Quantification of 2-aminothiazoline-4-carboxylic acid as a reliable marker of cyanide exposure using chemical derivatization followed by liquid chromatography–tandem mass spectrometry

Abstract

In this research, we have developed a novel and simple liquid chromatography coupled with electrospray ionization–tandem mass spectrometry (LC/ESI–MS/MS) method for quantification of 2-aminothiazoline-4-carboxylic acid (ATCA), which is produced by the direct reaction of cyanide (CN) with endogenous cystine. In forensic science, detection of CN is important because CN is a poison that is often used for murder or suicide, in addition to being produced by the thermal decomposition of natural or synthetic materials. However, because CN disappears rapidly from body tissue, ATCA is thought to be a more reliable indicator of CN exposure. For the method reported herein, human blood samples (20 μL) were subjected to protein precipitation followed by derivatization with 4-bromoethyl-7-methoxycoumarin. Blood spiked with ATCA at concentrations ranging from 50 to 1500 ng/mL was used to prepare a calibration curve (lower limit of quantification; 50 ng/mL, lower limit of detection; 25 ng/mL). Our method uses chemical derivatization, so unlike previously reported methods, it does not require tedious pretreatment procedures, hydrophilic interaction liquid chromatography columns, or specialized equipment. In addition, our method allows for repeatable and accurate quantification of ATCA, with intra- and inter-assay coefficients of variation of below 5.0% and below 6.0%, respectively. We used the method to analyze ATCA in postmortem human blood samples, including samples from people who had intentionally ingested CN or were fire victims. Blood ATCA concentrations were higher among people who had ingested CN or were fire victims than among people in a control group (P < 0.0001). The data reported herein demonstrate that our LC/ESI–MS/MS method can be used to detect and quantify ATCA in postmortem blood samples and that CN exposure strongly affects ATCA concentration, providing a useful tool for detection of CN poisoning.