CHIRAL SEPARATIONS BY HPLC AND TLC USING NEW DIASTEREOMERIZING REAGENTS

Abstract

It is well established fact that enantiomers can exhibit different physiochemical properties in natural and biological systems such as human body. The different responses of enantiomers in biological systems have led to intensive research in food, flavor, agrochemicals, biochemistry, and especially, in pharmaceuticals industries, where very selective and specific biological responses can be utilized and developed commercially. Regulatory authorities require determination of relative rates of metabolism and dissipation of the individual enantiomers in biological and environmental systems. Majority of therapeutics such as anti-inflammatory/analgesic, adrenergic and antihistamines drugs, agrochemicals and food additives are marketed as racemic mixtures. Now a days, greater attention is focused on developing methods for stereoselective and enantioselective synthesis. Hence chiral analytical methods remain important in analysis of enantiomeric purity of drugs during their different steps of synthesis, isolation from natural sources, and establishment of specifications for drug marketing as well as monitoring stereospecific fate of drugs. 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), gas-liquid chromatography (GLC), thin layer chromatography (TLC), supercritical fluid chromatography (SFC) and capillary electrophoresis (CE). Currently, there are two procedures used for chromatographic resolution of enantiomers. These are classified as direct and indirect. Direct method has several advantages over indirect method, e.g., the direct approach requires no chemical derivatization with a chiral reagent prior to chromatography. Indirect approach is the first widely used chromatographic method for enantiomeric separation of chiral drugs and related materials. Although development and commercial availability of chiral stationary phases have increased rapidly, indirect approach is still the method ofchoice in certain specific situations. a-Amino acids (proteinogenic and non-proteinogenic), baclofen (muscle relaxant), penicillamine (used for treatment of Wilson's disease, polyarthritis and cystinuria), aldehydes and ketones were selected for present studies because oftheir widespread applications in the field of chemistry, medicine, food science, forensic science and toxicology etc and easier availability. New chiral derivatizing reagents were synthesized, characterized and used for derivatization of above mentioned compounds. The present thesis comprises of six chapters. The first chapter is an introduction to chirality of pharmaceuticals, chiral chromatographic approaches, selection criteria of CDRs and their applications. 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, TLC and HPLC equipment. The third chapter deals with the synthesis of MR and its six structural variants, viz, FDNP-L-Phe-NH2, FDNP-L-Val-NH2, FDNP-L-Leu-NH2, FDNP-LPro- NH2, FDNP-L-Met-NH2 and FDNP-D-Phg-NH2. These were characterized and used for derivatization of DL-amino acids (eighteen proteinogenic and eight nonproteinogenic) to form diastereomeric pairs. The diastereomeric derivatives were separated on a reversed-phase C18 HPLC column using linear gradient of acetonitrile and aqueous trifluoroacetic acid (TFA) and results were compared. New chiral reagents gave better separation of diastereomers in comparison to Marfey's reagent in most of the cases. The reagent, FDNP-D-Phg-NH2 gave excellent separation for DL-serine and DL-asparagine. 11 The diastereomeric derivatives of proteinogenic DL-amino acids prepared with FDNP-L-Phe-NH2, FDNP-L-Val-NH2 and MR were separated on normal and reversed-phase TLC plates. Diastereomers were separated most effectively by normal-phase TLC with phenol-water, (3:1, v/v), as mobile phase. In reversedphase TLC the diastereomers were separated most effectively by use of mobile phases containing acetonitrile and triethylammonium phosphate buffer (50mM, pH 5.5). The separation efficiency ofthe structural variants was compared with that ofMR. In fourth chapter, ten chiral dichloro- and monochloro-s-triazines were prepared by the nucleophilic displacement of chlorine atom(s) in s-triazine chloride and its 6-methoxy derivative with different amino acid amides. Dichloro-s-triazines (DCTs) were used as CDRs for derivatization of twenty one a-amino acids under basic conditions at room temperature (30°C) and the resultant diastereomers were separated on a reversed-phase C)8 column using mixtures of acetonitrile and TFA. Optimization for derivatization yield, LOD, LOQ, linearity, accuracy and precision was carried out with respect to a DCT reagent. The results obtained with DCT reagents were compared with monochloro-s-triazine (MCT) reagents which were used for derivatization of thirteen a-amino acids. In most of the cases, DCT reagents provided better separation of diastereomers in comparison to MCT reagents. Effects of structural modifications in reagents on chromatographic properties were investigated. Separation mechanism of diastereomers was proposed in light of both MCT and DCT reagents. Fifth chapter presents TLC and HPLC studies of indirect resolution of baclofen and penicillamine enantiomers. It has been divided into two sections. Section A: A high-performance liquid chromatographic (HPLC) method has been developed for chiral assay of baclofen enantiomers in pharmaceutical formulations using indirect approach. Baclofen enantiomers were derivatized with Marfey's reagent (FDNP-L-Ala-NH2) and its structural variants FDNP-L-Phe-NH2, FDNP-L-Val-NH2, FDNP-L-Leu-NH2 and FDNP-L-Pro-NH2. The resultant diastereomers were separated on RP-TLC using triethylammonium phosphate iii buffer (pH 4.0, 50 mM)-acetonitrile, (50:50, v/v) and on a C18 column using linear gradient (45 min) of acetonitrile and 0.01M aq-TFA with UV detection at 340 ran. The differences in the retention times (At) of diastereomers due to the five chiral reagents were compared. The maximum and minimum difference in retention times between separated diastereomers was for FDNP-L-Leu-NH2 and FDNP-L-Pro-NH2, respectively. Effects of flow rate, acetonitrile content and TFA concentration, on resolution, were studied. The method was validated for linearity, repeatability, LOD and LOQ. Section B: TLC and HPLC methods were developed for indirect chiral separation of penicillamine enantiomers after derivatization with Marfey's reagent and two of its structural variants FDNP-L-Phe-NH2 and FDNP-L-Val-NH2. Binary mobile phase of phenol-water (3:1, v/v) and solvent combinations of acetonitrile and triethylammonium phosphate buffer were found to give the best separation in normal and reversed phase TLC, respectively. The diastereomers were also resolved on a reversed phase C18 HPLC column with gradient elution of acetonitrile and 0.01 M TFA. The results due to these three reagents were compared. The method was successful to check the enantiomeric impurity of L-penicillamine (L-PenA) in D-penicillamine. The method was validated for linearity, repeatability, LOD and LOQ. Sixth chapter deals with synthesis and application of chiral hydrazine reagents for liquid chromatographic enantioseparation of optically active carbonyl compounds. Five new CDRs were synthesized by straightforward two-step synthesis starting from l,5-difluoro-2,4-dinitrobenzene (DFDNB). Nucleophilic substitution of one fluorine atom in DFDNB with L-Ala-NH2 yielded Marfey's reagent and its structural variants were prepared by using L-Phe-NH2, L-Val-NH2, L-Leu-NH2, and L-Phg-NH2 in place of L-Ala-NH2. Further nucleophilic substitution of remaining fluorine atom in Marfey's reagent and its variants with hydrazine under basic conditions yielded the chiral hydrazine reagents, namely, 5-hydrazino-2,4-dinitrophenyl-L-alaninamide(HDNP-L-Ala-NH2), HDNP-L-Phe-NH2,

HDNP-L-Val-NH2> HDNP-L-Leu-NH2 and HDNP-D-Phg-NH2. These chiral reagents react quantitatively with chiral carbonyl compounds under mild conditions (30°C, 30 min) to form hydrazone diastereomers. The labeling reaction occurs only in the presence of acid which has a catalytic action and diastereomers have strong absorbance around 348 nm. The separation of diastereomers was tried on a reversed-phase C]8 HPLC column using different binary solvent combinations. Excellent separation was achieved in case of cyclic ketones having substitution at a-position. Optimization for derivatization yield, LOD, LOQ, linearity, accuracy and precision was carried out with respect to HDNP-L-Val-NH2. Studies related to effects of structural modification in reagents and analytes on chromatographic behavior of diastereomers were also analyzed.