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Mass Spectrometric Analysis
Published in Adorjan Aszalos, Modern Analysis of Antibiotics, 2020
The dilactonic mixture of antimycin A was studied by HPLC using various detectors, including mass spectrometry [132]. After derivatizing with dansyl chloride, four major and six minor components were separated and identified from their CI spectra.
Evaluating Performance Benefits of Conditioning Formulations on Human Skin
Published in Randy Schueller, Perry Romanowski, Conditioning Agents for Hair and Skin, 2020
Ronald L. Rizer, Monya L. Sigler, David L. Miller
In this method forearms are occlusively patched with 5% (w/w) dansyl chloride in petrolatum. Patches are removed after 6 hr and repatched with fresh dansyl chloride. After 24 hr, patches are removed and sites are gently cleansed with moist Webril pads and read in the dark for fluorescence using a Woods light. Subjects then treat their forearms as directed, and return to the clinic after 1 week to begin having their forearm sites graded for the presence of fluorescence. Subjects return every 2 days for grading until both forearms no longer have fluorescence at both patch sites (fluorescence extinction method). A treatment that produces a significant decrease in SC replacement time compared to either an untreated control or a placebo formula as measured by the disappearance of skin fluorescence is regarded as having a beneficial effect. There are variations in this method that involve treatment with product for one or more weeks followed by fluorescence staining of the SC and further treatment. In this case, the skin is preconditioned with the test product.
Methods for Sequence Determination
Published in Roger L. Lundblad, Chemical Reagents for Protein Modification, 2020
The aliquots removed at each cycle are dried in a vacuum desiccator over P2O5. Two successive 2-μl portions of 0.1 M NaHCO3 are added and dried to remove ammonia. Dansyl chloride solution (1 μl of a 1:1 mixture of water and a solution of 2.5 mg dansyl chloride per ml acetone) is added and the sample incubated at 37°C for 1 h. After drying, 5 μl of 6 N HCl is added. The tube is sealed and the peptide is hydrolyzed at 105°C for 16 h. The dansyl amino acids are identified by 2-dimensional thin-layer chromatography on 5 x 5 cm polyamide sheets.10 A fine capillary is used for sample application to keep the spot as small as possible. A mixture of standards is run on the reverse side of the sheet. The solvent for the first dimension is 1.5% (v/v) formic acid. In the second dimension benzene/acetic acid 9:1 (or less toxic toluene/acetic acid 10:1) is run, followed in the same direction by ethyl acetate/methanol/acetic acid 20:1:1. The plate is dried and examined under UV light after each step. Resolution of certain spots may require a fourth solvent, 0.05 M trisodium phosphate/ethanol 3:1, also run in the second direction. As in the subtractive method, glutamine and asparagine are deamidated during hydrolysis. The thiazolinones may be recovered from the organic solvent, converted to the phenylthiohydantoins, and identified by one of the methods described later.
Defect-induced electronic states amplify the cellular toxicity of ZnO nanoparticles
Published in Nanotoxicology, 2020
Indushekhar Persaud, Achyut J. Raghavendra, Archini Paruthi, Nasser B. Alsaleh, Valerie C. Minarchick, James R. Roede, Ramakrishna Podila, Jared M. Brown
RAECs were exposed to 20 µg/mL of pristine, annealed, oxidized, or reduced ZnO NPs for 6, 12, and 24 h. The cell fraction was collected and prepared for high-pressure liquid chromatography (HPLC) measurements of the thiol/disulfide couples concentration to determine the cellular redox potential. The procedures were based on that of Jones et al. (Jones and Liang 2009). Briefly, the cell pellet was resuspended in 0.5 mL of 5% perchloric acid (PCA)/0.2 M boric acid/10 μM ϒ-Glu-Cys and then sonicated. Samples were centrifuged at 13,000×g for 2 min and 300 µl of the supernatant was placed in a new tube. The tubes with the pellets were aspirated and stored for protein quantification by bicinchoninic acid (BCA) protein assay. Derivation of samples to extract the thiol/disulfide couples was performed on the collected supernatant. For each sample, 60 μL of 9.3 mg/mL iodoacetic acid was added, and the pH was adjusted to 8.8–9.2 with 1 M KOH. The samples were incubated for 20 min at room temperature after which 300 μl of 20 mg/mL of dansyl chloride was added to each sample. The samples were placed in the dark overnight. For each sample, 500 μL of chloroform was added, vortexed, and centrifuged at 13,000×g for 2 min to separate the aqueous and organic layers. HPLC was performed on an amino column with a Supelcosil™ LC-NH2 25 cm × 4.6 mm, 5 μm column (Supleco, Bellefonte, PA) with an Agilent 1200 HPLC equipped with a fluorimeter. The concentration of glutathione, its disulfide form GSSG, cysteine (Cys), and cystine (CySS) was determined from the scans. The Nernst equation was used to calculate the redox potential of the thiol/disulfide ratio:
Associations between Dietary Fiber, the Fecal Microbiota and Estrogen Metabolism in Postmenopausal Women with Breast Cancer
Published in Nutrition and Cancer, 2021
Ayse G. Zengul, Wendy Demark-Wahnefried, Stephen Barnes, Casey D. Morrow, Brenda Bertrand, Taylor F. Berryhill, Andrew D. Frugé
Assays for 17β-estradiol and estrone in serum were performed in the UAB Targeted Metabolomics and Proteomics Laboratory. Estrogen analyses were determined by isotope dilution HPLC-electrospray ionization-multiple reaction ion mass spectrometry adapted from the method of Tai and Welch (18). 17β-Estradiol and estrone standards were prepared in 0.05% BSA. Sera (500 µl) were diluted 1:1 with MilliQ H2O. Samples and standards were spiked with 0.5 ng/ml 13C6-estradiol (CIL, Tewksbury, MA) internal standard. Diluted samples (1 ml) and standards were loaded onto individual 30 mg Polymeric Strata-X Solid Phase Extraction cartridge columns (Phenomenex, Torrance, CA). The cartridges were washed with MilliQ H2O (1 ml) and 40% methanol (1 ml) followed by elution of 17β-estradiol with 1 ml of methanol. Sample eluents were dried under a gentle stream of N2. Sodium bicarbonate (50 µl, 100 mM, pH 10.5) and dansyl-chloride (50 µl, 1 mg/ml) in acetone were added to samples which were incubated at 60 °C for 10 min. Samples were dried once more under a gentle stream of N2 followed by reconstitution in 100 µl of 40% methanol/0.1% formic acid (FA). Chromatography was carried out using an Ace Excel C18-Aromatic 1.7 µm 50 x 3.0 mm IS column at 50 °C using a 20AD Prominence HPLC (Shimadzu, Kyoto, Japan) in tandem with 6500 Qtrap mass spectrometer (SCIEX, Framingham, MA). LC-MS operation and data collection were under the control of Analyst 1.6.2 software (SCIEX). The mobile phases were composed of (A) 0.1% FA and (B) acetonitrile 0.1% FA; the flow rate was 300 µl/min. Gradient starting conditions were 50% B which was held for 1 min, a linear increase of B to 100% B at 4 min, held at 100% B until 4.75 min, and returned to 50% B at 5 mins, to equilibrate to starting conditions until 7 min. LC flow was diverted to waste for the first 1.8 min to prevent salt contaminating the MS front end. The MS was operated in positive electrospray ionization mode with the following parameters: curtain gas 30, collision gas medium, temperature 500, ion spray voltage 5000, collision energy 25, GS1 60 and GS2 60. Mass transitions for multiple-reaction-monitoring mode were m/z 506/171 for dansyl-17β-estradiol, 504/171 for dansyl-estrone and m/z 512/171 for 13C6-dansyl-17β-estradiol. The standard curve ranged from 5 – 5000 pg/ml over seven points. All data were processed, and concentration factors were corrected using Multiquant 3.0.1 (SCIEX).