UV Spectrophotometric Method for the Estimation of Tolvaptan in Bulk and Pharmaceutical Formulations
K. Vijaya Sri*, S. Sruthi and l.D. Srinivas
ABSTRACT:
The main objective was to develop and validate the UV-spectrophotometric method for the estimation of tolvaptan in bulk and pharmaceutical formulations as per ICH guidelines. The initial stock solution of Tolvaptan was prepared in acetonitrile. The λmax of tolvaptan was found to be 267 nm it was proved linearity in the concentration range 1–10 μg/ml with a correlation coefficient value of 0.999. The accuracy studies of proposed method was performed at three different levels, i.e., 50%, 100%, and 150% and recovery was found to be in the range of 100.4%.The limit of detection (LOD) and limit of quantification (LOQ) were found to be 0.34 and 0.94 µg/ml, respectively.The % RSD less than 2 which indicates the accuracy and precise of the method. The above method was a rapid tool for routine analysis of tolvaptan in the bulk and in the pharmaceutical dosage form.
KEYWORDS: Tolvaptan, UV Spectroscopy, Development, Validation and Pharmaceutical formulation.
Tolvaptan is chemically 4'-[7-chloro-2,3,4,5-tetrahydro-5-hydroxy-1H-1-benzazepin1-yl)carbonyl]-o-tolu-m-toluidide. It is a selective vasopressin v2 receptor antogonist1. When taken orally tolvaptan antogonise 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. 2
Figure No.1 Structure of Tolvaptan
It is a diuretic agent. It is used to treat hyponatraemia associated with congestive heart failure, cirrhosis and syndrome of inappropriate antidiuretic hormone(SIADH) it is soluble in methanol and benzyl alcohol and insoluble in water and hexane at a wide range of pH. 2
Literature review reveals that there is UV method, 3,4 few methods based on HPLC5,6, UPLC7 and LC-MS/MS8 for its estimation in bulk and dosage forms. The present work describes the development and validation of UV spectrophotometric method, which can quantify the rilpivirine hydrochloride. An attempt was made to develop a simple, accurate, precise and rapid spectrophotometric method for the estimation of Tolvaptan in bulk and pharmaceutical dosage form. The method was validated as per International conference on Harmonization (ICH) guidelines.9,10 The present work describes the development and validation of UV spectrophotometric method, which can quantify the Tolvaptan. An attempt was made to develop a simple, accurate, precise and rapid spectrophotometric method for the estimation of Tolvaptan in bulk and pharmaceutical dosage form. The method was validated as per International conference on Harmonization (ICH) guidelines.
MATERIALS AND METHODS:
Materials
Tolvaptan was obtained as gift sample from Hetreo Laboratories Ltd. (Hyderabad, A.P, India). Acetonitrile All chemicals used were of analytical grade and purchased from Qualigens Fine Chemicals, Mumbai, India.
Instruments
A double beam UV-VIS spectrophotometer (UV-1800, Shimadzu, Japan) connected to computer loaded with spectra manager software UV Probe was employed with spectral bandwidth of 1nm and wavelength accuracy of ± 0.3 nm with a pair of 10 mm matched quartz cells.
Preparation of Standard stock solution of Tolvaptan
An accurately weighed quantity of Tolvaptan 50mg was transferred to 50ml volumetric flask, dissolved in 20ml , the final volume was made with acetonitrile to obtain standard solution having concentration of 1000 μg/mL. 1ml of this solution was transferred to 10ml volumetric flask, volume was made with acetonitrile. It gives 100 µg/ml. These stock solutions were used to prepare further dilutions.
Selection of wavelength for analysis of Tolvaptan
Appropriate volume 0.1 ml of standard stock solution of tolvaptan was transferred into a 10 ml volumetric flask, diluted to a mark with acetonitrile to give concentration of 1 μg/ml. The resulting solution was scanned in the UV range (200–400 nm).
VALIDATION OF PROPOSED METHOD:
The method was validated according to ICH guidelines in order to determine the linearity, precision, accuracy and ruggedness of the method.
Linearity
Linearity was assessed by performing measurement at several analyte concentration varying quantities of stock solution was diluted with the acetonitrile to give 0. 25, 0.5, 1, 1.5, 2, 2.5 and 3 µg/ml of Tolvaptan. The calibration curve was obtained by plotting absorbance against concentration (μg/ml).
Repeatability
Repeatability was determined by preparing six replicates of 1 µg/ml of Tolvaptan and the absorbance was measured at 280nm.
Precision
Precision studies were carried out to ascertain the reproducibility of the proposed method. Intraday precision study was carried out by preparing drug solution of three different concentrations (0.5, 1 and 2 µg/ml of Tolvaptan) and analyzing it at three different times in a day. Interday precision study was carried out by preparing drug solution of three different concentrations (0.5, 1 and 2 µg/ml of Tolvaptan) and analyzing it at three different days.
Accuracy
Accuracy of the proposed method was determined using recovery studies. The recovery studies were carried out by adding different amounts (50%, 100%, and 150%) of the pure drug to the pre-analysed formulation. The solutions were prepared in triplicates and the % recovery was calculated.
Limit of Detection and Limit of Quantitation:
The parameters LOD and LOQ were determined on the basis of response and slope of the regression equation. The limit of detection (LOD) and the limit of quantitation (LOQ) of the drug were derived by calculating the signal-to-noise ratio (S/N, i.e., 3.3 for LOD and 10 for LOQ) using the following equations designated by International Conference on Harmonization (ICH) guidelines.
LOD = 3.3 × σ/S
LOQ = 10 × σ/S
Where σ = the standard deviation of the response and S = slope of the calibration curve
Ruggedness Studies
Ruggedness studies were performed by preparing three replicates of 1µg/ml of Tolvaptan, analysing by two different analyst and on two different instruments and the results are reported as %RSD.
Application of the proposed method for pharmaceutical formulation
The solution was filtered through Whatman filter paper No.41. 0.5 ml this solution was transferred to 10ml volumetric flask and final volume was made with acetonotrile. It gives 0.5µg/ml. It was scanned on a spectrophotometer in the UV range 200–400 nm. The spectrum was recorded at 267 nm against blank solution of acetonitrile. Determine the amount of % Tolvaptan in tablet according to the following formula.
AT x WS × Sample D.F x Average Weight
% Assay =--------------------------------------------------- x PR
AR x Standard D.F x WT× LA
Where,
WS = weight of standard;
WT = weight of sample
Where,
AT = Absorbance of Tolvaptan in the test solution,
AR= Absorbance of Tolvaptan in the standard solution, Std.
DF=Standard dilution factor, Sample D.F = Sample dilution factor PR = Purity of working standard [%], LA = Labeled amount of Tolvaptan
RESULTS AND DISCUSSION:
In spectrum Tolvaptan shows maximum absorbance at 267 nm shown in figure 2.
Figure No. 2. Absorption Spectrum of Tolvaptan
Method validation
The proposed method was validated as per ICH guidelines. The solutions of the drugs were prepared as per the earlier adopted procedure given in the experiment.
Linearity
Standard solutions of Tolvaptan in the concentration range of 0.25 to 3 µg/ml were observed in UV spectroscopy at different wave lengths.
A graph of absorbance (on Y-axis) versus concentration (on X-axis) was plotted and calibration graph was shown in Figure 2 and 3. The regression equation was found to be Y=0.148x+0.075 , Correlation coefficient was 0.999.
Repeatability
Repeatability was determined by analyzing 1 μg/ml concentration of tolvaptan for six times with % RSD < 2 which shown in table no 1.
Table No. 1 Repeatability studies of Tolvaptan
|
Concentration [µg/ml] |
Absorbance at 267 nm |
Absorbance Mean |
SD |
%RSD |
|
6 |
0.641 |
0.646 |
0.008 |
1.295 |
|
6 |
0.657 |
|||
|
6 |
0.650 |
|||
|
6 |
0.651 |
|||
|
6 |
0.636 |
|||
|
6 |
0.638 |
Precision
The precision of the developed method was expressed in terms of % relative standard deviation (% RSD). These results show reproducibility of the assay. The % RSD values found to be less than 2 that indicate this method precise for the determination of the pure form. The inter and intraday precision results were mentioned in Table no.2.
Figure No. 3 Calibration curve of Tolvaptan at 267nm
Table No.2 Intraday and Interday precision of Tolvaptan
|
Conc. (µg/ml) |
Intraday Precision |
Interday Precision |
||
|
Absorbance mean± S.D. (n=3) |
%RSD |
Absorbance mean± S.D. (n=3) |
%RSD |
|
|
2 |
0.197 ± 0.001 |
1.015
|
0.196 ± 0.001
|
0.510
|
|
6 |
0.626 ± 0.006 |
0.962
|
0.627 ± 0.002
|
0.421
|
|
10 |
1.032 ± 0.004 |
0.477
|
1.033 ± 0.003
|
0.339
|
Accuracy
Accuracy shall be determined by performing recovery studies at 3 levels in which known amount of analyte shall be added and recovery shall be carried out in three replicates of each concentration level and the % recovery was calculated. The mean recovery was found between 100-101 % and %RSD between 0.7-1.0 The results are shown in Table No. 3.
Table No. 3 Recovery studies of Tolvaptan
|
Spiked level (%) |
Formulation Conc (µg/ml) |
Pure Drug Conc (µg/ml) |
Amount Conc recovered (µg/ml) |
% Recovery |
% Mean recovery SD |
%RSD |
|
50 |
2 |
1 |
3.01 |
100.6 |
99.24±1.311 |
0.32 |
|
2 |
1 |
2.97 |
99.0 |
|||
|
2 |
1 |
2.94 |
98.0 |
|||
|
100 |
2 |
2 |
4.02 |
100.7 |
100.24±0.491 |
0.490 |
|
2 |
2 |
3.99 |
99.7 |
|||
|
2 |
2 |
4.00 |
100.2 |
|||
|
150
|
2 |
3 |
4.96 |
99.2 |
99.74±0.487 |
0.488 |
|
2 |
3 |
4.99 |
99.8 |
|||
|
2 |
3 |
5.00 |
100.19 |
Limit of Detection and Limit of Quantitation
The parameters LOD and LOQ were determined on the basis of response and slope of the regression equation. LOD and LOQ values are 0.34 and 0.94 µg/ml.
Ruggedness Studies
This study was performed by analyzing 4 µg/ml of Tolvaptan by two different analysts and on two instruments, results of the study were given in Table no. 4 and % RSD obtained was less than two which is within the acceptance limits.
Table No. 4 Ruggedness of Tolvaptan
|
Parameter |
Conc. (µg/ml) |
Absorbance |
Absorbance mean ± S.D. (n=3) |
%RSD |
|
Different Analyst |
4
|
0.405 |
0.407± 0.0025 |
0.61 |
|
0.41 |
||||
|
0.407 |
||||
|
Different instrument |
4 |
0.404 |
0.408± 0.0036 |
0.88 |
|
0.409 |
||||
|
0.411 |
The percentage recovery for Tolvaptan tablet formulation was found to be 99.6 -101.06 % enlisted in table no.5. The results for assay are within acceptable limit.
Table no. 5 Assay of Tolvaptan
|
Labelled amount (mg) |
Amount found (mg) |
% Purity |
Mean % purity ± SD(n=3) |
%RSD |
|
15 |
15.07 |
100.49 |
100.16±0.788 |
|
|
15 |
15.10 |
100.73 |
0.787 |
|
|
15 |
14.88 |
99.26 |
|
The summary of proposed method results are shown in table no 6.
Table no 6: Summary of Validated parameters
|
Parameters |
Method |
|
λmax (nm) |
267 |
|
Beers law limit (µg/ml) |
1-10 |
|
Correlation coefficient (r2) |
0.999 |
|
Molar absorptivity (L mol-1 cm-2) |
3.41×104 |
|
Regression equation (y=mx+c) |
Y=0.1036x+0.0006 |
|
Slope(m) |
0.1036 |
|
Intercept(c) |
0.0006 |
|
accuracy |
99.24-100.24 |
|
precision |
0.339-1.015 |
|
LOD (µg/ml) |
0.310 |
|
LOQ (µg/ml) |
0.941 |
CONCLUSION:
The developed UV Spectrophotometric method was found to be simple, economic, easy, accurate, precise, reproducible and highly sensitive and can be used for routine estimation of Tolvaptan in bulk and formulations.
ACKNOWLEDGEMENTS:
The authors are grateful to, Mallareddy College of Pharmacy for providing necessary research facilities to carry out the research work and to hetero drugs, India for providing the gift sample of the drug.
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Received on 29.08.2014 Modified on 09.09.2014
Accepted on 13.09.2014 © AJRC All right reserved
Asian J. Research Chem. 7(9): September 2014; Page 773-776