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OBJECTIVES: Currently, no biomarker or scoring system could clearly identify patients at risk of progression to a severe coronavirus disease (COVID)-19. Even in patients with known risk factors, the fulminant course cannot be predicted with certainty. Analysis of commonly determined clinical parameters (frailty score, age, or body mass index) together with routine biomarkers of host response (C-reactive protein and viral nucleocapsid protein) in combination with new biomarkers neopterin, kynurenine, and tryptophan, could aid in predicting the patient outcome. METHODS: In 2021 and 2022, urine and serum samples were prospectively collected on 1st to 4th day after hospital admission in 108 consecutive COVID-19 patients hospitalized at the University Hospital Hradec Králové, Czech Republic. Delta and omicron virus variants were studied. Neopterin, kynurenine and tryptophan were determined by liquid chromatography. RESULTS: A significant correlation was observed between urinary and serum biomarker concentrations. Urinary and serum neopterin, kynurenine and kynurenine/tryptophan ratio were significantly (p≤0.05) higher in patients who subsequently needed oxygen therapy vs. patients without oxygen therapy. These parameters were also significantly increased in patients who died during the hospitalization compared to survivors. Complex equations have been derived using the investigated biomarkers and other clinical or laboratory parameters to predict the risk of subsequent oxygen therapy or death during hospitalization. CONCLUSIONS: Present data demonstrate that neopterin, kynurenine and kynurenine/tryptophan ratio in the serum or in the urine represent promising biomarkers in the management of COVID-19 that may help to guide important therapeutic decisions.
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Favipiravir is, an antiviral agent, used to treat diseases caused by RNA viruses like Ebola virus, SARS-CoV-2, Influenza virus, Polio measles etc. The prodrug Favipiravir enters the infected cells through endocytosis and undergo metabolism to become an active drug. An active form of Favipiravir selectively targets the catalytic domain of RNA-dependent RNA polymerase and then interrupts the nucleotide incorporation process during viral RNA replication. This dysregulation of viral RNA replication results in mutations where the replacement of guanine by adenine and cytosine by thymine occurs. This ultimately induces destructive mutagenesis in RNA viruses. Currently, Favipiravir is available in tablet and intravenous dosage forms. The following analytical methods have been carried over for Favipiravir: UV, HPLC, LC-MS/MS, Spectrofluorometric method, and HPTLC-densitometric techniques are reported as per literature. The present paper illustrates the review of analytical methods which involve the estimation of Favipiravir in bulk or dosage form. The review also describes the scope and limitations of many published analytical methods for the analysis of Favipiravir. This detailed review article will be of great help to the researcher who is working on Favipiravir.Copyright © 2023 Society of Pharmaceutical Sciences and Research. All rights reserved.
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Favipiravir is an antiviral drug with significant and widespread antiviral action. Favipiravir was crucial in the contest against the COVID-19 pandemic because of how well it treated the SARS-CoV-2 virus. It is well known that contemporary pharmaceutical analysis establishes green, stability-indicating analytical procedures. The current study aimed to develop and assess UV-spectrophotometric (zero order, first order, area under the curve) and RP-HPLC methods for estimating favipiravir in its pharmaceutical dose form, comparing them using ANOVA and an in-vitro dissolution analysis. A green solvents composition of methanol, ethanol, and water (25:35:40 v/v/v) is used for analysis as a mobile phase and diluent. Method A is a simple zero-order spectrophotometric method for determining favipiravir at 236 nm, and the correlation coefficient in the linearity study was found to be 0.9962, LOD, and LOQ are 0.18 and 0.55 mg/mL. Method B is a first-order spectrophotometric method for determining favipiravir at 227 nm, and the correlation coefficient in the linearity study was found to be 0.9964, LOD, and LOQ are 0.64 and 1.96 mg/mL. Method C is an area under the curve spectrophotometric method for determining favipiravir at 230 to 243 nm, and the correlation coefficient in the linearity study was found to be 0.9986, LOD, and LOQ are 0.32 and 0.96 mg/mL. Method D is the RP-HPLC method for the determination of favipiravir at the retention time of 7.216 min, a flow rate of 0.80 mL/min, column temperature of 25 degrees C, at 236 nm, Isocratic mode, and the correlation coefficient in the linearity study was found to be 0.9996, LOD, and LOQ are 0.52 and 1.56 mg/mL. All developed methods demonstrated good repeatability and recovery with %RSD < 2. The proposed established methods were assessed using one-way ANOVA. It was revealed that the Fcalculated value was lower than the Ftabulated value, with no discernible variation in the assay results. Studies on stress degradation show that oxidation and acid degradation mostly impact favipiravir solutions. The Analytical Eco-scale verified that these methods are the greenest and most environmentally friendly, enabling the suggested approach to use an effective green analytical methodology to measure favipiravir extensively. Phosphate buffer (pH 6.4) was the best dissolution medium after analysis of the favipiravir dissolution study in several dissolution media.
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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.
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In this study, a rapid and sensitive stability indicating reversed phase HPLC method was developed for quantitation of Nirmatrelvir and Ritonavir simultaneously in bulk and tablet formulation. Nirmatrelvir and ritonavir were separated on a Thermo C18 column with mobile phase containing 0.01M potassium dihydrogen phosphate buffer and acetonitrile (45:55, v/v). The flow rate was 1 mL/min and detection wavelength was 272 nm. Method linearity was established over a range of 75-225 μg/mL for nirmatrelvir and 50-150 μg/mL for ritonavir. Limit of quantification was 0.694μg/mL for nirmatrelvir and 0.820μg/mL for ritonavir. The recovery (%) was 99.96 to 100.45 (Nirmatrelvir) and 100.25 to 101.35 (Ritonavir). The method precisions were 0.11% (Nirmatrelvir) and 0.33% (Ritonavir). Method was suitable to assay nirmatrelvir and ritonavir in tablet formulation (Paxlovid). Stress degradation studies have shown that this method can be implemented to assay nirmatrelvir and ritonavir in the presence of its degradants. © 2023 World Research Association. All rights reserved.
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Background: Molnupiravir was granted approval by the UKS medicines and health product regulatory agency on 04 November 2021 and on 23 December 2021, granted emergency use of authorization by FDA. Objective: Provide a technique for measuring Molnupiravir in active pharmaceutical ingredients and formulations. Method: The wavelength maximum was found to be 236 nm. ICH guidelines were followed. The forced degradation study in the form of acidic, alkali, thermal, photolytic, hydrolytic, and oxidative stress conditions was carried out for Molnupiravir. Results: The method was linear, as measured by a coefficient of correlation (R2) of 0.9991 in the 10 to 50 µg/mL range. The %RSD for precision, accuracy, limit of detection (LoD), limit of quantitation (LoQ), ruggedness, and robustness was within acceptable limits per ICH Q2 (R1). Conclusion: HPLC equipped with a UV detector is used to create and verify the proposed method. An acetonitrile mobile phase component of 20% was used, demonstrating the more cost-effective technique. The extensive data of mobile phase optimization gives a complete idea of final chromatographic conditions, which can be further implemented for future analysis. Molnupiravir shows less than 4% degradation under different stress conditions. The forced degradation data helps show stability, indicating the behavior of Molnupiravir. © 2023, Dr. Yashwant Research Labs Pvt. Ltd.. All rights reserved.
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Two herbal plants, Akebia quinata D. leaf/fruit and Clitoria ternatea L. flower, well-known in traditional medicine systems, were investigated using a non-target effect-directed profiling. High-performance thin-layer chromatography (HPTLC) was combined with 11 different effect-directed assays, including two multiplex bioassays, for assessing their bioactivity. Individual active zones were heart-cut eluted for separation via an orthogonal high-performance liquid chromatography column to heated electrospray ionization high-resolution mass spectrometry (HPLC-HESI-HRMS) for tentative assignment of molecular formulas according to literature data. The obtained effect-directed profiles provided information on 2,2-diphenyl-1-picrylhydrazyl scavenging, antibacterial (against Bacillus subtilis and Aliivibrio fischeri), enzyme inhibition (tyrosinase, α-amylase, ß-glucuronidase, butyrylcholinesterase, and acetylcholinesterase), endocrine (agonists and antagonists), and genotoxic (SOS-Umu-C) activities. The main bioactive compound zones in A. quinata leaf were tentatively assigned to be syringin, vanilloloside, salidroside, α-hederin, cuneataside E, botulin, and oleanolic acid, while salidroside and quinatic acids were tentatively identified in the fruit. Taraxerol, kaempherol-3-rutinoside, kaempferol-3-glucoside, quercetin-3-rutinoside, and octadecenoic acid were tentatively found in the C. ternatea flower. This straightforward hyphenated technique made it possible to correlate the biological properties of the herbs with possible compounds. The meaningful bioactivity profiles contribute to a better understanding of the effects and to more efficient food control and food safety.
Subject(s)
Clitoria , Acetylcholinesterase/chemistry , Chromatography, Thin Layer/methods , Butyrylcholinesterase , Plant Extracts/chemistry , Spectrometry, Mass, Electrospray Ionization , Biological AssayABSTRACT
Introduction. Enoxaparin sodium-containing drugs are included in relevant protocols for COVID-19 therapy. An increase in the production volume of such drugs leads to a demand for available and precise methods of identification and quantitative measurement of the active ingredient in the preparations. Considering the fact that pharmacopoeial methods require significant amount of expensive standards and reagents that are unavailable for numerous laboratories, it is relevant to develop a more available method that will accelerate and make cheaper the process of quality control for enoxaparin sodium-containing preparations. Aim. To develop and validate a simple, economic, and precise method of gel-permeation HPLC with the application of a refractive index detector for the evaluation of enoxaparin sodium in preparations for injection. Materials and methods. Samples of enoxaparin sodium-containing substances and commercial preparations were studied. The identification and quantitative content of the active ingredient were performed by the method of gel-permeation HPLC using a 1260 Infinity (Agilent Technologies, USA) chromatograph equipped with a refractive index detector. Results and discussion. The authors analyzed standard validation characteristics: specificity, linearity, precision, and accuracy of the method. The analysis revealed high specificity and suitability of the proposed method to chromatographic symmetrical multiprocessing systems. The method is recommended for routine control of finished and semi-finished pharmaceutical preparations containing from 25 to 200 mg/ml of enoxaparin sodium-containing. Conclusion. The proposed method can be used for routine quality control of enoxaparin sodium-containing preparations for injection. © Pshenichnov E. A., Kondrasheva K. V., 2023.
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To treat mild to moderate COVID-19, an investigational drug called nirmatrelvir in combination with ritonavir is being researched for which the potential hazards with this are still unknown. Nirmatrelvir has been approved for immediate use by the US Food and drug intake in conjunction with the drug ritonavir for the treatment of mild to medium COVID-19 in grown ups and individuals of more than 12 years who test positive for the virus and are at a high risk to develop severe COVID-19.To quantify the drugs simultaneously in tablet dosage forms, a novel, sensitive and reproducible reverse phase liquid chromatography method has been developed. Thechromatographic separation was performed using Phenomenex (250x4.6mm,5micro particle size) column. The separation and elution were carried out at an ambient temperature using a mobile phase consisting of 0.1% trifluoro acetic acid &acetonitrile in the ratio of 50:50%v/v. The maximum absorbance by UV spectrophotometer shown at wavelength 258.3nm& 271.4nm for nirmatrelvir and ritonavir. Also, 266nm was selected as detector wavelength by a photodiode array detector for the HPLC chromatrographic method.Beer lambert's law obeyed in the linear range of 37.5-225microg/mL(R2=0.9998) for nirmatrelvir and 25-150 microg/mL(R2=0.9994) for ritonavir. The method shows method and system precision with % RSD less than 1%.The percentage mean recovery was found to be 99.9-100.2%& 100.0-100.2%. The LOD 1.5 microg/mL &1 microg/mL values indicates the method sensitivity. The proposed stability indicating method was validated for precision,accuracy, specificity, selectivity, robustness and stability studies according to ICH guidelines.Copyright © 2022, Anka Publishers. All rights reserved.
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Introduction. Coronavirus disease 2019 (COVID-19) is an infectious disease caused by the SARS-CoV-2 virus (severe acute respiratory syndrome-related coronavirus 2). COVID-19 is now expected to stay with us for many years as a recurring disease. Molnupiravir and favipiravir are oral antiviral drugs with anti-RNA polymerase activity. The Russian Health Ministry has approved molnupiravir and favipiravir for the treatment of COVID-19. The study describes development and validation of high-performance liquid chromatography – tandem mass spectrometry (HPLC-MS/MS) method for the simultaneous determination of β-D-N4-Hydroxycytidine and favipiravir in human blood plasma. The method could be applied in pharmacokinetic study of molnupiravir and favipiravir. Aim. The aim of this study is to develop and validate a HPLC-MS/MS bioanalytical method for the determination of β-D-N4-Hydroxycytidine and favipiravir in human plasma. Materials and methods. The determination of β-D-N4-Hydroxycytidine and favipiravir in human plasma by HPLC-MS/MS. The samples were processed by 0.1 % formic acid in acetonitrile. Internal standard: promethazine. Mobile phase: 0.01 mol/L Ammonium formate buffer solution (Eluent A), 0.1 % formic acid and 0.08 % aqueous ammonia in water/acetonitrile 10: 90 (Eluent B). Column: Shim-pack GWS C18, 150 × 4.6 mm, 5 μm. Analytical range: 50.00–10000.00 ng/mL for β-D-N4-Hydroxycytidine, 250.00–20000.00 ng/mL for favipiravir in human plasma. Ionization source: electrospray ionization. Detection conditions: 260.00 m/z → 82.10 m/z, 260.00 m/z → 111.00 m/z, 260.00 m/z → 127.95 m/z (β-D-N4-Hydroxycytidine);156.15 m/z → 65.95 m/z, 156.15 m/z → 85.00 m/z, 156.15 m/z → 113.10 m/z (favipiravir);285.05 m/z → 198.05 m/z (promethazine). Results and discussion. This method was validated by selectivity, suitability of reference standard, matrix effect, calibration curve, accuracy, precision, spike recovery, the lower limit of quantification, carry-over effect and stability. Conclusion. The HPLC-MS/MS method for quantitative determination of β-D-N4-Hydroxycytidine and favipiravir in human plasma was developed and validated. The analytical range was 50.00–10000.00 ng/mL for β-D-N4-Hydroxycytidine, 250.00–20000.00 ng/mL for favipiravir in human plasma. This method was applied to investigate the pharmacokinetics of molnupiravir and favipiravir. © Komarov T. N., Karnakova P. K., Archakova O. A., Shchelgacheva D. S., Bagaeva N. S., Shohin I. E., Zaslavskaya K. Ya., Bely P. A., 2023.
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Even though scientific communication and collaboration play critical roles in academic success, they often come in second to teaching laboratory fundamentals. COVID-19 associated capacity constraints forced our program to reexamine the traditional laboratory instruction paradigm by limiting physical attendance, as well as laboratory duration. Instead, we opted to turn these restrictions into opportunities to study peer-to-peer communication as a means to enhance in-person experimentation. Here, a two-week high performance liquid and gas chromatography (HPLC and GC) module uses centralized student communication between peer groups to not only maintain but increase quality laboratory experiences. Students rotate between two chromatography experiments to ensure each person gains exposure to foundational separations techniques. The module's first week focuses on method development, while the second week highlights method validation. Since HPLC and GC can accommodate similar experimental objectives, students receive comparable method development and validation experiences no matter which specific instrument they are assigned to that week. By integrating communication into the experimental process and adding peer accountability to the laboratory's design, the authors observed an increase in laboratory report words and figures when compared to the prior years' reports. Student questionnaires also indicated structured rotation and group communication increased student interest and investment in obtaining and reporting quality data. © 2023 Authors. All rights reserved.
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Ursodeoxycholic acid has gained increasing attention due to its recent discovery of the preventive effect on SARS-CoV-2 infection. Ursodeoxycholic acid has been included in various pharmacopoeias as an old drug, and the latest European Pharmacopoeia lists nine potential related substances (impurities Aâ¼I). However, existing methods in pharmacopoeias and literature can only quantify up to five of these impurities simultaneously, and the sensitivity is inadequate, as the impurities are isomers or cholic acid analogues lacking chromophores. Herein, a novel gradient RP-HPLC method coupled to charged aerosol detection (CAD) was developed and validated for the simultaneous separation and quantification of the nine impurities in ursodeoxycholic acid. The method proved sensitive and allowed the quantification of the impurities as low as 0.02 %. Relative correction factors of the nine impurities were all within the range of 0.8-1.2 in the gradient mode by optimizing chromatographic conditions and CAD parameters. In addition, this RP-HPLC method is fully compatible with LC-MS due to the volatile additives and high percentage of the organic phase, which can be directly used for the identification of impurities. The newly developed HPLC-CAD method was successfully applied to commercial bulk drug samples, and two unknown impurities were identified by HPLC-Q-TOF-MS. The effect of CAD parameters on the linearity and correction factors was also discussed in this study. Overall, the established HPLC-CAD method can improve the methods in current pharmacopoeias and literature and contributes to understanding the impurity profile for process improvement.
Subject(s)
COVID-19 , Ursodeoxycholic Acid , Humans , Chromatography, High Pressure Liquid/methods , SARS-CoV-2 , Respiratory Aerosols and Droplets , Drug Contamination/prevention & controlABSTRACT
Simple, direct, rapid, and sensitive HPLC and spectrophotometric methods were established for simultaneous estimation of a novel combination of budesonide and azelastine (BUD/AZL) in their laboratory-prepared mixture and dosage form according to the medicinally recommended ratio 1:4.28. Budesonide is an important inhalation corticosteroid that plays a vital role in the inhibition of COVID-19 replication and cytokine production. The first chromatographic method was created for the simultaneous estimation of BUD epimers in the presence of AZL with excellent efficiency in a relatively short chromatographic run (< 9 min). The separation of BUD epimers with AZL was carried out on a C18 column using acetonitrile: phosphate buffer of pH 3.5 adjusted by 0.2 M orthophosphoric acid (40:60, v/v) as a mobile phase, UV detection at 230 nm and a flow rate of regulated at 2 mL/min. Besides, three spectrophotometric methods were applied for the simultaneous determination of the provided mixture adopting zero order, first order derivative, and ratio first derivative approaches. The Zero-order spectrophotometry was used for the determination of AZL in presence of BUD, where BUD shows no absorbance at 290 nm. The first derivative amplitude at 265 nm (1D265) (zero-crossing of AZL) and the ratio of first derivative amplitudes at 270 nm (1DD270) using 10.0 µg mL-1 AZL as divisor was chosen for the simultaneous determination of BUD in the presence of AZL in the binary mixture. The proposed methods were found to be rectilinear in the concentration range of (0.4-40.0 µg mL-1) and (0.05-40.0 µg mL-1) for BUD and AZL, respectively in the HPLC method. Whereas the concentration range for AZL in the zero-order method was (1.0-35.0 µg mL-1) and for BUD in the first derivative and ratio derivative method was (6.0-20.0 µg mL-1). Validation of the suggested approaches according to the ICH criteria was performed. Furthermore, to ensure the proposed approaches' greenness, The AGREE and GAPI metrics were utilized, and the afforded results revealed an excellent greenness of the proposed approaches.
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d-Amino acids, rare enantiomers of amino acids, have been identified as biomarkers and therapeutic options for COVID-19. Methods for monitoring recovery are necessary for managing COVID-19. On the other hand, the presence of SARS-CoV2 virus in the blood is associated with worse outcomes. We investigated the potential of d-amino acids for assessing recovery from severe COVID-19. In patients with severe COVID-19 requiring artificial ventilation, the blood levels of d-amino acids, including d-alanine, d-proline, d-serine, and d-asparagine, which were lower than the normal range before treatment, quickly and transiently increased and surpassed the upper limit of the normal range. This increase preceded the recovery of respiratory function, as indicated by ventilation weaning. The increase in blood d-amino acid levels was associated with the disappearance of the virus in the blood, but not with inflammatory manifestations or blood cytokine levels. d-Amino acids are sensitive biomarkers that reflect the recovery of the clinical course and blood viral load. Dynamic changes in blood d-amino acid levels are key indicators of clinical course.
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A fast procedure obtained by the combination of fabric phase extraction (FPSE) with high performance liquid chromatography (HPLC) has been developed and validated for the quantification of favipiravir (FVP) in human plasma and breast milk. A sol-gel polycaprolactone-block-polydimethylsiloxane-block-polycaprolactone (sol-gel PCAP-PDMS-PCAP) coated on 100% cellose cotton fabric was selected as the most efficient membrane for FPSE in human plasma and breast milk samples. HPLC-UV analysis were performed using a RP C18 column under isocratic conditions. Under these optimezed settings, the overall chromatographic analysis time was limited to only 5 min without encountering any observable matrix interferences. Following the method validation procedure, the herein assay shows a linear calibration curve over the range of 0.2–50 µg/mL and 0.5–25 µg/mL for plasma and breast milk, respectively. The method sensitivities in terms of limit of detection (LOD) and limit of quantification (LOQ), validated in both the matrices, have been found to be 0.06 and 0.2 µg/mL for plasma and 0.15 and 0.5 µg/mL for milk, respectively. Intraday and interday precision and trueness, accordingly to the International Guidelines, were validated and were below 3.61% for both the matrices. The herein method was further tested on real samples in order to highlight the applicability and the advantage for therapeutic drug monitoring (TDM) applications. To the best of our knowledge, this is the first validated FPSE-HPLC-UV method in human plasma and breast milk for TDM purposes applied on real samples. The validated method provides fast, simple, cost reduced, and sensitive assay for the direct quantification of favipiravir in real biological matrices, also appliyng a well-known rugged and cheap instrument configuration. © 2022 Elsevier B.V.
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Hydrogen sulfide is a toxic gas but also established as a naturally occurring gaseous signaling molecule in humans, playing key physiological roles with particular involvement in lung disease including COVID-19. Thiosulfate is the conventional biomarker of hydrogen sulfide and is excreted in human urine at low micromolar levels. Thiosulfate is amenable to detection by the element-selective inductively coupled plasma tandem mass spectrometry (ICPMS/MS), but sulfur speciation in human samples at trace levels is challenging due to the high complexity of human sulfur metabolome and the utility of this detector under such settings has not been demonstrated. We report a method for thiosulfate determination in human urine at trace physiological levels by HPLC-ICPMS/MS. The method involved one-step derivatization to improve chromatographic behavior followed by direct injection. The instrumental limit of detection was 1.4 μg S L−1 (0.02 μM or 0.1 pmol). In a group of samples from volunteers (n = 24), measured thiosulfate concentrations in the diluted urine matrix were down to 8.0 μg S L−1 with a signal-to-noise ratio >10. The method was validated for recovery (80–110%), repeatability (RSD% <5%), and linearity (r2 = 0.9999, at a tested working concentration range of 0.01–1.0 mg S L−1), and the accuracy was assessed by comparing with HPLC-ESIMS/MS which showed agreement within ±20%. This work demonstrates the applicability of HPLC-ICPMS/MS for sulfur speciation at trace levels in a matrix with complex sulfur metabolome as human urine and provides a sensitive method for the determination of the hydrogen sulfide biomarker. © 2022 The Authors
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Favipiravir (FVP) structurally is an analog of pyrazine and showed its antiviral actions against a diverse species of viruses. Due to this fact, it was chosen as a potential candidate to be further investigated to cure COVID-19. A simple, accurate, rapid, precise, high-performance liquid chromatography (HPLC) method has been established for quality control assurance of FVP in pharmaceutical preparations. Column no C8 (150 mm x 4.6 mm;5 mu m) was used for such separation analysis. A combination of acetonitrile and phosphate buffer was used in 10:90 ratios as the mobile phase (90:10, v/v) having a flow rate of 1 mL / min. further, it was detected at 220 nm, and the temperature was maintained at 25 degrees C. The run time was then set at 10 min. A linear relationship of R-2 0.998 was confirmed while drawing a graph between FVP concentration and peak area that has a concentration range of 40-200 ppm. The developed method is sensitive (limits of detection and quantification are 1.83 ppm and 5.75 ppm respectively). Three injections at concentrations of 80,100 and 120 (ppm) were injected the same day to ensure the precision of the method. The prepared solution remains stable for 24 h. The suggested method has been successfully useful for the quantification of FVP in pharmaceutical formulations.
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SARS-CoV-2 has caused more than 596 million infections and 6 million fatalities globally. Looking for urgent medication for prevention, treatment, and rehabilitation is obligatory. Plant extracts and green synthesized nanoparticles have numerous biological activities, including antiviral activity. HPLC analysis of C. dirnum L. leaf extract showed that catechin, ferulic acid, chlorogenic acid, and syringic acid were the most major compounds, with concentrations of 1425.16, 1004.68, 207.46, and 158.95 µg/g, respectively. Zinc nanoparticles were biosynthesized using zinc acetate and C. dirnum extract. TEM analysis revealed that the particle size of ZnO-NPs varied between 3.406 and 4.857 nm. An XRD study showed the existence of hexagonal crystals of ZnO-NPs with an average size of 12.11 nm. Both ZnO-NPs (IC50 = 7.01 and CC50 = 145.77) and C. dirnum L. extract (IC50 = 61.15 and CC50 = 145.87 µg/mL) showed antiviral activity against HCOV-229E, but their combination (IC50 = 2.41 and CC50 = 179.23) showed higher activity than both. Molecular docking was used to investigate the affinity of some metabolites against the HCOV-229E main protease. Chlorogenic acid, solanidine, and catchin showed high affinity (-7.13, -6.95, and -6.52), compared to the ligand MDP (-5.66 Kcal/mol). Cestrum dinurum extract and ZnO-NPs combination should be subjected to further studies to be used as an antiviral drug.
Subject(s)
COVID-19 , Cestrum , Metal Nanoparticles , Nanoparticles , Zinc Oxide , Humans , Zinc Oxide/chemistry , Metal Nanoparticles/chemistry , Antiviral Agents/pharmacology , Molecular Docking Simulation , Zinc , SARS-CoV-2/metabolism , Nanoparticles/chemistry , Plant Extracts/pharmacology , Plant Extracts/chemistry , Anti-Bacterial Agents/chemistry , Microbial Sensitivity TestsABSTRACT
The COVID-19 pandemic triggered an unprecedented rate of development of mRNA vaccines, which are produced by in vitro transcription reactions. The latter has been the focus of intense development to increase productivity and decrease cost. Optimization of IVT depends on understanding of the impact of individual reagents on the kinetics of mRNA production and the consumption of building blocks, which is hampered by slow, low-throughput, end-point analytics. We implemented a workflow based on rapid at-line HPLC monitoring of consumption of NTPs with concomitant production of mRNA, with a sub-3 min read-out, allowing for adjustment of IVT reaction parameters with minimal lag. IVT was converted to fed-batch resulting in doubling the reaction yield compared to batch IVT protocol, reaching 10 mg/mL for multiple constructs. When coupled with exonuclease digestion, HPLC analytics for quantification of mRNA was extended to monitoring capping efficiency of produced mRNA. When HPLC monitoring was applied to production of an ARCA-capped mRNA construct, which requires an approximate 4:1 ARCA:GTP ratio, the optimized fed-batch approach achieved productivity of 9 mg/mL with 79% capping. The study provides a methodological platform for optimization of factors influencing IVT reactions, converting the reaction from batch to fed-batch mode, determining reaction kinetics, which are critical for optimization of continuous addition of reagents, thereby paving the way towards continuous manufacturing of mRNA. This article is protected by copyright. All rights reserved.
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As an empirical medicine of traditional Chinese medicine, Fuzhengjiedu Granules have shown an effect against COVID-19 in clinical and inflammatory animal models. It is formulated with eight herbs, including Aconiti Lateralis Radix Praeparata, Zingiberis Rhizoma, Glycyrrhizae Radix Et Rhizoma, Lonicerae Japonicae Flos, Gleditsiae Spina, Fici Radix, Pogostemonis Herba, and Citri Reticulatae Pericarpium. This study established a high-performance liquid chromatography-triple quadrupole mass spectrometry (HPLC-QQQ-MS/MS) method by simultaneously determining 29 active compounds in the granules with significant content differences. Separation by gradient elution using acetonitrile and water (0.1% formic acid) as mobile phases was performed on a Waters Acquilty UPLC T3 column (2.1 mm × 100 mm, 1.7 µm). A triple quadrupole mass spectrometer, operating in positive and negative ionization modes, was used for multiple reaction monitoring to detect the 29 compounds. All calibration curves showed good linear regression (r2 > 0.998). RSDs of precision, reproducibility, and stability of active compounds were all lower than 5.0%. The recovery rates were 95.4-104.9%, with RSDs< 5.0%. This method was successfully used to analyze the samples, and the results showed that 26 representative active components from 8 herbs were detected in the granules. While aconitine, mesaconitine, and hypaconitine were not detected, indicating that the existing samples were safe. The granules had the maximum and minimum content of hesperidin (27.3 ± 0.375 mg/g) and benzoylaconine (38.2 ± 0.759 ng/g). To conclude, a fast, accurate, sensitive, and reliable HPLC-QQQ-MS/MS method was established, which can simultaneously detect 29 active compounds that have a considerable difference in the content of Fuzhengjiedu Granules. This study can be used to control the quality and safety of Fuzhengjiedu Granules and provide a basis and guarantee for further experimental research and clinical application.