Caffeine is a widely consumed psychostimulant with several mechanisms of action and various positive and negative effects on organisms. Caffeine undergoes extensive hepatic metabolism to form main metabolites such as theobromine, theophylline, paraxanthine, and 1,3,7-trimethyluric acid. However, interspecies diversities have been observed in caffeine metabolism. In the present study, we developed a sensitive and straightforward ultra-high-performance liquid chromatography-tandem mass spectrometry method to quantify caffeine and its primary metabolites, namely theobromine, theophylline, paraxanthine, and 1,3,7-trimethyluric acid in rat plasma. After extraction of analytes using micro solid-phase extraction plate, analytes were separated by elution gradient on the Acquity UPLC HSS T3 (50 × 2.1 mm, 1.8 μm) column over 4 min. The detection was done on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring modes using a positive electrospray ionization interface.The method was successfully validated according to the European Medicine Agency guideline over a concentration range of 5-1500 ng/ml for caffeine, 5-1200 ng/mL for theobromine, and 2.5-1200 ng/mL for theophylline, paraxanthine, and 1,3,7-trimethyluric acid. The developed method was applied to analyze samples from animal experiments focusing on the metabolism and effects of caffeine and caffeine-containing beverages.

Rationale Tuberculosis (TB) remains a challenging global infectious disease, mainly affecting the lungs. First‐line anti‐TB drugs play a crucial role in slowing down the rapid spread of TB. In addition, the patient might benefit from therapeutic drug monitoring since it has become an accepted clinical tool for optimizing TB treatment. Methods A simple and sensitive liquid chromatography/tandem mass spectrometry method was developed to monitor the plasma level of isoniazid, ethambutol and pyrazinamide in plasma samples. A one‐step extraction procedure using an Ostro™ plate was applied, and extracts were analyzed by gradient elution followed by detection on a mass spectrometer by multiple reaction monitoring mode. Results The analytes were separated within 4.2 min and over the concentration range of 0.2–10 μg/ml for isoniazid and ethambutol and 1–65 μg/ml for pyrazinamide. The method was successfully validated according to the European Medicine Agency guideline for the selectivity, linearity and lower limit of detection, precision and accuracy, matrix effect, extraction recovery, carryover, dilution integrity and stability, and applied for quantification of analytes in clinical samples from TB patients. Conclusions The presented method allows sensitive and reproducible determination of selected anti‐TB drugs with advantages such as low sample volume requirement, short run time of analysis, one‐step sample preparation procedure with capabilities for phospholipids removal, and a low quantification limit as well as a high degree of selectivity.

Direct oral anticoagulants are widely used in many indications to prevent thromboembolic events. Routine therapeutic monitoring is not required; however, there is increasing evidence suggesting the benefit of plasma level measurement in some situations. In addition, laboratory monitoring might help improve patient and drug non‐compliance and thus individualize therapy. In the present study, we developed a sensitive and high throughput ultra‐high‐performance liquid chromatography‐tandem mass spectrometry method for simultaneous quantification of apixaban, dabigatran, edoxaban, and rivaroxaban in human plasma. A one‐step extraction procedure in 96‐well formate for phospholipid and protein removal was used for sample pre‐treatment, and analytes were separated using gradient elution over 4.2 min. Analytes were detected on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring mode. The method was validated according to the European Medicine Agency guideline for the selectivity, linearity, and lower limit of detection, precision and accuracy, matrix effects, extraction recovery, carryover, dilution integrity, and stability over a concentration range of 3.0–1000 ng/ml for all analytes. The validated method was applied to real clinical samples of patients treated with one of the drugs. Therefore, we can conclude that our method is suitable for therapeutic drug monitoring of direct oral anticoagulants.

Atranorin (ATR) is a secondary metabolite of lichens. While previous studies investigated the effects of this substance predominantly in an in vitro environment, in our study we investigated the basic physicochemical properties, the binding affinity to human serum albumin (HSA), basic pharmacokinetics, and, mainly, on the systematic effects of ATR in vivo. Sporadic studies describe its effects during, predominantly, cancer. This project is original in terms of testing the efficacy of ATR on a healthy organism, where we can possibly attribute negative effects directly to ATR and not to the disease. For the experiment, 24 Sprague Dawley rats (Velaz, Únetice, Czech Republic) were used. The animals were divided into four groups. The first group (n = 6) included healthy males as control intact rats (♂INT) and the second group (n = 6) included healthy females as control intact rats (♀INT). Groups three and four (♂ATR/n = 6 and ♀ATR/n = 6) consisted of animals with daily administered ATR (10mg/kg body weight) in an ethanol–water solution per os for a one-month period. Our results demonstrate that ATR binds to HSA near the binding site TRP214 and acts on a systemic level. ATR caused mild anemia during the treatment. However, based on the levels of hepatic enzymes in the blood (ALT, ALP, or bilirubin levels), thiobarbituric acid reactive substances (TBARS), or liver histology, no impact on liver was recorded. Significantly increased creatinine and lactate dehydrogenase levels together with increased defecation activity during behavioral testing may indicate the anabolic effect of ATR in skeletal muscles. Interestingly, ATR changed some forms of behavior. ATR at a dose of 10 mg/kg body weight is non-toxic and, therefore, could be used in further research.

Melanoma-bearing Libechov minipig (MeLiM) represents a large animal model for melanoma research. This model shows a high incidence of complete spontaneous regression of melanoma – a phenomenon uncommon in humans. Here, we present the first metabolomic characterisation of the MeLiM model comparing animals with progressing and spontaneously regressing melanomas. Plasma samples of 19 minipigs with progression and 27 minipigs with evidence of regression were analysed by a targeted metabolomic assay based on mass spectrometry detection. Differences in plasma metabolomics patterns were investigated by univariate and multivariate statistical analyses. Overall, 185 metabolites were quantified in each plasma sample. Significantly altered metabolomic profile was found, and 42 features were differentially regulated in plasma. Besides, the machine learning approach was used to create a predictive model utilising Arg/Orn and Arg/ADMA ratios to discriminate minipigs with progressive disease development from minipigs with regression evidence. Our results suggest that progression of melanoma in the MeLiM model is associated with alteration of arginine, glycerophospholipid and acylcarnitines metabolism. Moreover, this study provides targeted metabolomics characterisation of an animal model of melanoma with progression and spontaneous regression of tumours.