Study On Grayanane Toxins And Tropane Alkaloids Using Liquid Chromatography Coupled With Mass Spectrometry In Honey

As a natural sweet food,honey is known as a ?natural antibiotic?.However,in recent years,the frequent occurrence of honey poisoning in the southern region has caused panic among consumers,which has become an important risk in food safety.The poisonous nectar plants such as Rhododendron and Datura are widely distributed in China.When the nectar of the large honey plant is scarce,the poisonous nectar is easily collected by the bees,and the harmful substances are transferred and enriched in the honey to produce toxic honey.The grayanane toxins and tropane alkaloids are the main toxic components in rhododendron and mandala,respectively.Ingestion of trace amounts can lead human poisoning reaction,and even severe death.In view of this,a quantitative method was developed for the detection of grayanane toxins and tropane alkaloids in honey based on ultra performance liquid chromatography tandem mass spectrometry in this study,method validation was performed systematicly.The aim is to timely and effectively avoid the risk of toxic honey ingestion,protect the safety of consumers,and provide technical support for the quality and safety of bee products and risk assessment.This study is mainly divided into two parts.The first part establishes a UHPLC-MS/MS method for the determination of four grayanane toxins(Rhodojaponin II,Rhodojaponin III,Grayanotoxin I,Grayanotoxin III)in honey.Using methanol/water(20:80,v/v,containing 0.1% acetic acid)as extract solvents,0.1% acetic acid-water and 0.1% acetic acid-methanol as mobile phase for the gradient elution in this method.The detection limit of this method is 2.0 ?g/kg ~ 5.0 ?g/kg,and the limit of quantification is 5.0 ?g/kg~10.0 ?g/kg.In the range of 5.0~50.0 ?g/kg,the average recoveries of the four grayanane toxins were 77.94%~109.21%,the intra-day coefficient of variation was 4.06%~9.82%,and inter-day coefficient of variation was 4.94%~13.11%.Using the developed method,17 kinds of honey collected from parts of China were analysised for grayanane toxins.The results suggested that there was a risk of contamination of grayanane toxins in some honey in China,and the contaminated honey species were rhododendron honey,multifloral honey and some wild honey.However,this type of pollutant has not been detected in China's bulk commodity honey,which is relatively safe.In the second part,the study first identified the tropane alkaloids in the mandala nectar by UHPLC-Q Exactive Plus.A total of four tropane alkaloids(Hyoscyamine,Scopolamine,Anisodamine and N-demethyl-Anisodamine)were obtained.To determine whether tropane alkaloids in mandala nectar were transferred to honey by honeybees and produce toxic honey.By this method,the tropane alkaloids in the suspected positive honey samples were identified,and the results showed that there were four tropane alkaloids in agreement with the mandala nectar,and scopolamine accounted for more than 85.0%.In order to further clarify the content of tropine alkaloids in honey in China,this experiment developed a UHPLC-MS/MS quantitative detection method for four tropane alkaloids in honey.Using 0.1% formic acid-water and 0.1% formic acid-acetonitrile as the mobile phase and methanol/water(20:80,v/v)as the extract solvents,the detection limit of the method was 0.05 ?g/kg,and the limit of quantification was 0.1 ?g/ kg.In the range of 0.1~0.5 ?g/kg,the average recovery was 74.93%~103.42%,the intra-day coefficient of variation was 2.63%~10.81%,and the inter-day coefficient of variation was 3.78%~14.13%.