996 photodiode array pda detector

A Waters 1525µ HPLC (Milford, MA) was used for the chromatographic separation, performed with a Waters XBridge C18 (150 × 2.1 mm i.d.) 5 μm analytical column; A (MilliQ water/formic acid 5 mM) and B (acetonitrile/formic acid 5 mM) were used as mobile phase for the elution binary gradient (Petrucci et al. 2020b (link)) slightly modified. Briefly: 0–1 min, 5% B; 1–20 min, 16.5% B; 20–30 min, 40% B; 30–35 min, 60% B; 35–36 min, 80% B; 36–40 min, 80% B; 40–41 min, 5% B; 41–61 min, 5% B to equilibrate the column, flow rate of 0.20 mL/min. The Waters 996 photodiode array (PDA) detector was set for one spectrum/second, range 200–800 nm, resolution 1.2 nm. The Quattro Micro Tandem MS/MS with a Waters ESI source (Micromass, Manchester U.K.) acquired data in negative ionization ESI(-), capillary voltage 2.7 kV, cone voltage 27 V, source temperature 120 °C, desolvation temperature 350 °C, cone gas flow 40 L/h, desolvation gas flow 500 L/h (Petrucci et al. 2020b (link)). 16 separated channels for 16 different m/z value for the selected ions [M–H]⁻ were used to acquire spectral data in SIR mode, dwell cell value of 0.200 s. Data acquisition, data handling, and instrument control were performed by MassLynx Software 4.1 v (Data Handling System for Windows, Micromass, U.K.).

Tissues (lamina of the second-youngest fully expanded leaf) of the same sampled plants were frozen in liquid N₂ and freeze-dried to preserve metabolites. Organic solutes (sugars, sugar alcohols) were extracted twice from 0.1 g of a ground leaf tissue with 3 mL of ice-cold 5% (w/v) perchloric acid, following previously described methods [82 (link),83 (link)]. The supernatants from each extraction were combined and neutralized using K₂CO₃ until pH 3.0–3.5; the neutralized supernatants were collected after centrifugation at 15,000× rpm for 30 min. The neutralised extracts were analysed using high-performance liquid chromatography (HPLC), with a 600 E pump, 717 plus autoinjector, and 996 photodiode-array (PDA) detector (Waters, Milford MA, USA) with an evaporative light-scattering detector (ELSD) (Alltech, Deerfield, IL, USA). Separation was achieved with a Sugar-Pak column, as described elsewhere [82 (link)]. The Sugar-Pak column (300 × 6.5 mm i.d.) was held at 90 + 0.5 °C with separation achieved using a mobile phase of 2.5 mg L⁻¹ Ca-EDTA at 0.6 mL min⁻¹. Detection and quantification were undertaken with the PDA at 195 nm, with peak spectral and purity comparisons of samples made with the standards.