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Honey-Based Polyphenols: Extraction, Quantification, Bioavailability, and Biological Activities
Published in Megh R. Goyal, Arijit Nath, Rasul Hafiz Ansar Suleria, Plant-Based Functional Foods and Phytochemicals, 2021
Csilla Benedek, John-Lewis Zinia Zaukuu, Zsanett Bodor, Zoltan Kovacs
During elution, polar interfering substances are removed first, followed by elution of target analytes, which is usually achieved with methanol [23]. Dimitrova et al. proposed a two-stage sample conditioning, adjusted to the acid character of the sample: the C18 SPE column is conditioned first with methanol containing sodium hydroxide, then with the methanol containing hydrochloric acid. Finally, elution is performed using acetonitrile: tetrahydrofuran (1:1); and subsequently, reversed-phase HPLC is applied for separation [26].
High-Performance Liquid Chromatography
Published in Adorjan Aszalos, Modern Analysis of Antibiotics, 2020
Joel J. Kirschbaum, Adorjan Aszalos
The modern era of the treatment of disease can best be considered to have begun with the discovery of penicillin by Sir Alexander Fleming in 1929 and its meaningful application to therapy by Florey and Chain in 1940. Penicillin contains the β-lactam ring, a four-membered cyclic amide, which is responsible for interfering with cell wall synthesis by acylating a D-alanine transpeptidase and thus preventing the formation of peptide cross-links between two linear peptidoglycan chains. A second major class of β-lactam-containing antibiotics, the cephalosporins, appears to have a similar mode of action. HPLC has been used in every step of g-lactam research and production, from examining fermentation broths of natural antibiotics and reaction mixtures of synthetic and semisynthetic ones and testing stability and physical and chemical properties of bulk and formulated drugs, to quantifying contents in body fluids. First, assays capable of chromatographing several β-lactam antibiotics then methods suitable for one to three such compounds are described. Most of these methods involve reversed phase HPLC, typically using an octadecylsilane column with a mobile phase of methanol or acetonitrile-water or aqueous buffer and UV detection. Purified material is usually dissolved in a methanol or methanolic mobile phase prior to injection. Most formulations can be extracted or dissolved into similar solvents. (β-lactam antibiotics in body fluids are usually analyzed after deproteinization (typically by ultrafiltration or precipitation), dilution with buffered mobile phase, addition of an internal standard, and then injection.
Reactivities of Amino Acids and Proteins with Iodine
Published in Erwin Regoeczi, Iodine-Labeled Plasma Proteins, 2019
Reversed-phase HPLC can also be used for the separation of monoiodinated peptide hormones from their unlabeled and diiodinated counterparts.501 In the molecular weight range from 574 to 4400, the distance of separation between unlabeled and monosubstituted peptides is inversely proportional to peptide weights. Intriguingly, the system can sense, and separate, peptides containing oxidized methionyl residues.
Inhibition of Streptococcus mutans adhesion and biofilm formation with small-molecule inhibitors of sortase A from Juniperus chinensis
Published in Journal of Oral Microbiology, 2022
Eunji Cho, Ji-Yeon Hwang, Jae Sung Park, Daehyun Oh, Dong-Chan Oh, Hyeung-Geun Park, Jongheon Shin, Ki-Bong Oh
According to the results of 1H NMR analysis, the fractions eluted with water–methanol (50:50) and (40:60) were separated further. The water–methanol (50:50) fraction (32.7 g) was subjected to semi-preparative reversed-phase HPLC (YMC-ODS column, 250 × 10 mm; 2.0 mL/min; water–methanol, 68:32). The peak at tR = 43.3 min was purified through analytical HPLC (0.7 mL/min; water–acetonitrile gradient from 80:20 to 65:35 over 60 min) to yield compound 1 (tR = 47.5 min). The fraction (23.4 g) eluted with water–methanol (40:60) was separated through HPLC (1.8 mL/min; water–methanol, 50:50) to obtain compounds 2 (tR = 33.2 min) and 3 (tR = 46.5 min). Purification of an additional peak (tR = 44.7 min) through analytical HPLC (0.7 mL/min; water–acetonitrile, 75:25) produced compounds 4 (tR = 6.8 min) and 5 (tR = 60.1 min). The peak at tR = 70.8 min was purified through analytical HPLC (0.7 mL/min; water–acetonitrile, 70:30) to obtain compound 6 (tR = 46.3 min). The overall isolated amounts of compounds 1–6 were 13.2, 3.6, 3.3, 4.2, 4.8, and 1.1 mg, respectively.
Lipotoxicity-associated inflammation is prevented by guarana (Paullinia cupana) in a model of hyperlipidemia
Published in Drug and Chemical Toxicology, 2021
Jader B. Ruchel, Viviane M. Bernardes, Josiane B. S. Braun, Alessandra G. Manzoni, Daniela F. Passos, Lívia G. Castilhos, Fátima H. Abdalla, Juliana S. de Oliveira, Cinthia M. de Andrade, Emerson A. Casali, Ivana B. M. da Cruz, Daniela B. R. Leal
Serum purine (adenine nucleotides, adenosine, and inosine) levels were quantified by high performance liquid chromatography (HPLC). Perchloric acid (0.6 mol/L) was used to denature proteins. Next, samples were spun (14 000×g for 10 min) and the supernatants neutralized with 4 N KOH. A second centrifugation (14 000×g for 15 min) was performed to further purify the samples. An amount of 50 μL was applied to a reversed-phase HPLC system using a 25 cm C18 Shimadzu column (Shimadzu, Kyoto, Japan) at 260 nm with a mobile phase containing 60 mM KH2PO4, 5 mM tetrabutylammonium chloride, pH 6.0, in 30% methanol (Voelter et al.1980). Retention times were used to identify the peaks and standards were used for comparison and quantification of purines levels, which were expressed as nmoles of the purine tested per mL of serum.
Microbial biotransformation – an important tool for the study of drug metabolism
Published in Xenobiotica, 2019
Rhys Salter, Douglas C. Beshore, Steven L. Colletti, Liam Evans, Yong Gong, Roy Helmy, Yong Liu, Cheri M. Maciolek, Gary Martin, Natasa Pajkovic, Richard Phipps, James Small, Jonathan Steele, Ronald de Vries, Headley Williams, Iain J. Martin
Purification was performed by preparative and semi-preparative reversed-phase HPLC, using two or three orthogonal stationary phases. Initial fractionations typically employed a Waters NovaPak C18 40 × 100 mm RadPak column with a GuardPak C18 40 × 10 mm guard column, eluting with a flow rate of 50 mL/min and a linear gradient starting from 85/10/5% water/acetonitrile/200 mM ammonium formate +2% (v/v) formic acid in water, held for 2 min, then changed to 35/60/5% over the next 10 min and to 0/95/5% over the next 2 min. Fractions containing target metabolites were then subjected to further isocratic or gradient fractionation on a Waters Symmetry Shield RP8 19 × 100 mm column (with Symmetry Shield RP 8 Prep Guard Cartridge, 19 mm ×10 mm), eluting with a flow rate of 17 mL/min using water/acetonitrile acidified with 2%(v/v) formic acid. In cases where >90% purity was not achieved after these two steps, a third stationary phase was used, selected from either Waters Atlantis T3 C18, Waters Xterra C18, Waters XSelect C18, Waters XBridge Prep Phenyl or Agilent PLRPS polymeric phase columns. Occasionally, typically when target product amounts were <1 mg, analytical (4.6 mm diameter) HPLC columns using stationary phases listed above, were employed with fraction collection. UV, MS and evaporative light scattering detection (PL-ELS 2100 Ice, Agilent Polymer Laboratories, Marietta, GA) were used for the detection and purity confirmation of metabolites.