1. DRUG PROFILE. management of hypertension. It may also be used in patients with heart failure who are

Transcription

1 1. DRUG PROFILE 1.1 Valsartan Valsartan [Figure 3.1] is an angiotensin-ii receptor antagonist used in management of hypertension. It may also be used in patients with heart failure who are unable to tolerate ACE inhibitors [1]. Valsartan lowers blood pressure by antagonizing the Renin-Angiotensin-Aldosterone System (RAAS); it competes with angiotensin II for binding to the type-1 angiotensin II receptor (AT1) subtype and prevents the blood pressure increasing effects of angiotensin II [2]. Valsartan may be used to treat hypertension, isolated systolic hypertension, left ventricular hypertrophy and diabetic nephropathy. It may also be used as an alternative agent for the treatment of heart failure, systolic dysfunction, myocardial infarction and coronary artery disease [3-4]. Molecular formula : C 24 H 29 N 5 O 3 Molecular weight : Figure 3.1: Molecular structure of Valsartan Chemical name : N-(1-oxopentyl)-N-[[2'-(1H-tetrazol-5-yl) [1,1'-biphenyl]-4- yl]methyl]-l-valine. Solubility : Valsartan is freely soluble in ethanol, methanol and slightly soluble in water. 83

2 Table 3.1: List of brand names of formulations of Valsartan [5-6] S. No. Brand names Formulation Available strength 1 STARVAL Capsule 80 mg 160 mg Address of Manufacturer Ranbaxy Pharmaceuticals 2 VALENT Tablet 40 mg Capsule 80 mg 160 mg 40 mg 80 mg 160 mg Lupin Pharmaceuticals 2. LITERATURE SURVEY Several analytical methods have been reported for the determination of Valsartan in pure drug, pharmaceutical dosage forms and in biological samples using spectrophotometry [7-10], HPLC [11-17], HPTLC [18-19] and LC-MS [20] have been reported for the determination of Valsartan in pharmaceutical dosage forms. Nataraj et al (7) developed a simple, precise and accurate quantitative method development and validation of Valsartan in pure form and pharmaceutical dosage forms by UV spectroscopy. This method which is based on measurement of absorption of UV light, the spectra of Valsartan in methanol showed maximum wavelength at 250 nm and calibration graphs were plotted over the concentrations ranging from 2-20 µg/ml of Valsartan with correlation coefficient Validation was performed as per ICH 84

3 Q2(R1) guidelines for linearity, accuracy, precision and recovery. The limit of detection (LOD) and limit of quantification (LOQ) were found to be 0.15 µg/ml and µg/ml respectively by simple UV spectroscopy. The proposed method was validated. Kailash et al (8) developed and validated UV spectrophotometric methods for estimation of Valsartan in bulk and tablet dosage form. Valsartan is an angiotensin-ii receptor blocker drug used in hypertension and other cardiac diseases. Here they have developed two new, precise and simple UV spectrophotometric methods for estimation of Valsartan from bulk and tablet formulation in phosphate buffer ph 6.8. The drug obeyed the Beer s law with correlation coefficient and respectively for Method I and Method II. It showed absorption maxima at 250 nm and 220 nm respectively for Method I and Method II in phosphate buffer ph 6.8. The linearity was observed between 5-30 µg/ml. The results of analysis were validated by recovery studies, accuracy, precision, LOD, LOQ and ruggedness. The methods were found to be simple, accurate, precise, economical and robust. Gupta et al (9) developed two UV spectrophotometric methods for estimation of Valsartan in bulk and tablet dosage form. The zero order spectra of Valsartan in methanol shows λmax at 250 nm and estimation was carried out by 1% A 1cm and by comparison with standard (Method I). The second order spectra showed λmax at 241 nm where n=2 and estimation were carried out by comparison with standard (Method II). Calibration graphs were found to be linear (r 2 =0.999) over the concentration range of µg/ml. The proposed methods were validated for its accuracy, precision, specificity, ruggedness and robustness. Both the methods can be adopted in routine analysis of Valsartan in bulk and tablet dosage form. 85

4 Madhusudan et al [10] developed two simple, accurate and economical UV spectrophotometric methods have been developed for the estimation of Valsartan and Hydrochlorothiazide in bulk & pharmaceutical formulation. Literature survey revealed that only two methods reported for this combination. Hence rationale behind this work is to develop a novel method for estimation of Valsartan & Hydrochlorothiazide in Bulk and Pharmaceutical formulation. In Absorbance maxima method, the absorbance maxima for Valsartan and Hydrochlorothiazide in 0.1M NaoH was found to be 249 nm and 273 nm respectively. In First order derivative method, the absorbance was measured at maxima at 266 nm and minima at 217 nm for Valsartan and maxima at 283 nm and minima at 262 nm for Hydrochlorothiazide respectively, amplitude difference was measured for the respective concentration of standard and was plotted against concentration and regression equation was calculated. Linearity for detector response was observed in the concentration range of 2-24 µg/ml & 2-14 µg/ml for Valsartan and Hydrochlorothiazide respectively. The correlation coefficient was found to be & for Valsartan & Hydrochlorothiazide respectively. The results of analysis have been validated statistically and by recovery studies The value of standard deviation was satisfactory and recovery studies ranging from % for Valsartan and % for Hydrochlorothiazide were indicative of the accuracy and precision of the proposed method. The proposed methods were successfully applied for the determination of Valsartan and Hydrochlorothiazide in bulk & commercial pharmaceutical preparation. All two methods were validated statistically as per ICH guidelines. Piao et al (11) developed an improved analytical validation and pharmacokinetics of Valsartan using HPLC with UV detection. The primary objective of the study was to 86

5 validate a simple and sensitive method of determining Valsartan concentration in human plasma samples using high performance liquid chromatography (HPLC) combined with ultraviolet (UV) detection. Methanol appeared to be the best with a high recovery efficiency compared to other solvents such as acetonitrile, ethyl acetate and methyl-tertbutyl ether. After a simple protein precipitation using methanol, the analytes were separated on a Phenomenex Luna C18 column using 42% acetonitrile with 15 mm potassium dihydrogen phosphate in water (ph 2.0; adjusted with phosphoric acid) as the mobile phase at a flow rate of 1.2 ml/min. The standard calibration curve constructed in the concentration range of ng/ml showed good linearity (r 2 >0.9997). Spironolactone was used as an internal standard (IS). Valsartan and IS eluted at and min, respectively. The intra-day and inter-day precision and accuracy were satisfactory with relative standard deviations of less than 15%. No interference peaks or matrix effects were observed in human plasma. Valsartan concentration in human plasma was well established following a single 80 mg oral dose (Diovan capsule) to eight healthy volunteers. The current determination of Valsartan concentration by protein precipitation using methanol followed by analysis using HPLC with UV detection was rapid and sensitive, and provide an alternative to the analysis of Valsartan by studying its clinical applications. Thanusha et al (12) developed a validated RP-HPLC method for the quantitative estimation of Valsartan. It is simple, specific, accurate, precise and sensitive reverse phase High Performance Liquid Chromatographic method for the quantitation of Valsartan in both pure and pharmaceutical dosage forms. A Venusil XBP C18, 5 µm column having mm internal diameter in isocratic mode with mobile phase 87

6 containing 0.1M phosphate buffer: acetonitrile (20:80 v/v). The flow rate was 1.0 ml/ min and the effluents were monitored at 273 nm. The retention time was 4.95 min. The linearity was in the range of µg/ml. This method was validated for linearity, precision, limit of detection, limit of quantitation, accuracy, ruggedness and robustness. Statistical analysis proves that the method is reproducible and selective for the estimation of the drug. Rao et al (13) developed a stability-indicating HPLC assay method and validated for the determination of Valsartan in bulk drug and pharmaceutical dosage forms. An isocratic RP-HPLC was achieved on Waters 2695 using Symmetry C x 4.6 mm 5 µm column with the mobile phase consisting of 0.02 mm sodium dihydrogen orthophosphate, ph adjusted to 2.5 using orthophosphoric acid (solvent A), and acetonitrile (solvent B) in the ratio of 58:42 % v/v. The stress testing of Valsartan was carried out under acidic, alkaline, oxidative, thermal, and photolytic conditions. Valsartan was well resolved from its degradation products. The proposed method was validated as per ICH guidelines. The method was found to be suitable for the quality control of Valsartan in bulk and pharmaceutical dosage forms as well as the stability-indicating studies. Narendra Reddy et al (14) developed an isocratic RP-HPLC method for determination of Valsartan in tablet dosage form. The method was carried out using Thermohypersil ODS column mm i.d., 5 µm particle size with mobile phase comprised of water: acetonitrile: glacial acetic acid (500:500:01 v/v/v). The flow rate was set at 1.0 ml/min and effluent was detected at 273 nm. The retention time of Valsartan was found to be 4.6 minute. The method was validated for specificity, accuracy, precision, linearity, and limit of detection, limit of quantification, robustness, solubility 88

7 and stability. LOD and LOQ were found to be 2.72 µg/ml and 8.25 µg/ml respectively. The calibration curve was linear in the concentration range of µg/ml with coefficient of correlation The percentage recovery for the Valsartan was found to be and the %RSD was found to be less than 2%. The proposed method was successfully applied for quantitative determination of Valsartan in tablet dosage form. Patnaik et al (15) developed an HPLC method for estimation of Valsartan in tablet dosage form. Quantitative HPLC was performed with Shimadzu LC2010c HT with class- 10vp software with UV-Visible detector (SPD-IOA), PDA detector (PDA-10A) Pump (LC-IOAT) and (LCIOATvp). Microbondapak, C18 ( mm i.d., 5 µm particle size) column was used in the study. The mobile phase of Methanol: phosphate buffer of ph 3 (65:35 v/v) was used in this study. The conditions optimized were flow rate (1 ml/min), wavelength (210 nm) and run time was 20 min. Retention time was found to be 6.22 min. The linearity was found to be in the concentration range of µg/ml. The developed method was evaluated in the assay of commercially available tablets, Valzar and Valtan tablet containing 40 and 80 mg of Valsartan respectively. The amount of drug in tablet was found to be and mg/tab for the two brands. Results of analysis were validated statistically and by recovery studies. The recovery of 99.35% was indicative for the accuracy of proposed method. The precision was calculated as repeatability, inter and intraday variation for the drug. By using the method stability of drug is studied. Santosh et al (16) developed a sensitive liquid chromatographic method for estimation of Valsartan in pure and tablet formulation. The chromatographic method was standardised using Kromasil C18 column (250 x 4.6 mm i.d., 5 µm particle size) with UV 89

8 detection at 233 nm and flow rate of 1 ml/min. Mobile phase comprised of mixture of phosphate buffer and acetonitrile in 55:45 v/v is selected. The proposed method was validated for sensitivity, accuracy, precision and linearity. The retention time of Valsartan is min. The percentage recovery was within the range of %. The %RSD for precision and accuracy was found to be less than 2%. This method can be employed for routine quality control analysis of Valsartan in tablet dosage form. Parambi et al (17) developed a validated stability indicating HPLC method for the determination of Valsartan in tablet dosage forms. It is a simple, specific, rapid, precise and robust HPLC method for the quantitation of Valsartan in tablet dosage form on a C18 column (250 x 4.6 mm) using a mobile phase consisting of ammonium dihydrogen phosphate buffer: methanol (33.5:66.5 v/v) adjusted to ph 3 with formic acid at a flow rate of 1.0 ml/min and detection at 265 nm. The retention time of Valsartan was found to be at 11.9 min. The validation of above method was also done. Percentage label claim of the tablet formulations were found to be 100.8%. So the proposed method provides a faster and cost effective quality control tool for routine analysis of Valsartan from formulations. Shah et al [18] developed a simple, precise, accurate and rapid high performance thin layer chromatographic method has been developed and validated for the simultaneous estimation of Valsartan and Hydrochlorothiazide in combined dosage forms. The stationary phase used was precoated silica gel 60F254. The mobile phase used was a mixture of chloroform: methanol: toluene: glacial acetic acid (6:2:1:0.1 v/v/v/v). The detection of spots were carried out at 260 nm. The method was validated in terms of linearity, accuracy, precision and specificity. The calibration curve was found to be linear 90

9 between 300 to 800 ng/spot for Valsartan and 100 to 600 ng/spot for Hydrochlorothiazide. The limit of detection and the limit of quantification for the Valsartan were found to be 100 and 300 ng/spot respectively and for Hydrochlorothiazide 30 and 100 ng/spot respectively. The proposed method can be successfully used to determine the drug content of marketed formulation. Sunil et al [19] developed simple, selective, rapid, precise and stability indicating HPTLC method has been developed for the quantitative simultaneous estimation of Valsartan and Hydrochlorothiazide in combined pharmaceutical dosage form and validation was done. The proposed HPTLC method involves the use of HPTLC plates (Merck) pre coated with silica gel 60F254 on aluminium sheets and a mobile phase comprising of chloroform: ethyl acetate: acetic acid (5:5:0.2 v/v/v). Densiometric analysis of both the drugs was carried out in the absorbance mode at 248 nm. The method has been successfully applied for estimation of Valsartan and Hydrochlorothiazide in combined tablets dosage form. Both the drugs were subjected to acid alkali hydrolysis, oxidation and photolytic degradation and both of them were found to be susceptible to acid alkali hydrolysis, oxidation and photolytic degradation. Linearity of Valsartan was found to be within the range of ng/spot (r 2 =0.9996) and for Hydrochlorothiazide the range was found to be ng/spot (r 2 =0.9975), with significant high values of correlation coefficient for both the drugs. The limit of detection and limit of quantitation were found to be ng/spot and ng/spot for Valsartan and ng/spot and ng/spot for Hydrochlorothiazide. The percentage recovery of Valsartan and Hydrochlorothiazide was ranged from to 99.99% and to 99.98% respectively. The %R.S.D. values for intraday precision study were 91

10 <1.0% and for inter day study were <2.0%, confirming that the method was sufficiently precise. The validation studies were carried out fulfilling International Conference on Harmonisation (ICH) requirements. Hiten et al [20] developed a rapid and specific liquid chromatographic tandem mass spectrometric method is described for simultaneous analysis of Valsartan (VAL) and hydrochlorothiazide (HCTZ) in human plasma. VAL and HCTZ were chromatographed on a C8 column with 75:15:10 (v/v/v) acetonitrile, methanol 0.001% and aqueous ammonia as mobile phase. VAL and HCTZ were eluted at 0.69 min and 1.22 min, respectively, and, after electrospray ionization (ESI), detected in selectedreaction-monitoring mode. The precursor to product-ion transitions m/z and m/z were used to quantify VAL and HCTZ, respectively. Recovery by solid-phase extraction was >90% for both analytes and the internal standard. The method was suitable for application to a pharmacokinetic study after oral administration of tablet containing 160 mg VAL and 25 mg HCTZ to 18 healthy volunteers. 3. EXPERIMENTAL 3.1. Instrumentation The author had attempted to develop and validate a liquid chromatographic method for determination of Valsartan using an isocratic Waters HPLC system on an Xterra C18 column (100 mm x 4.6 mm, 5 µm). The instrument is equipped with a 2695 binary pump with inbuilt degasser, 2487 Dual absorbance detector and Rheodyne injector with 20 µl sample loop. A 20 µl Hamilton syringe was used for injecting the samples. Data was analysed using Waters Empower 2 software. A double-beam Elico SL-159 UV- Visible spectrophotometer was used for spectral studies. Degassing of the mobile phase 92

11 was done by using an ultrasonic bath sonicator. A Shimadzu balance was used for weighing the materials Chemicals and Solvents The reference sample of Valsartan (API) was obtained from Lupin Pharmaceutical Industries Ltd, Ahmadabad, India. The branded formulations (tablets) (VALENT and STARVAL tablets containing 80 mg of Valsartan) were procured from the local market. HPLC grade acetonitrile and analytical grade potassium dihydrogen phosphate was obtained from Qualigens Fine Chemicals Ltd, Mumbai, India. Hydrochloric acid, sodium hydroxide, hydrogen peroxide and orthophosphoric acid of analytical grade were obtained from Merck Chemicals Ltd, Mumbai, India. Milli-Q water was used throughout the experiment dispensed through 0.22 µ filter of the Milli-Q water purification system from Millipore, Merck KGaA, Darmstadt, Germany The Phosphate buffer solution Weigh about 7.0 grams of potassium dihydrogen phosphate and transfer to 1000 ml standard flask, add 400 ml of Milli-Q water mix and dilute to volume with Milli-Q water, sonicate for five minutes and cool to room temperature, measure the ph of above buffer solution and finally adjusted the ph to 3.0±0.05 with orthophosphoric acid solution and filtered through 0.45 µ nylon filter The mobile phase A mixture of potassium dihydrogen phosphate buffer ph 3.0 and acetonitrile in the ratio of 50:50 v/v was prepared and used as mobile phase. 93

12 3.5. The diluent The potassium dihydrogen phosphate buffer ph 3.0 and acetonitrile mixture in the ratio of 50:50 v/v was used as diluent Preparation of standard solution of the drug About 10 mg of Valsartan was accurately weighed and transferred into a 100 ml clean dry volumetric flask containing 50 ml of diluent. The solution was sonicated for 5 min and then volume was made up to the mark with a further quantity of the diluent to get a concentration of 100 µg/ml of Valsartan (Stock solution). Further pipette 1 ml of the above stock solution into a 10 ml volumetric flask and the volume was made up to the mark with the diluent Preparation of sample (tablet) solution Twenty tablets were weighed and finely powdered. An accurately weighed portion of powder sample equivalent to 10 mg of Valsartan was transferred to a 100 ml volumetric flask containing 50 ml of the diluent. The contents of the flask were sonicated for about 10 min for complete solubility of the drug and volume made up with further quantity of diluent. Then this mixture was filtered through 0.45 µ membrane filter. 5.0 ml of this filtrate was further diluted to 50 ml with mobile phase. Further pipette 1 ml of the above stock solution into a 10 ml volumetric flask and the volume was made up to the mark with the diluent. 94

13 4. METHOD DEVELOPMENT For developing the method, a systematic study of the effect of various factors was undertaken by varying one parameter at a time and keeping all other conditions constant. Method development consists of selecting the appropriate wavelength and choice of stationary and mobile phases. The following studies were conducted for this purpose Detection wavelength The spectrum of diluted solution of the Valsartan in diluent was recorded on UV spectrophotometer. The peak of maximum absorbance was observed. The spectra of Valsartan showed that a balanced wavelength was found to be at 210 nm Choice of stationary phase Preliminary development trials have performed with octadecyl and octyl columns with different types, configurations and from different manufacturers. Finally the expected separation and shapes of peak was succeeded in Xterra C18 column Selection of the mobile phase In order to get sharp peak and base line separation of the components, the author has carried out a number of experiments by varying the composition of various solvents and its flow rate. To effect ideal separation of the drug under isocratic conditions, mixtures of solvents like water, methanol and acetonitrile with or without different buffers in different combinations were tested as mobile phases on a C18 stationary phase. A mixture of Potassium dihydrogen phosphate buffer ph 3.0 and acetonitrile in the ratio of 50:50 v/v was proved to be the most suitable of all the combinations since the 95

14 chromatographic peaks obtained were better defined and resolved and almost free from tailing Flow rate Flow rates of the mobile phase were changed from ml/min for optimum separation. A minimum flow rate as well as minimum run time gives the maximum saving on the usage of solvents. It was found from the experiments that 1.0 ml/min flow rate was ideal for the successful elution of the analyte Run time No interference in blank and placebo solutions for the drug peak in the trail injections with a runtime of 7.0 min Optimized chromatographic conditions Chromatographic conditions as optimized above were shown in Table 3.2. These optimized conditions were followed for the determination of Valsartan in bulk samples and its combined tablet formulations. The chromatograms of standard and sample solutions of Valsartan were shown in Figure 3.2 and Figure 3.3. The chromatograms of stability studies of Valsartan were shown from in Figure 3.7 to Figure

15 Table 3.2: Optimized chromatographic conditions for the estimation of Valsartan in tablet dosage form Mobile phase : Potassium dihydrogen phosphate buffer ph 3.0:acetonitrile, 50:50 v/v Pump mode : Isocratic ph of Buffer : 3.0±0.05 Diluent : Potassium dihydrogen phosphate buffer: acetonitrile, 50:50 v/v Column : Xterra C18 column, 150 mm x 4.6 mm, 5 µm Column Temp Wavelength : Ambient : 210 nm Injection Volume : 20 µl Flow rate Run time : 1.0 ml/min : 7 min Typical t R :- Valsartan : 4.450±0.5 min 97

16 Figure 3.2: Chromatogram of standard solution of Valsartan Figure 3.3: Chromatogram of sample solution of Valsartan 98

17 5. VALIDATION OF THE PROPOSED METHOD The proposed method was validated as per ICH [21-22] guidelines. The parameters studied for validation were specificity, linearity, precision, accuracy, robustness, system suitability, limit of detection, limit of quantification and solution stability Specificity A study conducted to establish specificity of the proposed method involved injecting blank and placebo using the chromatographic conditions defined for the proposed method. It was found that there is no interference due to excipients in the tablet formulation and also found good correlation between the retention times of standard and sample. The specificity results are shown in Table 3.3. The chromatograms of blank and placebo for Valsartan was shown in Figure 3.4 and Figure 3.5. Table 3.3: Specificity study Name of solution Blank Retention time (min) No peaks Valsartan

18 Figure 3.4: Chromatogram showing no interference of blank for Valsartan Figure 3.5: Chromatogram showing no interference of placebo for Valsartan 100

19 5.2. Linearity Linearity was performed by preparing standard solutions of Valsartan at different concentration levels including working concentration mentioned in experimental condition from 5.0 to 25.0 µg/ml and twenty micro litres of each concentration was injected in duplicate into the HPLC system. The response was read at 210 nm and the corresponding chromatograms were recorded. From these chromatograms, the mean peak areas were calculated and linearity plot of concentration over the mean peak areas was constructed individually. The regression of the plot was computed by least square regression method. Linearity results were presented in Table 3.4 and linearity plot are shown in Figure 3.6. Table 3.4: Linearity study of Valsartan Level Concentration of Valsartan (µg/ml) Mean peak area Level Level Level Level Level Slope 8162 Intercept Correlation Coefficient

20 Figure 3.6: Linearity plot of Valsartan 5.3. Precision Precision is the degree of repeatability of an analytical method under normal operational conditions. Precision of the method was performed as system precision, method precision and intermediate precision System precision To study the system precision, five replicate standard solutions of Valsartan were injected. The percent relative standard deviation (%RSD) was calculated and it was found to be 0.23 for Valsartan, which is well within the acceptable criteria of not more than 2.0. Results of system precision studies were shown in Table

21 Table 3.5: System precision Injection number Area of Valsartan Acceptance criteria Mean The %RSD of peak area of Valsartan should not be more than 2.0 SD 2895 %RSD Method precision The method precision study was carried out on five preparations from the same tablet samples of Valsartan and percent amount of both were calculated. The %RSD of the assay result of five preparations for Valsartan in method precision study was found to be 0.24 which is well within the acceptance criteria of not more than 2.0. The results obtained for assay of Valsartan is presented in Table

22 Table 3.6: Method precision Sample number %assay Valsartan Mean SD %RSD Intermediate precision The intermediate precision study was carried out by different analysts, different columns, different reagents using different HPLC systems from the same tablet of Valsartan and the peak area of Valsartan was calculated. The %RSD of the peak areas of five preparations in intermediate precision study of Valsartan was 0.19 which is well within the acceptance criteria of not more than 2.0. The results of intermediate precision study are reported in Table

23 Table 3.7: Intermediate precision study of Valsartan Injection number Area of Valsartan Acceptance criteria Mean The %RSD of peak area of Valsartan should not be more than 2.0 SD 2403 %RSD Accuracy The accuracy of the method was determined by standard addition method. A known amount of standard drug was added to the fixed amount of pre-analyzed tablet solution. Percent recovery was calculated by comparing the area before and after the addition of the standard drug. The standard addition method was performed at three concentration levels of 50%, 100% and 150%. The solutions were analyzed in triplicate at each level as per the proposed method. The percent recovery and %RSD at each level was calculated and results are presented in Table 3.8. The percent recovery of Valsartan was 98.6% to 101.2% and the mean recovery was found to be 100.2% by the proposed method. This indicates that the proposed method was accurate. 105

24 Table 3.8: Recovery study for Valsartan %Concentration (at specification Level) Mean peak area Amount of Valsartan spiked (mg) Amount of Valsartan recovered (mg) %Recovery %Mean Recovery 50% % 100% % 100.2% 150% % 5.5. Robustness The robustness study was performed by slight modification in flow rate of the mobile phase and composition of the mobile phase. Sample of Valsartan at 10 µg/ml concentration was analyzed under these changed experimental conditions. It was observed that there were no marked changes in chromatograms, which demonstrated that the developed method was robust in nature. The results of robustness study are shown in Table

25 Table 3.9: Robustness study for Valsartan Condition Mean Peak area %Assay %Difference Unaltered Flow rate at 0.6 ml/min Flow rate at 1.0 ml/min Mobile phase: Buffer(55):Acetonitrile(45) Buffer(45):Acetonitrile(55) System suitability System suitability was studied under each validation parameters by injecting six replicates of the standard solution. The system suitability parameters are given in Table Table 3.10: System suitability for Valsartan Parameter Tailing factor Theoretical plates Specificity study Linearity study Precision study Robustness study Flow rate at 0.6 ml/min Flow rate at 1.0 ml/min Mobile phase: Buffer(55):Acetonitrile(45) Buffer(45):Acetonitrile(55)

26 5.7. Limit of detection and Limit of quantification Limit of detection (LOD) is defined as the lowest concentration of analyte that gives a detectable response. Limit of quantification (LOQ) is defined as the lowest concentration that can be quantified reliably with a specified level of accuracy and precision. For this study six replicates of the analyte at lowest concentration were measured and quantified. The LOD and LOQ of Valsartan are given in Table Table 3.11: LOD and LOQ of Valsartan Parameter Measured value (µg/ml) Limit of detection Limit of quantification Solution stability To determine the stability of Valsartan in solution, the standard and sample solution were observed under room temperature. Any change in the retention time, peak shape and variation in response was compared to the pattern of chromatogram of freshly prepared solution. The solution stability results are shown in the Table Table 3.12: Solution stability of Valsartan Standard solution Sample solution Time (hours) Response %Variation Time (hours) Response %Variation Initial Initial

27 5.9. Stability studies In order to demonstrate the stability of both standard and sample solutions during analysis, both solutions were analyzed over a period of 24 hours at room temperature. The results show that for both solutions, the retention time and peak area of Valsartan (%RSD less than 2.0) has no significant degradation within the indicated period, thus indicated that both solutions were stable for at least 24 hours, which was sufficient to complete the whole analytical process. Further forced degradation studies were conducted indicating the stability of proposed method. The results of the degradation studies are shown in the Table Table 3.13: Forced degradation study results for Valsartan Stress Degradation Valsartan Conditions Time (Hrs) %Assay %Degradation Control Acid Base Peroxide Thermal Control sample Twenty tablets were weighed and finely powdered. An accurately weighed portion of powder sample equivalent to 10 mg of Valsartan was transferred to a 100 ml volumetric flask containing 50 ml of the diluent. The contents of the flask were sonicated for about 10 min for complete solubility of the drug and volume made up with 109

28 further quantity of diluent. Then this mixture was filtered through 0.45 µ membrane filter. 5.0 ml of this filtrate was further diluted to 50 ml with mobile phase. Acid degradation sample Twenty tablets were weighed and finely powdered. An accurately weighed portion of powder sample equivalent to 10 mg of Valsartan was transferred to a 100 ml volumetric flask containing 50 ml of the diluent. The contents of the flask were sonicated for about 10 min for complete solubility of the drug. Then 10 ml of 5N acid (Hydrochloric acid) was added, refluxed for 60 minutes at 60 C, then cooled to room temperature, neutralized with 5N base (Sodium hydroxide) and diluted to volume with diluent. About 25 ml of the above sample solution was filtered through 0.45 µ membrane filter. 5 ml of the above filtered sample solution was pipetted into a 50 ml volumetric flask and diluted to volume with diluent. Typical chromatogram of acid degradation for Valsartan is shown in Fig Figure 3.7: Chromatogram of acid degradation showing Valsartan 110

29 Base degradation sample Twenty tablets were weighed and finely powdered. An accurately weighed portion of powder sample equivalent to 10 mg of Valsartan was transferred to a 100 ml volumetric flask containing 50 ml of the diluent. The contents of the flask were sonicated for about 10 min for complete solubility of the drug. Then 10 ml of 5N base (Sodium hydroxide) was added, refluxed for 60 minutes at 60 C, then cooled to room temperature, neutralized with 5N acid (Hydrochloric acid) and diluted to volume with diluent. About 25 ml of the above sample solution was filtered through 0.45 µ membrane filter. 5 ml of the above filtered sample solution was pipetted into a 50 ml volumetric flask and diluted to volume with diluent. Typical chromatogram of base degradation for Valsartan is shown in Fig Figure 3.8: Chromatogram of base degradation showing Valsartan 111

30 Peroxide degradation sample Twenty tablets were weighed and finely powdered. An accurately weighed portion of powder sample equivalent to 10 mg of Valsartan was transferred to a 50 ml volumetric flask containing 50 ml of the diluent. The contents of the flask were sonicated for about 10 min for complete solubility of the drug. Then 4 ml of 30% hydrogen peroxide was added, refluxed for 60 minutes at 60 C, then cooled to room temperature and diluted to volume with diluent. About 25 ml of the above sample solution was filtered through 0.45 µ membrane filter. 5 ml of the above filtered sample solution was pipetted into a 50 ml volumetric flask and diluted to volume with diluent. Typical chromatogram of peroxide degradation for Valsartan is shown in Fig Figure 3.9: Chromatogram of oxidative degradation showing Valsartan 112

31 Thermal degradation sample Twenty tablets were weighed and finely powdered. The powder is exposed to heat at 105 C for about 2 days. An accurately weighed portion of powder sample equivalent to 10 mg of Valsartan was transferred to a 100 ml volumetric flask containing 50 ml of the diluent. The contents of the flask were sonicated for about 10 min for complete solubility of the drug. About 25 ml of the above sample solution was filtered through 0.45 µ membrane filter. 5 ml of the above filtered sample solution was pipetted into a 50 ml volumetric flask and diluted to volume with diluent. Typical chromatogram of thermal degradation for Valsartan is shown in Fig Figure 3.10: Chromatogram of thermal degradation showing Valsartan 113

32 6. RESULTS AND DISCUSSION The present study was aimed at developing a simple, sensitive, precise and accurate HPLC method for the estimation of Valsartan from bulk samples and tablet dosage forms. A non-polar C18 analytical chromatographic column was chosen as the stationary phase for the separation and determination of Valsartan. Mixtures of commonly used solvents like water, methanol and acetonitrile with or without buffers in different combinations were tested as mobile phases. The choice of the optimum composition is based on the chromatographic response factor, a good peak shape with minimum tailing. A mixture of buffer and acetonitrile in the ratio of 50:50 v/v was proved to be the most suitable of all the combinations since the chromatographic peak obtained was well defined, better resolved and almost free from tailing. The retention time of Valsartan was found to be 4.45 min. The linearity was found satisfactory for the drug in the range µg/ml (Table 3.4). The regression equation of the linearity curve of Valsartan between concentrations over its peak areas was found to be Y=8162X (where Y is the peak area and X is the concentration of Valsartan in µg/ml). Precision of the method was studied by repeated injection of tablet solution and results showed lower %RSD values (Table ). This reveals that the method was quite precise. The percent recoveries of the drug solutions were studied at three different concentration levels. The percent individual recovery and the % RSD at each level were within the acceptable limits (Table 3.8). This indicates that the method was accurate. The absence of additional peaks in the chromatogram indicates non-interference of the commonly used excipients in the tablets and hence the method was specific. 114

33 The deliberate changes in the method have not much affected the peak tailing, theoretical plates and the percent assay. This indicates that the present method was robust (Table 3.9). The system suitability studies were carried out to check various parameters such as theoretical plates and tailing factor (Table 3.10). The lowest values of LOD and LOQ as obtained by the proposed method indicate that the method was sensitive (Table 3.11). The solution stability studies indicate that the drug was stable up to 24 hours (Table 3.12). The forced degradation studies indicate that the drug was stable in stability studies (Table 3.13). 7. CONCLUSION The proposed stability-indicating RP-HPLC method was simple, specific, sensitive, accurate and precise and can be used for analysis of Valsartan in bulk samples and its tablet dosage forms. 8. REFERENCES 1. Martindale, The Complete Drug Reference, 34 th Edn, Pharmaceutical press, p. 1018, (2005). 2. The Merck Index, 13 th Edn., Merck Research Laboratories, Merck & Co., White House Station, NJ, USA, p. 1767, (2001). 3. Wilson and Gisvolds, Text book of organic medicinal and pharmaceutical chemistry, 11 th Edn, Lippincott-Williams & Wilkins, Philadelphia. U.S.A, p. 649, (2004). 4. H.L.Sharma & K.K.Sharma, Principles of pharmacology, 2 nd Edn, Paras s medical publishers, Delhi, p. 256, (2012). 115

34 5. CIMS (Current Index of Medical Specialities), UBM Medica India Pvt. Limited, Bangalore, p. 104, Apr-Jul, (2012). 6. IDR Drug Triple i Compendium (Indian Drug Review), UBM Medica India Pvt. Limited, Bangalore, p. 219, 5, (2012). 7. Nataraj. K.S and Ramanjaneyulu. K. Simple quantitative method development and validation of Valsartan in pure form and pharmaceutical dosage forms by UV-spectroscopy. International Journal of Pharmaceutical Sciences, 1(3), (2011). 8. Kailash. T, Sachin. T and Manisha. J. Development and validation of UVspectrophotometric methods for estimation of Valsartan in bulk and tablet dosage form. Journal of Pharmaceutical Research, 1(2), (2012). 9. Gupta. K.R, Wadodkar. A.R and Wadodkar. S.G. UV-spectrophotometric methods for estimation of Valsartan in bulk and tablet dosage form. International Journal of Pharmaceutical Sciences, 2(8), (2010). 10. Madhusudan Deshpande. M, JaardanGondane. S, Prafulla Mahajan. M and Dinkar Sawant. S. Simultaneous estimation and validation of Valsartan & Hydrochlorothiazide by UV spectrophotometry. Journal of Pharmaceutical Research, 4(3), (2011). 11. Piao. Z.Z and Lee. E.S. Improved analytical validation and pharmacokinetics of Valsartan using HPLC with UV detection. Archival of Pharmaca Research, 8(4), (2008). 116

35 12. Thanusha. G and JoseGnanaBabu. C. Validated RP-HPLC method for the quantitative estimation of Valsartan in bulk and pharmaceutical dosage forms. Journal of Pharmaceutical Research, 2(2), ( 2010). 13. Rao. K.S and Jeena. Development and validation of a specific stability indicating high performance liquid chromatographic method for Valsartan. Journal of Chemical Research, 2(2), (2010). 14. Narendra Reddy. B and Chenna Reddy. U. RP-HPLC method development and validation of Valsartan tablet dosage form. Journal of Chemical and Pharmaceutical Research, 2(2), (2010). 15. Patnaik. A and Malikarjuna Shetty. A new RP-HPLC method for the determination of Valsartan in bulk and its pharmaceutical formulations with its stability indicative studies. International Journal of Chem Tech Research, 2(4), (2011). 16. Santosh Kumar. P.V, Sahu. M, Durga Prasad. K and Chandra shekhar. M. Development and validation of analytical method for the estimation of Valsartan in pure and tablet dosage form by RP-HPLC method. International Journal of Research in Pharmacy and Chemistry, 1(4), (2011). 17. Parambi. T, Grace. D, Mathew. M and Ganesan. V. A validated stability indicating HPLC method for the determination of Valsartan in tablet dosage forms. Journal of Applied Pharmaceutical Science, 1(4), (2011). 117

36 18. Shah. J.N, Suhagia. B.N, Shah. R.R and Patel. M.M. HPTLC method for the simultaneous estimation of Valsartan and Hydrochlorthiazide in tablet dosage form. Indian Journal of Pharmaceutical Sciences, 71(1), 72-74, (2009). 19. Sunil. S, Kuldeep. P, VipinKu. A and Seshank. C. Stability indicating HPTLC method for the simultaneous estimation of Valsartan and Hydrochlorthiazide in tablets. International Journal of Pharmacy and Pharmaceutical sciences, 4(4), (2012). 20. Hiten. J.S, Naresh. B.K, Gunta. S and Chagan. N.P. Simultaneous LC-MS-MS analysis of Valsartan and Hydrochlorothiazide in human plasma. Chromatographia, 69(9), (2012). 21. Alnajjar. A.O. Validation of a Capillary Electrophoresis method for the simultaneous determination of Amlodipine besylate and Valsartan in pharmaceuticals and human plasma. Journal of AOAC International, 94(2), (2011). 22. ICH Harmonised Tripartite Guideline. Validation of Analytical Procedures: Text and Methodology, Q2(R1), USA, 1-13 (2005). 23. ICH Harmonised Tripartite Guideline. Stability Testing of New Drug Substances and Products, Q1A(R2), USA, 1-18 (2003). 118