RESOLUTION OF CERTAIN RACEMIC PHARMACEUTICALS BY LIQUID CHROMATOGRAPHY

Abstract

It is well noticed in both academia and industry that a pair of enantiomers show different activity in the ‘chiral environment of our body’ and, in many countries, the regulatory agencies ask the industries to present full information on pharmacodynamics and pharmacokinetics of both the enantiomers including the stereoselective analytical methods before permitting a new drug to be registered and also insist on bringing (preferably) only the active enantiomer of a chiral drug to market. With such an awareness of these issues among those involved in the drug development, marketing and law enforcement efficient methods for verification of enantiomeric purity or to monitor stereoselective synthesis are increasingly required. Among the various available methods for establishing enantiomeric purity, liquid chromatographic techniques especially high performance liquid chromatography is commonly used. In recent years, applications of surfactant based aq. mobile phase for HPLC analysis (micellar liquid chromatography; MLC) of certain compounds has gained attention. But, the present thesis describes the application of the MLC for the first time for enantioseparations. From among the large number of β-adrenolytics in use, (RS)-propranolol, (RS)-metoprolol, (RS)-atenolol, (RS)-betaxolol, (RS)-carvedilol, (RS)-salbutamol and (RS)-bisoprolol, were selected for the development of sensitive methods of enantioseparation. Besides, (RS)-selenomethionine, (RS)-methionine, (RS)-cysteine and (RS)-penicillamine, were selected from among the pool of the amino acids considering the importance of sulphur containing amino acids specially. A chapter wise description is given below. Description of chapters The first chapter deals with preamble to present studies including introduction to enantiomers and their importance, their separation approaches and separation techniques. The chapter also includes present work, selection of chiral chromophoric moieties and experimental approach. The second chapter presents pharmaceutical importance and literature survey on enantioseparation of chosen β-adrenolytics and amino acids. Besides, physical and chemical properties of the chiral moieties and justification for choosing them for their application in presents studies has been described. ii The third chapter presents description of materials, equipment, preparation of stock solutions and extraction of active pharmaceutical ingredients from the commercial tablets, liquid chromatographic techniques, synthesis of chiral derivatizing reagents, and synthesis of diastereomeric derivatives (of racemic β-adrenolytics and amino acids) and their RP-HPLC, open column chromatography, detagging of diastereomeric derivative, characterization, calculation of chromatographic data and method validation. The fourth chapter deals with the enantioseparation of chosen racemic drugs, after derivatization with (S)-levofloxacin based chiral derivatizing reagents, using RP-HPLC and micellar liquid chromatography. It has been divided into three sections. Section A: Describes enantioseparation of (RS)-propranolol. “Diastereomeric derivatization of (RS)-propranolol were synthesized using (S)-levofloxacin-based new chiral derivatizing reagents (CDRs). Levofloxacin was chosen as the pure (S)-enantiomer for its high molar absorptivity (Ɛo ∼ 24000) and availability at a low price. Its -COOH group had N-hydroxysuccinimide and N-hydroxybenzotriazole, which acted as good leaving groups during nucleophilic substitution by the amino group of the racemic (RS)-propranolol; the CDRs were characterized by UV, IR, 1H-NMR, high resolution mass spectrometry (HRMS) and carbon, hydrogen, nitrogen, and sulphur elemental components analyser (CHNS). Diastereomeric derivatives were separated quantitatively using open column chromatography; absolute configuration of the diastereomeric derivatives was established and the reagent moiety was detagged under microwave-assisted acidic conditions. (S)- and (R)-propranolol as pure enantiomers and (S)-levofloxacin were separated, isolated and characterized. Optimized lowest-energy structures of the diastereomeric derivatives were developed using Gaussian 09 Rev. A.02 program and hybrid density functional B3LYP with 6-31G* basis set (based on density functional theory) for explanation of elution order and configuration. In addition, RP-HPLC conditions for separation of diastereomeric derivatives were optimized with respect to pH, concentration of buffer, flow rate of mobile phase and nature of organic modifier. HPLC separation method was validated as per International Conference on Harmonization guidelines. With the systematic application of various analytical techniques, absolute configuration of the diastereomeric derivatives (and the native enantiomers) of (RS)-propranolol was established”. Section B: Describes “An effective and simple method that successfully leads to liquid chromatographic enantioseparation of racemic β-adrenolytics by derivatization approach iii (with an example established with (RS)-metoprolol, and verified by using (RS)-atenolol). Chiral derivatizing reagents (CDRs) were synthesised using (S)-levofloxacin as the chiral moiety. The effectiveness of this method is not limited to enantioseparation but also to determine absolute configuration of diastereomeric derivatives. The method describes (i) synthesis of CDRs by reaction of (S)-levofloxacin with N-hydroxysuccinimide and N-hydroxybenzotriazole in presence of coupling reagent dicyclohexylcarbodiimide, (ii) synthesis of diastereomeric derivatives of racemic β-adrenolytics under microwave irradiation using the CDRs so synthesized, (iii) separation of diastereomeric derivatives by HPLC and open column chromatography, and (iv) determination of absolute configuration of diastereomeric derivatives. The (S)-Lfx based CDRs are efficient in chromatographic separation and provide very low limit of detection (LOD) and limit of quantitation (LOQ) they can be successfully used in trace analysis of several other racemic compounds which contain amino group in their structures (e.g., β-adrenolytics, amino acids etc)”. Section C: “The enantioseparation of a few commonly administered racemic β-adrenolytics (namely, betaxolol, carvedilol, salbutamol, and bisoprolol) has been achieved using a water micellar mobile phase containing surfactants (SDS and Brij-35) without organic solvents as a new approach in RP-HPLC. Two CDRs based on enantiomerically pure (S)-()-levofloxacin were synthesized using N-hydroxysuccinimide and N-hydroxybenzotriazole as the activation auxiliaries. Diastereomeric derivatives of the chosen β-adrenolytics were synthesized under microwave irradiation in a very short reaction time. The (S)-()-levofloxacin moiety enhanced molar absorbance of the diastereomeric derivatives resulting into very low LOD (1.618 ng mL−1 and 4.902 ng mL−1, respectively, for diastereomeric derivatives of (RS)-Bxl and better resolution with lower retention times (for all the analytes), in comparison to literature reports. There was 15-20 times less consumption of mobile phase because of lower retention time”. The fifth chapter deals with the enantioseparation of chosen racemic drugs, after derivatization with (S)-ketoprofen based chiral derivatizing reagents, using RP-HPLC and micellar liquid chromatography. It has been divided into two sections. Section A: Describes “the diastereomeric derivatives of racemic β-adrenolytic drugs [namely (RS)-propranolol, (RS)-metoprolol and (RS)-atenolol] were synthesized under microwave irradiation with (S)-ketoprofen based chiral derivatization reagents (CDRs) newly synthesized for this purpose. (S)-Ketoprofen was chosen for its high molar iv absorptivity (Ɛo ~ 40,000) and its availability as a pure (S)-enantiomer. Its -COOH group was activated with N-hydroxysuccinimide and N-hydroxybenzotriazole; these were easily introduced and also acted as good leaving groups during nucleophilic substitution by the amino group of the racemic β-adrenolytics. The CDRs were characterized by UV, IR, 1H-NMR, HRMS and CHNS. Separation of diastereomeric derivatives was achieved by RP-HPLC and open column chromatography. Absolute configuration of the diastereomeric derivatives was established with the help of 1H-NMR supported by developing their optimized lowest energy structures using Gaussian 09 Rev. A.02 program and hybrid density functional B3LYP with 6-31G* basis set (based on density functional theory), and elution order was established. RP HPLC conditions for separation were optimized and the separation method was validated. The limit of detection values were 0.308 and 0.302 ng mL1”. Section B: In this section “Micellar liquid chromatographic method has been developed for enantioseparation of four β-adrenolytics, namely, (RS)-salbutamol, (RS)-carvedilol, (RS)-bisoprolol, and (RS)-betaxolol. Both sodium dodecyl sulfate and Brij-35 were used as the surfactants in water as the mobile phase. Advantages for using both the surfactants in combination were investigated. Two (S)-ketoprofen-based activated esters were synthesized by activating its carboxylic group with N-hydroxybenzotriazole and N-hydroxysuccinimide, respectively. The esters were characterized by UV, IR, 1H-NMR, HRMS, and elemental analyses. These reagents were used for synthesis of diastereomeric derivatives of the chosen β-adrenolytics. These diastereomeric derivatives were enantioseparated on C18 column by high-performance liquid chromatography. Chromatographic conditions were optimized by varying concentration of surfactant and buffer, and pH. The method was validated according to International Conference of Harmonization guideline and the retention factor (k), selectivity factor (α), resolution factor (RS), and limit of detection and limit of quantification were calculated”. The sixth chapter describes enantioseparation of chosen racemic amino acids, after derivatization with (S)-ibuprofen, (S)-ketoprofen and (S)-levofloxacin based chiral derivatizing reagents, using Micellar liquid chromatography. “Micellar liquid chromatographic method has been developed for enantioseparation of four racemic amino acids, namely, (RS)-selenomethionine, (RS)-methionine, (RS)-cysteine and (RS)-penicillamine. The aqueous solution of sodium dodecyl sulphate and Brij-35 was prepared and used as mobile phase for HPLC analysis. Activated esters of (S)-ibuprofen, (S)-ketoprofen, and (S)-levofloxacin were synthesized by reacting them with N- v hydroxybenzotriazole. These esters were characterized by UV, IR, 1H-NMR, HRMS, and elemental analysis. These chiral reagents (activated esters) were used for the synthesis of diastereomeric derivatives of the chosen amino acids. The diastereomeric derivatives were separated on C18 column by micellar liquid chromatography. Chromatographic conditions were optimized by varying concentration of surfactant in aqueous solution, and by varying concentration and pH of the buffer. The method was validated according to ICH guidelines and the retention factor (k), selectivity factor (α), resolution factor (RS), and limit of detection and limit of quantification were calculated”.