Newborn screening by matrix-assisted laser desorption/ionization mass spectrometry based on parylene-matrix chip.

Newborn screening for diagnosis of phenylketonuria, homocystinuria, and maple syrup urine disease have been conducted by analyzing the concentration of target amino acids using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF MS) based on parylene-matrix chip. Parylene-matrix chip was applied to MALDI-ToF MS analysis reducing the matrix peaks significantly at low mass-to-charge ratio range (m/z < 500). Reproducibility of inter-spot and intra-spot analyses of amino acids was less than 10%. Methanol extraction was adopted for simple and rapid sample preparation of serum before mass spectrometric analysis showing 13.3 to 45% of extraction efficiency. Calibration curves for diagnosis of neonatal metabolic disorders were obtained by analyzing methanol-extracted serum spiked with target amino acids using MALDI-ToF MS. They showed good linearity (R2 > 0.98) and the LODs were ranging from 9.0 to 22.9 µg/mL. Effect of proteins in serum was estimated by comparing MALDI-ToF mass spectra of amino acids-spiked serum before and after the methanol extraction. Interference of other amino acids on analysis of target analyte was determined to be insignificant. From these results, MALDI-ToF MS based on parylene-matrix chip could be applicable to medical diagnosis of neonatal metabolic disorders.

Polyphenolic Profiling and Antioxidant Activity in Berry Extracts of Pyracantha Wild Varieties from the Mediterranean Region.

Pyracantha is a genus of wild perennial shrubs native in an area extending from Southwest Europe to Southeast Asia, and it is used in traditional medicine for the diuretic, cardiac, and tonic properties of its fruits, which can also be cooked to make jellies, jams, and sauces. This work aims to study and compare the antioxidant activity and the phenolic and anthocyanin composition of three varieties of Pyracantha coccinea: Red Column (PCR), Orange Glow (PCO), and Soleil d'Or (PCS), and one of Pyracantha angustifolia: Orange glow (PAO), collected from the spontaneous flora of the Mediterranean region (Southern Italy). Two different extraction processes were tested using methanol and an aqueous methanol solution (80% MeOH) to evaluate the polyphenolic content and antioxidant activity of freeze-dried berries. The highest total phenolic content was found in PCR and PAO berries (174.21 ± 0.149 and 168.01 ± 0.691 mg of gallic acid equivalent per gram of dry matter, respectively) extracted with an aqueous methanol solution (80% MeOH). Polyphenolic extracts analyzed by HPLC-DAD-ESI/MS confirmed the presence of rutin, quercetin hexose, neoeriocitrin, procyanidin B, and resveratrol. Moreover, the total antioxidant activity of the berries' extracts was measured by comparing two different spectrophotometric methods (ABTS and DPPH), showing that the varieties with the highest total phenolic content, PCR and PAO, also had the highest scavenging activity. Finally, a suitable extraction process was chosen for the evaluation of the anthocyanins' composition of all frozen berries, and in all MS spectra of Pyracantha varieties, two ionic species at 449 m/z attributable to two cyanidin derivatives were found.

UPLC-Q-TOF-MS, network analysis, and molecular docking to investigate the effect and active ingredients of tea-seed oil against bacterial pathogens.

Object: This research intended to probe the antibacterial effect and pharmacodynamic substances of Tea-Seed Oil (TSO) through the use of ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS) analysis, network analysis, and molecular docking. Methods: The major chemical components in the methanol-extracted fractions of TSO were subjected to UPLC-Q-TOF-MS. Network pharmacology and molecular docking techniques were integrated to investigate the core components, targets, and potential mechanisms of action through which the TSO exert their antibacterial properties. To evaluate the inhibitory effects, the minimum inhibitory concentration and diameter of the bacteriostatic circle were calculated for the potential active ingredients and their equal ratios of combinatorial components (ERCC) against Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans. Moreover, the quantification of the active constituents within TSO was achieved through the utilization of high-performance liquid chromatography (HPLC). Results: The methanol-extracted fractions contained a total of 47 chemical components, predominantly consisting of unsaturated fatty acids and phenolic compounds. The network pharmacology analysis and molecular docking analysis revealed that various components, including gallocatechin, gallic acid, epigallocatechin, theophylline, chlorogenic acid, puerarin, and phlorizin, have the ability to interact with critical core targets such as serine/threonine protein kinase 1 (AKT1), epidermal growth factor receptor (EGFR), a monoclonal antibody to mitogen-activated protein kinase 14 (MAPK14), HSP90AA1, and estrogen receptor 1 (ESR1). Furthermore, these components can modulate the phosphatidylinositol-3-kinase protein kinase B (PI3K-AKT), estrogen, MAPK and interleukin 17 (IL-17) signaling pathways, hereby exerting antibacterial effects. In vitro validation trials have found that seven components, namely gallocatechin, gallic acid, epigallocatechin, theophylline, chlorogenic acid, puerarin, and phloretin, displayed substantial inhibitory effects on E. coli, S. aureus, P. aeruginosa, and C. albicans, and are typically present in tea oil, with a total content ranging from 15.87∼24.91 µg·g-1. Conclusion: The outcomes of this investigation possess the possibility to expand our knowledge base concerning the utilization of TSO, furnish a theoretical framework for the exploration of antibacterial drugs and cosmetics derived from inherently occurring TSO, and establish a robust groundwork for the advancement and implementations of TOS products within clinical settings.

Study of the Chemical Composition and Biologically Active Properties of Glycyrrhiza glabra Extracts.

Glycyrrhiza glabra or licorice has long been known as a commonly used Ayurvedic herb. This study aims to investigate the effect of extraction methods on the chemical composition and biologically active properties of Glycyrrhiza glabra extract samples. The highest yield of the Glycyrrhiza glabra extract (21.31 ± 0.64 wt.%) was produced using the Soxhlet extraction method with methanol. The highest concentrations of biologically active substances (3,4-dihydroxybenzoic acid, n-coumaric acid, luteolin-7-glucoside, acacetin, apigenin-7-O-glucoside, chicoric acid, and hesperetin) were found in these samples of Glycyrrhiza glabra extracts. When applying the maceration method using a mixture of solvents methanol-NaOH, rosmarinic acid was identified, and catechin was found in large quantities with a mixture of methanol-trifluoroacetic acid (TFA). Growth inhibition zones were determined for Escherichia coli (13.6 ± 0.41 mm), Pseudomonas aeruginosa (10.8 ± 0.32 mm), Bacillus subtilis (16.1 ± 0.48 mm), and Candida albicans (13.2 ± 0.39 mm) when exposed to samples of Glycyrrhiza glabra extracts obtained by the Soxhlet method with methanol. The antioxidant activity of Glycyrrhiza glabra extract samples obtained by the Soxhlet method was 117.62 ± 7.91 µmol Trolox equivalent/g, using the ABTS method (highest value), and 23.91 ± 1.12 µmol Trolox equivalent/g according to the FRAP method (smallest). The antioxidant activity of the extract samples according to the DPPH method was an intermediate value of 58.16 ± 3.90 µmol Trolox equivalent/g. Antibacterial and antioxidant activities are manifested by the polyphenolic compounds and flavonoids contained in the samples of the methanol extract of Glycyrrhiza glabra produced using the Soxhlet method. These Glycyrrhiza glabra extract samples have the potential to become a natural alternative to existing therapies for the elimination of bacterial infections or the prevention of premature aging caused by free radicals and oxidative stress in the human body.

Effect of solvent type on ultrasound-assisted extraction of antioxidant compounds from Ficaria kochii: Optimization by response surface methodology.

The optimization of extraction conditions to recover antioxidant rich extract from plant of Ficaria Kochii with an ultrasound-assisted system was achieved by response surface methodology. The rotatable central composite design (RCCD) was used to optimize extraction parameters in terms of total phenolic and total flavonoid content and antioxidant activity. The optimum conditions for extraction by methanol were: ratio of solvent to raw material, 10%; extraction time, 50 min and extraction temperature, 60 °C. Under these conditions, the obtained total phenolic content 46.379 mg gallic acid equivalents/g dry matter and total flavonoid content 11.754 mg quercetin equivalents/g dry matter with antioxidant property (DPPH: the 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity) was 89.548%. Also, the optimum operating conditions for extraction by water were: ratio of solvent to raw material, 10%; extraction time, 30 min and extraction temperature, 40 °C. Under these conditions, the obtained total phenolic content 51.112 mg gallic acid equivalents/g dry matter and total flavonoid content 4.331 mg quercetin equivalents/g dry matter with antioxidant property was 97.004%. This study suggests that the antioxidant rich extract from plant of Ficaria Kochii can be a candidate for food antioxidant and Due to these features, this extract can be used in food storage.

Extraction of Metabolites from Cancer Cells.

Cancer cells possess an elevated demand for nutrients and metabolites due to their uncontrolled proliferation and need to survive in unfavorable conditions. Autophagy is a conservative degradation pathway that counters lack of nutrients and provides organelle and protein quality control, beyond maintenance of cellular metabolism.Mass spectrometry-based metabolomics is a powerful tool to study the metabolome of a cell. Such analysis requires proper sample preparation including the extraction of metabolites. Here, we provide a protocol for the extraction of metabolites from adherent cancer cells suitable for global metabolome profiling by mass spectrometry.

Adapting an Ergosterol Extraction Method with Marine Yeasts for the Quantification of Oceanic Fungal Biomass.

Ergosterol has traditionally been used as a proxy to estimate fungal biomass as it is almost exclusively found in fungal lipid membranes. Ergosterol determination has been mostly used for fungal samples from terrestrial, freshwater, salt marsh- and mangrove-dominated environments or to describe fungal degradation of plant matter. In the open ocean, however, the expected concentrations of ergosterol are orders of magnitude lower than in terrestrial or macrophyte-dominated coastal systems. Consequently, the fungal biomass in the open ocean remains largely unknown. Recent evidence based on microscopy and -omics techniques suggests, however, that fungi contribute substantially to the microbial biomass in the oceanic water column, highlighting the need to accurately determine fungal biomass in the open ocean. We performed ergosterol extractions of an oceanic fungal isolate (Rhodotorula sphaerocarpa) with biomass concentrations varying over nine orders of magnitude. While after the initial chloroform-methanol extraction ~87% of the ergosterol was recovered, a second extraction recovered an additional ~10%. Testing this extraction method on samples collected from the open Atlantic Ocean, we successfully determined ergosterol concentrations as low as 0.12 pM. Thus, this highly sensitive method is well suited for measuring fungal biomass from open ocean waters, including deep-sea environments.

Modelling and kinetic study of microwave assisted drying of ginger and onion with simultaneous extraction of bioactive compounds.

Onion and ginger are rich sources of bioactive compounds which are lost during conventional drying process. The present study was designed to optimize the novel Microwave Assisted Drying and Extraction technique (MADE) for simultaneous drying and extraction/recovery of bioactive compounds from model food products. The time required for drying of samples was 11 (onion) and 16 (ginger) minutes with recovery yield of 87% (onion) and 85% (ginger). The drying time was reduced to 100 times compared to hot air drying and moisture ratio of dried samples was best described by Midilli model. The diffusivities of onion and ginger slices were 1.27 e-11 and 1.43 e-11 m2/s, respectively. Moreover, microwave-based extraction was compared with conventional one. The results of antioxidant activity and total phenolic contents of condensates obtained through MADE were higher compared to conventional method. In short, MADE exhibited better yield of extraction and drying properties compared to conventional methods.

Subcritical Methanol Extraction of the Stone of Japanese Apricot Prunus mume Sieb. et Zucc.

The pits of Japanese apricot, Prunus mume Sieb. et Zucc., which are composed of stones, husks, kernels, and seeds, are unused by-products of the processing industry in Japan. The processing of Japanese apricot fruits generates huge amounts of waste pits, which are disposed of in landfills or, to a lesser extent, burned to form charcoal. Mume stones mainly consist of cellulose, hemicellulose, and lignin. Herein, we attempted to solubilize the wood-like carapace (stone) encasing the pit by subcritical fluid extraction with the aim of extracting useful chemicals. The characteristics of the main phenolic constituents were elucidated by liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR) analyses. The degrees of solubility for various treatments (190 °C; 3 h) were determined as follows: subcritical water (54.9%), subcritical 50% methanol (65.5%), subcritical 90% methanol (37.6%), subcritical methanol (23.6%), and subcritical isopropyl alcohol (14.4%). Syringaldehyde, sinapyl alcohol, coniferyl alcohol methyl ether, sinapyl alcohol methyl ether, 5-(hydroxymethyl)-2-furfural, and furfural were present in the subcritical 90% methanol extract. Coniferyl and sinapyl alcohols (monolignols) are source materials for the biosynthesis of lignin, and syringaldehyde occur in trace amounts in wood. Our current findings provide a solubilization method that allows the main phenolic constituents of the pits to be extracted under mild conditions. This technique for obtaining subcritical extracts shows great potential for further applications.

Uncertainties in thermal-optical measurements of black carbon: Insights from source and ambient samples.

Black carbon (BC) is important due to its complex influences on the environment and on climate in particular. However, reported BC data are largely dependent on measurement techniques due to the multitude of measurement principles. Here we focused on thermal-optical method which has been widely used to determine BC mass (as elemental carbon, EC). Several factors influencing EC measurement were investigated. Results from source samples representing vehicle engine emissions pointed to a continuum of EC components in thermal stability and provided direct observational evidence for the premature evolution of EC in inert atmosphere. It was also found that EC masses may be substantially underestimated for the vehicle exhaust samples if the adopted protocol requires an oxidizing atmosphere to define the split point between organic carbon (OC) and EC. Results from a field campaign conducted during winter in Beijing showed that the optical attenuation (ATN; i.e., the filter transmittance signal, I) was largely saturated for the samples with relatively high loadings, indicating their EC results were unreliable. Improved measurement of EC was achieved by extracting these heavily loaded filters using methanol, given that ATN was considerably reduced by the extraction and, moreover, saturation of ATN (or I) became not evident for the extracted samples. The methanol extraction also significantly reduced the transformation of OC to char-OC, by removing the majority (i.e., ~85%) of the deposited organic aerosols. Higher EC were measured for the extracted samples compared with the untreated ones, indicating that EC tends to be underestimated due to the charring-induced uncertainties. In addition, the methanol extraction largely reduced the inter-protocol discrepancy in the EC measurement results. Similar effects of methanol extraction have been observed during summer in Beijing, despite the seasonal variations of aerosol sources and compositions. This study indicates the potential benefits of methanol extraction for EC measurement.

Breast Cancer Metabolomics Using NMR.

Continued progress is being made in understanding the breast cancer metabolism using analytical magnetic resonance (MR)-based methods like nuclear magnetic resonance (NMR) and in-vivo MR spectroscopy (MRS). Analyses using these methods have enhanced the knowledge of altered biochemical pathways associated with breast cancer progression, regression, and pathogenesis. Comprehensive metabolic profiling of biological samples like tissues, cell lines, fine needle aspirate, and biofluids such as sera and urine enables identification of new biomarkers and abnormalities in biochemical pathways. These methods are not only useful for diagnosis, therapy monitoring, disease progression, and staging of cancer but also for the identification of new therapeutic targets and designing new treatment strategies. Additionally, in-vivo MRS studies have established choline-containing compounds (tCho) as biomarkers of malignancy, which is useful for enhancing the diagnostic specificity of magnetic resonance imaging (MRI). Recent technological developments related to in-vivo MRS such as increased magnetic field strength, multichannel phased array breast coils, and absolute quantification of tCho have provided a better understanding of the tumor heterogeneity, metabolism, and pathogenesis. This chapter focuses on providing the experimental aspects of in-vitro, ex-vivo, and in-vivo MR spectroscopy methods used for metabolomics studies of breast cancer.

An Efficient Single Phase Method for the Extraction of Plasma Lipids.

Lipidomic approaches are now widely used to investigate the relationship between lipid metabolism, health and disease. Large-scale lipidomics studies typically aim to quantify hundreds to thousands of lipid molecular species in a large number of samples. Consequently, high throughput methodology that can efficiently extract a wide range of lipids from biological samples is required. Current methods often rely on extraction in chloroform:methanol with or without two phase partitioning or other solvents, which are often incompatible with liquid chromatography electrospray ionization-tandem mass spectrometry (LC ESI-MS/MS). Here, we present a fast, simple extraction method that is suitable for high throughput LC ESI-MS/MS. Plasma (10 µL) was mixed with 100 µL 1-butanol:methanol (1:1 v/v) containing internal standards resulting in efficient extraction of all major lipid classes (including sterols, glycerolipids, glycerophospholipids and sphingolipids). Lipids were quantified using positive-ion mode LC ESI-MS/MS. The method showed high recovery (>90%) and reproducibility (%CV < 20%). It showed a strong correlation of all lipid measures with an established chloroform:methanol extraction method (R2 = 0.976). This method uses non-halogenated solvents, requires no drying or reconstitution steps and is suitable for large-scale LC ESI-MS/MS-based lipidomic analyses in research and clinical laboratories.

High-resolution delineation of chlorinated volatile organic compounds in a dipping, fractured mudstone: Depth- and strata-dependent spatial variability from rock-core sampling.

Synthesis of rock-core sampling and chlorinated volatile organic compound (CVOC) analysis at five coreholes, with hydraulic and water-quality monitoring and a detailed hydrogeologic framework, was used to characterize the fine-scale distribution of CVOCs in dipping, fractured mudstones of the Lockatong Formation of Triassic age, of the Newark Basin in West Trenton, New Jersey. From these results, a refined conceptual model for more than 55years of migration of CVOCs and depth- and strata-dependent rock-matrix contamination was developed. Industrial use of trichloroethene (TCE) at the former Naval Air Warfare Center (NAWC) from 1953 to 1995 resulted in dense non-aqueous phase liquid (DNAPL) TCE and dissolved TCE and related breakdown products, including other CVOCs, in underlying mudstones. Shallow highly weathered and fractured strata overlie unweathered, gently dipping, fractured strata that become progressively less fractured with depth. The unweathered lithology includes black highly fractured (fissile) carbon-rich strata, gray mildly fractured thinly layered (laminated) strata, and light-gray weakly fractured massive strata. CVOC concentrations in water samples pumped from the shallow weathered and highly fractured strata remain elevated near residual DNAPL TCE, but dilution by uncontaminated recharge, and other natural and engineered attenuation processes, have substantially reduced concentrations along flow paths removed from sources and residual DNAPL. CVOCs also were detected in most rock-core samples in source areas in shallow wells. In many locations, lower aqueous concentrations, compared to rock core concentrations, suggest that CVOCs are presently back-diffusing from the rock matrix. Below the weathered and highly fractured strata, and to depths of at least 50 meters (m), groundwater flow and contaminant transport is primarily in bedding-plane-oriented fractures in thin fissile high-carbon strata, and in fractured, laminated strata of the gently dipping mudstones. Despite more than 18 years of pump and treat (P&T) remediation, and natural attenuation processes, CVOC concentrations in aqueous samples pumped from these deeper strata remain elevated in isolated intervals. DNAPL was detected in one borehole during coring at a depth of 27 m. In contrast to core samples from the weathered zone, concentrations in core samples from deeper unweathered and unfractured strata are typically below detection. However, high CVOC concentrations were found in isolated samples from fissile black carbon-rich strata and fractured gray laminated strata. Aqueous-phase concentrations were correspondingly high in samples pumped from these strata via short-interval wells or packer-isolated zones in long boreholes. A refined conceptual site model considers that prior to P&T remediation groundwater flow was primarily subhorizontal in the higher-permeability near surface strata, and the bulk of contaminant mass was shallow. CVOCs diffused into these fractured and weathered mudstones. DNAPL and high concentrations of CVOCs migrated slowly down in deeper unweathered strata, primarily along isolated dipping bedding-plane fractures. After P&T began in 1995, using wells open to both shallow and deep strata, downward transport of dissolved CVOCs accelerated. Diffusion of TCE and other CVOCs from deeper fractures penetrated only a few centimeters into the unweathered rock matrix, likely due to sorption of CVOCs on rock organic carbon. Remediation in the deep, unweathered strata may benefit from the relatively limited migration of CVOCs into the rock matrix. Synthesis of rock core sampling from closely spaced boreholes with geophysical logging and hydraulic testing improves understanding of the controls on CVOC delineation and informs remediation design and monitoring.

Rapid and sensitive determination of propofol glucuronide in hair by liquid chromatography and tandem mass spectrometry.

A fast, sensitive and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the detection and quantitation of propofol glucuronide in human hair has been developed and validated. Propofol glucuronide was extracted from 10mg of hair using a simple methanol extraction method, with recovery greater than 91% at 3 quality control samples (15, 100, 4000 pg/mg). A reversed phase column (C8) was used to analyze and the mobile phase was composed of ammonium formate and acetonitrile gradient at a flow rate of 0.2 mL/min. The lower limit of quantitation (LLOQ) was 5 pg/mg and the assay was linear to 5000 pg/mg. The intra- and inter-day precision (% CV, coefficient of variation) ranged from 1.26 to 4.50% while the accuracy (% RE, relative error) were -4.24 to 4.4%. The matrix effects were monitored at 3 different concentrations and the %CV of the results for these concentrations was less than 10.6%. Propofol glucuronide was stable during processing and analysis in human hair. The procedure was validated and applied to the analysis of hair samples in human subjects previously administered in propofol.