Direct extraction of ten estrogens from milk samples with DVB/NVP-modified magnetic solid-phase extraction adsorbent followed by pre-column derivatization-UHPLC-MS/MS.

Estrogens and their analogues can cause harm to human health through the food chain. Ten estrogens in different milk samples were directly extracted by amphiphilic divinylbenzene/N-vinyl-2-pyrrolidone (DVB/NVP)-Fe3O4@SiO2-based magnetic solid-phase extraction (MSPE) followed by pre-column derivatization and ultra-high performance liquid chromatography tandem mass-spectrometry (UHPLC-MS/MS) detection. Under the optimal conditions, the limits of detection for ten analytes were in the range of 0.05-0.38 ng mL-1 in whole liquid milk matrix and 0.04-3.00 ng g-1 in milk powder matrix. The intra-/inter-day accuracy ranged in 83.4-113.8%, with RSDs in 2.5-15.0%. A total of 15 brands of liquid milk and milk powder samples were analyzed, and only estradiol was detected in three brands of boxed liquid milk within safe range. The proposed sample pretreatment eliminated the common protein precipitation process, improved the sample throughput, and has the potential for routine testing of estrogens and their analogues in market-sale milk samples.

Molecular targets of cisplatin in HeLa cells explored through competitive activity-based protein profiling strategy.

Cisplatin is widely used as anticancer drugs, and DNA is considered as the main target. Considering its high affinity towards cysteines and the important role of cystine containing proteins, we applied a competitive activity-based protein profiling strategy to identify protein cysteines that bind with cisplatin in HeLa cells. Living cells were treated with cisplatin at cytotoxic concentrations, then the protein was extracted. After labeling with desthiobiotin iodoacetamide (DBIA) probe, protein was precipitated, digested and isotopically labeled, subsequently the peptides were combined, and the biotinylated cysteine-containing peptides were enriched and quantified by LC-MS/MS. A total of 3571 peptides which originated from 1871 proteins were identified using the DBIA probe. Among them, 46 proteins were screened as targets, including proteins that have been identified as binding proteins by previous study. A novel cisplatin target, calpain-1 (CAPN1), was identified and validated as binding with cisplatin in vitro.

Identification of endogenous carbonyl steroids in human serum by chemical derivatization, hydrogen/deuterium exchange mass spectrometry and the quantitative structure-retention relationship.

Steroids are tetracyclic aliphatic compounds, and most of them contain carbonyl groups. The disordered homeostasis of steroids is closely related to the occurrence and progression of various diseases. Due to high structural similarity, low concentrations in vivo, poor ionization efficiency, and interference from endogenous substances, it is very challenging to comprehensively and unambiguously identify endogenous steroids in biological matrix. Herein, an integrated strategy was developed for the characterization of endogenous steroids in serum based on chemical derivatization, ultra-performance liquid chromatography quadrupole Exactive mass spectrometry (UPLC-Q-Exactive-MS/MS), hydrogen/deuterium (H/D) exchange, and a quantitative structure-retention relationship (QSRR) model. To enhance the mass spectrometry (MS) response of carbonyl steroids, the ketonic carbonyl group was derivatized by Girard T (GT). Firstly, the fragmentation rules of derivatized carbonyl steroid standards by GT were summarized. Then, carbonyl steroids in serum were derivatized by GT and identified based on the fragmentation rules or by comparing retention time and MS/MS spectra with those of standards. H/D exchange MS was utilized to distinguish derivatized steroid isomers for the first time. Finally, a QSRR model was constructed to predict the retention time of the unknown steroid derivatives. With this strategy, 93 carbonyl steroids were identified from human serum, and 30 of them were determined to be dicarbonyl steroids by the charge number of characteristic ions and the number of exchangeable hrdrogen or comparing with standards. The QSRR model built by the machine learning algorithms has an excellent regression correlation, thus the accurate structures of 14 carbonyl steroids were determined, among which three steroids were reported for the first time in human serum. This study provides a new analytical method for the comprehensive and reliable identification of carbonyl steroids in biological matrix.

Qualitative Distribution of Endogenous Cholesteryl Esters in Plasma of Humans and Three Rodent Species Using Stepwise UPLC-Q-Exactive-MS.

OBJECTIVE: Cholesteryl esters (CEs) are composed of various fatty acyl chains attached to the hydroxyl groups of cholesterol, and abnormalities in their metabolism are related to many diseases. This study aimed to develop an ultrahigh-performance liquid chromatography-quadrupole exactive mass spectrometry (UPLC-Q-Exactive MS) method to identify the CEs in plasma. METHODS: First, the MS fragmentation patterns were investigated using seven commercial CE standards. Then, the CEs in plasma were characterized through the accurate mass data of precursor ions and characteristic product ions. A strategy of step-by-step m/z scans in a narrow range was proposed to identify more trace CEs by the full-scan data-dependent MS/MS (ddMS2) mode. RESULTS: A total of 50 CE species consisting of 55 regioisomers were identified in human plasma. Among them, two species were reported for the first time. CONCLUSION: This study is the most comprehensive identification of CE species in human plasma to date. These results will contribute to the in-depth profiling of CEs in human plasma and provide guidance for animal model selection when studying lipid-related diseases.

Network pharmacology combined with metabolomics and lipidomics to reveal the hypolipidemic mechanism of Alismatis rhizoma in hyperlipidemic mice.

Alismatis rhizoma (AR), the dried rhizome of Alisma orientale (Sam) Juzep, is effective in treating hyperlipidemia, but the mechanisms involved require further exploration. This study evaluated the hypolipidemic properties of AR using an integrated strategy combining network pharmacology with metabolomics and lipidomics. Firstly, a hyperlipidemia mouse model induced by a high-fat diet was established to evaluate the therapeutic effects of AR. Secondly, plasma metabolomics and lipidomics were used to identify differential metabolites and lipids, and metabolic pathway analysis was performed using MetaboAnalyst. Thirdly, network pharmacology, based on the metabolic profile of AR in vivo, was used to discover potential therapeutic targets. Finally, key targets were obtained through a compound-target-metabolite network, which was verified by molecular docking and quantitative real-time PCR (qPCR). Biochemistry analysis and histological examinations showed that AR exerted hypolipidemic effects on hyperlipidemic mice. Seventy potential biomarkers for the AR treatment of hyperlipidemia were identified by metabolomics and lipidomics, which were mainly involved in lipid metabolism, energy metabolism and amino acid metabolism. Eighteen potentially active compounds were identified in the plasma of mice after oral administration of AR, which were associated with 83 potential therapeutic targets. The PPAR signaling pathway was considered a crucial signaling pathway of AR against hyperlipidemia by KEGG analysis. The joint analysis showed that 6 upstream key targets were regulated by AR, including ALB, TNF, IL1B, MMP9, PPARA and PPARG. Molecular docking showed that active compounds of AR had high binding affinity with these key targets. qPCR further demonstrated that AR could reverse the mRNA expression of these key targets in hyperlipidemic mice. This study integrates network pharmacology with metabolomics and lipidomics to reveal the regulatory effects of AR on endogenous metabolites and validates key therapeutic targets, and represents the most systematic and in-depth study on the hypolipidemic activity of AR.

Improved structural annotation of triterpene metabolites of traditional Chinese medicine in vivo based on quantitative structure-retention relationships combined with characteristic ions: Alismatis Rhizoma as an example.

As a fast, sensitive and selective method, liquid chromatography-tandem high-resolution mass spectrometry (LC-HRMS) has been used for studying the in vivo metabolism of traditional Chinese medicine (TCM). However, the rapid discovery and characterization of metabolites, especially isomers, remain challenging due to their complexity and low concentration in vivo. This study proposed a strategy to improve the structural annotation of prototypes and metabolites through characteristic ions and a quantitative structure-retention relationship (QSRR) model, and Alismatis Rhizoma (AR) triterpenes were used as an example. This strategy consists of four steps. First, based on an in-house database reported previously, prototypes and metabolites in biosamples were preliminarily identified. Second, the candidate structures of prototype compounds and metabolites were determined by characteristic ions, databases or potential metabolic pathways. Then, a QSRR model was established to predict the retention times of the proposed structure. Finally, the structures of unknown prototypes and metabolites were determined by matching experimental retention times with the predicted values. The QSRR model built by the genetic algorithm-multiple linear regression (GA-MLR) has excellent regression correlation (R2 = 0.9966). Based on this strategy, a total of 118 compounds were identified, including 47 prototypes and 71 metabolites, among which 61 unknown compounds were reasonably characterized. The typical compound identified by this strategy was successfully validated using a triterpene standard. This strategy can improve the annotation confidence of in vivo metabolites of TCM and facilitate further pharmacological research.

Quantitative proteomics of plasma and liver reveals the mechanism of turmeric in preventing hyperlipidemia in mice.

Hyperlipidemia is manifested by abnormal levels of circulating lipids and may lead to various cardiovascular diseases. Studies have demonstrated that turmeric supplemented in food can effectively prevent hyperlipidemia. The aim of this study is to elucidate the underlying mechanism. 27 male C57BL/6J mice were randomly divided into three groups, which were fed with a standard diet, a high-fat diet and a high-fat diet supplemented with turmeric powder (2.0% w/w), respectively. After eight weeks of feeding, turmeric intervention significantly reduced the plasma TC, TG, and LDL-C levels and the LDL-C/HDL-C ratio of mice compared with high-fat diet fed mice. TMT-based proteomic analysis showed that the expression of 24 proteins in mouse plasma and 76 proteins in mouse liver was significantly altered by turmeric, respectively. Bioinformatics analysis showed that differential proteins in the plasma were mainly involved in complement and coagulation cascades and the cholesterol metabolism pathway. The differential proteins in the liver were mainly involved in arachidonic acid metabolism, steroid hormone biosynthesis and the PPAR signaling pathway. Key differential proteins were successfully validated by western blot analysis. This study is the first to reveal the preventive mechanism of turmeric on hyperlipidemia from proteomics. The results showed that dietary turmeric could prevent hyperlipidemia through regulating the expression of proteins in metabolism pathways.

Systematic Identification of Proteins Binding with Cisplatin in Blood by Affinity Chromatography and a Four-Dimensional Proteomic Method.

Cisplatin is widely used for the treatment of various solid tumors. It is mainly administered by intravenous injection, and a substantial amount of the drug will bind to plasma proteins, a feature that is closely related to its pharmacokinetics, activity, toxicity, and side effects. However, due to the unique properties of platinum complexes and the complexity of the blood proteome, existing methods cannot systematically identify the binding proteome of cisplatin in blood. In this study, high-abundance protein separation and an ion mobility mass spectrometry-based 4D proteomic method were combined to systematically and comprehensively identify the binding proteins of cisplatin in blood. The characteristic isotope patterns of platinated peptides and a similarity algorithm were utilized to eliminate false-positive identification. Finally, 39 proteins were found to be platinated. Bioinformatics analysis showed that the identified proteins were mainly involved in the complement and coagulation cascade pathways. The binding ratio of some peptides with cisplatin was measured based on the area ratio of the free peptide using the parallel reaction monitoring method. This study provides a new method for systematically identifying binding proteins of metal drugs in blood, and the identified proteins might be helpful for understanding the toxicity of platinum anticancer drugs.

Interaction of Natural Compounds in Licorice and Turmeric with HIV-NCp7 Zinc Finger Domain: Potential Relevance to the Mechanism of Antiviral Activity.

Nucleocapsid proteins (NCp) are zinc finger (ZF) proteins, and they play a central role in HIV virus replication, mainly by interacting with nucleic acids. Therefore, they are potential targets for anti-HIV therapy. Natural products have been shown to be able to inhibit HIV, such as turmeric and licorice, which is widely used in traditional Chinese medicine. Liquiritin (LQ), isoliquiritin (ILQ), glycyrrhizic acid (GL), glycyrrhetinic acid (GA) and curcumin (CUR), which were the major active components, were herein chosen to study their interactions with HIV-NCp7 C-terminal zinc finger, aiming to find the potential active compounds and reveal the mechanism involved. The stacking interaction between NCp7 tryptophan and natural compounds was evaluated by fluorescence. To elucidate the binding mode, mass spectrometry was used to characterize the reaction mixture between zinc finger proteins and active compounds. Subsequently, circular dichroism (CD) spectroscopy and molecular docking were used to validate and reveal the binding mode from a structural perspective. The results showed that ILQ has the strongest binding ability among the tested compounds, followed by curcumin, and the interaction between ILQ and the NCp7 zinc finger peptide was mediated by a noncovalent interaction. This study provided a scientific basis for the antiviral activity of turmeric and licorice.

Fluorescent labeling of s2T-incorporated DNA and m5s2U-modified RNA.

We report herein comprehensive investigations of alkylation/sulfur exchange reactions of sulfur-containing substrates including nucleosides such as s2U, m5s2U, s4U, s2A and s2T-incorporated DNA enable by comprehensive screenings of the reagents (2a-2h). It has been proven that iodoacetamide (2a) displays the most promising feasibility toward sulfur-containing substrates including s2T, s2U, m5s2U, s4U and s2A. In sharp contrast, the alkylation process with S-benzyl methanethiosulfonate (BMTS, 2h) displays the best application potential only for s4U. Based on these results, the fluorescent labeling of s2T-incorporated DNA and m5s2U-modified RNA has been achieved.Supplemental data for this article is available online at https://doi.org/10.1080/15257770.2021.1942044 .

Qualitative distribution of endogenous sphingolipids in plasma of human and rodent species by UPLC-Q-Exactive-MS.

Sphingolipids (SLs) are endogenously bioactive molecules with diverse structures, and its metabolic disorders are involved in the progression of many diseases. In this study, an ultra-performance liquid chromatography quadrupole exactive mass spectrometry (UPLC-Q-Exactive-MS) method was established to comprehensively profile SLs in plasma. First, the fragment patterns of SL standards of each subclass were investigated. Then, the SL species in plasma were characterized based on the fragmentation rules. Finally, a total of 144 endogenous SL species consisting of 216 regioisomers were identified in plasma of human, golden hamster and C57BL/6 mice, which was the most comprehensive identification for SLs in plasma. In addition to the known species, 19 SL species that have never been reported were also identified. The profile of SLs in plasma of human and two rodent species was compared subsequently. It was worth noting that a total of 9 SL molecular species consisting of 11 regioisomers with low abundance were successfully identified in human plasma through comparison among species. Those findings contribute to a deeper understanding of SLs in human plasma and provide scientific basis for the selection of animal model. The established profile of SLs in plasma could be used for screening of lipid biomarkers of various diseases.

Comprehensive metabolic profiling of Alismatis Rhizoma triterpenes in rats based on characteristic ions and a triterpene database.

Alismatis Rhizoma (AR) is widely used in Chinese medicine, and its major bioactive components, triterpenes, reportedly possess various pharmacological activities. Therefore, it is very important to study the metabolism of triterpenes in vivo. However, the metabolism of AR triterpene extract has not been comprehensively elucidated due to its complex chemical components and metabolic pathways. In this study, an ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry method, which was based on the characteristic ions from an established database of known triterpenes, was used to analyze the major metabolites in rats following the oral administration of Alismatis Rhizoma extracts (ARE). As a result, a total of 233 constituents, with 85 prototype compounds and 148 metabolites, were identified for the first time. Hydrogenation, oxidation, sulfate and glucuronidation conjugation were the major metabolic pathways for triterpenes in AR. In addition, the mutual in vivo transformation of known ARE triterpenes was discovered and confirmed for the first time. Those results provide comprehensive insights into the metabolism of AR in vivo, which will be useful for future studies on its pharmacodynamics and pharmacokinetics. Moreover, this established strategy may be useful in metabolic studies of similar compounds.

The Protein-Binding Behavior of Platinum Anticancer Drugs in Blood Revealed by Mass Spectrometry.

Cisplatin and its analogues are widely used as chemotherapeutic agents in clinical practice. After being intravenously administrated, a substantial amount of platinum will bind with proteins in the blood. This binding is vital for the transport, distribution, and metabolism of drugs; however, toxicity can also occur from the irreversible binding between biologically active proteins and platinum drugs. Therefore, it is very important to study the protein-binding behavior of platinum drugs in blood. This review summarizes mass spectrometry-based strategies to identify and quantitate the proteins binding with platinum anticancer drugs in blood, such as offline high-performance liquid chromatography/inductively coupled plasma mass spectrometry (HPLC-ICP-MS) combined with electrospray ionization mass spectrometry (ESI-MS/MS) and multidimensional LC-ESI-MS/MS. The identification of in vivo targets in blood cannot be accomplished without first studying the protein-binding behavior of platinum drugs in vitro; therefore, relevant studies are also summarized. This knowledge will further our understanding of the pharmacokinetics and toxicity of platinum anticancer drugs, and it will be beneficial for the rational design of metal-based anticancer drugs.

Preparation of bovine serum albumin restricted access octadecyl/phenyl-mixed-functionalized magnetic silica nanoparticles for magnetic solid phase extraction of antidepressants in aquatic products followed by UHPLC-MS/MS.

Pharmaceuticals are widespread in aquatic ecosystem, which may pose a potential threat to fish and human. In the study, a robust and reliable magnetic solid-phase extraction (MSPE) based on bovine serum albumin (BSA) restricted access octadecyl/phenyl-mixed-functionalized magnetic silica nanoparticles (BSA-C18/Ph-Fe3O4@SiO2 NPs) as sorbent was developed for the extraction of venlafaxine, paroxetine, fluoxetine, norfluoxetine, sertraline and diphenhydramine from the muscle extracts of aquatic products followed by ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) detection. The homemade sorbent showed appropriate compatibility in aqueous solution and good performance in reducing matrix interference of snakehead muscle tissue extracts with absolute matrix effect ranging in 95.4%-105.5%. The protocol was validated in analyte-free snakehead muscle with favorable recoveries ranging in 91.6%-103.6% and relative standard deviations (RSDs) less than 9.5%. Limits of detection (LODs) of the drugs were lower than 0.018 µg kg-1. Short-term drug-exposure experiments at low and high doses were conducted on snakeheads, and the measured contents of analytes were in the range 0.029-9.58 µg kg-1 with appropriate recoveries (90.0%-114.0%). The approach was extensively applied for the analysis of twelve species of market-sale aquatic products (total 37 samples), and up to 1.868 and 0.521 µg kg-1 of diphenhydramine and venlafaxine were measured, respectively. The approach shows remarkable potential in biological complex samples.

Discrimination of turmeric from different origins in China by MRM-based curcuminoid profiling and multivariate analysis.

In this research, a three-step strategy was utilized for discriminating turmeric samples from different provinces and regions in China. Firstly, MRM-based UPLC-MS/MS method for chemical profiling of curcuminoids in turmeric samples was established. Then, response surface methodology was applied for optimizing the extraction process of targeted curcuminoids. Finally, multivariate analysis was conducted for systematic characterization of 66 curcuminoids in turmeric. Principal component analysis (PCA) and orthogonal projection to latent structure-discriminant analysis (OPLS-DA) revealed that turmeric samples from Sichuan and other regions could be classified into two distinct groups. Turmeric samples from the same group had similar curcuminoids content distribution. 25 differential curcuminoids were discovered through OPLS-DA, among which most curcuminoids were more abundant in Sichuan. Furthermore, turmeric samples from different provinces could be clearly discriminated based on hierarchical cluster analysis (HCA) using the screened differential curcuminoids.

Qualitative distribution of endogenous phosphatidylcholine and sphingomyelin in serum using LC-MS/MS based profiling.

PCs and SMs are the major types of glycerophospholipids and sphingophospholipids, the two main categories of phospholipids (PLs). To study the qualitative distribution of serum phosphatidylcholine (PC) and sphingomyelin (SM) in human and three rodent species, liquid chromatography-Orbitrap mass spectrometry (LC-Orbitrap-MS/MS) was used to identify them comprehensively through the accurate mass measurement of both precursor ions and their corresponding product ions. Based on the fragmentation rules of standards, the product ions at m/z 184.0733 were filtered to maximally screen possible PC and SM molecules. For PC, the fatty acid at sn-1 and sn-2 of the glycerol backbone was identified based on the product ions in negative mode. A total of 91 PCs and 31 SMs molecular species, consisting of 166 PCs and 39 SMs regioisomers, were detected in human serum, which is the most comprehensive identification of PC and SM species in serum. The qualitative distributions of PC in rat and SM in golden hamster, respectively, were more similar with that of human from an overall perspective. Those results provided guidance regarding to the animal model selection for mimicking lipid related-syndromes or diseases in human.

Magnetic solid-phase extraction modified Quick, Easy, Cheap, Effective, Rugged and Safe method combined with pre-column derivatization and ultra-high performance liquid chromatography-tandem mass spectrometry for determination of estrogens and estrogen mimics in pork and chicken samples.

In this study, conventional Quick, Easy, Cheap, Effective, Rugged and Safe (QuEChERS) method was modified by magnetic solid-phase extraction (MSPE) for purification/pre-concentration of eleven estrogens and estrogen mimics from the extracts of pork and chicken muscles, prior to dansyl chloride (DNS-Cl) derivatization coupled with ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) assay. Dual octadecyl- and 2-aminoethyl-3-aminopropyl- groups functionalized mesoporous silica core-shell magnetic nanoparticles (C18/NH2-Fe3O4@mSiO2 MNPs) were synthesized and employed as MSPE sorbent with remarkable aqueous compatibility in comparison with conventional C18 functionalized sorbent. The proposed MSPE is easier to handle than the traditional SPE purification process in QuEChERS method. The lab-prepared MNPs were characterized by transmission electron microscope (TEM), brunner-emmet-teller (BET), dynamic light scattering (DLS), Fourier transform infrared spectroscopy (FT-IR), thermo-gravimetric analysis (TGA) and vibrating sample magnetometer (VSM). Pre-column derivatization was conducted to significantly enhance the sensitivity of the analytes in MS/MS via analyzing their derivatives in positive ion mode instead of analyzing their original forms in negative ion mode. Under the optimal sample pretreatment and instrumental analysis conditions, the approach showed low limits of detection (LODs, 0.02‒3.00 µg kg-1), appropriate recoveries (81.1‒115.4%) and acceptable precisions (0.48‒15.1%, n = 6), with good feasibility and future prospect of trace compounds analysis in complex food samples.

The leaving group in Au(I)-phosphine compounds dictates cytotoxic pathways in CEM leukemia cells and reactivity towards a Cys2His2 model zinc finger.

Gold(i)-phosphine "auranofin-like" compounds have been extensively explored as anticancer agents in the past decade. Although potent cytotoxic agents, the lack of selectivity towards tumorigenic vs. non-tumorigenic cell lines often hinders further application. Here we explore the cytotoxic effects of a series of (R3P)AuL compounds, evaluating both the effect of the basicity and bulkiness of the carrier phosphine (R = Et or Cy), and the leaving group L (Cl-vs. dmap). [Au(dmap)(Et3P)]+ had an IC50 of 0.32 µM against the CEM cell line, with good selectivity in relation to HUVEC. Flow cytometry indicates reduced G1 population and slight accumulation in G2, as opposed to auranofin, which induces a high population of cells with fragmented DNA. Protein expression profile sets [Au(dmap)(Et3P)]+ further apart from auranofin, with proteolytic degradation of caspase-3 and poly(ADP-ribose)-polymerase (PARP), DNA strand-break induced phosphorylation of Chk2 Thr68 and increased p53 ser15 phosphorylation. The cytoxicity and observable biological effects correlate directly with the reactivity trend observed when using the series of gold(i)-phosphine compounds for targeting a model zinc finger, Sp1 ZnF3.

Novel PEP-PAN@PSF rods extraction of EDCs in environmental water, sediment, and fish homogenate followed by pre-column derivatization and UHPLC-MS/MS detection.

In the study, novel polysulfone hollow fiber membrane (PSF) supported polar enhanced phase (PEP) particles-polyacrylonitrile (PAN) polymer thin-film rods (PEP-PAN@PSF rods) were developed for the extraction of five steroidal endocrine disrupting chemicals (EDCs) (estrone (E1), estradiol (E2), estriol (E3), ethinyloestradiol (EE2), 2-methoxyestradiol (2ME2)) and four phenolic EDCs (bisphenol A (BPA), hexestrol (HEX), diethylstilbestrol (DES), dienestrol (DE)) in environmental water, sediment, and fish muscle homogenates, followed by pre-column derivatization and ultra-high performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS). The traditional preparation method for thin-film rods was improved by introducing a supporting membrane PSF between the external thin-film coating and the internal bare rod, avoiding the conventional pre-corrosion by strong acid/base. The commercial PEP prepared rods showed competitive capacity for both polar and nonpolar EDCs. In addition, pre-column derivatization with dansyl chloride (DNS-Cl) was adopted for the phenolic analytes prior to UHPLC-MS/MS detection, leading to a significant enhancement of sensitivity via analyzing the dansylated derivatives under positive electrospray ionization (ESI) mode instead of the analytes under negative ESI mode. The protocol was validated in four matrices including environmental water, sediment and two fish species. No matrix effects were observed in four matrices. The limits of detection (LODs) for the analytes were in the range of 0.002-0.072 µg L-1 for environmental water, 0.032-0.734 ng g-1 for sediment, and 0.011-0.435 ng g-1 for two fish species, respectively. Appropriate linearity was observed for all the analytes with correlation coefficients (R2) above 0.997. The intra-day trueness of the approach at low, medium and high levels was in the range of 86.6-116.1% with relative standard deviations (RSDs) lower than 15.4%. And the inter-day trueness was in the range of 84.6-114.8% with RSDs lower than 16.3%. The proposed method was successfully applied for the analysis of nine EDCs in environmental water, sediment, and fish muscle homogenates.

Spectrum-effect relationship for anti-tumor activity of shikonins and shikonofurans in medicinal Zicao by UHPLC-MS/MS and chemometric approaches.

Shikonin, shikonofuran and their derivatives are the main bioactive components of Zicao, a traditional Chinese medicine prepared with the dried roots of Lithospermum erythrorhizon, Arnebia euchroma or Arnebia guttata. To establish an efficient and sensitive method for studying material basis of Zicao, different scan modes of ultra-high performance liquid chromatography quadrupole time of flight tandem mass spectrometry (UHPLC-QTOF-MS/MS) and UHPLC triple quadrupole linear ion trap mass spectrometry (QTRAP-MS/MS) were incorporated to make full use of the sensitivity of multiple reaction monitoring (MRM) and overcome its disadvantages. A total of 73 shikonins and shikonofurans compounds were detected in Zicao utilizing various scanning modes. Thereafter the characteristic chemical profile for shikonins and shikonofurans was established based on UHPLC-QTRAP-MS/MS, which was subsequently used to study the spectrum-effect relationship by correlating the relative quantity of compounds and the anti-tumor activity. As a result, 27 compounds were screened as the main active components inhibiting HeLa cells by othogonal partial least square (OPLS). Among them, shikonin, acetylshikonin have been reported to inhibit HeLa cells previously, and ß, ß-dimethylacrylshikonin has been reported to be active component by other method. Those results showed that chemical characteristic profile combined with chemometric methods was efficient and reliable for discovery of material basis in TCM, especially trace active compounds.