New validated RP-HPLC method for the determination of Vilazodone hydrochloride – A serotonergic Anti-depressant

 

Mukthinuthalapati Mathrusri Annapurna*, Kunala Anusha, Afreen Shahena Sharief, Varanasi Sindhuri

Department of Pharmaceutical Analysis and Quality Assurance, GITAM Institute of Pharmacy, GITAM University, Visakhapatnam-530045, India

 

ABSTRACT:

A new simple liquid chromatographic method has been established for the determination of Vilazodone hydrochloride in pharmaceutical formulations. Vilazodone hydrochloride is a strong dopamine antagonist. Vilazodone hydrochloride was approved by the FDA for use in the United States to treat major depressive disorder in January 21, 2011. A mobile phase containing0.1M Ammonium formate: Methanol (20:80, v/v) with flow rate 0.7 mL/min was used for the present chromatographic study(UV detection 241 nm). Linearity was followed over the concentration range 0.1-120 µg/mL. Forced degradation studies were performed by exposing the drug Vilazodone hydrochloride to alkaline, acidic, and oxidation stress degradations. The method was validated as per ICH guidelines (ICH Guidelines 2005).

 

 

 

INTRODUCTION:

Vilazodone hydrochloride (Fig 1) chemically known as 5-(4-[4-(5-cyano-1H-indol-3-yl) butyl] piperazin-1-yl) benzofuran-2-carbox amide is a strong dopamine antagonist. It has high affinity for D2 dopaminergic receptors. It is a serotonergic antidepressant. Vilazodone hydrochloride (VLZD) was approved by the FDA for use in the United States to treat major depressive disorder in January 21, 2011. Vilazodone hydrochloride acts as a serotonin reuptake inhibitor and 5-HT1A receptor partial agonist¹. It has negligible affinity for other serotonin receptors such as 5- HT1D, 5-HT2A, and 5-HT2C. Vilazodone hydrochloride was quantitatively determined by spectrofluorimetric², spectrophotometric^3-5and HPLC^6-11 methods (Table 1).

 

In the present study the authors have proposed a stability indicating reverse phase liquid chromatographic method for the determination of Vilazodone hydrochloride and the method was validated as per ICH guidelines.

 

Fig 1: Chemical structure of Vilazodone hydrochloride(VLZD)

 

MATERIALS AND METHODS:

Instrumentation:

Chromatographic separation was achieved by using Shimadzu Model CBM-20A/20 Alite HPLC system, equipped with SPD M20A prominence photodiode array detector with Phenomenex C8 column (250 mm × 4.60 mm i.e., 5.0 µm particle size).

Table. 1. Details of reported analytical methods for Vilazodone hydrochloride

 

Chemical and Reagents:

The stock solution was prepared by transferring accurately 25 mg of Vilazodone hydrochloride(VLZD) in to a 25 mL volumetric flask and diluted with mobile phase (1000 μg/mL). Further dilutions were made from the stock with mobile phase and filtered through 0.45 μm membrane filter. Vilazodone hydrochloride is available with brand names VALZ (Torrent Pharmaceuticals Ltd, India) and VILANO (Sun Pharmaceutical Industries Ltd)  with label claim: 20 mg and 40 mg.

 

To prepare 0.1M ammonium formate 3.153 grams of ammonium formate was weighed into a 500 mLvolumetric flask and dissolved in HPLC grade water. This solution was sonicated for half an hour, filtered and finally made up to volume in 500 mL volumetric flask and the volume is made up to volume with HPLC grade water.

 

Optimized chromatographic conditions:

Isocratic elution was performed using 0.1M Ammonium formate: Methanol (20:80, v/v) with flow rate 0.7 mL/min was used for the present chromatographic study (UV detection 241 nm) and the overall run time was 10 min. The detection was carried at 241 nm. 20 µL of sample was injected each time into the HPLC system and the chromatographic study was performed at room temperature (25°C±2°C).

 

Method validation¹²:

A series of solutions (0.1–120 μg/mL) were prepared from the Vilazodone hydrochloride stock solution and 20 µL of each solution was injected in to the HPLC system and the peak area of the chromatogram was noted. Calibration curve was plotted by taking the concentration of the solutions on the x-axis and the corresponding peak area values on the y-axis.

 

The intra-day precision of the assay method was evaluated by carrying out 9 independent assays for Vilazodone hydrochlorideat three concentration levels (10, 50 and 100 μg/mL) (n=3) against a qualified reference standard and the %RSD was calculated. The inter-day precision study was performed on three different days i.e. day 1, day 2 and day 3 at three different concentration levels (10, 50 and 100 μg/mL) and the % RSD was calculated. The accuracy study was performed using standard addition method and recovery studies (80, 100 and 120%).

 

The limit of quantification and limit of detection were based on the standard deviation of the response and the slope of the constructed calibration curve (n=3), as described in ICH guidelines Q2 (R1) (ICH guidelines, 2005). Sensitivity of the method was established with respect to limit of detection LOD and LOQ for analytes.

 

Assay of marketed formulations (Tablets):

Twenty tablets of Vilazodone hydrochloride were procured from the medical store, weighed, crushed to a fine powder and powder equivalent to 25 mg of Vilazodone hydrochloride was transferred carefully into a 25 mL volumetric flask and made up to volume with mobile phase. The contents of the volumetric flask were sonicated for 30 min to enable complete dissolution and filtered. The filtrate was diluted with mobile phase as per the requirement. 20 μL of these solutions were injected into the HPLC system after filtering through 0.45 μm membrane and the peak area was recorded from the respective chromatogram.

 

Forced degradation studies:

Forced degradation studies were performed to evaluate the stability indicating properties and specificity of the method¹³. Forced degradation studies were performed to evaluate the stability indicating properties and specificity of the method. All solutions for stress studies were prepared at an initial concentration of 1 mg/mL of Vilazodone hydrochloride and refluxed for 20 min at 80ºC and then diluted with mobile phase. 

 

Acidic degradation was performed by treating the drug solution with 1 N hydrochloric acid and refluxed for 20 min in a thermostat maintained at 80 ºC. The stressed sample was cooled, neutralized with 1 N sodium hydroxide and then diluted with mobile phase and 20 µL of the solution was injected in to the HPLC system.

 

Alkaline degradation was performed by treating the drug solution with 1 N sodium hydroxide and refluxed for 20 min in a thermostat maintained at 80 ºC. The stressed sample was cooled, neutralized with 1 N hydrochloric acid and then diluted with mobile phase and 20 µL of the solution was injected in to the HPLC system.

 

Oxidation degradation was performed by treating the drug solution with 30% H₂O₂ and refluxed for 20 min in a thermostat maintained at 80 ºC. The stressed sample was cooled and diluted with mobile phase and 20 µL of the solution was injected in to the HPLC system. The drug solution mixture was cooled and then diluted with mobile phase as per the requirement and 20 µL of the solution was injected in to the HPLC system.

 

RESULTS AND DISCUSSION:

A new stability indicating RP-HPLC method was developed for the quantification of Vilazodone hydrochloride using a mixture of 0.1M Ammonium formate: Methanol (20:80, v/v) as mobile phase with flow rate 0.7 mL/min. The chromatogram of the mobile phase (blank) and the Vilazodone hydrochloride standard solution were shown in Fig 2A and Fig 2B respectively.

 

Fig 2: Typical chromatograms of Vilazodone hydrochloride A) Blank B) Standard (100 µg/mL) C) Brand I D) Brand II

 

Method Validation:

The method was validated for linearity, limit of quantitation (LOQ), limit of detection (LOD), precision, accuracy, (ICH guidelines, 2005).

 

Vilazodone hydrochloridehas shown linearity over a concentration range 0.1-120 μg/mL (Table 2) with linear regression equation y = 216381x + 125638 R² = 0.9994. The LOD and LOQ were found to be 0.028153 and 0.0853 μg/mL respectively. The % RSD is found to be 0.8-1.12 in precision studies and 0.75-1.28 in accuracy studies (Table 3) indicating that the method is precise and accurate.

 

Table.2. Linearity of Vilazodone hydrochloride

^*Mean of three replicates

Table. 3.Accuracy study of Vilazodone hydrochloride

*Mean of three replicates

 

Assay of marketed formulations (Tablets):

The percentage recovery was found to be 99.40-99.75 (Table 4) and the respected chromatograms for the marketed formulations (Tablets) were shown in Fig 2C and Fig 2D.

 

Table. 4. Assay of Vilazodone hydrochloride

*Mean of three replicates

 

Forced degradation studies:

The detailed report of the forced degradation studies was given in Table 5. The chromatograms obtained in these studies were shown in Fig 3. The drug is found to be more sensitive towards alkaline conditions as there is more than 15% (>32%) degradation. The theoretical plates are found to be more than 2000 and the tailing factor is found to be less than 1.5. 

 

Table 5: Forced degradation studies of Vilazodone hydrochloride

*Mean of three replicates

 

Fig4: Typicalchromatograms of Vilazodone hydrochloride (A) Blank (B) Standard (100 µg/mL) (C) Acidic (D) Alkaline (E) Oxidative degradation

 

CONCLUSION:

The proposed stability-indicating HPLC method was validated as per ICH guidelines and applied for the determination of Vilazodone hydrochloridein pharmaceutical dosage forms and can be successfully applied to perform long-term and accelerated stability studies of Vilazodone hydrochloride formulations. It was observed that Vilazodone hydrochlorideis more sensitive towards the alkaline environment during the forced degradation studies.

 

ACKNOWLEDGEMENT:

The authors are grateful to M/s GITAM University, Visakhapatnam for providing the research facilities. The authors have no conflict of interest. The authors are also thankful to Torrent Pharmaceuticals Ltd. for providing the gift samples of Vilazodone hydrochloride.

 

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Accepted on 28.04.2017           © RJPT All right reserved