Study On The LC-MS Determination Of Flavonoids Efficacy Ingredients In Dietary Supplements

ObjectiveA fast screening method for identification and quantitative analysis of the flavonoids efficacy ingredients in dietary supplements was established by liquid chromatography-mass spectrometry. This study also dedicated to provide new thoughts and methods for more comprehensive and precise quality control of dietary supplements, which needed to be applicable to the routine practice of enterprises, testing institue, even supervising authorities, finally, to fulfill the need for cracking down fake and inferior products and standardization of the dietary supplement market.Methods13 batches of ginkgo folium dietary supplements,13 batches of soy isoflavones dietary supplements, kudzu root hovenia dulcis softgels and propolis liquid were choosed for the demonstrative research.1、Samples collection:representative samples of different batches, different manufacturers, different packaging and different formulations of the same main raw materials of dietary supplements were collected from the market. Samples collected were evaluated according to the original standards before further experiments.2、An analytical method based on ultra performance liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry (UPLC-Q-TOF-MS) was developed for the rapid screening of flavonoid compounds in dietary supplements. Chromatography column, mobile phase, acquisition mode, parameters of MS such as ion source and collision energy were optimized to obtain higher sensitivity and more comprehensive information. The flavonoid compounds were identified by comparing retention time, UV spectra and characteristic molecular ions with those of references, or tentatively identified by MS/MS determination along with mass fragment software and literatures.3、A method for rapid simultaneous determination of the flavonoid compounds in dietary supplements by ultra performance liquid chromatography coupled with triple quadrupole mass spectrometry was established. Samples were detected by negative electrospray ionization tandem mass spectrometry in the SRM mode. Tube Lens offset, collision energy and ion-pairs were optimized, and the limit of detection (LOD), limit of quantitation (LOQ), linear range, precision, repeatability, stability and recovery rate were investigated to obtain precise and sensitive signal of each compound.4、The differences between the dietary supplements of same ingredients were compared by determinating their compositions and contents; results were used for evaluation of their efficacy.5、In order to obtain the accurate molecular weight, mass spectrometry (2nd) and retention time of each compound, fragmentation pathways of the flavonoid compounds had been studied by using electrospray ionization multi-stage tandem mass spectrometry (ESI-MSn).Results1、Refering to the original quality control method, the concentration of effective ingredients in some dietary supplements on market do not meet their labeled amount as enterprise standard described.2、A total number of 16 flavones were identified in 13 batches of ginkgo folium dietary supplements,6 isoflavones in 13 batches of soy isoflavones dietary supplements,20 flavones in kudzu root hovenia dulcis softgels and 16 flavones in propolis liquid by UPLC-Q-TOF-MS.3、6 isoflavones (daidzin, glycitin, genistin, daidzein, glycitein, genistein) in soy isoflavones dietary supplements were quantified simultaneously in 4 min by UPLC-MS. Comparing the results obtained by the proposed method to those of by conventional methods like HPLC, the former is fast, sensitive, reliable and selective.4、Packings of the same product usually changed by different dealers for their own profit. However, regardless of varying prices, no obvious difference was found in either quality or efficacy among those products. While the difference in the same products of diferent batches was significant, which can be attributed to the lack of strict quality control from the mamufacturer. Additionally, obvious difference was found between dietary supplements using the same main raw material. The results suggested that factors like raw material, processing methods and storage conditions are the keys to good quality of dietary supplements.5、The fragmentation pathways of 0-glycosidic flavonoid is different from C-glycosidic flavonoid. Analyzing the product ion spectra of flavonoids, products ions (i,jA- and i,jB-) were produced by the cleavage of C-ring including Retro Diels-Alder (RDA) fragmentations. And we observed some diagnostic neutral losses, such as CH3, CO, CO2, H2O, C3O2 and residue of glucose, which are very useful for the identification of the functional groups in the structures. And the results showed that the ion peaks of [M-H-120]-and [M-H-90]-were found in C-glycosidic flavonoid with hexose and the ion peaks of [M-H-60]-and [M-H-90]- were obtained in the spectrogram of ESI-MS-MS on C-glycosidic flavonoid with pentose.ConclusionA fast screening method for identification and quantitative analysis of the flavonoids efficacy ingredients in dietary supplements was established. By clarification of flavonoid compounds in dietary supplements and comparison of differences between products, current situation of this field can be carefully examined. It discloses that the quality of dietary supplements is not unified, suggesting that we should strengthen the basic research work of the effective ingredients in dietary supplements, and improve the detection analysis level. The method is accurate, rapid and sensitive, and can provide a reliable and effective technique for the quality control of dietary supplements.