Please use this identifier to cite or link to this item: http://localhost:8081/xmlui/handle/123456789/1508
Authors: Shivani
Issue Date: 2009
Abstract: Chirality plays an important role not only in pharmacodynamics but also in pharmacokinetics, involving the absorption, distribution, metabolism and excretion (ADME) of the drug. Usually the desired pharmacological activity resides in one enantiomer while the other enantiomer may be toxic, less active or possesses undesirable side effects. These days, substantial numbers of single isomer pharmaceuticals are entering the commercial market. United State Food and Drug Administration (US FDA), European Committee for Proprietary Medicinal Products and other drugs regulatory agencies have restricted the marketing of racemic drugs. Therefore, dosage concentration of a racemic drug is an illusion to the patients as normally 50% drug is active while the other 50% is inactive or may be toxic. Analytical methods for separating and determining enantiomers include fractional crystallization, chromatographic techniques such as high-performance liquid chromatography (HPLC), high-performance thin layer chromatography (HPTLC), gasliquid chromatography (GLC), thin layer chromatography (TLC), and supercritical fluid chromatography (SFC). The development of liquid chromatographic methods used for enantiomeric separation of racemates has represented an intensive area of research in the pharmaceutical industry. There are two major approaches for chiral separation, precolumn derivatization with a chiral reagent followed by separation of resulting diastereomers (the indirect approach) and the direct approach may use a chiral mobile phase additive (CMPA) or a chiral stationary phase (CSP) or the chiral selector is immobilized/ impregnated with the stationary phase. Chiral separations enjoy a great analytical importance in various fields and both from academic and industrial points of view. Therefore, studies have been carried out on direct and indirect enantiomeric resolution of some P-blockers, a-amino acids (proteinogenic and non-proteinogenic), penicillamine and mexiletine. These compounds have wide applications in the field of chemistry, biochemistry, medicine, etc. The first chapter is an introduction to chirality of pharmaceuticals, chiral chromatographic approaches, selection criteria of CDRs and their applications. The CDRs have been classified in different categories depending upon the nature of functional group present in analytes. The literature related to the class of compounds chosen has been cited in subsequent chapters. Second chapter deals with the description of the common experimental methods used for present studies. It includes materials, instrumentation, methods for synthesis of six new chiral derivatizing reagents (CDRs) and eleven others, and methods for synthesis of diastereomers. Of the six new CDRs, four were synthesized by substituting one of the fluorine atoms in l,5-difluoro-2,4-dinitrobenzene (DFDNB) with four optically pure amines, namely, [(#)-(-)-1-eyelohexylethylamine, (+)-dehydroabietylamine, (-)-cismyrtanylamine, and (S)-(-)-a,4-dimethylbenzylamine]. The other two were dinitrophenyl-L-Pro-N-hydroxysuccinimide ester and N-succinimidyl-(5)-2-(6- methoxynaphth-2-yI) propionate. The remaining ten CDRs were synthesized by substituting one of the fluorine atoms in l,5-difluoro-2,4-dinitrobenzene (DFDNB) with seven amino acid amides [LAla- NH2, L-Phe-NH2, L-Val-NH2, L-Leu-NH2, L-Met-NH2, L-Pro-NH2, and D-Phg- NH2], and three amino acids [L-Ala, L-Val and L-Leu]. In addition, one anhydride namely, [(S,S)-0,0'-d\-p-to\uoy\ tartaric anhydride] was synthesized and used as CDR. The characterization data for the new CDRs only is described. The details with respect to separation by TLC, HPLC and GC are described in different chapters along with results and discussions. The third chapter deals with enantioseparation of proteinogenic and nonproteinogenic a-amino acids by TLC and HPLC. Eight non-proteinogenic a-amino acids were derivatized with l-fluoro-2,4-dinitrophenyl-5-L-alaninamide (Marfey's reagent, MR, FDNP-L-Ala-NH2), FDNP-L-Phe-NH2, FDNP-L-Val-NH2, and FDNP-LPro- NH2. The resultant diastereomers were separated on a reversed-phase C8 column with gradient elution using mixture of aq trifluoroacetic acid (TFA) and acetonitrile (MeCN) with detection at 340 nm. The effects of flow rate and run time on HPLC separation were studied. The diastereomers of these amino acids prepared with FDNPL- Leu-NH2 and other four CDRs were also separated on NP and RP-TLC. Diastereomers of 18 proteinogenic amino acids were prepared with MR and FDNP-L-Leu-NH2; these were separated by NP and RP-TLC. The separation behavior of diastereomers prepared with different reagents was compared. Fourth chapter presents direct and indirect resolution of P-blockers. It has been divided into two sections. Section A: Direct enantioresolution of P-blockers by, (i) Impregnated TLC: Direct resolution of racemic atenolol and propranolol into their enantiomers was achieved by NP-TLC on silica gel plates impregnated with optically pure L-tartaric acid, (i?)-mandelic acid, and (-)-erythromycin as chiral selectors. Different solvent systems were worked out to resolve the enantiomers. Spots were detected using iodine vapour. The TLC method was validated for linearity and limit of detection (LOD). The influence of pH, temperature and concentration of chiral selector was studied. (ii) Ligand Exchange TLC: Different modes of loading/impregnating the silica plates with Cu(II) complexes of L-Pro, L-Phe, L-His, Af/V-dimefhyl-L-phenylalanine, and LTrp) were adopted for resolving atenolol, propranolol and salbutamol into their enantiomers. The approaches were, (A) using the Cu (II)-L-amino acid complex as chiral mobile phase additive with plain plates, (B)(i) mixing the Cu complex with silica gel before making thin layer plates, (B)(ii) ascending development of plain silica gel plates in the solutions of Cu complex, and (B)(iii) using a solution of Cu (II) acetate as mobile phase additive for the plates prepared by mixing L-amino acid with silica gel. Different solvent systems were found to be successful. Spots were located using iodine vapour. The effect oftemperature was investigated on enantiose lect ivity. Section B: Indirect enantioresolution of P-blockers, (i) via diastereomerization with 14 CDRs: Diastereomers of six P-blockers (atenolol, propranolol, bisoprolol, metoprolol, salbutamol, and carvedilol) were synthesized by reaction with 14 CDRs. The 84 diastereomeric pairs were analysed for separation by RP-HPLC. The method was validated for linearity, accuracy, LOD and LOQ. The CDRs used were, dinitrophenyl-L-Pro-N-hydroxysuccinimide ester and Nsuccinimidyl-( 5)-2-(6-methoxynaphth-2-yl) propionate while the other twelve CDRs were in which one ofthe fluorine atoms ofDFDNB was substituted with three optically pure amines [(i?)-(-)-l-cyclohexylethylamine, (+)-dehydroabietylamine, and (£)-(-)- in a,4-dimethylbenzylamine]; six amino acid amides [L-Ala-NH2, L-Phe-NH2, L-Val- NH2, L-Leu-NH2, L-Met-NH2 and D-Phg-NH2], and three amino acids [L-Ala, L-Val and L-Leu]. Derivatization with 12 CDRs was optimized and compared under conventional and microwave heating (ii) determination of atenolol enantiomers in rat plasma: An HPLC method was established for enantioseparation of (i?,5)-atenolol and determination of enantiomers in rat plasma using MR. The method was validated for linearity, repeatability, LOD and LOQ. Recovery at LOQ was92.8% for (R)- and 92.6% for (5)-isomer. Fifth chapter deals with enantioresolution of mexiletine via diastereomerization with eighteen CDRs using liquid chromatographic methods. Of these, 14 CDRs were the same as mentioned in Chapter-4. The other CDR that was synthesized included one anhydride namely, [(S,S)-0,(y-di-p-to\uoy\ tartaric anhydride] and the DNFB unit containing (-)-cis-myrtanylamine as the chiral selector. Besides, (S)-naproxen was used as such as the CDR. The diastereomers prepared with these 17 CDRs were separated by RP-HPLC. In addition, (tf)-a-methoxy-a- (trifluoromethyl)phenylacetyl chloride was used for enantioresolution of (R,S)-MEX by gas-chromatography mass-spectrometry (GC-MS). The methods for both HPLC and GC were validated for linearity, accuracy, LOD and LOQ. Sixth chapter deals with enantioseparation of DL-Penicillamine. The CDR, Nsuccinimidyl-( S)-2-(6-methoxynaphth-2-yl) propionate (SINP) was obtained in excellent yield (>92%) by the reaction of (5)-naproxen with N-hydroxysuccinimide in presence of dicyclohexyl carbodiimide. The diastereomers of DL-penicillamine prepared with SINP were resolved by RP-HPLC using TEAP buffer (pH-4.0, 5 mM)- MeCN (linear gradient (30 min) of MeCN from 30% to 70%). Excellent separation was achieved with gradient mobile phase. The detection limit was at pmol level.
Other Identifiers: Ph.D
Research Supervisor/ Guide: Bhushan, Ravi
metadata.dc.type: Doctoral Thesis
Appears in Collections:DOCTORAL THESES (chemistry)

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