LIQUID CHROMATOGRAPHIC STUDIES OF CERTAIN PHARMACEUTICALLY IMPORTANT COMPOUNDS

Abstract

It is an established fact that enantiomers have different biological, physiological and chemical behaviour both in natural and biological systems, including the human body as well as in the environment . Enantiomerically pure compounds are of prime importance in areas, such as chiral synthesis, mechanistic studies, pharmaceuticals, agrochemical industries, biochemistry and forensic science. Further, regulatory authorities have become aware that efficacy and toxicology of enantiomers may differ from each other and from the racemic mixture, and are increasingly asking for data on stereochemistry. Approximately 40% of the drugs including anti-inflammatory/analgesic, adrenergic and antihistaminic drugs, agrochemicals and food additives developed from organic synthesis possess stereogenic centres in structure, but only 12% are marketed as single enantiomers. Analytical methods capable of discriminating between enantiomers are, thus required for a variety of reasons including quality control, where the aim is to market only one enantiomer, monitoring chiral synthesis, determination ofenantiomeric drug in body fluids, for pharmacokinetic and metabolic studies, and testing the fate of agrochemicals in the environment. Methods commonly used for separating and determining enantiomers include crystallisation, chromatographic techniques, such as high performance liquid chromatography (HPLC), gas liquid chromatography (GLC). high performance thin layer chromatography (HPTLC), thin layer chromatography (TLC) and capillary electrophoresis (CE). (i) TLC provides a simple, sensitive, inexpensive and direct method for resolution and analytical control of enantiomeric purity. TLC also offers variety of modes for plate development including simple one dimensional, multiple, circular and multidimensional ones. The most advantageous feature of TLC is that a number of samples can be handled simultaneously. Availability of various detection techniques and use of modern instrumentation at all stages like, sample application, development units, detection equipment and documentation have increased its efficiency and reproducibility, while special techniques such as TLC/MS and TLC/FT-IR has further increased its usefulness. TLC allows formation of derivatives without resorting to extensive purification and recrystallisation. The present thesis comprises of five chapters. The First chapter is introductory one dealing with historical developments, theory and mechanism, nature of adsorbents, advantages and application of TLC, theory and mechanism of HPLC, and preamble to the present studies. The literature related to the class of compounds chosen has been cited in subsequent chapters. Second chapter deals with description ofthe common experimental method used for present studies. Experimental section includes material, equipment, preparation of plates, TLC of plates, development and detection of chromatogram and polarimetric experiments. Third chapter deals with enantiomeric separation of few important pharmaceuticals. Various methods used and explanations provided for the resolution of a variety of compounds into their enantiomers have been briefly discussed. The chapter is further divided into three sections. Section A incorporates (ii) enantioseparation of racemic amino acids using optically pure (-)-quinine as a chiral selector for impregnated TLC. The successful solvent systems were butanolchloroform- acetic acid (3:7:5, v/v) for DL-methionine; (6:8:4, v/v) for alanine; (10:1:4; v/v) for threonine and ethyl acetate-carbon tetrachloride-propionic acid (10.5:6.5:3.5, v/v) for valine. Minimum detection limits have been found to be different for each of the amino acids ranging between 0.9 ug to 3.7 ug. Effects of concentration of impregnating reagent, temperature and pH on resolution of enantiomers have been studied in details. Detection was done by using 0.2% ninhydrin in acetone. Section B gives a detailed study on enantiomeric resolution of some [3- adrenergic blocking agents viz., (±)-atenolol, (±)-propranolol and (±)-metoprolol, using TLC plates impregnated with optically pure L-aspartic acid as chiral selector. In all the cases different combinations of acetonitrile-methanol-water as solvent systems were tried to work out successful system. Investigations with respect to effect of temperature, concentration of the chiral selector and pH of the layer on enantioresolution were also carried out. Detection was done by using iodine vapours. The minimum detection limits of atenolol, metoprolol, propanolol were found to be 3.9, 1.9 and 1.9 ug, respectively for single isomer. Section C deals with enantiomeric resolution of (±)-ephedrine and atropine into their enantiomers which was achieved by normal-phase thin layer chromatography on silica gel plates impregnated with optically pure L-tartaric acid and L-histidine respectively, as chiral selectors. The mobile phases enabling (iii) successful resolution were different combinations ofacetonitrile-methanol-water. The spots were detected with iodine vapours and the detection limits were 2 and 6 ug, respectively, in terms of racemate. The effects of concentraton of impregnating reagent, temperature and pH on resolution have been studied. Dragendroff reagent or iodine vapours were used for detection. Fourth chapter presents studies on simple separation of aminoglycosides on plain silica gel Gthin layers and RP-C18 plates. Liquid chromatographic studies on the separation of aminoglycosides (streptomycin, kanamycin, gentamycin and tobramycin) were carried out using normal phase plain plates of silica gel and reversed phase TLC on C-18 precoated plates. Acetonitrile and sodium acetate buffer (5 mM) of pH 5.0 was found to be successful, while in case of normal phase TLC combination of acetone -2% sodium acetate-acetic acid- butanol (7.6:4:1, v/v) was worked out for separation. Fifth chapter describes the separation of mixture of vitamin B-complex and folic acid by HPLC, normal phase and RP-TLC. The vitamins studied were :B„ B2, B6, B12 and folic acid. HPLC was carried out with lichrospher® 100 RP-18 (5um) column, using binary combinations of acetonitrile-methanol and acetonitrile-borate buffer (5mM, pH 7.0), while aternary combination of acetonitrile-methanol-borate buffer was successful for RP-TLC. Normal phase TLC was carried out using thin silica-gel plates developed in butanol-chloroform-acetic acid-ammonia-water. The spots were either selfvisualised or located in iodine chamber.