The Influences of Perfluoroalkyl Substances on the Rheumatoid Arthritis Clinic

Objective. Perfluoroalkyl substances (PFASs) are a class of synthetic organic fluorine chemicals, which have been mass-produced and widely used in the past 60 years, and also have been shown to be one of the major pollutants affecting human health. The impact of fluoride on the development of RA is unclear. The objective of this study was to assess the relationship between perfluoroalkyl substances and the clinic of RA. Methods. A cohort of 155 patients with RA in Second Affiliated Hospital of Zhejiang University School of Medicine were investigated. Demographic and clinical data of these patients were recorded. The concentration of perfluoroalkyl substances were measured by ultra-performance liquid chromatography system (ACQUITY, UPLC) coupled to a tandem mass spectrometer that operated in negative ionization mode. The correlations between the clinical data and the concentration of perfluoroalkyl substances were analyzed.Results. There were 43 male patients and 112 female patients in the cohort. Some of perfluoroalkyl substances (PFOA, PFNA, PFTrA, PFOS) were correlated negatively with the Body Mass Index (BMI); some of them (PFOA, PFNA, PFTrA, PFOS, 8:2Cl-PFESA) were correlated positively with the Disease Activity Score 28 (DAS28); two (PFOA, PFOS) of them were correlated positively with the white blood cell count, and one (PFUnA) of them was correlated negatively with the hemoglobin; two (PFDA, PFUnA) of them were correlated negatively with the presence of interstitial lung disease. Conclusion. These data suggest that exposure to perfluoroalkyl substances may promote the disease activity of rheumatoid arthritis and the visceral lesions.

Simultaneous determination of three naphthoquinones from Arnebia euchroma (Royle) Johnst in beagle plasma by UPLC-MS/MS and application for pharmacokinetics study

A rapid, simple and efficient ultra-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UPLC-ESI-MS/MS) method was established to simultaneous determination of shikonin, isobutyryl shikonin, β, βʹ-dimethylacryl alkanin in beagle plasma and evaluated by using esculetin as internal standard. The electrospray ionization (ESI) source was operated in negative ionization mode. Multi-reaction monitoring (MRM) was used to quantitatively analyzed shikonin m/z 287.0 → 217.9, isobutyryl shikonin m/z 357.0 → 268.9, β, βʹ-dimethylacryl alkanin m/z 370.0 → 270.1 and esculetin m/z 177.0 → 89.0, respectively. The method was validated for selectivity, linearity, lower limit of quantification, precision, accuracy, recovery, matrix effect and stability. All validation parameters met the acceptance criteria according to regulatory guidelines. This method was successfully applied for the pharmacokinetic study of shikonin, isobutyryl shikonin, β, βʹ-dimethylacryl alkanin in beagle dogs plasma after oral administration of A. euchroma extract.

Conversion of Organic Dyes into Pigments: Extraction of Flavonoids from Blackberries (Rubus ulmifolius) and Stabilization

The blackberry’s color is composed mainly of natural dyes called anthocyanins. Their color is red–purple, and they can be used as a natural colorant. Anthocyanins are flavonoids, which are products of plants, and their colors range from orange and red to various shades of blue, purple and green, according to pH. In this study, the chemical composition of an extract obtained from blackberries was defined by LC-ESI/LTQOrbitrap/MS in positive and negative ionization mode. Furthermore, we investigated the adsorption process of blackberry extract using several inorganic fillers, such as metakaolin, silica, Lipari pumice, white pozzolan and alumina. The pigments exhibit different colors as a function of their interactions with the fillers. The analysis of the absorption data allowed the estimation of the maximum adsorbing capacity of each individual filler tested. Through thermogravimetric measurements (TGA), the thermal stability and the real adsorption of the organic extract were determined.

UHPLC-Orbitrap-MS Tentative Identification of 51 Oleraceins (Cyclo-Dopa Amides) in Portulaca oleracea L. Cluster Analysis and MS2 Filtering by Mass Difference

Oleraceins are a class of indoline amide glycosides found in Portulaca oleracea L. (Portulacaceae), or purslane. These compounds are characterized by 5,6-dihydroxyindoline-2-carboxylic acid N-acylated with cinnamic acid derivatives, and many are glucosylated. Herein, hydromethanolic extracts of the aerial parts of purslane were subjected to UHPLC-Orbitrap-MS analysis, in negative ionization mode. Diagnostic ion filtering (DIF), followed by diagnostic difference filtering (DDF), were utilized to automatically filter out MS data and select plausible oleracein structures. After an in-depth MS2 analysis, a total of 51 oleracein compounds were tentatively identified. Of them, 26 had structures, matching one of the already known oleracein, and the other 25 were new, undescribed in the literature compounds, belonging to the oleracein class. Moreover, based on selected diagnostic fragment ions, clustering algorithms and visualizations were utilized. As we demonstrate, clustering methods provide valuable insights into the mass fragmentation elucidation of natural compounds in complex mixtures.

UHPLC-Orbitrap-HRMS Identification of 51 Oleraceins (Cyclo-Dopa Amides) in Portulaca oleracea L. Cluster Analysis and MS2 Filtering by Mass Difference

Oleraceins are a class of indoline amide glycosides found in Portulaca oleracea L. (Portulacaceae), or purslane. These compounds are characterized with 5,6-dihydroxyindoline-2-carboxylic acid N-acylated with cinnamic acid derivatives, and many are glucosylated. Herein, hydromethanolic extracts of the aerial parts of purslane were subjected to UHPLC-Orbitrap-HRMS analysis, conducted in negative ionization mode. Diagnostic ion filtering (DIF), followed by diagnostic difference filtering (DDF), were utilized to automatically filter out MS data and select plausible oleracein structures. After an in-depth MS2 analysis, a total of 51 oleracein compounds were tentatively identified. Of them, 26 had structure matching one of already known oleraceins and the other 25 are new, undescribed in the literature structures, belonging to the oleracein class. Moreover, diagnostic fragment ions were selected, based on which clustering algorithms and visualizations were employed. As we demonstrate, clustering methods can provide valuable insights into the mass fragmentation elucidation of natural compounds in complex mixtures.

UHPLC-QQQ-MS/MS assay for the quantification of dianthrones as potential toxic markers of Polygonum multiflorum Thunb: applications for the standardization of traditional Chinese medicines (TCMs) with endogenous toxicity

Background The raw and processed roots of Polygonum multiflorum Thunb (PM) are commonly used in clinical practice to treat diverse diseases; however, reports of hepatotoxicity induced by Polygoni Multiflori Radix (PMR) and Polygoni Multiflori Radix Praeparata (PMRP) have emerged worldwide. Thus, it is necessary for researchers to explore methods to improve quality standards to ensure their quality and treatment effects. Methods In the present study, an ultra-high performance liquid chromatography triple quadrupole mass spectrometry (UHPLC-QQQ-MS/MS) method was optimized and validated for the determination of dianthrones in PMR and PMRP using bianthronyl as the internal standard. Chromatographic separation with a gradient mobile phase [A: acetonitrile and B: water containing 0.1% formic acid (v/v)] at a flow rate of 0.25 mL/min was achieved on an Agilent ZORBAX SB-C18 column (2.1 mm × 50 mm, 1.8 μm). The triple quadrupole mass spectrometer (TQMS) was operated in negative ionization mode with multiple reaction monitoring for the quantitative analysis of six dianthrones. Moreover, compounds 5 and 6 were further evaluated for their cytotoxicity in HepaRG cells by CCK-8 assay. Results The UHPLC-QQQ-MS/MS method was first developed to simultaneously determine six dianthrones in PMR and PMRP, namely, polygonumnolides C1–C4 (1–4), trans-emodin dianthrones (5), and cis-emodin dianthrones (6). The contents of 1–6 in 90 batches of PMR were in the ranges of 0.027–19.04, 0.022–13.86, 0.073–15.53, 0.034–23.35, 0.38–83.67 and 0.29–67.00 µg/g, respectively. The contents of 1–6 in 86 batches of commercial PMRP were in the ranges of 0.020–13.03, 0.051–8.94, 0.022–7.23, 0.030–12.75, 0.098–28.54 and 0.14–27.79 µg/g, respectively. Compounds 1–4 were almost completely eliminated after reasonable processing for 24 h and the contents of compounds 5 and 6 significantly decreased. Additionally, compounds 5 and 6 showed inhibitory activity in HepaRG cells with IC50 values of 10.98 and 15.45 μM, respectively. Furthermore, a systematic five-step strategy to standardize TCMs with endogenous toxicity was proposed for the first time, which involved the establishment of determination methods, the identification of potentially toxic markers, the standardization of processing methods, the development of limit standards and a risk–benefit assessment. Conclusion The results of the cytotoxicity evaluation of the dianthrones indicated that trans-emodin dianthrones (5) and cis-emodin dianthrones (6) could be selected as toxic markers of PMRP. Taking PMR and PMRP as examples, we hope this study provides insight into the standardization and internationalization of endogenous toxic TCMs, with the main purpose of improving public health by scientifically using TCMs to treat diverse complex diseases in the future.

Guide to Semi-Quantitative Non-Targeted Screening Using LC/ESI/HRMS

Non-targeted screening (NTS) with reversed phase liquid chromatography electrospray ionization high resolution mass spectrometry (LC/ESI/HRMS) is increasingly employed as an alternative to targeted analysis; however, it is not possible to quantify all compounds found in a sample with analytical standards. As an alternative, semi-quantification strategies are, or at least should be, used to estimate the concentrations of the unknown compounds before final decision making. All steps in the analytical chain, from sample preparation to ionization conditions and data processing can influence the signals obtained, and thus the estimated concentrations. Therefore, each step needs to be considered carefully. Generally, less is more when it comes to choosing sample preparation as well as chromatographic and ionization conditions in NTS. By combining the positive and negative ionization mode, the performance of NTS can be improved, since different compounds ionize better in one or the other mode. Furthermore, NTS gives opportunities for retrospective analysis. In this tutorial, strategies for semi-quantification are described, sources potentially decreasing the signals are identified and possibilities to improve NTS are discussed. Additionally, examples of retrospective analysis are presented. Finally, we present a checklist for carrying out semi-quantitative NTS.

Effect of Agroecological Conditions on Biologically Active Compounds and Metabolome in Carrot

Carrot serves as a source of health-beneficial phytochemicals for human diet whose content is affected by agroecological conditions. The effect of conventional, integrated and organic farming on ascorbic acid (AA) and α,β-carotene levels of new carrot cultivars Cortina F1 and Afalon F1 was investigated and their metabolomic profiles were measured by direct analysis in real time ion source coupled with a high-resolution mass spectrometer (DART-HRMS). Cortina and Afalon exhibited high levels of AA and total carotenes under all agroecological conditions tested that fluctuated in broad ranges of 215–539 and 173–456 mg AA.kg–1 dry biomass and 1069–2165 and 1683–2165 mg carotene.kg–1 dry biomass, respectively. The ratio of β- to α-carotene in both cultivars was about 1.3. The most important variable for the PCA and the partial least squares discriminant analysis (PLS-DA) models for ethyl acetate extracts measured in positive and negative ionization mode was 6-methoxymellein (6-MM). Total carotene content and 6-MM levels were higher in the organic carrot compared to the conventional one and were correlated with a higher level of spontaneous infection. Other important compounds identified were sitosterol, hexose and various organic acids including antioxidant ferulic and coumaric acids. The findings allow comparison of metabolomic profiles and the AA and carotene contents of both cultivars with those of other commercially used carrots.

Untargeted Metabolomic Profiling of Early Diet Energy Intake in C57BL/6 N Mice Reveals Differences Associated with Diet and Aberrant Crypt Foci

Objectives A high-fat (H) diet leads to obesity, a known risk factor for colorectal cancer (CRC). In contrast, calorie restriction (E) is associated with reduced CRC risk. However, the metabolome associated with H vs. E-associated CRC risk has never been directly compared. The different influences of these diets on the proximal (PC), medial (MC), and distal (DC) colon metabolome has also not been studied. Thus, the objective is to elucidate metabolites associated with abberant crypt foci (ACF) number, a marker of CRC risk, in each colon region after consumption of H, E, or a normocaloric control diet (C). Methods 3-week-old C57BL/6 N mice were fed a C, E, or H initiation diet for 13 weeks. In weeks 16–21, animals were injected with azoxymethane to initiate ACF formation, and switched to a C, E, or H progression diet (for a total of 9 diet groups: CC, CH, CE, HH, HC, HE, EE, EC, EH). Polar extracts of the colon regions (i.e., PC, MC, and DC) were analyzed using ultra-high performance liquid chromatography-high resolution mass spectrometry method (HRMS) and 1H NMR metabolomics methods. Linear models assessed the main effects of ACF, initiation diet, progression diet, as well as the diet * ACF interaction, on relative metabolite concentration in each colon region. Results Following HILIC-HRMS analysis of extracts in positive and negative ionization mode, 492 and 415 metabolites were detected, respectively. Linear models revealed 21 metabolites were significantly associated with initiation E diet * ACF (8 unique to MC, 13 unique to PC), 14 with initiation H diet * ACF (only in DC), 27 with progression H diet * ACF (14 unique to DC, 2 to MC, 11 to PC) and 20 with progression E diet * ACF (17 unique to DC, 1 to PC, and 1 common to both). Pathway integration and authentication of tentative metabolite identities with chemical standards is underway. Conclusions Diet * ACF interaction significantly influences multiple metabolite concentrations. Little to no overlap is observed between metabolites associated with ACF in a given colon region and the other regions tested, revealing that the diet * ACF interaction is region-specific. Future studies in humans will determine if these metabolites may serve as early biomarkers for CRC diagnosis. Funding Sources Sample analyses were supported by NIH Award Number Grant P30 CA016058, OSU, and OSUCCC.