Systematic Characterization Of Curcuminoids And Qualitative Analysis Of Endogenous Carbonyl-containing Steroid Hormones Based On LC-MS/MS

With the development of liquid chromatography-tandem mass spectrometry(LCMS/MS)technology,it is widely used in pharmaceutical analysis,drug metabolism,metabolomics and proteomics.Among the LC-MS/MS techniques,high-resolution mass spectrometer(HRMS)is mainly used for qualitative analysis,such as quadrupole time-of-flight(QTOF)and Orbitrap mass spectrometer.The triple quadrupole linear ion trap mass spectrometer(QTRAP)is a low-resolution mass spectrometer,which is widely used for quantitative analysis because of the high sensitivity and good selectivity of MRM scanning mode.LC-MS/MS technology has unique advantage in characterization of chemical component in traditional Chinese medicine and endogenous components.This study utilizes LC-QTRAP-MS/MS and high-resolution LC-MS/MS to analyze the curcuminoids in turmeric and endogenous carbonylcontaining steroid hormones in human serum and urine.The following results are achieved.Turmeric is a commonly used traditional Chinese medicine,and curcuminoids are one of the main active ingredients.Relevant study mainly focused on curcumin,demethoxycurcumin and bisdemethoxycurcumin.However,in addition to these three main curcuminoids,other minor curcuminoids in turmeric have significant bioactivities.Therefore,it is necessary to systematically characterize curcuminoids in turmeric.Based on previous study,an analytical strategy was constructed to predict potential curcuminoids according to the skeleton structures and possible aryl types of curcuminoids.Based on the predicted structures,a total of 89 curcuminoids were identified from turmeric samples by UPLC-QTOF-MS/MS analysis,among which 16 were novel compounds.Besides,the fragmentation rules of 15 different types of curcuminoids were summarized,which provided ideas for the identification of trace components in Chinese medicine.In China,turmeric is cultivated in a few regions including Sichuan,Yunnan,Fujian,Guangdong,Guangxi.Turmeric produced in Leshan of Sichuan is recognized as the geo-authentic herb.The total curcumin content was used to evaluate the quality of turmeric in the existing quality evaluation methods.However,they can't distinguish turmeric from different origins.In this study,based on the qualitative analysis results of curcuminoids,the profiling method was established by MRM scan mode of QTRAP,which can specifically characterize curcuminoids.Then,response surface methodology was applied for optimizing the extraction process of minor curcuminoids.The curcuminoids were extract under the optimized conditions.The profiling method was applied to relatively quantify the curcuminoids in turmeric from different origins.25 differential curcuminoids were screened through OPLS-DA,among which most differential compounds were more abundant in turmeric samples from Leshan of Sichuan.This study showed that the profiling method combined with multivariate analysis could effectively discriminate turmeric samples from geo-authentic origins and non-authentic origins,which can be used to evaluate the turmeric quality.Endogenous steroid hormones are an important class of small molecular metabolites in vivo.Most steroid hormones contain carbonyl groups.The carbonyl group-containing steroid hormones play an important role in endocrine regulation and immune regulation,and are related to a variety of diseases.Various studies were conducted to quantify human endogenous carbonyl-containing steroid hormones using LC-MS/MS.However,few studies focused on the qualitative analysis of such compounds by LC-MS/MS.As for the carbonyl-containing steroid hormones with low abundance in human body fluids,it is challenging to analyze them by LC-MS/MS.In this study,carbonyl-containing steroid hormone standards were derivatized by quaternary aminooxy(QAO)derivatization reagents to improve their responses,then the fragmentation rules of derivative products were summarized to guide the identification.Subsequently,the derivatized steroid hormones in human serum and urine samples were analyzed by UPLC-QE-HF-X MS/MS.Based on the fragmentation rules,a strategy was constructed to identify the QAO derivatives of the carbonylcontaining steroid hormones.Finally,106 kinds of carbonyl-containing steroid hormone compounds were identified from human serum and urine,among which 54 compounds were free steroid hormones,30 compounds were steroid glucuronide conjugates,and 22 compounds were steroid sulfate conjugates.This study is the most comprehensive characterization of endogenous carbonyl-containing steroid hormones in human body fluids so far,which provided basis for the analysis of these components in the human fluid.