Pakistan Journal of Science (Vol. 62 No.

3 September, 2010)

A VALIDATED HPLC METHOD FOR THE MEASUREMENT OF SILDENAFIL CITRATE IN DIFFERENT FORMULATIONS
K.T. Mahmood and B. Khan* Drug Testing Laboratory, Health Department, Punjab, Lahore *Department Of Chemistry, Lahore College for Women University, Lahore

ABSTRACT: Sildenafil citrate is a first oral medicine approved by Food and Drug Administration
(FDA), USA for treating erectile dysfunction in male. It is not registered but available in Pakistan through unlawful import. Furthermore, it is added in male sexual-enhancement products being sold fraudulently under the cover of alternate system without any medical supervision. The Drugs courts in Pakistan have acquitted such accused on the ground that no approved and validated method was available in Pakistan. The present HPLC method for the of measurement sildenafil citrate in different formulations was developed in this background. The solution at concentration of 10 g.mL-1 was prepared with external standard Sildenafil citrate. The injection volume was 10 L. The chromatographic separation was achieved by using a reversed phase C18 column (Phenomenex, particle size 5 m; 4.6 mm 150 mm). The mobile phase composed of methanol , acetonitrile and triethanolamine buffer solution pH 4.00 (33: 19: 50, v/v) was pumped at flow rate of 0.9ml/minute at 25 0C temperature.Sildenafil citrate was detected at 290 nm and eluted at retention time of 7.8 minutes .The method was validated with respect to linearity, accuracy, precision, sensitivity, reproducibility and robustness prior to the analysis of samples.It was successfully used for assay of sildenafil citrate in different formulations including products sold fraudulently in Pakistan, under the cover of alternate system of medicine. Key words: Sildenafil citrate; HPLC; pharmaceutical formulations: alternate system of medicines

INTRODUCTION
Sildenafil citrate (Viagra) was patented in1996 and launched in May 1998 as first oral drug approved by Food and Drug Administration (FDA) to treat erectile dysfunction (ED) in the United States. The penile prostheses, vacuum constriction devices, penile injection therapy, transurethral suppositories and professional counseling were primary alternatives prescription treatment for ED. It is also effective for treatment of pulmonary arterial hypertension (PAH). It is good for treatment of high altitude pulmonary edema. Recreational use and misuse of this drug is also common (Boolell et al., 1996, Kling, 1998, Andersson, 2001, Richalet et al., 2005, Smith and Romanelli, 2005, McCambridg et al., 2006, Fagenholz et al., 2007,Internet drug index 2010). More than $400 million worth of sildenafil citrate was sold in its first quarter on the U.S. market. More than 300,000 total prescriptions were written for Viagra in the first month after launching in USA (IMS, 1998, and National Prescription Audit, 1998). It is now being sold in more than fifty countries but not registered in Pakistan until today. Sildenafil citrate is a white to off-white crystalline powder with a solubility of 3.5 mg/mL in water and a molecular weight of 666.7 Dalton. Molecular formula is C22H30N6O4S. .Chemically, designated as 1[[3-(6,7-dihydro-1-methyl-7-oxo-3-propyl-1H

pyrazolo[4,3-d]pyrimidin-5-yl)-4ethoxyphenyl]sulfonyl]-4-methylpiperazine citrate. Its structural formula is given below in the fig.1 (Internet drug index 2010).

Figure.1: The structural formula of sildenafil citrate The parasympathetic nerves are stimulated when man arouses sexually, leading to penile erection as result of release nitric oxide (NO) which works by activation of the enzyme guanylate cyclase responsible for converting guanosine triphosphate (GTP) to 35 cyclic guanosine monophosphate (cGMP). The cGMP is a potent vasodilator vital erection of the penis. Sildenafil citrate selectively inhibits the enzyme PDE-5A (phosphodiesterase-5A) that hydrolyzes cGMP. Thus it increases level of cGMP by preventing it from breaking down. Consequently smooth muscle relaxation leads to vasodilation and increased inflow of blood into the spongy tissue of the penis causing an erection by fascinating the signaling actions of nitric oxide (NO) in penile smooth muscle, (Nicholas et al., 1996, Pfizer

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Pakistan Journal of Science (Vol. 62 No. 3 September, 2010) Viagra Product Monograph, 1998, Webb et al., 1999, Andersson, 2001). The most common side effects of Sildenafil citrate are headache, facial flushing, and upset stomach. Less commonly cyanopsia (bluish vision), blurred vision, or sensitivity to light may briefly occur, (Akash et al., 2005, Pomeranz and Bhavsar, 2005). It is contraindicated in patients receiving nitrates, in patients in whom vasodilation or sexual activity are inadvisable, or in patients with a previous history of non-arteritic anterior ischaemic optic neuropathy. It is also contraindicated in severe hepatic impairment. As a result of post marketing reports, in October 2007, the FDA announced that the labeling for all PDE5 inhibitors, including sildenafil, required a more prominent warning of the potential risk of sudden hearing loss (FDA Updates Labeling for Viagra 2005, Viagra prescribing informations 2010). Some HPLC methods for determination of sildenafil citrate in pharmaceutical formulations and plasma have been reported (Daraghmeh et al., 2001, Francis et al., 2003, Abd-Elbary et al., 2004, Reddy and Redy, 2008, Baselt, 2008). The basic aim of the present was to develop and validate a specific, precise accurate, reproducible, robust, efficient, economical and quicker HPLC method for measurement of sildenafil citrate in different formulation including products sold fraudulently in Pakistan, under the cover of alternate system of medicine and neutroceuticals. The work was done at Department of Pharmacy/Central Quality Assurance Laboratory, LCWU, Lahore. The HPLC method described below was different because high speed vortex mixing of the samples as well as mobile phase done, filtered through 0.22 m filter and better ultrasonication. 500mL buffer followed by addition of 190mL acetonitrile. High speed vortex mixing was done followed ultrasonication for 15minutes. Mobile phase was filtered through 0.22 m filter. Solutions of standard and samples Stock solution: Stock solution of standard sildenafil citrate was prepared in methanol at concentration of 1mg.mL-1. Mixing, ultrasonication and filtration was done in the same way as for mobile phase. Solutions of standard: The solution at concentration of 10gmL-1 was prepared with external standard Sildenafil citrate by diluting with mobile phase. The samples at concentration of 10.0, 9.0,8.0,6.0,4.0.2.0,1.5,1.0,0.5, 0.25 and 0.12 g.mL-1 were also prepared by diluting stock solution of external standard sildenafil citrate in mobile phase for constructing standard curve. All these samples were mixed well, ultrasonicated for 30 minutes and filtered through 0.22m. Solutions of Samples: Six tablets of the product sold as herbal product were weighed and powdered. Powder equivalent to one tablet was transferred to a 50 mL volumetric flask, followed by addition of 30 mL methanol. The contents in the flask mixed well and ultrasonicated for 30 minutes to complete dissolution and diluted to the mark with methanol and then filtered through 0.22 L filter. 0.25 mL sample solution was diluted to 25mL with mobile phase. The samples of Viagra 50mg and Indian brand Sildenafil 50mg tablets were also prepared.

MATERIALS AND METHODS

Experimental Drugs / Chemicals: Sildenafil citrate standard of the company Sigma, HPLC grade methanol, acetonitrile, triethanolamine, phosphoric acid, Viagra 50mg tablets Pfizer USA, Sildenafil tablets 50mg Indian brand were used for the present HPLC method. Three sex enhancement products sold under the cover of Alternate System of Medicines were purchased from local market of Lahore. The purified water prepared by using a MilliQ system was used for the preparation of buffer and other aqueous solutions. Statistical analysis: The software SPSS 13.0 was used for statistical analysis. The values in the raw data were expressed as range, mean, and standard deviation etc Preparation of mobile phase: The buffer was prepared by adding 70mL Triethanolamine in a purified water quantity sufficient to make 1000mL.The pH 4.0 was adjusted by use of 85% phosphoric acid. Buffer was filtered through 0.22 m filter. 330mL methanol added to

High performance liquid chromatography: The maximum absorbance max for sildenafil citrate external standard was determined by scanning in UV-Visible range of wave length ( max = 290nm).
10l of sample was injected in the HPLC system. The mobile phase composed of methanol, acetonitrile and triethanolamine buffer solution pH 4.00 (33: 19: 50, v/v) was pumped was pumped into Water 1525 Binary HPLC Pump at flow rate of 0.9mL/minute with oven temperature was set at 25 C. Separation was achieved by using a reversed phase C18 column (Phenomenex, particle size 5 m; 4.6 mm 150 mm).The samples were introduced through an injector valve with a 10 l sample loop. The drug sildenafil citrate was detected at 290nm by using a Water 2487 dual absorbance detectors. The distinct peak was visible in chromatograms of sildenafil citrate external standard. The retention time observed for sildenafil citrate was around 7.8 minutes. The representative chromatogram is shown in figure 2. Method validation is vital issue in any drug analysis and is also legal requirement USP_NF (2009). The validation ascertain suitably and reliability of a

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Pakistan Journal of Science (Vol. 62 No. 3 September, 2010) method for its intended use. The developed HPLC method was validated with respect to stability, linearity, accuracy, precision, sensitivity, and robustness The stability studies on stock solution of sildenafil citrate (1 mg.mL-1) were carried out for six weeks at 4 C and 25C. The stability studies on sildenafil citrate 10 g.mL-1 in mobile phase were carried out for 24h at 25C. The AUC of samples of sildenafil citrate at concentration of 10.0, 9.0, 8.0, 6.0, 4.0, 2.0,1.5, 1.0, 0.5, 0.25 and 0.12 g.mL-1 were determined for making calibration curve. The intraday and assays were carried out by testing three samples sildenafil citrate at the concentration 1.0, 5.0 and 10 g.mL-1.Six reading were taken for each samples. The assays were performed by different analyst to explore robustness in the method. 6 samples at the concentration of 10g.mL-1 were analyzed. The result was compared statistically (Wilcoxon paired test). The samples prepared from all the formulations were analyzed in the present method. interference peak. The representative chromatogram is shown in figure 4. So, method was specific The representative chromatogram of tablets of fraudulent products purported to be manufactured under alternate system of manufacturing are shown in figures 5 and 6. The peaks in these products were similar and distinct at same retention time 7.8 minutes, without any interference peak showing presence of sildenafil citrate. The chromatogram of tablets of an Indian product is shown in figure 7. The results regarding recovery/ percentage yield of sildenafil citrate in the patent tablets 50 mg are presented in table 1. The mean recovery of 199.84%% with C.V% (RSD) 0.5% had shown that HPLC method was accurate. The results of stability studies are presented in tables 2 and 3 .The results had shown that stock solution of analytes was stable for at least six weeks at 4 C and 25C. The analytes was stable in mobile phase at least for 24h.Thus, stability indicated reliability of analysis in the proposed procedure. The limit of detection (LOD) of the method was 0.0120g.mL-1 whereas the limit of quantitation (LOQ) for sildenafil was found to be 0.120g.mL-1 The intraday assays were carried out by testing three samples sildenafil citrate at the concentration 1.0, 5.0 and 10 microgram/ mL. Six readings were taken. The interday assays were carried out for five days, by testing three samples at concentration sildenafil citrate 1.0, 5.0 and 10 microgram/ mL. Six readings were taken. Results are presented in table.3. This result had shown that method was reproducible. The values of RSD for intraday (0.11%-0.73%) and inter-day (0.8%-1.91%) studies had indicated that method was precise. The method was cross checked by two different analysts. Results are presented in table 5.The statistical comparison (Wilcoxon paired test) had shown that there was no difference between results (p=0.217 > p=0.050). Therefore, the method was found to be robust.

RESULTS AND DISCUSSION

The maximum absorbance max for sildenafil citrate external standard was determined 290nm. The distinct peak was visible in chromatograms of sildenafil citrate external standard at retention time of 7.8 minutes when UV detector was set 290nm. There was no interference peak around this time. The representative chromatogram is shown in figure 2. There was no peak when placebo mobile phase was run as sample. The calibration curve shown in figure 3 was prepared by plotting AUC versus concentration of sildenafil citrate. The peak of sildenafil citrate in Viagra tablets was matched with the peak of standard. It was similar and distinct at same retention time 7.8 minutes, without any

Figure 2:Representative chromatogram of sildenafil citrate standard used in HPLC method for determination of sildenafil citrate (10.0microgram/mL

Figure.3 Standard calibration curve for HPLC method for determination of sildenafil citrate in different formulations.

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Pakistan Journal of Science (Vol. 62 No. 3 September, 2010)

Figure.4 HPLC method for determination of sildenafil citrate in different formulations. Representative chromatogram of Viagra tablet 50mg

Figure.5 Chromatogram of product-1 fraudulently manufactured and sold under alternate system of manufacturing

Figure.6 Chromatogram of product-2 fraudulently manufactured and sold under alternate system of manufacturing

Figure.7

Chromatogram of the product manufactured in India with label claim of Sildenafil Citrate 50mg

Table 1: HPLC method for determination of sildenafil citrate Percent yield/recovery in standard tablets 50mg Mean concentration sildenafil citrate recovered (mean +/-S.D.) % concentration sildenafil citrate recovered from tablets 50mg C.V% (RSD)
C.V = coefficient of variation, RSD = Relative standard deviation

49.92+0.16 99.84% 0.5%

Table.2: Stability studies on stock solution of sildenafil citrate at concentration of 1mg/mL Temp. 4 C 25 C Day1 100.0+ 0.044% 100.+ 0.044% Mean (%) concentration of sildenafil citrate recovered (mean +SD) Day 2 Day4 Day 8 Day 16 Day24 Day42 99.46+ 99.1+ 100.00+ 99.08+ 99.0+ 100.0+ 0.051% 0.044% 0.051% 0.50% 0.040% 0.036% 99.90+ 100.0+ 100.+ 99.88+ 99.7+ 99.73+ 0.040% 0.043% 0.045% 0.045% 0.043% 0.035% Day1 100..0+ 0.044% 100.+ 0.046%

Sildenafil citrate is not registered in Pakistan. The applications of many manufacturers are lying pending before Ministry of health, Government of Pakistan. There is big demand for this medicine in Pakistan. It is available in Pakistan through smuggling from other countries. Furthermore, many criminal are mixing/adulterating sildenafil citrate in male sexualenhancement supplements and selling these fraudulent

products under the cover of alternate system. Consequently, it is used in Pakistan without any medical supervision. The Government Analysts have identified sildenafil citrate in different samples sent by Drugs inspectors to two different Drugs Testing Laboratories in Punjab, Pakistan. However, the Drugs court Gujranwala based at Lahore has acquitted such accused on the ground that no approved assay method was available in Pakistan.

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Pakistan Journal of Science (Vol. 62 No. 3 September, 2010) Judgments of Drug Court Gujranwala H.Q. at Lahore, in complaints No.Jud.DC 649/2009 on 10.5.2010 complaint No. Jud. DC 869/2008 on 05.5.2010.This is a grave situation which is likely to endanger public health in Pakistan. Immediate interventions are required by concerned Health Authorities in Pakistan.

Table 3: Stability of sildenafil citrate in mobile phase at concentration of10g/ mL At 0h 100.+ 0.01% Mean+SD concentration of Sildenafil citrate in mobile phase at 25 C (n=6) At 2h At 6h At 8h At 12h At 18h 99.90+ 100..0+ 100..+ 99.88+ 99.7+ 0.015% 0.013% 0.032% 0.049% 0.044% At 24h 99.73+ 0.034%

Table.4: Intraday and intraday variability in HPLC method for determination of sildenafil citrate. Sildenafil citrate added Mean recovered +S.D. % sildenafil citrate recovered C.V%(RSD) intraday variability(n=6) 1 g/ mL 5 g/ mL 10g/ mL 4.97+ 0.99+ 0.0152 0.04087 9.99+ 0.0906 99% 99.94% 99.9.% 0.65.% 0.73% 0.11% Interday variability(n=6) 1 g/ mL 5 g/ mL 10g/ mL 0.992+ 4.95+ 9.956+ 0.010 0.031 0.023 99.2% 99% 99.56% 0.8% 1.61% 1.91.%

Table 5: HPLC determination of sildenafil citrate in 10 g/ mL solution done by two different analysts. Analysts Analyst.1 Analyst.2 Results Mean % concentration sildenafil citrate+S.D. (n=6) Mean % concentration sildenafil citrate+S.D. (n=6) 99.01%+0.027 100.04%+0.037

CONCLUSION: This method for measurement of sildenafil citrate was specific, precise, accurate, reproducible, robust, efficient, economical and quicker. It was successfully used for measurement of sildenafil citrate in pharmaceutical formulations and products sold fraudulently in Pakistan, under the cover of alternate system of medicine and neutroceuticals. The Government Analyst at different Drugs Testing Laboratories, in Pakistan may apply this method for assay of sildenafil citrate, after getting approval from the competent authorities under the Drugs Act 1976.

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