Ultra Pressure Liquid Chromatography Research Articles

Lipidomic changes occurring in platelets during extended cold storage.

Cold storage is being implemented as an alternative to conventional room-temperature storage for extending the shelf-life of platelet components beyond 5-7 days. The aim of this study was to characterise the lipid profile of platelets stored under standard room-temperature or cold (refrigerated) conditions. Matched apheresis derived platelet components in 60% PAS-E/40% plasma (n = 8) were stored at room-temperature (20-24°C with agitation) or in the cold (2-6°C without agitation). Platelets were sampled on day 1, 5 and 14. The lipidome was assessed by ultra-pressure liquid chromatography ion mobility quadrupole time of flight mass spectrometry (UPLC IMS QToF). Changes in bioactive lipid mediators were measured by ELISA. The total phospholipid and sphingolipid content of the platelets and supernatant were 44 544 ± 2915 μg/mL and 38 990 ± 10 880 μg/mL, respectively, and was similar over 14 days, regardless of storage temperature. The proportion of the procoagulant lipids, phosphatidylserine (PS) and phosphatidylethanolamine (PE), increased by 2.7% and 12.2%, respectively, during extended cold storage. Cold storage for 14 days increased sphingomyelin (SM) by 4.1% and decreased ceramide by 1.6% compared to day 1. Further, lysophosphatidylcholine (LPC) species remained unchanged during cold storage for 14 days. The concentration of 12- and 15-hydroxyeicosatetraenoic acid (HETE) were lower in the supernatant of cold-stored platelets than room-temperature controls stored for 14 days. The lipid profile of platelets was relatively unchanged during storage for 5 days, regardless of temperature. However, during extended cold storage (14 days) the proportion of the procoagulant lipids, PS and PE, increased, while LPC and bioactive lipids were stable.

A Quality by Design Approach for Developing SNEDDS Loaded with Vemurafenib for Enhanced Oral Bioavailability.

Vemurafenib (VMF) is a practically insoluble (< 0.1μg/mL) and least bioavailable (1%) drug. To enhance its oral bioavailability and solubility, we formulated a reliable self-nano emulsifying drug delivery system (SNEDDS). A Quality by Design (QbD) approach was used to optimize the ratio of Capryol 90, Tween 80, and Transcutol HP. VMF-loaded SNEDDS was characterized for its size, polydispersity index (PDI), zeta potential, drug content, and transmittance. The in vitro release profile of the drug loaded in SNEDDS was compared to the free drug in two media, pH 6.8 and 1.2, and the data obtained were analyzed with different mathematical models. A reverse-phase ultra-pressure liquid chromatography (UPLC) technique with high sensitivity and selectivity was developed and validated for the quantification of VMF in analytical and bioanalytical samples. Dissolution efficiency for SNEDDS was estimated using different models, which proved that the developed novel SNEDDS formulation had a better in vitro dissolution profile than the free drug. A 2.13-fold enhanced oral bioavailability of VMF-loaded SNEDDS compared to the free drug demonstrates the superiority of the developed formulation. This work thus presents an overview of VMF-loaded SNEDDS as a promising alternative to improve the oral bioavailability of the drug.

Relative validity of habitual sugar and low/no-calorie sweetener consumption assessed by food frequency questionnaire, multiple 24-h dietary recalls and urinary biomarkers: an observational study within the SWEET project

BackgroundStudies investigating associations between sweeteners and health yield inconsistent results, possibly due to subjective self-report dietary assessment methods. ObjectivesWe compared the performance of a food frequency questionnaire (FFQ), multiple 24-h dietary recalls (24hRs), and urinary biomarkers to estimate intake of sugars and low/no-calorie sweeteners (LNCSs). MethodsParticipants (n = 848, age 54 ± 12 y) from a 2-y observational study completed 1 semiquantitative FFQ and ≥ 3 nonconsecutive 24hRs. Both methods assessed intake of sugars (mono- and disaccharides, sucrose, fructose, free and added sugars) and sweetened foods and beverages (sugary foods, fruit juice, and sugar or LNCS-containing beverages [sugar-sweetened beverages and low/no-calorie sweetened beverages (LNCSBs)]); 24hRs also included LNCS-containing foods and tabletop sweeteners (low/no-calorie sweetened foods [LNCSFs]). Urinary excretion of sugars (fructose+sucrose) and LNCSs (acesulfame K+sucralose+steviol glucuronide+cyclamate+saccharin) were simultaneously assessed using ultrapressure liquid chromatography coupled to tandem mass spectrometry in 288 participants with 3 annual 24-h urine samples. Methods were compared using, amongst others, validity coefficients (correlations corrected for measurement error). ResultsMedian (interquartile range) FFQ intakes ranged from 0 (0–7) g/d for LNCSBs to 94 (73–117) g/d for mono- and disaccharides. LNCSB use was reported by 32% of participants. Median LNCSB+LNCSF intake using 24hRs was 1 (0–50) g/d and reported by 58%. Total sugar excretions were detected in 100% of samples [56 (37–85) mg/d] and LNCSs in 99% of urine samples [3 (1–10) mg/d]. Comparing FFQ against 24hRs showed VCs ranging from 0.38 (fruit juice) to 0.74 (LNCSB). VCs for comparing FFQ with urinary excretions were 0.25 to 0.29 for sugars and 0.39 for LNCSBs; for 24hR they amounted to 0.31–0.38 for sugars, 0.37 for LNCSBs, and 0.45 for LNCSFs. ConclusionsThe validity of the FFQ against 24hRs for the assessment of sugars and LNCSBs ranged from moderate to good. Comparing self-reports and urine excretions showed moderate agreement but highlighted an important underestimation of LNCS exposure using self-reports.

Variations in the Major Nutrient Composition of Dominant High-Yield Varieties (HYVs) in Parboiled and Polished Rice of Bangladesh.

Rice (Oryza sativa L.) is the principal staple food, a fundamental component of food security, a significant source of energy and major nutrients, and a key player in the overall nutritional status in Bangladesh. Parboiling is a common rice-processing treatment in Bangladesh. Recently, polishing has also become a common practice among millers seeking to attract consumers. Polishing may influence the nutrient composition of rice. The present study aimed to investigate the impact of parboiling and polishing on the nutritional content of the five High Yield Varieties (HYVs) of rice (BR11, BRRI dhan28, BRRI dhan29, BRRI dhan49, and BRRI dhan84) and their percent contributions to the Recommended Dietary Allowances (RDA) of vitamins and minerals. All of the rice samples were analyzed for proximate parameters, vitamins (B1, B2, B3, B6, and folate), and minerals (Ca, Mg, Fe, Zn, Na, K, P). Moisture, ash, fat, and total dietary fiber (TDF) were determined gravimetrically, according to the AOAC Official Methods; protein was measured by the Kjeldahl method; B-group vitamins were measured using Ultra Pressure Liquid Chromatography; and mineral content was determined by ICP-OES. The energy, protein, fat, and total dietary fiber (TDF) content of the samples ranged between 342-357 kcal/100 g, 6.79-10.74 g/100 g, 0.31-1.69 g/100 g, and 2.59-3.92 g/100 g respectively. Thiamin, riboflavin, niacin, pyridoxin, and folate content ranged from 0.11-0.25 mg/100 g, 0.01-0.05 mg/100 g, 2.82-6.42 mg/100 g, 0.12-0.30 g/100 g, and 5.40-23.95 g/100 g respectively. In a comparison of parboiling and polishing, macronutrients and vitamin retention were higher in parboiled unpolished rice than in polished unparboiled rice. The minerals (mg/100 g) Ca, Mg, Fe, Zn, Na, K, and P were in the ranges 32.82-44.72, 30.69-58.34, 0.51-0.70,1.83-2.79, 5.00-5.36, 106.49-112.73, and 162.23-298.03. Minerals of BRRI dhan84 were unaffected by polishing and parboiling. BRRI dhan84 contributed a higher percentage of RDA of all B vitamins and minerals. Therefore, to reduce nutrient loss in rice, industries and consumers should be encouraged to avoid polishing or limit polishing to 10% DOM and to consume unpolished rice, either parboiled or unparboiled.

Identification of polyphenols using UPLC-QTOF MS/MS, in-vitro photoprotective and antiaging activities of brown macroalga Padina tetrastromatica

Brown algae produce several bioactive compounds including unique secondary metabolites like polyphenols, which are known for various pharmacological activities. The present study focused on the cosmeceutical properties of polyphenol-rich fraction from brown macroalga (Padina tetrastromatica). Two fractions (ethyl acetate and aqueous) were obtained by liquid-liquid purification. Total phenolic and phlorotannin contents were assessed by in-vitro methods (Folin-Ciocalteu test and 2,4-dimethoxybenzaldehyde assay). Antioxidant properties were assessed through 2,2-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity, nitric oxide radical scavenging activity, and ferric-reducing antioxidant power assay. Ethyl acetate fraction showed high radical scavenging activity and was used for in-vitro photoprotective assays, cell viability tests, and anti-aging related enzyme inhibition tests (anti-tyrosinase, anti-elastase, anti-collagenase). Ethyl acetate fraction showed potential ultraviolet A absorption and a strong sun protection factor (SPF) value and was not toxic to the human skin cell line up to a concentration of 100 μg mL−1. Interestingly, this fraction also showed a strong tyrosinase inhibition and a significant inhibition of elastase and collagenase enzymes. Major polyphenols including phlorotannins present in the fraction were identified using Fourier transform infrared (FTIR) spectroscopy, Ultra pressure liquid chromatography (UPLC)-quadrupole-time of flight (QTOF) mass spectrometry (MS) and NMR spectroscopy. The findings suggest the ethyl acetate fraction of P. tetrastromatica as a promising source for cosmetic use with photoprotection and anti-aging properties.

Optimization and validation of an extraction method for the analysis of multi-class emerging contaminants in soil and sediment

Analytical methods for the determination of multi-class emerging contaminants are limited for soil and sediment while they are essential to provide a more complete picture of their distribution in the environment and to understand their fate in different environmental compartments. In this paper, we present the development and optimization of an analytical strategy that combines reliable extraction, purification and the analysis using ultra-pressure liquid chromatography triple quadrupole mass spectrometry (UPLC-MS/MS) of 90 emerging organic contaminants including pesticides, pharmaceuticals and personal care products, flame retardants, per- and polyfluoroalkyl substances (PFASs) and plasticizers in soil and sediment. To extract a wide range of chemicals, the extraction strategy is based on the QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) approach. A number of different options were investigated (buffer, acidification, addition of EDTA, different types and combinations of dispersive SPE etc.) and the effectiveness of the chemical extraction procedure and the clean-up was assessed for two matrices: soil (organic matter content of 9%) and sediment (organic matter content of 1.9%). The method was fully validated for both matrices, in terms of accuracy, linearity, repeatability (intra-day), reproducibility (inter-day), method limits of detection and quantification (LODs and MLOQs, respectively). The final performance showed good accuracy and precision (mean recoveries were between 70 and 120% with relative standard deviations (RSD) less than 20% in most cases), low matrix effects, good linearity for the matrix-matched calibration curve (R2≥0.991) and MLOQs ranged from 0.25 and 10 µg/kg. To demonstrate the applicability and suitability of the validated method, soil and sediment samples from Vietnam, France, Sweden and Mexico were analyzed. The results showed that of the 90 target compounds, a total of 33 were quantified in the sediment and soil samples analyzed. In addition to multi-target analysis, this strategy could be suitable for non-target screening, to provide a more comprehensive view of the contaminants present in the samples.

Interactions of legume phenols-rice protein concentrate towards improving vegan food quality: Development of a protein-phenols enriched fruit smoothie

Phenol-protein interaction is considered an effective tool to improve the functional properties of vegan proteins. The present work aimed to evaluate the covalent interaction between kidney bean polyphenols with rice protein concentrate and studied their characteristics for quality improvement in vegan-based foods. The impact of interaction on the techno-functional properties of protein was evaluated and the nutritional composition revealed that kidney bean was rich in carbohydrates. Furthermore, a noticeable antioxidant activity (58.11 ± 1.075 %) due to the presence of phenols (5.5 mg GAE/g) was observed for the kidney bean extract. Moreover, caffeic acid and p-Coumaric acid were confirmed using ultra-pressure liquid chromatography and the amount was 194.43 and 0.9272 mg/kg, respectively. A range of rice protein- phenols complexes (PPC0.025, PPC0.050, PPC0.075, PPC0.1, PPC0.2, PPC 0.5, PPC1) were examined and PPC0.2 and PPC0.5 showed significantly (p < 0.05) higher binding efficiency with proteins via covalent interaction. The conjugation reveals changes in physicochemical properties of rice protein, including, reduced size (178.4 nm) and imparted negative charges (-19.5 mV) of the native protein. The presence of amide Ⅰ, Ⅱ, Ⅲ, was confirmed in native protein and protein-phenol complex with vibration bands, particularly at 3784.92, 1631.07, and 1234 cm−1, respectively. The X-ray diffraction pattern depicted a slight decrease in crystallinity after the complexation and scanning electron microscopy revealed the alteration in morphology from less to improved smoothness and continuous surface characteristics for the complex. Thermo gravimetric analysis revealed high thermal stability of the complex with a maximum weight loss at a temperature range of 400–500 °C. Protein-phenol complex added fruit-based smoothie was developed and it was found to be acceptable in terms of various sensory attributes including color & appearance, textural consistency, and mouthfeel as compared to the control smoothie. Overall, this study provided novel insights to understand the phenol–protein interactions and the possible use of the phenol-rice protein complex in the development of vegan-based food products.

Development and validation of a UPLC-MS/MS method for the quantification of sugars and non-nutritive sweeteners in human urine

High-intensity sweeteners (‘sweeteners’), such as sucralose, saccharine, acesulfame, cyclamate and steviol, are replacing sugars in many food products, but biomarker-based data on their population-wide exposure, as well as analytical methods that can quantify urinary concentrations of sugars and sweeteners simultaneously, are lacking. Here, we developed and validated an ultra-pressure liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) method to quantify glucose, sucrose, fructose, sucralose, saccharine, acesulfame, cyclamate and steviol glucuronide in human urine. Urine samples were prepared by a simple dilution step containing the internal standards in water and methanol. Separation was achieved on a Shodex Asahipak NH2P-40 hydrophilic interaction liquid chromatography (HILIC) column using gradient elution. The analytes were detected using electrospray ionization in negative ion mode, and selective reaction monitoring was optimized using the [M−H]- ions. Calibration curves ranged between 34 and 19,230 ng/mL for glucose and fructose, and 1.8 to 1,026 ng/mL for sucrose and the sweeteners. The method has acceptable accuracy and precision, which depends on the application of appropriate internal standards. Storage of urine samples in lithium monophosphate gives the best overall analytical performance, and storage at room temperature without any preservatives should be avoided since this leads to reduced glucose and fructose concentrations. With the exception of fructose, all analytes were stable throughout 3 freeze–thaw cycles. The validated method was applied to human urine samples, demonstrating quantifiable concentrations of the analytes which were in the expected range. It is concluded that the method has acceptable performance to quantitatively determine dietary sugars and sweeteners in human urine.

Metabolomic Study of Flavonoids in Camellia drupifera under Aluminum Stress by UPLC-MS/MS.

Aluminum (Al) affects the yield of forest trees in acidic soils. The oil tea plant (Camellia drupifera Lour.) has high Al tolerance, with abundant phenolic compounds in its leaves, especially flavonoid compounds. The role of these flavonoids in the Al resistance of oil tea plants is unclear. In this metabolomic study of C. drupifera under Al stress, ultra-pressure liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS) was utilized to identify metabolites, while principal component analysis, cluster analysis, and orthogonal partial least squares discriminant analysis were applied to analyze the data on the flavonoid metabolites. The leaf morphology of C. drupifera revealed significant damage by excess aluminum ions under each treatment compared with the control group. Under Al stress at 2 mmol/L (GZ2) and 4 mmol/L (GZ4), the total flavonoid content in C. drupifera leaves reached 24.37 and 35.64 mg/g, respectively, which are significantly higher than the levels measured in the control group (CK) (p < 0.01). In addition, we identified 25 upregulated and 5 downregulated metabolites in the GZ2 vs. CK comparison and 31 upregulated and 7 downregulated flavonoid metabolites in GZ4 vs. CK. The results demonstrate that different levels of Al stress had a significant influence on the metabolite profile of C. drupifera. It was found that the abundance of the 24 differential flavonoid metabolites was gradually elevated with increasing concentrations of Al stress, including catechin, epicatechin, naringenin-7-glucoside, astilbin, taxifolin, miquelianin, quercitrin, and quercimeritrin. Moreover, the most significant increase in antioxidant activity (about 30%) was observed in C. drupifera precultured in leaf extracts containing 7.5 and 15 μg/mL of active flavonoids. The qRT-PCR results showed that the expression levels of key genes involved in the synthesis of flavonoids were consistent with the accumulation trends of flavonoids under different concentrations of Al. Therefore, our results demonstrate the key role of flavonoid compounds in the oil tea plant C. drupifera in response to Al stress, which suggests that flavonoid metabolites in C. drupifera, as well as other aluminum-tolerant plants, may help with detoxifying aluminum.

Sphingomyelin and Phosphatidylserine Derived Compounds Found into RBCs of Two Unrelated Individuals with Idiopathic Erythrocytosis with Altered Hemoglobin (Hb) Function Properties and No Hemoglobinopathies - Preliminary Results

Sphingomyelin and phosphatidylserine derived compounds were found into Red Blood Cells (RBCs) of two unrelated individuals with idiopathic erythrocytosis, whom were referred to the Laboratory of Hemoglobinopathies - State University of Campinas (State of São Paulo, Brazil) to investigate the presence of hemoglobin (Hb) variants with high oxygen affinity. Both unrelated individuals were 5-year-old age Caucasian Brazilian boys with the following counts: RBC (x 10/6mm3) 6.15 and 7.69, Hb (g/dL) 20.1 and 20, Htc (%) 54 and 58.7, respectively. Most common causes of erythrocytosis, including polycythemia vera (PV) and other secondary erythrocytosis; JAK2, VHL and EPOR mutational screen were previously excluded in the Centro Infantil Boldrini (Campinas, State of São Paulo, Brazil), where the children are followed. No hemoglobin or globin variants were detected by standard methods: alkaline electrophoresis in cellulose acetate (pH 8.9), acidic electrophoresis in agar gel (pH 5.8), cation-exchange HPLC (Variant II, Bio-Rad) and RP-HPLC (Alliance, Waters). Direct genome sequencing of HBA and HBB performed in automated sequencer (ABI PRISM™ 377 DNA Automated Sequencer, Applied BioSystems) after DNA amplification obtained from peripheral leukocytes (QIAamp DNA Blood, Qiagen) did not reveal any mutation. Familial study also did not show any alteration in blood count, hemoglobin or even globin gene mutation in their relatives. Deletional and non-deletional alpha thalassemia was also excluded by Multiplex-gap-PCR and restriction enzyme reaction (Hph and NcoI). Despite of normal results for Hb characterization, functional tests performed with Hb purified by exclusion (Sephadex G-25, Sigma-Aldrich) and ion exchange chromatography (Amberlite MB-3, Sigma-Aldrich) in Hepes buffer indicated some alteration. O2 affinity was evaluated by determining P50 with stripped lysates ([Hb]=70 μM/Heme) in pHs of 6.5, 7.0, 7.5, 8.0 and 8.5, for Bohr effect calculations. Heme-heme cooperativity was determined by Hill coefficient (n) and the activity of allosteric effector in the Hb-O2 ligation was also tested, using inositol hexaphosphate ([IHP]=1mM), a mimetic compound of the intraerythrocytic 2,3-BPG. P50 was slightly decreased (both patients, stripped Hb samples) at extreme acidic and alkaline pH (pH 6.5 and 8.5) leading to altered Bohr effect, in comparison to healthy and erythrocytosis control (caused by cardiopathy, with no Hb variant). P50 was also found decreased at pH 8.5 in the presence of IHP. Heme-heme cooperativity was very decreased at pHs 6.5, 7.0, 8.0 and 8.5, in stripped state as well as in the presence of IHP. In order to investigate the unexpected results mass spectrometry of the purified intraerythrocytic content was performed. RBC lysate was separated from stroma by ultracentrifugation (14.000rpm) and purified by molecular exclusion filtration (Amicon filter - Millipore, 10KDa cutoff) and ion exchange chromatography (Amberlite MB-3, Sigma-Aldrich) for desalinization. Quadrupole Mass spectrometry (MS) with 0.8 ppm MS and MS/MS accuracy (Impact High DefinitionTM, Bruker), preceded by UPLC (Ultra Pressure Liquid Chromatography) system (Shimadzu Nexera) with C18 Polymer RP-column (Supelco apHera C18 Polymer 150 x 2 mm, 5um) was used to separate and identify and compare the compounds to control sample (secondary erythrocytosis with no Hb function alteration). No alteration in peptide, hemoglobin or heme were found. Four small compounds, two derived of sphingomyelin and two derived of phosphatidylserine were identified in both patient samples (C39H79N2O6P, C42H80NO8P, C42H82NO8P, C44H84NO8P) - Figure 1. Sphingomyelin and phosphatidylserine are RBC membrane compounds rich in PO4-, which theoretically could bind b1b2 allosteric 2,3-BPG site in Hb. There is no evidence that these molecules were effectively bound to Hb except that were found in purified Hb samples with some function alteration, particularly with decreased heme-heme cooperativity. Some evidence suggests that analog compounds could be found in higher concentrations in PV or secondary erythrocytosis. To our knowledge it is the first-time that alteration in Hb function was observed in samples with these molecules. These preliminary results should be further validated and at least 10 other similar cases should be better characterized. (Financial support: Fapesp, Faepex-Unicamp, Educorp-Unicamp). Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Potassium penicillin and gentamicin pharmacokinetics in healthy conscious and anesthetized horses.

To determine the effects of general anesthesia on the safety and efficacy of co-administered potassium penicillin G (PEN) and gentamicin (GENT) in horses. Nonrandomized crossover. Six adult, Thoroughbred horses. Horses were administered PEN (22 000 IU/kg IV) and GENT (6.6 mg/kg IV). Plasma samples were collected over a 6 h period and synovial fluid was collected at 30 min and 6 h respectively. Drug administration and sample collection protocols were repeated after at least a 48 hour washout period and induction of anesthesia using xylazine/ketamine and maintenance with isoflurane gas. Drug concentrations were determined using ultrapressure liquid chromatography with mass spectrometry. A 2-compartment model was used to determine pharmacokinetics and differences were determined between conscious and anesthetized horses using paired t-tests (significance P< .05). Potassium penicillin g and GENT had higher minimum plasma concentrations (PEN 0.44 vs. 0.11 μg/mL, P= .002; GENT 3.0 vs. 1.9 μg/mL, P= .009), longer half lives (PEN 71 vs. 59 min, P= .018; GENT 149 vs. 109 min, P= .038), and slower clearances (PEN 3.41 vs. 5.1 mL/kg/min, P= .005; GENT 1.18 vs. 1.48 mL/kg/min, P= .028) in anesthetized horses vs. conscious horses. The PEN concentrations remained above the breakpoint minimum inhibitory concentration (MIC, 0.5 μg/mL) for 332 min in anesthetized vs. 199 min in conscious horses. The GENT concentrations reached 10 times higher than the breakpoint MIC (2 μg/mL) in all horses and were maintained for 58 vs. 59 min in anesthetized and conscious states, respectively. Synovial fluid concentrations were higher in conscious horses vs. anesthetized horses at 30 min for PEN (7.0 vs. 0.93 μg/mL, P< .001) and 30 (5.3 μg/mL vs. 0.79 μg/mL, P< .001) and 360 min (3.4 vs. 1.82 μg/mL, P < .003) for GENT. General anesthesia resulted in lower intrasynovial concentrations and delayed clearance of PEN/GENT in horses. Redosing healthy anesthetized horses with PEN prior to 4-5 h is not necessary. When administered to anesthetized horses, intravenous PEN/GENT may not reach adequate intrasynovial concentrations to treat or prevent common pathogens. The doses or dosing intervals of antimicrobials administered to horses undergoing anesthesia may need to be adjusted to ensure maintenance of safe and effective plasma concentrations.

Associations of perfluoroalkyl substances (PFAS) with non-targeted metabolomics across two independent cohorts of elderly individuals

BACKGROUND AND AIM: Perfluoroalkyl substances (PFAS) have been linked to adverse health effects including increased blood cholesterol levels and hepatic toxicity. Alterations in metabolism and oxidative stress are among the hypothesized mechanisms by which PFAS exposure may exert effects; however, studies of associations between PFAS and metabolome are scarce. The main aim of this study was to assess associations of PFAS exposure and metabolome by using non-targeted metabolomics in two independent cohorts of elderly individuals. METHODS: In two population-based cohorts of elderly individuals from Sweden and California (the Prospective Investigation of the Vasculature in Uppsala Seniors [PIVUS, n = 901, median age 70 years]; and the California Teacher Study [CTS, n = 325, median age 69 years]), plasma/serum PFAS concentrations were determined using isotope-dilution ultra-pressure liquid chromatography (UHPLC) coupled to tandem mass spectrometry. Non-targeted metabolomics was performed using UHPLC high-resolution mass spectrometry (QTOFMS). Associations of PFAS and metabolomics signatures was assessed using a discovery-validation approach with multivariable linear regression. RESULTS: In the discovery cohort 89 annotated metabolites out of 204 could be associated with levels of five PFAS following adjustment for age, sex, and correction for multiple testing. Eleven associations could then be replicated in the CTS. The metabolites being replicated in the CTS were primarily lipid related metabolites such as lysophosphatidylcholine, phosphatidylcholine, sphingomyelin as well as amino acids such as betaine. CONCLUSIONS: Using a non-targeted metabolomics discovery-validation approach, we the previously reported associations between PFAS and lipid related metabolites and identified and replicated novel associations. Our results encourage additional studies investigating the underlying mechanisms of our findings. KEYWORDS: PFAS, PFOS, PFOA, Metabolomics, Lipid metabolism

Analysis of Major Polyphenolic Compounds of Cydonia oblonga Miller (Quince) Fruit Extract by UPLC-MS/MS and Its Effect on Adipogenesis in 3T3-L1 Cells

Cydonia oblonga miller (quince) plant serves as a potential folk medicine for treating hypertension and cardiovascular diseases in China. However, to the best of our knowledge, no study has been conducted on the polyphenolic profile and anti-adipogenic effect of quince fruit grown in China. In the current study, we aimed to investigate the quince fruit extract’s major phenolic compounds, evaluate their antioxidant activity, and examine their effect on adipogenesis in 3T3-L1 cells. A rapid and sensitive analytical method was established for the simultaneous determination of major polyphenolic compounds by using ultra-pressure liquid chromatography coupled with a triple quadrupole mass spectrometer (UPLC-MS/MS). Among the 10 compounds, the cryptochlorgenic acid was noticed as the most abundant compound of both purified (242.44 ± 0.73 µg/mg dw) and unpurified extract (3.37 ± 0.01 µg/mg dw) followed by quercetin 3-rutinoside and chlorogenic acid. Alternatively, both extracts possessed a high quantity of phenolic acids (purified extract = 483.10 ± 5.16 µg/mg dw and unpurified extract = 7.89 ± 0.02 µg/mg dw). The purified extract exhibited a strong antioxidant capacity (DPPH: EC50 = 3.316 µg/mL, ABTS: EC50 = 36.38 µg/mL) as compared to the unpurified extract. Additionally, our results also showed that the extract at 100 µg/mL significantly suppressed the preadipocyte differentiation and decreased the lipid droplets up to 69% in mature adipocytes. The present study highlights an accurate and fast detection method for quince fruit extract polyphenolic compounds with its antioxidant and antiadipogenic effects. The study also provides the necessary information for the rational development and utilization of quince fruit extract as a source of phytochemicals.

Simultaneous Determination of Cortisol and Testosterone from Rat Serum by Liquid Chromatography-Tandem Mass Spectrometry

A liquid chromatography-electrospray ionization tandem mass spectrometry in a positive mode (LC-ESI-MS/MS) was developed to determine simultaneously the levels of cortisol and testosterone in rat serum. The MRM (Multiple Reaction Monitoring) was used to optimize ion precursor, fragmentor voltage, ion product, and collision energy. Analytical samples were treated by solid-phase extraction with Bond Elut C18 cartridge. The method was validated according to FDA guidance for bioanalytical method validation. The results indicated that this method showed a wide linearity range (0.1-100 ng/mL for cortisol and 0.025-100 ng/mL for testosterone), good linearity (r2 &gt; 0.999), low limit of quantification (LLOQ values of cortisol and testosterone were 0.1 ng/mL and 0.025 ng/mL, respectively), good recovery (90.4% - 108.0%), suitable repeatability (CV values were in the range of 2.7% - 14.8%), good stability in different conditions. The validated method was applied to quantify cortisol and testosterone levels in several rat blood samples.&#x0D; Keywords: LC-ESI-MS/MS, cortisol, testosterone, rat serum, solid-phase extraction.&#x0D; References&#x0D; [1] A. V. Rincon et al., Measuring Urinary Cortisol and Testosterone Levels in Male Barbary Macaques: A Comparison of EIA and LC-MS, General and Comparative Endocrinology, Vol. 281, 2019, pp. 117-125, https://doi.org/10.1016/j.ygcen.2019.05.017.[2] D. French, Advances in Bioanalytical Techniques to Measure Steroid Hormones in Serum, Bioanalysis, Vol. 8, No. 11, 2016, pp. 1203-1219, https://doi.org/10.4155/bio-2015-0025.[3] M. Kaleta et al., Analytical Methods for the Determination of Neuroactive Steroids, Biomolecules, Vol. 11, No. 4, 2021, pp. 1-23, https://doi.org/10.3390/biom11040553.[4] M. Závada, K. Šafarčik, O. Topolčan, Some Problems of Radioimmunoassay Control, Journal of Radioanalytical Chemistry, Vol. 46, No. 1, 1978, pp. 57-66.[5] K. S. Leung, B. M. Fong, LC-MS/MS in the Routine Clinical Laboratory: has its time come?, Anal Bioanal Chem, Vol. 406, No. 9-10, 2014, pp. 2289-2301, https://doi.org/10.1007/s00216-013-7542-5.[6] C. Shackleton, Clinical Steroid Mass Spectrometry: a 45-year History Culminating in HPLC-MS/MS Becoming an Essential Tool for Patient Diagnosis, J Steroid Biochem Mol Biol, Vol. 121, No. 3-5, 2010, pp. 481-490, https://doi.org/10.1016/j.jsbmb.2010.02.017.[7] K. Yamashita et al., Development of Sensitive Derivatization Method for Aldosterone in Liquid Chromatography–Electrospray Ionization tandem Mass Spectrometry of Corticosteroids, Journal of Chromatography A, Vol. 1200, No. 2, 2008, pp. 114-121, https://doi.org/10.1016/j.chroma.2008.05.034.[8] K. Yamashita et al., Highly Sensitive Determination of Estrone and Estradiol in Human Serum by Liquid Chromatography–Electrospray Ionization tandem Mass Spectrometry, Steroids, Vol. 72, No. 11-12, 2007, pp. 819-827, https://doi.org/10.1016/j.steroids.2007.07.003.[9] K. Yamashita et al., Development of Highly Sensitive Quantification Method for Testosterone and Hihydrotestosterone in Human Serum and Prostate Tissue by Liquid Chromatography-Electrospray Ionization Tandem Mass Spectrometry, Steroids, Vol. 74, No. 12, 2009, pp. 920-926, https://doi.org/10.1016/j.steroids.2009.06.007.[10] U. S. Department of Health and Human Services Food and Drug Administration, Center for Drug Evaluation and Research, Center for Veterinary Medicine, Bioanalytical Method Validation - Guidance for Industry, 2018.[11] M. Toukh, S. P. Gordon, M. Othman, Construction Noise Induces Hypercoagulability and Elevated Plasma Corticosteroids in Rats, Clin Appl Thromb Hemost, Vol. 20, No.7, 2014, pp. 710-715, https://doi.org/10.1177/1076029613483168.[12] L. H. Heywood, Testosterone Levels in the Male Laboratory Rat: Variation under Experimental Conditions, Int J Androl, Vol. 3, No. 5, 1980, pp. 519-529, https://doi.org/10.1111/j.1365-2605.1980.tb00140.x.[13] L. J. Mentzel, G. Wiedemann, Establishment of Reference Ranges for Cortisol in Neonates, Infants, Children and Adolescents, Eur. J. Clin. Chem. Clin. Biochem, Vol 31, No. 8, 1993, pp. 525-529, https://doi.org/10.1515/cclm.1993.31.8.525.[14] A. E. Kulle et al., A Novel Ultrapressure Liquid Chromatography Tandem Mass Spectrometry Method for the Simultaneous Determination of Androstenedione, Testosterone, and Dihydrotestosterone in Pediatric Blood Samples: Age- and Sex-specific Reference Data, J Clin Endocrinol Metab, Vol. 95, No. 5, 2010, pp. 2399-2409, https://doi.org/10.1210/jc.2009-1670.[15] A. L. Southren et al., Plasma Production Rates of Testosterone in Normal Adult Men and Women and in Patients with the Syndrome of Feminizing Testes, The Journal of Clinical Endocrinology &amp; Metabolism, Vol. 25, No. 11, 1965, pp. 1441-1450, https://doi.org/10.1210/jcem-25-11-1441.[16] P. Pearlmutter et al., Sweat and Saliva Cortisol Response to Stress and Nutrition Factors, Scientifc Reports, 2020, pp. 1-11, https://doi.org/10.1038/s41598-020-75871-3.[17] S. Clifton et al., Salivary Testosterone Levels and Health Status in Men and Women in the British General Population: Findings from the Third National Survey of Sexual Attitudes and Lifestyles (Natsal-3), J Clin Endo Metab, Vol. 101, No. 11, pp. 3939-51, https://doi.org/10.1210/jc.2016-1669.[18] J. Y. Moon et al., Reference Ranges for Urinary Levels of Testosterone and Epitestosterone, Which may Reveal Gonadal Function, in a Korean Male Population, The Journal of Steroid Biochemistry and Molecular Biology, Vol. 140, 2014, pp. 100-105, https://doi.org/10.1016/j.jsbmb.2013.12.001.

Highly Sensitive Simultaneous Measurement of Steroid Hormone in Human Serum by Liquid Chromatography-electrospray Ionization Tandem Mass Spectrometry

Highly Sensitive Simultaneous Measurement of Steroid Hormone in Human Serum by Liquid Chromatography-electrospray Ionization Tandem Mass Spectrometry

Metabolomics Study of Flavonoids of Taxilluschinensis on Different Hosts Using UPLC-ESI-MS/MS.

The goal of this study was to identify and compare the main biomarkers of Taxillus chinensis from different hosts. A metabolomics approach utilizing ultra-pressure liquid chromatography coupled with tandem mass spectrometry (UPLC-MS), including cluster analysis, sample correlation analysis and orthogonal partial least squares discriminant analysis, was used to explore the flavonoid metabolites of Taxillus chinensis growing on different hosts. Results: The total flavonoids content (up to 30.08 mg/g) in Taxillus chinensis from Morus alba (CSG) was significantly higher than that from growth on Liquidambar formosana (CFG) or Clausena lansium (CHG) (p < 0.01). There were 23 different metabolites between CSG and CHG, 23 different metabolites between CSG and CFG, and 19 different metabolites between CHG and CFG. The results demonstrated that different hosts exerted a large influence on the metabolites of Taxillus chinensis; it was found that CSG differed from CFG and CHG in eleven metabolic compounds, ten of which were upregulated and one of which was downregulated. Most of these metabolites derive from compounds contained in the host plant, white mulberry (Morus alba); many feature potent anti-cancer effects. Differences in host can influence the type and abundance of flavonoids in parasitic plants such as Taxillus chinensis, which is of great significance to researchers seeking to understand the formation mechanism of Taxillus chinensis metabolites. Therefore, attention should be paid to the species of host plant when studying the Taxillus chinensis metabolome. Plants grown on Morus alba offer the greatest potential for the development of new anti-cancer drugs.

Long-term elution of bisphenol A from dental composites

ObjectivesBPA release from composites on the short term has been reported in several in-vitro and in-vivo studies. However, it remains unclear whether these materials also leach BPA on the long term. Even though composites may release various (BPA-based) methacrylate monomers up to one year, quantitative data about BPA have not been reported due to the lack of a sensitive method to accurately quantify low levels of BPA. In this context, the aim of the study was to quantify the one-year release of BPA with an optimized analytical method. MethodsComposite disks (n = 6, 6 mm diameter and 2 mm height) from four commercial materials (G-ӕnial Posterior, Venus, Ceram.x mono and Filtek Supreme XTE) were immersed in 1 mL of water or ethanol as extraction solvent and stored in the dark at 37 °C. The extraction solvent was renewed weekly for a period of 52 weeks. Samples were derivatized with pyridine-3-sulfonyl chloride before analysis with ultra-pressure liquid chromatography tandem mass spectrometry (UPLC-MS/MS). ResultsDerivatizing BPA increased the sensitivity of the analytical method and allowed accurate quantification of very low levels of BPA (i.e. 0.78 pmol BPA). BPA eluted continuously in ethanol from all four tested composites over a period of one year. BPA elution was clearly higher when ethanol was used as extraction solution. In water, BPA eluted could be detected up to one year, but levels could not be accurately quantified anymore after several weeks. SignificanceComposites can be considered as a potential long-term source of BPA, and thus should not be neglected when assessing the overall exposure to endocrine disrupting chemicals.

Pharmacokinetic characteristics of psoralen,isopsoralen,psoralenoside and isopsoralenoside in rats after oral administration of Psoraleae Fructus extract

Coumarins are the main active components in Psoraleae Fructus. To study the multi-component pharmacokinetics of Psoraleae Fructus, this study established a sensitive and rapid ultra-pressure liquid chromatography coupled to tandem mass spectrometry(UPLC-MS/MS) method for simultaneous determination of psoralen, isopsoralen, psoralenoside, and isopsoralenoside in rat plasma. After validation, the method was applied to the investigation of pharmacokinetics of psoralen, isopsoralen, psoralenoside, and isopso-ralenoside in rats after single and multiple administration of Psoraleae Fructus extract. The results revealed that the exposure of psoralen and isopsoralen in rat plasma was high after a single intragastric administration of Psoraleae Fructus extract, with an AUC_(0-∞) of 443 619-582 680 and 167 314-276 903 ng·mL~(-1)·h~(-1), respectively. Compared with these two compounds, the exposure of psoralenoside and isopsoralenoside was lower with marked gender difference. After 7-day administration of Psoraleae Fructus extract to rats, the AUC_(0-∞) of psoralen and isopsoralen was 29 701-81 783 and 39 234-89 914 ng·mL~(-1)·h~(-1), respectively, which was significantly lower than that at the first day(P&lt;0.05), and that of psoralenoside and isopsoralenoside was 7 360-19 342 and 8 823-45 501 ng·mL~(-1)·h~(-1), respectively. There was no significant gender difference in exposure of psoralenoside and isopsoralenoside in male and female rats. However, the exposure of psoralenoside and isopsoralenoside in male rats was reduced(P&lt;0.05), and the t_(1/2) and mean residence time(MRT) were shortened, suggesting that the removal of these two compounds from the body was accelerated.

Determination of twenty herbicides in blood by ultrapressure liquid chromatography–tandem mass spectrometry

A sensitive and rapid method for the simultaneous determination of twenty herbicides (aclonifen, lactofen, terbutryn, butylate, carbetamide, fluazifop-P-butyl, propanil, prometryn, isoproturon, terbumeton, pretilachlor, pendimethalin, cycloxydim, tri-allate, metolachlor, diuron, alloxydim, prosulfuron, triflusulfuron-methyl, and acetochlor) in human blood is reported herein. Liquid-liquid extraction coupled with ultra-pressure liquid chromatography-tandem mass spectrometry was employed for the simultaneous analysis of all compounds in 15 min. Validation parameters were studied through the estimation of the limits of detection and quantification, calibration curves, sensitivity, spiked recovery and precision. The limits of detection ranged from 0.1 to 1.0 ng/mL. The limits of quantification ranged from 0.5 to 2.0 ng/mL. Good linearity was obtained for all compounds with R2> 0.99 in all cases. Furthermore, interday precision (< 15%) and intraday precision (< 15%) were shown to be satisfactory. Recoveries in spiked blood samples were evaluated, and acceptable values (88.0%~108.8%) were found. Finally, this method was successfully applied to the determination of fluazifop-P-butyl, isoproturon and acetochlor in blood samples from real forensic cases. These results suggest that this method is reliable for rapid forensic and clinical diagnosis.

Interactive effects of aging and aerobic capacity on energy metabolism\u2013related metabolites of serum, skeletal muscle, and white adipose tissue

Aerobic capacity is a strong predictor of longevity. With aging, aerobic capacity decreases concomitantly with changes in whole body metabolism leading to increased disease risk. To address the role of aerobic capacity, aging, and their interaction on metabolism, we utilized rat models selectively bred for low and high intrinsic aerobic capacity (LCRs/HCRs) and compared the metabolomics of serum, muscle, and white adipose tissue (WAT) at two time points: Young rats were sacrificed at 9 months of age, and old rats were sacrificed at 21 months of age. Targeted and semi-quantitative metabolomics analysis was performed on the ultra-pressure liquid chromatography tandem mass spectrometry (UPLC-MS) platform. The effects of aerobic capacity, aging, and their interaction were studied via regression analysis. Our results showed that high aerobic capacity is associated with an accumulation of isovalerylcarnitine in muscle and serum at rest, which is likely due to more efficient leucine catabolism in muscle. With aging, several amino acids were downregulated in muscle, indicating more efficient amino acid metabolism, whereas in WAT less efficient amino acid metabolism and decreased mitochondrial β-oxidation were observed. Our results further revealed that high aerobic capacity and aging interactively affect lipid metabolism in muscle and WAT, possibly combating unfavorable aging-related changes in whole body metabolism. Our results highlight the significant role of WAT metabolism for healthy aging.