Paper-based devices are always more gaining a relevant position in the field of sensors. The continuous demand for affordable, simple, sustainable, and portable devices, is making paper as the ideal basis towards the realization of analytical tools for the easy self-testing. In this work, we demonstrate, for the first time, the development of a disposable paper-based printed electroanalytical strip for reliable, rapid, and high-throughput detection of glutathione in blood. The detection is based on the thiol-disulfide exchange reaction, which produces a detectable compound easily oxidizable at a Prussian Blue/carbon black nanocomposite involving a favorable low-interference overpotential. This nanocomposite is mixed within a carbon-based conductive ink and successively screen-printed onto a wax-patterned filter paper. The employment of paper provides a reagent-free device, as a consequence of the reagents pre-loading within the testing area. After the experimental conditions have been optimized, glutathione has been detected up to 10 mM, with a detection limit of 60 μM, and a sensitivity of (0.102 ± 0.005) μA/mM. This sensor showed satisfactory repeatability (relative standard deviation equal to 10%, for detection of glutathione 1 mM), especially by considering the hand-made manufacturing process. The "real-world" applicability of this strip has been evaluated by quantifying blood glutathione at physiological levels and by recovery studies achieving satisfactory values.

There is a growing interest in the named “acidic sterolbiome” and in the genetic potential of the gut microbiome (GM) to modify bile acid (BA) structure. Indeed, the qualitative composition of BAs in feces correlates with the bowel microorganisms and their collective genetic material. GM is responsible for the production of BA metabolites, such as secondary and oxo-BAs. The specific BA profiles, as microbiome-host co-metabolic products, could be useful to investigate the GM-host interaction in animals under physiological conditions, as well as in specific diseases. In this context, we developed and validated an ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry method for the simultaneous analysis of up to 21 oxo-BAs and their 9 metabolic precursors. Chromatographic separation was achieved in 7 min with adequate analytical performance in terms of selectivity, sensitivity (LOQ from 0.05 to 0.1 µg/mL), accuracy (bias% < 5%), precision (CV% < 5%) and matrix effect (ME% < 10%). A fast solvent extraction protocol has been fine-tuned, achieving recoveries > 90%. In parallel, the gut microbiota assessment in farming animals was evaluated by 16S rRNA next-generation sequencing, and the correlation with the BA composition was performed by multivariate analysis, allowing to reconstruct species-specific associations between the BA profile and specific GM components.

Amino acids are ubiquitous components of mammalian milk and greatly contribute to its nutritional value. The compositional analysis of free amino acids is poorly reported in the literature even though their determination in the biological fluids of livestock animals is necessary to establish possible nutritional interventions. In the present study, the free amino acid profiles in mature swine milk, colostrum and plasma were assessed using a targeted metabolomics approach. In particular, 20 amino acids were identified and quantified via two alternative and complementary reversed-phase HPLC methods, involving two stationary phases based on core-shell technology, i.e., Kinetex C18 and Kinetex F5, and two detection systems, i.e., a diode array detector (DAD) and a fluorescence detector (FLD). The sample preparation involved a de-proteinization step, followed by pre-chromatographic derivatization with 9-fluorenylmethylchloroformate (FMOC-Cl). The two optimized methods were validated for specificity, linearity, sensitivity, matrix effect, accuracy and precision and the analytical performances were compared. The analytical methods proved to be suitable for free amino acid profiling in different matrices with high sensitivity and specificity. The correlations among amino acid levels in different biological fluids can be useful for the evaluation of physio-pathological status and to monitor the effects of therapeutic or nutritional interventions in humans and animals.

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