INTRODUCTION:

Vilazodone (‎C₂₆H₂₇N₅O₂) is a combined serotonin reuptake inhibitor and 5-HT1A receptor partial agonist and is used for the treatment of major depressive disorder^1,2,3. The two major human metabolites of vilazodone (M10 and M17) do not appear to have important serotonergic activity. Metabolite M10 is the product of vilazodone hydrolysis⁴. It has been shown to be equally efficacious as other antidepressants with similar gastrointestinal side effects and possibly with reduced sexual side effects and weight gain⁵. Vilazodone is an antidepressant agent⁶ that can use as an alternative for patients who cannot tolerate therapy with other antidepressant classes such as selective serotonin reuptake inhibitors⁷ or serotonin norepinephrine reuptake inhibitors. Vilazodone increases serotonin levels in the brain by inhibiting the reuptake of serotonin while acting as a partial agonist on serotonin-1A receptors⁸.

It has therefore been coined by scientists as a selective partial agonist and reuptake inhibitor (SPARI) ⁹. Because of its partial agonist activity for serotonin-1A, vilazodone helps to reduce anxiety¹⁰. The IUPAC¹¹ name of vilazodone is 5-(4-[4-(5-Cyano-1H-indol-3-yl) butyl] piperazin-1-yl) benzofuran-2-carboxamide

Fig-1: Structure of Vilazodone

A survey of literature^12-18 reveals that good analytical methods are not available for vilazodone. The present research manuscript describes innovative, simple, economical, accurate, specific, robust, rugged and rapid RP-HPLC method developed^19-23 in selected solvent system (Mobile Phase) and validated in accordance with International Conference on Harmonization (ICH) Guidelines Q2 (R1)^24,25, for the estimation of vilazodone in bulk drug and in its dosage forms.

MATERIALS AND METHODS:

Pharmaceutical grade working standard vilazodone were obtained from Syncorp Pvt. Laboratories, Hyderabad, India. All chemicals and reagents were HPLC grade and were purchased from S D Fine-Chem Limited and Loba Chemie Pvt Ltd, Mumbai, India^26,27.

Instrumentation:

The analysis was performed using HPLC (Waters-717 series) with PDA detector and data handling system EMPOWER2 software, UV-Visible double beam spectrophotometer (ELICO SL-159), analytical balance 0.1mg Sensitivity (SHIMADZU), pH meter (Labindia), ultra sonicator. The column used is Phenomenex Luna C₁₈, 100A, 5mm, 250mm x 4.6mm i.d. (as Stationary phase) with the flow rate 1.0ml/min (isocratic).²⁸

Sample and Standard Preparation for the Analysis:

25mg of Vilazodone standard was transferred into 25ml volumetric flask, dissolved and make up to volume with mobile phase. Further dilution was done by transferring 0.1ml of the above solution into a 10ml volumetric flask and make up to volume with mobile phase^29.

Selection of Wavelength:

The standard and sample stock solutions were prepared separately by dissolving standard and sample in a solvent in mobile phase diluting with the same solvent. (After optimization of all conditions) for UV analysis. It scanned in the UV spectrum in the range of 200 to 400nm. While scanning the Vilazodone solution we observed the maxima at 238nm³⁰.

Fig-2: UV Spectrum for Vilazodone

Method Development:

Preparation of 0.02M Phosphate buffer Solution:

About 2.72168 grams of Potassium dihydrogen orthophosphate was weighed and transferred into a 1000ml beaker, dissolved and diluted to 1000ml with HPLC water. The pH was adjusted to 7.60 with diluted Orthophosphoric acid.^31.

Preparation of Mobile Phase:

The mobile phase used in this analysis consists of a mixture of Phosphate Buffer (pH adjusted to 7.60 with ortho phosphoric acid) and Acetonitrile in a ratio of 30:70. 300mL (30%) of Phosphate Buffer (pH adjusted to 7.60 with ortho phosphoric acid) and 700mL of Acetonitrile (70%) of above prepared phosphate buffer were mixed well and degassed in ultrasonic water bath for 15 minutes. The solution was filtered through 0.45 µm filter under vacuum filtration.^32.

Summary of Optimized Chromatographic Conditions:

The Optimum Chromatographic conditions obtained from experiments can be summarized as below:

Table-1: Summary of Optimised Chromatographic Conditions

Mobile phase	Phosphate Buffer: Acetonitrile = 30:70 (pH-7.6)
Column	Phenomenex Luna C₁₈, 100A, 5mm, 250mmx4.6mm i.d.
Flow rate	1.0 ml/ min.
Wavelength	238nm
Sampling System	Automatic
Temp. of Auto sampler	Ambient
Volume of injection	20µl
Run time	08 min.
Mode of Separation	Isocratic

Fig-3: Chromatogram for Blank Preparation

Fig–4: Chromatogram of Vilazodone in Optimized Condition

Method Validation:

Linearity and Range:

Calibration standards at five levels were prepared by appropriately mixed and further diluted standard stock solutions in the concentration ranges from 6-16μg/mL for Vilazodone. Samples in triple injections were made for each prepared concentration. Peak areas were plotted against the corresponding concentration to obtain the linearity graphs. Chromatograms of each solution were recorded.^33.

Fig-5: Calibration Curve of Vilazodone (API)

Table-2: Concentration of Vilazodone

S. No.	Conc. (µg/ml)	Mean Peak Area
1	0	0
2	6	641233
3	8	844610
4	10	1052647
5	12	1250435
6	14	1465354
7	16	1662043

Accuracy:

To determine the accuracy of the proposed method, recovery studies were carried out by adding different amounts (80%, 100%, and 120%) of pure drug of Vilazodone were taken and added to the pre-analysed formulation of concentration 100mg/ml. From that percentage recovery values were calculated. The results were shown in table-3^34.

Table-3: Accuracy Readings of Vilazodone

Sample ID	Concentration (mg/ml)		Peak Area	% Recovery of Pure drug	Statistical Analysis
Sample ID	Amount Added	Amount Found	Peak Area	% Recovery of Pure drug	Statistical Analysis
S₁ : 80 %	8	8.105	93435	101.312	Mean= 100.0163% S.D. = 1.293505 % R.S.D.= 1.293294
S₂ : 80 %	8	7.898	91287	98.725
S₃ : 80 %	8	8.001	92356	100.012
S₄: 100 %	10	10.195	115135	101.95	Mean= 101.4033% S.D. = 0.613379 % R.S.D.= 0.60489
S₅ : 100 %	10	10.152	114687	101.52
S₆ : 100 %	10	10.074	113879	100.74
S₇: 120 %	12	12.171	135647	101.425	Mean= 100.6053% S.D. = 0.730041 % R.S.D. = 0.725649
S₈ : 120 %	12	12.044	134324	100.366
S₉ : 120 %	12	12.003	133897	100.025

Precision:

Repeatability:

The precision of each method was ascertained separately from the peak areas and retention times obtained by actual determination of six replicates of a fixed amount of drug. Vilazodone (API). The percent relative standard deviation was calculated for Vilazodone are presented in the Table-4.

Table-4: Repeatability Readings of Vilazodone

Replicates of Vilazodone	Area Under the Curve
Replicate – 1	1013546
Replicate – 2	1025824
Replicate – 3	1012351
Replicate – 4	1036584
Replicate – 5	1015419
Replicate – 6	1008572
Average	1018716
Standard Deviation	10495.73
% RSD	1.03029

Intermediate Precision:

The intra and inter day variation of the method was carried out and the high values of mean assay and low values of standard deviation and % RSD (% RSD < 2%) within a day and day to day variations for Vilazodone revealed that the proposed method is precise.

Table-5: Results of Intra-Assay and Inter-Assay

Conc. of Vilazodone (API) (µg/ml)	Observed Conc. of Vilazodone (µg/ml) by the proposed method
	Intra-Day		Inter-Day
	Mean (n=6)	% RSD	Mean (n=6)	% RSD
8	8.03	0.25	9.95	0.23
10	10.05	0.36	10.02	0.32
12	11.14	0.14	12.06	0.19

Method Robustness:

Influence of little changes in optimized chromatographic conditions like changes in flow rate (± 0.1ml/min), mobile phase ratio (±2%), Wavelength of detection (±2nm) and Acetonitrile content in mobile phase (±2%) studied to measure the robustness of the method are also in favour of (Table-36, % RSD < 2%) the developed RP-HPLC method for the analysis of Vilazodone (API).

Table-6: Results of Method Robustness Test

Change in Parameter	% RSD
Flow (1.1 ml/min)	1.06
Flow (0.9 ml/min)	0.97
Less Organic	0.82
More Organic	0.63
Wavelength of Detection (240 nm)	0.72
Wavelength of detection (236 nm)	0.61

LOD and LOQ:

The detection limit (LOD) and quantitation limit (LOQ) may be expressed as:

L.O.D. = 3.3(SD/S).

L.O.Q. = 10(SD/S)

Where, SD = Standard deviation of the response

S = Slope of the calibration curve

System Suitability Parameter:

System suitability testing is an integral part of many analytical procedures. The tests are based on the concept that the equipment, electronics, analytical operations and samples to be analyzed constitute an integral system that can be evaluated as such. Following system suitability test parameters were established. The data are shown in Table-7.

Table-7: Data of System Suitability Parameter

S. No.	Parameter	Limit	Result
1	Resolution	Rs > 2	4.85
2	Asymmetry	T £ 2	Vilazodone =0.99
3	Theoretical plate	N > 2000	Vilazodone =4896

Estimation of Vilazodone in Pharmaceutical Dosage Form:

Twenty tablets were taken and the I.P. method was followed to calculate the average weight. Above weighed tablets were finally powdered and triturated well. Some quantity of powder which is equivalent to 25 mg of drug was transferred to a clean and dry 25 ml volumetric flask, make and solution was sonicated for fifteen minutes. Then the volume was made up to 25 ml with the same Mobile Phase. Then 10 ml of the prepared above solution was diluted to 100 ml with the help of mobile phase. The resulted solution was filtered through a membrane filter (0.45 mm) and sonicated to degas. The final solution prepared was injected in 5 replicates into the HPLC system and the s are record the observations. Two duplicate injections of the standard solution were also injected into the HPLC system and the peak areas were recorded. The data are shown in Table-8.

Table 8: Assay of Vilazodone Marketed Formulation

Brand name of tablets

Labelled amount of Drug (mg)

Mean (±SD) amount (mg) found by the proposed method (n=6)

Mean

(± SD) Assay

(n = 6)

Vilano Tablet (Sun Pharmaceutical Industries Ltd)

19.869 (±0.07)

99.89 (±0.67)

Stability Studies:

The API (Vilazodone) was subjected to stress conditions in various ways to observe the rate and extent of degradation that is likely to occur in the course of storage and/or after administration to body. The various degradation pathways studied are acid hydrolysis, basic hydrolysis, thermal degradation, photolytic degradation and oxidative degradation.

Fig-6: Chromatogram for Acid Degradation

Fig-7: Chromatogram for Basic Degradation

Fig-8: Chromatogram for Thermal Degradation

Fig-9: Chromatogram for Photolytic Degradation

Fig-10: Chromatogram for Oxidation with 3% H₂O₂ Degradation

RESULTS:

Optimization of Developed Method:

The optimized chromatographic conditions were Phenomenex Luna C18, 100A, 5µm, 250mm x 4.6mm i.d.as stationary phase and mobile phase was prepared with a mixture of Phosphate Buffer: Acetonitrile = 30:70 (pH-7.6) flow 1.0ml/min, with Injection Volume 20µl, at detection wavelength 238nm and run time at 8.0 min. In these chromatographic conditions the peak was pure, sharp and symmetric and found a greater number of theoretical plates.

Validation of Developed Method:

The results obtained in method validation were:

Linearity and Range: The calibration curve showed good linearity in the range of 06-16µg/ml, for Vilazodone (API) with correlation coefficient (r²) of 0.999 (Fig-5). A typical calibration curve has the regression equation of y = 10380.x + 9304 for Vilazodone.

Accuracy:

The mean recoveries were found to be 100.01, 101.40 and 100.60% for Vilazodone. The limit for mean % recovery is 95-105% and as both the values are within the limit, hence it can be said that the proposed method was accurate.

Repeatability:

The repeatability study which was conducted on the solution having the concentration of about 10mg/ml for Vilazodone (n =6) showed %RSD of 1.03029%. It was concluded that the analytical technique showed good repeatability.

LOD and LOQ: The Minimum concentration level at which the analyte can be reliable detected (LOD) and quantified (LOQ) were found to be 0.126 and 0.383 µg/ml respectively.

Assay:

The assay in Vilano tablets containing Vilazodone was found to be 99.89 %.

Degradation studies: The results of the stress studies indicated the specificity of the method that has been developed. Vilazodone was more stable in acidic and in oxidation stress conditions as compare to other stress conditions. The result of forced degradation studies is given in table-9.

DISCUSSION:

To develop a precise, linear, specific RP-HPLC method for analysis of Vilazodone, different chromatographic conditions were applied and the results observed were compared with the methods available in literatures.

Lakshmi Narusu R, et al.¹² achieved separation by using acetonitrile: water in proportion of 60:40 v/v as mobile phase. Somsubhra Ghosh, et al¹³ developed method by using a mobile phase in combination of Methanol and Phosphate buffer adjusted at pH 7.4 in a ratio (40:60v/v) but we have used Phosphate Buffer: Acetonitrile = 30:70 (pH-7.6). As per Vivekkumar K Redasani, et al.¹⁴ used Qualisil BDS C18 reversed phase column, P. Ravisankar, et al.¹⁵ used Welchrom C18 isocratic column, (250mm × 4.6mm i.d., particle size 5μm, maintained at ambient temperature), is used as stationary phase where as we used Phenomenex Luna C₁₈, 100A, 5mm, 250mm x 4.6mm i.d. Sagar Suman Panda, et al¹⁶ applied the principles of quality by design (QbD) to quantify vilazodone hydrochloride (VLN) in pharmaceutical dosage form. Mukthinuthalapati Mathrusri Annapurna, et al.¹⁷ were fixed flow rate of 07 ml/min in their method and got the linearity in between concentration range of 0.1-120µg/mL where as the linearity in this developed method was obtained in the range of 06-16µg/mL, the flow rate was 1.0ml/min. Venkata Subbaiah. G, et al¹⁸ developed a method by using C8 (4.6x150mm, 3.5micron, make: Xterra) column using a mixture of Methanol: 10mM potassium dihydrogen phosphate (20:80pH 3.5 adjusted with acetic acid). Retention time of Vilazodone was found to be 2.5 min + or-0.5min. the retention time for the developed method in our research work is 3.68.

The result shows the developed method is yet another suitable method for assay which can help in the analysis of Vilazodone in formulations.

SUMMARY AND CONCLUSION:

A sensitive and selective stability indicting RP-HPLC method has been developed and validated for the analysis of Vilazodone in bulk form and marketed pharmaceutical dosage form. Based on peak purity results, obtained from the analysis of samples using described method, it can be concluded that the absence of co-eluting peak along with the main peak of Vilazodone indicated that the developed method is specific for the estimation of Vilazodone. Further the proposed RP-HPLC method has excellent sensitivity, precision and reproducibility.

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Received on 17.11.2022 Modified on 11.03.2023

Research J. Pharm. and Tech 2023; 16(9):4319-4324.

DOI: 10.52711/0974-360X.2023.00707

Stress condition	Time	Assay of active substance	Assay of degraded products	Mass Balance (%)
Acid Hydrolysis (0.1 M HCl)	Refluxed 4Hrs.	98.48	1.52	100.0
Basic Hydrolysis (0.I M NaOH)	Refluxed 4Hrs.	90.76	9.24	100.0
Thermal Degradation (50 ⁰C)	Refluxed 6 Hrs.	91.47	8.53	100.0
UV (254nm)	24 Hrs.	96.84	3.16	100.0
3 % Hydrogen peroxide	24Hrs.	97.61	2.39	100.0