3-Chloro-L-tyrosine

Quantification with satisfactory specificity and sensitivity of free 3-Nitro-l-tyrosine (3-NT), 3-Chloro-l-tyrosine (3-CT), and 3-Bromo-l-tyrosine (3-BT) in biological samples as potential inflammation, oxidative stress, and cancer biomarkers is analytically challenging. We aimed at developing a liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based method for their simultaneous analysis without an extract purification step by solid-phase extraction. Validation of the developed method yielded the following limits of detection (LOD) and quantification (LOQ) for 3-NT, 3-BT, and 3-CT: 0.030, 0.026, 0.030 ng/mL (LODs) and 0.100, 0.096, 0.098 ng/mL (LOQs). Coefficients of variation for all metabolites and tested concentrations were <10% and accuracy was within 95-105%. Method applicability was tested on colorectal cancer patients during the perioperative period. All metabolites were significantly higher in cancer patients than healthy controls. The 3-NT was significantly lower in advanced cancer and 3-BT showed a similar tendency. Dynamics of 3-BT in the early postoperative period were affected by type of surgery and presence of surgical site infections. In conclusion, a sensitive and specific LC-MS/MS method for simultaneous quantification of free 3-NT, 3-BT, and 3-CT in human plasma has been developed.

Chlorine gas exposure occurs in chemical warfare, industrial and household accidents. In forensic science, the generation of chlorine gas by mixing sodium hypochlorite detergent and strong acid detergent cannot be overlooked because of the possibility of suicide method (NaClO + 2HCl → NaCl + H2O + Cl2). Though typical autopsy findings are obtained in chlorine exposure, such as pulmonary edema, useful biomarkers don't exist. In this research, we developed an analytical method of 3-chloro-l-tyrosine (Cl-Tyr) in blood as a novel marker of chlorine poisoning utilizing gas chromatography-mass spectrometry (GC-MS). Cl-Tyr was purified using protein precipitation and cation-exchange solid phase extraction, derivatized by the silylation agent and subjected to GC-MS. The quantification range was 10-200 ng/mL and good reproducibility was obtained. We applied the developed method to analyze Cl-Tyr in autopsy sample, which is suspected of chlorine poisoning, and detected 59.7 ng/mL Cl-Tyr in left heart blood. To our knowledge, this is the first report of determination of the chlorinated biomolecule in the human autopsy sample from chlorine poisoning.

A simple and sensitive liquid chromatography coupled with electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) method for the determination of 3-chloro-L-tyrosine (Cl-Tyr) was developed and validated. For sample preparation, 50 μL of the body fluids or tissue extracts were processed by protein precipitation followed by the derivatization with dansyl chloride. The calibration curve was linear over the concentration range of 2.0-200 ng/mL blood or 4.0-400 ng/g tissue. Our method allowed the reproducible and accurate quantification. That is, the intra- and inter-assay coefficients of variation were below 7.73 and 6.94%, respectively in both the blood and lung. We applied the developed method to the analysis of Cl-Tyr in the human autopsy samples, which were suspected of chlorine poisoning, and detected 55.2 ng/mL and 206.6 ng/g Cl-Tyr in left heart blood and lung, respectively. Furthermore, in more than 20 autopsy samples, which were obtained from other causes of death including burn, drowning, hanging, internal disease, trauma and drug poisoning, Cl-Tyr was almost not detected in their both body fluids and organ tissues. In conclusion, the data here reported demonstrate that the LC/ESI-MS/MS method allows the Cl-Tyr in the autopsy samples and that chlorine exposure strongly affects its level, providing a basis for novel identification tool of chlorine poisoning.