Investigation of Different Analytical Techniques for the Quantitation of Some Selected Hypoglycemic Drugs

Abstract

Different methods for determination of some oral hypoglycemic drugs have been introduced in this thesis. These drugs are alogliptin benzoate, metformin hydrochloride, saxagliptin hydrochloride and vildagliptin.The chromatographic part consists of two sections, the first section of chromatographic methods with ultraviolet detection included determination of saxagliptin hydrochloride in bulk powder and in the presence of its major degradation products after their structure elucidation using LC/MS. While the second section chromatographic method with fluorimetric detection included determination of alogliptin benzoate in plasma. Spectrofluorimetric part included stability indicating assay based on the native fluorescence of alogliptin benzoate together with an indirect assay of vildagliptin after its reaction with dansyl chloride using statistical design of experiments to study the reaction conditions. Spectrophotometric part included determination of metformin in the presence of vildagliptin using zero order and first derivative methods and determination of vildagliptin in the presence of metformin using ratio derivative, ratio subtraction and mean centering methods. Finally, Factorial design spectrophotometric methods were proposed for saxagliptin hydrochloride either after its hantzsch reaction with acetyl acetone and formaldehyde or after its charge transfer complex with p-chloranil. Statistical comparison between the results obtained by the proposed methods and the reference methods was carried out. The thesis is presented in 190 pages and contains 56 tables, as well as 58 figures. The thesis includes five parts: PART(I): Introduction and Literature Review PART (II): High Performance Liquid Chromatographic Methods SECTION (A): Stability-indicating Liquid Chromatographic Method for Determination of Saxagliptin Hydrochloride and Structure Elucidation of the Major Degradation Products, using LC/MS Liquid chromatographic method is described for the determination of saxagliptin hydrochloride in bulk and in the presence of its major degradation products after their structure elucidation using LC/MS. Chromatographic separation was achieved on a Symmetry® Waters C18 column applying an isocratic elution using potassium dihydrogen xx phosphate buffer pH (4.6) - acetonitrile - methanol (40:30:30, v/v/v) as a mobile phase, with UV detection at 208 nm. SECTION (B): Determination of Alogliptin Benzoate in Plasma, using High Performance Liquid Chromatography with Fluorimetric Detection Liquid chromatographic method is described for the determination of alogliptin benzoate in plasma using fluorimetric detection based on the native fluorescence of the drug. Chromatographic separation was achieved on a Symmetry® cyanide column (150mm×4.6mm, 5μm) applying an isocratic elution based on potassium dihydrogen phosphate buffer pH (4.6) - acetonitrile (15:85, v/v) as a mobile phase. The fluorimetric detector was operated at 324 nm for excitation and 521 nm for emission. PART (III): Spectrofluorimetric Methods SECTION (A): Stability-Indicating Spectrofluorimetric Determination of Alogliptin Benzoate, Kinetic Study and Structure Elucidation of the Major Degradation Products, using LC/MS A direct spectrofluorimetric method is described for the determination of alogliptin benzoate in bulk, in its pharmaceutical preparation and in the presence of its major degradation products after their structure elucidation using LC/MS. The native fluorescence of the drug was measured at 521 nm after excitation at 324 nm. SECTION (B): Spectrofluorimetric Method for Determination of Vildagliptin After Dansylation, Using Factorial Design Experiment Indirect spectrofluorimetric method is described for the determination of vildagliptin after its reaction with dansyl chloride. The fluorescence intensity of the reaction product was measured at 431 nm after excitation at 348 nm. The reaction conditions were studied using statistical design of experiment. PART (IV): Spectrophotometric Methods SECTION (A): Spectrophotometric Methods for Determination of Vildagliptin and Metformin Hydrochloride in Binary Mixture xxi Two spectrophotometric methods are described for the determination of metformin hydrochloride in its binary mixture with vildagliptin, The proposed methods included zero order spectrophotometric method measuring the absorbance at 237.6 nm and also included first derivative spectrophotometric method measuring the amplitude at 247.4 nm. Three spectrophotometric methods are described for the determination of vildagliptin in its binary mixture with metformin hydrochloride; the proposed methods included ratio derivative spectrophotometric method measuring the amplitude at 265.8 nm and ratio subtraction spectrophotometric method measuring the absorbance at 204.4 nm and also mean centering spectrophotometric method measuring the amplitude at 387 nm. SECTION (B): Spectrophotometric Method for Determination of Saxagliptin Hydrochloride After Hantzsch Reaction and Charge Transfer Complexation Reaction, Using Design of Experiment (DOE) Spectrophotometric method is described for the determination of saxagliptin hydrochloride after the reaction of its primary amino group with acetyl acetone and formaldehyde. The absorbance of the hantzsch reaction product was measured at 342.5 nm. The reaction conditions were studied using factorial design. And another spectrophotometric method is described for the determination of saxagliptin hydrochloride after the reaction of its primary amino group with p-chloranil. The absorbance of the charge transfer product was measured at 530 nm. The reaction conditions were studied using factorial design. PART (V): General discussion References