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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.

Pistos C, Papoutsis I, Dona A, Stefanidou M, Athanaselis S, Maravelias C, Spiliopoulou C

University of Athens, Medical School, Laboratory of Forensic Medicine and Toxicology, 75M. Asias Str., Goudi 11527, Athens, Greece. cpistos@med.uoa.gr

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.

Published 2 June 2008 in Forensic Sci Int, 178(2): 192-8.
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