Liquid Chromatography-mass Spectrometry Methodology Research Articles

Characterising Alzheimer's disease through integrative NMR- and LC-MS-based metabolomics

BackgroundAlzheimer's Disease (AD) is a complex and multifactorial disease and novel approaches are needed to illuminate the underlying pathology. Metabolites comprise the end-product of genes, transcripts, and protein regulations and might reflect disease pathogenesis. Blood is a common biofluid used in metabolomics; however, since extracellular vesicles (EVs) hold cell-specific biological material and can cross the blood-brain barrier, their utilization as biological material warrants further investigation. We aimed to investigate blood- and EV-derived metabolites to add insigts to the pathological mechanisms of AD. MethodsBlood samples were collected from 10 AD and 10 Mild Cognitive Impairment (MCI) patients, and 10 healthy controls. EVs were enriched from plasma using 100,000×g, 1 h, 4 °C with a wash. Metabolites from serum and EVs were measured using liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR) spectroscopy. Multivariate and univariate analyses were employed to identify altered metabolites in cognitively impaired individuals. ResultsWhile no significant EV-derived metabolites were found differentiating patients from healthy individuals, six serum metabolites were found important; valine (p = 0.001, fold change, FC = 0.8), histidine (p = 0.001, FC = 0.9), allopurinol riboside (p = 0.002, FC = 0.2), inosine (p = 0.002, FC = 0.3), 4-pyridoxic acid (p = 0.006, FC = 1.6), and guanosine (p = 0.004, FC = 0.3). Pathway analysis revealed branched-chain amino acids, purine and histidine metabolisms to be downregulated, and vitamin B6 metabolism upregulated in patients compared to controls. ConclusionUsing a combination of LC-MS and NMR methodologies we identified several altered mechanisms possibly related to AD pathology. EVs require additional optimization prior to their possible utilization as a biological material for AD-related metabolomics studies.

Analysis of serum cytokine and protective vitamin D levels in severe cases of COVID-19.

In this study, we investigated the role and relationship between the cytokine profile and protective vitamin D by measuring their serum levels in COVID‐19 intensive care unit patients with severe illnesses. A total of 74 patients were included in our study. Patients were divided into two groups. Patients in the COVID‐19 group (n = 31) and individuals without a history of serious illness or infection were used as the control group (n = 43). The serum concentrations of interleukin‐1 (IL‐1), IL‐6, IL‐10, IL‐21, and tumor necrosis factor‐α (TNF‐α) were measured by enzyme‐linked immunosorbent assays. Levels of serum vitamin D were detected with Liquid chromatography–mass spectrometry methodologies. TNF‐α, IL‐1, IL‐6, IL‐10, IL‐21, and vitamin D levels were measured in all patients. The serum cytokine levels in the COVID‐19 patient group were significantly higher (151.59 ± 56.50, 140.37 ± 64.32, 249.02 ± 62.84, 129.04 ± 31.64, and 123.58 ± 24.49, respectively) than control groups. Serum vitamin D was also significantly low (6.82 ± 3.29) in patients in the COVID‐19 group than the controls (21.96 ± 5.39). Regarding the correlation of vitamin D with cytokine levels, it was significantly variable. Our study shows that COVID‐19 patients are associated with lower serum vitamin D and higher pro‐inflammatory cytokines associated with increased virus presence. Our data provide more evidence of the anti‐inflammatory effect of vitamin D on COVID‐19 patients and the protective effects of vitamin D on risk were demonstrated.

Initial Development toward Non-Invasive Drug Monitoring via Untargeted Mass Spectrometric Analysis of Human Skin.

Drug monitoring is crucial for providing accurate and effective care; however, current methods (e.g., blood draws) are inconvenient and unpleasant. We aim to develop a non-invasive method for the detection and monitoring of drugs via human skin. The initial development toward this aim required information about which drugs, taken orally, can be detected via the skin. Untargeted liquid chromatography-mass spectrometry (LC-MS) was used as it was unclear if drugs, known drug metabolites, or other transformation products were detectable. In accomplishing our aim, we analyzed samples obtained by swabbing the skin of 15 kidney transplant recipients in five locations (forehead, nasolabial area, axillary, backhand, and palm), bilaterally, on two different clinical visits. Untargeted LC-MS data were processed using molecular networking via the Global Natural Products Social Molecular Networking platform. Herein, we report the qualitative detection and location of drugs and drug metabolites. For example, escitalopram/citalopram and diphenhydramine, taken orally, were detected in forehead, nasolabial, and hand samples, whereas N-acetyl-sulfamethoxazole, a drug metabolite, was detected in axillary samples. In addition, chemicals associated with environmental exposure were also detected from the skin, which provides insight into the multifaceted chemical influences on our health. The proof-of-concept results presented support the finding that the LC-MS and data analysis methodology is currently capable of the qualitative assessment of the presence of drugs directly via human skin.

Rapid LC-MS Method for Accurate Molecular Weight Determination of Membrane and Hydrophobic Proteins.

Therapeutic target characterization involves many components, including accurate molecular weight (MW) determination. Knowledge of the accurate MW allows one to detect the presence of post-translational modifications, proteolytic cleavages, and importantly, if the correct construct has been generated and purified. Denaturing liquid chromatography-mass spectrometry (LC-MS) can be an attractive method for obtaining this information. However, membrane protein LC-MS methodology has remained relatively under-explored and under-incorporated in comparison to methods for soluble proteins. Here, systematic investigation of multiple gradients and column chemistries has led to the development of a 5 min denaturing LC-MS method for acquiring membrane protein accurate MW measurements. Conditions were interrogated with membrane proteins, such as GPCRs and ion channels, as well as bispecific antibody constructs of variable sizes with the aim to provide the community with rapid LC-MS methods necessary to obtain chromatographic and accurate MW measurements in a medium- to high-throughput manner. The 5 min method detailed has successfully produced MW measurements for hydrophobic proteins with a wide MW range (17.5 to 105.3 kDa) and provided evidence that some constructs indeed contain unexpected modifications or sequence clipping. This rapid LC-MS method is also capable of baseline separating formylated and nonformylated aquaporinZ membrane protein.

The High Prevalence of Vitamin D Insufficiency in Cord Blood in Calgary, Alberta (APrON-D Study)

Vitamin D is important in promoting healthy pregnancy and fetal development. We undertook this study to measure 25-hydroxyvitamin D in maternal and cord blood and to identify maternal factors related to vitamin D status in Calgary. Blood samples collected at the time of delivery from the Alberta Pregnancy Outcomes and Nutrition study cohort (ApronStudy.ca) participants were processed for plasma and assayed using liquid chromatography mass spectrometry methodology for 25(OH)D3. Ninety-two pairs of maternal and cord blood samples were obtained. The prevalence of 25(OH)D3 insufficiency-25(OH)D3 <75 nmol/L-was 38% and 80% in women and neonates, respectively. Vitamin D supplementation was the only clinical factor associated with 25(OH)D3 sufficiency, and the odds of sufficiency were 3.75 (95% CI 1.00 to 14.07) higher for women and 5.27 (95%CI 1.37 to 20.27) when over 2000 IU/day were used. Using liquid chromatography mass spectrometry, we demonstrated a very high prevalence of vitamin D insufficiency in cord blood and that the use of high dose vitamin D was associated with greater odds of sufficiency in pregnant women and cord blood in Alberta.

Are you also what your mother eats? Distinct proteomic portrait as a result of maternal high-fat diet in the cerebral cortex of the adult mouse.

Epidemiological studies suggest an association between maternal obesity and adverse neurodevelopmental outcomes in offspring. Our aim was to compare the global proteomic portrait in the cerebral cortex between mice born to mothers on a high-fat or control diet who themselves were fed a high-fat or control diet. Male mice born to dams fed a control (C) or high-fat (H) diet 4 weeks before conception and during gestation, and lactation were assigned to either C or H diet at weaning. Mice were killed at 19 weeks and their cerebral cortices were analysed using a two-dimensional liquid chromatography-mass spectrometry methodology. In total, 6 695 proteins were identified (q<0.01), 10% of which were modulated in at least one of the groups relative to controls. In silico analysis revealed that mice clustered based on the diet of the mother and not their own diet and that maternal high-fat diet was significantly associated with response to hypoxia/oxidative stress and apoptosis in the cerebral cortex of the adult offspring. Maternal high-fat diet resulted in distinct endophenotypic changes of the adult offspring cerebral cortex independent of its current diet. The identified proteins could represent novel therapeutic targets for the prevention of neuropathological features resulting from maternal obesity.

A fast liquid chromatography-mass Spectrometry methodology for membrane lipid profiling through hydrophilic interaction liquid chromatography

In this paper, we report the development of a new method based on HILIC-ESI-MS for the separation of several different membrane lipid classes and their detection on a triple quadrupole mass spectrometer using Precursor Ion (PIS) and Neutral Loss (NL) scanning in positive ion mode. Four different columns were tested for their ability to separate, under different conditions, a mixture of 14 lipid standards containing 7 glycerophospholipids (GPL), 2 glycosphyngolipids (GSL), 3 glycolipids (GL) and 2 betaine lipids (BL).The best separation was obtained using a Lichrosphere DIOL column as stationary phase and water (10mm ammonium acetate)/acetonitrile gradient elution as mobile phase which allows the separation of the 14 lipid classes within 35min runtime. Our method was successfully tested for the separation and analysis of crude lipid extracts obtained from a green alga (Jaoa bullata), a dinoflagellate (Peridinium cinctum) and a plant (Vitis vinifera cv. Corvina)

Liquid chromatography-mass spectrometry measurement of tacrolimus in finger-prick samples compared with venous whole blood samples

Finger-prick sampling is an alternative strategy for monitoring immunosuppressive drug concentrations that could be useful in reducing outpatient visits. We investigated the correlation between venous and finger-prick samples in a group of adult thoracic transplant patients. Blood samples (n = 65) for the measurement of whole blood tacrolimus were collected from adult heart (n = 18) and lung transplant (n = 20) recipients receiving tacrolimus-based immunosuppressive therapy and a finger-prick sample was taken at the same time. Between-batch assay imprecision (coefficient of variation [CV], %) for the last 12 months (n = 270) at concentrations of 3.5, 6.9 and 13.9 microg/L was 8.0%, 5.4% and 5.2%, respectively. Passing and Bablok regression analysis between finger-prick and venous blood showed finger-prick tacrolimus = 1.02 (venous blood tacrolimus) -0.06. Bland-Altman analysis showed good agreement with a bias of 0.114 microg/L and 95% limits of agreement from -1.0 to 1.2 microg/L. The liquid chromatography mass spectrometry methodology that we have developed has the potential to allow patients or their carers to collect finger-prick blood samples at home and send them to the laboratory using the routine mail service. We believe that finger-prick blood sampling has an important role to play in the care of transplant patients receiving immunosuppressive drugs, including tacrolimus.

Quantification of Focal Adhesion Kinase Activation Loop Phosphorylation as a Biomarker of Src Activity

A recently developed stable isotope dilution liquid chromatography-multiple reaction/mass spectrometry method to quantify focal adhesion kinase (FAK) activation loop phosphorylation was used to study endogenous Src kinase activity. This revealed that bis-phosphorylated pTyr(576)/Tyr(577)-FAK was a biomarker of Src activity and inactivation in vitro and in cell culture. Mouse embryonic fibroblasts (MEFs) expressing endogenous Src family kinases contained 65% unmodified Tyr(576)/Tyr(577), 33% mono-phosphorylated-pTyr(576)-FAK, and 6% bis-phosphorylated-pTyr(576)/pTyr(577)-FAK. In contrast, MEFs expressing oncogenic Y(529)FSrc contained 38% unmodified Tyr(576)/Tyr(577)-FAK, 29% mono-phosphorylated-pTyr(576)-FAK, and 19% bis-phosphorylated-pTyr(576)/pTyr(577)-FAK. This new method has made it possible to accurately determine the absolute amounts of FAK phosphorylation that occur after Src inhibition in cell culture and in vitro with increasing concentrations of the Src inhibitor N-(5-chloro-1,3-benzodioxol-4-yl)-7-[2-(4-methylpiperazin-1-yl)ethoxy]-5-(tetrahydro-2H-pyran-4-yloxy)quinazolin-4-amine (AZD0530). Phosphorylation of FAK at Tyr(576)/Tyr(577) was inhibited by AZD0530 in a dose-dependent manner both in cell culture and in vitro. However, there was a substantial difference in the ability of AZD0530 to inhibit Src that was constitutively activated in a cellular context (IC(50) = 2.12 muM) compared with the isolated enzyme (IC(50) = 0.14 muM). When normal MEFs and Y(529)FSrc-expressing MEFs were treated with pervanadate (a global phosphatase inhibitor), pTyr(576)/pTyr(577)-FAK accounted for almost 60% of the total FAK present in the cells. This suggests that activation loop phosphorylation is regulated by tyrosine phosphatases. These results confirm that FAK phosphorylation is a useful biomarker of Src inhibition in vivo. The accuracy and specificity of stable isotope dilution liquid chromatography-mass spectrometry methodology offers significant advantages over current immunochemical approaches for monitoring Src activity.

Application of Liquid Chromatography/Quadrupole-Linear Ion Trap Mass Spectrometry and Time-of-Flight Mass Spectrometry to the Determination of Pharmaceuticals and Related Contaminants in Wastewater

This paper describes an enhanced liquid chromatography-mass spectrometry (LC-MS) strategy for the analysis of a selected group of 56 organic pollutants in wastewater. This group comprises 38 pharmaceuticals and 10 of their most frequent metabolites, 6 pesticides, and 2 disinfectants. The LC-MS methodology applied is based in the use of a hybrid triple-quadrupole linear ion trap mass spectrometer (QTRAP) in combination with time-of-flight mass spectrometry (TOF-MS). The join application of both techniques provided very good results in terms of accurate quantification and unequivocal confirmation. Quantification was performed by LC-QTRAP-MS operating under selected reaction monitoring (SRM) mode in both positive and negative electrospray ionization. Unequivocal identification was provided by the acquisition of three SRM transitions per compound in most of the cases and by LC-TOF-MS analysis, which allows obtaining accurate mass measurements of the identified compounds with errors lower than 2 ppm. Additionally, the use of TOF-MS permits retrospective analysis, since the full spectrum is recorded at all times with a high sensitivity. Thus, review of recorded chromatograms looking for new compounds or transformation products suspected to be present in the samples is feasible allowing one to increase the scope of the method along the monitoring program. The analytical performance of the quantitative LC-QTRAP-MS method was evaluated in effluent wastewater samples. Linearity of response over 3 orders of magnitude was demonstrated for most compounds (R(2) > 0.99). Method limits of detection were between 0.04 and 50 ng L(-1). Finally, the methodology was successfully applied to a monitoring study intended to characterize wastewater effluents of six sewage treatment plants in Spain. The presence of most of compounds was detected at concentrations ranging from 9 ng L(-1) (atrazine) to 15 microg L(-1) (paraxanthine).

Advanced liquid chromatography-mass spectrometry (LC-MS) methods applied to wastewater removal and the fate of surfactants in the environment

This article is an overview of current methodologies for the analysis of different classes of surfactants by advanced liquid chromatography-mass spectrometry (LC-MS) technologies, including LC-tandem MS (LC-MS 2 ), ion trap, quadrupole-time-of-flight (Q-TOF) and matrix-assisted laser desorption/ionization (MALDI)-TOF. It also discusses the application of LC-MS methods in studies of the fate and the behavior of surfactants during wastewater treatment either using conventional activated sludge (CAS), membrane bioreactor (MBR) or advanced oxidation processes (AOPs). Using advanced LC-MS methodologies, it is possible to detect a variety of polar metabolites, such as carboxylic acids and short-chain sulphonic-acid derivatives of surfactants formed during the biodegradation processes in either the wastewater-treatment plants or the environment.