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

Experimental Conditions
Column	Ascentis® Express C18 150x4.6 mm, 5 µm	Injection volume	10 µL
Detection	UV @ 290 nm (analytical flow cell; 10 µL)	Flow rate	1.0 mL/min
Buffer	Dilute 7 mL of triethylamine with water to 1 L. Stir and adjust with phosphoric acid to a pH of 3.0 ± 0.1.	Temperature	30 °C
Mobile phase	Mix buffer, methanol and acetonitrile 58:25:17 (v/v/v)	Pressure drop	160 bar (2321 psi)
Diluent	Mobile phase
SST solution	Dissolve 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 solution	0.7 mg/mL of Sildenafil Citrate in mobile phase.
Diluted sample solution	1.4 µg/mL of Sildenafil Citrate in mobile phase from the sample solution
Sensitivity solution	0.35 µg/mL of Sildenafil Citrate in mobile phase from the diluted sample solution

Chromatographic Data (System Suitability Solution)

	Compound	Retention Time (min)	Resolution	Theoretical Plates	Tailing Factor
1	Sildenafil Citrate	6.0	-	11,607	1.1
2	N-oxide	7.0	3.7	11,499	1.1

Chromatographic Data (Sensitivity Solution)

	Compound	Retention Time (min)	Tailing factor	Theoretical Plates	Signal-to-noise ratio
1	Sildenafil Citrate	6.0	1.0	11,499	17.0

Validation and verification

1. Specificity: Inject solution and determine the retention time of desired analyte in presence of other component like impurities and excipient.
		Retention Time (min)	RRT	Tailing Factor
1	Sildenafil citrate	6.0	1.0	1.0
2	N-oxide	7.0	1.2	1.2
3	Related compound A	9.5	1.6	1.1

2. Standard Repeatability (0.35ppm)
STD 1	4881
STD 2	4900
STD 3	4797
STD 4	4842
STD 5	4860
Mean	4868
Standard Deviation	40.0
RSD (%)	0.8

3. Linearity
Concentration (ppm)	Area of Sildenafil Citrate
0.35	4783
1.4	20135
3.5	48171
7	94168
14	187369
22.4	299620
28	377224
33.6	461333
42	558476
LOQ	0.2 ppm (S/N 10)
LOD	0.06 ppm (S/N 3.0)