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Keywords = reversed phase chromatography (RPC)

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12 pages, 2259 KB  
Article
Charge Variants Characterization of Co-Formulated Antibodies by Three-Dimensional Liquid Chromatography–Mass Spectrometry
by Xiaoqing Jin, Luna Chen, Jianlin Chu and Bingfang He
Biomolecules 2024, 14(8), 999; https://doi.org/10.3390/biom14080999 - 13 Aug 2024
Cited by 2 | Viewed by 1548
Abstract
Co-formulated antibodies can bring clinical benefits to patients by combining two or more antibodies in a single dosage form. However, the quality analysis of co-formulated antibodies raises additional challenges, compared to individual antibodies, due to the need for accurate analysis of multiple antibodies [...] Read more.
Co-formulated antibodies can bring clinical benefits to patients by combining two or more antibodies in a single dosage form. However, the quality analysis of co-formulated antibodies raises additional challenges, compared to individual antibodies, due to the need for accurate analysis of multiple antibodies in one solution. It is extremely difficult to effectively separate the charge variants of the two co-formulated antibodies using one ion exchange chromatography (IEC) method because of their similar characteristics. In this study, a novel method was developed for the charge variants characterization of co-formulated antibodies using three-dimensional liquid chromatography–mass spectrometry (3D-LC-MS). Hydrophobic interaction chromatography (HIC) was used as the first dimension to separate and collect the two co-formulated antibodies. The two collections were then injected into the second-dimension IEC separately for charge variants separation and analysis. Subsequently, the separated charge variants underwent on-line desalting in the third-dimension reverse-phase chromatography (RPC) and subsequent mass spectroscopy analysis. The novel method could simultaneously provide a charge variants ratio and post-translational modification (PTM) data for the two co-formulated antibodies. Therefore, it could be used for release testing and stability studies of co-formulated antibodies, making up for the shortcomings of the existing approaches. It was the first time that charge variants of co-formulated antibodies were characterized by the 3D-LC-MS method, to the best of our knowledge. Full article
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15 pages, 4181 KB  
Article
The Role of the Preanalytical Step for Human Saliva Analysis via Vibrational Spectroscopy
by Beatrice Campanella, Stefano Legnaioli, Massimo Onor, Edoardo Benedetti and Emilia Bramanti
Metabolites 2023, 13(3), 393; https://doi.org/10.3390/metabo13030393 - 8 Mar 2023
Cited by 4 | Viewed by 2523
Abstract
Saliva is an easily sampled matrix containing a variety of biochemical information, which can be correlated with the individual health status. The fast, straightforward analysis of saliva by vibrational (ATR-FTIR and Raman) spectroscopy is a good premise for large-scale preclinical studies to aid [...] Read more.
Saliva is an easily sampled matrix containing a variety of biochemical information, which can be correlated with the individual health status. The fast, straightforward analysis of saliva by vibrational (ATR-FTIR and Raman) spectroscopy is a good premise for large-scale preclinical studies to aid translation into clinics. In this work, the effects of saliva collection (spitting/swab) and processing (two different deproteinization procedures) were explored by principal component analysis (PCA) of ATR-FTIR and Raman data and by investigating the effects on the main saliva metabolites by reversed-phase chromatography (RPC-HPLC-DAD). Our results show that, depending on the bioanalytical information needed, special care must be taken when saliva is collected with swabs because the polymeric material significantly interacts with some saliva components. Moreover, the analysis of saliva before and after deproteinization by FTIR and Raman spectroscopy allows to obtain complementary biological information. Full article
(This article belongs to the Special Issue Advances in Metabolic Profiling of Biological Samples)
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15 pages, 2771 KB  
Article
Supramolecular Chromatographic Separation of C60 and C70 Fullerenes: Flash Column Chromatography vs. High Pressure Liquid Chromatography
by Subbareddy Mekapothula, A. D. Dinga Wonanke, Matthew A. Addicoat, David J. Boocock, John D. Wallis and Gareth W. V. Cave
Int. J. Mol. Sci. 2021, 22(11), 5726; https://doi.org/10.3390/ijms22115726 - 27 May 2021
Cited by 2 | Viewed by 6012
Abstract
A silica-bound C-butylpyrogallol[4]arene chromatographic stationary phase was prepared and characterised by thermogravimetric analysis, scanning electron microscopy, NMR and mass spectrometry. The chromatographic performance was investigated by using C60 and C70 fullerenes in reverse phase mode via flash column and high-pressure [...] Read more.
A silica-bound C-butylpyrogallol[4]arene chromatographic stationary phase was prepared and characterised by thermogravimetric analysis, scanning electron microscopy, NMR and mass spectrometry. The chromatographic performance was investigated by using C60 and C70 fullerenes in reverse phase mode via flash column and high-pressure liquid chromatography (HPLC). The resulting new stationary phase was observed to demonstrate size-selective molecular recognition as postulated from our in-silico studies. The silica-bound C-butylpyrogallol[4]arene flash and HPLC stationary phases were able to separate a C60- and C70-fullerene mixture more effectively than an RP-C18 stationary phase. The presence of toluene in the mobile phase plays a significant role in achieving symmetrical peaks in flash column chromatography. Full article
(This article belongs to the Special Issue Supramolecular Materials)
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21 pages, 4352 KB  
Article
A Workflow towards the Reproducible Identification and Quantitation of Protein Carbonylation Sites in Human Plasma
by Juan Camilo Rojas Echeverri, Sanja Milkovska-Stamenova and Ralf Hoffmann
Antioxidants 2021, 10(3), 369; https://doi.org/10.3390/antiox10030369 - 1 Mar 2021
Cited by 15 | Viewed by 4044
Abstract
Protein carbonylation, a marker of excessive oxidative stress, has been studied in the context of multiple human diseases related to oxidative stress. The variety of post-translational carbonyl modifications (carbonyl PTMs) and their low concentrations in plasma challenge their reproducible identification and quantitation. However, [...] Read more.
Protein carbonylation, a marker of excessive oxidative stress, has been studied in the context of multiple human diseases related to oxidative stress. The variety of post-translational carbonyl modifications (carbonyl PTMs) and their low concentrations in plasma challenge their reproducible identification and quantitation. However, carbonyl-specific biotinylated derivatization tags (e.g., aldehyde reactive probe, ARP) allow for targeting carbonyl PTMs by enriching proteins and peptides carrying these modifications. In this study, an oxidized human serum albumin protein model (OxHSA) and plasma from a healthy donor were derivatized with ARP, digested with trypsin, and enriched using biotin-avidin affinity chromatography prior to nano reversed-phase chromatography coupled online to electrospray ionization tandem mass spectrometry with travelling wave ion mobility spectrometry (nRPC-ESI-MS/MS-TWIMS). The presented workflow addresses several analytical challenges by using ARP-specific fragment ions to reliably identify ARP peptides. Furthermore, the reproducible recovery and relative quantitation of ARP peptides were validated. Human serum albumin (HSA) in plasma was heavily modified by a variety of direct amino acid oxidation products and adducts from reactive carbonyl species (RCS), with most RCS modifications being detected in six hotspots, i.e., Lys10, Lys190, Lys199, Lys281, Lys432, and Lys525 of mature HSA. Full article
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14 pages, 2507 KB  
Communication
Detection and Isolation of Emetic Bacillus cereus Toxin Cereulide by Reversed Phase Chromatography
by Eva Maria Kalbhenn, Tobias Bauer, Timo D. Stark, Mandy Knüpfer, Gregor Grass and Monika Ehling-Schulz
Toxins 2021, 13(2), 115; https://doi.org/10.3390/toxins13020115 - 4 Feb 2021
Cited by 5 | Viewed by 5091
Abstract
The emetic toxin cereulide is a 1.2 kDa dodecadepsipeptide produced by the food pathogen Bacillus cereus. As cereulide poses a serious health risk to humans, sensitive and specific detection, as well as toxin purification and quantification, methods are of utmost importance. Recently, [...] Read more.
The emetic toxin cereulide is a 1.2 kDa dodecadepsipeptide produced by the food pathogen Bacillus cereus. As cereulide poses a serious health risk to humans, sensitive and specific detection, as well as toxin purification and quantification, methods are of utmost importance. Recently, a stable isotope dilution assay tandem mass spectrometry (SIDA–MS/MS)-based method has been described, and an method for the quantitation of cereulide in foods was established by the International Organization for Standardization (ISO). However, although this SIDA–MS/MS method is highly accurate, the sophisticated high-end MS equipment required for such measurements limits the method’s suitability for microbiological and molecular research. Thus, we aimed to develop a method for cereulide toxin detection and isolation using equipment commonly available in microbiological and biochemical research laboratories. Reproducible detection and relative quantification of cereulide was achieved, employing reversed phase chromatography (RPC). Chromatographic signals were cross validated by ultraperformance liquid chromatography–mass spectrometry (UPLC–MS/MS). The specificity of the RPC method was tested using a test panel of strains that included non-emetic representatives of the B. cereus group, emetic B. cereus strains, and cereulide-deficient isogenic mutants. In summary, the new method represents a robust, economical, and easily accessible research tool that complements existing diagnostics for the detection and quantification of cereulide. Full article
(This article belongs to the Special Issue Bacillus cereus Toxins)
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12 pages, 756 KB  
Article
Content/Potency Assessment of Botulinum Neurotoxin Type-A by Validated Liquid Chromatography Methods and Bioassays
by Bruna Xavier, Rafaela Ferreira Perobelli, Maurício Elesbão Walter, Francielle Santos da Silva and Sérgio Luiz Dalmora
Toxins 2019, 11(1), 35; https://doi.org/10.3390/toxins11010035 - 12 Jan 2019
Cited by 5 | Viewed by 6038
Abstract
Botulinum neurotoxin type-A (BoNTA) is one of the seven different serotypes (A to G) produced by Clostridium botulinum. A stability-indicating size-exclusion chromatography (SEC) method was developed and validated, and the specificity was confirmed by forced degradation study, interference of the excipients, and peaks [...] Read more.
Botulinum neurotoxin type-A (BoNTA) is one of the seven different serotypes (A to G) produced by Clostridium botulinum. A stability-indicating size-exclusion chromatography (SEC) method was developed and validated, and the specificity was confirmed by forced degradation study, interference of the excipients, and peaks purity. The method was applied to assess the content and high-molecular-weight (HMW) forms of BoNTA in biopharmaceutical products, and the results were compared with those of the LD50 mouse bioassay, the T−47D cell culture assay, and the reversed-phase chromatography (RPC) method, giving mean values of 0.71% higher, 0.36% lower, and 0.87% higher, respectively. Aggregated forms showed significant effects on cytotoxicity, as well as a decrease in the bioactivity (p < 0.05). The employment of the proposed method in conjunction with the optimized analytical technologies for the analysis of the intact and altered forms of the biotechnology-derived medicines, in the correlation studies, enabled the demonstration of the capability of each one of the methods and allowed for great improvements, thereby assuring their safe and effective use. Full article
(This article belongs to the Special Issue Characterization and Quantitative Analysis of Botulinum Neurotoxin)
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8 pages, 784 KB  
Article
A Validated Stability-Indicating HPLC Method for Simultaneous Determination of Amoxicillin and Enrofloxacin Combination in an Injectable Suspension
by Nidal Batrawi, Shorouq Wahdan and Fuad Al-Rimawi
Sci. Pharm. 2017, 85(1), 6; https://doi.org/10.3390/scipharm85010006 - 16 Feb 2017
Cited by 32 | Viewed by 10196
Abstract
The combination of amoxicillin and enrofloxacin is a well-known mixture of veterinary drugs; it is used for the treatment of Gram-positive and Gram-negative bacteria. In the scientific literature, there is no high-performance liquid chromatography (HPLC)-UV method for the simultaneous determination of this combination. [...] Read more.
The combination of amoxicillin and enrofloxacin is a well-known mixture of veterinary drugs; it is used for the treatment of Gram-positive and Gram-negative bacteria. In the scientific literature, there is no high-performance liquid chromatography (HPLC)-UV method for the simultaneous determination of this combination. The objective of this work is to develop and validate an HPLC method for the determination of this combination. In this regard, a new, simple and efficient reversed-phase HPLC method for simultaneous qualitative and quantitative determination of amoxicillin and enrofloxacin, in an injectable preparation with a mixture of inactive excipients, has been developed and validated. The HPLC separation method was performed using a reversed-phase (RP)-C18e (250 mm × 4.0 mm, 5 μm) column at room temperature, with a gradient mobile phase of acetonitrile and phosphate buffer containing methanol at pH 5.0, a flow rate of 0.8 mL/min and ultraviolet detection at 267 nm. This method was validated in accordance with the Food and Drug Administration (FDA) and the International Conference on Harmonisation (ICH) guidelines and showed excellent linearity, accuracy, precision, specificity, robustness, ruggedness, and system suitability results within the acceptance criteria. A stability-indicating study was also carried out and indicated that this method can also be used for purity and degradation evaluation of these formulations. Full article
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