ENANTIOSEPARATION OF CERTAIN PHARMACEUTICALS BY LIQUID CHROMATOGRAPHY

Abstract

Enantiomers of a compound show difference in physiological and chemical activity in biological systems. In general, one enantiomer of most of the racemic drugs shows significantly higher pharmacological activity than the other. In some cases one enantiomer can be totally inactive or in others, it can produce adverse side effects. Therefore, synthesis and establishing enantiomeric purity becomes important. Among the various available methods for establishing enantiomeric purity, liquid chromatographic techniques particularly HPLC and TLC are extensively used. Present thesis deals with studies on direct and indirect enantioseparation of certain pharmaceutical compounds which are marketed as racemic mixtures. These chiral compounds/ drugs are (RS)-betaxolol (Bet), (RS)-baclofen (Bac), (RS)-mexiletine (Mex), (RS)-ketamine (Ket), DL-carnitine (Ctn) and (RS)-isoprenaline (Ipn). For this purpose different chiral derivatizing reagents (CDRs) were synthesized and used, some of them were new with respect to their synthesis and characterization while others ones were used for the first time for derivatization and enantioseparation of these drugs. Description of Chapters The first chapter deals with introduction to present studies including introduction to enantiomers, their separation, and some important technical terms related to chromatographic separation. The chapter also includes discussion about liquid chromatography, HPLC and TLC. The CDRs used in the present have been discussed in brief. The second chapter describes common experimental procedures used in the present studies. Besides, it include description of materials, equipments, and preparation of TLC plates. In all, 18 CDRs were synthesized using 1, 5-difluoro-2, 4-dinitrobenzene (DFDNB), cyanuric chloride (CC), (S)-naproxen (Npx), and (S)-levofloxacin (Lfx) as starting materials. D- and L-amino acids (AAs) were used as chiral auxiliaries in DFDNB (1-10) and CC based CDRs (11-14). Three CDRs (15-17) based on (S)-Npx, namely N-succinimidyl-(S)-2-(6- methoxynaphth-2-yl) propionate, (S)-1-(1H-benzo(d)(1,2,3)triazol-1-yl)-2-(6- methoxynaphthalen-2-yl)propan-1-one and N-phthalimidyl-(S)-2-(6-methoxynaphth-2-yl) ii propionate were synthesized. Also, one Lfx based CDR (designated number 18) was synthesized. The method for extraction, isolation, and purification of active pharmaceutical ingredient from commercial formulations of drug analytes has also been described along with their characterization. The third chapter presents enantioseparation of (RS)-mexiletine (spiked in human plasma). In all, ten DFDNB based CDRs were synthesized by using D- and L-AAs as chiral auxiliaries and were further used for synthesis of diastereomers of (RS)-mexiletine in human plasma. The earlier studies mainly focused on the use of L-AAs or their amides as chiral auxiliaries in DFDNB. These ten reagents were characterized and used for synthesis of diastereomers of (RS)-mexiletine spiked in human plasma. Diastereomers were prepared under microwave irradiation and were separated on RP C18 column. CDRs having D-AAs as chiral auxiliaries were found to be better than L-AAs analogues. The best resolution was obtained using mobile phase consisting of MeCN and 0.1% TFA in gradient mode with detection at 340 nm. The method so developed was validated for linearity, accuracy and precision. The limit of quantitation (LOQ) was found to be 25.2 ng mL for each of the diasteromer. The fourth chapter describes preparative enantioseparation of (RS)-baclofen. Taking into account the characteristic properties of s-triazine and the anisotropy of s-triazine ring, the work involved (i) synthesis of a new monochloro-s-triazine reagent, namely, N-(4- chloro-6-piperidinyl-[1,3,5]-triazine-2-yl)-L-phenylalanine, (ii) synthesis of diastereomers of (RS)-Bac employing microwave irradiation, (iii) separation of diastereomers by both HPLC and preparative TLC, and isolation by TLC, (iv) establishment of molecular dissymmetry and determination of absolute configuration of diastereomers of (RS)-Bac (and thus the enantiomers), and (v) optimization of structures of the diastereomers have been executed using hybrid density functional B3LYP in conjunction with basis set 6-31G* using Gaussian 09 suite of programs for further verification of configuration and explanation of elution order. LOQ was found to be 0.168 and 0.183 ng mL, respectively, for the two diastereomers. The work presented herein has brought in a novel approach in the area of enantioseparation and this is the first report on enantioseparation including determination of absolute configuration of diastereomers of (RS)-Bac. iii The fifth chapter deals with enantioseparation of (RS)-isoprenaline (in human plasma) using CC based CDRs (CDR 11-13). L-isoleucine, D-phenylglycine and Lmethionine were introduced as chiral auxiliary in cyanuric chloride and three new monochloro-s-triazine reagents were synthesized. These reagents were characterized and used for synthesis of diastereomers of (RS)-isoprenaline spiked in human plasma. Isoprenaline was isolated from commercial pharmaceutical formulation and was used as the racemic sample. Diastereomers were synthesized employing microwave irradiation and were separated on C18 column. Mobile phase consisting of MeCN and 0.1 % TFA under gradient elution from 35-65 % of MeCN was found successful. The difference in retention times and resolution values was explained in terms of hydrophobicity of the alkyl side chain. Molecular structures of the diasteromers were optimized using Gaussian 09 at B3LPY/6- 31G* level of theory which showed the spatial orientation of hydrophobic groups on stereogenic centers in the diastereomers. The results were correlated with mechanism of separation and elution order. LOQ values were found to be 7.2 and 7.8 ng mL for the 1st and 2nd eluting diastereomers, respectively. The sixth chapter reports an efficient approach for recognizing chirality and determining absolute configuration of diastereomers of (RS)-betaxolol, which in turn is a measure of enantiomeric purity of Bet since the diastereomers were separated and isolated using preparative TLC. Besides, a RP-HPLC method was developed and validated for enantioseparation of (RS)-Bet. Diastereomers were synthesized, using Npx based CDR 14, under microwave irradiation. Absolute configuration of both the diastereomers was established with the help of 1H-NMR spectroscopy and by optimizing the lowest energy structures of diastereomers using the Gaussian 09 Rev. A02 program at B3LYP/6-31G* level of theory. Limits of quantitation were found to be 0.80 ng mL for the diastereomers. The seventh chapter describes enantiomeric resolution of DL-carnitine in human plasma using (S)-Npx based CDR. Following approach was planned and implemented, (i) (S)-Npx based CDR was introduced in samples of human plasma which was spiked with DL-Ctn, (ii) investigations were carried out using three different CDRs, (iii) the diastereomers so synthesized were separated using achiral RP-HPLC column, (iv) derivatization and separation was also performed in solutions without plasma to compare, validate and confirm the results, (v) structures of the diastereomers were optimized for iv lowest energy using the Gaussian 09 electronic program at B3LYP/6-31G* level of theory for verification of configuration and elution order, and (vi) the separation method was validated for linearity, accuracy, LOD and limit of quantification (LOQ). The novelty of the work lies in supplemental application of different techniques for determination and enantioseparation of DL-Ctn in human plasma by derivatization approach along with verification of configuration of the diastereomers so separated. The eighth chapter presents enantioseparation of (RS)-ketamine using newly synthesized (S)-levofloxacin based CDRs. The diastereomers of Ket were synthesized and separated on RP C18 column. Mobile phase containing MeCN and 0.1 % TFA under gradient elution from 35-65 % of MeCN resulted into the best separation among various other combinations tried. The LOD values were found to be 3.2 and 3.4 ng mL for first and second eluting diastereomers, respectively. The present work has demonstrated the versatility, flexibility and sensitivity of two LC techniques, namely HPLC and TLC, for enantioseparation. These techniques were successfully employed for separation of enantiomers of different categories of pharmaceuticals which are marketed in racemic form. Experiments were successful allowing very good enantioseparation with low LOD using derivatization approach. The optimized and validated RP-HPLC separation conditions described in the thesis (with the results already published) can be successfully applied for determination and control of enantiomeric purity of the said drugs routinely in industries and R&D laboratories (even without resorting to 1H NMR, and DFT, each time).