Yamaguchi_2026_Forensic.Toxicol__

PURPOSE: Authentic Novichok (A-series) nerve agents are highly restricted, making the rapid preparation of positive controls and reference materials challenging in forensic toxicology. This study aimed to evaluate p-nitrophenoxy analogs as surrogate compounds for Novichok-related biomarker verification by assessing their synthetic accessibility, ability to generate diagnostically relevant butyrylcholinesterase (BuChE) nonapeptide biomarkers, and functional relevance in cholinesterase inhibition and reactivation assays. METHODS: Six surrogate compounds representing phosphate- and phosphonate-type analogs were prepared using a revised synthetic sequence with scaffold-dependent optimization of the reaction and purification conditions. Their utility was evaluated using plasma BuChE inhibition assays, 2-pyridine aldoxime methochloride (2-PAM)-mediated reactivation studies, inhibition kinetics with horse BuChE and human acetylcholinesterase, and liquid chromatography-tandem mass spectrometry analysis of BuChE-derived nonapeptide adducts in plasma. Operational applicability was examined using the OPCW biomedical proficiency test (BioPT) evaluation criteria. RESULTS: All six surrogate compounds were obtained by structural class-dependent optimizations of their synthesis and purification. The plasma BuChE IC(50) values of authentic A230, A232, A234, A242, A262, and A2xx were 12.3, 9.6, 8.6, 10.1, 10.6, and 24.5 ng/mL, respectively, whereas those of the corresponding surrogates were 58.5, 38.6, 55.2, 3942, 3829, and 2575 ng/mL. 2-PAM-mediated BuChE reactivation showed similar qualitative patterns within each authentic/surrogate pair. All surrogates generated analytically equivalent BuChE nonapeptides and could be used to prepare qualitative reference materials for the OPCW BioPT-related workflow. CONCLUSION: p-Nitrophenoxy Novichok-related analogs provide a practical surrogate platform for preparing diagnostically relevant BuChE nonapeptide reference materials without using authentic A-series nerve agents.