Uv visible detector

The drug quantification in NE was conducted via high performance liquid chromatography (HPLC) on an LC6-2012HT model apparatus (Shimadzu, Kyoto, Japan) with a Prontosil C8 (120-5-C8 SH, 5 µm, 250 × 4.0 mm) column coupled with a UV-visible detector (Shimadzu, Kyoto, Japan). The flow rate was set to 1 mL/min, and the detection was measured at 206 nm. The mobile phase was a mixture of water and acetonitrile used in isocratic mode with a 90/10 acetonitrile/water ratio. The calibration curve line was linear, from 1 to 50 µg/mL, with a correlation coefficient of r² = 0.9996. All experiments were performed in triplicate.
The drug loading was calculated using the following equation: $$\mathrm{Drug} \mathrm{Loading}=\frac{\mathrm{Amount} \mathrm{of} \mathrm{IP}-\mathrm{DHA} \mathrm{added}}{\mathrm{Total} \mathrm{amount} \mathrm{of} \mathrm{IP}-\mathrm{DHA}+\mathrm{excipients}}$$
The percentage of IP-DHA in the nanoemulsions was determined according to the following equation: $$\% \mathrm{of} \mathrm{IP}-\mathrm{DHA} \mathrm{in} \mathrm{nanoemulsions} =\frac{\mathrm{Amount} \mathrm{of} \mathrm{IP}-\mathrm{DHA} \mathrm{added}}{\mathrm{Amount} \mathrm{of} \mathrm{IP}-\mathrm{DHA} \mathrm{dosed}}\times 100$$

The amount of LP in rabbit plasma samples was analyzed using a reversed-phase high-performance liquid chromatography (RP-HPLC) method. An HPLC system coupled with a UV–visible detector (Shimadzu, Japan) was used to analyze LP, utilizing valsartan as an internal standard (IS). The HPLC separation of LP and IS was carried out on an ODS C₁₈ column (250 mm × 4.6 mm, particle size 5 μm) maintained at 30 °C. The mobile phase used in this work was composed of phosphate buffer: acetonitrile (75:25, % v/v, pH 2.8). The mobile phase was delivered at a flow rate of 1.2 mL min⁻¹. The detection of LP was performed at 205 nm. The injection volume was 20 µL. The samples were prepared by the protein precipitation method using acetone. Plasma samples (200 µL) were transferred to 2.0 mL centrifuged tubes and 20 µL of IS (100 ng mL⁻¹ in acetonitrile) was added. The samples were vortexed for approximately one min and 400 µL of acetone was added. The samples were again gently vortexed for approximately two min and centrifuged at 14,000 rpm for 15 min. The supernatant was carefully taken and evaporated to dryness. The residue was reconstituted with an appropriate quantity of mobile phase and 20 µL was injected into the HPLC system for the analysis.

Chromatographic experiments were conducted on a Shimadzu (Shimadzu Corporation, Kyoto, Japan) HPLC instrument comprising quaternary LC-10A VP pumps, a variable wavelength programmable UV-visible detector, an SPD-10A VP column oven (Shimadzu Corporation, Kyoto, Japan), and a SCL 10A VP system controller. The instrument was controlled by use of Class VP 5.032 software (Shimadzu Corporation, Kyoto, Japan) installed with the equipment. Dried plant extract (10 mg) obtained by a rotatory evaporator was dissolved in 10 mL of HPLC grade methanol and sonicated for dissolving. This sample solution was filtered through a 0.22 µL syringe and then injected using a Rheodyne injector fitted with a 20 µL fixed loop. The separation was achieved using a column with dimensions of 15 × 4.6 mm, a particle size of 5 µm, and a C18 reverse phase column. The mobile phase used consisted of acetonitrile and water in the ratio of 50:50 v/v. All the analyses were performed at room temperature and chromatograms were monitored at wavelengths of 220, 254, and 300 nm using a UV visible detector (Shimadzu Corporation, Kyoto, Japan).