Pistos Constantinos, Papoutsis Ioannis, Dona Artemis, Stefanidou Maria, Athanaselis Sotiris, Maravelias Constantinos, Spiliopoulou Chara
University of Athens, Medical School, Laboratory of Forensic Medicine and Toxicology, 75M. Asias Str., Goudi 11527, Athens, Greece.
Forensic Sci Int. 2008 Jul 4;178(2-3):192-8. doi: 10.1016/j.forsciint.2008.03.018. Epub 2008 May 16.
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.
已验证一种简单的高效液相色谱法，可使用十八烷基硅胶（ODS）Hypersil柱测定人血中的西地那非及其活性代谢物（N-去甲基西地那非）。使用35:65（v/v）乙腈-0.015M磷酸氢二钠（Na₂HPO₄）、0.1%三乙胺、pH 7.4的流动相，流速为1 mL/min，并在230nm处进行紫外-可见检测时，色谱运行时间少于25分钟。该方法对每种分析物在10 - 500 ng/mL的浓度范围内呈线性（r>0.999，n = 5），相对标准偏差（R.S.D.）小于5.05%。日间和日内误差均≤11.94%。两种分析物的检测限和定量限分别为5.0 ng/mL（s/n>3）和10.0 ng/mL（s/n>10）。该方法应用于两份尸检人血样本，涉及希腊首次报道的两起因使用枸橼酸西地那非导致的致命病例，结果通过液相色谱-质谱联用（LC-MS）进一步得到证实。当质谱碎片不足无法提供符合确认标准的适当信息时，建议将该方法作为LC-MS的补充方法。
