Int J Pharm Pharm Sci, Vol 7, Issue 8, 57-61Original Article
DEVELOPMENT AND VALIDATION OF A STABILITY INDICATING RP-HPLC METHOD FOR THE DETERMINATION OF VALSARTAN
K. K. PRADHANa*, U. S. MISHRAb
aDepartment of Pharmaceutical Sciences & Technology, Birla Institute of Technology, MESRA, Ranchi 835215, bRoyal College of Pharmacy and Health Sciences, Berhampur, Odisha 76002
Email: kkpradhan@bitmesra.ac.in
Received: 07 Apr 2015 Revised and Accepted: 15 Jun 2015
ABSTRACT
Objective: A stability indicating RP-HPLC method was developed and validated for the determination of Valsartan using Telmisartan (10 µg/ml) as the internal standard.
Methods: In this procedure Phenomenex ODS C-18(250×4.6 mm, packed with 5 micron) column was used with a new mobile phase consisting of methanol: acetonitrile: water (70:15:15 v/v) and the pH was adjusted to 3 by 0.1% glacial acetic acid with a flow rate of 1 ml/min. The eluents were monitored at 249 nm. Valsartan was subjected to stress conditions including hydrolytic degradation in acidic, basic and neutral conditions, oxidation, photolytic, UV degradation and thermal degradation.
Results: Linearity was obtained in the concentration range of 10-90 µg/ml (R2 =0.999) and with a regression equation y=0.074x+0.005. The LOD and LOQ values were 0.261 and 0.791 µg/ml respectively. The drug had shown promising degradation in the acidic, basic, neutral, thermal and oxidative stress conditions.
Conclusion: The method was validated for accuracy, precision, linearity, specificity and robustness and revealed that it is specific, accurate, rapid, precise, reliable and reproducible enough to analyze commercial dosage forms as per ICH guidelines.
Keywords: Valsartan, Stability Studies, Stability Indicating RP-HPLC, Stress Degradation, ICH Guidelines.
INTRODUCTION
Valsartan is chemically 3-methyl-2-[pentanoyl-[[4-[2-(2H-tetrazol-5-yl) phenyl] phenyl]methyl]amino]-butanoic acid (fig. 1)[1], Angiotensin II receptor antagonist, acting on the AT1 subtype &used for treatment of high blood pressure, of congestive heart failure (CHF), and post-myocardial infarction (MI). By blocking the action of Angiotensin, Valsartan dilates blood vessels and reduces blood pressure [2]. The focus of the present study was to develop & validated a rapid, stable, & economic stability indicating HPLC method for the estimation of Valsartan in bulk and tablet dosage forms. As per ICH Q1A (R2) guidelines the purpose of stability testing is to provide evidence on how the quality of a drug substance or drug product varies with time under the influence of a variety of environmental factors, such as temperature, humidity, and light, and to establish a retest period for the drug substance or a shelf life for the drug product and recommended storage conditions.
Fig. 1: Chemical structure of valsartan
From the literature survey [3-10] it has been found that many HPLC methods are available for the determination of valsartanin bulk as well as in formulations along with stability indications. But with the use of this new mobile phase i.e. methanol: acetonitrile: water (70:15:15 v/v) pH adjusted to 3 by glacial acetic acid, the detection of degradant peaks of UV, Oxidative and Thermal stresses was found to be better and precise. It has also been found that using the new mobile phase, the developed RP HPLC method became more robust, accurate and highly précised. As the separation of the degradants using this mobile phase is quite good, isolation of the degradants with preparative techniques can also be achieved using this mobile phase. Further LC MS-MS analysis will help to deduce the structures of the degradants which can help to establish the possible degradation pathway of this drug. So this method can be economically very useful in both research and industrial aspect.
MATERIALS AND METHODS
Chemicals and reagents
Valsartan API was purchased from Cadila Healthcare Ltd, Ahmedabad, India. The HPLC grade solvents used were obtained from Merck (India) Ltd, Mumbai. HPLC grade water was prepared using Millipore System (Millipore, Molesheim France, and Model Elix-10). All other reagents were of HPLC grade.
Equipments
HPLC was performed on Shimadzu HPLC with LC-20AT pumps besides SPD-20A UV-Visible detector. Shimadzu spin crom-CFR software was used along with Phenomenex ODS C-18(250×4.6 mm, packed with 5 microns) for the separation.
Chromatographic conditions
Selected drug was injected to the column with different mobile phases of different ratios with different flow rates till sharp peaks without any interference peaks containing spectra were obtained. Final mobile phase was chosen as methanol: water: acetonitrile (70:15:15) and pH was adjusted to 3 with 0.1 N glacial acetic acid. The mobile phase was then sonicated for 10 minutes and filtered through 0.45μ membrane filter. C-18 column was equilibrated with the mobile phase. Mobile phase flow rate was maintained at 1 ml/min and eluent were monitored at 249 nm wavelength. The samples were injected using a 20 μl fixed loop. All determinations were performed at ambient temperature for a run time of 6 min.
Preparation of stock and working solutions
About 50 mg of valsartan was weighed accurately and was taken in 50 ml volumetric flasks. It was dissolved in the mobile phase and the volume was made up to the mark to prepare the 1000 µg/ml stock solution. From the above prepared stock solution of valsartan 5 ml was pipette out in to a 50 ml volumetric flask and the volume was made to up to the mark with mobile phase to yield the 100 µg/ml working solution of valsartan.
About 50 mg of telmisartan was weighed accurately and was taken in a 50 ml volumetric flask. It was dissolved in the mobile phase and the volume was made up to the mark to make a stock solution of 1000 µg/ml. From this stock, 5 ml was pipette out and diluted to 50 ml with the mobile phase to yield the working solution of Telmisartan (100 µg/ml).
Method validation
Validation of the established HPLC method was done as per the ICH guidelines to assess the accuracy, precision, specificity, ruggedness, robustness, LOD and LOQ values. The accuracy of the method was determined by calculating recoveries of drug by the method of standard drug. Known amounts of standard drug corresponding to 80%, 100% and 120% of the label claim was added to pre quantified sample solution and the amounts of drug were estimated by measuring the peak areas and by fitting these values to the straight line equation of calibration curve. The intraday and inter-day precision studies of the drugs were carried out by estimating the corresponding responses on the same day and consecutive three days respectively.
The results were reported in terms of standard deviation and % RSD. The specificity of the proposed RP-HPLC method was determined by complete separation of two peaks with parameters like retention time (Rt), resolution (RS) and tailing factor (T). Robustness of the method was studied by deliberate variations of the analytical parameters such as flow rate (1±0.2 ml/min), concentration of methanol (90±2%). Ruggedness is the degree of reproducibility of the results obtained under a variety of conditions, expressed as %RSD. These conditions include different laboratory conditions and different analysts.
Forced degradation studies
The force degradation studies were conducted on the sample using acid, alkaline, oxidative, thermal, and photolytic and UV degradations. Neutral stress was given by dissolving 10 mg of drug into 10 ml of methanol followed by volume made up with water and refluxing for 6 h. Acidic and basic stresses were given by using 0.1 N. 0.5 N and 1 N of hydrochloric acid and sodium hydroxide followed by 6 h reflux. Oxidative stress was put on by using 1 % w/v, 2 % and 3 % w/v hydrogen peroxide. Photo degradation was done upon exposure to sunlight for 12,24,72 h. UV illumination of 1.2×106lux h for 12,24,72 h was used as the stress factor for UV degradation. Thermal degradation was carried out at 70 °C dry heat for 10, 20 and 30 days.
RESULTS
Percentage recovery was calculated from differences between the peak areas obtained for fortified and unfortified solutions.
As shown from the data in table 1, good recoveries were made at each added concentration, confirming that the method was accurate. Data obtained from analysis of the samples on the same day (n = 6) and on consecutive days (n = 6) are given in table 1. As the % RSD values of the data obtained were well below 2%, thereby the values indicate that the method was sufficiently precise.
Linear calibration plots of each drug for the previously mentioned method were obtained over the calibration ranges 10–90 µg/ml; the correlation coefficient obtained was 0.999 (fig. 3). The chromatogram is shown in fig. 2. The results show that the good correlation existed between the peak area and concentration of the analyte. The LOD and LOQ values found for the analysis were 0.261 and 0.791 µg/ml respectively.
Robustness study was being performed by changing two parameter pH and flow rate. pH had been altered to 2.8 and 3.2 whereas flow rate was changed to 0.8 an 1.2. Ruggedness of the established method was assessed by performing 6 different injections of a particular concentration of the drug by two different analysts. Results obtained (shown in table 1-5) and % RSD values calculated clearly illustrate the ruggedness of the method.
Fig. 2: Linearity Chromatogram of valsartan along with I. S. telmisartan
Fig. 3: Calibration curve of valsartan
Table 1: Accuracy data of the method
| Samples | Concentration (μg/ml) | % Recovery | Statistical Analysis |
| Amount present in Formulation | Amount of drug added | ||
| S1: 80% | 30 | 24 | 100.02 |
| S2: 80% | 30 | 24 | 99.89 |
| S3: 80% | 30 | 24 | 99.75 |
| S4: 100% | 30 | 30 | 100.07 |
| S5: 100% | 30 | 30 | 99.79 |
| S6: 100% | 30 | 30 | 99.98 |
| S7: 120% | 30 | 36 | 99.59 |
| S8: 120% | 30 | 36 | 99.73 |
| S9: 120% | 30 | 36 | 100.04 |
Table 2: Intraday precision data
| S. No. | Concentration (μg/ml) | Peak Area Ratio | Calc. Amt. (μg/ml) | Statistical Analysis |
| 1 | 30 | 2.216 | 30.01 | Mean=29.99 SD=0.117 % RSD=0.390 |
| 2 | 30 | 2.209 | 29.91 | |
| 3 | 30 | 2.228 | 30.17 | |
| 4 | 30 | 2.207 | 29.89 | |
| 5 | 30 | 2.219 | 30.05 | |
| 6 | 30 | 2.212 | 29.95 |
Table 3: Inter-day precision data
| S. No. | Concentration (μg/ml) |
Day 1 | Day 2 | Day 3 | Statistical analysis |
| 1 | 30 | 2.231 | 2.236 | 2.232 | Mean=30.09 SD=0.076 % RSD=0.252 |
| 2 | 30 | 2.237 | 2.229 | 2.241 | |
| 3 | 30 | 2.205 | 2.208 | 2.201 | |
| 4 | 30 | 2.217 | 2.227 | 2.205 | |
| 5 | 30 | 2.211 | 2.206 | 2.209 | |
| 6 | 30 | 2.225 | 2.239 | 2.245 | |
| Mean Peak Area Ratio | 2.221 | 2.224 | 2.222 | ||
| Calc. Amt. (μg/ml) | 30.08 | 30.12 | 30.09 |
Table 4: Robustness data
| pH 2.8 | pH 3.2 |
| Conc. (μg/ml) | Peak Area Ratio |
| 30 | 2.207 |
| 30 | 2.218 |
| 30 | 2.211 |
| 30 | 2.226 |
| 30 | 2.207 |
| 30 | 2.219 |
Table 5: Ruggedness data
| Analyst-1 | Analyst-2 |
| Conc. (μg/ml) | Peak Area Ratio |
| 30 | 2.241 |
| 30 | 2.232 |
| 30 | 2.230 |
| 30 | 2.209 |
| 30 | 2.204 |
| 30 | 2.235 |
In case of acidic, neutral, basic, photo, UV and thermal stress 100 µg/ml solutions were made and injected into the system. For oxidative stress, 60 µg/ml solutions was made and injected. The chromatograms of the drug and degradants under different stress conditions are given in the fig. 4. Individual degradation study results are elaborated from table 6-12 and overall summary of the results of the degradation study is given in table 13.
Table 6: Hydrolytic degradation in neutral condition of valsartan
| S. No. | Reten. Time (min) | Area (Mv. s) | Height (Mv) | Area (%) | Height (%) | W05 (min) | Results |
| 1 | 2.120 | 280.393 | 25.156 | 10.0 | 13.0 | 0.11 | Drug Remaining= 85.85 μg/ml %Degradation= 14.15% |
| 2 | 2.173 | 68.824 | 4.843 | 2.5 | 2.5 | 0.16 | |
| 3 | 2.863 | 83.632 | 3.474 | 3.7 | 1.8 | 0.42 | |
| 4 | 3.637 | 32.965 | 1.598 | 1.1 | 0.8 | 0.27 | |
| 5 | 3.937 | 1614.188 | 154.788 | 79.4 | 80.3 | 0.16 | |
| 6 | 4.870 | 95.254 | 3.020 | 3.3 | 1.6 | 0.29 | |
| Total | 2175.256 | 192.879 | 100 | 100 | - |
Table 7: Degradation in acidic condition
| S. No. | Reten. Time (min) | Area (Mv. s) | Height (Mv) | Area (%) | Height (%) | W05 (min) | Results |
| 1 | 3.883 | 1544.773 | 140.349 | 81.6 | 84.9 | 0.16 | Drug Remaining= 82.12 μg/ml %Degradation= 17.88% |
| 2 | 4.393 | 281.473 | 17.391 | 18.4 | 14.1 | 0.17 | |
| Total | 1826.246 | 157.74 | 100 | 100 | - |
Fig. 4: Degradation chromatograms under different stress conditions
Table 8: Degradation in basic condition
| S. No. | Reten. Time (min) | Area (Mv. s) | Height (Mv) | Area (%) | Height (%) | W05 (min) | Results |
| 1 | 2.633 | 368.736 | 41.639 | 16.5 | 18.3 | 0.15 | Drug Remaining= 74.16 μg/ml %Degradation=25.84 |
| 2 | 3.603 | 35.599 | 1.663 | 1.3 | 0.7 | 0.45 | |
| 3 | 3.953 | 1396.637 | 127.092 | 74.2 | 74.7 | 0.16 | |
| 4 | 4.920 | 169.072 | 10.427 | 8.0 | 6.3 | 0.20 | |
| Total | 1907.044 | 180.821 | 100 | 100 | - |
Table 9: Thermal degradation
| S. No. | Reten. Time (min) | Area (Mv. s) | Height (Mv) | Area (%) | Height (%) | W05 (min) | Results |
| 1 | 1.913 | 61.636 | 4.333 | 1.6 | 1.8 | 0.13 | Drug Remaining= 72.14μg/ml %Degradation= 27.86% |
| 2 | 2.833 | 11.999 | 1.120 | 0.5 | 0.5 | 0.24 | |
| 3 | 3.423 | 1.760 | 0.279 | 0.1 | 0.1 | 0.11 | |
| 4 | 3.663 | 1359.045 | 159.154 | 72.14 | 71.6 | 0.13 | |
| 5 | 4.933 | 567.501 | 49.941 | 25.66 | 26.0 | 0.16 | |
| Total | 2001.941 | 214.827 | 100 | 100 | - |
Table 10: Oxidative degradation
| S. No. | Reten. Time (min) | Area (Mv. s) | Height (Mv) | Area (%) | Height (%) | W05 (min) | Results |
| 1 | 2.143 | 96.239 | 13.276 | 2.2 | 3.2 | 0.10 | Drug Remaining= 49.88μg/ml %Degradation= 16.86% |
| 2 | 2.473 | 34.131 | 3.271 | 0.8 | 0.8 | 0.19 | |
| 3 | 2.733 | 2175.281 | 214.507 | 53.8 | 50.1 | 0.16 | |
| 4 | 3.153 | 261.389 | 16.908 | 5.8 | 4.1 | 0.29 | |
| 5 | 3.397 | 132.315 | 15.605 | 3.0 | 3.8 | 0.16 | |
| 6 | 3.550 | 130.210 | 14.824 | 2.9 | 3.6 | 0.15 | |
| 7 | 3.683 | 170.121 | 15.579 | 3.8 | 3.8 | 0.22 | |
| 8 | 3.973 | 944.861 | 80.041 | 22.0 | 21.8 | 0.16 | |
| 9 | 4.943 | 380.859 | 31.115 | 5.7 | 8.8 | 0.18 | |
| Total | 4325.406 | 405.126 | 100 | 100 | - |
Table 11: Photolytic degradation
| S. No. | Reten. Time (min) | Area (Mv. s) | Height (Mv) | Area (%) | Height (%) | W05 (min) | Results |
| 1 | 1.990 | 21.850 | 1.748 | 1.1 | 0.9 | 0.15 | Drug Remaining= 94.16μg/ml %Degradation= 5.84% |
| 2 | 2.463 | 7.106 | 0.629 | 0.4 | 0.3 | 0.18 | |
| 3 | 2.847 | 5.013 | 0.604 | 0.3 | 0.3 | 0.15 | |
| 4 | 3.207 | 38.440 | 2.696 | 2.0 | 1.3 | 0.15 | |
| 5 | 3.693 | 7.383 | 0.728 | 0.4 | 0.4 | 0.17 | |
| 6 | 4.033 | 1768.837 | 191.886 | 95.3 | 96.5 | 0.14 | |
| 7 | 5.010 | 10.525 | 0.846 | 0.6 | 0.4 | 0.20 | |
| Total | 1859.154 | 199.136 | 100 | 100 | - |
Table 12: UV degradation
| S. No. | Reten. Time (min) | Area (Mv. s) | Height (Mv) | Area (%) | Height (%) | W05 (min) | Results |
| 1 | 1.993 | 24.852 | 2.215 | 1.2 | 1.0 | 0.15 | Drug Remaining= 98.44μg/ml %Degradation= 1.56% |
| 2 | 2.463 | 3.414 | 0.468 | 0.2 | 0.2 | 0.12 | |
| 3 | 2.847 | 8.294 | 0.868 | 0.4 | 0.4 | 0.18 | |
| 4 | 3.210 | 46.247 | 3.349 | 2.3 | 1.5 | 0.14 | |
| 5 | 3.700 | 11.733 | 1.162 | 0.6 | 0.5 | 0.17 | |
| 6 | 4.030 | 1848.488 | 206.241 | 93.7 | 95.0 | 0.13 | |
| 7 | 5.007 | 30.627 | 2.713 | 1.5 | 1.3 | 0.17 | |
| Total | 1973.657 | 217.016 | 100 | 100 | - |
Table 13: Overall summary of degradation study
| Stress condition | Stressing agents | Time (h) | Degradation |
| Neutral | WATER | 6 | 14.15% |
| Acidic | 0.1 N | 6 | Stable |
| 0.5 N | 6 | Stable | |
| 1 N | 6 | 17.88% | |
| Basic | 0.1 N | 6 | Stable |
| 0.5 N | 6 | Stable | |
| 1 N | 6 | 25.84% | |
| Oxidation | 1% H2O2 | 6 | Stable |
| 2% H2O2 | 6 | Stable | |
| 3% H2O2 | 6 | 16.86% | |
| Light | SUN LIGHT | 12 | Stable |
| 24 | Stable | ||
| 72 | 5.84% | ||
| Thermal | 70 °C HEAT | 240 | Stable |
| 480 | Stable | ||
| 720 | 27.86% | ||
| UV Radiation | 1.2×106 lux h (UV illumination at 256 nm) | 12 | Stable |
| 24 | Stable | ||
| 72 | 1.56% |
DISCUSSION
The developed RP-HPLC stability indicating assay method was found suitable for the analysis of drug in their pure form in presence of their respective degradants since the resolution between the drugs with its corresponding degradants was better. ADVANTAGE: Previously reported methods for analysis of valsartan in bulk are having retention times more than 4 minutes whereas in this method it is less than 4 minutes. More to that 99.94% accuracy and huge linearity range of 10-90 µg/ml with such a low LOD and LOQ values i.e. 0.261 and 0.791 µg/ml was not reported in earlier methods. Previously linearity was assessed in between 16-320 µg/ml range and LOD and LOQ values were also more than 10 µg/ml. The sensitivity and accuracy of the method were also ascertained by using internal standard telmisartan which is not reported earlier. With the use of the newly developed mobile phase and internal standard, further bio-analytical method can be developed. CONCLUSION: The results of stability studies of valsartan suggest that the drug is more prone to thermolytic, hydrolytic and oxidative degradations. So measures should be focused to reduce the exposure of valsartan to atmospheric conditions.
ACKNOWLEDGEMENT
Authors are thankful to Director cum Principal of Royal College of Pharmacy and Health Sciences, Berhampur, Odisha-76002 for providing the necessary facilities for this research work.
CONFLICT OF INTERESTS
Declared None.
REFERENCES
- Blake PS. Cardiovascular drugs. In: Brayfield; Alison. editors. Martindale the complete drug reference. 33rd ed. London: Pharmaceutical Press; 2002. p. 853.
- Tripathi KD. Essentials of medical pharmacology. New Delhi: Jaypee Brothers Medical Publishers; 2008. p. 875.
- Krishnaiah C, Reddy AR, Kumar R, Mukkanti K. Stability-indicating UPLC method for determination of Valsartan and their degradation products in active pharmaceutical ingredient and pharmaceutical dosage forms. J Pharm Biomed Anal 2010;53(3):483–9.
- Koseki N, Kawashita H, Hara H, Niina M, Tanaka M, Kawai R. Development and validation of a method for quantitative determination of valsartan in human plasma by liquid chromatography-tandem mass spectrometry. J Pharm Biomed Anal 2007;43:1769–74.
- Rizzo M, Ventrice D, Casale F, Caselli GF, Makovec F. Pharmacokinetic study of a new angiotensin-AT1 antagonist by HPLC. J Pharm Biomed Anal 2008;48:422–7.
- Shah NJ, Suhagia BN, Shah RR, Patel NM. Development and validation of HPTLC method for the simultaneous estimation of Valsartan and hydrochlorothiazide in tablet dosage form. Indian J Pharm Sci 2009;71(1):72–4.
- Uttamrao KS, Bhatia MS. Validation of Valsartan and its degradation products by HPLC. J Chem Metrol 2010;3(1):1-12.
- Chitlange SS, Bagri K, Sakarkar DM. Stability indicating RP-HPLC method for simultaneous estimation of Valsartan and Amlodipine in capsule formulation. Asian J Res Chem 2008;1(1):15-8.
- Daneshtalab N. High-performance liquid chromatographic analysis of angiotensin II receptor antagonist valsartan using a liquid extraction method. J Chromatogr B 2002;76(6):345–9.
- Sibel A, Ozakan SE, Gorger N, Altinay S, Zuhre S. Simultaneous determination of valsartan and hydrochlorthiazide in tablets by first-derivative UV spectrophotometry and LC. J Pharm Biomed Anal 2001;25(5):1009-13.