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RP-HPLC with Time Programmed FluorescenceDetection for Quantitation of Avanafil and DapoxetineHydrochloride; Application to Pharmaceutical DosageForm and Biological FluidM. Hegazya, A. Kessibab, M. Abdelkawyac & A. E. El-Gindyb

a Analytical Chemistry Department, Faculty of Pharmacy, Cairo University, Cairo, Egyptb Pharmaceutical Chemistry Department, Faculty of Pharmacy, Misr International University,Cairo, Egyptc Pharmaceutical Chemistry Department, Faculty of Pharmacy, Future University, New Cairo,EgyptAccepted author version posted online: 31 Aug 2015.

To cite this article: M. Hegazy, A. Kessiba, M. Abdelkawy & A. E. El-Gindy (2015): RP-HPLC with Time ProgrammedFluorescence Detection for Quantitation of Avanafil and Dapoxetine Hydrochloride; Application to Pharmaceutical DosageForm and Biological Fluid, Journal of Liquid Chromatography & Related Technologies, DOI: 10.1080/10826076.2015.1087860

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1

RP-HPLC with time programmed fluorescence detection for quantitation of

Avanafil and Dapoxetine Hydrochloride; application to pharmaceutical dosage form

and biological fluid

M. Hegazy1, A. Kessiba

2, M. Abdelkawy

1,3, A. E. El Gindy

2

1Analytical Chemistry Department, Faculty of Pharmacy, Cairo University, Cairo,

Egypt2Pharmaceutical Chemistry Department, Faculty of Pharmacy, Misr International

University, Cairo, Egypt3Pharmaceutical Chemistry Department, Faculty of Pharmacy,

Future University, New Cairo, Egypt

Corresponding author Dr. Maha A. Hegazy E-mail: mahahgazy@yahoo.com

Abstract

Avanafil (AVN) was recently co-formulated with dapoxetine HCl (DAP) for treatment of

erectile dysfunction and premature ejaculation. Sensitive and simple reversed-phase (RP)

high performance liquid chromatographic method (HPLC) was developed and validated

for their simultaneous determination using tadalafil (TAD) as an internal standard.

Isocratic separation was achieved within run time of only 7.0 min on Eclipse C18 column

(150 mm x 4.6 mm, 5µm particle size) using a mobile phase composed of acetonitrile:

0.15% triethylamine (40: 60, v/v) at pH=4.0 adjusted with o-phosphoric acid. The

analysis was performed at a flow rate of 1.0 mL/min with fluorescence detection at

236/370 nm for AVN, 236/410 nm for DAP and 236/330 for TAD using time

programming. The analytes were determined by their native fluorescence and the

response was linear over concentration ranges of 0.05- 40 and 0.01-30 µg/mL for AVN

and DAP, respectively, with limits of detection of 0.043 and 0.007 µg/mL in a respective

order. The developed method successfully determined AVN and DAP in bulk powder,

tablets and spiked human plasma.

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KEYWORDS: Avanafil; Dapoxetine; Tadalafil; HPLC; fluorescence detection; spiked

human plasma

1. INTRODUCTION

Avanafil, Figure (1a), is a new, highly selective and fast-acting phosphodiestrase type 5

inhibitor used for treatment of erectile dysfunction. It has a more rapid onset and a better

side effect profile relative to currently available PDE5 inhibitors. It exists as a single

enantiomer with S stereochemistry [1-5]. Dapoxetine, Figure (1b), is a short-acting

selective serotonin reuptake inhibitor (SSRI) developed for the treatment of premature

ejaculation [6,7]. Dapoxetine has a unique pharmacokinetic profile compared to other

SSRIs as it is rapidly absorbed and eliminated after oral adminstration [8-11]. Dapoxetine

doesn’t have pharmacokinetic interactions with PDE5 inhibitors so it is used in

combination with several PDE5 inhibitors for the treatment of premature ejaculation [12].

By reviewing literature, only two HPLC methods with UV detection [13, 14], two

spectrophotometric methods [15, 16] and a TLC method [17] were reported for the

simultaneous determination of AVN and DAP in their combined pharmaceutical

formulation. Upon comparison of the proposed HPLC method with the reported ones, the

proposed method is considered the first HPLC method with fluorescence detection for the

simultaneous determination of both drugs where DAP is reported to be fluorescent [18]

but there is no reference of AVN whether fluorescent or not. Furthermore, the proposed

method offers higher sensitivity that enables the determination of the studied drugs in

spiked human plasma.

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Thus, the present work develops a new, simple and sensitive RP-HPLC method with

fluorescence detection for the simultaneous determination of AVN and DAP using TAD

which is a PDE5 inhibitor as internal standard (chemically related material). The method

was validated according to ICH guidelines [19].

2. EXPERIMENTAL

2.1. HPLC Chromatograph

Samples were loaded into a manual Rheodyne injector (model G1328B, USA) equipped

with a 20-µL injector loop using a 100-µL Agilent syringe. HPLC separation and

quantitation were made on Eclipse C18 column (150 × 4.6 mm, 5 µm particle size) with a

mobile phase consisting of acetonitrile: 0.15 % TEA (40: 60, v/v), pH was adjusted to 4

using o-phosphoric acid. The mobile phase was filtered using 0.45 µm membrane filters

(Millipore, Milford, MA, USA) and degassed by ultrasonic vibrations for 15 minutes

prior to use. An isocratic pump model G1310A was used to deliver the mobile phase at a

flow rate of 1 mL/min. The fluorescence detector model G1321A was set at different

excitation/emission wavelengths using time programming. At 0 min, fluorescence

detection was set at 236/370 nm for AVN, at 4.3 min it was changed to 236/410 nm for

DAP and finally at 5.5 min, it was set at 236/330 nm for TAD. Data acquisition was

performed on Agilent LC ChemStation software. All determinations were performed at

room temperature.

2.2. Materials

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2.2.1. Pure Standard

Avanafil, Dapoxetine HCl and Tadalafil standard materials were supplied by El Andalus

Medical company, 6th

of October City, Giza-Egypt.

2.2.2. Pharmaceutical Formulations

Super -Avana® tablets (Batch No. E-827) are labeled to contain 100 mg of Avanafil and

60 mg of Dapoxetine HCl per tablet, produced by Sunrise Remedies Pvt. Ltd., India.

2.2.3. Chemicals And Reagents

All chemicals and reagents used throughout this work were of analytical grade and

solvents were of HPLC grade. Potassium dihydrogen phosphate (Adwic, Egypt), o-

phosphoric acid (Adwic, Egypt), methanol (Sigma – Aldrich, Germany), acetonitrile

(Sigma – Aldrich, Germany) and triethyl amine (Adwic, Egypt).

2.3. Solutions

Stock standard solutions of AVN, DAP and TAD stock standard solutions (100 µg/mL)

were prepared by accurately weighing 10 mg of each drug into 100-mL volumetric flask,

dissolved in and the volume completed with methanol.

2.4. Procedure

2.4.1. Construction Of Calibration Curves

Accurately measured aliquots of AVN and DAP stock standard solutions (100 µg/mL)

equivalent to 0.05 – 40 µg/mL and 0.01– 30 µg/mL, respectively, were transferred into

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two series of 10-mL volumetric flasks and the volumes were completed with the mobile

phase. An aliquot of 20-µL of each solution was injected in triplicate into the

chromatographic system using the previously described conditions.

2.4.2. Application To Pharmaceutical Formulations

Ten tablets of Super-Avana® [labeled to contain 100 mg AVN and 60 mg DAP] were

accurately weighed and finely powdered. An accurate weight equivalent to 10 mg AVN

and 6 mg DAP was transferred into a 100-mL volumetric flask, extracted with 50 mL

methanol in an ultrasonic bath for 10 minutes and diluted to volume with the same

solvent. The solution (100 µg/mL of Avanafil and 60 µg/mL of Dapoxetine ) was shaken

again for 10 minutes in the ultrasonic bath and filtered. An aliquot (1 mL) was transferred

into a 10-mL volumetric flask and the volume was completed with the mobile phase to

produce solution of concentrations equivalent to 10 µg/mL AVN and 6 µg/mL of DAP.

The procedure was completed as previously described and the concentrations of the drugs

were calculated from the corresponding regression equations.

2.4.3. Application To Spiked Human Plasma

Aliquots of 0.9 mL of human plasma were accurately transferred into a series of

centrifugation tubes and spiked with 0.1 mL of mixtures of AVN (1 – 20 µg/mL), DAP

(0.5 – 20 µg/mL) and TAD (5 µg/mL) so that the final concentrations of the drugs were

0.1 – 2 µg/mL, 0.05 – 2 µg/mL and 0.5 µg/mL, respectively. 1 mL of acetonitrile was

added as precipitating agent for proteins then centrifuged at 6000 rpm for 30 min. The

supernatant was then taken and filtered through syringe filters of pore size 0.45 µm into a

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series of eppendorf. An aliquot of 20-µL of each solution was injected into the

chromatographic system and processed according to the previously described conditions.

3. RESULTS AND DISCUSSION

Avanafil was very recently co-formulated with DAP for treatment of erectile dysfunction

and premature ejaculation. Both drugs show native fluorescence and there was no

reported method on their determination by HPLC based on their fluorescent nature. So,

our aim was to develop a simple and sensitive HPLC method with fluorescence detection

for the simultaneous determination of AVN and DAP in combined dosage form and

spiked human plasma using. Tadalafil, being a structurally related compound it was used

as internal standard.

In order to optimize the proposed HPLC method, several trials were carried out to obtain

good and optimum separation. As an approach to green chemistry, ethanol was tried with

water in different ratios instead of acetonitrile but it resulted in bad resolution of AVN

and DAP. Methanol was then tried but it resulted also in bad resolution and higher elution

time. Regarding the Pka values of AVN (Acidic Pka=11.84 and basic Pka=5.89) and

DAP (Pka=8.6), it was found that the maximum permissible pH limit is 4.0. Upon trying

lower pH values, the fluorescence of AVN decreased, so the optimum pH was found to

be 4.0. Triethylamine was added to decrease the tailing of the peak corresponding to DAP

and improve its symmetry, 0.15 %TEA improved the symmetry and at the same time it

resulted in better resolution of both drugs than 0.1 %TEA. Different excitation and

emission wavelengths were tried for both drugs along with the internal standard, it was

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found that the optimum excitation/ emission wavelengths were 236/370 nm, 236/410 nm

and 236/330 nm for AVN, DAP and TAD, respectively at which good detector response

was obtained with symmetrical peaks.

Finally, it was found that Acetonitrile: 0.15% TEA in the ratio (40: 60, v/v, pH adjusted

to 4 using o-phosphoric acid) using TAD as internal standard with a flow rate of 1

mL/min and fluorescence detection at the previously specified wavelengths was most

suitable to get resolved and sharp peaks, Figure (2).

Linear relationships were obtained for AVN and DAP between the relative peak areas

and the corresponding concentrations. The regression equations were computed and

found to be:

. . .1 1

A 0 1729 C 0 0068 r 0 9999

. . .2 2

A 1 0917 C 0 0187 r 0 9999

Where, A1 and A 2 are the relative peak areas, C1 and C2 are the concentrations in µg/mL

of AVN and DAP, respectively and r is the correlation coefficient.

System suitability was checked by calculating different parameters such as capacity

factor, tailing factor, column efficiency (N), selectivity and resolution factors, where the

system was found to be suitable relative to the reference values as shown in Table (I).

The method was validated as per ICH guidelines [19]. The regression and validation

parameters are summarized in Table (II).

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The proposed HPLC method was successfully applied for the determination of AVN and

DAP in pure powdered form with mean percentage recoveries of 101.13 ± 0.688 % and

99.83 ± 0.510 %, respectively, Table (III). It also has been successfully applied for the

determination of both drugs in Super-Avana® tablets. The validity of the method was

further assessed by applying the standard addition technique, Tables (IV, V).

The results obtained for the analysis of AVN and DAP in their pure forms by the

proposed method were statistically compared with those obtained by applying a reported

HPLC method [13]

. The calculated t and F values were less than the tabulated ones at 95

% confidence level, which reveals that there is no significant difference between the two

methods with respect to accuracy and precision, Table (VI).

The high sensitivity of the proposed method allowed the determination of the studied

drugs in spiked human plasma. The drugs were well resolved without interference from

other endogenous compounds, Figures (3 & 4). Linear relationships were obtained for

AVN and DAP between the relative peak areas and the corresponding concentrations in

spiked human plasma. The regression equations were computed and found to be:

. . .1 1

A 0 7847 C 0 0375 r 0 9995

. . .2 2

A 6 9646 C 0 0773 r 0 9997

Where, A1 and A 2 are the relative peak areas, C1 and C2 are the concentrations in µg/mL

of AVN and DAP, respectively and r is the correlation coefficient. The mean percentage

recoveries were 99.01 ± 2.379 % and 99.26 ± 2.405 % for both drugs, respectively, Table

(VII).

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4. CONCLUSION

The suggested method is simple, accurate, selective and sensitive. Application of the

proposed method to the analysis of Avanafil and Dapoxetine HCL in combined dosage

form showed that the excipients didn’t interfere with the determination. The method was

also successfully applied for the determination of the studied drugs in spiked human

plasma without interference from endogenous compounds indicating that the proposed

method could be applied successfully for the simultaneous determination of Avanafil and

Dapoxetine HCl in both pharmaceutical dosage forms and spiked human plasma.

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(11) Buvat, J., Tesfaye, F., Rothman, M., Rivas, D.A., Giuliano, F.; Dapoxetine for

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(15) Mangukiya, M.A., Patel, G.F., Rathod, B.G., Dhaduk, D.M., Maniya, J.B.;

Development and validation of spectroscopic methods for simultaneous estimation of

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(17) Chaudhari, H.H., Sen, D.J., Patel, C.N.; Development and validation of HPTLC

method for simultaneous estimation of avanafil and dapoxetine HCl in tablet dosage

form; World Journal of Pharmacy and Pharmaceutical Sciences, (2015); 4: 1566- 1575.

(18) Hamilton, C.L., Cornpropst, J.D.; Determination of dapoxetine, an

investigational agent with the potential for treating depression, and its mono- and di-

desmethyl metabolites in human plasma using column-switching high-performance liquid

chromatography; Journal of Chromatography B, (1993); 612: 253-261.

(19) International Conference of Harmonization of technical requirements for

registration of pharmaceuticals for human use (ICH), www.nihs.go.jp

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Table I System suitability parameters of the proposed HPLC method.

Parameters Avanafil Dapoxetine

HCl

Limit

Retention time (tR) 3.875 4.819

Resolution ( R ) 4.120 Rs > 1.5

Tailing factor (T) 0.950 1.250 T ≤ 2

Capacity factor (K') 1.923 2.692 K' > 2

Selectivity factor (α) 1.400 α > 1

Column efficiency (N) 5299 6204 N > 2000

Height equivalent to

theoretical plate (HETP)

0.028 0.024 As the HETP decreases, the

column efficiency increases

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Table II validation parameters for the determination of pure Avanafil and Dapoxetine

HCl by the proposed HPLC method.

Parameter Avanafil Dapoxetine HCl

Range 0.05 – 40 µg/mL 0.01 – 30 µg/mL

Slope 0.1729 1.0917

Intercept 0.0068 -0.0187

*SE of the slope 0.0007 0.0029

*SE of the intercept 0.0166 0.0483

Correlation coefficient( r ) 0.9999 0.9999

*LOD 0.0431 0.0070

*LOQ 0.1306 0.0212

Precision

Repeatability % 0.500 0.701

Intermediate precision % 0.063 0.464

*SE: Standard error.

*LOD(Limit of detection)=

33 SD

Slope.

*LOQ (Limit of quantitation)=

10 SD

Slope D

ownl

oade

d by

[10

5.19

3.10

.170

] at

12:

35 3

1 A

ugus

t 201

5

15

Table III Accuracy results of the proposed HPLC method for the determination of

Avanafil and Dapoxetine HCl pure samples.

Avanafil (µg/mL) *Recovery % Dapoxetine (µg/mL) *Recovery %

Taken Found Taken Found

1 1.02 102.00 1 0.99 99.00

3 3.03 101.00 3 2.99 99.67

8 8.11 101.37 7 7.01 100.14

12 12.01 100.08 12 12.02 100.17

25 25.30 101.20 25 25.04 100.16

Mean 101.13 Mean 99.83

RSD% 0.688 RSD% 0.510

* Average of three determinations

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Table IV Results obtained by applying the proposed HPLC method for the determination

of Avanafil and Dapoxetine HCl in Super-Avana® tablets.

Product Avanafil Dapoxetine HCl

Taken

(µg/mL)

Found *Recovery% Taken

(µg/mL)

Found *Recovery%

Super -Avana®

tablets (Batch

No. E-827)

10.00 10.03 100.30 6.00 5.90 98.33

9.92 99.20 6.01 100.17

10.18 101.80 6.08 101.33

Mean 100.43 Mean 99.94

RSD% 1.299 RSD% 1.514

*Average of three determinations.

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Table V Results obtained by applying the proposed HPLC method for the determination

of Avanafil and Dapoxetine HCl by the standard addition technique.

Avanafil Dapoxetine

Taken (µg/mL) Found *Recovery% Taken (µg/mL) Found *Recovery%

8.00 8.08 101.00 5.00 5.04 100.80

10.00 9.90 99.00 6.00 5.98 99.67

12.00 12.11 100.92 8.00 7.94 99.25

Mean 100.31 Mean 99.91

RSD% 1.129 RSD% 0.802

*Average of three determinations.

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Table VI Statistical comparison of the results obtained by the proposed HPLC method

and the reported HPLC method[13]

for the analysis of Avanafil and Dapoxetine HCl in

pure form.

Parameter Avanafil **Reported HPLC

method

Dapoxetine

HCl

**Reported HPLC

method

Mean 101.13 99.92 99.83 99.56

SD 0.696 0.982 0.509 0.917

N 5 5 5 5

Variance 0.485 0.964 0.259 0.842

Student’s

t

2.251

(2.365)*

0.580

(2.447)*

F 1.989

(6.388)*

3.253

(6.388)*

*The values in the parentheses are the corresponding tabulated values at p=0.05.

**HPLC method (C-18, using methanol: acetonitrile: TEA in the ratio of 95: 5: 0.5 v/v/v

at a flow rate of 1 mL/min and detection at 290 nm).

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Table VII Accuracy results of the proposed HPLC method for the determination of

Avanafil and Dapoxetine HCl in spiked human plasma.

Avanafil (µg/mL) *Recovery % Dapoxetine (µg/mL) *Recovery %

Taken Found Taken Found

0.2 0.20 100.00 0.1 0.10 100.00

0.4 0.41 102.50 0.4 0.41 102.50

0.7 0.68 97.14 0.7 0.68 97.14

0.8 0.79 98.75 0.8 0.80 100.00

1.2 1.16 96.67 1.5 1.45 96.67

Mean 99.01 Mean 99.26

RSD% 2.379 RSD% 2.405

*Average of three determinations

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Figure 1a Structure of Avanafil

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Figure 1b Structure of Dapoxetine HCl

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Figure 2 HPLC chromatogram of 10 µg/mL of pure Avanafil (tR = 3.875), 5 µg/mL of

Dapoxetine HCl (tR =4.819) and 3µg/mL of Tadalafil (tR =6.642).

Dow

nloa

ded

by [

105.

193.

10.1

70]

at 1

2:35

31

Aug

ust 2

015

23

Figure 3 HPLC chromatogram of blank plasma.

Dow

nloa

ded

by [

105.

193.

10.1

70]

at 1

2:35

31

Aug

ust 2

015

24

Figure 4 HPLC chromatogram of 0.5 µg/mL of Avanafil (tR = 3.189), 0.5 µg/mL of

Dapoxetine HCl (tR =4.450) and 0.5µg/mL of Tadalafil (tR =6.250) extracted from spiked

plasma.

Dow

nloa

ded

by [

105.

193.

10.1

70]

at 1

2:35

31

Aug

ust 2

015