Model 717 plus

Two milliliters of preheated PBS (37 °C) was added to the film substrates for in vitro release testing. Films were incubated under gentle agitation on an orbital shaker (150 rpm) at 37 °C. After each predetermined time interval, 1 mL of PBS was withdrawn from the vials and replaced with fresh fluid. This investigation was conducted in triplicate. DEX quantification in the samples was performed based on HPLC analysis using a Waters HPLC equipped with a pump and controller (Model 600), an autosampler (Model 717plus) and a UV absorbance detector (Model 486) (Waters, Milford, U.S.). The mobile phase consisted of water and acetonitrile (60:40 (v/v)), and an Equisil ODS C18 column with a precolumn (5 µm C18, 125 × 4 mm; Techlab, Braunschweig, Germany) was chosen as the stationary phase. The flow rate was 1 mL/min, and the detection wavelength was set at 238 nm. Twenty microliters of the samples were injected, and the DEX retention time was found to be ~3 min. Calibration was performed in the range from 0.2 to 20 µg/mL (r² > 0.999). Furthermore, the DEX degradation product 17-oxo-dexamethasone (17-oxo-DEX) was quantified. The peak-retention time for this compound was ~5 min, and calibration was conducted in the span between 0.2 and 26 µg/mL (r² > 0.999). Peak integration and evaluation were carried out with Clarity^TM 8.0 software (DataApex, Prague, Czech Republic).

The amount of MTK suspended in the preparations was determined by HPLC assay. Nanocrystal suspension (1 mL) was ultra-centrifuged at 13,000 rpm for 10 min to settle the nanocrystals in the aqueous vehicle. Methanol was then added to the precipitate and vortexed for 30 min to dissolve the drug nanocrystals. The concentration of MTK in the organic solvent or in the supernatant was analyzed using Waters HPLC system that is composed of a pump (Model 515 pump), a UV–VIS (ultraviolet–visible) detector (Model 486), and an autosampler (Model 717 plus). The mobile phase comprised acetonitrile and distilled water at a volume ratio of 4:6 with 0.15% v/v of trifluoroacetic acid, was flowed through the reversed-phase C18 column (4.6 mm × 50 mm, 1.8 µm, Agilent, Santa Clara, CA, USA) at a flow rate of 1.2 mL/min. The column temperature was set to 25 °C. A 20 µL aliquot was injected and the column eluent was monitored at a wavelength of 238 nm. The sharp peak of MTK was detected at 10.7 min. The least-square linear regression was linear in the MTK range from 1.0–100 μg/mL, with a coefficient of determination (r2) value of 0.997.

When maltotriose was used as substrate, the reaction products were analyzed by HPLC in an Ultimate 3000 equipped with a 300 RS Pump, a 3000 RS Autosampler and a RS Column Compartment, using an ELSD 2000ES Alltech detector. Both maltooligosaccharides and methyl-glucosides could be separated and detected using a Prevail Carbohydrate column (acetonitrile:water 70:30 as mobile phase, flow: 1 mL/min, column T: 30 °C, ELSD T: 80 °C, nitrogen Flow: 1.7 L/min). When starch was used as substrate, reaction products were analyzed in a Waters-Millipore 510 HPLC system equipped with an automatic sampler (model 717 Plus) (Waters Corp., MA, USA), a refractive-index detector (Waters 410) (Waters Corp., MA, USA) and a Hypersil GOLD™ Amino column (Thermo Scientific, Paisley, UK) using acetonitrile:water (80:20) as the mobile phase at a flow rate of 1.0 mL/min. The peak areas were measured and compared against those of a standard curve containing known amounts of methyl-glucoside.
To calculate the % conversion of the reaction, the following formula was used:
$\%Conversion=\frac{{\left[Maltotriose\right]}_{inicial}-{\left[Maltotriose\right]}_{final}}{{\left[Maltotrios\right]}_{inicial}}\times 100$