title: Sensitive and precise HPLC method with back-extraction clean-up step for the determination of sildenafil in rat plasma and its application to a pharmacokinetic study.
abstract: A sensitive HPLC method was developed and validated for the determination of sildenafil concentrations in rat plasma (200 μL) using a liquid-liquid extraction procedure and paroxetine as an internal standard. In order to eliminate interferences and improve the peak shape, a back-extraction into an acidic solution was utilized. Chromatographic separation was achieved on a cyanopropyl bonded-phase column with a mobile phase composed of 50 m m potassium dihydrogen phosphate buffer (pH 4.5) and acetonitrile (75:25, v/v), pumped at the flow rate of 1 mL/min. A UV detector was set at 230 nm. A calibration curve was constructed within a concentration range from 10 to 1500 ng/mL. The limit of detection was 5 ng/mL. The inter- and intra-day precisions of the assay were in the ranges 2.91-7.33 and 2.61-6.18%, respectively, and the accuracies for inter- and intra-day runs were within 0.14-3.92 and 0.44-2.96%, respectively. The recovery of sildenafil was 85.22 ± 4.54%. Tests confirmed the stability of sildenafil in plasma during three freeze-thaw cycles and during long-term storage at -20 and -80°C for up to 2 months. The proposed method was successfully applied to a pharmacokinetic study in rats.
authors: Department of Pharmacokinetics and Physical Pharmacy, Faculty of Pharmacy, Jagiellonian University, Medical College, Cracow, Poland.
authors: Beata
authors: B
authors: Strach
authors: Department of Pharmacokinetics and Physical Pharmacy, Faculty of Pharmacy, Jagiellonian University, Medical College, Cracow, Poland.
authors: Elżbieta
authors: E
authors: Wyska
authors: Department of Pharmacokinetics and Physical Pharmacy, Faculty of Pharmacy, Jagiellonian University, Medical College, Cracow, Poland.
authors: Krzysztof
authors: K
authors: Pociecha
authors: Department of Pharmaceutical Technology and Biopharmaceutics, Faculty of Pharmacy, Jagiellonian University, Medical College, Cracow, Poland.
authors: Anna
authors: A
authors: Krupa
authors: Department of Pharmaceutical Technology and Biopharmaceutics, Faculty of Pharmacy, Jagiellonian University, Medical College, Cracow, Poland.
authors: Renata
authors: R
authors: Jachowicz
mesh_terms: D000818:Animals; D002138:Calibration; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation / Q000379:methods*; D004355:Drug Stability; D005260:Female; D057230:Limit of Detection; D059625:Liquid-Liquid Extraction / Q000379:methods*; D008297:Male; D017208:Rats, Wistar; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D000068677:Sildenafil Citrate / Q000097:blood* / Q000493:pharmacokinetics*; D014466:Ultraviolet Rays
chemical_list: D000068677:Sildenafil Citrate
keywords: HPLC-UV; pharmacokinetics; rats; sample stability; sildenafil