SIMULTANEOUS DETERMINATION OF TELMISARTAN AND HYDROCHLOROTHIAZIDE IN TABLET DOSAGE FORM USING REVERSE PHASE HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

CHAPTER 5 SIMULTANEOUS DETERMINATION OF TELMISARTAN AND HYDROCHLOROTHIAZIDE IN TABLET DOSAGE FORM USING REVERSE PHASE HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

CHAPTER 5 Simultaneous determination of Telmisartan and Hydrochlorothiazide in tablet dosage form using reverse phase high performance liquid chromatography 5.0 INTRODUCTION The objective was to develop a single method for simultaneous determination of the Telmisartan and Hydrochlorothiazide tablet dosage form. The method, after development, was validated as per ICH guidelines Q2 (R1). The target for this research work was to present comprehensive methods of critical tests for each drug product. Thus this work can be treated as a monograph for the drug products. A brief introduction of each molecule is has already been included in the previous chapter. 5.1 LITERATURE SURVEY The literature survey reveals that, TE and HCTZ are reported in British Pharmacopoeia [6, 7]. There have been several publications describing analytical methods for the determination of HCTZ and TE individually or with other drugs as combination. However the finished product in combination dosage form was not official in any pharmacopeia (USP/ BP/ IP) @. Comparisons of official methods available are compiled in tables 5.1.1 and 5.1.2below. ----------------------------------------------------------------------------------------------------------- @ This search was performed at the start and during the course of the study. A draft monograph has been published in USP Forum Vol 36(3). The Monograph is now official as per USP 34, from December 2011. SPP SPTM, SVKM s NMIMS, Mumbai 85

Table 5.1.1: Assay Pharmacopeia methods for Drug Substance Method Telmisartan HCTZ BP 2011 USP 34 BP 2011 USP 34 Potentiometric Titration Potentiometric Titration Potentiometric Titration HPLC Column Not Applicable Not Applicable Not Applicable C18 50mm x 4.6mm: 3.5µ Column Temp Not Applicable Not Applicable Not Applicable 35 C Mobile Phase Not Applicable Not Applicable Not Applicable Solution A A mixture of acetonitrile and methanol (3:1) was prepared and degassed. Solution B A solution of anhydrous formic acid in water (5 in 1000) was prepared and degassed. SPP SPTM, SVKM s NMIMS, Mumbai 86

Gradient Not Applicable Not Applicable Not Applicable Time %A %B (min) 0 3 97 5 3 97 14 36 64 18 3 97 20 3 97 Flow Rate Not Applicable Not Applicable Not Applicable 1ml/min Wavelength Not Applicable Not Applicable Not Applicable 275nm Inj Vol (µl) Not Applicable Not Applicable Not Applicable 10 SPP SPTM, SVKM s NMIMS, Mumbai 87

Table 5.1.2: Assay Pharmacopeia methods for Drug Product CHAPTER - 5 Telmisartan HCTZ Telmisartan-HCTZ BP 2011 USP 34 BP 2011 USP 34 USP 35 Method Not Available HPLC UV HPLC HPLC Spectrophotometer Column Not Applicable C18 40mm x 4.0mm; 5µ Not Applicable C18 250mm x 4.6mm; C8 125mm x 4.0mm; 5µ Column Not Applicable 40 C Not Applicable Not Mentioned 40 C Temp Mobile Phase Not Applicable Buffer - 2.0 g/l ammonium dihydrogen phosphate was prepared. ph was adjusted with 1 M phosphoric acid to 3.0 Mobile Phase: Methanol and Buffer (7:3) Not Applicable A degassed mixture of 0.1 M monobasic sodium phosphate and acetonitrile (9:1) was prepared. ph was adjusted with phosphoric acid to 3.0 ± 0.1 Buffer: 2.0 g/l of ammonium dihydrogen phosphate was prepared. ph was adjusted with phosphoric acid to 3.0. Solution A: Methanol and Acetonitrile (1:1) SPP SPTM, SVKM s NMIMS, Mumbai 88

Gradient Not Applicable Isocratic (Run time Not Applicable Isocratic (Run time Time %Buffer %Solution not mentioned) not mentioned) (min) A 0 85 15 3.50 85 15 3.51 45 55 7.70 45 55 7.71 20 80 12.0 20 80 12.1 85 15 15.5 85 15 Flow Rate Not Applicable 0.7ml/min Not Applicable 2ml/min 1.2ml/min Wavelength Not Applicable 298nm 273nm 254nm 270-Telmisartan 298- HCTZ Inj Vol (µl) Not Applicable 5 Not Applicable 20 10 SPP SPTM, SVKM s NMIMS, Mumbai 89

5.2 PRESENT WORK AND DISCUSSION 5.2.1 Selection of Chromatographic Method Reverse Phase chromatography was the natural choice for method development because of its ease of handling and robust nature. All development was conducted using reverse phase methods. Official methods and methods published in literature for Telmisartan and Hydrochlorothiazide are based on reverse phase chromatographic (RPC) separation. 5.2.2 Selection of Stationary Phase USP, EP monograph and reported HPLC methods of Telmisartan or hydrochlorothiazide recommended use of C18 column for the purpose of determination of related impurities and assay. So C18 columns were preferred as stationary phase. However, one of the important criteria for assay method development was to have a short run time. Thus C8 columns were tried to reduce the retention of the major constituents. 5.2.3 Selection of Wavelength for Analysis The optimum wavelength selected was 270 nm which represents the wavelength where both components had suitable responses in order to permit simultaneous determination of Telmisartan and HCTZ. 270nm is also the wavelength which has absorption maxima for hydrochlorothiazide and absorption minima for Telmisartan. Thus at this wavelength, minor changes will not affect peak areas and thus the final result. This will in effect produce a robust method with respect to wavelength. Figure: 5.2.3.1 Overlaid UV spectrum of Telmisartan and Hydrochlorothiazide SPP SPTM, SVKM s NMIMS, Mumbai 90

5.2.4 Selection and Optimization of Mobile Phase From the experience with the retentively of the molecules as discussed in the previous chapter, a gradient run was the only viable option for a shorter run time. A high aqueous percentage was used in the initial period to retain the hydrochlorothiazide while a higher organic was used to elute the Telmisartan in the later period. Similar to the method for related substances, it was decided to proceed with ion pair chromatography. For assay experiment the primary use of ion pair reagent would be to retain the Hydrochlorothiazide peak and separate it from its main degradant which would, otherwise, eluted very close or could merge with the main peak. Unlike in method for impurities, in an assay method it would not cause any problem if the impurities elute in dead volume. During development of an assay, one of the main selectivity (specificity) criteria is that the degradants should not co-elute with the main analyte peak. The current experiment, being a multi component one, had the additional challenge of ensuring that a degradant of one constituent does not interfere with the other constituent. In other words a degradants due to Hydrochlorothiazide should not interfere with the Telmisartan peak and vice versa. Optimized Chromatographic Conditions: Instruments/Equipment : HPLC, Make Waters, Alliance, 2695 Separation Module, (UV/PDA), or equivalent. Analytical Balance, Make Mettler Toledo, Model- XS205DU, or equivalent. Column : Inertsil C8-3, 150 x 4.6 mm, 5µm or equivalent Flow rate : 2.0 ml/minute Column temperature : 30 C Wavelength : 270 nm Sample temperature : 20 C Injection volume : 20 l Run time : 15 minutes Diluent : 0.1N Methanolic NaOH SPP SPTM, SVKM s NMIMS, Mumbai 91

Diluent: 0.1N Methanolic NaOH Weigh accurately and transfer 4 gm of Sodium hydroxide in 1000 ml volumetric flask and add 40 ml water, sonnicate to dissolve and make the volume with Methanol. Mobile Phase A (Buffer): Mix 2 ml of Triethylamine in 1000 ml water. Adjust ph 3.0 with ortho-phosphoric acid. Mobile Phase B: Acetonitrile Preparation of Mobile Phase use for dilution: Prepare a mixture of Buffer: Acetonitrile in the ratio 85:15 v/v/v. Mix and degas. Gradient Program: Time in minutes Mobile Phase (A) % Mobile Phase (B) % 0 85 15 4 85 5 5 65 35 11 65 35 12 85 15 15 85 15 Mobile Phase A : Mix 2 ml of Triethylamine in 1000 ml water. Adjust ph 3.0 with ortho-phosphoric acid. Mobile Phase B : Acetonitrile SPP SPTM, SVKM s NMIMS, Mumbai 92

5.3 FORCED DEGRADATION STUDIES The forced degradation studies were carried out to achieve adequate degradation of the individual drugs. They were carried out on the higher strength tablets (80mg_12.5mg) and chromatographed along with a non-stressed sample (control). The placebo was treated similarly. Since the degradation probabilities of the individual drugs are very different from each other, the degradation conditions were kept different for target drug. Preparation of Sample stock solution: 10 tablets were transferred into 500ml volumetric flask. 300ml of diluent was added and sonnicated for 20 minutes; allowed to cool at room temperature. The volume was made up to the mark with diluent. Preparation of Placebo stock solution: Accurately weighed placebo similar to sample and transferred into 500ml volumetric flask. 300ml of diluent was added and sonnicated for 20 minutes; allowed to cool at room temperature. The volume was made up to the mark with diluent. 5.3.1 Hydrolytic conditions: acid-, base-induced degradation. Acid Induced degradation Telmisartan: 5ml of sample stock solution was transferred into 50 ml volumetric flask. 5 ml of 5N Hydrochloric acid was added. This solution was heated on the water bath at 70 C for 24 hours. This was cooled; neutralized with same volume and same strength alkali. This was made up the volume with diluent and filtered through 0.45 Nylon filter. Hydrochlorothiazide: 5ml of sample stock solution was transferred into 50 ml volumetric flask. 5 ml of 1N Hydrochloric acid was added. This solution was heated on the water bath at 70 C for 30 minutes. This was cooled; neutralized with same volume and same strength alkali. This was made up the volume with diluent and filtered through 0.45 Nylon filter. Placebo preparation: Placebos were prepared accordingly on the same lines with samples. SPP SPTM, SVKM s NMIMS, Mumbai 93

Base Induced degradation Telmisartan: 5ml of sample stock solution was transferred into 50 ml volumetric flask. 5 ml of 5N Sodium Hydroxide was added. This solution was heated in the oven at 70 C for 24 hours. This was cooled; neutralized with same volume and same strength acid. Made up the volume with diluent and filtered through 0.45 Nylon filter. Hydrochlorothiazide: 5ml of sample stock solution was transferred into 50 ml volumetric flask. 5 ml of 1N Sodium hydroxide was added. This solution was heated on the water bath at 70 C for 2 hours. This was cooled; neutralized with same volume and same strength acid. Made up the volume with diluent and filtered through 0.45 Nylon filter. Placebo preparation: Placebos were prepared accordingly on the same lines with samples. 5.3.2 Oxidative condition: hydrogen peroxide-induced degradation. Telmisartan and Hydrochlorothiazide: 5ml of sample stock solution was transferred into 50 ml volumetric flask. 5ml of 50% Hydrogen peroxide was added. This solution was heated in the oven at 70 C for 24 hours. This was cooled; made up the volume with diluent and filtered through 0.45 Nylon filter. Placebo preparation: Placebo was prepared accordingly on the same lines with samples. 5.3.3 Thermal degradation study. Telmisartan: 5ml of sample stock solution was transferred into 50 ml volumetric flask and heated at 70 C for 24 hours on the water bath. This was cooled; diluted up to the mark with diluent and filtered through 0.45 Nylon filter. SPP SPTM, SVKM s NMIMS, Mumbai 94

Hydrochlorothiazide: 5ml of sample stock solution was transferred into 50 ml volumetric flask and heated at 70 C for 2 minutes in an oven. This was cooled; diluted up to the mark with diluent and filtered through 0.45 Nylon filter. Placebo preparation: Placebos were prepared accordingly on the same lines with samples. 5.3.4 Photolytic degradation study. As per guidelines for photostability testing of new drug substances and products, samples should be exposed to light providing an overall illumination of not less than 1.2 million lx hours and an integrated near ultraviolet energy of not less than 200Wh/m2 to allow direct comparisons to be made between the drug substance and drug product. [19] 5.0ml of sample stock solution was exposed under UV and white light for 1.2 million lux hours in each of 50 ml of clear glass flask volumetric flask, amber colour 50 ml glass volumetric flask and flask covered with aluminium foil. This solution was diluted to 50 ml with diluent and filtered through 0.45 Nylon filter. Placebo preparation: Placebo was prepared accordingly on the same lines with samples. 5.3.5 Observations in forced degradation studies. Under all conditions the peak due to the actives were found to be pure. Telmisartan Telmisartan degrades significantly in acidic, thermal and oxidative conditions. The degradation is acid has already been discussed in the chapter on method for related substances, is due to the formation of Methanolic ester due to the presence of methanol in the diluent. The degradation observed is much higher when compared to the related substances method because of difference in concentration of analyte in solution and difference in the diluents. Thus, it is quite possible that the degradation had reached SPP SPTM, SVKM s NMIMS, Mumbai 95

secondary or tertiary stage. In other words, the impurities had also degraded further. However, primary concern in the assay method development is the peak purity of the main peak and the peak purity for the Telmisartan peak is comfortably passing in all cases. Hydrochlorothiazide Due to the already known fact of the instability of Hydrochlorothiazide, the degradation conditions were much milder than what Telmisartan was exposed to. Hydrochlorothiazide degraded in all conditions. However under photolytic conditions, where the conditions are predetermined as per ICH guidelines, the degradation was found to be major. Additionally it has to be understood that the solution stability for hydrochlorothiazide is low and storing the solution for a few days to allow it to be exposed to the necessary illumination had also contributed to the degradation. The impurity generated is very near to the main peak at this high level. Thus it is important to protect the solution from light. 5.4 EXPERIMENTAL WORK 5.4.1 Instrumentation Equipment Make Model HPLC Waters 2695Alliance Separation Module, (PDA/UV Detector) 2996/2487 Column GL Sciences Inc. Inertsil C8-3 150 x 4.6 mm, 5µm ph meter Thermo Orion-4star 1117000 Electron Corp. Analytical Balance Mettler Toledo XS205DU SPP SPTM, SVKM s NMIMS, Mumbai 96

5.4.2 Chemicals and Reagents Name Grade Manufacturer Triethylamine HPLC grade Merck Methanol Gradient grade Merck Acetonitrile Gradient grade Rankem Sodium Hydroxide GR Merck Ortho-phosphoric acid GR Merck Hydrochloric acid GR Merck Hydrogen peroxide GR Merck Water HPLC milli-q In-house 5.4.3 Working Standard Working Standard: Standard Lot.No. Potency (as is) Telmisartan TE0010108 98.7 Hydrochlorothiazide HCT/60914 99.5 Test Sample: Batch. No. Label claim (mg/tablets) Telmisartan Hydrochlorothiazide THT/80-12.5/034 80 12.5 THT/40-12.5/032 40 12.5 Placebo: Batch. No. THT/80-12.5/034 P THT/80-12.5/034 PH THT/80-12.5/034 PT SPP SPTM, SVKM s NMIMS, Mumbai 97

5.4.4 Solution Preparation Preparation of Standard solution: Standard Stock solution-telmisartan Accurately weigh and transfer about 80.0 mg of Telmisartan standard and transfer into a 50 ml volumetric flask, add 30 ml of diluent, sonnicate to dissolve and make up the volume with diluent. Standard Stock solution-hydrochlorothiazide Accurately weigh and transfer about 50.0 mg of Hydrochlorothiazide standard and transfer into a 100 ml volumetric flask, add 70 ml of Acetonitrile, sonnicate to dissolve and make up the volume with Acetonitrile. Standard Solution: Take 5 ml of Standard Stock solution of Telmisartan and 5 ml of Standard Stock solution of Hydrochlorothiazide in 100 ml volumetric flask; make the volume with mobile phase. Preparation of Sample solution: For 80-12.5 mg Weigh accurately 10 tablets and transfer these tablets into a 500ml volumetric flask. Add 300ml of diluent and sonnicate it for 20 minutes; allow it to cool at room temperature. Make up the volume up to the mark with diluent. Dilute 5 ml of this solution to 50 ml with mobile phase. Filter the sample solution through 0.45 Nylon filter. For 40-12.5 mg Weigh accurately 10 tablets and transfer these tablets into 500ml volumetric flask. Add 300ml of diluent and sonnicate it for 20 minutes; allow it to cool at room temperature. Make up the volume up to the mark with diluent. Dilute 5 ml of this solution to 50 ml with mobile phase. Filter the sample solution through 0.45 Nylon filter. Preparation of Placebo solution: Weigh accurately placebo similar to the sample and transfer these into 500ml volumetric flask. Add 300ml of diluent and sonnicate it for 20 minutes; allow it to cool SPP SPTM, SVKM s NMIMS, Mumbai 98

at room temperature. Make up the volume up to the mark with diluent. Dilute 5 ml of this solution to 50 ml with mobile phase. Filter the sample solution through 0.45 Nylon filter. (Use placebo preparation for only Validation) Evaluation of System suitability: Inject the Standard solution five times; the relative standard deviation of five replicate injections should not be more than 2.0% for Telmisartan and Hydrochlorothiazide. The USP tailing factor for Telmisartan and Hydrochlorothiazide peak should not be more than 2.0. The USP plates for Hydrochlorothiazide and Telmisartan should not be less than 4000 and 10,000 respectively. Procedure: Inject equal volumes of Blank (diluent), Standard solution (5 replicate) and sample solution (duplicate). Calculation: Calculate the amount of Telmisartan and Hydrochlorothiazide present in the Tablets as per give For Telmisartan AT WS 5 500 50 P % Assay = -------- x --------- x ------ x ----------- x -------- x -------- AS 50 100 10 Tab 5 LC For Hydrochlorothiazide AT WS 5 500 50 P % Assay = -------- x --------- x ------ x ----------- x -------- x -------- AS 100 100 10 Tab 5 LC Where, AT = Area of peak due to Active Ingredient in sample preparation. AS = Area of peak due to Active Ingredient in standard preparation. WS = Weight of Active Ingredient standard in mg. LC = Label claim of Active Ingredient per tablet in mg. P = Potency of Active Ingredient standard on as is basis. SPP SPTM, SVKM s NMIMS, Mumbai 99

5.5 VALIDATION OF THE DEVELOPED METHOD 5.5.1 Validation parameters and acceptance criteria The validation summary is given in the below table 5.5.1.1 Table 5.5.1.1: The validation summary Sr.No Parameters Acceptance criteria Result obtained 1.0 System suitability Telmisartan HCTZ % RSD for Standard solution USP Tailing USP Plate count NMT 2.0% NMT 2.0 Hydrochlorothiazide -NLT 4000. 0.14 1.08 21458 0.14 1.05 6579 Telmisartan-NLT 10,000. 2.0 2.1 Specificity Identification Results should be comparable with respect to the retention Retention time (min) 11.178 3.348 time. 2.2 Interference No interference from blank and placebo to main component. Complies 2.3 Peak purity Purity angle should be less than purity threshold. Sample Purity angle Purity Threshold Standard peak should be pure for working concentration level. Standard of Telmisartan Sample of Telmisartan Standard of HCTZ Sample of HCTZ 0.041 1.023 0.037 1.013 0.083 1.054 0.085 1.059 SPP SPTM, SVKM s NMIMS, Mumbai 100

Table 5.5.1.1: The validation summary (continued) Sr.No. Parameters 2.4 Forced degradation 3.0 Linearity Acceptance criteria The peak due to Telmisartan and Hydrochlorothiazide should be pure as shown on the PDA. Response should be Linear Correlation coefficient should not be less than 0.999. % Limit of Y- Intercept should be within ± 2.0% of the corresponding Y-co-ordinate of the working level. Result obtained Telmisartan Condition % Purity Purity Assay angle Threshold Control 96.4 0.037 1.013 Acid 17.2 0.168 1.170 Base 95.7 0.057 1.025 Peroxide 68.7 0.317 1.051 Heat 16.0 0.218 1.255 Photolytic 96.7 0.172 1.109 Hydrochlorothiazide Control 100.3 0.085 1.059 Acid 84.0 0.191 1.209 Base 84.4 0.147 1.156 Peroxide 81.4 0.734 1.205 Heat 89.5 1.260 1.701 Photolytic 48.9 1.937 2.287 Response is linear Telmisartan 80 mg 40 mg HCTZ 1.0000 0.9999 0.9999 0.80% -0.03% 0.88% SPP SPTM, SVKM s NMIMS, Mumbai 101

Table 5.5.1.1: The validation summary (continued) Sr. No Parameters Acceptance criteria Result obtained 4.0 Accuracy (Recovery) 5.0 5.1 Precision System Precision % RSD for Standard sol USP Tailing USP Plate count 5.2 Method Precision % RSD of six determinations Intermediate Precision (Ruggedness) % RSD for Standard sol Mean recovery should be in the range of 98.0%- 102.0%. NMT 2.0% Level % NMT 2.0 Hydrochlorothiazide -NLT 4000. Telmisartan-NLT 10,000. % Mean Recovery 80mg Telmisartan 40mg 50 99.0 99.9 100 98.5 99.0 150 98.8 99.0 HCTZ 50 99.9 100 99.9 150 99.6 Telmisartan 0.14 1.08 21458 HCTZ 0.14 1.05 6579 NMT 2.0%. For 80 mg Tablets 0.06 0.08 NMT 2.0%. For 40 mg Tablets 0.24 0.28 NMT 2.0% 0.22 0.18 USP Tailing NMT 2.0 0.96 0.98 USP Plate count Hydrochlorothiazide NLT 4000. Telmisartan-NLT 10,000. 20376 6196 RSD for % Assay NMT 2.0%. For 80 mg Tablets 0.11 0.18 NMT 2.0%. For 40 mg Tablets 0.46 0.25 Difference in pooled result ( Analyst-I and II ) The difference in the means should not be more than 2. 80 mg 0.8 0.7 40 mg 0.1 0.1 SPP SPTM, SVKM s NMIMS, Mumbai 102

Table 5.5.1.1: The validation summary (continued) Sr. No. Parameters Acceptance criteria Result obtained 6.0 Stability in analytical solution Filter 7.0 compatibility Robustness Change in Flow rate (± 0.2 ml/min) The % difference should not be more than 2. The % difference between centrifuged sample and filtered sample should not be more than 2. No significant change should be in System suitability parameters. % RSD should be less than 2%. Telmisartan HCTZ Sample is Sample is stable upto stable upto 24 15 hours, hours, stored stored at at 20 C. 20 C. Complies Complies No significant change. Compiles 8.0 Change in wavelength (± 5nm) No significant change should be in System suitability parameters. % RSD should be less than 2%. No significant change. Compiles Change in Buffer ph (± 0.2) Column oven temperature (± 5 C) No significant change should be in System suitability parameters. % RSD should be less than 2%. No significant change should be in System suitability parameters. % RSD should be less than 2%. No significant change. Compiles No significant change. Compiles SPP SPTM, SVKM s NMIMS, Mumbai 103

5.5.2 System suitability: Single injection of Blank (Diluent) and five replicate Standard solution were made on the system. The data obtained is summarized in Table. 5.5.2.1. The data demonstrate that the system suitability is within the acceptance criteria, thus the system is suitable. Table 5.5.2.1: System suitability Telmisartan Hydrochlorothiazide USP Tailing 1.08 1.05 USP Plates 21458 6579 Standard solution 1345962 938752 Area 1347933 938901 1346519 938816 1345207 937318 1342947 935798 Mean 1345714 937917 SD 1840.30 1353.02 %RSD 0.14 0.14 5.5.3 Specificity: The Specificity study included Identification of the main peak, Interference study and Peak Purity. Peak purity of degraded peak was also studied. Injections of standard solution, sample solution and placebo, as a directed in experimental section, were made onto the HPLC. The data demonstrate that there is no interference in blank and placebo peaks with the peaks due to Telmisartan and Hydrochlorothiazide. Purity angle is less than purity threshold for the main peaks. The data obtained is summarized in Table 5.5.3.1. Table 5.5.3.1: Specificity (Identification and Interference) Component Retention time (min) USP Plates USP Tailing Purity angle Purity threshold Standard solution Hydrochlorothiazide 3.348 6579 1.05 0.083 1.054 Telmisartan 11.178 21458 1.08 0.041 1.023 Sample solution Hydrochlorothiazide 3.358 6502 1.04 0.085 1.059 Telmisartan 11.101 21042 1.12 0.037 1.013 SPP SPTM, SVKM s NMIMS, Mumbai 104

Chromatograms of Blank (diluent), placebos, Standard solution and Sample solution are given below under n 5.5.3.1 to 5.5.3.4. Figure-5.5.3.1: Chromatogram of Blank. Figure-5.5.3.2: Chromatogram of Placebo without Telmisartan and Hydrochlorothiazide. SPP SPTM, SVKM s NMIMS, Mumbai 105

Figure-5.5.3.3: Chromatogram of Placebo with Telmisartan. Figure-5.5.3.4: Chromatogram of Placebo with Hydrochlorothiazide. SPP SPTM, SVKM s NMIMS, Mumbai 106

Figure-5.5.3.5: Chromatogram of Standard solution. Figure-5.5.3.6: Chromatogram of Sample solution. SPP SPTM, SVKM s NMIMS, Mumbai 107

Forced degradation: Under all degradation conditions, the peaks due to Telmisartan and Hydrochlorothiazide were found to be pure. 5.5.4 Linearity and Range: The Linearity of response was determined by preparing different concentrations of standard stock solution ranging from 50% to 150% of the working concentration. The data shows that the response is found to be linear; Correlation coefficient is more than 0.999. The data summarized in Table 5.5.4.1, 5.5.4.2 and 5.5.4.3. Table 5.5.4.1: Linearity of Telmisartan (80 mg). Level Concentration (%) Response 1 2 Mean 1 50.4 688313 687294 687804 2 63.0 858240 860365 859303 3 75.6 1032153 1032624 1032389 4 100.8 1351745 1360982 1356364 5 125.9 1708469 1707840 1708155 6 151.1 2034128 2033164 2033646 7 201.5 2725924 2722907 2724416 8 251.9 3402941 3401976 3402459 9 302.3 4059210 4058213 4058712 CORRELATION COEFFICIENT (r) 1.0000 SLOPE 13414 Y-INTERCEPT 13695 MEDIAN (AREA) 1708155 % LIMIT OF Y-INTERCEPT ± 2% OF MEDIAN 0.80 SPP SPTM, SVKM s NMIMS, Mumbai 108

Table 5.5.4.2: Linearity of Telmisartan (40 mg). Level Concentration (%) Response 1 2 Mean 1 25.2 322529 321286 321908 2 37.8 513367 514625 513996 3 50.4 688313 687294 687804 4 63.0 858240 860365 859303 5 75.6 1032153 1032624 1032389 6 100.8 1351745 1360982 1356364 7 125.9 1708469 1707840 1708155 8 151.1 2034128 2033164 2033646 CORRELATION COEFFICIENT (r) 0.9999 SLOPE 13522 Y-INTERCEPT -319 MEDIAN (AREA) 945846 % LIMIT OF Y-INTERCEPT ± 2% OF MEDIAN -0.03 Table 5.5.4.3: Linearity of Hydrochlorothiazide. Level Concentration (%) Response 1 2 Mean 1 50.0 473953 474514 474234 2 60.0 560479 561517 560998 3 79.9 751143 751493 751318 4 99.9 944510 944683 944597 5 119.9 1114941 1113915 1114428 6 139.9 1302274 1306179 1304227 7 159.9 1496577 1496190 1496384 CORRELATION COEFFICIENT (r) 0.9999 SLOPE 9286 Y-INTERCEPT 8271 MEDIAN (AREA) 944597 % LIMIT OF Y-INTERCEPT ± 2% OF MEDIAN 0.88 SPP SPTM, SVKM s NMIMS, Mumbai 109

Graph of concentration v/s area is attached as Figure-8.5.4.1, 8.5.4.2 and 8.5.4.3 Figure-8.5.4.1: Linearity for Telmisartan (80mg). Figure-8.5.4.2: Linearity for Telmisartan (40mg). SPP SPTM, SVKM s NMIMS, Mumbai 110

Figure-8.5.4.3: Linearity for Hydrochlorothiazide.. 5.5.5. Accuracy: The standard solution was spiked into the placebo at three different level, 50%, 100% and 150% from three different standard stock solutions and each level in duplicate were injected. From the amount added and the amount found, the percentage recovery was calculated. The results obtained are summarized in Table 5.5.5.1 to 5.5.5.3. The data shows that the % mean recovery at each level is within the acceptance criteria. Table 5.5.5.1: % Recovery for Telmisartan (80 mg) Level Mean % Mean recovery % response Recovery % 50 % 100 % 150 % 1350883 99.1 1349061 98.8 1352475 99.2 2677419 98.6 2678726 98.6 2674136 98.4 4017847 98.5 4033554 98.8 4041361 99.0 99.0 98.5 98.8 SPP SPTM, SVKM s NMIMS, Mumbai 111

Table 5.5.5.2: % Recovery for Telmisartan (40 mg) Level Mean % Mean recovery % response Recovery % 50 % 100 % 150 % 679906 100.0 680845 100.0 678742 99.6 1350883 99.1 1349061 98.8 1352475 99.2 2022307 99.1 2018274 98.8 2022569 99.2 99.9 99.0 99.0 Table 5.5.5.3: % Recovery for Hydrochlorothiazide. Level % Mean response % Recovery Mean recovery % 50 % 100 % 150 % 466666 100.0 467123 99.9 468201 99.8 922319 100.9 922231 99.8 920009 98.9 1385794 99.9 1390091 99.5 1393122 99.3 99.9 99.9 99.6 SPP SPTM, SVKM s NMIMS, Mumbai 112

5.5.6 Precision 5.5.6.1 System Precision: Single injection of Blank (Diluent) and five replicate injections of standard solution were made on the system. Please refer to Table 5.5.2.1 for system suitability of 80-12.5mg strength. Table 5.5.6.1.1 depicts the system suitability for 40-12.5 strength. All the data were acceptable as per the system suitability requirements. Table 5.5.6.1.1: System precision (40/12.5 mg Tablets) Telmisartan Hydrochlorothiazide USP Tailing 0.91 0.93 USP Plates 21336 6069 Standard solution 1351006 935866 Area 1328113 935101 1324715 937800 1323299 937637 1326007 939873 Mean 1330628 937255 SD 11528.44 1861.85 %RSD 0.87 0.20 5.5.6.2 Method Precision: Six sample solutions were prepared and injected in duplicate on the HPLC. The data obtained is summarized in Table 5.5.6.2.1 and 5.5.6.2.2. The data shows that % RSD is well within the acceptance criteria. SPP SPTM, SVKM s NMIMS, Mumbai 113

Table 14: Method precision (80/12.5 mg Tablets) Spl. No. Telmisartan Hydrochlorothiazide Response % Response % 1 2 Mean Assay 1 2 Mean Assay 1 2634290 2635581 2634936 96.4 946679 946628 946654 100.3 2 2634233 2635581 2634907 96.4 946116 948048 947082 100.4 3 2637308 2639255 2638282 96.5 948487 947625 948056 100.5 4 2638093 2635106 2636600 96.4 948390 947272 947831 100.5 5 2638719 2637763 2638241 96.5 947852 947504 947678 100.5 6 2638247 2639925 2639086 96.5 946799 948724 947762 100.5 Mean 96.5 100.5 SD 0.055 0.084 % RSD 0.06 0.08 Table 15: Method precision (40/12.5 mg Tablets) Spl. No. Telmisartan Hydrochlorothiazide Response % Response % 1 2 Mean Assay 1 2 Mean Assay 1 1344101 1333004 1333553 98.4 963149 963583 963366 102.1 2 1340650 1339613 1340132 98.9 971547 969227 970387 102.8 3 1331803 1332686 1332245 98.3 971810 963660 967735 102.5 4 1333804 1338005 1335905 98.6 968194 969696 968945 102.7 5 1332223 1333269 1332746 98.3 971515 970253 970884 102.9 6 1335520 1336135 1335828 98.6 968719 968259 968489 102.6 Mean 98.5 102.6 SD 0.232 0.283 % RSD 0.24 0.28 SPP SPTM, SVKM s NMIMS, Mumbai 114

5.5.6.3 Intermediate Precision (Ruggedness): Same procedure of system precision and method precision is followed by another Analyst on different instrument and on different day. The system suitability data obtained from Analyst-II are summarized in Table 5.5.6.3.1 and 5.5.6.3.2. The data demonstrate that the system complies. Thus the system was suitable and precise and % RSD is within the acceptance criteria. Table 5.5.6.3.1: System suitability (80/12.5 mg Tablets) Telmisartan Hydrochlorothiazide USP Tailing 0.96 0.98 USP Plates 20376 6196 Standard solution 1365710 961935 Area 1369717 958120 1369612 960145 1371561 962592 1373917 961235 Mean 1370103 960805 SD 3015.00 1753.57 %RSD 0.22 0.18 Table 5.5.6.3.2: System suitability (40/12.5 mg Tablets) Telmisartan Hydrochlorothiazide USP Tailing 1.11 1.09 USP Plates 20731 5867 Standard solution 1384468 979092 Area 1379496 971653 1380815 971487 1382211 972890 1388628 977139 Mean 1383124 974452 SD 3585.66 3457.42 %RSD 0.26 0.35 SPP SPTM, SVKM s NMIMS, Mumbai 115

The Data obtained by second analyst is given in table 5.5.6.3.3 and 5.5.6.3.4. The values are within acceptable limits. Table 5.5.6.3.3: Intermediate precision (80/12.5 mg Tablets) Spl. No. Telmisartan Hydrochlorothiazide Response % Response % 1 2 Mean Assay 1 2 Mean Assay 1 2709334 2702556 2705945 97.1 955967 952894 954431 99.7 2 2698924 2713819 2706372 97.2 950900 956218 953559 99.6 3 2711638 2703203 2707421 97.2 955949 952712 954331 99.7 4 2709442 2712576 2711009 97.3 954003 955478 954741 99.7 5 2708326 2715747 2712037 97.4 958315 958798 958557 100.1 6 2707315 2711003 2709159 97.3 955082 956413 955748 99.8 Mean 97.3 99.8 SD 0.105 0.175 % RSD 0.11 0.18 Table 5.5.6.3.4: Intermediate precision (40/12.5 mg Tablets) Spl. No. Telmisartan Hydrochlorothiazide Response % Response % 1 2 Mean Assay 1 2 Mean Assay 1 1386112 1388875 1387494 98.9 994157 994184 994171 102.8 2 1385105 1385003 1385054 98.7 994962 994282 994622 102.9 3 1381326 1381670 1381498 98.4 992945 992747 992846 102.7 4 1380493 1378797 1379645 98.3 994884 994554 994719 102.9 5 1378427 1377264 1377846 98.2 993722 992104 992913 102.7 6 1369241 1368657 1368949 97.6 988403 988144 988274 102.2 Mean 98.4 102.7 SD 0.451 0.261 % RSD 0.46 0.25 SPP SPTM, SVKM s NMIMS, Mumbai 116

The pooled data of twelve independent sample preparation obtained from Analyst-I and Analyst- II is summarized in Table 5.5.6.3.5 and 5.5.6.3.6. The results demonstrated that the values are within acceptable limits. Table 5.5.6.3.5: Pooled data (80/12.5 mg Tablets) Analyst Telmisartan % Assay Hydrochlorothiazide 96.4 100.3 96.4 100.4 I 96.5 100.5 96.4 100.5 96.5 100.5 96.5 100.5 Mean 96.5 100.5 97.1 99.7 97.2 99.6 II 97.2 99.7 97.3 99.7 97.4 100.1 97.3 99.8 Mean 97.3 99.8 % Difference between two means 0.80 0.70 SPP SPTM, SVKM s NMIMS, Mumbai 117

Table 5.5.6.3.6: Pooled data (40/12.5 mg Tablets) Analyst Telmisartan % Assay Hydrochlorothiazide 98.4 102.1 98.9 102.8 I 98.3 102.5 98.6 102.7 98.3 102.9 98.6 102.6 Mean 98.5 102.6 98.9 102.8 98.7 102.9 II 98.4 102.7 98.3 102.9 98.2 102.7 97.6 102.2 Mean 98.4 102.7 % Difference between two means 0.10 0.10 5.5.7 Stability in Analytical solution: The standard solution and sample solution were kept at sample temperature for 24 hours and were injected on to the HPLC time to time. The data obtained are summarized in Table 5.5.7.1 and 5.5.7.2. The data demonstrates that while Telmisartan sample solution was stable for at least 15 hours at the sample temperature, hydrochlorothiazide was stable for 24 hours. It was noted that the peak areas slowly increased over time. This suggested that there was slow evaporation of the solvent present in the diluent. SPP SPTM, SVKM s NMIMS, Mumbai 118

Table 5.5.7.1 Stability in analytical solution (Telmisartan) Time Mean area of standard Cumulative RSD for % Assay % Difference solution standard area Initial (control) 1339560-96.4-5 hrs 1345200 0.30 96.5 0.1 10 hrs 1348948 0.49 96.9 0.5 15 hrs 1353321 0.72 98.4 2.0 20hrs 1361186 1.13 98.9 2.5 24hrs 1369647 1.57 100.1 3.7 Table 5.5.7.2 Stability in analytical solution (Hydrochlorothiazide) Time Mean area of standard Cumulative RSD for % Assay % Difference solution standard area Initial (control) 935484-100.3-5 hrs 939675 0.32 100.5 0.2 10 hrs 940673 0.39 100.7 0.4 15 hrs 937563 0.16 101.4 1.1 20hrs 945933 0.79 101.3 1.0 24hrs 945323 0.74 101.9 1.6 5.5.8 Filter Compatibility: The Sample solution centrifuged and filtered through different filters was injected on to the HPLC. The data obtained are summarized in Table 5.5.8.1. The data shows that % difference is not more than 2. Thus all filters tested were compatible with the sample. SPP SPTM, SVKM s NMIMS, Mumbai 119

Table 5.5.8.1 Filter compatibility Filter Telmisartan Hydrochlorothiazide % Assay % Difference % Assay % Difference Centrifuged 96.5-100.5 - Glass Filter 96.5 0.0 100.3 0.2 Nylon Filter 96.6 0.1 100.5 0.0 Nylon + Glass Filter 96.3 0.2 100.2 0.3 PVDF 96.5 0.0 100.2 0.3 Teflon 97.3 0.8 100.6 0.1 Teflon + Glass Filter 97.2 0.7 100.4 0.1 5.5.9 Robustness: The changes in system suitability parameters and results, when deliberate controlled changes were made to the method, were studied in robustness. No significant changes in system suitability parameters or results were observed during robustness study proving the method to be considerable robust. The data obtained are summarized in Table 5.5.9.1 and 5.5.9.2. Table 5.5.9.1: Robustness (Telmisartan). Changes in parameters Values Retention time of Telmisartan USP Plates USP Tailing % RSD of standard area % Assay Cumulative % RSD Control As per method 11.178 21458 1.08 0.14 96.5 - Flow rate (ml/min) Wavelength (nm) Column temperature ( C) Buffer ph 1.8 11.673 21272 1.07 0.10 97.9 1.02 2.2 10.333 22146 1.06 0.28 97.7 0.87 265 11.179 21478 1.08 0.18 96.5 0.00 275 11.179 21580 1.08 0.20 96.6 0.07 25 10.774 20602 1.09 0.15 98.4 1.38 35 11.085 22448 1.05 0.30 98.1 1.16 2.8 9.902 23544 0.96 0.25 98.0 1.09 3.2 12.091 17954 0.91 0.30 97.8 0.95 SPP SPTM, SVKM s NMIMS, Mumbai 120

Table 5.5.9.2: Robustness (Hydrochlorothiazide). Changes in parameters Values Retention time of HCTZ USP Plates USP Tailing % RSD of standard area % Assay Cumulative % RSD Control As per method 3.348 6579 1.05 0.14 100.5 - Flow rate (ml/min) Wavelength (nm) Column temperature ( C) Buffer ph 1.8 3.710 6818 1.05 0.16 100.0 0.35 2.2 3.034 6311 1.03 0.12 100.4 0.07 265 3.348 6582 1.05 0.16 100.4 0.07 275 3.348 6567 1.05 0.18 100.2 0.21 25 3.639 5886 1.06 0.17 102.6 1.46 35 3.080 6665 1.03 0.09 100.6 0.07 2.8 3.477 6194 0.90 0.34 100.8 0.21 3.2 3.435 6275 0.92 0.13 100.1 0.28 5.5.10 Conclusions: The method has been shown to be specific for Telmisartan and Hydrochlorothiazide Tablets. The method has been shown to be Linear, precise and accurate across the suitable analytical range and stability indicating. Solution has been shown to be stable for at least 24 hours when stored at 20 C. The method has been shown to be robust towards deliberate minor changes in the method parameters. The method can be used in quality control laboratory for release of production batches. SPP SPTM, SVKM s NMIMS, Mumbai 121