Photodiode array detector

The experimental plant materials, including the leaves, stems, roots, transgenic root cultures, and control root cultures, were thoroughly dried at 40°C and then ground into a fine powder for use in the extraction of alkaloids (Xia et al., 2016 (link)) and their HPLC analysis (Wang et al., 2011 (link)). This alkaloid analysis was performed on the LC-20A system of HPLC with a photo-diode array detector (Shimadzu, Kyoto, Japan), while a CTP-ODS column (Phenomenex, Torrance, CA, United States) was used at 40°C with a 226-nm detecting wavelength. A sample of 20 μl was injected each time. The authentic samples included the hyoscyamine and scopolamine, as purchased from Sigma–Aldrich (St. Louise, MO, CA, United States).

Plasma malondialdehyde (MDA) was determined using high performance liquid chromatography (HPLC) with photodiode array detector (Shimadzu, Japan) as described by Pilz et al. [20 (link)] with some modifications. Briefly, samples (50 μL) were mixed with 200 μL of 1.3 M NaOH and incubated at 60°C for 30 min. After cooling the mixture, 100 μL of 35% HCIO₄ was added and centrifuged at 10 000 g for 10 min. Supernatant of the samples (300 μL) was transferred into 1.5 mL of HPLC tubes and 5 mM of DNPH solution (50 μL) was added into the mixture and incubated for 30 min at room temperature. Then, samples (40 μL) were injected into the HPLC.
The amount of MDA was expressed as concentration of MDA in nmol per mL plasma.

Lycopene was extracted from cultures and analyzed as described previously (McNerney and Styczynski, 2017a (link)). Briefly, 500 μL of bacterial culture was pelleted and resuspended in 50 μL of ultrapure water. Lycopene was extracted with 1 mL of acetone at 50°C for 20 min. Cellular debris was pelleted, and the supernatant was removed for analysis. Sudan I (TCI America, Portland, OR, United States) was used as an internal standard (Xu et al., 2006 (link)) and added to the acetone used for extractions at a concentration of 1 μg/mL.
All HPLC analysis was conducted on a Shimadzu Prominence UFLC using an Agilent C18 4.6 mm × 50 mm column with a 5 μm particle size and a Shimadzu photodiode array detector. A solvent ratio of 50:30:20 acetonitrile:methanol:isopropanol was used as the mobile phase (Lv et al., 2016 (link)) and run at a flow rate of 1 mL/min with a 25 μL sample injection volume. Absorption was detected at 471 nm. Retention times and peak intensities were compared to an analytical lycopene standard (Millipore Sigma, St. Louis, MO, United States) spiked into control extractions from DH10B cells, and the internal standard Sudan I was used to account for acetone evaporation during the extraction protocol and for instrument drift.

Twenty-five milligrams of GPE were mixed with 5 mL of 2% (v/v) methanol, placed in an ultrasonic bath for 10 min, shaken for 30 min, and centrifuged at 8000 rpm for 5 min. The supernatants were collected, and the precipitate was extracted twice by the foregoing procedure. Then, 1 mL supernatant was evaporated to dryness at 40 °C in the RapidVap evaporator, and the residue was redissolved in 1 mL of solution A (32:4:1 (v/v/v) water/acetonitrile/methanoic acid). The extracts were then passed through a 0.45 μm syringe filter. The target anthocyanins were determined using HPLC coupled with a photodiode array detector (Shimadzu Corp., Suzhou, China) and a Synergi Hydro-RP C18 column (250 mm × 4.6 mm, 4 µm; Phenomenex, Torrance, CA, USA). The test parameters used were previously published [29 (link)]. The mobile phase consisted of solutions A and B (16:20:1 (v/v/v) water/acetonitrile/methanoic acid), the detection wavelength was 520 nm, the column temperature was 35 °C, the flow rate was 1 mL/min, and the gradient was 0–10% B at 0–15 min, 10–20% B at 15–30 min, 20–35% B at 30–45 min, 35–100% B at 46–50 min, and 100% B at 50–51 min.