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Journal of AOAC International Jun 2024Reboxetine (RBX) is the first FDA-approved antidepressant drug of the selective noradrenaline reuptake inhibitors class. There is a serious need for a convenient...
Development of Two Green and High Throughput Microwell Spectrometric Platforms for Determination of Reboxetine, the First FDA-Approved Selective Noradrenaline Reuptake Inhibitor Antidepressant Drug.
BACKGROUND
Reboxetine (RBX) is the first FDA-approved antidepressant drug of the selective noradrenaline reuptake inhibitors class. There is a serious need for a convenient analytical tool for the quantitation of RBX in its dosage form.
OBJECTIVE
This study aims to the development and validation of two green and high throughput microwell spectrometric platforms for the pharmaceutical analysis of RBX.
METHODS
The two platforms, abbreviated as MW-AB and MW-FL, involved microwell-based analysis assisted with a multifunction microplate plate reader for measuring absorbance and fluorescence signals, respectively. The MW-AB and MW-FL platforms involved the formation of colored and fluorescent derivatives upon the reaction of RBX with oxidized pyrocatechol reagent (OPC) and tetracyanoquinodimethane (TCNQ), respectively. The absorbance of colored RBX-OPC derivative at 520 nm, and the fluorescence of RBX-TCNQ charge transfer complex at 283 nm and 484 nm for excitation and emission, respectively. The optimum conditions of both reactions were established, their molar ratios were determined, and reaction mechanisms were postulated.
RESULTS
Both platforms were optimized and validated according to the guidelines of the International Council on Harmonization. The limits of quantitation were 19.6 µg/mL and 27 ng/mL for MW-AB and MW-FL, respectively. Both platforms were applied with excellent reliability to the quantitation of RBX content in Edranox® tablets and their drug uniformity. The greenness levels of both platforms were assessed by two comprehensive tools, and the results confirmed the high level of greenness for both platforms.
CONCLUSIONS
Both platforms involved one-step reactions, adapted microwell analysis, and simultaneous handling of large number of samples. Therefore, they have the advantages of greenness and high throughput analysis.
HIGHLIGHTS
The proposed two platforms are valuable tools for the rapid quantitation of RBX.
PubMed: 38941495
DOI: 10.1093/jaoacint/qsae051 -
Luminescence : the Journal of... Jun 2024Pemigatinib (PGT) is a recently FDA-approved small molecule kinase inhibitor used for the treatment of relapsed or refractory myeloid/lymphoid neoplasms in adults. This...
A green and highly sensitive microwell spectrofluorimetric method with high throughput for the determination of pemigatinib based on dual fluorescence enhancement by photoinduced electron transfer blocking and micellization: Application to the analysis of tablets, content uniformity testing, and...
Pemigatinib (PGT) is a recently FDA-approved small molecule kinase inhibitor used for the treatment of relapsed or refractory myeloid/lymphoid neoplasms in adults. This study introduces the development of a first microwell spectrofluorimetric method (MW-SFM) for quantifying PGT in FDA-approved tablets and plasma samples. The method utilized the enhancement of PGT's weak native fluorescence by blocking photoinduced electron transfer (PET) and micellization with sodium lauryl sulfate (SLS). The MW-SFM was performed in 96-microwell plates, and fluorescence signals were measured using a fluorescence microplate reader with excitation at 290 nm and emission at 350 nm. The method exhibited a linear range of 2-250 ng mL, and a limit of quantitation was 6.5 ng mL. The accuracy and precision of the method were confirmed with recovery rates ranging from 96.5% to 102.8% and relative standard deviations of 1.52% to 3.51%. The MW-SFM successfully analyzed Pemazyre® tablets, assessed content uniformity, and analyzed PGT-spiked human plasma samples. The greenness of the MW-SFM was verified using three different metric tools. In conclusion, the proposed MW-SFM is a valuable tool in supporting quality assessment of dosage forms, conducting pharmacokinetic studies, and monitoring therapeutic outcomes.
Topics: Humans; Tablets; Spectrometry, Fluorescence; Fluorescence; Electron Transport; Micelles; Pyrimidines; Sodium Dodecyl Sulfate; Molecular Structure; Photochemical Processes
PubMed: 38922756
DOI: 10.1002/bio.4813 -
Journal of AOAC International Jun 2024Galidesivir (GDV) is a promising new antiviral drug for the potent and safe treatment of a broad spectrum of viral diseases, including COVID-19. In the literature, no...
BACKGROUND
Galidesivir (GDV) is a promising new antiviral drug for the potent and safe treatment of a broad spectrum of viral diseases, including COVID-19. In the literature, no analytical method exists for the determination of GDV in bulk and dosage form.
OBJECTIVE
The aim of this study was the development of versatile green and simple microwell spectrophotometric methods (MW-SPMs) for the determination of GDV in its bulk form and capsules.
METHODS
Three MW-SPMs were developed involving the oxidation of GDV by ammonium metavanadate (AMV), chromium trioxide (CTO), and potassium iodate (PIO) in an acid medium. The reactions were carried out in 96-well plates at room temperature and the absorbances of chromogenic reaction products were measured by an absorbance microplate reader at 780, 595, and 475 nm for AMV, CTO, and PIO, respectively. Variables influencing the reactions were carefully investigated and optimized.
RESULTS
Linear relations with excellent correlation coefficients (0.9991-0.9997) were found between the absorbances and GDV concentrations in a range of 25-500 µg/mL. The limits of detection were ≥8.3 µg/mL. The accuracy and precision of the three MW-SPMs were confirmed by recovery and replicate analysis, respectively. The recovery values were 98.6-101.2% and the relative standard deviations were ≤1.02%. The proposed MW-SPMs were successfully applied to the analysis of GDV in bulk drug and capsules with high accuracy and precisions. The greenness of MW-SPMs was confirmed by three comprehensive metric tools.
CONCLUSIONS
The proposed MW-SPMs combined the inherent advantages of microwell-based analysis and the use of common laboratory reagents for the involved reaction. These advantages include high-throughput, readily automation, reduced samples/reagents volume, precise measurements, and versatility. The advantages of the use of common laboratory reagents include availability, consistency, compatibility, safety, and cost-effectiveness.
HIGHLIGHTS
Overall, the proposed MW-SPMs are versatile valuable tools for the quantitation of GDV during its pharmaceutical manufacturing.
PubMed: 38870529
DOI: 10.1093/jaoacint/qsae047 -
Bacterial Aggregation in Cerebral Spinal Fluid: The Extent it Occurs and the Clinical Ramifications.Current Microbiology Jun 2024Bacteria can form aggregates in synovial fluid that are resistant to antibiotics, but the ability to form aggregates in cerebral spinal fluid (CSF) is poorly defined....
Bacteria can form aggregates in synovial fluid that are resistant to antibiotics, but the ability to form aggregates in cerebral spinal fluid (CSF) is poorly defined. Consequently, the aims of this study were to assess the propensity of four bacterial species to form aggregates in CSF under various conditions. To achieve these aims, bacteria were added to CSF in microwell plates and small flasks at static and different dynamic conditions with the aid of an incubating shaker. The aggregates that formed were assessed for antibiotic resistance and the ability of tissue plasminogen activator (TPA) to disrupt these aggregates and reduce the number of bacteria present when used with antibiotics. The results of this study show that under dynamic conditions all four bacteria species formed aggregates that were resistant to high concentrations of antibiotics. Yet with static conditions, no bacteria formed aggregates and when the CSF volume was increased, only Staphylococcus aureus formed aggregates. Interestingly, the aggregates that formed were easily dispersed by TPA and significant (p < 0.005) decreases in colony-forming units were seen when a combination of TPA and antibiotics were compared to antibiotics alone. These findings have clinical significance in that they show bacterial aggregation does not habitually occur in central nervous system infections, but rather occurs under specific conditions. Furthermore, the use of TPA combined with antibiotics may be advantageous in recalcitrant central nervous system infections and this provides a pathophysiological explanation for an unusual finding in the CLEAR III clinical trial.
Topics: Humans; Anti-Bacterial Agents; Cerebrospinal Fluid; Bacteria; Staphylococcus aureus; Tissue Plasminogen Activator; Drug Resistance, Bacterial; Microbial Sensitivity Tests
PubMed: 38831167
DOI: 10.1007/s00284-024-03727-4 -
Contact Lens & Anterior Eye : the... May 2024Midday fogging (MDF) occurs when particulate material accumulates in the fluid reservoir (FR) beneath scleral lenses (SL), and its impact on epithelial cells is unknown....
PURPOSE
Midday fogging (MDF) occurs when particulate material accumulates in the fluid reservoir (FR) beneath scleral lenses (SL), and its impact on epithelial cells is unknown. This study examines the in vitro pro-inflammatory effect of the FR on human corneal epithelial cells in varying degrees of MDF.
METHODS
Normal SL neophytes were recruited to wear SL 8 h daily for 4 days. Following 8 h on days 1 and 4, optical coherence tomography (OCT) images were acquired for MDF quantification using ImageJ, and the FR was collected. FR samples from the same eye were later pooled, diluted 2-fold and applied on human telomerase-immortalized corneal epithelial (hTCEpi) cells cultured on Terasaki microwell plates. Tumor necrosis factor (TNF)-α and culture media were used as positive and negative controls, respectively. After a 30-minute treatment, the nuclear factor-kappa B (NF-κB) pathway was measured by NF-κB-p65 immunofluorescence and images were analyzed with ImageJ. Pearson's correlation was conducted to determine the association between median nuclear fluorescence and MDF.
RESULTS
Fourteen FR samples with a mean volume of 22 ± 16 µl were tested. Mean MDF severity following 8 h of SL wear was 25 ± 17 units (range 7 - 64). The median nuclear fluorescence (NF-κB-p65 translocation) in cultured hTCEpi cells ranged from 31.43 to 45.16 while the negative and positive controls were 44.71 ± 1.72 and 108.77 ± 68.38, respectively. Although a potential positive trend between MDF and median nuclear fluorescence was observed, Pearson's correlation analysis revealed no significant association (r = +0.48, P = 0.09).
CONCLUSIONS
The results suggest that the FR can trigger NF-κB-p65 translocation in hTCEpi cells, which may be associated with MDF severity. This study introduces the use of Terasaki microwell plates for immunofluorescence studies of the FR. The technique is simple, minimizes sample usage, and does not require expensive instrumentation.
PubMed: 38762441
DOI: 10.1016/j.clae.2024.102187 -
RSC Advances May 2024Single use plasticware (SUP) in scientific, diagnostic, and academic laboratories makes a significant contribution to plastic waste generation worldwide. Polystyrene...
Single use plasticware (SUP) in scientific, diagnostic, and academic laboratories makes a significant contribution to plastic waste generation worldwide. Polystyrene (PS) microwell plates form a part of this waste. These plates are the backbone of high throughput colorimetric measurements in academic, research, and healthcare settings for detection/quantification of wide-ranging analytes including proteins, carbohydrates, nucleic acids, and enzyme activity. Polystyrene (PS) microwell plates serve as a platform for holding samples and reagents, where mixing initiates chemical reaction(s), and the ensuing color changes are quantified using a microplate reader. However, these plates are rarely reused or recycled, contributing to the staggering amounts of plastic waste generated in scientific laboratories. Here, we are reporting the fabrication of cellulose acetate (CA) microwell plates as a greener alternative to non-biodegradable PS plates and we demonstrate their application in colorimetric assays. These easy to fabricate, lighter weight, customizable, and environmentally friendly plates were fabricated in 96- and 384-well formats and made water impermeable through chemical treatment. The plates were tested in three different colorimetric analyses: (i) bicinchoninic acid assay (BCA) for protein quantification; (ii) chymotrypsin (CT) activity assay; and (iii) alkaline phosphatase (AP) activity assay. Color intensities were quantified using a freely available smartphone application, Spotxel® Reader (Sicasys Software GmbH). To benchmark the performance of this platform, the same assays were performed in commercial PS plates too and quantified using a UV/Vis microplate reader. The two systems yielded comparable linear correlation coefficients, LOD and LOQ values, thereby validating the CA plate-cell phone based analytical method. The CA microwell plates, coupled with smart phone optical data capture, provide greener, accessible, and scalable tools for all laboratory settings and are particularly well-suited for resource- and infrastructure-limited environments.
PubMed: 38741966
DOI: 10.1039/d4ra01317d -
Advanced Science (Weinheim,... Apr 2024Targeting "undruggable" targets with intrinsically disordered structures is of great significance for the treatment of disease. The transcription factor c-Myc controls...
Targeting "undruggable" targets with intrinsically disordered structures is of great significance for the treatment of disease. The transcription factor c-Myc controls global gene expression and is an attractive therapeutic target for multiple types of cancers. However, due to the lack of defined ligand binding pockets, targeted c-Myc have thus far been unsuccessful. Herein, to address the dilemma of lacking ligands, an efficient and high throughput aptamer screening strategy is established, named polystyrene microwell plate-based systematic evolution of ligands by exponential enrichment (microwell-SELEX), and identify the specific aptamer (MA9C1) against c-Myc. The multifunctional aptamer-based Proteolysis Targeting Chimeras (PROTAC) for proteolysis of the c-Myc (ProMyc) is developed using the aptamer MA9C1 as the ligand. ProMyc not only significantly degrades c-Myc by the ubiquitin-proteasome system, but also reduces the Max protein, synergistically inhibiting c-Myc transcriptional activity. Combination of the artificial cyclization and anti-PD-L1 aptamer (PA1)-based delivery system, circular PA1-ProMyc chimeras achieve tumor regression in the xenograft tumor model, laying a solid foundation for the development of efficacious c-Myc degrader for the clinic. Therefore, this aptamer-based degrader provides an invaluable potential degrader in drug discovery and anti-tumor therapy, offering a promising degrader to overcome the challenge of targeting intractable targets.
PubMed: 38682443
DOI: 10.1002/advs.202309639 -
Methods in Enzymology 2024There is intense interest in removing fluorinated compounds from the environment, environments are most efficiently remediated by microbial enzymes, and defluorinating...
There is intense interest in removing fluorinated compounds from the environment, environments are most efficiently remediated by microbial enzymes, and defluorinating enzymes are readily monitored by fluoride determination. Fluorine is the most electronegative element. Consequently, all mechanisms of enzymatic C-F bond cleavage produce fluoride anion, F. Therefore, methods for the determination of fluoride are critical for C-F enzymology and apply to any fluorinated organic compounds, including PFAS, or per- and polyfluorinated alkyl substances. The biodegradation of most PFAS chemicals is rare or unknown. Accordingly, identifying new enzymes, or re-engineering the known defluorinases, will require rapid and sensitive methods for measuring fluoride in aqueous media. Most studies currently use ion chromatography or fluoride specific electrodes which are relatively sensitive but low throughput. The methods here describe refashioning a drinking water test to efficiently determine fluoride in enzyme and cell culture reaction mixtures. The method is based on lanthanum alizarin complexone binding of fluoride. Reworking the method to a microtiter well plate format allows detection of as little as 4 nmol of fluoride in 200 μL of assay buffer. The method is amenable to color imaging, spectrophotometric plate reading and automated liquid handling to expedite assays with thousands of enzymes and/or substrates for discovering and improving enzymatic defluorination.
Topics: Fluorides; Drinking Water; Halogenation; Enzyme Assays
PubMed: 38658089
DOI: 10.1016/bs.mie.2023.12.020 -
Lab on a Chip May 2024Genetically modified (GM) food is still highly controversial nowadays. Due to the disparate policies and attitudes worldwide, demands for a rapid, cost-effective and...
Genetically modified (GM) food is still highly controversial nowadays. Due to the disparate policies and attitudes worldwide, demands for a rapid, cost-effective and user-friendly GM crop identification method are increasingly significant for import administration, market supervision, However, as the most-recognized methods, nucleic acid-based identification approaches require bulky instruments, long turn-around times and trained personnel, which are only suitable in laboratories. To fulfil the urgent needs of on-site testing, we develop a point-of-care testing platform that is able to identify 12 types of GM crops in less than 40 minutes without using laboratory settings. Our system integrates sample pre-treatment modules in a microfluidic chip, performs DNA amplification a battery-powered portable kit, and presents results eye-recognized colorimetric change. A paraffin-based reflow method and a slip plate-based fluid switch are developed to encapsulate and release amplification primers in individual microwells on demand, thus enabling identification of varied targets simultaneously. Our system offers an efficient, affordable and convenient tool for GM crop identification, thus it will not only benefit customs and market administration bureaus, but also satisfy demands of numerous consumers.
Topics: Plants, Genetically Modified; Crops, Agricultural; Point-of-Care Testing; Lab-On-A-Chip Devices; Nucleic Acid Amplification Techniques; Microfluidic Analytical Techniques
PubMed: 38644672
DOI: 10.1039/d4lc00040d -
Lab on a Chip Apr 2024Tumor spheroids are now intensively investigated toward preclinical and clinical applications, necessitating the establishment of accessible and cost-effective methods...
Tumor spheroids are now intensively investigated toward preclinical and clinical applications, necessitating the establishment of accessible and cost-effective methods for routine operations. Without losing the advantage of organ-chip technologies, we developed a rocking system for facile formation and culture of tumor spheroids in hydrogel microwells of a suspended membrane under microfluidic conditions. While the rocking is controlled with a step motor, the microfluidic device is made of two plastic plates, allowing plugging directly syringe tubes with Luer connectors. Upon injection of the culture medium into the tubes and subsequent rocking of the chip, the medium flows back and forth in the channel underneath the membrane, ensuring a diffusion-based culture. Our results showed that such a rocking- and diffusion-based culture method significantly improved the quality of the tumor spheroids when compared to the static culture, particularly in terms of growth rate, roundness, junction formation and compactness of the spheroids. Notably, dynamically cultured tumor spheroids showed increased drug resistance, suggesting alternative assay conditions. Overall, the present method is pumpless, connectionless, and user-friendly, thereby facilitating the advancement of tumor-spheroid-based applications.
Topics: Spheroids, Cellular; Humans; Lab-On-A-Chip Devices; Cell Culture Techniques; Diffusion; Microfluidic Analytical Techniques; Hydrogels; Cell Line, Tumor; Tumor Cells, Cultured; Equipment Design
PubMed: 38629978
DOI: 10.1039/d3lc01116j