Liquid Chromatography With Mass Spectrometry Research Articles

Latest Developments in Direct and Non-Direct LC-MS Methods Based on Liquid Electron Ionization (LEI).

Mass spectrometry (MS) enables precise identification and quantification of molecules, particularly when combined with chromatography. The advent of atmospheric pressure ionization (API) techniques allowed the efficient coupling of liquid chromatography with MS (LC-MS), extending analyses to nonvolatile and thermolabile compounds. API techniques present limitations such as low informative capacity and reproducibility of mass spectra, increasing instrument complexity and costs. Other challenges include analyzing poorly polar molecules and matrix effects (ME), which negatively impact quantitative analyses, necessitating extensive sample purification or using expensive labeled standards. These limitations prompted the exploration of alternative solutions, leading to the development of the Liquid Electron Ionization (LEI) interface. The system has demonstrated excellent robustness and reproducibility. LEI has been employed to analyze various compounds, including pesticides, drugs of abuse, phenols, polycyclic aromatic hydrocarbons (PAHs), phthalates, and many others. Its versatility has been validated with single quadrupole, triple quadrupole, and QToF detectors, operating in electron ionization (EI) or chemical ionization (CI) modes and with both reverse phase liquid chromatography (RPLC) and normal phase liquid chromatography (NPLC). LEI has also been successfully integrated with the Microfluidic Open Interface (MOI), Membrane Introduction Mass Spectrometry (MIMS), and Microfluidic Water-Assisted Trap Focusing (M-WATF), broadening its application scope and consistently demonstrating promising results in terms of sensitivity and identification power. The most recent advancement is the development of Extractive-Liquid Sampling Electron Ionization-Mass Spectrometry (E-LEI-MS), a surface sampling and real-time analysis technique based on the LEI concept. This review article offers a comprehensive and up-to-date picture of the potential of LEI.

Photodecomposition of antibiotics and their transformation products in wastewaters using ZnO and TiO2 with LED lamps

This work presents a study of the behavior of three antibiotics (erythromycin (ERY), clarithromycin (CLR) and sulfadiazine (SFZ)) and their transformation products (TPs), generated after photolytic and photocatalytic (TiO2 and ZnO) treatments, in wastewater irradiated with ultraviolet (UV) light emitting diode (LED) lamps as light source. For this, an analytical methodology for the antibiotics and their TPs was developed. Thus, samples were treated by salting-out assisted liquid–liquid extraction (SALLE) and identification and quantification was carried out by liquid chromatography with mass spectrometry (LC-MS) using quadrupole time-of-flight (QTOF) and triple quadrupole (QqQ) analyzers, respectively. The results revealed the presence of 14, 13 and 5 TPs of ERY, CLR and SFZ, respectively, being found in wastewaters spiked with the antibiotics during photo-oxidation treatment. Subsequently, the established analytical method was also applied, in a real case, to study of behavior of these pharmaceuticals (0.2 mg L-1) and their TPs generated in wastewater during a photocatalytic treatment. In this case, 13, 11 and 2 TPs were tentatively identified ERY, CLR and SFZ, respectively. In addition, the photocatalytic treatment using TiO2 exhibited higher degradation rate than photocatalytic treatment using ZnO. The ECOSAR programme was used to estimate the potential ecotoxicity of the identified TPs. The results showed a potential hazard of SFZ, CLR, ERY and some of their TPs to aquatic organisms. Furthermore, some of the identified TPs were found to be more toxic than their parent compounds.

Hydrophilic Interaction Liquid Chromatography-Hydrogen/Deuterium Exchange-Mass Spectrometry (HILIC-HDX-MS) for Untargeted Metabolomics.

Liquid chromatography with mass spectrometry (LC-MS)-based metabolomics detects thousands of molecular features (retention time-m/z pairs) in biological samples per analysis, yet the metabolite annotation rate remains low, with 90% of signals classified as unknowns. To enhance the metabolite annotation rates, researchers employ tandem mass spectral libraries and challenging in silico fragmentation software. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) may offer an additional layer of structural information in untargeted metabolomics, especially for identifying specific unidentified metabolites that are revealed to be statistically significant. Here, we investigate the potential of hydrophilic interaction liquid chromatography (HILIC)-HDX-MS in untargeted metabolomics. Specifically, we evaluate the effectiveness of two approaches using hypothetical targets: the post-column addition of deuterium oxide (D2O) and the on-column HILIC-HDX-MS method. To illustrate the practical application of HILIC-HDX-MS, we apply this methodology using the in silico fragmentation software MS-FINDER to an unknown compound detected in various biological samples, including plasma, serum, tissues, and feces during HILIC-MS profiling, subsequently identified as N1-acetylspermidine.

Phytomedicine Fructus Aurantii-derived two absorbed compounds unlock antidepressant and prokinetic multi-functions via modulating 5-HT3/GHSR

Ethnopharmacological relevanceFructus Aurantii (FA), a well-known phytomedicine, has been employed to evoke antidepressant and prokinetic multi-functions. Therein, systematically identifying bioactive components and the referred mechanism is essential for FA. Aim of the studyThis study was planned to answer “2 W” (What and Why), such as which components and pathways contribute to FA's multi-functions. We aimed to identify bioactive compounds as the key for opening the lock of FA's multi-functions, and the molecule mechanisms are their naturally matched lock cylinder. Materials and methodsThe phytochemical content of FA extract was determined, and the compounds were identified in rats pretreated with FA using liquid chromatography with mass spectrometry (LC-MS). The contribution strategy was used to assess bioactive compounds’ efficacy (doses = their content in FA) in model rats with the mechanism. The changes in functional brain regions were determined via 7.0 T functional magnetic resonance imaging-blood oxygen level-dependent (fMRI-BOLD). ResultEight phytochemicals’ content was detected, and merely six components were identified in rats in vivo. Meranzin hydrate + hesperidin (MH), as the primary contributor of FA, exerted antidepressant and prokinetic effects (improvement of indexes for immobility time, gastric emptying, intestinal transit, CRH, ghrelin, ACTH, DA, NA, 5-HT, CORT, and 5-HT3) by regulating 5-HT3/Growth hormone secretagogue receptor (GHSR) pathway. These results were validated by 5-HT2A, 5-HT3, and GHSR receptor antagonists combined with molecule docking. MH restored the excessive BOLD activation of the left accumbens nucleus, left corpus callosum and hypothalamus preoptic region. ConclusionAbsorbed MH accounts for FA's anti-depressant and prokinetic efficacy in acutely-stressed rats, primarily via 5-HT3/GHSR shared regulation.

Ultra-rapid determination of andrographolide and dehydroandrographolide in Andrographis Herba by solid phase extraction and liquid chromatography with mass spectrometry

AbstractAn ultra-rapid analytical method for determination of andrographolide and dehydroandrographolide in Andrographis Herba (AH) was developed by liquid chromatography with mass spectrometry (LC-MS). The sample was ultrasonically extracted with 10 mL 40% (v/v) methanol, and then purified with a C18 solid phase extraction column. The LC separation was performed on a Poroshell 120 EC-C18 column (30 × 2.1 mm, 2.7 μm) and eluted with 0.5 mmol L−1 ammonium acetate aqueous solution and acetonitrile (65:35) at a flow rate of 0.7 mL min−1, and detected by mass spectrometry (MS). The LC-MS analytical time was less than 1 min. The new developed method presented a good linearity (r > 0.9900), precision and repeatability (RSD < 2.0%). The recoveries for andrographolide and dehydroandrographolide were 93.5% (RSD = 2.2%) and 97.7% (RSD = 2.4%), respectively. The developed method was successfully applied in determination of andrographolide and dehydroandrographolide in seven batches of AH samples, and the contents of analytes in all samples were complied with the relative acceptance criteria in Chinese Pharmacopeia (>0.8%). This new developed LC-MS method is an ultra-rapid assay method for AH, which will help to improve the efficiency and reduce the cost of AH sample test.

Dysregulated Gln-Glu-α-ketoglutarate axis impairs maternal decidualization and increases the risk of recurrent spontaneous miscarriage

Recurrent spontaneous miscarriage (RSM) affects 1%-2% of fertile women worldwide and poses a risk of future pregnancy complications. Increasing evidence has indicated that defective endometrial stromal decidualization is a potential cause of RSM. Here, we perform liquid chromatography with mass spectrometry (LC-MS)-based metabolite profiling in human endometrial stromal cells (ESCs) and differentiated ESCs (DESCs) and find that accumulated α-ketoglutarate (αKG) derived from activated glutaminolysis contributes to maternal decidualization. Contrarily, ESCs obtained from patients with RSM show glutaminolysis blockade and aberrant decidualization. We further find that enhanced Gln-Glu-αKG flux decreases histone methylation and supports ATP production during decidualization. Invivo, feeding mice a Glu-free diet leads to a reduction of αKG, impaired decidualization, and an increase of fetal loss rate. Isotopic tracing approaches demonstrate Gln-dependent oxidative metabolism as a prevalent direction during decidualization. Our results demonstrate an essential prerequisite of Gln-Glu-αKG flux to regulate maternal decidualization, suggesting αKG supplementation as a putative strategy to rectify deficient decidualization in patients with RSM.

Tetragonal SnO2 Nanoparticles: An Efficient Photocatalyst for the Degradation of Hazardous Ionic Dyes

AbstractAn elegant approach was made to synthesize tetragonal shape SnO2 nanoparticles using solution combustion method and subjected to various characterization (X‐ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), UV‐Vis diffuse reflectance spectroscopy (UV‐DRS), Photoluminescence (PL) spectroscopy, Raman spectra, thermogravimetric analysis (TGA), differential thermal analysis (DTA), X‐ray photoelectron spectrometer (XPS), N2 adsorption‐desorption isotherms (BET), liquid chromatography with mass spectrometry (LCMS). Dynamic light scattering (DLS) and Zeta potential (ZP), field emission scanning electron microscopy (FESEM), energy dispersive X‐rays analysis (EDAX), and High‐resolution transmission electron microscopy (HRTEM)). Further, the as‐synthesized SnO2 nanoparticles were used for the degradation study of hazardous ionic dyes like cationic dye Malachite green (MG) and anionic dye Rose Bengal (RB), as well as its binary mixture (MG+RB) under the sunlight. Overall, it was observed that MG and RB degraded 100 % (80 min) and 99 % (90 min), while the mixture of (MG+RB) showed 97.5 % (140 min), respectively. Overall study shows that the degradation rate of MG in a mixture of both the dyes was higher than RB due to a positively charged surface. The time taken for degradation MG was 10 min less than that of RB. The fast degradation of MG was due to the cationic nature having a positive charge over the surface with the negatively charged surface of SnO2 nanoparticles, which is entirely different in the case of anionic dye RB.

An ultra-rapid and eco-friendly method for determination of loganic acid and gentiopicroside from Gentianae Macrophyllae Radix by vortex-assisted matrix solid-phase dispersion extraction and LC-MS

An ultra-rapid and eco-friendly method for the determination of loganic acid and gentiopicroside in Gentianae Macrophyllae Radix (GMR) was developed by vortex-assisted matrix solid-phase dispersion extraction (VAMSPD) and liquid chromatography with mass spectrometry (LC-MS). The optimized VAMSPD parameters are as follow: sample-dispersant (diatomaceous earth) ratio is 1:5, grinding for 0.5 min and whirling with 0.5 mL 15 % ethanol for 0.5 min. The LC separation is performed on a Poroshell 120 EC-C18 column (30 ×2.1 mm, 2.7 µm) and eluted by an eco-friendly mobile phase (14 % ethanol containing 0.1 % formic acid) at a flow rate of 0.5 mL min−1 in isocratic mode, and detected by mass spectrometry (MS). The developed method exhibits a good linearity for the analytes (r > 0.9990). The RSDs of precision and repeatability are less than 4.0 %, the recoveries for loganic acid and gentiopicroside are 106.5 % (RSD=3.6 %) and 95.7 % (RSD=8.0 %), respectively. The developed method was successfully applied in the analysis of loganic acid and gentiopicroside in GMR samples. The total analysis time is 2 min, including 1 min for sample extraction and 1 min for LC-MS analysis. In addition, the method only requires 0.3 mL of ethanol.

Direct detection of OXA-48-like carbapenemase variants with and without co-expression of an extended-spectrum β-lactamase from bacterial cell lysates using mass spectrometry

IntroductionAntibiotic-resistant Gram-negative bacteria are of a growing concern globally, especially those producing enzymes conferring resistance. OXA-48-like carbapenemases hydrolyze most β-lactam antibiotics, with typically low-level hydrolysis of carbapenems, but have limited effect on broad-spectrum cephalosporins. These are frequently co-expressed with extended spectrum β-lactamases, especially CTX-M-15, which typically shows high level resistance to broad-spectrum cephalosporins, yet is carbapenem susceptible. The combined resistance profile makes the need for successful detection of these specific resistance determinants imperative for effective antibiotic therapy. ObjectivesThe objective of this study is to detect and identify OXA-48-like and CTX-M-15 enzymes using mass spectrometry, and to subsequently develop a method for detection of both enzyme types in combination with liquid chromatography. MethodsCells grown in either broth or on agar were harvested, lysed, and, in some cases buffer-exchanged. Lysates produced from bacterial cells were separated and analyzed via liquid chromatography with mass spectrometry (LC-MS) and tandem mass spectrometry (LC-MS/MS). ResultsThe intact proteins of OXA-48, OXA-181, and OXA-232 (collectively OXA-48-like herein) and CTX-M-15 were characterized and detected. Acceptance criteria based on sequence-informative fragments from each protein group were established as confirmatory markers for the presence of the protein(s). A total of 25 isolates were successfully tested for OXA-48 like (2), CTX-M-15 (3), or expression of both (7) enzymes. Thirteen isolates served as negative controls. ConclusionsHere we present a method for the direct and independent detection of both OXA-48-like carbapenemases and CTX-M-15 β-lactamases using LC-MS/MS. The added sensitivity of MS/MS allows for simultaneous detection of at least two co-eluting, co-isolated and co-fragmented proteins from a single mass spectrum.

Comparative Characterization of Aspergillus Pectin Lyases by Discriminative Substrate Degradation Profiling.

Fungal genomes often contain several copies of genes that encode carbohydrate active enzymes having similar activity. The copies usually have slight sequence variability, and it has been suggested that the multigenecity represents distinct reaction optima versions of the enzyme. Whether the copies represent differences in substrate attack proficiencies of the enzyme have rarely been considered. The genomes of Aspergillus species encode several pectin lyases (EC 4.2.2.10), which all belong to polysaccharide lyase subfamily PL1_4 in the CAZy database. The enzymes differ in terms of sequence identity and phylogeny, and exhibit structural differences near the active site in their homology models. These enzymes catalyze pectin degradation via eliminative cleavage of the α-(1,4) glycosidic linkages in homogalacturonan with a preference for linkages between methyl-esterified galacturonate residues. This study examines four different pectin lyases (PelB, PelC, PelD, and PelF) encoded by the same Aspergillus sp. (namely A. luchuensis), and further compares two PelA pectin lyases from two related Aspergillus spp. (A. aculeatus and A. tubingensis). We report the phylogeny, enzyme kinetics, and enzymatic degradation profiles of the enzymes’ action on apple pectin, citrus pectin, and sugar beet pectin. All the pectin lyases exerted highest reaction rate on apple pectin [degree of methoxylation (DM) 69%, degree of acetylation (DAc) 2%] and lowest reaction rate on sugar beet pectin (DM 56%, DAc 19%). Activity comparison at pH 5–5.5 produced the following ranking: PelB > PelA > PelD > PelF > PelC. The evolution of homogalacturonan-oligomer product profiles during reaction was analyzed by liquid chromatography with mass spectrometry (LC-MS) detection. This analyses revealed subtle differences in the product profiles indicating distinct substrate degradation preferences amongst the enzymes, notably with regard to acetyl substitutions. The LC-MS product profiling analysis thus disclosed that the multigenecity appears to provide the fungus with additional substrate degradation versatility. This product profiling furthermore represents a novel approach to functionally compare pectin-degrading enzymes, which can help explain structure-function relations and reaction properties of disparate copies of carbohydrate active enzymes. A better understanding of the product profiles generated by pectin modifying enzymes has significant implications for targeted pectin modification in food and biorefinery processes.

NSAIDs detected in Iberian avian scavengers and carrion after diclofenac registration for veterinary use in Spain

Despite the now well recognised impact of diclofenac on vultures across the Indian subcontinent, this non-steroidal anti-inflammatory drug (NSAID) was registered in 2013 for livestock treatment in Spain, Europe’s main vulture stronghold. We assessed the risk of exposure to diclofenac and nine other NSAIDs in avian scavengers in the Iberian Peninsula (Spain and Portugal) after the onset of diclofenac commercialization. We sampled 228 livestock carcasses from vulture feeding sites, primarily pig (n = 156) and sheep (n = 45). We also sampled tissues of 389 avian scavenger carcasses (306 Eurasian griffon vultures, 15 cinereous vultures, 11 Egyptian vultures, 12 bearded vultures and 45 other facultative scavengers). Samples were analysed by liquid chromatography with mass spectrometry (LCMS). Seven livestock carcasses (3.07%) contained NSAID residues: flunixin (1.75%), ketoprofen, diclofenac and meloxicam (0.44% each). NSAID residues were only detected in sheep (4.44%) and pig (3.21%) carcasses. Fourteen dead avian scavengers (3.60%) had NSAID residues in kidney and liver, specifically flunixin (1.03%) and meloxicam (2.57%). Flunixin was associated with visceral gout and/or kidney damage in three (0.98%) dead Eurasian griffons. To date, diclofenac poisoning has not been observed in Spain and Portugal, however, flunixin would appear to pose an immediate and clear risk. This work supports the need for well managed carrion disposal, alongside appropriate risk labelling on veterinary NSAIDs and other pharmaceuticals potentially toxic to avian scavengers.

Characterization of Bispecific Antibody Production in Cell Cultures by Unique Mixed Mode Size Exclusion Chromatography.

Bispecific antibodies have received wide attention as promising immunotherapeutic agents because of their high specificity and the ability to target immune cells to tumors. However, analysis of bispecific antibodies is challenging because multiple forms of antibodies are potentially generated during production in cell culture. Most analyses of bispecific antibodies rely on liquid chromatography with mass spectrometry (LC-MS), which could miss detection or becomes less quantitative if those forms are not physically separated. Here, we report a novel and sensitive mixed mode size exclusion chromatography (MM SEC) coupled with multiangle light scattering (MALS) to analyze different forms of bispecific IgG molecules under native conditions. The method displayed great ability to separate various antibody forms with peak resolutions unmatched by other methods we tested, isolating desired bispecific molecules, parental homodimers, half molecules, and antibodies with mispaired light and heavy chains. Each peak was analyzed by online MALS and then identified and confirmed by intact and reduced LC-MS of isolated forms. MM SEC in this study performs by a novel mechanism through the interactions of resin with protein surface hydrophobic clusters distributed across CDRs of light chains. This novel MM SEC allows quantitative detection of even low abundance forms and provides a new tool for screening expression profiles of cell culture clones, monitoring purification, and evaluating drug substance purity.

Validation of a liquid chromatography coupled with tandem mass spectrometry method for the determination of drugs in wastewater using a three-phase solvent system.

Pharmaceuticals constitute one of the most important emerging classes of environmental pollutants. A three-phase solvent system of water, water containing 0.1% of formic acid and acetonitrile was successfully used to separate, by liquid chromatography with mass spectrometry (LC-MS), polarity-matched pharmaceuticals, that is, carbamazepine, clarithromycin, and erythromycin, as well as amoxicillin and metformin. Despite of polarity similarities, these pharmaceuticals were completely resolved in the analytical run time of 15 min. The optimized three-phase solvent system based-method was validated for the simultaneous analysis of six matched-polarity pharmaceuticals in wastewater samples. Good linearity (coefficient of determination more than 0.993) and precision (relative standard deviation less than 15.66%) were achieved. Recovery of analytes from the wastewater was between 0.70 and 1.18. Limits of detections ranged from 0.0001 to 0.5114µg/L. No significant matrix effect, evaluated by post extraction addition, was observed in the electrospray ionization (ESI) source. Then, this methodology has been successfully applied to environmental study of pharmaceutical residues occurring in influent and effluent wastewater samples, from the main wastewater treatment plant in Potenza (Basilicata, Southern Italy).

Liquid chromatographic methods for separation, determination, and bioassay of enantiomers of etodolac: A review.

The control of enantiomeric purity and determination of individual enantiomeric drug molecules remains the subject of importance for clinical, analytical, and regulatory purposes and to facilitate an accurate evaluation of the risks posed by them to human health. A large number of pharmaceuticals are marketed and administered as racemates. Etodolac is among such nonsteroidal anti-inflammatory drugs. Overall literature reports on its enantioseparation are scanty. Liquid chromatography (LC) methods of enantioseparation of (±)-etodolac, including certain unconventional ones, are well covered and discussed in this paper. Methods of direct approach without using chiral columns or chiral thin-layer chromatography plate and of indirect approach using certain chiral derivatizing agents such as (S)-naproxen and (S)-levofloxacin are described. Most interesting aspects include establishment of structure and molecular asymmetry of chemically different types of diastereomeric derivatives using liquid chromatography with mass spectrometry (LC-MS), 1 H NMR spectroscopy and by drawing conformations in three dimensional views by using certain software. The methods provide chirality recognition even in the absence of pure enantiomers. Besides, recovery of pure enantiomers by detagging or via solubility difference of chiral inducing reagent and the analyte, without racemization at any stage, has been achieved. The limits of detection and quantification are much lower than the industry benchmarks.

Characterization of Cell Glycocalyx with Mass Spectrometry Methods.

The cell membrane plays an important role in protecting the cell from its extracellular environment. As such, extensive work has been devoted to studying its structure and function. Crucial intercellular processes, such as signal transduction and immune protection, are mediated by cell surface glycosylation, which is comprised of large biomolecules, including glycoproteins and glycosphingolipids. Because perturbations in glycosylation could result in dysfunction of cells and are related to diseases, the analysis of surface glycosylation is critical for understanding pathogenic mechanisms and can further lead to biomarker discovery. Different mass spectrometry-based techniques have been developed for glycan analysis, ranging from highly specific, targeted approaches to more comprehensive profiling studies. In this review, we summarized the work conducted for extensive analysis of cell membrane glycosylation, particularly those employing liquid chromatography with mass spectrometry (LC-MS) in combination with various sample preparation techniques.

Influence of different types of acids and pH in the recovery of bioactive compounds in Jabuticaba peel (Plinia cauliflora).

Jabuticaba peel presents a high content of bioactive compounds such as phenolic acids, flavonoids, and anthocyanins, normally considered as a food residue. Nowadays, there is a great interesting in the recovery of bioactive compounds from food residue due to health benefits of the ingredients produced, environmental issues and economic aspects. For the success of phenolic compounds extraction, the solvent and pH influence recovery of these compounds. However, studies that evaluate the use of different weak acids bioactive compounds recovery are scarce. Thus, the aim of the present work was to evaluate the effect of formic, acetic, and phosphoric acids addition in the extraction solvent, to adjust the pH to 1.0, 2.0 and 3.0, in bioactive compounds recovery and antioxidant capacity of jabuticaba peel. The extracts were analyzed as antioxidant capacity (ORAC, FRAP), total phenols content monomeric anthocyanin's and a qualitative analysis of phenolics by Liquid Chromatography with mass spectrometry (LC-MS). The kind of acid used in the extraction process affected mainly in the extraction of anthocyanins. The acid that presented a better recovery of anthocyanin (3.4 mg/g raw material) and a better antioxidant capacity (ORAC) (841 μmol TE/g raw material) was formic acid in pH 1.0.

Dysregulated serum metabolites in staging of hepatocellular carcinoma

BackgroundCorrect staging of hepatocellular carcinoma (HCC) could help physicians to precisely select treatments for patients, such as surgery, chemotherapy, or their combination. The objective of this study was to explore potential metabolic markers for staging of hepatocellular carcinoma. MethodsBy liquid chromatography with mass spectrometry (LC-MS), the serum metabolic profiles of 60 pathologically confirmed hepatocellular carcinoma (HCC) patients were analyzed using the TNM staging system and Chinese staging system. ResultsThe serum levels of dihydrocortisol, lysophosphatidylcholine (LPC-18:0), lysophosphatidylethanolamine (LPE-16:0), taurine, uric acid, adipic acid, tetracosatetraenoic acid, and L-octanoylcarnitine differed significantly between staging I and non-stage I HCCs (p < 0.05) based on the HCC TNM staging system, and compared to stage I sera, non-stage I sera contained higher levels of dihydrocortisol, adipic acid, tetracosatetraenoic acid, and L-octanoylcarnitine. There are significant differences were observed in serum levels of LPC (22:6), alpha-linolenylcarnitine, estrone, LPE (16:0), LPE (18:2), and taurine between stage I and stage II HCCs (p < 0.05) based on the Chinese HCC staging system, and compared to stage I sera, stage II sera had a higher level of LPC (22:6). ConclusionThese dysregulated metabolites in sera of HCC patients potentially could be used as biomarkers for the clinical staging of HCC.

Algal\u2013bacterial synergy in treatment of winery wastewater

There is significant potential for employing algae in tertiary wastewater treatment, however, little is known about the contribution of algae-bacteria synergy toward treatment performance. This study demonstrates potential synergy in the treatment of three winery wastewater samples. Two strains of green algae, Auxenochlorella protothecoides and Chlorella sorokiniana were tested and each removed > 90% of nitrogen, > 50% of phosphate, and 100% of acetic acid in the wastewater. Both algae strains grew significantly faster on wastewaters compared to growth on minimal media. Organic carbon in the wastewater apparently played a limited role in algal growth enhancement. When cultured on sterile-filtered wastewater, A. protothecoides increased soluble COD loadings in two of the three wastewaters and C. sorokiniana secreted an insoluble film. Culturing algae with the native wastewater microbial community negated the secretion of algal photosynthate, allowing for simultaneous reductions in COD and nutrient concentrations. Both algae species stimulated bacterial growth in a strain-specific way, suggesting unique responses to algal photosynthate. Cofactor auxotrophy for thiamine, cobalamin, and biotin is widespread among algae and these cofactors are typically obtained from bacteria. Sequencing the wastewater microbial community revealed bacteria capable of synthesizing all three cofactors while liquid chromatography with mass spectrometry (LCMS) and bio-assays revealed the presence of thiamine metabolites in the wastewaters. These cofactors likely increased algal growth rates, particularly for A. protothecoides, which cannot synthesize thiamine de-novo but can salvage it from degradation products. Collectively, these results demonstrate that bacteria and algae provided synergistic growth benefits, potentially contributing to higher levels of wastewater treatment than either organism type alone.

Detection and Identification of the Vibrational Markers for the Quantification of Methionine Oxidation in Therapeutic Proteins.

Methionine oxidation is a major degradation pathway in therapeutic proteins which can impact the structure and function of proteins as well as risk to drug product quality. Detecting Met oxidation in proteins by peptide mapping followed by liquid chromatography with mass spectrometry (LC-MS) is the industry standard but is also labor intensive and susceptible to artifacts. In this work, vibrational difference spectroscopy in combination with 18O isotopic shift enabled us to demonstrate the application of Raman and FTIR techniques for the detection and quantification of Met oxidation in various therapeutic proteins, including mAbs, fusion proteins, and antibody drug conjugate. Vibrational markers of Met oxidation products, such as sulfoxide and sulfone, corresponding to S═O and C-S═O stretching frequencies were unequivocally identified based 18O isotoptic shifts. The intensity of the isolated νC-S Raman band at 702 cm-1 was successfully applied to quantify the average Met oxidation level in multiple proteins. These results are further corroborated by oxidation levels measured by tryptic peptide mapping, and thus the confirmed Met oxidation levels derived from Raman and mass spectrometry are indeed consistent with each other. Thus, we demonstrate the broader application of vibrational spectroscopy to detect the subtle spectral changes associated with various chemical or physical degradation of proteins, including Met oxidation as well as higher order structural changes.

4-hydrazinobenzoic acid as a derivatizing agent for aldehyde analysis by HPLC-UV and CE-DAD

Aldehydes are relevant analytes in a wide range of samples, in particular, food and beverages but also body fluids. Hydrazines can undergo nucleophilic addition with aldehydes or ketones giving origin to hydrazones (a group of stable imines) that can be suitably used in the identification of aldehydes. Herein, 4-hydrazinobenzoic acid (HBA) was, for the first time, used as the derivatizing agent in analytical methodologies using liquid chromatography aiming the determination of low-molecular aldehydes. The derivatization reaction was simultaneously performed along with the extraction process, using gas-diffusion microextraction (GDME), which resulted in a clean extract containing the HBA-aldehyde derivates. The corresponding formed imines were determined by both high-performance liquid chromatography (LC) with UV spectrophotometric detection (HPLC-UV) and capillary electrophoresis with diode array detection (CE-DAD). HBA showed to be a rather advantageous derivatization reagent due to its stability, relatively high solubility in water and other solvents, high selectivity and sensibility, reduced impurities, simple preparation steps and applicability to different separation and/or different detection techniques. Limits of detections (LODs) of the optimized methodologies (in terms of time and pH among other experimental variables) were all below 0.5 mg L−1, using both instrumental techniques. Furthermore, for the first time, the HBA-aldehyde derivatives were analyzed by LC with mass spectrometry (LC-MS), demonstrating the possibility of identification by MS of each compound. The developed methodologies were also successfully applied in the analysis of formaldehyde and acetaldehyde in several alcoholic beverages. This was also the first time GDME was combined with CE, showing that it can be a valuable sample preparation tool for electrophoresis, in particular by eliminating the interference of ions and inorganic constituents present in the samples.