Simultaneous determination of 19 Aconitum alkaloids in whole blood and urine by ultra-high performance liquid chromatography-tandem mass spectrometry.

A rapid and sensitive UPLC-MS/MS method was developed and validated for the simultaneous quantification of nineteen Aconitum alkaloids in human blood and urine. Sample preparation utilized Oasis PRIME MCX μElution plates, which enabled efficient extraction and minimized matrix effects. The method demonstrated excellent selectivity, linearity (R² > 0.99), precision (RSDs < 9.1 %), and accuracy (±9 %). Stability testing revealed that benzoylated alkaloids exhibited notable degradation at room temperature within 8 h, likely due to hydrolysis； other alkaloids demonstrated varying degrees of stability under freeze-thaw cycles and long-term frozen storage. Extracted samples remained mostly stable when stored at 4°C for 24 h in the autosampler, emphasizing the importance of prompt analysis to ensure analyte integrity. Application to 25 fatal aconite poisoning cases revealed significant inter-individual variability, with generally higher alkaloid concentrations detected in urine, highlighting its utility as a sensitive matrix for exposure confirmation. A unique mass poisoning incident was characterized by the detection of only alcohol amine-type alkaloids without the highly toxic diester diterpenoid alkaloids, underscoring the need to expand analyte panels in toxicological investigations. Due to overlapping alkaloid profiles among Aconitum species and the impact of environmental and sample-handling factors, chemical profiling alone is insufficient for definitive species identification. Integrated multidisciplinary strategies are recommended for accurate diagnosis and forensic interpretation. This validated method provides a robust analytical platform to enhance forensic and clinical assessments of Aconitum poisoning.