Grayanotoxins (GTXs) are naturally occurring toxins with diterpenoid structures, predominantly found in species of Rhododendron, and are responsible for food poisoning associated with honey, commonly referred to as mad honey. The Black Sea region, known for its rich Rhododendron species, is also the geographical area with the highest incidence of honey poisoning cases globally. In this study, the concentration of GTX-III was quantified in the honey and Rhododendron flower samples collected from the Black Sea region. An Ultra High Performance Liquid Chromatography-Orbitrap®-High Resolution Mass Spectrometry (UHPLC-Orbitrap®-HRMS) analytical method was validated for the precise measurement of GTX-III isomers in both honey and flower matrices. The GTX-III concentrations ranged from 0 to 6.59 mg/g in honey and 33.57-44.99 mg/g in flowers. Additionally, the concentration of GTX-III in honey was found to correlate with the density of Rhododendron pollen present in the honey. The study confirmed that the UHPLC-Orbitrap®-HRMS method is a reliable and sensitive tool for the accurate quantification of GTX-III. Using this sensitive and reliable method, the precise concentration of GTX-III in mad honey can be accurately determined
however, honey samples from case study samples are required for further investigation.