)-and (−)-enantiomers of lansoprazole. The analytes were extracted from serum as previously described for whole lansoprazole [K. Borner, Chromatographia 45, 450–452 (1997)]. The enantiomers were separated by chromatography on a CHIRAL-AGP^R column which contained covalently bound acid α₁-glycoprotein as chiral selector. In the pure drug the (−)/(+) ratio was 0.99:1.01. In serum of twelve human volunteers the concentration of the (−)-enantiomer was 3 to 5 times higher than that of the (+)-enantiomer. Both enantiomers differ remarkably in their pharmacokinetics.     

A Validated Chiral LC Method for the Determination of Enantiomeric Purity of Pemetrexed Disodium on an Amylose-Based Chiral Stationary Phase

A simple and new isocratic normal phase chiral HPLC method has been developed for the determination of enantiomeric purity of pemetrexed disodium (l-enantiomer) in bulk drugs with a short run time of about 20 min. Chromatographic separation of l and d-enantiomers of pemetrexed disodium was achieved on an amylose based chiral stationary phase using a mobile phase consists of hexane, ethanol and trifluoro acetic acid. The resolution between the enantiomers was found to be more than 2.0. The system precision and method precision were found to be within 5% RSD for the distomer (d-enantiomer) at its specification level (i.e. not more than 1.0% w/w). The limit of detection and limit of quantification of distomer were 1.6 and 5 μg mL⁻¹, respectively for 10 μL injection volume. The percentage recovery of distomer was ranged from 90.6 to 105.7 in bulk drug samples. The test solution was found to be stable in the diluent for 48 h. The method was found to be specific for the enantiomers of pemetrexed disodium and can be conveniently used for the quantification of undesired d-enantiomer present in the bulk drug samples of pemetrexed disodium.     

Chiral HPLC analysis of venlafaxine metabolites in rat liver microsomal preparations after LPME extraction and application to an in vitro biotransformation study

A three-phase LPME (liquid-phase microextraction) method for the enantioselective analysis of venlafaxine (VF) metabolites (O-desmethylvenlafaxine (ODV) and N-desmethylvenlafaxine (NDV) in microsomal preparations is described for the first time. The assay involves the chiral HPLC separation of drug and metabolites using a Chiralpak AD column under normal-phase mode of elution and detection at 230 nm. The LPME procedure was optimized using multifactorial experiments and the following optimal condition was established: sample agitation at 1,750 rpm, 20 min of extraction, acetic acid 0.1 mol/L as acceptor phase, 1-octanol as organic phase and donor phase pH adjustment to 10.0. Under these conditions, the mean recoveries were 41% and 42% for (−)-(R)-ODV and (+)-(S)-ODV, respectively, and 47% and 48% for (−)-(R)-NDV and (+)-(S)-NDV, respectively. The method presented quantification limits of 200 ng/mL and it was linear over the concentration range of 200–5,000 ng/mL for all analytes. The validated method was employed to study the in vitro biotransformation of VF using rat liver microsomal fraction. The results demonstrated the enantioselective biotransformation of VF.      

Validated Enantioselective LC Method, with Precolumn Derivatization with Marfey’s Reagent, for Analysis of the Antiepileptic Drug Pregabalin in Bulk Drug Samples

An enantioselective high-performance liquid chromatographic method, with precolumn derivatization with Marfey’s chiral reagent, sodium 2,4-dinitro-5-fluorophenyl-l-alanine amide, has been developed for resolution of the enantiomers of a new antiepileptic drug, pregabalin, in the bulk drug. The diastereomers of the pregabalin enantiomers were resolved to baseline on a reversed-phase ODS column with a 60:40 (v/v) mixture of aqueous 0.2% triethylamine (pH adjusted to 3.5 with dilute orthophosphoric acid) and acetonitrile as mobile phase. Resolution between the diastereomers was not less than five. The method was extensively validated and proved to be robust. The calibration plot was indicative of an excellent linear relationship between response and concentration over the range 750 (LOQ) to 7,500 ng L⁻¹ for the R enantiomer. The limits of detection and quantification of the R enantiomer were 250 and 750 ng L⁻¹, respectively, for an injection volume of 10 μL. Recovery of the R enantiomer from bulk drug samples of pregabalin ranged from 97.5 to 101.76%. Solutions of pregabalin in water and in the mobile phase were found to be stable for at least 48 h. The method was found to be suitable and accurate for quantitative determination of the R enantiomer in the bulk drug. It can be also used to test the stability of samples of pregabalin.     

A Validated Chiral LC Method for the Enantiomeric Separation of Repaglinide on Amylose Based Stationary Phase

A simple, isocratic, normal phase chiral HPLC method was developed and validated for the enantiomeric separation of repaglinide, (S)-(+)-2-ethoxy-4-N [1-(2-piperidinophenyl)-3-methyl-1-butyl] aminocarbonylmethyl] benzoic acid, an antidiabetic in bulk drug substance. The enantiomers of repaglinide were resolved on a ChiralPak AD-H (amylose based stationary phase) column using a mobile phase consisting of n-hexane: 2-propanol:trifluoroacetic acid (95:5:0.2 v/v/v) at a flow rate of 1.0 mL min⁻¹. The resolution between the enantiomers was found to be not >3.5 in optimized method. The presence of trifluoroacetic acid in the mobile phase played an important role, in enhancing chromatographic efficiency and resolution between the enantiomers. The developed method was extensively validated and proved to be robust. The calibration curve for (R)-enantiomer showed excellent linearity over the concentration range of 900 ng mL⁻¹ (LOQ) to 6,000 ng mL⁻¹. The limit of detection and limit of quantification for (R)-enantiomer were 300 and 900 ng mL⁻¹, respectively. The percentage recovery of the (R)-enantiomer ranged between 98.3 and 101.05% in bulk drug samples of repaglinide. Repaglinide sample solution and mobile phase were found to be stable up to 48 h. The developed method was found to be enantioselective, accurate, precise and suitable for quantitative determination of (R)-enantiomer in bulk drug substance.     

Experimental design methodologies to optimize the CE separation of epinephrine enantiomers

Summary A capillary electrophoretic method using a chiral selector was optimized by experimental design for the enantioresolution of epinephrine enantiomers. Two β-cyclodextrins derivatives, namely heptakis-2,6-di-O-methyl-β-cyclodextrin and carboxy-methyl-β-cyclodextrin, respectively neutral and charged, were used as chiral selectors employing an uncoated capillary. By using a statistical experimental design in which all factors are varied at the same time, it was possible to optimize the method with regard to the resolution between peaks and the two migration times. A fractional factorial design and a central composite design were used. A compromise between conflicting goals, such as maximization of resolution and minimization of analysis time, was found by means of a desirability function D. Balancing these goals against each other, the most acceptable solution to the problem was found and the optimized method gave a fast separation with complete resolution between the adrenaline enantiomers. The response surfaces obtained confirmed the robustness of the method.     

A Validated Chiral HPLC Method for the Determination of Enantiomeric Purity of R-β-amino-β-(4-methoxyphenyl) Propionic Acid

A normal phase chiral LC method for chiral purity evaluation of β-amino-β-(4-methoxyphenyl) propionic acid was developed on donor–acceptor (pirkle) column. The chiral stationary phase used was a 250 × 4.6 mm (R, R) Whelk-01 with 5 μm particle size, which was accompanied with a 1 cm long guard column. The “hybrid” pi-electron donor–acceptor based stationary phase (R, R) Whelk-01 was found to be enantiomeric selective for (R) and (S) enantiomers of β-amino-β-(4-methoxyphenyl) propionic acid with a resolution >2.5. The concentration of 2-propanol and TFA in the mobile phase plays an important role on the chrmatographic efficiency and resolution between the enantiomers. The limit of detection and limit of quantification of (S) enantiomer was 0.3 and 1.0 μg mL^-1 for 20 μL injection volume. The percentage RSD of the peak area of six replicate injections of (S) enantiomer at LOQ concentration was 4.5. The percentage recovery of (S) enantiomer from (R) enantiomer samples ranged from 92 to 100. The test solution was observed to be stable up to 24 h after the preparation. The developed method was also checked by different analysts and on different lots of columns, reagents and it was proved to be rugged. The developed normal phase chiral LC method can be used for the determination of the enantiomeric purity of R-β-amino-β-(4-methoxyphenyl) propionic acid.     

Enantiomer Separation of the Four Stereoisomers of 1-(4-Hydroxy-3-Methoxy)-Phenyl-2-[4-(1,2,3-Trihydroxy-Propyl)-2-Methoxy]-Phenoxy-1,3-Propandiol from Hydnocarpus annamensis by Capillary Zone Electrophoresis with HP-β-CD as Chiral Selector

A capillary zone electrophoresis method with HP-β-CD as chiral selector was established for the chiral separation of four stereoisomers of 1-(4-hydroxy-3-methoxy)-phenyl-2-[4-(1,2,3-trihydroxy-propyl)-2-methoxy]-phenoxy-1,3-propandiol for the first time, which were isolated from Hydnocarpus annamensis. The effects of chiral selector type and concentration, buffer composition, pH and concentration, and cartridge temperature on the enantioseparation were investigated. A baseline separation of the four stereoisomers was achieved in less than 18 min under the optimized conditions: 40 mmol L⁻¹ Borax–NaOH buffer (pH 10.02) in the presence of 100 mmol L⁻¹ HP-β-CD at 15°C and 30 kV. The experimental results showed that the method by capillary zone electrophoresis for the separation of four stereoisomers is powerful, sensitive and fast, requires less amounts of reagents, and can be employed as a reliable alternative to other methods.     

Intact Glycoform Characterization of Erythropoietin-α and Erythropoietin-β by CZE-ESI-TOF-MS

Glycosylation of recombinant human erythropoietin (rhEPO) is a post-translational process which depends on the type of cell in which rhEPO is synthesized, but also on the cell culture conditions and the final purification steps. These glycosylation modifications alter the biological activity, solubility and lifetime of rhEPO in blood. Thus, a rapid and simple method for the elucidation of the carbohydrate microheterogeneity of rhEPO is needed in order to evaluate a certain manufacturing process or assure the quality of the final product. Based on a recently developed method [1], the accurate mass determination of the intact glycoforms from two types of commercial rhEPO (epoetin-α and epoetin-β) by capillary electrophoresis-electrospray-time of flight-mass spectrometry is presented. The sample treatment consists of a fast and simple preconcentration step of the ready-to-use drug achieved by a centrifugal filter device. Characterization of the carbohydrate composition of each single glycoform is performed, in agreement with the results in glycan and glycopeptide analysis reported by other authors. The main differences between the carbohydrate structures of both epoetins are shown: the existence of two additional basic sialic acid isoforms for epoetin-β and the higher degree of acetylation for epoetin-α. The agreement of the main glycoforms for both epoetins is shown by molecular mass agreement. The high accuracy and reproducibility of the mass measurements with a standard deviation below 1 Da is proved by repeated analysis of European Pharmacopoeia rhEPO. Summarizing, the presented method enables the fast and reliable characterization of intact rhEPO in pharmaceutical products.Presented at: CE in the Biotechnology & Pharmaceutical Industries: 7th Symposium on the Practical Applications for the Analysis of Proteins, Nucleotides and Small Molecules, Montreal, Canada, August 12–16, 2005     

The preparative chromatographic enantioseparation of a racemic morphanthridine analog on a chiral stationary phase

Summary The preparative chromatographic enantioseparation of a chiral morphoanthridine analog has been performed on an analytical column using amylose-tris(3,5-dimethylphenylcarbamate) as chiral stationary phase. The racemate (100 mg) was resolved to baseline within 15 min. This paper describes the development of the method, estimation of the capacity of the chiral stationary phase and discussed the potential of the chromatography if performed under preparative conditions. From the results and calculations presented it seems likely that the resolution of 70 tons year⁻¹ could easily be achieved on 30 kg of stationary phase with a mobile-phase consumption of only 720 L day⁻¹.     

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.