Liquid chromatography system

The carotenoid composition of each sample was analysed by HPLC with a Waters liquid chromatography system equipped with a 600E pump and a photodiode array detector (DAD) model 2998, and Empower3 software (Waters, Spain). A C30 carotenoid column (250 × 4.6 mm, 5 μm) coupled to a C30 guard column (20 × 4.0 mm, 5 μm) (YMC, Teknokroma, Spain) was used. The chromatographic conditions are described in Lado et al. (2015) (link) and Rodrigo et al. (2015) (link). Absorbance spectra and retention time identified each carotenoid, peaks were integrated at their individual maximal wavelength, and their contents were calculated using the appropriate calibration curves, as described elsewhere (Lado et al., 2015 (link); Rodrigo et al., 2015 (link)).

The TP content of camellia seed oil was extracted by methanol–water solution (80%:20% v/v) and determined by Folin–Ciocalteu method according to the colorimetric method described previously by Delfan‐Hosseini, Nayebzadeh, Mirmoghtadaie, Kavosi, and Hosseini (2017). A calibration curve of gallic acid in methanol was carried out in the concentration ranges of 0.04–0.40 mg/ml. The results were expressed as μg gallic acid equivalent per gram of oil samples. Triplicate test was performed for each sample.
Tocopherols content of the extracted oils was determined using UPLC method with fluorescence detection. Waters liquid chromatography system equipped with a column heater, a photodiode array detector ACQ‐FLR, controlled by Waters Empower chromatographic software. In all analyses, an Acquity UPLC Waters BEH C18 column of 1.7 μm (2.1 × 50 mm) was used. The analysis was carried out at 35°C temperature under isothermal condition, and the mobile phase was composed of 100% acetonitrile. The volume of injection is 10 μl, and the flow rate was 0.5 ml/min. Using FLR to detect and quantify α‐tocopherol, the excitation wavelengths and emission wavelengths are 294 nm and 338 nm, respectively. A calibration curve of α‐tocopherol in toluene was performed in the concentration ranges of 0–300 mg/ml. Results were expressed in mg of α‐tocopherol per kilogram of oil.

Polyamine analysis was carried out as described by Flores [21 (link)], but with some modifications. An amount of 0.5 g anthers was weighed and the pollen collected with PBS buffer (0.1 M Na₂HPO₄ and 0.1 M NaH₂PO₄, pH = 7.0), then the pollen was mixed in 1 mL of extraction solution (5% HClO₄) and stirred into the homogenate. The solution was placed in an ice bath for 30 min and then centrifuged at 4 °C for 30 min; 1 mL of supernatant was removed and 7 μL of benzoyl chloride and 1 mL of 2 mol/L sodium hydroxide were added. After being vortexed for 30 s, it was placed in a 37 °C water bath for 30 min, 2 mL of saturated sodium hydroxide was added to terminate the reaction, and 1 mL of ether was added for extraction; it was then centrifuged at 15,000 rpm for 30 min, 1 mL of supernatant was removed, and a vacuum dryer was used to volatilize the ether. All reagents and samples were required to be filtered with a 0.22 μm filter membrane.
HPLC analysis was performed using a Waters liquid chromatography system (Milford, MA, USA). Polyamines were separated and analyzed via HPLC on a C18 column (4.6 × 100 mm, 3.5-Micron) with a mobile phase of methanol/double distilled water (64/36 v/v) at a flow rate of 0.7 mL/min. Absorbance was measured at 230 nm. Samples were tested using three biological replicates.

Carotenoid extraction and quantification was performed as previously described with modification [30 (link)]. Carotenoids were analyzed by reversed phase HPLC. Chromatography was carried out with a Waters liquid chromatography system equipped with a model 600E solvent delivery system, a model 2996 photodiode array detection (PAD) system, a model 717 plus autosampler, and an empower Chromatography Manager. Carotenoids were eluted with MeOH- Acetonitrile [75:25 v/v, eluent A] and MTBE [eluent B] using a C30 carotenoid column (15 × 4.6 mm; YMC, Japan). Carotenoids were identified by their characteristic absorption spectra, typical retention time, and comparison with authentic standards (Bern, Switzerland).

The shoot and root samples of L. album (1 g) were prepared using a pestle and mortar in liquid nitrogen, followed by extraction in methanol (80% v/v). The samples were then sonicated at room temperature for 1 h. Dichloromethane (4.0 mL) and water (4.0 mL) were added to obtain a partition of compounds between two layers, followed by centrifugation at 5000 rpm for 15 min. The dichloromethane fractions were then collected, dried, and dissolved in 1.0 mL of HPLC-grade methanol for HPLC analysis^{65 (link)}.
The Lignan content was determined using a Waters liquid chromatography system consisting of a 2695 Separations Module (USA) and a 2487 Dual Absorbance Detector (USA). Data acquisition and integration were carried out with Millennium32 software. The chromatographic separations were performed on a 250 × 4.6 mm Eurospher 100–5 C18 column (KNAUER company, Berline, Germany) with a reversed-phase matrix in a gradient system using acetonitrile (solvent A) and distilled water (solvent B) with a 1 mL min⁻¹ flow rate. A UV detector was set at 280 nm. The presence of PTOX, 6-MPTOX, and SECO was identified based on retention time and comparison of UV spectra with the authentic PTOX, 6-MPTOX, and SECO standards purchased from Sigma-Aldrich (Taufkirchen, Germany). Different concentrations of the three compounds (25, 50, 75, and 100 µg mL⁻¹) were used for the calibration curves.

VOO phenolic compounds were isolated by solid phase extraction (SPE) on a diol-bonded phase cartridge (Supelco, Bellefonte, PA, USA) based on the method by Mateos et al. [25 (link)] using p-hydroxyphenylacetic and o-coumaric acids as internal standards. Phenolic compounds were analyzed by HPLC on a Beckman Coulter liquid chromatography system equipped with a System Gold 168 detector, a solvent module 126 and a Waters column heater module following a previously described methodology [26 (link)]. A Superspher RP 18 column (4.6 mm i.d. × 250 mm, particle size 4 µm: Dr Maisch GmbH, Ammerbuch, Germany) at flow rate 1 mL min⁻¹ and a temperature of 35 °C was used. Tentative identification of compounds was conducted with their UV-Vis spectra and later confirmed with HPLC/ESI-qTOF-HRMS on a liquid chromatograph Dionex Ultimate 3000 RS U-HPLC liquid chromatograph system (Thermo Fisher Scientific, Waltham, MA, USA) equipped with a similar column and elution program. Mass spectra were acquired in MS fullscan mode and data were processed using TargetAnalysis 1.2 software (Bruker Daltonics, Bremen, Germany).