CORESTA Congress, Kunming, 2018, Agronomy/Phytopathology Groups, AP 40

Plant metabolism studies with 14C-maleic hydrazide (MH) in tobacco and evaluation of analytical methods

(1) Japan Tobacco Inc., Leaf Tobacco Research Center, Oyama, Tochigi, Japan; (2) Japan Tobacco Inc., Scientific and Regulatory Affairs Division, Tokyo, Japan

Maleic hydrazide (MH) is used as a sucker control agent for tobacco. Reportedly, absorbed MH in tobacco plants is metabolized to its glucoside conjugates and is converted to the bound form. However, how much original MH can be converted to those forms in tobacco plants has not been reported. Regarding analytical methods, present analytical methods including the ISO method and in-house methods exist to determine MH and, allegedly, its metabolites. Nevertheless, it remains uncertain whether all forms can be ascertained, or not, using the respective analytical methods. This study was conducted 1) to clarify the metabolite types and amounts and 2) to evaluate present MH analytical methods. 14C-labeled MH with inert ingredients was applied to flue-cured tobacco. Tobacco leaves were harvested 28 days after the application. Then, leaves were cured in a small curing chamber. After cured leaves were ground and extracted with MeOH/water, the radioactivity in these extracts/post-extraction solids (PES) was measured using a liquid scintillation counter. The respective radioactive compounds were characterized using HPLC radiochromatography and thin-layer co-chromatography. 1) Based on the results, the percentage of total radioactive residue (%TRR) was calculated for each compound. In the extract with MeOH/water of cured tobacco, MH-O-glucoside (MH-O-Glc) and MH-N-glucoside (MH-N-Glc) were observed as major metabolites with the TRR 13.6 % and 3.3 %, respectively. 2) When 14C-MH treated tobacco was extracted under present analytical conditions, these conjugates were not observed in each extract. Furthermore, the remaining radioactivity in PES differed under respective analytical methods. These results identify the precise %TRR of major metabolites and suggest that glycoside conjugates could be hydrolyzed or not extracted during the analytical process of the present MH analytical methods. %TRR of the parent compound, metabolites and/or non-extractable fraction should be examined when different analytical methods are applied.