[Component identification and analysis in vivo of Sanhan Huashi formula].

Sanhan Huashi formula(SHF) is the intermediate of a newly approved traditional Chinese medicine(TCM) Sanhan Huashi Granules for the treatment of COVID-19 infection. The chemical composition of SHF is complex since it contains 20 single herbal medicines. In this study, UHPLC-Orbitrap Exploris 240 was used to identify the chemical components in SHF and in rat plasma, lung and feces after oral administration of SHF, and heat map was plotted for characterizing the distribution of the chemical components. Chromatographic separation was conducted on a Waters ACQUITY UPLC BEH C_(18)(2.1 mm×100 mm, 1.7 µm) using 0.1% formic acid(A)-acetonitrile(B) as mobile phases in a gradient elution. Electrospray ionization(ESI) source was used to acquire data in positive and negative mode. By reference to quasi-molecular ions and MS/MS fragment ions and in combination with MS spectra of reference substances and compound information in literature reports, 80 components were identified in SHF, including 14 flavonoids, 13 coumarins, 5 lignans, 12 amino-compounds, 6 terpenes and 30 other compounds; 40 chemical components were identified in rat plasma, 27 in lung and 56 in feces. Component identification and characterization of SHF in vitro and in vivo lay foundations for disclosure of its pharmacodynamic substances and elucidation of the scientific connotation.

Evaluating the quality consistency of antiviral oral liquid by high-performance liquid chromatography five-wavelength matched average fusion fingerprint combined with electrochemical fingerprint and ultraviolet spectral quantum fingerprint.

The antiviral oral liquid (AOL) was an antiviral drug currently in clinical trials against coronavirus disease 2019. This study aimed to improve its quality consistency evaluation method using fingerprint techniques from several aspects. First, the five-wavelength matched average fusion fingerprint (FMAFFP) for HPLC, electrochemical fingerprint (ECFP), and ultraviolet spectral quantum fingerprint (UVFP) was established for 22 samples, respectively. Their quality was then assessed using the average linear quantitative fingerprint method, and 22 samples were classified into eight quality grades. OPLS and PCA were then used further to explore the characteristic parameters of these three fingerprints. Five compounds were quantified simultaneously for the first time, and then the relationship between the average linear quantitative similarity (PL) and the sum of the five quantitative components (P5c) was investigated. A linear correlation (r ≥ 0.9735) between PL and P5c suggested that PL may be used to predict chemical content. Finally, to investigate the antioxidant potential of the AOL, correlation analyses were performed for FMAFFP peaks-PEC and UVFP peaks-PEC, respectively, where the PEC value was defined as the quantitative similarity of ECFP. The Pearson correlation coefficient and gray correlation analysis were consistent, allowing us to initially explore the antioxidant capacity of the unidentified components of the samples. This study researched AOL using multidimensional fingerprints to provide a comprehensive and reliable method for quality consistency control of herbal compound preparations.

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 an LC-MS/MS method for quantification of favipiravir in human plasma.

Favipiravir (FVP) is a broad-spectrum antiviral that selectively inhibits viral RNA-dependent RNA polymerase, first trialled for the treatment of influenza infection. It has been shown to be effective against a number of RNA virus families including arenaviruses, flaviviruses and enteroviruses. Most recently, FVP has been investigated as a potential therapeutic for severe acute respiratory syndrome coronavirus 2 infection. A liquid chromatography tandem mass spectrometry method for the quantification of FVP in human plasma has been developed and validated for use in clinical trials investigating favipiravir as treatment for coronavirus disease-2019. Samples were extracted by protein precipitation using acetonitrile, using 13C, 15N- Favipiravir as internal standard. Elution was performed on a Synergi Polar-RP 150 × 2.1 mm 4 µm column using a gradient mobile phase programme consisting of 0.2% formic acid in water and 0.2% formic acid in methanol. The assay was validated over the range 500-50,000 ng/mL; this method was found to be precise and accurate and recovery of FVP from the matrix was high. Stability experiments confirmed and expanded on the known stability of FVP, including under heat treatment and for a period of 10 months at - 80 °C.

Characterization of in-vivo human metabolites of the oral nucleoside anti-COVID-19 drug VV116 using UHPLC-Orbitrap-MS.

VV116 is an oral nucleoside anti-COVID-19 drug undergoing clinical trials in China. We aimed to characterize its metabolites in plasma, urine, and feces of healthy Chinese male subjects after a single oral administration of 400 mg VV116, by using UHPLC-UV-Orbitrap-MS. After oral administration, VV116 was almost completely converted into the metabolite 116-N1. Seventeen other metabolites produced by the subsequent metabolism of 116-N1 were also detected, including 6 phase I metabolites and 11 phase II metabolites resulting from hydrolysis, oxidative deamination, oxidation, and CN-group removal and conjugations. The results were exploratory. The major metabolite of VV116 in human plasma and urine was 116-N1, the main metabolites in feces were M2 and 116-N1. We then synthesized a reference M2 standard and confirmed its structure by MS and NMR.

Development of HPLC-CAD method for simultaneous quantification of nine related substances in ursodeoxycholic acid and identification of two unknown impurities by HPLC-Q-TOF-MS.

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 A∼I). 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.

Development of UPLC method for simultaneous assay of some COVID-19 drugs utilizing novel instrumental standard addition and factorial design.

A green, rapid, and simple RP-UPLC method was developed and optimized by full factorial design for the simultaneous separation of oseltamivir phosphate, daclatasivir dihydrochloride, and remdesivir, with dexamethasone as a co-administered drug. The separation was established on a UPLC column BEH C18 1.7 µm (2.1 × 100.0 mm) connected with a UPLC pre-column BEH 1.7 µm (2.1 × 5.0 mm) at 25 °C with an injection volume of 10 µL. The detector (PDA) was set at 239 nm. The mobile phase consisted of methanol and ammonium acetate (8.1818 mM) in a ratio of 75.7: 24.3 (v/v). The flow rate was set at 0.048 mL min-1. The overall separation time was 9.5 min. The retention times of oseltamivir phosphate, dexamethasone, daclatasivir dihydrochloride, and remdesivir were 6.323 ± 0.145, 7.166 ± 0.036, 8.078 ± 0.124, and 8.572 ± 0.166 min (eight replicates), respectively. The proposed method demonstrated linearity in the ranges of 10.0-500.0 (ng mL-1) and 0.5-30.0 (µg mL-1) for oseltamivir phosphate, 50.0-5000.0 (ng mL-1) for dexamethasone, 25.0-1000.0 (ng mL-1) and 0.5-25.0 (µg mL-1) for daclatasvir dihydrochlorde, and 10.0-500.0 (ng mL-1) and 0.5-30.0 (µg mL-1) for remdesivir. The coefficients of determination (R2) were greater than 0.9999, with percentage recoveries greater than 99.5% for each drug. The limits of quantitation were 6.4, 1.8, 7.8, and 1.6 ng mL-1, and the limits of detection were 1.9, 0.5, 2.0, and 0.5 ng mL-1 for oseltamivir phosphate, dexamethasone, daclatasivir dihydrochloride, and remdesivir, respectively. The proposed method was highly precise, as indicated by the low percentage of relative standard deviation values of less than 1.2% for each drug. The average content and uniformity of dosage units in the studied drugs' dosage forms were determined. The average contents of oseltamivir phosphate, dexamethasone, daclatasivir dihydrochloride, and remdesivir were nearly 93%, 102%, 99%, and 95%, respectively, while the uniformity of dosage unit values were nearly 92%, 102%, 101%, and 97%. Two novel methods were established in this work. The first method was used to assess the stability of standard solutions. This novel method was based on the slope of regression equations. The second was to evaluate the excipient's interference using an innovative instrumental standard addition method. The novel instrumental standard addition method was performed using the UPLC instrument program. It was more accurate, sensitive, time-saving, economical, and eco-friendly than the classic standard addition method. The results showed that the proposed method can estimate the tested drugs' concentrations without interference from their dosage form excipients. According to the Eco-score (more than 75), the Green Analytical Procedure Index (GAPI), and the AGREE criteria (total score of 0.77), the suggested method was considered eco-friendly.

Determination of B Vitamins by Double-Vortex-Ultrasonic Assisted Dispersive Liquid-Liquid Microextraction and Evaluation of their Possible Roles in Susceptibility to COVID-19 Infection: Hybrid Box-Behnken Design and Genetic Algorithm.

OBJECTIVES: In this study, double-vortex-ultrasonic assisted dispersive liquid-liquid microextraction (DVUDLLME) was applied to determine the concentration of vitamin B9, 5-methyl tetrahydrofolate (5-MeTHF) and vitamin B12 in human serum samples. METHODS: High-performance liquid chromatography (HPLC) coupled with DVUDLLME was applied to analyze vitamins B in patients with Coronavirus disease (COVID-19). Then, significant variables were chosen and optimized using the hybrid Box-Behnken design and genetic algorithm. RESULTS: The detection limits of DVUDLLME-HPLC were 0.21 ng mL-1, 0.18 ng mL-1 and 55 pgmL-1 for vitamin B9, 5-MeTHF and vitamin B12, respectively. Subsequently, DVUDLLME-HPLC was applied to measure B vitamins and investigated their possible roles in susceptibility to COVID-19 infection. Fifty-seven percent of the patients without an underlying disease have significantly lower serum vitamin B12 levels in comparison to controls. CONCLUSIONS: The advantages of this method are low detection limit, simple preparation, low retention time and the use of a cheaper technique instead of expensive mass detectors. The results suggest that vitamin B12 deficiency may decrease the immune system defenses against COVID-19 patients without an underlying disease and cause the disease to become severe. However, these works need a large population and further research, such as a randomized trial and a cohort study.

A validated LC-MS/MS method for clinical pharmacokinetics and presumptive phase II metabolic pathways following oral administration of Andrographis paniculata extract.

Andrographis paniculata, a medicinal plant in Thailand national list of essential medicines, has been proposed for treatment of patients with mild to moderate coronavirus disease 2019. This study aims to develop a highly selective and sensitive liquid chromatography triple quadrupole tandem mass spectrometry method for quantitative determination of major diterpenoids in plasma and urine with application in pharmacokinetics. Chromatographic separation was performed on C18 column using a gradient mobile phase of water and acetonitrile. Mass spectrometry was analyzed using multiple reaction monitoring with negative ionization mode. This validated analytical method was very sensitive, less time consuming in analysis, and allowed the reliability and reproducibility on its application. The clinical pharmacokinetics was evaluated after single oral administration of A. paniculata extract (calculated as 60 mg of andrographolide). The disposition kinetics demonstrated that major diterpenoids could enter into systemic circulation, but they are mostly biotransformed (phase II) into conjugated glucuronide and sulfate metabolites. These metabolites are predominantly found in plasma and then extremely eliminated, in part through urinary excretion. The successful application of this analytical method supports its suitable uses in further clinical benefits after oral administration of A. paniculata.

Development and Validation of an Innovative Analytical Approach for the Quantitation of Tris(Hydroxymethyl)Aminomethane (TRIS) in Pharmaceutical Formulations by Liquid Chromatography Tandem Mass Spectrometry.

A novel COVID-19 vaccine (BriLife®) has been developed by the Israel Institute for Biological Research (IIBR) to prevent the spread of the SARS-CoV-2 virus throughout the population in Israel. One of the components in the vaccine formulation is tris(hydroxymethyl)aminomethane (tromethamine, TRIS), a buffering agent. TRIS is a commonly used excipient in various approved parenteral medicinal products, including the mRNA COVID-19 vaccines produced by Pfizer/BioNtech and Moderna. TRIS is a hydrophilic basic compound that does not contain any chromophores/fluorophores and hence cannot be retained and detected by reverse-phase liquid chromatography (RPLC)-ultraviolet (UV)/fluorescence methods. Among the few extant methods for TRIS determination, all exhibit a lack of selectivity and/or sensitivity and require laborious sample treatment. In this study, LC−mass spectrometry (MS) with its inherent selectivity and sensitivity in the multiple reaction monitoring (MRM) mode was utilized, for the first time, as an alternative method for TRIS quantitation. Extensive validation of the developed method demonstrated suitable specificity, linearity, precision, accuracy and robustness over the investigated concentration range (1.2−4.8 mg/mL). Specifically, the R2 of the standard curve was >0.999, the recovery was >92%, and the coefficient of variance (%CV) was <12% and <6% for repeatability and intermediate precision, respectively. Moreover, the method was validated in accordance with strict Good Manufacturing Practice (GMP) guidelines. The developed method provides valuable tools that pharmaceutical companies can use for TRIS quantitation in vaccines and other pharmaceutical products.

An efficient method for qualitation and quantitation of multi-components of the herbal medicine Qingjin Yiqi Granules.

Qingjin Yiqi Granules (QJYQ) is a Traditional Chinese Medicines (TCMs) prescription for the patients with post-COVID-19 condition. It is essential to carry out the quality evaluation of QJYQ. A comprehensive investigation was conducted by establishing deep-learning assisted mass defect filter (deep-learning MDF) mode for qualitative analysis, ultra-high performance liquid chromatography and scheduled multiple reaction monitoring method (UHPLC-sMRM) for precise quantitation to evaluate the quality of QJYQ. Firstly, a deep-learning MDF was used to classify and characterize the whole phytochemical components of QJYQ based on the mass spectrum (MS) data of ultra-high performance liquid chromatography quadrupole time of flight tandem mass spectrometry (UHPLC-Q-TOF/MS). Secondly, the highly sensitive UHPLC-sMRM data-acquisition method was established to quantify the multi-ingredients of QJYQ. Totally, nine major types of phytochemical compounds in QJYQ were intelligently classified and 163 phytochemicals were initially identified. Furthermore, fifty components were rapidly quantified. The comprehensive evaluation strategy established in this study would provide an effective tool for accurately evaluating the quality of QJYQ as a whole.

Nirmatrelvir increases blood tacrolimus concentration in COVID-19 patients as determined by UHPLC-MS/MS method.

OBJECTIVE: Transplant recipients have a higher risk of SARS-CoV-2 infection owing to the use of immunosuppressive drugs like tacrolimus (FK506). FK506 and nirmatrelvir (NMV) (an anti-SARS-CoV-2 drug) are metabolized by cytochrome P450 3A4 and may have potential drug-drug interactions. It is important to determine the effect of NMV on FK506 concentrations. PATIENTS AND METHODS: Following protein precipitation from blood, FK506 and its internal standard (FK506-13C,2d4) were detected by ultra-high performance liquid chromatography/tandem mass spectrometry (UHPLC-MS/MS). Total 22 blood samples (valley concentrations) from two coronavirus disease 2019 (COVID-19) patients were collected and analyzed for FK506 concentrations. RESULTS: Blood levels of FK506 (0.5-100 ng/mL) showed good linearity. The UHPLC-MS/MS method was validated with intra- and inter-batch accuracies of 104.55-107.85%, and 99.52-108.01%, respectively, and precisions of < 15%. Mean blood FK506 concentration was 12.01 ng/mL (range, 3.15-33.1 ng/mL). Five-day co-administration with NMV increased the FK506 concentrations from 3.15 ng/mL to 33.1 ng/mL, returning to 3.36 ng/mL after a 9-day-washout. CONCLUSIONS: We developed a simple quantification method for therapeutic drug monitoring of FK506 in patients with COVID-19 using UHPLC-MS/MS with protein precipitation. We found that NMV increased FK506 blood concentration 10-fold. Therefore, it is necessary to re-consider co-administration of FK506 with NMV.

Profiling aromatic constituents of Chimonanthus nitens Oliv. leaf granule using mass spectrometry.

RATIONALE: The chemical constituents of Chinese patent medicine are usually different from those of crude medicine because of specific preparation processes. Chimonanthus nitens Oliv. leaf granule is widely used for prevention against COVID-19 in China. However, no research has been reported on the chemical constituents of the granule and their variation during the preparation process. METHODS: Fragmentation patterns of reference compounds were investigated using electrospray ionization mass spectrometry, and the new gas-phase reaction was demonstrated by electronic and steric effects and calculated chemistry. Then, a strategy based on new fragmentation patterns was used to profile aromatic constituents. In addition, based on untargeted metabolomics analytical workflow, a comparison was made on the chemical constituents of the leaf and granule. RESULTS: New fragmentation patterns related to two competing reactions, ring-opening and ring-closing reactions for coumarin, have been proposed and investigated in depth. The newly established diagnostic ion at m/z 81.0331 worked strongly in the assignment of OH-7 and substituent at C-8 of coumarin. McLafferty rearrangement occurring in coumarin glycoside while sugar group locating at C-4 was first observed, and new diagnostic ions at m/z 147.0440, 119.0488, and 91.0543 were constructed. CONCLUSIONS: Aromatic constituents of the granule were first profiled. A total of 114 aromatic compounds were identified; of these 85 compounds were identified first. Kaempferol-7-O-neohesperidoside and its homologues were mostly enriched in the granule. Considering their reported bioactivities, these analogues possibly contribute greatly to clinical efficacy. Our results provided a new fragmentation theory for coumarins and a new material basis for the quality control of the granule.

A quality-by-design eco-friendly UV-HPLC method for the determination of four drugs used to treat symptoms of common cold and COVID-19.

An optimization approach based on full factorial design was employed for developing an HPLC-UV method for simultaneous determination of a quaternary mixture used for the treatment of symptoms related to common cold and COVID-19. The quaternary mixture is composed of paracetamol, levocetirizine dihydrochloride, phenylephrine hydrochloride and ambroxol hydrochloride. The developed technique is a green, fast and simple method that uses isocratic elution of mobile phase consisting of 20:5:75 (v/v) of ethanol: acetonitrile: 2.5 mM heptane-1-sulphonic acid sodium salt at pH 6.5 [Formula: see text] 0.02. The chromatographic separation was carried out using Hypersil BDS Cyano LC Column (250 × 4.6 mm, 5 µm) with 230 nm UV detection and 1.0 mL/min. flow rate. Avoiding the routine methodology and resorting to the modern technology-represented in the usage of experimental design-allows rapid determination of the four drugs using the optimum quantity of chemicals to avoid any waste of resources. The quaternary mixture was eluted in less than 9 min., where retention times of paracetamol, levocetirizine dihydrochloride, phenylephrine hydrochloride and ambroxol hydrochloride were found to be 2.2, 3.8, 6.6 and 8.8 min., respectively. The calibration graphs of the four drugs were linear over concentration ranges of 50.0-500.0, 0.5-20.0, 0.5-20.0 and 0.5-100.0 µg/mL for paracetamol, levocetirizine dihydrochloride, phenylephrine hydrochloride and ambroxol hydrochloride, respectively with correlation coefficients higher than 0.999. The method is accurate with mean recoveries between 99.87 and 100.04%, precise, as %RSD for the intraday and interday precision were between 0.61 and 1.64% and very sensitive with limit of detections (LOD)'s between 29 and 147 ng/mL and limit of quantification (LOQ)'s between 95 and 485 ng/mL. The proposed method was successfully applied for the analysis of the four drugs either in raw materials or in prepared tablet with the least amount of chemicals within short time. It is also validated following International Conference on Harmonization Guidelines. The proposed method was found to be green according to the most common greenness assessment tools; NEMI, GAPI, Analytical Eco-Scale and AGREE methods. The advantages of the proposed method qualify it for routine analysis of the studied drugs either in single or co-formulated dosage form in quality control labs.

Isocratic ion pair chromatography for estimation of novel combined inhalation therapy that blocks coronavirus replication in chronic asthmatic patients.

A rapid and sensitive isocratic ion-pair chromatographic method was developed for the accurate analysis of ternary mixtures of formoterol, tiotropium, and ciclesonide in their novel combined inhalation that is widely used for the symptomatic treatment of patients with chronic obstructive disease. Analytical separation was performed using a C8 column and ion pair mobile phase composed of acetonitrile: acidified deionized water (55: 45% v/v) containing 0.025% sodium dodecyl sulfate. The pH was adjusted to 3.0 using orthophosphoric acid and eluted isocratically at 2.0 mL/min and 40 °C applying UV detection at 237 nm. The calibration ranges were found to be 0.3-9.0 µg/mL for formoterol, 0.45-13.5 µg/mL for tiotropium, and 10.0-300.0 µg/mL concerning ciclesonide. The proposed method exhibited good repeatability, accuracy, and sensitivity (R.S.D. < 2.0%). The approach is rapid (run time does not exceed 15 min) and achieves satisfactory resolution (resolution factors = 7.45 and 5.3 between formoterol and tiotropium and tiotropium and ciclesonide respectively). The sensitivity and the efficiency of the proposed method permit their successful estimation with a recovery percentage ± SD of 99.33% ± 0.43 for formoterol, 99.15% ± 0.60 for tiotropium, and 99.90% ± 0.41 for ciclesonide.

Adjusted green HPLC determination of nirmatrelvir and ritonavir in the new FDA approved co-packaged pharmaceutical dosage using supported computational calculations.

The greening of analytical methods has gained interest in the quantitative analysis field to reduce environmental impact and improve safety health conditions for analysts. Nirmatrelvir plus ritonavir is a new FDA approved co-packaged medication developed for the treatment of COVID-19. The aim of this research was to develop green fitted HPLC method using pre experimental computational testing of different stationary phases as well as selecting mobile phase regarding to green analytical chemistry principles. Computational study was designed to test the physical interaction between nirmatrelvir and ritonavir and different columns (C8, C18, Cyano column). The study showed that the C18 column was better for simultaneous HPLC analysis of the cited drugs. Regarding to green point of view, mobile phase consisted of ethanol: water (80:20, v/v) provided an efficient chromatographic separation of nirmatrelvir and ritonavir within a short analytical run time, reasonable resolution and excellent sensitivity. Isocratic elution was performed on a selected C18 column and a green adjusted mobile phase at flow rate of 1 mL/min and UV detection at 215 nm. The chromatographic system allowed complete baseline separation with retention times of 4.9 min for nirmatrelvir and 6.8 min for ritonavir. The method succeeded to determine nirmatrelvir and ritonavir over the concentration range of 1.0-20.0 µg/mL in the pure form and in pharmaceutical dosage form. Greenness profiles of the applied HPLC method was assessed using analytical eco-scale, the green analytical procedure index and the AGREE evaluation method. The results revealed adherence of the described method to the green analytical chemistry principles. The authors hope to provide a promising challenge for achieving green goals through integrating computational tools and applying them with green assessment metrics.

HPLC-DAD quantification of favipiravir in whole blood after extraction from volumetric absorptive microsampling devices.

Favipiravir is a prodrug of T-1105 made by modifying the pyrazine group as a COVID-19 therapy. During the pandemic, a safe and comfortable biosampling technique is needed for the subject or patient. Volumetric Absorptive Microsampling (VAMS) is a biosampling technique with a small blood volume and minimum hematocrit effect. The aims of this study were to develop and validate an analytical method for quantifying favipiravir extracted from VAMS using High Performance Liquid Chromatography - Photodiode Array with remdesivir as an internal standard. Analysis of favipiravir was performed using a C18 column (Waters, Sunfire™ 5 µm; 250 × 4.6 mm), with injection volume of 50 µL, flow rate of 0.8 mL/min, column temperature 30 ℃, and wavelength 300 nm. The separation was conducted under gradient elution with mobile phase consists of acetonitrile-0.2 % formic acid-20 mM sodium dihydrogen phosphate pH 3.5 and run time 12 min. Sample preparation was carried out using a protein precipitation method with 500 µL of methanol as precipitating agent. Samples were mixed on vortex for 30 s, sonicated for 15 min, and centrifuged at 10,000 rpm for 10 min. Lower Limit of Quantification (LLOQ) obtained was 0.5 µg/mL and the calibration curve ranged from 0.5 to 160 µg/mL. Sensitivity, linearity, selectivity, carry-over, accuracy, precision, recovery, and stability were validated by the guideline from Food and Drug Administration 2018. The method developed has successfully met the full validation requirements by FDA 2018 with the LLOQ obtained was 0.5 µg /mL.

Extremely Simple and Rapid HPLC Analysis of Tocilizumab in Human Serum with Selective Precipitation Using Alkylamine.

An assay using HPLC with fluorescence (FL) detection method for monitoring native FL of tocilizumab (TCZ) in human serum combined with extremely simple and rapid pretreatment without any antigen-antibody reaction was developed. Good separation of TCZ was achieved within 13 min on a Presto FF-C18 column (100 × 4.6 mm i.d., 2 µm). Simple pretreatment with acetonitrile containing primary and secondary alkylamines having longer than C3 in the alkyl chain removed immunoglobulin G subclass 1 and TCZ could be recovered selectively. The spiked calibration curve of TCZ in human serum showed good linearity in the range of 40-1000 µg/mL (r > 0.997). The lower limit of quantitation (S/N = 10) of the TCZ was 19.7 µg/mL. The accuracy was within 103.5-114.9%, and the intra- and inter-day precisions as relative standard deviations were less than 5.3 and 7.8% (n = 5), respectively. The recovery of TCZ was 42.2 ± 3.4% (n = 3). The TCZ in pretreated sample was confirmed to be stable for 6 h (>95%) at room temperature and 24 h (>95%) at 4 °C. The proposed method is considered extremely superior to the previous methods in terms of time requirement for analysis. Therefore, the developed method may be more useful than conventional methods in urgent situations, such as confirming therapeutic efficacy of cytokine-release syndrome by 2019 coronavirus disease.

Combining Semi-Targeted Metabolomics and Machine Learning to Identify Metabolic Alterations in the Serum and Urine of Hospitalized Patients with COVID-19.

Viral infections cause metabolic dysregulation in the infected organism. The present study used metabolomics techniques and machine learning algorithms to retrospectively analyze the alterations of a broad panel of metabolites in the serum and urine of a cohort of 126 patients hospitalized with COVID-19. Results were compared with those of 50 healthy subjects and 45 COVID-19-negative patients but with bacterial infectious diseases. Metabolites were analyzed by gas chromatography coupled to quadrupole time-of-flight mass spectrometry. The main metabolites altered in the sera of COVID-19 patients were those of pentose glucuronate interconversion, ascorbate and fructose metabolism, nucleotide sugars, and nucleotide and amino acid metabolism. Alterations in serum maltose, mannonic acid, xylitol, or glyceric acid metabolites segregated positive patients from the control group with high diagnostic accuracy, while succinic acid segregated positive patients from those with other disparate infectious diseases. Increased lauric acid concentrations were associated with the severity of infection and death. Urine analyses could not discriminate between groups. Targeted metabolomics and machine learning algorithms facilitated the exploration of the metabolic alterations underlying COVID-19 infection, and to identify the potential biomarkers for the diagnosis and prognosis of the disease.

Metabolite profile of COVID-19 revealed by UPLC-MS/MS-based widely targeted metabolomics.

The metabolic characteristics of COVID-19 disease are still largely unknown. Here, 44 patients with COVID-19 (31 mild COVID-19 patients and 13 severe COVID-19 patients), 42 healthy controls (HC), and 42 patients with community-acquired pneumonia (CAP), were involved in the study to assess their serum metabolomic profiles. We used widely targeted metabolomics based on an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The differentially expressed metabolites in the plasma of mild and severe COVID-19 patients, CAP patients, and HC subjects were screened, and the main metabolic pathways involved were analyzed. Multiple mature machine learning algorithms confirmed that the metabolites performed excellently in discriminating COVID-19 groups from CAP and HC subjects, with an area under the curve (AUC) of 1. The specific dysregulation of AMP, dGMP, sn-glycero-3-phosphocholine, and carnitine was observed in the severe COVID-19 group. Moreover, random forest analysis suggested that these metabolites could discriminate between severe COVID-19 patients and mild COVID-19 patients, with an AUC of 0.921. This study may broaden our understanding of pathophysiological mechanisms of COVID-19 and may offer an experimental basis for developing novel treatment strategies against it.