Please use this identifier to cite or link to this item: http://hdl.handle.net/123456789/14644
Title: CHIRAL SEPARATION OF PHARMACEUTICAL COMPOUNDS USING LIQUID CHROMATOGRAPHY
Authors: Singh, Manisha
Keywords: Chiral Drugs
Desired Physiological
Active Ingredients
Pharmacologists
Issue Date: Jun-2016
Publisher: Dept. of Chemistry Engineering iit Roorkee
Abstract: In chiral drugs, “often only one of the enantiomers is responsible for the desired physiological effects while the other enantiomer is less active, inactive, or sometimes even causing adverse effects. Drugs composed of only one enantiomer can be developed to enhance the pharmacological efficacy” and eliminate some side effects. The regulatory agencies in many countries, “involved in the registration of new active ingredients, insist on registration of single enantiomer of a new drug and ask the pharmacologists to present full information on the stereochemistry and stereoselectivity of both the enantiomers including the necessary stereoselective analytical methods”. Therefore, efficient methods of enantioseparation are required to control the enantiomeric purity, or to separate the target molecule or one of its chemical precursors (obtained from conventional synthetic procedures), or for monitoring the completion of enantioselective reaction process (since the production of single enantiomer is a real difficult task). “Liquid chromatographic techniques especially thin layer chromatography and high performance liquid chromatography are commonly used”. Present thesis deals with the studies on direct and indirect enantioseparation of certain racemic compounds; these include some commonly administered and marketed drugs like (RS)-Atenolol, (RS)-Propranolol, (RS)-Baclofen, (RS)-Etodolac, ()-Bupropion, (RS)- Metoprolol, (RS)-Carvedilol, and certain didactic chiral aldehydes and ketones. A chapter wise brief account is given below. The first chapter deals with preamble to present studies including introduction to chirality, enantiomers and their separation. The chapter also includes discussion about liquid chromatography, HPLC and TLC. The CDRs, CSP and chiral selectors for TLC used in the present studies have been discussed in brief. The second chapter presents description of materials, equipments, preparation of stock solutions and extraction of active pharmaceutical ingredients from the commercial tablets. The third chapter describes formation of diastereomers of (RS)-Etodolac. Three pairs of diastereomers were synthesized using enantiomerically pure amines, namely, (R)-(+)-α- methyl benzyl amine, (S)-()-α,4-dimethylbenzylamine, (R)-()-1-cyclohexylethylamine. Separation of diastereomers was successful using C18 column and a binary mixture of methanol and triethyl ammonium phosphate buffer (TEAP) of pH 4.5 (80:20 v/v) as “mobile phase at a flow rate of 1 mL/min and UV detection at 223nm. Separation method was ii validated as per ICH guidelines. Derivatization reactions were carried out under conditions of stirring at room temperature (30 °C for 2 h) as well as under microwave irradiation (MWI) and the two types of diastereomers were compared”. Reaction conditions for derivatization were optimized with respect to mole ratio of CDR and (RS)-etodolac and MWI time. “Formation of diastereomers of (RS)-Etodolac was confirmed using LC-MS when [M+H]+ or [M]+ were recorded for the diastereomers. Lowest energy optimized structures of two diastereomers were drawn which confirmed three dimensional geometry of the diastereomers”. The fourth chapter describes the “synthesis of a new chiral derivatizing reagent from (S)- (+)-Naproxen and its application” for CN bond formation to prepare diastereomeric amides of (RS)-Propranolol, (RS)-Atenolol, (RS)-Carvedilol and (RS)-Metoprolol. Derivatization reactions were done at room temperature (30 °C for 30 min) under stirring conditions as well as under microwave irradiation (MWI). “Separation of diastereomers was achieved by open column chromatography”. 1H NMR spectra of the isolated and purified “diastereomers were recorded to establish the configurations of the first and second eluting diastereomers (and thus the elution order) and” to compare the chromatographic separation characteristics when the diastereomeric mixture was separated by “RP-HPLC (using C18 column and a binary mixture of MeCN and triethyl ammonium phosphate buffer of pH 3.5 (60:40 v/v) as mobile phase at a flow rate of 1 mL/min and UV detection at 230 nm). No racemization was observed throughout the study”. Test samples of -blockers were “isolated from commercial tablets and then were purified and characterized to be used as racemic standard. The conditions for derivatization and separation were optimized. Lowest energy optimized structures of the two diastereomers were developed using the Gaussian 09 Rev. A.02 program and hybrid density functional B3LYP with 6-31G basis set which supplemented 1H NMR interpretations and confirmed three dimensional geometry of the diastereomers. Separation method was validated as per ICH guidelines. The limit of detection and limit of quantification for each isomer were 0.4 ng/mL and 1.2 ng/mL, respectively”. The fifth chapter presents direct enantioresolution of (RS)-baclofen by ligand exchange TLC adopting two different approaches; (A) “TLC plates were prepared by mixing the ligand exchange reagent (LER) with silica gel slurry” and the chromatograms were developed with different achiral solvents or solvents having no chiral additive, and (B) the LER consisting of “Cu(II)-L-amino acid complex was used as chiral mobile phase additive and the plain plates iii of silica gel having no chiral selector were used. Cu(II) acetate and four L-amino acids (namely, L-tryptophan, L-histidine, L-proline and L-phenylalanine were used for the preparation of ligand exchange reagents. Spots were located by use of iodine vapour. Effect of temperature and mole ratio of Cu(II) to amino acid on enantioresolution was also studied. The results for the two methods have been compared and the issue of involvement of the Cu(II) cation for the best performance of the two methods has been discussed with respect to the same mobile phase”. The sixth chapter deals with enantioseparation of the chosen racemic drugs with a concept of using both achiral phases in TLC and HPLC. It has been divided into two sections. Section A: It deals with the enantioseparation of (RS)-Bupropion, (RS)-Baclofen and (RS)- Etodolac. It has been achieved by modifying the conventional ligand exchange approach. The Cu(II) complexes were first prepared with a few “L-amino acids, namely, L-proline, Lhistidine, L-phenylalanine, and L-tryptophan” and to these was introduced mixture of enantiomer pair of (RS)-Bupropion, or (RS)-Baclofen or (RS)-Etodolac. As a result, formation of a pair of diastereomeric complexes occurred by ‘chiral ligand exchange’ via the competition between the chelating L-amino acid and each of the two enantiomers from a given pair. The diastereomeric mixture formed in the pre-column process was loaded onto HPLC column. Thus, both the phases during chromatographic separation process were achiral (i.e., neither the stationary phase had any chiral structural feature of its own nor the mobile phase had any chiral additive). Separation of diastereomers was successful “using C18 column and a binary mixture of acetonitrile and TEAP buffer of pH 4.0 (60:40, v/v) as mobile phase at a flow rate of 1 mL/min and UV detection at 230 nm for (RS)-Bupropion, 220 nm for (RS)-Baclofen, and 223 nm for (RS)-Etodolac. Baseline separation of the two enantiomers was obtained with a resolution of 6.63 in less than 15 min. Section B: It deals with direct enantioresolution of (RS)-Etodolac involving both achiral phases in TLC. Enantiomerically pure “L-tryptophan, L-phenyl alanine, L-histidine, and Larginine, were used as chiral inducing reagents” (CIR); any of these was neither impregnated with silica gel (while making TLC plates) nor mixed with the mobile phase. The solvent system acetonitrile-dichloromethane-methanol, in different proportions, was found to be successful for enantioresolution. “Spots were located in iodine chamber. Effect of concentration of chiral inducing reagent and temperature on enantioresolution was studied”. iv The seventh chapter deals with a new method which has been developed involving, solid phase microwave-assisted conditions for synthesis of 2,4-dinitrophenyl hydrazone(s) of racemic carbonyl compounds wherein there occurred no inversion of configuration. The method provided high yields (91–95%) in short reaction time (4-6 min). The method proposed clearly has synthetic advantages over current practices. “The hydrazones were characterized by IR, 1H NMR and CHN analysis”. The hydrazones represent enantiomeric pairs tagged with a strong chromophore rather than diastereomers. The enantiomeric pairs were separated by HPLC using 1-acid glycoprotein column and the “best resolution of all the analytes was achieved with mobile phase” containing 0.5% 2-propanol in 10 mM citrate phosphate buffer at pH 6.5. The chromatographic peaks clearly showed base line separation with comparable peak areas and thus the results confirmed that there was no spontaneous inversion of configuration during derivatization.
URI: http://hdl.handle.net/123456789/14644
Appears in Collections:DOCTORAL THESES (chemistry)

Files in This Item:
File Description SizeFormat 
G25366-MANISHA-T.pdf3.21 MBAdobe PDFView/Open


Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.