Ultrahigh-performance Liquid Chromatography Quadrupole Orbitrap Research Articles

Metabolic and elemental profiling as potential discriminating features among the black mahlab seeds (Monechma ciliatum) grown in three different regions.

Black mahlab (Monechma ciliatum) seed is a rich source of metabolites and minerals and is usually believed to have a similar composition between different areas of cultivation. Until now, no studies have assessed changes in black mahlab seeds (BMSs) to determine those constituents that help to discriminate them according to geographical origin. The present study attempted to compare the metabolomics and elemental profiles of BMSs of different geographical origins and identified the potential markers using ultrahigh-performance liquid chromatography quadrupole Orbitrap tandem mass spectrometry (UHPLC-Q-Orbitrap-MS2), and inductively coupled plasma mass spectrometry (ICP-MS) techniques and established the chemometric model to identify the potential markers and discriminate them according to cultivation sites. In this work, data from metabolites analysis by UHPLC-Q-Orbitrap-MS2 and multi-elemental data obtained from ICP-MS were combined with chemometrics for tracing the geographical origin of BMSs. Principal component analysis (PCA) was used to evaluate the overall grouping of samples. In contrast, partial least squares-discriminant analysis (PLS-DA) and orthogonal partial least squares discriminant analysis (OPLS-DA) were employed for authentication. PLS-DA and OPLS-DA models were fully validated (R2Y and Q2 values > 0.5). Variable importance of various projections was applied to obtain valuable data about differential elements (seven markers were identified) and metabolites (23 markers were identified) with high discrimination potential. The outcomes presented in this study serve as an appropriate framework for developing novel discrimination approaches in food origin screening.

Renshen Baidu Powder Attenuated Intestinal Inflammation and Apoptosis in Ulcerative Colitis Rats through the Inhibition of PI3K/AKT/NF-κB Signaling Pathway.

Objective Renshen Baidu Powder (RBP) is a famous classic compound of traditional Chinese medicine (TCM) and is commonly used for treating ulcerative colitis (UC). However, the pharmacological mechanism of RBP in treating UC remains unclear. This study investigates the possible mechanism of RBP for UC treatment by network pharmacological analysis and rat validation. Methods First, the main chemical constituents of RBP were identified using ultrahigh-performance liquid chromatography quadrupole Orbitrap high-resolution mass spectrometry (UHPLC-Q-Orbitrap-HRMS). Then, we obtained targets of identified compounds from the SwissTargetPrediction database and targets associated with UC from GeneCards database. Furthermore, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were used to analyze the metabolism-related signaling pathways affected by RBP. Hematoxylin-eosin (HE) staining was used to observe the pathological change of colon in UC rats after treating RBP, and terminal deoxynucleotidyl-transferase (TdT)-mediated dUTP Nick end labeling (TUNEL) staining was used to detect apoptosis after RBP treatment. The enzyme-linked immunosorbent assay (ELISA) was employed to evaluate cytokine levels of TNF-α, IL-1β, and IL-6. The protein expressions of Bax, Bcl-2, PI3K, AKT, and NF-κB in colonic tissue were detected using immunohistochemistry (IHC). Real-time quantitative polymerase chain reaction (RT-QPCR) was employed to evaluate mRNA expression of PI3K, AKT, and NF-κB. Results We found a total of 24 main compounds and 329 potential targets related to UC. According to KEGG results, 3 main pathways were identified as responsible for UC, including PI3K-AKT, HIF-1, and VEGF signaling pathway. Animal experiments showed that RBP treatment significantly attenuated colon damage in rats with UC. Mechanistically, RBP could inhibit PI3K/AKT/NF-κB pathway; decrease cell apoptosis; and downregulate the expression of TNF-α, IL-1β, and IL-6. Conclusions This study demonstrated that RBP may exert anti-inflammatory and antiapoptotic therapeutic benefits in UC by regulating the PI3K/AKT/NF-κB signaling pathways, providing a scientific basis for understanding the mechanism of RBP against UC.

Multi-residue analysis of 206 pesticides in grass forage by the one-step quick, easy, cheap, effective, rugged, and safe method combined with ultrahigh-performance liquid chromatography quadrupole orbitrap mass spectrometry.

A novel method for detecting pesticide multi-residue in grass forage (alfalfa and oat) was established based on the one-step automatic extractionand purification technology of quick, easy, cheap, effective, rugged, and safe combined with ultrahigh-performance liquid chromatography quadrupole Orbitrap high-resolution mass spectrometry. The crushed sample was extracted with acetonitrile with 1% acetate, followed by a cleanup step with a primary-secondary amine, octadecylsilane, and graphitized carbon black. The extraction and purification were carried out using the one-step automatic pretreatment equipment. The target pesticides were acquired in positive ion electrospray ionization mode and full scan/data dependent secondary scan mode. The calibration curve shows good linearity over the corresponding concentration range, with the coefficient of determination greater than 0.99. The screening detection limits were 0.5-50μg/kg, and the limit of quantification for the 206 pesticides was set at 1-50μg/kg. At the spiking levels of one, two, and 10 times of limit of quantification, more than 95% of pesticides had recovery between 70-120%, with a relative standard deviation ≤20%. The method was proved to be simple, rapid, high-sensitivity, and could be routinely used for the high throughput screening and quantitative analysis of pesticide residues in alfalfa and oat.

Comparison of the Saponins in Three Processed American Ginseng Products by Ultra-High Performance Liquid Chromatography-Quadrupole Orbitrap Tandem Mass Spectrometry and Multivariate Statistical Analysis

A method with ultrahigh performance liquid chromatography Quadrupole-Orbitrap tandem mass spectrometry (UHPLC-Q-Orbitrap-MS/MS) was applied for the quality evaluation of different processing and drying of American ginseng, including natural drying (ND), steam drying (SD), and vacuum freeze-drying (VFD). A total of 51 saponins were successfully identified in three processed products. Three processed American ginseng products were well-differentiated in orthogonal partial least-squares discriminant analysis (OPLS-DA). The S-plot also identified the marker compounds in each product, while the major ginsenosides of ND (malonyl (M)-Rd, M-Rb1, Rg1), SD (20 (S)-Rg3, 20 (S)-Rg2), and VFD (M-Rd, M-Rb1) were found. The results indicate that the method by vacuum freeze-drying can retain the content of rare ginsenosides and malonyl-ginsenosides. The marker compounds selected will benefit the holistic evaluation of related American ginseng products.

Rapid determination of 93 banned industrial dyes in beverage, fish, cookie using solid-supported liquid–liquid extraction and ultrahigh-performance liquid chromatography quadrupole orbitrap high-resolution mass spectrometry

Banned industrial dyes are composed of a large number of chemicals with diverse physical and chemical properties, making their simultaneous determination a challenging task. A one-step extraction and purification of 93 banned industrial dyes from beverage, fish and cookie sample methods was proposed by using solid supported liquid–liquid extraction (SLE). The extract was analyzed by ultrahigh-performance liquid chromatography quadrupole orbitrap high–resolution mass spectrometry (UPLC-Q-Orbitrap-HRMS). The quantitative and qualitative mode adopts Q-Orbitrap-HRMS full scan MS (full scan MS1) and data-dependent MS/MS (dd-MS2) acquisition mode. The mass resolution was screened under 70,000 FWHM for full-scan MS1 and 35,000 FWHM for dd-MS2. Linearity was observed in the range of 0.01 ∼ 0.5 μg/mL and the limits of quantification were 0.04 ∼ 0.2 mg/kg for 93 dyes. The average recoveries were 70.5–105.8%, with RSD ≤ 10%. The proposed method has the ability to simultaneously screen many banned dyes in foods with high throughput, sensitivity and reliability.

Screening and elucidation of fragmentations of 23 diuretics in dietary supplements using UHPLC-Q-Orbitrap

Diuretics are used to treat the edematous state in cases of renal insufficiency, nephrotic syndrome, liver cirrhosis, and heart failure. These compounds are used by athletes to lose weight and are included in the list of prohibited substances by the World Anti-Doping Agency. They are also used by obese and overweight people for losing weight, and there are a number of recent reports on the contamination of dietary supplements with diuretics. Due to the alluring online marketing and blogging, there is an extensive misuse of products that are illegally adulterated with diuretics, which has seriously increased health risks. Therefore, it is essential to develop an analytical method for the detection of adulterants in such substances. In this study, 23 diuretics, categorized into four groups, namely, thiazide diuretics (e.g., bendroflumethiazide), loop diuretics (e.g., bumetanide), potassium-sparing diuretics (e.g., amiloride), and carbonic anhydrase inhibitors (e.g., acetazolamide), were analyzed using ultrahigh-performance liquid chromatography-quadrupole orbitrap (UHPLC-Q-Orbitrap). Their fragmentation was elucidated based on the MS/MS data. The 124 products were screened by the UHPLC-Q-Orbitrap (LC-HRMS) method, and the confirmed compounds were quantitated by a previously established LC-MS/MS method. Approximately 5% of the samples were found to be illegally contaminated with diuretics at a concentration of 0.051–162 mg/g. The high selectivity and sensitivity of the UHPLC-Q-Orbitrap (LC-HRMS) method, in combination with the established fragmentation, offer a new approach for the rapid and accurate screening of diuretics in adulterated products, which would be ultimately beneficial for the public health.

Serum concentrations of neonicotinoids, and their associations with lipid molecules of the general residents in Wuxi City, Eastern China

Exposure to neonicotinoid insecticides (NNIs) was proven to be harmful to organisms, however, there is a dearth of information regarding their occurrence and adverse effects on the general residents. Here, n = 120 human serum samples were collected from the same area of Wuxi city, Eastern China, and these serum samples were further analyzed for nine NNIs and nine target lipid molecules by use of ultrahigh performance liquid chromatography-quadrupole orbitrap high-resolution mass spectrometer. We observed that four out of nine NNIs exhibited relatively high detection frequencies (DF), and these NNIs were imidacloprid (IMI; DF = 28.3%), clothianidin (CLO; 16.7%), thiacloprid (THI; 14.2%), and acetamiprid (ACE; 12.5%), respectively, with 95th concentrations ranging from 32.0 to 427 pg/mL. Median concentrations of imidacloprid-equivalent total neonicotinoids (IMIeq) and ∑7NNI were 46.6 pg/mL and 26 pg/mL, respectively. Five out of nine lipid molecules exhibited higher levels, that were docosahexaenoic acid [FA(22:6)], 18:0 phosphocholine [LysoPC(18:0)], 18:0 phosphoethanolamine [LysoPE(18:0)], D18:1–18:0 sphingomyelin [SM(d18:1/18:0)], and 18:1–18:1 diglycerol [DG(18:1/18:1)], respectively. More interestingly, we observed statistically significant correlations (student’s t-test, one-way ANOVA, or Mann-Whitney test; p < 0.05) between NNI levels and population characteristics (i.e. age, smoking, and health status). Beyond that, we also observed statistically significant correlations between levels of selected NNIs (CLO, ACE, or THI) and lipid molecules [LysoPE(18:0), SM(d18:1/18:0), and DG(18:1/18:1)]. Collectively, for the first time, we provided the information on contamination levels of NNIs in serum samples of general residents in China and demonstrated the associations between concentrations of NNIs and levels of lipid molecular species.

Chemical Comparison of White Ginseng before and after Extrusion by UHPLC-Q-Orbitrap-MS/MS and Multivariate Statistical Analysis.

Ultrahigh-performance liquid chromatography Quadrupole-Orbitrap tandem mass spectrometry (UHPLC-Q-Orbitrap-MS/MS) was used to compare the composition of ginsenosides in white ginseng (WG) and extruded white ginseng (EWG). A total of 45 saponins, including original neutral ginsenosides, malonyl-ginsenosides, and chemical transformation of ginsenosides, were successfully identified in both WG and EWG. Multivariate statistical analyses including supervised orthogonal partial least squared discrimination analysis (OPLS-DA) and hierarchical clustering analysis (HCA) were used to analyze components of white ginseng before and after extrusion. As a result, three ginsenosides (malonyl (M)-Rb1, M-Rb2, and M-Rc) were found to be increased in WG, while three ginsenosides (Rb2, Rc, and Rg1) were elevated in EWG. In the OPLS-DA S-plot, the different compositions of ginsenoside that were distinguished between WG and EWG were screened out. Experimental results indicate that the UHPLC-Q-Orbitrap-MS/MS is a useful tool to characterize variations of ginsenosides in WG and EWG.

Investigation of Differentially Expressed Proteins Induced by Alteration of Natural Se Uptake with Ultrahigh-Performance Liquid Chromatography Quadrupole Orbitrap Uncovers the Potential Nutritional Value in Se-Enriched Green Tea.

Se-enriched green tea, with an increasing consumption, is the shoot of tea plants grown naturally in a seleniferous region. A label-free proteomic strategy based on ultrahigh-performance liquid chromatography quadrupole Orbitrap was applied to characterize and distinguish the difference between the Se-enriched and normal green tea with a total of 283 proteins identified and 264 proteins quantified, in which 96 proteins were observed different. The expressions of 10 proteins were upregulated and 40 proteins were downregulated (p < 0.05) in Se-enriched samples. Gene ontology, Kyoto Encyclopedia of Genes and Genomes pathway, and protein-protein interaction (PPI) network analysis results indicated that these differentially expressed proteins significantly interacted and were involved in secondary metabolites and inflammatory response biological processes. Furthermore, the expression of methyl-jasmonate- and ethylene-related genes changed significantly in Se-enriched green tea, and catalase proteins were employed as the center of the pathway that changed significantly in the PPI network. These results associating with the current knowledge of selenium in soil-plant cycling revealed that organic selenium was synthesized in green tea, which provided novel information on Se assimilation in Camellia sinensis and improved the understanding of Se-enriched green tea as a possible ideal selenium supplement in daily life.

A sensitive method for simultaneous determination of 12 classes of per- and polyfluoroalkyl substances (PFASs) in groundwater by ultrahigh performance liquid chromatography coupled with quadrupole orbitrap high resolution mass spectrometry

Per- and polyfluoroalkyl substances (PFASs) comprise a large group of chemicals with diverse physicochemical properties, which make their simultaneous determination a challenging task. A trace analytical method based on ultrahigh performance liquid chromatography-quadrupole Orbitrap high resolution mass spectrometry (UHPLC-Q-Orbitrap HRMS) was developed for simultaneous determination of 54 PFASs belonging to 12 classes in groundwater, including 24 perfluorocarbons and 30 precursors. This method provided good linearity of calibration standards (R2 > 0.99), excellent method limits of quantification (MLOQs) (0.5–250 pg/L), satisfactory matrix spiking recoveries (63%–148%), high precision (intra-day relative standard deviations (RSDs) 1.4–11.4%, inter-day RSDs 1.6–12.9%, and inter-week RSDs 2.1–12.7%), and short runtime (13 min), suitable for high throughput studies. The newly established method was successfully applied to detect PFASs in the groundwater samples collected from Hebei Province, China. Twenty PFASs were detected with the total concentration of 0.3–32.9 ng/L, indicating the contamination level similar to that in drinking water. The dominant PFASs were perfluorobutanesulfonate (PFBS), perfluorobutanoic acid (PFBA), perfluoropentanoic acid (PFPeA) and perfluorooctanoic acid (PFOA). In addition, 6:2 fluorotelomer phosphate diester (6:2 diPAP) and 6:2 fluorotelomer sulfonate (6:2 FTS) were found as the major precursors. The total PFAS concentrations were lower than the cumulative permissible limit of 70 ng/L for PFOS and PFOA recommended by the United States Environmental Protection Agency (USEPA) for drinking water in 2016. In a nutshell, this study provided a fast and sensitive method based on HRMS for the simultaneous analysis of a wide range of PFASs, present at trace levels in groundwater samples.

Exploring the metabolomic diversity of plant species across spatial (leaf and stem) components and phylogenic groups

BackgroundPlants have been used as an important source of indispensable bioactive compounds in various cosmetics, foods, and medicines. However, the subsequent functional annotation of these compounds seems arduous because of the largely uncharacterized, vast metabolic repertoire of plant species with known biological phenotypes. Hence, a rapid multi-parallel screening and characterization approach is needed for plant functional metabolites.ResultsFifty-one species representing three plant families, namely Asteraceae, Fabaceae, and Rosaceae, were subjected to metabolite profiling using gas chromatography time-of-flight mass spectrometry (GC-TOF-MS) and ultrahigh-performance liquid chromatography quadrupole orbitrap ion trap tandem mass spectrometry (UHPLC-Q-orbitrap-MS/MS) as well as multivariate analyses. Partial least squares discriminant analysis (PLS-DA) of the metabolite profiling datasets indicated a distinct clustered pattern for 51 species depending on plant parts (leaves and stems) and relative phylogeny. Examination of their relative metabolite contents showed that the extracts from Fabaceae plants were abundant in amino acids, fatty acids, and genistein compounds. However, the extracts from Rosaceae had higher levels of catechin and ellagic acid derivatives, whereas those from Asteraceae were higher in kaempferol derivatives and organic acids. Regardless of the different families, aromatic amino acids, branch chain amino acids, chlorogenic acid, flavonoids, and phenylpropanoids related to the shikimate pathway were abundant in leaves. Alternatively, certain amino acids (proline, lysine, and arginine) as well as fatty acids levels were higher in stem extracts. Further, we investigated the associated phenotypes, i.e., antioxidant activities, affected by the observed spatial (leaves and stem) and intra-family metabolomic disparity in the plant extracts. Pearson’s correlation analysis indicated that ellagic acid, mannitol, catechin, epicatechin, and quercetin derivatives were positively correlated with antioxidant phenotypes, whereas eriodictyol was positively correlated with tyrosinase inhibition activity.ConclusionsThis work suggests that metabolite profiling, including multi-parallel approaches and integrated bioassays, may help the expeditious characterization of plant-derived metabolites while simultaneously unraveling their chemodiversity.

Characterisation of hederacoside C metabolites using ultrahigh-performance liquid chromatography quadrupole Orbitrap mass spectrometry based on automatic fragment ion search.

Hederacoside C (HDC) is a bioactive natural triterpenoid saponins constituent originating from traditional Chinese medicines, playing an important role in the treatment of acute respiratory infections and chronic inflammatory bronchitis. Meanwhile, it is recognised by Korea as a botanical drug. In order to develop an integrated template approach to analysing screening and identification of the metabolites of traditional Chinese medicines. This study will provide available information for further pharmaceutical studies of HDC and other triterpene saponins. An analysis strategy based on ultrahigh-performance liquid chromatography quadrupole Orbitrap mass spectrometry (UHPLC-Q-Orbitrap-MS) technique combined with automatic fragment ion search (FISh) was firstly exploited for the characterisation metabolites of HDC in vivo and in vitro. Accurate full mass scan combined with an on-line FISh annotations approach was developed to rapidly identify all the potential metabolites of HDC. Furthermore, FISh accurately located the structure of the target compound in a large number of mass spectral data. A total of 34 metabolites were detected and tentatively identified by analysing comprehensive biological samples. The results clearly demonstrated that HDC underwent general metabolic reactions including dealkylation, reduction, oxidation, desaturation, dehydration, cysteine conjugation, GSH conjugation, taurine conjugation, and glycine conjugation to produce 26 phase I and eight phase II metabolites. In the present study, UHPLC-Q-Exactive Orbitrap MS technique combined with FISh provided a rapid and efficient platform to characterise metabolites of HDC in vivo and in vitro. The proposed method could develop an integrated template approach to screen and identify the constituents and metabolites of traditional Chinese medicines.

Target quantification and semi-target screening of halogenated carboxylic acids in drinking water using ultra-high performance liquid chromatography-quadrupole orbitrap high-resolution mass spectrometry

To monitor the existing and emerging halogenated carboxylic acids (HCAs) in drinking water, a sensitive and rapid ultra-high performance liquid chromatography-quadrupole orbitrap high-resolution mass spectrometry method for simultaneous target quantification of 10 haloacetic acids (HAAs) and semi-target screening of 19 HCAs was developed. After filtration, drinking water samples were injected into the instrument. HCAs were separated on an HSS T3 column and detected by a type of non-target scan in the electrospray ionization negative mode. For target quantification of 10 HAAs, good linearity was obtained and the correlation coefficients were higher than 0.995. The limits of detection were in the range of 0.050–2.0 µg/L. The recoveries were in the range of 89.7%-108%, 83.4%-121%, 77.1%-116% and 80.2%-104% at levels of 2.5, 5.0, 10 and 20 µg/L, respectively, with relative standard deviations of 1.26%-16.9%. For semi-target screening of 19 HCAs, several criteria including accurate m/z, predicted retention time, deduced fragment ions and simulated isotope pattern were used for identification. The method was applied to analyzing 41 drinking water samples successfully. Five HAAs were detected by target quantification, with dichloroacetic acid and trichloroacetic acid exceeding the limits suggested by the U.S. Environmental Protection Agency and the World Health Organization. Eight HCAs were preliminary identified by semi-target screening, and three of them were further confirmed with reference standards purchased later.

Residue Distribution and Depletion of Ractopamine in Goat Tissues After Exposure to Growth-Promoting Dose.

The objectives of the present study was to investigated the ractopamine (RAC) distribution and depletion process in various tissues of goat including liver, kidney, spleen, lung, heart, fat, bile, brain and the eyes. The experiment was carried out on 21 goats (18 treated and 3 controls). Treated goats were orally administered RAC in a dose of 1 mg/kg body mass per day for last 28 days and randomly sacrificed on withdrawal days of 0.25, 1, 3, 7, 14 and 21. RAC in all matrices were determined by ultra-high performance liquid chromatography-quadrupole orbitrap high resolution mass spectrometry. After 21 days treatment discontinuation, the levels of RAC in bile reached at 13.48 ± 3.36 mg/L, which was significantly higher than that in the other tissues. The concentrations of RAC were followed by kidney, the excretory organ and liver, the major metabolic organ (4.49 ± 0.16 mg/kg for kidney and 1.81 ± 0.11 mg/kg for liver, respectively). The residual concentration of the drug in the eyes of goat was less than that in bile, kidney, liver, lung and spleen on withdrawal days 0.25. RAC residues was higher than the limits of detection = 0.15 μg/mL in liver on Day 21. These findings demonstrated that liver can serve as an alimentary matrix and as a matrix for the control of RAC abuse hypothetically except for urine.

Application of an ultrahigh-performance liquid chromatography coupled to quadrupole-orbitrap high-resolution mass spectrometry for the rapid screening, identification and quantification of illegal adulterated glucocorticoids in herbal medicines.

In this work, the ultrahigh-performance liquid chromatography quadrupole orbitrap high resolution mass spectrometry (UHPLC-Q-Orbitrap HRMS) was applied to the rapid screening, identification and quantification of the illegal adulterated glucocorticoids in herbal medicines. The mass spectrometer was operated in positive ion mode and Full MS/dd-MS2 (data-dependent MS2) mode, where selected ions were subjected to a dd-MS2 scan with given fragmentation energy following a Full MS scan. The application of 70 000 FWHM mass resolution and narrow mass windows (5ppm) effectively improve the selectivity of the method, and a single injection was sufficient to perform the simultaneous screening and identification/quantification of 14 glucocorticoids in 15min. The method validation including selectivity, sensitivity, calibration curve, accuracy, precision, recovery, matrix effect and stability were evaluated. The results of all analytes showed excellent linear relationship while all coefficient of determination (r2) were>0.9990 over wide concentration ranges (e.g., 5-1000ng/mL for hydrocortisone butyrate, r2=1.0000). The recoveries were in the range of 86.1-102.7%, while the matrix effects ranged from 95.8%-105.8%. Accuracies and precisions were performed. The intra- and inter-day accuracies ranged from 90.6% to 108.9%, while the intra- and inter-day precisions were in the range of 0.5% to 8.5%. Finally, the established method was employed to detect illegal adulterated glucocorticoids in herbal medicines. It will provide more reliable technical basis for the drug quality supervision department and ensure public health.