Development And Application Of Detection Methods For Steroid Hormones In Royal Jelly

In recent years,"royal jelly hormone theory"has gained wide currency,so that consumers lose confidence in royal jelly,which also disturbs the bee product consumer market and brings a heavy blow to whole bee industry.Early studies have reported that trace amounts of testosterone,progesterone and estradiol and other endogenous hormones had been found in royal jelly using radioimmunoassay analysis.However,due to the technical limitations of the time,the method present false positive with relatively high probability;on the other hand,target compounds analysed covered the limited constitutes for early research.In this study,we used the new technology to study the types and contents of steroid hormones in royal jelly comprehensively,and explored their sources preliminarily.This work will restore the original turth of royal jelly and provide a theoretical basis for monitoring and precaution of steroid hormones in royal jelly.Three parts are included in this study.In the first part,a method for determination of 4 estrogens in royal jelly was developed by dispersive solid phase extraction-high performance liquid chromatography-tandem mass spectrometry(QuEChERS-HPLC-MS/MS).In the method,extraction,purification,derivatized and concentration were used to separate target compound efficiently,an appropriate instrumental method was used for qualitative and quantitative analysis of four compounds.In the linear range of 0.01?g/kg-10?g/kg,R~2>0.99,LOD=0.015?g/kg-0.02?g/kg,LOQ=0.1?g/kg.Under the three spiked levels(0.1?g/kg,1.0?g/kg and 10.0?g/kg),the matrix effect was not obvious ranged in 90.0%-120.0%,and the recoveries of 4 estrogens were 80.7%-113.0%,intra-day RSD?5.3%,inter-day RSD?8.8%.The developed method has the advantages of fast,efficient,high sensitivity,environmental-friendly.It is suitable the qualitative and quantitative analysis of estrogens in royal jelly.In the second part,a high-throughput method of QuECHERS-UPLC-MS/MS for 45 kinds of steroids in royal jelly was established.The target compounds included 4 estrogens,12 androgens,9progesterones,17 cortex hormones and 3 synthetic estrogen.Royal jelly samples were detected on the HPLC-MS/MS directly without derivatization after extraction,purification,concentration,which greatly shortening the pre-processing time.In the procedure of instrumental analysis,the mass spectrometer by changing the different ion monitoring mode was used to simultaneously analyse 45 kinds of compounds with the same pretreatment procedure,which achieved advantages of high efficiency and high throughput.By methodological validation,in a linear range,45 target compounds had R~2?0.99,LODs=0.003?g/kg-0.60?g/kg,LOQ=0.01?g/kg-0.20?g/kg,respectively.At three spiked levels of 1.0?g/kg,10.0?g/kg and 100.0?g/kg,the recoveries of 45 steroids were between66.49%-113.47%,and intra-day RSD?9.89%,inter-day RSD?13.75%within the acceptable range.The method developed is applicable to high-throughput screen of steroid hormones in royal jelly.Compared with the method for determination of 4 estrogens in royal jelly was developed QuEChERS-HPLC-MS/MS,the high-throughput method established in this part was expanded the range of target compounds screeneded significantly,and no derivatization during the pretreatment process.In the third part,237 batches of royal jelly samples collected in 2015 were analyzed by the first method,and 81 batches of royal jelly samples collected e in 2016 were analyzed by the second method.And the detection rate,steroid hormone detection types and content of statistical were analyzed.