Section (i): Brief account of Dexlansoprazole

Section (i): Brief account of Dexlansoprazole Dexlansoprazole (DLP) is the R-enantiomer of lansoprazole (a racemic mixture of the R- and S-enantiomers). It is chemically (R)-(+)2-([3-methyl-4-(2,2,2-trifluoroethoxy)pyridin-2- yl]methylsulfinyl)-1h-benzimidazole. Its empirical formula is: C 16 H 14 F 3 N 3 O 2 S, with a molecular weight of 369.36. The structural formula is: DLP is a white to nearly white crystalline powder which melts with decomposition at 140 C. DLP is freely soluble in dimethylformamide, methanol, dichloromethane, ethanol, and ethyl acetate; and soluble in acetonitrile; slightly soluble in ether; and very slightly soluble in water; and practically insoluble in hexane. DLP is stable when exposed to light. DLP is more stable in neutral and alkaline conditions than acidic conditions [1]. DLP exhibits polymorphism. It is available as both crystalline and amorphous forms. Amorphous forms are generally more unstable than crystalline forms. DLP is marketed with a brand name DEXILANT (earlier known as KAPIDEX) [2]. DEXILANT is the first proton pump inhibitor (PPI) with a Dual Delayed Release (DDR) formulation designed to provide two separate releases of medication upon oral administration. The capsules contain DLP in a mixture of two types of enteric-coated granules with different ph-dependent dissolution profiles. DEXILANT is available in two dosage strengths: 30 mg and 60 mg, per capsule. DEXILANT is a proton pump inhibitor that is marketed by Takeda Pharmaceuticals, Japan. DLP was approved by the U.S. Food and Drug Administration (FDA) on January 30, 2009 [3]. DLP DDR capsules approved for use of once-daily, oral treatment of heartburn 37

associated with symptomatic non-erosive gastro esophageal reflux disease (GERD), the healing of erosive esophagitis (EE) and the maintenance of healed EE. DEXILANT is based on a dual release technology, with the first quick release producing a plasma peak concentration about one hour after application, and the second retarded release producing another peak about four hours later [4]. DEXILANT works by turning off many of the millions of tiny pumps in stomach that produce acid. Clinical studies have shown that DEXILANT provides up to 24 hours of relief from heartburn due to acid reflux disease. Studies also showed DEXILANT heals damage (erosions) to the esophagus and keeps it from coming back. DLP drug substance and drug product is not official any pharmacopeia. Lansoprazole drug substance and drug product which is a recimic mixture of R and S enantiomers is official in USP. Literature survey revealed, LC-MS method have been reported for the quantitative determination of DLP in human plasma [5]. Few methods are reported for the quantification of lansoprazole and its impurities pharmaceutical preparations, biological fluids and in combination with other actives, these includes, colorimetry using dyes [6], UV spectrometry [7-9], HPLC [10-13] and chemo metric approach using HPLC [14] and TLC [15]. UPLC- MS/TOF[16] and HPLC[17] methods are reported for assay of lansoprazole oral suspension. A chiral LC method [18] reported for enantiomeric separation of DLP. Few papers are reported the synthesis, isolation, identification and characterization of the some of the impurities in lansoprazole [19-21]. So far no method is reported for determination of all 11 impurities of DLP. The reported assay methods of lansoprazole are not capable of quantifying DLP without interference from the other impurities. DLP is unstable when exposed to acid, base, peroxide and thermal degradations. Three unknown impurities (degradation products) present at a level below 0.05% in the initial samples of DLP capsules increased to a level of 0.5% in 3 M accelerated stability studies, i.e; 40 C/75%RH. The structures of these compounds are not reported in literature. This prompted the author to develop stability indicating HPLC methods for the assay and impurities. The three degradation impurities are enriched and isolated by using reversephase preparative liquid chromatography and characterized. 38

This chapter describes development and validation of a stability indicating methods for assay and impurities along with the identification, isolation and characterization of three unknown degradation products formed in DLP capsules during the stability studies. Though some of the impurities and degradation products were reported in the literature, identification, isolation and characterization of these degradation products is not reported to the best of our knowledge. Based LC-MS and NMR data the impurity I is characterized as 2-{(1-H- Benzoimidazol-2-ylsulfanyl)-[1-methyl-2-2,2,2-trifluoro-ethoxy)-4a,5,9b-triaza-indeno[2,1- a]inden-10-yl]-methyl}-3-methyl-pyridin-4-ol, with molecular formula C 30 H 23 F 3 N 6 O 2 S and molecular mass 588.16 Impurity-II is characterized as 10-{(1-H-Benzoimidazol-2- ylsulfanyl)-[3-methyl-4-(2,2,2-trifluoro-ethoxy)-pyridin-2-yl]-methyl}-1-methyl-2-(2,2,2- trifluoro-ethoxy)- 4a,5,9b-triaza-indeno[2,1-a]indene with molecular formula C 32 H 24 F 6 N 6 O 2 S and molecular mass 670.16. Impurity-III is characterized as 1-Methyl-2-(2,2,2-trifluoroethoxy)-4a,5,9b-triaza-indeno[2,1-a]indene with molecular formula C 16 H 12 F 3 N 3 O and molecular mass 319.09. 39

Section (ii): Stability Indicating HPLC Assay method for Dexlansoprazole Capsules. This section reports the various aspects relating to the development and validation of stability indicating HPLC method for assay of Dexlansoprazole (DLP) capsules. 1. Experimental 1.1. Chemicals Samples of DLP API are received from process R&D, Dr Reddy s Laboratories, Hyderabad, India. DLP Capsules of 30 and 60 mg & all excipients are received from formulation R&D, Dr Reddy s Laboratories, Hyderabad, India. HPLC grade acetonitrile, methanol, triethylamine, sodium hydroxide and potassium dihydrogen phosphate are supplied by Merck, Darmstadt, Germany. High purity water is prepared by using Millipore Milli-Q plus purification system. 1.2. Determination of appropriate UV wavelength The suitable wavelength for the determination of DLP in diluent is identified by scanning over the range 200 400 nm with a Shimadzu UV-160 (Shimadzu, Japan) double beam spectrophotometer. 1.3. Instrumentation and chromatographic conditions The M/s Waters HPLC System with a photo diode array detector is used for the method development and force degradation studies.the data is monitored and processed using Waters Empower Networking software. The HPLC system used for method validation is waters HPLC system with diode array detector and Agilent 1100 series LC system with variable wavelength detector (VWD). The data is monitored and processed by using waters Empower Networking Software. The chromatographic column used is an Xbridge C-18, 20mm x 4.6 mm column, with 5µ particle size with a Hypersil BDS C18, 10 mm x 4.6 mm guard column. The chromatographic condition follows a gradient program consisting of mixture of buffer and methanol in the ratio of 90:10 (v/v) as mobile phase A and of mixture of methanol and acetonitril in the ratio of 50:50 (v/v) as mobile phase B. The buffer is prepared as 0.05 M KH 2 PO 4 with 0.8% v/v triethylamine and finally ph is adjusted to 8.0. The gradient programme is : Time/%mobile phase A:% Mobile phase B is 0.0/75:25, 3.0/55:45, 4.0/55:45, 4.5/20:80, 5.5/20:80, 6.0/75:25, 8.0/75:25. The flow rate of the mobile phase is 1.2 ml min -1. 40

The column temperature is maintained at 30ºC and the detection wavelength is 285 nm. The injection volume is 20µl. 1.4. Diluent: 0.1N NaOH and methanol in the ratio 75:25(v/v) is used as diluent. 1.5. Preparation of standard drug solution: The stock solution of DLP standard (equivalent to 0.68 mg ml -1 of DLP) is prepared in diluent. The working standard solution (0.068mg ml -1 of DLP for both 30 mg and 60 mg sample analysis purpose) is obtained by dilution of the stock solution in diluent. The Specimen chromatograms of diluent and standard is shown in fig.2.2.1. Blank Standard Fig 2.2.1: Specimen chromatogram of diluent and DLP standard 0.068mg ml -1. 41

1.6. Test Preparation for pharmaceutical formulations: Contents of ten capsules of DLP are emptied and weighed the enteric coated pellets in the capsules. Enteric coated pellets equivalent to 600 mg of DLP is weighed and transferred into a clean dry 1000mL volumetric flask for 60mg and 500 ml volumetric flask for 30mg. 0.1N NaOH is added upto 20% of total volume and, swirled to avoid aggregation and sonicated with frequent intermediate shaking to disperse the pellets. Then diluent is added to make 60% total volume and sonicated for 20 min with intermediate shaking. The temperature of the water in the sonicator bath is maintained between 20 ºC and 25ºC to avoid heating up the solution during soniation. Then volume is made up to total volume and mixed well. The concentration of this test stock solution is 0.6 mg of DLP per ml. The resulting solution is centrifuged at 4000 rpm for 10 min. 5 ml of the centrifugate is then diluted to 50 ml in a volumetric flask with diluent. The final concentration of this test preparation is 60 µg ml -1 of DLP. Placebo sample is prepared in the same way by taking the placebo equivalent its weight present in a test preparation.the Specimen chromatogram of placebo and test sample are shown in fig.2.2.2. Placebo Test Fig 2.2.2: Overlay chromatogram of placebo and DLP Capsules. 42

1.7. Specificity: Regulatory guidances in ICH Q2A, Q2B, Q3B and FDA 21 CFR section 211, require the development and validation of stability-indicating potency of assays. However, the current guidance documents do not indicate detailed degradation conditions in stress testing. The forced degradation conditions, stress agent concentration and time of stress, are found to effect the % degradation. Preferably not more than 20% is recommended for active materials to make the right assessment of stability indicating nature of the chromatographic methods. The discovery of such stress conditions which can yield not more than 20% degradation is based on experimental studies. Chromatographic runs of placebo solution and samples subjected to force degradation are performed in order to provide an indication of the stability indicating properties and specificity of the method. The stress conditions employed are acid, base, neutral and oxidant media, moisture, heat and light. After the degradation treatments are completed, the samples are allowed to equilibrate to room temperature, neutralized with acid or base (as necessary), and diluted with diluent to get the working concentrations equivalent to test preparation. The samples are analyzed against a freshly prepared control sample (with no degradation treatment) and evaluated for peak purity by using photo diode array detector. Specific conditions are described below. 1.7.1. Placebo (excipients) interference: Placebo solutions are prepared by taking the weight of placebo approximately equivalent to its weight in the sample as described in the test preparation for DLP capsules dosage form. 1.7.2. Effect of acid hydrolysis : 300 mg of DLP pellets powder is treated with 100 ml of 1N HCl for 5 minutes on bench top with continuous shaking. The resulting solution is neutralized and then solution is prepared as per the test preparation to obtain a stock solution of 0.6 mg ml -1. Then the test stock is diluted with diluent to get the test preparation having final concentration of drug at about 60 µg ml -1. 43

1.7.3. Effect of base hydrolysis 300 mg of DLP pellets powder is treated with 100 ml of 2N NaOH at 50 C using a heating water bath for 24 hours. The resulting stress solution is neutralized, equilibrated to room temparature and then solution is prepared as per the test preparation to obtain a stock solution of 0.6 mg ml -1. Then the test stock is diluted with diluent to get the test preparation having final concentration of drug at about 60 µg ml -1. 1.7.4. Effect of neutral hydrolysis 300 mg of DLP pellets powder is treated with 100 ml water at 50 C using a heating water bath for 24 hours. The resulting stress solution is equilibrated to room temperature and then treated same as per the test preparation to obtain a stock solution of 0.6 mg ml -1. Then the test stock is diluted with diluent to get the test preparation having final concentration of drug at about 60 µg ml -1. 1.7.5. Effect of oxidation 300 mg of DLP pellets powder is treated with 100 ml of 10%H 2 O 2 for 15 minutes on bench top with continuous shaking. The resulting solution is neutralized and then solution is prepared as per the test preparation to obtain a stock solution of 0.6 mg ml -1. Then the test stock is diluted with diluent to get the test preparation having final concentration of drug at about 60 µg ml -1. 1.7.4. Effect of moisture and heat To evaluate the effect of moisture and heat, DLP pellets powder is distributed as thin layer over two glass plates. One plate is then exposed to 25ºC/90% relative humidity for 9 days. Similarly DLP pellets powder in another plate is exposed in an oven at 105ºC for 1 hour. Then, both the samples are subjected to sample preparation using diluents as described in test preparation. 1.7.5. Effect of UV and visible light To study the photochemical stability of the drug product, the DLP pellets powder is exposed to 1200 K Lux hours of visible light and 200 Watt hours/ meter 2 of UV light by using photo stability chamber. After exposure the samples are subjected to sample preparation using diluents as described in test preparation. 44

1.8. Method validation 1.8.1. Precision Precision (intra-day precision) of the assay method is evaluated by carrying out six independent assays of test sample of DLP capsules against qualified standard. The % RSD of six assays obtained is calculated. The intermediate precision (inter-day precision) of the method is also evaluated using two different HPLC systems and different HPLC columns in different days in the same laboratory. 1.8.2. Linearity Linearity study for assay method is made using six different concentration levels in the range of about 4-150 µg ml -1 of DLP (corresponding to 6.6 to 250% of assay of nominal sample concentration of 60 µg ml -1.). The data of peak area versus concentration is subjected to least-square regression analysis. 1.8.3. Accuracy A study of recovery of DLP from spiked placebo is conducted. Samples are prepared by mixing placebo with DLP API equivalent to about 20%, 50%, 70%, 100%, 120%, and 150% of the assay of nominal sample concentration. Sample solutions are prepared in triplicate for each spike level as described in the test preparation. The % recovery is then calculated. 1.8.4. Robustness To determine the robustness of the developed method, experimental conditions are purposely altered one after the other to estimate their effect. Five replicate injections of standard solution are injected under each parameter change. The effect of flow rate, ph, column temperature and organic phase composition in mobile phase (methanol in mobile phase A and acetonitrile and methanol in mobile phase B) on the tailing factor of DLP peak and the %RSD for peak areas of replicate injections of standard is studied at flow rates of 1.0 ml min -1 and 1.4 ml min -1, at ph of 7.8 and 8.2, at column temperatures of 25ºC and 35ºC and organic phase compositions in mobile phase at + 10% respectively. 45

1.8.5. Solution stability and mobile phase stability The solution stability of DLP in the assay method is carried out by leaving solutions of both the test preparation and reference standard preparation in tightly capped volumetric flasks at room temperature for 48 hours. The same sample solutions is assayed after every 24 hours during the study period. The mobile phase stability is also carried out by assaying freshly prepared sample solutions against freshly prepared reference standard solutions at 24 hours interval for 48 hours. Mobile phase prepared is kept constant during the study period. The % RSD of assay of DLP is calculated for the study period during mobile phase stability and solution stability experiments. 2. Results and discussion 2.1. Determination of suitable wavelength The UV spectrum of DLP recorded in the range 200-400 nm is illustrated in fig.2.2.3. The spectrum indicates that 285 nm gives a good sensitivity for the assay. Fig 2.2.3: UV Spectra of DLP. 46

2.2. Optimization of chromatographic conditions The HPLC procedure is optimized with a view to develop a stability indicating assay method. Pure drug and stressed samples are injected and run in different solvent systems. Selection of mobile phase ph is done based on stability of DLP. Drug is found to be not stable and peak area continuously decreases in mobile phase with ph less than 7.0. Due to problem in stability of standard and test solutions, after several experiments the diluent is finalized as 0.1N NaoH and methanol in the ratio of 75 : 25 v/v. The ph of the diluents is about 11. Normal silica based column stationary phases not workable at ph 8.0. Most of the C18 column peak shape goes quickly as the silica starts dissolving at ph 8.0. Hence a choice is made to work with hybrid silica based columns. After screening of columns which can withstand for higher ph conditions, a choice of the column is made to waters Xbridge C18 column. A guard column is also chosen with C18 stationary phase in order to increase the life of Xbridge column. DLP is prone to degradation upon stability, it generates number of impurities during upon storage. As several late eluting non-polar degradants are possible to be present in the sample, isocratic methods are found to be not feasible due to high run times in order to elute all the degradants. As in the pharmaceutical industry, lot of potency analysis is needed to check the quality of the formulated products, a study is conducted to get the stability indicating method with shorter runtime. A number of experiments are done with different lengths of the columns and different mobile phase compositions and with different gradient programmes to separate all the degradants from DLP peak within short time. Eventually, satisfactory peak shape and satisfactory separation is achieved using a 20 mm x 4.6 mm, Xbridge C18 column with 5 µm with mobile phase A consisting of mixture of buffer and methanol in the ratio of 90:10 (v/v) as mobile phase A and of mixture of methanol and acetonitril in the ratio of 50:50 (v/v) as mobile phase B with a gradient programme of : Time/%mobile phase A:% Mobile phase B is 0.0/75:25, 3.0/55:45, 4.0/55:45, 4.5/20:80, 5.5/20:80, 6.0/75:25, 8.0/75:25. The optimum flow rate and column temperatures are found to be 1.2 ml min -1 and 30ºC respectively. 47

2.3. Method validation 2.3.1. Precision Method repeatability (intra-day precision) is evaluated by assaying six samples, prepared as described in the test preparation. The mean % assay and % RSD for assay values are found to be 100.5 and 0.2 respectively. These are well with in the acceptance criteria i.e. mean % assay between 97.0-103.0 and RSD not more than 2.0 %. The intermediate precision (inter day precision) is performed by assaying six samples on different HPLC systems and different HPLC columns in different days as described in the sample preparation. The mean % assay and % RSD for assay values are found to be 100.1 and 0.2 respectively. The result shows good precision of the method (table 2.2.1). Table 2.2.1: Results of precision of test method Sample No. Assay of DLP as % of labeled amount Intra-day precision Inter-day precision 1 100.7 99.8 2 100.4 99.9 3 100.7 100.2 4 100.3 100.2 5 100.7 100.3 6 100.4 100.4 Mean 100.5 100.1 RSD 0.2 % 0.2 % 2.3.2. LOQ and LOD The LOQ and LOD are determined based on signal-to-noise ratios at analytical responses of 10 and 3 times the background noise, respectively. The LOQ is found to be 0.14 μg ml -1 with a resultant %R.S.D. of 0.6 (n = 5). The LOD is found to be 0.028 μg ml.-1 2.3.3. Linearity A linear calibration plot for assay of DLP is obtained over the calibration range 4-150 µg ml -1 and the correlation co-efficient is found to be greater than 0.999. The result shown in fig.2.2.4 indicates that an excellent correlation exists between the peak area and concentration of the analyte. 48

Fig.2.2.4: Linearity of detector response for DLP. 2.3.4. Accuracy The percentage recovery of DLP in pharmaceutical dosage form of capsules is shown in table 2.2.2 ranged from 98.2 to 100.7 and indicates high accuracy of the method. Table 2.2.2: Recovery results of DLP in DLP capsules Sample mg % level mg found No. added Recovery 1 20% 120.60 121.46 100.7 2 20% 120.60 121.23 100.5 3 20% 120.60 121.36 100.6 4 50% 301.50 296.04 98.2 5 50% 301.50 296.64 98.4 6 50% 301.50 297.48 98.7 7 70% 422.10 418.82 99.2 8 70% 422.10 418.66 99.2 9 70% 422.10 419.00 99.3 10 100% 603.00 604.23 100.2 11 100% 603.00 602.46 99.9 12 100% 603.00 604.14 100.2 13 120% 723.60 725.16 100.2 14 120% 723.60 725.38 100.2 15 120% 723.60 725.81 100.3 16 150% 904.50 907.72 100.4 17 150% 904.50 907.79 100.4 18 150% 904.50 907.77 100.4 Mean % Recovery 100.6 98.4 99.2 100.1 100.3 100.4 49

2.3.5. Robustness In all the deliberately varied chromatographic conditions studied (flow rate, column temperature, ratio of acetonitrile and methanol composition in mobile phase), the tailing factor and the % RSD for the DLP peak area for five replicate injections of standard is found to be within the acceptable limit of not more than 2.0%, illustrating the robustness of the method (table 2.2.3). Table2.2.3: Results of Robustness study Observed value Parameter Variation Tailing factor %RSD 1.Flow rate 0.8 ml min -1 1.1 0.04 1.0 ml min -1 1.0 0.04 1.2 ml min -1 1.0 0.1 2.Column temperature 25ºC 1.2 0.1 30ºC 1.0 0.04 35ºC 1.1 0.1 3.Mobile phase A 90% 1.2 0.1 (for methanol variation) 100% 1.0 0.04 110% 1.1 0.0 4. Mobile phase B 90% 1.1 0.2 (for acetonitrile variation) 100% 1.0 0.04 110% 1.1 0.3 5. Mobile phase B 90% 1.2 0.3 (for methanol variation) 100% 1.0 0.04 110% 1.1 0.1 6. ph 7.8 1.1 0.1 8.0 1.0 0.04 8.2 1.1 0.1 50

2.3.6. Solution stability and mobile phase stability The difference in % assay of DLP test and standard preparations upon storage on bench top is found to be less than 1.2% up to 48 hours. Mobile phase stability experiments showed that tailing factor and % RSD are less than 1.2 and 0.3% respectively up to 48 hours. The solution stability and mobile phase stability experimental data confirms that standard and test preparations and mobile phase used during assay determination are stable up to 48 hours. 2.3.7. Results of specificity studies All the placebo and stressed samples prepared are injected into the HPLC system with photodiode array detector as per the described chromatographic conditions. Chromatograms of placebo solutions have shown no peaks at the retention time of DLP. This indicates that the excipients used in the formulation do not interfere in estimation of DLP in capsules formulation dosage form. Degradation is not found to be significant when exposed to light and humidity. In acid hydrolysis, base hydrolysis, water hydrolysis, thermal stress and oxidative studies, significant degradation is observed. All degradant peaks are well resolved from DLP peak in the chromatograms of all stressed samples. The chromatograms of the stressed samples are evaluated for peak purity of DLP using Waters Empower Networking software. For all forced degradation samples, the purity angle (the weighted average of all spectral contrast angles calculated by comparing all spectra in the integrated peak against the peak apex spectrum) is found to be less than threshold angle (the sum of the purity noise angle and solvent angle, the purity noise angles across the integrated peak) for DLP peak (table 2.2.4). This indicates that there is no interference from degradants in quantitating the DLP in capsules dosage form. Thus, this method is considered "Stability indicating. The chromatogram and purity plots of all stressed samples are shown in figs 2.2.5 to 2.2.12. 51

Table 2.2.4: Table of results of specificity Stress condition % Assay of stressed sample Purity Angle Purity Threshold Kept on bench top for 5 minutes with 1N HCl solution. 84.3 0.111 0.302 Stressed at 50 C in water bath for 24 hrs with 2N NaOH solution. 92.4 0.107 0.301 Kept on bench top for 15 minutes with 10% H 2 O 2 solution. 97.1 0.130 0.327 Stressed at 50 C in water bath for 24 hrs with water. 91.5 0.116 0.299 Exposed to visible light for 1.2million lux hours and UV light at 200 Watt hours/ meter 2 99.7 0.117 0.318 Exposed to Dry heat at 105 C for about 1 hour. Exposed to humidity at 25 C, 90% RH for 9 days. 84.7 0.081 0.292 99.8 0.110 0.318 Fig 2.2.5: Chromatogram and purity plot of acid stressed DLP capsules. 52

Fig 2.2.6: Chromatogram and purity plot of base stressed DLP capsules. Fig 2.2.7: Chromatogram and purity plot of H 2 O 2 stressed DLP Capsules. 53

Fig 2.2.8: Chromatogram and purity plot of water stressed DLP capsules. Fig 2.2.9: Chromatogram and purity plot of light stressed DLP capsules. 54

Fig 2.2.10: Chromatogram and purity plot of Thermal stressed DLP capsules. Fig 2.2.11: Chromatogram and purity plot of humidity stressed DLP capsules. 55

3. Conclusion: A validated stability-indicating HPLC analytical method has been developed for the determination of DLP in delayed release capsules dosage form. The results of stress testing undertaken according to the International Conference on Harmonization (ICH) guidelines reveal that the method is selective and stability-indicating. The proposed method is simple, accurate, precise, specific and has the ability to separate the drug from degradation products and excipients of capsules dosage form. The method is suitable for the routine analysis of DLP in either bulk powder or in other pharmaceutical dosage forms. The HPLC procedure can be applied to the analysis of samples obtained during accelerated stability experiments to predict the expiry dates of DLP in bulk and in formulations. 56

Section (iii): Stability Indicating HPLC method for impurities Dexlansoprazole capsules. This section reports the various aspects relating to the development and validation of stability indicating HPLC method for impurities in DLP capsule dosage form. 1. Experimental 1.1. Chemicals Dexlansoprazole capsules (dual delayed release) are formulated in Dr Reddy s laboratories Ltd, Hyderabad, India. The standards of DLP and its impurities namely carboxylic acid, hydroxy benzimidazole, mercapto benzimidazole, N-oxide, nitrosulphoxide, sulphone, impurity I, sulphide, impurity II, impurity III and impurity M+467 are supplied by Dr. Reddy s laboratories limited, Hyderabad, India. The HPLC grade acetonitrile, ethanol and analytical grade KH 2 PO 4, NaOH and ortho phosphoric acid are purchased from Merck, Darmstadt, Germany. High purity water is prepared by using Milli Q Plus water purification system (Millipore, Milford, MA, USA). The chemical names and structures of DLP and its impurities are shown in the below. S.No Name of the Structure and IUPAC Name impurity 1 Dexlansoprazole 2 Carboxylic acid Impurity (R)-2-(((3-methyl-4-(2,2,2-trifluoroethoxy)pyridin-2- yl)methyl)sulfinyl)-1h-benzo[d]imidazole 3 Hydroxy- benzimidazole 1-(1H-benzo[d]imidazol-2-yl)-3-methyl-4-oxo-1,4- dihydropyridine-2-carboxylic acid 1H-benzo[d]imidazol-2-ol 57

4 Mercapto- benzimidazole 5 N-oxide 1H-benzo[d]imidazole-2-thiol 6 Nirosulphoxide (R)-2-(((1H-benzo[d]imidazol-2-yl)sulfinyl)methyl)-3-methyl-4- (2,2,2-trifluoroethoxy)pyridine 1-oxide 7 Sulphone 8 Cyclized dessulphur- des trifluoro ethyl sulphide adduct (Impurity I) 2-(((3-methyl-4-nitropyridin-2-yl)methyl)sulfinyl)-1Hbenzo[d]imidazole [[(1H-benzimidazole-2-yl)sulfinyl]methyl]-3-methyl-4-(2,2,2- trifluoroethoxy)-pyridine 1-oxide 2-(((1H-benzo[d]imidazol-2-yl)thio)(1-methyl-2-(2,2,2-58

trifluoroethoxy)benzo[4',5']imidazo[2',1':2,3]imidazo[1,5- a]pyridin-12-yl)methyl)-3-methylpyridin-4-ol. 9 Sulphide 10 Cyclized dessulphursulphide adduct (Impurity II) 2-(((3-methyl-4-(2,2,2-trifluoroethoxy)pyridin-2- yl)methyl)thio)-1h-benzo[d]imidazole 12-(((1H-benzo[d]imidazol-2-yl)thio)(3-methyl-4-(2,2,2- trifluoroethoxy)pyridin-2-yl)methyl)-1-methyl-2-(2,2,2- trifluoroethoxy)benzo[4',5']imidazo[2',1':2,3]imidazo[1,5- a]pyridine 11 Cyclized dessulphur (Impurity III) 1-methyl-2-(2,2,2- trifluoroethoxy)benzo[4',5']imidazo[2',1':2,3]imidazo[1,5- a]pyridine. 59

12 Cyclized dessulphurmercapto benzimidazole adduct (Impurity M+467) 12-((1H-benzo[d]imidazol-2-yl)thio)-1-methyl-2-(2,2,2- trifluoroethoxy)benzo[4',5']imidazo[2',1':2,3]imidazo[1,5- a]pyridine 1.2. Determination of appropriate UV wavelength The suitable wavelength for the determination of DLP and its impurities is identified by taking the overlay spectra from 200 400 nm of all impurities and DLP from PDA detector. 1.3. Instrumentation and chromatographic conditions The Waters HPLC System with a photo diode array detector is used for the method development and force degradation studies.the data is monitored and processed using Waters Empower Networking software. The HPLC system used for method validation is waters HPLC system with diode array detector and Agilent 1100 series LC system with variable wavelength detector (VWD). The chromatographic column used is an X-terra RP-18, 250mm x 4.6 mm column, with 3.5µ particle size with a 10 mm x 4.6 mm guard column. The chromatographic condition follows a gradient program consisting of mixture of buffer and acetonitrile in the ratio of 90:10 (v/v) as mobile phase A and of mixture of buffer and acetonitrile in the ratio of 30:70 (v/v) as mobile phase B. The buffer is prepared as 0.01 M KH 2 PO 4 (6.8g in 1000ml) in water with 1.0% v/v triethylamine and finally ph is adjusted to 7.0. The gradient program is Time/% Mobile phase B: 0.0/10, 50/60, 67/80, 85/80, 90/10, 100/10. The flow rate of mobile phase is 0.8 ml min -1. The column temperature is maintained at 25ºC and the detection wavelength is 285 nm. The injection volume is 10µl. Sample cooler temperature is used as 5 C. 60

1.4. Diluent: 0.1 N Sodium hydroxide and ethanol in the ratio of 3:5 (v/v) is used as a diluent. 1.5. Preparation of dexlansoprazole diluted standard solution: An accurately weighed amount of about 54 mg of DLP working standard is transferred into a 50 ml dried volumetric flask. 10 ml 0.1 N sodium hydroxide solution is added, sonicated to dissolve the material completely and diluted to volume with diluent and mixed well. This stock solution of DLP (equivalent to about 1.0 mg ml -1 of DLP) is then diluted to get a working DLP diluted standard solution (about 2 µg ml -1 of DLP) by dilution of the stock solution in with diluents along with 20% volume of 0.1N NaOH solution. The specimen chromatogram of diluent and DLP diluted standard solution is shown in fig.2.3.1. 1.6. Test Preparation for dexlansaprazole pharmaceutical formulations: Twenty capsules of DLP capsules (dual delayed release) are weighed and the contents are emptied and the pellets are transferred into a clean dry poly bag. Pellets equivalent to about 300 mg of DLP are transferred into a 500 ml volumetric flask. 100 ml of 0.1 N sodium hydroxide solution is first added and sonicated for about 10 minutes to disperse the pellets. Then 250 ml of diluent is added and, sonicated for 15 minutes with intermediate shaking. The temperature of water in sonicator bath is maintained between 20 C to 25 C. The volume is then made up with diluent and mixed well. A portion of the above solution is centrifuged in a centrifuge tube with cap at 10000 RPM for 5 minutes. The resultant clear supernatant solution is used for injection. Always freshly prepared solutions are used. Placebo sample is prepared in the same way by taking the placebo equivalent its weight present in a test preparation. The specimen chromatogram of placebo and test samples is shown in fig.2.3.2 and 2.3.3. 61

Diluent Diluted standard Fig 2.3.1: Specimen chromatograms of diluent and DLP diluted standard. Placebo Fig 2.3.2: Specimen chromatogram of placebo for DLP capsules. 62

Fig 2.3.3: Specimen chromatogram of DLP capsules test sample spiked with known impurities. 63

1.7. Specificity: Regulatory guidances ICH Q2A, Q2B, Q3B and FDA 21 CFR section 211, require the development and validation of stability-indicating impurities method for all pharmaceutical dosage forms. However, the current guidance documents do not indicate detailed degradation conditions in stress testing. The forced degradation conditions, stress agent concentration and time of stress, are found to effect the % degradation. Not more than 20% degradation is recommended for active materials to make the right assessment of stability indicating nature of the chromatographic methods. The optmisation of such stress conditions which can yield not more than 20% degradation is based on experimental study. Chromatographic runs of placebo and samples subjected to force degradation are performed in order to provide an indication of the stability indicating properties and to establish the specificity of the method. The stress conditions employed are acid, base, neutral and oxidant media, moisture, heat and light. After the degradation treatments are completed, the samples are allowed to equilibrate to room temperature, neutralized with acid or base (as necessary), and diluted with diluent to get the working concentrations equivalent to test preparation. The stressed samples are subjected to assay analysis to assess the mass balance. The samples are analyzed against a freshly prepared control sample (with no degradation treatment) and evaluated for peak purity by using photo diode array detector. Specific conditions are described below. 1.7.1. Placebo (excipients) interference: Placebo solutions are prepared in triplicate by taking the weight of placebo approximately equivalent to its weight in the sample as described in the test preparation for DLP capsules dosage form. 1.7.2. Effect of acid hydrolysis About 300 mg of DLP pellets powder is treated with 25 ml of 1N HCl for 1 minute on bench top with continuous shaking. The resulting solution is immediately neutralized and then solution is prepared as per the test preparation to obtain a solution having final concentration of drug at about 0.6 mg ml -1. 1.7.3. Effect of base hydrolysis About 300 mg of DLP pellets powder is treated with 25 ml 1N NaOH at 60 C using a heating water bath for 24 hrs. The resulting stress solution is neutralized, equilibrated to room 64

temperature and then solution is prepared as per the test preparation to obtain a solution having final concentration of drug at about 0.6 mg ml -1. 1.7.4. Effect of neutral hydrolysis About 300 mg of DLP pellets powder is treated with 25 ml water at 60 C using a heating water bath for 9.5 hours. The resulting stress solution is neutralized, equilibrated to room temperature and then treated same as per the test preparation to obtain a solution having final concentration of drug at about 0.6 mg ml -1. 1.7.5. Effect of oxidation About 300 mg of DLP pellets powder is treated with 25 ml of 3%H 2 O 2 for 30 minutes on bench top with continuous shaking. The resulting solution is neutralized and then solution is prepared as per the test preparation to obtain a solution having final concentration of drug at about 0.6 mg ml -1. 1.7.4. Effect of moisture and heat To evaluate the effect of moisture and heat, DLP pellets powder is distributed as thin layer over two glass plates. One plate is exposed to 25ºC/90% relative humidity for 7 days. Similarly DLP pellets powder in another plate is exposed in an oven at 105ºC for 1 hour. Then, both the samples are subjected to preparation using diluents as described in test preparation. 1.7.5. Effect of UV and visible light To study the photochemical stability of the drug product, DLP pellets powder is exposed to 1200 K Lux hours of visible light and 200 Watt hours/ m 2 of UV light by using photo stability chamber. After exposure, the samples are subjected to preparation using diluents as described in test preparation. 65

1.8. Method validation 1.8.1. Relative response factors of all Known impurities The relative retention times(rrts) and relative response factors (RRFs) of all known impurities are established against DLP. Different concentrations of DLP and its known impurities are injected into the chromatographic conditions developed. The linearity graphs are drawn for DLP and all its known impurities individually. The relative response factors are then calculated by dividing the slope of impurity by slope of DLP. The relative retention times(rrt s) and relative response factors (RRF s) of 11 known impurities are summarized in table 2.3.1. Table 2.3.1: RRT and RRF of known impurities of DLP. S.No Name of the impurity RRT RRF 1 Carboxylic acid Impurity 0.14 1.04 2 Hydroxy benzimidazole 0.25 0.92 3 Mercapto benzimidazole 0.36 2.66 4 N-oxide 0.71 1.21 5 Nitrosulphoxide 0.74 1.26 6 Sulphone 1.06 0.92 7 Impurity I 1.41 1.11 8 Sulphide 1.43 1.11 9 Impurity II 1.65 0.88 10 Impurity III 1.68 0.69 11 Impurity M+467 1.85 1.96 1.8.2. Precision Precision (intra-day precision) of the impurities method is evaluated by preparing six different solutions of test sample of DLP capsules spiked with known impurities and injected into the developed chromatographic conditions described above. % of impurities are calculated against a qualified DLP standard. RSD is then calculated for % of impurities individually obtained for six different preparations. The intermediate precision (inter day precision) of the method is also evaluated using different HPLC systems and different HPLC columns on different day in the same laboratory. 66

1.8.3. Limits of Detection (LOD) and Quantification (LOQ) The LOD and LOQ for DLP and its 11 known impurities are determined at a signalto-noise ratio of 3:1 and 10:1, respectively, by injecting a series of dilute solutions with known concentrations. Precision study is also carried out at the LOQ level by injecting six individual preparations and % RSD is calculated. 1.8.4. Linearity Linearity test solutions for DLP and all its known impurities are prepared by diluting stock solutions to the required concentrations. The solutions are prepared at different concentration levels from LOQ to equal to or more than 150% of the specification concentration level for DLP and its all known impurities. The solutions are then injected into the chromatographic conditions developed. The data of peak area versus concentration is subjected to least-square regression analysis. 1.8.5. Accuracy A study of recovery of DLP and its 11 known impurities from placebo is conducted. Samples are prepared by mixing placebo with DLP as per the formulation composition and then spiking all the known impurities at different spike levels starting from LOQ to 150% of the specification level. Sample solutions are prepared in triplicate for each spike level as described in the test preparation and injected into the chromatographic conditions developed. The % recovery is then calculated against DLP diluted standard and by using relative response factor and compared against the known amounts of impurities spiked. 1.8.6. Robustness To determine the robustness of the developed method, experimental conditions are deliberately altered and the elution patterns, tailing factor and resolution between its impurities are evaluated. The effect of flow rate is studied at 0.6 ml ml -1 and 1.0 ml min -1. The effect of the column temperature is studied at 20ºC and 30ºC instead of 25ºC. The effect of ph is studied using mobile phase containing buffer with ph 7.0 ± 0.2. The effect of the percent organic strength is studied by varying acetonitrile by 10 to +10% while other mobile phase components were held constant. 67

1.8.7. Solution stability and mobile phase stability The stability of DLP and its impurities in solution for the impurities method is determined by leaving spiked sample solution in a tightly capped volumetric flask at room temperature on bench top and refrigerator and by measuring the amounts of the impurities at different intervals. The stability of mobile phase is also determined by analysing freshly prepared solution of DLP and its impurities for 3 days by using the same mobile phase during the study period. 2. Results and discussion 2.1. Determination of suitable wavelength The UV spectrum of DLP and its 11 known impurities are extracted in PDA detector from 200-400 nm and is illustrated in fig.2.3.4. The spectrum indicates that 285 nm gives a good sensitivity for the all impurities of DLP. Fig 2.3.4: UV Spectra of DLP and its impurities. 2.2. Optimization of chromatographic conditions The HPLC procedure is optimized with a view to develop a stability indicating impurities method. Pure drug and stressed samples are injected and run in different solvent systems. Selection of mobile phase ph is done based on stability of DLP. Drug is found to be not stable and impurities peak area continuously decreases in mobile phases with ph less than 7.0. Due to the problem in stability of standard, test and impurity solutions, after several experiments the diluent is finalized as 0.1N NaOH and ethanol 75: 25 v/v. The ph of the diluent is about 11. Normal silica based column stationary phases not workable beyond ph 8.0. Most of the C18 column peak shape goes quickly as the silica starts dissolving beyond 68

ph 8.0. Hence a choice is made to work with hybrid silica based columns. After screening of columns which can withstand for higher ph conditions, a choice of the column is made to waters X-terra RP18 column. A guard column is also chosen with C18 stationary phase in order to increase the life of analytical column. DLP is prone to degradation upon stability, it generates number of impurities upon storage. As several late eluting non-polar degradants are found in the sample, isocratic method is found to be not feasible in order to elute all the degradants. As in the pharmaceutical industry, lot of stability analysis is needed to check the quality of the formulated products for shelf life determination, a study is conducted to get the stability indicating method which can separate all known unknown impurities satisfactorily. A number of experiments are done with different columns and different mobile phase compositions and with different gradient programmes to separate all the degradants from each other and from DLP peak. Eventually, satisfactory peak shape and satisfactory separation is achieved using a 250 mm x 4.6 mm, X-terra RP18 column with 3.5 µm with mobile phase consisting of mixture of buffer (ph 7.0) and acetonitrile in the ratio of 90:10 (v/v) as mobile phase A and of mixture of buffer (ph 7.0) and acetonitrile in the ratio of 30:70 (v/v) as mobile phase B with a gradient programme of Time/% Mobile phase B : 0.0/10, 50/60, 67/80, 85/80, 90/10, 100/10. The optimum flow rate and column temperatures are found to be 0.8 ml min -1 and 25ºC. As there are 11 known impurities which are differing in polarity very significantly, with carboxylic acid impurity being most polar and impurity M+467 being most non polar, it is learnt during the experiments that a run time of 100 min is necessary. Especially, the separation between impurity I & sulphone and the separation between impurity II and III is found to be critical. The column length reduction or making the faster gradient is not successful in reducing the run time. Different ph conditions are also experimented without success. Different buffers in mobile phase also explored but KH 2 PO 4 with triethylamine as ion pair is found to be showing the best separations especially between unknown and known impurities when samples of stress solutions are evaluated. The wavelength of 285 nm is found to be best suitable for all known and unknown impurities estimation, as all the impurities are having good response at this wavelength. A test concentration of 0.6 mg ml -1 with an injection volume of 10µl is found to provide adequate sensitivity to the method wherein the LOQ of DLP and its known impurities is less than the reporting threshold as per ICH. Due to limited stability of solution of test sample, selection of sample cooler temperature as 5 C is found necessary. 69

2.3. Method validation 2.3.1. Precision The precision of test method (intra-day precision) is evaluated by analyzing six test samples of DLP capsules by spiking test preparation with DLP impurities blend solution to get about 0.2 to 0.3% of carboxylic acid, hydroxy benzimidazole, mercapto benzimidazole, N- Oxide, nitrosulphoxide, impurity I, sulphide, impurity II, impurity III and impurity M+467 and about 0.4% of sulphone. Six different test preparations having placebo equivalent to test and spiked with about 0.25% of DLP are prepared and injected into the chromatographic condition developed. Relative standard deviations of % of DLP and its 11 known impurities were evaluated. Inter-day Precision study is conducted on a different day with different mobile phase, with different HPLC and different column. Six different test preparations of sample are prepared similar to intra-day precision and the relative standard deviations of % of DLP and its 11 known impurities are evaluated. The % RSD values are presented in table 2.3.2 and 2.3.3. % RSD values of less than 15% for DLP and its 11 known impurities shows that method is precise and work satisfactorily on different day, with different column and HPLC. 2.3.2. LOQ and LOD The limit of detection, limit of quantification are determined by following signal to noise ratio method. A precision study also conducted at LOQ level for DLP and its 11 known impurities. The results shows that method is sensitive enough to quantify impurities well below the ICH reporting threshold of 0.1%, as LOQ values are in the range of 0.02% to 0.05%. The data is summarized in table 2.3.4. 70

2.3.3. Linearity A linear calibration plot for DLP and its 11 known impurities is drawn over the calibration range LOQ to 150% of the specification levels. Correlation co-efficient for DLP and its 11 known impurities is found to be greater than 0.997. The regression analysis results are shown in table 2.3.4. The results indicates that an excellent correlation exists between the peak area and concentration of the analyte for DLP and its 11 known impurities. The linearity graphs are presented as figure 2.3.5 to 2.3.7. 2.3.4. Accuracy The percentage recovery of DLP and its 11 known impurities in presence of placebo matrix of DLP capsules from LOQ to 150% spike level are in the range of 90.9% to 109.2%. The LC chromatogram of test preparation spiked with all 11 known impurities is shown in Fig. 2.3.3. The % recovery values for DLP and impurities are presented in table 2.3.5. The data shows that the method is having capability to estimate accurately all 11 known impurities of DLP in DLP capsules. 71

Table 2.3.2: Results of Inter day precision of test method for DLP and its 11 known impurities. Sample No. DLP Carboxylic acid Impurity Hydroxy benzimi dazole Mercapto benzimid azole N- Oxide Nitro sulph oxide Sulph one Impurity I Sulphide Impurity II Impurity III Impurity M+467 1 0.256 0.237 0.180 0.234 0.181 0.178 0.444 0.268 0.276 0.256 0.295 0.212 2 0.248 0.232 0.187 0.240 0.187 0.182 0.456 0.267 0.286 0.248 0.305 0.210 3 0.257 0.237 0.187 0.239 0.186 0.184 0.457 0.271 0.286 0.249 0.296 0.210 4 0.255 0.238 0.186 0.242 0.188 0.184 0.456 0.265 0.286 0.248 0.301 0.213 5 0.249 0.236 0.190 0.230 0.185 0.178 0.465 0.268 0.287 0.249 0.301 0.207 6 0.246 0.237 0.189 0.231 0.182 0.176 0.461 0.265 0.284 0.249 0.304 0.206 Avg 0.252 0.236 0.187 0.236 0.185 0.180 0.457 0.267 0.284 0.250 0.300 0.210 %RSD 1.9 0.9 1.9 2.1 1.5 1.9 1.5 0.9 1.4 1.2 1.4 1.3 Table 2.3.3: Results of Intra-day precision of test method for DLP and its 11 known impurities. Sample No. DLP Carboxylic acid Impurity Hydroxy benzimi dazole Mercapto benzimid azole N- Oxide Nitro sulph oxide Sulph one Impurity I Sulphide Impurity II Impurity III Impurity M+467 1 0.252 0.227 0.200 0.197 0.205 0.171 0.437 0.235 0.254 0.244 0.225 0.191 2 0.246 0.229 0.205 0.195 0.197 0.176 0.440 0.241 0.249 0.232 0.239 0.188 3 0.251 0.226 0.214 0.201 0.193 0.166 0.437 0.231 0.261 0.228 0.242 0.191 4 0.235 0.231 0.193 0.194 0.190 0.171 0.427 0.252 0.252 0.221 0.238 0.183 5 0.245 0.239 0.195 0.190 0.184 0.171 0.441 0.226 0.274 0.226 0.241 0.204 6 0.248 0.234 0.207 0.192 0.181 0.169 0.418 0.229 0.251 0.230 0.237 0.188 Avg 0.250 0.231 0.202 0.195 0.192 0.171 0.433 0.236 0.257 0.230 0.237 0.191 %RSD 2.5 2.1 3.9 2.0 4.6 1.9 2.1 4.0 3.6 3.4 2.6 3.7 72

Table 2.3.4: LOD, LOQ data of DLP and its impurities Parameter DLP Carboxylic acid Impurity Hydroxy benzimida zole Mercapto benzimid azole N-Oxide Nitrosul phoxide Sulphone Impurity I Sulphide Impurity II Impurity III Impurity M+467 LOD In % 0.009 0.011 0.012 0.005 0.013 0.015 0.016 0.011 0.014 0.015 0.019 0.01 S/N ratio 2.82 3.117 2.93 2.95 3.01 3.04 3.01 3.464 3.01 2.815 2.370 3.26 LOQ In% 0.026 0.0347 0.037 0.016 0.039 0.044 0.049 0.033 0.042 0.044 0.048 0.029 S/N ratio 9.94 10.594 10.15 10.32 9.93 10.22 9.58 10.827 9.51 9.363 9.698 10.48 Precision at LOQ 3.0 2.6 2.5 7.7 3.5 2.1 3.1 7.7 5.5 2.4 1.6 1.9 (%RSD*) * RSD for 6 determinations. 73

Figure 2.3.5 : Linearity graphs of DLP and its impurities. 74

Figure 2.3.6 : Linearity graphs of DLP impurities. 75

Fig.2.3.7: Linearity graphs of DLP impurities. 76

Table 2.3.5: Recovery results of DLP impurities in pharmaceutical dosage forms Spike level DLP Carboxylic acid Impurity Hydroxy benzimida zole Mercapto benzimid azole N-Oxide Nitrosul phoxide Sulphone Impurity I Sulphide Impurity II Impurity III Impurity (M+467) LOQ 101.4(2.0) 102.2(5.4) 103.9(6.6) 103.4(0.0) 97.5(1.6) 90.9(5.4) 107.6(0.7) 96.4(4.6) 94.6(3.9) 96.7(3.2) 96.6(3.5) 99.9(1.9) 50% 101.1(1.2) 102.7(4.3) 102.9(0.6) 91.4(0.0) 100(1.2) 91.3(0.6) 97.7(1.6) 103.8(0.6) 102.0(1.1) 102.6(3.3) 107.3(1.4) 102.7(1.0) 75% 99.6(0.7) 101.5(3.8) 105.3(0.7) 91.1(0.9) 97.6(0.7) 92.3(1.3) 98.1(0.5) 102.6(0.8) 96.5(0.7) 100.7(3.7) 105.6(2.4) 101.1(0.7) 100% 99.0(0.7) 98.1(2.0) 104.5(0.5) 91.1(0.8) 97.1(0.4) 94.0(1.0) 97.3(0.5) 105.4(1.2) 97.1(0.7) 102.2(2.1) 109.2(2.4) 101.2(0.1) 125% 94.5(0.5) 99.6(3.1) 104.2(0.6) 93.7(0.8) 98.1(0.5) 95.0(0.6) 98.3(0.7) 101.0(1.1) 99.2(0.8) 101.9(1.5) 106.5(4.1) 101.3(0.6) 150% 94.1(1.4) 100.0(1.6) 99.0(0.6) 92.0(0.6) 96.6(0.7) 93.6(0.7) 96.6(0.6) 97.8(2.3) 96.5(0.9) 108.0(0.7) 108.5(0.5) 102.5(0.7) Values given in parenthesis represent standard deviation of triplicate results. 77