Stability Indicating High Performance Liquid Chromatographic Assay for the Determination of Sildenafil Tartrate

 

O.S.S. Chandana¹, R. Ravichandra Babu²

^{1, 2}Department of Chemistry, Institute of science, GITAM University, Visakhapatnam, Andhra Pradesh, India

 

ABSTRACT:

A simple, fast, accurate and specific reverse phase high pressure liquid chromatographic (HPLC) method has been developed for simultaneous determination of Sildenafil base and its impurities in the Sildenafil Tartrate (API). The method was developed by reversed phase chromatography equipped with C₁₈ (100 ´ 4.6 nm ´ 5.0 nm) column using mobile phase of (0.1%) formic acid and methanol (30:70 ratio) with a flow rate of 0.5 ml/min with ultraviolet detector configured at 290nm. The method was validated as per ICH guidelines.

 

 

 

INTRODUCTION:

Sildenafil (as citrate) marketed as Viagra was approved as a drug for treating male erectile dysfunction. sildenafil citrate was first reported as potent and selective inhibitor type 5PDE. Sildenafil is chemically known as 1-{[3-(6, 7-dihydro-1-methyl-7-oxo-3-propyl-1H-pyrazolo{4, 3-d}pyramidine-5-yl)-4-methylpiperazine citrate. Sildenafil citrate salt is the only drug popularly known as Viagra administered for erectile dysfunction. Hence an attempt has been made in the present study to synthesize an alternate form of Sildenafil as tartrate salt (supplied by M/S Ogene Systems (I) Pvt. Ltd.) and also developed a new method for its purity and also determination of impurities present during its synthesis.

 

Since Sildenafil is an unstable base, it has to be converted into a suitable salt. Literature survey reveals that many methods were reported for sidenafil citrate and its related substances [ 1-7] but there is no method was reported for sildenafil tartrate and its impurities to till now. Hence an attempt has been made to develop a new method for the determination of sildenafil tartrate and its impurities.

 

The synthetic route for the sildenafil tartrate is given in the figure 1.

 

MATERIALS AND METHODS:

Reagents and Materials

The reference sample of sildenafil were supplied as a gift sample from M/S ogene (I) pvt limited. Milli-Q-water was used throughout this research. All other analytical reagents such as Ammonium formate, Acetonitrile, Hydrochloric acid, Sodium hydroxide and Hydrogen peroxide (30%) were obtained from Merck specialty chemicals, Mumbai, India.

 

Instrumentation:

An HPLC unit equipped with photo diode 16990 detector modules and 2695 separation module (system water Alliance, U.K) was used.

 

 

Fig-1: Synthetic route of SFT and its impurities

 

 

Preparation of standard drug solution:

An accurately weighed quantity of 5.0 mg of sildenafil tartrate was dissolved in mobile phase and diluted quantitatively.

 

Diluent: Methanol

 

Chromatographic conditions:

Column used	:	C18 100 ´ 4.6 nm ´ 5.0 nm.
Flow rate	:	0.5 ml/min.
Mobile phase	:	0.1 % formic acid and methanol. (30: 70 ratio)
Wave length	:	290 nm
Retention time	:	30 mts.
Tailing factor	:	≤ 1.5

 

Procedure:

Equilibrate the system for 30 mins, inject exactly 20ml of the diluents as blank solution into the system and record the chromatogram for 30 mins duration. Similarly test solution also is performed under the same conditions as mentioned above and the chromatograms are recorded. The blend chromatogram of sildenafil tartrate and its impurities are given in the fig 2.

 

 

Fig-2: Blend chromatogram of SFT and its intermediates

 

 

RESULTS AND DISCUSSION:

The Chromatographic data, including the retention times (), retention factor (k), number of theoretical plates (N), tailing factor () and wavelength absorption maximum are given in Table-1

 

Linearity:

The linearity of calibration curves (peak area vs. concentration) for sildenafil tartrate is checked over the concentration range of about 25--150ug/ml found to be linear with correlation coefficients 0.999. Table-2 lists the linearity parameters of the calibration curves for sildenafil tartrate and related compounds and fig-3 represents the linearity graph of SFT and its impurities.

 

Table-1: Retention time and response data for SFT and potential impurities

Name of the compound	tR/min (Retention time)	K (retention factor)	N (theoretical plates)
Cyclised	18.93	10.21	56085
CSA	8.52	6.54	15049
Condensed	5.47	2.22	36248
Nitro	4.38	2.30	15137
Ammine	3.57	2.28	44179
Sildenafil	2.45	0.00	39287

 

 

Table-2 :Linearity results for Sildenafil Tartrate and related compounds

 

 

Fig-3: Linearity calibration graph of Sildenafil Tartrate and related compounds

 

Accuracy:

The Accuracy of the method was tested by analyzing different samples of sildenafil tartrate and all other

 

related compounds at various concentration levels. The results were expressed as percent recoveries in Table-3.

 

Table-3 ACCURACY OF THE METHOD

LEVEL (%)	Area of Linearity level	Area of Accuracy Level	Area of Linearity level	Area of Accuracy Level	Area of Linearity level	Area of Accuracy Level
LEVEL (%)	IMP-1		IMP-2		IMP-3
50.00	89180.97	84234.60	41227.76	39197.34	159507.13	143406.57
100.00	178361.94	164824.24	82455.52	80241.59	319014.25	299271.38
150.00	267542.91	243474.28	123683.28	110289.11	478521.38	457249.19

 

LEVEL (%)	Area of Linearity level	Area of Accuracy Level	Area of Linearity level	Area of Accuracy Level
LEVEL (%)	IMP-4		IMP-5
50.00	4271593.00	4092747.18	18274700.00	18028420.26
100.00	8543186.00	8315289.43	36549400.00	35294205.19
150.00	12814779.00	11716828.21	54824100.00	54182732.17

 

 

 

Fig 4: chromatogram of SFT and its impurities

 

 

Table-4: Solution stability results

	0hrs	48hrs	0hrs	48hrs	0hrs	48hrs
CONC	IMP-1		IMP-2		IMP-3
100.00	163361.94	149824.24	79455.52	171241.59	285014.25	2 264271.38

 

	0 hrs	48hrs	0hrs	48hrs
CONC	IMP-4		IMP-5
100.00	7925186.00	7124289.43	32159400.00	29185712.19

 

 

Table - 5: Precision of the method

 

STABILITY

To determine the stability of the Salt in mobile phase, the drug was stored in the mobile phase for 48 h and chromotographed on the following day. The solution stability of the test sample was stable up to 48hrs at 100% specification level) No significant change (≤ 1%) was observed in stock solution after 48 hrs. the results were given in the table -4.

 

Precision:

The precision of the method is determined by analyzing six samples associated with different impurities and the concentrations were determined. Good repeatability was observed over the concentration range. The R.S.D’s ranging from 0.27 to 2.17%.The precision results were given in the table-5.

 

Specificity:

Preparation of test Solution:

Weigh accurately about 5.0mg of Sildenafil tartarate and 5.0mg of each impurity into 5.0 mL of volumetric flask dissolved and dilute to the mark with diluent.

 

Observation:

There is no interference of impurities with sildenafil tartarate during its determination.

 

CONCLUSION:

In conclusion, the proposed HPLC method provides simple, accurate and reproducible method for routine analysis of sildenafil tartrate. The major advantage of this method is the quick sample analysis without prior separation. Simple sample preparation procedure and a short chromatographic time make this method suitable for processing of multiple samples in a limited amount of time.

CONFLICTS OF INTEREST:

“This manuscript has not been previously published and is not under consideration in the same or substantially similar form in any other peer-reviewed media.” The authors have no conflict of interest to declare with respect to financial, personal or other relationships with other people or organizations.

 

ACKNOWLEDGEMENT:

The author would like to thank M/S Ogene Systems (I) Pvt. Ltd for providing the gift sample of sildenafil tartrate and GITAM university for providing instrument facility and technical guidance.

 

REFERENCES:

1.       Liu YM, Yang HC, Miao J.R. ‘’Reversed-phase HPLC determination of Sildenafil citrate tablets. Yaowu. Fenxi. Zazhi 2000; 20: 61–62.

2.       Lee M, Min D, “Determination of Sildenafil citrate in plasma by high-performance liquid chromatography and a case for the potential interaction of grape fruit juice with Sildenafil citrate”. Ther. Drug Monit, 2001; 23(1):21-26.

3.       Reddy BPK, Reddy YR, “Validation and stability indicating RP-HPLC method for the determination of Sildenafil citrate in pharmaceutical formulation and human plasma” E-J Chem 2008; 5:1117-1122.

4.       Draghmen N, Al-omari M, Badwan AA, Jaber AM, “Determination of Sildenafil citrate and related substances in the commercial products and tablet dosage form using HPLC’’. J Pharm Biomed Ana 2001; 25:483-492.

5.       Reddy BP, Jaya prakash M, Sivaji K, Reddy SV, Reddy S V, “Validation and stability indicating RP-HPLC method for the determination of Sildenafil citrate in pharmaceutical formulations’’. Int J Applied Bio Pharm Tech 2010;1:104-111.

6.       Kuchekar BS, Thakkar SV, Chothe PP, Hiremath MR, Shinde DB, “Spectrophotometric estimation of Sildenafil citrate in tablets”. Ind J Pharma Sc 2005;1:749-751.

7.       Thangabalan B, Vadivel K, Sowjanya K, Tejaswi G, “Quantitative spectrophotometric determination of Sildenafil citrate in tablet formulation using urea as hydrotropic solubilizing agent”. Res J Pharm Tech 2011; 2(2); 235-239.

 

 

 

 

 

Received on 13.06.2017       Accepted on 19.07.2017     

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