Gonyautoxin III

The neurotoxins of the saxitoxin family can be the origin of the human neurological syndrome of paralytic shellfish poisoning via contaminated marine bivalve vectors. A pre-chromatographic oxidation method is the official testing method in the EU, also known as the 'Lawrence method'. It involves several steps, including toxin extraction, solid-phase clean-up, and oxidation to produce fluorescent derivatives. Despite the manual peroxide oxidation involving four pipetting steps, high coefficients of determination were commonly obtained for the calibration curves of the respective toxin oxidation products. However, the 11-hydroxysulphate toxins dcGTX2 + 3, C1 + 2 and GTX2 + 3, often had slightly lower coefficients of determination (i.e. R² < 0.998) than their non-11-hydroxysulphate counterparts dcSTX, GTX5 and STX (i.e. R² > 0.998). Deviations of ±10 s from the respective standard reaction times, caused significant alterations in fluorescent yield for toxins quantified with peroxide but not for those quantified with periodate. The oxidation is an endothermal reaction, and its reaction rate is toxin-specific, with the N11-hydroxysulphate toxins having a slower reaction rate. This was confirmed by incubating GTX2 + 3 and STX at different reaction times. The incubation of toxins in a bivalve matrix also slows down the reaction in comparison with oxidation in dilute acetic acid, giving lower recoveries, as studied here in detail for dcGTX2 + 3. Circumventing the matrix effect by dilution is not possible, as 10% of matrix is enough to cause a reduction to half of the fluorescence yield of dcGTX2 + 3. When heating the N1-H toxins with peroxide, the increase in fluorescence yield is inversely proportional to the recovery values commonly found for each toxin.

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.