Preparation And Application Of The Deuterated Flavonoids

Flavonoids,as polyphenols natural products,are widely distributed in nature,whose species number is up to tens of thousands.Due to their excellent pharmacological activities,flavonoids have been widely applied in medicine,health fields and so on,and were also used as quality control indexes of related pharmaceutical products.At present,a number of quantitative analysis methods for flavonoids in complex samples have been reported.The most common methods were high performance liquid chromatography（HPLC）and high performance liquid chromatography-mass/mass spectrometry（LC-MS/MS）.Although flavonoids in samples can be analyzed accurately and quantitatively by these methods,there were many disadvantages,such as high solvent consumption and low analytical throughput.Therefore,the development of a fast,low-cost and high-throughput method is crucial for the quality control of flavonoids.Paper spray mass spectrometry（PS-MS）,as one of the ambient mass spectrometry（AMS）,has many advantages,such as fast analysis speed（10²0 s）,high throughput,little or no sample preparation process,and solvent-saving.PS-MS has shown the great potential in the application of the biological analysis,drug research and development,and forensic science etc.However,the mutual inhibition or enhancement of ionization between components directly affected the accuracy of PS-MS analysis due to the lack of the separation of components before mass spectrometry analysis.With the development of AMS,it was found that isotopic internal standards could effectively eliminate the uncertainty of the ionization of target components,thus improving significantly the accuracy of AMS quantitative analysis.Therefore,isotopic internal standard has become the gold standard for AMS quantitative analysis.In this paper,ten deuterated flavonoids were successfully synthesized by H/D exchange with heterogeneous and acid-base catalysis considering the lack of isotopic internal standards in the rapid analysis of flavonoids in PS-MS.With the help of the deuterated flavonoids,two PS-MS quantitative methods for the rapid analysis of flavonoids in traditional Chinese medicines were developed.The main contents are as follows:1.Ten deuterium flavonoids were successfully synthesized using Pt/C-NaOH-D₂O as catalyst and deuterium source.The catalyst type,ratio of catalyst to substrate,alkali content,temperature and reaction time were optimized.Finally,NaOH and Pt/C were used as catalysts,and D₂O was selected as deuterium source,130 ^oC was the optimal reaction temperature.2.A rapid and accurate analysis method combined the quantitative of baicalin in Scutellaria baicalensis by PS-MS with baicalin-d₄ as internal standard assisted microwave assisted extraction was established.Sample pretreatment and the conditions of mass spectrometry analysis were optimized.PS-MS calibration curve linear coefficient was 0.9996,and LOD and LOQ were 0.1 mg/L and 0.05 mg/L,respectively.Standard addition recovery rate was between 98.25%and 104.36%.Compared with the quantitative results of HPLC method,there was no significant difference.In addition,the analysis time was shorten greatly by using PS-MS.This demonstrated that the PS-MS with deuterated internal standard could accurately and rapidly quantitative determine baicalin in Scutellaria baicalensis Georgi.3.A rapid quantitative analysis method of total flavonoids from ginkgo biloba by microwave-assisted hydrolysis assisted PS-MS was devolped.Isorhamnetin-d₃,kaempferol-d₄ and quercetin-d₅ were used as internal standards.The linear coefficients（R²）of kaempferol,quercetin and isorhamnetin were 0.9998,0.9997 and 0.9993,respectively.LOD were0.1,0.1 and 0.2 mg/L,and LOQ was less than 0.6 mg/L.Recovery rate was from 94.90%to 105.64%.The method was successfully applied in the analysis of total flavonoids in ginkgo biloba leaves,ginkgo preparations and ginkgo biloba extracts.Compared with the HPLC method,there were no significant difference in the quantitative results between the two methods.This indicated that the microwave-assisted hydrolysis PS-MS isotopic internal standard method could accurately quantify the total flavonoids in complex matrix.