2.4.4. In Vitro Hyt Release

The cumulative Hyt release from the hydrogel formulations was determined using the dialysis bag method in phosphate buffer saline (PBS, pH 7.4). The Hyt@tgels formulations (1 mL) were sealed in pre-swollen cellulose membrane dialysis bags (3.5–5.0 kDa cut-off, Spectrum) and immersed into 5 mL of PBS buffer (pH 7.4) in a water bath at 37 ◦C shaken at 100 rpm for 5 days. At set time intervals, 5 mL of the release media was collected for Hyt analysis and replaced with the same volume of fresh PBS to maintain the sink conditions. Hyt released in the PBS media from the hydrogels was measured with liquid chromatography–tandem mass spectrometry (LC-MS/MS) as reported by [37]. The LC-MS/MS system consisted of a Shimadzu NexeraXR UHPLC (Shimadzu Italy, Milan, Italy) coupled to an LCMS 8060 turbo spray ionization triple-quadrupole mass spectrometer (LCMS 8060, Shimadzu Italy, Milan, Italy). The whole system was controlled by Lab Solution software. Separation of analytes was achieved using a 2.6 μm Kinetex polar C18 column (Phenomenex, Torrance, CA, USA). The mobile phase included Buffer A (0.1% formic acid in water) and Buffer B (0.1% formic acid in acetonitrile) in isocratic flow. The total run time was 7 min for each injection. The mass spectrometer was operated in the turbo-spray mode with negative ion detection. The detection and quantification of Hyt was accomplished by multiple reaction monitoring (MRM) with the transitions *m*/*z* 153.05 → 123.0 (quantifier); 153.05 → 93.0 (qualifier). The instrumental parameters tuned to maximize the MRM signals were nebulizing gas flow 3 L/min, heating gas flow 10 L/min, interface temperature 370 ◦C, DL temperature 250 ◦C, heat block temperature 450 ◦C, and drying gas flow 10 L/min.
