Pakistan Journal of Science (Vol. 62No. 3September, 2010)
Viagra Product Monograph, 1998,Webb
.,1999,Andersson, 2001).The most common side effects of Sildenafilcitrate are headache, facial flushing, and upset stomach.Less commonlycyanopsia (bluish vision), blurred vision,or sensitivity to light may briefly occur,(Akash
2005,Pomeranz andBhavsar, 2005). It is contra-indicated in patients receiving nitrates, in patients inwhom vasodilation or sexual activity are inadvisable, or in patients with a previous history of non-arteritic anterior ischaemic optic neuropathy. It is also contraindicated insevere hepatic impairment.As a result of post marketingreports, in October 2007, the FDA announced that thelabeling for all PDE5 inhibitors, including sildenafil,required a more prominent warning of the potential risk of sudden hearing loss (FDA Updates Labeling for Viagra2005, Viagra prescribing informations2010).Some HPLC methods for determination of sildenafil citrate in pharmaceutical formulations and plasma have been reported(Daraghmeh
Francis et al
2004, ReddyandRedy,2008,Baselt, 2008).The basic aim of the present was to develop andvalidate a specific, precise accurate, reproducible, robust,efficient, economical and quicker HPLC method for measurement of sildenafil citrate in different formulationincluding products sold fraudulently in Pakistan, under the cover of alternate system of medicine andneutroceuticals.The work was done at Department of Pharmacy/Central Quality Assurance Laboratory,LCWU,Lahore.The HPLC method described below wasdifferent because high speed vortex mixingof thesamplesas well as mobile phasedone,filtered through0.22
and better ultrasonication.
MATERIALS AND METHODS
Experimental Drugs / Chemicals:
Sildenafil citratestandard of the company Sigma,HPLC grademethanol,acetonitrile,triethanolamine, phosphoric acid, Viagra50mg tablets Pfizer USA, Sildenafil tablets 50mg Indian brandwereused for the present HPLC method.Threesexenhancement products sold underthe cover of AlternateSystem of Medicines were purchased from local marketof Lahore. The purified water prepared by using a Milli-Q system was used for the preparation of buffer and other aqueous solutions.
The software SPSS 13.0 was usedfor statistical analysis. The values in the raw data wereexpressed as range, mean,and standard deviation etc
Preparation of mobile phase:
The buffer wasprepared by adding 70mLTriethanolaminein a purified water quantity sufficient to make 1000mL.The pH 4.0 wasadjusted by use of 85% phosphoric acid. Buffer wasfiltered through 0.22
.330mLmethanol added to500mLbufferfollowed by addition of 190mLacetonitrile.High speed vortex mixing was done followedultrasonication for 15minutes.Mobile phase was filteredthrough 0.22
Solutions of standardand samplesStock solution:
Stock solution of standard sildenafilcitrate was prepared in methanolat concentration of 1mg.mL
.Mixing,ultrasonication and filtration wasdone in the same way as for mobile phase.
Solutions of standard:
The solution at concentration of 10µgmL
was prepared with external standard Sildenafilcitrateby diluting with mobile phase.The samples at concentration of 10.0,9.0,8.0,6.0,184.108.40.206,1.5,1.0,0.5, 0.25 and 0.12 µg.mL
were also prepared by diluting stock solution of externalstandard sildenafil citrate in mobile phase for constructing standard curve.All thesesamples weremixed well,ultrasonicated for 30 minutes and filteredthrough 0.22
Solutions of Samples:
Six tablets of the product sold asherbal product were weighed and powdered. Powder equivalentto one tablet was transferred to a 50 mLvolumetric flask,followed by addition of30 mLmethanol. The contents in the flask mixed well andultrasonicated for 30minutesto complete dissolution anddiluted to the mark with methanol andthenfilteredthrough 0.22µLfilter.0.25 mLsample solutionwasdiluted to 25mLwith mobile phase.The samples of Viagra50mgandIndian brand Sildenafil 50mgtabletswere also prepared.
High performance liquid chromatography:
for sildenafil citrateexternal standardwas determined by scanning inUV-
Visible range of wave length (λ
10µl of sample was injected in the HPLCsystem.The mobile phase composed of methanol,acetonitrile and triethanolamine buffer solution pH 4.00(33: 19: 50, v/v) was pumped was pumped intoWater 1525 Binary HPLC Pump at flow rate of 0.9mL/minutewith oven temperature was set at 25C. Separation wasachieved by using a reversed phase C18 column(Phenomenex, particle size 5
mm×150mm).Thesamples were introduced through an injector valve with a10
l sample loop. The drug sildenafil citrate wasdetected at 290nmby using a Water 2487 dualabsorbance detectors.The distinct peak was visible inchromatograms of sildenafil citrate external standard. Theretention time observed for sildenafil citrate was around7.8minutes. The representative chromatogram is shownin figure 2.Method validation is vital issue in any druganalysis and is also legal requirement USP_NF (2009).The validation ascertain suitably and reliability of a