Waters autosampler model 717 plus

PAX level in the supernatant was determined using a previously published HPLC assay method with minor modification [29 (link)]. The HPLC system consisted of a Waters Model 1515 HPLC pump, Waters Autosampler Model 717 plus (Waters Inc., Bedford, MA, USA), and Waters 2487 dual absorbance UV detector (Waters Inc., Bedford, MA, USA) governed by a computer running Empower software (version 1154). Quantification of PAX was achieved via an isocratic elution using acetonitrile and water (70:30) as mobile phase delivered at a flow rate of 1 mL/min (at ambient temperature) through a C₁₈ analytical column (Sunfire^®; 250 mm length × 4. 6 mm i.d., 5 µm particle size). The UV detection was performed at a wavelength of 227 nm and the injection volume was 30 µL. Drug loading content and encapsulation efficiency were determined using the following equations: $\mathrm{PAX} \mathrm{loading} \left(\% w/w\right)=\frac{\mathrm{Amount} \mathrm{of} \mathrm{loaded} \mathrm{PAX} \left(\mathrm{mg}\right)}{\mathrm{Amount} \mathrm{of} \mathrm{polymer} \left(\mathrm{mg}\right)+ \mathrm{Amount} \mathrm{of} \mathrm{loaded} \mathrm{PAX} \left(\mathrm{mg}\right)}\times 100$
$\mathrm{Encapsulation} \mathrm{efficiency} (\%)=\frac{\mathrm{Amount} \mathrm{of} \mathrm{loaded} \mathrm{PAX} \left(\mathrm{mg}\right)}{\mathrm{Amount} \mathrm{of} \mathrm{PAX} \mathrm{added} \left(\mathrm{mg}\right)}\times 100$