UV Spectrophotometric Method Development and Validation for the Estimation of Sildenafil and Fluoxetine in Bulk Drug and its Pharmaceutical Formulation

Dr. B. Satya Prasad, Dr. J. N. Suresh Kumar, G. Jaswanth, G. Eswar, P. Nikhila, T. Uma, U. Dheeresh,

doi:10.5281/zenodo.18776897

Research Paper | Open Access
Volume 04 | Issue 02 | Article Id IJPS/260402438

UV Spectrophotometric Method Development and Validation for the Estimation of Sildenafil and Fluoxetine in Bulk Drug and its Pharmaceutical Formulation
Dr. B. Satya Prasad* Dr. J. N. Suresh Kumar G. Jaswanth G. Eswar P. Nikhila T. Uma U. Dheeresh
Narasaraopeta Institute of Pharmaceutical Sciences, Narasaraopet, Palnadu, Andhra Pradesh, India.

Abstract

A simple, rapid, precise and highly selective spectrophotometric method was developed for estimation of Sildenafil citrate and Fluoxetine Hydrochloride in bulk drug and in its tablet dosage form. This method, involves the measurement of absorbances of Sildenafil citrate and Fluoxetine Hydrochloride at the wavelengths of 280nm (?max of Sildenafil citrate) and 247 nm (?max of Fluoxetine Hydrochloride). The method was validated for the linearity, accuracy, precision, robustness, system suitability as per ICH guidelines. Linearity was observed in the concentration range of 10-18?g/ml for Sildenafil citrate and 3-8ug/ml for Fluoxetine Hydrochloride. The accuracy of the method was found to be 98.1% and 99.2% for Sildenafil citrate and Fluoxetine Hydrochloride respectively. The method showed good reproducibility and recovery with % RSD less than 2.

Keywords

Sildenafil citrate, Fluoxetine hydrochloride, Validation

Introduction

Sildenafil citrate is a medication used for the treatment of Erectile Dysfunction (ED) and Pulmonarty aterial hypertension. Chemically sildenafil citrate called as 1-[[3-(6,7-dihydro-1-methyl-7-oxo-3-propyl-1H-pyrazolo[3-d]pyrimidin-5-yl)-4-ethoxyphenyl]sulfonyl]-4-methylpiperazine citrate. ^(1,2) Molecular formula is C₂₂H₃₀N₆O₄S .C₆H₈O₇ and molecular weight is about 666.7g/mol. Sildenafil citrate is a Phosphodiesterase type-5 (PDE-5) Inhibitor, where PDE-5 is the enzyme responsible for the degradation of cyclic guanosine monophosphate (cGMP) in the corpus cavernosum and pulmonary vasculature.^(4,5) By this inhibition sildenafil citrate increases cGMP levels and causes smooth muscle relaxation and increased blood flow where these promotes erection.^(9,13,15) For the treatment of pulmonary aterial hypertension sildenafil causes pulmonary vasodilation by relaxing the smooth muscles. Sildenafil citrate shows several side effects like headache, flushing, dyspepsia, cyanopsia and some serious side effects like priapism, Non Arteritiic Anterior Ischemic Optic Neuropathy (NAION), hearing loss.^(16,17,21)

Fluoxetine is chemically known as (D, L-N-methyl-3-phenyl-3-[(tri-fluorop-tolyl) oxy]propylamine.^(6,10,12,19) Fluoxetine is an antidepressant drug of the selective serotonin reuptake inhibitor (SSRI) class. It was first documented in 1974 by scientists from Eli Lilly and Company. It was presented to the U.S. Food and Drug Administration in February 1977, with Eli Lilly receiving final approval to market the drug in December 1987.^(10,11,12) Fluoxetine went off-patent in August 2001. The molecular formula of fluoxetine hydrochloride is C₁₇H₁₈F₃NO. HCL and the molecular weight is about 345.79g/mol. Fluoxetine hydrochloride is used for the treatment of major depressive disorder, obsessive- compulsive disorder, bulimia nervosa, panic disorder, binge eating disorder, premenstual dysphoric disorder (PMDD).^(24,26) Fluoxetine increases the serotonin levels in the brain by selectively inhibiting the reuptake of serotonin at the presynaptic neuron in the central nervous system, which increases the serotonin availability in synaptic cleft.^{(10, 19)} Higher levels of serotonin helps to alleviate symptoms of depression, anxiety, and other disorders. Fluoxetine is also used in combination with sildenafil which promotes the therapeutic activity of sildenafil and relieves the depression caused by the erectile dysfunction.⁽¹²⁾

For both the drugs the method is developed by using UV spectroscopy. In this we analyze the formulation of sildenafil and fluoxetine and also involved in estimation of pure drugs and finally obtain calibration curve. Validation parameters like accuracy, precision, linearity, robustness are applied and validation is done according to ICH guidelines

Fg no.1: Structure of sildenafil citrate Fgno.2: Structure of fluoxetine hydrochloride

2. EXPERIMENTATION:

2.1 : Materials required :

2.1.1: Chemicals required :

Pure drug samples of sildenafil citrate and fluoxetine hydrochloride.
Marketed products or formulations of Sildenafil citrate (ZENEGERA-100) and Fluoxetine hydrochloride (FLUTOP-20)
Methanol
Hydrochloric acid
Distilled water ^{(16, 18)}

2.1.2 INSTRUMENTS :

UV-Visible double beam Spectrophotometer (Model No: ANALYTICAL TS2080 Plus)
Electronic Balance (Infra Digi - EI)
Whatman filter paper (no. 41)
Ultra Sonicator (Kshitis 1834) ^(26,27)

Fg no.3: UV-Visible double beam spectrophotometer Fg no.4: Electronic balance

2.2 METHOD DEVELOPMENT :

2.2.1 : Solvent Selection:

Sildenafil citrate and fluoxetine hydrochloride solutions can be prepared by using different solvents like methanol, ethanol, 0.1M HCl and 0.1M NaOH.^(7,8) UV spectrum of each were recorded by scanning between 200-400 nm. Mostly methanol is preffered because it shows better absorbance of both drugs without any distractions and errors.^(9,11)

2.2.2. Standard Solution of Sildenafil Citrate:

Accurately weighed 10mg of sildenafil citrate was transferred into clean, dry 100mL volumetric flask and dissolved with small volume of methanol. The volume was made upto 100mL with methanol to get concentration of 100 µg/mL.⁽¹⁴⁾

2.2.3. Standard Solution of Fluoxetine hydrochloride:

Accurately weighed 10mg of fluoxetine was transferred into clean, dry 100mL volumetric flask and dissolved with sufficient volume of methanol. The volume was made upto 100mL with methanol to get concentration of 100µg/mL.

2.2.4. Selection of Analytical wavelength:

Standard solutions of both the drugs which are previously prepared are dilued to appropriate concentrations (1, 1.2, 1.4, 1.6 ml in 10ml volumetric flasks and make up the voume upto 10ml with methanol ^(16,18) and 0.3, 0.4, 0.5, 0.6 in 10ml volumetric flasks and make up to volume upto 10ml with methanol for sildenafil and fluoxetine respectively, these are working standard solutions ) and these are scanned in the Uv range of 200 to 400nm, using methanol as blank.⁽²³⁾

Graph 1: Absorbance maxima of sildenafil citrate at 280nm

Graph 2: Absorbance maxima of fluoxetine hydrochloride at 247nm

2.2.5: DETERMINATION or CALIBRATION CURVE :

Working standard solutions of sildenafil citrate (10, 12, 14, 16, 18 µg/ml concentrations ) and fluoxetine hydrochloride (3, 4, 5, 6, 7 µg/ml ) subjected to test absorbance by using UV – Visible spectrophotometer at 280nm and 247nm respectively. Now plot a graph for the both drugs seperately by taking conentration on X- axis and mean response or absorbance on Y- axis. On Y- axis not only mean response we can also take absorbance.^{(10, 25)}

Table 1: Sildenafil citrate calibration values

Drug	Concentration	Calibration
Sildenafil	10	0.400
Sildenafil	12	0.490
Sildenafil	14	0.600
Sildenafil	16	0.700
Sildenafil	18	0.790

Table 2: Fluoxetine hydrochloride calibration values

Drug	Concentration	Calibration
Fluoxetine	3	0.270
Fluoxetine	4	0.325
Fluoxetine	5	0.375
Fluoxetine	6	0.420
Fluoxetine	7	0.463

Graph 3: Calibration graph of Sildenafil

Graph 4: Calibration graph of Fluoxetine

3. VALIDATION OF SPECTROPHOTOMERIC METHOD :

Validation is the process which ensures the procedure throughout the experimentation is accurate, precise for its intended purpose and it is typically follows ICH guidelines. Mainly validation is done by considering the different parameters like accuracy, precision, linearity, ruggedness, robustness, Limit of Detection and Limit of Quantification, etc.^(2,9)

3.1 Accuracy :

Accuracy expresses the closseness of measured values to the accepted true quantity and often it is expressed as percentage recovery.⁽¹⁸⁾ These are the recovery studies which are carried out by using standard addition method. 9.2 µg of fluoxetine is added which is equivalent to the formulation powder (10µg), take sildenafil about 10µg. A known quantity of sildenafil and fluoxetine were added at different percentages and analysed to calculate the % recovery and %RSD ( Relative Standard Deviation ) and values are reported in table 3.

Table 3 : Results of accuracy for sildenafil and fluoxetine

Drugs	Amount Taken (mg)	Amount added (mg)	Total amount found (mg)	% Recovery	% RSD
Sildenafil	100	20	120	98.45	0.12
		40	140	98.95	0.09
		60	160	99.75	007
Fluoxetine	30	10	40	98.75	0.16
		20	50	99.25	0.14
		30	60	99.85	0.18

3.2 Precision :

Precision measures the closeness of agreement independent or multiple test results from a homogenous sample and precision is expressed as %RSD.^{(9, 11)}

The precision can be quantified through different levels namely:

Intraday Precision (repeatability)
Interday Precision (reproducibilty)

3.2.1 Intraday Precision :

In this method, there is a assessment of test results consistency of the same sample conducted multiple times within a same day. Intraday precision measures the repeatability of the method.^(16,23)For the calculation 3 and 4 µg/ml concentrations of fluoxetine was taken and 10 and 12 µg/ml concentrations of sildenafil. Calculated and values are reported in table 4.

Table 4: Results of intraday precision for sildenafil and fluoxetine

Concentration (µg/ml)		Absorbance		% RSD
FLU	SIL	FLU	SIL	FLU	SIL
		247 nm	280 nm	247 nm	280 nm
3	10	0.285	0.338	0.20	0.32
		0.284	0.340
		0.284	0.338
4	12	0.335	0.470	0.17	0.48
		0.334	0.470
		0.334	0.473

3.2.2 Interday Precision :

Process of determination is as same like intraday precision where in intraday tests are done multiple times within a same day but to calculate interday precision process is continued or followed for six days and the calculated values are reported in table 5.

Table 5 : Results of interday precision for sildenafil and fluoxetine

Concentration (µg/ml)		Absorbance		*%RSD**
FLU	SIL	FLU	SIL	FLU	SIL
		247 nm	280 nm	247 nm	280 nm
3	10	0.283	0.339	0.191	0.323
		0.284	0.338
		0.284	0.339
4	12	0.335	0.470	0.18	0.481
		0.336	0.473
		0.337	0.471

Mean of Six Obsevations:

Table 5,6: Mean of six observations

Concentration (µg/ml)		Absorbance		*%RSD**
		FLU	SIL	FLU	SIL
		247 nm	280 nm	247 nm	280 nm
3	10	0.284	0.338	0.19	0.323
		0.284	0.338
		0.285	0.340
		0.285	0.339
		0.284	0.338
		0.284	0.340

Concentration (µg/ml)		Absorbance		*%RSD**
		FLU	SIL	FLU	SIL
		247 nm	280 nm	247 nm	280 nm
4	12	0.336	0.470	0.17	0.48
		0.336	0.471
		0.335	0.473
		0.336	0.473
		0.336	0.472
		0.335	0.473

3.3 Linearity :

Linearity measures that the method gives results which are directly proportional to the analyte concentration within the same range.⁽²⁶⁾ Usually calculated by taking atleast five concentrations of both drugs namely sildenafil citrate and fluoxetine hydrochloride. And linearity is demonstrated as correlation coefficient (R²), basically R² value is > 0.99 or should be equal to 1. The R² value of sildenafil is 0.9989 and value of fluoxetine is 0.9974

Graph 5 : Sildenafil linearity graphical representation

Graph 6 : Fluoxetine linearity graphical representation

3.4 : Ruggedness :

Ruggedness indicates the capacity of method to remain unchanged by small, deliberate changes in procedure or environment.^(16,22) Small changes may include different analyst, different instrument. Ruggedness is a part of method validation and it is mandatory to ensure that the method provides consistent and reliable results. By performing ruggedness it is easy to detect failures or errors and can be easily prevented or rectified.

Table 7 : Ruggedness results of sildenafil

Drug Sildenafil (nm)	Parameter	% Found	% RSD
280	Analyst-1	98.50	0.17
282	Analyst-1	99.15	0.19

Table 8 : Ruggedness results of fluoxetine

Drug (Fluoxetine) nm	Parameter	% Found	% RSD
248	Analyst-2	98.40	0.320
250	Analyst-2	99.30	0.345

4. ANALYSIS OF FORMULATION :

Sildenafil Citrate tablets :

Firstly involves the preparation of standard solution of reference standard by dissolving accurately weighed 10 mg of sildenafil in 100 ml ( 100 µg/ml ) volumetric flask by using solvent like methanol and make up volume. Now prepare working standard by pipetting 10ml of stock solution into 100 ml flask and make up with methanol upto the mark. ( 10 µg/ml )

Take 20 tablets ( each tablet contains 100 mg ) and calculate average weight.^(3,4) Now powder the tablet and weigh the tablet powder which is equivalent to the 10 mg of sildenafil citrate, then transfer it to 100 ml volumetric flask. Add nearly 60 ml of methanol and sonicate for 10 min. make up the volume with methanol upto the mark ( 100 µg/ml ) and filter the sample stock solution using whattman filter paper. Now pipette out 10ml from the sample stock and dissolve it in the 100ml volumetric flask and dilute upto the mark to get 10 µg/ml.

Set the UV Spectrophotometer at 280 nm and measure the absorbance of sample and standard against the blank.⁽⁴⁾

Now assay is Calculated by using Standard Comparison Method.

% Assay=A std? / A sam?? × 100

Where,

A std = Absorbance of Standard
A sam = Absorbance of Sample
% Assay = 238 / 241 × 100 = 98.75 %

Fluoxetine Hydrochloride tablets :

Firstly involves the preparation of standard solution of reference standard by dissolving accurately weighed 10 mg of Fluoxetine in 100 ml ( 100 µg/ml ) volumetric flask by using solvent like methanol and make up volume. ⁽⁵⁾ Now prepare working standard by pipetting 10ml of stock solution into 100 ml flask and make up with methanol upto the mark. ( 10 µg/ml)

Take 20 tablets ( each tablet contains 20 mg ) and calculate average weight. Now powder the tablet and weigh the tablet powder which is equivalent to the 10 mg of sildenafil citrate, then transfer it to 100 ml volumetric flask.⁽⁸⁾ Add nearly 60 ml of methanol and sonicate for 10 min. make up the volume with methanol upto the mark (100 µg/ml) and filter the sample stock solution using whattman filter paper. Now pipette out 10ml from the sample stock and dissolve it in the 100ml volumetric flask and dilute upto the mark to get 10 µg/ml.

Set the UV Spectrophotometer at 247 nm and measure the absorbance of sample and standard against the blank.⁽⁶⁾

Now assay is Calculated by using Standard Comparison Method.

% Assay=A std? / A sam?? × 100

Where,

A std = Absorbance of Standard
A sam = Absorbance of Sample
% Assay = 226 / 228 × 100 = 99.1 %

5. SUMMARY AND CONCLUSION :

The main objective of the experiment was the to develop analytical method and validation of UV spectroscopy studies for the drugs like sildenafil citrate and fluoxetine hydrochloride. The absorption maxima of sildenafil citrate is about 280nm and absorption maxima of fluoxetine hydrochloride is about 247nm. Assay of formulations of both drugs are conducted as per ICH guidelines and Indian Pharmacopoeia. Validation parameters like accuracy, precision, linearity, ruggedness were performed as ICH guidelines. A simple, accurate and reliable method is successfully developed. And the results are tabled above.

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