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HomeSmall Molecule HPLCSildenafil Citrate Method (HPLC) following USP Monograph on Ascentis® Express C18 with UV detection

Sildenafil Citrate Method (HPLC) following USP Monograph on Ascentis® Express C18 with UV detection

Sildenafil, sold under the brand name Viagra, among others, is a medication used to treat erectile dysfunction and pulmonary arterial hypertension. In 1994, a patent was filed covering the use of sildenafil to treat erectile dysfunction. This patent will expire in 2019.

Ascentis® Express C18 columns can be used for the HPLC methods within the sildenafil citrate USP Monograph, here illustrated with appropriate analytical data to verify that system suitability criteria are met per USP41-NF36 guidelines.

Same experimental conditions are used for both assay and organic impurities (related substances) but in the latter method the chromatograph should run for three (3) times the retention time of sildenafil. The suitability requirements for the assay method are defined reproducibility (not more than 0.85% for six replicates) and peak tailing factor (not more than 1.5) whereas for organic impurities the relative retention times for sildenafil, sildenafil N-oxide, and sildenafil related compound A are about 1.0, 1.2, and 1.7, respectively. The resolution should be not less than 2.5 between sildenafil N-oxide and sildenafil (using the system suitability solution), and the signal-to-noise ratio not less than 10 (using the sensitivity solution).

sildenafil-citrate

Sildenafil Citrate

Experimental Conditions
ColumnAscentis® Express C18 150x4.6 mm, 5 µmInjection volume10 µL
DetectionUV @ 290 nm (analytical flow cell; 10 µL)Flow rate1.0 mL/min
BufferDilute 7 mL of triethylamine with water to 1 L.
Stir and adjust with phosphoric acid to a
pH of 3.0 ± 0.1.
Temperature30 °C
Mobile phaseMix buffer, methanol and acetonitrile 58:25:17 (v/v/v)Pressure drop160 bar (2321 psi)
DiluentMobile phase  
SST solutionDissolve 70 mg of Sildenafil Citrate in 1 mL of a solution of hydrogen peroxide and anhydrous formic acid (2:1).
Allow to stand for at least 10 min to generate sildenafil N-oxide, and then dilute with mobile phase to 250 mL.
Sample solution0.7 mg/mL of Sildenafil Citrate in mobile phase.
Diluted sample solution1.4 µg/mL of Sildenafil Citrate in mobile phase from the sample solution
Sensitivity solution0.35 µg/mL of Sildenafil Citrate in mobile phase from the diluted sample solution
Sildenafil Citrate
sildenafil-citrate
sildenafil-citrate
 CompoundRetention
Time (min)
ResolutionTheoretical
Plates
Tailing
Factor
1Sildenafil Citrate6.0-11,6071.1
2N-oxide7.03.711,4991.1
Chromatographic Data (System Suitability Solution)
 CompoundRetention Time (min)Tailing factorTheoretical PlatesSignal-to-noise ratio
1Sildenafil Citrate6.01.011,49917.0
Chromatographic Data (Sensitivity Solution)
Chromatographic Data
1. Specificity: Inject solution and determine the retention time of desired analyte in presence of other component like impurities and excipient.
  Retention Time (min)RRTTailing Factor
1Sildenafil citrate6.01.01.0
2N-oxide7.01.21.2
3Related compound A9.51.61.1
Validation and verification
2. Standard Repeatability (0.35ppm)
STD 14881
STD 24900
STD 34797
STD 44842
STD 54860
Mean4868
Standard Deviation40.0
RSD (%)0.8
3. Linearity 
Concentration (ppm)Area of Sildenafil Citrate
0.354783
1.420135
3.548171
794168
14187369
22.4299620
28377224
33.6461333
42558476
LOQ0.2 ppm (S/N 10)
LOD0.06 ppm (S/N 3.0)
Materials
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