INTRODUCTION:

Tolvaptan¹ is chemically N-(4-{[(5R)-7-chloro-5-hydroxy-2,3,4,5-tetrahydro-1H-1-benzazepin-1-yl]carbonyl}-3-methylphenyl)-2-methylbenzamide and the chemical structure of Tolvaptan was shown in Fig. 1. Tolvaptan is selective, competitive nonpeptide arginine vasopressin V₂ receptor antagonist². Vasopressin acts on the V2 receptors found in the walls of the vasculature and luminal membranes of renal collecting ducts³. By blocking V2 receptors in the renal collecting ducts, aquaporins do not insert themselves into the walls thus preventing water absorption⁴. This action ultimately results in an increase in urine volume, decrease urine osmolality and increase electrolyte-free water clearance to reduce intravascular volume and an increase serum sodium levels. Tolvaptan is used in the treatment of congestive heart failure⁵.

Fig. 1: Chemical structure of Tolvaptan

Literature survey revealed that very few analytical methods such as UV^6,7, HPLC⁸, UPLC⁹ and LC-MS¹⁰ methods have been reported for the estimation of Tolvaptan in biological samples, bulk and pharmaceutical dosage forms. Hence we made an attempt to develop and validate a new stability-indicating HPLC method as per ICH guidelines^11,12 for the estimation of Tolvaptan in bulk and pharmaceutical dosage forms.

MATERIALS AND METHODS:

Instrumentation:

To develop a high pressure liquid chromatographic method for quantitative estimation of Tolvaptan using Waters HPLC system on Hypersil BDS C18 (100 mm x 4.6 mm I.D., 5 µm particle size) column was used. The instrument is equipped with an auto sampler and DAD or UV detector. A 20 μl rheodyne injector port was used for injecting the samples. Data was analyzed by using Empower 2 software.

Chemicals and solvents:

The working standard of Tolvaptan was provided as gift sample from Spectrum Labs, Hyderabad, India. The market formulation TOLVASCA tablets (Tolvaptan 30 mg) were procured from local market. HPLC grade water, methanol and acetonitrile were purchased from E.Merck (India) Ltd., Mumbai, India. Potassium dihydrogen orthophosphate, triethylamine and orthophosphoric acid of AR grade were obtained from S.D. Fine Chemicals Ltd., Mumbai, India.

Chromatographic conditions:

0.02M phosphate buffer pH 3.1 adjusted with orthophosphoric acid and acetonitrile in the ratio of 35:65 V/V was found to be the most suitable mobile phase for ideal chromatographic separation of Tolvaptan. The solvent mixture was filtered through a 0.45 μm membrane filter and sonicated before use. It was pumped through the column at a flow rate of 1.0 ml/min. Injection volume was 10 µl and the column was maintained at a temperature of 30⁰C. The column was equilibrated by pumping the mobile phase through the column for at least 30 minutes prior to the injection of the drug solution. The detection of the drug was monitored at 254 nm. The run time was set as 7 minutes.

Preparation of 0.02 M phosphate buffer pH 3.1:

2.72 grams of potassium dihydrogen orthophosphate was weighed accurately, transferred into a 1000 ml beaker and dissolved in 500 ml of HPLC grade water. 1 ml of triethyl amine was added to the above solution. The solution was sonicated for 30 minutes, degassed and then made to total volume with water. The pH of the resulting solution was adjusted to 3.1 with dilute orthophosphoric acid and filtered through 0.45 μm membrane filter.

Preparation of mobile phase and diluent:

The mobile phase was prepared by mixing 350 ml of 0.02M phosphate buffer pH 3.1 with 650 ml of acetonitrile. The solution was degassed in an ultrasonic water bath for 5 minutes and filtered through 0.45 µm filter under vacuum. The solvent methanol was used as diluent.

Preparation of standard solution:

10 mg of Tolvaptan was accurately weighed, transferred to 10 ml volumetric flask and is dissolved in 7 ml of the diluent. Sonicated the solution for few minutes and dissolved the drug completely. Then it is filtered through 0.45 μm filter and the volume is made up to 10 ml with diluent to get a concentration of 1 mg/ml stock solution. Further pipette 1.2 ml of the above stock solution into a 10 ml volumetric flask and diluted up to the mark with diluent to obtain required concentration of 120 µg/ml of Tolvaptan.

Preparation of sample solution:

Twenty tablets were weighed and finely powdered. An accurately weighed portion of powder sample equivalent to 10 mg of Tolvaptan was transferred to 10 ml volumetric flask and is dissolved in 7 ml of the diluent. Sonicated the solution for few minutes and dissolved the drug completely. Then it is filtered through 0.45 μm filter and the volume is made up to 10 ml with diluent to get a concentration of 1 mg/ml stock solution. Further pipette 1.2 ml of the above stock solution into a 10 ml volumetric flask and diluted up to the mark with diluent to obtain required concentration of 120 µg/ml of Tolvaptan.

Calibration plot:

About 10 mg of Tolvaptan was weighed accurately, transferred into a 10 ml volumetric flask and dissolved in 7 ml of a 35:65 V/V mixture of phosphate buffer pH 3.1 and acetonitrile. The solution was sonicated for 15 minutes and the volume made up to the mark with a further quantity of the diluent to get a 1000 μg/ml solution. From this, a working standard solution of the drug (100 μg/ml) was prepared by diluting with the above solution to 10 ml in a volumetric flask. Further dilutions ranging from 30-180 µg/ml were prepared from the solution in 10 ml volumetric flasks using the above diluent. The column was maintained at a temperature of 30^◦C. The pump pressure was set at 800 psi. The run time was set at 7 minutes. The column was equilibrated by pumping the mobile phase through the column for at least 30 minutes prior to the injection of the drug solution. Inject 10 μl of the standard, sample solutions six times into the chromatographic system at a flow rate of 1.0 ml/min and the corresponding chromatograms were obtained. From these chromatograms, the average area under the peak of each dilution was computed. The linearity curve constructed by plotting concentration of the drug against peak area was found to be linear in the concentration range of 30-180 μg/ml of the drug. The regression equation of this curve was computed. This regression equation was later used to estimate the amount of Tolvaptan in pharmaceutical dosage forms.

Method validation:

Linearity:

Several aliquots of standard solution of Tolvaptan were taken in different 10 ml volumetric flasks and diluted up to the mark with diluent such that the final concentrations of Tolvaptan were in the range of 30 to 180 µg/ml. The drug was eluted with UV detector at 254 nm, peak area was recorded for all the peaks.

Limit of detection and limit of quantification:

The limit of detection (LOD) and limit of quantification (LOQ) of the developed method were determined by injecting progressively low concentrations of the standard solution using the developed HPLC method.

Precision:

The precision was determined for Tolvaptan in terms of method precision and intermediate precision.

Accuracy:

The accuracy of the method was assessed by recovery study of Tolvaptan in the dosage form at three concentration levels. A fixed amount of pre-analyzed sample was taken and standard drug was added at 50%, 100% and 150% levels. Each level was repeated three times. The content of Tolvaptan was calculated.

System suitability:

System suitability parameters like retention time, theoretical plates and tailing factor were calculated and compared with standard values.

Ruggedness and robustness:

The ruggedness of the method was determined by carrying out the experiment on different instruments by different operators using different columns of similar types. The robustness of the method was determined by making slight changes in the chromatographic conditions such as flow rate and percent of composition of the mobile phase on the quantification of the drug substance and selectivity was studied.

Assay:

10 µl of sample solution was injected and from the peak area of Tolvaptan, amount of each drug in the sample were computed. The results were compared with the label claim of Tolvaptan.

Degradation studies:

Acid degradation studies:

To 1 ml of stock solution of Tolvaptan, 1 ml of 2N hydrochloric acid was added and refluxed for 30 minutes at 60⁰C. The resultant solution was diluted to obtain 120 µg/ml solution and 10 µl solution were injected into the system and the chromatograms were recorded to assess the stability of sample.

Alkali degradation studies:

To 1 ml of stock solution of Tolvaptan, 1 ml of 2N sodium hydroxide was added and refluxed for 30 minutes at 60⁰C. The resultant solution was diluted to obtain 120 µg/ml solution and 10 µl solution were injected into the system and the chromatograms were recorded to assess the stability of sample.

Oxidative degradation studies:

To 1 ml of stock solution of Tolvaptan, 1 ml of 20% hydrogen peroxide (H₂O₂) was added and refluxed for 30 minutes at 60⁰C. The resultant solution was diluted to obtain 120 µg/ml solution and 10 µl solution were injected into the system and the chromatograms were recorded to assess the stability of sample.

Thermal degradation studies:

The standard drug Tolvaptan solution was placed in an oven at 105⁰C for 6 hours. The resultant solution was diluted to obtain 120 µg/ml solution and 10 µl solution were injected into the system and the chromatograms were recorded to assess the stability of sample.

Photostability degradation studies:

The standard drug Tolvaptan solution was exposed to UV light by keeping the beaker in UV chamber for 7 days or 200 Watt hours/m²in photo stability chamber. The resultant solution was diluted to obtain 120 µg/ml solution and 10 µl solution were injected into the system and the chromatograms were recorded to assess the stability of sample.

Neutral degradation studies:

To 1 ml of stock solution of Tolvaptan, 1 ml of water was added and refluxed for 6 hours at 60⁰C. The resultant solution was diluted to obtain 120 µg/ml solution and 10 µl solution were injected into the system and the chromatograms were recorded to assess the stability of sample.

The percent of drug degraded in the presence of acidic, alkali, oxidative, thermal, photostability and neutral conditions were studied. The amount of drug recovered or degraded is calculated by comparing the area of the standard with that of the area of the degraded sample.

RESULTS AND DISCUSSION:

The stability-indicating RP-HPLC procedure was optimized with a view to develop an accurate method in tablet dosage form using Hypersil BDS C18 column (100 x 4.6 mm, 5 μm) in isocratic mode with mobile phase composition of 0.02M phosphate buffer pH 3.1 adjusted with orthophosphoric acid and acetonitrile in the ratio of 35:65 V/V. The use of phosphate buffer and acetonitrile in the ratio of 35:65 V/V resulted in peak with good shape and resolution. The flow rate was 1.0 ml/min and the drug component was measured with UV detector at 254 nm. The results of optimized chromatographic conditions were shown in Table 1. The quantification was linear in the concentration range of 30 to 180 µg/ml for Tolvaptan with a correlation coefficient of 0.999. The regression equation of the linearity plot of concentration of Tolvaptan over its peak area was found to be y=15781x+12246, where x is the concentration of Tolvaptan (μg/ml) and y is the corresponding peak area. The results show that an excellent correlation exists between peak area and concentration of drug within the concentration range indicated. The linearity results were shown in Table 2 and the linearity curve was shown in Fig. 2.

Table 1. Optimized chromatographic conditions of Tolvaptan

Parameter	Condition
Mobile phase	Phosphate buffer:acetonitrile (35:65 V/V)
pH	3.1
Diluent	Methanol
Column	Hypersil BDS C18 column (100 x 4.6 mm, 5 µm)
Column temperature	30⁰C
Wave length	254 nm
Injection volume	10 µl
Flow rate	1.0 ml/min
Run time	7 min.
Retention time	3.460 min.

Table 2. Linearity results of Tolvaptan

Concentration (μg/ml)	Mean peak area
30	499314
60	969173
90	1413275
120	1914031
150	2385600
180	2846076

Fig. 2: Linearity curve of Tolvaptan

The limit of detection and limit of quantification for Tolvaptan were found to be 0.312 µg/ml and 0.946 µg/ml respectively, which indicate the sensitivity of the method. The % RSD for method precision and intermediate precision for Tolvaptan were found to be 0.39% and 0.76% respectively (limit %RSD<2.0%) and hence the method is precise. The precision data of Tolvaptan were furnished in Table 3. The mean recovery of the drug Tolvaptan was 99.87% and the high percentage of recovery of Tolvaptan indicates that the proposed method is highly accurate. The results of recovery studies of Tolvaptan were shown in Table 4. The retention time for the drug Tolvaptan was 3.460 minutes. The number of theoretical plates calculated was 4820, tailing factor was 1.23, which indicates efficient performance of the column and the summary of system suitability parameters and validation parameters were shown in Table 5. Typical chromatograms for the drug Tolvaptan in standard and in sample were given in Fig. 3 and Fig. 4. The robustness studies indicated that no considerable effect on the determination of the drug. Therefore the test method is robust for the quantification of the drug.

Table 3. Precision data of the proposed HPLC method:

Concentration of Tolvaptan (120 μg/ml)	Peak area
Concentration of Tolvaptan (120 μg/ml)	Method precision	Intermediate precision
Injection-1	1778648	1791950
Injection-2	1778260	1757055
Injection-3	1778648	1785107
Injection-4	1765430	1794419
Injection-5	1765432	1783664
Injection-6	1766472	1789017
Mean	1772148	1783535
Standard deviation	6990.13	13586.78
% RSD	0.39%	0.76%

Validated method was applied for the determination of Tolvaptan in commercial formulations. The %assay of Tolvaptan was found to be 99.83%. No interfering peaks were found in the chromatogram of the formulation within the run time indicating that excipients used in tablet formulation did not interfere with the estimation of the drug Tolvaptan by the proposed HPLC method. The assay results are shown in Table 6. A study on degradation of the drug Tolvaptan in various stress conditions was conducted and the degradation of the drug was found to be negligible. Typical chromatograms for Tolvaptan in various degradation conditions were shown from Fig. 5 to Fig. 10. The degradation studies results are furnished in Table 7.

Fig. 3: Typical chromatogram of Tolvaptan standard

Fig. 4: Typical chromatogram of Tolvaptan sample

Table 4. Recovery studies of Tolvaptan

Fig. 10: Chromatogram of Tolvaptan showing degraded peaks under neutral conditions

CONCLUSION:

A simple, selective and sensitive stability-indicating RP-HPLC method with UV detection for Tolvaptan was developed and validated. The proposed study showed acceptable accuracy, precision and wide linear concentration range. The results of analysis proved that the method is suitable for the determination of Tolvaptan in bulk and tablet dosage forms without any interference from the degradation products and it is recommended for routine quality control analysis of the Tolvaptan in pharmaceutical dosage forms.

REFERENCES:

1. O.J.M. Neil. The Merck Index, An Encyclopedia of Chemicals Drug and Biologicals, 14^th Ed., Merck Research Laboratories, Division of Merck and Co. Inc., White House Station, NJ. 2006, 1639.

2. Indian Pharmacopoeia, Volume III, Government of India, Ministry of Health and Family Welfare, The Indian Pharmacopoeia Commission, Ghaziabad. 2014, 2892.

3. A. Cardenas, P. Gines, P. Marotta, F. Czerwiec, J. Oyuang, M. Guevara and N.H. Afdhal. Tolvaptan, an oral vasopressin antagonist, in the treatment of hyponatremia in cirrhosis. Journal of Hepatology, 2012, 56(3), 571-578.

4. B. Friedman and J. Cirulli. Hyponatremia in critical care patients: frequency, outcome, characteristics and treatment with the vasopressin V2-receptor antagonist Tolvaptan. Journal of Critical Care, 2013, 28(2), 219.

5. Y. Joko, N. Ikemura, K. Miyata, Y. Shiraishi, H. Tanaka, T. Yoshida, Y. Ikegami, J. Fuse, M. Sakamoto and Y. Momiyama. Efficacy of Tolvaptan in a patient with right-sided heart failure and renal dysfunction refractory to diuretic therapy. Journal of Cardiology Cases, 2014, 9(6), 226-229.

6. B.G. Chaudhari and C. Patel. Development and validation of UV spectrophotometric method for the estimation of Tolvaptan in bulk and tablet dosage form. International Journal for Pharmaceutical Research Scholars, 2012, 1(3), 193-198.

7. S. Murugan, N. Pavan Kumar, C. Kiran Kumar, V. Syam Sundhar, S. Harika and P. Anusha. Method development and validation for dissolution method of Tolvaptan in bulk and tablet dosage form by UV spectrophotometry. Indian Journal of Pharmaceutical Science and Research, 2013, 3(1), 17-19.

8. V. Kalyana Chakravarthy and D. Gowri Shankar. Development and validation of RP-HPLC method for estimation of Tolvaptan in bulk and its pharmaceutical formulation. Rasayan Journal of Chemistry, 2011, 4(1), 165-171.

9. L.A. Rama Prasad, J.V.L.N.S. Rao, P. Srinivasu, J. Vara Prasad and D. Naga Raju. Impurity profiling of Tolvaptan tablets using new stability indicating UPLC method. International Research Journal of Pharmacy, 2012, 3(11), 145-149.

10. Q. Pei, B. Zhang, H. Tan, L. Liu, X. Peng, Z. Li, P. Huang, M. Luo, X. Zuo, C. Guo and G. Yang. Development and validation of an LC-MS/MS method for the determination of Tolvaptan in human plasma and its application to a pharmacokinetic study. Journal of Chromatography B, 2013, 913-914, 84-89.

11. ICH Harmonised Tripartite Guideline, Validation of analytical procedures: Text and methodology, Q2(R1), International Conference on Harmonization. 2005, 1-13.

12. ICH Harmonised Tripartite Guideline, Stability Testing of New Drug Substances and Products, Q1A(R2), International Conference on Harmonization. 2003, 1-18..

Received on 18.06.2014 Modified on 25.06.2014

Asian J. Research Chem. 7(7): July 2014; Page 628-633

% Concentration level	Standard conc. (μg/ml)	Conc. added (μg/ml)	Conc. found (μg/ml)	% Recovery	% Mean recovery
50 %	150	60	60.06	100.1%	99.87%
100%	150	150	149.59	99.72%
150%	150	180	179.64	99.80%

System suitability	Results
Linearity range (μg/ml)	30-180
Correlation coefficient	0.999
Theoretical plates (N)	4820
Tailing factor	1.23
LOD (μg/ml)	0.312
LOQ (μg/ml)	0.946

Formulation	Label claim	Amount found	%Assay
TOLVASCA	30 mg	29.95 mg	99.83%

Degradation parameter	Peak area of sample	Peak area of standard	% Recovery	% Degradation
Standard	--	1759880	100	--
Acidic degradation	1666006	1759880	94.66	5.33
Alkali degradation	1676124	1759880	95.24	4.75
Oxidative degradation	1686360	1759880	95.82	4.17
Thermal degradation	1720609	1759880	97.76	2.23
Photostability degradation	1742681	1759880	99.02	0.97
Neutral degradation	1739552	1759880	98.84	1.15