Rapid RP-HPLC Method Development and Validation of Tolvaptan in Bulk and Pharmaceutical Dosage Form for an Internal Standard

 

K. Vijaya Sri*, S. Sruthi, M.A. Madhuri

Department of Pharmaceutical Analysis, Malla Reddy College of Pharmacy, (Affiliated to Osmania University) Maisammaguda, Secunderabad-500 014,

*Corresponding Author E-mail: vijayasree_2002@yahoo.co.in

ABSTRACT:

A simple, fast, precise and accurate RP-HPLC by internal standard method were developed and validated for the estimation of Tolvaptan as per ICH guidelines. Methanol: 0.2M phosphate buffer (70:30v/v) was used as the solvent. Sumatriptan was selected as an internal standard. The method was developed in Eclipse C₁₈ column (100 mm × 4.6 mm, 3.5 μm particle size). The method was found to be linear in the range of 5-100µg/ml with a correlation coefficient value of 0.999. The accuracy studies of RP-HPLC by internal standard method was performed at three different levels, i.e., 50%, 100%, and 150% and recovery was found to be in the range 99.01 to 100.7%. The % RSD is <2% which indicates the accuracy and precision of the method. The above method was a rapid tool for routine analysis of Tolvaptan in the bulk and in the pharmaceutical dosage form.

 

 

 

INTRODUCTION:

Tolvaptan is chemically 7 – chloro – 5 –hydroxy- 1- [2 – methyl – 4 – (2 – methyl benzoylamino) – 2, 3, 4, 5 – tetrahydro – 1H – 1 – benzazepine (Fig.1). It is a selective vasopressin V₂ receptor antagonist.^{1, 2} Tolvaptan antagonize the effect of vasopressin and causes an increase in urine water excretion that results in an increase in free water clearance, a decrease in urine osmolarity and a resulting increase in serum sodium concentrations. It is a diuretic agent. It is used to treat hyponatraemia associated with congestive cardiac failure. Literature review reveals that a few UV method, ^3-5 RP-HPLC method, ^6-8 UPLC⁹ and LC-MS/MS¹⁰ for its estimation in bulk and pharmaceutical dosage form but there is no reported method for the estimation of Tolvaptan by RP-HPLC method by using internal standard.

 

An attempt was made to develop a simple, accurate, precise and rapid RP-HPLC method for the estimation of tolvaptan in bulk and pharmaceutical dosage form. The method was validated as per International Conference on Harmonization (ICH) guidelines.¹¹ The present work describes the development and validation of RP-HPLC by using Sumatriptan as an internal standard method, which can quantify the tolvaptan.

 

Fig.1: Structure of Tolvaptan

 

MATERIALS AND METHODS:

Materials:

Tolvaptan was obtained as a gift sample from Hetero drugs Ltd. (Hyderabad, Telangana). Methanol, water (HPLC grade), Potassium dihydrogen ortho phosphate(GR grade) and sodium hydroxide (GR grade)  were used which were obtained from SD Fine chemicals.

 

Instrumentation and chromatographic conditions:

Reverse phase – high performance liquid chromatography (Agilent) is connected to computer loaded with Ezchrome software was used. UV detector and Eclipse C₁₈ (100 mm × 4.6 mm, 3.5 μm) was used. The flow rate was maintained at 1ml/min and the analysis was carried out at 267nm. The injection volume was 20µl and the run time was 5 min.

 

Preparation of stock solutions of Tolvaptan and Sumatriptan:

Standard Tolvaptan of 100mg was accurately weighed and simultaneously standard Sumatriptan drug of 100mg was also weighed and transferred into two separate 100ml volumetric flasks. About 40ml of methanol : 0.2 M phosphate Buffer (70:30 v/v) is added into both the flasks and are subjected to sonication for 20min and then the volume in both the flasks are made up to the mark with the mobile phase to give a concentration of 1000μg/ml. From Tolvaptan stock solution 1ml was pipette out and transferred into a 10ml volumetric flask to get a concentration of 100μg/ml. Similarly 1ml of Sumatriptan stock solution was pipette out and transferred into a 10ml volumetric flask to get a concentration of 100μg/ml. Now appropriate dilutions were made to get concentrations of 10μg/ml of Tolvaptan and 10μg/ml of Sumatriptan and injected.

 

Fig.2a Calibration curve for Tolvaptan                                      Fig.2b Chromatogram of Tolvaptan

 

System suitability testing (SST):

System suitability tests are an integral part of chromatographic method. System suitability standard solution which contained 10µg/ml of Tolvaptan and 10µg/ml of Sumatriptan was prepared by appropriately diluting and mixing the corresponding stock standard solutions. System suitability was determined from six replicate injections of the system suitability standard before sample analysis. Parameters such as a number of theoretical plates (N), tailing factor and retention time, resolution were calculated.

 

VALIDATION OF THE CHROMATOGRAPHIC METHOD:

Linearity and range:

Appropriate aliquots from standard Tolvaptan stock solutions and standard Sumatriptan stock solution were transferred into different volumetric flasks of 10ml capacity and the volume is adjusted to the mark to obtain Tolvaptan concentrations of 5, 10, 20, 30, 40, 50 and 100 µg/ml while a standard concentration of 10µg/ml of Sumatriptan is maintained in each of them.

Precision:

Repeatability studies were carried out by taking 100% test concentration and repeating it six times. Interday and intraday precision were done by taking 100% test concentration and repeating it six times.

 

Accuracy:

Recovery studies were carried out at 50%, 100% and 150% by adding known amount of standard drug solution, i.e. (10, 20, 30 µg/ml) to the sample solution whose concentration is maintained constant, i.e. 20µg/ml.

 

Robustness:

Robustness was studied by varying the wavelength and flow rate. Variation of wavelength by 2nm (265nm,267nm and 269nm) and mobile phase flow rate by 0.2ml (0.8ml, 1ml and 1.2ml/min) were carried out and the effect on the retention time and chromatographic response of the 20 µg/ml solution were studied.

 

 

Ruggedness:

To determine ruggedness, two different analysts performed in similar operational and environmental conditions using developed method.

 

Limit of quantification (LOQ) and limit of detection (LOD):

The limit of quantification (LOQ) and limit of detection (LOD) were based on the residual standard deviation of the response and the slope of the constructed calibration curve (n=3), as described in International Conference on Harmonization guidelines Q2 (R1).

 

ASSAY FOR THE TABLETS:

Twenty tablets were weighed and powdered. The powder equivalent to 15mg was calculated and transferred into a 10ml volumetric flask and 4ml of methanol: 0.2 M phosphate buffer (70:30 v/v) is added and sonicated for 30min. The volume was shaken and made up to the mark with the mobile phase to obtain a solution of 1000µg/ml. From this appropriate dilutions were made. The solution was filtered through Whatmann filter paper (No. 41). Table.8

 

RESULTS AND DISCUSSION:

Several mobile phases were tried for the analysis. The mobile phase methanol: 0.2 M phosphate buffer (70:30 v/v) showed good separation and good peak symmetry. By the proposed method, the retention time of Tolvaptan and internal standard was found to be 3.68 and 1.3 minutes. The System suitability parameters are shown in table 1.

 

Specificity:

The analyte was assessed in the presence of components and it was found that there was no interaction with the analyte.

 

Linearity:

Linearity was obeyed in the concentration range of 5-100μg/mL and the correlation 0.9998. The regression equation of Tolvaptan concentration over its peak area ratio was found to be y =0.514+0.225, where y is the mean peak area and x is the concentration of tolvaptan (μg/ml). The calibration curve and chromatogram are shown in Fig 2a and 2b and Linearity data are shown in Table 2.  The linearity was found in the concentration range of 5-100 µg/ml for the developed chromatographic method. The values for repeatability, intraday and interday were found to be 1.39, 0.92 and 0.88. The results show that the %RSD value for repeatability intraday and interday were <2.0% which indicate that they meet the acceptance criteria and hence the method is said to be precise. The results are shown in Table 3 and 4.

 

Table.1 System suitability parameters

Parameter	Retention time of olvaptan	Retention time of internal standard (Sumatriptan)	Asymmetric factor	Number of Theoretical plates	HETP
Results	3.68 min	1.31 min	0.94	6522	0.015

 

 

Table.2 Linearity for Tolvaptan at 267nm

Concentration of Tolvaptan (μg/ml)	Concentration of Sumatriptan (μg/ml)	AUC of Tolvaptan	AUC of Sumatriptan	Ratio of AUC of  tolvaptan to Sumatriptan
5	10	3952190	1365533	2.894
10	10	6928143	1351774	5.125
20	10	16044852	1496255	10.723
30	10	20795687	1311517	15.856
40	10	29609538	1418129	20.879
50	10	36167088	1409646	25.656
100	10	70555755	1312959	51.737

 

Table.3 Repeatability studies of Tolvaptan

 

Table.4 Precision studies of Tolvaptan

 

 

 

 

 

Table.5 Accuracy studies of Tolvaptan

Initial Concentration (μg/mL)	Excess drug added (μg/ml)	Amount added (μg/ml)	Amount recovered (μg/ml)	% Recovery ± SD(n=3)	% RSD
20	10	30	30.21	100.7± 0.25	1.548
20	20	40	39.606	99.01± 0.29	1.406
20	30	50	49.812	99.6± 0.39	1.622

 

Table.6 Robustness studies for varied wavelengths

 

 

Table.8 Assay studies of Tolvaptan

 

 

Table.7. Ruggedness results for different analyst

 

 

Accuracy:

The %recovery was calculated and was found to be in the range of 99.01-100.7. The results for accuracy indicate that the %recovery values are within the range which indicates that the method is accurate as it meets the necessary criteria. The results are shown in Table5.

 

Robustness:

The %RSD values for varied wavelengths and varied flow rates were found to be 1.59 and 1.34. The results indicate that the %RSD values for different analysts and different instruments was found to be within the range and indicates that they meet the acceptance criteria. The results are shown in Table 6.

Ruggedness:

Using developed method two different analysts performed on 30µg/ml in similar operational and environmental conditions. The results obtained were not affected and were in accordance with the results in original conditions. This shows the method was rugged and results are shown in Table no.7.

 

Limit of Detection and Limit of Quantization:

The LOD and LOQ were found to be 1.42 and 4.30 µg/ml.

 

RESULT:

The results show that the %purity was found to be 99.8% which indicates that the value was within the specified range and hence meets the necessary criteria. Assay chromatogram and results are shown in Fig.3 and Table no.8.

 

 

Fig.3 Assay chromatogram of Tolvaptan

 

CONCLUSION:

The developed RP-HPLC method was found to be simple, accurate, precise, reproducible and highly sensitive and can be use for the routine estimation of tolvaptan in bulk and pharmaceutical formulations.

 

ACKNOWLEDGEMENTS:

The authors are grateful to Malla Reddy College of Pharmacy for providing necessary research facilities to carry out the research work and to Hetero drugs Hyderabad, India for providing the gift sample.

 

REFERENCES:

1.     Australian public assessment report for Tolvaptan : 2012

2.     USFDA : Drug safety communications : 2013

3.     Murugan S, Pavan Kumar N, Kiran Kumar C, Syam Sundar V, Harika S and Anusha P: Method development and validation for dissolution method of Tolvaptan in bulk and tablet dosage form by UV – spectrophotometer. Ind J Phar sci and Research. 2013, 3(1), 17-19.

4.     Chaudhari B.G. and Patel C.: Development and validation of UV spectroscopic method for the estimation of Tolvaptan in bulk and tablet dosage form. Int J Pharm Res Scholars. 2012, 1(3),41-45

5.     Vijaya Sri. K., Sruthi. S, and Suresh. K.: UV Spectrophotometric method for the estimation of tolvaptan in bulk and pharmaceutical formulations, Asian J Research Chem, 2014, 7(9), 773-776.

6.     Prathyusha. B, Shirisha. B, Sriram, N. Ramathilagam, N.: Analytical method development and validation of Tolvaptan in bulk and tablet dosage form by RP-HPLC, Int J Phar analytical chemistry, 2013, 2(4), 32-36.

7.     Kalyan Chakravarthy .V and Gowri Shankar .D: Development and validation of RP – HPLC method for estimation of Tolvaptan  in bulk and its pharmaceutical formulation, Rasayan J Chem, 2011, 4(1), 1165 – 171.

8.     Lakshmi Aswini, G., Dachinamoorthy. D, Seshagiri rao, J.V.L.N.: A New Stability Indicating RP-HPLC method development and validation for the estimation of Tolvaptan with forced degradation studies in bulk and tablet dosage form, Int J Pharm Res Scholars, 2015, 4, I-2, 250-257.

9.     Ramprasad, L.A, Seshagiri rao J.V.L.N, Srinivasu, P., Vara Prasad. J, Naga Raju. D Impurity profiling of Tolvaptan tablets using new stability indicating UPLC method, Int Res J  Pharmacy, 2012 , 3(11).21-25.

10.   Pei. Q, Bikui Zhang, Hongyi Tan, Lihua Liv, Mi Luo: Development and validation of an LC-MS/MS method for the determination of Tolvaptan in human plasma and its application to a pharmacokinetic study, J of Chromatography. B, 2013, (913-914), 84-89.

11.   International Conference on Harmonization (2005). Validation of analytical procedure text and methodology in ICH Harmonized Tripartite Guidelines Q2 (R1) November

 

 

 

 

 

Received on 11.01.2017       Accepted on 11.02.2017     

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