Metabolomics of Chinese Hamster Ovary Cells.

Increasing demand of protein biotherapeutics produced using Chinese hamster ovary (CHO) cell lines necessitates improvement in the production yield of the bioprocess. Various cell engineering, improved media formulation and process-design based approaches utilizing the power of OMICS technologies, specifically, genomics and proteomics, have been employed; however, the potential of metabolomics largely remains unexplored. Metabolomics enables the detection, identification, and/or quantitation of small molecules, commonly known as metabolites, in and around the cells and may help to unlock the cellular molecular mechanism(s) that regulates cell growth and productivity in the bioprocess and improves cellular performance during the bioprocess. Currently, liquid chromatography (LC)/gas chromatography (CG)- coupled with mass-spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy are the most commonly used approaches for metabolomics. Therefore, in this chapter, we have discussed the standard procedures of investigating CHO metabolites using LC/GC-MS and/or NMR-based approaches.

Combining non-destructive devices and multivariate analysis as a tool to quantify the fatty acid profiles of linseed genotypes.

Linseed (Linum usitatissimum L.) and linseed oil, with a fatty acid profile rich in both macro and micro elements, are recognized as functional foods due to their valuable positive effects on health. Fatty acids composition (FAC) is a key indicator in assessing the quality of linseeds. The FAC of linseed is typically determined using chromatographic methods, yielding highly accurate results. However, chromatographic methods entail drawbacks such as requiring pre-chemical processes, generating chemical waste, and being both expensive and time-consuming, similar to chemical analyses. This study focused on the feasibility of colorimeter and FT-NIRS data to determine the FAC (%), protein (%) and neutral detergent fiber (NDF %) in linseed samples. By employing the PLSR analysis based on FT-NIRS, it was determined that the ratios of stearic (R2val = 0.74, RMSEP = 0.09 %), oleic (R2val = 0.75, RMSEP = 0.26 %), linoleic (R2val = 0.85, RMSEP = 0.58 %), linolenic (R2val = 0.71, RMSEP = 1.07 %), 8,11,14 eicosatrienoic (R2val = 0.77, RMSEP = 0.02 %), margaric (R2val = 0.71, RMSEP = 0.01 %), myristic (R2val = 0.75, RMSEP = 0.02 %), and behenic (R2val = 0.74, RMSEP = 1.12 %) in linseed could be successfully predicted. Furthermore, results demonstrated that the protein (R2val = 0.87, RMSEP = 0.9 %) and NDF (R2val = 0.90, RMSEP = 0.6 %) content in linseeds can be successfully predicted. PLSR demonstrated that FT-NIRS had relatively higher predictive capability compared to color models.

Promoting capillary microextraction of six organic nitrogen pesticides in water samples using versatile ZIF-8 hybrid monolithic column.

Because of rapid industrialization and agriculturalization, solving the pressing problems of environment pollution, especially water and food quality, requires innovative solutions. In this paper, a novel and versatile metal-organic framework (ZIF-8)-hybrid monolithic column (ZIF-HMC) was prepared for in-tube solid-phase microextraction (IT-SPME) of organic nitrogen pesticides (ONPs). The prepared monolithic columns had superior adsorption sites, high porosity, excellent permeability, and ideal specific surface area based on Fourier Transform Infrared Spectroscopy (FT-IR), X-ray Diffraction (XRD), Thermal Field Emission Scanning Electron Microscopy (SEM), Energy Dispersive Spectrometry (EDS), X-ray Photoelectron Spectroscopy (XPS), and N2 adsorption-desorption. The ZIF-HMC contained a large number of nitrogen and oxygen atoms, benzene rings and ZIF-8, which could synergistically promote the adsorption efficiency of ONPs through multiple interactions, such as hydrogen bonding, π-π accumulation, hydrophobic interactions, cation-π interactions, and pore adsorption by MOFs. Under the optimal conditions, a simple, efficient, and sensitive method for the analysis of six organic pesticides in environmental water samples was developed by using the ZIF-HMC as the extraction medium coupled with high performance liquid chromatography-ultraviolet (HPLC-UV). The method had a wide linear range (0.63-1000 µg L-1), a low detection limit (0.19-1.91 µg L-1) and satisfactory recoveries (87.4 %-110.2 %), the linear correlation coefficient was (R2) 0.9972-0.9995 and the relative standard deviation (RSD) was less than 2.64 %. The study had demonstrated the potential application of the developed method for the enrichment and analysis of organic pesticides in complex matrices of environmental samples, as well as the feasibility of MOFs materials for IT-SPME sample preparation.

Development of a hydrophilic-lipophilic-balanced copolymer@zirconium-based metal-organic framework-based solid-phase microextraction probe for the trace determination of organophosphorus pesticides in tea infusions.

Organophosphorus pesticides (OPPs) present in tea infusions pose a serious threat to human health. In this study, a sensitive method for the determination of OPPs was developed based on a direct-immersion solid-phase microextraction (DI-SPME) probe. By fine adjustment of the ratio and one-step polymerization of dihydroxy-functionalized zirconium-based metal-organic framework UiO-66-(OH)2 and divinylbenzene-N-vinyl pyrrolidone (DVB-NVP) microspheres, the DVB-NVP@ UiO-66-(OH)2 (D-N@U) composite with an optimal hydrophilic-lipophilic balance (HLB) was achieved. Furthermore, D-N@U was adhesively bonded to stainless-steel wires to fabricate a DI-SPME probe. OPPs, especially those with nonpolar properties characterized by a high octanol-water partition coefficient (log KOW), were selectively and efficiently enriched on the D-N@U-coated DI-SPME probe from tea infusions. Coupled with a gas chromatography-flame photometric detector, the as-fabricated D-N@U-coated DI-SPME probe achieved good performance for OPPs analysis with a wide linear dynamic range of 0.10-500.00 µg/L and low detection limits of 1.96-6.69 ng/L. Moreover, in spiked samples, the recoveries and relative standard deviations were in the ranges of 73.12%-101.20 % and 1.03%-6.56 %, respectively. Owing to its simple operation, high extraction efficiency, and high sensitivity, this approach has great potential for the rapid determination of multiple pesticide trace-level residues in food.

Cyano-functionalized porous hyper-crosslinked cationic polymers for efficient preconcentration and detection of phenolic endocrine disruptors in fresh water and fish.

Sensitively analyzing phenolic endocrine-disrupting chemicals (EDCs) in environmental substrates and aquatic organisms provides a significant challenge. Here, we developed a novel porous hyper-crosslinked ionic polymer bearing cyano groups (CN-HIP) as adsorbent for the highly efficient solid phase extraction (SPE) of phenolic EDCs in water and fish. The CN-HIP gave an excellent adsorption capability for targeted EDCs over a wide pH range, and the adsorption capacity was superior to that of several common commercial SPE adsorbents. The coexistence of electrostatic forces, hydrogen bond, and π-π interactions was confirmed as the main adsorption mechanism. A sensitive quantitative method was established by coupling CN-HIP based SPE method with high-performance liquid chromatography for the simultaneously determining trace bisphenol A, bisphenol F, bisphenol B and 4-tert-butylphenol in fresh water and fish. The method afforded lower detection limits (S/N = 3) (at 0.03-0.10 ng mL-1 for water and 0.8-4.0 ng g-1 for fish), high accuracy (the recovery of spiked sample at 88.0%-112 %) and high precision (the relative standard deviation < 8.5 %). This work provides a feasible method for detecting phenolic EDCs, and also opens a new perspective in developing functionalized cationic adsorbent.

Integrated interferometers as a new platform for low cost gas chromatography detection.

Gas chromatography is a reference method for gas analysis. As part of efforts to miniaturize gas chromatography systems, the miniaturization of detectors is essential. In this work, we report a new integrated photonic platform for gas chromatography analyte detection. The fabricated silicon die integrates Mach-Zehnder interferometers into low dead volume microfluidic channels, with coherent cost-effective detection scheme with a fixed 850 nm wavelength laser. A proof of concept is demonstrated with the separation and detection of three volatile organic compounds: heptane, octane, and toluene. Peaks' widths at half height range from 1 to 5 s. Peaks are very well resolved by our system, which acquires more than 100 points per second. From a heptane dilution range, we evaluate the limit of detection of our system to be the headspace of a 0.26 % heptane concentration solution. To our knowledge, these are the first integrated Mach-Zehnder interferometers reported for gas chromatography detection. This work could open new strategies for fast low cost and low limit of detection specific gas chromatography silicon micro-detectors.

Design and fabrication of acorn-like Janus molecularly imprinted materials for highly specific separation and enrichment of oxytetracycline from restaurant oily wastewater.

Molecularly imprinted polymer (MIP) is dedicated to the adsorption of target substances in the aqueous phase, but ignores the adsorption in a more complex environment (oily wastewater). In order to explore the application field of existing MIPs, acorn-like Janus particles were fabricated by photo-initiated seed swelling polymerization. A novel amphiphilic Janus-MIP was prepared with the acorn-like Janus particles as matrix, methacrylic acid, ethylene dimethacrylate and oxytetracycline (OTC) as functional monomers, crosslinking agents and template molecules via surface initiated-atom transfer radical polymerization (SI-ATRP). For comparison, the poly (glycidyl methacrylate-co-ethylene glycol dimethacrylate) (poly (GMA-co-EDMA)) microspheres were also utilized as the matrix to prepare common spherical-MIP. The adsorption capacity of Janus-MIP for OTC was 23.8 mg g-1 in oil-water system, while the adsorption capacity of spherical-MIP for OTC was only 12.6 mg g-1 in the same system. At the same time, through high performance liquid chromatography (HPLC) analysis, Janus-MIP can specifically recognize and adsorb trace OTC in restaurant oily wastewater samples, and the proposed method exhibited a lower limit of detection (LOD, 3 ng mL-1) and a higher OTC recovery rate (94.2 %-98.4 %). This work demonstrated great potential for the detection and control of OTC contamination from real samples in an oil-water mixed environment.

Development of new PDMS in tube extraction microdevice for enhanced monitoring of polycyclic aromatic hydrocarbons and their derivatives in water.

Contamination by polycyclic aromatic hydrocarbons (PAHs) is an urgent environmental concern, given its atmospheric dispersion and deposition in water bodies and soils. These compounds and their nitrated and oxygenated derivatives, which can exhibit high toxicities, are prioritized in environmental analysis contexts. Amid the demand for precise analytical techniques, comprehensive two-dimensional chromatography coupled with mass spectrometry (GCxGC/Q-TOFMS) has emerged as a promising tool, especially in the face of challenges like co-elution. This study introduces an innovation in the pre-concentration and detection of PAHs using an extraction fiber based on polydimethylsiloxane (PDMS), offering greater robustness and versatility. The proposed technique, termed in-tube extraction, was developed and optimized to effectively retain PAHs and their derivatives in aqueous media, followed by GCxGC/Q-TOFMS determination. Fiber characterization, using techniques such as TG, DTG, FTIR, and SEM, confirmed the hydrophobic compounds retention properties of the PDMS. The determination method was validated, pointing to a significant advancement in the detection and analysis of PAHs in the environment, and proved effective even for traces of these compounds. The results showed that the detection limits (LOD) and quantification limits (LOQ) ranged from 0.07 ng L-1 to 1.50 ng L-1 and 0.33 ng L-1 to 6.65 ng L-1, respectively; recovery ranged between 72 % and 117 %; and the precision intraday and interday ranged from 1 % to 20 %. The fibers were calibrated in the laboratory, with exposure times for analysis in the equilibrium region ranging from 3 to 10 days. The partition coefficients between PDMS and water were also evaluated, showing logarithm values ranging from 2.78 to 5.98. The fibers were applied to the analysis of real water samples, demonstrating high capacity. Additionally, given the growing demand for sustainable methods, the approach presented here incorporates green chemistry principles, providing an efficient and eco-friendly solution to the current chemical analysis scenario.

Isolation and Characterization of Exosomes from Ocular Samples.

Exosomes are microsize vesicles secreted by nearly all cells to the extracellular space. The vesicles transport cell signaling and communicate with other cells. Ultracentrifugation is the standard method to isolate exosomes from culture media or body fluid. Without ultracentrifuge, exosomes can be precipitated by polyethylene glycol or separated by size exclusion chromatography. After isolation, nanoparticle tracking analysis can help to estimate the size and concentration of exosome samples. Transmission electron microscopy can directly show the size and morphology of exosomes. Moreover, the sample should be characterized by the expression of several exosome biomarker proteins. Exosomal contents such as proteins and miRNAs could be profiled using appropriate technologies.

Lipidomics analysis reveals the effects of Schizochytrium sp. supplementation on the lipid composition of Tan sheep meat.

Schizochytrium sp. (SZ) can potentially be employed in nutritional strategies for producing high-quality sheep meat. However, the effects of SZ on the lipid composition of sheep meat are insufficiently understood. In this study, the effects of SZ supplementation on the lipid profile of Tan sheep meat were evaluated using non-targeted lipidomic techniques. Lipidomics analysis revealed 383 differential lipids (DLs) between the SZ and control groups, and there were six metabolic pathways associated with lipids, including glycerophospholipid metabolism, glycerolipid metabolism, α-linolenic acid metabolism, linoleic acid metabolism, glycine, serine and threonine metabolism, and arachidonic acid metabolism (P < 0.05). Glycerophospholipid metabolism was the core pathway of DLs; we found that phosphatidylcholine, phosphatidylserine, and lysophosphatidylcholine were the crucial lipid metabolites of this pathway. Dietary supplementation with SZ increased n-3 polyunsaturated fatty acid (PUFA), C22:6n-3, and C20:5n-3 (P < 0.05), while it decreased C18:0, saturated fatty acid (SFA), and SFA/PUFA (P < 0.05). These results indicate that SZ supplementation induces positive alterations in the lipid profile of Tan sheep meat, which is beneficial to meat quality and sheds valuable insights into the future development of functional lipids in sheep meat.

Stable Isotope Tracing Experiments Using LC-MS.

Metabolomics has emerged as a pivotal field in understanding cellular function, particularly in the context of disease. In numerous diseases, including cancer, alterations in metabolism play an essential role in disease progression and drug response. Hence, unraveling the metabolic rewiring is of importance to find novel diagnostic and therapeutic strategies. Isotope tracing is a powerful technique for delving deeper into the metabolic wiring of cells. By tracking an isotopically labeled substrate through biochemical reactions in the cell, this technique provides a dynamic understanding of cellular metabolism. This chapter outlines a robust isotope tracing protocol utilizing high-resolution mass spectrometry coupled to liquid chromatography in cell culture-based models. We cover essential aspects of experimental design and analyses, providing a valuable resource for researchers aiming to employ isotopic tracing.

Quantification of Total Ketone Bodies in Biological Matrices Using Stable Isotope-Labeled Internal Standards and RP-UHPLC-MS/MS.

Acetoacetate (AcAc) and D-beta-hydroxybutyrate (D-ßOHB), the two major ketone bodies found in circulation, are linked to multiple physiological and pathophysiological states. Therefore, analytical methodologies surrounding the quantification of total ketone body (TKB) concentrations in biological matrices are paramount. Traditional methods to quantify TKBs relied on indirect spectrophotometric assays with narrow dynamic ranges, which have been significantly improved upon by modern mass spectrometry (MS)-based approaches. However, the lack of stable isotope-labeled internal standards (ISs) for AcAc and the need to distinguish D-ßOHB from its closely related structural and enantiomeric isomers pose significant obstacles. Here, we provide a protocol to synthesize and quantify a [13C] stable isotope-labeled IS for AcAc, which, in conjunction with a commercially available [2H] stable isotope-labeled IS for ßOHB, allows TKBs to be measured across multiple biological matrices. This rapid (7 min) analysis employs reverse phase ultra-high performance liquid chromatography (RP-UHPLC) coupled to tandem MS (MS/MS) to distinguish ßOHB from three structural isomers using parallel reaction monitoring (PRM), providing excellent specificity and selectivity. Finally, a method is provided that distinguishes D-ßOHB from L-ßOHB using a simple one-step derivatization to produce the corresponding diastereomers, which can be chromatographically resolved using the same rapid RP-UHPLC separation with new PRM transitions. In summary, this method provides a rigorous analytical pipeline for the analysis of TKBs in biological matrices via leveraging two authentic stable isotope-labeled ISs and RP-UHPLC-MS/MS.

A Method for Analysis of Oxidized Phospholipids from Biological Samples Using Mass Spectrometry.

Oxidized phospholipids (oxPLs) are generated during innate immunity and inflammation, where they play a variety of biological roles, including regulation of autoimmunity and coagulation. Some are generated by enzymatic reactions, leading to stereo- and regiospecificity, while many others can be formed through nonenzymatic oxidation and truncation and can be used as biomarkers of oxidative stress. Mass spectrometry methods have been developed over many years for oxPL analysis, which can provide robust estimations of molecular species and amounts, where standards are available. Here we present a method used for the analysis of enzymatically-generated oxPL (eoxPL), which allows quantification of mono-hydroxy oxylipin-containing species. We also show profiling of many other partially characterized structures in tissue samples and provide typical chromatograms obtained.

Reversed-Phase Ultrahigh-Performance Liquid Chromatography-Mass Spectrometry Method for High-Throughput Lipidomic Quantitation.

Reversed-phase ultrahigh-performance liquid chromatography-mass spectrometry (RP-UHPLC/MS) method is optimized for the quantitation of a large number of lipid species in biological samples, primarily in human plasma and serum. The method uses a C18 bridged ethylene hybrid (BEH) column (150 × 2.1 mm; 1.7 µm) for the separation of lipids from 23 subclasses with a total run time of 25 min. Lipid species separation allows the resolution of isobaric and isomeric lipid forms. A triple quadrupole mass spectrometer is used for targeted lipidomic analysis using multiple reaction monitoring (MRM) in the positive ion mode. Data are evaluated by Skyline software, and the concentrations of analytes are determined using internal standards per each individual lipid class.

Long Chain Base Profiling with Multiple Reaction Monitoring Mass Spectrometry.

Sphingolipids (SLs) are essential lipids with important functions in membrane formation and cell signaling. The presence of a long chain base (LCB) structure is common to all SLs. De novo SL synthesis is initiated by the enzyme serine-palmitoyltransferase (SPT), which forms an LCB by the conjugation from serine and fatty acyl-CoAs. SPT can metabolize a variety of acyl-CoA substrates, which form diverse LCB structures within and across species. The LCB then undergoes further metabolic modifications resulting in an extraordinarily diverse spectrum of sphingolipids formed. SL analysis, using liquid chromatography-mass spectrometry (LC-MS)-based methods, poses challenges due to the diverse range of frequently isobaric species. This complexity complicates the identification of underlying LCB structures using standard lipidomics approaches. Here, we describe a simplified method to analyze the LCB profile in cells, tissue, and blood. The procedure involves chemical hydrolysis to remove the conjugated headgroups and N-acyl chains, allowing to specifically resolve the underlying LCB structures by LC-MS. This method can also be combined with an isotope labeling approach to determine in vivo SPT activity and total SL de novo synthesis over time.

Nonenzymatic Oxidized Polyunsaturated Fatty Acid Products in Human Plasma and Urine Samples by LC-QTOF-MS/MS.

Oxidative stress induces autooxidation of polyunsaturated fatty acids, producing numerous isoprostanoids and isofuranoids. These oxidized products are measurable in human plasma and urine and serve as oxidative stress biomarkers for chronic diseases. This chapter details the preparation and measurement of α-linolenic acid-derived phytoprostanes and phytofurans in human samples using liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (LC-QToF-MS/MS).

Quantification of Octadecanoids in Human Plasma Using Chiral Supercritical Fluid Chromatography-Tandem Mass Spectrometry.

Octadecanoids are a subset of oxylipins derived from 18-carbon fatty acids. These compounds have historically been understudied but have more recently attracted attention to their purported biological activity. One obstacle to the study of octadecanoids has been a lack of specific analytical methods for their measurement. A particular limitation has been the need for chiral-based methods that enable separation and quantification of individual stereoisomers. The use of chirality provides an additional dimension for distinguishing analytes produced enzymatically from those formed through autoxidation. In this chapter, we describe a comprehensive method using chiral supercritical fluid chromatography-tandem mass spectrometry (SFC-MS/MS) for the quantification of octadecanoids in human plasma. This method stands as an effective approach for quantifying octadecanoids and is applicable to diverse research applications including clinical research.

Rapid Lipid Mediator Profiling by Convergence Chromatography-MS/MS.

Bioactive lipid mediators derived from arachidonic acid constitute an attractive pool of metabolites that reflect cellular function and signaling, as well as potential biomarkers that may respond quantitatively to disease progression or pharmacological treatment. Their quantitative measurement in biological samples is complicated by the number of isomers that share common structural features, which are not easily distinguished by immunoassays or reverse phase chromatography-tandem mass spectrometry. Here, we present a method that enables the rapid analysis of a panel of over 25 biologically important eicosanoids in a 96-well format for cell culture supernatants, plasma, and organ tissues using convergence chromatography-tandem mass spectrometry to resolve these analytes of interest.

Ultrahigh-Performance Supercritical Fluid Chromatography-Mass Spectrometry in the Clinical Lipidomic Analysis.

Ultrahigh-performance supercritical fluid chromatography-mass spectrometry (UHPSFC/MS) method is optimized for the high-throughput quantitation of lipids in human serum and plasma with an emphasis on robustness and accurate quantitation. Bridged ethylene hybrid (BEH) silica column (100 × 3 mm; 1.7 µm) is used for the separation of 17 nonpolar and polar lipid classes in 4.4 min using the positive ion electrospray ionization mode. The lipid class separation approach in UHPSFC/MS results in the coelution of all lipid species within one lipid class in one chromatographic peak, including two exogenous internal standards (IS) per lipid class, which provides the optimal conditions for robust quantitation. The method was validated according to European Medicines Agency and Food and Drug Administration recommendations. UHPSFC/MS combined with LipidQuant software allows a semiautomated process to determine lipid concentrations with a total run time of only 8 min including column equilibration, which enables the analysis of 160 samples per day.

Rapid Analysis of Sterols in Blood-Derived Samples.

Dysregulations of cholesterol biosynthesis are known to be associated with several pathologies. Due to the rapid growth of clinical investigations in this research area, a specific, fast, and valid method for analyzing cholesterol, its precursors, and metabolites is required. Here, we describe a rapid method for sample preparation, separation, and quantification of sterols in blood-derived samples using polymeric solid phase extraction followed by gas chromatography-mass spectrometry. The validated method demonstrates a reliable quantification of cholesterol, its precursors, and metabolites.