Spd m20a diode

The ACE inhibition rate was determined using HPLC. A Shimadzu LC‐2010A HT system (Shimadzu) equipped with VP‐ODS C₁₈ column and SPD‐M20A diode array detector with detection wavelength of 283 nm was used. The mobile phase consisted of methanol and water (3:7, v/v,) at a flow rate of 1.0 ml/min and a column temperature of 30°C. Sample solution, ACE, and HHL were prepared using BBS (0.1mol/L, pH 8.3) containing 0.3 mol/L KCl as a solvent. Sample solution (40 μl) was transferred to a test tube, and 20 μl of ACE and 100 μl of HHL (5 mol/L) were added. The mixture was incubated for 1 hr at 37°C, and HCl (1 mol/L) was used to stop the reaction. The treated sample solution (10 μl) was filtered through a 0.45 μm microporous membrane and then injected into the HPLC to determine the amount of Hip. The ACE scavenging capacity was determined as follows. $\text{Inhibition}(\%)=\left(A_0-A_1\right)/A_1\times 100\%$ where A₀ is the peak area of Hip in the blank group and A₁ is the peak area of Hip of the sample. Peak areas were determined by the software that came with the instrument.

The products (Me esters) were separated using RP-HPLC on a Macherey–Nagel Nucleosil 5 ODS column (250 × 4 mm, 5 μm) using the solvent mixture methanol/water (linear gradient from 60:40 to 96:4, by volume) at a flow rate of 0.4 mL/min. The peaks of the products were collected and purified using NP-HPLC, as described in the previous section. All HPLC analyses were performed with a Shimadzu LC-20AB solvent delivery pump with UV spectral monitoring (190–370 nm) using a Shimadzu SPD-M20A diode array detector (Shimadzu, Kyoto, Japan). Separate products were collected after NP-HPLC separation, redissolved in [²H₆]benzene, and then, the NMR spectra were recorded. For additional qualitative information, the products (Me/TMS) were re-analyzed using GC-MS after hydrogenation over PtO₂ or after sequential NaBH₄ reduction, hydrogenation, methylation, and trimethylsilylation.
Product 1 collected after the NP-HPLC purification was dissolved in [²H₆]benzene and subjected to the NMR spectral records. Then, product 1 was reduced with NaBH₄ to compound 2, which was finally purified using NP-HPLC, as described in the previous section. Then, product 2 was dissolved in [²H₆]benzene, and its NMR spectra were recorded.

Individual carotenoid content was determined using RP-HPLC, which was performed using a Shimadzu LC-20AT with an SPD-M20A diode array detector (DAD) (Shimadzu Corporation, Kyoto, Japan), as described by Ghendov-Mosanu et al. [10 (link)]. Comparison of the UV–Vis spectra and the retention times of the sample peaks with those of the standard solutions and spectral data reported in the literature allowed for the identification of carotenoids from sea buckthorn samples (Table 5).