A liquid chromatography-tandem mass spectrometry method was developed and used to simultaneously detect 19 small peptide hormones prohibited in sport and their main metabolites in urine after solid-phase extraction. Detection was achieved using a triple-quadrupole mass spectrometric detector coupled with an electrospray ionization interface after chromatographic separation with an octadecyl column based on fused-core particle technology. Sample pretreatment was performed by solid-phase extraction. The extraction procedure was optimized by comparison of different sorbents and washing/elution protocols. The best results were obtained using a mixed-mode weak cation exchange sorbent, two washing steps (ultrapurified water and methanol), and elution using 300 mM ammonium formate in 25 % ammonia/methanol (10/90) or 25 % ammonia/10 % formic acid/methanol (8/12/80). The procedure was validated in terms of sensitivity (lower limits of detection: 0.05-2.0 ng/ml depending on the target analyte), specificity, recovery [[60 %, coefficient of variation (CV) \15 % except for TB500 17-23 fragment, AOD9604, and ARA290 for which recovery was \50 %), ion suppression/enhancement (\35 %), robustness, carryover, stability of the target analytes [stable for at least for 2 days (25°C, 2 weeks (4°C), 2 months (-20°C)], and repeatability of retention times (CV \0.1 %) and relative abundances of the selected ion transitions (CV \15 %).The suitability of the method was confirmed by analyzing spiked and excreted urines, the latter collected after intravenous injection of 0.1 mg of GHRP-2.
Aim: Development of fluorometholone-loaded PEG-PLGA nanoparticles (NPs) functionalized with cell-penetrating peptides (CPPs) for the treatment of ocular inflammatory disorders. Materials & methods: Synthesized polymers and peptides were used for elaboration of functionalized NPs, which were characterized physicochemically. Cytotoxicity and ability to modulate the expression of proinflammatory cytokines were evaluated in vitro using human corneal epithelial cells (HCE-2). NPs uptake was assayed in both in vitro and in vivo models. Results: NPs showed physicochemical characteristics suitable for ocular administration without evidence of cytotoxicity. TAT-NPs and G2-NPs were internalized and displayed anti-inflammatory activity in both HCE-2 cells and mouse eye. Conclusion: TAT-NPs and G2-NPs could be considered a novel strategy for the treatment of ocular inflammatory diseases of the anterior and posterior segment.
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