Model dgu 20a5

Quantitative analysis was performed using a Shimadzu high pressure liquid chromatography (HPLC) prominence system consisting of a degasser (Model DGU-20A5, Tokyo, Japan), a liquid chromatograph, an auto sampler, a diode array detector and a thermostatic oven. A modified, previously validated, method was used for the analysis [9 (link)]. The chromatograms were obtained and processed with the LC solutions software v. 1.21 SP1. In detail, an Athena CNW Technology C18, (250 × 4.6 mm and 5 μm internal diameter) at column temperature of 40 °C chromatographic workstation (CSW) was used for regression analysis and data acquisition. Flow rate was 1 mL/min and the injection volume were 20 μL. Mobile phase was phosphate buffer (10 mM, pH was adjusted to 3.5 using H₃PO₄)/ACN 80/20 v/v. Each sample was measured in triplicate.

Risperidone content was assayed using a Shimadzu Prominence HPLC system consisting of a degasser (Model DGU-20A5, Tokyo, Japan), a pump (Model LC-20AD, Tokyo, Japan), an autosampler (Model SIL-20AC, Tokyo, Japan), a UV-Vis detector (Model SPD-20A, Tokyo, Japan), and a column oven (Model CTO-20AC, Tokyo, Japan). Chromatographic analysis was performed with a C₁₈ column (CNW Technologies Athena, 120 Å, 5 μm, 250 mm × 4.6 mm, Tokyo, Japan) at 25 °C. Methanol/H₂O/triethylamine at a ratio of 80/19.5/0.5 v/v/v was used as mobile phase, while acetic acid was used to adjust the pH at 10.22. The flow rate was set at 1.0 mL/min, injection volume was 20 μL, while the risperidone analysis was performed at 254 nm.

The chromatographic equipment (Shimadzu-Prominence) consisted of an on-line degasser (Model DGU 20A5), solvent delivery module (Model LC-20AT), autosampler (Model SIL-20AHT), column oven (Model CTO-20A), fluorescence detector (Model RF-10AXL) and system controller CBM-20A. A reverse-phase C8 column Vydac of 5 μm, 4.6 mm × 250 mm (Thermo Fischer Scientific, Massachusetts, USA) with a pre-column of 5 μm, 4.6 mm × 50 mm (Thermo Fischer Scientific, Massachusetts, USA) was used. The mobile phase consisted of a mixture of 0.12 % (m:v) TFA in Milli-Q water (pump A) and methanol (pump B) at 40:60 (v/v) rendering an isocratic phase. Fluorimetric measurements were carried out in a 12-μL flow cell at 610 and 675 nm excitation and emission wavelengths, respectively. The injection volume was 5 μl and the flow rate was 1 ml/min at a working pressure of 135 kgf.cm⁻². Analyses were performed with a column temperature of 30 °C. LCsolution Software (Shimadzu, Tokyo, Japan) was used for data processing.
The calibration curve was generated with AlPc solutions with concentrations ranging from 0.01 to 8 μM. For AlPc quantification in nanoemulsions, sample aliquots were dissolved in ethanol (1:40, v/v), vortexed for 3 min, filtered through 0.22-μm nylon filters (Millex GN, Millipore, Darmstadt, Germany), and injected into the HPLC system.