CHAPTER-VII. Summary and Conclusions

Transcription

1 CHAPTER-VII

2 Drugs play a vital role in the progress of human civilization by curing and preventing diseases. Depending on mechanism of action, site of absorption, drugs are formulated into different pharmaceutical dosage forms like tablets, hard gelatin capsules, soft gelatin capsules, and injections. Depending on delivery mechanism needed, different kinds of excipients are used in formulations to achieve target release profile of the drug into human body. The quality of drug plays an important role in ensuring the safety and efficacy of the drugs. Quality assurance and quality control of pharmaceutical and chemical formulations is essential for ensuring the availability of safe and effective drug formulations to consumers. The quality and safety of a drug is generally assured by monitoring and controlling the assay (the potency of the drug) and impurities (safety aspect of the drug) effectively. The exports of drugs into regulatory market like USA and Europe is increasing day by day. To sustain in those markets manufacturers should provide drugs, free from all possible and potential impurities. The scenario of review in those regulatory authorities is also changed from conventional procedures to new analytical techniques. The descend in the existing analytical techniques are being critically reviewed by authorities and pharmaceutical industry. Thus, the analytical activities concerning impurities in drugs are among the most important issues in modern pharmaceutical analysis. So analysis of pure drug substances and their pharmaceutical dosage forms occupies a pivotal role in assessing the suitability to use in patients. Hence, novel chromatographic and spectroscopic studies on the identification and quantification of impurities in pharmaceutical ingredients have much significance. Analytical methodology should be capable of resolving all potential impurities in pure drug substances and their pharmaceutical dosage forms. A thorough literature search has revealed that different methods such as HPLC, UPLC, LC-MS, GC, TLC, UV, FT-IR, NMR, capillary electrophoresis, super fluid chromatography, powder X-ray diffractometer, etc. were used for the estimation of drugs and impurities present in the API and formulations. Powder X-ray diffraction, FT-IR, solid state NMR, DSC and TGA techniques were used for characterization and quantitative analysis of polymorphs. RP-HPLC or UPLC methods have been developed to monitor the stability of active pharmaceutical ingredients and pharmaceutical dosage forms during the investigational phase of drug development and also for the ongoing stability studies that must be conducted. Stress testing of the drug can help to identify the likely degradation products and the intrinsic stability of the molecules. It also 318

3 provides the stability indicating power of the analytical procedures used. Hence development of stability indicating analytical methods is very important for statutory certification of drugs and their formulations with the regulatory authorities. The proposed analytical techniques or procedures in this research study will help in finding pharmaceutical manufacturers and regulatory authorities are sensitive, specific, selective, reproducible, reliable, speed and cost effective quantitative techniques for the specified drug substances and products. An extensive literature review reveals that none of the stability indicting methods for quantifying the organic impurities and polymorphic impurities in the stated seven selected API s and dosage forms. The selected drugs for the present study have much potential in regulatory markets and dosage forms containing these active pharmaceutical ingredients have huge market value all around the world (Fig.7.1). Pharmaceutical dosage forms 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 are capable of identifying and quantifying for these products. i.e. the methods are stability indicative, specific and selective 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 drug substance. An attempt was made to provide the degradation products pathway for all the drug candidates, which should really ease the pharmaceutical development of drug products containing respective drug substances. Fig.7.1 Market Potential of Selected Drugs 319

4 The main objective of the present investigation is the development of advanced analytical methods for the separation of organic impurities, simultaneous assay of selected drugs and quantification of polymorphic impurities of the selected active pharmaceutical ingredients and dosage forms. Hence efforts were made to identify and elucidate the structure of those impurities using advanced techniques such as LC-MS and NMR. Hence, these methods will help to provide more insight on the quality of the products and also assist to improve or develop dosage forms containing the respective active substances. In this thesis we provide new analytical methodologies HPLC/UPLC/P-XRD that can be improves the analysis throughput and cycle times. The proficient research work has been divided in to six chapters. The research work presented in this thesis, is composed of six articles, seven drug substances and its pharmaceutical dosage forms. The author scrutinized new analytical methods in the current research work for the quantification of impurities, physical impurities like polymorphs and assay content in the selected drug substances as well as drug products and also presented the complete validation data for the said products which will be of great asset in investigating them. The performance of the developed new analytical methods for estimation of impurities and assay has been verified by applying the same to evaluate the quality of bulk samples of API during its stability studies. The developed new analytical methods were performed well for the quality evaluation of stability samples of API and its dosage forms. The primary chapter (Chapter-1) deals of a brief introduction on the need for development of new or novel analytical methods for drug substance and its dosage forms estimation, starting place of impurities in drug substances, positional isomers, recent or current quality guidelines, USP/EP/IP/Pharmacopeial norms, control of organic impurities and analogue impurities, way forward for development of performance stability-indicating analytical methods with chromatographic contents of method validation parameters and detailed discussion on how to develop a new method approaches on the common methodology for LC and P-XRD analysis. The left over chapter II-VI consists (HPLC/UPLC/P-XRD) of introduction, experimental, validation, results, discussion and conclusion. In the foreword part a concise account on the therapeutic activity, drug substance and require to establishing novel analytical method development has been discussed. In the sub-sequent sections, raw materials used for the analysis, model of the instruments can be used during new method development, working and reference standard samples, test solutions, a complete account on the steps involving due to developing for the original 320

5 methods for the resolve of related compounds, which are stability indicating and analytical method validation methodology were detail discussed. All supporting data, complete method validation results and the final conclusion was also furnished. The thorough results and discussions were discussed in the in each individual chapter. The chemical structures and chemical names of selected drugs were shown in Table7.1. Chapter-I: General Introduction Chapter-I consist of the study of impurities, a brief review on analytical techniques, analytical method development and validation, chromatographic parameters and statistical evaluation and profile of the selected drugs. It briefly explains the types of impurities, classification, formation, control of impurities and physical impurities like polymorphs. Different classes of impurities like organic impurities, inorganic impurities, residual solvents and contamination impurities, and different ways of formation of these impurities in API and finished product such as from starting materials, intermediates, stereochemistry related impurities, product side chains, reaction solvents, stability studies, dimerization, degradation of the API s (acid, base, oxidative, thermal and photolytic degradation), a detailed discussion about control of impurities, residual solvents and finally polymorphs are described in this chapter. Principle, instrumentation and applications of analytical techniques that are adopted for the present investigation such HPLC, UPLC, LC-MS and P-XRD is presented in experimental part. Development and optimization (detector selection, separation mode, mobile phase and ph, column, etc.) of analytical method, validation of analytical procedures (specificity, precision, accuracy, detection limit (DL), quantitation limit (QL), linearity, range, robustness and selectivity of the method) are discussed in development and validation part of chapter-i. The system suitability parameters such as resolution, capacity factor (k), column efficiency (N), peak asymmetry or tailing and selectivity and statistical evaluation of the experimental data (calibration, linear regression-method of least squares, standard error on estimation, standard deviation, slope, intercept, correlation coefficient) was given in chromatographic parameters and statistical evaluation part of chapter-i. A brief detailed drug profile of the selected drugs of present investigation such as solifenacin succinate, fesoterodine fumarate, darunavir ethanolate, levosimendan, sitagliptin phosphate, raltegravir potassium and irbesartan was given in profile of the selected drugs. Finally chapter-i is concluded by presenting the objective of the entire investigation. 321

6 Chapter-II: Development and Validation of a Specific Stability Indicating High Performance Liquid Chromatographic Methods for Related Compounds and Assay of Solifenacin Succinate Chapter-II opens with the drug introduction (chemical name, structure, empirical formula, molecular weight solubility, physical state, brand name, dosage forms, literature survey, etc.) and chemical synthetic route of solifenacin succinate (SFS). In experimental part, different analytical techniques (UV-Visible, FT-IR, NMR, Mass spectrometer, LC-MS and HPLC systems) that were adopted for the study of impurities, and assay were described. The source of solifenacin succinate reference standard, test samples, impurity-a, impurity-b, impurity-c and formulation tablets were discussed in materials and reagents part of experimental section. A detailed description about characterization of impurity-a, B & C by using UV, FT-IR, NMR [carbon ( 13 C) & proton ( 1 H)] and Mass techniques were presented. Development of analytical RP-HPLC method and optimization (selection of wavelength of maximum absorbance, selection of stationary phase, selection of mobile phase and mode of elution) was explained in detail. The use of LC-MS technique for the identification of degradation products during the degradation of solifenacin in presence of peroxide was pointed out. The major degradant formed under the oxidative stress condition identified as solifenacin N-oxide and it is characterized by using above said techniques. Other two impurities are also found in trace levels. These two also identified and characterized by using the above said techniques. A separate sensitive, specific, selective and stability indicating LC methods has been developed for quantification of solifenacin and its related impurities in active pharmaceutical ingredients and its all dosage forms. The chromatographic separation between solifenacin and its three impurities was achieved by injecting 10µl of sample in gradient mode (mobile phase-a and mobile phase-b) using symmetry shield RP x 4.6 mm. 5µm, at 35 C, wavelength of 220 nm, with flow rate of 1.0 ml/min for 40 minutes. The gradient programme (40 minutes) for related compounds was time/%mobile phase-b: 0.01/20, 20/40, 30/40 and 32/20 with a post run time of 8 minutes, where as for assay the gradient programme was optimized with shorter run time, which is time/%mobile phase-b: 0.01/30, 7/60 and 10/30 with a post run time of 5 minutes (The total run time of assay is 15 minutes). The typical retention times of solifenacin, impurity-a, B and C were about 16.5, 4.5, 6.2 and 18.5 respectively. A detailed study of stability of solifenacin in the presence of different stress conditions such as ambient (25+2 C), thermal (105 C), humidity (90% RH), photo light, water hydrolysis, acid, base and oxidation 322

7 was presented. The peak homogeneity data for solifenacin peak were confirmed with PDA detector in all the forced degradation samples. The major degradant mass number data was generated by using LC-MS method. The mass balance was in all degradation samples were near to 100.1%. A waters HPLC equipped with alliance 2695 separation module, thermostatic compartment connected with 2487 dual wavelength UV-visible (2996 PDA detector for method development and peak purity analysis) detector was used for the separation and quantitation of impurities in the present investigation. Empower-2 chromatography manager software was used for data collection and processing. Another HPLC system of shimadzu make 2010 series system equipped with quaternary gradient pump, auto sampler, column oven and dual wavelength UV-visible detector controlled with LC solutions software was used for ruggedness and assay studies. The developed method was validated as per ICH guidelines and reported the complete validation data in chapter-ii. The developed method was validated in terms of intraday precision, interday precision, accuracy, linearity, robustness and ruggedness. The precision is expressed as percent of relative standard deviation of five/six replicates, accuracy is evaluated by taking triplicate measurements at three different concentrations, linearity plot drawn between response of the detector and concentration of the analyte. The effect of small variation in method parameters is studied and a study is conducted for repeatability of results between different days, different columns and different labs. By using this method, few batches of bulk drug samples, innovator tablets and commercial formulation tablets were analyzed. The detection limits of impurity-a, B & C are 0.003, and 0.007% respectively. The quantitative limits of these three impurities are 0.01, 0.01 and 0.02% respectively. The developed RP-HPLC gradient method showed higher sensitivity for the determination of related compounds than previously reported methods. The developed method showed good precision (less than 5.0%) and accuracy (97.9 to 104.1%) at QL level. The assay and impurities showed good linearity with correlation coefficient The method was found to be simple, selective, precise, accurate, and robust. The method validation results showing satisfactory data for all the method parameters tested. The developed method is stability indicating and can be used for routine analysis of production samples and also to check the stability of bulk samples of solifenacin succinate. The HPLC conditions and brief validation results are summarized in Table.7.2 & 7.3 respectively. 323

8 Chapter-III (Section-A): A Validated Stability-Indicating HPLC Method for Determination of Fesoterodine Fumarate In section-a of chapter-iii, a validated stability indicating HPLC method for determination of fesoterodine fumarate in presence of its possible impurity i.e. impurity-a (5- hydroxymethyl tolterodine) was presented. This section starts with the profile of fesoterodine fumarate, literature review and objective of the present investigation. The same HPLC equipment as described in chapter-ii was used for the assay of the drug. The strength of mobile phase contains buffer concentration, organic modifier like methanol, buffer ph, column oven temperature and auto-sampler injection load was systematically verified in inertsil ODS-3V column (150 x 4.6 mm, length, particle size 5µm). Optimum results were obtained with mobile phase composition used was the 10mM ammonium dihydrogen orthophosphate ml triethylamine (ph adjusted to 3.0±0.05 with diluted orthophosphoric acid) and methanol in the ratio of 42:58 (v/v). The flow rate of the mobile phase was 1.0 ml/minute and detector wavelength was monitored at 210 nm. The column temperature was kept at 30 C and the injection volume was 10 µl. The developed method is specific for fesoterodine and its impurity (5-hydroxymethyl tolterodine). Fesoterodine fumarate was subjected to different ICH prescribed stress conditions (acid, base, oxidative, water, ambient, photo light and thermal). Fesoterodine is degraded into (5-hydroxymethyl tolterodine) in acid, base and oxidative conditions. There was no interference observed from degradants and blank solution at retention time of fesoterodine. This data indicated that method is stability indicative. The peak purity of fesoterodine peak was checked in all stress samples and found that the peak is homogeneous and no co-elution with other impurities, which is confirmed with PDA detector. The mass balance generated for drug substance in all stress degradation samples was near to 99.8%. The typical retention time of fumaric acid, impurity-a and fesoterodine are at about 1.9, 2.1 and 4.9 minutes respectively. In the developed method, the peak tailing factor is 1.19 and theoretical plates are about The developed new analytical method was validated as per current regulatory requirements or guidelines with respect to stress, detection, quantitation limits, accuracy, method precision, intermediate precision, linearity and robustness. The system and method precision (in terms of %RSD) of the method was 0.08 and 0.21 respectively. The %RSD of fesoterodine from intermediate precision study was The developed method show good linearity with correlation coefficient of in the concentration range of mg/ml. The linear regression equation is y= x The % recovery of fesoterodine is found between from to There was no 324

9 significant effect on assay content in robustness study. Mobile phase and solution stability is also carried out and sample and mobile phase stable up to 24 hrs. The same method was also suitable for tablet dosage form. The developed stability indicating isocratic RP-HPLC method for determination of fesoterodine fumarate in bulk was found to be specific, precise, accurate and robust. Hence the developed method was used for routine analysis of production samples and also to check the stability of bulk samples of Fesoterodine Fumarate. The HPLC conditions and brief validation results are summarized in Table.7.2 & 7.3 respectively. Chapter-III (Section-B): Stability indicating RP-HPLC Method for Quantitative Determination of Darunavir Ethanolate in bulk Active Pharmaceutical Ingredient and Tablet Dosage Forms In section-b of chapter-iii, A new stability indicating isocratic HPLC method was developed for the determination of darunavir ethanolate in presence of its closely eluted process related impurities is presented. The separation was achieved on X-Bridge C x 4.6 mm, 3.5 µm column, which was shown excellent selectivity for darunavir and its two impurities. The developed method is quite simple, sensitive, and reproducible. The mobile phase contained a mixture buffer (0.01M ammonium formate, adjusted ph of the solution to 3.0±0.05 with formic acid solution) and acetonitrile in the ratio of 55:45 (v/v) with a flow rate of 1.0 ml min -1, column temperature maintained at 30 C and the detector wavelength was kept at 265 nm. The injection volume was 20 µl. In the developed method, the tailing factor was less than 2.0, theoretical plates of the column was about The % RSD for peak area of darunavir from six replicate injections of system precision and method precision was found to be 0.35 and 0.24 respectively. The correlation coefficient of darunavir is over the concentration range of mg/ml with regression equation of y= x The intermediate precision of the method was 0.29 and the cumulative %RSD between method precision and intermediate precision study was The average percent recovery of darunavir from three levels (80, 100 and 120%, with respect test concentration i.e. 0.2 mg/ml) found in the developed method is between from Forced degradation studies were conducted to prove the stability indicating nature of the developed method as per ICH prescribed conditions. The mass balance generated for darunavir ethanolate drug substance in all stress samples was near to 99.6%. The validated RP-HPLC method for the assay of darunavir ethanolate in bulk active pharmaceutical ingredients and tablet dosage forms was found to be specific, precise, accurate and robust. Different pharmaceutical formulations were checked in the proposed method for its suitability and 325

10 found the results are satisfactory. Hence, the developed method can be used for routine and alternative method for assay determination of drug substance in bulk and dosage forms. The HPLC conditions and brief validation results are summarized in Table.7.2 & 7.3 respectively. Chapter-IV (Section-A): A Validated Chiral HPLC Method for the Enantiomeric Separation of Levosimendan in Bulk Drug Substances In section-a of chapter-iv begins with introduction of levosimendan, a new class of drugs, the calcium sensitizers which is used in the management of acutely decompensated congestive heart failure. A simple, sensitive, novel isocratic chiral HPLC method was developed for the separation of two enantiomers of levosimendan and determination of purity of levosimendan (drug substance) and quantification of unwanted S-isomer. The liquid chromatographic method was developed using polysaccharide derived based chiral stationary phase, chiralpak-ia with mobile phase contained a mixture of methyl-tert-butyl ether, ethanol and trifluoroacetic acid in the ratio of 65:35:0.1(v/v/v). The mobile phase was optimized in such a way to give maximum resolution between the components, which is critical to avoid merging/overlapping components. The flow rate of the mobile phase was 1.0 ml min -1. The column temperature was maintained at 30 C, injection volume was 10 µl and the eluted compounds were monitored at 383 nm. In the developed method, levosimendan drug was subjected to photolytic, thermal and humidity as per ICH prescribed conditions to show the stability indicating power of the method. Further, the developed HPLC method was evaluated for different parameters as per the ICH guidelines and found accurate, reproducible, specific and precise. This method utilizes a chiral pak IA column which is readily available in most of the quality control testing laboratories in the pharmaceutical industry and relatively simple to use. This method is sensitive enough to detect S-isomer at μg.ml -1 and can quantify from 0.075μg.ml -1. The precision of S-isomer at QL level is The method was linear in the concentration range of % with correlation coefficient of The trend line equation of S-isomer is y= x The precision and intermediate precision of the method at QL level is 7.0 and 9.68 respectively. The %recovery of S-isomer was found between from The S-isomer content was not affected in all robust parameters. The run time of the method was only 25 minutes, so it can reduce solvent consumption as well as analysis time. The HPLC conditions and brief validation results are summarized in Table.7.2 & 7.3 respectively. 326

11 Chapter-IV (Section-B): Chiral Separation of Sitagliptin Phosphate Enantiomer by HPLC Using Amylose Based Chiral Stationary Phase In section-b of chapter-iv described, sitagliptin phosphate, an oral antidiabetic agent that blocks dipeptidyl peptidase-4(dpp-4) activity. The drug substance and product is R- enantiomer, so that it contained unwanted enantiomer (S-isomer). So this isomer needs to be controlled in drug substance and product at specified levels with a validated stability indicating HPLC method. A novel new isocratic chiral HPLC method was developed for the separation of two enantiomers of sitagliptin phosphate and determination of purity of sitagliptin phosphate and quantification of unwanted enantiomer i.e S-isomer. The developed method is is quite simple, sensitive and reproducible. Chiralpak AD-H column has shown excellent selectivity for sitagliptin phosphate and its enantiomer. The chiral chromatographic method was developed using amylose carbamates derivitised CSP (chiral pak AD & AD-H) with mobile phase contained a mixture of n-heptane, ethanol and diethylamine in the ratio of 35:65:0.1 (v/v/v). The mobile phase was optimized in such a way to give maximum resolution between the components, which is critical to avoid merging/overlapping components. The flow rate was 1.0 ml min -1. The column temperature was maintained at 30 C, injection volume was 20 µl and the eluted compounds were monitored at 265 nm. The typical retention times of S and R isomers are 12.7 and 16.1 minutes respectively. In the developed method, sitagliptin phosphate was subjected to photolytic, thermal and humidity as per ICH prescribed conditions to show the stability indicating power of the method. The developed chiral HPLC method was validated as per international conference of harmonization (ICH) guidelines with respect to specificity, linearity, limit of detection, limit of quantification, accuracy, precision and robustness and found results satisfactory. In the developed method, the detection and quantitation limits are 150 and 400 ng/ml respectively. The precision (%RSD) of S-isomer at QL level is The method was linear in the concentration range of ng/ml with correlation coefficient of The trend line equation of S-isomer is y=246627x The precision and intermediate precision (%RSD) of the method is 8.67 and 6.83 respectively. The percent recovery of S-isomer was found to be between from In all the robust parameters, the S-isomer was not affected. The method can be used for the determination of enantiomeric purity of sitagliptin phosphate. The HPLC conditions and brief validation results are summarized in Table.7.2 & 7.3 respectively. 327

12 Chapter-V: Development and Validation of Stability Indicating Ultra Performance Liquid Chromatographic Method for Assay of Raltegravir Potassium In chapter-v, a simple, sensitive, reproducible stability indicating ultra performance liquid chromatographic (UPLC) method has been developed for estimation of raltegravir potassium in presence of six process related impurities in drug substance and as well as drug product was given. A waters acquity UPLC system, equipped with binary gradient pump, auto sampler, column oven and photo diode array detector (PDA) was employed for analysis. Chromatographic data was acquired using Empower II software. The chromatographic separation was achieved on waters acquity UPLC, BEH shield RP x 2.1 mm, 1.7 µm column. The separation was achieved in simple isocratic method. The mobile phase contained mixture of buffer and acetonitrile in the ratio of 65:35 (v/v). Buffer consists of 0.2 g sodium perchlorate dissolved in 1000 ml of water, ph adjusted to 2.5±0.05 with perchloric acid solution. The flow rate of the mobile phase was kept at 0.3 ml.min -1. The column temperature was maintained at 30 C and wavelength monitored at 240 nm. In the developed method, raltegravir potassium was subjected to acid, base, oxidative, photolytic, thermal and humidity as per ICH prescribed conditions to show the stability indicating power of the method. The peak homogeneity of raltegravir peak was confirmed with PDA detector in all the forced degradation samples. The mass balance was in all degradation samples were near to 99.2%. The system suitability parameters such as average tailing factor (0.91) and average theoretical plates (5332) are found to be within the acceptable limits. The typical retention time of the raltegravir was found to be 5.1 minutes. The method was linear with correlation coefficient The trend line equation of raltegravir is y= x The average percent recovery range is The newly developed RP-UPLC method for the quantitative determination of raltegravir potassium in bulk drug substances, in presence of its six related impurities, and its possible degradation products is precise, accurate, and specific. This method exhibited an excellent performance in terms of sensitivity and speed. The developed method is stability indicating and it can be conveniently used for routine production samples and also to check the stability of bulk samples to establish retest period for raltegravir potassium. Moreover, the lower solvent consumption, shorter run time of 10 minutes leads to cost effective to chromatographic method. The HPLC conditions and brief validation results are summarized in Table.7.2 & 7.3 respectively. 328

13 Chapter-VI: Physicochemical Characterization of Polymorphs of Irbesartan and Development &Validation of P-XRD Analytical Method for Quantitative Analysis of Irbesartan Form-B Content in Pure and Tablet Dosage Forms of Irbesartan Form-A Chapter-VI opens with physicochemical characterization of polymorphs of irbesartan, development and validation of P-XRD analytical method for quantitative analysis of irbesartan form-b content in pure and tablet dosage forms of irbesartan form-a was presented. Importance of bioavailability of drugs from the dosage form are depend on various factors such as physiological factors, formulation factors and physical and chemical properties of drugs is presented in introduction of chapter-vi. Irbesartan existing in three different polymorphic crystalline forms, amorphous and amorphous hydrate form, but only form-a is used in pharmaceutical formulations (Branded and generic formulations). Irbesartan form-a API can be manufactured reproducibly and form-a is the polymorph in the reference approved (innovator) product i.e Avapro. Irbesartan form-b is still under patent though the patent form-a is already expired in United States. The polymorphic form of irbesartan known to is change to form-a to less stable form-b during commercial manufacturing, and such as resulted market recall of product. In this chapter, evaluation of physicochemical properties of irbesartan form-a and form-b against the reported values are presented. The solubility profile of irbesartan form-a and form-b at different ph buffers as per BCS classification was presented. By using DSC, TGA, FT-IR and optical microscope, the physical properties and shape of the molecules of irbesartan form-a and B crystal forms were evaluated. Based on the DSC data of irbesartan form-a and form-b, form-b is found to be thermodynamically more stable than form A. Irbesartan form-a and form-b were found to be anhydrous as they exhibited no weight loss due to desolvation during TGA. In both forms, mass loss takes place after melting peak. The irbesartan form-a and form-b exhibited differences when viewed under optical polarized light. Irbesartan form-a appeared as bright needle shape. On the other hand, crystals of irbesartan form-b showed irregular or agglomerate in shape. Based on the results obtained from FT-IR data, it can be observed that irbesartan form-b has characteristic peaks at 745, 1200 and 1537 ± 5cm -1, which are not observed in form-a. From the results of the experiments, it is observed that the two forms of Irbesartan are found to be different in all aspects. According to the existing information available in USFDA website on product results recalls from market of irbesartan form-a, due to form-b contamination. Based on this, there 329

14 was a need to develop a quantitative analytical method for determination of irbesartan form-b contamination in irbesartan form-a drug substance and as well as drug product, either by P-XRD, DSC, TGA, Raman and solid state NMR. Here we selected a powder x-ray diffraction technique to quantify the form-b contamination in API and tablet dosage forms. From the powder X-ray diffraction pattern, the characteristic 2-theta values of irbesartan form-b are at 7.9, 14.1, 15.8, 17.6, 17.9 and Out of all these values, 2-theta 15.8±0.2 was selected for quantification of form-b in form-a, which is high intense characteristic peak. Due to particle size and preferred orientation in solid state, preferred orientation of both forms overcome by passed through different sieves. In the developed quantitative P-XRD method, the detection limit of form-b in form-a is about 0.3% and quantitation limit is 1.0%. The precision of form-b at quantitative level is The linearity graph of form-b peak area vs its concentration is linear with correlation coefficient of The average recovery at QL level is 98.0%. The method precision and intermediate precision was 0.90 and 2.16 respectively. The powder X-ray diffraction conditions for quantitative analysis of irbesartan form-b content in form-a, brief validation data are presented in Table.7.4 & 7.5 respectively. Conclusions The developed chromatographic and powder x-ray diffraction methods were found to be simple, sensitive, precise and accurate. The proposed methods were proved to be linear, robust and rugged. The pharmaceutical formulations were successfully analyzed by the proposed methods. Therefore these methods may be suggested as alternative methods for routine quality control. The present research will enrich practical subject knowledge significantly on novel chromatographic and spectroscopic studies on identification and quantification of impurities in pharmaceutical ingredients. It also expands the scope to which these tools 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. This research will add new dimensions to the existing literature on chromatographic and spectroscopic studies on pharmaceutical products. These developed methods are of great asset in quality monitoring of the selected products towards development of pharmaceutical dosage forms containing those active ingredients and may be claimed as a novel, noteworthy feature of the research work. 330

15 Table.7.1: Chemical structures, names and therapeutic activity of selected drugs Drug Name Chemical Names Structure of the compound Therapeutic Activity Solifenacin Succinate (1S)-(3R)-1-azabicyclo [2.2.2]oct-3-yl 3,4- dihydro-1-phenyl-2(1h)- iso-quinolinecarboxylate Overactive bladder Fesoterodine Fumarate isobutyric acid 2-((R)-3- diisopropyl ammonium- 1-phenylpropyl)-4- (hydroxymethyl) phenyl ester hydrogen fumarate Anti muscarinic Darunavir Ethanolate [(1S,2R)-3-[[(4-amino phenyl)sulfonyl](2- methylpropyl)amino)-2- hydroxy-1 (phenyl methyl)propyl]carbamic acid(3r,3as,6ar)-hexa hydrofuro[2,3-b]furan-3- yl ester mono ethanolate HIV-1 protease inhibitor Levosimendan (R)-[[4-(1,4,5,6- tetrahydro-4-methyl-6- oxo-3-pyridazinyl) phenyl]hydrazono] propanedinitrile] Congestive heart failure Sitagliptin Phosphate 7-[(3R)-3-amino-1-oxo- 4-(2,4,5-trifluorophenyl) butyl]-5,6,7,8- tetrahydro-3- (trifluoromethyl)-1,2,4 - triazolo[4,3-a]pyrazine phosphate(1:1)mono hydrate Antidiabetic Cond.. 331

16 Drug Name Chemical Names Structure of the compound Raltegravir Potassium N-[(4-Fluorophenyl) methyl]-1,6-dihydro-5- hydroxy-1-methyl-2-[1- [[(5-methyl-1,3,4- oxadiazol-2- yl)carbonyl]amino]ethyl]- 6-oxo-4-pyrimidine carboxamide monopotassium salt Therapeutic Activity Antiretroviral Irbesartan 2-butyl-3-[p-(o-1Htetrazol-5- ylphenyl)benzyl]-1,3-di azaspiro[4.4]non-1-en-4- one Anti hypertension 332

17 Table.7.2: Chromatographic conditions adopted for selected drugs Parameter Solifenacin Succinate Fesoterodine Fumarate Darunavir Ethanolate Levosimendan Sitagliptin Phosphate Raltegravir Potassium Chapter ID Chapter-II Chapter-III(A) Chapter-III(B) Chapter-IV(A) Chapter-IV(B) Chapter-V Working solution 0.5 mg/ml 0.2 mg/ ml 0.2 mg/ml 0.5 mg/ ml 2.0 mg/ml 0.2 mg/ ml Analytical column Buffer solution Mobile phase Symmetry shield RP x 4.6 mm, 5 µm Mobile Phase-A: 0.01M KH 2 PO 4, ph adjusted to 3.5±0.05 with H 3 PO 4 Mobile Phase-B: Acetonitrile and Water in the ratio of 90:10, v/v Gradient program: Mixture of mobile phase-a& B Inertsil ODS-3V 150 x 4.6 mm, 5 µm 0.01M ammonium dihydrogen orthophosphate+2.0 ml triethylamine, ph adjusted to 3.0±0.05 with H 3 PO 4 Buffer and methanol in the ratio of 42:58, v/v X-Bridge C x 4.6 mm, 3.5 µm 0.01M ammonium formate, ph adjusted to 3.0±0.05 with formic acid solution. Buffer and acetonitrile in the ratio of 55:45, v/v Chiralpak IA 250 x 4.6 mm, 5 µm Chiralpak AD-H 250x4.6mm, 5 µm MtBE, Ethanol, TFA (65:35:0.1, v/v/v) n-heptane, Ethanol, DEA (35:65:0.1, v/v/v) BEH shield 100 x 2.1 mm, 1.7 µm 0.2 grams of sodium perchlorate in 1000 ml of water. ph adjusted to 2.5±0.05, by adding few drops of perchloric acid Buffer and acetonitrile in the ratio of 35:65, v/v Wavelength nm Flow rate ml.min Injection volume 10 µl 10 µl 20 µl 10 µl 20 µl 3 µl Run time minutes Temperature C C 30 C 30 C 30 C 30 C Diluent Mobile phase-a and Acetonitrile (90:10, v/v) Mobile Phase Buffer and acetonitrile (50:50, v/v) Mobile Phase Methanol Mobile phase 333

18 Table.7.3: Summary of analytical method validation adopted for selected drugs Parameter Solifenacin Succinate Fesoterodine Fumarate Darunavir Ethanolate Levosimendan Sitagliptin Phosphate Raltegravir Potassium Chapter ID Chapter-II Chapter-III(A) Chapter-III(B) Chapter-IV(A) Chapter-IV(B) Chapter-V Test concentration (mg.ml -1 ) Average tailing Avg. Theoretical Plates Precision (% RSD) Linearity (Correlation coefficient) Detection limit (DL), % (Impurity-A,B & C, respectively) Quantitation limit (QL), % (Impurity-A,B & C, respectively) Intermediate precision (Cumulative % RSD) , and , 0.01 and 0.02 Not Applicable Not Applicable µg.ml -1 Not Applicable Not Applicable Not Applicable µg.ml -1 Not Applicable Accuracy at 100% concentration Robustness >2.8 (Resolution between solifenacin and impurity-c Not Applicable Not Applicable Resolution between R & S isomers was >9.0 Resolution between R & S isomers was >2.8 Not Applicable Test solution stability 48 hrs 24 hrs 24 hrs 48 hrs 72 hrs 24 hrs Mobile phase stability 48 hrs 24 hrs 24 hrs 48 hrs 72 hrs 24 hrs 334

19 Table.7.4: P-XRD conditions adopted for irbesartan form-b quantification in irbesartan form-a API and dosage forms Parameter Instrument & Model Make X-ray tube Detector Kβ filte Irbesartan Form-B quantitative analysis X-Ray diffractometer & D8 Advance Bruker axs Siemens K FL Cu 2K Lynx Eye Ni Voltage [kv] 40 Current [ma] 40 Wavelength [A ] Theta Start [ 2 Theta] Theta End [ 2 Theta] 16.7 Scan speed [ sec/step] 5 Increment [ ] 0.02 Scan Type-Locked coupled Continuous Rotation [rpm] ON [30] Divergence Slit type Fixed Divergence slit [ ] 0.3 Anti-scattering slit [mm] 3 Table.7.5: Summary of analytical method validation results of irbesartan form-b Parameter Result Detection limit (DL), % 0.3 Quantitation limit (QL), % 1.0 Precision at QL (% RSD) 3.15 Linearity (Correlation coefficient) Trend line equation Y=0.7799x Accuracy, %