PurposeForensic verification of cyanide (CN) poisoning by direct CN analysis in postmortem blood is challenging due to instability of CN in biological samples. CN metabolites, thiocyanate (SCN−) and 2-aminothiazoline-4-carboxylic acid (ATCA), have been proposed as more stable biomarkers, yet it is unclear if either is appropriate for this purpose. In this study, we evaluated the behavior of CN biomarkers in postmortem swine and postmortem blood to determine which serves as the best biomarker of CN exposure.MethodsCN, SCN−, and ATCA were measured in postmortem swine (N = 8) stored at 4 °C and postmortem blood stored at 25 °C (room temperature, RT) and 37 °C (typical human body temperature, HBT).ResultsFollowing CN poisoning, the concentration of each CN biomarker increased well above the baseline. In postmortem swine, CN concentrations declined rapidly (t1/2 = 34.3 h) versus SCN− (t1/2 = 359 h, 15 days) and ATCA (t1/2 = 544 h, 23 days). CN instability in postmortem blood increased at RT (t1/2 = 10.7 h) and HBT (t1/2 = 6.6 h). SCN− and ATCA were more stable than CN at all storage conditions. In postmortem swine, the t1/2s of SCN− and ATCA were 15 and 23 days, respectively. While both the t1/2s of SCN− and ATCA were relatively lengthy, endogenous levels of SCN− were much more variable than ATCA.ConclusionWhile there are still questions to be answered, ATCA was the most adept forensic marker of CN poisoning (i.e., ATCA produced the longest half-life, the largest increase above baseline levels, and most stable background concentrations).