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.

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.

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.

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.

Simultaneous quantification of seven repurposed COVID-19 drugs remdesivir (plus metabolite GS-441524), chloroquine, hydroxychloroquine, lopinavir, ritonavir, favipiravir and azithromycin by a two-dimensional isotope dilution LC-MS/MS method in human serum.

BACKGROUND: The present COVID-19 pandemic has prompted worldwide repurposing of drugs. The aim of the present work was to develop and validate a two-dimensional isotope-dilution liquid chromatrography tandem mass spectrometry (ID-LC-MS/MS) method for accurate quantification of remdesivir and its active metabolite GS-441524, chloroquine, hydroxychloroquine, lopinavir, ritonavir, favipiravir and azithromycin in serum; drugs that have gained attention for repurposing in the treatment of COVID-19. METHODS: Following protein precipitation, samples were separated with a two-dimensional ultra-high performance liquid chromatography (2D-UHPLC) setup, consisting of an online solid phase extraction (SPE) coupled to an analytical column. For quantification, stable isotope-labelled analogues were used as internal standards for all analytes. The method was validated on the basis of the European Medicines Agency bioanalytical method validation protocol. RESULTS: Detuning of lopinavir and ritonavir allowed simultaneous quantification of all analytes with different concentration ranges and sensitivity with a uniform injection volume of 5 µL. The method provided robust validation results with inaccuracy and imprecision values of ≤ 9.59 % and ≤ 11.1 % for all quality controls. CONCLUSION: The presented method is suitable for accurate and simultaneous quantification of remdesivir, its metabolite GS-441525, chloroquine, hydroxychloroquine, lopinavir, ritonavir, favipiravir and azithromycin in human serum. The quantitative assay may be an efficient tool for the therapeutic drug monitoring of these potential drug candidates in COVID-19 patients in order to increase treatment efficacy and safety.

Validation of a liquid chromatography tandem mass spectrometry method for the simultaneous determination of hydroxychloroquine and metabolites in human whole blood.

Objectives Hydroxychloroquine (HCQ) is an anti-malarial and immunomodulatory drug reported to inhibit the Corona virus, SARS-CoV-2, in vitro. At present there is insufficient evidence from clinical trials to determine the safety and efficacy of HCQ as a treatment for COVID-19. However, since the World Health Organisation declared COVID-19 a pandemic in March 2020, the US Food and Drug Administration issued an Emergency Use Authorisation to allow HCQ and Chloroquine (CQ) to be distributed and used for certain hospitalised patients with COVID-19 and numerous clinical trials are underway around the world, including the UK based RECOVERY trial, with over 1000 volunteers. The validation of a liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the simultaneous determination of HCQ and two of its major metabolites, desethylchloroquine (DCQ) and di-desethylchloroquine (DDCQ), in whole blood is described. Methods Blood samples were deproteinised using acetonitrile. HCQ, DCQ and DDCQ were chromatographically separated on a biphenyl column with gradient elution, at a flow rate of 500 µL/min. The analysis time was 8 min. Results For each analyte linear calibration curves were obtained over the concentration range 50-2000 µg/L, the lower limit of quantification (LLOQ) was 13 µg/L, the inter-assay relative standard deviation (RSD) was <10% at 25, 800 and 1750 µg/L and mean recoveries were 80, 81, 78 and 62% for HCQ, d4-HCQ, DCQ and DDCQ, respectively. Conclusion This method has acceptable analytical performance and is applicable to the therapeutic monitoring of HCQ, evaluating the pharmacokinetics of HCQ in COVID-19 patients and supporting clinical trials.