# Development and Validation of RP-HPLC Method for Quantitative Estimation of Vinpocetine in Pure and Pharmaceutical Dosage Forms

Authors: Subrata Bhadra; Sreedam Chandra Das; Sumon Roy; Shamsul Arefeen; Abu Shara Shamsur Rouf
Journal: Chromatography Research International
 (2011)

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## Abstract

A simple, precise, specific, and accurate reversed phase high performance liquid chromatographic (RP-HPLC) method was developed and validated for determination of vinpocetine in pure and pharmaceutical dosage forms. The different analytical performance parameters such as linearity, accuracy, specificity, precision, and sensitivity (limit of detection and limit of quantitation) were determined according to International Conference on Harmonization ICH Q2 (R1) guidelines. RP-HPLC was conducted on Zorbax C18 (150 mm length × 4.6 mm ID, 5 μm) column. The mobile phase was consisting of buffer (containing 1.54% w/v ammonium acetate solution) and acetonitrile in the ratio (40 : 60, v/v), and the flow rate was maintained at 1.0 mLmin−1. Vinpocetine was monitored using Agilent 1200 series equipped with photo diode array detector (λ = 280 nm). Linearity was observed in concentration range of 160–240 μgmL−1, and correlation coefficient was found excellent (R2 = 0.999). All the system suitability parameters were found within the range. The proposed method is rapid, cost-effective and can be used as a quality-control tool for routine quantitative analysis of vinpocetine in pure and pharmaceutical dosage forms.


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## Body

## 1. Introduction

Vinpocetine is a synthetic ethyl ester of apovincamine, a vinca alkaloid obtained from the leaves of the Lesser Periwinkle (Vinca minor) and discovered in the late 1960s [1]. It is a novel vasodilating agent widely used to treat acute and chronic stroke [2–5]. It also has a potential role in the treatment of Parkinson's disease and Alzheimer’s disease [6, 7]. Vinpocetine selectively inhibits voltage-sensitive Na+ channels, resulting in a dose-dependent decrease in evoked extracellular Ca++ ions in striatal nerve endings [8] and also inhibits IKK preventing IκB degradation and the following translocation of NF-κB to the cell nucleus [6, 7]. Chemically, it is 14-ethoxycarbonyl-(3α, 16α-ethyl-14, 15-eburnamenine) [9], as shown in Figure 1, with molecular formula of C22H26N2O2 and molecular weight of 350.454.Figure 1
Chemical structure of vinpocetine.From the literature survey, it was found that various methods were used for the estimation of vinpocetine such as spectrophotometric method, high performance liquid chromatographic (HPLC) method [10], and gas chromatographic-mass spectrometric (GC-MS) method [11] in laboratory-prepared mixture, pharmaceutical preparation, and biological matrices such as human plasma. However, the aim of the present work is to develop a simple, precise, specific, accurate, cost-effective, and validated RP-HPLC method according to USP and ICH guidelines [12, 13] for the estimation and routine analysis of vinpocetine in pure and pharmaceutical formulations.


## 2. Experimental

### 2.1. Reagents and Materials

Vinpocetine tablets were prepared from ACI Ltd., Narayanganj-1400, Bangladesh. Each tablet contained vinpocetine (5 mg, based on 100% potency) as an active ingredient, and microcrystalline cellulose (Avicel PH 102), sodium starch glycolate (SSG), colloidal silicon dioxide (Aerosil-200), and magnesium stearate as excipients. HPLC grade acetonitrile and reagent grade ammonium acetate were used for analytical purposes. Milli-Q water was used to prepare the mobile phase.


### 2.2. Preparation and Characterization of Vinpocetine Tablets (Avintol 5 mg Tablet)

#### 2.2.1. Tablets Preparation

The immediate release vinpocetine tablet was prepared by direct compression method. The required amount of the active drug, Avicel PH 102 (diluent), and SSG (super disintegrant) was mixed by geometrical order and then sieved the granules through no. 24 sized mesh. Finally the granules were mixed with Aerosil-200 (glidant) and magnesium stearate (lubricant) passing through no. 40 sized mesh. Then the granules were compressed in single rotary Mini compressed machine using “D”-Tooling of punch size 6.0 mm in diameter (round shape), 8 station. The tablets were then film coated with ethyl cellulose (EC) and hydroxypropyl methylcellulose (HPMC) mixed with the plasticizer polyethylene glycol 6000 and titanium dioxide as opacifier.


#### 2.2.2. Tablets Characterization

The tablets obtained were round and convex shaped with a break line on the upper side and the lower side was plain. The average weight of the tablets varied from 90 mg ± 7.5% [14]. The disintegration test of both core and coated tablets [15] and the friability test of core tablets [16] were found well within the British Pharmacopoeia (BP) acceptable limit.


### 2.3. Chromatographic Conditions

#### 2.3.1. Chromatographic Parameters

An Agilent HPLC model integrated with variable wavelength programmable photo diode array detector was employed for the investigation. The chromatographic analysis was performed on a Zorbax C18, 150 mm length × 4.6 mm ID with 5 μm particle size column. The mobile phase was buffer (containing 1.54% w/v ammonium acetate solution): acetonitrile (40 : 60, v/v) pumped at a flow rate of 1.0 mLmin−1. The column temperature was maintained at 30°C, and the detection wavelength was 280 nm. The injection volume was 10 μL, and the run time was 15 min for each injection. HPLC grade acetonitrile was used as diluent during the standard and test sample preparation.


#### 2.3.2. Preparation of Mobile Phase

6.16 gm of ammonium acetate was weighted and dissolved in 400 mL of distilled water. This solution was mixed with 600 mL of HPLC grade acetonitrile and mixed well. The resulting solution was sonicated for 5 min using ultrasonic bath, and finally this solution was filtered using 0.2μm filter.


#### 2.3.3. Preparation of Stock Solution of Standard Vinpocetine

The stock solution of vinpocetine was prepared by dissolving 200 mg of standard vinpocetine to 100 mL with acetonitrile to give a concentration of 2 mgmL−1. The solution was sonicated for 5 min using ultrasonic bath and then filtered through 0.2 μm disk filter.


#### 2.3.4. Preparation of Assay Sample Solution

For the analysis of the tablet dosage form, not less than 20 tablets were crushed and then powdered finely. To prepare assay sample solution, powdered sample equivalent to 10 mg of vinpocetine was weighed and transferred to a clean and dry 50 mL volumetric flask. About 30 mL of acetonitrile was added as diluting solution and shaken thoroughly to extract the drug from the excipients and then sonicated for 5 min for complete dissolution of drug. The solution was allowed to cool at room temperature and then the volume was made upto the mark with the same diluting solution. The solution was filtered through Whatman filter paper (No. 42) and then finally filtered through 0.2μm disk filter. The drug concentration of the resulting sample solution was determined by HPLC using the calibration curve of standard solution. All determinations were conducted in triplicate. To validate the proposed method, the different analytical performance parameters such as linearity, accuracy, specificity, precision, sensitivity (limit of detection and limit of quantitation), and system suitability were determined according to ICH guidelines [13].


### 2.4. Analytical Method Validation Parameters

#### 2.4.1. System Suitability

To assess system suitability of the method, the repeatability, theoretical plates, tailing factor and retention time of six replicate injections of standard vinpocetine of concentration 200μgmL−1 were used and the %RSD values were calculated in each case.


#### 2.4.2. Linearity

The linearity was analyzed through the standard curves ranging from 160 to 240μgmL−1 by diluting appropriate amounts of vinpocetine stock solution (2000 μgmL−1) with acetonitrile and prepared in triplicate. Three calibration curves were prepared in the same day with the following concentrations (160, 180, 200, 220, and 240 μgmL−1). The linearity was evaluated by linear regression analysis, which was calculated by the least-square regression analysis.


#### 2.4.3. Specificity

The specificity of the developed HPLC method for the determination of vinpocetine in bulk drug and pharmaceutical preparation (Avintol 5 mg tablet) was investigated by chromatographic analysis of the following.Noninterference of Placebo
To check the noninterference of placebo, placebo solution was prepared in the same way of the sample solution in the presence of all inactive ingredients of the Avintol 5 mg tablet formulation but without vinpocetine.Degradation Studies
For degradation studies, 10 mg of vinpocetine was accurately weighed and transferred to a 50 mL volumetric flask. To this, 1 mL 1N HCl (for acid degradation study) or 1 mL of 1N NaOH (for alkaline degradation study) was added and kept in a stability chamber (25 ± 2°C) for 8 h. The mixture was dissolved and made to volume with acetonitrile. For photolytic degradation, nominal standard solution of vinpocetine (200μgmL−1) was exposed to UV light at 254 nm for 1 h. The final solution was injected for analysis and the presence of interfering peak(s) eluted at/or near the retention time of vinpocetine was also checked. All determinations were conducted in triplicate.Peak Purity Evaluation
The peak purity tool was used to check the peak purity of the test solution.


#### 2.4.4. Accuracy

Accuracy study of the method was carried out for both drug and drug-matrix solutions. In case of drug solution, standard solutions of vinpocetine, corresponding to 80, 90, 100, 110, and 120% of the nominal analytical concentration of vinpocetine (200μgmL−1) were compared with reference standard solution of vinpocetine of known purity (200 μgmL−1), and the percent recoveries (mean ±  %RSD of three replicates) of vinpocetine in pure form were calculated. In case of drug-matrix solution, accuracy parameter was determined by the recovery test, which consisted of adding known amounts of vinpocetine to the samples’ solutions in the beginning of the process. This test was realized by assaying five different solutions, three replicates each, containing 160, 180, 200, 220, and 240 μgmL−1  of vinpocetine standard solution added to vinpocetine sample solution, corresponding to 80, 90, 100, 110, and 120% of the nominal analytical concentration of vinpocetine (200 μgmL−1), and the percent recoveries (mean ±  %RSD of three replicates) of vinpocetine in drug-matrix form were calculated.


#### 2.4.5. Precision

Precision of the method was determined by repeatability (intraday precision) and intermediate precision (interday precision) of both standard and sample solutions. Precision was determined in six replicates of both vinpocetine standard solution (200μgmL−1) and sample solution (200 μgmL−1) on the same day (intra-day precision) and daily for 6 times over a period of one week (inter-day precision). The results were expressed as %RSD of the measurements.


#### 2.4.6. Sensitivity

Limit of Detection (LOD) and Limit of Quantitation (LOQ) were determined using calibration curve method according to ICH Q2 (R1) recommendations. The LOD (k=3.3) and LOQ (k=10) of the proposed method were calculated using the following equation: (1)A=kσ/S,
where A is LOD or LOQ, σ is the standard deviation of the response, and S is the slope of the calibration curve.


#### 2.4.7. Ruggedness

Ruggedness of the current method was determined by analyzing six assay sample solutions of Avintol 5 mg tablet formulation having concentration of 200μgmL−1 by two analysts in the same laboratory to check the reproducibility of the test result. The % recovery and standard deviation were calculated in both cases.


#### 2.4.8. Robustness

To determine the robustness of the current method, the effect of flow rate was studied at 0.9 and 1.1 mLmin−1 instead of 1.0 mLmin−1. The effect of column temperature was studied at 25 and 35°C instead of 30°C. The effect of mobile phase composition was assessed at (Buffer : ACN = 38 : 62, v/v) and (Buffer : ACN = 42 : 58, v/v) instead of (Buffer : ACN = 40 : 60, v/v). The %RSD of robustness testing under these conditions was calculated in all cases.


## 2.1. Reagents and Materials

Vinpocetine tablets were prepared from ACI Ltd., Narayanganj-1400, Bangladesh. Each tablet contained vinpocetine (5 mg, based on 100% potency) as an active ingredient, and microcrystalline cellulose (Avicel PH 102), sodium starch glycolate (SSG), colloidal silicon dioxide (Aerosil-200), and magnesium stearate as excipients. HPLC grade acetonitrile and reagent grade ammonium acetate were used for analytical purposes. Milli-Q water was used to prepare the mobile phase.


## 2.2. Preparation and Characterization of Vinpocetine Tablets (Avintol 5 mg Tablet)

### 2.2.1. Tablets Preparation

The immediate release vinpocetine tablet was prepared by direct compression method. The required amount of the active drug, Avicel PH 102 (diluent), and SSG (super disintegrant) was mixed by geometrical order and then sieved the granules through no. 24 sized mesh. Finally the granules were mixed with Aerosil-200 (glidant) and magnesium stearate (lubricant) passing through no. 40 sized mesh. Then the granules were compressed in single rotary Mini compressed machine using “D”-Tooling of punch size 6.0 mm in diameter (round shape), 8 station. The tablets were then film coated with ethyl cellulose (EC) and hydroxypropyl methylcellulose (HPMC) mixed with the plasticizer polyethylene glycol 6000 and titanium dioxide as opacifier.


### 2.2.2. Tablets Characterization

The tablets obtained were round and convex shaped with a break line on the upper side and the lower side was plain. The average weight of the tablets varied from 90 mg ± 7.5% [14]. The disintegration test of both core and coated tablets [15] and the friability test of core tablets [16] were found well within the British Pharmacopoeia (BP) acceptable limit.


## 2.2.1. Tablets Preparation

The immediate release vinpocetine tablet was prepared by direct compression method. The required amount of the active drug, Avicel PH 102 (diluent), and SSG (super disintegrant) was mixed by geometrical order and then sieved the granules through no. 24 sized mesh. Finally the granules were mixed with Aerosil-200 (glidant) and magnesium stearate (lubricant) passing through no. 40 sized mesh. Then the granules were compressed in single rotary Mini compressed machine using “D”-Tooling of punch size 6.0 mm in diameter (round shape), 8 station. The tablets were then film coated with ethyl cellulose (EC) and hydroxypropyl methylcellulose (HPMC) mixed with the plasticizer polyethylene glycol 6000 and titanium dioxide as opacifier.


## 2.2.2. Tablets Characterization

The tablets obtained were round and convex shaped with a break line on the upper side and the lower side was plain. The average weight of the tablets varied from 90 mg ± 7.5% [14]. The disintegration test of both core and coated tablets [15] and the friability test of core tablets [16] were found well within the British Pharmacopoeia (BP) acceptable limit.


## 2.3. Chromatographic Conditions

### 2.3.1. Chromatographic Parameters

An Agilent HPLC model integrated with variable wavelength programmable photo diode array detector was employed for the investigation. The chromatographic analysis was performed on a Zorbax C18, 150 mm length × 4.6 mm ID with 5 μm particle size column. The mobile phase was buffer (containing 1.54% w/v ammonium acetate solution): acetonitrile (40 : 60, v/v) pumped at a flow rate of 1.0 mLmin−1. The column temperature was maintained at 30°C, and the detection wavelength was 280 nm. The injection volume was 10 μL, and the run time was 15 min for each injection. HPLC grade acetonitrile was used as diluent during the standard and test sample preparation.


### 2.3.2. Preparation of Mobile Phase

6.16 gm of ammonium acetate was weighted and dissolved in 400 mL of distilled water. This solution was mixed with 600 mL of HPLC grade acetonitrile and mixed well. The resulting solution was sonicated for 5 min using ultrasonic bath, and finally this solution was filtered using 0.2μm filter.


### 2.3.3. Preparation of Stock Solution of Standard Vinpocetine

The stock solution of vinpocetine was prepared by dissolving 200 mg of standard vinpocetine to 100 mL with acetonitrile to give a concentration of 2 mgmL−1. The solution was sonicated for 5 min using ultrasonic bath and then filtered through 0.2 μm disk filter.


### 2.3.4. Preparation of Assay Sample Solution

For the analysis of the tablet dosage form, not less than 20 tablets were crushed and then powdered finely. To prepare assay sample solution, powdered sample equivalent to 10 mg of vinpocetine was weighed and transferred to a clean and dry 50 mL volumetric flask. About 30 mL of acetonitrile was added as diluting solution and shaken thoroughly to extract the drug from the excipients and then sonicated for 5 min for complete dissolution of drug. The solution was allowed to cool at room temperature and then the volume was made upto the mark with the same diluting solution. The solution was filtered through Whatman filter paper (No. 42) and then finally filtered through 0.2μm disk filter. The drug concentration of the resulting sample solution was determined by HPLC using the calibration curve of standard solution. All determinations were conducted in triplicate. To validate the proposed method, the different analytical performance parameters such as linearity, accuracy, specificity, precision, sensitivity (limit of detection and limit of quantitation), and system suitability were determined according to ICH guidelines [13].


## 2.3.1. Chromatographic Parameters

An Agilent HPLC model integrated with variable wavelength programmable photo diode array detector was employed for the investigation. The chromatographic analysis was performed on a Zorbax C18, 150 mm length × 4.6 mm ID with 5 μm particle size column. The mobile phase was buffer (containing 1.54% w/v ammonium acetate solution): acetonitrile (40 : 60, v/v) pumped at a flow rate of 1.0 mLmin−1. The column temperature was maintained at 30°C, and the detection wavelength was 280 nm. The injection volume was 10 μL, and the run time was 15 min for each injection. HPLC grade acetonitrile was used as diluent during the standard and test sample preparation.


## 2.3.2. Preparation of Mobile Phase

6.16 gm of ammonium acetate was weighted and dissolved in 400 mL of distilled water. This solution was mixed with 600 mL of HPLC grade acetonitrile and mixed well. The resulting solution was sonicated for 5 min using ultrasonic bath, and finally this solution was filtered using 0.2μm filter.


## 2.3.3. Preparation of Stock Solution of Standard Vinpocetine

The stock solution of vinpocetine was prepared by dissolving 200 mg of standard vinpocetine to 100 mL with acetonitrile to give a concentration of 2 mgmL−1. The solution was sonicated for 5 min using ultrasonic bath and then filtered through 0.2 μm disk filter.


## 2.3.4. Preparation of Assay Sample Solution

For the analysis of the tablet dosage form, not less than 20 tablets were crushed and then powdered finely. To prepare assay sample solution, powdered sample equivalent to 10 mg of vinpocetine was weighed and transferred to a clean and dry 50 mL volumetric flask. About 30 mL of acetonitrile was added as diluting solution and shaken thoroughly to extract the drug from the excipients and then sonicated for 5 min for complete dissolution of drug. The solution was allowed to cool at room temperature and then the volume was made upto the mark with the same diluting solution. The solution was filtered through Whatman filter paper (No. 42) and then finally filtered through 0.2μm disk filter. The drug concentration of the resulting sample solution was determined by HPLC using the calibration curve of standard solution. All determinations were conducted in triplicate. To validate the proposed method, the different analytical performance parameters such as linearity, accuracy, specificity, precision, sensitivity (limit of detection and limit of quantitation), and system suitability were determined according to ICH guidelines [13].


## 2.4. Analytical Method Validation Parameters

### 2.4.1. System Suitability

To assess system suitability of the method, the repeatability, theoretical plates, tailing factor and retention time of six replicate injections of standard vinpocetine of concentration 200μgmL−1 were used and the %RSD values were calculated in each case.


### 2.4.2. Linearity

The linearity was analyzed through the standard curves ranging from 160 to 240μgmL−1 by diluting appropriate amounts of vinpocetine stock solution (2000 μgmL−1) with acetonitrile and prepared in triplicate. Three calibration curves were prepared in the same day with the following concentrations (160, 180, 200, 220, and 240 μgmL−1). The linearity was evaluated by linear regression analysis, which was calculated by the least-square regression analysis.


### 2.4.3. Specificity

The specificity of the developed HPLC method for the determination of vinpocetine in bulk drug and pharmaceutical preparation (Avintol 5 mg tablet) was investigated by chromatographic analysis of the following.Noninterference of Placebo
To check the noninterference of placebo, placebo solution was prepared in the same way of the sample solution in the presence of all inactive ingredients of the Avintol 5 mg tablet formulation but without vinpocetine.Degradation Studies
For degradation studies, 10 mg of vinpocetine was accurately weighed and transferred to a 50 mL volumetric flask. To this, 1 mL 1N HCl (for acid degradation study) or 1 mL of 1N NaOH (for alkaline degradation study) was added and kept in a stability chamber (25 ± 2°C) for 8 h. The mixture was dissolved and made to volume with acetonitrile. For photolytic degradation, nominal standard solution of vinpocetine (200μgmL−1) was exposed to UV light at 254 nm for 1 h. The final solution was injected for analysis and the presence of interfering peak(s) eluted at/or near the retention time of vinpocetine was also checked. All determinations were conducted in triplicate.Peak Purity Evaluation
The peak purity tool was used to check the peak purity of the test solution.


### 2.4.4. Accuracy

Accuracy study of the method was carried out for both drug and drug-matrix solutions. In case of drug solution, standard solutions of vinpocetine, corresponding to 80, 90, 100, 110, and 120% of the nominal analytical concentration of vinpocetine (200μgmL−1) were compared with reference standard solution of vinpocetine of known purity (200 μgmL−1), and the percent recoveries (mean ±  %RSD of three replicates) of vinpocetine in pure form were calculated. In case of drug-matrix solution, accuracy parameter was determined by the recovery test, which consisted of adding known amounts of vinpocetine to the samples’ solutions in the beginning of the process. This test was realized by assaying five different solutions, three replicates each, containing 160, 180, 200, 220, and 240 μgmL−1  of vinpocetine standard solution added to vinpocetine sample solution, corresponding to 80, 90, 100, 110, and 120% of the nominal analytical concentration of vinpocetine (200 μgmL−1), and the percent recoveries (mean ±  %RSD of three replicates) of vinpocetine in drug-matrix form were calculated.


### 2.4.5. Precision

Precision of the method was determined by repeatability (intraday precision) and intermediate precision (interday precision) of both standard and sample solutions. Precision was determined in six replicates of both vinpocetine standard solution (200μgmL−1) and sample solution (200 μgmL−1) on the same day (intra-day precision) and daily for 6 times over a period of one week (inter-day precision). The results were expressed as %RSD of the measurements.


### 2.4.6. Sensitivity

Limit of Detection (LOD) and Limit of Quantitation (LOQ) were determined using calibration curve method according to ICH Q2 (R1) recommendations. The LOD (k=3.3) and LOQ (k=10) of the proposed method were calculated using the following equation: (1)A=kσ/S,
where A is LOD or LOQ, σ is the standard deviation of the response, and S is the slope of the calibration curve.


### 2.4.7. Ruggedness

Ruggedness of the current method was determined by analyzing six assay sample solutions of Avintol 5 mg tablet formulation having concentration of 200μgmL−1 by two analysts in the same laboratory to check the reproducibility of the test result. The % recovery and standard deviation were calculated in both cases.


### 2.4.8. Robustness

To determine the robustness of the current method, the effect of flow rate was studied at 0.9 and 1.1 mLmin−1 instead of 1.0 mLmin−1. The effect of column temperature was studied at 25 and 35°C instead of 30°C. The effect of mobile phase composition was assessed at (Buffer : ACN = 38 : 62, v/v) and (Buffer : ACN = 42 : 58, v/v) instead of (Buffer : ACN = 40 : 60, v/v). The %RSD of robustness testing under these conditions was calculated in all cases.


## 2.4.1. System Suitability

To assess system suitability of the method, the repeatability, theoretical plates, tailing factor and retention time of six replicate injections of standard vinpocetine of concentration 200μgmL−1 were used and the %RSD values were calculated in each case.


## 2.4.2. Linearity

The linearity was analyzed through the standard curves ranging from 160 to 240μgmL−1 by diluting appropriate amounts of vinpocetine stock solution (2000 μgmL−1) with acetonitrile and prepared in triplicate. Three calibration curves were prepared in the same day with the following concentrations (160, 180, 200, 220, and 240 μgmL−1). The linearity was evaluated by linear regression analysis, which was calculated by the least-square regression analysis.


## 2.4.3. Specificity

The specificity of the developed HPLC method for the determination of vinpocetine in bulk drug and pharmaceutical preparation (Avintol 5 mg tablet) was investigated by chromatographic analysis of the following.Noninterference of Placebo
To check the noninterference of placebo, placebo solution was prepared in the same way of the sample solution in the presence of all inactive ingredients of the Avintol 5 mg tablet formulation but without vinpocetine.Degradation Studies
For degradation studies, 10 mg of vinpocetine was accurately weighed and transferred to a 50 mL volumetric flask. To this, 1 mL 1N HCl (for acid degradation study) or 1 mL of 1N NaOH (for alkaline degradation study) was added and kept in a stability chamber (25 ± 2°C) for 8 h. The mixture was dissolved and made to volume with acetonitrile. For photolytic degradation, nominal standard solution of vinpocetine (200μgmL−1) was exposed to UV light at 254 nm for 1 h. The final solution was injected for analysis and the presence of interfering peak(s) eluted at/or near the retention time of vinpocetine was also checked. All determinations were conducted in triplicate.Peak Purity Evaluation
The peak purity tool was used to check the peak purity of the test solution.


## 2.4.4. Accuracy

Accuracy study of the method was carried out for both drug and drug-matrix solutions. In case of drug solution, standard solutions of vinpocetine, corresponding to 80, 90, 100, 110, and 120% of the nominal analytical concentration of vinpocetine (200μgmL−1) were compared with reference standard solution of vinpocetine of known purity (200 μgmL−1), and the percent recoveries (mean ±  %RSD of three replicates) of vinpocetine in pure form were calculated. In case of drug-matrix solution, accuracy parameter was determined by the recovery test, which consisted of adding known amounts of vinpocetine to the samples’ solutions in the beginning of the process. This test was realized by assaying five different solutions, three replicates each, containing 160, 180, 200, 220, and 240 μgmL−1  of vinpocetine standard solution added to vinpocetine sample solution, corresponding to 80, 90, 100, 110, and 120% of the nominal analytical concentration of vinpocetine (200 μgmL−1), and the percent recoveries (mean ±  %RSD of three replicates) of vinpocetine in drug-matrix form were calculated.


## 2.4.5. Precision

Precision of the method was determined by repeatability (intraday precision) and intermediate precision (interday precision) of both standard and sample solutions. Precision was determined in six replicates of both vinpocetine standard solution (200μgmL−1) and sample solution (200 μgmL−1) on the same day (intra-day precision) and daily for 6 times over a period of one week (inter-day precision). The results were expressed as %RSD of the measurements.


## 2.4.6. Sensitivity

Limit of Detection (LOD) and Limit of Quantitation (LOQ) were determined using calibration curve method according to ICH Q2 (R1) recommendations. The LOD (k=3.3) and LOQ (k=10) of the proposed method were calculated using the following equation: (1)A=kσ/S,
where A is LOD or LOQ, σ is the standard deviation of the response, and S is the slope of the calibration curve.


## 2.4.7. Ruggedness

Ruggedness of the current method was determined by analyzing six assay sample solutions of Avintol 5 mg tablet formulation having concentration of 200μgmL−1 by two analysts in the same laboratory to check the reproducibility of the test result. The % recovery and standard deviation were calculated in both cases.


## 2.4.8. Robustness

To determine the robustness of the current method, the effect of flow rate was studied at 0.9 and 1.1 mLmin−1 instead of 1.0 mLmin−1. The effect of column temperature was studied at 25 and 35°C instead of 30°C. The effect of mobile phase composition was assessed at (Buffer : ACN = 38 : 62, v/v) and (Buffer : ACN = 42 : 58, v/v) instead of (Buffer : ACN = 40 : 60, v/v). The %RSD of robustness testing under these conditions was calculated in all cases.


## 3. Results and Discussion

### 3.1. Method Validation

#### 3.1.1. System Suitability

The results (Mean ±  %RSD of six replicates) of the chromatographic parameters are shown in Table1, indicating the good performance of the system.Table 1
Chromatographic characteristics of system suitability solution.
ParameterValue (Mean ±  %RSD)*Peak area3457550 ± 0.096Tailing factor1.109 ± 0.378Theoretical plate2945.667 ± 0.099Retention time10.581 ± 0.083*Mean and % Relative Standard Deviation of six replicates.


#### 3.1.2. Linearity

The regression equation for vinpocetine was foundy=17563x-50470 by plotting peak area (y) versus the concentration (x) studied from 160 to 240 μgmL−1, and the correlation coefficient (R2=0.999) was highly significant. The validity of the assay was verified by means of the ANOVA. According to it, there is linear regression and there is no deviation from linearity (P<0.05).


#### 3.1.3. Specificity

A typical HPLC chromatogram of vinpocetine standard preparation (a) and vinpocetine test sample (b) are shown in Figure2. The HPLC chromatograms recorded for the mixture of the inactive ingredients revealed no peaks within retention time around 10.5 minutes, and the peak purity was 99.99%. Figure 2 and the peak purity index show that vinpocetine is clearly separated from the response of any interfering peak(s).Typical chromatogram of (a) vinpocetine standard preparation and (b) vinpocetine test sample. (Chromatographic conditions-Zorbax C18, 150 mm length × 4.6 mm ID with 5 μm particle size column; mobile phase-buffer (containing 1.54% w/v ammonium acetate solution): acetonitrile (40 : 60, v/v); flow rate 1.0 mLmin−1; column temperature 30°C; wavelength 280 nm and injected volume 10 μL).
(a)
Chromatogram of vinpocetine standard preparation(b)
Chromatogram of vinpocetine test sampleUnder acidic condition (1N HCl for 8 h), it was found that 1.34% of vinpocetine content was decreased, but there was no detectable degradation peak(s). The samples submitted to alkaline condition (1N NaOH for 8 h) showed 4.57% degradation of vinpocetine content, and also there was no detectable degradation peak(s). In both cases, the peak purity was 99.99%. Again, when the sample was submitted to photolytic degradation (UV light at 254 nm for 1 h), the degradation of vinpocetine was found 0.97% and the peak purity was 99.99%. The results obtained from the peak purity tool demonstrated that the peak response of vinpocetine was pure in all cases and thus confirming the absence of other substance in the same retention time. The results of percentage of forced degradations are shown in Table2.Table 2
Results of vinpocetine exposed to different degradative pathways.
ParameterAmount added (μgmL−1)Amount recovered (μgmL−1)*Percentage of degradationAcidic degradation200197.321.34Alkaline degradation200190.864.57Photolytic degradation200198.060.97*Mean of three replicates.


#### 3.1.4. Accuracy

The results were expressed as percent recoveries of the particular components in the samples. The overall results of percent recoveries (mean ±  %RSD) of vinpocetine in pure and drug-matrix solutions are demonstrated in Table3, indicating good accuracy of the proposed HPLC method.Table 3
Accuracy studies of vinpocetine in standard and drug-matrix solutions.
Amount added (μgmL−1)Peak areaAmount recovered (μgmL−1)% RecoveryRecovery (Mean ±  %RSD)Standard solution1602758830160.12100.081602757741160.06100.04100.05 ± 0.0231602757692160.05100.041803108421180.41100.231803108988180.44100.25100.24 ± 0.0121803109134180.45100.252003463527201.02100.512003463889201.04100.52100.52 ± 0.0092003464117201.05100.532203811741221.23100.562203812077221.25100.57100.56 ± 0.0072203811562221.22100.562404168397241.93100.802404167556241.88100.78100.79 ± 0.0122404167502241.88100.78Drug-matrix solution1602773303160.96100.601602772297160.90100.56100.57 ± 0.0241602772063160.89100.561803119142181.03100.601803121001181.14100.66100.63 ± 0.0301803119725181.06100.622003477174201.81100.902003477005201.80100.90100.91 ± 0.0152003477987201.86100.922203831775222.39101.102203831000222.35101.08101.08 ± 0.0252203829872222.28101.052404185580242.92101.202404186378242.97101.22101.21 ± 0.0112404185596242.92101.20


#### 3.1.5. Precision

The values of %RSD for intraday and interday variation are given in Table4. In both cases, %RSD values were found well within 2% limit, indicating that the current method is repeatable.Table 4
Intraday and interday precision of HPLC method.
Sr. no.Concentration (μgmL−1)Standard solutionSample solutionIntra-day precisionInter-day precisionIntra-day precisionInter-day precision120034795013484566348133234892142200347900534841123481987348974132003478987348399134822443490301420034792273484977348151034897485200347980834850043481993348984962003479777348466934822143490488Mean3479384348455334818803489890SD367.100425.941375.626453.331%RSD0.0110.0120.0110.013


#### 3.1.6. Sensitivity

The LOD and LOQ of vinpocetine by the proposed method were found 0.0968μgmL−1 and 0.2904 μgmL−1, respectively. Figure 3 shows the sensitivity of the current method.Typical chromatogram of (a) blank, (b) LOD of vinpocetine, and (c) LOQ of vinpocetine. (Chromatographic conditions-Zorbax C18, 150 mm length × 4.6 mm ID with 5 μm particle size column; mobile phase-buffer (containing 1.54% w/v ammonium acetate solution): acetonitrile (40 : 60, v/v); flow rate 1.0 mLmin−1; column temperature 30°C; wavelength 280 nm and injected volume 10 μL).
(a)
Chromatogram of blank(b)
Chromatogram of LOD of vinpocetine(c)
Chromatogram of LOQ of vinpocetine


#### 3.1.7. Ruggedness

The results (% of Recovery ± Standard Deviation of six assay samples) are given in Table5, indicating the ruggedness of the current method.Table 5
Ruggedness of the method.
SampleTaken amount of standard vinpocetine (mg)Analyst-1Analyst-2Amount found (mg)% Recovery ± SD*Amount found (mg)% Recovery ± SD*Avintol 5 mg Tablet1010.04100.4 ± 0.0810.01100.1 ± 0.09*% of Recovery ± Standard Deviation of six samples (assay).


#### 3.1.8. Robustness

The % of RSD of robustness testing under different altered conditions is given in Table6, indicating that the current method is robust.Table 6
Robustness of the method.
ParameterAmount of vinpocetine added (μgmL−1)Amount of vinpocetine detected (Mean ± SD)*%RSDChange in mobile phase compositionBuffer : ACN = 38 : 62200200.33 ± 0.290.14Buffer : ACN = 40 : 60200200.47 ± 0.290.14Buffer : ACN = 42 : 58200200.56 ± 0.550.27Change in column temperature25°C200200.43 ± 0.710.3530°C200200.36 ± 0.500.2335°C200199.88 ± 0.560.28Change in flow rate0.9 mLmin−1200200.08 ± 0.650.331.0 mLmin−1200200.41 ± 0.310.151.1 mLmin−1200200.46 ± 0.610.31*Mean ± Standard Deviation of six replicates.


## 3.1. Method Validation

### 3.1.1. System Suitability

The results (Mean ±  %RSD of six replicates) of the chromatographic parameters are shown in Table1, indicating the good performance of the system.Table 1
Chromatographic characteristics of system suitability solution.
ParameterValue (Mean ±  %RSD)*Peak area3457550 ± 0.096Tailing factor1.109 ± 0.378Theoretical plate2945.667 ± 0.099Retention time10.581 ± 0.083*Mean and % Relative Standard Deviation of six replicates.


### 3.1.2. Linearity

The regression equation for vinpocetine was foundy=17563x-50470 by plotting peak area (y) versus the concentration (x) studied from 160 to 240 μgmL−1, and the correlation coefficient (R2=0.999) was highly significant. The validity of the assay was verified by means of the ANOVA. According to it, there is linear regression and there is no deviation from linearity (P<0.05).


### 3.1.3. Specificity

A typical HPLC chromatogram of vinpocetine standard preparation (a) and vinpocetine test sample (b) are shown in Figure2. The HPLC chromatograms recorded for the mixture of the inactive ingredients revealed no peaks within retention time around 10.5 minutes, and the peak purity was 99.99%. Figure 2 and the peak purity index show that vinpocetine is clearly separated from the response of any interfering peak(s).Typical chromatogram of (a) vinpocetine standard preparation and (b) vinpocetine test sample. (Chromatographic conditions-Zorbax C18, 150 mm length × 4.6 mm ID with 5 μm particle size column; mobile phase-buffer (containing 1.54% w/v ammonium acetate solution): acetonitrile (40 : 60, v/v); flow rate 1.0 mLmin−1; column temperature 30°C; wavelength 280 nm and injected volume 10 μL).
(a)
Chromatogram of vinpocetine standard preparation(b)
Chromatogram of vinpocetine test sampleUnder acidic condition (1N HCl for 8 h), it was found that 1.34% of vinpocetine content was decreased, but there was no detectable degradation peak(s). The samples submitted to alkaline condition (1N NaOH for 8 h) showed 4.57% degradation of vinpocetine content, and also there was no detectable degradation peak(s). In both cases, the peak purity was 99.99%. Again, when the sample was submitted to photolytic degradation (UV light at 254 nm for 1 h), the degradation of vinpocetine was found 0.97% and the peak purity was 99.99%. The results obtained from the peak purity tool demonstrated that the peak response of vinpocetine was pure in all cases and thus confirming the absence of other substance in the same retention time. The results of percentage of forced degradations are shown in Table2.Table 2
Results of vinpocetine exposed to different degradative pathways.
ParameterAmount added (μgmL−1)Amount recovered (μgmL−1)*Percentage of degradationAcidic degradation200197.321.34Alkaline degradation200190.864.57Photolytic degradation200198.060.97*Mean of three replicates.


### 3.1.4. Accuracy

The results were expressed as percent recoveries of the particular components in the samples. The overall results of percent recoveries (mean ±  %RSD) of vinpocetine in pure and drug-matrix solutions are demonstrated in Table3, indicating good accuracy of the proposed HPLC method.Table 3
Accuracy studies of vinpocetine in standard and drug-matrix solutions.
Amount added (μgmL−1)Peak areaAmount recovered (μgmL−1)% RecoveryRecovery (Mean ±  %RSD)Standard solution1602758830160.12100.081602757741160.06100.04100.05 ± 0.0231602757692160.05100.041803108421180.41100.231803108988180.44100.25100.24 ± 0.0121803109134180.45100.252003463527201.02100.512003463889201.04100.52100.52 ± 0.0092003464117201.05100.532203811741221.23100.562203812077221.25100.57100.56 ± 0.0072203811562221.22100.562404168397241.93100.802404167556241.88100.78100.79 ± 0.0122404167502241.88100.78Drug-matrix solution1602773303160.96100.601602772297160.90100.56100.57 ± 0.0241602772063160.89100.561803119142181.03100.601803121001181.14100.66100.63 ± 0.0301803119725181.06100.622003477174201.81100.902003477005201.80100.90100.91 ± 0.0152003477987201.86100.922203831775222.39101.102203831000222.35101.08101.08 ± 0.0252203829872222.28101.052404185580242.92101.202404186378242.97101.22101.21 ± 0.0112404185596242.92101.20


### 3.1.5. Precision

The values of %RSD for intraday and interday variation are given in Table4. In both cases, %RSD values were found well within 2% limit, indicating that the current method is repeatable.Table 4
Intraday and interday precision of HPLC method.
Sr. no.Concentration (μgmL−1)Standard solutionSample solutionIntra-day precisionInter-day precisionIntra-day precisionInter-day precision120034795013484566348133234892142200347900534841123481987348974132003478987348399134822443490301420034792273484977348151034897485200347980834850043481993348984962003479777348466934822143490488Mean3479384348455334818803489890SD367.100425.941375.626453.331%RSD0.0110.0120.0110.013


### 3.1.6. Sensitivity

The LOD and LOQ of vinpocetine by the proposed method were found 0.0968μgmL−1 and 0.2904 μgmL−1, respectively. Figure 3 shows the sensitivity of the current method.Typical chromatogram of (a) blank, (b) LOD of vinpocetine, and (c) LOQ of vinpocetine. (Chromatographic conditions-Zorbax C18, 150 mm length × 4.6 mm ID with 5 μm particle size column; mobile phase-buffer (containing 1.54% w/v ammonium acetate solution): acetonitrile (40 : 60, v/v); flow rate 1.0 mLmin−1; column temperature 30°C; wavelength 280 nm and injected volume 10 μL).
(a)
Chromatogram of blank(b)
Chromatogram of LOD of vinpocetine(c)
Chromatogram of LOQ of vinpocetine


### 3.1.7. Ruggedness

The results (% of Recovery ± Standard Deviation of six assay samples) are given in Table5, indicating the ruggedness of the current method.Table 5
Ruggedness of the method.
SampleTaken amount of standard vinpocetine (mg)Analyst-1Analyst-2Amount found (mg)% Recovery ± SD*Amount found (mg)% Recovery ± SD*Avintol 5 mg Tablet1010.04100.4 ± 0.0810.01100.1 ± 0.09*% of Recovery ± Standard Deviation of six samples (assay).


### 3.1.8. Robustness

The % of RSD of robustness testing under different altered conditions is given in Table6, indicating that the current method is robust.Table 6
Robustness of the method.
ParameterAmount of vinpocetine added (μgmL−1)Amount of vinpocetine detected (Mean ± SD)*%RSDChange in mobile phase compositionBuffer : ACN = 38 : 62200200.33 ± 0.290.14Buffer : ACN = 40 : 60200200.47 ± 0.290.14Buffer : ACN = 42 : 58200200.56 ± 0.550.27Change in column temperature25°C200200.43 ± 0.710.3530°C200200.36 ± 0.500.2335°C200199.88 ± 0.560.28Change in flow rate0.9 mLmin−1200200.08 ± 0.650.331.0 mLmin−1200200.41 ± 0.310.151.1 mLmin−1200200.46 ± 0.610.31*Mean ± Standard Deviation of six replicates.


## 3.1.1. System Suitability

The results (Mean ±  %RSD of six replicates) of the chromatographic parameters are shown in Table1, indicating the good performance of the system.Table 1
Chromatographic characteristics of system suitability solution.
ParameterValue (Mean ±  %RSD)*Peak area3457550 ± 0.096Tailing factor1.109 ± 0.378Theoretical plate2945.667 ± 0.099Retention time10.581 ± 0.083*Mean and % Relative Standard Deviation of six replicates.


## 3.1.2. Linearity

The regression equation for vinpocetine was foundy=17563x-50470 by plotting peak area (y) versus the concentration (x) studied from 160 to 240 μgmL−1, and the correlation coefficient (R2=0.999) was highly significant. The validity of the assay was verified by means of the ANOVA. According to it, there is linear regression and there is no deviation from linearity (P<0.05).


## 3.1.3. Specificity

A typical HPLC chromatogram of vinpocetine standard preparation (a) and vinpocetine test sample (b) are shown in Figure2. The HPLC chromatograms recorded for the mixture of the inactive ingredients revealed no peaks within retention time around 10.5 minutes, and the peak purity was 99.99%. Figure 2 and the peak purity index show that vinpocetine is clearly separated from the response of any interfering peak(s).Typical chromatogram of (a) vinpocetine standard preparation and (b) vinpocetine test sample. (Chromatographic conditions-Zorbax C18, 150 mm length × 4.6 mm ID with 5 μm particle size column; mobile phase-buffer (containing 1.54% w/v ammonium acetate solution): acetonitrile (40 : 60, v/v); flow rate 1.0 mLmin−1; column temperature 30°C; wavelength 280 nm and injected volume 10 μL).
(a)
Chromatogram of vinpocetine standard preparation(b)
Chromatogram of vinpocetine test sampleUnder acidic condition (1N HCl for 8 h), it was found that 1.34% of vinpocetine content was decreased, but there was no detectable degradation peak(s). The samples submitted to alkaline condition (1N NaOH for 8 h) showed 4.57% degradation of vinpocetine content, and also there was no detectable degradation peak(s). In both cases, the peak purity was 99.99%. Again, when the sample was submitted to photolytic degradation (UV light at 254 nm for 1 h), the degradation of vinpocetine was found 0.97% and the peak purity was 99.99%. The results obtained from the peak purity tool demonstrated that the peak response of vinpocetine was pure in all cases and thus confirming the absence of other substance in the same retention time. The results of percentage of forced degradations are shown in Table2.Table 2
Results of vinpocetine exposed to different degradative pathways.
ParameterAmount added (μgmL−1)Amount recovered (μgmL−1)*Percentage of degradationAcidic degradation200197.321.34Alkaline degradation200190.864.57Photolytic degradation200198.060.97*Mean of three replicates.


## 3.1.4. Accuracy

The results were expressed as percent recoveries of the particular components in the samples. The overall results of percent recoveries (mean ±  %RSD) of vinpocetine in pure and drug-matrix solutions are demonstrated in Table3, indicating good accuracy of the proposed HPLC method.Table 3
Accuracy studies of vinpocetine in standard and drug-matrix solutions.
Amount added (μgmL−1)Peak areaAmount recovered (μgmL−1)% RecoveryRecovery (Mean ±  %RSD)Standard solution1602758830160.12100.081602757741160.06100.04100.05 ± 0.0231602757692160.05100.041803108421180.41100.231803108988180.44100.25100.24 ± 0.0121803109134180.45100.252003463527201.02100.512003463889201.04100.52100.52 ± 0.0092003464117201.05100.532203811741221.23100.562203812077221.25100.57100.56 ± 0.0072203811562221.22100.562404168397241.93100.802404167556241.88100.78100.79 ± 0.0122404167502241.88100.78Drug-matrix solution1602773303160.96100.601602772297160.90100.56100.57 ± 0.0241602772063160.89100.561803119142181.03100.601803121001181.14100.66100.63 ± 0.0301803119725181.06100.622003477174201.81100.902003477005201.80100.90100.91 ± 0.0152003477987201.86100.922203831775222.39101.102203831000222.35101.08101.08 ± 0.0252203829872222.28101.052404185580242.92101.202404186378242.97101.22101.21 ± 0.0112404185596242.92101.20


## 3.1.5. Precision

The values of %RSD for intraday and interday variation are given in Table4. In both cases, %RSD values were found well within 2% limit, indicating that the current method is repeatable.Table 4
Intraday and interday precision of HPLC method.
Sr. no.Concentration (μgmL−1)Standard solutionSample solutionIntra-day precisionInter-day precisionIntra-day precisionInter-day precision120034795013484566348133234892142200347900534841123481987348974132003478987348399134822443490301420034792273484977348151034897485200347980834850043481993348984962003479777348466934822143490488Mean3479384348455334818803489890SD367.100425.941375.626453.331%RSD0.0110.0120.0110.013


## 3.1.6. Sensitivity

The LOD and LOQ of vinpocetine by the proposed method were found 0.0968μgmL−1 and 0.2904 μgmL−1, respectively. Figure 3 shows the sensitivity of the current method.Typical chromatogram of (a) blank, (b) LOD of vinpocetine, and (c) LOQ of vinpocetine. (Chromatographic conditions-Zorbax C18, 150 mm length × 4.6 mm ID with 5 μm particle size column; mobile phase-buffer (containing 1.54% w/v ammonium acetate solution): acetonitrile (40 : 60, v/v); flow rate 1.0 mLmin−1; column temperature 30°C; wavelength 280 nm and injected volume 10 μL).
(a)
Chromatogram of blank(b)
Chromatogram of LOD of vinpocetine(c)
Chromatogram of LOQ of vinpocetine


## 3.1.7. Ruggedness

The results (% of Recovery ± Standard Deviation of six assay samples) are given in Table5, indicating the ruggedness of the current method.Table 5
Ruggedness of the method.
SampleTaken amount of standard vinpocetine (mg)Analyst-1Analyst-2Amount found (mg)% Recovery ± SD*Amount found (mg)% Recovery ± SD*Avintol 5 mg Tablet1010.04100.4 ± 0.0810.01100.1 ± 0.09*% of Recovery ± Standard Deviation of six samples (assay).


## 3.1.8. Robustness

The % of RSD of robustness testing under different altered conditions is given in Table6, indicating that the current method is robust.Table 6
Robustness of the method.
ParameterAmount of vinpocetine added (μgmL−1)Amount of vinpocetine detected (Mean ± SD)*%RSDChange in mobile phase compositionBuffer : ACN = 38 : 62200200.33 ± 0.290.14Buffer : ACN = 40 : 60200200.47 ± 0.290.14Buffer : ACN = 42 : 58200200.56 ± 0.550.27Change in column temperature25°C200200.43 ± 0.710.3530°C200200.36 ± 0.500.2335°C200199.88 ± 0.560.28Change in flow rate0.9 mLmin−1200200.08 ± 0.650.331.0 mLmin−1200200.41 ± 0.310.151.1 mLmin−1200200.46 ± 0.610.31*Mean ± Standard Deviation of six replicates.


## 4. Conclusion

The developed RP-HPLC method for the determination of vinpocetine is simple, precise, accurate, reproducible, and highly sensitive. The developed method was validated based on USP and ICH guidelines [12, 13]. Hence, this method can be used for the routine determination of vinpocetine in pure and pharmaceutical formulations.



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*Source: 801656-2011-11-15.xml*