Research Article

Analytical method validation for estimation of avanafil and dapoxetine hydrochloride tablet dosage form by HPTLC method

Dhwani A. Shah*, Kunjal L. Vegad, Ekta D. Patel, Hitesh K. Prajapati, Ronak N. Patel, Yogesh K. Patel

Sharda School of Pharmacy, Gandhinagar, Pethapur, Gujarat, India

*For correspondence

Dhwani A. Shah,

Sharda School of Pharmacy, Gandhinagar, Pethapur, Gujarat, India.

Email: shahdhwani57 @gmail.com

 

Received: 02 May 2017

Revised: 19 May 2017

Accepted: 23 May 2017

ABSTRACT

Objective: A simple, specific, accurate and precise RP-HPTLC method has been developed and validated for simultaneous estimation of Avanafil and Dapoxetine.

Methods: The chromatographic separation was achieved on Aluminium plates precoated with Silica gel 60 F254 using chloroform: methanol: ethyl acetate: glacial acetic acid (5:2:3:0.2, v/v/v/v) as mobile phase detected at 279 nm.

Results: The correlation coefficient for RP-HPLC method was found to be 0.9987 for Avanafil and 0.9991 Dapoxetine and the linearity range was found to be 1040-3640 ng*spot-1 for Avanafil and 80-280 ng*spot-1 for Dapoxetine.

Conclusions: The developed method was successfully applied to marketed tablet dosage form and the results were found with higher confidence.

 

Keywords: Avanafil, Dapoxetine, RP-HPTLC, Validation

Introduction

Avanafil, a selective phosphodiesterase (PDE) inhibitor, vasodilating agent.1,2

Avanafil is indicated for treatment of erectile dysfunction. When sexual stimulation occurs, Nitric Oxide (NO) is released, which stimulates a specific enzyme to produce more cGMP, a biologically active compound that causes blood vessel dilation. This leads to filling of corpus cavernosum with blood and erection occurs.

However, the action of cGMP is terminated by PDE5, which destroys cGMP thus ending its action. Avanafil inhibits PDE5 and thus greatly prolonging the life of cGMP and causing a powerful erection in men suffering from erectile dysfunction.

Dapoxetine hydrochloride

Dapoxetine hydrochloride is white to off-white crystalline solid.3,4

Dapoxetine hydrochloride is selective serotonin reuptake inhibitor, inhibit the serotonin reuptake and prolong the ejaculation time. It inhibits serotonin reuptake centrally in nucleus paragigantocellularis and gives beneficial effect into premature ejaculation.

Introduction to dosage form5

The tablet super Avana contains the combination of Avanafil (100 mg) and Dapoxetine (60 mg). The Sunrise Remedies Pvt Ltd has manufactured the tablet which is used for treatment of erectile dysfunction.

The primary indication of Avanafil is treatment of erectile dysfunction (inability to sustain a satisfactory erection to complete intercourse). Data clearly indicates that men taking Dapoxetine for the treatment of PE experienced significant improvements in sexual function, as well as in ejaculatory control, satisfaction with sexual intercourse for men and their partners, and increases in IELT (intravaginal ejaculatory latency time).

These data collection were presented at the 100th Annual Scientific Meeting of the AUA (American Urological Association).

Materials and Methods

Preparation of standard stock solution of DAP

Accurately weighed 50 mg DAP was transferred to 50 ml volumetric flask, dissolved in methanol and diluted up to the mark with methanol to give a stock solution having strength 1.0 mg•mL-1 (1000 μg•ml-1).

Preparation of working standard solution of DAP

100 μg•ml-1 of DAP working standard solution was prepared by diluting 1.0 ml of stock solution to 10 mL with methanol.

Preparation of standard stock solution of AVN

Accurately weighed 50 mg AVN was transferred into 50 mL volumetric flask, dissolved in methanol and diluted up to the mark with methanol to give a stock solution having strength 1.0 mg•mL-1 (1000 μg•mL-1).

Preparation of working standard solution of AVN

100 μg•mL-1 of AVN working standard solution was prepared by diluting 1 mL of stock solution to 10 mL with methanol.

Preparation of combined standard solutions of DAP and AVN

Aliquot of 0.2 mL and 2.6 mL were taken from respective standard stock solution of DAP (1000 μg•mL-1) and AVN (1000 μg•mL-1) to 10 mL of volumetric flask and volume made up with methanol to produce mixture solution having the concentration of 20 and 260 μg•mL-1 of DAP and AVN respectively.

Preparation of sample solution of DAP and AVN

Twenty capsules were weighed and tablets were taken out. Tablets were crushed. Powder equivalent to 10 mg of DAP and 130 mg of AVN was transferred to 100 mL volumetric flask. 70 mL of methanol was added and the mixture was subjected to sonication. Volume made up to mark with methanol. The solution was filtered using Whatman filter paper no. 42. Aliquot of 2 mL was diluted up to 10 ml with methanol to achieve strength of 20 μg•mL-1 of DAP and 260 μg•mL-1 of AVN.

Chromatographic conditions

The chromatographic estimations were performed using following conditions:

Stationary phase: Aluminium plates precoated with Silica gel 60 F254 (10×10 cm2)

Mobile phase: Chloroform: Methanol: Ethyl acetate: Acetic acid (5:2:3:0.2)

Quantity of mobile phase: 10.2 mL

TLC Chamber saturation: 30 min

Migration distance: 90 mm

Applicator parameters

– Syringe: 100 μl

– Application rate: 150 nL·s-1

– Band width: 6 mm

– Distance from the plate edge: 15 mm

– Distance from the bottom of the plate: 15 mm

Scanning parameters

– Slit dimension: 4 mm × 0.30 mm

– Scanning speed: 20 mm·s-1

– Detection wavelength: 279 nm

– Lamp: D2

– Measurement mode: Absorption

Method Validation

Specificity

The peak purity of DAP and AVN were assessed by comparing respective spectrum of standard and sample at peak start, apex, and peak end positions of the spot. Good correlation (r =0.9990) was obtained between standard and sample spectra of DAP and AVN respectively.

Linearity and range

Calibration curve were plotted over a concentration range of 80-280 ng•spot-1 and 1040-3640 ng•spot-1 for DAP and AVN respectively. For the calibration curve combined standard solution of DAP (4, 6, 8, 10, 12, 14 μl) and AVN (4, 6, 8, 10, 12, 14 μl) were spotted on pre-coated TLC plate under nitrogen stream using Linomat-V automatic spotter. Linear ascending development was carried out in 10 × 10 cm2 twin trough glass chamber saturated with the mobile phase for 30 min. The plate was removed from the chamber, subsequently dried in a current of air. Densitometric scanning was performed on Camag TLC scanner IV in the reflectance-absorption mode at 279 nm. Peak areas were recorded for all the peaks. The same plate was developed in the second mobile phase as described. The calibration curves for DAP and AVN were constructed by plotting peak area versus concentration (ng•spot-1) corresponding to each spot. Each concentration was spotted five times on the TLC plate.

Precision

Repeatability

System precision

System precision was determined by preparing the standard solution of DAP (160 ng•spot-1) and AVN (2080 ng•spot-1) six times and analyzed as per the proposed method.

Method precision

Method precision was determined by preparing the standard solution of DAP (160 ng•spot-1) and AVN (2080 ng•spot-1) six times and analyzed each solution once as per the proposed method.

Intra-day precision

Combined standard solution of DAP (20 μg•mL-1) and AVN (260 μg•mL- 2) were analyzed three times on the same day.

Inter-day precision

Combined standard solution of DAP (20 μg•mL-1) and AVN (260 μg•mL- 2) were analyzed on three different days.

Accuracy

Recovery study was performed by standard addition method. Twenty capsules were weighed and from that tablets were taken, crushed. Powder equivalent to 25 mg of DAP and 325 mg of AVN was weighed and transfer into 25 mL of volumetric flask. Twenty mL of methanol was added. The mixture was subjected to sonication for 15 min and diluted up to mark with methanol. The solution was filtered using Whatman filter paper no. 42 and first few drops of filtrate were discarded. Aliquot of 1 mL was taken in 10 ml volumetric flask and diluted up to mark with methanol (100 μg•mL-1 of DAP and 1300 μg•mL-1). Two mL of solution was taken into 4 different volumetric flasks. Then 1.6, 2.0, 2.4 mL of solution of DAP (100 μg•mL-1) and 2.08, 2.60, 3.12 mL of solution of AVN (1000 μg•mL-1) were spiked in different 3 volumetric flask and dilute it up to 10 ml with methanol. 6 μL of the solution from each volumetric flask was spotted on TLC plate for analysis. Area of peak obtained with each solutionwas measured for DAP and AVN. The recovery experiments were carried out at 80%, 100% and 120%. The experiment was repeated three times.

Limit of detection (LOD) and limit of quantification (LOQ)

The limit of detection and the limit of quantification of the drugs were calculated using the formula given below. To perform the LOD and LOQ, average slope of five calibration curves and standard deviation of average intercept of five calibration curves were taken and LOD and LOQ were calculated using following equations as per International Conference on Harmonization (ICH) guidelines.

LOD = 3.3* σ/S

LOQ = 10 *σ/S

Where, "σ‟ is the standard deviation of the peak areas of the drug 'S' is the slope of the corresponding calibration curve

Analysis of the marketed formulations

Twenty tablets were weighed and were taken out, crushed. Powder equivalent to 10 mg of DAP or 130 mg of AVN was transferred to a 100 mL volumetric flask, dissolved and diluted up to 100 mL with methanol. The mixture was subjected to sonication for 15 min for complete extraction of the drugs and filtered. Aliquot of 2 ml was taken, diluted up to 10 mL with methanol to achieve concentration of 20 μg•mL-1 of DAP and 260 μg•mL-1 of AVN. 6 μL of the solution was spotted on the HPTLC plate for analysis. The amount of DAP and AVN per tablet was determined from the calibration curve.

Results and Discussion

Selection of wavelength

For the selection of wavelength, standard solution of DAP (10 μg•mL-1 in methanol) and AVN (10 μg•mL-1 in methanol) were prepared and scanned in the range of 200-400 nm. Both the drugs show reasonably good absorbance at 279 nm.

Method development

Figure 1: Overlain 3-D chromatogram of AVN (1040-3640 ng·spot-1) at 279 nm.

Figure 2: Comparison spectra of AVN in standard and in respective sample at peak start, peak apex and peak end.

Figure 3: Comparison spectra of DAP in standard and in respective sample at peak start, peak apex and peak end.

HPTLC method was optimized with a view to develop an assay method for the simultaneous estimation of DAP and AVN in bulk and combined dosage form. Both the pure drugs were spotted on the TLC plates and were run in different solvent systems. The mobile phase chloroform: methanol: ethyl acetate: glacial acetic acid (5:2:3:0.2, v/v/v/v) gave better resolution with improved characteristics with Rf values After the method was developed on small TLC plate, spotting was done on pre-coated silica gel aluminium HPTLC plate 60F254 (10×10 cm2) using Hamilton syringe (100 μl) on Linomat V automatic sample applicator. After spotting, it was dried for few minutes & placed in a twin-trough flat-bottom TLC chamber previously saturated with mobile phase. After development, scanning was performed in the reflectance-absorption mode using a UV detector in the range of 200-400 nm. The same plate was developed in second mobile phase and during this the run of the mobile was up to the mark below the spot run of AVN. The same procedure was followed as before. Well defined spots were obtained when the chamber was saturated with mobile phase for 30 min at room temperature. Overlain 3-D Chromatogram shown in Figure 1. Comparison spectra of standard and sample of both the drug shown in Figure 2 and 3.

Table 1: Data of peak purity of sample AVN and DAP.

Measurements

Substance (n=3)

r (s, m)

Sample AVN

Sample DAP

0.9998

0.9997

0.9997

0.9999

0.9999

0.9998

r (m, e)

0.9998

0.9997

0.9997

0.9996

0.9999

0.9996

Table 2. Data of peak purity of standard and sample of both the drug.

Measurements

Substance (n=3)

 

Standard AVN

Sample AVN

Standard DAP

Sample DAP

r (s, m)

0.9997

0.9996

0.9999

0.9999

r (m,e)

0.9994

0.9996

0.9999

0.9991

Table 3: Linearity data for DAP and AVN.

Sr. No.

Concentration

(ng•spot-1)

Area Mean ± SD (n=5)

% RSD

 

DAP

AVN

DAP

AVN

DAP

AVN

1

80

1040

1134.01 ± 19.42

8830.57 ± 178.12

1.71

2.01

2

120

1560

1551.61 ± 27.96

10292.15 ± 146.71

1.80

1.42

3

160

2080

1951.27 ± 40.47

11465.61 ± 159.86

2.07

1.39

4

200

2600

2314.77 ± 23.38

12912.56 ± 240.66

1.01

1.89

5

240

3120

2662.43 ± 30.17

14066.40 ± 209.70

1.01

1.49

6

280

3640

3081.84 ± 27.76

15338.64 ± 158.15

0.90

1.03

Table 4: Repeatability data for DAP and AVN.

DAP

concentration

(ng•spot-1)

AVN

concentration

(ng•spot-1)

System precision

Method precision

Peak area

Peak area

 

DAP

AVN

DAP

AVN

160

2080

1951.56

11489.35

1946.99

11426.31

160

2080

1942.69

11454.84

1955.64

11454.84

160

2080

1949.53

11435.52

1942.61

11412.56

160

2080

1952.98

11487.52

1925.45

11395.64

160

2080

1947.87

11495.85

1938.58

11597.82

160

2080

1942.53

11470.90

1959.62

11495.85

Mean

1946.99

11470.90

1946.90

11463.84

SD

4.91

23.73

13.75

74.49

%RSD

0.25

0.20

0.70

0.65

Method validation

Specificity

The peak purity of AVN and DAP was assessed by comparing respective spectra of standard and sample at peak start, apex and peak end positions of the spot. Specificity data is shown in.

Linearity and Range

The linearity study was carried out for both drugs at five different concentration levels. The linearity of DAP and AVN was in the range of 80 - 280 ng•spot-1 and 1040-3640 ng•spot-1 respectively. Calibration curves are depicted in Figure 4 (DAP) and 5 (AVN) and linearity data is shown in Table 3. Correlation co-efficient for calibration curve of DAP and AVN was found to be 0.9991 and 0.9987 respectively.

Figure 4: Calibration curve of DAP.

Figure 5: Calibration curve of AVN.

Table 5: Intraday precision data for estimation of DAP and AVN.

Sr. No. Concentration (ng•spot-1) Area Mean ± SD (n=5) % RSD
  DAP AVN DAP AVN DAP AVN
1 80 1040 1127.91±21.97 8787.82 ±130.18 1.94 1.48
2 120 1560 1567.29±25.22 10369.74±155.16 1.60 1.17
3 160 2080 1974.58±34.75 11461.75±214.85 1.76 1.87
4 200 2600 2327.46±19.61 13025.43±66.48 0.84 0.51
5 240 3120 2680.28±19.88 14132.95±119.26 0.74 0.84
6 280 3640 3099.80±17.62 15410.17±61.94 0.56 0.40

The regression line equation for DAP is: y= 9.596 x + 388.6

The regression line equation for AVN is: y= 2.489 x + 6325

Where, y= peak area, x= concentration of DAP or AVN (ng•spot-1).

Precision

A. Repeatability

The data for repeatability of peak area measurement for DAP and AVN are depicted in Table 4.

a. Intraday precision

The data for intraday precision for DAP and AVN is shown in Table 5. The %RSD for Intraday precision was found to be 0.56 – 1.94 for DAP and 0.40 – 1.87 for AVN.

b. Interday precision

The data for interday precision for DAP and AVN is shown in Table 6. The%RSD for interday precision was found to be 0.91 - 2.26 for DAP and 0.84 - 2.79 for AVN.

Accuracy

Accuracy of the method was confirmed by recovery study from marketed formulation (SUPERAVANA) at three level (80%, 100%, 120%) of standard addition. The results are shown in Table 7 for DAP and Table 8 for AVN. Percentage recovery for DAP and AVN was found to be in range of 99.46 – 101.04 and 99.00 – 100.21.

Table 6: Interday precision data for estimation of DAP and AVN.

Sr. No. Concentration (ng•spot-1) Area Mean ± SD (n=5) % RSD
  DAP AVN DAP AVN DAP AVN
1 80 1040 1142.87±19.97 08811.14±246.17 1.74 2.79
2 120 1560 1548.28±35.05 10200.66±86.083 2.26 0.84
3 160 2080 1934.49±31.99 11386.75±164.44 1.65 1.43
4 200 2600 2308.97±25.12 12812.13±279.24 1.08 2.18
5 240 3120 2656.28±38.82 13976.13±239.56 1.46 1.71
6 280 3640 3070.99±28.23 15297.79±200.61 0.91 1.31

Table 7: Recovery data for DAP.

DAP concentration in Sample (ng•spot-1) Amount of standard DAP added (ng•spot-1) Total concentration (ng•spot-1) Mean concentration recovered (ng•spot-1) % Recovery
120 0 120 119.83 99.86
120 96 216 216.63 100.29
120 120 240 242.50 101.04
120 144 264 262.57 99.46

Table 8: Recovery data for AVN.

AVN concentration in Sample (ng•spot-1) Amount of standard AVN added (ng•spot-1) Total concentration (ng•spot-1) Mean concentration recovered (ng•spot-1) % Recovery
1560 0 1560 1559.53 99.97
1560 1248 2808 2809.97 100.07
1560 1560 3120 3126.55 100.21
1560 1872 3432 3397.68 99.00

Table 9: Summary of validation parameters of HPTLC method.

Sr no. Parameters Result for DAP Result for AVN
1 Linearity Range (ng•spot-1) 80-280 1040-3640
2. Regression line equation y=9.596x+388.6 y=2.489x+6325
3. Correlation coefficient 0.9991 0.9987
4. Precision (%RSD) Repeatability System precision (n=6) 0.25 0.20
Method precision (n=6) 0.70 0.65
Intraday precision (n=3) 0.56-1.94 0.40-1.87
Interday precision (n=3) 0.91-2.26 0.84-2.79
5. Accuracy (%recovery) (n=3) 99.46-101.04 99.00-100.21
  LOD 10.43 159.68
  LOQ 31.62 483.89

LOD and LOQ

Calibration curve was repeated five times and the standard deviation (SD) of the intercepts was calculated. All the data related to LOD and LOQ is shown in table no 9 for both the drug. The LOD and LOQ were calculated as follows: -

LOD=3.3 × σ/S

LOQ=10 × σ/S

Where, σ = standard deviation of intercepts of five calibration curves, S= mean slope of five calibration curve.

Table 10: Analysis of DAP and AVN in formulation by proposed method.

Formulations

Label claim

(mg)

% Amount found DAP

Tablet

DAP

AVN

DAP

AVN

60

100

97.43

98.65

Assay

The proposed method was applied to analyze the combined dosage form of DAP and AVN. Marketed tablet preparation was analyzed by proposed method. The percentage of DAP and AVN was found to be 97.43% and 98.65% of labeled claim respectively in formulation. Data is shown in Table 10.

Validation of HPTLC method was found to be linear, accurate, precise, specific and robust according to acceptance criteria and with high level of LOD and LOQ.

Linearity of DAP and AVN is found be 0.9991 and 0.9987 respectively which is appropriate % RSD of all the other parameters is found <2 which is also within in the limit and the percentage of DAP and AVN was found to be 97.43% and 98.65% of labeled claim respectively.

Acknowledgements

The authors would like to thank Red son pharmaceutical Pvt. Ltd. India for providing a gift sample of standard Avanafil and Dapoxetine Hydrochloride.

Funding: No funding sources

Conflict of interest: None declared

References

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