title: Off-line HPLC method combined to LC-MS for the determination of sildenafil and its active metabolite in post-mortem human blood according to confirmation criteria.
abstract: A simple HPLC method has been validated for the determination of sildenafil and its active metabolite (N-desmethylsildenafil) in human blood, using an octadecyl silica (ODS) hypersil column. The chromatographic run time is less than 25 min using a mobile phase of 35:65 (v/v) acetonitrile-0.015 M disodium hydrogen phosphate (Na(2)HPO(4)), triethylamine 0.1%, pH 7.4 at 1 mL/min flow rate and UV-vis detection at 230 nm. The method is linear in the concentration range of 10-500 ng/mL (r>0.999, n=5) for each analyte, with relative standard deviation (R.S.D.) less than 5.05%. Interday and intraday errors were found to be < or =11.94%. The limits of detection and quantitation for both analytes were 5.0 ng/mL (s/n>3) and 10.0ng/mL (s/n>10), respectively. The method was applied in two post-mortem human blood samples, concerning two fatal cases from sildenafil citrate use, reported for the first time in Greece, and the results were further confirmed with LC-MS. The method is proposed as supplementary to LC-MS when inadequate mass fragmentation does not provide information appropriate to meet confirmation criteria.
authors.affiliation: University of Athens, Medical School, Laboratory of Forensic Medicine and Toxicology, 75M. Asias Str., Goudi 11527, Athens, Greece. cpistos@med.uoa.gr
authors.forename: Constantinos
authors.initials: C
authors.lastname: Pistos
authors.forename: Ioannis
authors.initials: I
authors.lastname: Papoutsis
authors.forename: Artemis
authors.initials: A
authors.lastname: Dona
authors.forename: Maria
authors.initials: M
authors.lastname: Stefanidou
authors.forename: Sotiris
authors.initials: S
authors.lastname: Athanaselis
authors.forename: Constantinos
authors.initials: C
authors.lastname: Maravelias
authors.forename: Chara
authors.initials: C
authors.lastname: Spiliopoulou
mesh_terms: D002851:Chromatography, High Pressure Liquid; D002853:Chromatography, Liquid; D004355:Drug Stability; D053593:Forensic Toxicology / Q000379:methods; D006801:Humans; D008297:Male; D013058:Mass Spectrometry; D008875:Middle Aged; D010726:Phosphodiesterase Inhibitors / Q000097:blood*; D010879:Piperazines / Q000097:blood*; D011687:Purines / Q000097:blood; D000068677:Sildenafil Citrate; D013048:Specimen Handling; D013450:Sulfones / Q000097:blood*
chemical_list: D010726:Phosphodiesterase Inhibitors; D010879:Piperazines; D011687:Purines; D013450:Sulfones; D000068677:Sildenafil Citrate