Residual Behavior Of Gibberellic Acid And Its Degradation In Tea

Gibberellic acid（GA₃）,as the typical plant growth regulator,has been widely used in various stages of tea plant growth and development in tea gardens in China.Due to the extremely low content of endogenous GA₃ in plants and the matrix effect caused by the tea matrix,accurate and sensitive analysis of GA₃ in tea is a challenge for tea safety and tea plant physiology concerning with GA₃.In addition,the behavior of GA₃ degradation in tea is still unclear,and its degradation products and residual behavior have not been reported,leading to the problem of the scientific use of exogenous GA₃ and the lack of maximum residue limit standard（MRL）in tea in China.Therefore,there is an urgence to establish an analytical method for determining GA₃ and its metabolites in tea,assess the safety of exogenous GA₃,and ensure the safety of tea.In this study,ultra high performance liquid chromatography-tandem mass spectrometry（UHPLC-MS/MS）was used to establish a simple,rapid,accurate,and highly sensitive method for the determination of GA₃ in tea.Q-Exactive Orbitrap mass spectrometry（Q-Exactive Orbitrap MS）was used to analyze degradation products of GA₃ stress degradation and their degradation mechanism.The degradation of GA₃ and its conversion into main metabolites were investigated during tea planting,manufacturing,and brewing.The results are as follows:（1）A highly sensitive analytical method for 13 acidic plant hormones（or plant growth regulators）including GA₃ in tea was established using UHPLC-MS/MS.An Agilent Eclipse Plus C18 column combined with the mobile phase A（acetonitrile）and B（water）was employed for high sensitivity and good chromatographic retention of 13 target compounds.Target acidic phytohormones were extracted using acidified methanol（2%formic acid）,and tea matrices were cleaned up by dispersive solid phase extraction（D-SPE）followed by polymer-based mixed mode cation-exchange solid phase extraction（SPE）,which achieved highly sensitive and accurate quantitative analysis of 13 phytohormones and their analogues in fresh tea leaves,including gibberellins,auxins,abscisic acid,etc.The method showed good linearity for all 13 analytes with regression coefficients（R²）>0.998.Satisfactory recoveries of 13analytes spiked with three levels ranged from 71.8%to 109.9%,while intra-day and inter-day relative standard deviations（RSDs）were below 20%.Limits of detection（LODs）and limits of quantitation（LOQs）for 12 acidic phytohormones were 0.1-4.2μg/kg and 0.3-13.9μg/kg,respectively.Finally,this method was firstly employed to analyze 13 analytes in fresh tea leaves（with the treatment of dormancy,light qualities,exogenous hormones and infestation of pests）,highlighting its sufficient capability for rapid analysis of multiclass phytohormones in agriculture field.（2）This study firstly investigated the degradation behavior of GA₃ and its degradation products using ultra high-performance liquid chromatography Q-Exactive Orbitrap MS（UHPLC Q-Exactive Orbitrap MS）.Different stress factors such as light,p H and temperatures were investigated through photolysis and hydrolysis experiments.Five degradation products were identified using UHPLC Q-Exactive Orbitrap MS.Three degradation products M273,M283-1 and M283-2 were produced through photolysis.GA₃ could be converted into its isomers through hydrolysis,while only one isomer gibberellenic acid（en-GA₃）was formed under acid conditions,and two isomers（iso-GA₃ and en-GA₃）were converted under alkaline conditions.In order to characterize each degradation product,complete mass fragmentation pathways of the GA₃ and its degradation products were initially established with the help of Q-Exactive Orbitrap MS accurate mass studies.These results could provide a practical reference for the safety of agricultural products and the guidance for scientific application and proposed storage conditions of GA₃.（3）Field experiments were conducted to explore the metabolic behavior of exogenous GA₃ during tea production.The metabolite iso-GA₃ was first discovered during the tea production chain.During tea planting,the half-lives of GA₃ dissipation ranged from 2.46 to 2.74 days in tea shoots.GA₃ was easy to degraded into iso-GA₃ in tea shoots,which had a longer residual period than GA₃.Meanwhile,the mechanism that application of external GA₃ could promote the rapid growth of tea plants was explained from the phytohormones level.During tea manufacturing,iso-GA₃ was quickly and massively converted from GA₃.Fixing（heat at 220-230°C）played an important role in the dissipation of GA₃ and iso-GA₃ during green tea manufacturing,but there were high residues of iso-GA₃ in black tea.High transfer rates（77.3%-94.5%）of GA₃ and iso-GA₃ were observed during tea brewing.These results could provide guidance for the scientific application of GA₃ in tea and other agricultural products.