Hplc elsd system

Simple sugars were analyzed using an HPLC-ELSD system (Shimadzu, Tokio, Japan) equipped with an evaporative light scattering detector (ELSD) LT III. Before injection, the samples were thermostated at 10 °C, and the injection volume was 4 μL. The separation was achieved using a Shodex Asahipak NH2P-40 3E (250 mm × 3.0 mm i.d.; 4 μm) column with a Shodex Asahipak NH2P-50G 3A (10 mm × 3.0 mm i.d.; 5 μm) precolumn thermostated at 40 °C. The mobile phase was composed of water (E) and acetonitrile (F), and the gradient elution was as follows: 80–85% F; 0.80–0.90 mL/min (0–5 min), 85–87% F; 0.90–0.95 mL/min (5–7 min), 87–80% F; 0.95–0.90 mL/min (7–10 min), 80% F; 0.90–0.80 mL/min (10–16.5 min). The analysis and results were controlled by LabSolutions™ Lite software version 5.106 SP1. Compounds were identified by comparison with the external standards of glucose and fructose. Results are presented as the mean ± standard deviation of three replications and expressed in mg/100 g dw of fruit.

To enable separation of individual disaccharides, the honey samples were chromatographed on a Shimadzu HPLC-ELSD system consisting of a Shimadzu Class VP Pump LC-10AD VP/Valve FCL-10AL VP/Degasser DGU-14A with a Class VP Software/ SCL-10A VP controller, SIL-10AD VP autosampler and a CTO-10A VP column oven operated at 40 °C. Separations were performed on a Phenomenex Luna 5 µm NH₂ 100 Å 250 × 4.6 mm column with an isocratic mobile phase comprised of 85% acetonitrile and 15% RO water at a flow rate of 2.5 mL/min. Eluted sugars were monitored with a Shimadzu ELSD- LT (Low Temperature) detector operated at 350 kPa and 45 °C. The elution flow was diverted to a collection tube when the target peak emerged and 5 mL (2 min) fractions collected before reconnection of the column eluant to the ELSD detector (to see the tail end of the peak). Collected fractions were analysed ‘as is’ by UPLC-MS/MS (as above), and a second portion freeze-dried and dissolved in D₂O for NMR analysis.