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SABYA SACHI DAS
PhD Scholar
Dept. Of Pharm. Sci. & Tech.
Birla Institute Of Technology
Mesra,Ranchi-835215,Jharkhand
INTRODUCTION
Sildenafil Citrate is 5-[2-ethoxy-5- (4-
methylpiperazin-1-ylsulfonyl)phenyl]-1methyl-3-
propyl-1, 6-dihydro-7H-pyrazolo [4, 3-d] pyrimidin-
7-one citrate .
 It is used as a smooth muscle relaxtant by enhancing
the effect of NO by inhibiting phosphodiesterase
type 5 (responsible for degradation of cGMP in
corpus cavernosum).
 It is used mainly for the treatment of erectile
dysfunction.
11/30/2017 sabya2049@gmail.com 2
OBJECTIVE
There is always a requirement of an accurate
and precise analytical method for determination of
drugs in bulk and pharmaceutical dosage form.
So, the objectives of this work are :
Develop a simple and novel RP-HPLC method
for estimation of Sildenafil citrate in bulk and
pharmaceutical dosage forms.
 Optimization of chromatographic (LC)
conditions.
 Validation of the developed method as per ICH
guidelines.
11/30/2017 sabya2049@gmail.com 3
MATERIALS AND METHODS
• 1] Instrumentation: Quantitative HPLC was
performed on Shimadzu HPLC with LC- 20AT pumps
besides SPD- 20A UV-Visible detector. Shimadzu
Spinchrom-CFR software is used along with inertsil
shield RP-C8 (150 mm × 3.9 mm), 5 µm column for the
separation. A calibrated electronic single pan of Mettler
Toledo was used.
• 2] Reagents Used: HPLC grade acetonitrile,triple
distilled water, pure drug & marketed formulation of
Sildenafil Citrate.
11/30/2017 sabya2049@gmail.com 4
HPLC conditions:
 Column : LiChroCART®LiChrospher® 100;C18(250 x 4 mm,5μm)
 Mobile phase : Acetonitrile: water at 50:50 (v/v)
 Flow Rate : 1 mL/min.
 Temperature : 300c
 Injection volume : 20 µL.
 Run time : 15min.
 Retention Time : 11.625 min.
 UV Détection Wavelength : 290nm
Fig.-1. Representative Chromatogram of Sildenafil Citrate in acetonitrile: water at 50:50; v/v 10μg/ml)
11/30/2017 sabya2049@gmail.com 5
DRUGPROFILE
SILDENAFIL
CITRATE
Pharmacokinetics data:
Bioavailability : Approximately 25%
Metabolism : Does not undergo metabolism , it is
excreted unchanged entirely in urine.
Half- life : 10-12 hours
Excretion : Renal
11/30/2017 sabya2049@gmail.com 6
validation
 The developed method was validated for linearity, accuracy,
intraday & interday precision,specificity,LOD,LOQ. The
results are shown in detail in the results and discussion section.
Results and discussion:
 Linearity: The method was found to be linear over a range
of 25-150μg/mL with a correlation coefficient of 0.9999.
 Analysis of commercial Sildenafil Citrate Tablet:
11/30/2017 sabya2049@gmail.com 7
 Accuracy:
 Precision:
 Specificity : No interference was observed from the excipients
present in tablet.
11/30/2017 sabya2049@gmail.com 8
 System Suitability:
 Stability: Bench top stability studies for 48hr of the sample
solution revealed no significant change in the concentration of
analyte. (% Recovery= 99.48-99.992%;n=5)
11/30/2017 sabya2049@gmail.com 9
Chemical data:
Chemical name : 5-[2-ethoxy-5- (4-methylpiperazin-1-
ylsulfonyl)phenyl]-1-methyl-3-propyl-1,6-
dihydro-7H-pyrazolo[4,3-d] pyrimidin-7-one
citrate
Empirical formula : C22H30N6O4S, C6H8O7
Molecular weight : 666.7 gm/mole
Physical stability:
Colour : White to off-white.
Solubility : Soluble in dimethylformamide, sparingly
soluble in acetic acid, slightly soluble in methanol.
Form : Crystalline powder
Usual strengths : 25mg;50mg;100mg
11/30/2017 sabya2049@gmail.com 10
Pharmacological action:
This Methylpiperazine compound inhibits the enzyme PDE 5 in
corpora caverosa of the penis,thereby prolonging the life of cGMP at
that site. This leads to better erection.
ADVERSE REACTION:
 The drug potentiates the hypotensive effects to nitrates, and hence its
administration to persons who are currently using nitrates in any
form is dangerous.
 There is also a degree of cardiac risk associated with sexual activity,
especially in elderly persons over the age of 60 years.
 The plasma levels of sildenafil increases in subjects over age
65,those with renal/hepatic impairment and in those who are being
concurrently treated with cytochrome P 450 inhibitors such as
erythromycin and ketoconazole.Hence,its adverse effects are likely
to be more frequent in these situations.
 Category : PDE 5 INHIBITOR (TREATMENT FOR SEXUAL IMPOTENCE)
11/30/2017 sabya2049@gmail.com 11
LITERATURE REVIEW OF SILDENAFIL
The Intraday and Interday precision for sildenafil and its active
metabolite were found to be 0.0946 and 0.1457 respectively.
Correlation co-efficient for sildenafil was found to be 0.9999 .
LOD and LOQ was found to be 0.1224 µg/ml and 0.4014 µg/ml
respectively.
RP-HPLC method was validated according to the guidelines of Food
and Drug Administration, showing to be accurate (bias within
±2.28%)over the concentration range of 25-150 μg/mL for sildenafil.
Λmax.( Abs. Max.) for sildenafil was monitored at 290 nm .
Tablet dosage form was estimated, and percentage recovery was
98.48-99.992%.
11/30/2017 sabya2049@gmail.com 12
CONCLUSIONS
 The proposed method was found to be simple, precise,
sensitive and accurate for determination of Sildenafil
Citrate in bulk and tablet dosage forms.
 The mobile phase is simple to prepare.
 The method is also validated as per ICH.
 Hence, this method can be easily and conveniently
adopted for routine analysis of Sildenafil Citrate in bulk,
pharmaceutical dosage forms and dissolution mediums.
11/30/2017 sabya2049@gmail.com 13
Precision
Theprecisionoftheproposedmethodwas
ascertainedbyactualdeterminationofsixreplicates
offixedconcentrationofthedrugwithintheBeer’s
rangeandfindingouttheabsorbance'sbythe
proposedmethod.
Fromtheseabsorbance'sMean,Standarddeviation
and%R.S.Dwascalculated.11/30/2017 sabya2049@gmail.com 14
RESULTS IN PRECISION
PRECISION
CONCENTRAT
ION
(µg/ml)
METHOD-I
(MEAN ± SD)
METHOD-II
(MEAN ± SD)
RSD(%)
METHOD-I METHOD-II
INTRADAY 20 0.213 ±
0.0028
20.112 ±
0.0121
1.314 0.0601
INTERDAY 20 0.204 ±
0.0019
20.011 ±
0.0221
0.0931 0.1104
11/30/2017 sabya2049@gmail.com 15
ACCURACY
Todeterminetheaccuracyoftheproposedmethod,
recoverystudieswerecarriedoutbytakingdifferent
amounts(80%,100%and120%)ofbulksamplesofA
sildenafilwithinthelinearityrangeandaddingto
thepre-analyzedformulationconcentration.
Fromthatpercentagerecoveryvalueswere
calculated.
11/30/2017 sabya2049@gmail.com 16
RESULT IN ACCURACY
SAMPLE ID
CONCENTRATION (µg/ml)
%RECOVERY OF PURE DRUG
STATISTICAL ANALYSIS
PURE DRUG FORMULATION METHOD-I METHOD-II METHOD-I METHOD-II
S1: 80% 8 10 98.45 98.69
MEAN= 98.36
SD =0.3286
%RSD = 0.3340
MEAN =99.23
SD =0.6709
%RSD =0.73
S2: 80% 8 10 98 99.89
S3: 80% 8 10 98.64 99.12
S4: 100% 10 10 100.12 101.21
MEAN= 100.45
SD = 0.6715
%RSD= 0.6684
MEAN =101.2
SD =1.014
%RSD= 1.065
S5: 100% 10 10 100.23 99.94
S6: 100% 10 10
100.02 100.25
S7: 120% 12 10 101.23 101.56 MEAN= 100.26
SD = 0.864
%RSD= 0.8617
MEAN = 101.7
SD = 1.214
%RSD = 1.12
S8:
120%
12 10
99.56 100.28
S9:
120%
12 10
100.01 101.8
11/30/2017 sabya2049@gmail.com 17
CONCLUSION
Theproposedmethodsaresimple,sensitiveand
reliablewithgoodprecisionandaccuracy.
Theproposedmethodsarespecificwhileestimating
thecommercialformulationswithoutinterference
ofexcipientsandotheradditives.
Hence,thismethodcanbeusedfortheroutine
determinationofsildenafilinbulksamplesand
Pharmaceuticalformulations.11/30/2017 sabya2049@gmail.com 18
REFERENCE
GRChatwal, S Anand, Instrumental Methods of Chemical Analysis;
2002:209-217.
 Scottish intercollegiate guidelines network (sign), control of pain in adults with
cancer-anationalclinicalguideline.Scotlandnationalhealthservice(nhs).2008:76-
89.
G. T. Warner and B. Jarvis, Olmesartan medoxomil, Drugs 62 (2002)
1345–1353.
 Sissmann j, bouroudian m, armagnac c, donazollo y, latreille m, and panis r.
Angiotensin ii blockade in healthy volunteers: tolerability and impact on renin
angiotensin system components of single and repeated doses of a new angiotensin
iireceptorantagonistsr47436(bms186295).Jhypertens1994;12:s92.
Metabocardforsildenafilretrieved29april2004:1010-1025.11/30/2017 sabya2049@gmail.com 19
11/30/2017 sabya2049@gmail.com 20

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Method development and validation technique

  • 1. SABYA SACHI DAS PhD Scholar Dept. Of Pharm. Sci. & Tech. Birla Institute Of Technology Mesra,Ranchi-835215,Jharkhand
  • 2. INTRODUCTION Sildenafil Citrate is 5-[2-ethoxy-5- (4- methylpiperazin-1-ylsulfonyl)phenyl]-1methyl-3- propyl-1, 6-dihydro-7H-pyrazolo [4, 3-d] pyrimidin- 7-one citrate .  It is used as a smooth muscle relaxtant by enhancing the effect of NO by inhibiting phosphodiesterase type 5 (responsible for degradation of cGMP in corpus cavernosum).  It is used mainly for the treatment of erectile dysfunction. 11/30/2017 sabya2049@gmail.com 2
  • 3. OBJECTIVE There is always a requirement of an accurate and precise analytical method for determination of drugs in bulk and pharmaceutical dosage form. So, the objectives of this work are : Develop a simple and novel RP-HPLC method for estimation of Sildenafil citrate in bulk and pharmaceutical dosage forms.  Optimization of chromatographic (LC) conditions.  Validation of the developed method as per ICH guidelines. 11/30/2017 sabya2049@gmail.com 3
  • 4. MATERIALS AND METHODS • 1] Instrumentation: Quantitative HPLC was performed on Shimadzu HPLC with LC- 20AT pumps besides SPD- 20A UV-Visible detector. Shimadzu Spinchrom-CFR software is used along with inertsil shield RP-C8 (150 mm × 3.9 mm), 5 µm column for the separation. A calibrated electronic single pan of Mettler Toledo was used. • 2] Reagents Used: HPLC grade acetonitrile,triple distilled water, pure drug & marketed formulation of Sildenafil Citrate. 11/30/2017 sabya2049@gmail.com 4
  • 5. HPLC conditions:  Column : LiChroCART®LiChrospher® 100;C18(250 x 4 mm,5μm)  Mobile phase : Acetonitrile: water at 50:50 (v/v)  Flow Rate : 1 mL/min.  Temperature : 300c  Injection volume : 20 µL.  Run time : 15min.  Retention Time : 11.625 min.  UV Détection Wavelength : 290nm Fig.-1. Representative Chromatogram of Sildenafil Citrate in acetonitrile: water at 50:50; v/v 10μg/ml) 11/30/2017 sabya2049@gmail.com 5
  • 6. DRUGPROFILE SILDENAFIL CITRATE Pharmacokinetics data: Bioavailability : Approximately 25% Metabolism : Does not undergo metabolism , it is excreted unchanged entirely in urine. Half- life : 10-12 hours Excretion : Renal 11/30/2017 sabya2049@gmail.com 6
  • 7. validation  The developed method was validated for linearity, accuracy, intraday & interday precision,specificity,LOD,LOQ. The results are shown in detail in the results and discussion section. Results and discussion:  Linearity: The method was found to be linear over a range of 25-150μg/mL with a correlation coefficient of 0.9999.  Analysis of commercial Sildenafil Citrate Tablet: 11/30/2017 sabya2049@gmail.com 7
  • 8.  Accuracy:  Precision:  Specificity : No interference was observed from the excipients present in tablet. 11/30/2017 sabya2049@gmail.com 8
  • 9.  System Suitability:  Stability: Bench top stability studies for 48hr of the sample solution revealed no significant change in the concentration of analyte. (% Recovery= 99.48-99.992%;n=5) 11/30/2017 sabya2049@gmail.com 9
  • 10. Chemical data: Chemical name : 5-[2-ethoxy-5- (4-methylpiperazin-1- ylsulfonyl)phenyl]-1-methyl-3-propyl-1,6- dihydro-7H-pyrazolo[4,3-d] pyrimidin-7-one citrate Empirical formula : C22H30N6O4S, C6H8O7 Molecular weight : 666.7 gm/mole Physical stability: Colour : White to off-white. Solubility : Soluble in dimethylformamide, sparingly soluble in acetic acid, slightly soluble in methanol. Form : Crystalline powder Usual strengths : 25mg;50mg;100mg 11/30/2017 sabya2049@gmail.com 10
  • 11. Pharmacological action: This Methylpiperazine compound inhibits the enzyme PDE 5 in corpora caverosa of the penis,thereby prolonging the life of cGMP at that site. This leads to better erection. ADVERSE REACTION:  The drug potentiates the hypotensive effects to nitrates, and hence its administration to persons who are currently using nitrates in any form is dangerous.  There is also a degree of cardiac risk associated with sexual activity, especially in elderly persons over the age of 60 years.  The plasma levels of sildenafil increases in subjects over age 65,those with renal/hepatic impairment and in those who are being concurrently treated with cytochrome P 450 inhibitors such as erythromycin and ketoconazole.Hence,its adverse effects are likely to be more frequent in these situations.  Category : PDE 5 INHIBITOR (TREATMENT FOR SEXUAL IMPOTENCE) 11/30/2017 sabya2049@gmail.com 11
  • 12. LITERATURE REVIEW OF SILDENAFIL The Intraday and Interday precision for sildenafil and its active metabolite were found to be 0.0946 and 0.1457 respectively. Correlation co-efficient for sildenafil was found to be 0.9999 . LOD and LOQ was found to be 0.1224 µg/ml and 0.4014 µg/ml respectively. RP-HPLC method was validated according to the guidelines of Food and Drug Administration, showing to be accurate (bias within ±2.28%)over the concentration range of 25-150 μg/mL for sildenafil. Λmax.( Abs. Max.) for sildenafil was monitored at 290 nm . Tablet dosage form was estimated, and percentage recovery was 98.48-99.992%. 11/30/2017 sabya2049@gmail.com 12
  • 13. CONCLUSIONS  The proposed method was found to be simple, precise, sensitive and accurate for determination of Sildenafil Citrate in bulk and tablet dosage forms.  The mobile phase is simple to prepare.  The method is also validated as per ICH.  Hence, this method can be easily and conveniently adopted for routine analysis of Sildenafil Citrate in bulk, pharmaceutical dosage forms and dissolution mediums. 11/30/2017 sabya2049@gmail.com 13
  • 15. RESULTS IN PRECISION PRECISION CONCENTRAT ION (µg/ml) METHOD-I (MEAN ± SD) METHOD-II (MEAN ± SD) RSD(%) METHOD-I METHOD-II INTRADAY 20 0.213 ± 0.0028 20.112 ± 0.0121 1.314 0.0601 INTERDAY 20 0.204 ± 0.0019 20.011 ± 0.0221 0.0931 0.1104 11/30/2017 sabya2049@gmail.com 15
  • 17. RESULT IN ACCURACY SAMPLE ID CONCENTRATION (µg/ml) %RECOVERY OF PURE DRUG STATISTICAL ANALYSIS PURE DRUG FORMULATION METHOD-I METHOD-II METHOD-I METHOD-II S1: 80% 8 10 98.45 98.69 MEAN= 98.36 SD =0.3286 %RSD = 0.3340 MEAN =99.23 SD =0.6709 %RSD =0.73 S2: 80% 8 10 98 99.89 S3: 80% 8 10 98.64 99.12 S4: 100% 10 10 100.12 101.21 MEAN= 100.45 SD = 0.6715 %RSD= 0.6684 MEAN =101.2 SD =1.014 %RSD= 1.065 S5: 100% 10 10 100.23 99.94 S6: 100% 10 10 100.02 100.25 S7: 120% 12 10 101.23 101.56 MEAN= 100.26 SD = 0.864 %RSD= 0.8617 MEAN = 101.7 SD = 1.214 %RSD = 1.12 S8: 120% 12 10 99.56 100.28 S9: 120% 12 10 100.01 101.8 11/30/2017 sabya2049@gmail.com 17
  • 19. REFERENCE GRChatwal, S Anand, Instrumental Methods of Chemical Analysis; 2002:209-217.  Scottish intercollegiate guidelines network (sign), control of pain in adults with cancer-anationalclinicalguideline.Scotlandnationalhealthservice(nhs).2008:76- 89. G. T. Warner and B. Jarvis, Olmesartan medoxomil, Drugs 62 (2002) 1345–1353.  Sissmann j, bouroudian m, armagnac c, donazollo y, latreille m, and panis r. Angiotensin ii blockade in healthy volunteers: tolerability and impact on renin angiotensin system components of single and repeated doses of a new angiotensin iireceptorantagonistsr47436(bms186295).Jhypertens1994;12:s92. Metabocardforsildenafilretrieved29april2004:1010-1025.11/30/2017 sabya2049@gmail.com 19