Lysophosphatidylcholine Biomarkers of Lung Cancer Detected by Ultra-performance Liquid Chromatography Coupled with Quadrupole Time-of-flight Mass Spectrometry

Centrifugal ultrafiltration after methanol extraction of whole plasma was used as an optimal condition for the preparation of blood plasma before metabonomic studies. The plasma samples from 102 lung cancer patients and 34 healthy volunteers were prepared with this approach. With ultra-performance liquid chromatography（UPLC） coupled with quadrupole time-of-flight mass spectrometry（Q-TOF MS） analysis, the samples were investigated in order to find potential disease biomarkers. After data acquisition, orthogonal signal correction partial least squares models were built to differentiate the healthy volunteers from lung cancer patients and to identify metabolites that showed significantly different expression between the two groups. Several metabolite ions were identified as potential biomarkers according to the variable importance in the project（VIP） value in both ion modes. Five lysophosphatidylcholines were further identified as specifically lysoPC 16：0, isomer of lysoPC 16：0, lysoPC 18：0, lysoPC 18：1 and lysoPC 18：2. These results suggest that UPLC coupled with Q-TOF MS is an effective technique for the analysis of plasma metabolites in metabonomic studies.

Detecting N-nitrosamines in water treatment plants and distribution systems in China using ultra-performance liquid chromatography-tandem mass spectrometry

N-nitrosodimethylamine （NDMA） and several other N-nitrosamines have been detected as disinfection by-products in drinking waters in many countries around the world. An ultra-performance liquid chromatography- tandem mass spectrometry method with solid phase extraction sample preparation was developed to study the occurrence of N-nitrosamines in several water treatment plants and distribution systems in China. Isotope labeled N-nitrosodi-n-propylamine-dl4 （NDPA-dl4） was selected as the internal standard for quantification. The solid phase extraction procedures including pH, enrichment process and MS/MS parameters including capillary voltage, cone gas flow, cone voltage, collision energy were optimized to give average recoveries of 26% to 112% for nine N- nitrosamine species. The instrument detection limits were estimated to range from 0.5 to 5μg.L-1 for the nine N- nitrosamine species. NDMA and several other N-nitrosa- mines were found at fairly high concentrations in several water treatment plants and distribution systems. NDMA was found in all locations, and the highest concentrations in cities B, G, T, and W were 3.0, 35.7, 21.3, and 19.7 ng. L 1, respectively. A wide range of N-nitrosamines concentrations and species were observed in different locations. Higher concentrations of N-nitrosamines were detected in distribution systems that were further away from the treatment plants, suggesting that the contact time between the residual disinfectant and natural organic matter may play an important role in the formation of these compounds.

Pharmacokinetic Comparison of Four Major Bio-Active Components of Naoxintong Capsule in Normal and Acute Blood Stasis Rats Using Ultra-Performance Liquid Chromatography Coupled with Triple-Quadrupole Mass Spectrometry

Objective: To compare the pharmacokinetic differences of the main components of Naoxintong capsule(NXTC) in normal and acute blood stasis rats. Materials and Methods: Rats were subcutaneously injected with adrenaline hydrochloride twice;during the two subcutaneous injections, the rats were placed in ice water for 4 min to reproduce the model rat of acute blood stasis. The normal and acute blood stasis rats were administrated a 5.04 g/kg dose of NXTC suspension. Then, blood samples were collected from the posterior retinal venous plexus at different time points. Plasma concentrations of four major bio-active components including caffeic acid, ferulic acid, formononetin, and tanshinone IIA in NXTC were measured using ultra-performance liquid chromatography coupled with triple-quadrupole mass spectrometry. Phoenix Win Nonlin v6.2 software was used to calculate the pharmacokinetic parameters. Results: Compared with the normal rats, the acute blood stasis rats showed a significant decrease in C_(max) of ferulic acid and formononetin, AUC_(all) of caffeic acid and ferulic acid, and AUC_(INF_obs) of ferulic acid. Conversely, an increase in the Vz_F_obs and MRT_(last) of ferulic acid and caffeic acid was observed. These findings demonstrate that the absorption of the four NXTC components was weakened in the acute blood stasis rats and that the elimination time was prolonged. Conclusions: The significant difference in some parameters of the four NXTC components between the normal and acute blood stasis rats might be caused by an increase in blood viscosity and the subsequent slowing down of blood flow in the acute blood stasis rats. The pharmacokinetic study conducted in pathological state can provide important information and scientific basis for further rational clinical application of NXTC.

Rapid Differentiation of Aconiti Kusnezoffii Radix from Different Geographic Origins Using Ultra-Performance Liquid Chromatography Coupled with Time-of-Flight Mass Spectrometry

Objective:There are different geographic origins of Aconiti Kusnezoffii Radixs(AKRs)sold in the market with different quality.This study aims to establish a rapid analysis method to distinguish the different geographic origins of AKRs and to realize the rapid evaluation of their quality.Methods:An ultra-performance liquid chromatography coupled with time-of-flight mass spectrometry(UPLC-Q-TOF MS)method was utilized to acquire the constituents'information of AKRs from different geographic origins.MSE data and Progenesis QI software were employed to identify the chemical constitutes.Principal component analysis(PCA)was applied to comparing MS data to find the chemical markers of AKRs from different geographic origins.Results:Twenty-three components were detected and 17 out of them were identified,including diester-diterpenoid alkaloids,monoester-diterpenoid alkaloids,and amine-diterpenoid alkaloids.Three pairs of isomers were detected and two of them were distinguished by the retention time of standard samples.Thirteen chemical markers were screened out through PCA and orthogonal partial least square discriminant analysis.Through detecting Napelline or isomer of Napelline(m/z 360.2530)and Aconifine(m/z 662.3170),AKRs from inner Mongolia autonomous could be screened.According to the existence of benzoylaconine(m/z 604.3108)and Indaconitine(m/z 630.3159),it could be confirmed that the AKRs are from Xinjiang Uygur autonomous.AKRs that cannot detect compounds above-mentioned could be from Liaoning or Shanxi Province.Conclusions:The chemical profile could be used not only to distinguish the AKRs from different geographic origins but also to identify the true and false of AKRs.This study lays a foundation for the study of efficacy and toxic of AKRs.

Simultaneous Determination of Six Compounds in Rat Plasma by Ultra-Performance Liquid Chromatography with Tandem Mass Spectrometry: Application in the Pharmacokinetic Study of Qing Gan-Shu Yu-Fang

A rapid and high selective ultra-performance liquid chromatography(UPLC)with tandem mass spectrometry method for simultaneous determination of six compounds including albiflorin,paeoniflorin,picroside I,picroside II,saikosaponin A,and saikosaponin D in rat plasma was developed and validated using butyl p-hydroxybenzoate as an internal standard.One-step direct protein precipitation with acetonitrile was used to extract the compounds from the rat plasma samples.Chromatographic separation was achieved using an ACQUITY UPLC BEH C18 column(100 mm×2.1 mm,1.7μm)at a flow rate of 0.4 m L/min,using gradient mode containing 0.1%formic acid in water and acetonitrile were used as the Mobile phase A and B.Electrospray ionization in negative ion mode and multiple reaction monitoring were used to identify and quantify active components.Calibration curves showed good linearity(R^2>0.9908)over a wide concentration range for all compounds.The intra-and interday precision(relative standard deviation)ranged 2.4%–7.0%and 2.6%–8.0%,respectively.The accuracy(relative error)was from-13.0%to 13.2%at all quality control levels.The recovery ranged from 81.1%to 92.5%.The validated method was successfully applied to pharmacokinetic study in rats after oral administration of Qing Gan-Shu Yu-Fang.The results show that one can draw a conclusion that these six active ingredients can be quickly absorbed and play a pharmacodynamic role rapidly in vivo.

An Automatic Solid Phase Extraction and Ultra-Performance Liquid Chromatography Tandem Mass Spectrometry for Determination of Seven Microcystins at Ultra-Trace Levels in Surface Water

A method was developed for the detection of seven microcystins(microcystin-LR, RR, YR, LA, LY, LW and LF) in surface water using automatic solid-phase extraction(A-SPE) coupled with ultra-performance liquid chromatography tandem mass spectrometry(UPLC-MS/MS). The automated solid-phase extraction system was used to extract microcystins(MCs) from water samples. UPLC-MS/MS was used to determine MCs concentrations in just 5 min. Method detection limits were from 0.3 to 0.9 ng/L, microcystin recoveries ranged from 83.8% to 114%, and the relative standard deviation(RSD) varied from 5.6% to 12.5%. This analytical approach was found to be simple, highly sensitive, accurate, which required little manual operation. Additionally, to validate this analytical method, A-SPE+UPLC-MS/MS was applied to characterize the concentration of MCs in Taihu Lake, Wuxi, China.

Ultra-performance Liquid Chromatography-Quadrupole/Time-of-Flight Mass Spectrometry Based Bile and Urine Metabonomics Study on the Ameliorative Effects of Curcuma wenyujin Rhizoma on Acute Blood Stasis in Rats

Background: Curcuma wenyujin rhizome(CWR) is a commonly used Chinese herbal medicine for treating blood stasis in China for 1000 of years. However, the underlying mechanism of CWR remains unclear. Aims and Objectives: The purpose of this study is to clarify the bioactive mechanism of CWR in treating blood stasis. Materials and Methods: In this study, pharmacological indexes, including hemorheology and four blood coagulation indexes were tested. Bile and urine metabolomics were engaged by UPLC-Q/TOF-MS. Multivariate statistical analysis were used to screen out differential endogenous metabolites. Results: The results indicated that CWR significantly ameliorated the hemorheology and coagulation functions of acute blood stasis(ABS) model rats. Moreover, 27 endogenous metabolites between the CWR group and the ABS group were screened, and the levels were all improved to certain degrees by CWR preadministration. Metabonomics results indicated that ABS was mainly related to linoleic acid metabolism, arachidonic acid metabolism, glycerophospholipid metabolism, sphingolipid metabolism, pentose and glucuronate intercereasonversions, steroid hormone biosynthesis, and primary bile acid biosynthesis. Conclusion: In a word, the metabolomics method is consistent with the holistic view of traditional Chinese medicine(TCM) that can be a powerful means to illustrate the biological activity mechanism of CWR in treating blood stasis and to offer research demonstration for further study on the effector mechanism of TCM.

Perfluoropentane-based oxygen-loaded nanodroplets reduce microglial activation through metabolic reprogramming

Microglia,the primary immune cells within the brain,have gained recognition as a promising therapeutic target for managing neurodegenerative diseases within the central nervous system,including Parkinson’s disease.Nanoscale perfluorocarbon droplets have been reported to not only possess a high oxygen-carrying capacity,but also exhibit remarkable anti-inflammatory properties.However,the role of perfluoropentane in microglia-mediated central inflammatory reactions remains poorly understood.In this study,we developed perfluoropentane-based oxygen-loaded nanodroplets(PFP-OLNDs)and found that pretreatment with these droplets suppressed the lipopolysaccharide-induced activation of M1-type microglia in vitro and in vivo,and suppressed microglial activation in a mouse model of Parkinson’s disease.Microglial suppression led to a reduction in the inflammatory response,oxidative stress,and cell migration capacity in vitro.Consequently,the neurotoxic effects were mitigated,which alleviated neuronal degeneration.Additionally,ultrahigh-performance liquid chromatography–tandem mass spectrometry showed that the anti-inflammatory effects of PFP-OLNDs mainly resulted from the modulation of microglial metabolic reprogramming.We further showed that PFP-OLNDs regulated microglial metabolic reprogramming through the AKT-mTOR-HIF-1αpathway.Collectively,our findings suggest that the novel PFP-OLNDs constructed in this study alleviate microglia-mediated central inflammatory reactions through metabolic reprogramming.

Serum metabolic profiling of targeted bile acids reveals potentially novel biomarkers for primary biliary cholangitis and autoimmune hepatitis

BACKGROUND Primary biliary cholangitis(PBC)and autoimmune hepatitis(AIH)are two unexplained immune diseases.The golden standard for diagnosis of these diseases requires a liver biopsy.Liver biopsy is not widely accepted by patients because of its invasive nature,and atypical liver histology can confuse diagnosis.In view of the lack of effective diagnostic markers for PBC and AIH,combined with the increasingly mature metabolomics technologies,including full-contour metabolomics and target.AIM To determine non-invasive,reliable,and sensitive biochemical markers for the differential diagnosis of PBC and AIH.METHODS Serum samples from 54 patients with PBC,26 patients with AIH and 30 healthy controls were analyzed by Ultra-high performance liquid chromatographytandem mass spectrometry serum metabolomics.The metabolites and metabolic pathways were identified,and the metabolic changes,metabolic pathways and inter-group differences between PBC and AIH were analyzed.Fifteen kinds of target metabolites of bile acids(BAs)were quantitatively analyzed by SRM,and the differential metabolites related to the diagnosis of PBC were screened by receiver operating characteristic curve analysis.RESULTS We found the changes in the levels of amino acids,BAs,organic acids,phospholipids,choline,sugar,and sugar alcohols in patients with PBC and AIH.Furthermore,the SRM assay of BAs revealed the increased levels of chenodeoxycholic acid,lithocholic acid(LCA),taurolithocholic acid(TLCA),and LCA+TLCA in the PBC group compared with those in the AIH group.The levels of BAs may be used as biomarkers to differentiate PBC from AIH diseases.The levels of glycochenodeoxycholic acid,glycochenodeoxycholic sulfate,and taurodeoxycholic acid were gradually elevated with the increase of Child-Pugh class,which was correlated with the severity of disease.CONCLUSION The results demonstrated that the levels of BAs could serve as potential biomarkers for the early diagnosis and assessment of the severity of PBC and AIH.

Determination of L-ergothioneine in food by UPLC-MS/MS method

L-Ergothioneine(L-EGT)possesses excellent antioxidant activity and has been used in the food,pharmaceuticals and cosmetics industries.In this study,a new efficient and sensitive ultra-performance liquid chromatography tandem mass spectrometry(UPLC-MS/MS)method was established for the quantitative determination of L-EGT in food.The sample was extracted with methanol-water(70:30,V/V),separated by hydrophilic interaction liquid chromatography(HILIC)and detected by triple-quadrupole mass spectrometry.Validation studies were carried out on different product and the limit of quantitation was 20μg/kg(milk,alcohol-free beverages,dairy products)and 40µg/kg(cereal bars,chocolate).Excellent linearity(correlation coefficient(R2)≥0.999)was achieved for L-EGT quantification in the range of 5–200 ng/mL.The recoveries of the method(83.7%−107.5%)and the relative standard deviation(RSD,0.88%−6.84%(n=6))meet the performance criteria required for the determination of L-EGT in food.Finally,the applicability of the method was tested by analysing actual samples.In general,the method developed is simple,reliable,accurate,and stable and could be useful for routine analyses of L-EGT in food.

Seed metabolomic study reveals significant metabolite variations and correlations among different soybean cultivars

Soybean[Glycine max（L.） Merr.]is one of the world＇s major crops,and soybean seeds are a rich and important resource for proteins and oils.While ＂omics＂ studies,such as genomics,transcriptomics,and proteomics,have been widely applied in soybean molecular research,fewer metabolomic studies have been conducted for largescale detection of low molecular weight metabolites,especially in soybean seeds.In this study,we investigated the seed metabolomes of 29 common soybean cultivars through combined gas chromatography-mass spectrometry and ultra-performance liquid chromatography-tandem mass spectrometry.One hundred sixty-nine named metabolites were identified and subsequently used to construct a metabolic network of mature soybean seed.Among the 169 detected metabolites,104 were found to be significantly variable in their levels across tested cultivars.Metabolite markers that could be used to distinguish genetically related soybean cultivars were also identified,and metabolitemetabolite correlation analysis revealed some significant associations within the same or among different metabolite groups.Findings from this work may potentially provide the basis for further studies on both soybean seed metabolism and metabolic engineering to improve soybean seed quality and yield.

An efficient resin for solid-phase extraction and determination by UPLCMS/MS of 44 pharmaceutical personal care products in environmental waters

In this study,a hydrophilic resin named GCHM was fabricated based on poly(N-vinyl pyrrolidone-codivinylbenzene),characterized,and applied as a solid-phase extraction(SPE)material.Up to 44 pharmaceuticals and personal care products(PPCPs)belonging to 10 classes were recovered in environmental water samples.Different variables affecting extraction,such as adsorbent amount,sample pH,and loading speed,were optimized.Under optimal conditions,the average absolute recovgy of 44 PPCPs was 75.6%using GCHM,indicating a better performance than the commercial Oasis^HLB.SPE with home-made hydrophilic polymeric sorbent followed by ultra-performance liquid chromatography and tandem mass spectrometry was validated,and the method achieved good linearity(r2>0.991,for all analytes).In addition,the method detection limits of target compounds ranged from 0.03 to 0.6 ng/L.The developed method was applied to determine PPCPs in 10 environmental water samples taken from the Yangtze River,Huaihe River,and Taihu Lake,1 groundwater sample from Changzhou in Jiangsu Province,1 wastewater sample from Xiamen and 2 seawater samples from the Jiulong River in Fujian Province,China.In these samples,22 compounds were determined at levels ranging from 0.5 to 1590 ng/L.

Analysis and Identification of Chemical Constituents of Fenugreek by UPLC-IT-MSn and UPLC-Q-TOF-MS

Fenugreek, a traditional Chinese medical plant, has been widely used as food ingredients because of its out- standing medicinal qualities. The hypoglycemic activity of fenugreek is one of its important pharmacological properties. Both of saponin and flavonoid components have the hypoglycemic activity and their contents in fenugreek are 4%--8% and 1% ---2%, respectively. This paper focused on these two types of components to carry out purification research and chemical analysis. The heating reflux extraction method and macroporous resin purification method were designed to prepare the saponins and the flavonoids components from defatted fenugreek seeds. Petroleum ether ultrasonic skim and 70% ethanol refluxing were used for the extraction of saponins and flavonoid from fenugreek. The column of DM130 macroporous resin and D101 macroporous resin were respectively used to prepare fenugreek saponin and flavonoid components, and the total contents of them were 78.56% and 62.28%, respectively. The saponin and flavonoids were subsequently analyzed by an ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry（UPLC-Q-TOF-MS） along with an ultra-performance liquid chromatography and ion-trap tandem mass spectrometry（UPLC-IT-MS＂）. As a result, 57 saponins and 19 flavonoid compounds were characterized. The obtained results will provide a theory basis for further research on fenugreek, as well as research and development of hypogly- cemic new drugs.

Analysis of Phytochemical Constituents of Zuogui Wan in Rat Serum and its Effects on Early Embryonic Development of Mice

Objective:Zuogui Wan(ZGW)has been used as a typical prescription for tonifying kidney essence in traditional Chinese medicine.The objective of this study is to elucidate the phytochemical constituents of ZGW-treated rat serum(ZGWRS)using ultra-performance liquid chromatography-electrospray ionization/quadrupole-time-of-flight high-definition mass spectrometry(UPLC-ESI-Q-TOF-MS).Methods:ZGW was administered to rats,and the phytochemical constituents in rat serum were determined using UPLC-ESI-Q-TOF-MS.MetaboLynx analysis in negative ion mode was adopted to characterize the chemical constituents of ZGWRS.Orthogonal partial least squares discriminant analysis was applied for the discovery of constituents of ZGW that entered the serum of rats.The fertilized eggs collected from the same experiment were randomly divided into four groups,including the normal,40 mmol/L glucose,40 mmol/L glucose 5%control rat serum,and 40 mmol/L glucose 5%ZGWRS groups.They were cultivated at 37°C and 5%CO2 in a saturated humidity CO2 incubator.The blastocyst rate and two-cell rate were used to evaluate the effects of ZGWRS on embryonic development.Results:Thirteen constituents were identified in the ZGWRS,among which sweroside,loganin,morroniside,loganic acid,and 8-epiloganic acid were from Fructus Corni(Shan Zhu Yu).5-Hydroxymethyl-2-furfural-glucuronide,2-H-5-hydroxymethyl-2-furfural-glucuronide,3-hydroxy-2,6,6-trimethyl-1-cyclohexene-1-carboxylic acid,andβ-D-ribofuranuronic acid methyl ester triacetate were from Radix Rehmanniae Preparata(Shu Di Huang).Coumaric acid was from Fructus Lycii(Gou Qi Zi).Kaempferol-3-beta-O-glucuronide and cuscutamine were from Semen Cuscutae(Tu Si Zi).The embryonic development was significantly inhibited using 40 mmol/L glucose.Compared with the normal group,the blastocyst rate of the glucose group was decreased.The blastocyst rate of the 40 mmol/L glucose 5%ZGWRS group was significantly higher than that of the glucose group,indicating that ZGWRS negates the effect of glucose on mouse embryonic development.Conclusion:The results verified that a rapid and robust UPLC-ESI-Q-TOF-MS-based platform had been successful for identifying multiple constituents of ZGW.ZGWRS is rich in active constituents of iridoid glycosides.The results of this study also showed that ZGWRS could negate the effect of glucose on mouse embryonic development.

Effect on serum metabolomics of rats with premature ovarian insufficiency by Zhibian(BL54) through Shuidao(ST28) acupuncture

OBJECTIVE:To analyze the serum metabolic targets of the"Zhibian(BL54)through Shuidao(ST28)"acupuncture technique in cyclophosphamide(CTX)-induced premature ovarian insufficiency(POI)model rats and to elucidate the potential molecular mechanism of acupuncture in improving POI.METHODS:We used an intraperitoneal injection of CTX to establish the POI rat model(POI group)and compared serum hormone levels and ovarian histopathological changes to evaluate the effect of the Zhibian(BL54)through Shuidao(ST28)technique(ZS+POI group)on ovarian function.Then,nontargeted metabolomics was performed using rat serum by ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry(UPLC-Q-TOF/MS).RESULTS:After acupuncture intervention,the serum hormone levels and ovarian pathological morphology of POI rats were effectively improved.Moreover,UPLC-Q-TOF/MS results showed that the ZS+POI group showed a significant reversal of the levels of 6 differential metabolites.Among them,the levels of four serum metabolic markers,divanillyltetrahydrofuran ferulate,trans-ferulic acid,tryptamine,and neuraminic acid,increased significantly.Further analysis of biological effects showed that all metabolites were involved in the regulation of reproductive hormone levels and antioxidant and antiapoptotic effects.CONCLUSIONS:The"Zhibian(BL54)through Shuidao(ST28)"acupuncture method may improve the ovarian function of POI rats by regulating serum metabolite markers to exert antioxidant and antiapoptotic effects,which provides a theoretical basis for the clinical application of acupuncture in the treatment of POI.