CHAPTERS 1, 2 and 3 CHAPTER-4 CHAPTER-5,

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1 319 Presently in the pharmaceutical industry, drug analysis plays a vital role in deciding the quality and potency of the drug. The selection of analytical method used to quantify the drugs and impurities in the dosage forms is a challenging problem. The methods should be sensitive, accurate, precise, reproducible and free from other interferences. The pharmaceutical industry is under intense pressure to increase productivity and to put new drugs on to the market in a shorter time period. To sustain in world market pharmaceutical manufacturers should provide drugs, free from all possible and potential impurities. The importation of drugs from overseas manufacturers is increasing year to year by the Global community by which is increasing the responsibility of the manufacturer and the pharmaceutical analyst to prove that pharmaceutical drugs are free from not only potential impurities but also possible health hazard impurities. The lapses in existing analytical techniques are being viewed critically by regulatory authorities and pharmaceutical industry. The proposed analytical techniques in this research study will help in finding pharmaceutical manufacturers and regulatory authorities reliable, reproducible, speed and cost effective quantitative techniques for impurities in the life saving pharmaceutical drug substances. The components considered for the present study have much potential in regulatory markets. In this context the author has selected the analytical techniques UPLC and LC/MS by using suitable stationary phase and chromatographic techniques for quantifying and qualifying impurities and the degradation products. Pharmaceutical active Pharmaceutical Ingredients of the considered drugs found to undergo facile degradation under different environmental conditions to yield different products which may have toxic or adverse effects. The study on these degradation products remained unidentified till date. Hence, the analytical methods capable of identifying and quantifying for these products i.e. the methods

2 320 stability indicative, specific and selectivity to perform the study have been developed. Stress studies were performed on drug candidates to assure the inherent stability of the drug and which intern extended and extrapolated towards the real time stability of the APIs. The author scrutinized new analytical methods in the current research work for the quantification of related substances (both process related and degradants) and active substances in the studied products. Also presented the validation and real time stability data for the said products which will be of great asset in investigating them. The CHAPTERS 1, 2 and 3 focuses on the theoretical aspects of impurities, degradation products, instrumentation of the new and Ultra Performance Liquid chromatography and LCMS, and literature survey CHAPTER 3 also highlights scope and significance of the research study. Degradation pathway for Gefitinib is established as per ICH recommendations by validated and stability indicating liquid chromatographic method in bulk drug, used for the Lung cancer is detailed in CHAPTER-4. This method is capable to detect the impurities of Gefitinib at a level of 0.01%. Significant degradation is observed in acid and base stress conditions. Two impurities are studied among which one impurity is found prominent degradant. In the developed RPLC method the resolution between Gefitinib and the potential impurities is found to be greater than 5.0. The developed RRLC method was validated with respect to linearity, accuracy, precision and robustness. In CHAPTER-5, the author describes a validated, specific and stabilityindicating reverse phase liquid chromatographic method for the quantitative determination of genotoxic impurities in the drug substance imatinib mesylate. This study includes a brief review of the toxicology of impurities in imatinib mesylate. The bulk drug imatinib mesylate containing the two genotoxic

3 321 impurities impurity-1 and impurity-2 are subjected to stress conditions of hydrolysis (acid and base), oxidation, photolysis and thermal degradation to show the stability-indicating power of the method. These two genotoxic impurities are also observed as degradation impurities during acid and base hydrolysis. The developed method is validated with respect to linearity, accuracy, precision and robustness. CHAPTER-6 presents the degradation pathway for Bortezomib by validated and stability indicating Ultra Performance Liquid Chromatographic method. The method is capable of separating five process related impurities and two degradation impurities. Bortezomib is subjected to stress conditions of acid, base, oxidation, thermal and photolysis. Significant degradation is observed in acid and base stress conditions. Five impurities are studied and the major degradant (RRT about 1.12) was identified by LC-MS and spectral analysis. This method is capable to detect the impurities of Bortezomib at a level of % with respect to test concentration of 2.0 mg ml-1 for an 8-μL injection volume. The developed Ultra Performance LC method is validated with respect to specificity, linearity & range, accuracy, precision and robustness for impurities determination. In CHAPTER-7, a single, short, stability indicating method was developed and validated for Eplerenone impurities. Eplerenone is subjected to stress conditions of acid, base, oxidation, and thermal and photolysis. Significant degradation is observed in base stress conditions. Four impurities are studied and the major degradant (RRT about 0.31) was identified by LC-MS and spectral analysis. In the developed UPLC method the resolution between Eplerenone and four potential impurities is found to be greater than 4.0. The developed UPLC method is validated with respect to specificity, linearity and range, accuracy, precision, and robustness for impurities determination.

4 322 In CHAPTER-8, a stability indicating Ultra Performance liquid chromatographic (UPLC) method has been developed for quantitative determination of Nilotinib Hydrochloride and an attempt has been made to identify the impurities found during the degradation employing advanced techniques. The method is capable of separating four process related impurities and two degradation impurities. It is clearly evident from the research investigation that advantage of adopted new analytical methods are considered to be significant factor over existing methods. UPLC gives faster results with better resolution. UPLC uses less of valuable solvents like Acetonitrile which lowers cost. The reduction of solvent use is more environmental friendly. Superior mechanical column strength, high efficiency, high ph stability and peak shape for bases saves time as well as cost for method development. The high resolution obtained in extremely short analysis times makes UPLC a very attractive tool for the pharmaceutical development Laboratory.

5 323 Advantages of the Proposed UPLC Methods Drug Proposed method Advantages Gefitinib Imatinib Mesylate Bortezomib Eplerenone Nilotinib HCl Stability indicating RRLC method for the determination of Gefitinib (III) in the presence of degradation product, its process related impurities were described. Stability indicating fast LC method for the determination of two impurities in the presence of degradation products was described. The Degradants were identified by LC-MS and other spectral analysis presented. Stability indicating Ultra performance LC method in the presence five process related impurities was described for the first time. The Degradants were identified by LC-MS and other spectral analysis presented. Ultra performance liquid chromatographic method was described in the presence four process related impurities and a Degradant was described. UPLC method for the determination of Nilotinib in the presence of degradation products, its four process related impurities and Degradants was described.the behaviour of Nilotinib (IV) under various stress conditions was studied. Analysis time reduced by four times. Degradation products and process. Able to quantify two process related impurities and degradation product. Analytical method capable of determining the genotoxic impurities in imatinib mesylate at the level of 10 ppm is determined. Able to detect and quantify five process related and two degradation impurities. Analysis time by using UPLC reduced to four times than normal method. Able to detect and process related and one degradation impurities. Analysis time by using UPLC reduced to four times than normal method. Able to detect and quantify four process related and one degradation impurities. Analysis time by using UPLC reduced to five times than normal method.

6 A parameter comparison graph between HPLC and proposed UPLC methods is given in figure below. COMPARISON OF HPLC AND UPLC METHODS Sensitivity of existing method Average time of HPLC methods COST of HPLC 1 Sensitivity of new method Average time of UPLC methods Cost of UPLC The proposed methods will save time, money and efforts for the manufacturing as all the pharmaceutical ingredients studied are life saving and important molecules these methods has got advantages as listed in below table over existing methods widely in use for respective impurities quantification.

7 325 Comparison between established HPLC method and proposed UPLC method Cost HPLC UPLC Remarks Run time 20 min 5 min 4 times faster, Manpower Rs 1500/Hr Rs.550(20 Rs.110 Solvent min) Rs.30(20 (4 min) Rs.15 Rs 1485/Injection ml) (8.0 ml) Disposal(Rs.110/Injection) Rs.5 < 50 NP Total Rs.585 Rs % Solvent consumption. 21% of cost in HPLC Cycle time 20 min cycle time 5 min cycle time Runs Approximate cost Rs.600 Rs.125 Approximate analysis cost Rs Cost 700 savings runs --- Rs Time 10 days 3 days Performance comparison of HPLC and UPLC with the proposed drugs Drug Substance Retention time Capacity Factor USP Tailing Resolution Peak capacity HPLC UPLC HPLC UPLC HPLC UPLC HPLC UPLC HPLC UPLC Gefitinib Imatinib Mesylate Bortezomib Eplerenone Nilotinib HCl

8 326 The present research investigation will add new dimensions to the existing literature in the field of Novel chromatographic and spectroscopic studies on pharmaceutical products. The research study will also enrich practical subject knowledge significantly on identification and Quantification of impurities present in Anticancer and Antihypertensive active pharmaceutical ingredients. The developed methods will be of great help to Quality Control and Assurance laboratories in a manner to analyze the samples for impurities in a sensitive, faster and cost effective way and to establish expiry of the drugs based on the stability studies. The validated stability indicating methods can be effectively employed towards screening of pharmaceutical products. The results in the present investigation are well supported by the research publications of reputed international journals. These developed methods are of great asset in quality monitoring of the selected products towards development of Active Pharmaceutical Ingredients and may be claimed as an advanced, noteworthy feature of the research work. *******************