Comprehensive Analysis and Forensic Investigation of Cyanide, Thiocyanate, and 2-Amino-2-thiazoline-4-carboxylic Acid in Ante- and Postmortem Blood utilizing Advanced Analytical Techniques-Abdullah Alluhayb

Abstract

Cyanide (CN), present as cyanide anion (CN-) or hydrogen cyanide (HCN), is a deadly poison. The main mechanism of CN toxicity is blocking terminal electron transfer, leading to cellular hypoxia, cytotoxic anoxia, and potential death. Determining blood CN concentrations along with its major metabolite, thiocyanate (SCN-), is crucial. A sensitive method for concurrent detection of CN and SCN- from human ante- and post-mortem blood via liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) analysis was developed. The method involved active microdiffusion with chemical modification using naphthalene-2,3 dicarboxaldehyde (NDA) and taurine, while monobromobimane (MBB) modification was used for SCN-. The method showed good sensitivity for CN with antemortem limits-of-detection (LODs) of 219 nM and 605 nM for CN and SCN-, respectively, and postmortem LODs of 352 nM and 509 nM. The method also produced good accuracy and precision, detecting elevated levels of CN and SCN- in both ante- and post-mortem blood samples from CN-exposed swine compared to non-exposed swine. Forensic verification of CN poisoning by direct CN analysis in postmortem blood is challenging due to the instability of CN in biological samples. CN metabolites, SCN- and 2-aminothiazoline-4-carboxylic acid (ATCA), have been proposed as more stable biomarkers, but their appropriateness for verification of poisoning remains unclear. The behavior of CN, SCN-, and ATCA in postmortem swine stored at 4°C and postmortem blood stored at room temperature (25°C; RT) and typical human body temperature (37°C; HBT) was evaluated. After poisoning swine with CN, the blood concentration of each CN biomarker increased well above the baseline. In postmortem swine, elevated CN concentrations declined rapidly, whereas SCN- and ATCA concentrations remained relatively stable. The decline in CN accelerated as the storage temperature increased, where CN was below baseline levels within 50, 74, and 120 h for postmortem swine at 4°C and postmortem blood at RT and HBT, respectively. The half-lifes (t1/2) of CN were 34.3 h, 10.8 h, and 6.6 h at 4 °C (in postmortem swine), RT, and HBT, respectively. SCN- and ATCA were more stable than CN at all storage conditions, producing t1/2s of 15-19 days and 23-26 days for SCN- and ATCA, respectively. ATCA exhibited the most extended t1/2 and most significant increase above baseline levels for the CN biomarkers tested, making it the most appropriate biomarker for confirming CN poisoning via postmortem blood analysis.