Bioactive compounds of Jingfang Granules against SARS-CoV-2 virus proteases 3CLpro and PLpro.

OBJECTIVE: Jingfang Granules have been recommended for the prevention and treatment of corona virus disease 2019 (COVID-19). Through chemical analysis and bioactivity evaluation, this study aims to elucidate the potential effective components of Jingfang Granules. METHOD(S): The inhibitory acti-vities of Jingfang Granules extract against 3-chymotrypsin-like protease (3CLpro), papain like protease (PLpro), spike protein receptor-binding domain (S-RBD) and human cyclooxygenase-2 (COX-2) were evaluated using enzyme assay. The antitussive effects were evaluated using the classical ammonia-induced cough model. The chemical constituents of Jingfang Granules were qualitatively and quantitatively analyzed by liquid chromatography-mass spectrometry (LC/MS). The 3CLpro and PLpro inhibitory activities of the major compounds were determined by enzyme assay, molecular docking, and site-directed mutagenesis. RESULT(S): Jingfang Granules exhibited 3CLpro and PLpro inhibitory activities, as well as COX-2 inhibitory and antitussive activities. By investigating the MS/MS behaviors of reference standards, a total of fifty-six compounds were characterized in Jingfang Granules. Sixteen of them were unambiguously identified by comparing with reference standards. The contents of the 16 major compounds were also determined, and their total contents were 2 498.8 mug/g. Naringin, nodakenin and neohesperidin were three dominating compounds in Jingfang Granules, and their contents were 688.8, 596.4 and 578.7 mug/g, respectively. In addition, neohesperidin and naringin exhibited PLpro inhibitory activities, and the inhibition rates at 8 mumol/L were 53.5% and 46.1%, respectively. Prim-O-glucosylcimifugin showed significant inhibitory activities against 3CLpro and PLpro, and the inhibitory rates at 8 mumol/L were 76.8% and 78.2%, respectively. Molecular docking indicated that hydrogen bonds could be formed between prim-O-glucosylcimifugin and amino acid residues H163, E166, Q192, T190 of 3CLpro (binding energy, -7.7 kcal/mol) and K157, D164, R166, E167, T301 of PLpro(-7.3 kcal/mol), respectively. Site-directed mutagenesis indicated amino acid residue K157 was a key active site for the interaction between prim-O-glucosylcimifugin and PLpro. CONCLUSION(S): Prim-O-glucosylcimifugin, neohesperidin, and naringin as the major compounds from Jingfang Granules could inhibit severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus proteases 3CLpro and PLpro. The results are valuable for rational clinical use of Jingfang Granules.

Dried blood spot (DBS) sample analysis for drug and metabolomic profiling in oncology clinical trials: Cost-effective decentralized sampling modality for precision Oncology

The COVID19 pandemic accelerated opportunities for innovation within the decentralization process of clinical trials with opportunities for implementation of patient-centric workflows for efficiency and cost-reduction. Decentralized sample collection, particularly whole blood using dried blood spots (DBS) provides the ideal mechanism for patient driven sample collection with ease of access to sample generation, drug level assessments and metabolomic prMegofiling, providing longitudinal real-time measure of drug specific pharmacodynamic readout for safety and efficacy. In this study, we report the development of a protocol for the capture and comprehensive profiling of metabolomics using dried blood spots from a cohort of 49 healthy volunteer donors. Using liquid chromatography combined with mass spectrometric (UPLC-MS/MS) methods an untargeted metabolomic approach resulted in the identification of >800 biochemicals of which a significant subset was found to be presented in corresponding matched plasma (from whole blood) samples. The biochemicals identified from the DBS samples included metabolites that were part of the lipid, amino acid, nucleotide, peptide, cofactors, carbohydrate and energy super pathways. A significant number of metabolites identified in the DBS samples were xenobiotics including those representing the biotransformation products of drugs. The overall metabolite profiles were analyzed for precision and accuracy of measure, variability in performance and dynamic range to establish benchmarks for evaluation. An additional cohort with a longitudinal sampling as part of the protocol provided the reproducibility of the analytic method for inter-day variability of metabolite performance over time. Although metabolomic profiles varied between individuals from a population perspective, there was minimal variation observed within individuals when samples were profiled longitudinally over several weeks. Thus, the protocols for DBS collection and the corresponding capture of a large set of metabolites with reproducible performance provides an opportunity for its implementation in oncological clinical trials as part of a de-centralized clinical trial solution.

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

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

Anastrozole dose escalation for optimal estrogen suppression in postmenopausal early stage breast cancer: A prospective trial

Introduction: We performed matched case-control studies utilizing cohorts of postmenopausal women with ER+ breast cancer receiving adjuvant aromatase inhibitors (AI) on MA.27 [anastrozole, exemestane] or PreFace [letrozole] to assess the association between estrogen suppression after 6 months of treatment and an early breast cancer (EBC) event within 5 years of AI initiation (Clin Cancer Res 2020;26:2986-98). We found a significant 3.0-fold increase in risk of an EBC event for those taking anastrozole with levels of estrone (E1) >=1.3 pg/mL and estradiol (E2) >=0.5 pg/mL, but not for exemestane or letrozole. Given these findings we designed a prospective pharmacodynamic (PD) study to evaluate the impact of anastrozole (1 mg/day: ANA1) on E1 and E2 levels, and among those with inadequate estrogen suppression (IES: E1 >=1.3 pg/mL and E2 >=0.5 pg/mL), to evaluate the safety and PD efficacy of high-dose anastrozole (10 mg/day: ANA10), which has been found to be safe in prior clinical trials (Cancer 1998;83:1142-52). Method(s): Post-menopausal women with stage I-III, ER >=1% positive/HER2-negative breast cancer who were candidates for anastrozole were eligible after completion of locoregional therapy and chemotherapy, as clinically indicated. Women who were pre-menopausal at diagnosis were not eligible. All patients received 8-10 weeks of ANA1, after which those with adequate estrogen suppression (AES: E1< 1.3 pg/mL or E2< 0.5 pg/mL) came off study. Those with IES went on to receive ANA10 for 8-10 weeks, followed by letrozole (2.5 mg/day: LET) for 8-10 weeks. All patients were managed at their treating oncologist's discretion following study discontinuation. E1 and E2 blood levels were measured pre-treatment and after completion of each treatment cycle by a CLIA-approved liquid chromatography with tandem mass spectrometry in the Immunochemical Core Laboratory at Mayo Clinic. With a sample size of 29 patients with IES after ANA1, a one-sided binomial test of proportions with a significance level of 0.05 will have an 87% chance of rejecting the proportion with AES after ANA10 is at most 25% (Ho) when the true proportion is at least 50%. Specifically, the null hypothesis is rejected if the number of women with AES after ANA10 is 12 or more. Data lock was July 6, 2022. Result(s): Of the 161 women enrolled from April 2020 through May 2022, 3 withdrew consent prior to start of ANA1 and 2 were ineligible;thus, 156 women comprised the study cohort. Median patient age was 64 years (range 44-86), 10% of patients were of Hispanic ethnicity and/or non-white race, and 15% received chemotherapy. Six patients remain on ANA1, and 10 discontinued ANA1 due to refusal (7), adverse event (AE) (2), or COVID-19 (1). Forty-one of the remaining 140 patients (29.3% 95%CI: 21.9- 37.6%) had IES with ANA1. Nine of these 41 patients did not go on to ANA10 due to refusal (6) or AE (3). Of the 32 patients who started ANA10, 8 remain on treatment, 5 discontinued due to refusal (3) or AE (1-grade 2 urinary tract infection;1-grade 1 palpitations), and 19 had a blood draw 45 days or more after starting ANA10. No grade 3-5 AEs or grade 2 hot flashes or arthralgias were reported. Of these 19 patients, 14 achieved AES with ANA10 (73.7% 95%CI: 48.8-90.9%). All 19 patients switched to LET of which 3 remain on treatment, 1 is missing E1/E2 data, and 15 had a blood draw 45 days or more after starting LET. Of these 15 patients, 10 maintained AES, 2 acquired AES with LET, and 3 no longer had AES. Anastrozole and letrozole drug levels will be reported at the meeting. Conclusion(s): Approximately 29% of postmenopausal women with ER+/HER2- BC receiving adjuvant anastrozole 1 mg/daily had IES. A majority of these patients achieved AES with dose escalation to ANA10 without tolerability issues. E1 and E2 levels are logical biomarkers given the mechanism of action of anastrozole, and further study utilizing them to determine the optimal dose of anastrozole for a given patient should be performed.

Single-Dose Bioequivalence Study of Rivaroxaban-Containing Medicinal Products in Healthy Volunteers.

Therapeutically, new oral anticoagulants (NOACs) are considered to be non-inferior or superior to vitamin K antagonists (warfarin). NOACs are included in current guidelines for the treatment of various cardiovascular diseases. Rivaroxaban medicinal products have been shown to effectively fight thrombotic complications of the new coronavirus infection, COVID-19. The wide clinical use of rivaroxaban products motivates the development of generics. The aim of the study was to compare the pharmacokinetics and safety of rivaroxaban medicinal products in a single-dose bioequivalence study in healthy volunteers under fasting conditions. Material(s) and Method(s): the bioequivalence study compared single-dose oral administration of Rivaroxaban, 10 mg film-coated tablets (NovaMedica Innotech LLC, Russia), and the reference product Xarelto, 10 mg film-coated tablets (Bayer AG, Germany), in healthy volunteers under fasting conditions. The open, randomised, crossover trial included 46 healthy volunteers. Each of the medicinal products (the test product and the reference product) was administered once;blood samples were collected during the 48 h after the administration. The washout between the study periods lasted 7 days. Rivaroxaban was quantified in plasma samples of the volunteers by high performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS). Result(s): no adverse events or serious adverse events were reported for the test and reference products during the study. The following pharmacokinetic parameters were obtained for Rivaroxaban and Xarelto, respectively: Cmax of 134.6 +/- 58.0 ng/mL and 139.9 +/- 49.3 ng/mL, AUC0-48 of 949.7 +/- 354.5 ngxh/mL and 967.6 +/- 319.9 ngxh/mL, AUC0- of 986.9 +/- 379.7 ngxh/mL and 1003.6 +/- 320.4 ngxh/mL, T1/2 of 8.2 +/- 3.2 h and 7.8 +/- 3.3 h. The 90% confidence intervals for the ratios of Cmax, AUC0-48, and AUC0- geometric means were 88.04-108.67%, 89.42-104.92% and 89.44-104.81%, respectively. Conclusion(s): the test product Rivaroxaban and the reference product Xarelto were found to have similar rivaroxaban pharmacokinetics and safety profiles. The study demonstrated bioequivalence of the medicinal products.Copyright © 2022 Obstetrics, Gynecology and Reproduction. All rights reserved.

Maackia amurensis Rupr. et Maxim.: Supercritical CO2 Extraction and Mass Spectrometric Characterization of Chemical Constituents.

Three types of extraction were used to obtain biologically active substances from the heartwood of M. amurensis: supercritical CO2 extraction, maceration with EtOH, and maceration with MeOH. The supercritical extraction method proved to be the most effective type of extraction, giving the highest yield of biologically active substances. Several experimental conditions were investigated in the pressure range of 50-400 bar, with 2% of ethanol as co-solvent in the liquid phase at a temperature in the range of 31-70 °C. The most effective extraction conditions are: pressure of 100 bar and a temperature of 55 °C for M. amurensis heartwood. The heartwood of M. amurensis contains various polyphenolic compounds and compounds of other chemical groups with valuable biological activity. Tandem mass spectrometry (HPLC-ESI-ion trap) was applied to detect target analytes. High-accuracy mass spectrometric data were recorded on an ion trap equipped with an ESI source in the modes of negative and positive ions. The four-stage ion separation mode was implemented. Sixty-six different biologically active components have been identified in M. amurensis extracts. Twenty-two polyphenols were identified for the first time in the genus Maackia.

Identification and high-throughput quantification of baicalein and its metabolites in plasma and urine

Ethnopharmacological relevance: Scutellaria baicalensis Georgi. contains varieties of function compounds, and it has been used as traditional drug for centuries. Baicalein is the highest amount of flavonoid found in Scutellaria baicalensis Georgi., which exerts various pharmacological activities and might be a promising drug to treat COVID-19. Aim of the study: The present work aims to investigate the metabolism of baicalein in humans after oral administration, and study the pharmacokinetics of BA and its seven metabolites in plasma and urine. Materials and methods: The metabolism profiling and the identification of baicalein metabolites were performed on HPLC-Q-TOF. Then a column-switching method named MPX™-2 system was applied for the high-throughput quantificationof BA and seven metabolites. Results: Seven metabolites were identified using HPLC-Q-TOF, including sulfate, glucuronide, glucoside, and methyl-conjugated metabolites. Pharmacokinetic study found that BA was extensively metabolized in vivo, and only 5.65% of the drug remained intact in the circulatory system after single dosing. Baicalein-7-O-sulfate and baicalein-6-O-glucuronide-7-O-glucuronide were the most abundant metabolites. About 7.2% of the drug was excreted through urine and mostly was metabolites. Conclusion: Seven conjugated metabolites were identified in our assay. A high-throughput HPLC-MS/MS method using column switch was established for quantifying BA and its metabolites. The method has good sensitivity and reproducibility, and successfully applied for the clinical pharmacokinetic study of baicalein and identified metabolites. We expect that our results will provide a metabolic and pharmacokinetic foundation for the potential application of baicalein in medicine. © 2022

Seasonal variation and ecological risk assessment of Pharmaceuticals and Personal Care Products (PPCPs) in a typical semi-enclosed bay — The Bohai Bay in northern China

The Bohai Bay as a typical semi-enclosed bay in northern China with poor water exchange capacity and significant coastal urbanization, is greatly influenced by land-based inputs and human activities. As a class of pseudo-persistent organic pollutants, the spatial and temporal distribution of Pharmaceuticals and Personal Care Products (PPCPs) is particularly important to the ecological environment, and it will be imperfect to assess the ecological risk of PPCPs for the lack of systematic investigation of their distribution in different season. 14 typical PPCPs were selected to analyze the spatial and temporal distribution in the Bohai Bay by combining online solid-phase extraction (SPE) and HPLC-MS/MS techniques in this study, and their ecological risks to aquatic organisms were assessed by risk quotients (RQs) and concentration addition (CA) model. It was found that PPCPs widely presented in the Bohai Bay with significant differences of spatial and seasonal distribution. The concentrations of ∑PPCPs were higher in autumn than in summer. The distribution of individual pollutants also showed significant seasonal differences. The high values were mainly distributed in estuaries and near-shore outfalls. Mariculture activities in the northern part of the Bohai Bay made a greater contribution to the input of PPCPs. Caffeine, florfenicol, enrofloxacin and norfloxacin were the main pollutants in the Bohai Bay, with detection frequencies exceeding 80 %. The ecological risk of PPCPs to algae was significantly higher than that to invertebrates and fish. CA model indicated that the potential mixture risk of total PPCPs was not negligible, with 34 % and 88 % of stations having mixture risk in summer and autumn, respectively. The temporary stagnation of productive life caused by Covid-19 weakened the input of PPCPs to the Bohai Bay, reducing the cumulative effects of the pollutants. This study was the first full-coverage investigation of PPCPs in the Bohai Bay for different seasons, providing an important basis for the ecological risk assessment and pollution prevention of PPCPs in the bay. © 2022 Elsevier B.V.

Presence and toxicity of drugs used to treat SARS-CoV-2 in Llobregat River, Catalonia, Spain.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a strain of coronavirus that causes COVID-19 (coronavirus disease 2019), the respiratory illness responsible for the on-going COVID-19 pandemic. In March 2020, it was declared global pandemic, causing millions of deaths. An evident tendency of global pharmaceutical consumption due to COVID-19 pandemic should be seen worldwide, and this increase might suppose an environmental threat. Pharmaceuticals administrated at home or in pharmacies are excreted by faeces and urine after consumption, and wastewater treatment plants (WWTPs) are not able to remove all pharmaceuticals residues that eventually will end up in the aquatic media (rivers and sea). For this reason, analytical techniques such as liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) have become prominent to identify and quantify pharmaceuticals residues in aquatic matrices. In view of the scarce data on the occurrence of pharmaceuticals used as COVID-19 treatment, the aim of the present study was to evaluate the presence of these class of pharmaceuticals in river water which were dexamethasone, prednisone, ciprofloxacin, levofloxacin, remdesivir, ritonavir, lopinavir, acetaminophen, hydroxychloroquine, chloroquine and cloperastine, their toxicity in the aquatic environment using D. magna and to perform an exhaustive risk assessment in seven points of the Llobregat river basin. Dexamethasone, cloperastine and acetaminophen were the pharmaceuticals with higher concentrations, showing mean levels between 313 and 859 ng L-1.

Monitoring Alcohol Consumption in Slovak Cities during the COVID-19 Lockdown by Wastewater-Based Epidemiology.

The consumption of alcohol in a population is usually monitored through individual questionnaires, forensics, and toxicological data. However, consumption estimates have some biases, mainly due to the accumulation of alcohol stocks. This study's objective was to assess alcohol consumption in Slovakia during the COVID-19 pandemic-related lockdown using wastewater-based epidemiology (WBE). Samples of municipal wastewater were collected from three Slovak cities during the lockdown and during a successive period with lifted restrictions in 2020. The study included about 14% of the Slovak population. The urinary alcohol biomarker, ethyl sulfate (EtS), was analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). EtS concentrations were used to estimate the per capita alcohol consumption in each city. The average alcohol consumption in the selected cities in 2020 ranged between 2.1 and 327 L/day/1000 inhabitants and increased during days with weaker restrictions. WBE can provide timely information on alcohol consumption at the community level, complementing epidemiology-based monitoring techniques (e.g., population surveys and sales statistics).

Enteropathy and bone marrow hypoplasia associated with presumptive albendazole toxicosis in a juvenile Boer goat.

Albendazole is a widely used anthelmintic drug that is labeled for the treatment of specific nematodes and flukes in ruminants. Albendazole is approved for the treatment of liver flukes in goats (10 mg/kg PO for a single dose), but is commonly used extra-label in situations in which parasite resistance is an issue. Albendazole toxicosis has been reported in pigeons, doves, alpacas, humans, dogs, and cats. Here we report an adverse event in a 6-mo-old goat associated with extra-label use of albendazole (35.7 mg/kg PO daily for 3 d). Clinicopathologic findings included severe diarrhea and death, with small intestinal crypt necrosis and dysplasia, and severe bone marrow hypoplasia. Microbial and molecular testing and transmission electron microscopy ruled out infectious organisms. The described pathologic changes are similar to those reported in other species that have experienced toxicosis associated with albendazole. To our knowledge, bone marrow and intestinal lesions associated with albendazole use in the goat have not been reported previously. Veterinarians should be aware of potential adverse events and toxicoses associated with anthelmintic drugs, especially as parasite resistance increases, and extra-label usage, and the use of such drugs without veterinary supervision, becomes more common.

COVID-19 patients with altered steroid hormone levels are more likely to have higher disease severity.

PURPOSE: This study aims to evaluate the correlations between the severity of the disease and serum steroid levels by analyzing the serum steroid levels in COVID-19 patients with different levels of disease progression and the control group. METHODS: Morning serum Aldosterone, 11-deoxycortisol, Androstenedione, 17-hydroxyprogesterone, Dihydrotestosterone (DHT), Dehydroepiandrosterone (DHEA), Corticosterone, Dehydroepiandrosterone sulfate (DHEAS), Estrone, Estradiol, Progesterone, 11-deoxycorticosterone, Cortisol, Corticosterone, Androsterone, Pregnenolone, 17-hydroxypregnenolone and 21-deoxycortisol levels were measured in 153 consecutive patients were grouped as mild, moderate, and severe based on the WHO COVID-19 disease severity classification and the control group. Steroid hormone levels were analyzed at once with a liquid chromatography-tandem mass spectrometric method (LC-MS/MS). RESULTS: In our study, nearly all steroids were statistically significantly higher in the patients' group than in the control group (p < 0.001). Also, DHEA was an independent indicator of the disease severity with COVID-19 CONCLUSIONS: Our study reveals that the alteration in steroid hormone levels was correlated with disease severity. Also, steroid hormone levels should be followed up during COVID-19 disease management.

Mass Spectrometry Multiplexed Detection of SARS-CoV-2.

Testing of large populations for virus infection is now a reality worldwide due to the coronavirus (SARS-CoV-2) pandemic. The demand for SARS-CoV-2 testing using alternatives other than PCR led to the development of mass spectrometry (MS)-based assays. However, MS for SARS-CoV-2 large-scale testing have some downsides, including complex sample preparation and slow data analysis. Here, we describe a high-throughput targeted proteomics method to detect SARS-CoV-2 directly from nasopharyngeal and oropharyngeal swabs. This strategy employs fully automated sample preparation mediated by magnetic particles, followed by detection of SARS-CoV-2 nucleoprotein peptides by turbulent flow chromatography coupled with tandem mass spectrometry.

A retrospective analysis of metabolic control in children with PKU in the COVID-19 era.

Background: Patients with phenylketonuria (PKU) must maintain a lifelong natural protein-restricted diet to prevent neuro-cognitive damage. Early diagnosis is established with newborn screening, with diet subsequently controlled by regular phenylalanine (Phe) monitoring. During the COVID-19 pandemic, significant lockdown measures were introduced that may have influenced the above. Aim of our study: To establish whether the diagnosis was delayed in neonates during the pandemic. In addition, metabolic control was further assessed during the COVID-19 pandemic era (CE) compared to the same period a year prior (non-COVID-19 era, NCE). The lockdown periods (LD) were also compared with unrestricted periods (URP). Patients methods: Six neonates born during the CE and eight neonates born during NCE were included in the newborn screening analysis. Seventy-two classical PKU patients aged 2-18 years and categorized as children (2-12 years; 51 patients) and adolescents (>13 years; 21 patients) were included in the metabolic control analysis. The frequency of dried blood spot (DBS) sampling and Phe levels were assessed according to the different periods. Results: There was no diagnostic or therapeutic delay in reaching the recommended Phe range in neonates born during CE compared to those born in NCE (median [interquartile range, IQR]: 23.5 [22.5-24] vs. 22 [18.0-27] days, p = NS). The cumulative DBS sampling frequency in children increased by 9.9% in the CE while no change was noted in the adolescent group. The median Phe level increased significantly in both age groups in the CE, but remained within the recommended target range. During CE, changes in Phe levels differed in the two age groups: children had the highest median Phe in the second lockdown period (LD2), while the adolescents had an increased Phe in URP.There were significant negative correlations between DBS sampling frequencies and Phe levels in both age groups in NCE (children: r - 0.43, p = 0.002; adolescents r = -0.37, p = 0.012), and in adolescents in CE (r = -0.62, p = 0.006). Conclusion: The pandemic did not impact newborn metabolic screening. The increased frequency of DBS sampling in CE and good target Phe levels suggest a better compliance in a very sensitive period. Since many factors may impact metabolic control in the different age groups, further studies are needed to analyse their respective role.

Potential biomarkers for the diagnosis and differential diagnosis of immune-mediated demyelinating diseases screened by tandem mass spectrometry technology

Objective To screen the potential biomarkers for the diagnosis and differential diagnosis of immune-mediated demyelinating diseases by tandem mass tags (TMT) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) technology. Methods Twenty patients with demyelinating diseases (demyelinating group) and 10 patients with noninflammatory neurological diseases (NND group) from Beijing Tiantan Hospital affiliated to Capital Medical University from January 2020 to January 2021 were enrolled in this study. The demyelinating group included 10 patients with Guillain-Barre syndrome (GBS subgroup) and 10 patients with multiple sclerosis (MS subgroup). TMT proteomics was used to screen out the different protein expression patterns between the demyelinating group and the NND group and between the GBS subgroup and the MS subgroup (difference>2 or<0.5 and with statistical significance), and String database was used to perform gene ontology (GO) analysis and Kyoto encyclopedia of gene and genomes (KEGG) analysis on the pathways involved in the differently expressed proteins between the groups. In addition, 80 demyelinating patients (demyelinating diseases validation group) and 40 healthy subjects (healthy control group) were selected for retrospective analysis of general lipid indexes. The demyelinating diseases validation group included 40 GBS patients (GBS validation group) and 40 MS patients (MS validation group). Receiver operating characteristic (ROC) curve was obtained to evaluate the value of general lipid indexes for the diagnosis of demyelinating diseases and the differential diagnosis between GBS and MS groups. Results A total of 362 proteins were detected by TMT proteomics. There were 101 differentially expressed proteins between the demyelinating group and the NND group, and 45 differentially expressed proteins between the GBS group and the MS group. Compared with the NND group, GO enrichment analysis showed that the top five enrichment pathways in the demyelinating group were macrophage colony stimulating factor and receptor complex, negative regulation of cholesterol input, negative regulation of very low density lipoprotein particle clearance, triglyceride-rich lipoprotein particle remodeling, and cholesterol reverse transport. Compared with MS group, the top five enriched pathways in GBS group were high-density lipoprotein particle receptor binding, negative regulation of very low density lipoprotein particle remodeling, negative regulation of cholesterol input, negative regulation of very low density lipoprotein particle clearance, and medium density lipoprotein particle. KEGG enrichment analysis results showed that differentially expressed proteins in the demyelinating group and the NND group were enriched in 8 pathways, including phosphatidylinositide 3-kinases-protein kinase B signaling pathway, complement and coagulation cascade reaction, extracellular matrix and its receptor interaction, Staphylococcus aureus infection, cholesterol metabolism, RAS signaling pathway, phagosome, and mitogen-activated protein kinase signaling pathway. Differentially expressed proteins in GBS group and MS group were enriched in 9 pathways: cholesterol metabolism, complement and coagulation cascade, platelet activation, peroxisome proliferators-activated receptors signaling pathway, vitamin digestion and absorption, novel coronavirus infection, fat digestion and absorption, axon guidance, and neutrophil extracellular trap formation pathway. The levels of triglyceride (TG), total cholesterol (TC), low density lipoprotein cholesterol (LDL-C) and apolipoprotein B (apoB) were significantly higher, while high density lipoprotein cholesterol (HDL-C) and apolipoprotein A1 (apoA1) levels were significantly lower in the demyelinating disease validation group than in the healthy control group (all P<0.05 or 0.01). Area under the curve (AUC) of TG, TC, HDL-C, LDL-C, apoA1 and apoB alone or in combination for the diagnosis of immune-mediated demyelinating diseases was 0.746, 0.643, 0.798, 0.703, 0.806, 0.708 and 0.868, respectively. The AUC of HDL-C, apoA1, LDL-C and apoB for differential diagnosis between GBS and MS was 0.692, 0.653, 0.632, 0.695 and 0.718, respectively. Conclusions There are differences in cerebrospinal fluid proteomics between patients with immune-mediated demyelinating disease and patients with NND, GBS and MS, and the differentially expressed protein patterns mainly exist in the pathways related to lipid metabolism. Lipid related indicators may be used as biomarkers for the diagnosis and differential diagnosis of immune-mediated demyelinating disease. © 2022 Chinese Medical Journals Publishing House Co.Ltd. All rights reserved.

Profiling SARS-CoV-2 Infection by High-Throughput Shotgun Proteomics.

A comprehensive cartography of viral and host proteins expressed during the different stages of SARS-CoV-2 infection is key to decipher the molecular mechanisms of pathogenesis. For the most detailed analysis, proteins should be first purified and then proteolyzed with trypsin in the presence of detergents. The resulting peptide mixtures are resolved by reverse phase ultrahigh pressure liquid chromatography and then identified by a high-resolution tandem mass spectrometer. The thousands of spectra acquired for each fraction can then be assigned to peptide sequences using a relevant protein sequence database, comprising viral and host proteins and potential contaminants from the growth medium or from the operator. The peptides are evidencing proteins and their intensities are used to infer the abundance of their corresponding proteins. Data analysis allows for highlighting the viral and host proteins dynamics. Here, we describe the sample preparation method adapted to profile SARS-CoV-2 -infected cell models, the shotgun proteomics pipeline to record experimental data, and the workflow for data interpretation to analyze infection-induced proteomic changes in a time-resolved manner.

Development of a tandem mass spectrometric method for the quantitation of dexamethasone levels

BACKGROUND AND AIM: Dexamethasone is one of the most potent glucocorticoid. It is used in the various medical conditions such as multiple sclerosis, allergies, inflammation, asthma, dermatitis and shock. Recently, dexamethasone has been found to be beneficial in patients with COVID-19 pneumonia requiring supplemental oxygen or mechanical ventilation. It can cause serious adverse effects such as adrenal suppression, hyperglycemia, and cardiac arrhythmia. Therefore, monitoring of dexamethasone levels is important. Our aim in this study is to develop an LC-MS/MS method for dexamethasone. METHODS: Dexamethasone was detected with ABSciex API 3200 tandem mass spectrometery in positive electrospray ionization mode. The selected ion transitions for dexamethasone and internal standard were m/z 289.5/97.3 and m/z 339.1/113.2, respectively. Briefly, 100 μL of internal standard (17-hydroxyprogesterone-d8) and 4 mL of diethylether were added to 250 μL of sample, and vortexed for 1 minute, then centrifuged at 4000 g for 10 minutes. The supernatant was taken into tubes and evaporated at 40 °C under nitrogen gas. The residues were dissolved in 200 μL of methanol:water (1:1, v/v%) and 25 μL was injected. RESULTS: The calibration curve was linear between 1.95 and 2000 ng/mL (r2>0.99). The limit of quantitation for dexamethasone was 1.95 ng/mL. Total run time was 5 minutes. Intra- and inter-assay imprecision values were less than 9%. The mean extraction recovery was 92.8%, and the matrix effect ranged from 3.6% to 9.8%. CONCLUSIONS: A sensitive and reproducible tandem mass spectrometry method was developed for dexamethasone. The method can be used in the analysis of dexamethasone.

Influence of traditional Chinese medicines on the in vivo metabolism of lopinavir/ritonavir based on UHPLC-MS/MS analysis.

A fast, reliable, and cost-effective liquid chromatography-tandem mass spectrometry method was established to determine the effects of the traditional Chinese medicine employed to treat coronavirus disease 2019, namely, Lianhua Qingwen granules, Huoxiang Zhengqi capsules, Jinhua Qinggan granules, Shufeng Jiedu capsules, and Angong Niuhuang pills, on the pharmacokinetics of lopinavir/ritonavir in rats. Blood samples were prepared using the protein precipitation method and atazanavir was selected as the internal standard (IS). Separation was performed on an Agilent ZORBAX eclipse plus C18 (2.1 mm × 50 mm, 1.8 µm) column using acetonitrile and water containing 0.1% formic acid as the mobile phase for gradient elution. The flow rate was 0.4 mL/min and the injection volume was 2 µL. Agilent Jet Stream electrospray ionization was used for mass spectrometry detection under positive ion multiple reaction monitoring mode at a transition of m/z 629.3→447.3 for lopinavir, m/z 721.3→296.1 for ritonavir, and m/z 705.4→168.1 for the IS. The method showed good linearity in the concentration range of 25-2500 ng/mL (r=0.9981) for lopinavir and 5-500 ng/mL (r=0.9984) for ritonavir. The intra-day and inter-day precision and accuracy were both within ±15%. Items, such as dilution reliability and residual effect, were also within the acceptable limits. The method was used to determine the effects of five types of traditional Chinese medicines on the pharmacokinetics of lopinavir/ritonavir in rats. The pharmacokinetic results showed that the half-life of ritonavir in the groups administered Lianhua Qingwen granules and Huoxiang Zhengqi capsules combined with lopinavir/ritonavir was prolonged by approximately 1.5- to 2-fold relative to that in the control group. Similarly, the pharmacokinetic parameters of lopinavir were altered. Overall, the results of this study offer important theoretical parameters for the effective clinical use of five types of traditional Chinese medicines combined with lopinavir/ritonavir to reduce the occurrence of clinical adverse reactions.

Development of a solid phase microextraction gas chromatography tandem mass spectrometry methodology for the analysis of sixty personal care products in hydroalcoholic gels - hand sanitizers - in the context of COVID-19 pandemic.

Because of the coronavirus pandemic, hydroalcoholic gels have become essential products to prevent the spread of COVID-19. This research aims to develop a simple, fast and sustainable microextraction methodology followed by gas chromatography tandem mass spectrometry (GC-MS/MS) to analyze simultaneously 60 personal care products (PCPs) including fragrances allergens, synthetic musks, preservatives and plasticizers in hand sanitizers. Micro-matrix-solid-phase dispersion (µMSPD) and solid-phase microextraction (SPME) were compared with the aim of obtaining high sensitivity and sample throughput. SPME demonstrated higher efficiency being selected as sample treatment. Different dilutions of the sample in ultrapure water were assessed to achieve high sensitivity but, at the same time, to avoid or minimize matrix effect. The most critical parameters affecting SPME (fibre coating, extraction mode and temperature) were optimized by design of experiments (DOE). The method was successfully validated in terms of linearity, precision and accuracy, obtaining recovery values between 80 and 112% for most compounds with relative standard deviation (RSD) values lower than 10%. External calibration using standards prepared in ultrapure water demonstrated suitability due to the absence of matrix effect. Finally, the simple, fast and high throughput method was applied to the analysis of real hydroalcoholic gel samples. Among the 60 target compounds, 39 of them were found, highlighting the high number of fragrance allergens, at concentrations ranging between 0.01 and 217 µg g-1. Most of the samples were not correctly labelled attending cosmetic Regulation (EU) No 1223/2009, and none of them followed the World Health Organization (WHO) recommendation for hand sanitizers formulation.

Untargeted detection of the carbonyl metabolome by chemical derivatization and liquid chromatography-tandem mass spectrometry in precursor ion scan mode: Elucidation of COVID-19 severity biomarkers.

Metabolomics (both targeted and untargeted) has become the gold standard in biomarker discovery. Whereas targeted approaches only provide information for the selected markers, thus hampering the determination of out-of-the-box markers, the common bottleneck of untargeted metabolomics is the identification of detected biomarkers. In this study, we developed a strategy based on derivatization and LC-MS/MS detection in a precursor ion scan for the untargeted determination of a specific part of the metabolome (carbonyl-containing metabolites). The usefulness of this guided metabolomics approach has been demonstrated by elucidating carbonyl-containing biomarkers of COVID-19 severity. First, the LC-MS/MS behavior of 63 model compounds after O-benzylhydroxylamine derivatization was studied. A precursor ion scan of m/z 91 was selected as a suitable approach for the untargeted detection of carbonyl-containing metabolites. The method was able to detect ≈300 potential carbonyl-containing molecules in plasma, including mono-/di-/tricarbonylic compounds with satisfactory intra-day and inter-day repeatability and RSDs commonly <15%. Additionally, the semiquantitative nature of the precursor ion scan method was confirmed by comparison with a fully validated targeted method. The application of the guided metabolomics method to COVID-19 plasma samples revealed the presence of four potential COVID-19 severity biomarkers. Based on their LC-MS/MS behavior, these biomarkers were elucidated as 2-hydroxybutyrate, 2,3-dihydroxybutyrate, 2-oxobutyrate and 2-hydroxy-3-methylbutyrate. Their structures were confirmed by comparison with reference materials. The alterations of these biomarkers with COVID-19 severity were confirmed by a target analysis of a larger set of samples. Our results confirm that guided metabolomics is an alternative approach for the untargeted detection of selected families of metabolites; this approach can accelerate their elucidation and provide new perspectives for the establishment of health/disease biomarkers.