HSPA5 Promotes Attachment and Internalization of Porcine Epidemic Diarrhea Virus through Interaction with the Spike Protein and the Endo-/Lysosomal Pathway.

Porcine epidemic diarrhea virus (PEDV) has caused huge economic losses to the global pig industry. The swine enteric coronavirus spike (S) protein recognizes various cell surface molecules to regulate viral infection. In this study, we identified 211 host membrane proteins related to the S1 protein by pulldown combined with liquid-chromatography tandem mass spectrometry (LC-MS/MS) analysis. Among these, heat shock protein family A member 5 (HSPA5) was identified through screening as having a specific interaction with the PEDV S protein, and positive regulation of PEDV infection was validated by knockdown and overexpression tests. Further studies verified the role of HSPA5 in viral attachment and internalization. In addition, we found that HSPA5 interacts with S proteins through its nucleotide-binding structural domain (NBD) and that polyclonal antibodies can block viral infection. In detail, HSPA5 was found to be involved in viral trafficking via the endo-/lysosomal pathway. Inhibition of HSPA5 activity during internalization would reduce the subcellular colocalization of PEDV with lysosomes in the endo-/lysosomal pathway. Together, these findings show that HSPA5 is a novel PEDV potential target for the creation of therapeutic drugs. IMPORTANCE PEDV infection causes severe piglet mortality and threatens the global pig industry. However, the complex invasion mechanism of PEDV makes its prevention and control difficult. Here, we determined that HSPA5 is a novel target for PEDV which interacts with its S protein and is involved in viral attachment and internalization, influencing its transport via the endo-/lysosomal pathway. Our work extends knowledge about the relationship between the PEDV S and host proteins and provides a new therapeutic target against PEDV infection.

Oligonucleotide mapping via mass spectrometry to enable comprehensive primary structure characterization of an mRNA vaccine against SARS-CoV-2.

Oligonucleotide mapping via liquid chromatography with UV detection coupled to tandem mass spectrometry (LC-UV-MS/MS) was recently developed to support development of Comirnaty, the world's first commercial mRNA vaccine which immunizes against the SARS-CoV-2 virus. Analogous to peptide mapping of therapeutic protein modalities, oligonucleotide mapping described here provides direct primary structure characterization of mRNA, through enzymatic digestion, accurate mass determinations, and optimized collisionally-induced fragmentation. Sample preparation for oligonucleotide mapping is a rapid, one-pot, one-enzyme digestion. The digest is analyzed via LC-MS/MS with an extended gradient and resulting data analysis employs semi-automated software. In a single method, oligonucleotide mapping readouts include a highly reproducible and completely annotated UV chromatogram with 100% maximum sequence coverage, and a microheterogeneity assessment of 5' terminus capping and 3' terminus poly(A)-tail length. Oligonucleotide mapping was pivotal to ensure the quality, safety, and efficacy of mRNA vaccines by providing: confirmation of construct identity and primary structure and assessment of product comparability following manufacturing process changes. More broadly, this technique may be used to directly interrogate the primary structure of RNA molecules in general.

An isotope dilution mass spectrometry assay to track Norovirus-like particles in vaccine process intermediates by quantifying capsid protein VP1.

The coronavirus disease (COVID-19) pandemic shows the rapid pace at which vaccine development can occur which highlights the need for more fast and efficient analytical methodologies to track and characterize candidate vaccines during manufacturing and purification processes. The candidate vaccine in this work comprises plant-derived Norovirus-like particles (NVLPs) which are structures that mimic the virus but lack any infectious genetic material. Presented here is a liquid chromatography-tandem mass spectrometry (LC-MS/MS) methodology for the quantification of viral protein VP1, the main component of the NVLPs in this study. It combines isotope dilution mass spectrometry (IDMS) with multiple reaction monitoring (MRM) to quantify targeted peptides in process intermediates. Multiple MRM transitions (precursor/product ion pairs) for VP1 peptides were tested with varying MS source conditions and collision energies. Final parameter selection for quantification includes three peptides with two MRM transitions each offering maximum detection sensitivity under optimized MS conditions. For quantification, a known concentration of the isotopically labeled version of the peptides to be quantified was added into working standard solutions to serve as an internal standard (IS); calibration curves were generated for concentration of native peptide vs. the peak area ratio of native-to-isotope labeled peptide. VP1 peptides in samples were quantified with labeled versions of the peptides added at the same level as that of the standards. Peptides were quantified with limit of detection (LOD) as low as 1.0 fmol µL-1 and limit of quantitation (LOQ) as low as 2.5 fmol µL-1. NVLP preparations spiked with known quantities of either native peptides or drug substance (DS) comprising assembled NVLPs produced recoveries indicative of minimal matrix effects. Overall, we report a fast, specific, selective, and sensitive LC-MS/MS strategy to track NVLPs through the purification steps of the DS of a Norovirus candidate vaccine. To the best of our knowledge, this is the first application of an IDMS method to track virus-like particles (VLPs) produced in plants as well as measurements performed with VP1, a Norovirus capsid protein.

Untargeted metabolomics of COVID-19 patient serum reveals potential prognostic markers of both severity and outcome.

INTRODUCTION: The diagnosis of COVID-19 is normally based on the qualitative detection of viral nucleic acid sequences. Properties of the host response are not measured but are key in determining outcome. Although metabolic profiles are well suited to capture host state, most metabolomics studies are either underpowered, measure only a restricted subset of metabolites, compare infected individuals against uninfected control cohorts that are not suitably matched, or do not provide a compact predictive model. OBJECTIVES: Here we provide a well-powered, untargeted metabolomics assessment of 120 COVID-19 patient samples acquired at hospital admission. The study aims to predict the patient's infection severity (i.e., mild or severe) and potential outcome (i.e., discharged or deceased). METHODS: High resolution untargeted UHPLC-MS/MS analysis was performed on patient serum using both positive and negative ionization modes. A subset of 20 intermediary metabolites predictive of severity or outcome were selected based on univariate statistical significance and a multiple predictor Bayesian logistic regression model was created. RESULTS: The predictors were selected for their relevant biological function and include deoxycytidine and ureidopropionate (indirectly reflecting viral load), kynurenine (reflecting host inflammatory response), and multiple short chain acylcarnitines (energy metabolism) among others. Currently, this approach predicts outcome and severity with a Monte Carlo cross validated area under the ROC curve of 0.792 (SD 0.09) and 0.793 (SD 0.08), respectively. A blind validation study on an additional 90 patients predicted outcome and severity at ROC AUC of 0.83 (CI 0.74-0.91) and 0.76 (CI 0.67-0.86). CONCLUSION: Prognostic tests based on the markers discussed in this paper could allow improvement in the planning of COVID-19 patient treatment.

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.

Diagnostic strategies in adrenal insufficiency.

PURPOSE OF REVIEW: Adrenal insufficiency (AI) is the clinical manifestation of deficient production of glucocorticoids with occasionally deficiency also in mineralocorticoids and adrenal androgens and constitutes a fatal disorder if left untreated. The aim of this review is to summarize the new trends in diagnostic methods used for determining the presence of AI. RECENT FINDINGS: Novel aetiologies of AI have emerged; severe acute respiratory syndrome coronavirus 2 infection was linked to increased frequency of primary AI (PAI). A new class of drugs, the immune checkpoint inhibitors (ICIs) widely used for the treatment of several malignancies, has been implicated mostly with secondary AI, but also with PAI. Salivary cortisol is considered a noninvasive and patient-friendly tool and has shown promising results in diagnosing AI, although the normal cut-off values remain an issue of debate depending on the technique used. Liquid chromatography-mass spectrometry (LC-MS/MS) is the most reliable technique although not widely available. SUMMARY: Our research has shown that little progress has been made regarding our knowledge on AI. Coronavirus disease 2019 and ICIs use constitute new evidence on the pathogenesis of AI. The short synacthen test (SST) remains the 'gold-standard' method for confirmation of AI diagnosis, although salivary cortisol is a promising tool.

Production and optimization of novel Sphorolipids from Candida parapsilosis grown on potato peel and frying oil wastes and their adverse effect on Mucorales fungal strains.

BRIEF INTRODUCTION: Mucormycosis disease, which has recently expanded with the Covid 19 pandemic in many countries, endangers patients' lives, and treatment with common drugs is fraught with unfavorable side effects. AIM AND OBJECTIVES: This study deals with the economic production of sophorolipids (SLs) from different eight fungal isolates strains utilizing potato peels waste (PPW) and frying oil waste (FOW). Then investigate their effect against mucormycetes fungi. RESULTS: The screening of the isolates for SLs production revealed the highest yield (39 g/100 g substrate) with most efficiency was related to a yeast that have been identified genetically as Candida parapsilosis. Moreover, the characterizations studies of the produced SLs by FTIR, 1H NMR and LC-MS/MS proved the existence of both acidic and lactonic forms, while their surface activity was confirmed by the surface tension (ST) assessment. The SLs production was optimized utilizing Box-Behnken design resulting in the amelioration of yield by 30% (55.3 g/100 g substrate) and ST by 20.8% (38mN/m) with constant level of the critical micelle concentration (CMC) at 125 mg/L. The studies also revealed the high affinity toward soybean oil (E24 = 50%), in addition to maintaining the emulsions stability against broad range of pH (4-10) and temperature (10-100℃). Furthermore, the antifungal activity against Mucor racemosus, Rhizopus microsporus, and Syncephalastrum racemosum proved a high inhibition efficiency of the produced SLs. CONCLUSION: The findings demonstrated the potential application of the SLs produced economically from agricultural waste as an effective and safer alternative for the treatment of infection caused by black fungus.

Contribution of voriconazole N-oxide plasma concentration measurements to voriconazole therapeutic drug monitoring in patients with invasive fungal infection.

BACKGROUND: Voriconazole (VRC), a widely used triazole antifungal, exhibits significant inter- and intra-individual pharmacokinetic variability. The main metabolite voriconazole N-oxide (NOX) can provide information on the patient's drug metabolism capacity. OBJECTIVES: Our objectives were to implement routine measurement of NOX concentrations and to describe the metabolic ratio (MR), and the contribution of the MR to VRC therapeutic drug monitoring (TDM) by proposing a suggested dosage-adjustment algorithm. PATIENTS AND METHODS: Sixty-one patients treated with VRC were prospectively included in the study, and VRC and NOX levels were assayed by LC-MS/MS. A mixed logistic model on repeated measures was implemented to analyse risk factors for the patient's concentration to be outside the therapeutic range. RESULTS: Based on 225 measurements, the median and interquartile range were 2.4 µg/ml (1.2; 4.2), 2.1 µg/ml (1.5; 3.0) and 1.0 (0.6; 1.9) for VRC, NOX and the MR, respectively. VRC Cmin <2 µg/ml were associated with a higher MR during the previous visit. MR values >1.15 and <0.48 were determined to be the best predictors for having a VRC Cmin lower than 2 µg/ml and above 5.5 µg/ml, respectively, at the next visit. CONCLUSIONS: Measurement of NOX resulted useful for TDM of patients treated with VRC. The MR using NOX informed interpretation and clinical decision-making and is very interesting for complex patients. VRC phenotyping based on the MR is now performed routinely in our institution. A dosing algorithm has been suggested from these results.

Serum lipid mediator profiles in COVID-19 patients and lung disease severity: a pilot study.

Coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 infection is highly heterogeneous, ranging from asymptomatic to severe and fatal cases. COVID-19 has been characterized by an increase of serum pro-inflammatory cytokine levels which seems to be associated with fatal cases. By contrast, the role of pro-resolving lipid mediators (SPMs), involved in the attenuation of inflammatory responses, has been scarcely investigated, so further studies are needed to understand SPMs metabolism in COVID-19 and other infectious diseases. Our aim was to analyse the lipid mediator metabolome, quantifying pro- and anti-inflammatory serum bioactive lipids by LC-MS/MS in 7 non-infected subjects and 24 COVID-19 patients divided into mild, moderate, and severe groups according to the pulmonary involvement, to better understand the disease outcome and the severity of the pulmonary manifestations. Statistical analysis was performed with the R programming language (R Foundation for Statistical Computing, Vienna, Austria). All COVID-19 patients had increased levels of Prostaglandin E2. Severe patients showed a significant increase versus controls, mild- and moderate-affected patients, expressed as median (interquartile range), in resolvin E1 [112.6 (502.7) vs 0.0 (0.0) pg/ml in the other groups], as well as in maresin 2 [14.5 (7.0) vs 8.1 (4.2), 5.5 (4.3), and 3.0 (4.0) pg/ml, respectively]. Moreover, 14-hydroxy docosahexaenoic acid (14-HDHA) levels were also increased in severe vs control and mild-affected patients [24.7 (38.2) vs 2.4 (2.2) and 3.7 (6.4) ng/mL, respectively]. Resolvin D5 was also significantly elevated in both moderate [15.0 (22.4) pg/ml] and severe patients [24.0 (24.1) pg/ml] versus controls [0.0 (0.0) pg/ml]. These results were confirmed by sparse partial least squares discriminant analysis which highlighted the contribution of these mediators to the separation between each of the groups. In conclusion, the potent inflammatory response to SARS-CoV-2 infection involves not only pro- but also anti-inflammatory lipid mediators that can be quantified in easily accessible serum samples, suggesting the need to perform future research on their generation pathways that will help us to discover new therapeutic targets.

Feasibility of Continuous Infusion of Cefiderocol in Conjunction with the Establishment of Therapeutic Drug Monitoring in Patients with Extensively Drug-Resistant Gram-Negative Bacteria.

BACKGROUND AND OBJECTIVE: Resistance to antibacterial substances is a huge and still emerging issue, especially with regard to Gram-negative bacteria and in critically ill patients. We report a study in six patients infected with extensively drug-resistant Gram-negative bacteria in a limited outbreak who were successfully managed with a quasi-continuous infusion of cefiderocol. METHODS: Patients were initially treated with prolonged infusions of cefiderocol over 3 h every 8 h, and the application mode was then switched to a quasi-continuous infusion of 2 g over 8 h, i.e. 6 g in 24 h. Therapeutic drug monitoring (TDM) was established using an in-house liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. RESULTS: Determined trough plasma concentrations were a median of 50.00 mg/L [95% confidence interval (CI) 27.20, 74.60] and steady-state plasma concentrations were a median of 90.96 mg/L [95% CI 37.80, 124]. No significant differences were detected with respect to acute kidney injury/continuous renal replacement therapy. Plasma concentrations determined from different modes of storage were almost equal when frozen or cooled, but markedly reduced when stored at room temperature. CONCLUSIONS: (Quasi) continuous application of cefiderocol 6 g/24 h in conjunction with TDM is a feasible mode of application; the sample for TDM should either be immediately analyzed, cooled, or frozen prior to analysis.

LC-MS/MS: A sensitive and selective analytical technique to detect COVID-19 protein biomarkers in the early disease stage.

INTRODUCTION: The COVID-19 outbreak has put enormous pressure on the scientific community to detect infection rapidly, identify the status of disease severity, and provide an immediate vaccine/drug for the treatment. Relying on immunoassay and a real-time reverse transcription polymerase chain reaction (rRT-PCR) led to many false-negative and false-positive reports. Therefore, detecting biomarkers is an alternative and reliable approach for determining the infection, its severity, and disease progression. Recent advances in liquid chromatography and mass spectrometry (LC-MS/MS) enable the protein biomarkers even at low concentrations, thus facilitating clinicians to monitor the treatment in hospitals. AREAS COVERED: This review highlights the role of LC-MS/MS in identifying protein biomarkers and discusses the clinically significant protein biomarkers such as Serum amyloid A, Interleukin-6, C-Reactive Protein, Lactate dehydrogenase, D-dimer, cardiac troponin, ferritin, Alanine transaminase, Aspartate transaminase, gelsolin and galectin-3-binding protein in COVID-19, and their analysis by LC-MS/MS in the early stage. EXPERT OPINION: Clinical doctors monitor significant biomarkers to understand, stratify, and treat patients according to disease severity. Knowledge of clinically significant COVID-19 protein biomarkers is critical not only for COVID-19 caused by the coronavirus but also to prepare us for future pandemics of other diseases in detecting by LC-MS/MS at the early stages.

An Unexpected Enzyme in Vascular Smooth Muscle Cells: Angiotensin II Upregulates Cholesterol-25-Hydroxylase Gene Expression.

Angiotensin II (AngII) is a vasoactive peptide hormone, which, under pathological conditions, contributes to the development of cardiovascular diseases. Oxysterols, including 25-hydroxycholesterol (25-HC), the product of cholesterol-25-hydroxylase (CH25H), also have detrimental effects on vascular health by affecting vascular smooth muscle cells (VSMCs). We investigated AngII-induced gene expression changes in VSMCs to explore whether AngII stimulus and 25-HC production have a connection in the vasculature. RNA-sequencing revealed that Ch25h is significantly upregulated in response to AngII stimulus. The Ch25h mRNA levels were elevated robustly (~50-fold) 1 h after AngII (100 nM) stimulation compared to baseline levels. Using inhibitors, we specified that the AngII-induced Ch25h upregulation is type 1 angiotensin II receptor- and Gq/11 activity-dependent. Furthermore, p38 MAPK has a crucial role in the upregulation of Ch25h. We performed LC-MS/MS to identify 25-HC in the supernatant of AngII-stimulated VSMCs. In the supernatants, 25-HC concentration peaked 4 h after AngII stimulation. Our findings provide insight into the pathways mediating AngII-induced Ch25h upregulation. Our study elucidates a connection between AngII stimulus and 25-HC production in primary rat VSMCs. These results potentially lead to the identification and understanding of new mechanisms in the pathogenesis of vascular impairments.

A Simultaneous Extraction/Derivatization Strategy for Quantitation of Vitamin D in Dried Blood Spots Using LC-MS/MS: Application to Biomarker Study in Subjects Tested for SARS-CoV-2.

Vitamin D plays a critical role in bone development and maintenance, and in other physiological functions. The quantitation of endogenous levels of individual vitamin D and its metabolites is crucial for assessing several disease state conditions. With cases of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) leading to the coronavirus disease 2019 (COVID-19) pandemic, there are several studies that have associated lower levels of serum vitamin D with severity of infection in COVID-19 patients. In this context, we have developed and validated a robust LC-MS/MS method for simultaneous quantitation of vitamin D and its metabolites in human dried blood spot (DBS) obtained from participants tested for COVID-19. The chromatographic separation for vitamin D and metabolites was performed using an ACE Excel C18 PFP column protected with a C18 guard column (Phenomenex, Torrance, CA, USA). The mobile phase consisted of formic acid in water (0.1% v/v) as mobile phase A and formic acid in methanol (0.1% v/v) as mobile phase B, operated at a flow rate of 0.5 mL/min. Analysis was performed utilizing the LC-MS/MS technique. The method was sensitive with a limit of quantification of 0.78 ng/mL for all analytes, and had a large dynamic range (200 ng/mL) with a total run time of 11 min. The inter- and intraday accuracy and precision values met the acceptance criteria per the US Food and Drug Administration guidelines. Blood concentrations of 25(OH)D3, vitamin D3, 25(OH)D2, and vitamin D2 over a range of 2-195.6, 0.5-121.5, 0.6-54.9, and 0.5-23.9 ng/mL, respectively, were quantified in 909 DBS samples. In summary, our developed LC-MS/MS method may be used for quantification of vitamin D and its metabolites in DBS, and may be applied to investigations of the emerging role of these compounds in various physiological processes.

Chronic Strongyloides stercoralis infection increases presence of the Ruminococcus torques group in the gut and alters the microbial proteome.

We explored the impact of chronic Strongyloides stercoralis infection on the gut microbiome and microbial activity in a longitudinal study. At baseline (time-point T0), 42 fecal samples from matched individuals (21 positive for strongyloidiasis and 21 negative) were subjected to microbiome 16S-rRNA sequencing. Those positive at T0 (untreated then because of COVID19 lockdowns) were retested one year later (T1). Persistent infection in these individuals indicated chronic strongyloidiasis: they were treated with ivermectin and retested four months later (T2). Fecal samples at T1 and T2 were subjected to 16S-rRNA sequencing and LC-MS/MS to determine microbial diversity and proteomes. No significant alteration of indices of gut microbial diversity was found in chronic strongyloidiasis. However, the Ruminococcus torques group was highly over-represented in chronic infection. Metaproteome data revealed enrichment of Ruminococcus torques mucin-degrader enzymes in infection, possibly influencing the ability of the host to expel parasites. Metaproteomics indicated an increase in carbohydrate metabolism and Bacteroidaceae accounted for this change in chronic infection. STITCH interaction networks explored highly expressed microbial proteins before treatment and short-chain fatty acids involved in the synthesis of acetate. In conclusion, our data indicate that chronic S. stercoralis infection increases Ruminococcus torques group and alters the microbial proteome.

Plasma Apolipoprotein Concentrations Are Highly Altered in Severe Intensive Care Unit COVID-19 Patients: Preliminary Results from the LIPICOR Cohort Study.

SARS-CoV-2 infection goes beyond acute pneumonia, as it also impacts lipid metabolism. Decreased HDL-C and LDL-C levels have been reported in patients with COVID-19. The lipid profile is a less robust biochemical marker than apolipoproteins, components of lipoproteins. However, the association of apolipoprotein levels during COVID-19 is not well described and understood. The objective of our study is to measure plasma levels of 14 apolipoproteins in patients with COVID-19 and to evaluate the relationships between apolipoprotein levels, severity factors and patient outcomes. From November to March 2021, 44 patients were recruited on admission to the intensive care unit because of COVID-19. Fourteen apolipoproteins and LCAT were measured by LC-MS/MS in plasma of 44 COVID-19 patients on admission to the ICU and 44 healthy control subjects. Absolute apolipoprotein concentrations were compared between COVID-19 patients and controls. Plasma apolipoproteins (Apo) A (I, II, IV), C(I, II), D, H, J and M and LCAT were lower in COVID-19 patients, whereas Apo E was higher. COVID-19 severity factors such as PaO2/FiO2 ratio, SO-FA score and CRP were correlated with certain apolipoproteins. Lower Apo B100 and LCAT levels were observed in non-survivors of COVID-19 versus survivors. To conclude, in this study, lipid and apolipoprotein profiles are altered in COVID-19 patients. Low Apo B100 and LCAT levels may be predictive of non-survival in COVID-19 patients.

Impact of COVID- 19 pandemic on antidepressants consumptions by wastewater analysis in Turkey.

The COVID-19 pandemic has been a major challenge worldwide, forcing countries to take restrictive measures beyond conventional methods in their fight against the spread of the disease. Followingly, many studies have been conducted on the effects of these measures on mental health. Wastewater-based epidemiology (WBE) was used in this study to monitor and estimate changes in antidepressant use under normal conditions (2019) and COVID-19 pandemic conditions (2020). Likewise, this study utilized wastewater-based epidemiology (WBE) to monitor and assess changing trends from the pre-pandemic period (2019) to COVID-19 pandemic conditions in antidepressant use (2020). Wastewater samples were collected from 11 cities in Turkey throughout six sampling periods covering the pre-pandemic and during-pandemic periods (June 2019-December 2020). Then, samples were analyzed via LC-MS/MS method. As a result, we observed that venlafaxine was the drug with the highest concentration (mean ± SD: 103.6 ± 112.1 mg/1000p/day). Moreover, city number 6 presented the highest venlafaxine use and the most dramatic increase during the pandemic period. Finally, this study revealed the potential of WBE to estimate the changing trends in mental health during the ongoing pandemic.

Characterization of serotonin as a candidate biomarker of severity and prognosis of COVID-19 using LC/MS analysis.

The coronavirus disease 2019 (COVID-19) pandemic has been associated with high mortality worldwide. Owing to its complicated pathophysiology, diagnostic and prognostic biomarkers for effective patient management remain scarce. We analyzed kynurenine, tryptophan, and serotonin levels in the serum of patients with COVID-19 via liquid chromatography/mass spectrometry analysis. Serum serotonin levels were decreased in patients with more severe COVID-19, along with increased kynurenine and decreased tryptophan concentrations. Patients with moderate disease who subsequently worsened showed significantly lower serotonin concentrations compared with those who did not experience severe disease. Serum serotonin levels may represent a valuable biomarker for COVID-19 severity and prognosis.

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.

LC-MS/MS-PRM Quantification of IgG Glycoforms Using Stable Isotope Labeled IgG1 Fc Glycopeptide Standard.

Targeted quantification of proteins is a standard methodology with broad utility, but targeted quantification of glycoproteins has not reached its full potential. The lack of optimized workflows and isotopically labeled standards limits the acceptance of glycoproteomics quantification. In this work, we introduce an efficient and streamlined chemoenzymatic synthesis of a library of isotopically labeled glycopeptides of IgG1 which we use for quantification in an energy optimized LC-MS/MS-PRM workflow. Incorporation of the stable isotope labeled N-acetylglucosamine enables an efficient monitoring of all major fragment ions of the glycopeptides generated under the soft higher-energy C-trap dissociation (HCD) conditions, which reduces the coefficients of variability (CVs) of the quantification to 0.7-2.8%. Our results document, for the first time, that the workflow using a combination of stable isotope labeled standards with intrascan normalization enables quantification of the glycopeptides by an electron transfer dissociation (ETD) workflow, as well as the HCD workflow, with the highest sensitivity compared to traditional workflows. This was exemplified by a rapid quantification (13 min) of IgG1 Fc glycoforms from COVID-19 patients.

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.