Development and validation of an analytical method for determination of bongkrekic acid in biofluids for toxin monitoring

Ying-Tzu Shieh^a,1, Te-I Weng^a,b,c,1, Ju-Yu Chen^a,d,1, I-Ting Wang^e, Kevin Shu-Leung Lai^f, Chia-Mo Lin^g,h, Chu-Yun Teng^a, Guan-Yuan Chen^a,b,*

^a Forensic and Clinical Toxicology Center, National Taiwan University Hospital, Taipei, Taiwan

^b Department and Graduate Institute of Forensic Medicine, College of Medicine, National Taiwan University, Taipei, Taiwanc Department of Emergency Medicine, National Taiwan University Hospital, Taipei, Taiwan

^d School of Pharmacy, College of Medicine, National Taiwan University, Taiwan

^e Department of Critical Care, Mackay Memorial Hospital, 10491, Taipei, Taiwan

^f Division of Critical Care Medicine, Taipei Medical University Hospital, Taipei 110, Taiwan

^g Chest Department, Shin Kong Memorial Wu Ho-Su Hospital, Taipei, Taiwan

^h Graduate Institute of Biomedical and Pharmaceutical Science, Fu-Jen Catholic University, New Taipei City, Taiwan

Bongkrekic acid (BKA) poisoning is a severe foodborne illness with a high mortality rate. This study aimed to identify BKA in postmortem tissues from poisoning victims in Taiwan and to develop a reliable analytical method for detecting BKA in biofluids to aid clinical diagnosis and treatment. BKA was identified in postmortem samples using high-reso-lution mass spectrometry (HRMS) and confirmed with an independent ultrahigh-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS) method. To meet clinical needs, a UHPLC-MS/MS method was developed and validated for BKA detection in plasma and urine. Method optimization included adjustments to ion source conditions for multiple reaction monitoring (MRM) transitions and avoidance of glass vials due to BKA adsorption onto free silanol groups. Chromatographic separation was achieved using a 50-mm Hypersil Gold C18 col-umn within a 6-min run time. The validated UHPLC-MS/MS method successfully detected BKA in biofluids, enabling its application in identifying victims of foodborne poisoning. The method demonstrated high accuracy and efficiency, facilitating timely diagnosis and aiding in treatment strategies for critically ill patients. The developed UHPLC-MS/MS method provides a reliable approach for detecting BKA in clinical and forensic settings. Its implementation enhances diagnostic capabilities, improves patient outcomes, and supports monitoring of toxin elimination in cases of BKA poisoning.

Keywords: Bongkrekic acid, Biofluids, Clinical analysis, Diagnosis, LC-MS/MS