Hplc dad

The cannabinoid profile analysis of the extract was studied with the use of ultra-high-performance liquid chromatography with the diode array detector (HPLC-DAD) validated method (Shimadzu Corp., Kyoto, Japan) [42 (link)]. The determination was performed with the use of a chromatographic column CORTECS Shield RP18, 2.7 µm; 150 mm × 4.6 mm. As a mobile phase, 0.1% trifluoroacetic acid (41%), and acetonitrile (41:59, v/v) were used. The flow rate was set at 2.0 mL/min, and the column temperature was set at 35 °C. The injection volume was 10.0 µL, the detection wavelength was 228 nm, and the analysis time was 50 min. The retention time of cannabinoids is presented in Table 2. The results were acquired and processed using LabSolutions LC software (version 1.86 SP2) from Shimadzu Corp. (Kyoto, Japan).

The extraction of LMWOAs was carried out based on the method described by Cayún et al. [18 (link)] with minor modifications; 0.5 g of rhizosphere soil was crushed and dissolved in 1 mL of 0.2 M calcium chloride, shaken, and centrifuged (Centurion Scientific Ltd, Bosham, UK) at 4000× g at 4 °C for 15 min. Afterward, the supernatant was filtered through 0.45 μm filters. The extraction procedure was repeated three times.
Chromatographic separation was performed using high-performance liquid chromatography with diode array detection (HPLC-DAD) (Shimadzu, Tokyo, Japan) equipped with a quaternary pump (LC-20AD), a degassing unit (DGU-20A5R), a column oven (CTO-20A), an autosampler (SIL-20A), and a UV–Vis diode array detector (SPD-M20A) using a C₁₈ column (Eclipse, 250 × 4.6 mm, 5 µm) and a C₁₈ precolumn (NovaPak, Waters, 22 × 3.9 mm, 4 µm). The mobile phase was 0.2 N phosphoric acid (pH 2.1) at a flow rate of 1.0 mL min⁻¹ with isocratic elution at 30 °C. Detection was performed at 210 nm with an analysis time of 15 min using oxalic acid (y = 12,295x + 11,179, detection limit (DL): 1.90 mg L⁻¹, quantification limit (QL): 6.34 mg L⁻¹, linear range (LR): 6.34 to 100 mg L⁻¹) and citric acid (y = 1274.2x + 3763.9, DL: 0.20 mg L⁻¹, QL: 0.66 mg L⁻¹, LR: 0.66 to 500 mg L⁻¹) as external calibration standards.

Ascorbic acid content was assessed by HPLC according to the method reported in literature [24 ]. Extraction was made directly in mobile phase; the mixture was centrifuged at 5000 rpm for 5 min and filtered through 0.45 µm PVDF syringe filters (Millex^®-GV, Merck, Darmstadt, Germany). Sample injection volume was 20 µL. The equipment was a HPLC-DAD Shimadzu (Tokyo, Japan), and a reverse phase C18 column (5 µm, 250 × 4.6 mm particle size) (Agilent Technologies, Santa Clara, CA, USA) was used. The solvent system consisted in 20% methanol and 80% phosphate buffer (20 mM, pH 4.0). The flow rate was set at 1 mL·min⁻¹, and detection wavelength was set at 240 nm. The column oven was set at 40 °C. All measurements were made in triplicate.