High-throughput Liquid Research Articles

Determination of the Polar Compounds in Vegetable Oil by Ultra-Performance Liquid Chromatography–Quadrupole-Time-of-Flight-Mass Spectrometry with Chemometrics

High-throughput ultra-performance liquid chromatography–quadrupole time-of-flight mass spectrometry was combined with chemometric tools for the rapid determination of polar components in camellia oil, rapeseed oil, and waste cooking oil. The results were analyzed by two unsupervised methods: principal component analysis (one-way ANOVA, p<.05) and volcano plot analysis (p<.05, fold change ≥2) and supervised method: partial least squares discriminant analysis. The results showed that the oils were correctly classified based on their polar components. The first three principal components reflected most detail with a cumulative contribution rate of 84.67% using principal component analysis. The prediction accuracy was close to 100% using partial least squares discriminant analysis. Nineteen components were screened by principal component analysis; twelve were preliminary identified as palmitamide, phytosphingosine, eicosasphinganine, 1-monopalmitin, glyceryl monooleate, glyceryl monostearate, 1α-hydroxyvitamin D2, 1-linoleoyl glycerol, oleamide, sphinganine, stearamide, and linoleic acid. The proposed method may be applied to effectively and accurately authenticate edible oils.

Impact of frit dispersion on gradient performance in high-throughput liquid chromatography

The impact of porous frits (2.1 mm i.d. × 1 mm thick, 0.2 μm porosity, 20% void, 0.69 μL) in short 0.5–5 cm long × 2.1 mm i.d. columns packed with sub-2 μm superficially porous particles on gradient performance was investigated using a low dispersive i-class ACQUITY UPLC system (25 cm × 75 μm outlet tube + 250 nL optical cell). In order to maximize data accuracy, the sample dispersion through a single frit was measured from four independent methods: (1) plate height subtraction, (2) peak capacity versus efficiency plot, (3) flow reversal, and (4) direct measurement. Frit dispersion increases non-linearly with increasing flow rate. The corresponding volume variances of small molecules were measured at 0.16 ± 0.04, 0.24 ± 0.05, and 0.31 ± 0.06 μL2 at flow rates of 0.1, 0.2, and 0.3 mL/min, respectively. These observed variances are lower than and consistent with the maximum volume variance of 0 . 692 ∼ 0.5 μL2 expected for a mixer-like behavior. The peak capacity of short columns were then calculated for mixtures of peptides using a general model of gradient elution by considering (or not) the actual analyte dispersion taking place in the outlet frit, in the post-column connecting tube, and in the detection cell. The results show that the sole presence of the outlet frit is responsible for about 50% loss in peak capacity (relative to the expected gradient performance in absence of frit and post-column tube) for a 1 cm long column operated under standard gradient steepness. The actual structure and volume of column frits needs adjustment if one wants to take full advantage of the true performance of short high-throughput columns packed with sub-2 μm particles.

Influence of surfactants on the electronic properties of liquid-phase exfoliated graphene

Graphene has been derived from graphite using a high-throughput liquid phase exfoliation technique, using two anionic surfactants with same polar head and different hydrophobic tail. Raman spectra indicate that the resulting samples are few-layer graphene, with large D-band, possibly arising from the surfactant-decorated edges. Scanning tunnelling spectroscopy (STS) demonstrates the linear density of states, with zero band gap to nearly 0.44 and 0.53 eV respectively for sodium dodecyl benzene sulfonate (SDBS) and dioctyl sodium sulfosuccinate (AOT) exfoliated graphene and the Fermi level has been slightly shifted towards the valence band due to the anionic surfactants. Thin film transistors (TFT’s) fabricated using these exfoliated graphene as channel layers exhibit ambipolar behaviour, with Dirac points shifted due to inherent charges in the layers. It is observed that the performance of transistors such as transconductance, carrier mobility, and Dirac point strongly depend on the surfactants used.

Metabolomic Applications in Radiation Biodosimetry.

Metabolomics, the collective assessment and quantification of small molecules in a given biofluid or tissue sample, has provided new ways in evaluating an individual's exposure level to ionizing radiation or other genotoxic stressors. Protocols that are routinely utilized for the preparation of samples from rodents to patients are presented here in order to be analyzed by high-throughput liquid chromatography-mass spectrometry techniques. These protocols are based on established methods in our laboratory that have been used extensively in radiation biodosimetry through metabolomics. These protocols are focused on general profiling of samples and therefore do not concentrate on extraction of specific classes on metabolites (e.g., eicosanoids).

A high-throughput liquid biopsy for rapid rare cell separation from large-volume samples.

Liquid biopsy techniques for rare tumor cell separation from body fluids have shown enormous promise in cancer detection and prognosis monitoring. This work established a high-throughput liquid biopsy platform with a high recovery rate and a high cell viability based on a previously reported 2.5D micropore-arrayed filtration membrane. Thanks to its high porosity (>40.2%, edge-to-edge space between the adjacent micropores <4 μm), the achieved filtration throughputs can reach >110 mL min-1 for aqueous samples and >17 mL min-1 for undiluted whole blood, only driven by gravity with no need for any extra pressure loading. The recoveries of rare lung tumor cells (A549s) spiked in PBS (10 mL), unprocessed BALF (10 mL) and whole blood (5 mL) show high recovery rates (88.0 ± 3.7%, 86.0 ± 5.3% and 83.2 ± 6.2%, respectively, n = 5 for every trial) and prove the high performance of this platform. Successful detection of circulating tumor cells (CTCs) from whole blood samples (5 mL) of lung cancer patients (n = 5) was demonstrated. In addition, it was both numerically and experimentally proved that a small edge-to-edge space was significant to improve the viability of the recovered cells and the purity of the target cell recovery, which was reported for the first time to the best of the authors' knowledge. This high-throughput technique will expand the detecting targets of liquid biopsy from the presently focused CTCs in whole blood to the exfoliated tumor cells (ETCs) in other large-volume clinical samples, such as BALF, urine and pleural fluid. Meanwhile, the technique is easy to operate and ready for integration with other separation and analysis tools to fulfill a powerful system for practical clinical applications of liquid biopsy.

High sensitive LC–MS/MS method for estimation of eluxadoline in human plasma and its application to pharmacokinetic study

A selective, sensitive and high throughput liquid chromatography – tandem mass spectrometry (LC–MS/MS) method was developed and validated for estimation of eluxadoline in human plasma. Plasma samples of analyte with internal standard (eluxadoline13CD3) were extracted using solid phase extraction on Orochem Panthera Deluxe cartridges. Chromatographic separation was performed on Ace Phenyl column (150 × 4.6 mm, 5 μm), using a mixture of buffer (2 mM ammonium acetate in 0.01% formic acid), acetonitrile and methanol (20:70:10, v/v/v) as mobile phase at a flow rate of 0.8 mL/min. The run time of analyte was 4.0 min. Tandem mass spectrometry, operating in positive ionization and multiple reaction monitoring modes was used for detection of analyte and internal standard. The method was established with a linear dynamic range of 25.0–5000 pg/mL for eluxadoline using 300 μL human plasma. The sample preparation procedure was consistent and reproducible (accuracy, 96.2–106.1%; precision (%CV), 0.8–6.6%), preventing the ex vivo hydrolysis of acyl glucuronide metabolite of eluxadoline to parent drug. The method was applied successfully to a clinical pharmacokinetic study in six healthy South Indian male subjects under fed conditions and the results were authenticated by incurred sample reanalysis.

A fully automated high-throughput liquid chromatography tandem mass spectrometry method for measuring creatinine in urine

Reliable creatinine measurements are important to evaluate kidney function and for creatinine correction to reduce biological variability of other urinary analytes. A high-throughput, accurate liquid chromatography tandem mass spectrometry method for quantitation of human urinary creatinine has been developed and validated.Sample preparation was fully automated including cryovial decapping, sample ID scanning and two serial dilution steps. Quantitation was performed using a stable isotope-labeled internal standard. Multiplexed chromatographic separation of creatinine was achieved within a one-minute analysis and followed by tandem mass spectrometry in positive electrospray ionization mode. The precursor and product ions of creatinine and D3-creatinine were monitored in selected reaction monitoring mode.Method validation results showed reproducibility with within-run precision of 3.59, 3.49 and 2.84% and between-run precision of 4.01, 3.28 and 3.57% for low, medium and high quality control materials prepared from pooled donor urine, respectively. The method showed excellent accuracy with a bias of −1.94%, −0.78% and −1.07% for three levels of certified reference material. The calibration curve was linear throughout a 7.50–300 mg/dL (0.663–26.5 mmol/L) measurement range (R2 = 0.999), with the mean slope of 0.0115 (95%CI, 0.0108–0.0122) and intercept of 0.0027 (95%CI, 0.0003–0.0051). The limit of detection (LOD) of the method was 3.17 mg/dL (0.280 mmol/L). Analytical specificity was achieved by chromatographically separating creatinine from potentially interfering creatine within a one-minute run and monitoring the Quantitation Ion/Confirmation Ion (QI/CI) ratios in samples.A simple, accurate, high-throughput method was successfully developed for measuring creatinine in human urine samples.

The Internet of Things in the Life Sciences Laboratory.

The Internet of Things in the Life Sciences Laboratory.

Isoform-Level Interpretation of High-Throughput Proteomics Data Enabled by Deep Integration with RNA-seq.

Cellular control of gene expression is a complex process that is subject to multiple levels of regulation, but ultimately it is the protein produced that determines the biosynthetic state of the cell. One way that a cell can regulate the protein output from each gene is by expressing alternate isoforms with distinct amino acid sequences. These isoforms may exhibit differences in localization and binding interactions that can have profound functional implications. High-throughput liquid chromatography tandem mass spectrometry proteomics (LC-MS/MS) relies on enzymatic digestion and has lower coverage and sensitivity than transcriptomic profiling methods such as RNA-seq. Digestion resultsin predictable fragmentation of a protein, which can limit the generation of peptides capable of distinguishing between isoforms. Here we exploit transcript-level expression from RNA-seq to set prior likelihoods and enable protein isoform abundances to be directly estimated from LC-MS/MS, an approach derived from the principle that most genes appear to be expressed as a single dominant isoform in a given cell type or tissue. Through this deep integration of RNA-seq and LC-MS/MS data from the same sample, we show that a principal isoform can be identified in >80% of gene products in homogeneous HEK293 cell culture and >70% of proteins detected in complex human brain tissue. We demonstrate that the incorporation of translatome data from ribosome profiling further refines this process. Defining isoforms in experiments with matched RNA-seq/translatome and proteomic data increases the functional relevance of such data sets and will further broaden our understanding of multilevel control of gene expression.

Mapping dynamic histone modification patterns during arsenic-induced malignant transformation of human bladder cells

Arsenic is a known potent risk factor for bladder cancer. Increasing evidence suggests that epigenetic alterations, e.g., DNA methylation and histones posttranslational modifications (PTMs), contribute to arsenic carcinogenesis. Our previous studies have demonstrated that exposure of human urothelial cells (UROtsa cells) to monomethylarsonous acid (MMAIII), one of arsenic active metabolites, changes the histone acetylation marks across the genome that are correlated with MMAIII-induced UROtsa cell malignant transformation. In the current study, we employed a high-resolution and high-throughput liquid chromatography tandem mass spectrometry (LC-MS/MS) to identify and quantitatively measure various PTM patterns during the MMAIII-induced malignant transformation. Our data showed that MMAIII exposure caused a time-dependent increase in histone H3 acetylation on lysine K4, K9, K14, K18, K23, and K27, but a decrease in acetylation on lysine K5, K8, K12, and K16 of histone H4. Consistent with this observation, H3K18ac was increased while H4K8ac was decreased in the leukocytes collected from people exposed to high concentrations of arsenic compared to those exposed to low concentrations. MMAIII was also able to alter histone methylation patterns: MMAIII transformed cells experienced a loss of H3K4me1, and an increase in H3K9me1 and H3K27me1. Collectively, our data shows that arsenic exposure causes dynamic changes in histone acetylation and methylation patterns during arsenic-induced cancer development. Exploring the genomic location of the altered histone marks and the resulting aberrant expression of genes will be of importance in deciphering the mechanism of arsenic-induced carcinogenesis.

Label-free quantitative proteomics analysis reveals distinct molecular characteristics in endocardial endothelium.

Endocardial endothelium, which lines the chambers of the heart, is distinct in its origin, structure, and function. Characterization studies using genomics and proteomics have reported molecular signatures supporting the structural and functional heterogeneity of various endothelial cells. However, though functionally very important, no studies at protein level have been conducted so far characterizing endocardial endothelium. In this study, we used endothelial cells from pig heart to investigate if endocardial endothelial cells are distinct at the proteome level. Using a high-throughput liquid chromatography-tandem mass spectrometry for proteome profiling and expression, we identified sets of proteins that belong to specific biological processes and metabolic pathways in endocardial endothelial cells supporting its specific structural and functional roles. The study also identified several transcription factors and cell surface markers, which may have roles in the specificity of endocardial endothelium. The detection of sets proteins preferentially expressed in endocardial endothelium offers new insights into its role in the regulation of cardiac function. Data are made available through ProteomeXchange with identifier PXD009194.

Rapid determination of polar pesticides and plant growth regulators in fruits and vegetables by liquid chromatography/tandem mass spectrometry

ABSTRACTA simple high-throughput liquid chromatography/tandem mass spectrometry (LC-MS/MS) multiresidue analysis method was developed for the simultaneous determination of polar pesticides, plant growth regulators and other polar compounds. These included amitrole, chlormequat, mepiquat, cyromazine, ETU, PTU, perchlorate, and daminozide using a mixed-mode column. A 10 g test portion was shaken with acidified methanol for 10 min. After centrifugation, the sample extract was injected and analyzed within 11 min by LC-MS-MS. This column eliminated the need for derivatization or the use of ion paring reagent. Two MS-MS transitions were monitored in the method for each target compound to achieve true positive identification. Eight isotopically-labeled internal standards corresponding to each analyte were used to correct for matrix suppression effect and/or instrument signal drift. The average recovery for all analytes at 20, 40, and 250 ng/g (n = 6) ranged from 73–136%, with a relative standard deviation of ≤ 20%.

A universal high-throughput liquid biopsy technique for rare tumor cells recovery from various body fluids.

e24224Background: Recently, micro/nanotechnology has been involved in developing highly sensitive liquid biopsy technique because of the manipulation capability at single tumor cell level. However,...

IoT for Real-Time Measurement of High-Throughput Liquid Dispensing in Laboratory Environments.

Critical to maintaining quality control in high-throughput screening is the need for constant monitoring of liquid-dispensing fidelity. Traditional methods involve operator intervention with gravimetric analysis to monitor the gross accuracy of full plate dispenses, visual verification of contents, or dedicated weigh stations on screening platforms that introduce potential bottlenecks and increase the plate-processing cycle time. We present a unique solution using open-source hardware, software, and 3D printing to automate dispenser accuracy determination by providing real-time dispense weight measurements via a network-connected precision balance. This system uses an Arduino microcontroller to connect a precision balance to a local network. By integrating the precision balance as an Internet of Things (IoT) device, it gains the ability to provide real-time gravimetric summaries of dispensing, generate timely alerts when problems are detected, and capture historical dispensing data for future analysis. All collected data can then be accessed via a web interface for reviewing alerts and dispensing information in real time or remotely for timely intervention of dispense errors. The development of this system also leveraged 3D printing to rapidly prototype sensor brackets, mounting solutions, and component enclosures.

Organic Solvent as Internal Standards for Quantitative and High-Throughput Liquid Interfacial SERS Analysis in Complex Media.

Liquid-state interfacial nanoparticle arrays for surface-enhanced Raman scattering (SERS) promises a practical, substrate-free, and rapid analysis but faces a great challenge to develop a batch and uniform fabrication strategy with stable internal standards (IS) because of the difficulties in precisely locating both the IS tags and analytes in the same local structure under the harsh conditions of biphasic liquid interface. Here, we develop a fast batch preparation of self-ordered dense Au nanoparticle (GNP) arrays on cyclohexane/water biphasic interface in 96-well plates with the assist of acetone as the phase-crossing inducer. The acetone can extract the pesticide molecules via a simple dipping sample peels and can rapidly capture and locate the pesticide molecule into the plasmonic hotspots. Meanwhile, this phase-crossing solvent, acetone itself, generates stable SERS signal and is used as the IS tags to calibrate the signal fluctuation. This platform presents an excellent uniformity with a relative standard deviation (RSD) of 5.9% compared to the RSD of 14.5% without the IS's correction and a good sensitivity with a limit of detection (LOD) of 1 nM thiram. This high-throughput strategy for analyzing pesticide residues at fruit peels reached detection levels of nanograms per square centimeter (ng/cm2). Combined with the 96-well plates, this platform greatly facilitates the self-assembly and multiplex sampling. The self-ordered arrays at two immiscible phases interface evidenced the detection of both the oil-soluble thiabendazole and the water-soluble thiram molecules and also realized the multiplex and two-phase detection of these two pesticides. This platform offers vast possibilities for on-site sensing of various analytes and paves a new way for the quantitative and high-throughput SERS analyzer just as convenient as the microplate reader.

Deciphering metabonomics biomarkers-targets interactions for psoriasis vulgaris by network pharmacology

Objectives: Psoriasis vulgaris is a chronic inflammatory and immune-mediated skin disease. 44 metabonomics biomarkers were identified by high-throughput liquid chromatography coupled to mass spectrometry in our previous work, but the roles of metabonomics biomarkers in the pathogenesis of psoriasis is unclear.Methods: The metabonomics biomarker-enzyme network was constructed. The key metabonomics biomarkers and enzymes were screened out by network analysis. The binding affinity between each metabonomics biomarker and target was calculated by molecular docking. A binding energy-weighted polypharmacological index was introduced to evaluate the importance of target-related pathways.Results: Long-chain fatty acids, phospholipids, Estradiol and NADH were the most important metabonomics biomarkers. Most key enzymes belonged hydrolase, thioesterase and acyltransferase. Six proteins (TNF-alpha, MAPK3, iNOS, eNOS, COX2 and mTOR) were extensively involved in inflammatory reaction, immune response and cell proliferation, and might be drug targets for psoriasis. PI3K-Akt signaling pathway and five other pathways had close correlation with the pathogenesis of psoriasis and could deserve further research.Conclusions: The inflammatory reaction, immune response and cell proliferation are mainly involved in psoriasis. Network pharmacology provide a new insight into the relationships between metabonomics biomarkers and the pathogenesis of psoriasis.KEY MESSAGES • Network pharmacology was adopted to identify key metabonomics biomarkers and enzymes. • Six proteins were screened out as important drug targets for psoriasis. • A binding energy-weighted polypharmacological index was introduced to evaluate the importance of target-related pathways.

Top-Down Characterization of Heavily Modified Histones Using 193 nm Ultraviolet Photodissociation Mass Spectrometry.

The characterization of protein post-translational modifications (PTMs) remains a significant challenge for traditional bottom-up proteomics methods owing to the lability of PTMs and the difficulty of mapping combinatorial patterns of PTMs based on analysis of small peptides. These shortcomings have accelerated interest in top-down MS/MS methods that focus on analysis of intact proteins. Simultaneous mapping of all PTMs requires extensive sequence coverage to confidently localize modifications. 193 nm ultraviolet photodissociation (UVPD) has been shown to generate unparalleled sequence coverage for intact proteins compared to traditional MS/MS methods. This study focuses on identification and localization of PTMs of histones by UVPD, higher-energy collisional dissociation (HCD), and the hybrid method electron-transfer/higher-energy collision dissociation (EThcD) via a high throughput liquid chromatography-mass spectrometry strategy. In total, over 500 proteoforms were characterized among these three activation methods with 46% of the identifications found in common by two or more activation methods. EThcD and UVPD afforded more extensive characterization of proteoforms than HCD with average gains in sequence coverage of 15% and C-scores that doubled on average.

LC-MS/MS based quantitation of ciprofloxacin and its application to antimicrobial resistance study in Balb/c mouse plasma, urine, bladder and kidneys

The authors proposed a simple, high throughput liquid chromatography-tandem mass spectrometric (LC-MS/MS) method for the determination of ciprofloxacin in small sample volumes of plasma, urine, bladder and kidneys of mice.

Liquid chromatography mass spectrometry-based profiling of phosphatidylcholine and phosphatidylethanolamine in the plasma and liver of acetaminophen-induced liver injured mice

BackgroundAcetaminophen (APAP) overdose is one of the most common causes of acute liver failure in many countries. The aim of the study was to describe the profiling of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) in the plasma and liver of Acetaminophen -induced liver injured mice.MethodsA time course study was carried out using C57BL/6 mice after intraperitoneal administration of 300 mg/kg Acetaminophen 1 h, 3 h, 6 h, 12 h and 24 h. A high-throughput liquid chromatography mass spectrometry (LC-MS) lipidomic method was utilized to detect phosphatidylcholine and phosphatidylethanolamine species in the plasma and liver. The expressions of phosphatidylcholine and phosphatidylethanolamine metabolism related genes in liver were detected by quantitative Reverse transcription polymerase chain reaction (qRT-PCR) and Western-blot.ResultsFollowing Acetaminophen treatment, the content of many PC and PE species in plasma increased from 1 h time point, peaked at 3 h or 6 h, and tended to return to baseline at 24 h time point. The relative contents of almost all PC species in liver decreased from 1 h, appeared to be lowest at 6 h, and then return to normality at 24 h, which might be partly explained by the suppression of phospholipases mRNA expressions and the induction of choline kinase (Chka) expression. Inconsistent with PC profile, the relative contents of many PE species in liver increased upon Acetaminophen treatment, which might be caused by the down-regulation of phosphatidylethanolamine N-methyltransferase (Pemt).ConclusionsAcetaminophen overdose induced dramatic change of many PC and PE species in plasma and liver, which might be caused by damaging hepatocytes and interfering the phospholipid metabolism in Acetaminophen -injured liver.

Development and Independent Laboratory Validation of an Analytical Method for the Direct Determination of Glyphosate, Glufosinate, and Aminomethylphosphonic Acid in Honey by Liquid Chromatography/Tandem Mass Spectrometry

A simple high-throughput liquid chromatography/tandem mass spectrometry (LC-MS/MS) method was developed for the determination of glyphosate, glufosinate and aminomethylphosphonic acid (AMPA) in honey using a reversed-phase column with weak anion-exchange and cation-exchange mixed-mode. One gram of sample was shaken with water containing ethylenediaminetetraacetic acid disodium salt and acetic acid for five minutes. After centrifugation, the supernatant was mixed with internal standard and directly injected and analyzed in ten minutes by LC-MS/MS with no sample concentration or derivatization steps. Two precursor/product ion transitions were monitored in the method for each target compound to achieve true positive identification. Three isotopically labelled internal standards corresponding to each analyte were used to correct for matrix suppression effects and/or instrument signal drift. The average recovery for all analytes at 25, 50, 100, and 500 ng/g (n=11) ranged from 87 to 111% with a relative standard deviation of less than 12%.https://doi.org/10.21423/jrs-v05n02p001 (DOI assigned 3/11/2019)