*Corresponding Author E-mail: raj_neetu78@rediffmail.com
ABSTRACT:
A simple, precise, rapid and accurate reverse phase HPLC method developed for the estimation of Vilazodone in Tablet dosage form by using a C8 Chromasil column (250 × 4.6 mm, 5μm particle size), with mobile phase containing mixture of phosphate buffer and methanol in ratio of 30:70, v/v with pH adusted to 5 with ortho-phosphoric acid was used. The flow rate was 1 ml/min and the effluents were monitored at 232 nm. The retention time was 6.008 min. The detector response was linear in the concentration of 4-20 mcg/ml. The respective linear regression equation being y = 66733x - 4974. The limit of detection and limit of quantification was 0.418and 1.267mcg/ml respectively. The percentage assay of Vilazodone was 100.15 %. The method was validated by determining its system suitability, precision, accuracy, and robustness parameters.. The results of the study showed that the proposed RP-HPLC method is simple, rapid, precise and accurate; this is useful for the routine determination of Vilazodone in bulk drug and in its pharmaceutical dosage form.
KEYWORDS:Vilazodone, Calibration curve, RP-HPLC, Estimation, and Tablets.
INTRODUCTION:
Vilazodone is the first in a new class of antidepressants that combine the effect of a serotonin selective reuptake inhibitors (SSRI) with 5-HT1A receptor partial agonist activity1. It was approved on January 21, 2011, by the Food and Drug Administration (FDA) for the treatment of major depressive disorder (MDD) in adult patients in the US. Vilazodone acts similarly to SSRIs in that it inhibits the serotonin transporter, desensitizes serotonin receptors, and consequently enhances serotonergic neurotransmission2. The drug is classified as a Selective Serotonin Reuptake Inhibitor (SSRI) and is manufactured by Forest Laboratories, Inc. in 10, 20, and 40 mg tablets. Vilazodone also has an inherent selectivity for serotonin-1A receptors, acting as a partial agonist which stimulates the production of serotonin3.
Chemically Vilazodone HCl (Figure 1) is 2-benzofurancarboxamide, 5-[4-[4-(5-cyano-1H-indol-3-yl) butyl]-1-piperazinyl]-, hydrochloride (1:1) with chemical formula C26H27N5O2. HCl. Vilazodone HCl is a white to cream coloured a chiral powder with molecular mass: 477.9 g/mol. The pKa of the drug is 7.1; the aqueous solubility is 0.32 mg/ml and it is freely soluble in methanol4,5.
Figure 1: Structure of Vilazodone HCl
Vilazodone HCl is a dual-acting and selective serotonin reuptake inhibitor and 5-HT1A receptor partial agonist. It is thought to optimise regulation of 5-HT circuitry at both pre-and postsynaptic sites to augment 5-HT neurotransmission, thereby producing an antidepressant effect6. Its clinical indication is for the treatment of the major depressive disorder. Vilazodone HCl is a new chemical entity belonging to the structural chemical group of the indol alkylamines 7. Vilazodone showed dose proportional pharmacokinetics in the dosage range of 5–80 mg after single and multiple administrations8, 9. Peak plasma concentrations were usually reached at 4 hours post-dose. Vilazodone is extensively metabolized by cytochrome P450 (CYP), principally by CYP3A4. Only 3% of the dose excreted from urine and feces as unchanged drug10,11.
In the present study, we developed and validated a rapid and sensitive HPLC method to determine vilazodone HCl in pharmaceutical formulation in a run-time of 6.008 min. The short run time enables efficient analysis of large number of formulation samples obtained for regular qualitative control analysis. To the best of our knowledge, this is the first report of a fully validated HPLC method for the quantification of vilazodone HCl in formulations. This method was successfully applied to the estimation studies of vilazodone HCl in marketed products. Existing literature reveals that there are only few methods for the assay of vilazodone in bulk and dosage forms12-14. Hence an attempt has been made to develop new simple, reliable, and reproducible, isocratic RP-HPLC methods to estimate the vilazodone in bulk and pharmaceutical formulation with good precision, accuracy, linearity and reproducibility respectively. Hence in present work, we developed a simple, rapid and inexpensive liquid chromatographic method for the analysis of vilazodone HCl in pure and pharmaceutical dosage form. The proposed HPLC method is validated using standard ICH guidelines15.
EXPERIMENTAL:
MATERIALS AND METHODS
The pure drug, Vilazodone HCl was procured from Jubilant Life Sciences, Ghaziabad, U.P., India. Marketed formulation Vilano 20 mg was procured from a local pharmacy. All the chemicals were used of analytical grade. HPLC grade methanol, disodium hydrogen phosphate, ortho-phosphoric acid and distilled water were procured from Merck Pharmaceutical Private Ltd., Mumbai, India used. Membrane filters 0.45 μm and 0.2 μm were procured from Millipore Pvt. Ltd. Bangalore, India.
Instrumentation
LC system used consist of pump (Model Shimadzu; LC-10 AT VP) with universal loop injector (Rheodyne 7725 i) of injection capacity 20 μl. Detector consists of photodiode array detector SPD-10 AVP. C8 Chromasil column (250 × 4.6 mm, 5μm particle size) was used. The HPLC system was equipped with LC solution software.
Preparation of the mobile phase:
The mobile phase was prepared by mixing of 0.02M disodium hydrogen phosphate buffer and methanol in ratio of 30:70, v/v. the pH of buffer was adjusted at 5 with ortho-phosphoric acid. It was filtered through 0.2 μm membrane filter and then sonicated for degassing. It was pumped from the respective solvent reservoirs to the column at a flow rate of 1.0 ml/min. Prior to the injection of the drug solution, the column was equilibrated for at least 30 min with the mobile phase flowing through the system. The eluent was monitored at 232 nm.
Preparation of stock and working standard solutions
Accurately weighed 20 mg pure drug of VLZ HCl was transferred in a 50 mL clean, dry volumetric flask and methanol was added and sonicated to dissolve. The volume was made up to the mark with methanol to prepare 400 μg/mL stock solution. 5mL of this solution was transferred into 50mL volumetric flask and volume was made up to the mark with mobile phase to prepare 40 μg/mL working standard solution. It was further diluted with mobile phase to prepare required concentration.
Preparation of Sample solution:
For the estimation of VLZ HCl in the tablet formulation, 20 tablets (label claim 20 mg, Sun Pharma) were accurately weighed and the average weight per tablet was calculated. The tablets were crushed and finely powdered in glass mortar. Powder equivalent to 20 mg of VLZ HCl was accurately weighed and transferred into a 50 mL volumetric flask and sonicated to dissolve. The volume was made up to the mark with methanol, mixed well to prepare 400 μg/mL stock solution. The solution was filtered using 0.2 μm membrane filter and degassed by sonication. 5mL of this solution was transferred into 50 mL volumetric flask and volume was made up to the mark with mobile phase to prepare 40 μg/mL test solution A. 3 ml of test solution A was transferred in 10 mL volumetric flask and volume was made up to the mark with mobile phase to prepare 12 μg/mL test solution. The resulting solution was used as the sample solution for chromatographic analysis. For accuracy study 2 ml of test solution A was transferred in 10 mL volumetric flask and volume was made up to the mark with mobile phase to prepare 8 μg/mL test solution. After setting the chromatographic conditions and stabilizing the instrument to obtain a steady baseline, the sample solution was loaded in the 20µl sample loop of the injection port. The solution was injected three times and the peak areas were recorded.
Chromatographic Conditions
The chromatographic separation was performed using a C8 Chromasil column (250 × 4.6 mm, 5μm particle size). The mobile phase consists of a mixture of phosphate buffer (pH 5) and methanol in ratio of 30:70, v/v. The mobile phase was set at a flow rate of 1 mL/min and the analytes were monitored at 232 nm. The column was maintained at 25°C temperature and injected volume was 20 μl. The total run time was 10 min. The mobile phase was filtered through 0.2 μm membrane filter prior to use. A typical chromatogram of VLZ HCl is shown in (Figure 2).
Figure 2: Chromatogram of standard solution of Vilazodone HCl
Method Validation
The method was validated according to ICH guidelinesto study system suitability, linearity, precision, accuracy, robustness, limit of detection (LOD) and limit of quantitation (LOQ).
System suitability
System suitability test parameters for VLZ HCl for the developed method are reported in (Table 1). % RSD for tailing factor, theoretical plate count, peak area and retention time for VLZ HCl were found to be within the limit of 2%, which indicates suitability of the system. The number of theoretical plates and tailing factor were found within the acceptance criteria of >2000 and ≤2.0, respectively, indicating good column efficiency and optimum mobile phase composition.
Table 1: Results from system-suitability study of Vilazodone HCl
|
Parameter |
VLZHCl (12µg/mL) |
|
|
Mean (n = 5) |
%RSD |
|
|
Retention time (tR) min |
6.012 |
0.152 |
|
Peak area (A) |
795923.2 |
0.025 |
|
Tailing factor (T) |
1.301 |
1.161 |
|
No. of theoretical plates (N) |
3661.33 |
1.824 |
Linearity
Linearity of this method was evaluated by linear regression analysis and calculated by least square method and studied by preparing standard solutions of VLZ HCl at different concentration levels. Peak area of resulting solutions was measured and the calibration curve was plotted between peak area and concentration of the drug (Figure 3). The response was found to be linear in the range 4-20 μg/ml for VLZ HCl. The data was given in (Table 2, 3).
Figure 3: Calibration curve for standard solution of Vilazodone HCl
Table 2: Linearity data of Vilazodone HCl
|
S.No. |
Conc. (µg/ml) |
* Peak Area |
|
1. |
4 |
267791 |
|
2. |
8 |
524480 |
|
3. |
12 |
795518 |
|
4. |
16 |
1053228 |
|
5. |
20 |
1338071 |
*Peak area mean of three replicates
Table 3: Results of regression analysis of Vilazodone HCl
Parameter |
VLZ HCl |
Linearity range (µg/ml) |
4-20 |
Regression equation (y = mx+c) |
y = 66733x - 4974 |
Slope (m) |
66733 |
Intercept (c) |
4974 |
Correlation coefficient (R˛) |
0.999 |
Limit of detection (μg/ml) |
0.418 |
Limit of quantitation (μg/ml) |
1.267 |
Accuracy /Recovery
Accuracy is the degree of agreement between a measured value and the accepted reference value. The accuracy of the method was tested by triplicate samples at 3 different concentrations equivalent to 80%, 100% and 120% of the active ingredient, by adding a known amount of VLZ HCl standard to a fixed amount of the pre-analysed sample of VLZ HCl. The recovered amount of VLZ HCl, % recovery and %RSD of each level is calculated to determine the accuracy. The data was given in (Table 4).
Table 4: Accuracy study for Vilazodone HCl
|
Accuracy Level (%) |
Amount Added (μg/ml) |
Amount Recovered (μg/ml) |
%Recovery
|
Mean
|
SD |
%RSD
|
|
80 |
6.4 |
6.45 |
100.79 |
100.34 |
0.45 |
0.45 |
|
6.4 |
6.42 |
100.34 |
||||
|
6.4 |
6.39 |
99.89 |
||||
|
100 |
8 |
8.03 |
100.38 |
100.48 |
0.39 |
0.39 |
|
8 |
8.01 |
100.15 |
||||
|
8 |
8.07 |
100.91 |
||||
|
120 |
9.6 |
9.62 |
100.18 |
100.45 |
0.30 |
0.30 |
|
9.6 |
9.64 |
100.39 |
||||
|
9.6 |
9.67 |
100.78 |
Precision
Repeatability
Five sample solutions of the same concentration were prepared and injected into the HPLC system as per test procedure. The results were given in (table 5).
|
S.No. |
VLZ HCl |
|
|
Conc. (µg/ml) |
Peak Area |
|
|
1. |
12.0 |
795754 |
|
2. |
795895 |
|
|
3. |
795747 |
|
|
4. |
794986 |
|
|
5. |
796234 |
|
|
|
|
|
|
Avg |
795923.2 |
|
|
SD |
200.63 |
|
|
% RSD |
0.025 |
|
Precision (Day to Day variability)
Intra-day precision was investigated by replicate applications and measurements of peak area for VLZ HCl for three times on the same day under similar conditions. Inter-day precision was obtained from % RSD values obtained by repeating the assay three times on different days. The percent relative standard deviation (% RSD) was calculated. The results were given in (Table 6).
Table 6: Precision study for Vilazodone HCl by HPLC
|
Conc. (μg/mL) |
Intra-day (n=3) |
Inter-day (n=3) |
||
|
Mean ± SD |
%RSD |
Mean ± SD |
%RSD |
|
|
8 |
524405±723 |
0.13 |
524505±910 |
0.17 |
|
12 |
796055±992 |
0.12 |
796389±1075 |
0.13 |
|
16 |
1055459±967 |
0.09 |
1057321±1192 |
0.11 |
Limit of detection and Limit of Quantification
LOD and LOQ were calculated from the average slope and standard deviation from the calibration curve as per ICH guidelines.
Robustness:
Robustness of an analytical method is a measure of its capacity to remain unaffected by small but deliberate variations in method parameters and provide an indication of its reliability during normal usage. Robustness can be determined by analysis of solution by changing physical parameters like composition of mobile phase and flow rate, In order to measure the extent of method robustness, the parameters were interchanged while keeping the other parameters unchanged and in parallel, the chromatographic profile was observed and recorded. The results by small variations in these parameters as shown in (Table 7).
Table 7: Robustness study for Vilazodone HCl by HPLC
|
S. No |
Parameter |
Optimized values |
Robust conditions |
Retention time (tR), min |
Plate count(N) |
Tailing factor (T) |
|
1 |
Flow rate |
1.0 mL/min |
1.1 mL/min 0.9 mL/min |
4.514 5.366 |
5852 6535 |
1.382 1.389 |
|
2 |
Mobile phase composition (phosphate buffer : methanol) |
250:750 |
225:775 275:725 |
4.782 5.052 |
6175 6309 |
1.383 1.386 |
Acceptance criteria: Tailing Factor (T) < 2.0, Plate count (N) > 2000, No significant change in Retention time (tR).
Assay
The developed method was applied to the assay of Vilazodone HCltablets. From the peak areas the amount of drug present in tablet was estimated. The drug content was calculated as an average of three determinations and assay results were shown in (Table 8). The results were very close to the labeled value of commercial tablets. The representative chromatogram of Vilazodone HClis shown in (Figure 4).
Table 8: Results of analysis of Tablet formulation of Vilazodone HCl
|
Formulation name |
Label Claim (mg) |
Amount found (mg) |
% Label claim |
|
Vilano |
20 |
20.093 |
100.46 |
|
20 |
20.021 |
100.20 |
|
|
20 |
19.976 |
99.88 |
|
|
Mean |
20.030 |
100.15 |
|
|
SD |
0.058 |
0.293 |
|
|
%RSD |
0.293 |
0.293 |
|
Figure 4: Chromatogram of Vilazodone HCl in tablet formulation
RESULTS AND DISCUSSION:
A reverse-phase HPLC method was proposed as a suitable method for the determination of Vilazodone HCl in tablet dosage form. The chromatographic conditions were optimized by changing the mobile phase composition. Different ratios were experimented to optimize the mobile phase. Finally, mixture of phosphate buffer and methanol in ratio of 30:70, v/v was used as mobile phase, which showed good resolution of Vilazodone HCl peak. The wavelength of detection selected was 232 nm, as the drug showed sharp and better peak shape at this wavelength. By developed method the retention time of Vilazodone HCl was about 6.008 min. In the present developed HPLC method, the standard and sample preparation required less time and no tedious extraction were involved. A good linear relationship (r2=0.999) was observed between the concentration range of 4-20 μg/mL. The assay of Vilazodone HCl in tablet dosage forms was found to be 100.15%. The statistical analysis of data and the drug recovery data showed that the method was simple, rapid, economical, sensitive, precise and accurate. It can thereby easily adopt for routine quality control analysis. The results of this analysis confirmed that the developed method was suitable for determination of drug in pharmaceutical formulation has no interference of additives. Hence the developed method can be applied for estimation of Vilazodone HCl in marketed formulation.
CONCLUSION:
The developed method is rapid, accurate and sensitive. It makes use of fewer amounts of solvents and change of set of conditions requires a short time. This method can be suitably analyzed for the routine analysis of Vilazodone HCl in tablet dosage form. It does not suffer from any interference due to common excipients present in pharmaceutical preparation and can be conveniently adopted for quality control analysis. The above proposed method obviates the need for any preliminary treatment and is the method could be of use for process development as well as quality assurance of Vilazodone HCl.
ACKNOWLEDGEMENTS:
The authors express their sincere gratitude to Jubilant Life Sciences, Ghaziabad, U.P., India for providing the pure drug sample of Vilazodone HCl and are also thankful to colleagues and authorities of Pacific University, Udaipur who helped us in this work.
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Received on 19.12.2017 Modified on 22.01.2018
Accepted on 26.02.2018 © AJRC All right reserved
Asian J. Research Chem. 2018; 11(2):427-431.
DOI:10.5958/0974-4150.2018.00078.0