Validation of an HPLC Method for Determining Sildenafil and NDMS Plasma Concentrations in Pulmonary Arterial Hypertension
Pranisa Wongwien, MD1, Wichittra Tassaneeyakul, PhD1, Sirimas Kanjanawart, PhD1, Thikhumporn Areesimpitak, MD1,
Nontaya Nakkam, PhD1, Burabha Pussadhamma, MD2, Suda Vannaprasaht, MD1
Affiliation : 1Department of Pharmacology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand, 2Cardiology Unit, Department of Medicine, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand
Background: Variability in sildenafil pharmacokinetics may impact pulmonary arterial hypertension (PAH) outcomes. According to the limitations of current methods.
Objective: The research developed a simple and accurate method to measure sildenafil and its metabolite (N-desmethyl sildenafil; NDMS).
Materials and Methods: High-performance liquid chromatography (HPLC) with a UV detector at 296 nm and a C18 Kinetex column was used. The mobile phase consisted of 0.05 M potassium dihydrogen phosphate, acetonitrile, and methanol (47: 23: 30 v/v/v), with a 50 μL injection volume and a flow rate of 1 mL/min. Glipizide served as the internal standard.
Results: A linear correlation was observed for sildenafil (10 to 1,500 ng/mL) and NDMS (10 to 500 ng/mL), with R² values of 0.9998 and 0.9995, respectively. The lower limit of detection was 5 ng/ml for both compounds. Retention times were 3.25 minutes for sildenafil and 6.48 minutes for NDMS.
Conclusion: This HPLC method with UV detection was validated for its simplicity, reliability, precision, recovery, stability, and specificity for measuring sildenafil and NDMS in plasma from PAH patients.
Keywords : HPLC; Sildenafil; N-desmethyl sildenafil; PAH
