LIQUID CHROMATOGRAPHIC CHIRAL SEPARATION OF CERTAIN IMPORTANT PHARMACEUTICALS

Abstract

Enantiomers are known to show different pharmacodynamic and pharmacokinetic properties after administration. Systematic investigations of biological activities of both enantiomers have become indispensable in areas, such as pharmacology, medicine, life sciences, asymmetric synthesis and studies of structure-function relationship. Various analytical methods have been used for separation of racemic mixtures into enantiomers; TLC and HPLC are widely used for resolution of a variety of chiral pharmaceuticals. Certain drugs/chiral compounds having wide applications in chemistry, biochemistry, medicine, etc have been chosen for present studies for separation of their enantiomers; these include, proteinogenic-α-amino acids, DL-selenomethionine (SeMet), β-blockers [(RS)-orciprenaline (Orc), (RS)-atenolol (Atl), (RS)-propranolol (Prl), (RS)-betaxolol (Bel) and (±)-isoxsuprine (ISP)] and (RS)-fluoxetine (Flx). Both direct and indirect approaches have been applied using different chiral derivatizing reagents (CDRs), chiral stationary phases (CSPs) and chiral selectors. The first chapter covers preamble to present studies and an introduction to chirality and various other related aspects such as enantiomers and their biological significance, chromatographic enantioseparation approaches, chiral selector (as chiral impregnating reagent, chiral mobile phase additive and chiral inducing reagent for TLC), CSPs, CDRs and their applications (for HPLC). The Second chapter presents a detailed description of common experimental procedures used for present studies; it includes instrumentation, materials, preparation of stock solutions, and extraction of active pharmaceutical ingredient from their drug formulations. The methods for synthesis of CDRs along with their characterization data, synthesis of diastereomers have been explained in detail. Separation of diastereomers using reversed phase-HPLC and TLC are described in subsequent chapters. A total of 17 CDRs were synthesized using DFDNB (1,5-Difluoro-2,4-dinitrobenzene), CC (cyanuric chloride; trichloro-s-triazine) and (S)-Naproxen ((S)-Nap), as starting materials. Characterization and determination of chiral purity of all the CDRs is also described in this chapter. Theoretical calculation using Gaussian 09 Rev A. 02 program and hybrid (ii) density functional B3LYP with 6-31G basis set was used for developing optimized structures of diastereomers to support the experimental results of elution sequence. Direct TLC approaches are described for separation of racemic compounds and isolation of their native enantiomers. The Third chapter is devoted to general introduction of β-blockers, literature survey on their enantioseparation by liquid chromatographic and to the present study on direct and indirect enantioseparation of β-blockers. It has been divided into three sections. Section A: This section describes use of DFDNB based CDRs (CDR 1-5 and 7-9) for synthesis of diastereomers of β-blockers, namely, Orc, Atl, Prl, Bel and ISP, along with their separation by RP-HPLC and RP-TLC. Following CDRs were used for synthesis of diastereomers of β-blockers: DFDNB as the Structural moiety CDR number Chiral auxiliary β-blockers 1. SMLC Orc, Atl, Prl, Bel 2. SBLC Orc, Atl, Prl, Bel, ISP 3. L-Met Orc, Atl, Bel 4. L-Leu 5. L-Val Orc, Atl, Bel, ISP 7. L-Ala 8. (S)-(+)-1-cyclohexylethylamine Orc, Atl, Prl, Bel 9. (R)-(+)-α-methyl benzylamine) These diastereomers were separated by RP-HPLC on a C18 column with detection at 340 nm using a linear gradient of acetonitrile and aq trifluoroacetic acid as the mobile phase components. The conditions of derivatization and chromatographic separation were optimized. The method was validated for accuracy, precision, limit of detection, and limit of quantification. The separation behavior (in terms of, retention time and resolution) of the diastereomers was compared. (iii) All the three pairs of diastereomers of ISP were also separated by RP-TLC on DC Kieselgel 60 RP*18 F254S stationary phase using MeCN and TEAP buffer as mobile phase. The influence of pH and temperature was examined on separation. The separated diastereomers were visible as bright yellow spots in ordinary light. LOD was estimated. Section B: This section deals with the experiment and results for development of RP-HPLC method for enantioseparation of β-blockers. Separation was carried out on certain CC based DCT reagents (CDR 10-15). These reagents were used for synthesis of eighteen pairs of diastereomers of β-blockers (Atl, Orc and Bel) under microwave irradiation and stirring method. Following CC based CDRs were used for synthesis of diastereomers of Atl, Orc, and Bel (the chosen β-blockers): CDR number Chiral auxiliary in CC moiety 10. SBLC 11. SMLC 12. L-met 13. L-Leu 14. L-Val 15. L-Ala These diastereomers were separated by RP-HPLC on a C18 column with detection at 230 nm using a linear gradient of acetonitrile and aq trifluoroacetic acid. Derivatization and chromatographic separation conditions were optimized. The method was validated in terms of accuracy, precision, limit of detection, and limit of quantification. Section C: This section presents direct TLC separation of enantiomers of β-blockers (Atl, Orc and Bel) using L-Glu as chiral selector. Direct TLC separation was (iv) performed using L-Glu as (i) chiral impregnating reagent, (ii) as chiral mobile phase additive (CMPA), and (iii) as chiral inducing reagent (CIR). Different binary, ternary and quaternary mixtures of a variety of solvents (such as CH3CN, CH3OH, H2O and CH2Cl2) were tried systematically to achieve enantiomeric resolution. Spots were located in iodine chamber. The developed TLC method was validated for precision (RSD) and LOD. The influence of pH, temperature and amount of chiral selector was examined on enantioseparation. Separated enantiomers were isolated and optical purity was determined. Optimized structures of the transient diastereomers of the three β-blockers were drawn by Gaussian 09 Rev A. 02 program, to confirm their migration order. The Fourth chapter deals with literature survey on liquid chromatographic enantioseparation of fluoxetine and its indirect enantioseparation by RP-HPLC and RP-TLC was carried out herein. It comprises of two sections. Section A: This section describes the synthesis of four DFDNB based CDRs (CDR 1, 2, 4 and 5) and their use for synthesis of diastereomers of Flx. All the four pairs of diastereomers were separated by RP-HPLC on a C18 column with detection at 340 nm using a linear gradient of mobile phase containing acetonitrile and aq trifluoroacetic acid. Method validation was carried out for accuracy, precision, limit of detection, and limit of quantification. Simultaneously, RP-TLC of diastereomers of Flx prepared with DFDNB based CDRs (CDR 4 and 5), has been carried out on DC Kieselgel 60 RP*18 F254S stationary phase using MeCN and TEAP buffer as mobile phase. The effect of pH and temperature was examined on separation. The separated diastereomers were detected in ordinary light. Section B: It deals with the application of four CC based CDRs (CDR 10, 11, 13 and 14), for synthesis of diastereomers of Flx. The four pairs of diastereomers, so synthesized, were separated by RP-HPLC on a C18 column with detection at 230 nm using a linear gradient of mobile phase containing acetonitrile and aq trifluoroacetic acid. (v) The fifth chapter presents background and literature survey on liquid chromatographic enantioseparation of SeMet. In view of the literature survey direct and indirect enantioseparation of selenomethionine was carried out. It has been divided into three sections. Section A: It describes the use of CDR 16 (Nap-Phth) for synthesis of diastereomers of selenomethionine prior to their separation. Resulting diastereomers were separated by RP-HPLC using C18 column and gradient eluting mixture of MeCN with TEAP buffer as mobile phase. Detection of separated diastereomers was carried out at 231 nm using PDA detector. Validation of the method was carried out in terms of linearity, accuracy, precision, recovery and LOD. Section B: It deals with separation of diastereomers of SeMet by RP-HPLC and RP-TLC. The diastereomers were synthesized using CDR 2, 3 and 6 under microwave irradiation and conventional heating. The three pairs of diastereomers were separated on C18 column using a linear gradient of MeCN and TFA, detection at 340 nm). Migration order of diastereomers of SeMet was supported by theoretical calculation. Simultaneously, these three pairs of diastereomers of SeMet were separated by RP-TLC on DC Kieselgel 60 RP*18 F254S stationary phase using the mobile phase (MeCN and TEAP buffer). Chromatographic conditions were optimized. The developed TLC method was validated in terms of linearity, accuracy, precision, recovery and LOD. The separated diastereomers were detected in ordinary light. Section C: This section presents direct TLC enantioresolution of SeMet using (−)-Quinine as Chiral Selector. Direct TLC separation was performed using (−)-quinine as (i) chiral impregnating reagent, (ii) as chiral mobile phase additive (CMPA), and (iii) as chiral inducing reagent. Different binary, ternary and quaternary mixtures of a variety of solvents (such as CH3CN, CH3OH, H2O and CH2Cl2) were tried systematically to achieve enantiomeric resolution. The spots were located by ninhydrin solution and also in iodine vapor. Precision (RSD) and LOD were determined for direct TLC method. The influence of pH, temperature and amount of chiral selector was examined on enantioseparation. Spots were scrapped from TLC plates and native enantiomers were isolated. (vi) The sixth chapter describes separation of enantiomers of proteinogenic-α-amino acids by HPLC using indirect method. CDR 17 was used as a CDR for the synthesis of diastereomers of eighteen amino acids. The diastereomers were separated by RP-HPLC using C18 column and gradient eluting mixture of MeCN with TEAP buffer. Detection was made at 231 nm using PDA detector. Chromatographic conditions were varied to obtain a good resolution. Method validation was carried out in terms of linearity, accuracy, precision, recovery and LOD.