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 Pakistan Journal of Science (Vol. 62No. 3September, 2010)
K.T. MahmoodandB. Khan
Drug Testing Laboratory,Health Department, Punjab, Lahore
Department Of Chemistry, Lahore College for WomenUniversity, Lahore
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 Pakistanthrough unlawful import. Furthermore, it is added in male sexual-enhancement products being soldfraudulently under the cover of alternate system without any medical supervision. The Drugs courts inPakistan have acquitted such accused on the ground that no approved and validated method wasavailable in Pakistan. The present HPLC method for the of measurement sildenafil citrate in differentformulations was developed in this background. The solution at concentration of 10 µg.mL
was prepared with external standard Sildenafil citrate. The injection volume was 10 µL. Thechromatographic separation was achieved by using a reversed phase C18 column (Phenomenex, particle size 5
μm; 4.6
mm×150mm). The mobile phase composed of methanol , acetonitrile andtriethanolamine buffer solution pH 4.00 (33: 19: 50, v/v) was pumped at flow rate of 0.9ml/minute at25
Ctemperature.Sildenafil citrate was detected at 290nm andelutedat retention time of 7.8 minutes.The method was validated with respect to linearity, accuracy, precision, sensitivity, reproducibilityand robustness prior to the analysis of samples.It was successfully used for assay of sildenafil citratein different formulations including products sold fraudulently in Pakistan, under the cover of alternate system of medicine.
Sildenafil citrate; HPLC;pharmaceutical formulations:alternate system of medicines
Sildenafilcitrate(Viagra)waspatented in1996and launchedin May 1998 as first oral drug approved byFood and Drug Administration (FDA)to treat erectiledysfunction (ED)in the United States.The penile prostheses, vacuum constriction devices, penile injectiontherapy, transurethral suppositoriesand professionalcounselingwere primary alternatives prescriptiontreatment for ED. It is also effective for treatment of  pulmonary arterial hypertension(PAH).It is good for treatment of high altitude pulmonary edema.Recreationaluse and misuse of this drug is also common (Boolell
et al.,
1996,Kling, 1998,Andersson,2001,Richalet
et al.,
2005, Smithand Romanelli, 2005,McCambridg
et al
.,2006, Fagenholz
et al.,
2007,Internet drug index 2010).More than $400 million worth of sildenafilcitrate 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 inUSA (IMS,1998, and National Prescription Audit, 1998).It is now being sold in more than fifty countriesbut not registeredin Pakistanuntil today.Sildenafil citrate is a white to off-whitecrystalline powder with a solubility of 3.5 mg/mLinwater and a molecular weight of666.7 Dalton. Molecular formula is C
Chemically,designated as 1-[[3-(6,7-dihydro-1-methyl-7-oxo-3-propyl-1
]pyrimidin-5-yl)-4-ethoxyphenyl]sulfonyl]-4-methylpiperazine citrate.Itsstructural formulais given below in the fig.1
Internetdrug index 2010).
Figure.1: The structural formula of sildenafil citrate
The parasympathetic nerves are stimulated whenman arouses sexually, leading to penile erection as resultof release nitric oxide (NO) which works by activation of the enzyme guanylate cyclase responsible forconvertingguanosine triphosphate (GTP) to 3’5’ cyclic guanosinemonophosphate (cGMP). The cGMP is a potentvasodilator vital erection of the penis.
Sildenafil citrate
selectively inhibits the enzyme PDE-5A(phosphodiesterase-5A) that hydrolyzes cGMP. Thus itincreases level of cGMP by preventing it from breakingdown. Consequentlysmooth muscle relaxation leads tovasodilation and increased inflow of blood into thespongy tissue of the penis causing an erectionbyfascinating the signaling actions of nitric oxide (NO) in penile smooth muscle,
et al
 Pakistan Journal of Science (Vol. 62No. 3September, 2010)
Viagra Product Monograph, 1998,Webb
et al
.,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
et al.,
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
et al.,
Francis et al
.,2003, Abd-Elbary
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
μm filter 
and better ultrasonication.
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.
Statistical analysis:
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
μm filter 
.330mLmethanol added to500mLbufferfollowed by addition of 190mLacetonitrile.High speed vortex mixing was done followedultrasonication for 15minutes.Mobile phase was filteredthrough 0.22
μm filter 
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,,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:
Themaximum absorbance
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
μm; 4.6
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
 Pakistan Journal of Science (Vol. 62No. 3September, 2010)
method for its intended use. The developed HPLCmethod was validated with respect to stability,linearity,accuracy, precision, sensitivity, and robustnessThe stability studies on stock solution of sildenafil citrate (1 mg.mL
)werecarried out for sixweeks at 4 ºC and25ºC.The stability studies on sildenafilcitrate 10µg.mL
in mobile phase werecarried out for 24hat 25ºC.The AUC of samples of sildenafil citrateatconcentration of 10.0, 9.0,8.0,6.0,4.0, 2.0,1.5,1.0,0.5,0.25 and 0.12 µg.mL
were determinedfor makingcalibration curve.The intradayandassays were carried out bytesting three samples sildenafilcitrate at theconcentration 1.0,5.0 and 10µg.mL
.Sixreading weretakenfor each samples.The assays were performed by different analystto explore robustness in the method. 6 samples at theconcentration of 10
were analyzed. The resultwas compared statistically(Wilcoxon paired test).The samples prepared from all the formulationswere analyzed in the present method.
The maximum absorbance λ 
for sildenafilcitrateexternal standardwas determined 290nm.Thedistinct peak was visible in chromatogramsof sildenafil citrate external standard at retention time of 7.8 minuteswhenUV detector was set 290nm.There wasno interference peak around this time. The representativechromatogram is shown in figure 2.There was no peak when placebo mobile phase was run as sample.Thecalibration curve shown infigure 3was prepared by plotting AUC versus concentration of sildenafil citrate.The peak of sildenafil citrate in Viagra tabletswas matched with the peak of standard. It was similar anddistinct at same retention time 7.8 minutes, without anyinterference peak. The representative chromatogram isshown in figure 4. So, method was specificThe representative chromatogram of tablets of fraudulent products purported to be manufactured under alternate system of manufacturingare shown in figures 5and 6. The peaks in these productswere similar anddistinct at same retention time 7.8 minutes, without anyinterference peak showing presence of sildenafil citrate.The chromatogram of tablets ofan Indian product isshown in figure7.The results regarding recovery/ percentage yieldof 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 methodwas accurate.The results of stability studies are presented intables 2 and 3 .The results had shown that stock solutionof analytes was stablefor at least six weeks at 4 ºC and25ºC. 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 was0.0120µg.mL
whereas the limit of quantitation (LOQ)for sildenafil was found to be 0.120µg.mL
The intraday assays were carried out by testingthree samples sildenafil citrate at the concentration1.0,5.0 and 10 microgram/ mL.Six readingswere taken. Theinterday assays were carried out for five days, by testingthree samples at concentration sildenafil citrate 1.0,5.0and 10 microgram/ mL.Six readingswere taken. Resultsare presented in table.3.This resulthad shown thatmethod was reproducible.Thevalues of RSD for intra-day(0.11%-0.73%)and inter-day (0.8%-1.91%) studieshadindicated that method was precise.The method wascross checked by two different analysts. Results are presented in table 5.The statisticalcomparison(Wilcoxon paired test) had shown that there was nodifference between results (p=0.217> p=0.050). Therefore,themethod was found to be robust.
Figure 2:Representative chromatogram of sildenafilcitrate standard used in HPLC method fordetermination of sildenafil citrate(10.0microgram/mLFigure.3Standard calibration curve for HPLCmethod for determination of sildenafilcitrate in different formulations.

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