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

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 (Na2HPO4), 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 ≤11.94%. The limits of detection and quantitation for both analytes were 5.0 ng/mL (s/n > 3) and 10.0 ng/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.