Sil 10a autosampler

QCT, LUT, and 3-O-MQ content into crude extract, nanoemulsions, and skin/mucosa after permeation studies were determined using a liquid chromatography equipment Shimadzu LC-10A, equipped with LC-10AD pump, CBM-10A system controller, SIL-10A autosampler, SPD-20AV UV/vis detector (set at 362 nm), and LC Solution software. The chromatographic system was composed by a Synergi Polar-RP 150 × 4.6 mm i.d., 4 μm (Phenomenex, Torrance, CA) column protected by precolumn packed with silica C18 Phenomenex (150 μm, 140 Å), temperature system of 30 ± 1°C, isocratic flux of 0.8 mL/min, and 20 μL as injection volume. The mobile phase consisted of methanol : 0.16 M phosphoric acid : acetonitrile (46 : 44 : 10, v/v/v) and the samples were diluted in methanol : phosphoric acid 16 mM (50 : 50, v/v) before analyses. The analytical method was previously validated for the determination of QCT, LUT, and 3-O-MQ in ethanolic extract and nanoemulsion [8 (link)], demonstrating to be specific, linear (0.25 to 10 µg/mL), precise, and accurate. In this paper, the revalidation of the analytical method was performed in terms of specificity and recovery of QCT, LUT, and 3-O-MQ from porcine ear skin and porcine esophageal mucosa. The limits considered acceptable for the evaluated parameters are in accordance with the “The Guidance for Industry: Bioanalytical Method Validation” (FDA).

The HPLC apparatus was as follows: a Shimadzu LC-10ATvp system HPLC (Shimadzu, Kyoto, Japan) equipped with an LC-10AT pump (Shimadzu), a Sil-10A autosampler (Shimadzu), an SPD-M10A DAD, and a Shimadzu Class-VP 6.12 chromatographic data processor was used. The analytical column was a C18 column (250 mm × 4.6 mm.d × 5 μm, Shimadzu). The mobile phase was a mixture of methanol and water (50/50, volume ratio), and the flow rate was 1.0 mL/min. The detection wavelength was 228 nm, and the injection volume was 20 μL [39 ].
ACE inhibitory activity was measured by high-performance liquid chromatography (HPLC) to determine the potential antihypertensive activity. Samples were prepared using 100 mmol/L sodium borate buffers (pH 8.3). The samples contained 30 μL of 5 mmol/L Hippuryl-His-Leu-OH (HHL) and 20 μL of the enzymatic hydrolysate as a substrate. After incubation for 3 min at 37 °C, 20 μL of ACE powder (0.1 units) was added to this tube. Finally, the mixture was incubated for 30 min at 37 °C, and the reaction was terminated by adding 60 μL of 1 mol/L HCl. Sodium borate buffers (100 mmol/L) were included as a blank control.
The ACE inhibitory activity was calculated using the following equation:
where A = peak area of hippuric acid in the blank group, and B = peak area of the reaction solution at the retention time of hippuric acid.

The LC-10A HPLC (Shimadzu, Kyoto, Japan) was equipped with LC-10ATvp binary pump (Shimadzu, Kyoto, Japan), SIL-10A autosampler (Shimadzu, Kyoto, Japan), and SPD-M10Avp detector (Shimadzu, Kyoto, Japan). The instrument was controlled by a computer and the Lab Solutions (Shimadzu, Kyoto, Japan) chromatography workstation was used to analyze the data. To obtain better chromatographic peaks of zeaxanthin and lutein, various mobile phase systems were optimized, and the optimal chromatographic separation conditions were determined. The Shimadzu^® C18 column (4.6 mm × 250 mm, 5 μm; Kyoto, Japan) was used for the analysis of the zeaxanthin and lutein contents. The optimum chromatographic conditions for the flow rate, detection wavelength, column temperature, and the injection volume were selected as 1.0 mL/min, 450 nm, 25 °C, and 20 μL, respectively. The mobile phase consisted of acetonitrile/dichloromethane (95:5, v/v).