HPLC determination of Sildenafil Tartrate and its related Substances along with some Supportive Studies using MS, XRD and NMR

 

O.S.S. Chandana¹, D. Swapna², R. Ravichandra Babu³

¹Aditya College of Engineering, Surampalem, E. G. Dist, A. P, India

²Dadi Institute of Engineering and Technology, Anakapalli, Visakhapatnam, A. P, India

³Department of Chemistry, GITAM University, Visakhapatnam, A. P, 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). Structural confirmation and characterization of Sildenafil Tartrate and its impurities was carried out by using mass spectrometry, XRD and NMR. Based on the spectroscopic studies five impurities were characterized as Impurity A- Amine, Impurity B-Nitro, Impurity C-condensed, Impurity D-CSA, Impurity E-Cyclised. The newly developed method was validated according to ICH guidelines to demonstrate specificity, precision, linearity and accuracy of the method. The proposed method enables us to determine purity of sildenafil tartrate and also the impurities associated with it in a single attempt. Mass identification has been made for the associated products during the synthesis of sildenafil tartrate. XRD study pattern reveals that polymorphic form of both citrate and tartrate salt of sildenafil are same, indicates that tartrate salt could be used as an alternate for citrate one. NMR Study pattern reveals that the formation of base peak and tartrate peak is significant in the final compound formation, of sildenafil as tartrate salt. The developed method is especially useful for routine pharmaceutical analysis.

 

 

 

 

1. 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 determination of sidenafil citrate and its related substances using HPLC[1-5], UV[6,7], MS[8-21] and NMR[22], 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.

 

 

 

Fig-1: Synthetic route of SFT and its impurities

 

2. MATERIALS AND METHODS:

2.1 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.

 

 

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

 

Mass spectrometry:

The mass spectral analysis was performed using Quattro micro ^TM API mass spectrometer (Manufactured by Quattro micro Mnchester, UK). A capillary voltage of 3.0kV and cone voltage of 25V were the analysis parameters with source, dessolvation gas temperatures of 100°C and 350°C and dessolvation gas flow fixed at450 L h-1. Mass spectral data was recorded by using Mass Lynxsoftware.

 

NMR spectroscopy:

1H spectra is recorded for base and salt in DMSO-d6 in order to establish the protonation site. The inspection of 1H chemical shifts showed that the differences between base and salt are significant. 

 

X-Ray diffraction analysis:

The X-rays used are of the Copper k a wavelength 1.54056 ´ 10^–10m, the scan is taken between 2 theta of 10° and 2 theta of 45° at increments of 0.04° with a count time of 4 seconds for each step. These angles have been selected as this is where the important reflections lie for hydroxyl apatite and other relevant impurities. The count time is selected as 4 seconds to give a good signal to noise ratio and to enable the analysis to take place over a reasonable period. The intensity of the X-Rays is measured on the Y- axis, and increasing values of 2 theta are shown on the X axis. The pattern was obtained using Nickel filters copper radiation. Copper K₂ A  x-ray tube with a Voltage of 40 KV, Current of 35 mA and a Wave length of 1.5406 A^O  using a Divergence slit of 0.3⁰  are the analysis parameters .

 

RESULTS AND DISCUSSION:

Optimization of chromatographic conditions:

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. Methanol is used as a diluent. The blend chromatogram of sildenafil tartrate and its impurities are given in the fig 3.

 

 

Fig-3: Blend chromatogram of SFT and its intermediates

 

 

Fig-4: NMR data of SFT and its intermediates

 

 

 

Identification of impurities by mass spectral studies:

Process impurities of sildenafil were identified as Impurity A- Amine, Impurity B-Nitro, Impurity C-condensed, Impurity D-CSA, Impurity E-Cyclised. It can be seen from fig-1 (synthesis route of sildenafil tartrate) the exact masses of sildenafil tartrate and its related compounds have been identified and determined.

 

 

 

Structural elucidation (NMR spectroscopy) :

1H spectra is recorded for base and salt in DMSO-d6 in order to establish the protonation site. The inspection of 1H chemical shifts showed that the the differences between base and salt are significant. NMR Study pattern reveals that the formation of base peak and tartrate peak is significant in the final compound formation, of sildenafil as tartrate salt.thge NMR data is shown in the fig 4

 

 

Fig 5: XRD peak of sildenafil tartrate

Table – 1: XRD data of sildenafil tartrate

Product name  : SF-TARTARATE – SF – IV-013-041

File name : C\DIFFDAT1\SF-TARTARATE-SF-IV-013-041. raw

S. No.	Angle 2 –theta°	D value angstrom	Intensity %
1	7.305	12.09119	33.6
2	8.035	10.9953	100
3	10.233	8.63748	59.3
4	10.818	8.17197	9.7
5	11.408	7.75013	10.4
6	13.111	6.7474	10
7	13.893	6.36935	22.1
8	14.362	6.162	53.8
9	14.673	6.03218	16.7
10	14.831	5.96841	6.8
11	15.313	5.78152	5.3
12	16.133	5.48936	13.3
13	16.404	5.3994	7.9
14	17.129	5.17245	8.3
15	17.38	5.09841	13.2
16	17.624	5.02817	20.7
17	18.023	4.9179	6.4
18	18.142	4.88595	7.1
19	19.059	4.65272	5.7
20	19.793	4.48197	37
21	20.059	4.42299	14
22	20.43	4.34348	5.4
23	20.463	4.29931	17.1
24	20.734	4.28064	20
25	21.903	4.05464	4.6
26	22.624	3.92711	20.1
27	22.96	3.87032	22.1
28	23.28	3.8179	3
29	24.34	3.65389	6.6
30	24.73	3.59714	5.6
31	25.28	3.52024	4.9
32	25.538	3.48524	7.1
33	25.967	3.42864	3.9
34	26.276	3.38901	4
35	26.73	3.33236	6.1
36	27.485	3.24259	4.8
37	28.431	3.13682	9.8
38	28.823	3.095	7.3
39	30.473	2.93106	5.3
40	30.814	2.89943	3.2
41	31.107	2.87274	4.9
42	33.962	2.63755	3.2
43	35.621	2.51842	4.7
44	37.36	2.40509	3.1
45	40.098	2.24694	3.7

 

The x-ray powder diffraction pattern of sildenafil tartrate was obtained using Bruker axs D8, Germany. The pattern was obtained using Nickel filters copper radiation. The full data summary is complied in Table-6. The XRD data pattern of tartrate obtained is compared with citrate compound of Sildenafil. From the data it can be concluded that the polymorphic forms of both citrate and tartrate salt are same.  Hence tartrate salt of sildenafil can also be used as an alternative for citrate salt, after passing through stringent clinical trials. The XRD data for sidenafil tartrate is given in the table -1 and the spectrum in the figure -5.

 

METHOD VALIDATION:

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-2

 

 

 

Table-2: 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
Amine	3.57	2.28	44179
Sildenafil	2.45	0.00	39287

 

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-3 lists the linearity parameters of the calibration curves for sildenafil tartrate and related compounds and fig-6 represents the linearity graph of SFT and its impurities.

 

 

Table-3 :Linearity results for Sildenafil Tartrate and related compounds

Linearity Level (%)	IMP-1	IMP-2	IMP-3	IMP-4	IMP-5
25	44590.49	20613.88	79753.56	2135796.50	9137350.00
50	89180.97	41227.76	159507.13	4271593.00	18274700.00
75	133771.46	61841.64	239260.69	6407389.50	27412050.00
100	178361.94	82455.52	319014.25	8543186.00	36549400.00
125	222952.43	103069.40	398767.81	10678982.50	45686750.00
150	267542.91	123683.28	478521.38	12814779.00	54824100.00

 

 

Fig-6:Linearity calibration graph of Sildenafil Tartrate and related compounds

 

Table-4-  ACCURACY OF THE METHOD

 

 

 

 

% RECOVERY	50%	100%	150%
IMP-1	94.45	92.41	91.00
IMP-2	95.08	95.08	89.17
IMP-3	89.91	89.91	95.55
IMP-4	95.81	95.81	91.43
IMP-5	98.65	98.65	98.83

 

Table-5 :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

 

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

 

 

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-4.

 

Solution stability studies:

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 upto 48hrs at 100%   specification level) No significant change (≤ 1%) was observed in stock solution after 48 hrs. the results were given in the table -5.

 

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-6.

 

Table-6 :Precision of the method

 

 

Fig 7: chromatogram of SFT and its impurities

 

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 . The blend chromatogram of SFT and its impurities is given in the figure 7.

Observation:

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

 

 

 

 

CONCLUSION:

A simple, accurate, stability indicating HPLC method was developed to determine purity of sildenafil tartrate and also the impurities associated with it in a single attempt. Mass identification has been made for the associated products during the synthesis of sildenafil tartrate. XRD study pattern reveals that polymorphic form of both citrate and tartrate salt of sildenafil are same, indicates that tartrate salt could be used as an alternate for citrate one. NMR study pattern reveals that the formation of base peak and tartrate peak is significant in the final compound formation of sildenafil as tartrate salt The developed method is especially useful for routine pharmaceutical analysis.

 

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.

 

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Accepted on 06.03.2018          © RJPT All right reserved

Research J. Pharm. and Tech 2018; 11(5):2086-2093.