Recovering from a COVID-19 Shutdown: Tips and Tricks for Starting Up, Part I

Aside from evaporation of water from buffer solutions, precipitation of buffer salts can also easily occur when a buffer solution comes into contact with a pure organic solvent. [...]one should avoid- at all costs-a situation where a LC system component containing an aqueous buffer is flushed immediately with a pure organic solvent. Solvents and Buffers Figure 1 shows a picture of a buffer bottle I observed in an LC laboratory about a year ago. Many of the aqueous buffer solutions used in LC (and even high-performance liquid chromatography [HPLC] grade water, if given enough time and exposure to aboratory dust) are environments quite favorable to microbes, particularly those in the middle of the pH range.

Complex presentations in people with diabetes: Does HbA1c help or hinder?

HbA1c measurement is widely used for diagnosis/ management/remission of diabetes with international schemes certifying comparability. A) 75 year-old Chinese female with type 2 diabetes was admitted in April 2020 with Covid-19 and diabetic ketoacidosis. Glucose was 35mmol/l and HbA1c 150mmol/mol with previous HbA1c of 45mmol/mol on metformin and alogliptin. She was treated for ketoacidosis and once-daily Lantus introduced along with supportive management of viral illness. B) 68 year-old Afro-Caribbean with type 2 diabetes on metformin before admission, presented with new onset, jerky ballistic movements of high amplitude in right arm, 10-15 movements every 5 min. Admission glucose was >33mmol/l, ketones 1.8mmol/l and HbA1c >217mmol/ mol. Hemichorea-hemiballism, a hyperglycaemia related movement was diagnosed and insulin commenced. Glucose decreased to 8-20mmol/ l, reaching 5-15mmol/ l by time of discharge. Ballistic movements resolved when glycaemic control improved with HbA1c 169mmol/mol, 25 days after discharge. C) Several days before admission, a female with diabetes over 20 years required attention from paramedics on four occasions for hypoglycaemia. Months beforehand metformin was replaced by gliclazide due to chronic kidney disease with HbA1c 50mmol/mol, and she was transfused six weeks before admission for microcytic anaemia. Gliclazide was discontinued and her diet modified which prevented further hypoglycaemic episodes. Variant haemoglobin, beta-thalassaemia which can overestimate glycaemia;undetected by HbA1c HPLC method, invalidated HbA1c as did the blood transfusion. These cases highlight that inadequate understanding of HbA1c can lead to acute presentations of dysglycaemia. As HbA1c accuracy can be affected by multiple factors, clinical assessment and triangulation are key to the management of such patients.

Synthesis and Analysis of the SARS-CoV-2 ORF7a Accessory Protein Transmembrane Domain and Determination of the Oligomerization State

The ongoing SARS-CoV-2 pandemic continues to sicken millions worldwide and fundamentally change the way people interact with each other. In order to better characterize the SARS-CoV-2 virus and potentially develop methods of inhibition for further spread of the disease, this research project focused on synthesizing and characterizing the trans-membrane region of the accessory protein ORF7a. ORF7a has been implicated in proper viral assembly, leading to the idea that inhibition of this protein could prevent viral copies from being produced and halt the spread of the virus. The goal of this project was to determine the oligomerization state of the protein through a fluorescence assay in order to better understand the quaternary structure of the ORF7a complex and how it folds. The fluorescence assay is performed using three different samples of the synthesized peptide: one labeled with a TAMRA fluorophore, one labeled with a NBD fluorophore, and the last is unlabeled. After determining the oligomerization state of the protein, potential inhibitors could be synthesized and tested for their efficacy at inhibiting the function of the protein. Further applications of these inhibitors on other viruses can be explored due to the highly conserved nature of transmembrane domains across multiple viral families. Synthesis of the protein was done using a Solid Phase Peptide Synthesis (SPPS) technique and multiple batches of all three samples of peptide have been generated. Characterization and purification were done using High Performance Liquid Chromatography (HPLC) as well as Liquid Chromatography Mass Spectrometry (LCMS). Current research focuses on the purification and quantification of purified ORF7a oligopeptide for implementation of the fluorescence assay. -Hampden-Sydney College Office of Undergraduate Research.Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.

VENDOR PERSPECTIVES

The people who work at companies that manufacture chromatography instruments and consumables are often well positioned to be aware of developments, needs, and trends that not everyone else sees, because they serve customers in a range of areas of focus and with diverse demands-such as academic researchers investigating fundamental aspects of separation science techniques, industrial analysts solving problems that they may not be allowed to talk about at conferences, or chemists working in government laboratories in areas like environmental research. Inaccurate data can be generated from variable, glass vial surface chemistries, which can lead to investigations or flawed decisions can be made from these results. The Reduced Surface Activity (RSA(tm)) Glass Technology was developed to address these issues, and to provide chemists with sample containers for LC-MS, MS, HPLC, GC, and CE that deliver reliable, consistent results by not adsorbing basic analytes, or adding metals to, or changing the pH of the diluent. The stringent RSA manufacturing processes are continued through to final contaminate-free packaging and quality control, where they are tested for adsorption, metal content, and residual materials.

In Situ Electrochemical Trapping and Unraveling the Mechanism of the Toxic Intermediate Metabolite N-Acetyl-p-Benzoquinone Imine of the Acetaminophen Drug and Its Biomimetic Mediated NADH Oxidation Reaction

The integrative study of the pharmacokinetics and dynamics of a drug has been of great research interest due to its authentic description of the biomedical and clinical pros and cons. Acetaminophen (N-acetyl-4-aminophenol, AcAP) is a well-known analgesic having a high therapeutic value, including the Covid-19 treatment. However, an overdose of the drug (>200 mg/kg of men) can lead to liver toxicity. An intermediate, N-acetyl-p-benzoquinone imine (NAPQI), metabolite formation has been found to be responsible for the toxicity. For the detection of NAPQI, several ex situ techniques based on electrochemical methods followed by nuclear magnetic resonance, high-performance liquid chromatography, and LC-MS were stated. For the first time, we report an in situ electrochemical approach for AcAP oxidation and NAPQI intermediate (Mw = 149.1 g mol-1) trapping on a graphitic nanomaterial, carbon black (CB)-modified electrode in pH 7 phosphate buffer solution (CB@NAPQI). The NAPQI-trapped electrode exhibited a surface-confined redox peak at E°′ = 0.350 ± 0.05 V vs Ag/AgCl with a surface excess value of 3.52 n mol cm-2. Physicochemical characterizations by scanning electron microscopy, Raman, FTIR, and in situ electrochemical quartz crystal microbalance (EQCM) techniques supported the entrapment of the molecular species. Furthermore, the scanning electrochemical microscopy (SECM) technique has been adopted for surface-mapping the true active site of the NAPQI-trapped electrode. As a biomimetic study, the mediated oxidation reaction of NADH by CB@NAPQI was demonstrated, and the mechanistic and quantitative aspects were studied using cyclic voltammetry, rotating disc electrode, amperometry, and flow injection analysis techniques. © 2023 American Chemical Society.

Changes in the proteomics of exhaled breath condensate under the influence of inhaled hydrogen in patients with post-COVID syndrome.

Aim. To study the effect of inhalation therapy with an active hydrogen (AH) on the protein composition of exhaled breath condensate (EBC) in patients with post-COVID syndrome (PCS). Material and methods. This randomized controlled parallel prospective study included 60 patients after coronavirus disease 2019 (COVID-19) with PCS during the recovery period and clinical manifestations of chronic fatigue syndrome who received standard therapy according to the protocol for managing patients with chronic fatigue syndrome (CFS). The patients were divided into 2 groups: group 1 (main) - 30 people who received standard therapy and AH inhalations (SUISONIA, Japan) for 10 days, and group 2 (control) - 30 medical workers who received only standard therapy. Patients in both groups were comparable in sex and mean age. All participants in the study were sampled with EBC on days 1 and 10. Samples were subjected to tryptic digestion and high-performance liquid chromatography combined with tandem mass spectrometry analysis using a nanoflow chromatograph (Dionex 3000) in tandem with a high-resolution time-of-flight mass spectrometer (timsTOF Pro). Results. A total of 478 proteins and 1350 peptides were identified using high resolution mass spectrometry. The number of proteins in samples after AH therapy, on average, is 12% more than before treatment. An analysis of the distribution of proteins in different groups of patients showed that only half of these proteins (112) are common for all groups of samples and are detected in EBC before, after, and regardless of hydrogen therapy. In addition to the qualitative difference in the EBC protein compositions in different groups, quantitative changes in the concentration of 36 proteins (mainly structural and protective) were also revealed, which together made it possible to reliably distinguish between subgroups before and after treatment. It is worth noting that among these proteins there are participants of blood coagulation (alpha-1-antitrypsin), chemokine- and cytokine-mediated inflammation, and a number of signaling pathways (cytoplasmic actin 2), response to oxidative stress (thioredoxin), glycolysis (glyceraldehyde-3- phosphate dehydrogenase), etc. Conclusion. The use of hydrogen therapy can contribute to the switching of a number of physiological processes, which may affect the success of recovery in PCS patients. In particular, the obtained results indicate the activation of aerobic synthesis of adenosine triphosphate in mitochondria by hydrogen therapy, which correlates well with the decrease in the blood lactate level detected by laboratory studies. At the same time, this therapy can inhibit pro-inflammatory activity, negatively affecting the coagulation and signaling pathways of integrins and apoptosis, and, in addition, activate protective pathways, tricarboxylic acid cycle, FAS signaling, and purine metabolism, which may be essential for effective recovery after COVID-19.Copyright © 2023 Vserossiiskoe Obshchestvo Kardiologov. All rights reserved.

Development of a spin-label model system for investigation of protein oligomerization states

The transmembrane domains of viral proteins are highly conserved and crucial to normal viral function. Oligomeric transmembrane domains present novel opportunities for drug development, as their disruption can prevent the assembly of the virus. The Reichart lab is particularly interested in developing retro-inverso peptide inhibitors. Retro-inverso peptides are peptides using D-amino acids mirroring a region of target protein, which allows the peptide to inhibit viral assembly, but they are also significantly less likely to be catabolized by natural metabolic or immunologic processes. The efficacy of these inhibitors is governed largely by the extent to which they mirror the target protein, making highly conserved regions, such as transmembrane domains, ideal target regions for these inhibitors. The primary technique in the literature for the investigation of oligomerization states uses fluorescence spectroscopy. We are now working on developing a novel alternative system to evaluate protein oligomerization using spin-labeled peptides that are directly incorporated into the peptide sequence. Direct incorporation of the spin-label into the peptide sequence is a more powerful technique than the standard procedures used in the literature. In particular, the ability to incorporate spin labels in various positions within the protein can give novel insights into the relative depth of the protein within a membrane, which is very difficult to study using other techniques and not possible using the fluorescence technique. The transmembrane domains of proteins with known and well-characterized monomer and trimer standard oligomerization states were synthesized using an Fmoc Solid- Phase Peptide Synthesis (SPPS) procedure incorporating an Fmoc-2,2,6,6-tetramethyl-N-oxyl-4-amino-4-carboxylic acid, (Fmoc-TOAC) instead of an alanine. Direct incorporation of stable N-oxide spin labels, which can be contrasted to labeling cysteine residues after the protein synthesis, has been used for the investigation of the secondary structure of proteins for decades, but the application of this spin labeling technique to study the oligomerization states of transmembrane domains of proteins is an understudied application. The products of SPPS were analyzed using a Liquid Chromatography Mass Spectroscopy instrument and purified using High Performance Liquid Chromatography. The spin-label was then deprotected and evaluated using Electron Spin Resonance (ESR) Spectroscopy. There are two primary future directions following this research project: first, the generation of viral proteins with spin labels incorporated in different positions to determine the relative depth of each position within the membrane;second, the incorporation of spin labels into SARS-CoV- 2 proteins to develop a model for in vitro evaluation of retro-inverso peptide assembly inhibitors. -Hampden-Sydney College Office of Undergraduate Research.Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.

Synthesis of the Transmembrane Domain of the Spike Protein from SARS-CoV-2 Using Solid Phase Peptide Synthesis and Determination of Its Oligomerization State

The spike protein in severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) is directly responsible for the binding to ACE2 receptors in host cells. While the spike protein overall is known to form trimers, the oligomerization state of the transmembrane domain of the spike protein in SARS-CoV-2 is unknown. It is believed to be essential for the function of this protein. Since the transmembrane domain of the spike protein is highly conserved in SARS-CoV-2 it is important to investigate its character and determine its relationship to the function of the protein as awhole. The goal of this project was to synthesize, characterize, and analyze the function of the transmembrane domain (TM) of the spike protein in SARS-CoV-2. The most practical method to synthesize the TM domain of the S protein is through solid phase peptide synthesis (SPPS). SPPS is a process in which peptides are made by linking amino acids, the monomers of proteins, one at a time until the full sequence is achieved. These peptide chains will then need to be purified using high-performance liquid chromatography (HPLC). The synthesized peptides will be analyzed using liquid chromatography- mass spectrometry (LCMS) to confirm the identity of the synthesized peptides as well as any potential impurities. The continued investigation of the S protein can lead to the discovery of small peptides capable of inhibiting key processes to the binding mechanism of SARS-CoV-2. The function of the S protein is believed to only present when the transmembrane domain forms a trimer. Therefore, the analysis of their oligomerization states will be investigated by synthesizing versions of the peptide that fluoresce when excited using dyes such as nitrobenzodiazole (NBD) and tetramethylrhodamine (TAMRA) in a fluorescence assay. -Hampden-Sydney College Office of Undergraduate Research.Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.

Using Synthetic Biology methods to construct a functional estrogen biosensor based on split Nanoluciferase activity

The presence of estrogenic compounds (endocrine-disruptors, EDCs) in the water supply raises concerns about human and aquatic health. Current methods for detecting estrogen contamination require expensive, time-consuming techniques such as liquid chromatography-mass spectrometry and high-performance liquid chromatography. Previously reported estrogen biosensors required multiple cloning and transformation steps for successful detection in bacteria. Synthetic biology allows for the construction of genetic devises composed of DNA sequences modified to be interchangeable and provide novel functions. New tools and devices are constantly needed to enhance the already extensive list of novel genetic parts. Our approach to the design of an estrogen responsive element uses methodology developed in the Wells lab (Elledge et al, 2021) to detect SARS-CoV-2 antibodies. This methodology takes advantage of the split Nanoluciferase (spLUC) protein divided into two functional domains (designated SmBit and LgBit). Based on rational engineering design we express dimerization dependent LgBit and SmBit fused to the Estrogen Receptor alpha protein (ERalpha) in bacteria cells. These two monomeric proteins will dimerize in the presence of estrogen, reconstitute the split luciferase enzyme and reestablish enzyme activity. Cells can be lysed, and luminescence detected to quantify estrogen present in the sample. We present here the construction strategy and proof of concept data demonstrating the efficiency of this dual-functional biosensor and its effectiveness for detection of estrogenic compounds in contaminated water. NSF-REU-1852150, REU Site: A multisite REU in Synthetic Biology, 2019.Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.

Estimating Moisture Content of Sausages with Different Types of Casings via Hyperspectral Imaging in Tandem with Multivariate

The moisture levels in sausages that were stored for 16 days and added with different concentrations of orange extracts to a modification solution were assessed using response surface methodology (RSM). Among the 32 treatment matrixes, treatment 10 presented a higher moisture content than that of treatment 19. Spectral pre-treatments were employed to enhance the model's robustness. The raw and pre-processed spectral data, as well as moisture content, were fitted to a regression model. The RSM outcomes showed that the interactive effects of [soy lecithin concentration] × [soy oil concentration] and [soy oil concentration] × [orange extract addition] on moisture were significant (p < 0.05), resulting in an R2 value of 78.28% derived from a second-order polynomial model. Hesperidin was identified as the primary component of the orange extracts using high-performance liquid chromatography (HPLC). The PLSR model developed from reflectance data after normalization and 1st derivation pre-treatment showed a higher coefficient of determination in the calibration set (0.7157) than the untreated data (0.2602). Furthermore, the selection of nine key wavelengths (405, 445, 425, 455, 585, 630, 1000, 1075, and 1095 nm) could render the model simpler and allow for easy industrial applications.

Predictive stability, novel HPLC-MS analysis and semi-automatic compounding process for the emergency implementation of a production line of pancuronium in injectable solution.

During the early months of the COVID-19 pandemic, the international medical product supply chain was tight, causing breaks in the availability of neuromuscular blocking agents essential for the treatment of patients in intensive care units. The present study describes the pharmaceutical development of an injectable 2 mg/mL solution of pancuronium bromide (PC) in a very short lapse of time. The sterile solution was compounded into a good manufacturing practice grade A clean room, filtered (0.2 µm) and filled into 10 mL type I glass, manually sealed with bromobutyl rubber stoppers. A novel HPLC-MS stability indicating method for pancuronium quantification and its degradation product was developed and validated. This fast, sensitive and straightforward method was used to study the stability of the formulation using a semi-predictive method, enabling a very fast attribution of a temporary shelf-life, which was confirmed by a classic prospective stability study. The production line and the analytical tools set-up were performed in six weeks and the semi-predictive stability study was conducted in 90 days, allowing us to predict a shelf life, which was successfully confirmed by prospective study. In conclusion, using innovative methods, we were able to rapidly overcome the shortage of a critical drug.

Development and Validation of a Novel Stability Indicating Reverse Phase High Performance Liquid Chromatographic Method for Related Substances and Assay Analysis of Molnupiravir Drug Substance and Drug Product

Molnupiravir, a broad-spectrum antiviral is an isopropyl ester prodrug of beta-D-N4-hydroxycytidine. Molnupiravir targets RNA-dependent RNA-polymerase enzyme of the viruses. A new stability-indicating HPLC-method was developed to determine related substances and assay of molnupiravir. Separation was achieved by using Shim-pack GWS C18 column. The method was validated according to current ICH requirements. The calibration plot gave a linear relationship for all known analytes over the concentration range from LOQ to 200%. LOD and LOQ for all known analytes were found in 0.05-0.08 microg mL-1 and 0.12-0.20 microg mL-1, respectively, the mean recovery was found to be 97.79-102.44 %. Study showed that the method, results of robustness, solution stability studies are precise and within the acceptable limits. Molnupiravir was found to degrade in acid, alkali, and oxidative conditions, and was stable in thermal, moisture, and photolytic degradation condition. The method is simple, accurate, precise, and reproducible for routine purity analysis of drug-samples.Copyright © 2022 Indian Drug Manufacturers' Association. All rights reserved.

An Rp-Hplc Method for the Quantitative Determination of Remdesivir in Intravenous Dosage Form

The worldwide epidemic of Coronavirus disease 2019 (COVID-19), caused by a new virus known as severe acute respiratory syndrome (SARS) coronavirus 2, has posed a growing threat to public health (SARS-CoV-2). The only antiviral drug authorized by the FDA for treating adult and pediatric patients hospitalized with a severe disease is remdesivir, which is given intravenously (IV). Although only a few methods for estimating remdesivir in pharmaceutical formulations using high-pressure liquid chromatography (HPLC) have been described, its determination still requires an accurate, precise, quick, and easy analytical methodology. The main goal of this study was to develop and validate a reliable and accurate HPLC method for quantitative estimation of remdesivir in its intravenous dosage formulation. The separation was performed on a C18 (4.6 mm x 150 mm, 5.0 microm) column with a flow rate of 0.7 mL/min and a total run duration of 6 minutes using a simple isocratic mobile phase of acetonitrile and 0.1 percent formic acid. The method was validated for the system suitability, linearity, precision, accuracy, robustness, and others as per the International Council for Harmonization (ICH) Q2 (R1) guideline. The results show that the method for measuring remdesivir using HPLC is simple, quick, sensitive, accurate, precise and robust. The described approach was successfully used to quantify remdesivir in a commercially available pharmaceutical formulation.Copyright All © 2023 are reserved by International Journal of Pharmaceutical Sciences and Research.

Central Nervous System Distribution of the Ataxia-Telangiectasia Mutated Kinase Inhibitor AZD1390: Implications for the Treatment of Brain Tumors

Effective drug delivery to the brain is critical for the treatment of glioblastoma (GBM), an aggressive and invasive primary brain tumor that has a dismal prognosis. Radiation therapy, the mainstay of brain tumor treatment, works by inducing DNA damage. Therefore, inhibiting DNA damage response (DDR) pathways can sensitize tumor cells to radiation and enhance cytotoxicity. AZD1390 is an inhibitor of ataxia-telangiectasia mutated kinase, a critical regulator of DDR. Our in vivo studies in the mouse indicate that delivery of AZD1390 to the central nervous system (CNS) is restricted due to active efflux by P-glycoprotein (P-gp). The free fraction of AZD1390 in brain and spinal cord were found to be low, thereby reducing the partitioning of free drug to these organs. Coadministration of an efflux inhibitor significantly increased CNS exposure of AZD1390. No differences were observed in distribution of AZD1390 within different anatomic regions of CNS, and the functional activity of P-gp and breast cancer resistance protein also remained the same across brain regions. In an intracranial GBM patient-derived xenograft model, AZD1390 accumulation was higher in the tumor core and rim compared with surrounding brain. Despite this heterogenous delivery within tumor-bearing brain, AZD1390 concentrations in normal brain, tumor rim, and tumor core were above in vitro effective radiosensitizing concentrations. These results indicate that despite being a substrate of efflux in the mouse brain, sufficient AZD1390 exposure is anticipated even in regions of normal brain. SIGNIFICANCE STATEMENT Given the invasive nature of glioblastoma (GBM), tumor cells are often protected by an intact blood-brain barrier, requiring the development of brain-penetrant molecules for effective treatment. We show that efflux mediated by P-glycoprotein (P-gp) limits central nervous system (CNS) distribution of AZD1390 and that there are no distributional differences within anatomical regions of CNS. Despite efflux by P-gp, concentrations effective for potent radiosensitization are achieved in GBM tumor-bearing mouse brains, indicating that AZD1390 is an attractive molecule for clinical development of brain tumors.Copyright © 2022 American Society for Pharmacology and Experimental Therapy. All rights reserved.

Review on Naringin: Method of Isolation, Analytical Development, and Its Recent Pharmacological Activities

Naringin is a flavonoid isolated from different citrus fruits like Grapefruit, Orange, Pomelo, Lemon, etc, which are commonly called local fruit. Naringin is found in the white spongy portion of citrus peel. Its content varies from 0.65 mg/gm in the mandarin peel to 14.40mg/gm in the grapefruit peel. Naringin is metabolized to the flavanone naringenin by the enzyme Naringinase present in the liver. It can be analyzed by using various analytical techniques such as HPLC, TLC, UV, HPTLC, mass spectroscopy, Liquid chromatography, chiral chromatography and LC/Mass spectroscopy. Naringin can act as an antioxidant and scavenge free radicals. Naringin mainly focuses on in-vitro and in-vivo animal studies showing its beneficial effects on cardioprotective, antioxidant, anti-inflammatory, antimicrobial, hypolipemiant, neurological, thermogenic, pulmonary disorders and antidiabetic. Naringin is also treated as a most promising treatment strategy against Covid-19 due to its antiviral and anti-inflammatory effects. Recently, Naringin has proven its activity in various molecular docking studies. Naringin keeps the body healthy against various illnesses and major lifestyle disorders.Copyright All © 2023 are reserved by International Journal of Pharmaceutical Sciences and Research.

A Review on Therapeutic Potential of Indian Herbal Plants to Counter Viral Infection and Disease Pathogenesis

Herbal plant extracts or purified phytocomponents have been extensively used to treat several diseases since ancient times. The Indian Ayurvedic system and Chinese traditional medicines have documented the medicinal properties of important herbs. In Ayurveda, the polyherbal formulation is known to exhibit better therapeutic efficacy compared to a single herb. This review focuses on six key ayurvedic herbal plants namely, Tinospora cordifolia, Withania somnifera, Glycyrrhiza glabra/Licorice, Zingiber officinale, Emblica officinalis and Ocimum sanctum. These plants possess specific phytocomponents that aid them in fighting infections and keeping body healthy and stress-free. Plants were selected due to their reported antimicrobial and anti-inflammatory effects in several diseases and effectiveness in controlling viral pathogenesis. An ad-vanced literature search was carried out using Pubmed and google scholar. Result(s): These medicinal plants are known to exhibit several protective features against various diseases or infections. Here we have particularly emphasized on antioxidant, anti-inflammatory, anti-microbial and immunomodulatory properties which are common in these six plants. Recent literature analysis has revealed Ashwagandha to be protective for Covid-19 too. The formulation from such herbs can exhibit synergism and hence better effectiveness against infection and related dis-eases. The importance of these medicinal herbs becomes highly prominent as it maintains the har-monious balance by way of boosting the immunity in a human body. Further, greater mechanistic analyses are required to prove their efficacy in fighting infectious diseases like Covid-19. It opens the arena for in-depth research of identifying and isolating the active components from these herbs and evaluating their potency to inhibit viral infections as polyherbal formulations.Copyright © 2023 Bentham Science Publishers.

A novel isothermal digital amplification system and its application for absolute quantification of respiratory infectious virus

Herein, we establish a novel isothermal digital amplification system termed digital nicking and extension chain reaction system-based amplification (dNESBA) by utilizing the isothermal NESBA technique and the newly developed miniaturized fluorescence monitoring system (mFMS). dNESBA enables parallel isothermal NESBA reactions in more than 10,000 localized droplet microreactors and read the fluorescence signals rapidly in 150 s by mFMS. This system could identify the genomic RNA (gRNA) extracted from target respiratory syncytial virus A (RSV A) as low as 10 copies with remarkable specificity. The practical applicability of dNESBA was also successfully verified by reliably detecting the gRNA in the artificial sputum samples with excellent reproducibility and accuracy. Due to the intrinsic advantages of isothermal amplifying technique including the elimination of the requirement of thermocycling device and the enhanced portability of the miniaturized read-out equipment, the dNESBA technique equipped with mFMS could serve as a promising platform system to achieve point-of-care (POC) digital molecular diagnostics, enabling absolute and ultra-sensitive quantification of various infectious pathogens even in an early stage.Copyright © 2023

Synthesis and crystal characteristics of nirmatrelvir

Several synthetic routes of nirmatrelvir (the ingredient of a new drug to treat COVID-19 made by Pfizer) have been reported. We focused on a second route to improve the synthetic method of nirmatrelvir with a methodology that included different steps. The first step was an analysis of reaction byproducts using acetonitrile as a solvent of the condensation reaction to improve the inversion rate. Then, we used isobutyl acetate as a crystalline solvent to obtain the key intermediate as a solvate, which was a stable crystal product with high purity. Complementarily, we also used trifluoroacetic anhydride as the primary-amide dehydrating agent, and 2-methyl tetrahydrofuran as the solvent to prepare nirmatrelvir, which led to an overall yield of 48% via four steps and a purity of 99.5% according to high-performance liquid chromatography. We also investigated the crystal form of nirmatrelvir: the single-crystal features and transformation from a crystal form to nirmatrelvir were dependent upon temperature. Our data have great value for study of the synthetic method and crystal stability of nirmatrelvir. © 2023 The Royal Society of Chemistry.

Development of a multivariate model with desirability-based optimization for simultaneous determination of five co-administrated drugs for the management of COVID-19 infection in their dosage forms via validated RP-HPLC method

More than 2.9 million people have died as a result of the global demographic impact of the coronavirus illness of 2019 (COVID-19). Numerous antiviral and anti-inflammatory medications have FDA approval to treat COVID-19 patients. For the simultaneous determination of COVID-19 utilized medications (Remdesivir, Moxifloxacin, Dexamethasone, Apixaban, and paracetamol) in their dosage forms, a sensitive technique has been developed and validated. The aforementioned medications were separated and quantified with the help of experimental design. The Box-Behnken design was used in the experiment to optimize the chromatographic method's analytical parameters. It employed RP-HPLC with a UV detector. An INERTSIL ODS-3 C18 column (5 µm, 250 × 4.6 mm) with mobile phase composed of acetonitrile: 30 mmoL potassium dihydrogen phosphate buffer (pH = 7.5) (50:50, v/v), at room temperature was employed to separate the aforementioned drugs. Paracetamol was linear over the concentration range (1–50 µg/mL), Moxifloxacin (5–70 µg/mL), Apixaban (5–70 µg/mL), Dexamethasone (1–100 µg/mL), and Remdesivir (5–100 µg/mL). According to ICH guidelines, the new approach underwent thorough validation. Between the proposed method's results and those from the reference or reported methods, there was no significant difference. The technique is simple to use in research of the cited medications in their dosage forms for quality control aspects.