Ultimate 3000 system

UPLC-DAD analyses were performed on an UltiMate 3000 system (DIONEX, Sunnyvale, CA USA) equipped with a pump, an autosampler, a column compartment and a diode array detector (DAD). Chromatographic separation was carried out on an ACQUITY UPLC HSS T3 column (2.1 × 100 mm, 1.8 µm, Waters Corp., Milford, MA, USA) at a column temperature of 30 °C, with a flow rate of 0.3 mL/min. Gradient elution of 0.1% formic acid solution (solvent A) and acetonitrile (solvent B) was employed: 0~2 min, 5% B; 2~12 min, 5~15% B; 12~20 min, 15~20% B; 20~25 min, 20~40% B; 25~30 min; 40~95% B. The injection volume was 5 µL, and the detection wave-length was set at 280 nm.

Blood was collected in heparin tubes and centrifuged for 5 min at 5000 r/min. Plasma was separated from blood cells, and about 1 mL was diluted with 2 mL water and loaded onto a tC18 Sep-Pak cartridge (Waters, Milford, MA), which was pre-activated by elution with 6 mL of methanol and 12 mL of water, respectively. The cartridge was washed with 3 mL water to collect the polar radioactive fraction. Thereafter, the tC18 Sep-Pak cartridge was eluted with 2 mL of methanol and 2 mL of water to collect the mixture of non-polar metabolites. This fraction was further analysed by HPLC using an Ultimate 3000 system (Dionex, Sunnyvale, CA) equipped with a 1-mL loop. As a stationary phase, a Gemini C18, 250 × 10 mm, 5 µm (Phenomenex, Torrance, CA) was used. The mobile phase was a gradient of A = acetonitrile and B = 0.1% trifluoroacetic acid in water. The gradient ran for 15 min, decreasing the concentration of eluent B from 90% to 40% in 11 min, followed by 1 min of elution with 40% B at a flow rate of 4 mL min⁻¹. The eluent was collected with a fraction collector (Teledyne ISCO Foxy Jr., Lincoln, NE), and the fractions were counted for radioactivity using a Wallac 2470 gamma counter (Perkin Elmer, Waltham, MA).

For lipidomics experiments, a Thermo Scientific Quadrupole-Orbitrap-high resolution Mass Spectrometry coupled to a Dionex UltiMate 3000 system was used, with an ESI ionization source in both positive- and negative-ion modes. All samples were maintained at 4 °C in the autosampler prior to injection (5 μL) onto the column in duplicate. Lipids were separated on a Phenomenon ACE Excel 1.7 C18-AR UPLC PR column (100 mm × 2.1 mm) at a mobile phase flow rate of 0.3 mL/min, and the temperature of the column was maintained at 40 °C. Mobile phase A was acetonitrile containing 0.1% acetic acid, and mobile phase B was 0.1% acetic acid in water. The gradient conditions were as follows: 0–5 min, 30% A; 5–30 min 30–90% A; 35–40 min 30% A. The mass spectrum parameters of the positive and negative model were as follows: capillary temperature and probe heater temperature to 320 °C, AGC target to MS 1 e⁶ to MS/MS 2 e⁵, sheath gas to 35 arb, and AUX gas to 10 arb. The positive-ion and negative-ion modes were set to 3.5 and 3.2 kV as well as an acquisition rate of 5 spectra/s. Mass spectrometric data were acquired in a full-scan mode (m/z 100–1000).

Glucose concentration was used as a measure of saccharification efficiency in all enzymatic experiments. Concentrations of glucose in all hydrolysates were determined on an UltiMate 3000 system from Dionex (Sunnyvale, CA) with a Rezex ROA-Organic Acid H + column (Phenomenex; Torrance, CA) running 0.5% sulfuric acid at 0.6 mL/min as the eluent.

HPLC analysis was performed using a Dionex UltiMate 3000 system (Dionex Corp., Sunnyvale, CA, USA) equipped with a binary pump, autosampler, column oven, and DAD. System control and data analysis were carried out using Dionex Chromelon software. Separation was carried out on a Luna C18 column (250 × 4.6 mm, 5 μm, Phenomenex, Torrance, CA, USA), with the column oven temperature kept at 30 °C, at a UV wavelength of 330 nm. The mobile phase consisted of water (solvent A) and acetonitrile (solvent B) with gradient elution of 0–5 min, 5–15% B; 5–12 min, 15–20% B; 12–30 min, 20–25% B; 30–40 min, 25–50% B; 40–60 min, 50% B; and 60–70 min, 50–80% B, at a flow rate of 1.0 mL/min. Before injection of the next sample, the column was re-equilibrated with the initial gradient of solvents for 10 min.

LC-(Q) TOF/MS analysis was performed on an UltiMate 3000 system (Dionex, Sunnyvale, CA, USA) comprising a pump, a UV detector (208 nm), and an autosampler cooled to 4 °C with an Inertsil ODS-3v column [5 µm, 4.6 × 250 mm (GL Science Inc., Tokyo, Japan)]. The two samples (each 50 µL) were directly injected into the HPLC column. The separation of tomatines was eluted with 20 mM ammonium acetate/acetonitrile (65:35, v/v) at a flow rate of 700 μL/min at 30 °C, and a MicroQ-TOF III mass spectrometer with an electrospray interface (ESI) source (Bruker Daltonics, Bremen, Germany). The interface voltage and current were 4.50 kV and 1.6 μA for the negative-ion mode. The flow rate of nebulizing gas was 1.5 L/min, and the N₂ drying pressure was 0.2 M Pa. The curved desorption line and heat block temperature were both at 200 °C. The detector voltage of the TOF analyzer was 1.68 kV. Ultrahigh-purity argon was used as the collision gas for collision-induced dissociation experiments. The relative energy in collisions was 100%. The sample injection volume was 50 μL. A direct valve was set to transmit and divert the HPLC eluent to waste. Mass spectral data were collected from m/z 160–1100. Data acquisition and processing were carried out with micrOTOFcontrol and dataAnalysis 4.0 (Bruker Daltonics, Bremen, Germany) software.