Study On The Determination Methods Of Bioactive Substances In Lonicerae Flos And The Quality Assessment

Lonicerae Flos was derived from the dried flower buds of Lonicera macranthoides H.–Mazz.,Lonicera hypoglauca Miq,Lonicera confusa DC.Lonicera fulvotomentosa Hsu et S*C.Cheng,is one of the widely used Traditional Chinese Medicines(TCMs)listed in Chinese Pharmacopoeia and the Medicinal and Edible Product Catalog.This thesis set Lonicerae Flos as object of study and the bioactive substances such as polyphenol acids,flavonoids and saponins were analyzed in the experiment.In addition,the analytical method focused on the potential risk substances like aflatoxins B1,B2,G1 and G2 was established according its growing environment and harvesting season,and the inorganic elements were determined using inductively coupled plasma mass spectrometry.A preliminary study on the quality and safety of shanyinhua was carried out.The main research content is as follows.(1)An ultrasonic-assisted extraction(UAE)and ultra high performance liquid chromatography/triple quadrupole mass spectrometry(UHPLC-QqQ-MS)method was developed for simultaneous determination of fourteen bioactive substances in Lonicerae Flos.Extraction parameters of fourteen analytes were optimized by Box-Behnken design,and the mathematical model studying the link between the factors and the extraction rate had been established.The optimized conditions were 62% methanol as extraction solvent,106 mL/g as liquid/solid ratio and 450 W as ultrasonic power.An Extend C18 column(100 mm×2.1mm i.d.1.7 ?m)and gradient elution were used during the analysis process.The detection was performed on a triple quadrupole mass spectrometer by multiple reaction monitoring(MRM)via electrospray ionization(ESI)source with negative ionization mode.Furthermore,the fragmentation pathway of polyphenol acids and flavonoids were analysed and the characteristic fragment ions were analysed.All calibration curves has good linearity(r2>0.9924)over each concentration ranges of the analyte.The intra-and inter-day precisions were all within 10% and the accuracy(relative standard deviation,RSD)all range from 84.94% to 116.42%.The method validation results demonstrated that the developed method was sensitive,time-saving and reliable,which was successfully applied to the analysise of Lonicera Flos samples.(2)An ultra high performance liquid chromatography /triple quadrupole mass spectrometry(UHPLC-QqQ-MS)method was developed for simultaneous determination of aflatoxins B1,B2,G1 and G2 in lonicerae flos,and the fragmentation pattern of the four aflatoxins and the major fragment ions were discussed in this paper.The sample was extracted with 60% acetonitrile and purified with 0.2 g primary secondary amine(PSA).The extraction solution obtained was evaporated to nearly dryness under nitrogen and redissolved.The separation and detection was performed in Agilent SB C18 column and multiple reaction monitoring(MRM)mode via electrospray ionization(ESI)source with negative ionization mode.The quantitation of target compounds was carried out by isotope-labeled internal standard method,and all calibration curves have good linearity(r2>0.9964)over each concentration range of the analyte.The average recoveries range from 82% to 109%,with the relative standard deviations(RSD)of 1.4%~8.8%.The characteristic fragment ions and their processes of fragmentation of each compound were discussed,respectively.This method is simple to operate,sensitive and repeatable,and can be used in the detection of aflatoxins in lonicerae flos.(3)12 inorganic elements in lonicerae flos were analyzed using ICP-MS.Nitric acid and hydrogen peroxide were added to the sample before microwave digestion,and the samples was analyzed by ICP-MS with the six interior label of Ge,In,Sc and so on.The 12 elements showed good linearity(R>0.999),and the method was sensitive enough to be used in the analysis of elements of lonicerae flos.