Liquid Chromatography Coupled With Mass Spectrometry Research Articles

The Inositol Pyrophosphate Biosynthetic Pathway of Trypanosoma cruzi.

Inositol phosphates (IPs) are phosphorylated derivatives of myo-inositol involved in the regulation of several cellular processes through their interaction with specific proteins. Their synthesis relies on the activity of specific kinases that use ATP as phosphate donor. Here, we combined reverse genetics and liquid chromatography coupled to mass spectrometry (LC-MS) to dissect the inositol phosphate biosynthetic pathway and its metabolic intermediates in the main life cycle stages (epimastigotes, cell-derived trypomastigotes, and amastigotes) of Trypanosoma cruzi, the etiologic agent of Chagas disease. We found evidence of the presence of highly phosphorylated IPs, like inositol hexakisphosphate (IP6), inositol heptakisphosphate (IP7), and inositol octakisphosphate (IP8), that were not detected before by HPLC analyses of the products of radiolabeled exogenous inositol. The kinases involved in their synthesis (inositol polyphosphate multikinase (TcIPMK), inositol 5-phosphate kinase (TcIP5K), and inositol 6-phosphate kinase (TcIP6K)) were also identified. TcIPMK is dispensable in epimastigotes, important for the synthesis of polyphosphate, and critical for the virulence of the infective stages. TcIP5K is essential for normal epimastigote growth, while TcIP6K mutants displayed defects in epimastigote motility and growth. Our results demonstrate the relevance of highly phosphorylated IPs in the life cycle of T. cruzi.

Simultaneous Estimation of Aflatoxins (B1, B2, G1 and G2) by Liquid Chromatography Coupled with Mass Spectrometry (LC-MS) in Corn Samples

Aflatoxins produced by Aspergillus flavins and Aspergillus pasasiticus have been investigated in the corn (maize) collected in different locations in Andhra Pradesh state, India. In the present study, 24 corn samples collected from different harvest, storage and dumping areas were analyzed for detection of aflatoxin contamination by liquid chromatography coupled with mass spectrometry (LC-MS). Mixed aflatoxin working standard solutions with a concentration of 10, 20, 30, 40, 50 and 60 ng/mL were prepared and followed by instrumental analysis and the retention times of the sample chromatograms were compared with the standard aflatoxins B1, B2, G1 and G2 retention times in order to determine the contamination. The findings revealed that among 24 collected samples, only 10 samples have been found positive for aflatoxins contamination when subjected to LC-MS analysis. Co-occurrence of aflatoxin was identified in the most of the samples. Even though 10 samples were tested positive total aflatoxin content in the most positive are found within the acceptable limits (30 μg/kg) and samples collected at dumping areas (S4, S8 and S10) show the presence of exceeding quantity than the limits described by FSSAI. Total aflatoxin content in the most positive were found within the acceptable limits (30 μg/kg) and the samples collected at dumping areas S4 (40.9 μg/kg), S8 (53.2 μg/kg) and S10 (71.86 μg/kg) had shown exceeding limits. The sample S7 collected from the storage was also found positive of presence of aflatoxin G2- 13.49 μg/kg and B2-21.14 μg/kg with total aflatoxin of 34.65 μg/kg. High contamination of these dumping areas may lead to contamination of storage points nearer to these areas. Present results showed that the corn samples at dumping areas were found to be contaminated with the Aspergillus flavins and Aspergillus pasasiticus.

Stability Analyses by HPLC-MS of Guanitoxin Isolated from Sphaerospermopsis torques-reginae

Guanitoxin (GNT) is a natural organophosphate produced by some species of freshwater cyanobacteria, which inhibits the active site of acetylcholinesterase, preventing the hydrolysis of cholinesterases and consequently causing serious disturbances in the neuromuscular system. Despite having a chemical structure like synthetic organophosphates, there is still no analytical standard available for environmental and freshwater monitoring. Therefore, this study investigated the stability of GNT under different storage conditions, pH, and temperature. The toxin is produced by the cyanobacterium Sphaerospermopsis torques-reginae and monitored by liquid chromatography coupled to mass spectrometry (LC-MS) and LC-MS/MS for the identification and verification of its stability. The main degradation product formed is the hydroxy-amino-guanidinic derivative of the toxin. The results also indicate that GNT is stable in acidic medium (pH = 3.0), but can gradually degrade at room temperature (> 23 ºC) over a period of 96 h. Lyophilized biomass of S. torques-reginae containing GNT remained stable when stored in a refrigerator below 4 ºC. In addition, the extraction yield is higher when prepared from fresh S. torques-reginae cells than from lyophilized material. Thus, the results shown here contribute with valuable information for studies that aim at the isolation, identification, and monitoring of GNT in samples of raw water and cyanobacterial blooms.

Determination of sweeteners in wine by liquid chromatography coupled with mass spectrometry (LC/MS)

Sweeteners are food additive substances that give a sweet taste to foods but their use in oenological practices is forbidden. Making use of the capabilities of liquid chromatography coupled with mass spectrometry, a method for wine analysis was developed and validated for the detection and quantitation of some of the most widely used sweeteners: aspartame, potassium acesulfame, sodium cyclamate, saccharin, sucralose and stevioside. A matrix-matched calibration was used for all compounds obtaining a linear concentration range from 50 μg/L to 1000 μg/L. The limit of detection ranged from 0.002 mg/L to 0.014 mg/L, and the limit of quantification varied between 0.005 mg/L and 0.048 mg/L. Precision and recovery were assessed for 50 μg/L, 250 μg/L and 1000 μg/L with repeatability and intermediate precision values from 0.6% to 21.6% and 2.7% to 26.4% respectively, and recoveries ranging from 60% to 126%. These results were achieved using minimal sample preparation with a fast and high throughput method that is applicable to a wide range of wine matrices.

Novel Method for the Extraction of Cocaine from Oral Fluid by Means of Disposable Pipette Modified with Restricted Access Material

This study proposes a new, efficient, and selective method for the analysis of cocaine in oral fluid. For this purpose, an extractive phase with restricted access materials (RAM) capable of extract and pre-concentrate low molecular weight analytes and simultaneously exclude macromolecules was developed for disposable pipette extraction (DPX) followed by liquid chromatography coupled to mass spectrometry (LC-MS) analyses. The proposed method was optimized using a 24 factorial design which pointed higher extraction efficiency under the following conditions: pH 9, 10 cycles of extraction (aspirate/dispense), 3 cycles of desorption (aspirate/dispense) and acetonitrile as desorption solvent. The DPX-RAM/LC-MS method showed linearity range from 10 to 100 ng mL-1 with determination coefficient (R2) = 0.999, accuracy values ranged from -0.9 to 4.3%, precision values ranged from 1.7 to 9.1% and recovery values ranged from 97.9 to 101.1%. The benefits of this method are that it can be performed in few minutes and that the analytical performances, including the limit of detection (3.13 ng mL-1), limit of quantitation (10 ng mL-1) and linear range were found to be in agreement with other methods used for similar analysis.

Phosphatase and Kinase Substrate Specificity Profiling with Pooled Synthetic Peptides and Mass Spectrometry.

Reversible phosphorylation is a pervasive regulatory event in cellular physiology controlled by reciprocal actions of protein kinases and phosphatases. Determining the inherent substrate specificity of kinases and phosphatases is essential for understanding their cellular roles. Synthetic peptides have long served as substrate proxies for defining intrinsic kinase and phosphatase specificities. Here, we describe a high throughput protocol to simultaneously measure specificity constants (kcat/KM) of many synthetic peptide substrates in a single pool using label-free quantitative mass spectrometry. The generation of specificity constants from a single pooled reaction provides a rigorous and rapid comparison of substrate variants to help define an enzyme's specificity. Equally applicable to kinases and phosphatases, as well as other enzyme classes, the protocol consists of three general steps: (1) reaction of enzyme with pooled peptide substrates, each ideally with a unique mass and at concentrations well below KM, (2) analysis of reaction products using liquid chromatography-coupled mass spectrometry (LC-MS), and (3) automated extraction and integration of elution peaks for each substrate/product pair. We incorporate an ionization correction strategy allowing direct calculation of reaction progress, and subsequently kcat/KM, from substrate and product peak areas in a single sample, obviating the need for stable isotope labeling. Peptide consumption is minimal, and high peptide purity and accurate concentrations are not required. Access to a high-resolution LC-MS system is the only nonstandard equipment need. We present an analysis pipeline consisting entirely of established open-source software tools, and demonstrate proof of principle with the highly selective cell cycle phosphatase Cdc14 from Saccharomyces cerevisiae.

Influence of Ni (II) oxime complex coupled with the combination of diverse sized ZnO nanoparticles on Photovoltaic Performance

The one side selective synthesis of quinoline carboxylic acid oxime complex was carried out successfully. The as-prepared quinoline carboxylic acid oxime complex was complexed with nickel (II) salts to form nickel (II) oxime complex. These complexes were further adsorbed onto ZnO films containing ZnO nanoparticles of various sizes. ZnO films containing a diverse proportion of ZnO nanoparticles were investigated to enhance the photovoltaic efficiency of the dye-sensitized solar cell. The as-synthesized complex was characterized by scanning electron microscopy (SEM), Ultra violet visible spectroscopy (UV-vis), Fourier Transform Infrared Spectroscopy (FT-IR) spectroscopy, 1Hydrogen Nuclear magnetic resonance spectroscopy (1HNMR), Liquid chromatography coupled with mass spectrometry (LC-MR), Brunauer–Emmett–Teller (BET), and Attenuated total reflection Infra-red spectroscopy (ATR-IR). The combination of large and small ZnO nanoparticles has significantly improves the photovoltaic efficiency. The optimum mixing ratio for the best performance (0.127%) of a dye-sensitized solar cell is achieved by mixing the small: large ZnO particles in a ratio 60:40. The increased efficiency is due to the harvesting of light caused by scattering effect from larger sized ZnO particles. The ZnO layer consisting of smaller particles which are very next to the ZnO bigger particles makes a good electronic contact between film electrode and the Indium-doped tin oxide glass substrate resulting in the increases in the dye molecules adsorption. The over-layered, large-sized ZnO particles enhance the light-harvesting by light scattering effect. Compared to the other mixtures of ZnO films, there is a decrease in the photovoltaic performance of the solar cell when ZnO particles (small and large in a ratio 1:1) were adsorbed onto the Ni (II) oxime complex, which are caused due to the decrease in the surface area and dye aggregation.

A Method for Diagnosing Organophosphate Pesticide Exposure in Humans using Liquid Chromatography Coupled Tandem Mass Spectrometry.

Organophosphate (OP) pesticides are commonly utilized worldwide for agricultural purposes and pose a health threat through air, ground, and water contamination. Here, we present a convenient method for diagnosing exposure to OP pesticides in humans. This immunoprecipitation method relies on extraction of butyrylcholinesterase (BChE), a biomarker of OP poisoning that adducts OP compounds, from human serum using agarose beads conjugated to anti-BChE antibodies. Extracted BChE was then digested with pepsin and analyzed for unadducted and OP-adducted peptides by high performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS). To characterize and validate this method, pooled human plasma was exposed to parathion and dichlorvos to form diethoxyphospho, aged ethoxyphospho and dimethoxyphospho adducts with BChE. Untreated plasma was also analyzed for unadducted peptides. Additionally, samples were analyzed using Ellman's assay to measure BChE functional activity. The percent inhibition of BChE was 53.5±5.76 and 95.2±0.37%, respectively, for plasma treated with parathion for 1 hour and 24 hours. The percent inhibition was 97.2±0.98 for plasma treated with dichlorvos for 1 hour. The percent inhibition was 97.9±0.41% when the plasma treated with parathion for 1 hour, parathion for 24 hour and dichlorvos for 1 hour were mixed. Individual adducts were quantified in a single chromatographic run. Untreated plasma contained 26.4±1.87 ng/mL of unadducted BChE and no adducted peptides. In contrast, the plasma sample treated with both pesticides contained no unadducted BChE, but did contain 9.46±1.10, 10.9±0.98 and 14.1±1.10 ng/mL of diethoxyphospho, aged-ethoxy, and dimethoxyphospho peptides, respectively. The ability to identify and measure BChE and BChE adducts to parathion and dichlorvos is expected to be useful for diagnosing human exposure to multiple OP pesticides.

Combining an effect-based methodology with chemical analysis for antibiotics determination in wastewater and receiving freshwater and marine environment

Two different methodologies were combined to evaluate the risks that antibiotics can pose in the environment; i) an effect-based methodology based on microbial growth inhibition and ii) an analytical method based on liquid-chromatography coupled to mass spectrometry (LC-MS). The first approach was adapted and validated for the screening of four antibiotic families, specifically macrolides/β-lactams, quinolones, sulfonamides and tetracyclines. The LC-MS method was applied for the identification and quantification of target antibiotics; then, the obtained results were combined with ecotoxicological data from literature to determine the environmental risk. The two methodologies were used for the analysis of antibiotics in water samples (wastewater, river water and seawater) and biofluids (fish plasma and mollusk hemolymph) in two monitoring campaigns undertaken in the Ebro Delta and Mar Menor Lagoon (both in the Mediterranean coast of Spain). Both approaches highlighted macrolides (azithromycin) and quinolones (ciprofloxacin and ofloxacin) as the main antibiotics in wastewater treatment plant (WWTP) effluents with potential risk for the environment. However, no risk for the aquatic life was identified in the river, lagoon and seawater as antibiotic levels were much lower than those in WWTP effluents. Fish from Ebro River were the organisms presenting the highest antibiotic concentration when compared with bivalves (mussels) from the Mediterranean Sea and gastropods (marine snails) from the Mar Menor Lagoon. The effect-based methodology successfully determined antibiotic risk in wastewater, but its applicability was less clear in environmental waters such as seawater, due to its high detection limits. Improving sample preconcentration could increase the method sensibility. Overall, combination of both methodologies provides comprehensive insights in antibiotic occurrence and risk associated in areas under study.

Multiresidue method for determination of pesticides in coconut (Cocos nucifera Linn.) endosperm by using GC–MS/MS and UHPLC–MS/MS analysis

Several pesticides are used in the cultivation of coconut palm, hence evaluation of the residues from these substances must be analyzed in the fruit, to provide and ensure the food safety. Therefore, the development and validation of comprehensive and effective methods becomes crucial, given the complexity of the matrix. Thus, a novel method was developed for the determination of 10 different pesticides residues in coconut endosperm. QuEChERS showed to be an efficient method for sample preparation to overcome the complexity of the matrix. To extend the analytical scope, including a wide range of polarities, ultrahigh-performance liquid chromatography coupled mass spectrometry (UHPLC-MS/MS) and gas chromatography coupled mass spectrometry (GC--MS/MS) were employed. Triple quadrupole spectrometer was used in selected reaction monitoring (SRM) on both detectors, providing high sensitivity and selectivity. These approaches showed to be optimal, considering the parameters of linearity (r ≥ 0.99), matrix effect (0–50 %), LOQ (5 μg kg−1, except for thiamethoxam (20 μg kg−1)), specificity, trueness (60–118 %) and precision (repeatability and within-laboratory reproducibility) ≤ 20 %. The concentration levels obtained were higher than the MRL recommended for thiamethoxam (EU regulation <10 μg kg−1), justifying the analysis of such residues in commercial coconut endosperm.

PSVII-9 Post transcriptional modifications may lead to changes in newborn goats’ skeletal muscle proteome as a consequence of maternal feed restriction at different stages of gestation

Abstract We aimed to investigate the impact of maternal feed restriction at different stages of gestation on proteomic profile in the skeletal muscle of newborn goats. A total of 14 pregnant dams were randomly divided into one of the follow dietary treatments: Animals fed at 50% of maintenance requirement from 8-84 d of gestation and then fed at 100% maintenance requirement from day 85 of gestation to parturition (RM, n = 6), and animals fed at 100% of maintenance requirement from 8-84 d of gestation and then fed at 50% maintenance requirement from day 85 of gestation to parturition (MR, n = 8). Longissimus muscle was sampled from male newborn goats and submitted to sarcoplasmic protein extraction and liquid chromatography coupled to mass spectrometry (LC-MS) analysis. The raw data were processed with MaxQuant (1.6.3.3) software with parameters set to default values. Label-free quantification (LFQ) was added and only protein ratios calculated from at least two unique peptides were considered. Our data showed 3 differentially expressed proteins down-regulated in RM (q-value &amp;lt; 0.05). Additionally, we observed proteins present exclusively in each treatment (RM= 137 proteins; MR= 41 proteins). The overall enriched pathways in RM newborn goats are associated with glycolysis (PKM), NADPH synthesis (PGD), lipid oxidation (ECHS1, ACAT1) and citrate cycle (ACO1, OGDH). While the overall enriched pathways in MR newborn goats are associated with glycolysis/gluconeogenesis (GAPDH, ENO) and citrate cycle (PDHA, IDH3A). In addition, correlation analysis between shotgun proteomics and RNAseq data from the same samples showed that there were no relationships between proteins and transcripts observed. These results indicate that maternal feed restriction during different stages of gestation alters enzymes and protein domains abundance associated with nucleotide metabolism in the skeletal muscle of newborn goats. Moreover, the lack of correlation between protein-transcript suggests the importance of post-transcriptional regulation of skeletal muscle metabolism as a consequence of maternal feed restriction at different stages of gestation.

Metabolomics in posttraumatic stress disorder: Untargeted metabolomic analysis of plasma samples from Croatian war veterans

Posttraumatic stress disorder (PTSD) is a severe, multifactorial and debilitating neuropsychiatric disorder, which can develop in a subset of individuals as a result of the exposure to severe stress or trauma. Such traumatic experiences have a major impact on molecular, biochemical and cellular systems, causing psychological and somatic alterations that affect the whole organism. Although the etiology of PTSD is still unclear, it seems to involve complex interaction between various biological genetic and environmental factors. Metabolomics, as one of the rapidly developing “omics” techniques, might be a useful tool for determining altered metabolic pathways and stress-related metabolites as new potential biomarkers of PTSD. The aim of our study was to identify metabolites whose altered levels allow us to differentiate between patients with PTSD and healthy control individuals. The study included two cohorts. The first, exploratory, group included 50 Croatian veterans with PTSD and 50 healthy control subjects, whereas a validation group consisted of 52 veterans with PTSD and 52 control subjects. The metabolomic analysis of plasma samples was conducted using liquid chromatography coupled with mass spectrometry (LC-MS), as well as gas chromatography coupled with mass spectrometry (GC-MS). The LC-MS analysis determined significantly different levels of two glycerophospholipids, PE(18:1/0:0) and PC(18:1/0:0), between control subjects and PTSD patients in both cohorts. The altered metabolites might play a role in multiple cellular processes, including inflammation, mitochondrial dysfunction, membrane breakdown, oxidative stress and neurotoxicity, which could be associated with PTSD pathogenesis.

Concurrent Metabolic Profiling and Quantification of Aromatic Amino Acids and Phytohormones in Solanum lycopersicum Plants Responding to Phytophthora capsici.

Pathogenic microorganisms account for large production losses in the agricultural sector. Phytophthora capsici is an oomycete that causes blight and fruit rot in important crops, especially those in the Solanaceae family. P. capsici infection is difficult to control due to genetic diversity, arising from sexual reproduction, and resistant spores that remain dormant in soil. In this study, the metabolomics of tomato plants responding to infection by P. capsici were investigated. Non-targeted metabolomics, based on liquid chromatography coupled to mass spectrometry (LC-MS), were used with multivariate data analyses to investigate time-dependent metabolic reprogramming in the roots, stems, and leaves of stem-infected plants, over an 8 day period. In addition, phytohormones and amino acids were determined using quantitative LC-MS. Methyl salicylate and 1-aminocyclopropane-1-carboxylate were detected as major signalling molecules in the defensive response to P. capsici. As aromatic amino acid precursors of secondary metabolic pathways, both phenylalanine and tryptophan showed a continuous increase over time in all tissues, whereas tyrosine peaked at day 4. Non-targeted metabolomic analysis revealed phenylpropanoids, benzoic acids, glycoalkaloids, flavonoids, amino acids, organic acids, and fatty acids as the major classes of reprogrammed metabolites. Correlation analysis showed that metabolites derived from the same pathway, or synthesised by different pathways, could either have a positive or negative correlation. Furthermore, roots, stems, and leaves showed contrasting time-dependent metabolic reprogramming, possibly related to the biotrophic vs. necrotrophic life-stages of the pathogen, and overlapping biotic and abiotic stress signaling. As such, the targeted and untargeted approaches complemented each other, to provide a detailed view of key time-dependent metabolic changes, occurring in both the asymptomatic and symptomatic stages of infection.

Evaluation of the Antioxidant Activity of Nigella sativa L. and Allium ursinum Extracts in a Cellular Model of Doxorubicin-Induced Cardiotoxicity.

Natural products black cumin—Nigella sativa (N. sativa) and wild garlic—Allium ursinum (AU) are known for their potential role in reducing cardiovascular risk factors, including antracycline chemotherapy. Therefore, this study investigates the effect of N. sativa and AU water and methanolic extracts in a cellular model of doxorubicin (doxo)-induced cardiotoxicity. The extracts were characterized using Ultraviolet-visible (UV-VIS) spectroscopy, Fourier-transform infrared (FT-IR) spectroscopy, Liquid Chromatography coupled with Mass Spectrometry (LC-MS) and Gas Chromatography coupled with Mass Spectrometry (GC-MS) techniques. Antioxidant activity was evaluated on H9c2 cells. Cytosolic and mitochondrial reactive oxygen species (ROS) release was evaluated using 2′,7′-dichlorofluorescin-diacetate (DHCF-DA) and mitochondria-targeted superoxide indicator (MitoSOX red), respectively. Mitochondrial membrane depolarization was evaluated by flow cytometry. LC-MS analysis identified 12 and 10 phenolic compounds in NSS and AU extracts, respectively, with flavonols as predominant compounds. FT-IR analysis identified the presence of carbohydrates, amino acids and lipids in both plants. GC-MS identified the sulfur compounds in the AU water extract. N. sativa seeds (NSS) methanolic extract had the highest antioxidant activity reducing both intracellular and mitochondrial ROS release. All extracts (excepting AU methanolic extract) preserved H9c2 cells viability. None of the investigated plants affected the mitochondrial membrane depolarization. N. sativa and AU are important sources of bioactive compounds with increased antioxidant activities, requiring different extraction solvents to obtain the pharmacological effects.

Determinación de la composición fenólica en granos ancestrales de uso alimentario. Comparación de métodos analíticos y de actividad antioxidante

El objetivo de este estudio fue cuantificar los compuestos fenólicos totales de diferentes granos ancestrales: dos variedades de tef (Eragrostis tef), trigo sarraceno (Fagopyrum esculentum) y trigo espelta (Triticum spelta) por diferentes métodos espectrofotométricos (Folin-Ciocalteu y Fast Blue BB) y comparar con los resultados obtenidos de estudiar la composición fenólica individualizada mediante cromatografía líquida de alta resolución acoplada a un sistema de detección de fotodiodos y espectrometría de masas (HPLC-DAD-MS). También se determinó la actividad antioxidante in vitro de los extractos de los granos ancestrales estudiados por el método TEAC (capacidad antioxidante equivalente de Trolox) y FRAP (poder antioxidante reductor del hierro). El uso de HPLC-DAD-MS permitió identificar un gran número de compuestos fenólicos: compuestos hidroxibenzoicos, hidroxicinámicos, flavonoles, flavonas y flavanoles. Las flavonas fueron la familia de compuestos fenólicos más abundante detectada en las muestras de espelta, teff, blanco y marrón. Sin embargo, en el trigo sarraceno son los flavanoles (64 %). La cuantificación de los compuestos fenólicos por HPLC-DAD-MS parece más oportuna que por los métodos espectrofotométricos. Con respecto a la actividad antioxidante, el trigo sarraceno mostró los valores más altos frente a su capacidad de captar radicales libres y poder reductor en relación a las otras muestras estudiadas, pudiéndose relacionar con su alto contenido en compuestos fenólicos.

Survey of protein-based sport supplements for illegally added anabolic steroids methyltestosterone and 4-androstenedione by UPLC-MS/MS

There is some evidence that marketable supplements contain hormones not declared on the product label. The presence of these androgenic anabolic steroids (AAS) in sports supplements can be considered an adulteration and affect the health of consumers, who are predominantly athletes. This study aimed to measure anabolic hormones (methyltestosterone and 4-androstenedione) in sport supplements. Ultra Performance Liquid chromatography coupled mass spectrometry (UPLC-MS/MS) with electrospray ionization (ESI) in positive mode was employed under the Multiple Reaction Monitoring (MRM) ion program. To overcome matrix effects and quantify the selected analyte, the calibration curve was made using Matrix Match method. The LOQ and LOD were 1 ng/g and 0.3 ng/g for both analytes. The recovery of 4-androstenedione and methyltestosterone was in the range of 86.87–107.35 and 77.31–113.98, respectively. In terms of reproducibility, CV % for 4-androstenedione and methyltestosterone ranged from 6.56 to 16.87% and 1.45–15.12%, respectively. 4-androstenedione was found in 11 samples including 9 whey as 1.578 ± 0.154 ng/g and 2 whey albumin samples with an amount of 1.134 ng/g and 1.474 ng/g. Consequently, continuous controlling of sport supplements comprising intentionally or unintentionally added androgens could be important for health and discuss in the context of compliance with anti-doping.

A Community-Based Study Identifying Metabolic Biomarkers of Mild Cognitive Impairment and Alzheimer's Disease Using Artificial Intelligence and Machine Learning.

Currently, there is no objective, clinically available tool for the accurate diagnosis of Alzheimer's disease (AD). There is a pressing need for a novel, minimally invasive, cost friendly, and easily accessible tool to diagnose AD, assess disease severity, and prognosticate course. Metabolomics is a promising tool for discovery of new, biologically, and clinically relevant biomarkers for AD detection and classification. Utilizing artificial intelligence and machine learning, we aim to assess whether a panel of metabolites as detected in plasma can be used as an objective and clinically feasible tool for the diagnosis of mild cognitive impairment (MCI) and AD. Using a community-based sample cohort acquired from different sites across the US, we adopted an approach combining Proton Nuclear Magnetic Resonance Spectroscopy (1H NMR), Liquid Chromatography coupled with Mass Spectrometry (LC-MS) and various machine learning statistical approaches to identify a biomarker panel capable of identifying those patients with AD and MCI from healthy controls. Of the 212 measured metabolites, 5 were identified as optimal to discriminate between controls, and individuals with MCI or AD. Our models performed with AUC values in the range of 0.72-0.76, with the sensitivity and specificity values ranging from 0.75-0.85 and 0.69-0.81, respectively. Univariate and pathway analysis identified lipid metabolism as the most perturbed biochemical pathway in MCI and AD. A comprehensive method of acquiring metabolomics data, coupled with machine learning techniques, has identified a strong panel of diagnostic biomarkers capable of identifying individuals with MCI and AD. Further, our data confirm what other groups have reported, that lipid metabolism is significantly perturbed in those individuals suffering with dementia. This work may provide additional insight into AD pathogenesis and encourage more in-depth analysis of the AD lipidome.

Separation of Permethylated O-Glycans, Free Oligosaccharides, and Glycosphingolipid-Glycans Using Porous Graphitized Carbon (PGC) Column.

Glycosylation is one of the most common and complex post-translational modifications of proteins. However, there are other carbohydrates such as free oligosaccharides and glycosphingolipids-glycans that are associated with important biological and clinical roles. To analyze these molecules using liquid chromatography coupled with mass spectrometry (LC-MS), the permethylation approach was utilized. Although permethylation is a commonly utilized glycan derivatization technique, separation of permethylated glycans released from glycosphingolipid (GSL) by LC-MS has never been previously demonstrated. Here, a nanoflow porous graphitized carbon (PGC) column coupled with a high-resolution mass spectrometer was used to achieve isomeric separation of these permethylated glycans. We demonstrate the separation of free reducing end and reduced end O-glycans, free oligosaccharides derived from human milk, and GSL glycans derived from the MDA-MB-231BR cancer cell line using PGC-LC-MS.

Rapid Analysis of Reduced Antibody Drug Conjugate by Online LC-MS/MS with Fourier Transform Ion Cyclotron Resonance Mass Spectrometry.

Antibody drug conjugates (ADCs), which harness the high targeting specificity of monoclonal antibodies (mAb) with the potency of small molecule therapeutics, are one of the fastest growing pharmaceutical classes. Nevertheless, ADC conjugation techniques and processes may introduce intrinsic heterogeneity including primary sequence variants, varied drug-to-antibody ratio (DAR) species, and drug positional isomers, which must be monitored to ensure the safety and efficacy of ADCs. Liquid chromatography coupled to mass spectrometry (LC-MS) is a powerful tool for characterization of ADCs. However, the conventional bottom-up MS analysis workflows require an enzymatic digestion step which can be time consuming and may introduce artifactual modifications. Herein, we develop an online LC-MS/MS method for rapid analysis of reduced ADCs without digestion, enabling determination of DAR, characterization of the primary sequence, and localization of the drug conjugation site of the ADC using high-resolution Fourier transform ion cyclotron resonance (FTICR) MS. Specifically, a model cysteine-linked ADC was reduced to generate six unique subunits: light chain (Lc) without drug (Lc0), Lc with 1 drug (Lc1), heavy chain (Hc) without drug (Hc0), and Hc with 1-3 drugs (Hc1-3, respectively). A concurrent reduction strategy is applied to assess ADC subunits in both the partially reduced (intrachain disulfide bonds remain intact) and fully reduced (all disulfide bonds are cleaved) forms. The entire procedure including the sample preparation and LC-MS/MS takes less than 55 min, enabling rapid multiattribute analysis of ADCs.

Evaluation of ELISA-based method for total anabaenopeptins determination and comparative analysis with on-line SPE-UHPLC-HRMS in freshwater cyanobacterial blooms

Anabaenopeptins (APs) are bioactive cyanopeptides of emerging concern produced by cyanobacteria. The research for analytical development has recently gained in importance due to their abundance in toxic cyanobacterial blooms. A new commercial enzyme-linked immunosorbent assay kit for the determination of total APs (APtot ELISA) has been released promising a rapid response with good cost efficiency for the routine monitoring of uncommon cyanopeptides. The present study explores the suitability of this new kit in comparison with a validated quantitative analytical method based on liquid chromatography coupled to mass spectrometry (LC-MS). The validation results were comparable with both methods for accuracy, precision, and calibration. Method detection limits were more sensitive using LC-MS specifically evaluated at 0.011 and 0.013 μg L−1 for AP-A and B respectively, compared to APtot ELISA evaluated at 0.10 μg L−1 for total of the two. For APtot ELISA, results were independent from the matrix; however, a systematic signal response was measured in blanks, requiring a blank subtraction in data treatment. Cross-reactivity of APtot ELISA was investigated by analyzing ten cyanopeptides selected for their abundance and diversity. Cyanopeptolin A (CP-A), nodularin-R (NOD), microcystin (MC)-RR, [Asp3]RR, and HilR showed cross-reactivity with an average overestimation going from 25 to 66%. Considering the contribution of cross-reactive cyanopeptides, thirteen lake samples out of fifteen showed higher concentrations using APtot ELISA with overestimation values up to 2261% compared to LC-MS. In light of this study results, LC-MS should still be preconized for the study and monitoring of APs when sensitivity and specificity are needed.