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Investigating the stability of a cerebral vasodilator drug using chromatographic methods: Evaluation of methods' practicality and environmental aspects.

A vinca alkaloid; vinburnine (VNB) is utilized as an effective vasodilator. As a cyclic amide-containing drug, it is likely susceptible to hydrolytic degradation. This study examined the degradation profile of VNB, findings indicated that VNB undergoes degradation solely in the presence of alkali, generating a carboxylic acid derivative (DEG). The present study aimed to design and apply green TLC-densitometric and RP-HPLC assays for concurrently measuring VNB and its degradation product for the first time. TLC-densitometric assay was carried out on silica gel 60 F254 TLC plates and a developing system of ethyl acetate: methanol: triethylamine (6:4:0.05, by volume) and detection at 230nm. RP-HPLC method depended on a C8 column and a mixture of methanol: water (95:5, v/v). The rate of flow was 1mL/min and UV detection at 230nm. The proposed assays were used for prediction of the degradation behavior of VNB under the mentioned conditions and then applied for quantitation of VNB in its commercially available capsules. Four distinct metric approaches; National Environmental Method Index (NEMI), Analytical Eco-Scale, Green Analytical Procedure Index (GAPI), and Blue Applicability Grade Index (BAGI) were utilized to assess the chromatographic method's ecological effect. Findings obtained from the provided methodologies were contrasted statistically with the stated HPLC method using Student's t and F-tests. The analysis revealed that there were no significant differences between them. The established methods were verified in accordance with the recommendations of the International Council for Harmonization (ICH), and all the outcomes were deemed to fall within the permissible limit.

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Development and validation of a robust RP-HPLC method to quantitate residual 2-mercaptoethylamine in drug product formulations containing amino acid additives.

Bispecific antibodies have a wide range of applications in cancer immunotherapy, some of which are manufactured by controlled Fab-arm exchange requiring the reductant 2-mercaptoethylamine (2-MEA). As a process impurity, monitoring the residual 2-MEA in bispecific antibody drug product process development is needed. A novel reversed phase-high performance liquid chromatography (RP-HPLC) method for measurement of residual 2-MEA that uses 7-fluorobenzofurazan-4-sulfonic acid ammonium salt (SBD-F) as a fluorescent-detection tag in drug product formulations containing high concentrations of arginine has been developed. Using a thiol tag for residual 2-MEA eliminates any potential interference from conventional tag binding to amine groups of the formulation arginine, and potentially resulting in overestimation of the amount of impurity in a given sample. The new method has been fully validated for specificity, linearity, accuracy, range, limit of quantitation, limit of detection, and robustness. This method therefore has potential to aid in detecting residual 2-MEA content for any process that utilizes 2-MEA for bispecific antibody manufacturing.

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Unraveling the molecular mechanism of aqueous extract of Sargentodoxa cuneata against ulcerative colitis from serum metabolomics and bioinformatics perspectives.

Symptoms of ulcerative colitis (UC) are like "intestinal carbuncle" in Chinese medicine. The aqueous extract of Sargentodoxa cuneata (AESc) has good therapeutic effects on UC, but the underlying mechanism needs to be further elucidated. The mechanism of AESc against UC was studied based on metabolomics and bioinformatics in mice with UC. Dextran sodium sulfate was applied to induce a mouse model of UC. After the intervention of AESc, the general condition of the animals was recorded, and efficacy-related indicators were measured. Information on serum metabolites was determined. Multivariate analysis combined with bioinformatics methods were used to identify the differential metabolites. Furthermore, "metabolite-target-disease" network was obtained, and differential metabolites of UC were screened, and further analysis of the metabolites were performed. Molecular docking validation was also carried out. AESc improved general conditions such as blood in stool, hair of animals, and weight loss, reduced disease activity index scores and shortening of colon length in mice with UC. A total of 3445 serum metabolites were obtained, and 64 differentiated metabolites of AESc against UC were screened. Enrichment analysis showed that arachidonic acid metabolism, bile secretion, drug metabolism-other enzymes, and tyrosine metabolism were associated with AESc in the treatment of UC. In addition, based on "metabolite-target-disease" network, the serum metabolites cholylleucine, 9,10,13-TriHOME, birabresib, anthramycin methyl ether, trans-hexadec-2-enoyl carnitine, and lucidumol A were found to have the therapeutic potential for UC. Further, 14 core targets were obtained, and lipids and atherosclerosis, rheumatoid arthritis and multiple immune-inflammatory pathways were associated with AESc for the treatment of UC. AESc corrects serum metabolic disturbances in UC mice, and multiple serum metabolites have therapeutic potential for UC. AESc may treat UC by regulating biological processes such as lipid metabolism, amino acid metabolism, thereby restoring normal physiological function of the intestine.

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Application of 1-octanol in the extraction and GC-FID analysis of volatile organic compounds produced in biogas and biohydrogen processes.

Information about the volatile organic compounds generated in biogas and hydrogen production bioreactors is essential to elucidate the metabolic routes and varying yields of CH4 and H2 processes. In this work, the determination of 12 compounds (acetone, methanol, ethanol, 1-propanol, 1-butanol, and acetic, propanoic, butyric, isovaleric, valeric, caproic, and lactic acids) was performed by gas chromatography, after a vortex-assisted liquid-liquid microextraction (VALLME) procedure using 1.2mL of crude sample and 400µL of 1-octanol. Optimization of the separation process was performed, considering the solvent viscosity. The analytical curves were validated using ANOVA, demonstrating satisfactory precision and accuracy. Selectivity was confirmed by GC-MS analysis, which allowed the detection of glycerol, 1,3-propanediol, and 1,3-butanediol in some samples. Methanol levels exceeded the upper limit of quantification, with acetic acid and ethanol being the predominant compounds in the analyzed reactors. An additional investigation was conducted to assess potential interferences for lactic acid. The developed method employs a biodegradable extraction solvent, without any need for a dispersing solvent, and involves a single chromatographic run, without any derivatization steps.

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Characterization of flours from the aroeira leaf (Schinus terebinthifolius Raddi), obtained by different drying methods

The present work aimed at the development and characterization of aroeira leaf flour (Schinus terebinthifolius Raddi), obtained by lyophilization and drying in an air circulation oven. The technological, physical, physico-chemical, morphological, functional, and microbiological aspects were analyzed. Physico-chemical analysis identified the following properties with values provided respectively for fresh leaves (FOin) and flours (FES and FLIO): low water activity (0.984, 0.370, 0.387 g/100 g), moisture (64.52, 5.37, 7.97 g /100 g), ash (2.69, 6.51, and 6.89 g/100 g), pH (0.89, 4.45, 4.48 g/100 g), lipids (0.84, 1.67, 5.23 g/100 g), protein (3.29, 8.23, 14.12 g/100 g), carbohydrates (17.02, 53.12, 33.02 g/100 g), ascorbic acid (19.70, 34.20, 36.90 mg/100 g). Sources of fiber from plant leaves and flours (11.64, 25.1, 32.89 g/100 g) showed increased levels of luminosity. For NMR, the presence of aliphatic and aromatic compounds with olefinic hydrogens and a derivative of gallic acid were detected. The most abundant minerals detected were potassium and calcium. Micrographs identified the presence of irregular, non-uniform, and sponge-like particles. The main sugars detected were: fructose, glucose, and maltose. Malic, succinic, citric, lactic, and formic acids were found. Fifteen phenolic compounds were identified in the samples, highlighting: kaempferol, catechin, and caffeic acid. The values ​​found for phenolics were (447, 716.66, 493.31 mg EAG/100 g), flavonoids (267.60, 267.60, 286.26 EC/100 g). Antioxidant activity was higher using the ABTS method rather than FRAP for analysis of FOin, FES, and FLIO. Since the flours of the aroeira leaf have an abundant matrix of nutrients with bioactive properties and antioxidant activity, they have a potential for technological and functional use when added to food.

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Applications of chromatographic methods in metabolomics: A review

Chromatography is a robust and reliable separation method that can use various stationary phases to separate complex mixtures commonly seen in metabolomics. This review examines the types of chromatography and stationary phases that have been used in targeted or untargeted metabolomics with methods such as mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy. General considerations for sample pretreatment and separations in metabolomics are considered, along with the various supports and separation formats for chromatography that have been used in such work. The types of liquid chromatography (LC) that have been most extensively used in metabolomics will be examined, such as reversed-phase liquid chromatography and hydrophilic liquid interaction chromatography. In addition, other forms of LC that have been used in more limited applications for metabolomics (e.g., ion-exchange, size-exclusion, and affinity methods) will be discussed to illustrate how these techniques may be utilized for new and future research in this field. Multidimensional LC methods are also discussed, as well as the use of gas chromatography and supercritical fluid chromatography in metabolomics. In addition, the roles of chromatography in NMR- vs. MS-based metabolomics are considered. Applications are given within the field of metabolomics for each type of chromatography, along with potential advantages or limitations of these separation methods.

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Improved lipid analysis using a 2D-LC-MS system with a novel injection procedure

The aim of this study was to improve analysis of nonpolar lipidomics sample extracts using reversed phase (RP) chromatography. A 4/3/3 (v/v/v) mixture of methanol/methyl tert-butyl ether/chloroform (MeOH/MTBE/CHCl3, MMC) was chosen for sample extraction solvent based on its proven extraction capability for several lipid classes. To avoid carry over, loss of analytes and peak distortion the loops and all capillaries of the presented LC system were flushed and filled up with methanol until the analytical column. The choice of methanol was due to its weak elution strength and being infinitely miscible with MMC and several other nonpolar solvents. This allowed injection of a 100 μl sample that was 20 μl nonpolar extraction solvent diluted fivefold with methanol. All lipids of 25 lipid classes were transferred quantitatively to the column head where the online dilution of methanol was carried out with aqueous eluent for focusing the lipid analytes. The weak elution strength of methanol prevented peak distortions. The consecutive reversed phase elution resulted in remarkably narrow peaks (full width at half maximum was 0.07–0.08 min typically) and enhanced sensitivity (limit of detection usually in sub nM region) because of increased sample injection volume and narrow peaks. Calibration and quality control samples made by diluting commercial lipid standards 200–50000 times confirmed the applicability of this approach both for targeted lipid quantification and for untargeted quantitative comparison of lipids from different sources.

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A dilute and shoot method for urinary free cortisol analysis by LC-MS/MS

24-hour urinary free cortisol (UFC) is considered as the first-line test for screening and diagnosis of Cushing’s syndrome. Although 24-hour UFC assay has been extensively studied by liquid chromatography-tandem mass spectrometry (LC-MS/MS), an accurate assay coupled with a reliable sample preparation procedure and a method-specific reference interval would be very important for reasonable diagnosis. In this study, a simple dilute and shoot method has been proposed for UFC determination by LC-MS/MS. Namely, 50 µL of urine sample was mixed with 200 µL of a 50 % methanol/water solution containing the internal standard cortisol-13C3. The mixture was centrifuged and the supernatant was used for direct analysis by LC-MS/MS. This method was validated with wide linear range from 0.625 to 500 ng/ml with coefficients of variation (CVs) ≤ 3.64 %, excellent precision (intra-day CVs ≤ 5.70 % and inter-day CVs ≤ 5.33 %) and good recovery in the range of 93.3–109 %. The preservatives were further evaluated for urine storage. It was recommended that no preservatives could be used in collection of 24-hour urine for good detecting peaks. The investigation of reference interval and diagnostic performance finally confirmed the potential usage of this LC-MS/MS assay in routing clinical testing.

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High purity and recovery of native filamentous hemagglutinin (FHA) from Bordetella pertussis using affinity chromatography

Filamentous hemagglutinin (FHA) is a critical adhesion molecule produced by Bordetella pertussis (BP), the causative agent of highly contagious respiratory infection known as whooping cough. FHA plays a pivotal role in the pathogenesis of whooping cough and is a key component of acellular pertussis vaccines (aPV). However, conventional purification methods for FHA often involve labor-intensive processes and result in low purity and recovery rates. Therefore, this study explores the use of monoclonal and polyclonal antibodies as specific tools to achieve highly pure and efficient FHA purification. To generate FHA-specific antibodies, polyclonal antibodies were produced by immunizing sheep and monoclonal antibodies (MAbs) were generated by immunizing mice with recombinant and native FHA. The MAbs were selected based on affinity, isotypes, and specificity, which were assessed through ELISA and Western blot assays. Two immunoaffinity columns, one monoclonal and one polyclonal, were prepared for FHA antigen purification. The purity and recovery rates of these purifications were determined using ELISA, SDS-PAGE, and immunoblotting. Furthermore, the MAbs were employed to develop an ELISA assay for FHA antigen concentration determination. The study's findings revealed that immunoaffinity column-based purification of FHA resulted in a highly pure antigen with recovery rates of approximately 57% ± 6.5% and 59% ± 7.9% for monoclonal and polyclonal columns, respectively. Additionally, the developed ELISA exhibited appropriate reactivity for determining FHA antigen concentration. This research demonstrates that affinity chromatography is a viable and advantageous method for purifying FHA, offering superior purity and recovery rates compared to traditional techniques. This approach provides a practical alternative for FHA purification in the context of aPV development.

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LC-MS/MS methods for simultaneous determination of youkenafil and its metabolite M1 in human seminal plasma and plasma: Application to evaluate the acute effect of youkenafil on semen quality and its pharmacokinetics in human

Youkenafil is a novel Phosphodiesterase type 5 inhibitor used for treating erectile dysfunction. N-desethyl compound of youkenafil (M1) is its main active metabolite. In this study, two methods were developed and validated for the simultaneous determination of youkenafil and M1 by HPLC-MS/MS in human matrices including seminal plasma and plasma, in which the multiple reaction monitoring and electrospray ionization in positive mode were adopted, and the deuterated youkenafil (youkenafil-d5) was selected as the internal standard. The collected semen sample was kept at room temperature for approximately 30 min until fully liquefied. The volume of the liquefied semen was measured and then divided into two parts. One part was centrifuged to obtain the seminal plasma for the content detection of youkenafil and M1, while the other part was used for routine semen analysis. The chromatographic separation was accomplished with the column of Poroshell 120 EC-C18 (5 × 2.1 mm, 2.7 μm, Agilent). Protein precipitation with methanol was used for the pretreatment of seminal plasma and plasma. The intra-run and inter-run precisions were less than 6.4 % (relative standard deviation) and accuracies were all within −4.7 %–6.8 % (relative error) in both matrices. All other validated bioanalytical parameters were within the acceptance criteria set by the FDA. The methods were successfully applied to different clinical studies of youkenafil. In the clinical study of the acute effect of youkenafil on semen quality in healthy males, the content of youkenafil in seminal plasma was extremely low. Concentrations of youkenafil and M1 in seminal plasma were lower than those in plasma, at 20.7 % and 4.49 % of the plasma concentration, respectively. There was no significant acute effect of youkenafil on semen quality. In the pharmacokinetic study of youkenafil after single dose-escalation administration, the exposure to youkenafil and M1 was non-linear with the dose in the range of 100–400 mg.

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