Validated Method for the Quantification of Buprenorphine in Postmortem Blood Using Solid-Phase Extraction and...

A highly sensitive and fully validated method was developed for the quantification of buprenorphine in postmortem blood. After a two-step protein precipitation process using acetonitrile, buprenorphine was purified using mixed-mode (C8/cation exchange) solid-phase extraction cartridges. Endogenous water-soluble compounds and lipids were removed from the cartridges before the samples were eluted, concentrated and derivatized using N-methyl-N-trimethylsilyltrifluoroacetamide. The samples were analyzed using two-dimensional gas chromatography-mass spectrometry (2D GC-MS) in selective ion-monitoring mode. A low polarity Rxi(®)-5MS (30 m × 0.25 mm I.D. × 0.25 µm) was used as the primary column and the secondary column was a mid-polarity Rxi(®) -17Sil MS (15 m × 0.32 mm I.D. × 0.25 µm). The assay was linear from 1.0 to 50.0 ng/mL (r(2) > 0.99; n = 6). Intraday (n = 6) and interday (n = 9) imprecisions (percentage relative standard deviation, % RSD) were <5% and the average recovery was 60%. The limit of detection (LOD) of the method was 0.5 ng/mL and limit of quantification was 1.0 ng/mL. 2D GC-MS improved the LOD of buprenorphine by 20-fold compared with analysis on a conventional GC-MS. The method was highly selective with no interference from endogenous compounds or from 62 commonly encountered drugs. To prove method applicability to forensic postmortem cases, 14 authentic postmortem blood samples were analyzed.