Kinetex polar c18 column

The quantitative profiling of the hydroalcoholic extracts of R. officinalis was performed via high HPLC-UV analysis using an Agilent instrument equipped with a Phenomenex Kinetex Polar C18 column (100 × 3.0 mm, 100 Å, 2.6 µm). The chromatographic profile was obtained using the same chromatographic method used for the LC-HRESIMSMS analysis. The injection volume was 10 µL, and the UV detection wavelength was set at 280 nm.
The calibration curve was obtained using salvigenin as internal standard at concentrations of 125, 250, 500, 1000, 2000, and 4000 ppm. The coefficient determination (R²) of the calibration curve had good fitness (0.9975–0.99874); salvigenin present in the extracts was quantified and used as reference to relatively quantify the other components by means of the % Area.

The qualitative analysis of three studied actinomycetes extracts was performed by high performance liquid chromatography-mass spectrometry (HPLC-MS) using an LTQ-XL Ion Trap mass spectrometer (Thermo Fischer Scientific Spa, Rodano, Italy) equipped with an Ultimate 3000 HPLC (Agilent Technology, Cernusco sul Naviglio, Italy). Chromatographic separation was obtained using a Kinetex Polar C₁₈ column (100 × 3.0 mm, 100 Å, 2.6 µm) (Phenomenex, Torrance, CA, USA). The injection volume was 0.5 mL/min and a mobile phase consisting of a combination of A (0.1% formic acid in water, v/v) and B (0.1% formic acid in acetonitrile MeCN); a linear gradient, which ranged between 5 and 60% B in 25 min, from 60 to 95% B in 10 min, and held at 95% B for 5 min, was used. The mass spectrometer was set in positive ion mode. ESI source conditions were the following: capillary voltage −48 V; tube lens voltage −176.47; capillary temperature 280 °C; sheath 15 and auxiliary gas flow (N₂) 5; sweep gas 0; and spray voltage 5. MS spectra were obtained at 30,000 resolutions by full-range acquisition with a scan range between 150 and 1500 m/z.

The C. sappan L. heartwood extracts were measured for brazilin content using reverse phase HPLC with the modified method of Yan-yan et al. [50 (link)]. The HPLC uses an Agilent 1200 equipped with a multi-wavelength detector. The detection wavelength was set at 280 nm. The assay was carried out using a Kinetex^® polar C18 column (4.6 mm × 150 mm, 2.6 µm particle diameters, Phenomenex Co., Ltd., Torrance, CA, USA) and 0.1% acetic acid in methanol and deionized water in the ratio of 25:75 was used for the mobile phase at the flow rate of 0.6 mL/min. The column temperature was set to 25 °C with an injected sample volume of 10 µL.

Olive leaf extracts were analyzed to determine the content of oleuropein by HPLC-UV analysis using a previous method with slight modifications [52 (link)]. The extracts were reconstituted in MeOH, and HPLC-UV analyses were performed on an UltiMate 3000 instrument equipped with an RS-3000 Diode Array Detector operating at 280 nm. Xcalibur software was used to acquire and process the data. A Kinetex Polar C18 column (150 × 2.1 mm, 2.6 µm, 100 Å; Phenomenex, Bologna, Italy) with a SecurityGuard C18 guard column (2 × 2.1 mm), thermostated at 35 ± 1 °C, was used for the chromatographic separation. The solvents used for the elution gradient were A (H₂O containing 0.1% formic acid) and B (acetonitrile containing 0.1% formic acid); the linear gradient was eluted from 5% to 50% of B (0–10 min), and from 50% to 95% of B (10–15 min). The injection volume was 3 μL, and the flow rate of 0.4 mL/min. The quantitative determination of oleuropein was carried out via external calibration. The calibration curve was acquired by injecting standard solutions in the linearity range of concentrations, 5–100 mg/L, obtaining the equation y = 1930.8x, R² = 0.9998. The LOD and LOQ were also determined as 1.00 and 3.00 mg/L, respectively. All samples were extracted and analyzed in triplicate (n = 9), and the results are expressed in milligrams per gram as mean concentration ± standard deviation.